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Cellular mechanism and regulation of KCl transport across an insect epithelium Hanrahan, John William 1982

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CELLULAR MECHANISM AND REGULATION OF KCl TRANSPORT ACROSS AN INSECT EPITHELIUM by - JOHN WILLIAM HANRAHAN B.Sc. (Hon.) DALHOUSIE UNIVERSITY, 1976 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n - THE FACULTY OF GRADUATE STUDIES DEPARTMENT OF ZOOLOGY We accept t h i s t h e s i s as conforming to the r e q u i r e d standard February, 1982 © John W i l l i a m Hanrahan, 1982 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or p u b l i c a t i o n of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of Zoology The University of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 D a t e March 25, 1982 •6 (2/79) i i ABSTRACT The c e l l u l a r mechanism and regulation of KC1 reabsorption across the rectum of the desert locust Schistocerca gregaria has been studied using tracer fluxes, ion-sensitive microelectrodes, and electrophysiological techniques. Serosal addition of 1 mM cAMP stimulates t r a n s e p i t h e l i a l short-C l c i r c u i t current (I ) and net Cl absorption (J ) 10-fold, increases sc r net tr a n s e p i t h e l i a l potential (V ) 4-fold, and reduces t r a n s e p i t h e l i a l resistance DY 40-65%. The properties of locust Cl transport are not consistent with NaCl cotransport models proposed i n other e p i t h e l i a : i ) Cl i s absorbed from nominally Na-free saline, i i ) there i s no correlation between trace amounts of Na contamination and the rate of Cl transport, i i i ) exposure to cAMP increases 36 22 Cl i n f l u x across the ap i c a l border into r e c t a l tissue without affecting Na in f l u x , iv) Cl-dependent I i s not inhibited by 1 mM ouabain (2 h) or 1 mil C l furosemide (1 h), v) J . i s not affected when the apical Na electrochemical net v gradient i s reduced by 85%, and v i ) there i s no relationship between Na and Cl net electrochemical gradients across the apical membrane. Cl/HCO^ exchange i s Cl also unlikely since i) Cl-transport i s electrogenic, i i ) J n e t i s insensitive to CO2- and HCO^-removal, and i i i ) Cl-dependent I i s not inhibited by 1 mM SITS or 1 mM acetazolamide after 1 h exposure. The cAMP-stimulated system i s Cl-selective: C l >> Br >> I,F,SCN,P0^,SO^.C^O.-,,urate. The halide sequence suggests a s i t e having high f i e l d strength. Cl-dependent I i s inhi b i t e d by Cl low mucosal pH and high osmotic pressure. ^ n e t °beys Michaelis-Menten-type ki n e t i c s . Mucosal K increases both the K and V of t r a n s e p i t h e l i a l C l m max 1 Cl absorption (K = 5.3 mM K). The active step i n J i s at the apical a ' Y net v membrane because net entry of C l occurs against a large, unfavourable i i i electrochemical gradient". * Serosal cAMP and mucosal K d i r e c t l y stimulate the CI act i v e step since both of these agents cause simultaneous increases i n J net and the electrochemical p o t e n t i a l opposing CI entry. Passive K transport i n the mucosa-to-serosa d i r e c t i o n i s favoured across a p i c a l and basal membranes. Most K absorption (^84%) i s e l e c t r i c a l l y coupled to active CI transport under open-circuit conditions, however a small active component i s apparent during exposure to cAMP. The response of V to t r a n s e p i t h e l i a l s a l t gradients depends strongly on the d i r e c t i o n of the gradients, suggesting that locust rectum i s a " t i g h t " epithelium. I n t r a c e l l u l a r current and fluorescent dye i n j e c t i o n s reveal strong coupling between r e c t a l c e l l s . Flat-sheet cable analysis indicates that locust rectum becomes " t i g h t e r " during cAMP exposure, when t r a n s c e l l u l a r conductance increases from 60 to 95% of the t o t a l t i s s u e conduc-tance. cAMP increases a p i c a l membrane K conductance and basal membrane CI conductance. K permeability i s i n h i b i t e d by high (physiological) K and osmotic concentrations. The d r i v i n g force of CI transport i s calculated by two independent methods and the r e s u l t s are interpreted i n terms of an equivalent e l e c t r i c a l c i r c u i t model for KCl reabsorption across locust rectum. i v -Table of Contents Page Abstract i i Table of Contents i v L i s t of Tables v i i L i s t of Figures x L i s t of Abbreviations xv Acknowledgments - x v i i i CHAPTER 1: GENERAL INTRODUCTION 1 A. Ion tr a n s p o r t 1 Previous s t u d i e s on l o c u s t rectum 2 Mechanisms of a c t i v e CI transport i n i n s e c t s 6 Models proposed f o r a c t i v e CI tr a n s p o r t i n other e p i t h e l i a 8 I o n i c p e r m e a b i l i t y i n e p i t h e l i a 17 Towards a c e l l u l a r model f o r i n s e c t CI transport 18 B. Or g a n i z a t i o n of the l o c u s t excretory system 20 CHAPTER 2: PROPERTIES OF TRANS EPITHELIAL CHLORIDE TRANSPORT 25 Summary 25 In t r o d u c t i o n 27 M a t e r i a l s and methods 29 Animals 29 Flux chambers 29 So l u t i o n s 29 E l e c t r i c a l methods 33 T r a n s e p i t h e l i a l t r a c e r f l u x e s 34 Tracer i n f l u x e s across the a p i c a l c e l l border 36 C a l c u l a t i o n s and s t a t i s t i c s . . . . . . 39 Results . '. 39 1) V e r i f i c a t i o n of a c t i v e CI transport 39 2) Exchange d i f f u s i o n 49 3) S e l e c t i v i t y of CI tr a n s p o r t 52 4) I o n i c dependencies of CI transport .. 53 a) Sodium dependence:. . . 53 i ) Prolonged removal of e x t e r n a l Na 55 i i ) Short-term removal of e x t e r n a l Na. 58 i i i ) U n i d i r e c t i o n a l i n f l u x e s of 36 C1 and 22fta across the a p i c a l border 60 i v ) E f f e c t s of i n h i b i t o r s of Na-coupled CI t r a n s p o r t . . • 63 b) Bicarbonate dependence: 65 i ) CI f l u x e s i n HCO3, C02"free s a l i n e . 66 i i ) Changes i n e x t e r n a l pH induced by a c t i v e CI t r a n s p o r t 68 Page i i i ) E f f e c t s of i n h i b i t o r s of HCC^-coupled CI transport 70 c) Potassium dependence:.... 7 1 i ) E f f e c t s of K-free s a l i n e 72 d) Dependence on divalent cations: 72 5) Regulation of CI transport by low pH, hyperosmocity and "second messengers" 72 i ) E f f e c t s of pH 72 i i ) E f f e c t s of high osmotic concentration 79 i i i ) Second messengers and hormonal stimulation 8 1 a) C y c l i c nucleotides.... 8 1 b) I n t r a c e l l u l a r Ca 84 Discussion 86 Comparison with vertebrate systems 86 Sodium-coupled models 87 Bicarbonate-coupled models 9 1 Potassium dependence of CI transport 94 Exchange d i f f u s i o n 95 Anion s e l e c t i v i t y . . . 95 Regulation of CI transport 97 CHAPTER 3: INTERRELATIONSHIP BETWEEN ACTIVE CHLORIDE TRANSPORT AND POTASSIUM 98 Summary 98 Introduction 100 Materials and methods 1 0 1 K i n e t i c s of t r a n s e p i t h e l i a l 36ci fluxes 1 0 1 Measurement of potassium dependence of chloride transport 102 T r a n s e p i t h e l i a l 42K fluxes 103 Results 103 1) K i n e t i c s of c h l o r i d e absorption 1 0 3 2) I s c and 3 6 C 1 fluxes i n "high K" s a l i n e 115 3) Apparent potassium a c t i v a t i o n constant (K a) of CI transport.... .' 117 4) S e l e c t i v i t y of K a c t i v a t i o n of CI transport 1 2 1 5) Sidedness of K a c t i v a t i o n 1 2 5 6) T r a n s e p i t h e l i a l 42K fluxes ( s h o r t - c i r c u i t conditions) 127 i ) Normal s a l i n e 127 i i ) C l - f r e e s a l i n e 130 7) T r a n s e p i t h e l i a l 42x<; fluxes (open-circuit conditions) 1 3 1 8) T r a n s e p i t h e l i a l 4 2 K fluxes i n "high K" s a l i n e 137 Discussion..... 139 Active CI transport: the e f f e c t s of K.... 139 Passive K transport 1 4 1 Properties of active K transport- 1 4 5 v i Page CHAPTER 4: ELECTROCHEMICAL POTENTIALS 147 Summary. 147 Introduction 149 Materials and methods 150 The r e c t a l preparation.. 150 E l e c t r i c a l measurements 152 Fabri c a t i o n and c a l i b r a t i o n of io n - s e n s i t i v e microelectrodes 153 Mannitol space 157 Calculations and s t a t i s t i c s 157 Results 158 1) Steady-state measurements of i n t r a c e l l u l a r chloride and potassium a c t i v i t i e s 158 a) Chloride 160 b) Potassium 165 2) Relationship between chloride and potassium e l e c t r o -chemical p o t e n t i a l s 167 3) E f f e c t s of a l t e r i n g the sodium electrochemical gradient across the a p i c a l membrane 179 4) Mannitol space , 184 Discussion 187 Meansurements of membrane potentials and i n t r a c e l l u l a r ion a c t i v i t i e s 189 Location and mechanism of act i v e chloride transport 190 Interpr e t a t i o n of potassium and sodium electrochemical p o t e n t i a l p r o f i l e s 195 CHAPTER 5: ELECTROPHYSIOLOGY 199 Summary 199 Introduction 201 Materials and methods.. 202 Ef f e c t s of t r a n s e p i t h e l i a l s a l t gradients 202 Voltage scanning 203 I n t r a c e l l u l a r i n j e c t i o n of fluorescent dye 204 E l e c t r i c a l coupling and f l a t - s h e e t cable analysis 205 T r a n s e p i t h e l i a l ^ 2K fluxes 208 Results 210 1) E f f e c t s of s a l t gradients on t r a n s e p i t h e l i a l p o t e n t i a l 210 2) E f f e c t s of cAMP exposure and Cl-removal on the voltage-d i v i d e r r a t i o 215 3) Voltage scanning 215 4) C e l l - c e l l interconnections 217 i ) Dye coupling 217 i i ) E l e c t r i c a l coupling.. 223 5) C e l l membrane and p a r a c e l l u l a r resistance 223 6) E f f e c t s of potassium concentration on the t r a n s e p i t h e l i a l permeability to 42K 239 v i i Page 7) E f f e c t s of mucosal potassium concentration on voltage-divider r a t i o 239 8) E f f e c t s of cAMP concentration on s h o r t - c i r c u i t current and t r a n s e p i t h e l i a l conductance 242 Discussion 244 Locust rectum: a " t i g h t " epithelium with low e l e c t r i c a l resistance 244 Regulation of passive permeability 249 i ) cAMP 249 i i ) I n h i b i t i o n of P K by high [K] . , 256 Equivalent e l e c t r i c a l c i r c u i t model 258 Relationship between " a c t i v e " and "passive" conductance... 265 CHAPTER 6: GENERAL DISCUSSION 271 References • ••• 276 APPENDIX: ELECTRICAL CIRCUIT USED TO VOLTAGE-CLAMP LOCUST RECTUM 295 v i i i L i s t of Tables Page Table 2.1 Table 2.2 Table 2.3 Table 2.4 Table 2.5 Table 2.6 Table 2.7 Table 3.1 Table 4.1 Table 4.2 Table 4.3 Table 4.4 Table 4.5 Composition of p h y s i o l o g i c a l f l u i d s and experimental s a l i n e s used to study i o n transport across l o c u s t rectum 31 35 SO^ f l u x e s as an i n d i c a t i o n of maximum p e r m e a b i l i t y through edge damage under c o n t r o l c o n d i t i o n s , and a f t e r s e q u e n t i a l cAMP and NaCl a d d i t i o n s 37 E f f e c t of removing mucosal CI on serosa-to-mucosa f l u x of CI under I c o n d i t i o n s 50 sc E f f e c t of s e q u e n t i a l a d d i t i o n of 1 mM ouabain and 1 mM cAMP on t r a n s e p i t h e l i a l CI f l u x e s and e l e c t r i c a l parameters 64 E f f e c t of i n h i b i t o r s a f t e r 1-2 h exposure on C l -dependent I across cAMP-stimulated r e c t a 71 r sc E f f e c t s of exposure to Ca-free or Mg-free s a l i n e on e l e c t r i c a l parameters 75 Neurosecretory dose/response i n normal and Ca-free s a l i n e 83 3 6 Influence of e x t e r n a l K on k i n e t i c s of net CI f l u x across cAMP-stimulated, s h o r t - c i r c u i t e d r e c t a 112 Liquid-ion-exchangers and s o l u t i o n s used i n f a b r i c a t -ing d o u b l e - b a r r e l l e d i o n - s e n s i t i v e microelectrodes -^4 E f f e c t s of adding cAMP and removing e x t e r n a l K on e l e c t r i c a l p o t e n t i a l s , i n t r a c e l l u l a r CI a c t i v i t y , and c a l c u l a t e d CI e l e c t r o c h e m i c a l gradients ^ 3 E f f e c t s of adding cAMP and removing e x t e r n a l K on e l e c t r i c a l p o t e n t i a l s , i n t r a c e l l u l a r K a c t i v i t y , and c a l c u l a t e d K e l e c t r o c h e m i c a l gradients 166 E f f e c t s of s e q u e n t i a l cAMP and i n c r e a s i n g [K] b i l a t e r a l l y on i n t r a c e l l u l a r p o t e n t i a l , K and CI a c t i v i t i e s and c a l c u l a t e d net e l e c t r o c h e m i c a l gradients under I c o n d i t i o n s 172 ° sc E f f e c t s of cAMP and mucosal [K] on a p i c a l membrane p o t e n t i a l , i n t r a c e l l u l a r K and CI a c t i v i t i e s , and c a l c u l a t e d e l e c t r o c h e m i c a l gradients of the a p i c a l membrane under o p e n - c i r c u i t c o n d i t i o n s 175 ix Page Table 4.6 E f f e c t of cAMP and high osmotic pressure on t o t a l t i s s u e water and non-mannitol space i n suspended recta.' " 188 Table 5.1 Relat i v e resistance of a p i c a l and basal membranes (a) as calculated from d e f l e c t i o n s i n a p i c a l and basal membrane po t e n t i a l s during t r a n s e p i t h e l i a l constant-current pulses 216 Table 5.2 E l e c t r i c a l parameters estimated by cable analysis i n normal s a l i n e 229 Table 5.3 Results of cable a n a l y s i s i n C l - f r e e s a l i n e before and a f t e r adding cAMP 235 Table 5.4 E l e c t r i c a l parameters estimated by cable analysis i n KCl-free s a l i n e 238 Table 5.5 a) Summary of previous tracer and microelectrode r e s u l t s used i n c a l c u l a t i n g p ermeabilities, and b) Permeabilities calculated by various methods 251 Table 5.6 C a l c u l a t i o n of steady-state equivalent electromotive forces ( E a and E^, resp e c t i v e l y ) across a p i c a l and basal membranes 260 X • L i s t of Figures Page Figure 1.1 C e l l u l a r models which have been proposed for active t r a n s e p i t h e l i a l Cl transport i n invertebrates 10 Figure 1.2 C e l l u l a r models which have been proposed for act i v e t r a n s e p i t h e l i a l C l transport i n vertebrates 14 Figure 1.3 Organization of the locust alimentary canal, h i s t o l -ogy of the rectum, and u l t r a s t r u c t u r e of r e c t a l c e l l s . . . . 22 Figure 2.1 The approach of i s o l a t e d unstimulated r e c t a l tissue to steady-state conditions 40 Figure 2.2 E f f e c t s of cAMP on C l fluxes and e l e c t r i c a l parameters under s h o r t - c i r c u i t current (I ) conditions i n normal s a l i n e . . . . 41 Figure 2.3 Responsiveness of I g c to cAMP as a function of time aft e r d i s s e c t i o n of recta from locusts 42 Figure 2.4 Chloride dependence of cAMP-stimulated I 44 ; Figure 2.5 E f f e c t s of sequential a d d i t i o n of 1 mM cAMP to the serosal side and mucosal addition of potassium on C l fluxes under open-circuit conditions 47 Figure 2.6 E f f e c t of adding anions on the differ e n c e between I s c and the u n i d i r e c t i o n a l f l u x of Cl from mucosa-to-serosa ( J ^ l ) 51 ms Figure 2.7 E f f e c t of 1 mM cAMP on I i n anion-substituted salines Figure 2.8 E f f e c t of cAMP on C l fluxes and e l e c t r i c a l parameters i n Na-free and HCO^-free sali n e s 56 Figure 2.9 Relationship between I s c and trace l e v e l s of Na i n the mucosal half-chamber during "Na-free" experiments.... 57 Figure 2.10 Stimulation of I s c a f t e r prolonged exposure to amino acid-free s a l i n e containing normal ion l e v e l s and 10 mM glucose 59 Figure 2.11 E f f e c t s of b i l a t e r a l Na removal on e l e c t r i c a l parameters across cAMP-stimulated locust rectum 61 3 6 Figure 2.12 E f f e c t s of cAMP on u n i d i r e c t i o n a l influxes of C l 0 0 f r\ and Na into r e c t a l tissue from the mucosal side "2 x i Page Figure 2.13 E f f e c t s of furosemide and SITS addition on I sc across locust rectum 67 Figure 2.14 Continuous recordings of I s c and external pH during exposure of rec t a to cAMP i n HCO^-free s a l i n e 69 Figure 2.15 E f f e c t s of cAMP on e l e c t r i c a l parameters and CI fluxes under K-free conditions 73 Figure 2.16 E f f e c t s of external pH on t r a n s e p i t h e l i a l e l e c t r i c a l parameters i n cAMP-stimulated recta 77 Figure 2.17 Influence of mucosal and serosal osmotic pressure on I and t r a n s e p i t h e l i a l conductance (G,.) 80 sc t Figure 2.18 E f f e c t s of sequential addition of theophylline ( 4 mM) and corpus cardiacum homogenate ( 1 gland p a i r / 5 ml) on I 85 Figure 3.1 Representative trace of I s c and t r a n s e p i t h e l i a l p o t e n t i a l (V^) during measurement of t r a n s e p i t h e l i a l 3 6 c i f l u x k i n e t i c s 1 0 3 CI Figure 3.2 Histogram of J m s measured at 15 min i n t e r v a l s under I conditions 106 Figure 3.3 The dependence of C l fluxes and I s c on CI concentration 108 Figure 3.4 The influence of external K concentration on the re l a t i o n s h i p between j C l and C l concentration of the s a l i n e HO Figure 3.5 Representative p l o t of data used i n c a l c u l a t i n g k i n e t i c s constants of r e c t a l C l transport 112 Cl Figure 3.6 H i l l p l o t s of J m s at external K concentrations of 0, 10 and 100 mM 1 1 3 Figure 3.7 E f f e c t s of 1 mM cAMP on u n i d i r e c t i o n a l C l fluxes and e l e c t r i c a l parameters i n "high K" s a l i n e Figure 3.8 E f f e c t s of stepwise b i l a t e r a l K additions on e l e c t r i c a l parameters under I conditions 119 r sc Figure 3.9 E f f e c t s of stepwise b i l a t e r a l Na additions on e l e c t r i c a l parameters under I conditions 120 sc Figure 3.10 Lineweaver-Burke plot of the r e l a t i o n s h i p between external K concentration and Cl-dependent I' 122 Figure 3.11 S p e c i f i c i t y of cation stimulation of Cl transport 124 x i i Page Figure 3.12 E f f e c t of adding K methylsulfate stepwise to one side only on Cl-dependent I 126 Figure 3.13 E f f e c t s of cAMP on I s c and K fluxes measured under I conditions i n normal s a l i n e 128 sc Figure 3.14 E f f e c t s of cAMP on K fluxes and I s c i n C l - f r e e s a l i n e 129 Figure 3.15 E f f e c t s of sequential a d d i t i o n of 1 mM cAMP to the s e r o s a l side and mucosal addition of potassium on K fluxes under open-circuit conditions 133 K K Figure 3.16 Comparison of K f l u x r a t i o s ( J m s / J s m ) under open-c i r c u i t conditions with those predicted by the Ussing f l u x r a t i o equation 135 Figure 3.17 E f f e c t s of cAMP on I s c and K fluxes measured under I conditions with "high-K" s a l i n e on both sides 138 Figure 4.1 Cross section of the chamber used i n microelectrode experiments 151 Figure 4.2 Representative traces obtained using double-b a r r e l l e d i o n - s e n s i t i v e microelectrodes 159 Figure 4.3 Time course of the e f f e c t s of cAMP on t r a n s e p i t h e l i a l p o t e n t i a l (Vt), a p i c a l membrane p o t e n t i a l (V a) and C l - s e n s i t i v e ( d i f f e r e n t i a l ) p o t e n t i a l (V-^ ) 161 Figure 4.4 D i s t r i b u t i o n of i n t r a c e l l u l a r C l and K a c t i v i t i e s i n r e c t a l and e p i t h e l i a l c e l l s under control conditions, and after sequential exposure to 1 mM cAMP, and to K-free s a l i n e with cAMP present 162 Figure 4.5 Time course of the decline i n i n t r a c e l l u l a r K a c t i v i t y (a£) and V a during b i l a t e r a l perfusion with K-free s a l i n e under open-circuit conditions 168 Figure 4.6 E f f e c t s of 1 mM cAMP and b i l a t e r a l K additions on i n t r a c e l l u l a r p o t e n t i a l , i n t r a c e l l u l a r a c t i v i t i e s of K and C l and calculated electrochemical potentials for K (Ay|/F) and C l (Aygl/F) across the a p i c a l membrane under I conditions. 171 sc Figure 4.7 E f f e c t s of sequential cAMP and mucosal K additions on membrane p o t e n t i a l s , i n t r a c e l l u l a r K and C l a c t i v i t i e s , , Ay^/F and AyQ]7F under open-circuit conditions during serosal perfusion with normal s a l i n e (10 mM K) 177 x i i i Page Figure 4.8 Representative traces obtained with double-b a r r e l l e d Na-sensitive microelectrode under I s c conditions during perfusion with normal s a l i n e (115 mM Na) and nominally Na-free s a l i n e (49 uM Na) 180 Figure 4.9 Relationship between Na electrochemical gradient (AH^ a/F) across the a p i c a l membrane and Cl fluxes across cAMP-stimulated recta when V was clamped at 0 mV 183 _a _ a Figure 4.10 Relationship between Aujj a/F and Aud/F i n cAMP-stimulated r e c t a with V clamped at 0 mV 186 Figure 5.1 Method used for measuring the r a d i a l spread of current i n r e c t a l epithelium 206 Figure 5.2 Equivalent c i r c u i t model of locust rectum 209 Figure 5.3 Representative recordings of V t during exposure to t r a n s e p i t h e l i a l NaCl or KCl gradients 211 Figure 5.4 Deflections i n Vj- r e s u l t i n g from exposure to t r a n s e p i t h e l i a l s a l t gradients 214 Figure 5.5 Voltage scans of the e p i t h e l i a l surface 219 Figure 5.6 Rectal c e l l s following i n j e c t i o n with the fluorescent dye L u c i f e r Yellow CH - 221 Figure 5.7 E l e c t r i c a l coupling between c e l l s i n the r e c t a l epithelium 224 Figure 5.8 Current-voltage r e l a t i o n of i n t r a c e l l u l a r injected current as measured i n a d i f f e r e n t c e l l . 225 Figure 5.9 E f f e c t of prolonged i n t r a c e l l u l a r i n j e c t i o n of large depolarizing current pulses on the voltage responses measured i n a d i f f e r e n t c e l l 226 Figure 5.10 Deflections i n V a as a function of distance from the point of i n t r a c e l l u l a r current i n j e c t i o n before and a f t e r a d d i t i o n of cAMP 228 Figure 5.11 E f f e c t s of cAMP on the de f l e c t i o n s i n a p i c a l and basal membrane potentials produced by t r a n s e p i t h e l i a l constant current pulses (voltage d i v i d e r r a t i o ) 232 Figure 5.12 E f f e c t s of cAMP on the voltage d i v i d e r r a t i o i n Cl - f r e e s a l i n e 233 x i v Page Figure 5.13 E f f e c t s of cAMP on def l e c t i o n s i n V a as a function of distance from, the point of i n t r a c e l l u l a r current i n j e c t i o n under C l - f r e e conditions 234 Figure 5.14 E f f e c t s of cAMP on the voltage d i v i d e r r a t i o under KCl-free conditions 237 Figure 5.15 Apparent t r a n s e p i t h e l i a l potassium permeability *P]£ and K backflux under I s c conditions as a function of b i l a t e r a l K concentration 240 Figure 5.16 Relationship between mucosal K concentration and the voltage d i v i d e r r a t i o i n cAMP-stimulated recta during serosal perfusion with normal s a l i n e 242 Figure 5.17 Log dose-response curve showing the eff e c t s of cAMP concentration on t r a n s e p i t h e l i a l conductance and I s c . . . . . . 245 Figure 5.18 E f f e c t of cAMP on apparent t r a n s e p i t h e l i a l potassium permeability (*P R) i n various salines 255 Figure 5.19 Equivalent c i r c u i t model of r e c t a l epithelium showing electromotive force (EMF) and conductance for each ion at the mucosal and serosal c e l l borders 262 Figure 5.20 Equivalent c i r c u i t model used by Yonath and Civan (1971) to c a l c u l a t e the electromotive force of active Na transport (E^a) and shunt conductance (k^). i n toad bladder epithelium 267 Figure 5.21 Relationship between I s c and t r a n s e p i t h e l i a l conduc-tance calculated from mean cAMP dose-response curves 269 Figure 6.1 A model summarizing the mechanism and control of KCl transport across locust rectum 273 Figure A . l E l e c t r i c a l c i r c u i t used to voltage-clamp locust rectum during tracer and microelectrode experiments 296 XV L i s t of Abbreviations K, C l , Na, Ca, Mg, HC0 3 > H, e t c e t e r a PA I J V sc 1 net R t SITS M S -a _b Au., Ay. ATP cAMP yEqcm h CTSH EDTA cpm J 1 , J 1 ms sm yM cm n [ i ] ( i ) - read as i o n s , valence not shown - microamp - s h o r t - c i r c u i t current - net, t r a n s e p i t h e l i a l absorption of ion " i " - t r a n s e p i t h e l i a l p o t e n t i a l ('i'mucosa-H'serosa) - t r a n s e p i t h e l i a l r e s i s t a n c e - 4-acetamide-4'-isothiocyano-stilbene-2, 2' -d i s u l f o n i c a c i d - mucosal or lumen-facing side of the epith e l i u m - s e r o s a l or hemocoel-facing side of the ep i t h e l i u m - gradient of e l e c t r o c h e m i c a l p o t e n t i a l of ion " i " across the a p i c a l and b a s a l membrane, r e s p e c t i v e l y - adenosine 5'-triphosphate - adenosine 3 ' : 5 ' - c y c l i c monophosphoric a c i d - microequivalents per square centimetre per hour - c h l o r i d e tran s p o r t s t i m u l a t i n g hormone - ethylenediamine t e t r a a c e t i c a c i d - counts per minute - t r a n s e p i t h e l i a l t r a c e r p e r m e a b i l i t y to " i " - u n i d i r e c t i o n a l f l u x of " i " from mucosa to serosa, and from serosa to mucosa, r e s p e c t i v e l y - micromoles per square centimetre per hour - c o n c e n t r a t i o n of " i " - a c t i v i t y of " i " x v i Z R T F W .„oc ^ s c A p A p C l ' C l EGTA G t mS Posm mV K J t C l max n C l n c s a., a ., a . 1 1 1 Hz a db K. . V V, - valence - gas constant - absolute temperature (°Kelvin) - Faradays number - work (calories/equivalent) 3 6 - t r a n s e p i t h e l i a l C l permeability under open-and s h o r t - c i r c u i t conditions, r e s p e c t i v e l y - ohms - e t h y l e n e g l y c o l - b i s ( 3 - a m i n o e t h y l ether)N,N'-t e t r a a c e t i c acid - t r a n s e p i t h e l i a l conductance - millisiemens - osmotic permeability - m i l l i v o l t s - [Cl] at hal f maximal J C l net - maximal J C l 'net - H i l l constant for act i v e C l transport - flux r a t i o exponent - a c t i v i t y of ion " i " i n the mucosal, i n t r a c e l l u -l a r and serosal compartments, res p e c t i v e l y - hertz - voltage d i v i d e r r a t i o , r a t i o of a p i c a l - t o -basal membrane resistance, R /R, a b - decibels - s e l e c t i v i t y of microelectrode for j as compared to i - a p i c a l membrane p o t e n t i a l (I'cell-H'mucosa) - basal membrane p o t e n t i a l ('l'cell-1'serosa) x v i i t r a n s e p i t h e l i a l square current pulse e l e c t r i c a l p o t e n t i a l d i f f e r e n c e between the reference- and i o n - s e n s i t i v e barrels of a microelectrode N-methyl-D-glucamine a c t i v i t y c o e f f i c i e n t of " i " r e s istance of a p i c a l and basal membranes, res p e c t i v e l y micrometre i n t r a c e l l u l a r l y injected current nanoamps resistance to i n t r a c e l l u l a r l y injected current t r a n s j u n c t i o n a l resistance (paracellular) conductance of a p i c a l and basal membranes, res p e c t i v e l y (1/R &, i / 1 ^ ) permeability of a p i c a l and basal membrane to " i " r e s p e c t i v e l y logarithmic mean a c t i v i t y of " i " i n a p i c a l and basal membranes guanosine 3 ' : 5 ' - c y c l i c monophosphoric acid net electromotive force of a p i c a l and basal membranes as calculated from measured voltages and resistances across a p i c a l and basal membranes due to a gradient of ion a c t i v i t y across the membrane t o t a l electromotive force of a p i c a l and basal memhrane calculated from ion a c t i v i t y measure-ments and estimates of p a r t i a l i o n i c conductance electromotive force net d r i v i n g force of t r a n s e p i t h e l i a l , a c t i v e C l transport x v i i i Acknowle dgment s I t i s a pleasure to thank Dr. J . P h i l l i p s f o r encouragement, suggestions and f i n a n c i a l support during the course of t h i s study. Joan Martin contributed d a i l y with h e l p f u l advice and expertise. I thank Dr. Mary E l l a Chamberlin (28) for many discussions, for introducing me to mar'garitas and the sun, and for brightening the past f i v e years with her refined sense of humour. I am g r a t e f u l to D. Brandys, F. Smith and Dr. P. Graystone for h e l p f u l suggestions i n constructing the voltage clamp, to Drs. W. Prince, N. W i l l s , S. Lewis and J . Steeves for i n s t r u c t i o n i n microelectrode methods, and to Drs. J . Gosline, J . Steeves, V. Palaty, A. M. Perks and A. Glass f o r comments on the manuscript. I have enjoyed numerous conversations with H. Bergen, R. Roxx-anne, and with Dr. D. Randall, S. Haswell, M. Denny, N. Scherer, J . Spring, J. Ballantyne, R. Shadwick, K. Black and many others to whom I express my appreciation. I e s p e c i a l l y wish to thank Caroline Smith for her friendship and support. 1 CHAPTER 1: GENERAL INTRODUCTION A. Ion t r a n s p o r t E p i t h e l i a are ultimately responsible for i o n i c and osmotic homeostasis i n most animals. The regulation of transport i s p a r t i c u l a r l y important i n these tissues since they must respond to the f l u c t u a t i n g requirements of the animal and at the same time maintain t h e i r own c e l l u l a r composition. The regulatory features of e p i t h e l i a are accentuated i n freshwater and t e r r e s t r i a l habitats where i o n i c stress i s greatest, e s p e c i a l l y for small animals such as insects, which have large surface-to-volume r a t i o s . Both active (energy-requiring) and passive fluxes of water and solutes across the gut, integument, r e s p i r a t o r y surface and excretory organs may be subject to con t r o l mechanisms. Most insects which have been studied to date are capable of regulating hemolymph ion l e v e l s (for general l i t e r a t u r e see reviews by Wigglesworth, 1972; Stobbart and Shaw, 1974; Edney, 1977). In the desert l o c u s t , Schisto- cerca gregaria, hemolymph ion l e v e l s remain r e l a t i v e l y constant i n animals which are fed on hypertonic s a l t solutions ( P h i l l i p s , 1964; Stobbart, 1968; Stobbart and Shaw, 1974) and also during the d r a s t i c reduction i n hemolymph volume which accompanies dehydration (Hanrahan, 1978; Chamberlin and P h i l l i p s , 1979). As i n the A u s t r a l i a n locust (Djajakusumah and Miles, 1966) and the American cockroach (Wall, 1970), feeding leads to rapid r e s t o r a t i o n of hemo-lymph volume i n locusts with only small, transient changes i n ion l e v e l s (Hanrahan, 1978; P h i l l i p s et a l . , 1980). The r o l e of the insect excretory system i n maintaining hemolymph composi-t i o n i s well established (reviewed by P h i l l i p s , 1970, 1977, 1980, 1981; Ramsay, 2 1971; Maddrell, 1971, 1980; Wall and Oschman, 1975). This system i s composed of two p r i n c i p a l e p i t h e l i a , the Malpighian tubules and the rectum. F i r s t , Malpighian tubules secrete an isosmotic primary urine containing most small hemolymph solutes into the gut. Then, according to the "recycling hypothesis" of Ramsay (1958), t h i s f l u i d passes to the rectum where selective reabsorption of ions, water and useful organic solutes occurs. Although the morphology of insect rectum was described i n 1737 by Swammerdam, i t was not u n t i l much l a t e r that evidence suggesting the reabsorption of water (Wigglesworth, 1931, 1932) and ions (Bong and Koch, 1942; Ramsay, 1953, 1955; Shaw, 1955) was obtained. However, early studies of r e c t a l ion transport were not d e f i n i t i v e because i ) volume changes were not measured as f l u i d passed through the rectum, and i i ) e l e c t r i c a l potentials were not measured. Previous studies on locust rectum Active ion transport across insect rectum was f i r s t shown unequivocally i n s i t u i n the locust by P h i l l i p s (1961, 1964b). He injected electrolyte 131 solutions containing an impermeant volume marker ( I-albumin) into ligated recta and measured the ion concentrations and r a d i o a c t i v i t y of the f l u i d as a function of time. Net absorption rates of ions and water were then calculated. I t was concluded that chloride and water are a c t i v e l y transported from the r e c t a l lumen and that sodium and potassium are also absorbed, at least p a r t i a l -l y by active transport. Since the insect remained v i r t u a l l y intact during these experiments and displayed normal a c t i v i t i e s , i t i s probable that net uptake rates were close to those which normally occur i n unperturbed insects. Nevertheless there were limitations to the approach: absorption could only be studied under non-steady-state conditions since measurements r e l i e d on changes i n the luminal f l u i d , i ndividual forward and back fluxes could not be measured 3 accurately, f l u i d composition could only be a l t e r e d on the lumen side, neither luminal nor hemolymph compartments were well s t i r r e d , and f i n a l l y , neural or hormonal factors which normally c o n t r o l r e c t a l reabsorption were unknown and could not be c o n t r o l l e d by the investigator. For these reasons, a good i n  v i t r o preparation was required for more de t a i l e d studies of transport mechan-isms and t h e i r regulation. Ion and water transport was studied across i s o l a t e d locust r e c t a by Irvine (1966; Irvine and P h i l l i p s , 1971), using a non-everted sac preparation. Active transport of K and C l was not observed, presumably because of inade-quate oxygenation of the luminal (or mucosal) surface. Transport of these ions was maintained i n l a t e r studies using everted sacs i n which the mucosal side was vigorously aerated (Goh, 1971; Goh and P h i l l i p s , 1978). Goh measured t r a n s e p i t h e l i a l p o t e n t i a l (V ), absorbate composition, and net water flux with time over 5-6 h, and showed that e i t h e r Na, K, or C l ions could support prolonged water absorption i n v i t r o . However, t h i s everted r e c t a l sac prepar-a t i o n was not amenable to the study of ion transport mechanisms per se, since u n i d i r e c t i o n a l ion fluxes could not be e a s i l y measured nor could V be c o n t r o l l e d . Herrera, Jordana and Ponz (1976; 1977) used a d i f f e r e n t sac preparation to study the e f f e c t s of ion substitutions and i n h i b i t o r s on V and short-c i r c u i t current (I » an i n d i r e c t measure of net active ion transport) across locust rectum. These workers found that C l removal r e s u l t s i n a 49% reduction i n I a f t e r 2 min, increasing to a 91% reduction a f t e r 12 min. However, a l l of sc > fa t h e i r experiments were performed under non-steady-state conditions ( i . e . during the f i r s t 20 min a f t e r dissection) when i n t r a c e l l u l a r ions are s t i l l e q u i l i b r a t -ing with the external s a l i n e and when active transport i s d e c l i n i n g r a p i d l y . Furthermore, non-steady-state ion substitutions may give misleading r e s u l t s . 4 For example, they observed large increases i n I when Na and K were removed from both sides (22 and 28%, r e s p e c t i v e l y ) . In chapters 2 and 3, I w i l l show that steady-state K removal a c t u a l l y reduces I and that the e f f e c t s report-ed by Herrera et a l . probably r e s u l t from passive d i f f u s i o n transients rather than a c t i v e transport. Also, i n t h e i r study, I was often measured when ion replacements were made only to one side of the epithelium, so that " l s c " w a s no longer a v a l i d measure of act i v e ion transport. Tracer fluxes, membrane potentials and i n t r a c e l l u l a r ion a c t i v i t i e s were not measured. I n i t i a l l y , Herrera et a l . made no serious errors i n the i n t e r p r e t a t i o n of t h e i r r e s u l t s . They proposed a c e l l u l a r model for ion transport across t h i s epithelium which featured a C l pump at the a p i c a l membrane (Herrera et a l . , 1976). However, the r e s u l t s i n t h i s paper did hot provide any information regarding pump lo c a t i o n , and t h i s scheme was revised i n a follow-up study i n which a ouabain-s e n s i t i v e C l pump was proposed at the basal membrane (Herrera et a l . , 1977). Their revised model i s not supported by the tracer and e l e c t r o - p h y s i o l o g i c a l data which w i l l be presented i n th i s t h e s i s . Vietinghoff et a l . (1969) made preliminary measurements of membrane pote n t i a l s i n recta of Carassius and Locusta using a sac preparation. In agreement with the present study on Schistocerca, they observed a p i c a l and basal membrane po t e n t i a l s of -59 and -47 mV, r e s p e c t i v e l y ( c e l l i n t e r i o r negative). However, i n the absence of tracer f l u x and i n t r a c e l l u l a r ion a c t i v i t y measurements, membrane potentials did not permit any new conclusions regarding the mechanism of ion transport. Williams (1975; Williams et a l . , 1978) developed a f l a t - s h e e t preparation and s h o r t - c i r c u i t i n g device f o r locust rectum i n order to measure t r a n s e p i t h e l -i a l tracer fluxes under I conditions as f i r s t described by Ussing and SC J O Zerahn (1951). The chamber, design was adapted from that of Wood (1972; Wood 5 and Moreton, 1978). Transrectal I declined exponentially during the f i r s t three hours although l e s s r a p i d l y than was observed by Herrera et a l . (1976). This decline was p a r a l l e l e d by a decrease i n net C l f l u x . Replacement of C l with other anions abolished the i n i t i a l decay i n I » suggesting that e l e c t r o -genic C l transport generated most, i f not a l l , of the i n i t i a l s h o r t - c i r c u i t cur-rent. Properties of the transport mechanism such as possible i o n i c coupling, k i n e t i c s , s e l e c t i v i t y , etc. were not investigated. Although a c e l l u l a r model of ion transport was not warranted, Williams (1976) did demonstrate the v i a b i l i t y of t h i s jLn v i t r o preparation. Rates of act i v e ion transport i n the Ussing chamber were comparable to those observed i n s i t u (Williams et a l . , 1978). P h i l l i p s (1961, 1964) measured the rate of net ion absorption from the r e c t a l lumen jLn s i t u as a function of concentration i n locusts fed on either tap water or on hypertonic s a l i n e . In s a l i n e - f e d l o c u s t s , s a l t absorption saturated at lower rates when compared with water-fed loc u s t s . He suggested that t h i s regulation could occur through changes i n e p i t h e l i a l permeability without excluding the p o s s i b i l i t y that changes might also occur i n the kinet-i c s of a c t i v e transport ( P h i l l i p s , 1964). Williams (1976; Williams et a l . , 1978) reported that electrogenic C l transport declined by 80% over the f i r s t few hours jLn. v i t r o and speculated that t h i s occurred because of removal from some neural or hormonal agent which normally stimulates a c t i v e transport i n the i n t a c t locust. Using t h i s prepar-ation, Spring (1979; Spring et a l . , 1978; Spring and P h i l l i p s , 1980a, b) showed that C l absorption and I could be restored to i n i t i a l l e v e l s i n v i t r o sc by adding small amounts of homogenized corpus cardiacum, a major neuroendo-cr i n e organ i n ins e c t s . 6 Evidence suggesting that the factor i s i n f a c t a natural hormone came from observations by the author that i n i t i a l t r a n s e p i t h e l i a l p o t e n t i a l i s s i g n i f i c a n t l y higher across recta from fed as compared to unfed locusts (Hanrahan, 1978; Spring et a l . , 1978). Also, hemolymph from recently fed locusts was found to increase Cl-dependent I and V and was more stimulatory than hemolymph from unfed locusts (see P h i l l i p s et a l . , 1981). Although the source of t h i s hemolymph factor was not established, i t was proposed that feeding (which i s known to cause massive neurosecretory release i n many insects i n c l u d i n g locusts; Highnam et a l . , 1966; Mordue, 1969; Highnam and West, 1971; Highnam and Mordue (Luntz), 1974) might t r i g g e r release of the factor from the corpus cardiacum (Hanrahan, 1978; Spring et a l . , 1978). In support of t h i s hypothesis, a b l a t i o n of the corpus cardiacum reduced the hemo-lymph t i t r e of t h i s f actor by 86%, and the stimulatory e f f e c t of hemolymph on C l absorption was l a t e r confirmed using tracers (Spring and P h i l l i p s , 1980c). Mechanisms of active Cl transport in insects The r e s u l t s described above suggest that C l absorption i s the predominant ion transport process i n locust rectum following hormonal stimulation. This r a i s e s the obvious question: How does Cl transport occur i n t h i s tissue? The c e l l u l a r mechanism of a c t i v e C l transport has not yet been examined i n insect e p i t h e l i a despite i t s widespread occurrence. Previous studies have demonstrated C l transport i n the c l a s s i c a l sense, but have not explored the membrane mechanisms using modern e l e c t r o p h y s i o l o g i c a l and tracer techniques. Evidence for net transport of Cl against a chemical gradient has been obtained i n i n t a c t Chironomus larvae (Hers, 1942), i n the anal p a p i l l a e of mosquito larvae (Koch, 1938; Wigglesworth, 1938; Stobbart, 1967; P h i l l i p s and Meredith, 1969) and marsh beetles (Treherne, 1954), i n the Malpighian tubules of Rhodnius (Maddrell, 1971), s a l i v a r y glands of t i c k s (Kaufman and P h i l l i p s , 7 1973; Sauer et a l . , 1976), anal canal of Drosophila (Gloor and Chen, 1950), midgut of aphids (Downing," 1980), i n the recta of several larvae including the caddis f l y ( S u t c l i f f e , 1961), mosquito (Bradley and P h i l l i p s , 1977) and blow-f l y (Prusch, 1974). In addition to locust rectum, active Cl transport has been measured d i r e c t l y _in v i t r o by tracer flux i n dragonfly rectum (Leader and Green, 1978) and the pupal integument of the hornworm (Cooper et a l . , 1980). Histochemical evidence for chloride c e l l s i s also very suggestive i n a variety of aquatic insects (reviewed by Komnick, 1977). Koch (1938) and Wigglesworth (1938) have shown that the anal papillae of dipteran larvae absorb Cl from d i l u t e NaCl although s p e c i f i c mechanisms were not proposed. A Cl/HCO^ exchange mechanism was suggested by Stobbart (.1967) to explain how mosquito larvae absorb C l from different solutions when they contain an impermeant cation ( i . e . from NaCl, KCl, CaC^ or NH^Cl). Chloride/ bicarbonate exchange has not been tested d i r e c t l y i n mosquito larvae, since base eff l u x has not been measured or correlated with C l uptake. However, the results do c l e a r l y indicate that some Cl absorption i s cation-independent. Potassium has been found to stimulate Cl absorption i n two insect recta, although t h i s effect may be explained by changes i n e l e c t r i c a l potential rather than KCl cotransport. P h i l l i p s (1964b) found that net Cl absorption by locust rectum was 7.5-fold faster when KCl rather than NaCl was injected into ligated locust recta. The enhanced absorption of Cl following KCl injection was attributed to a temporary reversal i n (lumen becoming negative) which would drive Cl out of the lumen. Prusch (1976) found that unidi r e c t i o n a l Cl flu x into the lumen of isolated maggot hindgut declined when K was removed from the hemolymph side. This might also"be explained as an e l e c t r i c a l effect of K, since the lumen became strongly negative under these conditions (-60 mV). 8 In s p i t e of the e l e c t r i c a l e f f e c t s of K on t r a n s e p i t h e l i a l p o t e n t i a l , the f a c t that C l i s absorbed from KC1 solutions when injected into the locust rectum _in s i t u suggests that C l transport does not require a high concentra-t i o n of sodium i n the lumen ( P h i l l i p s , 1964b). This i s not s u r p r i s i n g as the rectum contains a K C l - r i c h rather than NaCl-rich f l u i d ±n vivo. In conclusion, a c t i v e C l transport i s common i n insect e p i t h e l i a , but i t has not received d e t a i l e d study at the c e l l u l a r l e v e l . A Cl/HCO^ exchange has been proposed i n the anal p a p i l l a e of mosquitoes and clear evidence for electrogenic C l transport has been obtained i n the locust rectum and i n the pupal integument of the tobacco hornworm. However, there i s no compelling evidence for any p a r t i c u l a r a c t i v e C l transport model or coupling mechanism i n insect e p i t h e l i a . Models proposed for active Cl transport in other epithelia To a n t i c i p a t e possible mechanisms of C l transport i n i n s e c t s , i t i s useful to consider the various models which have been proposed for other e p i t h e l i a . Using t r a c e r s , Shaw (1960) showed that active absorption of C l by i n t a c t c r a y f i s h i s independent of external sodium. The uptake of external C l i s thought to occur by a countertransport mechanism which switches i n c h l o r i d e -depleted animals from C l / C l exchange to an exchange of external C l with some endogenous anion, presumably HCO^ (Fig- 1(a)). A s i m i l a r switchover has been suggested for C l / C l exchange i n brine shrimp (Smith, 1969). In contrast, C l absorption by prawn i n t e s t i n e i s Na-coupled (Fig. 1 ( b ) ; Ahearn and Tornquist, 1977; Ahearn, 1978). Also, i n t e s t i n a l C l absorption shows an "S-shaped" dependence on luminal C l concentration, sugg-estive of cooperative binding to the c a r r i e r . I t has not been shown that the a p i c a l entry step i s an energy-9 Figure 1.1 C e l l u l a r models which have been proposed for active transepi-t h e l i a l C l transport i n invertebrates. A p i c a l (external or mucosal "M") surface i s shown to the l e f t i n each model, basal ( i n t e r n a l or serosal "S") surface i s shown to the r i g h t . Large arrows ind i c a t e the d i r e c t i o n of net C l f l u x . Primary and secondary•active transports are shown by smaller arrows attached to c i r c l e s . E l e c t r o d i f f u s i o n and uncharacterized ion movements are represented as dashed arrows. a) Cl/HCO^ exchange entry - nymphs of L i b b e l l u l a and Aeschna (Krogh, 1939) - c r a y f i s h ( g i l l ; Shaw, 1960) - brine shrimp ( g i l l ; Smith, 1969) - earthworm (integument; Dietz, 1974) - freshwater mussel ( g i l l ; Dietz and Branton, 1975) - mosquito l a r v a (anal p a p i l l a ; Stobbart, 1967) b) Na,Cl coentry - prawn ( i n t e s t i n e ; Ahearn and Tornquist, 1977) c) Active C l e x i t _ Aplysia ( i n t e s t i n e ; Gerenscer and White, 1980) 10 M Cl Cl -HCO3 N a C l Ca N a - i C i s K C l N a T ! u c o s e — N ^ -ci N a 11 r e q u i r i n g process, but there i s evidence that t r a n s e p i t h e l i a l absorption i s a c t i v e (Ahearn, 1980). Active C l absorption i s also present i n e p i t h e l i a of molluscs and annelids. Electrogenic C l absorption across Aplysia i n t e s t i n e has been demon-strated using tracers ( F i g . 1( c ); Gerenscer et a l . , 1977). Furthermore, microelectrode data suggest that the a c t i v e step for C l absorption i s located at the basal membrane (Gerenscer and White, 1980; reviewed by Gerenscer, 1981). Freshwater mussels and annelids a c t i v e l y absorb C l from d i l u t e s a l t solutions independently of the cation and electr o n e u t r a l exchange mechanisms have been proposed for C l absorption by both of these animals ( F i g . 1(a); Dietz and Branton, 1976; Dietz, 1974).. Chloride absorption by earthworm integument i s i n h i b i t e d by i n j e c t i o n of the carbonic anhydrase i n h i b i t o r acetazolamide, suggestive of a Cl/HCO^ exchange mechanism (Dietz, 1974). Chloride/bicarbonate exchange was the f i r s t mechanism proposed for ac t i v e C l transport i n vertebrates. I t arose from the observation that C l uptake by frogs and other aquatic animals could occur from d i l u t e s a l t solutions i r r e s p e c t i v e of simultaneous cation absorption (Krogh, 1937). Krogh postulated that C l must exhange with an i n t e r n a l anion (HCO^) i n order to maintain charge balance when C l i s absorbed (see F i g . 2(a)). D i r e c t chemical coupling of t h i s type has been demonstrated rigorously with tracers i n toad skin ( L e s l i e et a l . , 1973) although a recent study using v e s i c l e s prepared from i n t e s t i n a l brush border suggests that Cl may be exchanged for hydroxy1 ions (Liedtke and Hopfer, 1980). Nevertheless, the model which proposes a Cl/HCO^ exchange at the a p i c a l plasma membrane (or C l entry step) i s s t i l l widely held. The g a s t r i c mucosa model shown i n F i g . 2(b ) has been suggested by Machen and Forte (1979) from t h e i r review of the l i t e r a t u r e . There are at l e a s t two d i f f e r e n t mechanisms for g a s t r i c Cl transport, one which i s a c i d -coupled, and the other which i s independent of proton secretion. The a c i d -coupled mechanism i s thought to involve a K-stimulated ATPase at the a p i c a l membrane and a Cl/HCO^ exchange at the basal membrane. The non-acidic compon-ent of C l secretion i s known to have a lower a f f i n i t y for C l , although the precise l o c a t i o n of t h i s pump i s uncertain. For a discussion of the contro-v e r s i a l aspects of g a s t r i c C l secretion, see the review by Machen and Forte (1979). Although chloride/bicarbonate exchange i s usually considered electroneut r a l , actual estimates of the coupling r a t i o ( i . e . r a t i o of the number of C l and HCO^ ions which are exchanged) vary somewhat. In skin of the frog Calyptocephallela, the coupling r a t i o has been calculated as ZCl/SHCO^ (Garcia Romeu et a l . , 1969) although r a t i o s of ZCl/lHCO^ i n the frog Rana esculenta (Ehrenfeld and Garcia Romeu, 1978) and 4C1/3HC03 i n g o l d f i s h g i l l s (DeRenzis and Maetz, 1973) have also been reported. The energetics of e p i t h e l i a l C1/HC0 exchange are not known; however, energy for " u p h i l l " C l absorption may be supplied by an ATPase to be discussed l a t e r i n t h i s section, or by the e f f l u x of HCO^ from the c e l l down i t s net electrochemical gradient. I t has been suggested that the mechanism of e p i t h e l i a l Cl/HCO^ exchange may resemble that of anion exchange across red blood c e l l membranes (reviewed by Gunn, 1979). A second general category of C l transport across e p i t h e l i a requires external sodium ( F i g . 2(c-e); reviewed by F r i z z e l l et a l . , 1979). In this model, coentry of NaCl across the c e l l membrane i s energized by inward movement of Na down i t s net electrochemical gradient (Au„ ) i n a manner ° Na. analogous to Na-coupled amino acid and sugar absorption (reviewed by Crane, 22 36 1977). Obligatory Na, Cl coentry across the a p i c a l c e l l membrane has been shown d i r e c t l y i n r a b b i t gallbladder ( F r i z z e l l et a l . , 1975; Cremaschi and Henin, 1975) and i n f i s h i n t e s t i n e ( F r i z z e l l et a l . , 1979; Ramos and 13 Figure 1.2 C e l l u l a r models which have been proposed for active C l trans-port across e p i t h e l i a i n vertebrate animals. See legend of Figure 1 for explanation. a) Cl/HCO^ exchange entry: e x i t unknown - f i s h g i l l - frog skin - t u r t l e bladder - small i n t e s t i n e - colon b) Cl/HCOg exchange entry: proton-coupled e x i t - g a s t r i c mucosa c) Na,Cl coentry: KC1 coexit - amphibian g a l l bladder d) Na,Cl coentry: Cl/HCO^ exchange ex i t - amphibian proximal tubule, i n t e s t i n e e) Na,Cl coentry: C l e x i t unknown, Na recycled p a r a c e l l u l a r l y - f i s h i n t e s t i n e f) Na,Cl coentry: e x i t by d i f f u s i o n - cornea -shark r e c t a l gland - f i s h operculum - small i n t e s t i n e (secreting) g) Na,K,Cl coupled entry mechanism (?): e x i t by d i f f u s i o n - MDCK cultured monolayers h) Electrogenic entry: e x i t by Cl/HCO^ exchange - amphibian small i n t e s t i n e 15 E l l o r y , 1981). T r a n s e p i t h e l i a l C l absorption would be ele c t r o n e u t r a l i f Na ions entering the c e l l through the a p i c a l plasma membrane were pumped out across the basal plasma membrane by the Na/K exchange pump (F i g . 2(c) and Fi g . 2(d)). A l t e r n a t i v e l y , t r a n s e p i t h e l i a l absorption of C l would be e l e c t r o -genic i f some Na recycles back through Na-selective t i g h t junctions from the l a t e r a l i n t e r c e l l u l a r spaces as proposed for flounder i n t e s t i n e ( Fig. 2(e); F i e l d et a l . , 1978). Chloride secretion i s stimulated by cAMP i n several vertebrate e p i t h e l i a including rabbit ileum ( F i e l d , 1 9 7 1 ) r a b b i t colon ( F r i z z e l l et a l . , 1976), cornea (C h a l f i e et a l . , 1972), shark r e c t a l gland ( S i l v a et a l . , 1977) and k i l l i f i s h operculum (Karnaky et a l . , 1977; Degnan et a l . , 1977). A widely accepted model for th i s process involves e l e c t r o n e u t r a l NaCl coentry across the basal membrane (Fig. 2 ( f ) . Sodium i s returned to the serosal side v i a the ubiquitous Na/K exchange pump ( i . e . "recycled") while C l leaves the c e l l down i t s net electrochemical gradient across the a p i c a l membrane. Chloride secretion across cornea epithelium i s stimulated by adrenaline, v i a an increase i n the C l conductance of the a p i c a l membrane (Klyce and Wong, 1977). A v a r i a -t i o n of NaCl coentry has recently been proposed i n cultured monolayers from dog kidney (MDCK c e l l s ; Simmons, 1981). Exposure to ATP on the serosal side stimulates C l secretion across the monolayer, as shown i n F i g . 2(g). This secretion i s i n h i b i t e d by adding furosemide or ouabain to the serosal side and by elevating the K concentration on the serosal side. Chloride entry across the basal membrane i s thought to occur by a NaK-coupled mechanism analogous to that found i n E h r l i c h a s c i t e s tumour c e l l s (Geek et a l . , 1980) and i n red blood c e l l s of ducks (Kregenow and Caryk, 1979) and humans (Dunham et a l . , 1980). In E h r l i c h c e l l s , C l entry i s el e c t r o n e u t r a l (2Cl:lNa:lK) although some other stoichiometry must ex i s t i n MDCK c e l l s . Geek and co-workers have suggested that the NaCl coentry mechanism which i s widespread i n e p i t h e l i a might a c t u a l l y be a Na,K,Cl-coupled system. A very d i f f e r e n t i n t e r p r e t a t i o n of Na dependence has been suggested for amphibian small i n t e s t i n e ( F i g . 2 ( h ) ; White, 1980). Chloride absorption i s dependent on both mucosal sodium and serosal bicarbonate i n th i s t i s s u e . Studies u t i l i z i n g i n h i b i t o r s , tracers and i o n - s e n s i t i v e microelectrodes suggest that C l entry occurs by primary active transport, and that i n t r a c e l l u -l a r C l i s exchanged f o r serosal HCO^ at the e x i t step. According to th i s model, HCO^ i s maintained at high l e v e l s i n the c e l l through the actions of an a p i c a l Na/H exchange and i n t r a c e l l u l a r carbonic anhydrase. I n t r a c e l l u l a r bicarbonate i s hypothesized to reduce the backflux of C l from c e l l to mucosal so l u t i o n . However, at present the most widely held model for Na-dependent C l transport involves an obligatory NaCl coentry into the e p i t h e l i a l c e l l s . Like the Cl/HCO^ exchange mechanism described e a r l i e r , t h i s cotransport system appears to be widespread i n vertebrate e p i t h e l i a (see F r i z z e l l et a l . , 1979; Ramos and E l l o r y , 1980). Both Na- and HCO^-coupled systems are examples of "secondary active transport" i n which the u p h i l l entry of Cl into the c e l l s i s coupled to the flow of a second solute down i t s net electrochemical gradient (reviewed by Aronson, 1981). Primary a c t i v e transport of C l has not yet been demonstrated i n e p i t h e l i a . Bicarbonate-stimulated ATPase a c t i v i t y has been reported i n g a s t r i c mucosa (Kasbekar and Durbin, 1965), pancreas (Simon et a l . , 1972) and kidney (Kinne-Saffran and Kinne, 1974) although these early preparations may have been contaminated with mitochondrial anion-stimulated ATPase a c t i v i t y . Results of recent studies i n r a t i n t e s t i n a l brush border (Humphreys and Chou, 1979) and f i s h g i l l (Bornancin et a l . , 1980) strongly suggest the presence of non-mitrochondrial ATPase a c t i v i t y which i s stimulated by both HCO^ and C l , 1 7 however t h i s conclusion has been disputed (Bonting, 1980). Anion-ATPase has been demonstrated i n r e c t a l tissue of dragonflies (Komnick et a l . , 1980) and HCO^-stimulated ATPase a c t i v i t y has been reported i n locust Malpighian tubules (Anstee and Fathpour, 1979) and rectum (Herrera et a l . , 1978) although mitochondrial contamination was not considered i n the l a t t e r study. In summary, l i t t l e i s known regarding the mechanism of C l transport i n insec t s . Based on studies of vertebrate e p i t h e l i a , secondary transport of C l coupled to HCO^ or Na movements are most common although the p o s s i b i l i t y of an active pump cannot be ruled out. In locust rectum, there i s l i t t l e d i r e c t information regarding possible i o n i c dependency, coupling or the k i n e t i c s of active C l absorption. Moreover, an understanding of C l transport at the c e l l u l a r l e v e l i s not possible since the l o c a t i o n of the ac t i v e step i s not known. Ionic permeability in epithelia Information regarding permeability i s c r u c i a l to understanding ion trans-port across any epithelium. With the exception of blowfly s a l i v a r y gland, where a Ca-stimulated increase i n C l conductance has been demonstrated (Berridge et a l . , 1975), studies of insect e p i t h e l i a have generally focused on active transport, ignoring the equally important permeability properties. A basic question which must be answered i s whether ions d i f f u s e passively across the epithelium t r a n s c e l l u l a r l y or by a p a r a c e l l u l a r route. The t i g h t junctions were o r i g i n a l l y considered to be impermeable b a r r i e r s which required ions to traverse the a p i c a l and basal c e l l membrane (see Ussing et a l . , 1974). While t h i s i s true of " t i g h t " e p i t h e l i a such as amphibian skin, the bladder and kidney d i s t a l tubule, i t i s now well known that i n many "leaky" e p i t h e l i a ions d i f f u s e l a r g e l y by a p a r a c e l l u l a r route ( i . e . i n t e r -18 c e l l u l a r spaces and tight.junctions) rather than through i n d i v i d u a l c e l l membrane (early evidence reviewed by FrOmter and Diamond, 1972). The e l e c t r i c a l p o t e n t i a l s observed across the epithelium and across each membrane are dependent on both the p a r a c e l l u l a r and membrane conductances. Despite t h i s importance, the r e l a t i v e contributions of c e l l u l a r and pa r a c e l l u -l a r routes to passive ion movements have not been d i r e c t l y measured i n any invertebrate epithelium. The need f o r a study of e p i t h e l i a l tightness i s indicated. Towards a cellular model for insect Cl transport This thesis examines some of the more important properties of C l trans-port and i t s r e g u l a t i o n i n locust r e c t a l epithelium. Chapter 2 describes the basic features of chloride absorption by measuring t r a n s e p i t h e l i a l tracer fluxes, s h o r t - c i r c u i t current and t r a n s e p i t h e l i a l p o t e n t i a l under various conditions. Several predictions of the Na,Cl cotransport and Cl/HCO^ exchange models proposed for vertebrates are tested using ion substitutions and w e l l -known transport i n h i b i t o r s . Also, several approaches are used: i ) to estab-l i s h whether C l / C l exchange d i f f u s i o n i s important i n t h i s epithelium as i t i s i n other arthropods such as c r a y f i s h , brine shrimp and mosquito larvae, and i i ) to examine the s e l e c t i v i t y of t r a n s e p i t h e l i a l transport f o r C l over other anions. F i n a l l y , the e f f e c t s of l o c a l factors' ( i . e . calcium, v a r i a t i o n s i n mucosal pH and osmotic pressure.) which might regulate active C l transport are studied i n v i t r o . During these i n i t i a l f l u x experiments, i t was discovered that potassium stimulates a c t i v e C l transport several f o l d . Also, e l e c t r i c a l r esistance measurements indicated that K might be the main counter ion for ac t i v e absorption. Thus the i n t e r r e l a t i o n s h i p between C l transport and K i s considered i n more d e t a i l i n Chapter 3. F i r s t , the e f f e c t s of K on t r a n s e p i t h e l i a l C l transport k i n e t i c s are measured under s h o r t - c i r c u i t condi-19 tions using tracers. I also determine the a c t i v a t i o n constant for K stimula-t i o n of C l absorption and the s p e c i f i c i t y of th i s stimulation for K as compared to other cations. The possible sidedness of K stimulation i s also tested. In a second series of experiments, the e f f e c t s of cAMP on transepi-t h e l i a l K transport are measured under open- and s h o r t - c i r c u i t conditions to determine whether K i s indeed the counter ion for C l transport and to f i n d out whether cAMP might have some e f f e c t on K transport i n addition to stimulating active C l absorption. In Chapter 4 , the act i v e step i n t r a n s e p i t h e l i a l C l transport i s l o c a l -ized by using double-barrelled microelectrodes to measure steady-state i n t r a c e l l u l a r ion a c t i v i t i e s and membrane potentials under open-circuit conditions. The e f f e c t s of cAMP addition and K removal on transmembrane electrochemical gradients are'also assessed under these conditions. Ion-se n s i t i v e microelectrodes are used to test further the p o s s i b i l i t y of Na-coupled C l transport by measuring i ) the r e l a t i o n s h i p between the Na 3 6 electrochemical gradient across the a p i c a l membrane and t r a n s e p i t h e l i a l C l fluxes, and i i ) steady-state Na and C l electrochemical gradients between the mucosal s o l u t i o n and the c e l l . A fter l o c a l i z i n g the act i v e step i n C l absorption, the permeability properties of the epithelium are studied i n Chapter 5 using e l e c t r o p h y s i o l o g i -c a l techniques. F i r s t , membrane and j u n c t i o n a l resistances are measured i ) to determine whether locust rectum i s a " t i g h t " or "leaky" epithelium, and i i ) to c a l c u l a t e the equivalent electromotive forces of a p i c a l and basal membranes. The e f f e c t s of cAMP during ion substitutions reveal s p e c i f i c permeability changes during hormone stimulation. Tracer and microelectrode r e s u l t s are used to construct an equivalent c i r c u i t model for KCl absorption across locust rectum. F i n a l l y , i n order to test the v a l i d i t y of measured 20 tracer fluxes, e l e c t r i c a l - p o t e n t i a l s , i n t r a c e l l u l a r ion a c t i v i t i e s and mem-brane resistance, a l l four types of data are combined to estimate the d r i v i n g force of C l transport. This value i s found to be i n s a t i s f a c t o r y agreement with that calculated using a second, independent method. In the General Discussion (Chapter 6) the main conclusions are assembled into a tentative model for KC1 transport and i t s regulation i n locust rectum. A few specula-tions regarding the p h y s i o l o g i c a l r o l e and s i g n i f i c a n c e of t h i s unusual transport system are also discussed. B. Organization of the locust excretory system The gross anatomy and histology of the gut have been thoroughly described i n locusts and grasshoppers (Chauvin, 1938; Hodge, 1939; Marshall, 1945; Albrecht, 1953; P h i l l i p s , 1961; B o c c e t t i , 1962; I r v i n e , 1966; P h i l l i p s , J a r i a l and I r v i n e , unpubl. man.; Jonas and V i e t i n g h o f f , 1975; reviewed by Wall and Oschman, 1975) . The purpose of this section i s to o u t l i n e those features of the excretory system which are relevant to this thesis and to b r i e f l y review some important aspects of r e c t a l u l t r a s t r u c t u r e . The locust excretory system consists of two transporting e p i t h e l i a , the Malpighian tubules and the rectum (Fig. 3). Approximately 250 tubules j o i n the alimentary canal at the junction between the midgut and ileum, and secrete a f l u i d into the gut which i s isosmotic to the hemolymph. Most of t h i s "primary urine" moves p o s t e r i o r l y to the rectira f o r s e l e c t i v e reabsorption of water and solutes ( P h i l l i p s , 1961; 1964a-c) . Some tubular f l u i d also moves a n t e r i o r l y to the midgut for f l u i d reabsorption (Dow, 1981). The locust rectum i s made up of s i x d i s c r e t e pads which are connected by narrow regions of reduced epithelium ( F i g . 3b). The intima, a thin porous layer of c u t i c l e , 2-5 ym thick, i s draped loosely over each pad. In cockroach 21 Figure 1.3 Diagram of the organization of the locust alimentary canal (a), histology of the rectum (b) and u l t r a s t r u c t u r e of r e c t a l pad c e l l s (c). Figure 3(c) incorporates features from Schistocerca rectum ( J a r i a l et a l . , unpubl. obs.) and rectum of the cockroach Periplaneta americana (Oschman and Wall, 1969; Wall and Oschman, 1975; Lane, 1979). a. 1) pharynx c. 22) intima 2) esophagus 23) sub i n t i m a l space 3) crop 24) desmosome 4) anterior midgut caeca 25) septate junction 5) posterior midgut caeca 26) gap junction 6) ventriculus 27) nucleus 7) proctodeal valve 28) tracheole 8) Malpighian tubule 29) sca l a r i f o r m junction 9) ileum 30) l a t e r a l i n t e r c e l l u l a r sinus 10) rectum 31) mitochondrion b. 11) inter-pad epithelium 32) tracheal sinus 12) r e c t a l pad c e l l 33) l a t e r a l i n t e r c e l l u l a r space 13) c i r c u l a r muscle 34) t i g h t , gap, and septate 14) r e c t a l lumen j u n c t i o n a l complex 15) sub-intimal space 35) s u b - e p i t h e l i a l space 16) intima 36) s u b - e p i t h e l i a l "secondary" 17) Type "B" c e l l c e l l s 18) nucleus 37) c i r c u l a r muscle 19) l a t e r a l membrane •20) trachea 21) l o n g i t u d i n a l muscle 22 s e r o s a l s i d e 23 rectum, which i s s t r u c t u r a l l y s i m i l a r and has been studied i n more d e t a i l , the intima attaches t i g h t l y bo. the a p i c a l membrane of s p e c i a l i z e d "sheath c e l l s " which are located at the seams between each pad (Noirot et a l . , 1979). The intima allows passage of water, ions and most organic solutes from the r e c t a l lumen into the sub-intimal space, but excludes solutes having a molecular weight greater than 400 ( P h i l l i p s and D o c k r i l l , 1968; Lewis, 1971). The r e c t a l pad consists mostly of large columnar " p r i n c i p a l " c e l l s 17 ym i n diameter and 100 ym long. These c e l l s are undoubtedly responsible for a c t i v e reabsorption of water and solutes from the r e c t a l f l u i d . Occasional small, goblet-shaped "Type B" c e l l s are also scattered throughout the pad. These c e l l s have few mitochondria and t h e i r function i s unknown (Peacock, 1979). Electron micrographs i n d i c a t e that r e c t a of locusts (Irvine, 1966; J a r i a l et a l . , unpubl. obs.; Jonas and V i e t i n g h o f f , 1975) and of cockroaches (Oschman and Wall, 1969; reviewed by Wall and Oschman, 1975) have many s t r u c t u r a l s i m i l a r i t i e s and i t i s reasonable to integrate findings from both tissue s . Both r e c t a are r i c h l y supplied with trachea and have an extraordinary number of mitochondria as might be expected for an a c t i v e l y transporting epithelium. In cockroach rectum, freez e - f r a c t u r e studies by Lane (1979) have shown the presence of many intramembranous p a r t i c l e s i n the l a t e r a l membrane and these may be involved i n transport. The l a t e r a l membrane of adjacent c e l l s i s held 20-25 nm apart (except for occasional d i l a t i o n s ) , apparently by i n t e r -c e l l u l a r columns which i n s e r t on some of these p a r t i c l e s . Gap junctions are numerous along the l a t e r a l c e l l border, suggestive of c e l l - c e l l coupling (Lane, 1979). I n t e r e s t i n g l y , the sheath c e l l s seem to lack these gap junctions (Noirot et a l . , 1979) and may i s o l a t e the i n t r a c e l l u l a r compartments of adjacent pads. Based on studies with i o n i c lanthanum, Lane (1979) has 24 suggested that occluding " t i g h t " junctions are located at the basal ends of the l a t e r a l i n t e r c e l l u l a r spaces i n cockroach rectum and that the more a p i c a l septate junctions do not block o f f access to the l a t e r a l space from the lumen. The exact l o c a t i o n of the p a r a c e l l u l a r b a r r i e r to t r a n s e p i t h e l i a l solute and water movement i s not known, however f l u i d and solutes which have been absorb-ed by the r e c t a l epithelium are thought to pass to the hemolymph v i a the l a t e r a l i n t e r c e l l u l a r spaces and tracheal sinuses ( F i g . 3c). A s i n g l e layer of secondary c e l l s , a layer of c i r c u l a r muscle 2-4 f i b e r s thick, and a bundle of l o n g i t u d i n a l muscle f i b e r s lay between the epithelium and the hemolymph, however these are penetrated by the trachea and do not form a continuous b a r r i e r . Secondary c e l l s are absent i n cockroach rectum. In summary, the epithelium has large transporting c e l l s with numerous mitochondria and extensively folded membranes. The serosal border (hemolymph-facing side of the tissue) i s extremely complex so that absorbate must pass through l a t e r a l spaces, sinuses and tracheal sinuses where separate secretory and reabsorptive processes have been proposed ( i . e . solute r e c y c l i n g ; P h i l l i p s , 1970; Wall and Oschman, 1970). The problems which r e s u l t from this complexity w i l l be noted i n the appropriate chapters. 25 CHAPTER 2: PROPERTIES OF TRANSEPITHELIAL CHLORIDE TRANSPORT Summary The mechanism and properties of t r a n s e p i t h e l i a l chloride transport across the locust rectum were examined In v i t r o . Serosal addition of 1 mM cAMP, a known stimulant of C l transport i n t h i s t i s s u e , increased s h o r t - c i r c u i t cur-Cl rent (I ) and net C l transport (J ) by 10-fold. Several predictions of Na-sc ^ net J v and HCO^-coupled models for t r a n s e p i t h e l i a l Cl transport were tested: ,C1-dependent I was not affected by Na-removal (<0.05 mM) during the f i r s t C l 75 min. Also, a large stimulation of J was e l i c i t e d by cAMP when recta were ° net 3 bathed f o r s i x hours i n Na-free s a l i n e (<0.001 mM-0.2 mM). In these experi-ments, there was no c o r r e l a t i o n between C l transport rate and the presence of micromolar quantities of Na contamination. Increased u n i d i r e c t i o n a l i n f l u x of 36 C l into r e c t a l t i s s u e a f t e r cAMP-stimulation was not accompanied by a 22 C l comparable uptake of Na. J^^was independent of exogenous CO2 and HCO^ • Rectal I was not affected by long-term exposure to 1 mM ouabain, furosemide, SITS or acetazolamide. In short, there i s no evidence that the major f r a c t i o n of C l transport across locust rectum occurs by the usual Na- or HCO^-coupled systems. C l transport did not exhibit an exchange d i f f u s i o n component, and was highly s e l e c t i v e for C l over a l l anions tested except Br. cAMP-stimulated Cl transport was strongly dependent on K but was i n s e n s i t i v e to removal of Ca or Mg during the f i r s t hour. Exposure to amino acid-free s a l i n e i ) lowered I from control l e v e l s , i i ) reduced the stimulatory e f f e c t s of cAMP, and i i i ) increased t r a n s e p i t h e l i a l C l permeability. I was not affected by serosal pH between 3.0 and 8.0 but was strongly i n h i b i t e d by low mucosal pH within the p h y s i o l o g i c a l range (pH 7.0-4.5). P h y s i o l o g i c a l hyperosmocity also reduced I and t i s s u e conductance. The r e s u l t s i n v i t r o suggest that r e c t a l 26 C l transport i s subject to several modes of regulation In vivo. Further d e t a i l s of K-dependence, and a c e l l u l a r model for KCl absorption are presented i n subsequent chapters. 27 Introduction E p i t h e l i a l transport of ch l o r i d e underlies many important regulatory processes i n animals. Studies of various vertebrate e p i t h e l i a have demonstrat-ed two general mechanisms of secondary C l transport: sodium-coupled systems, where C l entry into the e p i t h e l i a l c e l l s involves e l e c t r o n e u t r a l cotransport with Na (see chapter 1; reviewed by F r i z z e l l et a l . , 1979), and bicarbonate-coupled systems where C l entry or e x i t involves an ele c t r o n e u t r a l ( i . e . 1:1) exchange for HCO^ (Garcia Romeu et a l . , 1969; L e s l i e et a l . , 1973). Active C l transport i s also widespread i n invertebrate e p i t h e l i a , and has been i d e n t i -f i e d i n at l e a s t 15 species of in s e c t s . However, v i r t u a l l y nothing i s known regarding the properties and underlying mechanisms of chloride transport i n insects (see reviews by Harvey, 1981; P h i l l i p s , 1981). As might be expected, many functions of gut e p i t h e l i a i n insects p a r a l l e l those of vertebrate g a s t r o i n t e s t i n a l and renal systems (e.g. absorption of s a l t s , amino acids and water; reviewed by P h i l l i p s , 1980, 1981). Net active C l absorption across locust rectum i s well established i n s i t u ( P h i l l i p s , 1964b) and i n v i t r o (Williams, 1976; Williams et a l . , 1978). Also, absorption of C l i s normally regulated i n the i n t a c t locust i n response to s a l t loading or depletion ( P h i l l i p s , 1964b) and i s stimulated i n v i t r o by neuroendocrine gland homogenates (Spring et a l . , 1978; Spring and P h i l l i p s , 1980a,b), hemolymph from recently fed locusts (Hanrahan, 1978; Spring and P h i l l i p s , 1980c; P h i l l i p s et a l . , 1981), p u r i f i e d neuropeptide "chloride transport stimulating hormone" (CTSH; P h i l l i p s et a l . , 1980) and by cAMP (Spring et a l . , 1978; Hanrahan, 1978; Spring and P h i l l i p s , 1980b). After stimulation by CTSH or cAMP i n s a l i n e containing 200 mM C l , the rate of net C l absorption i s -2-1 approximately 7 uEq cm h (Spring and P h i l l i p s , 1980b) under approximate 28 s h o r t - c i r c u i t conditions ( i . e . no correction for s a l i n e r e s i s t a n c e ) . This value i s very s i m i l a r to that observed _in s i t u following i n j e c t i o n of 300 mM KCl i n t o the r e c t a l lumen ( P h i l l i p s , 1964b). Also, the spontaneous transepi-t h e l i a l p o t e n t i a l i n v i t r o (30 mV during stimulation; Spring et a l . , 1978; Hanrahan, 1978; Spring and P h i l l i p s , 1980a) i s s i m i l a r to that observed i n  s i t u ( P h i l l i p s , 1964b). In summary, the locust rectum transports C l at high rates In vivo and this a b i l i t y i s apparently preserved In v i t r o . The r e c t a l epithelium of the desert locust Schistocerca gregaria i s p a r t i c u l a r l y w ell suited for the d e t a i l e d study of ion transport mechanisms. Locust rectum has a high rate of s a l t absorption jLn vivo (potassium c h l o r i d e ; -2 -1 7.5 uEqcm h ; P h i l l i p s , 1964a-c), a large surface area when compared to most insect e p i t h e l i a (60 mm2 as a f l a t sheet), and i s s u f f i c i e n t l y robust to with-stand handling jLn v i t r o . A chitinous c u t i c l e which covers the mucosal surface acts as a natural support g r i d . The transporting columnar c e l l s are large (17 x 100 ym), accept microelectrode impalement r e a d i l y , and make up the great bulk of the epithelium. The purpose.of t h i s chapter i s to examine the properties of insect C l transport using t r a n s e p i t h e l i a l tracer f l u x and s h o r t - c i r c u i t current methods. Steady-state rates of a c t i v e C l transport across locust rectum are measured under control conditions and during cAMP stimulation using a voltage clamp which accurately s h o r t - c i r c u i t s the epithelium and a s a l i n e which i s based on the com-p o s i t i o n of locust hemolymph. Ion substitutions and i n h i b i t o r s are then used to test the various predictions of current models for C l transport across e p i -t h e l i a . Possible exchange d i f f u s i o n and anion s e l e c t i v i t y are also examined. F i n a l l y , several factors which might regulate r e c t a l ion transport including i n t r a - and e x t r a c e l l u l a r Ca, cGMP, external osmolarity and pH are investigated. 29 The major conclusions i n this chapter and those which follow (chapters 3-5) are summarized diagrammatically i n the General Discussion (chapter 6) . Some of the findings reported i n t h i s chapter have appeared i n abstracts (Hanrahan and P h i l l i p s , 1980a,b). Materials and methods Animals Desert locusts, Schistocerca gregaria Forskal,were obtained from a colony maintained at U.B.C. i n gregarious phase under a 15:9 hour lig h t : d a r k cycle, at 50% r e l a t i v e humidity, and with temperatures o s c i l l a t i n g between 40°C ( l i g h t ) and 26°C (dark). The colony was fed fresh l e t t u c e d a i l y and a dry mixture of a l f a l f a , bran, yeast and powdered milk. Unless otherwise indicated, adult female locusts 18-30 days beyond f i n a l moult were used i n a l l experi-ments because of t h e i r larger s i z e . Flux chambers I used modified Ussing-type chambers (Williams et a l . , 1978) which had been adapted from a design by Wood (1972). B r i e f l y , the tissue was mounted as a f l a t sheet over a collar-shaped opening and fastened on the outside by s i x small pins near the base of the c o l l a r . To form a s e a l , a rubber 0-ring was placed around the tiss u e on the c o l l a r so that i t f i t snuggly into a shallow groove above the pins. The area of the opening was 0.196 cm2. This c o l l a r design ensured that no pressure was exerted on the tissue when the two " p l e x i g l a s " half-chambers were clamped together. Solutions Five ml of s a l i n e were circulated, vigorously i n both half-chambers by a g a s - l i f t pump. Salines containing 10 mM HCO^ were bubbled with 95% 0 9:5% CO2 and remained at pH 6.8-7.0 during experiments. When HCO^-free s a l i n e was s t i r r e d with 100% 0 9, pH increased on the mucosal side from 7.0-7.4. This 30 change was ignored since I i s not affected by pH over t h i s range (see F i g . 16). Experiments were performed at 22±1°C, a value which i s within the d a i l y range of temperatures normally experienced by locusts i n the w i l d . The composition of experimental salines was based on analyses of locust hemolymph and Malpighian tubule f l u i d using a flame spectrophotometer (AA 120, Varian Techtron, PTY, Ltd., Melbourne, Aust.) on emission (Na, K) or absorp-t i o n mode (Mg, Ca). Samples of body f l u i d s were analysed a f t e r d i l u t i o n i n d i s t i l l e d H 20 (for Na measurements), 500 mM NaCl (K), 1.5% EDTA (Mg) or 0.5% LaCl^ (Ca). Chloride concentration was measured by t i t r a t i o n with AgNO^ according to Ramsay et a l . (1955). The pH of fresh locust hemolymph was measured using a thermostated pH microelectrode (PHM 71, Radiometer, Copen-hagen). To t a l CO2 was estimated using a Micro-Van Slyke method (Micro-C02 device, Harleco, Gibbstown, N.J.). Bicarbonate was calculated from the Henderson-Hasselbach equation assuming n e g l i g i b l e carbamino CO2, equilibrium between the dissolved gas and HCO^, and a pK i n locust hemolymph of ^ 6.1 (see Davenport, 1974). Hemolymph samples (10 ul) were co l l e c t e d from the neck and dissolved i n 0.5 ml of 3.75 (w/v) s u l f a s a l i c y l i c acid (pH 1.8, l i t h i u m c i t r a t e buffer) for analysis using an amino acid analyser (Model 118c, Beckman, Palo A l t o , Ca.). The i o n i c composition of p h y s i o l o g i c a l f l u i d s and experimental salines are given i n Table 1. A l l experimental sali n e s used i n th i s study contained ( i n mM): alanine (2.9), arginine (1.0), asparagine (1.3), glutamine (5.0), glycine (11.4), h i s t i d i n e (1.4), l y s i n e (1.4), pro l i n e (13.1), serine (1.5), tyrosine (1.9), v a l i n e (1.8). Glucose (10 mM) was also included. Sodium was completely replaced with choline i n Na-free s a l i n e , except where indicated otherwise. K- and HCO^-free salin e s were prepared by omitting the normally Table 2.1 Composition of p h y s i o l o g i c a l f l u i d s and experimental salines used to study ion transport across locus t rectum , . , Concentration (mEq L ) ody f l u i d s M (means ± s.e.) Cl K Na Mg Ca HC0 3 PH Hemolymph 106.7 12.2 103.0 24.4 18.4 13.0 7.1 ±14.7(6) ±1.6(8) ±7.4(9) ±5.0(5) ±5.2(5) ±1.0(6) ±0.04(9) Malpighian tubule f l u i d 87.6 165.1 . 46.9 39.3 13.1 -* t (obtained by l i g a t i o n ) ±14.3(11) ±15.1(11) • ±7 .9(13) ±8 .4(10) ±2.4(11) Food (fresh lettuce) 34(2) 114(2) 14(2) 5.2(2) 14.0(2) - - • Salines: Present study^ Normal s a l i n e 110-114 10 110-114 20 10 10 7.1 'High K' s a l i n e 50 140 50 20 10 10 7.1 (used i n chapter 3) 2 Salines: Previous studies Williams et a l . , 1978 63 9 55 26 4 10.5 6.7 (from Berridge, 1967) Spring et a l . , 1978; 201 11 209 5 5 24 7.0 Spring and P h i l l i p s , 1980a-c ''"See text (P. 30) for organic constituents common to these salines.. K-methylsulf ate s a l t was used i n "High K" s a l i n e . 2 For organic constituents, see o r i g i n a l references. 3 2 small amounts of these ions and adjusting s u l f a t e s a l t s and sucrose to main-ta i n constant Na, Mg, Ca, Cl and osmotic concentrations. A large anion d e f i c i t was observed i n Malpighian tubule f l u i d , perhaps the r e s u l t of dissolved urate or polyanions. Since the anion was not i d e n t i f i e d , I a r t i b r a r i l y used K-methylsulfate to prepare high K s a l i n e . Methylsulfate had no deleterious e f f e c t s on I when added to normal sa l i n e i n sc preliminary experiments. ... Osmotic concentration of sa l i n e s was r o u t i n e l y checked using an osmometer (Wide Range, Advanced Instr. Inc., Newton Highlands, Mass.) and for 15 d i f f e r -ent bulk preparations was 444.2±9.18 mOsm/l. As discussed i n l a t e r sections, s a l i n e osmolarity was increased on both sides during some experiments by additions of Na- or K-methylsulfate; however, these changes were well within the normal range experienced by t h i s tissue i n vivo (400-1100 mOsm/l, P h i l l i p s , 1964a). P r i o r to tracer f l u x measurements, tissues were exposed to Na, K or HCO^-free salines for 4 h and rinsed at l e a s t twice with fresh s a l i n e , except during Na-free experiments when s a l i n e was replaced three times. A f t e r turn-ing o f f the I s c> solutions were drained by suction and replaced using a syringe. I was not affected a r t i f a c t u a l l y by th i s method of changing solutions. To determine trace l e v e l s of contamination during flux studies using Na- and K-free s a l i n e s , these cations were r o u t i n e l y analysed by flame spectrophotometry as described above at the beginning and at the end of experiments. Maximal stimulation of r e c t a l transport (1 mM cAMP, f i n a l concen-tration) was achieved by adding 50 y l of a 101 mM cAMP solu t i o n to the serosal half-chamber. The sodium s a l t of th i s c y c l i c nucleotide was used except i n Na-free s a l i n e , when a c i d i c cAMP stock s o l u t i o n was t i t r a t e d to n e u t r a l i t y 33 with KOH. A stock s o l u t i o n of calcium ionophore was prepared i n absolute ethanol (400 yg/ml). In most experiments, A23187 was added to the chambers to a f i n a l concentration of 0.1-1.0 yg per ml s a l i n e so that the f i n a l ethanol concentration was l e s s than 0.25% v/v. At the highest dose used (5 yg/ml), i t -2 -1 was necessary to correct for a small increase i n I (1.12 ± 0.08 yEqcm h ) e l i c i t e d by ethanol. Calcium ionophore A23187 and furosemide were g i f t s of R. Dolman, E l i -L i l l y Canada, and J . Rees, Hoechst Pharm., r e s p e c t i v e l y . SITS (4-acetamido-4'- i s o t h i o c y a n o - s t i l b e n e - l , 2 ' - d i s u l f o n i c acid) was obtained from BDH Chem. Ltd., Poole, Eng. Amino acids and cAMP were obtained from Sigma. A l l s a l t s were reagent grade. E l e c t r i c a l methods T r a n s e p i t h e l i a l p o t e n t i a l (V ) was measured using a high input impedance 12 d i f f e r e n t i a l a m p l i f i e r (10 £2 5 4253 Teledyne P h i l b r i c k , Dedham, Mass.) connected to the chambers v i a calomel electrodes and 3 M KCl agar bridges. Operational amplifiers (725, National Semi-conductor Corp., Santa Clara, Ca., and 308, F a i r c h i l d Inc., Mountain View, Ca.) were used for voltage clamping and to measure lg C> r e s p e c t i v e l y . Both V and I were recorded on a s t r i p chart recorder (220, Soltec Corp., Sun Valley, Ca.). Corrections were made for asymetries between voltage-sensing electrodes and for resistance of the bathing s a l i n e by the method of Rothe et a l . (1969; also see appendix of t h i s t h e s i s ) . These corrections were e s s e n t i a l for several reasons: i ) the error i n I was ^10% under control conditions i f c o r r e c t i o n for s a l i n e resistance sc was not made; i i ) t h i s error changed unpredictably during K and C l additions or s u b s t i t u t i o n s since these ions affected t i s s u e conductance more than the conductivity of the external s a l i n e ; i i i ) cAMP greatly increased tissue con-ductance (^2-fold) without changing s a l i n e conductivity, thereby increasing 34 the s a l i n e resistance error p r o p o r t i o n a l l y . A d i f f e r e n t non-compensating voltage clamp has been used i n previous studies of locust rectum (Williams et a l . , 1978; Spring et a l . , 1978; Spring and P h i l l i p s , 1980a-c). In t h i s study, the normal protocol was to leave the rectum i n the short-c i r c u i t e d state and to measure open-circuit t r a n s e p i t h e l i a l p o t e n t i a l at 15 min i n t e r v a l s by b r i e f l y turning o f f I s c - Both I g c and V approached steady-state l e v e l s exponentially when the epithelium was voltage-clamped or undamped ( t x = 18 sec). To obtain accurate measurements of V , the tissue -5 t was undamped for 1.5 min. Open-circuit p o t e n t i a l was then 96.2 ± 0.63% (x ± s.e., n=8) of i t s steady-state value under continuous open-circuit conditions without cAMP and 92.2 ± 0.76% during exposure to cAMP. V measured a f t e r 1.5 min were s i m i l a r to those obtained i n l a t e r experiments under continuous open-circuit conditions using tracers (Fig. 5) and ion s e n s i -t i v e microelectrodes (Tables 2 and 3 i n chapter 4). T r a n s e p i t h e l i a l tracer fluxes 3 6 Cl (New England Nuclear, c a r r i e r - f r e e , 5.9 mCi/g Cl) was added as 36 36 H C l to control and Na-free s a l i n e s , and as Na C l to HCO^-free s a l i n e . These amounts were too small to cause s i g n i f i c a n t changes i n C l concentration (3% error) or pH. The protocol for measuring t r a n s e p i t h e l i a l tracer fluxes was as follows: Aliquots of stock isotope (10-50 pl) were added to one half-chamber referred to as the "hot side". A f t e r 10 min of vigorous mixing, 1 y l samples were taken i n duplicate from the hot side. These were placed into v i a l s containing 1 ml of "cold" s a l i n e and 10 ml of s c i n t i l l a t i o n f l u i d (ACS- Amer-sham Corp., O a k v i l l e , Ont.) for counting" with a l i q u i d s c i n t i l l a t i o n counter (Isocap, Nuclear Chicago). One ml samples were taken from the "cold s i d e " at 15 or 20 minute i n t e r v a l s and these were replaced with cold s a l i n e . Radio-35 a c t i v i t y of the hot side d i d not change measurably during f l u x experiments. The tracer a c t i v i t y of the cold side never exceeded 0.5% of that on the hot side, hence no correction was necessary for tracer backflux. U n i d i r e c t i o n a l f l u x was calculated using the formula (Williams et a l . , 1978): a -V-C 1 _^ 2 a «T-A -2 -1 where i s u n i d i r e c t i o n a l f l u x (uEqcm h ), a^ i s r a d i o a c t i v i t y of the hot side (cpm/ml), i s the increase i n r a d i o a c t i v i t y of the cold side (Acpm/ml), V i s volume of the cold side (5. ml), C i s t o t a l concentration of bulk and tracer isotopes (uEq/ml), T i s time between samples (h) and A i s tissue 2 surface area (0.196 cm ). Appropriate corrections were made for d i l u t i o n during sampling. I n i t i a l f l u x values (which included e q u i l i b r a t i o n with the t i s s u e pool) were not included i n the c a l c u l a t i o n s . No attempt was made to measure both u n i d i r e c t i o n a l fluxes on each tissue 36 77 with C l and Br since s e l e c t i v i t y experiments showed that Br i s transported at less than 1/2 the rate of C l . Paired tissues were not a v a i l a b l e . Never-theless, differences i n I , V and t r a n s e p i t h e l i a l resistance (R ) were sc t t n e g l i g i b l e between tissues used for forward arid back fluxes and these d i f f e r - ' ences w i l l be s p e c i f i e d In the r e s u l t s section. Although the method of mounting tissues used i n t h i s study has been described previously (Wood, 1972; Williams et a l . , 1978; Spring et a l . , 1978; Blankemeyer, 1978), no estimates of the permeability induced by edge damage have been published using t h i s technique. To estimate the magnitude of a r t i f a c t u a l shunting due to edge damage, 35 u n i d i r e c t i o n a l fluxes of SO^ were measured across recta i n "high SO^" s a l i n e (207 mEq/1; see simple S0 4 s a l i n e / / l , Spring and P h i l l i p s , 1980b) under control 36 conditions and following sequential additions of 1 mM cAMP and 114 mM NaCl • - 35 (Table 2). Forward and back fluxes of. SO^ are not s i g n i f i c a n t l y d i f f e r e n t (P >0.2) suggesting that l i t t l e i f any active SO^ transport occurs (see also 35 Williams et a l . , 1978). If a l l SO^ f l u x occurs through non-selective edge damage, then the calculated upper l i m i t of SO^ permeability for t h i s pathway —7 —1 36 3.7 x 10 cm sec . An upper l i m i t f o r C l f l u x due to edge damage can. the 35 be calculated from t h i s SO^ f l u x data using the r e l a t i v e f r e e - s o l u t i o n ux m o b i l i t i e s of C l and SO^. Such c a l c u l a t i o n s reveal that the maximum Cl f l due to edge damage i s <12% of the t o t a l passive C l backflux from serosa-to-mucosa and i s n e g l i g i b l e (1.5%) compared to the rate of net C l absorption. 36 A 2 In summary, I believe that C l and K f l u x measurements with this chamber design are not s i g n i f i c a n t l y influenced by edge damage. Independent e l e c t r o p h y s i o l o g i c a l data supporting t h i s view are presented i n chapter 5. Tracer influxes across the a p i c a l c e l l border Everted r e c t a l sacs cannulated with polyethylene tubing (PE 90, Parsip-pany, Dickinson and Co., N.J.) and containing control s a l i n e were prepared according to the method of Goh and P h i l l i p s (1978). This preparation was 22 36 convenient for measuring V and u n i d i r e c t i o n a l fluxes of Na and C l from the mucosal side into e p i t h e l i a l tissue under open-circuit conditions. The following protocol was used: everted sacs (mucosal surface f a c i n g outwards) were incubated with control s a l i n e on both sides for three hours. . Some sacs were injected with 10 mM cAMP for the f i n a l hour. V was measured using a high input impedance electrometer (602, Keithley I n s t r . , Cleveland, Oh.) connected v i a calomel electrode and 3 M KCl agar bridges. Tissues were then 3 1 placed i n c o n t r o l s a l i n e containing H-mannitol for 20 min, followed by 3 36 2 incubations i n s a l i n e containing H-mannitol and Cl or ~ Na for an a d d i t i o n a l 30-45 min at pH 4.5 to temporarily i n h i b i t C l transport. A f t e r a t i m e d e x p o s u r e to the t e s t s o l u t i o n (pH = 7.0, 35 Table 2.2 SO, fluxes as an i n d i c a t i o n of maximum permeability through edge damage 4 1 under control conditions, and a f t e r sequential cAMP and NaCl additions. Condition SO. J 4 ms SO. J 4 sm 2 SO. 4 3 *P .. calculated L i *p from SO. 4 4 *P p 1 observed i n normal s a l i n e Maximum f r a c t i o n of * P C 1 through -2 -1 (uMcm h ) -2 -1 (uMcm h ) (cm sec ^ x 10^) -1 7 (cm sec x 10 ) (cm sec ^ x 10^) edge damage Control 0.0804 0.1019 2.74 5.23 50.0 10.5% ±0.011 ±0.015 ±0.4 ±5.0 (12;4) (18;6) (18;6) ImM cAMP 0.1241 0.1326 3.47 6.62 57.0 11.61% added to ±0.012 ±0.012 ±0.54 ±9.0 serosal side . (12;4) (18;6) (18;6) cAMP + 114mM 0.1182 0.1843 4.97 9.43 - -C l added to ±0.012 ±0.030 ±0.79 both side (6;4) (12;6) (12:6) ! s o 4 S 0 4 Parentheses show (number of f l u x periods; number of animals); J and J are s u l f a t e fluxes from mucosa-to-serosa and serosa-to mucosa, res p e c t i v e l y . Recta were bathed i n s u l f a t e s a l i n e #1 SO, (Spring and P h i l l i p s , 1980a) under I ^ conditions. 2 35 t T r a n s e p i t h e l i a l SO^ permeability was calculated as /[SO^] 3 "Calculated *P " was obtained from * P c n by correcting f o r the diffe r e n c e between SO. and C l m o b i l i t i e s Cl 4 i n free solution, and therefore represents an upper l i m i t for C l permeability through non-selective edge damage. 38 3 36 22 H-mannitol plus C l or Na), tissues were dissected from the cannula, bl o t t e d on bibulous paper, weighed, macerated i n v i a l s containing 1 N KOH, and digested at 80°C over night. A f t e r n e u t r a l i z i n g with R^SO^, the v i a l s were 3 36 22 counted for H-mannitol and C l as previously described. Na was counted using an automatic gamma counter (model 4253, Searle). Tritium counts were corrected for a c t i v i t y due to other tracers. 3 H-mannitol was used to estimate e x t r a c e l l u l a r tissue space which was i n continuity with the mucosal side. To test whether mannitol i s metabolised by locust rectum, 6 tissues were incubated separately i n stoppered test tubes 14 with 1 ml of cont r o l s a l i n e containing C-mannitol (0.12 mM, approx. 4.5 x 10^ cpm). The bathing saline was a c i d i f i e d a f t e r 1 h to drive o f f dissolved 14 CO2 which was c o l l e c t e d on glass f i b e r f i l t e r s soaked i n hyamine hydroxide 14 and counted by l i q u i d s c i n t i l l a t i o n . Since no C - a c t i v i t y was detectable ( s e n s i t i v i t y of the method was estimated to be 5.4 x 10 ^ moles "^^CO^/h) i t was concluded that mannitol i s not metabolised. A layer of porous c u t i c l e covers the mucosal surface of the epithelium, trapping a large unstirred layer external to the a p i c a l membrane. In order to e q u i l i b r a t e tracers with this dead space, tissues were preincubated at low pH as described above. Very low counts were observed i n sac absorbate a f t e r 36 3 22 3 30 min exposure to pH 4.5. Ratios of C l : H and Na: H were s i m i l a r to those outside the sac i n d i c a t i n g i ) a non-selective shunt was probably responsible 3 for these fluxes, i i ) H-mannitol s t i l l provided a reasonable estimate of 36 2*^  22 36 e x t r a c e l l u l a r C l and "Na. In control experiments, Na and C l did not accumulate measurably inside r e c t a l sacs during 1-10 min exposures to tracers 36 at pH 7.0. F i n a l l y , cAMP stimulates the i n f l u x of Cl into r e c t a l tissue against an opposing e l e c t r i c a l gradient, providing further evidence for the v a l i d i t y of the technique ( F i g . 12a and discu s s i o n ) . 39 Calculations and s t a t i s t i c s In order to compare the instantaneous s h o r t - c i r c u i t current with tracer fluxes measured at i n t e r v a l s , I traces were integrated using a planimeter (model L30M, Lasico, Los Angeles, Ca.). Values are means ± standard errors unless stated otherwise; n = number of recta. S i g n i f i c a n t d i f f e r e n c e was determined using paired or unpaired t - t e s t s . Results 1) V e r i f i c a t i o n of active C l transport The mucosal (luminal) s o l u t i o n was e l e c t r i c a l l y p o s i t i v e with respect to the serosa at a l l times for locust r e c t a exposed to normal s a l i n e i n Ussing-type chambers under open-circuit conditions. Under control conditions, r e c t a l I ( i n d i c a t i n g net transport of anions to the serosal side or cations to the -2 -1 mucosal side) declined exponentially from 6 to 1.1 uEqcm h ( t i = 40 min). I approached a steady-state condition after 3 h ( F i g . l a ) . After an i n i t i a l increase, R remained constant for several hours (Fig. Id). These r e s u l t s agree with previous findings using everted sacs (Goh and P h i l l i p s , 1978) and f l a t sheet preparations of t h i s t i s s u e (Williams et a l . , 1978; Spring and C l P h i l l i p s , 1980b). Net C l f l u x (J t ) p a r a l l e l s I g c during the nine hour -2 -1 C l experimental period. The discrepancv of 1 uEqcm h between I and J ^ i n r r r • n sc net unstimulated recta may be due to some un i d e n t i f i e d electrogenic process (Williams et a l . , 1978; Baumeister et a l . , 1980). I n t e r - t i s s u e v a r i a b i l i t y i s probably not responsible for the discrepancy, since I g c> Vfc and Rfc were v i r t u a l l y i d e n t i c a l during forward and back f l u x experiments ( i . e . difference C l was not s i g n i f i c a n t at anv sample time, P >> 0.2). J was lower than ^ . f 5 n e t -2 -1 previous steady-state C l f l u x measurements (0.5 uEqcm h as compared to -2 -1 3-4 uEqcm h ; Williams et a l . , 197S). Two major differences from previous methods might explain these d i f f e r e n t r e s u l t s : i ) compensation was made for 40 a E o cf LU 3 . o in o \ , . CT LU 3 . V 1 i. > £ 20 10 H E o 300 < ^ 200 T T I I t„o-o'' 4 6 T i m e C h ) Figure 2.1 The approach of i s o l a t e d , unstimulated r e c t a l t i s s u e to steady-state conditions. (a) s h o r t - c i r c u i t current (T ) and net C l fl u x sc C l (J ); (b) u n i d i r e c t i o n a l C l fluxes measured under I conditions, from net sc mucosa to serosa, and from serosa to mucosa; (c) spontaneous transepithel-i a l p o t e n t i a l (V f c); and (d) t r a n s e p i t h e l i a l resistance ( R t ) . Experiments were performed during the time i n t e r v a l shown by the ho r i z o n t a l bracket as i n (a). Recta were bathed i n normal s a l i n e (Table.2.1) and were l e f t short-c i r c u i t e d except f or 90 second i n t e r v a l s when spontaneous V was measured. Means ± s.e.; n = 6 ( J ^ 1 , J C 1 ) ; n = 12 (I , V , R j . ms' ms sc' t t 41 T i m e C h ) Figure 2.2 E f f e c t s of cAMP on C l fluxes and e l e c t r i c a l parameters under """sc c o n d i t i o n s i n normal s a l i n e . Cyclic-AMP (1 mM) was added at the arrow to the serosal side. Time 0 was preceded by 4 h e q u i l i b r a t i o n i n order to achie' a steady-state. Other conditions and d e f i n i t i o n s as i n Figure 2.1. \ 6 0 - 1 0 | —i 1 — 1 1 1 — r 0 2 4 6 T i m e ( h ) Figure 2.3 Responsiveness of I to cAMP as a function of time a f t e r d i s s e c t i o n of recta from locu s t s . Cyclic-AMP (1 mM) was added at the arrows to the serosal side of two recta bathed i n normal s a l i n e (Table 2.1). 43 Figure 2.4 Chloride dependence of cAMP-stimulated I • Time 0 indicates time a f t e r d i s s e c t i o n . At the f i r s t arrow, 1 mM cAMP was added to the serosal side while the rectum was bathed i n normal s a l i n e . A l l C l was replaced b i l a t e r a l l y with gluconate at the second arrow; 1 mM cAMP was s t i l l present on the serosal side. T i m e (h) 45 the seri e s resistance of the s a l i n e , i i ) a complex s a l i n e based on normal hemolymph ion and amino acid l e v e l s was used i n flux experiments. For the purposes of th i s study, the important conclusion i s that no spontaneous increases i n l g c > C l fluxes, V^, or R occur i n v i t r o a f t e r the i n i t i a l decay i n C l transport. An approximate steady-state condition i s maintained between 4-8 hours a f t e r d i s s e c t i n g recta from locu s t s . Experiments were performed during t h i s period unless indicated otherwise. Figure 2 shows the e f f e c t s of adding 1 mM cAMP to the serosal (hemocoel) side on C l fluxes under I conditions, V and R . Zero time shown on t h i s sc t t and subsequent figures i s preceded by a four-hour e q u i l i b r a t i o n period under I conditions to ensure a steady-state; i . e . experiments were conducted between the 4th and 8th hour a f t e r d i s s e c t i o n . Cyclic-AMP caused very large C l increases i n I , and J , reduced R„ by ^50%, and' caused a s l i g h t elevation sc t ms t b Cl -2-1 i n Jsm ( f r o m 1-47 ± 0.14 to 1.68 ± 0.26 yEqcm h ). Maximal stimulation was obtained with 1 mM cAMP, a higher dose than previously reported (0.3 mM) by Spring (1979), who conducted studies between the second and fourth hour a f t e r d i s s e c t i o n . The e f f e c t s of cAMP are independent of sex since s i m i l a r stimulations of -2 -1 I (from 1.9 ± 0.12 to 8.8 ± 0.53 yEqcm h ; n = 6) were obtained using recta from male locu s t s . Responsiveness to cAMP i s also independent of time following d i s s e c t i o n . This i s i l l u s t r a t e d by the traces i n Figure 3. Even aft e r 6 hours, the I across i n v i t r o r e c t a increased a f t e r cAMP addition to ' sc -2 -1 the o r i g i n a l high values of 10 yEqcm h Active C l transport during cAMP stimulation was further confirmed by 0 replacing C l with anions which are not known to be transported i n other tissue s . Figure 4 shows that cAMP-stimulated I i s reduced by 91% when C l i s sc J replaced with gluconate. This r e s u l t i s consistent with those r e s u l t s obtain-46 ed by other workers using d i f f e r e n t salines and voltage clamps: Williams et a l . (1978) and Herrera et a l . (1976) found that high i n i t i a l I a f t e r dissec-sc tion i s abolished by C l replacement. Corpus cardiacum extracts did not stimulate t r a n s r e c t a l I when bathed i n salines containing s u l f a t e or n i t r a t e sc ° instead of C l (Spring and P h i l l i p s , 1980). As previously shown for unstimu--2 -1 lated r e c t a (Fig. 1), a small r e s i d u a l I (1 uEqcm h i n these experiments) was i n s e n s i t i v e to C l replacement a f t e r cAMP stimulation (Fig. 2). However, C l a l l Al a f t e r stimulation could be accounted for by AJ and t h i s i s true of sc J net a l l subsequent experiments i n th i s study. F i n a l l y i n a t h i r d s e r i e s of experiments )V and u n i d i r e c t i o n a l C l fluxes were measured under open-circuit conditions, and the observed f l u x r a t i o s were compared with those values calculated from V using the Ussing f l u x r a t i o equation and assuming that C l crosses the r e c t a l wall only by simple d i f f u s i o n and (Fig. 5). As i n previous experiments, cAMP caused a large increase i n V C l C l J . A f t e r adding cAMP, J was s i m i l a r under open-circuit and s h o r t - c i r c u i t ms ° sm r -2 -1 -2 -1 conditions (1.34 ± 0.06 uEqcm h and 1.71 ± 0.12 uEqcm h , r e s p e c t i v e l y ) , C l -2 -1 however J m g was s i g n i f i c a n t l y lower (4.75 ± 0.7 uEqcm h and 11.04 ± 0.64 -2-1 uEqcm h ; P < 0.001). When K methylsulfate was added to the mucosal side to r a i s e [K] from 10 to 100 mM and thereby mimic i n vivo conditions, V decreased by 92% (23 mV), J C 1 increased by 38% but J C 1 remained unchanged (P > 0.2). ms sm Observed f l u x r a t i o s were higher than those predicted from the f l u x r a t i o equation ( F i g . 5). Equations derived by Ussing (see Zerahn, 1956) may be used to c a l c u l a t e the minimal metabolic energy requirement for C l transport under each of these experimental conditions. The minimum work per equivalent - of C l transported i s : j C l W = ( v tF+ RT In 0.239 • ^sm 47 mucoial lOOmM K 1mM cAMP > E > E u cr UJ 3. 20 ~ 10 E 4 i 1 / / I observed predicted I r 1 2 T i m e C h ) Figure 2.5 E f f e c t s of sequential addition of 1 mM cAMP to the serosal side and mucosal a d d i t i o n of potassium on C l fluxes under open-circuit conditions. Potassium concentration was increased from 10 to 100 mM by adding K-methylsul-fate to the mucosal side. (a) T r a n s e p i t h e l i a l p o t e n t i a l ( • V ); 36 (b) u n i d i r e c t i o n a l C l fluxes from mucosa to serosa ( • m-^ s) , and serosa to mucosa ( A s-*m; n = 6) ; and (c) Cl fl u x r a t i o s ( • ) observed, and ( • ) predicted from V by the Ussing f l u x r a t i o equation. Means ± s.e.; n = 12 (V.), n = 6 ( J C 1 ) , n = 6 ( J C i ) . t ms sm 48 where W i s work (ca l o r i e s / e q u i v . ) , V i s t r a n s e p i t h e l i a l p o t e n t i a l ( v o l t s ) , and R, T and F have t h e i r usual meanings. Under open-circuit conditions, unstimulated recta i n normal s a l i n e must expend >3.1 x 10 c a l o r i e s cm h i n C l transport work (taking V = 0.008 V, J C 1 / J C 1 = 2.1, and J C 1 = 0.5 x 10~ 6 t ms sm net -2 -1 Eqcm h ). During cAMP stimulation, C l transport work increased by 2 0 - f o l d to 6.0 x 10~ 3 c a l o r i e s cnfV" 1 (V = 0.025 V, J C 1 / J C 1 = 4.1, J C 1 = 4.3 x 10~ 6 t ms sm net -2 -1 Eqcm h ). These c a l c u l a t i o n s i n 10 mM K s a l i n e are not representative of the C l transport work i n vivo where the lumen contains >100 mM K. Although addition of mucosal K increased both the f l u x r a t i o and net C l flux.42%, trans--3 -2-1 port work rose only 5% to 6.3 x 10 c a l o r i e s cm h due to the reduced Cl Cl -2 -1 t r a n s e p i t h e l i a l p o t e n t i a l (V = 0.002 V, J / J = 5.4, J = 6.1 yEqcm h ). t ms sm One i m p l i c a t i o n of the e f f e c t s of high mucosal K on r e c t a l C l transport i s that energy stored i n the K gradient between Malpighian tubule f l u i d and hemolymph i s p a r t i a l l y u t i l i z e d during C l reabsorption i n the rectum. In e f f e c t , the K pump i n the Malpighian tubules and the C l pump i n the rectum exhibit a form of energetic coupling. In summary, C l absorption across locust rectum f u l f i l l s c l a s s i c a l c r i t e r i a C l f or active transport reviewed by Koch, 1970): ^ n e t i - s maintained i n the absence of e l e c t r i c a l and chemical gradients ( i . e . under I conditions) and & sc f l u x r a t i o s at open-circuit are an order of magnitude higher than those predicted for simple d i f f u s i o n . The s e n s i t i v i t y of cAMP stimulated I to Cl anion substitutions and good agreement between I and J a f t e r cAMP addi-e ° sc net t i o n i n the present and previous studies (Williams et a l . , 1978; Spring and P h i l l i p s , 1980b) in d i c a t e that C l transport i s the predominant electrogenic process i n t h i s epithelium. Also, 1 mM cAMP causes a very large (^50%) decrease i n t r a n s e p i t h e l i a l r e s i stance. We s h a l l see in chapter 5 that t h i s stimulation of t r a n s e p i t h e l i a l conductance has two components: an enhanced K permeability and an "active C l transport" conductance. 49 2) Exchange d i f f u s i o n The presence of a large, electrogenic C l f l u x across locust rectum (Fig. -2) does not preclude an exchange d i f f u s i o n component. Exchange d i f f u -sion has been suggested i n other invertebrate e p i t h e l i a (see Krogh, 1939; Shaw, 1960; Stobbart, 1965; Smith, 1969; Dietz, 1974; Dietz and Branton, 1975). C l If J " s m occurs p a r t l y by exchange with mucosal C l , then the following behaviour ' C l i s predicted: i ) reduction of mucosal [Cl ] should decrease J (e.g. K r i s t e n -t- sm e sen and Larsen, 1978; Ques-von Petery et a l . , 1978; Biber et a l . , 1980), and C l i i ) estimates of t r a n s e p i t h e l i a l C l permeability calculated from J g m under I g c conditions should be higher than when calculated under open-circuit conditions, since e l e c t r o n e u t r a l exchange flux would be i n s e n s i t i v e to V (Lewis and Diamond, C l 1976). These predictions were not observed i n locust rectum. J was not r sm -2 -1 reduced, but increased s l i g h t l y from 0.62 ± 0.1 to 0.83 ± 0.15 yEqcm h (x ± s.e., n = 6 d i f f . not s i g n i f i c a n t at P > 0.1) under I conditions when ° sc C l mucosal C l was replaced with methylsulfate, i n d i c a t i n g that J was not stimulated by C l on the "trans" side (Table 3). Secondly, the apparent s c t r a n s e p i t h e l i a l C l permeability was s i m i l a r under s h o r t - c i r c u i t (/vP^) and oc sc open-circuit current (*^Q-^) conditions. As a f i r s t approximation, i s given by the equation *P^ !f = J C^/[cl] where J C 1 i s 4.7 x 10 ^ Eqcm 2 h ^ (from C l sm sm —6 —3 F i g . 2) and [ C l ] i s 114 x 10 Eqcm . To estimate C l permeability from the e f f e c t s of t r a n s e p i t h e l i a l p o t e n t i a l , the equation *P°!f = - R T . / C l passive Cl [ C l ] V zF, where V_ i s 24 mV (Fig. 5), and net passive flux (J . N t t ° r passive) was Vt=24 mV V =0 _ 2 , estimated as 2(J - J ) = 2.78 x 10 yEqcm h (Figs. 2 and 5). sm sm oc —6 —1 sc The value of *P (6.0 x 10 cm sec ) i s a c t u a l l y higher than *P p l (4.1 x 10 ^ cm sec ^ ) , a r e s u l t which is-not consistent with exchange d i f f u s i o n . C l Both of these c a l c u l a t i o n s assume that J i s passive ? independent, and that 50 Table 2.3 E f f e c t of removing mucosal C l on serosa-to-mucosa f l u x of C l under I conditions. sc serosa: 114 mM C l serosa: 114 mM Cl mucosa: 114 mM C l mucosa: 0 mM C l Cl -2 -1 J g m (yEqcm h ) 0.62 0.83 ±0.10 ±0.15 Both sides were i n i t i a l l y bathed i n normal s a l i n e (114 mM C l ; Table 2.1). C l was replaced on the mucosal side with methyl s u l f a t e and a small amount of s u l f a t e . Sucrose was adjusted to maintain isosmocity. Means ± s.e., n = 6; not s i g n i f i c a n t l y d i f f e r e n t (p >0.1). 51 the epithelium behaves as i f i t were a s i n g l e b a r r i e r to Cl movement. The val-i d i t y of these assumptions i s discussed l a t e r i n t h i s chapter and i n chapter 5. In summary, i f Cl exchange d i f f u s i o n i s present i n locust r e c t a l e p i t h e l -ium, i t must occur at an extremely low rate which does not contribute 3 6 s i g n i f i c a n t l y to C l fluxes under I conditions. 3) S e l e c t i v i t y of C l transport Two methods were used to examine the s e l e c t i v i t y of C l transport. The f i r s t method was semi-quantitative, but permitted anions to be tested i n the Cl presence of 20 mM C l on both sides of the epithelium. J was measured under r ms I conditions during cAMP stimulation. After three c o n t r o l f l u x periods, sc ° r > various unlabelled t e s t anions (A) were added to both sides as Na s a l t s ( f i n a l C l concentration of A was 20 or 50 mM). J and I were measured for a further ms sc three f l u x periods (3 x 15 minutes). If A (the added,unlabelled anion) i s transported e l e c t r o g e n i c a l l y , then i t should increase the difference between C l I and J since t h i s unmarked anion transport would contribute to I but sc ms r sc 3 6 not to the mucosa-to-serosa f l u x of C l . Moreover, the f o r e i g n anions should Cl depress J i f the transport s i t e s are nearly saturated with "substrate", ms C l P a r a l l e l decreases of s i m i l a r magnitude i n I and J would suggest i ) compe-& sc ms & & t i t i o n between test anion and Cl without A t r a n s l o c a t i o n or i i ) metabolic i n h i b i t i o n . Addition of unlabelled Cl under i d e n t i c a l conditions was used as a standard against which the e f f e c t s of other anions were compared. Figure 6 shows the e f f e c t s of nine test anions on the d i f f e r e n c e between 3 6 Cl s h o r t - c i r c u i t current arid C l forward f l u x (I - J ). As expected, the sc ms addition of 50 mM unlabelled Cl ( r a i s i n g t o t a l [Cl] to 70 mM) increased C l -2 -1 (I C - J m s ) by 4.3 and 4.5 yEqcm h in two preparations. Increases were part-Cl -2 -1 l y due to reductions i n J (-1.6 and -2.1 yEqcm h , r e s p e c t i v e l y ) . These -2 -1 tissues had normal values of I (7.1 and 8.5 yEqcm h ), consistent with 52 12 12 8H 'e • o cf 1U a. w 4 -U OJ 0-12-, 0 u E - 3 -c r Br" I-30 60 90 Time (min) 0 81 0 8n B ica rbonate o-i 81 0 J 30 60 90 Time (min) Figure 2.6 E f f e c t of adding anions on the d i f f e r e n c e between I and the . s c u n i d i r e c t i o n a l flux of C l from mucosa to serosa. Anions (20-50 mM) were added at the arrow to both sides of cAMP-stimulated r e c t a bathed i n normal s a l i n e (Table 2. ) i n which 90 mM C l had been replaced by gluconate. The C l shaded area i s (I - J ) f o r i n d i v i d u a l preparations. As a c o n t r o l (upper sc ms * v * v left-hand panel), 50 mM unlabelled C l was added at the arrow. 53 —2 —1 r e s u l t s which w i l l be reported i n chapter 3 at a [Cl] of 70 mM (7.3 yEqcm h Cl Bromide add i t i o n caused smaller increases i n ( I - J ) than did C l addition sc ms -2 -1 (3.8 and 2.7 yEqcm h i n two preparations). Fluoride addition produced a Cl gradual decline i n both I and J , suggesting a metabolic i n h i b i t i o n since b sc ms & t h i s ion i s often toxic to tissues (see Diamond and Wright, 1969). No other C l anions had si z a b l e e f f e c t s on I or J (n = 2, for each anion), suggesting sc ms l i t t l e competition for C l binding or tran s l o c a t i o n . In a second seri e s of experiments, recta were e q u i l i b r a t e d under I conditions and rinsed f o r 4 hours with salines i n which C l was completely replaced by other anions. I was measured before and a f t e r addition of 1 mM cAMP to the serosal side. Cyclic-AMP stimulated I s i g n i f i c a n t l y only when C l or Br was present -2-1 (Fig. 7). The s e l e c t i v i t y sequence, i n order of decreasing A I g c (yEqcm h ), was C l (5.4) > Br (2.7) > PO^.HPO^ (0.5) > I , acetate, thiocyanate, SO^, N0 3 > ( a l l l e s s than 0.2, i . e . not s i g n i f i c a n t l y d i f f e r e n t from unstimulated r e c t a ) . Urate was also unable to sustain the I • These data strongly suggest that C l i s the p r i n c i p a l anion which i s transported by t h i s system i n v i t r o . Over-a l l s e l e c t i v i t y could be determined at the Cl tr a n s l o c a t i o n step or at the binding s i t e . The l a t t e r i s suggested by the lack of i n h i b i t o r y e f f e c t of most anions on J ms 4) Ionic dependencies of C l transport a) Sodium dependence: Several experiments were performed to test whether Cl absorption requires external Na as predicted by the NaCl cotransport model (reviewed by F r i z z e l l et a l . , 1979). 54 cAMP 1-5 H 30 60 90 120 Time (min) Figure 2.7 E f f e c t of 1 mM cAMP on I i n anion-substituted s a l i n e s . — sc Tissues were pre-equilibrated f o r 4 h under I conditions i n s a l i n e i n which sc a l l C l was substituted with a test anion (see Table 1). Means ± s.e.; n = 5-6. 0 30 60 90 110 0 Time (min) 55 i ) Prolonged removal of external Na Recta were eq u i l i b r a t e d for 4 h i n nominally Na-free s a l i n e and were rinsed at l e a s t three times with fresh s a l i n e to remove Na which had leaked from the ti s s u e . F i n a l external Na concentration ranged from <l-200 uM. In Na-free s a l i n e , a d d i t i o n of 1 mM cAMP increased I (1.9 ± 0.44 to sc 8.8 ± 1.0 yEqcm~ 2h _ 1) and (0.1 ± 0.68 to 5.3 ± 2.8 uEqcm _ 2h - 1, ±95% con-Cl fidence i n t e r v a l ) and AJ s t i l l accounted for >75% of AI ( F i g . 8). sm sc C l Stimulated values of J and I were smaller than controls, but this net sc was not s t a t i s t i c a l l y s i g n i f i c a n t at P > 0.1 (compare Figs. 2 and 8). E l e c -t r i c a l parameters were s i m i l a r i n the groups of locusts used for forward and backfluxes. A f t e r 1 h of exposure to cAMP, I was 7.62 ± 0.47 vs 8.86 ± 0.8 -2 -1 yEqcm h , V was 27.93 ± 0.23 vs 31.29 ± 3.01 mV and R t was 136.78 ±7.89 " "2 vs 135.51 ± 11.81 0. cm for m->-s vs s-*m f l u x measurements, re s p e c t i v e l y . It was important to test whether Na-coupled Cl flux might p e r s i s t because of the presence of minute quantities of Na i n nominally Na-free s a l i n e . However, i t should be noted that such a mechanism would require an extremely high Na a f f i n i t y for a NaCl cotransport mechanism. Sodium contamination was measured at the beginning and at the end of most experiments as described i n the methods except that samples were not d i l u t e d i n d i s t i l l e d water. Figure 9 shows that there was no c o r r e l a t i o n between cAMP-stimulated I (which sc remained high) and trace l e v e l s of Na. In several preparations, Na could not be detected (<1 yM) at the end of the experiment when [Na] i s highest. Less 2 than 3% of the v a r i a t i o n i n I may be a t t r i b u t e d to [Na] (r = 0.02S8). Cl The 40% reduction i n average J n e t a f t e r long exposures (>5.5 h) to Na-free s a l i n e containing cAMP requires some comment. Much of t h i s decline may be due to the absence of Na-coupled amino acid absorption which has been demonstrated i n locust rectum (Balshin and P h i l l i p s , 1971). Consistent with 56 E 8 u C T L U 3. w 4 O in 0-> a —1 ! ^ 8 I SZ cr L U b . y\. / 1 2 Time Ch) 3 0 0 g 2 0 0 C w EE* 1 0 0 30 /-> 20 > E 10 o-J 1 1 I 1 2 3 Time (h) Figure 2.8 E f f e c t of cAMP on C l f l u x e s and e l e c t r i c a l parameters i n • Na-f r e e and • HCO^-free s a l i n e s . Cyclic-AMP (1 mM) was added to the s e r o s a l s i d e at the arrow. (a) S h o r t - c i r c u i t current (I )\ (b) u n i d i r e c t i o n a l C l C l C l f l u x e s ( J and J ): (c) t r a n s e p i t h e l i a l r e s i s t a n c e (R ); and (d) spontaneous ms sm t v C l C l t r a n s e p i t h e l i a l p o t e n t i a l (V ). Means ± s.e.: n = 6 (HC0~-free J , J ), v v t 3 ms sm n = 10 (Na-free J C 1 , J C 1 ) , n = 12 (HC0„-free I , V . R ), n = 20 (Na-fr.es I , ms sm j sc t t sc 57 10 - , V 8-4 CM i £ 3 cr o 6 4 2 0 0 2 0 4 0 6 0 Lumina l Na c o n c . C u M ) 8 0 Figure 2.9 Relationship between I and trace l e v e l of Na i n the mucosal half-chamber during Na-free experiments. Recta were exposed to nominally Na-free s a l i n e f o r 7-8 h. Cyclic-AMP (1 mM) was present on the serosal side during the l a s t 2 h. Each value i s for a d i f f e r e n t r e c t a l preparation. 58 t h i s suggestion, s a l i n e containing 10 mM glucose and normal sodium l e v e l s (114 mM), but lacking amino acids, does not maintain the s h o r t - c i r c u i t current or the stimulation produced by 1 mM cAMP (Fig. 10). Moreover, both Na-free and amino ac i d - f r e e s a l i n e s produced s i g n i f i c a n t l y higher serosa-to-36 mucosa C l f l u x and lower t r a n s e p i t h e l i a l resistance (compare Fi g s . 2, 8 and 10). I t w i l l be shown l a t e r that amino acids are necessary to maintain normal transport c a p a b i l i t y . Chamberlin (1981) has shown that p r o l i n e i s metabolised by t h i s t i s s u e and restores I most e f f e c t i v e l y when added to the mucosal J sc J side. In view of the importance of amino acids i n maintaining C l transport and also the presence of Na-coupled amino acid absorption, some reduction i n a c t i v e C l transport i s to be expected. The important fi n d i n g i s that a large -2 -1 cAMP-stimulated C l f l u x (6.0 yEqcm h ) i s observed a f t e r 5.5 h exposure to Cl nominally Na-free s a l i n e , conditions which abolish J and the " u p h i l l " C l net electrochemical gradient i n e p i t h e l i a where NaCl coentry are well established (Nellans et a l . , 1973; F r i z z e l l et a l . , 1975; Cremaschi and Henin, 1975; F r i z z e l l et a l . , 1979; Duffey et a l . , 1978; Reuss and Grady, 1979; Garcia-Diaz and Armstrong, 1980). To avoid the possible metabolic s i d e - e f f e c t s of prolonged exposure to Na-free s a l i n e , r e s u l t s of short-term removal are examined i n the following section. i i ) Short-term removal of external Na The r e s u l t s i n Figure 8 suggested that some Na might be needed with amino acids to maintain normal C l permeability and high transport rates. During the -2 -1 f i r s t 4 hours, I i n amino ac i d - f r e e s a l i n e (1.97 ± 0.36 yEqcm h ) i s not -2 -1 d i f f e r e n t from controls (1.62 ± 0.15 yEqcm h ). Also, Chamberlin (.1981) has found that non-cAMP-stimulated I i s maintained near control l e v e l s for 2 h when sc only endogenous substrates are a v a i l a b l e . To examine Na-dependency of locust C l 59 T i m e Ch ) Figure 2.10 E f f e c t of cAMP a f t e r prolonged exposure to amino ac i d - f r e e s a l i n e containing normal ion l e v e l s and 10 mM glucose. See Table f o r ion l e v e l s and Figure 2.1 for d e t a i l s and d e f i n i t i o n s . Cyclic-AMP was added to the serosal side at the arrow. Dotted l i n e shows data obtained i n normal s a l i n e containing amino acids (from F i g . 2.2). Means ± s.e., n = 6. 60 transport without substrate l i m i t a t i o n , the short-term e f f e c t s of Na-removal on Cl-dependent I were also measured. Chambers were rinsed thoroughly with Na-free s a l i n e so that [Na] dropped from 114 mM to l e s s than 0.05 mM. There was no change (P >> 0.2) i n C l -dependent 1 ' within the f i r s t 75 minutes (Fig. 11). This r e s u l t i s consis-tent with the notion that any i n h i b i t i o n during Na-removal i s slow, and i s probably not a d i r e c t a c t i o n on the transport mechanism per se. i i i ) Unidirectional influxes of Cl and ^^Na across the apical border 36 Figure 12 shows the accumulation of C l by r e c t a l t i s s u e at pH 7.0 a f t e r p r e - e q u i l i b r a t i o n with tracers at pH 4.5. V a r i a t i o n between preparations was reduced, but not eliminated by correcting for e x t r a c e l l u l a r tracers with 3 H-mannitol as described i n the methods. Control experiments showed that no time-dependent changes i n e x t r a c e l l u l a r volume occurred. In some experiments i n f l u x data had y-.intercepts which were l e s s than zero, suggesting a systemat-i c overestimate of e x t r a c e l l u l a r space. I t was not possible to use i n u l i n as a space marker since t h i s polysaccharide does not penetrate the layer of c u t i c l e which l i e s on the mucosal surface of the epithelium. Only the rates of tracer i n f l u x ( i . e . the number of counts as a function of time) are used i n t h i s study. I n j e c t i o n of 10 mM cAMP into sacs resulted i n s i g n i f i c a n t l y higher C l uni-d i r e c t i o n a l i n f l u x e s than for unstimulated controls (4.92 ± 1.57 vs 0.83 ± 1.01 nEq/mg/min; P < 0.05). V also increased from 15.5 ± 1.1 to 58.9 ± 2.1 mV (x ± s.e., n = 18-23). A higher concentration of cAMP was required to stimu-l a t e V maximally i n t h i s sac preparation than was necessary using the f l a t sheet preparation. This may r e f l e c t more rapid degradation of the nucleotide by r e c t a l t i s s u e since the sac contains only a small volume of f l u i d (20 pl) as compared to 5 ml i n the f l a t sheet preparation. For comparison with 61 18-, i -C CN I 16 -E U & LU 3. 14 -u to 1 2 -4 0 - i > £ 30 -> 2 0 -120-j CN 100 -£ c < ^ 8 0 • ac 6 0 -114 m M N a 0.049 m M N a 1 SI .5-— • i r o — i 1— 0.5 1.0 1.5 T i m e C h ) — I 2.0 Figure 2.11 E f f e c t s of b i l a t e r a l Na removal on e l e c t r i c a l parameters across cAMP-stimulated locust rectum. Normal s a l i n e (114 mM Na) was replaced with nominally Na-free N-methyl-D-glucamine sa l i n e at constant C l concentration (.114 mM). Means ± s.e., n = 6. 62 3 E c X r> u D 60 H . - 40 H 2 C H 20 4 40 A 60-J r 0 36 C| 2 4 6 8 T i m e C m i n ) 10 22 N a a C o n t r o l 6 8 T i m e C min ^  10 36 22 Figure 2.12 Ef f e c t s of cAMP on u n i d i r e c t i o n a l influxes of C l and Na into r e c t a l t i s s u e from the mucosal side. I n i t i a l u n i d i r e c t i o n a l i n f l u x of 36 22 C l (a) and Na (b) into t i s s u e of everted r e c t a l sacs was measured under control conditions ( O D ) and aft e r i n j e c t i o n with 10 mM cAMP ( • • ) . Each point i s the r e s u l t obtained from one animal. Tissue wet weight: 3-6 mg each. Non-zero Y-intercepts i n (b) indicate some overestimate of e x t r a c e l l u l a r space. 3 6 The diff e r e n c e between con t r o l and cAMP-stimulated i n f l u x of C l ( i . e . slopes 22 of regression l i n e s ) i s s i g n i f i c a n t at P < 0.05. In contrast, Na i n f l u x i s not d i f f e r e n t after exposure to cAMP (P >> 0.2). 63 2 t r a n s e p i t h e l i a l f l u x measurements, 1 mg tiss u e (wet weight) = 7 mm macro-scopic tissue area. . . 36 Cyclic-AMP stimulation caused a 6-fold increase i n C l i n f l u x into Cl r e c t a l sacs and a 4-fold increase i n J i n Ussing-type chambers. Absolute ms 36 C l C l i n f l u x and during cAMP exposure were also comparable (0.71 to • _2 - l 4.22 yEqcm h using sac preparations and from 1.52 ± 0.16 to 5.65 ± 0.77 -2 -1 yEqcm h , r e s p e c t i v e l y ) . Na influxes were low under these same conditions (Fig. 12b) and were not affected by exposure to 10 mM cAMP (P >> 0.2). In 36 22 contrast to cAMP-stimulated C l i n f l u x , Na i n f l u x was not s i g n i f i c a n t l y d i f f e r e n t from zero (P > 0.2). These lumen-to-tissue fluxes are not consistent with NaCl coentry across the a p i c a l membrane, and furthermore, they suggest that e l e c t r i c a l coupling between active C l absorption and Na i n f l u x i s minimal 22 since Na i n f l u x was not enhanced by the favourable V during cAMP stimulation. In the following chapter, I report that net K transport i s nearly C l i d e n t i c a l to J under open-circuit conditions. In other words, K i s the net . p r i n c i p a l counter-ion accompanying electrogenic C l transport across locust rectum, even when the Na concentration i s much higher than that of K (114 mM Na, 10 mM K) . iv) Effects of inhibitors of Na-coupled Cl transport I examined the s e n s i t i v i t y of C l transport to agents which i n h i b i t Na-coupled C l transport i n other e p i t h e l i a . Locust and other insect r e c t a l tissues contain a t y p i c a l ouabain-sensitive Na/K ATPase (Peacock, 1979; 1981) 3 which has been l o c a l i z e d by H-ouabain binding and autoradiography at the ba s o l a t e r a l c e l l border i n dragonfly recta (Komnick and Achenbach, 1979). Table 4 shows that the addition of 1 mM ouabain to both sides for 1 h did not C l a f f e c t J i n unstimulated recta or the stimulation of C l transport produced by ms sequential addition of 1 mM cAMP ( t o t a l exposure time = 2 h; P > 0.2; compare F i g . 2). In f a c t , 1 mM ouabain increased I s i g n i f i c a n t l y (P < 0.01) from Table 2.4 E f f e c t of sequential addition of 1 mM ouabain and 1 mM cAMP on t r a n s e p i t h e l i a l C l fluxes and e l e c t r i c a l parameters! I sc J C 1 ms J C 1 sm Cl net V t R t _2 (yEqcm h (yEqcm ^h yEqcm ^h ^  T-i -2,-1 yEqcm h (mV) (f2 cm ) Control 1.51 1.65 1.10 7.75 266.1 ±0.13 ±0.08 ±0.07 0.55 ±0.42 ±16.1 (48;12) (24;6) (24;6) (48;12) (48;12) 1 mM ouabain 1.84 • 1-73 1.32 ±0.21 ±0.08 ±0.07 0.61 9.01 ±0.55 241.0 ±17.1 (36;12) (18;6) (18;6) (36;12) (36;12) ouabain + 9.50 9.1 1.73 7.37 27.42 134.5 1 mM cAMP 1 ±0.54 ±0.51 ±0.12 ±0.79 ±10.0 (36;12) (18;6) (18;6) (36;12) (36;12) "Recta were bathed i n normal s a l i n e containing 10 mM K (see Table 1) and ouabain at 22°C for 1 hour p r i o r to addition of 1 mM cAMP. Tissues were pre-equilibrated under I conditions as described i n the text. Quasi-steady-state values are calculated from measurements between 15 minutes and 60 minutes under each condition. Means ± s.e.; (number of fl u x periods; number of animals). 65 -2 -1 C l 1.5 ± 0.3 to 1.84 ± 0.21 uEqcm h , and J showed a corresponding increase -2-1 from 1.65 to 1.93 uEqcm h- (P < 0.1). In two preparations exposed to 1 mM ouabain for 5 h at 22°C and [K] = 10 mM, addition of 1 mM cAMP produced stimu--2-1 l a t i o n s of 8.33 and 4.83 uEqcm h . However exposure to 10 mM ouabain under these conditions may reduce the stimulatory e f f e c t of cAMP n o n s p e c i f i c a l l y . In summary, C l transport i n locust rectum i s not s e n s i t i v e to high concentra-tions of ouabain, i n contrast to those vertebrate e p i t h e l i a where Na-dependent C l transport has been demonstrated (reviewed by F r i z z e l l et a l . , 1979). Na/K ATPase from locust rectum exhibits normal s e n s i t i v i t y to ouabain (K^ = 10 ^ M; Peacock, 1981) although the potency of t h i s i n h i b i t o r i n i n t a c t insect tissues i s c o n t r o v e r s i a l (reviewed by Anstee and Bowler, 1979). It was therefore d e s i r a b l e to test the e f f e c t s of another, more s p e c i f i c i n h i b i t o r of Na-coupled C l transport. The d i u r e t i c furosemide i s an e f f e c t i v e i n h i b i t o r of Na-coupled C l trans-port i n a v a r i e t y of e p i t h e l i a (see F r i z z e l l et a l . , 1979; Ramos and E l l o r y , 1981) . Furosemide (10 mM) i n h i b i t s electrogenic C l secretion across rabbit colon i n l e s s than 10 min ( F r i z z e l l and Heintze, 1979). In contrast, C l -dependent I i n locust rectum i s c l e a r l y i n s e n s i t i v e to 1 mM furosemide when added to both sides, even a f t e r a 75 min exposure (Fig. 13). There i s no obvious b a r r i e r which would l i m i t access of t h i s agent to the a p i c a l plasma membrane because the r e c t a l c u t i c l e was cut. This i n s e n s i t i v i t y to furosemide i s i n marked contrast to a l l Na-coupled systems which have been examined to date. b) Bicarbonate dependence: At present, i t i s p r a c t i c a l l y impossible to exclude the p o s s i b i l i t y of some Cl/HCO^ exchange i n i n t a c t t i s s u e since bicarbonate i s produced i n t r a -c e l l u l a r l y . Although p e r f e c t l y HCO^-free conditions cannot be ensured, 66 several d i f f e r e n t experiments provide strong i n d i r e c t evidence against such an exchange system as the ma*jor mechanism of locust C l transport, i ) Cl fluxes in ECO^ CO ^-fvee saline Since c e l l membranes are permeable to CO^ (Jacobs, 1940; see Gutknecht et a l . , 1977), removal of a l l exogenous HCO^ and CO2 should deplete c e l l s of HCO^, I^CO^ and CO2 v i a e f f l u x of these species down the i r enlarged e l e c t r o -chemical gradients. Any exchange process which involves i n t r a c e l l u l a r HCO^ should be i n h i b i t e d under these conditions. In s k e l e t a l muscle f i b e r s , HCO-"washout" occurs within 5 min (Bolton and Vaughan-Jones, 1977). When salines are s t i r r e d with 100% O2, any bicarbonate for a p o t e n t i a l Cl/HCO^ exchange process must be derived from aerobic metabolism. Removal of a l l external HCO^ and C0„ had no e f f e c t on I , u n i d i r e c t i o n a l C l fluxes, R^, or V across 2 sc t t stimulated locus t recta ( F i g . 8, compare with F i g . 2). Evidence that cytoplas-mic l e v e l s of HCO-j are indeed low during perfusion of the epithelium with CO2, HCO^-free s a l i n e comes from measuring i n t r a c e l l u l a r Cl a c t i v i t y with double-b a r r e l l e d i o n - s e n s i t i v e microelectrodes under these conditions. These experiments w i l l be described i n chapter 4, but some conclusions are relevant at t h i s point. After 3 h, apparent i n t r a c e l l u l a r C l a c t i v i t y was 4.84 ± 0.38 mM, not s i g n i f i c a n t l y d i f f e r e n t from the apparent Cl a c t i v i t y i n s a l i n e when glucon-ate and SO^ are used as replacement anions (5.2 mM). In addition to r e s i d u a l C l , some replacement anions are also probably sensed i n t r a c e l l u l a r l y . There-fore i n t r a c e l l u l a r HCO^ must be lower than 4.84 mM and i s probably close to zero. A s i m i l a r argument has been used by Garcia-Diaz and Armstrong (1980), who suggested that HCO^ i n c e l l s of Necturus gallbladder i s <1.0 mM i n CO2HCO.J-free s a l i n e . This view has been expressed by others using C l - s e n s i t i v e l i q u i d - i o n exchanger microelectrodes and HCO^-free salines (Brown, 1976; Bolton and Vaughan-Jones, 1977; Saunders and Brown, 1977). 67 F u r o s e m i d e Time (min) Figure 2.13 E f f e c t s of furosemide and SITS a d d i t i o n on I across l o c u s t sc rectum s t i m u l a t e d w i t h cAMP. Furosemide (1 mM) and 1 mM SITS were added to both s i d e s of l o c u s t r e c t a i n separate experiments. Tissues were bathed i n normal s a l i n e (see Table 2.1). The c u t i c l e was removed from the mucosal sur-face of r e c t a to allow access of the a p i c a l c e l l membrane to i n h i b i t o r s . Means ± s. e. ; n = 8. 68 i i ) Changes in external pH induced by active Cl transport Exchange of i n t r a c e l l u l a r HCO^ for external C l should r e s u l t i n a l k a l i n -i z a t i o n of the mucosal s a l i n e at rates commensurate with the rate of C l entry C l ( i . e . J ). Figure 14 shows continuous traces of mucosal and serosal pH ms (HCO^-free). In four r e c t a , average a l k a l i n i z a t i o n of the mucosal side was -2 -1 4.5 ± 0.5 yEqcm h or 66% of the simultaneous I • By comparison with C l Figure 2, t h i s rate of a l k a l i n i z a t i o n i s only 39% of J . Exposure to aceta-ms zolamide (1 mM) on both sides did not a f f e c t I but reduced the r a t e of sc mucosal a l k a l i n i z a t i o n by 10 and 30% i n two preparations. Addition of 1 mM SITS (4-acetamido-4'-isothiocyano-stilbene-2,2'-disulfonic acid) to both sides did not block mucosal a l k a l i n i z a t i o n ( F ig. 14). Also, Cl-dependent I increased with a much more rapid time course than did a l k a l i n i z a t i o n . One might postulate a coupling r a t i o of 3C1 to lHCO^ to account for the observed stoichiometry, however to my knowledge no such exchange has been reported and i t i s doubtful that such an exchange would be thermodynamically f e a s i b l e given the large electrochemical gradient opposing C l entry (46 mV, for each Cl) and the presumed gradient favouring HCO^ e f f l u x in.normal s a l i n e (maximum 76 mV; see d i s c u s s i o n ) . There i s also the problem of excess protons produced i n the reaction C 0 2 + H 2 0 i e = i H 2 C 0 3 ^ = ^ H H C 0 3 - Acetazolamide (1 mM) i s thought to i n h i b i t e p i t h e l i a l Cl/HCO^ exchange by i t s e f f e c t s on carbonic anhydrase (Maetz and Garcia Romeu, 1964; Maetz, 1971; Garcia Romeu et a l . , 1969; E r l i j , 1971) although other actions of t h i s drug are indicated at concentrations >1 mM (see Hogben and Karal, 1973; Radtke et a l . , 1972; Cousin and Motais, 1976; Bruus et a l . , 1976; reviewed by Maren, 1977). Mucosal pH was observed to increase i n 22 preparations, however serosal pH never decreased (Fig. 14c). A s i g n i f i c a n t f r a c t i o n of mucosal a l k a l i n i z a t i o n i n C02/HC0.j-free s a l i n e may be due to r e c t a l NH 3 production. Faecal NH^ i s a major route of 69 Figure 2.14 Continuous recordings of I and external pH during exposure of recta to cAMP i n HC0 3~free s a l i n e . Cyclic-AMP (1 mM), SITS (1 mM), acetazolamide (1 mM), and azide (^ 1 mM) were added b i l a t e r a l l y a f t e r the c u t i c u l a r intima had been removed from the mucosal surface of the t i s s u e . See text for d e t a i l s and Table 2.1 for composition of the s a l i n e . 70 nitrogen secretion i n cockroaches (Mullins and Cochran, 1972) and both locusts and grasshoppers are known- to excrete ammonia i n the faeces (see B u r s e l l , 1967). V o l a t i l e ammonia and other nitrogenous bases are produced by laboratory locust colonies ( B l i g h t , 1969) and a d e t a i l e d review by Cochran (1975) has emphasized the importance of ammonia excretion i n i n s e c t s . Chamberlin (1981) showed that amino acids are r a p i d l y metabolized by r e c t a l t i s s u e . To test whether NH^ might be produced by locust rectum jin v i t r o , accumu-l a t i o n of t o t a l NH, was "measured on both sides under I conditions i n HC0 o-4 sc 3 free s a l i n e (containing the usual 11 amino a c i d s ) . Before addition of cAMP, -2 -1 mucosal ammonium accumulated at a rate of 3.0 and 4.5 uMcm h , increasing to -2 -1 6 and 12 uMcm h during cAMP exposure. Chamberlin (1981) has measured very low rates of NH^ accumulation when p r o l i n e i s the only substrate a v a i l a b l e to suspended recta. However, glutamine i s the normal source of renal ammonia i n vertebrates and t h i s could also be true of locust rectum, p a r t i c u l a r l y i n view of the high glutamine concentration i n r e c t a l t i s s u e (44.5 mM; Chamberlin, 1981). i i i ) Effects of inhibitors of HCO ^-coupled Cl transport Table 5 summarizes the e f f e c t s of exposing recta to 1 mM SITS, acetazola-mide and azide for 1 h on Cl-dependent I c - Control periods for each preparation preceded add i t i o n of the i n h i b i t o r . SITS and acetazolamide exposure did not change I (P > 0.2). In contrast, azide v i r t u a l l y abolished I within 10-15 min (see also F i g . 14). sc & c) Potassium dependence: The e f f e c t s of K were of p a r t i c u l a r i n t e r e s t since most t e r r e s t r i a l insect r e c t a normally absorb ions and water from a K C l - r i c h f l u i d _in v i v o . For example, the primary urine entering the lumen of locust rectum from the Malpighian tubule contains 140 mM K as compared to 10 mM K i n the hemolymph. 71 Table 2.5 " E f f e c t of i n h i b i t o r s a f t e r 1-2 h exposure on Cl-dependent I across cAMP-stimulated sc r e c t a *P < 0.001 I n h i b i t o r (n) I sc % (1 mM) (yeq cm h ) I n h i b i t i o n C o n t r o l I n h i b i t o r SITS (8) 5.6±0.5 5.6±0.5 0 Ace t a z o l a -mide (4) 9.1±1.0 8.6±0.9 0 Azide (5) 7.5±1.1 0.29±0.1* 96 72 i ) Effects of K-free saline Lowering s a l i n e K from 10 mM ( c o n t r o l ) to 0 mM on both s i d e s had no C l s i g n i f i c a n t e f f e c t on I , J and V across unstimulated r e c t a , ' although R e sc' ms t to t was 25% higher under K-free c o n d i t i o n s ( F i g . 15, compare w i t h F i g . 2). Import-er a n t l y , 1 mM cAMP produced only small increases i n J , 1 , V„ and R . During J ' y J net sc t t ° exposure to 1 mM cAMP, r e s t o r i n g 10 mM K to both s i d e s (same as i n normal C l s a l i n e ) g r e a t l y enhanced I , J , V„ and reduced R„ to c o n t r o l l e v e l s ( F i g . 15). ° J sc' ms t t There i s c l e a r l y a strong dependence of a c t i v e C l transport on exogenous 36 K. Approximately 70% of the net C l absorption i s K-dependent w h i l e the remainder i s not a f f e c t e d by removing e x t e r n a l K. The nature of t h i s K-dependence i s examined i n chapters 3 and 4. d) Dependence on d i v a l e n t c a t i o n s : Omitting Mg from c o n t r o l s a l i n e d i d not a l t e r Cl-dependent I s i g n i f i -c a n t l y during the f i r s t hour (Table 6). S i m i l a r l y , when e x t e r n a l Ca was removed f o r 1 h there was no dete c t a b l e change i n cAMP-stimulated I . To sc ensure that e x t e r n a l Ca l e v e l s were <10 ^ M, 5 mM EGTA was a l s o included i n nominally Ca-free s a l i n e . Exposure to Ca-free c o n d i t i o n s f o r longer periods (>3 h) may reduce t r a n s p o r t c a p a b i l i t y , however t h i s was not i n v e s t i g a t e d . The l a c k of short-term i n h i b i t i o n of I suggests that C l transport by r e c t a l t i s s u e per se does not d i r e c t l y r e q u i r e exogenous Ca or Mg. Regulation of C l transport by low pH, hyperosmocity and "second messengers" i ) Effects of pH The pH of lumi n a l contents can be as low as 4.5 i n the l o c u s t rectum i n s i t u ( P h i l l i p s , 1964b; Speight, 1968).' Figure 16 shows the e f f e c t s of v a r y i n g mucosal pH on cAMP-stimulated"l s c"and Rfc. Abrupt re d u c t i o n of mucosal pH over the range 7.0 to 4.0 r e v e r s i b l y reduced I and V , and increased R . In & J sc t ' t 73 Figure 2.15 E f f e c t s of cAMP on e l e c t r i c a l parameters and C l fluxes under K-free conditions. Cyclic-AMP (1 mM) was added to the serosal side at the f i r s t arrow. Potassium methylsulfate (10 mM) was added to both sides at the second arrow. See Figure 2.1 for d e f i n i t i o n s . 7 4 Time Ch) Time (h ) 75 Table 2.6 E f f e c t s of exposure to Ca-free or Mg-free Saline on e l e c t r i c a l parameters.^" Sequential I V R M sc t t Condition , -2,-1.. , , , 2. (yEqcm h ) (mV) (ohms cm ) Calcium con t r o l 9.44 29.16 116.1 (24;6) ±0.28 ±0.85 ±3.1 Ca-free(lh) 10.13 31.25 114.9 (24;6) ±0.21 ±0.76 ±12.7 Ca^free + 5 mM EGTA 9.89 30.36 115.3 (0.5h) ±0.38 ±1.14 ±4.3 (12;6) Magnesium Control 8.67 29.98 131.9 (20;5) ±0.45 ±0.84 ±4.6 Mgtfree(lh) 9.01 28.53 121.4 (20;5) ±0.46 ±0.96 ±4.5 "Calcium-free and magnesium-free experiments were performed on d i f f e r e n t t i s s u e s . Means ± s.e., (number of observations I number of t i s s u e s ) . Differences between controls and experimentals were not s i g n i f i c a n t (p <.05). 76 Figure 2.16 E f f e c t s of external. pH on t r a n s e p i t h e l i a l e l e c t r i c a l parameters i n cAMP-stimulated r e c t a . Mucosal (m) and s e r o s a l (s) pH were v a r i e d i n separate experiments. l s c > V and were determined a f t e r 30 min exposure to each e x t e r n a l pH. Tissues were bathed b i l a t e r a l l y i n normal s a l i n e c o n t a i n i n g 20 mM phosphate. Means ± s.e.; n = 8 (ApH on mucosal s i d e ) , n = 6-8 (ApH on s e r o s a l s i d e ) . 77 12 4 pH 78 contrast, I was s u r p r i s i n g l y i n s e n s i t i v e to changes i n pH over a wide range (3.0-8.0) on the s e r o s a l side, even though hemolymph pH i s r e l a t i v e l y constant i n vivo (7.1 ± 0.04, x ± s.e., n = 6). Possible explanations for the e f f e c t s of mucosal pH on I include: i ) A passive net f l u x of protons from the a c i d i f i e d mucosal to more a l k a l i n e serosal side. This could cancel out some of the I which r e s u l t s from a c t i v e C l transport. i i ) High mucosal l e v e l s of protons might competitively i n h i b i t the K-activation s i t e s on a C l trans-port c a r r i e r (see chapters 3 and 4 for evidence regarding d i r e c t "enzyme-like" a c t i v a t i o n of a Cl-transport mechanism by K). Some other d i r e c t e f f e c t of low pH on the Cl transporter remains possible. i i i ) The e f f e c t s may be i n d i r e c t and mediated by changes i n i n t r a c e l l u l a r pH. The f i r s t of these p o s s i b i l i t i e s seems u n l i k e l y since r e v e r s a l of the pH gradient (serosal side a c i d i f i e d ; F i g . 16) did not have opposite e f f e c t s on I g c - According to the hypothesis ( i ) a very large increase i n I i s p r e d i c t -ed, comparable to the decrease i n I g c observed during mucosal a c i d i f i c a t i o n . Competition between protons and potassium ions ( p o s s i b i l i t y i i ) cannot alone account for the i n h i b i t i o n by low pH since mucosal a c i d i t y reduced I to a value which i s lower than the rate of C l transport under K-free conditions (compare I i n F i g . 16 and F i g . 15). Low i n t r a c e l l u l a r pH ( i . e . hypothesis i i i ) appears to mediate the i n h i b i t o r y e f f e c t s of low external pH on active Na transport across frog skin. In support of t h i s view, highly permeant a c i d i c buffer systems are more e f f e c t i v e i n i n h i b i t i n g transport than are l e s s permeant buffers (Funder et a l . , 1967; Mandel, 1978). The increase i n tissue resistance during exposure of locust recta to mucosal a c i d i t y could be due to two d i f f e r e n t processes: i ) a reduction i n conductance associated with the a c t i v e C l transport pathway, or i i ) a reduc-t i o n i n passive K permeability. In chapters 4 and 5, evidence based on the r e l a t i o n s h i p between I and active conductance w i l l suggest that most of the passive or "shunt conductance" i n locust rectum i s due to K permeability. 2 -2 The values of I (32.2 yAcm ) and (or 1/R„ = 5.9 mS cm ) observed sc t t during exposure to low pH are those predicted from the normal I g c / ^ t r e l a t i o n (see chapter 5); therefore, no ef f e c t of low pH on passive K permeability i s required i n order to explain AG^. i i ) Effects of high osmotic concentvation The locust rectum i s exposed to extraordinary f l u c t u a t i o n s i n osmotic concentration on the lumen side during the feeding-dehydration cycle. Rectal contents o s c i l l a t e between 0.4 Osaoles/1 a n d > l . l Osmoles/l depending on the state of hydration of locusts. It seemed possible that active C l transport or passive permeability to ions might be alt e r e d by these large changes i n luminal osmolarity. Figure 17 shows the response of I and R t when sal i n e osmolarity was adjusted by varying the sucrose concentration on both sides of the rectum or on the mucosal side only. Perfusion with hyposmotic sa l i n e on both sides (364 mOsm/l, measured) had no s i g n i f i c a n t e f f e c t on I and R • however, both I and R,_ showed a b sc t 5 1 sc t 40% decline when hyperosmotic s a l i n e (1,220 mOsm/l, measured) was present on -2 -1 both sides. In normal s a l i n e , when I c was 11.2 ± 1.5 yEqcm h , chloride a c t i v i t y was 81.9 mM as measured using an i o n - s e n s i t i v e microelectrode (see chapter 4 for methods). When osmotic concentration was increased to 1,220 mOsm/l with sucrose, C l a c t i v i t y decreased to 75.4 mM and I dropped -2 -1 from 11.2 ± 1.5 to 7.7 ± 0.2 yEqcm h . Based on the k i n e t i c s of C l trans-port across locust rectum (chapter 3), t h i s 30% i n h i b i t i o n of I cannot be attr i b u t e d simply to a reduction i n C l a c t i v i t y c o e f f i c i e n t from 0.718 to 0.661. Such a change should decrease I ^5%. S i m i l a r l y , a d d i t i o n a l passive backflux of C l due to small differences i n ion a c t i v i t i e s across the r e c t a l 80 O s m o t i c P r e s s u r e C m O s m . ! " 1 ) M u c o s a l 440 3 6 4 4 4 0 1 2 2 0 1 2 2 0 4 4 0 S e r o s a l 440 3 6 4 4 4 0 1220 4 4 0 4 4 0 4 J | . "I - i J , r 0 1 2 3 Time (h) Figure 2.17 Influence of mucosal and serosal osmotic pressure on I and sc t r a n s e p i t h e l i a l conductance (G t) . Mucosal and/or serosal osmotic pressure was elevated by adding sucrose. Dotted l i n e shows values of G which are predicted from the normal r e l a t i o n s h i p between I and G as described i n sc t chapter 5 (Fig. 5.21). The much lower values of G observed at high osmotic pressures i n d i c a t e that passive permeability of the epithelium i s reduced under these conditions. Means ± s.e.; n = 7. 81 wall under these conditions was too small to a l t e r I (see chapter 5). These r e s u l t s suggest that C l i s a c t i v e l y transported at a reduced rate during exposure to high osmolarity. A large decrease i n leak or "shunt" conductance must also occur under these conditions since R i s 70% higher than the value predicted by the r e -la t i o n s h i p between I and Gfc during cAMP stimulation (chapter 5). This e f f e c t i s not due simply to a decrease i n saline conductivity, since compensa-tio n for s a l i n e resistance was made during a l l experiments involving s h o r t - c i r c u i t current. In vivo, the osmotic concentration of r e c t a l contents gradually increases towards the end of the reabsorptive cycle. A decrease i n i o n i c permeability due to l o c a l hypertonicity would greatly reduce the work necessary to maintain i o n i c gradients across the epithelium. An analogous s i t u a t i o n e x i s t s with respect to osmotic permeability (P ), which i s lower osm i n the serosa-to-mucosa d i r e c t i o n than i n the mucosa-to-serosa d i r e c t i o n (Goh and P h i l l i p s , 1978). i i i ) Second messengers and hormonal stimulation a) C y c l i c nucleotides: Cyclic-AMP has been used i n the present study to mimic i n vivo hormonal control of C l transport. Corpus cardiacum extracts, cAMP (Spring et a l . , 1978; Spring and P h i l l i p s , 1980a,b) and hemolymph from fed locusts (Hanrahan, 1978; Spring et a l . , 1978; Spring and P h i l l i p s , 1980c) a l l produce comparable stimulations of I ,. and net C l transport. However, adding cAMP produces a more immediate stimulation of I s c> a n d more importantly, r e s u l t s i n a much larger decrease i n t r a n s e p i t h e l i a l resistance (25% according to Spring and P h i l l i p s , 1980b; 40-65%, t h i s study) than has been reported using natural stimulants (4%; Spring and P h i l l i p s , 1980a). In view of the large resistance change produced with cAMP, I re-examined the e f f e c t s of corpus cardiacum at higher doses than previously tested. 82 Exposure of locust recta to 0.08 gL pair/5 ml (a maximal or near-maximal dose according to Spring and P h i l l i p s , 1980) resulted i n a small drop i n R which was comparable to that observed previously (8%, not s i g n i f i c a n t at P > 0.1; Table 7). However, when the dose was increased approximately 10-fold to 1 g l pair/5 ml, Rfc decreased s i g n i f i c a n t l y (24%, P < 0.01) and I increased further (35%, P < 0.02; Table 7). No s i g n i f i c a n t change i n I (P > 0.2) was observed when the concentration of extract was increased from 1 gland p a i r / 5 ml to 2 gland pair/5 ml although R declined further to 70% of controls. In summary, large resistance changes do occur during exposure to corpus cardiacum extract, but only at higher concentrations. The dose (^0.08 gland pair 15 ml) which was previously reported to cause a maximum AI (but only 4% reduction i n R ) did not produce maximal stimulation i n t h i s study (Table 7). It should be noted that the l e v e l s of corpus cardiacum required to decrease R are not "unphysiological". For example, only 6% of the t o t a l active peptide i n the corpora cardiaca must be released into the hemolymph ( t o t a l volume 200-300 yl) i n order to achieve the maximal doses used i n my experiments. Highnam et a l . (1974) have calculated that 70% of the stored neurosecretory material i n these glands i s released within 10 min of feeding. Therefore, use of cAMP to study the d e t a i l s of Cl transport i n v i t r o seems j u s t i f i e d . Relative effectiveness Of cAMP and cGMP i n stimulating I across locust sc rectum was tested using c o n t r o l s a l i n e and conditions using the s a l i n e of Spring et a l . (see Table 1 of this chapter). Exposure to 1 mM cAMP caused an AI of 8.8 ± 1.4 yEqcm _ 2h _ 1 (x ± s.d., n = 5) while addition of 1 mM cGMP sc -2 -1 under i d e n t i c a l conditions increased I by 9.9 ± 0.71 yEqcm h . The d i f f e r e n c e i n AI caused by these two stimulants was not s t a t i s t i c a l l y s i g n i f i -sc cant (P > 0.2) i n marked contrast to blowfly s a l i v a r y gland (Berridge, 1973). 83 Table 2.7 Influence of corpora cardiaca on I across recta sc i n normal and Ca-free s a l i n e -2 -1 2 I g c (yEqcm h ) Vt(mV) R^ohms cm ) (a) Normal s a l i n e c o n t r o l 1.910.4 8.9+2.0 176.919.8 (7.7) 0.08 g l pair/5ml 7.112.0 . 29.317.0 162.7114.4 (7;7) 1 g l pair./5ml 9.612.2 33.9±7.1 132.8113.0 (7; 7) 2 g l pairs/5ml 9.312.0 3 0 . 9 1 6 . 6 124.1+11.7 (7;7) (b) Ca-free s a l i n e +EGTA(2.5 mM) 2.0 8.6 145.7 +A23187(lyg/ml) 10.1 ±1.6 110.8 (20;4) (4;4) (4;4) as above 10.0 26.7 100.2 +1 g l pair/5ml 10.5 11.9 18.1 (20;4) (4;4) (4;4) Means 1 s.e., (n = number of observations; animals). 84 Tissue cAMP l e v e l s increase ^2.6-fold when i s o l a t e d locust recta are exposed to extracts of corpus cardiacum (Spring et a l . , 1978; Spring and P h i l l i p s , 1980a) possibly due to c y c l i c - n u c l e o t i d e synthesis, although the re s u l t s could also be explained by a reduction i n the rate of cAMP degradation (Wells and Hardman, 1977). Moreover, i f cAMP i s normally produced at a low rate i n the absence of hormonal stimulation, i n t r a c e l l u l a r l e v e l s should increase at a rate which i s proportional to the rate of synthesis when degrad-ation of the c y c l i c - n u c l e o t i d e i s blocked. The r e l a t i v e rates of cAMP synthesis under control conditions and during hormonal stimulation were estimated semi-quantitatively by comparing the i n i t i a l r i s e i n I ( d l ^ / d t ) a f t e r a d d i t i o n of the phosphodiesterase i n h i b i t o r theophylline. The i n i t i a l increase i n I i n the absence of corpus cardiacum extract must be due to the sc accumulation of cAMP from "basal" cAMP synthesis (Fig. 18). After addition of corpus cardiacum extract, d l ^ / d t increased 5-fold from 448.4 ± 56.7 nAcm -1 -2 -1 -min to 2640.8 ± 373.5 nAcm min (x ± s.e., n = 4). Note that complete i n h i b i t i o n of phosphodiesterase i s not necessary for this c a l c u l a t i o n , only that the f r a c t i o n a l i n h i b i t i o n of the enzyme remains constant during exposure to corpus cardiacum extract. This r e s u l t provides further evidence that the rate of cAMP synthesis increases during hormonal stimulation. b) I n t r a c e l l u l a r calcium: I n t r a c e l l u l a r Ca i s known to regulate C l permeability and active C l transport i n e p i t h e l i a (Prince and Berridge, 1973; Candia et a l . , 1977; F r i z z e l l , 1977; Bolton and F i e l d , 1979). Although the C l transport system has no d i r e c t and immediate requirement for external Ca (Table 6), i t i s possible that following hormone binding, an increase i n c y t o s o l i c Ca released from i n t r a c e l l u l a r stores accompanies the r i s e i n i n t r a -T i m e Ch) Figure 2.18 E f f e c t s of sequential addition of theophylline (4 mM) and corpus cardiacum homogenate (1 gland pair/5 ml) on I . Both agents were to the serosal side. I n i t i a l d l /dt i s indicated by str a i g h t l i n e s . 86 c e l l u l a r cAMP. If t h i s were true, i n v i t r o addition of cAMP might bypass normal calcium requirements. To further test whether i n t r a c e l l u l a r Ca i s involved i n the hormone response, recta were placed i n Ca-free s a l i n e contain-ing 2.5 mM EGTA (ethyleneglycol-bis-(g-amino-ethyl ether)N,N'-tetra-acetic acid; a Ca-chelating agent) and also containing the Ca ionophore A23187 (1 yg/ml), After 1 h exposure to both ionophore and EGTA, corpus cardiacum extract (1 gland pair/5 ml) was added to the bath. The A I g c e l i c i t e d by hormone extract under Ca-free conditions was i d e n t i c a l to that i n normal saline (compare Tables 7a and 7b; 1 gland pair/5 ml). F i n a l l y , i f cytoplasmic Ca a c t i v i t y i n maintained at low l e v e l s (10 ^ M) as i n most c e l l s , increasing membrane Ca permeability i n the presence of normal external Ca l e v e l s (5 mM) should have the e f f e c t of increasing i n t r a -c e l l u l a r Ca l e v e l s , depending on the Ca buffering capacity of the c e l l s . However, there was no change i n I when A23187 was added to both sides of locust r e c t a (0.1, 1.0 and 5.0 yg/ml) bathed i n normal s a l i n e (5 mM Ca). These r e s u l t s provide no evidence that i n t r a c e l l u l a r Ca i s involved i n regulating C l transport across locust rectum although i n t r a c e l l u l a r calcium measurements are needed to exclude this p o s s i b i l i t y d e f i n i t i v e l y . Discussion. Comparison with vertebrate systems Results presented i n this chapter indicate that chloride absorption by locust r e c t a l epithelium i s s u b s t a n t i a l l y d i f f e r e n t from mechanisms previously reported for several well-studied systems. Two general mechanisms of i o n i c coupling have been proposed for a c t i v e C l transport across these e p i t h e l i a : one involves NaCl coentry into the c e l l s i n a manner analogous to Na-coupled amino acid and sugar transport (reviewed by F r i z z e l l et a l . , 1979). 87 Sodium-coupled models: The Na-coupled coentry model a l l o w s several testable predictions: .1) removal of external Na should i n h i b i t C l entry and t r a n s e p i t h e l i a l C l transport and there should be a p o s i t i v e c o r r e l a t i o n between the amount of Na contamination and the rate of C l transport; 2) a l t e r a t i o n s i n the rate of C l i n f l u x across the a p i c a l membrane should be p a r a l l e l e d by changes i n the rate of Na i n f l u x ; 3) serosal a d d i t i o n of ouabain should reduce C l transport i n d i r e c t l y through elimination of the favourable Na electrochemical gradient across the a p i c a l membrane; 4) mucosal addition of furosemide should block coentry of Na and C l as i n other c e l l s (see F r i z z e l l et a l . , 1979). The r e s u l t s i n locust rectum are not consistent with any of these predic-tions. No s i g n i f i c a n t i n h i b i t i o n of Cl-dependent I occurred during the f i r s t 75 min a f t e r replacing c o n t r o l s a l i n e (^114 mM Na) with nominally Na-free s a l i n e . Also, large s h o r t - c i r c u i t currents and net C l fluxes (60% of controls) were measured during prolonged exposure to nominally Na-free s a l i n e . Although C l incubation i n Na-free s a l i n e f o r periods exceeding 6 hours depressed ^ n e t by approximately 40%, th i s e f f e c t was highly v a r i a b l e and hence was not s t a t i s t i -c a l l y s i g n i f i c a n t , even at the P < 0.1 l e v e l . As described i n the r e s u l t s section, some reduction i n transport i s expected i n Na-free s a l i n e since i ) Na-coupled amino acid absorption has been demonstrated i n locust rectum (Balshin and P h i l l i p s , 1971), i i ) amino acids are necessary to maintain a c t i v e Cl transport at normal l e v e l s ( F i g . 10), and i i i ) amino acids ( p a r t i c u l a r l y proline) are preferred metabolic substrates for supporting C l transport (Chamberlin, 1981). Na removal could also .have other s i d e - e f f e c t s : 1) Na/H exchange may be important i n maintaining i n t r a c e l l u l a r pH about one pK unit higher than expected for the passive d i s t r i b u t i o n of protons across the c e l l membrane (reviewed by Roos and Boron, 1981). 2) There i s evidence for Na/Ca 88 exchange at the basal membrane of at l e a s t three e p i t h e l i a ( G r i n s t e i n and E r l i j , 1978; Lee et al.,"1980; Chase and Al-Awqati, 1981) and a r i s e i n i n t r a -c e l l u l a r Ca a c t i v i t y might depress C l transport a f t e r extended periods. In Cl summary, the fi n d i n g that J n e t w a s p a r t i a l l y reduced by the rather d r a s t i c removal of Na for >6 h i s not s u r p r i s i n g . The more important observation i s -2 -1 that a large net f l u x of C l (6 yEqcm h ) p e r s i s t s under these conditions. During tracer f l u x experiments, some sodium leached from the ti s s u e , increasing the average Na contamination of the mucosal half chamber from 36 53.9 ± 12.2 yM at the s t a r t of C l fl u x measurements to 94.6 ± 47.3 yM aft e r 3.5 h. At these l e v e l s , NaCl coentry i s u n l i k e l y due to the "turnover" rate of Na that would be required, p a r t i c u l a r l y i n those preparations where Na contamination was not detected (<1 yM). For example, based on average Na contamination, a l l mucosal Na ions would need to recy c l e through the a p i c a l plasma membrane >200,000 times per second to sustain 1:1 NaCl coentry during some experiments. This frequency i s higher than expected for carrier-mediated translocations (^104 ions sec "S Armstrong, 1975; Lindemann and Van Dreissche, 1977). Turnover numbers of anion c a r r i e r s i n erythrocyte membrane, for example, have been estimated at 2 x 10 4 sec ^  (H. Passow, c i t e d by Lindemann and Van Driessche, 1977) si m i l a r to that of the mobile K + " c a r r i e r " valinomycin i n a r t i f i c i a l systems (LSuger, 1972). Such c a r r i e r mechanisms would be too slow to permit Na r e c y c l i n g i n the present system. Sodium/potassium ATPase i s seen only on the basal membrane i n dragonfly r e c t a and there i s considerable evidence f o r this l o c a t i o n i n locust rectum ( P h i l l i p s , 1980, and chapter 4 of C l this t h e s i s ) . To account f o r the equivalence of I and J ^ i n the absence 1 sc net of an a p i c a l Na pumping from c e l l to mucosa, Na entering the c e l l s with Cl would have to "r e c y c l e " back to the mucosal side by a mechanism analogous to that proposed by F i e l d et a l . (1978) f or flounder i n t e s t i n e . In th i s model, 89 Na ions which enter the c e l l s with C l are pumped into the l a t e r a l i n t e r c e l l u -l a r spaces by a b a s o l a t e r a l Na/K pump and then leak back to the lumen p a r a c e l l u l a r l y through permselective junctions (see chapter 1). However, i n locust rectum the back-diffusion of Na v i a p a r a c e l l u l a r shunts to the mucosal side would be r a t e - l i m i t i n g during experiments i n which tissues were exposed to nominally Na-free s a l i n e . From known dimensions of locust r e c t a l c e l l s (see chapters 1 and 5) the d i f f u s i o n distance for Na ions would, on average, exceed 5 ym. Assuming the minimum r e c y c l i n g frequency of 1000 Hz for Na C l 20 (based on an average J of 2.89 x 10 C l ions/0.196 cm sec) to be matched net by a t o t a l of 2.85 x 10"^ Na ions i n bulk s o l u t i o n , Na ions moving p a r a c e l l u -l a r l y would need a mean v e l o c i t y of 0.5 cm sec \ Since e l e c t r i c a l gradients -1 -3 -4 -1 of 1 v o l t cm r e s u l t i n mean v e l o c i t i e s of only 10 -10 cm sec (Robinson and Stokes, 1959), an absurdly high d r i v i n g force equivalent to 2.5-25 v o l t s would be required between the l a t e r a l i n t e r c e l l u l a r space and the mucosal s a l i n e f or t h i s mechanism to operate i n locust rectum. Furthermore, at low l e v e l s of Na contamination, there was no c o r r e l a t i o n between trace amounts of -2 -1 Na and I ( F i g . 9); s h o r t - c i r c u i t currents of ^10 yEqcm h were observed when no sodium was detected in the bath. Most contamination resulted from slow leakage of Na from ti s s u e to s a l i n e a f t e r several hours of exposure under nominally Na-free conditions. This observation alone suggests that the net electrochemical gradient for sodium under these conditions must favour Na e f f l u x from the c e l l s rather than coentry with C l . In chapter 4 we w i l l see that when the Na electrochemical gradient across the a p i c a l membrane i s reduced to near zero (as measured using Na-selective microelectrodes), net 36 \ C l flux and transmembrane Cl electrochemical gradients are not affected. 22 There i s also no c o r r e l a t i o n between u n i d i r e c t i o n a l i n f l u x of Na and 36 C l from the lumen into r e c t a l tissue using an everted sac preparation 90 (Fig. 12). Technically, the experiments were d i f f i c u l t because of small tissue s i z e and the presence of a large unstirred compartment on the lumen side between the a p i c a l membrane and c u t i c l e . However, since C l transport was found to be quickly and r e v e r s i b l y blocked by lowering mucosal pH w i t h i n the p h y s i o l o g i c a l range ( F i g . 16), tissues were pre-incubated at low pH so as to 36 22 allow C l and Na to e q u i l i b r a t e i n the subcuticular compartment p r i o r to s t a r t i n g C l transport by r a i s i n g pH. Most of the f l u i d i n t h i s external 3 compartment was quickly removed at the end of experiments. H-mannitol space 36 22 was measured i n order to correct for any e x t r a c e l l u l a r Cl and Na, although this c o r r e c t i o n met only l i m i t e d success. Nevertheless, the stimulatory 36 e f f e c t of cAMP on C l uptake by t h i s preparation suggests that i t i s a reasonable estimate of u n i d i r e c t i o n a l i n f l u x . P a r a c e l l u l a r fluxes are prob-ably low, due to h i g h j u n c t i o n a l resistance (see chapter 5). When 10 mM 3 6 cAMP was injected into the sac, the i n f l u x of C l increased s i g n i f i c a n t l y by 22 6-fold (P < 0.01) while that of Na did not change (P > 0.2; F i g . 12). The r e s u l t s are very d i f f e r e n t from those of s i m i l a r experiments on mammalian i n t e s t i n e (Nellans et a l . , 1973), gallbladder ( F r i z z e l l et a l . , 1975; Crem-aschi and Henin, 1975) a nd f i s h i n t e s t i n e ( F r i z z e l l et a l . , 1979; Ramos and E l l o r y , 1980), where interdependent NaCl influxes have been observed. The lack of c o r r e l a t i o n between ^Na and " ^ C l influxes suggests that t h e i r move-ments are neither chemically nor e l e c t r i c a l l y coupled. In chapter 3 I show that K i s normally the p r i n c i p a l counter ion for active Cl transport across locust rectum. Further evidence for Na-independence i s provided by i n s e n s i t i v i t y of locust Cl transport to known i n h i b i t o r s . Furosemide, which blocks Na-dependent Cl transport in other tissues (Candia, 1973; Humphreys, 1976; Degnan et a l . , 1977; S i l v a et a l . , 1977; F r i z z e l l et a l . , 1979), has no e f f e c t on locust r e c t a l C l transport. Since the r e c t a l c u t i c l e was cut i n my experiments, there was no obvious b a r r i e r to furosemide d i f f u s i o n to a postulated NaCl c a r r i e r on the 10 um-long folds i n the a p i c a l membrane. Locust rectum was also i n s e n s i t i v e to ouabain, a known i n h i b i t o r of Na-coupled C l transport i n other tissu e s . Although ouabain s e n s i t i v i t y i n in t a c t insect tissues i s c o n t r o v e r s i a l , Na-K ATPase i s o l a t e d from locust rectum —6 i s s e n s i t i v e to ouabain (K^ = 10 M; Peacock, 1981). In p r i n c i p l e , the eff e c t s of i n h i b i t o r s alone do not e s t a b l i s h the presence or absence of a par-t i c u l a r transport mechanism, since the same mechanism might vary i n s e n s i t i v i t y between t i s s u e s . For example, i t could be argued that an unusual furosemide-i n s e n s i t i v e NaCl coentry process may exist i n locust rectum. Nevertheless, when combined with tracer f l u x r e s u l t s ( t h i s chapter) and io n - s e n s i t i v e microelectrode data (chapter 4), r e s u l t s of i n h i b i t o r studies do provide c i r -cumstantial evidence against known mechanisms. Bicarbonate-coupled models: A second type of act i v e chloride transport, Cl/HCO^ exchange, has been demonstrated or inf e r r e d i n a v a r i e t y of tissues (see chapter 1). In most of these cases there i s some requirement for exogenous C0 2 and HCO^- Presumably, i n t r a c e l l u l a r HCO^ production i s inadequate to sustain the exchanger. In locust rectum, net C l absorption has no requirement for exogenous C0 2 or HCO^ (Fig. 8). Therefore, under C0 2jHCO^-free conditions, i n t r a c e l l u l a r HCO^ for a possible Cl/HCO^ exchange must necessarily o r i g i n a t e from metabolic CO,-,. However, i t appears that aerobic metabolism by locust rectum i s too low to produce s u f f i c i e n t C0 2. When recta are f r e e l y suspended i n s a l i n e , i t may be calculated that V must be less than 31 mV due to the small tissue dimensions and s a l i n e conductivity. Oxygen consumption by recta under these approximate s h o r t - c i r c u i t conditions has been measured by Chamberlin (1981) and was found to be (2.8 ymoles/rectum/h). Assuming that the tissue has an RQ of 1 (maximum), that 100% of Che metabolically produced CO 2 i s converted to HCO^, then the maximum bicarbonate which could possibly be supplied for 1:1 exchange -2 -1 with Cl i s 3.2 yEqcm h . However, measured Cl transport i s 3-4 times greater than t h i s value under I conditions. This scenario requires p e r f e c t l y e f f i c i e n t CO2 hydration and d i r e c t i o n of HCO^ to the a p i c a l side of the c e l l and would probably require the enzyme carbonic anhydrase. The presence of this enzyme has been shown i n locust rectum (Hanrahan, unpubl. obs.), however acetazolamide had no e f f e c t on r e c t a l C l transport as indicated by I a f t e r cAMP stimulation ( F i g . 14). If mucosal C l i s exchanged for cytoplasmic bicarbonate, the lumen should C l become a l k a l i n e at a rate commensurate with J . Some mucosal a l k a l i n i z a t i o n ms was observed but the rate was only 39% of the u n i d i r e c t i o n a l mucosa-to-serosa Cl f l u x measured under the same conditions (Fig. 14). In f a c t , there i s no evidence that t h i s a l k a l i n i z a t i o n i s due to HCO^ e f f l u x . The anion exchange i n h i b i t o r SITS (1 mM) had no e f f e c t on Cl-dependent I or on the rate of base sc e f f l u x (Fig. 14). Acetazolamide (1 mM) caused only small reductions i n the rate of a l k a l i n i z a t i o n (10-30%) and did not a f f e c t I . These r e s u l t s are not sc consistent with the usual one-for-one Cl/HCO^ exchange. A stoichiometry of 3C1 moving into the c e l l for each HCO^ moving out would not seem to be ener-g e t i c a l l y f e a s i b l e i n normal s a l i n e given the normal range of i n t r a c e l l u l a r HCO-j concentrations which have been measured or calculated i n other c e l l s i n bicarbonate salines (7.6-20.2 mM; Khuri et a l . , 1974; Fujimoto et a l . , 1980; see Roos and Boron, 1981). Assuming a concentration gradient of 20 mM c e l l [HCO^] to 10 mM s a l i n e [HCO^] and membrane p o t e n t i a l of -58 mV under I g c conditions (measured i n chapter 4), HCO^ e f f l u x would provide an electrochemi-c a l d r i v i n g force of 76 mV as compared to a t o t a l of 138 mV opposing entry of 93 3C1. Moreover, hydration of CO^ produces both HCO^ and H. At steady-state, the f l u x of HCO-j into the lumen of s h o r t - c i r c u i t e d recta would require a s i m i l a r f l u x of protons to the hemolymph side. In r e a l i t y , hemolymph side pH does not change measurably, i n f a c t there i s a s l i g h t a l k a l i n i z a t i o n rather than a c i d i f i c a t i o n of the serosal side (Fig. 14). A p i c a l Na/H or K/H "exchanges might also explain luminal a l k a l i n i z a t i o n , however these mechanisms seem inappropriate since they should ultimately a c i d i f y the serosal side as w e l l . Moreover, i n preliminary experiments, a l k a l i n i z a t i o n continued a f t e r removal of a l l K from the bathing s a l i n e , further suggesting that K/H exchange i s not responsible for mucosal a l k a l i n i z a -t i o n . Ammonia production by locust rectum c e l l s i s one p l a u s i b l e explanation for mucosal a l k a l i n i z a t i o n (Fig. 14). NH^ i s excreted i n the faeces of many insects including Schistocerca (reviewed by B u r s e l l , 1967). Deamination of amino acids i s the most l i k e l y source of r e c t a l ammonia. Pro l i n e and glycine are a c t i v e l y accumulated from the mucosal side (Balshin, 1973) and both p r o l i n e (Balshin, 1973) and glutamine (Chamberlin, 1981) are found at high concentrations i n r e c t a l tissue (65 and 45 mM, r e s p e c t i v e l y ) . I found that amino acids are necessary to maintain I across locust rectum (Fig. 10), and oxidation of p r o l i n e has been shown d i r e c t l y by Chamberlin (1981). Ammonia could be generated by deamination of glutamine as i s the case i n vertebrate kidney. Balshin (1973) measured a s i g n i f i c a n t l e v e l of NH^ i n locust r e c t a l t i s s u e (10 mM). Proximity of mitochondria to the a p i c a l membrane would cause the NH.j which i s produced to d i f f u s e p r e f e r e n t i a l l y to the lumen, r a i s i n g the pH on the mucosal side, as shown i n Figure 14. Further studies are required to support t h i s hypothesis. 94 In summary, I tested the major predictions which have been used to substantiate NaCl cotransport and Cl/HCO^ exchange systems i n other e p i t h e l i a and found that neither mechanism could adequately explain C l transport by this insect epithelium. It should also be emphasized that both these entry mechanisms (by themselves) are e l e c t r i c a l l y s i l e n t whereas there i s strong evidence that C l entry i n locust rectum i s a "rheogenic" process ( i . e . i t d i r e c t l y separates charge; see chapter 4). Potassium dependence of C l transport Chloride absorption by locust rectum i s strongly dependent on K. After Cl prolonged exposure to K-free s a l i n e , 1 mM cAMP stimulated J to only 1/3 of net control rates. This " K - i n s e n s i t i v e " component i s not due to imperfect K s e l e c t i v i t y ( i . e . Na taking the place of K ions i n K-free saline) because i n control experiments, addition of cAMP stimulated I when both Na and K were substituted with N-methyl-D-glucamine, choline or tetramethyl ammonium ions. Cl In.other words, the K-independent component of A I g (and J fc) I s completely independent of cations. This finding has been confirmed using i o n - s e n s i t i v e microelectrodes. K-dependent C l movements have been demonstrated i n E h r l i c h tumour c e l l s (Geek et a l . , 1980), red blood c e l l s (Kregenow and Caryk, 1979; Dunham et a l . , 1980), and are thought to function i n c e l l volume regulation (reviewed by Kregenow, 1981). Also, an electro n e u t r a l KCl transport mechanism has been demonstrated i n oxyntic c e l l a p i c a l membrane (Wolosin and Forte, 1981). Sodium-K-Cl coupling has been suggested at the basal membrane of cultured MDCK c e l l monolayers (Simmons, 1981). However the r e s u l t s presented i n chapters 3 and 4 of t h i s thesis w i l l suggest yet another K-dependent C l transport system i s present i n the locust rectum; potassium i s proposed to have a d i r e c t , stimulatory e f f e c t on the Cl pump at the external surface of th a p i c a l membrane. 95 Exchange d i f f u s i o n A C l / C l exchange d i f f u s i o n process which i s s i m i l a r to that found i n red blood c e l l membranes (reviewed by Gunn, 1979) has been suggested i n a v a r i e t y of e p i t h e l i a including g a s t r i c mucosa (Heinz and Durbin, 1958), frog skin (Bruus et a l . , 1976; Ques-von Petery et a l . , 1978), across the g i l l s of cray-f i s h (Shaw, 1960), and brine shrimp (Smith, 1969) and the anal p a p i l l a e of mosquitoes (Stobbart, 1967). C l f l u x v i a an exchange process should 1) require the presence of C l on the opposite or "trans" side, and 2) reduce apparent C l permeability at open-circuit because of i t s i n s e n s i t i v t y to e l e c t r i c a l g r a d i -C l ents. Neither of these properties were exhibited by J i n locust rectum. In sm Cl f a c t , J g m increased s l i g h t l y when mucosal C l was replaced with methyl s u l f a t e under I conditions, although t h i s change was not s t a t i s t i c a l l y s i g n i f i c a n t (P > 0.1). Also, the apparent t r a n s e p i t h e l i a l C l permeability was enhanced rather than depressed i n the presence of a spontaneous t r a n s e p i t h e l i a l p o t e n t i a l (see p. 49). F i n a l l y , i f C l / C l exchange i s a carrier-mediated process, one might expect a saturation e f f e c t when chloride concentration i s elevated on C l mucosal and serosal side. In chapter 3, i t w i l l be shown that J i s l i n e a r sm over the range of 2-114 mM. In summary, exchange d i f f u s i o n i s not apparent i n C l locust rectum, and J must occur v i a some other pathway. sm Anion s e l e c t i v i t y Chloride i s probably the only anion transported by the cAMP-stimulated mechanism i n locust rectum. Of the 9 anions tested, only Br addition increased C l C l (I - J ) by simultaneously increasing I and decreasing J , suggestive of sc ms sc ms C l competition with Cl (Fig. 6). This increase i n ( l s c - ^ m s ^ w a s ^50% of that obtained by adding the same amount of C l and i s consistent with a c t i v e Br transport. This i n t e r p r e t a t i o n i s supported by r e s u l t s of a second series of experiments i n which the C l i n normal s a l i n e was replaced with various test anions. In Br s a l i n e , the stimulation e l i c i t e d by cAMP was 49% of that i n normal C l s a l i n e . S i g n i f i c a n t stimulations were not observed when other anions were used to replace C l . Neither of these experiments can d i s t i n g u i s h between the s e l e c t i v i t i e s of the'binding and translocation steps. For example, although f l u o r i d e i s not C l transported, F i n h i b i t i o n of J i n Figure 7 could r e s u l t from competitive or noncompetitive interactions with the binding s i t e or translocation mechanism, or from i n h i b i t i o n of C l tra n s l o c a t i o n i n d i r e c t l y v i a metabolic e f f e c t s . Further investigations are required to d i s t i n g u i s h between these p o s s i b i l i t i e s . Whether determined by C l binding, or by some step i n translocation, s e l e c t i v i t y w i l l ultimately depend on s i m i l a r types of interatomic forces (Wright and Diamond, 1977). Some properties of the s i t e s may be in f e r r e d from the anion " s e l e c t i v i t y sequence". According to the theory of Eisenman (1961), the s e l e c t i v i t y sequence r e s u l t s from the r e l a t i v e energies of hydration and of e l e c t r o s t a t i c . i n t e r a c t i o n s of the ion with s e l e c t i v i t y s i t e s . The anion sequence obtained f o r locust rectum suggests s e l e c t i v i t y s i t e s with high f i e l d strength. I f F i s omitted on the basis of i t s being a r e s p i r a t o r y i n h i b i t o r , s e l e c t i v i t y r a t i o s may be f i t q u a n t i t a t i v e l y to the isotherms of Wright and Diamond to give sequence VI for a s i t e having very high f i e l d strength. I f F i s not omitted, then the C l > Br > I,F i s "sequence IV", expected for a s i t e of moderately high f i e l d strength. In the l a t t e r case, the r e l a t i v e anion s e l e c t i v i t i e s are not i n quantitative agreement with those calculated by Diamond and Wright (1969). The s e l e c t i v i t y sequence C l > Br > I i s not unusual for membrane C l transfer i n animals; some examples include frog skin (Kristensen, 1972; Kristensen and Larsen, 1978), stomach (Durbin, 1964), rabbit ileum 97 F r i z z e l l et a l . , 1973), red blood c e l l anion exchange (Dalmark, 1973a,b) ' and cation+ Cl-cotransport (Kregenow and Caryk, 1979), i n dog trachea (Widdi-comb et a l . , 1979), frog cornea (Zadunaisky et a l . , 1971), elasmobranch e l e c t r i c organ ( M i l l e r and White, 1980). S e l e c t i v i t y s i t e s may be very d i f f e r e n t for act i v e and passive anion transport i n insect e p i t h e l i a . Koch (1938) demonstrated that C l uptake i n  vivo by anal p a p i l l a e of mosquito larvae was 2.5 times fa s t e r than Br uptake, i n s a t i s f a c t o r y agreement with the present study which reports a Cl:Br selec-t i v i t y of two. In contrast, Berridge (1969) found that anions passively support f l u i d s e cretion by blowfly Malpighian tubules according to sequence I of Wright and Diamond (I > Br > C l > F), a find i n g which implies s e l e c t i v i t y s i t e s having a weak f i e l d strength. Regulation of C l transport Results described i n th i s chapter suggest that C l transport by locust rectum i s probably regulated at several l e v e l s . O v e r a l l control of r e c t a l absorption i s hormonal (Spring et a l . , 1978; Hanrahan, 1978; Spring and P h i l l i p s , 1980a-c; P h i l l i p s et a l . , 1980; and t h i s chapter). At present there i s no evidence to implicate Ca i n th i s response although data are s t i l l very l i m i t e d . When stimulated hormonally, C l transport i s also probably modified by l o c a l f a c t o r s , p a r t i c u l a r l y luminal K concentration (Fig. 15), pH (Fig. 16) and osmotic pressure (Fig. 17), which are known to vary over a wide range i n  vivo. The r e l a t i o n s h i p between K and active Cl transport i s examined i n more d e t a i l i n the following chapter. 98 CHAPTER 3: INTERRELATIONSHIP BETWEEN ACTIVE CHLORIDE TRANSPORT AND POTASSIUM Summary The r e l a t i o n s h i p between transmural chloride and potassium transport has been examined i n an insect epithelium. U n i d i r e c t i o n a l C l f l u x from mucosa-to-C l serosa (J ) and s h o r t - c i r c u i t current (I ) both increase h y p e r b o l i c a l l v as ms sc v chloride concentration i s raised b i l a t e r a l l y from 0 to 114 mM. In contrast, Cl the serosa-to-mucosa f l u x (J ) i s a l i n e a r function of [ C l ] . When [K] i s sm raised b i l a t e r a l l y from 0 to 100 mM, both the C l concentration required for Cl Cl half maximal Cl absorption (K. ) and the maximum rate of C l absorption (J ) t r max Cl C l increase dramatically due to stimulation of J . H i l l plots of J data are ms v net li n e a r and have slopes near one, i n d i c a t i n g non-cooperative C l in t e r a c t i o n s . C l Increases i n J ^ account for Al at a l l K concentrations a f t e r cAMP stimula-net sc tio n . By analogy with enzyme-catalyzed reactions, the external potassium Cl concentration required for half-maximal stimulation of J i s 5.3 mM. The net Cl s e l e c t i v i t y sequence for cation a c t i v a t i o n of J i s K > Rb > Cs > Na, which net i s consistent with a s i t e having moderately weak f i e l d strength. Addition of external Na has l i t t l e e f f e c t on Cl-dependent I . Low concentrations of K sc are stimulatory only when present on the mucosal side. In the absence of cAMP K there i s no J under I conditions, suggesting that active K transport i s net sc •> oo o i K K n e g l i g i b l e under control conditions. However, the r a t i o of J / J was ms sm higher than that predicted from the Ussing f l u x r a t i o equation for passive, independent ion transport suggesting the presence of non-independent ion d i f f u s i o n i n the mucosal or serosal membrane. T r a n s e p i t h e l i a l potassium permeability, i s reduced by high K l e v e l s i n the s a l i n e and enhanced >4-fold during exposure to 1 mM cAMP under I conditions. J equals J during sc net H net cAMP exposure under open-circuit conditions, however under I conditions sc K C l J . i s less than 10% of J . These observations indicate that t r a n s e p i t h e l -net net ^ i a l K absorption i s e l e c t r i c a l l y coupled to C l transport. Cotransport of K and C l by a common c a r r i e r i s u n l i k e l y because t r a n s e p i t h e l i a l K fluxes are not affected by C l removal under I conditions. The r e s u l t s are consistent J sc with the stimulation of an electrogenic C l pump by mucosal K ions. 100 Introduction In the previous chapter, i t was shown that C l Is a c t i v e l y transported across locust rectum by an unusual mechanism which apparently does not require sodium or bicarbonate and which.is strongly stimulated by K . In this chapter, the r e l a t i o n s h i p between C l transport and K absorption i s studied i n more d e t a i l . Interdependence between t r a n s e p i t h e l i a l C l and K movements has been suggested i n the r e c t a l epithelium of the desert locust, Schistocerca gregaria ( P h i l l i p s , 1964b) and the blowfly, Sarcophaga b u l l a t a (Prusch, 1976); however, no evidence for s p e c i f i c chemical or e l e c t r i c a l i n teractions between these ion movements has been reported. In the Malpighian tubule of the blowfly, Calliphora erythrocephala, electrogenic K secretion i s dependent on the presence of permeant anions bathing the basal membrane and varies inversely with the hydrated radius of the anion (Berridge, 1969). In Malpighian tubules, and s a l i v a r y glands, an i n d i r e c t dependent of f l u i d secretion (driven by active K secretion) on passive C l movements has been demonstrated (Berridge, 1969, 1980). In l i g h t of the K stimulation of a c t i v e C l transport i n locust rectum, I undertook a study of K absorption and i t s r e l a t i o n s h i p to C l transport i n order to better understand "this i n t e r a c t i o n between K and C l movements. Moreover, the rate of K absorption and the dependence of C l transport on C l and K concentrations are important p h y s i o l o g i c a l l y since both [Cl] and [K] i n r e c t a l f l u i d decrease d r a s t i c a l l y during the reabsorptive cycle ( P h i l l i p s , 1964b). Previous studies suggested that K transport across this tissue might be active since luminal K reached a f i n a l concentration of only 0.2 mM ( P h i l l i p s , 1964b). While approaching this f i n a l concentration, net K absorption would have to occur against a net electrochemical gradient of 72 ± 9.39 mV (x ± s.e., n = 5; 101 calculated from P h i l l i p s , 1964b). However, Williams et a l . (1978) reported only low rates of K transport under co n t r o l I conditions. No evidence for cAMP stimulation of active K transport was obtained by Spring and P h i l l i p s (1980b) who found that could be accounted for by an increase i n C l absorp-t i o n alone. Nevertheless, the e f f e c t s of cAMP on K transport were not d i r e c t l y measured and a small electrogenic K fl u x or a large e l e c t r o n e u t r a l absorption could be of great importance i n understanding the mechanism of K-stimulated C l transport. In this chapter, I i ) inve s t i g a t e the k i n e t i c s of t r a n s e p i t h e l i a l C l absorption and determine whether K stimulates C l absorption by a l t e r i n g the Cl maximal rate of transport (^ m a x) o r the apparent a f f i n i t y of the mechanism for Cl C l (K^ ), i i ) quantify K requirements of C l transport and determine the sidedness and K s e l e c t i v i t y of the stimulation, and i i i ) test whether the active components of net C l and K absorptions are interdependent by measuring 42 the e f f e c t s of cAMP and C l omission on t r a n s e p i t h e l i a l K fluxes. These studies also provide the background information which i s needed to int e r p r e t the electrochemical p o t e n t i a l p r o f i l e s , and conductance data presented i n the following two chapters. Materials and methods Experimental animals and methods were s i m i l a r to those described i n the preceding chapter and only a d d i t i o n a l experimental protocols used i n th i s study are described below. 36 K i n e t i c s of t r a n s e p i t h e l i a l C l fluxes Tissues x^ere e q u i l i b r a t e d i n normal s a l i n e (Table 1 of chapter 1) under I conditions. After 3 h, the external medium was replaced b i l a t e r a l l y with c h l o r i d e - f r e e s a l i n e which was prepared by replacing Cl with methylsulfate. 102 1 was near zero under these conditions (Fig. 1). Approximately 0.5 h l a t e r , the tissue was rinsed three times with fresh C l - f r e e s a l i n e and cAMP was added to the serosal side at a f i n a l concentration of 1 mM. Small aliquots of 2 M NaCl were added to both sides, r a i s i n g [ C l ] stepwise from 0 to 2, 4, 10, 36 40 and 114 mM. C l fluxes were measured during each step as described i n chapter 1. As w i l l be shown i n the r e s u l t s section, a new steady-state flux C l from mucosa-to-serosa (J ) i s reached between 15 and 30 min a f t e r a change i n ms ° [ C l ] , although tracer mixing r e s u l t s i n lower fluxes during the f i r s t 15 min. Only data from the second flux period were used i n c a l c u l a t i o n s . C l The dependence of J on C l concentration was determined using salines •ms & containing 0, 10 and 100 mM potassium i n separate experiments using d i f f e r e n t animals. [K] was adjusted with K-methylsulfate. Corrections were made in a l l experiments for the asymmetry between voltage-measuring electrodes and for series resistance of the sa l i n e , which changes s i g n i f i c a n t l y during ion additions. Measurement of potassium dependence of chloride transport C l The increase i n I was used as a measure of AJ while adding K ions sc net & b i l a t e r a l l y as methylsulfate s a l t . The equivalence of these two parameters i n cAMP-stimulated tissues has been established for K concentrations between 0 and 140 mM (Fig. 15 of chapter 2; F i g . 7, th i s chapter). That i s , elevating s a l i n e [K] did not r e s u l t i n electrogenic transport of ions other than C l . Tissues were equ i l i b r a t e d for 3-4 h i n K-free saline (see methods in chapter 2) under I conditions, and rinsed p e r i o d i c a l l y with fresh K-free s a l i n e . When tissues were judged to be i n a steady-state condition, 1 mM cAMP was added to the serosal side. I increased to a new steady-state l e v e l sc J within 1 h. Aliquots of K-methylsulfate (2 M) were then added to both sides 103 6 0 5 0 H CN i g 4 0 O CD O ^ 3 0 O < 3L U CO 20 10 H o - J C l c o n c e n t r a t i o n C m M ) 2 4 10 4Q 114 w 1 - 3 0 r- 20 ^ /-s 3 < 10 w L - 0 T i m e Ch) Figure 3.1 Representative trace of I g c and t r a n s e p i t h e l i a l p o t e n t i a l (V ) 3 6 during measurement of t r a n s e p i t h e l i a l C l f l u x k i n e t i c s . After e q u i l i b r a t i n g r e c t a i n normal s a l i n e (114 mM C l , 10 mM K) under 1 conditions for >3 h, the sc ' t i s s u e was rinsed twice on both sides with C l - f r e e (methylsulfate) s a l i n e . A f t e r a t h i r d r i n s e , 1 mM cAMP was added to the serosal side. Chloride was 3 6 added as NaCl stepwise every 30 min under I conditions. V. and Cl fluxes sc t were measured at 15 min i n t e r v a l s as in Figure 2.1. I traces were integra t -& sc ° ed by planimetry over each 15 min i n t e r v a l for comparison with the correspond-ing tracer f l u x . 104 at 0.5 h i n t e r v a l s , to f i n a l K concentrations of 0, 2, 4, 10, 40, 100, 140 and 200 mM. An i d e n t i c a l protocol was followed during control experiments i n which Na-methylsulfate (2 M) was added. T r a n s e p i t h e l i a l p o t e n t i a l and resistance were determined at 15 min i n t e r v a l s as described previously i n the methods section of chapter 2. 42 T r a n s e p i t h e l i a l K fluxes 42 K (New England Nuclear Corp., Lachine, P.Q.; 0.13-0.15 Ci/g) was added 42 42 as KC1 to normal sa l i n e or as K^CO^ to C l - f r e e s a l i n e . Samples were taken at i n t e r v a l s as previously described and counted using an automatic gamma counter %(1085, Nuclear Chicago; see chapter 2, methods). I n i t i a l r a d i o a c t i v i t y of the l a b e l l e d side served as a reference i n order to corrent for tracer 42 decay during experiments ( t o t a l sampling and counting time: 11 hours; K half-l i f e : 12.42 hours). Fluxes were calculated as described i n the preceding chapter. Results I) K i n e t i c s of chloride absorption 36 T r a n s e p i t h e l i a l C l fluxes were measured under I conditions over the sc range of chloride concentrations which occur in vivo. Figure 1 i s a represent-ative trace of instantaneous I g c and V obtained during these experiments with 10 mM K. I usually decayed over the f i r s t 2-4 h i n normal s a l i n e , reaching a value which includes baseline C l transport and any other electrogenic transport processes. I g c decays further during exposure to C l - f r e e s a l i n e (methylsulfate s u b s t i t u t i o n ) . Addition of 1 mM cAMP under these conditions -2 -1 _ usually increased I very s l i g h t l y (AT.sc = 0.15 ± 0.02 yEqcm h ; x ± s.e., n = 28; P « 0.01). It i s not clear whether this stimulation r e s u l t s from trace amounts of Cl contamination or to some minor cAMP-sensitive transport system. These small 105 currents under C l - f r e e conditions were ignored since they averaged only 1.5% o f t h e I i n normal (114 mM Cl) s a l i n e . Figure 2 shows that J C 1 reached new sc ms steady-state l e v e l s within 30 min a f t e r increasing chloride concentration. Therefore only two 15 min f l u x periods were required at each C l concentration to measure approximate steady-state flux rates and only the second of these Cl C l was used i n these c a l c u l a t i o n s . S h o r t - c i r c u i t current, J and J increased ms net h y p e r b o l i c a l l y as the C l concentration was raised on both sides (Fig. 3). In C l contrast, J increased l i n e a r l y , and showed no evidence of saturation. Since sm J C l C l C l « J and J could not be measured on the same tissues, J was calculated by ms sm ' net C l C l subtracting the mean J (n = 6 animals) from J for i n d i v i d u a l t i s s u e s . sm ms Cl As expected, J and I were nearly i d e n t i c a l at a l l C l concentrations r ' net sc (Fig. 3). 36 Forward fluxes of C l were s i g n i f i c a n t l y lower i n K-free salines (P << 0.01) and higher i n 100 mM K s a l i n e (P << 0.01) when compared to values C l i n normal sa l i n e (10 mM K) . Figure '4 shows that J armroaches s a t u r a t i o n i ms c c l o w e r r a t e s ^ i n K-free s a l i n e as compared to normal s a l i n e . I w i l l show that J i s sm s i m i l a r whether tissues are bathed i n s a l i n e containing 10 or 140 mM K, j u s t i -C l C l C l fying the use of J obtained i n normal s a l i n e to calculate J ( i . e . J -sm net ms — C l x J ) at other K l e v e l s . Data from each preparation, as exemplified by C l Cl Figure 4 and corrected for J to obtain J , were f i t t e d to the Michaelis-° sm net Menten (MM) equation using standard l i n e a r regression. The Woolf transformation (Haldane, 1957) was used: [C l ] K 1 net max max 106 C l c o n e . ( m M ) 5 10 20 40 80 12 4 10 H T-• . C CN I E o C T U J = L \ ^ in U E -3 8 H 6 H 4 J 2 -4 0 —' T 3 "1 I T i m e Ch) Figure 3.2 Histogram of J measured at 15 min i n t e r v a l s under I ms sc conditions. Chloride concentration of the s a l i n e was abruptly increased ever^ Cl 45 minutes to the values shown. Since J approached new steady-state l e v e l s ms - J a f t e r the f i r s t f l u x period, only 2 f l u x i n t e r v a l s were used at each [Cl] i n subsequent experiments and the f i r s t of these was disregarded i n c a l c u l a t i o n s . 107 Figure 3.3 The dependence of C l fluxes and I on C l concentration. Cl • u n i d i r e c t i o n a l C l f l u x from mucosa to serosa (J ); • u n i d i r e c t i o n a l C l ms Cl Cl flu x from serosa to mucosa (J ); o calculated net C l f l u x (J ); o short-sm net c i r c u i t current ( I s c ) • Experimental protocol as i n Figure 3.1. Potassium concentration = 10 mM at a l l times. Means ± s.e.; n = 10. Cl c o n e . (mM) 109 Figure 3.4 The influence of external K concentration on the r e l a t i o n s h i p C l between J and C l concentration of the s a l i n e . Tissues were stimulated by ms J 1 mM cAMP on the se r o s a l side under I conditions. Potassium concentration sc was increased b i l a t e r a l l y from 0 mM K ( a ) to • 10 mM K, or A 100 mM K by adding K methylsulfate. Means ± s.e.; n =6-10. I l l where, [Cl] = chloride concentration J C 1 = net C l f l u x net Cl K. = [Cl] at h a l f maximal J t L net TC1 . TC1 J = maximum J max net This method i s considered to be less s e n s i t i v e to measurement errors than other methods (Blunck and Mommsen, 1978). N o n - t r i v i a l weighting was not used since the type of error (absolute vs r e l a t i v e ) was unknown. Woolf plots had l i n e a r r e l a t i o n s h i p s as indicated i n Figure 5. For the ten preparations used to calculate K and J C 1 at 10 mM K, 98.2 ± 0.65% of the v a r i a t i o n i n J C 1 was t max net a t t r i b u t a b l e to the r e l a t i o n s h i p with [ C l ] . Table 1 summarizes the values of C l K. and J at each concentration of K for 6-10 preparations. It i s clear t net tr f C l that both K. and J increase s i g n i f i c a n t l y (P << 0.001) as the K concentra-t max J t i o n i s r a i s e d from 0 to 100 mM. The e f f e c t s of K addition are reminiscent of enzyme a c t i v a t i o n . In the following chapter, I report that K e f f e c t s are probably due to d i r e c t stimulation of the C l pump rather than to an i n d i r e c t e l e c t r i c a l e f f e c t on the p o t e n t i a l opposing C l transport. F i n a l l y , cooperative i n t e r a c t i o n s between C l binding s i t e s have previously been demonstrated for the transporting epithelium of prawn i n t e s t i n e (Ahearn, 1979) and the mosquito posterior rectum (Bradley and P h i l l i p s , 1977). In contrast, C l absorption by locust rectum increases h y p e r b o l i c a l l y with increasing [ C l ] (Fig. 4) and i s describable by Michaelis-Menten k i n e t i c s C l Figure 6 shows that H i l l plots of mean J had slopes near 1 at a l l ° net concentrations of K (e.g. 1.09 for OmM K; 0.91 for 10 mM K; and 0.99 for 100 mM K). The slope i s a measure of the H i l l constant or the number of i n t e r -acting s i t e s (see Segel, 1975). These results" suggest that the r a t e - l i m i t i n g 112 Table 3.1 Influence of external K on k i n e t i c s 36 of net Cl fl u x across cAMP-stimulated s h o r t - c i r c u i t e d r e c t a a 1 K cone. . K t J C 1 max (mM) (mM Cl) (n) (yeq C l cm~2h'~1) 0 22.7 ±4.0 (6) 3.5 ±0.7 10 60.2 • ±8.7 (10) 14.9 ±1.9 100 99.6 ±13.4 (7) 23.1 ±5.3 ^ e a n ± s.e.m. Note: Cl K and J were t max determined for i n d i v i d u a l recta using Woolf plots ( [ C l ] / j C \ vs. 1 net 1 "Cl ] ) . The mean J C 1 s.m Cl was subtracted from J at ms each chloride concentration t o C l c a l c u l a t e J net 113 ^ 14 CM i E D" L U — CD U c 12 -4 10 A 8 A E 6 c o o o 03 6 -( O 4 4 2 4 I. 0 ~f o p r e p a r a t i o n 3 r 2 = 0 . 9 9 8 0 y = 0 . 0 7 5 4 x + 4 .766 J m a x = 1 3 . 3 p E q . c m ^ r f 1 K t = 6 3 . 2 m M T T 1 1 25 5 0 75 100 Cl cone. (mM) 12 Figure 3^5 Representative p l o t of data used i n c a l c u l a t i n g k i n e t i c 3 6 constants of r e c t a l C l transport. Net C l fluxes (at 10 mM K) were c a l c u -C l l a t e d f o r each C l concentration by subtracting mean J (n = 6 recta, see sm F i g . 3.3) from i n d i v i d u a l l y measured forward fluxes. Data were f i t t e d by l i n e a r regression to the Michaelis-Menten equation according to the Woolf transformation. 114 — <D O c I X — o O c ~3 o -0.5 1 ,ci Figure 3.6 H i l l p l o t s of J at external K concentrations of 0, 10 and ms 100 mM. Net fluxes were calculated under I conditions i n the presence of sc r 1 mM cAMP on the serosal side, and p l o t t e d according to the H i l l equation for homotropic coope r a t i v i t y : l o g ( J C 1 / J C 1 - J C 1 ) = n„, log [cl] - log K . max max C l • t Symbols in d i c a t e mean values obtained from Figure 3.4 as described i n the text. 115 step i n t r a n s e p i t h e l i a l C l absorption does not involve cooperative i n t e r a c t i o n between s i t e s . 2) i s C C 1 f l u x e s iS. "high K" s a l i n e Normally i n vivo, the rectum contains a K - r i c h f l u i d secreted by the Malpighian tubules (140 mM; P h i l l i p s , 1964b; see also Table 1 of chapter 2 of t h i s t h e s i s ) . Nevertheless, a l l previous studies of a c t i v e transport across the tissue and other insect recta have employed high Na, low K s a l i n e s . 3 6 T r a n s e p i t h e l i a l C l fluxes were measured i n s a l i n e containing 140 mM K C l a) to determine i f Al equals AJ , as i t does i n normal s a l i n e , b) to meas-sc n net ' C1 ure the possible e f f e c t s of high [K] on J , and c) to examine whether sm responsiveness to cAMP i s altered by this p h y s i o l o g i c a l l e v e l of K. C l Both J and I increased during exposure to 1 mM cAMP from 1.55 ± 0.2 ms sc to 10.28 ± 0.99 yEqcm" 2h - 1 and from 2.59 ± 0.36 to 8.53 ± 1.07 yEqcm~ 2h _ 1, 2 r e s p e c t i v e l y . (Fig.- 7). Mean t r a n s e p i t h e l i a l r e s i s t a n c e was 40 0cm higher i n high K s a l i n e (140 mM K) than i n normal s a l i n e (10 mM K) during cAMP stimula-t i o n (P << 0.01). Results of subsequent tracer and microelectrode experiments (chapters 4 and 5) also suggest that K permeability of the epithelium varies inversely with s a l i n e [K]. The passive backflux of c h l o r i d e i n high K s a l i n e (140 mM K, 50 mM Cl) C l may be compared with J g m at normal K l e v e l s (10 mM K, 50 mM C l ) . From C l —2 —1 Figure 3 i t may be seen that at [ C l ] = 50 mM, J was 1.3 yEqcm h , i n good -2 -1 agreement with values observed with 140 mM K and 50 mM C l (0.9-1.3 yEqcm h ). 3 6 T r a n s e p i t h e l i a l C l permeability (as indicated by C l backflux) i s apparently C l not affected by external [K] over t h i s range, since J was i d e n t i c a l i n 10 and sm C l 140 mM K s a l i n e . Therefore i t was necessary to measure J at only one sm C l external concentration of K. In summary, A l equals AJ when external [K] J sc net 116 4 A 2 H o-J cAMP T/1 a f I I • r r ? / I r-200 H 150 CN £ o <^  100 50 cAMP \ 1 1 1 1 1 1 1 T — I 10 H 8H - 2 cAMP T • i / / l^H 1 1 1 1 b m»s -1 r -1 2 T i m e C h ) - i i — i 3 40 30 H > £ ~ 20 10 4 cAMP T i LU-U-I • I T T T ] " - 1 — I 3 T i m e C h ) Figure 3.7 E f f e c t s of 1 mM cAMP on u n i d i r e c t i o n a l C l fluxes and e l e c t r i -c a i parameters i n "high-K" s a l i n e . (a) I ; (b) t r a n s e p i t h e l i a l C l fluxes from mucosa to serosa (m-*s) , and i n the reverse d i r e c t i o n (s->m) ; (c) t r a n s e p i t h e l i a l r e s i s t a n c e R ; and (d) t r a n s e p i t h e l i a l p o t e n t i a l d i f f e r -ence V . See Table 2.1 for composition of "high-K" s a l i n e . Means ± s.e.; n = 12 (V R ), n = 6 ( J ^ , j";) . t t ms sm 117 i s 140 mM (F i g . 7) or 0 .or 10 mM (F i g . 15 of chapter 2) and act i v e Cl trans-port i s the major electrogenic process i n locust rectum under a l l these conditions. 3) Apparent potassium a c t i v a t i o n constant (K ) o_f C l transport Since A l g c i s a v a l i d measure of act i v e C l transport regardless of the concentration of K i n the s a l i n e , the apparent K & of K stimulation can be estimated from the e f f e c t s of K on Al i n the presence of cAMP, as described i n the methods section. To ensure that the K-independent component of AI G C (see F i g . 15 of chapter 2) was not p a r t l y the r e s u l t of increases i n osmotic pressure and i o n i c strength, Na methylsulfate was added i n p a r a l l e l experi-ments under i d e n t i c a l conditions. Figure 8a shows that addition of cAMP -2-1 -2 -1 caused I to increase from 0.86 ± 0.09 yEqcm h to 2.56 ± 0.38 yEqcn h , i n excellent agreement with previous r e s u l t s i n K-free s a l i n e ( F i g . 14 of chapter 2). As external [K] was then elevated by stepwise addition of KCH^SO^ to both sides, I increased s i m i l a r l y to a maximum of 12.68 ± 0.67 -2-1 yEqcm h at 100 mM K. Above th i s l e v e l , I decreased r e v e r s i b l y (Fig. 8a). V increased from 7.2 ± 0.5 to 16.8 ± 1.8 mV when 1 mM cAMP was added to K-free 3 6 sa l i n e i n close agreement with values obtained during C l flux experiments (8.1 ± 2.0 to 16.8 ± 3.0 mV, F i g . 14 of chapter 2). R remained constant when [K] was raised from 10 to 200 mM (Fig. 8c), a s u r p r i s i n g r e s u l t considering the normally high K permeability of this epithelium at low external K l e v e l s . As argued i n the preceding section, t r a n s e p i t h e l i a l K permeability must decrease at high K l e v e l s i n order for e l e c t r i c a l conductance to remain constant as external K concentration i s r a i s e d . Results obtained during NaCH^SO^ addition were very d i f f e r e n t (Fig. 9a-c). Na addition did not produce large s t e p - l i k e increases i n I . I reached a ^ t> f sc sc -2 -1 -2 -1 maximum at ^ 5 yEqcm h as compared to 13 yEqcm h when K was added 118 Figure 3.8 " E f f e c t s of stepwise b i l a t e r a l K additions on e l e c t r i c a l parameters under I conditions. Recta were e q u i l i b r a t e d i n K-free s a l i n e for 4 hours. Cyclic-AMP (1 mM) was added to the serosal side at the arrows. A f t e r 1 h of cAMP exposure, K-methylsulfate was added to both sides to give the f i n a l concentrations shown. Means ± s.e.; n = 9-10. 119 • E o cf LU w O CO 12 J 10 H 8 6 J _w 4 c A M P Increase in K c o n c . (mM) I 2 | 4 | 10 | 40 I10o|l40|200| 100 | U'Hv 1 T e 1 1 V 11 l / l Ki/1 300 ~ 200 E u c cT 100 I—" c A M P o F I 'TIN cv [ 1 i V o 1. \ T o 9"6 O - O . o - O * 1 1 i * D " T T i O " 0 0 J I 1 1 , - T r-0 1 2 3 4 5 Time (h) 1 2 0 I n c r e a s e in Na c o n e . ( m M ) I 2 | 4 | 10 j 40 | 1 0 0 | 1 4 0 | 2 0 0 | 100 | i JC CN I E o cr U J 3 . o 0) 5 4 3 2 1 0 4 0 0 c A M P 3 0 0 4 CM E o 2 0 0 C 100 1 T ! * B 1 •••.j.I, i i V ^ T |M T 7*' T 1 i i D-O^T T 1 i * i T • iSTn T T T/1 1 i T iju • - o - i 0 2 3 T i m e Ch) 5 Figure 3.9 E f f e c t s of stepwise b i l a t e r a l Na additions on e l e c t r i c a l parameters under I conditions. Experimental protocol was i d e n t i c a l to that described i n Figure 3.8 except that Na-methylsulfate was added to both sides instead of K-methylsulfate. Means- ± s.e.; n = 6. (P << 0.01). Also, high Na l e v e l s (>100 mM) did not i n h i b i t I , i n marked contrast to the e f f e c t s of elevated K l e v e l s . T r a n s e p i t h e l i a l resistance declined i n a predictable manner when sa l i n e [Na] was increased above 10 mM (Fig. ..9c), i n contrast to the r e l a t i v e l y constant R observed during K addit i o n over the same range. The di f f e r e n c e between mean I obtained during K and Na additions was sc b Cl used as a measured of K-dependent C l transport, since i ) A I g c equals A J n e t at a l l K concentrations (previous section and chapter 2), and i i ) I i s not strongly Na-dependent (Fig. 8 of chapter 2). Figure 10 shows a conventional Lineweaver-Burke pl o t of I stimulation which was used to determine the maximal K-dependent I (K-dependent C l transport) and the K concentration giving h a l f maximal stimulation (K ). cL Potassium a c t i v i t y of the sa l i n e s was also measured using a l i q u i d ion exchanger-type K-sensitive electrode (see chapter 4). A l i n e a r r e l a t i o n s h i p was obtained between [ K +] ^  and (K-dependent-I ) ^ when [K +] was greater than 2 2 mM (r = 0.9992). K was calculated to be 5.3 mM K concentration or 3.6 mM a -2 -1 K a c t i v i t y . Maximum K-dependent I was 8.3 yEqcm h These c a l c u l a t i o n s r e l y on the assumption that Na does not stimulate C l transport s i g n i f i c a n t l y since 1) Na (110 mM) was present when the e f f e c t s of K addi t i o n were tested, and 2) NaCH^SO^ addition was used as a con t r o l for osmotic and i o n i c e f f e c t s on the K-dependent component of I • In the next section the s e l e c t i v i t y of K a c t i v a t i o n i s examined. 4) S e l e c t i v i t y of K a c t i v a t i o n of C l transport Malpighian tubule f l u i d entering the locust r e c t a l lumen i n vivo contains Na and probably NH^ ions i n addi t i o n to the much higher l e v e l s of K ( P h i l l i p s , 1964b; Maddrell and Klunsuwan, 1973; see also Table 1 of chapter 2). The cation s e l e c t i v i t y of K-stimulation of active C l transport was determined i n 122 c o n c e n t r a t i o r 2 = 0 .9992 1 / K c o n c e n t r a t i o n or ' a c t i v i t y C m M ) Figure 3.10 Lineweaver-Burke plot of the r e l a t i o n s h i p between external K concentration and Cl-dependent I • Recta were exposed to 1 mM cAMP on the serosal side throughout t h i s experiment. The difference between I i n sc Figures 3.8 and 3.9 was used as a measure of K-stimulated C l transport. The K concentration r e s u l t i n g i n half-maximal stimulation of I i s 5.3 mM. sc External [Cl] was constant at 114 mM.( 123 order to f i n d out whether other cations, p a r t i c u l a r l y NH^, might substitute for K i n enhancing active C l transport. I t was also important to test whether the K-dependent component of I during cAMP stimulation r e s u l t s from the presence of Na (110 mM) i n K-free s a l i n e . If t h i s were so, lack of s e l e c t i v -i t y for K would r e s u l t i n a large error i n my estimate of the K for cl potassium " a c t i v a t i o n " of C l transport. Tissues were e q u i l i b r a t e d f o r 3-4 h under I conditions i n K-free s a l i n e and were then exposed to 1 mM cAMP. Aft e r 2-3 h exposure to cAMP, various test cations were added b i l a t e r a l l y so t h a t the c o n c e n t r a t i o n of cation was rai s e d by 40 mM (Fig. l l a - e ) . A s e l e c t i v i t y sequence was deter-mined by comparing A I s c a f t e r 1 h of exposure to the test cation. Arranged i n order to decreasing potency, the sequence was: 1.0 K > 0.58 Rb > 0.49 Cs > 0.08 NH^, 0.03 Na. During a d d i t i o n of K, Rb, Cs and NH^, the concentration of these cations increased from 0 to 40 mM whereas the Na l e v e l increased from 114 to 154 mM during the experiment. Therefore, r e s u l t s do not include the p o s s i b i l i t y that low l e v e l s of Na stimulate C l transport during exposure to cAMP, i . e . the possible stimulating e f f e c t of Na ions might be maximal at <110 mM and might not be enhanced further by addi t i o n of 40 mM Na. To te s t t h i s hypothesis, the ef f e c t of 1 mM cAMP on I was measured i n two tissues when choline was the only monovalent cation present for 3 h. The stimulations -2 -1 obtained i n the absence of both Na and K were 4.0 and 4.5 yEqcm h , i d e n t i c a l to those observed with ^200 mM Na present (Fig. 9). Moreover, cAMP stimula-tions of t h i s magnitude were also observed when Na and K were replaced with tetramethyl ammonium. Even when Na, K, Ca and Mg i n normal s a l i n e (Table 1 of chapter 2) were completely replaced by choline for 3 h so that choline was the only cation present above 1 mM, cAMP stimulated I from 1.33 to 2.62 and r ' sc -2 -1 0.91 to 2.66 yEqcm h i n two re c t a . These r e s u l t s suggest that the I -C CM I E o cf U J 2. w O 10 8 A 6 A 4 A 124 1 1 * b / I Q I I 6 -i Cs / 4 4 3 I • U . J / Na 1 11 : ; - ; - r ; - t - ! - r T • A 1 1 0 0.5 1.0 1.5 2.0 T ime (h) Figure 3.11 S p e c i f i c i t y of c a t i o n s t i m u l a t i o n of C l t r a n s p o r t . Cyclic-AMP-s t i m u l a t e d r e c t a were e q u i l i b r a t e d under I c o n d i t i o n s f o r 2-4 h i n K-free sc s a l i n e (Table 2.1). When I reached an approximate steady-state value, concentrated s o l u t i o n s of various c a t i o n s were added b i l a t e r a l l y as s u l f a t e s a l t s i n order to achieve a f i n a l t e s t c a t i o n concentration of.40 mM (K, Rb, Cs, NH 4) or 150 mM (Na). Means ± s.e.; n = 5-6. 125 K-independent component of cAMP-stimulated i s independent of cations i n the external s a l i n e . Sequence I (K > Rb > Cs > Na) observed f o r cation stimulation of locust C l transport i s the series expected f o r s e l e c t i v i t y s i t e s having moderately weak f i e l d strength (Eisenman, 1961). A s i m i l a r sequence i s observed i n the serosal membrane of frog skin (Lindley and Hoshiko, 1964) and Malpighian tubules of the blowfly (Berridge, 1969; for reviews see Diamond and Wright, 1969). 5) •• Sidedness of K a c t i v a t i o n To determine whether K " a c t i v a t i o n " of C l transport occurred s p e c i f i c a l l y at one side of the tis s u e , I was measured during stepwise addition of K methylsulfate to eit h e r mucosal or serosal side. Recta were f i r s t e q u i l i b r a t -ed under I conditions i n K-free s a l i n e for 3-4 h and then exposed to 1 mM sc r cAMP. After I reached a new steady-state, low concentrations (2-10 mM) of K methylsulfate were added to the mucosal or serosal side. Only low concentra-tions of K were used i n order to minimize the K d i f f u s i o n current caused by a t r a n s e p i t h e l i a l K gradient and also the rate of contamination of the K-free side. To measure passive K currents, I was recorded during asymmetrical K additions using recta poisoned with 1 mM azide. Currents produced by K gradients i n Cl- f r e e s a l i n e were s i m i l a r to those observed i n azide-poisoned tissues. Correction of I for K d i f f u s i o n ranged from 0% to 21% i n the sc b presence of a 10 mM -> 0 mM (mucosa-to-serosa) K gradient. The mean K d i f f u -sion currents measured i n this way were subtracted from the I measured i n sc unpoisoned tissues i n order to c a l c u l a t e true Cl-dependent I in the sc presence of a K gradient. Figure 12 shows the eff e c t s of adding K methylsulfate to either side of the epithelium. After corrections, I a t t r i b u t a b l e to active Cl transport U J CJ co u cu o u c 126 K cone. C m M ) 0 | 2 4 10 V 6 H _c OJ I E 4 4 2 A 0 -—' T a • i CM • CT LU "D CD o u 6 A 4 4 0 - J / m / / / / / / / / I ' ' ^ 1 0 0.5 1.0 Time CrO Figure 3.12 E f f e c t of adding K-methylsulfate stepwise to one side only on Cl-dependent I . A f t e r e q u i l i b r a t i n g recta 3 h under I conditions in K-sc n ° sc free s a l i n e , 1 mM cAMP was added to the serosal side and I was monitored for sc 1 hour. Aliquots of K-methylsulfate were then added to the mucosal or serosal chambers to give the concentrations -indicated. The apparent A I g c produced by asymmetrical K addi t i o n i n a z i d e / l ^ s a l i n e was subtracted to correct for K d i f f u s i o n a l current as described i n the text. Means ± s.e.; n = 7. 127 -2 -1 -2 -1 increased from 1.55 yEqcm h to 6.85 yEqcm h when 10 mM K was added to the mucosa. In contrast*, w a s not changed s i g n i f i c a n t l y by serosal addition of K (P >> 0.2). In chapter 4, I w i l l show that K enters K-depleted r e c t a l c e l l s much more e f f e c t i v e l y from the serosal side than from the lumen, presumably because of a Na/K exchange pump at the basal c e l l border. Potas-sium must act at the external surface of the mucosal membrane rather than at some i n t r a c e l l u l a r s i t e , because serosal K addition f a i l s to stimulate C l transport i n s p i t e of the fact that i n t r a c e l l u l a r K concentration i s maintain-ed at high l e v e l s (^60 mM) when mucosal [K] i s 0 mM and serosal [K] i s 10 mM (see F i g . 6 of chapter 4). 42 6) T r a n s e p i t h e l i a l K fluxes ( s h o r t - c i r c u i t conditions) i ) Normal saline Active K absorption has been reported across locust rectum i n vivo ( P h i l l i p s , 1964b) and i n v i t r o (Williams et a l . , 1978), however l i t t l e i s known regarding i t s i o n i c requirements or hormonal regulation or the r e l a t i v e s i z e of active and passive components of K absorption. Considering the dependence of active Cl transport on external K, i t was important to study the properties of a c t i v e K absorption, p a r t i c u l a r l y the e f f e c t s of cAMP and possible Cl dependence. For example, does KC1 cotransport occur i n t h i s epithelium as i n red blood c e l l s (Kregenow and Caryk, 1979) and E h r l i c h tumour c e l l s (Geek et a l . , 1980)? Figure 13 shows the e f f e c t s of 1 mM cAMP on a) t r a n s e p i t h e l i a l fluxes of 42 K K and b) J _ and across r e c t a bathed i n normal s a l i n e (114 mM C l , 10 mM K). net Values of and V were i d e n t i c a l to those shown i n Figure 2 of chapter 2 and K K therefore are not shown. Upon adding I mM cAMP, both J and J increased r & ms sm _? _ i from about 0.35 to 2.08 ± 0.22 and 1.56 ± 0.18 yEqcm h , r e s p e c t i v e l y . K Under steady-state, cAMP-st imulated conditions, J (0.632 + 0.26) was 128 I E o cf LU 3 . ~3 T -!c CM 'E o cf LU 3 . ^ c -3 O CO 2.5 H 2.0 1.5 -1.0 -0.5 -0 --0.5 --1.0 --1.5 -10 • 8 6 4 2 0 / i c A M P • m -»-s s -*-m • • — 5 — 5 . \ \ T T l ^ o T—^ —T A l c A M P I / 1 i I . t — t ^ i i Isc J K J n e t — I — 1.5 0.5 1.0 T i m e (h) 2.0 2.5 Figure 3.13 E f f e c t s of cAMP on I and K fluxes measured under I condi-2 sc sc 42 tions i n normal s a l i n e . (a) U n i d i r e c t i o n a l K f l u x from mucosal-to-serosal side ( • , m->-s) and i n the reverse d i r e c t i o n ( • , s-*m) , (b) d ^ e t ( ° ) and I ( A ) across s h o r t - c i r c u i t e d recta bathed i n normal saline (114 mM C l , sc 10 mM K). Tissues were pre-equilibrated 4 h under I conditions. Means ± r ^ sc s.e.; n = 6 ( J K , J K ), n = 12 (I ). ms sm sc B i l a t e r a l C l c o n c . ( m M ) I 0 1 1T4 1.5 -1 I • • • ' - r -0 0.5 1.0 1.5 2.0 2.5 Time Ch) Figure 3.14 E f f e c t s of 1 mM cAMP on K fluxes and I i n C l - f r e e s a l i n e . s c C l was replaced with methylsulfate; basic s a l i n e as described i n Table 2.1 (10 mM K, 110 E J M Na). NaCl was added to both sides in order to restore normal (114 mM) C l concentration. Means ± s.e.; n = 6 ( J K , J K ), n = 12 (I ). ms sm' sc 130 i -u \r u . u x ; D U C was xess tnan ru or t measured under these conditions s i g n i f i c a n t l y d i f f e r e n t from zero (P << 0.01) but l h 7% f J net K K The large (400%) stimulation of both J and J suggests that cAMP ms sm increases t r a n s e p i t h e l i a l permeability to potassium (P t r). Using the approxi-K mation *P„ =-J / [ K ] , where [K] i s 10 mM and J i s assumed to be passive,*P„ K sm sm R K increases from 0.98 ± 0.14 to 4.0 ± 0.62 x 10 ^ cm sec 1 (x ± s.e., n = 6) a f t e r adding cAMP. Quantitatively, t h i s method of c a l c u l a t i n g t r a n s e p i t h e l i a l 42 P may lead to some error because K must penetrate two membranes i n series whereas t h i s approach assumes the tissue i s a s i n g l e b a r r i e r (see chapter 5). Nevertheless, these r e s u l t s c l e a r l y indicate a large increase i n P during cAMP stimulation and t h i s i n t e r p r e t a t i o n i s supported by e l e c t r o p h y s i o l o g i c a l r e s u l t s described i n chapter 5. In summary, cAMP has two major e f f e c t s on t r a n s e p i t h e l i a l K movements under I conditions: I t causes i ) a very large (^4-fold) increase i n 42 t r a n s e p i t h e l i a l permeability to K, and i i ) the appearance of a small net K -2 -1 absorption of approximately 0.8 yEqcm h ( i . e . a small active K transport component). i i ) Cl-free saline 42 Figure 14a shows the e f f e c t s of- 1 mM cAMP on t r a n s e p i t h e l i a l K fluxes before and a f t e r r e s t o r i n g normal chloride l e v e l s to C l - f r e e s a l i n e . After 1 h of exposure to cAMP both u n i d i r e c t i o n a l fluxes increased by about 4-fold, J K from 0.45 ± 0.13 to 1.91 ± 0.2 yEqcm"2!"!""1 and J K frozi 0.31 ± 0.06 to ms sm 1.25 ± 0.19 yEqcm~ 2h _ 1. (0.66 ± 0.39 yEqcm~ 2h _ 1) was s i g n i f i c a n t l y greater than zero (P < 0.01). When Cl was restored to normal l e v e l s on both K K sides (114 mM), there was no change in J or J (P > 0.2) although I b sm ms sc increased 6-fold following C l addition to values not d i f f e r e n t from those obtained i n experiments with normal sa l i n e (compare Figs. 14 and 13). 131 The present r e s u l t s show that neither the K permeability increase caused by cAMP nor the small net.K transport are affected by C l removal. In view of t h i s independence, i t seems u n l i k e l y that there i s s t r i c t chemical coupling between C l and K movements at the a p i c a l or basal membranes. This conclusion K Cl i s supported by the previous observation that J _ i s l e s s than 8% of J C l under I conditions (Fig. 13) and approximately 35% of J i s cation-sc 3 net independent. 42 7) T r a n s e p i t h e l i a l K fluxes (open-circuit conditions) Chloride absorption across locust rectum i s electrogenic and must, under normal open-circuit conditions, be matched by a s i m i l a r flow of cations from mucosa-to-serosa or a l t e r n a t i v e l y , a flow of anions in the reverse d i r e c t i o n . To determine whether K moves to maintain e l e c t r o n e u t r a l i t y during C l trans-42 port, open-circuit t r a n s e p i t h e l i a l K fluxes were measured sequentially; f i r s t i n normal s a l i n e ( i . e . under control conditions) and then during cAMP-stimulation (10 mM K b i l a t e r a l l y ) . F i n a l l y , mucosal [K] was raised to 100 mM i n the presence of 1 mM cAMP to mimic normal in.vivo K gradients (10:1) across the locust rectum (Fig. 15). In control (10 mM K) s a l i n e before stimulation, V ranged between 8-10 mV, i n close agreement with previous r e s u l t s (Fig. 5, chapter 2). U n i d i r e c t i o n a l 42 -2 -1 K fluxes were less than 1 yEqcm h (Fig. 15a,b). Serosal addition of cAMP (1 mM) increased from 8 to 28 mV (see also F i g . 5, chapter 2), K K enhanced J 5-fold, and produced a small but s i g n i f i c a n t increase in J ms r 6 sm -2 -1 ( s i g n i f i c a n t P < 0.01). Net K f l u x ranged from 4.5 to 5.0 yEqcm h . I t i s Cl K noteworthy that J _ equals J at open-circuit before and during cAMP net n net r b exposure (compare Fig. 15b of t h i s chapter with F i g . 5 of chapter 2). This r e s u l t indicates that K i s the main counter ion during active C l transport even when the r a t i o of [Na]/[K] i s 11/1 in the external s a l i n e . 132 Figure 3.15 E f f e c t s of sequential a d d i t i o n of 1 mM cAMP to the serosal side and mucosal add i t i o n of potassium on K fluxes under open-circuit conditions. Recta were i n i t i a l l y bathed i n normal s a l i n e (10 mM K, 110 mM Na, 110 mM C l ) . Time "0" was preceded by 4 h e q u i l i b r a t i o n under op e n - c i r c u i t conditions. After 75 min exposure to cAMP, mucosal [K] was increased from 10 to 100 mM by adding concentrated K-methylsulfate s o l u t i o n . Means ± s.e.; n = 16 (V ), n = 8 ( J K , J K ). ms sm 133 m u c o s a l 1 0 0 m M K 1 m M c A M P 30 H 25 A 2 0 ^ > £ 15 10 H 5 H o —' 1 1 1 1 i i r T r 46 20 18 16 ~ 14 -j I •= 12 CM d 10 cf U J 8 6 ^ 4 2 0 fl T/T\; • B B / • /I m -*-s s m | ' ' 1 ' " I 1 0 1 2 3 T i m e C h ) V 134 declined from 28 mV to about 5 mV when mucosal [K] was raised from 10 to 100 mM (Fig. 15a,b). - J increased concurrently by more than 3-fold to to net J J -2 -1 C l ^16 yEqcm h . This t r a n s e p i t h e l i a l flux rate i s twice that of J observed net -2 -1 under these conditions (8 yEqcm h ; Fi g . 5, chapter 2). It i s not yet known which ion(s) maintain charge balance when mucosal [K] i s elevated. Prelimin-ary experiments suggest that K/H exchange i s not involved: Mucosal pH was monitored using phenol red as mucosal [K] was raised from 10 to 100 mM i n HCC>2/C02-free s a l i n e . From t i t r a t i o n curves i t was calculated that mucosal pH would decrease 0.3-0.4 pH units within 1 h i f protons were exchanged 1:1 with K across the mucosal membrane during cAMP stimulation under I conditions. Contrary to t h i s p r e d i c t i o n , mucosal pH increased K during exposure to high mucosal [K]. Other fluxes which might balance J o r ° ms under these conditions of very high mucosal [K] include serosa-to-mucosa fluxes of some other cation such as Ca, Mg or perhaps NH^. These p o s s i b i l i -t i e s were not tested. According to the flux r a t i o n equation (Ussing, 1949) for passive, independent movements of an ion such as K, Jls _^m e x ? ( V t F / R T ) ( 2 ) / " ( K ) s sm K K where J and J are the forward and back fluxes measured with tracers, ms sm (K) and (K) are mucosal and serosal K a c t i v i t i e s , V\ i s the t r a n s e p i t h e l i a l m s t po t e n t i a l , and F, R and T have t h e i r usual meanings. 42 In normal s a l i n e , r a t i o s of u n i d i r e c t i o n a l K fluxes at open-circuit are much higher than those predicted from the fl u x r a t i o equation (Fig. 16). 42 Under con t r o l conditions (normal s a l i n e , no cAMP) the K flux r a t i o s were between 4 and 6, whereas the predicted r a t i o was only 1.4. It i s u n l i k e l y that t h i s discrepancy could a r i s e from a c t i v e transport since no net flux of 135 m u c o s a l 1 0 0 m M K 1 m M c A M P T i m e ( h ) K K Figure 3.16 Comparison of K f l u x r a t i o s J / J under op e n - c i r c u i t condi-— = r ms sm ^ tions with those predicted from the Ussing f l u x - r a t i o equation. See Figure 3.15 f o r o r i g i n a l f l u x data and experimental protocol. Note log scale. Also shown are values of the t r a n s e p i t h e l i a l flux r a t i o exponent (n') calculated as RT , , ( K ) m ' J s B , n = — — In (• — ) " V ( K ) J K s P.S where ( K ) m , (K) a r e K a c t i v i t i e s on tha ,.:^osal and serosal sides, respective-K K l y : R, T, F, V , J and J have their usual meanings, t sm ms 136 potassium was observed when tissues were s h o r t - c i r c u i t e d in the absence of cAMP. Rather, the discrepancy i s suggestive evidence for non-independence of K movements across the tissue as discussed i n a l a t e r section. As expected, flux r a t i o s increased further a f t e r addition of cAMP, a manoeuvre which resulted in a small active f l u x under I conditions. However, sc a step increase i n mucosal [K] from 10 to 100 mM increased the steady-state flux r a t i o to >100:1, much larger than the predicted value of 10.4. This larger discrepancy i s not due to enhanced active transport because i t was j£ l a t e r found that J _ under I conditions i s s i m i l a r whether s a l i n e contains net sc 10 mM or 140 mM K. These r e s u l t s are again consistant with non-independence 42 between transmural K fluxes. A measure of non-independence may be obtained by r a i s i n g the right-hand side of equation 2 to some power n', the flux r a t i o exponent. According to the".single-file theory", n' i s related to the number of s i t e s within a channel (Hodgkin and Keynes, 1955). Values of n' for t r a n s e p i t h e l i a l fluxes (n^) were calculated a f t e r correcting the f l u x r a t i o for the small measured under net I conditions as, sc K RT . / (K)m* Jsm n^ = In C -V "* \ (K) • J K / s ms Values of n' are shown i n Figure 16. Under control conditions, ranged between 4.0 and 5.10 and decreased to 1.97-2.30 during exposure to 1 nM cAMP. When [K] was elevated on the mucosal side, n^ increased to 10.9-13.2. Although high values of n^ do indicate non-independence i n t r a n s e p i t h e l i a l K fluxes, i n t e r p r e t a t i o n of n^ . i s complicated i n e p i t h e l i a by the presence of two b a r r i e r s i n series since the c e l l u l a r compartment contains tracer at some unknown a c t i v i t y . This w i l l be discussed i n a l a t e r section. 137 4 2 8) T r a n s e p i t h e l i a l K fluxes i n "high-K" s a l i n e 4 2 Figure 17 shows the e f f e c t s of 1 mM cAMP on u n i d i r e c t i o n a l K fluxes under I conditions when recta were bathed b i l a t e r a l l y i n "high-K" s a l i n e sc ' 42 (140 mM K, 50 mM C l ; see Table 1 of chapter 2). U n i d i r e c t i o n a l K fluxes 2 2_ 4-2 2 1 (1.0-1.7 yEqcm h ) and net K fluxes (-0.1 to +0.5 uEqcm h ) were s u r p r i s i n g l y s i m i l a r to those observed i n normal s a l i n e i n which [K] was 14-f o l d lower. As before, J m g increased s i g n i f i c a n t l y during cAMP exposure (P < 0.05). Although J increased s t e a d i l y over the course of the expert-ly ment, addition of 1 mM cAMP did not increase J (P >> 0.2), i n marked sm contrast to the e f f e c t s observed i n normal (10 mM K) s a l i n e . Several conclusions regarding active K transport may be drawn from these data. F i r s t , a c t i v e K transport saturates at low concentrations of K K —2 —1 (K < 10 mM) since J ^ was 0.8-1.0 uEqcm h under I conditions whether t net n sc [K] of the s a l i n e was 10 mM (normal saline) or 140 mM (high-K s a l i n e ) . Second, when t r a n s e p i t h e l i a l K permeability (*Pjr) i s calculated from J (see P-295), must decrease s e v e r a l - f o l d at high [K]: J 1 ^ does not increased proportionately at higher K l e v e l s , as shorn by comparing J i n Figure 13a and Figure 16a. Rather, *P i s apparently 3- to 4-fold higher when tissues are bathed i n normal s a l i n e (10 mM K, 114 mM Cl) than i n "high-K" s a l i n e (140 mM K, 50 mM Cl) . Chloride concentration was lower i n "high-K" s a l i n e than i n normal s a l i n e (50 mM and 110 mM, r e s p e c t i v e l y ) ; however, this could not account for differences i n potassium permeability because *P V i n C l - f r e e s a l i n e (10 mM K) i s also 4-fold higher than i n "high-K" s a l i n e ; i . e . J ^ i s not proportionally higher i n Figure 17 (140 mM K, 50 mM Cl) as compared to J ^ m i n Figure 14 (10 mM K, 0 mM C l ) . Moreover, addition of 1 mM cAMP does not increase *P„ 4-fold as i t does eg i E o cf LU ~~> JZ CM '£ o cf LU n> V c O CO 2 H -1 H -2 4 138 c A M P V m — s s -» m 10 -8 -6 -4 -2 0 -2 — ' n e t — i — 0.5 — i — 1.0 — I — 1.5 — I — 2.0 2.5 Time Ch) Figure 3.17 E f f e c t s of cAMP on K fluxes and I across recta bathed i n • sc "high-K" s a l i n e on both sides. High-K s a l i n e contained 140 m>! and 50 mM C l (see Table 2.1 for d e t a i l s ) . Tissues were e q u i l i b r a t e d for 3-4 h under I. sc conditions p r i o r to adding 1 mM cAMP to the serosal side. Means ± n = 12 (I ) , n = 6 ( J K , J K ). sc ms sm s.e, 139 i n normal or C l - f r e e salines containing 10 mM K. This i s consistent with the e a r l i e r unexpected observation that t r a n s e p i t h e l i a l e l e c t r i c a l resistance was higher i n "high-K" s a l i n e than i n normal s a l i n e (compare present F i g . 8c and F i g . 2d of chapter 2) and also with the find i n g that R did not decrease as [K] was increased stepwise from 10 mM to 200 mM (Fig. 9c). The simplest explana-t i o n for a l l these r e s u l t s i s that high external [K] lowers t r a n s e p i t h e l i a l K permeability. E l e c t r o p h y s i o l o g i c a l r e s u l t s to be described i n chapter 5 support t h i s conclusion. Discussion The data i n t h i s paper provide evidence for an unusual i n t e r r e l a t i o n s h i p between K and C l during KC1 reabsorption i n locust rectum. Although the largest component of active C l absorption requires potassium i n the mucosa, K does not act simply as a permeant counterion because most ac t i v e Cl transport (70%) i s stimulated by external K even when recta are s h o r t - c i r c u i t e d by extern a l l y applied current. On the other hand, t r a n s e p i t h e l i a l K absorption under open-circuit conditions i s mostly passive (>80%), and e l e c t r i c a l l y coupled to act i v e Cl absorption. The much smaller a c t i v e f l u x of K to the serosal side during cAMP stimulation i s not Cl-dependent. Active C l transport: the e f f e c t s of K The e f f e c t s of K on t r a n s e p i t h e l i a l C l fluxes were examined over the normal p h y s i o l o g i c a l range of C l concentrations. The r e l a t i o n s h i p between Cl steady-state J and external [ C l ] i s well described by the Michaelis-Menten J net . . - I r e l a t i o n s h i p f o r enzyme-catalysed reactions, although other hyperbolic functions could also f i t the data. Potassium addition had the e f f e c t of Cl increasing both the Cl concentration causing half maximal J n e t (^t) a u <^ also C l the maximal rate of active C l transport (J )• These parameters could depend 140 on the properties of the transfer process for C l across the basal membrane ( l o c a l i z e d using i o n - s e n s i t i v e microelectrodes, i n the following chapter), but they are more l i k e l y to r e f l e c t the properties of'the C l pump i n the Cl a p i c a l membrane. There i s some i n d i r e c t evidence that K and J are proper-t max t i e s of the a p i c a l entry step: The net electrochemical gradient opposing C l entry across the mucosal membrane va r i e s with transport rate while the gradient favouring C l e x i t across the serosal membrane remains constant. This f i n d i n g suggests that the pump at the a p i c a l membrane i s r a t e - l i m i t i n g for Cl transport; however, more d i r e c t evidence for t h i s i n t e r p r e t a t i o n i s required. If the a p i c a l C l pump i s an ATPase, one convincing approach would be to compare the properties of is o l a t e d enzyme (K m and potassium K ) with the Kfc and K for K-stimulation of t r a n s e p i t h e l i a l Cl transport. Mucosal K might 3. depolarize the mucosal membrane, thereby reducing the l o c a l electrochemical gradient against which the a p i c a l C l pump must work. This type of e l e c t r i c a l coupling would not be obvious from t r a n s e p i t h e l i a l flux measurements which treat the epithelium as a black box. Nevertheless, in order to explain the Cl K 10-fold difference between J and J under I conditions, e l e c t r i c a l net net sc coupling across the a p i c a l membrane between K and active C l transport would require that K i s a c t i v e l y pumped from the c e l l to mucosal side ( i . e . recy-cled) . No evidence was found for K secretion or r e c y c l i n g at the mucosal c e l l border by using tracer fluxes ( t h i s chapter) or by i o n - s e n s i t i v e microelec-trode techniques (chapter 4). A s i m i l a r argument may be made against c a r r i e r -mediated coentry of K and Cl under I conditions. This w i l l be discussed sc further i n chapter 4. By what mechanism does K stimulate net C l transport under I conditions? In the absence of KC1 coentry, I propose that mucosal K at the external surface of the a p i c a l membrane enhances C l transport by stimulating the C l 141 pump without a c t u a l l y being transported, analogous to an enzyme-activator which accelerates a reaction but i s not altered (or transported) by i t . To C l explain the K - i n s e n s i t i v e component of cAMP-stimulated J n g t > the a p i c a l membrane may contain a single population of Cl pump s i t e s having a graded response to mucosal K or a l t e r n a t i v e l y two populations of C l pumps may e x i s t ; one which operates without K and the other which i s stimulated as [K] i s increased. Further studies are required to d i s t i n g u i s h between these a l t e r n a t i v e hypotheses. Passive K transport Most t r a n s r e c t a l K flux i s passive under open-circuit conditions. Net K K C l f l u x ( J ) i s only 8% of J when locust rectum i s s h o r t - c i r c u i t e d , however net J net K Cl under open-circuit conditions J equals J . K acts as the maior counter net n net J ion for electrogenic Cl transport even when much higher concentrations of Na are present i n the experimental s a l i n e bathing the mucosal side (114 mM Na, 10 mM K ) . Moreover, the predominance of K as a counter ion i s ensured i n vivo for two reasons: i ) normal K l e v e l s (140 mM) are much higher than Na (20-40 mM) i n the r e c t a l lumen; i i ) cAMP increases apparent t r a n s e p i t h e l i a l K permeability by more than 4-fold. When high concentrations of K are added to the mucosal side under open-c i r c u i t conditions to simulate natural i n vivo K gradients, the mucosal side should become e l e c t r i c a l l y negative with respect to the serosal side i f ti s s u e potassium conductance is. high. However, no reversal of V was observed. Stimulation of electrogenic C l absorption by K and cAMP, as described previous-l y , tends to balance the reduction i n V by K d i f f u s i o n from mucosal-to-serosal side. It w i l l be shown in appendix 3 that V reverses ( i . e . lumen becomes negative with respect to hemolymph) i n the presence of a normal mucosa-to-serosa K gradient under C l - f r e e conditions both i n s i t u and i n v i t r o . 142 Several types of experiments reported i n t h i s chapter suggest that trans-e p i t h e l i a l P„ i s lowered-by high l e v e l s of K. F i r s t , when Na methylsulfate was added stepwise to both sides of the epithelium, R decreased i n a predictable manner as s a l t concentration was increased to 200 mM. In contrast, R remained constant when K methylsulfate concentration was raised under i d e n t i c a l conditions. Second, the observation that K i s the counter ion for electrogenic C l transport indicates that K conductance of the rectum i s high r e l a t i v e to other ions. If P.. remained constant i n the presence of high [K], increasing K methylsulfate concentration from 10 to 200 mM should have reduced t i s s u e resistance (R ) d r a s t i c a l l y . Since Rfc did not decrease when [K] was elevated, t h i s r e s u l t indicates that P declines as [K] increases. Concentration-dependent permeability i s to be expected whether ion permeation occurs v i a c a r r i e r s or channels (reviewed by Heckmann, 1973; H i l l e , 1979; Lauger, 1980) and an inverse r e l a t i o n s h i p between mucosal [Na] and the rate of Na entry at the a p i c a l membrane has been reported i n frog skin (Biber and Curran, 1970; Rotunno et a l . , 1970; E r l i j and Smith, 1971; Moreno et a l . , 1973; Rick et a l . , 1975; Lindemann, 1977; Mandel, 1978) and i n proximal tubule (Spring and Giebisch, 1977). A l t e r n a t i v e l y , elevated i n t r a c e l l u l a r K a c t i v i t y during exposure to high external [K] may exert a negative feedback e f f e c t on a p i c a l P„. There i s evidence for such a feedback system regulating Na entry i n frog skin (MacRobbie and Ussing, 1961; Biber, 1971; E r l i j and Smith, 1973; Rick et a l . , 1975), toad bladder (Finn, 1975), rabbit bladder (Lewis et a l . , 1976) and rabbit colon (Turnheim et a l . , 1978; F r i z z e l l , 1979). I n h i b i t i o n of P^ by external [K] (seems more l i k e l y than negative feedback exerted by i n t r a c e l l u l a r K, since r a i s i n g external [K] b i l a t e r a l l y from 10 to 40 mM under I conditions produces only a small increase in i n t r a c e l l u l a r K 143 a c t i v i t y (^ 7 mM; F i g . 6 of chapter 4), whereas "P.. declines more than 70% under these conditions. Calculating "P., from J under I conditions should r e s u l t i n an over-K sm sc estimate of t r a n s e p i t h e l i a l potassium permeability (see equation 14 of Schultz and F r i z z e l l , 1976). This p r e d i c t i o n i s confirmed in chapter 5; however, i t w i l l also be shown i n chapter 5 that *P i s a reasonable indic a t o r of changes Is. i n t r a n s e p i t h e l i a l P.. during cAMP stimulation. Although *P„ i s dependent on membrane p o t e n t i a l s , the A*P„ observed at high [K] can not be accounted for by membrane depolarization. I n t r a c e l l u l a r p o t e n t i a l has been measured as a function of external [K] under I conditions (chapter 4). Using a p i c a l and basal membrane K permeabilities (estimated from cable a n a l y s i s ; chapter 5), the error which r e s u l t s from considering the epithelium as a s i n g l e b a r r i e r was calculated to be <40% (see Schultz and F r i z z e l l , 1976). In other words, 60% of the observed A*P„ must be due to a r e a l decline in membrane K K permeability. Also, an error i n *P would not explain whv cAMP stimulates K J 4-fold i n normal s a l i n e (10 mM K) but not i n high-K s a l i n e (140 mM K). sm b E l e c t r o p h y s i o l o g i c a l r e s u l t s described in chapter 5 w i l l confirm that p o t a s s i -um permeability i s influenced by cAMP and external [K] and that changes i n P occur mainly at the mucosal c e l l border. When electrogenic Cl transport and counter ion permeability (P„) are enhanced simultaneously in normal s a l i n e , l e s s work must be done by the C l pump per mole of C l absorbed (see chapter 2 for c a l c u l a t i o n s ) . What advantages might a r i s e from K-sensitive P 1 When the rectum contains unmodified K Malpighian tubule f l u i d , a very large concentration gradient (140:12 mM K) favours movement of t h i s cation from mucosal to serosal side. In s p i t e of t h i s , the lumen does not become e l e c t r i c a l l y negative to the serosal side, presumably due to the decline i n P„ as mentioned above. Maintenance of a mucosal-144 p o s i t i v e V would be advantageous i n that i t would minimize Na loss from the hemolymph (110 mM Na) into the lumen (40 mM) and hence the work of Na trans-port. Active Na transport i s low (20%) compared to C l absorption even when mucosal Na l e v e l s are high (90-200 mM; Williams et a l . , 1978; Spring and P h i l l i p s , 1980b). The maximum t r a n s e p i t h e l i a l electrochemical gradients for Na developed across the rectum of hydrated locusts _in vivo are also smaller than for C l or K ( P h i l l i p s , 1964b). A large net potassium f l u x from K-rich r e c t a l f l u i d would r e s u l t i n a flow of anions from mucosal-to-serosal side and/or a flow of cations (Na) i n the reverse d i r e c t i o n under open-circuit conditions. Conserving hemolymph Na i n t h i s way may be s i g n i f i c a n t for an insect which feeds on fresh plant matter which i s low i n Na (14 mM) versus K (114 mM ; for l e t t u c e ) . 42 P r i o r to stimulation by cAMP, open-circuit K fluxes deviated from the Ifesing f l u x r a t i o equation for simple d i f f u s i o n . Deviation from the predicted flux r a t i o s i n c o n t r o l r e c t a i s probably not the r e s u l t of active transport, since J was zero under I conditions, net sc Passive t r a n s e p i t h e l i a l K fluxes apparently do not occur independently across locust rectum although non-independence can not be l o c a l i z e d to a s p e c i f i c c e l l membrane since t r a n s e p i t h e l i a l K fluxes depend on steady-state tracer a c t i v i t y within the epithelium, which i s determined by K permeabilities 42 of both a p i c a l and basal membranes. If i n t r a c e l l u l a r K a c t i v i t y i s i d e n t i c a l to that of the serosal chamber, then the f l u x r a t i o exponent (n') w i l l be a measure of i n t e r a c t i o n s between K ions as they move through the mucos-42 a l membrane. Conversely, i f K l e v e l s are s i m i l a r i n mucosal and i n t r a c e l l u l a r compartments, n' w i l l be determined by the properties of the serosal membrane. According to the s i n g l e - f i l e d i f f u s i o n model with "knock-on" c o l l i s i o n s 145 (Hodgkin and Keynes, 1955; Hladky and Har r i s , 1967), n' i s the number of s i n g l e - f i l e s i t e s i n the membrane channel or one plus the number of s i t e s depending on whether a l l s i t e s are f i l l e d . The maximum value of n' places an upper l i m i t on the number of ions permitted within the channel ( H i l l e and Schwarz, 1978). In the case of 2 s e r i e s membranes, t r a n s e p i t h e l i a l values of n 1 w i l l be determined by mucosal, se r o s a l , or both membranes according to the r e l a t i v e permeabilities of musocal and serosal membranes. The present data suggest that either mucosal or serosal membrane may have a K f l u x r a t i o exponent i n d i c a t i v e of s i n g l e - f i l i n g through channels with at le a s t 5 s i t e s . Further studies of n' value i n t h i s tissue might involve measurement of steady-state tracer a c t i v i t y within the e p i t h e l i a l c e l l s , of the use of ionophores to greatly increase the permeability of one membrane so that the properties of the other might be studied with voltage clamping techniques. The f i r s t evidence for s i n g l e - f i l i n g i n e p i t h e l i a l K channels was obtained recently for the Ba-sensitive K channels i n the ba s o l a t e r a l membrane of t u r t l e bladder (Kirk and Dawson, 1981). Concentration-dependent K permeability has also been shown i n the p a r a c e l l u l a r pathway of rabbit colon (Fromm and Schultz, 1981). Properties of ac t i v e _K transport 42 A net f l u x of K from mucosal-to-serosal side of locust rectum was measured under I conditions during cAMP stimulation. The presence of a sc & F small active absorption of K across the r e c t a l epithelium i s consistant with e a r l i e r findings that K concentration i n the r e c t a l lumen i s maintained f a r from electrochemical equilibrium i n salt-depleted (hydrated) locusts ( P h i l l i p s , 1964b). In the present study, no net flux of K was observed u n t i l cAMP was added. This d i f f e r s from the r e s u l t s obtained by Williams et a l . (1978). They reported a small net K absorption by locust rectum using the blowfly 1 4 6 s a l i n e of Berridge (1966) and a voltage clamp which did not correct for series resistance. . . Two c h a r a c t e r i s t i c s of a c t i v e K absorption are apparent from this study. F i r s t , the mechanism for active K absorption must have a K of l e s s than K 10 mM K, because J ^ was s i m i l a r whether the bathing s a l i n e contained e i t h e r ' net 6 10 or 140 mM K. This low rate of a c t i v e K absorption i s probably responsible for reducing [K] i n the r e c t a l f l u i d to low l e v e l s i n vivo (<0.5 mM; P h i l l i p s , 1964). Second, when C l was omitted from the s a l i n e , there was no reduction i n 42 the active net K f l u x e l i c i t e d by cAMP. Whereas the major component of active C l transport i s K-dependent, a l l of the active K transport i s C l -independent. The l a t t e r finding strongly suggests that obligatory KCl cotransport entry i s not involved i n t r a n s e p i t h e l i a l K absorption and by implication K must enhance C l absorption by some other means. Further evidence against KCl cotransport was obtained using i o n - s e n s i t i v e microelec-trodes and i s reported i n the following chapter. 147 CHAPTER 4:; ELECTROCHEMICAL POTENTIALS Summary Double-barrelled l i q u i d ion-exchanger electrodes were used to measure c c c i n t r a c e l l u l a r K, C l and Na a c t i v i t i e s (a T r, a n 1 and a„T , respectively) and K C l Na membrane potentials i n locust r e c t a l epithelium. Steady-state net electrochem-i c a l gradients for C l and K across a p i c a l and basal membranes were calculated during exposure to 1 mM cAMP, and during ion substitutions under open-circuit conditions. Under co n t r o l conditions, a ^ (30.7 ± 1.1 mM) was 3.5 times higher than that predicted for passive equilibrium across the mucosal membrane. Serosal addition of 1 mM cAMP resulted i n i ) hyperpolarization of the mucosal membrane (V ), i i ) d e p o l a r i z a t i o n of the serosal membrane (V, ), and a b i i i ) a 50% increase i n a . The net electrochemical gradient (Ay /F) opposing C l entry from the mucosal side increased from -32.0 ± 1.2 mV (unstimu-lated control) to -49.8 ± 0.5 mV during cAMP stimulation, whereas the net gradient favouring C l e x i t across the basal membrane Ay^ /F did not change. When cAMP-stimulated recta were exposed to K-free conditions for 1 hour, a b Ay^/F declined to control l e v e l s while Ay^/F again was unchanged. Measure-ments of a^ and membrane pot e n t i a l s as a function of external [K] under s h o r t - c i r c u i t current conditions indicate that cAMP and low concentrations of K d i r e c t l y stimulate active C l entry across the a p i c a l membrane. The hyper-p o l a r i z a t i o n of V i s consistent with electrogenic Cl entry. No c o r r e l a t i o n 3. exists between Ay^/F and the net electrochemical gradient across the a p i c a l membrane for sodium (Ay^/F) , or between Ay^/F and t r a n s e p i t h e l i a l net f l u x 36 of C l measured under s h o r t - c i r c u i t (I ) conditions. The net electrochemical sc —Si gradient favouring K entry across the a p i c a l membrane (Ay /F) i s only 25% of K 148 Ay ../F at open-circuit, and i s n e g l i g i b l e under I conditions when C l trans-LiX S C -2 -1 port i s high (10 yEqcm h- ). In summary, net absorption involves an act i v e entry step at the a p i c a l membrane which i s stimulated by cAMP, and by low le v e l s of K on the mucosal side, but i s not energized by Ay /F or Ay v/F. 149 Introduction In the preceding chapter, t r a n s e p i t h e l i a l f l u x experiments revealed that a c t i v e chloride transport across locust rectum occurs by a mechanism which d i f f e r s from the well established Na- and HCO^-coupled systems found i n vertebrate e p i t h e l i a . T r a n s e p i t h e l i a l C l absorption i n locust rectum i s electrogenic, i s stimulated by a neuropeptide from the corpus cardiacum and by cAMP (Spring et a l . , 1978; Spring and P h i l l i p s , 1980a,b; P h i l l i p s et a l . , 1980; and chapter 2 of t h i s t h e s i s ) , and i s greatly enhanced by addition of K on the mucosal side (chapter 3). Furthermore, addition of 1 mM cAMP to the serosal side under I conditions r e s u l t s i n a very small net fl u x of K from mucosal to serosal sc J side and a large increase i n t r a n s e p i t h e l i a l K permeability. In t h i s chapter, the c e l l u l a r mechanism of C l absorption i s examined using double-barrelled i o n - s e n s i t i v e microelectrodes. S p e c i f i c a l l y , I attempt i ) to e s t a b l i s h whether the active step for t r a n s r e c t a l C l transport i s located at the mucosal or the serosal c e l l border, i i ) to determine whether K stimulates active Cl transport d i r e c t l y , or i n d i r e c t l y through changes i n membrane p o t e n t i a l , and i i i ) to examine whether active C l absorption might be driven by transmembrane electrochemical gradients f o r Na or K through coupled ion movements ( i . e . "secondary" a c t i v e transport). Answers to these questions are obtained by comparing previous tracer flux r e s u l t s i n chapters 2 and 3 with i o n - s e n s i t i v e microelectrode measurements made under i d e n t i c a l conditions. The r e s u l t s suggest that the entry mechanism for C l across the a p i c a l membrane i s active, electrogenic, stimulated r j i r e c t l y by cAMP and also by mucosal K, but i s not energized by Na or K net electrochemical gradients across the a p i c a l membrane. 150 The electrochemical p o t e n t i a l p r o f i l e s observed i n t h i s study and elec-t r o p h y s i o l o g i c a l experiments to be described i n chapter 5 allow estimation of membrane permeability properties and thus d r i v i n g force of active chloride transport i n locust rectum. Materials and methods The r e c t a l preparation Locust recta were dissected and mounted as f l a t sheets i n a p l e x i g l a s s perfusion chamber (shown i n F i g . 1) i n the same manner as previously describ-ed for tracer f l u x experiments (see chapter 2). The chamber design permitted independent perfusion of both sides of the tis s u e and allowed microelectrodes to be positioned on the mucosal side. The preparation was observed through a glass window at the end of the chamber, using a di s s e c t i n g microscope (Zeiss, Jena, GDR) at 25-100x magnification, and was illuminated front and rear by f i b e r optics (Intralux 150H, V o l p i AG, Urdorf, Switz.). Electrodes were advanced manually at an angle of 30°-40° to the plane of the epithelium using L e i t z micromanipulators (Wetzlar, F.R.G.). The tis s u e was continuously perfused by gravity-feed from well-gassed r e s e r v o i r s . The basic s a l i n e i s described i n chapter 2 (Table 1) and was modified as follows i n t h i s study: i ) N-methyl D glucamine was substituted f o r Na, and gluconate for C l , during microelectrode experiments because choline and methylsulfate are sensed by K and C l l i q u i d ion-exchangers, r e s p e c t i v e l y ; i i ) a l l salines were HCO^-free and were oxygenated with 100% 0 2 i n order to minimize HCO^ interference during i n t r a c e l l u l a r Cl measurements. Previous tracer flux measurements showed that Cl transport and t r a n s e p i t h e l i a l e l e c t r i c a l properties are not affected by removal of a l l external C0 2 and HC0 q (chapter 2). 151 Figure 4.1 Cross section of the chamber used i n microelectrode experi-ments. A, locust rectum with c u t i c l e removed; B, "0"-ring; C, pin; D, i n l e t for p o t e n t i a l sensing agar bridge; E, suction o u t l e t ; F, current-passing s i l v e r f o i l electrode; G, support stand; H, f i b e r optic l i g h t ; I, perfusate i n l e t ; J , glass window; K, d i s s e c t i n g microscope; L, double-barrelled microelectrode. 152 E l e c t r i c a l measurements T r a n s e p i t h e l i a l p o t e n t i a l (V ) was measured as i n chapter 2, with the exception that calomel h a l f - c e l l s were replaced with Ag/AgCl wires. The p o t e n t i a l difference between reference and i o n - s e n s i t i v e b a r r e l s of the double-barrelled microelectrode ( v^) was measured using a d i f f e r e n t i a l e l ec-trometer with very high input impedance (^10^ ft; FD223, WP Instruments, New Haven, Conn.). The p o t e n t i a l d i f f e r e n c e between the reference b a r r e l within the c e l l and external mucosal and serosal agar bridges (V and V,, Si D respectively) were measured i n d i v i d u a l l y using high input impedance operation-12 a l a m p l i f i e r s at unity gain (10 ft; 4253, Teledyne P h i l b r i c k , Dedham, MA). T r a n s e p i t h e l i a l resistance ( R t) was monitored as the de f l e c t i o n s i n V produced by t r a n s e p i t h e l i a l current pulses to i n d i c a t e possible ti s s u e damage during mounting and also ti s s u e v i a b i l i t y . The r a t i o of voltage d e f l e c t i o n s across a p i c a l and basal membranes r e s u l t i n g from t r a n s e p i t h e l i a l current pulses ( i . e . voltage-divider r a t i o "a") was measured during measurements under open-circuit conditions as a test f o r possible impalement damage. Constant current pulses (^ 1 sec duration, 0.3 Hz) were supplied by waveform/pulse generators (Type 160 Series, Tektronix, Beaverton, Ore.). Current was measured as the voltage drop across a series r e s i s t o r . A f t e r f i l t e r i n g (3 db at 5 Hz), V , V , V, , V. and T were recorded & ' t ' a b ' i t simultaneously using a 6-channel pen recorder (Brush 260, Gould Inc., St. Louis, Mo.) or, when fewer parameters were required, on a 2-channel recorder (7402A, Hewlett-Packard, San Diego, C a l i f . ) . Signals were also monitored using a storage o s c i l l o s c o p e (D15, Tektronix) and V was always displayed d i g i t a l l y (616, Keithley Instr. Inc., Cleveland, OH). 153 F a b r i c a t i o n and c a l i b r a t i o n of i o n - s e n s i t i v e microelectrodes The techniques used-for constructing double-barrelled microelectrodes were s i m i l a r to those of Fujimoto and Kubota (1976). C a p i l l a r y glass (1.0 mm O.D., Frederick Haer and Co., Brunswick, Maine) was cleaned for at least 2 h i n cone. HNO^, thoroughly rinsed with d i s t i l l e d water, dried i n an oven, and stored dust-free at 0% r e l a t i v e humidity. Two glass c a p i l l a r i e s were heated i n a v e r t i c a l p u l l e r (PE 2, Narishige S c i e n t i f i c Instr. Lab., Tokyo), rotated one-half turn, and drawn to a f i n a l t i p diameter of less than 1 ym. A f t e r b a c k - f i l l i n g the reference b a r r e l with acetone (ACS, Eastman Kodak Co., Rochester, N.Y.) electrodes were dipped into a 0.1% s o l u t i o n v/v of Dow Corning 1107 s i l i c o n e o i l i n acetone (1107 was a generous g i f t of D. Jenkins, Dow Corning, Vancouver) for approximately 10 sec i n order to s i l a n i z e the i o n - s e n s i t i v e b a r r e l . Electrodes were then cured on a hot plate at ^300°C for 15 min. After the electrode shaft was reinforced with f a s t -drying epoxy, a 2-4 mm column of l i q u i d - i o n exchange r e s i n was injected into the s i l a n i z e d b a r r e l from a syringe through f i n e polyethylene tubing and coaxed to the t i p using a cat whisker. When r e s i n reached the t i p , the electrode was b a c k - f i l l e d with one of the solutions l i s t e d i n Table 1. 9 The resistance of the i o n - s e n s i t i v e electrodes ranged between 2 x 10 and 5 x 1 0 1 0 Q. Electrodes were ca l i b r a t e d frequently during the course of experiments i n e l e c t r o l y t e solutions encompassing the e n t i r e range of i n t r a - and e x t r a c e l l u l a r ion a c t i v i t i e s . In most experiments 5, 50, 120, 500 mM KCl (cone.) solutions were used for c a l i b r a t i n g K and C l electrodes except during perfusion with nomin-a l l y K- or C l - f r e e s a l i n e s , when 1 mM KCl was also included i n the c a l i b r a t i o n s e r i e s . Sodium electrodes were c a l i b r a t e d i n pure solutions containing 1, 10, 100, 120 mM NaCl and mixed solutions containing 10 mM NaCl and 110 mM KCl. Ion 154 Table 4.1 L i q u i d ion exchangers and solutions used i n f a b r i c a t i n g double-barrelled i o n - s e n s i t i v e microelectrodes. Ion s e n s i t i v e b a r r e l Electrode Resin Backing Reference B a r r e l e l e c t r o l y t e K Corning 477317 0.5M KCl 1.0M Na acetate Corning Med. Prod., - Medfield Mass. Cl Orion 92-17102 0.5M KCl 1.0M Na acetate Orion Res. Na Monensin ~10% w/w 0.49M NaCl 0.5M KCl i n Corning 477317 at pH=3.0 (see Kotera et a l . Membr. Biochem. (0.1M c i t r a t e vol.2:323-338,1979) buffer) 155 a c t i v i t i e s i n c a l i b r a t i n g solutions were calculated using the modified Debye-Huckel equation (Robinson and Stokes, 1970). Plots of electrode electromotive force (mV) against the logarithm of 2 — the ion a c t i v i t y (mM) yielded the following slopes (s) and r values (x ± s.e.): C l , s = 57.77 ± 0.87 mV, r 2 = 0.9950 ± 0.0013, 29 electrodes; K, s = 52.44 ± 0.62 mV, r 2 = 0.9961 ± 0.0008, 26 electrodes; Na, s = 54.08 ± 1.4 mV, r 2 = 0.9988 ± 0.0007, 11 electrodes. These values compare well with those obtained i n previous studies (Walker, 1971; Fujimoto and Kubota, 1976; Spring and Kimura, 1978; Reuss and Weinman, 1979; Duffey et a l . , 1979; Fujimoto et a l . , 1980; and Garcia-Diaz and Armstrong, 1980). Response time of the i o n - s e n s i t i v e b a r r e l was usually about 1-3 seconds except for Na-se n s i t i v e electrodes which occasionally required 15-20 seconds to reach a stable value. S e l e c t i v i t y of C l electrodes over gluconate using the separate s o l u t i o n method of Moody and Thomas (1971) gave values f o r gluconate-to-Cl s e l e c t i v i t y ) of 0.039 ± 0.006 and K 0 1 of 0.1324 ± 0.0215. The anion Cl,gluconate C1,S0^ interference observed during perfusion with nominally C l - f r e e s a l i n e ( i . e . "apparent" C l a c t i v i t y a f t e r gluconate + s u l f a t e substitution) was 5.2 mM, as predicted using the microelectrode s e l e c t i v i t y c o e f f i c i e n t s . Perfusion for 3 h with C l - f r e e s a l i n e resulted i n apparent a = 4.84 ± 0.38. Under this unnatural condition, the c e l l s may contain some r e s i d u a l C l , replacement ions ( i . e . gluconate, SO^) i n addition to the -interfering anions which are normally present. The v a l i d i t y of subtracting "apparent a ^ " observed i n nominally C l - f r e e s a l i n e i s debatable when the i d e n t i t y of the i n t r a c e l l u l a r anion i s not known under normal conditions. An i d e n t i c a l "apparent C l a c t i v i t y " has recently been reported i n heart muscle a f t e r prolonged exposure to methylsulfonate s a l i n e in the absence of HCO^ (4.8 ± 0.6 mM, Baumgarten and Fozzard, 1981). Measurement of r e s i d u a l i n t r a c e l l u l a r Cl was not attempted since no method having the required s e n s i t i v i t y was a v a i l a b l e ( t o t a l non-mannitol space of the tissue i s 3.61 ± 0.24 y l ) . I t i s u n l i k e l y that i n t r a c e l l u l a r HCO^ ( K „ T . = 0.2-0.09) accounts for a l l of the "apparent C l a c t i v i t y " since salines were HCO^-free and vigorously s t i r r e d with 100% The values reported i n t h i s thesis are not "corrected" for possible anion interference. Regardless, since a ^ normally ranged between 30-60 mM, such interference would not be s u f f i c i e n t to change any of the conclusions i n this study. It i s also u n l i k e l y that Na interference was serious during i n t r a c e l l u -l a r K measurements since 1) electrodes showed high s e l e c t i v i t y ( K ^ ^ <0.02) and measured i n t r a c e l l u l a r Na a c t i v i t i e s were low (a„ = 8.0 ± 0.41 mM, Na 125 c e l l s i n 11 recta under I conditions), and 2) i n t r a c e l l u l a r K was , sc reduced to approximately 1 mM a f t e r prolonged perfusion with K - f r e e s a l i n e (time course shown i n F i g . 5). Results obtained with Na-sensitive micro-electrodes required corrections for K interference. S e l e c t i v i t y for Na over K i n eleven electrodes was 11.2 ± 1.1. Double-barrelled microelectrodes were c used to measure a^ immediately before or a f t e r Na measurements on the same tissu e under each experimental condition. Successful impalements were characterized by i ) abrupt monotonic d e f l e c -tions in voltage, i i ) stable i n t r a c e l l u l a r p o t e n t i a l which remained within ± 1 mV, i i i ) constant voltage d i v i d e r r a t i o s , and iv) return to the o r i g i n a l baseline p o t e n t i a l s upon r e t r a c t i o n of the electrodes. No evidence of impale-ment damage was obtained using double-barrelled as compared to s i n g l e -b a r r e l l e d microelectrodes, probably due to the large s i z e of the columnar e p i t h e l i a l c e l l s (^ -17 x 90 ym) and extensive i n f o l d i n g of the c e l l membrane which, judging from electron micrographs, r e s u l t s in a 9- to 200-fold increase i n membrane area i n various regions of the c e l l . 157 Mannitol space Since changes i n ion' a c t i v i t y during cAMP stimulation might r e s u l t from a l t e r a t i o n s i n c e l l volume rather than i o n i c fluxes, mannitol space of recta was measured i n normal s a l i n e , with or without 1 mM cAMP. Tissues were 3 placed i n small v i a l s of vigorously oxygenated s a l i n e containing H-mannitol. After 1 h of incubation, three 1 u l samples of s a l i n e were counted by l i q u i d s c i n t i l l a t i o n to estimate external H a c t i v i t y . Tissues were blotted dry on bibulous paper and wet weight was determined to within ± 0.1 mg. After drying to constant weight i n a desiccating oven at 60°C, tissues were digest-ed i n 1 N KOH at 60°C overnight, neutralized with cone. H^SO^, and counted as described i n chapter 2. The difference between wet and dry weights was used as an estimate of t o t a l t i s s u e water, and i n t r a c e l l u l a r volume was calculated as the differe n c e between t o t a l tissue water and mannitol space, assuming that mannitol d i s t r i b u t e s homogeneously throughout the e x t r a c e l l u l a r space, does not enter the c e l l s , and does not adsorb to the tissue surface. Similar estimates of mannitol were obtained a f t e r 1 h or 1.5 h, suggesting that 1 h 3 was adequate for mannitol d i s t r i b u t i o n to reach a steady-state. H-mannitol • 14 space could not be checked using C - i n u l i n because the c u t i c u l a r intima i s v i r t u a l l y impermeable to t h i s polysaccharide ( P h i l l i p s and D o c k e r i l l , 1968). Mannitol i s not metabolised by locust rectum. Ca l c u l a t i o n and s t a t i s t i c s Voltage divider r a t i o s , transmembrane e l e c t r i c a l p o t e n t i a l d i f f e r e n c e s , and equilibrium ion a c t i v i t i e s were calculated with corrections for saline,, resistance and i o n i c a c t i v i t i e s by computer (PDP-11). C a l i b r a t i o n curves f o r ion - s e n s i t i v e microelectrodes were determined by l i n e a r regression analysis. Net electrochemical gradients were calculated as: i 158 p -rn q Ay./F = RT(ln aT - In a.' )/F + zV , x x x a,b c * m s where a. i s the a c t i v i t y of ion " i " i n the c e l l , a.' i s the a c t i v i t y of ion 1 J x " i " i n mucosal or serosal s o l u t i o n , V and V, are a p i c a l or basal membrane a b v p o t e n t i a l s , r e s p e c t i v e l y , and z, F, R and T have the i r usual meanings. " U p h i l l " net electrochemical gradients are given as negative values;favourable gradients are p o s i t i v e i n r e l a t i o n to the d i r e c t i o n of net transport of the ion i n question. S t a t i s t i c a l comparisons were made using standard paired or unpaired t - t e s t s . Results 1) Steady-state measurements of i n t r a c e l l u l a r chloride  and potassium a c t i v i t i e s Figure 2 shows o r i g i n a l traces of V t, V , and i n t r a c e l l u l a r ion a c t i v -i t y obtained during impalements with double-barrelled K- and C l - s e n s i t i v e microelectrodes. Values were usually stable and showed l i t t l e v a r i a b i l i t y between d i f f e r e n t c e l l s i n any p a r t i c u l a r t i s s u e . A p i c a l , basal and trans-e p i t h e l i a l p o t e n t i a l changes due to applied current pulses are also shown. After correcting for series resistance, the d e f l e c t i o n s i n V & and V^ were used to c a l c u l a t e the voltage d i v i d e r r a t i o (R / R , or "a"), and served as an a D i n d i c a t o r of impalement damage. Values of a w i l l be discussed i n more d e t a i l i n chapter 5, which deals with membrane permeability changes. Transient d e f l e c t i o n s i n the Cl a c t i v i t y trace are a r t i f a c t s r e s u l t i n g from the d i f f e r e n t e l e c t r i c a l time constants of i o n - s e n s i t i v e and reference b a r r e l s . K-sensitive microelectrodes usually had resistances that were one order of magnitude lower than did Na or C l electrodes, and generally did not show these transient d e f l e c t i o n s . 159 C l V m r r Innrr 1 min K V + ._ o + - 4 - o 20 mV 40 mV (II i ii I! UUUUlUU o--innnr hnnnf Innnr uu i l l IJUul, l i l t j JUUUL ULH 1 1 1 1 40 mV 40 mV Sill -li Jul ULflJi U U 0 — n 20 MA 1 M U Figure 4.2 Representative traces obtained using double-barrelled i o n -s e n s i t i v e microelectrodes. Simultaneous recording of t r a n s e p i t h e l i a l poten-t i a l (V ), a p i c a l membrane p o t e n t i a l (V ), basal membrane p o t e n t i a l (V, : t a b also shows V when the electrode i s withdrawn into the mucosal half-chamber), p o t e n t i a l difference between reference and ion - s e n s i t i v e barrels of double-b a r r e l l e d i o n - s e n s i t i v e microelectrodes (V^) under open-circuit conditions i n normal saline (Table 2.1). T r a n s e p i t h e l i a l current pulses are shown at bottom (I ) 160 V was monitored continuously a f t e r mounting tissues i n the perfusion chamber. As i n previous"open-circuit f l u x experiments (Figures and chapters 1 and 2, r e s p e c t i v e l y ) , V was i n i t i a l l y 25-40 mV mucosal-side p o s i t i v e , but decayed to approximately 8 mV a f t e r 2-3 hours. Control i n t r a c e l l u l a r measurements were made when V had declined to t h i s l e v e l (Table 2). Approximately 30 minutes was required for measured parameters across recta to reach new steady-state following addition of 1 mM cAMP or external K (indicated by t r a n s e p i t h e l i a l resistance and p o t e n t i a l , F i g . 3). Therefore, steady-state i n t r a c e l l u l a r measurements were made following 30-60 min e q u i l i b r a t i o n under each new condition. Figure 4 shows histograms c c of a ^ and a R i n control preparations (unstimulated, 10 mM K), and during exposure to 1 mM cAMP. Both C l and K a c t i v i t i e s appear normally d i s t r i b u t e d about their mean values, suggesting that a s i n g l e c e l l population was sampled or that there i s e f f e c t i v e c e l l - c e l l coupling i f d i f f e r e n t populations do exist (see chapter 5). a) Chloride Table 2 summarizes r e s u l t s obtained i n one series of experiments using double-barrelled C l - s e n s i t i v e microelectrodes. Under co n t r o l (open-circuit) conditions, a ^ was 30.7 ± 1.1 mM, approximately 3.5-fold higher than the value predicted for passive d i s t r i b u t i o n across the.apical membrane (8-9 mM). 3 6 As shown previously (chapter 2), there i s a net f l u x of C l from mucosa to serosa under these conditions; therefore an active step or "pump" must be postulated at the a p i c a l membrane while exi of C l across the basal membrane may occur passively down a favourable electrochemical gradient (Apj^). The e f f e c t s of cAMP exposure provide further evidence for an a p i c a l C l pump. It was shown previously (chapters 2 and 3) that the mucosal side becomes ^20 mV more p o s i t i v e with respect to the serosal side and net C l 161 % -20 >°-40 -60 0 i jtrnimimnrr r WiiiniumuiuiiTOiiTiiiiiiiiniiiiniiintrnininniiiinininiiinn £ 80 E 60 > 40 mmmm r 2 0 r ? " - 4 0 ~q~ fc 8 0 | 5 min Figure 4.3 Time course of the e f f e c t s of cAMP on t r a n s e p i t h e l i a l poten-t i a l (V ), a p i c a l membrane p o t e n t i a l (V ) and C l - s e n s i t i v e p o t e n t i a l (V., t a i d i f f e r e n t i a l ) . See legend of Figure 4.2 for d e f i n i t i o n s . Deflections i n V 2 and Vfl are due to t r a n s e p i t h e l i a l current pulses (20 uA/0.1962 cm ), and ind i c a t e t r a n s e p i t h e l i a l resistance. The regular d e f l e c t i o n s i n are a r t i f a c t s which r e s u l t from the large d i f f e r e n c e between time constants of io n - s e n s i t i v e and reference b a r r e l s . As indicated bv V and V., the electrode a i ' became dislodged a f t e r 8 min (at *) and a second c e l l was impaled. cu _ Q E z 3 0 H 2 5 2 0 15 10 5 J. "5 0 2 0 - , 15 1 10 H 5 J K Normal saline K + c A M P T 2 0 4 0 6 0 8 0 I n t r a c e l l u l a r i o n a c t i v i t y C m M ) 1 0 0 Figure 4.4 D i s t r i b u t i o n of i n t r a c e l l u l a r Cl and K a c t i v i t i e s i n r e c t a l pad e p i t h e l i a l c e l l s under control conditions, and a f t e r sequential exposure to 1 mM cAMP, and to K-free saline with cAMP present. Tissues were i n open-c i r c u i t e d state. Chloride and potassium "leasuroraents were made on rec t a from d i f f e r e n t animals (n = 6-10). 163 Table 4.2 E f f e c t s of adding cAMP and removing external K on e l e c t r i c a l p o t e n t i a l s , i n t r a c e l l u l a r C l a c t i v i t y , and calculated C l electrochemical gradients. Sequential Condition V t V a V b a C l Ay^/F Ay^/F (mV) (mV) (mV) (mM) (mV) (mV) Control +8.6 -57.0 -48.7 30.7 -32.0 +23.3 (6;40) ±0.3 ±0.8 ±0.9 ±1.1 ±1.2 ±1.4 + ImM cAMP +29.8 -63.8 -34.0 46.6 -49.8 + 20.1 (6;42) ±0.5 ±0.6 ±0.6 ±0.8 ±0.5 ±0.6 + cAMP, +4.8 K-free, ±0.4 a f t e r 1 hour (6;42) -79.6 -74.8 11.8 -23.8 +23.5 ±4.8 ±5.0 ±0.9 ±4.2 ±4.4 V t r a n s e p i t h e l i a l p o t e n t i a l ; V a p i c a l membrane p o t e n t i a l ; t, a, c _ a V, basal membrane p o t e n t i a l ; a p, i n t r a c e l l u l a r Cl a c t i v i t y ; A y ,/F, o, L.X C l Ay^/F, C l net electrochemical gradients calculated for a p i c a l and basal membranes. Sign convention: V mucosal side r e l a t i v e to s e r o s a l ; V and V, i n t r a c e l l u l a r p o t e n t i a l r e l a t i v e to mucosal . a b, a b and s e r o s a l side, r e s p e c t i v e l y ; A y^/F and Ay^/F, a p o s i t i v e sign indicates gradients favouring passive net C l movement i n the mucosal-to-serosal d i r e c t i o n , negative sign indicates u p h i l l gradient. Means ± s.e., (number of re c t a ; number of c e l l s ) f o r i n t r a c e l l u l a r measurements; (number of r e c t a ; number of obser-vations) for measurements o f V f 164 C l absorption (J ) increases 9-fold when 1 mM cAMP i s added to the sero s a l side net under open-circuit conditions. Similar changes i n t r a n s e p i t h e l i a l p o t e n t i a l were observed i n the present microelectrode experiments (Table 2). Serosal addition of 1 mM cAMP r e s u l t s i n both hyperpolarization of V by 3. ^6 mV, and a 50% elevation of a ^ . If a cAMP-stimulated C l pump were located at the serosal c e l l border, one would expect a ^ to either decrease or remain constant during cAMP exposure: an increase i n a ^ i s not consistent with a pump at the basal membrane. When combined with the e a r l i e r f i n d i n g that Cl steady-state J i s elevated 9-fold under these conditions (chapter 2), these net data i n d i c a t e that a cAMP-stimulated Cl pump i s located at the a p i c a l membrane. b F i n a l l y i t i s i n t e r e s t i n g that A u ^ did not change s i g n i f i c a n t l y follow-ing cAMP addition. This suggests that conductance of the basal membrane may also be stimulated by cAMP i f C l exits by e l e c t r o d i f f u s i o n , since the net electrochemical gradient for C l across the serosal c e l l border i s not changed despite a 9-fold increase i n net C l flux through the membrane. This predic-t i o n i s confirmed i n chapter 5 using f l a t sheet cable a n a l y s i s . I t w i l l be shown that the increase i n basal membrane conductance i s larger than i s needed to account for the observed flux of C l by e l e c t r o d i f f u s i o n . No ele c t r o n e u t r a l exchange or cotransport mechanisms need be postulated at the Cl e x i t step. Following K-removal from both sides, and both hyperpolarized while V increased temporarily from 29.8 ± 0.5 mV to ^ 45 mV and then declined to Cl 4.8 ± 0.4 mV, consistent with the reduced J shown previously ( F i g . 15 of ' net 1 3 6 chapter 2). Membrane po t e n t i a l s immediately hyperpolarized to -130 mV (V ) 3. and -85 mV (V^) and then declined exponentially to ^ -80 mV a f t e r 1 hour. Most importantly, Ap^/F decreased i n K-free s a l i n e from -49.8 ± 0.5 to -28.3 ± 4.2 mV, i n d i c a t i n g that K d i r e c t l y stimulated Cl entry. Note 165 TC1 Clthat i f potassium-stimulated J resulted from K " s h o r t - c i r c u i t i n g " V net 6 a (thereby reducing the work required for C l entry), then the u p h i l l step for C l entry would have increased following K removal. F i n a l l y , i t i s worth pointing out that the net electrochemical gradient for C l across the a p i c a l membrane i s at l e a s t p a r t l y determined by processes at the serosal c e l l border. For example, both enhancing C l entry and blocking _a Cl e x i t would both increase Ap^. However a decrease i n C l e x i t would also increase the favourable C l electrochemical gradient for e x i t across the basal membrane. Contrary to t h i s p r e d i c t i o n , perfusion with K-free s a l i n e has no e f f e c t on the C l electrochemical gradient across the serosal c e l l border (Table 2). Taken together, these r e s u l t s i n d i c a t e that C l i s pumped into the e p i t h e l -i a l c e l l s across the a p i c a l membrane by an active mechanism which i s stimulated by both cAMP and potassium ions. b) Potassium Table 3 shows r e s u l t s obtained using double-barrelled K-sensitive micro-electrodes during experiments s i m i l a r to those j u s t described for C l : control unstimulated, and cAMP stimulated, i n the presence and then absence of external K for 1 hour. Values of V . V , and V, were s i m i l a r to those obtain-t a' b ed with C l - s e n s i t i v e microelectrodes (compare Tables 2 and 3). The mean i n t r a c e l l u l a r K a c t i v i t y (a^) was s l i g h t l y below that predicted for equilibrium across the a p i c a l membrane; however,this was not s i g n i f i c a n t (P < 0.05). In order for V to be a t t r i b u t e d s o l e l y to a K d i f f u s i o n p o t e n t i a l , a would have a K to be above equilibrium across the a p i c a l membrane. Therefore some a d d i t i o n a l electromotive force must be proposed at the a p i c a l membrane (such as an inwards-directed, electrogenic C l pump) i n order to explain V^. 166 Table 4.3 E f f e c t s of adding cAMP and removing external K on e l e c t r i c a l p o t e n t i a l s , i n t r a c e l l u l a r K a c t i v i t y and calculated K electrochemical gradients Condition V t V a V b 4. A yK/F A yK/F (mV) (mV) (mV) (mM) (mV) (mV) Control +7.2 -57.8 -50.7 61.4 +3.3 +3.9 [10;97] ±0.3 ±0.5 ±0.3 ±0.7 ±0.5 ±0.3 + lmM cAMP +32.2 -70.2 38.0 70.3 +12.4 +19.8 [9;8l] ±0.8 ±0.6 ±1.0 ±1.1 ±0.8 ±1.2 + cAMP +9.8 -84.1 -74.3 13.9 <-55.2 >+65.0 K-free, ±0.7 ±3.4 ±3.3 ±1.2 ±2.0 ±1.9 af t e r 1 hour [5;58] See footnote to Table 4.2 for explanations 167 Small electrochemical gradients (<5 mV) favouring K net absorption from mucosa to serosa were observed across mucosal Ay^/F and serosal Ay^/F membrane under control conditions. This f i n d i n g i s consistent with passive t r a n s e p i t h e l i a l K absorption, but alone does not exclude the presence of an 42 ad d i t i o n a l a c t i v e f l u x . In chapter 3 evidence was presented that K movements are l a r g e l y passive i n the absence of cAMP: i . e . there was no J net i n unstimulated recta under s h o r t - c i r c u i t conditions ( F i g . 13 of chapter 3). Serosal addition of cAMP dramatically increased both Ay^ and Ay^, mostly K K through changes i n membrane p o t e n t i a l s . The a c t i v e step i n K transport i s d i f f i c u l t to l o c a l i z e under these conditions. _a _b It may: be noted, that Ay /F increased s i g n i f i c a n t l y less than AyT./F during cAMP exposure (P < 0.05), however t h i s may simply r e f l e c t a higher K conductance at the a p i c a l membrane. Regardless, under open-circuit conditions, active K transport i s very small compared to the net passive f l u x which i s e l e c t r i c a l l y coupled to ac t i v e C l absorption (<20%, chapter 3). Locust r e c t a l c e l l s contained some potassium af t e r perfusion with K-free s a l i n e for 1 hour (Table 3): a continues to decline for several hours (see F i g . 5). Although K the conditions are not s t r i c t l y steady-state with respect to a a f t e r 1 h, this does not influence my conclusions regarding the mode of action of K on chloride transport, because these are based on C l net fluxes and net e l e c t r o -chemical gradients when t r a n s e p i t h e l i a l C l transport i s approximately stready-state as judged by V^and Moreover, i n the next section, very s i m i l a r r e s u l t s are reported f o r experiments which begin a f t e r recta were more nearly depleted of i n t r a c e l l u l a r K (<5 mM). 2) Relationship between chloride and potassium electrochemical potentials Chloride-dependent I increases dramatically when K i s added stepwise to both sides of K-depleted tissues ( F i g . 8 of chapter 3). From the t r a n s e p i t h e l -168 T i m e C h ) Figure 4.5 Time course of the decline i n i n t r a c e l l u l a r K a c t i v i t y (a£) and K V during b i l a t e r a l perfusion with K-free s a l i n e under open-circuit conditions. cL Each point i s the mean ± s.e. of 6 measurements using a double-barrelled microelectrode in one tissue. Compare with a = 61.4 ± 0./ under co n t r o l K. conditions. 169 i a l measurements above, i t i s not possible to determine whether this i s due to i n d i r e c t e l e c t r i c a l e f f e c t s ( i . e . changes i n membrane p o t e n t i a l s ) , to d i r e c t stimulation of a C l pump, or to both. Direct stimulation of the Cl pump i s indicated i n the preceding section because removal of 10 mM external K under open-circuit conditions reduces the net electrochemical gradient opposing C l entry. Nevertheless, increasing the K a c t i v i t y on the mucosal side should depolarize the a p i c a l membrane and reduce the work required for Cl entry. I attempted to evaluate t h i s e f f e c t . Also, i t i s of some i n t e r e s t to compare a p i c a l and basal membrane Ay /F and Ay .. /F under I conditions. Such a JN. U J_ s c comparison would ind i c a t e whether C l movements could be energized by coupled movements with K ( i . e . cotransport). Figure 6 i l l u s t r a t e s i n t r a c e l l u l a r potassium and chloride a c t i v i t i e s and p o t e n t i a l differences i n one r e c t a l preparation (means ± s.e.; = under I conditions; i o n i c a c t i v i t i e s and electrochemical gradients, n = 9-10; i n t r a c e l l u l a r p o t e n t i a l s , n = 18-20). Table 4 summarizes values from 5 prepara-tio n s . Data were obtained sequentially under the following conditions: unstimulated (K-free s a l i n e ) , cAMP-stimulated (K-free s a l i n e ) , cAMP-stimulated (with stepwise increases i n K concentration on both sides; [K] = 0, 2, 4, 10, 40, 100 and 140 mM). Measurements were made at least 0.5 h a f t e r exposure to cAMP and a f t e r each change i n [K] ( i . e . steady-state conditions were approxi-mated during measurements). Also shown are the K and C l net electrochemical gradients during these experiments, calculated from the data i n the upper panels. Several points should be noted: i) Prolonged exposure (>2.5 h) to K-free s a l i n e resulted in reduction of a^ to <5 mM and mucosal and serosal membrane depolarization to 23 mV (compare with F i g . 5 ) . 170 Figure 4.6 E f f e c t s of 1 mM cAMP and b i l a t e r a l K additions on i n t r a c e l l u -l a r p o t e n t i a l , i n t r a c e l l u l a r a c t i v i t i e s of K and C l , and calculated - a -a electrochemical potentials for K (Ay /F) and C l (Ay ../F) across the a p i c a l K Cl membrane under I conditions. Cyclic-AMP was added at the arrows under sc J K-free conditions. Note that i ) i n t r a c e l l u l a r p o t e n t i a l changes about 50 mV/ decade change i n external K a c t i v i t y , i i ) a i n K-depleted tissue increases from 3 mM to near control l e v e l s (65 mM) when recta are re-exposed to 2 mM K, c _a i i i ) a , decreases during hyperpolarization of V , , iv) Ay /F i s approximate-Cl a,b K l y 0 mV at a l l external K a c t i v i t i e s i n d i c a t i n g that l i t t l e energy i s _a a v a i l a b l e i n the K gradient for KCl coentry; Ay^/F becomes more negative when 36 external K i s increased. When combined with C l f l u x and I measurements sc under comparable conditions (chapters 2 and 3), t h i s l a t t e r observation suggests a d i r e c t stimulation by K of active C l entry. Results shown are from c c one preparation. Means ± s.e.; n = 20 obs. (V , ), n = 10 obs. (a , a , a,D K LI Ay^/F, Ayf, /F). See Table 4.4 for complete data. K L . i 1 7 1 1 m M c A M P | i 1 r — — i 1 1 1 1 j — 0 1 2 4 6 8 10 2 0 4 0 6 0 8 0 Bi l a t e r a I K a c t i v i t y ( m M ) 172 Table 4.4 E f f e c t s of sequential cAMP addition and increased [K] b i l a t e r a l l y on i n t r a c e l l u l a r p o t e n t i a l , ion a c t i v i t i e s and net electrochemical gradients measured with double-barrelled micro-electrodes under I conditions sc Condition ' (mV) (mM) (mM) (mV) (mV) OmM K -40.1 37. 8 1.55 -18. 11 -31. 89 (30-60;3) ±0.4 ±3. 1 ±0.1 ±2. 0 ±3. 3 + ImM cAMP -54.7 50. 0 1.2 -40. 6 -18. 2 (40-90;5) ±2.5 ±2. 8 ±0.1 ±1. 8 ±2. 8 2mM K -89.5 20. 8 52.0 -55. 3 -5. 4 (30-70;4) ±1.3 ±0. 7 ±1.3 ±6. 2 ±1. 2 4mM K -76.2 30. 7 59.1 -48. 3 -0. 9 (38-87;5) ±0.5 1. 1 2.0 ±1. 5 ±0. 6 lOmM K -57.5 40. 3 74.8 -39. 5 -0. 1 (26-66;4) 0.4 0. 9 1.2 ±0. 9 ±0. 6 40mM K -29.8 58. 5 90.6 -19. 2 +1. 5 (30-70;4) ±0.53 ±1. 2 ±1.6 ±1. 3 0. 6 lOOmM K -10.6 74. 4 101.3 -7. 9 +0. 9 (30-70;4) ±0.4 ±1. 2 ±2.3 ±1. 1 ±0. 2 14 OmM K -10.2 84. 2 117.8 -3. 62 +0. 4 (34-64;4) ±0.6 ±2.' 7 ±1.1 ±1. 3 ±0. 4 Means ± s.e., (number of observations! number of ti s s u e s ) . See legend of F i g . 4.6 and text for further explanation. 173 i i ) Addition of 1 mM cAMP to the serosal side of K-depleted tissues increased a ^ by 12 mM, hyperpolarized the mucosal and serosal membranes by 15 mV (both changes s i g n i f i c a n t at P < 0.05), and elevated the net e l e c t r o -chemical gradient opposing C l entry across the mucosal membrane by 12 mV. Each of these observations i s consistent with an a p i c a l electrogenic C l pump and with the previous observation that some C l transport i s stimulated by cAMP i n K-free saline (v35%; F i g . 15 of chapter 2). i i i ) Addition of K (2 or 4 mM) to both sides caused recovery of a to K c o n t r o l l e v e l s and membrane r e p o l a r i z a t i o n within 0.5 h (see F i g . 6, K a c t i v i t i e s of 1.44 and 2.89 mM). Under these I conditions, both V and V, sc ' a b were v i r t u a l l y i d e n t i c a l to E , the equilibrium p o t e n t i a l for K. This i s not K s u r p r i s i n g since the top panel shows that when sa l i n e [K] i s increased, V and Si vary approximately 50 mV per decade change i n external K a c t i v i t y , i n -di c a t i n g extremely high s e l e c t i v i t y of both c e l l borders to K (note that c e l l borders are e s s e n t i a l l y studied i n p a r a l l e l under these I conditions). iv) Potassium (2-4 mM cone.) on both sides of the epithelium increased the electrochemical gradient opposing C l entry (Au^/F) , as shown previously by removal of 10 mM K from both sides (Table 2). As explained e a r l i e r , this p a r t i c u l a r r e s u l t cannot be explained as a simple " s h o r t - c i r c u i t i n g " of the a p i c a l membrane p o t e n t i a l by external K; thus a more d i r e c t action of K on Cl entry must be postulated ( i . e . d i r e c t pump stimulation). However, when [K] i s increased further (from 10 to 100 mM cone, or 7.2 to 66.7 mM a c t i v i t y ) , u p h i l l Ay^/F va r i e s d i r e c t l y with i n t r a c e l l u l a r p o t e n t i a l , suggesting that at high K concentrations b i l a t e r a l l y under I conditions, simple membrane depolarization may contribute to the enhanced rate of C l transport observed at these high l e v e l s (Fig. 8 of chapter 3). 174 v) There i s no r e l a t i o n s h i p between Ay /F and Ay /F when external K i s K C l varied, or between Ay /F*and Cl-dependent I (compare F i g . 6 of t h i s chapter K s c with Fig. 9 of chapter 3). C l e a r l y C l entry cannot be energized by cotransport with K, since Ay../F i s not s i g n i f i c a n t l y d i f f e r e n t from zero under I K ° J sc conditions (P > 0.2), over the range 4-140 mM K concentration. Although useful for examining mechanisms, the above experiment was per-formed under highly unnatural conditions; i . e . t r a n s e p i t h e l i a l p o t e n t i a l (V ) was clamped at zero and [K] was varied over a wide range on both sides. In vivo, the lumen i s normally p o s i t i v e with respect to the hemolymph and [K] fluctuates only i n the lumen. The experiment described above was repeated under more p h y s i o l o g i c a l conditions: i ) tissues were l e f t open-circuited, i i ) serosal [K] was 10 mM throughout the experiment while [K] was increased from 0 to 140 mM on the mucosal side only. Table 5 summarizes data from 5 preparations and Figure 7 i l l u s t r a t e s r e s u l t s obtained from one rectum. The top panel of Figure 7 shows a p i c a l (V ) and basal (V, ) membrane potentials SL D (note that V no longer equals V, ). Transepithelal p o t e n t i a l i s simply the cl D difference between V and V, . The following observations are of i n t e r e s t : a b i ) When mucosal [K] equaled zero, addition of 1 mM cAMP to the serosal side caused a 20 mV hyperpolarization of V and an increase i n a r 1 , consistent a (_, J_ with the stimulation of an electrogenic C l pump in the a p i c a l membrane. i i ) a i s near control l e v e l s (-60 mM) when the mucosal side i s K-free for >2.5 h. Under these conditions i n t r a c e l l u l a r K must have been acquired from the serosal side, perhaps by the ubiquitous Na/K exchange pump. Na/K_ ATPase a c t i v i t y has been demonstrated i n t h i s tissue (see Peacock, 1981). i i i ) K d i f f u s i o n from the c e l l across the mucosal membrane hyperpolarizes V , thereby increasing the electrochemical gradient opposing Cl entry (compare SL Ay r 1/F i n Figs. 6 and 7). Also, since a p i c a l and basal membranes are not 175 Table 4.5 E f f e c t s of cAMP and mucosal [K] on a p i c a l membrane p o t e n t i a l , i n t r a c e l l u l a r K and C l a c t i v i t i e s , and net electrochemical gradients across the a p i c a l membrane under open c i r c u i t conditions* Condition V a J L a£ Au* /F Avu/F OmM K -91. 1 21. 1 39. 4 -56.9 -63. 1 (29-59;3) ±2. 8 ±0. 6 ±2. 6 ±4.4 ±3. 8 + cAMP -110. 8 32. 4 56. 1 -86.4 -51. 1 (28-69;4) ±1. 2 ±1. 2 ±1. 3 ±2.1 ±1. 1 2mM K -94. 2 41. 8 51. 5 -77.4 0. 4 (38-87;5) ±1. 0 ±1. 3 ±1. 8 ±1.8 ±0. 5 4mM K -86. 4 40. 9 57. 0 -68.8 5. 0 (39-87;5) ±0. 9 ±1. 0 ±1. 6 ±1.5 ±1. 3 lOmM K -70. 0 46. 2 66. 9 -57.1 11. 9 (40-79;5) ±1. 1 ±1. 3 ±1. 7 ±1.5 ±1. 1 40mM K -47. 7 48. 5 83. 4 -37.5 15. 5 (40-90;5) ±0. 7 ±2. 4 ±2. 6 ±1.3 ±0. 6 lOOmM K -40. 5 58. 2 84. 0 -32.0 26. 8 (39-87-5) ±0. 8 ±1. 6 ±1. 6 ±1.5 ±0. 9 14 OmM K -41. 8 59. 8 82. 3 -33.3 35. 2 (38-87;5) ±0. 9 ±1. 8 ±4. 6 ±1.4 ±1. 0 * serosal side contained lOmM under a l l conditions x ± s.e. (number of observations; number of t i s s u e s ) , see text. 176 Figure 4.7 E f f e c t s of sequential cAMP and mucosal K additions on membrane a ._ —a. p o t e n t i a l s , i n t r a c e l l u l a r K and C l a c t i v i t i e s , Au /F .and Au_,/F under open-Is. or c i r c u i t conditions during serosal perfusion with normal s a l i n e (10 mM K). For d e f i n i t i o n s see legends of Figures 4.2 and 4.6. Measurements were made 30-60 min a f t e r exposure to each condition. Note that a i s near normal l e v e l s when the mucosal side i s K-free and serosal [K] =10 mM. Also, the electrochemical gradient opposing C l entry declines progressively as mucosal [K] i s elevated (compare F i g . 4.6). Results are from one preparation. Means ± s.e.; n = 20 obs. (V , V, ), n = 10 obs. (a£, a^., Avu/F, Ay* /F). See Table a t ) R C l K L_L 4.5 for complete data. 177 > E > -40 •80 • 1 m M c A M P -120-1 | 8 0 l o 60 i 60 E - 40 H D t .9 25 > £ O * -25 -50 H -25 > E -50 °u -75 H -100 A —r~ 2 0 1 - i 1 1 — i — 4 6 8 10 - T 1 1 1 1 20 40 60 80 M u c o s a l K a c t i v i t y ( m M ) 178 clamped at the same p o t e n t i a l , i s lower at open-circuit than under I conditions (compare Figs.. 6 and 7 when mucosal K a c t i v i t y i s 7.2 #1; at s h o r t - c i r c u i t equals -58 mV, but at open-circuit equals -34 mV). It w i l l be shown i n the following chapter that depolarization of alone would cause some increase i n a ^ , since basal membrane C l conductance i s greater than that of the a p i c a l membrane. Since any increase i n a ^ which might be induced by depolarization of would also increase Ay^/F, i t i s clear that unknown active or passive processes at the basal membrane may, under ce r t a i n conditions, a f f e c t the electrochemical gradient across the ap i c a l membrane and complicate the i n t e r p r e t a t i o n of the electrochemical p o t e n t i a l p r o f i l e (see dicussion). iv) When 10 mM K (concentration) i s present on the serosal side as i n the present experiment, increasing mucosal [K] from 0 to 10 mM causes a decrease i n Ay^/F rather than an increase as was observed when [K] was raised symmetrically on both sides of the epithlium. In summary, the r e s u l t s of varying [K] symmetrically under I condi-tions or asymmetrically under open-circuit conditions show that 1) both mucosal and serosal c e l l borders are highly K - s e l e c t i v e , 2) K i s always near electrochemical equilibrium across both membranes under I conditions and sc therefore Ay„/F could not energize Cl entry, and 3) high concentrations of a potassium reduce Ay^/F by depolarizing a p i c a l and basal membranes. Under I conditions, addition of low l e v e l s of K (2-4 mM) increases the unfavour-sc ' able Ap^ across the a p i c a l membrane while stimulating net Cl transport, i n d i c a t i n g that K d i r e c t l y stimulates the active mechanism. However, under open-circuit conditions i n which serosal [K] i s " p h y s i o l o g i c a l " ( i . e . 10 mM c o n e ) , t h i s e f f e c t on Ay i s obscured, presumably by a large (hyperpolariz-ing)K d i f f u s i o n p o t e n t i a l at the a p i c a l membrane which r e s u l t s from the high l e v e l of K in the c e l l and K-free mucosal s o l u t i o n . 179 3) E f f e c t s of a l t e r i n g the sodium electrochemical gradient  across the a p i c a l membrane Previous r e s u l t s indicated that Cl transport across locust rectum i s r e l a t i v e l y i n s e n s i t i v e to Na removal (chapter 2). Nevertheless, even trace quantities of sodium might sustain NaCl coentry i n the presence of a favour-able Na electrochemical gradient across the a p i c a l membrane (Ay^ a) provided that the Na a f f i n i t y of the c a r r i e r i s exceptionally high and Na i s recycled. Therefore the r e l a t i o n s h i p between t r a n s e p i t h e l i a l Cl fluxes and a Ap /F was d i r e c t l y examined during cAMP exposure (see chapter 2 f or fl u x 36 methods)• Cl fluxes were measured while mucos a l [Na] was varied and V was clamped at zero (mV). Corrections were made f o r s a l i n e resistance and for asymmetrical l i q u i d junctions at the potential-sensing agar bridges. The serosal side contained normal sa l i n e throughout the experiment and Na le v e l s were measured to ensure no s i g n i f i c a n t changes occurred ([Na] = 115.3 3 6 ± 0 . 9 mM). T r a n s e p i t h e l i a l Cl fluxes i n both d i r e c t i o n s (n = 7) and Na contamination on the mucosal side (n = 14) were measured before and a f t e r the mucosal side was rinsed with N-methyl-D-glucamine s a l i n e . The measured Na l e v e l was 48.8 ± 0.32 pM i n nominally Na-free s a l i n e . T h i r t y minutes aft e r the f i r s t wash, mucosal [Na] was 6.81 ± 0.98 mM (n = 14). This con-tamination l e v e l was due to i ) r e s i d u a l Na which had not been completely out of the chamber and i i ) N a which had been leached from the serosal chamber (115 mM [Na]),and represents the maximum Na contamination during the f i r s t two "low-Na" flux periods. Th i r t y minutes a f t e r the second ri n s e with nominally Na-free s a l i n e on the mucosal side, Na contamination was 2.18 ± 0.74 mM (n = 14). Again t h i s value represents the maximum Na l e v e l on the mucosal side during the t h i r d and fourth "low-Na" C l flux periods. 180 M u c o s a l N a c o n e . 115" m M M u c o s a l N a c o n e . 4 9 u M 4 i A t A t -59 mV -5 7 mV - 6 0 mV -62 ! mV n r 6 3 mV 8.4 m M r—H 5 0 m V 10.9 m M 7.4 mM 7.7 mM jT*- — — ' 8.6 M i • i 1 m i n Figure 4.8 Representative traces obtained with double-barrelled Na-se n s i t i v e microelectrode under I conditions during perfusion of mucosal sc b r side with normal s a l i n e (115 mM Na) and nominally Na-free s a l i n e (49 uM Na). Tissue was bathed i n normal s a l i n e containing 1 mM cAMP on the serosal side throughout the experiment. V was clamped at 0 mV. Values of i n t r a -c e l l u l a r p o t e n t i a l (V , ) are shown after c o r r e c t i o n for series resistance. a, o I n t r a c e l l u l a r Na a c t i v i t y ( a^ &) i s corrected for electrode s e l e c t i v i t y using K a c t i v i t i e s measured i n the same tissue under both conditions. As shown at lower r i g h t , the d i f f e r e n t i a l Na-sensitive trace goes o f f - s c a l e when the microelectrode i s retracted into nominally Na-free s a l i n e (N-methyl-D-glucamine substituted). Arrows indicate impalement (+) and r e t r a c t i o n (+) from several c e l l s of one r e c t a l preparation. 181 Double-barrelled Na-sensitive microelectrodes were then used to measure c a,, and i n t r a c e l l u l a r p o t e n t i a l under I conditions when the mucosal side Na v sc was perfused with Na l e v e l s i d e n t i c a l to those measured i n f l u x experiments. It i s important to note that by using the f i n a l Na contamination during each "low sodium" f l u x period, we undoubtedly overestimate the AH^ favouring inward movement during the flux period. Correcting for t h i s error, i f such correction was possible, would further strengthen the conclusion that inward Na gradient does not drive C l entry. Also, a was measured immediate-ly l y before or a f t e r Na measurements i n every tissue and at each mucosal [Na]. The mean a from at l e a s t 3 impalements was used to correct for the s e l e c t i v i t y properties of the sodium electrodes as determined with each c a l i b r a t i o n (mean K^ - = 0.089, see methods). Figure 8 shows t y p i c a l recordings of impalements made using double-b a r r e l l e d Na-sensitive microelectrodes under I conditions. Tissues were sc perfused with normal s a l i n e and were exposed to 1 mM cAMP on the serosal side. Under these conditions, a„ averaged S.01 ± 0.41 mM and i n t r a c e l l u l a r Na a p o t e n t i a l was 58.06 ± 0.54 (x ± s.e.,.125 c e l l s , 11 t i s s u e s ) . Large net electrochemical gradients favour movement of Na into the c e l l s across both c e l l membranes. Also shown are measurements of a„, obtained when mucosal Na [Na] was 49 yM. 3 6 Figure 9 shows C l fluxes and i n t r a c e l l u l a r measurements made under the same conditions i n p a r a l l e l experiments. Not shown are periods (>5 min) when c a^ was measured, electrodes were c a l i b r a t e d and solutions were changed. Replacement of mucosal Na with N-methyl-D-glucamine had no detectable e f f e c t c on Vfl or on a^ , suggesting that Na permeability of the mucosal membrane i s —a. low. Importantly, reducing -Ay /F from 127.6 ± 3.2 mV (x ± s.e., 59 c e l l s , 6 animals) to 22.3 ± 1.55 (x ± s.e., 60 c e l l s , 5 animals) had no e f f e c t on 182 Figure 4.9 R e l a t i o n s h i p between Na e l e c t r o c h e m i c a l gradient (Ap^/F) across the a p i c a l membrane and C l f l u x e s across cAMP-stinulated l o c u s t r e c t a when V was-clamped at 0 mV. Na i n normal s a l i n e (bathing both sides i n i t i a l l y ) was repl a c e d stepwise on the mucosal s i d e with N-methyl-D-glucamine, and e x t e r n a l Na l e v e l s were measured. Recta were repeatedly impaled w i t h d o u b l e d - b a r r e l l e d microelectrodes to measure a p i c a l membrane p o t e n t i a l (V ) a. c c and i n t r a c e l l u l a r Na a c t i v i t y ( a^j a> c o r r e c t e d f o r measured a^) under these c o n d i t i o n s . Values of Au„ /F were c a l c u l a t e d from these data. A t y p i c a l Na 3 6 experiment i s shown where each p o i n t ( $ , n ) represents one c e l l . C l f l u x e s were measured i n i d e n t i c a l experiments and were independent of the inward sodium gra d i e n t across the a p i c a l membrane. 183 L u m i n a l N a * c o n e . (mM) 115 | 6\8 ] 2.2 | 115 -75i 0 0.5 HI-1.0 T i m e (h) 1.5 2.0 184 3 6 t r a n s e p i t h e l i a l C l fluxes. This r e s u l t i s not consistent with predictions of NaCl cotransport i f energized by the flow of Na down i t s electrochemical _a p o t e n t i a l gradient across the mucosal membrane. The e f f e c t s of Ay.T /F on Na 3 6 t r a n s e p i t h e l i a l Cl fluxes have not yet been d i r e c t l y measured i n vertebrate _a -a C l e p i t h e l i a : however, based on comparisons of Ay„,/F and Ay„, /F, J should r > > * KC1 "Na ' net _a decrease l i n e a r l y as Ay /F i s reduced (Garcia-Diaz and Armstrong, 1980). Since t r a n s e p i t h e l i a l C l transport i s not i n h i b i t e d by d r a s t i c reduction ( i . e . 83%) i n Ay^ /F, i t follows that the active C l entry and thus ci a Ay ./F should also be i n s e n s i t i v e to Ay . This suggestion i s confirmed i n \j-L IN a Figure 10, which shows the r e l a t i o n s h i p between mucosal [Na], Ay^ /F and Ay n / F measured under I conditions. The sodium gradient across the a p i c a l L* J. S C membrane var i e d d i r e c t l y with mucosal [Na], as would be expected i f the —a a p i c a l membrane had low Na permeability. In contrast, Ay^/F did not change s i g n i f i c a n t l y (P > 0.05) when mucosal [Na] was decreased from 115 mM to 49 yM. Chloride was above electrochemical equilibrium by more than 38 mV, —a a even when Ay.T was reversed. The " u p h i l l " Ay_ n/F observed i n these and i n Na p C l previous experiments i s probably not due to a r t i f i c i a l l y high estimates of i n t r a c e l l u l a r C l a c t i v i t y since l e s s than 3.7% of Ay^/F may be accounted for by assuming normal i n t r a c e l l u l a r anion interference (-5 mM) and less than 14% by assuming very high (10 mM) interference during impalements. 4) Mannitol space Although i t i s clear that cAMP increases a and the electrochemical gradient opposing C l entry, both of these e f f e c t s might be simply explained without postulating e f f e c t s on the Cl pump i f cAMP exposure r e s u l t s i n tis s u e shrinkage. To examine t h i s p o s s i b i l i t y , i n t r a c e l l u l a r volume was estimated i n controls and during cAMP exposure by comparing t o t a l t i s s u e water with t i s s u e r a d i o a c t i v i t y a f t e r e q u i l i b r a t i n g recta for 1.5 h i n s a l i n e containing 185 Figure 4.10 Relationship between hy. /¥ and Ay^/F i n cAMP-stimulated —a recta with V clamped at 0 mV. The value of Ay,T declined l i n e a r l y 58 mV/ t r Na decade change i n mucosal Na concentration and reversed when mucosal [Na] _< 1 mM. In contrast, Ay* /F changed l i t t l e when mucosal [Na] was va r i e d . a The shaded area shows e n e r g e t i c a l l y f e a s i b l e t r a j e c t o r i e s for Ay ^/F i f C l were to enter across the a p i c a l membrane by "secondary" active transport a —a energized by Ay^/F. The dashed l i n e shows the predicted values of Ay^/F i f C l i n f l u x i s coupled 1:1 with that of Na. A depolarizing entry s t o i c h i -ometry (>1 Na per Cl) would be indicated.by the shaded area below the dashed l i n e . A hyperpolarizing entry process ( i . e . l e s s than 1 Na:Cl) would l i e i n the shaded region above the dashed l i n e . Note that Ay^/F f a l l s completely outside the shaded region. Mean ± s.e.; 58-125 impalements, 6-11 r e c t a . 186 -100 J i — i . « . 1 100 10 1.0 0.1 Mucosa l Na concent ra t ion (mM) 187 3 H-mannitol (see methods f or j u s t i f i c a t i o n ) . Results are summarized i n 3 Table 6. There was no s i g n i f i c a n t d i f f e r e n c e i n H-mannitol space or c e l l volume between controls ( c e l l volume: 3.61 ± 0.24 y l ; and cAMP-treated tissues: 3 3.79 ± 0.24 y l ) . H - i n u l i n space was not determined because i t does not penetrate the c u t i c u l a r intima which covers the mucosal surface of the epithelium ( P h i l l i p s and D o c k e r i l l , 1968). However, the estimates of c e l l volume from "non-mannitol space" agree well with those predicted from the dimensions of the c e l l s measured a f t e r i n j e c t i o n with the fluorescent dye L u c i f e r Yellow CH (chapter 5) and from electron micrographs ( J a r i a l et a l . , unpublished). Discussion Several observations confirm the v i a b i l i t y of the preparation used i n t h i s study as well as the v a l i d i t y of the microelectrode techniques. Trans-e p i t h e l i a l p o t e n t i a l across recta i n the microelectrode chamber was i n i t i a l l y high (25-40 mV, lumen p o s i t i v e ) and declined exponentially to reach an approximate steady-state t r a n s e p i t h e l i a l p o t e n t i a l of about +8 mV. These changes i n V are s i m i l a r to those observed i n previous flux experi-ments where long-term v i a b i l i t y of the ti s s u e i s well established (see chapter 2). The salin e s and the method of mounting the tiss u e onto the chamber were i d e n t i c a l i n fl u x and microelectrode experiments. In micro-electrode experiments i n which tissues were s h o r t - c i r c u i t e d , currents of -2 250-300 yA cm were not unusual. Stimulation of V from ^8 to ^ 30 mV and reduction of R during cAMP exposure provides further evidence that tissue properties were s i m i l a r during microelectrode and tracer flux experiments. Evidence that l i t t l e t i s s u e damage i s caused by mounting the preparation i s 35 provided by measurements of very low SO^ permeability (chapter 2) and also 188 Table 4. 6 E f f e c t of cAMP and h i gh osmotic pressure on t o t a l tissue water and non-mannitol space i n suspended i r e c ta Condition (n) Wet weight Tot a l Tissue Water 2 H-mannitol space-* Non-Calculated . -Manmitol space (mg) (yl) (yl) (yl) Control (6) 7.07 4.87 1.26 3.61 ±0.20 ±0.22 ±0.05 ±0.24 ImM cAMP (7) 7.10 5.07 1.28 3.79 ±0.27 ±0.21 ±0.07 ±0.24 T T - ^ • 2 High osmotic pressure (6) 5.60 ±0.30 2.80 ±0.23 1.97 ±0.04 0.43 ±0.11 "Tissues were incubated for 1-1.5h suspended i n v i a l s containing 3 H-mannitol-labelled normal s a l i n e , or normal s a l i n e + ImM cAMP, or normal s a l i n e containing 600mM sucrose. Taken as the dif f e r e n c e between wet weight and dry weight. Used as an estimate of e x t r a c e l l u l a r volume. Used as an estimate of the i n t r a c e l l u l a r volume. 189 from e l e c t r o p h y s i o l o g i c a l data (cable analysis and voltage scanning) to be presented i n chapter 5. In any one preparation, membrane po t e n t i a l s and i n t r a c e l l u l a r ion a c t i v i t i e s showed l i t t l e v a r i a t i o n between c e l l s . P o t e n t i a l changes were abrupt a f t e r impalement, and pot e n t i a l s and ion a c t i v i t i e s r a r e l y changed during the course of measurements. Recordings were occasionally made from one c e l l for more than 30 min without detectable changes i n membrane p o t e n t i a l s , ion a c t i v i t i e s , or voltage d i v i d e r r a t i o s . No consistent differences were observed between r e s u l t s obtained with s i n g l e - or double-barrelled electrodes. Double-barrelled microelectrodes are thought to cause serious impalement damage a r t i f a c t s i n some e p i t h e l i a (see Lewis and Graf, 1979; Delong and Civan, 1978; Reuss and Weinman, 1979). The apparent lack of damage i n the present study i s probably due i ) to the large si z e of locust r e c t a l c e l l s , and i i ) to the high degree of membrane i n f o l d i n g which greatly increases c e l l membrane area, and probably reduces the importance of shunting caused by leaky impalements. No gradients were observed when electrodes were advanced deep into the epithelium. Only one e l e c t r i c a l p o t e n t i a l "well" was observed when electrodes were pushed through the tiss u e and only one population of c e l l s was evident from the d i s t r i b u t i o n of i n t r a c e l l u l a r ion a c t i v i t i e s (Fig. 4). The under-l y i n g secondary c e l l s do not constitute a continuous layer (they are penetrated by tracheoles)and therefore they probably do not contribute greatly to R . Measurements of membrane pot e n t i a l s and i n t r a c e l l u l a r ion a c t i v i t i e s Membrane potentials observed i n th i s study are " t y p i c a l " for many verte-brate and invertebrate c e l l s , and agree well with values reported for i s o l a t e d recta of Locusta migratoria (V = -65 to -59 mV, V, = -45 to -47 mV over the v a ^ f i r s t 4 h; Vietinghoff et a l . , 1969). Lower membrane'potentials i n 190 Schistocerca recta were observed by P h i l l i p s (1964b; V = -50, V, = -37), cL D however his measurements were made In s i t u using microelectrodes with large t i p diameters, thus differences might be due to the d i f f e r e n t experimental conditions used, or more probably, to impalement damage. The s t r u c t u r a l complexity of the serosal c e l l border makes i t d i f f i c u l t to i n terpret r e s u l t s s t r i c t l y i n terms of the basal membrane (see chapter 1). Localized regions of ion r e c y c l i n g at the l a t e r a l membrane would not be detected using these microelectrode techniques. It i s noteworthy that membrane pot e n t i a l s i n insect e p i t h e l i a are not always s i m i l a r to those i n v e r t e b r a t e preparations; basal membrane potentials of ^ 1 mV have been described i n K-transporting c e l l s of silkworm midgut epithelium (Blankemeyer and Harvey, 1978). Ion a c t i v i t i e s have not previously been measured in locust r e c t a l c e l l s , however,intracellular a c t i v i t i e s of C l (30-46 mM), K (60-70 mM) and Na (8-10 mM) are s i m i l a r to most vertebrate e p i t h e l i a , with the possible exception of a which i s perhaps s l i g h t l y lower than average. Somewhat c c higher a (133 mM) and lower anl (9-10 mM) have been reported i n blowfly s a l i v a r y gland, although i n t h i s preparation, the mucosal surface i s normally bathed with K C l - r i c h f l u i d secreted by the gland rather than normal "low-K" sa l i n e (Berridge and Schlue, 1978; Berridge, 1980). In the lepidoteran midgut, which has 2 major c e l l types, Blankemeyer and Duncan (1980) reported that the "low PD" c e l l s (V^ ~ 0) have a^ of 36.2 mM (approximately equal to that of the bathing saline) whereas i n "high PD" c e l l s (V - -25 mV) , have af. of 75.7 mM. The "low PD" c e l l s are thought to secrete K from serosa to mucosa. I n t r a c e l l u -l a r ion a c t i v i t i e s i n the present study are in reasonable agreement with concentrations measured chemically i n homogenates of whole r e c t a l tissue (55 mM Na, 88 mM K, 39 mM C l ; recta from water-fed locusts; P h i l l i p s , 1964b) 191 and i n very good agreement with concentrations measured by electron microprobe i n blowfly r e c t a l p a p i l l a e (23-47 mM Na, 65-85 mM K, 23-43 mM C l ; Gupta et a l . , 1980). Comparison of ion concentrations measured i n homogenates of whole tissue with i n t r a c e l l u l a r ion a c t i v i t i e s obtained using microelec-trodes i s complicated by the presence of ions in the e x t r a c e l l u l a r space. This could explain why [Na] i s so much higher i n r e c t a l homogenates compared to microelectrode measurements. Differences i n concentrations (electron microprobe) and ion a c t i v i t i e s ( t h i s study) are well within the range which can be expected based on wide range of a c t i v i t y c o e f f i c i e n t s which have been reported i n other tissues (see reviews by Edzes and Berendsen, 1975; Lev and Armstrong, 1975; Civan, 1978, 1980). The data a v a i l a b l e for locust rectum do not allow r e l i a b l e c a l c u l a t i o n s of i n t r a c e l l u l a r a c t i v i t y c o e f f i c i e n t s . However, microelectrode. and electron microprobe measurements have been made i n blowfly s a l i v a r y glands under s i m i l a r conditions ( i . e . during peritubular perfusion of tubules with saline and i n tubules suspended in s a l i n e (Gupta et a l . , 1978). The r e s u l t s i n d i c a t e that the K a c t i v i t y c o e f f i c i e n t i s near one as i n amphibian oocytes (Palmer et a l . , 1978; Horowitz et a l . , 1979), however approximately 30-40% of i n t r a c e l l u l a r Cl may be bound. Low a c t i v i t y c o e f f i c i e n t s have been reported i n amphibian oocytes using both microelectrode (y = 0.08 ± 0.02; Palmer et a l . , 1978) and reference phase analysis ( y v = Na Na 0.22; Horowitz et a l . , 1979); however, NMR data are not consistent with, i n t r a -c e l l u l a r Na binding (evidence reviewed by Civan, 1978). Location and mechanism of active c h l o r i d e transport Chloride entry across the a p i c a l membrane occurs against a net e l e c t r o -chemical gradient under both open- and1 s h o r t - c i r c u i t conditions. Net C l f l u x across both membranes i s i n the mucosa-to-serosa d i r e c t i o n as judged by previous tracer measurements under both open- and s h o r t - c i r c u i t conditions. 192 Consequently, C l entry i s an active process for which energy could be obtained through coupled ion movements such as NaCl cotransport or a l t e r -n a t i v e l y , from an exergonic reaction such as ATP hydrolysis. In vertebrates, microelectrode studies of Cl-absorbing e p i t h e l i a also indicate an a p i c a l a c t i v e step (Duffey et a l . , 1978; Spring and Kimura, 1978; Reuss and Weinman, 1979; Reuss and Grady, 1979; Duffey et a l . , 1979; Garcia-Diaz and Armstrong, 1980). Results from t h i s wide v a r i e t y of vertebrate e p i t h e l i a are consistent with a model involving sodium-coupled C l entry energized by the inward f l u x of Na down i t s net electrochemical gradient (Ap ), as suggested by Quay and IN a. Armstrong (1969), Nellans et a l . (1973) and by F r i z z e l l and coworkers, and reviewed by F r i z z e l l et a l . (1979). In contrast, the i n s e n s i t i v i t y of C l _a J ^ to a very large reduction i n Au„T i n locust rectum, and the lack of net J b Na c o r r e l a t i o n between A y ^ and A p ^ provides strong evidence that C l does not enter by t h i s mechanism, as indicated previously by other observations (see chapter 2). Cl Both 1 mM cAMP and low concentrations of K stimulate steady-state J , net — 3. Cl-dependent I , and the Au r, opposing C l entry across the a p i c a l membrane. * SC CjJL The e f f e c t s of K on A p ^ are not compatible with stimulation of C l transport s o l e l y by e l e c t r i c a l coupling across either the a p i c a l membrane or the ent i r e r e c t a l wall. Instead, K must stimulate the active entry mechanism. Also, small quantities of K stimulate I only when added to the mucosal side. In the presence of 10 mM K on the serosal side of locust rectum, C l -dependent I remains very low (Fig. 13 of chapter 3), a i s maintained at sc K cont r o l l e v e l s (74.8 mM) under these conditions (Table 4). This r e s u l t strongly suggests that mucosal potassium acts at the external surface of the a p i c a l membrane when stimulating active C l transport, and not through some c action on a„. 193 I n t r a c e l l u l a r i o n a c t i v i t i e s are determined by the properties of both c e l l membranes. For example, i f the C l conductance of the serosal border i s much higher than that of the a p i c a l membrane (as w i l l be shown i n chapter 5), c c changes i n a ^ w i l l l a r g e l y depend on changes i n V^, even when a ^ i s maintain ed above equilibrium across the serosal membrane by the action of an a p i c a l C l pump. S h o r t - c i r c u i t current, which i n t h i s tissue hyperpolarizes and deporalizes V by approximately equal amounts, w i l l therefore tend to reduce Q a ^ ; i . e . , more Cl w i l l " be driven out of the c e l l by hyperpolarization of V i - , than w i l l be drawn i n by de p o l a r i z a t i o n of V . In f a c t , s i g n i f i c a n t l y higher a c lev e l s of a ^ were observed under open-circuit conditions (46.2 ± 1.3, 49 c e l l 5 tissues) than under I conditions (40.3 ± 0.9, 40 c e l l s , 4 tissues; sc P < 0.01). Changes i n c e l l volume may a l t e r ion a c t i v i t i e s and electrochemical gradients independently of ion transport. If cAMP causes a 50% reduction i n c a c e l l volume, this might account for the changes i n a ^ and A y ^ observed i n t h i s study. The r e s u l t s shown i n Table 6 suggest that c e l l volume i s not greatly affected by cAMP. Total tissue water, e x t r a c e l l u l a r volume (space 3 l a b e l l e d by H-mannitol) and i n t r a c e l l u l a r volume (non-mannitol space, NMS) were not d i f f e r e n t i n controls and cAMP-treated tissues (P > 0.2). Two observations suggest that NMS provides a reasonable estimate of c e l l volume. F i r s t , exposing tissues to high osmotic pressure (1.2 osm/1) d r a s t i c a l l y reduced NMS and increased mannitol space as expected i f water were drawn out of the c e l l s . Second, we may predict approximately what the i n t r a c e l l u l a r volume of the epithelium should be, based on c e l l dimensions. As described i n chapter 1, the columnar e p i t h e l i a l c e l l s are approximately 17 ym by 80-100 ym (judging from c e l l s f i l l e d with fluorescent dye and from photomicrographs of r e c t a l t i s s u e ) - Assuming a cylinder with a 20 ym diameter base and height of 80 ym, I estimate the volume of one c e l l to be 2.5 x 10 ~* mm^  and the. number 194 5 2 of c e l l s / t i s s u e area to be ^2 x 10 cells/cm . Since the area of one stretched 2 - -5 male locust rectum i s 0.66 ± 0.02 cm (x ± s.e., n = 6) and (2.5 x 10 ) x 5 2 (2 x 10 ) = 5 yl/cm , then the t o t a l c e l l volume i s calculated to be 3.3 y l , i n good agreement with the measured NMS of 3.61 ± 0.24 y l . The s l i g h t l y larger value of NMS may be due to other c e l l types such as muscles and "secondary" 3 c e l l s which would also exclude the H-mannitol. In summary, NMS appears to give reasonable estimates of c e l l volume. Since no changes i n NMS were measur-a able during cAMP exposure, i t i s u n l i k e l y that the changes i n a ^ and A y ^ observed i n th i s study are due to c e l l shrinkage but instead are due to ion transport processes. Also, one might expect volume changes to be temporary as the ions e q u i l i b r a t e , or perhaps were corrected by ac t i v e c e l l volume regula-t i o n . I t must be emphasized that events at the serosal border do not a f f e c t conclusions regarding the l o c a t i o n of the active step or the stimulatory e f f e c t of cAMP and K on the active process; these are based on measurements of both _a C l Ay„., and J measured i n tissues under the same steady-state conditions. By Cl net d e f i n i t i o n of the steady-state, the net fl u x of C l across a p i c a l and basal membranes and the ent i r e epithelium are equal. Consequently, measurements of t r a n s e p i t h e l i a l C l flux and Ay*^ are s u f f i c i e n t to c a l c u l a t e the energetic requirements of C l entry across the a p i c a l membrane. A potassium-coupled i n f l u x could supply l i t t l e i f any energy for C l transport across the a p i c a l membrane. In contrast, sodium entry i s strongly favoured when mucosal Na i s a r t i f i c i a l l y a high (115 mM, normal s a l i n e ; A y ^/F - 122 mV; Fig. 9) and i s more than _a adequate to energize C l entry with 1:1 stoichiometry (Ay^/F = -45 mV) . —a C l —a However, no interdependences between Ay„ /F and J (Fig. 9) or Ay.. /F and - , Na net Na Ay^/F ( F i g . 10) were observed and there i s much evidence that Cl fluxes and Cl-dependent I are i n s e n s i t i v e to sodium removal (chapter 2). A l t e r n a t i v e l y , Cl entry might be coupled to that of H and energized by an inward electrochemi-195 c a i gradient for protons (Au^ = 60 mV i n most c e l l s ) . This seems very u n l i k e l y since Cl-dependent I i s not affected by r a i s i n g s a l i n e pH from 6.2 to 8.0 (F i g . 16 of chapter 2), a manoeuvre which should reverse or at l e a s t greatly diminish Au^ since the pH i n most c e l l s i s i n the range 7.1-7.4 (reviewed by Roos and Boron, 1981). Furthermore, reducing mucosal pH i n h i b i t s rather than stimulates I as would be predicted for HCl coentry (half maximal i n h i b i t i o n at approximately pH 4.7). F i n a l l y , any coupled entry mechanism must involve active r e c y c l i n g of the unknown ion back to the same side of the membrane i n order to explain the C l equivalence of I and J There i s much evidence that C l transport across sc net• the locust rectum i s dependent on aerobic metabolism; extensive tracheation of the ti s s u e , very abundant mitochondria, s e n s i t i v i t y of I to i n h i b i t o r s of aerobic r e s p i r a t i o n (Williams et a l . , 1978; Baumeister et a l . , 1981; Chamber-l i n , 1981; th i s t h e s i s , chapter 1). A Cl-ATPase pump should be considered i n locust rectum. Anion-stimulated ATPase a c t i v i t y has been reported i n e p i t h e l i -a l plasma membrane preparations i n which great care was taken to remove mitochondrial contamination (Humphreys and Chow, 1978; Bornancin et a l . , 19S0) although even recent reports have been c r i t i c i z e d (Bonting et a l . , 1980). Plants from s a l t marshes which a c t i v e l y secrete C l are known to have a Cl-stimulated ATPase ( H i l l and H i l l , 1973; H i l l and Hanke, 1979). There i s some recent (but less convincing) evidence for anion-stimulated ATPases i n recta of locusts (Herrera et a l . , 1978) and dragonfly larvae (Komnick et a l . , 1978). In these reports, no marker enzymes were assayed to check against mitochondrial contam-ina t i o n , although electron microscopic examination and p a r a l l e l Na/K ATPase assays were employed i n the l a t t e r study. Interpretation of potassium and sodium electrochemical p o t e n t i a l p r o f i l e s T r a n s e p i t h e l i a l absorption of K across locust rectum under open-circuit conditions i s l a r g e l y passive, and e l e c t r i c a l l y coupled to active C l absorption 196 (^80%, chapter 3). I t w i l l be shown i n chapter 5 that ions move mainly through the c e l l s rather than by a p a r a c e l l u l a r route. I t i s clear from Table 3 that both a p i c a l and basal membrane net electrochemical gradients K favour passive K movement i n the mucosa-to-serosa d i r e c t i o n . A small J net was observed previously under I conditions; however, i t i s d i f f i c u l t to _a ( _b l o c a l i z e the ac t i v e step i n the present experiments because Ay /F and Ay /F is. K are extremely small when recta are s h o r t - c i r c u i t e d ( F i g . 6). High s e l e c t i v i t y of the membranes for K presumably causes i n t r a c e l l u l a r potentials to remain very close to the equilibrium p o t e n t i a l for K when rec t a K —2 are s h o r t - c i r c u i t e d . F i n a l l y , a small J under I conditions (0.8 yEqcm net sc h "S could r e s u l t from solvent drag since water i s a c t i v e l y absorbed with ion by t h i s t i s s u e . I f a "solvent-drag" K f l u x e x i s t s and i s p a r a c e l l u l a r , no " u p h i l l " step would be observed across either c e l l membrane using i on-se n s i t i v e microelectrodes. Further experiments are required to characterize the active step f o r the small K transport component. There i s evidence f o r a c t i v e sodium absorption across locust rectum ( P h i l l i p s , 1964b; Williams et a l . , 1978; Spring and P h i l l i p s , 1980), however th i s net f l u x must be e l e c t r i c a l l y s i l e n t since Na removal does not a f f e c t I J sc Cl and J equals I . Some Na i s taken up at the a p i c a l membrane with amino net sc acids (Balshin and P h i l l i p s , 1971). The present study suggests two s i m i l a r i -t i e s between locust r e c t a l epithelium and t y p i c a l Na-transporting e p i t h e l i a l i n vertebrates. F i r s t l y , passive Na entry across the a p i c a l membrane i s favoured by a large net electrochemical gradient of 120 mV when a high NaCl s a l i n e i s present on both sides. Secondly, i o n - s e n s i t i v e microelectrode data are consis-tent with a basal Na/K exchange since i ) for t r a n s e p i t h e l i a l Na absorption, any Na entering the c e l l s from the mucosal sides must be a c t i v e l y pumped across 197 the basal membrane against a large Au^ /F (M.20 mV) as i n vertebrates, and i i ) K-depleted r e c t a l c e l l s r a p i d l y accumulate K from the se r o s a l , but not from the mucosal side. This serosal Na/K exchange, i f i t e x i s t s , may be r e l a t i v e l y i n s e n s i t i v e to ouabain. Membrane potentials and a„, were measured (n = 10 c e l l s ) at 1 h r Na i n t e r v a l s for 3 h during perfusion of the serosal side (temperature = 22°C) with 1 mM ouabain and no changes were observed (Hanrahan and Black, unpubl. obs.). There i s some controversy regarding the e f f e c t s of ouabain i n insect tissues, although many e p i t h e l i a do appear to be i n s e n s i t i v e (see reviews by Jungreis, 1977; Anstee and Bowler, 1979; Harvey, 1981). However ouabain may not reach the Na/K pump s i t e s i n the l a t e r a l spaces because of the "sweeping away" e f f e c t of net water absorption, the complexity of the basal c e l l border, and the presence of s u b - e p i t h e l i a l tissue (Irvine and P h i l l i p s , 1969). I t may be argued that a "sweeping out" e f f e c t due to f l u i d transport through l i m i t e d e x i t points impedes ouabain penetration of the serosal border. However, the f a c t that cAMP which i s s i m i l a r i n s i z e to ouabain stimulates recta within minutes, makes t h i s i n t e r p r e t a t i o n u n l i k e l y unless cAMP acts more externally i n the ti s s u e . D i f f u s i o n of ouabain to s i t e s i n the l a t e r a l i n t e r c e l l u l a r spaces may be more d i f f i c u l t than movement of cAMP into the c e l l s across the basal borders of the e p i t h e l i a l c e l l s . Access of l a b e l l e d ouabain to the pump s i t e s must be demonstrated i n t h i s tissue before p h y s i o l o g i c a l s e n s i t i v i t y to ouabain can be ruled out, p a r t i c u l a r l y since Na/K-dependent ATPase i n homogen-ates of locust rectum i s highly s e n s i t i v e to ouabain (Peacock, 1981) . I t i s s t i l l unclear whether this Na/K ATPase i s present i n muscle or e p i t h e l i a l layers or both. The presence of a K-stimulated, rather than a Na-coupled mechanism for Cl transport may be advantageous given that the f l u i d entering the r e c t a l lumen i s 198 K-rich (140 mM) but low i n Na (20-40 mM). Since [Cl] i n t h i s f l u i d i s high (^88 mM), coupled NaCl cotransport could recover only 25-50% of the t o t a l C l present i n the r e c t a l lumen. Moreover, Na-coupled amino acid absorption would further reduce the amount of Na a v a i l a b l e f o r NaCl cotransport. For example, pr o l i n e i s a c t i v e l y secreted by the Malpighian tubules at high concentrations (38 mM) and i s reabsorbed i n the rectum where i t serves as the main r e s p i r a -tory substrate to sustain Cl transport i n the rectum(Chamberlin, 1981). I t has been suggested that most of the Na i n Malpighian tubule f l u i d i s reabsorbed by a coupled process with organic substrates i n the primary urine ( P h i l l i p s , 1981). It i s c l e a r l y more e f f i c i e n t to absorb most K from the r e c t a l lumen by a passive process since p o t e n t i a l energy i s a v a i l a b l e i n the K electrochemical gradient created by tubular secretion (see chapter 3 for c a l c u l a t i o n s ) . Because the r e c t a l wall i s highly K-selective, V immediately reverses p o l a r i t y i f C l i s removed from the mucosal side. This r e s u l t has been obtained i n  v i t r o and also during perfusion of recta i n vivo (unpubl. obs.). No net K absorption can occur i n the absence of electrogenic C l transport since V under these conditions i s exactly equal to the t r a n s e p i t h e l i a l equilibrium p o t e n t i a l for K (-67.4 mV). The function of active C l transport may therefore be to cause passive K reabsorption by making V more p o s i t i v e than the e q u i l -ibrium p o t e n t i a l for K. Direct modulation of the C l pump by K would then represent an i n t e r e s t i n g control mechanism for passive K absorption analogous to enzyme-catalysed reactions which have substrate "feed-forward" a c t i v a t i o n . 199 CHAPTER 5: ELECTROPHYSIOLOGY Summary Membrane permeability and i t s regulation by cAMP has been studied i n locust rectum using e l e c t r o p h y s i o l o g i c a l and tracer methods. T r a n s e p i t h e l i a l 2 resistance (Rfc) i s - 220 ficm under control conditions, t y p i c a l of moderately "leaky" e p i t h e l i a . However, the mucosal and serosal surfaces have very d i f f e r e n t permeability properties as judged by the response of t r a n s e p i t h e l i a l p o t e n t i a l to s a l t gradients. This asymmetry i s suggestive of a t r a n s c e l l u l a r route for passive ion movements. Voltage scanning d i d not reveal current leaks between the c e l l s , between the r e c t a l "pads" or at the edge of the ti s s u e . Extensive c e l l - c e l l coupling was demonstrated by dye iontophoresis and by measuring voltage d e f l e c t i o n s i n neighbouring c e l l s during i n t r a c e l l u l a r current i n j e c t i o n . Resistances of the-apical and basal membranes (R and R^) and the p a r a c e l l u l a r pathway (Rj) were obtained by f l a t - s h e e t cable a n a l y s i s . "Tightness" of the epithelium increased during exposure to cAMP. Both R and a. R^ decreased by 80%, Rfc declined by more than 50%, and R^  was not changed. The cAMP-induced AR^ was abolished i n C l - f r e e s a l i n e , suggesting that AR^ re s u l t s from a massive increase i n basal membrane C l conductance. When compared with previous measurements of the Cl electrochemical gradient, this increase i n conductance i s more than adequate to allow C l ex i t from the c e l l by e l e c t r o d i f f u s i o n when t r a n s e p i t h e l i a l f l u x i s enhanced 10-fold. In contrast, cAMP-stimulated AR i s i n s e n s i t i v e to C l removal, but i s a abolished under K-free conditions. Therefore, AR i s interpreted as a cAMP-a induced K conductance i n the a p i c a l membrane, a conclusion which i s consistent 42 with the previous f i n d i n g that cAMP stimulates t r a n s e p i t h e l i a l K permeability. 200 F i n a l l y , an inverse r e l a t i o n s h i p between a p i c a l membrane K permeability and external [K] was observed: 42 r i i ) t r a n s e p i t h e l i a l P R calculated from K backflux declined as [_KJ was elevated on both sides of the epithelium; 42 i i ) K fluxes i n both mucosa-to-serosa, and serosa-to-mucosa, d i r e c t i o n s were stimulated 4 - f o l d by cAMP i n normal s a l i n e , but not i n "high-K" s a l i n e ; i i i ) when mucosal [K] was elevated from 40 to 140 mM, R remained constant while R a / R k increased 5 - f o l d , i n d i c a t i n g a decrease i n a p i c a l membrane conductance. The r e s u l t s are interpreted using an equivalent e l e c t r i c a l c i r c u i t model for t r a n s e p i t h e l i a l KC1 transport. 201 Introduction The preceding chapters provide evidence for an unusual chloride transport mechanism i n the rectum of the desert locust. In order to understand ion transport across e p i t h e l i a , i t i s also e s s e n t i a l to know the permeability properties of the c e l l membranes and tight junctions. E p i t h e l i a are c l a s s i f i e d into two categories according to the r e l a t i v e importance of t r a n s c e l l u l a r and pa r a c e l l u l a r routes for passive ion movements. "Tight" e p i t h e l i a are those i n which the c e l l junctions e f f e c t i v e l y seal o f f the p a r a c e l l u l a r pathway so that fluxes occur predominantly through the c e l l s . In contrast, junctions i n "leaky" e p i t h e l i a have low e l e c t r i c a l resistance and cause most passive fluxes to occur p a r a c e l l u l a r l y (see Frb'mter and Diamond, 1972). Locust r e c t a l e p i -thelium has some properties associated with both types of junctions. This epithelium generates large e l e c t r i c a l (35 mV), chemical (20-fold), and osmotic (600 mOsm/l "*") gradients i n d i c a t i v e of "tightness", but has low e l e c t r i c a l r esistance consistent with moderately "leaky" e p i t h e l i a . Since the r e l a t i v e resistances of t r a n s c e l l u l a r and j u n c t i o n a l (paracellular) routes have not been d i r e c t l y measured i n locust rectum, the f i r s t part of t h i s chapter investigates the passive permeability of locust rectum using e l e c t r o p h y s i o l o g i -c a l methods. The e f f e c t s of cAMP on membrane conductances are then measured during ion substitutions i n order to i d e n t i f y which ion permeabilities are stimulated. D e t a i l s of a c t i v e C l absorption, including the d r i v i n g force and i n t e r n a l resistance of the transport mechanism, are deduced from membrane resistances and i n t r a c e l l u l a r ion a c t i v i t i e s using a simple equivalent c i r c u i t model, and compared to independent estimates based on the r e l a t i o n s h i p between s h o r t - c i r c u i t (I ) and t r a n s e p i t h e l i a l conductance (G ) . The r e s u l t s i n t h i s sc t and preceding chapters are incorporated into a model for t r a n s e p i t h e l i a l ion transport and i t s regulation i n locust rectum. In addition, this study 202 provides an opportunity to test several e l e c t r o p h y s i o l o g i c a l methods and th e i r underlying assumptions on a very unusual epithelium. Materials and methods D e t a i l s of the d i s s e c t i o n and chambers are given i n chapters 2 and 4. The microelectrode chamber (chapter 4) was used only when i n t r a c e l l u l a r measurements were required. A l l other experiments were performed with the f l u x chambers of Williams et a l . (1978; chapter 2). Bathing sal i n e s were -i d e n t i c a l to those described i n chapter 2, except when the e f f e c t s of si n g l e s a l t gradients were measured (see next secti o n ) . T r a n s e p i t h e l i a l p o t e n t i a l (V.), resistance (PO and s h o r t - c i r c u i t current (I ) were measured as t t sc previously described (chapters 2 and 4). Ef f e c t s of t r a n s e p i t h e l i a l s a l t gradients V was measured during exposure of the tissue to t r a n s e p i t h e l i a l concen-t r a t i o n differences of various s a l t s . Salines containing 200 mM NaCl, KCl, choline C l , or K-methylsulfate were mixed separately with sucrose (400 mM) i n order to give intermediate concentrations of each s a l t (2, 8, 40 and 120 mM). A l l solutions also contained the normal eleven amino acids as l i s t e d i n chapter 2 as energy sources. After tissues had equ i l i b r a t e d i n HCO^-free sal i n e for 3 hours, both sides were exposed to the f u l l - s t r e n g t h s a l t solutions described above and r e - e q u i l i b r a t e d for a further 30 minutes. The mucosal or serosal side was then r a p i d l y rinsed with lower concentrations of the same s a l i n e , a l t e r n a t i n g with the 200 mM f u l l - s t r e n g t h solutions (e.g. sequence: 200, 2, 200, 8, 200, 40, 200, 120, 200 or reverse order). A vacuum pump was used to drain the chambers as fresh s a l i n e was injected by syringe. Solutions changes were completed within 10 seconds. Control experi-ments showed that neither Vfc nor R were alt e r e d a r t i f a c t u a l l y by the technique of changing s o l u t i o n . Also, V returned to approximately the same value 203 between each t e s t s o l u t i o n . The average d i f f e r e n c e before and aft e r was +0.32 ± 0.32 mV following"mucosal substitutions and +0.34 ± 0.12 mV after serosal substitutions (x ± s.e., n = 214). Net l i q u i d junction potentials were measured by replacing the rectum with a short 3 M KCl agar bridge. It i s clear that correcting for such errors w i l l strengthen our conclusion that t r a n s e p i t h e l i a l permeability properties are asymmetrical, since l i q u i d junc-t i o n p o t entials at the agar bridges would be of s i m i l a r magnitude when testing gradients i n opposite d i r e c t i o n s , and would therefore tend to increase the symmetry of V responses. When solutions were changed on the mucosal side, new quasi-steady-state t r a n s e p i t h e l i a l p o t e n t i a l s were observed within 10 sec. Responses were slower when solutions were changed on the serosal side, often requiring several minutes to reach a stable value when the largest gradients were tested. This delay i s probably due to s u b e p i t h e l i a l d i f f u s i o n b a r r i e r s (secondary c e l l s , and muscle bands) and the unstirred layer associated with them; however, the p o s s i b i l i t y of changes i n i n t r a c e l l u l a r composition cannot be ruled out during these slow transients at the serosal side. Voltage scanning This technique was used to explore the surface of the epithelium for regions of high conductance (see Fromter, 1972; Higgins et a l . , 1975; Lewis et _2 a l . , 1976). Current (250 uAcm ) was passed t r a n s e p i t h e l i a l l y while the p o t e n t i a l d i f f e r e n c e between a "scanning" microelectrode (which was moved over the ti s s u e surface) and the mucosal 3 M KCl agar bridge was monitored i n order to determine the uniformity of the e l e c t r i c a l f i e l d . The voltage between scanning electrode and agar bridge was measured using a d i f f e r e n t i a l electromet-er (FD 223, W.P. I n s t r . , New Haven, Conn.). This s i g n a l was f i l t e r e d , displayed on a storage o s c i l l o s c o p e , and recorded using a pen recorder as described i n chapter 4. 204 I n t r a c e l l u l a r I n j e c t i o n of fluorescent dye The fluorescent dye"Lucifer Yellow CH was injected i n t r a c e l l u l a r l y by iontophoresis. A 3-5 mm column of dye was placed into the t i p of a s i n g l e -b a r r e l l e d microelectrode by immersing the blunt end of the electrode into a 5% solution of L u c i f e r Yellow CH i n d i s t i l l e d water (approx. 0.1 M) . Electrode t i p s were less than 1.0 p i n diameter and had resistances of 25-40 Mft or 10-15 Mft a f t e r b e v e l l i n g i n a stream of abrasive according to the method of Ogden et a l . (1978). C e l l s were impaled at an angle of 30° to the plane of the epithelium as described previously. Hyperpolarizing current was passed through the microelectrode by a constant current source (M 701, W.P. Instr., New Haven, Conn.), driven by waveform and pulse generators (Type 160 s e r i e s , Tektronix, Beaverton, Ore.). A switch allowed passage of d i r e c t current or pulses. Current was monitored continuously using a storage o s c i l l o s c o p e . As a precaution, a second electrometer (616, Keithley I n s t r . , Cleveland, Ohio) was used to measure current flowing through the microelec^ trode during dye i n j e c t i o n and this was displayed d i g i t a l l y . Continuous impalement during dye i n j e c t i o n s was ensured by switching to pulse mode and observing the membrane p o t e n t i a l between pulses. Also, a standard bridge was occasionally used to i n j e c t current and measure membrane p o t e n t i a l simul-taneously through the same electrode. Sudden increases i n "apparent" input resistance during L u c i f e r Yellow CH i n j e c t i o n indicated t i p blockage by dye p a r t i c l e s . This problem was usually remedied by switching from d i r e c t current to pulse mode (1 sec duration, 0.5 Hz) for 0.5-1.0 min. Currents ranged between -5 and -50 nA during dye iontophoresis i n d i f f e r e n t experiments. Two c e l l s , at least 1 mm apart, were each 1 injected for 30-45 min. This protocol allowed dye from the f i r s t i n j e c t i o n to continue d i f f u s i n g as the second c e l l was being f i l l e d ; however, t h i s should not a f f e c t the r e s u l t s noticeably since the dye spreads within seconds by d i f f u s i o n (^200 p/sec estimated by Stewart, 205 1978) and then reacts p r e f e r e n t i a l l y with the n u c l e i i n locust r e c t a l c e l l s . The preparation was perfused on both sides with oxygenated normal s a l i n e during i n j e c t i o n s . For processing, recta were pinned onto thin wafers of polymerized 2 2 Sylgard 184 (Dow Corning Corp., Midland, Mich.; 2.3 cm with 0.2 cm hole cut i n the centre) and fixed i n 4% paraformaldehyde buffered with 0.1 M phosphate at pH 7.2. Aft e r at l e a s t 1 h of f i x a t i o n , r e c t a were dehydrated i n ethanol (50, 80, 100% for 5, 5, and 10 min, respectively) and then cleared for 5 min in methyl s a l i c y l a t e . Fluorescent c e l l s were observed microscopically i n whole mounts with incident l i g h t e x c i t a t i o n at 125x or 500x magnification (Orthoplan, L e i t z Wetzlar, W. Germany). Photomicrographs were taken using an automatic camera attachment (Orthomat W, L e i t z Wetzlar) and 35 mm Kodak Ektachrome 160 (Tungsten) f i l m . E l e c t r i c a l coupling and f l a t - s h e e t cable analysis Two s i n g l e - b a r r e l l e d microelectrodes were used to study c e l l - c e l l e l e c t r i -c a l coupling and to perform cable analysis. Current pulses (200 nA, frequency = 0.3 Hz, duration = 1 sec) were passed i n t r a c e l l u l a r l y through one microelectrode, and the r e s u l t i n g voltage d e f l e c t i o n s were measured i n other c e l l s using the second microelectrode ( F i g . 1). Hyperpolarizing currents were usually used although depolarizing pulses gave s i m i l a r r e s u l t s . The distance between c u r r e n t - i n j e c t i n g and voltage-sensing microelectrodes was measured using a ca l i b r a t e d eyepiece micrometer. Voltage responses were displayed on a storage oscilloscope a f t e r f i l t e r i n g (3 db at 5 Hz) and were measurable when greater than ^0.3 mV. The mucosal and serosal sides are e f f e c t i v e l y short-c i r c u i t e d with respect to i n t r a c e l l u l a r current i n these experiments since the t o t a l resistance of the epithelium i s much less than the resistance of the basal membrane i n the region of current spread (<0.5% i n locust rectum; see also Fromter, 1972; Lewis et a l . , 1976). The voltage spread adjacent to the 206 Figure 5.1 Method used for measuring the r a d i a l spread of current i n r e c t a l epithelium. Deflections i n a p i c a l membrane p o t e n t i a l (V ) were measured by microelectrode "a" while current pulses were injected i n t r a c e l l u -l a r l y through microelectrode "b". See text for d e t a i l s . 2 0 7 s i t e of current i n j e c t i o n i s described by the d i f f e r e n t i a l equation (see Eisenberg and Johnson, 1 9 7 0 ; Shiba, 1 9 7 1 ; Fromter, 1 9 7 2 ; Reuss and Finn, 1 9 7 4 , 1 9 7 5 ; Lewis et a l . , 1 9 7 6 ) : j 2 x dx , 2 dx X where V i s the voltage d e f l e c t i o n at some distance x, and A i s the space constant defined as YR /R R i s the e f f e c t i v e input resistance ( r e s i s -Z X z 2 tance to ground: R R, / (R +R, ) i n Qcm and R i s the resistance to current ° a D a b x flow w i t h i n the e p i t h e l i a l sheet i n Kfi. Under the condition V -»• 0 at x = «>, the s o l u t i o n of Eq. ( 1 ) i s V = A K Q ( x / A ) , where K q i s the zero-order modified Bessel function, A i s an in t e g r a t i o n constant (mV), and x i s distance (ym). Deflections i n a p i c a l membrane p o t e n t i a l were measured as a function of distance and compared to a set of curves obtained by drawing the Bessel func-tion at 1 4 v alues of A between 5 0 and 8 0 0 ym from published.tables (Olver, 1 9 6 7 ) . Data were f i t t e d by inspection to give values of A and A (see F i g s . 1 0 and 1 3 ) which were then used to c a l c u l a t e R z according to R = 2 n A A 2/I z o where I i s the current injected i n t r a c e l l u l a r l y (yA) and symbols are described as above. The r a t i o of a p i c a l to basal membrane resistances (a) was calcul a t e d , af t e r corrections for serie s resistance, from the deflections i n a p i c a l and basal membrane pot e n t i a l s produced by t r a n s e p i t h e l i a l current pulses (I = 2 0 yA/ 2 0 . 1 9 6 cm , frequency = 0 . 3 Hz, duration = 1 sec) as a = AV a/AV b = R a/R b ( 2 ) 208 Resistance of the l a t e r a l i n t e r c e l l u l a r space should not cause a s i g n i f i -cant underestimate of R /R, i n t h i s tissue since AV /AV, < 2 and since the a b a b — r a t i o of a p i c a l membrane:paracellular resistance i s less than 4 (see Boulpaep and Sackin, 1980). T r a n s e p i t h e l i a l resistance was determined as R t = (AV a + A V b ) / I t (3) where I i s t r a n s e p i t h e l i a l current as described above. A p i c a l membrane resistance (R ), basal membrane resistance (R, ), and a b ju n c t i o n a l resistance (Rj) were then determined using the standard equations: R p = (1 + a)R (4) R b = (1 + a)R z/a (5) R j = ( R t R a + R t V / ( R a + R b " V ( 6 ) which follow from the "lumped" equivalent e l e c t r i c a l c i r c u i t model usually applied to e p i t h e l i a l tissues (Fig. 2). For further d e t a i l s regarding f l a t -sheet cable a n a l y s i s , see references for equation (1). Equivalent electromo-t i v e forces f o r a p i c a l and basal membranes are calculated from mean resistances and membrane po t e n t i a l s by a c i r c u i t analysis described i n the discussion section. 42 T r a n s e p i t h e l i a l K fluxes 42 K The u n i d i r e c t i o n a l f l u x of K from serosa to mucosa (J ) may be used as sm a measured of t r a n s e p i t h e l i a l K permeability (see chapter 2 for assumptions). In order to quantify the e f f e c t s of [K] on A P „ , the following protocol was used: Recta were e q u i l i b r a t e d under I conditions for 3-4 hours i n K-free s a l i n e and 1 sc then exposed to 1 mM cAMP on the serosal side. A f t e r a new steady-state I was obtained, K-methylsulfate was added to both sides of the epithelium so that K cone, increased stepwise (0, 2, 4, 10, 40, 100, 140, 200 mM). The serosa-209 Figure 5.2 Equivalent c i r c u i t model of locust rectum. Shown are r e s i s -tances of the a p i c a l and basal membranes (R &, R^), and tight junctions ( Rj)> and net electromotive forces at each of these b a r r i e r s (E , E, and E ). M, a' b j S and C in d i c a t e mucosal, serosal and i n t r a c e l l u l a r compartments, respective l y . The resistance of the cytoplasm and i n t e r c e l l u l a r junctions to current within the epithelium (R ) i s also indicated diagrammatically. !Note that unlike e p i t h e l i a i n vertebrates, gap junctions between insect recta c e l l s ar d i s t r i b u t e d along the entire i n t e r c e l l u l a r space; see Lane, 1979, 1981). 210 to-mucosa f l u x of ~*^ K was measured at each concentration for two 15-min periods. Previous experiments showed that the tracer f l u x measured during the f i r s t period included tracer e q u i l i b r a t i o n and was therefore not used i n c a l c u l a t i o n s . Sampling and counting methods were i d e n t i c a l to those described i n chapter 3. Results 1) E f f e c t s of s a l t gradients on t r a n s e p i t h e l i a l p o t e n t i a l Junctional complexes form passive b a r r i e r s to d i f f u s i o n across e p i t h e l i a and probably do not have i n t r i n s i c r e c t i f y i n g properties. If the p a r a c e l l u l a r pathway i s "leaky" and constitutes the major r o u t e for t r a n s e p i t h e l i a l ion d i f f u s i o n , the e f f e c t s of s a l t gradients on V should be symmetrical and determined p r i m a r i l y by the properties of t h i s b a r r i e r rather than by the permeability of a p i c a l and basal c e l l membranes. Conversely, i f the junections are i n f a c t " t i g h t " , then V responses may be very d i f f e r e n t depending on the d i r e c t i o n of the s a l t gradient. Figure 3 shows representative traces of t r a n s e p i t h e l i a l p o t e n t i a l obtain-ed i n the presence of s a l t gradients. In this case, the mucosal side was exposed to various concentrations of NaCl while serosal [NaCl] remains constant at 200 mM. When NaCl and KCl solutions were used, V reached a maximum with i n seconds and then declined. When choline solutions were used,the i n i t i a l spike did not occur although V s t i l l declined i n the same manner. Only the i n i t i a l d e f l e c t i o n s were used for c a l c u l a t i n g permeability properties. As may be seen i n Figure 3b, d e f l e c t i o n s i n V were less abrupt when solutions were changed on the serosal side. Transients which sometimes occur at the serosal border were not studied i n d e t a i l ; nevertheless AV during mucosal s o l u t i o n changes are considered more r e l i a b l e simply because they occurred i n s t a n t l y , with no opportunity for a l t e r a t i o n s i n i n t r a c e l l u l a r ion l e v e l s . Figure 5.3 Representative recordings of V during exposure to t r a n s e p i -t h e l i a l NaCl or KCl gradients. (a) Serosal (S) composition held constant (200 mM NaCl) and the mucosal side (M) was exposed to s o l u t i o n of i n c r e a s i n [NaCl] as indicated. (b) Mucosal [KCl] was maintained at 200 mM while the serosal side was exposed to various .concentrations of KCl. Sucrose was added to solutions having low s a l t concentration i n order to minimize strea ing p o t e n t i a l s . 212 Figure 4 summarizes the r e l a t i o n s h i p between the logarithm of the imposed gradient, and the change i n V produced across locust rectum by each s a l t s o l u t i o n . The e f f e c t s of choline c h l o r i d e , NaCl and KCl gradients are c l e a r l y asymmetrical, i . e . changes i n mucosal or serosal concentrations produce d i f f e r e n t changes i n AV . This r e s u l t implies that t r a n s e p i t h e l i a l properties are determined by two b a r r i e r s with d i f f e r e n t properties ( i . e . c e l l membranes) rather than by a s i n g l e , symmetrical b a r r i e r (the j u n c t i o n s ) . The s e l e c t i v i t y of mucosal and serosal surfaces may be assessed semi-quantitatively by treating AVt as a l i q u i d junction p o t e n t i a l i n free s o l u t i o n and c a l c u l a t i n g the appar-ent transference numbers for cation and anion using the modified Nernst-Planck-Henderson equation: where t + and t_ are the transference numbers of the cation and anion, a^ and a2 are the i o n i c a c t i v i t i e s on either side of the epithelium, and AV , R, T and F have t h e i r usual meanings. Transference numbers were calculated as described i n the methods s e c t i o n and are also shown i n Figure 4. It i s c l e a r that the mucosal surface i s s e l e c t i v e for cations over C l , contrary to predictions based on l i m i t i n g equivalent conductances of C l , K and Na (Weast, 1978). Assuming that C l permeability i s equal under each condition, the permeability r a t i o s based on mucosal s o l u t i o n changes are Na - choline - K > MeSO^ > Cl i n cAMP-stimulated recta. One s t r i k i n g observation i s that a p i c a l C l permeability i s extremely low, even when compared to the large cation choline. Also, a p i c a l membrane K permeability also seems low r e l a t i v e to Na although i t w i l l be shown l a t e r that high external [K] i n h i b i t s K permeability of the apical.membrane. In contrast to the high cation s e l e c t i v i t y of the mucosal border, the serosal border i s only s l i g h t l y c a t i o n - s e l e c t i v e (K > Na = C l > MeSO^ >> choline). The symmetry i n AV produced by^ , K-methylsulf ate gradients i s interpreted as low 213 Figure 5.4 Deflections i n V r e s u l t i n g from exposure of cAMP-stimulated r e c t a to t r a n s e p i t h e l i a l s a l t gradients. (a) sodium chl o r i d e , (b) potassium chloride,_ (c) choline c h l o r i d e , (d) potassium methylsulfate. See legend of Figure 3 .and text f o r d e t a i l s of the method. S a l t concentration was reduced either on the mucosal side (to the l e f t of the ordinate) or on the serosal side (to the r i g h t of the ordinate). Sucrose was added to maintain approxi-mately isosmotic conditions at low-salt concentrations. A l l solutions contained the standard amino acids l i s t e d i n Table 2.1. Similar r e s u l t s were obtained using unstimulated r e c t a and azide-poisoned t i s s u e s . Means ± s.e.; n = 10 recta (NaCl), n = 6 (KC1), n = 6 (choline C l ) , n = 8 (K-methylsulfate). Apparent transference numbers were calculated by l i n e a r regression as described i n the text. 214 C h o l i n e C l d h-60 1 AVtCmV) 215 methylsulf ate permeability s i n c e a l l . c h l o r i d e s a l t s gave asymmetrical r e s u l t s . Asymmetry implies that AVt i s determined by two b a r r i e r s i n s e r i e s , each having d i f f e r e n t permeability properties. These b a r r i e r s are almost c e r t a i n l y the a p i c a l and basal membranes; underlying muscle and second-ary c e l l s do not form a continuous layer due to penetration by tracheoles. 2) E f f e c t s of cAMP exposure and Cl-removal on the voltage-divider r a t i o Results i n the previous section suggested that the mucosal surface has very low C l permeability compared to cations whereas Cl permeability i s r e l a t i v e l y h i g h a t the serosal border. I f this i n t e r p r e t a t i o n i s correct, then the r a t i o of apical-to-basal membrane resistance (voltage-divider r a t i o or "a") should decrease when C l i s replaced by an impermeant anion. Table 1 shows the e f f e c t s on a of sequential cAMP additions (1 mM to serosal side) and - gluconate s u b s t i t u t i o n . Double-barrelled microelectrodes were used i n t h i s p a r t i c u l a r experiment. Addition of cAMP had no s i g n i f i c a n t e f f e c t on a (P > 0.2) although tissue conductance increases dramatically during cAMP exposure (chapters 2 and 3). I t w i l l be shown that both a p i c a l and basal membrane resistances decrease by s i m i l a r amounts under these conditions so that the r a t i o R^R^ remains constant. When Cl was removed from both sides, a decreased from 2.01 ± 0.34 to 0.52 ± 0.09. This f o u r - f o l d increase i n R /R, i s consistent with low a p i c a l a b v membrane C l permeability and strongly suggests that the e f f e c t s of transepi-t h e l i a l s a l t gradients on AV observed i n the preceding section were determined by the s e l e c t i v e permeability of the c e l l membranes rather than by the tight junctions. 3) Voltage scanning When current was passed through the epithelium by s i l v e r f o i l electrodes at opposite ends of the chamber,a "scanning" microelectrode was used 216 Table 5.1 Relative resistance of a p i c a l and basal membranes (a) as calculated from d e f l e c t i o n s i n a p i c a l and basal membrane pot e n t i a l s during t r a n s e p i t h e l i a l constant-current pulses Normal s a l i n e Normal s a l i n e C l - f r e e s a l i n e (control) + ImM cAMP1 + ImM cAMP1 2.48 (137) a 2.01 (123) 0.52 (71) ±0.35 ±0.34 ±0.09 1 ImM cAMP added to serosal side only Means ± 95% confidence i n t e r v a l ; (number of c e l l s ) 217 to sense v a r i a t i o n s i n the e l e c t r i c a l f i e l d due to l o c a l i z e d regions of current flow through the- epithelium. No current leaks were observed when the microelectrodes were moved over the epithelium proper, over the reduced epithelium between each r e c t a l pad (see chapter 1) or at the edge of the tissue where i t was fastened to the chamber ( F i g . 5a-c). However, experimentally damaged areas ( F i g . 5d) were e a s i detected because of the large sizes of the hole (^50 pm diameter). The d e f l e c t i o n i n scanning voltage was also very large (13 mV, as compared to a l i m i t of s e n s i t i v i t y = 0.3 mV), i n d i c a t i n g that a 7.6 pm hole would have been detectable (compare with c e l l diameter of vL5 ym). F i n a l l y , the absence of 35 low-resistance shunts i s consistent with previous S0^ f l u x studies (chapter 2); these measurements showed that l i t t l e , i f any, t r a n s e p i t h e l i a l permeability i s a t t r i b u t a b l e to nonselective "leak" pathways-4) C e l l - c e l l interconnections Since e p i t h e l i a l c e l l - c e l l coupling was demonstrated by Loewenstein and coworkers (1965; see review by Loewenstein, 1981), c e l l u l a r interconnections ( i . e . gap junctions) have been found i n most e p i t h e l i a . To study locust rectum with e l e c t r o p h y s i o l o g i c a l methods, i t i s important to know whether current injected into one c e l l flows mostly into adjacent c e l l s or passes d i r e c t l y to the external solutions through the membranes of the i n j e c t e d c e l l . Both v i s u a l and e l e c t r i c a l methods were used to examine the p o s s i b i l i t y of c e l l - c e l l c o u p l i n g ; f i r s t , b y observing the l a t e r a l d i f f u s i o n of fluorescent dye af t e r i n j e c t i n g i t into an e p i t h e l i a l c e l l and second, by measuring voltage d e f l e c t i o n s i n neighbouring c e l l s during i n t r a c e l l u l a r current i n j e c t i o n . i ) Dye coupling Figure 6 shows the e f f e c t of L u c i f e r Yellow CH i n j e c t i o n into locust r e c t a l c e l l s as observed by fluorescence microscopy. In recta from eleven l o c u s t s , eight out of t h i r t e e n experiments showed extensive d i f f u s i o n of dye 218 Figure 5.5 Voltage scans of the e p i t h e l i a l surface. Current was passed t r a n s e p i t h e l i a l l y from serosa to mucosa (250 yA cm ) while a blunt microelec-trode was dragged across the e p i t h e l i a l surface to record inhomogeneities i n the e l e c t r i c a l f i e l d . Traces (a)-(c) show the p o t e n t i a l d i f f e r e n c e between the microelectrode and a mucosal agar bridge, as the electrode was located i n the region indicated i n the photograph ( d i f f e r e n t t i s s u e was photographed). Trace (d) was obtained by moving the electrode over a hole (^50 ym diameter) which had been made by d e l i b e r a t e l y damaging the t i s s u e with the microelec-trode. 2 1 5 25 C — mV 220 Figure 5 . 6 Rectal c e l l s following i n j e c t i o n with the fluorescent dye L u c i f e r Yellow CH. (a) Broken microelectrode t i p remains i n c e l l during processing of the tissue. (b) The n u c l e i of 20-30 c e l l s s t a i n intensely a f t e r i n j e c t i o n of dye into one c e l l . (c) C e l l margins are c l e a r l y v i s i b l e i n the region of dye i n j e c t i o n when the surface of the tissue i s i n focus. C a l i b r a t i o n l i n e s : 50 ym. 221 222 from the in j e c t e d c e l l into adjacent c e l l s . The cen t r a l c e l l was usually the brig h t e s t , and was presumed to be the one i n which the microelectrode was situated. C e l l s t a i n i n g became progressively f a i n t e r with distance away from the centre c e l l . No p r e f e r e n t i a l routes of coupling were observed, i . e . the stained area was c i r c u l a r and did not s t a i n p a r t i c u l a r pathways or c l u s t e r s of o c e l l s . The average distance of dye spread ( i . e . radius of stained area) was 78.9 ± 13.8 ym(x + s.e.). O v e r a l l , an average of 28 ± 4 c e l l s were stained when "coupled" c e l l s were i n j e c t e d . C e l l dimensions were e a s i l y measured a f t e r dye i n j e c t i o n . When the microscope was focused on the surface of the tiss u e , a fluorescent " f i s h n e t " pattern corresponding to c e l l margins was observed (see F i g . 6c). The base of the columnar c e l l was 15.2 ± 1.3 ym(x ± s.e., n = 6 l o c u s t s ) . When the plane of focus was lowered into the ti s s u e , n u c l e i were observed at a depth of 53.0 ± 7.5 pm from the mucosal surface. These values agree with those obtained from electron micrographs ( J a r i a l et a l . , unpubl. obs.) and from observations on unfixed tissues under a d i s s e c t i n g microscope (Hanrahan, unpubl.). Serious f i x a t i o n a r t i f a c t s are u n l i k e l y ; a recent study i n which gallbladders were observed continuously during f i x a t i o n i n OsO^ and dehydration i n alcohol showed l i t t l e change i n c e l l dimensions during these procedures (Rostgaard and Frederiksen, 1981). Out of t h i r t e e n c e l l s i n j e c t e d with dye, f i v e appeared to be uncoupled. However, of these uncoupled c e l l s , four were within 200 ym of the edge of r e c t a l pads, the s i t e where the c u t i c u l a r intima attaches to the rectum. I t i s possible that c e l l s near the edge of the pad were damaged and became uncoupled during d i s s e c t i o n of >the intima. In summary, under normal conditions, most ( i f not a l l ) of the e p i t h e l i a l c e l l s are probably interconnected by low resistance pathways. This i s confirm-ed i n the next section using e l e c t r i c a l methods. 223 i i ) Electrical coupling Figure 7 shows the e f f e c t s of current i n j e c t i o n into one c e l l on the a p i c a l membrane p o t e n t i a l of a second c e l l located 42 p from the point of i n j e c t i o n . In order to produce these d e f l e c t i o n s , current must flow i n t r a -c e l l u l a r l y between the two c e l l s , i . e . the c e l l s must be e l e c t r i c a l l y coupled (F i g . 1). I t may be noted that c e l l - c e l l coupling i n locust rectum i s independent of the d i r e c t i o n of current flow, since voltage responses were i d e n t i c a l when negative (hyperpolarizing) or p o s i t i v e (depolarizing) currents were i n j e c t e d . The current-voltage r e l a t i o n of i n t r a c e l l u l a r l y injected current measured i n d i f f e r e n t c e l l s was reasonably l i n e a r under various conditions (Fig. 8), i n contrast to many e p i t h e l i a i n which c e l l s are uncoupled by depolarization (Socolar and P o l i t o f f , 1971; Lewis et a l . , . 1976; reviewed by Loewenstein, 1981). To test whether very large current pulses might cause time-dependent uncoupling or d e t e r i o r a t i o n of membrane p o t e n t i a l , very large depolarizing current pulses (300 nA) were in j e c t e d into an e p i t h e l i a l c e l l for 20 minutes (Fig. 9). Membrane p o t e n t i a l and voltage d e f l e c t i o n s recorded i n a second c e l l were not altered ( i n t e r e l e c t r o d e distance =42 p) . This i n s e n s i t i v i t y to large i n t r a c e l l u l a r current i n j e c t i o n s made f l a t - s h e e t cable analysis of the epithelium f e a s i b l e despite very low membrane resistances. Currents of 50 and 100 nA have been i n j e c t e d i n studies of Chironomous Malpighian tubules and s a l i v a r y glands(Loewenstein et a l . , 1965). 5) C e l l membrane and p a r a c e l l u l a r resistance I f c e l l s within the epithelium are e l e c t r i c a l l y coupled, then i t i s not possible to estimate membrane resistances by i n j e c t i n g current and observing the voltage response, i n the same c e l l . . Instead, voltage d e f l e c t i o n s must be measured as a function of distance from the point of current i n j e c t i o n i n 224 — On < £ -50-y-ioo-llr-"' > 0 < 40 1 min ~ ^oo^ < S 50 4- p u ^ i i i T i i i r i iiiiiiiiiiiii II m i || I! lilih Figure 5.7 E l e c t r i c a l coupling between c e l l s i n the r e c t a l epithelium. Hyperpolarizing (~^-QJ upper traces) and depolarizing currents (+IQ> lower traces) were passed from an adjustable constant-current source (10-100 nA, ^1 sec duration, square pulses) into a c e l l through one microelectrode, and the r e s u l t i n g d e f l e c t i o n s i n a p i c a l membrane p o t e n t i a l (AV ) were measured a using a second microelectrode located 42 p from the point of current i n j e c -t i o n . This distance represents 2- to 3 - c e l l separation between electrodes ( c e l l diameter =17 pm). Note that s i m i l a r r e s u l t s were obtained with hyper-and depolarizing currents. 1 225 V a CmVD 15 -300 -200 j — -100 A A _ O 9 © • © O o o o o • o o M 0 ^5 9 O • A A 2 ! * A 0 h-5 X 100 200 L Cn A D 10 300 L 15 Figure 5.8 Current-voltage r e l a t i o n of i n t r a c e l l u l a r l y injected current as measured i n a d i f f e r e n t c e l l . Recta were perfused with ( A ) normal s a l i n e , ( A. ) normal s a l i n e containing 1 mM cAMP on the se r o s a l side, ( O ) C l - f r e e s a l i n e , ( © ) C l - f r e e + cAMP. Results from d i f f e r e n t tissues are shown under each condition. 226 > " 3 0 3 J -50 ->° -70 3 Figure 5.9 E f f e c t of prolonged i n t r a c e l l u l a r i n j e c t i o n of large depolar i z i n g current pulses on the voltage response measured in a d i f f e r e n t c e l l . The distance between current i n j e c t i n g ana voltage sensing electrodes was 42 ym. See Figure 5.7 for d e f i n i t i o n s and explanation. 227 order to c a l c u l a t e e f f e c t i v e input resistance (R )• as described i n the methods section and Figure 1. T r a n s e p i t h e l i a l resistance (R ) and the r e l a t i v e r e s i s -tances of a p i c a l and basal membranes (a)were measured as described previously. Once R , R and a were obtained, a p i c a l and basal membrane resistances (R and et a l . , . 1974; Reuss and Finn, 1974, 1975; Lewis et a l . , 1976; methods of t h i s chapter). Figure 10 shows d e f l e c t i o n s i n a p i c a l membrane p o t e n t i a l as a function of distance from the current i n j e c t i n g microelectrode before and a f t e r serosal ad d i t i o n of 1 mM cAMP i n normal s a l i n e . The b e s t - f i t Bessel functions are also shown as s o l i d l i n e s . Table 2 summarizes the r e s u l t s of cable analysis under control conditions, and during cAMP exposure. Also shown are p a r a c e l l u l a r pathway (R^). The r e s u l t s suggest that locust rectum i s a " t i g h t " epithelium, p a r t i c u l a r l y during cAMP exposure, when approximately 90-95% of passive ion movements across the tissue occur t r a n s c e l l u l a r l y rather than .around the c e l l s . Compared to other e p i t h e l i a l membranes, the e l e c t r i c a l 2 resistance of locust r e c t a l c e l l membranes i s extremely low (36-40 ftcm during cAMP stimulation). T y p i c a l values for e p i t h e l i a l membrane resistance are 2 3,000-5,000 Qcm based on macroscopic area (Fromter, 197 2; Reuss and Finn, 2 1974, 1975) or 7,000-23,000 S]cm when normalized to true membrane area using capacitance measurements (Lewis and Diamond, 1976). This discrepancy w i l l be discussed l a t e r i n t h i s chapter. Addition of 1 mM cAMP causes a large decline i n t r a n s e p i t h e l i a l resistance (.65%) and 80% decreases i n both a p i c a l and basal membrane resistance. The space constant decreased s i g n i f i c a n t l y a f t e r cAMP addition from 420.0 ± 40.6 to 218.8 ± 27.7 pm (P < 0.01). Junctional resistance was d i f f i c u l t to measure i n a R^) and j u n c t i o n a l resistance (R.) could be calculated (Fromter, 1972; Spenney the calculated resistances of a p i c a l and basal membranes and the 2 2 8 D i s t a n c e ( j j r n ) Figure 5.10 Deflections i n V as a function of distance from the point of a i n t r a c e l l u l a r current i n j e c t i o n before and a f t e r addition of cAMP. Measure-ments during cAMP exposure were made at l e a s t 30 min a f t e r addition of 1 mM cAMP to the serosal perfusate. The s o l i d l i n e indicates the best f i t t i n g Bessel function", constants "A" and "A" were obtained by f i t t i n g the data as described i n the text. Table 5.2 E l e c t r i c a l parameters estimated by cable analysis i n normal s a l i n e Preparation R t (Q, cm2) a R z (fi cm2) R X (Kfi) A (mV) A (ym) R a (Q cm2) h (n cir h R J (JJ cm2) Current D i r e c t i o n number of c e l l s Control 1 213.9 0.80 70.0 34.6 1.10 450 126.0 184. 6 687.0 - 14 2 280.4 0.91 90.3 44.6 1.42 400 183.3 178. 0 1252.0 - 16 3 213.0 1.44 95.0 31.4 1.00 550 232.0 160. 8 469.4 - 29 5a 299.4 1.17 118.8 131.9 4.20 300 257.8 220. 3 801.0 + 28 • 6a 256.6 1.35 128.2 8.0 2.55 400 301.3 223. 2 502.4 + 20 6b 1 256.6 1.35 115.6 7.2 2.30 400 271.7 201. 3 560.9 + 21 x ± s.e. 252.7 1.13 100.5 50.3 2.05 420.0 220.1 193. 4 742.4 n = 5 ±17.4 ±0.12 ±10.4 ±21.2 ±0.60 ±40.6 ±30.3 ±12. 2 ±141.1 ho VO + . ImM cAMP 4 63.7 2.05 17.6 44.0 1.4 200 53.7 26.2 314.2 - 26 5b 2 77.9 0.95 15.4 50.2 1.6 175 : 30.1 31.6 801 + 15 6c 3 82.7 0.67 12.6 31.4 1.0 200 21.1 31.3 . 560.9 + 15 7 109.3 1.30 31.1 34.6 1.10 300 71.5 55.0 803.9 + 22 : s.e. 76.7 1.24 19.2 40.1 1.27 218.8 44.10 36.0 559.1 • 2,4 ±15.0 ±0.3 ±4.1 ±4.3 ±0.14 ±27.7 ±11.4 ±6.5 244.9 230 Table 5.2 (cont.) 1 cable analysis was repeated i n order to test the r e p r o d u c i b i l i t y of measurements. 2 and 3 R^ was extremely low during cAMP exposure (15.4, 12.6 Q, cm 2), and approached the l i m i t s of s e n s i t i v i t y of the method. Since small errors resulted i n non-sensical values of R. R and R, i t J a 1>, was necessary to c a l c u l a t e R^ and R^ i n two recta from R^ and a measured during cAMP stimulation and R_. (measured under control conditions),, assuming that R^  was not affected by cAMP. This assumption seems j u s t i f i e d since no consistant e f f e c t of cAMP was observed i n preparations i n which R^ was high enough to allow d i r e c t measurement of Rj (see Table 5.3). 231 cAMP-treated tissue i n normal s a l i n e , however no large changes i n R_. were observed i n Table 2 or i n C l - f r e e s a l i n e when R. was e a s i l v measured. J Figure 11 shows the de f l e c t i o n s i n a p i c a l (AV ) and basal (AV, ) membrane a b potentials during t r a n s e p i t h e l i a l current pulses before and a f t e r adding 1 mM cAMP to the serosal side. Both AV & and AV^ were reduced during cAMP exposure. In order to i d e n t i f y which ions are involved i n these resistance changes, the e f f e c t of cAMP on a was determined i n Cl-free-(gluconate) s a l i n e ( F i g . 12). Only the a p i c a l membrane resistance decreased during cAMP exposure under these conditions, i n d i c a t i n g that resistance changes at the serosal border are C l -dependent. I t may be noted that AV^ a c t u a l l y increased during cAMP exposure. However, t h i s i s to be expected because more current flows t r a n s c e l l u l a r l y when R declines and the increase i n current should cause larger- d e f l e c t i o n s a & i n the basal membrane p o t e n t i a l even though R^ remains constant. Table 3 shows the e f f e c t s of cAMP on resistances under C l - f r e e conditions as determined by cable a n a l y s i s . As i n normal s a l i n e , a p i c a l membrane r e s i s -tance declined by 80% during cAMP exposure. Obviously this change must be due to an increase i n the permeability of the a p i c a l membrane to some ion other than C l . Basal membrane resistance also decreased s l i g h t l y . When expressed as a change i n conductance (AG, ), where R, i s basal membrane resistance before and a f t e r cAMP addition, G, of b b -2 the basal membrane increases 22.6 mmhos cm i n normal s a l i n e (114 mM Cl) and -2 only 0.4 mmhos cm i n C l - f r e e s a l i n e . Since more than 98^ of the cAMP-induced conductance of the basal membrane i s abolished by Cl-removal, the cAMP-induced conductance of the basal membrane probably represents an increase i n C l permeability. (8) 232 C O N T R O L c A M P 5 0 m V 1 m i n Figure 5.11 E f f e c t s of cAMP on the d e f l e c t i o n s i n a p i c a l and basal membrane pote n t i a l s produced by t r a n s e p i t h e l i a l current pulses (voltage d i v i d e r r a t i o ) . Control conditions are shown at l e f t . Results shown at the r i g h t were obtained 30-60 min aft e r adding cAMP to the serosal side. Voltage d e f l e c t i o n s 2 were produced by t r a n s e p i t h e l i a l constant-current pulses (20 pA/0.196 cm ). The numbers shown at l e f t i d e n t i f y the preparation. 233 C l - f r e e c A M P t 50 mV 1 min Figure 5.12 E f f e c t s of cAMP on the voltage d i v i d e r r a t i o i n C l - f r e e s a l i n e . See legend of Figure 5.11 for d e f i n i t i o n s and conditions. 1 n>I cAMP was added to the serosal side at the arrow. 234 0 100 200 300 4 0 0 D i s t a n c e C p m ) Figure 5.13 Effects of cAMP on d e f l e c t i o n s i n V as a function of distance — e a from the point of i n t r a c e l l u l a r current i n j e c t i o n under C l - f r e e conditions. Solid l i n e indicates the b e s t - f i t t i n g Bessel function calculated as described i n the text. Table 5.3 Results of cable analysis i n Cl-f r e e s a l i n e before and a f t e r adding ImM cAMP Preparation R t (n cm2) a R z (n cm 2) R X A (mV) X (pm) R a (Q cm2) *b (Q cm2) R. J (n cm2) Current D i r e c t i o n number of c e l l s Unstimulated 8a 370.7 0 .773 254.5 125.7 4 450 451.2 583.7 577.6 + 22 9a 295.6 1 .160 248.8 69.1 2.2 600 537.4 463.3 419.5 - 15 10a 347.9 0 .386 93.3 149.2 4.75 250 129.3 334.9 1388.6 - •17 11a 390.7 0 .473 159.3 150.8 4.8 325 234.6 496.0 839.8 - 17 12a 438.1 0 .222 138.2 86.4 2.75 400 168.6 759.5 832.0 - 12 x ± s.e. 368.7 0 .603 178.8 116.2 3.70 405 304.2 527.5 811.5 ±23.6 ±0.166 ±31.6 ±16.56 ±0.527 ±59.37 ±80.57 ±70.42 ±164.7 ImM cAMP added 8b 210.1 0.111 33.3 44 1.4 275 37.0 333.3 485.7 + 17 9b 231.0 0.286 127.2 62.8 2.0 450 163.6 572.0 336.7 - 21 10b 243.3 0.118 25.4 113.1 3.6 150 28.4 240.7 2537.7 - 17 12b 350.4 0.0631 33.9 84.8 2.70 200 36.04 571.2 828.4 - 9 x ± s.e. 258.7 0.145 55.0 74.2 2.43 268.8 66.3 429.3 1047.1 n = 4 ±31.3 ±0.049 ±24.2 ±14.9 ±0.47 ±65.7 ±32.5 ±84.3 ±507.4 236 Which i o n i c conductance increases i n the a p i c a l membrane during cAMP 42 stimulation? From measurements of K backflux, i t was concluded i n chapter 3 that t r a n s e p i t h e l i a l potassium permeability ("P„) increases during cAMP K exposure. It seemed reasonable that a p i c a l membrane K permeability (P3.) might increase during cAMP exposure, and that t h i s might account for the increase i n 42 both t r a n s e p i t h e l i a l K permeability and AR under these conditions. cl Figure 14 shows the e f f e c t s of cAMP when both K and C l are removed from the s a l i n e . In contrast to the r e s u l t s i n normal and C l - f r e e s a l i n e (compare Figs. 12 and 13) neither AV nor AV, change following cAMP addition. Table 4 a. D summarizes the r e s u l t s of cable analysis under these conditions. Only one complete cable analysis was successful during cAMP stimulation, although e f f e c t s of cAMP on a were measured i n two preparations. cAMP-induced AR a was abolished under K-free conditions, suggesting that i ) the stimulation of a p i c a l membrane conductance i s due to increased K permeability, i i ) cAMP-42 induced t r a n s e p i t h e l i a l K permeability probably r e s u l t s from this a p i c a l K conductance. I t may be noted that R^ measured under KCl-free conditions i s 2 lower than when the s a l i n e i s only C l - f r e e (107 versus 304 ficm ). The reason for t h i s inconsistency i s that R and R^ are underestimated under KCl-free a t conditions because V & does not reach steady-state values during 1 sec current pulses. This e f f e c t cannot be att r i b u t e d to membrane capacitance since a p i c a l membrane area would have to be 50,000 times greater than the macroscopic 2 tissue area (assuming 1 uF/cm of membrane) i n order to explain the observed time constant. A more de t a i l e d analysis w i l l be required to determine whether the transient i s due to membrane p o l a r i z a t i o n , voltage-dependent conductance or some other mechanism (see Reuss and, Finn, 1977). At present, the important fin d i n g i s that K-removal blocks AR during cAHP-exposure. In summary, the locust rectum i s a tight epithelium with low t r a n s e p i t h e l -i a l resistance. The r e s u l t s show that cAMP exposure produces a K-dependent 237 50 mV 1 min Figure 5.14 E f f e c t s of cAMP on the voltage d i v i d e r r a t i o under KCl-free conditions. See legend of Figure 5.11 for d e f i n i t i o n s and conditions. Table 5.4 E l e c t r i c a l parameters estimated by cable analysis i n KCl-free s a l i n e R t (tt cm2) a R z (tt cm2) R X (Ktt) A (mV) A R a (pm)(tt cm2 R. ) (tt a b a2) R. j (tt cm2) Current D i r e c t i o n number of c e l l s KCl-free 13a 476.4 0. 493 148.4 659.7 21 150 221.6 449 .4 1462.9 - 15 14a 693.0 0. 228 120.2 534.0 17 150 147.6 647 .4 5401.3 - 10 15a 687.3 0. 197z 116.6 518.4 16.5 150 139.6 707 .9 3636.0 - 22 x n=3 618.9 0. 306 128.4 570.5 18.2 150 169.6 601 .6 3560.1 ± s.e. + 71.3 ±0. 094 ±10.1 ±44.5 ±1.4 ±26.1 ±78 .1 ±1085.6 + ImM cAMP 13b 427.3 0. 3820 190.5 292.0 9.7 250 263.3 689 .2 775.0 - 15 See text for d e f i n i t i o n s . 239 increase i n a p i c a l membrane conductance and a Cl-dependent increase i n basal membrane conductance. 6) E f f e c t s of potassium concentration on the t r a n s e p i t h e l i a l 42 permeability to K The concentration-dependence of apparent potassium permeability w a s determined from the mucosa-to-serosa f l u x under I conditions (Fig. 15). The sc ° calculated *P was approximately 5 x '10 cm sec i n the range 0-10 mM [K] , however P„ declined d r a s t i c a l l y when [K] was raised higher. The decrease i n *P„ was half-maximal between 10 and 40 mM [K], and was maximal at 100 mM [K] (Fig. 15). This tracer method of c a l c u l a t i n g K permeability overestimates P is. due to the e f f e c t s of 2 b a r r i e r s i n serie s (see discussion and Schultz and F r i z z e l l , 1976). Nevertheless, such error could not account for the d r a s t i c decline observed i n *P„. The present r e s u l t s are consistent with previous findings that t r a n s e p i t h e l i a l K backflux i s lower than expected i n "high K" saline (chapter 3), and that K addition does not reduce R as anticipated (chapter 2). A decline i n *P K could occur at the a p i c a l or basal membrane or both, since K was added to both sides of the tissue i n the present experiments. In the next section, r e s u l t s of microelectrode experiments i n d i c a t e that the ap i c a l membrane i s the s i t e of concentration-dependent potassium permeability, and that high [K] may exert i t s i n h i b i t o r y e f f e c t s at the luminal surface of the membrane. 7) E f f e c t s of_ mucosal potassium concentration on voltage d i v i d e r r a t i o The e f f e c t s of mucosal [K] on voltage divider r a t i o s , membrane potentials c c and i n t r a c e l l u l a r C l and K a c t i v i t i e s (a... and a ) were determined using an ion-L i K. s e n s i t i v e microelectrode (chapter 4). Although not pertinent i n chapter 4, the voltage d i v i d e r r a t i o (a, or r a t i o of a p i c a l to basal membrane resistance) was measured during these experiments to give insight into the [K.]-dependent changes i n potassium permeability. I n t u i t i v e l y , one would expect a p i c a l 240 Q J E 8 4 4 2 A o J 0 5 0 1 0 0 1 5 0 B i l a t e r a l K c o n e . C m M ) 2* m o Figure 5.15 Apparent t r a n s e p i t h e l i a l potassium permeability *P„ and K •— K. backflux under I conditions as a function of b i l a t e r a l concentration. *P„ sc K was calculated using equation 13 i n text. Tissues were e q u i l i b r a t e d i n K-free s a l i n e and exposed to 1 mM cAMP on the serosal side. Serosa-to-mucosa 42 flux of K was measured during two 15-min i n t e r v a l s under I conditions, sc however only the second f l u x period at each K concentration was used i n c a l c u l a t i o n s . Potassium concentration was elevated by adding K-methylsulfate b i l a t e r a l l y under I conditions to give the concentrations shown on the sc ° abscissa. Means ± s.e.; n = 6. 241 membrane resistance (and a) to decline when [K] i s elevated on the mucosal side. In this section, a-simple equation i s derived to allow comparison of the measured values of a with those predicted i f K and C l permeability c o e f f i c i e n t s remained constant. Deviations from the predicted behaviour i s then used as an i n d i c a t i o n of changes i n i o n i c permeability. Sodium was not included i n these c a l c u l a t i o n s since Na conductance i s a minor f r a c t i o n of a p i c a l and basal membrane conductance (less than 15% and 7%, re s p e c t i v e l y , according to cable a n a l y s i s ) . Further evidence for high K permeability comes from the observation that i n t r a c e l l u l a r p o t e n t i a l varies approximately 50 mV/decade change i n s a l i n e K a c t i v i t y under I conditions, close to the value predicted for a perfect K electrode (Table 5 of chapter 4). As a r e s u l t of low i n t r a c e l l u l a r Na a c t i v i t y and membrane permeability, sodium would not be expected to a f f e c t a s i g n i f i c a n t l y . If K and C l account for most of the a p i c a l membrane conductance, then that f r a c t i o n due to K i s ^ J ^ c V = 1 + P K / P C 1 . <9> where P^ P3, are K and C l permeabilities of the a p i c a l membranes (cm sec "*") . K C l Similar expressions are obtained for both ions at mucosal and serosal membranes. During cAMP exposure, P^ - 16 P^ and P^ = 9 P5, according to the r e s u l t s of K L i L i K. cable analysis (Tables 2 and 3). Values of a are then predicted as a = [<1 + P j / P ^ K + V + # P 2 l ) S C l ] / C ( 1 + PK / PC1 ) 5K + < 1 > + PC1 / PK ) 5K <10> a b where P., P. are the permeabilities of the a p i c a l and basal membranes to ion i i _a -b " i " ; a., a. are the logarithmic mean a c t i v i t i e s of ion " i " i n the a p i c a l and i i basal membranes, calculated as v c a. - a. l l In a? l a. I 2 4 2 where a. and a. are i n t r a c e l l u l a r and e x t r a c e l l u l a r a c t i v i t i e s of ion " i " , r e s p e c t i v e l y . This r e l a t i o n s h i p predicts a i f i ) most of the a p i c a l and basal membrane conductance i s due to K and C l , and i i ) permeability c o e f f i c i e n t s do not change.during addition of K to the mucosal side. Figure 1 6 shows the e f f e c t s of mucosal K addition on a and the values of a predicted using equation ( 1 0 ) . The values of a and i n t r a c e l l u l a r ion a c t i v i t i e s used i n the c a l c u l a t i o n are those i n Figure 6 of chapter 4 . When mucosal K a c t i v i t y was elevated, a increased 5 - f o l d instead of decreasing as predicted i f permeability c o e f f i c i e n t s remained constant. I t might be argued that higher values of a could r e s u l t from a decline i n rather than an increase i n R &. Ce r t a i n l y some decline i n R^ i s expected since i n t r a c e l l u l a r ion a c t i v i t i e s increase when mucosal [ K] i s elevated. Regardless, there must s t i l l be an increase i n R when K i s added, even i f R, does decline, because a '• b ' R remains constant at 1 1 0 - 1 2 0 Qcm under these conditions when [ K ] i s between 6 and 1 0 0 mM ( F i g . 1 6 ) . Since v i r t u a l l y a l l of the a p i c a l membrane conductance i s due to K ( ^ 9 6 % during cAMP stimulation, from Table 2 ) , the increase i n R a. during mucosal K addition indicates a decline i n K permeability of the a p i c a l membrane. 8) E f f e c t s of cAMP concentration on s h o r t - c i r c u i t current  and t r a n s e p i t h e l i a l conductance S h o r t - c i r c u i t current (I c ) and t r a n s e p i t h e l i a l conductance (G ) were measured as a function of cAMP concentration for several reasons. F i r s t , i f stimulations of G and I by cAMP r e f l e c t two d i f f e r e n t processes, this might t sc become obvious i f G and I had d i f f e r e n t dose-response curves. Second, a t sc 1 dose-response r e l a t i o n s h i p for the Cl-dependent e f f e c t s could be calculated by comparing the r e s u l t s i n normal and C l - f r e e s a l i n e . F i n a l l y , the r e l a t i o n s h i p between I g c and G may be used to determine the d r i v i n g force of C l transport and the "shunt" conductance under c e r t a i n conditions. 243 E u G Qi 3 0 0 -\ 200 H 100 H >«•>« 5 -\ D 0) > O) o ~o > 2 H 1 i o - J — ! « 1 — r - i I  i i 11—• r — — i — i — i — r 0 1 2 6 10 20 •1 n 60 100 M u c o s a l K a c t i v i t y ( m M ) Figure 5.16 Relationship between mucosal K concentration and the voltage divider r a t i o i n cAMP-stimulated recta during serosal perfusion of recta with normal saline. Voltage divider ratios were predicted using equation (10) of the text and i ) measured values of i n t r a c e l l u l a r ion a c t i v i t i e s , and i i ) par-t i a l ionic conductances estimated by cable analysis during ion substitutions. Results are from one ty p i c a l preparation; see text for f u l l d e t a i l s . Means ± s.e.; n = 20 observations. 244 Figure 17 shows the e f f e c t s of cAMP concentration on G and I i n normal t sc and C l - f r e e s a l i n e . Tissues were kept under s h o r t - c i r c u i t conditions except at 15 min i n t e r v a l s , when I was turned o f f for 1.5 min to allow measurement sc of t r a n s e p i t h e l i a l p o t e n t i a l (V ) . Conductance was calculated as l s c / v t * Both I and G had "S" shaped dose-response curves with s i m i l a r threshold doses (5 x 10 ~* M cAMP) and maximal doses of approximately 1 mM cAMP. These r e s u l t s i n d i c a t e that Cl-dependent and Cl-independent conductances have s i m i l a r cAMP s e n s i t i v i t y (with the reasonable assumption that membrane permeability to cAMP i s not affected by C l removal). The value of R obtained under C l - f r e e condi-tions i n th i s experiment compares well with that obtained by cable analysis (see Table 3). In summary, these data i n d i c a t e that AR^ during cAMP exposure r e s u l t s from an increase i n a p i c a l membrane K conductance, a find i n g which i s consistent with the cAMP-induced increase i n t r a n s e p i t h e l i a l potassium permea-42 b i l i t y observed i n chapter 3 using K fluxes. Discussion We begin t h i s s ection by discussing whether locust rectum i s a " t i g h t " or "leaky" epithelium. Three factors which have been found to regulate permeabil-i t y i n th i s tissue are described: cAMP, K concentration, and osmotic pressure. An equivalent e l e c t r i c a l c i r c u i t model i s then derived- from measurements of fluxes, i n t r a c e l l u l a r ions and membrane potentials (chapter 4) and cable analysis (this chapter), and the model i s used to predict the dr i v i n g force of Cl Cl transport (E ). This p r e d i c t i o n i s then tested by an independent method. Locust rectum: a. " t i g h t " epithelium with low e l e c t r i c a l resistance Several observations suggest that locust rectum i s a " t i g h t " epithelium. F i r s t , d e f l e c t i o n s i n V produced by t r a n s e p i t h e l i a l s a l t gradients were strongly dependent on the d i r e c t i o n of the gradient, i n d i c a t i v e of two b a r r i e r s i n s e r i e s having d i f f e r e n t properties. When s a l t concentration was lowered on 245 CM ' E o to E 6 J 4 J O 2 0 1 2 CM 'E u CT LU 3. 1 0 H 8 i 6 H 4 H 0 -J I r •5 N o r m a l J J© S a l i n e ©l® I •II1 C l - f r e e S a l i n e N o r m a l — i --4 - 3 l o g c A M P c o n e . C M ) Figure 5.17 Log dose-response curve showing the r e l a t i o n s h i p between cAMP concentration and both t r a n s e p i t h e l i a l conductance (G ) and s h o r t - c i r c u i t current (I ). Tissues were bathed i n normal and C l - f r e e s a l i n e s (Table 2.1 sc and gluconate-substituted). Means ± s.e.; n = 6 recta i n normal s a l i n e , 4 r e c t a i n C l - f r e e s a l i n e . 246 the mucosal side, AV indicated high cation s e l e c t i v i t y over C l . However, the e f f e c t s of lower serosal s a l t concentration suggest that the permeability of the basal border i s more C l - s e l e c t i v e . Such asymmetry i s not expected i f ion d i f f u s i o n occurs through a s i n g l e ( p a r a c e l l u l a r ) b a r r i e r , since tight junctions are not known to " r e c t i f y " (e.g. proximal tubule; Boulpaep and Seely, 1971; Fromter et a l . , 1971). In contrast, e p i t h e l i a which are known to be t i g h t i n v a r i a b l y have asymmetrical responses to t r a n s e p i t h e l i a l s a l t gradients (for references see Fromter and Diamond, 1972). In order to minimize possible streaming p o t e n t i a l s , sucrose was added to maintain isosmocity when the con-centration of s a l t was reduced on one side. The e f f e c t i v e osmotic pressure i n mixed solutions i s not known with c e r t a i n t y since the osmotic r e f l e c t i o n c o e f f i c i e n t at the t i g h t j unction probably varies for each solute. Also, some acti v e water absorption might entrain K even i f mucosal and serosal sides were isosmotic. However, the f a c t that nearly symmetrical r e s u l t s were obtained with K-methylsulfate gradients, which also employed sucrose as an osmotic e f f e c t o r , suggests that the asymmetry i s not some a r t i f a c t of using sucrose. No attempt was made to measure streaming potentials due to active water uptake which i s thought to occur by l o c a l i z e d r e c y c l i n g of solutes i n the l a t e r a l i n t e r c e l l u l a r spaces ( P h i l l i p s , 1970; Wall and Oschman, 1970; Goh and P h i l l i p s , 1978) . Permeability r a t i o s c a l c ulated from Figure 4 probably underestimate the s e l e c t i v i t y of the c e l l membranes, since even s l i g h t p a r a c e l l u l a r shunting would place an upper l i m i t on transference numbers. For example, the mucosal r a t i o of t / t n (3.0) i s lower than t / t (4.7), which would suggest that K Cl Ma C l Na permeability of the a p i c a l membrane i s greater than that of K. However, cable analysis (Tables 2-4) and the e f f e c t s of K on membrane p o t e n t i a l s , i n d i c a t e that the opposite i s true (P /P < 0.06). In summary, transference 247 numbers i n these experiments are not quantitative measures of permeability because of f i n i t e p a r a c e l l u l a r shunting, however, they do suggest that a s i g n i f i c a n t f r a c t i o n of t r a n s e p i t h e l i a l ion d i f f u s i o n occurs through the c e l l s . Voltage scanning did not detect any low-resistance regions ( F i g . 5). The same negative r e s u l t has been obtained i n tight e p i t h e l i a including urinary bladders of Necturus (Higgins et a l . , 1975) and rabbi t (Lewis and Diamond, 1976). This technique i s q u a l i t a t i v e i n nature, and there i s the p o s s i b i l i t y that small leaks could go undetected (see r e s u l t s ) . However, p a r a c e l l u l a r shunting i s detectable i n at le a s t one leaky epithelium, Necturus gallbladder (Fromter and Diamond, 1972; Frb'mter, 1972). Cable analysis of the epithelium indicates that locust rectum i s moderate-l y " t i g h t " under c o n t r o l , unstimulated conditions, when ^60% of the transepi-t h e l i a l conductance i s t r a n s c e l l u l a r . The epithelium becomes " t i g h t e r " during cAMP stimulation, .when t r a n s c e l l u l a r conductance ranges between 89 and 96% of the t o t a l . The f r a c t i o n a l conductance of the t r a n s c e l l u l a r pathway of locust rectum under these conditions resembles that of other t i g h t e p i t h e l i a (53%, toad urinary bladder, Reuss and Finn, 1975; 65-96%, rabbit urinary bladder, Lewis and Diamond, 1976; 80%, Necturus stomach, Spenney et a l . , 1974; 31%, rabbit cornea, Marshall and Klyce, 1981) but i s much higher than that of leaky e p i t h e l i a (^4%, Necturus gallbladder, Fromter, 1971; ^ 1%, Necturus proximal tubule, A s t e r i t a and Boulpaep, 1978; see Boulpaep, 1979, for references). Further evidence that locust rectum i s " t i g h t " comes from the large de-crease i n t r a n s e p i t h e l i a l resistance during cAMP stimulation (50-65%). cAMP i s not known to increase tight j u n c t i o n a l conductance so d r a s t i c a l l y i n leaky e p i -t h e l i a , although a small, cAMP-dependent reduction of t i g h t - j u n c t i o n a l conduc-tance has been reported i n Necturus gallbladder (Duffey et a l . , 1981; see review 35 by Powell, 1981). F i n a l l y , t r a n s e p i t h e l i a l S0 A permeability i s extremely low 248 both i n unstimulated r e c t a , and a f t e r exposure to 1 mM cAMP, a fin d i n g which i s inconsistent with d i f f u s i o n through w a t e r - f i l l e d channels of low or moderate s e l e c t i v i t y ( i . e . t i g h t j u n c t i o n s ) . Results of early studies suggested that septate and gap junctions occlude the l a t e r a l i n t e r c e l l u l a r space i n insect r e c t a l e p i t h e l i a (Gupta and Berridge, 1966; Noirot-Timothee and Noirot, 1967; Oschman and Wall, 1969), although more recent work suggests that these junc-tions are patent to i o n i c lanthanum i n cockroach and blowfly rectum (Lane, 1979). True t i g h t junctions have also been reported at the basal ends of the l a t e r a l i n t e r c e l l u l a r spaces i n these tissues (Lane, 1979). Most passive ion fluxes are believed to occur t r a n s c e l l u l a r l y i n insect e p i t h e l i a , although this, has not been measured d i r e c t l y . Berridge et a l . (1975) found the t r a n s e p i t h e l i a l resistance of blowfly s a l i v a r y gland to be 2 2 approximately 80 flcm at r e s t , d e c l i n i n g to 5.5 Hem during stimulation with 5 HT. Such enormous resistance changes would not be expected i f p a r a c e l l u l a r shunting was s i g n i f i c a n t . Furthermore, i f blowfly s a l i v a r y glands have s p e c i f i c membrane resistances on the same order as calculated for basal 2 membranes of other insect s a l i v a r y glands (Drosophila, ^10,000 ttcm , normalized to membrane area as estimated from electromicrographs, Loewenstein and Kanno, 2 1964; and Chironomus, ^9,000 ftcm , normalized, Loewenstein et a l . , 1965), then most conductance would be p a r a c e l l u l a r . Exposure of blowfly glands to 5-HT r e s u l t s i n a 20 mV depo l a r i z a t i o n of V but only a s l i g h t d e p o l a r i z a t i o n of V, (2-3 mV) (Berridge and Prince, 1972). This observation suggests that the b epithelium i s reasonably t i g h t , since large p a r a c e l l u l a r currents would be expected to deporalize more dramatically, although the small change in basal membrane p o t e n t i a l i s i n the r i g h t d i r e c t i o n to be explained by pa r a c e l l u l a r current. Furthermore, the small AV^ does not necessarily show high j u n c t i o n a l resistance since 5 HT might a l t e r the equivalent EMF at the basal membrane so as to counteract a depolarizing p a r a c e l l u l a r current. 249 In s h o r t , these observations provide i n d i r e c t evidence that the p a r a c e l l u l a r pathway i s " t i g h t " i n insect s a l i v a r y glands. Malpighian tubules are also thought to have low j u n c t i o n a l permeability (see Maddrell, 1980) and Loewen-s t e i n et a l . (1965) have measured high basal membrane resistance i n Chironomus 2 Malpighian tubules (3,000 Hem , normalized to membrane area). Lepidopteran midgut i s also presumed to be a tight epithelium with low 2 e l e c t r i c a l resistance (^150 Hem ; Wood and Moreton, 1978). Also, from Blanke-meyer's data (1978), one may c a l c u l a t e a f l u x r a t i o of ^ 50 i n th i s t i s s u e , a value which i s si m i l a r to other t i g h t e p i t h e l i a when mounted without edge damage (e.g. >100, frog skin, O'Neil and Helman, 1976; 30, rabbit bladder, Lewis and Diamond, 1976a). F i n a l l y , there i s one other report of e p i t h e l i a l cable analysis i n the s e n s i l l a of the waxmoth c a t e r p i l l a r , although the data have not yet been published ( E r l e r and Thurm, i n preparation, c i t e d i n Thurm and Kuppers, 1980). B e c a u s e of its" sensory function the sensillum might not be t y p i c a l of insect transporting e p i t h e l i a . Tightness may be a general c h a r a c t e r i s t i c of insect e p i t h e l i a , which generally have high r a t i o s of membrane/junctional area due to large transporting c e l l s and microscopic i n f o l d i n g of membrane, p a r t i c u l a r l y of the l a t e r a l c e l l border which i s usually very complex and associated with mitochondria. I t must be re-emphasized that the present study cannot d i s t i n g u i s h l o c a l secre-ti o n and absorption processes at the l a t e r a l border. Further studies involving lanthanum t r a c e r / e l e c t r o n microscope and"electron microprobe are needed to answer these questions i n locust r e c t a l epithelium. Regulation of passive permeability i ) cAMP Addition of cAMP, cGMP or theophylline stimulates I (10-fold) and trans-e p i t h e l i a l conductance (>60%). When the voltage d e f l e c t i o n s across a p i c a l and basal membranes during t r a n s e p i t h e l i a l current pulses are monitored before and 250 a f t e r cAMP addition, both a p i c a l and basal voltage d e f l e c t i o n s are greatly-reduced, suggesting a decline i n a p i c a l and basal membrane resistances. This conclusion was confirmed by measuring the r a d i a l spread of i n t r a c e l l u l a r current. Since AR^ was nearly abolished i n C l - f r e e s a l i n e whereas AR & was In-dependent, the simplest explanation for these observations i s that cAMP -2 increases a p i c a l membrane conductance to K by 18.1 mmhos cm , and increases -2 basal membrane Cl-conductance by approximately 19.3 mmhos cm How do these cAMP stimulations of membrane conductance compare with membrane and t r a n s e p i t h e l i a l permeabilities calculated using tracer f l u x measurements? Table 5 shows rough estimates of membrane and t r a n s e p i t h e l i a l permeability to K and C l , before and during exposure to 1 mM cAMP. Three methods were used to c a l c u l a t e membrane perm e a b i l i t i e s . These are i l l u s t r a t e d using potassium conductance of the a p i c a l membrane as an example: exp(V F/RT) - 1 Method 1: Tv = J K ^  £ ± - - (11) K net V F c / T T „ / T, m. m a a exp(V aF/RT) - a R where J i s the net f l u x of K . under open-circuit conditions, V i s the net r a c m ap i c a l membrane p o t e n t i a l , a^ and are potassium a c t i v i t i e s i n the c e l l and mucosal solutions, r e s p e c t i v e l y , and R, T and F have t h e i r usual meanings. A c t i v i t i e s of K and C l i n the s a l i n e were 7.2 and 82 mM, res p e c t i v e l y , as measured using i o n - s e n s i t i v e microelectrodes (chapter 4). A l l other parameters used i n the c a l c u l a t i o n are l i s t e d i n Table 6a. G K Method 2: PT = ?±-=—± (12) K 2 2 _a Z F a K where G i s the potassium conductance of the membrane as estimated from cable a r _a a n a l y s i s , and a i s the logarithmic mean a c t i v i t y of K i n the a p i c a l membrane calculated as (a^ - a™)/(In a^ - In a™). K K. K K Table 5.5(a) Summary of previous tracer and microelectrode results used i n calculating permeabilities Tk Cl „ ac c - K Cl rK Cl rK Cl K „C1 J J V V, a a a a. J ^ J G G G, G, G G , . . sm sm a b K Cl K Cl net net a a b b t t Condition _2 _^ _2 _i -2 (yEqcm h ) (mV) (mM) (pEqcm s ) (mScm ) Control 0.351 1.47 -58 +51 61 31 25.18 52.4 2.39 131 3.2 1.2 1.9 3.3 1.19 0.88 cAMP added 1.564 1.67 -70 +38 70 47 27.60 62.9 1250 1194 21.3 1.4 5.2 22.6 4.2 1.3 (b) Permeabilities (PxlO cm sec ) calculated by various methods Apical Membrane Basal Membrane Transepithelial P a P a Cl P b P b Cl PK p c i Method 1 2 1 2 1 2 1 2 1 2 3 2 3 Control cAMP added 0.85 3.0 14.8 20.2 - 0.6 - 0.5 8.7 2 5.1 4.9 0.29 1.64 2.14 9.38 0.43 ' 3.80 1.20 3.94 1.35 ±0.19 6.03 0.439 0.50 ±0.05 0.546 0.57 ±0.69 ±0.09 1 Method 1: Permeabilities were calculated from the Goldman constant f i e l d equation for net fl u x using membrane potentials (V V ), ion a c t i v i t i e s (a.), and net tracer . a, D I fluxes (open-circuit conditions, J n e t ) i n Table 5.6(a) Method 2: calculated according to the conductance equation using logarithmic mean a c t i v i t i e s (a) and p a r t i a l i o n i c conductances (G 1 G?"), i n Table 5.6(a) a. $ u Method 3: calculated from t r a n s e p i t h e l i a l backflux under s h o r t - c i r c u i t conditions K C l — (J J ) and a c t i v i t i e s of each ion i n the s a l i n e , x ± s.e., n=6 recta sm, sm See text for further explanation. ro ho 253 Method 3: The simplest (but le a s t accurate) approach allows c a l c u l a t i o n of t r a n s e p i t h e l i a l permeability only: * ? v = J K I at (13) K sm K where i s the serosa-to-mucosa f l u x under I conditions and af, i s the sm sc K a c t i v i t y of K on the serosal side. A p p l i c a t i o n of method 1 (Ml) assumes a constant f i e l d i n the membrane (Goldman, 1943; Hodgkinand Katz, 1949), method 3 (M3) assumes a lack of isotope i n t e r a c t i o n s (Essig and L i , 1975) , and a l l three methods require ion independence, i . e . a lack of coupling to the flows of solvent or to other solutes and also the absence of s i g n i f i c a n t a c t i v e transport. A p i c a l membrane K permeability increases dramatically a f t e r cAMP addition according to methods 1 and 2 (Table 5b). P ^ changes very l i t t l e although i t was not possible to use method 1 to c a l c u l a t e P since the net f l u x across this membrane i s due to an active pump (shown i n chapter 4). In contrast to the a p i c a l membrane, P^ changed l i t t l e during cAMP exposure (decreasing somewhat according to Ml, increasing somewhat according to M2), whereas both methods revealed a 6-fold increase i n basal membrane C l permeability. More experiments are needed to explain why P^ i s 4-fold higher when calculated from the conduc-tance equation (12; M2) than when the Goldman equation i s used (11; Ml) before and a f t e r addition of cAMP. The important f i n d i n g i s that C l conductance of the basal membrane -2 -2 -1 increases ^21 mS cm . Net Cl fl u x increases by approximately 4-5 pEqcm h during cAMP exposure under open-circuit conditions (chapter 2), while the net electrochemical gradient favouring C l ex i t across the basal membrane A p ^ -2 -1 i s ^20 mV (chapter 4). Converting the net C l fl u x to current, 5 pEqcm h _2 becomes 134 uA cm . The minimum increase i n basal membrane C l conductance which would be required f o r passive Cl e f f l u x by e l e c t r o d i f f u s i o n i s 254 (134.1 x 10 A cm" )/(20 x 10" V) = 6.7 mS cm" or only 30% of the observed increase i n basal membrane conductance during cAMP exposure. Even af t e r sub-t r a c t i n g the possible error caused by i n t e r f e r i n g i n t r a c e l l u l a r anions on a^ (4.9 mM) and allowing for a 36% higher rate of t r a n s e p i t h e l i a l Cl transport which i s indicated by the low value of R i n these experiments (from -2 -1 -2 F i g . 21, I would be 13.6 pEqcm h at G = 13.0 mS cm as compared to S C L -2 -1 10 pEqcm h obtained previously i n chapter 2), the increase i n basal membrane conductance according to cable analysis i s s t i l l more than twice that required for passive C l e x i t by e l e c t r o d i f f u s i o n . a b During cAMP exposure, estimates of P and P from a p p l i c a t i o n of Ml and K. K M2 agree more c l o s e l y than under c o n t r o l conditions. In using method 1, the 4^ 2 36 assumption was made that a l l of the net flux of K and C l under open-circuit conditions i s t r a n c e l l u l a r . Ignoring the transjunctional fluxes should cause some error i n unstimulated r e c t a since 35-40% of a l l passive ion movements occur p a r a c e l l u l a r l y . However, during cAMP stimulation t h i s j u n c t i o n a l component of t r a n s e p i t h e l i a l permeability drops to ^5% of the t o t a l , and the assumption that net K f l u x i s t r a n s c e l l u l a r i s then r e a l i s t i c . In summary, permeability estimates based on net fluxes (method 1, the Goldman equation) w i l l improve as the epithelium e f f e c t i v e l y becomes " t i g h t e r " during cAMP stimulation. T r a n s e p i t h e l i a l K permeabilities compare well when determined using d i f f e r -ent methods (Table 5b). T r a n s e p i t h e l i a l P,. was calculated from P = P a P b/P a + P b (14) K K V K K K J using method 1 and method 2. As expected, P„ i s higher according to M3 than by is. methods 1 and 2. Schultz and F r i z z e l l (1976) have predicted that an o v e r e s t i -mate of t r a n s e p i t h e l i a l permeability should occur using M3 because the method 2 5 5 T i m e C h 3 Figure 5 . 1 8 E f f e c t of cAMP on apparent t r a n s e p i t h e l i a l potassium permeabil-i t y ( " F j r ) i n various s a l i n e s . A l l sali n e s were present b i l a t e r a l l y ; high-K ( 1 4 0 mM K), C l - f r e e ( 1 0 mM K) and normal ( 1 0 mM K). Note that the 4 2 e f f e c t of cAMP on *P^ i s blocked i n high-K s a l i n e . O r i g i n a l K flux data are shown i n Figures 3 . 1 3 , 3 . 1 4 and 3 . 1 7 . Means ± s.e.; n = 6 . 256 considers the epithelium as a single d i f f u s i o n b a r r i e r rather than two mem-branes i n s e r i e s . Nevertheless, by comparison with r e s u l t s obtained using Ml and M2,fluxes under s h o r t - c i r c u i t conditions do provide a good i n d i c a t i o n of the e f f e c t s of cAMP and an excellent estimate of P Q ^ - Also, the fact that t r a n s e p i t h e l i a l K and C l permeabilities can be estimated c l o s e l y by the conductance equation strongly suggests that e l e c t r i c a l l y " s i l e n t " pathways such as Cl/HCO^ exchange and NaCl cotransport are not s i g n i f i c a n t . Further evidence that tracer backflux under I conditions i s a reasonable measure of sc t r a n s e p i t h e l i a l permeability w i l l be described i n the next section. • i i ) Inhibition of by high [K] Several observations are consistent with the idea that t r a n s e p i t h e l i a l K permeability i s reduced when K concentration i s increased to normal physio-l o g i c a l l e v e l s (140 mM i n primary urine secreted by Malpighian tubules Chapter 2). F i r s t , t r a n s e p i t h e l i a l resistance remains constant when [ K] i s rais e d on both sides from 40 to 200 mM, although R would be expected to 42 decline as i o n i c strength increases.. Second, K backflux. i n high K (140 mM) s a l i n e does not increase during cAMP stimulation as observed i n normal (10 mM) s a l i n e . This observation suggests that cAMP-induced AP^ i s blocked by 2 2 high [ K] . Third, *P (as estimated from the serosa-to-mucosa f l u x of K ) i s is. much lower i n " h i g h - K " s a l i n e than i n normal (10 mM K ) s a l i n e . Figure 18 shows the e f f e c t s of cAMP on *P as calculated from f l u x data (see Fig s . 14 is. and 18 i n chapter 3). When compared to normal s a l i n e , *P i s 70% lower i n " h i g h - K " s a l i n e , and i s not stimulated by cAMP. When the error due to trea t i n g the epithelium as a single b a r r i e r i s incorporated into *P„, this is. apparent drop i n permeability might be diminished by ^40% as a r e s u l t of membrane depolarization, but.this would s t i l l not account for the d r a s t i c reduction i n *P_, observed i n h i g h - K s a l i n e . F i n a l l y , an i n d i r e c t i n h i b i t o r y 257 e f f e c t on metabolism by high [K] i s u n l i k e l y since a c t i v e C l transport responds normally to cAMP i n "high-K" s a l i n e (see F i g . 7 i n chapter 2). The fact that cAMP-stimulated P K i s blocked at high [K] whereas cAMP-stimulated C l transport C l i s not affected implies that AP„ and AJ are independent processes which are K net turned on by cAMP. Microelectrode data also i n d i c a t e that concentration-dependent P„ occurs K at the a p i c a l membrane. The e f f e c t s of mucosal K addition on t r a n s e p i t h e l i a l resistance (Rfc) and a may be calculated from equation (12) i f i n t r a c e l l u l a r ion a c t i v i t i e s are measured and membrane permeabilities remain constant. S u r p r i s i n g l y , when K was added to the mucosal side, a increased (at external [K] = 40-140 mM) while R- remained constant. This shows that a p i c a l membrane conductance declined i n the presence of high mucosal [K]. Since potassium conductance i s ^ 96% of the t o t a l a p i c a l membrane conductance, t h i s decline i n membrane conductance must r e s u l t from a decrease i n K conductance at the a p i c a l membrane. As explained i n chapter 3, t h i s regulatory mechanism would prevent the r e c t a l lumen from becoming e l e c t r i c a l l y negative to the'hemolymph. One possible advantage of maintaining a p o s i t i v e lumen could be to reduce Na loss from the hemolymph to the faeces. Na i s low i n dietary plant matter (see chapter 2). F i n a l l y , any change i n K permeability under open-circuit conditions w i l l a l t e r the rate of C l absorption since K i s probably the only s i g n i f i c a n t counter-ion for C l transport i n vivo. These r e s u l t s together i n d i c a t e that P., i s "up-regulated" by cAMP and "down-regulated" by high mucosal [K]. By varying the conductance of the a p i c a l membrane, these factors would provide a t h i r d , i n d i r e c t mechanism for regulat-ing active C l transport i n addi t i o n to d i r e c t stimulation by cAMP and mucosal K as described i n chapters 2 and 3. 258 Equivalent e l e c t r i c a l c i r c u i t model The e l e c t r i c a l properties of e p i t h e l i a are u s e f u l l y described using an equivalent e l e c t r i c a l c i r c u i t model. Figure 2 shows the simplest model applicable to e p i t h e l i a . The contribution of the c u t i c u l a r intima to transepi-2 t h e l i a l r esistance i s low i n i n t a c t t i s s u e (^ 8 Qcm ; Hanrahan, unpub. obs.). Since the layer was dissected off during cable analysis, i t i s not included i n the following c a l c u l a t i o n s . S i m i l a r l y , muscle and secondary c e l l layers were also not included since the series resistance of t h i s layer i s probably low because i ) they are penetrated by tracheoles and are not continuous, i i ) they allow cAMP to penetrate within seconds as indicated by the almost instantaneous response of I a f t e r cAMP addition. Each of the three major b a r r i e r s ( a p i c a l and basal membranes and t i g h t junctions) are replaced with a r e s i s t o r and an electromotive force, or "Thevenin equivalent". The a p i c a l and basal membrane potentials (V and V, ) and t r a n s e p i t h e l i a l p o t e n t i a l (V ) are: . V a = [ E a ( R b + V + R a ( E b + E j ) ] / R a + R b + R j ( 1 5> V b " [ E b ( R a + V + \ <Ea + E j ^ / R a + R b + R j <16> V t " ( V a - V R j / R a + \ + R j <17> with p o l a r i t i e s of V^, Vfe and E b r e l a t i v e to the i n t r a c e l l u l a r compartment, and Vfc and E r e l a t i v e to the serosal side. With these p o l a r i t i e s , the equivalent electromotive force across a p i c a l (E ) and basal (E, ) membranes i s a b E a - V a " \ V R j ^ E b = V b + \ R b / R j (19) It i s assumed i n the following c a l c u l a t i o n s that E. =0, which i s reason-3 able because i d e n t i c a l solutions were used on both sides of the epithelium. However, a small E^ would e x i s t i f ions are more concentrated i n the l a t e r a l 259 space. There are several reports of elevated [ K ] i n the l a t e r a l spaces of insect r e c t a . Wall and Oschman ( 1 9 7 0 ) found higher [ K] i n the i n t e r c e l l u l a r sinuses of cockroach rectum than i n hemolymph, probably the r e s u l t of l o c a l K 'secretion into the space as part of a solute r e c y c l i n g mechanism or a l t e r n a t i v e l y , e q u i l i b r a t i o n with K - r i c h f l u i d i n the lumen through imperfectly tight junctions. Also, i n r e c t a l p a p i l l a e of hydrated blowfly, [ K] of the i n t e r c e l l u l a r sinus i s higher than both lumen and hemolymph according to electron microprobe r e s u l t s (animal I; Gupta et a l . , 1 9 8 0 ) , again suggesting a l o c a l s e c r e t i o n of K . These studies used f r e s h l y dissected tissues whereas the present microelectrode study was performed i n v i t r o using vigorously perfused locust recta; i . e . conditions which might d i s s i p a t e normal i n vivo gradients. No unusually high ion a c t i v i t i e s i n d i c a t i v e of l a t e r a l spaces were observed i n thousands of impalements. One might expect to impale the sinuses occasionally despite t h e i r small s i z e (<5 pm). Nevertheless the r e s u l t s i n this study provide no d i r e c t evidence for or against the existence of high concentrations of s a l t i n the l a t e r a l spaces. If concentration gradients between l a t e r a l spaces produce a s i g n i f i c a n t E^ ., t h i s EMF might be expected to "run down" gradually during i n h i b i t i o n of metabolism by azide. The component of V. and I which i s due to E. would t sc 2 also be expected to decay more gradually than an active pump mechanism per se. However, azide r a p i d l y and completely abolishes V^ . and I (see chapter 2), which suggests that any E. which does e x i s t i s either very small, or extremely s e n s i t i v e to metabolic i n h i b i t o r s . Table 6 shows the equivalent EMF's for a p i c a l and basal membranes under control conditions, during exposure to 1 mM cAMP, and cAMP-stimulation i n C l -free s a l i n e . Membrane and j u n c t i o n a l resistances from Table 2 and Table 3 were used to c a l c u l a t e R /R., and RK/R^• Since s l i g h t l y d i f f e r e n t values of V a 260 Table 5.6 . .Calculation of steady-state equivalent electromotive forces (E and E respectively) a D , across a p i c a l and basal membranes Saline a b t a j l i j a b (mV) (mV) a) Control 57.0 48.4 8.6 0.2965 0.2605 54.5 50.6 +cAMP 63.8 34.0 29.8 0.0711 0.0581 61.7 32.3 Cl - f r e e 45.7 41.0 4.7 0.0633 0.4100 45.4 42.9 +cAMP  b) Control 57.8 50.6 7.2 55.7 52.5 +cAMP 70.2 38.0 32.2 67.9 39.9 Cl - f r e e 56.7 50.4 6.3 56.3 53.0 +cAMP  a) voltages from Table 4.2, resistance r a t i o s calculated from Table 5.2 b) voltages from Table 4.3, resistance r a t i o s from Table 5.3 1 Recta bathed i n normal sa l i n e (see Table 2.1) and C l - f r e e s a l i n e i n which a l l C l was replaced with gluconate. V V a p i c a l and basal membrane p o t e n t i a l r e s p e c t i v e l y , a, b, (with respect to the i n t r a c e l l u l a r compartment) , (with respect to the serosal s i d e ) . 261 and were obtained during C l and K microelectrode experiments (chapter 4, Tables 2 and 3), membrane,- E & , and EMF's were calculated separately for the two sets of data using equations (18) and (19). In both cases, addition of 1 mM cAMP increased E by 7-12 mv ( i . e . i n t r a c e l l u l a r side of the a p i c a l membrane became more negative) as would be expected i f an absorptive Cl pump was stimulated. Upon removing Cl from cAMP-stimulated recta E declined by a. 16 mV (Table 6a) or 12 mV (Table 6b). These values are s i m i l a r to those obtained under control conditions. In normal s a l i n e , exposure to cAMP resulted i n a decline i n E, , consistent with enhanced C l e x i t out of the c e l l s across b the basal membrane. This could be explained simply by an increase i n conduc-tance; i . e . i n t r a c e l l u l a r C l i s above electrochemical equilibrium across the basal membrane (chapter 4) so that any increase i n C l conductance would enhance C l d i f f u s i o n from the c e l l and lower E, as observed i n Table 6. This b explanation for cAMP-induced AE, i s f e a s i b l e since -AE, i s less than the b b electrochemical gradient d r i v i n g C l out of the c e l l ( i . e . _AE^ ranges between _b -18.3 and -12.6 mV i n Table 6 as compared to a Au /F = 23.3 ± 1.4 mV; chapter 4). F i n a l l y , Auj^/F does not decline when C l conductance of the basal membrane increases, presumably because Cl i s pumped into the c e l l across the opposite (apical) membrane 10 times f a s t e r during cAMP exposure. The C l pump would maintain A y ^ at a constant l e v e l (20.1 ± 0.6 mV, chapter 4) as E^ declined (chapters 2 and 4). It i s now possible to derive an equivalent e l e c t r i c a l c i r c u i t model for cAMP-stimulated C l transport across locust rectum. This exercise w i l l also test the v a l i d i t y of most of the methods used i n t h i s thesis since i t r e l i e s on data from i o n - s e n s i t i v e microelectrode experiments, f l a t - s h e e t cable analy-s i s , and tracer flux measurements. 262 M u c o s a o-G C i , Eci G K E K A M G N a , E N A AM II EK J E N E C I G C I HvV G K S e r o s a -o ' N a HAr W\AA G ; Figure 5.19 Equivalent c i r c u i t model of r e c t a l epithelium showing e l e c t r o -motive force (EMF) and conductance for each ion at the mucosal and serosal c e l l borders. EMF's were calculated from i n t r a c e l l u l a r ion a c t i v i t y measure-ments described i n chapter 4. E C 1 (25.1 mV), E K (54.7 mV) , E N a (57.2 mV) under control conditions and E C 1 (14.4 mV), E K (58.2 mV), and E N a (57.2 mV) during exposure to 1 mM cAMP. See Table 5.5a for^summary of conductances used i n c a l c u l a t i n g membrane equivalent EMF's. 263 Figure 19 shows the relevant c i r c u i t parameters. The electromotive force for each ion was calculated from i n t r a c e l l u l a r ion a c t i v i t i e s measured i n chapter 4 using the Nernst equation. For example, under control conditions: E K = | ^ In (^|) = 54.7 mV (20) I n t r a c e l l u l a r ion a c t i v i t i e s used i n c a l c u l a t i n g EMF's under control conditions were ( i n mM): Cl (30.7), K (61.4), Na (8.0), and with cAMP: C l (46.6), K (70.3) and Na(8.0). The ion a c t i v i t i e s i n s a l i n e were C l (82), K (7.2) and Na (75.2). The EMF d r i v i n g C l entry also includes an active pump EMF, which w i l l be estimated l a t e r i n th i s discussion. Since a p i c a l and basal membranes have very low sodium conductance, Na was not included i n c a l -culations of the net equivalent EMF's of the a p i c a l and basal membranes. Using i o n - s e n s i t i v e microelectrode data and the r e s u l t s of ion s u b s t i t u t i o n during cable a n a l y s i s , membrane EMF's are obtained'-" K K C l Cl E * = E a G a + E a G a (21) a  a K K C l C l E b = b b *t) b • - (22) Gb where E* and E* are the t o t a l electromotive forces of a p i c a l and basal c e l l a b r borders as calculated from the Nernst potentials f o r K and C l . Since the net EMF of the epithelium i s E* - E*, then t r a n s e p i t h e l i a l p o t e n t i a l ( v t) may be calculated as V t = (E* - E*)R. (23) R + R, + R. a b 3 Using E* and Eg calculated before and during exposure to cAMP i n conjunc-ti o n with i o n i c conductances from Table 2, we predict that under c o n t r o l conditions, 264 V = (46.6 - 35.9) 742/(220 + 193 + 742) = 6.9 mV and during cAMP exposure," V = (55.5 - 22.6) 620/(44 + 36 + 620) = 29.1 mV These predictions are i n agreement w i t h e x p e r i m e n t a l l y o b s e r v e d values, 8.6 ± 0.3 mV and 29.8 ± 0.5 mV (Table 2 of chapter 4). This provides i n d i r e c t evidence f o r the v a l i d i t y of the i o n - s e n s i t i v e microelectrode data, for the p a r t i a l i o n i c conductances, and also for the assumption that sodium contributes l i t t l e towards E* or Eg and may be sa f e l y ignored. Also, values of E* and E* calculated i n control and cAMP-stimulated tissues also compare well with values of E^ and E^ calculated by c i r c u i t a n alysis i n Table 6, although t h i s i s less s u r p r i s i n g since both methods r e l y p a r t i a l l y on measure-ments of membrane resistance (equations 18, 19, 21 and 22). What i s the EMF of active C l transport i n locust rectum? A f t e r convert-e r -2 -1 ing net t r a n s e p i t h e l i a l Cl f l u x to current ( J n e t = 4.3 yEqcm h under open-c i r c u i t conditions during cAMP exposure), the t o t a l d r i v i n g force required for Cl entry i s ca l c u l a t e d from the C l conductance of the a p i c a l membrane as Epump = jfet F _ C l _ a a a G* 3.6 x 10 = 81.1 - (14.4 - 63.8) = 130.5 mV By assuming that a l l of the Cl conductance of the a p i c a l membrane resides i n the pump pathway, the o v e r a l l d r i v i n g force of C l transport i s EC1 = Epum P + EC1 _ VC1 _ EC1 + VC1 a a a b b = 130.5 + 14.4 - 63.8 - 14.4 + 34.0 = 100.7 mV 265 This EMF i s s l i g h t l y lower than that of active Na transport i n most vertebrate e p i t h e l i a (103-158 mV: Ussing and Zerahn, 1951; Civan et a l . , 1966; Civan, 1970; Hong and E s s i g , 1976; Feig et a l . , 1977; Yonath and Civan, 1971; Lewis et a l . , 1978; Saito et a l . , 1974; Chen and Walser, 1975; Schultz et a l . , 1977; Canessa.et al,,1978), and i s considerably lower than the 200 mV c a l c u l a t -ed for a c t i v e K transport i n lepidopteran midgut by three methods (reviewed by C l Harvey, 1981). E i s probably an underestimate, since the true EMF can be measured accurately only when the transport system does not work, i . e . when no C l C l current i s drawn from the E "battery". However, assuming that the true E C l i s near 100.7 mV, then the energy required for Cl transport i s FE =9.72 kJ/ mole or 2.32 kcal/mole C l transported. I t has been estimated that ^20 C l ions are transported per mole of O2 consumed based on the increase i n O2 consumption when C l i s added to cAMP-stimulated recta (Chamberlin, 1981). If the C l pump i s an ATPase, t h i s means that ^3.3 C l are transported per ATP and the energy required per mole of ATP to transport 3.3 C l ions i s 7.7 k c a l . This estimate i s lower than the energetic e f f i c i e n c y of active Na transport across toad bladder (via a known ATPase pump), which requires 11.7 kcal/mole of ATP (Canessa et a l . , 1978). Therefore, the energetics of locust C l transport would not require an unusually e f f i c i e n t coupling between metabolism and an Cl ATPase pump. ^ n e P r e s e n t estimate of E i s subject to error since i t r e l i e s on data obtained by a v a r i e t y of d i f f e r e n t experimental techniques. An Cl independent method of c a l c u l a t i n g E i s used i n the next section which con-firms the above c a l c u l a t i o n s . Relationship between " a c t i v e " and "passive" conductance The r e l a t i o n s h i p between s h o r t - c i r c u i t current (I ) and tissue conductance r - sc (Gj.) may be used to c a l c u l a t e both the d r i v i n g force of active ion transport and also the shunt conductance. (In this context, shunt conductance includes 266 c e l l u l a r and p a r a c e l l u l a r "leak" pathways.) Representing active Na transport across toad bladder by ttie~equivalent c i r c u i t shown i n Figure 20, Yonath and Civan (1971) found that a plot of G t versus I during vasopressin exposure Na gave a s t r a i g h t l i n e with slope = 1/E and a "y i n t e r c e p t " equal to the shunt conductance "k ". The r a t i o n a l e of the method i s as follows. Under I L sc conditions, the voltage between mucosal and serosal sides i n 0 mV, so there i s no net current flow through k^ (see F i g . 20). By analogy with Ohms Law, Na I g c = E k^. Further, t i s s u e conductance i s given by Gfc = + k^. Substi-Na tuting I /E for k y i e l d s the equation for a s t r a i g h t l i n e G = k + (I / S C A t ij S C Na E ) having slope = 1/E^ which i s the inverse d r i v i n g force of a c t i v e sodium transport and an intercept of k^, the shunt conductance (Yonath and Civan, 1971) . This method i s a p p l i c a b l e to electrogenic ion transport across any epithelium provided that i ) I g c and G can be made to vary over a large range, and i i ) the rate of a c t i v e transport of ion " i " i s varied by changing k^ rather than E 1 or k^. The cAMP dose-response curves shown i n Figure 17 i n d i c a t e that both I and G^ are strongly dependent on [cAMP], increasing seven-fold and three-fold r e s p e c t i v e l y when maximal doses are added. However, from chapter 4 i t i s clear that active entry of C l across the a p i c a l membrane i s d i r e c t l y C l stimulated by cAMP, suggesting that both E and G increase during cAMP exposure. Also, tracer fluxes and cable analysis experiments have shown that cAMP stimulates passive permeability of the rectum to potassium. In view of these complications, the method of Yonath and Civan seems inappropriate since Cl i t requires that E and k^ remain constant during exposure to cAMP. Neverthe-Cl l e s s , i f cAMP stimulates k A much more than E , then the I /G r e l a t i o n r\ S C L C l -1 should s t i l l be l i n e a r and approximate (E ) . S i m i l a r l y , i f the e f f e c t s of cAMP on potassium permeability can be measured and subtracted so that changes 267 S e r o s a Q M u c o s a Figure 5.20 Equivalent c i r c u i t model used by Yonath and Civan (1971) to c a l c u l a t e the electromotive force of a c t i v e Na transport (E„ ) and shunt Na conductance (k^) i n toad bladder epithelium. Conductance of the active transport pathway (k^) was v a r i e d by exposing the bladder to vasopressin. 268 i n do not influence the c a l c u l a t i o n s , then the Yonath and Civan method should be v a l i d . - -Figure 21 shows the r e l a t i o n s h i p between t r a n s e p i t h e l i a l conductance and I . Also shown i s the "corrected" or "Cl-dependent" G /I r e l a t i o n which sc t sc was calculated by subtracting the AGfc produced by cAMP i n C l - f r e e s a l i n e . I was taken from that i n normal s a l i n e ( Fig. 17) for both regressions. P r i o r to co r r e c t i n g for Cl-independent conductance, the apparent d r i v i n g force of Cl Cl (E ) was 43.4 mV, much"lower than calculated perviously from i n t r a c e l l u l a r measurements (100.7 mV). However, the shunt conductance (k ) was 4.55 mS -2 2 cm (resistance = 219.8 flcm ), i n close agreement with the t r a n s e p i t h e l i a l 2 resistance observed under control conditions (200-250 f!cm without cAMP). Afte r subtracting the cAMP-induced conductance measured i n C l - f r e e s a l i n e (presumably r e s u l t i n g from an increase i n P ) -the e s t i m a t e d EMF increased from 43.4 to 92.6 mV and the shunt conductance k decreased from -2 2 4.55 to 0.88 mS cm (resistance = 1,138.0 ftsm ). This "corrected" estimate C l of E (92.6 mV) i s i n reasonable agreement with the value (100.7 mV) obtained independently from i n t r a c e l l u l a r ion a c t i v i t i e s and cable analysis as described above. From the l i n e a r i t y of the I s c , / G t r e l a t i o n , i t i s l i k e l y that changes Cl i n E during cAMP stimulation are small when compared to the stimulation of active conductance. This i s not s u r p r i s i n g since cable analysis showed that cAMP increases the C l conductance of the basal membrane by approximately seven-Cl f o l d . A small increase i n E (<50%) would presumably be obscured by such a large increase i n conductance. F i n a l l y , comparison of the shunt conductances calculated from the two regressions shown i n Figure 21 reveals that Cl-independent shunt conductance ^ t o t a l _ ^ C l ^ ^ ^ t o t a l ^ 100%) i s 81% of the t o t a l tissue conductance. This f i n d i n g i s consistent with h i g h potassium permeability of the epithelium 269 1 2 1 0 CM 'E u to £ O 8 9 / / / / y = 0 . 0 2 3 x + 4 . 5 5 r 2 = 0 . 9 6 9 0 E C I = 4 3 . 4 2 mV C u n c o r r e c t e d ) • / 4 4 o o o Q, " O y = 0 . 0 1 0 8 x + 0 . 8 7 8 7 r 2 = 0 . 8 7 2 3 E C I = 9 2 . 6 mV C c o r r e c t e d ) o o 0 -0 3 0 0 4 0 0 1 0 0 2 0 0 l s c C p A c m " 2 ) Figure 5.21 Relationship between I g c and t r a n s e p i t h e l i a l conductance (G ) calculated from mean cAMP dose-response curves. See Figure 5.17 for o r i g i n a l data and v a r i a b i l i t y . The upper l i n e shows the r e l a t i o n s h i p between G t and I i n normal s a l i n e but includes the stimulatory e f f e c t of cAMP on K conduc-tance. The lower l i n e i s the corrected, Cl-dependent G t / T s c r e l a t i o n obtained by subtracting the cAMP-induced AG C observed i n C l - f r e e s a l i n e from that observed i n normal s a l i n e . 270 as indicated by 1) e f f e c t s of external potassium on membrane pot e n t i a l s (V50 mV/decade change in* [K], chapter 4),and 2) cable analysis (^76% of trans-c e l l u l a r conductance due to K). C l In summary, cAMP may cause some increase i n E , but th i s i s small i n comparison to the increase i n C l conductance of the basal membrane. As explained previously, d e p o l a r i z a t i o n of during cAMP-stimulation may be at t r i b u t e d to the d i f f u s i o n a l e x i t of Cl from the c e l l across the basal membrane when C l conductance of t h i s membrane i s increased by cAMP. The over-a l l EMF d r i v i n g t r a n s e p i t h e l i a l C l transport i s probably between 92.6 and 100.7 mV. The f i r s t of these two estimates i s derived from i ) i n t r a c e l l u l a r ion a c t i v i t i e s measured using i o n - s e n s i t i v e microelectrodes, i i ) membrane p a r t i a l i o n i c conductances from cable analyses during ion s u b s t i t u t i o n s , and i i i ) net C l absorption rate from tracer f l u x measurements under open-circuit C l conditions. In contrast, the second estimate of E i s based simply on measurements of s h o r t - c i r c u i t current and t r a n s e p i t h e l i a l resistance. C l Agreement between these two independent estimates of E provides circumstan-t i a l evidence for the r e s u l t s obtained i n this t h e s i s . 271 CHAPTER. 6: GENERAL DISCUSSION The properties of active C l absorption across locust rectum are not con-s i s t e n t with models which have been proposed for C l transport across other e p i t h e l i a . In t h i s chapter I b r i e f l y summarize the c e l l u l a r mechanism of KC1 transport and i t s regulation i n locust rectum as deduced from the work i n th i s t h e s i s . The experimental r e s u l t s have already been discussed and integrated. The reader i s re f e r r e d to the appropriate chapters for d e t a i l s and supporting evidence. Electrogenic absorption of C l by i n v i t r o locust rectum i s now fi r m l y C l established from i ) J ^ measured under true I conditions, i i ) u n i d i r e c t i o n a l net sc flux r a t i o s measured under open-circuit conditions, and i i i ) the e f f e c t s of C l removal on I during cAMP stimulation (chapter 2; see also Williams et a l . , 1978, and Spring and P h i l l i p s , 1980b). Chloride entry into the c e l l s i s active (chapter 4), but i s not driven by the Na-coupled mechanism found i n vertebrate C l -2 -1 e p i t h e l i a : i ) J and I both exceed 5.5 pEqcm h during cAMP stimulation r net sc ^ b after 6 h exposure to nominally Na-free s a l i n e (chapter 2); i i ) there i s no c o r r e l a t i o n between Cl-dependent I and mucosal Na concentration (chapter 2); 36 i i i ) cAMP stimulates u n i d i r e c t i o n a l C l i n f l u x into r e c t a l t i s s u e seven-fold 22 but does not increase the i n f l u x of Na (chapter 2); iv) I i s not i n h i b i t e d by 1 mM furosemide, a potent i n h i b i t o r of NaCl cotransport i n other c e l l s C l (chapter 2). J and I are also i n s e n s i t i v e to 1 mM ouabain a f t e r 2-5 h * net sc exposure (chapter 2) and Na/K ATPase i s o l a t e d from locust rectum has normal C l s e n s i t i v i t y to t h i s i n h i b i t o r (Peacock, 1981); v) J n e t i s not affected by reducing the Na electrochemical gradient- across the a p i c a l membrane by 85% from 128 mV to 22 mV (chapter 4); v i ) there i s no dependence of the C l electrochemi-_a c a i gradient across the a p i c a l membrane (Ap^) on the p a r a l l e l Na gradient, 272 —a, contrary to predictions of NaCl cotransport models i n which Ap n i s maintained v i a Na-coupled i n f l u x (i.je, secondary a c t i v e transport; chapter 4). There i s strong i n d i r e c t evidence that C l does not enter the c e l l s i n exchange for HCO^: i) t r a n s e p i t h e l i a l C l transport i s d e f i n i t e l y electrogenic (chapter 2), and C l entry may also be rheogenic (charge-separating) since cAMP produces a marked hyperpolarization of V , even i n K,HC0,. ,C0„-free s a l i n e (chapter 4); i i ) meta-a j z b o l i c CO^ production would be inadequate to provide HCO^ for 1:1 exchange with C l Cl at the rates of J observed under HC0 o,C0„-free conditions (calculated i n ms 3 2 chapter 2 from data of M. Chamberlin); i i i ) the rate of a l k a l i n i z a t i o n of the Cl mucosal side i s too low under HC0~,C0„-free conditions to account for J and 3 2 net may be explained by NH^ secretion (chapter 2); iv) Cl-dependent I i s not i n h i b i t e d by exposure to the potent anion exchange i n h i b i t o r SITS (1 mM) or to 1 mM acetazolamide f o r 1 h. Cotransport of Cl with protons i s also u n l i k e l y since i ) Cl-dependent I i s not i n h i b i t e d by r a i s i n g mucosal pH from 7.0 to 8.0, a manoeuvre which should reduce or abolish the proton electrochemical gradient across the a p i c a l membrane i f the i n t r a c e l l u l a r pH i n locust rectum i s s i m i l a r to that i n other tissues (chapter 2); i i ) low mucosal pH i n h i b i t s rather than stimulates 1 » contrary to expectations f o r a HCl coentry process. Figure 6.1 summarizes my i n t e r p r e t a t i o n of the data i n th i s thesis and i s the simplest model which i s consistent with a l l of the experimental r e s u l t s . Cl entry i s act i v e (chapter 4) and i s stimulated by mucosal K (chapters 3, 4). C l i s probably not cotransported with K since: i ) 35% of I during cAMP stimulation i s independent of potassium (chapter 2); i i ) the a p i c a l membrane hyperpolarizes during exposure to cAMP a f t e r prolonged exposure to K-free s a l i n e , i n d i c a t i n g electrogenic Cl entry (chapter 4); i i i ) t r a n s e p i t h e l i a l K fluxes are completely independent of C l cl M U C O S A 4$2> * 273 C E L L \ • ' t i l © ^ Us f t g S E R O S A TT, H + i c r 3 ^ K + K , T T N a + a m i n o a c i d s jcr °net IK+ °net c A M P t k l C . T . S . H . ~7T - •« s i / 7 - K + — 0 . 5 - ^ 4 . 3 pEqu i v . c m 2 h ~ 1 0 . 9 - ^ 4 . 5 57*—67 R a 260* - 70 m V O c m - 4 9 * - - 3 9 200*~55 Rj 8 0 0 r Figure 6^1 Summary diagram of the c e l l u l a r mechanism and r e g u l a t i o n of KCl transport across l o c u s t rectum (TT high osmotic c o n c e n t r a t i o n ) . 274 and provide no i n d i c a t i o n of r i g i d KC1 cotransport; iv) C l entry i s d e f i n i t e l y - a C l not energized by coentry-with K, since ApTr - 0 under I conditions when J K sc net -2 -1 i s 10 pEqcm h , and i s also low (<15 mV) under open-circuit conditions. The simplest explanation f o r the e f f e c t s of K on cAMP-stimulated C l transport i s that external K i n t e r a c t s i n some manner with a rheogenic C l pump i n the a p i c a l membrane, stimulating C l entry without a c t u a l l y being cotrans-ported. A v a i l a b l e data do not exclude the p o s s i b i l i t y that two "primary" C l pumps are present, one which i s K-independent and electrogenic, and a second KC1 cotransport s y s t e m . Although t h i s hypothesis would explain the In-dependent and independent components, i t requires that K be a c t i v e l y "recycled". at the a p i c a l membrane to account f o r the small J observed under I r net sc K conditions . Also, i t i s not consistent with the observation that J i s C l -ms independent (chapter 3). Further studies w i l l be necessary to understand the d e t a i l s of K-stimulation. For example, i s o l a t i o n of a K + Cl-stimulated ATPase from the a p i c a l membrane would provide strong i n d i r e c t evidence for thi s hypothesis. Balshin (1971) showed that glycine uptake into r e c t a l tissue i s Na-dependent. Although Na-cotransport with amino acids was not d i r e c t l y demon-strated, coupled transport with organic solutes may be an important entry mechanism for Na into r e c t a l c e l l s . Na conductance of the a p i c a l membrane i s probably low compared to K since: i ) V var i e s approximately 50 mV/decade a. external K a c t i v i t y (chapter 4); i i ) V i s not affected by replacing mucosal a. Na with N-methyl-D-glucamine. I n t r a c e l l u l a r l e v e l s of Na and K are consistent with the presence of a Na/K ATPase pump i n the basal membrane. Net Na absorp-t i o n under I conditions would involve Na entry across the a p i c a l membrane down a large (>120 mV) electrochemical gradient and require e x i t across the basal membrane against the same gradient. 275 In a d d i t i o n to stimulating the C l pump, cAMP reduces ~R^. Results i n chapter 5 suggest that locust rectum becomes a very " t i g h t " epithelium due to cAMP-induced K conductance at the a p i c a l membrane and cAMP-induced C l conductance at the basal membrane. These cAMP-stimulated conductances may be channels as shown i n Figure 6. The increase i n basal membrane C l conductance i s more than adequate to allow C l e x i t from the c e l l by e l e c t r o d i f f u s i o n down i t s measured electrochemical gradient (chapters 4 and 5). Most K may exit the c e l l s by e l e c t r o d i f f u s i o n during cAMP exposure, although some other e l e c t r o -neutral mechanism might also be involved since both basal membrane K electrochemical gradient and K conductance are lower than those for C l . K entry i s almost c e r t a i n l y by e l e c t r o d i f f u s i o n as a counter ion for C l trans-port (chapter 3). This e l e c t r i c a l coupling i s greatly enhanced by the high K s e l e c t i v i t y of the a p i c a l membrane and the large cAMP-induced K conductance i n the a p i c a l membrane (chapters 4 and 5); There i s a small active f l u x of K (17% of the t o t a l under open-circuit conditions). This active component has high a f f i n i t y f o r K ,(Kfc < 10 mM) and i s probably responsible f o r reducing luminal K to very low concentrations i n vivo i n salt-depleted animals. F i n a l l y , i n addi t i o n to "up-regulation" of C l absorption by CTSH and by mucosal K ions, "down-regulation" i s exerted by high osmotic pressure and low mucosal pH (chapter 2), which are observed i n vivo near the end of the normal reabsorptive c y c l e . The r e s u l t s also i n d i c a t e that a p i c a l membrane K conduc-tance i s "down-regulated" by high mucosal K concentration and osmotic pressure (chapters 3 and 5). In summary, the mechanism of KC1 reabsorption i s unlike any system which has been described previously. Although KC1 reabsorption i s stimulated by a neuropeptide hormone (on-off c o n t r o l ) , the absorption rate may be controlled l o c a l l y i n vivo by several i n t e r a c t i n g factors including mucosal K concentration, pH and osmotic pressure. 276 References Ahearn, G. A. 1978. A l l o s t e r i c cotransport of sodium, chloride, and calcium by the i n t e s t i n e of freshwater prawns. ^J. Membrane B i o l . 42: 281-300. Ahearn, G. A. 1980. I n t e s t i n a l electrophysiology and transmural ion trans-port i n freshwater pawns. Am. ^J. P h y s i o l . 239: C1-C10. Ahearn, G. A. and Tornquist, A. 1977. A l l o s t e r i c cooperativity during i n t e s t i n a l cotransport of sodium and chloride i n freshwater prawns. Biochim. Biophys. Acta 471: 273-279. Albrecht, F. 0. 1953. The Anatomy of the Migratory Locust. Athlone Press, London. 2+ Anstee, J. H. and Fathpour, H. 1979. The presence and properties of a Mg dependent HCO^ stimulated ATPase i n the Malpighian tubules of Locusta  migratoria. Insect Biochem. 9: 383-388. Anstee, J . H. and Bowler, K. 1979. Ouabain-sensitivity of insect e p i t h e l i a l t i s s u e s . Comp. Biochem. Physiol. 62A: 763-769. Armstrong, C. M. 1975. Ionic pores, gates and gating currents. Quart. Rev. Biophysics,7: 179-210. Aronson, P. S. 1981. I d e n t i f y i n g secondary a c t i v e solute transport i n e p i t h e l i a . Am. J_. P h y s i o l . 240: F l - F l l . B a c c e t t i , B. 1962. Ricerche s u l l ' u l t r a s t r u c t t u r a d e l l i n t e s t i n o d e g l i i n s e t t i IV l e p a p i l l e r e c a l l i i n un ortottero adulto. Redia 47: 105-118. Balshin, M. 1973. "Absorption of Amino Acids In V i t r o by the Rectum of the Desert Locust Schistocerca gregaria." Ph.D. Thesis, U n i v e r s i t y of B r i t i s h Columbia. Balshin, M. and P h i l l i p s , J. E. 1971. Active absorption of amino acids i n the rectum of the desert locust (Schistocerca gregaria). Nature (New Biology) 233: 53-55. Baumeister, T., Meredith, J . , J u l i e n , W. and P h i l l i p s , J. 1981. Acetate transport by locust rectum iri v ivo. J^ . Insect Physiol. 27: 195-201. Baumgarten, C. M. and Fozzard, H. A. 1981. I n t r a c e l l u l a r chloride a c t i v i t y i n mammalian v e n t r i c u l a r muscle. Am. J_. Physiol. 241: C121-C129. Berridge, M. J. 1966. Metabolic pathways of i s o l a t e d Malphigian tubules of the blowfly functioning i n an a r t i f i c i a l medium. J^. Insect P h y s i o l . 12: 1523-1538. Berridge, M. J. 1969. Urine formation by the Malpighian tubules of C a l l i -phora. I I . Anions. ^J. Exp. B i o l . 50: 15-28. 277 Berridge, M. J. 1980. The r o l e of c y c l i c nucleotides and calcium i n the regulation of chloride transport. Ann. N_.Y. Acad. S c i . 341: 156-169. Berridge, M. J . , Lindley, B. C. and Prince, W. T. 1975. Membrane permeabil-i t y changes during stimulation of i s o l a t e d s a l i v a r y glands of Calliphora by 5-hydroxytryptamine. J_. Physiol. 244: 549-567. Berridge, M. J . and Schlue, W. R. 1978. Ion-selective electrode studies on the e f f e c t s of 5-hydroxytryptamine on the i n t r a c e l l u l a r l e v e l of potas-sium i n an insect s a l i v a r y gland. Exp. B i o l . 72: 203-216. Biber, T. U. L. 1971. E f f e c t of changes i n t r a n s e p i t h e l i a l transport on the uptake of sodium across the outer surface of the frog skin. J_. Gen. Physiol. 58: 131-144. Biber, T. U. L. and Curran, P. F. 1970. Direct measurement of uptake of sodium at the outer surface of the frog skin. J^. Gen. Physiol. 56: 83-99. Biber, T. U. L., Walker, T. C. and Mullen, T. L. 1980. Influence of extra-c e l l u l a r C l concentration on C l transport across i s o l a t e d skin of Rana  pipiens. ^J. Membrane B i o l . 54: 191-202. Blankemeyer, J. T. 1978. Demonstration of a pump-mediated e f f l u x i n the e p i t h e l i a l potassium a c t i v e transport system of insect midgut. Biophys. J. 23: 313-318. Blankemeyer, J. and Harvey, W. 1978. I d e n t i f i c a t i o n of active c e l l i n potassium transporting epithelium. ^J. Exp. B i o l . 77: 1-13. Bl i g h t , M. M. 1969. V o l a t i l e nitrogenous bases emanating from laboratory reared colonies of the desert locust Schistocerca gregaria. J^. Insect  Physiol. 15: 259-272. Blunck, M. and Mommsen, T. P. 1978. Systematic errors i n f i t t i n g l i n e a r transformation of the Michaelis-Menten equation. Biometrika 65: 363-368. Bolton, T. B. and Vaughan-Jones, R. D. 1977. Continuous d i r e c t measurement of i n t r a c e l l u l a r c hloride and pH i n frog s k e l e t a l muscle. J_. Ph y s i o l . (Lond.) 270: 801-833. Bone, G. J. and Koch, H. J . 1942. Le r o l e des tubes de malpighi et du rectum dans l a regulation ionique chez les i n s e c t s . Ann. Roy. Zool. Belg. 73: 73-87. Bonting, S. L., de Pont, J . J . H. H. M., van Amelsvoort, J . M. M. and Schrijen, J. J. 1980. Transport ATPases in anion and proton transport. Ann. N.Y. Acad. S c i . 341: 335-356. Bornancin, M., De Renzis, G. and Naon, R. 1980. C l -HC03-ATPase i n g i l l s of the rainbow trout: evidence for i t s microsomal l o c a l i z a t i o n . Am. J^. Physiol. 238: R251-R259. ~ 278 Boulpaep, E. L. 1979. Electrophysiology of the kidney. In: Membrane Transport i n Biology, edited by G. Giebisch, D. C. Tosteson and H. H. Ussing. Springer-Verlag, New York, v o l . IVA. Boulpaep, E. L. and Sackin, H. 1980. E l e c t r i c a l analysis of i n t r a e p i t h e l i a l b a r r i e r s . In: Current Topics i n Membranes and Transport, edited by F. Bronner and A. K l e i n z e l l e r . Academic Press, New York, v o l . 13, chapter 12, pp. 169-197. Boulpaep, E. L. and Seely, J. F. 1971. Electrophysiology of proximal and d i s t a l tubules i n the autoperfused dog kidney. Am. J_. Physiol. 221: 1084-1096. Bradley, T. J. and P h i l l i p s , J. E. 1977. The l o c a t i o n and mechanism of hyperosmotic f l u i d secretion i n the rectum of the saline-water mosquito larvae Aedes taeniorhynchus. ^J. Exp. B i o l . 66: 111-126. Brown, H. M. 1976. I n t r a c e l l u l a r Na , K and Cl a c t i v i t i e s i n Balanus photoreceptors. _J. Gen. P h y s i o l . 68: 281-296. Bruus, K., Kristensen, P. and Larsen, E. H. 1976. Pathways for chloride and sodium transport across toad skin. Acta Physiol. Scand. 97: 31-47. B u r s e l l , E. 1967. The excretion of nitrogen i n i n s e c t s . In: Advances i n  Insect Physiology, edited by J . W. L. Beament, J. E. Treherne, V. B. Wigglesworth. Academic Press, London. Candia, 0. A. 1973. S h o r t - c i r c u i t current re l a t e d to active transport of chloride i n frog cornea: e f f e c t s of furosemide and ethacrynic a c i d . Biochim. Biophys. Acta 298: 1011-1014. Canessa, M., Labarca, P., Dibona, D. R. and Leaf, A. 1978. Energetics of sodium transport i n toad urinary bladder. Proc. Nat. Acad. S c i . (U.S.A.) 75: 4591-4595. C h a l f i e , M., Neufeld, A. H. and Zadunaisky, J . A. 1972. Action of epine-phrine and other c y c l i c AMP-mediated agents on the chloride transport of the frog cornea. Invest. Ophthalmol. 11: 663-650. Chamberlin, M. 1981. "Metabolic Studies on the Locust Rectum." Ph.D. Thesis, U n i v e r s i t y of B r i t i s h Columbia. Chamberlin, M. E. and P h i l l i p s , J . E. 1979. Regulation of hemolymph free amino acids i n the desert locust. Fed. Proc. 38: 970. Chase, H. S. and Al-Awqati, Q. 1981. Regulation of the sodium permeability of the luminal border of toad bladder by i n t r a c e l l u l a r sodium and calcium. J. Gen. Physiol. 77: 693-712. Chauvin, R. 1938. Anatomie et h i s t o l o g i e du tube d i g e s t i f de Schistocerca  gregaria. B u l l . Soc. H i s t . Nat. A f r . Nord. 28: 488-499. 279 Chen, J. S. and Walser, M. 1975. Sodium fluxes through the a c t i v e transport pathway i n toad bladder. J_. Membrane B i o l . 21: 87. Civan, M. M. 1970. E f f e c t s of a c t i v e sodium transport on current-voltage r e l a t i o n s h i p of toad bladder. Am. ^J. P h y s i o l . 219: 234-245. Civan, M. M. 1978. I n t r a c e l l u l a r a c t i v i t i e s of sodium and potassium. Am. J. P h y s i o l . 234(4): F261-F269. Civan, M. M. 1980. Potassium a c t i v i t i e s i n e p i t h e l i a . Fed. Proc. 39: 2865-2870. Cochran, A. N. 1975. Excretion i n i n s e c t s . In: Insect Biochemistry and Function, edited by D. J . Condry and B. A. K i l b y . Chapman and H a l l , London. Cooper, P. D., Beaton, L. E. and Jungreis, A. M. 1980. Chloride transport during resorption of molting f l u i d across the pharate pupal integument -of tobacco hornworms, Manduca sexta. ^J. Gen. P h y s i o l. 76: 13a-14a. Cousin, J . L. and Metais, R. 1976. The r o l e of carbonic anhydrase i n h i b i t o r s on anion permeability into ox red blood c e l l s . ^J. P h y s i o l . (Lond.) 256: 61-80. Crane, R. K. 1977. The gradient hypothesis and other models of c a r r i e r -mediated a c t i v e transport. Rev. P h y s i o l. Biochem. Pharmacol. 78: 99-159. Cremaschi, D. and Henin, S. 1975. Na + and C l t r a n s e p i t h e l i a l routes i n rabbit g a l l bladder. Tracer analysis of the transports. Pfluegers  Arch. 361: 33-41. Davenport, H. W. 1974. The ABC of Acid-Base Chemistry, 6th E d i t i o n . The U n i v e r s i t y of Chicago Press, Chicago. De Renzis, G. and Maetz, J. 1973. Studies on the mechanism of chloride absorption by the g o l d f i s h g i l l : r e l a t i o n with acid-base r e g u l a t i o n . J. Exp_. B i o l . 59: 339-358. Degnan, K. J . , Karnaky, K. J . and Zadunaisky, J. A. 1977. Active chloride transport i n the i_n v i t r o opercular skin of a teleost (Fundulus hetero- c l i t u s ) , a g i l l - l i k e epithelium r i c h i n chloride c e l l s . J_. Physiol. 271: 155-191. Delong, J. and Civan, M. M. 1978. D i s s o c i a t i o n of c e l l u l a r K + accumulation from net Na + transport by toad urinary bladder. J_. Membrane B i o l . 42: 19-43. Diamond, J. M. and Wright, E. M. 1969. B i o l o g i c a l membranes: The physical basis of ion and nonelectrolyte s e l e c t i v i t y . Ann. Rev. P h y s i o l . 31: 582-646. 280 Dietz, T. H. 1974. Active chloride transport across the skin of the earth-worm Lumbricus t e r r e s t r i s . Comp. Biochem. Ph y s i o l . A. Comp. Phy s i o l . 49: 251-258. Dietz, T. H. and Branton, W. D. 1975. Ionic regulation i n the fresh water mussel Ligumia subrostrata. J_. Comp. Ph y s i o l . _B. Metab. Transp. Funct. 104: 19-26. Djajakusumah, T. and Miles, P. W. 1966. Changes i n the r e l a t i v e amounts of soluble protein and amino acid i n the hemolymph of the locust, Chortoicetes terminifera Walker (Orthoptera: Acididae), i n r e l a t i o n to dehydration and subsequent hydration. Aust. ^J. B i o l . S c i . 19: 1081-1094. Dow, J. A. T. 1981. L o c a l i z a t i o n and ch a r a c t e r i z a t i o n of water uptake from the midgut of the locust, Schistocerca gregaria. J_. Exp. B i o l . 93: 269-281. Downing, N. 1980. The regulation of sodium, potassium and chloride i n an aphid subjected to i o n i c s t ress. J^. Exp. B i o l . 87: 343-349. Duffey, M. E., Thompson, S. M., F r i z z e l l , R. A. and Schultz, S. G. 1979. I n t r a c e l l u l a r chloride a c t i v i t y and active chloride absorption i n the' i n t e s t i n a l epithelium of the winter flounder. J_. Membrane B i o l . 50: 331-341. Duffey, M. E., Turnheim, K., F r i z z e l l , R. A. and Schultz, S. G. 1978. Intra-c e l l u l a r chloride a c t i v i t i e s i n rabbit gallbladder: d i r e c t evidence for the r o l e of the sodium-gradient i n energizing " u p h i l l " c hloride transport. J . Membrane B i o l . 42: 229-245. Dunham, P. B., Stewart, G. W. and E l l o r y , J . C. 1980. Chloride-activated passive potassium transport i n human erythrocytes. Proc. Nat. Acad. S c i . (U.S..A.) 77: 1711-1715. Durbin, R. P. 1964. Anion requirements for g a s t r i c acid secretion. J_. Gen. Phy s i o l . 47: 735-748. Edney, E. B. 1977. Water Balance i n Land Arthropods. Springer-Verlag, New York. Edzes, H. T. and Berendsen, H. J. C. 1975. The physi c a l state of d i f f u s i b l e ions i n c e l l s . Ann. Rev. Biophys. Bioeng. 4: 265-285. Ehrenfeld, J . and Garcia-Romeu, F. 1978. Coupling between c h l o r i d e absorp-t i o n and base excretion i n i s o l a t e d skin of Ran a esculenta. Am. J_. Phy s i o l . 235: F33-F39. Eisenberg, R. S. and Johnson, E. A. 1970. Three-dimensional e l e c t r i c a l f i e l d problems i n physiology. In: Progress i n Biophysics and Molecular B i o l - ogy, edited by J. A. V. Butler and D. Nobel. Pergamou Press, Toronto, v o l . 20, pp. 1-65. 281 Eisenman, G. 1961. On the elementary atomic o r i g i n of equilibrium i o n i c s p e c i f i c i t y . In: Symposium on Membrane Transport and Metabolism, edited by'A. K l e i n z e l l e r and A. Kotyk. Academic Press, New York, pp. 163-179. E r l i j , D. 1971. Salt transport across i s o l a t e d frog skin. P h i l . Trans. Roy. Soc. Lond. B 262: 153-161. E r l i j , D. and Smith, M. W. 1973. Sodium uptake by frog skin and i t s m o d i f i -cation by i n h i b i t o r s of t r a n s e p i t h e l i a l sodium transport. J_. Phy s i o l . 228: 221-239. Feig, P. U., Wetzel, G. and F r a z i e r , H. S. 1977. Dependence of the d r i v i n g force of the sodium pump on rate of transport. Am. J^. Phy s i o l . 232: F448-F454. F i e l d , M. 1971. Ion transport i n rabbi t i l e a l mucosa. I I . E f f e c t s of c y c l i c 3\5'-AMP. Am. J. Physiol. 221: 992-997. F i e l d , M. , Karnaky, K. J. J r . , Smith, P. L. , Bolton, J. E.. and Kinter, W. B. 1978. Ion transport across the i s o l a t e d i n t e s t i n a l mucosa of the winter flounder, Pseudopleuronectes americanus. I. Functional and s t r u c t u r a l properties of c e l l u l a r and p a r a c e l l u l a r pathways for Na and C l . J_. Membrane B i o l . 41: 265-293. Finn, A. L. 1975. Action of ouabain on sodium transport i n toad urinary bladder. Evidence for two pathways for sodium entry. J^_. Gen. Ph y s i o l . 65: 503-514. F r i z z e l l , R. A. 1979. Autoregulation of amiloride-sensitive sodium entry i n mammalian colon. In: Amiloride and E p i t h e l i a l Sodium Transport, edited by A. W. Cuthbert, G. M. F a n e l l i , J r . and A. Scriabine. Urban & Schwarz-enburg, Inc., Baltimore. F r i z z e l l , R. A., Dugas, M. C. and Schultz, S. G. 1975. Sodium chloride transport by rabbit gallbladder. D i r e c t evidence for a coupled NaCl i n f l u x process. J^. Gen. Physiol. 65: 769-795. F r i z z e l l , R. A., F i e l d , M. and Schultz, S. G. 1979. Sodium-coupled chloride transport by e p i t h e l i a l t i s s u e s . Am. J^. Phy s i o l . 236(1): F1-F8. F r i z z e l l , R. A. and Heintze, K. 1979. Electrogenic chloride secretion by mammalian colon. In: Mechanisms of I n t e s t i n a l Secretion, edited by H. J. Binder. Alan L i s s , Inc., New York, Kroc Foundation Series, v o l . 12. F r i z z e l l , R. A., Koch, M. J. and Schultz, S. G. 1976. Ion transport by rabbit colon. I. Active and passive components. J_. Membrane B i o l . 27: 297-316. F r i z z e l l , R. A., Nellans, H. N. , Rose, R. C , Markscheid-Kaspi, L. and Schultz, S. G. 1973. I n t r a c e l l u l a r C l concentrations and influ x e s across the brush border of rabbit ileum. Am. _J. P h y s i o l . 224: 328-227. 282 F r i z z e l l , R. A., Smith, P. L., Vosburgh, E. and F i e l d , M. 1979. Coupled sodium-chloride i n f l u x across brush border of flounder i n t e s t i n e . J. Membrane B i o l . 46: 27-39. Fromm, M. and Schultz, S. G. 1981. Potassium transport across rabbit descending colon In v i t r o : Evidence for s i n g l e - f i l e d i f f u s i o n through a p a r a c e l l u l a r pathway. J_. Membrane B i o l . 63: 93-98. Fromter, E. 1972. The route of passive ion movement through the epithelium of Necturus gallbladder. J. Membrane B i o l . 8: 259-301. Fromter, E. and Diamond, J. 1972. Route of passive ion permeation i n e p i -t h e l i a . Nature New B i o l . 235: 9-13. Fromter, E., Muller, C. W. and Wick, T. 1971. Permeability properties of the proximal tubular epithelium of the rat kidney studied with e l e c t r o -p h y s i o l o g i c a l methods. In: Electrophysiology of E p i t h e l i a l C e l l s : Symposia Medica Hoechst, edited by F. K. Schattauer. Verlag GmbH, Stuttgart, Germany. Fujimoto, M. and Kubota, T. 1976. Physiochemical properties of a l i q u i d ion exchanger microelectrode and i t s a p p l i c a t i o n to b i o l o g i c a l f l u i d . Jap. J. P h y s i o l . 26: 631-650. Fujimoto, M., Naito, K. and Kubota, T. 1980. Electrochemical p r o f i l e for ion transport across the membrane of proximal tubular c e l l s . Memb. Biochem. 3: 67-97. Garcia-Diaz, J . F. and Armstrong, W. McD. 1980. The steady-state r e l a t i o n -ship between sodium and chloride transmembrane electrochemical p o t e n t i a l differences i n Necturus gallbladder. J^. Membrane B i o l . 55: 213-222. Garcia Romeu, F., Saliban, A. and Pezzani-Hernandez, S. 1969. The nature of the in vivo sodium and chlo r i d e uptake mechanisms through the epithelium of the Chilean frog Calyptocephalella gayi (Dum. et Bibr.,1841). J^. Gen. Phys i o l. 53: 816-835. Geek, P., Pietrzyk, C , Burckhardt, B. C , P f e i f f e r , B^ and Heinz, E. 1980. E l e c t r i c a l l y s i l e n t cotransport of Na +, K + and Cl i n E h r l i c h c e l l s . Biophys. Biochim. Acta 600: 432-447. Gerencser, G. A. 1978. Enhancement of sodium and chloride transport by mono-saccharides i n Aplysia c a l i f o r n i c a i n t e s t i n e . Comp. Biochem. Physiol. 61A: 203-208. Gerencser, G. 1981. I n t e s t i n a l p o t e n t i a l s . Comp. Biochem. Physiol. 69A: 15-21. Gerencser, G. A., Hong, S. K. and Malvin, G. 1977. Metabolic dependence of transmural p o t e n t i a l d i f f e r e n c e and s h o r t - c i r c u i t current i n i s o l a t e d Aplysia J u l i a n a i n t e s t i n e . Comp. Biochem. Ph y s i o l . 56A: 519-523. Gerencser, G. A. and White, J . F. 1980. Membrane potentials and chloride a c t i v i t i e s i n e p i t h e l i a l - c e l l s of Aplysia i n t e s t i n e . Am. J_. Phy s i o l . 239: R445-R449. 283 Gloor, H. and Chen, P. S. 1950. Uber ein Anal organ bei Drosophila-Larven. Rev. Suisse Zool. 57: 570-576. Goh, S. L. 1971. "Mechanism of Water and Salt Absorption i n the In V i t r o Locust Rectum." M.Sc. Thesis, University of B r i t i s h Columbia. Goh, S. and P h i l l i p s , J . E. 1978. Dependence of prolonged water absorption by i_n v i t r o locust rectum on ion transport. J^_. Exp. B i o l . 72: 25-41. Graf, J. and Giebisch, G. 1979. I n t r a c e l l u l a r sodium a c t i v i t y and sodium transport i n Necturus gallbladder epithelium. _J. Membrane B i o l . 47: 327-355. Grinstein, S. and E r l i j , D. 1978. I n t r a c e l l u l a r calcium and the regulation of sodium transport i n the frog skin. Proc. Roy. Soc. Land. B. 202: 353-360. Gunn, R. B. 1979. Anion transport i n red c e l l s : An asymmetric, ping-pong mechanism. In: Mechanisms of I n t e s t i n a l Secretion, edited by H. J . Binder. Alan L i s s , Inc., New York, Kroc Foundation Series, v o l . 12. Gupta, B. L. and Berridge, M. J . 1966. Fine s t r u c t u r a l organization of the rectum i n the blowfly, C a l l i p h o r a erythrocephala (Meig.) with s p e c i a l reference to connective t i s s u e , trachaea and neurosecretory innervation i n the r e c t a l p a p i l l a e . J . Morph. 120: 23-82. Gupta, B. L., Wall, B. J., Oschman, J . L. and H a l l , T. A. 1980. Direct microprobe evidence of l o c a l concentration gradients and r e c y c l i n g of e l e c t r o l y t e s during f l u i d absorption i n the r e c t a l p a p i l l a e of C a l l i p h o r a . _J. Exp_. B i o l . 88: 21-47. Haldane, J . B. S. 1957. Graphical methods i n enzyme chemistry. Nature 179: 832. Hanrahan, J . W. 1978. Hormonal regulation of chloride i n l o c u s t s . The  P h y s i o l o g i s t 21: 50. Hanrahan, J . W. and P h i l l i p s , J . E. 1980a. Na +-independent C l transport i n an insect. Fed. Proc. 39: 285. Hanrahan, J. W. and P h i l l i p s , J . E. 1980b. Characterisation of locust C l transport. Am. Zool• 20: 938. Harvey, W. R. 1981. Membrane physiology of i n s e c t s . In: Membrane P h y s i o l -ogy of Invertebrates, edited by R. P. Podesta and S. F. Timmers. Dekker, New York (in press). Heckmann, K. 1972. Single f i l e d i f f u s i o n . In: Biomembranes, edited by F. Kreuzer and J . F. G. Siegers. Plenum Press, New York, v o l . 3, pp. 125-153. 284 Herrera, L., Lopes-Moratalla, N., Santiago, E., Ponz, F. and Jordana, R. 1978. E f f e c t of bicarbonate on chloride-dependent transmural p o t e n t i a l and ATPase a c t i v i t y "in.the r e c t a l w a l l of Schistocerca gregaria. Revta  Esp. F i s i o l . 34: 219-224. Herrera, L., Jordana, R. and Ponz, F. 1977. E f f e c t of i n h i b i t o r s on ch l o r i d e -dependent transmural p o t e n t i a l i n the r e c t a l wall of Schistocerca  gregaria. J_. Insect Physiol. 23: 677-682. Hers, M. J . 1942. Anaerobiose et regulation minerale chez l e s larves de Chironomus. Ann. Soc. Roy. Zool. Belg. 72: 173-179. Higgins, J . T. J r . , Cesaro, L., Gebler, B. and Fromter, E. 1975. E l e c t r i c a l properties of amphibian urinary, bladder e p i t h e l i a . I. Inverse r e l a t i o n -ship between p o t e n t i a l d i f f e r e n c e and resistance i n t i g h t l y mounted preparations. Pfliigers Arch. 358: 41-56. Highnam, K. C , H i l l , L. and Mordue, W. 1966. The endocrine system and oocyte growth i n Schistocerca i n r e l a t i o n to starvation and f r o n t a l ganglionectomy. J^. Insect P h y s i o l . 12: 977-994. Highnam, K. C. and Mordue, A. J. (Luntz). 1974. Induced changes i n neuro-secretory a c t i v i t y of adult female Schistocerca gregaria i n r e l a t i o n to feeding. Gen. Comp. Endo. 22: 519-525. Highnam, K. C. and West, M. W. 1971. The neuropilar neurosecretory res e r v o i r of Locusta migratoria migratorioides. Gen. Comp. Endo. 16: 574-585. H i l l , B. S. and Hanke, D. E. 1979. Properties of the chloride-ATPase from Limonium s a l t glands: A c t i v a t i o n by, and binding to, s p e c i f i c sugars. J . Membrane B i o l . 51: 185-194. H i l l , B. S. and H i l l , A. E. 1973. ATP-driven chloride pumping and ATPase a c t i v i t y i n the Limonium s a l t gland. J^. Membrane B i o l . 12: 145-158. H i l l e , B. 1979. Rate theory models for ion flow i n i o n i c channels of nerve and muscle. In: Membrane Transport Processes, edited by C. F. Stevens and R. W. Tsien. Raven Press, New York, v o l . 3. H i l l e , B. and Schwarz, W. 1978. Potassium channels as multi-ion s i n g l e - f i l e pores. J . Gen. Ph y s i o l . 72: 409-442. Hladky, S. B. and Ha r r i s , J . D. 1967. An ion displacement membrane model. Biophys. J_. 7: 535-543. Hodge, C. 1939. The anatomy and histology of the alimentary t r a c t of Locusta  migratoria L. J_. Morph. 64: 375-400. Hodgkin, A. and Keynes, R. 1955. The potassium permeability of a giant nerve f i b e r . J . Phy s i o l. (Lond.) 116: 61-88. Horowitz, S. B., Paine, P. L., Tlucek, L. and Reynhout, J. K. 1979. Refer-ence phase analysis of free and bound i n t r a c e l l u l a r solutes. I. Sodium and potassium i n amphibian oocytes. Biophys. J_. 25: 33-44. 285 Humphreys, M. H. 1976. I n h i b i t i o n of NaCl absorption from perfused rat ileum by furosemide.. Am. _J. Physiol. 230: 1517-1523. Humphreys, M. H. and Chou, L. Y. N. 1979. Anion-stimulated ATPase a c t i v i t y of brush border from r a t small i n t e s t i n e . Am. _J. P h y s i o l . 236: E70-E76. Irvine, H. B. 1966. "In v i t r o Rectal Transport and Rectal Ultrastructure i n the Desert Locust, Schistocerca gregaria." M.Sc. Thesis, University of B r i t i s h Columbia. Irvine, H. B. and P h i l l i p s , J. E. 1971. E f f e c t s of respiratory i n h i b i t o r s and ouabain on water transport by i s o l a t e d locust rectum. J_. Insect  P h y s i o l . 17: 381-393. Jungreis, A. M. 1977. Comparative aspects of invertebrate e p i t h e l i a l trans-port. In: Water Relatons i n Membrane Transport i n Plants and Animals, edited by A. M. Jungreis, T. K. Hodges, A. K l e i n z e l l e r and S. G. Schultz. Academic Press, Inc., Toronto, pp. 89-96. Karnaky, K. J . J r . , Degnan, K. J . and Zadunaisky, J . A. 1977. Chloride transport across i s o l a t e d opercular epithelium of k i l l i f i s h : A membrane r i c h i n chloride c e l l s . Nature 195: 203-205. Kasbekar, D. K. and Durbin, R. P. 1965. An adenosine triphosphatase from frog g a s t r i c mucosa. Biochim. Biophys. Acta 105: 472-482. Kaufman, W. R. and P h i l l i p s , J . E. 1973. Ion and water balance i n the Ixodid t i c k Dermacentor andersoni. I I I . Influence of monovalent ions and osmotic pressure of s a l i v a r y secretion. _J. Exp. B i o l . 58: 549-564. Khuri, R. N., Bogharian, K. K. and Agulian, S. K. 1974. I n t r a c e l l u l a r b i -carbonate i n s i n g l e s k e l e t a l muscle f i b r e s . Pflligers Arch. 349: 285-294. Kinne-Saffran, E. and Kinne, R. 1974. Presence of bicarbonate stimulated ATPase i n the brush border m i c r o v i l l u s membranes of the proximal tubule. Proc. Soc. Exp. B i o l . Med. 146: 751-753. Kirk, K. L. and Dawson, D. C. 1981. Bas o l a t e r a l potassium channel i n t u r t l e colon: Interaction of permeating ions. Fed. Proc. 40: 357. Klyce, S. D. and Wong, R. K. S. 1977. S i t e and mode of adrenalin action on chloride transport across the rabbit corneal epithelium. J_. Ph y s i o l . 266: 777-799. Koch, A. R. 1970. Transport equations and c r i t e r i a for active transport. Am. Zool. 10: 331-346. Koch, H. J . 1938. The absorption of chloride ions by the anal p a p i l l a e of diptera larvae. _J. Exp. B i o l . 15:. 152-160. Komnick, H. 1977. Chloride c e l l s and chloride e p i t h e l i a of aquatic insects. In: Int. Rev, of Cytology, edited by G. H. Bourne and J . F. D a n i e l l i , v o l . 49, pp. 285-329. 286 Komnick, H. and Achenbach, U. 1979. Comparative biochemical, histochemical and autoradiographic- studies of Na+/K+-ATPase i n the rectum of dragonfly larvae (Odonata, Aeshnidae) . Eur. J_. C e l l . B i o l . 20: 92-100. Kregenow, F. M. and Caryk, T. 1979. Co-transport of cations and C l during the volume regulatory responses of duck erythrocytes. The P h y s i o l o g i s t 22: 73. Kristensen, P. 1972. Chloride transport across i s o l a t e d frog skin. Acta  Physiol. Scand. 84: 338-346. Kristensen, P. and Larsen, E. H. 1978. Relation between chloride exchange d i f f u s i o n and a conductive chloride pathway across the i s o l a t e d skin of the toad (Bufo bufo). Acta Physiol. Scand. 102: 22-34. Krogh, A. 1937. Osmotic re g u l a t i o n i n the frog (R. esculenta) by active absorption of C l ions. Skand. Arch. P h y s i o l . 76: 6-74. Krogh, A. 1939. Osmotic Regulation i n Aquatic Animals. Cambridge University Press, New York. Lane, N. J . 1979. Freeze-fracture and tracer studies on the i n t e r c e l l u l a r junctions of insect r e c t a l tissues. Tissue C e l l 11: 481-506. Lane, J. J . 1981. Tight junctions i n anthropod tissu e s . International Rev, of C y t o l . 73: 243-318. LMuger,,P. 1972. Carrier-mediated ion transport. Science 178: 24-30. LSuger, P. 1980. K i n e t i c properties of ion c a r r i e r s and channels. J_. Membrane B i o l . 57: 163-178. Leader, J. P. and Green, L. B. 1978. Active transport of chloride and sodium by the r e c t a l chamber of the larvae of the dragonfly, Uropetala carovei. J. Insect P h y s i o l . 24: 685-692. Lee, C. 0., Taylor, A. and- Windhager, E. E. 1980. Cytosolic calcium ion a c t i v i t y i n e p i t h e l i a l c e l l s of Necturus kidney. Nature 287: 859-861. L e s l i e , B. R., Schwartz, J. H. and Steinmetz, P. R. 1973. Coupling between C l - absorption and HCO3 secretion i n t u r t l e urinary bladder. Am. _J. P h y s i o l . 225: 610-617. Lev, A. A. and Armstrong, W. McD. 1975. Ionic a c t i v i t i e s i n c e l l s . In: Current Topics i n Membranes and Transport, edited by F. Bronner and A. K l e i n z e l l e r . Academic Press, New York, v o l . 6, pp. 59-123. Lewis, S. A. 1971. "Charge Properties and Ion S e l e c t i v i t y of the Rectal Intima of the Desert Locust." M.Sc. Thesis, University of B r i t i s h Columbia. Lewis, S. A. and Diamond, J. M. 1976. Na + transport by rabbit urinary bladder, a tight epithelium. J_. Membrane B i o l . 28: 1-40. 287 Lewis, S. A., Eaton, D. C. and Diamond, J. M. 1976. The mechanism of Na"*" transport by rab b i t urinary bladder. J_. Membrane B i o l . 28: 41-70. Lewis, S. A. and Graf, J . 1979. Assessment of impalement damage. In: I n t r a c e l l u l a r Sodium A c t i v i t y and Sodium Transport i n Necturus g a l l b l a d -der Epithelium, j;. Membrane B i o l . 47: 327-355. Lewis, S. A., W i l l s , N. K. and Eaton, D. C. 1978. Basolateral membrane p o t e n t i a l of a t i g h t epithelium: Ionic d i f f u s i o n and electrogenic pumps. _J. Membrane B i o l . 41: 117-148. Ques-von Petery, M. V., Rotunno, C. A. and C e r e i j i d o , M. 1978. Studies on ch l o r i d e permeability of the skin of Leptodactylus o c e l l a t u s . I. Na + and C l - e f f e c t on passive movements of C l - . J^. Membrane B i o l . 42: 317-330. Liedtke, C. M. and Hopfer, U. 1980. Chloride-sodium symport versus chloride/ hydroxide antiport or chloride uniport as mechanisms for chloride transport across r a t i n t e s t i n a l brush border membranes. Fed. Proc. 39: 734. Lindemann, B. and Van Driessche, W. 1977. Sodium-specific membrane channels of frog skin are pores: Current f l u c t u a t i o n s reveal high turnover. Science 195: 292-294. Loewenstein, W. R. 1981. Junctional i n t e r c e l l u l a r communication: The c e l l -t o - c e l l membrane channel. P h y s i o l . Rev. 61: 829-913. Loewenstein, W. R., Socolar, S. J . , Higashimo, S., Kanno, Y. and Davidson, N. 1965. I n t e r c e l l u l a r communication: Renal, urinary bladder, sensory and s a l i v a r y gland c e l l s . Science 149: 295-298. Machen, T. E. and Forte, J . G. 1979. Gastric secretion. In: Membrane Transport i n Biology, edited by G. Giebisch, D. C. Tosteson and H. H. Ussing. Springer-Verlag, New York, v o l . IVA, chapter 13, pp. 693-748. Maddrell, S. H. P. 1971. The mechanisms of insect excretory systems. In: Advances i n Insect Physiology, edited by J . W. L. Beament, J. E. Treherne and V. B. Wigglesworth. Academic Press, London and New York, v o l . 8, pp. 199-331. Maddrell, S. H. P. 1980. C h a r a c t e r i s t i c s of e p i t h e l i a l transport i n insect Malpighian tubules. In: Current Topics i n Membranes and Transport, edited by F. Bronner and A. K l e i n z e l l e r . Academic Press, Toronto. Maddrell, S. H. P. and Klunsuwan, S. 1973. F l u i d secretion by i n v i t r o preparations of the Malpighian tubules of the desert locust Schistocerca  gregaria. _J. Insect P h y s i o l . 19: 1369-1376. Maetz, J. 1971. The f i s h g i l l s : mechanisms of s a l t transfer i n fresh water and sea water. P h i l . Trans. Roy. Soc. Lond. B 262: 209-249. 288 Maetz, J. and Garcia-Romeu, F. 1964. The mechanisms of sodium and chloride uptake by the g i l l s ' o f fresh water f i s h , Carassius auratus. I I . Evidence for NH+/Na"+" and HCO3/CI" exchange. J. Gen. P h y s i o l . 47: 1209-1227. Mandel, L. J . 1978. E f f e c t s of pH, Ca, ADH and theophylline on the k i n e t i c s of Na entry i n frog skin. Am. j;. P h y s i o l . 235: C35-C48. Maren, T. H. 1977. Use of i n h i b i t o r s i n p h y s i o l o g i c a l studies-of carbonic anhydrase. Am. j;. P h y s i o l . 232: F291-F297. Marshall, W. G. 1945. The r e c t a l sac of the red-legged grasshopper Melano-plus fermur-rubrum De Geer. Ann. Ent. Soc. Amer. 38: 460-461. Marshall, W. S. and Klyce, S. D. 1981. Membrane resistances i n r a b b i t corneal epithelium. Fed. Proc. 40: 370. M i l l e r , C. and White, M. M. 1980. A voltage-dependent ch l o r i d e conductance channel from Torpedo electroplax membrane. Ann. N.Y. Acad. S c i . 341: 534-551. Moody, G. J . and Thomas, J. D. R. 1971. Selective Ion Sensitive Electrodes. Merrow, Watford, England. Mordue, W. 1969. Hormonal control of Malpighian tube and r e c t a l function i n the desert locust, Schistocerca gregaria. ^J. Insect P h y s i o l . 15: 273-285. Moreno, J . H., R e i s i n , I. L., Rodriguez-Boulan, E., Rotunno, C. A. and C e r e i j i d o , M. 1973. B a r r i e r s to sodium movements across frog s k i n . J_. Membrane B i o l . 11: 99-115. Mullins, D. E. and Cochran, D. G. 1972. Nitrogen excretion i n cockroaches: Uric acid i s not a major product. Science 177: 699-701. Nellans, H. N., F r i z z e l l , R. A. and Schultz, S. G. 1973. Coupled sodium-chloride i n f l u x across the brush border of rabbit ileum. Am. J^. P h y s i o l . 225: 467-475. Noirot, C , Smith, D. S., Cayer, M. L. and Noirot-Timothee, C. 1979. The organisation and i s o l a t i n g function of insect r e c t a l sheath c e l l s : A greeze-fracture study. Tissue C e l l 11: 325-336. Noirot-Timothee, C. and Noirot, C. 1967. L i a i s o n des mitochondries aves des zones d'adhesion i n t e r c e l l u l a i r e s . J^. Microscopie 6: 87-90. O'Neil, R. G. and Helman, S. I. 1976. Influence of vasopressin and amilor-ide on the shunt pathway of skin. Am. J_. P h y s i o l . 231: 164-173. Ogden, T. E., C i t r o n , M. C. and Pierantoni, R. 1978. The j e t stream micro-beveler: An inexpensive way to bevel u l t r a - f i n e glass micropipettes. Science 201: 469-470. 289 Olver, F. W. J . 1967. Bessel functions of integer order. In: Handbook of  Mathematical Functions, edited by M. Abramowitz and J. A. Stegun. National Bureau of Standards, Washington, pp. 355-422. Oschman, J . L. and Wall, B. J . 1969. The structure of the r e c t a l pads of Periplaneta americana L. with regard to f l u i d transport. ^J. Morph. 127: 475-509. Palmer, L. G., Century, T. J . and Civan, M. M. 1978. A c t i v i t y c o e f f i c i e n t s of i n t r a c e l l u l a r Na + and K + during development of frog oocytes. J^. Membrane B i o l . 40: 25-38. Peacock, A. J . 1979. : TLtrastructure of the type "B" c e l l s i n the r e c t a l pad epithelium of Locusta migrator i a . J_. Morph. 159: 221-232. Peacock, A. J . 1981. Further studies of the properties of locust r e c t a l Na +-K +-ATPase, with p a r t i c u l a r reference to the ouabain s e n s i t i v i t y of the enzyme. Comp. Biochem. Ph y s i o l . 68C: 29-34. P h i l l i p s , J . E. 1961. "Studies on the Rectal Absorption of Water and Salts i n the Locust, Schistocerca gregaria, and the Blowfly, C a l l i p h o r a erythro- cephala." Ph.D. Thesis, University of Cambridge. P h i l l i p s , J . E. 1964a. Rectal absorption i n the desert locust, Schistocerca  gregaria ForskSl. I. Water. J^. Exp. B i o l . 41: 15-38. P h i l l i p s , J. E. 1964b. Rectal absorption i n the desert locust, Schistocerca gregaria Forsk&l. I I . Sodium, potassium and chloride. J_. Exp. B i o l . 41: 39-67. P h i l l i p s , J . E. 1964c. Rectal absorption i n the desert l o c u s t , Schistocerca gregaria ForskSl. I I I . The nature of the excretory process. J^. Exp. B i o l . 41: 67-80. P h i l l i p s , J . E. 1970. Apparent transport of water by insect excretory systems. Am. Zool. 10: 413-436. P h i l l i p s , J. E. 1977. Excretion i n i n s e c t s : function of gut and rectum i n concentrating and d i l u t i n g the urine. Fed. Proc. 36: 2480-2486. P h i l l i p s , J . E. 1980. E p i t h e l i a l transport and control i n re c t a of t e r r e s -t r i a l i n s e c t s . In: Insect Biology i n the Future. Academic Press, pp. 145-177. P h i l l i p s , J . 1981. Comparative physiology of insect renal function. Am. J^. Phy s i o l. 241: R241-R257. P h i l l i p s , J . E. and D o c k r i l l , A. A. 1968. Molecular sieving of hydrophilic molecules by the r e c t a l intima of the desert locust (Schistocerca gregar-i a ) . J . Exp_. B i o l . 48: 521-532. P h i l l i p s , J . and Meredith, J . 1969. Active sodium and chloride transport by anal p a p i l l a e of a s a l t water mosquito la r v a (Aedes compestris). Nature (London) 222: 168-169. 290 P h i l l i p s , J . E., Mordue, W. , Meredith, J . and Spring, J . 1980. P u r i f i c a t i o n and c h a r a c t e r i s t i c s of the chloride transport stimulating factor from the locust corpora cardiaca: a new peptide. Can. J_. Zool. 58: 1851-1860. P h i l l i p s , J . E., Spring, J . , Hanrahan, J . , Mordue, W. and Meredith, J . 1981. Hormonal c o n t r o l of s a l t reabsorption by the excretory system of an insect: i s o l a t i o n of a new protein. In: Neurosecretion: Molecules, C e l l s , Systems, edited by D. S. Farmer and K. Lederis. Plenum, New York, pp. 373-382. Powell, D. W. 1981. B a r r i e r function of e p i t h e l i a . Am. J_. P h y s i o l . 241: G275-G288. Prusch, R. D. 1974. Active ion transport i n the l a r v a l hindgut of Sarcophaga  b u l l a t a (Diptera:Sarcophagidae). _J. Exp. B i o l . 61: 95-109. Prusch, R. D. 1976. U n i d i r e c t i o n a l ion movements i n the hindgut of l a r v a l Sarcophaga b u l l a t a (Diptera: Sarcophagidae) . J_. Exp . B i o l . 64: 89-100. Quay,