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Cellular mechanism of bicarbonate regulation and excretion in an insect inhabiting extremes of alkalinity Strange, Kevin 1983

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CELLULAR MECHANISM OF BICARBONATE REGULATION AND EXCRETION IN AN INSECT INHABITING EXTREMES OF ALKALINITY by KEVIN STRANGE B . S c , U n i v e r s i t y of C a l i f o r n i a , Davis, 1977 M.A., U n i v e r s i t y of C a l i f o r n i a , Davis, 1978 A THESIS SUBMITTED IN PARTIAL FULFILMENT 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 THE UNIVERSITY OF BRITISH COLUMBIA May 1983 (c) Kevin Strange, 1983 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 available for 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. I t i s understood that copying or publication 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 ~Zc>o The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 DE-6 (3/81) 1 ABSTRACT The s a l t w a t e r mosquito l a r v a , Aedes dorsa l i s , i s one o f the only organisms capable o f i n h a b i t i n g h y p e r s a l i n e lakes composed a lmost e n t i r e l y o f high concen t ra t i ons o f NaHC03 and Na 2C03 s a l t s . Under l a b o r a t o r y cond i t i ons l a r vae su rv i ved and developed normal ly i n s a l i n e media w i th pH va lues up to p 1 0 . 5 , HC03~ concen t ra t i ons up to 250 mM, or C 0 3 t - concen t ra t i ons up to 100 mM. Desp i te i n g e s t i o n o f these a l k a l i n e media a t ra tes e q u i v a l e n t to 130% o f l a r v a l body weight per day, these i n s e c t s regu la ted hemolymph pH (7 .55 -7 .70 ) and HCO3" concen t ra t i ons (8.0 - 18.5 mM) w i t h i n narrow p h y s i o l o g i c a l l i m i t s . Micropuncture and mic rocannu la t ion s t u d i e s on the r e c t a l s a l t g land demonstrated tha t t h i s organ was an impor tant s i t e of pH and HC03~ r e g u l a t i o n . M ic rocannu la ted s a l t g lands sec re ted a s t rong l y p hyperosmot ic f l u i d c o n t a i n i n g 402 mM HCO3" and 41 mM C 0 3 t a t a ra te of 38 n l - h " 1 . Lumen-to-bath HC03~ and C 0 3 2 " g rad ien ts of 21:1 and 2 4 1 : 1 , r e s p e c t i v e l y , were generated by the s a l t g land e p i t h e l i u m a g a i n s t 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 o f -25 mV (lumen nega t i ve ) demonstrat ing c l e a r l y the a c t i v e nature of HCO3" s e c r e t i o n . To study the mechanisms of HCO3" t r a n s p o r t , an i n v i t r o mic roper fused r e c t a l s a l t g land p repa ra t i on was developed. Net t o t a l CO2 C0o t r a n s p o r t ( J p e | ) as measured by m ic roca lo r ime t r y i n per fused s a l t g lands was una f fec ted by b i l a t e r a l N a + or K + and se rosa l C l " s u b s t i t u t i o n s , or by s e r o s a l a d d i t i o n o f 1.0 mM ouaba in , 2.0 mM a m i l o r i d e or 0.5 mM SITS. - CO Removal of luminal C l " i n h i b i t e d J n g | by 80%, wh i l e se rosa l a d d i t i o n of 1.0 rn mM acetazo lamide or 0 .5 mM DIDS i n h i b i t e d by 80% and 40%, r e s p e c t i v e l y . P e r f u s i o n of the a n t e r i o r and p o s t e r i o r r e c t a l segments demon-s t r a t e d c l e a r l y t ha t the a n t e r i o r rectum was the s i t e of CO2 s e c r e t i o n i n i i the microper fused s a l t g l and . Net C l ~ reabso rp t i on in the a n t e r i o r segment was measured by e l e c t r o n microprobe a n a l y s i s and was e q u i v a l e n t to the ra te of CO2 s e c r e t i o n . In a d d i t i o n , C l " r eabso rp t i on i n the a n t e r i o r segment was complete ly i n h i b i t e d by b i l a t e r a l l y r e p l a c i n g C02 and HC03~ w i th a phosphate or HEPES bu f fe red s a l i n e . These data prov ide s t rong q u a n t i t a t i v e ev idence f o r the presence of a 1:1 C T / H C C ^ " exchange mechanism l o c a t e d i n the a n t e r i o r r e c t a l s a l t g land segment. M i c rocannu la t i on s tud ies on the i n d i v i d u a l s a l t g land segments demonstrated tha t both r e c t a l segments are capable of s e c r e t i n g a hyperosmot ic f l u i d c o n t a i n i n g N a + , C l ~ and HCO3". Based on these r e s u l t s and the r e s u l t s of s t u d i e s i n which the e f f e c t s of se rosa l ion s u b s t i t u t i o n s on s a l t g land f l u i d s e c r e t i o n were examined, i t has been suggested t e n t a t i v e l y t ha t both segments sec re te a N a C l - r i c h f l u i d and tha t f l u i d s e c r e t i o n i s d r i ven by coupled NaCl t r a n s p o r t . I t i s f u r t h e r suggested t ha t once t h i s f l u i d en te rs the s a l t g land lumen i t s compos i t ion i s mod i f i ed by ion exchange and reabso rp t i ve processes which are dependent upon the i o n i c r egu la to r y needs of the an ima l . In l a r vae i n h a b i t i n g low C l " , NaHCC^-CC^ l a k e s , t h i s m o d i f i c a t i o n i n v o l v e s a 1:1 exchange of lumina l C l ~ f o r se rosa l HCO3". The c e l l u l a r mechanisms of a n t e r i o r s a l t g land HCC^ - and C l ~ t r a n s p o r t were examined us ing ion and v o l t a g e - s e l e c t i v e m ic roe lec t rodes i n con junc t i on w i th a mic roper fused a n t e r i o r segment p repa ra t i on which a l lowed complete changes i n se rosa l and mucosal s a l i n e composi t ion to be made i n <5-10 seconds. A d d i t i o n of DIDS or acetazo lamide to or removal of CO2 and HC03~ from the se rosa l bath caused l a r g e , 20-50 mV h y p e r p o l a r i z a t i o n s of V a and had l i t t l e e f f e c t on . Rapid changes i n luminal C l " concen t ra t i on a l t e r e d V a i n a r a p i d , s tep -w ise manner. The s lope of the i i i r e l a t i o n s h i p between V a and luminal C l " a c t i v i t y was 42.2 mV/decalog a^-j-( r = 0 .992 ) . I n t r a c e l l u l a r C l ~ a c t i v i t y was 23.5 mM and was approx imate ly 10 mM lower than t ha t p r e d i c t e d f o r a pass i ve d i s t r i b u t i o n a t the a p i c a l membrane. Changes i n se rosa l C l " concen t ra t i on had no e f f e c t on i n d i c a t i n g an e l e c t r i c a l l y s i l e n t b a s o l a t e r a l C l " e x i t s t ep . I n t r a c e l l u l a r pH i n a n t e r i o r r e c t a l c e l l s was 7.67 and the c a l c u l a t e d a^Q 0 - w a s 1 4 * 4 m M * 3 These r e s u l t s show t ha t under con t ro l c o n d i t i o n s HC03~ en te rs the a n t e r i o r r e c t a l c e l l by an a c t i v e mechanism aga ins t an e l ec t r ochem ica l g rad ien t o f 77.1 mV and e x i t s the c e l l a t the a p i c a l membrane down a f avo rab le e l ec t r ochem ica l g rad ien t o f 27.6 mV. Based on these r e s u l t s , a t e n t a t i v e c e l l u l a r model has been proposed i n which C l " en te rs the a p i c a l membrane of the a n t e r i o r r e c t a l c e l l s by p a s s i v e , e l e c t r o d i f f u s i v e movement through a C l ~ - s e l e c t i v e channe l , and HCO3" e x i t s the c e l l by an a c t i v e or pass i ve e l e c t r o g e n i c t r a n s p o r t mechanism. The e l e c t r i c a l l y s i l e n t nature of b a s o l a t e r a l C l " e x i t and HC03~ e n t r y , and the e f f e c t s of se rosa l a d d i t i o n of - - ro the C1~/HC0 3 ~ exchange i n h i b i t o r DIDS on J n e | and V t e suggest s t r o n g l y t ha t the b a s o l a t e r a l membrane i s the s i t e o f a d i r e c t c o u p l i n g between C l " and HCO3" movements v i a a C1~/HC03~ exchange mechanism. i v TABLE OF CONTENTS Page ABSTRACT i TABLE OF CONTENTS i v LIST OF TABLES v i LIST OF FIGURES v i i LIST OF ABBREVIATIONS x ACKNOWLEDGEMENTS x i i CHAPTER I: GENERAL INTRODUCTION 1 A. Ac id -Base Regu la t i on 1 1. Ve r teb ra te Ac id -Base E x c r e t i o n 2 2. I nve r teb ra te Ac id -Base E x c r e t i o n 8 B. O rgan i za t i on o f La rva l Exc re to ry System 11 CHAPTER I I : BICARBONATE REGULATION AND EXCRETION 17 A. I n t r oduc t i on 17 B. M a t e r i a l s and Methods 20 Animals 20 C a p i l l a r y pH E lec t r odes 23 Hemolymph pH Measurements 26 Hemolymph H C O 3 " Measurements 26 Rec ta l Micropuncture S tud ies 27 Cannulated i n v i t r o Rec ta l P r e p a r a t i o n 27 A n a l y s i s o fTTec ta l S e c r e t i o n s 31 T r a n s e p i t h e l i a l P o t e n t i a l 31 C a l c u l a t i o n s 32 C. Resu l t s 32 D. D i s c u s s i o n 50 1. Rec ta l H C O 3 " S e c r e t i o n 53 CHAPTER I I I : IONIC REQUIREMENTS OF C 0 2 TRANSPORT IN THE MICROPERFUSED RECTAL SALT GLAND 56 A. I n t roduc t i on 56 B. M a t e r i a l s and Methods 57 Animals 57 M i c r o p e r f u s i o n System 58 Determinat ion of Net Tota l C O 2 F l ux 61 S a l i n e s 62 I n h i b i t o r S t u d i e s 65 V Page C. R e s u l t s 66 D. D i s c u s s i o n 78 CHAPTER IV: SITE OF CL~/HC03 _ EXCHANGE AND FUNCTION OF ANTERIOR AND POSTERIOR SALT GLAND SEGMENTS 83 A . I n t r oduc t i on 83 B. M a t e r i a l s and Methods 84 Animals 84 M i c r o p e r f u s i o n and M i c r o c a n n u l a t i o n S tud ies 84 Sample A n a l y s i s 85 S a l i n e s 85 C. R e s u l t s 89 D. D i s c u s s i o n 102 1. S i t e o f C T / H C O 3 - Exchange 102 2 . Exc re to ry Func t ions of A n t e r i o r and P o s t e r i o r S e g m e n t s . . . . 103 CHAPTER V: CELLULAR MECHANISM OF BICARBONATE AND CHLORIDE TRANSPORT... 110 A . I n t r oduc t i on 110 B. M a t e r i a l s and Methods 110 Animals 110 M i c r o p e r f u s i o n System 110 Bath and Luminal S o l u t i o n Changes I l l E l e c t r i c a l Measurements 115 M i c r o e l e c t r o d e Impalements 118 S a l i n e s 118 C. Resu l t s 120 1. M i c r o e l e c t r o d e Impalements 120 2 . E l e c t r o g e n i c HC03" T ranspor t 128 3 . E l e c t r o g e n i c C l " Reabsorpt ion 149 D. D i s c u s s i o n 158 1. C e l l u l a r En t ry and E x i t Steps 158 2 . E l e c t r o c h e m i c a l Grad ien ts 173 CHAPTER V I : GENERAL DISCUSSION 179 REFERENCES 197 LIST OF TABLES Page Tab le 2.1 Composi t ion of a r t i f i c i a l l ake waters i n which l a r v a e were reared or a c c l i m a t e d . 21 Tab le 2.2 Composi t ion o f hemolymph and p h y s i o l o g i c a l s a l i n e s f o r l a r vae acc l ima ted to d i f f e r e n t a r t i f i c i a l l ake wa te r s . 22 Tab le 2.3 Values of p K ^ ' , p K 2 ' and S used f o r c a l c u l a t i o n o f C 0 2 , HC03~ and C0^~ c o n c e n t r a t i o n s . 33 Table 2.4 Concen t ra t i ons of C a 2 + , M g 2 + , t o t a l s u l f u r and t o t a l phosphorus, and pH i n r e c t a l s e c r e t i o n s c o l l e c t e d by mic ropunc tu re . 46 Tab le 3.1 Composi t ion o f p h y s i o l o g i c a l s a l i n e s . 63 Table 3.2 Changes i n t o t a l C O 2 and C l " concen t ra t i ons i n c o l l e c t e d p e r f u s a t e s . 64 Tab le 3.3 Summary of c h i t i n a s e exper iments . 81 Table 4.1 Composi t ion of p h y s i o l o g i c a l s a l i n e s . 88 Tab le 4.2 Measured f l u i d s e c r e t i o n ra tes f o r whole, cannu la ted r e c t a l s a l t g lands bathed i n va r i ous s a l i n e s . 98 Tab le 4.3 Ion ic compos i t ion of r e c t a l s e c r e t i o n s . 99 Tab le 4.4 C a l c u l a t e d ra tes of N a + and C l " e x c r e t i o n f o r cannu la ted a n t e r i o r and p o s t e r i o r segments. 105 Tab le 5.1 Composi t ion of p h y s i o l o g i c a l s a l i n e s . 119 Tab le 5.2 C a l c u l a t e d C l " e l ec t rochemica l g rad ien ts f o r a p i c a l and b a s o l a t e r a l c e l l membranes bathed by con t ro l se rosa l and mucosal s a l i n e s . 156 Tab le 5.3 C a l c u l a t e d HC03~ e lec t rochemica l g rad ien ts f o r a p i c a l and b a s o l a t e r a l c e l l membranes bathed by con t ro l se rosa l and mucosal s a l i n e s . 176 Tab le 5.4 I n t r a c e l l u l a r pH and c a l c u l a t e d a p i c a l membrane e l ec t r ochem ica l g rad ien ts f o r H + and H C O 3 " . 177 v i i LIST OF FIGURES Page F i g u r e 1.1 O r g a n i z a t i o n of l a r v a l gut and exc re to ry system. 12 F i gu re 1.2 O r g a n i z a t i o n and u l t r a s t r u c t u r e o f r e c t a l s a l t g l and . 14 F i g u r e 2.1 U l t r a m i c r o i n t e r n a l c a p i l l a r y pH e l e c t r o d e . 25 F i gu re 2.2 M ic rocannu la ted r e c t a l p r e p a r a t i o n . 30 F i g u r e 2 .3 S u r v i v a l and development of f ou r th i n s t a r l a r v a e i n a l k a l i n e env i ronments. 35 F i g u r e 2.4 D r i n k i n g ra tes es t imated by ^ C - i n u l i n i n g e s t i o n f o r f ou r th i n s t a r l a r vae acc l ima ted to a l k a l i n e env i ronments . 38 F i g u r e 2.5 Hemolymph pH r e g u l a t i o n i n f ou r th i n s t a r l a r vae du r ing a c c l i m a t i o n to h igh HC03~ and C0^~ media. 40 F i gu re 2.6 Hemolymph HC03~ c o n c e n t r a t i o n . 42 F i g u r e 2.7 Osmo la l i t y and concen t ra t i ons of major ions i n r e c t a l s e c r e t i o n s c o l l e c t e d by micropuncture from l a r v a e acc l ima ted to th ree d i f f e r e n t a l k a l i n e media. 45 F i g u r e 2.8 Osmo la l i t y and concen t ra t i ons of major ions i n r e c t a l s e c r e t i o n s c o l l e c t e d from cannu la ted rec ta o f an imals acc l ima ted to 250 mM H C O 3 " medium. 49 F i g u r e 2 .9 T r a n s e p i t h e l i a l p o t e n t i a l ac ross cannula ted r e c t a from animals acc l ima ted to 250 mM HC03~ medium. 52 F i gu re 3.1 M ic rope r fused r e c t a l s a l t g land p r e p a r a t i o n . 60 F i gu re 3.2 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 t ime course de te rmina t ions of J ^ g | i n microper fused r e c t a l s a l t g l and . 68 F i g u r e 3 .3 E f f e c t s o f b i l a t e r a l N a + o r K + s u b s t i t u t i o n s on Jpg|« 70 F i g u r e 3.4 E f f e c t s of luminal and s e r o s a l C l " s u b s t i t u t i o n s on Jpg|« 73 F i gu re 3.5 E f f e c t s o f se rosa l a d d i t i o n o f 1.0 mM ouabain or 2.0 mM a m i l o r i d e on J ^ £ # 75 net F i g u r e 3.6 E f f e c t s o f se rosa l a d d i t i o n o f 0 .5 mM SITS, 0.5 mM DIDS or 1.0 mM acetazo lamide on J ^ ? ? . 77 net v i i i Page F i gu re 4.1 Arrangement of p i p e t s and l i g a t u r e s f o r m i c rope r f us i on and m ic rocannu la t i on o f i n d i v i d u a l s a l t g land segments. 87 F i g u r e 4.2 Changes i n pe r fusa te t o t a l C O 2 concen t ra t i on f o r whole s a l t g lands and i n d i v i d u a l segments. 91 F i g u r e 4.3 E f f e c t s of lumina l C l " s u b s t i t u t i o n on the change i n pe r fusa te t o t a l C O 2 concen t ra t i on from whole s a l t g lands and a n t e r i o r segments. 93 F i g u r e 4 .4 Changes i n a n t e r i o r segment pe r fusa te t o t a l C O 2 and C l " concen t ra t i ons and e f f e c t s of b i l a t e r a l C O 2 and HC03" s u b s t i t u t i o n w i th HEPES or phosphate bu f fe red s a l i n e s on C l " r e a b s o r p t i o n . 95 F i gu re 4 .5 Rates of f l u i d s e c r e t i o n i n whole , mic rocannu la ted s a l t g lands and a n t e r i o r and p o s t e r i o r segments. 101 F i g u r e 4 .6 Summary of major exc re to ry and t r anspo r t f u n c t i o n s of i n d i v i d u a l s a l t g land segments. 107 F igu re 5.1 Schematic diagram of m ic roper fused a n t e r i o r r e c t a l s a l t g land segment and double p e r f u s i o n p i p e t arrangement. 113 F i g u r e 5.2 Examples o f acceptab le c e l l u l a r impalements ob ta ined w i th v o l t a g e - s e l e c t i v e m i c r o e l e c t r o d e s . 122 F igu re 5.3 Examples of acceptab le impalements ob ta ined w i th H + and C l " - s e l e c t i v e m i c r o e l e c t r o d e s . 125 F i g u r e 5.4 D i s t r i b u t i o n of VD-| and i n t r a c e l l u l a r pH and C l " a c t i v i t y i n a n t e r i o r r e c t a l s a l t g land c e l l s . 127 F i g u r e 5.5 E f f e c t s of r a p i d a d d i t i o n of 1.0 mM acetazo lamide to the se rosa l ba th ing s a l i n e on V - ^ 130 F igu re 5.6 E f f e c t s of a d d i t i o n of 0 .5 mM DIDS to the se rosa l ba th ing s a l i n e on V ^ e i n 4 separate a n t e r i o r r e c t a l s a l t g land p r e p a r a t i o n s . 132 F i g u r e 5.7 E f f e c t s of se rosa l C O 2 and H C O 3 " replacement w i th a 5.0 mM HEPES bu f fe red s a l i n e on V t e . 135 F i g u r e 5.8 E f f e c t s of r e p l a c i n g se rosa l C O 2 and HC03~ w i th the l i p i d s o l u b l e b u f f e r , g l y c o d i a z i n e , on V t e . 138 F igu re 5.9 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 V D i dur ing and a f t e r a d d i t i o n o f 1.0 mM acetazo lamide to the se rosa l ba th ing s a l i n e . 140 i x Page F i gu re 5.10 E f f e c t s o f se rosa l C O 2 and HC03~ replacement w i th the l i p i d s o l u b l e b u f f e r , g l y c o d i a z i n e , on V D ] and V t e - 142 F i g u r e 5.11 Changes i n V51 and V^e dur ing and a f t e r replacement of se rosa l C O 2 and H C O 3 - w i th a 5.0 mM HEPES bu f fe red s a l i n e . 144 F i g u r e 5.12 E f f e c t s o f h igh se rosa l H C 0 3 " concen t ra t i on a t cons tan t pH on Vfci and V t e . 146 F i g u r e 5.13 E f f e c t s o f lumina l N a + or K + s u b s t i t u t i o n s on and V b l . 148 F i g u r e 5.14 E f f e c t of se rosa l C 0 2 and H C O 3 " s u b s t i t u t i o n on V^ i and V t e du r ing p e r f u s i o n of s a l t g land lumen w i th C l ~ -f r e e s a l i n e s . 151 F i g u r e 5.15 E f f e c t s of r a p i d changes i n lumina l C l ~ concen t ra t i on on V t e and V ^ i . 153 F i g u r e 5.16 R e l a t i o n s h i p between the l og o f the lumina l C l " a c t i v i t y and V a . 155 F igu re 5.17 E f f e c t s o f r a p i d changes i n se rosa l C l " concen t ra t i on on V D i • 1 6 0 F igu re 5.18 E f f e c t s of r a p i d changes i n se rosa l K + concen t ra t i on on V D i . !62 F i g u r e 5.19 T e n t a t i v e c e l l u l a r model o f HC03~ and C l " en t ry and e x i t s teps i n a n t e r i o r r e c t a l s a l t g land c e l l s . 164 F i g u r e 5.20 E f f e c t s o f b i l a t e r a l C O 2 and HC03~ replacement w i th e i t h e r a phosphate, HEPES or g l y c o d i a z i n e bu f fe red s a l i n e on the change i n a n t e r i o r segment pe r fusa te C l " c o n c e n t r a t i o n . 169 F igu re 5.21 T e n t a t i v e scheme showing proposed proton s h u t t l i n g e f f e c t s of g l y c o d i a z i n e b u f f e r . 172 X LIST OF ABBREVIATIONS PCO2 - p a r t i a l p ressure o f CO2 gas . A - read a s , change i n . [ i ] - concen t ra t i on of " i " . a^ - chemical a c t i v i t y o f an ion ( " i " ) . a ] , a $ , a? - chemical a c t i v i t y o f an ion i n the l u m i n a l , se rosa l and i n t r a c e l l u l a r compartments, r e s p e c t i v e l y . pH c - i n t r a c e l l u l a r pH. J n e t - net f l u x of an ion ( " i " ) . V t e - 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 p o t e n t i a l (^mucosa - ^ s e r o s a ) . V b- | - b a s o l a t e r a l membrane p o t e n t i a l ( ^ c e l l - ^ s e r o s a ) . V a - a p i c a l membrane p o t e n t i a l ( Y c e l l - ^mucosa). - e l ec t rochemica l g rad ien t f o r an ion ( " i " ) . A j j a , A p ^ - e l ec t rochemica l g rad ien t f o r an ion ( " i " ) a t the a p i c a l and b a s o l a t e r a l membranes, r e s p e c t i v e l y . V.j - vo l tage output from an i o n - s e l e c t i v e m i c r o e l e c t r o d e . ft - ohms. y - m ic ron . nl - n a n o l i t e r . mV - m i l l i v o l t . K j ' - the o v e r a l l apparent e q u i l i b r i u m cons tan t between d i s s o l v e d C0 2 and H + + HCO3". p K j ' - the negat ive l oga r i t hm of K ^ ' . pK2* - the negat ive l oga r i t hm of the apparent e q u i l i b r i u m cons tan t between HCO3" and C0 3 2~ + H + . S - C0 2 s o l u b i l i t y c o e f f i c i e n t . I .D. - i nne r d iameter . O.D. - ou te r d iameter . x i SITS - 4 - a c e t a m i d o - 4 ' - i s e t h i o c y a n o s t i l b e n e - 2 , 2 ' - d i s u l f o n i c a c i d (an i n h i b i t o r of anion exchange mechanisms). DIDS - 4 , 4 ' - d i i s o t h i o c y a n o - 2 , 2 1 - d i s u i f o n i c a c i d (an i n h i b i t o r of anion exchange mechanisms). HEPES - N-2-hydroxyethy l p i p e r a z i n e - N " - 2 - e t h a n e s u l f o n i c a c i d (a weak a c i d b u f f e r i n g compound). x i i ACKNOWLEDGEMENTS I would l i k e to thank my research s u p e r v i s o r , Dr . John P h i l l i p s , f o r p r o v i d i n g me w i th the academic freedom and f i n a n c i a l means to pursue my own ideas and f o r use fu l comments and d i s c u s s i o n throughout the course of t h i s work. I am a l s o indebted to and would l i k e to thank the f o l l o w i n g people: Dr . Wa l te r Boron f o r va luab le i n s t r u c t i o n i n m ic roe lec t rode techn iques and the many i n d i v i d u a l s a t Y a l e U n i v e r s i t y who shared w i th me t h e i r ex tens i ve knowledge of i n s i t u and i n v i t r o m ic rope r fus ion methods; Dr . G . G . E . Scudder f o r purchas ing and a l l o w i n g me e x c l u s i v e use of the P icapnotherm; Dr . Tom Mommsen f o r many use fu l d i s c u s s i o n s and f o r sugges t ing the use of and c o l l e c t i n g s p i d e r d i g e s t i v e j u i c e s ; Dr . Mary Chamber l in f o r her i n v a l u a b l e d i s t o r t i o n s of r e a l i t y , f o r making the l abo ra to r y a s t i m u l a t i n g environment dur ing the f i r s t three yea rs of my stay a t U . B . C . , and f o r i n t r o d u c i n g me to Mr. P. Head; Mr. P. Head f o r h i s t e c h n i c a l suppor t and e x c e p t i o n a l l y wise c o u n s e l ; Joan Mar t i n f o r use fu l comments and d i s c u s s i o n , f o r sugges t ing the use of the 10 /i l i g a t u r e s , and f o r her companionship; My research committee, D r s . B. M i l som, J . G o s l i n e and D. Randal l f o r use fu l comments concern ing the f i n a l form of t h i s manuscr ip t ; C h r i s t i n e McCaf f rey and Dr . G.A. Quamme f o r e l e c t r o n microprobe a n a l y s e s ; Mr. Ken Pope of Vancouver General H o s p i t a l f o r h i s t r u l y superb job and cra f tsmansh ip i n c o n s t r u c t i n g the m ic rope r fus ion p i p e t ho lde r desc r ibed i n Chapter V; Don Brandys and Fergus O'Hara f o r b u i l d i n g me coun t l ess p ieces of equipment; x i i i Kathy Gorko f f f o r her ou ts tand ing job i n t yp i ng t h i s manuscr ip t and f o r he lp i ng me f i n i s h on t ime ; Bob La t race f o r be ing here and having S to res open a t 7:30 a . m . ! F i n a l l y , I would p a r t i c u l a r l y l i k e to thank Tom Baumeis te r , Katy F a r l e y , Matt and Joey J o n e s , R ich Moore, T e r r i Scemons and Lynn Vas ington f o r t h e i r f r i e n d s h i p and suppor t , and f o r p r o v i d i n g me wi th the a l l - t o o - i n f r e q u e n t i n c e n t i v e s to p lay i n s t e a d of work. - 1 -CHAPTER I. GENERAL INTRODUCTION A. Ac id -Base Regu la t i on Almost every b iochemica l r e a c t i o n tha t occurs i n a l i v i n g organism i s a f f e c t e d by the H + concen t ra t i on of the surrounding medium. The a c i d -base s ta tus of both 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 f l u i d s must be mainta ined w i t h i n a very narrow range i n order f o r these r e a c t i o n s to proceed a t opt imal r a tes and to a l l ow t h e i r i n t e g r a t i o n i n t o complex p h y s i o l o g i c a l f u n c t i o n s . Shor t - te rm defense aga ins t changes i n ac id -base balance i s p rov ided f i r s t by 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 b u f f e r s . In a d d i t i o n , b u f f e r i n g by organ systems such as bone (Robinson, 1975) , ca lca reous exoske le tons (Henry, Kormanik, Smatr isk and Cameron, 1981; Randa l l and Wood, 1981) , the l ime sacs of f rogs (Rober tson, 1972) and ca l ca reous s h e l l s of mo l luscs (Duga l , 1939; Bu r t on , 1976) and t u r t l e s (Jackson and U l t s c h , 1982) a i d i n c o r r e c t i n g ac i d -base d i s t u r b a n c e s . Long-term pH r e g u l a t i o n , however, must be mediated by e l i m i n a t i o n of a c i d s and bases . In s i n g l e c e l l e d organisms and i n d i v i d u a l c e l l s t h i s process i s c a r r i e d out by we l l de f i ned membrane t r a n s p o r t mechanisms and p o s s i b l y by s p e c i f i c metabo l i c pathways (Cohen and l i e s , 1975; Roos and Boron , 1981; Sanders and Slayman, 1982). Long-term e x t r a c e l l u l a r ac i d -base homeostasis i n m u l t i c e l l u l a r organisms i s main ta ined by the r e s p i r a t o r y and exc re to ry systems, and perhaps by metabo l i c f u n c t i o n s of the gu t , musc le , and l i v e r or analogous o rgans . The i n d i v i d u a l r o l e s p layed by these va r ious organ and bu f f e r systems i n ac id -base r e g u l a t i o n are h i g h l y complex and dependent upon an organ ism's p h y s i o l o g i c a l s t a t e . Attempts to summarize these mechanisms are beyond the scope of the cu r ren t work and i n s t e a d , only a d i s c u s s i o n of exc re to r y ac id -base r e g u l a t i o n i s war ran ted . - 2 -1) Ver teb ra te A c i d - B a s e E x c r e t i o n The r o l e o f the mammalian k idney i n ac i d -base homeostasis has been s t u d i e d e x t e n s i v e l y ( f o r recen t reviews see Ma ln i c and G i e b i s c h , 1979; Warnock and R e c t o r , 1979, 1981; M a l n i c , 1981; Berry and Warnock, 1982) and the k idney has been shown c l e a r l y to respond to c o n d i t i o n s of a c i d o s i s and a l k a l o s i s i n a compensatory manner ( rev iewed by Cogan, Rec to r and S e l d i n , 1981). C o n t r o l l e d reabso rp t i on of f i l t e r e d H C O 3 " by the nephrons and concomitant u r i ne a c i d i f i c a t i o n i s q u a n t i t a t i v e l y the most impor tant means of u r i n a r y ac i d -base e x c r e t i o n . The bulk (80-90%) of H C O 3 " r eabso rp t i on occurs i n the proximal convo lu ted tubu le wh i l e s m a l l e r amounts are reabsorbed i n the proximal s t r a i g h t and d i s t a l t u b u l e s . The c o l l e c t i n g tubu le a l s o p lays a r o l e i n ac id -base r e g u l a t i o n and i s capable o f a c i d i f y i n g (McKinney and Bu rg , 1977a, 1978a; Lombard, Jacobson and Kokko, 1979; R ichardson and Kunau, 1981; DuBose, 1982; Koeppen and Helman, 1982) or a l k a l i n i z i n g (McKinney and B u r g , 1977a, 1978b) the f i n a l u r i ne depending upon p h y s i o l o g i c a l c o n d i t i o n s . The k idney a d d i t i o n a l l y f u n c t i o n s i n ac id -base r e g u l a t i o n by e x c r e t i n g protons bound to n o n v o l a t i l e b u f f e r s such as phosphate and ammonia. Indeed, t h i s i s thought to be q u a n t i t a t i v e l y f a r more impor tant than e x c r e -t i o n of unbound H + s i nce the maximal u r i ne to b lood pH g rad ien t generated by mammalian k idneys i s only approx imate ly 2.0 pH u n i t s . As o r i g i n a l l y suggested by P i t t s (1948), ammonia i s the pr imary b u f f e r i nvo l ved i n u r i n a r y ac id -base e x c r e t i o n . Ammonia i s syn thes i zed i n the tubu le c e l l as N H 3 from glutamine and o ther amino a c i d s . Because of i t s smal l s i z e and l ack of charge, N H 3 i s thought to d i f f u s e f r e e l y i n t o the u r i n e and p e r i t u b u l a r b l ood . S i nce the u r i n e i s more a c i d i c than e i t h e r the b lood or c e l l i n t e r i o r , N H 3 i s t rapped i n the tubu le lumen as i t r a p i d l y b u f f e r s sec re ted H + to form N H 4 + . Th i s ' a c i d t r a p p i n g ' of N H 3 as N H 4 + - 3 -ma in ta ins a cons tan t downhi l l g rad ien t f o r N H 3 d i f f u s i o n a t the lumina l c e l l membrane and r e s u l t s i n net Nh^"1" e x c r e t i o n (reviewed by P i t t s , 1974; R e c t o r , 1976; Warnock and R e c t o r , 1981). R e c e n t l y , the concept of ammoniagenesis and ' a c i d t r a p p i n g ' has been quest ioned by A tk inson and Camien (1982) . These authors were apparent ly unaware of an ex tens i ve l i t e r a t u r e d e s c r i b i n g severa l decades of i n tense research on the sub jec t (reviewed by Ba lagu ra -Ba ruch , 1971; Cohen and Kamm, 1976; Hems, 1975; S t o f f , E p s t e i n , Nar ins and Relman, 1976; K l a h r and Schoo lwer th , 1977; Tannen, 1978; Warnock and R e c t o r , 1981) and i ns tead have based t h e i r c r i t i c i s m on the s imple obse rva t i on tha t g lutamine ca tabo l i sm to glutamate produces e q u i v a l e n t amounts of N H 3 and H + . As such , the authors po in t out t ha t there can be no net a c i d e x c r e t i o n by the kidney and they c l a i m the k idney p lays l i t t l e r o l e i n ac id -base r e g u l a t i o n . These authors f a i l e d to c o n s i d e r , however, the obvious p o s s i b i l i t y of f u r t h e r g lutamine metabol ism. In r e a l i t y , g lutamine i s f i r s t conver ted to glutamate and then t o « < - k e t o g l u t o r a t e produc ing two N H 3 and two H + . A l p h a -k e t o g l u t o r a t e i s metabo l i zed p r i m a r i l y to g lucose or c a t a b o l i z e d to C O 2 and H 2 0 (see f o r example, Krebs and V inay , 1975; Cheema-Dhadli and H a l p e r i n , 1978; Venay, Lemieux and Gorugoux, 1979). Dur ing the process the two H + are consumed r e s u l t i n g i n net N H 3 s y n t h e s i s and p r o v i d i n g the b a s i s f o r net u r i n a r y ammonia and a c i d e x c r e t i o n as N H ^ . Ac id -base e x c r e t i o n in amphibians depends on the coord ina ted f u n c t i o n s of the k i dney , u r i n a r y b ladde r , s k i n and in some c a s e s , the g i l l s . L i t t l e i s known about the r o l e of the amphibian kidney i n ac i d -base regu-l a t i o n which i s s u r p r i s i n g g iven the u t i l i t y of t h i s organ i n s t u d i e s of c e l l u l a r mechanisms of rena l t r a n s p o r t . E a r l y micropuncture s t u d i e s in the f r og and Necturus (Montgomery and P i e r c e , 1937; Walker , 1940) suggested tha t - 4 -the k idney was i n v o l v e d i n u r i na ry a c i d i f i c a t i o n and ammonia e x c r e t i o n . Yoshimura, Y a t a , Yuasa and Wolbach (1961) have shown tha t the f r o g kidney responds i n a compensatory manner to a c i d o s i s . More r e c e n t l y , O'Regan, Ma ln i c and G i e b i s c h (1982) have shown u r i n a r y a c i d i f i c a t i o n i n Necturus p rox -imal tubu le and Boron and Boulpaep (1983a,b) have desc r ibed a c e l l u l a r mecha-nism i n Ambystoma proximal tubu le which they b e l i e v e i s i nvo l ved i n both i n t r a c e l l u l a r pH r e g u l a t i o n and u r i na ry a c i d i f i c a t i o n (d i scussed i n Chapter V I ) . The r o l e of the amphibian s k i n i n ac id -base ba lance can be i n f e r r e d from i t s a b i l i t y to exc re te H + (Huf, P a r r i s h and Weather ford , 1951; F l e m i n g , 1957; Garc ia-Romeu, S a l i b i a n and Pezzan i -Hernandez , 1969; E m i l i o , Machado and Menano, 1970; Eh ren fe ld and Garc ia-Romeu, 1977) , N H 4 + ( F a n e l l i and G o l d s t e i n , 1964; Vanat ta and F r a z i e r , 1980) and H C 0 3 " or OH" (Garc ia-Romeu, 1971; Eh ren fe l d and Garc ia-Romeu, 1978; Vanat ta and F r a z i e r , 1981) , and by the a b i l i t y of the animal to a l t e r t h i s e x c r e t i o n i n response to ac i d -base d is tu rbances (Vanatta and F r a z i e r , 1980, 1981). Amphibian g i l l s are s i m i l a r l y capable of e x c r e t i n g HH^+ and H C O 3 " o r OH" (D ie tz and A l v a r a d o , 1974) and may a l s o be i nvo l ved i n ac i d -base homeostas is . The most e x t e n s i v e l y s tud ied a c i d - b a s e exc re to ry organ i n amphibians i s the toad u r i na ry b ladder (reviewed by S te i nme tz , 1974; L e a f , 1982). Th is organ exc re tes H + , N H 4 + and HC0 3 ~ or OH" ( F r a z i e r and V a n a t t a , 1971, 1972, 1973; Ludens and F a n e s t i l , 1972; F i t z g e r r e l and V a n a t t a , 1980), and compensatory changes i n the e x c r e t i o n of these ions are observed dur ing ac i d -base imbalances ( F r a z i e r and V a n a t t a , 1971, 1973; F i t z g e r r e l and V a n a t t a , 1980). In a d d i t i o n , a c i d and base e x c r e t i o n may be under hormonal and neural con t ro l ( F r a z i e r and Z a c h a r i a h , 1979; F r a z i e r and - 5 -V a n a t t a , 1980) i n the b ladder f u r t h e r i m p l i c a t i n g i t as an important pH regu la to r y s i t e . Only a few i n d i r e c t s t u d i e s have examined renal a c i d and base e x c r e t i o n i n r e p t i l e s and the r e s u l t s suggest t ha t the k idney may be i nvo l ved i n ac i d -base homeostas is . Fo r example, i n the a l l i g a t o r and c r o c o d i l e k i d n e y s , N H 4 + and H C 0 3 ~ are sec re ted i n exchange f o r N a + , K + and C l ~ , and the major s a l t i n the f i n a l u r i ne i s N H 4 H C O 3 (Coulson and Hernandez, 1959; Schmid t -N ie l sen and Skadhauge, 1967). The e x c r e t i o n of N H 4 H C O 3 i s thought to be i nvo l ved p r i m a r i l y i n e l i m i n a t i o n of excess water and i n s a l t conse rva t i on s i nce the rena l tubu les of these animals have a l i m i t e d capac i t y f o r e s t a b l i s h i n g and ma in ta in i ng a h igh u r i n e - t o - p l a s m a osmot ic g rad ien t (Schmid t -N ie lsen and Skadhauge, 1967). Observa t ions of Coulson and Hernandez (1959, 1961) sugges t , however, t ha t e x c r e t i o n of N H 4 H C O 3 may a l s o be l i n k e d to ac id -base r e g u l a t i o n . D a n t z l e r (1969) has observed pa t te rns of u r i n a r y a c i d i f i c a t i o n i n f reshwater snakes s i m i l a r to those observed i n mammalian nephrons and he has suggested tha t the tubu le c e l l s p lay an impor tant r o l e i n H + e x c r e t i o n and HC03~ r e a b s o r p t i o n . In a d d i t i o n , M inn ich (1972) has p rov ided ev idence which suggests t ha t u r i na ry a c i d e x c r e t i o n i n r e p t i l e s i s i n f l u e n c e d by d i e t i n a pa t te rn s i m i l a r to t ha t observed f o r mammals (Long and G i e b i s c h , 1979). U r i c a c i d i s the most important end product of n i t r ogen metabol ism i n r e p t i l e s ( D a n t z l e r , 1978) and the pH of the u r i c a c i d / u r a t e bu f f e r system (ca . 5.8) suggests t ha t i t cou ld p lay a r o l e i n u r i n a r y ac i d -base e x c r e t i o n . In support of t h i s i d e a , D a n t z l e r (1968) found tha t induced a l k a l o s i s i n the f reshwater snake N a t r i x s ipedon i nc reased u ra te s e c r e t i o n i n the form of monobasic u r a t e . E x c r e t i o n of t h i s form of u ra te would serve to e l i m i n a t e excess base from the b lood and regu la te pH. - 6 -Other organs tha t may be i n v o l v e d i n r e p t i l e a n a c i d - b a s e homeostasis are the c l o a c a which exc re tes both ura te and u r i c a c i d (House, 1974, c i t e d i n M i n n i c h , 1979) and a c i d i f i e s the f i n a l u r i ne (Green, 1969, c i t e d i n M i n n i c h , 1979) , and the s a l t g land of herb ivorous r e p t i l e s which may be a major s i t e f o r the e x c r e t i o n of d i e t a r y base (see M i n n i c h , 1979). In the t u r t l e k idney the renal medul la and d i s t a l nephron are poor l y developed and u r i ne a c i d i f i c a t i o n and s a l t and water ba lance are i n -s tead c a r r i e d out by the u r i n a r y b ladder . The b ladder of f reshwater t u r t l e s has been the most e x t e n s i v e l y s tud ied r e p t i l e a n ac id -base exc re to ry organ and i s the pr imary s i t e of u r i ne a c i d i f i c a t i o n (reviewed by S te inme tz , 1974) or a l k a l i n i z a t i o n (Brodsky, Durham and Ehrenspeck, 1980; Sa take , Durham and Brodsky , 1982) depending on the an ima l ' s ac i d -base s t a t u s . In a d d i t i o n , i n  v i t r o p repa ra t i ons of the b ladder have p rov ided the bulk of c u r r e n t i n fo rma-t i o n on molecu la r and c e l l u l a r mechanisms of u r i n a r y a c i d t r a n s p o r t (reviewed by A l - A w q a t i , 1978; Ste inmetz and Anderson, 1982; d i scussed i n Chapter V I ) . Only a few s t u d i e s have been conducted on renal a c i d and base e x c r e t i o n i n b i r d s . F i n a l u r ine to b lood pH g rad ien ts generated by the av ian k idney are s i m i l a r to those observed i n mammals (Long, 1982) and u r i n a r y a c i d i f i c a t i o n has been shown to be i n f l u e n c e d by the a n i m a l ' s ac i d -base s ta tus (Wolbach, 1955; Sauveur, 1968; S i m k i s s , 1970) and d i e t (Okumura and T a s a k i , 1968). The av ian k idney may a l s o c o n t r i b u t e to a c i d - b a s e balance by e x c r e t i o n of H + bound to n o n v o l a t i l e b u f f e r s . Some b i r d spec ies have been shown to have high u r i n a r y ammonia e x c r e t i o n r a t e s , but the major end product of n i t rogen metabol ism i s u r i c a c i d (Long, 1982) which may be i n v o l v e d i n e x c r e t i o n of ac i ds and bases i n a manner s i m i l a r to tha t suggested f o r r e p t i l e s (see p rev ious d i s c u s s i o n ) . Prashad and Edwards (1973) have suggested tha t e x c r e t i o n and t i t r a t i o n of phosphate may be a means of a c i d - 7 -e x c r e t i o n i n b i r d s dur ing a c i d o s i s induced by eggshe l l c a l c i f i c a t i o n . Ac id -base r e g u l a t i o n has been s tud ied e x t e n s i v e l y i n f i s h . The g i l l s perform both ac i d -base exc re to ry and r e s p i r a t o r y f u n c t i o n s and the b ranch ia l e p i t h e l i u m appears to be the major s i t e of a c i d , base and ammonia e l i m i n a t i o n (reviewed by Evans, 1975, 1980; K i r s c h n e r , 1979; Evans, C l a i b o r n e , Farmer, M a l l e r y and Krasny , 1982; H e i s l e r , 1982). Fur thermore, b ranch ia l a c i d and base movements respond to a c i d o t i c and a l k a l o t i c c o n d i t i o n s i n a compensatory manner ( rev iewed by Cameron, 1978a; H e i s l e r , 1980, 1982) i n d i c a t i n g tha t the f u n c t i o n of the g i l l s i s c r u c i a l f o r ac i d -base homeostas is . Comparat ive ly l i t t l e work has been done on the f u n c t i o n o f o ther f i s h organ systems i n a c i d and base e x c r e t i o n . The sk i n and the r e c t a l g land appear to p lay n e g l i g i b l e r o l e s i n ac id -base r e g u l a t i o n a l though few s t u d i e s have been conducted on these organs (see H e i s l e r , 1982). The f u n c t i o n of the k idney i n pH balance may vary cons ide rab l y between s p e c i e s . For example, i n the marine t e l e o s t , Opsanus b e t a , Evans (1982) found tha t the k idney p lays a n e g l i g i b l e r o l e i n a c i d e x t r u s i o n . In the f reshwater c a t f i s h , however, Cameron (1980) and Cameron and Kormanick (1982) demonstrated t ha t renal e l i m i n a t i o n of a c i d and base was c r u c i a l f o r recovery from ac i d -base d i s t u r b a n c e s . S i m i l a r l y , Wood and Ca ldwe l l (1978) suggested t ha t renal e x c r e t i o n o f a c i d i n the f reshwater t r o u t cou ld account comple te ly f o r the observed recovery from a f i x e d a c i d l o a d . These spec ies d i f f e r e n c e s may be r e l a t e d to the f a c t t ha t marine t e l e o s t s g e n e r a l l y have lower u r i ne f low ra tes than f reshwater f i s h and have a f i x e d u r ine pH which prevents M g 2 + p r e c i p i t a t i o n and a l l ows cont inued M g 2 + e x c r e t i o n (see f o r example, P i t t s , 1934; Sm i t h , 1939) . - 8 -2) Inver teb ra te Ac id -Base E x c r e t i o n The only group of i n v e r t e b r a t e s i n which the exc re to ry r e g u l a t i o n of ac id -base ba lance has been s y s t e m a t i c a l l y s tud ied are the C r u s t a c e a . In c r u s t a c e a n s , as i n f i s h e s , the b ranch ia l e p i t h e l i u m performs both r e s p i r a t o r y and ac id -base exc re to ry f u n c t i o n s . Net e x c r e t i o n of a c i d ( K i r s c h n e r , Greenwald and K e r s t e t t e r , 1973; E h r e n f e l d , 1974; T rucho t , 1979) , base ( E h r e n f e l d , 1974; T rucho t , 1979) and ammonia (Mangum, S l i v e r t h o r n , H a r r i s , Towle and K r o l l , 1976; P r e s s l e y and Graves , 1980; Kormanick and Cameron, 1981; Pequeux and G i l l e s , 1981) occurs at the g i l l and f l u x e s of a c i d and base change i n a compensatory manner dependent upon the ac i d -base s ta tus of the animal (Cameron, 1978b, 1979; T rucho t , 1979). The r o l e p layed by the antennal g land i n ac i d -base r e g u l a t i o n may vary cons ide rab l y between s p e c i e s . Fo r example, i n the antennal glands of C a l l i n e c t e s (Cameron and B a t t e r t o n , 1978) and Carc inus (Truchot , 1979) , a c i d , base and ammonia e x c r e t i o n are n e g l i g i b l e . In the Dungeness c r a b , however, the antennal g land exc re tes l a r g e q u a n t i t i e s of HC03~ and ammonia, and t h i s organ may be c r i t i c a l l y important i n pH r e g u l a t i o n (Wheat ly, 1982). In i n s e c t s , the mechanisms of ac i d -base r e g u l a t i o n are e s s e n t i a l l y unknown. The few p rev ious s tud ies of i n s e c t ac id -base phys io logy were mainly concerned w i th determin ing hemolymph pH, b u f f e r i n g c a p a c i t y and C O 2 content under a s i n g l e p h y s i o l o g i c a l c o n d i t i o n (C ra ig and C l a r k , 1938; Hast ings and Pepper , 1943; Levenbook, 1950a,b) . The i n s e c t exc re to ry system most l i k e l y p lays the predominant r o l e i n ac id -base homeostas is . Un l i ke the v e r t e b r a t e lung and b ranch ia l g i l l o f aqua t i c organisms, the i n s e c t r e s p i r a t o r y system outwardly appears to be poor l y designed f o r ac i d -base regu la to ry f u n c t i o n s . A s e r i e s of a i r - f i l l e d tubes , the t r ac hae , d e l i v e r 0 2 d i r e c t l y to i n s e c t t i s s u e s and c e l l s . The - 9 -t rachae open on the ex te rna l sur face of the animal through the s p i r a c l e s and gas exchange i n the t r a c h e a l system i s e f f e c t e d by mass f low down p ressure g rad ien ts and d i f f u s i o n a long concen t ra t i on g rad ien ts (reviewed by S c h e i d , Hook and B r i d g e s , 1981) . Gas exchange i n l a rge i n s e c t s i s a l s o mediated by v e n t i l a t o r y movements of the abdomen w a l l . Insec t hemolymph p lays no d i r e c t r o l e i n gas t r a n s p o r t and exchange. Hemolymph c i r c u l a t i o n in i n s e c t s i s r e l a t i v e l y slow and the c i r c u l a t o r y system i s 'open ' w i th a s i n g l e , d o r s a l l y l o c a t e d vesse l which f u n c t i o n s as a h e a r t . Fur thermore, i n s e c t s possess no s t r u c t u r e s analagous to e r y th rocy tes which are e s s e n t i a l f o r gas t r anspo r t and r e s p i r a t o r y maintenance of a c i d - b a s e balance in v e r t e b r a t e s . Resp i r a to r y pigments are a l s o l a c k i n g i n most i n s e c t s . Noted excep t ions are a few spec ies of e n d o p a r a s i t i c i n s e c t s , ch i ronomid l a r vae and some d i v i n g Hemipterans such as Anisops and Buenoa which possess va r i ous types of hemoglobin. In the e n d o p a r a s i t e , G a s t r o p h i l u s i n t e s t i n a l i s , hemoglobin f u n c t i o n s as an 0 2 s to re under anox ic c o n d i t i o n s ( K e i l i n and Wang, 1946) , wh i l e i n Chironomus hemoglobin f unc t i ons as an 0 2 s to re and t r a n s p o r t e r a t low 0 2 t ens ions (Ewer, 1942; Walshe, 1950). Hemoglobin i n the backswimmer, An isops a s s i m i l i s , f u n c t i o n s un ique ly i n the maintenance of neu t ra l buoyancy dur ing pro longed d i ves by r e l e a s i n g 0 2 i n t o and r e g u l a t i n g the s i z e of the ex te rna l gas bubble ( W e l l s , Hudson and B r i t t a i n , 1981). A c i d and base e x c r e t i o n have been s tud ied only very s u p e r f i c i a l l y i n i n s e c t s . A few i n d i r e c t observa t ions suggest tha t the rectum and Ma lp igh ian tubu les may be ac id -base regu la to r y s i t e s . For example, Waterhouse (1940) found t ha t i n L u c i l i a the Ma lp igh ian tubu le f l u i d was s l i g h t l y a l k a l i n e compared to the hemolymph wh i l e the rectum had a pH between 4 . 8 to 5 . 3 . S i m i l a r obse rva t i ons were made on D ix ippus (Ramsay, 1956) where - 10 -the r e c t a l f l u i d had a pH of 3.5 to 4 . 5 . In the deser t l o c u s t the pH of Ma lp igh ian tubu le f l u i d i s about the same as hemolymph pH ( S p e i g h t , 1967) , however, P h i l l i p s (1961) and Spe ight (1967) found tha t the rectum a c t i v e l y ma in ta ins an a c i d pH g rad ien t of 2-3 u n i t s . Hanrahan (1982) has shown luminal a l k a l i n i z a t i o n i n s h o r t - c i r c u i t e d l o c u s t r ec ta s t imu la ted w i th cAMP. R e c e n t l y , Sz ibbo and Scudder (1979) have shown tha t segment II of the Ma lp igh ian tubu les of Cenocor ixa b i f i d a produces an a l k a l i n e f l u i d dur ing cAMP s t i m u l a t i o n . M a r t i n and Strange (unpub l ished obse rva t i ons ) demonstrated t h a t t h i s a l k a l i n i z a t i o n i s due to HC03~ s e c r e t i o n and suggested tha t t h i s may represent an important ac i d -base regu la to ry mechanism s i nce t h i s spec ies i s normal ly found i n N a H C ^ - C t ^ s a l t l akes (Scudder, 1969). An ac i d -base regu la to ry r o l e of the i n s e c t exc re to ry system can a l s o be i n f e r r e d from i t s a b i l i t y to t r a n s p o r t t i t r a t a b l e b u f f e r s such as ammonia (P rusch , 1971, 1972, 1975; Chamber l i n , 1982; Hanrahan, 1982), phosphate (reviewed by M a d d r e l l , 1977; P h i l l i p s , 1980) and u r i c a c i d (Wiggelswor th , 1931; O ' D o n n e l l , Maddre l l and G a r d i n e r , 1983) . The major goal o f the present work was to study the problem of i n s e c t ac i d -base r e g u l a t i o n a t the whole a n i m a l , organ and c e l l u l a r l e v e l s , and to study fundamental mechanisms of e p i t h e l i a l H + and H C O 3 " t r a n s p o r t . Larvae of the sa l twa te r mosquito Aedes dorsa l i s were chosen f o r t h i s study f o r two reasons . F i r s t , /\. dorsa l i s normal ly i n h a b i t s hype rsa l i ne NaHC0 3 -C0 3 waters (Scudder, 1969; S t range , P h i l l i p s and Quamme, 1982) c l e a r l y r e q u i r i n g the animal to possess powerful ac id -base regu la to ry mechanisms. And second, the exc re to ry system of sa l twa te r mosquito l a r vae has been f a i r l y we l l c h a r a c t e r i z e d (d i scussed be low) . - 11 -B. O rgan i za t i on o f L a r v a l Excre to ry System Sa l twa te r mosquito l a r vae can s u r v i v e i n a range o f environments from f r esh water to 300% sea water , and i n a t h a l a s s o h a l i n e s a l t l akes where the predominant s a l t s may be mix tures o f MgSO^ Na2S04 or NaHC.03 and ^ 0 0 3 . Under these environmental c o n d i t i o n s hemolymph i o n i c and osmotic concen t ra t i ons are regu la ted w i t h i n narrow l i m i t s by the coo rd ina ted f u n c t i o n s of the Ma lp igh ian t u b u l e s , anal p a p i l l a e and r e c t a l s a l t g land ( F i g . 1 .1 ) . The Ma lp igh ian tubu les of s a l t w a t e r l a r vae f u n c t i o n much the same as those i n o ther i n s e c t s by produc ing a p r imary , i sosmo t i c s e c r e t i o n o r ' f i l t r a t e . 1 of the hemolymph which i s impor tant f o r waste e x c r e t i o n . In a d d i t i o n , the Ma lp igh ian tubu les of Aedes campest r is have the unique a b i l i t y 2- 2+ to a c t i v e l y sec re te S 0 4 (Maddrel l and P h i l l i p s , 1975) and Mg ( P h i l l i p s and M a d d r e l l , 1974) , which i s an impor tant adapta t ion f o r l a r v a e i n h a b i t i n g Na 2 S04 and MgS0 4 l a k e s . A c t i v e S O 4 s e c r e t i o n has a l s o been demonstrated i n the Ma lp igh ian tubu les of Aedes taeniorhynchus (Maddrel l and P h i l l i p s , 1978) . The anal p a p i l l a e of s a l t w a t e r l a r v a e i n h a b i t i n g d i l u t e media have been shown to be s i t e s of N a + , C l " ( P h i l l i p s and M e r e d i t h , 1969) and K + (Sche re r , 1977) up take. The f unc t i on of these organs i n s a l i n e wate r , however, i s u n c e r t a i n . Balance sheets o f whole animal ion and water movements ( P h i l l i p s and B r a d l e y , 1977; P h i l l i p s , Brad ley and M a d d r e l l , 1978) and u l t r a s t r u c t u r a l s t u d i e s on the anal p a p i l l a e of l a r v a e i n h a b i t i n g hyperosmot ic media (Meredi th and P h i l l i p s , 1973a,b) suggest i n d i r e c t l y t ha t these organs may p lay a r o l e i n ion e x c r e t i o n . In a d d i t i o n , these organs cou ld be i nvo l ved i n the uptake of ions p resent i n low concen t ra t i ons i n a t h a l a s s o h a l i n e media. F igu re 1.1 O rgan iza t i on of l a r v a l gut and exc re to ry system. - 13 -Both t e r r e s t r i a l ( P h i l l i p s , 1980, 1981) and s a l t w a t e r (S tobbar t and Shaw, 1974) i n s e c t s exc re te a f i n a l u r i ne which i s s t r ong l y hyperosmotic to the hemolymph. P roduc t i on of hyperosmot ic exc re ta i n t e r r e s t r i a l i n s e c t s i s an important means of water conse rva t i on and i s c a r r i e d out by the rectum which s e l e c t i v e l y reabsorbs water and s o l u t e s from the i sosmo t i c Ma lp igh ian tubu le f l u i d (reviewed by P h i l l i p s , 1980, 1981). Sa l twa te r mosquito l a r v a e , on the o ther hand, i n g e s t l a rge q u a n t i t i e s of hyperosmotic media (reviewed by P h i l l i p s and B r a d l e y , 1977) to rep lace water l o s t by osmosis and d i f f u s i o n ac ross the c u t i c l e . As such , s a l t w a t e r mosquito l a r vae are not faced w i th the problem of water c o n s e r v a t i o n , but i n s t e a d , of r i d d i n g themselves of i nges ted i o n s . The f i r s t s t u d i e s on the f u n c t i o n of the rectum i n s a l t w a t e r mos-q u i t o l a r vae were c a r r i e d out by Mered i th and P h i l l i p s (1973c) . These i n v e s -t i g a t o r s examined r e c t a l u l t r a s t r u c t u r e i n the f reshwater l a r v a , Aedes  a e g y p t i , and i n the s a l t w a t e r s p e c i e s , A . campes t r i s . In A . campest r is i t was found t ha t the rectum lacked the e l a b o r a t e l y f o l ded l a t e r a l c e l l membranes tha t are a s s o c i a t e d w i th hyperosmot ic u r ine fo rmat ion i n t e r r e s -t r i a l i n s e c t s . I ns tead , the rectum was d i v i d e d i n t o two d i s t i n c t segments, each c o n s i s t i n g of a s i n g l e c e l l t ype . C e l l s of the a n t e r i o r r e c t a l segment, which resembled r e c t a l c e l l s i n the f reshwater s p e c i e s , A_. a e g y p t i , had we l l developed a p i c a l and basal membrane i n f o l d i n g s and mi tochondr ia d i s t r i b u t e d evenly throughout the cytop lasm ( F i g . 1 .2 ) . The u l t r a s t r u c t u r e of the p o s t e r i o r segment found only i n the s a l t w a t e r spec ies was cons ide rab l y more e l a b o r a t e , however. C e l l s of t h i s segment were shown to be approx imate ly tw ice as t h i c k as those of the a n t e r i o r rectum and most of the mi tochondr ia were a s s o c i a t e d w i th the a p i c a l i n f o l d i n g s which extended ac ross 60% o f the c e l l . Based on these o b s e r v a t i o n s , Mered i th and P h i l l i p s (1973c) pos tu l a ted - 14 -Posterior Anterior Segment Segment Cuticle-' Lumen F igu re 1 .2 . O r g a n i z a t i o n and u l t r a s t r u c t u r e o f r e c t a l s a l t g l and . - 15 -t ha t s a l t w a t e r mosquito l a r vae produce a hyperosmotic u r i ne by s e c r e t i o n of ions or a concent ra ted f l u i d across the wa l l of the rectum. D i r e c t con f i rma t i on of t h i s hypo thes is was p rov ided by Brad ley and P h i l l i p s (1975) us i ng A .^ taeniorhynchus l a r v a e and e labo ra ted upon i n f u r t h e r s t u d i e s (Bradley and P h i l l i p s , 1 9 7 7 a , b , c ) . B r i e f l y , i f the animal i s l i g a t e d p o s t e r i o r l y and a n t e r i o r l y to the rectum, the organ s w e l l s w i th a s t r ong l y hyperosmotic s e c r e t i o n . N a + , K + , C l " and M g + + are a l l sec re ted i n t o the rectum a g a i n s t 2 - to 1 8 - f o l d concen t ra t i on g r a d i e n t s . Th i s obse rva t i on p l us measurements o f 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 e n c e s i n d i c a t e d tha t a l l f ou r ions are t r anspo r t ed by a c t i v e mechanisms (Bradley and P h i l l i p s , 1977c) . The r o l e p layed by the rectum i n i o n i c r e g u l a t i o n du r ing a c c l i m a t i o n of s a l t w a t e r mosquito l a r vae to d i f f e r e n t ex te rna l media was examined by Brad ley and P h i l l i p s (1977a,b) . In g e n e r a l , the concen t ra t i ons of N a + , M g + + and C l " sec re ted i n t o the rectum tended to resemble the concen t ra t i ons o f these ions i n the e x t e r n a l medium to which the animal had been a c c l i m a t e d . Rec ta l K + c o n c e n t r a t i o n s , however, were always more concent ra ted than those i n the a n i m a l ' s environment. These authors a l s o noted t h a t when l a r vae were reared i n hyposmotic media, the rectum f a i l e d to swe l l w i th s e c r e t i o n . Only very smal l amounts of hyposmotic u r i ne cou ld be c o l l e c t e d under these c o n d i t i o n s . S ince no u l t r a s t r u c t u r a l changes were observed i n r e c t a of l a r vae acc l ima ted to hyposmotic or hyperosmot ic c o n d i t i o n s (Meredi th and P h i l l i p s , 1973c) , these s t u d i e s i n d i c a t e d an e l abo ra te r e o r g a n i z a t i o n of membrane c a r r i e r s and/or neural and hormonal c o n t r o l of t r a n s p o r t p rocesses . - 16 -Bradley and P h i l l i p s (1977c) s t u d i e d i n d i r e c t l y the f u n c t i o n s of the a n t e r i o r and p o s t e r i o r r e c t a l segments. The r e s u l t s of t h e i r work and u l t r a s t r u c t u r a l s t u d i e s o f Mered i th and P h i l l i p s (1973c) suggested tha t the p o s t e r i o r rectum was the s i t e of hyperosmot ic f l u i d s e c r e t i o n and tha t the a n t e r i o r rectum was a s i t e of s e l e c t i v e ion r e a b s o r p t i o n . The p resent work i s o rgan ized i n t o s i x chapters w i th the exper imenta l r e s u l t s presented i n Chapters II through V. In Chapter II the degree of hemolymph pH and HCG^ - r e g u l a t i o n i n l a r vae i n h a b i t i n g extremes of a l k a l i n i t y i s examined. Th is chapter a l s o demonstrates c l e a r l y tha t the r e c t a l s a l t g land i s an impor tant s i t e of a c t i v e H C O 3 " e x c r e t i o n and a c i d - b a s e r e g u l a t i o n . Chapter I I I examines the i o n i c requirements f o r HC03~ s e c r e t i o n us ing a new microper fused s a l t g land p repa ra t i on i n con junc t i on w i th convent iona l ion s u b s t i t u t i o n and t r a n s p o r t i n h i b i t o r s t u d i e s . Chapter IV p rov ides f u r t h e r ev idence on the mechanism of HCC^" s e c r e t i o n , demonstrates the s i t e of HCC^" s e c r e t i o n i n the mic roper fused s a l t g land and d e s c r i b e s the f u n c t i o n of both a n t e r i o r and p o s t e r i o r r e c t a l segments. In Chapter V the c e l l u l a r mechanism of H C O 3 " s e c r e t i o n i s s t u d i e d us ing i n t r a c e l l u l a r ion and v o l t a g e - s e n s i t i v e m ic roe lec t rodes i n con junc t i on w i th a mic roper fused a n t e r i o r r e c t a l segment p repa ra t i on which permi t ted r a p i d changes i n bath and luminal f l u i d compos i t i on . A summary of the r e s u l t s i s presented i n Chapter VI a long w i th a comparat ive d i s c u s s i o n of c e l l u l a r mechanisms of ac id -base t r a n s p o r t i n ve r teb ra te e p i t h e l i a . - 17 -CHAPTER I I . BICARBONATE REGULATION AND EXCRETION A . I n t r oduc t i on Hype rsa l i ne l a k e s found i n a r i d reg ions of the wor ld are among the most extreme na tu ra l aqua t i c environments known. The i o n i c composi t ion of these l akes v a r i e s w ide ly and can range from sa tu ra ted b r i nes w i th ion r a t i o s s i m i l a r to sea water to a t h a l a s s o h a l i n e MgSO^, Na2S04 and NaHC03~C03 lakes (Topping and Scudder , 1977). Organisms i n h a b i t i n g such environments are p o t e n t i a l l y va luab le model systems f o r s tudy ing fundamental p h y s i o l o g i c a l and b iochemica l p r o c e s s e s . H a l o p h i l i c organisms can p rov ide impor tant i n s i g h t s i n t o molecu la r s t r u c t u r e and metabo l i c f u n c t i o n s by r e v e a l i n g unde r l y i ng b iochemica l d i f f e r e n c e s i n these parameters which a l l ow the organism to w i ths tand the environmental s t r e s s of s a l t l a k e s . S a l t - l o v i n g b a c t e r i a , f o r example, have been used e x t e n s i v e l y to study p r o t e i n and c e l l membrane s t r u c t u r e and f u n c t i o n (reviewed by Hochachka and Somero, 1973; Langworthy, 1982) . C l e a r l y , the va lue of an organism such as Ha lobacter ium halobium i n s t u d i e s of pho tosyn the t i c mechanisms, membrane p r o t e i n s t r u c t u r e and H + t r a n s p o r t cannot be overes t imated (see f o r example, S t o e c k e n i u s , 1976; S t o e c k e n i u s , L o z i e r and Bogomoln i , 1979; Krumrn and Dw ived i , 1982; Ha ines , 1983) . H a l o p h i l i c organisms make good model systems a l s o by v i r t u e of the f a c t t ha t many of t h e i r p h y s i o l o g i c a l p rocesses are exaggerated and the re fo re e a s i l y s t u d i e d . Fo r example, v i r t u a l l y no th ing i s known about the mechanisms 2+ of t r a n s e p i t h e l i a l Mg t r anspo r t and at tempts to study t h i s phenomenon i n convent iona l model e p i t h e l i a such as the mammalian k idney have been 2+ thwarted by extremely low t u b u l a r Mg f l u x e s (Su t ton , Quamme and D i r k s , 1979). C e r t a i n spec ies of s a l t w a t e r mosquito l a r v a e , however, normal ly - 18 -i n h a b i t hype rsa l i ne MgS0 4 l akes and by n e c e s s i t y must exc re te l a r g e q u a n t i -2+ t i e s o f Mg . Th i s e x c r e t i o n i s c a r r i e d out by both the Ma lp igh ian tubu les ( P h i l l i p s and M a d d r e l l , 1974) and r e c t a l s a l t g land (Brad ley and P h i l l i p s , 1977a ,b ) , e p i t h e l i a which can now be i s o l a t e d complete ly i n v i t r o (Wi l l i ams and Beyenbach, 1982; S t range , t h i s study) and which are composed of homogeneous, r e l a t i v e l y l a rge t r a n s p o r t i n g c e l l s t ha t g r e a t l y s i m p l i f y t r a n s p o r t s t u d i e s . For s a l t w a t e r organisms, NaHCC^-CC^ l akes p resent both i o n i c and ac id -base regu la to r y problems. Soda l akes o f ten have pH va lues exceeding 10 and H C O 3 " and C O 3 concen t ra t i ons as high as 1 to 2.4 M ( B l i n n , 1969; Topping and Scudder , 1977). Only a few organisms are capable of s u r -v i v i n g under these severe a l k a l i n e c o n d i t i o n s , the most prominent of which are i n s e c t s , c r u s t a c e a n s , h a l o p h i l i c b a c t e r i a and a lgea ( B l i n n , 1969; Scudder , 1969). L i t t l e i s known about the p h y s i o l o g i c a l adap ta t ions which a l l ow organisms to s u r v i v e i n these a l k a l i n e env i ronments. A c i d - b a s e and i o n i c r e g u l a t i o n have been s tud ied p r e v i o u s l y i n the f i s h T i l a p i a grahami which normal ly i n h a b i t s a l k a l i n e Lake Magadi i n Kenya 's R i f t V a l l e y ( R e i t e , Ma lo iy and Aasehaug, 1974; Johansen, Ma lo iy and Lykkeboe, 1975; Maetz and DeRenz is , 1978, M a l o i y , Lykkeboe, Johansen and Bamford, 1978). The f u n c t i o n a l cha rac -t e r i s t i c s and adap ta t ions of T i l a p i a hemoglobin have been i n v e s t i g a t e d by Lykkeboe, Johansen and Ma lo iy (1975). These authors demonstrated tha t T i l a p i a hemoglobin had a very low s e n s i t i v i t y to changes i n pH and i o n i c s t reng th which permi t ted normal hemoglobin f u n c t i o n over the high and v a r i -ab le range of pH and i o n i c s t reng th found i n the an ima l ' s p lasma. A very h igh temperature s e n s i t i v i t y of the oxy-hemoglobin e q u i l i b r i u m was - 19 -a d d i t i o n a l l y observed which f a c i l i t a t e s both 0 2 l oad ing and un load ing i n the h i gh l y v a r i a b l e Lake Magadi environment. P h y s i o l o g i c a l s t u d i e s have a l s o been conducted on seve ra l spec ies o f a l k a l o p h i l i c b a c t e r i a . These organisms have been shown to possess e x t r a o r d i n a r i l y h igh l e v e l s and complex assortments of membrane cytochromes which may represen t an important adap ta t ion to the b i o e n e r g e t i c cos t s of l i f e a t h i gh l y a l k a l i n e pH va lues (Lewis , P r i n c e , Du t ton , Knof f and K r u l w i c h , 1981; K r u l w i c h , 1982). In a d d i t i o n , a l k a l o p h i l i c b a c t e r i a have evo lved N a V s o l u t e co t ranspo r t mechanisms (Bonner, Mann, Gu f fan t i and K r u l w i c h , 1982) s i m i l a r to those observed i n e u k a r y o t i c t i s s u e s (Schu l t z and C u r r a n , 1970; Aronson, 1981) f o r ion coupled s o l u t e t r a n s p o r t . T h i s form of so l u te uptake i s unique to a l k a l o p h i l i c b a c t e r i a as most o ther b a c t e r i a l spec ies u t i l i z e H + / s o l u t e c o t r a n s p o r t systems which are energ ized by an inward ly d i r e c t e d proton g rad ien t such tha t [H + ]ou t > [ H + ] i n (see f o r example, F l agg and W i l s o n , 1977; Konings and B o o n s t r a , 1977). The need of a l k a l o p h i l i c b a c t e r i a to ma in ta in cy top lasm ic pH a c i d i c to ex te rna l pH, however, reverses the g rad ien t ( i . e . [ H + ] i n > [H + ] ou t ) and thus prevents i t from e n e r g i z i n g s o l u t e uptake. To study the c e l l u l a r mechanisms of i n s e c t ac i d -base r e g u l a t i o n , l a r v a e of the mosquito Aedes dorsa l i s were chosen. Aedes do rsa l i s i s a common s a l t w a t e r spec ies found throughout much of western North America and i n one of the only metazoan organisms capable of i n h a b i t i n g hype rsa l i ne NaHC03~C03 l a k e s . In the present chapter the extremes of a l k a l i n i t y under which l a r vae can develop normal ly and the ex ten t of hemolymph pH and HC03~ r e g u l a t i o n i n these environments i s examined. I t i s a l s o shown tha t the r e c t a l s a l t g land i s an impor tant s i t e of HC03~ e x c r e t i o n and pH - 20 -r e g u l a t i o n , and r e c t a l HC0 3 ~ s e c r e t i o n i s demonstrated to occur by an e n e r g y - r e q u i r i n g mechanism. B. M a t e r i a l s and Methods An ima ls . Aedes dorsa l i s eggs were ob ta ined from the Department of B iomedica l and Envi ronmenta l Hea l th S c i e n c e s , U n i v e r s i t y of C a l i f o r n i a , B e r k e l e y . Eggs were hatched as desc r ibed p rev i ous l y (B rad ley , 1976) and l a r vae were reared a t 25°C i n an a l k a l i n e r e a r i n g medium (Table 2 .1 ) resembl ing na tu ra l l ake waters (Topping and Scudder , 1977). Larvae were fed d a i l y w i th dry f i s h food (Tetramin S t a p l e Food) and the r e a r i n g media were aera ted gent ly and changed p e r i o d i c a l l y to prevent s t a g n a t i o n . Maintenance of the co lony was o therw ise s i m i l a r to t h a t desc r ibed by Brad ley (1976). S u r v i v a l s t u d i e s were conducted by abrupt ly t r a n s f e r r i n g t h i r d and f ou r th i n s t a r l a r vae f ou r days a f t e r ha tch ing to media w i th h igh H C O 3 " and C O 3 / C l r a t i o s . M o r t a l i t y and development o f l a r vae were observed f o r f ou r days a f t e r t r a n s f e r . Based on these r e s u l t s , three exper imenta l media were chosen f o r use i n the remainder of t h i s study and are r e f e r r e d to as 2_ Rear ing medium, 250 mM HC03~ medium, and 100 mM C O 3 medium (Table 2 . 1 ) . A l l exper iments were conducted on f ou r th i n s t a r l a r vae acc l ima ted to these a r t i f i c i a l l ake waters f o r a t l e a s t th ree days. La rva l d r i n k i n g ra te was es t imated a t 25°C us ing the * 4 C - i n u l i n uptake method of B rad ley and P h i l l i p s (1975). Samples of l a r v a l hemolymph were c o l l e c t e d and N a + , K + , M g 2 + , C a 2 + , C l " and osmot ic concen t ra t i ons were measured as desc r i bed by Brad ley and P h i l l i p s (1975, 1977b). P h y s i o l o g i c a l s a l i n e s (Table 2 .2) used to bathe i s o l a t e d r e c t a were based on measured i o n i c and osmotic concen t ra t i ons of na tu ra l hemolymph. Free amino a c i d concen t ra t i ons i n l a r v a l hemolymph were determined us ing a - 21 -Tab le 2.1 Composi t ion of a r t i f i c i a l l ake waters i n which l a r v a e were reared or a c c l i m a t e d . Rear ing 250 mM 1Q0 mM C o n s t i t u e n t (mM) Medium HC0 3 ~ Medium C 0 3 ~ Medium N a + 361.50 361.50 398.00 K + 2.50 2.50 2.50 C a 2 + 0.03 0.03 0.03 M g 2 + 0.50 0.50 0.50 C l " 246.06 39.56 97.06 2 -S O 4 10.00 10.00 10.00 H C 0 3 " 43.50 250.00 87.00 C 0 3 2 " 29.00 29.00 100.00 PH 9.50 8.85 9.75 mOsm 638.00 597.00 576.00 - 22 -Tab le 2.2 Composi t ion of hemolymph (mean + S . E . , n = 6-10) and p h y s i o l o g i c a l s a l i n e s f o r l a r v a e acc l ima ted to d i f f e r e n t a r t i f i c i a l l ake wa te rs . A l l th ree s a l i n e s a l so con ta ined the f o l l o w i n g ( i n mM): p r o l i n e 20 , a l a n i n e 5 , g l y c i n e 3 , g lutamine 4 , succ ina te 7 . 4 , c i t r a t e 2 . 5 , g lucose 10. L a r v a l A c c l i m a t i o n Medium Rear ing 250 mM 100 mM 3 - 2 -C o n s t i t u e n t (mM) Medium HC0 Medium C0„ Medium Hemolymph S a l i n e Hemolymph S a l i n e Hemolymph S a l i n e N a + 163+2.4 164.5 191+7.2 189.5 182+2.0 180 K + 9.5+0.7 9 9.4+0.9 9 11.7+0.5 9 M g 2 + 3.8+0.2 4 4.8+0.4 4 5.5+0.4 4 C a 2 + 8.7+0.5 4 12.7+0.8 4 14.2+0.9 4 s o 4 2 ~ — 5 — 5 — 5 C l " 56+1.8 56 39.1+2.3 39 49+1.6 49 cy lcamate" — 89 125 108.5 H C 0 3 - 8.1+0.5 12.5 18.5+0.6 18.5 12.1+0.3 15.5 pH 7.55+0.03 .7.51 7.70+0.02 7.70 7.70+0.02 7.60 mOsm 359+1.5 380 432+3.0 427 401+12 420 a S e e Tab le 2.1 f o r compos i t ion of a r t i f i c i a l l ake wa te rs . - 23 -Beckman 118 C automat ic amino a c i d ana l yze r and the fou r major hemolymph amino ac i ds were i n c l u d e d i n the s a l i n e s a t p h y s i o l o g i c a l c o n c e n t r a t i o n s . The l a r g e anion d e f i c i t observed i n l a r v a l hemolymph was s imu la ted by i n c l u d i n g sodium cyclamate i n the s a l i n e s . A l l s a l i n e s were gassed wi th 98% 0 2 - 2% C 0 2 to g ive a C 0 2 concen t ra t i on s i m i l a r to t ha t measured i n hemolymph (see R e s u l t s ) . C a p i l l a r y pH E l e c t r o d e s . To min imize C 0 2 l o s s from samples o f hemolymph and r e c t a l s e c r e t i o n dur ing pH measurements, u l t r a m i c r o i n t e r n a l c a p i l l a r y pH e l e c t r o d e s were used ( F i g . 2 . 1 ) . These e l e c t r o d e s were mod i f i ed from K h u r i , A g u l i a n and Harik (1968) and requ i red a t o t a l sample volume o f 10 to 30 n l . E l e c t r o d e s were c a l i b r a t e d a t 25°C i n four bu f f e r s (pH 6.4 to 9.0) o f cons tan t i o n i c s t reng th s i m i l a r to t ha t o f na tura l hemolymph or r e c t a l s e c r e t i o n . The pH of these bu f fe rs was determined before each experiment us ing a Radiometer Model 27 pH meter and Radiometer pH e l e c t r o d e c a l i b r a t e d w i th Radiometer b u f f e r s a t 25°C. Only e l e c t r o d e s w i th f u l l response t imes of l e s s than 60 seconds and a c a l i b r a t i o n curve s lope of 55 to 61 mV/pH U n i t were used. The mean (+S.E.) c a l i b r a t i o n curve s lope and c o r r e l a t i o n c o e f f i c i e n t f o r a l l e l e c t r o d e s used throughout t h i s study were 58.32+0.19 and 1.00+0.00 (n=34), r e s p e c t i v e l y . One d i f f i c u l t y exper ienced w i th these e l e c t r o d e s was a smal l downward d r i f t o f the vo l tage recorded i n any c a l i b r a t i o n bu f f e r a f t e r pH measurements on b i o l o g i c a l f l u i d s . The cause of t h i s d r i f t was unce r ta i n and c o u l d not be c o r r e c t e d by r i n s i n g w i th chromic a c i d or by repea ted ly ' e x e r c i s i n g ' the e l e c t r o d e i n two b u f f e r s . To moni tor t h i s prob lem, e l e c t r o d e s were r e c a l i b r a t e d i n two b u f f e r s f o l l o w i n g each pH measurement. Subsequent pH measurements on hemolymph or r e c t a l s e c r e t i o n samples.were then - 24 -F i g u r e 2.1 U l t r a m i c r o i n t e r n a l c a p i l l a r y pH e l e c t r o d e . - 25 -to Syringe - 26 -c o r r e c t e d f o r t h i s d r i f t us ing the new c a l i b r a t i o n cu rve . E l e c t r o d e s showing d e v i a t i o n s i n c a l i b r a t i o n s lope of more than +_ 2mV/pH u n i t were d i s c a r d e d . Hemolymph pH Measurements. Regu la t i on of hemolymph pH was examined 2 -by t r a n s f e r r i n g t h i r d and fou r th i n s t a r l a r vae to high HC0 3 and C 0 3 media four days a f t e r h a t c h i n g . Hemolymph samples were c o l l e c t e d by b r i e f l y r i n s i n g l a r v a e i n d i s t i l l e d water , b l o t t i n g them dry and p l a c i n g them under heavy p a r a f f i n o i l (MCB Reagents; Saybo l t v i s c o s i t y 340-355) . The c u t i c l e was then to rn w i th fo rceps and the drop of exuded hemolymph c o l l e c t e d immediately i n a m e r c u r y - f i l l e d m i c r o p i p e t . Th is sample was t r a n s f e r r e d r a p i d l y to a heavy p a r a f f i n o i l bath (25°C) and a c a p i l l a r y pH and 3M K C 1 mic roe lec t rode were lowered i n t o the hemolymph drop v i s u a l i z e d under a d i s s e c t i n g mic roscope. The p o t e n t i a l d i f f e r e n c e between the e l e c t r o d e s was measured w i th a K i e t h l e y Model 602 e lec t rome te r and Model 6013 pH e l e c t r o d e adaptor and the vo l tage recorded on a F i s h e r S e r i e s 5000 R e c o r d a l l . Hemolymph H C O 3 " Measurements. Hemolymph H C 0 3 ~ was measured us ing the u l t r a m i c r o As t rup method of Karlmark and S o h t e l l (1973) . To e q u i l i b r a t e p a r a f f i n o i l baths w i th gasses o f d i f f e r e n t C O 2 c o n t e n t , h igh gas f low ra tes (2-3 Kmin"-*-) and an e q u i l i b r a t i o n t ime of a t l e a s t one hour were used before samples were added. Karlmark and S o h t e l l (1973) found tha t a minimum gas f low ra te of 450 ml .m in"^ and a 30 minute e q u i l i b r a t i o n t ime were necessary f o r complete gas to o i l e q u i l i b r a t i o n . Th is was conf i rmed us ing an Or ion PC0 2 e l e c t r o d e , but the h igher f low ra tes were chosen to assure complete e q u i l i b r a t i o n under a l l c o n d i t i o n s . In a d d i t i o n , i t was found t ha t complete gas to o i l e q u i l i b r a t i o n a t 25°C was on ly ob ta ined when l i g h t p a r a f f i n o i l (MCB Reagents; Saybo l t v i s c o s i t y 90) was used. - 27 -Samples of hemolymph were c o l l e c t e d f o r de te rmina t ion of H C 0 3 " as desc r i bed above and a l lowed to e q u i l i b r a t e w i th COg under o i l f o r 2 0 to 30 minutes before pH measurements were made w i th c a p i l l a r y e l e c t r o d e s a t 25°C. Gas f low was turned o f f b r i e f l y ( l e s s than 1 min) dur ing sample l o a d i n g and pH measurements. D i f f e r e n t hemolymph samples were e q u i l i b r a t e d w i th f ou r gasses of va ry i ng C O 2 content ( 0 . 5 to 10% C O 2 , ba lance N^; Canadian L i q u i d A i r Company, c e r t i f i e d a n a l y s i s ) . R e s u l t i n g pH versus log P C 0 2 t i t r a t i o n curves were p l o t t e d us ing the l e a s t squares method of l i n e a r r e g r e s s i o n . Rec ta l M ic ropunc ture S t u d i e s . The micropuncture p repa ra t i on used i n t h i s study was mod i f i ed cons ide rab l y from tha t desc r i bed by Brad ley and P h i l l i p s (1975). B r i e f l y , the rectum was i s o l a t e d by l i g a t i n g a l a r v a j u s t a n t e r i o r to the seventh abdominal segment and a l s o a t the te rmina l anal segment. The p o r t i o n of the l a r v a a n t e r i o r to the f i r s t l i g a t u r e , the c u t i c l e over the rectum, and the t r achea l connect ions were then d i s s e c t e d away. Us ing the te rm ina l l i g a t u r e , the rectum was suspended i n 1 0 to 15 ml of the app rop r ia te p h y s i o l o g i c a l s a l i n e main ta ined a t 25°C and gassed w i th 98% 0 2 - 2% C O 2 . A f t e r 90 minutes the rectum had swol len n o t i c e a b l y w i th s e c r e t i o n and was removed gent ly from the ba th , touched l i g h t l y to b ibu lous paper to remove adher ing ex te rna l f l u i d , and p laced under p a r a f f i n o i l . Ten to 40 nl of r e c t a l s e c r e t i o n were c o l l e c t e d by punc tu r ing r e c t a w i th sha rp -ened and b e v e l l e d ac id-washed m ic rop ipe ts f i l l e d w i th Sudan b l a c k - s t a i n e d p a r a f f i n o i l . M ic ropunc ture p i p e t s were b e v e l l e d a t an angle o f 30° to a t i p d iameter of 1 0 - 1 5 JJ us ing a K .T . Brown type m ic roe lec t rode bevel e r (Su t t e r Instrument Company, San F r a n c i s c o , C a l i f o r n i a ) . Cannulated In V i t r o Rec ta l P r e p a r a t i o n . To examine r e c t a l H C 0 3 ~ s e c r e t i o n i n g rea te r d e t a i l , a mic rocannu la ted p repa ra t i on was - 28 -developed which permi t ted r a p i d and complete c o l l e c t i o n of sec re ted r e c t a l f l u i d . Diagrams of the Sy lga rd r e s i n (Dow Corn ing) ba th ing chamber and the cannu la ted rectum are shown i n F i gu re 2 . 2 . A l l cannu la t i on exper iments were conducted a t room temperature (21-23°C) us ing l a r vae acc l ima ted to 250 mM H C 0 3 " medium. Mic rocannu lae were p u l l e d from g l a s s tub ing over a smal l f lame to y i e l d t i p d iameters o f 35 to 50 u. The t i p s of the p i pe t s were broken to the app rop r i a te leng th w i th fo rceps and the broken end h e a t - p o l i s h e d w i th a m i c ro fo rge . P i p e t s were s i l i c o n i z e d w i th Dow Corn ing 1107 f l u i d , f i l l e d wi th Sudan b l a c k - s t a i n e d p a r a f f i n o i l and mounted on a N a r i s h i g e micromanipu la tor us ing a m ic rop ipe t ho lde r ( L e i t z , W e t z l a r , Germany) a t tached to a 50 ml s y r i n g e . Recta were i s o l a t e d by p l a c i n g a l a r v a i n the ba th ing chamber and t y i n g a f i n e l i g a t u r e around the te rmina l anal segment. The ba th ing chamber was f i l l e d and per fused con t inuous ly w i th p h y s i o l o g i c a l s a l i n e and the rectum, i leum and a p o r t i o n of the midgut were d i s s e c t e d f r ee from the l a r v a . Approx imate ly 20 to 30 nl of s a l i n e were then drawn up i n t o a microcannula and the p i pet i n s e r t e d down the midgut and i n t o the rectum. The s a l i n e was i n j e c t e d i n t o the rectum and then withdrawn complete ly and e x p e l l e d i n t o the b a t h . A f t e r a s s u r i n g t ha t the rectum was complete ly empty the microcannula was r e i n s e r t e d and t i e d i n t o p lace us ing two u l t r a f i n e l i g a t u r e s (diameter c a . 10 J J ; see F i g . 2 . 2 B ) . Dur ing the course of these exper iments the ba th ing chamber was per fused w i th s a l i n e gassed w i th 98% 0 2 - 2% C 0 2 and r e c t a were a l lowed to swel l w i th s e c r e t i o n f o r 90 minutes before f l u i d was drawn i n t o the cannula us ing the a t tached s y r i n g e . A t the end of the exper iments the bath - 29 -F i g u r e 2.2 M ic rocannu la ted r e c t a l p r e p a r a t i o n . A) Sy lga rd r e s i n ba th ing chamber. B) Cannulated r e c t a l s a l t g land . - 30 -Agar Bridge - 31 -was d r a i n e d , the rectum removed from the p i pet and the sample of s e c r e t i o n prepared f o r a n a l y s i s . A n a l y s i s o f Rec ta l S e c r e t i o n s . Rec ta l s e c r e t i o n volume was determined by measuring drop diameters under p a r a f f i n o i l as desc r i bed by Brad ley and P h i l l i p s (1975). Osmotic concen t ra t i ons of r e c t a l s e c r e t i o n s were measured w i th a C l i f t o n n a n o l i t e r osmometer. Concen t ra t i ons of N a + , K + , M g 2 + , C a 2 + , C l " , t o t a l phosphorous and t o t a l s u l f u r were measured i n each sample of s e c r e t i o n by e l e c t r o n microprobe a n a l y s i s us ing a Cameca model MBX microprobe and methods desc r i bed p r e v i o u s l y (Morel and R o i n e l , 1969; R o i n e l , 1975). Because of the high ion concen t ra t i ons i n r e c t a l f l u i d , samples were d i l u t e d two to three t imes w i th d i s t i l l e d water before p repa ra t i on f o r microprobe a n a l y s i s . C h l o r i d e concen t ra t i ons were a l s o measured by the e l e c t r o m e t r i c t i t r a t i o n procedure of Ramsay, Brown and Croghan (1955) on r e c t a l s e c r e t i o n s pooled from four to f i v e an ima l s . B ica rbona te concen t ra t i ons i n r e c t a l s e c r e t i o n s c o l l e c t e d by micropuncture were es t imated by measuring the pH of samples e q u i l i b r a t e d under p a r a f f i n o i l a t 25°C w i th 98% 0 2 - 2% C 0 2 accord ing to the methods desc r i bed above. In l a t e r exper iments , t o t a l C 0 2 and pH were measured i n r e c t a l s e c r e t i o n s c o l l e c t e d by m i c r o c a n n u l a t i o n . S e c r e t i o n pH was measured a t 25°C immediately a f t e r c o l l e c t i o n wh i l e t o t a l C 0 2 concen t ra t i ons were determined by m ic roca lo r ime t r y (P icapnotherm; M i c r o a n a l y t i c Ins t rumen ta t i on , Be thesda, Maryland) as desc r ibed by Vurek, Warnock and Corsey (1975). A l i q u o t s of r e c t a l s e c r e t i o n were t r a n s f e r r e d to the Picapnotherm by cons tan t volume n a n o l i t e r p i p e t s (2 to 3 n l ) s i m i l a r to those of P r a g e r , Bowman and Vurek (1965). T r a n s e p i t h e l i a l P o t e n t i a l . Rec ta 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 (TEP) was measured us ing a microcannula (see above) w i th 3M KC l -Aga r i n the t i p and - 32 -the remainder of the p i p e t f i l l e d w i th 3M KC1 s o l u t i o n . The microcannula and bath chamber made con tac t v i a s a l t b r idges w i th calomel e l e c t r o d e s and TEP was measured us ing a K i e t h l e y Model 602 e lec t r ome te r . T r a n s e p i t h e l i a l p o t e n t i a l was recorded con t inuous ly f o r 90 minutes on a F i s h e r S e r i e s 5000 R e c o r d a l l w i th subsequent c o r r e c t i o n s f o r j u n c t i o n and asymmetry p o t e n t i a l s . p C a l c u l a t i o n s . Concen t ra t ions of C 0 2 , HCO3 and CO3 were c a l c u l a t e d from the Henderson-Hasse lba lch equat ion a f t e r c o r r e c t i n g p K j ' , p K 2 ' , and C 0 2 s o l u b i l i t y c o e f f i c i e n t s (S) f o r i o n i c s t reng th us i ng the equat ions o f Has t ings and Sendroy (1925) and McGee and Has t ings (1948). Values o f p K ^ ' , p K 2 ' and S used throughout t h i s study are shown i n Tab le 2 . 3 . S t a t i s t i c a l ana lyses were conducted us ing p a i r e d or unpa i red t - t e s t s i n t h i s chapter and throughout the t h e s i s . C. R e s u l t s In p r e l i m i n a r y s t u d i e s I examined the a b i l i t y of A . dorsa l i s l a r vae to s u r v i v e i n s a l i n e media bu f fe red w i th low concen t ra t i ons of HC03~ and p CO3 a t h igh pH v a l u e s . La rva l s u r v i v a l and development were normal i n media w i th pH va lues up to 10 .5 . A pH 11.0 s u r v i v a l was s t i l l normal but pupat ion and p u p a l - t o - a d u l t e c l o s i o n were g r e a t l y reduced. S ince the pH of a l k a l i n e lakes r a r e l y r i s e s above 10.5 due to the inheren t nature of t h e i r b u f f e r i n g systems, f a c t o r s o ther than pH per se must l i m i t the d i s t r i b u t i o n o f l a r v a e i n na tu ra l a l k a l i n e wa te rs . F u r t h e r s u r v i v a l s t u d i e s were t h e r e f o r e conducted by r e p l a c i n g NaCl i n the Rear ing medium w i th d i f f e r e n t concen t ra t i ons of NaHC0 3 and/or N a 2 C 0 3 . F i g u r e 2.3 demonstrates tha t s u r v i v a l and development are normal i n media c o n t a i n i n g 250 mM HCO3" or 100 mM C 0 3 2 " . S l i g h t l y h igher concen t ra t i ons of these two ions (300 mM p HC0 3 ~ and 150 mM CO^ ) r e s u l t e d i n g r e a t l y reduced s u r v i v a l (32 to - 33 -Tab le 2 .3 Values of p K j ' , p K 2 ' and S used f o r c a l c u l a t i o n o f C 0 2 , H C O 3 " and C O 3 2 " c o n c e n t r a t i o n s . L a r v a l A c c l i m a t i o n Medium 9 Constant Rear ing Medium 250 mM HC0 3 ~ Medium 100 mM C 0 3 2 " Medium Hemolymph p K i ' 6.12 6.11 6.11 Hemolymph S 0.0340 0.0338 0.0339 Secre t ion* 3 pKi' 6.04 6.04 6.04 Secre t ion* 5 S O'.O 329 0.0329 0.0329 S e c r e t i o n 0 P K i ' — 6.00 — S e c r e t i o n 0 p K 2 ' — — — 9.64 a See Table 2.1 f o r compos i t ion o f a c c l i m a t i o n media. b Rec ta l s e c r e t i o n c o l l e c t e d by mic ropuncture . c Rec ta l s e c r e t i o n c o l l e c t e d by m i c r o c a n n u l a t i o n . - 34 -F i g u r e 2 . 3 . S u r v i v a l and development of f ou r th i n s t a r l a r vae i n a l k a l i n e env i ronments. Refer to Table 2.1 f o r media compos i t i on . - 36 -46% a f t e r four days) and f a i l u r e of any of the l a rvae to develop pas t the pupal s tage . Ion i c and osmot ic concen t ra t i ons of hemolymph from animals acc l ima ted to h igh HCG^" and C O 3 environments are shown i n Tab le 2 . 2 . Hemolymph osmotic concen t ra t i ons i nc reased s l i g h t l y but s i g n i f i c a n t l y (0.005 < P < 0.01) from 359 mOsm to 401-432 mOsm f o l l o w i n g t r a n s f e r of l a r vae from the Rear ing medium to e i t h e r 250 mM H C 0 3 " o r 100 mM 0 0 3 * - medium. In a d d i t i o n , hemolymph N a + concen t ra t i on i nc reased s i g n i f i c a n t l y (P< 0.001) from 163 mM to 181-190 mM, w h i l e C a 2 + i nc reased from 8.5 mM to 12 .5 -14 .0 mM (P < 0 .001 ) . Hemolymph C l " concen t ra t i on decreased s l i g h t l y but s i g n i f i c a n t l y (0.001 < P < 0.005) as ex te rna l C l " was rep laced by H C 0 3 " and C 0 3 2 " . F l u i d i n g e s t i o n ra te was measured to es t imate the l oad imposed on pH and H C O 3 " r egu la to r y mechanisms i n the l a r vae and to determine whether l a r vae cou ld reduce t h i s l oad by reduc ing d r i n k i n g r a t e . F i g u r e 2.4 shows tha t d r i n k i n g ra te (53-56 n l .mg"^ .h~^) was independent of ex te rna l H C O 3 " and C 0 3 2 " l e v e l s . To determine how we l l l a r vae regu la te hemolymph a c i d - b a s e s ta tus when faced w i th such high i n g e s t i o n r a t e s , hemolymph pH was measured f o r four consecu t i ve days f o l l o w i n g t r a n s f e r of l a r v a e from the Rear ing medium to e i t h e r h igh HC0 3 ~ or h igh C 0 3 2 " environments ( F i g . 2 . 5 ) . Hemolymph pH i nc reased on ly s l i g h t l y from 7.55 to 7.70 dur ing the f i r s t day and remained constant t h e r e a f t e r . Hemolymph HC03~ concen t ra t i on was determined i n l a r v a e a c c l i -mated to a l l th ree a r t i f i c i a l l ake waters ( F i g . 2 . 6 ) . A t y p i c a l t i t r a t i o n curve of hemolymph pH versus l og hemolymph PC0 2 i s shown i n F i g . 2 .6A. T i t r a t i o n curves f o r the three groups of l a r vae were l i n e a r w i th c o r r e l a t i o n c o e f f i c i e n t s va ry i ng between -0 .98 and - 0 . 9 9 . Hemolymph P C 0 2 was - 37 -F i g u r e 2.4 D r i n k i n g ra tes es t imated by i H C - i n u l i n i n g e s t i o n f o r fou r th i n s t a r l a r v a e acc l ima ted to a l k a l i n e environments (mean _+ S . E . , n = 1 0 - 1 2 ) . Refer to Table 2.1 f o r media compos i t i on . 75 50 H 25 -\ T Rearing Medium - 39 -F i g u r e 2.5 Hemolymph pH r e g u l a t i o n in f ou r th i n s t a r l a r vae dur ing a c c l i m a t i o n to h igh HCOo" and CO^ 2 - media. Animals were t r a n s f e r r e d from the Rear ing medium to 250 mM HCOo" or 100 mM C0o2~ media a t zero t ime (means +_ S.E., n = 6-97. Refer to Tab le 2.1 f o r media compos i t i on . I 1 I T T 0 1 2 3 4 Days after transfer - 41 -F i g u r e 2.6 Hemolymph HCG^ - c o n c e n t r a t i o n . A) pH v s . l og PCO2 t i t r a t i o n curve f o r hemolymph from animals acc l ima ted to the Rear ing medium. S o l i d c i r c l e s are means _+ S . E . (n = 8-9) of hemolymph samples t i t r a t e d w i th one of four d i f f e r e n t CO0-N2 gas m i x t u r e s . The l i n e i s the l i n e a r r eg ress i on c a l c u l a t e d f o r these po in t s (y = -0 .51x + 8 . 0 3 ; r = - 0 . 9 9 4 ) . S o l i d squares are pH va lues of na t i ve hemolymph (see F i g . 2 . 5 ) . The va lues were f i t t e d to the l i n e us ing the c a l c u l a t e d r e g r e s s i o n equa t i on . B) Concen t ra t ion of hemolymph HCO3" i n animals acc l ima ted to a l k a l i n e media. PCO2 va lues were obta ined from pH v s . l og PCO2 t i t r a t i o n curves and HC03~ concen t ra t i on c a l c u l a t e d us ing the Henderson-Hasse lba lch equat ion (means ;+ S . E . , n = 9 -29 ) . Re fe r to Table 2.1 f o r media compos i t i on . - 42 -E x t e r n a l Medium - 43 -determined us ing these t i t r a t i o n curves and the measured hemolymph pH va lues i n F i g . 2 . 5 . P C O 2 va lues were ( i n mm Hg, mean _+ S . E . , n = 9-29) 9.05 +_ 1 .10, 14.79 +_ 1.33 and 9.44 + 0 . 5 0 f o r an imals acc l ima ted to the R e a r i n g , 250 mM H C O 3 " and 100 mM C O 3 media, r e s p e c t i v e l y . Hemolymph P C O 2 was s i g n i f i c a n t l y h igher (P<0.0001) i n animals acc l ima ted to 250 mM H C O 3 " medium. B ica rbona te concen t ra t i ons c a l c u l a t e d from these data are shown i n F i g . 2 .6B . Desp i t e l a r g e i n c r e a s e s i n ex te rna l H C O 3 " and C O 3 l e v e l s , hemolymph HC03~ concen t ra t i ons remain low and only i nc rease from 8.0 to 12 .0 -18 .5 mM. As p r e v i o u s l y desc r ibed by Brad ley and P h i l l i p s (1975, 1977a) f o r Aedes taeniorhynchus and A . campest r is l a r v a e from o ther h y p e r s a l i n e wa te rs , the l i g a t e d rectum of h. do rsa l i s a l s o s w e l l s w i th s e c r e t i o n when bathed i n a r t i f i c i a l hemolymphs. The osmotic and i o n i c concen t ra t i ons of s e c r e t i o n s c o l l e c t e d by micropuncture o f i s o l a t e d r e c t a are shown i n F i g . 2 . 7 . Mean N a + (270-300 mM) and osmot ic (700-735 mOsm) concen t ra t i ons were e s s e n t i a l l y the same f o r l a r vae acc l ima ted to the th ree ex te rna l media. Mean c h l o r i d e concen t ra t i ons i n r e c t a l s e c r e t i o n s i nc reased from 50 mM to 135 mM, be ing p l owest i n animals acc l ima ted to high HC03~ and 0 0 3 ^ media. Concen t ra t ions of K + i n r e c t a l s e c r e t i o n s were between 27 and 46.5 mM. C a 2 + and M g 2 + con -c e n t r a t i o n s i n s e c r e t i o n s were very low (0 .3 - 1.0 mM) w h i l e t o t a l phosphorus v a r i e d from 1.5 to 4.6 mM and t o t a l s u l f u r was c a . 8.0 mM (Table 2 . 4 ) . Osmotic and i o n i c concen t ra t i ons of r e c t a l s e c r e t i o n s c o l l e c t e d from microcannu la ted r e c t a o f l a rvae acc l ima ted to 250 mM HC03~ medium are shown i n F i g . 2 .8 and were s i g n i f i c a n t l y h igher than those of s e c r e t i o n s c o l l e c t e d by micropuncture ( c f . F i g . 2 . 7 ) . Mean osmotic concen t ra t i on was 1030 mOsm, w h i l e mean N a + , K + and C l ~ concen t ra t i ons were 413 mM, 31.4 mM and 44.9 mM, r e s p e c t i v e l y , Concen t ra t i ons of Ca and Mg" were - 44 -F i g u r e 2.7 Osmo la l i t y and concen t ra t i ons of major ions i n r e c t a l s e c r e t i o n s c o l l e c t e d by micropuncture from la r vae acc l ima ted to three d i f f e r e n t a l k a l i n e media (means _+ S . E . , n = 5 - 1 2 ) . See Tables 2.1 and 2.2 f o r composi t ion of a c c l i m a t i o n media and p h y s i o l o g i c a l s a l i n e s . 400 H 1-800 X ImOsmj 300—1 Na+ CT LU ,E c o ra Z 200 c 0> u c o o o o 100 X [HCO; cv Rearing Medium X Na + JHCO3 X ImOsml X Na + (mOsml I -600 WCCv C l Cl 250 mM H C O 3 Medium 100 mM C O 3 Medium -1-0 b CM X E co O E, c o (0 -p. 1-400 -c 0) u c o o I- 200 ~ o E co O - 46 -Tab le 2.4 Concen t ra t i ons of C a 2 , Mg 2 , t o t a l s u l f u r and t o t a l phosphorus and pH in r e c t a l s e c r e t i o n s c o l l e c t e d by mic ropunc tu re . pH va lues were determined on s e c r e t i o n s e q u i l i b r a t e d w i th 2% C 0 2 (mean + S . E . , n = 7 -11 ) . La rva l a c c l i m a t i o n medium 3 Rear ing 250 mM 100 mM C o n s t i t u e n t (m M) Medium H C 0 3 " Medium C 0 32 " Medium 0.77+0.11 1.04+0.26 — C a 2 + 0.34+0.04 0.76+0.22 — Tota l S u l f u r 7.85+0.81 8.90+1.84 — Tota l Phosphorus 4.63+1.03 3.61+1.64 1.49+0.30 PH 8.43+0.04 8.72+0.05 8.72+0.03 a See Tab le 2.1 f o r composi t ion of a c c l i m a t i o n media. - 47 -again very low ( 0 . 5 - 0 . 8 mM) wh i l e t o t a l phosphorus l e v e l s were 2 .4 mM and t o t a l s u l f u r was 13.4 mM (data not shown). The major o b j e c t i v e of the micropuncture s t u d i e s was s imply to determine whether the rectum was an impor tant s i t e of H C O 3 " e x c r e t i o n and r e g u l a t i o n . B i ca rbona te concen t ra t i ons were thus i n i t i a l l y es t imated by measuring the pH a t a known C 0 2 t ens ion of r e c t a l s e c r e t i o n s c o l l e c t e d by mic ropunc ture . Th i s method i s the same as tha t used i n e a r l y work on the k idney (Go t t scha l k , L a s s i t e r and M y l l e , 1960; V i e r a and M a l n i c , 1968) and pancreas (Swanson and Solomon, 1973) and i s based on the assumption tha t lumina l P C 0 2 i s the same as tha t i n the hemolymph ( i . e . 2.0% C 0 2 ) . The concen t ra t i ons of HC03~ c a l c u l a t e d from the Henderson-Hasse lba lch equat ion us ing the measured pH and known P C 0 2 are shown i n F i g . 2.7 and ranged between 150 to 240 mM. I t must be s t r e s s e d here tha t the c a l c u l a t e d HC03~ l e v e l s shown i n F i g . 2 .7 are s l i g h t overes t imates of ac tua l c o n c e n t r a t i o n s . A t the pH va lues of the r e c t a l s e c r e t i o n s (8.4 - 8 . 7 ; Table 2 . 4 ) , a s i g n i f i c a n t f r a c t i o n of the t o t a l C 0 2 pool i s p resent as C 0 3 2 " which cannot be c a l c u l a t e d d i r e c t l y from the Henderson-Hasse lba lch equat ion us ing only known pH and p PC0 2 v a l u e s . To determine ac tua l H C O 3 and C O 3 concen t ra t i ons i n r e c t a l s e c r e t i o n t hen , and to c a l c u l a t e t r a n s e p i t h e l i a l H C O 3 " and p C O 3 g rad ien ts across the r e c t a l w a l l , I subsequent ly measured t o t a l C 0 2 i n s e c r e t i o n s c o l l e c t e d from cannu la ted r e c t a of animals acc l ima ted to 250 mM H C O 3 " medium. The t o t a l C 0 2 concen t ra t i on i n these s e c r e t i o n s was 444 + 25 mM (mean + S . E . , n = 6) w h i l e pH was 8.65 + 0.05 (mean + S . E . , n = 7 ) . Us ing these measured v a l u e s , concen t ra t i ons of C 0 2 , HC03~ and p C O 3 were c a l c u l a t e d from the Henderson-Hasse lba lch equat ion and were 0 .90 mM, 402 mM and 41 mM, r e s p e c t i v e l y (see F i g . 2 . 8 ) . - 48 -F i g u r e 2.8 Osmo la l i t y and concen t ra t i ons of major ions i n r e c t a l s e c r e t i o n s c o l l e c t e d from cannula ted r e c t a of animals acc l ima ted to 250 mM H C O 3 " medium (see Table 2 . 1 ) . Va lues are means +_ S . E . , n = 8 - 9 . Concen t ra t i ons of HC03~ and C O 3 are c a l c u l a t e d from measurements o f t o t a l C O 2 and pH as desc r ibed i n the t e x t . 600 - J or UJ c 400 o mmm CO c <D O c o o Na + mOsml 200-1 O 1200 O) • E CO O c o (0 C <D O C o o [-800 c o K + J - 0 o E CO O - 50 -S e c r e t i o n r a te was es t imated i n cannu la ted r e c t a by measuring the volume of c o l l e c t e d f l u i d and was 37.6 +_ 6.4 nl . h " 1 . r e c t u m " 1 (mean +_ S . E . , n = 6 ) . The c a l c u l a t e d t o t a l C 0 2 s e c r e t i o n ra te i n cannu la ted rec ta was 16.7 nMoles t o t a l C 0 2 . h _ 1 . r e c t u m - 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 s ac ross cannu la ted r e c t a are shown i n F i g . 2 . 9 . Immediately a f t e r l i g a t i o n , mean TEP was -14 .2 mV (lumen n e g a t i v e ) . Dur ing the course of the exper iment TEP g radua l l y i nc reased as the rectum swe l led w i th s e c r e t i o n and reached a value of - 2 5 . 3 mV (lumen negat i ve ) a f t e r 90 minu tes . D. D i s c u s s i o n The r e s u l t s o f s u r v i v a l s t u d i e s i n d i c a t e t ha t A. dorsa l i s l a r vae s u r v i v e and develop normal ly i n concent ra ted NaHC03-C0 3 media and tha t the d i s t r i b u t i o n of t h i s spec ies i n a l k a l i n e lakes i s l i m i t e d not by high pH p per s e , but i n s tead by the concen t ra t i ons of HC0 3 and C 0 3 i n these env i ronments. Larvae i n h a b i t i n g a l k a l i n e waters i nges t the ex te rna l medium a t a ra te e q u i v a l e n t to c a . 130% o f t h e i r body weight per day. P r e -v ious s tud ies (Wigge lswor th , 1933a,b; K i cen iuk and P h i l l i p s , 1974; Maddre l l and P h i l l i p s , 1975) have shown tha t the bulk of inges ted f l u i d and ions i s absorbed i n t o the hemolymph across the midgut and caecae. The degree of hemolymph pH and HC03~ r e g u l a t i o n e x h i b i t e d by these i n s e c t s ( F i g s . 2.5 and 2 .6 ) i s remarkable c o n s i d e r i n g the a c i d - b a s e regu la to ry problems which must be imposed by such h igh ra tes o f f l u i d i n g e s t i o n . The e l eva ted hemolymph PC0 2 observed i n animals acc l ima ted to 250 mM HC0 3 ~ medium (see R e s u l t s ) i s d i f f i c u l t to e x p l a i n a t p resen t , but may represen t an attempt of the l a r vae to r e t a i n C 0 2 so as to lower hemolymph pH. Th is cou ld be a va l uab le regu la to ry response e s p e c i a l l y i f the exc re to ry system i s not capable of e l i m i n a t i n g HC03~ r a p i d l y enough to ma in ta in - 51 -F i g u r e 2.9 T r a n s e p i t h e l i a l p o t e n t i a l (lumen r e l a t i v e to haemocoel s ide ) ac ross cannu la ted r e c t a from animals acc l ima ted to 250 mM H C O 3 medium (see Table 2 . 1 ) . Va lues are means + S . E . , n = 6. - 3 0 0 - L Time (minutes) after cannulation I 9 0 - 53 -hemolymph pH w i t h i n p h y s i o l o g i c a l l i m i t s . A l t e r n a t i v e l y , t h i s e l eva ted P C O 2 va lue cou ld a r i s e from pass i ve processes and l a r g e l y be a r e s u l t of a l e f t w a r d s h i f t o f the CG^-HCC^ - e q u i l i b r i u m as inges ted HCO3" r a p i d l y en te rs the hemolymph. La rva l hemolymph e x h i b i t s a l a r g e i no rgan i c anion d e f i c i t (Table 2 .2 ) which i s c h a r a c t e r i s t i c of d ip te ran hemolymph and i s due to the presence of l a rge concen t ra t i ons of o rgan ic ac i ds ( F l o r k i n and Jeun iaux , 1964). Th is i n o r g a n i c anion d e f i c i t i nc reased as hemolymph N a + concen t ra t i ons i nc reased and C l " l e v e l s dropped when l a r vae were acc l ima ted to h igh HCC^ - and CO3 2" media (Table 2 . 2 ) . I f o rgan ic a c i d s account f o r t h i s d e f i c i t then an i nc rease i n these compounds would not on ly ma in ta in hemolymph e l e c t r o n e u t r a l i t y , but may a l s o enhance hemolymph b u f f e r i n g c a p a c i t y necessary to counter shor t - te rm a l k a l i n e s h i f t s i n hemolymph pH. 9+ Hemolymph Ca concen t ra t i ons (Table 2.2) a l s o i nc reased when l a r vae were acc l ima ted to 250 mM HC03~ or 100 mM C 0 3 2 " media. The 9+ 9+ r i s e i n Ca l e v e l s may r e f l e c t changes i n the amount of Ca bound to o rgan i c mo lecu les . In a d d i t i o n , e l e v a t e d hemolymph pH may i n c r e a s e 9+ Ca complexat ion and p r e c i p i t a t i o n , making i t necessary f o r l a r v a e to 9+ i n c r e a s e t o t a l hemolymph Ca concen t ra t i on as a means of r e g u l a t i n g 9+ Ca*- a c t i v i t y . 1) Rec ta l H C O 3 " S e c r e t i o n The f u n c t i o n o f the rectum of s a l i n e water mosquito l a r v a e i n osmoregulat ion was d i scussed i n Chapter I. B r i e f l y , the rectum i s composed of two u l t r a s t r u c t u r a l l y d i s t i n c t segments (Meredi th and P h i l l i p s , 1973c; c f . F i g . 2.2B) and i s f u n c t i o n a l l y analogous to av ian and r e p t i l e a n s a l t g l ands . When l a r v a e are acc l ima ted to hype rsa l i ne media, the r e c t a l s a l t g land produces s t rong l y hyperosmot ic s e c r e t i o n s w i th ion r a t i o s and t o t a l osmotic - 54 -concen t ra t i ons r e f l e c t i n g those found i n the ex te rna l environment (Bradley and P h i l l i p s , 1975, 1977a,b) . Th i s organ i s be l i eved to be the major s i t e of Na , Mg and C l " r e g u l a t i o n i n s a l i n e waters (Bradley and P h i l l i p s , 1977a). Data from micropuncture s t u d i e s ( F i g . 2.7 and Table 2 .4) demonstrate t ha t the r e c t a l s a l t g land i s a l s o an important s i t e o f H C O 3 " e x c r e t i o n and r e g u l a t i o n as pos tu l a ted by Brad ley and P h i l l i p s (1977a). Larvae acc l ima ted to 250 mM HC03~ and 100 mM C O 3 2 " media produce r e c t a l s e c r e t i o n s w i th s i g n i f i c a n t l y h igher concen t ra t i ons of HC03~ than animals acc l ima ted to the Rear ing medium. The t o t a l osmotic and i o n i c concen t ra t i ons of s e c r e t i o n s c o l l e c t e d by m ic rocannu la t i on were cons ide rab l y h igher than those of s e c r e t i o n s c o l l e c -ted by micropuncture ( c f . F i g s . 2.7 and 2 . 8 ) . The most l i k e l y exp lana t i on f o r t h i s d isc repancy i s r e l a t e d to the d i f f e r e n c e s between the two i s o l a t e d r e c t a l p repa ra t i ons used. With the micropuncture p repa ra t i on i t i s p o s s i b l e t ha t smal l amounts of r e l a t i v e l y d i l u t e midgut and Ma lp igh ian tubu le f l u i d remain i n the rectum a f t e r l i g a t i o n even though the l a r vae normal ly empty the rectum when handled (Bradley and P h i l l i p s , 1975; S t range , unpub l ished obser -v a t i o n s ) . When us ing the cannula ted p r e p a r a t i o n , however, i t was always pos-s i b l e to r i n s e and then empty the rectum complete ly before the s t a r t of any exper iment . Th i s presumably r e s u l t s i n the c o l l e c t i o n of pure r e c t a l s e c r e t i o n . Data from mic rocannu la t i on s t u d i e s a l lowed de te rmina t ion o f t r a n s -r e c t a l H C O 3 " and C 0 3 2 ~ g r a d i e n t s . Lumen-to-hemocoel s ide H C O 3 " p and C0%~ r a t i o s of 21:1 and 2 4 1 : 1 , r e s p e c t i v e l y , were c a l c u l a t e d us ing r e s u l t s shown i n F i g . 2 . 8 . In a d d i t i o n , the c a l c u l a t e d lumina l C O 2 concen-t r a t i o n (see R e s u l t s ) was s l i g h t l y h igher than tha t i n the bath g i v i n g a - 55 -lumen-to-hemocoel C O 2 r a t i o o f 1 . 8 : 1 . p To generate the observed H C O 3 " and C O 3 g rad ien ts by pass i ve mechanisms, 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 of +69 mV to +76 mV (lumen p o s i t i v e ) would be requ i red as c a l c u l a t e d from the Nernst e q u a t i o n . The measured r e c t a l TEP ( F i g . 2 .9) v a r i e d between -14 mV to -25 mV (lumen negat i ve ) demonstrat ing c l e a r l y t ha t H C O 3 " t r anspo r t occurs by an energy-dependent p r o c e s s . In c o n c l u s i o n , i t i s c l e a r t ha t A,, dorsa l i s l a r v a e can i n h a b i t extremes of a l k a l i n i t y and y e t regu la te t h e i r ac id -base s ta tus w i t h i n narrow p h y s i o l o g i c a l l i m i t s . The r e c t a l s a l t g land mediates a l a rge component of ac i d -base r e g u l a t i o n by e x c r e t i n g HCO^" a g a i n s t remarkably l a r g e e l e c t r o -chemical g r a d i e n t s . In the f o l l o w i n g chapter the e p i t h e l i a l mechanism of H C O 3 " e x c r e t i o n i s examined us ing a mic roper fused s a l t g land p r e p a r a t i o n . - 56 -CHAPTER I I I - IONIC REQUIREMENTS OF C O 2 TRANSPORT IN THE MICROPERFUSED RECTAL SALT GLAND A. I n t roduc t i on Mechanisms of H + /OH" and HC03~ t r a n s p o r t have been s t u d i e d i n a wide v a r i e t y of ve r teb ra te e p i t h e l i a i n c l u d i n g f i s h g i l l s , pancreas , rena l t u b u l e s , s a l i v a r y g l a n d s , amphibian and r e p t i l e a n u r i n a r y b l a d d e r s , g a s t r i c mucosa, i n t e s t i n e s , amphibian s k i n , g a l l b ladder and cho ro id p l exus . D e t a i l e d c e l l u l a r mechanisms of a c i d and base movements have been most e x t e n s i v e l y s tud ied i n the t u r t l e u r i n a r y b ladder (reviewed by S te inmetz , 1974; Ste inmetz and Anderson, 1982; see a l s o G luck , Cannon and A l - A w q a t i , 1982) , g a s t r i c mucosa (reviewed by F o r t e , Machen and Obr ink , 1980; Sachs , F a l l e r and Rabon, 1982) and proximal tubu le (reviewed by Warnock and Rec to r , 1979, 1981; see a l s o Boron and Boulpaep, 1983a,b; I ves , Yee and Warnock, 1983; Sasak i and B e r r y , 1983). S tud ies i n s i n g l e c e l l s such as sea u rch in eggs , s n a i l and squ id neurons, and c r a y f i s h and barnac le muscle f i b e r s (reviewed by Roos and Boron , 1981) have p rov ided d e t a i l e d i n fo rma t ion on i n v e r t e b r a t e a c i d and base t r a n s p o r t mechanisms. V i r t u a l l y no th ing i s known, however, about H + / 0 H " and H C O 3 " t r a n s p o r t i n i n v e r t e b r a t e e p i t h e l i a . I n d i r e c t s t ud ies on L i b b e l l u l a and Aeschna nymphs (Krogh, 1939) , f reshwater b i v a l v e s (D ie tz and B ran ton , 1975; D i e t z , 1978; Scheide and D i e t z , 1982) , anal p a p i l l a e of f reshwater mosquito l a r v a e (S tobbar t , 1967, 1971) , earthworm integument (D ie tz and A l v a r a d o , 1970; D i e t z , 1974) and s n a i l integument (de W i t h , Wit teveen and van der Woude, 1980; de With and van der Scho rs , 1982) suggest the presence of N a + / H + (or N H 4 + ) and C T / H C O 3 " exchange mechanisms i n these t i s s u e s . In c rus tacean g i l l s the d i r e c t measurement o f both a c i d and base, and N a + and C l " f l u x e s , and the demonstrat ion of the interdependence of these f l u x e s has p rov ided good evidence f o r the presence - 57 -o f N a + / H + and C 1 " / H C 0 3 ~ exchangers which mediate t r a n s e p i t h e l i a l ac id -base movements ( K i r s c h n e r , Greenwald and K e r s t e t t e r , 1973; E h r e n f e l d , 1974; Pequeux and G i l l e s , 1981) . The s t r u c t u r a l complex i ty of the g i l l ep i t he l i um and problems a s s o c i a t e d wi th both i n v i vo and i n v i t r o s t u d i e s , however, has prevented f u r t h e r c h a r a c t e r i z a t i o n of a c i d and base t r a n s p o r t mechanisms. The mosquito l a r v a , Aedes do rsa l i s , i s one of the few organisms capable of i n h a b i t i n g extremely a l k a l i n e s a l t l akes c o n t a i n i n g h igh concen t ra t i ons of HCG^" and C O 3 and having pH values up to 10.5 (see Scudder , 1969; Topping and Scudder , 1977) . In Chapter II i t was demonstrated tha t the r e c t a l s a l t g land i s an important s i t e of pH and H C O 3 " r e g u l a t i o n i n these l a r v a e . I s o l a t e d s a l t g lands sec re te f l u i d c o n t a i n i n g 402 mM H C O 3 " and 41 mM C 0 3 2 " a t a ra te of 38 n l - h " 1 . Lumen-to-bath H C O 3 " and C 0 3 2 " g rad ien ts o f 21:1 and 2 4 1 : 1 , r e s p e c t i v e l y , are generated by the s a l t g land e p i t h e l i u m a g a i n s t 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 o f -25 mV (lumen n e g a t i v e ) . To i n v e s t i g a t e the t r a n s p o r t mechanisms r e s p o n s i b l e f o r the p maintenance of these e x c e p t i o n a l l y l a rge HCO3 and C O 3 g r a d i e n t s , an i n v i t r o mic roper fused s a l t g land p repa ra t i on was deve loped. The work i n the present chapter examines the i o n i c requirements of CO2 t r a n s p o r t us ing convent iona l ion replacement exper iments and wel l -known i n h i b i t o r s of ac i d -base t r a n s p o r t mechanisms. B. M a t e r i a l s and Methods An ima ls . Larvae were reared i n a low a l k a l i n i t y r e a r i n g medium and the a d u l t co lony main ta ined as desc r ibed p r e v i o u s l y (Chapter I I ) . Three days a f t e r ha tch ing l a r v a e were t r a n s f e r r e d to 250 mM H C O 3 " medium c o n t a i n i n g ( i n mM): 361.5 N a + , 2 .5 K + , 0.03 C a 2 + , 0.50 M g 2 + , 39.6 C l " , 10.0 S 0 4 2 " , - 58 -250 H C 0 3 ~ , 29.0 C 0 3 2 ~ , pH 8 . 9 . A l l exper iments were conducted a t room temperature (21 - 24°C) us ing fou r th i n s t a r l a r vae acc l ima ted to t h i s medium f o r th ree to s i x days. M i c r o p e r f u s i o n System. Diagrams of the bath chamber and m ic ro -per fused s a l t g land are shown i n F i g . 3 . 1 . P e r f u s i o n p i p e t s were p u l l e d from acid-washed g l ass tub ing (1.0 mm O .D . , 0 .8 mm I .D . ; Drummond S c i e n t i f i c Company, B r o o m a l l , Pennsy lvan ia ) over a smal l f lame to y i e l d ou te r diameters of 35 - 50 j i . The p i p e t t i p s were broken to an app rop r ia te leng th w i th fo rceps and the t i p opening reduced to a diameter o f 3 - 8 and heat p o l i s h e d us ing a m i c ro fo rge . C o l l e c t i o n p i p e t s were p u l l e d from g l a s s tub ing (3.0 mm O . D . , 1.8 mm I .D. ) on a m ic rop ipe t p u l l e r (Model PE2, Na r i sh i ge Ins t ruments , Tokyo) . The t i p s o f the p i p e t s were broken o f f w i th fo rceps and a g l a s s cannula (2.0 - 3 .0 mm l o n g , 70 - 90 JJ O . D . , 50 - 70 JJ I .D.) was sea led i n t o the t i p wi th a long column of epoxy. Th i s epoxy seal was e l e c t r i c a l l y i n s u l a t e d and waterproofed us ing Sy lga rd 184 r e s i n (Dow C o r n i n g ) . The bath chamber was f a b r i c a t e d from po lymer ized S y l g a r d 184 r e s i n molded i n t o the bottom of a 55 mm diameter p l a s t i c p e t r i d i s h . To ta l chamber volume was 1.5 to 1.8 m l . Rec ta l s a l t g lands were i s o l a t e d by p inn ing a l a r v a to the bottom of the bath chamber us ing a smal l ' s t a p l e ' fash ioned from an i n s e c t p i n . The ba th ing chamber was f i l l e d and super fused con t inuous ly w i th oxygenated s a l i n e and the anal c a n a l , r e c t a l s a l t g l a n d , i leum and a smal l po r t i on of the midgut were d i s s e c t e d f r e e . S a l t g lands were approx imate ly 1.0 - 1.2 mm long and 200 - 300 |i i n d iameter . The t o t a l length of the p repa ra t i on w i th midgut , i leum and anal canal a t tached was approx imate ly 3.0 - 3.5 mm. - 59 -F igu re 3.1 A) Diagram of Sy lga rd bath chamber showing arrangement of c o l l e c t i o n and pe r f us i on p i p e t s . Bath super fus ion was i n the oppos i te d i r e c t i o n to luminal pe r f us i on and the ba th ing s a l i n e f lowed con t inuous ly over a g l a s s l i p a t the bath o u t l e t i n t o a waste c o n t a i n e r . B) Diagram of microper fused r e c t a l s a l t g l and . Note a n t e r i o r and p o s t e r i o r r e c t a l segments. Flu id Co l lec t ion P i p e t S a m p l i n g P ipet Bath Inlet B B a t h S u p e r f u s i o n F l u i d C o l l e c t i o n P i p e t S a m p l i n g P ipet Poster ior Segment C o l l e c t e d P e r f u s a t e Per fus ion P ipet 10 JJ L i g a t u r e s Anterior Segment N a n o l i t e r P e r f u s i o n P u m p P e r f u s i o n P i p e t - 61 -A f t e r an i n i t i a l 2 - 2.5 hour e q u i l i b r a t i o n pe r i od (see D i s c u s s i o n ) , s a l t g lands were per fused by i n s e r t i n g a p e r f u s i o n p i pet down the midgut and i leum i n t o the a n t e r i o r segment, and by i n s e r t i n g a c o l l e c t i o n p i p e t i n t o the p o s t e r i o r r e c t a l segment v i a the anal c a n a l . Both the c o l l e c t i o n p i p e t ho lde r and pe r f us i on pump (see below) were mounted on N a r i s h i g e micromanipu-l a t o r s (Model MM-3). The s a l t g land was l i g a t e d onto the c o l l e c t i o n and p e r f u s i o n p ipe t s us ing two or three 10 u l i g a t u r e s which were made by t e a s i n g apa r t 5-0 s i l k s u r g i c a l su ture th read (E th i con I n c . , S o m e r v i l l e , N . J . ) under minera l o i l . These l i g a t u r e s were p a r t i a l l y t i e d under the o i l , b l o t t e d on b ibu lous paper and dampened before use . Recta l s a l t g lands were per fused a t a ra te of 10 n l *m in~* us i ng a n a n o l i t e r p e r f u s i o n pump (WPI Model 1400, New Haven, C T ) . The bath chamber was super fused by g r a v i t y at a ra te of 4 - 5 m l ' m i n - * . Dur ing exper iments and e q u i l i b r a t i o n per iods the bath chamber was covered wi th the bottom of a p l a s t i c p e t r i d i sh and a stream of 98% 0 2 - 2% C 0 2 was d i r e c t e d immediately over the bath ing s a l i n e . Determinat ion o f Net t o t a l C 0 2 F l u x . C o l l e c t e d pe r fusa tes were sampled by i n s e r t i n g an o i l - f i l l e d m ic rop ipe t a t tached to a l eng th o f PE 100 tub ing down the bore of the c o l l e c t i o n p i p e t . S a l t g lands were i n i t i a l l y pe r fused f o r a 20 - 25 minute pe r i od a f t e r se t -up and the c o l l e c t e d pe r fusa te d i s c a r d e d . Timed c o l l e c t i o n s (20 - 25 minutes) of pe r fusa te were then made f o r de termina t ion of t o t a l C 0 2 content us ing m ic roca lo r ime t r y (Picapnotherm; M i c r o a n a l y t i c I ns t rumen ta t i on , Bethesda, MD) as desc r ibed by by Vurek , Warnock and Corsey (1975). Volumes of c o l l e c t e d pe r fusa te were determined by measuring drop diameters under o i l w i th an eyep iece micrometer . - 62 -, COo, The net ra te of t o t a l CO? t r a n s p o r t (J c ) was c a l c u l a t e d c net acco rd ing to the e q u a t i o n : J C ° 2 = C c V_ - C D V_ / L net c c P P where C c and Cp are the t o t a l C 0 2 concen t ra t i ons i n the c o l l e c t e d and per fused f l u i d s , r e s p e c t i v e l y , Vc i s the p e r f u s i o n c o l l e c t i o n r a t e , Vp i s the pump pe r fus i on ra te and L i s the s a l t g land l e n g t h . Net f l u i d movement across the per fused s a l t g land e p i t h e l i u m was measured under a v a r i e t y o f exper imenta l c o n d i t i o n s us ing H - i n u l i n (New England Nuc lear C o r p . ) as a volume marker or by determin ing the d i f f e r e n c e s between the t imed pe r fusa te c o l l e c t i o n ra te and the c a l i b r a t e d pump r a t e . Mean i n u l i n recovery under a v a r i e t y of exper imenta l c o n d i t i o n s was 97.0 +_ 0.6% (+_ S . E . , n = 26) and the mean d i f f e r e n c e between Vc and Vp was -0 .09 +_ 0.08 n l - m i n " * ( j ^ S . E . , n = 31 ) . In s topped- f low m ic rope r fus ion e x p e r i -ments the ra te of s a l t g land f l u i d s e c r e t i o n was only 0.3 0.07 n l - m i n " * (mean + S . E . , n = 3) demonstrat ing tha t net f l u i d movements were c l e a r l y below accura te d e t e c t i o n l i m i t s i n g lands per fused a t a ra te of 10 n l - m i n " * ( i . e . a t these f l u i d s e c r e t i o n and p e r f u s i o n ra tes a maximal volume change o f only 3 - 4 % would occur i n the c o l l e c t e d p e r f u s a t e s ) . T h e r e f o r e , f o r a l l c a l c u l a t i o n s Vc was assumed to be equal to Vp. For compar ison, the ac tua l changes i n t o t a l C 0 2 concen t ra t i ons of c o l l e c t e d pe r fusa tes are shown in Table 3 . 2 . S a l i n e s . The composi t ion of p h y s i o l o g i c a l s a l i n e s used throughout t h i s study are shown i n Table 3 . 1 . The con t ro l bath s a l i n e was based on measured hemolymph i o n i c and f r ee amino a c i d concen t ra t i ons and on measured pH, PC0 2 and H C 0 3 " va lues (Chapter I I ) . The con t ro l pe r fusa te was i sosmo t i c to the ba th ing s a l i n e and had high N a + , K + and C l " concen t ra t i ons Table 3.1. Composi t ion of p h y s i o l o g i c a l s a l i n e s . A l l s a l i n e s were gassed w i t h 98% Op - 2% COp and conta ined the f o l l o w i n g ( i n mM): p r o l i n e 20 , a l an ine 5 , g l y c i n e 3, glutamine 4 , succ ina te 7 . 4 , c i t r a t e 2 . 4 , g lucose 10. Component Bath S a l i n e s P e r f u s i o n S a l i n e s (mM) Cont ro l Na + -F ree K + - F r e e C l T r e e Cont ro l N a + - F r e e K + - F r e e C l " - F r e e N a + 189.5 - 198.5 189.5 148.5 - 198.5 148.5 K + 9 9 - 9 50 50 - 50 M g 2 + 4 4 4 4 4 4 4 4 C a 2 + 4 4 4 4 4 4 4 4 c r 39 39 39 - 164 164 164 -H C O 3 - 18.5 18.5 18.5 18.5 18.5 18.5 18.5 18.5 s o 4 2 _ 4.5 4.5 4.5 4.5 5 5 5 5 I se th i ona te " 125 - 125 148 - - - -Sucrose - 250 - - - - - -Choline" 1" - 64.5 - - - 148.5 - -M e t h y l s u i f a t e - - - - - - - 148 N O 3 - - - - 16 - - - 16 L issamine Green 0.05% - 64 -Tab le 3 . 2 . Changes i n t o t a l C O 2 and C l ~ concen t ra t i ons i n c o l l e c t e d p e r f u s a t e s . Values are means +_ S . E . (n = 4 - 1 2 ) . Experiment Tota l C 0 2 (mM) C l " (mM) Cont ro l 70.6 + 5.3 -81 .6 + B i l a t e r a l N a + - f r e e 68.3 + 4.4 -B i l a t e r a l K + - f r e e 68.3 + 5.1 -1.0 mM se rosa l ouabain 75.8 + 6.8 -2.0 mM se rosa l a m i l o r i d e 73.1 + 1.3 -Serosa l C l ~ - f r e e 75.6 + 3.4 -Luminal C l " - f r e e 17.0 + 2.4 -B i l a t e r a l C l " - f r e e 17.0 + 2.4 -0 .5 mM se rosa l SITS 69.7 + 4 .8 -0.5 mM serosa l DIDS 43.3 + 4.1 -0.1 mM serosa l acetazo lamide 14.5 + 1.3 -1.0 mM se rosa l acetazo lamide 14.7 + 0.9 _ - 65 -s i m i l a r to l a r v a l Aedes campest r is Ma lp igh ian tubu le f l u i d ( P h i l l i p s and M a d d r e l l , 1974). A l l pe r fusa tes were M i l l i p o r e f i l t e r e d (0.45 u) two t imes before use . Both p e r f u s i o n and bath ing s a l i n e s were gassed w i th 98% 0 2 -2% C 0 2 and had mean pH va lues of 7.64 and 7 .63 , r e s p e c t i v e l y . Note tha t the l a r g e i no rgan i c anion d e f i c i t observed i n l a r v a l hemolymph (Chapter I I ) was s imu la ted i n the ba th ing s a l i n e s us ing sodium i s e t h i o n a t e . I n h i b i t o r S t u d i e s . The mechanisms of C 0 2 t r a n s p o r t were examined by adding the f o l l o w i n g i n h i b i t o r s to the s e r o s a l medium (see D i s c u s s i o n ) : 2.0 mM a m i l o r i d e (Merck, Sharp and Dome, K i r k l a n d , Quebec), 0 .5 mM SITS ( 4 - a c e t a m i d o - 4 ' - i s o t h i o c y a n o s t i l b e n e - 2 , 2 ' - d i s u l f o n i c a c i d , BDH Chemicals L t d . , Vancouver, B r i t i s h Co lumb ia ) , 0 .5 mM DIDS (4 , 4 ' - d i i s o t h i o c y a n o - 2 , 2 ' - d i s u l f o n i c a c i d , Sigma Chemical C o . , S t . L o u i s , MO), 1.0 mM ouabain (Sigma) or 10"^ - 1 0 " ^ M acetazo lamide (Sigma). S a l t g lands were d i s s e c t e d and prepared f o r pe r f us i on i n con t ro l s a l i n e s as desc r i bed above. Immediately a f t e r the s t a r t of lumina l p e r f u s i o n , the bath was changed to c o n t r o l s a l i n e c o n t a i n i n g one of the i n h i b i t o r s . A f t e r a 20 - 25 minute p e r f u s i o n p e r i o d , pe r fusa tes were c o l l e c t e d and d isca rded and the s a l t g land per fused f o r an a d d i t i o n a l 25 minutes. To ta l exposure t ime to the i n h i b i t o r was 45 - 55 minutes . Both SITS and DIDS were d i s s o l v e d i n amino a c i d - f r e e s a l i n e ( rep laced w i th s u c r o s e ) . Weighing and p repa ra t i on o f SITS and DIDS and p e r f u s i o n exper iments w i th SITS and DIDS s a l i n e s were a l l c a r r i e d out i n a darkened room. In a d d i t i o n , the bath pe r fus ion r e s e r v o i r was wrapped i n f o i l and the bath chamber was covered w i th a f o i l - w r a p p e d p e t r i d i s h . A m i l o r i d e was d i s s o l v e d i n bath s a l i n e s i n which K 2 S 0 4 had been rep laced by KC1 and M g C l 2 rep laced by Mg(N03) 2 . A l l i n h i b i t o r s were made up f r esh before each exper iment . - 66 -C. Resu l t s Resu l t s shown i n F i g . 3.2 demonstrate the v i a b i l i t y o f the in v i t r o s a l t g land p r e p a r a t i o n . M ic roper fused whole s a l t glands ma in ta in a high and s t a b l 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 of -40 to -50 mV (lumen nega t i ve ) f o r a t l e a s t 7 - 8 hours and t h i s p o t e n t i a l i s r a p i d l y abo l i shed by cyanide ( F i g . 3.2A). In a d d i t i o n , mic roper fused s a l t g lands sec re te t o t a l C O 2 a t very CO high and s t a b l e ra tes ( F i g . 3.2B). Mean J 2 under con t ro l c o n d i t i o n s was net 799 + 57 p M o l e - m i n ' ^ m m " 1 (+ S.E., n = 9). The c o n t r i b u t i o n o f metabo l ic C O 2 to t r a n s e p i t h e l i a l C02 f l u x i n CO t h i s t i s s u e i s n e g l i g i b l e . Mean J \ i n r e c t a l s a l t g lands bathed b i l a t e r -3 3 net 3 a l l y w i th C02 and HC0 3-free s a l i n e s ( rep laced w i th HEPES bu f fe red s a l i n e ) was 23.2 _+ 0.7 pMo le -m in^ 'm iT f 1 (+_S.D., n = 2). The mean change i n c o l l e c -ted pe r fusa te t o t a l C O 2 concen t ra t i on was 1.9 +_ 0.1 mM which i s c l o s e to the l i m i t s of accura te d e t e c t i o n us ing m ic roca lo r ime t r y (Vurek e t _al_., 1975). Ion s u b s t i t u t i o n s t u d i e s were conducted to determine the i o n i c requi rements f o r C O 2 t r a n s p o r t . Rec ta l s a l t g lands were e q u i l i b r a t e d i n the va r i ous i o n - f r e e s a l i n e s f o r 2 - 2 . 5 hours p r i o r to pe r f us i on to minimize the problem of i on leakage and r e c y c l i n g through the e p i t h e l i a l c e l l s . F i g . 3.3 and Table 3.2 show t ha t b i l a t e r a l N a + or K + s u b s t i t u t i o n s COo COo + + had no s i g n i f i c a n t (P > 0.25) e f f e c t on J c . Mean J L i n Na and K - f r e e net net s a l i n e s were 771 _+ 54 pMole'im'n ^mm * (+_ S.E., n = 9) and 757 +_ 57 pMole*min ^-mm * (+_S.E., n = 10), r e s p e c t i v e l y . Q u a l i t a t i v e , m ic ro -scop i c obse rva t i ons showed tha t removal of se rosa l and mucosal K + caused both a n t e r i o r and p o s t e r i o r r e c t a l c e l l s to swel l s l i g h t l y , w h i l e b i l a t e r a l N a + s u b s t i t u t i o n caused only a smal l amount of s w e l l i n g i n a n t e r i o r r e c t a l c e l l s . Removal of se rosa l N a + or K + o n l y , caused excess i ve s w e l l i n g of - 67 -F i g u r e 3.2 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 recorded in m ic roper fused r e c t a l s a l t g l and . The s a l t g land was per fused by g r a v i t y w i th the p e r f u s i o n p i p e t f u n c t i o n i n g as the luminal e l e c t r o d e (Chapter V ) . B) Time course de te rmina t ions of con t ro l J 2 i n m ic ro -net per fused s a l t g lands . The open and c l o s e d squares and c i r c l e s rep resen t r e s u l t s ob ta ined from i n d i v i d u a l s a l t g land p r e p a r a t i o n s . - 68 --50 -40 _ -30 1 * -20 -10 0 + 10 L 0 10 3 M KCN 4 6 Time (hours) 8 1200 B E 800 \ -o E a 400 CM O O -3 O -O - o -o i. 20 40 Time (min) 60 - 69 -F i g u r e 3.3 E f f e c t s of b i l a t e r a l N a + o r K + s u b s t i t u t i o n s on J C ° 2 . Values net are means + S . E . (n = 9 -10 ) . Bath Lumen 1000 h 750 h 500 CM O 250 188.5 Na+ 9 K + 148.5 Na+ 50 K + 0 0-Na + 9 K + 0 N a + 50 K + 188.5 Na+ 0-K + 148.5 Na+ 0 K + - 71 -the a n t e r i o r r e c t a l c e l l s and u s u a l l y r e s u l t e d in luminal o c c l u s i o n . S u b s t i t u t i o n o f se rosa l C l " w i th i s e t h i o n a t e or m e t h y l s u l f a t e ro ro ( F i g . 3 . 4 , Table 3.2) had no s i g n i f i c a n t (P > 0.25) e f f e c t on J 2 (x J 2 = net net -1 -1 861 +_ 78 pMole*min -mm ; +_S.E. , n = 5 ) . Removal of lumina l C l " , however, decreased J ^ 2 by approx imate ly 80% (P < 0.0005) to 178 + 24 p M o l e - m i n ' ^ m m - 1 (mean +_ S . E . , n = 6 ) . S u b s t i t u t i o n of both se rosa l and mucosal C l " had no COo , COo -1 -1 f u r t h e r i n h i b i t o r y e f f e c t on J f (x J L = 175 + 25 pMole*min -mm ; net net -+_ S . E . , n = 6; see F i g . 3.4 and Table 3 . 2 ) . In two exper iments both C l " and S O ^ 2 " were removed from the se rosa l and mucosal s a l i n e s . The mean J ^ 2 o f 144 + 44 pMole*min *Tim * net — {+ S . D . , n = 2) was not s i g n i f i c a n t l y d i f f e r e n t (0.10 < P < 0 .25) from tha t CO observed w i th mucosal and se rosa l C l " s u b s t i t u t i o n s alone (x J 2 = 191 + net — 69 pMole-min *mm ; +_S.D., n = 4 ) . These r e s u l t s were t he re fo re pooled and are shown i n F i g . 3 . 4 . Removal of SO4 and/or C l " had no obvious e f f e c t on c e l l volume i n e i t h e r a n t e r i o r o r p o s t e r i o r s a l t g land segments. To examine the mechanisms of C 0 2 t r anspo r t f u r t h e r , we l l known i n h i b i t o r s of a c i d - b a s e t r anspo r t mechanisms were added to the se rosa l medium. Net t o t a l C 0 2 t r a n s p o r t r a tes were 849 _+ 80 pMole-min '^ -mm" 1 and 761 +_ 32 p M o l e - m i n ' ^ m m " 1 (_+ S . E . , n = 4-5) i n the presence of 1.0 mM se rosa l ouabain or 2.0 mM serosa l a m i l o r i d e , r e s p e c t i v e l y ( F i g . 3.5 and Table CO 3 . 2 ) , and were not s i g n i f i c a n t l y d i f f e r e n t (P > 0.25) from con t ro l J 2 . net S i m i l a r l y , a d d i t i o n o f 0 .5 mM SITS ( F i g . 3 .6 and Table 3 .2) to the bath ing CO CO -1 -1 s a l i n e had no e f f e c t (P > 0.25) on J 2 (x J 2 = 750 + 45 pMole-min -mm ; net net — +_S .E . , n = 4 ) . A d d i t i o n o f 0 .5 mM DIDS ( F i g . 3 . 6 ) , however, caused a s i g n i -C0 f i c a n t (0.0025 < P < 0.005) decrease of approx imate ly 40% i n J 2 to a va lue net - 72 -F igu re 3.4 E f f e c t s of lumina l and se rosa l C l " s u b s t i t u t i o n s on J Values are means + S . E . (n = 5-9). Bath : 39 Cl" 0 - C f 39 Cl" O-CI L u m e n : 164 Cl" 164 Cl" O-CI" O-CI 1000 h - 74 -F i gu re 3.5 E f f e c t s o f se rosa l a d d i t i o n of 1.0 mM ouabain or 2.0 mM a m i l o r i d e on J _ ? . Values are means + S . E . (n = 4 - 9 ) . 1000 — 750 E E E — 500 o E CM o o ~* 250 0 Control 1 mM Serosal Ouabain 2 mM Serosal Amiloride - 76 -F i g u r e 3.6 E f f e c t s of se rosa l a d d i t i o n of 0 .5 mM SITS, 0 .5 mM DIDS or 1.0 mM acetazo lamide on J „ „ ? . Va lues are means + S . E . (n = 4 - 9 ) . net — 1000 1 mM Serosal Acetazolamide - 78 -o f 500 + 42 pMole*min~l 'mnf* (_+ S . E . , n = 4 ) . The lack of any i n h i b i t o r y e f f e c t of SITS i n d i c a t e d t ha t DIDS i n h i b i t i o n was not due to a n o n s p e c i f i c e f f e c t o f the amino a c i d - f r e e s a l i n e (see M a t e r i a l s and Methods) . Net C 0 2 s e c r e t i o n was i n h i b i t e d by approx imate ly 80% (P < 0.0005) when the ca rbon ic anhydrase i n h i b i t o r , ace tazo lamide , was added to the bath a t a concen t ra t i on of 10" 4 M (Table 3.2) or 10" 3 M ( F i g . 3.6 and Tab le 3 . 2 ) . D. D i s c u s s i o n The mic roper fused r e c t a l s a l t g land of A. dorsa l i s p rov ides an e x c e l l e n t model system i n which to study the mechanisms of t r a n s e p i t h e l i a l H + and HC03~ t r a n s p o r t . M ic roper fused s a l t g lands are remarkably v i a b l e and main ta in a l a r g e and very s t a b l 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 f o r a t l e a s t 7 to 8 hours ( F i g . 3 . 2 A ) . T h i s p o t e n t i a l i s comple te ly poisoned by KCN demonstrat ing t ha t i t i s due to a c t i v e t r anspo r t p r o c e s s e s . S a l t g lands per fused i n v i t r o sec re te t o t a l C 0 2 a t very h igh and e a s i l y measurable r a t e s . P r e l i m i n a r y s t u d i e s (unpubl ished obse rva t i ons ) CO i n d i c a t e d tha t J 2 d e c l i n e d s t e a d i l y when s a l t g lands were per fused net immediately a f t e r d i s s e c t i o n . When s a l t g lands were e q u i l i b r a t e d f o r 2 to CO 2.5 hours i n ba th ing s a l i n e before p e r f u s i o n , however, J 2 remained s t a b l e net dur ing the 1 hour c o l l e c t i o n pe r i od ( F i g . 3 . 2 B ) . The r e s u l t s of the present study suggest t ha t N a + / H + and K + / H + exchange mechanisms p lay no r o l e i n t r a n s e p i t h e l i a l HC03~ movements. Complete b i l a t e r a l s u b s t i t u t i o n of N a + or K + ( F i g . 3 . 3 ) , or se rosa l a d d i t i o n CO o f 1.0 mM ouabain ( F i g . 3 .5) have no e f f e c t on J 2 . B ica rbona te t r a n s p o r t net i s a l s o una f fec ted by se rosa l a d d i t i o n of 2.0 mM a m i l o r i d e ( F i g . 3 . 5 ) , a d i u r e t i c which has been shown c l e a r l y to be a potent i n h i b i t o r of N a + / H + - 79 -exchange mechanisms i n a wide v a r i e t y of c e l l s and e p i t h e l i a (reviewed by Benos, 1982). CO Removal of se rosa l C l ~ had no e f f e c t on J 2 wh i l e lumina l C l " net removal i n h i b i t e d C 0 2 t r a n s p o r t by approx imate ly 80% ( F i g . 3 . 4 ) . In CO a d d i t i o n , the C T / H C O o " exchange i n h i b i t o r , DIDS, decreased J 2 by 40% J net when added to the se rosa l s i de of the e p i t h e l i u m ( F i g . 3 . 6 ) . These r e s u l t s suggested tha t H C O 3 " t r a n s p o r t i s mediated by a C1~/HC03~ exchange mechanism l o c a t e d a t the b a s o l a t e r a l c e l l membrane. To t e s t t h i s idea C l f u r t h e r , net C l " t r a n s p o r t ( J n e t ) was examined by e l e c t r o n microprobe a n a l y s i s as desc r i bed i n the f o l l o w i n g chap te r . A l a rge net C l " abso rp t i on was C l -1 -1 observed and t h i s J of -888 + 57 pMole^min -mm (+ S . E . , n = 12) was net — — not s i g n i f i c a n t l y d i f f e r e n t (P > 0.10) from the ra te of C 0 2 s e c r e t i o n (see R e s u l t s and Table 3 . 2 ) . These r e s u l t s t he re fo re p rov ide good q u a n t i t a t i v e ev idence f o r the presence of a 1:1 C1"/HC03~ exchange system i n the r e c t a l s a l t g l a n d . Carbon ic anhydrase f u n c t i o n has been shown to be of major importance in a lmost a l l ac id -base t r a n s p o r t i n g e p i t h e l i a s t u d i e d to date ( rev iewed by Maren, 1967; Pa rsons , 1982) . The pronounced i n h i b i t o r y e f f e c t of se rosa l acetazo lamide ( F i g . 3 .6) i n d i c a t e s tha t ca rbon ic anhydrase f u n c t i o n a l s o p lays a c r i t i c a l r o l e i n s a l t g land H C O 3 " s e c r e t i o n . Fu r the r s t u d i e s are needed to determine i f ca rbon i c anhydrase i s l o c a t e d i n the cy top lasm, and a p i c a l and b a s o l a t e r a l c e l l membranes (see Dobyan and B u l g e r , 1982) , and whether acetazo lamide d i r e c t l y i n h i b i t s c e l l u l a r HC03~ ent ry and e x i t s teps (see f o r example Fromter and S a t o , 1976; Boron and Fong , 1983). Exper iments designed to examine mechanisms of s a l t g land HC03~ t r a n s p o r t us ing lumina l ion t r anspo r t i n h i b i t o r s were comp l i ca ted by the - 80 -presence of the lumina l c u t i c l e (Meredi th and P h i l l i p s , 1973c) . S tud ies i n the l o c u s t rectum ( P h i l l i p s and D o c k r i l l , 1968; Lew is , 1971) have shown t ha t the i n s e c t h indgut c u t i c l e con ta ins pores w i th a rad ius o f 6.5 A and f u n c t i o n s as a mo lecu la r s i eve p reven t ing l a rge molecules (> 200 M.W.) i n the lumen from i n t e r a c t i n g d i r e c t l y w i th the a p i c a l c e l l membrane. The p e r m e a b i l i t y p r o p e r t i e s of the s a l t g land c u t i c l e were examined i n d i r e c t l y i n the present study by use of the v i t a l dye Janus Green. Th i s dye has a mo lecu la r weight o f 511 and s t a i n s c e l l s an in tense b l u e . S a l t g lands per fused w i th s a l i n e s c o n t a i n i n g > 1% Janus Green showed no s t a i n i n g , wh i l e a d d i t i o n of the dye to the se rosa l s i de caused c e l l s to immediately s t a i n a dark b l u e . S i nce Janus Green c l e a r l y cou ld not pass through the c u t i c l e i t was apparent tha t the t r a n s p o r t i n h i b i t o r s used i n the present study (M.W. o f 222 to 724) would not l i k e l y be ab le to i n t e r a c t w i th c a r r i e r molecules a t the a p i c a l membranes. Severa l attempts (summarized i n Tab le 3.3) were t he re fo re made to a l t e r c u t i c u l a r pe rmeab i l i t y us ing Janus Green as a pe rmeab i l i t y i n d i c a t o r . Sp ide r d i g e s t i v e j u i c e s were c o l l e c t e d as desc r i bed by C o l l a t z and Mommsen (1974) and Mommsen (1978a). These j u i c e s con ta in powerful c h i t i n a s e enzymes s p e c i f i c a l l y adapted to f u n c t i o n on ar thropod c u t i c l e (Mommsen, 1980) . In a d d i t i o n , the j u i c e s c o n t a i n powerful p r o t e a s e s , ca rbohydrases , e s t e r a s e s , phosphatases, nuc leases and e m u l s i f i e r s ( C o l l a t z and Mommsen, 1974; Mommsen, 1 9 7 8 a , b , c ) . U n f o r t u n a t e l y , none of the t reatments desc r i bed i n Table 3.3 had any e f f e c t on apparent c u t i c u l a r pe rmeab i l i t y and thus prevented lumina l i n h i b i t o r s t u d i e s . The r e s u l t s do suggest i n d i r e c t l y , however, tha t the s t r u c t u r e of the h indgut c u t i c l e may be cons ide rab l y d i f f e r e n t from tha t of the i n s e c t e x o s k e l e t o n . - 81 -Tab le 3.3 Summary of c h i t i n a s e exper iments . - P e r f u s i o n of the lumen w i th s a l i n e c o n t a i n i n g h igh concen-t r a t i o n s of commerc ia l ly a v a i l a b l e c h i t i n a s e enzymes (1-2 m g . / m l . ; S igma). - P e r f u s i o n of the lumen w i th s a l i n e c o n t a i n i n g smal l a l i q u o t s of d i g e s t i v e j u i c e s c o l l e c t e d from funnel -weaver sp i de rs (Tegenar ia a t r i c a ) . - P r e - p r e f u s i o n of the lumen w i th 1% T r i t o n X-100 fo l l owed by a 45-55 minute pe r f us i on w i th pure d i g e s t i v e j u i c e s c o l l e c t e d from South American t a r a n t u l a s ( D u g e s i e l l a s p . ) . - 82 -To summarize, the r e c t a l s a l t g land of A_. dorsa l i s l a r v a e can be s u c c e s s f u l l y mic roper fused i n v i t r o . S a l t g lands from l a r vae i n h a b i t i n g extremely a l k a l i n e environments sec re te t o t a l C 0 2 a t h igh and s t a b l e r a t e s , and the s e c r e t i o n appears to be mediated p r i m a r i l y by a 1:1 C T / H C G ^ " exchange mechanism. In the f o l l o w i n g chapter f u r t h e r ev idence i s p rov ided f o r a C1" /HC03 _ exchange system, the a n t e r i o r r e c t a l segment i s demonstrated to be the s i t e of t h i s exchange and the major t r a n s p o r t and exc re to r y f u n c t i o n s of the a n t e r i o r and p o s t e r i o r r e c t a l segments are examined. - 83 -CHAPTER IV. SITE OF C L " / H C 0 3 " EXCHANGE AND FUNCTION OF ANTERIOR AND POSTERIOR SALT GLAND SEGMENTS A . I n t roduc t i on The r e c t a l s a l t g land of s a l t w a t e r mosquito l a r vae sec re tes a s t r o n g l y hyperosmotic f l u i d (Bradley and P h i l l i p s , 1975) and i s a major s i t e o f N a + , K + , M g 2 + , C l " (Brad ley and P h i l l i p s , 1977a,b) and H C 0 3 " (Chapter I I ) e x c r e t i o n and r e g u l a t i o n . Rec ta l s a l t g lands are composed of two segments l i n k e d together by a s i n g l e row of s m a l l , i n d i s t i n c t j u n c t i o n a l c e l l s , and each segment i s composed of a s i n g l e c e l l t ype . Morpho log ica l s t u d i e s on the s a l t g land (Meredi th and P h i l l i p s , 1973c) showed tha t the u l t r a s t r u c t u r e of a n t e r i o r r e c t a l c e l l s was s i m i l a r to c e l l s found i n r e c t a of o b l i g a t o r i l y f reshwate r l a r v a e which produce a hyposmotic u r i ne by r e c t a l s o l u t e r e a b s o r p t i o n . The l a r g e r p o s t e r i o r r e c t a l c e l l s , found only i n s a l t w a t e r s p e c i e s , have an e l a b o r a t e l y f o l d e d a p i c a l membrane w i th i n f o l d i n g s ex tend ing ac ross 60% of the cy top lasm. The bulk of the p o s t e r i o r r e c t a l c e l l ' s m i tochondr ia are a s s o c i a t e d d i r e c t l y w i th these i n f o l d i n g s . Based on these obse rva t i ons Mered i th and P h i l l i p s (1973c) suggested t ha t the p o s t e r i o r segment was the ac tua l s i t e of hyperosmotic f l u i d s e c r e t i o n and t ha t the a n t e r i o r segment was i n v o l v e d in s e l e c t i v e so l u te r e a b s o r p t i o n . Exper imenta l ev idence f o r t h i s hypo thes is was p rov ided by Brad ley and P h i l l i p s (1977c) . These i n v e s t i g a t o r s t i e d r e l a t i v e l y l a rge l i g a t u r e s around i n t a c t l a r vae to ab la te e i t h e r the a n t e r i o r or p o s t e r i o r r e c t a l segments. The c u t i c l e over the segment of i n t e r e s t was d i s s e c t e d away and the segment incubated i n a r t i f i c i a l hemolymph. Photographic ev idence suggested tha t the p o s t e r i o r rectum had swe l l ed s l i g h t l y w i th f l u i d a l though the ra te o f s e c r e t i o n was seve re l y i n h i b i t e d compared to t ha t observed i n the whole s a l t g land ( c f . Brad ley and P h i l l i p s , 1975). No s w e l l i n g and f l u i d - 84 -s e c r e t i o n were apparent i n a n t e r i o r r e c t a l segments. F l u i d c o l l e c t e d by micropuncture from the p o s t e r i o r segment was hyperosmot ic , wh i l e the very smal l amounts of f l u i d c o l l e c t e d from a n t e r i o r segments was s l i g h t l y hyposmotic to the ba th ing s a l i n e . In the p rev ious chapter i t was shown tha t H C O 3 " s e c r e t i o n i n the r e c t a l s a l t g land of A . dorsa l i s i s mediated by a 1:1 exchange of luminal C l ~ f o r se rosa l H C O 3 " . The work i n the present chapter demonstrates the l o c a t i o n o f t h i s C T / H C G ^ - exchange us ing microper fused a n t e r i o r and p o s t e r i o r segment p repa ra t i ons and desc r i bes d i r e c t l y the major exc re to ry f u n c t i o n s of a n t e r i o r and p o s t e r i o r r e c t a l s a l t g land c e l l s . B. M a t e r i a l s and Methods An ima ls . Exper imenta l an ima l s , and r e a r i n g and a c c l i m a t i o n media were s i m i l a r to those desc r ibed in the p rev ious chap te r . M i c r o p e r f u s i o n and M ic rocannu la t i on S t u d i e s . P e r f u s i o n and c o l l e c t i o n p i p e t s were c o n s t r u c t e d , and s a l t glands d i s s e c t e d and e q u i l i b r a t e d as desc r i bed p r e v i o u s l y (Chapter I I I ) . I n d i v i d u a l s a l t g land segments were per fused by cannu la t i ng w i th pe r f us i on and c o l l e c t i o n p i p e t s l i g a t e d i n t o p lace us ing two to three 10 JJ l i g a t u r e s (see Chapter I I I ) t i e d around the i l eum, anal canal and d i r e c t l y between the two segments a t the j u n c t i o n a l reg ion ( F i g . 4 . 1 A ) . Segments were per fused a t 10 n l *min~* us ing a n a n o l i t e r p e r f u s i o n pump (see Chapter I I I ) . A p r e - c o l l e c t i o n pe r iod of 10 minutes fo l l owed by a per fusa te c o l l e c t i o n pe r iod of 10-20 minutes was used i n a l l s t u d i e s . M i c r o c a n n u l a t i o n s t u d i e s were performed by cannu la t i ng i n d i v i d u a l segments w i th microcannulae (same c o n s t r u c t i o n as pe r f us i on and c o l l e c t i o n p i p e t s ) f i l l e d w i th Sudan B l a c k - s t a i n e d p a r a f f i n o i l . L i g a t u r e s were t i e d - 85 -around the i l eum, anal canal and one c e l l row ( ca . 50 u) on e i t h e r s ide of the j u n c t i o n a l reg ion (see F i g . 4 . I B ) . Both o i l - f i l l e d microcannulae were mounted on N a r i s h i g e micromanipu la tors (Model MM-3), and the a n t e r i o r cannula (see F i g . 4 . I B ) was he ld i n a m i c rop ipe t t e ho lder ( L e i t z , W e t z l a r , West Germany) a t tached to a 50 ml s y r i n g e . F l u i d sec re ted by the i n d i v i d u a l segments was c o l l e c t e d con t inuous ly i n the a n t e r i o r p i p e t us ing the a t tached s y r i n g e . A f t e r 60-90 minutes the bath chamber was d r a i n e d , the rectum removed and the s e c r e t i o n sample prepared f o r a n a l y s i s (see be low) . The bath chamber design and super fus ion were the same as those desc r i bed i n the p rev ious chap te r . A l l exper iments were conducted a t room temperature (21 - 24°C) Sample A n a l y s i s . Volumes of c o l l e c t e d pe r fusa tes and r e c t a l s e c r e t i o n were determined by measuring drop diameters under minera l o i l w i th an eyep iece micrometer . To ta l C O 2 i n these f l u i d s was measured by m ic roca lo r ime t r y (P icapnotherm; M i c r o a n a l y t i c Ins t rumen ta t i on , Bethesda, Mary land) as desc r i bed by Vurek, Warnock and Corsey (1975). Concen t ra t i ons of N a + , K + , M g 2 + , C a 2 + , C l " , t o t a l phosphorus and t o t a l s u l f u r were measured i n pe r fusa te and s e c r e t i o n samples by e l e c t r o n microprobe a n a l y s i s us ing a Cameca model MBX microprobe and methods desc r i bed p r e v i o u s l y (Morel and R o i n e l , 1969; R o i n e l , 1975). Because of the h igh ion concen t ra t i ons i n these f l u i d s , samples were d i l u t e d two to th ree t imes w i th d i s t i l l e d water before p repa ra t i on f o r microprobe a n a l y s i s . S a l i n e s . The composi t ion o f c o n t r o l and HCO-j ' - f ree s a l i n e s used i n t h i s study are shown i n Tab le 4 . 1 . Sodium and C l " - f r e e s a l i n e s were the same as those desc r ibed i n the p rev ious chap te r . B i c a r b o n a t e - f r e e s a l i n e s were bu f fe red w i th HEPES (N-2-hydroxyethy l p i p e r a z i n e - N 1 - 2 -e t h a n e s u l f o n i c a c i d ) or phosphate and were gassed v i go rous l y w i th 100% 0 2 - 86 -F i g u r e 4.1 A) Arrangement of l i g a t u r e s and p ipe t s f o r m ic rope r fus ion of i n d i v i d u a l s a l t g land segments. B) Arrangement of o i l - f i l l e d cannulae and l i g a t u r e s f o r c o l l e c t i o n of s e c r e t i o n s from i n d i v i d u a l s a l t g land segments. - 8 7 -Posterior Segment 10 u Ligatures Collection Pipet Perfusion Pipet Anterior Segment Table 4 . 1 . Composi t ion of p h y s i o l o g i c a l s a l i n e s . A l l s a l i n e s a l s o con ta ined the f o l l o w i n g ( in mM): p r o l i n e 20, a l an ine 5 , g l y c i ne 3 , g lutamine 4 , succ ina te 7 . 4 , c i t r a t e 2 . 4 , g lucose 10. Bath ing S a l i n e s P e r f u s i o n S a l i n e s Component (mM) Cont ro l C 0 2 - F r e e (Phosphate) C 0 2 - F r e e (HEPES) Con t ro l C 0 2 - F r e e (Phosphate) C 0 2 - F r e e (HEPES) N a + 189.5 189.2 189.5 148.5 148.2 148.5 K + 9 9 9 50 50 50 M g 2 + 4 4 4 4 4 4 C a 2 + 4 4 4 4 4 4 C l " 39 39 39 164 180 171 HC0 3 - 18.5 0 0 18.5 0 0 s o 4 2 _ 4.5 4.5 4 .5 5 5 5 I s e t h i o n a t e " 125 141 140 - - -HEPES - - 10 - - 10 H 2 P 0 4 - - 0.2 - - 0.2 -H P 0 4 2 " - 1.0 - - 1.0 -pH 7.7 7.7 7.7 7.7 7.7 7.7 c o 2 [%) 2 0 0 2 0 0 o 2 (%) 98.0 100 100 98 100 100 - 89 -w h i l e a l l o ther s a l i n e s were gassed w i th 98% 0 2 - 2% C 0 2 . S a l i n e pH was ad jus ted to 7.7 us ing concent ra ted H N O 3 o r NaOH. C. R e s u l t s R e s u l t s i n F i g u r e 4.2 demonstrate c l e a r l y t ha t the a n t e r i o r segment i s the s i t e o f C 0 2 s e c r e t i o n i n the mic roper fused s a l t g l and . The inc rease i n pe r fusa te t o t a l C 0 2 concen t ra t i on dur ing pe r f us i on of the a n t e r i o r s e g -ment was 67.4 + 5 .9 mM (mean + S . E . , n = 7) and was not s i g n i f i c a n t l y d i f f e r -ent (P > 0.25) from the va lue of 70.6 + 5.3 mM (mean + S . E . , n = 9) observed dur ing p e r f u s i o n of the whole g land . F u r t h e r ev idence i n support of t h i s obse rva t i on i s shown i n F igu re 4 . 3 . Removal of luminal C T i n per fused whole g lands or per fused a n t e r i o r segments i n h i b i t e d t o t a l C 0 2 s e c r e t i o n by 80-85% (see a l s o the p rev ious c h a p t e r ) . The change i n pe r fusa te t o t a l C 0 2 concen t ra t i on when whole s a l t g lands were per fused w i th C l " - f r e e s a l i n e s (17.0 +_ 2.4 mM; mean + S . E . , n = 6) was not s i g n i f i c a n t l y d i f f e r e n t (0.1 < P < 0.25) from tha t of a n t e r i o r segments per fused w i th C l " - f r e e s a l i n e s (12.9 +_ 2 .6 mM; mean + S . E . , n = 4 ) . Note tha t there i s a l s o a very smal l and h igh l y v a r i a b l e component of C 0 2 s e c r e t i o n i n the per fused p o s t e r i o r segment ( F i g . 4 . 2 ) . The mean i n c r e a s e i n p o s t e r i o r pe r fusa te t o t a l C 0 2 concen t ra t i on was 4 .9 +_ 1.7 mM (+_ S . E . , n = 5 ) . F igu re 4.4 demonstrates t ha t C l " reabso rp t i on ( A [ C l " ] = -77.1 + 6 . 3 mM; mean +_ S . E . , n = 9) i n per fused a n t e r i o r segments 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 (0.1 < P < 0.25) from t o t a l C 0 2 s e c r e t i o n as p r e v i o u s l y shown f o r per fused whole s a l t glands (Chapter I I I ) . Fur thermore , e q u i l i b r a -t i o n of s a l t g lands f o r 2.5 hours i n C 0 2 and HC0 3 ~- f r ee ba th ing s a l i n e s p r i o r to p e r f u s i o n of a n t e r i o r segments w i th C 0 2 and H C 0 3 ~ - f r e e pe r fusa tes - 90 -F i g u r e 4.2 Changes i n pe r fusa te t o t a l C 0 2 concen t ra t i on f o r whole s a l t g lands and i n d i v i d u a l segments. Values are means +_ S . E . (n = 5 - 9 ) . Whole Salt Gland Posterior Segment - 92 -F i g u r e 4.3 E f f e c t s o f lumina l C l " s u b s t i t u t i o n on the change i n pe r fusa te t o t a l C 0 2 concen t ra t i on from whole s a l t g lands and a n t e r i o r segments. Va lues are means + S . E . (n = 4 - 9 ) . 2 E 75 c g ro hm c <D O J 50 CM O o CO 0) (0 (0 3 «*-mm 0) 0 . 25 Control Whole Salt Gland i CO I Cl-free Lumen I Cl-free Lumen l > Whole Salt Anterior Gland Segment - 94 -F i gu re 4.4 Changes i n a n t e r i o r segment pe r fusa te t o t a l COo and C l " concen t ra t i ons and e f f e c t s of b i l a t e r a l C 0 2 and HCOo" s u b s t i t u t i o n w i th HEPES or phosphate bu f fe red s a l i n e s (see Table 4.1) on C l " r e a b s o r p t i o n . Values are means + S.E. (n = 5 - 9 ) . 75 Control | | A Total C 0 2 A cr 50 25 Control P 0 4 = HEPES - 96 -(HEPES or phosphate s u b s t i t u t i o n ) complete ly i n h i b i t e d C l " r e a b s o r p t i o n . The change i n pe r fusa te C l " concen t ra t i ons w i th b i l a t e r a l HEPES or phosphate bu f fe red s a l i n e s were 4 .7 + 5.1 mM and - 1 . 7 + 2 .6 mM (mean +_ S . E . , n = 5 - 9 ) , r e s p e c t i v e l y , and these va lues were 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 (0.1 < P < 0 .25) In e a r l y s t u d i e s on s a l t g land f u n c t i o n i t was observed t ha t f l u i d s e c r e t i o n i n who le , cannu la ted s a l t g lands was dependent upon the presence of N a + and C l " i n the ba th ing s a l i n e . Rates of f l u i d s e c r e t i o n f o r s a l t g lands p r e - e q u i l i b r a t e d f o r 30 minutes i n N a + - f r e e ( rep laced by c h o l i n e ) or C l " - f r e e ( rep laced by i s e t h i o n a t e or N 0 3 " ) s a l i n e s and then cannu la ted w i th o i l f i l l e d p i p e t s are shown i n Table 4 . 2 . F l u i d s e c r e t i o n was i n h i b i t e d by 92% i n N a + - f r e e s a l i n e and by 57-68% i n C l ~ - f r e e s a l i n e . These va lues were s i g n i f i c a n t l y d i f f e r e n t from con t ro l r a t es of f l u i d s e c r e t i o n (P < 0 .001 ) . Data i n Tab le 4.2 are c o n s i s t e n t w i th coup led NaCl s e c r e t i o n s i n c e both ions are necessary f o r normal f l u i d s e c r e t i o n ra tes assuming h igh pe rmeab i l i t y of the s u b s t i t u t e d c o - i o n . I t should be noted tha t s e c r e t i o n s c o l l e c t e d from glands bathed i n C l " - f r e e s a l i n e s s t i l l con ta ined a s i g n i f i c a n t amount of C 1 " ( [ C 1 " ] = 28.9 +_ 2.1 mM) sugges t ing C l " leakage and r e c y c l i n g through the e p i t h e l i a l c e l l s and /o r a C l " t r a n s p o r t mechanism w i th a very h igh C l " a f f i n i t y . Cont ra ry to these r e s u l t s were the f i n d i n g s tha t f l u i d s e c r e t i o n was r e l a t i v e l y independent of bath C 0 2 and H C 0 3 " (Table 4 . 2 ) . Mean f l u i d s e c r e t i o n ra tes i n phosphate or HEPES bu f fe red ba th ing s a l i n e s were 61.0 + 1.8 n l - h " 1 and 26.8 + 3 . 4 n l - h " 1 , r e s p e c t i v e l y . In a d d i t i o n , under C 0 2 and H C 0 3 _ - f r e e c o n d i t i o n s r e c t a l s e c r e t i o n s are a lmost pure , concen t ra ted NaCl (see Tab le 4 . 3 ) . The h igher ra te of f l u i d s e c r e t i o n i n phosphate versus HEPES bu f fe red s a l i n e may be due to an i n d i r e c t e f f e c t of - 97 -p P O 4 on c e l l u l a r energy metabolism. Regardless, the rates and h y p e r t o n i c i t y of the NaCl s e c r e t i o n from glands bathed i n CC^-free s a l i n e are comparable to those of NaHC03~rich s e c r e t i o n s from glands bathed i n c o n t r o l s a l i n e (see Table 4.3). To determine where i n the s a l t gland t h i s hyperosmotic f l u i d s e c r e t i o n was o c c u r r i n g (see D i s c u s s i o n ) , and to examine the major excretory f u n c t i o n s of a n t e r i o r and p o s t e r i o r s a l t gland segments, the microcannulated preparations shown i n F i g u r e 4.IB were used. F l u i d s e c r e t i o n r a t e i n the cannulated p o s t e r i o r segment was 29.5 + 3.4 n l * h - 1 (mean + S.E., n = 8), which was s l i g h t l y but not s i g n i f i c a n t l y lower (0.1 < P < 0.25) than the value of 37.6 + 6.4 n l * h _ 1 (mean +_ S.E., n = 6; data from Chapter I I ) observed f o r whole s a l t glands ( F i g . 4.5). A n t e r i o r segments a l s o secrete f l u i d but at a much lower rate of 9.9 +_ 1.7 nl • h - 1 (mean +_ S.E., n = 5; F i g . 4.5). The i o n i c composition of s e c r e t i o n s c o l l e c t e d from whole s a l t glands (data from Chapter I I ) and the i n d i v i d u a l segments bathed i n co n t r o l s a l i n e are shown i n Table 4.3. A l l three s e c r e t i o n s are c l e a r l y hyperosmotic to the bathing s a l i n e (427 mOsm). The Na + concentrations f o r these three s e c r e t i o n s were not s i g n i f i a n t l y d i f f e r e n t (P > 0.25) and ranged between 384 and 413 mM. C h l o r i d e concentrations were about two to three times higher i n s e c r e t i o n s from p o s t e r i o r segments than e i t h e r s e c r e t i o n s from the whole s a l t gland (P < 0.0005) or the a n t e r i o r segment (P < 0.005). In a d d i t i o n , p o s t e r i o r segment s e c r e t i o n s had a l a r g e anion d e f i c i t i n d i c a t i n g a s i g n i f i c a n t component of C O 2 s e c r e t i o n by p o s t e r i o r r e c t a l c e l l s when C02/HC03~ was present i n the bathing s a l i n e (see D i s c u s s i o n ) . - 98 -Table 4 . 2 . Measured f l u i d s e c r e t i o n r a tes f o r whole, cannu la ted r e c t a l s a l t g lands bathed i n va r i ous s a l i n e s . Values are means +_ S . E . or S .D . Exper iment F l u i d s e c r e t i o n ra te ( n T h - 1 ) n Con t ro l 37.6 + 6.4 6 N a + - f r e e bath 3.0 + 0.6 4 ( cho l i ne ) C T - f r e e bath 16.2 + 4.1 3 ( N 0 3 _ ) C T - f r e e bath 12.2 + 2.4 5 ( i s e t h i o n a t e ) C 0 2 - f r e e bath 6 1 . 0 + 1 . 8 2 (pnosphate) C 0 2 - f r e e bath 26.8 + 3.4 4 (HEPES) Table 4 . 3 . I on i c composi t ion o f r e c t a l s e c r e t i o n s . Data f o r who le , cannu la ted s a l t g lands are from Chapter I I . Concent ra t ions of H C O 3 " and C O 3 2 - were c a l c u l a t e d from measurements o f t o t a l C O 2 and pH as desc r i bed p rev ious l y (Chapter I I ) . Calc ium and M g 2 + concen t ra t i ons were always < 1.0 mM. Values are means + S . E . , n = 5-9 (*mean _+S.D. , n = 2 ) . A l l p repa ra t i ons were bathed i n con t ro l s a l i n e (see Tab le 4.1) un less otherwise s ta ted in t h i s t a b l e . To ta l To ta l P r e p a r a t i o n N a + K + C l " S u l f u r Phosphorus H C O 3 " C 0 3 Whole, cannu la ted 412.7 + 31.5 s a l t g land Cannu la ted a n t e r i o r 384.4 + 17.0 segment Cannu la ted p o s t e r i o r 409.2 _+ 42.1 segment *Whole, cannu la ted 640.3 + 99.7 s a l t g land - C02- f ree bath (phosphate b u f f e r ) 31.4 + 3.4 20.9 + 1.8 5.9 + 3.5 6.0 + 0.9 44.9 + 4.7 86.0 + 4.0 141.8 + 13.8 632.6 + 79.8 13.4 + 2.8 14.7 + 1.7 27.3 + 4 .8 9.2 + 4 .8 2 . 4 0 + 0 . 8 402 41 0.64 + 0 . 4 0.30 + 0.07 0.50 + 0 . 4 Whole, cannu la ted 2 6 9 . 6 + 4 0 . 5 1 8 . 6 + 4 . 6 2 9 8 . 5 + 3 1 . 7 1 5 . 0 + 6 . 4 0 . 4 8 + 0 . 1 s a l t g land - C02~free bath (HEPES bu f fe r ) - 100 -Figure 4.5 Rates of f l u i d secretion in whole, microcannulated salt glands and anterior and posterior segments. Values are means +_ S.E. (n = 5-8). 45 £ 30 CC 15 i t—> o I—> I Whole Salt Gland Anterior Segment Posterior Segment - 102 -D. D i s c u s s i o n 1) S i t e of C T / H C O 3 " Exchange Dur ing i n i t i a l s t u d i e s when the mic roper fused p repa ra t i on desc r ibed i n the p rev ious chapter was being deve loped, attempts were made to per fuse the i n d i v i d u a l s a l t g land segments. 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 i n per fused a n t e r i o r and p o s t e r i o r segments were observed to d e c l i n e s low ly w i th t ime sugges t ing tha t the long- te rm v i a b i l i t y of these p repa ra t i ons was ra the r poor and t h e r e f o r e they were not i d e a l l y s u i t e d f o r s t u d i e s o f ion t r a n s p o r t mechanisms. Desp i te t h i s , the use of these p repara t ions f o r the shor t - te rm exper iments of the p resen t i n v e s t i g a t i o n has shown c l e a r l y t ha t the a n t e r i o r rectum i s the ac tua l s i t e o f C O 2 s e c r e t i o n i n the per fused s a l t g land ( F i g s . 4 .2 and 4 . 3 ) . Net C l " reabso rp t i on i s equ i va l en t to t o t a l C 0 2 s e c r e t i o n i n the a n t e r i o r segment ( F i g . 4 .4 ) as i t i s i n the whole s a l t g land (see Chapter I I I ) . Fur thermore, b i l a t e r a l C 0 2 s u b s t i t u t i o n w i th e i t h e r a phosphate or HEPES bu f fe red s a l i n e comple te ly i n h i b i t s a n t e r i o r segment C l " r eabso rp t i on ( F i g . 4 . 4 ) . These r e s u l t s toge ther w i th data presented in the p rev ious chap te r p rov ide good ev idence f o r the e x i s t e n c e o f a 1:1 C 1 ~ / H C 0 3 ~ exchange mechanism loca ted i n the a n t e r i o r s a l t g l and . I t should be noted a t t h i s t ime tha t the ac tua l i o n i c spec ies t r anspo r ted ( i . e . H C O 3 " a n Q , / ° r H + /0H~) dur ing C 0 2 s e c r e t i o n are not known. Indeed, the unequivocal demonstrat ion of H + / 0 H " versus H C O 3 " t r a n s p o r t i n i n t a c t e p i t h e l i a has not been p o s s i b l e (reviewed by A l - A w q a t i , 1978) . Experiments w i t h the l i p i d s o l u b l e b u f f e r g l y c o d i a z i n e , d i scussed i n Chapter V , however, suggest i n d i r e c t l y t ha t H + and/or OH" movements are i n v o l v e d i n C l " and HC03~ t r a n s p o r t . - 103 -2) Exc re to ry Func t i ons of A n t e r i o r and P o s t e r i o r Segments In e a r l i e r s t u d i e s on whole, cannu la ted s a l t g lands i t was observed t ha t f l u i d s e c r e t i o n was dependent c r i t i c a l l y on the presence of N a + and C l " i n the bath ing s a l i n e and r e l a t i v e l y independent of bath C 0 2 and HC03~ (Table 4 . 2 ) . Indeed, s a l t g lands bathed i n C 0 2 - f r e e s a l i n e s sec re ted s t rong l y hyperosmotic f l u i d s which were almost pure NaCl (Table 4 . 3 ) . Th i s suggested i n d i r e c t l y t ha t the s a l t g land might normal ly sec re te a N a C l - r i c h f l u i d and t ha t once t h i s f l u i d entered the s a l t g land lumen i t s compos i t ion was mod i f i ed by ion exchange and reabso rp t i ve processes dependent upon the a n i m a l ' s i o n i c regu la to ry needs. In l i g h t of these o b s e r v a t i o n s , i t was of i n t e r e s t to examine an hypothes is suggested r e c e n t l y by Brad ley and P h i l l i p s (1977c; see a l s o Mered i th and P h i l l i p s , 1973c) . S tud ies on the s a l t w a t e r mosquito l a r v a , A_. taen io rhynchus , conducted by these i n v e s t i g a t o r s suggested i n d i r e c t l y t ha t the p o s t e r i o r s a l t g land segment was the s i t e of hyperosmot ic f l u i d s e c r e t i o n and tha t the a n t e r i o r segment was i nvo l ved i n s e l e c t i v e so lu te and f l u i d r e a b s o r p t i o n . To study t h i s hypothes is d i r e c t l y , i n d i v i d u a l s a l t g land segments were cannu la ted w i th o i l - f i l l e d p i p e t s . In i n i t i a l s t u d i e s l i g a t u r e s were t i e d around the i leum or anal canal and d i r e c t l y between the two segments as shown f o r the per fused p repa ra t i on i n F i g . 4 .2A . The ra te of f l u i d s e c r e t i o n was found to be q u i t e low i n e i t h e r a n t e r i o r or p o s t e r i o r segments which aga in suggested poor long- te rm v i a b i l i t y o f t h i s p repa ra t i on as d i scussed above. To c i rcumvent t h i s problem, the l i g a t u r e technique was r e f i n e d and w i th g rea t care i t was p o s s i b l e to t i e l i g a t u r e s about one c e l l row or approx imate ly 50 u on e i t h e r s i de of the j u n c t i o n a l reg ion ( F i g . 4 . 2 B ) . Resu l t s ob ta ined us ing t h i s p repa ra t i on are shown i n F i g . 4 .5 and Tab les 4 .3 and 4.4 and demonstrate c l e a r l y t ha t the p o s t e r i o r segment i s the - 104 -major s i t e of hyperosmot ic f l u i d s e c r e t i o n as o r i g i n a l l y suggested by Brad ley and P h i l l i p s (1977c) . Cont ra ry to t h e i r o r i g i n a l h y p o t h e s i s , however, i t was found tha t the a n t e r i o r segment of A. do rsa l i s i s a l s o capable of s e c r e t i n g a hyperosmot ic f l u i d a t a ra te e q u i v a l e n t to approx imate ly 25% o f t ha t observed i n the whole gland ( F i g . 4 . 5 , Table 4 . 3 ) . Data i n Tab le 4.4 show tha t the c a l c u l a t e d ra tes of N a + and C l " e x c r e t i o n in the cannu la ted p o s t e r i o r segment are about 3 to 5 t imes h igher than those observed i n the cannula ted a n t e r i o r segment. These r e s u l t s p lus the obse rva t i on tha t the C l " concen t ra t i ons of p o s t e r i o r s e c r e t i o n s are 2 to 3 t imes h igher than those i n e i t h e r a n t e r i o r or whole g land s e c r e t i o n s (Table 4 . 3 ) , suggests t ha t one of the major f unc t i ons of the p o s t e r i o r segment i s e x c r e t i o n of a N a C l - r i c h f l u i d . S ince f l u i d f low through the i n v i v o s a l t g land i s not cont inuous and u n i d i r e c t i o n a l ( i . e . the i n v i v o r e c t a l s a l t g land empties on ly when i t becomes f i l l e d wi th s e c r e t i o n and feces) as i t i s i n the per fused or cannu la ted p r e p a r a t i o n , f l u i d sec re ted by the p o s t e r i o r segment would come i n t o con tac t w i th a n t e r i o r r e c t a l c e l l s . The c l e a r demonstrat ion of a C1"/HC03~ exchange mechanism i n the a n t e r i o r s a l t g land ( F i g . 4 . 4 ) i n d i c a t e s tha t the a n t e r i o r segment i s a t l e a s t one s i t e where m o d i f i c a t i o n of the compos i t ion of r e c t a l s e c r e t i o n s can occu r . F i g u r e 4.6 summarizes the major exc re to ry f unc t i ons of a n t e r i o r and p o s t e r i o r s a l t g land segments. F u r t h e r s tud ies are needed to determine the i o n i c requirements of f l u i d s e c r e t i o n i n the i n d i v i d u a l segments and to determine i f both segments sec re te a N a C l - r i c h f l u i d when bathed i n CO2 and H C 0 3 _ - f r e e s a l i n e . The r e l a t i v e r o l e s p layed by the i n d i v i d u a l segments i n HCO3" e x c r e t i o n and pH r e g u l a t i o n are u n c e r t a i n . Data i n Table 4 .3 show tha t s e c r e t i o n s from p o s t e r i o r segments bathed i n con t ro l s a l i n e s c o n t a i n i n g - 105 -Tab le 4 . 4 . C a l c u l a t e d ra tes o f N a + and C T e x c r e t i o n f o r cannu la ted a n t e r i o r and p o s t e r i o r segments. Segment J N a J c l P o s t e r i o r 12.1 n M o l e - h - 1 4 .2 n M o l e - h - 1 A n t e r i o r 3.8 n M o l e - h " 1 0.85 n M o l e - h - 1 - 106 -F i gu re 4 .6 Summary of major exc re to ry and t r anspo r t f u n c t i o n s of i n d i v i d u a l s a l t g land segments. - 108 -CO2/HCO3" have a l a r g e anion d e f i c i t . P a r t of t h i s d e f i c i t can be a t t r i b u t e d to s u l f u r - c o n t a i n i n g compounds such as i s e t h i o n a t e which may p a s s i v e l y en ter the lumen down a f avo rab le e lec t rochemica l g rad ien t (lumen TEP p o s i t i v e , unpub l ished obse rva t i ons ) and/or a smal l amount of SO4 s e c r e t i o n (see Brad ley and P h i l l i p s , 1977a). The remainder of the d e f i c i t p can most l i k e l y be a t t r i b u t e d to HC03~ and CO3 s i nce e a r l i e r unpub l ished obse rva t i ons i n d i c a t e d tha t the smal l volumes of s e c r e t i o n s c o l l e c t e d from p o s t e r i o r segments l i g a t e d a t the j u n c t i o n a l reg ion conta ined approx imate ly 150 mM t o t a l Thus, both segments are capable o f e x c r e t i n g CO2 a t h igh r a t e s . The i n a b i l i t y to measure s i g n i f i c a n t C 0 2 s e c r e t i o n i n the per fused p o s t e r i o r segment, however ( F i g . 4 . 2 ) , cou ld i n d i c a t e t ha t the ra te of p o s t e r i o r CO2 t r a n s p o r t i s f a r lower than t ha t i n the a n t e r i o r segment. Indeed, i n the i n t a c t animal one can e n v i s i o n the p o s t e r i o r segment s e c r e t i n g a N a C l - r i c h f l u i d . Given the nature o f f l u i d movements through the i n v i vo s a l t g l a n d , C l ~ cou ld be r a p i d l y removed from the s e c r e t i o n i n the a n t e r i o r segment i n exchange f o r se rosa l 1-03"*. The p genera t ion of l a rge t r a n s e p i t h e l i a l HCO3" and 003^" g rad ien ts by the a n t e r i o r segment would then d i r e c t l y a f f e c t the ra te and degree of p o s t e r i o r segment HC03~ t r a n s p o r t . A l t e r n a t i v e l y , i t i s e n t i r e l y p o s s i b l e tha t u n i d i r e c t i o n a l pe r fus ion of the s a l t g land w i th the i sosmot i c s a l i n e s used i n t h i s study s imply i n h i b i t s p o s t e r i o r segment 1-03" s e c r e t i o n . To conc lude , these s tud ies demonstrate c l e a r l y t ha t the a n t e r i o r segment i s the s i t e of C l ' / H C ^ " exchange i n the mic roper fused r e c t a l s a l t g l and . In a d d i t i o n , t h i s work has shown tha t both s a l t g land segments sec re te a hyperosmotic f l u i d c o n t a i n i n g N a + , C l ~ and HC03~. Thus i t can no longer be s a i d w i th c e r t a i n t y t ha t e i t h e r s a l t g land segment i s - 109 -per forming one s p e c i f i c f u n c t i o n in v i v o as o r i g i n a l l y suggested by Mered i th and P h i l l i p s (1973c) and Brad ley and P h i l l i p s (1977c) . I ns tead , i t i s t e n t a t i v e l y suggested t ha t both segments sec re te a N a C l - r i c h f l u i d i n v i vo and tha t the compos i t ion of t h i s f l u i d i s mod i f i ed once i t en te rs the s a l t g land lumen by ion exchange and reabso rp t i ve p rocesses . To t e s t t h i s hypothes is f u r t h e r i t would be va luab le to examine the mechanisms of p o s t e r i o r and a n t e r i o r segment f l u i d s e c r e t i o n i n animals i n h a b i t i n g a v a r i e t y of environments such as MgSO^, Nao^O^ and NaCl s a l t l a k e s . In Chapter V the a p i c a l and b a s o l a t e r a l membrane en t ry and e x i t s teps f o r C l " and H C O 3 " are examined us ing i n t r a c e l l u l a r ion and v o l t a g e - s e l e c t i v e m i c r o e l e c t r o d e s . - 1 1 0 -CHAPTER V - CELLULAR MECHANISM OF BICARBONATE AND CHLORIDE TRANSPORT A . I n t r oduc t i on C e l l u l a r mechanisms of H + and H C O 3 " t r anspo r t have not p r e v i o u s l y been s tud ied i n an i n v e r t e b r a t e e p i t h e l i u m . In chapters I I I and IV I have shown tha t a c t i v e H C O 3 " t r a n s p o r t i n the microper fused r e c t a l s a l t g land of Aedes dorsa l i s l a r v a e i s mediated by a 1:1 exchange of lumina l C l " f o r se rosa l H C O 3 " and tha t t h i s exchange i s l o c a t e d i n the a n t e r i o r s a l t g land segment. The work i n the present chapter examines the c e l l u l a r ent ry and e x i t s teps i nvo l ved i n H C O 3 " and C l " t r a n s p o r t us ing i n t r a c e l l u l a r ion and v o l t a g e - s e n s i t i v e m ic roe lec t rodes and a per fused a n t e r i o r segment p repara t i on which a l lowed both the bath and lumina l f l u i d compos i t ion to be changed r a p i d l y . B. M a t e r i a l s and Methods An ima ls . Larvae were reared i n a low a l k a l i n i t y r e a r i n g medium and the a d u l t co lony main ta ined as desc r ibed p r e v i o u s l y (Chapter I I ) . Three days a f t e r ha tch ing l a r v a e were t r a n s f e r r e d to 250 mM H C 0 3 " medium c o n t a i n i n g ( i n mM): 361.5 N a + , 2 .5 K + , 0.03 C a 2 + , 0 .50 M g 2 + , 39.6 C T , 1 0 . 0 S 0 4 2 " , 250 H C O 3 " , 29.0 C 0 3 2 ~ , pH 8 . 9 . A l l exper iments were conducted a t room temperature ( 2 1 - 24 °C) us ing fou r th i n s t a r l a r vae acc l ima ted to t h i s medium f o r th ree to s i x days . M i c r o p e r f u s i o n System. C o l l e c t i o n p i pe t s were cons t ruc ted as desc r i bed i n Chapter I I I . P e r f u s i o n p i p e t s were f a b r i c a t e d by s e a l i n g a smal l g l ass cannula (O.D. 50-60 u, I .D. 35-40 J J , t i p opening 2 0 - 2 5 ji) w i th a long column of epoxy i n t o a g lass p i p e t p u l l e d from f l i n t g l ass tub ing (O.D. 1.0 mm, I .D. 0 .8 mm; Drummond G lass C o . , B r o o m a l l , PA) . The epoxy seal was waterproofed and e l e c t r i c a l l y i n s u l a t e d us ing cured Sy lga rd 184 r e s i n (Dow - I l l -C o r n i n g ) . Bath chambers were cons t ruc ted from molded Sy lga rd 184 r e s i n and were s i m i l a r to those desc r ibed i n Chapter I I I except t ha t two super fus ion i n l e t s were p o s i t i o n e d on e i t h e r s ide of the s a l t g land p repa ra t i on and the t o t a l chamber volume was reduced to approx imate ly 0.6 m l . Recta l s a l t g lands were d i s s e c t e d as desc r ibed i n Chapter I I I . In these s t u d i e s the a n t e r i o r segment a lone was per fused by cannu la t i ng the midgut and i leum wi th a pe r fus ion p ipe t and the anal canal and p o s t e r i o r segment w i th a c o l l e c t i o n p i p e t (see F i g . 5 . 1 ) . Two or th ree 10 u l i g a t u r e s were t i e d around the i leum and anal canal to l i g a t e these p i p e t s i n t o p l a c e . An a d d i t i o n a l one or two 10 u l i g a t u r e s were t i e d one c e l l row or approx imate ly 50 u p o s t e r i o r to the j u n c t i o n a l reg ion to i s o l a t e the a n t e r i o r s a l t g l and . The 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 n per fused a n t e r i o r segments was a l lowed to s t a b i l i z e a f t e r se t -up f o r 30-40 minutes before exper iments were begun. Bath and Luminal S o l u t i o n Changes. The bath chamber was normal ly super fused by g r a v i t y a t a ra te of 3 to 5 m l • m i n " 1 . Rapid changes (< 5 sees) i n bath compos i t ion were made by us ing a 6-way ro ta ry va lve ( A l t e x , R a i n i n Instrument Co. I n c . , Woburn, MA) and by i n c r e a s i n g the ra te of supe r fus ion to 20-30 m l ' m i n - 1 . Whi le t h i s h igh super fus ion ra te tended to cause a smal l amount of mechanical d i s tu rbance to the p r e p a r a t i o n , i t was neve r the less p o s s i b l e to ma in ta in a h igh number of s t a b l e m ic roe lec t rode impalements dur ing these s o l u t i o n changes (see R e s u l t s ) . Ba th ing s a l i n e s were d e l i v e r e d to the ro ta ry va lve and bath chamber through po lye thy lene tub ing (PE 240) . Rapid changes i n lumina l s o l u t i o n compos i t ion were made us ing a double p i p e t arrangement shown s c h e m a t i c a l l y i n F i g . 5 . 1 . P e r f u s i o n p i p e t s were sea led by a gasket arrangement i n t o a s p e c i a l l y designed p l e x i g l a s s - 112 -F i gu re 5.1 Schematic diagram of m ic roper fused a n t e r i o r r e c t a l s a l t g land segment and double p e r f u s i o n p i p e t arrangement. co P E l O T u b i n g - 114 -p i p e t ho lder (shown s c h e m a t i c a l l y i n F i g . 5 . 1 ) . P e r f u s i o n s a l i n e s were d e l i v e r e d by g r a v i t y through ^ - i m p e r m e a b l e Saran tub ing (C la rkson Equipment and C o n t r o l s , D e t r o i t , MI) to a 6-way ro ta ry va l ve ( A l t e x , R a i n i n ) . A leng th of po l ye thy lene tub ing (PE 240) l e d from the ro ta r y va l ve to a p l a s t i c Y - j u n c t i o n . One branch of the Y - j u n c t i o n l ed to a waste con ta i ne r through po lye thy lene tub ing (PE 240) which was clamped w i th a smal l s e r r e f i n e c lamp. Th is branch i s r e f e r r e d to as a l i n e d ra i n (see F i g . 5 . 1 ) . Approx imate ly 1 cm of a 6 to 7 cm length of po lye thy lene tub ing (PE 10) was sea led i n t o the o ther branch of the Y - j u n c t i o n by a long column of epoxy. The PE 10 tub ing entered the rear of the p i p e t ho lder and was i n s e r t e d down the bore of the p e r f u s i o n p i p e t to a p o s i t i o n j u s t behind the p e r f u s i o n cannula (see F i g . 5 . 1 ) . A gasket arrangement was used to sea l the PE 10 tub ing i n t o the rea r of the p i p e t ho lder (see F i g . 5 . 1 ) . The p e r f u s i o n ra te through the PE 10 tub ing was approx imate ly 0.6 m l • m i n - 1 and the bulk of p e r f u s i o n s o l u t i o n f lowed up the p i p e t and out a d ra in on the p i p e t ho lder ( F i g . 5 .1) to a waste c o n t a i n e r . An a d j u s t a b l e p inch clamp on the l i n e d r a i n i n g the p i p e t ho lde r a l lowed adjustment of luminal p e r f u s i o n to ra tes o f 150-250 n l - m i n - 1 . Changes i n lumina l f l u i d compos i t ion were made by choos ing one of f ou r pe r fusa tes us ing the ro ta ry va lve and then opening the l i n e d ra in f o r 3 to 4 seconds to f l u s h the pe r f us i on l i n e . When the d ra i n was c l o s e d a new pe r fusa te almost i ns tan taneous l y began f l u s h i n g the p e r f u s i o n p i p e t and s a l t g land lumen. The t o t a l t ime f o r a complete luminal s o l u t i o n change was < 10 seconds. A l though t h i s procedure n e c e s s a r i l y caused very smal l luminal p ressure t r a n s i e n t s when the l i n e d ra in was opened and c l o s e d , i t was p o s s i b l e to ma in ta in an acceptab le number of m ic roe lec t rode impalements by impa l ing c e l l s c l o s e to the pe r f us i on and c o l l e c t i o n p i p e t s . - 115 -Both the p e r f u s i o n and c o l l e c t i o n p i p e t ho lders were mounted on N a r i s h i g e mic roman ipu la to rs (Model MM-3, Na r i sh i ge Instrument L a b o r a t o r i e s , Tokyo) . Pe r fusa tes c o l l e c t i n g in the c o l l e c t i o n p ipe t were con t i nuous l y drawn o f f to waste by vacuum suc t i on through a g l ass m ic rop ipe t a t tached to a l eng th o f po l ye thy lene tub ing (PE 100; see F i g . 5 . 1 ) . 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 t e ) was measured w i th re fe rence to the bath ing s a l i n e us ing calomel h a l f - c e l l s and a K i e t h l e y Model 616 d i g i t a l e lec t romete r ( K i e t h l e y Instruments I n c . , C l e v e l a n d , OH). The calomel h a l f - c e l l s made con tac t v i a 3 M KC1 - 4% Agar b r idges w i th the bath s a l i n e through a por t i n the bath chamber and w i th the p e r f u s i o n s a l i n e through a gaske t - sea led po r t on the p e r f u s i o n p i p e t ho lder ( F i g . 5 . 1 ) . Changes i n agar b r idge j u n c t i o n p o t e n t i a l s which occur red dur ing s o l u t i o n changes were measured sepa ra te l y aga ins t a grounded calomel h a l f - c e l l . Data were c o r r e c t e d f o r these changes i n p o t e n t i a l d i f f e r e n c e s on ly when they were ^ 1 . 0 mV. T r a n s e p i t h e l i a l p o t e n t i a l was recorded con t i nuous l y on 1 channel of a 2-channel c h a r t recorder (Model 7402A, Hew le t t -Packa rd , San D iego , CA) . V o l t a g e - s e n s i t i v e m ic roe lec t rodes were p u l l e d on a N a r i s h i g e v e r t i c a l p i p e t p u l l e r (Model PE 2 , N a r i s h i g e ) from c a p i l l a r y g l ass (1 .0 mm O . D . , F r e d e r i c k Haer and C o . , Brunswick , ME). These m ic roe lec t r odes had r e s i s t a n c e s of e i t h e r 10-12 Mn or 30-50 Mn. The lower r e s i s t a n c e m ic ro -e l e c t r o d e s were used i n l a t e r s t u d i e s s i nce they prov ided s t a b l e r impalements and v i r t u a l l y e l i m i n a t e d the problem of changing t i p p o t e n t i a l s which can occur dur ing and a f t e r c e l l puncture. Both h igh and low r e s i s t a n c e m ic ro -e l e c t r o d e s gave the same va lues f o r b a s o l a t e r a l membrane p o t e n t i a l (V b - | ) , were f i l l e d w i th 0.5 M or 3.0 M KC1 and had t i p p o t e n t i a l s < 5 .0 mV. Vo l tage output from these e l e c t r o d e s was measured us ing a Model M701 Microprobe - 116 -System (WP Ins t ruments , New Haven, CT) or a d i f f e r e n t i a l e l ec t rome te r (Model FD 223, WP Ins t ruments ) , monitored on a s torage o s c i l l o s c o p e (Model D15, T e k t r o n i x , Beaver ton , OR) and recorded con t inuous ly on 1 channel of a 2-channel cha r t recorder (Model 7402A, Hew le t t -Packa rd ) . The a p i c a l membrane p o t e n t i a l ( V a ) was determined s imply as the d i f f e r e n c e between V b l and v t e -In my hands, I cou ld not c o n s t r u c t r e l i a b l y d o u b l e - b a r r e l l e d l i q u i d ion-exchanger m ic roe lec t rodes (see Fu j imoto and Kubota , 1976) w i th t i p d iameters < 1.0 u. M i c r o e l e c t r o d e s w i th t i p d iameters t h i s l a rge were i n e f f e c t i v e in impa l ing a n t e r i o r r e c t a l c e l l s probably because of the presence of a r a the r t h i c k basement membrane i n t h i s t i s s u e (Meredi th and P h i l l i p s , 1973c) . T h e r e f o r e , i t was necessary to use s i n g l e - b a r r e l l e d i o n - s e l e c t i v e m i c roe lec t r odes which had t i p d iameters tha t c o n s i s t e n t l y measured _< 0.5 u . These e l e c t r o d e s were p u l l e d from ac id-washed c a p i l l a r y g l a s s on a N a r i s h i g e v e r t i c a l p i p e t p u l l e r , s i l a n i z e d by d ipp ing t h e i r t i p s f o r 10-12 seconds i n t o a 0.2% s o l u t i o n (v /v ) o f Dow Corn ing 1107 s i l i c o n e o i l i n acetone ( A r i s t a r , BDH Chemicals L t d . , P o o l e , England) and baked a t approx imate ly 300°C on a ho tp la te f o r 15-25 minutes. C h l o r i d e - s e n s i t i v e m ic roe lec t rodes were cons t ruc ted by i n j e c t i n g a smal l column of Cl~-exchange r e s i n (Or ion 92-17102, Or ion Research) i n t o the e l e c t r o d e shank w i t h a sy r i nge and drawn-out po l ye thy lene t u b i n g . The m ic roe lec t rodes were p laced t i p down i n t o 0.5 M KC1 overn igh t and the r e s i n a l lowed to f i l l the e l e c t r o d e t i p . The f o l l o w i n g day e l e c t r o d e s were b a c k - f i l l e d w i th 0 .5 M KC1 and b e v e l l e d by j e t stream abras ion (Ogden, C i t r o n and P i e r a n t o n i , 1978) a t a 45° angle f o r approx imate ly 30 seconds. C h l o r i d e -s e n s i t i v e e l e c t r o d e s were u s u a l l y f u n c t i o n a l f o r 3 to 4 days . - 117 -Hyd rogen -sens i t i ve m ic roe lec t rodes were cons t ruc ted i n a s i m i l a r manner us ing a H + -exchange r e s i n which was a generous g i f t from Dr . W. Simon (see Ammann, L a n t e r , S t e i n e r , S c h u l t h e s s , S h i j o and Simon, 1981). Th i s r e s i n was s to red con t i nuous l y under 100% CO2. A f t e r f i l l i n g the e l ec t r ode shank w i th a smal l column of the r e s i n , e l e c t r o d e s were p laced t i p down in a pH 7.0 b u f f e r and l e f t overn igh t i n a d e s s i c a t o r under 100% C 0 2 . The m ic roe lec t r odes were b a c k - f i l l e d the next day w i th pH 7.0 b u f f e r (see Ammann, e t a l _ . , 1981) and b e v e l l e d as desc r ibed above. The H + -exchange m ic roe lec t rodes were u s u a l l y only f u n c t i o n a l f o r 1 day. The vo l tage outputs of i o n - s e n s i t i v e m ic roe lec t rodes were measured us ing a d i f f e r e n t i a l e lec t romete r w i th 1 0 1 5 n i npu t r e s i s t a n c e (Model FD 223, WP Ins t ruments ) , and the s igna l was f i l t e r e d before r eco rd i ng us ing a low-pass f i l t e r . E l e c t r o d e s were c a l i b r a t e d i n smal l w e l l s c o n t a i n i n g t e s t s o l u t i o n s tha t made con tac t w i th the bath calomel h a l f - c e l l v i a a 3 M KC1 -4% Agar b r i d g e . The C l " - s e n s i t i v e m ic roe lec t rodes were c a l i b r a t e d i n 3 to 4 s o l u t i o n s c o n t a i n i n g 1, 5 , 50 or 150 mM K C 1 . Ion a c t i v i t i e s were c a l c u l a t e d us ing the Debye-HUckel equat ion (Robinson and S t o k e s , 1965) and ion s i z e parameters from K i e l l and (1937). These e l e c t r o d e s had s lopes o f 50-60 mV/decalog C l " a c t i v i t y and s e l e c t i v i t i e s f o r C l " over HCO3" rang ing between 3 and 5. Hyd rogen -sens i t i ve m ic roe lec t rodes were c a l i b r a t e d i n 3 b u f f e r s (pH 6.4 - 8 .3) of cons tan t i o n i c s t reng th s i m i l a r to the ba th ing s a l i n e and had s lopes of 55-60 mV/pH u n i t . The pH of c a l i b r a t i o n bu f f e r s was determined before each exper iment us ing a Radiometer Model 27 pH meter (Radiometer , Copenhagen) and a Radiometer pH e l e c t r o d e c a l i b r a t e d w i th Radiometer b u f f e r s . As desc r ibed p r e v i o u s l y (Ammann, e t a l . , 1981) , these H + -exchanger - 118 -m ic roe lec t rodes showed n e g l i g i b l e s e n s i t i v i t i e s to N a + or K + i n concen t ra t i ons up to 100 mM w i t h i n the pH range of 6.4 - 8 . 3 . Both H + and C l " - s e n s i t i v e m ic roe lec t rodes had r e s i s t a n c e s of l O 1 1 ^ and f u l l response t imes to step changes i n s o l u t i o n of < 5-8 seconds. M i c r o e l e c t r o d e Impalements. The per fused a n t e r i o r r e c t a l s a l t g land was viewed through a d i s s e c t i n g microscope ( Z e i s s , J e n a , Eas t Germany) and i l l u m i n a t e d from above by f i b e r o p t i c s ( I n t r a l u x Model 150 H, Vol p i AG, U r d o r f , S w i t z e r l a n d ) . E l e c t r o d e s were advanced manually a t an angle of 45°-60° to the c e l l su r face us ing a L e i t z micromanipu la tor (We tz la r , West Germany) u n t i l the m ic roe lec t rode t i p j u s t began to touch the c e l l membrane. The m ic roe lec t rode was a l lowed to ' r u b ' a g a i n s t the c e l l membrane f o r approx imate ly 20 seconds and the c e l l was then impaled by tapp ing the t a b l e g e n t l y . In e a r l y f ou r t h i n s t a r l a r vae i t was p o s s i b l e to remotely advance m ic roe lec t rodes d i r e c t l y i n t o c e l l s us ing a h y d r a u l i c m i c r o d r i v e (Model MO-8, N a r i s h i g e ) . Accep tab le c e l l u l a r impalements were c h a r a c t e r i z e d by an abrup t , monotonic change i n m ic roe lec t rode v o l t a g e , a p o t e n t i a l which remained s t a b l e f o r a t l e a s t 1 minute and a re tu rn of the m ic roe lec t rode vo l tage to w i t h i n +_ 2 .0 mV of b a s e l i n e a f t e r the e l e c t r o d e was withdrawn from the c e l l . S a l i n e s . The composi t ion of va r i ous bath ing and p e r f u s i o n s a l i n e s used throughout t h i s study are shown i n Table 5 . 1 . The c o n t r o l ba th ing s a l i n e was based on measured hemolymph i o n i c , osmotic and f r e e amino a c i d concen t ra t i ons (Chapter I I ) wh i le the c o n t r o l pe r fusa tes resembled l a r v a l Ma lp igh ian tubu le f l u i d ( P h i l l i p s and M a d d r e l l , 1974). Note tha t the l a rge anion d e f i c i t observed i n l a r v a l hemolymph (Chapter I I ) was s imu la ted i n the ba th ing s a l i n e w i th sodium i s e t h i o n a t e . Concen t ra t ions of N a + , K + and C l " were v a r i e d i n bath and pe r fus ion s a l i n e s by r e p l a c i n g these ions w i th Table 5 . 1 . Composi t ion of p h y s i o l o g i c a l s a l i n e s . A l l s a l i n e s a l s o con ta ined the f o l l o w i n g ( i n mM): p r o l i n e 20 , a lan ine 5, g l y c i n e 3 , glutamine 4 , succ ina te 2 . 4 , c i t r a t e 2 . 4 , g lucose 10. Bath ing S a l i n e s P e r f u s i o n S a l i n e s Component (mM) Cont ro l C 0 2 - f r e e (HEPES) C 0 2 - f r e e (G lycod iaz ine ) 65 mM H C O 3 " 113 mM H C O 3 " ' Cont ro l C 0 2 - f r e e (HEPES) A r t i f i c i a l S e c r e t i o n N a + 189.5 189.5 189.5 189.5 189.5 148.5 148.5 502 K + 9 9 9 9 9 50 50 35 M g 2 + 4 4 4 4 4 4 • 4 0.5 C a 2 + 4 4 4 4 4 4 4 0.5 C l " 39 39 39 39 39 164 164 37 H C 0 3 " 18.5 - - 65 113 18.5 - 400 C 0 3 2 - - - - - - - - 40 so 4 2 " 4.5 4 .5 4 .5 4.5 4 .5 5 5 -I s e t h i o n a t e " 125 141.5 125 78.5 30.5 - 16 -HEPES - 5 - - - - 5 -G l y c o d i a z i n e " - - 18.5 - - - - -pH 7.7 7.7 7.7 7.7 7.7 7.7 7.7 8.75 co2 (%) 2 0 0 6 11 2 0 2 0 2 (%) 98 100 100 94 89 98 100 98 L issamine Green (%) - - - - - 0.05 0.05 0.05 - 120 -c h o l i n e + , N a + or i s e t h i o n a t e " , r e s p e c t i v e l y (see R e s u l t s ) . G l y c o d i a z i n e (sodium g lymid ine) was a generous g i f t from Mr. H. Wehner (Pentagone Labs L t d . , V a u d r e u i l , Quebec). Acetazo lamide (Sigma Chemical C o . , S t . L o u i s , MO) and DIDS ( 4 - 4 ' - d i i s o t h i o c y a n o - 2 - 2 ' d i s u l f o n i c a c i d , Sigma) - c o n t a i n i n g bath s a l i n e s were made up f r esh before each exper iment . DIDS was d i s s o l v e d i n amino a c i d - f r e e s a l i n e ( rep laced w i th sucrose) and the p repa ra t i on o f DIDS s a l i n e was c a r r i e d out i n a darkened room. In a d d i t i o n , the bath super fus ion r e s e r v o i r and l i n e s were wrapped in f o i l and the t i s s u e was exposed to DIDS f o r 1 hour i n complete darkness . C. R e s u l t s 1) M i c r o e l e c t r o d e Impalements Examples of accep tab le vo l tage m ic roe lec t rode impalements are shown i n F i g . 5 . 2 . Mean c o n t r o l va lues f o r V t e (lumen n e g a t i v e ) , V b l and V g ( c e l l nega t i ve ) were -45 .5 + 0 . 5 , -75 .6 + 0.2 and -30.1 + 0.4 mV (+ S . E . , n = 150 impalements on 39 p r e p a r a t i o n s ) , r e s p e c t i v e l y . Measurements of Vb-j showed very l i t t l e v a r i a t i o n between i n d i v i d u a l c e l l s and i n d i v i d u a l a n t e r i o r segment p repa ra t i ons ( F i g . 5 . 4 ) . B a s o l a t e r a l membrane p o t e n t i a l measurements were remarkably s t a b l e and i t was o f ten p o s s i b l e to ma in ta in impalements wi th vo l t age e l e c t r o d e s f o r a t l e a s t 60-90 minu tes . In a d d i t i o n , any g iven p repa ra t i on cou ld be repeated ly impaled w i th vo l tage m ic roe lec t rodes wi th no apparent de t r imenta l e f f e c t s to the t i s s u e as evidenced by very s t a b l 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 s which changed by no more than _+ 3 .0 mV over the course of a normal 6-8 hour exper iment . S t a b l e impalements and t i s s u e v i a b i l i t y are most l i k e l y f a c i l i t a t e d by the l a rge s i z e of a n t e r i o r r e c t a l c e l l s (30-40 JJ i n d iameter , 30-40 JJ long) and the ex tens i ve i n f o l d i n g of the - 121 -F i g u r e 5.2 Examples of acceptab le c e l l u l a r impalements ob ta ined w i th v o l t a g e - s e l e c t i v e m i c r o e l e c t r o d e s . - 122 -V J j s o (A"J) , qA - 123 -basal membrane which g r e a t l y i nc reases membrane sur face area (see Mered i th and P h i l l i p s , 1973c) . Acceptab le impalements ob ta ined w i th H + and C l " - s e n s i t i v e e l e c t r o d e s are shown i n F i g . 5 . 3 . S ince s i n g l e - b a r r e l l e d i o n - s e l e c t i v e m ic roe lec t rodes were used i n these s t u d i e s , v b l i n any g iven p repa ra t i on was determined a t the beg inn ing or end of an experiment and t h i s va lue sub t rac ted from the observed vo l tage outputs from e i t h e r the H + (VH+) or C l " (VQ - j - ) - s e l e c t i v e m i c r o e l e c t r o d e s . Mean i n t r a c e l l u l a r pH (pH c ) and C l " a c t i v i t y U Q I - ) f o r p repara t ions bathed and per fused w i th con t ro l s a l i n e s were 7.67 + 0.03 ( + S . E . , n = 39 c e l l s , 4 p repa ra t i ons ) and 23.5 + 1.4 mM ( j ^ S . E . , n = 23 c e l l s , 7 p r e p a r a t i o n s ) , r e s p e c t i v e l y , and v a r i e d l i t t l e between i n d i v i d u a l c e l l s ( F i g . 5 . 4 ) . I n t r a c e l l u l a r pH was a l s o measured i n a n t e r i o r segments bathed on t h e i r lumina l su r faces by h igh HC0 3 ~ s o l u t i o n s (Table 5 . 4 ) . Mean pH c was 7.99 HH 0.02 (+_S.E . ; n = 8 c e l l s , 2 p r e p a r a t i o n s ) f o r a n t e r i o r segments per fused w i th an a r t i f i c i a l , hyperosmot ic s e c r e t i o n (see Tab le 5.1) and 7.84 + 0 .04 (+^S .E . ; n = 14 c e l l s , 3 p r e p a r a t i o n s ) f o r s a l t g lands bathed on t h e i r lumina l su r face w i th t h e i r own s e c r e t i o n s . Attempts to impale 2 c e l l s s imu l taneous ly w i th a vo l tage and i o n -s e l e c t i v e m ic roe lec t rode gene ra l l y f a i l e d and were t e c h n i c a l l y very d i f f i c u l t to c a r r y out . P r e l i m i n a r y exper iments us ing a ' bender - t ype ' p i e z o e l e c t r i c e l e c t r o d e advancing system (Boron and Bou lpaep, 1982) to impale c e l l s were u n s u c c e s s f u l . Th i s may have been due to the ra the r t h i c k basement membrane p resen t i n t h i s t i s s u e (Meredith and P h i l l i p s , 1973c) , and i t may the re fo re be use fu l i n f u tu re m i c roe lec t r ode s t u d i e s to attempt impalements us ing f a s t e r - 124 -F igu re 5 . 3 Examples of accep tab le impalements obta ined w i th H + and C l ~ -s e l e c t i v e m i c r o e l e c t r o d e s . The t r a c e marked w i th an a s t e r i s k shows the e f f e c t o f se rosa l CO? and H C 0 3 " removal ( rep laced w i th a HEPES bu f fe red s a l i n e ) on Vu+. I nc reas ing n e g a t i v i t y o f Vu+ i n d i c a t e s i n t r a c e l l u l a r a l k a l i n i z a t i o n . Note a l s o tha t v H+ becomes more p o s i t i v e ( i . e . dec reas ing pH) as the H - e l e c t r o d e approaches the c e l l membrane. Th is obse rva t i on i s c o n s i s t e n t w i th the presence of an a c i d i c u n s t i r r e d l a y e r produced by b a s o l a t e r a l H + e x t r u s i o n or H C O 3 " uptake (see Resu l t s and D i s c u s s i o n ) . The ac tua l b a s e l i n e i s t ha t seen upon wi thdrawal of the H + - m i c r o e l e c t r o d e from the ee l 1. out A - 126 -F i g u r e 5 . 4 D i s t r i b u t i o n o f V 5 1 and i n t r a c e l l u l a r pH and C l " a c t i v i t y i n a n t e r i o r r e c t a l s a l t g land c e l l s . Measurements o f V b ] , pH c and a^-j_ were ob ta ined from 3 9 , 4 and 7 separa te p r e p a r a t i o n s , r e s p e c t i v e l y . N u m b e r of C e l l s - 128 -and more powerful p i e z o e l e c t r i c d r i v e systems such as the ' s tack-a r rangement ' desc r ibed by Boron and Boulpaep (1982). As w i th the vo l tage m ic roe lec t rodes d i scussed above, c e l l s cou ld be repeated ly impaled w i th H + - s e n s i t i v e m ic roe lec t rodes w i th no apparent d e l e t e r i o u s e f f e c t s to the t i s s u e . The r e s u l t s obta ined w i th C T - s e n s i t i v e e l e c t r o d e s , however, were cons ide rab l y d i f f e r e n t . In g e n e r a l , i t was p o s s i b l e to make only 5-6 at tempts a t impalements, s u c c e s s f u l or u n s u c c e s s f u l , on any g iven p repa ra t i on before V t e would s t a r t to r a p i d l y d e p o l a r i z e . S ince the problem never occur red w i th vo l tage or H + - s e l e c t i v e m i c r o e l e c t r o d e s , which had the same t i p dimensions as C l " - e l e c t r o d e s , the only l i k e l y exp lana t i on i s a d i r e c t e f f e c t of the C l " -exchange r e s i n on the t i s s u e i t s e l f . Th i s e f f e c t cou ld be the r e s u l t of r e s i n t o x i c i t y to c e l l u l a r metabol ism or i n t r i g u i n g l y , to a d i r e c t e f f e c t of the r e s i n on e l e c t r o g e n i c H C 0 3 " t r anspo r t o r a p i c a l C l " conductance (see be low) . Any p repa ra t i on which d i s p l a y e d t h i s r a p i d l y d e p o l a r i z i n g V t e o r had a V t e which dropped below -40 mV was immediately d i s c a r d e d . 2) E l e c t r o g e n i c H C O 3 " T ranspor t Severa l obse rva t i ons suggested t ha t HC03~ t r a n s p o r t i n the a n t e r i o r r e c t a l s a l t g land was e l e c t r o g e n i c i n na tu re . Rap id a d d i t i o n of 1.0 mM acetazo lamide to the ba th , which i n h i b i t s J n e | i n t h i s t i s s u e (Chapter I I I ) , caused a l a rge d e p o l a r i z a t i o n of V t e ( F i g . 5 . 5 ) . A f t e r a 60 minute exposure to acetazo lamide V t e had s t a b i l i z e d a t a new va lue of - 7 . 3 _+ 3.9 mV (mean +_S.E. , n = 5 ) . Removal of acetazo lamide from the ba th ing s a l i n e caused V t e to re tu rn to con t ro l va lues w i t h i n 30 minutes ( F i g . 5 . 5 ) . The e f f e c t s o f 0 .5 mM se rosa l DIDS on V t e are shown i n F i g . 5 . 6 . In 3 o f the 4 p repa ra t i ons s t u d i e d , the t i s s u e was i n i t i a l l y exposed to amino - 129 -F i g u r e 5.5 E f f e c t s o f r a p i d a d d i t i o n o f 1.0 mM acetazo lamide to the se rosa l ba th ing s a l i n e on V t e . Values are means +_ S . E . (n = 5 p r e p a r a t i o n s ) . - 131 -F i gu re 5.6 E f f e c t s o f a d d i t i o n o f 0 .5 mM DIDS to the se rosa l ba th ing s a l i n e on V t e i n 4 separate a n t e r i o r r e c t a l s a l t g land p r e p a r a t i o n s . A n t e r i o r segments were exposed to DIDS s a l i n e f o r 60 minutes and then re tu rned to con t ro l s a l i n e f o r 30 minutes . The i n s e t shows the e f f e c t s of amino a c i d - f r e e s a l i n e (0-AA; see M a t e r i a l s and Methods) a lone on V t e i n 3 of the 4 p repa ra t i ons s t u d i e d . T i ssues were f i r s t exposed to amino a c i d - f r e e s a l i n e f o r 60 minutes and then re tu rned to con t ro l s a l i n e f o r 30 minutes before exposure to DIDS (see R e s u l t s ) . The open and c l o s e d squares and c i r c l e s represent r e s u l t s ob ta ined from a s i n g l e p r e p a r a t i o n . - 133 -a c i d - f r e e ba th ing s a l i n e (see M a t e r i a l s and Methods) fo r 60 minutes and then re tu rned to con t ro l s a l i n e f o r 30 minutes before a d d i t i o n o f 0 .5 mM DIDS to the ba th . The i n s e t i n F i g . 5.6 shows tha t V t e hype rpo la r i zed by approx imate ly 8-10 mV i n amino a c i d - f r e e ba th ing s a l i n e s and then re turned to c o n t r o l va lues i n c o n t r o l s a l i n e s . Exposure of the s a l t g land to se rosa l DIDS i n h i b i t e d ^ n e ^ (Chapter I I I ) and caused a d e p o l a r i z a t i o n o f ( F i g . 5 . 6 ) . The time course of t h i s d e p o l a r i z a t i o n v a r i e d cons i de rab l y between i n d i v i d u a l p repa ra t i ons and was comp l i ca ted by the h y p e r p o l a r i z i n g e f f e c t s of the amino a c i d - f r e e s a l i n e . In a d d i t i o n , V t e was not s t a b l e a f t e r the 60 minute DIDS exposure pe r i od and was i n a l l cases con t i nu i ng to d e c l i n e r a p i d l y . N e v e r t h e l e s s , the mean V t e of - 25 .8 + 3.5 mV (+ S . E . , n = 4) a t t h i s t ime was s i g n i f i c a n t l y d i f f e r e n t (0.005 < P < 0.01) from the con t ro l va lue of -44.1 +_ 0.8 mV (+_ S . E . , n = 4 ) . In 3 o f the p r e p a r a t i o n s , V t e re tu rned to va lues near con t ro l a f t e r DIDS was removed from the bath ( F i g . 5 . 6 ) . Rapid removal of C O 2 and H C O 3 " from the bath and replacement w i th a 5 .0 mM HEPES bu f fe red s a l i n e caused a r a p i d and l a rge 30-40 mV d e p o l a r i z a t i o n of V t e ( F i g . 5 . 7 ) . Th i s d e p o l a r i z a t i o n was complete w i t h i n 1 minute (V^ e = -12+_ 1 .7 , mean +_ S . E . , n = 6; c f . F i g . 5.11) and was then f o l l owed by a p a r t i a l r e p o l a r i z a t i o n of v a r i a b l e t ime course ( c f . F i g . 5 . 1 4 ) . A f t e r anywhere from 10-90 minutes V t e s t a b i l i z e d a t a new mean va lue of -31.0 +_ 1.1 mV ( j ^ S . E . , n = 6) which was s i g n i f i c a n t l y lower (P < 0.0005) than the c o n t r o l V t e of -44 .6 +_ 1.1 mV (mean _+ S . E . , n = 6 ) . Th i s r e p o l a r i z a t i o n phase cou ld have been due to cont inued e l e c t r o g e n i c HC03~ s e c r e t i o n s i nce C 0 2 and HC03~ were s t i l l p resent i n the mucosal s a l i n e i n t h i s exper iment . Removal of lumina l C 0 2 and HC03~ and replacement w i th HEPES s a l i n e , however, caused only a very smal l 1-3 mV h y p e r p o l a r i z a t i o n of V t e i r r e s p e c t i v e of the - 134 -F i g u r e 5.7 E f f e c t s o f se rosa l C 0 2 and HCO3" replacement w i th a 5.0 mM HEPES bu f fe red s a l i n e on V t e . Values are means +_ S . E . (n = 6 ) . Removal o f se rosa l C 0 2 and HC03~ caused a r a p i d d e p o l a r i z a t i o n of V t e which was complete w i t h i n 1 minute (T]_; c f . F i g . 5 . 1 1 ) . Th i s d e p o l a r i z a t i o n was f o l l owed by a p a r t i a l r e p o l a r i z a t i o n u n t i l a new s tab l e V t e was reached a f t e r anywhere from 10-90 minutes ( T ^ - 9 0 ' s e e R e s u l t s a n d D i s c u s s i o n ) . -50 - 136 -presence or absence of CO2 and HCG^ - i n the se rosa l medium (data not shown). These exper iments i n d i c a t e tha t the r e p o l a r i z a t i o n phase i s not due to a p i c a l CO2 r e c y c l i n g and con t inued e l e c t r o g e n i c HCC^" t r a n s p o r t . 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 hype rpo la r i zed t r a n s i e n t l y by approx imate ly 10 mV and then re tu rned to con t ro l va lues a f t e r C 0 2 and HCO3" were added back to the bath (data not shown). Replacement of C 0 2 and HCO3" w i th the l i p i d s o l u b l e b u f f e r , g l y c o d i a z i n e , caused V t e to r a p i d l y d e p o l a r i z e and to reverse ( i . e . lumen p o s i t i v e ; F i g . 5 . 8 ) . Th i s new p o s i t i v e V t e o f 2.2 + 2.6 mV (mean + S . E . , n = 3) remained s t a b l e f o r a t l e a s t 60 minutes ( V t e = 1.4 _+ 1.2 mV a f t e r 60 min; mean _+ S . E . , n = 3 ) . A d d i t i o n of C 0 2 and HC0 3 ~ back to the bath caused V t e to r e p o l a r i z e r a p i d l y and u s u a l l y h y p e r p o l a r i z e by 5-10 mV ( F i g . 5 . 8 ; c f . F i g . 5 . 1 0 ) . To determine a t which c e l l membrane the e l e c t r o g e n i c HC03~ t r a n s -po r t step was l o c a t e d , the exper iments w i th ace tazo lamide , HEPES and g l y c o -d i a z i n e were repeated wh i l e s imu l taneous ly measuring V^-j. A d d i t i o n of acetazo lamide to the bath or replacement of se rosa l CO2 and HC03~ w i th g l y c o d i a z i n e caused to hype rpo la r i ze s l i g h t l y by 1-3 mV ( F i g s . 5.9 and 5 . 1 0 ) . Replacement of CO2 and HC0 3~ w i th HEPES caused a s m a l l , t r a n s i e n t (2-4 mV) d e p o l a r i z a t i o n of ( F i g . 5 . 1 1 ) . Rapid i nc reases of se rosa l CO2 and HCO3" concen t ra t i ons a t cons tant pH caused V b i to d e p o l a r i z e s l i g h t l y by 2-4 mV and had a smal l h y p e r p o l a r i z i n g e f f e c t on V t e of 3-4 mV ( F i g . 5 . 1 2 ) . Taken together these r e s u l t s i n d i c a t e t ha t HCO3" ent ry i n t o the c e l l a t the b a s o l a t e r a l membrane i s l a r g e l y an e l e c t r i c a l l y s i l e n t p r o c e s s , and tha t HCO3" e x i t a t the a p i c a l membrane i s mediated by an e l e c t r o g e n i c mechanism. Data shown i n F igu re 5.13 demonstrate t ha t the observed e f f e c t s of these exper imenta l pe r t u rba t i ons on V a cannot be exp la ined by an i n d i r e c t - 137 -F i g u r e 5.8 E f f e c t s o f r e p l a c i n g se rosa l C 0 2 and H C 0 3 " w i th the l i p i d s o l u b l e b u f f e r , g l y c o d i a z i n e , on V t p . Values are means + S . E . (n = 3 ) . - 139 -F i gu re 5.9 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 dur ing and a f t e r a d d i t i o n o f 1.0 mM acetazo lamide to the serosa l ba th ing s a l i n e . A t o t a l number of 12 s i m i l a r exper iments were conducted on 4 separate a n t e r i o r segment p r e p a r a t i o n s . Bath: 1 mM I Control |Acetazolamide| 801 J _ - 4 0 H 0 « -Control o - 141 -F i g u r e 5.10 E f f e c t s o f se rosa l C O 2 and HC03~ replacement w i th the l i p i d s o l u b l e b u f f e r , g l y c o d i a z i n e , on and V t g . E i g h t s i m i l a r exper iments were conducted on 3 separa te a n t e r i o r segment p r e p a r a t i o n s . Bath: I C | Glycodiazine I Control I -80* -40 - 143 -F i gu re 5.11 E f f e c t s o f replacement of se rosa l C O 2 and HC03~ w i th a 5.0 mM HEPES bu f fe red s a l i n e on VD-| and V^g . Seventeen s i m i l a r exper iments were performed on 5 separate a n t e r i o r segment p r e p a r a t i o n s . Bath: - 145 -F i g u r e 5.12 E f f e c t s of h igh se rosa l HCC>3~ concen t ra t i on a t cons tan t pH on Vbi and V^e- F i v e s i m i l a r exper iments were conducted on 2 separate a n t e r i o r segment p r e p a r a t i o n s . Bath HC03": 18.5, 65 | 113 | 18.5 | 113 i 18.5 | -80 E — -40 _ -60 h > E ~ -30 CD ST 0 I 1 min | - 147 -F i g u r e 5.13 E f f e c t s of lumina l N a + or K + s u b s t i t u t i o n s on V ^ e and V51. A t o t a l o f 4-5 s i m i l a r exper iments were conducted on 2 separate a n t e r i o r segment p repa ra t i ons f o r e i t h e r the N a + o r K + s u b s t i t u t i o n s t u d i e s . 148 -Lumen Na +: -80 h > J _ - 4 0 H 1149, 95 41 i 149 Lumen K + = 50 | 0 | 25 | 100 | 50 | -80 h-> E ~_ -401 _ -601-> J -30 0 1 min - 149 -i n h i b i t o r y e f f e c t of changes i n pH c on an a p i c a l , e l e c t r o g e n i c c a t i o n reabso rp t i ve p r o c e s s . Rap id decreases i n lumina l N a + or K + c o n c e n t r a t i o n s , or complete removal o f lumina l N a + or K + have v i r t u a l l y no e f f e c t on V g or Vjji i n d i c a t i n g t ha t the a p i c a l membrane has a very low c a t i o n conduc-tance . S i m i l a r l y , the observed a l t e r a t i o n s i n V g cannot be due to an i nc reased a p i c a l C l " conductance (see below) a r i s i n g from changes i n p H c . P e r f u s i o n of the lumen w i th C l ~ - f r e e s a l i n e f o l l owed by removal of C O 2 and H C O 3 " from the se rosa l bath has the same h y p e r p o l a r i z i n g e f f e c t on V Q as observed i n glands per fused w i th con t ro l s a l i n e s ( F i g . 5 . 1 4 ; c f . F i g . 5 . 1 1 ) . 3 ) E l e c t r o g e n i c C l " Reabsorp t ion Data i n F i gu re 5.15 show tha t r a p i d decreases i n lumina l C l " con-c e n t r a t i o n cause a r a p i d , s tep -w ise h y p e r p o l a r i z a t i o n of v t e . Simultaneous measurements of i n d i c a t e d tha t these p o t e n t i a l changes were l o c a t e d a lmost e n t i r e l y a t the a p i c a l membrane ( F i g . 5 . 1 5 ) . The r e l a t i o n s h i p between the l oga r i t hm of the lumina l C l " a c t i v i t y a ^ i - and v a was found to be l i n e a r between C l " concen t ra t i ons of 16 to 164 mM ( F i g . 5 . 1 6 ) . The s lope of the l i n e f o r exper iments conducted on 11 d i f f e r e n t p repa ra t i ons was 42.2 mV/decalog a^-]- and the c o r r e l a t i o n c o e f f i c i e n t was 0 .992 . C h l o r i d e concen t ra t i ons below 16.0 mM caused a r e p o l a r i z a t i o n of V f l . A t a luminal C l " concen t ra t i on of 4 .0 mM, mean V g was 0.01 +_ 1.1 mV (+_ S . E . ; n = 17 impalements, 4 p r e p a r a t i o n s ; see F i g . 5 . 1 6 ) . Mean V f l i n the presence of C l " - f r e e mucosal s a l i n e was 0.2 + 1.1 mV (+_ S . E . ; n = 5 impalements, 1 p r e p a r a t i o n ) . These r e s u l t s suggested t ha t the a p i c a l membrane had a h igh C l " p e r m e a b i l i t y and t ha t C l " en te rs the c e l l , a t l e a s t i n p a r t , by a p a s s i v e , e l e c t r o d i f f u s i v e mechanism. To t e s t t h i s idea f u r t h e r , a^-j- was measured under con t ro l c o n d i t i o n s . C a l c u l a t e d C l " e l ec t rochemica l g rad ien ts (^y^-) - 150 -F i gu re 5.14 E f f e c t s o f se rosa l C O 2 and HC03~ removal ( rep laced w i th HEPES bu f fe red s a l i n e ) on V D i and Vf, e dur ing p e r f u s i o n of the a n t e r i o r segment w i th C l ~ - f r e e s a l i n e s . Seven s i m i l a r exper iments were conducted on 4 a n t e r i o r segment p r e p a r a t i o n s . Lumen: I -80 h 1 Control | o - c r 1 l Control I HEPES 1 Control | J -40 h > o«-I I—» c n t—* i - 152 -F i gu re 5.15 E f f e c t s o f r a p i d changes i n lumina l C l " concen t ra t i on on Vf.e a r , d Vbl • S i m i l a r exper iments were conducted on a t o t a l of 11 separate a n t e r i o r segment p r e p a r a t i o n s . - 1 5 4 -F igu re 5 . 1 6 R e l a t i o n s h i p between the l og o f the lumina l C l " a c t i v i t y U Q - | - ) and V A . Each po in t i s the mean _+ S . E . o f 9 - 1 7 impalements ob ta ined from a t l e a s t 2 separate a n t e r i o r segment p r e p a r a t i o n s . A t o t a l of 1 1 a n t e r i o r segment p repa ra t i ons were used i n these s t u d i e s . The s t r a i g h t l i n e drawn through the po in t s was determined us ing the l e a s t squares method of l i n e a r r e g r e s s i o n . The po in t a t the arrow i s the va lue of V A i n the presence of 4 mM luminal C l " (see R e s u l t s ) . +20 - 156 -Tab le 5.2 C a l c u l a t e d C l " e l ec t rochemica l g rad ien ts f o r a p i c a l and b a s o l a t e r a l c e l l membranes bathed by c o n t r o l se rosa l and mucosal s a l i n e s Net C l " f l u x i s from lumen to bath (Chapter I I I and I V ) . Negat ive s igns i n d i c a t e a f avo rab le A J J C - J - f o r pass ive C l " movement. The A X I Q ] - was c a l c u l a t e d us ing the mean V^ i of -75.6 mV (see R e s u l t s ) and a se rosa l C l " a c t i v i t y of 27.4 mM. Values are means + S . E . (n = 23 c e l l s , 7 p r e p a r a t i o n s ) . A ? C 1 " 23.5 + 1.4 mM -30 .8 + 1.1 mV -10 .7 + 2.2 mV -71 .7 mV - 157 -f o r a p i c a l and b a s o l a t e r a l c e l l membranes are shown i n Table 5 . 2 . C h l o r i d e a c t i v i t y i n the c e l l was 23.5 mM and the Apf c l- f o r the a p i c a l membrane was 10.7 +_ 2.2 mV f a v o r i n g pass i ve C l ~ e n t r y . I t should be noted a t t h i s t ime tha t i s e t h i o n a t e was used as an an ion s u b s t i t u t e f o r C l " and to s imu la te the anion d e f i c i t observed i n l a r v a l hemolymph (Chapter I I ) . G e n e r a l l y , g luconate, i s used as the anion s u b s t i t u t e of cho ice i n exper iments i n which a^-j- i s measured s i n c e C l " r e s i n s tend to be l e s s s e n s i t i v e to t h i s compound than most o ther anion s u b s t i t u t e s . In exper iments i n which g luconate rep laced i s e t h i o n a t e , however, V t e hyper-p o l a r i z e d by approx imate ly 10-15 mV and e x h i b i t e d a con t i nua l o s c i l l a t i o n o f approx imate ly +_ 5.0 mV (data not shown). T h i s obse rva t i on i s c o n s i s t e n t w i th the f a c t t h a t g luconate i s gene ra l l y more permeable than e i t h e r C l " or many o ther an ion s u b s t i t u t e s i n c e r t a i n c e l l s and t i s s u e s (see f o r example, Brown and Saunders , 1977). I n t r a c e l l u l a r C l " measurements i n t i s s u e s and c e l l s bathed f o r pro longed per iods i n C l " - f r e e s a l i n e s g ive ' apparen t ' a^-j- va lues of 4 to 6 mM ( e . g . Sp r i ng and K imura , 1978; Baumgarten and F o z z a r d , 1981; Hanrahan, 1982). Th i s r e s i d u a l C l " a c t i v i t y i s thought to be due to the presence o f i n t e r f e r i n g anions in the c e l l and most measurements o f a^-are assumed to be overes t imates of ac tua l v a l u e s . No attempt was made i n the present study to. measure t h i s r e s i d u a l anion a c t i v i t y p r i m a r i l y because i t was a n t i c i p a t e d t ha t the concen t ra t i on o f the major i n t r a c e l l u l a r i n t e r f e r i n g a n i o n , H C O 3 " , would be a l t e r e d by removing lumina l and bath C l " . I t must be s t r e s s e d , however, t ha t any overes t imate o f a ^ - would only s t rengthen the major conc lus ions of t h i s study (see D i s c u s s i o n ) . Table 5.2 demonstrates tha t there i s a l a rge A J T Q I - a t the baso-l a t e r a l membrane of 71.7 mV f a v o r i n g pass i ve C l " e x i t from the c e l l . Un l i ke - 158 -the a p i c a l C l " en t ry s t e p , however, C l " e x i t a t the b a s o l a t e r a l c e l l membrane i s e l e c t r i c a l l y s i l e n t . F igu re 5.17 shows the r e s u l t s o f 3 separa te exper iments i n which se rosa l C l " concen t ra t i on was v a r i e d r a p i d l y from a c o n t r o l va lue o f 39 mM to va lues o f 5 , 100 or 164 mM. These s o l u t i o n changes had v i r t u a l l y no e f f e c t on V ^ . The smal l p o t e n t i a l changes which d i d occu r , and which are seen most e a s i l y i n the lower t r a c e of F igu re 5 .17 , are c l e a r l y i n the wrong d i r e c t i o n to be exp la ined by an e l e c t r o g e n i c , b a s o l a t e r a l C l " e x i t s t e p . Data i n F i gu re 5.18 demonstrate t ha t the V b-| i n a n t e r i o r r e c t a l c e l l s i s p r i m a r i l y a K + d i f f u s i o n p o t e n t i a l as i s observed i n many e p i t h e l i a . Changes i n se rosa l K + concen t ra t i on a l t e r V b i i n a r a p i d , l i n e a r f a s h i o n . The s lope of the r e l a t i o n s h i p between V b i and se rosa l K + a c t i v i t y (a|+) i s 54.2 mV/decalog aj<+ and the c o r r e l a t i o n c o e f f i c i e n t i s 0.9999 ( F i g . 5 . 1 8 ) . Decreases i n se rosa l N a + concen t ra t i on from 189.5 to 63 mM had a s m a l l , 1-3 mV d e p o l a r i z i n g e f f e c t on V ^ (data not shown). D. D i s c u s s i o n 1) C e l l u l a r Ent ry and E x i t Steps The r e s u l t s of the present study are summarized i n the c e l l u l a r model shown i n F i gu re 5 .19 . As demonstrated i n Chapters I I I and IV , HCO3" s e c r e t i o n i n the a n t e r i o r r e c t a l s a l t g land i s mediated by a 1:1 exchange of lumina l C l " f o r se rosa l HC03~ i o n s . Data i n F igu res 5.5 to 5.11 show ro c l e a r l y tha t exper imenta l c o n d i t i o n s which i n h i b i t J n g | i n t h i s t i s s u e (see Chapter I I I ) cause l a r g e h y p e r p o l a r i z a t i o n s of V f l and have l i t t l e e f f e c t on V b - | . These r e s u l t s are c o n s i s t e n t w i th the hypothes is t ha t H C 0 3 " e x i t a t the a p i c a l c e l l membrane i s mediated by an e l e c t r o g e n i c HCO3" o r H + /0H~ t r a n s p o r t mechanism. - 159 -F i gu re 5.17 E f f e c t s o f r a p i d changes i n se rosa l C l ~ concen t ra t i on on V b - | . Each t r a c e i s the r e s u l t from an experiment conducted on a separa te a n t e r i o r segment p r e p a r a t i o n . A t o t a l number of 8-15 s i m i l a r exper iments were conducted on 2-3 a n t e r i o r segments. - 160 -Bath Cl -80 > J -40 39 0 L -39 1 3 9 I -80 h > E -40 h-39 100 39 80 1 -40 o1-39 1641 39 1 min 164 | 39 | - 161 -F i g u r e 5.18 The upper panel shows the e f f e c t s of r a p i d changes i n se rosa l K concen t ra t i on on V ^ . The r e l a t i o n s h i p between the l o g of the se rosa l K a c t i v i t y and V^- i is shown i n the lower p a n e l . Each p o i n t i s the mean +_ S . E . o f 10-13 impalements ob ta ined from 4 separa te a n t e r i o r segment p r e p a r a t i o n s . The s t r a i g h t l i n e drawn through the po in t s was determined us i ng the l e a s t squares method of l i n e a r r e g r e s s i o n . In a l l cases the S . E . bar was s m a l l e r than the s i z e of the ac tua l p o i n t . - 162 -Bath K+= 19! 4.5 | 32 1 58 l32i 4.5 | 9 t -90 h £ -60 :>? -30 0 « -1 min -log a* + - 163 -c F igu re 5.19 Ten ta t i ve c e l l u l a r model o f H C O 3 " and C l " en t ry and e x i t s teps i n a n t e r i o r r e c t a l s a l t g land c e l l s ( c a . = ca rbon ic anhydrase) . - 164 -Apical Membrane Lumen 31 mV + HC0 3 "^ 23.5 mM Clj pHj 7.67 14.4 mM HC03". - • Cl" H HCO3 < H 20 c o 2 Basolateral Membrane Hemolymph 76 mV - 165 -R e c e n t l y , B i a g i , Kubota, S o h t e l l and G i e b i s c h (1981) have shown t ha t decreases i n s e r o s a l H C O 3 " concen t ra t i on and pH d e p o l a r i z e V b i and i n h i b i t b a s o l a t e r a l K + conductance i n per fused r a b b i t prox imal tubu les (see a l s o S t e e l s and Bou lpaep, 1976). These changes i n K + conductance are presumably due to e f f e c t s o f changes i n pH c on b a s o l a t e r a l K + channels and cou ld suggest f a l s e l y a h igh H C O 3 " p e r m e a b i l i t y of the b a s o l a t e r a l c e l l membrane. S i m i l a r e f f e c t s cannot e x p l a i n the r e s u l t s i n F i g u r e s 5 .5 to 5 . 1 1 . Rapid changes i n or complete removal of luminal N a + o r K + have l i t t l e e f f e c t on V a demonstrat ing tha t the a p i c a l membrane has a very low c a t i o n p e r m e a b i l i t y ( F i g . 5 . 1 3 ) . In a d d i t i o n , the a l t e r a t i o n s of V a seen i n F igu res 5.5 to 5.11 are not due to i n c r e a s i n g a p i c a l C l " conductance. Removal of C 0 2 and H C 0 3 ~ from the bath s t i l l causes a l a rge d e p o l a r i z a t i o n o f V t e when s a l t g lands are per fused w i th C l " - f r e e s a l i n e ( F i g . 5 . 1 4 ) . Decreases i n lumina l C l " concen t ra t i on from 164 to 16 mM d e p o l a r i z e s V a i n a r a p i d , s tep -w ise manner ( F i g . 5 . 15 ) . The r e l a t i o n s h i p between V g and lumina l C l " a c t i v i t y i s l i n e a r w i th a s lope of 42.2 mV/decalog a C l " ( F l 9 * 5 .16 ) . These r e s u l t s i n d i c a t e t ha t the a p i c a l membrane has a very h i g h , pass i ve C l " conductance. Fur thermore, measurement of con t ro l a£-|- demonstrated t ha t the e lec t rochemica l g rad ien t f o r C l " a t the a p i c a l membrane favo rs pass i ve en t ry of C l " i n t o the a n t e r i o r r e c t a l c e l l s (Table 5 . 2 ) . Taken t oge the r , these data suggest s t r ong l y tha t a p i c a l C l " ent ry i s media ted, a t l ease i n p a r t , by p a s s i v e , e l e c t r o d i f f u s i v e movement of C l " through a C l " - s e l e c t i v e channe l . A t lumina l C l " concen t ra t i ons below 16 mM, V Q begins to r e p o l a r i z e . Mean V a i n the presence of 4 mM luminal C l " was c l o s e to 0 mV (see F i g . 5 . 1 6 ) . The nature of t h i s r e p o l a r i z a t i o n phase i s u n c e r t a i n , but i t may be due to an i n h i b i t o r y e f f e c t of low lumina l C l " concen t ra t i on on e l e c t r o g e n i c - 166 -HC03~ s e c r e t i o n . The b a s o l a t e r a l c e l l membrane i s the most l i k e l y s i t e f o r a d i r e c t 1:1 c o u p l i n g between C l " and HCC^" movements. B a s o l a t e r a l C l " e x i t and HC03~ en t ry are both l a r g e l y e l e c t r i c a l l y s i l e n t processes (see F i g s . 5.9 to 5.12 and F i g . 5.17 ). In a d d i t i o n , the C T / H C O 3 " exchange i n h i b i t o r ro DIDS, which i n h i b i t s J n e | i n t h i s t i s s u e (Chapter I I I ) , d e p o l a r i z e s when when added to the se rosa l ba th ing s a l i n e ( F i g . 5 . 6 ) . The a n t e r i o r r e c t a l c e l l V b l appears to be p r i m a r i l y a K + d i f f u s i o n p o t e n t i a l ( F i g . 5 . 1 7 ) . The mechanism of c e l l u l a r K + up take, however, i s u n c e r t a i n a t p resen t . Super fus ion of the t i s s u e w i t h 1.0 mM se rosa l ouabain f o r up to 1 hour had no e f f e c t on V b-| or V t e (data not shown). Th i s may be due to an i n s e n s i t i v i t y o f a pu ta t i ve N a + / K + ATPase to ouabain a t room temperature as observed f o r o ther i n s e c t N a + / K + pumps (reviewed by Anstee and Bowle r , 1979). A l t e r n a t i v e l y , i t cou ld s imply represent f a i l u r e of the ouabain to permeate the basement membrane and i n t e r a c t d i r e c t l y w i th pump s i t e s . S ince N a + / K + pumps have been found i n a number of i n s e c t t i s s u e s (see Anstee and Bow le r , 1979; P h i l l i p s , 1981) and are p resent i n a lmost a l l ve r teb ra te e p i t h e l i a s tud ied in d e t a i l , i t i s t e n t a t i v e l y suggested t ha t a n t e r i o r r e c t a l c e l l K + accumulat ion i s mediated by a b a s o l a t e r a l N a + / K + ATPase (see F i g . 5 . 1 9 ) . Exper iments us ing g l y c o d i a z i n e suggest , a l b e i t very i n d i r e c t l y , t ha t b a s o l a t e r a l C l " e x i t and H C O 3 " en t ry may a t l e a s t p a r t i a l l y be mediated by an HCI co t ranspo r t or C1~/0H" exchange mechanism. G l y c o d i a z i n e (sodium g lymid ine) i s a bu f f e r which has a high l i p i d s o l u b i l i t y . The pK a of g l y c o d i a z i n e ( 5 . 7 ; U l l r i c h , Radtke and Rumr ich, 1971) i s s i m i l a r to tha t f o r the C02/HC03~ e q u i l i b r i u m (6.1) and hence t h i s compound has been used - 167 -as a C O 2 and HCC^" replacement . In the proximal t u b u l e , i t has been shown t ha t g l y c o d i a z i n e i s reabsorbed from the tubu le lumen a t a r a te equal to the ra te o f H + s e c r e t i o n ( M a l n i c , Costa S i l v a , C a m p i g l i a , de M e l l o A i r e s and G i e b i s c h , 1980). U l l r i c h e t j i T (1971) have shown tha t g l y c o d i a z i n e supports normal i s o t o n i c f l u i d reabso rp t i on i n the proximal tubu le i n the absence of ambient C O 2 and HC03~. Fur thermore, rena l g l y c o d i a z i n e t r a n s p o r t d i s p l a y s many c h a r a c t e r i s t i c s s i m i l a r to those of H + t r anspo r t such as Na + -dependence, i n h i b i t o r s e n s i t i v i t y and adapta t ion to ch ron i c a l k a l o s i s ( U l l r i c h , Rumrich and Baumann, 1975). In the pancreas , g l y c o d i a z i n e p a r t i a l l y s t imu la tes f l u i d and s o l u t e s e c r e t i o n i n the absence of exogenous C O 2 and H C O 3 " ( S c h u l z , 1976, 1981; Schu lz and U l l r i c h , 1979). S ince the l a rge g l y c o d i a z i n e molecule (M.W. 308) per se i s u n l i k e l y to be t r anspo r ted on a membrane c a r r i e r , the exper iments in the proximal tubu le and pancreas have been taken as evidence f o r the e x i s t e n c e of a H + /0H~ t r a n s p o r t e r r a t h e r than a HC03~ c a r r i e r i n v o l v e d i n t r a n s e p i t h e l i a l ac id -base movements. For example, i n the proximal t u b u l e , r eabso rp t i on o f H C O 3 " o r the g l y c o d i a z i n e an ion i s dependent upon t u b u l a r H + s e c r e t i o n and bu f f e r t i t r a t i o n fo l l owed by p a s s i v e d i f f u s i o n of uncharged C O 2 and H 2 C O 3 o r g l y c o d i a z i n e molecules from lumen to s e r o s a . In the p resent s tudy , no attempt was made to i n v e s t i g a t e g l y c o d i a z i n e t r a n s p o r t per se p r i m a r i l y because of the d i f f i c u l t y o f o b t a i n i n g r a d i o a c t i v e l y l a b e l l e d forms of t h i s compound. The e f f e c t s o f g l y c o d i a z i n e on C l " r eabso rp t i on and e l e c t r o p h y s i o l o g y of the a n t e r i o r s a l t g l a n d , however, were examined. F igu re 5.20 i s reproduced i n pa r t from the p rev ious chapter and shows tha t C l " r eabso rp t i on i n the per fused a n t e r i o r segment i s complete ly i n h i b i t e d by b i l a t e r a l l y r e p l a c i n g C 0 2 and HC03~ w i th an impermeant phosphate o r HEPES b u f f e r . When the l i p i d s o l u b l e bu f fe r g l y c o d i a z i n e i s used as a CO2 and H C O 3 " rep lacement , however, t h i s compound - 168 -F i g u r e 5.20 E f f e c t s o f b i l a t e r a l C O 2 and HC03~ replacement w i th e i t h e r a phosphate, HEPES or g l y c o d i a z i n e bu f fe red s a l i n e on the change i n a n t e r i o r segment pe r fusa te C l " c o n c e n t r a t i o n . Values are means + S . E . (n = 5 -12 ) . Th is f i g u r e i s reproduced i n pa r t from Chapter IV. 75 h ^ 50 2 25 2 -25 ta (0 < -50 Control Control -75 | | A Total C 0 2 E U A C I -HEPES >4 Glycodiazine - 170 -ma in ta ins a smal l and c o n s i s t e n t component of C l ~ r e a b s o r p t i o n . The change i n pe r fusa te C l " concen t ra t i on w i th g l y c o d i a z i n e was -8 .8 +_ 2 .8 mM (mean _+ S . E . , n = 5) and was s i g n i f i c a n t l y d i f f e r e n t (0.01 < P < .025) from z e r o . In a d d i t i o n , when C 0 2 and H C 0 3 " are removed from the se rosa l s a l i n e and rep laced w i th g l y c o d i a z i n e , V t e r a p i d l y d e p o l a r i z e s and r e v e r s e s , and the s m a l l , p o s i t i v e V t e remains s t a b l e f o r a t l e a s t 60 minutes ( F i g . 5 . 8 ) . When the impermeant HEPES bu f f e r rep laces se rosa l C 0 2 and HCO3" , however, V t e i n i t i a l l y d e p o l a r i z e s by 20-30 mV and i s then fo l l owed by a slow r e p o l a r -i z a t i o n phase u n t i l a new s t a b l e V t e , 10-15 mV lower than con t ro l v a l u e s , i s reached ( F i g . 5 . 7 ) . The nature of t h i s r e p o l a r i z a t i o n i s u n c e r t a i n (see R e s u l t s ) , but i t cou ld be due to changes i n i n t r a c e l l u l a r ion concen t ra t i ons w i th subsequent changes i n a Donnan or C l ~ - d i f f u s i o n p o t e n t i a l . In order f o r g l y c o d i a z i n e to support C l " r eabso rp t i on i n the a n t e r i o r s a l t g land i t i s necessary to propose the scheme shown i n F igu re 5 . 2 1 . S h u t t l i n g of protons across the c e l l membrane would r e q u i r e tha t both the a n i o n i c and u n d i s s o c i a t e d forms of g l y c o d i a z i n e be l i p i d s o l u b l e . Indeed, many weak a c i d anions are qu i t e permeable to the c e l l membrane w i th the a n i o n i c form moving down a f avo rab le e lec t rochemica l g rad ien t c rea ted by the membrane p o t e n t i a l and the i n t r a c e l l u l a r d i s s o c i a t i o n of the uncharged a c i d (see f o r example McLaughl in and D i l g e r , 1980; Roos and Boron , 1981; Boron , 1983). Measurement of dur ing r a p i d changes in se rosa l f l u i d compos i t ion suggests i n d i r e c t l y tha t the g l y c o d i a z i n e anion permeates the c e l l membrane. G l y c o d i a z i n e added to the bath e i t h e r i n the presence (18.5 mM g l y c o d i a z i n e p lus normal C 0 2 and H C 0 3 " c o n c e n t r a t i o n s ; data not shown) or absence ( F i g . 5.10) of C 0 2 and HCO3" causes a s m a l l , t r a n s i e n t 2-3 mV h y p e r p o l a r i z a t i o n of V^-j. Replacement of se rosa l C 0 2 and HC03~ w i th the impermeant HEPES b u f f e r , however, causes a s m a l l , t r a n s i e n t - 171 -F i gu re 5.21 T e n t a t i v e scheme showing proposed proton s h u t t l i n g e f f e c t s o f g l y c o d i a z i n e b u f f e r . - 173 -d e p o l a r i z a t i o n of V b- | ( F i g . 5.17) which i s c o n s i s t e n t w i th a very smal l outwardly d i r e c t e d H C O 3 " o r inwardly d i r e c t e d H + p e r m e a b i l i t y (note tha t removal o f se rosa l C 0 2 and H C 0 3 " causes i n t r a c e l l u l a r a l k a l i n i z a t i o n ; see F i g . 5 . 3 ; Roos and Boron , 1981). The e f f e c t s of g l y c o d i a z i n e on C l " reabsorp t ion and V t e i n the a n t e r i o r segment are admi t ted ly s m a l l , but never the less support the hypo thes is t ha t t h i s compound main ta ins a component of e l e c t r o g e n i c C l " r eabso rp t i on i n the absence of exogenous C 0 2 and HCO^". I t may be p a r t i c u l a r l y i n s t r u c t i v e i n fu tu re i n v e s t i g a t i o n s to examine the e f f e c t s C l " o f g l y c o d i a z i n e on J n g t and e l e c t r o p h y s i o l o g y by va ry ing the r a t i o s of charged and uncharged s p e c i e s . In p a r t i c u l a r , a h igher concen t ra t i on of the u n d i s s o c i a t e d g l y c o d i a z i n e molecule may be more e f f e c t i v e i n ma in ta in i ng C l " reabso rp t i on by genera t ing a h igher ra te of proton s h u t t l i n g . 2) E lec t rochem ica l Grad ien ts In the p resent s tudy , c e l l u l a r HCG^ - concen t ra t i on was c a l c u l a t e d us i ng the Henderson-Hasse lba lch equat ion and by assuming tha t P C 0 2 , K j ' , C 0 2 s o l u b i l i t y c o e f f i c i e n t s and H C O 3 " a c t i v i t y c o e f f i c i e n t s were the same i n s i d e the c e l l as i n f r e e s o l u t i o n . I t i s u n c e r t a i n whether these assumptions a c t u a l l y ho ld i n v i v o . A t p resen t , however, c a l c u l a t i o n of H C O 3 " concen t ra t i on p rov ides the best means of e s t i m a t i n g i n t r a c e l l u l a r H C O 3 " a c t i v i t y . Given the above assumption i t f o l l o w s tha t K i ! o = L H + 3 o [ H C O 3 - ] o (1) [co2]o K l ! c = [ H + ] c [ H C 0 3 - ] c ( 2 ) [co2]c K i ; o [ C 0 2 ] o = C H + ] o [ H C 0 3 - ] o , ( 3 ) - 174 -K i ; c [ C 0 2 ] c = £ H + ] c [ H C 0 3 - ] c ' ( 4 ) Since i t was assumed t ha t ^i'Q = K j _ ' c and [ C 0 2 ] o = t C 0 2 ^ c t h e n K l ! o C C 0 2 ] o = K l ! c C C 0 2 ] c , ( 5 ) [ H + ] o [ H C 0 3 ] o = [ H + ] c [ H C 0 3 - ] c ^ (6) [ H + ] Q = [ H C 0 3 - ] c (7) WTQ C H C 0 3 - ] o and t he re fo re aJJL|+ = AMHCO " * ^ 3 Tab le 5.3 shows the c a l c u l a t e d - ^ o r a p i c a l and b a s o l a t e r a l 3 c e l l membranes under c o n t r o l c o n d i t i o n s . B icarbonate en t ry i n t o the c e l l or the e q u i v a l e n t p rocess a t the se rosa l membrane i s aga ins t an un favorab le e l ec t r ochem ica l g rad ien t of 77.1 mV and t he re fo re must be mediated by an a c t i v e t r a n s p o r t p r o c e s s . I t i s u n c e r t a i n a t p resen t , however, whether the C 1 " / H C 0 3 ~ exchanger i s a p r imary , ATP-d r i ven pump, or whether i t i s a secondary a c t i v e t r a n s p o r t process w i th the b a s o l a t e r a l C l " e l ec t rochemica l g rad ien t of 71.7 mV (Table 5.2) e n e r g i z i n g a c t i v e , b a s o l a t e r a l H C 0 3 " en t r y . Even i f the ove res t ima t i on of afa- which i s inheren t i n m ic roe lec t rode measurements i s i g n o r e d , and i t i s assumed t ha t there i s 100% c o u p l i n g e f f i c i e n c y between C l " and H C 0 3 " movements, there s t i l l does not appear to be s u f f i c i e n t energy s to red i n the a JJQ ] - to d r i v e HC0 3 ~ i n t o the c e l l . Under c o n t r o l c o n d i t i o n s H C 0 3 " e x i t a t the a p i c a l c e l l membrane i s down a f avo rab le e l ec t r ochem ica l g rad ien t o f 27.6 mV (Table 5 . 3 ) . These exper iments a lone do not demonstrate t ha t a p i c a l HC0 3 ~ t r a n s p o r t i s normal ly a downhi l l p rocess and f u r t h e r s t u d i e s are needed to determine i f A ^ H C 0 3 ~ f a v o r s pass i ve HC0 3 ~ movement under a v a r i e t y of exper imenta l c o n d i t i o n s . - 175 -I t i s i n s t r u c t i v e to po in t out tha t under c o n d i t i o n s which are perhaps more p h y s i o l o g i c a l to the s a l t g land ( i . e . the lumina l membrane bathed by a s t r ong l y hyperosmotic NaCl or NaHCO^-r ich f l u i d ; see Chapter II and I V ) , t ha t i t can c l e a r l y no longer be assumed tha t K j / , C 0 2 s o l u b i l i t y c o e f f i c i e n t s and H C O 3 " a c t i v i t y c o e f f i c i e n t s are equ i va len t i n the luminal and i n t r a c e l l u l a r f l u i d s . The re fo re , under these c o n d i t i o n s AJJ^+ f AJ^QQ _ 3 and i t would be necessary to determine the ac tua l i o n i c spec ies i nvo l ved i n H C O 3 " s e c r e t i o n be fore i t cou ld be determined t ha t the a p i c a l s tep was a c t i v e or p a s s i v e . Data i n Table 5.4 i l l u s t r a t e t h i s problem. Measurement o f a n t e r i o r segment c e l l pH i n r e c t a l s a l t g lands bathed by t h e i r own s e c r e t i o n s or i n a n t e r i o r segments per fused w i th a r t i f i c i a l s e c r e t i o n s c o n t a i n i n g 400 mM H C O 3 " and 40 mM C 0 3 2 " (pH 8 . 7 5 ; Table 5.1) y i e l d e d c a l c u l a t e d a p i c a l , e l ec t rochemica l g rad ien ts which would f avo r pass i ve H + ent ry i n t o the c e l l o r r equ i r e the presence of a pr imary e l e c t r o g e n i c H C O 3 " pump i n the a p i c a l membrane. S ince the unequivocal demonstrat ion o f H + / 0 H " versus H C O 3 " t r a n s p o r t i n an i n t a c t ep i t he l i um i s d i f f i c u l t and always sub jec t to ques t ion (see f o r example A l - A w q a t i , 1978; M a l n i c , 1980), i t may be more f r u i t f u l i n f u tu re i n v e s t i g a t i o n s to attempt to vo l tage clamp the a n t e r i o r segment a t va r ious va lues of V t e . Measurement o f pH c and net 7 + ~ f l u x e s of HC03~, C O 3 and H , and c a l c u l a t i o n of a><HQQ - under c o n d i t i o n s 3 of a l t e r e d V^. e may prov ide a c l e a r e r p i c t u r e than measurement o f these p a r a -meters under c o n d i t i o n s of a l t e r e d t r a n s e p i t h e l i a l chemical g rad ien ts ( i . e . a n t e r i o r segments per fused w i th hyperosmot ic NaCl or NaHC03-r ich f l u i d s ) . Tab le 5.3 C a l c u l a t e d HCC^" e lec t rochemica l g rad ien ts f o r a p i c a l and b a s o l a t e r a l c e l l membranes bathed by con t ro l serosa l and mucosal s a l i n e s . Net C 0 2 f l u x i s from bath to lumen. Negat ive s igns i n d i c a t e a f avo rab le A J J H C Q - f o r p a s s i v e ' H C 0 3 " movement. The ApHC03~ W 3 S c a l c u 1 a t e d u s i n g the mean V b l of -75 .6 mV (see R e s u l t s ) . The a c t i v i t y c o e f f i c i e n t f o r i n t r a c e l l u l a r HC03~ was assumed to be the same as i n the bath ing and pe r fus ion s a l i n e s ( i . e . 0 .733 ) . Va lues are means +_ S . E . (n = 39 c e l l s 4 p r e p a r a t i o n s ) . Note tha t g iven the assumptions used in c a l c u l a t i n g [HC03~] C (see D i s c u s s i o n ) t ha t A P H C O 3 - = A £ H + -PH C CHC0 3 ] c a f i c 0 3 - Va A p ^ - A J J ^ -7.67 + 0.03 19.6 + 1.0 mM 14.4 + 0.7 mM -27 .3 + 0.5 mV -27 .6 + 1.3 mV 77.1 mV Tab le 5 . 4 . I n t r a c e l l u l a r pH and c a l c u l a t e d a p i c a l membrane e lec t rochemica l g rad ien ts f o r H + and HCC^" . Net H C O o " f l u x i s from bath to lumen and net H f l u x i s from lumen to ba th . Negat ive s i gns i n d i c a t e a f a v o r a b l e e lec t rochemica l g rad ien t f o r pass ive ion movement. S e c r e t i o n pH and H C O 3 " concen t ra t i on were assumed to be 8.65 and 402 mM, r e s p e c t i v e l y (see Chapter I I ) , f o r s a l t g lands w i th lumina l membranes bathed by t h e i r own s e c r e t i o n s . I n t r a c e l l u l a r K ^ ' , S and H C O 3 " a c t i v i t y c o e f f i c i e n t s were assumed to be the same as those in the ba th . Values are means +_ S . E . of 8-14 impalements on 2-3 p r e p a r a t i o n s . Exper iment [ H C 0 3 - ] c Va A n t e r i o r segments 7 . 9 9 + 0 . 0 2 36.9 + 1.7 mM 27.1 + 1.2 mM -51 .2 + 1.5 mV 8.3 + 2 .5 mV - 7 . 0 + 2.5 mV per fused w i th a r t i f i c i a l s e c r e t i o n . Rec ta l s a l t g lands 7.84 + 0.04 29.4 + 2.6 mM 21.6 + 1.9 mM - 4 6 . 3 + 1.0 mV 20.9 + 2 .9 mV - 0 . 6 + 2.8 mV ^ bathed on t h e i r ^ lumina l su r face ""J wi th t h e i r own 1 s e c r e t i o n s . - 178 -In summary, C l " en te rs the a p i c a l c e l l membrane of the a n t e r i o r s a l t g l a n d , a t l e a s t i n p a r t , by p a s s i v e , e l e c t r o d i f f u s i v e movement through a C l " s e l e c t i v e pathway, and HCC^" e x i t s the c e l l by an a c t i v e or pass i ve e l e c t r o g e n i c t r a n s p o r t mechanism. The b a s o l a t e r a l c e l l membrane i s the s i t e of d i r e c t coup l i ng between C l " and HCC^" movements v i a a C T / H C C ^ " exchange or HCI co t r anspo r t mechanism. In the l a s t chapter the r e s u l t s of t h i s t h e s i s are summarized and HCC^" t r a n s p o r t i n the a n t e r i o r s a l t g land i s compared to ac i d -base t r a n s p o r t i n ve r t eb ra te e p i t h e l i a and i n v e r t e b r a t e s i n g l e c e l l s . - 179 -CHAPTER VI - GENERAL DISCUSSION Mechanisms of a c i d and base t r a n s p o r t have been s t u d i e d i n a v a r i e t y of ve r teb ra te g a s t r o i n t e s t i n a l and exc re to ry e p i t h e l i a , the cho ro id p l e x u s , and i n severa l types of i n v e r t e b r a t e n o n - e p i t h e l i a l c e l l s . Th i s f i n a l chapter i s devoted to a comparat ive d i s c u s s i o n of these mechanisms and to a summary of the work i n t h i s t h e s i s . Hydrogen and HC03~ t r anspo r t i n g a s t r o i n t e s t i n a l e p i t h e l i a p lays a number of important and s p e c i a l i z e d r o l e s i n c l u d i n g p r o v i d i n g the proper medium f o r d i g e s t i o n of f o o d s t u f f s , n e u t r a l i z a t i o n of g a s t r i c a c i d s , p r o t e c -t i o n of the mucosal su r face from d i g e s t i v e j u i c e s and f a c i l i t a t i o n of the abso rp t i on of n u t r i e n t s such as NH 3 and v o l a t i l e f a t t y a c i d s . In the duct e p i t h e l i u m of the mammalian s a l i v a r y g l a n d , H C 0 3 " i s a c t i v e l y sec re ted i n concen t ra t i ons i n excess o f 100 rrM (reviewed by Young and van Lennep, 1979) and i s probably impor tant f o r ma in ta in i ng opt imal f u n c t i o n of s a l i v a r y d i g e s t i v e enzymes. S tud ies on the mic roper fused r a t s a l i v a r y duct have p rov ided evidence f o r the presence of an a p i c a l , e l e c t r o n e u t r a l K + / H + exchange or KHCO3 c o t r ans po r t mechanism which mediates HCO3" s e c r e t i o n (Knauf and Lubcke, 1975; Knauf , Lubcke, Kreu tz and Sachs , 1982) . The a p i c a l K + / H + exchanger has been pos tu l a ted to be a seconda r i l y a c t i v e t r a n s p o r t p rocess which i s coup led to the a p i c a l , downh i l l K + g rad ien t mainta ined by a b a s o l a t e r a l N a + / K + ATPase (Knauf e t a l _ . , 1982). B a s o l a t e r a l H C 0 3 " en t ry i n t o s a l i v a r y duct c e l l s i s thought to be mediated by a pass i ve HC03~ conductance and a N a + / H + exchanger (Knauf e_t a l_ . , 1982). I t i s i n t e r e s t i n g to note t ha t the l a b i a l g land of s a t u r n i i d moths produces a K H ^ - r i c h f l u i d dur ing e c d y s i s of the a d u l t moth from i t s cocoon ( K a f a t o s , 1968). S e c r e t i o n of HC0 3 ~ i s necessary to ma in ta in an a l k a l i n e pH f o r opt imal f u n c t i o n of the - 180 -cocoon-opening enzyme (Kafatos and W i l l i a m s , 1964) and cou ld conce ivab ly be mediated by a K + / H + exchange mechanism s i m i l a r to t ha t of the duct e p i t h e l i u m . The ve r t eb ra te g a s t r i c mucosa has been one of the most e x t e n s i v e l y s t u d i e d ac id -base t r a n s p o r t i n g organs and has been the sub jec t of numerous recen t rev iew a r t i c l e s (see Sachs, Spenney and Lewin , 1978; Machen and F o r t e , 1979; F o r t e , Machen and Obr ink , 1980; Sachs , F a l l e r and Rabon, 1982; Diamond and Machen, 1983). I t i s now f a i r l y we l l e s t a b l i s h e d tha t lumina l a c i d s e c r e t i o n and genera t ion o f t r a n s e p i t h e l i a l H + g rad ien ts o f up to 10 7 : 1 are mediated by a p r imary , e l e c t r o n e u t r a l H + / K + ATPase mechanism (reviewed by Sachs e t a]_., 1978, 1982; Sachs , Rabon, S tewar t , P i e r c e , Smolka and Saccomani , 1980) . These r e s u l t s a r e , however, d i f f i c u l t to r e c o n c i l e w i th exper iments i n dog stomach and f r og g a s t r i c mucosa which suggest tha t H + s e c r e t i o n i s e l e c t r o g e n i c (reviewed by Machen and F o r t e , 1979; Fo r te e t a l . , 1980). A t p r e s e n t , i t appears t ha t the H + pump may be capable of ope ra t i ng i n e i t h e r an e l e c t r o g e n i c or e l e c t r o n e u t r a l mode depending on the exper imenta l c o n d i t i o n s employed. The process of g a s t r i c H + s e c r e t i o n n e c e s s a r i l y produces i n t r a c e l l u l a r 0H~. Th i s OH" i s n e u t r a l i z e d by C 0 2 i n a ca rbon ic anhydrase-dependent r e a c t i o n to y i e l d H C 0 3 " which e x i t s the b a s o l a t e r a l membrane of the o x y n t i c c e l l v i a a C T / H C O 3 " exchange mechanism (Rehm and Sanders , 1975) . S t i m u l a t i o n o f g a s t r i c a c i d s e c r e t i o n by hormones i s accompanied by s p e c t a c u l a r changes i n the u l t r a s t r u c t u r e of the oxyn t i c c e l l s (see Diamond and Machen, 1983, f o r an e x c e l l e n t r e v i e w ) . In the uns t imu la ted c e l l , the sec re to r y c a n a l i c u l i are g r e a t l y reduced i n volume, a p i c a l m i c r o v i l l i are s c a r c e , and the c e l l cy top lasm i s f i l l e d w i th an ex tens i ve system of - 181 -t u b u l o v e s i c l e s . Upon s t i m u l a t i o n of the o x y n t i c c e l l w i th a compound such as h i s t am ine , however, there i s a pronounced expansion of the c a n a l i c u l i and a dramat ic i nc rease i n a p i c a l m i c r o v i l l i w i th a concomitant decrease i n t u b u l o v e s i c l e s . These u l t r a s t r u c t u r a l changes may be a s s o c i a t e d w i th i n s e r t i o n of new ion t r a n s p o r t e r s i n t o the a p i c a l membrane i n a manner s i m i l a r to t ha t proposed f o r the a p i c a l H + pumps of the t u r t l e b ladder (G luck , Cannon and A l - A w q a t i , 1982; d i scussed be low) . In a d d i t i o n to s e c r e t i n g H + , the fund ic and an t ra l p o r t i o n s of the g a s t r i c mucosa are a l s o capable o f s e c r e t i n g H C O 3 - . Severa l s t u d i e s have suggested tha t H C O 3 " s e c r e t i o n i s an a c t i v e , e l e c t r o n e u t r a l process which i s mediated by a C1~/HC03~ exchange mechanism l o c a t e d i n the luminal membrane of su r face c e l l s ( reviewed by Flemstrom and Garne r , 1982). G a s t r i c HCG^ - s e c r e t i o n appears to be c r i t i c a l l y impor tant i n a l k a l i n i z i n g an a p i c a l mucous l a y e r and u n s t i r r e d boundary which serves to p r o t e c t the mucosal e p i t h e l i u m from high i n t r a l u m i n a l a c i d i t y (Flemstrom and Garne r , 1981; see a l s o P o w e l l , 1981). The mammalian pancreas produces an a l k a l i n e s e c r e t i o n c o n t a i n i n g HC03~ concen t ra t i ons up to 130 mM. Th i s a l k a l i n e s e c r e t i o n i s necessary to n e u t r a l i z e the h i g h l y a c i d i c d i g e s t i v e j u i c e of the stomach before i t en te rs the i n t e s t i n e and to ma in ta in an opt imal pH f o r f u n c t i o n o f panc rea t i c enzymes. G e n e r a l l y , i t i s be l i eved t ha t a c t i v e HC03~ s e c r e t i o n occurs both i n the a c i n i and e x t r a l o b u l a r ducts of the pancreas (reviewed by Schu lz and U l l r i c h , 1979). The ac tua l c e l l u l a r mechanism of HC03~ s e c r e t i o n i s u n c e r t a i n , however, because of the hetergeneous nature of the e p i t h e l i u m and the d i f f i c u l t i e s a s s o c i a t e d w i th i s o l a t i n g the va r ious segments of the pancreas ducta l system i n v i t r o . S tud ies w i th weak o rgan ic ac i ds such as s u l f a m e r a z i n e , g l y c o d i a z i n e , a c e t a t e , fo rmate , p rop r iona te and bu ty ra te - 182 -(reviewed by S c h u l z , 1976, 1981; Schu lz and U l l r i c h , 1979; see a l s o Chapter V) suggest t ha t the ac tua l i o n i c spec ies t r anspo r ted i n HCC^" s e c r e t i o n i s H + / 0 H ~ . Th is o b s e r v a t i o n , p lus s t u d i e s on e x t r a l o b u l a r ducts which demonstrated t ha t a p p l i c a t i o n o f ouabain to or removal of N a + from the se rosa l ba th ing medium i n h i b i t s HCO^ - s e c r e t i o n and a l t e r s i n t r a c e l l u l a r pH (Swanson and Solomon, 1972, 1975), suggested the presence of N a + / H + exchange i n the pancreas . Swanson and Solomon (1975) p o s t u l a t e d tha t a p i c a l proton reabso rp t i on was mediated by a p r imary , ATP-dr i ven N a + / H + exchanger and t h a t b a s o l a t e r a l H + e x i t was e f f e c t e d by a N a + / H + exchanger seconda r i l y coup led to the downh i l l en t ry o f N a + i n t o the c e l l . Two o ther models have been proposed by Schu lz and U l l r i c h (1979) based on the observed N a + dependence and ouabain s e n s i t i v i t y o f HC0 3 ~ t r a n s p o r t . These i n c l u d e : 1) a b a s o l a t e r a l N a + / H + exchange and a p a s s i v e , conduct ive H C O 3 " e x i t s tep a t the a p i c a l membrane s i m i l a r to tha t p o s t u l a t e d f o r the b a s o l a t e r a l membrane of the r a t proximal tubu le (see Burckhard t and Fromter , 1980) ; 2) a s e c o n d a r i l y a c t i v e reabso rp t i on of H + coupled to downhi l l K + e x i t w i th t i g h t K + r e c y c l i n g a t the a p i c a l membrane and a p a s s i v e , conduc t i ve e x i t of H + a t the b a s o l a t e r a l c e l l border . Very l i t t l e d i r e c t exper imenta l evidence e x i s t s f o r any of these models and f u r t h e r s t u d i e s us ing more s o p h i s t i c a t e d t e c h n i c a l approaches are c l e a r l y needed. The a c t i v e s e c r e t i o n of H C O 3 " by the duodenal e p i t h e l i u m of the smal l i n t e s t i n e i s necessary to n e u t r a l i z e g a s t r i c a c i d , p r o t e c t the i n t e s t i n a l s u r f a c e , and to p rov ide an opt imal a l k a l i n e pH f o r f u n c t i o n o f p a n c r e a t i c enzymes. E a r l y s t ud ies by F l o r e y , J e n n i n g s , Jenn ings and O'Connor (1939) suggested t ha t HC03~ s e c r e t i o n o r i g i n a t e d i n the Brunners g land . L a t e r i n v e s t i g a t i o n s , however, have shown ex tens i ve luminal a l k a l i n i z a t i o n i n duodena from spec ies l a c k i n g Brunners g lands and i n duodenal segments - 183 -prox imal and d i s t a l to the Brunners g lands i n spec ies such as dog, c a t , human, guinea p i g , r a t and r a b b i t ( reviewed by Flemstrom and Garner , 1982). These s t u d i e s i n d i c a t e t ha t the su r face e p i t h e l i a l c e l l s are the s i t e s of H C O 3 " s e c r e t i o n . S t u d i e s on the i s o l a t e d b u l l f r o g duodenum by Simson, Meshav and S i l e n (1981a,b) have shown tha t uns t imu la ted H C O 3 " t r a n s p o r t i s e l e c t r o g e n i c , s e n s i t i v e to se rosa l ouabain and dependent upon the presence of N a + i n the se rosa l ba th ing s a l i n e . A c c o r d i n g l y , the authors p o s t u l a t e d a NaHC03 co t ranspo r t o r N a + / H + exchange mechanism to mediate H C O 3 " en t ry i n t o the c e l l a t the b a s o l a t e r a l membrane and a p a s s i v e , conduc t i ve e f f l u x of HC03~ a t the lumina l c e l l border . S t u d i e s on duodenal e p i t h e l i a exposed to g lucagon, however, showed tha t s t imu la ted HCG^" s e c r e t i o n was e l e c t r o n e u t r a l and was i n h i b i t e d by furosemide and lumina l C l " removal sugges t ing the presence of a C 1 " / H C 0 3 ~ exchange mechanism (Flemstrom, Hey l i n g s and Garner , 1982). In Amphiuma duodenum, HCC^" s e c r e t i o n requ i res the presence of se rosa l N a + and mucosal C l " (White and Imon, 1982) and i s i n h i b i t e d by se rosa l ace tazo lam ide , ouabain and SITS (Imon and Whi te , 1981) . White and Imon (1982) p o s t u l a t e t ha t H C O 3 " en te rs the c e l l a t the b a s o l a t e r a l c e l l membrane by p a r a l l e l N a + / H + and Cr /HCC^" exchangers and e x i t s v i a an a p i c a l conduct ive pathway. The ve r teb ra te jejunum i s capable o f reabsorb ing HCC^" and a c i d i f y i n g the lumina l contents i n v i v o , however, few s t u d i e s have examined the ac tua l mechanisms of a c i d i f i c a t i o n . E a r l y s t ud ies on the human jejunum suggested tha t HCC^" may be reabsorbed by an a c t i v e , e l e c t r i c a l l y s i l e n t N a + / H + exchanger (Ford t ran and Rec to r , 1968; Turnberg, F o r d t r a n , C a r t e r and R e c t o r , 1970). More r e c e n t l y , Imon and White (1983) have p rov ided ev idence which i n d i c a t e s t ha t a c t i v e H C O 3 " reabsorp t ion i n Amphiuma jejunum i s mediated by a K + / H + exchange or K H C O 3 co t r anspo r t mechanism. - 184 -White and Imon (1983) have a l s o shown tha t b a s o l a t e r a l H C 0 3 " e x i t i n j e j u n a l c e l l s i s f a c i l i t a t e d by an anion exchanger which exchanges i n t e r n a l H C O 3 " f o r ex te rna l C l " , B r " , I", or S O 4 i n a manner s i m i l a r to t ha t observed i n the e ry th rocy te membrane (Knauf, 1979) . A l though the mammalian i leum has been used e x t e n s i v e l y as a model f o r s t u d i e s of N a + and C l " t r a n s p o r t , r e l a t i v e l y few i n v e s t i g a t i o n s have examined the mechanisms of t r a n s e p i t h e l i a l H + and HCG^" movements. S tud ies by Hubel (1967, 1969) ; Turnberg , B i e b e r d o r f , Morawski and Fo rd t ran (1970); and F i e l d , Fromm and McCol l (1971) have shown tha t i l e a l H C O 3 " s e c r e t i o n i s an a c t i v e process r e q u i r i n g luminal C l " , thus suggest ing the presence of a C T / H C O 3 " exchanger i n t h i s t i s s u e . Sheer in and F i e l d (1975) have presented a very s p e c u l a t i v e model of i l e a l HC03~ t r a n s p o r t which pos tu la tes a b a s o l a t e r a l N a + / H + exchanger media t ing HC03~ ent ry a t t h i s membrane and an a p i c a l C 1 ~ / H C 0 3 _ exchange e x i t s t e p . Fu r t he r s t ud ies of H + and HC0 3 ~ t r anspo r t are c l e a r l y needed i n both the i l e a l and j e j u n a l i n t e s t i n a l segments. S e c r e t i o n of H C O 3 " by the c o l o n i c e p i t h e l i u m has been shown to be an a c t i v e , e l e c t r o n e u t r a l process which i s dependent upon the presence of C l " i n the mucosal ba th ing s a l i n e . These r e s u l t s , p lus exper iments showing a c l o s e r e c i p r o c a l r e l a t i o n s h i p between net C l " and H C O 3 " movements i n t h i s t i s s u e , are i n d i c a t i v e of a luminal C 1 " / H C 0 3 ~ exchange mechanism (reviewed by P o w e l l , 1979; S c h u l t z , 1981a). Recen t l y , Duffey and Bebern i t z (1983) have s tud ied C1~/HC03~ exchange i n r a b b i t co lon us ing i n t r a c e l l u l a r H + and C l ~ - s e l e c t i v e m i c r o e l e c t r o d e s . T h e i r r e s u l t s demonstrated tha t a p i c a l C l " ent ry was an a c t i v e t r a n s p o r t process tha t cou ld f e a s i b l y be ene rg i zed by the movement o f HC03~ down a f avo rab le lumina l e l ec t rochemica l g r a d i e n t . Emmer and Duffey (1983) have shown tha t a p p l i c a t i o n of se rosa l - 185 -DIDS to the r a b b i t co lon s t imu la tes a H C O 3 " - dependent s h o r t - c i r c u i t c u r r e n t suggest ing t ha t t h i s compound unmasks an e l e c t r o g e n i c HCG^" sec re to r y p r o c e s s . Regu la t ion o f the ac id -base ba lance of the ce reb rosp ina l f l u i d (CSF) may be c r i t i c a l l y important i n c o n t r o l l i n g the f u n c t i o n o f r e s p i r a t o r y neurons which i n tu rn would i n f l u e n c e d i r e c t l y e x t r a c e l l u l a r pH and H C O 3 " homeostas is . U n f o r t u n a t e l y , r e l a t i v e l y few s tud ies have been conducted on the mechanisms of c h o r o i d p lexus H + and HC03~ t r a n s p o r t , p r i m a r i l y because of the d i f f i c u l t y of i s o l a t i n g and work ing w i th t h i s t i s s u e i n v i t r o . E a r l y s t u d i e s by S i e s j o and K j a l l q u i s t (1969) suggested tha t CSF pH was main ta ined by a pass i ve p r o c e s s . More r e c e n t l y , however, B l e d s o e , Eng and Hornbein (1981) have presented good ev idence i n d i c a t i n g t ha t c h o r o i d p lexus H + t r a n s p o r t i s mediated by an a c t i v e mechanism. Based l a r g e l y on i n d i r e c t ev idence , Wright (1972, 1977) p o s t u l a t e d t ha t HCC^ - s e c r e t i o n i n the c h o r o i d p lexus was mediated by a p a s s i v e , conduct ive HCC^ - e x i t s tep a t the a p i c a l membrane and tha t HCC^ - en tered the c e l l v i a a b a s o l a t e r a l N a + / H + exchanger. S a i t o and Wright (1982, 1983) have p rov ided d i r e c t ev idence i n support o f an a p i c a l HCC^" conductance i n the cho ro id p lexus and have shown t ha t HCC^" s e c r e t i o n i n t o the CSF may be c o n t r o l l e d by hormones which s t i m u l a t e the adeny la te c y c l a s e system. As desc r i bed i n Chapter I, the g i l l ep i t he l i um of f i s h e s i s an impor tant s i t e of ac i d -base e x c r e t i o n and r e g u l a t i o n . Numerous s tud ies have p rov ided s t rong ev idence f o r the presence of N a + / H + , N a + / N H 4 + and C T / H C O 3 " exchange mechanisms i n the a p i c a l membranes of g i l l s from f reshwater -adapted f i s h (reviewed by Evans , 1975, 1980; P o t t s , 1977; K i r s c h n e r , 1979; Evans, C l a i b o r n e , Farmer, M a l l e r y and Krasny , 1982). Because of the heterogeneous nature of the b ranch ia l e p i t h e l i u m and the - 186 -problems a s s o c i a t e d w i th i n v i t r o g i l l p r e p a r a t i o n s , however (see f o r example Karnaky, 1980; Evans e t ^ a K , 1982), more d e t a i l e d c h a r a c t e r i z a t i o n s of a c i d and base t r a n s p o r t have not been p o s s i b l e . The use of techn iques to i s o l a t e and p o s s i b l y c u l t u r e s p e c i f i c c e l l types i n con junc t ion w i th s t u d i e s on i s o l a t e d a p i c a l and b a s o l a t e r a l membrane v e s i c l e s from f i s h g i l l s may be extremely use fu l i n f u r t h e r s tud ies of f reshwater b ranch ia l H + / 0 H " and HC0 3 ~ t r a n s p o r t s t e p s . The use of the ope rcu la r e p i t h e l i u m cou ld a l s o be ext remely va luab le i n i n v e s t i g a t i o n s of ac i d -base t r a n s p o r t . Opercu la r sk i ns are composed of the fou r major c e l l types which form the s a l t - t r a n s p o r t i n g e p i t h e l i u m of the g i l l s (Karnaky and K i n t e r , 1977), and the o p e r c u l a r e p i t h e l i u m can be i s o l a t e d and s h o r t - c i r c u i t e d as a f l a t sheet i n conven t iona l Uss ing chambers making pH s t a t s t u d i e s f e a s i b l e . In a d d i t i o n , the use of ion and v o l t a g e - s e l e c t i v e m ic roe lec t rodes i n the ope rcu la r e p i t h e l i u m may p rov ide important i n s i g h t s i n t o c e l l u l a r en t ry and e x i t s teps i n v o l v e d i n t r a n s e p i t h e l i a l a c i d and base movements. Exchanges of ex te rna l N a + f o r i n t e r n a l H + or Nr^"1" are a l s o impor tant mechanisms of a c i d e x c r e t i o n in marine t e l e o s t s and elasmobranchs ( rev iewed by Evans, 1975, 1980, 1982). Whi le t h i s uptake of ex te rna l N a + n e c e s s a r i l y imposes an a d d i t i o n a l i o n i c l oad on marine f i s h e s , i t i s b e l i e v e d to be over r idden by the pr imary need of the animal to r egu la te i t s ac i d -base ba lance (Evans, 1982). The same techn iques d i scussed above shou ld a l s o be use fu l i n c h a r a c t e r i z i n g c e l l u l a r mechanisms of ac i d -base t r a n s p o r t i n seawater t e l e o s t s and elasmobranchs. The amphibian s k i n i s an impor tant s i t e of ac i d -base e x c r e t i o n and homeostas is , as was d i scussed i n Chapter I. In many r e s p e c t s , the mechanisms of H + t r a n s p o r t i n the i n v i t r o f r og s k i n appear to be very s i m i l a r to tha t observed i n the t u r t l e and toad u r i na ry b ladder w i th an a c t i v e , e l e c t r o g e n i c - 187 -H + pump l o c a t e d a t the a p i c a l c e l l membrane (see f o r example, F l em ing , 1957; Eh ren fe l d and Garc ia-Romeu, 1977; reviewed by S te inme tz , 1974). In a d d i t i o n , a number of i n v e s t i g a t o r s , s tudy ing H + and HC0 3 ~ t r a n s p o r t i n the i n v i v o f r o g s k i n bathed e x t e r n a l l y by d i l u t e s o l u t i o n s , have prov ided conv inc i ng ev idence f o r the presence of a p i c a l N a + / H + and C T / H C O 3 " exchangers which mediate net a c i d and base e x c r e t i o n (reviewed by K i r s c h n e r , 1983). The t u r t l e u r i n a r y b ladder has been s tud ied both i n i t s own r i g h t and as a model system f o r the mammalian c o l l e c t i n g duct . Numerous s tud ies have shown c l e a r l y t ha t H + s e c r e t i o n i s mediated by a p r imary , a p i c a l , e l e c t r o g e n i c H + pump (reviewed by S te inmetz , 1974; Ma ln i c and S te inme tz , 1976; A l - A w q a t i , 1978; Ste inmetz and Anderson, 1982). The l o c a t i o n of e l e c t r o g e n i c H + s e c r e t i o n i s gene ra l l y b e l i e v e d to be con f i ned to a smal l popu la t i on o f m i t o c h o n d r i a - r i c h , ca rbon i c anhydrase -con ta in ing c e l l s which make up approx imate ly 10-15% of the b ladder e p i t h e l i u m (Schwar tz , Rosen and S te i nme tz , 1972; Hus ted , M u e l l e r , Kesse l and S te inmetz , 1981). I t i s i n t e r e s t i n g to note t ha t d i r e c t con f i rma t i on of t h i s hypothes is may be p o s s i b l e us ing the v i b r a t i n g probe techn ique which was o r i g i n a l l y developed to l o c a l i z e ion cu r ren t s i n s i n g l e c e l l s ( J a f f e and N u c c i t e l l i , 1974). More r e c e n t l y , Foske t t and Schef fey (1982) have a p p l i e d t h i s powerful method to the ope rcu la r e p i t h e l i u m of a seawater t e l e o s t and have p rov ided the f i r s t unequivocal ev idence t ha t the c h l o r i d e - c e l l i s the s i t e of a c t i v e , e l e c t r o g e n i c C l ~ s e c r e t i o n i n t h i s t i s s u e . The nature o f the O H ' / H C ^ - e f f l u x or H + en t ry mechanism a t the b a s o l a t e r a l membrane of the t u r t l e b ladder i s unce r t a i n a t p resen t . Ste inmetz and Anderson (1982) have suggested t ha t H C O 3 " e f f l u x may be mediated by a conduc t i ve pathway or v i a a C T / H C O 3 " exchanger (see a l s o - 188 -Ehrenspeck and B rodsky , 1976; Cohen, M u e l l e r and S te i nme tz , 1978; F i s c h e r , Husted and S te inme tz , 1981). Severa l i n v e s t i g a t i o n s have p rov ided good evidence i n d i c a t i n g tha t H + s e c r e t i o n i n the b ladder i s mediated by a H + -ATPase . Dixon and A l -Awqa t i (1979) were ab le to induce ATP syn thes i s i n t u r t l e b ladder c e l l s by app l y i ng an adverse proton e l ec t rochemica l g rad ien t to the e p i t h e l i u m (see a l s o Dixon and A l - A w q a t i , 1980). Fu r the r b iochemica l s t u d i e s have shown c l e a r l y t ha t an e l e c t r o g e n i c H + -ATPase i s l o c a t e d i n lumina l microsomal membrane f r a c t i o n s ob ta ined from the t u r t l e b ladder (see f o r example, Pa rk , Campen, Dar re l and F a n e s t i l , 1981; G luck , K e l l y and A l - A w q a t i , 1982; Youmans, Worman and Brodsky , 1982, 1983; Gluck and A l - A w q a t i , 1983). Recent s t u d i e s have shown impor tant c o r r e l a t i o n s between H + s e c r e t i o n and the u l t r a s t r u c t u r e of the m i t o c h o n d r i a - r i c h c e l l s of the b ladder e p i t h e l i u m . These a c i d s e c r e t i n g c e l l s are c h a r a c t e r i z e d by prominent luminal m i c r o p l i c a e and by an ex tens i ve popu la t i on of s u b c e l l u l a r v e s i c l e s l oca ted j u s t below the a p i c a l plasma membrane. Husted e^ t al_. (1981) demonstrated tha t i n h i b i t i o n o f H + s e c r e t i o n i n the b ladder reduced the number of c e l l s hav ing m i c r o p l i c a e from 12.7% to 0.5% and suggested tha t the m i c r o p l i c a e represented the a c t i v e s t a t e o f the H + s e c r e t i n g c e l l . S te tson and Ste inmetz (1982) used u l t r a s t r u c t u r a l morphometry to demonstrate t ha t C02-mediated s t i m u l a t i o n o f H + s e c r e t i o n caused a dramat ic i n c r e a s e i n a p i c a l membrane su r face area and a decrease i n the volume f r a c t i o n o f s u b c e l l u l a r v e s i c l e s . These s t u d i e s suggested tha t the s t i m u l a t i o n o f H + s e c r e t i o n was p a r t i a l l y dependent upon f u s i o n of s u b c e l l u l a r v e s i c l e s w i th lumina l membranes. Gluck e r t a K (1982; see a l s o Reeves, Gluck and A l - A w q a t i , 1983) have demonstrated more d i r e c t l y us ing f l uo rescence microscopy and i n t r a c e l l u l a r dye techn iques tha t these s u b c e l l u l a r v e s i c l e s con ta i n H + - 189 -pumps and tha t ves ic le- to-membrane f u s i o n regu la tes u r i na ry a c i d i f i c a t i o n by i n s e r t i o n of new pumps i n t o the a p i c a l c e l l border . The f u s i o n of i n t r a c e l l u l a r v e s i c l e s c o n t a i n i n g s p e c i f i c ion t r a n s p o r t e r s w i th c e l l membranes i s an e x c i t i n g d i scovery and may represent a ub iqu i t ous means of r e g u l a t i n g ion and f l u i d t r a n s p o r t i n c e l l s and e p i t h e l i a (see f o r example Wade, 1980; K a r n i e l e , Za rnowsk i , H i s s i n , Simpson, Sa lano , and Cushman, 1982; Lewis and de Moura, 1982; Sp r ing and E r i c s o n , 1982). The t u r t l e b ladder i s a l s o capable o f a l k a l i n i z i n g the f i n a l u r i ne depending upon the ac i d -base s ta tus of the animal (see Cohen, 1980) . S tud ies by Ste inmetz and co l l eagues ( L e s l i e , Schwartz and S te inme tz , 1973; Husted, Cohen and S te inme tz , 1979; Husted and Mahadeva, 1983) suggest t ha t u r i na ry a l k a l i n i z a t i o n i s mediated by an e l e c t r o n e u t r a l C 1 " / H C 0 3 " exchanger , whereas B r o d s k y ' s group (reviewed by Brodsky , Durham and Ehrenspeck, 1980; Sa take , Durham and Brodsky , 1981) has p o s t u l a t e d an e l e c t r o g e n i c H C O 3 " s e c r e t o r y mechanism. The mechanism of a c i d e x c r e t i o n i n the nephron has been of major i n t e r e s t to rena l p h y s i o l o g i s t s s i nce the e a r l y micropuncture s t u d i e s o f Montgomery and P i e r c e (1937) and G o t t s c h a l k , L a s s i t e r and M y l l e (1960) suggested t ha t the proximal tubu le was the major s i t e of f i l t e r e d H C O 3 " r e a b s o r p t i o n . The measurement of i n t r a l u m i n a l d i s e q u i l i b r i u m pH va lues and t r a n s e p i t h e l i a l P C O 2 g rad ien ts has prov ided good evidence i n d i c a t i n g tha t u r i ne a c i d i f i a t i o n i n the proximal tubu le i s mediated by H + s e c r e t i o n r a t h e r than reabso rp t i on of H C O 3 " per se ( rev iewed by A l - A w q a t i , 1978; M a l n i c , 1980; Warnock and R e c t o r , 1981). Numerous i n s i t u and i n v i t r o s t u d i e s have demonstrated t ha t the bulk of proximal tubu le H + s e c r e t i o n i s mediated by a luminal N a + / H + exchanger as ev idenced by the s e n s i t i v i t y o f the a c i d i f i c a t i o n mechanism to mucosal a m i l o r i d e and se rosa l ouaba in , and to - 190 -i t s dependence on the presence of N a + i n the pe r fus ion s a l i n e (reviewed by Ma ln i c and G i e b i s c h , 1979; Warnock and R e c t o r , 1979, 1981). In a d d i t i o n , s t u d i e s on i s o l a t e d renal membrane v e s i c l e s have shown c l e a r l y t ha t an e l e c t r o n e u t r a l N a + / H + exchanger i s l o c a t e d i n the brush-border membrane (reviewed by Murer and K i n n e , 1980; A ronson , 1981; Kinne and K i n n e - S a f f r a n , 1981). Severa l s t u d i e s have a l s o demonstrated t ha t there i s a smal l component o f proximal tubu le H + s e c r e t i o n which i s independent o f N a + t r a n s p o r t (Burg and Green , 1977; McKinney and Burg , 1977b; Chan and G i e b i s c h , 1981). Two i n d i r e c t s t u d i e s have suggested tha t t h i s component may be mediated by an a c t i v e , e l e c t r o g e n i c H + pump (Fromter and Gessner , 1975; B i c h a r a , P a i l l a r d , L e v i e l , P r i g e n t and G a r d i n , 1983). Fu r t he r s t u d i e s are needed, however, to con f i rm t h i s h y p o t h e s i s . The mechanism by which H C O 3 " e x i t s the b a s o l a t e r a l c e l l membrane of the prox imal tubu le i s u n c l e a r . M i c r o e l e c t r o d e s t u d i e s i n r a t proximal tubu le have i n d i c a t e d the presence of a s i g n i f i c a n t b a s o l a t e r a l HC03~ conductance (Burckhardt and Fromter , 1980). S tud ies i n r a b b i t proximal tubu le suggest t ha t HC03~ e x i t s by both a conduc t i ve pathway and an e l e c t r o n e u t r a l C 1 " / H C 0 3 ~ exchange (Sasak i and B e r r y , 1983; see a l s o B i a g i , Kubota , S o h t e l l and G i e b i s c h , 1981) . Ex tens ive vo l tage and i o n - s e l e c t i v e m i c roe lec t r ode s t u d i e s by Boron and Boulpaep (1983a,b) have p rov ided a d e t a i l e d c e l l u l a r model o f H + and HC03~ t r a n s p o r t i n Ambystoma proximal t u b u l e . These i n v e s t i g a t o r s demonstrated t ha t N a + / H + exchangers were p resent on both the a p i c a l and b a s o l a t e r a l c e l l membranes. In a d d i t i o n , t h e i r r e s u l t s i n d i c a t e d the presence of an e l e c t r o g e n i c , Na + -dependent , b a s o l a t e r a l H C O 3 " e f f l u x which cou ld be cons ide red as the s imul taneous e x i t o f 1 N a + and 2 H C O 3 " on a c a r r i e r mechanism, or - 191 -the e l e c t r o d i f f u s i v e e x i t of an ion p a i r such as N a C C ^ - . Boron and Boulpaep (1983b) c l a i m t ha t the a p i c a l and b a s o l a t e r a l N a + / H + exchangers account f o r i n t r a c e l l u l a r pH r e g u l a t i o n and t ha t the b a s o l a t e r a l HCG^ -e f f l u x i s the asymmetric component of the system necessary f o r net t r a n s e p i t h e l i a l a c i d t r a n s p o r t . These i n v e s t i g a t o r s f u r t h e r c l a i m tha t u r ine a c i d i f i c a t i o n i s secondary to the need of the c e l l to regu la te i n t r a c e l l u l a r pH. I t must be no ted , however, t ha t prox imal tubu le u r i ne a c i d i f i c a t i o n has never been demonstrated i n Ambystoma. As such , t h i s model of a c i d e x c r e t i o n has been c r i t i c i z e d by mammalian renal p h y s i o l o g i s t s . In support o f these c r i t i c i s m s , I ves , Yee and Warnock (1983) have prov ided i n d i r e c t ev idence us ing renal v e s i c l e p repa ra t i ons which i n d i c a t e s tha t the N a + / H + exchanger i s con f i ned to the luminal membrane of r a b b i t prox imal t u b u l e . Mammalian c o l l e c t i n g tubu les are capable i n e i t h e r a c i d i f y i n g (McKinney and B u r g , 1977a, 1978a; Lombard; Jacobson and Kokko, 1979; R ichardson and Kunau, 1981; DuBose, 1982; Koeppen and Helman, 1982) or a l k a l i n i z i n g (McKinney and Burg , 1977a, 1978b) the f i n a l u r i ne depending upon the ac id -base s ta tus of the an ima l . The ac tua l mechanisms of H + and HC03~ t r anspo r t i n the c o l l e c t i n g tubu le are qu i t e complex and poor ly understood a t p resen t . Koeppen and Helman (1982; see a l s o L a s k i , Morgan and Kurtzman, 1983) have r e c e n t l y p rov ided good evidence f o r an a c t i v e , e l e c t r o g e n i c H + sec re to r y mechanism i n the r a b b i t c o r t i c a l c o l l e c t i n g t u b u l e . S tud ies on the mechanisms of c o l l e c t i n g tubu le H C O 3 " s e c r e t i o n have suggested very i n d i r e c t l y the presence of an e l e c t r o g e n i c H C O 3 " t r a n s p o r t and/or an e l e c t r o n e u t r a l C T / H C O 3 " exchange process (reviewed by Ber ry and Warnock, 1982) . I t i s i n t e r e s t i n g to note t ha t the t u r t l e u r i n a r y b ladder performs many of the same f u n c t i o n s as the c o l l e c t i n g tubu le and has been s tud ied - 192 -e x t e n s i v e l y as a model o f the d i s t a l nephron. The two major c e l l types of the t u r t l e b l adde r , g ranu la r and m i t o c h o n d r i a - r i c h c e l l s , are morpho log i ca l l y s i m i l a r to the p r i n c i p a l and i n t e r c a l a t e d c e l l s of the c o l l e c t i n g tubu le e p i t h e l i u m ( K a i s s l i n g and K r i z , 1979). R e c e n t l y , A l -Awqa t i (personal communication) has observed a popu la t i on of i n t r a c e l l u l a r v e s i c l e s i n the c o l l e c t i n g duct which s t a i n w i th a c r i d i n e orange i n a manner s i m i l a r to the H + pump-conta in ing v e s i c l e s i n the t u r t l e b ladder (Gluck e t a l _ . , 1982). Thus, both e p i t h e l i a may possess s i m i l a r mechanisms f o r r e g u l a t i n g u r i na ry a c i d i f i c a t i o n . As d i scussed i n Chapter I I I , the only i n v e r t e b r a t e e p i t h e l i u m i n which H + and H C 0 3 " t r a n s p o r t has been s tud ied i s the c rus tacean g i l l . Good ev idence i n d i c a t e s tha t a c i d and base e x c r e t i o n i n the b ranch ia l e p i t h e l i u m i s mediated by N a + / H + and C 1 " / H C 0 3 ~ exchange mechanisms (see K i r s c h n e r , Greenwald and K e r s t e t t e r , 1973; E h r e n f e l d , 1974; Pequeux and G i l l e s , 1981) , a l though f u r t h e r i n v e s t i g a t i o n s have not been p o s s i b l e because of the problems a s s o c i a t e d w i th s tudy ing t h i s t i s s u e i n v i t r o . D e t a i l e d c e l l u l a r mechanisms of H + and HC03~ t r a n s p o r t have, however, been s t u d i e d i n s i n g l e , n o n - e p i t h e l i a l i n v e r t e b r a t e c e l l s . Indeed, i n v e s t i g a t i o n s on the s n a i l neuron, squ id axon and barnac le muscle f i b e r have prov ided the bulk o f our c u r r e n t unders tanding of i n t r a c e l l u l a r pH r e g u l a t i o n . S tud ies by Thomas' group and Boron and co-workers ( reviewed by Thomas, 1980, 1982; Roos and Boron , 1981; Bo ron , 1983) have p rov ided s t rong ev idence f o r the presence of an a c i d e x t r u s i o n mechanism i n these c e l l s which requ i res H C O 3 " , ex te rna l N a + and i n t e r n a l C l " , and i s b locked almost comple te ly by SITS and o the r d i s t i l b e n e d e r i v a t i v e s such as DIDS and DNDS. A c c o r d i n g l y , i t has been pos tu la ted tha t the e l e c t r o n e u t r a l f l u x e s of N a + , HC03~, C l " and p o s s i b l y H + are a l l coupled on the same c a r r i e r . - 193 -Al though ve r t eb ra te i n t r a c e l l u l a r pH regu la to ry mechanisms have not been s tud ied as e x t e n s i v e l y as those i n i n v e r t e b r a t e c e l l s , i t i s worth no t ing t ha t the mechanisms so f a r proposed on ly s u p e r f i c i a l l y resemble those seen i n squ id axons, s n a i l neurons and barnac le muscle f i b e r s . In sheep c a r d i a c P u r k i n j i e f i b e r s , Vaughan-Jones (1979, 1982) has p o s t u l a t e d a Na + - i ndependen t , C T / H C C ^ " exchange i n t r a c e l l u l a r pH regu la to ry mechanism. A i c k i n and Thomas (1977) suggest t ha t a c i d e x t r u s i o n i n the mouse so leus muscle f i b e r i s mediated by p a r a l l e l N a + / H + and C T / H C O 3 " exchangers . C l e a r l y , f u r t h e r s tud ies need to be focused on the mechanisms of ve r teb ra te i n t r a c e l l u l a r pH r e g u l a t i o n . One of the major o b j e c t i v e s o f the work i n t h i s t h e s i s was to u t i l i z e the s a l t water mosquito l a r v a , Aedes dorsa l i s , as a model system i n which to study fundamental c e l l u l a r mechanisms of e p i t h e l i a l H + and HC0 3 ~ t r a n s p o r t . As such , t h i s i n v e s t i g a t i o n has prov ided the f i r s t d e t a i l e d study of ac i d -base r e g u l a t i o n i n an i n s e c t spec ies and the f i r s t study of c e l l u l a r mechanisms of H + and H C 0 3 " t r anspo r t i n an i n v e r t e b r a t e e p i t h e l i u m . Aedes dorsa l i s i s one of the only organisms capable of i n h a b i t i n g s a l t l akes composed a lmost e n t i r e l y o f high concen t ra t i ons of NaHC03 and Na2C03 s a l t s . Under these extreme a l k a l i n e c o n d i t i o n s the r e c t a l s a l t g land regu la tes e x t r a c e l l u l a r HC03~ concen t ra t i on and pH w i t h i n narrow p h y s i o l o g i c a l l i m i t s by e x c r e t i n g HC03~ a g a i n s t remarkably l a r g e e l e c t r o -chemical g r a d i e n t s . In v i t r o m ic rope r fus ion exper iments have demonstrated c l e a r l y t h a t a c t i v e HC03~ s e c r e t i o n i s l o c a t e d i n the a n t e r i o r segment o f the per fused s a l t g land and i s mediated by a 1:1 exchange of lumina l C l " f o r se rosa l H C O 3 " . S tud ies w i th ion and v o l t a g e - s e l e c t i v e m i c r o e l e c -t rodes and the an ion exchange i n h i b i t o r , DIDS, have shown tha t the - 194 -C T / H C C ^ " an t i po r t mechanism i s l oca ted a t the b a s o l a t e r a l c e l l membrane. C h l o r i d e ent ry i n t o the a n t e r i o r r e c t a l c e l l a t the a p i c a l membrane i s media ted, a t l e a s t i n p a r t , by p a s s i v e , e l e c t r o d i f f u s i v e movement of C l " through a C l " - s e l e c t i v e pathway or channe l . The e x i t of HCO^" from the c e l l i s f a c i l i t a t e d by an a c t i v e or p a s s i v e , e l e c t r o g e n i c H C O 3 " t r a n s p o r t mechanism. The c e l l u l a r model f o r a n t e r i o r segment HCC^" s e c r e t i o n shown i n F i gu re 5.19 most c l o s e l y resembles t ha t proposed f o r the HCG^" sec re to r y mechanism i n Amphiuma duodenum (d iscussed above) . White and Imon (1982) p o s t u l a t e d a b a s o l a t e r a l C T / H C O 3 " exchanger and a p a s s i v e , conduc t i ve HCG^" e f f l u x a t the a p i c a l membrane of the duodenal c e l l s . As i n the case f o r the r e c t a l s a l t g l and , C l " reabsorp t ion i n the duodenum i s mediated by an a p i c a l , e l e c t r o g e n i c C l " en t ry s t e p . In the duodenum, however, t h i s mechanism appears to be an a c t i v e C l " pump ra the r than e l e c t r o d i f f u s i v e C l " movement as shown f o r the a n t e r i o r r e c t a l s a l t g land i n Chapter V. Whi le the r e c t a l s a l t g land does not p rov ide a use fu l model system f o r i n v e s t i g a t i n g some presumably l e s s e a s i l y s tud ied e p i t h e l i u m , i t c l e a r l y p rov ides an e x c e l l e n t t i s s u e i n which to study bas i c membrane p h y s i o l o g i c a l p r o c e s s e s . The work i n t h i s t h e s i s has revea led two important areas which warrant f u r t h e r i n v e s t i g a t i o n . Luminal ion s u b s t i t u t i o n s t u d i e s i n the a n t e r i o r r e c t a l s a l t g land have shown tha t the a p i c a l membrane p o t e n t i a l i s p r i m a r i l y a C l " conductance which i s p a r t i a l l y s h o r t - c i r c u i t e d by an e l e c t r o g e n i c HCC^" t r a n s p o r t mechanism. As such , the p u t a t i v e , a p i c a l C l " channel o f the a n t e r i o r segment i s analogous to the N a + channel i n the N a + - s e l e c t i v e lumina l membranes of t i g h t , N a + reabsorb ing e p i t h e l i a . A l though ex tens ive - 195 -s t u d i e s have been conducted on c a t i o n - s e l e c t i v e channels from nerve , muscle and e p i t h e l i a l c e l l s , r e l a t i v e l y l i t t l e work has been done on a n i o n - s e l e c t i v e channe l s . White and M i l l e r (reviewed by M i l l e r , 1982) have s tud ied s i ng le - channe l cu r ren t s a t high t ime r e s o l u t i o n i n Torpedo e l e c t r o p l a x C l " channels r e c o n s t i t u t e d i n t o p lanar l i p i d membranes. These e legan t s tud ies have prov ided the f i r s t , and to da te , only d i r e c t exper imenta l ev idence e l u c i d a t i n g the mo lecu la r mechanisms of a C l " d i f f u s i o n pathway. Given the h igh C l " s e l e c t i v i t y of the a p i c a l membrane of a n t e r i o r r e c t a l c e l l s i t i s reasonable to suggest t ha t there i s a very h igh dens i t y o f lumina l C l " channels i n t h i s t i s s u e . Using m i c r o s u r g i c a l techn iques which I have developed dur ing the course of t h i s t h e s i s work, i t i s p o s s i b l e to expose the a p i c a l membrane to the se rosa l ba th ing medium and to remove the lumina l c u t i c l e . As such , the mechanisms and con t ro l of the a p i c a l C l " channels can f e a s i b l y be s tud ied us ing i s o l a t e d c e l l membrane patch-c lamp techn iques (see H a m i l l , Mar ty , Neher, Sakmann and S igwor th , 1981) . An area which has been of major i n t e r e s t to e p i t h e l i a l p h y s i o l o g i s t s i n recen t yea rs i s the nature o f the mechanisms by which e p i t h e l i a l c e l l s ma in ta in i n t r a c e l l u l a r homeostasis i n the face of r a p i d , p h y s i o l o g i c a l l y - i n d u c e d v a r i a t i o n s o f t r a n s e p i t h e l i a l ion and f l u i d t r a n s p o r t . The processes by which c e l l s con t ro l cy top lasmic compos i t ion have been termed i n t r i n s i c or homoce l lu la r r egu la to r y mechanisms ( S c h u l t z , 1981b). Very l i t t l e i s known about these mechanisms a t present and they represent an extremely important area of f u tu re e p i t h e l i a l r e s e a r c h . One of the most obvious ques t ions which a r i s e s from t h i s t h e s i s work i s how do the r e c t a l s a l t g land c e l l s regu la te i n t r a c e l l u l a r ac i d -base balance wh i l e s imu l taneous ly e f f e c t i n g a l a rge t r a n s c e l l u l a r C 0 2 f l u x and genera t ing remarkable t r a n s e p i t h e l i a l H C 0 3 " and CO^ g rad ien ts? Are the - 1 9 6 -b a s o l a t e r a l C T / H C C ^ " exchange and a p i c a l , e l e c t r o g e n i c HCC^" t r a n s p o r t e r i n v o l v e d i n i n t r a c e l l u l a r pH r e g u l a t i o n , or does the c e l l possess a d d i t i o n a l ion t r a n s p o r t e r s such as N a + / H + exchangers f o r c o n t r o l l i n g ac i d -base balance? I f these separate mechanisms e x i s t , how are they c o n t r o l l e d ? Such ques t ions are of obvious importance i n a l l a c i d -base t r a n s p o r t i n g e p i t h e l i a , e s p e c i a l l y those which u t i l i z e e l e c t r o g e n i c H + and H C O 3 " t r a n s p o r t mechanisms. The homogeneous nature of the r e c t a l s a l t g land e p i t h e l i u m , the l a rge s i z e of the s a l t g land c e l l s and the a b i l i t y to i s o l a t e t h i s organ comple te ly i n v i t r o makes i t an e x c e l l e n t system i n which to answer these types of q u e s t i o n s , p a r t i c u l a r l y when compared to more s t r u c t u r a l l y complex t i s s u e s such as the pancreas , g a s t r i c mucosa, t u r t l e b ladder and c o l l e c t i n g t u b u l e . - 197 -REFERENCES A i c k i n , C . C . and Thomas, R .C . (1977). An i n v e s t i g a t i o n o f the i o n i c mechanism of i n t r a c e l l u l a r pH r e g u l a t i o n i n mouse so leus muscle f i b e r s . J . P h y s i o l . 273: 295-316. 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