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Transmembrane and intracellular distrubution of chloride and potassium in single striated muscle fibers… Gayton, David Charles 1970

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THE TRANSMEMBRANE AND INTRACELLULAR DISTRIBUTION OF CHLORIDE AND POTASSIUM IN SINGLE STRIATED MUSCLE FIBERS OF THE GIANT BARNACLE  by  DAVID CHARLES GAYTON B.Sc,  U n i v e r s i t y o f B r i t i s h Columbia, 1 9 6 6  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY i n t h e Department o f Anatomy  We a c c e p t t h i s t h e s i s as c o n f o r m i n g t o t h e r e q u i r e d standards  THE UNIVERSITY OF BRITISH COLUMBIA September, 1 9 7 0  In  presenting  this  an a d v a n c e d d e g r e e the I  Library  further  for  agree  in p a r t i a l  fulfilment  of  at  University  of  Columbia,  the  make  it  freely  that permission  this  representatives. thesis  for  It  financial  for  of  gain  Anatomy  The U n i v e r s i t y o f B r i t i s h V a n c o u v e r 8, C a n a d a  for  extensive by  the  Columbia  shall  not  the  requirements  reference copying of  Head o f  is understood that  written permission.  Department  British  available  s c h o l a r l y p u r p o s e s may be g r a n t e d  by h i s of  shall  thesis  I  agree  and this  be a l l o w e d  that  study. thesis  my D e p a r t m e n t  copying or  for  or  publication  w i t h o u t my  i  ABSTRACT  The  initial  transmembrane striated The  bution  and i n t r a c e l l u l a r  muscle  study  fibers  +  than predicted  i s probably  the  resting fiber.  analytic free  the  of C l ~ i n single  barnacle,  Balanus  t h e transmembrane o f t h e Donnan  t o measure C l  -  relation.  the a c t i v i t y  activity  from the Nernst  distributed passively  was j u s t  equation, across  i n t h e myoplasm.  that Most  were of C l  i n  slightly  i n d i c a t i n g that  t h e membrane o f  Comparison of the electrode  r e s u l t s suggested  nubilus.  distri-  s e n s i t i v e Ag-AgCl microelectrodes  The m y o p l a s m i c  Cl  is  of the giant  into single fibers  myoplasm.  higher  distribution  and a c o n s i d e r a t i o n  Chloride  the  t h e s i s was t o s t u d y  was e x p a n d e d t o i n c l u d e  of K  inserted  aim of t h i s  results  with  less than h a l f of the f i b e r C l ~ of this  extra  C l  -  was l o c a t e d i n  14 an  extracellular  and  a Cl  Electron large the  d e t e r m i n e d , by  washout method, t o occupy micrographs  clefts  indicate that  and s m a l l e r  tubules  C-sorbitol  about this  penetration  5% o f t h e f i b e r  space  volume.  i s comprised of  which penetrate  deeply  into  fiber. In  Cl  a second  exchange  solutions results about free  space,  with  and t h e o u t f l o w reduced  indicate that  equally  series of experiments,  of i n t r a c e l l u l a r  C l " concentrations the I n t r a c e l l u l a r  bound  or compartmentalized.  were  Cl  fraction  exchanging  The  divided which i s  fraction  Furthermore,  of  into  studied.  C l " c a n be  between a r a p i d l y exchanging  i n t h e myoplasm and a s l o w l y  either  the kinetics  which i s  the free  Ii  myoplasmic  Cl  appears  intracellular  In K  +  of  and  Cl  t o be  series  i n t h e myoplasm  e q u i l i b r i u m were  F i b e r s were  was  i n three  ways-:  and  +  but  x  [Cl]  [ C l ] and o  osmolarity for  muscle also 45%  K  were  fiber  o  +  that  K  +  same  i n fibers  the  [C1]  constant; results  that,  under  water  Q  K  w  a  varied  s  0  rigorous  proof  e q u i l i b r i u m cont h e membrane  earlier  which  , [ C l ] , and o' o'  relation.  a  micro-  v a r i e d but  [ ^  provide most  states  solution  were  [K]  across  from  from  experiments  The  of a  results  excluded  from  f i n d i n g s from  this  smaller  fraction  are described  distribution  barnacles  I t i s suggested  that  and  i s bound  of K  collected  +  and N a  i n two  thatalthough  or compartmentalized  differences i n the results  variation  i n Ringer  constant;  t o t h e Donnan  two  the i n t r a c e l l u l a r  +  of i o n sensitive  The  i s excluded  that  Na  kept  at d i f f e r i n g  of  of the  water.  Finally,  plus  kept  the a c t i v i t i e s  the f i n d i n g . t h a t C l " i s apparently  intracellular  locations.  fibers  and  are d i s t r i b u t e d  of the i n t r a c e l l u l a r  laboratory  o  theory  Cl  [K]  [Na]  [Na]  according  confirm  of single  equilibrated  increased. held  and  +  [K]  [K]  the widely  ditions,  45% o f t h e  about  of experiments,  m e a s u r e d by means  electrodes.  [K]  from  water.  the t h i r d  modified  excluded  i n t h e amount  could  o f each  be  due  cation that  which +  i s not the  different  t h e sum may  show  be  t o an  of the  K  +  constant, inverse  i s bound  or  compartmentali zed.  The  results  of this  study  are i n accord  with  recent  iii  findings i n this  and o t h e r l a b o r a t o r i e s w h i c h i n d i c a t e  monovalent i n o r g a n i c  i o n s and w a t e r a r e d i s t r i b u t e d  g e n e o u s manner i n t h e s t r i a t e d Cl  of the b a r n a c l e muscle  muscle  that  must be e i t h e r bound t o p r o t e i n s plasmic reticulum; t o s e l e c t one  fiber.  intracellular  or accumulated i n the  at p r e s e n t t h e r e i s not s u f f i c i e n t  sarcoevidence  or the other (or both) of these p o s s i b i l i t i e s .  i s o r d e r e d by t h e c e l l p r o t e i n s  from water t h a t the n e g a t i v e l y  i n a hetero-  i s not f r e e i n t h e myoplasm  I t i s proposed t h a t the f r e e myoplasmic C l water that  The  that  i s e x c l u d e d from and p a r t i a l l y  l i e s w i t h i n the e l e c t r i c a l f i e l d charged  proteins.  that  excluded  surrounds  iv . TABLE OF CONTENTS  CHAPTER I  THE PHYSICAL STATE OF THE CELL  CHAPTER I I  THE DISTRIBUTION ,OF CHLORIDE AND POTASSIUM IN THE CELL A. B. C. D. E. F.  CHAPTER I I I  CHAPTER IV  CHAPTER V  B o y l e and'Conway -•The. Donnan E q u i l i b r i u m More Recent Work on the R e l a t i o n between Transmembrane I o n D i s t r i b u t i o n and Membrane P o t e n t i a l . . . The D i s t r i b u t i o n o f C h l o r i d e i n t h e Muscle F i b e r The D i s t r i b u t i o n o f P o t a s s i u m (and Sodium) i n t h e Muscle F i b e r The S t a t e o f Water i n t h e Muscle F i b e r .. Summary . . . . ..........  10 10 16 26 27 32  ELECTRODE AND CHEMICAL DETERMINATION OF FIBER CHLORIDE .•  40  A. B. C. D.  40 4l 49 60  Introduction Methods Results Discussion  .•  ;  THE HETEROGENEOUS DISTRIBUTION OF CHLORIDE IN THE BARNACLE MUSCLE  66  A. B. C. D.  66 67 73 79  Introduction Methods Results Discussion  TRANSMEMBRANE POTASSIUM AND CHLORIDE ACTIVITY GRADIENTS . • A. B. C. D.  CHAPTER V I  1  Introduction Methods Results Discussion  THE DETERMINATION OF a MICROELECTRODE STUDIES A. B.  ,  M  38  88 88 90 97 107  FROM POTASSIUM 112  Introduction . 112 V a r i a t i o n s i n I o n i c Content o f B a r n a c l e s from D i f f e r e n t Sources 113 C. I o n i c Changes o f F i b e r s i n H y p e r t o n i c Solution 118 D . Discussion ^ ....... 122  V  CHAPTER- V I I  CONCLUDING DISCUSSION  BIBLIOGRAPHY  ......  APPENDIX ' I  ............  .  126 132  t  145  vi L I S T OF TABLES  TABLE I  C o m p o s i t i o n o f B a r n a c l e .Ringer s o l u t i o n s  (mM)..  TABLE I I  C o m p a r i s o n , o f C o t l o v e and S a n d e r s o n methods of c h l o r i d e a n a l y s i s i  TABLE I I I  The m y o p l a s m i c a c t i v i t y (aQ]_) and t o t a l . concentration [ C 1 ] of chloride i n single muscle f i b e r s  51  TABLE I V  The e x t r a c e l l u l a r s p a c e o f s i n g l e m u s c l e f i b e r s by t h e c o n s t a n t p r o d u c t c h l o r i d e w a s h o u t method  54  TABLE V  A c o m p a r i s o n o f t h e 3 methods o f s a m p l e p r e p a r a t i o n f o r 36ci~ c o u n t i n g  ; .  TABLE V I I TABLE V I I I  49  m  T  TABLE V I  42  The e f f e c t o f d e c r e a s e d [ 3 6 c i ] ^ exchange  [Cl]  0  • 71  on [ C l ] i and 72  S i z e and e x c h a n g e r a t e s o f C l c o m p a r t m e n t s i n s i n g l e f i b e r s from b a r n a c l e muscle -  80  K and C l a c t i v i t y c o e f f i c i e n t s f o r some b a t h i n g s o l u t i o n s c a l c u l a t e d f r o m K m i c r o e l e c t r o d e and C l m i c r o e l e c t r o d e measurements 94 +  -  +  -  TABLE I X  K a c t i v i t y a n d c h e m i c a l a n a l y s i s on s i n g l e fibers equilibrated i n solutions with [ K ] x [Cl] constant o C l ~ a c t i v i t y a n d c h e m i c a l a n a l y s i s on s i n g l e f i b e r s e q u i l i b r a t e d i n s o l u t i o n s with [K] x [Cl] constant ,?..... o K a c t i v i t y a n d , c h e m i c a l a n a l y s i s on s i n g l e fibers equilibrated In solutions with elevated [K] and c o n s t a n t [ C l ] o o C l ~ a c t i v i t y on s i n g l e f i b e r s e q u i l i b r a t e d (20 h r ) i n s o l u t i o n s w i t h e l e v a t e d [ K ] a n d constant [ C l ] ? +  0  TABLE X  TABLE X I  TABLE X I I  TABLE X I I I  TABLE X I V  97  99  +  103  104  K and C l ~ a c t i v i t i e s on s i n g l e f i b e r s e q u i l i b r a t e d (20 h r ) i n s o l u t i o n s w i t h h i g h KC1 and c o n s t a n t [Na]  107  Summary o f i n t e r n a l i o n c o n t e n t o f s i n g l e muscle f i b e r s from giant b a r n a c l e s  115  +  vii  TABLE XV  TABLE X V I TABLE X V I I  The f r a c t i o n s , o f b o u n d N a and water i n d i f f e r e n t groups o f barnacles  117  A c o m p a r i s o n o f t h e same h y p e r t o n i c on t h e t w o t y p e s o f l o c a l b a r n a c l e  119  +  experiment  The a c t i v i t y c o e f f i c i e n t s o f . C l " , N a and i n a number o f b a r n a c l e R i n g e r s o l u t i o n s +  3  K  +  147  viii  L I S T OF  Figure Figure  Figure  1 2  3  The m a i n , s t e p s microelectrode  i n the  FIGURES  construction, of  a  Ag-AgCl .•  Diagram of a cannulated inserted microelectrode  muscle  fiber  with  an  ;  46  C o m p a r i s o n between the. c o n c e n t r a t i o n o f C l t h e f i b e r w a t e r , and t h e a c t i v i t y o f C l in m y o p l a s m i n f i b e r s s o a k e d f o r up t o 30 m i n constant product s o l u t i o n s  in the in  -  Figure  Figure Figure Figure  4  5 6 7  Figure Figure  Figure  Figure  8 9 10  11  12  53  Time c o u r s e o f the e q u i l i b r a t i o n o f " ^ C - s o r b i t o l i n the bath w i t h the e x t r a c e l l u l a r space of single fibers  56  A c r o s s - s e c t i o n of muscle  the  57  A c r o s s - s e c t i o n of muscle  the  R e l a t i o n between b a t h and t h e C l  -  Figure  43  The l o s s solution  surface interior  the C l content  of  the  of  barnacle ....  the  barnacle 59  concentration of the f i b e r  of  the 74  of C l ~ from f i b e r s bathed i n Ringer i n w h i c h t h e C l " has b e e n r e p l a c e d  Exchange of i n t r a c e l l u l a r C l ~ w i t h Cl f i b e r s bathed i n normal Ringer s o l u t i o n Comparison of the l o s s of f r o m f i b e r s b a t h e d i n NO-, exchange of i n t r a c e l l u l a r  ...  in 76  intracellular Cl R i n g e r and the C l w i t h 3orji -  75  78  -  R e l a t i o n between transmembrane K and C l g r a d i e n t s and t h e r e s t i n g p o t e n t i a l f o r f i b e r s e q u i l i b r a t e d i n constant product s o l u t i o n s ....  100  R e l a t i o n between transmembrane K and C l g r a d i e n t s and t h e r e s t i n g p o t e n t i a l f o r . f i b e r s equilibrated i n solutions with elevated [K] and c o n s t a n t [ C l ] '.'  105  +  +  Figure  13  The e l e c t r i c a l , p o t e n t i a l s o f a K a C l " e l e c t r o d e i n KC1 s o l u t i o n s  electrode  Figure  14  The e l e c t r i c a l , p o t e n t i a l s o f a N a a C l electrode i n NaCl s o l u t i o n s  +  -  electrode  and and  G L O S S A R Y OF  X = C l . K,  a  ( a  X m  ( a  X o  SYMBOLS  o r Na  activity  of  the  i o n i n the" m y o p l a s m  activity  of  the  i o n i n the bathing  fraction  of  the  fiber  water  that  i s bound  m  fraction myoplasm  of  the  fiber  water  that  i s free  o  fraction cellular  of the f i b e r space  water  that  i s i n t h e extra-  }  }  B  a a  solution  i nt h e  B-£  c o n c e n t r a t i o n o f t h e bound o r c o m p a r t m e n t a l i z e d i o n per kilogram of f i b e r water  E  membrane  m  potential ^  E-£  chemical p o t e n t i a l o f the i o n across membrane; or the potential recorded sensitive electrode  F  Farraday's  constant  Y  m  mean a c t i v i t y  Y  Q  mean a c t i v i t y c o e f f i c i e n t  Y-£  individual solution  P  the fiber on t h e i o n  coefficient  o f t h e myoplasm of the bathing  activity coefficient  solution  o f t h e I o n i nt h e  pressure  P^-  permeability  R  gas  T  absolute  V  volume  [X].  concentration intracellular  (X)  c o n c e n t r a t i o n - o f t h e i o n t h a t i s free, i n t h e myoplasm (measured w i t h an i o n s e n s i t i v e microelectrode)  m  constant  of the i o n  constant temperature  of i n t r a c e l l u l a r i o nper kilogram water  of  concentration  of  the  ion  in  t o t a l c o n c e n t r a t i o n of the kilogram of f i b e r water  the ion  bathing i n the  solution  fiber  per  ACKNOWLEDGEMENTS  I critic,  and f r i e n d ,  rewarding Mrs. Mr.  R. A l l e n ,  Canada  I also  thank  M r s . Mary Weir,  a n d M r . T. W i l s o n  assistance;  electron  helpful  h a s made t h e l a s t  Mahlberg,  J . Lewis,  their  t o D r . J.A.M.  and enjoyable.  Sheila  technical  Dr.  am g r a t e f u l  D r . W.  Hinke,  who a s m e n t o r ,  four years  both  Mrs. Irene the late  for their  very  M r . C.  W e b b e r a n d D r . A. S p i r a f o r  D r . S. M c L a u g h l i n ,  for their  a n d D r . V. P a l a t y  and t h e M e d i c a l Research  financial  Lemon,  capable  microscopic studies of the barnacle  discussions;  Ingraham,  support.  muscle; for their  Council of  CHAPTER  I  THE P H Y S I C A L S T A T E OP THE  Over t h e l a s t have been advanced properties continues or  of the l i v i n g t o be f a v o u r e d  together  apparent feature  of  to describe  more c o r r e c t l y , g r o u p  classed  the  century,  of the l a t t e r  alone  The o l d e r  of theories  and t h a t  ignore  o r d i s c r e d i t t h e arguments who q u e s t i o n e d  cytoplasm  postulated is  does  The u n i f y i n g a l l of  by t h e ' s o r p t i o n i s t s ' . of the c e l l  membrane a n d a h e t e r o g e n e o u s  o f a membrane i s f o r the  and experiments  existence  they  tended  of these  o f t h e membrane  raised are s t i l l  are demonstrating  i n fact possess  a more c o m p l e x v i e w  to explain  many b i o l o g i s t s h a v e  the v a l i d i t y  of the questions studies  theory,  restrictively  developed  as t h e e v i d e n c e  to  the  (often  the existence  membrane h a s i n c r e a s e d ,  Recent  The s e c o n d  Is the postulation that  a cell  answered.  theory'  c a n be a t t r i b u t e d t o t h e p r o p e r t i e s  Unfortunately,  e v e n t h o u g h many  'membrane  i n t h e membrane t h e o r y .  theories  theories  and p h y s i o l o g i c a l  by most b i o l o g i s t s .  of  investigators  divergent  the physical  cell.  of the c e l l  the cytoplasm  unnecessary.  two w i d e l y  as t h e s o r p t i o n . t h e o r y ) ,  inconsistencies  properties  CELL  theory  un-  unequivocally  that  some o f t h e p r o p e r t i e s Emerging  from these  requiring a  studies  semi-permeable  cytoplasm, both with  unusual  properties.  The formulated de  Vries  basic  p r i n c i p l e s o f t h e membrane t h e o r y  at the turn  (1888)  of the century.  observed  that  t h e volume  Pfeffer  were  (1877)  of the vacuole  and of a  plant the be  cell  was i n v e r s e l y p r o p o r t i o n a l t o t h e o s m o t i c  bathing  solution.  surrounded  totally 1902a)  the  b y a membrane p e r m e a b l e  impermeable extended  osmotic  principles  and s i m i l a r  of a strong  macromolecules  The universal  first  discovered driven  Na  maintain  +  t h e membrane  the  ofthe  a  limiting  i n t o and out o f  the properties  properties  sugars  against  chemical  gradients  when i t was  t o N a , an energy +  t h e membrane was p o s t u l a t e d t o Na  +  concentration  concentrations  matters,  almost  experimental  Por instance,  (Dean 1 9 4 1 ;  plicate  enjoys  o f t h e membrane  as more a n d more  Krogh  with  S i m i l a r mechanisms  low i n t r a c e l l u l a r  properties  physico-chemical  membrane became a n a c t i v e o r g a n e l l e suming a c t i v i t i e s .  Prom  on t h e c e l l s ,  of materials  I s permeable  t h e low i n t r a c e l l u l a r gradient  cells.  were e s t a b l i s h e d :  explanation.  pump o r c a r r i e r . i n  electrochemical  and animal  two p r i n c i p l e s s t i l l  even though  required  that  a l a r g e , number o f  and n o n - e l e c t r o l y t e s ) .  have been a l t e r e d c o n s i d e r a b l y observations  (1895,  Overton  e l e c t r o l y t e s o l u t i o n (even though i t  of these  acceptance  or  s t u d i e s , two fundamental  a cytoplasm with  t o those  must  t h e membrane m u s t be l i p i d i n  c o n t r o l s t h e passage  cytoplasm;  contains  substances.  the permeability  o f t h e membrane t h e o r y  membrane, w h i c h  similar  that  cell  t o water but r e l a t i v e l y  to include  to categorize  From t h e s e  the plant  e f f e c t s o f the substances  membrane a n d d e d u c e  nature.  the  studies  that  and a v a r i e t y o f p l a n t  osmotic  O v e r t o n was a b l e cell  t o water-soluble  these  substances  relative  P f e f f e r concluded  strength of  (Ling 1962),  1946).  intrinsic  + +  energy  and M g  can pass  a  large  Thus t h e  are necessary  of Ca  and amino a c i d s  against  + +  con-  to explain .  T o com-  into the c e l l  necessitating the  3 existence  of  reviewed-in (1969))by  inward recent  These  geneous The  strong  no  evidence  The  studies  of  the  of  Boyle  of  and  be  cell  (1941) the  i f the  free  support  the  (1890)  surface  g e n e r a t e d by  of  the  This  original  ions  and  this possibility  extracted  his  proteins.  that  and  "very  that by  of  the  found  little,  the  osmotic  assuming  completely  that  the  intracellular  speculated  dissolved  the  concentration  by  i960;  the  the  were  cell  assumed  amply Caldwell  also  membrane.  p o t e n t i a l at cell  gradients  of  the  homogeneity.  electrical  a p o t e n t i a l across ion  of  by  are  already  biologists  properties  that  that  distribution  water  Robinson  intracellular  century,  vapor  further strengthened  Conway 1957; of  found  famous w a t e r  Numerous p a p e r s ,  electrical  inorganic  biologists.  f r o g m u s c l e membrane i s d e s c r i b e d  concept  m i g h t be  v i e w was  homo-  for the  membrane.  c y t o p l a s m but  to  are  is a  basis  described  muscle  cell  amongst  concluded  best  considered  the  considered  'bound'"  when t h e y  1951;  turn  is  are  solution.  the  the  investigating  cell  muscle  water.  (Hodgkin  At  Ostwald  of  of  cytoplasm  published He  still  prevalent  (1929)  f r o g muscle.  across  i n one  1968),  are  cation binding  r e s t i n g muscle  Conway  Cl  reviewed  still  (1930)  constituents  total  and  the  is  Lakshminarayanaiah  capacity  that  Hober  Hill  water  Donnan r e l a t i o n to  cited.  of  the  soluble the  w i t h i n the  significant  study  any,  properties  w h i c h were the  f o l l o w i n g year.  properties  +  t o be  and  p r o t e i n - c a t i o n i n t e r a c t i o n i n the  pressure  K  many o t h e r  (membrane t r a n s p o r t  (1968)  Stein  solution is also  have been  of  in  and  systems  second p r i n c i p l e  free  osmotic  belief  if  b o o k s by  most b i o l o g i s t s The  transport  the  membrane i n the  same  manner t h a t branes.  he h a d o b s e r v e d  Since  appreciable  K  was t h e o n l y  +  that  mainly  according  boundary  potentials: E  t o Nernst's  = RT  where E  m  of K  Parraday's muscle  following  changes  equation  Hodgkin and Katz  In [K]  (1).  -  (Boyle  19^9 ) •  basic tenets  observations  and N a , t h i s +  o f these  aspect  that the frog  in  hyposmotic  solutions,  to  be o s m o t i c a l l y i n a c t i v e .  I n Chapter  (1922) c l a i m e d  early  ions  theory  on t h e r e s t i n g  Goldman  19^3;  to question  F o r example,  m u s c l e was n o t a p e r f e c t  about  20%  of the fiber  Overton  will  II.  l e d some i n v e s t i g a t o r s  must be b o u n d t o t h e l a r g e m o l e c u l e s Rubner  that the  o f t h e membrane t h e o r y  o f t h e membrane t h e o r y .  (1902a) f o u n d  o f E^ o f  were i n q u a l i t a t i v e  a n d C o n w a y 19^1; This  R i s  and F i s  When i t b e c a m e a p p a r e n t  to C l  d i s c u s s e d more t h o r o u g h l y Early  respectively,  temperature,  to include the effects  membrane p o t e n t i a l  the  and t h e c e l l  McDonald's rough measurements  membrane i s p e r m e a b l e  was m o d i f i e d  be  N ( 1 )  T i s the absolute  constant.  agreement w i t h cell  (1892) f o r p h a s e  n r r 1  i n the bath  +  gas c o n s t a n t ,  frog  across the  i s t h e membrane p o t e n t i a l , [ K ] a n d [ K ] . a r e t h e c o n ^ ' o 1  centrations the  be  l n  F -  M  equation  to small  must  concentration gradient  +  at  and McDonald  a n d t h e membrane p o t e n t i a l  by t h e K  membrane  (1902, 1912)  t h e membrane must be p e r m e a b l e  but not anions  generated  inorganic i o n i n the c e l l  concentration, Bernstein  (1900) r e a s o n e d cations  i n a r t i f i c i a l s e m i p e r m e a b l e mem-  claimed  t h e same f o r t h e 23%  osmometer;  water  that this  o f the muscle of fiber  Overton  appeared water  fiber. water  that  did  not  freeze  questioned that  the  cytoplasm could  Moore and  onto  in  Rubner's  the c e l l even  cell  i f this  Overton's  i t need  inactive.  exactly  stituents  not  Hill  and  t o be  virtually  Several a model o f the  were  chemistry. Jong  pension  based  Bungenberg  de  a t -20°C  had  that  20%  r u n down a n d  water become  r e s t i n g muscle  can  t h e known s o l u b l e  i n i t s 'free' water.  these constituents  with  30  Hill,  Kruyt  years.  of  (1930) a n d  of hydrophilic  s e p a r a t e i n t o two  They p o i n t e d  most  out t h a t  s u c h as w a t e r  of the c o l l o i d s ,  molecules, resemble  Lepeschkin  ability  a  sus-  phases,  c a l l e d the coacervate.  many o f t h e p r o p e r t i e s  c o n t e n t and  colloid  Bungenberg  certain conditions,  can  Hill's  formulated  s p e c i a l knowledge  and  con-  not  o f t h e membrane t h e o r y , to  of coacervates,  to concentrate or exclude  the properties  (1930) a n d F i s c h e r a n d S a u r  of cytoplasm.  (1938) a l s o  be  There  are  (1932) o b s e r v e d t h a t , u n d e r  containing  Hill  (1930) a l s o c o n c l u d e d  assuming  their  colloids  and  somehow d i f f e r e n t i a t e d  unchallenged for close  Jong  effects  the  dissolved  on  the  argued  chemists, contemporary  cell  (1930)  exclude ions.  Amongst p r o p o n e n t s  went  enhanced  Kupalov  a c c o u n t e d f o r by  1907;  necessarily  l a t i t u d e f o r supposing that  arguments  small  be  selective  Moore  that  also  suggestin  However, H i l l  the grounds  i n fibers that  of muscle  dissolved.".  one,  ( F i s c h e r and  "the osmotic pressure of frog's  almost  de  on  r e s u l t s but  osmotically  no  colloids  on w a t e r m i g h t  water  is  to ion distribution,  f r a c t i o n of water  cell,  investigators  c o n t r o l i o n a c c u m u l a t i o n by  findings  represented  that  A number o f e a r l y  N e u s c h l o s z 1926).  colloids  the l i v i n g  confirmed  the  1908;  Roaf  questioned  that  C.  t h e membrane a p p r o a c h  adsorption  of  a t -20  proposed  6 phase models which  dispensed  have been developed  into  Russian  (Nasonov  the of  cytologists  cell  t o be  colloids  bound t o to  a  of  the  opposite  cell  water  cell'Colloids. a is  matter  of  realm  1962;  Troshin  charge). and  (one  The  acts  cytoplasm  their  selective by  fairly  the  membrane.  sorption theory  as  s o r p t i o n by metabolic  old, this  water  the  has  on  consider  coacervation  poor  solvent  their  of the  of  solubility  binding to  This  living  advanced  in the is  +  selectivity cell.  beyond  the  of speculation.  has  induction  been developed  by  hypothesis.  Ling  exchange r e s i n , In  They  for instance, K  A much more r i g o r o u s d e s c r i p t i o n cell  of  i s almost a l l  colloids.  not  ideas  concentration  chemical of,  enzymes  theory  1966).  The  These  a group  a relatively  depends  adsorption or  by  f o r m e d by  cell  S e l e c t i v e accumulation  maintained  Although  cell  s o l u b l e i n water.  i n the and  the  coacervate  colloids  normally  small molecules the  complex  cell  substances  the  with  such  water  the  a matrix  approaches  Ling  In t h i s  between  c a t i o n s and  fixed  1926).  Taking  account  forces  between anion  numbers anion,  (up  and  to three)  of  sites  i n the  According  the  to  five  alkali  matrix  at  L i n g , the  anion  the  and  various and  anion  sites  cation  charged  and  between  sequences  i n the  the  field  of  (Bjerrum  allowing for  of  proteins.  freedom  i s favoured  cations with  various values  a  strong association  attractive  selectivity earth  to  rotational  environment, sites  cation,  cell  non-membranous  association-  composed o f  of water molecules  L i n g p r e d i c t e d the  ation  compares  translational  unity.  into  (1962) i n h i s  exchange m a t r i x  the  of the  repulsive integral  cation  and  f o r the fixed  associ-  anion  strength.  cytoplasm  of  the  7 resulting  cell  binding.  Transient  selectivity Ling it  except  changes such  i t s binding This  bulk-phase  strength which  i n field  as o b s e r v e d  during  a l 1957;  to  the selective  (with  theoretical  forK  (1966) b u t  i s higher  +  f o r Ling's  but uptake  arguments  I960, 1962).  Eisenman  binding  glasses  approach. anion  potential. claims  than i n  claim that  of other  small  up-  molecules  limited.  et  Ling's  +  account f o r  an a c t i o n  f o rbinding  r e i n f o r c e d by t h e f i n d i n g s o f Eisenman's  selective  K  i n s t r u c t u r e and f u n c t i o n  account  limited  favours  s t r e n g t h would  o f a membrane  specificity  should  i s surface  +  Ling's are  a field  the presence  phase.  of K  have  the rest of the c e l l  that  bulk  take is  changes  acknowledges  i s like  the  must  of the a l k a l i  group  earth  of various  on g l a s s e s field  (Eisenman  thermodynamic cations  y i e l d e d t h e same s e l e c t i v i t y Experiments  sites  Their  specificity  to cation  sequences  of various  strengths)  approach  have  as  composition confirmed  the  theoretical predictions.  Several totally  bound  has argued  ions  phase, which in  a three  i n muscle;  cellular  that  phase  which  should  system  the bulk  and K  +  Since  i n muscle  (1957>  1959)  to explain the distribution phase,  i s i n equilibrium with phase,  or  be m e n t i o n e d .  Simon and co-workers  resembling  of the fiber. magnetic  and N a , has considered +  have p a r t i a l l y  of the water  an e x t r a c e l l u l a r  and an ordered comprises  phase  i n t e r p r e t i n g h i s nuclear  water  most  ( s e e E r n s t 1963).  have proposed of  laboratories that  r e j e c t e d t h e membrane t h e o r y  1930j E r n s t  is  other  a free  the extracellular  Ling's  cell  Recently,  resonance  the cell  intra-  phase,  Cope  studies  as a n o n - l i q u i d  (1967a), of  cell  phase  consisting  of macromolecules This  the  small  membrane t h e o r y  This  i s not  evidence The  with  the  capacitance  micrographs model of protein  the  the  presence  of  1964;  thickness  were a l s o  a  the  lipid  1965;  1968).  Recent work  Mueller  and  Rudin  on  artificial  (1963,  b r a n e s p o s s e s s most  Korn  of  1967,  the  most  the  biologists. of  the  membrane.  revealed  C u r t i s and  giant  bilayer  questioned  weight  1957)  p r e d i c t e d by  matrix.  have  a versatile  squid  a (1938)  Cole  axon.  The (1935)  Danielli-Davson  sandwiched between  two  intensive research, .further  membrane s t r u c t u r e has  Kavanau  the  (Robertson  However, d e s p i t e  the  of  consistent with  membrane as  ice-like  i n f l u e n c e on  considers  m e a s u r e m e n t s on  layers.  detailing (Lucy  little  s u r p r i s i n g when one of  i n as  i n v e s t i g a t o r s who  electron micrographs  partition from  of  have had  i n favour  first  group  embedded  been mostly  1966;  1966;  Finean  lipid  1968),  electrical  speculative  bilayers,  Bangham  developed  indicate that  these  properties  the  of  by  memcell  membrane. Ironically, of the  anion  may  prove  membrane t h e o r y .  lishment of  sites  Ling's  of  the  specificity  standing action  of  the  role  very  As  field  provides  a key  to  of  ionic  excretion which  terms  of  binding  biology  elementary  force  fields  involved.".  Similarly,  B u n g e n b e r g de  sorptionists  draw h e a v i l y , has  only  pointed  the  be  the  Jong, out  of  estab-  s i n e qua an  non  under-  resting potentials,  ionic  of  as  toward  s u c h phenomena as accumulation,  context  s t r e s s e d , "The  strength  u l t i m a t e l y can  specificity  w i t h i n the  (i960)  Eisenman anion  origin  on  important  of  potentials, ionic  the  reasoning  exclusion, accounted atoms and  and for  molecules  f r o m whose work that  our  in  the  understanding  9 of  the structure  the  and f u n c t i o n  cytoplasm w i l l  chemistry  depend  of the c e l l  membrane a s w e l l  on o u r u n d e r s t a n d i n g  ( B u n g e n b e r g de J o n g  19^9;  Booij  1956).  find  an a l t e r n a t i v e t o t h e membrane t h e o r y ,  contributed  and t h e s o r p t i o n i s t s , i n t r y i n g t o  s o l u t i o n i s on f i r m e r  membrane I t s e l f .  about  the complexity  naive  to dismiss  electrical)  the p o s s i b i l i t y may  Even though both  theory  appear  equally  fifteen  naive  that  they  water  more c o m p a t i b l e  chemistry  cell  and  Interinorganic  and a s s o c i a t i o n - i n d u c t i o n have  stimulated  studies  have  a few  of the cytoplasm. attempted t o  necessarily  is  (and  the possible  without  these  i t i s  t h e membranes o f  t h e p h y s i c a l and p h y s i o l o g i c a l p r o p e r t i e s  from  knowledge  concentration  the physical nature many  homo-  rejection of  structure,  to ignore  the sorption  years,  i s a  of our present  explain  Emerging  i n fact  of t h e second  their  macromolecules with  untenable,  to reconsider  the last  than  e x i s t across  I t i s equally  ions.  In  have  the c e l l  o f i n t e r n a l membrane  of i n t r a c e l l u l a r  biologists  grounds  In the light  gradients  organelles. actions  may  and t h e s o r p t i o n i s t ' s s c e p t i c i s m  a s s u m p t i o n by t h e membrane t h e o r i s t s t h a t  the  de  toi t .  Ling  geneous  colloid  and Bungenberg  Jong  Thus, L i n g  of  as  of the  cell  p o s t u l a t i n g homogeneity  of the cytoplasm.  studies  of the c e l l  with  i s a physiology  the structure  of the cytoplasm.  of the c e l l  which  and t h e  10 CHAPTER I I THE D I S T R I B U T I O N OF C H L O R I D E AND STRIATED  The  initial  of the C l  of  barnacle;  the giant  -  thesis  i o n i n single  was t o s t u d y t h e  striated  muscle  fibers  first,  the distribution  of C l  across  membrane a n d s e c o n d ,  the distribution  of C l  within  the  fiber  the  fiber.  the  distribution  Of n e c e s s i t y ,  examination these  MUSCLE  aim of t h i s  distribution  P O T A S S I U M I N THE  of K  this  across  +  w o r k was e x p a n d e d t o i n c l u d e t h e membrane a n d a c a r e f u l r e -  o f t h e Donnan e q u i l i b r i u m .  topics  i s given  i n this  chapter  A detailed with  review of  emphasis  on  skeletal  muscle.  A.  Boyle  a n d Conway - The Donnan For  pletely as  excluded  accurately  washed  cellular  findings volume with  Cl  space  see a l s o  space  increased fiber  into  isotonic  be u s e d  that  that  of frog  was  sucrose  com-  reported  that,  rapidly  solution.  t o be i n t r a c e l l u l a r .  He  1936)  confirmed  Fenn  Overton's  t h e C l ~ s p a c e was 1 4 . 7 % o f t h e fiber.  This  Hermann (1888) sartorius  was i n a g r e e m e n t h a d made o f t h e  muscle  from  frozen  Fenn e t a l observed  that  the C l "  t o 33% o f t h e f i b e r  i n Ringer  (1902b)  Cl  as a measure o f t h e e x t r a -  by Fenn  dissected  of the muscle.  that  m e a s u r e , a l l o f t h e N a C l was  review  estimate  thought  Overton  none a p p e a r e d  of the freshly  sections  the-  could  since  t h e 1^.5%  space  muscle  and e s t i m a t e d  extracellular  of  as he c o u l d  that  a l (193^;  years, biologists  from t h e c e l l .  out o f frog  suggested  et  many  Equilibrium  solution.  volume  They  following  attributed  cross  soaking  this  increase  11  to  an  entry  changes little  of  i n anion  1924;  damaged  fibers. i n the  p o t e n t i a l of  or  no  anion  the  studies  investigators. Cs ,  Rh ,  +  of  hydrated  radii  measuring  Bernstein  potential  change was  only (1)  of  biologists  favoured  favour  the  i s free  of  had  Anson  showed t h a t  their  K'  1905;  about  but  considered  the  of  (Fenn 1936),  fibers  of  fold  cations  Sugi  change that  1934)  found  was  a  +  authors on  +  the  injury  that  from  K  +  the  to  the  Although a l l of  most the  still  argued  basis  of  significant  the  external  due  (1930) t h a t  several  maintained  K ,  larger  and  i n the  methods.  Hill  K  to  with  expected  discrepancy  bound  many  A number o f i n v e s t i g a t o r s ,  potential recording view  (1900)  permeable  i n frog muscle,  h a l f of  felt  the  to  1928).  a l 1934)  in  studies  fraction  of  + after perfusion  solutions  (Neuschlosz  Takacs  1931; In  were  Seo  u p o n by  +  penetrate  that  membrane,  McDonald  improved  ( F i s c h e r 1924;  v a r i a b l e amounts  which  and  found  relatively  (Hober  (1902) and and  were  following a ten  inaccuracy  +  also  f r o g muscle  impermeable  potentials  equation  K  but  +  (Hegnauer et  concentration  fiber  H  fibers  (Mond and  salt  potentials  Nernst  Muscle  NH^"*", a n d  +  bath  permeability  i n f r o g muscle were r e p e a t e d  +  I t was  1934).  The K  the  little  Sugi  Into  -  concentrations  e f f e c t on  indicating  on  C l  1939,  the  soaked  or m i l d  1926;  Ernst  Mitchell  and  Conway  f r o g muscle  Pricker  and  (1941) m e a s u r e d  the  a  v a r i e t y of markers  high  concluded  Horton  discovered  -free 1930;  that  C l  a m o u n t s when t h e K  extracellular  and  1928;  K  Ernst  1934).  Boyle  i n large  i n solutions with  al  extraction with  +  that  of  did  muscles  concentrations. space  -  Boyle  f r o g muscle  i t occupied  about  et  with 13%  12 of the  fiber  solution the if  volume and  proposed  classic  by  paper  Fenn et  of Boyle  m u s c l e s were p l a c e d (1.3  [K]  muscle  -  2.5  (in relation  increased to  ion  content  exhibit  Na  to  and  the  10-12  C l " entered  With  They  with and  at  further  Ringer  led to found  that  physiological K  left  +  dissected fiber).  (for fibers  were m i n i m a l .  in  These papers  solutions  freshly  mM/1  expansion  Conway i n 1941.  and  +  the  (1934).  al  i n Ringer  mM/1),  was  d i d not  2°C),  the If  the  [K]  changes  i n c r e a s e s i n [K]  in  (and o  [Cl]  )  K  3  and  +  relatively duct  of  Cl  entered  constant.  the  muscle while Na  Furthermore,  intracellular  intracellular  the  w a t e r ) was  K  and  +  equal  K  according  Donnan e q u a t i o n  the in  to the  injury  potentials  accord w i t h the  from  the  and  Cl  of  of  fibers  K  least  from  to  one  Kirkwood system two  we  phase and  and  colloids  are  which  starting  are  of  Cl  (Donnan  separated  by  most  contained  Nernst  the  membrane.  components  the  Overbeek  of  were  coexistent pass  1956;  1969).  The  consists  of  i s permeable  to  the  charged  phases.  Donnan e q u a t i o n  (1929)  membrane  equation  cannot  common s y s t e m ,  of the  the  the  impermeable to  i n one  extracellular  solutions  a membrane w h i c h  s m a l l i o n s but  w i t h Guggenheim's  KC1  b e t w e e n two  1911;  pro-  Measurements  Lakshminarayanaiah  i n , the  of  across  ionic  across  the  (mM/Kg  of the  1911).  high  i s s e t up  the  1961;  derivation  product  (Donnan i n the  and  +  other  interested  certain  The  the  Oppenheim  aqueous phases  water  one  concentrations  -  p r e d i c t e d by  A Donnan e q u i l i b r i u m p h a s e s when a t  solutions  were d i s t r i b u t e d  potentials  distribution  C l  to the  concentrations;  +  i n these  remained  +  is simplified  expression f o r the  electro-  by  13 chemical  potential .  n.  = y.  3  3  where y . i s t h e valence  chemical  potential  i s the  fraction  of  At  the  +  FE  z.  potential F  phase.  i s expanded = y!  3  3  + RTln  standard  of the  ions w i l l  x.  V.,  the  equal  s u p e r s c r i p t s o and  ideal  Combining librium V. s  3  by  +  I n x? 3  state (5)  and I  and  an  (5)  and  the  (3)  potential partial  and  x.  i s the  molar volume  mole  of j ,  P. electrochemical potentials  i n the  two  phases  of  (represented  the by  ............(4)  + 'RT  equation  solution,  the  (2)-(4) f o r a l l permeant s p e c i e s a t e q u i -  equations  The  is  3  solutions  3  E  I):  in ideal  + P°V.  the  3  n? = r , i 3  is  follows:  the  3  z.  c o n s t a n t , and  In the  + PV  3  pressure  be  as  chemical  equilibrium,  species j ,  of  i s Farraday's  of the  y .  (2)  3  s p e c i e s j , and  independent  permeant  species j :  sign),  potential  where y.  of a  chemical  (including  electrical  is  (n)  across  z.FE° = y ! 3  3  of t h i s the  z.  x?  3  3  ideal  the +  P V. X  o  =  i z.  3  system  c o n d i t i o n of =  semipermeable  +  z.FE ..(5)  i s described  completely  + RT  In  3  1  3  electro-neutrality.  (6)  x:  3  membrane,  2  3  I n an i d e a l sysfern w i t h a permeant u n i - u n i v a l e n t s a l t impermeant i o n q of v a l e n c e n i n phase ( i ) , equations (6)  result (P  1  - P°)V  i n the  following  + F(E  X  - E°)  equations: = RT  o lnf+ X " + 3  (7)  14 (P  - P°)V_ - F ( E  1  X  o - E ° ) = RT l n f j x  (8)  1  1 , 1 1 x. + n x = x + q x  brane  (10 )  = x_  +  where + and - r e p r e s e n t The  c a t i o n and anion.  distribution  c a n be e x p r e s s e d  m*+*°  of the permeant.ions  by a d d i n g  .. <  -  F  (P  v  - P°) t e r m  equation  (11)  the  skeletal  the  muscle  that  c a n be n e g l e c t e d becomes  partment.  (ii)  zero.  i o n , q, i s s m a l l , t h e  and t h e r i g h t - h a n d  I n t h e s y s t e m we  side of the  are' i n t e r e s t e d i n ,  m u s c l e , t h e c o n c e n t r a t i o n o f impermeant  i s relatively gradient  Conway 1 9 5 7 ) .  exclusion  (8):  RT  a pressure  1936;  and  t h e mem-  v  t h e c o n c e n t r a t i o n o f t h e impermeant 1  (7)  equations  across  ° » + - ->  F  xjx^  If  fn\  (9;  and y e t there  exists  across  This  o f t h e main Equation  large,  i s made  then  the fiber  inside  evidence  membrane  (Penn  p o s s i b l e by t h e r e l a t i v e  external cation,  (11)  i s no  ions  N a , from t h e i n s i d e +  becomes:  i i o o x x = x x_ +  / -, o \ (12 ;  +  I n a system where t h e s o l u t i o n s on e i t h e r are  non-ideal, equation  activities trations). (12)  (12)  (a) r a t h e r than I f K  +  and C l  com-  should  mole  s i d e o f t h e membrane  be e x p r e s s e d  fractions  (or molar  a r e t h e major permeant  c a n be r e w r i t t e n i n t h e f o r m  familiar  i n terms  concen-  ions,  t o most  ofi o n  equation  biologists:  15  which Cl  i s t h e Donnan e x p r e s s i o n  across  the muscle Similarly,  difference  across  E  i  - E  o  fiber  f o r the distribution  t h e membrane R  T  expression  R  T  (  a  equation,  potential (7)  equations  l  (14)  o  (1),  and i s s t i l l  (8).  and  }  c  i s t h e same a s e q u a t i o n  boundary p o t e n t i a l  and  °V  C l i = =^ln , F (a )  }  K  This  f o r the e l e c t r i c a l  f o l l o w s from  K o (a ).  ( a  +  membrane.  an e q u a t i o n  = =-ln  of K  Nernst's  called  phase  the  Nernst  equation. It siderations by  should  do n o t a p p l y  that the  attracts  counter  ions  out that  these  t o a two phase  theoretical system  F o r i n s t a n c e , an but p a r t i a l l y  con-  separated  ion-exchange  excludes  co-ions,  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 s e t up b e t w e e n t h e r e s i n surrounding  medium w h i c h , (13)  Donnan e q u a t i o n the surface  rise  t o an e l e c t r i c a l  could  exhibit  the equilibrium  Because ionic  activities,  equations  (13)  (y)  and C l  of K  +  1962,  they  a n d (14),  chapter  according  fixed  5;  to equation  (Ling  a n d Conway h a d no means had t o use molar assuming that  and  1962).  Ling  give (14).  cell  properties that  membrane  so  to the  charge model o f the  potential  to the c e l l  Boyle  conform  the concentration gradients  potential  of Ling's  Conway a t t r i b u t e  at equilibrium,  (Helfferich  of the resin,  Thus, t h e s u r f a c e  and  just  a s e m i p e r m e a b l e membrane.  resin  At  be p o i n t e d  1962,  Boyle  p.  271).  of measuring  concentrations the activity  (C) i n  coefficients  a r e t h e same I n t h e m y o p l a s m a s i n t h e b a t h  16 (C  = a/y).  appeared muscle  In the light  valid.  equation cult in  this  i n the freshly  This  dissected  amounts  The  1/40.  to only  be l e s s t h a n  about  10%  detected  by e a r l y a n a l y t i c m e t h o d s .  tissue of  (1923)  Slyke  1928;  Sunderman Shenk  analyzable  Cl  Heilbrunn  were b i n d i n g binding  appreciable (Conway  to Ag . +  Cl  1935)  Recent  Ion  Distribution Boyle  of C l  of C l  with  was a l l  was  -  ratio  water.  i n the extra-  not  easily  Many.investigators  acid followed  1933;  by  that  Heilbrunn  used  precipitation  that  the c e l l u l a r  have  mild  Work on the' R e l a t i o n B e t w e e n  macromolecules  unavailable avoided  micro-diffusion  a very  yields  digestion.  l e a v i n g the C l  their  and  and  acid digestion  a n d C o n w a y may  by e x t r a c t i n g w i t h  More  with  low pH,  Boyle  binding  B.  solutions  found  concluded  C l " at very  Cl  3mM/Kg i n t r a f i b e r  than n e u t r a l or a l k a l i n e  and H a m i l t o n  diffi-  of i n v e s t i g a t o r s (Wilson  and W i l l i a m s 1950)  i t i s not  involved digestion of the  nitric  A number  +  19^2;  Hamilton lower  Ag .  Donnan  I f the [ C l ^ / C C l ]  amount  method w h i c h  i n hot, concentrated  the C l ~ with  Ball  for  and such a s m a l l  to the  fiber  o f t h e amount  space  Van  according  frog muscle,  cellular  the  on i o n d i s t r i b u t i o n i n  [K] / [ K ] ^ ratio." i n the i n v i v o  i s at least  [Cl]^ will  assumption  assumption.  space.  muscle  t h e same,  studies  this  e a r l y i n v e s t i g a t o r s thought  the e x t r a c e l l u l a r skeletal  results,  and C l ~ a r e d i s t r i b u t e d  +  t o s e e why  frog is  A l lsubsequent  have f o l l o w e d I f K  of t h e i r  acid  technique solution.  Transmembrane  and Membrane P o t e n t i a l and Conway's  [K]  at least  e x p e r i m e n t s were four  times  done  the normal  In  bathing  physio-  17 logical the in  concentration.  Donnan  relation  interpreting  potential  In Ringer  d i d n o t seem  these  results  They t e n t a t i v e l y  able  to maintain this  [K]  b e c a u s e o f some d e f f i c i e n c y  solved with  by L i n g  relation  of less  than  fibers  without  micropipettes  filled  with  liquid  potential (they [K] of  junction  [K]  and  Gerard  filled  than  calculated  carefully  findings  was  [K]  damage.  With the most o f  the resting  solution  forthis  substitution  membrane  was - 9 7 - 5  mV  experiment).  When  f o rN a ) , the plot +  s l o p e o f 44  had a constant  s l o p e o f 58  (1950),  muscle from  repeated  including  those  i n 2.5  mM  using the Ling that  (Ling  [K]  the Nernst  w a s - 8 8 mV,  equation.  and c o n f i r m e d most of Boyle  of these  a n d Conway.  the chemical potential Finally,  calculated  Hodgkin and Horowicz  mV  (1956,  found  d i d not agree  from  (1959b)  lower  early  He a l s o  with  +  11  Adrian  a t l o w [ K ] , t h e m e a s u r e d membrane p o t e n t i a l of K ,  and Gerard  the resting  that  equation.  w i t h low  eliminating  found  w i t h 3 M KC1, found  of frog  i960)  were n o t  could penetrate  the theoretical  and Hodgkin  potential they  that  1950). Nastuk  electrodes  than  solutions  l y which  v s . membrane p o t e n t i a l less  concluded  They p u l l e d m i c r o p i p e t t e s  they  (by e q u i m o l a r  > 5 mM,  for  hampered  membrane p o t e n t i a l  i n Ringer  do n o t r e p o r t t h e e x a c t  l o g [K]  were  i n the Ringer.  3 M KC1, t h u s  soaked  [K] ,  the muscles  causing v i s i b l e  potential,  of fibers  was v a r i e d ,  i n Ringer  (1949).  muscle  the  but that  of measuring  and Gerard  t i p diameters  single  t o h o l d but they  relation held i n vivo  The p r o b l e m  with lower  by t h e i n a c c u r a c y o f t h e i r  r e c o r d i n g methods.  t h e Donnan  solutions  the Nernst  showed  that  18 if  [K] o  L  and [ C l ] o  J  kept  constant,  according but  than  brane  t h e membrane p o t e n t i a l  < 10 mM/1,  expected.  i s twice  ations  from  Adrian  (1956)  [K]  found  equilibration).  > 10. mM/1  The e x p e r i m e n t s (I960)  and Kimura  basis.  Upon r e t u r n i n g  potential.  the K  Ling  K +  +  changes  allowed  only  depleted  fibers  of their  results  and  due t o 10 m i n . f o r and  o n t h e same solution,  t h e membrane  reveals  by t h e C l  devi-  following  to Ringer  d i d not equal  was  (1950)  (1957)  be c r i t i c i z e d  was b r a c k e t e d  The  partly  of Stephenson  potential  mM/1  m u s c l e mem-  and Gerard  can also  Re-examination  membrane p o t e n t i a l  the frog +  (they  varied  > 10  i f [K]  were p r o b a b l y  of C l  muscle  as i t i s t o K .  that  + [Na] o  i n membrane p o t e n t i a l  f o r membrane p o t e n t i a l  Koketsu  that  equations  to Cl  and [ K ] o  of the frog  showed t h a t  equation  equilibration  found  also  as permeable  the Nernst  x [Cl] o  the variation  They  changes a t [K]  incomplete  they  but [K] o  t o t h e Donnan and N e r n s t  when [ K ]  less  were, v a r i e d ,  and K  +  that  the  chemical  potentials. These across  under these authors  [K]  conditions,  excluded  this  effect  i s low.  that muscle  deviation  +  according  +  permeability  1941).  field  Hodgkin  equation  C l , and N a -  range  +  t o the Nernst and  that  i s not v a l i d .  on t h e p o t e n t i a l  a n d Conway b e l i e v e d  (Dean  constant  of K ,  i s not d i s t r i b u t e d  f r o m Donnan e q u i l i b r i u m  f r o m t h e myoplasm u n d e r most  Goldman's  +  t h e Donnan r e l a t i o n  of Na  Boyle  soon d i s c o u n t e d  K  i s i n the physiological  attribute  significant  effects  Indicate  t h e membrane o f t h e f r o g  e q u a t i o n when  [K]  studies  that  Na  conditions and Katz  ( G o l d m a n 1943)  permeability  +  was  These to the  when  totally  but t h i s  was  (1949) u s e d to include  on t h e r e s t i n g  the  membrane  19 potential: E  =  p-ln[  V ^ o where  ( ). a n d ( ) 1  the  cell  the  ions.  stants the  W  +  ^  o  are the ionic  0  a n d P^, ^Na.'  a n c  ^ ^Cl  a  r  activities P  e  The r e l a t i v e m a g n i t u d e s  depend  on t h e m o b i l i t i e s  e  In deriving gradient  this  the  electric  the  relative permeabilities i n the. s o l u t i o n s  Hodgkin  and Katz were a b l e  field  equation  constants. 1964;  of  equation  is  only  fitted Hodgkin  (15).  forg  from t h e squid  )  ^ ^ y constants of  studies 1967)  i n t h e membrane a n d s o l u t i o n and  on e i t h e r  (1967)  that  with  axon t o t h e constant i n the  have  considerably  side  permeability  (Baker  shown t h a t  et a l  the relative following  o f t h e membrane.  and M a c G i l l i v r a y  t o t h e membrane p o t e n t i a l  and  that  (1942)  and Cole's  on t h e axon  can change  con-  o f t h e membrane.  small, adjustments  a rough approximation;  (1969)  and Hare  l i m i t a t i o n s on t h e a p p l i c a t i o n stressed  that  this  equation  as such i t i s s u c c e s s f u l l y readings  on f r o g  muscle  by  (1959b).  the chemical potentials  from t h e e l e c t r i c a l  muscle  :  do n o t c h a n g e  side  Hodgkin and Katz  and Horowicz  l  t h e a s s u m p t i o n s were  to f i t Curtis  out the t h e o r e t i c a l  Since differ  on e i t h e r  of the ions  i n the solutions  have p o i n t e d  a  between  of the ions  and W a l l i n  Sandblom and Eisenman  e  of the ions  equation,  However, r e c e n t  permeabilities changes  data  by m a k i n g  Strickholm  m  and o u t s i d e  t h r o u g h t h e membrane i s c o n s t a n t  changes  membrane p o t e n t i a l  r  inside  of the permeability  p a r t i t i o n c o e f f i c i e n t s of the ions  membrane.  (15)  ^l^cA  +  potential  under p h y s i o l o g i c a l  o f Na  across  conditions,  and K  appear t o  t h e membrane o f t h e these  chemical  20 potentials a pump. any  m u s t be m a i n t a i n e d  According  measurable  diffusion  to Geduldig's  electrogenicity  currents which (15).  of"  equation  one  direction  the  constant  b y some a c t i v e calculation  should  i s too great  across field  into  a component  d e s c r i b e d by e q u a t i o n  on t h e o p e r a t i o n  genic  pumps h a v e b e e n d e s c r i b e d  Slayman  1966; 1968;  Alving  criticisms, should  1962; Hinke  The  effect  membrane p o t e n t i a l  the et the  but the data  skeletal a l 1957).  field  there  muscle They  +  and a  1963;  1967;  neuron  component pump.  +  Electro-  equation,  A d r i a n and  Carpenter  In the light  has been  of  and these  i fapplied at a l l , of i t s limitations  studied i n other  from these muscle.  An A u s t r a l i a n  found  muscle  on  muscle  under p h y s i o l o g i c a l  of these  bundle.  on t h e a v e r a g e r e a d i n g s  b;  Simon  conditions, that t o the average  equation  parameters  They  extensive  group has studied-  c o u l d be r e l a t e d the Nernst  resting  prepar-  i s n o t as  (Shaw e t a l 1 9 5 6 a ,  of the toad  correlation  individual  correlation  preparations  concentration through  was l i t t l e  each  were  i n a number o f o t h e r  an u n d e r s t a n d i n g  a v e r a g e membrane p o t e n t i a l K  (15)  of inorganic i o n distribution  i t i s f o r the frog  internal  on  et a l 1969).  the constant  the use o f  of the molluscan  and Rybova  and McLaughlin  Taylor  invalidate  1967).  (Grundfest  as  of  r i g o r o u s use  o f an e l e c t r o g e n i c N a  Keynes  be a p p l i e d w i t h  ations  imbalance  to justify  potential  dependent  (Kernan  "almost  Marmor a n d Gorman (1970)  equation.  the resting  tissues  indicate  t h e membrane w i l l  to separate  +  (1968),  A m e c h a n i s m w h i c h pumps a n e t c h a r g e i n  able  Na  mechanism; i . e .  concluded  from  but that readings  that the  was f o r t u i t o u s .  However,  21 since  t h e y w e r e r e c o r d i n g membrane p o t e n t i a l s  fibers the  but analyzing theK  average  results  results  from  content  +  areprobably  individual  bundles.  [K]  = 1 3 mM/1,  fold  increase i n [K] but that o  was l o w e r  expected  crepancy  was i n f l a t e d ,  chemical  results  values cell  which  average,  Table  They  this  o f inadequate  bath.  +  wet w e i g h t ;  o f t h e toad muscle  had e a r l i e r  this  author's  predicted  opinion,  i s based  this  on r a t h e r  their  the K  +  of the  I fthese  results  i n terms  i s 75% o f t h e  water  theC l  by e q u a t i o n ( 1 4 )  Their results and N a  +  for Cl  content o f fashion.  f o r t h e e x i s t e n c e o f an NaCl  a t about  t h e same  con-  I t c o u l d s i m p l y have been a space  i t w a s 15% o f t h e f i b e r they  dis-  c o n c e n t r a t i o n s a r e , on t h e  extracellular  (Tasker e t a l 1959),  they  theory  K  containing  reflection  space  This  expresse'd  matter.  (assuming  as evidence  as t h e e x t e r n a l  cellular  concentration  considerably but i na p a r a l l e l  compartment  that  +  I V , p. 271) a r e r e c a l c u l a t e d  centration  study,  they  above  58 mV p e r t e n  equation.  10% o f t h e v a l u e s p r e d i c t e d  interpreted  guessed  K  bundle,  than t h e  that  t o o l o w by t h e f r a c t i o n  up o f s o l i d  water  preparation varied  (they  the Nernst  arenot conclusive;  intracellular  found  dropped  r e c o r d e d membrane p o t e n t i a l s .  distribution their  also  theinternal  weight), thefiber  within  their  They  however, because  i s made  o f mM/Kg i n t r a c e l l u l a r intracellular  o f more s i g n i f i c a n c e  a s mM/Kg i n t r a c e l l u l a r  (Shaw e t a l 1 9 5 6 b ,  from  from  areelectrochemically  weight  o f t h e whole muscle  t h e membrane p o t e n t i a l  than  o n l y on s u r f a c e  found  that  was a b o u t  determination  volume);  i na  t h e average 50% l a r g e r  later  extrathan  and that  i tvaried  considerably.  group's  rejection  o f t h e Donnan  weak e x p e r i m e n t a l  evidence.  In  22 • Little muscle. into of  (1945)  Wilde  live  work has been done on mammalian s k e l e t a l  rats  and found  the s k e l e t a l muscle  theory.  with  the  He f o u n d  rat.  space  that  sucrose  and i n u l i n  slowly  filled  was  no i n t r a c e l l u l a r  C l .  t h e dog but supported Cotlove's  penetrating perfused [K]  felt  this  the hind  limb  latter  (Conway  there  experiment  I t seems  likely  to the fibers or  1957).  of the cat with  of C l  c a s e was  c a r r i e d o u t t h e same  markers were e i t h e r b i n d i n g  with  indicate that  t h e Donnan t h e o r y .  into the fibers  deter-  equilibrated quickly  a Donnan d i s t r i b u t i o n  Cotlove  -  t h e Donnan  on t h e s k e l e t a l m u s c l e o f  a space which would  Walsen e t a l (1954)  correct.  that  with  with  e x t r a c e l l u l a r space  the markers  was c o n s i s t e n t  then  t h e c o r r e l a t i o n between [ C l ] ^  d i di n vivo  that  but  on  that  and e x t r a c e l l u l a r markers  +  a n d [ K ] was c o n s i s t e n t o  (195*0  Cotlove  minations  a  injected K  e t a l (1957)  Pillat  solutions  a n d o b s e r v e d membrane p o t e n t i a l c h a n g e s  of  varying  consistent  with  equation (14). (1969)3  Usherwood the  membrane p r o p e r t i e s  order  insects  are very  f r o g muscle. 1953;  level  of the striated  muscles  o f most  s i m i l a r t o t h e membrane p r o p e r t i e s  Janiszewski  Wood  of [Cl]  (Hagiwara  lower of  and Watanabe  1967)  and Skubalanka  that  have  o f membrane p o t e n t i a l on [ K ] i n  (1965)  n o t seem t o f o l l o w  logical  concludes  a dependence  these muscles. did  review,  A number o f i n v e s t i g a t o r s  Wood 1 9 6 3 ;  demonstrated  i n a recent  found  equation  that  the C l "  (14) (except  ) i n locust  distribution  at the physio-  and cockroach  f i b e r s but  o Usherwood to  claims  that  Wood's m u s c l e  come t o e q u i l i b r i u m .  Usherwood's  preparations (1967a,  b)  were n o t a b l e electrophysio-  23 logical of  studies  o n t h e same t i s s u e s  these muscles  muscle (14)  behaves  f o l l o w i n g changes  i n [K]  K ,  divalent  +  potentials played  insects  cations,  Grundfest  1962;  Usherwood  1969).  Of  ions  1967;  striated  i t was s i m i l a r t o f r o g m u s c l e  Shaw  (1955a,  Donnan d i s t r i b u t i o n Shaw's v a l u e s  working of K  +  thesis  found  i n other  found  that  h i s values  found  K , +  crustacean  that  they C l  -  assumed t h a t  and  evidence  muscle  held  e t a l (1964a,  (1959)  indicated t o [K] . for a  However,  fibers. not  Also,  long  i s substantial,  and Hinke  1968)  so  e t a l (1968)  Hays  i n the brackish-water  crab  substantial fractions of the intrafiber  the crayfish striated Horowicz  (which  be t o o h i g h .  , and w a t e r were n o t f r e e  Zacher of  muscle  tissue.  crustacean  t h e Donnan r e l a t i o n  (1953)  and Katz's  resting p o t e n t i a l are  fibers)(Gayton  f o r [ C l ] ^ may  and  a r e t h e membrane  Fatt  enough t o c l e a r t h e e x t r a c e l l u l a r space even i n s i n g l e  (Belton  roles  1968;  30 s e c . w a s h i n i s o t o n i c s u c r o s e w a s p r o b a b l y  his  if  1967,  and C l ~i n t h i s  f o r [ K ] ^ and t h e normal  than  r e s t i n g membrane  i n i t s sensitivity  on c r a b ,  The  but the r e l a t i v e  o f the r e s t i n g p o t e n t i a l o f crab  that  lower  Their  Huddart  muscle.  and  concentrations  has n o t been determined  special interest to this  b) a l s o  insects.  e x h i b i t s high  and amino a c i d s .  Grundfest  on c r u s t a c e a n  measurements  much  order  seem t o be m u l t i - i o n i n o r i g i n  by t h e v a r i o u s  studies  i n higher  (13)  Equations  Q  often  t h e membrane  t o t h e membrane o f t h e f r o g  and [ C 1 ] .  do n o t seem t o be f o l l o w e d  haemolymph o f t h e s e of  similarly  indicated that  muscle  had s t u d i e d  i n t h e myoplasm.  b) s t u d i e d  t h e membrane  i n t h e same m a n n e r t h a t the f r o g muscle  and  properties Hodgkin  obtained  2H  virtually were Na  +  t h e same r e s u l t s .  compatible permeability  potential. and  with  1969)  Rudel  muscle  i s about  t h e Donnan r e l a t i o n w h i l e  also  This  appeared  and other indicated equally  to effect  that  the inhibitory postsynaptic  fish  muscle  also  indicates  according the  permeability  zing  striated  muscle  ions  revealed  K  and C l  +  respectively  mM/1,  membrane Dudel  Investigation  (IPSP) of the cray1961)  Dudel and K u f f l e r across  t h e membrane,  Impulse  increases  membrane t o C l ~ ,  a t o r a few m i l l i v o l t s  (1961)  -  stabili-  above t h e  and C l ~i n t h e  +  the t o t a l concentrations  out of the f i b e r with o f 138  results  of these  potential  were  i n [K]  i n favour  of the lobster  (Gainer  10  IPSP phenomenon as t h e c r a y f i s h  mM/1  gradients  Thus,  mM/1 predict  muscles  Robertson's  o f a Donnan r e l a t i o n . reacts  1968)  The  typically to  and e x h i b i t s t h e  membrane  a  - 7 0 mV w h i c h i s  crustacean  muscle  and G r u n d f e s t  press,  a n d 530  14) o f about  o f most  a  and 35.7  concentration  d i d n o t measure p o t e n t i a l s ) .  membrane p o t e n t i a l  of  on t h e i n t r a c e l l u l a r m u s c l e  concentrations  c a n be i n t e r p r e t e d  lobster  t h e Donnan r e l a t i o n d i d n o t a p p l y .  (from equation  to the resting  K  from  of h i s data squeezed  Both  membrane p o t e n t i a l  (Robertson  measured  ( [ K ] and [ C l ]  respectively).  same  and C l ~ .  +  an i n h i b i t o r y  i n the f i b e r , that  the liquid  changes  1958;  and c o n c l u d e d ,  "juice",  results  < 10  potential.  However, e x a m i n a t i o n  close  f o r [K]  the resting  potential  of the postsynaptic  Robertson  these  (14);  t h e membrane p o t e n t i a l  resting  to K  C l ~i s d i s t r i b u t e d  to equation  results  t h e membrane o f t h e c r a y f i s h  permeable  ( B o i s t e l and.Fatt  these  (Reuben e t a l 1962;  studies  into  that  > 10 mM/1  For [K]  (Grundfest  et a l  25 1959;  Motokizawa et  Gainer  (1962, 1968)  exchangeable expected 3a,  4a)  et  these  was  highly at  that, to  permeabilities  fairly  possibility  the  to  the  Hagiwara et [K]  i n normal C l ~ as  of  Cl  and  K  barnacle  striated  affected  later the  K .  At  +  resting  study  the  the  4,  is  the  under a  few  internal C l ~ concentration  concentration  predicted  by  the  by  (Hagiwara  membrane pH  2,  in  slightly  were r e v e r s e d ;  +  is  The  Ringer,  i t i s to  Cl  (1964).  al  A  than  (1968, F i g .  this  giant  and  the  higher  data  that  and  of  membrane p o t e n t i a l h y p e r p o l a r i z e d  i n d i c a t i n g that  close  of  s e n s i t i v e to  permeable  conditions  by  concentration  physiological [K]^.  revealed  millivolts, be  the  membrane p r o p e r t i e s studied  h a n d , Dunham  However, t h e i r  The  as  relative  (14). out  other  l o b s t e r m u s c l e was  compartment.  a l 1968) 1/6  the  the  the  one  permeability  about  On  that  of  rule  have been  potential +  Cl  from equation  more t h a n  Na  found  fiber  does not  muscle  a l 1969).  must  Nernst  equation.  In on  crustacean  free  K  +  and  described [K] been may  summary, most  ).  muscle Cl  by  are  the  Many o f  the  described  under  d i s t r i b u t e d across  chemical but,  evidence  in this  electrophysiological studies  indicate that,  as  p o s i t i v e evidence  than negative  the  Donnan r e l a t i o n  contradictory be  of  the  on  these  tissues  with  for  these  The  as low  have results  heterogeneity  Donnan r e l a t i o n .  t h e s i s were d e s i g n e d  conditions,  membrane  been i n d i c a t e d , these  for intracellular  f o r the  fiber  (with modifications  studies  has  equilibrium  rather  experiments  ideas  in  mind.  26 C.  The D i s t r i b u t i o n Since  muscle  that  r e c o n c i l e these and placed  without  a significant  Carey  depended  extra  Cl  down  authors could  a n d Conway  enters fibers)  have  10 -  suggested  this  that  plasma.  that  1957;  solution,  (19.63)  +  potential  a n d Conway i nfiber C l  being  space  -  when  (and N a )  almost  explanation  extra C l  that  this  (made u p o f  A number o f  (along with  Na ) +  i n t r a c e l l u l a r compartment  volume  (Carey  1959;  Simon  -  membrane  substantiated.  this  I960,  authors d i d  C l  increase  Fenn's  t h e mem-  frog muscle,  Carey  extracellular  i n a separate  Harris  partmentalized 36  found  20% o f t h e f i b e r  1963).  that  considerable  19*11;  h a s n o t been  Edwards and H a r r i s Harris  gains  have  1956,  However, these  a n d Conway  an expanded  be c o n t a i n e d  prising  1959b).  of the bathing  i nheparinized  studies  (Adrian  change i n t h e r e s t i n g  Boyle  the frog  passively across  i nnormal Ringer  i nRinger,  on t h e nature  eliminated  showed t h a t  findings t o the fact  excised  run  i sdistributed  Hodgkin and Horowicz  195**).  (19*11)  t o C l , a number o f o t h e r  bathed  (Fenn e t a l 1934;  i n t h e Muscle" F i b e r  a n d Conway  Cl  of thefiber  1961; not  Boyle  i spermeable  demonstrated brane  of Chloride  could  195*);  a n d Conway  et a l 1963;  Conway  not differentiate  C l " from t h e e x t r a c e l l u l a r  C l  com-  -  this  i n short  com-  term  — Cl  efflux  freely Cl will  permeable  under  be l e f t  compartment would  i nvitro  conditions.  Further  until  -  conditions  have  t o be  but free of  discussion of this  theory  Chapter IV.  already  Robertson  this  under  a heterogeneous  fibers.  thus  to C l  i nvivo  As for  studies;  indicated, there distribution  (1961)  i sconsiderable  o f ions  found that  i n crustacean  evidence muscle  the C l ~concentration of  27 his  l o b s t e r muscle  the  total  the  intrafiber  analyzable  binding.  was. t h e . c o n c e n t r a t i o n  fiber  water;  Cl  was u n i f o r m l y  1968)  and evidence t h a t  ments.  One c o m p a r t m e n t ,  the C l  there  of lobster,  f o u n d a much h i g h e r  tration  expected I f  dissolved  t h u s he c o n c l u d e d t h a t  However, i n a n o t h e r s p e c i e s  (1962,  Gainer  "juice"  intrafiber  about  was no C l "  Dunham a n d  was i n a t l e a s t  containing  i n a l l of  C l  -  two  concen-  compart-  30 m M C l ~ / K g  intrafiber  36 water, would  not exchange  [Cl]  bathing  their  exchangeable  solutions.  results they  concluded in  also  that  found- a l a r g e  60 mM  that  the free  35%  of the intrafiber  the  heterogeneous  crustacean  water.  muscle,  there  muscle.  Their  location  of this  D.  The D i s t r i b u t i o n  have  t h e Donnan r e l a t i o n ) w h i c h intracellular  dissolved  K  +  no d i r e c t  Their  Horowicz  (1959a),  also  about  evidence f o r C l "i n  evidence to indicate  (and Sodium) i n t h e Muscle  already  been  reinforce  In a l lof the intracellular s h o u l d be m e n t i o n e d .  results  Cl".  i n the striated  evidence  f r a c t i o n but  C l " was e x c l u d e d f r o m  non-myoplasmic  Numerous p a p e r s  preliminary  w a t e r was n o t f r e e  of the intrafiber  of Potassium  compartment.  Hays e t a l (1968)  Finally,  Despite a l lof this  i s s t i l l  that  non-exchangeable C l "  of Cl~/Kg fiber  distribution  the  the  a single  of the exchangeable  myoplasmic  zero  i t i s not certain  t h e myoplasm o f t h e b r a c k i s h - w a t e r crab.  indicated  of  striated  up t h e s t u d y .  about  o r wash o u t i n t o  i s ,i n fact,  heterogeneity  d i d not follow  -  As m e n t i o n e d ,  i n the crayfish  indicated  ^ C l  compartment  Dunham e t a l ( 1 9 6 4 ) compartment  with  Keynes  cited  Hill's  muscle  Fiber  (see discussion  claim  that  a l l of  i s uniformly  water. (1954)  Other  pertinent  and Hodgkin and  42 + comparing  influx  and e f f l u x  rates  of  K  i n  28 frog muscle, reached of  this  (say  10  -  15%  K  +  the  the  of  K  fiber.  small  i n the  +  to  the  and  the  longi-  the  80  dynamic  K  K  fiber  100%  +  (1964),  Lev  the  -  myoplasmic  respectively.  determined of  heterogeneity  that  measured  f r o g muscles  most  of  (1959) a n d  Hinke  chemically  accuracy  f r o g m u s c l e was  microelectrodes,  crab  the  degree  (1953) f o u n d  Finally,  indicated that  These  concentrations  +  K  was  +  free  in  myoplasm.  Early picture  i n v e s t i g a t o r s who  of muscle  and  co-workers  and  Sjodin  to  Harris  to  exchange  m  normal Ringer  100%  fixed  exchange  In  need not  reflect  ion binding.  tightest  binding  should  from  reached  the  Harris  1956;  fiber  K  Harris of  +  frog  this  author's  view,  a  sites.  increased.  slow  Given  eventually  distribution  was  Q  charge  of  K  +  However,  McLennan skeletal  exchange r a t e  enough  time,  K  may  even  but the  exchange.  i n mammalian c a r d i a c  Weatherall  muscle  (1958, 1962a, b ) ,  + K  studies, the  Steinbach  a l l of  negative  been w i d e l y i n v e s t i g a t e d . 42  More r e c e n t l y ,  same p h e n o m e n a i n r a t  muscle.  has  simple  s o l u t i o n , have p r o p o s e d  i f [K]  f o u n d much t h e  The  and  this  +  intracellular  (1955, 1956)  with  + K  they, a c h i e v e d  disagreed  have been mentioned.  +  1961), u n a b l e  muscle w i t h binding  K  ( H a r r i s 1953;  42  of  a  Harris  labelled  the  compared  fibers,  out  although  solution, indicating little  +  i n the  activities  results, of  K  of  sensitive glass  +  rule  binding).  +  in a  association K  K  diffusion  f a s t as  with  same c o n c l u s i o n  method would not  tudinal as  the  influx produced  and  convincing  rabbit auricle the  efflux  studies  evidence  i s i n the  same c o n c l u s i o n  and  from f r a c t i o n a t i o n  that  about  mitochondria.  from a v a r i e t y of  20%  Page  of  the  (1967)  experiments  K  +  has on  cat  heart.  K  This mitochondrial K  influx  under  experiments  steady  (Carmeliet  mitochondrial  than  1961;  than  K  K  fraction  +  20%  because  c a r d i a c muscle.  d o e s n o t s h o w up i n  because  exchange under these  +  i n skeletal skeletal  of a  have  fast  conditions.  muscles would  muscles  The b a r n a c l e  1965)  Goerke and Page  s t a t e c o n d i t i o n s , presumably  mitochondrial-myoplasmic  smaller  fraction  likely  fewer  muscle has very  A  be  mitochondria  few m i t o -  chondria.  Potassium parations.  Robertson's  concentration about al  25%  has been  than  muscle  t h e whole  of the fiber  (1968)  studied i n several crustacean  indicated  K  fiber  66 mM  of the fiber  i n t h e myoplasm and t h a t t h e r e s t  from  30%  (1966)  (1969a,  and Hinke  activity  was h i g h e r  analysis  of fiber  evidence  f o r exclusion of K  if  water;  there  that  this  fraction  also yield  that  there  K . +  25%  +  from  K  at least  The f a c t  K  was n o t .  was  excluded  +  and Hinke  from t h e  of non-solvent  little  that  (30%)  sensitive  +  +  K  +  glass chemical  have been i n t e r p r e t e d as  non-solvent  i s very  +  K  o f Hays e t  the myoplasmic  expected  These r e s u l t s  +  were any bound  techniques suggests  K .  K  McLaughlin  found  than  suggesting  of the K  o f t h e b a r n a c l e , measured w i t h  microelectrodes,  fiber  water.  1970)  a lower  The r e s u l t s  free  o-f t h e i n t r a c e l l u l a r  contained  (lobster),  was b o u n d .  +  that  "juice"  pre-  that  25%  water would other  water +  of the intraincrease  experimental  (Hinke  1970)  binding i n the barnacle  muscle.  The strong  measurement  evidence  sorptionists  against  (Nasonov  o f high myoplasmic  the theories of Ling  1962;  Troshin  1966),  K  +  activities i s  (1962), the and t h e o t h e r  30 (Simon 1959;  authors cell  interior  (with  .196.3) who b e l i e v e  Ernst few f r e e  ions).  electrode  r e s u l t s on t h e grounds  sensitive  to intracellular  ponents that  have been  found  Considering  which  other  1969).  Ling  (Hinke  causes  releases  the rate  of K  +  1953),  K  and Keynes  diffuse  away r a p i d l y , r e d u c i n g  stant the  (Dick  criticisms  the question  have  and  Na  barnacle  Hinke either  1970)  (10 al  -  the K  chemical  +  1969;  1968;  Keynes  at the t i p of the  remain remarkably  conof  observations).  support.  Na  +  distribution  i s of  relevance  of K .  Direct  +  microelectrodes,  and Hinke  and t r a c e r  (Harris. 1953;  following penetration  (Hinke  50%  argued  the electrode.  and'personal  sensitive glass  over  has a l s o  com-  w o u l d be e x p e c t e d t o  of intracellular  o f crab  be  no s u c h  +  (1969)  activity  +  o f time  (McLaughlin  K ;  might  damage t o t h e f i x e d  readings  1959) 1966;  of the  (Lev 1964),  frog  A l l e n and  of the intrafiber  or compartmentalized.  Na  +  S i m i l a r r e s u l t s have studies  been  1961;  (Robertson  and S t e i n h a r d t  i s  1968;  A l l e n and  1970).  Nuclear muscle  +  indicate that  from  K  no e x p e r i m e n t a l  activities  Dunham a n d G a i n e r Hinke  Na  than  i n the c e l l  of the intracellular  muscle  bound  obtained  +  free  length  electrodes  +  ordered  c r i t i c i z e d the  to interact with  electrode  discussion  measurements, w i t h myoplasmic  this  and McLaughlin  A short to  +  f o r an i n d e f i n i t e  cell  Ling's  However, K  +  local  diffusion  Hodgkin  electrode.  the K  components  the t i p of the electrode  charge matrix  (1966)  Ling  that  i n a highly  magnetic  resonance  have been I n t e r p r e t e d 15  1969;  mM)  of the fiber  Na  (NMR) s t u d i e s  t o indicate that +  i s free  C z e i s l e r . e t a l 1970).  Since  (Cope none  only  1967a;  on  frog  30 -  65%  Martinez  o f t h e NMR  et  measure-  31 merits  were  corrected  may r e f l e c t  free Na  extracellular  puzzling, 9 mM was  lost.  loss  K  of fiber This  the Na  has  with  similar  sucrose  and  K . +  found  rapidly the  secondary  (1962)  argues  importance in  time,  (1957)  mainly  and t e r t i a r y that  more t h a n  2/1  i f the  water  matter).  of fiber  studies  most mobile  (1967b) Na  should  +  be  Into treated  ground.  evidence  binding  field  myosin,  amounts this,  myosin  structure  strength  of the protein matrix).  over  K  also  (Ling i s o f prime  and s t r u c t u r i n g c a p a c i t y and  Lewis  and S a r o f f  of extracted +  +  decreased  of the protein.  specificity  of Na  may b e d e p e n d e n t o n  the binding  +  that  observed  of extracted  of Na  case, the  that  Cope  considerable  and thus  i n favour  assume  i s bound b u t very  and t e r t i a r y  the binding  +  from t h e e x t r a c e l l u l a r  who f i r s t  anion  Na  a n d i n t e r p r e t a t i o n o f NMR  structure  secondary  selectivity that  this  biochemical  capacity  i n determining  found  space  T h e s e NMR  (19^7),  I  to the conclusion  i n d i c a t i n g that  the surrounding  binding  was  the binding  with  mM  Ringer,  +  +  p r o t e i n s , binds  Szent-Gyorgyi  that  volume and  the details,  t i s s u e i s on f i r m  i s strong  other  water  o r no N a , i n w h i c h  the calibration  from b i o l o g i c a l  most  (12.5  o f NMR-detectable  r e s u l t s f o rt h e l o s s  solution.  until  There unlike  little  et a l d i dnot discuss  caution  signals  d i dnot give  was p r o b a b l y  +  1 mM  but only  i n t e r p r e t a t i o n leads  reported  isotonic  they  contained  Na  fluid  f o r 10 m i n . i n K  soaked  i n the extracellular  +  (Czeisler  were  was l o s t  +  Although Ringer  +  space.  Na  of the fiber  estimates  The f i n d i n g s o f C z e i s l e r e t a l a r e somewhat  i f muscles  of fiber  their  of  i s 12.5%  space  space, these  i n the extracellular  +  = 100 mM/1).  [Na]  f o re x t r a c e l l u l a r  myosin  and t h e b i n d i n g  32 capacity cation  water  10  per  binding  7-3  a t pH  gm.  would  40 m o l e s  of myosin. 50  mM  of monovalent  In the barnacle of Na  plus  +  K  to avoid  cation binding  They  decreases  as  the  also  to g l y c e r i n a t e d muscle.  indicate that  ionic  strength  or  that  of  the g l y c e r i n a t e d  cations  Further muscle  Kg  this  of  fiber  the d i s r u p t i o n of e x t r a c t i o n ,  r e a s o n a b l e q u a n t i t a t i v e agreement w i t h  Saroff.  muscle,  per  +  inorganic  1968).  (1957),  Fenn  in  about  amount t o  (McLauchlin  studied  was  3  His r e s u l t s are  t h o s e by  Lewis  e i t h e r the binding  increases  and  capacity  ( w h i c h seems u n l i k e l y )  a r e - e x c l u d e d from  a fraction  of the  water  muscle.  evidence f o r cation binding  comes f r o m t h e  s t u d y by  Hinke  and  i n the  McLaughlin  barnacle (1967). +  They  found  increased  that about  the myoplasmic 10  mM/1  shortening  analyzable  +  decreasing. released changes. evidence  and  They  from  K  argued  the myosin  Whatever the f o r an  i n the barnacle  E.  The  State  the  Most  matter.  pointed  even of the  bound N a  during  the  explanation,  +  Na  and  though fiber  irreversible  the  chemically  were K )  was  +  configurational  these r e s u l t s are of Na  K  induced  (and perhaps  distribution  +  strong  (and  perhaps  muscle.  of Water i n the Muscle  Water has biology.  that  heterogeneous  K ) +  of the muscle  concentrations  +  of both  f o l l o w i n g a temperature  irreversible Na  activities  +  Fiber  once more become a c o n t r o v e r s i a l s u b j e c t  biologists  felt  that  Hill  However, as M c L a u c h l i n and  out, a correction i n H i l l ' s  (1930) Hinke  had  (1966)  settled have  c a l c u l a t i o n s ( f o r water  in  shifts  and  bound  water  1  figure  extracellular from  than  (Overton 1902a;  (1963)  has  also  of  s t u d y on water  cell  25%.  to at l e a s t  1922)  extensively  Hill  must  or ordering  f o u n d by was  criticized  i s being written  has  the  experiments.  bound  water;  the p o s s i b l e  ion distribution  b e e n k n o w n f o r some t i m e  room t e m p e r a t u r e (1933)  Fowler  lattice  water.  structural  suggested that  and  The  exhibits  that  most  water  extensive  effects  i n the  i s a disordered  form  structuring  of Frank  that  bonding  i s c o - o p e r a t i v e so t h a t  hydrogen  bond would  breakage  o f one  water  will  (having Scheraga  spans  (1962a),  I structure,  disrupt  "flickering of 10 "^  -  -  assuming have  about  70%  of the water  water  to structure  (1957).  They  10 " " -  that  itself  be  may  1  that,  be  Nemathy  clusters a t room  i n clusters.  In (see  one  Likewise,  of structured  s e c ) .  these  calculated will  1  the  reasoned  n e i g h b o u r i n g bonds.  clusters"  and  formation of  to formation of others.  bond would  contain  life  lead  Wen  at  of  bonding p e r s i s t s  i s that  hydrogen  water  Bernal  f o r water  and  pure  properties.  hydrogen  a c c e p t a b l e model  that  Kavanau 1964)  ice  latter  criticizing.  Hill's  about  consider  on  This  water.  It  ice  h i s estimate of  the bound water  ion distribution  structuring  increases  Rubner  At p r e s e n t a g r e a t d e a l any  5%  i s i n agreement w i t h  authors Ernst  less  space)  will  Thus water  and favour the  temperature,  This tendency  of  extremely important i n the  *Many a u t h o r s p a r e n t h e s i z e t h e ' b o u n d ' o f ' b o u n d w a t e r ' t o e m p h a s i z e t h a t t h e y d o n ' t r e a l l y know I f i t i s b o u n d o r n o t (or t h e y don't r e a l l y t h i n k I t ' s bound). This practice w i l l n o t be f o l l o w e d i n t h i s t h e s i s , n o t b e c a u s e t h e a u t h o r k n o w s w h e t h e r t h i s w a t e r i s t r u l y b o u n d b u t b e c a u s e he f i n d s t h e practice tiring.  34 cell,  especially The  i fthe s t r u c t u r i n g  effects  o f macromolecules  been w i d e l y s t u d i e d . been mentioned,  such  sidered  colloids  water.  Other  from  Several  colloid  as Bungenberg  on w a t e r  (1932),  de J o n g  studies,  forcolloid  Jones  and G o r t n e r 1932).  types  of water  water  Since this  structuring  large  time were a l s o  around  span.  structure  c h e m i s t s have  capable of adsorbing rather  chemists of this  freezing  i s of longer l i f e  alreadywho  evidence, 1932;  (Briggs  time, at least  macromolecules  con-  amounts o f  finding  binding  has  three  have  been  considered.  When n o n - p o l a r s o l u t e s a decrease negative  i n t h e volume  Nemathy that  water  by F r a n k  and Scheraga  around  (1962a,  s h o u l d o c c u r when w a t e r  water  molecule with  energy  level  (called  an i c e b e r g ) .  attributed  structured  molecules with  hydrogen  will  have  that  water  density  This latter  solutes. changes  0-4 hydrogen  bonds  concluded that have  of ice-like  water.  the water  a  lowered Thus water  o f volume i n m i x i n g i s  of the non-polar solutes  s  the solute i s  t h e volume  structured  i s n o t t h e same a s b u l k  to penetrate  Kauzmann (1959 )  around  and t h a t  of this  a  only  a non-polar solute.  a coating  fact  the energy  bonds would  The d e c r e a s e  but not i c e - l i k e  the increased this  four  large  as evidence f o r  b ) , considering  of the ice-like  however, has argued  (1945)  non-polar solute,  to the a b i l i t y  interstices  case,  and an e x c e s s i v e l y  the non-polar  when i n c o n t a c t w i t h  the non-polar solute  to  and Evans  structuring  come i n c o n t a c t w i t h  the  o f the system  with water, there i s  change i n t h e e n t r o p y o f t h e system.  was i n t e r p r e t e d enhanced  are mixed  change I s due  water.  water.  In either  35 Water w i l l molecules.  interact with  Water b i n d i n g  at i n t e r c h a i n hydrogen  side  bonds  concluded that  (excluding of  amides)  f i x e d water  estimated site  around  that  that  resins this  has a g r e a t e r  collagen water  triple  mutual for  fitting  the large  This  type  secondary thus  an  (Rich  bonds  amount  argument  of loss  also  i n an i c e - l i k e  state.  occur i f the water  repeating  distance  of the  the expected  length  of s i x  and C r i c k  (Berendsen and water  1961). at  theory).  regular collagen  1962).  be v e r y  o f t h e i r 'in v i v o  water  Such  has  a  may  account  (Kuntz e t a l 1969).  b y RNA  could  These  structure  (Katchman  proteins  i s central t o Ling's  charge  i n s o l u t i o n so  into a structured  held  structure  shell  W a t e r t a k e n up b y  than water  s t r u c t u r i n g would  on e x t r a c t e d  because  protein  s t r u c t u r i n g around  studies  o f water  and t e r t i a r y  of a  to the collagen  o f macromolecule  of water  studies  results  fits  The e x p e c t e d w a t e r NMR  chain  and Breese  s t r u c t u r i n g may  helix i s exactly  confirmed with  Bull  resins.  the axially  can form hydrogen  intervals. been  of water  molecules i n a chain  chains  helix  H e l f f e r i c h (1962)  density  o f a macromolecule F o r example,  1945),  (Pauling  molecules, building a  i s not l i k e l y  type  linkages  m o l e c u l e s a r e bound t o each  i o n exchange  bound water  lattice.  side  the protein.  s i x water  A third geometry  polar  o f macro-  at peptide  chains  1968).  binds s i x water  i n polystyrene  these  each  groups  i n the t r i p l e  (Ramachandran and Chandrasekharan (1968)  polar  has been proposed  e t a l 19-40), a t p o l a r  (Sponsler and  also  dependent  and McLaren  yield  on 1957),  low bound  configurations  water (such  The  amount  varies  considerably  to  that  say  of water from  of these  1968,  1970;  studies  Kuntz  study to study. amounts t o 0.1  bound water  (most  bound t o e x t r a c t e d  are  e t a l 1969;  gm/gm o f  (Bull  and  of  structured  1964).  T h i s bound water  i c e but  becomes more i m m o b i l i z e d as  tration  of the macromolecule  operative does not (Kuntz  action freeze  i s also  A variety  is  i n the  a measure  of the  as  is  a realistic  described  1968).  a normal  by  i t i s not  i n a l l cases, the degree  to which  solution. term;  Thus  oldest  amount  the  cell  "water  t y p e o f bound water  (Overton 1902a).  indicate  the  some t i m e  freezable  20  inactive  1970).  cell  that  water  -  30%  (Blinks  1957; water water  1922).  Mazur does not  1963)  but  binding  does  not  i n the  cell  be  cell  is  Overton's the  results  intracellular  water  is  1965;  G a i n e r -1968;  Rome  1968;  water  has  also  been  More r e c e n t s t u d i e s indicate  freeze.  i n muscle  water  of the  Non-freezable c e l l (Rubner  of water  i n the  have been r e p e a t e d numerous t i m e s ;  Rosenberg  water  detects i t .  experiments  for  this  should  inactive  Hinke  co-  to study  binding"  osmotically  osmotically  concen-  ice-like  water  the anomalous water  the procedure which  The  still  the  That  o f t e c h n i q u e s have been used  cell;  behave not  rigidly  s u g g e s t i n g some  Sterling  evidence that  as  1969).  et a l  binding  increases,  (Masazuwa and  layer  i s not  1967,  that  water  as  one  protein  or  are  immobilized  least  safe  Breese  macromolecules (Kavanau  at  1.0  e t a l 1970)  Gent  c o v e r e d by  I t i s probably  -  on p r o t e i n )  macromolecules  that  Pocsik  only  (1969)  studied  (Wood  about found  at the temperature  he  10% 30%  used  and of non-  37 (-4°C  and  water  binding  This  - 7 ° C ) , Mazur  latter  i s the major  author also  water  does not  can't  be  very  muscle  Hinke  (1970)  about  i t has  water  has  the  also  same a s  osmotically  inactive  non-solvent  water  sign,  and  different  can  signal  from  about  all).  Cope  strongly  10%  (I969)  and  of  NMR  evidence  et a l (1969)  frog  that  b e ' p o i n t e d out  structuring  also  as  muscle  f o r ions  K  +  the show,  of opposite  same i o n b e t w e e n  showed a  loss  signal  D^O  forms  state  the  split  forms  that  loss  that  of the  of  of water  more  1968).  (Fritz  at  his  deuterium i s a  Emerson  freedom  deuterium  fiber  from hydrogen,  in  the  signal  of study with except  of  of water  a l l of  small  normally displays and  the  ( p r o d u c i n g no  from  signal  (Holtzer  e v i d e n c e f o r two  found  same r e s u l t s  than the  t h a n H^O  f o r two  same-type  the  that  interpreted  a m o u n t e d t o 27%  ordered fraction  of s t r u c t u r i n g  and  +  for  study w i l l  to investigate  et a l (1965)  obtained the  gauge  this  f o r the  b e i n g used  made t h e  Cope a r g u e s  1970).  Hinke  f o r Na  1966;  Hinke  non-solvent water  considerably  showed a l a r g e  While  also  this  of  barnacles.  Hazlewood  while  muscle  freezing.-  a fraction  ( M c L a u g h l i n and  +  However, as  differ  muscle  protons of the  that  Hays e t a l 1968;  water.  of  water  in  not  the non-freezable  been demonstrated  excludes K  Bratton  frog  muscle.  since  the non-solvent water  i s also  water.  membrane and  i n supercooled muscles, i t  shown t h a t  can  batches  muscle  cell  to i n t r a c e l l u l a r  that  vary s i g n i f i c a n t l y  NMR  the  stresses  M c L a u g h l i n 1967,  and  is  barrier  the  ice-like.  fiber  Hinke  claims  nucleate freezing  Recently the  (1970)  water. better  i t should evidence There  and'Swift  i s 1967)  38 and  water  structuring  However, K l e i n and evidence while not al  Cope's work  indicate (1968),  from  As  water  results  are  pretation  does  water  the water  with  consistent  studies  water.  and  around  the  does not  behave  as  assumed  proteins.  that,  in  compact manner t h a n a r e  lattice it  (Mazur  i f this  1970;  i s wrong to t h i n k  distinct tinuous  states; spectrum  also  +  agree  reported protein  these  studies  until  on  on m u s c l e  about  water  the  the  inter-  cell  water.  water  more  offer  of states  in  simple  state  of  this  structured  e v i d e n c e we  i s structured,  have  i t is  structured  the molecules i n the i c e  water  likely,  In a  i t i s somehow  Gustafson 1970). of c e l l  the  et  and  s i g n a l from  What l i t t l e  indicates a more  Na ,  i f i t were  know l i t t l e  a u t h o r s have  cell  i t does  reservation  studies  that,  Abetsedarskaya  of c e l l s  the a u t h o r s can  no  state  of water,  cell  (or deuterium)  found  a s i g n i f i c a n t f r a c t i o n of the water  H o w e v e r , we  Most  on  i n nerve.  also  measurements, have  considered with  of the proton  solution.  They  forms  1967) have  i s structured.  t h e NMR  s h o u l d be  muscle  D^O,  structuring i n a variety  evidence that  skeletal  using  indicate'two  Taken t o g e t h e r , these strong  McLauchlan  i n nerve.  NMR-spin echo  water  solutions. cell  that  (1969),  Phelps  for structured  negligible  on  (Chapman and  the  As  Cope has  suggested,  as b e i n g i n a number cell  from r i g i d  water  of  exists in a  structuring to  con-  free  solution.  F.  Summary Most  performed these  on  studies  of the early  f r o g muscle. contradict  studies Only  on  s k e l e t a l muscle  r a r e l y d i d the r e s u l t s  the b e l i e f that  the  cell  were of  interior  is  39 a  free,  homogeneous  However, as have been it  has  often  large  behave  the  electrical results  as  Neglect effect  of  increasing  that water are  of t h i s  studies.  support  the  especially  ments, which  should  the  monovalent  on  the  chemical equations  distribution  that these  muscles.  Donnan  of  on  will  an  assumption  theory.  the  studies should  free  of  led to  be  measureions hold for  become a p p a r e n t  have  the  the  equations  As  barnacle muscle would of the  thesis,  two  in  ions  biologists  invalidate  Nernst  homogeneity  rejection  Inorganic  distributed  example, the  in this  erroneous  techniques,  ion distribution  the work p r e s e n t e d i n the  of  s i n c e most e l e c t r o p h y s i o l o g i c a l  depend  skeletal  variety  h e t e r o g e n e i t y by  Donnan and  water.  muscle p r e p a r a t i o n s  m e m b r a n e may  For  membrane, i n d i c a t e  t h e membrane o f  and  proteins in  heterogeneously  p r o p e r t i e s of the  not  skeletal  of transmembrane  re-examined,  across  o f i o n s and  ever  i f they  of these  do  an  apparent  fiber.  studying  variety  s t u d i e d by  become  muscle  which  a  solution  from  Internal an  40 CHAPTER I I I  E L E C T R O D E AND  CHEMICAL DETERMINATION FIBER  A.  distribution muscle  first of C l  fiber.  chemical  Cl  objective across  determination  should  and'Na  +  data  fiber  C l  (Hinke  useful tools  1959;  Lev 1964).  of the giant  chemical  measurements  barancle;  them t o e s t i m a t e  i n t h e myoplasm  that  water.  of  fiber  i s apparent, e s p e c i a l l y since unit  be c o n t e n t  of K  +  itself free  glass and Hinke with  and N a  20%  +  i n  of the fiber from  of a similar  no C l  -  Na  25 - 4 0 %  and not  f o r t h e Donnan  selective  glass  the less s a t i s f a c t o r y  electrode.  The  Cl  activity  of axoplasm  +  examination  activity,  o f measure  with  the free  combination of  i s excluded  Unfortunately,  a v a i l a b l e a n d one must  Ag-AgCl  +  The d e s i r a b i l i t y  i s the pertinent  equations.  K  only  the fiber  concentration,  and t h a t  this  of  Cl  by  i n t h e myo-  McLaughlin  free  is  of C l  f o rmeasuring  o f the myoplasmic a c t i v i t i e s  permitted  Nernst  content  directly.  combined c o n v e n t i o n a l  fibers  striated  f o r one t o c a l c u l a t e  is  and  -  o f muscle has been t h e c a t i o n s e n s i t i v e  measurements single  of a single  Ideally, the activity  o f t h e most  microelectrode (1966)  was t o e x a m i n e t h e  d i s c u s s i o n has i n d i c a t e d ,  information  be m e a s u r e d  One  study  t h e membrane  of the t o t a l  enough  i n t h e myoplasm.  plasm  of this  As t h e p r e c e e d i n g  does n o t p r o v i d e  +  CHLORIDE  Introduction The  K  OF  has b e e n m e a s u r e d by a  41  number o f and  1965;  Wallin  trodes.  Kerkut  Technical  electrodes  has  inhibited  In  culties,  relatively  and  only  barnacle,  B.  order  on  their  avoid large  the  using  Strickholm  Ag-AgCl  in miniaturizing a p p l i c a t i o n on  some o f Ag-AgCi  very  1966)  Meech  1963;  Keynes  large  these  elec-  these  most  muscle  construction  electrodes  were  single fibers  diffi-  constructed  from the  giant  obtained  in  hub11us.  Methods  waters  and  months.  Barnacle.  stored  Their  Cornwall  1955).  barnacle  was  (1963a).  The  20  mg.  sea  water  described  muscle In  5  average weight  of  fibers  fibers  from  other  i n normal Ringer  s o l u t i o n (see  stored  at  10°C,  dissection sites,  they  without  localized  swelling,  electrode  fibers  could  could  any  be  signs  s w e l l i n g or  be  of  the  the  dissected Table  f o r up  and  I for  to  12  Smyth 2  and  free  from  a  barnacle; hr  after  ulceration, shortening,  The  from others  reported  i n the  differs  Ag-AgCl  i n design  literature  and  (Mauro 1952;  damage  generalized  MlcroeTectrode  study  each  composition).  changes i n i o n  in this  study  from  c h a n g e i n membrane p o t e n t i a l ,  used  mm  post-  deterioration (visible  Construction.  by  in this  lateral  three  giant  and  used  local  1916;  i n length  obtained  used of  cm  to  determined  Hoyle  to  muscles were  50  by  range,up  Single  f o r up  (Pilsbry  fiber  detail  s c u t a l depressor  about  10°C  at  n u b i l u s , was  erior  Usually  were  descriptions  striated  fibers  The  barnacles  Balanus  documented  Single  about  i n aerated  first  diameter.  Giant  species,  comparison with  was  to  Balanus  The  in  and  difficulties  fibers.  used  1952;  i n v e s t i g a t o r s (Mauro  content).  microconstruction Keynes  42 TABLE I Composition  of barnacle NaCl  KC1  Ringer  CaCl„  solutions  MgCl ••••  d  (mM)  Tris (Cl) p H 7. 6  Q  d  NaCH^SO, i  5  Normal R i n g e r *  450  8  20  10  25  (19 C l )  50%  [ C 1 ] Ringer  174  16  20  10  25  (19  Cl)  268  25%  [ C 1 ] Ringer  32  20  10  25  (19  Cl)  402  Q  ^Modified  1963; The  from Hoyle  Strickholm  glass  except  and AgCl.  A piece 2 mm  of  then  o f 25 u d i a m e t e r diameter  fused  1  lead  puller.  construction.  cm f r o m about  i s coated  with  (see P i g .  through  a  1).  length  Two m i c r o c a p i l l a r i e s a r e  by means o f a n  automatic  I f the microcapillary i s long,  straight,  at i t s t i p , i t i s acceptable f o r  (10  mg)  i s attached  around  the Pt wire  t o t h e end  the glass  Pt wire  75 u o f w i r e  protruding  from the glass  t h e AgO t o A g .  with  After  moist  of  i s cut o f f ,  and then heated  insulation. t o con-  s e v e r a l a p p l i c a t i o n s o f AgO, t h e  f o r 5 min t o ensure the electrode  AgO  i s heated  f o r a distance  The e x c e s s  vert  before  insulated  the t i p .  Pt t i p I s coated  hours  i s threaded  By m e a n s o f a m i c r o f o r g e ,  This  i s heated  Pt wire  a r e as f o l l o w s  tubing.  1966).  and Meech  The m i c r o c a p i l l a r y i s p o s i t i o n e d ,  collapsed uniformly  leaving  tip  details  and a s m a l l weight  the Pt wire.  t o 1.5  Kerkut  exposed t i p which  Pt wire  glass  to the Pt wire  vertically,  and  f o r a small  drawn out from t h e t u b i n g  further  of  and W a l l i n 1965,  Construction  microelectrode and  (1963a).  and Smyth  c o n s i s t s o f a 25 u d i a m e t e r  electrode  with Ag  24  Q  complete  conversion.  Several  i s t o be u s e d , t h e t i p i s c o a t e d  43 with  A g C l by i m m e r s i n g  cases,  A g C l was  i t i n a bead  deposited  electrodes.  unstable, final  they  microcapillaries  and more  When t h e e l e c t r o d e s  c a n be r e j u v e n a t e d  t i pdiameter  I n some  by an e l e c t r o l y t i c ; method.  f o r m e r m e t h o d p r o d u c e d more d u r a b l e stable  of molten AgCl,  o f these  electrically  become  electrically  by r e c o a t i n g w i t h  electrodes  i s about  w i d e n t o a maximum d i a m e t e r  The  AgCl.  40 u .  o f 100 y  The  The  glass  2 cm  from the e l e c t r o d e t i p .  B  PASS Pt-Ag MAKE  BRUSH M O I S T  LONG  TIP  THROUGH  G L A S S - P t SEAL  A g O PPT. O N  BEAD O F  T H E N CUT Pt T O  Pt TIP A N D BAKE  MOLTEN  DESIRED TIP  TO  LENGTH  AgCl  SILVER-GRAY e-30-4QA  AgO ON  MICROFORGE  .MOLTEN AgCl  BRUSH  WIRE  |-«—MICRO WEIGHT  P i g . 1. The m a i n s t e p s i n t h e c o n s t r u c t i o n o f a A g - A g C l microelectrode: (A) g l a s s c a p i l l a r y i s f u s e d t o Pt w i r e ; ( B ) AgO i s a p p l i e d t o P t t i p a n d c o n v e r t e d t o A g b y h e a t i n g ; (C) A g - c o a t e d P t t i p i s i m m e r s e d i n b e a d o f m o l t e n A g C l .  Microelectrode by  Performance.  The p o t e n t i a l  t h e A g - A g C l e l e c t r o d e , ^Q-^, i s d e s c r i b e d  equation:  by t h e  recorded  following  44 E  where  (a  and  c  ci  =  ci  E  +  c i °g( ci 1  a  ) i s the activity  l  and  each  s  dard  solutions  were r o u t i n e l y  NaNO^;  ( b ) 10 mM  and  40 mM  NaNO^-  These  similar  tested  t o be d e t e r m i n e d f o r  or barnacle  t o 0.5  equivalent  difference  between  a l l the C l  that  standard  yielded  M KNC>  solutions  KC1  microelectrodes steady  KC1  generally  t o o k no l o n g e r  d i d not vary  during  experiments.  i s possible  be a f f e c t e d  microelectrode NaCl,  that  10 mM  the following:  Tris(pH NH-,  7.4),  coeffi-  mV  found  10 mM  (from  to  than  4 mV  The  30 s e c t o come t o Usually  i n a given  a Cl standard  performance A  containing  and i n c r e a s i n g  1M)  solutions,  for calibration.  and p r o t e i n s .  arginine,  of  56 t o 59  from  solutions.  i n solutions  ratio  +  t h e dominant  the microelectrode  by amino a c i d s  was t e s t e d  +  When i t w a s  used  more t h a n  KNO^,  a K /Na  as i n t h e above  were o f t e n  3  The p o t e n t i a l  varied  solutions  stan-  KN0 ,  an a c t i v i t y  solutions  constants  microelectrode  It  Since  sensitive microelectrodes.  p o t e n t i a l i n the standard  a day's  have  mM  3 8 5 mM  KC1,  (y = 0 . 5 5 ) .  3  475  s i m i l a r to that  assigned  t h e two s o l u t i o n s  t h e same e l e c t r o d e  simpler  25 mM  9 0 mM  solution.  c a l i b r a t i o n i n unbuffered  might  (Tris)Cl,  (Tris)Cl,  Ringer  f i b e r , the  calibrated i n the following  i s KNO^j t h e s t a n d a r d s w e r e  cient  o f each  t o myoplasm and an i o n i c s t r e n g t h  water  salt  of  have  ( a ) 10 mM  a t p H 7-3:  40 mM  a  which  and a f t e r t h e impalement  and  the  i n the solution being  electrode.  microelectrodes  for  ( l 6 )  of C l  are constants  Before  sea  )  Ag-AgCl 2 5 0 mM  KC1,  amounts o f one  l y s i n e , protamine  and  45 albumen.  The c h e m i c a l and e l e c t r o d e  were I d e n t i c a l albumen. amounts is  i n a l l solutions  In the latter, of C l  as t h e albumen  Cl  significantly that  (where  by s m a l l  t h e gram w e i g h t  26  This error  c  fibers  were  before  electrode  Br  other in  (a  groups,  c  l  albumen b i n d s  ratio  corresponds  i n Br~-free  Ringer  )results  t o be  4 mM  calculated  a measured  t o an a c t u a l  (a  c  l  )of  i n our experiments solution  because  for2 -  5 hr  S i n c e no c o r r e c t i o n f o r i ti s possible too high.  that the  There  may  be  interact  with the C l ~ electrode  t h e m y o p l a s m b u t no a t t e m p t  w a s made  to estimate the  o f such  Fiber  mounting  (20 -  of the fiber  described Hinke  interference.  Impalement.  room t e m p e r a t u r e  i n detail  1969b).  25°C).  A l l experiments  negligible.  were p e r f o r m e d  The e x p e r i m e n t a l  and impalement  elsewhere  Briefly  I t i s probably  procedure  have  (McLaughlin and Hinke  (Fig. 2),  t h e tendon  fiber,  b e a r i n g the weight  was s u s p e n d e d solution.  vertically  The C l  from  of a small  been 1966;  of a single  stone  fiber  junction.  at i t s origin,  the cannula i n the bathing  m i c r o e l e c t r o d e was a d v a n c e d  at  assembly,  was c a n n u l a t e d w i t h o u t d a m a g i n g t h e m y o - t e n d i n o u s The  influenced  (1969a)  Hinke  1952).  a s S~ w h i c h  magnitude  such  are 2 -  finding  i s t h e same a s i n s e a  r e a d i n g s were t a k e n . has been attempted,  This  C l ~ (Carr  i s 0.0033),  s h o u l d be r e d u c e d  soaked  interference  myoplasmic  that  Br / C l r a t i o  o f ( a - ^ ) = 30 mM  containing  content increased.  amounts o f B r ~ .  reading mM.  i n those  m i c r o e l e c t r o d e s were found  i ft h e myoplasmic  water  except  t h e m i c r o e l e c t r o d e s measured s m a l l e r  compatible with the fact  The  determinations of C l  downward  46  Chloride-selective microelectrode •Glass cannula -Platinum wire ^  Silk tie -Tendon  Open-tipped microelectrode;  F i g . 2. Diagram microelectrode.  of a cannulated  —•—Single muscle fibre •— <-500/l 1  muscle  fiber  with  an  inserted  through t h e cannula, 1,5  cm b e y o n d  were tip  but these  were used selected  tate  open-tipped  total  t o ensure  error  water.  tendinous  was  filled  C l  i s used  throughout  this  fibers,  [C1]  [Cl]^,  was d e t e r -  companion  d i d not f a c i l i -  was 7 5 - 3 +  T  1.2  fiber  w a s 7 4 . 9 +^ 1 . 4  T  that  t o themicroelectrode  j u n c t i o n and abolished  measured  t h e s i s ) mM/Kg  [C1]  These r e s u l t s demonstrate itself  criterion  and non-impaled  fibers,  were  i nthe fiber.  t h e impalement procedure  I n 22 i m p a l e d  3 M KC1  with  These e l e c t r o d e s  concentration,  impaled  con-  discarded.  o f t i p p o t e n t i a l by A d r i a n ' s  fiber  that  the fiber  mM/Kg  membrane  a t t h e myo-  t h e leakage  pathway  c r e a t e d by  impalement.  Analytic analysis methods  of total involve  Procedure. fiber  C l  -  thetitration  drying  sucrose,  Two m e t h o d s  concentration  each method, s i n g l e f i b e r s  isotonic  The  I fgeneralized  microelectrodes  a n d i n 32 n o n - i m p a l e d  must h a v e . f u s e d  Por  of the electrode  t o t h e t i po f t h e C l ~ microelectrode  (standard  the  1 min.  t h e t i p was  contractures  The membrane p o t e n t i a l was a l w a y s  C l ~entry.  fiber  area  impalement, t h e f i b e r  (see below) on b o t h  water,  until  Small  t o m e a s u r e membrane p o t e n t i a l .  The  fibers  junction.  within  during  on t h e b a s i s  adjacent  fiber  i n t h e immediate  disappeared  1956).  (Adrian  mined  observed  occurred  Conventional  and muscle  t h e myo-tendinous  sometimes  traction  tendon,  of C l  -  with  were washed  blotted twice,  f o r the chemical were t e s t e d . Ag  +  t o produce  AgCl.  f o r 30 s e c i n  and weighed before  (24 h r a t 105°C) t o d e t e r m i n e  Both  t h eweight  and a f t e r  of fiber  30 s e c w a s h t i m e w a s c h o s e n a f t e r a s e r i e s o f w a s h  water.  studies  48 showed  that  washing.  [ C l ] ^ , became r e l a t i v e l y  The  two  (a) in  analyzed  the  sample v i a l  difference and  The  NaCl'in  were  2 ml  0.4  M HNO^.  Two  milliliters  and  titrated  electrometrically  The  results  o f two  d e s c r i b e d by  delivered The  m e a s u r e d on a curve  to  potential  was  Vibron  recorded The  on  a  experimental  o f 8 x 10~^  solutions  The  C l ~ content  and  of the f i b e r (1964).  was  Single  M NaOH, a n d t h e  protein  q u a n t i t y o f 4% Z n S O ^ i n  of the supernatant on an a u t o m a t i c  and s t a n d a r d s never  was  were  was  acidified  Buchler-Cotlove  t r e a t e d i n t h e same 2% d e v i a t i o n f r o m  showed more t h a n  mean.  analytic  The  was  an e q u a l  standards  The  gave  method  d e s c r i b e d by C o t l o v e  by a d d i n g  Blanks  soaked  C l ~ content  M AgNO^ was  d i g e s t e d i n 3 ml o f hot 0.6  Chloridometer.  were  acid.  precipitated  the  Their  microsyringe.  sigmoid  was  manner.  fibers  sealed i n the t i p of the syringe  method:  by a method  20 s e c  follows:  laboratory recorder.  50% a c e t i c  Cotlove  analyzed  fibers  resulting  compared w i t h  (b) also  by a m o t o r - d r i v e n  a n d Lomb V . 0 . M . - 5  x 10~^  dried  hr.  of 0.1  A solution  i n the sample v i a l  electrometer.  samples were  The  f o r 24  between a Pt wire  a Ag.wire  Bausch  acid  after  a r e as  by a p o t e n t i o m e t r i c t i t r a t i o n  (1952).  Sanderson  procedures  Sanderson method:  2 m l o f 50% a c e t i c  then  16  analytic  constant  methods  slightly,  a r e shown i n T a b l e  but not s i g n i f i c a n t l y  C o t l o v e m e t h o d was  because  i t gave  generally  more  comparisons  less  I I .  b e t w e e n t h e two The  Sanderson  higher, results  s e l e c t e d f o r subsequent  variation  convenient  than  on s t a n d a r d  for [Cl]^.  experiments  samples and  the Sanderson  method  method.  was  TABLE I I Comparison o f Cotlove  and Sanderson methods  of chloride analysis  Cotlove  Sanderson  mM/Kg f i b e r  water  75.1+1.1  Summer 66  75-9+2.0  (54)* 67  Spring  (15)  + 1.0  64.7  67.8 +  (75)  (82) *Pigures  i nbrackets  Electron fixed  refer  Microscopic  buffered  t o pH 7-3 w i t h  solution  by adding  fixed  i nalcohol,  tome,  stained with  C.  Procedure.  Single fibers  were  NaH^PO.^and' made i s o t o n i c  NaCl and glucose.  The f i b e r s  s e c t i o n e d on a P o r t e r - B l u m lead citrate  with  Ringer  were  post-  MT-2 u l t a m i c r o -  and u r a n y l a c e t a t e , and viewed  Results  experiments  t h e summer  spring  two groups  of results  on b a r n a c l e s  o f 1 9 6 6 , a n d the- e x p e r i m e n t s on b a r n a c l e s  o f 1967.  collected  reflect  seasonal  sea  water.  None o f t h e c o n c l u s i o n s  seasonal  The co.llected  i nthe other  i nthewinter  group  and early  The s m a l l d i f f e r e n c e s b e t w e e n t h e two g r o u p s  may  these  arepresented.  i n one group were p e r f o r m e d  were p e r f o r m e d  by  fibers.  E.M. 2 0 0 .  Often  in  t o number o f e x p e r i m e n t a l  l e n g t h a t 1 0 ° C f o r 2 h r i n 1% g l u t a r a l d e h y d e  at resting  on a P h i l l i p s  1.2  changes i n e i t h e r  changes.  thebarnacles  i nthis  paper  or the  areaffected  50 After usually  stabilized  noticed, often  impalement, the C l to a constant  however, t h a t  began  to drift  after  i n the standard  experiments  the C l  impalements  before  results  results.  The  solution plasmic  of  Cl  22 mM  and  44  and  from  a  fiber  water.  (Cl)  , which  total  Cl  f o r three  recoated; '66  i n normal  Ringer  calculated  myo-  i n summer b a r n a c l e  I f the a c t i v i t y  (y = 0 . 6 5 ) ,  millimolar  content  [ C 1 ] , which T  Notice  the  fibers  coefficient  calculated  concentrations, (Cl) ,  indicates  findings,  extracellular  i s the t o t a l  that  than  within  not measured  penetrate  1963a).  extracellular  cleft  One  i n the  way  system  Space.  into  [ C l ] ^ and of f i b e r  I n view  a relatively which  i n t h e myoplasm.  deeply  i n  C l ~ per kilogram  fraction  the f i b e r  (seen  i s listed  Cl  myoplasm.  t o expect  contain large,clefts  some o f w h i c h  -  fibers  between  a significant  i t i s logical  -  fiber  of Extracellular  compartment  of the C l  o f companion  the large difference  rrr  Smyth  average  to  '67  t h e summer  fibers  ^9 mM  fibers.  to free  Determination  and  s  solution  l o c a t e d somewhere o t h e r  do i n f a c t  a  The  slow  35-  I I I as  most  w  m  used  than  resting  III.  ( Q^ >  only  discarded or  mean r e s u l t s  correspond  Table  above  either  i n spring barnacle  The  is  were  more r e l i a b l e  or Ringer  We  and were  In the s p r i n g  are probably  activity,  sea water  activities of  solutions.  a r e shown i n T a b l e  sec.  s e v e r a l impalements, the electrodes  m i c r o e l e c t r o d e s were they  potential  60  reading within  n e g a t i v e l y i n t h e myoplasm  recalibrate  these  microelectrode  may  the f i b e r  to estimate i s t o reduce  contain fibers  microscopy),  interior  t h e volume i t sCl  large  Single  under l i g h t  of the  of  (Hoyle  this  content  by  TABLE I I I The m y o p l a s m i c  activity • ( a ^) of  Group  ^C^m Myoplasmic (mM)  Summer 66  28.8 +1.2  (Cl)  * m Myoplasmic (mM)  44.3  22.4 +0.6  67  34.5  (22)  * Assuming  y  =  O.65  and t o t a l  [ci] ** T  Total (mM/Kg w a t e r )  method o f C l  Membrane Potential (mV)  •68.6 +0_. 8  (54)  (3D  66.8 + 1.3  •73-9 + 0.6  (60)  (22)  analysis  [C1] , T  fibers  75.1 + 1.1  i n myoplasm.  * * D e t e r m i n e d by C o t l o v e  concentration,  c h l o r i d e i n s i n g l e muscle  (3D  Spring  m  C1 Predicted (mV) E  -63.5  -70.9  washing in  thefiber  thetrue  [Cl]  extracellular  space  simplified  extracellular  this  solution.  ideal  product  space.  condition  i nthelow-Cl  the product  i nnormal  itself  The a n a l y s i s  i f the loss  of C l  solution Ringer  to the  o f such  an e x p e r i -  i sconfined to  -  A c c o r d i n g t o t h e Donnan  should exist  -  Obviously, the C l  should adjust  of thebathing solution.  ment i s g r e a t l y the  i na low-Cl  relation,  when t h e [ K ] x [ C l ] o o  is.held  solution  constant and equal t o  by a d d i n g  excess [ K ]  (see T a b l e I ) . 3 summarizes t h e r e s u l t s  Figure  f o r 30 m i n i n 50% [ C l ]  brated in  which  [K]  x [Cl]  microelectrodes ( C]_)  m  reduced;• further curves  this  change  was c o n s t a n t .  of Fig. 3).  reduction  slightly  so that  average  values f o r [C1]  of t h e e x t r a c e l l u l a r min f o r C l  experiments reduced in  which  from  analysis  i nwhich  values  fibers  50% [ C l ]  Ringer  a r egiven i nTable •  water  [Cl]™ r e s u l t s  a t each  have  i n 30 m i n .  large  been  the extent  time).  In four  was u s e d ,  fibers.  arerelatively  The upper  a t zero and  was u s e d ,  IV together with •  o b t a i n e d on companion  15 m i n a n d n o  correspond t o the  were t a k e n  water.  solution  solutions  was g r e a t l y  To d e t e r m i n e  solution  65 t o 47 mM/Kg f i b e r  25% [ C l ]  T  curves which  end p o i n t s  (5-10 f i b e r s  6 3 t o 36 mM/Kg f i b e r  results  the  from  space,  [C1]  i n about  g i v e n below.  T  equili-  occurs i n  15 a n d 6 0 m i n .  washout  their  Ringer  no change  was c o m p l e t e  i nFig. 3 aretypical  fibers  By means o f t h e C l ~  However,  took p l a c e between  shifted  30  a n d 25% [ C l ]  i t was c o n f i r m e d t h a t  (lower l i n e  a  from  In eight  [ C l ] ^ was experiments  [ C l ] m was r e d u c e d These  average  t h e average  (a„-, ) Clm  The s t a n d a r d e r r o r s i n because  of the variation  53  F i g . 3. Comparison between the c o n c e n t r a t i o n of C l i n the f i b e r w a t e r , [ C l ] , and t h e a c t i v i t y o f C l ~ i n t h e myoplasm, A^, i n m u s c l e f i b e r s s o a k e d f o r up t o 30 m i n i n c o n s t a n t [K]  x[Cl]  normal  product  [C]  .  The  solutions with A  readings  m  are  50% the  (a)  and  average  25% of  (b)  of  five  the  fibers  i n (a) and n i n e f i b e r s i n ( b ) . The i n t e r m e d i a t e p o i n t s o n [ C l ] curves are the average of three - four f i b e r s . These curves are f u r t h e r e x p l a i n e d i n the t e x t .  in  the  little a  starting  variation,  given  fiber  T  i n different  however, i n the  barnacles.  T h e r e was  very  [ C l ] ^ obtained  from  fibers  strongly indicate  that  the  in  barnacle. The  of  [C1]  the  Cl  data was  i n Table  confined  to  IV an  extracellular  compartment.  loss Not  TABLE The  extracellular  space  product  of single  chloride  IV muscle  washout  [ci] Bathing solution  50%  [Cl]  Ringer  start  30  30 + 1.1  min,  31 +1.7  At  25%  [Cl]  Ringer  29 + 2,  30  the  constant  65.3 + 3.8  63.2 + 6.8 (4)  Extracellular Space (% f i b e r w a t e r )  min.  47-5 + 3-6  6.6 +0.7 (8)  35.6 + 5-4  6.8 + 0.9 (4)  (8)  29 + 3-0  (9)  T  start  (5)  by  method  Total (mM/Kg w a t e r )  Myoplasmic (mM) At  fibers  55 shown h e r e constant  i s the additional  throughout  fact  the C l  washout  assumed t h a t  the concentration  space  [ C l ] , the fraction  equals  cellular  space,  (  a 0  ) j  c  • A [ d ]  % where in  a  of C l  weight, the  this  fiber In  i n the  remained  I f i t i s  extracellular  water  i n the  extra-  from t h e r e l a t i o n  e  T  ( 1 7 )  are the reductions  and the b a t h i n g s o l u t i o n  space  the fiber  -  of fiber  above e q u a t i o n and t h e d a t a i n T a b l e  of  water  experiment.  t> c a l c u l a t e d  n  A [ C 1 ] ^ and A[C1]  cellular  fiber  ATCTI-;  =  the fiber  that  was c a l c u l a t e d  water.  IV, the size  t o comprise  space  concentration  respectively.  Since the fiber  extracellular  i n Cl  of the  between  contains  With the  6.6  and  75% water  corresponds t o about  extra6.8%  by  5% o f  volume. the course  o f some o t h e r e x p e r i m e n t s , t h e  extracellular  14 space  was a l s o  Bundles  of fibers  containing  0.2  90 m i n , f o u r sucrose  m e a s u r e d b y means o f t h e u p t a k e  fibers  and a n a l y z e d f o r  (2) i n t h e Method  were t r e a t e d  (where  by t h e C l  the size  were examined  i n 50 m l o f n o r m a l  of ' " ^ C - s o r b i t a l sorbital  space,  the  v a l u e o f 6.6%  ( f o r counting procedure  possible)  o r a companion  washout method, d e s c r i b e d space.  Fibers bundle  above,  4 shows t h a t  complete  i n 40 m i n .  the uptake The s i z e  water, agrees w e l l  d e t e r m i n e d by t h e C l  -  washout  to  Four b a r n a c l e s  Figure  of the fiber  solution  f o r 30 s e c i n i s o t o n i c  of chapter IV).  manner.  6.4%  Ringer  section  was v i r t u a l l y  the  C  of the extracellular  i n this  C-sorbital.  A t 1 0 , 2 0 , 4 0 , 60, a n d  were removed, washed 14  f r o m t h e same b u n d l e  determine  soaked  yCi/ml of ^ C - s o r b i t a l .  solution,  see method  were  of  method.  of  with Hinke  56  7 -  TIME  (MIN) I  . 14 F i g . 4. The t i m e c o u r s e o f t h e e q u i l i b r a t i o n o f C sorbital i n the b a t h w i t h t h e e x t r a c e l l u l a r space o f s i n g l e f i b e r s . Each p o i n t r e p r e s e n t s a t l e a s t 16 f i b e r s .  ( 1 9 6 9 a ) has f o u n d a n ' i n u l i n  of f i b e r water f o r the  barnacle muscle.  The  is  to determine e x t r a c e l l u l a r  used r o u t i n e l y  Cl  s p a c e o f 6.5%  -  washout method, because  i t i s simple,  space i n t h i s  laboratory. A clear space i n a s i n g l e  i d e a o f what c o n s t i t u t e s t h e muscle  f i b e r i s o b t a i n e d from the p a r t i a l  c r o s s - s e c t i o n of a b a r n a c l e muscle  fiber  shown i n F i g . 5 .  surface.of the f i b e r i s covered with a t h i n and n o n c e l l u l a r e l e m e n t s .  extracellular  In addition,  layer  The  of c e l l u l a r  the surface  communicates  57  F i g . 5. A c r o s s - s e c t i o n of the s u r f a c e of the b a r n a c l e muscle, showing a l a r g e c l e f t p e n e t r a t i n g the f i b e r . Small tubules a r i s e f r o m t h e c l e f t and a p p a r e n t l y f r o m t h e f i b e r s u r f a c e . C o n n e c t i v e t i s s u e c o v e r s t h e s u r f a c e and f i l l s t h e c l e f t . M a g n i f i c a t i o n x 14,000.  58 with  clefts  which  communicate w i t h transverse muscle cleft  the  tubular  fibers.  of c l e f t s  a volume  by  the  30  be  attributed  system  and  tubules 5%  a l lthe  to this  E^,  RT  (a , )  coefficient passively  Table  across with be  mean r e s t i n g was  the  second  an  -63.5  average  spring  '67  elements  Potential.  5%  can  space  It is i s removed probably  Knowing  t h e myoCl  (14)  a  T^T; i n normal  Ringer  0.65.  other  i o n which  to the r e s t i n g  the average  potentials.  group  ( s p r i n g '67)» mV.  i s not  and  average was  the  group  coupled  thermo-  transported,  -70.9  t h e most  '66),  o f -68.6 mV  E^.  corresponding  (summer  E^  activity  distributed  Por reasons already  considered  i f the  membrane p o t e n t i a l ,  In the f i r s t an  is  i s actively  values  compared w i t h  solution  If Cl  any  are  this  surface.  membrane a n d  results  extracellular  volume.  surface  fiber  o f -73-5  striated  c a l c u l a t e the transmembrane  mV  E^  the  ^ Cl^m  P  mM  fiber  the  equal  III lists  E ^  -  ln  i s a s s u m e d t o be  dynamically should  ci  i s 350  1  can  of  Presumably,  a p o r t i o n of the  Chloride  one  o f an  to  the e x t r a c e l l u l a r  of the t o t a l  from equation  E  fiber.  i n the  tubules  correspond  example  m a k e s up  Cl  Small  i n a large variety  m a t e r i a l on t h e  Cl" activity,  r  seen  s e c w a s h , so t h a t  potential,  where  and p r o b a b l y  i n the muscle  Transmembrane plasmic  the f i b e r .  6 shows an  of about  d o u b t f u l whether  into  clefts  Figure  l o c a t e d deep  system with  penetrate  mV.  In  compared  with  given,  reliable.  the  59  F i g . 6. A c r o s s - s e c t i o n showing a l a r g e b r a n c h i n g c l e f t , deep i n the muscle f i b e r . This c l e f t gives r i s e to a smaller c l e f t (or t u b u l e ? ) . M a g n i f i c a t i o n , x 3 0 , 0 0 0 .  60 D.  Discussion In  n e v e r be  a l l intracellular  absolutely certain  measure what cularly  difficult an  to  of  cleft  should  be  membrane a c c o r d i n g w h i c h we fiber is  used  usually a The  was  potential  open-tipped (a -,)  decreased No  net  ments. to  l o s s or The  and  gain  f o r the  (a  the  in  with  some  was  o  the  In  the  Cl  Cl  of  the  KC1,  water  Cl  or  so  that  the the  of water.  e l e c t r o d e was  microelectrode  20  [K]  o  x[Cl]  micro-  when t h e in  in  solution  electrical and  the  monitored or  hypertonic solutions. i n these  experi-  i t was  expected  concentration  Not  only  i n the was  to  Cl  increased  e x a c t l y as or  there  10  (1969a)  occurred  of'dilution  impaled  parallel  microelectrode  fiber  in  observation  drop  Hinke  often  the  experiments  a l l cases,  changed  accuracy.  The  proof  hypotonic  shift  across  When t h e  increased  Finally,  )  degree  show t h a t that  p 1  One  displays a  magnitude  to  i t is  recorded  containing 3 M  myoplasm.  of myoplasmic  the  indicates p  [K]  i n which  measured to  the  follows.  additional  exposure  parti-  depolarization lasting  myoplasm.  rate  m y o p l a s m c a u s e d by experiment  an  identical  during  i s as  always  microelectrode.  according  (a ,)  time  and  offer  i n experiments  p  to  fact  myoplasm or  i s distributed  -  a micropipette  i n the  was  cases  a Donnan e q u i l i b r i u m .  lowered  changed, the  t i p i s In the  i n both  i f i t i s i n the  constant  electrode was  to  with  was  o  First,  the  can  does i n  microelectrode.  same i f C l  C l ~ microelectrode  [Cl]  remained  the  microelectrode m u s t be  small transitory  depolarization which  Cl  w o r k , one  One  because  from time  i s jabbed  sec.  the  the  t o measure.  determine whether  extracellular  potential  that  i t i s supposed  sceptical  microelectrode  does  of  the  this  myoplasm, i t a l s o  recording  the  changes  o  It  i sd i f f i c u l t  microelectrodes normal  fiber.  a r emeasuring  electrical  Any  ionic  of  with our  )  should  with  o f Ag.  They  seem t o have  micropipette exchanger. measure  With  It such as B r  -  successful a  plugged  thus  that  performance  t h emyoplasm  on t h e e l e c t r i c a l  Tasaki-and  Ag-AgCl e l e c t r o d e  will  Singer  from a  be p o s s i b l e t o  any c e l l .  intracellular  increasing theestimation More  (1970).  chloride ion  i t should  of virtually  be s m a l l .  i s the C l ~  by W a l k e r a n d Brown  microelectrode,  l  electrode i n  promising  a liquid  c  at the t i p  a n d S~ may c o n t r i b u t e t o t h e r e c o r d e d  error should  electrode.  More  i s given  i n other  to duplicate this  almost p e r f e c t  has been mentioned  t h e myoplasm,  This  developed  micropipettes  developed  a ^ i nthei n t e r i o r  d i s p e r s e d by  sensitive electrodes  (1966) r e p o r t e d  at thet i p with  this  o f these  microelectrode  have been  not successful.  achieved  filled  contractures and  be t h e n  Chloride  Attempts  microelectrode  during  n o t be changed f r o m n o r m a l i f  a n d Meech  ordinary  l a b o r a t o r y were  should  thet i p of theC l  Kerkut  a deposit  sensitive  of  p 1  less d i s r u p t i v e dimensions  measurements  of  (a  large and  membranes  as l o c a l  of the  t h e damage i s q u i c k l y r e p a i r e d .  t o r e p a i r and e q u i l i b r a t e .  laboratories.  arer e l a t i v e l y  damage i n t e r n a l  changes i n t h e myoplasm Thus,  the C l  activity  The r a p i d d i s a p p e a r a n c e  indicates that  myoplasm.around  time  t h emyoplasmic C l  damage i s w i t n e s s e d  instability.  diffusion. the  undoubtedly  this  disturbances  how a c c u r a t e l y  These m i c r o e l e c t r o d e s  rough, therefore impalement;  t o assess  significant properties  moieties C l ~potential  of  ( a ^ ) ^  may b e t h e e f f e c t of theC l  -  micro-  (1968) h a v e p o i n t e d o u t t h a t a  n o t come t o a n e x a c t  equilibrium i n the  presence give  of polyelectrolytes that  rise  t o a continuous  electrode.  The t e n d e n c y  loss of Ag  t h e myoplasm a f t e r s e v e r a l  by  a thinning  o f the AgCl  be e l i m i n a t e d  with  +  Ag ; from  t h eb i n d i n g  the surface  of ourelectrodes  in  also  bind  to drift  will  of the  negatively  i m p a l e m e n t s may h a v e b e e n  surface.  Walker  This  source  a n d Brown's  caused  of error  liquid  would  i o n exchange  microelectrode.  The will The  both  possible  increase  (a ) v a l u e s u _i_ m  errors  o f 28.8 a n d 22.4 mM  potentials using  errors small  involved,  the Nernst  t h eBoyle  distributed equilibrium.  t o attach  and E ^ .  a n d Conway p r o p o s a l  passively  across  t h ef i b e r  t o a state, of r e l a t i v e l y  (by  t h e pH o f t h eb a t h i n g  brane p o t e n t i a l e i t h e r remained i fi t had been d e p o l a r i z e d  hyperpolarized that, Ejyj. to  to this  level.  i nt h e d e p o l a r i z e d Most  likely,  t h elowered  which maintains diffusion  the recorded I n view  E^.  E ^ simply  mem-  of the  (1941) t h a t  these  results  C l " i s  membrane i n a D o n n a n - t y p e when t h e b a r n a c l e  of relatively  high  Cl"  low C l ~  permeability  s o l u t i o n ) , t h e r e s t i n g mem-  steady during  a t b e t w e e n -70 a n d -73 mV the handling  procedure,  The h y p e r p o l a r i z a t i o n  fibers,  .  significance to the  Therefore,  from a s t a t e  permeability  or,  o f (a„,) Cl m I I I ) a r e 4.5 a n d  H a g i w a r a e t a l (1968) f o u n d t h a t  m u s c l e membrane was c h a n g e d  reducing  from  equation.  i ti s d i f f i c u l t  paragraph  estimation  (Table  than predicted  d i f f e r e n c e s between  support  i nthe last  theC l microelectrode  3 mM r e s p e c t i v e l y h i g h e r brane  considered  E ^ was s l i g h t l y  larger  h a d n o t h a d enough time  However, t h ee x i s t e n c e  indicates than  t o adjust  o f an a c t i v e  mechanism  (a„,) s l i g h t l y l o w e r t h a n e x p e c t e d f r o m p a s s i v e Cl m ^ ^ ( a s p r o p o s e d b y M o t o k i z a w a e t a l (1969) f o r t h e  63 postsynaptic ruled the  out.  membrane o f t h e l o b s t e r m u s c l e ) c a n n o t Whatever t h e e x p l a n a t i o n ,  conclusion  plasmic  C l  millimoles  activity  nitrate in  [C1]  fibers.  t h e range  not  any d e t a i l s  cellular  space,  results.  i ti sd i f f i c u l t and Hinke  t o be t i t r a t e d  acidic.  Their  a t a very  proteins  i sbased  present  results  Cotlove's  the is  f o r[C1]  T  study  (1965)  Hagiwara d i d  of extra-  C l  -  10 mM/Kg  only  so that  I t i spossible  i s bound by c o n t r a c t i l e Ag  of C l " with  of the similar  (the Cotlove  +  Ag ).  cells  t h e volume  The  +  first  results  A small  (Fig. 5).  latter  a n d was e x c e s s i v e l y  be r e l i a b l e ;  tested.  fiber  the final  o f h i s a n a l y t i c procedure  compared w i t h  mercuric  Hagiwara  was f a r t o o l o w .  should  the fiber  a  those  However, t h i s  proteins  located i nthe small  surrounding  found  nubilus.  on a p r e c i p i t a t i o n  two a n a l y t i c procedures  tissue  T  than  water  Since  to precipitate with  and second because  undoubtedly  (1966)  contained  of [C1]  equation.  32 mM/Kg  or estimation  low pH, c o n s i d e r a b l e  thorough  paper,  a n a l y t i c method  and i snot free  method  1964),  value  myo-  to explain thevariation i n  o f Balanus  Cotlove's  solution  that  using  was o n l y  T  In a later  about washing  i nsingle fibers  the free  arehigher  T  McLaughlin  group m o d i f i e d  [C1]  support  i sw i t h i n a few  o f 40 - 6 0 ' mM f o r [ C 1 ] .  reported  water  measured here  a n a l y t i c method, found  give  muscle  Hagiwara, e t a l (1964),  reported.  single barnacle  that  p r e d i c t e d by t h e N e r n s t  results  T  Hagiwara's r e s u l t s  experiments  of thebarnacle  of theactivity  The previously  from t h e present  be d e f i n i t e l y  because o f (Cotlove  obtained  fraction  from  of [C1]  T  o f the connective  Since  cells  i sminute  their  c o n t r i b u t i o n t o [Cl]„ c a n b e i g n o r e d . -  t h e volume  o f t h emuscle  o f these  fiber,  64 The  existence  of the s i n g l e cally  fiber  determined  single  tissue  fibers 1966)  may a l s o  m  T  the  If  a  i nthefiber,  of free  space  hence,  large  [Ci] ,  c a n be  each  s e to f r e s u l t s .  C l  i n t h e myoplasm  of fiber  water i n  then from Table I I I ,  respectively.  i nTable  III  a  This i salmost  i f a l lof theintracellular  a l lof theintracellular  water.  i t i ssimply fortuitous.  m  C l  will  f o rt h e = 0, 0.92 f o r  be about thevalue  i sdissolved  to rule  expected  uniformly  C o n v i n c i n g as t h i s  As a l r e a d y  l e d t o an i n f l a t e d  water  I f we a s s u m e  exactly  seem, t h e r e i s no e v i d e n c e h e r e  may h a v e  T  f o r i n these two compartments.  = 537 mM/1,  results  the (Cl) results m  errors  that  (18)  to  from  that  indicate  and t h e myoplasm r e s p e c t i v e l y , and  m  might  as t h e crab  manner:  C l ~ not accounted  summer a n d s p r i n g  in  -  ( C l ) + B ] i s 4 0 . 3 a n d 3 2 . 0 mM/Kg f i b e r m C l  (0.935)  micrographs  n  = O.O65 a n d [ C l ]  Q  pre-  = a [ C l ] + a (Cl) + B„ o o m m C l  extracellular any f i b e r  m  C l  ] , a and a arethefractions in o m  is  [a  analyzable  m  v i m  a  analyzable  Electron  and t u b u l e s and,  ( C l ) i s theconcentration p  of thetotal  ( B r a n d t e t a l 1965)  clefts  i nthefollowing  ( a - , ) /y  a multifiber  o t h e r crustacenas such  large  total  [Cl]  [=  like  that  spaces.  The  where  fraction  and c r a y f i s h have  extracellular  expressed  from  o c c u p y i n g 5%  I t i s now a p p a r e n t  i snot i n t r a c e l l u l a r .  +  space  t h e u s e f u l n e s s o f chemi-  must be t r e a t e d  a large  C l " and N a  (Peachey  decreases  [ C l ] ^ , a n d [Na],p.  i nwhich  of muscle  they  volume  barnacle fiber  paration  o f an e x t r a c e l l u l a r  agreement  out the p o s s i b i l i t y  indicated,  value f o r (Cl) .  a number Also  of  McLaughlin a  m  and Hinke  i s between 0.6  eating  K  cellular nature  water.  of this  to  believe  if  Cl  and Hinke  forK  since  found  fraction  necessarily  that indi-  of the i n t r a -  t h e r e i s no e v i d e n c e a s t o t h e  n o n - s o l v e n t w a t e r , t h e r e i s no a p r i o r i  i t must  water,  have  3  from a s i g n i f i c a n t  However,  (1969a)  i n the barnacle fiber,  exclude C l  i s excluded from a s i g n i f i c a n t  cellular wrong.  and 0.7  exclusion  +  (1966)  then the assumption  -  also.  fraction  that  reason  Obviously,  of the i n t r a -  = 0 will  also  be  66 CHAPTER I V  THE HETEROGENEOUS D I S T R I B U T I O N OP IN  A.  THE B A R N A C L E  contradictory offer  direct  Nernst  that  fiber  results  Conway  Cl  .  Casteels 1968),  water.  which  cellular  could  evidence  others  muscle  results  results  t h e membrane  according  to the  this  i f equal  -  this  (Wood  Dunham  the assumption  have  1965;  and of  Gainer intra-  i o n (Buck and Goodford  I n Chapter fractions  1968;  1966;  I I If o r homogeneity  of intracellular C l ~ Since  kind of heterogeneity 1962,  muscle  a n a l y s i s o f muscle  equation  from t h e myoplasm.  (Dunham a n d G a i n e r  i960).  Adrian  1967;  Huddart  with  (Boyle and  some i n v e s t i g a t o r s  of the Nernst  The e v i d e n c e  are excluded  with  a l l of  f i tperfectly  s t u d i e s on o t h e r  evidence,  f o r the C l  that  dissolved i n a l l  muscle  1959b;  have q u e s t i o n e d  homogeneity  f o rjust  a r e many  1966;  be c o i n c i d e n t a l  water  solution  i n the frog  of this  and Kuriyama  et a l 1968).  Hays  These  are- i n c o m p a t i b l e  the v a l i d i t y  while  across  and f r e e l y  Hodgkin and Horowicz  In the light  questioned  Ringer  i s uniformly  However, t h e r e preparations  The e l e c t r o d e  c a n be i n t e r p r e t e d t o i n d i c a t e  s t u d i e s on C l  1941;  I I I a r e i n n o way  C l "i s d i s t r i b u t e d  i n normal  of the i n t r a c e l l u l a r classic  i n Chapter  The a g r e e m e n t b e t w e e n t h e e l e c t r o d e a n d t h e  intracellular  the  presented  t o t h e membrane t h e o r y .  equation.  chemical the  results  proof  of the muscle  and  MUSCLE  Introduction The  Cl  CHLORIDE  Dunham  there i s  i n crustacean e t a l 1964;  67 Hays et Cl  in  1968;  al the  barnacle The  compartment rate  of  in  22+Na  a  the  of  -  the  chapter a  of  is  low  to  necessary  in  a  out  contained  the  on  single  system.  the at  and  least  the  fibers.  large  the  i n t r a c e l l u l a r water.  fraction  between the III  was  B.  Methods  chemical  indeed  The  ceeding  of  group  on  by  fibers  potential  of  single The  the  Cl  from  single  C l " of  a  [Cl].,  fibers  fibers  cellular  of  Cl" analysis  the  was  of  results  outflow 3  6  Cl"  indicate  bound  reported  and  or  from  a  agreement  in  Chapter  f i b e r to  extracellular  been d e s c r i b e d  also  course  f i b e r was a  has  space  each b a r n a c l e .  during  Cl  the  Thus, the  results  +  compart-  C l " i s excluded  w a s h o u t m e t h o d , on  fibers  centration,  electrode  extracellular  of  analyzable  myoplasmic  Na  coincidental.  chapter.  determined,  and  dissection,  determination  group  of  two  intracellular Cl" i s either free  and  study  i n which  The  and  the  Allen  kinetics  compartmentalized  one  Intracellular  experiments  bath  follow  microelectrode  in  study  more t h a n  system i s to of  to  means.  illustrating  show t h a t  [Cl]  on  the  or  of  sensitive  +  reports  studied  half  ion  into  a Na  muscle  Cl" into  about  ion  an  i n f l u x study  exchange were that  for  i t was  some o t h e r  common m e t h o d  combined  This  fiber  m u s c l e by  state  barnacle  ments. of  or  (1970)  Hinke with  most  flow  1-969),  Richards  The  the  routinely obtain  pre-  single  a  representative  resting on  fibers  a  representative  from  Extrathe  i n t r a c e l l u l a r Cl  i n mM/Kg i n t r a c e l l u l a r w a t e r .  was  membrane  experiment.  subtracted  the  the  of  measured  of  in  space  con-  Employing  68 the  terms  and  free  of  (18)  equation  water  and  Cl  i  not  distinguishing  between  bound  :  [Cl]™ ^  but  .-. a [ . C l V  ( 1 - , ) "  =  (  1  9  )  o  Experiment The  C l ~ content  I:  Effect  of Ringer  on  solution  [ C l ] ^ when  [Cl]. i s  (see  I ) was  substituting  i t with methanesulfonate  basis.  solutions  Pour  mM/1  were used.  5 hr  at  10°C  with a  Groups  i n Ringer  of  Each  group  transferred  to  similar  ion  and  4°C. 1  a l l the  The  hr  day  by  chemical  the  Some f i b e r s w e r e degree  of  content  of  20  mM  was  12  and  were N0~ 24  [ C l ] ) was  c o n t a i n i n g 0.2 to  equilibrate  before  ^Cl~  f o r Na the  overnight  K  +  [Cl]  then Cl at  t o w a r m up  to  for  for [Cl]^ method).  check  the  The  ion  experiment.  i n Ringer  low  overnight  tracer  and  +  for  uCi/ml  they--were a n a l y z e d  h a l f by  zero  soaked  i n zero  f i b e r s were a l l o w e d  and  and  that  preliminary experiments, r e m o v e d more r a p i d l y  s m a l l a m o u n t o f N0~  fibers  67,  solution  was  observed  that  constant.  In Cl  soaked  134,  by  equimolar  of these  left  also analyzed  fibers  269,  an  i n each  i o n exchange throughout  remarkably  fiber  method  of  reduced  and  solution  at room t e m p e r a t u r e  (half  a  (except  groups were  following  (CH^SO") on  d i s s e c t e d f i b e r s were  solution  solutions.  a  [Cl]  Table  reduced.  + hr  was  equilibrated 517  mM  for Cl  i n zero  present. in a  zero  CH^SO" a n i o n s analysis.  i t was  To [Cl]  and  [Cl]  Ringer  document t h i s Ringer  removed  at  when  observation,  solution  with  0,  4,  1,  2,  8,  6 9  —  Experiment' solution. stoney in  At l e a s t  baseplate)  a 50 m l b a t h  isotope. 4°C  least  at  0,  50  from  Influx  of  The b a t h  was  12  4 fibers  one b a r n a c l e  kept  hr.  of fiber  constant  from  A t 0.5,  0 t o 24 h r .  (attached to  1,  Ringer their  12  2, 3 , 4, 8 , 11, ^f — Cl  4 fibers  time  uCi/ml ^ C l ~  f o r the f i r s t  h r and and 24 h r  isotope analysis,  and  were removed f o r c h e m i c a l  F i b e r C l ~ was  -  normal  c o n t a i n i n g 0.2  were removed f o r  C l .  from  were p l a c e d a t z e r o  a t 10°C  8, a n d 2 4 h r a t l e a s t  analysis  Cl  dissected fibers  of normal Ringer  f o r the next  at  II:  found  t o remain  F i v e b a r n a c l e s were used  nearly  i n this  experiment. Experiment Ringer  solution  I I I :  Comparison  and C l ~ l o s s  Companion groups  of fibers  into  from  of ^ C l ~ influx  N0~ Ringer  five  solution.  barnacles 9  normal Ringer Ringer 1,  2,  solution  solution 3,  c o n t a i n i n g 0.2  were p l a c e d i n f  uCi/ml  — Cl  ( a l l t h e C l " r e p l a c e d by N 0 ~ ) .  and 5 h r f o u r f i b e r s  from  were removed from  — or i n  A t 0.25, normal  N0^ 0.5,  Ringer  for C l a n a l y s i s and f o u r f i b e r s were removed from N 0 ^ Ringer f o r [ C l ] ^ analysis. At 0 and 5 h r , f i b e r s were a l s o •~,c  removed  from  _  Cl  Ringer  5 hr p e r i o d i n the ^ C l [Cl]^  rose  increase  from  was  26  and a n a l y z e d solution  for  Cl  over  were washed i n e x a c t l y  chemical  analysis.  beta  counting  fiber  This  Fibers for  C l ~  t h e same m a n n e r a s f i b e r s f o r  Three methods  i n a liquid  water.  the  the 5 hr period.  counting.  counting  During  ( a t room t e m p e r a t u r e ) ,  t o 30 mM/Kg i n t r a c e l l u l a r  assumed g r a d u a l  Procedure  for [Cl]^.  of preparing the f i b e r f o r  scintillator  were  tried.  70 (1)  The d r i e d  90°C f o r 45 m i n .  cedure 1.5  The p r o t e i n  2 m l o f 4% Z n S o ^ i n 0 . 4  with  as used  i n the digest M HNO^  precipitated  ( t h e same d i g e s t i o n  I n t h e Cotlove method).  solution:  60 gm n a p h t h a l e n e , 4 gm  100 m l m e t h a n o l ,  After  pro-  centrifugation,  20 m l e t h y l e n e  2,5-diphenyloxazole  l,4-bis-[2-(5-phenyloxazolyi)]-benzene dioxane (2)  (Bray  A fiber (3)  fiber  water  ghost  persisted  100 mg w e t t i s s u e  seldom  dissolved  fibers  was m e a s u r e d water  10 m l o f B r a y ' s  ml bath  The d r i e d  and  gm/1  with  fiber  and t h e water  1  added.  (1 m l S o l u e n e p e r  and t h e m i x t u r e  was n o t u s e d  because i t  The w e t w e i g h t  volume  ml  calculated  of these from the  The f i b e r - S o l u e n e  w i t h 15  ml o f toluene c o n t a i n i n g  samples  were  4 gm/1  PPO  POPOP.  All  Cl  means o f a N u c l e a r C h i c a g o channel r a t i o  t h e energy samples  gm  solution.  c o n t e n t . o f companion f i b e r s .  was d i l u t e d  fiber  s o l u t i o n was  solution)  i n t h e Soluene-100.  mixture  that  a n d 0.2  o v e r n i g h t i n 0.5  i n the counting  o r 0.1  overnight.  A double  glycol,  (POPOP) t o 1 l i t e r  was p l a c e d i n S o l u e n e - 1 0 0  shaken  0.2  was e q u i l i b r a t e d  and then  The wet f i b e r  percentage  (PPO),  scinti-  I960).  The d r i e d  distilled  1  was  M NaOH a t  m l o f t h e s u p e r n a t a n t w a s a d d e d t o 10 m l o f B r a y ' s  llation  by  ml o f 0.6  f i b e r , w a s d i g e s t e d i n 2.0  levels  counted  a t about  scintillation  85%  counter  efficiency  (Series  720).  c o u n t i n g m e t h o d was e m p l o y e d t o i n s u r e o f t h e b e t a e m i s s i o n s w e r e t h e same  as from b a t h  samples.  The s o l u t i o n  from  S o l u e n e - 1 0 0 i s a m i x t u r e o f q u a t e r n a r y ammonium b a s e s m a n u f a c t u r e d by P a c k a r d I n s t r u m e n t Company, I n c .  from  the three  i n toluene  71 methods were days.  clear  and  30  fibers,  solution,  fiber  order  fibers  Cl  results  Although  statistically e r r on  that is The 2  are  were  the  be  a reflection values (before  the  low  highest  for  i n Table  the  results  side relative  the  rate  best  by  method  comparison  of  the  Method  standard  the  for  VI,  of  on the of  be  sample  Fiber Cl exchanged (mm/Kg f i b e r w a t e r )  error  + 2.1  46.0  +  1.8  49.1  +  1.3  *  ways.  of  not  the  the  2  third  reasoning  bath  sample)  the  sample.  determined  counting  47.3  three  amount  That  — Cl  Ringer  -  t h a t method  V  3 methods  C l  means a r e  3.  to  rate.  extracellular  3 i n experiments  36 preparation  to  dissolving  the  for  o b t a i n e d ) , may  TABLE A  as  geometry  i n Table  was  in  suggest  (relative  for  of  i s based  counting  C l ~ exchange  were p r e p a r e d  V  to method  most r e l i a b l e  counting of  overnight  d i f f e r e n c e between the  the  3 began  methods  C l " (uncorrected  Soluene-100  the  three  t r e a t e d i n one  listed  significant,  should  the  Fibers  fibers  four  a reduction i n counting  compare the  exchanged w i t h  space).  results  to  f o r about  s o l u t i o n s from method  were e q u i l i b r a t e d  10  then  average  c o n s i s t e n t counts  often demonstrated  In  may  gave  T h e r e a f t e r , sample  discolor  The  and  5-10%  by too  method low.  I I and I I I .  TABLE The e f f e c t  Bath [Cl] mM/1  of decreased  Water Loss  o  Intrafiber [Cl],  VI  [ C l ] on  [Cl]  j L  and [  Concentration [36 i], C  mM/Kg  3 6  C 1 ] exchange  Intrafiber Concentration x Volume [C1].V t [36 ] v mM C 1  H 0 2  1  n > 20 537 (Ringer)  25.9 +0.8  3 7 . 4V  7.6  34.1 +0.4  20.9 +0.8  31.5V  134  9  30.5 +0.6  14. 7 +0.6  2 7 . 7V  67  10  25. 3 +0.8  8.6 +0.6  23.  12  16. 5 + 1.0  * extracellular V = V  o  x  (1 -  Cl , %  w  a  o  o  19.3V  o  13.4V  o  0  ov  7.7V 0  1 4 . 5V 0  C l , and water s u b t r a c t e d  ^ 100 l  o  fibers.  Expressed  Cl~  has been l o s t  that  —  25.9V 0  269  0  t  37-4 + 1.2  s  5)  w h  i n this from  ere  V  manner,  i s the volume o f water o [ C l ] ^ V shows  the f i b e r .  i n the Ringer  t h e a b s o l u t e amount  &  of  Q  C.  Results Table  content Not  after  [Cl]  Since the  the  mean c h a n g e s  equilibration  observations  l o s s of  C l " was  [ K ] ^ remained  Cl  ions  (1964,  al  occurred  i n the  drop  and  fibers  were  i n 67  Repolarization  [C1]  must  and  have  [Cl]  5 and  20  occurred  Cl  ions.  reflection necessary (V[C1] ) ±  fibers [Cl] a  This  of to  the  reduction  i n Table  VI  7 to  in Fig.  Cl  loss.  67  have As  and  0  night. left  found  with by  depolarization For  example,  observed  when  solutions respectively. since  no  (average  absolute to  Included  exchange w i t h  exchangeable  Only  a  significant  difference  -77  observed  fibers  mV)  was  Cl  real  that  fiber of  about  Cl  the 15  hr  water  are  not  as  well  an  exact  It i s , therefore, intracellular  losses are  the  Cl"  i n Table VI the  .  q u a n t i t i e s of  outside  lost  [ C l ] ^ values  These v a l u e s  fraction  even a f t e r  the  show t h e  the  the  in fiber  illustrate  f u n c t i o n of  solutions.  that  means t h a t  include  Also of  i n Table VI  i n each, s o l u t i o n .  linear  [Cl]  hr.  Notice as  must  solutions.  [Cl]  mM  i n the  i n p o t e n t i a l were 0  and  through the  ions  +  solutions.  Cl  134  and  i n 5 hr;  water  I n mean p o t e n t i a l s b e t w e e n g r o u p s at  269  water  [Cl]  l o s s of  a transitory  drop  Q  of  continued  the  i n low  8 mV  an  rate  some K  to  1968),  fibers  5 mV  Cl  i n [ C l ] ^ and  i n reduced  In the  constant,  in proportion  et  on  complete  s o l u t i o n s , l o s s of  Hagiwara  a  the  in resting potential.  solutions, mM  lists  overnight  shown a r e  changes  VI  of  C l  plotted  fiber  Cl  from  -  Cl the  against  content  is  not  concentration.  and  Fig.  i n four fiber of  7 are of  the  the  five  C l " appeared  equilibration  results  to at  bathing be 4°C.  Fig. and  f.  Relation  the intracellular  cellular soaked the  Cl~  including ( 5 1 7 mM this  Clearly,  Pig.  with  8,  3  ^C1~,  space  CH^SO" + 20 mM experiment; there  -  solutions  The f i b e r s and then  of intracellular Cl~)  into  rates  [Cl].  a 0[C1]  [  c  l ]  were  overnight i n  C l Q  -  loss (not  solution  Two b a r n a c l e s  contains  data  from  from t h e f i b e r .  i s associated  Since  of the bath,  added.  each p o i n t  a r e two e f f l u x  Cl~.  Vl^ci ]  N0~) i s s h o w n .  = 0 h r , the slow r a t e  intracellular  ^ Cl  the rate  extracellular  concentration  0  c o n t e n t , V [ C 1 ] ^ , o r t h e amount o f i n t r a -  exchanged w i t h  same s o l u t i o n s  for  Cl  -  f o r 5 h r i n the low [Cl]  In  t  between t h e C l  with  about  w a s 3 2 . 8 mM/Kg  were 8  used  fibers.  At  55% o f t h e intracellular  • 75 water slowly  at the  start  of this  experiment, then the  e x c h a n g i n g p o o l o f C l ~ was  P i g . 8. The i n which the 20 mM NO-,.  18  size  of  the  mM.  l o s s of Cl. from f i b e r s bathed i n Ringer s o l u t i o n C l ~ h a s b e e n r e p l a c e d b y 517 mM CH SO" and 5  5  The influx Fig.  9-  results  from normal  o f t h e second  Ringer solution  As i n F i g . 8 ,  this  8  experiment was m e a s u r e d  data reveals  12  16  TIME (HOURS)  i n which  i s shown i n  at least  20  Cl  two  exchang  24  F i g . 9Exchange o f i n t r a c e l l u l a r C l w i t h ^ C l i n f i b e r s b a t h e d i n n o r m a l R i n g e r s o l u t i o n a t 10°C. Percent C l ~ unexchanged = 1 - ( s p e c i f i c a c t i v i t y i n t r a c e l l u l a r C l ) / (specific activity Ringer Cl~). Each p o i n t represents t h e mean o f a t l e a s t 2 0 f i b e r s . -  77 rates  forintracellular  either  incomplete  estimation [Cl]^  cooling  of the size  was r e l a t i v e l y  reason  C l .  i sprobably  The. d a s h e d  f o r t h e slow  contains plots  about  could  of theextracellular.space.  small i nthis  correct.  1 0 . 4 mM o f C l  o f t h e two curves  experiment,  The a v e r a g e water.  i s 39%,  rate  indicate  t o 10°C a t t = 0 h r o r a n u n d e r -  was 2 6 . 6 mM/Kg i n t r a c e l l u l a r line  line  -  As t h e i n t e r c e p t exchanging  time of the  pool  From t h e s l o p e s o f s e p a r a t e  i n F i g . 9,  k^) f o r t h e two compartments . -1  t h e former  [ C l ] ^a t zero  the slowly  .  Since  therate  a r e 1.1  x 10  constants  -2  (k-^ a n d  a n d 4 . 6 x 10  -4  m m  The the  rates  with the  size NO^  o f exchange  results  two curves  of fiber  are very  to theright).  same i n b o t h  Separate  plotting  revealed  k  similar  1  exchanging  of the fast  t o b e 2 . 3 5 x 10  f o r both It  (abscissa  i n which  NO^ ( c u r v e A ) a n d  f o r curve  B has been  water).  The  i s 1 3 mM f o r t h e C l -  curve (B).  and slow p a r t s t o curves -1 min  Notice  [ C l ] ^ a t t = 0 h r was  compartment  A and B -3  and k  2  t o be 1.9  x 10  curves.  s h o u l d be mentioned  i n zero  returned  with bath  ( 2 6 . 2 mM/Kg i n t r a c e l l u l a r  -2  potential  -  experiment  ( a ) a n d 1 2 . 4 mM f o r t h e C l " - ^ C l  curve  placed  C l  A l s o t h e average  groups  of the slowly  min  of thefinal  ( c u r v e B) a r e compared a n d shown i n F i g . 1 0 .  C l  shifted the  average  C l (NO^) R i n g e r  i n c r e a s e d about t o normal  within  potential  i sconsistent  permeable  t o NO^ t h a n  5 mV  that  when t h e f i b e r  solution,  theresting  was  first  membrane  ( - 7 7 t o - 8 2 mV) b u t g r a d u a l l y  an hour.  This transitory  with the idea that  to C l " ions.  change i n  t h e membrane i s more  78  P i g . . 10. Comparison of the l o s s of i n t r a c e l l u l a r C l from fibers b a t h e d i n NO, R i n g e r ( A ) a n d t h e e x c h a n g e o f i n t r a c e l l u l a r C l 36 — with Cl i n f i b e r s b a t h e d i n n o r m a l R i n g e r . ( B ) a t 24°C. Each p o i n t r e p r e s e n t s t h e m e a n o f a t l e a s t 18 f i b e r s . -  J  79 D.  Discussion Single  compartments, external  C l  compartment Fig.  8,  -  each w i t h  t h e NO" i o n ;  10  were  clearly  the  rates  and  9,  the C l " i o n (  probably  (24°C i n F i g . It  because  or the slow  f r e e , myoplasmic  (about  Cl .  likely with  Cl  our i n f l u x  micro-injected, Cl  ) exchange  be n o t e d ,  this  rate that  from F i g . 8  operating  temperature  from  this  data  Cl"  whether  represents  w a s made t o m e a s u r e  3  )  t o permit  o f (a„ ) . Cl m n  by R i c h a r d s  and Richard's  microelectrodes  an a c c u r a t e  The f a s t i n f l u x  (1969) fiber  rate rate  following  efflux rate  compartment.  estimate  micro-  o f t h e crab.  Since  c o m p a r t m e n t was s u r e l y  compartment.  a fast  C l " i n t o NO" R i n g e r b u t t h e  i n t o the muscle rate  from  however,  the rates  the C l " sensitive ri  t h e same i n t r a c e l l u l a r  plasmic  -1  a t 17°C) c a n be c o m p a r e d t o t h e e f f l u x  of  of  8 and 9 ) .  An a t t e m p t  a t l o w (a.  (0.044 a t 17°C) o b t a i n e d injection  I n a l l cases,  min  apparent  of fiber  of decrease  0.017  -3  of the increased  were i n c o n c l u s i v e ;  were t o o e r r a t i c  estimates  c a n be o b t a i n e d  fraction of intracellular  (a^,-, ) d u r i n g t h e l o s s Cl m °  the rate  10  f a s t e r than  i s not immediately  the  of  Cl").  10 a n d 10°C i n F i g .  fast  the four  with  9 and 10B, i n t r a c e l l u l a r  I t should  10 w e r e  the  results  (ca.  identifiable.  from F i g .  which  i n Fig.  -1 min. ) and a slow  (ca.  VII lists  o f exchange  8 and 10A, i n t r a c e l l u l a r C l "  In Fig.  exchanged w i t h -2  Table  s i z e and exchange r a t e  9 and 10.  two i n t r a c e l l u l a r C l  a different rate  (or N0~).  exchanged w i t h Cl  f i b e r s a p p e a r t o have  Most  are associated Richards  i n the free  myo-  80  TABLE V I I Size in  Figure  and exchange single  517 C H S 0 3  Temp (°C)  3  of C l  -  compartments  f i b e r s from barnacle  Bath Anion (mM/1)  rate  muscle  Chloride Compartments mM/Kg K-.X10 mM/Kg Intra H 0 . -1 I n t r a H 0 2 mm 2  K xl0 . -1 mm  2  o  3  p  o  10  14.8  0.9  18.0  0.3  10  16.2  1.1  10.4  0.5  24  13.8  2.3  12.4  1 . 9  24  13.2  2.3  13.0  1 . 9  2 0 NO"  9  537 C l "  (  3 6  C1")  10A  537 N 0  10B  537 C l "  (  3 6  3  C1")  The a s s o c i a t i o n  of a small  the  loss  of the fast  the  fast  f r a c t i o n i sthe free  The  similarity  either  C l  internal finding  C l  c a n exchange w i t h  1969).  with  suggests  that  myoplasmic C l " compartment. i n F i g . 10 i n d i c a t e s  that  a n d e f f l u x a r e n o t l i n k e d i n a n y way o r  i sconsistent  (Richards  f r a c t i o n i n F i g . 4A a l s o  o f t h e two c u r v e s  influx  hyperpolarization  with  NO^ a s w e l l  Richards'  There i s evidence  as with  findings that  Cl  Cl .  on crab influx  This  muscle  and e f f l u x  are  linked  see  Moore  the to  muscle  o f thebarnacle  i n other  not provide  exchanging  (0.4  V I I (these  any o f t h e i r  t h e slow  exchanged  fraction  constant  authors,  'slow'  fraction).  contains  equally efficiently 3  min"  1  a t 10°C a n d 1 . 9  not  exchange  as r e a d i l y  the  fiber  fiber  (1970)  contains  Our r e s u l t s  min"  3  the fiber  an anion  ( e . g . CH^SO" s u b s t i t u t i o n  and Hinke  with  had d i f f i c u l t y i n  C l " , and t h a t  x 10~  must be l o c a t e d i n s i d e  Allen  reports  indicate intra-  i t c a n be  e x t e r n a l C l " o r N0~  fraction  with  earlier  50% ( 1 3 - 5 mM/Kg  nearly  with  C l " has been  f o r comparison  i nfact,  Cl  barnacle  permeable  (Dunham e t a l 1 9 6 4 ;  muscles  water) o f the i n t r a c e l l u l a r  x 10~  more  of internal  Unfortunately, these  a clear-cut rate  i n Table  cellular  indicates that  i sslightly  fraction  crustacean  1968)/.  Dunham a n d G a i n e r  that  study  The  toCl".  demonstrated  those  i nthis  muscle  A slowly exchanging  do  m u s c l e membrane i s l e s s  h y p e r p o l a r i z a t i o n observed  N0~ t h a n  1961)(however  Adrian  t o C l " (Hutter and Padsha 1959).  t o N0~ t h a n  membrane  1958;  (Harris  1969) and t h e t h e f r o g  permeable small  i nfrog  which  an i n t e r n a l  Na  +  a t 24°C).  because  i t does  at  a rate  Cl both  the size  internal may  (0.3 x 10"  exchange  fractions  water) which  recently that the  fraction  exchanges w i t h  min"  3  (0.4 x 10~  3  1  ( 1 2 mM/Kg  e x t e r n a l Na  a t 4°C) q u i t e s i m i l a r  min  -  1  compartment  f o r some r e a s o n  i t i stempting  such  +  (  t o imagine  NaCl.  This  +  Na )  t o t h e slow  and C l  as t h e s a r c o p l a s m i c  concentrate  22  a t 10°C) r e p o r t e d h e r e .  and r a t e constants o f t h e N a  are similar,  7).  V I and P i g .  + intracellular  This  does n o t p e n e t r a t e  i nTable  reported  1  Since  slow  a common  r e t i c u l u m which  i d e a has been  82 advanced before Simon 1959; Harris  15%  of  1967;  view  that  concentration  such  f o r the  to  Attempts  1966),  not  penetrate  antimonate, what  they  around they Ca  + +  of  .  Ca  + +  ,  ever,  skinned  an  the  that  of  the  sarcoplasmic  precipitated  with  i n the  sarcoplasmic  of  that  Ca  reticulum.  reticulum,  Na  and and  +  f i b e r s and  i t is is  C l  -  of  1962;  be.  pyro-  observed  NaSb(OH)g Apparently,  precipitates  i n the  of  i f Na  did  with  observed p r e c i p i t a t i o n  dog  is necessarily  C l " may  in no  applied  terminal  myocardium.  electron micrographs  Even  a  Conway  (Komnick  also  they  reticulum  + +  be  f r o g muscle  reticulum.  pyroantimonate  their  at  frog muscle;  p r e c i p i t a t i o n of  pyroantimonate  Na ,  support  NaCl  seems t o  (1966)  Engel  claim  1969)  p r e c i p i t a t i n g agents  sarcoplasmic  (1969)  Podolsky  stressed  (Carey  f r o g muscle  abundant  Langer  author  and  and  contain  i n vivo  should  reticulum  H o w e v e r , as  the  muscle,  Peachey's  Davey  intracellular  Tice  not  this  the  fibers.  inspection  may  solution  because  +  and  r e s u l t s when the  Ringer  but  close  convince  and  outside.  probably  realize  Legato  Birks  have been u n s u c c e s s f u l  membranes o f  d i d not  the  localize  was  (Costantin  i n the  frog  compartment  i s concerned, there  of  Sb(OH)g, to  thought  the  cisternae  the  the  the  explain  muscle h i s t o c h e m i c a l l y  For  sarcoplasmic  reticulum  compartment  to  the  1968;  sarcoplasmic  Cl  of  1957;  al  agrees with  studies  the  for this  Zadunaisky  volume  Steinhardt  f a r as  1963-  i n t e r n a l NaCl  More r e c e n t  i n certain kinds  1954).  an  Simon et  Harris  water which  s i m i l a r to  required  placed  a l 1963;  fiber  1954;  Conway  and  I I , as  evidence only  the  1965).  Keynes  Chapter  that  (13%)  estimation (Peachey  and  Conway e t  estimated  contain  the  (Carey  +  The  the  i s not two  does  only  Hownot  cation  present parallels  in  83 between t h e two slow the  p a i r i n g o f t h e two i o n s  If in  barnacle frog  was a t t e m p t e d ,  partment  (see P i g .  would  could,  t o be much  NaCl  verse  tubules  which  C l  1965)j  otherwise  of course,  be p a r t  -  compartment w i t h  Although  reticulum than  i nthe  of this  com-  the permeability and B i r k s  i s leaving the fiber  several  other  of  and Davey  i t w o u l d be i n c l u d e d i n space  (the sarcoplasmic extracellular  and  i960;  The Cl  +  that  myosin  membrane a n d Gage  conditions  the literature  forCl  d i d not locate  While et a l  of the C l muscle  -  (Hodgkin  1969). f o r the slow  exchange  1947). binding  rate  I t has been known f o r  small'quantities of C l  (Szent-Gyorgyi  a d d i t i o n a l evidence  of this  to protein.  can bind  conditions i n  (Foulks  most  of a  the trans-  ( G i r a r d i e r et a l 1963).  alternative explanation  (and Na ) i s b i n d i n g  some t i m e in .vitro  Eisenberg  that  s w e l l under  indicate that  i s i n the surface  Horowicz  the observation  i n the f r o g muscle  studies  no  i n the  o f t h e measured  o f t h e c r a y f i s h muscle  permeability  of  (1963)  of the extracellular  same h a s b e e n o b s e r v e d  mised  water.  The p e r m e a b i l i t y  by H a r r i s  were  i n the bath, i t  smaller  lower than  muscle  I t w o u l d be i n t e r e s t i n g t o r e l a t e t h e p o s s i b i l i t y  reticular  for  of NaCl  i ti s certainly  proposed  measurements  space).  i n the barnacle  of the.sarcoplasmic  5 and 6 ) .  evidence f o r  compartment.  t o 3% o f t h e f i b e r  f o r the frog muscle;  reticulum  the  have  compartment  (1969) the  o f t h e volume  muscle  i n one  at the concentration  be d i s s o l v e d i n 2 . 5  estimation  i s not conclusive  the non-myoplasmic NaCl  a compartment  would  the  compartments  Saroff  under  (1957).pro-  t o myosin but a  such a paper.  Certainly,  search  84 other  proteins  recently, various  NMR  are has  capable been used  complexing  Zeppezauer  et  also  NMR,  using  agents  (1969)  al  of b i n d i n g  report  to  on  Unfortunately,  live  Thus the  neither the  binding  slowly  nor  Cl  they  1967;  anion  a v a i l a b l e evidence  Cl"  1969).  Ward and  (1970),  Forsen  binding  to  a  number  studied n e i t h e r myosin  compartmentalization  exchanging  More  association with  Happe  significant  1952)..  (Carr  -  G i l l b e r g - L a Force  of p r o t e i n s . tissue.  detect  (Ward and  and  C l  at as  present the  nor  favours  explanation  for  fraction.  96  From the entries  i n Table fast  two  C l ~ isotope  V I I ) , the  estimated  i s 14.7  (see  average  concentration  Cl"  i n the  The  a v e r a g e r e s t i n g m e m b r a n e p o t e n t i a l was  537  mM/1  i n these  distributed to  the  C-l  fraction  Nernst  does  about  55%  i n fact of  the  the  But  membrane o f  equation  concentration,  phase  mM/Kg i n t r a c e l l u l a r  experiments.  across  (E  [Cl] , i  =  m  59  the  -77  mV  barnacle  about  26  mM.  to  can  be  the  solvent  was  be  according myoplasmic  I f the Cl  of  [Cl]  muscle  free myoplasmic  water  and  ), the  i  10B  water.  C l " appears  [Cl] /[C1]  be  the  intracellular  since  log  should  represent  9 and  studies  fast  , then  only  a c t i n g as i t s  solvent. Several in  the  be  C h a p t e r V) 55%  and  85%  estimated of  another  r e p o r t , Hinke  solvent  water  from  estimates  myoplasm compartment  (1970; to  other  71  to  78%  i n the of  the  are the  of  available. solvent  intracellular (1970)  water  described  myoplasm.  The  intracellular  Hinke  water  water and  content Gayton  f o r C l " and  respectively.  seven estimates  numerical water  of  values the  K  +  Inof  the  ranged  fiber.  Working  85 with  the brackish-water  solvent  o f muscle  C l " i o n a n d 71%  the In  water  a l lthese  crab.  studies,  +  be  or Na  +  data.  the estimation  excluded  the  entry  that  of K  which  the  +  will  This  i n the v i c i n i t y  attract cations  region  of  the charge  of  the ions  o f the double  region.  (see  Conway a n d G o r d o n  In hexagonal depends  a recent  close  will,  paper,  i n turn,  diffraction  pH, i o n i c  On t h e o t h e r filaments  may  which  permits represent  charged  of the double excluded  layer from  excluded  i s known a b o u t o r about  pro-  In the  l a y e r and p a r t i a l l y  the density the state  layer, i t i s impossible  of C l " exclusion  (1968)  Elliot  of the thick  forces  between t h e f i l a m e n t s . X-ray  t h e C l " i o n may  anions.  to  i n a muscle  postulated  contractile  on a b a l a n c e between a t t r a c t i v e f o r c e s  force  than  cell  1969).  packing  Waals) and r e p u l s i v e  i n the  rather  could  i s totally  i n the double  a theoretical estimation  with  model  As so l i t t l e  make  by  and r e p e l  on t h e c o n t r a c t i l e p r o t e i n s  and water  water  of negatively  (1963)  and K a t c h a l s k y  the outer  latter  s p e c i a l water  water f o r +  on C l " d a t a  water  the  f o r the K i o n .  suggests that  a p o l y e l e c t r o l y t e , the coion  inner  from  and Na ..  +  water  Alexandrowicz around  l o w when b a s e d observation  water  of solvent  from a f r a c t i o n of the f i b e r  solvent  teins  This  estimated  t o b e 6H% o f t h e i n t r a c e l l u l a r  of the intracellular  myoplasm i s r e l a t i v e l y K  e t a l (1968)  Hays  (electric depend  This  view  double  layer).  and c a t i o n  valence  hand, t h e e l e c t r i c a l . f i e l d be s u f f i c i e n t l y  strong  der  This  environment  o n Rome's  of interfilament distance  strength  filaments  (London-van  on t h e f l u i d i s based  that the  measurements  and i t s v a r i a t i o n  (Rome 1 9 6 7 ,  density  to polarize  1968).  between t h e significant  86 quantities Because  of water  a t pH 7 • 3 ) >  exclusion  surrounding may  have  ions, the in  on t h e t h i c k  selective  1967b).  Cope  filaments  attraction  of  cations  o f a n i o n s i s t o be e x p e c t e d i n t h e w a t e r  the filament.  a large  effect  Furthermore, the structured  upon t h e i n t e r a c t i o n  enhancing t h e chances  evidence found  i n this  t h e b a r n a c l e muscle  interactions  1965;  Bernal  o f the net negative, charge  (myosin p r o t e i n and  196:2;.  (Ling  between  may  of binding laboratory  of the protein 1962).  (Ling  with  Much o f  i n favour of heterogeniety  be a r e f l e c t i o n  the charged  water  of these  filaments  and t h e  mutual fluid  environment.  This the  ionic  chemical  study i l l u s t r a t e s  content o f a muscle analysis.  than  expected  data  would  tion  of C l  the  from  have  the Nernst  across the fiber  found  [ C l ] ^ = 36 mM  with  found  t h e m y o p l a s m he c o u l d  a  all be  of the fiber  C l  -  was  by means o f s i m p l e  the fraction of the figures  free.  fraction  as t h a t  i n this  fraction  study  greater  By c o i n c i d e n c e ,  conclusion  about  distribution  this  the d i s t r i b u -  of C l .  i n the lobster o f about  squeeze  concentration  containing  4 mM  i s just  from  muscle  which i s He  the muscle  also with  concluded  H o w e v e r , t h e same r e s u l t s  the free  Cl .  squeezed  exchanges  that might  o u t were  As c a n be s e e n  chapter, the d e f i n i t i o n which  Robertson  - 7 0 'mv".  and t h u s  o b t a i n e d i n t h e b a r n a c l e i f t h e myoplasm  from any  which  a membrane p o t e n t i a l  p r e s s h a d t h e same C l  investigating  membrane b u t w i t h o u t k n o w l e d g e o f  consistent that  of  o f [ C l ] ^i n t h i s  equation.  intracellular  that  solely  measure  water  l e d to the right  heterogeneous  (1961)  fiber  The a v e r a g e  3 0 . 8 mM/Kg i n t r a c e l l u l a r  was  the danger  of a  from  free  over a set i n t e r v a l  of time  may a l s o b e m i s l e a d i n g .  fraction lobster data  of C l  that  m u s c l e may  with  and Brown  Dunham a n d G a i n e r  c o n s i s t o f more t h a n  does n o t negate  some o f t h e s e cable  -  this  possibility.  muscle preparations  the C l  (1970).  The l a r g e  sensitive  exchangeable  (1968) f o u n d  i nthe  one c o m p a r t m e n t . A re-examination  i s d e s i r a b l e a n d now  microelectrode  developed  Their of  practiby W a l k e r  88 CHAPTER TRANSMEMBRANE P O T A S S I U M AND  V CHLORIDE  ACTIVITY  GRADIENTS  A.  Introduction The  the  simplest  and most w i d e l y  r e s t i n g p o t e n t i a l of muscle  difference  between t h e I n s i d e  from t h e c o n c e n t r a t i o n Chapter turn,  II).  These g r a d i e n t s  fiber  ^ K^o a  (  a  K i  anions  across  the Nernst a  theory,  I960),  has subsequently  Hays e t a l 1968)  ( Cl^l In jy(a ) c  originally Hodgkin  based  1  3  )  ,,. (14) n  Q  on d a t a  on o t h e r  Zachar  and n e g a t i v e  Wood 1 9 6 5 ;  l  and Horowicz  been t e s t e d  b o t h p o s i t i v e (Shaw 1 9 5 5 ;  1961;  ions  t h e membrane a s f o l l o w s :  a  1  (  a n d Conway 1 9 4 1 ;  Robertson  and C l ~ , these  +  equation:  n  (Boyle  1967;  to K  Since  e q u i l i b r i u m c o n d i t i o n s be r e l a t e d t o t h e membrane  K  with  inside the fiber.  (  RT ( K^o RT = =- l n v = m F ( a ) . F  This  on t h e s y s t e m by t h e  ^ C ^ o  through „ E  ions (see  ^ C l ^ i  }  u n d e r most  potential  =  themselves  arises  t h e membrane r e s u l t , i n  membrane i s p e r m e a b l e  distribute  of a fiber  o f t h e permeant  across  organic  explanation f o r  the e l e c t r i c a l p o t e n t i a l  and o u t s i d e  gradients  o f impermeant  muscle  should  and  i s that  f r o m a Donnan e q u i l i b r i u m imposed  presence the  accepted  Huddart  from  frog  1959b; muscle  e t a l 1964;  Adrian preparations Usherwood  (Shaw e t a l 1 9 5 6 ; 1967)  results.  muscle  Two studies. in  F i r s t , concentrations  of  and  water vely  of  fiber.  challenged  1964;  Lev  1970)  et  al  (1968)  on  muscle  from the  of  the  were not  fiber  this  K , +  the  ion sensitive  measured  d i r e c t l y , thus  (14)  and  intracellular  muscle  are  bath.  can  fiber  water  from the  strong  of 1959;  they  Hays  and  (14)  postulated quantities.  experiments  designed  the  use  of  and  support  despite  By  (a,-,,),  were  concentrations  to  to  Nernst  giant barnacle. (a„).  and  1969;  that  (13)  activity coefficient  membrane t h e o r y  heterogeneity.  the  effecti-  Dunham  Cope  unless  microelectrodes,  give  be  in significant  reported  avoiding  now  1967;  equations  from  analyzable  I t i s noteworthy  crab  the  analyzable  (Hinke  a l 1969;  confirm  the  identical  i n favour  ions  Cope  et  used  Second,  a l l the  evidence  1966;  these  were d e t e r m i n e d  Donnan e q u i l i b r i u m and  i n v e n t i n g an  results  the  to  , and  chapter  means o f  and  Hinke  that  myoplasm i s  d i s s o l v e d i n a l l the  muscle.  Cl  f o r the  The  [Cl]^)  of monovalent  able  equation  myoplasm.  outside  brackish-water  v a l i d i t y of  n e c e s s i t y of  i n the  (Hazlewood  in skeletal  In test  water  assumption  i n the  accumulating  and  i n a l l of  a c t i v i t i e s were  l a t t e r assumption  distribution  and  Hinke  binding  the  McLaughlin  1968)  Gainer  by  This  the  assumption that  i s homogeneously the  evident  -  ( [ K ] ^ and  a n a l y s i s w i t h the  Cl  on  (or C l )  +  concentrations  heterogeneous  the  K  are  i n s t e a d of  (14), based  i t s activity coefficient  chemical +  and  coefficient  myoplasmic  K  (13)  equations  activity to  fundamental weaknesses  for  and  the  equations  f u r t h e r evidence  (13) for  90. B.  Methods K  Microelectrodes  +  o f pK m i c r o e l e c t r o d e s 1969a, b ) . Corning  pipette  the  W o r k s , New Y o r k . glass  lead  insulating  The' d i a m e t e r  filled  with  0.1 m K C 1 a n d s t o r e d  2 weeks o r u n t i l variation  the electrode  i n a standard  electrodes  E  K  E  K  +  S  K  l 0  S  ( a  K  +  W  t e s t e d and t h e other  McLaughlin  0.05  M KC1 p l u s  plus  0.05  0.2  terms  stable  Some K  M NaCl.  0.4  of K  +  and N a  E^,  0.2  0.2  constants.  by H i n k e M KC1,  0.05 M NaCl, and 0.3  o f a mixed  were  t o pH 7-3 ( t h e  M K C 1 , 0.02  0.1 M N a C l ,  2 0 )  i n the solutions  determined  M KC1 p l u s  coefficient  +  '  microelectrodes  +  muscle,  10 mM T r i s :  M KC1 p l u s  The a c t i v i t y  micro-  (  of the K  of the barnacle  0.05 M NaCl,  M NaCl,  +  (1 mV  microelectrode,  +  are electrode  constants  1968) w i t h  i s then  s o l u t i o n f o r about  i n thefollowing solutions, buffered  o f t h e myoplasm  and  The e l e c t r o d e  W  being  pH  the length of  equation:  T  determined  down b e t w e e n  20 u w h i l e  by t h e K  a , a n d a,, a r e t h e a c t i v i t i e s K Na  electrode  The g l a s s - g l a s s  beeswax  i n this  where  The  micro-  f o r up t o 6 m o n t h s .  by t h e f o l l o w i n g  =  from  micro-  the t i p of a  i selectrically  The p o t e n t i a l r e c o r d e d described  walled  solution i n 5 hr).  have been used  1967,  of the sensitive t i p atthe  t i p v a r i e s b e t w e e n 100 a n d 3 0 0 u .  the  (Hinke  was o b t a i n e d  glass.  by r u n n i n g  g l a s s - g l a s s . j u n c t i o n i s u s u a l l y about  is  thin  i s jammed t h r o u g h  at the t i p I s sealed  two g l a s s e s .  A very  The c o n s t r u c t i o n  i n detail  (NAS27-4)  +  of this  Analysis.  has been d e s c r i b e d  made f r o m 0120  junction  +  The K . s e n s i t i v e g l a s s  Glass  capillary  and K  0.4  M KC1  M KC1  plus  s o l u t i o n was  91 estimated and  NaCl  tration  as b e i n g  between t h e a c t i v i t y  s o l u t i o n s (Robinson according  1958,  Chapter  (1961); were not  almost  to  14), using  done  the K  by  reading  +  each  K  change i n K  constant, and  10/1  Using  amounted  , f o rthese a n d was q u i t e  this  were  The r e s u l t s  S  activity,  +  K  +  K  by R o b i n s o n  activity  +  to estimate  figure,  the Na  was  +  that correction  2%.  calibrated  before and  were r e j e c t e d i f E° v a r i e d  of the electrodes  , was 53 -  58 mV.  microelectrodes  constant  a n d Owen  b u t , f r o m t h e many  t o more t h a n  The r e s p o n s e  standard  d e t e r m i n e d by t h e two methods  l a b o r a t o r y , i ti s safe  never  1 mV.  determined  experiments  microelectrodes  +  KC1  concen-  three  (Harned  The m y o p l a s m i c N a  o f these  impalement.  more t h a n  fold  i n most  i n this  The after  coefficients  w a s c l o s e t o 10 mM.  )  of equal  of the last  the constants  e x a c t l y t h e same.  determined  M  coefficients  c a l c u l a t e d by Harned's r u l e  the activity  studies (a  were  1959)  and Stokes  o f pure  t o t h e p r o p o r t i o n o f each i o n i n the s o l u t i o n .  As a c h e c k , t h e a c t i v i t y solutions  coefficients-  to a ten  The  selectivity  v a r i e d between  from day t o day f o r a  3/1  given  electrode. All calomel define (a all  K  +  microelectrode  electrode the K  +  v i a a saturated  electrode potential  2 mV e r r o r r e p r e s e n t s electrical  potential  standard  or bathing 1907;  In order t o  as p r e c i s e l y  as p o s s i b l e  m  o f about  10  mM),  corrected f o r variationsi n  between t h e s a t u r a t e d solutions, using  Bates  referred to a  KC1 b r i d g e .  an e r r o r i n ( a ^ )  measurements were  junction  (Henderson  p o t e n t i a l s were  1964, page  KC1 b r i d g e  the Henderson  40).  and t h e equation  The l i m i t i n g c o n -  ductivities  of the ions  from Robinson  and-Stokes  for methanesulfonate was u s e d  no v a l u e  (CH^SO~). t h e v a l u e  was  taken  available  f o r a c e t a t e '('CH CO")-  d i d not a l t e r  the f i n a l  calculation  o f (a„) Km  by more J  3%. details  fibers  of fiber  impalement  w a s t h e same a s d e s c r i b e d  microelectrodes.  Both  were a n a l y z e d  Impaled  for K  and then  concentrated diluted  This  was e s t i m a t e d  content.  A fiber  companion was c u t  sucrose After  drying  and d i g e s t e d i n  d i g e s t was n e u t r a l i z e d w i t h  NH^OH  a n a l y s i s on a Unicam  The e x t r a c e l l u l a r  on companion f i b e r s  micro-  I I If o r the C l "  bottle.  was r e w e i g h e d  a p p r o p r i a t e l y f o r flame  spectrophotometer.  +  and non-impaled  i n a preweighed  the fiber  HNO^.  the K  f o r 30 s e c i n i s o t o n i c  weighed  24 h r a t 105°C  +  with  i n Chapter  fibers  and N a  +  i t s b a s e p l a t e , washed  solution,  and  Since  were,  corrections forjunction  electrode  for  (1959).  calculations  A l l these  The  from  f o r these  i n i t s place.'  potential than  used  space  SP 9 0 0  of the single  by t h e C l " w a s h o u t  fiber  method  (Chapter I I I ) .  From time activity,  (  a  ^  a  )  m  3  w  a  t o time s  i n this  measured w i t h  (Corning  NAS11-18) m i c r o e l e c t r o d e s .  of  electrodes i s similar  these  that  the glass-glass seal  with  heat.  They  stabilize  h a v e t o be d i s c a r d e d impedance. glass,  k,  T  after  study, Na  sensitive  +  The  to the K  the myoplasmic  c o n s t r u c t i o n and microelectrode  +  about  overnight  soaking  a week b e c a u s e  Fortunately the selectivity „, i s a l w a y s . g r e a t e r t h a n  +  glass  i s made b y f u s i n g t h e t w o with  Na  design  except  glasses  but g e n e r a l l y  of increasing  constant  100/1 so t h a t  of the Na accurate  +  93 measurement of  K  +  of (  procedures  )  m  i s possible  f o r the C l  (a )  In this  fibers  o f t h e Ag-AgCl also  which  ditions,  the electrodes  drift  distribution  Solutions. under  i n t h r e e ways.  , and [ C l ]  O  +  [Na] remained o  a  substitute  pitation  t h e second NaCl  calculated  used  used  high  electrode  ( Qj_)  and each  a  m  Under  response  only  these time  con-  and d i d  constant. -  so t h a t  experiment  [K]  +  conditions, the  (see Table  In the f i r s t  altered  t h e transmembrane  of equilibrium  solution  Q  IX).  i o n was u s e d  A small  (see Table increased  0.4  by s u b s t i t u t i n g  X).  In the  as  preci-  w h e n CH^SO" e x c e e d e d  was i n c r e a s e d  and [ C 1 ] were  ,  Q  [ K ] x [ C l ] and [ K ] O o o  i o n (see Table  [K]  I f o r composition)  experiment, [ K ]  The m e t h a n e s u l f o n a t e  on an e q u i m o l a r b a s i s  normal  the K  were  Ringer  f o r the C l  M.  KC1  final  s i m p l y by a d d i n g  KC1  Ringer. The  of  m i c r o e l e c t r o d e s were  o f M g C C H ^ S O ^ ^ was o b s e r v e d  experiment, to  discussed i n Chapter  I n order t o study  a variety  was a l t e r e d  for  amply  maintained a fast  o f normal  In  The e x p e r i m e n t a l  i n Chapter I I I .  three impalements.  composition  O  preponderance  w h i l e i n t h e myoplasm.  Bathing  [Na]  been  had a r e l a t i v e l y  f o r no more t h a n  ion  the  microelectrodes, especially  s t u d y , t h e Ag-AgCl  was u s e d  not  despite  i o n have been d e t a i l e d  i s l o w , have  ri  III.  a  M i c r o e l e c t r o d e s and C l ~A n a l y s i s .  inaccuracies  when  in  N  i n t h e myoplasm.  Cl  The  a  K  +  and C l " a c t i v i t y  coefficients  f o r some o f t h e b a t h i n g s o l u t i o n s and C l  f o r these  -  microelectrodes.  calculations;  Equations  the ionic  (Y  from  k  and Y  C  1  ) were  the potentials  (16) a n d (20) a r e  activity  i s r e p l a c e d by  94 TABLE V I I I K  and  +  C l  activity  -  solutions  calculated  Cl  Solution Code  coefficients from  K  microelectrode  Bath [K]  microelectrode  +  L  1  J  0  8  Y  450  537  and  Activity Coefficients* E q u a t i o n (20) Equation  c l  mM/1  [K] [ C l ] o 0 Const.  bathing  measurements  Concentrations [ ^ [ ^o N a  f o r some  K  =  Y  Na  Y  (16)  C1  0. 667  —  5  128  330  34  o. 650  (0.700)  6  229  229  19  0. 640  (0.705)  11  128  330  537  0. 640  0. 647  Const.  12  229  229  537  0.636  0.651  KCl Added  14  128  450  657  0.621  0.624  [ci]  o  * j1 u n c t i o n p o t e n t i a l c o r r e c t i o n s t o calculating Y and Y Q ^  E^  and  E ^  w e r e made  before  k  the  activity  C1  '- -'o^'  Y  C1  calculated 0.2  [Na]  values value is  not  values  ).  coefficient I f  Y  K  a  n  d  from the Table  f o r some  (6  Y  Na  times a  r  a  modified  s  s  u  m  e  concentration d  equal, (20)  equation  then  the  calculated  out  the  bathing  of  14)  when  [Kj  Ringer '<<  f o r s o l u t i o n s 5 and  have been a  few  information  i n mind, a  of  millimolar  [Na] 6 may  higher  i s not . be  common v a l u e  of  can  be  Y  k  Y  k  listed. 0.65  r i  [K] Y  and  )  =  > C  1  Y  A  K  (20)  since equation  relatively  discounted  than  (&  solutions.  listed  The  (e.g.  (provided  VIII contains  f o r normal barnacle reliable  e  the  high  because With  for y  and  Y  C  [Cl]  1  may  this y  was  assigned ionic  to barnacle  strength  presented  study  (solutions  which  solutions.  effect  L~ ]Q K  L"C1]  K  constant.  order  was  an  always  three  from  solutions  within  impaled  solutions.  Each  subsided  analysis  5 min before constant One  removing  geometry  i s then  these  always  fibers.to  equilibrated  cation  was  further  content  were  e  n  with  similar  I , evidence  °t  equal  i s  i n these  on t h e r e s u l t s  of  this  Chapter.  f i b e r was  through  fibers  to  solutions [ K ] ).  of the K  the f i r s t  impaled  equili(the  In the  spontaneously microelectrode,  +  three  i n each of the l a s t  was  after  solutions. three  contraction  f o r a r e a d i n g a t 30 m i n . which  gently  pulled  were  i n equating  justified from  destined f o r chemical  to their  them i n o r d e r  t o be  allowed  product  contracted then  fibers  solutions, found  r  (Ringer) to high  taken  i n normal  subtractions  equilibrating  q  the e x t r a c e l l u l a r  fibers  cellular  test  resting  to maintain  to the e x t r a c e l l u l a r  justified  a a  D of this  equilibrated  and i n time  were  Y^  To a v o i d b r e a k a g e  of these  Contracted  ^  of the constant  only  f i b e r was  had  The  low [ K ]  10 m i n .  A different  In Appendix  inequality  the f i b e r  f i b e r was  solutions  Procedures.  f o r 30 m i n i n e a c h  relaxed  a n  be d i s c u s s e d i n s e c t i o n  brate  last  that  of this  Experimental I.  and a l l o t h e r  1 t o 12)..  indicates  The  will  Ringer  cleft  length  a relatively  system  of the f i b e r .  the e x t r a c e l l u l a r space  Ringer  solution.  when, a f t e r  different  determined  space on  This  simplifiextra-  i n the s i x  the i n t r a c e l l u l a r i o n content constant.  companion  six different  fibers  of  and  water  96 II.  High  attached seven  I.Kj , c o n s t a n t [ C . l ] . . A g r o u p o  t o a common  solutions  usually  only  b a s e p l a t e was e q u i l i b r a t e d  (Table X I ) f o r about  four  groups  were  two b a r n a c l e s were r e q u i r e d  These  fibers  chemical  either  i t was a s s u m e d t h a t  intracellular. of  fibers.  After  water  demonstrated  Impalement  or  an i n c r e a s e  i n water  t h e a c c u m u l a t e d w a t e r was a l l  swelling  Harris  o f KC1.  overnight  impaled with The  one  volume p e r f i b e r  s p a c e was t a k e n t o be c o n s t a n t f o r a l l  i n which  1961;  Addition  brated  f o r at least  f o relectrode  assumption has proven  Adrian  III.  KC1.  This  on m u s c l e  1959;  an experiment.  P u t a n o t h e r way, t h e a b s o l u t e  the extracellular  ments  t o complete  analysis. When t h e s e f i b e r s  content  Since  f r o m one b a r n a c l e , a t  were warmed a t room t e m p e r a t u r e  b e f o r e t h e y were used  fibers  i n one o f t h e  20 h r a t 4°C.  obtainable  least  hour  of dissected,  1963;  adequate  In other  was i n v o l v e d Hinke  (Tasker et a l  1969a).  As i n I I , a g r o u p  of fibers  a t 4°C i n a R i n g e r s o l u t i o n  was  containing  warming t o room t e m p e r a t u r e , f i b e r s electrodes  experi-  were  equiliexcess  either  o r a n a l y z e d f o r i o n and water c o n t e n t .  content of these fibers  was s i m i l a r  t o the water  1  content  of fibers  cellular used  equilibrated  s p a c e was m e a s u r e d  on t h e f i b e r s  junction  measured  b e f o r e and a f t e r  The r e s t i n g  with  extraR i n g e r and  solutions.  1 cm f r o m t h e  permanent  The  i n normal  i n the other  impalement  The a v e r a g e  Ringer.  on f i b e r s  D u r i n g Impalement.  was r o u t i n e l y  microelectrode.  only  equilibrated  Alteration potential  i n normal  membrane  myotendinous  an i o n s e n s i t i v e  drop  i n membrane  potential  of  fiber.s  d r o p was less  soaking 4 mV.  drop.  fiber  the as  Fibers  The  fibers  membrane  with  because  was. 2 mV  resting  recorded  potentials  results  Throughout  before  and i s t h e p o t e n t i a l  and. t h e m a x i m u m displayed  d i d n o t seem t o a l t e r t h e  the analytic  w e r e t h e same.  potential  most v a l i d  lower  small depolarization  composition  non-impaled  i n Ringer. s o l u t i o n  on i m p a l e d these  experiments  i m p a l e m e n t was  used  and  accepted  for calculations.  C. • R e s u l t s Constant from  [K]  x [ C l ] i n Bathing  the major p o r t i o n  of this  experiment  Solution. are l i s t e d  The  results  i n Table  IX.  TABLE I X K  +  activity  equilibrated  Bath . Concentration [K] [Cl] o o mM/1 J  and c h e m i c a l  analysis  i n solutions  with  Intrafiber Concentrations* [K]. [Cl]. mM/Kg1 H 0 2  n  on s i n g l e  [K] x [ C l ]  fibers  constant  K"" Microelectrode (a .) Km mM/1  = 16  1  T  n  Membrane Potential E m (mV)  = 8  1  8  537  175 +4  32.6 + 1.2  130 +4  -79.9 + 1.1  2  16  269  175 + 2. 3  31.4 + 0.7  133 +4  -64.3  178 ±3  30.9 + 0.5  132 +6  -45.1 +1.5 -26.4 +1.3  + 1.0  3  32  134  4  64  67  177 • +1.8  28.6 + 0.7  132 +7  5  128  34  175 + 2.4  26.8 + 0.9  132 ±5  •  172 + 3.6  25. 8 + 0.9  136 +4  +1.1 • +0.9  6  229  19  * extracellular  fiber  K , Cl  and water  excluded  -10. 7 +1.1  98' The'Cl in  results  Chapter  different  i n Table  I I I ;  X are .from  the C l  results  set of barnacles.  centrations  ([K]  1  the experiments  I n Table  The v a l u e s  Notice  the  membrane p o t e n t i a l  that  ( a „ J o _L m  is  x  (a  III  two  n  i  K  Q  which  x  constant.  (a  c  l  )  Q  Notice  = 1820;  ( a  K  ) x  (a  m  c  l  )  (a^)  values  O  of these =  0.65,  x 29 = 3 8 2 0 )  be c o n f i r m e d  by t h i s  (a ) i s the least ox in (13) ( s e e C h a p t e r I I I ) .  i n equation  p  that the ( a ) readings Cl m ° agreement w i t h t h e value expected  than  p  OX  data  i n one o f t h e m e a s u r e m e n t s .  o p i n i o n , the term  n  x (a ,)  O  ( a s s u m i n g Yi  = 132  m  cannot  introducing a correction  the four terms  when  one c a n s a y t h a t t h e p r o d u c t ,  different  (13)  also  I X ) was c o n s t a n t  Unfortunately, the numerical  unfortunate in  mV.  when t h e p r o d u c t  are significantly  In the author's of  - 8 0 t o +1  from  Iv  means t h a t e q u a t i o n  without  water.  ITI  products  (a )  varied  ) , i s a constant  OX  held  and  68),  IX that  Therefore,  T  iv  Ions  con-  19 . ( p a g e  by e q u a t i o n  (Table X) b u t n o t [ C l ] . (Table x  (a ,) was c o n s t a n t . j\ m (a^)  a  (a„) a s w e l l a s [ K ] . was Km I i n a l l o f t h e s i x e q u i l i b r a t i n g s o l u t i o n s even though  constant  i n Table  IX a r e from  f o r the i n t r a f i b e r  and [ C l ] ^ ) , determined  have been 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  presented  n  n  reliable I ti s  i n this from  experiment are less ^ the Nernst equation  are the average ( a ) readings l i s t e d i n Table I I I . • Cl m ° Furthermore, the r e s t i n g p o t e n t i a l i n normal Ringer o f the fibers  n  used  potential  n  for (a ) readings ox m p  l  i n normal Ringer  w a s - 8 0 mV.  I f Cl  was - 7 1 . 5  of the fibers  Is distributed  membrane, t h e e x p e c t e d  ( Q]_) a  a m  t  mV w h i l e t h e r e s t i n g used  passively  -71.5  mV  f o r (a-^) across  i s 8.5  mM  readings  the fiber higher  than  e x p e c t e d a t - 8 0 mV. T h e r e f o r e , i t seems q u i t e p o s s i b l e t h a t t h e (a„,) u s e d i n t h e above c a l c u l a t i o n i s t o o h i g h by a f a c t o r Cl m  99 TABLE X Cl  a c t i v i t y and chemical  equilibrated  i n solutions  Bath Concentration [K]  [ C l ]  D  8  2  o  269  32  3  mM/Kg 537  16  134  * extracellular t  of  numbers  two  11 w h e r e  [K] x [ C l ]  plotted  constant  Membrane Potential E m (mV)  Cl m mM/1  2  30.2 t +0.5(60)  29-5 +1.5  (14)  -71.5 + 0.8  31.6 +0.6(32)  31.0 +1.7  (5)  -58.4 + 0.8  28.5 +0.8(23)  29. 0 + 3.0  (9)  -  -46.6  + 0.8  and water excluded  refer  t o number o f  determinations  and t h e c o n c e n t r a t i o n a r e drawn  l i n e i s the least  part  gradients  only  squares  E ^ = 57-4  to the f i r s t  gradients are  from the t h e o r e t i c a l  and C l  f i t f o r t h e mean K  of equation  and Y - i n t e r c e p t  +  Both the are  f o r the a c t i v i t y data.  Q  K  (14).  and ( a m  different  ionic  forK  log (a ) /(a )^  t h e r e l a t i o n between E  (43 mV)  illustratedi n  t h e m e a s u r e d membrane p o t e n t i a l s .  but l i n e s  defines  (14) i s b e s t  t h e l o g o f t h e transmembrane  has t h e e q u a t i o n  slope  fibers  Cl Microelectrode  H 0  v a l i d i t y of equation  against  activity  close  on s i n g l e  times.  plotted  solid  fiber Cl  i n brackets  The Pig.  with  Intrafiber Cl Concentrations* [Cl].  mM/1 1  analyses  (-26  mV)  (59 mV  ). Cl l n n  +0.4  ( a  K  ) data m  which  The d a s h e d (Table  which  The and  i s very line  X) and has a  are quite  slope- and zero  intercept).  100  Log of Ion  Gradient  F i g . 11. R e l a t i o n between transmembrane K and C l gradients and t h e r e s t i n g p o t e n t i a l when f i b e r e q u i l i b r a t e d (30 m i n ) i n s o l u t i o n s i n w h i c h [ K ] [ ^ p r o d u c t was m a i n t a i n e d c o n s t a n t C 1  0  (Table  I X and X ) .  closed  circles,  [Clj^/CCl] the is  solid  log [K] /[K]^ Q  squares.  The s o l i d  the s t a t i s t i c a l  a slope of -57.4  (a  (13)  ni  )  ^  o  r  t  o f 14  line  through  technique  instead  be s a t i s f i e d .  I I I and those  h  e  f o r t h e open c i r c l e s , l o g  o f Hagiwara  the closed o f +0.4  of 29-30,  f  o  r  circles  f o r l e a s t squares  mV a n d a y - i n t e r c e p t  had a value  and (14) would  Chapter  i s l o g (^^o^^K^m  f o r t h e open t r i a n g l e s and l o g ( a ^ i ^ n / ^ c i ^ o  drawn.by  has  If  The a b s c i s s a  f i t and  mV.  then both  equations  The r e s u l t s p r e s e n t e d i n et.al  (1969)  indicate  that  101 the  free myoplasmic  the  concentration  line to  (open c i r c l e s )  f o r the  the  can  p r e d i c t e d by i n F i g . 11  Notice points  K  shown t h a t  ment.  Now  from  from  (a. )  that  and  -  a )[K].  (18)  equation  know  [K]„  (inserting  the  If a  to  the  gradient  left  line  of  is  activity  the  fitted line,  /[K]^ for this  a [K] o  i t  experi-  Q  (K)  for C l " ) , since  +  to  that:  K  close  concentration  +  [K] we  very  equation.  points.  =  =  o  K  compared  (19)  equation  Nernst  the  gradient  data  i s i n fact  consistently to  (a-^c/^K^i  (1  the  deviate  activity  +  concentration  be  and  Cl . concentration  =  /y  v  Km  'm  / \ (a ,)  a (1 when b o u n d  a  o  )  [  K  ] .  and  a  i s the  T  =  l  Y  B^. = 0 , where Y  K , +  coefficient  _  'm i s the  M  fraction  of  myoplasmic fiber  activity  water  a c t i n g as °  m + solvent  f o r myoplasmic  K  .  Therefore  (a ,)  (a,,)  o  l  T  Since to  a  the  0.065  =  Q  ratio  of  the  s o l u t i o n s by  a  0.795  a  m about  activity =  0.795  J  m acting  as  and  solvent  permissible  'm  for single fibers,  side =  a  15%  of  f o r the  becomes r e l a t e d  coefficients  of  the  Y / Y • 'm ' o the f i b e r  for fiber K. :,  then  A  +  C l " data  B ^  ^  The  0  (see  since  Chapter  intracellular  in  the  constant  product  30  min  in solution 2  (16  out-  Clearly, i f Y 'Y , t h e n ' 'm 'o' w a t e r [ 1 - ( a +a ) ] i s n o t m o s i m i l a r c a l c u l a t i o n i s not (1  =  J  -  a  ¥• a  )[C1]  o because  i n s i d e and  simply  o  (a -, ) p  i/i m  m  /y  in '  IV). Na  +  content,  s o l u t i o n s was mM[K]  , 269  [Na]^,  of  fibers  also determined. mM[Cl] ),  [Na]  ±  had  soaked  Following dropped  102 from t h e normal Ringer  l e v e l , o f . 22. mM/Kg i n t r a c e l l u l a r  17 mM/Kg i n t r a c e l l u l a r  water.  Table  drop mM  I n a separate  sensitive  +  i n [Na] .  come f r o m  results  a N  a  )  (n=6).  space  theextracellular  i s seen i n f i b e r s  16 mM  [ K ] but normal o  the doubling • High  this  from  l  with a  and'Hinke  must  compartmentalized  1970).  These  immobilized i n  content  access  o f t h e myoplasm A similar  drop i n  f o r 30 m i n i n a s o l u t i o n The N a  +  loss  [Cl]  theproduct,  ( ^)  x Q  with  must be due e i t h e r drop i n E . ^ m  I n Bathing  a  +0.7  have  i n a compartment w i t h  [ K ] , Constant Q  e (  t o 23.5  +  or  change i n [ N a ] ^ ) .  soaked  ^  concen-  8.8 + 0 . 8 t o  bound  (theionic  [Cl] . o  r e c o r <  i nfiber.Na  of [K] or to therelated o  experiment,  s  must be e i t h e r  +  or held  this  [Na]^  to  Na  space  during  Allen  a  29.6 + 0 . 9  slightly  i seither  1966;  that this  extracellular  does n o t v a r y  that  w m  (see  t h e p e r i o d o f t h e observed  Thus, t h e drop  and Hinke  indicate  a  from  i  increased  m  -( Na.)  during  [Na] fell  thefraction  (McLaughlin  to  Again,  ±  9.4 + 0.9 mM  experiment,  microelectrode  (n=20) b u t (  the  solutions  I X ) , [Na]_^ g r a d u a l l y i n c r e a s e d t o i t s o r i g i n a l  tration. Na  In thefollowing  water t o  ( ci^o a  l  s  Solution. a  ^  o  w  e  (  ^  In  ^°  increase  as [ K ] i s i n c r e a s e d ; KC1 s h o u l d e n t e r t h e f i b e r i n o o r d e r t o i n c r e a s e t h e p r o d u c t (a ,) x ( a ^ , ) . T a b l e s X I a n d ^ Km Clm XII l i s t t h e m e a s u r e m e n t s t a k e n f r o m f i b e r s a f t e r 20 h r o f T  equilibrium  i na given  equilibrium, M  3  duct  equals  128 mM Y  'o  both that  •  solution.  As e x p e c t e d  (a,,) a n d ( a ~ ) K m Clm n  y  i nthe outside  [K] solution, o 5  (a„) K o  increase  solution.  x (a~,, ) C lo  degree  T  o f agreement  so that  Clm  their pro^  P o r example,  = 2.90 x 10  = 0 . 6 5 f o r e a c h i o n ) a n d ( a , ) x (a„, ) Km  u n d e r t h e Donnan  4  i n the  (assuming  = 2.98 x 10^.  i s e x c e p t i o n a l when one c o n s i d e r s  This  the fact  103 TABLE X I K  +  activity in  and c h e m i c a l  solutions with  Bath Concentration [K] o mM/1  a n a l y s i s on s i n g l e  elevated  7  4  1  1  8  8  32  10  64  = 16  128  12  229  t.hat t h e (a„) Km Also,  fiber  (a„-, ) w i t h Cl m  less  tration  gradients  potential  -89.6 +1.6  ±3  35.4 +1  + 2.9  -79.2 + 0.6  182 + 1.3  +1.8  38.9  143 + 4.1  -64.3 +1.2  51.3  134  +1.5  +2  145 + 6.7  -47. 3 + 0.8  232  78.1 +2  + 4.7  177  -35.9 + 0.4  +4  274  142 +4  196 + 4.2  -23-2  287 +7  171 +6  204 + 2.9  -9.6 + 0.6  K , Cl  the C l  and'water  come  from d i f f e r e n t  are plotted against The s o l i d  must be  barnacles.  measuring  c o n c e n t r a t i o n exceeds  t h e transmembrane  as i n P i g . 11.  + 0.2  excluded  microelectrode  e r r o r when t h e C l  I n P i g . 12  8  + 2.6  a n d (a„, ) v a l u e s Cl m  apparently  =  116  29.5  185  * extracellular  n  Membrane Potential E m (MV)  + 1.4  ±3 11  +  1  179  ' 16  9  K Microelectrode (a„) Km mM/1  mM/Kg H O  167 ±3  equilibrated  [ K ] and constant [ C l ] o o  Intrafiber Concentrations* [K]. [ C l ] .  n  fibers  activity  and concen-  t h e measured  line  0.1  membrane  i s drawn a c c o r d i n g  to  M.  104 TABLE X I I Cl  a c t i v i t y on  in  solutions with  Bath Concentration  single  elevated  64  94 +2  11  128  12  229  least  equation ^ the in  three Table  validity  squares E  m  order (14). fiber  to  Cl  XII,fall  water,  equations  K , +  value. (13) The  gradient  log  -21. 4  -21. 7 + 0.3  204 +6  -13-5  -11.3 + 0.4  f o r the ( a ^ )  gradients  on t h e K  +  m  data  - 2.2.  (solid  activity  and d e s c r i b e s  Notice  boxes),  gradient  now  how  close  from the line.  the  data  Thus, the  (14) i n d e f i n i n g t h e e q u i l i b r i u m s t a t e i s  extent  In addition, this a fiber  the relations  F o r example,  Membrane Potential E m (mV)  ^C^m .349  1  152 +5  confirmed.  t o what  J  -34. 2 + 0.6  T  of equation  L  (mV)  l o g (a ,) /(a,,) K o Km  activity  to satisfy  absolute  59  [Cl] o  -33.6  technique  = 54.4  satisfactorily ustrates  constant  Cl m mM/1  10  the  [ K ] and o  Cl~ Microelectrode  mM/1  (20 h r )  fibers equilibrated  after  and C l  -  will  take  expressed  experiment  up i o n s  had i n c r e a s e d  The r e s u l t  was  and w a t e r i n  i n equations  20 h r i n t h e 229 mM 1.4x,  i l l -  [K] 2.3x,  (13)  and  solution, the a n d 7x  a r e s t i n g state which  In  satisfied  and ( 1 4 ) . concentration  (solid  circles)  gradient  points  for K  (open c i r c l e s ) +  fall  and  activity  closer together  i n  105  F i g . 12. R e l a t i o n between transmembrane K and C l g r a d i e n t s a n d t h e r e s t i n g p o t e n t i a l w h e n t h e f i b e r was e q u i l i b r a t e d (20 h r ) i n s o l u t i o n s w i t h e l e v a t e d [ K ] a n d c o n s t a n t [ C l ] (Table  X I and X I I ) .  circles,  The  abscissa  l o g .[K] / [ K ] ^ f o r open  open t r i a n g l e s  and  i s log (a ) /'(a ) R  circles,  l o g ( c]_^rr/ ^ Cl^o a  /  a  ^  K  m  f o r closed  l o g [C.1]^/[C1]  o r  s o  '- -l 1  for  squares.  d  The s o l i d l i n e , d r a w n by t h e l e a s t s q u a r e s f i t t e c h n i q u e , h a s a s l o p e o f - 5 4 . 4 mV a n d a y - i n t e r c e p t o f - 2 . 2 mV. The d a s h e d l i n e i s drawn f r e e h a n d .  this  experiment  experiment to  the l e f t  [1 -  ( a +a  ( F i g . 12) compared t o t h e c o n s t a n t  ( F i g .11) b u t t h e f o r m e r of the l a t t e r  points.  )] should decrease  points The  i n fibers  still  product  deviate  bound water  slightly  fraction  equilibrated  i n high  106 [K]  solutions  solution)  to balance  Unlike centration from first  the K  activity  +  experiment,  even though Cl  may  the osmotic  triangles)  remained  p  have been  from  the fraction  bound o r c o m p a r t m e n t a l i z e d second  experiment,  clearly and  reveals  ( Q-^) a  between  m  (solid  i n Chapter  between  x ( -[_) a  c  0  product  that  i s either  IV).  of  In the i n F i g . 12 triangles) outlined  value.  Table  i n one  (a^)  However, f i b e r  XIII  (128  mM  As i n t h e p r e v i o u s  i s increased  t o t a k e up K C l u n t i l  solution.  states;  measured.  In this  new r e s t i n g  x 10  x ( Q]_)  the analysis  [K] ), both  (a )  a  ls  equiliwere  p  m  0x  x  7  x 10  u  a n d (a,)  v  t h e (a)  = 3-23  x (a„-)  i  o f two  Am  state,  c  of the  (a )  product  p1  j\ m 2}  w h e n '(a„)  e m  does n o t i n c r e a s e  of the hypertonicity lists  and t h e  a  m  water  4  2.98  loss  [ C l ] ^ (open  o  is  -  This  i s n o t as s i m p l e as t h e one  K C l accumulation because  brium  C l  of KCl Bathing Solution.  t h e (a-^)  i s required  bathing  decreased,  (Table X ) . of fiber  In the  T  new e x t e r n a l  with  and 12).  [ K ] . a n d (a,) . l Km  experiment,  the  significantly  between t h e two l i n e s  the r e l a t i o n  squares)  Addition  fiber  a comparison  that  deviate  (described  Q  t h e C l ~ con-  (Table I X ) as [ C l ] constant  [K]  o f the K C l uptake.  ( F i g . 11  gradient lines  [ C l ] ^f e l l  (a ,)  effects  concentration gradients,  +  g r a d i e n t s (open  the K  (Mo%. i n t h e 229: mM  a s t h e y , t a k e up w a t e r  ox  m  (assuming  y  =  K o C l o 'o 0.62, Table V I I I ) . F u r t h e r m o r e , t h e p r e d i c t e d membrane p o t e n t i a l i s - 2 3 mV f r o m t h e {&.„) v a l u e a n d - 2 5 mV f r o m t h e ( a ) value. to  p  A. m  Both of  of these  - 2 3 . 3 mV.  states  calculations  1  ux  are close  Thus, equations  (13)  i f t h e y become e s t a b l i s h e d  t o t h e measured  and (14)  also  at increased  m  potential  define  resting  osmolarity.  In  107 TABLE K  +  and C l  a c t i v i t i e s on s i n g l e  (20. h r ) i n s o l u t i o n s  Bath. Concentration  [ci]  with  o  561  32 128  167 ±3  fiber  same s o l u t i o n ,  [K]  (-17  D.  mV)  K , +  the measured  [Cl]  ±  }  = 6  129  -77.2 + 1.2  135  -46  135 +3  197 ±3  and water  (-41  mV)  Membrane Potential E m (mV)  +4  +4  Cl  Cl m  ( a  n  +1.0151 +^  -23-3  +4  excluded  t h e membrane p o t e n t i a l s  and from  predicted  from  are s i g n i f i c a n t l y  different  potential.  Discussion This  favour  study  provides  of the widely  activities can  }  mM/1  44.7  +6  this  from  K m  • +1.8  249  657  * extracellular  1  ( a  29-3 + 1.0  ±3  [Na]  Myoplasmic Activities  = 12  148  537  equilibrated  constant  2  8  14  high-KCl,  mM/Kg H 0 n  13  fibers  Intrafiber Concentrations* [Cl].  mM/1  1  XIII  held  i n t h e myoplasm  be d e f i n e d  t h e most  theory  that  direct  proof,  the free  o f a muscle f i b e r  K  at  +  to date, i n  and C l equilibrium  by t h e e q u a t i o n :  ( a  K o }  < K>m a  ( a  =  Cl'm  < Cl>o a  V =  of the proof rests  S  X  P  The  strength  the  i o n a c t i v i t i e s o f t h e myoplasm  R  T  on t h e d i r e c t of single  measurements fibers  of  b y means o f  108 ion  s e n s i t i v e ' m i c r o e l e c t nodes'.  reality fact  measuring  ionic  That these, e l e c t r o d e s  activity  are i n  i n the myoplasm.is based  that  the p o t e n t i a l of these  electrodes  activity,  not the concentration,  o f an i o n i n s o l u t i o n .  the on  above e q u a t i o n the binding  fiber.  the  the troublesome  coefficients  of course,  membrane;  i s a function of the  making  of ions question  any  Thus,  assumption  or water  i n the  on t h e i o n i c  i n t h e myoplasm has been avoided.  assumed t h a t  microelectrode  fiber  without  or compartmentalization  Furthermore,  activity have,  c a n be t e s t e d  on t h e  We  the solution i n equilibrium  with  i s the solution i n equilibrium with the  the results offer strong  support  f o r this  assumption.  Unfortunately, devoid  of assumption  calibrated  and weakness.  i n standard  such an e l e c t r o c h e m i c a l electrode well in  be t h e b a s i s  that  electrical  of the K  t h e myoplasm.  selectivity expected ^  [ C l ] ^ was  when  appears  (K/Na < 10), t h e K  t o measure  Fortunately,  this  v  +  i n myoplasm i s The  J  of i t s  i s u s u a l l y always  when  probably  electrical  seem t o be a l t e r e d imperfect  microelectrode  (a,,) r e l i a b l y Km  reliable  indicates that the  does-not  because  p  t o give  0 . 1 0 mM.  microelectrode  may  (a -, ) values o _L in ( T a b l e X ) . The  0.035 M  X I I and- X I I I  Of c o u r s e ,  high  that  f o r t h e same  of assumption  false  less than  [ C l ] ^ exceeds  +  when a n e l e c t r o d e i s  i s valid  weakness  deviation of the electrode  insignificant  by  This  f o r the apparently  i n Tables  ni  behavior  standardization  the C l ~ microelectrode  (a. ) values ui. m  First,  i s by no means  s o l u t i o n s , one assumes i m p l i c i t l y  i n t h e myoplasm.  m y o p l a s m when  fact  our a n a l y t i c approach  (a .) K'm T  t h e case  can only  be  > (a, ) . Na m T  i n myoplasm.  109 Oddly of the  bathing  coefficient VIII, was Y  enough, i n t h i s  the  solution  of the  determined = Y^a'  K  H  this  w  e  v  that y  revealed If  o  e  r  >  j  a  study at  i s N a  of  K  (20)  equation  of Y„ Na  value  In the  coefficient  from  activity,  i s mor.e o f a p r o b l e m t h a n  myoplasm.  activity  study,, the  a Na .  0.70  i s used  f o r these  i n the  (20),  6,  11,  not  then  12.  and  electrode  s o l u t i o n s (Appendix o f V„  calculation  y  the  Theoretically  Y^  increase  [Na] . gradient 59-  of  curves None o f  qualitatively  K  the In  m  to  the  < 10  attributed  K  +  mM.  Fig.  12  this  F i g . 11  x  [Cl]  This  to the  equation  points  as  A  show a  [K]  12  becomes  Q  to  tilt  towards  and  small 12,  slight  [K]  is  small  vs.  of p a s s i v e  1943, [K]  slope  log  of  Na  Hodgkin  d e v i a t i o n and  [K]  decreases Nernst  K  compared  to  activity  +  study  slope  would  the  curve  be  obtained  rapidly  when  equation,  Katz  (uppermost undoubtedly  kind,  curves,  f o r i n the  and  relating  this  p e r m e a b i l i t y on  +  accounted  solution  15),  theoretical  in this  [K] : o o  Chapter  I n most membrane s t u d i e s o f  i s roughly  4 mM  >>  are  change.  the  solutions,  effect  [Na]  low  the  the  5,  for solutions  1959,  Stokes  d e v i a t i o n from the  (Goldman  f o r the  and  tend  o f membrane p o t e n t i a l [K]  from  glass microelectrodes  +  conclusions reached  a f f e c t e d by  membrane p o t e n t i a l , field  Y ^  and  m  constant  K  0.62  about  solutions with  i n k  would  k  E . i s constant.  slopes  [K]  towards  i n F i g ' . 11  In both (a )  to  (Robinson  change i n Y  This  Q  of Y  value  unknown. should  reduced  i s K  enough t o m e a s u r e Y„ a c c u r a t e l y when 'K  consequently,  that  K  U n f o r t u n a t e l y , the  selective  Table  solutions  1  equation  in  assumption  sensitive  +  activity  [K]  in•the high  +  the  presented  under the  with  least  study  coefficient  the  constant  1949). circles) this  generally  The  two  in  deviation  I)  110 a s E.Kj  would  increase  these  c o n d i t i o n s , the muscle  state  so t h a t  ion  4 mM is Na  (P^g/P^  results the  Nernst  field  (filled  With  this  i n this  [Cl]^  C l  -  Chapter  there  curves  a  zation [Cl]^  the results  equation,  then  the passive  exception, the  are adequately  results  i s a simple  described  confirm  Nernst  there  m  of Chapter  appreciable  fraction  ( j^)  i n the  p e r m e a b i l i t y and  +  one u n c e r t a i n  microelectrode  I I I that  since  deviation the  circle)  by  the finding  relation  I s n o t as s i m p l e  of the Intracellular  of fiber  by b i n d i n g  to the right C l  of the ( -  a  from part  ^ ) curve m  surprising.  excludes  and C l  -  since  , the  I n F i g . 12, t h e to the left  or  The d e v i a t i o n o f t h e l o w e r  -  exclusion of fiber  discrepancy  water  a  triangles).  i s bound  of t h e [ C l ] ^ curve  c a n be e x p l a i n e d C l .  i s not  Cl  a r e more o r l e s s a s e x p e c t e d .  curve  curve  IV, this  Intracellular  o f t h e upper part  between  However, i t i s '  b e t w e e n membrane p o t e n t i a l a n d [ C l ] ^ ( o p e n  In-fact,  devi-  equation.  Considering  large  an e q u i l i b r i u m  m u s c l e membrane I s r e l a t i v e l y  f r o m F i g . 11 a n d 12 t h a t  relation  under  I f the slight  r e s t i n g m e m b r a n e p o t e n t i a l a n d (&„-,) . • ^ Cl m  obvious  by  )•  presented  Chapter  the  point ^  v  of the barnacle  1°  =  The of  to assess.  o f t h e (a ) Km  by t h e c o n s t a n t  permeability  +  does n o t r e a c h  s o l u t i o n i n F i g . 12 i s d u e t o N a  described  low  a  i s difficult  from t h e curve [K]  fiber  .Unfortunately,  t h e r e l a t i o n s h i p between..membrane p o t e n t i a l a n d  distribution  ation  approached, z e r o .  compartmentalipart  can only  of the fiber  of  of the be  water.  explained I fthe  between t h e [ C l ] . and (a„ ) r e s u l t s i n t h e 6 4 , 128, x Cl m [ K ] s o l u t i o n s i s t r e a t e d i n t h e same w a y t h a t t h e n  3  and  229  mM  discrepancy  between  E'K], a n d (a,,) r e s u l t s l Km  was t r e a t e d  earlier  3  I l l (except  as.suming B„  excluded  from about  These r e s u l t s the  the  intracellular  e x c l u s i o n of  of these  results  assumption In  the  of  light  Hinke  1970)  in  these  as  well  the  the  of  the  that  to  the  been  for  i n the  and of  the  i s not frog  of  and  ions  there and  muscle.  On  relation  distribution  the  other  (i960),  i n the  hand,  would  study  of  the  compartmentalization ion  studies, i t is possible  is significant i n the  ion  i n t r o d u c e d by  i n chemical  1968,  al  However, s i n c e  b i n d i n g and  water  McLaughlin  Donnan  Adrian  f r o m D o n n a n s t u d i e s on  that  i fa l l  1961;  the  a problem  heterogeneity  water  of  followed.  Hays e t  ion distribution.  i n t r o d u c e d by  fiber  study  (1941)  half  intracellular  consider intracellular  heterogeneity  results  distribution  evidence  had  1968;  Conway  IV  e x p l a i n e d i f the  Gainer  and  ( a +a. ) ] . m o  contemplating  homogeneity  a  -  C l ~ from almost  adequately  that  Cl~ is  i n Chapter  Dunham a n d  from  fortuitous  presented  (Robertson  c a n c e l out  exclusion  [1  water  I t i s worth  amount o f  and  muscle  Donnan r e l a t i o n  tends  fiber  0.5  =  i n crustacean  Boyle  heterogeneity  that  water.  transmembrane  of  the  evidence  i t i s obvious  as  Indicate  the  then, a  intracellular  m u s c l e s must  work  of  c o u l d be  1966;  Hinke  mM),.  intracellular  heterogeneity and  45%  support  for  10  =  n  f r o g muscle  are  heterogeneity  frog  muscle.  in  the  112 CHAPTER V I  THE D E T E R M I N A T I O N OF a ^ FROM P O T A S S I U M MICROELECTRODE STUDIES  A.  Introduction In  Chapter  heterogeneity  I I (section  of intracellular  laboratory,  i t has been shown,  measurement  of K  intracellular fraction  activity,  +  water  E ) , ways o f d e m o n s t r a t i n g t h e  w a t e r were  described.  by means o f  that  this  intracellular  a significant  o f t h e b a r n a c l e muscle  In  fraction  of the  excludes K .  The  +  of the fiber  w a t e r , a , which a c t s as s o l v e n t f o r ' m i s c a l c u l a t e d from the equation: 5  intracellular  K  +  (21)  [K]™ = a [ K ] + a ( K ) + B „ T o o m m K J  where  ( K ) = m  Ringer  ^^m^m'  solution  B  following •  = 0,  m  then  (22)  (K) m  m  will  values of a  and M c L a u g h l i n  m  be s m a l l e r . f o rfibers  0.58  By t h i s  i n normal  1967),  -.685  chapter).  Hinke  strength  of a  determined  from  ion  exclusion  calculations and osmotic  Ringer °  studies,  solution  1966),  (Hinke 1969a),  V ) , and 0 . 8 l ( t h i s m  method, t h e  (McLaughlin and Hinke  (Chapter to  0 i n normal  -  o  = T^rpr—  have been o b t a i n e d ; (Hinke  R  Q  [K]  ^ 0, then a  K  Since a [ K ]  a  and i fB  a If  ^ K ^rr/°' ^ '  0.73  0.795  (1970), seven  on t h e  different  concluded that a  -  0.68  m and  that  about  25% o f t h e i n t r a c e l l u l a r  muscle  i s somehow b o u n d .  latter  results,  I n view  I t i s puzzling  water  i n the barnacle  of the consistency  that  of the  t h e two r e c e n t d e t e r m i n a t i o n s  1.13 of a  by. t h e K  m  microelectrode  +  J  (indicating  12  only  measured by t h e K  in  the area  tion given  from which  m  with  the barnacles  different  to indicate  of bound N a B.  microelectrodes  +  two s t u d i e s a r e p r e s e n t e d  i n a  were  V a r i a t i o n s i n I o n i c Content  change i n  real  a change  collected.  In this  associate the varia-  colonies.  i s probably  high •  coincides with  which  barnacle  This  the q u a n t i t a t i v e variation  and water  +  are rather  - .15% w a t e r b i n d i n g ) . .  as  chapter,  method  Arguments a r e i n the estimations  r a t h e r than  of Barnacles  from  artifactual.  Different  Sources All in  this  of the K  thesis  were  +  microelectrode  from  fibers  measurements  of barnacles  Vancouver while  the e a r l i e r  K  laboratory  from  of barnacles  Campbell  were  River.  The e v i d e n c e  studies  (McLaughlin  (1968a,  b;  value  and Hinke  f o rNa 1966)  values  obtained  Sjodin  1967;  internal  Na  by o t h e r s  Brinley  barnacles  Biomarine  Supply  was r e a l ,  barnacles  1968a).  Bay, C a l i f o r n i a  obtained  analyzed  niques  o f t h i s l a b o r a t o r y (Gayton  California  Brinley  than  apparently (Pacific discrepancy and  single  and e l e c t r o c h e m i c a l t e c h -  et a l 1969).  analysis,  were washed  whether t h i s  from C a l i f o r n i a  using the chemical  barnacles  by  Beauge' and  These i n v e s t i g a t o r s  fibers  Before- chemical  near  because the a n a l y t i c  e t a l 1964;  To d e t e r m i n e  were  collected  c o n c e n t r a t i o n was h i g h e r  from Monterey  Co.).  studies i n this  was c r i t i c i z e d  (Hagiwara  near  b i n d i n g from the e a r l y  +  1968b)  see a l s o McLaughlin  fortotal  obtained  fibers  collected  microelectrode  +  reported  the single  fibers  from the  f o r 30 s e c o r 10 m i n i n a n  114 isotonic 20  mM  sucrose  Ca(N0 > 3  remove j u s t all  was  during  determined  In  these  of  the fiber  Ion  laboratory  space  agreement w i t h  this +  same s o u r c e .  fibers  (5,6)  i n this  barnacles paper  there  California  obtained  Therefore,  California selective  discrepancy  i s no d i s c r e p a n c y and Vancouver  The  activities  barnacles  method.  s p a c e was 6.1%  were  these (7)  from  similar results  data  from  California  and  was t a k e n o f  o f N a , K , and +  +  barnacles  are i n  using barnacles  the analytic  techniques  from  used i n  introduced the discrepancy i n and Campbell  River  must be r e a l .  Notice,  i n Na  between t h e  +  content  however,  barnacles.  of Na  and K  +  +  i n t h e myoplasm o f t h e  a l s o measured w i t h  glass microelectrodes.  The a v e r a g e  the Na  +  (a., )  and K was  IN cl ITI  9.6 + 0.1 ( 1 7 )  this  other  of McLaughlin  The v a l u e s  by o t h e r s  space  results  no a c c o u n t  c o n c e n t r a t i o n between C a l i f o r n i a This  space  washout  and present  work. from  yielded  XIV l i s t s  The e a r l i e s t  l a b o r a t o r y c o u l d n o t have  barnacles. that  those  Extracellular  by t h e C l  Table  concentration f o rfibers  the  a c t i v i t y d i d n o t change  (1966) I s n o t i n c l u d e d because  extracellular  depleting the intra-  extracellular  on C a l i f o r n i a  (2,3,4).  laboratories  Cl  fibers.  with previous  and data  remove  When 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  t h e 10 m i n w a s h e d  (1) t o g e t h e r  +  fibers  t h e 30 s e c w a s h e d  content,  Hinke  Na  b a r n a c l e s , t h e average water.  should  t h e l o n g wash s h o u l d  t h e 10 m i n w a s h ) .  on companion  TrisNO^,  The s h o r t wash  ions without  (the myoplasmic  significantly  Na  ions while  25 mM  sucrose, 2  surface  ions  ( 6 5 5 mM  a n d 10 mM MgCNO^ > )•.  2 5  of the extracellular  cellular  to  solution  and t h e average  (a )  w a s 1 2 4 + 3 mM  (15).  +  TABLE X I V Summary  of internal  i o n content of single Wash Method  Barnacle Source  1.  California  [Na]  1  muscle f i b e r s  from giant  [K].  10 m i n .  water)*  21. 8 + 1.6 34  160 +3 34  36.0 +1 35  + 2.6 11  24.5  161 ±3 11  —  2.  California  unknown  22  168  35  3.  California  10 m i n .  22  162  —  4.  California  wash c u r v e , extrapolation to zero  27  160  —  5.  Campbell River  30 s e c , c o r r e c t e d for extracellular space  44  169  38  6.  Campbell River  10 m i n .  38  175  —  7-  Vancouver  30 s e c , c o r r e c t e d for extracellular space  23  175  30  * Water  i n extracellular  **Number o f  fibers  space  (6% o f f i b e r w a t e r ) n o n  References  [Cl].  (mM/Kg i n t r a c e l l u l a r  30 s e c , c o r r e c t e d for extracellular space  barnacles  included.  Hagiwara  1964  et a l ,  Beauge' a n d S j o d i n , 1967 —  Brinley,  Hinke,  1968  1969a  M c L a u g h l i n and H i n k e , 1968 Chapter V  IV and  116  The  former  types  result  of local  i s consistent with the results  barnacle.  The  (a„)  result,  K slightly and in  lower  than  found  considerably lower Campbell  of Na  and K  +  mM/Kg w a t e r  +  fiber  K  K  +  similar  fractions  i s lower  are very  Campbell  variation  riubilus. ruled  +  (which  the California  similar,  but both  +  the t o t a l  i s excluded  (Table XV).  both  types  Brinley  of local  K ) and  from  exactly than  the free t h e same  found i n  Thus, c h e m i c a l l y '  and t h e Vancouver differ  considerably  (1968a)  t h a t one o f t h e l o c a l term  from  type  collected  h a s shown t h a t  species  There  i s no r e a s o n t o  barnacle  types  i s not  differences  Balanus  can a l s o  of barnacle demonstrated  f o r up t o 3 m o n t h s i n a e r a t e d  at  between them i s n o t  factor.  environmental  out because n e i t h e r  b a r n a c l e s were  the differences  i s not an i m p o r t a n t  Short  changes  Na  excludes  Na  concentration  +  i s higher than  barnacles are almost  of the year,  seasonal.  believe  Na  a free  River barnacles.  Since times  barnacles are  i n having  the total  191  and  intracellular  River barnacles.  barnacles  concentrations  (which  o f bound water  electrochemically,  simply  than  1966;  t o b e 15  i n V a n c o u v e r b a r n a c l e s b u t much l o w e r  Campbell  various  barnacles  found  the calculated  i n the California  the  the free  V)  Quantitatively,  water)  and  1 9 0 mM)  and Hinke  = 0.65,  c o n c e n t r a t i o n which  or compartmentalized  found  -  (Chapter  Thus, t h e C a l i f o r n i a  bound  as  (McLaughlin  to local  concentration.  +  study  i n t h e myoplasm a r e c a l c u l a t e d  c o n c e n t r a t i o n which a free  3  (150  the values  Assuming t h a t Y  respectively.  qualitatively  and  than  both  however, i s 3  i n the present  River barnacles  1969a).  Hinke  m  from  sea water.  The  be ionic  effect  117  TABLE XV  The f r a c t i o n s  o f bound N a  different  groups  +  and water i n  of barnacles  Percentage, b i n d i n g o f intracellular H 0 Na +  Barnacle Source  References  2  California  16  present  study  12-15  Chapter  V  45  Vancouver  42-49  Allen 70-80  Campbell River  McLaughlin 1966  20-35  Hinke 1968  of  long term  cannot  environmental differences  1970  i s unknown and t h e r e f o r e  be r u l e d o u t .  McLaughlin  and Hinke  analyzable  Na  Na  addition, +  and K  lated  +  that  variations has from  Hinke,  1969a  Changes i n t h e b a r n a c l e w i t h age w a r r a n t s  In  and  and M c L a u g h l i n ,  Hinke, Hinke  1970  and H i n k e ,  also  +  and K  they  i n fiber  presumably  observed  i n single  ([K]  wide  observed  +  observed  content this  (1966)  m  fibers  a good  - O.78  variation content  higher Na  a wide from  linear  +  variation i n different  relation  [ N a ] = const.). T  They  anions.  concentrations i n small  young b a r n a c l e s .  barnacles.  between  i n i o n content might  o f impermeable  consideration.  fiber  specu-  be due t o The  author  fibers  Consistent with this  observ-  118 at i o n  i s .the f a c t  that the California  study yielded, f i b e r s fibers  from  appreciable barnacle nate  local  barnacles.  difference.in  has  been  demolished.  C.  Ionic  Part (Hinke  the Campbell  Vancouver  barnacles.  [C1]  T  (chemical  problem  River  and a  Q  a r e compared here  further  the difference  Each least  were  about was  (K)  b a r n a c l e s were  soaked  i n normal  o f 750 mM/1  complete  that  this  water  loss  that  the increase  1966-67  i n  Ringer  parameters  were  [ K ] , [ N a ] , and T  m  T  sensitive  T h e 0, 10, a n d 30 m i n  (Table XVI) i n order t o emphasize  result  16 d e t e r m i n a t i o n s .  nearly  obtained  between t h e two t y p e s o f b a r n a c l e s .  electrode  50% o f i t s w a t e r  that  sucrose.  and ( N a ) ( c a t i o n  C-sorbital).  8 d e t e r m i n a t i o n s and each  least  I t i s unfortu-  i s concerned)  (wet-dry weight),  (  local  b a r n a c l e s was r e p e a t e d o n  by a d d i t i o n  content  results  in  River  analysis),  microelectrodes),  at  possible).  0, 5, 10, 20, a n d 30 m i n , t h e f o l l o w i n g water  i s no  i n Hypertonic Solution  Fibers  made h y p e r t o n i c  determined:  than the usual  o f an e x p e r i m e n t p e r f o r m e d by Hinke  1969a) o n C a m p b e l l  solution  of this  Changes o f F i b e r s  larger  b e t w e e n t h e two t y p e s o f  i s s t i l l  (as f a r as e l u c i d a t i o n from which  50 - 100%  i n this  On t h e o t h e r hand,, t h e r e  size  (an age d i f f e r e n c e  the p i e r  At  which were  barnacles: used  i n T a b l e X V I i s t h e mean o f a t chemical result  In both experiments the f i b e r  to the hypertonic  within  i s t h e mean o f  30 m i n .  solution  The i n c r e a s e  was f r o m t h e i n t r a c e l l u l a r  of [K]^ i n the hypertonic  lost  and the l o s s i n a  Q  water.  solution  indicates Notice  was.less  exp. 1 (Hinke). than i n exp. 2 (Gayton) but the i n c r e a s e i n  TABLE X V I A comparison  o f t h e same h y p e r t o n i c  1.  Hinke's  2.  Gayton's  Parameter  experiments  experiment  on Campbell  River  experiments on Vancouver  Symbol  Experiment  on t h e two t y p e s o f l o c a l  barnacles  barnacles.  barnacles.  Time 0  i nhypertonic  s o l u t i o n (min)  10  0.56V  30  0. 58V  0. 50V o 0. 5 2 V  0 . 065  0.121  0.142  1  159  224  235  2  168  266  302  (K) . m (mM/1)  1  235  406  447  2  207  333  338  myoplasmic water f r a c t i o n  a. m  1  0 . 678  0.551  0.525  2  0 . 812  0.796  0.89  bound water fraction  aB  1  0.257  0 . 327  0.333  2  0.123  0. 083  -0.032  volume H 0  of fiber  1  V  2  extracellular water f r a c t i o n total fiber K cone.  2  [K]  +  myoplasmic K cone. +  1  a  (mM/Kg  T H 0) 2  & 2  o V o V  c  TABLE XVI ( C o n t i n u e d )  Parameter  Symbol  Time  Experiment  i n hypertonic  0 fiber K content  [K] V T  ~V (mM/Kg  bound  *  125  117  2  168  156  156  2  assumed 2  a  Na  1)  27  48  64  2  (Na) V m V o (mM/Kg H 0 ) m  Na V o (mM/Kg  (a from m  = 0  H 0)  2  bound  159  H 0)  V  myoplasmic Na+  30  1  K o (mM/Kg  10  s o l u t i o n (min)  7.1  7.1  10. 4  2)  9-5  9-3  18. 0  1)  8.1  6.4  10. 2  34.5  17. 6  22. 4  2)  19-6  15. 8  10. 5  1)  21.0  18.6  18.1  1 2  (a from m (a from m  2 1 2  H 0) 2  (a from m (a from m  2  * The l a s t f o u r t e r m s r e p r e s e n t t h e a b s o l u t e amount o f t h e i o n ( c o n e , x v o l . ) d i v i d e d by t h e v o l u m e o f t h e f i b e r a t z e r o t i m e (V ) . T h i s g i v e s a n i d e a o f i o n m o v e m e n t i n t o o r out o f t h e compartment. 0  121  (K)  was g r e a t e r  calculated from  i n exp. 1 than  myoplasmic water  0.68 ( R i n g e r )  increased  from  otg = [ 1 -  (  surprising 1970).  a  m  +  0  ^  exp.  1 but only  was or  increased  3  t o zero  7% i n e x p . 2.  so t h a t  + +  slightly  amount o f f i b e r  n o t a c c o m p a n i e d by a l o s s Mg  K  m  )]_  +  must have  decreased  but ( a ) m  Thus, t h e f r a c t i o n o f bound i n exp. 1 which  i s not osmotically  H o w e v e r , otg d e c r e a s e d  the absolute  a  t o 0.525. ( 3 0 m i n i n h y p e r t o n i c )  i f bound water  that  As a r e s u l t , t h e  f r a c t i o n i n e x p . 1, (  0.81 t o O.89. a  i n e x p . 2.  active  large  of C l left  i s not  (Hinke  dropped  T  This  water,  i n e x p . 2. • N o t i c e  K , ([K] V),  -  loss  of K  o r an uptake  the fiber  with  2  +  also  26% i n  i n exp. 1  of Na , +  an a n i o n  Ca  + +  ,  other  than C l " .  Since  both  o f these  experiments  were  u n d e r t h e same c o n d i t i o n s ,  the differences  represent  i n the barnacles.  claims, fiber,  differences  ( )-j_ i s equal  to the osmotically  a  m  the loss  roughly P,  real  of water  proportionate  from t h i s  c a r r i e d out  i n t h e r e s u l t s must I f , as Hinke  active water  compartment  to the increase  should  i n the osmotic  i n the be pressure,  i n the bath •H _ ^ m^ ^ R P " ( a V)„  /  a  Q \  0  K  d  i  c  R where  m  the subscripts  solution.  H  H and R r e f e r t o h y p e r t o n i c  The o s m o t i c  pressure  ratio,  o s m o m e t e r , was 2200/930 = 2.37. indicating equation  that  (23).  amount o f s o l u t e  slightly This  T h e (a )  drop  i n a "V i n e x p . 2 w a s o n l y m  with  i n e x p . 1. r  the  m  1  on a  ratio  more w a t e r was l o s t  i s consistent  was l o s t  measured  and Ringer  than  the fact  Piske  was 2.56, expected that  a  from  small  The ( a ) r a t i o was 1.75; . m 2 ' a b o u t 2/3 t h e d r o p e x p e c t e d 0  )  122 from the  the osmotic a  determined  m  equal  b y the' K  results  that  B^. = 0 w a s  used  i n e x p . 2, t h e n  water  f o r the fibers  amount, bound solution that in  i n e x p . 2.  +  the  (21), B  Ringer  K  = 27 mM/Kg  solution  doubles  i n the hypertonic of  speculation,  [ a (Na)V] m  that  and bound N a  +  Na  +  and bound N a  further  +  supports  myoplasmic D.  e q u a t i o n 21 reason  f o r Na  t o expect  during osmotic the v a l i d i t y  changes  water  of (a )-^ m  loss,  i f t h e (oc^) -,  to calculate  instead  +  d i d not  (B„ V ) i n e x p . 2 Na ^  I n s t e a d o f t h e (o^)2 v a l u e s a r e used  i s no o b v i o u s  i s equal  the absolute  values  there  +  T  constant during the hypertonic experiment  (using  K  o f [ K ] V i n exp. 1 which Interesting  fiber  and i n a b s o l u t e  remain  parameters  assumption  I f the ( c ^ ) ^ values are  or compartmentalized  I t i s also Na  osmotic  as a measure o f  Perhaps  N o t i c e , f o r t h e sake  to the decrease  of free  ( o ^ ^ -  equation  i n normal  and  more p l a u s i b l e ^  n  (B^V) more t h a n  +  results  +  i n e x p . 2.  i n c r e a s e i n bound  magnitude  amounts  than  from  (Table X V I ) .  this  occur  K  the K  (a ) m l  erroneous  does n o t  water.  compatibility•of  myoplasmic water  barnacles,  m i c r o e l e c t r o d e method  +  active  i n exp. 1 renders ^  free  T h u s , i n the. V a n c o u v e r  J  the osmotically The  the  gradient.  of K ) .  these  Since  +  i n the t o t a l this  free  observation  as a measure  of the free  water.  Discussion Both  strate  of the studies  the v a r i a b i l i t y  microelectrode barnacles  studies.  (bathed  of a  presented  chapter  when i t i s d e t e r m i n e d  m In both  i n normal  i n this  California  Ringer &  solution)  and a m  by  demon-  K  +  Vancouver - 0.80  and, '  123 at .least  i n the  osmotically  latter,  .a  d o e s not- s e e m t o  a c t i v e w a t e r . . On  the  other  be' r e l a t e d t o  hand, the  (0.68)  a m  5  from Campbell River  barnacles  fiber  d e t e r m i n e d t o be  water  that  Is  alternative  techniques  experiments  w e r e done on  For 0.68  as  for  the  total  that  fiber  the  the  K  of  fiber  )  m  and  K  the  fiber  K .  et  (1967,  Fig.  4)  al  or  water  even though  (1968)  be  a  due  to  0.73  and  Hinke  K  +  The  i n the  barnacle  kinetics  whether the the  (a =  (in preparation)  compartment.  if  B^.  of  were  muscle w i t h  small rise  expected  increase  k2  K  +  to  Unfortunately,  i t i s not  r e m a i n i n g . 'K -would have  experiment  +  had  Biochemical  been  (10  mM)  could  m  of  this  K  +  with  heating  have  been  Allen  8l% of  was  a  of  the  50  hr.  single  c l e a r from t h e i r  exchanged  at  the  the  shortening.  experiment). exchange  and  in  the  increase K  of  Hinke  over a period  exchange i n d i c a t e d that  25%  shortening  (« )  in this  that,  least  during  to  in  able  the  barnacles.  irreversible  p r o t e i n d i s r u p t i o n , the  to  have p o s t u l a t e d  decreased  of  solvent  of  compartmentalized.  and  of  value  as  15%  r e s p e c t i v e l y , at  [K]„  bound w a t e r would not  a release  latter  the  acts  (about  Vancouver  observed  thermally-Induced  muscle  of  these  accept  water which  and  muscles  i s e i t h e r bound  +  a number  of  barnacles).  mM/Kg f i b e r  crab  fraction  I t t h e n becomes n e c e s s a r y  +  Hays  f r e e by  a r g u m e n t , l e t us  in California  +  during  barnacle Since  K )  the  (several of  Vancouver  of  - 25  R  l o b s t e r and  McLaughlin a  B  (1961)  Robertson  (  sake  fraction  1970)  (Hinke  free intracellular  postulate  in  the  approximates  the  data  same  rate  continued.  evidence  f o r monovalent  cation  binding  124 t o . m y o s i n was. c i t e d and  1957;  Saroff  Sar o f f ,  i n Chapter 19-5.7).  Fenn  (1970)  Hinke  35 - 40 mM.  and  ( i n which  (McLaughlin  specificity  As  indicate  Allen  barnacles that  1968;  w o u l d be  i n Campbell N  River  = 3 0 - 3 5  &  1969a).  Hinke  mM/Kg I f K  +  by t h a t  that  Campbell River  selectivity  certainly  N  a  t h r o u g h membranes;  /  a  /  p  p  =  1  0  K  = K  barnacles.  11-and  10  —3 )•  of the ions 12 o f C h a p t e r  Hinke  V  (unpublished  f o rthe surface  I t i sp o s s i b l e  membranes c o u l d  that  or compartmentalized..  (equation  a  o f t h e membrane o f a n i n t r a -  Fig.  p o s s i b l e , even probable,  definition  specificity i s  21),that  membrane  that the  also  vary.  a system as complex as t h e muscle  e i t h e r bound  apparent  N  differences  membrane o f V a n c o u v e r b a r n a c l e s i s  p  p  of internal  In  o f ions  organelle.  + forK (  I ,binding  change t h e p r o f i l e  the surface  specific  f o rtheionic  barnacle.  specificity  could  data) calculated that  is  Since  capacity  1 5 mM i n V a n c o u v e r  = 0), B  K  out i n Chapter  i n t h e passage  organelle  accumulated  account  of local  i nthebinding  cellular  of  and Hinke  could  pointed  important  highly  = 10 -  a  i t i s assumed B  between t h e two types  change  N  thebinding  a r e bound t o t h e c o n t r a c t i l e p r o t e i n s , a change I n  +  binding  also  B  I t i s'Interesting t o note  water  Na  that  v  3  fiber  fount  Lewis  o f Lewis and  water.  t h e sum o f B „ + B i n t h e s e Na K  barnacles,  barnacles  the. d a t a  50 mM/Kg f i b e r  o f m u s c l e w o u l d be about and  Using  (196.8a)^ c a l c u l a t e d t h a t  McLaughlin  19-47;  I I (Szent-G.yorgyi  fiber,  a small  i t i s  amount o f K  +  I t i s i n e v i t a b l e , by  a variation i nB  as a v a r i a t i o n i n the c a l c u l a t e d a .  K  would  Since  become  0.68  125 s e e m s t o be  t h e most r e l i a b l e  fiber  which  water  solvent B  K  =  f o r free  0 must be  barnacles.  equilibrated reasonable  above  a  + B^  i n normal  but  variable  cations  capacity  proteins the  way  such  = const., Ringer  i n a state  living  cell.  may  as  valid  solution.  Such  system  include H , +  that  Ca  + +  To  with  ,  be  and  close  a  and  + +  as that  California  further  i n barnacle  muscle  a relationship i s binding  more p r e c i s e ,  Mg ..  of other  the com-  What i s n e e d e d  i s a d e t a i l e d study  specificity  i s as  acts  constant  the b i n d i n g  laboratory  binding  that  be  specificity.  i n this and  (and w h i c h  then the assumption  the r e s u l t s suggest  i n a physicochemical  i s under  binding  in  B^  cations)  f r a c t i o n of  at l e a s t i n the Vancouver  r e l a t i o n s h i p should  petitive what  invalid,  for-the  i n the myoplasm  monovalent,  Moreover,  relationship,  capacity  is. free  estimate  of  and the  of the c o n t r a c t i l e  as p o s s i b l e  to their  state  126 CHAPTER V I I  CONCLUDING  The e x p e r i m e n t a l expand that the It  previous  barnacle  muscle  I s now a p p a r e n t  equally  water  distributed  that  thesis  evidence from t h i s  and monovalent  myoplasmic  myoplasmic  from about  cellular  water.  suggest  fraction  of the i n t r a c e l l u l a r  The r e s u l t s a l s o K  results are i n keeping with  others  which  indicate  Na  least  two phases water  (McLaughlin Hinke  and Hinke  1970).  either  neither  cations  1966;  a free  It incorporates  +  a small,  variable  i n t h e myoplasm. muscle  inactive 1970;  and Hinke  agreement  with  and'association-  of the I n t r a c e l l u l a r environo f the barnacle muscle i s  solution nor a highly  to consider  a theory  f r o m t h e membrane t h e o r y  theories.  t o t h e Donnan  Allen  or the sorption  i s now p o s s i b l e  "non-membrane"  i s passively  i s d i s t r i b u t e d between a t  1969a;  environment  homogeneous  ideas  -  and i s o s m o t i c a l l y  Hinke  on t h e n a t u r e  The i n t r a c e l l u l a r  almost  on t h e b a r n a c l e  These r e s u l t s a r e not i n t o t a l  theories  manner.  a substantial f r a c t i o n of the i n t r a -  t h e membrane t h e o r y  induction ment.  intracellular  excludes  of  45% o f t h e i n t r a -  that  i s not free  +  These  and that  C l  membrane a c c o r d i n g  b u t seems t o be e x c l u d e d  cellular  ions  f r a c t i o n a n d a b o u n d o r com-  The f r e e  the fiber  and  laboratory  inorganic  relation  that  confirm  Intracellular C l "Is divided  fraction.  across  of this  are d i s t r i b u t e d i n a heterogeneous  between a f r e e  partmentalized  findings  and c o i n c i d i n g  the i n t r a c e l l u l a r  DISCUSSION  ordered  which  and t h e  Pew b i o l o g i s t s d i s p u t e  matrix.  various  the presence  12 7 of  the c e l l  actively, The  m e m b r a n e o r i t s . a b i l i t y , to: c o n t r o l , p a s s i v e l y a n d  t h e movement o f s u b s t a n c e s  results presented  under'most across  in  Chapter  I I from  parations. the  i spredictable  water not  o f water  teins,  then  those  within  proteins  ions  solution.  the evidence  skeletal  a portion of  I . e . I n a manner  water  I fthis  m u s c l e do  heterogeneity  claimed  studies  the intracellular  The r e s u l t s f r o m  monovalent  by L i n g .  on m y o s i n  for the binding  possible  v a r i a t i o n ) may w e l l b e f o u n d  theories  of Ling  Lehninger  and Eisenman  Scott  1968;  1968)  pro-  contains (Carvalho  (I960;  other  They  (Lewis  also' i n d i c a t e ,  specificity  bound. (and i t s  i n the i o nassociation  1962).  I t should  Carvalho  and'mucopolysaccharides  Dougherty  1957;  and S a r o f f  potential sites  e t a l 1963]  this  cations i s  +  However, t h e e x p l a n a t i o n  a s membranes  i s the  of the intracellular  and t o bind  +  such  theory.  of the intracellular  of the barnacle  the capacity  the capacity  muscle  pre-  o f t h e "non-membrane" t h e o r i s t s , e s p e c i a l l y  indicate that  that  cited  muscle  1957), t h a t N a , a n d n o t K , i s p r e f e r e n t i a l l y  noted  to the  o f t h e membrane  and i o na s s o c i a t i o n w i t h  do t h e b i o c h e m i c a l  Fenn  and  according  solution;  (1962), may b e p e r t i n e n t .  than  surface.  ared i s t r i b u t e d  -  indicate that  the concepts  inorganic  t o organize  much l e s s  other  substantial portions  t h e Ideas  of Ling  laboratory  muscle  results also  as i f i n f r e e  result  on most  as i f i n free  and monovalent  behave  and C l  +  i s compatible with  studies  The p r e s e n t  However,  as  This  myoplasm.behaves  that  K  of the barnacle  Donnan e q u i l i b r i u m .  the cell  i n Chapter V strongly i n d i c a t e . t h a t ,  equilibrium conditions,  t h e membrane  across  1969)3 w h i c h may a l s o  besides 1966;  (Farber bind  be myosin, Gear  e t a l 1957;  monovalent  12c cations, fiber  but the concentration  i s rather  excretion  may  have  of  exercise  of materials  c o n t r o l over  and  water  primary  I n t h e case  must be m o d i f i e d  association with  i n the muscle of myosin.  c o n t r o l over the uptake  from the f i b e r ,  the composition  the•fiber interior.  membrane t h e o r y  substances  l o w c o m p a r e d to. t h e c o n c e n t r a t i o n  W h i l e t h e m e m b r a n e may and  .of t h e s e  the protein  matrix  of a substantial f r a c t i o n  of the barnacle  to include  muscle, the  the effects of i o n  proteins  on t h e i n t r a c e l l u l a r  and t h e nature  o f t h e i o n and water  environment.  The ation is  with  extent  proteins  i n vitro  i t certain.that the findings  polated  to i n vivo  whether  i o n and water  all  muscle. IV)  and N a  not  been  +  composition fractions  could  nuclei  may  valent  cations  1970)  to accumulate  (Lehninger Azzone  et a l 1969),  only  organelle  of significant  reticulum.  I t i s now g e n e r a l l y  the  membrane  surface  or a l l of these  -  (Chapter  slow  While mitochondria small  these  volume  organelles  i s the  that  and  a m o u n t s o f mono-  of the barnacle  agreed  o f the muscle  barnacle  muscle has  Antonov e t a l 1965;  n e g l i g i b l e f r a c t i o n o f t h e volume  The  i n the  i n the  c a n b e made a b o u t t h e  and b i n d  1965;  can account f o r  i n the barnacle  Part  Nor  extra-  fractions of C l  no c o n c l u s i o n  fractions.  c a n be  observed  and water  be c o m p a r t m e n t a l i z e d .  be a b l e  1968;  Lehninger  so t h a t  ions  clarified.  i t i s not c e r t a i n  proteins  o f the slow  ( A l l e n and Hinke  of these  studies  of the heterogeneity  exchange  obtained  not been  Therefore,  association with  o f the monovalent  Complete  s t i l l  o f such  conditions.  o r even a major part  distribution  a  have  associ-  Gear and comprise muscle.  sarcoplasmic  depolarization of  i s transmitted  down t h e t r a n s -  verse, t u b u l e s cisternae (Huxley  to  a c t i v a t e , r e l e a s e . o f Ca  which-in  and  is  r e l e a s e d by  cisternae,  expect  nature  as  membrane; low  Ca  + +  i n the  chemical  accumulate  Na .  amounts  bound  (Carvalho  is  possible that Na  and  +  the  myoplasm.  IV)  have  K  +  i n the  that  and  Leo  the  be  exist  reasonable  across  the  C l  ,  the  bulk  1967;  Cl  of the  other  Ion  1968).  muscle which  authors  possibility direct  (see  of NaCl  evidence  a  at  of  be  as  the  able  accumulates  i s believed  to  Thus, i t  reticulum contains  barnacle  r e t i c u l u m but  this  Carvalho  sarcoplasmic  A number o f  of  reticulum  and  -  well  f  sarcoplasmic  same  surface  Na -K' -A,TPase as  r e t i c u l u m may  to  the  r e t i c u l u m , or  +  + +  across t h i  concentration of  Since  Ca  terminal  membrane o f  sarcoplasmic  the  the  If  potential  then  sarcoplasmic  the + +  1964;  m i c r o s o m a l membrane f r a c t i o n  the  Ca  membrane o f  this  a high  i n the  of  considered  sarcoplasmic  across  process  Sandow 1 9 6 5 ) .  I t would  c o u l d be  While  +  be  cellular  state.  gradients  1968),  (Carvalho  terminal  Huxley  electrical  terminal cisternae. i s found  substantial  an  gradients  i . e . there  -ATPase  muscle to  resting  concentration of  least  a l 1965;  C o s t a n t i n et  chemical  the  a l 19&3;  Weber e t  t h e r e m u s t be  membrane i n t h e also  1958;  a d e p o l a r i z a t i o n of the  then  the  t u r n a c t i v a t e s the. c o n t r a c t i l e  Taylor  Hasselbach.1964;  from  the  i s not  intrafree  s e c t i o n D,  accumulation for this  in  Chapter in  the  proposal  is  lacking.  It reticulum cellular and  is in relatively space v i a the  i s part  small  i s also possible that  of.the  molecules  as  transverse  C l  least  a p o r t i o n of  free communication w i t h  extracellular  such  at  -  and  tubules space  the  ( B I r k s and  Luft  extra1969)  Davey  t h a t , i s measured  "^C-sorbital.  the  with  (1964)  and  130 Kelly  (1.96.9) have, f o u n d  penetrates as  the. t e r m i n a l  ferritin  diad  such  junctions  Spira  between t h e t e r m i n a l  appear  1965;  t o be s i m i l a r  between e p i t h e l i a l f o rintracellular  Bullivant  and Loewenstein  ions  freely  pass  Much l a r g e r m o l e c u l e s  unpublished results).  tubules (Fahrenbach  1969)  pathways  cisternae.  a n d h o r s e r a d i s h p e r o x i d a s e do n o t e n t e r t h e t e r m i n a l  junctions  transverse Kelly  = 550)  (Huxley 1964;  cisternae and  t h a t ..the. d y e r u t h e n i u m .red- (M.W.  account  f o rthe fact  (Ling  and Kromash. 1 9 6 7 ) .  graphic sizes  study  into  greater  finding  fraction  Hays  picture.of  is  structured,  excluded the  extra  surrounding not  that  C l  than  large  I f small, i t would molecules  molecules  be an  appears  autoradio-  water  t o be e x c l u d e d f r o m  than i s K  another  +  environment.  I f K  water  water.  I n Chapter  (15%  +  i s excluded  because  this  Cl" will  water)  might  exclusion..from the negative e l e c t r i c a l  exclusion  from  proteins. 15%  into the  water also  I V , i t was s u g g e s t e d  of the fiber  This,  of the fiber  a  (Chapters IV and  complication  i t i s r e a s o n a b l e t o assume t h a t  the i n t r a c e l l u l a r  be a t o t a l  -  introduces  C l " exclusion  electrostatic  junctions.  of tagged molecules of various  of the intracellular  from t h i s  inorganic  fiber.  the intracellular  from a f r a c t i o n  monovalent  Of p e r t i n e n c e w o u l d  of the fiber  e t a l 1968)  1966;  (Loewenstein  these junctions,  space  1965;  to the septate  s u p p o s e d l y Impermeant  of the penetration  t h e muscle  The  V;  small,  triad  a c t as low r e s i s t a n c e  and diad  m o l e c u l e s were a b l e t o c r o s s  extracellular  Peachey  Perhaps  across the t r i a d  a larger  19&5;  which  neutral  indicate  and t h e  Birks  iontransfer 1968).  that  cisternae  i n structure  cells  The  that  be due t o field  of course, water  be  but a  would  131 graded the  exclusion  field)  strength is  from  a larger  proportionate  to the strength  percentage, o f the, f i b e r  a s e s t i m a t e d by. E l l i o t  excluded from  other  hand,  field  and hence  field  would  a l l . of the water  cations  (1968),  should accumulate  be s t r o n g e s t .  Kallay  C l ~could  i n t h e A band.  s h o u l d be a t t r a c t e d  of  water.  o f t h e e l e c t r o s t a t i c , f i e l d . between . the t h i c k  as s t r o n g  ally  (inversely  I f the  filaments be  parti-  On t h e  by a n e g a t i v e  i n t h e A band where  electrical this-  and T i g y i - S e b e s (1969),  from ho  an  autoradiographic study of frog muscle, 2k  and I  were  band.  K  to  accumulation of K  as bound  +  rise  of the variety  intracellular  water  just  free  T h u s , i t may  water, through p a r t i a l l y  bound water.  the  as e x i s t i n g  similar  binding,  which  ions  structured  may  from  structure  be.further  water i s  t o imagine  to  depending  ideal firmly  will  of heterogeneity i n i o n distribution  Ion d i s t r i b u t i o n  existing  of states,  water,  may naive  intracellular  environment,  from  112).  page  i t i s perhaps  i n a variety  This heterogeneity i n water  degree  water.  that  have t h e  i s calculated  be more r e a l i s t i c  t h e m o l e c u l a r and m a c r o m o l e c u l a r  solvent  a  water  would  i n increasing the  and i n t r a c e l l u l a r  It i s unlikely  intracellular  than i n the  of the conditions  i n two s t a t e s . o r bound.  +  that  t o heterogeneity i n the muscle,  imagine  this  (equation 21,  only  on  like  myoplasmic water,  microelectrode results view  +  or•compartmentalized K  of a , the free  In give  50% m o r e c o n c e n t r a t e d i n t h e A b a n d  A regional  same e f f e c t  the  K  + Na  value  concluded that  +  define  within  c o m p l i c a t e d by  c o m p a r t m e n t a l l z a t i o n , and. e l e c t r o s t a t i c  interactions.  132 BIBLIOGRAPHY 1.  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I.  145 APPENDIX  The  study  o f e l e c t r o l y t e s o l u t i o n s has always  hampered by t h e i n a b i l i t y of  the individual  sensitive  limited liquid  by t h e a c c u r a c y  is  activity 1965;  Shatkay  thesis,  solutions  in  the course  of this  sensitive electrode  (e.g.  a Na  only  +  glass  coefficients  1969;  electrode  (vs.a calomel  coefficient  AgCl  the potentials of a Na  electrode  concentration. junction trode  were  recorded  A l lreadings  +  i n NaCl were  results)).  reported  i n this  on a v a r i e t y o f calibrations).  glass  electrode  electrode  were  corrected.for  p o t e n t i a l between t h e s o l u t i o n - a n d  was r e p e a t e d  several  times.  recorded  ( a t 24°C).  and a Ag-  s o l u t i o n s o f t h e same the liquid  the calomel  b y means o f t h e H e n d e r s o n ( 1 9 0 7 ) . e q u a t i o n .  readings  this  (Eisenman  reference)  glass  by  methods  0 . 0 5 , 0 . 1 , 0 . 2 , 0 . 4 , 0 . 6 , a n d 1.0 M K C l s o l u t i o n s  Similarly,  vs. a  t o t h e mean  to get precise +  an  close  w e r e made  the potentials of a K  a Ag-AgCl electrode  opposite  obtained  unpublished  studies  ( u s u a l l y i n an e f f o r t  one e x p e r i m e n t ,  method i s  t h e mean a c t i v i t y  of the experiments  electrode  t o measure  the magnitude o f the  m e a s u r e d by c l a s s i c  and Lerman  numerous  the activity  t h e numerous i o n  and K C l s o l u t i o n s a r e very  coefficients  In  and  a cation  ( t h e mean a c t i v i t y  f o rNaCl  been  The j u n c t i o n p o t e n t i a l c a n be  e l e c t r o d e ) , but then  measured  method  In  of estimating  sensitive electrode  Ag-AgCl  With  but the accuracy  junction potential. by u s i n g  t o measure  now a v a i l a b l e , i t i s p o s s i b l e  ionactivities  eliminated anion  of chemists  ioni n solution.  electrodes  individual  I  elec-  Each s e t o f  146 In-Fig. (B)  potentials  activitiesj  K  C  1  K  +  electrode  (A) and.the C l ~  i n the' K C l . s o l u t i o n s ' a r e  calculated  Y  where Y  13i t h e  KC1  =  Y  p l o t t e d against  the i o n i c  from the e q u a t i o n :  K  =  Y  C1.  i s the' mean a c t i v i t y  ( R o b i n s o n a n d - S t o k e s 1959).  c o e f f i c i e n t o f the- K C l s o l u t i o n s  The s l o p e s  (S) o f both curves a r e  c o n s t a n t and t h e changes i n p o t e n t i a l p e r t e n f o l d activity  electrode  a r e as e x p e c t e d f o r t h e s e e l e c t r o d e s  change i n  a t 24°C.  The N a  +  +100-  -20 0.01  1  1  •  •  1 1 1 1 1  0.1  IONIC A C T I V I T I E S  1  1  1—1—1—1—1—1—1—  1.0  (mM)  F i g . 13. The e l e c t r i c a l p o t e n t i a l s o f a K e l e c t r o d e ( A ) a n d - a C l ~ e l e c t r o d e (B.) '.(vs. a c a l o m e l e l e c t r o d e ) i n K C l s o l u t i o n s . S and S r e p r e s e n t t h e changes i n p o t e n t i a l p e r t e n f o l d N a  C 1  change i n i o n i c  activityi  .147 electrode  (A) and t h e C l  solutions  a r e shown i n F i g . 14.  obtained  i f we  assume  Y  The  less has  than  than  expected  expected  Cl  then,  i n NaCl  T  Na  =  Y  i n NaCl  the slopes  almost  C1  =  Y  KC1  a  = 60.5) and  the activity K  C  than  1  Y  N  a  It  coefficient  C  1  Since  -  1  •  l  a  n  d  +  Y  , Na  (wY  y  =  NaCl  K  C  ;  )  2  1  are calculated i n this  electrode  ( S  N  a  = 59)  as t h e o r e t i c a l l y and' L e r m a n  activity  predicted  (1969)  (open  (+_59).  have r e p o r t e d  ion activities coefficient,  manner,  and t h e C l ~  potentials vs. activities  Thus, i n d i v i d u a l  t o t h e mean  N  (Sp^ = - 5 6 . 5 ) .  electrode  Y  ( S  l e t  of the Na  and Shatkay  electrode  closer to  coefficients  exactly  findings. equal  c  ( S ^ = -58.5)  electrode  (I967)  Na  T  -  +  change i n a c t i v i t y i s  1919) t h a t  ' ^NaCly  solution,  the activity  then  (Mclnnes  _  c i r c l e s are-  C1  f o r the Na  i s probably  Y  are  The f i l l e d  that  f o r the C l  been suggested  Y  If  =  '(B) p o t e n t i a l s i n t h e N a C l  change i n p o t e n t i a l p e r t e n f o l d  greater  of  NaCl  electrode  circles)  Garrels similar  are not necessarily  even i n simple  solutions.  It coefficient position)  i s generally.assumed o f sea water  i s O.65  coefficients in  Table  VIII  (and Ringer  and t h a t  are close  that  t h e mean  solutions of similar  the individual  to this  value.  (Chapter V) s u p p o r t  activity  this  com-  ion activity  The r e s u l t s p r e s e n t e d assumption.  However,  148  _l  o.oi  I  I i _  0.1  1.0  IONIC ACTIVITIES (mM)  Fig._l4. The e l e c t r i c a l p o t e n t i a l s o f a N a e l e c t r o d e (A) and a C l . e l e c t r o d e (B) i n N a C l s o l u t i o n s . The o p e n c i r c l e s w e r e +  b y a s s u m i n g YQ-J_ = Y ^ d  obtained  a n <  ^  ^NaCl^ YKCl  Y Na  2  The  YNaCl' f i l l e d c i r c l e s were o b t a i n e d by a s s u m i n g y YQJ_ O n l y one p o i n t i s shown a t t h e two l o w e s t c o n c e n t r a t i o n s b e c a u s e b o t h p o i n t s .are v i r t u a l l y identical. =  Na  Y  K  was  Y  K  = Y j ^ i n .these  the  c a l c u l a t e d from equation a  solutions.  (20) w i t h  This  study  f o l l o w i n g electrode, combinations:  calomel, corrected sented  Na  +  vs. calomel,  and.K  +  for junction potential,  i n Table  XVII.  Notice  the assumption was  that  expanded t o i n c l u d e  Cl  vs. calomel,  K  vs. C l .  The r e s u l t s ,  roughly  where necessary-,  t h a t . y. T  > 0.7  +  vs.  are pre-  f o r a l l of  these  TABLE The  activity  Solution  coefficients  Ion [C1]  Q  o f C l , Na  Concentration [Na] [K] Q  and K  y  Q  XVII  c  i n a number o f b a r n a c l e  Y  l  YK  N a  mM/1  (  34  330  128  .  (.700)  19  229  229  (.705)  11  537  330  128  .647  12  537  229  229  651  14  657  450  128  624  .712  .  Y  K  =  Y  Ringer  K Na  )  (  Y  Na  =  Y  0  -  7  1  )  (  Y  K  solutions  KC1 =  Y  Y  Na  450  667  (  Y  KC1  Na  =  0  -  .65  .615  .656  .645  .713  64  .627  .672  .665  .716  64  .601  .625  .610  .713  .636  .61  626  .618  621  .575  .613  .595  .708  Y  537  )  .716  NaCl .685  7  1  )  . 15-0 solutions  than  solutions;  in  the bathing  drawn from on  K  m  slopes  more p r e c i s i o n individual  l  r e c a l c u l a t e d from  s  then  y^  solutions.  i s considerably  Unfortunately,  K  +  a c c u r a t e l y i n low [K]  i n Chapter [K] . o  V , y. T  While  should  such  tend  more  a variation  of  y„ K  s o l u t i o n s would n o t change t h e c o n c l u s i o n s  the present  the ( a )  [K]  cannot measure  w h e n [ N a ] >> o  M  Y^  = 0..71,  t h a t y^  as mentioned  y 'Na  ^  a  0.65 i n t h e h i g h  glass.electrodes  toward  ? ,Yfj *  K  (20). a s s u m i n g  equation lower  and t h e r e f o r e y  than  ionic  Donnan s t u d y ,  i n F i g . 11  and 12.  the present  activities  i twould  have  an  I n any s t u d y  one, these  would  have  effect requiring  variations i n  t o be t a k e n  into  account.  REFERENCES 1.  E i s e n m a n G. ( 1 9 6 5 ) . I n Advances i n a n a l y t i c a l chemistry and i n s t r u m e n t a t i o n . V o l . 4. E d . R e i l l e y C.N. W i l e y I n t e r s c i e n c e , New Y o r k .  2.  Shatkay  3.  Henderson,P.  4.  R o b i n s o n R.A. a n d S t o k e s solutions. Butterworths  5.  Mclnnes  6.  G a r r e l s R.M. ( 1 9 6 7 ) . I n Glass e l e c t r o d e s f o r hydrogen and other cations. E d . E i s e n m a n G. M a r c e l D e k k e r , I n c . , New Y o r k .  A. a n d L e r m a n A.  D.A.  (1907).  (1919).  (1969).  Z. p h y s i k .  Anal.  Chem.  Chem. 5 9 ,  4l,  514.  118.  R.H. ( 1 9 5 9 ) . Electrolyte S c i e n t i f i c Publications,.-London.  J . Am.  Chem. S o c . 4_1,  1086.  

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