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Distribution and fluxes of sodium and hydrogen in crustacean muscle cells Menard, Michael Reald 1980-12-31

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DISTRIBUTION AND  FLUXES  OF SODIUM AND HYDROGEN IN CRUSTACEAN MUSCLE' CELLS by MICHAEL REALD MENARD B.Sc,  University of B r i t i s h  M.Sc, University M.D.,  Columbia,  o f Toronto,  1971  1972  U n i v e r s i t y o f B r i t i s h Columbia, 1979  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Department o f Anatomy We a c c e p t t h i s  t h e s i s as  to the r e q u i r e d  conforming  standard  THE UNIVERSITY OF BRITISH COLUMBIA February,  1980  © M i c h a e l R e a l d Menard, 1980  In p r e s e n t i n g t h i s  thesis  an advanced degree at  further  fulfilment  of  the  requirements  the U n i v e r s i t y of B r i t i s h Columbia, I agree  the L i b r a r y s h a l l make it I  in p a r t i a l  freely  available  for  agree t h a t p e r m i s s i o n for e x t e n s i v e copying o f  of  this  representatives. thesis for  It  financial  this  thesis  of  gain s h a l l not be allowed without my  Anatomy  The U n i v e r s i t y of B r i t i s h Columbia 2075 Wesbrook P l a c e V a n c o u v e r , Canada V6T 1W5  Date  A p r i l 22, 1980  or  i s understood that copying or p u b l i c a t i o n  written permission.  Department  that  reference and study.  f o r s c h o l a r l y purposes may be granted by the Head of my Department by h i s  for  ABSTRACT A new efflux  technique was  d e v i s e d f o r measurement o f the u n i d i r e c t i o n a l sodium  from s i n g l e s t r i a t e d muscle c e l l s  nubilus.  It involves  the  the myoplasm w i t h an  calculated It  barnacle,  can  same c e l l  i n s i d e the be  by a s t a n d a r d method.  c e l l , which are  calculated  directly.  s o l u t i o n i s the  that  the  d e t e c t e d by  the  I t was  was  muscle c e l l s  several  normal R i n g e r ' s s o l u t i o n was content.  used to l o a d  cell  However, s a t u r a t i o n  the myoplasm o f  new not  The  results.  regions.  Saturation  of the  efflux  into  apparent even i n c e l l s w i t h v e r y h i g h sodium  o f the  e f f l u x into potassium-free s o l u t i o n  and  l e v e l s of i n t r a -  sodium. e f f l u x i n t o sodium-free s o l u t i o n was  Ringer's s o l u t i o n .  The  decline  i n the  workers to occur i n t h i s s i t u a t i o n was of the  longitud-  sodium e f f l u x from i n t a c t s i n g l e  i n t o o u a b a i n - c o n t a i n i n g s o l u t i o n o c c u r r e d a t r e l a t i v e l y low cellular  single  from i n j e c t e d t o n o n i n j e c t e d  technique to measure the  revealed  the  glycocalyx.  n e c e s s a r y to take i n t o account the  i n a l d i f f u s i o n of t r a c e r i n s i d e the new  with  i n t r a c e l l u l a r sodium-specific microelectrode  Intracellular microinjection  o f the  l i t e r a t u r e , are  sodium a s s o c i a t e d  e x t r a c e l l u l a r space i n a s s o c i a t i o n w i t h the  c e l l s w i t h radiosodium.  extracellular  S e v e r a l experiments  t o g e t h e r w i t h r e s u l t s from the  w i t h the h y p o t h e s i s that most of the  i n the  Use  can  only pool of i n t r a c e l l u l a r  sodium i n s o l u t i o n i n the myoplasm.  which t e s t t h i s assumption,  resides  been ..  sodium e f f l u x  sodium o f a p p r e c i a b l e s i z e which exchanges r a p i d l y w i t h the  c e l l y e t not  Changes  l a r g e r than had  Thus the  the  accurately.  i s assumed i n these c a l c u l a t i o n s  consistent  Balanus  i n t r a c e l l u l a r sodium-specific microelectrode, during  i n the s p e c i f i c a c t i v i t y  be  giant  continuous measurement of the a c t i v i t y of sodium i n  c o l l e c t i o n o f r a d i o s o d i u m from the  thought p r e v i o u s l y ,  o f the  s i m i l a r to t h a t  i n t o normal  sodium e f f l u x r e p o r t e d by found to be  sodium content o f the myoplasm which o c c u r s .  due No  to the  other  rapid  decline  'sodium-sddium  iii  exchange' was appears  found.  to be due  Most of the sodium e f f l u x under normal c o n d i t i o n s  t o a mechanism which i s not s e n s i t i v e t o e x t e r n a l ouabain  or  potassium. The  sodium e f f l u x i n b a r n a c l e muscle was  shown to be e l e c t r o g e n i c .  c o r r e l a t i o n between the measured v a l u e s of the a c t i v e sodium e f f l u x and e l e c t r o g e n i c p o r t i o n o f the membrane p o t e n t i a l was was  found.  c o n s i s t e n t w i t h the p r e d i c t i o n s o f a phenomenological  l e a d i n g model f o r the membrane p o t e n t i a l ,  The  the  correlation  e x t e n s i o n o f the  the Goldman-Hodgkin-Katz e q u a t i o n .  The e f f l u x o f hydrogen ions from the c e l l can o n l y be measured from changes i n the i n t r a c e l l u l a r pH.  A  indirectly,  Measurements o f the i n t r a c e l l u l a r  w i t h an i n t r a c e l l u l a r p H - s p e c i f i c g l a s s m i c r o e l e c t r o d e r e v e a l e d no  pH  'pH  t r a n s i e n t s ' of the type r e p o r t e d by o t h e r workers i n d i f f e r e n t p r e p a r a t i o n s of b a r n a c l e muscle.  Measurements of the i n t r a c e l l u l a r pH made w i t h the  e l e c t r o d e and w i t h an i n d i c a t o r method were i n c l o s e agreement. d i s t r i b u t i o n o f the i n d i c a t o r DMO unusual  micro-  However, the  (5,5-dimethyl-2,4-oxazolidinedione) e x h i b i t e d  b e h a v i o r not p r e v i o u s l y r e p o r t e d .  which takes t h i s b e h a v i o r i n t o account  A refinement o f the DMO  i s described.  method  iv  TABLE OF CONTENTS , 11  Abstract L i s t o f Tables  ..vi  L i s t o f Figures  .vn  Acknowledgements S e c t i o n 1.  S e c t i o n 2.  ..x  General  Introduction: A. Scope o f the t h e s i s B. H i s t o r i c a l notes  ...4  Transmembrane Fluxes o f Sodium and Hydrogen Ions: A. G e n e r a l c o n s i d e r a t i o n s B. S t a t e s o f water and ions i n c e l l s C. The sodium e f f l u x ( i ) normal R i n g e r ' s s o l u t i o n ( i i ) potassium-free s o l u t i o n ( i i i ) sodium-free s o l u t i o n (iv) ouabain-containing s o l u t i o n D. Membrane p o t e n t i a l E. Summary o f problems t o be addressed F. Summary o f models ( i ) sodium e f f l u x from a whole c e l l ( i i ) steady s t a t e d i s t r i b u t i o n o f c a t i o n s ( i i i ) e l e c t r o g e n i c membrane p o t e n t i a l  S e c t i o n 3.  The  S t a t e s o f Sodium i n C e l l s ; Introduction G e n e r a l methods A. Increase o f c e l l sodium B. Decrease o f c e l l sodium  S e c t i o n 4.  M i c r o i n j e c t i o n o f Radiosodium i n t o S i n g l e Muscle Methods Results Discussion  S e c t i o n 5.  Survey o f the Sodium E f f l u x from S i n g l e Muscle Methods Results: ( i ) normal R i n g e r ' s s o l u t i o n ( i i ) potassium-free s o l u t i o n ( i i i ) sodium-free s o l u t i o n (iv) ouabain-containing s o l u t i o n Discussion  S e c t i o n 6.  Comparison o f Sodium E l e c t r o d e Methods Results Discussion  S e c t i o n 7,  E l e c t r o g e n i c Sodium Methods Results Discussion  Transport  ,10 .19 .31 .33 .34 .36 .44 .48 .56 .59 .69 .71  ...75 ...77 .. .85 ...95 Cells  Cells  ..100 ..107 ..117 ..133 . .137 . .138 ..141 ..145 ..148 ..153 ..157  and Radiosodium Measurements ..172 ..173 ..174 ..184 ..187 ..189 ..189 ..196  V  S e c t i o n 8.  D i s t r i b u t i o n o f Hydrogen Ions D u r i n g Steady C o n d i t i o n s Methods Results Discussion  ...201 ...2 02 ...204 ...212  S e c t i o n 9.  Comparison o f the I n t r a c e l l u l a r pH Measured by DMO and by m i c r o e l e c t r o d e s Methods Results Discussion  ...217 ...218 ...223 ...230  S e c t i o n 10. S i g n i f i c a n c e o f the R e s u l t s F u r t h e r Work Bibliography  and Suggestions f o r ...237 ...242  LIST OF TABLES Table I  Composition o f s o l u t i o n s .  Table I I  a. Summary o f measurements on loaded c e l l s  passively-  b. Ion content o f the myoplasmic and nonmyoplasmic compartments. Table I I I  I n t r a c e l l u l a r pH and membrane p o t e n t i a l i n c r u s t a c e a n muscle.  Table IV  Mean water and e l e c t r o l y t e  Table V  Calculation  Table VI  S e n s i t i v i t y o f pH(DMO) to e r r o r s  of flux j  m  content o f t e s t  cells,  from the data o f F i g . 29. i n measurement.  LIST OF FIGURES F i g u r e 1.  Models o f the c e l l used fluxes.  i n calculation of ion  F i g u r e 2.  Insertion  F i g u r e 3.  Changes i n the sodium content o f c e l l s d u r i n g immersion i n sodium-free lithium-substituted Ringer's s o l u t i o n .  F i g u r e 4.  Microinjection  F i g u r e 5.  Perfusion  F i g u r e 6.  Vacuum  F i g u r e 7.  E f f l u x o f m i c r o i n j e c t e d radiosodium i n t o normal R i n g e r ' s s o l u t i o n .  F i g u r e 8.  'Slope R a t i o ' f o r a c e l l versus myoplasmic sodium activity.  F i g u r e 9.  E f f l u x o f p a s s i v e l y - l o a d e d radiosodium c e l l i n t o normal Ringer's s o l u t i o n .  F i g u r e 10.  Summary o f the raw data and reduced r e s u l t s f o r a t y p i c a l experiment.  F i g u r e 11.  E f f l u x o f sodium from a c e l l i n t o normal R i n g e r ' s s o l u t i o n , uun c o r r e c t e d f o r Na£^.^, versus myoplasmic sodium a c t i v i t y .  F i g u r e 12.  E f f l u x o f sodium from a c e l l i n t o normal R i n g e r ' s s o l u t i o n , c o r r e c t e d f o r Na* versus myoplasmic \cell sodium a c t i v i t y .  of microelectrodes into  cell.  apparatus.  apparatus.  system. from a c e l l  from a  J  r  F i g u r e 13.  E f f l u x o f sodium from a c e l l i n t o p o t a s s i u m - f r e e a solution, uncorrected f o r versus myoplasmic ce sodium a c t i v i t y .  F i g u r e 14.  E f f l u x o f sodium from a c e l l i n t o p o t a s s i u m - f r e e s o l u t i o n , c o r r e c t e d f o r Na" v e r s u s myoplasmic cell sodium a c t i v i t y .  ® n>  1  l  5  J  F i g u r e 15.  The e f f e c t o f sodium-free  solutions  on  M^  F i g u r e 16.  E f f l u x o f sodium from a c e l l i n t o sodium-free s o l u t i o n , uu n c o r r e c t e d f o r ^ ' ^ > v e r s u s myoplasmic sodium a c t i v i t y .  a<  a  ce  F i g u r e 17.  E f f l u x o f sodium from a c e l l i n t o sodium-free s o l u t i o n , c o r r e c t e d f o r Na* ,., versus myoplasmic j. • • c e l l sodium a c t i v i t y .  Vlll  LIST OF F i g u r e 18.  FIGURES  (cont'd)  E f f l u x o f sodium from a c e l l i n t o normal R i n g e r ' s s o l u t i o n which c o n t a i n s ouabain, versus myoplasmic sodium a c t i v i t y .  ...155  F i g u r e 19.  Summary of r e s u l t s  ...162  F i g u r e 20.  F a l l of the myoplasmic sodium a c t i v i t y upon exposure of the c e l l to sodium-free l i t h i u m substituted solution.  ...175  S i z e of the r a p i d f a l l i n the myoplasmic sodium a c t i v i t y upon exposure o f the c e l l to sodiumfree lithium-substituted s o l u t i o n .  ...177  Rate of f a l l o f the myoplasmic sodium a c t i v i t y immediately a f t e r exposure o f the c e l l to sodiumf r e e s o l u t i o n , versus the myoplasmic sodium a c t i v i t y .  ...178  R a t i o o f the sodium e f f l u x to the r a t e o f l o s s o f sodium from the myoplasm, f o r e f f l u x i n t o sodiumfree s o l u t i o n .  ...182  Change i n membrane p o t e n t i a l on exposure o f the c e l l to p o t a s s i u m - f r e e or o u a b a i n - c o n t a i n i n g solution.  ...190  R e l a t i o n s h i p between the change i n membrane p o t e n t i a l and the change i n sodium e f f l u x which o c c u r i n response to ouabain.  ...193  R e s t i n g membrane p o t e n t i a l o f c e l l s loaded w i t h sodium by m i c r o i n j e c t i o n , v e r s u s myoplasmic sodium a c t i v i t y .  ...195  Response o f membrane p o t e n t i a l and Ringer's s o l u t i o n .  pH^  ...207  Response of membrane p o t e n t i a l Ringer's s o l u t i o n .  pH.  F i g u r e 21.  F i g u r e 22.  F i g u r e 23.  F i g u r e 24.  F i g u r e 25.  F i g u r e 26.  F i g u r e 27.  F i g u r e 28.  F i g u r e 29.  Relationship pH  and  the  F i g u r e 31.  Uptake of i n d i c a t o r solution.  F i g u r e 34.  to  C02  NH^...208 of  transmembrane d i s t r i b u t i o n of DMO.  ...211 ...220  compounds i n normal R i n g e r ' s ...224  Uptake of i n d i c a t o r compounds i n CO^ solution.  Ringer's  Uptake o f i n d i c a t o r solution.  Ringer's  Correlation  _  r e s t i n g membrane p o t e n t i a l .  Model of the  F i g u r e 33.  and  to  between transmembrane g r a d i e n t  F i g u r e 30.  F i g u r e 32.  f o r e f f l u x experiments.  compounds i n NH^  between pH(DMO) and  .. .226  . . .227 pH(electrode).  ...229  LIST OF SYMBOLS A ( ^ ) a  a  c(x) cpm dpm E m F  m  2 a r e a o f membrane (cm ) myoplasmic sodium a c t i v i t y  Page f i r s t us (mM)  c o n c e n t r a t i o n o f c a t i o n a t d i s t a n c e x from o r i g i n counts p e r minute o f r a d i o a c t i v i t y d i s i n t e g r a t i o n s per minute o f r a d i o a c t i v i t y membrane p o t e n t i a l ( m i l l i v o l t s ) charge o f a mole o f e l e c t r o n s (96,520 coulomb/mole) c o n d u c t i v i t y t o potassium ions (coulomb2/joule-cm^-sec)  I  c u r r e n t o f potassium ions (coulomb/cm^-sec) K. j(x) i o n f l u x a t d i s t a n c e x from o r i g i n ; j^=passive, j = a c t i v e k r a t e c o n s t a n t ( u n i t s depend on context, u s u a l l y min"-'-) M„ u n i d i r e c t i o n a l sodium e f f l u x (mole/cm2-sec) Na M n e t c a t i o n e f f l u x i n e l e c t r o g e n i c t r a n s p o r t (mole/cm -sec] m „ ,m„,m • n e t p a s s i v e f l u x a c r o s s membrane (mole/cm^-sec) Na K C l mc m i l l i c u r i e of r a d i o a c t i v i t y mV millivolt "k 22 Na moles Na c o l l e c t e d i n 5 min i n an e f f l u x experiment * 22 Na „ moles Na i n s i d e the c e l l cell * 22 Na .moles Na i n s o l u t i o n i n the myoplasm m Na ,, moles Na i n s i d e the c e l l cell (Na) , = Ba , ., /V : apparent c o n c e n t r a t i o n o f Na i n c e l l 'cell cell o.d. o u t s i d e diameter (mm o r |j) pes picomoles/cm^-sec P^ p e r m e a b i l i t y o f membrane to s p e c i e s x (cm/sec) m  2  2  3  r  (R. c o u p l i n g r a t i o o f o u a b a i n - s e n s i t i v e Na-K exchange R gas c o n s t a n t per mole (8.3 x lO'' erg/mole-°K) SA s p e c i f i c a c t i v i t y o f sodium Slope R a t i o T a b s o l u t e temperature (°K) t time u m o b i l i t y (erg-cm/mole-sec) U numerator o f l n term o f Goldman-Hodgkin-Katz e q u a t i o n V volume o f r e g i o n i n d i c a t e d by s u b s c r i p t W denominator o f l n term o f Goldman-Hodgkin-Katz e q u a t i o n z valence o f i o n 6 p a r t i t i o n c o e f f i c i e n t between s o l u t i o n and membrane y mean i o n i c a c t i v i t y c o e f f i c i e n t +  X "j u pi $ ( )  microlitre e l e c t r o c h e m i c a l p o t e n t i a l (erg/mole) micrometre microlitre e l e c t r i c a l p o t e n t i a l (joule/coulomb) concentration (mole/litre)  subscripts: i = intracellular o,e = e x t r a c e l l u l a r m = myoplasmic  X  ACKNOWLEDGEMENTS  The work d e s c r i b e d  i n this  t h e s i s was c a r r i e d out i n 1973-1976 as p a r t  of a combined M.D.-Ph.D. program. Dr. J.A.M. Hinke f o r the a d v i c e course o f t h i s program. Dr. V. P a l a t y  I w i s h t o thank my t h e s i s  and encouragement  supervisor  he o f f e r e d throughout the  I a l s o wish t o thank Dr. S.M. Friedman and  f o r t h e i r a s s i s t a n c e , and f o r the support they p r o v i d e d  Dr. Hinke moved to the U n i v e r s i t y o f Ottawa,,  Skilled  after  technical assistance  w i t h the DMO experiments and some o f the flame photometry was p r o v i d e d by Ms. Edwina Nee Wong and Mr. L a u r i e N i c o l .  1  SECTION 1.  A.  GENERAL INTRODUCTION  SCOPE OF THE THESIS  A p r i n c i p a l f u n c t i o n o f the c e l l and  molecules.  membrane i s the t r a n s l o c a t i o n o f ions  I t has been e s t i m a t e d t h a t between o n e - f i f t h and o n e - t h i r d  o f the r e s t i n g energy p r o d u c t i o n which t r a n s p o r t s  o f the c e l l  i s devoted t o the mechanism  sodium and potassium ions a l o n e ( B r i n l e y & M u l l i n s 1968;  Whittam 1975; but see Chinet,  Clausen, & G i r a r d i e r 1977).  The transmembrane  d i s t r i b u t i o n o f ions and molecules i s f a r d i f f e r e n t from t h a t which would occur i f they a l l were i n e q u i l i b r i u m . medium i s poor i n s o l u b l e o r g a n i c medium.  Altogether,  there  I n p a r t i c u l a r , the e x t r a c e l l u l a r  molecules r e l a t i v e t o the i n t r a c e l l u l a r  i s always present  water and e l e c t r o l y t e s i n t o t h e c e l l .  a f o r c e which tends t o move  For c e l l s which l a c k r i g i d  walls,  the amount o f o s m o t i c a l l y a c t i v e i n t r a c e l l u l a r s o l u t e must be r e g u l a t e d i f osmotic l y s i s The  i s t o be prevented and the c e l l  a c t i v e e x t r u s i o n o f sodium i s f e l t  content o f the c e l l The  t o be the major c o n t r o l o f the water  (eg. T o s t e s o n 1964; MacKnight & L e a f 1977).  transmembrane d i s t r i b u t i o n o f sodium i s a l s o a s t o r e o f energy,  s u i t a b l e f o r u t i l i z a t i o n by e n e r g y - r e q u i r i n g c e l l membrane.  order  r e a c t i o n s and processes a t the  Important examples a r e those processes which e f f e c t t r a n s -  p o r t o f substances a c r o s s transmit  i s t o be enabled t o e x i s t .  the c e l l membrane, and those which r a p i d l y  s i g n a l s t o another p a r t o f t h e c e l l  or to a d i f f e r e n t c e l l i n  t o t r i g g e r chemical r e a c t i o n s . Of course, whatever the other  functions  i t serves,  the r e g u l a t e d  c o m p o s i t i o n o f the i n t e r i o r o f the c e l l appears t o be r e q u i r e d  ionic  f o r the  e f f e c t i v e f u n c t i o n i n g o f the m e t a b o l i c machinery o f the c e l l . This  t h e s i s i s concerned w i t h the e x p e r i m e n t a l measurement o f i o n  2  t r a n s p o r t , and w i t h ions  c e r t a i n aspects  o f the t r a n s p o r t of sodium and  i n whole c e l l s o f s t r i a t e d muscle.  i s much more s t r a i g h t f o r w a r d w i t h whole c e l l s .  However, w i t h  t h a t i n t a c t membranes can be  techniques,  isolated  the t r a n s p o r t p r o p e r t i e s o f these two  and  data  than w i t h  i t i s o n l y from a few  f o r study: red blood c e l l s ,  g i a n t axons such as t h a t o f the s q u i d .  differences.  Interpretation of transport  i s o l a t e d membrane p r e p a r a t i o n s  current  hydrogen  and  There are many s i m i l a r i t i e s c e l l types,  but  cells  between  t h e r e are a l s o many  Of n e c e s s i t y , then, whole c e l l s must be examined so the n a t u r e  importance of the v a r i o u s  t r a n s p o r t processes  can be  discovered.  In a d d i t i o n , t h e r e are p o s i t i v e reasons f o r examining membrane t r a n s p o r t p r o p e r t i e s i n whole c e l l s .  The  c e l l s of a s p e c i a l i z e d t i s s u e w i l l  possess a r e p e r t o i r e o f t r a n s p o r t mechanisms s u i t a b l e f o r the t i s s u e ' s function.  A p a r t i c u l a r t r a n s p o r t mechanism, present  might be prominent i n a p a r t i c u l a r c e l l  type and  i n most c e l l  types,  so be more e a s i l y  studied  there. The o n l y be  chemical  s p e c i e s which mediates a g i v e n aspect  identified  in isolation  i f i t s behavior  must be deduced from study o f the behavior d e t a i l e d p r o p e r t i e s o f an plays  of ion transport  i s known.  This  o f the whole c e l l .  behavior Once the  i d e n t i f i e d s p e c i e s are known, the p a r t t h a t  i n the complete t r a n s p o r t system o f the c e l l can be deduced.  behavior  due  to other  to i s o l a t e them.  Eventually  to ion transport w i l l concert  t r a n s p o r t s p e c i e s can be deduced and  can  species  Then the  an attempt made  i t i s hoped t h a t a l l s p e c i e s which c o n t r i b u t e  be c h a r a c t e r i z e d , and  t h e i r behavior  when a c t i n g i n  understood.  F i n a l l y , abnormalities  o f the t r a n s p o r t systems can be a cause o f or a  f e a t u r e of pathology of the t i s s u e ( B o l i s , Hoffman, &•Leaf 1976). o t h e r areas of p h y s i o l o g y , mechanisms can  a d e t a i l e d knowledge of c e l l u l a r  l e a d to the f o r m u l a t i o n  As  with  transport  o f a r a t i o n a l treatment p l a n .  An  3  example i s the u s e i n the treatment o f c h o l e r a o f one t r a n s p o r t mechanism to bypass another which i s deranged ( F i e l d  1977).  There a r e many reasons, then, why the c a p a b i l i t y to study t r a n s p o r t i n whole c e l l s and m u l t i c e l l u l a r p r e p a r a t i o n s Several  should  be developed.  problems a r e addressed i n t h i s t h e s i s .  The f i r s t  i s the t e c h -  n i c a l problem o f measuring the trans-membrane f l u x i n whole c e l l s . main concern i s the measurement o f sodium f l u x e s . hydrogen i o n f l u x e s p r e s e n t s p r a c t i c a l nature. In order  The  The measurement o f  d i f f e r e n t problems, o f both a c o n c e p t u a l  and a  I t i s o f secondary concern here.  t o r e s o l v e the t e c h n i c a l problem o f measuring the f l u x , an  i n v e s t i g a t i o n o f the s t a t e s o f water and ions c a r r i e d out.  The  This  i n s i d e the c e l l had t o be  i s an a c t i v e area o f r e s e a r c h  i n i t s own r i g h t .  t e c h n i q u e developed f o r f l u x measurement, which i n v o l v e s  neous use o f an i o n - s p e c i f i c i n t r a c e l l u l a r m i c r o e l e c t r o d e  simulta-  and r a d i o i s o t o p e s ,  was a p p l i e d t o a b r i e f overview o f the k i n e t i c s o f sodium t r a n s p o r t  ina  whole c e l l . Then, two s p e c i f i c problems were i n v e s t i g a t e d : the sodium e f f l u x i n t o sodium-free s o l u t i o n s , o f the type p r e v i o u s l y seen i n f r o g s k e l e t a l muscle; and  the e l e c t r o g e n i c properties Finally,  and  o f t h e sodium t r a n s p o r t .  i n d i r e c t measurements o f t h e e x t r u s i o n o f a c i d by the c e l l  o f the h e t e r o g e n e i t y  o f the i n t r a c e l l u l a r pH were made.  In t h i s  the use o f an i n d i c a t o r f o r the measurement o f pH was e v a l u a t e d . is a question The  cell  o f great  The l a t t e r  practical interest.  chosen f o r t h i s work i s t h e v e r y  of the g i a n t b a r n a c l e  context,  Balanus n u b i l u s .  Darwin i n 1854, but i t was o n l y neuromuscular p h y s i o l o g y  This crustacean  cell  was d e s c r i b e d by  i n 1963 t h a t Hoyle and Smyth d e s c r i b e d i t s  and suggested t h a t  t i o n f o r f u r t h e r such study.  l a r g e s t r i a t e d muscle  Since  i t would be a v a l u a b l e  then,, work has been p u b l i s h e d  preparaon i t s  4  u l t r a s t r u c t u r e , on the s t a t e s of i t s water and and  e l e c t r i c a l properties  i o n s , on the  o f i t s membranes, and  on  i t s ion  permeability transport  mechanisms. Its  l a r g e s i z e makes i t e s p e c i a l l y s u i t a b l e f o r impalement by  electrodes I t was  (which i t t o l e r a t e s f o r long p e r i o d s )  d e s i r e d t o use  t i v e l y and  B.  for microinjection.  these techniques to sample the c e l l  to l o a d the c e l l  be e x p l a i n e d  and  more f u l l y  interior selec-  i n t e r i o r with radioisotope s e l e c t i v e l y ,  as  will  later.  HISTORICAL NOTES  The  study o f the movement o f substances  as o l d as  the c e l l  theory  l i g h t microscope, but demonstrated.  itself.  The  1855).  i n t o and  out  of c e l l s  i s almost  c e l l membrane cannot be seen w i t h  permeation o f s o l u t e s and  i n f e r r e d the presence of a  permeability  ( N a g e l i & Cramer  P f e f f e r (1877) proposed from h i s work w i t h a r t i f i c i a l  semipermeable  films that a f i l m with s i m i l a r properties  surrounded the c e l l .  (1899) measured the p e r m e a b i l i t y  to many substances, and  of c e l l s  t h a t a l a y e r o f l i p i d on the s u r f a c e of the c e l l was  Overton proposed  the p r i n c i p a l b a r r i e r  penetration. I t was  the p l a n t p h y s i o l o g i s t s who  single cells. substances, and respect  l e d the way  i n these s t u d i e s  on  They found t h a t p l a n t c e l l s a c t u a l l y accumulated c e r t a i n seemed to e x i s t i n a  (Hoagland & Davis 1929;  the Donnan e f f e c t as  'non-equilibrium  Brooks 1929).  the  osmotic e f f e c t s are r e a d i l y  N a g e l i , a student o f S c h l e i d e n ' s ,  c e l l membrane from h i s s t u d i e s of p l a n t c e l l  to  micro-  condition' i n this  Osterhout (1931) r e j e c t e d  the cause o f the a c c u m u l a t i o n o f ions, and  proposed  5  t h a t the continuous p r o d u c t i o n  o f a c i d by t h e c e l l  l e d t o the p a s s i v e  inflow  o f p o t a s s i u m and c h l o r i d e . Brooks (1938) appears t o have been the f i r s t t o employ r a d i o i s o t o p e s i n 1 the study o f i o n a c c u m u l a t i o n by i n d i v i d u a l c e l l s .  He employed a r a d i o i s o -  tope o f p o t a s s i u m t o q u a n t i t a t e t h e a c c u m u l a t i o n o f p o t a s s i u m by a c e l l , and expressed t h e i n t r a c e l l u l a r c o n c e n t r a t i o n  i n terms o f the t o t a l c e l l  water  (the d i f f e r e n c e between the wet and d r y weights o f the 'protoplasm').  He  observed a r a p i d p e n e t r a t i o n o f potassium a g a i n s t the g r a d i e n t o f p o t a s s i u m concentration.  He a t t r i b u t e d t h i s t o i o n exchange (Brooks 1940).  (1940) noted t h a t the p o t a s s i u m a c c u m u l a t i o n t h e o r i e s then c u r r e n t low  permeability  trates the c e l l  t o sodium, w h i l e quite r e a d i l y .  mechanism p r e s e n t plasm."  He f e l t  "physico-chemical  The  -He remarked t h a t " t h e r e must be some  f o r pumping out t h e sodium which wanders i n t o the p r o t o t h a t the i o n d i s t r i b u t i o n as a whole was due t o a balance between t h e p r o t o p l a s m and the medium, w i t h the  o f a r a d i o i s o t o p e o f sodium was measured by L e v i  (1949) and l a t e r by o t h e r s .  which should  be obeyed by p a s s i v e  that according nerve  subordinate  role."  e f f l u x from c e l l s  and U s s i n g  required  experiments had shown t h a t sodium pene-  p e r m e a b i l i t y c h a r a c t e r i s t i c s o f the membrane p l a y i n g o n l y a structural  Steinbach  Ussing  fluxes.  to Ussing's r e l a t i o n ,  (1949) d e r i v e d a r e l a t i o n  Hodgkin and Keynes (1954) found  sodium was a c t i v e l y e x p e l l e d  from  cells.  """The f i r s t use o f r a d i o i s o t o p e i n uptake s t u d i e s was much e a r l i e r . Hevesy (1923) measured the uptake by p l a n t s o f an i s o t o p e o f l e a d o b t a i n e d as a n a t u r a l breakdown p r o d u c t , o f thorium. The use o f r a d i o i s o t o p e s became more common a f t e r the development o f the c y c l o t r o n and r a d i o a c t i v a t i o n i n about 1936. I t was E.O. Lawrence o f the U n i v e r s i t y o f C a l i f o r n i a at B e r k e l e y and N i e l s Bohr o f t h e I n s t i t u t e o f T h e o r e t i c a l P h y s i c s i n Copenhagen who s u p p l i e d l o c a l p h y s i o l o g i s t s w i t h r a d i o i s o t o p e s o f phosphorous and potassium.  6  Keynes and Lewis (1951) e x p l i c i t l y animal c e l l  formulated  the 'bag model' of the  f o r f l u x s t u d i e s , wherein the i n t r a c e l l u l a r r e g i o n was  to comprise a s i n g l e homogeneous compartment w i t h i n a c l o s e d permeable membrane.  assumed  selectively-  The r e s u l t s o f t h e i r experiments on s q u i d axon seemed  to be c o n s i s t e n t w i t h t h i s  formulation,  but the r e s u l t s  f o r muscle c e l l s  were more d i f f i c u l t  to i n t e r p r e t .  c a t e d models h a v i n g  s e v e r a l c e l l u l a r compartments among which ions can move  ( f o r example, Keynes & S t e i n h a r d t  The t r e n d has been to employ more compli-  1968; Rogus & Z i e r l e r 1973).  An enzymatic b a s i s f o r the a c t i v e t r a n s p o r t o f sodium and potassium across  the c e l l membrane was d i s c o v e r e d by Skou (1957) i n the form o f a  sodium- and p o t a s s i u m - a c t i v a t e d ,  magnesium-dependent  phosphohydrolase (the (Na + K)ATPase).  triphosphate  T h i s enzyme has come t o be c a l l e d  "the sodium pump" (Glynn & K a r l i s h 1975).  Other mechanisms  p o r t o f sodium have been p o s t u l a t e d , as w i l l mechanisms  adenosine  be d i s c u s s e d  f o r the t r a n s -  l a t e r , and many  f o r the t r a n s p o r t o f o t h e r s p e c i e s have been p o s t u l a t e d .  The a b i l i t y o f t i s s u e s to generate an e l e c t r i c p o t e n t i a l d i f f e r e n c e has been r e c o g n i z e d Reymond 1843). and  f o r w e l l over a hundred years  ( M a t t e u c c i 1840; Du B o i s -  The e a r l y work i n v o l v e d r a t h e r gross  the p o t e n t i a l d i f f e r e n c e s were c a l l e d  i n j u r y to the t i s s u e s ,  'injury potentials'.  They were  thought to be due to the f r e e i n g o f i n o r g a n i c ions through chemical i n the i n j u r e d t i s s u e .  The e q u i l i b r i u m theory o f Donnan (1910) p r o v i d e d  one model f o r the o r i g i n o f 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 membrane, w h i l e the t h e o r e t i c a l d e s c r i p t i o n by T e o r e l l Sievers  the c e l l  (1935) and Meyer and  (1936) o f the p o t e n t i a l d i f f e r e n c e a t the boundary between  s o l u t i o n s o f d i f f e r e n t composition Osterhout  reactions  or c o n c e n t r a t i o n p r o v i d e d  two  another.  (1931) measured e l e c t r i c a l p o t e n t i a l d i f f e r e n c e s a c r o s s the  'protoplasm' of s i n g l e p l a n t c e l l s ,  and formulated  as d i f f u s i o n p o t e n t i a l s c h i e f l y due to potassium.  a model f o r t h e i r He f e l t  t h a t phase  origin  7  boundary p o t e n t i a l s for  and the Donnan p o t e n t i a l would be too s m a l l t o account  h i s measured v a l u e s , and that o x i d a t i o n - r e d u c t i o n  be measured w i t h h i s apparatus. and  Henderson d e s c r i b i n g  tial  differences  that  diffusion potentials  the equations o f Nernst  "enable us t o p r e d i c t  w i t h s u f f i c i e n t a c c u r a c y t o j u s t i f y t h e i r use q u i t e  from a l l o t h e r c o n s i d e r a t i o n s . " ities  He s t a t e d  p o t e n t i a l s would n o t  as the key f e a t u r e  He i d e n t i f i e d d i f f e r e n c e s  of d i f f u s i o n potentials,  model by measuring the m o b i l i t i e s o f the ions  potenaside  i n i o n i c mobil-  and s e t . o u t t o t e s t the  i n the "nonaqueous  layers"  (membranes) o f c e l l s . In the t h e o r e t i c a l d e s c r i p t i o n and  Sievers  formulated by T e o r e l l  (1936) f o r the c o n c e n t r a t i o n p o t e n t i a l developed a c r o s s  membranes s e p a r a t i n g  two e l e c t r o l y t e s o l u t i o n s , therewas a Donnan p o t e n t i a l  a t each i n t e r f a c e and a d i f f u s i o n p o t e n t i a l S t e i n b a c h (1940) noted t h a t requires  (1935) and Meyer  i n the membrane.  the maintenance o f a d i f f u s i o n p o t e n t i a l  " c o n t i n u e d p r o d u c t i o n o f e l e c t r o l y t e s , and as such i s l i n k e d t o  the metabolism o f the c e l l . " B o y l e and Conway (1941) a n a l y z e d the a c c u m u l a t i o n o f potassium by muscle and concluded t h a t sodium p e r m e a b i l i t y  i t must be due t o a Donnan e q u i l i b r i u m ,  w h i l e the  o f the muscle c e l l membrane must be extremely low.  Goldman (1943) a p p l i e d  the theory o f d i f f u s i o n p o t e n t i a l s  to s i m p l i f i e d  models o f the c e l l membrane, and o b t a i n e d a good q u a l i t a t i v e d e s c r i p t i o n o f the r e c t i f i c a t i o n and membrane p o t e n t i a l  i n squid  axon.  Hodgkin and Katz (1949) used Goldman's equations under the assumption that  the r e s t i n g s q u i d  axon membrane was more permeable t o potassium than  to sodium, w h i l e the sodium p e r m e a b i l i t y  could  increase  g r e a t l y to bring  about the r e v e r s a l o f p o l a r i z a t i o n o f the membrane which occurs d u r i n g an action potential.  The passage o f sodium a c r o s s the membrane was proposed  to occur " i n combination w i t h a l i p o i d - s o l u b l e c a r r i e r i n the membrane  8  which i s o n l y  f r e e t o move when the membrane i s d e p o l a r i z e d . "  regarded t h e i r e x p r e s s i o n  f o r the v a l u e  more than a rough a p p r o x i m a t i o n . "  They  o f the membrane p o t e n t i a l as "no  However, i t was s u f f i c i e n t l y simple and  f l e x i b l e that  i t c o u l d be a p p l i e d t o almost any r e s u l t under q u i t e r e a s o n a b l e  assumptions.  Goldman's e q u a t i o n and v a r i a t i o n s o f i t c o n t i n u e t o be used t o  describe  the membrane p o t e n t i a l (eg. Schwartz 1971).  C e l l water came under s c r u t i n y v e r y of c e l l s felt  e a r l y on, because o f the f a i l u r e  t o a c t as p e r f e c t osmometers ( f o r example, Overton 1902).  t h a t "a c o n s i d e r a b l e  I t was  p o r t i o n o f the water i n the c e l l o r body i s  physically  'bound' i n the c o l l o i d a l s t r u c t u r e o f the p r o t o p l a s m and must be  considered  an i n t e g r a l p a r t o f the l i v i n g system" (Sharp 1934).  The  presence o f 'bound i o n s ' was i n d i c a t e d by t h e presence o f s l o w l y - e x c h a n g i n g fractions  i n i o n uptake and d e p l e t i o n s t u d i e s .  i o n - s p e c i f i c electrodes  small  & Whitaker 1927; C a l d w e l l  With t h e c o n s t r u c t i o n o f  enough t o be p l a c e d  into single c e l l s  (Taylor  1954; Hinke 1959; Walker 1971) i t became p o s s i b l e  to study the i n t e r i o r o f the c e l l d i r e c t l y , and i t was apparent t h a t not all  o f the ions measured by chemical a n a l y s i s o f the c e l l were present i n  f r e e s o l u t i o n i n s i d e the c e l l . asymmetrical  Some workers have concluded t h a t the  i o n d i s t r i b u t i o n s and osmotic b e h a v i o r o f the c e l l a r e due t o  the a s s o c i a t i o n o f the ions w i t h f i x e d charge groups i n c y t o p l a s m i c macromolecules and t o o r g a n i z a t i o n o f the c e l l water, w i t h the membrane p l a y i n g only a passive Ochsenfeld  r o l e ( T r o s c h i n 1961; L i n g 1962; L i n g , M i l l e r &  1973).  A problem addressed by many present-day i n v e s t i g a t o r s i s the e l u c i d a t i o n o f the d e t a i l e d mechanism o f i o n t r a n s p o r t  in cells.  Most a t t e n t i o n  i s a d d r e s s e d t o the c e l l membrane, but f o r the i n t e r p r e t a t i o n o f s t u d i e s o f the t r a n s p o r t p r o p e r t i e s o f the membrane i n whole c e l l s ,  i t i s necessary t o  9  characterize  the s t a t e s o f the  so i s d e s c r i b e d  i n the next  i n t r a c e l l u l a r water and  section.  ions.  Why  this  is  10  SECTION 2.  A.  TRANS-MEMBRANE FLUXES OF SODIUM AND HYDROGEN IONS  GENERAL CONSIDERATIONS  Almost a l l o f the observed passage o f sodium ions and indeed o f a l l l i p i d - i n s o l u b l e i n o r g a n i c ions a c r o s s the c e l l membrane p r o t e i n s . sodium  membrane i s a s s o c i a t e d w i t h  The p e r m e a b i l i t y o f a pure p h o s p h o l i p i d b i l a y e r t o  i s s e v e r a l o r d e r s o f magnitude lower than t h a t o f a c e l l  ( J a i n 1972; Lauger & Neumcke 1973).  membrane  The t r a n s l o c a t i o n o f the ions can thus  be regarded as an enzyme-mediated r e a c t i o n i n which one product i s the translocated ion. The e f f l u x o f sodium from c e l l s n o r m a l l y i n v o l v e s a c o n s i d e r a b l e i n c r e a s e i n the e l e c t r o c h e m i c a l p o t e n t i a l o f the t r a n s l o c a t e d ions, and so r e q u i r e s energy.  T h i s energy must come u l t i m a t e l y from metabolism.  (Exchange and the p a s s i v e u n i d i r e c t i o n a l  f l u x a r e c o n s i d e r e d below.)  In  theory, the d i r e c t source o f energy f o r sodium e f f l u x c o u l d be o t h e r ions (or  sodium) which pass spontaneously t o a r e g i o n o f lower  electrochemical  p o t e n t i a l , o r i t c o u l d be the h y d r o l y s i s o f 'high-energy' phosphate compounds or the  o t h e r p r o d u c t s o f metabolism.  A mechanism i n v o l v i n g the cytochromes o f  e l e c t r o n t r a n s p o r t system d i r e c t l y has a l s o been proposed  (Mitchell  1969). Experiments have i n d i c a t e d t h a t most and perhaps a l l o f the metabolismdependent for  energy  sodium e f f l u x depends d i r e c t l y on adenosine t r i p h o s p h a t e (ATP) (Dunham 1957; Whittam 1958; C a l d w e l l 1960; Hoffman  1960).  I n o s i n e t r i p h o s p h a t e (ITP), guanosine t r i p h o s p h a t e (GTP), u r i d i n e  triphos-  phate (UTP), p h o s p h o a r g i n i n e (PA), c y t i d i n e t r i p h o s p h a t e (CTP), a c e t y l phosphate  (AcP), phospho(enol) p y r u v a t e (PEP), D-glyceraldehyde-3-phosphate  (G-3-P), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) do  11  not support sodium e f f l u x , w h i l e deoxyadenosine t r i p h o s p h a t e supports about h a l f o f the normal sodium e f f l u x  (d-ATP)  (Hoffman 1960; B r i n l e y &  M u l l ins 1968). When the ATP c o n c e n t r a t i o n i n s q u i d axon i s reduced t o v e r y low l e v e l s by i n t e r n a l d i a l y s i s ,  the sodium e f f l u x approaches the r a t e expected from  p a s s i v e mechanisms ( B r i n l e y & M u l l i n s 1968). c y a n i d e (2 mM.  CN f o r 1 - 3  I f the axon i s t r e a t e d w i t h  hours) and then d i a l y z e d ,  the ATP c o n c e n t r a t i o n  i s reduced t o about 2 JAM. and t h e sodium e f f l u x i s reduced t o the v a l u e e s t i m a t e d f o r p a s s i v e movement ( B r i n l e y & M u l l i n s 1967). little  immediate e f f e c t on t h e sodium e f f l u x  & Keynes  CN a l o n e has  (Keynes & M a i s a l 1954; Hodgkin  1956; Carey, Conway, 6c Kernan 1959).  An ATPase  found i n the c e l l membrane and a c t i v a t e d by sodium and  p o t a s s i u m has come t o be regarded as being l a r g e l y s i b l e f o r the metabolism-dependent sodium e f f l u x  i f not e n t i r e l y respon-  (Skou 1965), a t l e a s t i n  1 red blood c e l l s . (Glynn & K a r l i s h  I t i s c u r r e n t l y b e i n g r e f e r r e d t o as " t h e " sodium pump 1975).  F i v e modes o f b e h a v i o r have been d e s c r i b e d f o r t h i s  (Na+K)ATPase:  ( i ) exchange o f i n t e r n a l sodium f o r e x t e r n a l potassium, r e q u i r i n g ATP and accompanied by a n e t h y d r o l y s i s o f ATP; ( i i )  reversal of ( i ) ;  (iii)  exchange  o f i n t e r n a l sodium f o r e x t e r n a l sodium, r e q u i r i n g ATP and ADP but accom-  ^Sodium has a l s o been a l l e g e d t o be i n v o l v e d i n the e f f l u x o f c a l c i u m from n e r v e and muscle, where i n t e r n a l c a l c i u m i s exchanged f o r e x t e r n a l sodium w i t h no n e t h y d r o l y s i s o f ATP (Baker 1972; Requena, D i P o l o , B r i n l e y , & M u l l i n s 1977), and i n the e f f l u x o f magnesium, where once a g a i n sodium e n t e r s and t h e r e i s no n e t h y d r o l y s i s o f ATP (Baker 6e Crawford 1972; A s h l e y 6c E l l o r y 1972; M u l l i n s , B r i n l e y , Spangler, 6c Abercrombie 1977). Sodium e f f l u x a p p a r e n t l y i s a s s o c i a t e d w i t h the t r a n s p o r t o f sugars ( S c h u l t z 6e C u r r a n 1970; Kimmich 1972) and amino a c i d s ( C o l o m b i n i 6c Johnstone 1974; Johnstone 1974). A sodium-hydrogen exchange has been suggested but i s not c l e a r (Keynes " 1965; B i r o 1965; B i t t a r e t a l . 1973, 1976). An a s s o c i a t i o n o f sodium and i o d i d e t r a n s p o r t has a l s o been suggested (Skou 1965).  12  panied by no net h y d r o l y s i s o f ATP; (a s e p a r a t e sodium-sodium found i n nerve and muscle i s d i s c u s s e d l a t e r ;  exchange  i t i s thought t o comprise as  much as h a l f o f the sodium e f f l u x in. muscle c e l l s and i s i n s e n s i t i v e t o inhibitors);  ( i v ) exchange o f i n t e r n a l p o t a s s i u m f o r e x t e r n a l potassium,  r e q u i r i n g i n o r g a n i c phosphate and ATP but accompanied by no n e t h y d r o l y s i s of ATP; t h i s a p p a r e n t l y i s a s s o c i a t e d w i t h o p e r a t i o n i n mode ( i ) ;  and (v)  e f f l u x o f sodium w i t h o u t c o u p l i n g t o i n f l u x o f another i o n , r e q u i r i n g ATP and p r o b a b l y accompanied by a n e t h y d r o l y s i s o f ATP ( o r much l e s s tively,  CTP, ITP, o r UTP: t h i s mode i s much l e s s  others)  (Glynn & K a r l i s h 1974, 1975).  effec-  f a s t i d i o u s than the  The c o n t r i b u t i o n o f a g i v e n mode o f the (Na+K)ATPase o r o f o t h e r t r a n s p o r t mechanisms t o the n e t f l u x might be d i f f i c u l t  t o deduce from simple  experiments, s i n c e a l t e r a t i o n o f the s u b s t r a t e c o n c e n t r a t i o n s s h o u l d cause a r e d i s t r i b u t i o n among a l l  o f the modes, r a t h e r than s i m p l y  an a l t e r a t i o n o f the c o n t r i b u t i o n o f a s i n g l e mode. literature  (ions and ATP)  An example  from the  i s . t h a t i n the absence but n o t i n the presence o f e x t e r n a l  potassium, both the i n f l u x and e f f l u x o f sodium i n f r o g s k e l e t a l muscle a r e reduced by ouabain (Keynes & S t e i n h a r d t 1968).  That i s , removal o f e x t e r n a l  p o t a s s i u m d i s a b l e s the sodium-potassium exchange but unmasks a ouabains e n s i t i v e sodium-sodium exchange n o t apparent i n s o l u t i o n s which c o n t a i n potassium.  T h i s type o f o c c u r r e n c e must be acknowledged  i n the i n t e r p r e t a -  t i o n o f experiments. Nevertheless,  experiments i n which the i n t r a - o r e x t r a c e l l u l a r  sodium  or p o t a s s i u m i o n c o n c e n t r a t i o n i s a l t e r e d and the e f f e c t on the sodium e f f l u x i s observed a r e o f v a l u e i n t h e study o f the sodium e f f l u x .  Results  from such experiments and v a r i a t i o n s on them a r e known f o r many c e l l s , and by c o n s i d e r a t i o n o f t h i s abundance  o f d a t a i t i s hoped t h a t t h e g e n e r a l  f e a t u r e s o f the d i f f e r e n t routes o f sodium e f f l u x can be deduced.  13  L i k e the e f f l u x o f sodium, t h e e f f l u x o f hydrogen ions from c e l l s i n v o l v e s an i n c r e a s e  i n the electrochemical  ions, and so r e q u i r e s  energy.  v i n c i n g evidence f o r s p e c i f i c  p o t e n t i a l o f the t r a n s l o c a t e d  I t i s o n l y r e l a t i v e l y r e c e n t l y t h a t cont r a n s p o r t mechanisms has been found.  study o f the e f f l u x mechanism f o r hydrogen ions  i s more d i f f i c u l t  the case f o r sodium because processes e q u i v a l e n t appear t o be o p e r a t i n g , Also,  the c e l l  difficulties Mitchell  i n a d d i t i o n t o the e x p u l s i o n  i s not c o n s e r v a t i v e  with respect  o f a more t e c h n i c a l n a t u r e w i l l  o f protons  than i s o f protons  themselves.  t o hydrogen i o n s .  be d i s c u s s e d  (1969) proposed a model, o r i g i n a l l y  the e l e c t r o n t r a n s p o r t  (Further  later.)  f o r m i t o c h o n d r i a , wherein  system o f a e r o b i c metabolism was embedded i n t h e  membrane, and t h e energy d e r i v e d system was used d i r e c t l y t o expel He  to the expulsion  The  from t h e flow o f e l e c t r o n s protons t o t h e outer  through  this  s i d e o f the membrane.  assumed t h a t t h e r e was a membrane-bound ATPase which had t h e c a p a b i l i t y  of e x p e l l i n g protons as i t hydroly.zed  ATP.  wards' as protons passed i n t o t h e c e l l from ADP and i n o r g a n i c phosphate.  I t was thought to 'run back-  through i t , and ATP was thus formed  This  i s c a l l e d t h e chemiosmotic  hypothesis. Rehm (1972) proposed t h a t t h e a c i d i f i c a t i o n o f the lumen o f the stomach was  due t o an e l e c t r o g e n i c hydrogen i o n pump d i r e c t e d toward t h e lumen.  I n a d d i t i o n , a c h l o r i d e pump was thought t o be d i r e c t e d toward t h e lumen and  a sodium pump toward t h e blood.  A c i d i f i c a t i o n would occur when t h e  hydrogen i o n pump became a c t i v e and t h e sodium pump became i n a c t i v e . Coupling  o f t h e pumps was n o t r i g i d .  The r e s p i r a t o r y c h a i n was invoked  d i r e c t l y t o pump protons, as i n t h e M i t c h e l l model, and i t was thought ATP  was n o t d i r e c t l y  that  involved.  S t o e c k e n i u s and co-workers ( O s t e r h e l t & Stoeckenius 1973; Danon & S t o e c k e n i u s 1974; S t o e c k e n i u s 1976) found t h a t t h e 'purple membrane' o f the  14  bacterium Halobacterium  h a l o b i u m c o u l d e x p e l protons  from the b a c t e r i u m  when exposed t o l i g h t , and t h a t the a c t i o n spectrum f o r ATP p r o d u c t i o n i n response  t o l i g h t was s i m i l a r t o the a b s o r p t i o n spectrum o f the p u r p l e  membrane. conditions.  The b a c t e r i u m c o u l d a l s o expel protons Stoeckenius  i n the dark under a e r o b i c  proposed t h a t the b a c t e r i u m c o n t a i n e d t h r e e membrane-  bound systems c a p a b l e o f e x p e l l i n g p r o t o n s ;  ( i ) a p u r p l e membrane p r o t e i n  which c o u l d e x p e l protons when exposed t o l i g h t ;  ( i i ) a respiratory  chain  which c o u l d e x p e l protons u s i n g the energy from a e r o b i c metabolism; and (iii)  an ATPase which c o u l d e x p e l protons u s i n g the energy from the hydro-  lysis  o f ATP, but which u s u a l l y operated  ATP  i n the o p p o s i t e sense, s y n t h e s i z i n g  from ADP and i n o r g a n i c phosphate u s i n g the energy s t o r e d i n the 'proton  g r a d i e n t ' c r e a t e d by the o t h e r two systems. but  i t p r o v i d e d evidence  per se e x i s t s . of  T h i s seems t o be a unique case,  t h a t an ATPase c a p a b l e o f c a u s i n g p r o t o n t r a n s p o r t  Nature has tended  t o employ extended and improved v e r s i o n s  p r i m i t i v e c e l l u l a r mechanisms i n the more s o p h i s i t i c a t e d c e l l s which  evolved l a t e r .  I t would not be too s u r p r i s i n g i f the 'proton pump' o f  nucleated c e l l s  i s found  t o be b u i l t  on these mechanisms.  A sodium-hydrogen exchange was suggested 1965;  by many workers  (Keynes  B i r o 1965; B i t t a r e t a l . 1973, 1976) but o n l y r e c e n t l y has the  dependence o f t h e a l k a l i n i z a t i o n o f the c e l l  on e x t r a c e l l u l a r sodium been  demonstrated, i n mouse s k e l e t a l muscle c e l l s  ( A i c k i n & Thomas 1977),  neurone (Thomas 1977), and b a r n a c l e muscle c e l l s  snail  (Boron & Ross 1978).  F i n a l l y , a c h l o r i d e - b i c a r b o n a t e exchange has been proposed.  The  exchange o f i n t e r n a l c h l o r i d e f o r e x t e r n a l b i c a r b o n a t e would be e q u i v a l e n t to  the e x t r u s i o n o f HC1, s i n c e a t a g i v e n CC^ t e n s i o n excess  bicarbonate  would q u i c k l y combine w i t h a p r o t o n t o y i e l d c a r b o n i c a c i d and then, c a t a l y s i s by c a r b o n i c anhydrase, water and CC^cell  passively.  under  The CC^ would l e a v e the  S i m i l a r systems had been proposed f o r the c e r e b r o s p i n a l  15  fluid  (eg. review by S i e s j o 1972).  Good e v i d e n c e f o r c h l o r i d e - b i c a r b o n a t e  exchange has been found i n s n a i l neurone (Thomas 1976), s q u i d axon (Boron & DeWeer 1976a), mouse s k e l e t a l muscle ( A i c k i n & Thomas 1977), and b a r n a c l e muscle  (Boron & Roos 1978).  A p a r t i c u l a r mechanism f o r sodium or hydrogen t r a n s p o r t by i t s k i n e t i c b e h a v i o r . kinetics  i s characterized  Much more work has been done on the sodium e f f l u x  than on the p r o t o n e f f l u x k i n e t i c s .  The c o n n e c t i o n between the  e f f l u x and the k i n e t i c s o f each o f the sodium t r a n s p o r t systems w i l l discussed  i n part  (C) o f t h i s  be  section.  I n the g e n e r a l case, the c o n n e c t i o n i s made v i a a chemical r e a c t i o n model  i n which the s u b s t r a t e S binds w i t h an enzyme E t o form a complex ES,  which then d i s s o c i a t e s  i n t o enzyme and products P r , w i t h no back r e a c t i o n : k  k-  1  E + S V  T  s.  ES  2  E + Pr  -1  The o v e r a l l r a t e , and hence the e f f l u x r a t e i f t h i s models mechanism f o r sodium e x t r u s i o n , complex,  (ES).  the dominant  i s p r o p o r t i o n a l to the c o n c e n t r a t i o n o f the  I t i s assumed t h a t the d i s s o c i a t i o n o f t h i s complex to y i e l d  products i s so much slower than t h e - r e a c t i o n s which form i t t h a t the reactions preceding t h i s d i s s o c i a t i o n are e s s e n t i a l l y at e q u i l i b r i u m (steady s t a t e assumption). The r e a c t i o n mechanism f o r the (Na+K)ATPase has been c h a r a c t e r i z e d i n some d e t a i l through the use o f ixi v i t r o p r e p a r a t i o n s o f the enzyme, but a s i m p l i f i e d model w i l l  be adopted here.  i s assumed t o be v a r i a b l e , and the r e s t In t h i s ,  Only one parameter, (S) = (Na), i s c o n c e a l e d i n the r a t e c o n s t a n t s .  the elementary 'Michaelis-Menten' model o f enzyme k i n e t i c s ,  (ES) can be expressed i n terms o f (S) and the e q u i l i b r i u m c o n s t a n t  f o r the  r e a c t i o n which forms the complex, and the o v e r a l l r a t e , eg. o f sodium e f f l u x M^ , a  depends  on (Na) as  16  M  =  M  'Na  / (1 +  k  )  max  For t h r e e sodium ions b i n d i n g s u c c e s s i v e l y to E a t e q u i v a l e n t dent  sites, a realistic  a similar  treatment  model o f the (Na+K)ATPase (Glynne & K a r l i s h  1975),  yields (Na)  (Na)  where k of course has a meaning d i f f e r e n t  (Na)  the r e l a t i o n i s  a different  be d e s c r i b e d l a t e r .  t h a t the e f f e c t i v e number o f b i n d i n g s i t e s  case.  3  ( M u l l i n s & Frumento 1963), where a g a i n k has More c o m p l i c a t e d v e r s i o n s w i l l  (Na)  from t h a t i n the p r e v i o u s  I f the t h r e e sodium ions b i n d s i m u l t a n e o u s l y ,  concentrations,  indepen-  is different  significance. I t i s conceivable at d i f f e r e n t  sodium  f o r example.  I t s h o u l d be noted a t t h i s p o i n t t h a t Baker, B l a u s t e i n , Keynes et al_. (1969) and Garay and Garrahan (1973) appeared to take a d i f f e r e n t i n t h a t they assumed i n s t e a d t h a t t h r e e sodium ions had lent  independent s i t e s on the enzyme, and  to b i n d to e q u i v a -  t h a t the e f f l u x was  proportional  to the f r a c t i o n o f the independent enzyme u n i t s which were f u l l y by t h r e e sodium i o n s . probability  o c c u p i e d w i t h sodium a t one  is  (E) but they took  +  (ES)  +  (ES )  +  2  (ES ) 3  i t to be  (E) for k = (S)(E)/(ES).  occupied  T h i s f r a c t i o n they took to be the cube o f the  of h a v i n g an enzyme u n i t  This p r o b a b i l i t y  approach,  IES1 + (ES)  Since  1  +  k/(S)  site.  17  k  (S)  =  (E)  (S) (ES)  =  (ES)  (S)  (ES )  (ES„)  (ES )  2  3  then (E)  +  (ES)  +  (ES )  +  2  (E) Writing  (ES) =  (ES )  =  3  T l + x§I L k  (S) (E) / k, one  +  (  X§1 ) k  2  +  sees t h a t the p r o b a b i l i t y  ( isi  )  k  3 J  is actually  1 k (S) f o r h a v i n g one  +  1  <£t  +  k  +  ( <£L " k  )  2  s i t e o c c u p i e d by sodium, w h i l e the p r o b a b i l i t y o f h a v i n g a l l  three s i t e s occupied i s  (ES3) (E)  +  (ES)  +  (ES )  +  2  (ES ) 3  1 1  +  JL. (S)  or j u s t as f o r the M i c h a e l is-Menten had  +  ( JL_  (S)  )  +  2  ( Ji_ )  3  (S)  case f o r t h r e e e q u i v a l e n t s i t e s .  to be so, of course, s i n c e the assumptions were e q u i v a l e n t .  t h a t a good f i t t o the experimental  data was  o b t a i n e d w i t h the  The  This fact  incorrect  model i l l u s t r a t e s  the ease w i t h which a smooth curve can be approximated  a p o l y n o m i a l , and  the l i m i t a t i o n s of t h i s s o r t of m o d e l l i n g .  by  C o n t i n u i n g i n t h i s v e i n , the a p p l i c a b i l i t y of t h i s type o f model to the (Na+K)ATPase can be c o n s i d e r e d .  In r e a l i t y ,  the steps f o l l o w e d by  the  enzyme t o t r a n s p o r t sodium out o f the c e l l v i a the (Na+K)ATPase i n v o l v e the b i n d i n g of ATP, phosphorylated  magnesium, and and  "n" sodium i o n s .  The  enzyme becomes  the c o n f o r m a t i o n a l changes r e q u i r e d t o make the  sodium a v a i l a b l e t o the o u t s i d e o f the c e l l  occur.  I t has g e n e r a l l y  been assumed t h a t these steps of c o n f o r m a t i o n a l change w i l l limiting,  so t h a t the steady s t a t e a p p r o x i m a t i o n  R e c e n t l y , Mardh and P o s t  (1977) found evidence  l i g a n d t o E, the c o n f o r m a t i o n  be r a t e  can be a p p l i e d .  t h a t w i t h each b i n d i n g of  s h i f t e d s i g n i f i c a n t l y towards the  "potent"  18  complex which can proceed to p h o s p h o r y l a t i o n r a t e of p h o s p h o r y l a t i o n magnesium, and  of the enzyme was  o f E.  That i s , the  increased  over t h a t when  sodium were a l l made a v a i l a b l e a t once, i f one  l i g a n d s were added f i r s t , and  then the m i s s i n g  initial ATP,  or two  l i g a n d s were added.  suggests t h a t the s t e a d y s t a t e assumption cannot be a p p l i e d w i t h  of  the  This  impunity  i n the case of the sodium e f f l u x . Nevertheless,  i t should  be p o s s i b l e to o b t a i n s e m i - q u a n t i t a t i v e  ment w i t h the data i f the b a s i c n o t i o n o f a ions b i n d i n g  'dominant mode' w i t h n sodium  is correct.  Thus, the approach has sodium t r a n s p o r t out such as the above. it  been to compare the k i n e t i c curves of  range.  the  of c e l l s w i t h the curves produced by k i n e t i c models Even w i t h j u s t two  adjustable  parameters, k  and  i s u s u a l l y easy to get a r e a s o n a b l e f i t f o r data over most o f  concentration  agree-  I t i s a t low  substrate  the  (sodium) c o n c e n t r a t i o n  that  d i f f e r e n c e s between models are most apparent. It  i s important, then, t o measure as a c c u r a t e l y as p o s s i b l e  concentration  o f the r e a c t a n t  the  (eg. sodium) i n the t r a n s p o r t r e a c t i o n .  (In  f a c t , the q u a n t i t y o f i n t e r e s t i s the chemical p o t e n t i a l , but  discussion  o f such refinements w i l l  concentration  at  the  point  not  be p r e s e n t e d here.)  i n t e r n a l r e a c t i o n s i t e s o f the t r a n s p o r t that a complication  As  i s the  enzymes, and  i t i s on  this  arises.  noted above, the c e l l  found t h a t the water and  This  i s a heterogeneous s t r u c t u r e , and  solutes  i n s i d e the c e l l  do not  i t has  behave as  been  i f they  were i n a simple aqueous s o l u t i o n bounded by a p r o t e i n - l i p i d membrane. B e f o r e the e x p e r i m e n t a l work on current barnacle  notions  f l u x e s c o u l d be done, i t was  about the s t a t e s o f water and  muscle c e l l had  to be c l a r i f i e d .  sodium ions  felt  inside  that the  In p a r t i c u l a r , the amount  d i s t r i b u t i o n of the c e l l u l a r sodium which w i l l  the  and  participate in flux studies,  19 and the c o n c e n t r a t i o n of sodium i n the s o l u t i o n which bathes the i n t e r n a l s u r f a c e o f the c e l l membrane must be known.  This i n t e r e s t i n g  general  problem i s reviewed next.  B.  STATES OF WATER AND  IONS IN CELLS  The c u r r e n t wiew o f the s t a t e o f water and ions i n s i d e l i v i n g c e l l s be summarized  b r i e f l y as f o l l o w s  ( T a i t & Franks 1971; Hinke, C a i l l e ,  Gayton 1973; P a l a t y & Friedman 1973; Cooke & Kunta 1974;  Berendsen  can  &  1975;  Lee & Armstrong 1974; Edzes & Berendsen 1975; Lev & Armstrong 1975). Water i n c e l l s behaves of  l a r g e l y as i t does i n bulk s o l u t i o n s .  75 to 90%  the water has normal l i q u i d p r o p e r t i e s as f a r as d i f f u s i o n o f ions and  molecules, osmotic e f f e c t s , and s o l v a t i o n a r e concerned, and responds b u l k water i n NMR, studies.  like  i n f r a r e d s p e c t r o s c o p y , and x - r a y and neutron d i f f r a c t i o n  About 1% i s t i g h t l y bound to macromolecules as ' s t r u c t u r a l water'.  The r e m a i n i n g 8 - 24% i s i n f l u e n c e d by the macromolecules and the s t r u c t u r a l water, a p p a r e n t l y through the f o r m a t i o n o f s h o r t - l i v e d extended c l u s t e r s of water m o l e c u l e s , by means o f hydrogen bonding, i n the s o - c a l l e d 'hydrophobic interaction'.  This  f r a c t i o n i s of s p e c i a l  that i t s behavior i s d i f f e r e n t about i t s exact s i z e .  interest  i n that i t i s conceivable  from t h a t o f b u l k water.  Techniqes which r e f l e c t  i n d i v i d u a l water m o l e c u l e s , such as NMR  yield  There i s disagreement  the freedom o f motion o f the lower e s t i m a t e s , w h i l e  measurements o f s o l v e n t p r o p e r t i e s y i e l d the h i g h e r e s t i m a t e s . Water passes the c e l l u l a r membranes v e r y r e a d i l y , and q u i c k l y flows t o or  from any r e g i o n o f the c e l l where i t s c h e m i c a l p o t e n t i a l d e v i a t e s  t h a t o f the r e s t o f the c e l l , the  or indeed o f the v i c i n i t y o f the c e l l .  from Because  c e l l membranes can t r a n s p o r t substances and are s e l e c t i v e l y - p e r m e a b l e ,  however, t h i s  i s not the case f o r the major i n o r g a n i c i o n s .  They can be  20  c o n f i n e d w i t h i n or b a r r e d i n s i d e the c e l l .  from the c e l l  Further,  or membrane d e l i m i t e d  organelles  i n a given compartment i n s i d e the c e l l  or i n the  e x t r a c e l l u l a r space they can be i n f r e e s o l u t i o n , o r a s s o c i a t e d w i t h or s m a l l o r g a n i c r j n o l e c u l e s v i a s p e c i f i c or n o n s p e c i f i c b i n d i n g .  large  Finally,  even though an i o n can pass from a f r e e s t a t e t o one o f the n o n - f r e e s t a t e s listed,  i n response t o a n o n - u n i f o r m i t y i n i t s chemical p o t e n t i a l , the  c h a r a c t e r i s t i c time o f the exchange might be v e r y other  cell  processes,  notably  slow r e l a t i v e t o that o f  d i f f u s i o n i n bulk s o l u t i o n and transmembrane  transport. The  a c t i v i t y o f some ions  s p e c i f i c microelectrodes. o f the i o n i n s i d e the c e l l , ion  i n s i d e the c e l l  can be measured w i t h i o n -  These a c t u a l l y r e f l e c t  the chemical p o t e n t i a l  but the a c t i v i t y and c o n c e n t r a t i o n  o f the f r e e  can be e s t i m a t e d under r e a s o n a b l e assumptions about the s o l v e n t  t i e s o f t h e water i n which the f r e e i o n i s thought t o r e s i d e .  proper-  I f the volume  o f d i s t r i b u t i o n o f t h e f r e e i o n can be estimated, then t h e amount o f f r e e ion  i n the c e l l This  can be c a l c u l a t e d .  i s the conceptual  heart  o f the question.  The ions  i n solution  a r e no more f r e e than those p a r t i c i p a t i n g i n i o n p a i r i n g , d u r i n g conditions, groups.  i n the sense t h a t the c h e m i c a l p o t e n t i a l i s the same f o r the two  The d i s t i n c t i o n i s made because both a r e measured by chemical  a n a l y s i s , w h i l e o n l y the s o l v a t e d trode  steady  studies.  i o n i s assumed to be measured i n m i c r o e l e c -  The assumption i n v o l v e d  i s t h a t a l l o f the f r e e i o n i s i n  a homogeneous compartment as f a r as c o n c e n t r a t i o n  i s concerned, a l t h o u g h i t  i s c l e a r that near charge inhomogeneities on membranes or macromolecules, considered  as a group, the c o n c e n t r a t i o n  w i l l be d i f f e r e n t from t h a t i n the  b u l k even though the chemical p o t e n t i a l i s the same. The  total  i o n content o f the c e l l ,  i n c l u d i n g that  l o c a t i o n s , can be determined q u i t e a c c u r a t e l y  in extracellular  from chemical a n a l y s i s o f the  21  ashed t i s s u e .  The  i o n content  to determine, because f i x e d  o f the e x t r a c e l l u l a r space i s o f t e n  negatively-charged  the p o l y s a c c h a r i d e - r i c h g l y c o c a l y x . then by s u b t r a c t i o n one i n t r a c e l l u l a r but not sodium, which has The  If this  i o n f r a c t i o n can be  i n free solution.  This  a h i g h e x t r a c e l l u l a r but  low  than o u t s i d e  the c e l l ) .  intracellular  i n turn.  membrane, but a t v e r y d i f f e r e n t  the chemical  estimated,  is especially d i f f i c u l t  for  concentration.  They a l l can pass  (that i s , during  steady  p o t e n t i a l f o r sodium ions i s lower i n s i d e the No  have been found i n a n a l y s e s  local  the  rates.  Sodium i s a c t i v e l y e x p e l l e d from the c e l l conditions  s i t e s abound i n .  can c a l c u l a t e the amount o f i o n which i s t r u l y  i n d i v i d u a l ions can be c o n s i d e r e d  resting cell  difficult  cell  i n t r a c e l l u l a r accumulations of sodium  of s u b c e l l u l a r f r a c t i o n s .  (This w i l l  below.)  However, a l a r g e amount i s thought to be a s s o c i a t e d w i t h  negative  s i t e s on  i n t r a c e l l u l a r macromolecules, i n c o m p e t i t i o n  be  reviewed  fixed  with  other  cations. Hydrogen i s a c t i v e l y e x p e l l e d from the c e l l . b u f f e r e d by the  i n t r a c e l l u l a r p r o t e i n s , and  Hydrogen ions a r e a l s o produced and  The  by phosphate and  o f the c e l l  potential  bicarbonate.  consumed i n many r e a c t i o n s  P o t a s s i u m i s a c t i v e l y accumulated by the c e l l , bility  i n t r a c e l l u l a r pH i s  cell.  but because the permea-  membrane to potassium i s r e l a t i v e l y h i g h the  f o r p o t a s s i u m ions  i n the  i s about the same i n s i d e and  chemical  o u t s i d e many  cells.  A q u a n t i t y of potassium which i s s m a l l r e l a t i v e to the amount o f p o t a s s i u m i n s o l u t i o n probably  associates with  fixed negative  C a l c i u m i s a c t i v e l y e x p e l l e d from the c e l l . sequestered  i n mitochondria  and  sites  i n s i d e the  cell.  It is also actively  i n the s a r c o p l a s m i c  r e t i c u l u m o f muscle.  An a d d i t i o n a l b u f f e r i n g mechanism o f v e r y l a r g e c a p a c i t y appears to e x i s t (Brinley, T i f f e r t ,  Scarpa, & M u l l ins 1977).  Magnesium i s a c t i v e l y e x p e l l e d from the c e l l .  About, h a l f of  the  22  i n t r a c e l l u l a r magnesium i s bound to ATP & Tiffert  i n b a r n a c l e muscle ( B r i n l e y , Scarpa,  1977).  The s i t u a t i o n w i t h c h l o r i d e i s not c l e a r .  M i c r o e l e c t r o d e measurements  i n d i c a t e t h a t t h e r e i s a s l i g h t a c c u m u l a t i o n o f c h l o r i d e i n s i d e the c e l l (see a l s o B o l t o n & Vaughan Jones 1977; Dulhunty 1978). show l i t t l e  chloride associated with fixed  bound f r a c t i o n s have been r e p o r t e d .  Diffusion  intracellular sites,  C h l o r i d e , l i k e sodium,  studies  but l a r g e  i s abundant i n  the e x t r a c e l l u l a r space, and t h i s makes a c c u r a t e a l l o c a t i o n o f c h l o r i d e to compartments  difficult.  The s i t u a t i o n i n a g i v e n c e l l generalizations.  This thesis  i n b a r n a c l e muscle  type o f t e n  differs  in detail  from these  i s p r i m a r i l y concerned w i t h sodium and hydrogen  cells.  The hydrogen i o n e x e m p l i f i e s the problems o f i n t e r p r e t a t i o n o f measurements o f the c h e m i c a l p o t e n t i a l .discussed above. in several First,  The hydrogen i o n d i f f e r s  fundamental r e s p e c t s from the sodium i o n i n l i v i n g  the c e l l  systems.  i s not c o n s e r v a t i v e w i t h r e s p e c t to hydrogen: hydrogen  p a r t i c i p a t e as r e a c t a n t and product i n many c h e m i c a l r e a c t i o n s  ions  i n the c e l l .  Changes i n these r e a c t i o n s might occur w i t h any m a n i p u l a t i o n o f the c e l l or i t s environment.  Second, hydrogen i s b u f f e r e d by the bicarbonate-C02  system and, more i m p o r t a n t l y i n s i d e the c e l l , the p r o t e i n system. s o l u t i o n a t pH 7.0  Only 0.001% o f the a v a i l a b l e hydrogen i o n i s f r e e i n (Waddell & Bates 1969).  To c a l c u l a t e changes  amount o f hydrogen ions w i t h any m a n i p u l a t i o n , measure o n l y pH changes.  i n the  i t i s not s u f f i c i e n t to  The. b u f f e r i n g c a p a c i t y a t each s t a g e o f the  m a n i p u l a t i o n must be known. solvent  by the phosphate system and  T h i r d , hydrogen i s a l a b i l e p a r t o f the  (water) i n which the e n t i r e c e l l u l a r system i s embedded.  The  e f f e c t i v e t r a n s l o c a t i o n o f hydrogen ions can o c c u r by the forming and b r e a k i n g o f hydrogen bonds and hydrogen-oxygen  bonds i n the water.  This i s  23  much more r a p i d than the s e l f - d i f f u s i o n o f hydrogen, and means t h a t no r a d i o i s o t o p e can be used t o measure f l u x e s . hydrogen ions i n s o l u t i o n i n the c e l l  Finally,  the c o n c e n t r a t i o n o f  i s u s u a l l y about 10^ times  smaller  than t h a t o f sodium. The defined  q u e s t i o n o f the r e l a t i o n s h i p between measurements o f pH (which i s i n terms o f the p o t e n t i a l d i f f e r e n c e developed  chemical chemists, all  electro-  c e l l ) and 'hydrogen i o n c o n c e n t r a t i o n ' t r o u b l e s even the p h y s i c a l and they m a i n t a i n t h a t the q u a n t i t y o f p r a c t i c a l  contexts  processes  i n a standard  i s the.chemical  potential  (Waddell & Bates  interest  1969).  i n almost  Nevertheless,  e q u i v a l e n t t o the movement o f hydrogen ions do occur a c r o s s the  c e l l membrane, and i t i s r e a s o n a b l e hydrogen ions i n v o l v e d .  t o c a l c u l a t e the e f f e c t i v e q u a n t i t y o f  The r e a s o n a b l e approximation  t h a t pH i s the  n e g a t i v e o f the l o g a r i t h m o f the hydrogen i o n a c t i v i t y w i l l be adopted i n these q u a l i t a t i v e d i s c u s s i o n s . The  q u e s t i o n o f the inhomogeneity o f the pH i n s i d e t h e c e l l  e a r l y because measurement o f the d i s t r i b u t i o n o f an i n d i c a t o r or weak base) was the u s u a l technique  arose  (weak a c i d  f o r measuring pH (Fenn & Maurer 1935).  When these measurements i n d i c a t e d t h a t the pH was too h i g h i n s i d e  cells,  the e x i s t e n c e o f a l k a l i n e o r g a n e l l e s was suggested  Otherwise  the a c t i v e e x t r u s i o n o f protons  as t h e cause.  from the c e l l would have had t o be  postulated. R e c e n t l y , Garthwaite by a c i d i c  (1977) examined the d i f f e r e n c e i n t h e pH measured  (DM0 - see s e c t i o n 9) and b a s i c ( n i c o t i n e ) i n d i c a t o r s  t i s s u e s , w i t h r e f e r e n c e t o t h e number o f m i t o c h o n d r i a  present.  i n various A weak a c i d  w i l l y i e l d a pH v a l u e c l o s e r t o the h i g h e r pH i n the inhomogeneous  tissue,  and a weak base w i l l y i e l d a pH v a l u e c l o s e r t o the lower pH (Waddell Bates  1969).  The d i f f e r e n c e ( p H  a c i d  - pH  b a s e  )  &  i n the pH r e s u l t s o f the two  i n d i c a t o r s was 1.0 i n brown f a t , which has many m i t o c h o n d r i a ,  about 0.8 i n  24  most c e l l s , organelles. cells DNP  and about 0.08  In mature r e d blood c e l l s , which have no  M i t o c h o n d r i a a r e thought  t o have a h i g h pH.  the d i f f e r e n c e i n the measured pH v a l u e s was  (dinitrophenol).  T h i s would uncouple  F u r t h e r , i n most  reduced  by exposure t o  oxidative phosphorylation i n  m i t o c h o n d r i a and presumably prevent them from m a i n t a i n i n g a h i g h P  internal  H. For b a r n a c l e muscle c e l l s ,  DMO  has  the pH measured w i t h the a c i d i c  indicator  been r e p o r t e d t o be h i g h e r than t h a t measured w i t h the b a s i c i n d i -  c a t o r methylamine by about 0.1  (Boron & Roos 1976).  These v a l u e s were  lower  than the v a l u e measured w i t h an i n t r a c e l l u l a r e l e c t r o d e , which i s c o n s i s t e n t w i t h the e x i s t e n c e o f an a c i d i c  intracellular  compartment.  T h i s phenomenon p r e s e n t s a p r a c t i c a l problem, i n t h a t the pH  intracellular  f o r most t i s s u e s can o n l y be measured w i t h i n d i c a t o r s , and the meaning  of what they measure i s not c e r t a i n .  For t h i s reason,  done, as p a r t o f the work p r e s e n t e d ' i n t h i s t h e s i s , measured w i t h DMO,  experiments  were  i n which the pH  as a measure o f the pH o f the whole c e l l  i n the  was  sense  d i s c u s s e d above, and w i t h a pH m i c r o e l e c t r o d e , as a measure o f the pH o f the major aqueous i n t r a c e l l u l a r compartment, over a wide range of c e l l u l a r pH,  i n i d e n t i c a l l y - p r e p a r e d b a r n a c l e muscle c e l l s .  d i s c u s s e d i n s e c t i o n 9, was pH(electrode).  t h a t pH(DMO) was  This i n i t s e l f  The  c o n s i s t e n t l y higher  intraresult, than  i s c o n s i s t e n t w i t h the e x i s t e n c e of an  a l k a l i n e i n t r a c e l l u l a r compartment, but the t e c h n i c a l d i f f i c u l t i e s i n d i c a t o r method a r e such t h a t t h i s cannot  o f the  be s t a t e d w i t h any degree of  certainty. A t t e n t i o n w i l l now  be turned t o the s t a t e s o f water and sodium i n  b a r n a c l e muscle. Hinke (1970) adopted c e l l s wherein  a working h y p o t h e s i s  the i n t r a c e l l u l a r water was  f o r s i n g l e b a r n a c l e muscle  divided  i n t o two  fractions:  one  25  ( " i d e a l water") was c o m p l e t e l y l i k e bulk water; the o t h e r was n o t behaving as b u l k water i n t h a t i t d i d n o t a c t as s o l v e n t f o r sodium, p o t a s s i u m o r c h l o r i d e , and was not o s m o t i c a l l y a c t i v e .  H i s experiments i n d i c a t e d  the b u l k water comprised about 68% o f the water i n a b l o t t e d c e l l ,  that  which i s  about 73%, o f the i n t r a c e l l u l a r water s i n c e about TL o f the c e l l water i n t h e e x t r a c e l l u l a r space.  lies  The measured s i z e o f the e x t r a c e l l u l a r space  depends on the t e c h n i q u e o f b l o t t i n g . .  I n the same study, i t was found t h a t  the mean i o n i c a c t i v i t y c o e f f i c i e n t  i n the myoplasm was 0.65, the v a l u e  i n a bulk s o l u t i o n a t the i o n i c s t r e n g t h o f normal b a r n a c l e R i n g e r ' s solution.  A s i m i l a r v a l u e can be deduced from the data o f Hagiwara,  C h i c h i b u , and Naka (1964). The volume o f d i s t r i b u t i o n o f the f r e e i o n was assumed t o be t h e volume o f " i d e a l water", so f r e e i o n contents c o u l d be determined from m i c r o e l e c trode measurements.  I t was found t h a t o n l y a p a r t o f the i n t r a c e l l u l a r  sodium, potassium, and c h l o r i d e measured is  by chemical a n a l y s i s o f whole  cells  i n f r e e s o l u t i o n i n the " i d e a l water" (McLaughlin & Hinke 1966; Hinke,  C a i l l e , & Gayton 1973).  The " m i s s i n g f r a c t i o n s " were t y p i c a l l y 13% o f the  potassium, 73% o f the sodium, and 31% o f t h e c h l o r i d e One e s t i m a t e was t h a t  fully  (Hinke e t al.  1973).  83% o f the i n t r a c e l l u l a r sodium c o u l d be  i n a c c e s s i b l e t o the sodium m i c r o e l e c t r o d e (Hinke 1969b). The r e s u l t s o f m i c r o e l e c t r o d e s t u d i e s by o t h e r workers i n other have been s i m i l a r  (reviewed by L e v & Armstrong 1975).  allowance f o r 'bound water' i s seldom made.  However,  cells  explicit  L e e & Armstrong (1972;1974)  made no a l l o w a n c e f o r 'bound water' i n t h e i r c a l c u l a t i o n s o f f r e e i o n concentrations  i n f r o g s k e l e t a l muscle, a l t h o u g h they acknowledge  and the p h y s i c a l meaning o f the c a l c u l a t e d c o n c e n t r a t i o n s .  the concept  They based t h e i r  c o n c l u s i o n s about t h e e x i s t e n c e o f s e q u e s t e r e d sodium and p o t a s s i u m on t h e observed changes  i n the apparent a c t i v i t y c o e f f i c i e n t  ( a ) / ( N a ) i when the N a  26  sodium c o n t e n t o f the c e l l was sodium deduced of  a l t e r e d , where ( a ^ )  the a c t i v i t y o f  1 S  a  from the m i c r o e l e c t r o d e measurement and (Na)^ i s the q u o t i e n t  the t o t a l a n a l y z e d c e l l u l a r sodium and the t o t a l water c o n t e n t o f the  cell,  e x c l u d i n g the e x t r a c e l l u l a r space. The l o c a t i o n o f the 'missing sodium' i n b a r n a c l e muscle c e l l s  i s not  c e r t a i n , but some c o n c l u s i o n s can be drawn from a c r i t i c a l review o f m o r p h o l o g i c a l and p h y s i o l o g i c a l s t u d i e s . muscle c e l l was  examined  The u l t r a s t r u c t u r e o f the b a r n a c l e  by Hoyle et ai.(/9?3).The s t r u c t u r e i s q u a l i t a t i v e l y  s i m i l a r to t h a t o f v e r t e b r a t e s t r i a t e d muscle, but t h e r e are s e v e r a l unusual features.  The c e l l membrane i s deeply furrowed by an e x t e n s i v e , unordered  system o f c l e f t s .  These were c l a s s i f i e d as "major c l e f t s " ,  deep furrows  opening d i r e c t l y i n t o the b a t h i n g s o l u t i o n a l l a l o n g t h e i r l e n g t h ,  and  "minor c l e f t s " , branches opening i n t o the major c l e f t s or the b a t h i n g s o l u t i o n o n l y a t t h e i r ends.  The c l e f t s c o n t a i n e d " m u c o p o l y s a c c h a r i d e - l i k e "  m a t e r i a l , and comprised about 8% o f the t o t a l c e l l volume as measured micrographs.  from  A system o f f l a t t e n e d t u b u l e s o r i e n t e d both l o n g i t u d i n a l l y  and r a d i a l l y , and d e v o i d o f the 'mucopolysaccharide', comprised l e s s than 17o o f the t o t a l volume. of  The l a t t e r system was  i d e n t i f i e d as the analogue  the t r a n s v e r s e t u b u l a r system (TTS) o f v e r t e b r a t e s t r i a t e d muscle.  t u b u l e s open i n t o c l e f t s or t o the exposed s u r f a c e o f the c e l l .  The  s a r c o p l a s m i c r e t i c u l u m i s s m a l l , c o m p r i s i n g about 0.57 o f the t o t a l o  volume.  By comparison,  s a r t o r i u s muscle  cell  i t i s about 13% of the t o t a l c e l l volume f o r f r o g  (Peachey 1965).  elements, and d i a d i c  The  (rarely:  There a r e l o n g i t u d i n a l and  cisternal  t r i a d i c ) c o n t a c t s a r e made w i t h TTS.  M i t o c h o n d r i a and n u c l e i a r e l o c a t e d j u s t under the exposed and the membrane o f the major c l e f t s .  Together the l a t t e r  sarcolemma  organelles  p r o b a b l y comprise l e s s than 1% o f the c e l l volume. The remaining almost 90% o f the c e l l volume i s o c c u p i e d by the  27  c o n t r a c t i l e p r o t e i n s and the myoplasmic It  solution.  i s among a l l of these s t r u c t u r e s t h a t the compartments o f a  flux  model s h o u l d f i n d c o u n t e r p a r t s . I f the sodium not d e t e c t e d i n the myoplasm by the m i c r o e l e c t r o d e i s isolated  i n the other i n t r a c e l l u l a r compartments, v e r y h i g h c o n c e n t r a t i o n s  must be a t t a i n e d . available.  Few  s t u d i e s on the i o n i c content o f o r g a n e l l e s a r e  S i z e a l o n e was  c o n s i d e r e d t o r u l e out the n u c l e i and  mitochondria  2 o f b a r n a c l e muscle c e l l s as s i g n i f i c a n t r e p o s i t o r i e s of sodium. membrane i t s e l f  i s probably s l i g h t l y more important,  The  s i n c e sodium,  potassium,  magnesium, and c a l c i u m i n t e r a c t c o m p e t i t i v e l y w i t h the membrane p o l a r The  cleft  system i s d i r e c t l y open t o the b a t h i n g s o l u t i o n , and  solution f i l l i n g solution.  The  cell  groups.  so the  i t w i l l have the s o d i u m - r i c h c o m p o s i t i o n o f the b a t h i n g  s o l i d m a t e r i a l i n the c l e f t s  i s the n e g a t i v e l y - c h a r g e d  p o l y s a c c h a r i d e o f the g l y c o c a l y x and w i l l have sodium a s s o c i a t e d w i t h i t , perhaps i n l a r g e q u a n t i t i e s 1977).  ( H a r r i s & S t e i n b a c h 1956;  B r a d i n g & Widdicombe  The amount o f sodium a s s o c i a t e d or adsorbed w i l l  c o n c e n t r a t i o n o f sodium i n the b a t h i n g s o l u t i o n . p r o t e i n i n b a r n a c l e c e l l s has not been determined. b i n d i n g c a p a c i t y can be found  i n experiments  the  The amount of g l y c o An  i n d i c a t i o n of i t s  on smooth muscle, where a t  l e a s t h a l f of the e x t r a c e l l u l a r space c a t i o n content was s o l u t i o n i n the s u c r o s e space  depend on  found not to be i n  ( B r a d i n g & Widdicombe 1977).  C o r r e c t i o n o f the t o t a l a n a l y z e d c e l l  sodium by assuming t h a t a l l  e x t r a c e l l u l a r sodium i s i n a volume o f f l u i d equal to the i n u l i n  (or o t h e r  o  Some sodium i s sequestered i n the n u c l e i and m i t o c h o n d r i a of f r o g s k e l e t a l muscle ( S o r o k i n a & Kholodova 1970) and i n the n u c l e i o f r a t hepatocytes (Hooper & Dick 1976). However, e l e c t r o n microprobe a n a l y s i s has shown t h a t n u c l e a r and c y t o p l a s m i c sodium c o n c e n t r a t i o n s a r e the same i n toad oocytes (Dick 197 8), and some accumulation of sodium i n n u c l e i and m i t o c h o n d r i a o f thymus and l i v e r c e l l s has been r e p o r t e d i n d i f f e r e n t s p e c i e s ( I t c h & Schwarta 1957; T h i e r s , Reynolds, & V a l e e 1960).  28  marker) space, is  a t the c o n c e n t r a t i o n of the b a t h i n g s o l u t i o n ,  caused  of c r a y f i s h muscle has  T h i s occurs when the e x t r a c e l l u l a r c h l o r i d e or potassium  t i o n i s reduced  so t h a t potassium  not d u r i n g an osmotic t i o n s i s kept  c h l o r i d e i s caused  s t r e s s when the product  constant so t h a t potassium  conditions. indeed  I t appears to open d i r e c t l y  307„ o f the c e l l c h l o r i d e , and  (K)bath (^-'-)bath °^ x  sucrose.  volume under some  i n t o the e x t r a c e l l u l a r  solution,  i n d i c a t e d t h a t 15 -  1967,  1968;  Vinogradova,  Nikolsky, & Troshin  Rogus & Z i e r l e r 1973) However, N e v i l l e  (1979) has  s a r c o p l a s m i c r e t i c u l u m , and Somlyo, Shuman, & Somlyo (1977a) found  shown be no  o f sodium i n the s a r c o p l a s m i c r e t i c u l u m on e l e c t r o n microprobe  a n a l y s i s o f t o a d f i s h s t r i a t e d muscle.  I t i s p o s s i b l e t h a t the  compartments o f f l u x s t u d i e s a r e a r t i f a c t s In any case, the TTS  special  o f analysis..  o f the b a r n a c l e c e l l s used i n the experiments  i n t h i s t h e s i s were s u b j e c t e d to treatment  s w e l l i n g i n o n l y a few  The  1967;  indicated that  from the k i n e t i c behavior o f t h i s " s p e c i a l r e g i o n " t h a t i t cannot  the TTS  sucrose  The work o f B i r k s and Davey p l u s , t h a t o f many  the s a r c o p l a s m i c r e t i c u l u m .  described  cell.  a " s p e c i a l r e g i o n " f r e e l y a c c e s s i b l e to sodium,  S p e r e l a k i s , Shigenobu, & Rubio 1978;  accumulation  but  concentra-  c h l o r i d e does not l e a v e the  F l u x s t u d i e s by H a r r i s (1963) had  volume was  (Vinogradova  t h i s was  concentra-  i n f r o g s k e l e t a l muscle i s a c c e s s i b l e to e x t r a c e l l u l a r  ( B i r k s & Davey 1969).  Grundfest  to l e a v e the c e l l ,  t h e r e f o r e might comprise more than VL o f the c e l l  TTS  others  been shown to s w e l l when c h l o r i d e i s  to e n t e r i t from the myoplasm ( G i r a r d i e r , Reuben, Brandt, &  1963).  and  probably  inadequate. The TTS  The  thus  s p e c i a l cases.  which would cause  Altogether, i t i s very u n l i k e l y that  i n b a r n a c l e c o n t a i n s much of the  'missing sodium'.  c o n t r a c t i l e p r o t e i n s form a l a r g e compartment, and have not yet  been c o n s i d e r e d here.  I t might r e a s o n a b l y  be expected  t h a t most of the  29  i n t r a c e l l u l a r sodium not d e t e c t e d by the e l e c t r o d e i s a s s o c i a t e d as c o u n t e r ion  w i t h the f i x e d a n i o n i c s i t e s on the p r o t e i n s i n t h i s compartment  e_t al_. 1973) . 1947;  Myosin i s known t o a s s o c i a t e  with cations  Fenn 1957; Lewis & S a r o f f 1957), and i s unique  p r o t e i n s o f the c e l l potassium.  (Hinke  (Szyent-Gyorgi  among the major  i n showing a modest p r e f e r e n c e f o r sodium over  S t u d i e s o f the d i f f u s i o n o f ions and molecules  inside barnacle  muscle c e l l s have i n d i c a t e d t h a t the muscle p r o t e i n has s i t e s which can s e q u e s t e r c a t i o n s but admit t o v e r y r a p i d exchange w i t h the c a t i o n s which are free i n s o l u t i o n The  i n s i d e the c e l l  t o t a l c a p a c i t y o f these s i t e s  ( C a i l l e & Hinke 1972, 1973, 1974).  f o r sodium and potassium was e s t i m a t e d t o  be about 68 m i l l i m o l e s per k i l o g r a m o f d r y weight. w i t h o n l y v e r y r a p i d l y exchanging  Again, a simple model,  s i t e s , was assumed, so t h e c a p a c i t y might  be l a r g e r i f some s i t e s have l o n g e r r e s i d e n c e times. Experiments  with a d i f f e r e n t  time r e s o l u t i o n ,  i n which the c e l l  membrane  was d e s t r o y e d and t h e p r o t e i n a l l o w e d t o e q u i l i b r a t e v i a a j a c k e t o f porous glass, with bathing solutions of d i f f e r e n t  sodium and potassium  y i e l d e d c a p a c i t i e s about twice as l a r g e (Fenn 1957; McLaughlin e t al_. 1973).  1968; Hinke  About h a l f o f the d r y weight o f a b a r n a c l e muscle  a p p a r e n t l y i s due t o s o l u b l e o r g a n i c molecules  cell  (M.E. C l a r k , p e r s o n a l  communication), a c c o u n t i n g f o r the l a r g e r apparent experiments  content,  c a p a c i t y , but the  w i t h membrane-damaged c e l l s might r e f l e c t  compartmentalization  w i t h l e s s - r a p i d exchange. From these m o r p h o l o g i c a l and p h y s i o l o g i c a l s t u d i e s , then, r e a s o n a b l e t o conclude  t h a t , i n b a r n a c l e muscle c e l l s ,  the c e l l  i t seems membrane  and membrane-delimited o r g a n e l l e s ' w h i c h s e q u e s t e r sodium and a r e not d i r e c t l y open t o the e x t r a c e l l u l a r space c o n t a i n o n l y a s m a l l p o r t i o n o f the i n t r a c e l l u l a r sodium not d e t e c t e d by the m i c r o e l e c t r o d e ( i n the model o f the cell  o u t l i n e d above).  Most o f t h i s s m a l l p o o l o f sodium should.engage i n  30  r a p i d exchange w i t h sodium i n f r e e s o l u t i o n i n the c e l l and f l u x experiments, but  i t appears t h a t a c c u r a t e  c e l l u l a r sodium might be a more s i g n i f i c a n t The was  so  influence  measurement of the  extra-  problem.  p l a n o f e x p e r i m e n t a l i n v e s t i g a t i o n o f t h i s problem f o r t h i s  thesis  to f o l l o w changes i n the sodium content o f the compartment measured  the m i c r o e l e c t r o d e , I t was  found t h a t  as the t o t a l sodium content o f the c e l l was indeed a great  d e a l o f the  r e s i d e i n the e x t r a c e l l u l a r space, but s e q u e s t e r e d i n s i d e the c e l l section  as w e l l .  'missing  manipulated.  sodium' appears to  t h a t there appears to be some These experiments are d e s c r i b e d  in  3.  I t was  concluded from these experiments t h a t the  a c t i v i t y measured by a s o d i u m - s p e c i f i c  microelectrode  parameter a g a i n s t which the sodium e f f l u x should studies.  by  An  a d d i t i o n a l advantage o f the use  w i t h the m i c r o e l e c t r o d e a c t i v i t y c o u l d be  was  followed.  was  sodium  the most s u i t a b l e  be compared i n k i n e t i c  o f continuous measurements  t h a t r a p i d changes i n the I t was  intracellular  intracellular  sodium  a n t i c i p a t e d that these might occur i n  sodium-free s o l u t i o n s under c e r t a i n c o n d i t i o n s ,  as they had  in frog skeletal  muscle (White & Hinke 1976). I t was  a l s o concluded t h a t an attempt should  o f the c e l l w i t h r a d i o s o d i u m s e l e c t i v e l y , c e l l u l a r space would not  conceal  & Hinke 1976), a l t h o u g h t h i s  any  i s not  be made to l o a d the  so t h a t e f f l u x from the  this  i s described  information later.  i n s e c t i o n 4.  extra-  p a r t of the transmembrane f l u x (White the o n l y way  to accomplish t h i s  T h i s meant t h a t a study o f the e f f e c t s o f m i c r o i n j e c t i o n had and  inside  end.  to be done,  M i c r o i n j e c t i o n s t u d i e s can a l s o  about the s t a t e s of sodium i n s i d e the c e l l ,  as w i l l  be  yield  described  31  C.  THE SODIUM EFFLUX  As noted above, the g e n e r a l has  e x p e r i m e n t a l approach t o the sodium e f f l u x  been t o compare the data w i t h the p r e d i c t i o n s o f k i n e t i c models i n hope  o f d e t e r m i n i n g the g e n e r a l systems.  k i n e t i c p r o p e r t i e s o f the sodium t r a n s p o r t  The b e h a v i o r o f the t r a n s p o r t systems when the c e l l s a r e i n  p h y s i o l o g i c a l s a l i n e i n v i t r o should is  l i k e l y t h a t there  be c l o s e t o t h a t i n v i v o .  However, i t  i s more than one t r a n s p o r t mode, so the r e s u l t s i n the  p h y s i o l o g i c a l s a l i n e (normal R i n g e r ' s s o l u t i o n ) p r o b a b l y r e f l e c t the contributions  o f s e v e r a l modes.  I t has been o f i n t e r e s t to compare the k i n e t i c s i n normal Ringer's s o l u t i o n w i t h the k i n e t i c s i n s o l u t i o n s where one p o s s i b l e mode has been altered.  For example, a sodium-potassium exchange mode should  be markedly  reduced i f potassium i s omitted from the s a l i n e , and the c o n t r i b u t i o n o f other  modes t o the sodium e f f l u x seen i n t h i s I t bears r e p e a t i n g  situation will  be g r e a t e r .  t h a t t h i s maneuver p o s s i b l y does not j u s t reduce  the sodium-potassium exchange mode, but r a t h e r causes i n a d d i t i o n a change i n the c o n t r i b u t i o n o f the other  modes t o the e f f l u x .  Neither  the s i z e o f  the sodium-potassium exchange mode nor t h e s i z e o f the c o n t r i b u t i o n o f the other  modes i n normal R i n g e r ' s s o l u t i o n can be measured.  i s possible to obtain  information  from experiments such as these.  example from t h e l i t e r a t u r e i s t h e f i n d i n g t h a t the r e d u c t i o n  Nevertheless, i t An  i n i n v e r t e b r a t e g i a n t axons,  i n the sodium e f f l u x which f o l l o w s removal o f e x t e r n a l  sium can be a p p r e c i a b l y  greater  potas-  than the s i z e o f the potassium i n f l u x .  This  was s t r o n g support f o r the h y p o t h e s i s t h a t potassium ions a c t as a c t i v a t o r s o f the sodium e f f l u x as w e l l as engaging i n exchange f o r sodium (Hodgkin & Keynes 1955a; S j o d i n & Beauge 1967; M u l l i n s & B r i n l e y 1967, 1969; Baker, B l a u s t e i n , Keynes, M a n i l ,  Shaw & S t e i n h a r d t  1969).  32  Examination o f the k i n e t i c s o f the t o t a l sodium e f f l u x has  been done f o r e f f l u x i n t o sodium-free  traditionally  s o l u t i o n s , because some e x p e r i -  ments i n d i c a t e d t h a t a g r e a t d e a l o f sodium-for-sodium  exchange o c c u r r e d  a c r o s s the c e l l membrane (Keynes & Swan 1959, but see M u l l i n s & Frumento 1963).  T h i s was c o n s i d e r e d t o be e n t i r e l y s e p a r a t e from the ' a c t i v e  which was t h e e f f l u x o f most i n t e r e s t h a r d t 1968).  Such experiments  efflux',  (Keynes 1966, but see Keynes & S t e i n -  d e f i n e d the problem  o f the dependence o f the  e f f l u x on t h e i n t e r n a l sodium c o n c e n t r a t i o n , so they w i l l  be reviewed  b r i e f l y here w i t h r e f e r e n c e t o t h i s q u e s t i o n . Keynes and Swan (1959) found t h a t a p l o t ,  f o r s e l e c t e d experiments, o f  the e f f l u x o f r a d i o s o d i u m from f r o g s k e l e t a l muscle versus t h e t o t a l amount 23 of radiosodium remaining out i n t o sodium-free  i n t h e muscle, as r a d i o s o d i u m and  Na were washed  l i t h i u m - s u b s t i t u t e d Ringer's s o l u t i o n ,  i m p l i e d a power  law r e l a t i o n s h i p between e f f l u x and sodium c o n t e n t . the c a r r i e r  They suggested  i n the membrane can o n l y operate when "n" sodium ions were  bound t o i t , where n appeared  t o be t h r e e .  dent o f the sodium-sodium exchange seen  T h i s was assumed t o be indepen-  i n normal Ringer's s o l u t i o n .  model was a t t r a c t i v e because i t r e q u i r e d o n l y one i n t r a c e l l u l a r hence no c o m p l i c a t e d exchanges o f sodium between i n t r a c e l l u l a r and y e t e x p l a i n e d t h e data f a i r l y w e l l . account  o f s a t u r a t i o n o f t h e t r a n s p o r t system,  compartment, compartments,  and should f i t best a t v e r y  Keynes and Swan found d e v i a t i o n s  a t low r a t h e r than a t h i g h l e v e l s o f sodium. these experiments  This  Of course, a power law takes no  low v a l u e s o f the c e l l u l a r sodium c o n t e n t .  extended  that  M u l l i n s and Frumento (1963)  t o h i g h e r v a l u e s o f sodium c o n t e n t .  They  found  a "cube law" f i t best a t low sodium c o n c e n t r a t i o n , but t h a t the 'power decreased as t h e sodium content  i n c r e a s e d , and t h a t s a t u r a t i o n o c c u r r e d .  (At v e r y h i g h sodium content, a v e r y r a p i d e f f l u x was seen. d i s c u s s e d w i t h t h e sodium-free  1  e f f e c t below.)  T h i s w i l l be  They used a r e l a t i o n v e r y  33  s i m i l a r t o the M i c h a e l i s - M e n t e n taneously,  case o f t h r e e sodium ions b i n d i n g s i m u l -  d i s c u s s e d above, but d i d not use the M i c h a e l is-Menten model.  L a t e r , Keynes and S t e i n h a r d t because t h e l a r g e s a r c o p l a s m i c described  (1968) r e c a n t e d on the power law model  r e t i c u l u m o f the f r o g muscle c e l l had been  (Peachey 1965), p r o v i d i n g a m o r p h o l o g i c a l  ment models.  b a s i s f o r two-compart-  I n a d d i t i o n , the l a r g e f r a c t i o n o f the i n t r a c e l l u l a r  sodium  not d e t e c t e d by m i c r o e l e c t r o d e s had been d e s c r i b e d f o r f r o g muscle (Lev 1964). and  A " s e r i e s - p a r a l l e l " model w i t h  f i r s t - o r d e r k i n e t i c s was proposed,  e x p l a i n e d some o f the o b s e r v a t i o n s . However, t h e p r o p e r t i e s o f t h e (Na+K)ATPase had been f u r t h e r e l u c i d a t e d  i n t h e meantime, and i t appeared t h a t about t h r e e sodium ions were t r a n s p o r t e d per ATP molecule h y d r o l y z e d Sen & Post  1961, 1964).  c o u l d n o t be ignored. verified  The mechanism suggested  by Keynes and Swan thus  (eg. Glynn & K a r l i s h 1975), but attempts t o c l a r i f y t h e k i n e t i c s  the sodium e f f l u x  to  r e v e a l added c o m p l i c a t i o n s  Sodium e f f l u x The  1963;  The k i n e t i c s o f the v a r i o u s modes o f the ATPase were  of  (i)  (Glynn 1962; Bonting & Caravaggio  from i n t a c t c e l l s o t h e r than e r y t h r o c y t e s tended i n s t e a d (eg. White & Hinke 1976).  i n t o normal Ringer's  e f f l u x o f sodium M^  a  solution.  i n t o normal R i n g e r ' s  solution,  t h a t i s , where  the e x t r a c e l l u l a r sodium c o n c e n t r a t i o n has n o t been reduced, has been measured i n s e v e r a l c e l l  types, and the q u e s t i o n o f how the e f f l u x v a r i e s  w i t h the i n t e r n a l sodium c o n c e n t r a t i o n (Na) In the s q u i d axon, M^  a  is a strict  has been c o n s i d e r e d .  l i n e a r f u n c t i o n o f the i n t r a c e l l u l a r  sodium c o n c e n t r a t i o n over t h e range o f 1 t o 220 mM S j o d i n & Beauge 1967; B r i n l e y & M u l l i n s 1968).  (Hodgkin & Keynes 1956;  No s a t u r a t i o n was seen.  It  i s known t h a t nerve c e l l s have h i g h c o n c e n t r a t i o n s o f the (Na+K)ATPase (eg. Bonting,  Simon, & Hawkins 1961), but i t i s n o t c l e a r why such a l a r g e  34  pumping c a p a c i t y i s needed by s q u i d axon. In s n a i l neurones, t h e r a t e o f f a l l (ajj ) f o l l o w i n g i o n t o p h o r e t i c  i n j e c t i o n o f sodium ions i s an a f f i n e  a  o f (a ) , that Na v  o f the i n t r a c e l l u l a r sodium a c t i v i t y function  i s , l i n e a r above a t h r e s h o l d v a l u e o f (a, ) (Thomas 1972b). Na' T  v  7  In f r o g v e n t r i c u l a r muscle, no s a t u r a t i o n was seen over the range o f sodium content s t u d i e d , but M^  a  r o s e as (Na)? f o r n between 1.0 and 1.6 i n  d i f f e r e n t experiments (Van d e r K l o o t & Dane 1964). In r e d b l o o d c e l l s  (Garay & Garrahan 1973) and i n f r o g s k e l e t a l  ( H a r r i s 1965) the r e l a t i o n s h i p between Mjj In b a r n a c l e muscle c e l l s ,  Brinley  a  muscle  and (Na) ^ i s s i g m o i d a l .  (1968) found s a t u r a t i o n o f M^  a  at  h i g h (Na)^, w i t h t h e s h o u l d e r a t c a . 20mM. The experiments t o be d e s c r i b e d i n s e c t i o n 5 of. t h i s t h e s i s  revealed  t h a t the t r u e b e h a v i o r o f the sodium e f f l u x from b a r n a c l e muscle i n t o normal R i n g e r ' s s o l u t i o n i s s i m i l a r t o t h a t from the s q u i d axon and s n a i l  neurone.  S a t u r a t i o n does n o t occur a t t h e low l e v e l found by B r i n l e y (1968).  ( i i ) Sodium e f f l u x i n t o p o t a s s i u m - f r e e s o l u t i o n . S t e i n b a c h (1940).showed  t h a t when f r o g s k e l e t a l muscle i s soaked i n  potassium-free Ringer's s o l u t i o n ,  i t l o s e s p o t a s s i u m and gains  Return o f p o t a s s i u m t o the s o l u t i o n enables the c e l l s accumulated sodium. efflux  t o e x t r u d e some o f the  The e f f e c t was a s c r i b e d t o a r e d u c t i o n i n t h e sodium  i n p o t a s s i u m - f r e e media.  c e l l s by H a r r i s and M a i z e l s (1954).  A s i m i l a r e f f e c t was found i n r e d blood  (1951), and i n g i a n t axons by Hodgkin and Keynes  I t was n o t due t o p e r m e a b i l i t y changes o r t o changes  potential.  sodium.  H a r r i s and M a i z e l s  i n the membrane  (1952)-proposed t h a t the p o t a s s i u m i n f l u x and  the sodium e f f l u x i n r e d c e l l s were l i n k e d .  As d e s c r i b e d above,  linked  sodium and p o t a s s i u m t r a n s p o r t was found t o be the p r i n c i p a l mode o f the 'sodium pump' under normal c o n d i t i o n s .  The s t o i c h i o m e t r y o f t h e c o u p l i n g  35  appears  to be f i x e d  i n red c e l l s  i n f r o g s k e l e t a l muscle  (Garrahan & Glynn 1967c) but t o be v a r i a b l e  (Cross, Keynes, Rybova 1965)  ( M u l l i n s & B r i n l e y 1969).  and  i n s q u i d axon  However, the decrease i n the sodium e f f l u x  from  s q u i d axon upon removal o f the e x t e r n a l potassium can f a r exceed the magnitude o f the p o t a s s i u m i n f l u x , as d i s c u s s e d  by S j o d i n  that potassium ions a c t as a c t i v a t o r s f o r sodium In b a r n a c l e results  muscles,  E l e v a t i o n o f the p o t a s s i u m c o n c e n t r a t i o n between 8 (the normal v a l u e ) and 40 mM,  ( B r i n l e y 1968;  o f the b a t h i n g  A s i m i l a r e f f e c t was (1965a,b),  1972). value  efflux  greater  than  caused  efflux.  found i n f r o g s k e l e t a l muscle by Horowicz  and  and they proposed t h a t the i n c r e a s e i n sodium e f f l u x  was  by the c o i n c i d e n t a l t e r a t i o n s o f the membrane p o t e n t i a l due t o the  that f o r f r o g s k e l e t a l muscle  cells  Beauge and S j o d i n  i n which E  m  changes i n the e x t e r n a l p o t a s s i u m c o n c e n t r a t i o n potassium-rich  almost i d e n t i c a l sium-activated stimulated  (1976) have shown  i s made u n r e s p o n s i v e to by prolonged i n c u b a t i o n i n  s o l u t i o n s , changes i n the e x t e r n a l potassium  between 0 and 10 mM  concentration  a c t i v a t e the sodium e f f l u x a l o n g an a c t i v a t i o n c u r v e  to t h a t o b t a i n e d  for untreated  sodium e f f l u x a l s o was  control cells.  shown t o d i f f e r  sodium e f f l u x , which a l s o was  independent  The potas-  from the a z i d e o f membrane p o t e n t i a l  Beauge and S j o d i n suggest t h a t e x t e r n a l p o t a s s i u m a c t i v a t e s the  sodium pump i n f r o g muscle least  s o l u t i o n , to a  had no e f f e c t on the sodium  change i n the potassium c o n c e n t r a t i o n .  changes.  B i t t a r et,.al.  However, f u r t h e r i n c r e a s e s above 40 mM  a marked s t i m u l a t i o n o f the sodium  mediated  transport.  even though the c e l l s c o n t r a c t a t c o n c e n t r a t i o n s  20mM. ( B i t t a r et a l . 1972).  Gerber  I t i s implied  too, removal o f the e x t r a c e l l u l a r potassium  i n a decrease i n the sodium e f f l u x  in barnacle,  (1971).  by a l t e r i n g the t r a n s p o r t enzyme d i r e c t l y , a t  f o r external potassium concentrations  between 0 and 10 mM.  a t which a c t i v a t i o n by potassium occurs i s d i s t i n c t  The  from the c a t a l y t i c  site site  36  for potassium transport.  The a c t i v a t i o n a t v e r y h i g h c o n c e n t r a t i o n s o f  e x t e r n a l p o t a s s i u m remains t o be e x p l a i n e d . Only the e f f e c t o f r e d u c t i o n o f the e x t e r n a l p o t a s s i u m c o n c e n t r a t i o n was  examined e x p e r i m e n t a l l y i n the p r e s e n t work, as d e s c i r b e d i n s e c t i o n 5.  I t was  found t h a t the e f f l u x i n t o p o t a s s i u m - f r e e s o l u t i o n s behaved much  l i k e the e f f l u x i n t o o u a b a i n - c o n t a i n i n g R i n g e r ' s s o l u t i o n .  ( i i i ) Sodium e f f l u x  i n t o sodium-free s o l u t i o n .  The removal o f sodium from the e x t r a c e l l u l a r medium r e v e r s e s the g r a d i e n t o f e l e c t r o c h e m i c a l p o t e n t i a l which i s the d r i v i n g f o r c e f o r ' p a s s i v e ' sodium i o n movement a c r o s s the c e l l membrane. o f sodium from the c e l l  T h i s should make the e f f l u x  l e s s c o s t l y i n terms o f energy, and so r e s u l t  i n c r e a s e both i n the p a s s i v e and i n the a c t i v e e f f l u x .  i n an  However, f o r enzyme-  mediated t r a n s p o r t o f i n t r a c e l l u l a r sodium t o occur, sodium might be r e q u i r e d a t an e x t e r n a l n o n t r a n s p o r t s i t e .  I t has a l s o been suggested t h a t  an exchange o f i n t e r n a l f o r e x t e r n a l sodium ions might occur, w i t h no net f l u x o f ions and no net consumption o f energy. There a r e two c l a s s e s o f 'sodium-free e f f e c t s ' on the b a s i s o f the time course.  The d i f f e r e n c e between the steady sodium e f f l u x  i n t o normal  Ringer's s o l u t i o n and the s t e a d y sodium e f f l u x i n t o sodium-free s o l u t i o n has been the one more s t u d i e d .  There i s a l s o a t r a n s i e n t r a p i d l o s s o f  sodium from the myoplasm o f f r o g s k e l e t a l muscle and c r a b s t r i a t e d  muscle  and from the s n a i l neurone upon removal o f sodium from the b a t h i n g  solution.  The better-known e f f e c t w i l l Ussing  (1947, 1949)  be reviewed f i r s t .  first  suggested the p o s s i b i l i t y t h a t a one-for-one  exchange o f i n t r a c e l l u l a r sodium f o r e x t r a c e l l u l a r sodium c o u l d occur, w i t h no net e x p e n d i t u r e o f m e t a b o l i c energy. t a t i o n of radiosodium fluxes.  T h i s would c o m p l i c a t e the i n t e r p r e -  Subsequently i t was  found (Keynes & Swan  37  1959)  t h a t the r a d i o s o d i u m e f f l u x from f r o g s k e l e t a l . m u s c l e was r e v e r s i b l y  reduced t o about h a l f when the e x t e r n a l sodium was r e p l a c e d by l i t h i u m o r choline, with  e x t e r n a l potassium unchanged.  l e s s when t h e sodium content appropriate  The f r a c t i o n a l r e d u c t i o n was  o f the muscle was e l e v a t e d by i n c u b a t i o n i n an  s o l u t i o n , but c o u l d be r e s t o r e d by l o w e r i n g  a g a i n by a f u r t h e r i n c u b a t i o n .  the sodium  content  E x t e r n a l potassium d i d n o t a f f e c t the  sodium-free response. In s q u i d axon, however, c h o l i n e - o r l i t h i u m - s u b s t i t u t e d sodium-free s o l u t i o n s caused an i n c r e a s e i n t h e e f f l u x o f r a d i o s o d i u m (Hodgkin & Keynes 1955;  M u l l i n s e t a l . 1962).  Mullins  (appendix t o M u l l i n s & Frumento 1963)  suggested t h a t a c c u m u l a t i o n o f incoming sodium near t h e i n t e r n a l s i t e o f an a c t i v e t r a n s p o r t enzyme, due t o r e s t r i c t e d d i f f u s i o n from t h i s l o c a t i o n t o t h e b u l k cytoplasm, c o u l d account f o r both o f these o b s e r v a t i o n s , the  i n t r a c e l l u l a r sodium c o n c e n t r a t i o n  (Na)  was t h e d e t e r m i n i n g  where  factor.  Subsequently i t was shown t h a t removal o f e x t e r n a l sodium does  indeed  cause a decrease i n the r a d i o s o d i u m e f f l u x from s q u i d axons o f low sodium content  (lowered  by s t i m u l a t i o n o f the axon i n l i t h i u m s o l u t i o n )  (Frumento  & M u l l i n s 1967; M u l l i n s & B r i n l e y 1967; S j o d i n & Beauge 1968a), and an i n c r e a s e i n t h e r a d i o s o d i u m e f f l u x from f r o g s k e l e t a l muscle o f h i g h content  (loaded by s o a k i n g  f o r 20 hours a t 2 deg.C i n p o t a s s i u m - f r e e  s o l u t i o n ) ; (Keynes 1965; Beauge & S j o d i n 1968). of the e f f e c t d i f f e r s  content  (The g l y c o s i d e  sensitivity  i n t h e two t i s s u e s ( S j o d i n & Beauge 1968b; Baker,  B l ^ u s t e i n , Keynes, M a n i l , t h e r e was l i t t l e  sodium  Shaw & S t e i n h a r d t  1969)).  I n toad oocyte,  however,  e f f e c t o f removal o f e x t e r n a l sodium, whatever the sodium  (Dick & L e a 1964).  A c a r d i a c g l y c o s i d e - s e n s i t i v e sodium-sodium exchange o f the type seen i n r e d blood  cells  (Garrahan & Glynn 1967) ( t h e r e i s no g l y c o s i d e i n -  s e n s i t i v e sodium-sodium exchange i n r e d b l o o d  c e l l s ) was thought t o be  38  responsible  f o r o n l y p a r t o f the e x t e r n a l sodium-dependent e f f l u x seen i n  muscle s i n c e the e f f e c t s o f g l y c o s i d e s appeared t o be  independent and  & Beauge 1968a). about  the removal o f e x t e r n a l  a d d i t i v e (Horowicz 1965;  1968;  by g l y c o s i d e  decreased both sodium e f f l u x and  Keynes 1966), as  f o r red blood  s k e l e t a l muscle i n the same way (Beauge & S j o d i n 1968).  influx  The  cells  was  the a c t i v e sodium e f f l u x from f r o g  t h a t potassium, rubidium, and  s t i m u l a t i o n was  abolished  cesium  by c a r d i a c  do  glycosides.  sodium  proposed: (a) removal of- e x t r a c e l l u l a r sodium prevents the  dependent e f f l u x , and  (b)  l i t h i u m stimulates  l i k e potassium e x t e r n a l l y . pump r a t e w i l l Eventually  by  (Garrahan & Glynn 1967).  e f f e c t " o f l i t h i u m - s u b s t i t u t e d sodium-free s o l u t i o n s on  e f f l u x was  Sjodin  i n p o t a s s i u m - c o n t a i n i n g s o l u t i o n (Keynes & S t e i n -  L i t h i u m appeared to s t i m u l a t e  A "dual  sodium  Keynes 1966;  207» i n f r o g muscle i n potass ium-free s o l u t i o n but the i n f l u x  unaffected hardt  Glycoside  and  r i s e , and  efflux will  acting  i n t e r n a l sodium content r i s e s ,  so the s t i m u l a t o r y  this stimulated  l a c k of e x t e r n a l  Thus as the  the sodium e f f l u x by  sodium-  e f f e c t of l i t h i u m w i l l  surpass the  the  increase.  i n h i b i t o r y e f f e c t of  the  sodium.  A complication  o f these s t u d i e s  i n whole muscle p r e p a r a t i o n s  i n p o t a s s i u m - f r e e s o l u t i o n s , potassium which leaves  the c e l l s p a s s i v e l y  accumulate i n the e x t r a c e l l u l a r space i n s u f f i c i e n t amounts to sodium e f f l u x a p p r e c i a b l y  ( S j o d i n & Beauge 1973).  i s that can  stimulate  Beauge (1975) e s t i m a t e d  t h a t almost h a l f of t h e - s t i m u l a t i o n o f the sodium e f f l u x seen i n sodiumf r e e , p o t a s s i u m - f r e e , l i t h i u m - s u b s t i t u t e d s o l u t i o n i n f r o g muscle was  due  to t h i s r e a c c u m u l a t i o n of potassium. Sachs (1977) has e x t r u s i o n by r e d blood end  competitive  the  (Na+K)ATPase.  shown t h a t the e f f e c t s o f e x t e r n a l sodium on  sodium  c e l l s are c o n s i s t e n t with' sodium a c t i n g as a "dead-  i n h i b i t o r and  as a h e t e r o t r o p i c a l l o s t e r i c  e f f e c t o r " on  39  I t was  found t h a t a s m a l l  g l y c o s i d e - s e n s i t i v e increase  e f f l u x from f r o g s k e l e t a l muscle was  obtained  i n the sodium  i f c a l c i u m or magnesium were  used to r e p l a c e e x t e r n a l sodium, a s . w e l l as when l i t h i u m was (Horowicz, T a y l o r , & Waggoner 1970).  I t was  so used  concluded t h a t most o f the  g l y c o s i d e - i n s e n s i t i v e sodium e f f l u x r e q u i r e d e x t e r n a l sodium, but o f the g l y c o s i d e - s e n s i t i v e sodium e f f l u x , p a r t can be i n h i b i t e d by e x t e r n a l  sodium  (?sodium-sodium exchange).  The former p a r t o f the g l y c o s i d e - s e n s i t i v e e f f l u x  appears when (Na)^ i s h i g h ,  and the l a t t e r when (Na)^ i s low.  r e s u l t s were o b t a i n e d  by another worker  ( S j o d i n 1971).  Similar  That the g l y c o s i d e -  s e n s i t i v e e f f e c t s a r e due to the (Na+K)ATPase i s supported by r e s u l t s on the i s o l a t e d enzyme (Robinson 1975).  Further,  demonstrated a s t r o p h a n t h i d i n - s e n s i t i v e  Kennedy and DeWeer (1976) have  increase  i n sodium e f f l u x r e q u i r i n g  e x t e r n a l sodium but not potassium, i n f r o g s k e l e t a l muscle i n which the ATP/ADP r a t i o had been lowered by p o i s o n i n g .  A  strophanthidin-sensitive  sodium i n f l u x o f s i m i l a r s i z e a l s o occurs under t h e s e c o n d i t i o n s . In s q u i d axons, Baker, B l a u s t e i n , Keynes sodium exchange under c o n d i t i o n s r e s u l t s o f Frumento and M u l l i n s Poisoning  w i t h CN or DNP,  et a l . (1969) saw  of p a r t i a l poisoning, (1964).  sodium-  c o n t r a r y t o the  The exchange was g l y c o s i d e - s e n s i t i v e .  or i n j e c t i o n o f apyrase (which h y d r o l y z e s  ATP) a l l  g i v e r r i s e t o an e x t e r n a l - p o t a s s i u m - i n d e p e n d e n t , external-sodium-dependent sodium e f f l u x (DeWeer 1970, concentrations r e d blood Since  cells  1974).  i s the c r i t i c a l  A p p a r e n t l y the presence o f ADP  factor.  This  i s s i m i l a r t o the r e s u l t s i n  (Garrahan & Glynn 1967b; Glynn & Hoffman  1971).  the p o t a s s i u m - f r e e and s o d i u m - f r e e e f f e c t s a r e h i g h l y c o r r e l a t e d  ( S j o d i n & Beauge 1969; DeWeer 1970), i t was  concluded t h a t the 98% o f the  sodium e f f l u x from s q u i d axon which i s not p a s s i v e stimulated  i n high  sodium e f f l u x , i n h i b i t e d by e x t e r n a l  i s due t o : (a) potassium-  sodium ( a s c r i b e d to the  sodium-potassium exchange mode o f the pump), and (b) e x t e r n a l - s o d i u m -  40  s t i m u l a t e d sodium e f f l u x ,  i n h i b i t e d by e x t e r n a l p o t a s s i u m ( a s c r i b e d  sodium-sodium exchange mode p l u s a d i f f e r e n t ,  to the  glycoside-insensitive  mechanism) . Sodium-sodium exchange c o n c e i v a b l y c o u l d occur by a mechanism other than a c l o s e l y - l i n k e d  one-for-one exchange.  measured sodium i n f l u x i n t o calculated  For example,  i f from the  f r o g muscle one s u b t r a c t s the p a s s i v e  influx  i n the c o n s t a n t f i e l d a p p r o x i m a t i o n , t h e r e remains a component  of the sodium i n f l u x which i s about 38% o f the t o t a l ( i n normal R i n g e r ' s solution)  (Venosa 1974).  I f the c e l l  i s maintaining a constant  content, t h i s must be b a l a n c e d by sodium e x t r u s i o n , flux).  I f this  influx  sodium  (as i s the p a s s i v e i n -  i s v i a an enzyme, the net r e s u l t  i s enzyme-mediated  sodium-sodium exchange c o m p r i s i n g 38% o f the t o t a l sodium e f f l u x , need not be one-for-one exchange by. one enzyme.  The n o t i o n o f one-for-one  exchange o f sodium ions e l i m i n a t e s the need t o s p e c i f y but  c l e a r l y c r e a t e s other d i f f i c u l t i e s .  a c o u n t e r i o n motion,  I t might be t h a t one enzyme mediates  sodium i n f l u x and a nearby enzyme mediates sodium e f f l u x . i s how  f a r a p a r t can such a p a i r o f d i f f e r e n t  'thermodynamically p e r m i s s i b l e  The key q u e s t i o n  enzymes be y e t o p e r a t e i n a  manner.  1  Perhaps another i n d i c a t i o n  o f the e x i s t e n c e o f a s e p a r a t e sodium  t r a n s p o r t enzyme i s the e f f e c t o f e t h a c r i n i c a c i d appears to i n h i b i t  acid  on muscle.  e x t e r n a l - p o t a s s i u m - s e n s i t i v e sodium  ( E r l i j & L e b l a n c 1971), but o n l y i n g l y c o s i d e - t r e a t e d muscles: a c i d a l o n e s t i m u l a t e s the sodium e f f l u x . sodium e f f l u x  Ethacrinic  efflux  ethacrinic  a c i d has no e f f e c t  on  from b a r n a c l e muscle normally, but p r e v e n t s the i n c r e a s e  i n sodium e f f l u x which u s u a l l y 1972).  Ethacrinic  the external-sodium-dependent sodium i n f l u x w i t h o u t  r e d u c i n g the g l y c o s i d e - s e n s i t i v e ,  the  but i t  f o l l o w s exposure t o  ( D a n i e l s o n et^ a l .  41  In summary, i t appears t h a t  i n nerve (under some c o n d i t i o n s ) and i n  muscle ( n o r m a l l y ) , t h e r e p r o b a b l y i s a component o f the sodium e f f l u x which r e q u i r e s e x t e r n a l sodium. exchange o f i n t e r n a l  I t i s u s u a l l y regarded as s t r i c t one-for-one  f o r e x t e r n a l sodium.  On the b a s i s o f the e f f e c t o f  p o i s o n s , i t would appear t h a t much o f t h i s exchange proceeds by a mechanism o t h e r than the (Na+K)ATPase, ase  a l t h o u g h sodium"sodium exchange o f the (Na+K)ATP-  type can be demonstrated under some c o n d i t i o n s .  S i n c e removal o f  e x t e r n a l sodium causes a decrease i n t h e sodium e f f l u x when (Na) but  i s low,  an i n c r e a s e when (Na)^ i s h i g h , t h e r e appears t o be a mechanism by which  e x t e r n a l sodium can i n h i b i t  the sodium e f f l u x .  p o t a s s i u m mode o f the (Na+K)ATPase.  T h i s c o u l d be the sodium-  The sodium-free e f f e c t i s the combined  r e s u l t o f ( a t l e a s t ) these two e f f e c t s , and the net e f f e c t observed i n experiments i n v i t r o depends on t h e e x p e r i m e n t a l c o n d i t i o n s  ( S j o d i n 1971).  In f r o g s k e l e t a l muscle, 20 t o 507=, o f the sodium e f f l u x under normal conditions the  i s o f the sodium-sodium exchange type.  I n s q u i d axon, none o f  sodium e f f l u x under normal c o n d i t i o n s i s o f the sodium-sodium  type.  The s i t u a t i o n  i n b a r n a c l e muscle was not c l e a r .  exchange  Brinley,(1968)  found t h a t replacement o f e x t e r n a l sodium by l i t h i u m reduced the r a d i o s o d i u m e f f l u x by 677  OJ  w h i l e replacement by sucrose reduced i t by 477o.  f r e e s o l u t i o n s reduce the r a d i o s o d i u m e f f l u x by 517».)  (Potassium-  In c e l l s with higher  sodium content, an i n c r e a s e i n e f f l u x was seen, but the absence o f e x t e r n a l sodium always caused a c o n t r a c t u r e , so the e f f e c t s c o u l d not be measured. Experiments on t h i s type o f sodium-free e f f e c t a r e d e s c r i b e d i n s e c t i o n 5.  Both an i n h i b i t o r y and a s t i m u l a t o r y e f f e c t o f the removal o f e x t e r n a l  sodium were seen i n b a r n a c l e muscle c e l l s , characteristics  but on t h e whole t h e k i n e t i c  o f the e f f l u x i n t o sodium-free s o l u t i o n were the same as  those o f the e f f l u x i n t o normal R i n g e r ' s s o l u t i o n . 'sodium-free e f f e c t s '  That i s , much o f the  i n b a r n a c l e muscle i s due t o changes i n t h e sodium  42  content o f the c e l l s which occur when they a r e p l a c e d  i n sodium-free  solution. A second 'sodium-free e f f e c t - has been r e p o r t e d . s k e l e t a l muscle t o sodium-free s o l u t i o n s causes a l a r g e r a p i d f a l l  ( s u b s t i t u t e d w i t h l i t h i u m or t r i s )  of the i n t r a c e l l u l a r sodium a c t i v i t y measured w i t h  an i n t r a c e l l u l a r m i c r o e l e c t r o d e (White & Hinke 1976). has.  Exposure o f f r o g  A similar rapid  r e c e n t l y been r e p o r t e d i n c r a b muscle (Vaughan-Jones  effect  1977), and a  s i m i l a r but slower e f f e c t e n t i r e l y c o n s i s t e n t w i t h the c o n t i n u a t i o n o f the normal sodium e f f l u x  i n the absence o f i n f l u x , was  found i n s n a i l  neurone  (Thomas 1972b). For  f r o g muscle (White & Hinke 1976), the time c o u r s e o f the f a l l  be f i t t e d by a sum o f two e x p o n e n t i a l terms. unchanged  The two r a t e c o n s t a n t s were  by ouabain treatment, but the ' c a p a c i t y ' o f the k i n e t i c reduced.  to  i d e n t i f i e d as " p a s s i v e " leakage.  more r a p i d r a t e was c o n s t a n t was the  The slower r a t e was  compartment  d e f i n e d by the more r a p i d r a t e was t h a t f o r sodium i n f l u x , and was  could  comparable The  i d e n t i f i e d as a c t i v e sodium e x t r u s i o n , and the r a t e  the same as t h a t found f o r the washout o f l a b e l l e d sodium from  e x t r a c e l l u l a r space. With a muscle which had been loaded w i t h r a d i o s o d i u m by p a s s i v e uptake,  the  washout o f r a d i o s o d i u m from the e x t r a c e l l u l a r space would mask such a  r a p i d e f f l u x u n l e s s the e x t r a c e l l u l a r space was b e f o r e the muscle was  cleared of radiosodium  exposed t o sodium-free s o l u t i o n  (White & Henke 1976).  Chemical a n a l y s i s o f the muscles i n d i c a t e d t h a t the f a l l sodium a c t i v i t y  i n myoplasmic  i s due to movement o f sodium ions out o f the c e l l  Hinke 1976), but t h i s has not been c o n f i r m e d . some s i g n i f i c a n c e to the i n i t i a l  (White &  Some e a r l i e r workers a t t a c h e d  r a p i d exchange o f r a d i o s o d i u m i n whole  f r o g muscle (Carey & Conway 1954).  Others d i s c a r d e d the f i r s t  twenty  minutes o f the i s o t o p e e f f l u x data from s i n g l e muscle c e l l s , which they  43  q u i t e r e a s o n a b l y assumed to r e p r e s e n t washout of the e x t r a c e l l u l a r space almost e x c l u s i v e l y  (Hodgkin & Horowicz 1959).  The s i t u a t i o n i n crab muscle i s q u i t e d i f f e r e n t The r a p i d  fall  (Vaughan-Jones  1977).  o f the myoplasmic sodium a c t i v i t y on exposure o f the c e l l to  sodium-free l i t h i u m - s u b s t i t u t e d or t r i s - s u b s t i t u t e d s o l u t i o n was  unaffected  by ouabain, the removal o f e x t r a c e l l u l a r potassium, c a l c i u m , or magnesium, or  by changes  i n the e x t r a c e l l u l a r pH.  I t was  b l o c k e d by manganese, c o b a l t ,  and lanthanum, which a r e known t o b l o c k the movement o f d i v a l e n t  cations  a c r o s s membranes, but not by D600 or Verapamil, which b l o c k c a l c i u m f l u x e s in  nerve.  However, lanthanum and, t o a l e s s e r extent, manganese themselves  o f t e n caused a r a p i d f a l l of  external If  nor  o f the myoplasmic  sodium a c t i v i t y  sodium.  the r a p i d e f f l u x o f sodium i s not accompanied by p o t a s s i u m i n f l u x ,  by c h l o r i d e e f f l u x  (the myoplasmic c h l o r i d e a c t i v i t y  low e x t e r n a l sodium s o l u t i o n s ) ,  the  found.  i s not changed i n  one would expect a c o n s i d e r a b l e e l e c t r o g e n i c  c o n t r i b u t i o n t o the membrane p o t e n t i a l to occur. t i o n was  i n the presence  Only a s l i g h t  depolariza-  Perhaps t h e r e i s some c o u n t e r i o n or c o - i o n t r a n s p o r t , or  p e r m e a b i l i t y to l i t h i u m might be g r e a t e r than t h a t t o sodium and a  c o n c u r r e n t d e p o l a r i z a t i o n thus r e s u l t  i n the r e s t i n g membrane p o t e n t i a l ,  a p p r o x i m a t e l y c o u n t e r i n g the e l e c t r o g e n i c  effect.  C a l c i u m i n f l u x i s suggested by the r e s u l t s w i t h manganese, c o b a l t , and lanthanum, and by the s i m i l a r time c o u r s e o f the r i s e o f ( C a ) ^ i n c r a b muscle and s q u i d axon under s i m i l a r c o n d i t i o n s .  Removal o f e x t e r n a l  calcium  prevents the r i s e o f the sodium e f f l u x n o r m a l l y seen i n s q u i d axon when exposed t o l i t h i u m s o l u t i o n s  (Baker, B l a u s t e i n , Hodgkin et a l .  1969).  However, w i t h c r a b muscle removal o f e x t r a c e l l u l a r c a l c i u m and/or magnesium had no e f f e c t .  F u r t h e r , a sudden r a p i d  c o n t r a c t i o n , and t h i s d i d not occur.  i n f l u x o f c a l c i u m would t r i g g e r a  I t might be t h a t the e x t r a c e l l u l a r  44  space o f the whole-muscle p r e p a r a t i o n used was not c o m p l e t e l y c l e a r e d o f c a l c i u m o r potassium, but t h i s i s u n l i k e l y because l o n g washout used.  times were  The s i m i l a r i t y o f the response t o lanthanum and removal o f e x t e r n a l  sodium was noted above.  Lanthanum can d i s p l a c e q u i t e l a r g e q u a n t i t i e s o f  membrane-bound c a l c i u m , and i n t r a c e l l u l a r c a l c i u m s t i m u l a t e s sodium e f f l u x from b a r n a c l e muscle ( B i t t a r et a l . 1972, 1973).  A l t o g e t h e r , though, the  p r o c e s s e s which l e a d t o a d e c l i n e i n the myoplasmic sodium a c t i v i t y remain unknown. Measurements  w i t h the i n t r a c e l l u l a r m i c r o e l e c t r o d e can o n l y d e t e c t  l o s s o f sodium from the major i n t r a c e l l u l a r compartment. the f a t e o f the l o s t sodium.  They cannot r e v e a l  T h e r e f o r e an experiment i n which the micro-  e l e c t r o d e measurements were combined w i t h r a d i o s o d i u m e f f l u x measurements was d e v i s e d , as d e s c r i b e d i n s e c t i o n 6.  I t was found t h a t a r a p i d f a l l i n  the myoplasmic sodium a c t i v i t y s i m i l a r t o t h a t  i n f r o g and crab muscle  occurs i n b a r n a c l e muscle under c e r t a i n c o n d i t i o n s , and t h a t i t i s accompanied by a r a p i d l o s s o f sodium from t h e c e l l .  As w i t h s n a i l neurone,  t h i s was found t o be due t o the c o n t i n u i n g normal o p e r a t i o n o f the sodium e f f l u x i n t h e absence o f sodium  influx.  ( i v ) The e f f e c t o f ouabain on the e f f l u x o f sodium. C a r d i a c g l y c o s i d e s , p r i n c i p a l l y ouabain ( g - s t r o p h a n t h i n ) and i t s aglycone s t r o p h a n t h i d i n , have l o n g been known t o i n h i b i t the t r a n s p o r t o f sodium and p o t a s s i u m i n r e d b l o o d c e l l s and f r o g s k e l e t a l muscle 1953; M a t c h e t t & Johnson 1954).  (Schatzmann  Some such a c t i o n had been suspected because  t o x i c doses o f the drugs were known t o cause a l o s s o f p o t a s s i u m from h e a r t muscle  (Schatzmann & W i t t 1954).  I t was found l a t e r t h a t c a r d i a c  glycosides  a l s o i n h i b i t t h e a c t i v i t y o f t h e (Na+K)ATPase (Skou 1965). The c u r r e n t theory o f the a c t i o n o f c a r d i a c g l y c o s i d e s  (Schwartz e t a l .  45  1975;  Glynn & K a r l i s h 1975)  on the  (Na+K)ATPase which i s s e p a r a t e  binding s i t e and  i s t h a t they b i n d s p e c i f i c a l l y to a s i n g l e s i t e  there  from the c a t a l y t i c s i t e s .  The  i s exposed o n l y a t the e x t e r n a l s u r f a c e of the c e l l membrane,  i s a p a r t i c u l a r c o n f o r m a t i o n o f the enzyme which favours  o f the g l y c o s i d e . t i o n of the  The  binding i s very strong.  i s o l a t e d enzyme-glycoside complex  The  h a l f time f o r d i s s o c i a -  a t 37°C i s about 2.5  a l t h o u g h the p h y s i o l o g i c a l e f f e c t o f g l y c o s i d e s can be r e v e r s e d rapidly.  The  binding  b i n d i n g does not render the enzyme c o m p l e t e l y  hours  much more  inactive.  A l t h o u g h the sodium-potassium exchange appears to be prevented, some p a r t i a l or s i d e r e a c t i o n s can s t i l l  occur  (Glynn  et a l . 1974).  t h a t the e f f e c t s o f g l y c o s i d e are d i f f e r e n t  f o r the  It is  conceivable  i s o l a t e d and  the  in  s i t u enzyme, however. In whole c e l l s , In squid.axons w i t h sodium e f f l u x  c a r d i a c g l y c o s i d e s can promote some modes of i o n low ATP  content,  strophanthidin  ( B r i n l e y & M u l l i n s 1968).  i n c r e a s e d the r a t e o f  Strophanthidin  p o t a s s i u m e f f l u x from f r o g s k e l e t a l muscle ( H a r r i s 1957; 1968a) and The  flux.  a l s o increases  the  S j o d i n & Beauge  s q u i d axons ( M u l l i n s & B r i n l e y 1969).  i n h i b i t i o n o f the sodium e f f l u x by s t r o p h a n t h i d i n  increases  with  i n c r e a s i n g sodium content  i n 'aged' f r o g s a r t o r i u s muscle ( S j o d i n & Beauge  1968a) but  i n c r e a s i n g sodium content  decreases w i t h  in freshly-dissected  f r o g s a r t o r i u s muscle (Horowicz et al_. 1970) . Dependence o f f l u x e s on ATP tivity  to g l y c o s i d e s  does not always c o r r e l a t e w i t h  ( M u l l i n s & B r i n l e y 1969).  the s e n s i -  In ATP-depleted s q u i d axon,  s t r o p h a n t h i d i n causes a marked i n c r e a s e i n the sodium e f f l u x , a g a i n s t gradient of electrochemical  p o t e n t i a l , while  the  l e a v i n g the sodium i n f l u x  unchanged ( M u l l i n s 1972). Recent experiments on red blood  c e l l ghosts (Bodeman & Hoffman  1976)  r e v e a l e d t h a t i n the presence o f e x t e r n a l potassium, the r a t e a t which  46  ouabain  bound decreased when e i t h e r the i n t e r n a l sodium o r t h e i n t e r n a l  potassium was r a i s e d . variations  When e x t e r n a l potassium was not present,  i n i o n content had no e f f e c t on ouabain  experiments,  the f i n a l amount o f ouabain  In other  bound was n o t a f f e c t e d by such  (Schwartz eit a l . 197 5), and i t i s n o t known i f an i n c r e a s e i n  manipulations the i n t e r n a l  binding.  such  i o n c o n c e n t r a t i o n s can promote the d i s a s s o c i a t i o n from the  enzyme o f ouabain which i s a l r e a d y bound t o the enzyme. The  e f f e c t s o f s t r o p h a n t h i d i n on t h e sodium e f f l u x from b a r n a c l e muscle  c e l l s were s t u d i e d by B r i n l e y  (1968).  He found  t h a t t h e r e was l i t t l e  or no  -8 i n h i b i t i o n a t 10  M. s t r o p h a n t h i d i n and t h a t as the c o n c e n t r a t i o n o f s t r o p h -  a n t h i d i n was i n c r e a s e d t o 10 ^M, the percent  i n h i b i t i o n i n c r e a s e d . The  maximum i n h i b i t i o n was about 907 and o c c u r r e d f o r c o n c e n t r a t i o n s o f s t r o p h o  a n t h i d i n g r e a t e r than o r equal t o 10 "'M. content,  t h e r e was a delayed  I n one c e l l o f v e r y h i g h sodium  i n c r e a s e i n the sodium e f f l u x a f t e r an i n i t i a l  -4 fall  a t 10  M strophanthidin.  effective. content.  Ouabain appeared t o be s l i g h t l y  The i n h i b i t i o n was l e s s  i n c e l l s which had a l a r g e r sodium  S t r o p h a n t h i d i n produced a g r e a t e r r e d u c t i o n o f sodium e f f l u x  than d i d removal o f e x t e r n a l sodium and/or potassium, sodium and potassium treated The  less  and removal o f e x t e r n a l  d i d not i n c r e a s e the i n h i b i t i o n i n s t r o p h a n t h i d i n -  cells. e f f e c t s o f ouabain  on t h e sodium e f f l u x from b a r n a c l e muscle  were s t u d i e d by B i t t a r e t a l . (1973). by exposing  isolated cells  They o b t a i n e d dose-response  cells  curves  t o i n c r e a s i n g c o n c e n t r a t i o n s o f ouabain, as  B r i n l e y had done, and o b t a i n e d a s i m i l a r c u r v e but w i t h a maximum i n h i b i t i o n of  about 707o.  They used t h e r e d u c t i o n i n the f r a c t i o n o f r a d i o s o d i u m  lost  per u n i t time as t h e i r measure o f i n h i b i t i o n , w h i l e B r i n l e y had c a l c u l a t e d  _3 the s i z e o f the sodium e f f l u x u s i n g an e s t i m a t e d v a l u e f o r (Na)^. a f t e r about twenty minutes o f exposure t o ouabain,  A t 10  M,  the e f f l u x o f radiosodium  47  began t o r i s e a g a i n . The i n h i b i t i o n was g r e a t e r i n c e l l s which had " s l o p e r a t i o s " c l o s e t o 1, t h a t  i s , i n c e l l s h a v i n g a lower sodium content  t i o n 4 ) . I n j e c t i o n o f ouabain i n t o the c e l l s  (see sec-  caused no change i n the sodium  e f f l u x . The i n c r e a s e i n the sodium e f f l u x caused by d e p o l a r i z a t i o n o f the c e l l s , by r a i s i n g the e x t e r n a l p o t a s s i u m c o n c e n t r a t i o n or by i n j e c t i n g C a C ^ , was not i n h i b i t e d by ouabain. treatment.  Nor was the i n c r e a s e i n the sodium e f f l u x caused by C O 2  B i t t a r et a l .  concluded t h a t t h e r e a r e a t l e a s t two s e p a r a t e  sodium e x t r u s i o n systems, l o c a t e d i n d i f f e r e n t p a r t s o f the membrane. The e f f e c t s o f s e v e r a l c a r d i a c aglycones on the sodium e f f l u x muscle c e l l s have a l s o been s t u d i e d  ( B i t t a r & Brown 1977). They a l l  i n barnacle appear t o  b i n d t o the c a r d i a c g l y c o s i d e s i t e and t o have the same e f f e c t s as c a r d i a c glycosides.  Only the potency d i f f e r s .  Three a s p e c t s o f the e f f e c t o f ouabain on the sodium e f f l u x muscle were examined First,  i n this  thesis.  the e f f e c t on the dose-response c u r v e o f the use o f the sodium e l e c t r o d e  to measure the r i s i n g was i n v e s t i g a t e d . sult  e x p e r i m e n t a l l y as p a r t o f the work r e p o r t e d  i n barnacle  i n t r a c e l l u l a r sodium a c t i v i t y a f t e r ouabain begins t o a c t  Impariment  i n an immediate  o f the e x t r u s i o n mechanism by ouabain s h o u l d r e -  i n c r e a s e i n the sodium content o f the c e l l as the p a s s i v e  i n f l u x c o n t i n u e s unchanged,  and t h e i n c r e a s e d sodium content s h o u l d be r e f l e c t e d  i n the measured sodium e f f l u x .  Second, the k i n e t i c c h a r a c t e r i s t i c s o f the  sodium e f f l u x i n c e l l s which a r e maximally i n h i b i t e d by ouabain were t o be measured. I t turned out t h a t t h i s  f l u x i s an i n c r e a s i n g f u n c t i o n o f the i n t r a -  c e l l u l a r sodium a c t i v i t y a t low l e v e l s o f i n t e r n a l sodium, but reaches a p l a t e a u at higher l e v e l s .  These two a s p e c t s a r e d e s c r i b e d  i n s e c t i o n 5. T h i r d , an  e l e c t r o g e n i c c o n t r i b u t i o n o f the sodium f l u x t o the membrane p o t e n t i a l was sought. tial  Experiments which demonstrate t h e e x i s t e n c e o f an e l e c t r o g e n i c poten-  i n b a r n a c l e muscle w i l l  be d e s c r i b e d  i n s e c t i o n 7.  A l t h o u g h the membrane p o t e n t i a l has o n l y now been mentioned i n c o n n e c t i o n  48  w i t h the i o n f l u x e s , of  i t i s o f course i n t i m a t e l y i n v o l v e d w i t h them.  the e a r l y work on i o n i c c u r r e n t s ,  adopted o p e r a t i o n a l l y . and c i r c u i t to  I n much  the models o f e l e c t r i c a l c i r c u i t s were  The r e s u l t has been a mixture o f m o l e c u l a r p h y s i c s  theory, w i t h emphasis on t h e l a t t e r .  I t was f e l t  t o be worthwhile  review t h e o r i g i n s o f the most commonly used models o f the membrane poten-  t i a l and i o n i c c u r r e n t s , and t o i n d i c a t e how they c o u l d be improved. review i s p r e s e n t e d i n t h e next p a r t o f t h i s s e c t i o n , and completes duction.  I t i s f o l l o w e d by a summary o f t h e problems  t h e s i s , and a d e s c r i p t i o n o f t h r e e models used l a t e r of  D.  experiments  a l l cells  Almost always,  measurable  the e l e c t r i c a l p o t e n t i a l measured  i s n e g a t i v e w i t h r e s p e c t t o t h a t measured i n  bulk s o l u t i o n bathing the c e l l . In  a number o f c e l l s  pump i s suddenly a l t e r e d , brane p o t e n t i a l . t i o n o f ouabain,  the  i n the i n t e r p r e t a t i o n  t h e r e i s an e l e c t r i c a l p o t e n t i a l d i f f e r e n c e  in the i n t e r i o r o f the c e l l  An  t o be addressed i n t h i s  but gathered t o g e t h e r f o r the convenience o f the r e a d e r .  a c r o s s the c e l l membrane.  the  the i n t r o -  THE TRANSMEMBRANE DIFFERENCE IN ELECTRICAL POTENTIAL  In  the  This  i t has been found t h a t t h e r e i s an immediate  i s accompanied  interior,  by a d e p o l a r i z a t i o n o f the c e l l .  That i s ,  becomes l e s s n e g a t i v e w i t h r e s p e c t t o the o u t s i d e .  i n c r e a s e i n t h e pump r a t e ,  Kernan  change i n t h e r e s t i n g mem-  A decrease i n t h e pump r a t e , caused f o r example by a p p l i c a -  i n s i d e o f the c e l l  cell  i f t h e r a t e o f t h e sodium  caused  i s accompanied  f o r example by i n j e c t i o n o f sodium  by a h y p e r p o l a r i z a t i o n  (see reviews by  1970; Thomas 1972; a l s o DeWeer & G e d u l d i g 1973; DeWeer 1974).  D i f f e r e n t modes o f the pump appear  t o have d i f f e r e n t degrees o f  into  49  electrogenicity  (DeWeer 1974).  The s t o i c h i o m e t r y o f the net sodium-  potassium exchange by t h e t r a n s p o r t enzymes appears t o be f i x e d f o r r e d b l o o d c e l l s a t 3Na : 2K, but to vary K a r l i s h 1975).  from 1:1 t o 3:1 i n s q u i d axon (Glynn &  A s e p a r a t i o n o f charge such as i s i m p l i e d by t h i s c o u l d  be e f f e c t e d w i t h a v e r y l a r g e investment o f energy. the charge s e p a r a t i o n necessary  only  The q u e s t i o n o f how  t o produce t h e observed change i n membrane  p o t e n t i a l comes about i s e s s e n t i a l l y t h e same as t h e q u e s t i o n o f how a c t i v e sodium e x t r u s i o n comes about. The  development o f ideas on t h e e l e c t r o g e n i c i t y o f t h e sodium pump has  been reviewed by Thomas (1972).  Theories  o f the o r i g i n o f the c o n t r i b u t i o n  of the e l e c t r o g e n i c sodium pump to t h e r e s t i n g membrane p o t e n t i a l a r e phenomenological extensions and  Katz (1949).  o f the approach o f Goldman (1943) and Hodgkin  The n o t i o n s  o f i o n i c p e r m e a b i l i t y and conductance, and  the i o n i c mechanisms i n v o l v e d i n t h i s approach, o f t e n a r e employed i n other contexts. It  i s generally f e l t  that t h e membrane p o t e n t i a l E  m  can be regarded  as b a s i c a l l y a d i f f u s i o n p o t e n t i a l , which a r i s e s i n nerve and s t r i a t e d muscle because o f t h e 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 c e l l membrane to p o t a s s i u m and the e l e v a t e d i n t r a c e l l u l a r potassium c o n c e n t r a t i o n  created  by a c t i v e t r a n s p o r t o f ions.  (Other  value are considered  KC1 tends t o l e a k out o f the c e l l a c r o s s t h e  below.)  l i k e l y c o n t r i b u t i o n s t o the measured  membrane, but s i n c e the m o b i l i t y i n the membrane o f the potassium ions i s g r e a t e r than t h a t o f the c o u n t e r i o n c h l o r i d e (the m o b i l i t i e s a r e almost equal  i n bulk s o l u t i o n ) , a s m a l l l o c a l charge s e p a r a t i o n o c c u r s .  charge s e p a r a t i o n r e s u l t s ion  i n an e l e c t r i c a l  This  f o r c e which r e t a r d s the potassium  movement and promotes t h e c h l o r i d e i o n movement.  A steady  a t t a i n e d where the u n i d i r e c t i o n a l e f f l u x o f K and C l a r e e q u a l .  state i s This i s  j u s t d i k e _the f a m i l i a r l i q u i d j u n c t i o n - o r d i f f u s i o n p o t e n t i a l (eg. • „ Lakshminarayanaiah 1969), but there  i s no n e t l o s s o f K because o f the  50  sodium-potassium exchange pump, there, i s no v a l u e o f the by  resting.potential,  the membrane, l i k e the  and  net  loss-of Cl at'the  steady  the d i f f u s i o n f r o n t i s f i x e d i n space  "constrained  liquid junction"  of P l a n c k  (Lakshminarayanaiah 1969). From a model of the c e l l membrane as a homogeneous l a m e l l a , t e r i z e d by m o b i l i t i e s d r i v i n g force)  i n which the  an e x p r e s s i o n f o r the  over the  electric  The  i n t e g r a t i o n can  and  when the  _ m  RT F  J K (_P  •  P  l  n  K  e x t r a c e l l u l a r , and  i o n by  integrating,  f l u x and  the  f o r sodium, potassium, and  chloride  o f Na . (K)i + P ( K )  ?  ' < o . (Na)i  C1 " + P  N a )  P  +  N a  permeability  i 1 . (Cl)  ( C 1 )  C 1  0  J  i r e f e r s to i n t r a c e l l u l a r and of the membrane t o the  (This w i l l be r e f e r r e d  to as  the  'GHK  ion  o to (essen-  equation .) 1  C l e a r l y t h i s model o f d i f f u s i o n through a homogeneous s l a b does c o i n c i d e w i t h the  s i t u a t i o n i n a r e a l membrane.  the membrane o n l y a t c e r t a i n l o c a t i o n s , At  o f the  e q u a t i o n must d e s c r i b e  The states  ions  seen i n d i f f u s i o n i n a bulk s o l u t i o n .  -the membrane p o t e n t i a l v e r y w e l l .  basis  that  the  In r e a l i t y ,  the  ie. in association with  the m o l e c u l a r l e v e l the movement o f the  to that  ions,  i s zero,  P i s the  the m o b i l i t y ) .  mobilities  i n the membrane (the N e r n s t - P l a n c k e q u a t i o n ) .  be c a r r i e d out  t o t a l current  to  i s c o n s t a n t , Goldman (1943)  transmembrane f l u x o f an  (Hodgkin & Katz 1949), where s u b s c r i p t  tially  field  t h i c k n e s s of the membrane, a r e l a t i o n between the  d r i v i n g f o r c e at each p o i n t  E  r a t i o s of average d i f f u s i o n v e l o c i t y  f o r each major i o n which are much lower than the  i n bulk s o l u t i o n and derived  (empirical  charac-  bears no Yet  T h e r e f o r e the  what i s o c c u r r i n g  the  ions  not pass  proteins.  simple r e l a t i o n  equation describes  essential physical a t the m o l e c u l a r  basis  level.  o f the e q u a t i o n i s the N e r n s t - P l a n c k e q u a t i o n , which s i m p l y f l u x of an  ion is proportional  to the d r i v i n g f o r c e .  The  51  d r i v i n g f o r c e f o r a system a t c o n s t a n t and u n i f o r m p r e s s u r e and  temperature  can be deduced from an e x p r e s s i o n f o r the energy change accomplished the r e s u l t i n g flow, the flow b e i n g easy to d e f i n e . (where G i s the Gibbs i s -^/u,  wherey^=  f o r the i o n .  by  The energy change i s -dG  f r e e energy) and the f o r c e conjugate to a flow o f ions +  RT"ln(a)  +  zF<f> i s the e l e c t r o c h e m i c a l p o t e n t i a l  (a) = a c t i v i t y o f the i o n , <f> = e l e c t r i c a l p o t e n t i a l .  (The  d r i v i n g f o r c e can a l s o be deduced i n the framework o f i r r e v e r s i b l e thermodynamics,  from the entropy p r o d u c t i o n - eg. see K a t c h a l s k y & Curran 1967.)  T h i s "phenomenological  f o r c e " i s an approximation, u s e f u l i n a macroscopic  r e p r e s e n t a t i o n o f a system i n which  ' a l l gradients are s u f f i c i e n t l y gradual.'  The thermodynamic f u n c t i o n s cannot a c t u a l l y be d e f i n e d a t each p o i n t i n space, s i n c e they r e p r e s e n t the i n t e r a c t i o n s o f a l a r g e number o f p a r t i c l e s , and u n l e s s the system can be regarded as an aggregate o f m a c r o s c o p i c a l l y s m a l l volume elements,  each c o n t a i n i n g a l a r g e number o f p a r t i c l e s ,  the  r e p r e s e n t a t i o n s t a t e d above cannot be a p p l i e d w i t h any e x p e c t a t i o n o f success.  Goldman (1943) s t a t e d t h a t "the c u r r e n t c a r r i e r s pass  more or l e s s randomly d i s t r i b u t e d which i s assumed u n i f o r m normal was  interstices  i n the s t r u c t u r e  to the d i r e c t i o n o f f l o w . "  through (membrane),  The  integration  a l o n g a d i r e c t i o n p a r a l l e l to the d i r e c t i o n o f flow, p a s s i n g through a  pore.  In the pore the ions were regarded as d i f f u s i n g as they do i n bulk  s o l u t i o n , but i n one dimension and w i t h much lower m o b i l i t y . not s u r p r i s i n g l y , was junction . 1  The  result,  s i m i l a r t o t h a t o f P l a n c k f o r a. ' c o n s t r a i n e d l i q u i d  Hodgkin and Katz wrote the s o l u t i o n f o r the major ions and  s o l v e d f o r the transmembrane p o t e n t i a l d i f f e r e n c e a t zero net c u r r e n t , as s t a t e d above. A m e c h a n i s t i c model a t the m o l e c u l a r l e v e l f o r the o r i g i n o f the membrane p o t e n t i a l can be envisaged.  The p h y s i c a l o r i g i n o f the p o t e n t i a l  d i f f e r e n c e i s indeed s i m i l a r to t h a t i n the case o f a l i q u i d j u n c t i o n .  In  52  the c e l l membrane, the p r o t e i n channels thought  through which c a t i o n s pass a r e  to be l i n e d w i t h e l e c t r o n e g a t i v e m o i e t i e s , such as c a r b o n y l o r  c a r b o x y l groups,  so t h a t the c a t i o n i c charge can be p a r t i a l l y or c o m p l e t e l y  balanced when i t i s i n the channel.  (These models a r e c a l l e d the " n e u t r a l  p o l a r pore" (Eisenman 1968; M u e l l e r & Rudin pore"  (Eisenman 1968)).  channel.  There  1967) and the " f i x e d  charge  i s a f i n i t e c o n c e n t r a t i o n o f c a t i o n i n the  When a c a t i o n e n t e r s the channel,  i t l e a v e s i t s c o u n t e r i o n behind.  T h i s can occur o n l y o c c a s i o n a l l y on the m o l e c u l a r s c a l e o f space and time, s i n c e a l o c a l c o n c e n t r a t i o n o f n e g a t i v e charge would r e t a r d f u r t h e r of c a t i o n  ( v i a the channel o r o t h e r w i s e ) .  Such an inhomogeneity  egress  o f charge  c o u l d be b a l a n c e d by a movement o f c a t i o n s from the r e g i o n t o which the cations  i n t h e channel a r e heading,  i n a one-for-one  but i t i s more l i k e l y t h a t the anions w i l l path i s a v a i l a b l e .  exchange on average,  be drawn a f t e r the c a t i o n s i f a  The a n i o n c l e a r l y cannot  r e a d i l y f o l l o w through the  c a t i o n c h a n n e l , so t h e r e i s assumed t o be an a n i o n channel nearby. anions n o r m a l l y would pass through the channel  i n a manner s i m i l a r t o t h a t  o f the c a t i o n s , but the c a t i o n s can do so more r e a d i l y the membrane"). difference  The  ("higher m o b i l i t y i n  A s e p a r a t i o n o f charge o c c u r s , and an e l e c t r i c a l  potential  results.  The GHK e q u a t i o n has proved  t o be a good q u a l i t a t i v e and q u a n t i t a t i v e  or s e m i q u a n t i t a t i v e d e s c r i p t i o n o f the membrane p o t e n t i a l  i n many s i t u a t i o n s .  However, t h e r e a r e s e v e r a l ways i n which a q u a n t i t a t i v e r e l a t i o n between 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 membrane and the d r i v i n g f o r c e f o r t h e flow o f ions c o u l d be formulated.  The n e t e f f e c t a l o n e can be c o n s i d e r e d ,  so t h a t the membrane channels a r e regarded as 'black boxes' c h a r a c t e r i z e d by a r e s i s t a n c e or m o b i l i t y .  Then t h e c u r r e n t equals the q u o t i e n t o f the  v o l t a g e and a r e s i s t a n c e , o r the f l u x v e l o c i t y equals t h e product o f the net d i f f e r e n c e i n chemical p o t e n t i a l and a p e r m e a b i l i t y ( o r m o b i l i t y ,  53  denoted u ) .  Such a r e l a t i o n u n d e r l i e s the u s u a l d e f i n i t i o n o f membrane  conductance (Hodgkin & Horowicz 1959b) and the u s u a l c o n c e p t i o n  o f the  ' e l e c t r o g e n i c component' o f t h e r e s t i n g membrane p o t e n t i a l (Hodgkin & Keynes 1955a). I f one regards  the potassium f l u x i n t h i s manner, f o r example,  then  2 Mj, (moles/cm sec)  =  -u  R  . (K) ^ . &jui  =  -u  K  . ( K ) . ( R-T-In ( ) i + R  ±  09 2 Ij, (coulomb/cm sec) = g£ • ( m  while  E  = S  • (m E  K  F E  m  )  o  ~ K ) E  " ^  F  *  l  > (K)i  n  2 and  s i n c e Ij^ =  Mjr . F, the conductance g ^ would be F  Ujr ( K ) ^ .  In fact,  Hodgkin and Horowicz (1959b) r e l a t e d g£ t o Pj^ by s u b s t i t u t i n g from t h e constant  field  s o l u t i o n f o r Ijr i n Ijr = gjr ( E  m  - E  K  ).  They mixed t h e i r  models, i n a sense, and t h e r e s u l t i n g r e l a t i o n s h i p between g^ and P^ i s a complicated  function of E  m  and the c o n c e n t r a t i o n s .  the pure " n e t e f f e c t " model i s a l s o a c o m p l i c a t e d concentrations, process  Thus the m o b i l i t y i n function of E  m  and the  as might have been expected when the c o m p l i c a t i o n s  o f the  a r e f o r c e d i n t o the m o b i l i t y as a p r o p o r t i o n a l i t y f a c t o r .  The Goldman-Hodgkin-Katz (GHK) treatment, as a l r e a d y s t a t e d , the ions as d i f f u s i n g through a regime o f reduced but c o n s t a n t a f t e r e n t e r i n g the channel by an u n s p e c i f i e d p r o c e s s .  regards  mobility,  The e n t r y i n t o the  channel i s i n c l u d e d i n t h e p e r m e a b i l i t y P as a p a r t i t i o n c o e f f i c i e n t  (^ ) :  the c o n c e n t r a t i o n o f i o n i n the membrane i s (3 times the c o n c e n t r a t i o n i n the b u l k s o l u t i o n .  A more d e t a i l e d model would t r e a t e n t r y i n t o and e x i t  from t h e channel as a m a s s - a c t i o n s i t u a t i o n , to occur  i n the channel.  Donnan e q u i l i b r i u m .  a g a i n w i t h d i f f u s i o n assumed  F o r example, T e o r e l l  (1935) regarded  t h i s as a  54  T h i s s o r t o f treatment was ion  used by Eisenman et a l . (1968) to  t r a n s p o r t v i a n e u t r a l mobile c a r r i e r s i n the membrane, but  can be taken over to the channel model almost i n t a c t . GHK  r e s u l t i s the  by N i c o l s k y  s u r f a c e s , which p r o v i d e m a t r i x of the g l a s s separately,  i n Eisenman 1967).  the s e l e c t i v i t y ,  and  ( d e s c r i b e d by a constant  A still  occurs v i a an e x t e r n a l  The  ( a r t i c l e s by Doremus, i n t e r a c t i o n s at  the d i f f u s i o n through m o b i l i t y ) are  to y i e l d a r e l a t i o n l i k e the GHK  transport  The  ;  type e q u a t i o n would s t i l l  further characterized,  be o b t a i n e d .  The  expression  counter-  "single-file"  Lakshminarayanaiah  but a l i q u i d - j u n c t i o n -  e s s e n t i a l feature of a l l of  models i s the d i f f e r e n t m o b i l i t y o f the c a t i o n and The u s u a l  the  circuit.  b e t t e r r e p r e s e n t a t i o n would take i n t o account the  m o b i l i t y would be  approach taken to i n c l u d e e l e c t r o g e n i c pumping i n the  for E  m  has  been to i n c l u d e the  f l u x e s are d e s c r i b e d  Noda 1963; arises  Moreton 1969;  i n the same way  a sense, the is  f l u x o f ions which occurs through  Schwartz 1971).  The  t h a t the r e s t o f the membrane p o t e n t i a l a r i s e s .  ' m o b i l i t y ' o f sodium ions  &  e l e c t r o g e n i c p o t e n t i a l then In  i n the membrane ( f o r outward movement) i s an apparent net  o f c a t i o n s , a p o t e n t i a l develops i n the manner d e s c r i b e d  If  the  by the N e r n s t - P l a n c k e q u a t i o n (eg. M u l l i n s  enhanced by the pump, so t h a t when t h e r e  movement  the  anion.  the sodium pump as a phenomenological term i n a f l u x balance wherein passive  the  treated  equation,' a l t h o u g h the  e f f e c t s which must occur i n a pore (Hodgkin & Keynes 1955; 1969).  been  Perhaps a b e t t e r example i s the model proposed  the p o t e n t i a l developed i n a g l a s s m i c r o e l e c t r o d e  by Eisenman, and  ion  the r e s u l t  e q u a t i o n plus an e x t r a term, where the p a r t i t i o n c o e f f i c i e n t has  characterized e x p l i c i t l y . for  The  describe  expulsion  above as  counterion  occurs. t h i s h y p o t h e s i s f o r the o r i g i n of the e l e c t r o g e n i c c o n t r i b u t i o n to  the r e s t i n g membrane p o t e n t i a l i s e s s e n t i a l l y c o r r e c t , a measurement o f  the  55  e l e c t r o g e n i c p o t e n t i a l i s a measurement o f the n e t i o n c u r r e n t sodium pump. to keep E pump.  Similarily,  i n a voltage-clamped c e l l ,  the current  steady i s a measurement o f the n e t i o n c u r r e n t  m  With t h e simultaneous use o f r a d i o i s o t o p e s  electrodes,  through t h e required  through t h e sodium  and i n t r a c e l l u l a r micro-  i t i s p o s s i b l e t o measure t h e sodium f l u x and the membrane  p o t e n t i a l s i m u l t a n e o u s l y , and t o d e t e c t  t h e simultaneous changes i n t h e two  when t h e sodium pump i s s e l e c t i v e l y impaired by exposure o f the c e l l t o ouabain.  Such experiments a r e d e s c r i b e d  i n s e c t i o n 7, and i t i s shown t h a t  the two measurements can .be r e l a t e d i n a t h e o r e t i c a l model l i k e described  above, t o y i e l d measurements o f p e r m e a b i l i t i e s  r a t i o o f the sodium pump. described  by o t h e r workers a r e a l s o  t h i s d i s c u s s i o n o f the membrane p o t e n t i a l , f u r t h e r  must be made o f the c o n t r i b u t i o n o f other p o t e n t i a l s t o the  measured v a l u e o f the membrane p o t e n t i a l . p o t e n t i a l s of micropipette problems.  or o f the c o u p l i n g  i n s e c t i o n 7.  Before concluding mention  V o l t a g e .clamp s t u d i e s  those  electrodes  The l i q u i d j u n c t i o n and t i p  a r e w e l l known, and a r e t e c h n i c a l  S e v e r a l workers have found t h a t an e l e c t r i c a l p o t e n t i a l d i f f e r e n c e  q u a l i t a t i v e l y and q u a n t i t a t i v e l y l i k e a Donnan p o t e n t i a l can be measured i n muscle c e l l s which have been ' c h e m i c a l l y g l y c e r o l or with detergent J.A.  Hinke - p e r s o n a l  s k i n n e d ' by e x t r a c t i o n w i t h  ( C o l l i n s & Edwards 1971; Pemrick & Edwards 1974;  communication).  The Donnan p o t e n t i a l due t o f i x e d  charges on t h e l a t t i c e o f c o n t r a c t i l e p r o t e i n s  must be c o n f i n e d  to a region  o f a t most a few hundred Angstroms diameter around t h e charges, y e t i t appears t o i n f l u e n c e the i n t r a c e l l u l a r The  microelectrode.  membrane p o t e n t i a l measured w i t h i n t r a c e l l u l a r t n i c r o e l e c t r o d e s  thus  might be d i f f e r e n t from the e l e c t r i c a l p o t e n t i a l d i f f e r e n c e between the bulk i n t r a c e l l u l a r s o l u t i o n and the e x t e r n a l Tasake and S i n g e r  solution.  (1968) review s e v e r a l problems i n v o l v e d  in electrical  56  measurements  o f b i o l o g i c a l systems.  electrochemist  They remark t h a t "no r i g h t - m i n d e d  would even attempt to p e r f o r m meaningful measurements  the complex c o n d i t i o n s which a r e r e q u i r e d t o m a i n t a i n l i v i n g systems,!' but conclude t h a t meaningful measurements "proper p r e c a u t i o n s  E.  under  biological  can be made i f the  a r e observed."  SUMMARY OF THE PROBLEMS TO BE ADDRESSED  The p r i n c i p a l o b j e c t  of  this  t h e s i s i s the measurement  and i n t e r p r e t a -  t i o n o f the sodium and hydrogen i o n e f f l u x from whole c e l l s .  Hydrogen i o n  e f f l u x can o n l y be measured i n d i r e c t l y ; most o f the work t o be  described  concerns t h e sodium e f f l u x . It  i s c u r r e n t l y b e l i e v e d t h a t i t i s mainly the c e l l membrane which  c o n t r o l s the i o n content o f the c e l l , and  by i t s p a s s i v e  i t s a b i l i t y t o t r a n s l o c a t e ions a g a i n s t  permeability  properties  the f o r c e s which e f f e c t p a s s i v e  flow. The membrane t r a n s p o r t  r e a c t i o n s can be viewed as enzyme r e a c t i o n s and  c h a r a c t e r i z a t i o n of the t r a n s p o r t  can be c a r r i e d out i n the context  of  enzyme k i n e t i c s . One enzyme system, the (Na+K)ATPase, has been i s o l a t e d i n an a c t i v e form.  D i s c u s s i o n o f sodium t r a n s p o r t tends t o be dominated by d i s c u s s i o n  of the (Na+K)ATPase,  but t h e r e appear t o be other membrane mechanisms  which sodium can be t r a n s p o r t e d  i n whole c e l l s .  the a c t i v i t y o f the enzyme i n the whole c e l l  by  The c h a r a c t e r i z a t i o n o f  i s required before  a  component  of the measured f l u x can be a s c r i b e d t o a p a r t i c u l a r enzyme which has been e x t r a c t e d and s t u d i e d  in isolation.  57  The a c t u a l measurement o f the sodium e f f l u x from whole c e l l s s e v e r a l problems. characterized,  The s t a t e of^sodium i n s i d e the c e l l has not been w e l l  a prominent problem b e i n g the e s t i m a t i o n  sodium which r e s i d e s  i n e x t r a c e l l u l a r regions,  the c e l l membrane per se.  that  o f the amount o f  i s , i n regions  The sodium c o n c e n t r a t i o n  bathes .the i n t e r n a l s u r f a c e it  involves  outside  i n the s o l u t i o n which  o f the c e l l membrane should be measured,  because  i s the c o r r e c t parameter t o use f o r the i n t e r p r e t a t i o n o f experiments i n  the c o n t e x t o f the enzyme k i n e t i c s model. microelectrode  An i n t r a c e l l u l a r  sodium-specific  w i l l measure t h i s parameter, even when i t changes r a p i d l y .  In the l a s t p a r t o f t h i s s e c t i o n i s d e s c r i b e d  a r e v i s e d e q u a t i o n by  which the sodium e f f l u x from whole b a r n a c l e muscle c e l l s can be c a l c u l a t e d from simultaneous m i c r o e l e c t r o d e  and r a d i o i s o t o p e  measurements.  Two  other  models, r e l a t e d to the steady s t a t e c a t i o n d i s t r i b u t i o n and t o the r e l a t i o n between t h e sodium e f f l u x and the membrane p o t e n t i a l , a r e a l s o  described  there. In s e c t i o n 3 a r e d e s c r i b e d sodium-specific altered.  microelectrode  measurements made w i t h an  on c e l l s whose sodium content had been  The r e s u l t s and those o f o t h e r workers a r e c o n s i s t e n t w i t h a simple  model o f the s t a t e s o f sodium i n the c e l l which i n c l u d e d and e x t r a c e l l u l a r p o o l s o f s e q u e s t e r e d In s e c t i o n 4 i s d e s c r i b e d microinjection.  T h i s was  An a l t e r n a t e  M i c r o i n j e c t i o n was the  intracellular  sodium.  an i n v e s t i g a t i o n o f the use o f i n t r a c e l l u l a r  b a r n a c l e muscle c e l l s  interpreted  i n terms o f a model  the s t a t e s o f sodium i n the c e l l which c o n t r a d i c t s  i n s e c t i o n 3.  both  c a r r i e d out because o t h e r workers had  t h e i r r e s u l t s on m i c r o i n j e c t e d for  intracellular  the model  described  i n t e r p r e t a t i o n of t h e i r r e s u l t s i s presented.  a l s o s t u d i e d because i t was  i n t e r i o r of c e l l s with radioisotope  desired  t o use i t to l o a d  r a p i d l y and s e l e c t i v e l y , as a  convenience i n c a r r y i n g out e f f l u x experiments.  M i c r o i n j e c t i o n i s shown t o  58  be r e l a t i v e l y , cell  a l t h o u g h not e n t i r e l y benign as  i s concerned.  f a r as the b a r n a c l e muscle  I t i s shown t o be e q u i v a l e n t to a p a s s i v e method f o r  l o a d i n g the c e l l w i t h r a d i o i s o t o p e , a s i d e from i t s f a i l u r e to l o a d the e x t r a c e l l u l a r space,  once c e r t a i n c o r r e c t i o n s a r e a p p l i e d .  In s e c t i o n 5 a survey o f the sodium e f f l u x from b a r n a c l e muscle c e l l s i s presented.  I t i s shown t h a t the dependence of the sodium e f f l u x on  the  i n t r a c e l l u l a r sodium c o n c e n t r a t i o n i s s i m i l a r to t h a t i n s q u i d axon and s n a i l neurone. not occur over sodium-free Ringer's and  S a t u r a t i o n o f the e f f l u x  i n t o normal Ringer's  the wide range o f sodium content  studied.  The  efflux  into  s o l u t i o n i s shown to be v e r y s i m i l a r to t h a t i n t o normal  solution.  The  b e h a v i o r of the sodium e f f l u x i n t o  potassium-free  i n t o o u a b a i n - c o n t a i n i n g s o l u t i o n s i s shown to be almost  results  s o l u t i o n does  f o r barnacle d i f f e r  identical.  The  i n s e v e r a l r e s p e c t s from those of p r e v i o u s  workers because o f d i f f i c u l t i e s w i t h the m i c r o i n j e c t i o n and r a d i o i s o t o p e techniques which have not been r e c o g n i z e d b e f o r e . In s e c t i o n 6 the measurements w i t h the s o d i u m - s p e c i f i c m i c r o e l e c t r o d e and w i t h r a d i o s o d i u m a r e compared f o r e f f l u x i n t o sodium-free C o n s i s t e n c y o f the model and i s demonstrated, and  techniques  developed  the nature of the sodium-free  solutions.  i n the p r e c e d i n g s e c t i o n s effect  is considerably  clarified. In s e c t i o n 7 an e l e c t r o g e n i c c o n t r i b u t i o n to the membrane p o t e n t i a l b a r n a c l e muscle i s demonstrated. membrane p o t e n t i a l and  of  The c o r r e l a t i o n between the e l e c t r o g e n i c  the a c t i v e sodium e f f l u x i s measured, and  p r e t e d i n terms o f an e x t e n s i o n of the GHK  is inter-  model f o r the membrane p o t e n t i a l .  In s e c t i o n 8 the r e s u l t s of measurement of the i n t r a c e l l u l a r pH u s i n g p H - s p e c i f i c g l a s s m i c r o e l e c t r o d e s a r e presented.  I t i s shown t h a t i n the  b a r n a c l e muscle c e l l p r e p a r a t i o n used i n the p r e s e n t work, there a r e no t r a n s i e n t s " o f the type r e p o r t e d by o t h e r workers.  Also, a relationship  "pH  59  between t h e steady d i s t r i b u t i o n o f hydrogen ions and the r e s t i n g membrane potential ion  i s d e s c r i b e d , and an e s t i m a t e o f the s i z e o f t h e a c t i v e hydrogen  e f f l u x i s made. In  s e c t i o n 9 i s p r e s e n t e d a d i r e c t comparison  o f measurements o f the  i n t r a c e l l u l a r pH made w i t h the m i c r o e l e c t r o d e and w i t h an i n d i c a t o r method Such a comparison  can r e v e a l the e x i s t e n c e o f s u b c e l l u l a r compartments  h a v i n g a r e l a t i v e l y low o r h i g h pH, but the most important r e s u l t s o f t h i s study turned out t o be those c o n c e r n i n g t h e a p p l i c a b i l i t y o f the i n d i c a t o r method. In  s e c t i o n 10 i s p r e s e n t e d a b r i e f d i s c u s s i o n o f the s i g n i f i c a n c e o f  the r e s u l t s and some suggestions f o r f u r t h e r work.  F.  SUMMARY OF MODELS  P a r t o f the development o f the models p r e s e n t e d here r e l i e s on r e s u l t s which have not y e t been d e s c r i b e d .  The r e a d e r might wish t o proceed  d i r e c t l y t o s e c t i o n 3 and t o r e f e r back t o t h i s s e c t i o n when r e f e r e n c e s t o these models a r e encountered.  (i)  E f f l u x o f sodium from a whole c e l l . I t was d e s i r e d t o formulate an e x p r e s s i o n by which the sodium e f f l u x  from a whole c e l l In  c o u l d be c a l c u l a t e d  from experimental measurements.  the model o f the c e l l used by e a r l y workers  (Keynes & Lewis 1951;  Keynes 1951), i t was assumed t h a t the i n t r a c e l l u l a r medium i s a simple s o l u t i o n o f s a l t s and o r g a n i c molecules b a t h i n g the i n t e r n a l s u r f a c e o f the cell  membrane, and t h a t o n l y a n e g l i g i b l y s m a l l f r a c t i o n o f the transmembrane  60  passage o f ions occurs by simple d i f f u s i o n .  I n such a s i t u a t i o n ,  i f one  c o u l d r e p l a c e some o f the i n t r a c e l l u l a r sodium w i t h l a b e l l e d sodium i o n s , the  unidirectional  e f f l u x o f sodium ions from the c e l l  from the net e f f l u x )  c o u l d be deduced from the e f f l u x o f the l a b e l l e d  into a large bathing solution assumed t h a t the l a b e l l e d as  (which i s d i f f e r e n t  c o n t a i n i n g no l a b e l l e d  sodium i o n s .  Thus: the s p e c i f i c a c t i v i t y o f sodium i n t h e c e l l  A  22 Na 23 moles Na 22 A moles Na moles N a _ moles  -  2 3  N a = N a  i n the c e l l i n the c e l l in cell in cell  +  moles  can be approximated  Na i n the c e l l  . „  cell 22  (moles o f  c e l  (moles o f N a 2 3  Na i n the c e l l ) i s much s m a l l e r than N a - Q ce  i n the c e l l ) i n p r a c t i c e .  A s h o r t i n t e r v a l o f time " t " i s c o n s i d e r e d . discrete  22  22 23 Na and Na.  cell  * where N a  It is  i o n behaves j u s t as the abundant i o n does as f a r  i o n movements a r e concerned, an assumption s a t i s f i e d by  ^ cell  ions  collection intervals  any i n s t a n t  used i n p r a c t i c e  (The e x t e n s i o n from the  to a function a p p l i c a b l e a t  i s assumed i m p l i c i t l y i n t h i s development.)  I f , i n time t , Na  moles o f r a d i o s o d i u m l e a v e the c e l l w i t h no b a c k f l u x o f radiosodium, then the  t o t a l number o f moles o f sodium l e a v i n g the c e l l  the  efflux density i s Mjj (moles/cm -sec) = a  Na SA  .  1 A  c e l l  .  i s Na  / S A - Q and ce  1  t  ...(1)  where A i s the area o f the membrane a c r o s s which the sodium ions pass. I f V i s tMh e volume = _JL_ o •f_ £ the S _ i n t r a•c ev»«v l l u l ell ar fluid, Na A-t * cell Y_ • Na* . (Na) C  N  where ( N a )  c e l l  = 'Na  w a  cell  then  n i  /V.  ...(2)  61  In an a c t u a l experiment, Mjj c o l l e c t i o n o f Na •^ cell a  a  t  t  *  a  as a f u n c t i o n o f time i s determined  f o r each of a s u c c e s s i o n  b e g i n n i n g o f each c o l l e c t i o n p e r i o d  i e  *  a d d i t i o n " o f the Na  values  o f the experiment.  not  change much d u r i n g a c o l l e c t i o n  this off  It is required  that  The  i s found by  to the r a d i o a c t i v i t y l e f t  end  The  o f time p e r i o d s .  by  value  of  "back-  i n the c e l l a t  ( N a ) - Q be known, and  the  that i t  C£  period.  r e s u l t s o f experiments on s q u i d axons seemed to be c o n s i s t e n t w i t h  formulation.  The  r a t e a t which r a d i o s o d i u m came out of the c e l l  as a s i m p l e e x p o n e n t i a l  w i t h time, and was  fell  a p p r o x i m a t e l y equal t o  the  i n f l u x r a t e , which can be measured d i r e c t l y from s e p a r a t e i s o t o p e uptake experiments (Keynes & Lewis 1951; c e l l s are more d i f f i c u l t complicated  to i n t e r p r e t .  illustrated  r e s u l t s f o r muscle been t o employ more exchange  (eg. Keynes &  Steinhardt  been c o m p l e t e l y s u c c e s s f u l ,  but  i n p a r t the c u r r e n t view o f the heterogeneous s t r u c t u r e o f  the  the s t a t e o f ions and water i n s i d e the c e l l which would seem to  make the above model u n t e n a b l e f o r most The  t r e n d has  i n F i g . 1(b)  These i n t e r p r e t a t i o n s have not  recognize c e l l and  The  The  models h a v i n g s e v e r a l c e l l u l a r compartments which can  sodium i o n s , such as t h a t 1968).  Keynes 1951).  cells.  f e a t u r e s which p r o b a b l y g i v e r i s e to e r r o r are as  the r e l e v a n t c o n c e n t r a t i o n s  a r e those o f the  follows.  First,  'myoplasm', the l a r g e aqueous  compartment i n s i d e the c e l l which i s much l i k e a bulk aqueous s o l u t i o n and which i s assumed t o bathe the s t r u c t u r a l l a y e r deep to the p a r t o f the concentration  i n t e r n a l s u r f a c e o f the c e l l membrane. 'bimolecular  ' f u n c t i o n a l membrane'.)  l e a f l e t ' w i l l be assumed t o  ( N a ) | ^ must be r e p l a c e d  by  c e  o f sodium i n f r e e s o l u t i o n i n the myoplasm (Na)  .  (Any be  the The  latter  m can be measured r e l i a b l y as to be 0.65.  This  (Na)  m  = (ajja)m / tf+ •  The  value  for y  i s taken  i s j u s t i f i e d by the concept o f the myoplasmic compartment  as a s o l u t i o n l i k e a bulk s o l u t i o n , and  by an e x p e r i m e n t a l  determination  62  F i g u r e 1,  Models o f the c e l l used i n c a l c u l a t i o n  of i o n fluxes.  (A) Model on which e q u a t i o n ( 2 ) i s based. A l l of the i n t r a c e l l u l a r sodium i s i n s o l u t i o n i n a l l o f the i n t r a c e l l u l a r water, and can only exchange w i t h the e x t r a c e l l u l a r compartment (arrows). (B) Current model. I n t r a c e l l u l a r sodium can r e s i d e i n a nonmyoplasmic compartment exchanging only w i t h the myoplasmic compartment, or i n a nonmyoplasmic compartment which a l s o exchanges w i t h the e x t r a c e l l u l a r compartment. ' N o n p a r t i c i p a t o r y ' sodium and water a r e not shown, nor a r e a l l p o s s i b l e exchanges f o r a multicompartment system.  63  (Hinke  1970).  It  i s suggested by p r e v i o u s work (and c o n f i r m e d by the p r e s e n t e x p e r i -  ments) t h a t Mj^  a  ing of ( a j j ) a  changes,  m  i s a nondecreasing  function of ( a j j ) . a  d u r i n g e f f l u x experiments,  Continuous  m  e s p e c i a l l y those i n which  s h o u l d y i e l d a more r e l i a b l e measurement o f M^  a  monitor(aj\j ) a  m  than i s p o s s i b l e  from c h e m i c a l a n a l y s i s o f t o t a l c e l l u l a r sodium and b a c k - a d d i t i o n o f changes due  to transmembrane t r a n s p o r t . Na*  i s r e a d i l y and u n e q u i v o c a l l y measured as the amount o f r a d i o i s o t o p e  collected  i n the s o l u t i o n ..bathing the c e l l d u r i n g a g i v e n time  However, N a ^ n  i s a problem.  As  interval.  f a r as can be measured w i t h the p r e s e n t  t e c h n i q u e s , i n j e c t e d r a d i o s o d i u m i s d e p o s i t e d o n l y i n the myoplasm and  in a  s m a l l compartment which exchanges sodium w i t h the myoplasm v e r y q u i c k l y . T h i s was  concluded from the c l o s e s i m i l a r i t y o f the sodium e f f l u x from c e l l s  loaded w i t h r a d i o s o d i u m by m i c r o i n j e c t i o n and from c e l l s loaded by  immersion  i n normal R i n g e r ' s s o l u t i o n which c o n t a i n e d radiosodium, as d e s c r i b e d i n section  4.  M o r p h o l o g i c a l and p h y s i o l o g i c a l s t u d i e s r u l e out the o r g a n e l l e s as important s i t e s o f s e q u e s t r a t i o n o f sodium. a l a r g e enough c a p a c i t y , nor. b a r n a c l e muscle  membrane-delimited They do not have  r a p i d enough exchange w i t h the myoplasm i n  cells.  The c o n t r a c t i l e p r o t e i n s a r e known t o b i n d sodium and to exchange r a p i d l y w i t h the myoplasm.  T h e i r t o t a l c a p a c i t y , ca. 68 m i l l i m o l e s per kg 3  d r y weight  f o r r a p i d l y exchanging  potassium and hydrogen  sites,  would l a r g e l y be taken up  by  s i n c e they show o n l y a modest p r e f e r e n c e f o r sodium  over p o t a s s i u m and the i n t r a c e l l u l a r c o n c e n t r a t i o n o f potassium i s much  moles Na kg dry weight  moles Na kg c e l l water  ,  7 water 1 - 7 water 0  0  64  g r e a t e r than t h a t o f sodium. From experiments c e l l s was  altered  They c e r t a i n l y s h o u l d b i n d some sodium,  though.  i n which the t o t a l sodium content o f b a r n a c l e muscle  (section 3 ) ,  i t was  concluded t h a t o f the t o t a l amount o f  sodium a s s o c i a t e d w i t h the c e l l a f t e r account had been taken o f e x t r a c e l l u l a r sodium by the u s u a l t e c h n i q u e s , a l a r g e amount o f e x t r a c e l l u l a r sodium was still  included.  I f this  i s taken i n t o account, then about  c e l l u l a r sodium i s not a c c e s s i b l e t o the m i c r o e l e c t r o d e .  3 0 7  The most generous  p r o b a b l y l e s s than 1 0 7 o )  e s t i m a t e was  intra-  207o  experiments  t h a t l e s s than 1 5 7 o  (and  o f the i n t r a c e l l u l a r sodium i s i n r a p i d exchange  w i t h the f r e e sodium, where the f r e e sodium r e p r e s e n t s about sodium which i s t r u l y  o f the  About  exchanges so s l o w l y as t o be n o n p a r t i c i p a t o r y i n the f l u x d i s c u s s e d here.  o  707°  o f the  intracellular.  More important i s the e x i s t e n c e o f an e f f e c t i v e i n t r a c e l l u l a r s i n k o f i n j e c t e d r a d i o s o d i u m due t o l o n g i t u d i n a l d i f f u s i o n o f r a d i o s o d i u m i n i n j e c t e d c e l l s , . a s d i s c u s s e d i n s e c t i o n 4. c o n t e n t was  For the c e l l s  not r a i s e d by m i c r o i n j e c t i o n o f NaCl s o l u t i o n s ,  r a d i o s o d i u m a s s o c i a t e d w i t h the c o n t r a c t i l e p r o t e i n s , appears  to be e q u i v a l e n t t o about  ments r e p o r t e d here.  The e f f e c t  157  D  t h i s plus  i n a r e a l i s t i c estimate,  o f i n j e c t e d r a d i o s o d i u m i n the e x p e r i -  i s g r e a t e r when NaCl  is injected.  However, the amount l o s t t o the s i n k i s d i f f e r e n t and  i n which the sodium  in different  i n c r e a s e s from the i n i t i a l v a l u e o f z e r o as the experiment  Thus a v a l u e Na* of N a J  e l l  cells,  progresses.  f o r the 'myoplasmic r a d i o s o d i u m ' s h o u l d be used i n p l a c e  .  A c o r r e c t i o n can be a p p l i e d t o the data to account d e f i n i t i o n o f the e f f l u x d e n s i t y ( e q u a t i o n ( 1 ) ) amount o f sodium which comes out o f the c e l l  for this.  In the  i t i s assumed t h a t  the  i n u n i t time i s r e l a t e d t o the  amount o f r a d i o s o d i u m which comes out i n u n i t time, Na  , v i a the  specific  a c t i v i t y o f r a d i o s o d i u m i n a homogeneous i n t r a c e l l u l a r compartment c o n t a i n -  65  ing  o n l y exchangeable  assumption appears  sodium.  An e q u i v a l e n t  statement o f t h i s  o f the t r a c e r method i s t h a t the r a t e a t which  i n t h e bath, Na*/t,  exchangeable  radiosodium  i s d i r e c t l y p r o p o r t i o n a l t o the amount o f  radiosodium i n s i d e the c e l l , Na . m  via  fundamental  Thus  t  membrane  under steady c o n d i t i o n s , where the i n t e r i o r o f the c e l l  i s w e l l - m i x e d and  the s i z e o f the e f f l u x doesn't change too much over a c o l l e c t i o n p e r i o d t = 5 minutes.  Note t h a t i t i s assumed i n a d d i t i o n t h a t the c o n t i n u i n g  loss of  r a d i o s o d i u m from the myoplasmic compartment t o the i n t r a c e l l u l a r s i n k i s entirely The  independent o f the l o s s o f r a d i o s o d i u m a c r o s s the c e l l  membrane.  'constant' k i s then the i n s t a n t a n e o u s s l o p e o f the p l o t o f InNa  time.  I t bears r e p e a t i n g  t h a t the a p p r o x i m a t i o n w i l l  versus  be c l o s e s t t o r e a l i t y  where the s e m i l o g p l o t ( I n Na* v e r s u s time) i s l i n e a r and n o t too s t e e p , for  then i t i s most l i k e l y  t h a t the myoplasmic compartment  and t h a t the u s e o f a time r e s o l u t i o n o f 5 minutes w i l l  i s well-mixed,  n o t i n t r o d u c e too  much e r r o r . The e f f e c t o f the c o n t r a c t i l e p r o t e i n s c o u l d be c o r r e c t e d  for v i a a  model f o r the c o m p e t i t i o n o f sodium and p o t a s s i u m f o r the s i t e s on the protein,  i f such a r e l a t i v e l y s m a l l c o r r e c t i o n were f e l t  Another problem a r i s e s w i t h V/A. i s s i m i l a r t o t h a t a t the s u r f a c e the s y n c y t i a l  muscle  fibre  t o be n e c e s s a r y .  The membrane i n the c l e f t s  presumably  ( t h e two develop from the same source as  i s formed,  and f u n c t i o n a l l y i t i s r e a s o n a b l e  t h a t they s h o u l d have s i m i l a r p r o p e r t i e s , a l t h o u g h the membrane o f the TTS p r o b a b l y i s d i f f e r e n t - G i r a r d i e r e t a l . 1963). cleft  I f t h e f u n c t i o n o f the  system is_ t o ensure t h a t a l l p a r t s o f t h e i n t e r i o r o f the c e l l a r e  within a c e r t a i n distance  from some p a r t o f the c e l l membrane, the s u r f a c e -  66  to-volume r a t i o might w e l l be  independent  o f the diameter  of the c e l l .  A  f u r t h e r c o m p l i c a t i o n i s t h a t the volume r e p r e s e n t e d by V s h o u l d be t h a t o f the myoplasmic compartment, as d i s c u s s e d above and I t was  concluded  i n the  t h a t r e a s o n a b l e approximations  i n the f o r m u l a t i o n o f a p r a c t i c a l e q u a t i o n .  introduction.  would have to be made  Thus: conductance measurements  i n d i c a t e t h a t the v a l u e s f o r the membrane c a p a c i t a n c e and r e s i s t a n c e f o r the b a r n a c l e muscle c e l l  can l a r g e l y be r e c o n c i l e d w i t h those  membranes of o t h e r c e l l s  i f the e f f e c t i v e a r e a o f homogeneous c e l l  i s about ten times  the apparent  (Hagiwara et a l . 1964;  f o r the  s u r f a c e a r e a of the c y l i n d r i c a l  B r i n l e y 1968).  membrane  cell  For a c y l i n d e r of r a d i u s r , V/A  =  r/2 so here V/A w i l l be taken t o be equal to r/20. Thus: M  / i / 2 \ (moles/cm^sec)  =  =  i  r 20  1 300sec  2.56  x 10"  1 0  Na* —— Na  v  m  (a„ ) Na m 0.65  litre cm  r  3  where r (cm)  i s taken to be the average  perpendicular directions, i n moles/cm -sec.  Na / N a  and m  (a^ ) a  m  of the c e l l  V  .  ...(4)  r a d i i measured i n  is in millimoles/litre,  i s the r a t i o of counts  p e r f u s a t e sample t o c a l c u l a t e d counts  Na*_ Na*  to y i e l d  per minute i n a 300  M^  a  second  per minute i n the c e l l a t the s t a r t  of  the c o l l e c t i o n p e r i o d f o r those c e l l s which have a " s l o p e r a t i o " c l o s e to unity,  i e . i n essence  those c e l l s not m i c r o i n j e c t e d and some o f  i n j e c t e d w i t h s o l u t i o n s of v e r y low sodium c o n c e n t r a t i o n . Na*/Na* s h o u l d be c a l c u l a t e d time,  =  k  5  For o t h e r  from the s l o p e k of a p l o t of l n Na*  i d e a l l y o n l y where such a p l o t  Na  those  minutes.  i s l i n e a r and not too steep,  cells,  versus as  67  This l i m i t s effluxes.  the use o f m i c r o i n j e c t e d c e l l s  Such w i l l  occur  i n t o normal Ringer's  i n t o sodium-free s o l u t i o n , but s o l u t i o n s the c e l l w i l l (a^ ) a  m  i n c r e a s e s , and  cases,  s o l u t i o n , and  i n potassium-free  or  g a i n sodium c o n t i n u o u s l y .  A reasonable  but  steady  i n most  cases  ouabain-containing The  the s l o p e of the p l o t o f In Na  z e r o or even p o s i t i v e . made i n these  to measurements of  e f f l u x increases versus  as  time can become  e s t i m a t i o n o f Na*/Na* u s u a l l y can  the r e s u l t i n g c a l c u l a t e d v a l u e o f Mjj  a  be  i s more  u n c e r t a i n than i t i s f o r steady e f f l u x e s . Nevertheless, convenience, and  m i c r o i n j e c t i o n does have some v i r t u e s .  economy o f the m i c r o i n j e c t i o n technique  degree the u n c e r t a i n t y due  to l o n g i t u d i n a l d i f f u s i o n .  i n t r a c e l l u l a r compartments appear to be loaded, but  the e x t r a c e l l u l a r space i s not  loaded.  i s o t o p e i s r e q u i r e d f o r each experiment. a f t e r d i s s e c t i o n , when they should be The  determination  of N a  m  The  selectivity,  o f f s e t to some  A l l 'participatory'  j u s t as w i t h p a s s i v e  loading,  Only a s m a l l amount o f r a d i o Finally,  c e l l s can be used s h o r t l y  i n a s t a t e most l i k e t h a t i n v i v o .  would be more a c c u r a t e  p a r t o f a l o n g segment of i n j e c t e d c e l l were p e r f u s e d experiments.  The  s t r a t e g y adopted i n the present  i f o n l y the c e n t r a l in microinjection  experiments was  to  perfuse  o n l y a l o n g i n j e c t e d segment o f the c e l l r a t h e r than o f p e r f u s i n g a l o n g cell ful  o n l y p a r t o f which had  T h i s was  only p a r t i a l l y  i n e l i m i n a t i n g the problem o f l o n g i t u d i n a l d i f f u s i o n .  e l i m i n a t e d by the use d e a l o f i s o t o p e and and  been i n j e c t e d .  of p a s s i v e l o a d i n g , but  long periods  'time constant  problem i s  t h i s method r e q u i r e s a great  o f i n c u b a t i o n o f the c e l l a f t e r d i s s e c t i o n ,  loads the e x t r a c e l l u l a r space w i t h The  The  success-  isotope.  f o r exchange' Na*/Na* ^^ has e  been taken to r e f l e c t  the o p e r a t i o n o f the t r a n s p o r t systems o f the membrane most d i r e c t l y  (eg.  Dick & Lea  efflux  1967;  B r i n l e y 1968).  should be c a l c u l a t e d .  I t can be asked why  the s i z e of the  Q u i t e a s i d e from the problems o f u s i n g Na  /Na£ ;Q e  68  for microinjected c e l l s ,  i t can be seen  from e q u a t i o n (4) t h a t the sodium  content o f the myoplasm must be taken i n t o account  i f the a c t i v i t y o f the  t r a n s p o r t mechanisms i s t o be deduced from i s o t o p e measurements. v a l u e s of Na*/Na* i n c e l l s /pumping r a t e s ' .  o f d i f f e r e n t sodium content r e f l e c t  Identical  different  As can be seen from the manner i n which e q u a t i o n  (2) i s  d e r i v e d from the d e f i n i t i o n o f f l u x d e n s i t y ( e q u a t i o n ( 1 ) ) , the appearance o f the  'time c o n s t a n t ' i n the e f f l u x e q u a t i o n i s i n i n t i m a t e a s s o c i a t i o n  w i t h the s p e c i f i c a c t i v i t y o f r a d i o s o d i u m Similarily, misleading. Mjj  a  this  on  a  m  i s not so.  cell.  the appearance o f (aNa)m e x p l i c i t l y  I t might appear t h a t t h i s  (ajj ) .  i n s i d e the  Again, M^  a  examination  and  ( ^ )  imposes a s p u r i o u s dependence o f  o f equations  (1) and  (2) r e v e a l s t h a t  m u t u a l l y dependent i n s e v e r a l ways i n  a  a  i n e q u a t i o n (4) i s  m  the c o n t e x t of a l i v i n g c e l l d u r i n g an e f f l u x experiment (4) simply s e p a r a t e s  f o u r measurable q u a n t i t i e s  in vitro.  from which M^  a  Equation  can  be  calculated. I t must be asked a t t h i s p o i n t what advance a l l o f t h i s method o f Keynes and Lewis (1951). . r e a l i s t i c model of the c e l l and  Conceptually,  i s over  the  i t i s c e r t a i n l y a more  the i o n movements.  The main advance i s the  i n t r o d u c t i o n o f the s o d i u m - s p e c i f i c m i c r o e l e c t r o d e .  T h i s permits measure-  ment o f the t r u e s p e c i f i c a c t i v i t y  The  a l s o enables one  i n s i d e the c e l l .  t o measure the e f f l u x i n experiments  microelectrode  where ( a j j ) a  m  changes  rapdily. S e v e r a l fundamental problems remain, to-volume r a t i o cannot  however.  be taken f u r t h e r without  m o r p h o l o g i c a l measurements, which themselves  The  the performance of e x a c t i n g  a r e plagued w i t h u n c e r t a i n t i e s  i n the form of changes i n c e l l volume d u r i n g f i x a t i o n . s i g n i f i c a n t and d i f f i c u l t  q u e s t i o n of s u r f a c e -  The much more  q u e s t i o n o f the homogeneity of the c e l l  w i t h r e s p e c t to t r a n s p o r t p r o p e r t i e s has not been pursued  at a l l .  membrane In  69  practice,  the best  strategy  i s t o use c e l l s o f about the same s i z e , as was  done here, so any e r r o r due t o these f a c t o r s w i l l be about the same f o r each c e l l .  Finally,  the q u e s t i o n  of film-controlled d i f f u s i o n  l a y e r s " ) has not been addressed, a s i d e  from the e x p l i c i t  assumption t h a t boundary l a y e r s a r e c o n s i d e r e d membrane'.  ("unstirred  statement o f the  t o be p a r t o f the ' f u n c t i o n a l  I f a r e a l l y good e s t i m a t e o f the magnitude o f the sodium e f f l u x  i s t o be o b t a i n e d , these d i f f i c u l t  problems must be s o l v e d .  In the remainder o f t h i s t h e s i s , e q u a t i o n (4) i s used t o c a l c u l a t e the sodium e f f l u x from b a r n a c l e muscle c e l l s , whether loaded w i t h  radio-  sodium by m i c r o i n j e c t i o n o r by immersion i n a s o l u t i o n c o n t a i n i n g  radio-  sodium.  The a p p r o p r i a t e  c o r r e c t i o n should be a p p l i e d  i n the former case.  ( i i ) Steady s t a t e d i s t r i b u t i o n o f c a t i o n s . In the experiments d e s c r i b e d o f hydrogen ions measured d u r i n g  i n s e c t i o n 8 c o n c e r n i n g the d i s t r i b u t i o n steady c o n d i t i o n s  (the use o f the term  'steady s t a t e ' i n t h i s c o n t e x t has been c r i t i c i z e d ) ,  a r e l a t i o n s h i p between  the membrane p o t e n t i a l and the transmembrane d i f f e r e n c e i n pH was found. Such a r e l a t i o n s h i p had been sought by o t h e r workers, but had n o t been found.  I n t h e d i s c u s s i o n o f t h e r e s u l t s , the r e l a t i o n s h i p d e r i v e d here from  elementary t h e o r y w i l l  be employed.  ions as w e l l as hydrogen  These c o n s i d e r a t i o n s  a p p l y t o sodium  ions.  Assume the c e l l membrane i s a l a m e l l a u n i f o r m i n the y and z d i r e c t i o n s of a Cartesian  coordinate  to the membrane s u r f a c e . density jP(x)  system h a v i n g the x a x i s d i r e c t e d  A t a p o i n t x, i n the membrane, the n e t f l u x  j ( x ) (moles/cm sec)  i s assumed t o be the sum o f a f l u x  due t o d i f f u s i o n o f hydrogen ions as d e s c r i b e d  equation, and an a d d i t i o n a l f l u x d e n s i t y specified.  Thus  perpendicular  density  by t h e N e r n s t - P l a n c k  j ( x ) which i s not f u r t h e r m  70  j(x)  =  j ( x ) - u(x) • c(x) |R • T 9_  I n c(x) + F 9 p(x) j  m  where u(x) and c ( x ) a r e the m o b i l i t y and c o n c e n t r a t i o n hydrogen ions  (or a c t i v i t y ) o f  f r e e t o d i f f u s e a t x, (6(x) i s the e l e c t r i c a l p o t e n t i a l a t x,  R i s the gas content, T i s the a b s o l u t e constant.  ...(5)  temperature, and F i s t h e Faraday  Note t h a t an e f f l u x i s p o s i t i v e i n s i g n .  To o b t a i n a r e l a t i o n  between the net transmembrane f l u x d e n s i t y and the transmembrane d i f f e r e n c e o f c and p, e q u a t i o n (5) must be i n t e g r a t e d across steady c o n d i t i o n s  are considered  the membrane.  here, so t h e t o t a l  independent o f x i n t h e membrane, a l t h o u g h j  m  Only  flux density j i s  need not be.  Multiplying  both s i d e s by £exp(Fp(x)/RTj)^i(x) and making use o f the i d e n t i t y  £—\  ^*  c(x) exp(F(6(x)/RT/  =  L  one f i n d s  j = _ 1 _ £ M - RT £ c ( a ) - c(0) e x p ( F E / R T ) J j m  where  * Q  "  exp[_JL  I x=b %  M  exp(Fp(x)/RT) U_c(x) + c ( x ) F ? (6(x) 1** R-T ^x" -  .  ( p(x) - p(a) )]  J  I RT  . dx  j ( x ) e x p f ^ . ( p ( ) -.(6(a) .)] = j l_RT J_ . dx * u(x) x=o ' m  x  v  and  E  m  = (6(0) - (6(a) i s the membrane p o t e n t i a l , and i s g e n e r a l l y  Also, Q>0  and i f j ( x ) r e p r e s e n t s  an e f f l u x , M>0.  m  negative.  ( T h i s r e l a t i o n has  been d e r i v e d by Schwartz (1971) ) . I f a t l e a s t one compartment vanish  is finite,  i n the steady s t a t e , so M = RT \_ c(a)  the net t o t a l f l u x d e n s i t y - c(0) exp F (6/RTj .  must  (Note  t h a t t h i s argument c o u l d have been a p p l i e d a t e q u a t i o n (6).) I f the i n t e r n a l pH i s pH^ = -log^Q c ( 0 ) and the e x t e r n a l pH i s p H arrangement y i e l d s  Q  = -log-^Q c ( a ) , r e -  71  P  H  - p  e  - log  H l  1 0  Note t h a t the c o n s t r a i n t taken to be  M,  and  with  on M i s now  - _I_  E  0<M<RT'c(a).  i n the e x t e r n a l  be c o n s i d e r e d w i t h r e s p e c t  the v a l u e s of  (pH  - pH^)  Q  m  ( 6 )  Also,  x = 0 can  o f the membrane,  and  b a t h i n g s o l u t i o n , so s u r f a c e  to (6 or c except i n the  and  be  E  evaluation  i n e q u a t i o n (6) are  m  effects of  those measured  microelectrodes. The  c o r r e s p o n d i n g r e l a t i o n s h i p f o r sodium ions i s :  - log  logoff.)!  (iii)  - —Hj-]  i n the myoplasm, away from the s u r f a c e  x = a s i m i l a r i l y to be need not  fl  1 0 ( H  Electrogenic  a)  -  o  log  contribution  1 0  (l - —M-^)  to the  „  I  0  i  n  f  E  The  usual strategy  i s t o c a r r y out a d e r i v a t i o n o f  membrane p o t e n t i a l as a d i f f u s i o n p o t e n t i a l , w i t h the b a l a n c e of an e x t r a  f l u x o f ions due  of Moreion (1969) w i l l  in section  i n the a n a l y s i s  The  transmembrane f l u x e s  (m^)  = -  calculated  f o r sodium ( n ^ ) > p o t a s s i u m (m^),  approximation: Na  . u F  mjr, VOQ-^ s i m i l a r i l y .  d dx  of  7.  from the N e r n s t - P l a n c k e q u a t i o n i n the c o n s t a n t  m  flux  derivation  o f the r e s u l t s  Moreton (1969) adopted the Goldman-Hodgkin-Katz model, and the p a s s i v e  by  the  i n c l u s i o n i n the  to a c t i v e t r a n s p o r t .  be adapted f o r use  the experiments d e s c r i b e d  chloride  electrogenic  to the r e s t i n g membrane p o t e n t i a l have been made (see review  Thomas 1972a).  ( 7 )  r e s t i n g membrane p o t e n t i a l .  A number o f p h y s i o c h e m i c a l models o f the c o n t r i b u t i o n of transport  ...  m  (  -  }  '  (  }  .  d N  a  0  dx  and  field  72  I f t h e r e a r e no f l u x e s but these, then i t must happen t h a t  "^a  +  s i n c e t h e c e l l cannot  =  k  m  0  accumulate a n e t charge.  The r e s u l t  i s t h e Goldman-  Hodgkin-Katz equation, as d i s c u s s e d above. I f t h e r e i s an a d d i t i o n a l component M o f i o n f l u x due, f o r example, t o an a c t i v e exchange o f sodium f o r potassium which i s not one-for-one,  then  the sum o f t h i s and t h e p a s s i v e f l u x e s i s z e r o :  ^Na and  +  m  K  "  ""Cl  +  =  M  0  the f o l l o w i n g expression i s obtained  P  Em  = E l  (K)  K  D  + P  N a  (Na)  (Moreton 1969)  Q  + P  In L  P  00 i + P a (  K  N a  N  c l  (Cl)i + R T F E„ m  > i + C 1 < >o + f - | P  U + — F  S  E  This  R_T  In  -c.  m  W +  E  m  JL1  F E m.  E  or  M  C1  F  M  m  M M  for  convenience.  i s t h e f a m i l i a r GHK equation, w i t h an a d d i t i o n a l term r e p r e s e n t i n g t h e  net pumped c a t i o n e f f l u x . the membrane p o t e n t i a l  When t h e n e t e f f l u x i n c r e a s e s , t h e magnitude o f  i n c r e a s e s because t h e n u m e r i c a l v a l u e o f t h e numera-  t o r i s l e s s than t h a t o f t h e denominator. This  i s not p r e s e n t e d as an exact d e s c r i p t i o n o f what occurs  but r a t h e r i s developed approximations  i n the s p i r i t  can be made t o f a c i l i t a t e comparison o f t h i s  w i t h experimental r e s u l t s . due  o f the GHK f o r m u l a t i o n .  t o t h e pump, i e . U «  Some f u r t h e r  relationship  Most o f t h e r e s t i n g membrane p o t e n t i a l W, so i t i s a good approximation  pump term i n t h e denominator t o y i e l d  in reality,  i s not  to neglect the  73  U  +  RJL M  , In  W When the c e l l i s exposed to ouabain, the pump term changes from M to a new value M ' , and the membrane potential changes from E  4E  E'  s  "m M  RT  ln  U  +  R T  U  +  R Tm  F E'  Thus:  F E„  M' M  >  0,  where i t i s assumed that U and W are unchanged complete the measurement of E^. R T In  4E. m  to E'.  'm  m =  M  Upon rewriting this as R T ln  M'l  1 +  UFE;  i n the time i t takes to  R-T  1 +  U  j  F Em  Ml  i t i s seen that the second term i n the argument of each of the logarithmic functions i s much less than unity.  I t i s thus a reasonable approximation  to expand each of the logarithmic functions i n a Taylor series and r e t a i n only terms to f i r s t order i n the small quantities R T U  F Em.  4E  where  m  AM  IF/ =  /R T\^  and  I  U/  UF E i  This yields:  M.  }  M  1  uE M <  M m  0  i *  . M' and  R T  /R TN^  has been assumed to be equal to /  O - E ;  h  1 '  ( F / U -  I  m  as  j  a matter of convenience. With one further step, the measured change i n the sodium efflux on exposure of the c e l l to ouabain can be introduced.  I f the coupling r a t i o  of sodium to potassium transported by the pump i s introduced:  74  (R*  -_^a  o  >  then the n e t pumped c a t i o n  M  AE  and  = =  ^ a  *K  +  flux is  "  ( 1  /R T\ . 1  -P  "  ^ a  . (1 - 1).  2  . . . (8) ..  4M  m This  the change i n membrane p o t e n t i a l 4 E  approximate e x p r e s s i o n r e l a t e s  to t h e change i n sodium e f f l u x  AMj^  a  m  i n a r e l a t i v e l y simple manner, i n the  context o f the u s u a l f o r m u l a t i o n f o r the o r i g i n o f the membrane p o t e n t i a l . r a t i o (R. o r the p e r m e a b i l i t i e s  E i t h e r the c o u p l i n g  i n U can be e v a l u a t e d  from e x p e r i m e n t a l data i f one or the o t h e r i s a l r e a d y known from independent measurements. The  proportionality  f a c t o r between the unbalanced c a t i o n e f f l u x and  the change i n E i s decreased i f the c e l l m c o n c e n t r a t i o n g r a d i e n t s a r e reduced. o f the pumped f l u x e s  This  on the Nernst-Planck  to oppose the charge s e p a r a t i o n  balance.  r e f l e c t s the  equation.  imposition  The f o r c e s which tend l i k e w i s e oppose t h e  because o f how the l a t t e r has  mobility  the e x i s t e n c e o f t h i s e f f l u x i s f i r s t  boundary c o n d i t i o n  or i f the  Although mechanistically  pumped c a t i o n e f f l u x i s l i k e an i n c r e a s e d the c e l l ,  feature  which a r i s e s p a s s i v e l y  unbalanced f l u x added phenomenological-ly; been imposed i n t h e c u r r e n t  i s hyperpolarized  the unbalanced  f o r sodium going out o f  apparent o n l y as a s o r t o f  o f the steady s t a t e p a s s i v e f l u x e s .  A b e t t e r and  p o t e n t i a l l y v e r y u s e f u l model would s p e c i f y some d r i v i n g f o r c e a c t i v e e f f l u x , such as the a f f i n i t y o f a chemical r e a c t i o n ,  f o r the  and would  acknowledge the i n t e r a c t i o n between the a c t i v e and p a s s i v e f l u x e s . repeating  that  active cation  such a model would r e f l e c t transport.  I t bears  the mechanism which b r i n g s about  75  SECTION 3.  THE STATES OF SODIUM IN CELLS  The c e l l  i s known t o be composed o f s e v e r a l d i s t i n c t  compartments.  These were d e s c r i b e d i n the I n t r o d u c t i o n .  i n t h e c e l l a r e d i s t r i b u t e d among these compartments.  morphological The sodium i o n s  I f i n an experiment  some l a b e l l e d sodium ions a r e i n t r o d u c e d i n t o one compartment, of  the exchange  sodium ions v i a d i f f u s i o n a l and n o n - d i f f u s i o n a l processes which occurs  c o n t i n u o u s l y between communicating compartments  will  tend t o b r i n g about a  steady d i s t r i b u t i o n i n which the s p e c i f i c a c t i v i t y o f sodium i s t h e same i n every  compartment. The r a t e a t which sodium exchange occurs i s n o t the same f o r every  communicating p a i r o f compartments.  One can c o n c e i v e o f a v e r y r a p i d  exchange, as between sodium ions i n the myoplasmic compartment  and t h e  sodium ions which a r e a c t i n g as c o u n t e r i o n s t o the f i x e d n e g a t i v e  charges  on a macromolecule i n a p r o t e i n m a t r i x immersed  compart-  ment.  On the o t h e r hand, one can c o n c e i v e o f a v e r y slow exchange, as  between myoplasmic  sodium ions and sodium ions which a r e c o u n t e r i o n s  i s o l a t e d by t h e hydrophobic In  i n the myoplasmic  b a r r i e r o f a c o i l e d and f o l d e d macromolecule.  the l a t t e r case, the exchange r a t e i s p r o b a b l y so slow t h a t , as f a r as  in v i t r o  i s o t o p e f l u x experiments a r e concerned,  such sodium ions a r e non-  participatory. Between these extremes a r e known t o l i e most o f the i o n t r a n s p o r t and exchange p r o c e s s e s o f the c e l l membrane. experiments,  In order to i n t e r p r e t  one would l i k e t o know the exchange r a t e s between  i n t r a c e l l u l a r compartments, c o n t a i n e d i n each  flux communicating  as w e l l as the f r a c t i o n o f the c e l l  sodium  compartment.  I o n - s p e c i f i c g l a s s m i c r o e l e c t r o d e s o f t h e type used t o make c e l l u l a r measurements a r e assumed t o sample the myoplasmic  intra-  compartment.  76  T h i s i s the i n t r a c e l l u l a r compartment  which behaves v e r y much l i k e a bulk  S o l u t i o n and i s not e n c l o s e d by s u b c e l l u l a r membranes, (see, f o r example, Hinke, C a i l l e , & Gayton 1973 and the d i s c u s s i o n f o l l o w i n g i t , and Edzes & Berendsen 1975). of  M i c r o e l e c t r o d e measurements have i n d i c a t e d t h a t o n l y a p a r t  the i n t r a c e l l u l a r sodium, potassium, and c h l o r i d e measured by chemical  a n a l y s i s o f whole c e l l s  i s i n f r e e s o l u t i o n i n the bulk water o f the  myoplasm (McLaughlin & Hinke 1966; D i c k & M c L a u g h l i n 1969; Lee & Armstrong 1972; Hinke et a l . not  1973; L e v & Armstrong 1975).  The s i z e o f the f r a c t i o n  a c c e s s i b l e t o the m i c r o e l e c t r o d e i n each case i s not c e r t a i n , because of  u n c e r t a i n t y about the volume o f the myoplasmic compartment for  and,  especially  sodium, because o f g r e a t u n c e r t a i n t y about the p o r t i o n o f the ions  which i s e x t r a c e l l u l a r ,  i n s o l u t i o n or s e q u e s t e r e d  One extreme e s t i m a t e i s t h a t f u l l y  (Lev & Armstrong 1975).  837, o f the i n t r a c e l l u l a r sodium can be  i n a c c e s s i b l e t o the sodium m i c r o e l e c t r o d e  (Hinke 1969b).  The sodium content o f the b a r n a c l e muscle c e l l can be i n c r e a s e d by immersion o f the c e l l  i n a potassium-free s o l u t i o n  (Beauge & S j o d i n 1967),  and can be decreased by immersion i n a sodium-free s o l u t i o n ( A l l e n & Hinke 1971).  I t was  expected t h a t the d i s t r i b u t i o n o f sodium i n s i d e the c e l l  would a l s o change d u r i n g such m a n i p u l a t i o n s . of the  myoplasmic and  'nonmyoplasmic'  Thus, changes i n the amount  i n t r a c e l l u l a r sodium were measured as  t o t a l sodium content o f the c e l l was  changed.  77  METHODS  Specimens. The specimens Georgia S t r a i t . Sound.  used i n a l l o f t h e Na experiments were o b t a i n e d from  Those used i n t h e pH experiments were o b t a i n e d from Puget  The morphology  d e s c r i b e d by P i l s b r y  o f t h e d i f f e r e n t s p e c i e s o f g i a n t b a r n a c l e s has been  (1916).  He s t a t e s t h a t t h e o v e r a l l form i s h i g h l y  v a r i a b l e , and t h a t t h e shape o f t h e o p e r c u l a r v a l v e s , the d e t a i l s o f t h e s t r u c t u r e o f t h e p l a t e s o f the w a l l , and t h e s t r u c t u r e o f t h e f e e t a r e t h e important c h a r a c t e r i s t i c s .  The l a r g e s t specimens, which a r e s p e c i f i c a l l y  chosen f o r m i c r o i n j e c t i o n work, a r e Balanus n u b i l i s , and o c c a s i o n a l l y B. aquila.  (These a r e t h e l a r g e s t N o r t h American  water.)  As noted by P i l s b r y ,  shell  b a r n a c l e s found i n s h a l l o w  these l a r g e r b a r n a c l e s a r e o l d , so t h e i r  i s worn and o f t e n r i d d l e d by b o r i n g animals.  affect  T h i s does n o t appear t o  t h e h e a l t h o f t h e b a r n a c l e , but makes the' i d e n t i f i c a t i o n o f a s p e c i e s  difficult.  B. n u b i l i s  i s unique i n t h a t as i t grows i t i n c r e a s e s i t s  i n t e r n a l volume by e x c a v a t i o n o f t h e b a s i s , and t h i s e x c a v a t i o n i s e a s i l y seen d u r i n g d i s s e c t i o n . between t h e muscle  A p p a r e n t l y t h e r e i s no p h y s i o l o g i c a l  difference  f i b r e s o f these two s p e c i e s .  Specimens were o b t a i n e d by d i v e r s , and kept i n a h o l d i n g tank a t t h e Vancouver P u b l i c Aquarium. the  tank c o n t i n u o u s l y .  Seawater  drawn from B u r r a r d I n l e t was r u n through  The o s m o l a r i t y o f t h i s seawater v a r i e d somewhat  over t h e year, (950+50 mOsm on a F i s k e osmometer). the  tank was n o t c l o s e l y monitored, but never exceeded t h r e e months.  B a r n a c l e s were moved t o a c o n t r o l l e d a r t i f i c i a l Ocean) a t t h e l a b o r a t o r y 1-3 weeks p r i o r t o use. was  The r e s i d e n c e time i n  seawater aquarium The a r t i f i c i a l  (Instant seawater  made up t o 960 mOsm (the v a l u e f o r normal R i n g e r ' s s o l u t i o n ) and main-  t a i n e d a t 10-12° C.  The b a r n a c l e s a c t i v e l y extended t h e i r c i r r i i n both  78  aquaria.  Dissection. Great c a r e was fibres.  taken t o minimize  The b a r n a c l e was  q u i c k l y k i l l e d by c u t t i n g through the o p e r c u l a r  adductor and removing the c i r r i , mass.  This l e f t  the trauma e x p e r i e n c e d by the muscle  and  the d i g e s t i v e and r e p r o d u c t i v e organ  the s i x l a r g e depressor muscles a t t a c h e d t o the o p e r c u l a r  p l a t e s and t o the b a s i s .  In the e a r l y experiments  w i t h bone shears to i s o l a t e each muscle bundle its  f i b r e s a t t a c h e d a t one  end d i r e c t l y  the s h e l l was  intact  cracked  (Hoyle 1963), w i t h  (without v i s i b l e tendon) t o a  fragment of the b a s i s , and a t the other end v i a tendons to a fragment o f the o p e r c u l a r p l a t e . saw was  used.  For most o f the experiments,  T h i s was  however, a  f a r s u p e r i o r , and enabled one  lapidarist's  to i s o l a t e a muscle  bundle v e r y quickly,, w i t h a minimum o f m a n i p u l a t i o n , on a compact of the b a s i s .  The  i s o l a t e d bundle was  immediately  fragment  suspended by the fragment  of operculum, i n a beaker o f normal b a r n a c l e R i n g e r ' s a t 5 - 10° C. muscles used.were the Depressor t e r g a l depressor was  Scutorum L a t e r a l i s and R o s t r a l i s .  to  S i n g l e muscle c e l l s were t y p i c a l l y 1.0  another,  To f a c i l i t a t e  a bundle was  The  not used because i t s f i b r e s were h e a v i l y i n v e s t e d w i t h  c o n n e c t i v e t i s s u e and were more d i f f i c u l t  cm i n l e n g t h .  The  isolate. - 1 ^,5 mm  i n diameter  and  the s e p a r a t i o n of these s i n g l e f i b r e s  4-5  from  one  a t t a c h e d by the o p e r c u l a r and b a s a l fragments to a  frame which h e l d the bundle h o r i z o n t a l l y a t about the r e s t i n g l e n g t h , i n a dish of c o l d Ringer's s o l u t i o n .  Fat and c o n n e c t i v e t i s s u e were c a r e f u l l y  removed w i t h j e w e l l e r ' s f o r c e p s and  i r i d e c t o m y s c i s s o r s , under a d i s s e c t i o n  microscope.  the tendon o f an a c c e s s i b l e , f i b r e  To  i s o l a t e the f i b r e s ,  grasped w i t h the f o r c e p s and cut from the operculum. the other f i b r e s  The connections  ( c o n n e c t i v e t i s s u e and a few s m a l l nerve  was with  f i b r e s ) were then  79  cut,  p r o c e e d i n g from tendon t o b a s i s .  (Damage t o the f i b r e membrane would  become apparent immediately as a l o c a l c o n t r a c t i o n . ) c a r r i e d out to as c l o s e to the b a s i s as was  This d i s s e c t i o n  was  p o s s i b l e , and the f i b r e was  left  a t t a c h e d t o the b a s i s , s i n c e removal  from the b a s i s w i t h o u t c a u s i n g damage  to  the c e l l membrane i s i m p o s s i b l e .  T h i s procedure was  of  the f i b r e s  at  5 - 10° C f o r 1 - 3  i n the bundle, and then the bundle was  repeated f o r each  left  i n normal R i n g e r ' s  hours b e f o r e b e i n g used.  T h i s procedure a s s u r e d t h a t any damaged f i b r e s would be even i f the damage was (ie.  slight.  identified,  Only f i b r e s which were u n i f o r m i n contour  w i t h o u t c o n t r a c t u r e s ) and u n i f o r m i n t r a n s l u c e n c y were chosen f o r  experiments.  F i b r e s were o n l y removed from the b a s i s a t the end o f the  experiment, when they were taken f o r weighing and chemical a n a l y s i s . use o f i n t a c t (Hagiwara,  fibres  is different  C h i c h i b u , & Naka 1964;  Tong 1972), who  This  from the p r a c t i c e o f o t h e r workers B r i n l e y 1968;  B i t t a r , Chen, D a n i e l s o n , &  c u t the c e l l s o f f a t the b a s i s and then c a n n u l a t e d the cut  end o f the c e l l .  Solutions. The a r t i f i c i a l seawater was to  960 mOsm.  prepared from I n s t a n t Ocean i n g r e d i e n t s ,  The R i n g e r ' s s o l u t i o n s were as i n T a b l e I.  (Schwartz-Mann) s t o c k s o l u t i o n (100 mM) all  solutions  was  A ouabain  prepared and used t o prepare  f o r ouabain experiments, by a d d i t i o n o f the a p p r o p r i a t e  amount o f ouabain s t o c k s o l u t i o n i n making up one o f the s o l u t i o n s shown i n T a b l e I.  80  TABLE I COMPOSITION OF SOLUTIONS A l l v a l u e s a r e mM.  Normal  Na  450  K  Zero-K  458  Li  Li-%C1  Choline  --  --  8  A l l s o l u t i o n s a r e 960±5 mOsm.  --  8  16  8  Tris**  Sucrose  Rinse  8  --  -8  Ca  20  20  20  20  20  20  20  20  Mg  10  10  10  10  10  10  10  10  Cl  543  543  522  269  543  543  93  85  Tris  25  25  25  25  25  475  25  25  Li  --  429  421  --  --  --  --  Choline  —  --  --  --  450  Sucrose  --  --  --  117  117  675  665  --  --  268  --  --  --  --  CH„S0. 3 4  Tris  --  i s tris-hydroxymethyl  --  aminomethane  T r i s C l i s not completely d i s s o c i a t e d : a t pH 7.6 t h e r e a r e 19 mM C l " f o r 25 mM T r i s C l (Gayton & Hinke 1968).  Microelectrodes. Sodium-specific  g l a s s m i c r o e l e c t r o d e s were c o n s t r u c t e d and c a l i b r a t e d  by the method o f Hinke (1967; 1969a), except  t h a t the s e n s i t i v e g l a s s was  drawn by hand, and the f i n a l g l a s s - t o - g l a s s s e a l a t the t i p was performed w i t h t h e e l e c t r o d e h e l d h o r i z o n t a l l y i n the m i c r o f o r g e and the h e a t e r  wire  brought i n h o r i z o n t a l l y t o touch and melt the t i p o f the s e n s i t i v e g l a s s .  81  The  heat was s u f f i c i e n t t o make the g l a s s - t o - g l a s s s e a l , and when the h e a t e r  w i r e was then withdrawn h o r i z o n t a l l y the s e n s i t i v e g l a s s was drawn i n t o a f i n e t i p and s e a l e d .  The o u t s i d e diameter o f the i n s u l a t i n g g l a s s a t the  s e a l was t y p i c a l l y 2 0 - 2 5 ^ , and the l e n g t h o f the s e n s i t i v e t i p was  typi-  c a l l y 75^u.  before  they l o s t  These e l e c t r o d e s were q u i t e durable,  t h e i r Na s e l e c t i v i t y .  and u s u a l l y broke  With use, the response time lengthened,  but an e l e c t r o d e c o u l d be ' r e - a c t i v a t e d ' by a 10 sec. immersion o f the t i p i n 0.1 M h y d r o f l u o r i c a c i d . * T h i s treatment made.the trips more Conventional puller,  micropipette  fragile.  e l e c t r o d e s were p u l l e d on a mechanical  e i t h e r from the same l e a d g l a s s used i n the c o n s t r u c t i o n of the Na  e l e c t r o d e , o r from b o r o s i l i c a t e g l a s s between 0.2 M NaCl and 0.2 M KC1 was f o r use ( A d r i a n 1956).  New  (Hinke 1969).  The p o t e n t i a l change  l e s s than 2 mV  f o r electrodes  e l e c t r o d e s were c o n s t r u c t e d  accepted  the day b e f o r e o r  the day o f each experiment. Potassium-specific the method o f Walker  i o n exchanger m i c r o e l e c t r o d e s  were c o n s t r u c t e d  (1971), and t e s t e d by the method o f Hinke  by  (1969a).  For most o f the experiments, the sodium or potassium e l e c t r o d e was r e f e r r e d to an e x t r a c e l l u l a r calomel e l e c t r o d e . was measured by a Cary 401 e l e c t r o m e t e r , The  micropipette  The p o t e n t i a l d i f f e r e n c e  and recorded  on a c h a r t  recorder.  e l e c t r o d e was a l s o r e f e r r e d to the calomel e l e c t r o d e , and  the p o t e n t i a l d i f f e r e n c e was monitored by a V i b r o n 33B e l e c t r o m e t e r a K i e t h l e y 616 e l e c t r o m e t e r .  For a few experiments the sodium e l e c t r o d e  was r e f e r r e d d i r e c t l y t o the i n t r a c e l l u l a r m i c r o p i p e t t e Electrical  o r by  i n t e r f e r e n c e was more o f a problem w i t h  electrode.  the l a t t e r method, but  the two methods y i e l d e d s i m i l a r r e s u l t s . The and  experimental  a l l electrometers,  apparatus was housed i n a s m a l l m e t a l - w a l l e d recorders,  l i g h t sources,  room,  and power s y r i n g e s were  o u t s i d e the room, except f o r the pre-amp o f the Cary e l e c t r o m e t e r .  The  82  s e p a r a t i o n o f the above equipment from the e x p e r i m e n t a l chamber was  less  than one metre.  through  Leads,  t u b i n g , and  f i b r e o p t i c c o n d u i t were passed  s m a l l p o r t s c u t i n the w a l l o f the s h i e l d e d room.  With t h i s  arrangement  and a p p r o p r i a t e grounding, the e l e c t r o m e t e r r e a d i n g s were v e r y s t a b l e throughout. The a x i a l  i n s e r t i o n o f the i o n - s p e c i f i c e l e c t r o d e i n t o the c a n n u l a t e d  c e l l and the r a d i a l i n F i g . 2. activities was  i n s e r t i o n o f the m i c r o p i p e t t e e l e c t r o d e a r e d e p i c t e d  The c a l c u l a t i o n o f the i n t r a c e l l u l a r sodium and from the p o t e n t i a l d i f f e r e n c e s  by the method o f Hinke  potassium  i n the m i c r o e l e c t r o d e c i r c u i t s  (1969a).  Chemical A n a l y s i s . In  a p a r t i c u l a r experiment, a c e l l was  basis, rinsed paper. the  cut near i t s c o n n e c t i o n t o the  f o r 30 sec i n s u c r o s e r i n s e s o l u t i o n , and b l o t t e d on  A s h o r t segment o f the c e l l was  cut from the tendon end and  o t h e r end, and the remaining c e n t r a l p a r t was  stoppered v i a l .  The wet weight was  measured a f t e r d r y i n g o f the c e l l was  then wet  tion  ashed  for analysis  pre-weighed  measured, and the d r y weight  fragment  was  i n an oven o v e r n i g h t .  f o r sodium and potassium by atomic  The  cell  absorp-  Space.  The volume o f the e x t r a c e l l u l a r space was same l o t as those used 3 of  placed i n a  from  spectrophotometry.  Extracellular  the  filter  ( H ) i n u l i n or (  measured f o r b a r n a c l e s from  i n the experiments as the volume o f d i s t r i b u t i o n  14 C)sorbitol  The c o m p o s i t i o n o f the f l u i d  (New  England N u c l e a r ) , by s t a n d a r d methods.  i n t h i s volume was  assumed t o be t h a t o f the  b a t h i n g s o l u t i o n , and the t o t a l sodium and potassium c o n t e n t s o f the  cell  as determined by c h e m i c a l a n a l y s i s were c o r r e c t e d f o r the c o n t r i b u t i o n o f  83  cation - selective microelectrode micropipette electrode glass  cannula  silk t i e tendon  single  muscle  cell  F i g u r e 2. C o n f i g u r a t i o n of the m i c r o e l e c t r o d e s an experiment. Not to s c a l e .  and  cannulated  cell  during  84  this extracellular  fluid  i n the u s u a l manner.  Myoplasmic and Nonmyoplasmic For  a given c e l l ,  Intracellular  Sodium.  the myoplasmic sodium a c t i v i t y ( a j \ j ) a  m  i n millimoles/  l i t r e and the i n t r a c e l l u l a r sodium c o n c e n t r a t i o n (Na)^ i n ( m i l l i m o l e s c e l l u l a r s o d i u m ) / ( k i l o g r a m i n t r a c e l l u l a r water) were measured. amount o f i n t r a c e l l u l a r sodium i s thus (Na)^ x of  i n t r a c e l l u l a r water.  i s the weight  i n t h e e x t r a c e l l u l a r space i s  The myoplasmic sodium content i s ( /. ) x (a, ) x V , where «Y+ t Na' m m A T  J  i s taken as 0.65  and V , the volume o f the myoplasmic s o l v e n t water, i s  taken as the s o l v e n t water f r a c t i o n (0.73  The t o t a l  T h i s amount i s u n c e r t a i n i n s o f a r as t h e f r a c t i o n  o f the c h e m i c a l l y a n a l y z a b l e sodium r e s i d i n g uncertain.  where  intra-  measured  x V ^ ) , r a t h e r than the h i g h e r f i g u r e  general figure  for cells.  The c a l c u l a t e d  might be an underestimate.  for. the barha'cle (Hirike 1970),  quoted i n the I n t r o d u c t i o n as a myoplasmic sodium content thus  A similar calculation  can be done f o r potassium.  The form i n which the sodium content o f each compartment  w i l l be  p r e s e n t e d , f o r the purpose o f d i r e c t comparison o f the amount,rather than the  c o n c e n t r a t i o n , i n each compartment,  of water i n the c e l l  i s a r r i v e d a t as f o l l o w s .  i s V = (wet weight) - (dry w e i g h t ) .  space i s assumed t o be 67o o f t h e t o t a l water. normal R i n g e r ' s s o l u t i o n extracellular  i s 450 mM.  space i s thus 0.06  The weight  The e x t r a c e l l u l a r  The sodium c o n c e n t r a t i o n i n  The sodium content i n s o l u t i o n  i n the  x V ( k g ) x 0 . 4 5 0 ( m o l e / l i t r e ) . The t  t a b u l a t e d t o t a l i n t r a c e l l u l a r sodium c o n c e n t r a t i o n (Na)^ = (mmoles i n t r a c e l l u l a r Na)/(kg i n t r a c e l l u l a r water ) = [ ( t o t a l a n a l y z e d Na) - ( e x t r a c e l l u l a r Na)J/(0.94 x V ) . fc  'Analyzed' r e f e r s  t o flame photometry.  The v a l u e s i n T a b l e I I p a r t b and i n F i g . 6 as 'sodium c o n t e n t ' have been n o r m a l i z e d by d i v i s i o n by V t  compartment  Thus the sodium content o f the myoplasmic  i s (moles Na i n myoplasm)/V  t  = ( a ^ ) 0 . 6 8 / 0 . 6 5 and the sodiumconteht a  m  85  of the nonmyoplasmic compartment i s (moles Na myoplasm)/V  Two  t  i n s i d e c e l l but not i n  = 0.94(Na)^ - (sodium content o f the myoplasmic compartment).  s e p a r a t e experiments  w i l l be d e s c r i b e d i n t u r n .  Methods, R e s u l t s , and D i s c u s s i o n f o r each experiment  For c l a r i t y ,  the  w i l l be p r e s e n t e d  separately.  A.  INCREASE OF CELL SODIUM  Methods. C e l l s were loaded w i t h sodium by immersion o v e r n i g h t i n p o t a s s i u m - f r e e Ringer's s o l u t i o n . dissected  Four muscle bundles  as d e s c r i b e d above.  to be c o n t r o l s . from each group.  ( N )m' ( a  a  N a  )i>  Two  from the same b a r n a c l e were  were a s s i g n e d to be e x p e r i m e n t a l and  (a ) , K  m  a n  d  ( K ) ^ were measured on s i x c e l l s  The r e m a i n i n g e x p e r i m e n t a l c e l l s were then p l a c e d i n  p o t a s s i u m - f r e e R i n g e r ' s s o l u t i o n a t 2° C f o r 20 hours. c o n t r o l c e l l s were kept A f t e r 20 hours,  remaining  the e x p e r i m e n t a l c e l l s were t r a n s f e r r e d  ( K ) ^ were measured f o r s i x more c e l l s . six control  The  i n normal Ringer's s o l u t i o n a t 2° C.  f r e e R i n g e r ' s s o l u t i o n a t room temperature  for  and  (ajj ) , a  m  to  potassium-  (Na) ^, ( a j r ) , m  and  S i m i l a r measurements were a l s o done  cells.  H a l f of the r e m a i n i n g e x p e r i m e n t a l c e l l s were then s e t i n normal R i n g e r ' s s o l u t i o n a t 10° C f o r 18 hours, and h a l f were s e t i n normal R i n g e r s s o l u t i o n to which had been added ouabain t o 10  M,  and  l e f t at  o 10  two  C f o r 18 hours.  The remaining c o n t r o l c e l l s were kept o R i n g e r s s o l u t i o n a t 10 C.  i n normal  86  At the end o f 18 hours, measurements o f (a.. ) , (Na)., (a„) , and ( K ) . ' Na'm' ' 1' K'm' ' i x  were performed  on c e l l s  from each  v  v  s  group.  The sodium and potassium content o f the two i n t r a c e l l u l a r were c a l c u l a t e d  compartments  f o r each c e l l as d e s c r i b e d i n Methods.  T h i s procedure was modelled a f t e r a method f o r measurement o f a sodium e x t r u s i o n dependent on e x t e r n a l potassium ( K ) ( S t e i n b a c h 1940; Beauge & S j o d i n 1967).  Q  and i n h i b i t e d  by.ouabain  The f e a t u r e s r e l e v a n t t o the  p r e s e n t problem a r e the changes i n i o n content o f the myoplasmic and nonmyoplasmic compartments.  Results. The r e s u l t s o f t h i s experiment w i t h sodium by immersion  i n which c e l l s were ' p a s s i v e l y loaded'  i n p o t a s s i u m - f r e e s o l u t i o n i n the c o l d and then  were a l l o w e d t o r e c o v e r i n normal Ringer's s o l u t i o n , a r e d e t a i l e d  i n Table  II. The c o n t r o l c e l l s , which were m a i n t a i n e d i n normal R i n g e r ' s throughout  the ca. 40 hours o f the experiment,  underwent a continuous  i n sodium c o n t e n t , amounting t o almost 407, o v e r a l l . was  unchanged over the f i r s t  over the f i n a l 20 hours.  rise  The potassium content  22 hours, but showed an i n c r e a s e o f about 47o  I t had been a n t i c i p a t e d t h a t a d e c l i n e i n the  potassium content o f the c e l l s would accompany the r i s e  i n t h e sodium  content.  The membrane p o t e n t i a l was v e r y c l o s e t o the potassium  potential  f o r b a r n a c l e muscle c e l l s  change d u r i n g t h i s  solution  l o n g experiment.  equilibrium  (Hinke & Gayton 1971), and i t showed no The water content o f a l l c e l l s i n -  c r e a s e d s l i g h t l y over the f i n a l 20 hours o f the experiment. The changes i n t h e t o t a l amount o f sodium and potassium i n the e x p e r i mental  cells  (where c o r r e c t i o n was made f o r e x t r a c e l l u l a r sodium and  potassium on t h e b a s i s o f an e x t r a c e l l u l a r space c o n t a i n i n g 67, o f the c e l l  87  TABLE  Ha  SUMMARY OF MEASUREMENTS ON PASSIVELY-LOADED CELLS  Condition  % wat:er  ( Na)m  (Na) . l  (a ) m  Control n = 6  7.08 (1.00)  14. 57 (1- 82)  158. 07 (16. 99)  195. 75 (4. 47)  72.3 (1.2)  74. 5 (0. 3)  Experiment n = 6  10.12 (2.45)  13. 05 (1. 7 8)  139. 47 (21. 58)  193. 88 (2. 25)  71.4 (2.7)  74. 9 (0. 3)  a  (K)  K  i  E  m  Initial:  Loaded: Control n = 6  7.82 (2.30)  16. 98 (2. 56)  149. 68 (24. 28)  194. 47 (6. 21)  72.5 (4.4)  74. 4 (0. 4)  Experiment n = 6  15.47 (4.18)  25. 82 (4. 44)  113. 68 (37. 41)  178. 17 (1. 91)  88.6 (9.4)  74. 8 (0. 2)  Control n = 4  9.58 (4.09)  20. 35 (3. 45)  127. 85 (34. 74)  202. 45 (5. 88)  60.6 (15.0)  75. 1 (0. 3)  Experiment n = 4  5.83 (0.79)  12. 60 (1. 21)  136. 30 (10. 28)  196. 05 (5. 11)  69.1 (2.1)  75. 5 (0. 1)  Ouabain n = 4  25.48 (7.20)  50. 48 (9. 93)  106. 83 (22. 89)  159. 98 (12. 97)  55.4 (12.0)  75. 5 (0. 4)  Recovered:  (a,, ) Na'm v  and (a ) K'm x  v  a r e m i l l i m o l e s / l i t r e myoplasmic water. • J  (Na)^ and ( K ) ^ a r e m i l l i m o l e s / k g c e l l water, c o r r e c t e d f o r e x t r a c e l l u l a r space ions as d e t a i l e d i n Methods. E  m  i s membrane p o t e n t i a l , i n - m i l l i v o l t s . Loaded-Experiment c e l l s was  The numbers i n p a r e n t h i s e s values,  Note t h a t the measurement on the  c a r r i e d out i n p o t a s s i u m - f r e e s o l u t i o n .  a r e the s t a n d a r d  and n i s the number o f c e l l s  d e v i a t i o n o f the measured  examined.  88  TABLE l i b ION CONTENT OF THE MYOPLASMIC AND NONMYOPLASMIC COMPARTMENTS  Condition  Myopl asmic Na  K  Nonmyoplasmic Na  Total  K  Na  K  Initial: Control Experiment  7.4  165  6.3  19  13.7  184  10.6  146  1.7  36  12.3  182  8.2  157  7.8  26  16.0  183  16.2  119  8.1  49  24.3  168  4.8  -19  4.9  6  9.7  -14  10.0  134  9.1  56  19.1  190  6.1  143  5.7  41  11.8  184  -11.9  47  -3.7  -38  -15.6  9  26.6  112  20. 8  38  47.4  150  8.6  16  -41  20.0  -25  Loaded: Control Experiment Change  Recovered: Control Experiment Change Ouabain Change  11.4  A l l v a l u e s a r e m i l l i m o l e s / k g t o t a l c e l l water, as d e t a i l e d i n Methods. The change i n t h e i o n content o f the e x p e r i m e n t a l c e l l s was c o r r e c t e d by s u b t r a c t i o n o f the c o r r e s p o n d i n g change i n t h e c o n t r o l  cells.  89  water, as determined  by i n u l i n and s o r b i t o l uptake  on b a r n a c l e s from the  same l o t u s i n g t h e same b l o t t i n g technique) were s i m i l a r t o those found by Beauge and S j o d i n (1967) i n a s i m i l a r experiment o n l y chemical a n a l y s i s was done.  on b a r n a c l e muscle where  When the changes i n i o n content o f the  c o n t r o l c e l l s were s u b t r a c t e d from t h e changes i n t h e e x p e r i m e n t a l over t h e c o r r e s p o n d i n g time p e r i o d , i t was found t h a t t h e t o t a l and p o t a s s i u m l o s s was about potassium-free s o l u t i o n .  one-for-one  cells  sodium g a i n  on i n c u b a t i o n o f t h e c e l l s i n  When c e l l s which had been i n c u b a t e d i n potassium-  f r e e s o l u t i o n were a l l o w e d t o r e c o v e r i n normal R i n g e r ' s s o l u t i o n ,  sodium  was l o s t and p o t a s s i u m gained, a g a i n v e r y r o u g h l y on a one-for-one  basis.  When companion c e l l s which a l s o had been immersed i n p o t a s s i u m - f r e e  solution  were s e t t o 'recover' i n normal Ringer's s o l u t i o n t o which ouabain had been -4 added ( t o 10  M), t h e r e was no r e c o v e r y .  Rather, a f u r t h e r g a i n o f sodium  and l o s s o f potassium o c c u r r e d , a g a i n on about a one-for-one  basis.  The changes i n t h e sodium and potassium content o f t h e myoplasmic compartment, c a l c u l a t e d cellular  from t h e measurements made w i t h i o n - s p e c i f i c  e l e c t r o d e s as d e s c r i b e d i n Methods, were f o r t h e most p a r t j u s t  T i k e those f o r t h e e n t i r e c e l l . free solution,  On immersion o f the c e l l s  i n a potassium-  sodium was gained and potassium l o s t by the myoplasmic  compartment on about a 4Na:IK b a s i s .  When c e l l s which had been immersed i n  a p o t a s s i u m - f r e e s o l u t i o n were a l l o w e d t o r e c o v e r i n normal Ringer's tion, but  intra-  solu-  sodium was l o s t and potassium gained by t h e myoplasmic compartment,  i n t h i s case on about a lNa:4K b a s i s .  i n t h e s o l u t i o n which c o n t a i n e d ouabain,  F o r those c e l l s s e t t o 'recover' sodium was not l o s t ,  but r a t h e r a  further gain occurred.  The potassium content o f the myoplasmic compartment  a l s o showed an apparent  i n c r e a s e , but i t s h o u l d be noted that t h e r e was a  rather large correction applied This  f o r the behavior of the c o n t r o l  cells.  i s t h e o n l y i n s t a n c e i n which the c o r r e c t i o n f o r changes i n t h e i o n  90  content o f the c o n t r o l c e l l s caused a change i n the q u a l i t a t i v e r e s u l t f o r the e x p e r i m e n t a l c e l l s .  The u n c o r r e c t e d data show a r o u g h l y  one-for-one  sodium g a i n and potassium l o s s by the myoplasmic compartment f o r c e l l s which were s e t t o r e c o v e r i n a normal Ringer's s o l u t i o n which c o n t a i n e d  ouabain.  The changes i n the sodium and potassium content o f the nonmyoplasmic compartment, c a l c u l a t e d as the d i f f e r e n c e between the change i n i o n content o f the whole c e l l and different.  On  t h a t o f the myoplasmic compartment, were q u a l i t a t i v e l y  immersion i n p o t a s s i u m - f r e e s o l u t i o n ,  t h e r e was  an equal  g a i n o f sodium and o f potassium by the nonmyoplasmic compartment. r e c o v e r y i n normal Ringer's s o l u t i o n ,  t h e r e was  a l o s s both o f sodium and  potassium by the nonmyoplasmic compartment, on about-a  lNa:10K b a s i s .  those c e l l s s e t to 'recover' i n the s o l u t i o n which c o n t a i n e d sodium was  On  For  ouabain,  gained and potassium l o s t by the nonmyoplasmic compartment, on '  r o u g h l y a lNa:4K b a s i s . The different  initial  sodium d i s t r i b u t i o n i n the e x p e r i m e n t a l group was  from t h a t  i n the c o n t r o l group.  quite  Large r e c i p r o c a l d i f f e r e n c e s i n  the sodium and potassium content o f b a r n a c l e muscle c e l l s have been noted b e f o r e (McLaughlin & Hinke 1966).  T h i s and  the d i f f e r e n c e s  i n the t o t a l i o n  c o n t e n t o f b a r n a c l e s from d i f f e r e n t , p o p u l a t i o n s ( B r i n l e y 1968; A l l e n , & Hinke 1969)  a p p a r e n t l y a r e normal.  I t i s c e r t a i n l y c o r r e c t to t r y  to i s o l a t e changes i n the e x p e r i m e n t a l c e l l s which a r e due e x p e r i m e n t a l m a n i p u l a t i o n , but the unexpected c o n t r o l c e l l s over the f i n a l 20 hours  bility  s o l e l y to the  g a i n o f potassium by  o f the experiment  suggests  p o s s i b i l i t y o f a s i m i l a r c a p r i c e by the e x p e r i m e n t a l c e l l s , to the e x p e r i m e n t a l m a n i p u l a t i o n .  Gayton,  For t h i s reason,  i t was  the  the  quite unrelated felt  that  credi-  s h o u l d be assumed o n l y f o r the q u a l i t a t i v e changes d e s c r i b e d here.  Thus: the myoplasmic compartment behaved, l i k e the c e l l s as a whole. The b e h a v i o r i s c o n s i s t e n t w i t h the model o f the myoplasmic compartment as  91  a compartment whose i o n content i s governed by a sodium-potassium mechanism which ouabain.  exchange  i s s t i m u l a t e d by e x t r a c e l l u l a r p o t a s s i u m and i n h i b i t e d by  The nonmyoplasmic  compartment, on the o t h e r hand, gains sodium  and potassium t o g e t h e r i n the absence o f e x t e r n a l potassium, so t h e r e must i n e f f e c t be a s h i f t o f p o t a s s i u m from t h e myoplasmic compartment.  The nonmyoplasmic  t o the nonmyoplasmic  compartment l o s e s sodium and p o t a s s i u m  t o g e t h e r when e x t e r n a l p o t a s s i u m i s r e s t o r e d .  Finally,  t h e nonmyoplasmic  compartment g a i n s sodium but l o s e s p o t a s s i u m on exposure t o R i n g e r ' s s o l u t i o n which c o n t a i n s ouabain but i s o t h e r w i s e normal.  Discussion. The main problem o f concern here i s t h e l o c a t i o n o f t h e sodium not d e t e c t e d by the s o d i u m - s p e c i f i c i n t h e above c a l c u l a t i o n s ,  i n t r a c e l l u l a r electrode.  The model  i n terms o f s o l v e n t water and p a r t i t i o n o f ions,  i s due t o Hinke (McLaughlin & Hinke 1966; Hinke e t a l . 1973). t h a t the nonmyoplasmic barnacle  employed  ions were a s s o c i a t e d w i t h p r o t e i n s  He proposed  i n s i d e the  cell.  Experiments on i n t a c t c e l l s and on membrane-damaged c e l l s t h a t t h i s compartment has a maximum c a p a c i t y  indicate  f o r ions o f about 68 m i l l i -  e q u i v a l e n t s / k i l o g r a m d r y weight o f i n t a c t c e l l  (Hinke e t a l . 1973),^ and a  b i n d i n g p r e f e r e n c e f o r sodium ions over p o t a s s i u m i o n s .  In e a r l i e r  experi-  ments, i t was assumed that a l l o f the p o t a s s i u m was f r e e i n the myoplasm (Hinke 1970), but f u r t h e r experiments i n d i c a t e d t h a t some p o t a s s i u m was  The membrane-damaged p r e p a r a t i o n has l o s t s o l u b l e o r g a n i c molecules, which account f o r about h a l f o f the d r y weight o f the b a r n a c l e c e l l . The b i n d i n g c a p a c i t y found i n experiments on membrane-damaged c e l l s i s r o u g h l y twice t h a t found f o r i n t a c t c e l l s , when no account i s taken o f the d i f f e r ence i n t h e d r y weight i n t h e two s i t u a t i o n s .  92  'bound' as w e l l  (Hinke et a l .  associated with fixed the  though, because the myoplasmic p o t a s s i u m  i s much g r e a t e r than the myoplasmic sodium a c t i v i t y .  t o t a l amount o f nonmyoplasmic proteins  The a c t u a l amount o f potassium  i n t r a c e l l u l a r a n i o n i c s i t e s should be g r e a t e r than  amount o f sodium so bound,  activity  the  1973).  i n whole c e l l s .  nonmyoplasmic  c a t i o n was  However, the  about twice the c a p a c i t y o f the  Thus t h e r e p r o b a b l y i s an a d d i t i o n a l component o f  compartment,  which c o n t a i n s more sodium than potassium.  Experiments i n which the water content o f the b a r n a c l e c e l l was by exposure o f the c e l l  t o h y p e r t o n i c or h y p o t o n i c s o l u t i o n s  most o f the nonmyoplasmic  sium was unchanged when the c e l l water was  to  c e l l water was  indicated  that  sodium i n b a r n a c l e muscle c e l l s remained immobile  d e s p i t e l a r g e changes i n c e l l water (Hinke 1969b).  the  changed  decreased.  The myoplasmic p o t a s -  i n c r e a s e d , but decreased when  C o m p e t i t i v e b i n d i n g o f sodium and p o t a s s i u m  i n t r a c e l l u l a r p r o t e i n s s h o u l d f o l l o w a mass a c t i o n r u l e , so s h o u l d not  be changed by a change i n the amount o f water i n the myoplasmic  compartment.  It  differently  i s i n t e r e s t i n g , however, t h a t the p o t a s s i u m behaved s l i g h t l y  from the sodium i n these experiments, as i t d i d i n the p r e s e n t experiments. The e f f e c t of p o t a s s i u m - f r e e s o l u t i o n on the myoplasmic sodium and potassium c o n t e n t o f f r o g s k e l e t a l muscle has been r e p o r t e d Lee 1971; Lee & Armstrong 1974).  The r e s u l t s d i f f e r e d from those found here  i n b a r n a c l e muscle, i n t h a t the nonmyoplasmic  compartment  l o s t p o t a s s i u m on i n c u b a t i o n i n p o t a s s i u m - f r e e s o l u t i o n . of  the myoplasmic and nonmyoplasmic  t a t i v e l y the same.  (Armstrong &  However, i t was  compartments found t h a t  o f f r o g muscle Thus the b e h a v i o r  i n f r o g muscle was  i f the p o t a s s i u m - f r e e  used f o r i n c u b a t i o n c o n t a i n e d much l e s s c a l c i u m than f r o g R i n g e r ' s does, then a l l o f the sodium gained by the c e l l compartment,  a l t h o u g h p o t a s s i u m was  and nonmyoplasmic  compartments.  still  lost  qualisolution  solution  entered the myoplasmic from both the myoplasmic  F u r t h e r , exposure t o the c a l c i u m - p o o r  93  solution resulted compartment.  i n a decrease i n the c a p a c i t y o f the  F i n a l l y , when the l o a d i n g p e r i o d was  the myoplasmic compartment  nonmyoplasmic  extended to 48 hours,  accounted f o r most o f the a c c u m u l a t i o n o f sodium  over the f i n a l 24 hours, as i f the nonmyoplasmic  compartment  had become  saturated. A s i d e from t h e p o t a s s i u m l o s s by t h e nonmyoplasmic the b e h a v i o r o f f r o g muscle was  compartment,  the same as t h a t of b a r n a c l e muscle on  exposure t o p o t a s s i u m - f r e e s o l u t i o n .  The e f f e c t s o f c a l c i u m a r e p a r t i c u l a r l y  r e l e v a n t to the second l i k e l y s i t e o f r e s i d e n c e o f nonmyoplasmic the p o l y s a c c h a r i d e s  then,  sodium,  i n the e x t r a c e l l u l a r space.  The p o s s i b i l i t y t h a t the sodium not d e t e c t e d by the m i c r o e l e c t r o d e might be e x t r a c e l l u l a r was mentioned by C a l d w e l l  (1968).  S t e i n b a c h (1956) had measured- c a t i o n b i n d i n g by sugars.  H a r r i s and B r a d i n g and  Widdicombe (1977) r e c e n t l y p u b l i s h e d a c a r e f u l study o f the c a p a c i t y o f the e x t r a c e l l u l a r cation-exchanging s i t e s used the t r i v a l e n t  i n mammalian smooth muscle.  They  i o n lanthanum to d i s p l a c e sodium, potassium, magnesium,  and c a l c i u m from the t i s s u e , and c a l c u l a t e d the c o n t r i b u t i o n o f the i n t r a c e l l u l a r and e x t r a c e l l u l a r space t o the e f f e c t . f o r a n i o n i c b i n d i n g s i t e s o u t s i d e the c e l l . cation displaced  These c a t i o n s a l l compete  The amount o f e x t r a c e l l u l a r  from s p e c i f i c s i t e s by lanthanum s h o u l d be l e s s than the  t o t a l amount o f c a t i o n bound t o e x t r a c e l l u l a r s i t e s . 4 mmole.potassium weight  I t amounted to about  per kg d r y weight, and about 60 mmole sodium per kg d r y  (assuming 807* water) .  I t was  a l s o found t h a t lanthanum reduced t h e  p a s s i v e sodium and ( t o a l e s s e r extent) p o t a s s i u m movement a c r o s s the c e l l membrane, but d i d not appear t o a f f e c t the a c t i v e i o n movements. suggested t h a t the b i n d i n g o f c a t i o n s t o e x t r a c e l l u l a r s i t e s p a s s i v e transmembrane  I t was  i s a stage o f  passage o f the c a t i o n s , and t h a t p o t a s s i u m behaves  q u i t e d i f f e r e n t l y from sodium i n i t s p a s s i v e passage o f the c e l l membrane.  94  If  the e x t r a c e l l u l a r p o l y s a c c h a r i d e i s s i m i l a r  seems l i k e l y , compartment,  i n b a r n a c l e muscle, as  t h e r e i s then a c r e d i b l e second component to the c o n t a i n i n g mostly sodium and h a v i n g an i o n - b i n d i n g  s i m i l a r t o t h a t o f the i n t r a c e l l u l a r p r o t e i n s .  sodium (mostly e x t r a c e l l u l a r ) and p o t a s s i u m (mostly i s not c l e a r how  o f the  potassium i n  b a r n a c l e muscle can be accounted f o r w i t h b i n d i n g t o these  compartment  i n the transmembrane  from t h a t  p o t a s s i u m i s removed  t o the TTS  transport of  f o r sodium, but t h i s  c o u l d not be c o n c l u d e d from the present experiments a l o n e . l i k e l y that potassium s h i f t s  compartments  I t might be t h a t the e x t r a c e l l u l a r  i s indeed i n v o l v e d d i r e c t l y  p o t a s s i u m by a method d i f f e r e n t  nonmyoplasmic  intracellular).  the b e h a v i o r o f the nonmyoplasmic  a c c o r d i n g t o the mass a c t i o n r u l e .  capacity  Together, these two compo-  nents appear t o have a c a p a c i t y adequate t o c o n t a i n a l l  It  nonmyoplasmic  certainly  I t seems more  from the myoplasm when e x t e r n a l  ( B i r k s & Davey 1969), w h i l e b i n d i n g o f p o t a s s i u m t o  f i x e d charges i s l e s s important. The b e h a v i o r o f the nonmyoplasmic of  the nonmyoplasmic  compartment  sodium i s accounted f o r by a model  as two r e g i o n s which can b i n d sodium,  i n t r a c e l l u l a r and c o n t a i n i n g r e l a t i v e l y l i t t l e  one  sodium i n comparison w i t h  potassium, and one e x t r a c e l l u l a r , c o n t a i n i n g r e l a t i v e l y l i t t l e  potassium i n  comparison w i t h sodium. At  l e a s t some o f t h i s e x t r a c e l l u l a r sodium s h o u l d engage i n r a p i d  exchange w i t h the sodium i n the b a t h i n g s o l u t i o n , a l t h o u g h i n smooth these c a t i o n s were m o b i l i z e d o n l y when lanthanum was  introduced.  muscle  Such  exchange might be r e v e a l e d i n an experiment i n which the e f f l u x o f sodium i n t o sodium-free s o l u t i o n s are  d e s c r i b e d next.  i s measured.  The r e s u l t s o f such an experiment  95  B.  DECREASE OF CELL SODIUM  Methods. In a s e p a r a t e s e r i e s o f experiments, d e p l e t e d o f sodium by immersion Ringer's s o l u t i o n  i n i s o t o n i c sodium-free  as d e s c r i b e d  and (Na). were performed on s e v e r a l  a t room temperature.  above, and measurements o f i n normal R i n g e r ' s  solution  The bundle was then immersed i n a sodium-free  lithium-  1  substituted  lithium-substituted  (Table.I).  A muscle bundle was d i s s e c t e d ) am  s i n g l e b a r n a c l e muscle c e l l s were  cells  R i n g e r ' s s o l u t i o n f o r 30 seconds,  volume o f t h i s s o l u t i o n .  Measurements o f ( a  then t r a n s f e r r e d N a  )  m  and ( N a )  i  to a large  were performed  on each o f a s u c c e s s i o n o f c e l l s over the next t h r e e hours. This  procedure was c a r r i e d out on t h r e e d i f f e r e n t muscle bundles,  b a r n a c l e s from the same l o t . a f t e r 16 hours o f immersion  from  I n one case, some measurements were made  i n sodium-free s o l u t i o n , where the c e l l s were  k e p t a t 10° C between t h e t h i r d and s i x t e e n t h  hours.  The sodium c o n t e n t o f the myoplasmic and nonmyoplasmic compartments was c a l c u l a t e d ,  as d e s c r i b e d  above.  Results. The r e s u l t s a r e p r e s e n t e d i n F i g . 3 as the change i n the sodium c o n t e n t o f the myoplasmic ( c l o s e d symbols)  and nonmyoplasmic (open symbols)  partments w i t h time, w h i l e the c e l l s were immersed i n sodium-free substituted of  Ringer's s o l u t i o n .  immersion.  com-  lithium-  The zero o f time corresponds t o the moment  The l i n e s were drawn by eye as a v i s u a l a i d .  Under c e r t a i n c o n d i t i o n s ,  a large rapid  a c t i v i t y can o c c u r i n f r o g s k e l e t a l muscle c r a b s t r i a t e d muscle  (Vaughan-Jones 1977).  f a l l o f the myoplasmic  sodium  (White 6c Hinke 1976) and i n Such an e f f e c t had been sought  96  o 14« »  12 o E 10 E  "E o o o z  •  8 t  •  o •a  o O  a o •  30  60  90  120  150  f80  210  240  1000  time (min.)  F i g u r e 3. Changes i n the sodium content o f c e l l s d u r i n g i n c u b a t i o n i n sodium-free l i t h i u m - s u b s t i t u t e d s o l u t i o n . Correction for extracellular sodium, by standard methods (see t e x t ) , was made f o r the c e l l s a t zero time (normal R i n g e r ' s s o l u t i o n ) . Each p o i n t r e p r e s e n t s one c e l l , where the c l o s e d symbol a t a g i v e n time r e p r e s e n t s the myoplasmic sodium, and the open symbol a t t h a t time r e p r e s e n t s the nonmyoplasmic i n t r a c e l l u l a r sodium. The three d i f f e r e n t symbol shapes r e p r e s e n t t h r e e d i f f e r e n t experiments. The i r r e g u l a r l y broken l i n e r e p r e s e n t s the myoplasmic i n t r a c e l l u l a r sodium, and the r e g u l a r l y broken l i n e r e p r e s e n t s the nonmyoplasmic sodium. The l i n e s were drawn by eye to summarize the t h r e e e x p e r i m e n t a l runs.  97  and found i n b a r n a c l e s t r i a t e d muscle, and i s d e s c r i b e d i n s e c t i o n  6.  However, i t i s o n l y seen i n b a r n a c l e muscle c e l l s which have an e l e v a t e d sodium c o n t e n t .  Two  o f the muscle bundles used i n t h i s experiment had  r e l a t i v e l y low sodium content  ( t r i a n g u l a r and square symbols  i t was not expected t h a t the e f f e c t would be seen i n them. initial  fall  i n F i g . 3), and The  i n the c a l c u l a t e d sodium content o f the..myoplasmic  small compartment,  shown i n F i g . 3, might r e f l e c t a r a p i d e f f l u x from the t h i r d muscle bundle (round symbols), whose i n i t i a l  sodium content was  A s i d e from t h i s v a r i a t i o n , myoplasmic compartment  higher.  the d e c l i n e o f the sodium c o n t e n t o f the  w i t h time was  r a t h e r slow.  Even a f t e r 16 hours o f  immersion i n the sodium-free s o l u t i o n (much o f which time was as  noted above), the myoplasmic compartment  spent a t 10° C  had r e t a i n e d h a l f o f i t s i n i t i a l  sodium. The d e c l i n e o f the sodium content o f the nonmyoplasmic markedly d i f f e r e n t . of  There was  a l a r g e f a l l over the f i r s t  muscle c e l l s on b r i e f has been measured 1971).  sodium a c t i v i t y  ( l e s s than 60 min)  30 to 40 minutes  by o t h e r workers  They found t h a t  ( ^ ) a  a  m  for  (ajj ) a  m  i n barnacle  immersion i n sodium-free s o l u t i o n  (McLaughlin & Hinke 1968; A l l e n & Hinke  increased i n i t i a l l y ,  However, the l i t h i u m s o l u t i o n they used was  then decreased.  prepared by s u b s t i t u t i n g L i C l  NaCl on a one-for-one b a s i s ( M c L a u g h l i n & Hinke 1968, T a b l e I ) .  s o l u t i o n i s h y p e r t o n i c , so the i n i t i a l '  b e h a v i o r o f (a. ) Na'm  J t r  movement o f water out o f the myoplasm. to  was  immersion, but then almost no l o s s over the next 15 hours. The v a r i a t i o n o f the myoplasmic  a  compartment  v  T  reflects  the  T h i s by i t s e l f has been found not  a f f e c t the sodium content o f the myoplasmic and nonmyoplasmic  ments (Hinke 1969b).  Such  compart-  A s i m i l a r experiment o f 25 minutes' .duration u s i n g  i s o t o n i c sodium-free s u c r o s e - s u b s t i t u t e d R i n g e r ' s s o l u t i o n showed b e h a v i o r s i m i l a r to that  i n F i g . 3 (Hinke 1969b).  98  A rough a p p r o x i m a t i o n o f the s i z e o f the v a r i o u s sodium can be made from F i g . myoplasmic  3.  I f the r a p i d l y - l o s t  fractions of c e l l u l a r f r a c t i o n o f t h e non-  sodium i s a s s i g n e d t o the e x t r a c e l l u l a r space, t h e r e remains  about 307o o f the i n t r a c e l l u l a r sodium n o t a c c e s s i b l e t o the m i c r o e l e c t r o d e (in  the model d e s c r i b e d e a r l i e r ) .  T h i s i s about 10 m i l l i m o l e / k g d r y weight.  The d a t a f o r v e r y l o n g time o f immersion suggest t h a t  i n b a r n a c l e muscle  perhaps 207 o f the c e l l u l a r sodium i s both not a c c e s s i b l e t o the microo  e l e c t r o d e and so slow t o exchange as t o be n o n p a r t i c i p a t o r y i n the type o f i n v i t r o experiments d e s c r i b e d i n t h i s  thesis.  I t has o f t e n been found t h a t a f r a c t i o n o f the c e l l  sodium exchanges  o n l y v e r y s l o w l y w i t h r a d i o a c t i v e l y - l a b e l l e d sodium i n t h e b a t h i n g  solution  (Conway & Cary 1955; T r o s c h i n 1961; Dunham & G a i n e r 1968; A l l e n & Hinke 1970).  I n p a r t i c u l a r , A l l e n and Hinke (1970)  agreement  found good  quantitative  i n b a r n a c l e muscle f o r the amount o f c e l l u l a r sodium which ex-  changes s l o w l y as c a l c u l a t e d f r o m i s o t o p e f l u x s t u d i e s w i t h t h e amount o f 'bound'  (nonmyoplasmic)  sodium c a l c u l a t e d  from m i c r o e l e c t r o d e s t u d i e s .  Discussion. In  the f i r s t experiment, where the sodium content o f the c e l l was  i n c r e a s e d by immersion o f the c e l l  i n potassium-free s o l u t i o n ,  i t was found  t h a t the b e h a v i o r o f the nonmyoplasmic  sodium c o u l d be accounted f o r by a  model o f the nonmyoplasmic  as two r e g i o n s which can b i n d  sodium: one i n t r a c e l l u l a r , and one e x t r a c e l l u l a r ,  compartment  c o n t a i n i n g a r e l a t i v e l y s m a l l amount o f sodium;  containing a r e l a t i v e l y  The second experiment showed t h a t can  be washed out v e r y r a p i d l y  l a r g e amount o f sodium.  indeed much o f the nonmyoplasmic  sodium  i n sodium-free s o l u t i o n , w h i l e some cannot  be washed out even w i t h l o n g immersion i n s o d i u m - f r e e s o l u t i o n .  I t seems  r e a s o n a b l e t o a s s i g n the former t o the e x t r a c e l l u l a r component o f the non-  99  myoplasmic compartment, the  nonmyoplasmic  and the l a t t e r t o the i n t r a c e l l u l a r component  compartment,  a l t h o u g h some o f the e x t r a c e l l u l a r  of  sodium  p r o b a b l y i s t i g h t l y - b o u n d , as i n smooth muscle. To summarize  the rough q u a n t i t a t i v e e s t i m a t e s , about 307> o f the sodium  which i s t r u l y i n t r a c e l l u l a r i s not a c c e s s i b l e to the m i c r o e l e c t r o d e . of t h i s  (about 207>) cannot be washed out d u r i n g l o n g immersion o f the c e l l  i n sodium-free s o l u t i o n . the  cell  Most  I f t h i s 207. i s i d e n t i f i e d w i t h the f r a c t i o n o f  sodium which exchanges v e r y s l o w l y w i t h r a d i o i s o t o p i c  then the amount o f nonmyoplasmic  sodium,  i n t r a c e l l u l a r sodium which exchanges  r a p i d l y w i t h the myoplasmic sodium i s p r o b a b l y l e s s than 107 o f the i n t r a o  c e l l u l a r sodium.  T h i s c o n c l u s i o n i s important because i t a l l o w s the model  f o r the measurement o f the sodium e f f l u x from b a r n a c l e muscle c e l l s to be r e l a t i v e l y simple, as d i s c u s s e d  i n section  2.D.  The p o s s i b i l i t y t h a t the e x t r a c e l l u l a r nonmyoplasmic p o t a s s i u m i s w i t h i n the o v e r a l l mechanism membrane was  raised  by which these ions pass the c e l l  i n the d i s c u s s i o n o f the f i r s t experiment.  p o s s i b l e t h a t some o f the e x t r a c e l l u l a r nonmyoplasmic d i r e c t l y w i t h the myoplasmic sodium. the  sodium and  s i m p l e r h y p o t h e s i s f i r s t , and t e s t  next two s e c t i o n s support t h i s  choice.  It is  sodium can exchange  However, i t seems prudent to adopt i t in practice.  The r e s u l t s  o f the  100  SECTION 4.  MICROINJECTION OF RADIOSODIUM INTO SINGLE MUSCLE CELLS  In the p r e p a r a t i o n o f a c e l l  f o r an experiment  i n which the e f f l u x o f  r a d i o s o d i u m i s t o be measured, i t i s u s u a l l y necessary to immerse the f o r some time i n a s o l u t i o n which c o n t a i n s radiosodium. c e l l u l a r and experiment  the e x t r a c e l l u l a r sodium become l a b e l l e d , and  intra-  i n the  subsequent  both the i n t r a c e l l u l a r and the e x t r a c e l l u l a r sodium c o n t r i b u t e to  the measured r a d i o s o d i u m The  Both the  cell  efflux.  e x t r a c e l l u l a r r a d i o s o d i u m i s l o s t v e r y r a p i d l y , and a f t e r a s h o r t  p e r i o d o f time o n l y the i n t r a c e l l u l a r r a d i o s o d i u m c o n t r i b u t e s a p p r e c i a b l y to the observed  efflux.  i n t e r i o r o f the c e l l It  However, the b e h a v i o r o f the e f f l u x from the  immediately  i s not a simple matter  a f t e r the experiment  In l a r g e c e l l s , by m i c r o i n j e c t i o n .  begun i s masked.  to s u b t r a c t the e s t i m a t e d c o n t r i b u t i o n o f the  e x t r a c e l l u l a r sodium to the t o t a l r a d i o s o d i u m e f f l u x , from e x t r a c e l l u l a r s i t e s  has  because the  i s not always simple (eg. Rogus & Z i e r l e r  the i n t e r i o r can be loaded w i t h r a d i o s o d i u m As  efflux  first  selectively  d e s c r i b e d by Hodgkin and Keynes (1956;  a l s o C a l d w e l l & W a l s t e r 1963), a f i n e c y l i n d r i c a l g l a s s needle was axially  i n t o a s q u i d axon, and  the t i p .  The  injected  fluid  then removed w h i l e f l u i d was  filled  the space v a c a t e d by the  1973).  see inserted  e j e c t e d from withdrawing  needle. The  technique i s o f i n t e r e s t here i n two  respects.  First,  the  distri-  b u t i o n o f the i n j e c t e d r a d i o s o d i u m among i n t r a c e l l u l a r p o o l s o f sodium can p r o v i d e a t e s t o f the model f o r the c e l l u l a r sodium d e s c r i b e d i n s e c t i o n The  i n t e r p r e t a t i o n by B i t t a r and coworkers o f sodium m i c r o i n j e c t i o n  ments i n b a r n a c l e muscle seems t o be a t v a r i a n c e w i t h the model Chen, D a n i e l s o n , Hartmann, & Tong 1972), as w i l l Second, the t e c h n i q u e i s convenient  3.  experi-  (Bittar,  be d e s c r i b e d f u l l y below.  i n t h a t the c e l l can be loaded w i t h  101  radiosodium quickly,  the e x t r a c e l l u l a r sodium p o o l can be bypassed,  sodium content o f the c e l l All of  and the  can be r a i s e d by i n j e c t i o n o f sodium s o l u t i o n s .  o f t h i s can be done w i t h p a s s i v e techniques as w e l l ,  but a t the expense  l o n g immersion times, o f t e n i n n o n p h y s i o l o g i c a l s o l u t i o n s . Of course, the e f f e c t s o f m i c r o i n j e c t i o n on t h e c e l l and i n p a r t i c u l a r  on the sodium t r a n s p o r t out o f the c e l l must be known b e f o r e the technique can be adopted In  as a convenience  t h e remainder  of this  in efflux  experiments.  i n t r o d u c t o r y passage,  the r e s u l t s o f o t h e r  workers on the q u e s t i o n s o f the e f f e c t o f m i c r o i n j e c t i o n on b a r n a c l e muscle cells,  and the d i s t r i b u t i o n o f i n j e c t e d r a d i o s o d i u m among i n t r a c e l l u l a r  sodium pools a r e d i s c u s s e d . c o n s i s t s o f the comparison  The e x p e r i m e n t a l p o r t i o n o f t h i s  o f the e f f l u x o f r a d i o s o d i u m from c e l l s  by m i c r o i n j e c t i o n , w i t h t h a t  from c e l l s  M i c r o i n j e c t i o n was f i r s t used and Naka (1964).  loaded  loaded p a s s i v e l y .  i n b a r n a c l e muscle by Hagiwara, C h i c h i b u ,  A l a r g e needle was used  (200 - 500yU o.d.),  i n j e c t e d as the n e e d l e was advanced down t h e a x i s o f the c e l l , f l u i d was i n j e c t e d t o double the diameter o f the c e l l . e x c i t a b i l i t y o f the c e l l  section  membrane was unimpaired,  f l u i d was and enough  Even so, the  and the r e s t i n g membrane  p o t e n t i a l showed a dependence on the transmembrane d i f f e r e n c e i n t h e potassium concentration s i m i l a r to that of i n t a c t , noninjected barnacle cells  (Hagiwara Brinley  e t a l . 1964; Hinke 1970).  (1968) used the technique o f Hodgkin and Keynes, but w i t h a  m i c r o i n j e c t o r n e e d l e which s e r v e d as an open-tipped for  i n t r a c e l l u l a r electrode  measurement o f the membrane p o t e n t i a l as the needle was b e i n g advanced  down the a x i s o f the c e l l  ( B r i n l e y & M u l l i n s 1965).  He found t h a t t h e r e  was a t r a n s i e n t membrane p o t e n t i a l d e p o l a r i z a t i o n o f 2 o r 3 m i l l i v o l t s (mV) w i t h each advance o f the i n j e c t o r needle, w i t h r e c o v e r y o c c u r r i n g  102  w i t h i n a few seconds  a f t e r the advance was  halted.  He  interpreted  this  as b e i n g due t o the t e a r i n g and r a p i d r e s e a l i n g o f the c l e f t s and t r a n s v e r s e t u b u l e s , which a r e open to the b a t h i n g s o l u t i o n and p e n e t r a t e deeply the c e l l  (Hoyle 1973).  B r i n l e y found  i t difficult  into  to o b t a i n s t a b l e membrane  p o t e n t i a l r e a d i n g s i n h i s p r e p a r a t i o n , even when u s i n g c o n v e n t i o n a l micropipette electrodes.  The mean v a l u e he found was  v a r y w i t h temperature  (16 t o 22° C).  (1964) r e p o r t e d -73.5  mV;  on s e l e c t e d McLaughlin  ( C a l d w e l l & W a l s t e r 1963).  Hagiwara et a l .  from the muscle bundle; on i n t a c t  and  original  Brinley,  from the b a s i s a t the p o i n t o f They found t h a t  the  u s u a l l y u n a f f e c t e d by the process o f m i c r o i n j e c t i o n ,  a l t h o u g h t h e i r mean v a l u e f o r the r e s t i n g p o t e n t i a l was t o -72  mV  cells.  L i k e Hagiwara e t al_. and  to the b a s i s , and c a n n u l a t e d the c u t end.  membrane p o t e n t i a l was  -42  i t d i d not  Chen, D a n i e l s o n , Hartmann, and Tong (1972) used the  they c u t s i n g l e b a r n a c l e muscle c e l l s attachment  and  (1963) r e p o r t e d -74 t o -96  and Hinke (1966) r e p o r t e d -71 mV  Bittar, technique  By comparison,  Hoyle and Smith  i n t a c t c e l l s not d i s s e c t e d  -68 mV,  o n l y -56 mV  (range  mV).  B i t t a r et: a_l. found t h a t the e f f l u x o f i n j e c t e d r a d i o s o d i u m from the cell  i n t o normal Ringer's s o l u t i o n d e c l i n e d e x p o n e n t i a l l y w i t h time.  f r a c t i o n o f the t o t a l  i n t r a c e l l u l a r r a d i o s o d i u m l o s t p p e r u n i t time  s l o w l y over the f i r s t  60 minutes i n the m a j o r i t y o f the c e l l s  but was  more s t a b l e t h e r e a f t e r .  The  declined  they s t u d i e d ,  They found t h a t the s l o p e o f the s e m i l o g  p l o t versus time o f the amount o f r a d i o s o d i u m l o s t  from the c e l l  per u n i t tiVe  ,d_ l n . d „ * . . was g r e a t e r than the s l o p e o f the s e m i l o g p l o t versus Mt ' ' W e e l l " time o f the amount o f r a d i o s o d i u m l e f t i n the c e l l /d « * \, as Mt cell did  Hodgkin and Keynes.(1956) i n i n j e c t e d s q u i d axon.  That  i s , the amount  of  r a d i o s o d i u m i n the c e l l  d i d not d e c l i n e w i t h time a t a r a t e commensurate  to  the d e c l i n e w i t h time o f the r a t e a t which r a d i o s o d i u m appeared  i n the  103  bath.  T h i s was  not expected under the c o n d i t i o n s o f the experiment.  the r a t e o f u n i d i r e c t i o n a l sodium e f f l u x occurs  i n s i d e the c e l l ,  dependence o f t h i s the f a l l nential and  i s c o n s t a n t , and r a p i d  mixing  then whatever the k i n e t i c r e l a t i o n d e s c r i b i n g the  e f f l u x r a t e on the i n t r a c e l l u l a r c o n c e n t r a t i o n o f sodium,  o f the t o t a l r a d i o s o d i u m content o f the c e l l f u n c t i o n o f time.  That  i s , the t o t a l  i s a simple  i s i n normal R i n g e r ' s  s o l u t i o n , but the p o o l o f r a d i o s o d i u m p r e s e n t a t the i n i t i a l 22  expo-  i n t r a c e l l u l a r sodium content  d i s t r i b u t i o n s h o u l d be constant, w h i l e the c e l l  d e p l e t e d as.  If  time i s  23 Na  exits with  Na by a random process which i s slow r e l a t i v e 22  to  diffusion  i n bulk s o l u t i o n s .  D i l u t i o n o f the i n t r a c e l l u l a r  Na  by  23 to  Na occurs (where mixing i n s i d e the c e l l i s assumed to be r a p i d compared the e f f l u x r a t e ) , and the r a t e a t which the t o t a l r a d i o a c t i v i t y Na* cell  due to  to r a d i o s o d i u m  i n the c e l l  the amount p r e s e n t a t t h a t dNa* -I -i ce_L_L dt  The  d e c l i n e s a t each i n s t a n t  is proportional  instant:  = -k-Na* ,, . cell  r a t e c o n s t a n t k depends on the r a t e o f u n i d i r e c t i o n a l sodium e f f l u x ,  which i n t u r n depends, i n p a r t i c u l a r ,  on the a c t i v i t y o f sodium i n the  s o l u t i o n b a t h i n g the i n t r a c e l l u l a r s i t e s o f the t r a n s p o r t mechanisms. Thus: Na* and  d_  s i n c e dk dt (^_ dt  =  e l l  l  n  N  a  *  Na* e  U  e l l  (t=0)  =  _  i s assumed t o be z e r o .  In N a * e l l ) / ( — In — Na^ n)' dt dt  . exp(-kt)  = d_  k  l  n  d _  B i t t a r et: al. w  h  i  c  e  h  t  h  e  y  a  *  e  U  r e f e r to the " s l o p e r a t i o "  found  Hodgkin and Keynes (1956) c o n s i d e r e d and e x p l a n a t i o n t h a t the sodium e f f l u x was  N  to be l e s s than u n i t y ,  r e j e c t e d as a p o s s i b l e  not v e r y s e n s i t i v e to changes i n  104  the i n t r a c e l l u l a r sodium c o n c e n t r a t i o n to  raise  (Na)^,  (Na)± caused an a p p r e c i a b l e r i s e  considered  i n the sodium e f f l u x .  a  was d i r e c t l y p r o p o r t i o n a l t o ( N a ) ^  each i n s t a n t but the p r o p o r t i o n a l i t y c o n s t a n t  slowly declined with  They demonstrated t h a t a t any g i v e n time the sodium e f f l u x strict  They  i t p o s s i b l e t h a t t h e i r p r e p a r a t i o n was s l o w l y d e t e r i o r a t i n g , so  t h a t the u n i d i r e c t i o n a l sodium e f f l u x M^ at  s i n c e i n j e c t i o n o f sodium  time.  increased i n  p r o p o r t i o n t o the amount o f sodium i n j e c t e d i n t o the axon.  B i t t a r e t al_. (1972) mentioned the p o s s i b i l i t y t h a t the "sodium pump" was r u n n i n g  down i n t h e i r p r e p a r a t i o n , but argued t h a t t h e s m a l l  slope  r a t i o was a c t u a l l y due t o damage done t o the " i n t e r n a l membrane system" by the passage o f the m i c r o i n j e c t o r .  An examination o f i n j e c t e d f i b r e s  w i t h the e l e c t r o n microscope had r e v e a l e d l o c a l d i s r u p t i o n o f the s a r c o plasmic  r e t i c u l u m and c l e f t s a l o n g the i n j e c t i o n t r a c k .  t h a t sodium and c a l c i u m were compartmentalized c r e a t e d by t h e i n j e c t i o n . sodium be sequestered  hypothesized  i n microsome-like v e s i c l e s  T h i s r e q u i r e s t h a t some o f the i n j e c t e d r a d i o -  a t the time o f i n j e c t i o n and exchange o n l y v e r y  w i t h the f r e e i n t r a c e l l u l a r sodium. out,  They  As Dick  t h i s would cause the r a t i o o f d_ i  n  slowly  and L e a (1967) have p o i n t e d  Na* ll e  t  o  iL_ l n i l _ Na* n  t  o  e  c  l  u  a  i  dt ' dt dt the f r a c t i o n o f the i n t r a c e l l u l a r r a d i o s o d i u m which i s f r e e i n the myoplasm c  (assuming t h a t d_ Na* i i ^ dt c  e  l  s  li  n  e  a  r  ly  i  p r o p o r t i o n a l t o the amount o f f r e e  From such a c a l c u l a t i o n , . B i t t a r jet aT. conclude  t h a t on average about 30%. o f the i n j e c t e d r a d i o s o d i u m  i s sequestered,  some experiments an average o f about 75% i s sequestered.  they p o s t u l a t e t h a t the c e l l s  w i t h time, form two d i s t i n c t p o p u l a t i o n s Some p h a r m a c o l o g i c a l  and  In addition,  i n which the f r a c t i o n o f i n j e c t e d  l o s t per u n i t time does n o t f a l l w i t h time, and those  workers.  i  1  l a b e l , as d i s c u s s e d above).  in  e  radiosodium  i n which i t does  o f normal b a r n a c l e muscle  fall  cells.  experiments were a l s o done by B i t t a r and co-  They were i n t e r p r e t e d i n terms o f bound sodium, and so w i l l be  105  d i s c u s s e d here.  B i t t a r and T a l l i t s c h  (1975, 1976)  showed t h a t exposure  to  a l d o s t e r o n e o f muscles from a b a r n a c l e which had been exposed to a l d o s t e r o n e over the p r e v i o u s n i g h t r e s u l t s  i n a h a l t o f the d e c l i n e o f the f r a c t i o n o f  the i n j e c t e d r a d i o s o d i u m l o s t per u n i t time. the f r a c t i o n resuming  T h i s e f f e c t was  i t s d e c l i n e when a l d o s t e r o n e was  b a t h i n g s o l u t i o n a f t e r an exposure  of less  reversible,  removed from the  than 30 minutes.  They  t h a t , a f t e r the pretreatment w i t h a l d o s t e r o n e , acute exposure to  a l d o s t e r o n e caused a r e v e r s i b l e r e l e a s e o f r a d i o s o d i u m from  binding  proposed  o f the  cell  intracellular  sites.  The a c t o f i n j e c t i n g s o l u t i o n s o f NaCl a f t e r r a d i o s o d i u m had injected  been  ( i n t o a l d o s t e r o n e - p r e t r e a t e d c e l l s ) a l s o caused a c e s s a t i o n o f  the d e c l i n e o f the f r a c t i o n o f the i n j e c t e d r a d i o s o d i u m l o s t per u n i t even w i t h s o l u t i o n s o f NaCl t r a t i o n was  so d i l u t e t h a t the i n t r a c e l l u l a r sodium  r a i s e d by o n l y 1 mM.  Subsequent acute exposure  had no e f f e c t u n l e s s the i n j e c t e d NaCl had c e l l u l a r sodium c o n c e n t r a t i o n .  concen-  to a l d o s t e r o n e  c o n s i d e r a b l y i n c r e a s e d the  steady  level.  i n the o v e r n i g h t pretreatment w i t h a l d o s t e r o n e a c t i n o m y c i n D  included,  i t was  found t h a t no c e l l  a c u t e exposure It  was  showed a d e c l i n e w i t h time o f the  f r a c t i o n o f i n j e c t e d r a d i o s o d i u m l o s t per u n i t time. o n l y w i t h a l d o s t e r o n e was  intra-  In the l a t t e r case, the r a t e o f l o s s o f  r a d i o s o d i u m r o s e s l o w l y t o a new If  time,  I f a c e l l pretreated  exposed a c u t e l y t o s p i r o n o l a c t o n e , subsequent  t o a l d o s t e r o n e was  i s i n t e r e s t i n g that  without  effect.  in a later publication, Bittar,  Chambers,  and  S h u l t z (1976) found t h a t the f r a c t i o n o f i n j e c t e d r a d i o s o d i u m l o s t per u n i t time was  c o n s t a n t i n almost a l l cases  the f i g u r e s ) .  ( j u d g i n g from the data p r e s e n t e d i n  T h e i r specimens were o b t a i n e d from Puget Sound, w h i l e f o r  the p r e v i o u s work b a r n a c l e s both from Puget Sound and used.  D i f f e r e n c e s i n the i o n content o f these two  from C a l i f o r n i a were  p o p u l a t i o n s have been  106  reported The  ( B r i n l e y 1968; Gayton, A l l e n , & Hinke 1969). acute.exposure t o a l d o s t e r o n e  a l s o caused a delayed  of aldosterone-pretreated  cells  t r a n s i e n t s t i m u l a t i o n o f the r a d i o s o d i u m e f f l u x .  T h i s e f f e c t was a b o l i s h e d by a c u t e treatment w i t h a c t i n o m y c i n DPH ( d i p h e n y l h y d a n t o i n ) ,  D, ouabain,  or i n j e c t e d e t h a c r y n i c a c i d , but was  by maneuvers which would i n c r e a s e the i n t r a c e l l u l a r supply  stimulated  o f ATP.  To account f o r these f i n d i n g s , B i t t a r and coworkers suggested t h a t aldosterone  induces s y n t h e s i s  muscle c e l l ,  o f new p r o t e i n r e c e p t o r s  i n the barnacle  some o f which cause the r e v e r s i b l e r e l e a s e o f "bound" i n t r a -  c e l l u l a r sodium ( a l l e g e d i n the e a r l i e r paper t o be sequestered  i nvesicles  c r e a t e d by the m i c r o i n j e c t o r ) and some o f which cause a delayed  stimulation  o f the ATP-dependent sodium e f f l u x , upon subsequent exposure t o a l d o s t e r o n e . A d i f f e r e n t explanation  f o r the observed " s l o p e r a t i o " ,  i n terms o f  an e f f e c t i v e i n t r a c e l l u l a r s i n k f o r i n j e c t e d radiosodium, w i l l be d e s c r i b e d i n the d i s c u s s i o n o f the experimental  portion of this section.  account f o r the e f f e c t s o f a l d o s t e r o n e but  I t cannot  r e p o r t e d by B i t t a r and coworkers,  i t seems l i k e l y t h a t the main e f f e c t o f a l d o s t e r o n e  i s on t h e t r a n s p o r t  systems i n the membrane r a t h e r than on the s t a t e o f t h e i n t r a c e l l u l a r sodium.  The s u g g e s t i o n  t h a t over h a l f o f the exchangeable  sodium can be r e v e r s i b l y sequestered  does not seem reasonable,  knowledge o f t h e morphology and i o n - s e q u e s t e r i n g It  intracellular  i s p o s s i b l e t h a t i n some c e l l s  the supply  g i v e n our  p r o p e r t i e s o f the c e l l . of metabolic  energy i n a  s u i t a b l e form f o r u t i l i z a t i o n by the sodium t r a n s p o r t systems i s n o t o p t i m a l , so that the system i s indeed  'running  down'.  Aldosterone  specifically  promotes the t r a n s p o r t o f sodium i n some c e l l s by a mechanism which the s y n t h e s i s  o f new p r o t e i n by the c e l l .  involves  T h i s c o u l d cause a c t i v a t i o n o f  the t r a n s p o r t enzymes, p r o v i d e a d d i t i o n a l energy f o r the t r a n s p o r t enzymes, provide  a d d i t i o n a l t r a n s p o r t enzymes, o r y i e l d a combination o f these  107  effects  (Feldman,  Funder, & Edelman 1 9 7 2 ) .  METHODS  D i s s e c t i o n o f b a r n a c l e muscle bundles has been d e s c r i b e d P r e v i o u s work on the sodium e f f l u x from b a r n a c l e muscle  i n s e c t i o n 3.  c e l l s was  w i t h c e l l s which had been c u t o f f a t the b a s i s , as mentioned  done  above.  Since  t h i s cannot be done w i t h o u t damaging the c e l l membrane, s p e c i a l measures were r e q u i r e d to prevent the r a p i d o c c u r r a n c e o f d e t e r i o r a t i o n o f the c e l l . Brinley oil  (1968)  immersed the t e r m i n a l 5 - 1 0 mm  f o r a t l e a s t 3 0 minutes  o f the c u t end o f the c e l l i n  b e f o r e c a n n u l a t i o n and  a l . a p p a r e n t l y adopted a s i m i l a r procedure  injection.  (Bittar 1966).  B i t t a r et  In the p r e s e n t  work the c e l l s were kept i n t a c t throughout, w i t h the tendon a l o n e b e i n g cannulated. or  The c e l l  membrane was  the sodium e l e c t r o d e through the tendon end, and by the m i c r o p i p e t t e  e l e c t r o d e r a d i a l l y about 2 0 mm ing  from the tendon end  ( F i g . 2 ) . No  o i l was-used, y e t o n l y r a r e l y d i d these m a n i p u l a t i o n s cause  damage t o the c e l l ^ N a C l was t i l l e d water. the  breached o n l y by the i n j e c t i o n n e e d l e  22  (and thereby cause the c e l l  o b t a i n e d from New  NaCl r e d i s s o l v e d i n d i s t i l l e d  visible  t o be d i s c a r d e d ) .  England N u c l e a r , c a r r i e r - f r e e ,  B e f o r e use f o r i n j e c t i o n ,  insulat-  the water was  water or i n  23 NaCl  in dis-  evaporated o f f and solution.  I n j e c t i o n Apparatus. A H a m i l t o n m i c r o s y r i n g e was of the  1.0 m i c r o l i t r e  used throughout.  ( A ) , a Chaney adaptor, and a metal c o l l a r  p r o x i m a l p a r t o f the n e e d l e (model NCH  from 4 mm  o.d.  T h i s had a nominal volume  7001).  protecting  A f i n e n e e d l e was  drawn  l e a d g l a s s , on a mechanical m i c r o p i p e t t e p u l l e r w i t h a l o n g  108  throw (Hinke 1969a).  S u f f i c i e n t l y d u r a b l e needles had c y l i n d r i c a l  shaft  w i t h o.d. 110-120yU and l e n g t h from t h e top o f t h e s h o u l d e r t o t h e t i p o f 38 mm. was  The t i p was broken o f f t o t h i s  beveled s l i g h t l y .  l e n g t h i n such a manner t h a t t h e t i p  New g l a s s needles were made each day.  The g l a s s n e e d l e was a t t a c h e d t o t h e s y r i n g e n e e d l e w i t h s t i c k y wax as f o l l o w s  (Fig. 4).  With t h e p l u n g e r withdrawn from t h e t i p s l i g h t l y ,  a s m a l l c o l l a r o f h o t s t i c k y wax was put on t h e metal s y r i n g e needle the t i p , and a l l o w e d t o harden.  near  D i s t i l l e d water was then drawn up i n t o the  s y r i n g e t o 0 . 9 A , t h e t i p was d r i e d by b l o t t i n g , and t h e p l u n g e r withdrawn to  0 . 9 5 ^ so no water was a t the t i p .  The metal n e e d l e was then  i n t o t h e g l a s s n e e d l e , up t o t h e s h o u l d e r .  inserted  The s h o u l d e r (where t h e wax  c o l l a r c o n t a c t e d the i n s i d e o f the g l a s s ) was b r i e f l y passed through a g e n t l e flame so t h a t the s t i c k y wax melted,  and t h e g l a s s needle was then  g e n t l y pushed f u r t h e r onto the metal needle, u n t i l stopped by t h e t a p e r i n g s h o u l d e r o f the g l a s s . s e a l the needles t o g e t h e r w i t h o u t bubbles. which extends  t h e metal needle was  The s t i c k y wax flowed to  The stem o f t h e g l a s s needle,  i n s i d e t h e metal p r o t e c t i v e c o l l a r , was then f i x e d t o the  metal c o l l a r w i t h d e n t a l i m p r e s s i o n compound. e x p e l l e d i n t o t h e g l a s s needle, and u s u a l l y  The d i s t i l l e d water was then  filled  i t w i t h o u t bubbles.  all  a i r had been e x p e l l e d from t h e g l a s s n e e d l e , the assembled  was  mounted v e r t i c a l l y on t h e i n j e c t o r  n e e d l e submerged i n d i s t i l l e d The  injector  ( F i g . 4)  water.  c o n s i s t e d o f a brace which f i r m l y h e l d the micromanipulator  so t h a t t h e e n t i r e m i c r o s y r i n g e c o u l d be moved i n t h e v e r t i c a l  direction  ( ' p o s i t i o n i n g ' ) , and so t h a t t h e b a r r e l o f t h e m i c r o s y r i n g e c o u l d  be moved r e l a t i v e t o the p l u n g e r ( ' i n j e c t i n g ' ) . used  microsyringe  (see below) w i t h t h e t i p o f t h e g l a s s  m i c r o s y r i n g e by t h e b a r r e l and by t h e plunger, and a P r i o r rebuilt  When  to p o s i t i o n  the glass needle i n the c e l l ,  The former movement was and t h e l a t t e r t o withdraw  F i g u r e 4. M i c r o i n j e c t o r . The b a s i c f e a t u r e s are shown, not to s c a l e . I n s e t : d e t a i l of the c o n n e c t i o n of the g l a s s i n j e c t i o n needle to the Hamilton s y r i n g e n e e d l e . Not to s c a l e .  110  the needle w h i l e clamp h o l d i n g  e x p e l l i n g a column of i n j e c t i o n f l u i d  the s y r i n g e was  adjustable  i n two  t h a t the g l a s s n e e d l e c o u l d be a l i g n e d w i t h  i n t o the c e l l .  The  h o r i z o n t a l d i r e c t i o n s , so  the v e r t i c a l d e f i n e d by  the  motion of the m i c r o m a n i p u l a t o r .  C a l i b r a t i o n of the M i c r o i n j e c t o r . To  t e s t the u n i f o r m i t y o f the d e l i v e r y of f l u i d  from the s y r i n g e ,  a  22 solution of and  ejected  NaCl i n water was  drawn up  i n t o gamma counter g l a s s c o u n t i n g  d i s t i l l e d water, i n a l i q u o t s n o m i n a l l y a l i q u o t s c o u l d be e j e c t e d b e f o r e total  f o r each of the p r e c e d i n g  m i x i n g o f the working f l u i d w i t h which i t was  of 0 . 1 ^ .  , of  F i v e s u c c e s s i v e 0.1  "X  the counts i n the tube dropped below tubes.  The  d r o p o f f presumably was  from (nominally)  0.05/\  the  due  to  the c a l i b r a t e d f l u i d  In s e v e r a l such t e s t s , and to 0.5^,  trial  ejections  the e j e c t e d volume  from the amount o f r a d i o s o d i u m e j e c t e d agreed w i t h  volume to w e l l w i t h i n the u n c e r t a i n t y due than 47o) .  ^  tubes c o n t a i n i n g 5 ml  ( d i s t i l l e d water) w i t h  i n contact.  o f amounts v a r y i n g calculated  i n t o the s y r i n g e , to 0.75  to c o u n t i n g  the nominal  of the i s o t o p e  I n j e c t i o n s i n t o c e l l s d u r i n g experiments were c a r r i e d out  (less within  these l i m i t s o f u n i f o r m d e l i v e r y . The  absolute value  of the i n j e c t e d volume was  a s o l u t i o n of known r a d i o a c t i v i t y , and was value  to w i t h i n the u n c e r t a i n t y o f  C o l l e c t i o n of The  but  found to agree w i t h  the nominal  counting.  Isotope.  chamber used d u r i n g  p e r f u s i o n f l u i d was  t e s t e d by e j e c t i o n s o f  i n j e c t i o n of i n t a c t  s i m i l a r to t h a t d e s c r i b e d  considerably modified  ( F i g . 5).  The  held v e r t i c a l l y at i t s r e s t length, with  f i b r e s and  of  by A l l e n and Hinke (1970)  cannulated the  collection  fibre  (see below)  was  fragment o f the b a s i s r e s t i n g  Ill  F i g u r e 5. Apparatus The moveable b l o c k s , the o u t f l o w channel, fragment of b a s i s i s  f o r i s o l a t i o n of a segment of a c e l l f o r p e r f u s i o n . one c o n t a i n i n g the i n f l o w channel and the other c o n t a i n i n g a r e shown i n the opened p o s i t i o n . A c e l l a t t a c h e d to a outlined.  112  on the f a l s e f l o o r o f the lower chamber.  The h e i g h t o f the f a l s e f l o o r was  a d j u s t e d so t h a t the c a n n u l a t e d tendon was a t the d e s i r e d h e i g h t above the top  o f the chamber w h i l e undue t e n s i o n was not e x e r t e d on the f i b r e .  The  movable P l e x i g l a s b l o c k s were r e t r a c t e d and t h e chamber f i l l e d w i t h R i n g e r ' s s o l u t i o n f o r i n j e c t i o n o f i s o t o p e and placement  of microelectrodes.  The  movable b l o c k s were then brought t o g e t h e r by t u r n i n g the thumb screws, while the displaced grooves m i l l e d  f l u i d was withdrawn  through the s u c t i o n tubes.  The  i n the movable b l o c k s formed a c y l i n d r i c a l chamber around  the  f i b r e when the b l o c k s were brought t o g e t h e r .  the  lower chamber, c o n t a i n i n g t h e fragment o f b a s i s , by a s e a l o f p e t r o l e u m  jelly  (Vaseline).  T h i s was s e p a r a t e d from  V a s e l i n e was a l s o used t o s e a l the movable b l o c k s t o the  s t a t i o n a r y p a r t s o f the chamber.  The grease s e a l which s e p a r a t e d the upper  and lower chambers was r o u t i n e l y t e s t e d by r a i s i n g the f l u i d lower chamber and o b s e r v i n g the f a i l u r e o f f l u i d or  by f i l l i n g  level  i n the  t o e n t e r the upper chamber,  the upper chamber and o b s e r v i n g no l e a k i n t o t h e lower  chamber. P e r f u s i o n f l u i d was d e l i v e r e d  from a Braun s y r i n g e pump, f i t t e d w i t h  a 50 ml s y r i n g e ( o r two 50 ml; s y r i n g e s i n p a r a l l e l ) , 1 ml/min.  at a constant rate of  Two such pumps were used a l t e r n a t e l y , so t h a t s o l u t i o n  changes  were accomplished by d i s c o n n e c t i n g t h e d e l i v e r y tube from one s y r i n g e and c o n n e c t i n g i t t o the o t h e r . the  T h i s s w i t c h takes l e s s than two seconds, so  i n t e r r u p t i o n o f the p e r f u s i o n was n e g l i g i b l e . P e r f u s i o n f l u i d was drawn o f f through t h r e e exhaust p o r t s l o c a t e d near  the  top o f the upper chamber, and c o l l e c t e d d i r e c t l y  i n a g l a s s gamma  c o u n t i n g tube ( F i g . 6 ) . The c o l l e c t i o n p e r i o d was 5 min. tube was changed manually.  This  The c o l l e c t i o n  i n t e r r u p t e d the c o l l e c t i o n f o r 2 t o 3  seconds, but caused no l o s s o f f l u i d . The volume o f the upper chamber, which housed  the i n j e c t e d p o r t i o n o f  113  bleeder suction  from washout chamber rubber stopper  glass gamma counter tube  F i g u r e 6. Vacuum system. The p e r f u s a t e from the washout chamber i s c o l l e c t e d i n the g l a s s gamma-counter tube.  ( F i g . 5)  114  the f i b r e , was  1.0 ml.  The washout time o f the chamber was t e s t e d , w i t h a  g l a s s r o d p l a c e d where the f i b r e would n o r m a l l y be, and was 87% complete w i t h i n 2 minutes, exponential loss  found  to be  c o r r e s p o n d i n g to a time constant f o r  o f l a b e l o f about 1 min. ^.  The e f f e c t o f the f i n i t e washout time o f the chamber can be e s t i m a t e d from a simple model. and  For the myoplasm  the c o l l e c t i o n tube  •ic (Na^), the washout chamber  ic (Na2),  ic (Na^) c o n s i d e r e d as t h r e e compartments i n s e r i e s  w i t h no b a c k f l u x , and n  _l  =  -k  2  =  k  1  3  =  k  2  Na*  x  dt d N a  Na*  -  k  Na*  2  dt d N a  Na*  dt ic  ic  where a t t = 0, Na-^ — (Na^)^ and N a  s 2  Na-j = 0. The g e n e r a l s o l u t i o n f o r  t h i s simple l i n e a r system i s Na (t) x  = (NaJ)  Na*(t) =  Q  expC-^t)  ^ ^ f J ^ C  1  "  e x  P(" lt)) k  " k ( l - exp(-k t))] . x  2  That i s , ic ^ f i dt while  =  k ^ N a ^ o exp(-k ) l t  k.k^Na?),) r  dNao  so i f dNa^/dt (which must be much l e s s  ~\  i s measured) i s to approximate -dNa^/dt c l o s e l y ,  than k . 2  S i n c e k^ i s t y p i c a l l y 0.01 min"- - and k  about 1 m i n " , t h i s c o n d i t i o n i s w e l l - s a t i s f i e d , 1  1  and except  2  k^  is  f o r the i n t e r v a l  115  j u s t a f t e r t = 0 the f a l l approximates  the f a l l  i n the amount o f l a b e l c o l l e c t e d w i t h time  closely  i n the amount o f l a b e l l e a v i n g the muscle c e l l w i t h  time. The 5 ml samples  o f p e r f u s i o n f l u i d were counted on a w e l l - t y p e gamma  counter (Nuclear Chicago). d u r i n g the experiment c o u n t i n g was  I n i t i a l c o u n t i n g f o r 1 min. per sample was  t o monitor the p r o g r e s s o f the e f f l u x .  r e p e a t e d a t 20 min. per  Later,  done  the  sample.  Sample tubes were r e - u s e d a f t e r washing w i t h d e t e r g e n t and then chromic sulfuric acid.  Backgrounds f o r each tube were determined by a 10  min.  count and s u b t r a c t e d i n d i v i d u a l l y from the c o r r e s p o n d i n g sample count. T h i s economy was  p o s s i b l e because glassware c l e a n i n g and u n l i m i t e d counter  time were a v a i l a b l e . background  Sample counts were almost always more than 10 times  (the e x c e p t i o n s b e i n g samples  o f low a c t i v i t y a t the end of a  l o n g e f f l u x experiment), and the background was  equal t o t h a t o f new  o f the c h e m i c a l l y c l e a n e d tubes  tubes.  M i c r o i n j ec t ion. C a n n u l a t i o n o f the tendon o f the muscle  fibre  w i t h o u t a f f e c t i n g the c e l l membrane ( F i g . 2 ) . positioned v e r t i c a l l y the  s i l k t i e was  chambers.  accomplished  The c a n n u l a t e d f i b r e  was  i n the P l e x i g l a s chamber as d e s c r i b e d above, so t h a t  about 3 mm  The cannula was  The P l e x i g l a s chamber was  above the l e v e l o f the f l u i d held v e r t i c a l l y ,  form o f a second Palmer  screw stand.  down through the cannula and  the  by a P r i o r m i c r o m a n i p u l a t o r .  p o s i t i o n e d on the h o r i z o n t a l The g l a s s n e e d l e was  i n t o the muscle  the l o n g a x i s o f the f i b r e .  a b i n o c u l a r microscope,  filling  p o s i t i o n e d on the h o r i z o n t a l p l a t f o r m o f a Palmer  screw stand, and the m i c r o i n j e c t o r was  to  is readily  plat-  then  advanced  f i b r e , as c l o s e as  possible  The advance o f the needle was  viewed  through  from the f r o n t and, v i a a s m a l l m i r r o r p o s i t i o n e d  116  at  a 45 degree angle near the muscle f i b r e ,  diameters  of the f i b r e i n these  micrometer.  I l l u m i n a t i o n was  two  from the s i d e .  The  views were measured w i t h an  q u i t e v i s i b l e i n the opaque muscle f i b r e , and was  the c e n t r a l a x i s o f the muscle The  the needle w h i l e e x p e l l i n g the i n j e c t i o n f l u i d 22 mm  long, corresponding  t i o n was of  terminated  the f l u i d was  having  5 mm  t o 0.4^,  then used to withdraw  i n t o the c e l l .  i n most cases, but  injection  was  f a r enough down  the t i p d e v i a t e from the a x i a l l i n e .  i n s i d e the  With the more c o n c e n t r a t e d  Injec-  cell.  i n j e c t i o n s o l u t i o n s , t h e r e would o f t e n be a  the i n j e c t e d r e g i o n , and  by the time the m i c r o e l e c t r o d e s were put continued.  The  from the p o i n t of impalement, to ensure t h a t a l l  deposited  s l i g h t c o n t r a c t u r e over  needle  always kept c l o s e to  s h o r t e r when i t proved i m p o s s i b l e to advance the needle the muscle c e l l w i t h o u t  The  fibre.  i n j e c t i n g movement o f the m i c r o i n j e c t o r was  t r a c k was  eyepiece  p r o v i d e d by a 500 watt lamp ( V o l p i ) , d i r e c t e d  o b l i q u e l y at the muscle f i b r e by a f i b r e o p t i c l i g h t c o n d u i t . was  outside  i f t h i s had not  i n t o p l a c e , the experiment was  I n most cases, and w i t h the more d i l u t e  almost always, the i n j e c t i o n was  disappeared  injection  not  fluids  w e l l t o l e r a t e d by the muscle f i b r e , as f a r  as c o u l d be d e t e c t e d by o b s e r v a t i o n through the microscope. The m i c r o i n j e c t o r was was  removed and  p l a c e d i n the c e l l a x i a l l y ,  the s o d i u m - s p e c i f i c  through the cannula,  via  manipulations  s i m i l a r to those used f o r p o s i t i o n i n g the i n j e c t o r needle. tip of  o f the e l e c t r o d e was the muscle f i b r e .  microelectrode  The  sensitive  p l a c e d i n about the c e n t r e o f the i n j e c t e d  The m i c r o p i p e t t e e l e c t r o d e t i p was  region  passed o b l i q u e l y  a c r o s s the c e l l membrane so i t r e s t e d a t the l e v e l o f the sodium e l e c t r o d e . The movable b l o c k s were then brought t o g e t h e r the grease  s e a l was  t e s t e d , and  to form the e f f l u x chamber,  the p e r f u s i o n was  these maneuvers, the f i r s t p e r f u s i o n f l u i d was  started.  W i t h a l l of  c o l l e c t e d 10 to 12 minutes  117  a f t e r the a c t u a l  injection.  Passive Loading  Experiments.  From a s i n g l e d i s s e c t e d muscle bundle, c e l l s were i s o l a t e d ,  t h r e e s m a l l groups o f muscle  each group on a fragment  o f the b a s i s .  These were 22  p l a c e d i n a v e s s e l w i t h 10 ml o f normal R i n g e r ' s s o l u t i o n t o which had been added, t o 20,930 cpm per m i c r o l i t r e , The next day,  and kept a t 5° C o v e r n i g h t .  two o f the s m a l l groups were used  d e s c r i b e d above f o r m i c r o i n j e c t e d c e l l s , t h i r d group was  NaCl  f o r e f f l u x experiments,  but they were not i n j e c t e d .  loaded f o r 48 hours b e f o r e b e i n g used  as The  f o r e f f l u x measurement.  RESULTS  E f f e c t s o f m i c r o i n j e c t i o n on the The cell  cell.  e f f l u x o f m i c r o i n j e c t e d r a d i o s o d i u m from a s i n g l e b a r n a c l e muscle  i s shown i n F i g . 7.  A similar plot  for a c e l l  loaded p a s s i v e l y ,  immersion i n a s o l u t i o n which c o n t a i n e d radiosodium, (page 128).  The  by  i s p r e s e n t e d as F i g . 9  f a l l of the r a d i o s o d i u m content o f the i n j e c t e d c e l l  time and the f a l l o f the e f f l u x o f r a d i o s o d i u m w i t h time can each be matched by a simple e x p o n e n t i a l f u n c t i o n . from the p a s s i v e l y - l o a d e d c e l l ,  A f t e r the i n i t i a l r a p i d  a s c r i b e d to the e x t r a c e l l u l a r space  with closely  efflux as  d i s c u s s e d below, the p l o t s can each be matched by a simple e x p o n e n t i a l f u n c t i o n d u r i n g the e f f l u x  i n t o normal R i n g e r ' s s o l u t i o n  r a t e constants are s i m i l a r  f o r the two  cases.  The  ( F i g . 9).  The.  'slope r a t i o ' e f f e c t i s  d i s c u s s e d below. In  F i g . 10  (page 140)  i s p r e s e n t e d a summary o f the raw data and  reduced  118  F i g u r e 7. S e m i l o g a r i t h m i c p l o t of the amount o f r a d i o s o d i u m c o l l e c t e d i n the p e r f u s a t e from a c e l l loaded w i t h i s o t o p e by m i c r o i n j e c t i o n (upper t r a c e ) , and the amount of r a d i o s o d i u m remaining i n the c e l l a t the s t a r t o f each c o l l e c t i o n p e r i o d , c a l c u l a t e d by b a c k - a d d i t i o n (lower t r a c e ) , v e r s u s time. The c e l l was p e r f u s e d w i t h normal R i n g e r ' s s o l u t i o n . L i n e s r e p r e s e n t the ^ l i n e a r r e g r e s s i o n l n y = l n a + bx. Upper: a = 23,112 cpm, b = -0.00928 m i n , r = 0.97. Lower: a = 548,947 cpm, b = -0.00786 m i n " , r = 1.00. -  ?  1  2  119  results  for a typical  experiment  i n the present  series.  Measurements w i t h the s o d i u m - s p e c i f i c m i c r o e l e c t r o d e show t h a t myoplasmic sodium a c t i v i t y  (afj ) a  m  r i s e s a f t e r the i n j e c t i o n i n most  sometimes t a k i n g 30 minutes to r e a c h a steady v a l u e . in noninjected c e l l s was  i s t y p i c a l l y about  l e s s marked i n c e l l s o f a l a r g e r Further,  10 mM  cells,  (The v a l u e o f  - see T a b l e I I ) .  (a^ ) a  This r i s e  immersion o f a b a r n a c l e muscle  1968;  sodium content almost unchanged (McLaughlin & Hinke 1966;  Brigden, S p i r a , & Hinke 1971;  sodium-loaded  the  ' r a p i d ' sodium-free  c e l l s has been mentioned i n s e c t i o n 2 and  in s e c t i o n 6).  When a c e l l was  kept  Ringer's s o l u t i o n immediately  experiments that  after  much l e s s marked.  injection,  is discussed f u l l y  a  a  used  The o b s e r v a t i o n by B r i n l e y  m  after  i n a number o f the  I t s h o u l d be mentioned  no data was  used  for calculation  had become steady, except where noted  m  i n normal  the r i s e o f ( a j j )  T h i s maneuver was  i n the p r e s e n t s e r i e s o f experiments, (a^j )  Brinley  i n sodium-free s u c r o s e - s u b s t i t u t e d  t o o b t a i n data a t low sodium l e v e l s ;  of fluxes u n t i l  intra-  effect in  R i n g e r ' s s o l u t i o n f o r 5 to 10 minutes p r i o r to i n j e c t i o n , and  i n j e c t i o n was  cell  s u c r o s e - s u b s t i t u t e d R i n g e r ' s s o l u t i o n f o r 5 t o 10 minutes  washes out most o f the e x t r a c e l l u l a r sodium w h i l e l e a v i n g the t o t a l cellular  m  diameter.  i t has been r e p o r t e d that  i n sodium-free  the  (1968) o f a s l i g h t t r a n s i e n t  explicitly. depolarization  o f the membrane p o t e n t i a l w i t h each advance o f the i n j e c t o r n e e d l e was i n the I n t r o d u c t i o n .  B i t t a r ejt al_.  (1972) r e p o r t e d t h a t the t o t a l  content o f the c e l l was  r a i s e d by i n j e c t i o n but not by c a n n u l a t i o n .  suggested  due  t h a t t h i s was  noted  sodium  to damage to the membrane o f the c l e f t s  They along  the i n j e c t i o n t r a c k , c a u s i n g e x t r a c e l l u l a r sodium t o be r e l e a s e d i n t o  the  myoplasm. In F i g . 7 i t can be seen t h a t the f i r s t  two p o i n t s i n the p l o t o f the  measured amount o f r a d i o s o d i u m l e a v i n g the c e l l per u n i t time  (upper  plot)  120  seem h i g h .  In many experiments, the  c o n c e i v a b l y c o u l d be  due  f i r s t few  t o a s m a l l l o s s of  injection fluid  c l e f t s d u r i n g the a c t u a l  injection.  s l i g h t d e l a y between the  i n j e c t i o n i t s e l f and  B i t t a r et a l . (1972) noted that o f the  e f f l u x of previously  r e p o r t e d that  the  p o i n t s were lower.  This  i n t o damaged  I t a l s o c o u l d s i m p l y be due the  to  i n i t i a t i o n of  a second i n j e c t i o n d i d not  the  perfusion.  a l t e r the  i n j e c t e d radiosodium, a l t h o u g h B r i n l e y  c e l l would not  t o l e r a t e a second  course  (1968)  injection.  3 The  e x t r a c e l l u l a r space marker ( H ) i n u l i n does not  pass the  intact  cell  3  membrane. no  When a s o l u t i o n c o n t a i n i n g ( H ) i n u l i n was  l a b e l was  d e t e c t e d subsequently i n the  hand, f o l l o w i n g  i n j e c t i o n of  bathing solution.  bathing solution.  I t was  readily,  sodium (and  On  the  the  cell, other  l a b e l appeared  concluded t h a t any  sodium through damaged membrane d u r i n g m i c r o i n j e c t i o n Altogether,  into a  (^CJDMO, ( 5 , 5 - d i m e t h y l - 2 , 4 - o x a z o l i d i n e d i o n e ) ,  which i s known t o c r o s s the c e l l membrane q u i t e promptly i n the  injected  i t seems almost c e r t a i n t h a t  c a l c i u m - B i t t a r et. a_l. 1972)  the  loss of  must be  radio-  quite  small.  increase i n i n t r a c e l l u l a r  seen a f t e r  i n j e c t i o n i s due  to a  t r a n s i e n t i n f l u x of e x t r a c e l l u l a r f l u i d d u r i n g i n j e c t i o n . The membrane p o t e n t i a l E i s the most s e n s i t i v e i n d i c a t o r o f m  the  i n t e g r i t y of the  10  to  minutes)  to  r  c e l l membrane.  As  a rule, E  m  was  d e p o l a r i z e d by  m i l l i v o l t s a f t e r i n j e c t i o n , then r e c o v e r e d s l o w l y (20 assume a s t e a d y v a l u e c l o s e the  pre-injection  tials  for several Altogether,  benign, the  value.  t o but  Injected  generally cells  s l i g h t l y more p o s i t i v e  hours a t room temperature i n normal Ringer's i t was  concluded t h a t w h i l e m i c r o i n j e c t i o n  for several  hours a f t e r  than  then showed s t a b l e membrane poten-  injected c e l l s recover quickly  i n j e c t e d c e l l s do  to 30  15  and  solution.  i s not  appear to behave as  injection.  entirely non-  121  The  'slope r a t i o ' .  For  the experiment  In Na  and In  ^  a c e  p r e s e n t e d i n F i g . 7, the l i n e a r r e g r e s s i o n s o f  n on time i n d i c a t e that almost a l l o f the s c a t t e r can be 2 i n the sampling and c o u n t i n g ( r =0.97  accounted f o r by random e r r o r 1.00  r e s p e c t i v e l y ) , and y i e l d e d time c o n s t a n t s 0.00928 min"-'" and 0.007 86  min "'" r e s p e c t i v e l y . -  d_ ^ j d_ i d_ dt dt dt ' s l o p e s ' y i e l d e d by the l i n e a r r e g r e s s i o n s ,  The s l o p e r a t i o  c a l c u l a t e d as the r a t i o o f the is  and  n  n  0.85. The range o f 'slope r a t i o s '  i n F i g . 8. cell.  f o r a number o f experiments  is indicated  They a r e p l o t t e d versus the myoplasmic sodium a c t i v i t y  U s u a l l y the d u r a t i o n o f exposure  f o r the  t o normal R i n g e r ' s s o l u t i o n  was  o  s h o r t e r than i n F i g . 7 (50 minutes) and the v a l u e f o r r (0.90 to 0.95  i n almost a l l c a s e s ) .  Overall,  was  slightly  lower  these 'slope r a t i o s ' were  c l o s e r to u n i t y than those r e p o r t e d by B i t t a r et a l . (1972). With r e s p e c t to the 'slope r a t i o s ' as i n t e r p r e t e d by B i t t a r el: a_l., p r i n c i p a l q u e s t i o n i s how cell.  the i n j e c t e d r a d i o s o d i u m d i s t r i b u t e s  For convenience, the assumptions  employed i n t h i s t h e s i s w i l l A fundamental  assumption  be  adopted  the  i n s i d e the  i n the model o f the  cell  restated.  o f t h i s model i s t h a t the  c o n c e n t r a t i o n s which a r e o f d i r e c t  importance  intracellular  f o r transmembrane e f f l u x a r e  those o f the myoplasm ( g i v e n c o n s t a n t e x t r a c e l l u l a r c o n d i t i o n s ) .  By  "membrane" i s meant the b a r r i e r t o d i f f u s i o n , and any boundary.layer o f g l y c o c a l y x or o f water which  is  strongly  i n f l u e n c e d by the p r o t e i n - l i p i d  lamina can be i n c l u d e d as p a r t o f the membrane. differ tial  i n the myoplasm and the boundary  cannot,  (sodium)  l a y e r , but e l e c t r o c h e m i c a l poten-  i t b e i n g assumed t h a t transmembrane t r a n s p o r t  slow r e l a t i v e to m i x i n g i n s i d e the c e l l . ion  Ion c o n c e n t r a t i o n s can  activities  do not d i f f e r  i s almost  always  I t can be demanded f o r m a l l y  i n the myoplasm and the  boundary  that  122  1.2  slope ratio 1.0 0.8 0.6 0.4 0.2  20 Na>m  (a  F i g u r e 8.  'Slope R a t i o '  i s the r a t i o  ( —  40 (mM)  In N a  60  c e l l  )/( —  ln —  Na  c g l l  ),  which i s the r a t i o of the s l o p e o f the lower l i n e to t h a t o f the upper l i n e i n F i g . 7. Each p o i n t r e p r e s e n t s one e f f l u x experiment i n normal R i n g e r ' s solution. Not a l l o f the experiments done i n the p r e s e n t s e r i e s are represented. P o i n t s o m i t t e d f o r c l a r i t y f e l l i n the lower range o f (a ) .  123  l a y e r , as The across  l o n g as  t h i s conception  myoplasm bathes the  i n t e r n a l s u r f a c e o f t h i s membrane, and  t h i s membrane, v i a v a r i o u s  e f f l u x from the c e l l  occurs.  sodium e f f l u x i n t o the TTS R e c a l l that  i t was  i f and  mechanisms, t h a t the bulk of the  i t is sodium  That i s , f o r the moment i t i s assumed t h a t  and  the SR  i s unimportant under most c o n d i t i o n s .  s t a t e d above t h a t  — In Na i dt cell  In — dt  —  dt  Na cell  only i f  4-  Na*  dt where d  „  =  The  ,  -k Na*  cell  k = 0.  plasmic  cell  '  s l o p e r a t i o statements must be made to r e f e r to  parameters: the amount of r a d i o s o d i u m i n the myoplasm N a  stituted tuted  of the "membrane" i s kept i n mind.  f o r Na^ -Q, and e  f o r the  the myoplasmic sodium a c t i v i t y  i n t r a c e l l u l a r sodium c o n c e n t r a t i o n  r a t e o f u n i d i r e c t i o n a l sodium e f f l u x . i t was  postulated  as  (  a  j T  a  )  m  m  myo-  is  sub-  is substi-  the determinant o f  Given previous  the  experimental r e s u l t s ,  t h a t the r a t e o f u n i d i r e c t i o n a l sodium e f f l u x i s an  i n c r e a s i n g f u n c t i o n o f the myoplasmic sodium a c t i v i t y over a wide range. Thus as  l o n g as  constant,  the net  model d e s c r i b e d The  the r a t e o f u n i d i r e c t i o n a l sodium e f f l u x i s found to f l u x o f sodium must be  i n the p r e c e d i n g  r a t e constant  I t has cell  been s t a t e d t h a t  the s l o p e r a t i o w i l l conditions If  do not  there  1  the  T h i s r e l a t i o n s h i p i s u s u a l l y expressed as  a  concentration.  i f the r a d i o s o d i u m i s well-mixed i n s i d e the  the r a t e of u n i d i r e c t i o n a l sodium e f f l u x i s  be equal to u n i t y .  o b t a i n can be  i s a net  'membrane-flux  paragraph.  i n t r a c e l l u l a r sodium  at a l l times, and  g i v e n the  k i s , i n p a r t i c u l a r , a f u n c t i o n of the r a t e o f  u n i d i r e c t i o n a l sodium e f f l u x . p o l y n o m i a l i n the  zero,  be  increase  The  constant,  s i t u a t i o n s i n which these  listed.i n the sodium content of the myoplasm w i t h  124  time, the s p e c i f i c a c t i v i t y o f the myoplasm S A  m  will  fall  more q u i c k l y  would be expected a c c o r d i n g t o t h e observed e f f l u x o f radiosodium. of  SA w i l l  A  than fall  decrease d w * , s i n c e a s m a l l e r f r a c t i o n o f the u n i d i r e c t i o n a l ~T~ m dt sodium e f f l u x w i l l c o n s i s t of radiosodium. On the o t h e r hand, as ( a j j ) m  a  M  a  m  r i s e s t h e r a t e o f u n i d i r e c t i o n a l sodium e f f l u x w i l l r i s e , and t h e r e w i l l be an i n c r e a s e i n d — dt specified In  Na* • m  The n e t e f f e c t on the s l o p e r a t i o cannot be  i n the absence  o f a s p e c i f i c model f o r the sodium e f f l u x .  any event, the sodium c o n t e n t o f i n j e c t e d c e l l s has been monitored  w i t h a s o d i u m - s p e c i f i c m i c r o e l e c t r o d e , as noted above. sodium content i n c r e a s e s upon i n j e c t i o n , extracellular stabilize,  fluid,  The i n t r a c e l l u l a r  due t o a t r a n s i e n t  influx of  but t h e sodium content takes a t most 30 minutes t o  and u s u a l l y much l e s s .  T h i s cannot account  f o r the observed  slope r a t i o s . It might  i s c o n c e i v a b l e t h a t the r a t e o f the u n i d i r e c t i o n a l sodium e f f l u x  change i n d e p e n d e n t l y .  energy  from metabolism  F o r example, i t might  i n the i s o l a t e d c e l l  fall  i s depleted.  as the s u p p l y o f The c o n d i t i o n o f  the 'pump' can be i n f e r r e d from o b s e r v a t i o n o f the e f f l u x over a l o n g p e r i o d i n normal  Ringer's  solution.  I t was found t h a t t h e r e was no obvious d e v i a t i o n from l i n e a r i t y " i n the s e m i l o g p l o t s d e p i c t i n g t h e l o s s o f r a d i o s o d i u m from i n j e c t e d c e l l s i n experiments the  o f l o n g d u r a t i o n ( f o r example, F i g . 7 ) .  This indicates  that  'pump' i s n o t r u n n i n g down. The  final possibility  i s t h a t t h e i n j e c t e d r a d i o s o d i u m becomes com-  p a r t m e n t a l i z e d i n s i d e the c e l l .  B i t t a r e_t al_.  (1972) adopted  t h e model o f  D i c k and L e a (1967), wherein some o f the r a d i o s o d i u m i n s i d e t h e c e l l was s e q u e s t e r e d and exchanged w i t h the f r e e i n t r a c e l l u l a r r a d i o s o d i u m o n l y a t a n e g l i g i b l y slow r a t e .  As noted above, t h e s l o p e r a t i o i s equal t o the  f r a c t i o n o f the r a d i o s o d i u m l e f t  i n the c e l l which i s f r e e .  This i s not a  125  c o n s t a n t , but r a t h e r d e c l i n e s w i t h time as the f r e e r a d i o s o d i u m out of the Fig.  i s washed  cell. 7 i n d i c a t e s t h a t the s l o p e d_ i dt  n  jj *  does not d e c l i n e n o t i c e a b l y  a  c  e  l  1  w i t h time, c o n t r a r y t o the D i c k and Lea model i f the s i z e of the f r a c t i o n i s not t o be n e g l i g i b l e .  Indeed, B i t t a r e_t a l . c a l c u l a t e d  f r a c t i o n as up t o 707 o f the i n j e c t e d o  The a l t e r n a t i v e  sequestered this  radiosodium.  i s a compartmental model i n which the i n t e r n a l  ments can exchange sodium.  Many mathematical  treatments  compart-  o f such models  have been p u b l i s h e d , but q u a l i t a t i v e c o n s i d e r a t i o n s can narrow the range o f poss i b i l i t i e s . The rapidly.  i n j e c t i o n loads the myoplasmic compartment, s e l e c t i v e l y and  very  I f t h e r e were an i n t r a c e l l u l a r compartment o f f i n i t e s i z e which  exchanged sodium w i t h the myoplasm w i t h a r a t e c o n s t a n t f o r exchange comparable to the rate- c o n s t a n t f o r the transmembrane-flux, then the  efflux  curve f o r i n j e c t e d c e l l s would not be so c l o s e t o a s i m p l e e x p o n e n t i a l f o r all  times  from near z e r o .  The  compartment would l o a d from, then empty  the myoplasm i n the course of the  into  experiment.  C o n s i d e r a t i o n of both the s i z e o f the h y p o t h e t i c a l compartment and r a t e a t which i t exchanges sodium w i t h the myoplasm i s important  the  i n drawing  this conclusion. For the range o f e f f l u x r a t e c o n s t a n t s found experiments, (as of  i n the present s e r i e s  any such compartment o f a p p r e c i a b l e but not too l a r g e a  e x p l a i n e d i n the next paragraph) whose r a t e c o n s t a n t was  of  size  w i t h i n an o r d e r  magnitude o f the r a t e c o n s t a n t f o r the transmembrane f l u x would  be  d e t e c t a b l e as a d e v i a t i o n from l i n e a r i t y i n a s e m i l o g p l o t such as F i g . 7. A s m a l l compartment which exchanges sodium r a p i d l y w i t h the myoplasm would not be seen as a d e v i a t i o n from l i n e a r i t y , the p r e s e n t c o n t e x t .  The  and  i s o f no concern i n  c o n s i d e r a t i o n s o f s e c t i o n 3 suggest  t h a t the  126  sodium a s s o c i a t e d w i t h i n t r a c e l l u l a r ment.  fixed anionic sites  i s such a compart-  A l a r g e r compartment which exchanges v e r y s l o w l y w i t h the myoplasm  would not be loaded w i t h i n j e c t e d r a d i o s o d i u m i n experiments whose d u r a t i o n was  a few hours, a l t h o u g h i t might be loaded i n an experiment whose d u r a t i o n  was  tens o f hours.  The sodium which exchanges o n l y v e r y s l o w l y , such as  t h a t r e p o r t e d f o r b a r n a c l e muscle by A l l e n and Hinke termed  (1971), and the sodium  " i n e x c h a n g e a b l e " i n s e c t i o n 3, which p r o b a b l y i s the same p o o l ,  should be such a compartment.  I t a l s o i s o f no concern i n the p r e s e n t  context. A v e r y l a r g e compartment would l o a d w i t h i n j e c t e d r a d i o s o d i u m from the myoplasmic compartment throughout  the d u r a t i o n o f the experiment,  not be seen as a d e v i a t i o n from l i n e a r i t y i n the s e m i l o g p l o t  and  might  i fits  exchange w i t h the myoplasm can be d e s c r i b e d by a simple e x p o n e n t i a l f u n c t i o n o f time, even i f the exchange were r e l a t i v e l y r a p i d .  I t would c o n s t i t u t e  an i n t r a c e l l u l a r s i n k f o r radiosodium, and the t o t a l amount o f r a d i o s o d i u m i n the c e l l would not d e c l i n e w i t h time a t a r a t e commensurate t o the d e c l i n e w i t h time o f the r a t e a t which r a d i o s o d i u m appeared  i n the bath.  That  i s , the two s l o p e s would each be c o n s t a n t , but the s l o p e r a t i o would be  less  than u n i t y . In t h i s c o n n e c t i o n , comparison  that  from c e l l s  loaded w i t h r a d i o s o d i u m by immersion  contains radiosodium i s relevant. cells  o f the e f f l u x from i n j e c t e d c e l l s w i t h i n a s o l u t i o n which  Three e f f l u x experiments were done on  from a s i n g l e b a r n a c l e which were loaded w i t h r a d i o s o d i u m by  a t l e s s than 5° C i n normal  Ringer's s o l u t i o n which c o n t a i n e d some r a d i o -  sodium, as d e s c r i b e d i n Methods.  Two  experiments were done a f t e r a 24  l o a d i n g p e r i o d , and one a f t e r a 48 hour n a t i o n was  hour  l o a d i n g . p e r i o d . The e f f l u x d e t e r m i -  c a r r i e d out as f o r i n j e c t e d c e l l s ,  The s e m i l o g p l o t s  immersion  but no  i n j e c t i o n was  f o r the e f f l u x from p a s s i v e l y loaded c e l l s  done. differed  127  from those f o r i n j e c t e d c e l l s . shown i n F i g . 9.  The p l o t s  f o r the second experiment a r e  They were l i n e a r f o r time g r e a t e r than 50 minutes when  p e r f u s i o n was w i t h normal R i n g e r ' s s o l u t i o n .  A l i n e a r r e g r e s s i o n was per-  formed on the p a r t o f the c u r v e from 50 t o 135 minutes.  The r e g r e s s i o n  l i n e was e x t r a p o l a t e d back t o z e r o time, and t h e d i f f e r e n c e between t h e data and t h i s l i n e over the i n t e r v a l shown i n F i g . 9.  from 0 t o 50 minutes was r e - p l o t t e d , as  This operation y i e l d e d a simple exponential.  It is  r e a s o n a b l e t o a s c r i b e t h i s r a p i d e f f l u x t o the washout o f r a d i o s o d i u m from the e x t r a c e l l u l a r space, as w i l l  be d i s c u s s e d below.  An e f f l u x o f comparable  r a t e from the i n t e r i o r o f t h e c e l l has been i m p l i e d by measurements i n f r o g and c r a b muscle, as noted i n s e c t i o n 2, but o n l y i n sodium-free s o l u t i o n . No such r a p i d e f f l u x i n t o normal R i n g e r ' s s o l u t i o n has ever been r e p o r t e d . The s l o p e r a t i o s o b t a i n e d i n the t h r e e experiments were 1.30 and 0.98 f o r a 24 hour l o a d i n g p e r i o d , and 0.99 f o r a 48 hour l o a d i n g p e r i o d . was noted a t the c o n c l u s i o n o f the f i r s t slightly  i r r e g u l a r i n appearance.  It  experiment t h a t the c e l l was  The c o r r e s p o n d i n g s l o p e r a t i o f o r f r o g  s k e l e t a l muscle loaded p a s s i v e l y i s a l s o u n i t y  (Keynes & Swan  1959).  There i s one d i f f e r e n c e between t h e two l o a d i n g methods which might account f o r t h e r e s u l t s .  P a s s i v e l o a d i n g loads the e n t i r e c e l l , w h i l e  i n j e c t i o n d e p o s i t s t h e r a d i o s o d i u m a l o n g a t r a c k which does not extend r i g h t t o the tendon end o f the c e l l and which p o r t i o n o f the c e l l which extends beyond sodium ions  i n b a r n a c l e muscle c e l l s  i s a c c e s s i b l e t o the u n i n j e c t e d t h e grease s e a l .  Diffusion of  i s about as r a p i d as t h a t  s o l u t i o n ( C a i l l e & Hinke 1972), but t h e d i f f u s i o n f r o n t s t i l l hours t o t r a v e l one c e n t i m e t r e .  i n a bulk takes s e v e r a l  There i s thus a slow continuous d i l u t i o n  o f r a d i o s o d i u m throughout even t h e l o n g e s t e f f l u x experiment on i n j e c t e d cells.  The r e s u l t s h o u l d be a s l i g h t l y more r a p i d  f a l l o f f o f the r a d i o -  sodium e f f l u x as t h e i n t r a c e l l u l a r d i f f u s i o n p r o g r e s s e s .  S i n c e the t o t a l  128  t  0  i • \' •  30  •  i • «  60  «  i  I I  i  90 120 time (min.)  I  l l  I  150  I — l I—I—  180  210  F i g u r e 9. S e m i l o g a r i t h m i c p l o t of the amount of r a d i o s o d i u m c o l l e c t e d i n the p e r f u s a t e from a c e l l loaded w i t h i s o t o p e by i n c u b a t i o n o v e r n i g h t ('passive l o a d i n g ' ) , and the amount of r a d i o s o d i u m remaining i n the c e l l , v e r s u s time. The c e l l was p e r f u s e d w i t h normal R i n g e r ' s s o l u t i o n i n i t i a l l y . At c a . 140 minutes p e r f u s i o n was begun w i t h sodium-free l i t h i u m - s u b s t i t u t e d solution. At c a . 180 minutes t h i s was r e p l a c e d w i t h p o t a s s i u m - f r e e s o l u t i o n (Table I ) . R e s o l u t i o n o f the lower curve i n t o the sum of two e x p o n e n t i a l s is indicated: y = A exp(-ax) + B exp(-bx) ; A = 22,837 cpm, a = 0.125 m i n ~ l , B = 12,830 cpm, b = 0.0072 m i n ~ l . L i n e a r r e g r e s s i o n o f upper c u r v e , 55 - 135 minutes, as y = B exp(-bx) i s shown: B = 460 cpm, b = 0.0073 min "'". -  129  i n t r a c e l l u l a r r a d i o s o d i u m a t each i n s t a n t of the  radiosodium c o l l e c t e d  taken i n t o account. fall  too  than u n i t y ,  unity  perfusate,  the  s l o p e r a t i o was  e s p e c i a l l y when h y p e r t o n i c s o l u t i o n s  to r a i s e  than i n the  tend through the  grease s e a l and i n j e c t e d and  (ajr ) a  m  (see  c a p t i o n to F i g .  l e n g t h , and successively methods and  often  somewhat l e s s  8), but  o v e r a l l was  experiments r e p o r t e d by B i t t a r et a l . (1972).  i n j e c t i o n track  i n the  p r e s e n t experiments was  e n t i r e p e r f u s e d l e n g t h of the  i n t o the  few  millimetres  which were kept above the  (1972) i n j e c t e d o n l y a 1 cm  c e l l whenever  c o l l e c t e d the  column of  r a d i o s o d i u m by  into a s e r i e s of v i a l s difference  fluid,  as noted above.  The  i n t e r i o r of the  cell.  Bittar  entire  in  intra-  results  loaded  injected  i s d e p o s i t e d i n the  cells.  cells.  myoplasm,  e f f l u x o f t h i s r a d i o s o d i u m r e p r e s e n t s e f f l u x from It exhibits  a s i m p l e e x p o n e n t i a l dependence on  f o r e f f l u x i n t o normal R i n g e r ' s s o l u t i o n s e m i l o g p l o t f o r the  (Fig.  the time  7).  e f f l u x o f sodium from p a s s i v e l y  i n t o normal R i n g e r ' s s o l u t i o n  i s more c o m p l i c a t e d  F i g . 9), as noted above.  initial  The  £t  in  proposal that between the  the  cell  This difference  loaded and  ex-  not  i n t o c e l l s 3 to 5 cm  difference  cell  explained  c e l l past  perfusate.  support to the  to  possible.  those o b t a i n e d w i t h p a s s i v e l y  i n t o the  As  made to  o f the  immersion o f the  C o m p a r a b i l i t y of sodium e f f l u x i n p a s s i v e l y radiosodium i n j e c t e d  closer  tendon end which were  of perfusate.  i n r e s u l t s lend  o b t a i n e d w i t h i n j e c t e d c e l l s and  a t the  part  l e v e l o f the  c e l l u l a r d i f f u s i o n i s r e s p o n s i b l e f o r the  The  not  of sodium c h l o r i d e were  I n t r a c e l l u l a r d i f f u s i o n would thus occur i n t o the  The  d i l u t i o n effect is  measured r a d i o s o d i u m c o n t e n t o f the myoplasm w i l l  p r e s e n t experiments, the  i n Methods, the  al.  from b a c k - a d d i t i o n  slowly.  In the  injected  The  i n the  is calculated  loaded  (time 0 to 135  r a p i d e f f l u x was  ascribed  cells  minutes i n to the wash-  130  out o f r a d i o s o d i u m from the e x t r a c e l l u l a r  space, and the slower e f f l u x t o  the e f f l u x o f r a d i o s o d i u m from the i n t e r i o r o f the c e l l . s e p a r a t i n g the two  components, known as  The process o f  'curve p e e l i n g ' ,  i s commonly used,  and q u i t e r e a s o n a b l e as l o n g as one can be a s s u r e d t h a t the components o f the net e f f l u x which i s measured can each be d e s c r i b e d by a simple exponential,  and t h a t the r a t e c o n s t a n t s f o r the two  components d i f f e r by a t l e a s t  an o r d e r o f magnitude. Indeed,  application  o f the procedure  t o any smooth curve o f a p p r o x i -  mately the shape o f a washout curve (no i n f l e c t i o n p o i n t s , and decrease  i n the magnitude o f the slope) w i l l y i e l d a sum  terms whose r a t e c o n s t a n t s d i f f e r by about  steady  of exponential  an order o f magnitude.  The  process i s i n essence the d e t e r m i n a t i o n by a process o f s u c c e s s i v e a p p r o x i mations o f the power s e r i e s  expansion o f the p l o t t e d  function.  I t s success  i n a p a r t i c u l a r case does not i n i t s e l f c o n s t i t u t e p r o o f o f the nature o f the e x p e r i m e n t a l system, model, i t can be v e r y  but as a t e s t o f a model, e s p e c i a l l y a simple  useful.  The model i n the c o n t e x t o f which the e f f l u x curve i n F i g . 9 i s to be t e s t e d i s t h a t the r a d i o s o d i u m i s washed out from two the i n t e r i o r o f the c e l l ,  and the e x t r a c e l l u l a r  space.  be some exchange between the i n t e r i o r o f the c e l l and space,  e s p e c i a l l y deep i n the c l e f t system.  correction'  takes account  refinement here. rapid  The  of t h i s  independent Actually, the  q u e s t i o n s to. be,addressed  1960)  'Huxley  but i s an  compartment which g i v e s r i s e to the r a p i d the e x t r a c e l l u l a r Very r a p i d  unnecessary  a r e whether the r a t e o f the  component i s c o n s i s t e n t w i t h e f f l u x from an e x t r a c e l l u l a r  not w i t h e f f l u x from an i n t r a c e l l u l a r s i t e ,  t h e r e must  extracellular  The well-known  (A.F. Huxley  pools:  s i t e but  and whether the s i z e o f the  e f f l u x i s s i m i l a r t o the s i z e o f  space as measured by o t h e r means.  e f f l u x from the i n t e r i o r o f the c e l l has been seen i n f r o g  131  s k e l e t a l muscle (White & Hinke 1976) and c r a b s t r i a t e d muscle (Vaughan-Jones 1977),  but o n l y d u r i n g e f f l u x i n t o sodium-poor s o l u t i o n .  observed  A c t u a l l y , what was  i n these two cases was r a p i d d i s a p p e a r a n c e o f sodium from the  myoplasm and t h e r a t e c o n s t a n t s f o r t h i s process have a d i f f e r e n t cance -- see s e c t i o n 6. muscle,  The demonstration t h a t , a t l e a s t  signifi-  i n barnacle  such a r a p i d disappearance o f myoplasmic sodium i s indeed accompanied  by a r a p i d e f f l u x o f sodium from the c e l l  i s d e s c r i b e d i n s e c t i o n 6.  In  normal R i n g e r ' s s o l u t i o n , o n l y e f f l u x e s w i t h r a t e c o n s t a n t o f o r d e r 0.01 min  ^ a r e seen.  Yet t h i s  f i g u r e i s the product o f p r e c i s e l y t h e model i t  is desired to test. I t c a n be argued, however, t h a t t h e assignment r a p i d e f f l u x t o the i n t r a c e l l u l a r p o o l ,  o f a component o f the  f o r e f f l u x i n t o normal  s o l u t i o n , w h i l e the t o t a l sodium c o n t e n t o f the c e l l  Ringer's  i s steady,  requires  t h a t the i n f l u x and e f f l u x r a t e s change t o g e t h e r when t h e l o a d i n g b a t h 22 (normal Ringer's s o l u t i o n c o n t a i n i n g  Na) i s r e p l a c e d by a bath  i n a l l r e s p e c t s except f o r t h e absence  o f radiosodium.  exhibit a similar  initial  Influx  identical  experiments  r a p i d component, so t h e same argument can be  a p p l i e d t h e r e , and the c e l l  supposed  t o sense and respond t o the i n c l u s i o n 22  i n or o m i s s i o n from the b a t h i n g s o l u t i o n o f  Na.  T h i s does not seem  reasonable. The s i z e o f the compartment which y i e l d s  the r a p i d e f f l u x o f radiosodium  i n t o normal R i n g e r ' s s o l u t i o n i n F i g . 9 i s a p p r o x i m a t e l y 5.5% o f the volume of  the p o r t i o n o f the c e l l which was b e i n g p e r f u s e d .  T h i s i s an u n d e r e s t i -  mate, s i n c e a p o r t i o n o f the tendon end o f t h e c e l l was n o t w e l l p e r f u s e d , and because  t h e l o s s o f r a d i o s o d i u m from t h e e x t r a c e l l u l a r space  is diffu-  s i v e , but agrees w e l l w i t h the v a l u e s o f 6 to. 1% found by v a r i o u s techniques for  the s i z e o f t h e e x t r a c e l l u l a r space  i n s e c t i o n 3.  i n b a r n a c l e muscle c e l l s ,  as noted  132  Altogether,  i t seems r e a s o n a b l e to a s c r i b e  r a d i o s o d i u m e f f l u x e n t i r e l y to the nent to the  c e l l s can the  loaded c e l l s and  thus be compared.  r a t e constant  (0.00729 min  .  However, the  account f o r the  the  lower t r a c e  function  a  loaded c e l l  too The  rate  In F i g . 12  (ca. 20 mM  (page 143)  seen to l i e i n the r e g i o n  was  versus 12 mM),  a  calculated  from  and  are p r e s e n t e d the  the  caclulated  i n normal R i n g e r ' s s o l u t i o n , as  o f the myoplasmic sodium a c t i v i t y loaded c e l l s are  injected c e l l  difference.  for a l l injected c e l l s ,  three passively  is declining  l a r g e r than the  b e s t comparison i s of the sodium e f f l u x Mjj  r a d i o s o d i u m e f f l u x data.  be  s i n g l e component i n i n j e c t e d  sodium content o f the  t h i s alone could  v a l u e s o f Mjr  slow  slow e f f l u x i n t o normal R i n g e r ' s s o l u t i o n i n F i g . 9  o f the p a s s i v e l y  can  r a t e c o n s t a n t s f o r the  i s comparable to but  h i g h e r than t h a t  The  the  slow compo-  i n t r a c e l l u l a r s i n k o f r a d i o s o d i u m noted above.  (0.00928 min  c o n s t a n t f o r the  The  the  the  In F i g . 7 i t i s the upper t r a c e which r e f l e c t s  e f f l u x a c r o s s the membrane, s i n c e  s l o w l y because o f the  r a p i d component o f  e x t r a c e l l u l a r space, and  i n t r a c e l l u l a r compartments.  component i n p a s s i v e l y  the  included defined  (ajj ) . a  m  The  r e s u l t s from  as open diamond symbols. by  the  a the  They  results for injected  cells  o f comparable sodium content. Brinley passively but  (1968) mentioned t h a t  loaded c e l l s was  d i d not  report  the  within  the magnitude o f the  the range observed w i t h i n j e c t e d  r e s u l t s on p a s s i v e l y  (1970) r e p o r t e d an average r a t e c o n s t a n t sodium e f f l u x from p a s s i v e l y 15°  C.  loaded c e l l s .  f o r the  cells,  A l l e n and  slower component o f  loaded b a r n a c l e muscle c e l l s  Hinke the  o f 0.0085 min  B i t t a r e_t al_. (1972) r e p o r t e d an average r a t e c o n s t a n t f o r t h i s -1  component o f about 0.010 muscle  sodium e f f l u x from  cells.  to 0.015  min  o a t 23  C for injected  barnacle  -1  at  133  DISCUSSION  B i t t a r and coworkers b a r n a c l e muscle c e l l s  (1972) found that the amount o f r a d i o s o d i u m i n  loaded by m i c r o i n j e c t i o n d i d not d e c l i n e w i t h time a t  a r a t e commensurate t o t h e d e c l i n e w i t h time o f the r a t e a t which appeared  i n the bath.  They i n t e r p r e t e d t h i s  i n terms o f a model i n which a  l a r g e p o r t i o n o f the i n j e c t e d r a d i o s o d i u m was s e q u e s t e r e d from fragments injector. the c e l l of  radiosodium  in vesicles  formed  o f t h e c e l l membrane c r e a t e d by t h e i n s e r t i o n o f t h e micro-  T h i s sodium d i d not exchange a t a l l w i t h t h e f r e e sodium i n s i d e over t h e c o u r s e o f t h e experiment,  the c e l l  but c o u l d be r e l e a s e d by  exposure  to aldosterone.  That so much o f the i n j e c t e d sodium c o u l d be s e q u e s t e r e d a t the moment 23 of  injection,  (so  t h a t a concomitant  the apparent  'bound' sodium),  amount o f  Na c o u l d a l s o be s e q u e s t e r e d  f r a c t i o n o f 'bound' r a d i o s o d i u m r e f l e c t e d t h e f r a c t i o n of and t h a t t h e c e l l membrane fragments  which form these  v e s i c l e s c o u l d become so impermeable t o sodium t h a t sodium exchange a c r o s s them i s n e g l i g i b l y slow compared t o sodium exchange a c r o s s the r e s t o f the cell  membrane, were not c o n s i d e r e d t o be r e a s o n a b l e  hypotheses.  On t h e o t h e r hand, i t seems v e r y r e a s o n a b l e t h a t l o n g i t u d i n a l of  diffusion  i n j e c t e d r a d i o s o d i u m i s r e s p o n s i b l e f o r t h e observed s l o p e r a t i o s .  s l o p e r a t i o s were f a r l e s s than u n i t y i n c e l l s  The  i n which the l e n g t h o f the  i n j e c t i o n t r a c k was much l e s s than the l e n g t h o f the c e l l which was p e r f u s e d ( B i t t a r et a l . 1972). u n i t y when an attempt  The s l o p e r a t i o s were c l o s e r t o but s t i l l  than  was made t o p e r f u s e o n l y t h e r e g i o n o f the c e l l which  c o n t a i n e d the i n j e c t i o n t r a c k ( p r e s e n t s t u d y ) . to u n i t y i n c e l l s  less  The s l o p e r a t i o s were equal  loaded w i t h r a d i o s o d i u m by immersion,  l o n g i t u d i n a l d i f f u s i o n o f r a d i o s o d i u m i s expected  i n which no n e t  t o occur ( p r e s e n t study  and r e s u l t s o f o t h e r workers f o r f r o g s k e l e t a l muscle).  Finally,  the s l o p e  134  o f the semilog p l o t s of sodium e f f l u x versus time a r e c o n s t a n t over e n t i r e d u r a t i o n o f l o n g experiments  in injected c e l l s ,  the  c o n t r a r y t o the  e x p e c t a t i o n t h a t the magnitude o f the s l o p e should d e c l i n e i f a f i x e d amount o f the i n j e c t e d r a d i o s o d i u m The  i s sequestered.  e f f e c t s o f a l d o s t e r o n e remain t o be e x p l a i n e d , but p r o b a b l y  reflect  an a c t i o n on the t r a n s p o r t systems i n the c e l l membrane. The b e h a v i o r o f the sodium e f f l u x microinjected c e l l s cells  ( F i g . 12,  i n t o normal Ringer's  solution  i s i n d i s t i n g u i s h a b l e from t h a t from p a s s i v e l y  page 143).  A d e t a i l e d study of the response  component i n p a s s i v e l y loaded c e l l s  loaded  o f the slow  t o ouabain and t o changes i n the  c o m p o s i t i o n of the b a t h i n g s o l u t i o n was  not c a r r i e d out, but i n the e x p e r i -  ments which were done the b e h a v i o r o f the p a s s i v e l y loaded c e l l s q u a l i t a t i v e l y and  from  q u a n t i t a t i v e l y the same as f o r i n j e c t e d  was  cells.  The use o f m i c r o i n j e c t i o n leads to a c o m p l i c a t i o n i n the measurement o f the sodium e f f l u x , however, due q u a n t i t y which appears i s Na*/Na* m  t o the l o n g i t u d i n a l d i f f u s i o n .  The  i n the e f f l u x equation, e q u a t i o n (4) o f s e c t i o n  the r a t i o o f the amount o f r a d i o s o d i u m which leaves the  2.F,  cell  d u r i n g a c o l l e c t i o n i n t e r v a l t o the amount o f r a d i o s o d i u m i n the myoplasmic compartment a t the s t a r t o f t h a t i n t e r v a l .  The myoplasm i s c o n t i n u o u s l y  l o s i n g r a d i o s o d i u m t o l o n g i t u d i n a l d i f f u s i o n as w e l l as to the b a t h i n g i  solution. The r a t i o Na*/Na* would be equal t o the s l o p e of the p l o t o f In versus  time i f the l o s s of r a d i o s o d i u m by l o n g i t u d i n a l d i f f u s i o n from  myoplasm b e i n g p e r f u s e d were e n t i r e l y  independent  o f the l o s s a c r o s s  Na* the the  c e l l membrane, and the r a t e o f the l o s s a c r o s s the c e l l membrane was p r o p o r t i o n a l t o the amount o f r a d i o s o d i u m Then the s l o p e o f a l i n e drawn through  i n the myoplasm a t each  instant.  the data p o i n t s o f the s e m i l o g  c o u l d be assumed t o be equal t o Na*/Na*!, and used  i n equation  (4) to  plot  135  c a l c u l a t e Mjj , but o n l y over time a  very l i t t l e  i n t e r v a l s d u r i n g which the s l o p e changed  ( i e . dk = 0 ) . dt  T h i s has  been done f o r the e f f l u x  the experiment d e p i c t e d i n F i g . 10  i n t o normal Ringer's  (page 140).  curve averages out s m a l l v a r i a t i o n s  i n the raw  The data.  solution for  drawing of a smooth The  net e f f e c t  is a  v a l u e f o r Mjj which i s about 107o h i g h e r than the average o f the v a l u e s a  calculated directly was  0.95.  The  from e q u a t i o n  (4).  slope r a t i o f o r t h i s  e f f e c t of i g n o r i n g the i n t r a c e l l u l a r s i n k due  d i f f u s i o n w i l l be g r e a t e r i n c e l l s r a t i o tends  The  to be lower  to  experiment longitudinal  loaded w i t h sodium, f o r which the s l o p e  ( F i g . 8).  T h i s c o r r e c t i o n to the c a l c u l a t e d s i z e o f the sodium e f f l u x from i n jected c e l l s i n the raw  i s s y s t e m a t i c , and  data b e f o r e M^  employ e q u a t i o n  a  i n v o l v e s a severe a v e r a g i n g of  can be c a l c u l a t e d .  (4) as w r i t t e n , and  u n c e r t a i n t y i n Na* and m J  results  data.  i n V /A d u r i n g d i s c u s s i o n s to which the a b s o l u t e m ° r a t h e r than to a p p l y c o r r e c t i o n s  However, comparison o f the u n c o r r e c t e d and  of most experiments r e v e a l e d a profound  on the data, as w i l l be d i s c u s s e d i n d e t a i l Finally,  the apparent  by the e f f l u x curves value obtained  I t would be p r e f e r a b l e to  then c o n s i d e r the e f f e c t s of the  s i z e o f the sodium e f f l u x i s important, to the raw  fluctuations  the c o r r e c t e d  e f f e c t o f the above e f f e c t s  later.  s i z e of the e x t r a c e l l u l a r p o o l o f sodium y i e l d e d  f o r the p a s s i v e l y loaded c e l l s must be mentioned.  (about  5.57») was  s i m i l a r to the i n u l i n space.  However, the  p o o l of r a p i d l y exchanging e x t r a c e l l u l a r sodium might be expected much l a r g e r than t h i s .  The  s i z e o f the r a p i d l y exchanging  The  to appear  extracellular  sodium f r a c t i o n proposed i n s e c t i o n 3 i s about 12 m i l l i m o l e s Na/kg c e l l water ( F i g . 3 ) . if  the l a t t e r  0.06  The  amount o f sodium i n s o l u t i o n i n the e x t r a c e l l u l a r  i s taken as 67, o f the c e l l volume, i s a p p r o x i m a t e l y  or 27 m i l l i m o l e s Na/kg c e l l water.  Thus one might expect  450  space, mM  the s i z e  x of  the  e x t r a c e l l u l a r space deduced  larger occur.  than the i n u l i n space. First,  free d i f f u s i o n , is the  free  the l o s s  from the r a d i o s o d i u m washout to be somewhat There a r e two main reasons why  o f r a d i o s o d i u m from the e x t r a c e l l u l a r space i s by  the same process which mixes the e x t r a c e l l u l a r sodium  i n solution.  T h i s causes the y - i n t e r c e p t  s i z e o f the e x t r a c e l l u l a r  i n F i g . 9 t o be low,  space to be u n d e r e s t i m a t e d .  e x t r a c e l l u l a r nonmyoplasmic c a t i o n s are not a l l h i g h l y experiments on smooth muscle, the  t h i s does not  lanthanum was  z e r o o f time i s not p r e c i s e l y  f o r the e x t r a c e l l u l a r  definable.  Second,  mobile.  used t o f r e e  them.  which and  the  I n the In  addition,  In a l l , the v a l u e o b t a i n e d  space i s not u n r e a s o n a b l e .  137  SECTION 5.  SURVEY OF THE SODIUM EFFLUX FROM SINGLE MUSCLE CELLS  In t h i s s e c t i o n ,  the dependence  o f the sodium e f f l u x from s i n g l e whole  b a r n a c l e muscle c e l l s on t h e myoplasmic sodium a c t i v i t y serves f i r s t  i s surveyed.  o f . a l l as a t e s t o f the techniques d e s c r i b e d  measuring the sodium e f f l u x , b a r n a c l e muscle c e l l s  This  i n s e c t i o n 2.F f o r  s i n c e the r e s u l t s o f s i m i l a r experiments on  loaded w i t h r a d i o s o d i u m by m i c r o i n j e c t i o n a r e a v a i l -  a b l e f o r comparison ( B r i n l e y 1968; B i t t a r £t aT. 1972). The r e s u l t s o f s e c t i o n 3 suggest t h a t t h e a p p l i c a t i o n o f the new t e c h n i q u e w i l l not y i e l d r e s u l t s markedly d i f f e r e n t  from those found w i t h  the u s u a l t e c h n i q u e s , because most o f the nonmyoplasmic exchange r a p i d l y w i t h the myoplasmic the s o d i u m - s p e c i f i c  sodium.  sodium does not  T h i s means t h a t the use o f  i n t r a c e l l u l a r e l e c t r o d e w i l l o n l y improve the e s t i m a t e  o f the s i z e o f t h e i n t e r n a l sodium c o n c e n t r a t i o n on which t h e e f f l u x The n a t u r e o f the dependence  depends.  should be t h e same w i t h e i t h e r method.  However, t h e use o f m i c r o i n j e c t i o n has been shown t o g i v e r i s e t o a s i g n i f i c a n t uncertainty, injected  into a c e l l  i n t h a t most but not a l l o f the r a d i o s o d i u m  i s a v a i l a b l e f o r exchange w i t h e x t r a c e l l u l a r  when the u s u a l techniques a r e employed.  sodium  There w i l l be an u n d e r e s t i m a t e o f  the s i z e o f t h e sodium e f f l u x , as e x p l a i n e d  i n s e c t i o n s 2.F and 4.  Further,  t h i s u n d e r e s t i m a t e w i l l be g r e a t e r i n m i c r o i n j e c t e d c e l l s w i t h an e l e v a t e d sodium content, as i n d i c a t e d by F i g . 8. T h e r e f o r e , t h e n a t u r e o f the dependence sodium content o f the c e l l  o f the sodium e f f l u x on t h e  found by o t h e r workers u s i n g m i c r o i n j e c t e d  cells  might be i n c o r r e c t . The dependence o f the sodium e f f l u x Mjj activity  (ajj ) a  m  i n muscle c e l l s .  reflects  a  on t h e myoplasmic  sodium  the c o n t r i b u t i o n o f more than one t r a n s p o r t system  The prominent systems a r e thought t o be the  (Na+K)ATPase,  138  and a system which mediates  sodium-sodium exchange. The b e h a v i o r o f t h e  e f f l u x can r e a s o n a b l y be expected t o be d i f f e r e n t d u r i n g c o n d i t i o n s which favour one o r another t r a n s p o r t mode. The dependence o f M ^  a  on ( a j * ) a  m  was measured i n normal Ringer's  solu-  t i o n , which s h o u l d correspond c l o s e l y t o t h e normal c o n d i t i o n s o f t h e c e l l in vivo;  i n p o t a s s i u m - f r e e s o l u t i o n , where t h e major mode o f t h e (Na+K)ATPase  s h o u l d be d i s a b l e d ;  i n sodium-free  s o l u t i o n s , where t h e sodium-sodium  exchange mode r e p o r t e d i n muscle should be d i s a b l e d ; and i n t h e presence o f ouabain, where almost a l l o f t h e r e a c t i o n s o f the (Na+K)ATPase should be disabled. I t was found t h a t a p p l i c a t i o n o f t h e c o r r e c t i o n t o N a £ - Q f o r c e l l s e  loaded w i t h r a d i o s o d i u m by m i c r o i n j e c t i o n changed t h e r e s u l t s a p p r e c i a b l y .  METHODS  The method o f p r e p a r i n g c e l l s and t h e use o f the s o d i u m - s p e c i f i c m i c r o e l e c t r o d e t o measure (a, ) were d e s c r i b e d i n s e c t i o n 3. Na m T  The method  o f i n j e c t i n g , c o l l e c t i n g , and c o u n t i n g t h e r a d i o s o d i u m was d e s c r i b e d i n s e c t i o n 4. The c a l c u l a t i o n o f t h e sodium e f f l u x Mjr (4) o f s e c t i o n 2.F, both w i t h and w i t h o u t i n s e c t i o n s 2.F and 4. reasons.  First,  a  was c a r r i e d out v i a e q u a t i o n  t h e c o r r e c t i o n t o Na* -Q d e s c r i b e d e  Steady c o n d i t i o n s a r e o f i n t e r e s t here f o r two  t h e response  time o f t h e t r a n s p o r t mechanisms t o changes  i n t h e myoplasmic sodium a c t i v i t y  i s n o t known, so t h e most r e l i a b l e  s h o u l d be o b t a i n e d d u r i n g steady c o n d i t i o n s .  data  Second, t h e c o r r e c t i o n t o  ic N c e l l can o n l y be made w i t h c o n f i d e n c e d u r i n g steady c o n d i t i o n s , as exa  139  plained  i n s e c t i o n s 2.F and 4.  ness o f (^a),^ over a t l e a s t  L i n e a r i t y o f t h e s e m i l o g p l o t and s t e a d i -  f o u r c o l l e c t i o n p e r i o d s was the c r i t e r i o n f o r  the s e l e c t i o n o f d a t a . Experiments  i n which  the sodium e f f l u x  i s impaired, as by removal o f  e x t r a c e l l u l a r p o t a s s i u m o r by exposure t o ouabain, r e s u l t i n t h e myoplasmic sodium a c t i v i t y as the sodium  i n a steady r i s e  i n f l u x i s no l o n g e r adequ-  a t e l y c o u n t e r e d by sodium e x t r u s i o n ( f o r example, F i g . 10 o f t h i s  section).  T h i s u s u a l l y caused no problem but i n a few cases the e f f l u x f e l l  to a  minimum, then r o s e s l o w l y as ( a j j ) a  be made about to r e f l e c t  the v a l u e o f M^  m  rose.  to extract  a  A s u b j e c t i v e judgment then had t o for analysis.  The v a l u e judged  the maximum e f f e c t o f t h e e x p e r i m e n t a l m a n i p u l a t i o n was e x t r a c t e d ,  a l o n g w i t h the v a l u e o f ( a ^ ) a  m  was q u i t e s m a l l , but the e f f e c t  a t t h a t time.  The u n c e r t a i n t y due t o t h i s  i s o f i n t e r e s t , as w i l l  be d i s c u s s e d below  i n c o n n e c t i o n w i t h the dose-response curve f o r ouabain.  Use o f Day-old  Cells.  For some experiments,  such as l o a d i n g w i t h r a d i o s o d i u m by i n c u b a t i o n  i n r a d i o s o d i u m - c o n t a i n i n g s o l u t i o n s , measurements on t h e d i s s e c t e d cannot be performed u n t i l 24 t o 48 hours a f t e r the d i s s e c t i o n .  cell  Dissected  c e l l s kept i n normal R i n g e r ' s s o l u t i o n a t l e s s than 5° C a r e found t o maint a i n t h e i r i o n g r a d i e n t s and membrane p o t e n t i a l : f o r s e v e r a l days Table I I ) .  In p i l o t  experiments,  ( f o r example,  t h e b e h a v i o r o f the e f f l u x o f i n j e c t e d  r a d i o s o d i u m from b a r n a c l e muscle c e l l s was not n o t i c e a b l y d i f f e r e n t i n c e l l s which were i n j e c t e d 24 hours a f t e r d i s s e c t i o n from t h a t were i n j e c t e d a few hours a f t e r d i s s e c t i o n . was noted i n a few c e l l s cells  in cells  Some d i f f e r e n c e (enhanced  t e s t e d a t 48 hours a f t e r d i s s e c t i o n .  which M^ ) a  The use o f  from a b a r n a c l e d i s s e c t e d t h e p r e c e d i n g day makes much more e f f i c i e n t  use o f the a v a i l a b l e specimens,  and saves a c o n s i d e r a b l e amount o f time.  140  E  30  normol  I  | K-free |  normal  |  I0" M. ouabain 4  Q.  O  •o  ••  20 10  •  0>  o 10>  ••••  0.5  tt> o o 0.2H 26  I  I  I  I  I.  I  ••••••  18 10 2H |  24  «E  201-  o  3  r  16 1  0  1  30  60  90  120 150 time (min.)  180  210  240  F i g u r e 10. Summary o f the raw d a t a and reduced r e s u l t s f o r a t y p i c a l experiment. Upper t r a c e : l o g a r i t h m of the amount of r a d i o s o d i u m c o l l e c t e d i n each 5 minute c o l l e c t i o n p e r i o d , i n counts per minute; lower t r a c e : myoplasmic sodium a c t i v i t y as measured by a s o d i u m - s p e c i f i c g l a s s m i c r o e l e c t r o d e , i n mM; m i d d l e t r a c e : sodium e f f l u x deduced from the data v i a e q u a t i o n ( 4 ) , p l o t t e d i n picomoles/cm sec ( p e s ) . For the i n t e r v a l i n normal R i n g e r ' s s o l u t i o n , the c o r r e c t e d v a l u e o f M ^ i s i n d i c a t e d as a dashed l i n e .  141  About h a l f o f the experiments cells.  The  r e p o r t e d on here were done on such  data o b t a i n e d from such c e l l s  is indicated  s i m i l a r b e h a v i o r of f r e s h and d a y - o l d c e l l s  'day-old'  i n the f i g u r e s .  (eg. F i g . 11)  The  is. discussed  below.  RESULTS  In Fig.- 10 a summary o f the raw experiment  i s presented.  data and reduced  Reference w i l l be made t o t h i s  (a) Sodium e f f l u x i n t o normal R i n g e r ' s The  results  as p l o t s o f M  Na  from 58 experiments versus  result  (a- ) . N a  m  for a  figure  typical  later.  solution.  are presented  In F i g . 11, M^  a  i n F i g . 11 and F i g . 12  was  c a l c u l a t e d a c c o r d i n g to  ic equation  (4) u s i n g ^  a c e  ic  n'  i  c o r r e c t e d v a l u e o f Na / N a In F i g . 11,  the same data was  m  was  used  (ajj )  a  (1968) used  s a t u r a t i o n appears (Na)^ r a t h e r than  ducted h i s experiments  employed but  the  i n equation (4).  the r e l a t i o n s h i p between Mjj and  s l i g h t l y sigmoidal: Brinley  In F i g . 12,  ***  a t 0° C.  a  appears  m  to be  to occur a t h i g h e r v a l u e s of  (Na)  m  i n c a l c u l a t i n g Mj^,  and  (ajj ) . a  m  con-  I t had been a n t i c i p a t e d t h a t the p r e s e n t  r e s u l t s would be s i m i l a r to h i s ( a f t e r c o r r e c t i o n f o r s u r f a c e a r e a by a f a c t o r o f 10), but s h i f t e d t o lower v a l u e s on the a b s c i s s a s i n c e ( a j j ) a  used due  i n s t e a d o f (Na)  T h i s was  r e l a t i o n s h i p i s shown as a broken l i n e  I t appears  was  , and w i t h a l a r g e r e f f l u x a t a g i v e n sodium c o n t e n t  t o the h i g h e r temperature.  In F i g . 12,  m  as was  found  Brinley's empirical  i n F i g . 11.  the r e l a t i o n s h i p between  t o be a f f i n e ,  found.  and  (  a  j T  a  )  m  i  s  quite different.  i n s n a i l neurone by Thomas  (1972).  142  F i g u r e 11. Sodium e f f l u x i n t o normal Ringer's s o l u t i o n , c a l c u l a t e d from e q u a t i o n (4), w i t h o u t c o r r e c t i o n f o r Na* , ., . S o l i d c i r c l e s : c e l l s dissected ——— cell on the day o f the experiment. S o l i d diamonds; c e l l s d i s s e c t e d on the day b e f o r e t h a t o f the experiment. Open t r i a n g l e s : c e l l s loaded w i t h radiosodium by immersion o v e r n i g h t i n l a b e l l e d s o l u t i o n . S o l i d c u r v e : model c a l c u l a t i o n f o r t h r e e sodium ions b i n d i n g s u c c e s s i v e l y to e q u i v a l e n t independent s i t e s per c y c l e o f the t r a n s p o r t enzyme (k = 15.75 mM, = 45 p e s ) . Dashed curve: e x p e r i m e n t a l data o f B r i n l e y (1968) as M„  Ma  versus  i  (Na)., where  x  (Na). i s on the same n u m e r i c a l s c a l e as 'i  (a„ ) . Na m  143  F i g u r e 12. Sodium e f f l u x i n t o normal R i n g e r ' s s o l u t i o n , c a l c u l a t e d from equation (4), with c o r r e c t i o n f o r * -QSymbols as i n F i g . 11. Solid l i n e : curve to which k i n e t i c models were f i t t e d by t r i a l and e r r o r . Dashed l i n e : e x p e r i m e n t a l d a t a o f B r i n l e y (1968), as i n F i g . 11. Elevation of (a„ ) above the normal range ( c a . 10 mM) was accomplished by i n j e c t i o n of Na m NaCl i n t o the myoplasm. Na  e  144  No  s a t u r a t i o n i s evident.  difficult  to o b t a i n .  A c c e p t a b l e data a t h i g h e r sodium content  The v a l u e of (ajyj ) u s u a l l y d i d not become steady a  m  when l a r g e amounts o f 5 M NaCl were i n j e c t e d , so i t was p e r m e a b i l i t y o f the c e l l membrane had been compromised. were seen  i n such cases.  concluded  t h a t the  Very r a p i d  High v a l u e s o f (Na)^ (about 70 mM)  a l l e g e d to unmask pre-formed  have been  i t i s hard to imagine  t h a t such a  c h a l l e n g e would ever occur i n a l i v i n g animal.  In one a c c e p t a b l e  at  found  a  a  prolonged  m  = 70 mM,  a r e l a t i v e l y h i g h e f f l u x was  immersion o f the c e l l s  not  however, t h a t w h i l e F i g . 10 suggests  that ( a j ^ ) a  m  note,  m  over  20  s o l u t i o n i s not v e r y g r e a t .  ( s o l i d diamonds) c e l l s  the dependence o f the response  for fresh ( s o l i d  i n F i g . 12.  circles)  I t has been r e p o r t e d t h a t  o f f r o g s k e l e t a l muscle to ouabain on  sodium content o f the c e l l d i f f e r s  i n f r e s h and  T a y l o r , & Waggoner 1970), as does the response (Keynes 6c Swan 1959;  of  can be r a i s e d e a s i l y i n a  No d i f f e r e n c e i s seen between the r e s u l t s  of  In t h i s c o n n e c t i o n ,  s o l u t i o n , T a b l e I I shows t h a t the i n c r e a s e i n ( a ^ )  hours i n p o t a s s i u m - f r e e  Fig.  The use  i n v e s t i g a t e d , but s h o u l d be a  b e t t e r method f o r l o a d i n g the c e l l w i t h sodium.  and d a y - o l d  ( F i g . 11).  experiment  i n p o t a s s i u m - f r e e s o l u t i o n as a means o f  p a s s i v e l y r a i s i n g the sodium content was  potassium-free  effluxes  t r a n s p o r t enzymes i n f r o g s k e l e t a l muscle  ( E r l i j & G r i n s t e i n 1976a,b), a l t h o u g h  ( N )  was  'aged' c e l l s  the  (Horowicz,  t o removal o f e x t e r n a l sodium  Keynes 6c S t e i n h a r d t 1968) .  12 does not r e v e a l any d e f i n i t i v e  i n f o r m a t i o n about the k i n e t i c s  the e x t r u s i o n o f sodium from the c e l l , because o f the s c a t t e r o f the  d a t a but more i m p o r t a n t l y because o f the u n a v a i l a b i l i t y of data f o r low values of ( ^ ) a  a  m  w i t h the p r e s e n t t e c h n i q u e s .  the behavior r e v e a l e d w i t h the new u s u a l method.  I t i s c l e a r , however, t h a t  method d i f f e r s  from t h a t o b t a i n e d w i t h  the  145  (b) Sodium e f f l u x i n t o p o t a s s i u m - f r e e s o l u t i o n . The r e s u l t s o f 20 experiments  i n which the sodium e f f l u x i n t o  potassium-  f r e e s o l u t i o n was measured a r e p r e s e n t e d i n F i g s . 13 and 14 as p l o t s o f M^ versus  C -^)^ 3  I  n  F i g - 13,  was c a l c u l a t e d  from e q u a t i o n (4) w i t h o u t  the c o r r e c t i o n t o N a * ^ , w h i l e i n F i g . 14, Mjj was c a l c u l a t e d w i t h e  a  a  this  correction. The b e h a v i o r o f M^  a  i s similar  i n the two p l o t s , a s i d e from the  c o r r e c t i o n i n F i g . 14 o f the underestimate o f the s i z e o f M^ The d e f i n i t e p l a t e a u i s markedly Ringer's s o l u t i o n ,  F i g . 12.  different  i n F i g . 13.  a  from the b e h a v i o r found i n normal  The sodium e x t r u s i o n mechanism which does not  r e q u i r e e x t e r n a l p o t a s s i u m appears  t o have a l i m i t e d c a p a c i t y ,  over the ' p h y s i o l o g i c a l range' o f ( a ^ ) a  m  i t responds  although  t o an i n c r e a s e i n  (a. ) by i n c r e a s i n g i t s r a t e . Na'm ° v  T  3  Further,  i t appears  t h a t the sodium e x t r u s i o n mechanism which does  r e q u i r e e x t e r n a l potassium does n o t s a t u r a t e a t myoplasmic sodium a c t i v i t i e s up t o 70 mM.  By comparison,  Keynes and Swan (1959) found i n f r o g  muscle t h a t the r e d u c t i o n i n t h e sodium e f f l u x caused by removal  striated of external  potassium was g r e a t e r as (Na)^ was r a i s e d . Two o t h e r o b s e r v a t i o n s on.the can be made.  e f f e c t o f removal  of external  As i l l u s t r a t e d by F i g . 10, the e f f e c t o f removal  p o t a s s i u m i s r e v e r s e d by r e s t o r a t i o n o f e x t e r n a l potassium. noted that B i t t a r ejt al_.  potassium  of external  I t s h o u l d be  (1972) d e s c r i b e a r i s e o f the sodium e f f l u x t o a  l e v e l above t h a t o b t a i n e d b e f o r e e x t e r n a l p o t a s s i u m was removed i f e x t e r n a l potassium i s subsequently r e s t o r e d .  This occurred only f o r c e r t a i n  cells,  those f o r which they c a l c u l a t e d a l a r g e " s e q u e s t e r e d f r a c t i o n " o f sodium by the s l o p e r a t i o method.  Such c e l l s a r e found t o have h i g h myoplasmic  sodium a c t i v i t y , as d i s c u s s e d i n s e c t i o n 4.  B i t t a r e t al_. r e p o r t e d no  a p p r e c i a b l e change i n the i o n content o f c e l l s  incubated i n p o t a s s i u m - f r e e  146  50k  ^ i J m  (  m  M  )  F i g u r e 13. Sodium e f f l u x from the c e l l i n t o a p o t a s s i u m - f r e e b a t h i n g s o l u t i o n , c a l c u l a t e d from e q u a t i o n (4) w i t h o u t c o r r e c t i o n f o r Na* ^^, v e r s u s myoplasmic g  sodium a c t i v i t y a t the time >of the change from p o t a s s i u m - c o n t a i n i n g t o potassium-free s o l u t i o n . C i r c l e s : c e l l s d i s s e c t e d the day o f the experiment. Diamonds: c e l l s d i s s e c t e d the day b e f o r e t h a t o f the experiment. S o l i d l i n e drawn by eye. Dashed l i n e r e p r e s e n t s the sodium e f f l u x i n t o normal R i n g e r ' s s o l u t i o n c a l c u l a t e d i n a s i m i l a r manner ( F i g . 11). Note: the o r d i n a t e i s d i f f e r e n t from that i n F i g . 11 and F i g . 12. (  147  F i g u r e 14. Sodium e f f l u x from the c e l l i n t o a p o t a s s i u m - f r e e b a t h i n g s o l u t i o n c a l c u l a t e d from e q u a t i o n (4) w i t h c o r r e c t i o n f o r N a * ^ , versus myoplasmic e  sodium a c t i v i t y a t the time o f the change from p o t a s s i u m - c o n t a i n i n g to potassium-free s o l u t i o n . Symbols as i n F i g . 13. S o l i d l i n e : k i n e t i c model f o r three sodium ions b i n d i n g s u c c e s s i v e l y t o e q u i v a l e n t independent s i t e s per c y c l e o f the t r a n s p o r t enzyme (k = 15 mM, ^ ^0 p e s ) . Dashed l i n e : =  m  efflux  i n t o normal Ringer's  solution,  a  x  from F i g . 12.  148  s o l u t i o n f o r 50 t o 70 minutes, (Na)^ must r i s e , and treatment.  a l t h o u g h i t i s c l e a r from T a b l e I I that  from F i g . 10 t h a t  B i t t a r et a l . a s s e r t  sodium e f f l u x i s a l t e r e d ,  that  (ajj ) a  m  will  be i n c r e a s e d  i n these c e l l s  but i t seems c l e a r t h a t  by  such  the b e h a v i o r o f the  the " e x t r a  e f f l u x " can  r e a s o n a b l y be a t t r i b u t e d to the r a i s e d myoplasmic sodium a c t i v i t y . have not demonstrated external As  that  changes i n the sodium e f f l u x caused by removal  potassium a r e not  (page 190),  the membrane p o t e n t i a l does  p o t a s s i u m i s removed.  steady i s a d e p o l a r i z a t i o n  The  e f f e c t when E  in this particular c e l l ,  although  m  becomes immediately  a f t e r the s o l u t i o n change t h e r e i s a t r a n s i e n t h y p e r p o l a r i z a t i o n . the c e l l s cells  tested,  the net e f f e c t was  i n which ( a ^ ) a  s  w a m  i n p o t a s s i u m - f r e e s o l u t i o n t o that 1.05  for ( a j j )  the r a t i o was  0.06).  g r e a t e r than or equal t o 40 mM  (Note:  times was  t h e r e a r e fewer than 22 p o i n t s  s t a b l e when M^  a  was  solutions  i n normal  (n = 11, SD = 0.02) 1.10  while  (n = 11, SD =  i n F i g . 13 because E  some-  m  not.)  (c) Sodium e f f l u x i n t o sodium-free Sodium-free  For  p r i o r t o the s o l u t i o n change, the  R i n g e r ' s s o l u t i o n p r i o r t o the change was m  I n most o f  found t o be a h y p e r p o l a r i z a t i o n .  l e s s than 40 mM  r a t i o o f the membrane p o t e n t i a l  a  of  reversible.  i l l u s t r a t e d by F i g . 24  change when e x t e r n a l  They  substituted  s u c r o s e were employed ( T a b l e I ) .  solution. with lithium,  tris,  choline,  or  The e f f e c t s on the sodium e f f l u x o f r e -  placement  o f the normal Ringer's s o l u t i o n b a t h i n g a c e l l by one o f the above  solutions  a r e shown i n F i g . 15.  out c o r r e c t i o n  M^  f o r Na* -Q, s i n c e  a  was  i s the r a t i o o f M^  from e q u a t i o n (4) w i t h -  the c o r r e c t i o n cannot be a p p l i e d  e  c o n f i d e n c e when the e f f l u x i s not steady. value plotted  calculated  a  To make comparison  with  easier,  the  a t each time t o the s t e a d y v a l u e o f  H^  found b e f o r e the change from normal R i n g e r ' s s o l u t i o n .  Both  a  i n h i b i t o r y and  149  0.50r •  0  i  i  20  1  i  i  40  time after solution change  •  •  60 (min.)  F i g u r e 15. The e f f e c t on the sodium e f f l u x o f removal o f sodium from the e x t r a c e l l u l a r medium. A t time zero minutes (arrow), the e x t r a c e l l u l a r s o l u t i o n was changed from normal R i n g e r ' s s o l u t i o n t o a sodium-free s o l u t i o n , substituted as i n d i c a t e d . Sodium e f f l u x has been n o r m a l i z e d to 1.0, so each c e l l s e r v e s as i t s own c o n t r o l . I t can be seen from F i g . 16 that the c e l l - t o - c e l l v a r i a t i o n i n the s i z e o f the e f f l u x i n t o normal R i n g e r ' s s o l u t i o n and i n t o the v a r i o u s sodium-free s o l u t i o n s i s so g r e a t t h a t the d i f f e r e n t response to d i f f e r e n t sodium-free s o l u t i o n s i s obscured.  150  s t i m u l a t o r y e f f e c t s can be seen, and the t r a n s i e n t e f f e c t s appear to be different  f o r the d i f f e r e n t s u b s t i t u t e i o n s .  Lithium. in  a  i n 17 o f 22 experiments.  f o l l o w i n g the s o l u t i o n change, M^ an i n i t i a l abrupt f a l l .  a  changed  In the time  fall  immediately  e r r a t i c a l l y , y e t t h e r e was  The r e l a t i v e r e d u c t i o n was  T h i s almost always s e t t l e d It  i n turn.  The replacement o f e x t e r n a l sodium by l i t h i u m caused a  the v a l u e o f M^  17).  They can be c o n s i d e r e d  by 0.30  always  (SD = 0.17,  n =  i n t o a slow d e c l i n e as ( a ^ ^ f e l l .  seemed l i k e l y t h a t both a t r a n s i e n t s t i m u l a t o r y and a  sustained  i n h i b i t o r y e f f e c t r e s u l t e d from the replacement o f the e x t e r n a l sodium by lithium.  The p o r t i o n s o f F i g . 15 which d e a l w i t h the time p e r i o d  immediately  a f t e r the change t o sodium-free s o l u t i o n a r e o n l y p r e s e n t e d as q u a l i t a t i v e results. in  The t r a n s i e n t s i n the sodium e f f l u x w i l l  s e c t i o n 6.  Only the s u s t a i n e d  be d e s c r i b e d  i n h i b i t o r y e f f e c t w i l l be c o n s i d e r e d  I n one experiment, a t v e r y h i g h  ( jr ) , a  as was  here.  the s o l u t i o n change was  a  m  f o l l o w e d by an abrupt drop and then a marked i n c r e a s e t r a c t i o n o f the c e l l ,  separately  found by B r i n l e y (1968).  i n M^,  p l u s a con-  R e t u r n o f the c e l l  t o normal R i n g e r ' s s o l u t i o n seemed t o reduce the e f f l u x o f radiosodium, but M^j  a  c o u l d not be c a l c u l a t e d because the c o n t r a c t i o n d i s l o d g e d  Baker, B l a u s t e i n , Hodgkin  ejt al.  the e l e c t r o d e s .  (1969) have suggested t h a t c o n t r a c t i o n s i n  t h i s s i t u a t i o n might be due to an i n c r e a s e d e n t r y o f c a l c i u m  i n t o the c e l l  via  to i t s usual  the sodium-calcium exchange mechanism o p e r a t i n g o p p o s i t e  manner due t o the absence o f e x t e r n a l sodium. in  squid  They observed such an  effect  axon.  Choline. s o l u t i o n was  I n f i v e experiments where c h o l i n e - s u b s t i t u t e d sodium-free used, the i n i t i a l b e h a v i o r was  abrupt drop i n M^,  again e r r a t i c .  There was  f o l l o w e d by a r i s e to a l e v e l above the i n i t i a l  ( r e l a t i v e i n c r e a s e by 0.17,  SD = 0.14,  n = 5).  Again, the s u s t a i n e d  an  level effect  151  was  a slow d e c l i n e  Tris. was  of the e f f l u x .  I n two experiments where t r i s - s u b s t i t u t e d sodium-free  used, t h e r e was an abrupt r i s e  i n Mj- ( r e l a t i v e i n c r e a s e by 0.36 on a  average), w h i l e a g a i n t h e s u s t a i n e d e f f e c t was a slow d e c l i n e  Sucrose.  solution  I n two experiments where s u c r o s e - s u b s t i t u t e d  o f the e f f l u x .  sodium-free  s o l u t i o n was used, t h e r a d i o s o d i u m e f f l u x r o s e a f t e r t h e s o l u t i o n Measurements o f ( a ^ )  change and  then d e c l i n e d  slowly.  experiments.  The q u a l i t a t i v e b e h a v i o r o f the sodium e f f l u x p r o b a b l y can be  a  m  were n o t done i n these two  deduced from examination o f t h e r a d i o s o d i u m e f f l u x a l o n e i n t h i s case.  As the  indicated  i n Fig.  15, M^  removal o f e x t e r n a l sodium.  decline  t o the f a l l  solution.  of ( a j j ) a  a  seldom a t t a i n e d  a low steady v a l u e  I t seems r e a s o n a b l e t o a t t r i b u t e  as c e l l u l a r sodium i s l o s t  m  i n F i g . 16.  s e c t i o n s 2.F and 4.  a  a t high values o f  Note, however, that  and  solutions.  free solution  f o r Na -Q, and the r e s u l t s ce  (ajr ) , a  m  This  the  (ajr ) a  m  a  choline,  i s t h e b e h a v i o r a f t e r l o n g immersion i n sodium-  (beyond 30 minutes) w h i l e F i g .  v a r i a t i o n o f M^  than the  t h e b e h a v i o r i s t h e same f o r l i t h i u m ,  15 shows i n a d d i t i o n t h e  b e h a v i o r immediately a f t e r t h e change t o sodium-free  range o f  as e x p l a i n e d i n  The v a l u e s a t 30 and 35 mM a r e l e s s c e r t a i n  others.  The  to the bathing  The r e s t r i c t i o n s o f t h i s c o r r e c t e d c a l c u l a t i o n make i t  i m p o s s i b l e t o e s t i m a t e Mjj  tris  t h i s slow  The s i z e o f t h e sodium e f f l u x d u r i n g t h i s slow d e c l i n e was  e s t i m a t e d from e q u a t i o n (4) u s i n g t h e c o r r e c t i o n plotted  after  with  (a^ ) a  m  solution.  c a n be p r e s e n t e d over a s l i g h t l y w i d e r  i f e q u a t i o n (4) i s employed w i t h o u t c o r r e c t i o n .  e f f l u x o f sodium i n t o sodium-free l i t h i u m - s u b s t i t u t e d  p r e s e n t e d as t h e change i n M^  a  I n F i g . 17,  solution i s  o f i n d i v i d u a l c e l l s as sodium i s l o s t  them i n t o t h e sodium-free b a t h i n g s o l u t i o n .  T h i s i s an approximate  from calcula-  152  50 A**  (pes) 25  A nl  0  9 o  •,.<> 0 I  10  20 (° ) N a  30  "40  (mM)  m  F i g u r e 16. Sodium e f f l u x from the c e l l i n t o a sodium-free s o l u t i o n , c a l c u l a t e d from e q u a t i o n (4) w i t h c o r r e c t i o n f o r Na* -^, versus myoplasmic sodium a c t i v i t y a t the time the e f f l u x was c a l c u l a t e d . e  Diamonds: l i t h i u m - s u b s t i t u t e d s o l u t i o n . Circles: Tris-substituted solution. Open symbols: c e l l s d i s s e c t e d on the day o f the experiment. C l o s e d symbols: c e l l s d i s s e c t e d on the day b e f o r e t h a t o f the experiment. Dashed l i n e : e f f l u x i n t o normal Ringer's s o l u t i o n , from F i g . 12. Note: s c a l e i s d i f f e r e n t from F i g . 12.  153  t i o n because o f the time c o n s t a n t s i n v o l v e d and t h e use o f N a £ - Q , £  been d i s c u s s e d i n s e c t i o n 2.F.  A p p l i c a t i o n of the c o r r e c t i o n f o r Na -Q ce  would r a i s e t h e e s t i m a t e o f M^ , a  The range o f C  3  as has  ^ ^ remains  e s p e c i a l l y a t h i g h e r sodium c o n t e n t . j u s t s h o r t o f the r e g i o n o f most  interest,  about 40 mM, a t which  the e f f l u x into potassium-free s o l u t i o n e x h i b i t s a  shoulder.  i n t o sodium-free s o l u t i o n seems not t o have a s h o u l d e r ,  The e f f l u x  and so t o be s i m i l a r t o t h e e f f l u x i n t o normal R i n g e r ' s s o l u t i o n ,  F i g . 12,  but u n f o r t u n a t e l y t h i s cannot be a l l e g e d w i t h c e r t a i n t y on the b a s i s o f t h i s d a t a over t h e f u l l range i n which s o l u t i o n has been measured. ^ N a ^ i t o ( £[ ) i ^ dt a  a  n  a  the e f f l u x i n t o normal R i n g e r ' s  A s i m i l a r i t y between t h e r e l a t i o n s h i p o f  sodium-free and s o d i u m - c o n t a i n i n g s o l u t i o n has been  found i n s n a i l neurone by Thomas (1972b). b a r n a c l e muscle  i s quite s i m i l a r to that  The b e h a v i o r found here f o r found i n s n a i l  (d) Sodium e f f l u x i n t o s o l u t i o n s c o n t a i n i n g In was  t e s t e d on a p a r t i c u l a r c e l l . in (a^)^,  In sodium  a  a  s h o u l d i n c r e a s e as ( a ^ )  r a p i d d e c l i n e i n M^  (a ) c o u l d Na m  t o ouabain causes a  e f f l u x depended on ( ^ ) a  e x p l a i n e d i n Methods, a v a l u e f o r M^  initial of  a  Exposure o f t h e c e l l  as shown i n F i g . 10, and i t was n o t known how  o u a b a i n - i n s e n s i t i v e sodium  submaximal i n h i b i t i o n , M^ As  ouabain.  t h e p r e s e n t s e r i e s o f experiments, o n l y one c o n c e n t r a t i o n o f ouabain  continuing r i s e the  neurone.  a  &  m  m  l  n  increases  the case o f ( F i g . .12).  c o u l d be c a l c u l a t e d o n l y a f t e r t h e  was completed,  so data a t low c o n c e n t r a t i o n s  not. he o b t a i n e d .  F i g . 18, t h e dependence o f t h e sodium e f f l u x on the myoplasmic activity  i n t h e presence o f ouabain i s p r e s e n t e d .  from e q u a t i o n (4) w i t h t h e c o r r e c t i o n f o r Na -Q. ce  ouabain (,- .r c i r c l e s ) ,  Mjj was c a l c u l a t e d a  I n the presence o f 10  t h e sodium e f f l u x i n t o normal R i n g e r ' s s o l u t i o n i s  s i m i l a r t o but s m a l l e r than t h a t i n t o normal R i n g e r ' s s o l u t i o n  which  M  154  F i g u r e 17. Sodium e f f l u x from the c e l l i n t o sodium-free s o l u t i o n , c a l c u l a t e d from e q u a t i o n (4) w i t h o u t c o r r e c t i o n  lithium-substituted f o r N a * ^ , versus e  myoplasmic sodium a c t i v i t y . Each l i n e r e p r e s e n t s a s i n g l e c e l l , and the change i n the sodium e f f l u x as the myoplasmic sodium a c t i v i t y d e c l i n e d d u r i n g immersion o f the c e l l i n the sodium-free s o l u t i o n . T h i s i s an approximate c a l c u l a t i o n , as e x p l a i n e d i n the t e x t . The dashed l i n e i s the c o r r e s p o n d i n g approximate c a l c u l a t i o n f o r e f f l u x i n t o normal R i n g e r ' s s o l u t i o n ( F i g . 11).  155  F i g u r e 18.  Sodium e f f l u x from the c e l l i n t o normal R i n g e r ' s s o l u t i o n t o -6 -4 which had been added 10 M ( c i r c l e s ) o r 10 M (diamonds) ouabain, v e r s u s myoplasmic sodium a c t i v i t y . Open symbols r e p r e s e n t c e l l s d i s s e c t e d on the day o f the experiment. C l o s e d symbols r e p r e s e n t c e l l s d i s s e c t e d the day before t h a t o f the experiment. S o l i d l i n e : k i n e t i c model f o r three sodium ions b i n d i n g s u c c e s s i v e l y to e q u i v a l e n t independent s i t e s per c y c l e o f the t r a n s p o r t enzyme (k = 15 mM, M = 5 5 p e s ) . Dashed l i n e : e f f l u x i n t o normal max  *  R i n g e r s s o l u t i o n , from F i g u r e 12. i n s t a n c e i s e x p l a i n e d i n the t e x t .  C o r r e c t i o n f o r Na  used i n t h i s  156  c o n t a i n s no ouabain ( F i g . 12 and broken l i n e  i n F i g . 18).  I n the presence  -4 of  10  M ouabain ('  diamonds  ), the sodium e f f l u x d i f f e r s  b e i n g much reduced a t h i g h e r sodium  markedly,  concentrations.  The sodium e f f l u x i n t o normal R i n g e r ' s s o l u t i o n i n the presence o f 10 M ouabain shows o n l y a weak dependence on ( a j j ) . I t i s v e r y s i m i l a r to a  -4  m  -4 the  e f f l u x i n t o p o t a s s i u m - f r e e s o l u t i o n ( F i g . 14).  almost t o t a l which  I f 10  M ouabain y i e l d s  i n h i b i t i o n o f the (Na+K)ATPase, F i g . 18 shows the sodium  i s mediated by o t h e r t r a n s p o r t mechanisms. The c o n s t r u c t i o n o f a dose-response curve i s made d i f f i c u l t  f a c t t h a t the e f f l u x depends q u i t e s t r o n g l y on ( £ j ) a  a  m  sodium t r a n s p o r t system can r e s u l t  i n h i b i t i o n of  i n appreciable increases i n ( a j j ) . a  A dose-response curve c o u l d be c o n s t r u c t e d by comparing for  by the  a t ouabain c o n c e n t r a -  t i o n s which y i e l d submaximal i n h i b i t i o n , w h i l e even p a r t i a l the  a  curve s h o u l d r e p r e s e n t c e l l s so much s c a t t e r  m  the e f f l u x measured  i n the data, t h i s endeavour was  dose-response  Because  there i s  thwarted by the absence o f  i n a s m a l l range o f (ajja^m"  Q u a l i t a t i v e o b s e r v a t i o n s can be made. c o n t a i n i n g 10"^,  The e n t i r e  o f s i m i l a r sodium c o n t e n t .  a l a r g e number o f e x p e r i m e n t a l p o i n t s  10"**, or 10"^ M ouabain.  There was  no e f f e c t o f s o l u t i o n s  The e f f e c t o f 10"^ M ouabain i s  seen i n F i g . 18 t o be s l i g h t , w h i l e the e f f e c t o f 10"^ M ouabain was The b i n d i n g o f ouabain to an enzyme r e n d e r s i t unable to t r a n s p o r t  marked. sodium,  the many enzymes which do not have ouabain bound t o them c o n s t i t u t e a  v e r y l a r g e f u n c t i o n a l r e s e r v e o f sodium e x t r u s i o n . responded o n l y t o the myoplasmic of  m  a g i v e n c o n c e n t r a t i o n o f ouabain w i t h the e f f l u x i n t o normal R i n g e r ' s  s o l u t i o n a t the same v a l u e o f (a-j| ) > v i a F i g . 12.  but  efflux  ( ^ ) a  a  m  sodium a c t i v i t y ,  Yet i f the enzyme the e f f l u x a t a g i v e n v a l u e  i n the presence o f enough ouabain t o b i n d t o the enzymes a p p r e c i -  ably should r e s u l t case i n F i g . 18.  i n a lower t o t a l sodium e f f l u x .  T h i s appears t o be  I n o r d e r to c h a r a c t e r i z e the dose-response t o ouabain  the  157  p r o p e r l y , more data Brinley  f o r 10 ° M and  10  3  (1968) used the technique  M ouabain must be o f exposing  obtained.  the c e l l  to a s e r i e s o f  s o l u t i o n s each w i t h a g r e a t e r c o n c e n t r a t i o n of s t r o p h a n t h i d i n than the preceding  one.  He  a l s o d i d experiments i n which o n l y a s i n g l e c o n c e n t r a t i o n  of s t r o p h a n t h i d i n was maximal i n h i b i t i o n  used on a c e l l ,  (about  5 x 10 ~* M) .  of the sodium e f f l u x to be g r e a t e r content for  (Na) „  This  a t the c o n c e n t r a t i o n judged to y i e l d  i s confirmed  He  found the f r a c t i o n a l  in cells  of lower estimated  by F i g . 18.  inhibition sodium  B r i n l e y found t h a t the dose  half-maximal i n h i b i t i o n o f the sodium e f f l u x by s t r o p h a n t h i d i n v a r i e d  from about 1 x 10 ^ M f o r c e l l s  B i t t a r at al.  to about 5 x 10 ^ M  cells  for  half-maximal i n h i b i t i o n by ouabain o f about 5 x 10 ^ f o r b a r n a c l e 18  sodium content.  sodium content  for  Fig.  of h i g h e r  of low  (1973) r e p o r t e d a dose  i n d i c a t e s t h a t the a c t u a l dose o f ouabain f o r half-maximal  i s g r e a t e r than 10  -6  M except a t r e l a t i v e l y low sodium  muscle.  inhibition  content.  DISCUSSION  Microinjection. I t was  noted i n s e c t i o n 4 t h a t m i c r o i n j e c t i o n causes o n l y t r a n s i e n t  changes i n the p e r m e a b i l i t y , b a r n a c l e muscle c e l l . NaCl a r e  An  i o n content,  and  e x c e p t i o n might be when c o n c e n t r a t e d  t e c h n i c a l problem has  been i d e n t i f i e d ,  l o n g i t u d i n a l d i f f u s i o n of i n j e c t e d radiosodium  the c e l l  Mixing  (a^ ) . a  m  i n what appears to  i n t o non injected?;-, r e g i o n s  i n the r a d i a l d i r e c t i o n s a l o n g the  appears to be q u i t e r a p i d .  the  s o l u t i o n s of  i n j e c t e d , as t h i s o f t e n r e s u l t e d i n a s u s t a i n e d r i s e i n  A significant  the c e l l .  t r a n s p o r t p r o p e r t i e s of  i n j e c t e d portion of  be of  158  An  attempt was made t o i n j e c t a l o n g segment o f the c e l l ,  radiosodium only along t h i s longitudinal  region.  T h i s reduced the e f f e c t o f  d i f f u s i o n , but d i d not e l i m i n a t e i t .  A correction This  injected  and t o c o l l e c t  f o r the e f f e c t o f l o n g i t u d i n a l  d i f f u s i o n has been d e s c r i b e d .  i n v o l v e s no assumptions beyond those i m p l i c i t i n the statement o f the 23  r e l a t i o n between the f l u x o f r a d i o s o d i u m and the f l u x o f fundamental assumption o f the t r a c e r the  technique.  Na, that  I t can o n l y be a p p l i e d i n  form p r e s e n t e d when the r a t e a t which sodium i s e x p e l l e d  is constant.  i s , the  from the c e l l  C o r r e c t i o n a t other times r e q u i r e s a c a l c u l a t i o n  o f the r a t e  o f change o f the sodium e f f l u x . Calculation  o f t h e sodium e f f l u x was a v o i d e d d u r i n g t h e i n t e r v a l s i n  which the sodium content o f the c e l l was changing r a p i d l y ,  such as immed-  i a t e l y a f t e r c e r t a i n changes i n the c o m p o s i t i o n o f the e x t e r n a l because i t was not c l e a r whether the i n t e r i o r o f the c e l l to be w e l l - m i x e d a t such times.  Similarly,  c o u l d be assumed  the a p p l i c a b i l i t y o f the f l u x  model when the sodium e f f l u x was v e r y r a p i d was not known. that limit  an ' u n s t i r r e d  i n such c i r c u m s t a n c e s .  In t h i s c o n n e c t i o n , the data o f F i g s .  (Fig.  I t i s thought  l a y e r ' a t the i n t e r n a l s u r f a c e o f the c e l l membrane c o u l d  the sodium e f f l u x  flux calculated  a t each i n s t a n t  16 and 17 a r e o f i n t e r e s t .  conditions  a  a  ( F i g . 16).  o f the e f f l u x by an u n s t i r r e d  layer  a  a  lithium  m  s h o u l d l a g as w e l l  m  calculated  I f t h e r e were a p p r e c i a b l e  limitation  a t the i n t e r n a l s u r f a c e o f the c e l l  membrane, the former should be l e s s than t h e l a t t e r . value of ( j g )  The  d u r i n g r e l a t i v e l y r a p i d changes i n ( ] ; j )  17) appears i f a n y t h i n g t o exceed the c o r r e s p o n d i n g f l u x  during steadier  solution,  Of course, the measured  during f i l m - c o n t r o l l e d  d i f f u s i o n , and  i s thought t o be a b l e t o s t i m u l a t e the sodium e f f l u x as potassium  does, but a l t o g e t h e r i t appears that which i s d i f f u s i v e ,  r a d i a l mixing o f ions i n s i d e  the c e l l ,  i s n o t a g r e a t problem w i t h t h e t i m e . r e s o l u t i o n a t t a i n -  159  able  i n the p r e s e n t The  implications of this,  the d i s t a n c e is  small.  so.  flux studies. to continue t h i s s p e c u l a t i o n further, i s that  from any p o i n t i n the i n t e r i o r o f the c e l l  t o the c e l l  I t i s known t h a t the c l e f t system i n b a r n a c l e  Further,  membrane  muscle makes t h i s  i t i s i m p l i e d t h a t the t r a n s p o r t p r o p e r t i e s  o f the membrane  l i n i n g the deep c l e f t s a r e s i m i l a r t o those o f the r e s t o f the c e l l membrane. T h i s seems r e a s o n a b l e from a f u n c t i o n a l p o i n t o f view, but o f course cannot be  concluded w i t h c e r t a i n t y from these  considerations.  In p r a c t i c a l terms, one c o u l d attempt t o reduce the e f f e c t s o f l o n g i t u d i n a l d i f f u s i o n f u r t h e r by i n j e c t i n g a longer r e g i o n o f the c e l l and c o l l e c t i n g i s o t o p e o n l y a t the c e n t r e  of the i n j e c t e d region.  t e c h n i q u e o f m i c r o i n j e c t i o n more d i f f i c u l t , of l o n g i t u d i n a l d i f f u s i o n n e g l i g i b l e .  T h i s would make the  and might not make the e f f e c t  O v e r a l l , passive  l o a d i n g seems  p r e f e r a b l e f o r sodium e f f l u x s t u d i e s , even though i t r e q u i r e s  prolonged  immersion o f the d i s s e c t e d c e l l s p r i o r t o the performance o f the experiment. The  c o r r e c t i o n devised  here f o r the m i c r o i n j e c t i o n t e c h n i q u e i s as f u l l y  j u s t i f i e d as t h e use o f the t r a c e r t e c h n i q u e i t s e l f . rule,  i t seems d e s i r a b l e  to design  experiments so t h a t the l e a s t manipula-  t i o n o f the raw data must be done b e f o r e i n v e s t i g a t i o n can be  Aged The  However, as a g e n e r a l  an answer t o the q u e s t i o n  under  obtained.  cells. maintenance o f d i s s e c t e d b a r n a c l e  muscle c e l l s  s o l u t i o n has been shown t o a f f e c t the c e l l s but l i t t l e .  i n normal R i n g e r ' s The c e l l s  tend t o  g a i n sodium and l o s e potassium, but t h e membrane p o t e n t i a l and water c o n t e n t remain constant  (eg. T a b l e I I ) .  I n the e f f l u x experiments i t has been  found t h a t the b e h a v i o r o f f r e s h and day-old 14;  16 w i t h l e s s c e r t a i n t y ; and 18).  cells  i s t h e same ( F i g s . 11;  The d i f f e r e n c e i n the response o f  160  ' f r e s h ' and  'aged' f r o g s k e l e t a l muscle c e l l s noted above i s p r o b a b l y due to  differences  i n the sodium content, as noted f o r example by Keynes and  S t e i n h a r d t (1968).  . Modes o f sodium e x t r u s i o n . The dependence o f the sodium e f f l u x on the sodium content o f the b a r n a c l e muscle, f o r e f f l u x  i n t o normal Ringer's s o l u t i o n ,  i s s i m i l a r to  t h a t r e p o r t e d i n s n a i l neurone (Thomas 1972b) and i n s q u i d axon (Hodgkin & Keynes 1956; S j o d i n & Beauge 1967; B r i n l e y & M u l l i n s 1968). from t h a t r e p o r t e d  i n r e d blood c e l l s  s k e l e t a l muscle ( H a r r i s 1965)  (Garay & Garrahan 1973) and i n f r o g  i n the f a i l u r e to d e t e c t s a t u r a t i o n even a t  r e l a t i v e l y h i g h l e v e l s o f i n t r a c e l l u l a r sodium content. from the r e s u l t s o f B r i n l e y muscle c e l l s ,  It is different  (1968) and o f B i t t a r e t al.  I t also  differs  (1972) f o r b a r n a c l e  almost c e r t a i n l y because o f the problems i n v o l v e d i n working  w i t h m i c r o i n j e c t i o n , as d i s c u s s e d a t l e n g t h above and i n s e c t i o n 4. The s t r i k i n g  f e a t u r e o f the dependence  i s the apparent absence o f  s a t u r a t i o n a t myoplasmic sodium a c t i v i t i e s up t o 50 mM and perhaps up to 70 mM.  I t i s not clear-why the b a r n a c l e muscle c e l l  l a r g e f u n c t i o n a l r e s e r v e f o r sodium e x t r u s i o n .  s h o u l d have such a  A c t i o n p o t e n t i a l s do not  propagate i n the b a r n a c l e muscle c e l l membrane under o r d i n a r y c o n d i t i o n s , so  t h i s cannot be a l a r g e source o f sodium i n f l u x _in v i v o .  Perhaps a  sodium-calcium exchange a c r o s s the c e l l membrane i s r e q u i r e d f o r r e l a x a t i o n of  the muscle, s i n c e i t s s a r c o p l a s m i c r e t i c u l u m i s so s m a l l .  Frog  striated  muscle, by comparison, shows s a t u r a t i o n a t r e l a t i v e l y low sodium content ( s h o u l d e r a t about 10 m i l l i m o l e sodium per kg t i s s u e - H a r r i s  1965).  The e f f l u x i n t o normal R i n g e r ' s s o l u t i o n i s thought t o be composed o f s e v e r a l components.  The dominant ones a r e thought to be sodium-potassium  exchange v i a the (Na+K)ATPase and sodium-sodium exchange v i a some other  161  mechanism, as noted i n t h e I n t r o d u c t i o n .  A s m a l l c o n t r i b u t i o n appears to  be made by t h e sodium-sodium exchange mode o f t h e (Na+K)ATPase i n potassiumf r e e s o l u t i o n and i n e n e r g y - d e p l e t e d c e l l s , sodium exchange has been r e p o r t e d conditions  (Keynes & S t e i n h a r d t  since a ouabain-sensitive  sodium-  i n f r o g s k e l e t a l muscle under these  1968; Kennedy & De Weer 1976).  Thus the  i n t e r p r e t a t i o n o f F i g . 12 must be c a r r i e d out by comparison w i t h the c o r r e s ponding p l o t s when one o r another o f t h e sodium e x t r u s i o n mechanisms i s disabled.  For t h i s purpose, t h e curves f i t t e d  by eye t o F i g s . 12, 14, 16,  and 18 a r e c o l l e c t e d i n F i g . 19. The dependence  o f M^  on ( ^ ) a  a  a  i  n  t  n  m  e  absence o f e x t e r n a l p o t a s s i u m  and t h a t i n the.presence o f 10"^ M ouabain a r e c l o s e l y c o r r e l a t e d , but d i f f e r from t h e dependence saturation.  This  i n normal R i n g e r ' s s o l u t i o n i n t h a t they show a d e f i n i t e  i s c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t a dominant mode o f  sodium e x t r u s i o n i s t h e sodium-potassium exchange mode o f t h e (Na+K)ATPase. The e f f l u x which remains i n the presence o f ouabain o r i n t h e absence of external potassium r e f l e c t s  t h e maximum c o n t r i b u t i o n t h a t other mechan-  isms can make t o the t o t a l sodium e f f l u x . great- as. t h e e f f l u x range'.  T h i s appears t o be almost as  i n t o normal R i n g e r ' s s o l u t i o n over t h e ' p h y s i o l o g i c a l  However, i t i s w i d e l y h e l d t h a t t h i s  i s mostly t i g h t l y - l i n k e d  sodium-sodium exchange, and so i n e f f e c t u a l as f a r as sodium r e g u l a t i o n i s concerned.  Further,  seems t o f a l l the  influx.  even i f t h i s were a l l e f f e c t i v e sodium e x t r u s i o n , i t  s h o r t o f what i s needed under normal c o n d i t i o n s  Thus exposure t o ouabain o r removal o f e x t e r n a l p o t a s s i u m w i l l  cause the sodium e f f l u x t o f a l l  below t h e sodium i n f l u x .  s t e a d i l y , and t h e s p e c i f i c a c t i v i t y o f t h e myoplasm w i l l will  to balance  cause a r e d u c t i o n This reduction  ( Na^m ' a  w:  ii  be reduced.  r  i  i n the e f f l u x o f r a d i o s o d i u m c o u l d be i n t e r p r e t e d as i n the sodium e f f l u x ,  i f the myoplasmic  e  This  i n the e f f l u x o f radiosodium.  representative of a f a l l  s  sodium  162  F i g u r e 19. Summary o f sodium e f f l u x from the c e l l i n t o v a r i o u s s o l u t i o n s , e x t r a c t e d from F i g u r e s 12 (normal Ringer's s o l u t i o n - s o l i d c u r v e ) , 14 ( p o t a s s i u m - f r e e s o l u t i o n - lower dashed c u r v e ) , 16 (sodium-free s o l u t i o n upper dashed curve, r e p r e s e n t i n g c h o l i n e ) , and 18 (ouabain i n normal Ringer's s o l u t i o n a t 10 ^ M - l i n e as f o r p o t a s s i u m - f r e e s o l u t i o n ) . The r e l a t i v e p o s i t i o n o f the d i f f e r e n t curves over the p h y s i o l o g i c a l range, (approx. 10 - 20 mM) was deduced form the b e h a v i o r o f a c e l l compared t o i t s e l f as c o n t r o l , f o r a g i v e n t e s t s o l u t i o n . The s c a t t e r o f the grouped data does not permit the r e l a t i v e p o s i t i o n s t o be d i s t i n g u i s h e d over t h i s range. Thus; the e f f l u x a t a g i v e n v a l u e o f (a., ) i n c r e a s e s when the ° Na m e x t e r n a l s o l u t i o n i s changed from normal R i n g e r ' s s o l u t i o n to sodium-free c h o l i n e - s u b s t i t u t e d s o l u t i o n , but decreases when i t i s changed t o a p o t a s s i u m - f r e e s o l u t i o n or to one c o n t a i n i n g ouabain. The curves f o r the -4 l a t t e r two cases (with 10 M ouabain) are almost i n d i s t i n g u i s h a b l e i n the p r e s e n t s e r i e s o f experiments, and the e f f l u x i n t o p o t a s s i u m - f r e e ouabainc o n t a i n i n g s o l u t i o n was not examined. The curve f o r sodium-free l i t h i u m s u b s t i t u t e d s o l u t i o n would be p a r a l l e l t o the c h o l i n e curve, but below the normal curve. b  163 activity  i s not monitored f o r the purpose o f c a l c u l a t i n g the sodium e f f l u x  from r a d i o i s o t o p e movement.  That i s ,  the f a l l  i n the sodium e f f l u x due t o  i n h i b i t i o n o f the pump might have been o v e r e s t i m a t e d i n the p a s t . If  the mechanism which remains o p e r a t i o n a l i n the absence o f e x t e r n a l  potassium and i n the presence o f ouabain does indeed r e p r e s e n t exchange,  sodium-sodium  then i t s h o u l d be markedly reduced, i f n o t e l i m i n a t e d , by the  removal o f sodium from the b a t h i n g s o l u t i o n .  I t has been found by o t h e r  workers, however, t h a t removal o f e x t e r n a l sodium and potassium, even when combined w i t h exposure t o ouabain, does n o t reduce the sodium e f f l u x t o the l e v e l expected i f o n l y p a s s i v e f l u x e s a r e p r e s e n t  (eg. B r i n l e y 1968).  Only  when ATP i s almost c o m p l e t e l y removed from the c e l l and i n h i b i t o r s o f metabolism a r e a p p l i e d a r e the f l u x e s reduced t o the p a s s i v e r a t e s , as d i s c u s s e d i n s e c t i o n 2. It  appears from F i g . 19 t h a t f o r b a r n a c l e muscle c e l l s  the e f f l u x o f  sodium i n t o sodium-free s o l u t i o n i s v e r y s i m i l a r to the e f f l u x i n t o normal Ringer's s o l u t i o n .  Further,  t h i s appears t o be independent o f the c a t i o n  used i n p l a c e o f sodium i n the b a t h i n g s o l u t i o n but  hardly l i k e l y  ( F i g . 16).  t h a t these c a t i o n s c o u l d each s u b s t i t u t e  Is i s c o n c e i v a b l e f o r sodium i n a  sodium-sodium exchange, o r e n t e r the c e l l by some other path, t o y i e l d the b e h a v i o r observed. solution reflects  I f i t i s assumed t h a t the e f f l u x i n t o normal R i n g e r ' s  the normal o p e r a t i o n o f the c e l l ,  i t appears t h a t a sodium  e f f l u x which depends on e x t e r n a l sodium c o n t r i b u t e s l i t t l e in  b a r n a c l e muscle  to the normal  flux  cells.  Through the use o f the i n t r a c e l l u l a r s o d i u m - s p e c i f i c m i c r o e l e c t r o d e , the net  r e d u c t i o n i n the sodium e f f l u x seen when the b a t h i n g s o l u t i o n i s changed  from one c o n t a i n i n g sodium to one n o t c o n t a i n i n g sodium has been r e v e a l e d to be due t o the consequent f a l l of  (a„ ) and somewhat beyond. Na'm v  J  in ( ^ ) ' ° a  a  m  v  e  r  the ' p h y s i o l o g i c a l range'  That the e f f l u x  i n t o sodium-free s o l u t i o n  seems t o be s l i g h t l y  g r e a t e r i n magnitude  than the e f f l u x  into  sodium-  containing solutions  i s c o n s i s t e n t w i t h the f i n d i n g o f o t h e r workers  164  t h a t e x t e r n a l sodium can i n h i b i t l i t h i u m probably r e f l e c t  the sodium e f f l u x .  i n a d d i t i o n the a b i l i t y  l i k e potassium (eg. Beauge  The  first  o f l i t h i u m to s t i m u l a t e  1975).  The e x i s t e n c e o f sodium-sodium exchange was (1949).  The r e s u l t s w i t h  first  suggested by U s s i n g  evidence i n f a v o u r o f i t s e x i s t e n c e i n muscle c e l l s  was  p r e s e n t e d by Keynes and Swan (1959) and t h e i r r e s u l t s demand c l o s e r c o n s i d e r a tion.  In t h e i r experiments, whole muscles  o f f r o g s were loaded w i t h r a d i o -  24 sodium  (  Na)  by immersion  f o r about 3.5  s o l u t i o n which c o n t a i n e d radiosodium. 5 o r 10 minute  muscle d u r i n g each i n t e r v a l  The muscle was  f r o g Ringer's  then t r a n s f e r r e d a t  The amount of r a d i o a c t i v i t y  ( c a l l e d Na*  c o r r e c t e d f o r decay o f t h i s s h o r t - l i v e d a g a i n s t time.  i n normal  i n t e r v a l s through a s u c c e s s i o n o f t e s t - t u b e s c o n t a i n i n g  i n a c t i v e R i n g e r ' s or other s o l u t i o n . the  hours  i n t h i s t h e s i s ) was  i s o t o p e , and p l o t t e d  leaving  measured,  logarithmically  T h e i r e s t i m a t e o f changes i n t h e . r a t e o f l o s s o f sodium,  23 that i s , of of  Na,  from the muscle c e l l s were c a l c u l a t e d from displacements  t h i s curve caused by changes i n the i o n i c c o m p o s i t i o n o f the medium. They found t h a t the s l o p e "k£" o f the p l o t o f l n Na  c o n s t a n t d u r i n g e f f l u x i n t o normal s l o p e "k|" of the p l o t o f l n N a * was  frog Ringer's s o l u t i o n . versus time was  c a l c u l a t e d by b a c k - a d d i t i o n as was  experiments —l/—1  e l l  W  a  S  v  e  on b a r n a c l e muscle r  y  c  l°  s e  versus time t o be L i k e w i s e , the  c o n s t a n t , where N a *  done i n the p r e s e n t s e r i e s o f  (see Methods, s e c t i o n 4 ) .  The s l o p e r a t i o  to u n i t y , as expected f o r p a s s i v e l y - l o a d e d  When the e x t e r n a l s o l u t i o n was  e l l  changed from normal  t i o n to sodium-free l i t h i u m - s u b s t i t u t e d s o l u t i o n ,  cells.  frog Ringer's s o l u -  the v a l u e o f Na  decreased.  The p l o t o f In Na* versus time assumed a s t e a d i e r , almost l i n e a r s l o p e ii  "k^" a f t e r about 20 minutes. time e x h i b i t e d a break, substituted solution.  The s l o p e o f the p l o t o f l n N a  to a new  c e  ii  s m a l l e r s l o p e " ^ " f o r sodium-free  Keynes and Swan found t h a t k^/loj was  versus lithium-  c l o s e t o 3.  165  I t was t h i s d i s c r e p a n c y  t h a t gave r i s e t o t h e i r enquiry  models f o r the n a t u r e o f the sodium e f f l u x . Na*  into different  They c o n s i d e r e d  the p l o t o f  v e r s u s Na* -Q f o r e f f l u x i n t o sodium-free s o l u t i o n , which i s l i k e a e  power f u n c t i o n w i t h power t h r e e , and f i n a l l y s e t t l e d on a model i n which sodium i s t r a n s p o r t e d at  the r e a s o n f o r the v a l u e  i s q u i t e simple.  as  from a s i n g l e homogeneous compartment,  a r a t e p r o p o r t i o n a l t o t h e cube o f the sodium content o f the c e l l . In f a c t ,  the  out o f t h e c e l l  2 2  2 4  2  transported  Then no matter what the mechanism by which sodium i s  out o f the compartment, as l o n g as the r a t e o f sodium t r a n s p o r t  amount o f t r a c e r l e f t  i n the compartment  be p r o p o r t i o n a l o n l y t o the  (assuming no b a c k f l u x ) .  = -k x Na* where k i s a p o s i t i v e constant. cell ^  the e x p o n e n t i a l constant  than u n i t y  i s i n a w e l l - m i x e d compartment, and behaves j u s t  i s steady the r a t e o f e f f l u x o f t r a c e r w i l l  dNa -z—cell at  greater  The fundamental assumption o f the t r a c e r technique i s t h a t  t r a c e r ( N a o r N a) the - % a does.  o f k^/k^ being  function :  This equation ^  Na* -Q = N a * ^ ^ ( t = 0 ) . e x p ( - k t ) . e  Thus,  e  defines  The 'rate  k f o r r a d i o s o d i u m e f f l u x i s determined by t h e r a t e o f the sodium  1  efflux.  When t h i s r a t e i s steady, k i s c o n s t a n t  versus time i s l i n e a r . s o l u t i o n f o r frog,as  and the p l o t o f In Na ;Q ce  T h i s occurs f o r e f f l u x i n t o normal Ringer's  i t does f o r b a r n a c l e  muscle, but n o t f o r sodium-free  solution. If  t h e r a t e o f mixing i n the compartment i s always r a p i d compared t o  the r a t e o f t r a n s p o r t out o f the compartment  (which i s an assumption o f  the t r a c e r method), then a s i m i l a r s i t u a t i o n should o f time even when the r a t e o f sodium t r a n s p o r t Na*  e l l  (t)  =  i s changing:  Na* (t=0)-exp(-k(t)-t)  where now the 'rate c o n s t a n t ' The  occur a t each i n s t a n t  e U  k i s a f u n c t i o n o f time. •k  r a t e o f change o f N a - Q must r e f l e c t both the r a t e o f sodium ce  166  t r a n s p o r t and for  the r a t .  o f change o f the sodium t r a n s p o r t , and  e  the v a l u e o f ^ 2 ^ 2  f° d  dNa* dt  =  e l l  then  ic  d dt  versus  i  n  experimentally  of  'pseudo-calculus' • ic  time  i s a p l o t o f In d N a e l l versus dt C  dNa* -Q; t h i s dt e  is collected  - Na*/dt  where a g a i n the shorthand Na  i n s i d e the c e l l i s  i n i n t e r v a l A t of time an amount o f r a d i o s o d i u m Na*  i n the b a t h i n g s o l u t i o n ,  accounts  i n frog.  u n  More f o r m a l l y , i f the t o t a l amount of r a d i o s o d i u m N ^ c e l l ' and  this  i s "k"' of Keynes and Swan.  i s employed. time, and  A plot of l n  i t s slope is  A p l o t of l n N a * l l e  versus  ic  time has forward  s l o p e d_ In dt  ^ ' nl a  this  ce  i s "k" o f Keynes and Swan.  Straight-  d i f f e r e n t i a t i o n o f the above e x p r e s s i o n f o r N a - ^ ^ ( t ) r e v e a l s : ce  ( k ( t ) ..+ t ^ , ) dt  2  ( k(t) and  k  =  ( k(t)  +  t ^ dt  "  ( 2 ^ + dt  +  t ^ )  )  ) .  When k ( t ) i s c o n s t a n t , as i n normal Ringer's  s o l u t i o n , where (Na)^ i s  steady,  been noted  then k' = k = k and k'/k  = 1, as has  However, when the muscle i s i n sodium-free r o u g h l y as an e x p o n e n t i a l f u n c t i o n o f time 1976), and  so k ( t ) w i l l  fall. (  k'/v  It  = "  solution,  (Na)^ w i l l  fall  i n f r o g muscle (White & Hinke  Then dk  ?  before.  d k 2  1  i s not unreasonable  (  k  ( t )  +  t f )  2  *  to assume s o l e l y f o r the purposes of t h i s d i s c u s s i o n  t h a t the d e c l i n e o f k ( t ) i s a l s o d e s c r i b a b l e by an e x p o n e n t i a l f u n c t i o n :  167  k(t)  k  Q  exp(-bt) where b i s a p o s i t i v e c o n s t a n t , r o u g h l y equal t o  min"''" a f t e r the f i r s t  0.01  20 minutes f o r f r o g muscle (White & Hinke 1976).  Then k'/  ~  k  1  -  b ( b-t - 2 ) k ( 1 - b-t )  and k'/k. w i l l  exceed u n i t y w h i l e b-1<  t a t i v e example i s d u r i n g the f i r s t  2  2, which i n t h i s v e r y rough,  200  The c o n c l u s i o n t o be drawn, then,  semiquanti-  minutes. i s t h a t the " d i s c r e p a n c y " which l e d  Keynes and Swan (1959) and Keynes and S t e i n h a r d t (1968) to p o s t u l a t e the e x i s t e n c e o f more than one of  i n t r a c e l l u l a r compartment was  a misinterpretation  the t r a c e r data. Similarily,  the " p l a t e a u s " found by M u l l i n s and  Frumento (1963) f o r  r a d i o s o d i u m e f f l u x from f r o g s k e l e t a l muscle i n t o sodium-free be accounted  f o r by the above e x p r e s s i o n f o r k',  they proposed  need not be  and  which occur as  can  the e l e c t r i c a l c o u p l i n g  invoked.  F u r t h e r , a l l o f the r e s u l t s on the sodium-free can be accounted  solution  effect  i n f r o g muscle  f o r i n terms o f the changes i n the r a t e o f sodium e f f l u x  (Na) ^ changes.  I t i s i n t e r e s t i n g t h a t Keynes and Swan (1959)  and Keynes and S t e i n h a r d t (1968) t r i e d t o account  for their results  by  p o s t u l a t i n g t h a t sodium-sodium exchange o c c u r r e d a t low v a l u e s o f (Na)^ but not a t h i g h v a l u e s .  That  l a t i o n s o f the muscle c e l l sodium e x p u l s i o n on  i s , they acknowledged the e f f e c t o f t h e i r manipuon  (Na)^, and the dependence o f the r a t e o f  (Na)^, but d i d not take these f e a t u r e s i n t o account  t h e i r a n a l y s i s o f t h e i r t r a c e r data. (Na). c o u l d d e c l i n e so p r e c i p i t o u s l y R e c o g n i t i o n o f t h i s e f f e c t has  A p p a r e n t l y they d i d not f e e l i n sodium-free  1977).  that  (Keynes 1965).  come from measurements w i t h s o d i u m - s p e c i f i c  i n t r a c e l l u l a r m i c r o e l e c t r o d e s (Thomas 1972bJones  solution  in  White & Hinke 1976;  Vaughan-  168  The r e s u l t s  imply t h a t the b e h a v i o r o f the sodium t r a n s p o r t  i s much more s t r a i g h t f o r w a r d than p r e v i o u s l y had been thought. sodium-sodium exchange which was  The  linked  supposed to be o u a b a i n - i n s e n s i t i v e and to  comprise almost h a l f o f the sodium t r a n s p o r t measured occur i n b a r n a c l e muscle.  i n muscle  i n muscle does not  Evidence p r e v i o u s l y presented f o r i t s existence  i n f r o g muscle has been shown t o be i n c o r r e c t .  In t h i s connection, i t i s  i n t e r e s t i n g t h a t when (Na)^ was m a i n t a i n e d by i n t e r n a l d i a l y s i s  i n squid  axon, sodium-free s o l u t i o n s caused no r e d u c t i o n i n the sodium e f f l u x as l o n g as ATP was  i n c l u d e d i n the d i a l y s i s  s o l u t i o n ( M u l l i n s & B r i n l e y 1967).  The v a r i o u s e f f e c t s on the sodium e f f l u x o f ouabain or changes i n the i o n i c c o m p o s i t i o n o f the e x t e r n a l s o l u t i o n , r e p o r t e d h e r e f o r b a r n a c l e and by o t h e r s f o r f r o g muscle, appear almost a l l o f F i g . 19.  Most o f the r e s u l t s  I t . almost-seems  t o be e x p l a i n e d by the curves  i n nerve can be e x p l a i n e d  t h a t the overwhelming dominant mode o f sodium t r a n s p o r t  i n muscle and nerve, and perhaps i n most c e l l s ,  under normal c o n d i t i o n s , i s  the sodium-potassium exchange mode o f the (Na+K)ATPase.  However, i t would  have to be proposed i n a d d i t i o n t h a t exposure o f the c e l l s cannot i n h i b i t a l l  i n l i k e manner.  t o ouabain  o f the t r a n s p o r t enzymes i n the c e l l membrane, as  suggested, for.example, by B r i n l e y which l e a k s out o f the c e l l  (1968), and t h a t  ' r e c y c l i n g ' o f potassium  i n t o p o t a s s i u m - f r e e s o l u t i o n can occur, as  s u g g e s t e d , J f o r example, by. Beauge (1975).  The c o n t r i b u t i o n o f sodium t r a n s -  p o r t modes i n v o l v i n g c a l c i u m , amino a c i d s ,  e t c . , should be r e l a t i v e l y  normally.  C e r t a i n l y no p r o o f o f t h i s h y p o t h e s i s  small  i s c l a i m e d here, but these  c o n s i d e r a t i o n s seem to be a worthwhile b a s i s on which to p l a n  further  experiments t o f i n d the mechanism o f sodium e x p u l s i o n i n t o p o t a s s i u m - f r e e and i n t o o u a b a i n - c o n t a i n i n g  solutions.  169  Kinetics. Much of the c o n t r i b u t i o n o f the (Na+K)ATPase t o the sodium e f f l u x be e l i m i n a t e d s e l e c t i v e l y by exposure o f the c e l l s e f f l u x can be assumed to be due mostly mation,  and  to ouabain.  The  to one mechanism as a f i r s t  i t s k i n e t i c c h a r a c t e r i s t i c s examined.  Of course,  can  remaining approxi-  t h i s might  r e p r e s e n t more than one mechanism, but t h e r e i s no good reason to assume this  now. I f a simple two-parameter model o f the type d i s c u s s e d i n s e c t i o n 2 i s  a p p l i e d to the experimental data, a r e a s o n a b l e v a l u e s o f the parameters.  f i t can be found  f o r some  S e l e c t i o n o f the most a p p l i c a b l e model  then  depends on a knowledge o f the v a l u e s these parameters can assume, s i n c e each has a p h y s i c a l  interpretation.  MJJ^JJ i s the maximum v a l u e the e f f l u x can a t t a i n , and can be q u i t e w e l l from the data a t h i g h e r v a l u e s o f ( a ^ ) a  I f s a t u r a t i o n cannot any degree o f  be d e t e c t e d i n the data, M  the apparent  sodium complex when used  cannot  be e s t i m a t e d w i t h  d i s s o c i a t i o n c o n s t a n t o f the enzyme-  i n t h i s context, r e f l e c t s  the b i n d i n g  can be q u i t e h i g h s i n c e the b i n d i n g r e s u l t s  i n the c o n f o r m a t i o n  are complicated  energy.  in a slight  o f the enzyme, as d i s c u s s e d i n s e c t i o n  For a c y c l i c a l c a r r i e r system, the apparent  reactions  i f s a t u r a t i o n occurs.  assurance.  The v a l u e o f k,  T h i s energy  m a x  m  estimated  change  2.  dissociation  constants  f u n c t i o n s o f the r a t e c o n s t a n t s which d e s c r i b e the v a r i o u s  i n the c y c l i c sequence.  actual a f f i n i t i e s  ( C a l d w e l l 1969).  t i o n c o n s t a n t s a t one  They can be v e r y d i f f e r e n t I t appears  from  t h a t the apparent  s u r f a c e o f the membrane a r e independent  the  dissocia-  of changes i n  the c o m p o s i t i o n of the s o l u t i o n b a t h i n g the o p p o s i t e s u r f a c e under most c o n d i t i o n s , even though such changes would a l t e r some o f the r e a c t i o n r a t e s i n the c y c l i c sequence (Hoffman & T o s t e s o n  1971;  Garay & Garrahan  1973;  170  C h i p p e r f i e l d & Whittan  1976).  It is likely,  then,  t h a t the  apparent  a f f i n i t i e s o b t a i n e d v i a a good model w i l l r e f l e c t the t r u e a f f i n i t i e s o f the sodium s i t e s . From s t u d i e s of the a c t i o n of sodium i n p r o t e c t i n g the (NaH-K)ATPase from i n a c t i v a t i o n by DCCD ( d i c y c l o h e x y l c a r b o d i - i m i d e ) , Robinson (1974) a b l e to estimate the d i s s o c i a t i o n c o n s t a n t  f o r the enzyme-sodium complex.  In the absence o f o t h e r l i g a n d s , the v a l u e was  2.3  mM.  In the c e l l ,  sium w i l l compete w i t h sodium f o r the sodium s i t e s , and so the d i s s o c i a t i o n c o n s t a n t w i l l be too l a r g e . sodium s i t e f o r potassium  was  potas-  apparent  However, s i n c e the a f f i n i t y of the  i s much l e s s than t h a t f o r sodium, the  apparent  v a l u e w i l l probably be of the c o r r e c t order of magnitude. With r e s p e c t to the models d i s c u s s e d i n s e c t i o n 2, f o r "n" ions b i n d i n g b e f o r e t r a n s p o r t occurs, the v a l u e o f k which gives a good f i t to the data is  lower The  f o r h i g h e r v a l u e s of n. data f o r ouabain  potassium-free 1/M^  solution  y i e l d s k = 105  mM,  ( F i g . 18) a r e almost  ( F i g . 14). M^^  = 116  10"^ M oubain,  and k = 116 mM,  free solution.  These v a l u e s  \  i a K  i d e n t i c a l to the data f o r  A l i n e a r regression of l / ( ^ ) a  a  pes  pes.  M  '  =60  max  = 124 pes  f o r the e f f l u x i n t o  f o r k a r e too l a r g e , however.  pes.  r  For f o u r ions i t was  For t h r e e ions i t was  k = 15 mM,  above d i s c u s s i o n , as a f i r s t  on  f o r the e f f l u x i n the presence  b i n d i n g s u c c e s s i v e l y , the best f i t to the data, by t r i a l and w i t h k = 20 mM,  m  approximation  = 55 pes.  potassium-  For two error,  k = 15 mM,  of  ions was  M „„ = max  60  On the b a s i s of the  only, the data a r e f i t t e d best  by  a model w i t h a t l e a s t t h r e e sodium ions b i n d i n g s u c c e s s i v e l y to e q u i v a l e n t independent s i t e s .  The key  low l e v e l s of ( f j ) j a  a  i n normal Ringer's saturation.  m  a s  to a more p r e c i s e c o n c l u s i o n i s good data a t  d i s c u s s e d p r e v i o u s l y , plus e x t e n s i o n o f the data  s o l u t i o n and  sodium-free  s o l u t i o n to the r e g i o n o f  171  C o n t i n u i n g i n t h i s r a t h e r s p e c u l a t i v e v e i n , i t can be noted a g a i n t h a t the e f f l u x  i n t o sodium-free  normal Ringer's  solution  s o l u t i o n i s v e r y s i m i l a r t o the e f f l u x  ( F i g . 19).  into  I f i t i s assumed t h a t the dominant  mode o f sodium t r a n s p o r t n o r m a l l y  i s sodium-potassium exchange, the two-  parameter models can be a p p l i e d .  There i s no good i n d i c a t i o n o f M ^ ^  a good f i t can be a c h i e v e d pes;  f o r one i o n b i n d i n g w i t h k = 200 mM, M^.^ = 400  f o r two ions w i t h k = 50 mM,  w i t h k = 30 mM, 200 pes.  That  but  M^^j = 225 pes; f o r t h r e e ions b i n d i n g  = 175 pes; and f o r four ions w i t h k = 27.5 mM,  M^^  =  i s , t h e f i t i s b e t t e r f o r h i g h e r numbers o f ions b i n d i n g  successively. Thus the two modes d e f i n e d o p e r a t i o n a l l y here appear t o d i f f e r i n their kinetic characteristics  i n the c o n t e x t o f the two-parameter models.  For no v a l u e o f n i s i t found  t h a t k i s the same but M  i s lower  for cells  i n normal  Ringer's  max i n which the (Na+K)ATPase i s i n h i b i t e d compared t o c e l l s solution. and  T h i s suggests  t h a t t h e e f f l u x observed  i n the absence o f e x t e r n a l potassium  o f fewer t r a n s p o r t enzymes. o f ( N a ) can be o b t a i n e d , m  i n the presence  o f ouabain  i s not simply due t o the o p e r a t i o n  When more data a t v e r y low and v e r y h i g h  i t will  levels  be i n t e r e s t i n g t o perform a more d e t a i l e d  k i n e t i c a n a l y s i s i n terms o f i n h i b i t i o n o f d i f f e r e n t  types.  172  SECTION 6.  Two  COMPARISON OF SODIUM ELECTRODE AND  RADIOSODIUM MEASUREMENTS  'sodium-free' e f f e c t s have been r e p o r t e d i n sodium e f f l u x  i n muscle, as summarized The better-known e f f e c t sodium i s removed  i n s e c t i o n 2.C.  They d i f f e r  i n t h e i r time course.  i s a s u s t a i n e d r e d u c t i o n i n the sodium e f f l u x when  from the b a t h i n g s o l u t i o n .  I n the l a s t s e c t i o n ,  r e p o r t e d t h a t t h i s e f f e c t appears to be due t o the f a l l sodium a c t i v i t y a l o n e , a t l e a s t  i n b a r n a c l e muscle  The second 'sodium-free e f f e c t '  i t was  i n the myoplasmic  cells.  is a large rapid f a l l  c e l l u l a r sodium a c t i v i t y measured w i t h an i n t r a c e l l u l a r microelectrode.  studies  i n the i n t r a -  sodium-specific  T h i s e f f e c t has been observed i n f r o g s k e l e t a l  muscle  (White & Hinke 1976) and i n c r a b s t r i a t e d muscle (Vaughan-Jones  1977).  s l i g h t l y different rapid This rapid f a l l  f a l l was  in (ajq ) a  m  A  found i n s n a i l neurone (Thomas 1972b).  c o i n c i d e s r o u g h l y w i t h the t r a n s i e n t  stimula-  t i o n o f the sodium e f f l u x seen i n F i g . 15 w i t h c h o l i n e and t r i s , and w i t h the to the  'biphasic t r a n s i e n t ' with l i t h i u m .  The change from s o d i u m - c o n t a i n i n g  sodium-free s o l u t i o n i s not i n s t a n t a n e o u s , and as noted i n s e c t i o n 4, e x p e r i m e n t a l apparatus was  However, i t was  felt  not d e s i g n e d to measure t r a n s i e n t phenomena.  t o be worthwhile t o compare the i n f o r m a t i o n from the  i n t r a c e l l u l a r m i c r o e l e c t r o d e and the r a d i o i s o t o p e , p a r t i c u l a r l y d u r i n g the p e r i o d where the t r a n s i e n t s In  occur.  the experiments on f r o g muscle, c h e m i c a l a n a l y s i s i n d i c a t e d  the  fall  i n the myoplasmic sodium a c t i v i t y  out  o f the c e l l  (White & Hinke 1976).  that  i s due to movement o f sodium ions  S t u d i e s o f the e f f l u x o f r a d i o s o d i u m  from f r o g muscle i n t o sodium-free s o l u t i o n have r e v e a l e d some t r a n s i e n t r a p i d f l u x e s , but these were u s u a l l y a s c r i b e d t o the e x t r a c e l l u l a r space and ignored (eg. Hodgkin & Horowicz 1959). Measurements  w i t h the i n t r a c e l l u l a r m i c r o e l e c t r o d e a l o n e can o n l y  173  r e v e a l a l o s s o f sodium from the major i n t r a c e l l u l a r compartment, myoplasm.  They cannot r e v e a l the f a t e o f the l o s t sodium.  the  d a t a was  are  combined,  the  A treatment o f  thus d e v i s e d i n which m i c r o e l e c t r o d e and r a d i o i s o t o p e  results  so t h a t changes i n the sodium content o f the myoplasm c o u l d be  compared w i t h the simultaneous l o s s o f sodium from the c e l l . ments were conducted p r i o r to the p u b l i c a t i o n o f the r e s u l t s  The  experi-  f o r crab  muscle. I t was to  found t h a t a r a p i d f a l l  i n the myoplasmic sodium a c t i v i t y  t h a t seen i n s n a i l neurone and i n f r o g and crab muscle occurs  muscle under c e r t a i n c o n d i t i o n s , and t h a t  similar  i n barnacle  i t i s accompanied by a commensu-  r a t e l o s s o f sodium from the c e l l . The t r a n s i e n t changes  i n the sodium e f f l u x which occur immediately  a f t e r the-change t o sodium-free s o l u t i o n appear to r e f l e c t a d i r e c t  effect  on the t r a n s p o r t mechanism, as w e l l as an e f f e c t due t o r a p i d changes i n the  sodium content, o f the c e l l s .  METHODS  D i s s e c t i o n o f s i n g l e muscle c e l l s ,  i n j e c t i o n and c o l l e c t i o n o f r a d i o -  sodium, and measurements w i t h m i c r o e l e c t r o d e s have been d e s c r i b e d i n p r e v i o u s sections. latter  The f l u i d  injected contained  22  NaCl and, sometimes,  i n o r d e r to r a i s e the myoplasmic sodium a c t i v i t y .  23  NaCl, the  174  RESULTS  In  F i g . 20 i s p r e s e n t e d the time course o f the d e c l i n e i n ( a ^ ) a  three c e l l s of d i f f e r e n t sodium-free  initial  sodium content, d u r i n g immersion i n a  lithium-substituted solution.  The b e h a v i o r  d i f f e r e n t f o r c e l l s of d i f f e r e n t sodium c o n t e n t . mic.  A simple e x p o n e n t i a l f a l l o f ( j j ) a  initial  t h a t seen  v a l u e of ( . ^ ) a  A  m  plot  m  rapid  initial  Jones 1977), was  seen  and  a  i n c e l l s having a higher i n i t i a l  c e l l s h a v i n g low i n i t i a l  f a l l of ( a  by M c L a u g h l i n  v a l u e of  )  M  s i m i l a r to  i n t h e i r experiments ^  discussed i n section  i n frog  was  (% ) a  m  seen even i n  Ul  is different  from t h a t r e p o r t e d  and Hirtke (1968) and by A l l e n and Hinke (1971).  they had used a h y p e r t o n i c sodium-free  s o l u t i o n , and  However,  the i n i t i a l  behavior of  i n c l u d e d the e f f e c t s of water movement, as '  3.B.  time c o u r s e o f the f a l l  in (ajj ) a  m  i n the c e l l s h a v i n g a h i g h  sodium content c o u l d be f i t t e d q u i t e w e l l by the sum but f o r the c e l l s h a v i n g a lower or  m  having  v a l u e s of (a„ ) . Na m  T h i s b e h a v i o r of b a r n a c l e muscle c e l l s  The  in cells  f a l l , of (aji ) >  IN a ,  v  seen  i n c r a b s t r i a t e d muscle (Vaughan-  f r o g and crab muscle, a l a r g e i n i t i a l  (a„ ) Na'm  is semilogarith-  i n s n a i l neurone (Thomas 1972b) but not u n l i k e t h a t seen  s k e l e t a l muscle (White & Hinke 1976)  In  The  is qualitatively  w i t h time was  a  a lower  for  m  initial  of two  initial  exponentials,  sodium content a s i n g l e e x p o n e n t i a l  o c c a s i o n a l l y even a l i n e a r f u n c t i o n s u f f i c e d . The hazards  of 'curve p e e l i n g ' were mentioned i n s e c t i o n 4.  no r e a s o n to propose t h a t the b e h a v i o r o f the c e l l s c o n t e n t r e f l e c t s the sum likely  o f two  independent  There i s  of h i g h i n i t i a l  processes.  sodium  I t seems e q u a l l y  t h a t a s i n g l e mechanism i s o p e r a t i n g but the r a t e c o n s t a n t  p l a y i n g a dependence on the g r a d i e n t o f the c h e m i c a l p o t e n t i a l When r a t e c o n s t a n t s f o r the r a p i d e f f l u x were c a l c u l a t e d by  is dis-  f o r sodium.  'curve p e e l i n g ' ,  175  0  20  40  60  time (min.)  F i g u r e 20. F a l l o f the myoplasmic sodium a c t i v i t y upon exposure o f the c e l l to sodium-free l i t h i u m - s u b s t i t u t e d s o l u t i o n . S o l u t i o n change from normal R i n g e r ' s s o l u t i o n o c c u r r e d a t time 5 minutes. P r i o r to t h a t time, the steady v a l u e o f the myoplasmic sodium a c t i v i t y o f each c e l l i n normal R i n g e r ' s s o l u t i o n i s shown. The p l o t i s s e m i l o g a r i t h m i c . For the c e l l which s t a r t e d w i t h the h i g h e s t sodium c o n t e n t (upper c u r v e ) , i t i s shown how the ' s i z e of the i n i t i a l r a p i d f a l l was c a l c u l a t e d f o r F i g . 21, by e x t r a p o l a t i o n back t o zero time o f the l i n e a r t a i l of the c u r v e . 1  176  the v a l u e s were found t o show c o n s i d e r a b l e s c a t t e r and t o be p o o r l y c o r r e l a t e d with the i n i t i a l  value o f ( ^ ) a  a  m  I t appeared  t h a t above a c e r t a i n  t h r e s h o l d v a l u e o f (a„ ) o f about 15 mM t h e f a s t r a t e was p r e s e n t , w h i l e ^ Na m T  y  below t h i s t h r e s h o l d i t was not. was  The mean v a l u e o f t h e l a r g e r r a t e c o n s t a n t  comparable t o t h e v a l u e which d e s c r i b e s the washout o f t h e e x t r a c e l l u l a r  space  (0.15 min"-'-, SD = 0.06 min"''' f o r 9 c e l l s  f i t t e d by two e x p o n e n t i a l s ; a  s i m i l a r v a l u e was r e p o r t e d by White & Hinke 1976 f o r f r o g muscle). d i f f e r e n t s i g n i f i c a n c e o f the r a t e constant sidered  i n t h e two cases w i l l be con-  i n the Discussion.  Vaughan-Jones (1977) noted of  The  t h a t t h e change o f ( j j )  d u r i n g 15 minutes  a  a  m  immersion o f crab muscle i n sodium-poor s o l u t i o n c o r r e l a t e d w e l l w i t h the  initial  v a l u e o f (a^ ) T  i n the c e l l s .  A s i m i l a r r e s u l t was found i n  b a r n a c l e muscle, as shown i n F i g . 21.  The / s i z e o f t h e r a p i d  was  i n F i g . 20 top t r a c i n g .  c a l c u l a t e d by t h e method i n d i c a t e d  j u s t confirms  t h e i m p r e s s i o n gained  sodium-free  solution.  This  fall  really  i s 'switched o f f when  t o ca. 15 mM d u r i n g immersion o f t h e c e l l  x  i n mM  from F i g . 20 t h a t , whatever t h e i n i t i a l  v a l u e o f (ajjg^jjj above about 15 mM, t h e r a p i d the v a l u e o f (a ) f a l l s Na'm  fall'  The ' t h r e s h o l d ' v a l u e o f ( - ^ ) a  a  m  i  n  i n the  crab muscle was  about 2 mM (Vaughan-Jones 1977). A d i f f e r e n t and b e t t e r approach i s t o measure t h e i n i t i a l tracings of ( j j ) a  a  fall.  m  v e r s u s time,  to y i e l d a s i n g l e rate d e s c r i b i n g the rapid  When t h i s r a t e i s p l o t t e d versus r  the f a l l ,  a c o r r e l a t i o n i s seen  slope o f the  the v a l u e o f (a, ) a t t h e s t a r t o f Na m  ( F i g . 22).  T  The dependence i s v e r y  similar,  even i n t h e s l i g h t d i f f e r e n c e i n s l o p e f o r d i f f e r e n t c e l l s a t a g i v e n v a l u e of ( j j ) J t o t h a t d i s p l a y e d i n F i g . 17 f o r t h e e f f l u x M^ ( c a l c u l a t e d from e q u a t i o n (4) w i t h o u t c o r r e c t i o n f o r Na* ,,) versus (a„ ) . T h i s i n t u r n * cell Na m a  a  m  a  n  is  s i m i l a r t o t h e c o r r e c t e d v a l u e F i g . 16, as noted In  i n s e c t i o n 5.  theory, t h e r e l a t i o n s h i p between t h e two i s V * ^ ^ ^ m = A-M^ , a  m  a  177  (o  N o  )  m  (mM.)  F i g u r e 21. S i z e o f the r a p i d f a l l i n the myoplasmic sodium a c t i v i t y upon exposure o f the c e l l to sodium-free l i t h i u m - s u b s t i t u t e d s o l u t i o n (see F i g . 20 and t e x t ) , v e r s u s the steady v a l u e o f the myoplasmic sodium a c t i v i t y i n the c e l l p r i o r to the change from normal R i n g e r ' s to sodium-free s o l u t i o n . One data p o i n t was excluded from the l i n e a r r e g r e s s i o n r e p r e s e n t e d by the l i n e , and i s shown i n p a r e n t h e s e s .  178  0.7 0.6 c E  2  0.5  E  0.4  o  0.3 3 0.2 0.1  i_ 10  20 Na'm  30 (mM.)  40  F i g u r e 2 2 . Rate o f f a l l o f the myoplasmic sodium a c t i v i t y immediately a f t e r exposure o f the c e l l t o sodium-free l i t h i u m - s u b s t i t u t e d s o l u t i o n , versus the s t e a d y v a l u e of the myoplasmic sodium a c t i v i t y i n normal R i n g e r ' s s o l u t i o n p r i o r to the change to sodium-free s o l u t i o n . The p o i n t i n parentheses i s from the c e l l excepted i n F i g . 21. C i r c l e s ; c e l l s d i s