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Charge properties and ion selectivity of the rectal intima of the desert locust Lewis, Simon Andrew 1971

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CHARGE PROPERTIES AND ION SELECTIVITY OF THE RECTAL INTIMA OF THE DESERT LOCUST. by SIMON ANDREW LEWIS B . S c , U n i v e r s i t y of B r i t i s h Columbia, 1970. A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE i n the 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 September, 1971. In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the Head of my Department or by his representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of ~£ oo\o 2V The University of British Columbia Vancouver 8, Canada Date ^ j U * ^ ^ W » i ABSTRACT The r e c t a l i n t i m a of the desert l o c u s t was found to possess f i x e d negative charges, r a t h e r than f i x e d n e u t r a l s i t e s . I t was sug-gested that the molecular species r e s p o n s i b l e f o r the negative s i t e s might be a c i d i c amino a c i d s . The s e l e c t i v e p e r m e a b i l i t y of the i n t i m a as estimated from d i f -+2 +2 +2 +2 f u s i o n p o t e n t i a l s , f o r d i v a l e n t c a t i o n s was 3a > Ca > Sr > Mg > Mn +^, f o r monovalent c a t i o n s was NH^ + > Rb + > C s + > K + > N a + > L i + > TEA + and f o r monovalent anions was HC03~ > CN~ > F~ > NO^" > CL~ > CH3COO~ > Br~ > H2P04~ > I ~ . C a t i o n a f f i n i t y f o r the f i x e d charged +2 ++ + + s i t e was found to be i n the order of Ca > Mg » K > Na . S i m i l a r i t y of e f f e c t s of pH and i o n c o n c e n t r a t i o n on streaming and d i f f u s i o n p o t e n t i a l s i n d i c a t e d that i o n movement and water flow might take p l a c e through the same route. The i n t i m a was found to act as an osmotic compartment such that at h i g h e x t e r n a l osmotic p r e s s u r e s , the r a t e of water flow was r e -duced due to a shrinkage of the e f f e c t i v e pore s i z e i n the i n t i m a , however the r e l a t i v e p e r m e a b i l i t y of ions d i d not seem e f f e c t e d by membrane dehydration. U n s t i r r e d l a y e r s at the membrane-solution i n t e r f a c e s were found to have a minimal e f f e c t on d i f f u s i o n p o t e n t i a l s , however h a l f of the value f o r streaming p o t e n t i a l s was found to be due to a d i f f u s i o n p o t e n t i a l caused by an i o n c o n c e n t r a t i o n d i f f e r e n c e i n opposing u n s t i r r e d l a y e r s . i i Calcium -45 f l u x across the i n t i m a at pH 5.5 ( i . e . p o s s e s s i n g f i x e d charge) was found to be 81 times g r e a t e r , a t a c o n c e n t r a t i o n of 10 mM/l CaCl^j than c a l c i u m f l u x at the same co n c e n t r a t i o n across the uncharged membrane (pH 2.2). The same e f f e c t was not s i g n i f i c a n t f o r rubidium. Conversely, the removal of f i x e d charge enhanced anion f l u x . Calcium permeation r a t e was found to be a f u n c t i o n of i t s d i s s o c i a t i o n r a t e from the f i x e d charge and d i d not c o r r e l a t e i n a simple manner w i t h the membrane b i n d i n g c a p a c i t y f o r calcium. A trans e f f e c t on c a l c i u m f l u x was a l s o found i n the i n t i m a and i s b e l i e v e d to be a f u n c t i o n of the d i s s o c i a t i o n r a t e of c a l c i u m from the f i x e d negative s i t e . I t was concluded t h a t electro-osmosis was not the mode of water movement across the rectum, however p h y s i o l o g i c a l advantage of e l e c t r o - o s m o s i s was di s c u s s e d . F l u x experiments p o s s i b l e i n d i c a t e t h a t the i n t i m a might be the r a t e l i m i t i n g step f o r K + r e a b s o r p t i o n i n a hydrated animal. i i i TABLE OF CONTENTS TITLE PAGE INTRODUCTION 1 MATERIALS AND METHODS M a t e r i a l s 13 Methods A. IN VITRO p r e p a r a t i o n of the r e c t a l c u t i c l e . 13 B. P e r f u s i o n chambers 13 C. P r e p a r a t i o n of S o l u t i o n s 16 D. A n a l y t i c a l Methods 17 1. Recording system f o r measuring p o t e n t i a l d i f f e r e n c e s 17 2. E s t i m a t i o n of p e r m e a b i l i t y from Radio-isotope f l u x 18 3. Membrane a n a l y s i s and F i x e d charge d e n s i t y 19 4. E l e c t r i c a l Resistance 19 CHAPTER I - PROPERTIES OF THE FIXED CHARGE I n t r o d u c t i o n 21 Re s u l t s A. Membrane Resistance 22 B. Anion: C a t i o n P e r m e a b i l i t y as a Function of Concentration 23 C. Estimate of the Concentration of Fi x e d Charges i n the Intima 24 D. Amino A c i d A n a l y s i s of C u t i c u l a r P r o t e i n 25 i v TITLE PAGE Discussion 26 CHAPTER I I - PARAMETERS INFLUENCING POTENTIAL DIFFERENCE ACROSS THE INTIMA. Introduction 31 Results A. R e l a t i v e permeability of the intima to monovalent and d i v a l e n t cations and monovalent anions 33 B. Competition between ions f o r f i x e d negative s i t e s 35 C. Re l a t i v e permeability of the intima as measured by streaming p o t e n t i a l s 37 D. E f f e c t of pH on membrane p o t e n t i a l s 38 E. E f f e c t s of high osmotic pressure on d i f f u s i o n p o t e n t i a l s 39 F. E f f e c t s of osmotic gradient on streaming p o t e n t i a l s 39 G. Ion concentration e f f e c t on streaming and d i f f u s i o n p o t e n t i a l s 42 H. Possible e f f e c t s of u n s t i r r e d layers on membrane p o t e n t i a l s 44 I. Additive and c a n c e l l i n g properties of streaming and d i f f u s i o n p o t e n t i a l s 50 Discussion 52 CHAPTER I I I - ION FLUX ACROSS THE INTIMA. Introduction 63 Results A. Ion fluxes at various concentrations and pH values 65 B. Membrane binding capacity 67 C. Trans e f f e c t of the intima 68 D i s c u s s i o n SUMMARY APPENDIX A APPENDIX B LITERATURE CITED ACKNOWLEDGEMENTS I would l i k e to thank Dr. P h i l l i p s f o r having me as a graduate student i n h i s Laboratory, and s i n c e r e thanks f o r h i s many h e l p f u l d i s c u s s i o n s d uring the course of the research and p r e p a r a t i o n of t h i s T h e s i s . I must express many thanks to Dr. V. P a l a t y f o r help i n de t e r -mining the number of a c i d i c groups i n the i n t i m a and f o r h i s thorough rea d i n g and c r i t i c i s m of t h i s T h e s i s . I am indebted to Dr. I . Tayl o r f o r conducting the amino a c i d a n a l y s i s of the p r o t e i n o f the r e c t a l i n t i m a , and a l s o f o r h i s c r i t i c a l r e a d i n g of t h i s T h e s i s . I would l i k e to thank the members of my Masters Committee f o r t h e i r suggestions d u r i n g the course of the rese a r c h , and a l s o my f e l l o w students i n the Laboratory. A very s p e c i a l thanks goes out to Bet t y Coldbeck and Bob Schroeder f o r the t y p i n g and p r o o f r e a d i n g of t h i s Thesis on such s h o r t n o t i c e . v i i GLOSSARY A - area of the membrane s u r f a c e A g - r a d i o a c t i v i t y per u n i t volume of c o l l e c t e d p e r f u s a t e Ap - r a d i o a c t i v i t y per u n i t volume of e x t e r n a l medium C - c o n c e n t r a t i o n of t e s t molecule D - d i e l e c t r i c constant E - p o t e n t i a l d i f f e r e n c e (Eo - E i ) AE 1/2 - h a l f maximal p o t e n t i a l d i f f e r e n c e AE 0 0 - maximal steady s t a t e p o t e n t i a l AEo - i n i t i a l p o t e n t i a l d i f f e r e n c e b efore s t i r r i n g F - Faraday constant H - p o t e n t i a l d i f f e r e n c e developed i n streaming p o t e n t i a l s i - cu r r e n t flow d u r i n g electro-osmosis J v - volume flow of water i n electro-osmosis I J - t o t a l volume flow of p e r f u s a t e i n time t K i ' - K + c o n c e n t r a t i o n on i n s i d e of membrane. K i " - K + c o n c e n t r a t i o n on ou t s i d e of membrane. K' - K + c o n c e n t r a t i o n on i n s i d e of membrane before s t i r r i n g . K i , C l i , X i - a c t i v i t i e s of s o l u t i o n on s i d e one of the membrane. Ko, C l o , Xo - a c t i v i t i e s of s o l u t i o n on s i d e two of the membrane, k - s p e c i f i c c o n d u c t i v i t y i n r e c i p r o c a l s t a t ohms, k' - u n i t s i n r e c i p r o c a l mv. Om - o s m o l a r i t y i n musocal b a t h i n g s o l u t i o n . Os - o s m o l a r i t y i n s e r o s a l b a t h i n g s o l u t i o n . v i i i P - osmotic d i f f e r e n c e causing osmotic pressure. P i - membrane p e r m e a b i l i t y to i o n i . pHb - pH of pore l i q u i d . pHs - pH of b a t h i n g s o l u t i o n . R - gas constant. Ro' - r e s i s t a n c e to water flow w i t h u n i t s of m osm/mv. t - time. 6 - t h i c k n e s s of u n s t i r r e d l a y e r , n - c o e f f i c i e n t of v i s c o s i t y of water i n p o i s e s . <J> - f l u x (meles/unit time) a) - p e r m e a b i l i t y c o e f f i c i e n t . £ - z e t a p o t e n t i a l . INTRODUCTION The desert l o c u s t , being a t e r r e s t r i a l l n s e c t has to be able to conserve water and regulate body s a l t s . This i s achieved by the mal-pighian t u b u l e - r e c t a l system (reviewed by Stobbart and Shaw, 1964). The malpighian tubules (259 i n number i n the l o c u s t Shistocerca gregaria  f o r s k a l ; Savage, 1956) produce a primary f i l t r a t e , which i s isosmotic to the haemolymph (Ramsay, 1953). The primary f i l t r a t e i s formed by the a c t i v e s e c r e t i o n of K + i n t o the lumen of the tubule thus forming a electrochemical gradient and osmotic pressure d i f f e r e n c e whereby water, e l e c t r o l y t e s andronelectrolytes flow i n t o the tubule lumen down t h e i r respective gradients (Ramsay, 1956). This primary f i l t r a t e passes down the malpighian tubule, through the hindgut and i n t o the rectum where s e l e c t i v e reabsorption of e s s e n t i a l metabolites, ions and water occurs i n the required amounts (Reviewed by Stobbard and Shaw, 1964). The reabsorptive processes include a c t i v e transport of C l , Na +, K + ( P h i l l i p s , 1964, b)'and amino acids (Balshin and P h i l l i p s , 1971). Net water absorption i s independent of simultaneous movement of solute across the r e c t a l w a l l as a whole and can lead to formation of a hyperosmotic urine. These s e l e c t i v e reabsorptive processes are regulated i n response to changes i n blood concentration ( P h i l l i p s , 1964 a,b,c). The rectum consists of an e p i t h e l i a l l a y e r and a c u t i c u l a r layer ( i . e . the chitinous intima) separating the r e c t a l pad c e l l s from the f e c a l m a terial present i n the r e c t a l lumen ( F i g . 1). Noirot and Noirot-Timithee (1966) studied the u l t r a s t r u c t u r e of the r e c t a l c u t i c l e i n i n s e c t s . The e l e c t r o n microsopic studies showed that i n general the i n t e s t i n a l c u t i c l e of Cephalotermes rectangularis F i g . 1 A l i g h t m i c r o s c o p i c p i c t u r e of the cross s e c t i o n of the l o c u s t rectum where: P - r e c t a l pad c e l l s . I - Intima. 2 SI (termite) v a r i e d from one segment to another. The e p i c u t i c l e appears to be greatly reduced to a very t h i n s u p e r f i c i a l l a y e r , and also to a dense uniform layer corresponding to the c u t i c u l i n ( C h i t i n - p r o t e i n complex as described by Locke, 1964). The e p i c u t i c l e according to Weis-Fogh (1970) i s t o t a l l y devoid of c h i t i n . The fliickness of the e p i c u t i c l e v a r i e s from 200-1800 A*. The p r o c u t i c l e which i s defined as ex o c u t i c l e and endocuticle i s even more v a r i a b l e . D i r e c t l y below the e p i c u t i c l e , Noirot (1966) observed s e v e r a l lamellae made up of a fiberous c h i t i n p r o t e i n complex, which i s often replaced by dense granules. Histo chemical studies with A l c i a n blue (Stoward, 1967 a,b, c,d) suggests that a c i d mucopolysaccharides e x i s t i n the p r o c u t i c l e . They found that mucopolysaccharide s t a i n i n g i n t e n s i t y decreased as the pH decreased which i n d i c a t e s that the mucopolysaccharides are moderately a c i d i c due to carboxyl groups. The layer of the p r o c u t i c l e s t a i n i n g f o r a c i d i c mucopolysaccharides can be many microns t h i c k , and using d i f f e r e n t s t a i n i n g techniques , i t was shown to possess very f i n e and i r r e g u l a r l y anastomosing filaments. A histochemical and electromicros-copic study of the e p i c u t i c l e using hexamethylene-tetramine s i l v e r , i n d i c a t e s without doubt the presence of polyphenols. An a l t e r a t i o n of the above technique also showed s i l v e r grains i n the procuticle, again i n d i c a t i n g a c i d i c mucopolysaccharides (Noirot and Noirot-Timithee, 1966). The r e c t a l c u t i c l e has been shown ( P h i l l i p s and D o c k r i l l , 1968) to act as a molecular s i e v e , allowing by s e l e c t i v e reabsorption a ra p i d exchange of water and small solute molecules between the lumen and e p i t h e l i a l c e l l s . The r e l a t i v e impermeability of the intima to large 3 organic molecules caused the l a t t e r to accumulate i n the rectum as a consequence of f l u i d r e c i r c u l a t i o n through the malpighian t u b u l e - r e c t a l system. P h i l l i p s (1964 b) found that the net rate of reabsorption of K +, Na + and C l was regulated by the s a l t concentration of the haemolymph. In s a l i n e fed locusts a maximum rate of transport or net reabsorption was noticed, the curve following t y p i c a l Michaelis-Menten k i n e t i c s . S a l t depleted locusts do not e x h i b i t the Michaelis-Menten k i n e t i c s , and the rate of reabsorption increased i n a l i n e a r fashion f o r the three ions i n v e s t i g a t e d . Since i n s a l t depleted animals no Michaelis-Menten k i n e t i c s were obvious, then one of the composite parts of the rectum ( c u t i c u l a r l a y e r or epithelium) must be rate l i m i t i n g . P h i l l i p s (1964 b) found that the a d d i t i o n a l absorption of C l i n s a l t depleted over s a l i n e fed animals (at high concentrations) could be a t t r i b u t e d to a large d i f f u s i o n component. I f t h i s was true f o r the other ions, then r e g u l a t i o n of absorption could involve a c o n t r o l of passive perme-a b i l i t y of ions rather than changed i o n a f f i n i t i e s and concentration of c a r r i e r s i n the r e c t a l pad c e l l s . The p h y s i c a l b a s i s of s e l e c t i v i t y by the intima was thought to be the presence of water f i l l e d pores with apparent radius of 6.5 A* and l i n e d with f i x e d negative charges. The former was postulated be-cause the r e l a t i v e rate of pen t e t r a t i o n of h y d r o p h i l i c non-ionic molecules depends on t h e i r hydrated s i z e ( P h i l l i p s and D o c r i l l , 1968) as p r e d i c t e d by Renkin (1954) equation. Supporting evidence f o r t h i s hypothesis includes ( P h i l l i p s and D o c k r i l l , 1968): (a) f a i l u r e of molecules with r a d i i greater than 6 X to penetrate the membrane, 4 (b) osmotic p e r m e a b i l i t y t w e n t y - f i v e times greater than t h a t estimated by i s o t o p i c d i f f u s i o n of t r i t i a t e d water (suggesting t h a t water move-ment occurs by laminar flow through pores) and (c) l a c k of a c o r r e l a t i o n between p e r m e a b i l i t y and l i p i d s o l u b i l i t y of s o l u t e s . F i x e d negative charges were f i r s t p o s t u l a t e d when p r e l i m i n a r y measurements of i o n f l u x e s across the i n t i m a suggested that p e r m e a b i l i t y +2 to c a t i o n s ( e s p e c i a l l y Ca ) might be higher than p r e d i c t e d by the Renkin equation based on previous observations w i t h uncharged molecules. - -2 Anion p e n e t r a t i o n (CI and PO ^)has been measured ( P h i l l i p s , unpublished o b s e r v a t i o n s ) and found to conform to the p a t t e r n f o r uncharged molecules. In the i n t i m a one or a l l of the three l a y e r s could c o n t a i n the p o s t u l a t e d f i x e d charges: ( i ) the e p i c u t i c l e composed e n t i r e l y of p r o t e i n , ( i i ) the t h i n l a y e r of p r o c u t i c l e compoxed of a c h i t i n - p r o t e i n complex or ( i i i ) the deeper l a y e r i n the p r o c u t i c l e thought to be composed of a a c i d i c mucopolysaccharide. Ion permeation i n porous membranes i s governed by a s e r i e s of f a c t o r s : ( i ) the a v a i l a b l e pore ar e a , ( i i ) t o r t u o s i t y i n the pores, and such s p e c i f i c i n t e r a c t i o n s of the ions w i t h the pore w a l l s as ( i i i ) i o n -s i t e i n t e r a c t i o n ( i v ) i o n - m a t r i x i n t e r a c t i o n (v) s o l v e n t drag and pos-s i b l y ( v i ) s t r u c t u r a l and chemical changes of the membrane that are dependent on the f u n c t i o n a l s t a t e of the i o n exchange. According to the f i x e d charge theory of porous charged i o n i c membranes ( T e o r e l l , 1953) the pore w a l l s i n h e r e n t l y c a r r y a c e r t a i n number of a n i o n i c d i s s o c i a b l e ( a c i d i c ) groups, or c a t i o n i c d i s s o c i a b l e ( b a s i c ) groups. The permeant species act as c o u n t e r - i o n s , which e f f e c t i v e l y n e u t r a l i z e the f i x e d charge of the s i t e s . 5 Eisenman, et a l (1967) c l a s s i f i e d fixed s i t e s into two groups: (i ) fixed dissociated, i n which the pores are so wide that the i n t e r -action between ions i n the pores and fixed charges are n e g l i g i b l e , ( i i ) f ixed associated, here an arbitrary maximal diameter of 10 $ i s given for the pore, and under these conditions a l l . membrane water i s i n a bound state. In the fixed dissociated state only pore area, tortuosity and solvent drag are of importance i n determining ion permeation. The co-ion and counter-ion content i s dependent on a Donnan equilibrium with the external solutions. Current voltage relationships are characterized by free solution m o b i l i t i e s , and are a function of the charge density. Ion permeation i s in s e n s i t i v e to swelling and shrink-ing of the membrane. The most distinguishing feature of a fixed dissociated system i s the p o s s i b i l i t y of solvent drag and e l e c t r o k i n e t i c phenomena. The f l u i d i n the pores contain more ions of charge opposite that of the fixed charge, giving the f l u i d i n the pore a net charge. An applied water flow w i l l then sweep out the f l u i d containing the excess charges thereby creating a p o t e n t i a l difference across the membrane which i s called a streaming p o t e n t i a l . As pore size narrows i o n - s i t e and ion-matrix interactions become of greater importance. Specific interactions between the mobile species and the s i t e s a l t e r the mob i l i t i e s and standard chemical potentials from those found i n aqueous solutions. Upon decreasing the pore diameter a stage i s reached where there i s no free water, thus eliminating solvent drag while pore area and tortuosity become unimportant. Donnan boundary 6 c o n d i t i o n s are repl a c e d by boundary c o n d i t i o n s c h a r a c t e r i s t i c of an i o n exchange e q u i l i b r i u m i n whfch i n c r e a s e d s e l e c t i v i t y from amongst ions of the same charge become apparent. Membrane s t r u c t u r e at s m a l l pore diameters can be a l t e r e d by pressure g r a d i e n t s o r volume changes as a f u n c t i o n of the concentra-t i o n of a p a r t i c u l a r counter-ion species a t a given p o i n t . Since m o b i l i t i e s and chemical gradients are l i k e l y to be s e n s i t i v e to the degree of h y d r a t i o n , a s t r o n g dependence of m o b i l i t i e s on the con-c e n t r a t i o n p r o f i l e s of the counter-ions i s to be expected. (Eisenman, et a l . 1967). The a c t u a l mode of i o n permeation of m o b i l i t y through a charged membrane i s not known, but one of the acceptable t h e o r i e s o f f e r e d i s tha t each c o u n t e r - i o n i n a pore once attached to a f i x e d charge w i l l swing f r e e l y about t h i s charge (Shean and S o l l n e r (1966) c a l l t h i s an o s c i l l a t i o n c e l l ) by thermal motion. The d i r e c t i o n of motion i s dependent upon the c o n c e n t r a t i o n gradients so that t h i s f o r c e w i l l t h e n move the i o n from one o s c i l l a t i o n c e l l to another. This w i l l occur i f the i o n can o b t a i n s u f f i c i e n t k i n e t i c energy to jump out of i t s absorp-t i o n s i t e , the mean f r e e path a l s o b e i n g r e s t r i c t e d by the dense crowding of water molecules. Because of the energy requirements a second system of m i g r a t i o n i s o f f e r e d by L i n g (1962). I f a second counter-ion approaches the d i p o l a r p a i r ( i . e . f i x e d charge p l u s primary counter-ion) an e q u i l i b r i u m s t a t e might be reached i n which both counter-ions are changed to a h i g h e r c o n f i g u r a t i o n r e s u l t i n g i n l e s s k i n e t i c energy b e i n g r e q u i r e d to desorb. H a n i (1970) adds to t h i s by suggesting that i f i o n movement c o n s i s t s of jumps from one s i t e to an adjacent vacant s i t e , 7 the permeation r a t e of the i o n w i l l be determined by the a c t i v a t i o n energy of the jump and the p r o b a b i l i t y that an adjacent s i t e i s vacant, vacancies a l s o b e i n g formed by ions moving from a f i x e d s i t e to an i n t e r s t i t i a l p o s i t i o n . A change i n m o b i l i t y w i t h a change i n the membrane composition might be due to the change i n the number of vacancies i n the membrane; ions being more mobile i n a membrane which contains more vacancies. P a r t i a l or complete co-ion e x c l u s i o n by a charged membrane i m p l i e s a s e l e c t i v i t y by the membrane. Such a s e l e c t i v i t y under proper c o n d i t i o n s would l e a d to membrane p o t e n t i a l s . I f one s i d e of the membrane contains a higher c o n c e n t r a t i o n of an e l e c t r o l y t e there w i l l be an i o n flow from h i g h c o n c e n t r a t i o n to low c o n c e n t r a t i o n r e s u l t i n g i n a " d i f f u s i o n p o t e n t i a l " , (due to co-ion e x c l u s i o n ) a s t e a d y - s t a t e p o t e n t i a l b e i n g reached when co-ion e x c l u s i o n i s balanced by the a t t r a c t i v e f o r c e of the t o t a l membrane p o t e n t i a l . T o t a l p o t e n t i a l across an i o n exchange membrane i n v o l v e s 3 d i s -c r e t e components, two at the s o l u t i o n membrane i n t e r f a c e (Donnan p o t e n t i a l s ) and one through the membrane ( d i f f u s i o n p o t e n t i a l ) . The i n t e r f a c i a l p o t e n t i a l s are determined by s e l e c t i v i t y of the f i x e d charges. The d i f f e r e n c e between i n t e r f a c i a l p o t e n t i a l s i s d e f i n e d and determined by the concentrations on each s i d e and by the s e l e c t i v i t y f a c t o r , w h i l e the d i f f u s i o n p o t e n t i a l (h the membrane) i s determined by the i o n m o b i l i t y . H a n i (1970) a f t e r measuring membrane s e l e c t i v i t y , m o b i l i t y and e l e c t r o n e g a t i v i t y i n a b i - i o n i c c e l l , found no c o r r e l a t i o n between 8 s e l e c t i v i t y and t o t a l trans-membrane p o t e n t i a l nor between m o b i l i t y and p o t e n t i a l and consequently concluded that d i f f u s i o n p o t e n t i a l s are determined by both s e l e c t i v i t y and m o b i l i t y . I n general terms the f l u x of an i o n species i n s i d e the membrane i s p r o p o r t i o n a l to i t s m o b i l i t y and to the d i f f e r e n c e i n e l e c t r o c h e m i c a l a c t i v i t y between the membrane i n t e r f a c e s ( T e o r e l l , 1953). The i n t e r n a l m o b i l i t y of counter-ions through the membrane i s a f u n c t i o n of the d i s -s o c i a t i o n r a t e of the counter-ion from the f i x e d charge and the p r o b a b i l i t y that an adjacent f i x e d s i t e i s vacant. On the assumption th a t c o u n t e r - i o n m o b i l i t y i s g r e a t e r through a charged membrane than through a s i m i l a r uncharged membrane, the Donnan e q u i l i b r i u m has the a b i l i t y to concentrate the mobile i o n species and w i l l i n c r e a s e the f l u x p r o p o r t i o n a t e l y to the f i x e d charge d e n s i t y ( t h i s i s only v a l i d i f the f i x e d charge d e n s i t y i s much grea t e r than the e x t e r n a l con-c e n t r a t i o n ) . Eisenman's theory (Eisenman, 1962) p r e d i c t s the r e l a t i v e s e l e c t i v i t y to c a t i o n s o f a membrane c o n t a i n i n g f i x e d negative charges. The theory was t e s t e d and confirmed on n o n - b i o l o g i c a l m a t e r i a l ( i . e . glass e l e c t r o d e s ) ; only r e c e n t l y has i t been a p p l i e d to b i o l o g i c a l m a t e r i a l (membranes and a c t i v e b i n d i n g s i t e s on enzymes; Diamond and Wright, 1969). The b a s i c f a c t o r s determining s p e c i f i c i t y of a f i x e d charge f o r ions i s the f r e e energies of i n t e r a c t i o n of the ca t i o n s w i t h water and w i t h the f i x e d a n i o n i c s i t e s i n the membranes. These i n t e r a c t i o n s of 9 cation-water and c a t i o n - s i t e w i l l c o n t r o l : ( i ) the e q u i l i b r i u m i o n exchange p r o p e r t i e s of the membrane, (consequently the phase boundary p o t e n t i a l s ) , ( i i ) the a c t i v a t i o n energies which govern c a t i o n m i g r a t i o n from the s o l u t i o n i n t o the membrane and ( i i i ) the m i g r a t i o n of the c a t i o n i n the pore regions of the membrane. The competing i n t e r a c t i o n s of c a t i o n s w i t h the membrane versus water can be represented by an i o n exchange r e a c t i o n : I (memb.) + J (aqueous) J (memb.) + I (aqueous) + A The dependence of the f r e e energy change,A F^j° °f t n e above r e a c t i o n can be w r i t t e n : o _ - hyd. - memb. - memb. - memb. A F i j " F I + " F J + + F J + " F I + where F^^ 1^" symbolizes the p a r t i a l m o l a l f r e e energy of h y d r a t i o n of the i o n 1+ w h i l e ^ ^ a e i^ 3' represents the p a r t i a l m olal f r e e energy of 1+ w i t h the membrane. Therefore the i o n which undergoes the great e s t f r e e energy change from i t s hydrated s t a t e to the bound s t a t e w i l l be s e l e c t e d over an i o n w i t h a s m a l l e r p o s i t i v e f r e e energy change. I n more general terms then, c a t i o n s which can most c l o s e l y approach a f i x e d charge w i l l be p r e f e r e n t i a l l y bound, and w i l l cross the membrane by r a p i d s i t e to s i t e t r a n s f e r (as on an i o n exchange column). The closeness of approach of the ions i s dependent on the e l e c t r o s t a t i c f i e l d s t r e n g t h of the f i x e d charge and the a f f i n i t y of each i o n f o r i t s s h e l l of water molecules (energy of h y d r a t i o n ) . A f i x e d charge w i t h f i e l d s t r e n g t h g r e a t e r than a l l the h y d r a t i o n energies of the ions concerned w i l l p r e f e r e n t i a l l y b i n d ions according 10 + + + + + to i n c r e a s i n g dehydrated s i z e of the ions ( L i > Na > K > Rb > Cs "f*2 | ^  j ^  and Mg > Ca > Sr > Ba ). F i x e d charges w i t h f i e l d s t r e n g t h i n t e r - m e d i a t e between Hydration energies of va r i o u s ions w i l l have other s e l e c t i v e sequences, the t o t a l number i s 13 f o r the f i v e a l k a l i n e metal c a t i o n s and seven f o r the f o u r a l k a l i n e e a r t h d i v a l e n t c a t i o n s . The f i e l d s t r e n g t h i s the p r i n c i p l e f a c t o r governing the sequence of s e l e c t i v i t y among the monovalent c a t i o n s ; however the sequence of s e l e c t i v i t y among the d i v a l e n t c a t i o n s can be s h i f t e d at constant f i e l d s t r e n g t h w i t h a change i n the s i t e s p a c i n g . I f the s i t e spac-i n g i s i n c r e a s e d the s e l e c t i v i t y p a t t e r n w i l l move from the sequence where the i o n w i t h the s m a l l e s t i o n i c r a d i u s i s p r e f e r r e d to one i n which the i o n w i t h the l a r g e s t i o n i c r a d i u s i s s e l e c t e d (Eisenman, 1962). The degree of s w e l l i n g (hydration) of a membrane w i l l not a f f e c t the s e l e c t i v i t y sequence but w i l l a f f e c t the magnitude of s e l e c t i v i t y between d i f f e r e n t c a t i o n s . P r o c u n i e r (1966) i n p r e l i m i n a r y experiments report e d t h a t stream-i n g p o t e n t i a l s t h a t were developed across the r e c t a l i n t i m a of the l o c u s t were n e g l i g i b l e i n s i z e , thus throwing some doubt on the ' f i x e d charge' hy p o t h e s i s . This discrepancy between hypothesis and experimental r e s u l t s was r e - i n v e s t i g a t e d by Lewis (1970). I n i t i a l experiments u s i n g 1 0 - f o l d KCI c o n c e n t r a t i o n g r a d i e n t s showed that the r e c t a l c u t i c l e was c a t i o n s e l e c t i v e . This s e l e c t i v i t y immediately suggested that the p o s t u l a t e d pores i n the i n t i m a c o n t a i n f i x e d negative s i t e s . These nega-t i v e s i t e s were shown to be s e l e c t i v e l y permeable to 5 monovalent c a t i o n s , the 11 s e l e c t i v i t y corresponding to Eisenman's second sequence (Rb > Cs > K + > N a + > L i + ) . The p e r m e a b i l i t y r a t i o P / P , as c a l c u l a t e d from d i f f u s i o n K u l p o t e n t i a l s i s 10/1, t h i s p e r m e a b i l i t y r a t i o was shown to decrease to u n i t y as the pH of the b a t h i n g s o l u t i o n was lowered from 5.5 to a value 2.3. S e l e c t i v i t y was a l s o lowered by i n c r e a s i n g concertration. (Lewis, 1970). Streaming p o t e n t i a l s developed when osmotic gradients were a p p l i e d across the i n t i m a u s i n g sucrose. This i s c o n s i s t e n t w i t h the p o s t u l a t e d negative f i x e d charges being l o c a t e d on the w a l l s of the membrane pore. As the average osmotic pressure was i n c r e a s e d , the streaming p o t e n t i a l would decrease, t h i s phenomena of n o n - l i n e a r osmosis was p o s t u l a t e d to be caused by the pores a c t i n g as osmotic compartments. U n t i l r e c e n t l y , s e l e c t i v e p e r m e a b i l i t y of a membrane to i o n s , was assumed to be caused by a f i x e d charge and the system l i k e n e d to a i o n exchange membrane; the evidence f o r t h i s assumption was i n c o n c l u s i v e . The f i r s t p o i n t to be c l a r i f i e d i n t h i s study was whether the s e l e c t i v i t y of the i n t i m a f o r c a t i o n over anions was due to a f i x e d charged s i t e as i n a i o n exchanger; or whether the s e l e c t i v i t y was determined by a f i x e d n e u t r a l s i t e as proposed by B a r r y , Diamond and Wright (1971) f o r the r a b b i t g a l l - b l a d d e r . The i d e n t i f i c a t i o n of f i x e d s i t e type was based on d i s t i n g u i s h i n g c r i t e r i a o f f e r e d by Wright, Barry and Diamond (1971). The p o s s i b l e membrane components r e s p o n s i b l e f o r i o n s e l e c t i v i t y were a l s o considered. 12 The composition and volume of the primary f i l t r a t e e n t e r i n g the rectum w i l l vary depending on the p h y s i o l o g i c a l s t a t e of the animal. Such v a r i a b l e f a c t o r s as, i o n c o n c e n t r a t i o n , i o n r a t i o s , pH, osmotic pressure and net water f l o w , might a f f e c t membrane s e l e c t i v i t y by a l t e r i n g the charge or membrane s t r u c t u r e . I t was t h e r e f o r e of p h y s i o l o g i c a l importance to study the e f f e c t s of these v a r i a b l e s on the i n t i m a (Chapter 2 ) . The response of the i n t i m a to these v a r i o u s per-t u r b a t i o n s a l s o allowed a comparison to be made between behavior of t h i s membrane and v a r i o u s current models f o r f i x e d charged membranes. +2 P h i l l i p s (unpublished observation) found that the f l u x o f Ca across the i n t i m a was 50 times g r e a t e r than f o r sucrose, even though both chemical s p e c i e s have the same hydrated s i z e . This o b s e r v a t i o n provoked three questions: ( i ) to what extent does the membrane f i x e d charge a f f e c t permeation of ions of d i f f e r e n t charge and valency through +2 the membrane? ( i i ) Can the p r e v i o u s l y observed h i g h f l u x r a t e f o r Ca be completely a t t r i b u t e d to the f i x e d charge? ( i i i ) What i s the +2 r e l a t i o n s h i p between b i n d i n g of Ca i n the i n t i m a and the f l u x r a t e of the i o n across the l a t t e r membrane? These questions are considered i n Chapter 3. 13 METHODS AND MATERIALS M a t e r i a l s Mature male l o c u s t s , S c h i s t o c e r c a g r e g a r i a f o r s k a l , were used i n a l l experiments. The o r i g i n a l stock was obtained from the A n t i - l o c u s t Research Center, London, and the animals were bred i n cages a t 28°C and 50% r e l a t i v e h umidity, on a d i e t of bran and l e t t u c e , at the Department of Zoology, U n i v e r s i t y of B r i t i s h Columbia. Methods A) IN VITRO p r e p a r a t i o n of the r e c t a l i n t i m a . The l o c u s t s were a n a e s t h e t i z e d w i t h CO^, the extremeties removed so t h a t the exoskeleton of the abdominal r e g i o n could be s l i t up the mid-d o r s a l l i n e . The rectum was l o c a t e d and s l i t open p o s t e r i o r to a n t e r i o r , and a l l f e c a l m a t e r i a l removed. The rectum then was cut at the a n t e r i o r and p o s t e r i o r ends, l i f t e d from the body, and p l a c e d i n d i s t i l l e d water f o r 4 to 5 hours to l y s e the r e c t a l pad c e l l s and to p a r t i a l l y detach the t i s s u e l a y e r s from the i n t i m a . The p r e p a r a t i o n was then removed from the d i s t i l l e d water and the t i s s u e l a y e r s removed u s i n g needle p o i n t forceps under a b i n o c u l a r microscope. The i n t i m a was checked f o r major damage m i c r o s c o p i c a l l y a f t e r being r e p l a c e d i n the water bath. B) P e r f u s i o n Chambers The p e r f u s i o n chambers f o r p o t e n t i a l measurements were made of persex, c o n t a i n i n g 3 v e r t i c a l w e l l s opening a t the top and j o i n e d by a t r a v e r s e channel which opened at the s i d e ( F i g . 2 ) . An overflow tube F i g . 2 Schematic diagram of apparatus used to measure the d i f f u s i o n and streaming p o t e n t i a l s across the r e c t a l i n t i m a . 1. Voltmeter 2. P e r f u s i o n apparatus 3. Calomel e l e c t r o d e s 4. Overflow tubes 5. P o s i t i o n of the r e c t a l i n t i m a 6. Rubber '0' r i n g Enlarged view of the membrane p o s i t i o n i n the p e r f u s i o n b l o c k s . 6 5 14 was l o c a t e d i n the upper s e c t i o n of the l a s t w e l l . Two of these cham-bers f i t together so th a t the o r i f i c e s of the transverse spaces were opposed. The membrane, mounted on ruber "0" r i n g s , separate the t r a n s -verse spaces of the two chambers. A t i g h t s e a l was f a c i l i t a t e d by the rubber "0" r i n g s . The two h a l f - c e l l s w i t h the i n t i m a between were h e l d together in a bench v i s e . I n i n i t i a l experiments, s t i r r i n g of s o l u t i o n i n the c e l l s was achieved u s i n g c a l i b r a t e d b u r e t t e tubes. S o l u t i o n was allowed to flow from the l a t t e r i n t o both chambers. The f l u i d was d e f l e c t e d by the membrane up the l a s t v e r t i c a l w e l l and out the overflow tube. This flow p a t t e r n would cause laminar flow at the membrane s u r f a c e , p o s s i b l y r e s u l t i n g i n an inadequate s t i r r i n g due to a s m a l l dead space next to the membrane. This method of s t i r r i n g was l a t e r replaced by a second. Two "LKB" v a r i a b l e speed p e r i s t a l t i c pumps were used to d i r e c t a flow of f l u i d through t e f l o n t u b i n g , the o r i f i c e of which was p l a c e d w i t h i n 2 mm o f , and p e r p e n d i c u l a r t o , the membrane. The flow of f l u i d was thus d i r e c t l y a g a i n s t the membrane sur f a c e e l i m i n a t i n g the laminar flow produced by the b u r e t t e s . The p e r i s t a l t i c pump rat e s could be v a r i e d from 0.45 to 2.8 ml/minute. These r a t e s were s u f f i c i e n t to maintain a constant c o n c e n t r a t i o n of s o l u t i o n s on both s i d e s of the membrane i n the presence of e i t h e r i o n co n c e n t r a t i o n or osmotic g r a d i e n t s . This was checked u s i n g a c o n d u c t i v i t y meter (Lewis, 1970). Two types of p e r f u s i o n chambers were used f o r measurements of is o t o p e f l u x ( F i g . 3 ) . One s e t of bl o c k s were made us i n g t e f l o n , the other s et out of persex. T e f l o n was used because of i t s i n e r t n e s s to a c i d i c s o l u t i o n s . The r a d i o - i s o t o p e was contained i n a s i n g l e v e r t i c a l w e l l (2.6 cm i n diameter, 3.0 cm i n depth) h o l d i n g 14 ml of s o l u t i o n . F i g . 3 Schematic diagram of the apparatus used f o r measurements of r a d i o a c t i v e t r a c e r f l u x across the i n t i m a . 1. A i r hose f o r s t i r r i n g s o l u t i o n . 2. P e r f u s i o n tube. 3. P o s i t i o n of r e c t a l i n t i m a . 4. C o l l e c t i n g tube f o r p e r f u s a t e . 5. C o l l e c t i n g v i a l f o r p e r f u s a t e . 15 The s i n g l e v e r t i c a l w e l l was j o i n e d to a sh o r t transverse space which opens to the s i d e ( F i g . 3 ) . The other type of chamber had two channels of 3 mm diameter, j o i n i n g p e r p e n d i c u l a r l y to form a s m a l l t r a n s v e r s e space opening to the s i d e . These two chambers f i t t e d together so that the aperture of the tra n s v e r s e space i n the two chambers were apposed. They were h e l d i n t h i s p o s i t i o n by a bench v i s e . The membrane f i t s between these two chambers s e p a r a t i n g the transverse spaces. A t i g h t s e a l and simple mount f o r the membrane was f a c i l i t a t e d by rubber "0" r i n g s . R a d i o a c t i v e f l u i d (5 m i l l i l i t e r s ) was p l a c e d i n the s i n g l e v e r t i c a l w e l l , by s y r i n g e , to i n s u r e t h a t no a i r bubbles form over the membrane s u r f a c e . The r a d i o a c t i v e s o l u t i o n was s t i r r e d by b u b b l i n g a i r i n the v i c i n i t y of the membrane. The top of the chamber was s e a l e d , u s i n g "Scotch Brand Tape", to prevent s p l a s h i n g of i s o t o p e caused by bu b b l i n g . A P o r t a b l e i n f u s i o n withdrawal pump ("Model 1100" Harvard Apparatus Company) w i t h a d e l i v e r y r a t e of 0.042 ml/minute was used to perfuse n o n - r a d i o a c t i v e s o l u t i o n through the second w e l l past the mem-brane. To achieve t h i s , the s y r i n g e was connected to the v e r t i c a l channel by a l e n g t h of P.E. 90 tu b i n g . The tub i n g proceeded down the channel and was f i x e d i n such a p o s i t i o n that i t would discharge the n o n - r a d i o a c t i v e s o l u t i o n d i r e c t l y onto the top of the membrane. The s o l u t i o n then p i c k e d up any r a d i o - i s o t o p e l e a v i n g the membrane and flowed along the other channel to be c o l l e c t e d i n a v i a l which was changed every h a l f hour to provide s e r i a l samples. 16 To check f o r submicroscopic t e a r s i n the i n t i m a , a f t e r b e i n g mounted i n one of the above p e r f u s i o n chambers, the dye amaranth ( P h i l l i p s , 1964 a) was p l a c e d on one s i d e of the membrane and l e f t f o r 12 hours. I f there was no submicroscopic damage, the dye d i d not appear on the opposite s i d e . Previous experiments i n d i c a t e d t h a t the p r o p e r t i e s of the i n t i m a were not a l t e r e d s i g n i f i c a n t l y by t h i s type of p r e p a r a t o r y technique ( P h i l l i p s and D o c k r i l l , 1968; Lewis, 1970). C) P r e p a r a t i o n of S o l u t i o n s The s a l i n e s o l u t i o n s ( u s u a l l y of s i n g l e s a l t s ) were made to the d e s i r e d c o n c e n t r a t i o n s , and the pH adjusted (to pH 5.5 unless o t h e r -wise sta t e d ) u s i n g 9 N HC1. Some experiments i n v o l v i n g i o n concentra-t i o n g r a d i e n t s r e q u i r e d t h a t s o l u t i o n s be i s o m o t i c ; to achieve t h i s an a p p r o p r i a t e amount of sucrose ( c a l c u l a t e d from p u b l i s h e d values of m o l a l f r e e z i n g p o i n t depressions; Handbook of Chemistry and P h y s i c s , 1968-69) was added to the s i d e w i t h low s a l t c o n c e n t r a t i o n . To study streaming p o t e n t i a l s i n the absence of i o n c o n c e n t r a t i o n g r a d i e n t s , osmotic gradients were created by adding sucrose at the d e s i r e d f i n a l c o ncentrations to stock s a l i n e s o l u t i o n s . R a d i o - i s o t o p e s , i n the chemical forms and at s p e c i f i c a c t i v i t i e s shown below, were purchased from New England Nuclear (Canada Ltd.) and added to the a p p r o p r i a t e stock s o l u t i o n s of the same compound. These s o l u t i o n s were s t o r e d at -20° ^ (to a v o i d b a c t e r i a l contamination). 17 Isotope Chemical Form S p e c i f i c A c t i v i t y Urea 4.7 mc/mM Ca 45 350 mc/mM C l 36 KCI Rb 86 RbCl 2.22 mc/mM D) A n a l y t i c a l Methods 1. Recording System f o r measuring P o t e n t i a l D i f f e r e n c e s . A "Radiometer" pH meter (model 25 w i t h expanded s c a l e ) and two calomel e l e c t r o d e s ( c o n t a i n i n g s a t u r a t e d KCI s o l u t i o n ) which were plac e d immediately adjacent and on both s i d e s of the membrane, were used to measure membrane p o t e n t i a l d i f f e r e n c e s . The p o t e n t i a l s were recorded every two minutes to i n s u r e a steady s t a t e p o t e n t i a l was a t t a i n e d . I n i n i t i a l experiments, measurements of membrane p o t e n t i a l under s e l e c t e d c o n d i t i o n s were repeated f o r two to three hours per day f o r three days, to t e s t the change i n p e r m e a b i l i t y p r o p e r t i e s of the i n t i m a w i t h time. No s i g n i f i c a n t change occurred over a p e r i o d of two days (Lewis, 1970). When a continuous time course of membrane p o t e n t i a l development was needed, the elect r o m e t e r was coupled to a "Photovolt Movel 43" l i n e a r / l o g . paper chart r e c o r d e r . The d i f f u s i o n p o t e n t i a l d i f f e r e n c e caused by i o n c o n c e n t r a t i o n g r a d i e n t s were used to determine the r e l a t i v e p e r m e a b i l i t y of the i n t i m a to monovalent and d i v a l e n t c a t i o n s and monovalent anions u s i n g a modi f i e d form of the Goldman constant f i e l d equation ( H a n i , 1970): 18 (Equation 1) E = RT/F l n Pk ( k i ) + Pel (Clo) + 4m Px i ( X i ) Pk (ko) + PCI ( C l i ) + 4m Px + 2o(xo + 2)exp.(-FE/RT WHERE: (Equation 2) m= 1-exp. (-FE/RT) 1-exp. (-2FE/RT) Assumptions and d e r i v a t i o n of t h i s equation are o u t l i n e d i n Appendix A. 2. E s t i m a t i o n of p e r m e a b i l i t y from Radio-Isotope F l u x . R a d i o a c t i v e samples (0.5 ml) from f l u x s t u d i e s , depending on t h e i r emission type were e i t h e r p l a c e d on a pl a n c h e t , d r i e d and subsequently counted on a Nuclear Chicago Model 4318 planchet counter; or the sample 14 35 (C , SO^ ) was t r a n s f e r r e d to 10 ml of Bray's s o l u t i o n (Bray, 1960) and counted i n a "Nuclear Chicago Mark 1" L i q u i d s c i n t i l l a t i o n counter. Standards f o r each r a d i o - i s o t o p e were made by p i p e t t i n g 0.5 ml of c o l d s o l u t i o n i n t o a planchet or s c i n t i l l a t i o n v i a l and adding to t h i s one lambda of the r a d i o a c t i v e s o l u t i o n . The r a d i o a c t i v i t y of the c o l l e c t e d p e r f u s a t e was below 2% of t h a t i n the r a d i o a c t i v e s o l u t i o n from which d i f f u s i o n occured; hence back d i f f u s i o n of the is o t o p e across the membrane was n e g l i g i b l e . The f l u x of the t e s t molecules was then c a l c u l a t e d u s i n g the f o l l o w i n g equation (modified from Shaw, 1955): (Equation 3) $ = A ZJC The p e r m e a b i l i t y can be c a l c u l a t e d from the f l u x r a t e ( f ) by F i c k ' s law (Davson, 1964): 19 (Equation 4) $ = oaAC To ensure that a steady f l u x value had been reached, f l u x rates were recorded at h a l f hour i n t e r v a l s f o r 4 hours. F i g . 4 i l l u s t r a t e s that 45 a f t e r the f i r s t h a l f hour period, the f l u x rate of Ca had reached a constant value. Thereafter f l u x values were determined a f t e r 1 hour pre-incubation i n radio-isotopes. 3. Membrane Analysis and Fixed charge Density. The l o c u s t c u t i c l e was hydrolysed i n 2ml of 6 N HCl, containing 6% V/V t h i s g y l o c o l i c a c i d (Matsubara and Sasaki, 1969) f o r 18 hours at 105 i n vacuo. The sample hydrolysate was sealed i n vacuo and analyzed using a "Beckman" 120C Automatic Amino Acid A n a l y s i s . The number of f i x e d negative charges was determined using a "Radiometer Copenhagen" T i t r i g r a p h . Six Milligrams of r e c t a l c u t i c l e were prepared as o u t l i n e d i n Section A and then placed i n a large volume of 0.5 N HCl, and then centrifuged 6 times i n d i s t i l l e d water. The membranes were then shredded using needle point forceps, and added to 1 ml of d i s t i l l e d water. This preparation was then t i t r a t e d with carbonate free NaOH i n the presence of a nitrogen atmosphere. 4. E l e c t r i c a l Resistance. The resistance of the membrane was measured using calomel e l e c t -rodes connected to the electrometer and placed on both sides of the membrane to measure p o t e n t i a l differences as previously described. A set of s i l v e r wire electrodes were used to apply a v a r i a b l e current across the membrane from two dry c e l l b a t t e r i e s (3 v o l t s ) i n s e r i e s with a K e i t h l e y ammeter (Model 601) and v a r i a b l e resistance (Heathkit Decade / F i g . 4 45 Time course of Ca f l u x across the r e c t a l i n t i m a . Each p o i n t represents counts per minute i n the constant volume of p e r f u s a t e that was sampled at h a l f hour i n t e r v a l s . 200 -I a U 150 i o cu +-> q 100 -4 u o a CQ -t-> « o 50 0 0 " T 2 T i m e i n h o u r s 20 Resistance Model IN-17). The current was v a r i e d and the r e s u l t i n g poten-t i a l d i f f e r e n c e across the membrane (bathed on both s i d e s w i t h the same KC1 or CaCl^ s o l u t i o n of v a r i o u s m o l a r i t i e s ) was measured. The i n t i m a was then cut and the above procedure repeated. By s u b t r a c t i o n of the r e s i s t a n c e of the r e c o r d i n g system alone, from the r e s i s t a n c e of the membrane plu s the r e c o r d i n g system, the membrane r e s i s t a n c e was c a l c u -l a t e d u s i n g Ohm's law. 21 CHAPTER I PROPERTIES OF THE FIXED CHARGE INTRODUCTION Before i n v e s t i g a t i n g the e f f e c t s of v a r i o u s f a c t o r s of p h y s i o -l o g i c a l importance on i o n permeation across the l o c u s t i n t i m a , i t was u s e f u l t o determine whether i o n s e l e c t i v i t y of t h i s membrane was due to a f i x e d charge system (with p r o p e r t i e s c l o s e l y approximating those of an i o n exchange r e s i n ) o r whether the i n t i m a possessed f i x e d n e u t r a l s i t e s . I d e n t i f i c a t i o n of the type of s i t e a l l o w d i f f e r e n t p r e d i c t i o n s to be made. C r i t e r i a have been suggested by Wright, Barry and Diamond (1971) f o r d i f f e r e n t i a t i n g between f i x e d charges and f i x e d n e u t r a l s i t e s . These c r i t e r i a i n c l u d e the nature of the r e l a t i o n s h i p between i o n con-c e n t r a t i o n of the b a t h i n g media and both the membrane r e s i s t a n c e and the P v : P - r a t i o . Both these r e l a t i o n s h i p s were considered i n t h i s chapter Jv O JL f o r the l o c u s t i n t i m a . The p o s s i b l e chemical nature of the f i x e d charges was then considered. The approach was to f i r s t determine the pK of the i n t i m a by t i t r a t i o n f o r comparison w i t h known values of pK f o r va r i o u s r a d i c a l s (eg. amino acid s i n i n t a c t p r o t e i n ) . Assuming the p r o b a b i l i t y that the f i x e d charge was a s s o c i a t e d w i t h the p r o t e i n of the i n t i m a , an amino a c i d a n a l y s i s of the l a t t e r was c a r r i e d out to determine i f an excess of an amino a c i d w i t h the r e q u i r e d pK value was present. 22 RESULTS A. Membrane Resistance H e l f f e r i c h (1962) has shown th a t i n an i o n exchange membrane the c o n d u c t i v i t y of the exchanger bed i s independent of the i o n concentra-t i o n when the b a t h i n g s o l u t i o n c o n c e n t r a t i o n i s l e s s than the concentra-t i o n of f i x e d s i t e s . Wright, Barry and Diamond (1971) r e i t e r a t e d t h i s p o s t u l a t e and a l s o added that i f the membrane contained f i x e d n e u t r a l s i t e s the conductance of the membrane should be l i n e a r l y r e l a t e d to i o n c o n c e n t r a t i o n r e g a r d l e s s of the co n c e n t r a t i o n range. With these p r e d i c t i o n s i n mind, the conductance of the i n t i m a was measured as the c o n c e n t r a t i o n of the b a t h i n g s o l u t i o n (both s i d e s ) was i n c r e a s e d from 0.1 to 1000 mM/l CaC^. The i n t i m a seemodto act as an i o n exchanger r a t h e r than a membrane w i t h f i x e d n e u t r a l s i t e s ( F i g . 5) because at low i o n concentrations (0.1 to 10 mM/l CaCl^) the conductance i s r e l a t i v e l y independent of the i o n co n c e n t r a t i o n ( i . e . a very s m a l l decrease i n r e s i s t a n c e of 3 - f o l d f o r a 100-fold i n c r e a s e i n i o n concentration) . At high i o n concentrations (1000 mM/l CaC^) , the r e s i s t a n c e of the i n t i m a does not f a l l i n p r o p o r t i o n to decrease i n the i o n c o n c e n t r a t i o n . At high c o n c e n t r a t i o n s , the membrane might have reached a s a t u r a t i o n c a p a c i t y f o r the i o n , such that any i n c r e a s e i n i o n c o n c e n t r a t i o n of the e x t e r n a l s o l u t i o n would not r e s u l t i n an i n -crease of the membrane i o n c o n c e n t r a t i o n . When the above experiment was repeated using a KCI s o l u t i o n ( F i g . 5 ) , a l i n e a r r e l a t i o n s h i p was observed between r e s i s t a n c e and co n c e n t r a t i o n (except below 1 mM/l KCI). This might i n d i c a t e that the F i g . 5 The s o l i d c i r c l e s represent the r e l a t i o n s h i p between KC1 c o n c e n t r a t i o n of the b a t h i n g s o l u -t i o n and membrane r e s i s t a n c e . The s o l i d squares represent the r e l a t i o n s h i p between the ba t h i n g s o l u t i o n c o n c e n t r a t i o n of CaCl^ and the membrane r e s i s t a n c e . See Appendix B Table 8 f o r i n d i v i d u a l v a l u e s . 5 -, W -J , 1 1 1 0.1 1 10 100 1000 Concentration of bathing solution in m M / l 23 K i n the membrane e x i s t s i n two phases, one a s s o c i a t e d w i t h a f i x e d charge, and the other i n bulk s o l u t i o n . Such a system according to Eisenman (1967) c o n s t i t u t e s a "Fixed D i s s o c i a t e d System" i n which the c o n d u c t i v i t y i s a f u n c t i o n of the path of l e a s t r e s i s t a n c e . The d i f -+2 fe r e n t r e s u l t s w i t h Ca might be r e l a t e d to i t s l a r g e r hydrated radius (only s l i g h t l y s m a l l e r than the pore s i z e ) so that i t i s e s s e n t i a l l y only i n one phase, i . e . a s s o c i a t e d w i t h a charge ( i . e . F i x e d A s s o c i a t e d system of Eisenman, et a l . , 1967) except at very high c o n c e n t r a t i o n s . I t w i l l be seen i n Chapter 3 that the f i x e d charge of the i n t i m a has a +2 + s e l e c t i v e a f f i n i t y f o r Ca r e l a t i v e to K . The c o n d u c t i v i t y measure-ments at low i o n concentrations i n a f i x e d a s s o c i a t e d system would be dependent on the f i x e d charge c o n c e n t r a t i o n . B. Anion: Cation p e r m e a b i l i t y r a t i o as a f u n c t i o n of c o n c e n t r a t i o n . The e f f e c t of i o n co n c e n t r a t i o n on c a t i o n versus anion s e l e c t i v i t y , provides a t e s t of the p o s s i b i l i t y that the r e c t a l i n t i m a behaves as a i o n exchange membrane as described by H e l f f e r i c h (1962) and T e o r e l l (1953). T e o r e l l (1953) showed that d i f f u s i o n p o t e n t i a l s were dependent on the average concentrations of the bathing s o l u t i o n s ; i . e . as the average c o n c e n t r a t i o n i n c r e a s e d the d i f f u s i o n p o t e n t i a l decreased . Wright, Barry and Diamond (1971) s t a t e that a l i n e a r conductance - concentra t i o n curve should r e s u l t i n a constant p c l / p k r a t i o r e g a r d l e s s of the ba t h i n g s o l u t i o n c o n c e n t r a t i o n both of these phenomena being i n d i c a t i v e of a f i x e d n e u t r a l s i t e . F i g . 6 / The p e r m e a b i l i t y r a t i o P ^ /P^ as a f u n c t i o n of KC1 c o n c e n t r a t i o n . Average KC1 concentration in mM/l 24 D i f f u s i o n p o t e n t i a l s were measured a t a constant c o n c e n t r a t i o n r a t i o (10:1 KC1) w h i l e the average c o n c e n t r a t i o n of the e x t e r n a l b a t h i n g s o l u t i o n was v a r i e d . The p e r m e a b i l i t y r a t i o P ^ /P^ was c a l c u l a t e d ( F i g . 6) u s i n g the Goldman-Hodgkin-Katz constant f i e l d equation (see Appendix A ) . The P ^ /P^ r a t i o was dependent on the average concentra-t i o n of KC1, such t h a t the r a t i o approached u n i t y as the average con-c e n t r a t i o n i n c r e a s e d . This experiment i n d i c a t e s that the i n t i m a acts as a f i x e d negative s i t e r a t h e r than a f i x e d n e u t r a l s i t e . C. Estimate of the Concentration of F i x e d Charges i n the Intima. An estimate of the number of f i x e d charges and the apparent pKa and p i of the f i x e d charges i n the i n t i m a was obtained d i r e c t l y by performing an acid-base t i t r a t i o n on the i n t i m a . The p i value f o r the f i x e d charges was found to be 2.4, w h i l e the pKa value was estimated to be 4.8 (using the method of H e l f f e r i c h , 1962). An estimate of the number of f i x e d charges was a l s o made ( H e l f f e r i c h , 1962) by p l o t t i n g the pH change versus the amount of N a + t i t r a t e d ( F i g . 7). The c a p a c i t y of the membrane and thus the number of a c i d i c groups per u n i t weight i s equal to the amount of t i t r a n t added up to the p o i n t of i n f l e c t i o n on the t i t r a t i o n curve. The charge d e n s i t y of the i n t i m a was estimated i n t h i s manner to be approximately 0.045 micro-equivalents of charge per m i l l i g r a m (dry weight) of i n t i m a . +2 Another estimate of f i x e d charge d e n s i t y was obtained from Ca s o r p t i o n isotherms. The membranes were bathed i n 1 and lOmM/1 CaCl^ +2 at pH 2.3 and 5.5 To o b t a i n the amount of Ca present i n the membrane i n the bound form at a pH of 5.5, the assumption was made that the amount F i g . 7 pH t i t r a t i o n curve of the r e c t a l i n t i m a . 5 1 25 ' e'o ' 120 ' lfeo ' 2bo microequivalents NaOH per gram of tissue Table 1 The b i n d i n g c a p a c i t y of the r e c t a l i n t i m a +2 f o r Ca was measured f o r 6 i n t i m a prepara-t i o n s . The intimas were bathed i n 10 mM/l or AS 1 mM/l Ca l a b e l l e d C a C l 2 s o l u t i o n s at pH 5.5 or 2.3. Membranes were counted i n a planchet counter. Solution Concentration of C a C l 2 10 mM/l 1 mM/l pH 5.5 0.0678 i 0.018 0.0371 1 0.0065 uM Ca + 2/mg ± S.D. ( intima Dry Weight) pH2.3 Bound C a + 2 0.025 ±0.0028 0.0428 ± 0.011 0.0026*0.0008 0.0345 ± 0.007 % water - S.D. pH 5.5 pH 2.3 66.8 t 2.5 71.7* 2.5 25 of Ca present i n the membrane at t h i s pH was i n two s t a t e s , one being a s s o c i a t e d w i t h a f i x e d charge, the other b e i n g f r e e i n the pore f l u i d +2 (the amount of f r e e Ca being the amount found i n the membrane at a +2 pH of 2.3). The d i f f e r e n c e i n Ca content at the two pH values was +2 then the amount of bound Ca . Table 1 shows that the amount of bound +2 Ca at c o n c e n t r a t i o n of 1 and 10 mM/l C a C l 2 are 0.034 and 0.0428 +2 micromoles o f Ca per m i l l i g r a m dry weight of i n t i m a r e s p e c t i v e l y . Both of these values agree w e l l w i t h the number of charges found by acid-base t i t r a t i o n . D. Amino A c i d A n a l y s i s of C u t i c u l a r P r o t e i n . In an attempt to i d e n t i f y the chemical groups which might be r e s p o n s i b l e f o r the observed f i x e d charge i n the i n t i m a , the amino a c i d composition of the i n t i m a was determined. The r e s u l t s are given i n Table 2. The t o t a l amino a c i d content was approximately 57.7% by weight (dry) of the membrane. The most l i k e l y p o t e n t i a l source of negative charges were the d i c a r b o x y l i c amino a c i d s . The i n t i m a con-t a i n e d 567 nanomoles of f l u t a m i c a c i d and 370 nanomoles of a s p a r t i c a c i d per m i l l i g r a m dry weight of i n t i m a . On the other hand, the con-c e n t r a t i o n s of the three b a s i c amino acid s observed, l y s i n e , a r g i n i n e and h i s t i d i n e , were 218, 198 and 167 nanomoles per m i l l i g r a m dry weight of the i n t i m a r e s p e c t i v e l y . The t o t a l c o n c e n t r a t i o n of a c i d i c amino acids (937 nanomoles per m i l l i g r a m of intima) t h e r e f o r e exceeds the t o t a l c o n c e n t r a t i o n of b a s i c amino a c i d s (586 nanomoles p e r m i l l i g r a m of i n t i m a ) by 351 nanomoles per m i l l i g r a m of i n t i m a . There i s , i n c o n c l u s i o n , an excess of negative groups i n c u t i c u l a r p r o t e i n which might be the source of the observed f i x e d charge p r o p e r t i e s . Table 2 Amino a c i d a n a l y s i s of the r e c t a l i n t i m a . Amino Aci d Amino Acid classification Glycine Neutral Glutamic Acidic Proline Imino Aspartic Acidic Tyrosine Aromatic Arginine Basic Lysine Basic Threonine Neutral Isoleucine Neutral Serine Neutral Leucine Neutral Histidine Basic Phenylalanine Aromatic Valine Aromatic Quantity of Amino A c i d mg/gm of cuticle nm/gm of cuticle 94 1644 73 567 57 588 42.6 371 32.4 199 30.8 198 27.8 218 25.8 245 24.8 220 24.2 279 23.8 211 23 168 20 136 17 173 26 DISCUSSION Using the c r i t e r i a proposed by Wright, Barry and Diamond (1971) i t i s p o s s i b l e to decide whether the f i x e d s i t e i s e i t h e r , n e u t r a l ( i . e . a d i p o l e w i t h one charged end a s s o c i a t e d w i t h the pore w h i l e the other end i s e f f e c t i v e l y b u r i e d i n the matrix) or charged ( i . e . a s i t e which has to be a s s o c i a t e d w i t h a mobile counter-ion so as to maintain e l e c t r o n e u t r a l i t y ) . The experimental observations which d i s t i n g u i s h between these two p o s s i b i l i t i e s are: 1) the r e l a t i o n s h i p between mem-brane conductance and i o n c o n c e n t r a t i o n of the medium, and 2) the degree of change of P ^ /P^ r a t i o as a f u n c t i o n of KCI c o n c e n t r a t i o n . C o n s i d e r i n g the f i r s t r e l a t i o n s h i p , Wright et a l . (1971) found f o r the g a l l b l a d d e r , t h a t the conductance i n c r e a s e d l i n e a r l y w i t h i o n c o n c e n t r a t i o n of the medium f o r both of the monovalent s a l t s t e s t e d . They a l s o found a s i m i l a r r e l a t i o n s h i p f o r r a b b i t i n t e s t i n e and f r o g c h o r o i d p l e x u s . These r e s u l t s c o n t r a s t w i t h those f o r i o n exchange r e s i n s . I n a simple case of an i o n exchanger where the only c o n d u c t i v i t y i s through the pores, ( t o t a l l y d i s r e g a r d i n g the p o s s i b l e conductance pathways through the bed of the i o n exchanger; H e l f f e r i c h , 1962) the c o n d u c t i v i t y i s almost independent of the e x t e r n a l i o n c o n c e n t r a t i o n except at h i g h e r c o n c e n t r a t i o n s . Since the i o n exchanger must remain e l e c t r i c a l l y n e u t r a l , there w i l l always be r e s i d e n t counter-ions present i n the membrane to equal i n c o n c e n t r a t i o n the number of f i x e d s i t e s , r e g a r d l e s s of the e x t e r n a l c o n c e n t r a t i o n . The c o n d u c t i v i t y i n the exchanger w i l l not change u n t i l the e x t e r n a l c o n c e n t r a t i o n exceeds that 27 of the f i x e d charge c o n c e n t r a t i o n . In the case of f i x e d n e u t r a l s i t e s , however, the s i t e s can mainta i n e l e c t r o n e u t r a l i t y without engaging c o u n t e r - i o n s , thus the i o n c o n c e n t r a t i o n i n the pore w i l l c l o s e l y approximate that found i n the e x t e r n a l s o l u t i o n . The r e s u l t was t h a t c o n d u c t i v i t y was t o t a l l y dependent on the e x t e r n a l i o n c o n c e n t r a t i o n . Two types of concentration-conductance curves were observed i n the present study. When the bat h i n g media c o n s i s t e d of a KC1 s o l u t i o n , the curve was approximately l i n e a r over most of the co n c e n t r a t i o n range used ( i . e . above 1 mM/l). When CaCl^ s o l u t i o n s were used, however, the r e s i s t a n c e of conductance was r e l a t i v e l y independent of i o n concentra-t i o n a t low s a l t c o ncentrations but becomes more dependent on concentra-t i o n as the C a C ^ c o n c e n t r a t i o n i n c r e a s e d , p o s s i b l y i n d i c a t i n g a s a t u r a -t i o n of a membrane charge. The C a C ^ conductance seemed to i n d i c a t e a f i x e d charge system w h i l e the KC1 conductance f i t t e d a n e u t r a l s i t e model (or uncharged pore model), except at very low co n c e n t r a t i o n s . Turning to the second c r i t e r i o n ( i . e . the P ^ /P r a t i o ) , Wright e t a l . (1971) found only a s l i g h t change i n t h i s r a t i o (0.05 per con-c e n t r a t i o n change o f 100 mM/l) w i t h an i n c r e a s e i n the average concentra-t i o n of the b a t h i n g s o l u t i o n s when the con c e n t r a t i o n r a t i o across the g a l l bladder was kept constant. The very s l i g h t change i n the r a t i o observed was i n s u f f i c i e n t to produce a s i g n i f i c a n t n o n - l i n e a r i t y i n conductance-concentration curves. By c o n t r a s t , the r a t i o P . /P f o r an i o n exchanger w i l l vary c o n s i d e r a b l y as the t o t a l i o n c o n c e n t r a t i o n i s changed, w h i l e m a i n t a i n -i n g a constant c o n c e n t r a t i o n r a t i o ( T e o r e l l , 1953). This can be ex-p l a i n e d by c o n s i d e r i n g the membrane i n two phases, both v a r i a b l e and 28 dependent on the i o n c o n c e n t r a t i o n . When the membrane charge d e n s i t y i s much grea t e r than the e x t e r n a l i o n c o n c e n t r a t i o n , the p o t e n t i a l d i f f e r e n c e i s almost completely determined by aDonnan e q u i l i b r i u m p o t e n t i a l which f o r a 1 0 - f o l d gradient approaches 58 mv at 20°. When the co n c e n t r a t i o n of the membrane charge i s i n s i g n i f i c a n t compared to the e x t e r n a l i o n c o n c e n t r a t i o n , the Donnan e q u i l i b r i u m p o t e n t i a l d i m i n i s h e s and the poten-t i a l d i f f e r e n c e i s determined by a d i f f u s i o n p o t e n t i a l . The P /P r a t i o f o r the l o c u s t i n t i m a was observed to be dependent on the average c o n c e n t r a t i o n of the bathing media ( F i g . 6 ) . A 100 mM/l change i n average c o n c e n t r a t i o n of KCI i n the ba t h i n g media e l i c i t e d a change of 0.25 i n the p e r m e a b i l i t y r a t i o . This i m p l i e d a s i g n i f i c a n t decrease i n c a t i o n conductance o r an in c r e a s e i n anion con-ductance, or both, w i t h i n c r e a s i n g s a l t c o n c e n t r a t i o n of the media. The magnitude of t h i s p e r m e a b i l i t y change should be s u f f i c i e n t to produce a s i g n i f i c a n t n o n - l i n e a r i t y change i n the conductance-concentration r e l a t i o n s h i p . This experiment agrees w i t h r e s u l t s expected f o r a f i x e d charged membrane. A t h i r d l i n e of evidence i n d i c a t e , the f i x e d chargewa S not a +2 n e u t r a l s i t e . The amount of bound Ca i n the intimawas s i m i l a r f o r 1 and 10 mM/l C a C ^ media (Table 1 ) . That i s , i n order to maintain +2 e l e c t r o n e u t r a l i t y , the membrane c o n c e n t r a t i o n of Ca tended to be +2 independent of the e x t e r n a l Ca l e v e l . Such e l e c t r o n e u t r a l i t y i n d i c a t e d a f i x e d charge s i t e r a t h e r than a f i x e d n e u t r a l s i t e . Assuming t h a t the f i x e d charge was a s s o c i a t e d w i t h the p r o t e i n moiety i n the c u t i c l e , the former could be due to e i t h e r the fr e e c a r b o x y l or amino groups on the end of each p r o t e i n c h a i n , or to 29 d i c a r b o x y l i c or diamine groups. Alpha carboxyl groups are usually bound i n peptide linkages, thus w i l l not play a s i g n i f i c a n t r o l e as charged groups. In general, the t i t r a t a b l e a c i d i c groups i n proteins are the free terminal groups of glutamic a c i d and a s p a r t i c a c i d . T i t r a t i o n of the intima gave a pKa value of approximately 4.8 with an i s o e l e c t r i c point of 2.4. The c h a r a c t e r i s t i c pK values of various groups i n i n t a c t proteins are l i s t e d below: Groups pK (25°C) Carboxyl (alpha) 3.0-3.2 Carboxyl (aspartyl) 3.0-4.7 Carboxyl (glutamyl) ca. 4.4 Phenolic-hydroxyl (diiodotyrosine) 6.5 Phenolic-hydroxyl (tyrosine) 9.8-10.4 S y l f h y d r y l 9.1-10.8 (Page 445, Cohn and E d s a l l - Proteins, Amino Acids and Peptides). The most l i k e l y source f o r the observed negative charges i n the intima were a s p a r t i c glutamic a c i d . This suggestion was supported by an excess of the two d i s c a r b o x y l i c acids over a l l b a s i c amino acids. The p o s s i b i l i t y that the charge could be associated with a c i d i c muco-polysaccharides which reside i n the p r o c u t i c l e (Noiret et a l . 1966) cannot be overlooked. The l a t t e r authors suggest that the a c i d i c mucopolysaccharides might contribute f i x e d charges because a reduction i n mucopolysaccharides (detected histochemically) occurs as the pH of the s t a i n i n g s o l u t i o n was reduced from 4.2 to 2. 30 I suggest that the i n t i m a contains f i x e d negative s i t e s r a t h e r than n e u t r a l s i t e s and t h a t the chemical nature of the s i t e s could be e i t h e r a c i d i c mucopolysaccharides, or more probably a c i d i c amino a c i d s , 31 CHAPTER I I PARAMETERS INFLUENCING POTENTIAL DIFFERENCE ACROSS THE INTIMA. INTRODUCTION P r e l i m i n a r y s t u d i e s (Lewis, 1970) demonstrated that the i n t i m a was s e l e c t i v e l y permeable to c a t i o n s and was capable of forming both d i f f u s i o n and streaming p o t e n t i a l s under the appropriate c o n d i t i o n s . This chapter i s then a l o g i c a l e x t e n s i o n of these p r e l i m i n a r y e x p e r i -ments . The previous study (Lewis, 1970) e s t a b l i s h e d that the presence of f i x e d charges i n the i n t i m a could account f o r the s e l e c t i v i t y to 5 monovalent c a t i o n s . One of the o b j e c t i v e s of t h i s Thesis was to extend t h i s study to i n c l u d e the measurements of s e l e c t i v i t y of the i n t i m a f o r d i v a l e n t c a t i o n s , monovalent anions and a d d i t i o n a l monovalent c a t i o n s , so as to e s t a b l i s h more p r e c i s e l y the p h y s i c a l b a s i s f o r s e l e c t i v i t y , and i n order to e s t a b l i s h more completely the r o l e of the i n t i m a i n s e l e c t i v e r e a b s o r p t i o n and r e g u l a t i o n of blood i o n l e v e l s . Various parameters of the primary f i l t r a t e (eg. t o t a l i o n con-c e n t r a t i o n , i o n r a t i o s , pH, osmotic pr e s s u r e , and net water movement) be i n g dependent on the p h y s i o l o g i c a l s t a t e of the animal, w i l l vary w i d e l y i n the l o c u s t rectum. Since these f a c t o r s might be expected to a l t e r the s e l e c t i v i t y of a f i x e d charge or a l t e r the membrane s t r u c t u r e , i t was of immediate p h y s i o l o g i c a l importance to study t h e i r e f f e c t s on the i n t i m a . Such experiments a l s o provide f u r t h e r data on the proper-t i e s of f i x e d charged membranes under a v a r i e t y of c o n d i t i o n s f o r a comparison w i t h other membrane systems and various h y p o t h e t i c a l models which p r e d i c t the behavior of b i o l o g i c a l membrane. 32 Recent observations on the e f f e c t of u n s t i r r e d layers on i o n movement and p o t e n t i a l development across various membranes (Dainty and House, 1966) i n d i c a t e the l a t t e r could be of considerable importance i n r e c t a l reabsorption. These observations (Dainty and House, 1966) then necessitated a re-evaluation of some previous experiments on streaming and d i f f u s i o n p o t e n t i a l s . 33 RESULTS A. R e l a t i v e P e r m e a b i l i t y of the i n t i m a to Monovalent and D i v a l e n t Cations and Monovalent Anions. During the s e l e c t i v e r e a b s o r p t i o n of ions from the r e c t a l lumen (which i s u l t i m a t e l y r e s p o n s i b l e f o r the r e g u l a t i o n of i o n concentra-t i o n s i n the blood) the f i r s t b a r r i e r that the ions must cross i s the r e c t a l i n t i m a . P r e l i m i n a r y s t u d i e s (Lewis, 1970) on p e r m a s e l e c t i v i t y of the i n t i m a was r e s t r i c t e d to 5 monovalent a l k a l i metal i o n s , f o r which the r e l a t i v e s e l e c t i v e p e r m e a b i l i t y was shown to correspond to Eisenman's second sequence f o r a f i x e d negative charge a s s o c i a t e d w i t h aqueous channels. The present experiments were undertaken to extend these s t u d i e s to i n c l u d e monovalent anions, d i v a l e n t c a t i o n s and some a d d i t i o n a l monovalent c a t i o n s , and i n p a r t i c u l a r to see i f the r e l a t i v e p e r m e a b i l i t i e s agree w i t h Eisenman's theory f o r monovalent anions and Sherry's p r e d i c -t i o n s f o r d i v a l e n t c a t i o n s i n the presence of f i x e d charges. To measure r e l a t i v e p e r m e a b i l i t y , the l u m i n a l s i d e of the i n t i m a was bathed i n 10 mM/l KC1 and the haemocoel s i d e was bathed i n 5 mM/l of a second s a l t plus 5 mM/l KC1. This arrangement gave r i s e to con-c e n t r a t i o n gradients f o r e i t h e r K + or C l and a second c a t i o n or anion. R e l a t i v e p e r m e a b i l i t y , f o r anions and c a t i o n s , as measured by p o t e n t i a l d i f f e r e n c e s i n t h i s Thesis were i n a steady s t a t e c o n d i t i o n . The c a l c u -l a t i o n of r e l a t i v e p e r m e a b i l i t y u s i n g the Goldman constant f i e l d equation (see Appendix A) r e q u i r e d e q u i l i b r i u m c o n d i t i o n s , however the c a l c u l a -t i o n s were v a l i d as a f i r s t approximation. Table 3 R e l a t i v e p e r m e a b i l i t y of the i n t i m a f o r mono-v a l e n t anions and c a t i o n s and d i v a l e n t c a t i o n s , measured u s i n g d i f f u s i o n p o t e n t i a l s . D i f f u -s i o n p o t e n t i a l s were formed u s i n g a 2 f o l d KC1 c o n c e n t r a t i o n g r a d i e n t (10 mM/l -5 mM/l), 5 mM/l of a second s a l t was added to the s i d e of low KC1 c o n c e n t r a t i o n . Values were c a l c u l a t e d u s i n g the mo d i f i e d Goldman constant f i e l d equation (see Appendix A ) . Each value i s the mean f o r 6 p r e p a r a t i o n s . Ionic Species Relative Permeability - S.D. N H / 4 101 t 0.08 Rb + 1.05 ± 0.03 C s + 1.01 t 0.01 K+ 1.00 ± 0.01 N a + 0.67 t 0.01 L i + 0.55 ± 0.04 T E A + 0.062 ± 0.05 B a + + 0.354 ± 0.05 C a + + 0.29 ± 0.03 S r + + 0.28 ± 0.03 Mg++ 0.23 t 0.05 M n + + 0.23 ± 0.05 HCOg" 0.128 t 0.012 CN'. 0.121 ± 0.01 F" 0.102 ± 0.03 N 0 3 " 0.101 ± 0.02 c r 0.097 t 0.06 C H 3 C 0 0 " 0.095 t 0.006 B r ' 0.084 t 0.008 2 4 0.082 t 0.005 I" 0.067 ± 0.008 34 Monovalent Cations The r e c t a l intima has the a b i l i t y to s e l e c t f o r monovalent cations i n a p r e f e r e n t i a l order of NH* > Rb + > C s + > K + > Na + > L i + > TEA, which i s sequence two of Eisenman, i n d i c a t i n g widely spaced nega-t i v e f i x e d charges of weak f i e l d strength (Eisenman, 1962). That i s , the rate of penetrationwas i n v e r s e l y p r o p o r t i o n a l to the hydrated radius of ions aswas the case f o r uncharged h y d r o p h i l i c molecules ( P h i l l i p s and D o c k r i l l , 1968). Monovalent Anions The r e c t a l intima also has the a b i l i t y to s e l e c t f o r monovalent anions i n a p r e f e r e n t i a l order of: HCO~ > CN~ > F~ > N0~ > C l ~ > CH^COO > Br > E^PO^ > I. which i s sequence seven of Eisenman (F > C l ~ > Br~ > I~ ). In the l a t t e r sequence, F~ i s the smallest unhydrated i on with the strongest energy of hydration; while I i s the la r g e s t unhydrated i on with the weakest energy of hydration. That i s , the sequence of permeability was according to the decreasing hydrated radius. Divalent Cations The intima can s e l e c t for dival e n t cations i n a p r e f e r e n t i a l +2 "r2 +2 +2 +2 order of: Ba > Ca > Sr > Mg > Mn , which i s sequence two of Sherry, i n d i c a t i n g (as d i d the sequence f o r the monovalent cations) negative f i x e d charges, e i t h e r of weak f i e l d strength (Diamond and Wright, 1969), but c l o s e l y spaced, or stronger f i e l d strength with the 35 charges widely spaced. I t seems that the former configuration was the most l i k e l y , since weak f i e l d strength was indicated from the above studies on monovalent cations. Both the monovalent and divalent sequences indicate that s e l e c t i v i t y was mediated by the same fixed charge. The sequence of permeability for divalent ions was according to increasing hydrated radius. B. Competition Between Ions for Fixed Negative Sites. The preliminary studies were concerned with pure solutions of one or two s a l t s , which were used to determine the p r i n c i p l e s regulating ion permeability of the intima. In the i n vivo rectum of the locust there i s a mixture of ions, the levels of which may be very high (eg. exceeding 500 mM/l NaCl or KCI in'the dehydrated animal). In such circumstances the ions must compete for the fixed negative s i t e s which are l i m i t e d i n number ( i . e . the s i t e s can be saturated at high concentra-tions) . The effects of increasing the concentration of monovalent and divalent cations (as C l s a l t s ) on the s e l e c t i v i t y of the intima for K + r e l a t i v e to Cl was investigated (K + and Cl are the p r i n c i p l e ions normally entering the locust ( P h i l l i p s , 1964 b) ). These studies indicate properties of the fixed charge. The procedure used was to keep a constant KCI concentration gradient (10 mM/l - 5 mM/l) across the intima. Sucrose was added to the side of low KCI to maintain equal osmotic pressures. The competing s a l t was added equally to both sides of the bathing solutions, making f i n a l concentrations for the second s a l t equal to 1, 10 or 100 mM/l. F i g . 8 The e f f e c t of two d i v a l e n t and two monovalent c a t i o n s on a KC1 d i f f u s i o n p o t e n t i a l . Concen-t r a t i o n of KC1 on lumenal s i d e was twice that of haemocoel s i d e i n a l l experiments. Values are presented as a f r a c t i o n of the p o t e n t i a l d i f f e r e n c e i n the absence of a second s a l t . S a l t s were added at equal concentrations on both s i d e s . Values represent the average of s i x p r e p a r a t i o n s . • - NaCl • - KC1 A - M g C l 2 O - C a C l 2 See Appendix B Table 9 f o r i n d i v i d u a l v a l u e s . Concentration of added chloride salt in mM/l 36 In a n a l y s i n g the e f f e c t s of the added s a l t , the r e s u l t s were expressed as a f r a c t i o n of the d i f f u s i o n p o t e n t i a l observed f o r a given p r e p a r a t i o n i n the absence of a competing s a l t . The r e s u l t s of the experiment can be observed i n F i g . 8. A f t e r adding ImM/l of CaCl^ a p o t e n t i a l drop of 40% occurs, w h i l e 1 mM/l of MgCl^, KC1 and NaCl pro-duced decreases of 25%, 18% and 16% r e s p e c t i v e l y . Adding 10 mM/l C a C l 2 , MgCl 2,KCl and NaCl l e d to decreases of 87%, 67%, 61% and 47% r e s p e c t i v e l y . The e f f e c t of 100 mM/l C a C l 2 and M g C l 2 was to cause a r e -v e r s a l of p o t e n t i a l amounting to 14% and 15% r e s p e c t i v e l y of the i n i t i a l p o t e n t i a l d i f f e r e n c e : 100 mM/l KC1 and NaCl reduce the p o t e n t i a l by 98% and 94% r e s p e c t i v e l y ; i e . the chargewas s a t u r a t e d so t h a t s e l e c t i v i t y f o r c a t i o n over anion was a b o l i s h e d . The most s t r i k i n g o b s e r v a t i o n was t h a t 1 mM/l C a C l 2 can reduce the KC1 d i f f u s i o n p o t e n t i a l as e f f e c t i v e l y as approximately 10 mM/l NaCl; i . e . t h i s d i v a l e n t i o n was 10 times as e f f e c t i v e at masking f i x e d charge as a monovalent i o n . These experiments i n d i c a t e d t h a t f i x e d negative charge was an important f a c t o r e f f e c t i n g i o n s e l e c t i v i t y i n the i n t a c t animal at lower c o n c e n t r a t i o n s , such as was observed i n hydrated l o c u s t s . 37 C. Relative Permeability of the Intima as Measured by Streaming P o t e n t i a l s . In previous experiments the properties of the f i x e d charge have been studied i n the absence of f l u i d flow. The membrane model f or the intima, as developed so f a r , consists of f l u i d f i l l e d p o r e s l i n e d with f i x e d negative charges. The e f f e c t of f l u i d movement ( r e s u l t i n g i n solute drag) must then be considered with respect to the s e l e c t i v e movement of ions. This was done by observing the streaming p o t e n t i a l developed by a 400 mill i o s m o l a r sucrose gradient, when the s a l t con-ce n t r a t i o n (10 mM/l) was the same on both sides of the membrane. As can be seen from Table 4, four of the preparations (1,3,5,6) gave a sequence of L i + > Na + > K + > C s + > Rb +, one of the preparations + + + + + (2) gave a sequence of L i > Na > Cs > K > Rb , and preparation 4 gave + + + + a sequence of L i > Na > Cs > Rb > K. The f i r s t sequence l i s t e d above f or streaming p o t e n t i a l s vias exactly opposite that f o r d i f f u s i o n p o t e n t i a l s , i . e . the sequence f o r d i f f u s i o n p o t e n t i a l s was Rb > Cs > K + > Na +> L i + . I i n t e r p r e t t h i s as follows: the drag e f f e c t between water and ions should be pr o p o r t i o n a l to the hydrated s i z e , being maximal f o r L i + and l e a s t f o r Rb . I f the drag e f f e c t i s large enough, t h i s w i l l become more important than f i x e d charge i n determining s e l e c t i v i t y f o r ions. From the other sequences noted above, we can imply that the order of s e l e c t i v i t y would correspond to Eisenman's t h i r d and fourth s e r i e s . For preparation two, Rb + or K + w i l l have the smallest hydrated s i z e . The d i f f e r e n c e i n sequence may i n d i c a t e the v a r i a b i l i t y of charge strength of the intima. Such v a r i a b i l i t y might be associated with the density of the f i x e d charges i . e . as the charge density increases, the s e l e c t i v i t y sequence s h i f t s from that of inc r e a s i n g hydrated s i z e to Table 4 S e l e c t i v i t y of the i n t i m a f o r monovalent c a t i o n s as shown by streaming p o t e n t i a l s . A 400 m i l l i o s m o l a r sucrose s o l u t i o n was on the lumenal s i d e . 10 m i l l i m o l a r s a l t con-c e n t r a t i o n s were used. Sign of p o t e n t i a l i s w i t h reference to the lumenal s i d e . Salts used for Streaming Potentials Streaming Potentials in millivolts Individual preparations 1 2 3 4 5 6 Mean ± S.D. L i 28 29 21 25 25 20 24.6 t 3.6 Na 20 23 16 15.5 15 15 17.4 t 3.2 K 13 13 12 11 13 13 12.5 ± 0.8 Cs 13 14 8.5 15 13 9 12.1 t 2.7 Rb 11 13 8 13 12 8 10.8 - 2.3 38 t h a t of i n c r e a s i n g dehydrated s i z e . The d i f f u s i o n a l p e r m e a b i l i t y to Rb + has been shown to be greater than to L i + , so that d i f f u s i o n down any co n c e n t r a t i o n g r a d i e n t caused by u n s t i r r e d l a y e r s (see s e c t i o n H of t h i s Chapter) would be gr e a t e s t + + f o r Rb and l e a s t f o r L i , i . e . u n s t i r r e d l a y e r s w i l l not account f o r the r e l a t i v e order of the c a t i o n s as determined from streaming p o t e n t i a l s . D. E f f e c t of pH on Membrane p o t e n t i a l s . I f the f i x e d charge was due to a weakly d i s s o c i a t e d a c i d group, such as an amino a c i d (pK 3.5-4.7) as i n d i c a t e d i n Chapter 1, the con-c e n t r a t i o n of f i x e d charges and hence the s e l e c t i v i t y of the i n t i m a f o r i o n s should be pH dependent. I f the same f i x e d c h a r g e r s r e s p o n s i b l e f o r both streaming and d i f f u s i o n p o t e n t i a l s , we would expect the same q u a n t i t a t i v e dependence of these two types of p o t e n t i a l s on pH, thus implying" t h a t both i o n and water movement occur through the same route. This experiment was designed to observe whether a change of pH from a maximum of 5.5 to a minimum value of 2.2 would a f f e c t streaming of d i f f u s i o n p o t e n t i a l s ( i . e . c a t i o n s e l e c t i v i t y ) , i n the same way. As shown i n F i g . 9, the p o t e n t i a l s which were u s u a l l y caused by KC1 co n c e n t r a t i o n g r a d i e n t s ( d i f f u s i o n p o t e n t i a l s ) or osmotic gradients (streaming p o t e n t i a l s ) at a pH of 5.5, disappear at a pH of 2.2, w i t h a pK i n both cases of 3.7. These r e s u l t s support the previous evidence (Lewis, 1970) that the pores were l i n e d w i t h negative f i x e d charges due to weakly a c i d i c groups, w i t h the pK as estimated by t i t r a t i o n ( a p p r o x i -mately 4.8; Chapter 1). Both types of p o t e n t i a l d i f f e r e n c e s seem to be a s s o c i a t e d w i t h the same f i x e d charges. This suggested that the main F i g . 9 Open c i r c l e s represent the e f f e c t s of pH change on streaming p o t e n t i a l s developed across the i n t i m a . The osmotic pressure d i f f e r e n c e was kept constant w i t h 400 m i l l i o s m o l a r sucrose on the haemocoel s i d e and no sucrose on the lumen s i d e . Both s i d e s were bathed i n 10 mM/l KCI. Sign i s w i t h reference to the haemocoel. P o i n t s represent mean value f o r 6 p r e p a r a t i o n s . S o l i d c i r c l e s represent the e f f e c t s of pH change on d i f f u s i o n p o t e n t i a l s developed across the i n t i m a . A 2 f o l d KCI c o n c e n t r a t i o n g r a d i e n t (10 mM/l on the haemocoel s i d e and 5 mM/l on the l u m i n a l s i d e ) was used to i n i t i a t e d i f f u -s i o n p o t e n t i a l s . Sign i s w i t h reference to the haemocoel. P o i n t s represent mean value f o r 6 p r e p a r a t i o n s . See Appendix B Tables 10 and 11 f o r i n d i v i d u a l v a l u e s . Potential difference (mv) a cr 5" o o _ i oo _i 3 i CO CJ1-J Oi O • to 1 OS O • O • O F i g . 10 The a f f e c t s of h i g h sucrose concentrations on d i f f u s i o n p o t e n t i a l s . D i f f u s i o n p o t e n t i a l s were i n i t i a t e d by a 2 f o l d c o n c e n t r a t i o n d i f f e r e n c e of KC1 (p.01M-0.005M). High con-c e n t r a t i o n of KC1 on the lumen s i d e . Sucrose was p l a c e d i n equal concentrations on both s i d e s of the membrane. The s i g n i s w i t h reference to the haemocoel s i d e . V e r t i c a l bars represent standard d e v i a t i o n s f o r 6 pre-p a r a t i o n s . See Appendix B Table 12 f o r i n -d i v i d u a l v a l u e s . Concentration of sucrose in milliosmolar 39 d i f f u s i o n pathway f o r ionsv&s the same pores through which water flow and hence streaming p o t e n t i a l s occurred. E. E f f e c t s of High Osmotic Pressure on D i f f u s i o n P o t e n t i a l s . A high and v a r i a b l e osmotic pressure i s n a t u r a l l y observed i n the l o c u s t rectum i n v i v o . A hydrated animal normally possesses a low i o n concentration, but a high osmotic pressure due to organic molecules. I t has already been shown that high ion concentration decreases the eff e c t i v e n e s s of the f i x e d charge; i . e . high i o n concentration reduces c a t i o n ; anion d i s c r i m i n a t i o n by the f i x e d charge (Chapter 1). I t would be of i n t e r e s t then to know i f high osmotic pressure alone w i l l e f f e c t the f i x e d charge properties as opposed to high ion concentrations. A two-fold KC1 concentration gradient was created (10 mM -5 mM ) across the intima. Sucrose was added to the side with low KC1 to main-t a i n equal osmotic pressures. Further sucrose was added i n equivalent amounts to both solutions to create t o t a l concentrations of 100, 200, 400, 600, 800 or 1200 mill i o s m o l a r on both sides of the membrane. As can be observed from F i g . 10, therewas no s i g n i f i c a n t change i n the d i f f u s i o n p o t e n t i a l with an increase i n osmotic pressure, as predicted for i o n exchange membranes ( H e l f f e r i c h , 1962). Thus the osmotic pressure as such does not a f f e c t the a v a i l a b i l i t y of f i x e d negative charges as i n d i c a t e d by ca t i o n s e l e c t i v i t y . F. E f f e c t s of Osmotic Pressure Difference (A IT) The osmotic pressure of the r e c t a l contents, as previously F i g . 11 Streaming p o t e n t i a l s were developed u s i n g suc-rose c o n c e n t r a t i o n g r a d i e n t s , w i t h 10 mM/l KCI on both s i d e s . In both curves, the concentra-t i o n d i f f e r e n c e s were kept constant, but the absolute c o n c e n t r a t i o n was changed. The upper curve ( s o l i d c i r c l e s ) represents a 200 m i l l i o s -molar d i f f e r e n c e . The lower curve ( s o l i d squares) a 100 m i l l i o s m o l a r d i f f e r e n c e . The s i g n i s w i t h reference to the haemocoel s i d e . V e r t i c a l bars represent standard d e v i a t i o n s f o r 6 p r e p a r a t i o n s . Values f o r i n d i v i d u a l p r e-p a r a t i o n s are given i n the Appendix B Table 13. 14 -, 12 J 0 -I , , . 0 200 400 600 800 Average sucrose concentration in milliosmolar 40 mentioned, vary g r e a t l y . An i n c r e a s e i n the osmotic p r e s s u r e , which might dehydrate the membrane, has been shown to r e s t r i c t the water p e r m e a b i l i t y f o r the r e c t a l i n t i m a ( P h i l l i p s and Beaumont, 1971). Diamond (1966) and Lewis (1970) found a n o n - l i n e a r r e l a t i o n s h i p between i n c r e a s i n g osmotic pressure d i f f e r e n c e and the r a t e of water flow asso-c i a t e d w i t h the g a l l bladder and the i n t i m a r e s p e c t i v e l y . Was t h i s non-l i n e a r i n c r e a s e i n water flow as r e f l e c t e d by streaming p o t e n t i a l s , a f u n c t i o n of the r a t e of water flow (i.e.was the streaming p o t e n t i a l independent of t o t a l osmotic p r e s s u r e , but dependent on the osmotic pressure d i f f e r e n c e ) orvas i t a f u n c t i o n of the average o s m o l a r i t y of the b a t h i n g s o l u t i o n ( i . e . due to an osmotic compartment e f f e c t ) ? One of the explanations o f f e r e d f o r the n o n - l i n e a r water flow (Brodsky and S c h i l b , 1965) was t h a t the r a t e of water flow could r e -v e r s i b l y deform the membrane s t r u c t u r e . I f the osmotic pressure differencewas kept constant, but the average o s m o l a r i t y was changed then t h i s i n t e r p r e t a t i o n predicted that there should be no change i n the streaming p o t e n t i a l v a l u e . This hypothesis was t e s t e d by keeping the osmotic pressure d i f f e r e n c e constant at 100 m i l l i o s m o l a r w h i l e v a r y i n g the average o s m o l a r i t y of the b a t h i n g s o l u t i o n ( c o n t a i n i n g 10 mM/l MCI on both s i d e s ) from 50 to 350 m i l l i o s m o l a r w i t h sucrose. This experiment was a l s o repeated keeping the osmotic gradient constant at 200 m i l l i o s -molar. As can be observed from F i g . 11, the streaming p o t e n t i a l ' s not constant, which Was c o n t r a r y to the p r e d i c t i o n made by the membrane de-formation theory. Rather the p o t e n t i a l d i f f e r e n c e decreased r a p i d l y as the average o s m o l a r i t y increased; F i g . 12 Resistance of the r e c t a l i n t i m a to water flow as r e f l e c t e d i n the r a t i o of osmotic pressure d i f f e r e n c e and streaming p o t e n t i a l d i f f e r e n c e . Each p o i n t represent represents mean value f o r 6 o b s e r v a t i o n s . Values c a l c u l a t e d from upper curve of F i g . 20. Osmotic pressure difference/ streaming potential (mv) Table 5 Percentage of water by weight i n the i n t i m a when bathed i n a s o l u t i o n of d i s t i l l e d water and i n a 400 m i l l i o s m o l a r sucrose s o l u t i o n . Percentage of water by weight Bathing Distilled Sucrose solution water solution 77 72 73 71 69 54 72 63 66 57 72 68 Mean ± S.D. 71.5 i 3.7 64.2 t 7.3 41 I f the r e s i s t a n c e to water flow across the i n t i m a ( d e f i n e d as the r a t i o o f the osmotic pressure d i f f e r e n c e and the streaming p o t e n t i a l ) wasrelated to the average o s m o l a r i t y , a l i n e a r r e l a t i o n s h i p i s observed ( F i g . 12); i . e . an i n c r e a s e i n the average o s m o l a r i t y w i l l e l e c i t a p r o p o r t i o n a t e i n c r e a s e i n the membrane r e s i s t a n c e . The most acceptable i n t e r p r e t a t i o n of t h i s behavior at present was that the membrane behaves as an osmotic compartment. That i s , waterwas withdrawn from the mem-brane at h i g h o s m o l a r i t i e s , reducing the e f f e c t i v e pore s i z e and con-sequently i n c r e a s i n g the r e s i s t a n c e to water flow. A q u a l i t a t i v e t e s t f o r membrane dehydration was c a r r i e d out. S i x membrane p r e p a r a t i o n s were pl a c e d i n d i s t i l l e d water f o r one hour. A f t e r that time i n t e r v a l they were removed, excess water removed and the i n t i m a were weighed. The same 6 membranes were then p l a c e d i n 400 m i l l i o s m o l a r sucrose f o r 1 hour, e x t e r n a l water removed and the pr e p a r a t i o n s re-weighed. The membranes were then d r i e d i n an oven u n t i l a constant weight had been reached. The percentage water by weight f o r both experimental c o n d i t i o n s were c a l c u l a t e d . The r e s u l t s (Table 5) i n d i c a t e t h a t the membrane pla c e d i n the 400 m i l l i o s m o l a r s o l u t i o n had a lower percent of water by weight than d i d membranes i n d i s t i l l e d water. The d i f f e r e n c e i n percent v a r i e d from 2.2 to 15.2; however i n a l l cases, membranes i n d i s t i l l e d water contained more water than membranes i n sucrose s o l u t i o n (p<0.025). A s i m i l a r behavior has been reporte d f o r i o n exchange membranes, where h i g h e x t e r n a l osmotic pressures s h r i n k the membranes, r e s u l t i n g i n a denser m a t r i x system (reducing the pore s i z e ) and consequently reducing the d i f f u s i o n c o e f f i c i e n t s ( H e l f f e r i c h , 1962). 42 G. Ion Concentration E f f e c t on Streaming and D i f f u s i o n P o t e n t i a l s . The i o n c o n c e n t r a t i o n i n the r e c t a l lumen i n the i n t a c t animal v a r i e s g r e a t l y . A previous i n v e s t i g a t i o n (Results 5) i n d i c a t e d that the osmotic pressure per se d i d not a f f e c t d i f f u s i o n p o t e n t i a l s . T h i s , however, was not the case f o r streaming p o t e n t i a l s , here the p o t e n t i a l was a f u n c t i o n of the average osmotic pressure ( l a s t s e c t i o n ) . The e f f e c t o f i o n c o n c e n t r a t i o n per se on streaming p o t e n t i a l d i f f e r e n c e was important i n understanding s e l e c t i v e r e a b s o r p t i o n under d i f f e r i n g p h y s i o l o g i c a l c o n d i t i o n s . Such a study would t e s t f u r t h e r whether the same f i x e d charge was r e s p o n s i b l e f o r streaming and d i f f u s i o n p o t e n t i a l s , by determining whether both types of p o t e n t i a l s respond i n a s i m i l a r manner to an i n c r e a s e i n i o n c o n c e n t r a t i o n of the b a t h i n g s o l u t i o n . T e o r e l l (1953) s t a t e s that as the i o n c o n c e n t r a t i o n i n c r e a s e d (keeping a constant gradient) the d i f f u s i o n p o t e n t i a l decreased, t h i s T e o r e l l c a l l e d the " c o n c e n t r a t i o n e f f e c t . " This e f f e c t i s caused by a masking of the f i x e d charge r e s u l t i n g i n an i n c r e a s e i n co-ion con-ductance; that i s , s e l e c t i v i t y f o r c a t i o n s r e l a t i v e to anions decreased. A d r a s t i c decrease i n the P . /P w i t h an i n c r e a s e i n the average KCI c o n c e n t r a t i o n (at constant c o n c e n t r a t i o n r a t i o ) has already been reported (Chapter 1, F i g . 6 ) . When c o n s i d e r i n g the e f f e c t of Ion c o n c e n t r a t i o n on streaming p o t e n t i a l s two v a r i a b l e s must be considered, one the i o n c o n c e n t r a t i o n e f f e c t i n masking the charge ( p r e v i o u s l y mentioned f o r d i f f u s i o n poten-t i a l s ) and the second was an osmotic e f f e c t imported by the ions ( F i g . 11, osmotic pressure e f f e c t on streaming p o t e n t i a l s ) . As can be observed i n F i g . 13, as the i o n c o n c e n t r a t i o n i n c r e a s e d , the streaming p o t e n t i a l de-creased f o r any given osmotic g r a d i e n t . At a c o n c e n t r a t i o n of 1 mM/l F i g . 13 Streaming p o t e n t i a l s measured across the i n t i m a due to osmotic pressure d i f f e r e n c e created by the use of sucrose. The sucrose c o n c e n t r a t i o n was v a r i e d from 0 to 400 m i l l i o s m o l a r on the haemocoel s i d e , w h i l e keeping sucrose at 0 m i l l i o s m o l a r on the lumen s i d e . The concentra-t i o n of KCI was the same on both s i d e s . The s i g n i s w i t h reference to the haemocoel s i d e . V e r t i c a l bars represent standard d e v i a t i o n s f o r 6 p r e p a r a t i o n s . See Appendix B Table 14 f o r i n d i v i d u a l p r e p a r a t i o n s . 40i Concentration of sucrose in milliosmolars F i g . 14 Streaming p o t e n t i a l s were developed u s i n g suc-rose c o n c e n t r a t i o n g r a d i e n t s , w i t h 1 mM/l KC1 on both s i d e s . The sucrose c o n c e n t r a t i o n g r a d i e n t was kept constant, but the absolute con-c e n t r a t i o n was changed. The sucrose concentra-t i o n gradient was 200 m i l l i o s m o l a r . V e r t i c a l bars represent standard d e v i a t i o n s f o r 6 p r e -p a r a t i o n s . Values f o r i n d i v i d u a l p r e p a r a t i o n s are given i n the Appendix B Table 15. 30 -, 25 -1000 1200 Average sucrose concentration in milliosmolar 43 KC1, when the osmotic e f f e c t of 1 mM/l KC1 was i n s i g n i f i c a n t , the poten-t i a l d i f f e r e n c e i n c r e a s e d a s y m p o t i c a l l y w i t h i n c r e a s i n g osmotic gradients due to the osmotic compartment e f f e c t . At 10 mM/l KC1, the p o t e n t i a l s were approximately 50% of those f o r 1 mM/l KC1. Was t h i s p o t e n t i a l drop caused by i o n c o n c e n t r a t i o n (masking) e f f e c t , osmotic e f f e c t ( i . e . a reduced water p e r m e a b i l i t y ) , or both? I f the decrease were caused s o l e l y by an osmotic e f f e c t , then 1 mM/l KC1 s o l u t i o n brought up to the same osmotic pressure as that of the 10 mM/l KC1 s o l u t i o n ( u s i n g sucrose) should show the same decrease i n p o t e n t i a l . This was not the case. I t can be shown from F i g . 14 that a 1 mM/l s o l u t i o n w i t h o s m o l a r i t y r a i s e d (using sucrose) r e s u l t e d i n streaming p o t e n t i a l i n the order of 20 mv, whereas 10 mM/l KC1 s o l u t i o n r e s u l t e d i n a p o t e n t i a l of 11 mv. This i n d i c a t e d t h a t a r e d u c t i o n i n p o t e n t i a l a t a concentra-t i o n of 10 m M/l KC1 r e l a t i v e to 1 mM/l KC1 was caused s o l e l y by the i o n c o n c e n t r a t i o n e f f e c t . At low i o n c o n c e n t r a t i o n s , then the decrease i n the streaming p o t e n t i a l was caused by a masking of the charges. In a s i m l a r manner, i t was e s t a b l i s h e d that at 100 mM/l KC1, the osmotic e f f e c t of the KC1 reduced the streaming p o t e n t i a l by 25% and the i o n c o n c e n t r a t i o n e f f e c t reduced the p o t e n t i a l by 75%. At h i g h i o n con-c e n t r a t i o n s (1000 mM/l KC1) the r e d u c t i o n of the streaming p o t e n t i a l could be accounted f o r completely by an osmotic e f f e c t . To summarize, at low i o n concentrations the r e d u c t i o n i n stream-i n g p o t e n t i a l s (as i o n concentrations are g r a d u a l l y r a i s e d ) was due completely to an i o n c o n c e n t r a t i o n e f f e e t . At intermediate concentra-t i o n s (100 mM/l KCl) the r e d u c t i o n of streaming p o t e n t i a l s seems to be F i g . 15 The e f f e c t of i o n c o n c e n t r a t i o n per se on streaming and d i f f u s i o n p o t e n t i a l s . The average c o n c e n t r a t i o n of KCI b a t h i n g s o l u t i o n f o r d i f f u s i o n p o t e n t i a l s i s v a r i e d but a con-s t a n t c o n c e n t r a t i o n g r a d i e n t i s maintained. Values f o r d i f f u s i o n p o t e n t i a l s , represented by s o l i d squares, are presented as a f r a c t i o n of the p o t e n t i a l d i f f e r e n c e a t the lowest aver-age i o n c o n c e n t r a t i o n . A constant osmotic p r e s -sure of 200 m i l l i o s m o l a r i s maintained f o r streaming p o t e n t i a l u s i n g sucrose. Values f o r streaming p o t e n t i a l s , represented by s o l i d c i r c l e s , are presented as a f r a c t i o n of the streaming p o t e n t i a l a t a b a t h i n g c o n c e n t r a t i o n of 1 mM/l KCI and where the osmotic e f f e c t of the i o n c o n c e n t r a t i o n i s maintained by sucrose. V e r t i c a l bars represent standard d e v i a t i o n s f o r 6 p r e p a r a t i o n s . Values c a l c u l a t e d from F i g . 6 f o r d i f f u s i o n p o t e n t i a l s and F i g . 13 and 14 f o r streaming p o t e n t i a l s . Average concentration of KC1 bathing solution in mM/l 44 i n f l u e n c e d by both osmotic and i o n co n c e n t r a t i o n s , w h i l e a t high i o n c o n c e n t r a t i o n the r e d u c t i o n i n streaming p o t e n t i a l s seemed to be caused almost completely by an osmotic e f f e c t . I f both streaming and d i f f u s i o n p o t e n t i a l s were dependent, i n t h e i r f o r m a t i o n , on the same f i x e d charge, as was i n d i c a t e d by the e f f e c t of pH on these two p o t e n t i a l d i f f e r e n c e s (see s e c t i o n D), one would expect e x t e r n a l i o n co n c e n t r a t i o n to a f f e c t streaming and d i f f u s i o n p o t e n t i a l s i n a s i m i l a r manner. As can be seen i n F i g . 15, both poten-t i a l d i f f e r e n c e s decreased as the i o n co n c e n t r a t i o n i n c r e a s e d . When the i o n c o n c e n t r a t i o n of the b a t h i n g s o l u t i o n f o r streaming p o t e n t i a l s was 10 mM/l KCI, the p o t e n t i a l d i f f e r e n c e decreased 50%, f o r a s i m i l a r decrease i n d i f f u s i o n p o t e n t i a l s , the average i o n c o n c e n t r a t i o n of the ba t h i n g s o l u t i o n was 55 mM/l KCI ( F i g . 15); i . e . d i f f u s i o n p o t e n t i a l changes were 5 times l e s s s e n s i t i v e to the e x t e r n a l i o n c o n c e n t r a t i o n than were streaming p o t e n t i a l changes. H. P o s s i b l e E f f e c t s of U n s t i r r e d Layers on membrane P o t e n t i a l s . Immediately adjacent to a l l membrane surfaces are u n s t i r r e d boundary l a y e r s (Dainty and House, 1966). Nerst (1904) was one of the f i r s t to develop the concept of u n s t i r r e d l a y e r s . An u n s t i r r e d l a y e r r e f e r s to a t h i n l a y e r of s o l u t i o n i n which the only form of mixing i s slow laminar flow p a r a l l e l to the liquid-membrane i n t e r f a c e , so that s o l u t e t r a n s f e r i s i n e f f e c t only by d i f f u s i o n . Wedner and Diamond (1969) found that a l a r g e component of streaming p o t e n t i a l s i n the g a l l 45 bladder was a boundary d i f f u s i o n p o t e n t i a l , owing to water flow which produces l o c a l s a l t c o n c e n t r a t i o n gradients i n opposing u n s t i r r e d l a y e r s . Since the f l u i d flow i n the rectum, at b e s t , would be q u i t e s l u g g i s h , the i n t i m a could c o n t a i n u n s t i r r e d l a y e r s of consi d e r a b l e magnitude. In p r e l i m i n a r y s t u d i e s (Lewis, 1970) streaming p o t e n t i a l s were observed, caused by osmotic gradients across the r e c t a l i n t i m a . The opp o r t u n i t y was taken i n t h i s study to observe the e f f e c t of u n s t i r r e d l a y e r s on both streaming and d i f f u s i o n p o t e n t i a l s . I n a l l i n i t i a l experiments, s o l u t i o n concentrations were maintained by p e r f u s i o n from b u r e t t e s w i t h a flow r a t e of 4 ml per minute. I t should be noted t h a t t h i s s t i r r i n g r a t e probably g r e a t l y exceeds those found i n v i v o . The ends of the buret were 3 cm from the i n t i m a . Thus there e x i s t e d the p r o b a b i l i t y that t h i s flow r a t e d i d not remove the u n s t i r r e d l a y e r s , so th a t c e r t a i n e f f e c t s noted i n previous experiments (eg. time course of development of streaming p o t e n t i a l s ) might be p a r t l y r e l a t e d to these l a y e r s r a t h e r than the membrane. To i n v e s t i g a t e t h i s p o s s i b i l i t y , i n s t e a d of u s i n g b u r e t t e tubes, LKB p e r i s t a l t i c pumps were used to d i r e c t the s o l u t i o n flow a t the surface of the membrane. The r a t e s of p e r f u s i o n of s o l u t i o n were 0.45, 0.8, 1.5 or 2.8 ml/minute. P o t e n t i a l d i f f e r e n c e s were recorded u s i n g a "Ratiometer" pH meter coupled to a paper chart recorder. U n s t i r r e d l a y e r e f f e c t on d i f f u s i o n p o t e n t i a l s . D i f f u s i o n p o t e n t i a l s were i n i t i a t e d u s i n g 2 - f o l d KCI concentra-t i o n g r a d i e n t s (10 - 5 mM/l KCI). In a n a l y z i n g the r e s u l t s f o r F i g . 16 The e f f e c t of p e r f u s i o n r a t e ( d i r e c t i n g f l u i d at the membrane surf a c e ) on a KC1 d i f f u s i o n p o t e n t i a l . Values f o r each p r e p a r a t i o n are expressed as a % of value observed f o r the pre-p a r a t i o n at a maximum s t i r r i n g r a t e s . S t i p p l e d area represents d i f f u s i o n p o t e n t i a l s observed u s i n g b u r e t t e s as the p e r f u s i o n apparatus. V e r t i c a l bars represent standard e r r o r f o r 6 obse r v a t i o n s . .3 a cu -*-> O a O •r-t CO <t-l cu cu 0.6 0.4 H 0.2 H .0 0 T 2 Perfusion rate in ml/min. 46 d i f f e r e n t p e r f u s i o n r a t e s , the d i f f u s i o n p o t e n t i a l s ( f o r i n d i v i d u a l p r e p a r a t i o n s ) were expressed as a f r a c t i o n of the value observed f o r the same p r e p a r a t i o n at the maximum s t i r r i n g r a t e . The r e s u l t s are shown i n F i g . 16, where the. s t i p p l e d area represent d i f f u s i o n p o t e n t i a l s p r e v i o u s l y observed u s i n g b u r e t t e s as t h e . p e r f u s i o n apparatus. The s o l i d l i n e represents values f o r d i f f u s i o n p o t e n t i a l s u s i n g the LKB p e r i s t a l t i c pumps. There was a s l i g h t i n c r e a s e i n the d i f f u s i o n poten-t i a l at i n c r e a s e d p e r f u s i o n r a t e s . I f the r a t e of d i f f u s i o n across the i n t i m a was g r e a t e r than the r a t e of d i f f u s i o n i n the u n s t i r r e d l a y e r , an i o n - d e p l e t e d l a y e r w i l l occur on the s i d e of h i g h i o n c o n c e n t r a t i o n , w h i l e a l o c a l excess of ions w i l l develop on the s i d e of low i o n con-c e n t r a t i o n . This change i n i o n c o n c e n t r a t i o n w i l l lower the concentra-t i o n g r a d i e n t across the membrane and consequently the d i f f u s i o n p o t e n t i a l . This may be what happened across the i n t i m a , s i n c e there i s a s l i g h t i n c r e a s e i n d i f f u s i o n p o t e n t i a l s as s t i r r i n g was i n c r e a s e d ; however the e f f e c t was q u i t e s m a l l . However i n the i n t a c t animal where the s t i r r i n g r a t e s may be q u i t e low, i t i s not c l e a r how important t h i s e f f e c t may be. U n s t i r r e d l a y e r E f f e c t on Streaming p o t e n t i a l s . Water flow caused by e i t h e r an osmotic or h y d r o s t a t i c g r a d i e n t would drag along i o n s . I f the r e f l e c t i o n c o e f f i c i e n t of the membrane f o r the ions was zero, then the ions wjuld flow s t r a i g h t through the membrane t o t a l l y unhindered. I f , however, the r e f l e c t i o n c o e f f i c i e n t (o) was l e s s than one but grea t e r than zero, then some of the ions would pass through but a l s o some ions would be r e f l e c t e d by the membrane and a hi g h i o n c o n c e n t r a t i o n wjuld s t a r t to form i n u n s t i r r e d l a y e r s on the F i g . 17 Chart recorder record f o r a t y p i c a l p r e p a r a t i o n showing the time course f o r development of a p o t e n t i a l d i f f e r e n c e caused by osmotic water flow. A 200 m i l l i o s m o l a r osmotic pressure d i f f e r e n c e was caused by sucrose, the i o n b a t h i n g c o n c e n t r a t i o n was 10 mM/l KCI. Streaming potentials (mv) F i g . 18 Chart recorder records showing the time course f o r development of streaming p o t e n t i a l during p e r f u s i o n of a t y p i c a l i n t i m a l p r e p a r a t i o n , at d i f f e r i n g r a t e s . The time r e f e r s to that f o l l o w i n g the s t a r t of p e r f u s i o n at the s t a t e d r a t e w i t h the system, i n a l l cases, p r e v i o u s l y i n the u n s t i r r e d c o n d i t i o n . The osmotic pressure g r a d i e n t , i n i t i a t i n g stream-i n g p o t e n t i a l s , of 200 m i l l i o s m o l a r was caused by sucrose w i t h an i o n b a t h i n g c o n c e n t r a t i o n of 10 mM/l KCI. Perfusion Rate (ml/min) Time (minutes) 47 s i d e from which water i s f l o w i n g . S i m i l a r l y , ionswould be swept away from the u n s t i r r e d l a y e r i n the s i d e to which water was f l o w i n g , so that i n t h i s r e g i o n the i o n c o n c e n t r a t i o n wmld be l e s s than i n the bulk of s o l u t i o n . Ions d i f f u s e down these l o c a l c o n c e n t r a t i o n gradients between the u n s t i r r e d l a y e r s , and could produce d i f f u s i o n p o t e n t i a l s even when the i o n l e v e l s i n the bulk of the solutionwere the same on both s i d e s . Since the water flow and i o n flow were i n the same d i r e c t i o n , then d i f f u s i o n p o t e n t i a l and streaming p o t e n t i a l were a d d i t i v e . In p r e l i m i n a r y s t u d i e s , (Lewis, 1970) the time course f o r development of streaming p o t e n t i a l s was measured. The i n i t i a l poten-t i a l d i f f e r e n c e s t a r t e d at zero and only b u i l t up to a maximum s i z e of 15 mv a f t e r 15 minutes. This could p o s s i b l y mean that the observed streaming p o t e n t i a l s might be the r e s u l t of a co n c e n t r a t i o n gradient forming across the membrane i n the u n s t i r r e d l a y e r s as a consequence of an osmotic flow of water (Schmid and Schwarz, 1952). A l t e r n a t i v e l y , the membrane p r e p a r a t i o n might not have been completely d r i e d p r i o r to use, so that a l a y e r of d i s t i l l e d water was i n i t i a l l y present on e i t h e r s i d e of the membrane. Thus the delay i n development of streaming p o t e n t i a l s could r e f l e c t the time r e q u i r e d f o r ions to move through t h i s f i l m of water. A number of d i f f e r e n t experiments were performed to e l u c i d a t e whether a l l o r p a r t of the streaming p o t e n t i a l phenomena was caused by u n s t i r r e d l a y e r s as r e c e n t l y suggested f o r the g a l l bladde by Wedner and Diamond (1969). The f i r s t experiment was to prepare the membrane i n the normal f a s i o n , and then dab dry both s i d e s of the mem-brane w i t h kleenex. The b a t h i n g s o l u t i o n s were then r a p i d l y p l a c e d on both s i d e s of the membrane, the potentiometer and paper chart recorde F i g . 19 The e f f e c t of p e r f u s i o n r a t e on streaming p o t e n t i a l , w i t h 10 mM/l KC1 on both s i d e s of the membrane and an osmotic gradient due to sucrose of 200 m i l l i o s m o l a r . The r e s u l t s are expressed as a f r a c t i o n of the p o t e n t i a l d i f -ference observed f o r i n d i v i d u a l p r e p a r a t i o n s i n the absence of mixing. The s t i p p l e d area represents the " i n s t a n t a n e o u s l y " developed streaming p o t e n t i a l when b a t h i n g s o l u t i o n s are added to a "dry" membrane. V e r t i c a l l i n e s represent standard e r r o r s f o r 6 observations. P e r f u s i o n rate ( m l / m i n ) 48 being turned on simultaneously. The b a t h i n g s o l u t i o n s used were 400 m i l l i o s m o l a r sucrose-10 mM/l KCI on the lumenal s i d e and 10 mM/l KCI on the haemocoel s i d e . As shown i n F i g . 17 an instantaneous streaming p o t e n t i a l of 7 mv developed across the membrane. The time r e q u i r e d to sw i t c h on the recorders and add the s o l u t i o n s took approximately 3 seconds. This instantaneous p o t e n t i a l was p o s t u l a t e d to be whol l y caused by streaming p o t e n t i a l s , w h i l e the steady but slow r i s e i n the p o t e n t i a l which f o l l o w e d could be a d i f f u s i o n p o t e n t i a l a s s o c i a t e d w i t h u n s t i r r e d l a y e r s . Such a p o t e n t i a l would be expected to be of the same s i g n as the streaming p o t e n t i a l . The previous experiment i n d i c a t e d that p a r t of the steady s t a t e streaming p o t e n t i a l measured might be a d i f f u s i o n p o t e n t i a l due to un-s t i r r e d l a y e r s . To f u r t h e r t e s t t h i s h y p o t h e s i s , the e f f e c t of v a r y i n g the t h i c k n e s s of the u n s t i r r e d l a y e r s (by v a r y i n g the p e r f u s i o n r a t e s ) on the s i z e of the "streaming p o t e n t i a l " was s t u d i e d . The experimental c o n d i t i o n s were the same as i n the previous experiment, except t h a t per-f u s i o n r a t e s were v a r i e d . Both s i d e s of the membrane were perfused u s i n g 2 LKB v a r i a b l e speed p e r i s t a l t i c pumps. The p e r f u s i o n r a t e being used at any one time was the same on both s i d e s of the membrane. The time course f o r the development of a steady p o t e n t i a l d i f f e r e n c e f o l l o w -i n g a change i n the p e r f u s i o n r a t e i s shown i n F i g . 18. P e r f u s i o n causes the p o t e n t i a l to drop r a p i d l y to a steady base l e v e l (shown as a f u n c t i o n of p e r f u s i o n r a t e i n F i g . 18). As the p e r f u s i o n r a t e i n -creased, the l a g time to reach a steady s t a t e p o t e n t i a l decreased. I t i s e v i d e n t from F i g . 19 that as the r a t e of p e r f u s i o n i n c r e a s e d , the F i g . 20 Upper curve represents n o n - l i n e a r i n c r e a s e i n streaming p o t e n t i a l s w i t h an i n c r e a s e i n osmotic pressure without s o l u t i o n p e r f u s i o n . Lower curve represents a n o n - l i n e a r i n c r e a s e i n streaming p o t e n t i a l s at a s o l u t i o n p e r f u s i o n r a t e of 2.8 m i l l i l i t e r s per minute. V e r t i c l e l i n e s are S.D. f o r 6 ob s e r v a t i o n s . See Appendix B Table 16 f o r i n d i v i d u a l v a l u e s . 14-, Concentration of sucrose in milliosmolars (water flow > ) F i g . 21 Resistance of the r e c t a l i n t i m a to water flow as r e f l e c t e d i n the r a t i o of osmotic pressure d i f f e r e n c e and streaming p o t e n t i a l d i f f e r e n c e . Each p o i n t represents mean value f o r 6 o b s e r v a t i o n s , f o r p e r f u s i o n of the i n -tima. Value c a l c u l a t e d from lower curve of F i g . 20. CQ A v e r a g e o s m o l a r i t y 49 steady s t a t e p o t e n t i a l d i f f e r e n c e decreased a s y m p o t i c a l l y and approached the value f o r the " i n s t a n t a n e o u s l y " developed p o t e n t i a l d i f f e r e n c e r e -corded i n the preceding type of experiment ( F i g . 17). The p o t e n t i a l s measured at the two h i g h e s t p e r f u s i o n r a t e s f e . l l w i t h i n t h i s band. This seened to i n d i c a t e that atthese p e r f u s i o n r a t e s the u n s t i r r e d l a y e r s had been l a r g e l y removed, so t h a t only true streaming p o t e n t i a l s were observed. I t was concluded then that water flow through the mem-brane caused not only a streaming p o t e n t i a l , but a l s o a d i f f u s i o n poten-t i a l . The d i f f u s i o n p o t e n t i a l i s beieved t o account f o r approximately 45% of what was p r e v i o u s l y designated as "streaming p o t e n t i a l " ( i . e . the p o t e n t i a l d i f f e r e n c e caused by water flow as opposed to a p o t e n t i a l d i f f e r e n c e caused by e x t e r n a l l y a p p l i e d c o n c e n t r a t i o n g r a d i e n t s . Since the osmotic flow seemed to produce two types of p o t e n t i a l d i f f e r e n c e ( i . e . a streaming p o t e n t i a l and a d i f f u s i o n p o t e n t i a l ) some of the o r i g i n a l experiments had to be repeated. Of immediate i n t e r e s t was whether e l i m i n a t i o n of u n s t i r r e d l a y e r s would a l t e r the conclusions concerning the evidence of the n o n - l i n e a r r a t e of water flow (where the r e s i s t a n c e to water flow i n c r e a s e s i n a l i n e a r manner w i t h the average o s m o l a r i t y ) . With t h i s i n mind the previous experiment ( F i g . 13) was repeated by p l a c i n g a 10 mM/l KC1 s o l u t i o n on both s i d e s of the i n t i m a , and 100, 200, 300 or 400 m i l l i o s m o l a r sucrose one one s i d e o n l y . The p e r f u s i o n r a t e was i n c r e a s e d to 2.8 ml/minute to a b o l i s h u n s t i r r e d l a y e r s (as suggested by r e s u l t s i n F i g . 19). In F i g . 20, the upper curve represents "streaming p o t e n t i a l s " without p e r f u s i o n w h i l e the lower curve represents streaming p o t e n t i a l s at a p e r f u s i o n r a t e of 2.8 ml/minute. Comparing the two curves, the r a t i o of the p o t e n t i a l s f o r 50 p e r f u s i o n versus no p e r f u s i o n was constant at approximately 0.5; i . e . the r a t i o of the two p o t e n t i a l s was independent of the osmotic g r a d i e n t . Thus i n the i n t a c t animal, the p o t e n t i a l d i f f e r e n c e produced by osmotic water flow under s i m i l a r c o n d i t i o n s would vary by a maximum of two-fold depending on the r a t e of f l u i d mixing. Most probably the values found f o r unperfused membranes were c l o s e s t to those i n v i v o . The n o n - l i n e a r i t y of osmosis was i n f a c t more accentuated w i t h p e r f u s i o n . Using Diamond's (1966) method f o r c a l c u l a t i n g the r e s i s t a n c e of the membrane to water f l o w from streaming p o t e n t i a l s , i t can be shown ( F i g . 21) that the r e s i s t a n c e to water flow remained a l i n e a r f u n c t i o n of the average o s m o l a r i t y of the b a t h i n g media. I . A d d i t i v e and C a n c e l l i n g P r o p e r t i e s of Streaming and D i f f u s i o n P o t e n t i a l s . The previous experiment i n d i c a t e d that both streaming and d i f -f u s i o n p o t e n t i a l s weie a s s o c i a t e d w i t h u n s t i r r e d l a y e r s . Itwas not c l e a r however, whether these two types of p o t e n t i a l s were q u a n t i t a t i v e l y a d d i t i v e or not. In order to make p r e d i c t i o n s concerning p o t e n t i a l d i f f e r e n c e s across the i n t a c t i n t i m a under v a r i o u s c o n d i t i o n s i n f u t u r e experiments, i t was of importance to have t h i s type of i n f o r m a t i o n . The approach taken i n answering t h i s q u e s tion was to create streaming and d i f f u s i o n p o t e n t i a l s simultaneously across the same membrane. The observed p o t e n t i a l d i f f e r e n c e could then be compared w i t h that p r e d i c t e d i f the two p o t e n t i a l s were s t r i c t l y a d d i t i v e , u s i n g data from previous experiments i n which each type of p o t e n t i a l F i g . 22 The a d d i t i v e and s u b t r a c t i v e p r o p e r t i e s of streaming p o t e n t i a l s and d i f f u s i o n p o t e n t i a l s created simultaneously across the same mem-brane. A 2 f o l d c o n c e n t r a t i o n d i f f e r e n c e of KC1 (10 mM/1-5 mM/l) was used to i n i t i a t e the d i f f u s i o n p o t e n t i a l i n a l l cases ( i . e . con-s t a n t ) . High c o n c e n t r a t i o n of KC1 was place d on the lumen s i d e . Sucrose was used to cause streaming p o t e n t i a l s of va r i o u s s i z e s . Suc-rose, when placed on the haemocoel s i d e caused streaming p o t e n t i a l s to be added to d i f f u s i o n p o t e n t i a l s (represented i n the graph by s o l i d c i r c l e s ) , and when pl a c e d on the lumen s i d e caused them to be su b t r a c t e d (represented i n the graph by open c i r c l e s ) . The s i g n i s w i t h reference to the haemocoel s i d e . V e r t i -c a l bars represent standard d e v i a t i o n s of 6 readings. See Appendix B, Table 17 f o r i n d i v i d u a l p r e p a r a t i o n s . Concentration of sucrose in milliosmolars 51 d i f f e r e n c e was measured s e p a r a t e l y under s i m i l a r c o n d i t i o n s . To c a r r y out t h i s experiment the membrane separated s o l u t i o n s w i t h concentrations of 10 and 5 mM/l KCI. Sucrose was added to the s i d e of low i o n c o n c e n t r a t i o n to e l i m i n a t e any osmotic g r a d i e n t s . To t e s t f o r the a d d i t i v e e f f e c t s of streaming p o t e n t i a l s on d i f f u s i o n p o t e n t i a l s , sucrose was added to the s i d e of low i o n c o n c e n t r a t i o n i n f i n a l c o n c e n t r a t i o n s of 100, 200, 300 or 400 m i l l i o s m o l a r . To t e s t f o r the s u b t r a c t i v e e f f e c t s of streaming p o t e n t i a l s on d i f f u s i o n poten-t i a l s , sucrose was added to the s i d e of h i g h i o n c o n c e n t r a t i o n i n f i n a l c o n c e n t r a t i o n s of 100, 200, 300 or 400 m i l l i o s m o l a r . In F i g . 22, i t can be seen t h a t streaming p o t e n t i a l s and d i f f u s i o n p o t e n t i a l s ware q u a n t i t a t i v e l y a d d i t i v e . I t can a l s o be seen t h a t streaming p o t e n t i a l s rare not e x a c t l y s u b t r a c t i v e from the d i f f u s i o n p o t e n t i a l s , but the discrepancy between observed and p r e d i c t e d values Wis only about 11%. 52 DISCUSSION Previous work on the i n t i m a ( P h i l l i p s and D o c k r i l l , 1968 and Lewis, 1970) i n d i c a t e d that the c h i t i n o u s membrane contains pores of 6-7 A* i n ra d i u s w i t h a p o s s i b i l i t y that laminar flow of water may occur through these pores. This was supported by the demonstration of stream-i n g p o t e n t i a l s i n the present study. Both streaming and d i f f u s i o n poten-t i a l s i n d i c a t e t h a t the membrane can s e l e c t f o r c a t i o n s over anions, were both of these p o t e n t i a l s caused by the same f i x e d charge, or must a separate channel he hypothsized f o r water flow and i o n movement? The s e l e c t i v i t y ( f o r monovalent c a t i o n s ) a s s o c i a t e d w i t h f i x e d charges as measured by streaming and d i f f u s i o n p o t e n t i a l s (Results 1 and 3)were q u a l i t a t i v e l y the same. Ion s e l e c t i v i t y i n both cases has been shown to decrease i n a s i m i l a r manner w i t h an i n c r e a s i n g i o n concentra-t i o n , however f o r a s i m i l a r decrease i n p o t e n t i a l d i f f e r e n c e , the i o n c o n c e n t r a t i o n was 5 times greater i n the b a t h i n g s o l u t i o n f o r d i f f u s i o n p o t e n t i a l s as compared to streaming p o t e n t i a l s ( F i g . 1 5 ) . High i o n con c e n t r a t i o n s masks the f i x e d charges and hence the s e l e c t i v i t y f o r c a t i o n over anion was decreased ( T e o r e l l , 1953), and disappears when anion and c a t i o n were of the same hydrated s i z e (as was the case f o r KC1). As the pH of a s o l u t i o n decreases, so does the s e l e c t i v i t y , as was a l s o found by Lannoye, Tarr and Dainty (1970 ) i n Chara a u s t r a l i s . The probable mechanism i s t h a t low pH decreases the c o n c e n t r a t i o n of negative charges on or i n the membrane (Dainty, Hore and Denby, 1960). The pKa and p i values of the f i x e d charges i n the r e c t a l i n t i m a as measured by streaming p o t e n t i a l s gave values of 3.7 f o r the pKa and 2.2 53 f o r the p i ; the same values were found f o r d i f f u s i o n p o t e n t i a l s (pKa of 3.7 and p i of 2.2). This supports the hypothesis t h a t s e l e c t i v i t y as measured by d i f f u s i o n and streaming p o t e n t i a l s i n v o l v e the same f i x e d charges, p o s s i b l y i n d i c a t i n g that f i x e d charges are l o c a t e d on w a l l s of the p o s t u l a t e d pores. Experiments w i t h d i v a l e n t s e r i e s of c a t i o n s suggested that the f i x e d charges were p o s s i b l y weak and w i d e l y spaced. The g r e a t e r +2 (10-fold) e f f i c i e n c y of Ca i n masking of the f i x e d charge r e l a t i v e to the e q u i v a l e n t c o n c e n t r a t i o n of monovalent c a t i o n s suggests t h a t the i n t e r a c t i o n of counter-ions w i t h the f i x e d charges may i n v o l v e a s e l e c t i v e b i n d i n g . This was e x p e r i m e n t a l l y supported when i t was shown +2 that at h i g h Ca c o n c e n t r a t i o n s the d i f f u s i o n p o t e n t i a l s were a c t u a l l y +2 +2 reversed. Such a r e v e r s a l i m p l i e d that the Ca and Mg i o n not only b l o c k the e x i s t i n g charges, but a l s o must a s s o c i a t e themselves w i t h the f i x e d charge and thus form a membrane w i t h a net excess of p o s i t i v e + +2 f i x e d charge. Whether the K conductance was decreased at h i g h Ca c o n c e n t r a t i o n or the C l conductance was i n c r e a s e d (or both) under these c o n d i t i o n s was not known; however, the K + to C l p e r m e a b i l i t y +2 r a t i o decreased to u n i t y as the amount of Ca was i n c r e a s e d i n the s o l u t i o n s . Wright and Diamond (1968) u s i n g the g a l l b l a d d e r , Cassidy and T i d b a l l (1967) usi n g l o b s t e r nerve and van Preeman (1968) usi n g p h o s p h o l i p i d - c h o l e s t e r o l a r t i f i c i a l membranes have a l l shown a s i m i l a r +2 Ca e f f e c t on d i f f u s i o n p o t e n t i a l s due to monovalent i o n s . Curran +2 and G i l l J r . (1961) showed that when Ca was added e x t e r n a l l y to f r o g s k i n , i t caused a decrease i n net sodium t r a n s p o r t . They b e l i e v e that +2 Ca decreased the p e r m e a b i l i t y of the outward f a c i n g membrane of the t r a n s p o r t i n g c e l l s . T a r r , Lannoye and Dainty (1970) found that changes 54 i n i o n i c p e r m e a b i l i t y d u r i n g a c t i o n p o t e n t i a l s i n Chara a u s t r a l i s can be +2 mediated by Ca . Van Preemen (1968) usi n g p h o s p h o l i p i d - c h o l e s t e r o l +2 a r t i f i c i a l membranes found that the a d d i t i o n of Ca reduced the d i f -f u s i o n p o t e n t i a l d i f f e r e n c e by 50% and the change i n the p o t e n t i a l was r e v e r s i b l e . Van Preemen b e l i e v e s that the d e p o l a r i z a t i o n i n d i c a t e s a l o s s i n c a t i o n p e r m s e l e c t i v i t y , which may be due to e i t h e r a decrease i n the number of f i x e d charged s i t e s or to an i n c r e a s e i n pore diameter ( H e l f f e r i c h , 1962). He e l i m i n a t e d the l a t t e r p o s s i b i l i t y because i n +2 the presence of Ca , the membrane r e s i s t a n c e i n c r e a s e d ; i . e . the pore diameter had decreased r a t h e r than i n c r e a s e d i n s i z e . Wright and Diamond (1968) came to the same c o n c l u s i o n i n t h e i r study w i t h the g a l l b l a d d e r , where they found that an i n c r e a s e i n the Ca*c"oncentration caused the Na^conductance to decrease and the Cl~conductance to i n c r e a s e . T h e i r c o n c l u s i o n , concerning the high Ca+£oncentration and low pH e f f e c t on the g a l l b l a d d e r , was that probably both b l o c k the same f i x e d s i t e s (Wright, Barry and Diamond, 1971), s i n c e the e f f e c t s of these two f a c t o r s on p e r m e a b i l i t y and conductance were q u a n t i t a t i v e l y s i m i l a r . The e f f e c t of d i v a l e n t c a t i o n s on the P^ / ^ r j i r a t i ° f ° r t n e +2 +2 r e c t a l i n t i m a i n d i c a t e s a competitive a f f i n i t y sequence of Ca > Mg As i n the experiments of Bungenburg De Jong's on c o l l o i d s and Wright and Diamond (1968) on g a l l b l a d d e r , the e q u i l i b r i u m s e l e c t i v i t y of the r e c t a l i n t i m a was independent of m o b i l i t y s e l e c t i v i t y . The p e r m s e l e c t i v i t y of the r e c t a l i n t i m a , f o r both monovalent and d i v a l e n t c a t i o n s was 55 + + + + + +2 +o 4-9 4-9 Rb > Cs > K > Na > L i and Ba > Ca > Sr > Mg but the equilibrium selectivity for monovalent and divalent cations was + + +2 +2 K > Na : Ca > Mg . This seemedto imply that the greater the af f i n i t y between ion and s i t e , the greater was the relative permeability across the membrane. Streaming potentials were observed to form across the rectal intima. The relationship between the latter and the osmotic gradient suggested the occurance of non-linear osmosis. Diamond (1966) proposed five different explanations for non-linearity of osmotic water flow across the ga l l bladder: i . membrane structure is deformed by water flow. i i . unstirred layers reduce the proportionality constant between osmotic gradient and water flow. i i i . force flow relation may be non-linear for an asymetrical system composed of several dissimilar membrane layers i n series (i.e. heterogeneous membrane), even though the relation may be linear for each individual membrane. iv . concentration of impermeant molecules might exert a non-osmotic effect on the membrane by altering chemical or physical inter-actions within the membrane i t s e l f (i.e. changes in the membrane structure). v. the concentration of molecules might exert an osmotic effect upon the membrane, altering the volume of f l u i d - f i l l e d channels and thereby changing the permeability. 56 The f i r s t of these p o s s i b i l i t i e s had been shown to be i n v a l i d s i n c e the streaming p o t e n t i a l was not dependent on the osmotic pressure g r a d i e n t but r a t h e r on the average o s m o l a r i t y of the two b a t h i n g s o l u -t i o n s . The p o s s i b i l i t y of u n s t i r r e d l a y e r s e f f e c t i n g streaming poten-t i a l s i n a n o n - l i n e a r manner can a l s o be e l i m i n a t e d , s i n c e i t was found th a t the streaming p o t e n t i a l s e x h i b i t e d greater n o n - l i n e a r i t y when u n s t i r r e d l a y e r s were mostly removed than when they were maximal ( i . e . no s t i r r i n g ) . The p o s s i b i l i t y t h a t the f o r c e - flow r e l a t i o n might be n o n - l i n e a r f o r an asymmetrical system, can be e l i m i n a t e d , s i n c e i t was found t h a t streaming p o t e n t i a l s across the membrane d i d not vary w i t h the d i r e c t i o n of flow under any one set of c o n d i t i o n s (Lewis, 1970). A l s o P h i l l i p s and Beaumont (1971) found no s u b s t a n t i a l d i f f e r e n c e i n the osmotic p e r m e a b i l i t y c o e f f i c i e n t w i t h d i r e c t i o n of water flow across the l o c u s t i n t i m a . Diamond (1966) found that v a r i o u s impermeant molecules a l l caused n o n - l i n e a r water flow suggesting that chemical i n t e r a c t i o n s which changed membrane s t r u c t u r e were not i n v o l v e d . Experiments i n t h i s chapter a l s o i n d i c a t e d that n o n - l i n e a r osmosis was not dependent on the type of molecule used. This leaves only the osmotic e f f e c t of molecules on membrane h y d r a t i o n . The d i r e c t evidence which supported t h i s a l t e r n a t i v e was that streaming p o t e n t i a l s (and thus water flow) were dependent on the average o s m o l a r i t y and not the osmotic g r a d i e n t , such that the r a t e of water flow could be expressed by the equation: (Equation 5) (0m-0s) / (Ro' + 1/2 K'(Om + Os) ) 57 Furthermore, i t was shown d i r e c t l y by determination of water content that i n c r e a s e i n o s m o l a r i t y of the b a t h i n g s o l u t i o n reduced the amount of the water i n the i n t i m a . P h i l l i p s and Beaumont (1971) found that the f l u x r a t e of water through the i n t i m a decreased approximately 50% w i t h 1.1 osmolal sucrose b a t h i n g the membrane as compared to d i s t i l l e d water. The n o n - l i n e a r i t y can be t e n t a t i v e l y a s c r i b e d to the membrane passages a c t i n g as osmotic compartments so t h a t dimensions of the pores were i n -fluenced by the c o n c e n t r a t i o n of the b a t h i n g medium. I t was found t h a t membrane dehydration d i d not a f f e c t the values of d i f f u s i o n p o t e n t i a l s caused by KCI g r a d i e n t s . H e l f e r r i c h (1962) suggests t h a t i n weakly c r o s s - l i n k e d i o n exchange membranes the d i f f u -s i o n c o e f f i c i e n t of the counter-ion may be increased by p a r t i a l dehydra-t i o n s i n c e dehydration would i n c r e a s e the charge d e n s i t y , thus reducing the d i s t a n c e that an i o n has to jump from one f i x e d charge to another, assuming-the same hydrated s i z e f o r both i o n s . I f the hydrated s i z e of the anion and c a t i o n were d i f f e r e n t , then the osmotic pressure might a f f e c t the m o b i l i t i e s of both ions through the membrane and thus the p o t e n t i a l d i f f e r e n c e . P h i l l i p s (1964 a) p o s t u l a t e d , among other mechanisms, e l e c t r o -osmosis as a p o s s i b l e f o r c e behind net water flow across the r e c t a l w a l l . F l u i d i n membrane pores c a r r y a net e l e c t r i c charge of the same s i g n as t h a t of the counter i o n s . Hence the water w i l l flow i n the same d i r e c t i o n as the counter i o n i . e . towards the s i d e of opposite charge. Electro-osmosis and streaming p o t e n t i a l s are r e l a t e d by the f o l l o w i n g equation ( d e r i v e d by i r r e v e r s i b l e thermodynamics; Dainty, 1966): 58 (Equation 6) H/P = J v / i In the present study the value of H observed ( f o r a c t u a l stream-i n g p o t e n t i a l s i n the absence of u n s t i r r e d l a y e r s ) was 3.5 mv, and the -12 value of P was 100 m i l l i o s m o l a r . This gives a value of 4.87x10 2 statvolts/dyne/cm f o r (H/P) (Smyth and Wright, 1966). The r a t e of net water a b s o r p t i o n across the r e c t a l w a l l as a whole i n the absence of 2 —6 2 osmotic gradients i s 17 ml/hr/cm or 4.72x10 ml/sec/cm i n the i n t a c t animal ( P h i l l i p s , 1964 a ) . I t i s of i n t e r e s t to determine whether t h i s net water movement across t h e i n t i m a observed i n v i v o could occur by e l e c t r o - o s m o s i s , or whether a l t e r n a t i v e l y , i t i s necessary to p o s t u l a t e an osmotic g r a d i e n t across the i n t i m a . I f t h i s net water movement i s 5 2 completely due to e l e c t r o - o s m o s i s , a current ( i ) of 9.67x10 statamps/cm ( i = JvP/H) i s r e q u i r e d according to the above equation. Using the average r e s i s t a n c e value f o r the membrane bathed i n 10 mM/l KC1 i s 2 145.3 ft cm . The p o t e n t i a l d i f f e r e n c e across the i n t i m a necessary to create a current of t h i s magnitude i s t h e r e f o r e 47 mv. Doing the con-verse c a l c u l a t i o n ( i . e . the r a t e of water flow which can be induced by a p o t e n t i a l d i f f e r e n c e across the i n t i m a of 3.5 mv, w i t h a membrane 2 4 r e s i s t a n c e of 145.3 ftcm ) gives a value f o r current ( i ) of 7.24x10 2 statamps/cm . Using the equation which r e l a t e s electron-osmosis to stream--12 i n g p o t e n t i a l s (Jv = iH/P) and p l a c i n g H/P equal to 4.87x10 s t a t -2 —6 2 volts/dyne/cm , a value f o r water flow of 0.352x10 ml/sec/cm , or 2 1.27 ul/hr/cm i s obtained. P h i l l i p s (1964 a) and V i e t i n g h o f f (1969) have observed a two step p o t e n t i a l d i f f e r e n c e across the r e c t a l w a l l . Present experiments 59 i n d i c a t e that the p o t e n t i a l d i f f e r e n c e between lumen and c e l l i n t e r i o r c ould be due i n p a r t to a p o t e n t i a l d i f f e r e n c e across the i n t i m a due to s e l e c t i v e d i f f u s i o n of cat i o n s across the l a t t e r i n s e r i e s w i t h a c a t i o n pump i n the e p i t h e l i a l l a y e r . However i f t h i s i s true then water flow from the lumen i n t o the s u b i n t i m a l space can not be due to e l e c t r o - o s m o s i s , s i n c e such a flow should be i n the opposite d i r e c t i o n . This i s because the s u b - i n t i m a l space would have to be negative f o r electro-osmosis to occur toward the haemocoel s i d e , w h i l e the present study suggested that net d i f f u s i o n of KCI toward the e p i t h e l i a l l a y e r would make that s i d e p o s i t i v e . In essence t h e r e f o r e electro-osmosis may occur from the sub-i n t i m a l space to the lumen. This suggests that when the r e c t a l lumen contains a low c o n c e n t r a t i o n of i o n s , so th a t i o n pumps i n the e p i t h e l i a l l a y e r m a i n t a i n a c o n c e n t r a t i o n gradient across the i n t i m a , s e l e c t i v e a b s o r p t i o n of ions w i l l occur but water flow w i l l be reduced due to the opposing e l e c t r o - o s m o t i c e f f e c t . The f i x e d charge p r o p e r t i e s of the i n t i m a could thus be of adaptive advantage to hydrated animals s i n c e s e l e c t i v e r e a b s o r p t i o n of ions but not water would be favored. On the other hand i n dehydrated animals w i t h excess body s a l t (and h i g h i o n conc e n t r a t i o n s i n the lumen; P h i l l i p s , 1964 b) the c a t i o n s e l e c t i v i t y of the i n t i m a should decrease very s u b s t a n t i a l l y , thus reducing i o n t r a n s f e r and f a c i l i t a t i n g water r e a b s o r p t i o n (by removal of the opposing e l e c t r o - o s m o s i s ) . Charged i o n i c groups present on the w a l l of the aqueous channels i n the i n t i m a create a z e t a p o t e n t i a l between the i o n i c s o l u t i o n i n the pore and the f i x e d groups. C a l c u l a t i o n of the zeta p o t e n t i a l f o r the 60 i n t i m a bathed i n e l e c t r o l y t e s o l u t i o n s i s p o s s i b l e by the f o l l o w i n g equation (Smyth and Wright; 1966): (Equation 7) . 4 im H/DP Using values of 0.01 poises f o r n and 80 f o r D at 20°C (Hand-book of Chemistry and P h y s i c s , 49th E d i t i o n ) , and c a l c u l a t e d values f o r -3 -12 2 n equal to 2.12x10 mhos and H/P = 4.87x10 statvolts/dyne/cm , t h i s equation y i e l d s a value of 4.36 mv f o r the zeta p o t e n t i a l . This i s a negative value because the pore i s a n i o n i c . Electro-osmosis or conversely streaming p o t e n t i a l s are important when the z e t a p o t e n t i a l i s l a r g e . From the c a l c u l a t e d z e t a p o t e n t i a l of the i n t i m a , at a b a t h i n g s o l u t i o n pH of 5.5, i t can be concluded that the i o n i c groups of the pore impart a s i g n i f i c a n t net charge to the water and thus can cause s i g n i f i -cant water movement by e l e c t r o - o s m o s i s . Davies, Hayden and R i d e a l (1956) found that the zeta p o t e n t i a l i s not a f f e c t e d by osmotic pressure. This i s i n agreement w i t h present observations that osmotic pressure does not" a l t e r the P /P^ r a t i o of the r e c t a l i n t i m a . The pH of the s o l u t i o n i n the immediate v i c i n i t y of the f i x e d charge i s a f f e c t e d by the z e t a p o t e n t i a l , as described by the f o l l o w i n g equation ( H a r t l y and Roe, 1940): (Equation 8) pHs = pHb + (•£ /60) at 25°C. This y i e l d s a value of 5.43 (approximately) f o r t h i s micro-c l i m a t e , i . e . pHb (Smyth and Wright, 1966). This s m a l l pH d e v i a t i o n w i l l not a f f e c t the pore charge, s i n c e the value i s s t i l l w e l l above the pKa f o r the f i x e d charge. An u n s t i r r e d l a y e r can be represented as an e q u i v a l e n t membrane i n s e r i e s w i t h the a c t u a l membrane, w i t h p e r m e a b i l i t y c o e f f i c i e n t "P" 61 given by: (Equation 9) w = D/6 The thi c k n e s s of an u n s t i r r e d l a y e r which could completely account f o r the r a t e of i o n movement across the i n t i m a can be estimated by l e t t i n g the value f o r the p e r m e a b i l i t y c o e f f i c i e n t be 45.3x10 ^cm/sec (Chapter 3) f o r i o n movement across the i n t i m a , and the value f o r -5 2 D^ be 1x10 cm /sec (Kidder e t a l . 1964). The thickness of an u n s t i r r e d l a y e r , which would be r a t e c o n t r o l l i n g f o r i o n movement across the i n t i m a i s estimated to be 2210 microns. An estimate f o r the thic k n e s s of u n s t i r r e d l a y e r s can a l s o be obtained from the time course f o r development of d i f f u s i o n p o t e n t i a l d i f f e r e n c e as des c r i b e d by Dainty and House (1966). These c a l c u l a -t i o n s were completed u s i n g two f o l d c o n c e n t r a t i o n gradients of KC1 which were allowed to " s i t " u n t i l the p o t e n t i a l went to zero. The zero poten-t i a l i n d i c a t e d a c o n c e n t r a t i o n gradient of u n i t y had been a t t a i n e d . The i o n c o n c e n t r a t i o n i n the u n s t i r r e d l a y e r s on both s i d e s of the i n t i m a were assumed to equal the i o n c o n c e n t r a t i o n of the b a t h i n g s o l u -t i o n c o n t a i n i n g the h i g h e s t i o n c o n c e n t r a t i o n . Using the equation derivedby Dainty and House (1966) f o r the c a l c u l a t i o n of A E 1/2 the p o t e n t i a l d i f f e r e n c e a f t e r t 1/2 had elapsed was: (Equation 10) A E 1/2 = A Eo + (A E °° - Eo) l o g ( 2 ( K i ' ) / { ( K i ' ) (Ki")>) l o g ( ( K ' ) / ( K i " ) ) the f o l l o w i n g observed values f o r the i n t i m a (A Eo = 0, A E 0 0 = 16 mv, K i ' = 10 mM/l, K i " = 5 mM/l and K' = 10 mM/l) y i e l d a value f o r the A E 1/2 p o t e n t i a l of 6.64 mv. The time r e q u i r e d f o r the p o t e n t i a l to reach h a l f maximum value i s then 3 seconds, when the p e r f u s i o n r a t e f o r the s o l u t i o n was 2.8 ml/minute. 62 Using the equation d e r i v e d by Dainty and House (1966) from data by Olson and Schulz (1942) f o r c a l c u l a t i o n of u n s t i r r e d l a y e r s . (Equation 11) t 1/2 = 0.38 <S2 Dk and s u b s t i t u t i n g the observed values f o r the i n t i m a ( t 1/2 3 seconds -5 2 and Dk = 1x10 cm /sec.) i n equation 11, the value of 6 i s 88.9 microns. Work done on a c a t i o n exchange membrane (Kidder et a l . 1964) i n d i c a t e d t h a t the u n s t i r r e d l a y e r s were approximately 20-25 microns i n t h i c k n e s s , i f a value f o r the d i f f u s i o n c o e f f i c i e n t of K i n aqueous -5 2 s o l u t i o n of 1x10 cm /sec. was used. Kidder e t a l . (1964) used the d i f f u s i o n c o e f f i c i e n t i n aqueous s o l u t i o n because the p o t e n t i a l d i f f e r -ence developed across t h i s type of membrane arose mainly at the s u r f a c e due to a Donnan d i s t r i b u t i o n of c a t i o n s ( T e o r e l l , 1953). Then the time course of the p o t e n t i a l d i f f e r e n c e change should only r e f l e c t d i f f u s i o n through t h i s u n s t i r r e d l a y e r . Whether or not the u n s t i r r e d l a y e r s p l a y an important r o l e i n membrane t r a n s p o r t depends p r i m a r i l y on the p e r m e a b i l i t y of the membrane i t s e l f to the molecular species being t r a n s p o r t e d . The importance of s t u d y i n g the u n s t i r r e d l a y e r s i s t h a t the movement of r a p i d l y permeating s o l u t e s might be r a t e - l i m i t e d by these l a y e r s r a t h e r than by the mem--7 2/sec brane. The measured D f o r the i n t i m a i s approximately 4.53x10 cm (Chapter 3 ) , which i s approximately 20 f o l d l e s s than the D i n f r e e s o l u t i o n . This d i f f e r e n c e then makes i t u n l i k e l y that the u n s t i r r e d l a y e r s are dominant i n c o n t r o l l i n g the r a t e of d i f f u s i o n across the r e c t a l i n t i m a at s t i r r i n g r a t e s used i n t h i s study. 63 CHAPTER I I I ION FLUX ACROSS THE INTIMA INTRODUCTION P h i l l i p s (unpublished observation) found that f l u x of Ca across the r e c t a l c u t i c l e was 50 times greater than t h a t of sucrose at normal r e c t a l pH, even though both chemical species have the same hydrated r a d i u s . This dienomena appeared c o n t r a r y to evidence p r e v i o u s l y found by P h i l l i p s and Beaumont (1968), which showed t h a t the r e l a t i v e r a t e of movement of n o n - i o n i c molecules across the i n t i m a i s r e l a t e d to t h e i r hydrated s i z e as d e s c r i b e d by the Renkin equation (1954). This can be e x p l a i n e d i n two ways; e i t h e r the l a t t e r r e l a t i o n s h i p does not h o l d f o r the r e c t a l i n t i m a or some other f a c t o r s i n f l u e n c e the movements of i o n s , w h i l e e x e r t i n g l i t t l e e f f e c t on n o n - i o n i c molecules. The present study (Chapter 1) i n d i c a t e s that the r e c t a l c u t i c l e possesses a f i x e d negative charge, r e s u l t i n g i n the membrane being c a t i o n s e l e c t i v e . This f a c t gives r i s e to two questions, ( i ) Generally speaking, how does the membrane f i x e d charge e f f e c t permeation of ions w i t h d i f f e r e n t charge (anion versus c a t i o n ) and valency (monovalent versus d i v a l e n t ) ; i . e . how much does permeation r a t e f o r ions d e v i a t e from that found f o r n o n - i o n i c molecules of s i m i l a r hydrated s i z e ? ( i i ) S p e c i f i c a l l y , can the presence of the f i x e d charge account completely f o r the p r e v i o u s l y observed 5 0 - f o l d discrepancy between p r e d i c t e d and observed values f o r +2 Ca f l u x across the intima? Previous experiments (Chapter 2) i n d i c a t e that at a pH of 5.5 or above, the d i f f u s i o n p o t e n t i a l across the i n t i m a does not i n c r e a s e 64 in size, implying that the net membrane charge was fu l l y dissociated, while at a pH of 2.3 the membrane was not cation selective, implying that the net charge was abolished. Using this information, i t was pos-sible to study the influence of fixed charge on the ion permeation across the intima by comparing the flux rate of any ion across the same preparation of these two pH values. The pH effect on basic membrane properties, excluding fixed charge, was considered i n a similar study using the uncharged molecule urea. +2 Results i n Chapter 2 indicated that Ca was 10 times as effective in masking the fixed charge as was a monovalent ion such as K+. The best explanation, previously, was that the a f f i n i t y of the +2 charge is greater for Ca than for the monovalent cations. It was i n i t i a l l y d i f f i c u l t to reconcile the fact that both selective binding +2 (i.e. association of Ca with the fixed charge) and accelerated move-+2 ment of Ca could be caused simultaneously by a fixed charge. The opportunity was therefore taken to compare the relationship between flux rate and binding capacity of the membrane as the charge density was varied by changing the membrane pH. +2 Since Ca movement across the intima might involve an inter-action with a series of binding sites, then flux might be expected to be influenced to a degree by the counter-ion flux i n the opposing direction as described by van Breemen (1968). Such a 'trans' effect was studied +2 by observing the influence of Ca concentration on the 'trans' side on +2 unidirectional flux rate of radioactive Ca 65 RESULTS A. Ion Fluxes a t Various Concentrations and pH v a l u e s . 45 F l u x r a t e s of Ca at fo u r d i f f e r e n t concentrations of C a C l 2 and at 2 pH values (5.5 and 2.3) are shown i n F i g . 23. At a pH of 5.5 the f l u x r a t e s d i d not in c r e a s e i n a d i r e c t l y p r o p o r t i o n a l manner w i t h i n -+2 +2 c r e a s i n g Ca c o n c e n t r a t i o n s ; i . e . Ca p e r m e a b i l i t y tended to decrease +2 at very h i g h Ca conc e n t r a t i o n s ( F i g . 24) probably due to masking of f i x e d charges. At pH 2.3 ( F i g . 24) probably due to masking of f i x e d charges. At pH 2.3 ( i . e . no f i x e d charge) however ( F i g . 23) the f l u x +2 r a t e was d i r e c t l y p r o p o r t i o n a l to the Ca c o n c e n t r a t i o n ; i . e . there was +2 +2 a l i n e a r r e l a t i o n s h i p between Ca p e r m e a b i l i t y and Ca c o n c e n t r a t i o n ( F i g . 24). At a c o n c e n t r a t i o n of 1000 mM/l, pH had no e f f e c t on the +2 +2 Ca f l u x . However, as Ca c o n c e n t r a t i o n was decreased the e f f e c t of +2 pH on Ca f l u x became i n c r e a s i n g l y pronounced. Thus at a concentra-t i o n of 100 mM/l the f l u x a t pH 5.5 was 6.5 times as great as at pH 2.3, w h i l e t h i s f a c t o r reached 81 times at a c o n c e n t r a t i o n of 10 mM/l C a C l 2 . These d i f f e r e n c e s i n f l u x r a t e s at the two pH values might be a t t r i b u t a b l e to a r e v e r s i b l e s t r u c t u r a l change i n the membrane s t r u c t u r e r a t h e r than to f i x e d charge d e n s i t y . This p o s s i b i l i t y was t e s t e d by measuring the f l u x r a t e of a n o n - e l e c t r o l y t e , urea, at the same two pH values (Table 6; urea does not become an e l e c t r o l y t e u n t i l 14 pH 0.1). The f l u x r a t i o (pH 5.5:2.3) f o r urea-C was 1.52 which was +2 very s m a l l when compared to the f l u x r a t i o of 81 f o r Ca at 10 mM/l C a C l 2 . I t appearedthat the presence of f i x e d charges can completely F i g . 23 Measurement of Ca f l u x across the i n t i m a at four d i f f e r e n t CaCl^ concentrations (same on both s i d e s ) and two pH v a l u e s . The u n i d i r e c t i o n a l f l u x was measured u s i n g Calcuim-45, and the c o n c e n t r a t i o n of b a t h i n g s o l u t i o n was v a r i e d from 1000 mM/l to 1 mM/l C a C l 2 . The pH values used were 5.5 and 2.3. S o l i d c i r c l e s represent values of ca l c i u m f l u x a t pH of 5.5, w h i l e open c i r c l e s are c a l c i u m f l u x e s at pH 2.3. V e r t i c a l bars represent standard d e v i a t i o n s f o r 6 p r e p a r a t i o n s . See Table 6 f o r va l u e s . F i g . 24 Calcium p e r m e a b i l i t y c o e f f i c i e n t f o r the i n t i m a at four d i f f e r e n t CaCl^ concentrations and two pH val u e s . The p e r m e a b i l i t y c o e f f i c i e n t s were 45 c a l c u l a t e d from u n i d i r e c t i o n a l Ca f l u x measurements. The c o n c e n t r a t i o n of both b a t h i n g s o l u t i o n s were the same and were v a r i e d from 1000 to 1 mM/l. The pH values used were 5.5 and 2.3. S o l i d c i r c l e s represent Ca p e r m e a b i l i t y c o e f f i c i e n t s at a pH of 2.3. Each p o i n t represents mean value f o r 6 p r e -p a r a t i o n s . V e r t i c a l l i n e s i n d i c a t e the standard d e v i a t i o n where i t exceeds the width of the symbols. See Table 6 f o r values. Table 6 Flux rates and permeability c o e f f i c i e n t s o monovalent and d i v a l e n t anions and cations expressed as the mean value ± standard deviation of 6 observations. Radio Isotope Concentration pH (mM/l) 4 5 C a C l 2 1000 5.5 4 5 C a C l 2 1000 2.3 4 5 C a C l 2 100 5.5 4 5 C a C l 2 100 2.3 4 5 C a C l 2 10 5.5 4 5 C a C l 2 10 2.3 4 5 C a C l 2 1 5.5 45 CaCl 2-l Mordue 10 5.5 CaCl 2-2 Mordue io 5.5 Urea C-14 10 5.5 Urea C-14 10 2.3 Na 2 S0 4 10 5.5 Na 2 3 5S0 4 10 2.3 8 6RbCl 10 5.5 8 6RbCl 10 2.3 K 3 6C1 10 5.5 K 3 6C1 10 2.3 fflux Ratio of Fluxes PxlO cm/sec *S.D (uM/hr/cm2tSX>.) (pH 5.5/2.3) 20.27 * 9.54 0.83 5.6 + 2,65 24.65 t 9.34 6,8 + 2.6 5.06 ± 0.74 6.54 14.06 + 2.06 0.83 - 0.22 2.3 + 0.61 1.39 ± 0.41 81.0 38.6 + 11.4 0.02 t 0.01 0.56 + 0.28 0.12 ± 0.11 33.3 + 30.3 0.88 ± 0.19 24.4 + 5.28 0.63 t 0.17 17.5 + 2.73 1.52 t 1.25 1.52 42.2 + 34.8 0.98 ± 1.09 27.2 + 30.3 0.01 - 0.007 0.27 0.28 + 0.29 0.04 t 0.023 1.11 + 0.65 1.63 t i 0.36 1.25 45.3 + 10.0 1.30 - 0.25 36.1 + 6.85 0.40 ± 0.12 0.42 11.1 + 3.34 0.96 t 0.31 26.7 + 8.6 66 +2 account for the greater movement of Ca r e l a t i v e to uncharged molecules +2 of the same s i z e . In the absence of the f i x e d charge Ca movement was reduced to that predicted by Renkin's equation (1954) for uncharged molecules. Since the concentration of monovalent ions i n the locust rectum was often very high (eg. 500 meg/L) the p o s s i b i l i t y that high i on con-+2 centrations might abolish the s e l e c t i v e permeability to Ca i n the +2 presence of f i x e d charges (pH 5.5) was considered. The Ca f l u x at a concentration of 10 mM/l C a C ^ was measured i n the presence of both normal and double strength Mordue^s Ringers (See Table 7 f o r the composi-tion) on both sides of the membrane at a pH of 5.5. The r e l a t i v e l y +2 minor drop i n Ca f l u x when Mordue's Ringer was added (Table 6) in d i c a t e s +2 that the a f f i n i t y f o r the f i x e d charges f o r Ca i s much greater than most of the other ions i n the ri n g e r . The s l i g h t decrease i n f l u x might r e s u l t i n part from an osmotic e f f e c t (eg. on pore area) rather than from the masking of the charged s i t e s by other ions. The effects of f i x e d negative charges on the f l u x of a monovalent cation and anion and a di v a l e n t anion were also considered. For the monovalent cation (Rb +, Table 6), the pH did not s i g n i f i c a n t l y a f f e c t the f l u x r a t e s . The f l u x rate of C l increased 2.5 times when the pH -2 was decreased from 5.5 to 2.3, while the f l u x rate of SO . increased 4 approximately 4 times. The influence of the fi x e d charge i n a l l these +2 cases i s r e l a t i v e l y small compared to that f o r Ca , suggesting a +2 r e l a t i v e l y s p e c i f i c a f f i n i t y of the negative charge f o r Ca or at l e a s t , d i v a l e n t cations. Table 7 Mordue's Ringer Chemical species grams/liter Moles/liter NaCl 9.82 0.168 KC1 0.48 6.44xl0"3 MgCl2-6H 20 0.73 3.6xl0"3 CaCl 2-6H 20 0.01 NaH 2P0 4'H 20 0.84 6.1xl0"3 NaHCOg 0.18 2.14xl0"3 glucose 3.0 16.6xl0~3 67 B. Membrane B i n d i n g Capacity. +2 The Ca f l u x a t a c o n c e n t r a t i o n of 10 mM/l C a C ^ and a pH of 5.5 was 8 1 - f o l d g r e a t e r than at a pH of 2.3. One e x p l a n a t i o n for t h i s +2 i n c r e a s e i n Ca movement might be that the f i x e d charge i n the mem-brane creates a Donnan d i s t r i b u t i o n i n the pores such that the con-+2 c e n t r a t i o n of Ca i s 81 times higher at pH 5.5 than at pH 2.3. F l u x +2 ra t e might then simply be p r o p o r t i o n a l to membrane Ca c o n c e n t r a t i o n . To t e s t t h i s p o s s i b i l i t y , membranes were soaked i n a 10 mM/l r a d i o -45 45 a c t i v e C a C ^ s o l u t i o n a t pH of 5.5 and 2.3, damped dry and the Ca 45 content of the i n t i m a determined (Table 1 ) . At pH 2.3 the Ca con-c e n t r a t i o n i n the i n t i m a per l i t e r of membrane water was approximately the same as i n the b a t h i n g s o l u t i o n . At normal r e c t a l pH of 5.5 the 45 Ca c o n c e n t r a t i o n i n the i n t i m a was only 3 times h i g h e r . The r a t e of +2 i n c r e a s e i n Ca f l u x w i t h pH in c r e a s e (81 times) would appear not to be simply r e l a t e d to the membrane co n c e n t r a t i o n of Ca (3 f o l d i n c r e a s e ) . These i n i t i a l o bservations suggested two question s : (a) When +2 the pH value i s inc r e a s e d beyond 5.5, how w i l l the uptake of Ca be a f f e c t e d ? (b) What i s the r e l a t i o n s h i p between membrane b i n d i n g of +2 +2 Ca and Ca f l u x rate? To answer these questions 6 membrane prepara-45 t i o n s were p l a c e d i n 1 mM/l r a d i o a c t i v e Ca s o l u t i o n at each of the f o l l o w i n g pH v a l u e s : 3.5, 3.8, 5, 6, 7 or 8, w i t h a constant concentra-+2 +2 t i o n of Ca i n the medium. The Ca b i n d i n g c a p a c i t y of the membranes as a f u n c t i o n of pH i s shown i i F i g . 25b. At a pH of 2.2 the b i n d i n g again was n e g l i g i b l e . As the pH was in c r e a s e d from 2.2 to 5, the F i g . 25 A. Ca f l u x as a f u n c t i o n of ba t h i n g s o l u -t i o n pH. Both s i d e s of the membrane contained 10 mM/l C a C ^ and one s i d e was i n i t i a l l y 45 l a b e l l e d w i t h Ca . V e r t i c a l bars i n d i c a t e standard d e v i a t i o n f o r 6 p r e p a r a t i o n s . P o i n t at pH9 was the mean of two p r e p a r a t i o n s . B. The i n t i m a was allowed to soak i n ImM/l 45 Ca s o l u t i o n . The pH was v a r i e d from an i n i t i a l value of 2.2 to a f i n a l value of 8. +2 The Ca uptake was p l o t t e d as a f u n c t i o n of pH. V e r t i c a l l i n e s represent standard d e v i a -t i o n s of 6 p r e p a r a t i o n s . See Appendix B Table 18 f o r i n d i v i d u a l v a l u e s . pH of CaCl 2 bathing solution 68 +2 b i n d i n g of Ca i n c r e a s e s s i g m o i d a l l y by 1 2 - f o l d . From pH 5 to 6 the +2 amount of i n c o r p o r a t e d Ca d i d not i n c r e a s e ( F i g . 25b). However, +2 when the pH was i n c r e a s e d from 6 to 8, the amount of Ca i n the mem-+2 brane again i n c r e a s e d 2 - f o l d . The t o t a l i n c r e a s e i n membrane Ca between pH 2.2 and 8 was 2 4 - f o l d . +2 The i n c r e a s e i n membrane Ca might be caused by two f a c t o r s : ( i ) an i n c r e a s e d number of f i x e d negative s i t e s over f i x e d p o s i t i v e s i t e s as the pH i n c r e a s e d and ( i i ) an i n c r e a s e d a f f i n i t y of the e x i s t -+2 i n g negative s i t e s f o r Ca , each s i t e u l t i m a t e l y b i n d i n g more than one +2 Ca i o n ( L i n g , 1960). One p o s s i b l e means of d i f f e r e n t i a t i n g between 45 these p o s s i b i l i t i e s was to extend the measurement of Ca f l u x as a f u n c t i o n of pH ( F i g . 25a). The f l u x r a t e i n c r e a s e d as the pH i n -creased from a pH of 2.3 to 5.5, but then decreased as the pH i n -+2 creased above 5.5, even though Ca b i n d i n g i n c r e a s e d . Obviously there i s no simple d i r e c t r e l a t i o n s h i p between the degree of b i n d i n g and the f l u x r a t e . C. Trans E f f e c t of the Intima. +2 The i n c r e a s e i n Ca b i n d i n g of the i n t i m a as the pH i n c r e a s e s from pH 2.2 to 8, suggests that the f l u x r a t e i s l i m i t e d by the a b i l i t y +2 of Ca to d i s s o c i a t e i t s e l f from the f i x e d charge. I f the f i x e d charge +2 +2 i s a r a t e l i m i t i n g step f o r Ca movement then Ca f l u x might be ex-pected t o e x h i b i t a trans e f f e c t ; i . e . f l u x r a t e should be i n f l u e n c e d +2 +2 by a Ca c o n c e n t r a t i o n on the s i d e to which f l u x occurs. Ca f l u x was t h e r e f o r e measured w i t h 10 mM/l C a C ^ on both s i d e s of the membrane, or F i g . 26 Accumulated f l u x of Ca45 w i t h time. S o l i d +2 squares i n d i c a t e f l u x w i t h 10 mM/l Ca on one s i d e of the membrane and b u f f e r e d sucrose s o l u -t i o n on the other s i d e . Open c i r c l e s i n d i c a t e +2 f l u x w i t h 10 mM/l Ca on both s i d e s of the membrane. A s t e r i c k represents s i d e of the i n t i m a which contains the r a d i o i s o t o p e . Each p o i n t represents the range of two obs e r v a t i o n s . "frlO mM CaCl ! Sucrose ^TIO mM CaCl 9 | 10 mM CaCl 0 2 ; I 2 8 -, CM ^ 6 S o O 4 H 2 J 0 0 2 3 4 5 Hours "3 69 w i t h the i o n i c s o l u t i o n on the trans s i d e r e p l a c e d by a sucrose s o l u t i o n of equal o s m o l a r i t y to eliminate water flow . F i g . 26 shows th a t there +2 2 was a Ca f l u x of 0.51 uM/hr/cm when there was no CaCl^ on the trans s i d e . With a CaCl^ s o l u t i o n on both sides of the membrane, the f l u x 2 r a t e i n c r e a s e d to a value of 1.14 uM/hr/cm . This experiment seems to +2 i n d i c a t e the e x i s t e n c e of a trans e f f e c t f o r Ca movement. This would +2 be e x p l a i n e d i f Ca becomes d i s s o c i a t e d from the f i x e d s i t e moiE e a s i l y +2 i f another Ca i o n i s a v a i l a b l e to replace i t . Exchange d i f f u s i o n i s u s u a l l y a t t r i b u t a b l e to a h y p o t h e t i c a l c a r r i e r molecule. I t i s of i n t e r e s t that the same e f f e c t ( i . e . exchange d i f f u s i o n ) can be observed i n a membrane posse s s i n g f i x e d charged s i t e s . 70 DISCUSSION The s e l e c t i v i t y of the negative s i t e s i n the i n t i m a has been estimated from d i f f u s i o n p o t e n t i a l s from which the r e l a t i v e p e r m e a b i l i t y of the membrane to v a r i o u s ions can be c a l c u l a t e d u s i n g the Goldman f i e l d e quation (Appendix A ) . R e l a t i v e p e r m e a b i l i t y was a l s o determined by i s o t o p e f l u x as o u t l i n e d i n t h i s Chapter. The f l u x r a t i o of Rb:Cl was found to be 4 (range 3.8-4.5) at a b a t h i n g media c o n c e n t r a t i o n of 10 mM/l. The :P^j. rat-^-° estimated from d i f f u s i o n p o t e n t i a l measurements at the same average c o n c e n t r a t i o n of b a t h i n g media ( F i g . 6) was approximately 5 (range 4-7). Thus there was good agreement between the two d i f f e r e n t methods of measuring r e l a t i v e p e r m e a b i l i t y to i o n s . While good agreement was obtained by comparing r e l a t i v e p e r m e a b i l i t y v a l u e s , there i s an i n h e r e n t danger i n comparing absolute values be-cause c o n d i t i o n s may d i f f e r i n the two types of experiments. For example u n s t i r r e d l a y e r s could a l t e r the f l u x r a t i o i f they represent a s u b s t a n t i a l f r a c t i o n o f the t o t a l r e s i s t a n c e to i o n f l u x i n one but not i n the other type of experiment. P h i l l i p s and Beaumont (1971) showed a decrease i n f l u x r a t e of water across the i n t i m a w i t h an i n c r e a s e i n the e x t e r n a l osmotic p r e s -sure. The same may be true f o r i o n f l u x e s . The e f f e c t s of h i g h osmotic pressure on d i f f u s i o n p o t e n t i a l s was considered i n Chapter 2. Although there was no s t a t i s t i c a l l y s i g n i f i c a n t decrease i n the d i f f u s i o n p o t e n t i a l there was a g e n e r a l downward trend. However t h i s only i n d i c a t e s an e f f e c t on the r e l a t i v e r a t h e r than the absolute p e r m e a b i l i t y . The P k : P C 1 r a t i o decreased by 0.09 per 100 m i l l i o s m o l a r change. This 71 i n d i c a t e d t h a t r e l a t i v e p e r m e a b i l i t y i s only s l i g h t l y changed w i t h an i n c r e a s e i n osmotic p r e s s u r e . The b a t h i n g media used f o r measuring C l f l u x c o n s i s t e d of a KCI s o l u t i o n . Some of the C l might have crossed the i n t i m a as a molecule of KCI. Under the assumption that a molecule of KCI can d i f f u s e across the membrane as e a s i l y as a n e u t r a l molecule (u r e a ) , and assuming a value f o r the a c t i v i t y c o e f f i c i e n t of the KCI s o l u t i o n of 0.925 (Lange's Handbook of Chemistry), then approximately 7.5% of the f l u x w i l l occur as a n e u t r a l molecule of KCI. This would change the 86 36 f l u x r a t i o (Rb :C1 ) to a new value of 5.6:1, approximately that found f o r the :PC1 r a t i o . Rb The r a t i o of Rb^^ to Ca^"* f l u x was found to be one, w h i l e the r e l a t i v e p e r m e a b i l i t y as estimated from d i f f u s i o n p o t e n t i a l s gave a J?Rb SPQ r a t i o of 3.8. However i n Chapter 2 i t was reported t h a t the +2 + presence of Ca decreased the conductance of the i n t i m a ID K or i n c r e a s e d the conductance to C l , the p o s s i b l e mechanism being that +2 Ca binds to the negative s i t e . Such b i n d i n g might then i n c r e a s e the C l movement and reduce the e l e c t r o p o t e n t i a l d i f f e r e n c e , thereby reduc-+2 i n g the apparent r e l a t i v e p e r m e a b i l i t y to Ca as measured by d i f f u s i o n p o t e n t i a l s . This may account f o r the discrepancy i n r e l a t i v e p e r m e a b i l i t y +2 + values of Ca and Rb as determined by the two methods. Changes i n the s w e l l i n g of an i o n exchanger c h i e f l y a f f e c t s the i n d i v i d u a l d i f f u s i o n c o e f f i c i e n t s . The d i f f u s i o n i s more s t r o n g l y hindered by the m a t r i x when the f r e e s o l v e n t c o n c e n t r a t i o n i s low ( H e l f f e r i c h , 1962). The degree of s w e l l i n g i s determined by the chemi-c a l nature of the membrane, the degree of cross l i n k i n g of the m a t r i x and the d e t a i l s of the network s t r u c t u r e , the c o n c e n t r a t i o n of the i o n i z a b l e 72 groups i n the membrane, the nature of the ions a s s o c i a t e d w i t h these groups, the s o l u t e c o n c e n t r a t i o n of the b a t h i n g s o l u t i o n and the temperature. S w e l l i n g i s r e s t r i c t e d by any t e n s i o n which i s set up i n the m a t r i x . The u l t i m a t e amount of s w e l l i n g i s dependent on the type of c o u n t e r - i o n present. The former tends to be grea t e r i n the presence +2 of h i g h l y hydrated i o n s , such as Mg , r a t h e r than a r e l a t i v e l y non-hydrated i o n such as C s + or Rb +. Rice and Nagasawa (1961) and F l e t t and Meares (1966) found t h a t t h i s was not n e c e s s a r i l y true i n a l l cases. They found, i n c e r t a i n cases, that when the f i x e d ions are monovalent and the counter ions are d i v a l e n t , the o v e r a l l e l e c t r o s t a t i c e f f e c t i s to r e s t r i c t r a t h e r than i n c r e a s e the s w e l l i n g . This was i l l u s t r a t e d +2 by F l e t t and Meares (1966) f o r a a n i o n i c membrane, i n which Ca r e -s t r i c t e d the amount of w e l l i n g to a gr e a t e r extent that d i d K. +2 L e i t c h and Tobias (1964) reported that Ca decreased the amount of s w e l l i n g of a p h o s p h o l i p i d - c h o l e s t e r o l membrane, whereas monovalent io n s ( K + and Na +) a c t u a l l y i n c r e a s e d s w e l l i n g . D i v a l e n t i o n s o r p t i o n i n the l a t t e r a r t i f i c i a l membrane probably caused a decrease i n the wi d t h of the aqueous channels p e n e t r a t i n g the membrane, r e s u l t i n g i n a decrease i n s o l u t i o n movement. Thus exchanging monovalent f o r d i v a l e n t ions w i l l a f f e c t the membrane p e r m e a b i l i t y . Such an a f f e c t has been shown by van Breemen (1968) i n p h o s p h o l i p i d - c h o l e s t e r o l membranes i n which he +2 found t h a t Ca reduced the membrane h y d r a t i o n and consequently i o n i c p e r m e a b i l i t y . I t i s b e l i e v e d that a s i m i l a r dehydration e f f e c t i s +2 present i n the i n t i m a ; i . e . the presenceof Ca reduces the pore s i z e and subsequently the t o t a l amount of water present i n the i n t i m a , as i s demonstrated i n F i g . 1. The i n t i m a , when bathed i n 10 mM/l CaCl_ a t 73 pH 5.5 had a water content of 67% by weight, w h i l e the same membrane bathed i n 10 mM/l C a C l 2 at pH 2.2 had a water c a p a c i t y of 72%, these values being s t a t i s t i c a l l y s i g n i f i c a n t (p < 0.05). This p o s s i b l y ex-p l a i n s why at h i g h C a C ^ concentrations of the ba t h i n g media (1000 mM/l) the f l u x r a t e at pH 2.2 was greater than the f l u x r a t e a t a pH of 5.5 ( F i g . 23). H e l f f e r i c h (1962) showed that at low pH a n e g a t i v e l y charged membrane sw e l l e d l e s s than at a h i g h pH, and s w e l l i n g decreased as the b a t h i n g s o l u t i o n c o n c e n t r a t i o n i n c r e a s e d . This would r e s u l t i n a r e -du c t i o n of the d i f f u s i o n c o e f f i c i e n t or p e r m e a b i l i t y c o e f f i c i e n t as found f o r the i n t i m a . The f l u x of urea (a n o n - e l e c t r o l y t e and thus u n a f f e c t e d by charge) was greater at h i g h pH than at low pH. At h i g h +2 Ca co n c e n t r a t i o n s however, the l a t t e r e f f e c t was not observed ( F i g . 23; +2 at low pH the Ca f l u x was g r e a t e r than at high pH). This suggests +2 tha t the Ca i n the membrane at high pH reduces the pore s i z e to a gre a t e r extent than does a simple e l i m i n a t i o n of the f i x e d charge. The s e l f - d i f f u s i o n c o e f f i c i e n t of counter ions i n a i o n exchange r e s i n has been found to be very s e n s i t i v e to the c o n c e n t r a t i o n of the e x t e r n a l s o l u t i o n ( H e l f f e r i c h , 1962). The d i f f u s i o n c o e f f i c i e n t of +2 Ca i n c r e a s e d markedly as the s o l u t i o n c o n c e n t r a t i o n i n c r e a s e d , es-p e c i a l l y i n d i l u t e s o l u t i o n s , as i s shown i n F i g . 24. However the d i f f u s i o n c o e f f i c i e n t becomes r e l a t i v e l y constant at higher concentra-t i o n s and i n very concentrated s o l u t i o n s i t begins to decrease due presumably to the osmotic shrinkage of the r e s i n ( H e l f f e r i c h , 1962). D i f f u s i o n then seems to be retarded mainly by the o b s t r u c t i n g e f f e c t s of the ma t r i x and p o s s i b l y the v i s c o s i t y of the i n t e r s t i t i a l f l u i d . 74 Van Breemen and van Breemen (1966) showed i n the r a t uterus that +2 +2 Ca f l u x was reduced by removal of Ca from the s i d e to which i s o t o p i c +2 d i f f u s i o n was o c c u r i n g . However, EDTA or Sr added to the trans s i d e +2 completely prevented t h i s decrease i n e f f l u x r a t e w h i l e Pa only p a r t i a l l y prevented i t . Van Breemen (1968) showed usi n g a r t i f i c i a l porous p h o s p h o l i p i d - c h o l o s t e r o l membranes, that the membrane concentrated +2 + + ca t i o n s w i t h s e l e c t i v i t y f o r Ca ions over Na and K . They a l s o +2 +2 noted that Ca e f f l u x was slowed down by Ca f r e e media on the 'trans ' +2 +2 s i d e , w h i l e a d d i t i o n of Ca s t i m u l a t e d the Ca f l u x . Luxoro and +2 Yanez (1968) showed that the r a t e l i m i t i n g step i n the e f f l u x of Ca +2 from a p e r f o r a t e d c o l l o d i u m tube f i l l e d w i t h Ca l a b e l l e d axoplasm was not the b a r r i e r o f f e r e d by the p e r f o r a t e d membrane, but r a t h e r by +2 the r a t e of d i s s o c i a t i o n of the complex formed between Ca ions and axoplasmic p r o t e i n s . Van Breemen and van Breemen (1969) found two i n -+2 f l e c t i o n p o i n t s f o r s o r p t i o n isotherms of Ca i n p h o s p h o l i p i d -c h o l e s t e r o l membranes; i e . as the pH i n c r e a s e d from 2.2 to 4.5 the +2 Ca content of the membrane i n c r e a s e d and then plateaued u n t i l pH 6, +2 +2 at which p o i n t the Ca content s t a r t e d to i n c r e a s e again. Ca f l u x i n c r e a s e d to a maximum at a pH of 5.5 and then decreased as the pH inc r e a s e d above the value of 5.5. Papahadjopoulos (1968) found that the c a r b o x y l groups of p h o s p h a t i d y l s e r i n e were d i s s o c i a t e d at a pH of +2 5 and the amino groups at a pH of 9. The b i n d i n g of Ca to such a f i l m (Rojas and Tobias, 1965) corresponds to i t s s o r p t i o n isotherm. +2 At s a t u r a t i o n approximately one molecule of Ca was bound as the 75 c a r b o x y l i c a c i d d i s s o c i a t e s and a second as the amino group was d e p o l a r i z e d ; i . e . another negative charge was exposed. However measurement of the surf a c e p o t e n t i a l of the f i l m , a t d i f f e r i n g pH values and i n the +2 +2 presence of Ca , i n d i c a t e d an i n c r e a s e d Ca b i n d i n g w i t h i n i t i a l d i s s o c i a t i o n of the a c i d i c groups, but gave no evidence of e x t r a b i n d i n g on d i s s o c i a t i o n of the amino groups. This paradox might be +2 ex p l a i n e d by m u l t i p l e b i n d i n g of Ca w i t h both the a c i d i c groups and theceprotonated amino groups. This experiment and others l e d Luxoro and Yanez (1968) to the same c o n c l u s i o n ; i . e . f l u x from the membrane was +2 c o n t r o l l e d by the r a t e of d i s s o c i a t i o n of Ca from the membrane. +2 This same a f f e c t was found i n the i n t i m a . Ca movement through the charged membrane at low i o n concentrations probably occurs by s i t e to s i t e t r a n s f e r , s i n c e f l u x a t hig h pH was 81 times g r e a t e r than a t low pH v a l u e s . As the c o n c e n t r a t i o n i n c r e a s e d the simple d i f f u s i o n component becomes predominant over s i t e to s i t e t r a n s f e r . The r a t e l i m i t i n g step +2 +2 f o r Ca f l u x seemed to be the r a t e of d i s s o c i a t i o n of Ca from the f i x e d charge, s i n c e ( i ) an i n c r e a s e i n membrane b i n d i n g above a c e r t a i n +2 +2 value r e s u l t e d i n a decreased Ca f l u x and ( i i ) Ca f l u x i s inc r e a s e d +2 i f Ca was present on both s i d e s of the membrane. The f l u x of monovalent c a t i o n s seemed to be r e l a t i v e l y u n a f f e c t e d by the presence of charge; i . e . they d i f f u s e through a n e u t r a l membrane as r a p i d l y as through a charged membrane, i n d i c a t i n g that e i t h e r the a f f i n i t y of the membrane f o r monovalent c a t i o n s was q u i t e low or the d i f f u s i o n through the membrane can occur at a r a t e equal to the s i t e to s i t e t r a n s f e r r a t e through the membrane. 76 Turning to the s i g n i f i c a n c e of these observations to the i n t a c t +2 animal, Ramsay (1953) found t h a t the Ca content i n the haemolymph and malpighian tubules of the s t i c k i n s e c t Dixippus morosus was 7 and 2 m.eq./l r e s p e c t i v e l y . Since the l a t t e r i n s e c t secretes i t s own content of water per day and reabsorbs back 95% of the ions and water i n the +2 rectum, r a p i d r e a b s o r p t i o n of Ca i n the rectum was e s s e n t i a l f o r main-+2 tenance of blood Ca l e v e l s . The pore s i z e of the i n t i m a was such as to a l l o w only very slow movement of uncharged molecules the s i z e of +2 +2 Ca . Assuming that Ca c o n c e n t r a t i o n and turnover was of the same order ofmagnitude i n the l o c u s t , then the f i x e d charge on the i n t i m a +2 was immediately r e s p o n s i b l e f o r p e r m i t t i n g the re a b s o r p t i o n of Ca at the high r a t e r e q u i r e d f o r the maintenance of homeostatis. -<•<-' + 2 The r o l e of Ca i n the i n t i m a could be of great p h y s i o l o g i c a l importance i n the r e g u l a t i o n of i o n movement across the i n t i m a . In a +2 hydrated animal ( i . e . i n a s a l t depleted s t a t e ) the r e c t a l Ca con-+2 c e n t r a t i o n may o f t e n be low, r e s u l t i n g i n a f l u x of Ca across the +2 membrane a t a r a t e g r e a t e r than by simple d i f f u s i o n . Low Ca i o n conce n t r a t i o n s might be expected to a i d i n the movement of C l across +2 the membrane. L e i t c h and Tobias (1964) showed th a t Ca w i l l not i n t e r -+ + f e r e w i t h the movement of K , i f the K c o n c e n t r a t i o n was g r e a t e r than +2 Ca c o n c e n t r a t i o n . This would a l s o seem t o be true f o r the i n t i m a , s i n c e the r a t e of K + or N a + movement seems to be r e l a t i v e l y independent of whether the membrane was charged or not. I f the animal was i n a dehydrated s t a t e , then the c o n c e n t r a t i o n +2 +2 of Ca might become very h i g h . High c o n c e n t r a t i o n of Ca would have a many-fold e f f e c t on the membrane p e r m e a b i l i t y p r o p e r t i e s . One would 77 be to dehydrate the membrane, both by an osmotic e f f e c t and a b i n d i n g e f f e c t . This dehydration should l e a d to a r e s t r i c t i o n i n both i o n and +2 water movement. Ca movement under such c o n d i t i o n s would most l i k e l y be by simple d i f f u s i o n . C a t i o n and anion movement across the i n t i m a should be r e s t r i c t e d by the membrane dehydration and by competition +2 w i t h Ca and other i o n s f o r the pore. L e i t c h and Tobias (1964) found +2 + that Ca w i l l compete w i t h K ,such that at equal c o n c e n t r a t i o n s , 2.5 +2 + times more Ca w i l l t r a v e r s e the membrane than K . These c o n s i d e r a t i o n s i n d i c a t e t h a t there w i l l be a s e l f r e g u l a -+2 +2 t i o n of Ca movement across the i n t i m a ; i . e . as Ca s e c r e t i o n s by the m a l p i g h i a tubules i n c r e a s e d l e a d i n g to h i g h r e c t a l c o ncentrations of * +2 +2 '-Ca , then the Ca p e r m e a b i l i t y of the i n t i m a should be d r a s t i c a l l y +2 .reduced. Ca a l s o might a c t as a f i n e c o n t r o l of i o n permeation and p o s s i b l y of water movement across the i n t i m a . L e i t c h and Tobias (1964) +2 showed t h a t a l i n e a r i n c r e a s e i n Ca c o n c e n t r a t i o n would e l i c i t a l o g a r i t h m i c decrease i n water flow across a p h o s p h o l i p i d - c h o l e s t e r o l membrane. P h i l l i p s (1964, b) found that the r a t e s of r e a b s o r p t i o n of K +, Na + and C l were r e g u l a t e d i n response to i o n l e v e l s i n haemolymph. In s a l i n e - f e d l o c u s t s ( i . e . dehydrated) uptake f o l l o w e d Michaelis-Menten k i n e t i c s , w h i l e s a l t - d e p l e t e d animals d i d not show r a t e - l i m i t i n g k i n e t i c s ; r a t h e r the rate of r e a b s o r p t i o n i n c r e a s e d l i n e a r l y w i t h the r e c t a l c o n c e n t r a t i o n of i o n s . At any r e c t a l c o n c e n t r a t i o n , except very low v a l u e s , the r a t e of r e a b s o r p t i o n i n s a l t - d e p l e t e d animals was hi g h e r than i n the s a l i n e - f e d l o c u s t s . 78 Studies of d i f f u s i o n p o t e n t i a l d i f f e r e n c e (Chapter 2) i n d i c a t e that K + and N a + w i l l have a f l u x r a t e very s i m i l a r to that f o r Rb + (then P f c 'v- P N a ^ P ) , which was found to be 1.6 u eq/hr/cm ). P h i l l i p s (1964 b) c a l c u l a t e d r e c t a l r e a b s o r p t i o n r a t e s of 0.25, 2 + + -2.0 and 0.25 u eq/hr/cm f o r Na , K and C l r e s p e c t i v e l y i n s a l t de-p l e t e d animals, when the r e c t a l concentrations were 10 mM/l f o r a l l three i o n s . Comparing the l a t t e r values to those f o r f l u x across the i n t i m a alone, i n d i c a t e s t h a t the f l u x value of K + f o r the whole r e c t a l w a l l i n v i v o was almost i d e n t i c a l w i t h the value obtained f o r the i n t i m a alone. This i n d i c a t e d that r e a b s o r p t i o n of K + was l i m i t e d p r i m a r i l y by the i n t i m a . 79 SUMMARY 1. The r e c t a l intima of the desert l o c u s t was found to possess f i x e d negative charges, rather than f i x e d n e u t r a l s i t e s . I t was sug-gested that the molecular species responsible for the negative s i t e s might be a c i d i c amino acids. 2. The s e l e c t i v e permeability of the intima as estimated from d i f f u -+2 +2 +2 sio n p o t e n t i a l s f o r di v a l e n t cations was Ba > Ca > Sr > ~^"2 "4*2 *4" "f* | | Mg > Mn , f o r monovalent cations was NH^ > Rb > Cs > K > Na + > L i + > TEA + and f o r monovalent anions was MCO^ > CN > F~ >N0~> CL~> CHoC00~ > Br~ > H„PO ~ > i " . J J 2 4 3. Cation a f f i n i t y f o r the f i x e d charged s i t e was found to be i n the *4*2 | | -^ -|_ order of Ca > Mg » K > Na . 4. S i m i l a r i t y of e f f e c t s of pH and ion concentration pn streaming and d i f f u s i o n p o t e n t i a l s i n d i c a t e d that ion movement and water flow might take place through the same route. 5. The intima was found to act as an osmotic compartment such that at high external osmotic pressures, the rate of water flow was reduced due to a shrinkage of the e f f e c t i v e pore s i z e i n the intima, however the r e l a t i v e permeability of ions d i d not seem ef f e c t e d by membrane dehydration. 6. Un s t i r r e d layers at the membrane-solution i n t e r f a c e s were found to have a minimal e f f e c t on d i f f u s i o n p o t e n t i a l s , however h a l f of the value f o r streaming p o t e n t i a l s was found to be due to a d i f f u s i o n p o t e n t i a l caused by an ion concentration d i f f e r e n c e i n opposing u n s t i r r e d l a y e r s . 80 7. Calcium -45 f l u x across the i n t i m a at pH 5.5 ( i . e . p o s s e s s i n g f i x e d charge) was found to be 81 times g r e a t e r , at a concentra-t i o n of 10 mM/l CaC^, than c a l c i u m f l u x a t the same concentra-t i o n , across the uncharged membrane (pH 2.2). The same e f f e c t was not s i g n i f i c a n t f o r subidium. Conversely, the removal of f i x e d charge enhanced anion f l u x . 8. Calcium permeation r a t e was found to be a f u n c t i o n of i t s d i s s o -c i a t i o n r a t e from the f i x e d charge and d i d not c o r r e l a t e i n a simple manner w i t h the membrane b i n d i n g c a p a c i t y f o r calcium. 9. A trans e f f e c t on cal c i u m f l u x was a l s o found i n the i n t i m a and i s b e l i e v e d to be a f u n c t i o n of the d i s s o c i a t i o n r a t e of cal c i u m from the f i x e d negative s i t e . 10. I t was concluded that electro-osmosis was not the mode of water movement across the rectum, however p h y s i o l o g i c a l advantage of electro-osmosis was di s c u s s e d . 11. F l u x experiments p o s s i b l y i n d i c a t e that the i n t i m a might be the + r a t e l i m i t i n g step f o r K r e a b s o r p t i o n i n a hydrated animal. A P P E N D I X A 81 This s e c t i o n contains a b r i e f d e s c r i p t i o n of the way that the constant f i e l d equation may be derived so that permeability c o e f f i c i e n t s can be c a l c u l a t e d simultaneously for monovalent and dival e n t cations and anions. The treatment i s b a s i c a l l y the same as that of Goldman (1943) and Hodgkin and Katz (1949) and i s s i m i l a r to the equation i l l u s t r a t e d by H a n i (1970). The b a s i c assumptions made i n the d e r i v a t i o n are (Hodgkin and Katz, 1949): 1. Ions i n the membrane move under the influence of d i f f u s i o n and the e l e c t r i c f i e l d i n a manner which i s e s s e n t i a l l y s i m i l a r to that i n free s o l u t i o n . 2. The e l e c t r i c f i e l d may be regarded as constant through out the membrane. 3. The concentrations of ions at the edges of the membrane are d i r e c t l y p r o p o r t i o n a l to those i n the aqueous solutions bounding the membrane. 4. The membrane i s homogeneous. Assumption 1. leads to the following i n t e r g r a t e d equations f o r the current c a r r i e d by ions: (Equation 1) I z = Z FV (C Az)o - (C z )a e " z V F / R T ' - Z VF/RT a 1 - e Now the concentration (C z ) at the outer edge of the membrane A i s p r o p o r t i o n a l to the concentration (A )o i n the external s o l u t i o n (assumption 3). 82 ( C A z ) o = B A z ( A Z ) o and ( C ^ a = B^z (A Z) i . where B^z i s the p a r t i t i o n c o e f f i c i e n t between membrane and the aqueous phase; (A ) i i s the co n c e n t r a t i o n on the opposite s i d e of the membrane. Thus (Equation 1) becomes f o r K, C l , and Ca. (Equation 2) I f c = P k F 2V (k)o - ( k ) i e " V F / R T (Equation 3) IQ1 = PQ1 F 2 V ( C l ) i - ( C l ) o e ~ V F / R T — ! _ e ~VF/RT (Equation 4) I = 4P_ F 2V (Ca)o - ( C a ) i e ~ 2 V F / R T — . ^ ^ V P / R T where P z = U z B z RT/ZaF A A A Then f o r P, = 11 B, RT/af and f o r Pea = M B RT/„ „ k k k ca ca 2aF The t o t a l i o n i c c u r r e n t d e n s i t y through the membrane i s t h e r e f o r e given by: (Equation 5) I = F 2V P f c W - Y e " V F / R T 1 - e " V F / R T where W = Ko + p c l / p k ( C l ) i + p C a / p k 4m (Ca)o Y = K i + P c l / P k ( C l ) o + p C a / P k 4m ( C a ) i e " V F / R T M _ , -VF/RT , M = 1 - e a n d " j ^WRT 1 - e The p o t e n t i a l d i f f e r e n c e across the membrane i n the absence of i o n i c c u r r e n t w i l l be designated as E where E= Eo - E i . V=E when 1=0 Then E = RT l o g £ Y/W E = RT l n P, ( K ) i + p ( C l)o + 4m P. ( C a ) i e E F / R T _k C l Ca F P, (K)o + P ( C l ) i + 4m P (Ca)o k c l ca A P P E N D I X B Table 8 R e l a t i o n s h i p between c o n c e n t r a t i o n of e x t e r -n a l s o l u t i o n ( C a C ^ and KC1) and the mem-brane r e s i s t a n c e . See F i g . 5. Bathing Solution Concentration of Solution Membrane bathing solution resistance resistance (mM/l) (ohms) (ohms) KC1 0.1 3.17xl05 25xl0 3 0.5 1.06xl05 8.1xl03 1.0 50.8xl03 10.5xl03 5.0 11.6xl03 1.69xl03 10 6.2xl03 472 50 1.21xl03 131 100 602 86 500 134 18.8 1000 68.7 7.4 C a C l 2 0.1 91xl0 3 62.5xl03 1.0 23.3xl03 21.9xl03 10 3.47xl03 6.96xl03 100 504 351 1000 70.2 62 Table 9 The a f f e c t of two d i v a l e n t and two monovalent cati o n s on a p o t e n t i a l d i f f e r e n c e caused by a 2 f o l d KC1 c o n c e n t r a t i o n d i f f e r e n c e . S a l t s were added at equal concentrations on both s i d e s . See F i g . 8. Concentration of added salt (mM/l) CaCl 2 0 1 10 100 MgCl 2 0 1 10 100 NaCl 0 1 10 100 KC1 0 1 10 100 Diffusion potentials in millivolts Individual preparations 1 2 3 4 5 6 Mean ± S.D. 4 4 8 9 7.25 11 7.50 ± 2.8 2 2 5 6 7 9 5.4 + 3.2 1 1 2.5 2.5 2 2 1.11 + 1.3 -1.5 • -1.5 -1 -0.5 0 -1 -1.17 + 0.71 4 5 8 9 7.25 11 7.4 + 2.6 4 4 7 6 6 7 5.7 + 1.4 0 -3 2 3.5 2 4 2.4 + 1.4 -1 -4 -1 -1 0 0 -1.16 + 1.47 4 6 4 9 7.25 11 6.9 + 2.8 4 5.5 3 9 6 7 5.75 + 2.19 4 4.5 2 3.5 4 4 3.67 ± 0.88 1 1 0.5 0 0 0 0.42 + 0.5 12 11 10 12 9.5 10 11 + 1.5 10 10 8 10 8 8 9 + 1 4 4.5 3.5 5 4 5 4.35 + 0.6 0.5 0.5 0 0.5 0 0 0.25 + 0.3 Table 10 The a f f e c t of pH on streaming p o t e n t i a l s . See F i g . 9. p H of bathing so lut ion 1 5.5 14 5.0 14 4.5 14 4.0 7.5 3.5 3 • 3.0 0 2.5 0 2.2 0 S t r e a m i n g potent ia ls i n m i l l i v o l t s Indiv idual p r e p a r a t i o n s 2 3 4 5 6 M e a n S . D . 9 11 12 13 12.5 11.9 + 1.74 9 11 12 13 12.5 11.9 + 1.74 9 11 12 13 12.5 11.9 + 1.74 5 9 9 10 9 8.3 + 1.78 2.5 7 6 4 5 4.6 + 1.74 0 2.5 1.5 2 3 1.5 + L27 0 0.5 0.5 0 1.5 0.4 + 0.59 0 0 0 0 0 0.0 + 0.00 Table 11 The a f f e c t of pH on d i f f u s i o n p o t e n t i a l s . See F i g . 9. pH of bathing solution 5.5 5.0 4.5 4.0 3.5 3.0 2.5 2.3 Diffusion potentials in millivolts Individual preparations 1 2 3 4 5 6 Mean + S.D 14 12 10 10 10 13 11.5 + 1.-75 13 12 10 10 10 13 11.3 + 1.5 12 11 9.5 6 8 13 9.9 + 2.6 11 6 8 4 6 11 7.7 + 2.8 9 4 6 4 6 9 6.3 + 2.3 5 2 3 0 4 6 3.3 + 2.2 2 0 1 -1.5 1 2 .75 + 1.3 0 0 0 0 0 0 0 + 0 Table 12 The a f f e c t of h i g h sucrose concentrations on d i f f u s i o n p o t e n t i a l s . See F i g . 10. KCI concentration gradient Concentration gradient of sucrose Lumen Haemocoel 1 0.01 M KCI - 0.005 M KCI 000 m. osm.-OOO m. osm. 9.5 100 m. osm.-100 m. osm. 9 200 m. osm.-200 m. osm. 8 400 m. csm.-400 m. osm. 7 600 m. osm.-600 m. osm. 6 800 m. osm.-800 m. osm. 6 Potential difference in millivolts Individual preparations 2 3 4 5 6 Meant S.D 9.5 8.5 15 10.5 12.5 10.9± 2.42 10 9 12 10 10.5 10.1+ 1.11 9 9.5 10 9 10.5 9.3 ±0.88 9 8.5 9.5 5.5 11 8.4 ± 1.93 9 9 9 9.5 10 8.8 ± 1.41 9.5 9 8 8 8.5 8.2 ± 1.21 Table 13 Streaming p o t e n t i a l s were developed u s i n g sucrose c o n c e n t r a t i o n d i f f e r e n c e s , 10 mM/l KCI on both s i d e s . In both i n s t a n c e s the osmotic pressure d i f f e r e n c e was kept con-s t a n t but the average sucrose c o n c e n t r a t i o n changed. See F i g . 11. Milliosmolars of Average sucrose sucrose concentration Haemocoel Lumen 100 000 50 6.5 200 100 150 3.5 300 200 250 2.5 400 300 350 .5 200 000 100 9 400 200 300 4 600 400 500 3 800 600 700 0 Streaming potentials in millivolts Individual preparations 2 3 4 5 6 Mean + S.D. 8 6 9 7 6.5 7.2 t 1.13 4 3 5 3.5 2.5 3.6 t .86 2 2 3 1.5 2 - 2.2 ± .52 1.5 1 2 1 2 1.3 ± .61 10 10 14 10 10.5 10.6± 1.74 3 2 3 4 4.5 3.4 t .92 1.5 1.5 1 2 2.25 1.9 t .90 1.5 1.5 .5 1 2 1.1 ± .54 Table 14 Streaming p o t e n t i a l s measured across the i n t i m a due to osmotic gradients created by the use of sucrose. The sucrose concentra-t i o n was v a r i e d from 0 to 400 m i l l i o s m o l a r on the haemocoel s i d e , w h i l e keeping the sucrose at 0 m i l l i o s m o l a r on the lumen s i d e . The c o n c e n t r a t i o n of KC1 was the same on both s i d e s . See F i g . 13. Concentration of KCI bathing solution Milliosmolar concentration gradient of sucrose Haemocoel Lumen IM KCI 100 m. osm. 000 m. osm. 200 m. osm. 000 m. osm. 300 m. osm. 000 m. osm. 400 m. osm. 000 m. osm. .IM KCI 100 m. osm. 000 m. osm. 200 m. osm. 000 m. osm. 300 m. osm. 000 m. osm. 400 m. osm. 000 m. osm. .01M KCI 110 m. osm. 000 m. osm. 210 m. osm. 000 m. osm. 410 m. osm. 000 m. osm. Streaming potentials in millivolts Individual preparations 1 2 3 4 5 6 Mean ± S.E .75 1 .75 .75 .5 .75 .75 t .16 1.5 1 1 1.5 1 1.25 1.21 t .25 1 1.5 1.25 2 1.25 1 1.33 t .38 1 2 1.5 2.25 1.5 1.25 1.58 t .47 5 2 2 2 2 2.25 2.54 1 1.21 4 3.5 3 3 3.25 4 3.46 ± .46 4 5 4 4.5 5 5 4.58 t .49 5 5 4.5 4.5 5.5 5.5 5.00 ± .45 8 7 7.5 8 7.63 t .47 11 11 12 10 11.0 t .50 14 14 15 13 14.0 ± .50 Concentration of KC1 bathing solution Milliosmolar concentration gradient of sucrose Haemocoel Lumen .001M KC1 100 m. osm. 000 m. osm. 200 m. osm. 000 m. osm. 300 m. osm. 000 m. osm. 400 m. osm. 000 m. osm. Streaming potentials in millivolts Individual preparations 1 2 3 4 5 6 Mean + S.D. 25 22 12 14 13 18.8 17.46 + 5.3 32 29 14 19 19 20 22.17 + 6.9 23 31 22 26 24 24 25.00 + 3.23 24 33 24 31 26 26 27.33 + 3.78 Table 15 Streaming p o t e n t i a l s were developed u s i n g sucrose c o n c e n t r a t i o n d i f f e r e n c e s , w i t h lmM/1 KCI on both s i d e s . The sucrose c o n c e n t r a t i o n d i f f e r e n c e was kept constant, but the absolute c o n c e n t r a t i o n was changed. The sucrose c o n c e n t r a t i o n d i f f e r e n c e was 200 m i l l i o s m o l a r . See F i g . 14. Milliosmolars of Average sucrose sucrose concentration Haemocoel , Lumen 1 200 0 100 32 400 200 300 14 600 400 500 11 800 600 700 3 Streaming potentials in millivolts Individual preparations 2 3 4 5 6 Mean- S.D 29 16 19 21 18 22.5 ± 6.5 9 8 13 17 11 12 t 3.3 4.5 3.5 5 6 5 6 1 2.7 2 2.5 3 3 3 2.75 t 0.4 Table 16 Non-linear i i c r e a s e i n streaming p o t e n t i a l s w i t h an i n c r e a s e i n the osmotic pressure. A. Represents n o n - l i n e a r i n c r e a s e i n the streaming p o t e n t i a l s w i t h an i n c r e a s e the osmotic pressure without p e r f u s i o n of s o l u t i o n . B. Represents n o n - l i n e a r i n c r e a s e i n the streaming p o t e n t i a l s w i t h an i n c r e a s e i n the osmotic pressure, w h i l e the membrane i s being perfused at a r a t e of 2.8 ml/min. See. F i g . 20. Milliosmolars of sucrose Haemocoel Lumen 1 A 100 0 7 200 0 10 300 0 12 400 0 13 B 100 0 4 200 0 6 300 0 7 400 0 8 Streaming potentials in millivolts Individual preparations Mean - S.D. 8 6 5 4 7 6.2 + 1.5 9.5 8 7 7 10 8.6 + 1.4 11 11 9.5 12 12 11.3 + 1.0 13 12 11 13 13 12.5 + 0.8 4 4 3 2 4 3.5 + 0.8 4.5 5 4.5 3.5 5 4.8 + 0.8 7 8 6 4 6 6.3 + 1.4 8 7 7 5 6.5 6.9 + 1.1 Table 17 The a d d i t i v e and s u b t r a c t i v e p r o p e r t i e s of streaming p o t e n t i a l s and d i f f u s i o n p o t e n t i a l s created simultaneously across the same mem-brane. A 2 f o l d c o n c e n t r a t i o n d i f f e r e n c e of KCI (10 mM/l - 5 mM/l) was used to i n i t i a t e the d i f f u s i o n p o t e n t i a l i n a l l cases. High c o n c e n t r a t i o n of KCI was pl a c e d on the lumen s i d e . Sucrose was used to cause streaming p o t e n t i a l s of v a r i o u s s i z e s . Sucrose, when pla c e d on the haemocoel s i d e caused stream-i n g p o t e n t i a l s to be added to d i f f u s i o n poten-t i a l s , and when plac e d on the lumen s i d e caused them to be su b t r a c t e d . Sign i s w i t h reference to the haemocoel s i d e . See F i g . 22. KC1 concentration gradient Concentration gradient of sucrose 0,01 M KC1 0.005 M KC1 a 000 m. osm. 100 m. osm. 200 m. osm. 300 m. osm. 400 nr. osm. b 000 m. osm. 100 m. osm. 200 m. osm. 300 m. osm. 400 m. osm. Potential difference in millivolts Individual preparations 1 2 3 4 5 6 Mean ± S.E 11 14 9 10 11 7 10.3 ± 2.34 18 17 19 16 17 17 17.3 + 1.03 23 20 23 19 22 22 21.5 + 1.64 26 22 26 25 25 23 24.5 + 1.64 27 23 28 28 27 23 26.0 + 2.37 11 14 9 10 11 7 10.3 + 2.34 4.5 8 '4.5 6 2 3 4.7 + 2.14 3.5 7 1.5 2 2 1 2.8 + 2.21 -1 6 -1 -2 -1 -1 0.0 + 2.97 -2 5 -4 -4 -3 -4 -2.0 + 3.52 Table 18 The i n t i m a was allowed to soak i n 1 mM/l Ca s o l u t i o n . The pH was v a r i e d from an i n i t i a l v alue of 2.2 to a f i n a l value of 8. See F i g . 25 B. pH of CaCl 2 bathing solution 1 2 2.2 1.9 2.7 3.5 2.4 1.7 3.8 3.8 6.8 5.0 31.7 36.7 6.0 40.0 28.4 7.0 56.2 37.2 8.0 57.8 57.8 uM Ca + V gm of dry membrane Individual preparations 3 4 5 6 Mean + S.D. 2.4 3.1 4.4 2.5 2.84 + 0.86 3.3 2.3 4.1 3.4 2.87 + 0.88 3.3 2.7 4.9 3.4 4.13 + 1.5 35.7 47.9 30.4 40.0 37.1 + 6.4 35 35 38 42.3 36.5 + 4.9 39.7 68.5 50.7 63.6 52.7 + 12.6 47.7 86 65 74.6 64.8 + 13.7 83 LITERATURE CITED B a l s h i n , M. and P h i l l i p s , J.E. (1971) Amino A c i d Absorption by the Rectum of Locusts. Nature ( i n press) Bray, G.A. (1960) A Simple E f f i c i e n t L i q u i d S c i n t i l l a t o r f o r Counting Aqueous S o l u t i o n i n a L i q u i d S c i n t i l l a t i o n Counter. A n a l . Biochem. I : 279-285. Brodsky, W.A. and S c h i l b , T.P. (1965) Osmotic P r o p e r t i e s of I s o l a t e d T u r t l e Bladder. Am. J . P h y s i o l . 208: 46-57. 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