UBC Theses and Dissertations

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UBC Theses and Dissertations

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.Sc,  U n i v e r s i t y o f B r i t i s h C o l u m b i a , 1970.  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  i n t h e Department of Zoology  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e required 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 B r i t i s h 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 B r i t i s h Columbia Vancouver 8, Canada  Date  ^ j U * ^  ^  W»  i  ABSTRACT  The r e c t a l i n t i m a o f t h e d e s e r t l o c u s t was found t o p o s s e s s 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 .  gested that the molecular  I t was s u g -  species responsible f o r the negative  sites  m i g h t 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 o f t h e i n t i m a as e s t i m a t e d  +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 Mn ^, f o r monovalent c a t i o n s was NH^ +  TEA  +  +  > Rb  +  > Ca  > Cs > K +  +2  > Sr > Na  +  from d i f -  +  +2  > Mg  > L i  +  >  >  and f o r monovalent a n i o n s was HC0 ~ > CN~ > F~ > NO^" > CL~ > 3  CH COO~ > B r ~ > H P0 ~ > I ~ . C a t i o n a f f i n i t y f o r t h e f i x e d charged 3  2  4  +2  s i t e was found t o be i n t h e o r d e r o f Ca  > Mg  ++  »  K  +  +  > Na .  S i m i l a r i t y o f e f f e c t s o f pH and i o n c o n c e n t r a t i o n on s t r e a m i n g 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 t h a t i o n movement and w a t e r f l o w might take p l a c e through t h e same r o u t e . The i n t i m a was found t o a c t as an o s m o t i c compartment such t h a t at high e x t e r n a l osmotic pressures, duced due t o a s h r i n k a g e  t h e r a t e o f w a t e r f l o w was r e -  o f 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 t h e r e l a t i v e p e r m e a b i l i t y o f i o n s d i d n o t 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 a t t h e membrane-solution i n t e r f a c e s were found t o have a m i n i m a l 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 o f t h e v a l u e f o r s t r e a m i n g p o t e n t i a l s was found t o be due t o 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 o p p o s i n g u n s t i r r e d layers.  ii  C a l c i u m -45 f l u x a c r o s s t h e i n t i m a a t pH 5.5 ( i . e . p o s s e s s i n g f i x e d c h a r g e ) was found t o 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 o f 10 mM/l C a C l ^ j than c a l c i u m f l u x at t h e same c o n c e n t r a t i o n a c r o s s t h e uncharged membrane (pH 2.2). f o r rubidium. anion f l u x .  The same e f f e c t was n o t s i g n i f i c a n t  C o n v e r s e l y , t h e removal o f f i x e d charge enhanced Calcium permeation  r a t e was found t o be a f u n c t i o n o f i t s  d i s s o c i a t i o n r a t e from t h e f i x e d charge and d i d n o t c o r r e l a t e i n a s i m p l e manner w i t h t h e membrane b i n d i n g c a p a c i t y f o r c a l c i u m .  A  t r a n s 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 t h e i n t i m a and i s b e l i e v e d t o be a f u n c t i o n o f the d i s s o c i a t i o n r a t e o f c a l c i u m f r o m the f i x e d negative  site.  I t was c o n c l u d e d  t h a t e l e c t r o - o s m o s i s was n o t t h e mode o f  w a t e r movement a c r o s s t h e r e c t u m , however p h y s i o l o g i c a l advantage o f e l e c t r o - o s m o s i s was d i s c u s s e d .  F l u x experiments  possible indicate  t h a t t h e i n t i m a might be t h e r a t e l i m i t i n g s t e p f o r K i n a hydrated  animal.  +  reabsorption  iii TABLE OF CONTENTS  TITLE  PAGE  INTRODUCTION  1  MATERIALS AND METHODS Materials  13  Methods A.  IN VITRO p r e p a r a t i o n o f t h e 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.  Preparation of Solutions  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 differences  2.  E s t i m a t i o n o f p e r m e a b i l i t y from  17 Radio-isotope  flux  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 Introduction  21  Results A.  Membrane R e s i s t a n c e  B.  A n i o n : C a t i o n P e r m e a b i l i t y as a F u n c t i o n o f Concentration  C.  D.  Estimate  22  23  o f t h e C o n c e n t r a t i o n o f F i x e d Charges  i n the Intima  24  Amino A c i d A n a l y s i s o f C u t i c u l a r P r o t e i n  25  iv 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 p e r m e a b i l i t y o f t h e i n t i m a t o monovalent and  d i v a l e n t c a t i o n s and monovalent anions  B.  C o m p e t i t i o n between i o n s f o r f i x e d n e g a t i v e  C.  Relative permeability  33 sites  35  o f t h e i n t i m a as measured by  streaming p o t e n t i a l s  37  D.  E f f e c t o f 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  39  F.  E f f e c t s o f o s m o t i c g r a d i e n t on s t r e a m i n g p o t e n t i a l s  G.  Ion concentration  on d i f f u s i o n p o t e n t i a l s  e f f e c t on s t r e a m i n g and d i f f u s i o n  potentials H.  42  P o s s i b l e e f f e c t s o f u n s t i r r e d l a y e r s on membrane potentials  I.  39  44  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 o f s t r e a m i n g and diffusion potentials  50  Discussion  52  CHAPTER I I I - ION FLUX ACROSS THE INTIMA. Introduction  63  Results A.  Ion f l u x e s a t various  concentrations  B.  Membrane b i n d i n g  C.  Trans e f f e c t o f the i n t i m a  capacity  and pH v a l u e s  65 67 68  Discussion  SUMMARY APPENDIX A APPENDIX B LITERATURE CITED  ACKNOWLEDGEMENTS  I w o u l d l i k e t o thank D r . P h i l l i p s f o r h a v i n g me as a g r a d u a t e s t u d e n t i n h i s L a b o r a t o r y , 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 u r i n g t h e c o u r s e o f t h e r e s e a r c h and p r e p a r a t i o n o f t h i s Thesis. I must e x p r e s s many thanks t o D r . V. P a l a t y f o r h e l p i n d e t e r mining  t h e number o f a c i d i c groups i n t h e i n t i m a and f o r h i s t h o r o u g h  r e a d i n g and c r i t i c i s m o f t h i s T h e s i s . I am i n d e b t e d t o D r . I . T a y l o r f o r c o n d u c t i n g  t h e amino a c i d  a n a l y s i s o f t h e p r o t e i n o f t h e 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 reading of this  Thesis.  I w o u l d l i k e t o thank t h e members o f my M a s t e r s Committee f o r t h e i r suggestions  d u r i n g t h e course o f t h e r e s e a r c h , and a l s o my  fellow students i n the Laboratory. A v e r y s p e c i a l thanks goes o u t t o B e t t y Coldbeck and Bob S c h r o e d e r f o r t h e t y p i n g and p r o o f r e a d i n g o f t h i s T h e s i s on such short notice.  vii GLOSSARY  A A  g  -  a r e a o f t h e membrane s u r f a c e  -  r a d i o a c t i v i t y p e r u n i t volume o f 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 p e r u n i t volume o f e x t e r n a l medium  C  -  concentration of t e s t molecule  D  -  dielectric  E  -  potential difference  AE 1/2 AE  00  -  AEo -  -  constant  h a l f maximal p o t e n t i a l  maximal s t e a d y s t a t e  -  Faraday constant  H  -  potential difference  i  -  current  IJ  -  difference  potential  i n i t i a l p o t e n t i a l difference before  F  Jv -  (Eo - E i )  stirring  developed i n streaming p o t e n t i a l s  flow during electro-osmosis  volume f l o w o f w a t e r i n e l e c t r o - o s m o s i s t o t a l volume f l o w o f p e r f u s a t e i n t i m e t  Ki' - K  +  c o n c e n t r a t i o n on i n s i d e o f membrane.  Ki" - K  +  c o n c e n t r a t i o n on o u t s i d e o f membrane.  K' -  +  c o n c e n t r a t i o n on i n s i d e o f membrane b e f o r e s t i r r i n g .  K  K i , C l i , X i - a c t i v i t i e s o f s o l u t i o n on s i d e one o f t h e membrane. Ko, C l o , Xo - a c t i v i t i e s o f s o l u t i o n on s i d e two o f t h e membrane, k - s p e c i f i c conductivity  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 serosal bathing solution.  viii  P  -  osmotic d i f f e r e n c e causing osmotic pressure.  Pi  -  membrane p e r m e a b i l i t y t o i o n i .  pHb -  pH of p o r e  pHs -  pH o f b a t h i n g  R  gas  -  liquid. solution.  constant.  Ro' -  r e s i s t a n c e t o w a t e r f l o w w i t h u n i t s o f m osm/mv.  t  -  time.  6  -  thickness 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)  -  permeability coefficient.  £  -  zeta potential.  INTRODUCTION  The  d e s e r t l o c u s t , b e i n g a t e r r e s t r i a l l n s e c t has  conserve water and  r e g u l a t e body s a l t s .  p i g h i a n t u b u l e - r e c t a l system (reviewed malpighian tubules  (259  f o r s k a l ; Savage, 1956)  electrochemical  by  Stobbart  The  gradient  and  osmotic p r e s s u r e flow i n t o the  (Ramsay, 1956).  s e l e c t i v e reabsorption  reabsorptive processes  (i.e.  i o n s and w a t e r  Stobbard and  ( B a l s h i n and  Shaw, 1964).  Phillips,  1971).  Net  i s independent o f s i m u l t a n e o u s movement o f s o l u t e lead to formation  These s e l e c t i v e r e a b s o r p t i v e p r o c e s s e s concentration  ( P h i l l i p s , 1964  f e c a l m a t e r i a l present N o i r o t and  separating  K  occurs The  +  (Phillips,  water across  of a hyperosmotic  are r e g u l a t e d i n response a,b,c).  rectum c o n s i s t s o f an e p i t h e l i a l l a y e r and  the c h i t i n o u s i n t i m a )  the  i n t o the rectum where  +  can  the r e c t a l pad  a cuticular layer cells  from  the  i n the r e c t a l lumen ( F i g . 1 ) .  Noirot-Timithee  the r e c t a l c u t i c l e i n i n s e c t s .  (1966) s t u d i e d the u l t r a s t r u c t u r e o f  The  to  lumen down t h e i r  i n c l u d e a c t i v e t r a n s p o r t of C l , N a ,  the r e c t a l w a l l as a whole and  The  gregaria  thus f o r m i n g a  tubule  of e s s e n t i a l metabolites,  b ) ' a n d amino a c i d s  to changes i n b l o o d  The  T h i s p r i m a r y f i l t r a t e passes down  i n the r e q u i r e d amounts (Reviewed by  urine.  Shaw, 1964).  d i f f e r e n c e whereby water,  the m a l p i g h i a n t u b u l e , through the h i n d g u t and  absorption  to  the mal-  p r i m a r y f i l t r a t e i s formed by  i n t o the lumen o f the t u b u l e  electrolytes andronelectrolytes  1964,  and  by  able  produce a p r i m a r y f i l t r a t e , which i s i s o s m o t i c  +  respective gradients  This i s achieved  i n number i n the l o c u s t S h i s t o c e r c a  the haemolymph (Ramsay, 1953). a c t i v e s e c r e t i o n of K  to be  e l e c t r o n microsopic  s t u d i e s showed  t h a t i n g e n e r a l the i n t e s t i n a l c u t i c l e of Cephalotermes r e c t a n g u l a r i s  Fig.  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 o f the s e c t i o n o f the l o c u s t r e c t u m where: P  -  r e c t a l pad  I  -  Intima.  cells.  cross  2  SI ( t e r m i t e ) v a r i e d from one segment t o a n o t h e r .  The e p i c u t i c l e  appears  to be g r e a t l y reduced t o a v e r y t h i n s u p e r f i c i a l l a y e r , and a l s o t o a dense u n i f o r m l a y e r c o r r e s p o n d i n g t o the c u t i c u l i n complex as d e s c r i b e d by Locke, 1964). Weis-Fogh  The e p i c u t i c l e a c c o r d i n g t o  (1970) i s t o t a l l y d e v o i d o f c h i t i n .  e p i c u t i c l e v a r i e s from 200-1800 A*.  The fliickness o f the  The p r o c u t i c l e which  e x o c u t i c l e and e n d o c u t i c l e i s even more v a r i a b l e . e p i c u t i c l e , Noirot  (Chitin-protein  i s d e f i n e d as  D i r e c t l y below the  (1966) o b s e r v e d s e v e r a l l a m e l l a e made up o f a  f i b e r o u s c h i t i n p r o t e i n complex, which i s o f t e n r e p l a c e d by dense granules.  H i s t o c h e m i c a l s t u d i e s w i t h A l c i a n b l u e (Stoward, 1967 a,b,  c,d) s u g g e s t s t h a t a c i d m u c o p o l y s a c c h a r i d e s e x i s t i n the p r o c u t i c l e . They found t h a t m u c o p o l y s a c c h a r i d e s t a i n i n g i n t e n s i t y d e c r e a s e d as the pH d e c r e a s e d which i n d i c a t e s t h a t t h e m u c o p o l y s a c c h a r i d e s a r e moderately a c i d i c due t o c a r b o x y l groups. f o r a c i d i c mucopolysaccharides  The l a y e r o f the p r o c u t i c l e can be many microns  staining  t h i c k , and u s i n g  d i f f e r e n t s t a i n i n g t e c h n i q u e s , i t was shown t o p o s s e s s v e r y f i n e and i r r e g u l a r l y anastomosing  filaments.  A h i s t o c h e m i c a l and e l e c t r o m i c r o s -  c o p i c s t u d y o f t h e e p i c u t i c l e u s i n g hexamethylene-tetramine i n d i c a t e s w i t h o u t doubt  the p r e s e n c e o f p o l y p h e n o l s .  silver,  An a l t e r a t i o n o f  the above t e c h n i q u e a l s o showed s i l v e r g r a i n s i n the p r o c u t i c l e , a g a i n i n d i c a t i n g a c i d i c mucopolysaccharides  ( N o i r o t and N o i r o t - T i m i t h e e , 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)  t o a c t as a m o l e c u l a r s i e v e , a l l o w i n g by s e l e c t i v e r e a b s o r p t i o n a r a p i d exchange o f water epithelial cells.  and s m a l l s o l u t e m o l e c u l e s between the lumen and The r e l a t i v e i m p e r m e a b i l i t y o f the i n t i m a t o l a r g e  3  o r g a n i c m o l e c u l e s caused the l a t t e r t o accumulate i n the rectum as a consequence o f f l u i d  r e c i r c u l a t i o n through the m a l p i g h i a n  tubule-rectal  system. Phillips Na  +  and C l  In s a l i n e  (1964 b) found t h a t the n e t r a t e o f r e a b s o r p t i o n o f K , +  was r e g u l a t e d by the s a l t  f e d l o c u s t s a maximum r a t e o f t r a n s p o r t o r n e t r e a b s o r p t i o n  was n o t i c e d , the curve depleted  c o n c e n t r a t i o n o f the haemolymph.  f o l l o w i n g t y p i c a l Michaelis-Menten  l o c u s t s do n o t e x h i b i t the M i c h a e l i s - M e n t e n  kinetics.  k i n e t i c s , and the  r a t e o f r e a b s o r p t i o n i n c r e a s e d i n a l i n e a r f a s h i o n f o r the t h r e e investigated.  Since i n s a l t  k i n e t i c s were o b v i o u s ,  depleted  Michaelis-Menten  must be r a t e l i m i t i n g .  b) found t h a t the a d d i t i o n a l a b s o r p t i o n o f C l  o v e r s a l i n e f e d animals  (at high  to a l a r g e d i f f u s i o n component. then r e g u l a t i o n of absorption ability  ions  then one o f the composite p a r t s o f the rectum  ( c u t i c u l a r l a y e r or epithelium) (1964  animals no  concentrations)  Phillips  i n salt  depleted  c o u l d be a t t r i b u t e d  I f t h i s was t r u e f o r the o t h e r  ions,  c o u l d i n v o l v e a c o n t r o l o f p a s s i v e perme-  o f i o n s r a t h e r than changed i o n a f f i n i t i e s  and c o n c e n t r a t i o n o f  c a r r i e r s i n the r e c t a l pad c e l l s . The be and  p h y s i c a l b a s i s o f s e l e c t i v i t y by the i n t i m a was thought t o  the p r e s e n c e o f water f i l l e d p o r e s w i t h l i n e d with  f i x e d negative  charges.  apparent r a d i u s o f 6.5 A*  The former was p o s t u l a t e d be-  cause the r e l a t i v e r a t e o f p e n t e t r a t i o n o f h y d r o p h i l i c n o n - i o n i c m o l e c u l e s depends on t h e i r h y d r a t e d as p r e d i c t e d by Renkin hypothesis  includes  molecules with  Salt  size  (1954) e q u a t i o n .  (Phillips  ( P h i l l i p s and D o c r i l l , Supporting  evidence  and D o c k r i l l , 1968): (a)  r a d i i g r e a t e r than 6 X t o p e n e t r a t e  1968)  f o r this  f a i l u r e of  the membrane,  4  (b) o s m o t i c p e r m e a b i l i t y t w e n t y - f i v e times g r e a t e r 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 w a t e r ( s u g g e s t i n g t h a t w a t e r movement o c c u r s by l a m i n a r f l o w t h r o u g h p o r e s ) and between p e r m e a b i l i t y and Fixed negative  (c) l a c k o f a c o r r e l a t i o n  l i p i d s o l u b i l i t y of s o l u t e s .  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 o f i o n f l u x e s a c r o s s  the i n t i m a s u g g e s t e d t h a t p e r m e a b i l i t y  +2 t o c a t i o n s ( e s p e c i a l l y Ca  ) might be h i g h e r t h a n p r e d i c t e d by  R e n k i n e q u a t i o n b a s e d on p r e v i o u s -  observations  the  w i t h uncharged  molecules.  -2  Anion penetration  (CI and PO  observations)  found t o c o n f o r m t o the p a t t e r n f o r uncharged  and  I n the i n t i m a one  ^)has been measured ( P h i l l i p s ,  unpublished  o r a l l o f the t h r e e l a y e r s c o u l d c o n t a i n the  f i x e d charges: ( i )  molecules. postulated  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 o r ( i i i ) the deeper l a y e r i n the p r o c u t i c l e thought t o be  composed o f  a a c i d i c mucopolysaccharide. I o n p e r m e a t i o n i n porous membranes i s governed by a s e r i e s o f factors:  (i)  the a v a i l a b l e p o r e a r e a ,  ( i i ) t o r t u o s i t y i n the p o r e s ,  s u c h s p e c i f i c i n t e r a c t i o n s o f the i o n s w i t h the p o r e w a l l s as  ( i i i ) ion-  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 d r a g and sibly  ( v i ) s t r u c t u r a l and  chemical  and  changes of the membrane t h a t  pos-  are  dependent on the f u n c t i o n a l s t a t e o f the i o n exchange. According  t o the f i x e d charge t h e o r y 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 o f a n i o n i c d i s s o c i a b l e ( a c i d i c ) g r o u p s , o r 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 s p e c i e s a c t as c o u n t e r - i o n s ,  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  sites.  which  5  Eisenman, et a l (1967) c l a s s i f i e d f i x e d s i t e s i n t o two groups: (i)  f i x e d d i s s o c i a t e d , i n which the pores are so wide that the i n t e r -  a c t i o n between ions i n the pores and f i x e d charges are n e g l i g i b l e , (ii)  f i x e d associated, here an a r b i t r a r y maximal diameter of 10 $  is  given f o r the pore, and under these conditions a l l . membrane water i s i n a bound s t a t e . In the f i x e d d i s s o c i a t e d s t a t e only pore area, t o r t u o s i t y and solvent drag are of importance i n determining i o n permeation.  The  co-ion and counter-ion content i s dependent on a Donnan e q u i l i b r i u m w i t h the e x t e r n a l s o l u t i o n s .  Current voltage r e l a t i o n s h i p s are  characterized by free s o l u t i o n m o b i l i t i e s , and are a f u n c t i o n of the charge density.  Ion permeation i s i n s e n s i t i v e to s w e l l i n g and s h r i n k -  ing of the membrane.  The most d i s t i n g u i s h i n g feature of a f i x e d d i s s o c i a t e d  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 f i x e d charge, g i v i n g 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 c r e a t i n g a p o t e n t i a l d i f f e r e n c e across the membrane which i s c a l l e d a streaming p o t e n t i a l . As pore s i z e narrows i o n - s i t e and ion-matrix i n t e r a c t i o n s become of greater importance.  S p e c i f i c i n t e r a c t i o n s between the mobile species  and the s i t e s a l t e r the m o b i l i t i e s and standard chemical p o t e n t i a l s from those found i n aqueous s o l u t i o n s .  Upon decreasing the pore diameter a  stage i s reached where there i s no free water, thus e l i m i n a t i n g solvent drag while pore area and t o r t u o s i t y become unimportant.  Donnan boundary  6 c o n d i t i o n s a r e r e p l 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 o f 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 i o n s of  t h e same charge become a p p a r e n t . Membrane s t r u c t u r e a t s m a l l pore d i a m e t e r s c a n be a l t e r e d by  p r e s s u r e g r a d i e n t s o r volume changes as a f u n c t i o n o f t h e c o n c e n t r a t i o n of a p a r t i c u l a r counter-ion species a t a given point.  Since  m o b i l i t i e s and c h e m i c a l g r a d i e n t s a r e l i k e l y t o be s e n s i t i v e t o t h e degree o f h y d r a t i o n , a s t r o n g dependence o f m o b i l i t i e s on t h e conc e n t r a t i o n p r o f i l e s o f t h e c o u n t e r - i o n s i s t o be e x p e c t e d . et  al.  (Eisenman,  1967).  The a c t u a l mode o f i o n p e r m e a t i o n o f m o b i l i t y t h r o u g h a c h a r g e d membrane i s n o t known, b u t one o f t h e a c c e p t a b l e t h e o r i e s o f f e r e d i s t h a t each c o u n t e r - i o n i n a p o r e once a t t a c h e d t o a f i x e d charge w i l l s w i n g 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 t h e r m a l m o t i o n .  The d i r e c t i o n o f m o t i o n i s  dependent upon t h e c o n c e n t r a t i o n g r a d i e n t s so t h a t t h i s f o r c e w i l l t h e n move t h e i o n from one o s c i l l a t i o n c e l l t o a n o t h e r . the  This w i l l occur i f  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 t o jump o u t o f i t s a b s o r p -  t i o n s i t e , t h e mean f r e e p a t h a l s o b e i n g r e s t r i c t e d by t h e dense c r o w d i n g of w a t e r m o l e c u l e s . of the  Because o f the energy r e q u i r e m e n t s a second  m i g r a t i o n i s o f f e r e d by L i n g ( 1 9 6 2 ) . dipolar pair (i.e.  system  I f a second c o u n t e r - i o n approaches  f i x e d charge p l u s p r i m a r y c o u n t e r - i o n ) an  e q u i l i b r i u m s t a t e might be r e a c h e d i n w h i c h b o t h c o u n t e r - i o n s a r e changed t o 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. ion  Hani  (1970) adds t o t h i s by s u g g e s t i n g t h a t i f  movement c o n s i s t s o f jumps from one s i t e t o an a d j a c e n t v a c a n t s i t e ,  7  the p e r m e a t i o n r a t e o f t h e i o n w i l l be d e t e r m i n e d by t h e a c t i v a t i o n energy o f the jump and t h e p r o b a b i l i t y t h a t an a d j a c e n t s i t e i s v a c a n t , vacancies  a l s o b e i n g formed by i o n s moving from a f i x e d s i t e t o an  i n t e r s t i t i a l position. membrane c o m p o s i t i o n vacancies  A change i n m o b i l i t y w i t h a change i n t h e  might be due t o t h e change i n t h e number o f  i n t h e membrane; i o n s b e i n g more m o b i l e i n a membrane  w h i c h c o n t a i n s more v a c a n c i e s . P a r t i a l o r complete c o - i o n 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 t h e membrane.  Such a s e l e c t i v i t y under p r o p e r  c o n d i t i o n s would l e a d t o membrane p o t e n t i a l s .  I f one s i d e o f t h e  membrane c o n t a i n s a h i g h e r c o n c e n t r a t i o n o f an e l e c t r o l y t e t h e r e be an i o n f l o w from h i g h c o n c e n t r a t i o n t o low c o n c e n t r a t i o n  will  resulting  i n a " d i f f u s i o n p o t e n t i a l " , (due t o c o - i o n 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 r e a c h e d when c o - i o n e x c l u s i o n i s b a l a n c e d  by t h e  a t t r a c t i v e f o r c e o f t h e 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 a c r o s s 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 a t t h e 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 t h r o u g h t h e 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 a r e d e t e r m i n e d by s e l e c t i v i t y o f t h e 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  d e t e r m i n e d by t h e c o n c e n t r a t i o n s on each s i d e and by t h e s e l e c t i v i t y f a c t o r , w h i l e t h e d i f f u s i o n p o t e n t i a l (h t h e membrane) i s d e t e r m i n e d by t h e ion mobility.  Hani  (1970) a f t e r m e a s u r i n g 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 n o r between m o b i l i t y and p o t e n t i a l and c o n s e q u e n t l y c o n c l u d e d t h a t d i f f u s i o n p o t e n t i a l s a r e d e t e r m i n e d by b o t h s e l e c t i v i t y and m o b i l i t y . I n g e n e r a l terms t h e f l u x o f an i o n s p e c i e s  i n s i d e t h e membrane  i s p r o p o r t i o n a l t o i t s m o b i l i t y and t o t h e 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 t h e membrane i n t e r f a c e s ( T e o r e l l , 1953). m o b i l i t y of counter-ions  t h r o u g h t h e membrane i s a f u n c t i o n o f t h e d i s -  s o c i a t i o n rate o f the counter-ion  f r o m t h e f i x e d charge and t h e  p r o b a b i l i t y t h a t an a d j a c e n t f i x e d s i t e i s v a c a n t . that counter-ion  The i n t e r n a l  On t h e a s s u m p t i o n  m o b i l i t y i s g r e a t e r t h r o u g h a charged membrane than  t h r o u g h a s i m i l a r uncharged membrane, t h e Donnan e q u i l i b r i u m has t h e ability  to concentrate  the mobile i o n species  and w i l l i n c r e a s e t h e  f l u x p r o p o r t i o n a t e l y t o t h e f i x e d charge d e n s i t y if  t h e f i x e d charge d e n s i t y i s much g r e a t e r  ( t h i s i s only  valid  than t h e e x t e r n a l con-  centration) . Eisenman's t h e o r y  (Eisenman, 1962) p r e d i c t s t h e r e l a t i v e  t o 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 n e g a t i v e  charges.  selectivity The  t h e o r y was t e s t e d and c o n f i r m e d on n o n - b i o l o g i c a l m a t e r i a l ( i . e . g l a s s e l e c t r o d e s ) ; o n l y r e c e n t l y has i t been a p p l i e d t o 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 W r i g h t , 1969).  The b a s i c f a c t o r s d e t e r m i n i n g ions i s the free energies with  s p e c i f i c i t y o f a f i x e d charge f o r  o f i n t e r a c t i o n o f t h e c a t i o n s w i t h w a t e r and  the f i x e d a n i o n i c s i t e s i n t h e membranes.  These i n t e r a c t i o n s o f  9  c a t i o n - w a t e r and c a t i o n - s i t e w i l l c o n t r o l : ( i ) t h e 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 o f t h e membrane, ( c o n s e q u e n t l y potentials),  t h e phase  boundary  ( i i ) t h e a c t i v a t i o n e n e r g i e s w h i c h govern c a t i o n m i g r a t i o n  from t h e s o l u t i o n i n t o t h e membrane and ( i i i ) t h e m i g r a t i o n o f t h e c a t i o n i n t h e pore r e g i o n s o f t h e membrane. The competing i n t e r a c t i o n s o f c a t i o n s w i t h t h e membrane  versus  w a t e r c a n be r e p r e s e n t e d by an i o n exchange r e a c t i o n : I  (memb.) + J  (aqueous)  J  (memb.) + I  (aqueous) +  The dependence o f t h e f r e e energy change,A ^ j ° °f F  t  n  e  A  above  r e a c t i o n c a n be w r i t t e n : A  F  o _ - h y d . - memb. i j " I+ " J+ J+ F  F  +  where F ^ ^ ^ " s y m b o l i z e s a e i  memb. " I+  memb.  F  t h e p a r t i a l m o l a l f r e e energy o f h y d r a t i o n o f  1  the i o n 1+ w h i l e ^ ^  F  ^ ' r e p r e s e n t s t h e p a r t i a l m o l a l f r e e energy o f 3  1+ w i t h t h e membrane. Therefore from i t s h y d r a t e d  t h e i o n w h i c h undergoes  t h e g r e a t e s t f r e e energy change  s t a t e t o t h e bound s t a t e w i l l be s e l e c t e d o v e r 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 g e n e r a l terms t h e n , c a t i o n s w h i c h 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 t o s i t e t r a n s f e r (as on an i o n exchange column).  The c l o s e n e s s o f approach o f t h e i o n s i s dependent  on t h e  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 o f t h e f i x e d charge and t h e a f f i n i t y o f each i o n f o r i t s s h e l l o f w a t e r m o l e c u l e s (energy o f 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 t h e h y d r a t i o n e n e r g i e s o f t h e i o n s concerned w i l l p r e f e r e n t i a l l y b i n d i o n s  according  10  +  +  +  +  +  to  i n c r e a s i n g d e h y d r a t e d s i z e o f t h e i o n s ( 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 H y d r a t i o n e n e r g i e s o f v a r i o u s i o n s w i l l have o t h e r s e l e c t i v e sequences, t h e t o t a l number i s 13 f o r t h e f i v e  alkaline  m e t a l c a t i o n s and s e v e n f o r t h e 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  t h e sequence o f  s e l e c t i v i t y among t h e monovalent c a t i o n s ; however t h e sequence o f s e l e c t i v i t y among t h e d i v a l e n t c a t i o n s can be s h i f t e d a t c o n s t a n t f i e l d s t r e n g t h w i t h a change i n t h e s i t e s p a c i n g . ing  I f the s i t e  spac-  i s i n c r e a s e d t h e s e l e c t i v i t y p a t t e r n w i l l move f r o m t h e sequence  where t h e i o n w i t h t h e 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 t o one i n w h i c h t h e i o n w i t h t h e 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 o f s w e l l i n g ( h y d r a t i o n ) o f a membrane w i l l n o t  a f f e c t t h e s e l e c t i v i t y sequence b u t w i l l a f f e c t t h e magnitude o f 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 . Procunier ing  (1966) i n p r e l i m i n a r y e x p e r i m e n t s r e p o r t e d t h a t s t r e a m -  p o t e n t i a l s t h a t were d e v e l o p e d a c r o s s t h e r e c t a l i n t i m a o f t h e l o c u s t  were n e g l i g i b l e i n s i z e , thus t h r o w i n g hypothesis.  some doubt on t h e ' f i x e d c h a r g e '  T h i s d i s c r e p a n c y between h y p o t h e s i s  r e s u l t s was r e - i n v e s t i g a t e d by L e w i s (1970).  and e x p e r i m e n t a l  I n i t i a l experiments u s i n g  1 0 - f o l d K C I c o n c e n t r a t i o n g r a d i e n t s showed t h a t the r e c t a l c u t i c l e was cation selective.  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 contain f i x e d negative s i t e s .  These nega-  t i v e s i t e s were shown t o be s e l e c t i v e l y permeable t o 5 monovalent c a t i o n s , the  11  s e l e c t i v i t y c o r r e s p o n d i n g t o Eisenman's second sequence (Rb K  +  > Na  +  permeability  p o t e n t i a l s i s 10/1,  ratio P / K  P  , as c a l c u l a t e d from d i f f u s i o n ul  this permeability  u n i t y as t h e pH of the b a t h i n g S e l e c t i v i t y was  (Lewis,  >  >Li ).  +  The  2.3.  > Cs  r a t i o was  s o l u t i o n was  shown t o d e c r e a s e t o  l o w e r e d f r o m 5.5  a l s o l o w e r e d by i n c r e a s i n g  to a value  concertration.  1970). S t r e a m i n g p o t e n t i a l s d e v e l o p e d when o s m o t i c g r a d i e n t s  a p p l i e d a c r o s s the i n t i m a u s i n g s u c r o s e .  were  This i s c o n s i s t e n t w i t h  p o s t u l a t e d n e g a t i v e f i x e d charges b e i n g l o c a t e d on the w a l l s o f membrane p o r e .  As  the average o s m o t i c p r e s s u r e was  increased,  s t r e a m i n g p o t e n t i a l w o u l d d e c r e a s e , t h i s phenomena o f osmosis was  postulated  t o be caused by  the the  the  non-linear  the p o r e s a c t i n g as  osmotic  compartments. U n t i l recently, selective permeability was  assumed t o be  o f a membrane t o i o n s ,  caused by a f i x e d charge and  the s y s t e m l i k e n e d t o a  i o n exchange membrane; the e v i d e n c e f o r t h i s a s s u m p t i o n was 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 s t u d y was  o f the i n t i m a f o r c a t i o n o v e r a n i o n s was as i n a i o n e x c h a n g e r ;  due  inconclusive.  whether the  selectivity  t o a f i x e d charged s i t e  o r whether the s e l e c t i v i t y was  d e t e r m i n e d by  a  f i x e d n e u t r a l s i t e as p r o p o s e d by B a r r y , Diamond and W r i g h t (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 o f f i x e d s i t e type  b a s e d 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 W r i g h t , B a r r y (1971). were a l s o  The  p o s s i b l e membrane components r e s p o n s i b l e  considered.  and  was  Diamond  for ion selectivity  12  The  c o m p o s i t i o n and volume o f t h e p r i m a r y f i l t r a t e e n t e r i n g t h e  r e c t u m w i l l v a r y d e p e n d i n g on t h e p h y s i o l o g i c a l s t a t e o f t h e a n i m a l . Such v a r i a b l e f a c t o r s a s , 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, o s m o t i c p r e s s u r e and n e t w a t e r 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 t h e charge o r membrane s t r u c t u r e . p h y s i o l o g i c a l importance i n t i m a (Chapter 2 ) .  I t was t h e r e f o r e o f  t o s t u d y t h e e f f e c t s o f these v a r i a b l e s on t h e  The response  o f the i n t i m a t o these v a r i o u s p e r -  t u r b a t i o n s a l s o a l l o w e d a c o m p a r i s o n t o be made between b e h a v i o r o f t h i s membrane and v a r i o u s c u r r e n t models f o r f i x e d charged  membranes. +2  Phillips  ( u n p u b l i s h e d o b s e r v a t i o n ) found t h a t t h e f l u x o f Ca  a c r o s s t h e i n t i m a was 50 t i m e s g r e a t e r t h a n f o r s u c r o s e , even though b o t h c h e m i c a l s p e c i e s have t h e same h y d r a t e d s i z e . provoked three questions: charge a f f e c t p e r m e a t i o n  This observation  ( i ) t o what e x t e n t does t h e membrane f i x e d o f i o n s o f d i f f e r e n t charge and v a l e n c y  through +2 t h e membrane? ( i i ) Can t h e 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 c o m p l e t e l y a t t r i b u t e d t o t h e f i x e d charge? ( i i i ) What i s t h e +2 r e l a t i o n s h i p between b i n d i n g o f Ca  i n t h e i n t i m a and t h e f l u x r a t e o f  the i o n a c r o s s t h e l a t t e r membrane?  These q u e s t i o n s a r e c o n s i d e r e d i n  Chapter  3.  13  METHODS AND MATERIALS  Materials 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 s t o c k was o b t a i n e d from t h e A n t i - l o c u s t  R e s e a r c h C e n t e r , London, and t h e a n i m a l s were b r e d i n cages a t 28°C and 50% r e l a t i v e h u m i d i t y , on a d i e t o f b r a n and l e t t u c e , a t t h e 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 o f t h e 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^, t h e e x t r e m e t i e s removed so t h a t t h e e x o s k e l e t o n o f t h e abdominal r e g i o n c o u l d be s l i t up t h e mid-dorsal l i n e .  The r e c t u m was l o c a t e d and s l i t open p o s t e r i o r t o  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  The r e c t u m then was c u t a t  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 w a t e r f o r 4 t o 5 h o u r s t o l y s e t h e r e c t a l pad c e l l s and t o p a r t i a l l y d e t a c h t h e t i s s u e l a y e r s from t h e i n t i m a .  The p r e p a r a t i o n  was t h e n removed from t h e d i s t i l l e d w a t e r and t h e t i s s u e l a y e r s removed u s i n g n e e d l e p o i n t f o r c e p s under a b i n o c u l a r m i c r o s c o p e .  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 b e i n g 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 o f p e r s e x , c o n t a i n i n g 3 v e r t i c a l w e l l s o p e n i n g a t t h e t o p and j o i n e d by a t r a v e r s e c h a n n e l w h i c h opened a t t h e s i d e  ( F i g . 2 ) . An o v e r f l o w tube  Fig. 2  Schematic d i a g r a m o f a p p a r a t u s used t o measure the d i f f u s i o n and s t r e a m i n g p o t e n t i a l s a c r o s s 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  4.  O v e r f l o w tubes  5.  P o s i t i o n o f the r e c t a l i n t i m a  6.  Rubber '0' r i n g  electrodes  E n l a r g e d v i e w o f t h e membrane p o s i t i o n i n the perfusion blocks.  6 5  14  was l o c a t e d i n t h e upper s e c t i o n o f t h e l a s t w e l l .  Two o f t h e s e cham-  b e r s f i t t o g e t h e r so t h a t t h e o r i f i c e s o f t h e t r a n s v e r s e spaces were opposed.  The membrane, mounted on r u b e r "0" r i n g s , s e p a r a t e  v e r s e spaces o f t h e two chambers. the r u b b e r "0" r i n g s .  the t r a n s -  A t i g h t s e a l was f a c i l i t a t e d by  The two h a l f - c e l l s w i t h t h e i n t i m a between were  h e l d t o g e t h e r i n a bench v i s e . I n i n i t i a l experiments, achieved  s t i r r i n g o f s o l u t i o n i n t h e c e l l s was  u s i n g c a l i b r a t e d b u r e t t e tubes.  f r o m the l a t t e r i n t o b o t h chambers.  S o l u t i o n was a l l o w e d t o f l o w  The f l u i d was d e f l e c t e d by t h e  membrane up t h e l a s t v e r t i c a l w e l l and o u t t h e o v e r f l o w t u b e .  This  f l o w p a t t e r n w o u l d cause l a m i n a r f l o w a t t h e 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 i n a d e q u a t e s t i r r i n g due t o a s m a l l dead space n e x t t o the membrane.  T h i s method o f s t i r r i n g was l a t e r r e p l a c e d 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 t o d i r e c t a f l o w o f f l u i d t h r o u g h t e f l o n t u b i n g , t h e o r i f i c e o f w h i c h 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 , t h e membrane.  The f l o w o f f l u i d was  thus d i r e c t l y a g a i n s t t h e membrane s u r f a c e e l i m i n a t i n g t h e l a m i n a r f l o w p r o d u c e d by t h e b u r e t t e s .  The p e r i s t a l t i c pump r a t e s c o u l d be  v a r i e d f r o m 0.45 t o 2.8 ml/minute. maintain a constant  These r a t e s were s u f f i c i e n t t o  c o n c e n t r a t i o n o f s o l u t i o n s on b o t h s i d e s o f t h e  membrane i n t h e p r e s e n c e o f e i t h e r i o n c o n c e n t r a t i o n o r o s m o t i c gradients.  T h i s was checked u s i n g a c o n d u c t i v i t y meter ( L e w i s , 1970).  Two types o f p e r f u s i o n chambers were used f o r measurements o f i s o t o p e f l u x ( F i g . 3 ) . One s e t o f b l o c k s were made u s i n g t e f l o n , t h e other s e t out o f persex. acidic solutions.  T e f l o n was used because o f i t s i n e r t n e s s t o  The r a d i o - i s o t o p e was c o n t a i n e d 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 d i a m e t e r ,  3.0 cm i n d e p t h ) h o l d i n g 14 m l o f s o l u t i o n .  Fig.  3  S c h e m a t i c d i a g r a m o f the a p p a r a t u s used f o r measurements o f r a d i o a c t i v e t r a c e r f l u x a c r o s s the i n t i m a . 1.  A i r hose f o r s t i r r i n g  solution.  2.  Perfusion  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 perfusate.  tube.  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 t o a s h o r t t r a n s v e r s e space w h i c h  opens t o t h e s i d e ( F i g . 3 ) . The o t h e r type o f chamber had two c h a n n e l s o f 3 mm d i a m e t e r ,  j o i n i n g p e r p e n d i c u l a r l y t o form a s m a l l  space o p e n i n g t o t h e s i d e .  transverse  These two chambers f i t t e d t o g e t h e r s o t h a t  the a p e r t u r e o f t h e t r a n s v e r s e space i n t h e 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 . between these  The membrane f i t s  two chambers s e p a r a t i n g t h e t r a n s v e r s e s p a c e s .  A tight  s e a l and s i m p l e mount f o r t h e membrane was f a c i l i t a t e d by r u b b e r "0" rings.  Radioactive 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 t h e 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 , t o i n s u r e t h a t no a i r b u b b l e s f o r m o v e r t h e 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 t h e v i c i n i t y o f t h e membrane.  The t o p o f the chamber was s e a l e d ,  u s i n g " S c o t c h Brand Tape", t o p r e v e n t bubbling.  s p l a s h i n g o f i s o t o p e caused by  A Portable i n f u s i o n withdrawal  pump ("Model 1100" H a r v a r d  A p p a r a t u s Company) w i t h a d e l i v e r y r a t e o f 0.042 ml/minute was used t o p e r f u s e n o n - r a d i o a c t i v e s o l u t i o n t h r o u g h t h e second w e l l p a s t t h e membrane.  To a c h i e v e  t h i s , t h e s y r i n g e was c o n n e c t e d t o t h e v e r t i c a l  c h a n n e l by a l e n g t h o f P.E. 90 t u b i n g . channel  The t u b i n g proceeded down t h e  and was f i x e d i n s u c h a p o s i t i o n t h a t i t w o u l d d i s c h a r g e t h e  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 t h e t o p o f t h e membrane.  The  s o l u t i o n t h e n 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 t h e membrane and f l o w e d a l o n g the o t h e r c h a n n e l  t o be c o l l e c t e d i n a v i a l w h i c h was  changed e v e r y h a l f h o u r t o p r o v i d e s e r i a l samples.  16  To check f o r s u b m i c r o s c o p i c mounted i n one ( P h i l l i p s , 1964 12 h o u r s .  t e a r s i n the i n t i m a , a f t e r  o f the above p e r f u s i o n chambers, the dye a) was  I f t h e r e was  p l a c e d on one  amaranth  s i d e o f the membrane and l e f t f o r  no s u b m i c r o s c o p i c  appear on t h e o p p o s i t e s i d e .  being  damage, the dye d i d not  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 o f 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 preparatory  technique  ( P h i l l i p s and D o c k r i l l , 1968;  Lewis,  1970).  C)  Preparation of Solutions  The  s a l i n e s o l u t i o n s ( u s u a l l y o f s i n g l e s a l t s ) were made t o  d e s i r e d c o n c e n t r a t i o n s , and  the pH a d j u s t e d  w i s e s t a t e d ) u s i n g 9 N HC1.  ( t o pH 5.5  unless  Some e x p e r i m e n t s i n v o l v i n g i o n  concentra-  ( c a l c u l a t e d from p u b l i s h e d values  m o l a l f r e e z i n g p o i n t d e p r e s s i o n s ; Handbook o f C h e m i s t r y and 1968-69) was  added t o the s i d e w i t h low s a l t  the  other-  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 ; t o a c h i e v e a p p r o p r i a t e amount o f s u c r o s e  of  this  an  of  Physics,  concentration.  To s t u d y s t r e a m i n g p o t e n t i a l s i n the absence o f 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 , o s m o t i c g r a d i e n t s were c r e a t e d by a d d i n g s u c r o s e desired f i n a l concentrations  at  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 c h e m i c a l shown b e l o w , were p u r c h a s e d f r o m New  forms and a t s p e c i f i c  England Nuclear  activities  (Canada L t d . )  added t o the a p p r o p r i a t e s t o c k s o l u t i o n s o f the same compound. s o l u t i o n s were s t o r e d a t -20°  the  ^  (to avoid b a c t e r i a l  and  These  contamination).  17  Isotope  C h e m i c a l Form  Specific Activity 4.7 mc/mM  Urea Ca  45 350 mc/mM  Cl Rb  36  KCI  86  RbCl  2.22 mc/mM  D)  A n a l y t i c a l Methods  1.  R e c o r d i n g System f o r m e a s u r i n g P o t e n t i a l D i f f e r e n c e s .  A " R a d i o m e t e r " pH meter (model 25 w i t h expanded s c a l e ) and two calomel  electrodes  ( 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 ) w h i c h were p l a c e d  immediately adjacent  and on b o t h s i d e s o f t h e membrane, were used t o  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  e v e r y two minutes t o i n s u r e a s t e a d y 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 e x p e r i m e n t s , measurements o f 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 r e p e a t e d  f o r two t o t h r e e h o u r s p e r day f o r t h r e e d a y s ,  t o 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 o f t h e i n t i m a w i t h  time.  No s i g n i f i c a n t change o c c u r r e d o v e r a p e r i o d o f two days ( L e w i s , 1970). When a c o n t i n u o u s t i m e c o u r s e o f membrane p o t e n t i a l development was needed, t h e e l e c t r o m e t e r was c o u p l e d l i n e a r / l o g . paper c h a r t The  t o a "Photovolt Movel 43"  recorder.  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 t o d e t e r m i n e t h e r e l a t i v e p e r m e a b i l i t y o f t h e i n t i m a t o monovalent and d i v a l e n t c a t i o n s and monovalent a n i o n s u s i n g a modified  f o r m o f t h e Goldman c o n s t a n t  f i e l d equation  (Hani,  1970):  18  ( E q u a t i o n 1)  E = RT/F  l n Pk ( k i ) + P e l ( C l o ) + 4m P x Pk  i(Xi  )  (ko) + P C I ( C l i ) + 4m P x o ( x o ) e x p . ( - F E / R T + 2  + 2  WHERE: ( E q u a t i o n 2)  m= 1-exp. (-FE/RT) 1-exp. (-2FE/RT)  Assumptions and d e r i v a t i o n o f t h i s e q u a t i o n a r e o u t l i n e d i n A p p e n d i x A. 2.  Estimation  Radioactive  of permeability  from R a d i o - I s o t o p e  Flux.  samples (0.5 ml) f r o m f l u x s t u d i e s , depending on t h e i r  e m i s s i o n type were e i t h e r p l a c e d on a p l a n c h e t , d r i e d and s u b s e q u e n t l y c o u n t e d on a N u c l e a r C h i c a g o Model 4318 p l a n c h e t c o u n t e r ; o r t h e sample 14 (C  35 , SO^  ) was t r a n s f e r r e d t o 10 ml o f Bray's s o l u t i o n ( B r a y , 1960) and  c o u n t e d i n a " N u c l e a r C h i c a g o Mark 1" L i q u i d s c i n t i l l a t i o n S t a n d a r d s f o r each r a d i o - i s o t o p e  counter.  were made by p i p e t t i n g 0.5 ml o f c o l d  s o l u t i o n i n t o a p l a n c h e t o r s c i n t i l l a t i o n v i a l and a d d i n g t o t h i s one lambda o f t h e 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 o f the c o l l e c t e d perfusate  was below 2%  o f t h a t i n t h e r a d i o a c t i v e s o l u t i o n f r o m w h i c h d i f f u s i o n o c c u r e d ; hence back d i f f u s i o n o f the i s o t o p e  a c r o s s t h e membrane was n e g l i g i b l e .  f l u x o f t h e t e s t m o l e c u l e s was t h e n c a l c u l a t e d u s i n g equation (modified ( E q u a t i o n 3) The  permeability  (Davson, 1964):  The  the f o l l o w i n g  from Shaw, 1955): $  = A ZJC  can be c a l c u l a t e d f r o m t h e f l u x r a t e  ( f ) by F i c k ' s l a w  19  (Equation  4)  $  = oaAC  To ensure t h a t a steady recorded  f l u x v a l u e had  been r e a c h e d , f l u x r a t e s were  a t h a l f hour i n t e r v a l s f o r 4 h o u r s .  Fig. 4 illustrates  that  45 a f t e r the constant  f i r s t h a l f hour p e r i o d , the f l u x r a t e of Ca value.  had  reached a  T h e r e a f t e r f l u x v a l u e s were determined a f t e r 1 hour  pre-incubation i n radio-isotopes.  6% V/V 105  3.  Membrane A n a l y s i s and F i x e d charge  The  l o c u s t c u t i c l e was  hydrolysed  The  sample h y d r o l y s a t e was  u s i n g a "Beckman" 120C The  number o f f i x e d n e g a t i v e  were p r e p a r e d  S a s a k i , 1969)  containing  f o r 18 hours a t  s e a l e d i n vacuo and  charges was  analyzed  determined u s i n g  S i x M i l l i g r a m s of r e c t a l  as o u t l i n e d i n S e c t i o n A and  N H C l , and  of 6 N H C l ,  A u t o m a t i c Amino A c i d A n a l y s i s .  "Radiometer Copenhagen" T i t r i g r a p h .  o f 0.5  i n 2ml  t h i s g y l o c o l i c a c i d (Matsubara and  i n vacuo.  Density.  a  cuticle  then p l a c e d i n a l a r g e volume  then c e n t r i f u g e d 6 times i n d i s t i l l e d water.  The  membranes were then shredded u s i n g n e e d l e p o i n t f o r c e p s , and  added to  1 ml o f d i s t i l l e d water.  with  T h i s p r e p a r a t i o n was  then t i t r a t e d  c a r b o n a t e f r e e NaOH i n the p r e s e n c e o f a n i t r o g e n  4.  Electrical  The  r e s i s t a n c e o f the membrane was  atmosphere.  Resistance.  rodes connected to the e l e c t r o m e t e r  and  measured u s i n g c a l o m e l  p l a c e d on b o t h s i d e s o f  electthe  membrane t o measure p o t e n t i a l d i f f e r e n c e s as p r e v i o u s l y d e s c r i b e d . set  of s i l v e r wire  across  e l e c t r o d e s were used to apply  the membrane from two  A  a variable current  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 w i t h  a K e i t h l e y ammeter (Model 601)  and v a r i a b l e r e s i s t a n c e ( H e a t h k i t Decade  /  Fig.  4  45 Time c o u r s e o f Ca intima.  f l u x across the r e c t a l  Each p o i n t r e p r e s e n t s counts  per  minute i n the c o n s t a n t volume of p e r f u s a t e t h a t was sampled a t h a l f h o u r  intervals.  a  U  200  -I  150  i  o  cu +->  q  100 -4  u  o a  CQ  -t-> « o  50  0  " T  0  2 Time in hours  20  Resistance  Model I N - 1 7 ) . The c u r r e n t was v a r i e d and t h e r e s u l t i n g p o t e n -  t i a l d i f f e r e n c e across KC1  the membrane (bathed on b o t h s i d e s w i t h t h e same  o r C a C l ^ s o l u t i o n o f v a r i o u s m o l a r i t i e s ) was measured.  was t h e n c u t and t h e above p r o c e d u r e r e p e a t e d .  The i n t i m a  By s u b t r a c t i o n o f t h e  r e s i s t a n c e o f the r e c o r d i n g system alone, from the r e s i s t a n c e o f the membrane p l u s t h e r e c o r d i n g system, t h e 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 l a w .  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 o f various  factors of physio-  l o g i c a l i m p o r t a n c e on i o n p e r m e a t i o n a c r o s s t h e l o c u s t i n t i m a , i t was u s e f u l t o d e t e r m i n e whether i o n s e l e c t i v i t y o f t h i s membrane was due t o a f i x e d charge s y s t e m ( w i t h p r o p e r t i e s  c l o s e l y a p p r o x i m a t i n g those o f  an i o n exchange r e s i n ) o r w h e t h e r t h e i n t i m a p o s s e s s e d f i x e d n e u t r a l sites.  I d e n t i f i c a t i o n o f the type o f s i t e allow d i f f e r e n t p r e d i c t i o n s  t o be made.  C r i t e r i a have been s u g g e s t e d by W r i g h t , B a r r y  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  sites.  These c r i t e r i a i n c l u d e t h e n a t u r e o f t h e r e l a t i o n s h i p between i o n conc e n t r a t i o n of the bathing P  v  Jv  :P- ratio.  media and b o t h the membrane r e s i s t a n c e and t h e  B o t h t h e s e r e l a t i o n s h i p s were c o n s i d e r e d  i n this  chapter  O JL  f o r the l o c u s t intima.  The p o s s i b l e c h e m i c a l n a t u r e o f t h e f i x e d  c h a r g e s was t h e n c o n s i d e r e d .  The approach was t o f i r s t d e t e r m i n e t h e  pK o f t h e 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 v a l u e s o f pK f o r various  r a d i c a l s ( e g . amino a c i d s i n i n t a c t p r o t e i n ) .  Assuming t h e  p r o b a b i l i t y t h a t t h e f i x e d charge was a s s o c i a t e d w i t h t h e p r o t e i n o f the i n t i m a , an amino a c i d a n a l y s i s o f t h e l a t t e r was c a r r i e d o u t t o d e t e r m i n e i f an e x c e s s o f an amino a c i d w i t h t h e r e q u i r e d pK v a l u e was p r e s e n t .  22  RESULTS  A.  Membrane R e s i s t a n c e Helfferich  (1962) has shown t h a t i n an i o n exchange membrane t h e  c o n d u c t i v i t y o f t h e exchanger bed i s i n d e p e n d e n t o f t h e i o n c o n c e n t r a t i o n when t h e 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 t h a n t h e c o n c e n t r a tion of fixed s i t e s .  W r i g h t , B a r r y and Diamond (1971) r e i t e r a t e d  this  p o s t u l a t e and a l s o added t h a t i f t h e membrane c o n t a i n e d f i x e d n e u t r a l s i t e s t h e conductance o f t h e membrane s h o u l d be l i n e a r l y r e l a t e d t o 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 o f the c o n c e n t r a t i o n range. W i t h t h e s e p r e d i c t i o n s i n mind, t h e conductance o f t h e i n t i m a was measured as t h e c o n c e n t r a t i o n o f t h e b a t h i n g s o l u t i o n (both was i n c r e a s e d f r o m 0.1 t o 1000 mM/l C a C ^ .  sides)  The i n t i m a seemodto a c t as  an i o n e x c h a n g e r 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 a t low i o n c o n c e n t r a t i o n s  (0.1 t o 10 mM/l C a C l ^ ) t h e  conductance i s r e l a t i v e l y i n d e p e n d e n t o f t h e i o n c o 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 o f 3 - f o l d f o r a 100-fold i n c r e a s e i n ion concentration) .  At high i o n concentrations  (1000 mM/l C a C ^ ) , t h e  r e s i s t a n c e o f t h e i n t i m a does n o t f a l l i n p r o p o r t i o n t o d e c r e a s e i n t h e ion concentration.  A t h i g h c o n c e n t r a t i o n s , t h e membrane might have  r e a c h e d a s a t u r a t i o n c a p a c i t y f o r t h e i o n , such t h a t 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 o f t h e e x t e r n a l s o l u t i o n w o u l d n o t r e s u l t i n an i n c r e a s e o f the membrane i o n c o n c e n t r a t i o n . When t h e above e x p e r i m e n t was r e p e a t e d u s i n g a K C I 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 o b s e r v e d between r e s i s t a n c e and c o n c e n t r a t i o n (except below 1 mM/l K C I ) .  T h i s might i n d i c a t e t h a t t h e  Fig.  5  The s o l i d c i r c l e s r e p r e s e n t  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 o f t h e b a t h i n g t i o n and membrane r e s i s t a n c e . squares represent  solu-  The s o l i d  t h e r e l a t i o n s h i p between t h e  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 o f C a C l ^ and the membrane r e s i s t a n c e . 8 f o r i n d i v i d u a l values.  See A p p e n d i x B T a b l e  5 -,  W  -J  ,  0.1  1  1  10  1  100  1  1000  Concentration of bathing solution in m M / l  23  K  i n the membrane e x i s t s i n two p h a s e s , one a s s o c i a t e d w i t h a f i x e d  c h a r g e , and the o t h e r i n b u l k s o l u t i o n .  Such a s y s t e m a c c o r d i n g t o  Eisenman (1967) c o n s t i t u t e s a " F i x e d D i s s o c i a t e d System" i n w h i c h t h e c o n d u c t i v i t y i s a f u n c t i o n o f the p a t h o f l e a s t r e s i s t a n c e .  The d i f -  +2 f e 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 t o i t s l a r g e r h y d r a t e d  radius  ( o n l y s l i g h t l y s m a l l e r than t h e p o r e s i z e ) so t h a t i t i s e s s e n t i a l l y o n l y 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 s y s t e m o f Eisenman, e t a l . , 1967) e x c e p t a t v e r y h i g h  concentrations.  I t w i l l be seen i n C h a p t e r 3 t h a t t h e f i x e d charge o f t h e 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 a t low i o n c o n c e n t r a t i o n s i n a f i x e d a s s o c i a t e d s y s t e m w o u l d be dependent on t h e f i x e d charge c o n c e n t r a t i o n . B.  Anion: The  C a t i o n 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 o f c o n c e n t r a t i o n . e f f e c t o f i o n c o n c e n t r a t i o n on c a t i o n v e r s u s  anion  selectivity,  p r o v i d e s a t e s t o f the p o s s i b i l i t y t h a t the r e c t a l i n t i m a behaves as a i o n exchange membrane as d e s c r i b e d by H e l f f e r i c h (1953).  (1962) and T e o r e l l  T e o r e l l (1953) showed t h a t d i f f u s i o n p o t e n t i a l s were dependent  on the average c o n c e n t r a t i o n s o f t h e b a t h i n g s o l u t i o n s ; i . e . as t h e average c o n c e n t r a t i o n i n c r e a s e d t h e d i f f u s i o n p o t e n t i a l d e c r e a s e d . W r i g h t , B a r r y and Diamond (1971) s t a t e t h a t a l i n e a r conductance t i o n curve s h o u l d r e s u l t i n a c o n s t a n t  p c l  /  p k  r a t i o regardless o f 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 b o t h o f these phenomena b e i n g of a f i x e d n e u t r a l s i t e .  concentra  indicative  Fig.  / The  6  permeability ratio P ^  o f KC1  concentration.  /P^ as a f u n c t i o n  Average KC1 concentration in  mM/l  24  Diffusion r a t i o (10:1 KC1) s o l u t i o n was  p o t e n t i a l s were measured a t a c o n s t a n t c o n c e n t r a t i o n w h i l e t h e average c o n c e n t r a t i o n o f the e x t e r n a l b a t h i n g  varied.  The p e r m e a b i l i t y r a t i o P ^ /P^ was  calculated  ( F i g . 6) u s i n g t h e Goldman-Hodgkin-Katz c o n s t a n t f i e l d e q u a t i o n Appendix A ) . t i o n o f KC1,  The P ^  /P^ r a t i o was  (see  dependent on the average c o n c e n t r a -  such t h a t the r a t i o approached u n i t y as the average  centration increased.  T h i s experiment  con-  i n d i c a t e s t h a t the i n t i m a a c t s  as a f i x e d n e g a t i v e 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.  E s t i m a t e o f the C o n c e n t r a t i o n o f F i x e d Charges i n the I n t i m a . An e s t i m a t e o f the number o f f i x e d charges  pKa and p i o f the f i x e d charges  and t h e  apparent  i n t h e i n t i m a was  o b t a i n e d d i r e c t l y by  p e r f o r m i n g an a c i d - b a s e t i t r a t i o n on the i n t i m a .  The p i v a l u e f o r the  f i x e d charges was be 4.8  found t o be 2.4, w h i l e the pKa v a l u e was  ( u s i n g the method o f H e l f f e r i c h , 1962).  number o f f i x e d charges was  An e s t i m a t e o f the  a l s o made ( H e l f f e r i c h , 1962)  pH change v e r s u s the amount o f N a  +  estimated to  t i t r a t e d ( F i g . 7).  by p l o t t i n g  The  the  capacity of  the membrane and thus the number o f a c i d i c groups p e r u n i t w e i g h t i s e q u a l t o the amount o f t i t r a n t added up t o the p o i n t o f i n f l e c t i o n on the t i t r a t i o n c u r v e .  The  charge d e n s i t y o f the i n t i m a was  estimated i n  t h i s manner t o be a p p r o x i m a t e l y 0.045 m i c r o - e q u i v a l e n t s o f charge p e r m i l l i g r a m (dry weight) of i n t i m a . A n o t h e r e s t i m a t e o f f i x e d charge d e n s i t y was sorption isotherms. a t pH 2.3  and 5.5  The membranes were b a t h e d  +2 o b t a i n e d from Ca  i n 1 and lOmM/1 C a C l ^  +2 To o b t a i n the amount o f Ca p r e s e n t i n the membrane  i n the bound form a t a pH o f 5.5,  the a s s u m p t i o n was  made t h a t t h e amount  Fig. 7  pH t i t r a t i o n c u r v e o f the r e c t a l i n t i m a .  5  1  25  '  e'o  ' 120  '  lfeo  microequivalents NaOH per gram  '  2bo  of tissue  Table 1  The b i n d i n g c a p a c i t y o f t h e r e c t a l i n t i m a +2 f o r Ca tions.  was measured f o r 6 i n t i m a  prepara-  The i n t i m a s were b a t h e d i n 10 mM/l o r AS  1 mM/l Ca  labelled CaCl  pH 5.5 o r 2.3. planchet  2  solutions at  Membranes were counted i n a  counter.  uM C a / m g ± S.D. ( intima D r y Weight) +2  Solution Concentration of C a C l 2  pH 5.5  pH2.3  Bound C a  % water - S.D. + 2  10 mM/l  0.0678 i 0.018  0.025 ±0.0028  0.0428 ± 0.011  1 mM/l  0.0371 1 0.0065  0.0026*0.0008  0.0345 ± 0.007  pH 5.5 66.8 t 2.5  pH 2.3 71.7* 2.5  25  o f Ca  p r e s e n t i n the membrane a t t h i s pH was i n two s t a t e s , one b e i n g  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  fluid  +2 (the amount o f f r e e Ca b e i n g the amount found i n t h e membrane a t a +2 pH o f 2 . 3 ) . The d i f f e r e n c e i n Ca c o n t e n t a t the two pH v a l u e s was +2 t h e n t h e amount o f bound Ca  .  T a b l e 1 shows t h a t the amount o f bound  +2 Ca  at concentration  o f 1 and 10 mM/l C a C l  2  a r e 0.034 and 0.0428  +2 m i c r o m o l e s o f Ca  per m i l l i g r a m dry weight o f i n t i m a r e s p e c t i v e l y .  B o t h o f t h e s e v a l u e s agree w e l l w i t h t h e number o f charges found by acid-base D.  titration.  Amino A c i d A n a l y s i s o f C u t i c u l a r P r o t e i n . I n an a t t e m p t t o i d e n t i f y t h e c h e m i c a l groups w h i c h might be  responsible  f o r the o b s e r v e d f i x e d charge i n the i n t i m a , t h e amino a c i d  c o m p o s i t i o n o f the i n t i m a was d e t e r m i n e d . T a b l e 2.  The r e s u l t s a r e g i v e n i n  The t o t a l amino a c i d c o n t e n t was a p p r o x i m a t e l y 57.7% by  w e i g h t ( d r y ) o f the membrane.  The most l i k e l y p o t e n t i a l s o u r c e o f  n e g a t i v e 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 o f f l u t a m i c a c i d and 370 nanomoles o f 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 . centrations  On the o t h e r hand, t h e con-  o f the t h r e e b a s i c amino a c i d s o b s e r v e d , 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 p e r m i l l i g r a m d r y w e i g h t o f the i n t i m a r e s p e c t i v e l y . acids  The t o t a l c o n c e n t r a t i o n  (937 nanomoles p e r m i l l i g r a m o f i n t i m a )  t o t a l concentration  o f b a s i c amino a c i d s  t h e r e f o r e exceeds the  (586 nanomoles  o f i n t i m a ) by 351 nanomoles p e r m i l l i g r a m o f i n t i m a . conclusion,  o f a c i d i c amino  permilligram  There i s , i n  an e x c e s s o f n e g a t i v e groups i n c u t i c u l a r p r o t e i n w h i c h  m i g h t be the s o u r c e o f t h e o b s e r v e d 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 o f the r e c t a l  intima.  Amino A c i d  Amino A c i d classification  Quantity of Amino A c i d mg/gm of cuticle nm/gm of cuticle  Glycine  Neutral  94  1644  Glutamic  Acidic  73  567  Proline  Imino  57  588  Aspartic  Acidic  42.6  371  Tyrosine  Aromatic  32.4  199  Arginine  Basic  30.8  198  Lysine  Basic  27.8  218  Threonine  Neutral  25.8  245  Isoleucine  Neutral  24.8  220  Serine  Neutral  24.2  279  Leucine  Neutral  23.8  211  Histidine  Basic  23  168  Phenylalanine  Aromatic  20  136  Valine  Aromatic  17  173  26  DISCUSSION  U s i n g the c r i t e r i a p r o p o s e d by W r i g h t , B a r r y and Diamond (1971) i t i s p o s s i b l e t o d e c i d e 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 p o r e w h i l e  the  o t h e r end i s e f f e c t i v e l y b u r i e d i n the m a t r i x ) o r charged ( i . e . a s i t e w h i c h has  t o be a s s o c i a t e d w i t h a m o b i l e c o u n t e r - i o n so as t o  electroneutrality). between these  The  experimental  two p o s s i b i l i t i e s a r e :  maintain  observations which d i s t i n g u i s h 1)  the r e l a t i o n s h i p between mem-  b r a n e conductance and i o n c o n c e n t r a t i o n o f the medium, and 2) o f change o f P ^ /P^ r a t i o as a f u n c t i o n o f KCI  the degree  concentration.  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 , W r i g h t e t a l . (1971) found for  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 o f the medium f o r b o t h o f 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 choroid plexus. resins.  frog  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  I n a s i m p l e case o f an i o n exchanger where the o n l y c o n d u c t i v i t y  i s t h r o u g h the p o r e s ,  ( 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 t h r o u g h the bed  o f the i o n e x c h a n g e r ; 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 a l m o s t i n d e p e n d e n t o f 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 higher concentrations.  S i n c e the i o n exchanger must r e m a i n  e l e c t r i c a l l y n e u t r a l , t h e r e w i l l always be r e s i d e n t c o u n t e r - i o n s i n the membrane t o e q u a l i n c o n c e n t r a t i o n the number o f f i x e d r e g a r d l e s s o f the e x t e r n a l c o n c e n t r a t i o n .  The  present  sites,  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 t h a t  27  o f t h e f i x e d charge c o n c e n t r a t i o n .  I n t h e case o f f i x e d n e u t r a l  s i t e s , however, t h e s i t e s c a n m a i n t a i n e l e c t r o n e u t r a l i t y w i t h o u t c o u n t e r - i o n s , thus t h e i o n c o n c e n t r a t i o n i n t h e p o r e w i l l a p p r o x i m a t e t h a t found i n t h e e x t e r n a l s o l u t i o n .  engaging  closely  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 t h e 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 t y p e s o f c o n c e n t r a t i o n - c o n d u c t a n c e present study.  When t h e b a t h i n g media c o n s i s t e d o f a KC1 s o l u t i o n , t h e  c u r v e was a p p r o x i m a t e l y (i.e.  c u r v e s were o b s e r v e d i n t h e  above 1 mM/l).  l i n e a r o v e r most o f t h e c o n c e n t r a t i o n range used  When C a C l ^ s o l u t i o n s were u s e d , however, t h e  r e s i s t a n c e o f conductance was r e l a t i v e l y independent o f i o n  concentra-  t i o n a t low s a l t c o n c e n t r a t i o n s b u t becomes more dependent on c o n c e n t r a t i o n as t h e C a C ^  concentration increased, possibly indicating a satura-  t i o n o f a membrane c h a r g e .  The C a C ^ conductance seemed t o i n d i c a t e a  f i x e d charge s y s t e m w h i l e t h e KC1 conductance f i t t e d a n e u t r a l s i t e model ( o r uncharged pore m o d e l ) , e x c e p t a t v e r y l o w c o n c e n t r a t i o n s . Turning  t o t h e second c r i t e r i o n ( i . e . t h e P ^ /P  e t a l . (1971) found o n l y a s l i g h t change i n t h i s r a t i o  r a t i o ) , Wright (0.05  p e r 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 t h e average  concentra-  t i o n o f t h e b a t h i n g s o l u t i o n s when t h e c o n c e n t r a t i o n r a t i o a c r o s s t h e g a l l b l a d d e r was k e p t c o n s t a n t .  The v e r y s l i g h t change i n t h e r a t i o  o b s e r v e d was i n s u f f i c i e n t t o 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 , t h e r a t i o P . /P  f o r an i o n exchanger w i l l  c o n s i d e r a b l y as t h e 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 ing  a constant  concentration ratio  ( T e o r e l l , 1953).  vary  maintain-  T h i s can be e x -  p l a i n e d by c o n s i d e r i n g t h e membrane i n two p h a s e s , b o t h v a r i a b l e and  28  dependent on t h e i o n c o n c e n t r a t i o n .  When t h e membrane charge d e n s i t y i s  much g r e a t e r than t h e e x t e r n a l i o n c o n c e n t r a t i o n , i s almost completely  the p o t e n t i a l d i f f e r e n c e  d e t e r m i n e d by aDonnan e q u i l i b r i u m p o t e n t i a l w h i c h  f o r a 1 0 - f o l d g r a d i e n t approaches 58 mv a t 20°. When t h e c o n c e n t r a t i o n o f t h e membrane charge i s i n s i g n i f i c a n t compared t o t h e e x t e r n a l i o n concentration,  t h e 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 t h e p o t e n -  t i a l d i f f e r e n c e i s d e t e r m i n e d 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 t h e l o c u s t i n t i m a was o b s e r v e d t o be  dependent on t h e average c o n c e n t r a t i o n  o f t h e b a t h i n g 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  o f KCI i n the b a t h i n g  e l i c i t e d a change o f 0.25 i n t h e p e r m e a b i l i t y r a t i o .  media  This i m p l i e d a  s i g n i f i c a n t d e c r e a s e i n c a t i o n conductance o r an i n c r e a s e i n a n i o n conductance, o r both, w i t h i n c r e a s i n g s a l t concentration  o f t h e media.  The  magnitude o f t h i s p e r m e a b i l i t y change s h o u l d be s u f f i c i e n t t o 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 t h e c o n d u c t a n c e - c o n c e n t r a t i o n relationship.  T h i s e x p e r i m e n t agrees w i t h r e s u l t s e x p e c t e d f o r a f i x e d  c h a r g e d membrane. A t h i r d l i n e o f evidence i n d i c a t e , the f i x e d chargewa not a S  neutral site. and  +2 The amount o f bound Ca i n the intimawas s i m i l a r f o r 1  10 mM/l C a C ^ media (Table 1 ) .  That i s , i n o r d e r t o m a i n t a i n  e l e c t r o n e u t r a l i t y , t h e membrane c o n c e n t r a t i o n +2 i n d e p e n d e n t o f t h e e x t e r n a l Ca  level.  +2 o f Ca tended t o be  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 t h a n a f i x e d n e u t r a l s i t e . Assuming t h a t t h e f i x e d charge was a s s o c i a t e d w i t h t h e p r o t e i n m o i e t y i n t h e c u t i c l e , t h e f o r m e r c o u l d be due t o e i t h e r t h e f r e e c a r b o x y l o r amino groups on t h e end o f each p r o t e i n c h a i n , o r t o  29  d i c a r b o x y l i c o r diamine groups. i n peptide groups.  linkages,  In general,  free terminal the i n t i m a  proteins  groups a r e u s u a l l y bound  thus w i l l n o t p l a y a s i g n i f i c a n t r o l e as charged t h e t i t r a t a b l e a c i d i c groups i n p r o t e i n s  groups o f glutamic  gave a pKa v a l u e  p o i n t o f 2.4.  Alpha carboxyl  a c i d and a s p a r t i c a c i d .  a r e the  T i t r a t i o n of  o f a p p r o x i m a t e l y 4.8 w i t h an i s o e l e c t r i c  The c h a r a c t e r i s t i c pK v a l u e s  of various  groups i n i n t a c t  a r e l i s t e d below: Groups  pK (25°C)  C a r b o x y l (alpha)  3.0-3.2  Carboxyl (aspartyl)  3.0-4.7  Carboxyl  c a . 4.4  (glutamyl)  Phenolic-hydroxyl  (diiodotyrosine)  6.5  Phenolic-hydroxyl  (tyrosine)  9.8-10.4  Sylfhydryl  9.1-10.8  (Page 445, Cohn and E d s a l l - P r o t e i n s , Amino A c i d s The  most l i k e l y  and P e p t i d e s ) .  s o u r c e f o r the observed n e g a t i v e  i n t i m a were a s p a r t i c g l u t a m i c  acid.  This suggestion  charges i n the  was s u p p o r t e d by  an excess o f the two d i s c a r b o x y l i c a c i d s o v e r a l l b a s i c amino a c i d s . The  possibility  polysaccharides  t h a t t h e charge c o u l d be a s s o c i a t e d w i t h a c i d i c mucowhich r e s i d e i n the p r o c u t i c l e ( N o i r e t e t a l . 1966)  cannot be o v e r l o o k e d .  The l a t t e r a u t h o r s suggest t h a t the a c i d i c  m u c o p o l y s a c c h a r i d e s might c o n t r i b u t e i n mucopolysaccharides  f i x e d charges because a r e d u c t i o n  (detected histochemically)  the s t a i n i n g s o l u t i o n was  reduced from 4.2 t o 2.  o c c u r s as the pH o f  30 I s u g g e s t t h a t the i n t i m a c o n t a i n s t h a n n e u t r a l s i t e s and  f i x e d negative  sites  rather  t h a t the c h e m i c a l n a t u r e o f the s i t e s c o u l d  be  e i t h e r a c i d i c m u c o p o l y s a c c h a r i d e s , o r more p r o b a b l y a c i d i c amino a c i d s ,  31  CHAPTER I I PARAMETERS INFLUENCING POTENTIAL DIFFERENCE ACROSS THE INTIMA. INTRODUCTION  Preliminary studies  ( L e w i s , 1970) demonstrated t h a t t h e i n t i m a  was s e l e c t i v e l y permeable t o c a t i o n s and was c a p a b l e o f f o r m i n g b o t h d i f f u s i o n and s t r e a m i n g p o t e n t i a l s under t h e a p p r o p r i a t e This chapter i s then a l o g i c a l extension  conditions.  o f these p r e l i m i n a r y  experi-  ments . The  previous  s t u d y ( L e w i s , 1970) e s t a b l i s h e d t h a t t h e p r e s e n c e  o f f i x e d charges i n the i n t i m a c o u l d account f o r t h e s e l e c t i v i t y t o 5 monovalent c a t i o n s .  One o f t h e o b j e c t i v e s o f t h i s T h e s i s was t o e x t e n d  t h i s s t u d y t o i n c l u d e t h e measurements o f s e l e c t i v i t y o f t h e 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 a n i o n s and a d d i t i o n a l monovalent c a t i o n s , so as t o e s t a b l i s h more p r e c i s e l y t h e 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  t o e s t a b l i s h more c o m p l e t e l y  selective reabsorption Various  the r o l e o f the i n t i m a i n  and r e g u l a t i o n o f b l o o d  ion levels.  parameters o f the primary f i l t r a t e  c e n t r a t i o n , i o n r a t i o s , pH, o s m o t i c p r e s s u r e ,  ( e g . t o t a l i o n con-  and n e t w a t e r movement)  b e i n g dependent on the p h y s i o l o g i c a l s t a t e o f t h e a n i m a l , w i l l w i d e l y i n t h e l o c u s t rectum.  vary  S i n c e t h e s e f a c t o r s might be e x p e c t e d t o  a l t e r t h e s e l e c t i v i t y o f a f i x e d charge o r a l t e r t h e membrane s t r u c t u r e , i t was o f immediate p h y s i o l o g i c a l i m p o r t a n c e t o s t u d y t h e i r e f f e c t s on the i n t i m a .  Such e x p e r i m e n t s a l s o p r o v i d e  f u r t h e r d a t a on t h e p r o p e r -  t i e s o f f i x e d charged membranes under a v a r i e t y o f c o n d i t i o n s f o r a c o m p a r i s o n w i t h o t h e r membrane systems and v a r i o u s h y p o t h e t i c a l models w h i c h p r e d i c t t h e b e h a v i o r o f b i o l o g i c a l membrane.  32  Recent o b s e r v a t i o n s  on the e f f e c t o f u n s t i r r e d l a y e r s on i o n  movement and p o t e n t i a l development  a c r o s s v a r i o u s membranes  ( D a i n t y and  House, 1966) i n d i c a t e the l a t t e r c o u l d be o f c o n s i d e r a b l e importance in r e c t a l reabsorption.  These o b s e r v a t i o n s  ( D a i n t y and House, 1966)  then n e c e s s i t a t e d a r e - e v a l u a t i o n o f some p r e v i o u s e x p e r i m e n t s on s t r e a m i n g 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 o f the i n t i m a t o Monovalent and Cations  Divalent  and Monovalent A n i o n s .  D u r i n g the s e l e c t i v e r e a b s o r p t i o n  o f i o n s 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 b l o o d ) the f i r s t b a r r i e r t h a t the i o n s must c r o s s i s the rectal intima.  Preliminary studies  o f the i n t i m a was  ( L e w i s , 1970)  on  r e s t r i c t e d t o 5 monovalent a l k a l i  w h i c h 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  permaselectivity metal ions, f o r  shown t o c o r r e s p o n d t o  Eisenman's s e c o n d sequence f o r a f i x e d n e g a t i v e  charge a s s o c i a t e d  with  aqueous c h a n n e l s . The  p r e s e n t e x p e r i m e n t s were u n d e r t a k e n t o e x t e n d t h e s e s t u d i e s  t o i n c l u d e monovalent a n i o n s ,  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 t o 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 t h e o r y  f o r monovalent a n i o n s 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 p r e s e n c e of f i x e d c h a r g e s . 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 o f the was  b a t h e d i n 10 mM/l  KC1  and  o f a second s a l t p l u s 5 mM/l centration gradients  the haemocoel s i d e was KC1.  for either K  bathed i n 5  mM/l  T h i s arrangement gave r i s e t o con+  or C l  R e l a t i v e p e r m e a b i l i t y , f o r a n i o n s and  and  a second c a t i o n o r  anion.  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 T h e s i s were i n a s t e a d y s t a t e c o n d i t i o n . 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 c o n s t a n t  approximation.  The  field  (see A p p e n d i x 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 t i o n s were v a l i d as a f i r s t  intima  calcuequation  calcula-  Table 3  R e l a t i v e p e r m e a b i l i t y o f t h e i n t i m a f o r monov a l e n t a n i o n s 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 .  Diffu-  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 o f a second s a l t was added t o t h e s i d e o f l o w 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 t h e m o d i f i e d Goldman constant  f i e l d equation  (see A p p e n d i x A ) .  Each v a l u e i s t h e mean f o r 6 p r e p a r a t i o n s .  Ionic Species  Relative Permeability - S.D.  t 0.08  NH/  1 1  Rb  +  1.05 ± 0.03  Cs  +  4  0  1.01  t 0.01  1.00 ± 0.01  K+ Na  +  0.67  Li  +  0.55 ± 0.04  TEA  t 0.01  0.062 ± 0.05  +  Ba  + +  0.354 ± 0.05  Ca  + +  0.29 ± 0.03  Sr  0.28 ± 0.03  + +  Mg++  0.23 t 0.05  Mn  0.23 ± 0.05  + +  HCOg"  0.128 t 0.012  CN'.  0.121 ± 0.01  F"  0.102 ± 0.03  N0 "  0.101 ± 0.02  cr  0.097 t 0.06  CH3C00"  0.095 t 0.006  Br'  0.084 t 0.008  3  2 I"  4  0.082 t 0.005 0.067 ± 0.008  34  Monovalent The  Cations  r e c t a l i n t i m a has the a b i l i t y  to s e l e c t  c a t i o n s i n a p r e f e r e n t i a l o r d e r o f NH* > R b  +  > Cs  f o r monovalent +  > K  +  > Na  +  > L i > +  TEA,  which i s sequence two o f Eisenman, i n d i c a t i n g w i d e l y spaced  tive  f i x e d charges  o f weak f i e l d  the r a t e o f p e n e t r a t i o n w a s  and D o c k r i l l ,  That i s ,  i n v e r s e l y p r o p o r t i o n a l t o the h y d r a t e d  r a d i u s o f i o n s aswas the case (Phillips  s t r e n g t h (Eisenman, 1962).  nega-  f o r uncharged h y d r o p h i l i c m o l e c u l e s  1968).  Monovalent Anions The anions  to s e l e c t  f o r monovalent  i n a p r e f e r e n t i a l o r d e r o f : HCO~ > CN~ > F~ > N0~ > C l ~ >  CH^COO (F  r e c t a l i n t i m a a l s o has the a b i l i t y  > Br  > E^PO^ > I.  > C l ~ > Br~ > I~ ).  unhydrated  i o n with  l a r g e s t unhydrated  which i s sequence seven o f Eisenman  I n t h e l a t t e r sequence, F~ i s the s m a l l e s t  the s t r o n g e s t energy o f h y d r a t i o n ; w h i l e I i o n w i t h the weakest energy o f h y d r a t i o n .  the sequence o f p e r m e a b i l i t y was a c c o r d i n g to the d e c r e a s i n g  i s the  That i s , hydrated  radius.  Divalent The order of:  Cations  i n t i m a can s e l e c t  +2  Ba  Sherry, i n d i c a t i n g negative  "r2  > Ca  +2  > Sr  f o r divalent cations i n a p r e f e r e n t i a l > Mg  +2  > Mn  +2  , which i s sequence two o f  (as d i d the sequence f o r the monovalent c a t i o n s )  f i x e d charges,  e i t h e r o f weak f i e l d  W r i g h t , 1 9 6 9 ) , b u t c l o s e l y spaced,  s t r e n g t h (Diamond and  or stronger f i e l d  strength with the  35 charges widely spaced.  I t seems that the former c o n f i g u r a t i o n was  most l i k e l y , since weak f i e l d strength was studies on monovalent cations.  the  i n d i c a t e d from the above  Both the monovalent and d i v a l e n t  sequences i n d i c a t e that s e l e c t i v i t y was mediated by the same f i x e d charge.  The sequence of permeability f o r d i v a l e n t ions was  according  to i n c r e a s i n g hydrated radius.  B.  Competition Between Ions f o r Fixed Negative S i t e s . The preliminary studies were concerned with pure s o l u t i o n s 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 r e g u l a t i n g ion permeability of the i n t i m a .  In the i n vivo rectum of the l o c u s t  there i s a mixture of i o n s , the l e v e l s 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 f o r the f i x e d 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-  t i o n s ) . The e f f e c t s of i n c r e a s i n g the concentration of monovalent and d i v a l e n t cations (as C l r e l a t i v e to C l  s a l t s ) on the s e l e c t i v i t y of the intima f o r K  was i n v e s t i g a t e d (K  +  and C l  are the p r i n c i p l e ions  normally entering the l o c u s t ( P h i l l i p s , 1964 b) ). i n d i c a t e properties of the f i x e d charge.  Sucrose was  osmotic pressures.  These studies  The procedure used was  a constant KCI concentration gradient (10 mM/l intima.  - 5 mM/l)  across  to keep the  added to the side of low KCI to maintain equal The competing s a l t was  added equally to both sides  of the bathing s o l u t i o n s , making f i n a l concentrations s a l t equal to 1, 10 or 100  mM/l.  +  f o r the second  Fig. 8  The e f f e c t o f 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 . t r a t i o n o f KC1 on l u m e n a l s i d e was  Concen-  twice that  o f haemocoel s i d e i n a l l e x p e r i m e n t s .  Values  are p r e s e n t e d as a f r a c t i o n o f the p o t e n t i a l d i f f e r e n c e i n t h e absence o f a second  salt.  S a l t s were added a t e q u a l c o n c e n t r a t i o n s on both sides.  V a l u e s r e p r e s e n t the average  of  s i x preparations. •  - NaCl  •  -  A  - MgCl  2  O  - CaCl  2  KC1  See Appendix B T a b l e 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  I n a n a l y s i n g the e f f e c t s o f the added s a l t , t h e r e s u l t s were e x p r e s s e d as a f r a c t i o n o f the d i f f u s i o n p o t e n t i a l o b s e r v e d f o r a g i v e n p r e p a r a t i o n i n the absence o f a competing s a l t . e x p e r i m e n t can be o b s e r v e d i n F i g . 8.  The r e s u l t s o f the  A f t e r a d d i n g ImM/l o f C a C l ^ a  p o t e n t i a l drop o f 40% o c c u r s , w h i l e 1 mM/l  o f MgCl^, KC1 and  duced d e c r e a s e s o f 25%, 18% and 16% r e s p e c t i v e l y .  A d d i n g 10  NaCl p r o mM/l  C a C l , M g C l , K C l and N a C l l e d t o d e c r e a s e s o f 87%, 67%, 61% and 47% 2  2  respectively.  The e f f e c t o f 100 mM/l  was  t o cause a r e -  v e r s a l o f p o t e n t i a l amounting t o 14% and 15% r e s p e c t i v e l y  of the i n i t i a l  potential difference:  100 mM/l  98% and 94% r e s p e c t i v e l y ;  CaCl  2  2  and M g C l  2  KC1 and N a C l reduce t h e p o t e n t i a l  i e . the chargewas  f o r c a t i o n o v e r a n i o n was a b o l i s h e d . t h a t 1 mM/l  CaCl  s a t u r a t e d so t h a t  by  selectivity  The most s t r i k i n g o b s e r v a t i o n was  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 a p p r o x i m a t e l y 10 mM/l  NaCl; i . e . t h i s divalent  i o n was 10 t i m e s as  e f f e c t i v e a t masking f i x e d charge as a monovalent i o n . These e x p e r i m e n t s i n d i c a t e d t h a t f i x e d n e g a t i v e charge was an i m p o r t a n t 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 a n i m a l a t l o w e r c o n c e n t r a t i o n s , such as was o b s e r v e d i n h y d r a t e d l o c u s t s .  37  C.  Relative Permeability In p r e v i o u s  o f the Intima as Measured by Streaming P o t e n t i a l s .  experiments the p r o p e r t i e s  been s t u d i e d i n the absence o f f l u i d  flow.  o f the f i x e d charge have  The membrane model f o r the  i n t i m a , as developed so f a r , c o n s i s t s o f f l u i d f i l l e d p o r e s l i n e d w i t h f i x e d negative  charges.  The e f f e c t o f f l u i d movement ( r e s u l t i n g i n  s o l u t e drag) must then be c o n s i d e r e d movement o f i o n s .  with respect  T h i s was done by o b s e r v i n g  to the s e l e c t i v e  the s t r e a m i n g p o t e n t i a l  developed by a 400 m i l l i o s m o l a r s u c r o s e g r a d i e n t , when the s a l t centration As  (10 mM/l) was the same on b o t h s i d e s o f the membrane. can be seen from T a b l e 4, f o u r o f the p r e p a r a t i o n s  gave a sequence o f L i  +  > Na  +  (2) gave a sequence o f L i  + a sequence o f L i  +  > K  +  +  > Na  +  > Na  > Cs  > Cs  +  +  > Cs  (1,3,5,6)  > R b , one o f the p r e p a r a t i o n s  +  +  +  > K  +  > Rb , and p r e p a r a t i o n  > Rb  > K.  The f i r s t sequence  listed  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 +  > Na > +  L i  +  .  I i n t e r p r e t t h i s as f o l l o w s :  water and i o n s s h o u l d for L i  +  >  Cs >  the d r a g e f f e c t between  be p r o p o r t i o n a l t o the h y d r a t e d s i z e , b e i n g  and l e a s t f o r Rb .  4 gave  +  above f o r s t r e a m i n g p o t e n t i a l s vias e x a c t l y o p p o s i t e  K  con-  maximal  I f the d r a g e f f e c t i s l a r g e enough, t h i s  will  become more i m p o r t a n t than f i x e d charge i n d e t e r m i n i n g s e l e c t i v i t y f o r ions.  From the o t h e r  sequences n o t e d above, we can imply t h a t the o r d e r  o f s e l e c t i v i t y would c o r r e s p o n d t o Eisenman's t h i r d and f o u r t h s e r i e s . For preparation The  two, R b  +  or K  +  w i l l have the s m a l l e s t h y d r a t e d  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  strength density  o f the i n t i m a .  size.  o f charge  Such v a r i a b i l i t y might be a s s o c i a t e d w i t h the  o f the f i x e d charges i . e . as the charge d e n s i t y  s e l e c t i v i t y sequence s h i f t s  i n c r e a s e s , the  from t h a t o f i n c r e a s i n g h y d r a t e d s i z e t o  Table 4  S e l e c t i v i t y o f t h e i n t i m a f o r monovalent cations  as shown by s t r e a m i n g p o t e n t i a l s .  A 400 m i l l i o s m o l a r  s u c r o s e s o l u t i o n was on  the l u m e n a l s i d e .  10 m i l l i m o l a r s a l t  centrations  were used.  con-  Sign of p o t e n t i a l  i s w i t h r e f e r e n c e to the lumenal s i d e .  Salts used for Streaming Potentials  Streaming Potentials in millivolts Individual preparations 1  2  3  4  5  6  Li  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  Mean  ±  S.D.  38 t h a t o f 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 t o R b than to L i , +  +  has been shown t o be g r e a t e r  so t h a t d i f f u s i o n down any c o 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 f o r Rb  +  ( s e e s e c t i o n H o f t h i s C h a p t e r ) w o u l d be g r e a t e s t  + 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 n o t a c c o u n t f o r t h e  r e l a t i v e o r d e r o f t h e c a t i o n s as d e t e r m i n e d from s t r e a m i n g p o t e n t i a l s .  D.  E f f e c t o f pH on Membrane p o t e n t i a l s . I f t h e f i x e d charge was due t o a w e a k l y d i s s o c i a t e d a c i d  group,  s u c h as an amino a c i d (pK 3.5-4.7) as i n d i c a t e d i n C h a p t e r 1, t h e conc e n t r a t i o n o f f i x e d charges and hence t h e s e l e c t i v i t y o f t h e i n t i m a f o r i o n s s h o u l d be pH dependent.  I f t h e same f i x e d c h a r g e r s  responsible  f o r b o t h s t r e a m i n g and d i f f u s i o n p o t e n t i a l s , we w o u l d e x p e c t t h e same q u a n t i t a t i v e dependence o f these two t y p e s o f p o t e n t i a l s on pH, thus i m p l y i n g " t h a t b o t h i o n and w a t e r movement o c c u r through t h e same r o u t e . T h i s e x p e r i m e n t was d e s i g n e d t o o b s e r v e whether a change o f pH from a maximum o f 5.5 t o a minimum v a l u e o f 2.2 would a f f e c t s t r e a m i n g 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 t h e same way.  As shown i n F i g . 9, t h e p o t e n t i a l s w h i c h were u s u a l l y caused by KC1 concentration 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 ( s t r e a m i n g p o t e n t i a l s ) a t a pH o f 5.5, d i s a p p e a r a t a pH o f 2.2, w i t h a pK i n b o t h cases o f 3.7.  These r e s u l t s s u p p o r t t h e p r e v i o u s e v i d e n c e  ( L e w i s , 1970) t h a t t h e p o r e s were l i n e d w i t h n e g a t i v e f i x e d charges due t o w e a k l y a c i d i c groups, w i t h t h e pK as e s t i m a t e d by t i t r a t i o n m a t e l y 4.8; C h a p t e r 1 ) .  (approxi-  Both t y p e s o f p o t e n t i a l d i f f e r e n c e s seem t o be  a s s o c i a t e d w i t h the same f i x e d c h a r g e s .  T h i s suggested t h a t t h e main  Fig. 9  Open c i r c l e s r e p r e s e n t on s t r e a m i n g intima.  t h e e f f e c t s o f pH change  p o t e n t i a l s developed across the  The o s m o t i c p r e s s u r e  d i f f e r e n c e was  k e p t c o n s t a n t w i t h 400 m i l l i o s m o l a r s u c r o s e on the haemocoel s i d e and no s u c r o s e side.  on t h e lumen  B o t h s i d e s were b a t h e d i n 10 mM/l K C I .  S i g n i s w i t h r e f e r e n c e t o t h e haemocoel. P o i n t s r e p r e s e n t mean v a l u e 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  t h e e f f e c t s o f pH change  on d i f f u s i o n p o t e n t i a l s d e v e l o p e d a c r o s s t h e intima.  A 2 f o l d KCI c o n c e n t r a t i o n  gradient  (10 mM/l on t h e haemocoel s i d e and 5 mM/l on the l u m i n a l s i d e ) was used t o i n i t i a t e sion potentials. haemocoel.  diffu-  Sign i s w i t h reference to the  P o i n t s r e p r e s e n t mean v a l u e f o r  6 preparations.  See A p p e n d i x B T a b l e s  11 f o r i n d i v i d u a l v a l u e s .  10 and  Potential difference (mv)  oo _i  _i  to 1  3i  a  CO  cr  O  •  5" o o  O CJ1-J  •O •O  Oi  OS  Fig.  The  10  a f f e c t s of h i g h sucrose concentrations  diffusion potentials.  on  Diffusion potentials  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 o f KC1  (p.01M-0.005M).  c e n t r a t i o n o f KC1  on the lumen s i d e .  was  H i g h con-  p l a c e d i n e q u a l c o n c e n t r a t i o n s on  s i d e s o f the membrane.  Sucrose both  The s i g n i s w i t h  r e f e r e n c e t o the haemocoel s i d e .  Vertical  bars represent standard d e v i a t i o n s f o r 6 preparations.  See A p p e n d i x B T a b l e 12 f o r i n -  dividual values.  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 w a t e r  flow  and hence s t r e a m i n g p o t e n t i a l s o c c u r r e d .  E.  E f f e c t s o f High Osmotic P r e s s u r e on D i f f u s i o n  Potentials.  A h i g h and v a r i a b l e o s m o t i c p r e s s u r e 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  i o n c o n c e n t r a t i o n , b u t a h i g h osmotic  animal n o r m a l l y p o s s e s s e s p r e s s u r e due t o o r g a n i c  a low  molecules.  I t has a l r e a d y been shown t h a t h i g h i o n c o n c e n t r a t i o n decreases t h e e f f e c t i v e n e s s o f the f i x e d charge;  i . e . high i o n concentration  c a t i o n ; a n i o n d i s c r i m i n a t i o n by the f i x e d charge be o f i n t e r e s t  (Chapter 1 ) .  reduces I t would  then t o know i f h i g h o s m o t i c p r e s s u r e a l o n e w i l l  effect  the f i x e d charge p r o p e r t i e s as opposed t o h i g h i o n c o n c e n t r a t i o n s . A t w o - 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 was c r e a t e d (10 mM -5 mM ) across the i n t i m a .  Sucrose was added t o t h e s i d e w i t h low KC1 t o main-  t a i n equal osmotic p r e s s u r e s .  F u r t h e r s u c r o s e was added i n e q u i v a l e n t  amounts t o b o t h s o l u t i o n s t o c r e a t e t o t a l c o n c e n t r a t i o n s o f 100, 200, 400,  600, 800 o r 1200 m i l l i o s m o l a r on both  can be observed  s i d e s o f the membrane.  As  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 w i t h an i n c r e a s e i n osmotic p r e s s u r e , as p r e d i c t e d f o r i o n exchange membranes ( H e l f f e r i c h , 1962). as such  does n o t a f f e c t  the a v a i l a b i l i t y  Thus t h e o s m o t i c  of f i x e d negative  charges as  i n d i c a t e d by c a t i o n s e l e c t i v i t y .  F.  E f f e c t s o f Osmotic P r e s s u r e D i f f e r e n c e (A IT) The  osmotic  pressure  p r e s s u r e o f the r e c t a l c o n t e n t s , as p r e v i o u s l y  Fig.  11  S t r e a m i n g p o t e n t i a l s were d e v e l o p e d u s i n g  suc-  r o s e 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 b o t h s i d e s .  I n b o t h c u r v e s , the  concentra-  t i o n d i f f e r e n c e s were k e p t c o n s t a n t , b u t  the  a b s o l u t e c o n c e n t r a t i o n was  upper  curve  (solid circles)  molar d i f f e r e n c e .  The  changed.  represents  The  a 200  l o w e r curve  millios-  (solid  s q u a r e s ) 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 r e f e r e n c e t o the haemocoel s i d e . V e r t i c a l bars represent standard deviations f o r 6 preparations.  Values f o r i n d i v i d u a l pre-  p a r a t i o n s are g i v e n i n the A p p e n d i x B T a b l e  13.  14 -,  12  0  J  -I  ,  ,  .  0  200  400  600  Average  sucrose  concentration  in  800  milliosmolar  40  mentioned, v a r y g r e a t l y .  An i n c r e a s e i n the o s m o t i c p r e s s u r e , w h i c h  might d e h y d r a t e the membrane, has been shown t o r e s t r i c t the w a t e r 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 L e w i s (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 o f w a t e r f l o w  c i a t e d w i t h the g a l l b l a d d e r and the i n t i m a r e s p e c t i v e l y . Was  asso-  t h i s non-  l i n e a r i n c r e a s e i n w a t e r f l o w as r e f l e c t e d by s t r e a m i n g p o t e n t i a l s , a f u n c t i o n o f the r a t e o f w a t e r f l o w (i.e.was  the s t r e a m i n g p o t e n t i a l  i n d e p e n d e n t o f t o t a l o s m o t i c p r e s s u r e , b u t dependent on the o s m o t i c p r e s s u r e d i f f e r e n c e ) orvas  i t a f u n c t i o n o f the average o s m o l a r i t y  the b a t h i n g s o l u t i o n ( i . e . due One (Brodsky  of  t o an o s m o t i c compartment e f f e c t ) ?  o f the e x p l a n a t i o n s o f f e r e d f o r the n o n - l i n e a r w a t e r f l o w  and  S c h i l b , 1965)  v e r s i b l y deform differencewas  was  t h a t the r a t e o f w a t e r f l o w c o u l d r e -  the membrane s t r u c t u r e .  I f the o s m o t i c  pressure  k e p t c o n s t a n t , 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 p r e d i c t e d t h a t t h e r e s h o u l d be no change i n the streaming  p o t e n t i a l value.  osmotic pressure  T h i s h y p o t h e s i s was  d i f f e r e n c e constant  t e s t e d by k e e p i n g  a t 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 o f 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 on b o t h s i d e s ) f r o m 50 t o 350 m i l l i o s m o l a r w i t h s u c r o s e . was  a l s o repeated keeping  molar.  the  the o s m o t i c g r a d i e n t c o n s t a n t  MCI  This experiment a t 200  millios-  As can be o b s e r v e d from F i g . 11, the s t r e a m i n g p o t e n t i a l ' s n o t  c o n s t a n t , w h i c h Was c o n t r a r y t o the p r e d i c t i o n made by the membrane deformation theory.  R a t h e r the p o t e n t i a l d i f f e r e n c e decreased  the average o s m o l a r i t y i n c r e a s e d ;  r a p i d l y as  Fig.  12  R e s i s t a n c e o f the r e c t a l i n t i m a t o w a t e r f l o w as r e f l e c t e d i n the r a t i o o f o s m o t i c  pressure  d i f f e r e n c e and s t r e a m i n g 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 r e p r e s e n t r e p r e s e n t s mean v a l u e f o r 6 observations. curve o f F i g . 20.  Values  c a l c u l a t e d f r o m upper  Osmotic pressure difference/ streaming  potential  (mv)  Table 5  P e r c e n t a g e o f w a t e r by w e i g h t i n the when b a t h e d i n a  intima  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 solution  Mean ± S.D.  Distilled water  Sucrose solution  77  72  73  71  69  54  72  63  66  57  72  68  71.5 i 3.7  64.2 t 7.3  41  I f t h e r e s i s t a n c e t o w a t e r f l o w a c r o s s t h e i n t i m a ( d e f i n e d as the  r a t i o o f t h e o s m o t i c p r e s s u r e d i f f e r e n c e and the s t r e a m i n g p o t e n t i a l )  w a s r e l a t e d t o t h e 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 o b s e r v e d (Fig.  1 2 ) ; i . e . an i n c r e a s e i n t h e 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 t h e membrane r e s i s t a n c e .  The most a c c e p t a b l e  i n t e r p r e t a t i o n o f t h i s b e h a v i o r a t p r e s e n t was t h a t t h e membrane behaves as an o s m o t i c compartment.  That i s , waterwas w i t h d r a w n from t h e mem-  b r a n e a t h i g h o s m o l a r i t i e s , r e d u c i n g t h e e f f e c t i v e p o r e s i z e and consequently i n c r e a s i n g the r e s i s t a n c e t o water flow. for  membrane d e h y d r a t i o n was c a r r i e d o u t .  p l a c e d i n d i s t i l l e d w a t e r f o r one h o u r .  A qualitative  test  S i x membrane p r e p a r a t i o n s were A f t e r that time i n t e r v a l they  were removed, e x c e s s w a t e r removed and t h e i n t i m a were w e i g h e d .  The  same 6 membranes were t h e n p l a c e d i n 400 m i l l i o s m o l a r s u c r o s e f o r 1 h o u r , e x t e r n a l w a t e r removed and t h e p r e p a r a t i o n s r e - w e i g h e d .  The membranes  were then d r i e d i n an oven u n t i l a c o n s t a n t w e i g h t h a d been r e a c h e d . The p e r c e n t a g e w a t e r by w e i g h t f o r b o t h e x p e r i m e n t a l c o n d i t i o n s were calculated. The r e s u l t s  ( T a b l e 5) i n d i c a t e t h a t t h e membrane p l a c e d i n t h e  400 m i l l i o s m o l a r s o l u t i o n had a l o w e r p e r c e n t o f w a t e r by w e i g h t t h a n did  membranes i n d i s t i l l e d w a t e r .  The d i f f e r e n c e i n p e r c e n t v a r i e d  from 2.2 t o 15.2; however i n a l l c a s e s , membranes i n d i s t i l l e d  water  c o n t a i n e d more w a t e r t h a n membranes i n s u c r o s e s o l u t i o n (p<0.025). A s i m i l a r b e h a v i o r has been r e p o r t e d f o r i o n exchange membranes, where h i g h e x t e r n a l o s m o t i c p r e s s u r e s s h r i n k t h e membranes, r e s u l t i n g  ina  d e n s e r m a t r i x s y s t e m ( r e d u c i n g t h e pore s i z e ) and c o n s e q u e n t l y r e d u c i n g the  diffusion coefficients  ( H e l f f e r i c h , 1962).  42  G.  Ion Concentration The  varies  E f f e c t on S t r e a m i n g and D i f f u s i o n  Potentials.  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  greatly.  A previous i n v e s t i g a t i o n  ( R e s u l t s 5) i n d i c a t e d  o s m o t i c p r e s s u r e p e r se d i d n o t 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 . however, was was  n o t the case f o r s t r e a m i n g  a f u n c t i o n o f the average o s m o t i c p r e s s u r e  important  physiological  i n understanding conditions.  same f i x e d charge was  potential  (last section).  The  difference  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  Such a s t u d y would t e s t f u r t h e r whether the  r e s p o n s i b l e f o r streaming  by d e t e r m i n i n g w h e t h e r b o t h types o f 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 , respond i n a s i m i l a r  manner t o 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 o f the b a t h i n g  solution.  T e o r e l l (1953) s t a t e s t h a t as the i o n c o n c e n t r a t i o n (keeping a constant  t h a t the  This,  p o t e n t i a l s , h e r e 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 p e r se on s t r e a m i n g p o t e n t i a l was  animal  increased  g r a d i e n t ) the d i f f u s i o n p o t e n t i a l d e c r e a s e d , 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 . "  T h i s e f f e c t i s caused by  a  masking o f 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 c o - i o n cond u c t a n c e ; t h a t 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 t o anions A d r a s t i c d e c r e a s e i n the P . /P concentration (at constant (Chapter  w i t h an i n c r e a s e i n the average  c o n c e n t r a t i o n r a t i o ) has  KCI  a l r e a d y been r e p o r t e d  1, F i g . 6 ) .  When c o n s i d e r i n g the e f f e c t o f Ion c o n c e n t r a t i o n on potentials  two v a r i a b l e s  must be c o n s i d e r e d , one  e f f e c t i n masking the charge ( p r e v i o u s l y t i a l s ) and the second was osmotic pressure  mentioned f o r d i f f u s i o n p o t e n -  an o s m o t i c e f f e c t i m p o r t e d  e f f e c t on s t r e a m i n g  f o r any  potentials).  given osmotic gradient.  streaming  the i o n c o n c e n t r a t i o n  by the i o n s ( F i g . 11, As can be o b s e r v e d 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 s t r e a m i n g creased  decreased.  potential  At a concentration of 1  mM/l  de-  Fig.  13  S t r e a m i n g p o t e n t i a l s measured a c r o s s t h e i n t i m a due t o o s m o t i c p r e s s u r e d i f f e r e n c e c r e a t e d by t h e use o f s u c r o s e .  The s u c r o s e c o n c e n t r a t i o n  was v a r i e d from 0 t o 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 k e e p i n g s u c r o s e a t 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 o f KCI was t h e same on b o t h s i d e s .  The  s i g n i s w i t h r e f e r e n c e t o t h e 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 preparations.  See A p p e n d i x B T a b l e 14 f o r  individual preparations.  40i  Concentration of sucrose  in milliosmolars  Fig.  14  S t r e a m i n g p o t e n t i a l s were d e v e l o p e d u s i n g 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 KC1  on b o t h s i d e s .  g r a d i e n t was  The  sucrose  suc-  mM/l  concentration  k e p t c o n s t a n t , b u t the a b s o l u t e  c e n t r a t i o n was  changed.  t i o n g r a d i e n t was  The  sucrose  200 m i l l i o s m o l a r .  con-  concentraVertical  bars represent standard deviations f o r 6 preparations .  Values f o r i n d i v i d u a l  are g i v e n i n the A p p e n d i x B T a b l e  preparations 15.  30 -,  25 -  1000  Average  sucrose concentration in milliosmolar  1200  43  KC1, when the o s m o t i c 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 p o t e n -  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 t o the o s m o t i c compartment e f f e c t .  A t 10 mM/l  were a p p r o x i m a t e l y 50% o f t h o s e f o r 1 mM/l  KC1.  KC1, t h e p o t e n t i a l s  Was  this potential  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 , o s m o t i c e f f e c t ( i . e . a r e d u c e d w a t e r p e r m e a b i l i t y ) , o r both? caused s o l e l y by an o s m o t i c e f f e c t , t h e n 1 mM/l  I f the d e c r e a s e were KC1 s o l u t i o n  up t o t h e same o s m o t i c p r e s s u r e as t h a t o f the 10 mM/l  KC1  solution  ( u s i n g s u c r o s e ) s h o u l d show the same d e c r e a s e i n p o t e n t i a l . the c a s e .  I t can be shown from F i g . 14 t h a t a 1 mM/l  brought  T h i s was n o t  solution with  o s m o l a r i t y r a i s e d ( u s i n g s u c r o s e ) r e s u l t e d i n s t r e a m i n g p o t e n t i a l i n the o r d e r o f 20 mv, whereas 10 mM/l o f 11 mv.  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  This indicated that a reduction i n p o t e n t i a l at a concentra-  t i o n o f 10 m M / l KC1 r e l a t i v e t o 1 mM/l ion concentration effect.  caused s o l e l y by  the  A t low i o n c o n c e n t r a t i o n s , t h e n t h e d e c r e a s e  i n the s t r e a m i n g p o t e n t i a l was a s i m l a r manner, i t was  KC1 was  caused by a masking o f the c h a r g e s .  e s t a b l i s h e d t h a t a t 100 mM/l  KC1,  In  the o s m o t i c  e f f e c t o f the KC1 reduced the s t r e a m i n g 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%. c e n t r a t i o n s (1000 mM/l  KC1)  A t h i g h i o n con-  the r e d u c t i o n o f the s t r e a m i n g p o t e n t i a l  c o u l d be a c c o u n t e d f o r c o m p l e t e l y by an o s m o t i c e f f e c t . To summarize, a t low i o n c o n c e n t r a t i o n s the r e d u c t i o n i n s t r e a m ing potentials  (as i o n c o n c e n t r a t i o n s are g r a d u a l l y r a i s e d ) was  c o m p l e t e l y t o an i o n c o n c e n t r a t i o n e f f e e t . t i o n s (100 mM/l  due  At intermediate concentra-  K C l ) the r e d u c t i o n o f s t r e a m i n g p o t e n t i a l s seems t o be  Fig.  15  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 p e r se s t r e a m i n g and d i f f u s i o n p o t e n t i a l s .  on  The  average c o n c e n t r a t i o n o f KCI b a t h i n g s o l u t i o n for diffusion potentials  i s v a r i e d b u t a con-  stant concentration gradient 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 s q u a r e s , are p r e s e n t e d as a f r a c t i o n o f the p o t e n t i a l d i f f e r e n c e age i o n c o n c e n t r a t i o n .  a t the l o w e s t a v e r -  A constant osmotic p r e s -  s u r e o f 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 using sucrose.  Values f o r  s t r e a m i n g p o t e n t i a l s , r e p r e s e n t e d by  solid  c i r c l e s , a r e p r e s e n t e d as a f r a c t i o n o f the streaming p o t e n t i a l at a bathing concentration of 1 mM/l  KCI and where t h e o s m o t i c e f f e c t o f  the i o n c o n c e n t r a t i o n i s m a i n t a i n e d 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 preparations.  Values c a l c u l a t e d  for d i f f u s i o n potentials f o r streaming  potentials.  from F i g . 6  and F i g . 13 and  14  Average concentration of KC1  bathing  solution in  mM/l  44  influenced  by b o t h o s m o t i c and i o n c o n c e n t r a t i o n s ,  concentration the reduction  while a t high i o n  i n s t r e a m i n g p o t e n t i a l s seemed t o be caused  almost c o m p l e t e l y by an o s m o t i c e f f e c t . I f b o t h s t r e a m i n g 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 t h e same f i x e d c h a r g e , as was i n d i c a t e d by t h e e f f e c t o f pH on t h e s e 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  w o u l d e x p e c t e x t e r n a l i o n c o n c e n t r a t i o n t o a f f e c t s t r e a m i n g 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. t i a l differences  As can be seen i n F i g . 15, b o t h p o t e n -  d e c r e a s e d as t h e 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 .  When t h e  i o n c o n c e n t r a t i o n o f t h e b a t h i n g s o l u t i o n f o r s t r e a m i n g p o t e n t i a l s was 10 mM/l K C I , t h e p o t e n t i a l d i f f e r e n c e d e c r e a s e d 50%, f o r a s i m i l a r d e 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 , t h e average i o n c o n c e n t r a t i o n o f t h e b a t h i n g s o l u t i o n was 55 mM/l KCI ( F i g . 1 5 ) ; 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 t o t h e e x t e r n a l  i o n concentration  than were s t r e a m i n g p o t e n t i a l changes.  H.  Possible  E f f e c t s o f U n s t i r r e d L a y e r s on membrane P o t e n t i a l s .  I m m e d i a t e l y a d j a c e n t t o a l l membrane s u r f a c e s a r e u n s t i r r e d boundary l a y e r s  ( D a i n t y and House, 1966).  N e r s t (1904) was one o f t h e  f i r s t t o develop t h e concept o f 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  layer  r e f e r s t o a t h i n l a y e r o f s o l u t i o n i n w h i c h t h e o n l y form o f m i x i n g i s s l o w l a m i n a r f l o w p a r a l l e l t o t h e liquid-membrane i n t e r f a c e , so t h a t solute  t r a n s f e r i s i n e f f e c t o n l y by d i f f u s i o n .  Wedner and Diamond  (1969) found t h a t a l a r g e component o f s t r e a m i n g p o t e n t i a l s i n t h e g a l l  45  b l a d d e r was a boundary d i f f u s i o n p o t e n t i a l , owing t o w a t e r f l o w w h i c h produces l o c a l s a l t c o n c e n t r a t i o n g r a d i e n t s i n opposing u n s t i r r e d l a y e r s . S i n c e the f l u i d f l o w i n t h e rectum, a t 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 contain u n s t i r r e d l a y e r s of considerable magnitude.  I n p r e l i m i n a r y s t u d i e s ( L e w i s , 1970) s t r e a m i n g p o t e n t i a l s  were o b s e r v e d , caused by o s m o t i c g r a d i e n t s a c r o s s t h e r e c t a l i n t i m a . The o p p o r t u n i t y was t a k e n i n t h i s study l a y e r s on b o t h s t r e a m i n g  t o observe the e f f e c t of u n s t i r r e d  and d i f f u s i o n p o t e n t i a l s .  In a l l i n i t i a l experiments,  s o l u t i o n c o n c e n t r a t i o n s were  by p e r f u s i o n from b u r e t t e s w i t h a f l o w r a t e o f 4 m l p e r m i n u t e . s h o u l d be n o t e d t h a t t h i s s t i r r i n g r a t e p r o b a b l y found i n v i v o .  maintained It  g r e a t l y exceeds  those  The ends o f t h e b u r e t were 3 cm f r o m t h e i n t i m a .  Thus  t h e r e e x i s t e d t h e p r o b a b i l i t y t h a t t h i s f l o w r a t e d i d n o t remove t h e u n s t i r r e d l a y e r s , so t h a t c e r t a i n e f f e c t s n o t e d i n p r e v i o u s (eg. time c o u r s e  o f development o f s t r e a m i n g  experiments  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 t o these l a y e r s r a t h e r t h a n t h e 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 o f u s i n g b u r e t t e t u b e s , LKB p e r i s t a l t i c pumps were used t o d i r e c t t h e s o l u t i o n f l o w a t t h e s u r f a c e o f t h e membrane.  The  r a t e s o f p e r f u s i o n o f s o l u t i o n were 0.45, 0.8, 1.5 o r 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 r e c o r d e d u s i n g a " R a t i o m e t e r " coupled  t o a paper chart  pH meter  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 c o n c e n t r a t i o n gradients  (10 - 5 mM/l K C I ) . I n a n a l y z i n g the r e s u l t s f o r  Fig.  16  The e f f e c t o f p e r f u s i o n r a t e ( d i r e c t i n g at  fluid  t h e membrane s u r f a c e ) on a KC1 d i f f u s i o n  potential. expressed  V a l u e s f o r each p r e p a r a t i o n a r e as a % o f v a l u e observed  p a r a t i o n a t a maximum s t i r r i n g  f o r the p r e -  rates.  area represents d i f f u s i o n p o t e n t i a l s  Stippled 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 a p p a r a t u s . V e r t i c a l bars represent standard e r r o r f o r 6 observations.  .3  a cu  -*-> O  0.6  a  O  •r-t CO  <t-l  0.4 H  cu cu  0.2  H  .0 0  T  2  Perfusion rate in ml/min.  46  d i f f e r e n t perfusion 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 e x p r e s s e d  as a f r a c t i o n o f the v a l u e o b s e r v e d f o r  the same p r e p a r a t i o n a t t h e maximum s t i r r i n g r a t e .  The r e s u l t s a r e  shown i n F i g . 16, where the. s t i p p l e d a r e a r e p r e s e n t 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 o b s e r v e d 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 a p p a r a t u s .  The  s o l i d l i n e r e p r e s e n t s v a l u e s 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 t h e 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 t h e d i f f u s i o n p o t e n -  t i a l at increased perfusion rates.  I f the r a t e o f 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 t h e r a t e o f d i f f u s i o n i n t h e 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 o c c u r on t h e s i d e o f 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 e x c e s s o f i o n s w i l l d e v e l o p on t h e s i d e o f low i o n concentration.  T h i s change i n i o n c o n c e n t r a t i o n w i l l l o w e r the c o n c e n t r a -  t i o n g r a d i e n t a c r o s s t h e membrane and c o n s e q u e n t l y potential.  the d i f f u s i o n  T h i s may be what happened a c r o s s t h e 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 t h e e f f e c t was q u i t e s m a l l .  However i n t h e i n t a c t a n i m a l where  the s t i r r i n g r a t e s may be q u i t e l o w , i t i s n o t c l e a r how i m p o r t a n t  this  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 S t r e a m i n g p o t e n t i a l s . Water f l o w caused by e i t h e r an o s m o t i c o r 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 . for  I f t h e r e f l e c t i o n c o e f f i c i e n t o f t h e membrane  t h e i o n s was z e r o , then t h e i o n s wjuld f l o w s t r a i g h t t h r o u g h t h e  membrane t o t a l l y u n h i n d e r e d .  I f , however, t h e r e f l e c t i o n  coefficient  (o) was l e s s than one b u t g r e a t e r than z e r o , then some o f t h e i o n s would p a s s t h r o u g h b u t a l s o some i o n s would be r e f l e c t e d by the membrane and a h i g h i o n c o n c e n t r a t i o n wjuld s t a r t t o f o r m i n u n s t i r r e d l a y e r s on t h e  Fig.  17  Chart recorder r e c o r d f o r a t y p i c a l p r e p a r a t i o n showing t h e time c o u r s e f o r development o f a p o t e n t i a l d i f f e r e n c e caused by o s m o t i c w a t e r flow.  A 200 m i l l i o s m o l a r o s m o t i c p r e s s u r e  d i f f e r e n c e was  caused by s u c r o s e , 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)  Fig.  18  C h a r t r e c o r d e r r e c o r d s showing the time f o r development o f s t r e a m i n g  course  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 differing rates.  The  time r e f e r s to t h a t  f o l l o w i n g the s t a r t o f p e r f u s i o n a t  the  s t a t e d r a t e w i t h the s y s t e m , i n a l l c a s e s , 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 . 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 , o f 200 m i l l i o s m o l a r was by s u c r o s e w i t h an i o n b a t h i n g o f 10 mM/l  KCI.  The  caused  concentration  Perfusion Rate  (ml/min)  Time  (minutes)  47  s i d e from w h i c h w a t e r i s f l o w i n g .  S i m i l a r l y , i o n s w o u l d be swept away  from t h e u n s t i r r e d l a y e r i n t h e s i d e t o w h i c h w a t e r was f l o w i n g , so t h a t in  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 w m l d be l e s s t h a n i n the b u l k o f  solution.  Ions d i f f u s e down t h e s e l o c a l c o n c e n t r a t i o n g r a d i e n t s between  the  u n s t i r r e d l a y e r s , and c o u l d 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 t h e b u l k o f t h e s o l u t i o n w e r e the same on b o t h s i d e s .  S i n c e t h e w a t e r f l o w and i o n f l o w were i n t h e same d i r e c t i o n , t h e n d i f f u s i o n p o t e n t i a l and s t r e a m i n g 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 , ( L e w i s , 1970) t h e time c o u r s e f o r development  o f s t r e a m i n g p o t e n t i a l s was measured.  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 s t a r t e d a t z e r o and o n l y b u i l t up t o a maximum s i z e of  15 mv a f t e r 15 m i n u t e s .  T h i s c o u l d p o s s i b l y mean t h a t t h e o b s e r v e d  s t r e a m i n g p o t e n t i a l s might be t h e r e s u l t o f a c o n c e n t r a t i o n g r a d i e n t f o r m i n g a c r o s s t h e membrane i n the u n s t i r r e d l a y e r s as a of the  an o s m o t i c f l o w o f w a t e r (Schmid and Schwarz, 1952).  consequence Alternatively,  membrane p r e p a r a t i o n might n o t have been c o m p l e t e l y d r i e d p r i o r t o  u s e , so t h a t a l a y e r o f d i s t i l l e d w a t e r was i n i t i a l l y p r e s e n t on e i t h e r s i d e o f t h e membrane.  Thus the d e l a y i n development  of streaming  p o t e n t i a l s c o u l d r e f l e c t the time r e q u i r e d f o r i o n s t o move t h r o u g h t h i s f i l m of water. to  A number o f d i f f e r e n t e x p e r i m e n t s were p e r f o r m e d  e l u c i d a t e whether a l l o r p a r t o f t h e s t r e a m i n g 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 s u g g e s t e d f o r t h e g a l l b l a d d e by Wedner and Diamond (1969).  The f i r s t e x p e r i m e n t was t o p r e p a r e t h e  membrane i n t h e normal f a s i o n , and t h e n dab d r y b o t h s i d e s o f t h e membrane w i t h k l e e n e x .  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 b o t h s i d e s o f t h e membrane, t h e p o t e n t i o m e t e r and paper c h a r t r e c o r d e  F i g . 19  The e f f e c t o f p e r f u s i o n r a t e on s t r e a m i n g p o t e n t i a l , w i t h 10 mM/l KC1 on b o t h s i d e s o f the membrane and an o s m o t i c g r a d i e n t due t o s u c r o s e o f 200 m i l l i o s m o l a r . expressed  The r e s u l t s a r e  as a f r a c t i o n o f t h e p o t e n t i a l  ference observed  dif-  f o r i n d i v i d u a l preparations  i n t h e absence o f m i x i n g .  The s t i p p l e d  represents the "instantaneously"  area  developed  s t r e a m i n g 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 t o a " d r y " membrane.  Vertical  lines  represent standard e r r o r s f o r 6 observations.  Perfusion  rate  (ml/min)  48  b e i n g t u r n e d on s i m u l t a n e o u s l y .  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 s u c r o s e - 1 0 mM/l K C I on t h e l u m e n a l s i d e and 10 mM/l K C I on the haemocoel s i d e .  As shown i n F i g . 17 an i n s t a n t a n e o u s  p o t e n t i a l o f 7 mv d e v e l o p e d a c r o s s s w i t c h on t h e r e c o r d e r s This instantaneous streaming  t h e membrane.  streaming  The time r e q u i r e d t o  and add t h e s o l u t i o n s took a p p r o x i m a t e l y  p o t e n t i a l was p o s t u l a t e d t o be w h o l l y  3 seconds.  caused by  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  w h i c h f o l l o w e d c o u l d 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 layers.  Such a p o t e n t i a l would be e x p e c t e d t o be o f t h e same s i g n as  the s t r e a m i n g p o t e n t i a l . The streaming  previous  experiment i n d i c a t e d that p a r t o f the steady s t a t e  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 t o un-  stirred layers.  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 o f 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 on t h e s i z e o f t h e " s t r e a m i n g  p o t e n t i a l " was s t u d i e d .  c o n d i t i o n s were t h e same as i n t h e p r e v i o u s f u s i o n r a t e s were v a r i e d .  The  rates)  experimental  experiment, except that per-  Both s i d e s o f t h e membrane were  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.  perfused  The p e r f u s i o n r a t e  used a t any one time was t h e same on b o t h s i d e s o f t h e membrane.  being The  time c o u r s e f o r t h e development o f a s t e a d y 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 t h e p e r f u s i o n r a t e i s shown i n F i g . 18.  Perfusion  causes t h e p o t e n t i a l t o drop r a p i d l y t o a s t e a d y base l e v e l (shown as a f u n c t i o n o f p e r f u s i o n r a t e i n F i g . 1 8 ) . As t h e p e r f u s i o n r a t e i n creased,  t h e l a g time t o r e a c h a s t e a d y s t a t e p o t e n t i a l d e c r e a s e d .  i s evident  It  from F i g . 19 t h a t as t h e r a t e o f p e r f u s i o n i n c r e a s e d , t h e  Fig.  20  Upper curve r e p r e s e n t s 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 o s m o t i c  pressure without s o l u t i o n perfusion. curve r e p r e s e n t s  Lower  a non-linear increase 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 o f 2.8 m i l l i l i t e r s p e r minute. l i n e s a r e S.D.  f o r 6 observations.  Verticle See  Appendix B T a b l e 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  >)  Fig.  21  R e s i s t a n c e o f the r e c t a l i n t i m a t o w a t e r f l o w as r e f l e c t e d i n the r a t i o o f  osmotic  p r e s s u r e d i f f e r e n c e and s t r e a m i n g  potential  difference.  Each p o i n t r e p r e s e n t s mean v a l u e  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 o f the i n tima. Fig.  V a l u e c a l c u l a t e d from l o w e r curve 20.  of  CQ  Average  osmolarity  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 the v a l u e f o r the " i n s t a n t a n e o u s l y "  perfusion  T h i s seened to i n d i c a t e t h a t a t t h e s e l a y e r s had  approached  developed p o t e n t i a l d i f f e r e n c e r e -  c o r d e d i n the p r e c e d i n g t y p e o f e x p e r i m e n t ( F i g . 1 7 ) . measured a t the two h i g h e s t  and  The  potentials  r a t e s f e . l l w i t h i n t h i s band.  p e r f u s i o n r a t e s the u n s t i r r e d  been l a r g e l y removed, so t h a t o n l y t r u e s t r e a m i n g p o t e n t i a l s  were o b s e r v e d .  I t was  c o n c l u d e d t h e n t h a t w a t e r f l o w t h r o u g h the mem-  b r a n e caused n o t o n l y a s t r e a m i n g p o t e n t i a l , but a l s o a d i f f u s i o n p o t e n tial.  The  d i f f u s i o n p o t e n t i a l i s b e i e v e d t o account f o r a p p r o x i m a t e l y  45% o f what was  p r e v i o u s l y d e s i g n a t e d as " s t r e a m i n g 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 w a t e r f l o w as opposed t o 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 S i n c e the o s m o t i c f l o w seemed t o produce two difference  ( i . e . a s t r e a m i n g p o t e n t i a l and  o f the o r i g i n a l e x p e r i m e n t s had was  types of p o t e n t i a l  a d i f f u s i o n p o t e n t i a l ) some  t o be r e p e a t e d .  Of immediate i n t e r e s t  whether e l i m i n a t i o n o f u n s t i r r e d l a y e r s would a l t e r the  c o n c e r n i n g the e v i d e n c e o f the n o n - l i n e a r r e s i s t a n c e to water flow increases osmolarity).  100,  200,  300  o r 400  p e r f u s i o n r a t e was layers  rate of water flow  (where the  i n a l i n e a r manner w i t h the  curve represents  KC1  average  s o l u t i o n on b o t h s i d e s o f the  m i l l i o s m o l a r s u c r o s e one  increased  (as s u g g e s t e d by  t o 2.8  one  intima, The  ml/minute t o a b o l i s h u n s t i r r e d  r e s u l t s i n F i g . 19).  I n F i g . 20,  streaming p o t e n t i a l s at a p e r f u s i o n  Comparing the two  was  side only.  the upper  "streaming p o t e n t i a l s " without perfusion while  lower curve represents ml/minute.  conclusions  W i t h t h i s i n mind the p r e v i o u s e x p e r i m e n t ( F i g . 13)  r e p e a t e d by p l a c i n g a 10 mM/l and  gradients.  the  r a t e of  2.8  c u r v e s , the r a t i o o f the p o t e n t i a l s f o r  50  p e r f u s i o n v e r s u s no p e r f u s i o n was c o n s t a n t a t a p p r o x i m a t e l y 0.5; r a t i o o f t h e two p o t e n t i a l s was independent  i . e . the  o f the o s m o t i c g r a d i e n t .  Thus i n the i n t a c t a n i m a l , the p o t e n t i a l d i f f e r e n c e produced  by  osmotic  w a t e r f l o w under s i m i l a r c o n d i t i o n s would v a r y by a maximum o f t w o - f o l d depending on the r a t e o f f l u i d m i x i n g . for of  Most p r o b a b l y the v a l u e s  u n p e r f u s e d membranes were c l o s e s t t o those i n v i v o . osmosis was i n f a c t more a c c e n t u a t e d w i t h p e r f u s i o n .  The  found  non-linearity  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 o f the membrane to w a t e r f l o w from s t r e a m i n g p o t e n t i a l s , i t can be shown ( F i g . 21) t h a t the r e s i s t a n c e t o w a t e r  f l o w remained a l i n e a r f u n c t i o n o f the average  o s m o l a r i t y o f t h e 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 o f S t r e a m i n g and  Diffusion  Potentials. The p r e v i o u s experiment  i n d i c a t e d t h a t b o t h s t r e a m i n g and  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 .  dif-  Itwas n o t  clear  however, whether t h e s e two types o f 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.  I n o r d e r t o make p r e d i c t i o n s c o n c e r n i n g p o t e n t i a l  d i f f e r e n c e s a c r o s s 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 e x p e r i m e n t s , i t was The  approach  of importance  t o have t h i s type o f i n f o r m a t i o n .  taken i n answering  t h i s q u e s t i o n was  to create  s t r e a m i n g and d i f f u s i o n p o t e n t i a l s s i m u l t a n e o u s l y a c r o s s the same membrane.  The o b s e r v e d p o t e n t i a l d i f f e r e n c e c o u l d then be compared  w i t h t h a t 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 d a t a from p r e v i o u s e x p e r i m e n t s  i n w h i c h each type o f 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 o f s t r e a m i n g 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  a c r o s s t h e same mem-  brane. A 2 f o l d concentration d i f f e r e n c e o f KC1 (10 mM/1-5 mM/l) was used t o i n i t i a t e t h e 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 . constant) .  H i g h c o n c e n t r a t i o n o f KC1 was p l a c e d  on t h e lumen s i d e .  S u c r o s e was used t o cause  streaming p o t e n t i a l s o f various s i z e s . r o s e , when p l a c e d on t h e haemocoel caused s t r e a m i n g  Suc-  side  p o t e n t i a l s t o be added t o  d i f f u s i o n p o t e n t i a l s ( r e p r e s e n t e d i n the graph by s o l i d c i r c l e s ) , and when p l a c e d on t h e lumen s i d e caused them t o be s u b t r a c t e d  (represented  i n t h e graph b y open c i r c l e s ) .  The s i g n i s  w i t h r e f e r e n c e t o t h e haemocoel  side.  Verti-  c a l bars represent standard deviations o f 6 readings.  See A p p e n d i x B, T a b l e 17 f o r  individual preparations.  Concentration  of  sucrose  in m i l l i o s m o l a r s  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 e x p e r i m e n t the membrane s e p a r a t e d with  concentrations  o f 10 and  s i d e of low i o n c o n c e n t r a t i o n  5 mM/l  KCI.  Sucrose was  to eliminate  any  solutions  added t o  the  osmotic gradients.  To  t e s t f o r the a d d i t i v e e f f e c t s o f s t r e a m i n g 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 , s u c r o s e was f i n a l concentrations for  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  of 100,  200,  300  o r 400  milliosmolar.  To  in  test  the s u b t r a c t i v e e f f e c t s o f s t r e a m i n g 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 , s u c r o s e was concentrations  added t o 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  o f 100,  200,  300  o r 400  in final  milliosmolar.  I n F i g . 22, i t can be s e e n t h a t s t r e a m i n g p o t e n t i a l s and 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 s e e n  s t r e a m i n g p o t e n t i a l s rare n o t e x a c t l y s u b t r a c t i v e p o t e n t i a l s , but Wis o n l y about  that  from the d i f f u s i o n  the d i s c r e p a n c y between o b s e r v e d and p r e d i c t e d 11%.  diffusion  values  52  DISCUSSION  P r e v i o u s 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 L e w i s , 1970) i n d i c a t e d t h a t the c h i t i n o u s membrane c o n t a i n s p o r e s o f 6-7 A* i n r a d i u s w i t h a p o s s i b i l i t y t h a t l a m i n a r f l o w o f w a t e r may through these pores.  occur  T h i s was s u p p o r t e d by t h e d e m o n s t r a t i o n o f s t r e a m -  i n g p o t e n t i a l s i n the present study.  Both s t r e a m i n g 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 t h a t the membrane can s e l e c t f o r c a t i o n s o v e r a n i o n s , were b o t h o f t h e s e p o t e n t i a l s caused by the same f i x e d c h a r g e , o r must a s e p a r a t e c h a n n e l he h y p o t h s i z e d f o r w a t e r f l o w 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 s t r e a m i n g and d i f f u s i o n p o t e n t i a l s 3)were q u a l i t a t i v e l y the same.  ( R e s u l t s 1 and  I o n s e l e c t i v i t y i n b o t h cases has been  shown t o d e c r e a s e 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 c o n c e n t r a t i o n , however f o r a s i m i l a r d e c r e a s e i n p o t e n t i a l d i f f e r e n c e , t h e i o n c o n c e n t r a t i o n was 5 times g r e a t e r i n t h e 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 t o s t r e a m i n g p o t e n t i a l s  (Fig.15).  High i o n  c o n 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 o v e r a n i o n was d e c r e a s e d ( T e o r e l l , 1953), and d i s a p p e a r s when a n i o n and c a t i o n were o f the same h y d r a t e d s i z e  (as was the case f o r K C 1 ) .  As t h e pH o f a s o l u t i o n d e c r e a s e s , so does t h e s e l e c t i v i t y , as was a l s o found by Lannoye, T a r r and D a i n t y (1970 ) i n Chara a u s t r a l i s . The p r o b a b l e mechanism i s t h a t low pH d e c r e a s e s the c o n c e n t r a t i o n o f n e g a t i v e charges on o r i n the membrane ( D a i n t y , Hore and Denby,  1960).  The pKa and p i v a l u e s o f t h e f i x e d charges i n the r e c t a l i n t i m a as measured by s t r e a m i n g p o t e n t i a l s gave v a l u e s o f 3.7 f o r the pKa and 2.2  53  for  the p i ; the same v a l u e s were found f o r d i f f u s i o n p o t e n t i a l s  o f 3.7  and  p i o f 2.2).  T h i s s u p p o r t s the h y p o t h e s i s  as measured by d i f f u s i o n and  that  (pKa  selectivity  s t r e a m i n g p o t e n t i a l s i n v o l v e the same f i x e d  c h a r g e s , p o s s i b l y i n d i c a t i n g t h a t f i x e d charges are l o c a t e d on w a l l s the p o s t u l a t e d  of  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 f i x e d charges were p o s s i b l y weak and w i d e l y s p a c e d .  The  the  greater  +2 ( 1 0 - f o l d ) e f f i c i e n c y o f Ca t o the e q u i v a l e n t  i n masking o f the f i x e d charge r e l a t i v e  concentration  the i n t e r a c t i o n of c o u n t e r - i o n s selective binding.  T h i s was  o f monovalent c a t i o n s s u g g e s t s t h a t w i t h the f i x e d charges may  experimentally  involve a  s u p p o r t e d when i t was  shown  +2 t h a t a t h i g h Ca reversed.  concentrations  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  Such a r e v e r s a l i m p l i e d t h a t the Ca  and Mg  i o n not  only  b l o c k the e x i s t i n g c h a r g e s , b u t a l s o must a s s o c i a t e t h e m s e l v e s w i t h the f i x e d charge and  thus f o r m a membrane w i t h a n e t e x c e s s of p o s i t i v e + +2 f i x e d c h a r g e . Whether the K conductance was d e c r e a s e d a t h i g h Ca c o n c e n t r a t i o n o r the C l conductance was i n c r e a s e d ( o r b o t h ) under t h e s e c o n d i t i o n s was n o t known; however, the K  +  to C l  permeability  was  i n c r e a s e d i n the  +2 r a t i o d e c r e a s e d t o u n i t y as the amount o f solutions. and  Ca  W r i g h t and Diamond (1968) u s i n g the g a l l b l a d d e r ,  Cassidy  T i d b a l l (1967) u s i n g l o b s t e r n e r v e and van Preeman (1968) u s i n g  phospholipid-cholesterol 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  and  t o monovalent i o n s . Curran +2 G i l l J r . (1961) showed t h a t when Ca was added e x t e r n a l l y t o f r o g  s k i n , i t caused a d e c r e a s e i n n e t 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 o f the outward f a c i n g membrane o f  transporting cells.  T a r r , Lannoye and D a i n t y  the  (1970) found t h a t 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 m e d i a t e d by Ca .  Van Preemen (1968) u s i n g  phospholipid-cholesterol +2  a r t i f i c i a l membranes found t h a t t h e a d d i t i o n o f Ca  reduced t h e 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 t h e change i n t h e p o t e n t i a l was reversible.  Van Preemen b e l i e v e s t h a t t h e 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 , w h i c h may be due t o e i t h e r a d e c r e a s e i n t h e number o f f i x e d c h a r g e d s i t e s o r t o an i n c r e a s e i n p o r e d i a m e t e r ( 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 p r e s e n c e o f Ca  , t h e membrane r e s i s t a n c e i n c r e a s e d ; i . e . t h e p o r e  d i a m e t e r had d e c r e a s e d r a t h e r than i n c r e a s e d i n s i z e .  W r i g h t and  Diamond (1968) came t o t h e same c o n c l u s i o n i n t h e i r s t u d y w i t h t h e g a l l b l a d d e r , where they found t h a t an i n c r e a s e i n t h e  Ca*c"oncentration  caused t h e Na^conductance t o d e c r e a s e and t h e C l ~ c o n d u c t a n c e t o i n c r e a s e . T h e i r c o n c l u s i o n , c o n c e r n i n g t h e h i g h Ca £oncentration and low pH +  e f f e c t on t h e g a l l b l a d d e r , was t h a t p r o b a b l y b o t h b l o c k t h e same fixed sites  (Wright,  B a r r y and Diamond, 1971), s i n c e t h e e f f e c t s o f  t h e s e 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 similar. The  e f f e c t o f d i v a l e n t c a t i o n s on t h e P^ / ^ r j i  r a t  i° f°  r  t  n  e  +2 r e c t a l intima indicates a competitive  a f f i n i t y sequence o f Ca  +2 > Mg  As i n t h e e x p e r i m e n t s o f Bungenburg De Jong's on c o l l o i d s and W r i g h t 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 o f t h e  r e c t a l i n t i m a was i n d e p e n d e n t o f 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  o f the r e c t a l i n t i m a , f o r b o t h 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 s e l e c t i v i t y for monovalent and divalent cations was K  +  + +2 +2 > Na : Ca > Mg .  This seemedto imply that the greater the  a f f i n i t y between i o n and s i t e , the greater was the r e l a t i v e permeability across the membrane. Streaming p o t e n t i a l s were observed to form across the r e c t a l intima.  The r e l a t i o n s h i p between the l a t t e r and the osmotic gradient  suggested the occurance of non-linear osmosis.  Diamond (1966) proposed  f i v e d i f f e r e n t explanations for n o n - l i n e a r i t y of osmotic water flow across the g a l l bladder: i.  membrane structure i s deformed by water flow.  ii.  u n s t i r r e d layers reduce the p r o p o r t i o n a l i t y constant between osmotic gradient and water flow.  iii.  force flow r e l a t i o n may be non-linear f o r an asymetrical system composed of several d i s s i m i l a r membrane layers i n s e r i e s ( i . e . heterogeneous membrane), even though the r e l a t i o n may be l i n e a r for each i n d i v i d u a l membrane.  iv.  concentration of impermeant molecules might exert a non-osmotic e f f e c t on the membrane by a l t e r i n g chemical or p h y s i c a l i n t e r actions within the membrane i t s e l f ( i . e . changes i n the membrane structure).  v.  the concentration of molecules might exert an osmotic e f f e c t upon the membrane, a l t e r i n g 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 o f t h e s e p o s s i b i l i t i e s had been shown t o be  s i n c e the s t r e a m i n g p o t e n t i a l was  invalid  not dependent on the o s m o t i c  pressure  g r a d i e n t b u t r a t h e r on the average o s m o l a r i t y o f the two b a t h i n g tions.  The  solu-  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  t h a t the s t r e a m i n g p o t e n t i a l s e x h i b i t e d g r e a t e r 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 m o s t l y removed t h a n when they were maximal ( i . e . no s t i r r i n g ) . non-linear  The  p o s s i b i l i t y t h a t the f o r c e - f l o w r e l a t i o n might be  f o r an a s y m m e t r i c a l s y s t e m , 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 d i r e c t i o n o f f l o w under any A l s o P h i l l i p s and  one  the membrane d i d n o t v a r y  set of conditions  (Lewis,  with  1970).  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 o s m o t i c p e r m e a b i l i t y the l o c u s t i n t i m a .  c o e f f i c i e n t with d i r e c t i o n of water flow  Diamond (1966) found t h a t v a r i o u s  across  impermeant  m o l e c u l e s a l l caused n o n - l i n e a r w a t e r f l o w s u g g e s t i n g  that chemical  i n t e r a c t i o n s w h i c h changed membrane s t r u c t u r e were n o t  involved.  E x p e r i m e n t s i n t h i s c h a p t e r a l s o i n d i c a t e d t h a t n o n - l i n e a r osmosis n o t dependent on the t y p e o f m o l e c u l e used.  This leaves only  o s m o t i c e f f e c t o f m o l e c u l e s on membrane h y d r a t i o n . w h i c h s u p p o r t e d t h i s a l t e r n a t i v e was  The  the  d i r e c t evidence  t h a t s t r e a m i n g p o t e n t i a l s (and  w a t e r f l o w ) were dependent on the average o s m o l a r i t y  equation: 5)  (0m-0s) / (Ro' + 1/2  K'(Om  thus  and n o t the o s m o t i c  g r a d i e n t , such t h a t the r a t e o f w a t e r f l o w c o u l d be e x p r e s s e d by  (Equation  was  + Os)  )  the  57  Furthermore,  i t was  shown d i r e c t l y by d e t e r m i n a t i o n o f w a t e r  t h a t i n c r e a s e i n o s m o l a r i t y o f the b a t h i n g s o l u t i o n reduced of  the w a t e r i n the i n t i m a .  f l u x r a t e of water 1.1  content the amount  P h i l l i p s and Beaumont (1971) found t h a t the  t h r o u g h the i n t i m a d e c r e a s e d a p p r o x i m a t e l y 50% w i t h  o s m o l a l s u c r o s e b a t h i n g the membrane as compared t o 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 t h e membrane passages a c t i n g as o s m o t i c compartments so t h a t dimensions  o f the pores were i n -  f l u e n c e d by the c o n c e n t r a t i o n o f the b a t h i n g medium. I t was of  found t h a t membrane d e h y d r a t i o n d i d n o t a f f e c t t h e v a l u e s  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 .  suggests  t h a t i n weakly  Helferrich  (1962)  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 o f the c o u n t e r - i o n may  be i n c r e a s e d by p a r t i a l  dehydra-  t i o n s i n c e d e h y d r a t i o n would i n c r e a s e the charge d e n s i t y , thus r e d u c i n g the d i s t a n c e t h a t an i o n has t o jump from one f i x e d charge t o a n o t h e r , assuming-the same h y d r a t e d s i z e f o r b o t h i o n s .  I f the h y d r a t e d s i z e o f  the a n i o n and c a t i o n were d i f f e r e n t , t h e n the o s m o t i c p r e s s u r e might a f f e c t the m o b i l i t i e s o f b o t h i o n s through the membrane and thus  the  potential difference. Phillips  (1964 a) p o s t u l a t e d , among o t h e r mechanisms, e l e c t r o -  osmosis as a p o s s i b l e f o r c e b e h i n d n e t w a t e r f l o w a c r o s s the wall.  rectal  F l u i d i n membrane p o r e s c a r r y a n e t e l e c t r i c charge o f the same  s i g n as t h a t o f the c o u n t e r i o n s .  Hence the water w i l l f l o w i n the  same d i r e c t i o n as the c o u n t e r i o n i . e . towards the s i d e o f o p p o s i t e charge.  E l e c t r o - o s m o s i s and s t r e a m i n g p o t e n t i a l s a r e r e l a t e d by  the  f o l l o w i n g e q u a t i o n ( d e r i v e d by i r r e v e r s i b l e thermodynamics; D a i n t y , 1966):  58  ( E q u a t i o n 6) In ing  H/P  = Jv/i  the p r e s e n t s t u d y the v a l u e o f H o b s e r v e d  ( f o r a c t u a l stream-  p o t e n t i a l s i n the absence o f u n s t i r r e d l a y e r s ) was  3.5  mv,  and  the  -12 v a l u e o f P was  100 m i l l i o s m o l a r . T h i s g i v e s a v a l u e o f 4.87x10  2 s t a t v o l t s / d y n e / c m f o r (H/P) (Smyth and W r i g h t , 1966). The r a t e o f n e t w a t e r a b s o r p t i o n a c r o s s the r e c t a l w a l l as a whole i n the absence o f o s m o t i c g r a d i e n t s i s 17 ml/hr/cm a n i m a l ( P h i l l i p s , 1964  a).  2  o r 4.72x10  —6  ml/sec/cm  2  i n the i n t a c t  I t i s o f i n t e r e s t t o determine w h e t h e r t h i s  n e t w a t e r movement a c r o s s t h e i n t i m a o b s e r v e d  i n v i v o could occur  e l e c t r o - o s m o s i s , o r whether a l t e r n a t i v e l y , i t i s n e c e s s a r y an o s m o t i c g r a d i e n t a c r o s s the i n t i m a .  by  to postulate  I f t h i s n e t w a t e r movement i s 5  2  c o m p l e t e l y due t o e l e c t r o - o s m o s i s , a c u r r e n t ( i ) o f 9.67x10 ( i = JvP/H) i s r e q u i r e d a c c o r d i n g t o the above e q u a t i o n .  statamps/cm  Using  a v e r a g e r e s i s t a n c e v a l u e f o r the membrane b a t h e d i n 10 mM/l  the  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 a c r o s s the i n t i m a n e c e s s a r y  c r e a t e a c u r r e n t o f t h i s magnitude i s t h e r e f o r e  47 mv.  D o i n g the  v e r s e c a l c u l a t i o n ( i . e . t h e r a t e o f w a t e r f l o w w h i c h can be  to con-  induced  by a p o t e n t i a l d i f f e r e n c e a c r o s s the i n t i m a o f 3.5 mv, w i t h a membrane 2 4 r e s i s t a n c e o f 145.3 ftcm ) g i v e s a v a l u e f o r c u r r e n t ( i ) o f 7.24x10 2 statamps/cm . U s i n g the e q u a t i o n w h i c h r e l a t e s electron-osmosis t o s t r e a m -12 i n g p o t e n t i a l s ( J v = iH/P) and p l a c i n g H/P e q u a l t o 4.87x10 stat2 —6 2 v o l t s / d y n e / c m , a v a l u e f o r w a t e r f l o w o f 0.352x10 ml/sec/cm , o r 2 1.27  ul/hr/cm Phillips  i s obtained. (1964  a) and V i e t i n g h o f f (1969) have observed  s t e p p o t e n t i a l d i f f e r e n c e a c r o s s the r e c t a l w a l l .  Present  a  two  experiments  59  i n d i c a t e t h a t t h e 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  interior  c o u l d be due i n p a r t t o a p o t e n t i a l d i f f e r e n c e a c r o s s t h e i n t i m a due t o s e l e c t i v e d i f f u s i o n of cations 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 t h e e p i t h e l i a l l a y e r .  However i f t h i s i s t r u e then w a t e r f l o w  f r o m t h e lumen i n t o the s u b i n t i m a l space can n o t be due t o e l e c t r o - o s m o s i s , s i n c e s u c h a f l o w s h o u l d be i n t h e o p p o s i t e d i r e c t i o n . the s u b - i n t i m a l space would have t o be n e g a t i v e  T h i s i s because  for electro-osmosis to  o c c u r toward t h e haemocoel s i d e , w h i l e t h e p r e s e n t s t u d y suggested t h a t n e t d i f f u s i o n o f KCI toward t h e e p i t h e l i a l l a y e r w o u l d make t h a t s i d e positive.  I n e s s e n c e t h e r e f o r e e l e c t r o - o s m o s i s may o c c u r f r o m t h e sub-  i n t i m a l space t o t h e lumen.  T h i s s u g g e s t s t h a t when t h e r e c t a l lumen  c o n t a i n s a l o w c o n c e n t r a t i o n o f i o n s , so t h a t i o n pumps i n t h e e p i t h e l i a l l a y e r maintain a concentration 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 o f i o n s w i l l o c c u r b u t w a t e r f l o w w i l l be r e d u c e d due t o t h e 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 o f t h e  i n t i m a c o u l d thus be o f a d a p t i v e advantage t o h y d r a t e d  animals  s e l e c t i v e r e a b s o r p t i o n o f i o n s b u t n o t w a t e r w o u l d be f a v o r e d . o t h e r hand i n d e h y d r a t e d animals w i t h e x c e s s body s a l t  since On t h e  (and h i g h i o n  c o n c e n t r a t i o n s i n t h e lumen; P h i l l i p s , 1964 b) t h e c a t i o n s e l e c t i v i t y o f the i n t i m a s h o u l d d e c r e a s e v e r y s u b s t a n t i a l l y , thus r e d u c i n g i o n t r a n s f e r and f a c i l i t a t i n g w a t e r r e a b s o r p t i o n (by r e m o v a l o f t h e o p p o s i n g electro-osmosis). Charged i o n i c groups p r e s e n t on t h e w a l l o f t h e aqueous c h a n n e l s i n t h e i n t i m a c r e a t e a z e t a p o t e n t i a l between t h e i o n i c s o l u t i o n i n t h e p o r e and t h e f i x e d groups.  C a l c u l a t i o n o f the z e t a p o t e n t i a l f o r t h e  60  i n t i m a b a t h e d 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 W r i g h t ;  ( E q u a t i o n 7)  .  4 im  U s i n g v a l u e s o f 0.01 book of C h e m i s t r y n  1966): H/DP  p o i s e s f o r n and 80 f o r D a t 20°C (Hand-  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 v a l u e s f o r  e q u a l t o 2.12x10  -3  mhos and H/P  t h i s e q u a t i o n y i e l d s a v a l u e o f 4.36  = 4.87x10 mv  -12  statvolts/dyne/cm  f o r the z e t a p o t e n t i a l .  i s a n e g a t i v e v a l u e because the p o r e i s a n i o n i c .  2  ,  This  Electro-osmosis  or  c o n v e r s e l y s t r e a m i n g p o t e n t i a l s a r e i m p o r t a n t when the z e t a p o t e n t i a l i s large.  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 o f the i n t i m a , a t a b a t h i n g  s o l u t i o n pH o f 5.5,  i t can be c o n c l u d e d  t h a t the i o n i c groups o f the p o r e  i m p a r t a s i g n i f i c a n t n e t charge t o the w a t e r and thus can cause s i g n i f i c a n t w a t e r movement by e l e c t r o - o s m o s i s .  D a v i e s , Hayden and R i d e a l  (1956) found t h a t the z e t a p o t e n t i a l i s not a f f e c t e d by o s m o t i c T h i s i s i n agreement w i t h p r e s e n t o b s e r v a t i o n s t h a t o s m o t i c does not" a l t e r the P  pressure.  pressure  /P^ r a t i o o f the r e c t a l i n t i m a .  The pH o f the s o l u t i o n i n the immediate v i c i n i t y o f the  fixed  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 d e s c r i b e d by the f o l l o w i n g equation  ( H a r t l y and Roe,  ( E q u a t i o n 8)  pHs  1940): = pHb  + (•£ /60)  T h i s y i e l d s a v a l u e o f 5.43 c l i m a t e , i . e . pHb  a t 25°C.  (approximately)  (Smyth and W r i g h t , 1966).  for this  micro-  T h i s 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 c h a r g e , s i n c e the v a l u e 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 r e p r e s e n t e d 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  g i v e n by: ( E q u a t i o n 9)  w  = D/6  The t h i c k n e s s o f an u n s t i r r e d l a y e r w h i c h c o u l d c o m p l e t e l y account  f o r t h e r a t e o f i o n movement a c r o s s t h e i n t i m a can be e s t i m a t e d  by l e t t i n g t h e v a l u e f o r t h e 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 a c r o s s t h e i n t i m a , and t h e v a l u e f o r -5 D^ be 1x10  2 cm / s e c ( K i d d e r e t a l . 1964).  The t h i c k n e s s o f an u n s t i r r e d  l a y e r , w h i c h w o u l d be r a t e c o n t r o l l i n g f o r i o n movement a c r o s s t h e i n t i m a i s e s t i m a t e d t o be 2210 m i c r o n s . An e s t i m a t e f o r t h e t h i c k n e s s o f u n s t i r r e d l a y e r s can a l s o be o b t a i n e d f r o m t h e time c o u r s e f o r development o f 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 d e s c r i b e d by D a i n t y 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 g r a d i e n t s o f KC1 w h i c h were a l l o w e d t o " s i t " u n t i l t h e p o t e n t i a l went t o z e r o .  The z e r o  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 g r a d i e n t o f 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 t h e u n s t i r r e d l a y e r s on b o t h s i d e s o f t h e i n t i m a were assumed t o e q u a l t h e i o n c o n c e n t r a t i o n o f t h e 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  d e r i v e d b y D a i n t y and House (1966) f o r t h e c a l c u l a t i o n o f A E 1/2 t h e 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 e l a p s e d was: ( E q u a t i o n 10)  A E 1/2 = A Eo + (A E °° log  the f o l l o w i n g observed  Eo) l o g ( 2 ( K i ' ) / { ( K i ' )  ((K')/(Ki"))  v a l u e s f o r t h e 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 v a l u e f o r t h e A E 1/2 p o t e n t i a l o f 6.64 mv.  The time r e q u i r e d f o r t h e p o t e n t i a l t o  r e a c h h a l f maximum v a l u e i s then 3 s e c o n d s , when t h e 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.  (Ki")>)  62  U s i n g the e q u a t i o n d e r i v e d by D a i n t y and House (1966) from by O l s o n and S c h u l z (1942) f o r c a l c u l a t i o n o f u n s t i r r e d ( E q u a t i o n 11)  t 1/2  = 0.38  data  layers.  <S  2  Dk and s u b s t i t u t i n g the o b s e r v e d v a l u e s f o r the i n t i m a ( t 1/2 -5 and Dk = 1x10  3 seconds  2 cm /sec.) i n e q u a t i o n 11, t h e v a l u e o f  6 is  88.9  microns. Work done on a c a t i o n exchange membrane ( K i d d e r e t 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 a p p r o x i m a t e l y 20-25 microns i n t h i c k n e s s , i f a v a l u e f o r the d i f f u s i o n c o e f f i c i e n t o f K i n aqueous -5 s o l u t i o n o f 1x10  2 cm / s e c . was  used.  K i d d e r e t a l . (1964) used t h e  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  differ-  ence d e v e l o p e d a c r o s s t h i s t y p e o f membrane a r o s e m a i n l y a t the s u r f a c e due t o a Donnan d i s t r i b u t i o n o f c a t i o n s ( T e o r e l l , 1953).  Then the  c o u r s e o f the p o t e n t i a l d i f f e r e n c e change s h o u l d o n l y r e f l e c t through t h i s u n s t i r r e d  time  diffusion  layer.  Whether o r n o t the u n s t i r r e d l a y e r s p l a y an i m p o r t a n t 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 o f the membrane i t s e l f t o the m o l e c u l a r s p e c i e s b e i n g t r a n s p o r t e d .  The i m p o r t a n c e  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 o f 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 t h e s e l a y e r s r a t h e r than by the -7 brane.  The measured D f o r the i n t i m a i s a p p r o x i m a t e l y 4.53x10  of  mem2/sec cm  ( C h a p t e r 3 ) , w h i c h i s a p p r o x i m a t e l y 20 f o l d l e s s than the D i n f r e e solution.  T h i s d i f f e r e n c e then makes i t u n l i k e l y t h a t the  unstirred  l a y e r s a r e 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 a c r o s s the r e c t a l i n t i m a a t 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  Phillips  ( u n p u b l i s h e d o b s e r v a t i o n ) found t h a t f l u x o f  a c r o s s the r e c t a l c u t i c l e was  Ca  50 times g r e a t e r than t h a t o f s u c r o s e  at  n o r m a l r e c t a l pH, even though b o t h c h e m i c a l s p e c i e s have the same hydrated radius.  T h i s dienomena appeared c o n t r a r y t o e v i d e n c e p r e v i o u s l y  found by P h i l l i p s and Beaumont ( 1 9 6 8 ) , w h i c h showed t h a t t h e r a t e o f movement o f n o n - i o n i c m o l e c u l e s  relative  a c r o s s the i n t i m a i s r e l a t e d t o  t h e i r h y d r a t e d s i z e as d e s c r i b e d by the R e n k i n e q u a t i o n  (1954).  This  can be e x p l a i n e d i n two ways; e i t h e r t h e l a t t e r r e l a t i o n s h i p does n o t h o l d f o r the r e c t a l i n t i m a o r some o t h e r f a c t o r s i n f l u e n c e the movements o f 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 m o l e c u l e s . present study  ( C h a p t e r 1) i n d i c a t e s t h a t the r e c t a l c u t i c l e  The possesses  a f i x e d n e g a t i v e charge, r e s u l t i n g i n the membrane b e i n g c a t i o n s e l e c t i v e . T h i s f a c t g i v e s r i s e t o two q u e s t i o n s , ( i )  G e n e r a l l y s p e a k i n g , how  t h e membrane f i x e d charge e f f e c t p e r m e a t i o n  of ions w i t h d i f f e r e n t  charge ( a n i o n v e r s u s c a t i o n ) and v a l e n c y (monovalent v e r s u s i . e . how  much does p e r m e a t i o n  n o n - i o n i c molecules the presence observed  does  divalent);  r a t e f o r i o n s d e v i a t e from t h a t found f o r  of s i m i l a r hydrated s i z e ?  o f the f i x e d charge account  (ii) Specifically,  can  c o m p l e t e l y f o r the p r e v i o u s l y  5 0 - f o l d d i s c r e p a n c y between p r e d i c t e d and observed  values f o r  +2 Ca  f l u x a c r o s s the i n t i m a ? P r e v i o u s experiments  (Chapter 2) i n d i c a t e t h a t a t a pH o f  5.5  o r above, the d i f f u s i o n p o t e n t i a l a c r o s s the i n t i m a does not i n c r e a s e  64  i n s i z e , implying that the net membrane charge was f u l l y d i s s o c i a t e d , while at a pH of 2.3 the membrane was not cation s e l e c t i v e , implying that the net charge was abolished.  Using this information, i t was pos-  s i b l e to study the influence of fixed charge on the ion permeation across the intima by comparing the f l u x rate of any i o n across the same preparation of these two pH values.  The pH e f f e c t on b a s i c membrane  properties, excluding f i x e d charge, was considered i n a s i m i l a r study using the uncharged molecule urea. +2 Results i n Chapter 2 indicated that Ca  was 10 times as  e f f e c t i v e i n masking the fixed charge as was a monovalent i o n such as K.  The best explanation, previously, was that the a f f i n i t y of the +2 charge i s greater f o r Ca than for the monovalent cations. I t 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 s e l e c t i v e binding +2 +  ( i . e . association of Ca  with the fixed charge) and accelerated move-  +2 ment of Ca  could be caused simultaneously by a f i x e d charge.  The  opportunity was therefore taken to compare the r e l a t i o n s h i p between f l u x 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 i n t e r -  action with a series of binding s i t e s , then flux might be expected to be influenced to a degree by the counter-ion flux i n the opposing d i r e c t i o n as described by van Breemen (1968). Such a 'trans' e f f e c t was studied +2 by observing the influence of Ca concentration on the 'trans' side on +2 u n i d i r e c t i o n a l f l u x rate of radioactive Ca  65  RESULTS  A.  I o n F l u x e s a t V a r i o u s C o n c e n t r a t i o n s and pH v a l u e s . 45 F l u x r a t e s o f Ca  a t 2 pH v a l u e s  at four different  concentrations of C a C l  (5.5 and 2.3) a r e shown i n F i g . 23.  2  and  A t a pH o f 5.5 t h e  f l u x r a t e s d i d n o t i n 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 t o d e c r e a s e +2 a t v e r y h i g h Ca f i x e d charges.  c o n c e n t r a t i o n s ( F i g . 24) p r o b a b l y due t o masking o f A t pH 2.3 ( F i g . 24) p r o b a b l y due t o masking o f f i x e d  charges.  A t pH 2.3 ( i . e . no f i x e d charge) however ( F i g . 23) t h e 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 t o t h e Ca c o n c e n t r a t i o n ; i . e . t h e r e 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 concentration ( F i g . 2 4 ) . A t a c o n c e n t r a t i o n o f 1000 mM/l, pH had no e f f e c t Ca  +2  flux.  However, as Ca  +2  c o n c e n t r a t i o n was d e c r e a s e d  on t h e  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 a t a c o n c e n t r a -  t i o n o f 100 mM/l t h e f l u x a t pH 5.5 was 6.5 times as g r e a t as a t pH 2.3, w h i l e t h i s f a c t o r  reached  81 times a t a c o n c e n t r a t i o n o f 10 mM/l  CaCl . 2  These d i f f e r e n c e s i n f l u x r a t e s a t t h e two pH v a l u e s might be attributable  to a r e v e r s i b l e  structural  change i n t h e membrane  s t r u c t u r e r a t h e r than t o f i x e d charge d e n s i t y .  T h i s p o s s i b i l i t y was  t e s t e d by m e a s u r i n g t h e f l u x r a t e o f a n o n - e l e c t r o l y t e , u r e a , a t t h e same two pH v a l u e s  ( T a b l e 6; u r e a does n o t 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 w h i c h was +2 v e r y s m a l l when compared t o t h e f l u x r a t i o o f 81 f o r Ca CaCl . 2  I t appearedthat  the presence  o f f i x e d charges  a t 10 mM/l  can c o m p l e t e l y  Fig.  Measurement o f Ca at f o u r d i f f e r e n t  23  f l u x across the i n t i m a CaCl^ concentrations  b o t h s i d e s ) and two pH v a l u e s .  The  (same on  unidirectional  f l u x was measured u s i n g C a l c u i m - 4 5 , and the c o n c e n t r a t i o n o f b a t h i n g s o l u t i o n was f r o m 1000 mM/l t o 1 mM/l C a C l . 2  used were 5.5 and 2.3.  varied  The pH v a l u e s  Solid circles  represent  v a l u e s o f c a l c i u m f l u x a t pH o f 5.5, w h i l e open c i r c l e s a r e c a l c i u m f l u x e s a t pH 2.3. V e r t i c a l bars represent standard d e v i a t i o n s for 6 preparations.  See T a b l e 6 f o r v a l u e s .  F i g . 24  Calcium permeability c o e f f i c i e n t f o r the i n t i m a a t f o u r d i f f e r e n t C a C l ^ c o n c e n t r a t i o n s and two pH v a l 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 f r o m u n i d i r e c t i o n a l Ca measurements.  flux  The c o n c e n t r a t i o n o f b o t h  b a t h i n g s o l u t i o n s were t h e same and were v a r i e d f r o m 1000 t o 1 mM/l. 5.5 and 2.3.  The pH v a l u e s used were  S o l i d c i r c l e s r e p r e s e n t 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 a t a pH o f 2.3. Each p o i n t r e p r e s e n t s mean v a l u e f o r 6 p r e parations.  V e r t i c a l l i n e s i n d i c a t e the  s t a n d a r d d e v i a t i o n where i t exceeds t h e w i d t h o f t h e symbols.  See T a b l e 6 f o r v a l u e s .  Table  6  F l u x r a t e s and  permeability  monovalent and  d i v a l e n t anions and  e x p r e s s e d as the mean v a l u e d e v i a t i o n of 6  observations.  coefficients  ±  o  cations  standard  Radio Isotope 4 5  4 5  4 5  Concentration (mM/l)  pH  fflux (uM/hr/cm tSX>.) 2  +  2,65  24.65 t 9.34  6,8  +  2.6  5.5  5.06 ±  14.06  2  1000  5.5  20.27 * 9.54  CaCl  2  1000  2.3  100 2  PxlO cm/sec *S.D  5.6  CaCl  CaCl  Ratio of Fluxes (pH 5.5/2.3)  0.74  0.83  6.54  + +  2.06  CaCl  2  100  2.3  0.83 - 0.22  2.3  CaCl  2  10  5.5  1.39 ± 0.41  38.6  CaCl  2  10  2.3  0.02 t  CaCl  2  1  5.5  0.12 ± 0.11  33.3 + 30.3  45 C a C l - l Mordue  10  5.5  0.88 ± 0.19  24.4  CaCl -2 Mordue  io  5.5  0.63 t  0.17  17.5 + 2.73  Urea C-14  10  5.5  1.52 t  1.25  42.2 +  34.8  Urea C-14  10  2.3  0.98 ±  1.09  27.2 +  30.3  Na  10  5.5  0.01 - 0.007  10  2.3  0.04  RbCl  10  5.5  1.63 t  RbCl  10  2.3  1.30 - 0.25  36.1 +  K C1  10  5.5  0.40 ± 0.12  11.1  K C1  10  2.3  0.96  4 5  4 5  4 5  4 5  81.0  0.01  +  0.56 +  +  2  2  Na 8 6  8 6  S0  2  3 5 2  S0  36  36  4  4  t  0.27  0.28  +  0.023  1.11 +  0.36  45.3  i  t  1.52  0.31  1.25  0.42  +  +  0.61 11.4 0.28  5.28  0.29 0.65 10.0 6.85 3.34  26.7 + 8.6  66  +2 f o r the g r e a t e r movement o f Ca r e l a t i v e t o uncharged  account  molecules  +2 of the same s i z e . reduced  I n the absence o f the f i x e d charge  t o t h a t p r e d i c t e d by Renkin's  equation  Ca  movement was  (1954) f o r uncharged  molecules. S i n c e the c o n c e n t r a t i o n o f monovalent i o n s i n the l o c u s t  rectum  was o f t e n v e r y h i g h (eg. 500 meg/L) the p o s s i b i l i t y  t h a t h i g h i o n con+2 c e n t r a t i o n s might a b o l i s h the s e l e c t i v e p e r m e a b i l i t y t o Ca i n the  +2 presence  o f f i x e d charges  (pH 5.5) was c o n s i d e r e d .  The Ca  c o n c e n t r a t i o n o f 10 mM/l C a C ^ was measured i n the p r e s e n c e normal and double s t r e n g t h Mordue^s R i n g e r s  The r e l a t i v e l y  +2 minor drop i n Ca f l u x when Mordue's R i n g e r was added +2 f o r the f i x e d charges  most o f the o t h e r i o n s i n t h e r i n g e r . r e s u l t i n p a r t from an osmotic e f f e c t  o f both  (See T a b l e 7 f o r the composi-  t i o n ) on both s i d e s o f the membrane a t a pH o f 5.5.  t h a t the a f f i n i t y  f l u x at a  f o r Ca The s l i g h t  ( T a b l e 6) i n d i c a t e s  i s much g r e a t e r than decrease i n f l u x might  ( e g . on pore area) r a t h e r than  from the masking o f the charged s i t e s by o t h e r i o n s . The e f f e c t s o f f i x e d n e g a t i v e charges on the f l u x o f a monovalent c a t i o n and a n i o n and a d i v a l e n t a n i o n were a l s o c o n s i d e r e d . monovalent c a t i o n the f l u x r a t e s .  For the  ( R b , T a b l e 6 ) , t h e pH d i d n o t s i g n i f i c a n t l y +  affect  i n c r e a s e d 2.5 times when the pH -2 was d e c r e a s e d from 5.5 t o 2.3, w h i l e the f l u x r a t e o f SO . i n c r e a s e d approximately  The f l u x r a t e o f C l  4  4 times.  The i n f l u e n c e o f the f i x e d charge i n a l l these +2 cases i s r e l a t i v e l y s m a l l compared t o t h a t f o r Ca , s u g g e s t i n g a +2  relatively specific affinity least, divalent  cations.  o f the n e g a t i v e charge  f o r Ca  or at  Table 7  Mordue's Ringer Chemical species  grams/liter  Moles/liter  NaCl  9.82  0.168  KC1  0.48  6.44xl0"  0.73  3.6xl0"  MgCl -6H 0 2  2  3  0.01  CaCl -6H 0 2  2  NaH P0 'H 0  0.84  6.1xl0"  NaHCOg  0.18  2.14xl0"  glucose  3.0  16.6xl0~  2  4  3  2  3  3  3  67  B.  Membrane B i n d i n g The  Capacity.  +2 Ca f l u x a t a c o n c e n t r a t i o n o f 10 mM/l C a C ^ and a pH o f  5.5 was 8 1 - f o l d g r e a t e r than a t a pH o f 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 t h a t t h e f i x e d charge i n t h e mem-  b r a n e c r e a t e s a Donnan d i s t r i b u t i o n i n t h e p o r e s such t h a t t h e c o n +2 c e n t r a t i o n o f Ca i s 81 times h i g h e r a t pH 5.5 than a t pH 2.3. F l u x +2 r a t e might t h e n s i m p l y be p r o p o r t i o n a l t o membrane Ca concentration. 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 active C a C ^ s o l u t i o n a t pH o f 5.5 and 2.3, damped d r y and t h e Ca 45  content  o f the i n t i m a determined (Table 1 ) .  c e n t r a t i o n i n the i n t i m a p e r l i t e r  A t pH 2.3 t h e Ca  o f membrane w a t e r was  the same as i n t h e b a t h i n g s o l u t i o n .  con-  approximately  A t n o r m a l r e c t a l pH o f 5.5 t h e  45  Ca  c o n c e n t r a t i o n i n t h e i n t i m a was o n l y 3 times h i g h e r .  The r a t e o f  +2 i n c r e a s e i n Ca  f l u x w i t h pH i n c r e a s e (81 t i m e s ) w o u l d appear n o t t o  be s i m p l y r e l a t e d t o t h e membrane c o n c e n t r a t i o n o f Ca (3 f o l d i n c r e a s e ) . These i n i t i a l o b s e r v a t i o n s s u g g e s t e d two q u e s t i o n s : the pH v a l u e i s i n c r e a s e d beyond 5.5, how w i l l affected? +2 Ca  (a) When +2 t h e uptake o f Ca be  (b) What i s t h e r e l a t i o n s h i p between membrane b i n d i n g o f +2  and Ca  f l u x rate?  To answer these q u e s t i o n s  6 membrane p r e p a r a -  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 a t each o f t h e f o l l o w i n g pH v a l u e s : 3.5, 3.8, 5, 6, 7 o r 8, w i t h a c o n s t a n t c o n c e n t r a +2 +2 t i o n o f Ca i n t h e medium. The Ca b i n d i n g c a p a c i t y o f t h e membranes as a f u n c t i o n o f pH i s shown i i F i g . 25b. A t a pH o f 2.2 t h e b i n d i n g a g a i n was n e g l i g i b l e .  As t h e pH was i n c r e a s e d f r o m 2.2 t o 5, t h e  F i g . 25  A.  Ca  t i o n pH.  f l u x as a f u n c t i o n o f b a t h i n g s o l u B o t h s i d e s o f t h e membrane c o n t a i n e d  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  indicate  standard d e v i a t i o n f o r 6 preparations.  Point  a t pH9 was t h e mean o f two p r e p a r a t i o n s . B.  The i n t i m a was a l l o w e d t o soak i n ImM/l 45  Ca  solution.  The pH was v a r i e d from an  i n i t i a l v a l u e o f 2.2 t o a f i n a l v a l u e o f 8. +2 The Ca pH.  uptake was p l o t t e d as a f u n c t i o n o f  V e r t i c a l l i n e s represent standard devia-  tions of 6 preparations.  See Appendix B  T a b l e 18 f o r i n d i v i d u a l v a l u e s .  pH  of C a C l  2  bathing solution  68  b i n d i n g o f Ca  +2  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 .  amount o f i n c o r p o r a t e d Ca when the pH was  the  +2  d i d not i n c r e a s e ( F i g . 25b). However, +2 i n c r e a s e d from 6 t o 8, the amount o f Ca i n the mem+2  brane again i n c r e a s e d 2 - f o l d . between pH 2.2  From pH 5 t o 6  and 8 was  The  t o t a l i n c r e a s e i n membrane Ca  24-fold. +2  The (i)  i n c r e a s e i n membrane Ca  might be caused by two f a c t o r s :  an i n c r e a s e d number o f f i x e d n e g a t i v e s i t e s o v e r 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 +2 i n g n e g a t i v e s i t e s f o r Ca  ( i i ) an i n c r e a s e d a f f i n i t y o f the  exist-  , 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).  t h e s e p o s s i b i l i t i e s was f u n c t i o n o f pH creased  One  t o e x t e n d the measurement o f Ca  ( F i g . 25a).  f r o m a pH o f 2.3  c r e a s e d above 5.5,  p o s s i b l e means o f d i f f e r e n t i a t i n g between 45  The  f l u x as  f l u x r a t e i n c r e a s e d as the pH i n -  t o 5.5,  b u t t h e n d e c r e a s e d as the pH i n +2  even though Ca  binding increased.  Obviously  there  i s no s i m p l e d i r e c t r e l a t i o n s h i p between the degree o f b i n d i n g and C. Trans E f f e c t o f the I n t i m a . flux rate. +2 The  i n c r e a s e i n Ca  from pH 2.2  a  b i n d i n g o f the i n t i m a as the pH  t o 8, s u g g e s t s t h a t the f l u x r a t e i s l i m i t e d by  the  increases the  ability  +2 o f Ca  t o d i s s o c i a t e i t s e l f f r o m the f i x e d c h a r g e . +2  i s a r a t e l i m i t i n g s t e p f o r Ca pected by a Ca  I f the f i x e d charge  +2 movement then Ca  f l u x might be  ex-  t o e x h i b i t a t r a n s e f f e c t ; i . e . f l u x r a t e s h o u l d be i n f l u e n c e d +2  c o n c e n t r a t i o n on the s i d e t o w h i c h f l u x o c c u r s .  t h e r e f o r e measured w i t h 10 mM/l  CaC^  Ca  +2  flux  was  on b o t h s i d e s o f the membrane, or  F i g . 26  A c c u m u l a t e d f l u x o f Ca45 w i t h t i m e .  Solid +2  s q u a r e s 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 o f t h e membrane and b u f f e r e d s u c r o s e t i o n on t h e o t h e r s i d e . +2 f l u x w i t h 10 mM/l Ca membrane.  solu-  Open c i r c l e s i n d i c a t e on b o t h s i d e s o f t h e  A s t e r i c k represents side of the  i n t i m a which contains the r a d i o i s o t o p e . point represents  Each  t h e range o f two o b s e r v a t i o n s .  "frlO mM  CaCl  ! Sucrose 2 ;  ^TIO  mM  CaCl  9  I  | 10 mM  8 -,  ^ CM  6  S  o  4 H  O  2 J  0  0  2  3 Hours  4  5  "3  CaCl 2  0  69  w i t h t h e i o n i c s o l u t i o n on t h e t r a n s s i d e r e p l a c e d by a s u c r o s e s o l u t i o n o f e q u a l o s m o l a r i t y t o eliminate w a t e r f l o w .  F i g . 26 shows t h a t  there  +2 2 was a Ca f l u x o f 0.51 uM/hr/cm when t h e r e was no C a C l ^ on t h e t r a n s side.  W i t h a C a C l ^ s o l u t i o n on b o t h s i d e s o f the membrane, t h e f l u x  2 r a t e i n c r e a s e d t o a v a l u e o f 1.14 uM/hr/cm .  T h i s e x p e r i m e n t seems t o  +2 i n d i c a t e the e x i s t e n c e o f a t r a n s e f f e c t f o r Ca movement. +2 be e x p l a i n e d i f Ca  T h i s would  becomes d i s s o c i a t e d from t h e f i x e d s i t e moiE e a s i l y  +2 i f a n o t h e r 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 t h a t t h e same e f f e c t ( i . e . exchange d i f f u s i o n ) can be o b s e r v e d i n a membrane p o s s e 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 o f the n e g a t i v e s i t e s i n the i n t i m a has been  e s t i m a t e d f r o m d i f f u s i o n p o t e n t i a l s f r o m w h i c h the r e l a t i v e p e r m e a b i l i t y o f the membrane t o v a r i o u s i o n s can be c a l c u l a t e d u s i n g the Goldman f i e l d equation  (Appendix A ) .  R e l a t i v e p e r m e a b i l i t y was  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  a l s o d e t e r m i n e d by  f l u x r a t i o of Rb:Cl  found t o be 4 (range 3.8-4.5) a t a b a t h i n g media c o n c e n t r a t i o n o f mM/l.  The  was 10  : P ^ j . -^-° e s t i m a t e d f r o m d i f f u s i o n p o t e n t i a l measurements rat  a t the same average c o n c e n t r a t i o n o f b a t h i n g media ( F i g . 6) approximately  5 (range 4-7).  was  Thus t h e r e was good agreement between  the two d i f f e r e n t methods o f m e a s u r i n g r e l a t i v e p e r m e a b i l i t y t o i o n s . W h i l e good agreement was  o b t a i n e d 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 , t h e r e i s an i n h e r e n t danger i n comparing a b s o l u t e v a l u e s cause c o n d i t i o n s may  d i f f e r i n the two types o f e x p e r i m e n t s .  example u n s t i r r e d l a y e r s c o u l d a l t e r the f l u x r a t i o i f they  be-  For 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 t o i o n f l u x i n one b u t i n the o t h e r type o f  not  experiment.  P h i l l i p s and Beaumont (1971) showed a d e c r e a s e i n f l u x r a t e o f w a t e r a c r o s s 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 o s m o t i c  pres-  sure.  osmotic  The  same may  be t r u e f o r i o n f l u x e s .  p r e s s u r e on d i f f u s i o n p o t e n t i a l s was  The  e f f e c t s of high  c o n s i d e r e d i n C h a p t e r 2.  Although  t h e r e 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 d e c r e a s e i n the d i f f u s i o n p o t e n t i a l  t h e r e was  a g e n e r a l downward t r e n d .  However t h i s o n l y 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 a b s o l u t e p e r m e a b i l i t y . P  k  : P  C1  r  a  t  i  o  decreased  by 0.09  p e r 100 m i l l i o s m o l a r change.  The 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 o n l y s l i g h t l y changed w i t h increase i n osmotic The  pressure.  b a t h i n g media used f o r m e a s u r i n g C l  KCI s o l u t i o n .  Some o f the C l  m o l e c u l e o f KCI. d i f f u s e across  might have c r o s s e d  86 (Rb  Rb  ) t o a new  :PC1  t o Ca^"*  r a t i o o f 3.8. +2  and  then a p p r o x i m a t e l y  7.5%  of  the  T h i s w o u l d change the  v a l u e o f 5.6:1, a p p r o x i m a t e l y  that  ratio.  r a t i o o f Rb^^  p r e s e n c e o f Ca  can  36 :C1  f l u x was  r e l a t i v e p e r m e a b i l i t y as e s t i m a t e d J?Rb S P Q  a  f o r the a c t i v i t y c o e f f i c i e n t o f the KCI s o l u t i o n o f  f l u x w i l l o c c u r as a n e u t r a l m o l e c u l e o f KCI.  The  the i n t i m a as  the membrane as e a s i l y as a n e u t r a l m o l e c u l e ( u r e a ) ,  0.925 (Lange's Handbook o f C h e m i s t r y ) ,  found f o r the  f l u x c o n s i s t e d of a  Under the a s s u m p t i o n t h a t a m o l e c u l e o f KCI  assuming a v a l u e  flux ratio  an  found t o be one, w h i l e  the  from d i f f u s i o n p o t e n t i a l s gave a  However i n C h a p t e r 2 i t was  reported that  d e c r e a s e d the conductance o f the i n t i m a ID K  +  i n c r e a s e d the conductance t o C l , the p o s s i b l e mechanism b e i n g  the  or that  +2 Ca Cl  binds  t o the n e g a t i v e  movement and  site.  Such b i n d i n g might then i n c r e a s e  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 , t h e r e b y  the  reduc-  +2 i n g the a p p a r e n t r e l a t i v e p e r m e a b i l i t y t o Ca potentials.  T h i s may +2  v a l u e s o f Ca  as measured by  a c c o u n t f o r the d i s c r e p a n c y  diffusion  i n relative permeability  + and Rb  as d e t e r m i n e d by  the two  methods.  Changes i n the s w e l l i n g o f 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 . h i n d e r e d by  d i f f u s i o n i s more s t r o n g l y  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). c a l nature  The  The  degree o f s w e l l i n g i s d e t e r m i n e d by the  chemi-  o f the membrane, the degree o f c r o s s l i n k i n g o f the m a t r i x  and  the d e t a i l s o f 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 o f the i o n i z a b l e  72  groups i n t h e membrane, t h e n a t u r e o f t h e i o n s a s s o c i a t e d w i t h groups, the s o l u t e c o n c e n t r a t i o n temperature. the m a t r i x .  these  o f t h e b a t h i n g s o l u t i o n and t h e  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 w h i c h i s s e t up i n The u l t i m a t e amount o f s w e l l i n g i s dependent on t h e type  of counter-ion  present.  The f o r m e r tends t o be g r e a t e r i n t h e p r e s e n c e +2  of h i g h l y h y d r a t e d i o n s , such as Mg h y d r a t e d i o n s u c h as C s  +  or Rb . +  , r a t h e r than a r e l a t i v e l y non-  R i c e and Nagasawa (1961) and F l e t t  and Meares (1966) found t h a t t h i s was n o t n e c e s s a r i l y t r u e i n a l l c a s e s . They f o u n d , i n c e r t a i n c a s e s , t h a t when t h e f i x e d i o n s a r e 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 t o r e s t r i c t r a t h e r than i n c r e a s e t h e s w e l l i n g .  T h i s 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 w h i c h Ca  re-  s t r i c t e d t h e amount o f w e l l i n g t o a g r e a t e r e x t e n t t h a t d i d K. +2 L e i t c h and T o b i a s (1964) r e p o r t e d  t h a t Ca  d e c r e a s e d t h e amount  o f s w e l l i n g o f 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 ions  ( K and N a ) 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 . +  +  Divalent i o n sorption  i n t h e l a t t e r a r t i f i c i a l membrane p r o b a b l y caused a d e c r e a s e i n the w i d t h o f t h e aqueous c h a n n e l s p e n e t r a t i n g d e c r e a s e i n s o l u t i o n movement.  t h e membrane, r e s u l t i n g i n a  Thus e x c h a n g i n g monovalent f o r d i v a l e n t i o n s  w i l l a f f e c t t h e 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 v a n 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 w h i c h he +2 found t h a t Ca r e d u c e d t h e membrane h y d r a t i o n and c o n s e q u e n t l y i o n i c permeability. 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 p r e s e n t i n t h e i n t i m a ; i . e . t h e p r e s e n c e o f Ca and  reduces t h e p o r e s i z e  s u b s e q u e n t l y the t o t a l amount o f w a t e r p r e s e n t i n t h e i n t i m a , as i s  d e m o n s t r a t e d i n F i g . 1.  The i n t i m a , when b a t h e d i n 10 mM/l CaCl_ a t  73  pH 5.5 h a d a w a t e r c o n t e n t o f 67% by w e i g h t , w h i l e t h e same membrane bathed  i n 10 mM/l C a C l  2  a t pH 2.2 had a w a t e r c a p a c i t y o f 72%, these  values being s t a t i s t i c a l l y  significant  (p < 0.05).  This p o s s i b l y ex-  p l a i n s why a t h i g h C a C ^ c o n c e n t r a t i o n s o f t h e b a t h i n g media (1000 mM/l) t h e f l u x r a t e a t pH 2.2 was g r e a t e r than t h e f l u x r a t e a t a pH o f 5.5 ( F i g . 2 3 ) . Helfferich  (1962) showed t h a t a t low pH a n e g a t i v e l y  membrane s w e l l e d l e s s than a t a h i g h pH, and s w e l l i n g bathing solution  concentration increased.  charged  decreased  as t h e  T h i s would r e s u l t i n a r e -  d u c t i o n o f t h e d i f f u s i o n c o e f f i c i e n t o r 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 o f u r e a ( 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 g r e a t e r a t h i g h pH t h a n a t low pH.  At high  +2 Ca  c o 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 n o t o b s e r v e d  ( F i g . 23;  +2 a t low pH t h e Ca  f l u x was g r e a t e r than a t h i g h pH).  This  suggests  +2 t h a t t h e Ca  i n the membrane a t h i g h pH reduces  g r e a t e r e x t e n t than does a s i m p l e e l i m i n a t i o n  the pore s i z e to a  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 o f c o u n t e r i o n s i n a i o n exchange r e s i n has been found t o be v e r y s e n s i t i v e external solution  ( H e l f f e r i c h , 1962).  to the concentration o f the  The d i f f u s i o n c o e f f i c i e n t o f  +2 Ca  i n c r e a s e d markedly as t h e s o l u t i o n  pecially i n dilute solutions,  concentration increased, es-  as i s shown i n F i g . 24.  However t h e  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 c o n s t a n t a t h i g h e r tions  concentra-  and i n v e r y c o n c e n t r a t e d s o l u t i o n s i t b e g i n s t o d e c r e a s e due  presumably t o t h e o s m o t i c s h r i n k a g e o f t h e r e s i n Diffusion  (Helfferich,  1962).  then seems t o be r e t a r d e d m a i n l y by the o b s t r u c t i n g e f f e c t s  o f the m a t r i x and p o s s i b l y the v i s c o s i t y o f the i n t e r s t i t i a l  fluid.  74  Van Ca  +2  Breemen and v a n Breemen (1966) showed i n t h e r a t u t e r u s  f l u x was r e d u c e d by removal o f Ca  d i f f u s i o n was o c c u r i n g . completely  +2  from the s i d e t o which i s o t o p i c  +2 However, EDTA o r S r added t o t h e t r a n s s i d e +2  prevented t h i s decrease i n e f f l u x r a t e w h i l e Pa  p a r t i a l l y prevented i t .  that  Van Breemen (1968) showed u s i n g  only  artificial  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, t h a t t h e membrane c o n c e n t r a t e d +2 + + c a t i o n s w i t h s e l e c t i v i t y f o r Ca i o n s o v e r Na and K . They a l s o +2 +2 n o t e d t h a t Ca e f f l u x was s l o w e d down by Ca f r e e media on t h e ' t r a n s ' +2 +2 s i d e , w h i l e a d d i t i o n o f Ca s t i m u l a t e d t h e Ca flux.  L u x o r o and  +2 Yanez (1968) showed t h a t t h e r a t e l i m i t i n g s t e p i n t h e e f f l u x o f Ca +2 f r o m 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 n o t t h e b a r r i e r o f f e r e d by t h e p e r f o r a t e d membrane, b u t r a t h e r by +2 the r a t e o f d i s s o c i a t i o n o f t h e complex formed between Ca i o n s and axoplasmic p r o t e i n s .  Van Breemen and v a n 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  o f Ca  i n phospholipid-  c h o l e s t e r o l membranes; i e . as t h e pH i n c r e a s e d from 2.2 t o 4.5 t h e +2 Ca c o n t e n t o f the membrane i n c r e a s e d and then p l a t e a u e d u n t i l pH 6, +2 +2 a t w h i c h p o i n t the Ca content s t a r t e d to increase again. Ca flux increased  t o a maximum a t a pH o f 5.5 and t h e n d e c r e a s e d as t h e pH  i n c r e a s e d above t h e v a l u e o f 5.5.  P a p a h a d j o p o u l o s (1968) found t h a t  the c a r b o x y l groups o f 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 a t a pH o f 5 and t h e amino groups a t a pH o f 9. f i l m (Rojas and T o b i a s ,  +2 The b i n d i n g o f Ca t o such a  1965) c o r r e s p o n d s t o i t s s o r p t i o n  At s a t u r a t i o n approximately  isotherm.  +2 one m o l e c u l e o f Ca was bound as t h e  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 . a n o t h e r n e g a t i v e charge was exposed.  However measurement o f t h e  s u r f a c e p o t e n t i a l o f t h e f i l m , a t d i f f e r i n g pH v a l u e s and i n t h e +2 +2 p r e s e n c e o f Ca , i n d i c a t e d an i n c r e a s e d Ca binding with  initial  d i s s o c i a t i o n o f t h e a c i d i c groups, b u t gave no e v i d e n c e o f 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 o f t h e amino groups.  T h i s paradox might be  +2 e x p l a i n e d by m u l t i p l e b i n d i n g o f Ca t h e c e p r o t o n a t e d amino groups.  w i t h b o t h t h e a c i d i c groups and  T h i s e x p e r i m e n t and o t h e r s l e d Luxoro and  Yanez (1968) t o t h e same c o n c l u s i o n ; i . e . f l u x from t h e membrane was +2 c o n t r o l l e d by t h e r a t e o f d i s s o c i a t i o n o f Ca from t h e membrane. +2 T h i s same a f f e c t was found i n t h e i n t i m a .  Ca  movement t h r o u g h t h e  charged membrane a t low i o n c o n c e n t r a t i o n s p r o b a b l y o c c u r s by s i t e t o s i t e t r a n s f e r , s i n c e f l u x a t h i g h pH was 81 times g r e a t e r than a t low pH v a l u e s .  As t h e c o n c e n t r a t i o n i n c r e a s e d t h e s i m p l e d i f f u s i o n component  becomes predominant o v e r s i t e t o 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 s t e p +2 +2 f o r Ca f l u x seemed t o be t h e r a t e o f d i s s o c i a t i o n o f Ca from t h e f i x e d c h a r g e , 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 v a l u e r e s u l t e d i n a d e c r e a s e d Ca f l u x and ( i i ) Ca f l u x i s increased +2 i f Ca  was p r e s e n t on b o t h s i d e s o f t h e membrane. The f l u x o f monovalent  c a t i o n s seemed t o be r e l a t i v e l y u n a f f e c t e d  by t h e p r e s e n c e o f c h a r g e ; i . e . they d i f f u s e t h r o u g h 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 t h a t e i t h e r t h e a f f i n i t y o f t h e membrane f o r monovalent  c a t i o n s was q u i t e low o r t h e  d i f f u s i o n t h r o u g h t h e membrane can o c c u r a t a r a t e e q u a l t o t h e s i t e t o s i t e t r a n s f e r r a t e t h r o u g h t h e membrane.  76  Turning  t o t h e s i g n i f i c a n c e o f these o b s e r v a t i o n s  to the i n t a c t  +2 a n i m a l , Ramsay (1953) found t h a t t h e Ca malpighian  c o n t e n t i n the haemolymph and  t u b u l e s o f t h e s t i c k i n s e c t D i x i p p u s morosus was 7 and 2  m.eq./l r e s p e c t i v e l y .  S i n c e t h e l a t t e r i n s e c t s e c r e t e s i t s own  content  o f w a t e r p e r day and r e a b s o r b s  back 9 5 % o f t h e i o n s and w a t e r i n t h e +2 r e c t u m , r a p i d r e a b s o r p t i o n o f Ca i n t h e rectum was e s s e n t i a l f o r main+2 tenance o f b l o o d Ca levels. The pore s i z e o f the i n t i m a was such as t o a l l o w o n l y v e r y s l o w movement o f uncharged m o l e c u l e s t h e s i z e o f Ca  +2  .  Assuming t h a t Ca  +2  c o n c e n t r a t i o n and t u r n o v e r was o f t h e same  o r d e r ofmagnitude i n t h e l o c u s t , t h e n t h e f i x e d charge on t h e i n t i m a was i m m e d i a t e l y  +2 r e s p o n s i b l e f o r p e r m i t t i n g t h e r e a b s o r p t i o n o f Ca  a t t h e h i g h r a t e r e q u i r e d f o r t h e maintenance o f h o m e o s t a t i s . -<•<-' 2 The r o l e o f Ca i n t h e i n t i m a c o u l d be o f g r e a t p h y s i o l o g i c a l +  i m p o r t a n c e i n t h e r e g u l a t i o n o f i o n movement a c r o s s t h e i n t i m a .  In a  +2 ( i . e . i n a s a l t d e p l e t e d s t a t e ) t h e 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 l o w , r e s u l t i n g i n a f l u x o f Ca across the +2 membrane a t a r a t e g r e a t e r t h a n by s i m p l e d i f f u s i o n . Low Ca i o n hydrated  animal  c o n c e n t r a t i o n s might be e x p e c t e d t o a i d i n t h e movement o f C l the membrane.  across  +2 L e i t c h and T o b i a s (1964) showed t h a t Ca w i l l not i n t e r -  + f e r e w i t h the movement o f K , i f t h e 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  concentration.  since the rate o f K  T h i s w o u l d a l s o seem t o be t r u e f o r t h e i n t i m a , +  or Na  +  movement seems t o be r e l a t i v e l y  independent  o f whether t h e membrane was charged o r n o t . I f t h e a n i m a l was i n a d e h y d r a t e d s t a t e , then t h e c o n c e n t r a t i o n +2 +2 o f Ca might become v e r y h i g h . H i g h c o n c e n t r a t i o n o f Ca w o u l d have a m a n y - f o l d e f f e c t on t h e 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  t o d e h y d r a t e t h e membrane, b o t h by an o s m o t i c e f f e c t and a b i n d i n g  effect.  This dehydration  s h o u l d l e a d t o a r e s t r i c t i o n i n b o t h i o n and  +2 Ca movement under such c o n d i t i o n s would most l i k e l y  w a t e r movement.  be by s i m p l e d i f f u s i o n .  C a t i o n and a n i o n movement a c r o s s t h e i n t i m a  s h o u l d be r e s t r i c t e d by t h e membrane d e h y d r a t i o n  and by c o m p e t i t i o n  +2 w i t h Ca and o t h e r i o n s f o r t h e p o r e . L e i t c h and T o b i a s (1964) found +2 + t h a t Ca w i l l compete w i t h K ,such t h a t a t e q u a l 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 t h e 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 t h e r e w i l l be a s e l f r e g u l a +2 +2 t i o n o f Ca movement a c r o s s t h e i n t i m a ; i . e . as Ca s e c r e t i o n s by t h e malpighia tubules increased leading to high r e c t a l concentrations of * +2 '-Ca  +2 , t h e n t h e Ca  p e r m e a b i l i t y o f t h e i n t i m a s h o u l d be d r a s t i c a l l y  +2 .reduced. Ca a l s o m i g h t a c t as a f i n e c o n t r o l o f i o n p e r m e a t i o n and p o s s i b l y o f w a t e r movement a c r o s s t h e i n t i m a . L e i t c h and T o b i a s (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. Phillips Na  +  (1964, b) found t h a t t h e r a t e s o f r e a b s o r p t i o n o f K , +  and C l were r e g u l a t e d i n r e s p o n s e t o i o n l e v e l s i n haemolymph.  s a l i n e - f e d l o c u s t s ( i . e . d e h y d r a t e d ) uptake f o l l o w e d 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  In  Michaelis-Menten  d i d n o t 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 reabsorption increased l i n e a r l y w i t h the r e c t a l concentration of ions.  A t any r e c t a l c o n c e n t r a t i o n , e x c e p t v e r y  low v a l u e s , t h e r a t e o f 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 h i g h e r than i n t h e s a l i n e - f e d l o c u s t s .  78  Studies that K  and N a  +  (then P  fc  Phillips  'v- P  N a  o f 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 +  w i l l have a f l u x r a t e v e r y s i m i l a r t o t h a t f o r R b ^ P  ) , w h i c h was found t o be 1.6 u eq/hr/cm ) .  (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 2  2.0 and 0.25 u eq/hr/cm  + f o r Na , K  +  r a t e s o f 0.25,  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 a n i m a l s , when the r e c t a l c o n c e n t r a t i o n s three i o n s .  Comparing t h e l a t t e r v a l u e s  were 10 mM/l f o r a l l  t o t h o s e f o r f l u x a c r o s s the  i n t i m a a l o n e , i n d i c a t e s t h a t the f l u x v a l u e o f K  +  f o r t h e whole r e c t a l  w a l l i n v i v o was a l m o s t i d e n t i c a l w i t h t h e v a l u e o b t a i n e d alone.  This i n d i c a t e d that reabsorption  by the i n t i m a .  +  of K  +  f o r the i n t i m a  was l i m i t e d p r i m a r i l y  79 SUMMARY  1.  The r e c t a l i n t i m a o f the d e s e r t l o c u s t was found t o p o s s e s s negative gested  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 .  t h a t the m o l e c u l a r  fixed  I t was s u g -  species responsible f o r the negative  s i t e s might be a c i d i c amino a c i d s . 2.  The s e l e c t i v e p e r m e a b i l i t y o f t h e i n t i m a as e s t i m a t e d  from  diffu-  +2 +2 +2 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 Ba > Ca > Sr > ~^"2 "4*2 *4" "f* Mg > Mn , f o r monovalent c a t i o n s was NH^ > Rb > Na  +  F~ 3.  >L i  +  >N0~> J  > TEA  and f o r monovalent anions was MCO^  +  CL~> CH C00~ > B r ~ > H„PO ~ > i " J 2 4  Cation a f f i n i t y  o  |  K  > CN  > >  .  f o r t h e f i x e d charged s i t e was found t o be i n t h e  *4*2 | | o r d e r o f Ca > Mg » 4.  |  Cs >  K  ^-|_ > Na .  S i m i l a r i t y o f e f f e c t s o f pH and i o n c o n c e n t r a t i o n pn s t r e a m i n g 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 t h a t i o n movement and w a t e r  f l o w might take p l a c e through t h e same r o u t e . 5.  The i n t i m a was found t o a c t as an o s m o t i c compartment such t h a t at high e x t e r n a l osmotic pressures, reduced due t o a s h r i n k a g e  t h e r a t e o f w a t e r f l o w was  o f the e f f e c t i v e pore s i z e i n t h e  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 o f i o n s d i d n o t seem e f f e c t e d by membrane 6.  dehydration.  U n s t i r r e d l a y e r s a t t h e membrane-solution i n t e r f a c e s were found t o have a m i n i m a l 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 o f t h e value  f o r streaming  p o t e n t i a l s was found t o be due t o 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 o p p o s i n g unstirred layers.  80  7.  C a l c i u m -45 f l u x a c r o s s t h e i n t i m a a t pH 5.5 ( i . e . p o s s e s s i n g f i x e d c h a r g e ) was found t o 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 o f 10 mM/l C a C ^ , than c a l c i u m f l u x a t t h e same c o n c e n t r a t i o n , a c r o s s the uncharged membrane (pH 2.2). was n o t s i g n i f i c a n t f o r s u b i d i u m . f i x e d charge enhanced a n i o n  8.  Calcium permeation  The same e f f e c t  C o n v e r s e l y , the r e m o v a l o f  flux.  r a t e was found t o be a f u n c t i o n o f i t s d i s s o -  c i a t i o n r a t e f r o m t h e f i x e d charge and d i d n o t c o r r e l a t e i n a s i m p l e manner w i t h t h e membrane b i n d i n g c a p a c i t y f o r c a l c i u m . 9.  A t r a n s 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 t h e i n t i m a and i s b e l i e v e d t o be a f u n c t i o n o f t h e d i s s o c i a t i o n r a t e o f c a l c i u m from the f i x e d n e g a t i v e  10.  I t was c o n c l u d e d  site.  t h a t e l e c t r o - o s m o s i s was n o t t h e mode o f w a t e r  movement a c r o s s t h e r e c t u m , however p h y s i o l o g i c a l advantage o f e l e c t r o - o s m o s i s was d i 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 t h a t t h e i n t i m a might be t h e  + rate l i m i t i n g step f o r K  reabsorption i n a hydrated  animal.  A P P E N D I X  A  81  This s e c t i o n contains constant  field  a b r i e f d e s c r i p t i o n o f the way t h a t the  e q u a t i o n may be d e r i v e d  can be c a l c u l a t e d s i m u l t a n e o u s l y and  anions.  so t h a t p e r m e a b i l i t y c o e f f i c i e n t s  f o r monovalent and d i v a l e n t  cations  The treatment i s b a s i c a l l y the same as t h a t o f Goldman  (1943) and Hodgkin and K a t z (1949) and i s s i m i l a r t o the e q u a t i o n i l l u s t r a t e d by H a n i The  (1970).  b a s i c assumptions made i n the d e r i v a t i o n a r e (Hodgkin and  K a t z , 1949): 1.  Ions i n the membrane move under the i n f l u e n c e o f d i f f u s i o n and  the e l e c t r i c  field  i n a manner w h i c h 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  field  may be r e g a r d e d as c o n s t a n t  through o u t  the membrane. 3.  The c o n c e n t r a t i o n s  o f i o n s a t the edges o f the membrane  are d i r e c t l y p r o p o r t i o n a l t o those i n the aqueous s o l u t i o n s bounding the membrane. 4.  The membrane i s homogeneous.  Assumption 1. l e a d s the c u r r e n t  t o the f o l l o w i n g i n t e r g r a t e d e q u a t i o n s f o r  c a r r i e d by i o n s :  (Equation  1)  I  z  =  Z FV  A  - (C ) a z  e "  z  V  F  /  R  T  ' - Z VF/RT 1 - e  a Now the c o n c e n t r a t i o n  (C z)o  (C z ) a t the o u t e r  edge o f the membrane  A  i s p r o p o r t i o n a l t o the c o n c e n t r a t i o n (assumption 3 ) .  (A )o i n the e x t e r n a l s o l u t i o n  82  (C z)o = B z A  ( A ) o and ( C ^ a = B^z Z  A  (A ) i . Z  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 t h e aqueous phase; (A ) i i s t h e c o n c e n t r a t i o n on t h e o p p o s i t e s i d e o f t h e membrane. Thus ( E q u a t i o n 1) becomes f o r K, C l , and Ca. ( E q u a t i o n 2)  I  ( E q u a t i o n 3)  I  f c  = P  F V  = P  Q1  (k)o - ( k ) i "  2  k  F  Q1  ( C l ) i- (Cl)o  2 V  — ( E q u a t i o n 4)  I  = 4P_  B z  A  A  A  e  ~  V F / R T  !_~  VF/RT  e  F V  (Ca)o - ( C a ) i  2  — where P z = U z  V F / R T  e  e  ~  2 V F / R T  .^^VP/RT  RT/ZaF  Then f o r P, = 11 B, RT/af k k k  and f o r P e a = M  ca  B  ca  RT/„ „ 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 t h r o u g h t h e membrane i s t h e r e f o r e g i v e n by: ( E q u a t i o n 5)  where  W = Ko +  p c l  Y = Ki+ P  , a n d  _ M =  M  /  c l  I = F V P 2  (Cl)i +  p k  /P  k  p  (Cl)o +  C a  fc  /  p C a  V F / R T  1 - e "  V F / R T  4m (Ca)o  p k  /P  W - Y e "  k  4m ( C a ) i  e  "  V F / R T  , -VF/RT 1 - e  " j  ^WRT  1 - e The p o t e n t i a l  d i f f e r e n c e a c r o s s t h e membrane i n t h e absence o f  i o n i c c u r r e n t w i l l be d e s i g n a t e d as E where E= Eo - E i . V=E when 1=0 Then  E = RT  log  £  Y/W  E = RT l n P, ( K ) i + p ( C l ) o + 4m P. ( C a ) i e _k Cl Ca F  P, (K)o + P ( C l ) i + 4m P (Ca)o k cl ca  E  F  /  R  T  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 o f e x t e r nal s o l u t i o n (CaC^ brane r e s i s t a n c e .  and KC1)  and  See F i g . 5.  the mem-  Bathing Solution  KC1  CaCl  2  Concentration of bathing solution (mM/l)  Solution resistance (ohms)  Membrane resistance (ohms)  0.1  3.17xl0  5  25xl0  0.5  1.06xl0  5  8.1xl0  1.0  50.8xl0  5.0  3  3  3  10.5xl0  11.6xl0  3  1.69xl0  10  6.2xl0  472  50  1.21xl0  131  100  602  86  500  134  18.8  1000  68.7  7.4  0.1  91xl0  1.0  23.3xl0  10  3.47xl0  100  504  351  1000  70.2  62  3  3  3  3  62.5xl0  3  3  3  21.9xl0  3  6.96xl0  3  3  Table 9  The  a f f e c t o f two  cations  d i v a l e n t and  two  on a p o t e n t i a l d i f f e r e n c e  a 2 f o l d KC1  concentration  See  Fig.  caused by  difference.  S a l t s were added a t e q u a l on b o t h s i d e s .  monovalent  concentrations 8.  Diffusion potentials in millivolts Individual preparations  Concentration of added salt (mM/l) 1 CaCl  2  2  KC1  4  5  6  Mean ± S.D.  4  4  8  9  7.25  11  7.50  ± 2.8  1  2  2  5  6  7  9  5.4  +  10  1  1  2.5  2.5  2  2  1.11  -1  -0.5  0  -1  -1.17  -1.5 • -1.5  +  3.2 1.3  +  0.71  +  2.6  +  1.4  0  4  5  8  9  7.25  11  7.4  1  4  4  7  6  6  7  5.7  10  0  -3  2  3.5  2  4  2.4  -1  -4  -1  -1  0  0  -1.16  0  4  6  4  9  7.25  11  6.9  1  4  5.5  3  9  6  7  5.75  10  4  4.5  2  3.5  4  4  3.67  ± 0.88  100  1  1  0.5  0  0  0  0.42  + 0.5  0  12  11  10  12  9.5  10  11  1  10  10  8  10  8  8  9  + 1  10  4  4.5  3.5  5  4  5  4.35  + 0.6  100  0.5  0.5  0  0.5  0  0  0.25  + 0.3  100 NaCl  3  0  100 MgCl  2  + +  1.4 1.47  +  2.8  +  2.19  +  1.5  T a b l e 10  The a f f e c t o f pH on s t r e a m i n g See F i g . 9.  potentials.  Streaming potentials i n m i l l i v o l t s  p H of b a t h i n g solution  Individual preparations 1  2  3  4  5.5  14  9  11  5.0  14  9  4.5  14  4.0  S.D.  Mean  5  6  12  13  12.5  11.9  +  1.74  11  12  13  12.5  11.9  +  1.74  9  11  12  13  12.5  11.9  +  1.74  7.5  5  9  9  10  9  8.3  +  1.78  3.5  3 •  2.5  7  6  4  5  4.6  +  1.74  3.0  0  0  2.5  1.5  2  3  1.5  +  L27  2.5  0  0  0.5  0.5  0  1.5  0.4  +  0.59  2.2  0  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  Fig.  9.  pH  of bathing  Diffusion potentials in millivolts Individual preparations  solution  Mean +  S.D  11.5  +  1.-75  13  11.3  +  1.5  8  13  9.9  +  2.6  4  6  11  7.7  + 2.8  6  4  6  9  6.3  +  2.3  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  4  5  6  10  10  10  13  12  10  10  10  12  11  9.5  6  4.0  11  6  8  3.5  9  4  3.0  5  2.5 2.3  1  2  3  5.5  14  12  5.0  13  4.5  +  0  Table  The  12  a f f e c t of h i g h sucrose concentrations  diffusion potentials.  See F i g . 10.  on  Potential difference in millivolts  KCI concentration gradient Concentration gradient of sucrose  Individual preparations 4  5  6  8.5  15  10.5  12.5  10.9± 2.42  10  9  12  10  10.5  10.1+ 1.11  8  9  9.5  10  9  10.5  9.3 ±0.88  400 m. csm.-400 m. osm.  7  9  8.5  9.5  5.5  11  8.4 ± 1.93  600 m. osm.-600 m. osm.  6  9  9  9  9.5  10  8.8 ± 1.41  800 m. osm.-800 m. osm.  6  9.5  9  8  8  8.5  8.2 ± 1.21  1  2  000 m. osm.-OOO m. osm.  9.5  9.5  100 m. osm.-100 m. osm.  9  200 m. osm.-200 m. osm.  Lumen  Haemocoel  3  Meant S.D  0.01 M KCI - 0.005 M KCI  T a b l e 13  S t r e a m i n g p o t e n t i a l s were d e v e l o p e d u s i n g s u c r o s e 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 KCI on b o t h s i d e s .  mM/l  I n both i n s t a n c e s the  o s m o t i c p r e s s u r e d i f f e r e n c e was k e p t  con-  s t a n t b u t the average s u c r o s e c o n c e n t r a t i o n changed.  See F i g . 11.  Milliosmolars of sucrose Haemocoel  Streaming potentials in millivolts Individual preparations  Average sucrose concentration  Lumen  2  3  4  5  6  Mean + S.D.  100  000  50  6.5  8  6  9  7  6.5  7.2 t 1.13  200  100  150  3.5  4  3  5  3.5  2.5  3.6 t .86  300  200  250  2.5  2  2  3  1.5  2  400  300  350  .5  1.5  1  2  1  2  1.3 ± .61  200  000  100  9  10  10  14  10  10.5  10.6± 1.74  400  200  300  4  3  2  3  4  4.5  3.4 t .92  600  400  500  3  1.5  1.5  1  2  2.25  1.9 t .90  800  600  700  0  1.5  1.5  .5  1  2  1.1 ± .54  -  2.2 ± .52  T a b l e 14  S t r e a m i n g p o t e n t i a l s measured a c r o s s t h e i n t i m a due t o o s m o t i c g r a d i e n t s the use o f s u c r o s e .  created  by  The s u c r o s e c o n c e n t r a -  t i o n was v a r i e d f r o m 0 t o 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 k e e p i n g t h e s u c r o s e a t 0 m i l l i o s m o l a r on t h e lumen s i d e . The c o n c e n t r a t i o n both sides.  o f KC1 was the same on  See F i g . 13.  Concentration of KCI bathing solution  Streaming potentials in millivolts  Milliosmolar concentration gradient of sucrose Haemocoel  Individual preparations 1  Lumen  2  3  4  5  6  Mean  ±  S.E  IM KCI 100 m. osm.  000 m. osm.  .75  1  .75  .75  .5  .75  .75  t  .16  200 m. osm.  000 m. osm.  1.5  1  1  1.5  1  1.25  1.21 t  .25  300 m. osm.  000 m. osm.  1  1.5  1.25  2  1.25  1  1.33 t  .38  400 m. osm.  000 m. osm.  1  2  1.5  2.25  1.5  1.25  1.58 t  .47  2.25  2.54 1  1.21  .IM KCI 100 m. osm.  000 m. osm.  5  2  2  2  2  200 m. osm.  000 m. osm.  4  3.5  3  3  3.25 4  3.46 ±  .46  300 m. osm.  000 m. osm.  4  5  4  4.5  5  5  4.58 t  .49  400 m. osm.  000 m. osm.  5  5  4.5  4.5  5.5  5.5  5.00 ±  .45  .01M KCI 110 m. osm.  000 m. osm.  8  7  7.5  8  7.63 t  .47  210 m. osm.  000 m. osm.  11  11  12  10  11.0  t  .50  410 m. osm.  000 m. osm.  14  14  15  13  14.0 ±  .50  Concentration of KC1 bathing solution  Streaming potentials in millivolts  Milliosmolar concentration Individual preparations  gradient of sucrose Lumen  1  2  3  4  5  6  Mean +  S.D.  100 m. osm.  000 m. osm.  25  22  12  14  13  18.8  17.46 +  5.3  200 m. osm.  000 m. osm.  32  29  14  19  19  20  22.17 +  6.9  300 m. osm.  000 m. osm.  23  31  22  26  24  24  25.00 +  3.23  400 m. osm.  000 m. osm.  24  33  24  31  26  26  27.33 +  3.78  Haemocoel .001M KC1  T a b l e 15  S t r e a m i n g p o t e n t i a l s were d e v e l o p e d u s i n g sucrose lmM/1  concentration differences, with  KCI on b o t h s i d e s .  The  c o n c e n t r a t i o n d i f f e r e n c e was  sucrose kept  constant,  b u t the a b s o l u t e 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 sucrose Haemocoel  Average sucrose concentration  Streaming potentials in millivolts Individual preparations  , Lumen  1  2  3  4  5  6  Mean- S.D  200  0  100  32  29  16  19  21  18  22.5 ± 6.5  400  200  300  14  9  8  13  17  11  12  t 3.3  600  400  500  11  4.5  3.5  5  6  5  6  1 2.7  800  600  700  3  2  2.5  3  3  3  2.75  t 0.4  T a b l e 16  Non-linear iicrease i n streaming  potentials with  an i n c r e a s e i n the o s m o t i c p r e s s u r e . A.  R e p r e s e n t s 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  the o s m o t i c p r e s s u r e w i t h o u t  increase perfusion  of s o l u t i o n . B.  R e p r e s e n t s n o n - l i n e a r i n c r e a s e i n the s t r e a m i n g 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 o s m o t i c p r e s s u r e , w h i l e the membrane i s b e i n g p e r f u s e d a t a r a t e o f 2.8  See.  F i g . 20.  ml/min.  Streaming potentials in millivolts Individual preparations  Milliosmolars of sucrose Haemocoel  Mean -  S.D.  Lumen  1  100  0  7  8  6  5  4  7  6.2  +  1.5  200  0  10  9.5  8  7  7  10  8.6  +  1.4  300  0  12  11  11  9.5  12  12  11.3  +  1.0  400  0  13  13  12  11  13  13  12.5  100  0  4  4  4  3  2  4  3.5  200  0  6  4.5  5  4.5  3.5  5  4.8  300  0  7  7  8  6  4  6  6.3  400  0  8  8  7  7  5  6.5  6.9  A  +  0.8  B +  0.8  +  0.8  +  1.4  +  1.1  T a b l e 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 o f  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 brane. KCI  a c r o s s t h e same mem-  A 2 f o l d concentration difference of  (10 mM/l - 5 mM/l) was used t o 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 c a s e s .  High  c o n c e n t r a t i o n o f KCI was p l a c e d on t h e lumen side.  S u c r o s e was used t o cause  potentials of various sizes.  streaming  S u c r o s e , when  p l a c e d on t h e haemocoel s i d e caused s t r e a m i n g p o t e n t i a l s t o be added t o d i f f u s i o n p o t e n t i a l s , and when p l a c e d on t h e lumen s i d e caused them t o be s u b t r a c t e d .  Sign i s with  r e f e r e n c e t o t h e haemocoel s i d e .  See F i g . 22.  KC1 concentration gradient  Potential difference in millivolts  Concentration gradient of sucrose  Individual preparations  0.005 M KC1  1  2  3  4  5  6  Mean ±  000 m. osm.  11  14  9  10  11  7  10.3  100 m. osm.  18  17  19  16  17  17  17.3  200 m. osm.  23  20  23  19  22  22  21.5  300 m. osm.  26  22  26  25  25  23  24.5  400 nr. osm.  27  23  28  28  27  23  26.0  000 m. osm.  11  14  9  10  11  7  10.3  100 m. osm.  4.5  8  '4.5  6  2  3  4.7  200 m. osm.  3.5  7  1.5  2  2  1  2.8  300 m. osm.  -1  6  -1  -2  -1  -1  0.0  400 m. osm.  -2  5  -4  -4  -3  -4  -2.0  0,01 M KC1  S.E  a ± 2.34 + + + +  1.03 1.64 1.64 2.37  b + 2.34 + +  2.14 2.21  + 2.97 +  3.52  Table  18  The i n t i m a was a l l o w e d t o soak i n 1 mM/l solution.  The pH was v a r i e d from an  v a l u e o f 2.2 t o a f i n a l v a l u e o f 8. F i g . 25 B.  Ca  initial See  pH of  CaCl  uM C a V gm of dry membrane +  2  bathing solution  Individual preparations Mean +  S.D.  2.84  +  0.86  3.4  2.87  +  4.9  3.4  4.13  47.9  30.4  40.0  37.1 +  35  35  38  42.3  36.5  +  37.2  39.7  68.5  50.7  63.6  52.7  +  57.8  47.7  86  65  74.6  64.8  4  1  2  3  2.2  1.9  2.7  2.4  3.1  4.4  2.5  3.5  2.4  1.7  3.3  2.3  4.1  3.8  3.8  6.8  3.3  2.7  5.0  31.7  36.7  35.7  6.0  40.0  28.4  7.0  56.2  8.0  57.8  5  6  +  +  0.88 1.5 6.4 4.9 12.6 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 A b s o r p t i o n by t h e Rectum o f L o c u s t s . Nature ( i n press) B r a y , 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 C o u n t i n g 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. B r o d s k y , W.A. and S c h i l b , T.P. (1965) Osmotic P r o p e r t i e s o f I s o l a t e d Turtle Bladder. Am. J . 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