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The movement of potassium ions in normal and dystrophic mouse muscle Burr, Lawrence Herbert 1961

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THE  MOVEMENT OP POTASSIUM IONS  IN NORMAL AND DYSTROPHIC MOUSE MUSCLE  by LAWRENCE HERBERT BURR B.A., U n i v e r s i t y of B r i t i s h Columbia, 1 9 5 8 .  A t h e s i s submitted i n p a r t i a l f u l f i l l m e n t of the requirements f o r the degree o f  MASTER OP SCIENCE i n the Department of '  PHYSIOLOGY  We accept t h i s t h e s i s as conforming to the r e q u i r e d standard.  THE UNIVERSITY OP BRITISH COLUMBIA OCTOBER 1961  In p r e s e n t i n g  t h i s thesis i n p a r t i a l f u l f i l m e n t of  the r e q u i r e m e n t s f o r an advanced degree a t t h e British  Columbia, I agree t h a t the  a v a i l a b l e f o r reference  and  study.  University  of  L i b r a r y s h a l l make i t f r e e l y I f u r t h e r agree t h a t  permission  f o r e x t e n s i v e copying o f t h i s t h e s i s f o r s c h o l a r l y purposes may g r a n t e d by  the  Head o f my  It i s understood t h a t f i n a n c i a l gain  Department o r by h i s  representatives.  c o p y i n g or p u b l i c a t i o n o f t h i s t h e s i s f o r  s h a l l not  be a l l o w e d w i t h o u t my  Department The U n i v e r s i t y o f B r i t i s h Vancouver 8, Canada. Date  be  Columbia,  OofoMjLj f .  /*76f  written  permission.  ii  ABSTRACT 42 The radioactive isotope K was used to measure the rate of potassium exchange in muscle from 129 strain mice. 42 The results followed an unique course i f plotted as K  up-  take versus ( e x t e r n a ! p o t a s s i u * concentration • * ! » ) * ,  and  42 corresponded to the result predicted for K  uptake mediated  by an' ion-exchange compartment in the muscle.  Variations in  external potassium concentration did not affect the uptake rate i f plotted this way, but sodium ion exerted some effect 42 on the rate.  Dystrophic mouse tissue accumulated K  more  rapidly than did normal tissue, and the effect of varying the external potassium concentration did not alter this rate. The effects of sodium variation were more pronounced than in normal, tissue. Inulin space of muscle was measured in vivo as well 42 as in vitro, to enable a correction for K  in the extracellu-  lar space to be made. The inulin space was found to decrease with increasing muscle size, and this was thought to be related to the development of the muscle. Dystrophic muscle exhibited more of a dependance of inulin space on muscle size than did normal muscle.  The suggestion was made that the dystrophic  muscle membrane might be abnormally permeable to inulin. Muscles were excised and assayed by flame photometry for sodium and potassium content. They were assayed when freshly excised, and also following incubation in a variant of Locke's solution.  The muscle cations were stable for the f i r s t  two hours of incubation, but after this time, intracellular  iii  sodium rose and potassium f e l l .  Fresh d y s t r o p h i c mouse  muscle had lower potassium and higher sodium content normal f r e s h muscle. t i o n resembled  than  The c a t i o n changes f o l l o w i n g incuba-  those found f o r normal muscle.  The changes  42 i n i n t r a c e l l u l a r c a t i o n s were c o r r e l a t e d with the K take r e s u l t s ,  and discussed i n some d e t a i l .  up-  iv  ACKNOWLEDGEMENT It help  i s a n h o n o u r and a p l e a s u r e  and guidance g i v e n  t o me o v e r  the past  D r . Hugh McLennan o f t h e Department unfailing interest t o me.  Department in  of constant  His  inspiration  t o t h a n k t h e o t h e r members o f t h e f o r t h e i r g e n e r o s i t y and a s s i s t a n c e  a l l matters p e r t a i n i n g to t h i s t h e s i s .  grateful of  of Physiology  three y e a r s hy  of Physiology.  has been a source  I would a l s o l i k e  to acknowledge the  I am e s p e c i a l l y  t o D r . D.H. Copp f o r p e r m i t t i n g me t h e u n l i m i t e d u s e  the Departmental f a c i l i t i e s , . a n d  t o Mr. K u r t Henze f o r t h e  e x c e l l e n t p r e p a r a t i o n o f t h e g r a p h s and t a b l e s . My s i n c e r e , t h a n k s go t o D r . E»..J. H a r r i s , Department of B i o p h y s i c s , U n i v e r s i t y C o l l e g e , London, f o r the use of much o f h i s m a t e r i a l p r i o r  to p u b l i c a t i o n .  Thanks a r e a l s o e x t e n d e d t o D r . J . R. M i l l e r , ment  o f N e u r o l o g i c a l R e s e a r c h , a n d M i s s Ann C o o p e r , Department  of Pathology 129  Depart-  strain  f o r a constant  final  o f n o r m a l and d y s t r o p h i c  mice.  Finally, patience  source  I w i s h t o t h a n k my w i f e , M a r g a r e t , f o r h e r  and e n c o u r a g e m e n t , a n d f o r h e r h e l p  i n p r e p a r i n g the  d r a f t , a n d M i s s Tanya Dournovo f o r t y p i n g t h e f i n a l  copy.  i  TABLE OF CONTENTS  SECTION I :  INTRODUCTION  1  SECTION I I :  METHODS  25  SECTION I I I :  RESULTS  38  SECTION IV:  DISCUSSION  69  SECTION V:  CONCLUSIONS AND SUMMARY  85  SECTION VI:  BIBLIOGRAPHY  88  SECTION I  INTRODUCTION  A.  Purposes o f the study  B.  General aspects of b i o e l e c t r i c i t y  G  #  D.  1.  The composition of tissues  2.  Ionic e q u i l i b r i a  3.  Maintenance of i o n i c gradients  Tissue compartments E l e c t r o l y t e s and tissues 1,  Nerve tissue  2. Muscle tissue E.  Interpretation of r e s u l t s : muscle models  F.  Muscular dystrophy: human and murine 1.  Electrophysiological changes i n dystrophic muscle  2,  Histology  3«  E l e c t r o l y t e studies  of dystrophy  1  A.  PURPOSES OF THE STUDY The investigations reported i n this thesis centre around two objectives: to investigate and measure the rates of movement of potassium i n t o mammalian s k e l e t a l muscle under varying environmental conditions, with the a i d of the radioactive isotope K^ , and 2  to compare the rates of movement of the ion i n normal and dystrophic mouse s k e l e t a l muscle. The r e s u l t s were to be expressed as m. equiv. i n t r a c e l l u l a r K moving per (K • t ) 2 u n i t .  Thus, several problems required solvingj  the non-inulin space volume had to be determined, involving an e s t i mation of the e x t r a c e l l u l a r i n u l i n space and a determination  of the  dry weight of the musclej the normal concentration of K i n f r e s h muscle tissue also had to be known.  As the experiments would involve incuba-  t i o n i n a modified Locke's solution f o r several hours, a knowledge of the i n t r a c e l l u l a r ionic concentration changes due to incubation was 1  necessary.  Net alterations i n K l e v e l during incubation could then be  appropriately corrected. The concentrations of sodium and potassium i n some of the incubation f l u i d s were altered, t o see i f differences i n e x t r a c e l l u l a r concentrations of the ions would a f f e c t the rate of K entry into the muscle f i b r e . Concomitant observations of muscle resting potentials under s i m i l a r experimental  conditions have been investigated separately by  Professor H. McLennan.  2 B.  GENERAL ASPECTS OF BIOELECTRICITT 1.  The Composition of Tissues I t i s a c h a r a c t e r i s t i c of the excitable c e l l to exhibit  a r e s t i n g p o t e n t i a l difference between i t s environment and the c e l l interior.  I t i s also c h a r a c t e r i s t i c f o r protoplasm and tissue f l u i d  to contain d i f f e r i n g concentrations of c e r t a i n i o n i c species, the most obvious examples being the a l k a l i metal ions sodium and potassium (Na and K), which are present i n high concentrationsj this i s i l l u s t r a t e d i n Table I .  Other substances are also d i s t r i b u t e d un-  equally between the c e l l i n t e r i o r and environment; Table I I , from Conway (1950), shows values f o r some substances found i n f r o g and r a t muscle. To maintain the i o n i c balance of excised tissue, a r t i f i c i a l f l u i d s were devised to enable the tissue, when incubated i n the f l u i d , to survive and function normally . ,l  ,,  Sydney Ringer (e.g. (1883)) i n -  t e n s i v e l y studied the e f f e c t of ions on the frog heart. that small quantities of KC1 and CaClg added to a 0.1%  He observed  NaCl bathing  s o l u t i o n would enable the heart to beat for hoursj without these two s a l t s , i t stopped in a few minutes.  Locke (1895) demonstrated  glucose had the same e f f e c t as addition of the s a l t s .  that  He also altered  the t o n i c i t y , and used the f l u i d f o r perfusing mammalian heart, oxygenat i n g and warming i t to body temperature (Locke 1904).  Many "physiolo-  g i c a l * solutions have been devised since then f o r s p e c i f i c t i s s u e s j 1  Ringer-Tyrode f o r gut tissue, Ringer-Krebs-Hensleit f o r brain tissue, and Ringer-Conway f o r f r o g muscle are a few common ones.  These f l u i d s  d i f f e r p r i n c i p a l l y i n the concentrations of t h e i r constituents, or have special substances added to them; they are a l l supposedly i n osmotic equilibrium with the tissues f o r which they are designed.  TABLE I:  K, Na, and RESTING POTENTIALS OP EXCITABLE TISSUES*. Ion values expressed as m.equiv. per l i t r e of c e l l or plasma water. R.P. values i n -mV. Calculated values from Nernst Equation.  TISSUE  K  Na  R.P.  CELL  ENVIRON  CELL  ENVIRON  CALC.  ACTUAL  FROG  - Skel. muscle  124.0  2.2  3.6  104.0  98  92  RAT  - Skel. muscle  152.0  4.7  3.0  150.0  87  74  CRAB  - Muscle  146.0  12.9  54.0  513.0  61  72  GRAB  - Nerve  112.0  12.1  54.0  468.0  60  82  SQUID - Giant axon  369.0  13.0  44.0  498.0  83  65  Values from Shanes (1958), Ling and Gerard (1949), Hodgkin and Krynes (1955), Conway (1950), and Z i e r l e r (1959).  TABLE I I ;  DISTRIBUTION OF SUBSTANCES IN FROG AND RAT MUSCLE. Values expressed as m.equiv. per l i t r e f i b r e or plasma water.  SUBSTANCE  H" 4  expressed as pH.  FROG SKELETAL MUSCLE FIBRE  PLASMA  RAT SKELETAL MUSCLE FIBRE  PLASMA  3.6  104.0  3.0  150.0  124.0  2.2  152.0  6.4  4.9  2.0  1.9  3.1  14.0  1.2  16.1  1.5  1.5  74.3  5.0  119.0  12.4  25.4  16.0  24.3  PHOSPHATE  7.3  3.1  10.6  2.3  SULPHATE  0.4  1.9  -  AMINO ACIDS  8.8  6.9  14.7  3.2  Ca. 6.9  7.4  Ca. 6.2  7.4  SODIUM POTASSIUM CALCIUM MAGNESIUM CHLORIDE BICARBONATE  H+  -  5 2.  Ionic E q u i l i b r i a D i f f u s i o n i s the movement of p a r t i c l e s from a region of  higher concentration to one of a lower concentration, due to the random migrations of thermally agitated solute p a r t i c l e s .  In a  f i x e d structure such as NaCl c r y s t a l , t h i s movement does not occurj i n the c r y s t a l l a t t i c e , each sodium ion i s surrounded by s i x c h l o r ides, f i x e d by e l e c t r o s t a t i c forces i n a face-centered cube form. In s o l u t i o n , the ions dissociate from tiie c r y s t a l i n equal numbers, and at any point i n a homogenous s o l u t i o n , a s i t u a t i o n s i m i l a r to the l a t t i c e arrangement w i l l tend to arise, the hydrated sodium ions surrounded by a cloud of hydrated chloride ions, and vice versa*  In  t h i s s o l u t i o n , there w i l l be no p o t e n t i a l difference between any  two  points ( P r i n c i p l e of Microscopic E l e c t r o n e u t r a l i t y ) , f o r the random movements of the ions cancel any transient imbalance.  However, i f  these ion species are d i f f u s i n g , the rate of d i f f u s i o n w i l l be prop o r t i o n a l to the square root of t h e i r hydrated r a d i i j a chloride ion, with a smaller radius than a sodium ion, w i l l diffuse more r a p i d l y ; but as i t moves, the r a t e w i l l tend to be retarded by the a t t r a c t i o n of the p o s i t i v e sodium ions l e f t behind, and a small but measurable difference i n d i s t r i b u t i o n of e l e c t r i c a l charges between various parts of the s o l u t i o n w i l l a r i s e .  A region of high s a l t concentration w i l l  be more p o s i t i v e than a region of lower concentration, and a d i f f u s i o n p o t e n t i a l may  be measured; t h i s p o t e n t i a l w i l l run down as d i f f u s i o n  proceeds, and w i l l disappear when d i f f u s i o n a l equilibrium i s attained. To maintain a p o t e n t i a l between two compartments, e i t h e r there must be no net movement of the ions causing the p o t e n t i a l , or there must be an i n d i f f u s i b l e (impenetrable)  ion i n one of them.  Donnan equilibrium rule states that i n a system ifaere  The Gibbs-  an impermeable  6  anion i s on one side of a f i x e d membrane, and a solution permeable to the membrane (e.g. NaCl) bathes both sides of i t , the ions w i l l become d i s t r i b u t e d unequally i n the two compartments, with more cation and l e s s d i f f u s i b l e anion i n the i n d i f f u s i b l e anion compartment than i n the otherj two opposing tendencies w i l l arise i n this system: the movement of ions down chemical gradients and tiie maintenance of e l e c t r i c n e u t r a l i t y i n the s o l u t i o n s . The i n d i f f u s i b l e anion compartment w i l l be s l i g h t l y negative with r e s p e c t to the other, thus cations w i l l t r y to move i n to i t j t h i s , however, i s against the concentration gradient, and does not occur5 also the movement would be retarded by the excess anion i n that compartment.  The d i s t r i b u t i o n i s complicated  i n l i v i n g c e l l s by the presence of more than one cation.  The differences  i n ion concentrations of l i v i n g c e l l s and t h e i r surroundings  (Tables I  and II) are p a r t i a l l y due to a Donnan systemj there are unequal numbers of ions i n the solutions, and the tendency to reach e l e c t r i c n e u t r a l i t y i s opposed by the concentration p o t e n t i a l across the membrane.  I f an  electrode i s placed on each side of the membrane i n a Donnan system, a p o t e n t i a l of magnitude  E =  — In l°lbZ F  (c )  (Eq. 1)  +  2  can be measured; R i s the gas constant, C the cation concentration, z i t s valency and F i s Faraday's constant. The muscle membrane was formerly thought t o be impermeable to Na and Gl; this concept would s a t i s f a c t o r i l y explain the r e s t i n g potent i a l as a r i s i n g from passive d i s t r i b u t i o n of K by the Donnan system, and account f o r the dependence of the magnitude of the p o t e n t i a l on the external K concentration ( K ) ; the p o t e n t i a l of Eq. 1 adapted f o r potase  7 slum i s E =  RT  In  F  (K )i +  (Eq. 2)  The r e s t i n g p o t e n t i a l i s l i n e a r l y r e l a t e d to the l o g K values of K , Q  e  f o r most  but f o r muscle the slope of p l o t t e d experimental  observations i s not the same as the slope of the Nernst equation values.  Boyle & Conway (1941) showed that the membrane i s permeable  to chloride ions; Steinbach (1940) showed chemically and Heppel (1940) with radioisotopes that frog muscle was permeable to Na.  In accord-  ance with these observations, McLennan (1961), working with mouse muscle, found that whereas K r e s t i n g p o t e n t i a l , i t was  e  had the greatest influence on the  also affected by the Na ion r a t i o ; h i s  data were best f i t t e d by the equation  E -  Min  K  i  +  0.005 Nai 0.005 Na  3.  (Eq. 3)  Q  Maintenance of Ionic Gradients The d i s t r i b u t i o n of muscle K (or Na) during steady state  conditions can be explained electrochemically; the r esting p o t e n t i a l of 60-80 mV w i l l influence the K ions by attracting them; the ions w i l l flow "passively" from a region of high electrochemical p o t e n t i a l to a lower one, even though the chemical gradient be opposing the movement; the K ions may move against a 2 5 t l r a t i o , and s t i l l be moving "passively".  This i s d i s t i n c t l y d i f f e r e n t from d i f f u s i o n , f o r the  l a t t e r occurs down a concentration gradient, not " u p h i l l " against a gradient.  The term "passive transport" has been coined to describe  t h i s phenomenon of ion movement by e l e c t r i c forces against a d i f f u s i o n gradient.  This passive transport does not require metabolic energy  8 and should not be stopped by metabolic  inhibitors.  The Nernst electrochemical p o t e n t i a l equation consists of two terms, an osmotic, and an e l e c t r i c , which are opposed; f o r i n transferring an ion against the concentration gradient, osmotic work i s done on the system, while e l e c t r i c forces concomitantly push the ion back down the gradient.  t r y to  I f the two terms are equal,  work i s zero, and no transfer occurs; the osmotic term RT n K i / K  e  i s equal to the p o t e n t i a l term zFE. Sodium ions do not follow t h i s Nernst r e l a t i o n s h i p ; then, does one  how,  account f o r the Na ion d i s t r i b u t i o n ? A mechanism,  termed "active transport" was postulated f o r maintaining the  distri-  bution (Dean 1941); a simple d e f i n i t i o n would be "transport against an electrochemical gradient, using energy derived from c e l l u l a r metabolism"; consequently,  -the sodium ion was  extruded as f a s t as i t  penetrated the c e l l , keeping the i n t r a c e l l u l a r Na concentration low, and providing a f u n c t i o n a l impermeability to the i o n . to passive transport, energy was  In d i s t i n c t i o n  required f o r this process; a wealth  of studies (e.g. references page 60 of Ussing (I960)) showed a dependance of the continued extrusion on a continuous metabolic supply of energy; i f Na extrusion could thus be maintained, K ions would tend to e q u i l i b r a t e with the electrochemical gradient of the tissue, i . e . be d i s t r i b u t e d by a Donnan system. This theory i s able to account f o r most of the phenomena observed i n excitable t i s s u e s . However, i t has been suggested (e.g. Ling (1953)) that active transport i s an unnecessary hypothesis,  and  that a series of f i x e d s i t e s that bind K and Na (the binding r e q u i r i n g continuous metabolic energy) would e x h i b i t the properties experiment a l l y observed.  9  C.  TISSUE COMPARTMENTS In Part B, i t was seen that i n t r a c e l l u l a r ion concentrations d i f f e r from e x t r a c e l l u l a r concentrations.  Tissue f l u i d ion  concentrations i n turn d i f f e r from those i n plasma; thus, i t would be misleading to draw o f f a sample of blood, analyse the plasma, and represent t h i s plasma value as being equivalent to the tissue f l u i d concentration of the ion, f o r the plasma sample i s not a true representation of the i n t e r s t i t i a l f l u i d , i . e . , the f l u i d bathing the c e l l s . The tissue f l u i d i s e s s e n t i a l l y an u l t r a f i l t r a t e i t contains some p r o t e i n .  of plasma, although  A Gibbs-Donnan equilibrium i s established  between the plasma and tissue f l u i d s ; since the plasma protein concentration i s greater than the tissue f l u i d protein concentration, there w i l l be s l i g h t l y less Na and K, and s l i g h t l y more C l i n the i n t e r s t i t i a l f l u i d than i n the plasma; the r a t i o tissue f l u i d K/plasma K i s t h e o r e t i c a l l y 0,90-0.95; some experimental values reported were as low as 0,75 (Manery 1954); i f these l a t t e r plasma samples were used t o determine e x t r a c e l l u l a r K concentrations, they could have been 25% i n error. Caution must also be used when estimating e x t r a c e l l u l a r Ca or Mg ion; these ions may be p a r t i a l l y bound to plasma proteins, f o r t h e i r d i s t r i b u t i o n r a t i o s are much lower than the K or Na values. Tissue f l u i d i s scant i n muscular t i s s u e ; i t i s d i f f i c u l t to sample, f o r there are no normal accumulations o f i t ; i t was estimated that i n a muscle b e l l y with an e x t r a c e l l u l a r space (ECS) of 10$, the tissue f l u i d would be only a 15 micron f i l m covering the surface of the cells.  Maurer (1938) punctured frog muscles and drew o f f small quanti-  t i e s of f l u i d that he regarded as normal i n t e r s t i t i a l f l u i d ; h i s C l analyses corresponded c l o s e l y to the calculated values reported by  10  Gamble  (1950).  Edema f l u i d has been analysed (Manery  1954);  the  i o n i c concentrations depended upon the amount of protein i n the f l u i d , and corresponded to tissue f l u i d i n ion content only i f the quantity of protein i n the two f l u i d s was the same. Accurate i n t r a c e l l u l a r concentrations could not be calculated unless the ECS was known; Na and C l were considered to be exclusively e x t r a c e l l u l a r ions, and since they appeared not to enter the tissue c e l l s , they were often used as Indicators f o r ECS estimations.  The  basis f o r t h i s was that i f Na and C l were found only i n the e x t r a c e l l u l a r f l u i d , then the amount of C l (or Na) i n tissue and plasma samples could be used to calculate the ECS of the t i s s u e .  However, muscle c e l l s  have been shown to contain both these ions and the p o s s i b i l i t y of t h e i r c e l l u l a r uptake was suggested t o account f o r the higher values calcul a t e d f o r these spaces.  C l and Na spaces are now used l e s s often f o r  estimating ECS than i n the past,  Thiocyanate (SON), a negative i o n  s i m i l a r i n mobility to chloride (but not normally found i n the body), has also been used to determine the ECS of tissues; values f o r t h i s space were 4% too large according to Pappius and E l l i o t t  (1956),  for  t h i s amount was taken up by the tissue c e l l s . And " i d e a l " indicator f o r ECS determinations (either t i s s u e or whole-body ECS) would be a substance that was not harmful to the animal or i t s tissues, was d i s t r i b u t e d evenly i n the e x t r a c e l l u l a r f l u i d , d i d not penetrate i n t o or adsorb onto the c e l l , assayed.  and was  readily  The disaccharides maltose and sucrose (MW 360 and 342)  and  the polysaccharide i n u l i n (Ml 991) s a t i s f i e d these c r i t e r i a ; with the 1  disaccharides, there was the p o s s i b i l i t y of metabolic u t i l i z a t i o n , whereas i n u l i n was not metabolized, but excreted from the body unaltered. Radioiodinated serum albumin (RISA) has also been used for ECS studies  11  (e.g. Tasker, Simon, Johnstone, Shankly and Shaw (1959)); r e s u l t s using t h i s tracer corresponded c l o s e l y to i n u l i n space estimations. The values f o r other space estimations varied according t o the i n d i cator used; f o r amphibian s a r t o r i i , the spaces measured with Na, C l , CNS, sucrose, i n u l i n , and R1SA decreased i n that order; i t was suggested that the higher values obtained may have been due to the tracer entering the c e l l s (Tasker, e t a l , l 9 5 9 ) . The volume of muscle ECS must be known to calculate the muscle f i b r e or non-ECS volume, and i d e a l l y i t should be determined for each muscle used.  This i s often impractical, and many investiga-  tors have excised paired muscles such as sartorius, analysed one muscle f o r ions, and assayed the other f o r ECS. There i s usually a v a r i a t i o n between the ECS values of the p a i r , but the value f o r the companion muscle i s a better estimation of the ECS than are group averages.  Tasker et a l (1959), showed a s i g n i f i c a n t v a r i a t i o n of  the average ECS i n s a r t o r i i from d i f f e r e n t batches o f toads;  they  also found a seasonal v a r i a t i o n i n ECS and tissue i o n content.  The  authors concluded that applying group ECS values to i n d i v i d u a l muscles was not a v a l i d procedure because of the large v a r i a t i o n s among and within groups of muscles. Each muscle seems to have a c h a r a c t e r i s t i c ECS value, which varies with s i t e , age, weight, and condition.  McLennan (1956) reported  r a t diaphragm ECS to be 25%, gastrocnemius 12%, and extensor digitorum longus to be 15%. Tasker et a l (1959) found a negative c o r r e l a t i o n between ECS and muscle weight i n excised toad s a r t o r i i ; t h i s f i n d i n g was confirmed by Burr and McLennan (I960) f o r mouse muscle.  The l a t t e r  authors assayed dystrophic tissue as w e l l , and found the ECS to be greater than i n normal muscle.  12  D.  ELECTROLYTES AND 1.  TISSUES  Nerve Tissue Individual mammalian nerve f i b r e s are too small f o r con-  venient study; consequently, many investigators of cation movements i n nerve have used giant nerve f i b r e s - the squid and giant axons that are 0.2-1.0 mm.  diameter.  h i b i t e d a resting p o t e n t i a l of 40-80 mv  cuttlefish  These nerve f i b r e s ex-  (inside negative),  apparently  maintained by an active transport of Na and Kj when studied i n v i t r o the axons were l i a b l e to lose K and gain Na.  Hodgkin (1957), Shanes  (1955) (1958), and Keynes (1951) wrote review a r t i c l e s on the b i o e l e c t r i c and i o n i c phenomena observed i n nervous t i s s u e .  Ion movements  associated with nerve a c t i v i t y w i l l not be considered i n t h i s t h e s i s . I f nerve i s exposed to suitable low-K solutions, i t loses K and gains Na;  i n high-K media, the reverse occurs.  The d i s t r i b u t i o n  of K between the f i b r e and i t s environment seemed to be a Donnan d i s t r i b u t i o n , and i t was proposed that Na was ted passively. that there was axons.  pumped out and K e q u i l i b r a -  On the other hand, Hodgkin and Keynes (1955a) showed an apparent active transport of K inwards i n giant  The i n h i b i t o r s of Na active transport also reduced the i n f l u x  of K from sea-water.  This drop i n K i n f l u x equalled the change i n Na  e f f l u x , suggesting a coupled exchange of the two ions; however, some Na extrusion p e r s i s t e d i n K-free solution, and the active Na f l u x exceeded the active K f l u x .  In these cases, the excess Na must be  accompanied by a negative ion, or a cation must enter the c e l l to balance the e l e c t r i c a l l o s s . The r e s t i n g p o t e n t i a l d i d not change when K and Na decreased a f t e r poisoning  fluxes  the nerve (1955a); apparently the active  transport per se does not a f f e c t the p o t e n t i a l .  The p e r s i s t i n g fluxes  13  may have been due to passive d i f f u s i o n o f the ions; this passive movement d i d not vary as expected when the r e s t i n g p o t e n t i a l was a r t i f i c i a l l y altered; the f l u x change was much greater than predicted (Hodgkin & Keynes 1955), and always occurred down the K electrochemical gradient.  Hodgkin and Keynes (1955) proposed a  "long-pore" membrane model that adequately accounted f o r the observed phenomena.  Excess K ions moving out may have diminished the K i n f l u x  by competing f o r s i t e s i n the membrane.  2.  Muscle Tissue Approximately h a l f the weight of the human body i s s k e l e t a l  muscle; the i n d i v i d u a l muscles are composed of groups of c e l l s 1 to 40 mm long and 10 to 40 micra i n diameter;  simultaneous contraction  of the f i b r e s produces a v i o l e n t shortening of the muscle. Each f i b r e i s a complete c e l l , multinucleate, with a membrane enclosing i t .  In the space between the f i b r e s i s tissue f l u i d , the  m i l i e u i n t e r i e r of the body.  A p o t e n t i a l difference of 60-80 mv  (inside negative) exists across the membrane of these c e l l s , due mainly to d i f f e r i n g concentrations of K inside the c e l l and i n the tissue f l u i d ; most animal c e l l s , including muscle f i b r e s , are high i n K and low i n Na (see Table I ) . Recent publications by Harris (i960), Conway (1957), Ussing (i960), and Harris and Sjodin (1961) discuss K i n muscle t i s s u e . The permeability of muscle to K was f i r s t demonstrated i n 1916 by Meigs and Atwood, who found that i n high KC1 solutions, muscles would swell and take up KC1.  In 1939, Heppel showed that muscle c e l l s  were also permeable to Na, which could replace some of the K i n r a t muscle; he confirmed t h i s a year l a t e r using radioactive Na (Heppel 1940).  14 Excised frog muscles soaked i n ordinary Ringer s o l u t i o n l o s t K and gained Na; t h i s was prevented  i f the K concentration i n  the bathing f l u i d was higher than normal.  Soaking s a r t o r i i i n K-free  f l u i d r e v e r s i b l y depleted the i n t r a c e l l u l a r K (the K^). accompanied by a r e v e r s i b l e increase i n Na^« strated (Creese 1952)  was  I t was further demon-  that the addition of bicarbonate to the normal  medium decreased K l o s s . and gained KC1.  This  In high-KCl solutions, the f i b r e s swelled  These solutions r e s u l t e d i n the replacement of some  of the Naj_ by K; however, the resting p o t e n t i a l f e l l due to the lowered Kj/K  e  ratio.  Chloride therefore entered the c e l l more r e a d i l y ,  and K plus an osmotic equivalent of water moved with i t , to maintain e l e c t r i c a l and osmotic n e u t r a l i t y ; consequently, I f non-penetrating  anions such as methyl-sulphate  the c e l l s swelled. were used, the  swelling was markedly reduced. Dean (1941) proposed the Na pump" theory to explain the rt  maintenance of Na and K gradients; Keynes (1954) suggested that K  was  the exchange partner f o r the pump (resting p o t e n t i a l must be kept constant), f o r a reduction of Na e f f l u x occurred i n K-free bathing f l u i d s , and an increase i n K-rich f l u i d s . an optimum K  e  Steinbach (1952) showed  for Na extrusion i n Na-loaded muscle; he suggested that  K d i f f u s i o n i n the muscle was the l i m i t i n g f a c t o r i n the exchange of Na f o r K. The low K. of i n t e r s t i t i a l f l u i d could a f f e c t K exchange with muscle f i b r e s , because a small r i s e i n K„ could l e a d to an * e appreciable back d i f f u s i o n into the f i b r e ;  t h i s f a c t o r could lead to  incorrect i n t e r p r e t a t i o n of tracer f l u x c a l c u l a t i o n s .  Harris and  Burn (1949) found the fibre-interspace exchange to be about equal i n rate to the interspace-soaking s o l u t i o n , i f the muscle f i b r e s impede  15  the movements of the ions. McLennan (1956) (1956a) (1957), working with mammalian muscle discussed the problem at some length, and concluded that in high phosphate solutions the interspace K diffusion rate was one-twentieth of the free solution diffusion values. With similar assumptions regarding the interspace K diffusion rate, Harris and Burn calculated the K ion flux across frog muscle membrane to be 1.1 x 10"^ m. equiv./cm /min, close to McLennan*s value of 1.4 for 2  rat muscle.  The latter found that K^  efflux from equilibrated  2  muscles could be resolved into two exponentials xcLth different time constants; there appeared to be an inexchangeable fraction of muscle K (more at low temperature), but an increased K to exchange.  would enable a l l K  e  The presence of phosphate in the bathing solution appear-  ed to prevent K loss, but impeded K diffusion;  McLennan suggested the  phosphate might have formed a complex that could bind K, and the slow dissociation of the complex resulted in an abnormally low apparent diffusion rate for K.  Creese (i960) showed that deeper diaphragm  fibres exchanged less K than more superficial ones; the turnover of total muscle K, at 38°C was apparently complete, but individual cells exchanged faster than the total muscle (Creese, Weil and Stephenson 1956). Frog muscle studies using K^  -2  tracer showed a rapid phase of  movement of an amount of K, too large to be simply that of the extracellular space, and less than the total fibre K. this fast portion seemed to vary with the K  Q  The amount of K i n  of the bathing solution  (Carey and Conway (1954)). The authors concluded that seme of the K and most of the fibre Na was in a "special region", one that had unique ion exchange properties.  Na that replaced K i n tissue appeared to  differ from "normal" Na:  the efflux into non-electrolyte solutions was  slower, High K  e  solutions increased the efflux more than usual, and the  16  temperature dependance was  greater (Conway & Carey 1956).  Possibly  t h i s Na had replaced K i n t h i s " s p e c i a l region"; i f only a l i m i t e d number of s i t e s to bind cations were available, competition f o r them might have retarded K or Na movement i n t o or out of the c e l l .  E.  INTERPRETATION OF RESULTS:  MUSCLE MODELS  The " c l a s s i c a l " concept of a muscle c e l l involved a protoplasmic mass enclosed by a thin, r e s i s t i v e membrane, one with no capac i t y to store ions, and through which ions passed when entering or leaving the c e l l ; the r e s i s t a n c e supposedly v a r i e d f o r each i o n i c species, as some ion species could enter more e a s i l y than others; the membrane was  considered as the r a t e - l i m i t i n g step f o r ion movement  i n t o or out of the c e l l .  I t was  assumed that e x t r a c e l l u l a r d i f f u s i o n  would maintain the tissue f l u i d i o n i c composition constant, and that i n t r a c e l l u l a r d i f f u s i o n was more r a p i d than the rate of movement through the membrane into the c e l l ; consequently,  the i n t r a c e l l u l a r ions became  homogenously d i s t r i b u t e d , and new ions entering the c e l l would r e a d i l y diffuse and e q u i l i b r a t e within i t .  The c h a r a c t e r i s t i c s of ion uptake  therefore depended upon the r a t e of i n t e r n a l mixing and the resistance of the membrane, assuming that the membrane contained no ions; thus the uptake should have followed f i r s t - o r d e r k i n e t i c r e l a t i o n s , i . e . , have a simple exponential time course.  But i t was necessary to use two or  three exponential terms to interpret experimental observations of ion movements instead of a single one; various "fractions"' of muscle K were assigned to each exponential, but the t o t a l K i n the f r a c t i o n s did  not always equal the t o t a l muscle K.  I f muscles were incubated i n  radioactive K solution, and i f f u l l exchange had not been reached,  there  appeared to be a portion of the muscle K that d i d not undergo exchange;  17 however, the amount of exchangeable K increased i f the K bathing solution was above normal.  e  of the  Radioactive K also f a i l e d to  become uniformly mixed with frog muscle K a f t e r l i m i t e d exposure to the tracer (Harris & Steinbach 1956)j the s p e c i f i c a c t i v i t i e s of successive extracts of these muscles diminished r a p i d l y ; the inhomogeneity of the K seemed to be i n t r a c e l l u l a r , and not due to a greater degree of exchange of s u p e r f i c i a l than deep f i b r e s .  The membrane  theory i n f e r r e d that the tracer e f f l u x from loaded muscles would be independent of the time of loading, but this r e s u l t has not been confirmed;  muscles exposed f o r a short time appeared to have more  K that would exchange r a p i d l y than others incubated f o r a longer time (Harris 1953).  Apparently this t h i n r e s i s t i v e membrane model  d i d not adequately i n t e r p r e t the k i n e t i c s of i o n movements;  Ling,  Shaw et a l , and Harris have each presented hypotheses i n an attempt to explain t h e i r observations more completely. Ling (1952) (1955) (i960) proposed a "fixed-charge" hypothesis to explain s e l e c t i v e K accumulation by muscle c e l l s .  The  hypothesis maintained that free moving ions (such as K and Na) tended to approach f i x e d anions (e.g. protein chains) as c l o s e l y as t h e i r charge would allow; since hydrated K i s smaller than hydrated Na, i t moved closer to the f i x e d anion;  this represented a lower energy  state f o r K than f o r Na, and the K would be held i n a more stable manner. Since a system w i l l tend to a t t a i n i t s lowest energy state, the model proposed would take up K rather than Na from a solution containing both ions.  Ling further stated that i h i s K was not chemically "bound", but  could detach from the anion by acquiring enough thermal energy, or by replacement by another ion of the same species.  Ions with a greater  "absorption energy" would replace a "bound" cation (absorption energy varies as l/hydrated r a d i u s ) ; but Rb (with a smaller hydrated radius  18  than K) was found to compete f o r these f i x e d s i t e s , instead of replacing the already bound K.  The anionic s i t e s were maintained  by energy from c e l l u l a r metabolism;  poisoning the muscle l e d to  K l o s s and Na gain, but poisoning of an "active transport" system was  not necessary to explain t h i s s h i f t ; a lack of ion binding  gave the same r e s u l t . The theory could not adequately  explain cation s h i f t s  i n cooled muscle; neither d i d i t account f o r Na replacing K i n t r a c e l l u l a r l y , nor the sequence of r e l a t i v e cation permeability of the muscle. - Shaw and h i s collaborators exhaustively investigated the r e l a t i o n s of Na, K and Gl of toad s a r t o r i i (Shaw, Simon & Johnstone 1957)  (Simon, Uohnstone, Shankly & Shaw 1959)  1959)  (Simon, Shaw, Bennett & Muller 1957);  K d i d not exchange as expected when K  Q  (Frater, Simon & Shaw they found that muscle  was a l t e r e d , but that Na and  Cl movements were independent of K movements or concentrations.  The  muscles d i d not swell i n hypertonic KC1, but d i d i n high NaCl s o l u tions; also, there was no replacement of Na by K i n low Na s o l u t i o n s . To account f o r these findings, the authors postulated a "three-phase theory", c o n s i s t i n g of e x t r a c e l l u l a r , free i n t r a c e l l u l a r , and phases.  ordered  I t was assumed that ions accumulated by the c e l l were adsorb-  ed to the ordered phase, and that other species were excluded from i t . The free i n t r a c e l l u l a r phase was i n d i f f u s i o n a l equilibrium with the e x t r a c e l l u l a r phase; the apparent c e l l u l a r concentration gradient due to ion exclusion from the ordered phase.  was  No energy was thus nec-  essary to maintain the c e l l i n t h i s manner. Harris (1953) (1957) postulated a permeation-diffusion model to account f o r tissue K exchange; this d i f f e r e d i n several respects from  19  the models presented above. stage process:  Ion accumulation was regarded as a two-  exchange between ions i n the tissue f l u i d with others  i n an outer region of the c e l l , and subsequent d i f f u s i o n from t h i s outer region i n t o the c e l l i n t e r i o r .  This second step would be b a l -  anced by d i f f u s i o n from the i n t e r n a l region to the outer one; consequently, there would be no net change i n the K concentration of the cell.  The model was thus a three compartment one, with the compart-  ment between i n t r a - and e x t r a - c e l l u l a r f l u i d s having i o n exchange properties;  K movement i n t h i s compartment was slow, due to binding  by f i x e d anionic charges.  Harris and Sjodin (1961) have shown that  there are two types of K exchange possible i n this model:  (1) when  the outer-layer ions exchange with those i n the s o l u t i o n more r a p i d l y than they exchange with i n t e r n a l ions, and (2) when the i n t e r n a l d i f f u sion i s so r a p i d that the rate of passage through the outer region, rather than e q u i l i b r a t i o n within i t , determines the rate of exchange. Condition (2) was the " c l a s s i c a l " r e s i s t i v e membrane that gave r i s e t o f i r s t - o r d e r k i n e t i c s of K movement; condition (1) followed ordinary d i f f u s i o n laws. This model can s a t i s f a c t o r i l y account f o r the k i n e t i c phenomena observed i n K exchange experiments.  The model can be used to  distinguish between K—K exchange and net c e l l u l a r gain of K, whether the l a t t e r i s with an anion or i n exchange f o r Na;  the K—K exchange  seems to r e l y on thermal a g i t a t i o n and d i f f u s i o n , while the net movement may be associated with expenditure of metabolic energy.  The Harris  model f a c i l i t a t e s an explanation of the phenomena observed by Harris and Steiribach (1956) regarding K movement following incomplete exchange with tracer K.  The e f f e c t on K uptake of adding other cations to the  incubation media may be due to a competition f o r s i t e s i n this interme-  20 diate compartment; gain or loss of  w i l l also a f f e c t the rate  of K# uptake, e s p e c i a l l y i f added ions a l t e r the K^.  Metabolic  poisons would tend to weaken the binding of the cations i n the s p e c i a l region, and lead t o a loss of K and gain of Na by the cells.  Temperature would a f f e c t the uptake rate, by increasing  the thermal a g i t a t i o n of the p a r t i c l e s , thus speeding up the exchange process; increased temperature would give an increased rate of K# uptake. Since the permeation-diffusion model appears to follow ordinary d i f f u s i o n laws, Harris and Sjodin could show that K e q u i l i b r a t i o n i s independent of the i n t r a c e l l u l a r d i f f u s i v i t y (D) if r  2  /D i s less than unity ( r i s the radius of a cylinder, and  a constant dependant upon K ) ; 0  the r a t i o i s usually ca. 0.5 f o r muscle  c e l l s ; the authors further showed that p l o t t i n g exchange against • t (or K ° t ) w i l l approximate a l l curves to one. e  Any K—K exchange  w i l l f a l l along t h i s curve, but i f the muscle i s gaining or l o s i n g K the values w i l l be above or below the exchange curve.  I f the K  1  uptake i s p l o t t e d against ( K * t ) , much of the exchange w a l l follow 2  e  a linear relationship. . The experimental r e s u l t s f o r K exchange presented i n t h i s thesis w i l l be interpreted with the a i d of the Harris permeation-diffusion muscle model, f o r i t i s f e l t that this model can best a i d i n the explanation and i n t e r p r e t a t i o n of the k i n e t i c data recorded. F.  MUSCULAR DYSTROPHY:  HUMAN AND MURINE  U n t i l the nineteenth century, medical p r a c t i t i o n e r s generall y believed that a l l muscular disorders, weaknesses, and atrophies were of neurogenic o r i g i n , consequent upon interruption or damage of the nerve supply.  In 1848,  G. B. Duchenne, a French neurologist,  21  described various muscle disorders (Duchenne 1848)  and introduced  the term "myopathy'*, meaning a muscular weakness not due to a demonstratable pathological a l t e r a t i o n i n the nervous system.  The  modern term progressive muscular dystrophy i s applied to those myopathies characterised by an hereditary pattern of occurrence, symmetry of muscle wasting, and gradual involvement of the muscle groups. In 1951,  Ann Michelson of Bar Harbour, Maine, discovered  a myopathy i n her colony of s t r a i n 129 mice (reported i n Michelson, Russell & Harmon 1955)J i t was & Southard 1957) 1.  subsequently  shown (Stevens, Russell  to be s i m i l a r to the human muscular dystrophies.  E l e c t r o p h y s i o l o g i c a l Changes i n Dystrophic Mouse Muscle Dystrophic mice have been used to study e l e c t r i c a l a l t e r a -  tions i n muscle.  Conrad and Glaser (1959) reported micro-electrode  studies showing no change i n r e s t i n g potentials i n dystrophy,  but  reported an increased e x c i t a b i l i t y of the dystrophic muscle to e l e c t r i c a l stimulation, as w e l l as an increase i n Na conductance. This l a t t e r observation was  confirmed by McLennan ( I 9 6 I ) , who  suggested that the s i m i l a r i t y between the potentials from the types of muscle was because r e s t i n g potentials could only be from sound f i b r e s , and not from degenerate ones.  also two recorded  Sandow and Brust (1958)  reported dystrophic muscles to be weaker ( l / l O to 1/5  the strength),  the "active s t a t e " duration to be 1/3 shorter, and the relaxation peri o d three times longer than f o r the muscles of normal l i t t e r m a t e s . 2.  Histology of Dystrophy Adams et a l (1954) reviewed the histology of dystrophy;  great v a r i a t i o n i n f i b r e s i z e and the large proportion of f a t and f i b r e c e l l s were s t r i k i n g features of dystrophic muscle sections. Fibres were found randomly scattered i n t h e i r bundles, with swollen  22  and a t r o p h i e d f i b r e s mixed. i s e v i d e n t l y due  The hypertrophy observed  clinically  to f a t t y i n f i l t r a t i o n of the bundles; the f i b r e  n u c l e i are swollen and more numerous, and v a c u o l a t i o n and l a t i o n are t y p i c a l i n t r a f i b r e changes. Garvin 1957)  has  granu-  Tissue c u l t u r e (Geiger &  shown that the c e l l s develop hypertrophy  and  begin to degenerate a f t e r 3 to 4 weeks of growth. The 129  s t r a i n mice show s i m i l a r microscopic  changes.  Michel son et_ a l (1955;) could see no p a t h o l o g i c a l a l t e r a t i o n i n any nervous t i s s u e examined, but muscle f i b r e preparations  revealed  changes s i m i l a r to those found i n human muscle. 3i  Electrolyte Pew  Studies  s t u d i e s have been done on the e l e c t r o l y t e  a s s o c i a t e d with human muscular dystrophy,  levels  Danowski (1955) reported  s l i g h t l y e l e v a t e d calcium and phosphorous l e v e l s and  decreased  l e v e l s i n serum samples of c h i l d r e n with dystrophy;  other i n d i c e s  such as serum Na,  K, p r o t e i n , and NPN  Cl  were e s s e n t i a l l y normal.  Dowben and H o l l e y (1959) d e s c r i b e d erythrocyte e l e c t r o l y t e  levels  i n dystrophic patientss Sodium:  Normals Dystrophics  13©1 nu 15©5  Potassium:  Normals Dystrophics  87*2 91•O  equiv./Kg " " "  T h i s e l e v a t e d K l e v e l i s i n c o n t r a s t to the usual lowered muscle K l e v e l s a s s o c i a t e d with dystrophy;  i t may  indicate a reciprocal  r e l a t i o n s h i p between muscle and plasma potassium, with the  possibi-  l i t y of the normal r a t i o being a l t e r e d by any changes i n muscle membrane p e r m e a b i l i t y .  23  Williams (1957) used neutron a c t i v a t i o n a n a l y s i s to assay f o r muscle Na and K, and found that the Na l e v e l  was  e l e v a t e d (normal 1 1 3 m. equiv./Kg dry weight, dystrophic  191  m. e q u i v . ) , and that the K l e v e l was lowered (normal 376  m.  equiv., d y s t r o p h i c 181 m. e q u i v . ) , c o n f i r m i n g the r e s u l t s r e p o r ted  by Horvath, Berg, Cummings & Shy  (1955).  Studies on d y s t r o p h i c mice r e v e a l e d e l e c t r o l y t e v a r i a t i o n s s i m i l a r to those found i n human dystrophic  subjects.  Potassium concentrations i n thigh muscle of the animals were sub-normal:  normal K was;-100 m. equiv./Kg f r e s h muscle, whereas  d y s t r o p h i c K was 76 m. equiv./Kg f r e s h muscle.  Sodium determina-  t i o n s i n d i c a t e d an i n c r e a s e of ca. 43$ i n the sodium v a l u e s , from a normal average of 46 m. equiv./Kg f r e s h muscle  to 66  m.  equiv./Kg; the d e f i c i t i n muscle K of 24 m. equiv. i s almost balanced by the 20 m. equiv. i n c r e a s e i n muscle Na.  The authors  suggested that t h i s may be due to replacement of i n t r a c e l l u l a r space by e x t r a c e l l u l a r space, with the r e s u l t a n t i n c r e a s e i n Na and decrease i n K l e v e l s , or to the l e a k of K from the c e l l , with compensatory  g a i n of Na.  U n f o r t u n a t e l y the authors d i d not take  i n t o account the problem of e x t r a c e l l u l a r space; i t i s to  difficult  draw c o n c l u s i o n s about i n t r a c e l l u l a r e l e c t r o l y t e l e v e l s from  their data. Young, Young, and Bdelman (1959) analysed s k e l e t a l and c a r d i a c muscle from d y s t r o p h i c mice f o r Na, K and l i p i d content. They r e p o r t e d a 19% decrease i n d y s t r o p h i c s k e l e t a l muscle Na, no d i f f e r e n c e i n c a r d i a c muscle K between the normal and  and  dystrophic  24 animals;  cardiac Na v a l u e s were not r e p o r t e d .  allowance f o r e x t r a c e l l u l a r space was  Again, no  mentioned.  SECTION I I  METHODS  A.  Experimental Animals  B.  Incubation s o l u t i o n s 1.  2e  E x t r a c e l l u l a r space a.  Inulin solutions  b«  Thiocyanate s o l u t i o n s  R a d i o a c t i v e Isotope  C«  D i s s e c t i o n technique  D.  Chemical methods  E.  experiments  experiments  1.  E x t r a c e l l u l a r space e s t i m a t i o n s  2»  Dry weight determinations  3»  Non-radioactive i n c u b a t i o n experiments  R a d i o a c t i v e Isotope techniques  25  EXPERIMENTAL ANIMALS The animals used i n t h i s i n v e s t i g a t i o n were pure i n b r e d mice of the Bar Harbour 129 s t r a i n , some of which develop an i n h e r i t a b l e muscle weakness s i m i l a r to human p r o g r e s s i v e muscular dystrophy.  They were obtained from the Roscoe B. Jackson Labora-  tory, Bar Harbour, Maine, and from the colony maintained by Dr. James R. M i l l e r of the Department U n i v e r s i t y of B r i t i s h Columbia.  of N e u r o l o g i c a l Research,  Purebred normal mice, with no  pedigree h i s t o r y of dystrophy, and l i t t e r m a t e s of a f f l i c t e d animals (those which d i d not develop muscular weakness) p r o v i d e d c o n t r o l animals. The dystrophic animals were 2-J to 8 weeks o l d , of v a r y i n g body weights, and i n v a r y i n g stages of d i s a b i l i t y due to the dystrophy.  The c o n t r o l s were used only i f i n p e r f e c t  condition,  and ranged from about 2 weeks to several months of age.  The  weight increase with age i s p r e d i c t a b l e i n the normal animals; the d y s t r o p h i c s , however, do not grow as r e g u l a r l y nor as q u i c k l y ; they l a g behind the normal animals by about 35-50% i n t h e i r development;  a three-week o l d normal mouse may  weigh 15-20  while a d y s t r o p h i c l i t t e r m a t e may be only 8-9 gm.  gm.,  i n weight.  At  maturity the normal animals weigh 25-35 gnu, depending on sex, while the d y s t r o p h i c s are only 15-18 gm.  i n weight.  The animals were kept i n the departmental animal room f o r s e v e r a l days before b e i n g used.  During t h i s time the d y s t r o -  p h i c s were kept separately from t h e i r normal l i t t e r m a t e s , and were maintained on powdered commercial chow and water, both ad l i b i t u m .  26 The  chow and water were p l a c e d i n s p e c i a l low feeding  for  ease of reach by the d y s t r o p h i c s .  t r e a t e d s i m i l a r l y , with the exception were provided,  containers  The normal animals were that no s p e c i a l containers  and the food was i n p e l l e t form.  INCUBATION SOLUTIONS Locked  s o l u t i o n , as mentioned e a r l i e r , i s an a r t i f i c i a l  extracellular f l u i d .  I n the majority  of the experiments performed,  t h i s s o l u t i o n , or v a r i a n t s of i t , was used f o r the i n v i t r o t i o n of s k e l e t a l muscles.  incuba-  For,convenience, stock i s o t o n i c s o l u t i o n s  (0.154 M. f o r s a l t s d i s s o c i a t i n g i n t o two i o n s , 0.11 M. f o r those h i e l d i n g three) were prepared.  Table I I I l i s t s  the stock  solutions  used. From these c o n s t i t u e n t s the experimental s o l u t i o n s of d e s i r e d i o n i c composition could r e a d i l y be prepared, and were f r e s h l y made up before  each experiment.  The f i n a l i o n i c concen-  t r a t i o n s i n the v a r i o u s s o l u t i o n s used are shown i n Table IV.  TABLE I I I ISOTONIC STOCK COMPONENT SOLUTIONS NaCl  90.0  gm/litre*  Choline C l  21.49 g m / l i t r e  KC1  11»47  gm/litre  15.41 g m / l i t r e  NaHCOj, j  12.49  gm/litre  KHCO, j KH P0  21.25  gm/litre  CaCl  15.62  gm/litre  MgCl  Na HP0  4  NaH P0  4  2  2  *10 times normal strength.  2  2  2  4  19.94 g m / l i t r e 16.18 g m / l i t r e 22.34 g m / n t r e  TABLE IV:  COMPOSITION OP EXPERIMENTAL BATHING SOLUTIONS. P i n a l i o n i c concentrations o f c o n s t i t u e n t substances expressed i n m.equiv. p e r l i t r e . 3 gm. o f glucose p e r l i t r e  was added i n a l l  cases, and the s o l u t i o n s e q u i l i b r a t e d 95$ 0 - 5 % 0 0 2  SOLUTION  Na  K  Cl  1  144  6  2  150  3  2  with  gas mixture.  HCO*  Oa  Mg  Choline  Phosphate  134  24  2  1  —  —  -  134  24  2  1  -  -  138  12  134  24  2  1  254  24  2  1  -  -  4  265  6  5  18  6  134  24  2  1  126  -  6  144  6  7  _  2  1  _  151  28  lo  E x t r a c e l l u l a r Space Experiments (a)  I n u l i n Space E s t i m a t i o n s :  used f o r the i n v i t r o i n c u b a t i o n space e s t i m a t i o n s , concentration.  I n u l i n was  d i s s o l v e d i n hot 0.154 cooled  normal S o l u t i o n 1  was  of excised muscles f o r i n u l i n  with d r i e d B. D. H.  ture, so the appropriate  had  The  I n u l i n added to 1%  (w/v)  r e l a t i v e l y i n s o l u b l e at room tempera-  amount was  weighed out a c c u r a t e l y  M NaCl s o l u t i o n .  and  A f t e r the i n u l i n - s a l i n e  s l i g h t l y , the other c o n s t i t u e n t s  of the S o l u t i o n 1  were added. Por  the i n v i v o i n j e c t i o n of i n u l i n i n t o nephrecto-  mised mice, a concentrated s o l u t i o n was prepared i n hot 0.9%  t i o n was  saline.  d i s s o l v e d r e a d i l y i n the hot NaCl, but ture s o l i d i f i e d completely.  needed.  A 25%  T h i s amount of  Gentle reheating  ml.  as  in a  completely. estimation  needed; the D i l u t e I n u l i n Standard  was  of the i n u l i n - c o n t a i n i n g bathing f l u i d d i l u t e d i n a  volumetric f l a s k to 100 of 100  inulin  resulted f i r s t  A standard s o l u t i o n f o r the c o l o u r i m e t r i c  1.0  solu-  on c o o l i n g to room tempera-  milky f l u i d , which upon f u r t h e r heating c l e a r e d  of muscle i n u l i n was  (w/v)  ml.  microgm. i n u l i n / m l .  T h i s provided a reference The  standard  reagents f o r a n a l y s i s were prepared  follows: Resorcinol:  125  mgm.  r e s o r c i n o l d i s s o l v e d i n 100  ml.  of a l c o h o l ; 30%  HCl:  80 ml.  of concentrated HCl  20 ml.  of water;  Trichloroacetic Acid: 100  ml.  12 gm.  of TCA  was  were added to  dissolved i n  of demineralised water.  29 (b)  Thiocyanate Space Estimations:  Estimations of t h i o -  cyanate space were done i n v i t r o as a check for the i n u l i n space determinations.  The thiocyanate bathing solution was  prepared with a CNS"" concentration of 48ra.e q u i v . / l . r e placing chloride i n the normal bathing s o l u t i o n . A dilute standard reference solution for the colourimetric estimation of muscle thiocyanate was prepared by d i l u t i n g 1,0 ml. o f the bathing solution to 100 ml., giving a CMS" concent r a t i o n of 10 microgm. CNS" per ml.  The 12$ TCA reagent used i n  the CNS" estimation i s the same as f o r the i n u l i n ; f e r r i c n i t r a t e reagent was prepared by dissolving 5.0 gm. of F e ^ O j ) ^ • 9H 0 i n 2  2«5 ml. of concentrated n i t r i c acid, and this was made up to 100 ml. with d i s t i l l e d water'.  2,  Radioactive Isotope Solutions There were two problems i n preparing the K^  2  incubation  solutions; f i r s t , the s p e c i f i c a c t i v i t y of the stock 0.154 M. K^  2  C l solution was usually not too high, and second, the r e q u i r e d concentrations of potassium ions i n the various bathing solutions was  only about 1/25 that of the sodium concentrations; thus, most  of the KC1 composing the bathing solutions had to be radioactive, or e l s e the s p e c i f i c a c t i v i t y o f the f l u i d would be too low t o be useful. Each shipment of the powdered K  4 2  gCOj arrived by a i r from  Amersham, England, encapsuled i n two nestled screw-top aluminum containers, sealed inside a thick lead c a s t l e .  The screw-top con-  tainers, were opened with tongs and p l i e r s , and the container and i t s contents (6.65 gm. of K  42 2  C 0 ^ , powdered) were c a r e f u l l y lowered  into a large beaker o f water.  The powder was washed out o f the  30  container, and dissolved i n the water. the  K ' CQ converted t o K C 1 with LOW 4  2  42  2  5  Indicator was added, and HCl.  This s o l u t i o n was  then d i l u t e d to 0.154 M and used as a stock solution f o r making up the experimental incubation f l u i d s . The f l u i d s were prepared with the K^ C1 2  l a s t ingredient;  added as the  48 ml. of KCl-free non-active s o l u t i o n of the  desired type was placed i n a small glass b o t t l e ; to t h i s was added 2 . 0 of the isotonic K^ C1, completing the ionic requirements. 2  C.  DISSECTION TECHNIQUE The m. gastrocnemius of the 129 s t r a i n mice has been used almost e x c l u s i v e l y i n these experiments. The mice were s a c r i f i c e d by c e r v i c a l f r a c t u r e of the spine.  The s k i n of the thigh was cut completely around with a  small p a i r of sharp scissors and peeled down the l e g and over the heel, exposing the muscles, tendons and f a s c i a e .  The skinned l e g  was then gently washed free of h a i r cuttings, etc., with a cotton pledget soaked with 37°C. Solution 1.  A small pair of forceps was  c a r e f u l l y inserted between the tendon of A c h i l l e s and the t i b i a , and moved back and f o r t h to create a small patent space.  Another  p a i r of forceps was then used to s t r i p away the f a s c i a l coverings of the gastrocnemius and the nearby muscles, s t a r t i n g from this space.  The p o p l i t e a l nerves were severed i n the p o p l i t e a l space,  close to the body of the gastrocnemius; the muscle was  then freed  by blunt dissection from those bordering i t , the tendon of A c h i l l e s severed near the caleaneous, and the entire muscle l i f t e d upwards by p u l l i n g on the freed tendon. ted  The soleus muscle was then separa-  from the gastrocnemius, and the l e g was cut through completely  just above the knee; the preparation was placed on the stage of a  31  d i s s e c t i n g microscope, on a warm, moistened paper towel.  Great  care was exercised during the entire procedure to avoid tearing, cutting, stretching or otherwise damaging the musclej i t was moistened frequently with 3 7 ° G . Solution 1 to prevent drying of the  tissues. Using f i n e forceps and a sharp s c a l p e l , excess f a t and  nervous tissue was stripped and cut from the preparation.  The  tendon ( A c h i l l e s ) was trimmed free of f a t and r e s i d u a l shreds of soleus muscle, and the excess length of tendon was c a r e f u l l y cut off.  However, i t was not cut o f f too short so as to damage any  muscle f i b r e s , and was l e f t long enough so there was  sufficient  free tendon to accomodate a firm grasp by forceps, to enable i t to be handled e a s i l y .  Now  the knee j o i n t was c a r e f u l l y trimmed  and scraped free of a l l muscle insertions except the gastrocnemius, the preparation bathed i n more warmed f l u i d , and then the tendinous ends of the gastrocnemius gently freed from the femur, with a minimum  amount of damage to the muscle.  The completely dissected  muscle was then weighed immediately on a Roller-Smith Precision Torsion Balance, and t r a n s f e r r e d immediately to the t e s t tube containing the bathing f l u i d .  Fow a few of the i n u l i n space studies,  m. peroneus longus was s i m i l a r l y excised by loosening and cutting the  tendinous ends. Small pieces of t h i n coloured thread were t i e d around  the  tendon of each gastrocnemius f o r i d e n t i f i c a t i o n purposes while  being incubated.  I t was determined that this thread did not appre-  c i a b l y a f f e c t the radioactive counting. CHEMICAL METHODS 1.  E x t r a c e l l u l a r space estimations The polysaccharide i n u l i n has been used i n t h i s study to  32 estimate the volume of e x t r a c e l l u l a r space i n muscle.  Muscles,  dissected and weighed as above, -were incubated f o r 4 hours i n a 1% i n u l i n bathing s o l u t i o n .  At the end of the incubation per-  iod, the muscles were rinsed b r i e f l y i n demineralised  water,  b l o t t e d , and weighed r a p i d l y ; they were then placed i n small clean mortars, 1.0 ml. of 12% TCA and some acid-washed sand added, and ground with a p e s t l e .  The mixture was placed i n a  graduated centrifuge tube, the mortar rinsed three times with 1,0 ml. portions of demineralised  water, and the whole mixture  centrifuged. For estimation of the muscle uptake of i n u l i n i n vivo, b i l a t e r a l l i g a t i o n of the renal pedicle was ether anaesthesia,  c a r r i e d out under  and 0.3 ml. of 25% i n u l i n s o l u t i o n i n j e c t e d  under the skin of the back.  After a variable period of time  to 3 j hours), the animals were k i l l e d by exsanguination from the heart, and the gastrocnemii and/or peroneii l o n g i i r o u t i n e l y excised, rinsed, b l o t t e d , weighed, and further treated as above.  The blood sample taken from each animal was  described  placed i n a  tube containing a small known amount of heparin solution, c e n t r i fuged, and 0.1 ml. aliquots of the plasma treated with TCA  and  assayed f o r i n u l i n . Aliquots of the muscle supernatant solutions were taken and analysed by the method of Hubbard and Loomis (1942).  A stan-  dard i n u l i n s o l u t i o n was prepared from the incubation solution, and portions of this were further d i l u t e d to give varying concentrations of i n u l i n for use as standards f o r colourimetric a n a l y s i s . Five standard tubes were prepared, with 1.0,  0.75,  0.50,  0*25, and  0,0 ml. of d i l u t e i n u l i n standard made up to 1,0 ml, with.deminera-  33 l i s e d water.  One ml. of each of the muscle homogenate super-  natant solutions were pipetted i n t o t e s t tubes also.  To each  tube was added 0 . 2 5 ml. 12% TCA, 1.0 ml. r e s o r c i n o l , and 3.0 of 30% HCL.  ml  8  This mixture was shaken well, heated i n an 80°C.  water bath f o r 8 minutes, and then placed to cool f o r 30 minutes i n i c e water.  The contents of a l l the tubes were d i l u t e d to 10 ml.,  mixed w e l l by inversion, and colourimetric values read a t 510 m i l l i microns i n a Klett-summerson Photoelectric Colourimeter. As a check on the e x t r a c e l l u l a r spaces estimated by the i n u l i n method, estimations of thiocyanate space were done on some normal mouse muscles, to see i f there was  a s i g n i f i c a n t difference  between the apparent i n u l i n spaces and the apparent thiocyanate spaces.  The m. gastrocnemii were r o u t i n e l y excised, weighed, and  incubated i n v i t r o i n the thiocyanate bathing s o l u t i o n f o r a peri o d of two hours.  The muscles were then ground and centrifuged,  and assayed f o r thiocyanate by the method of Crandall and Anderson (1934).  Standards were prepared by making 2.0, 1.0,  0.50,  and  0 . 2 5 ml. portions of d i l u t e thiocyanate standard (1.0 ml. bathing s o l u t i o n diluted to 100.0 ml. with water) up to 5.0 ml. with demineralised water.  The unknown solutions were prepared by  adding 4.0 ml. of water to 1.0 ml. of the supernatant s o l u t i o n . To every test tube 2.0 ml. of f e r r i c n i t r a t e reagent was added, and the mixture shaken w e l l .  The r e s u l t i n g colour was read immed-  i a t e l y at 510 milljjnicrons i n a K l e t t Summerson Colourimeter.  The  thiocyanate content of the muscles could then be calculated with reference to the standard s o l u t i o n s . 2.  Dry Weight Determinations For the determination of percentage water of representa-  34  t i v e muscles, the following procedure was employed: (a)  Small metal planchets were washed, dried, heated  for 2 hours at 1 1 0 ° C , and cooled overnight i n a dessicator. These were then weighed on a Sartorius Selecta balance, reheated for one hour, cooled and weighed to constant weight.  The planchets  were stored i n the dessicator u n t i l used. (b)  Each muscle was excised i n the routine way, placed  on the planchet, and r a p i d l y weighed on the same balance. (c)  Wiien a number o f muscles were ready, they were  placed i n a 110°C, constant temperature oven, dried f o r one hour, cooled i n a dessicator, and weighed c a r e f u l l y .  They were then  reheated, cooled and weighed t o constant weight. 3.  Non-Radioactive Incubation Experiments The mice were s a c r i f i c e d , and the gastrocnemii excised  as previously described; the muscles were then immediately weighed, and transferred t o the selected bathing s o l u t i o n .  The bathing  f l u i d was contained i n large glass test tubes suspended i n a constant temperature (33°G.) water bath.  The bathing solution  was oxygenated and c i r c u l a t e d i n the t e s t tubes with a  35fd0 ~^ 2 cC0  2  gas mixture. After a variable time of soaking, the muscles were r e t r i e v e d from the bottom of the tubes, immersed b r i e f l y (less than 2 seconds) i n d i s t i l l e d water to remove any adhering drops of b athing solution, b l o t t e d l i g h t l y on dry f i l t e r paper, transferred to acid rinsed b o r o - s i l i c a t e t e s t tubes, and digested with 4 drops of concentrated n i t r i c acid. analysis.  The tubes were kept t i g h t l y corked u n t i l ready f o r  35  The samples were heated s l i g h t l y to ensure the complete digestion of the muscles.  The n i t r i c a c i d solutions were d i l u t e d  with a few ml. of demineralised water, and transferred into graduated "Pyrex" cylinders; an additional few ml. were added to the digestion tube, and these rinsings added to the c y l i n d e r .  The  solution was d i l u t e d to 5.0 ml. with water, and f i l t e r e d through Whatman #4-0 f i l t e r paper i n small p l a s t i c funnels i n t o t e s t tubes. Two 1.0 ml. aliquots of the f i l t r a t e were placed i n separate t e s t tubes; to one was added demineralised water to give the amount of d i l u t i o n necessary t o bring the concentration  of sodium into a  range suitable f o r analysis (usually 2 t o 10 ml.); to the other 1.0 ml. aliquot 1.0 ml. of 0.25 M NaCl and the same amount of water f o r the K determination were added.  The amounts of Na and  K i n these solutions were determined with the a i d of an Evans Electroselenium  Flame Photometer.  A l l a n a l y t i c a l r e s u l t s are  expressed i n terms of m i l l i e q u i v a l e n t s of the appropriate ion per kilogram fresh weight of muscle.  E.  RADIOACTIVE ISOTOPE TECHNIQUES Radioisotope tracer experiments formed the major part of the experimental work done i n t h i s investigation, with the soaking experiments, e x t r a c e l l u l a r space determinations, and dry weight analyses a l l a n c i l l a r y to the isotope f l u x studies. isotope K^  2  The  was used as a tracer; whenever t h e radioactive solu-  tions were t o be handled, gloves were worn to protect the hands from contact, and a l l work was done over an enamel tray l i n e d with t i n f o i l and absorbent paper.  A f t e r each experiment or each d i l u -  ting operation involving the radioisotope  storage b o t t l e s , the  36  entire area was Geiger counter.  thoroughly monitored with an end-window portable At a l l times, extreme care was used to l i m i t any  contamination by the active s o l u t i o n of equipment used (such as glassware) to a bare minimum. The main disadvantage of using the K* half-life.  2  was i t s short  Every 24 hours 74$ of i t disintegrated, and a f t e r  4 days, the l e v e l of a c t i v i t y would be down to 0,40$ of the o r i g i n a l value.  Thus ike experiments had to be done i n the f i r s t  three or four days a f t e r r e c e i p t of a shipment of the K^ .  There  2  was no disposal problem, f o r the radioactive solutions were kept f o r 3 weeks a f t e r the termination of the run of experiments,  and  then treated as being non-radioactive. The solutions were prepared i n the usual non-active form, and the 2.0 ml, portion of K^  2  stock solution added to the 48 ml.  of the bathing f l u i d to be used. medium f o r the i n f l u x studiesj  This active solution was  the  i t was placed i n a "Pyrex" t e s t  tube i n a 33 C. water bath, and mixed and oxygenated with a 95$0 -5$CO gas mixture. 2  2  The muscles were r o u t i n e l y excised,  r a p i d l y weighed, and placed i n the solution to incubate;- a f t e r approximately 10, 30, 60, 90, 120, and 150 minutes of incubation the muscles were removed from the s o l u t i o n with a curved glass rod, and placed f o r 1,0 minutes i n a beaker containing 200 ml. of nonactive s o l u t i o n of the same composition as the incubation medium. This short soaking period e f f e c t i v e l y reduced the e x t r a c e l l u l a r K^  2  ing  to ca. 50$ of i t s previous l e v e l (McLennan 1955), thus lessenthe error i n the counting of the f i b r e K.  The muscles were then  b l o t t e d b r i e f l y on f i l t e r paper, and arranged on a p l a s t i c counting  37  tray i n a standard reproducible p o s i t i o n .  The t r a y and muscles  were then placed under a Geiger-Muller end-window counter and counted f o r three one-minute i n t e r v a l s .  The impulses from the  counter were transmitted to an e l e c t r o n i c scaler and mechanical recorder.  The scaler and counter were Nuclear Chicago equipment,  the scaler a 161A model, and the counter a D-34 model, with a thin mica window.  At the end of the experiment, the muscles were weighed  and placed i n b o r o - s i l i c a t e t e s t tubes with 0.1 ml. of concentrated n i t r i c acid, to digest.  To this digest was then added 0.9 ml. of  demineralised water, the tube shaken, and the mixture poured into a metal counting planchet.  The tube was rinsed with 1.0 ml. of  the water, and this was added to the planchet.  The planchet plus  d i l u t e d digest was s e t i n a sample holder tray, and placed under the Geiger counter t o be assayed f o r r a d i o a c t i v i t y .  After the  counting p e r i o d was f i n i s h e d , the planchet was removed from the sample holder, and the digested muscle mixture poured back i n t o the b o r o - s i l i c a t e t e s t tubej  the planchet was rinsed twice with  2.0 ml. portions of demineralised water, these added t o the test tube, and the tube t i g h t l y corked u n t i l used f o r flame a n a l y s i s . A 0.1 ml. aliquot of the bathing solution was placed i n a metal planchet, diluted with 1.9 ml. water, and s i m i l a r l y assayed f o r radioactivity.  Thus, a r e l a t i o n s h i p between the amount of K^" i n 2  the muscle and that i n the bathing s o l u t i o n would be established. After 2 to 3 weeks, when the r a d i a t i o n was at a low l e v e l , the muscles were assayed f o r t o t a l sodium and potassium by flame photometry.  SECTION I I I RESULTS A.  B.  C.  D  «  E x t r a c e l l u l a r Space Estimations 1.  Muscle Inulin Space:  Normal S t r a i n 129 mice  2.  Muscle I n u l i n Space:  Dystrophic S t r a i n 1 2 9 Mice  3.  Muscle Inulin Space:  Swiss Albino Mice  4.  Muscle Thiocyanate Space:  Normal S t r a i n 129 Mice  Dry Weight and Non-Inulin Space Water Estimations 1.  Dry Weight Determinations  2.  Non-Inulin Water Estimations  Chemical  Analyses  1.  Fresh Muscle  2.  Incubated Muscle:  3«  Incubated Muscle: Summary of Results of incubation i n solutions 2 to 6  4»  Conculsions  Results of K 1.  4 2  Solution 1  Uptake Studies  Normal Mouse S k e l e t a l Muscle a) b)  E f f e c t s of Varying K E f f e c t s of Varying Na^  c)  E f f e c t s of Varying K  e  and  e  Na  2.  Dystrophic Mouse S k e l e t a l Muscle  3.  a) E f f e c t s of Varying E b) E f f e c t s of Varying Na Summary of K Results e  4 2  e  e  38  A.  EXTRACELLULAR SPACE ESTIMATIONS I n t r a c e l l u l a r ion concentrations cannot be estimated without making adequate allowance f o r the ion content of the extracellular f l u i d .  To make this correction, the volume of the  e x t r a c e l l u l a r space (ECS) and the concentration of the i o n i n the e x t r a c e l l u l a r f l u i d must be known.  The calculated amount of i o n  present i n the ECS can then be subtracted from the t o t a l muscle content to give the i n t r a c e l l u l a r value.  Muscles from normal and  dystrophic animals were assayed f o r ECS as described on page  28  but the averaged r e s u l t s from groups of muscles were found to be variable from group to group;  small muscles seemed to have l a r g e r  ECS values than larger ones, and muscles from the dystrophic a n i mals seemed to have larger ECS values than comparable muscles from normal animals. When the percent ECS was plotted against fresh muscle weight, the graphs .resembled F i g . I, i . e . , there was a negative c o r r e l a t i o n between muscle weight and magnitude of the ECS. 1.  Muscle I n u l i n Space:  Normal S t r a i n 129 Mice  Gastrocnemii from normal s t r a i n 1 2 9 mice were soaked f o r a standard time of four hours i n an inulin-containing s o l u t i o n ; the muscles were analysed f o r i n u l i n content, the raw data corrected f o r muscle swelling during incubation, and the r e s u l t s divided by the muscle weights to give the amount of i n u l i n per gm. of muscle; t h i s was expressed as . mg. i n u l i n per gm. fresh muscle ————————_____________________  . X lOCyo  mg. i n u l i n per ml. soaking s o l u t i o n F i g . I i s a graph obtained when the i n u l i n spaces of 34 muscles were plotted against the f r e s h muscle weights.  On inspec-  Muscle  Fresh  Weight,  mg  40  t i o n , there was an obvious r e l a t i o n s h i p between the s i z e of the muscle and i t s i n u l i n space, with the space decreasing with i n creasing muscle s i z e .  The calculated regression l i n e (as drawn ±  i n F i g . I) i s y - 44,7 - 0.43x (S.E.~ 1.1). However, i t was considered that excised muscles incubated i n an a r t i f i c i a l f l u i d might not have the same i n u l i n space as muscles i n s i t u .  In order to examine this p o s s i b i l i t y , normal  s t r a i n 129 mice were nephrectomised t i o n subcutaneouslyj  and injected with i n u l i n s o l u -  a f t e r a variable time, gastrocnemii and/or  peronei l o n g i were excised and analysed.  (Peroneus longus was  a  small muscle, with an ECS s i m i l a r to the gastrocnemius, and i t provided a u s e f u l check on the values previously found f o r the gastrocnemii.) content;  Plasma samples were taken and assayed f o r i n u l i n  each plasma value was used as the d i v i s o r f o r muscles  from the same animal, i n place of the soaking solution value. Results were calculated as described above, and p l o t t e d s i m i l a r l y . F i g . I I i l l u s t r a t e s the r e s u l t s obtained;  values from  both the gastrocnemius and the peroneus longus muscles are p l o t t e d ; the calculated regression l i n e of the 23 points i s y = 54.0 - 0.472x (S.E.* 2.8). cantly.  The l i n e s i n F i g s . I and I I do not d i f f e r s i g n i f i -  This r e s u l t indicates that incubated muscles have i n u l i n  spaces s l i g h t l y less than but not s i g n i f i c a n t l y d i f f e r e n t from those of fresh muscles;  a t l e a s t part of the swelling noted during incu-  bation appears to occur i n a region of the muscle that i s not available to i n u l i n . 2.  Muscle Inulin Space:  Dystrophic S t r a i n 129 Mice  There was only a l i m i t e d number of dystrophic mice a v a i l able f o r t h i s investigation, and i n the l i g h t of the previous r e s u l t s  41  Fig.JL  Inulin Space of normal Mouse Muscle: Injection Method. * Gastrocnemii x Peronei Longi  Figures in parentheses between injection and  show the number of hours sacrifice of the animals.  x(3)  (3) X(|/2)  X(2)  #  (2 1/2)  (3)  (3  1/2)  •(1/2)  •  (l)»x(2) •(2  —i  12)  (3)«  1/2)  1  1  20 40 Muscle Fresh Weight, mg.  1  1—  60  42  with normal mice, i t was decided to pool the r e s u l t s obtained on dystrophic tissue by the incubation and i n j e c t i o n methods.  Since  the peronei longi were too small to excise i n t a c t , only the gastrocnemii of these dystrophic animals were used;  F i g . I l l i s a graph  of the dystrophic muscle i n u l i n spaces plotted against the muscle weight.  The negative c o r r e l a t i o n described f o r normal muscles i s  also observed i n these 17 dystrophic muscles, but there i s a greater dependance of the i n u l i n space on muscle weight. calculated regression l i n e i s y » 94.6  The  - l 6 5 x (S.E.~ 2.8). a  It  should be noted that some of the smallest muscles had i n u l i n spaces as high as 30%;  this may have been due to a permeability of the  muscle membrane to i n u l i n .  I f t h i s was the case, and the i n u l i n  could penetrate i n t o the degenerating muscle c e l l s i t would account f o r the high i n u l i n space values observed. 3.  Muscle I n u l i n Space:  Swiss Albino Mice  Inherited muscular dystrophy i s unknown i n Swiss albino mice.  Some analyses of gastrocnemii from Swiss mice were done t o  eliminate the p o s s i b i l i t y that the previously observed r e s u l t s were a p e c u l i a r i t y of the 129  strain.  Twenty-three muscles were excised,  soaked, and analysed i n the usual way.  A relationship between muscle  weight and i n u l i n space s i m i l a r to that observed i n the s t r a i n 129 muscles was noted (see F i g . HT).  The calculated regression l i n e i n +  F i g . 17 i s y o 39.8- - 0.249x (S.E." 1.2).  This l i n e i s not s i g n i -  f i c a n t l y d i f f e r e n t from that of either F i g . I or F i g . I I . 4.  Muscle Thiocyanate Space;  Normal S t r a i n 129 Mice  Some thiocyanate (SCN) estimations were done on normal s t r a i n 1 2 9 mice, as a check on the i n u l i n space r e s u l t s .  Twenty-  three muscles were excised and incubated as usual, and analysed f o r  43  o + 0  i 10 Muscle  1  1  1  20  30  40  Fresh  Weight,  mg  -i 50  0  _ 0  1  ~JZ  20  r—  i  1  40  Muscle  r~  1 60  Fresh  Weight,  1  80 mg.  1  r— w  45 SCN.  These r e s u l t s are i l l u s t r a t e d i n F i g . V;  the calculated  regression l i n e drawn i s y = 35.7 - 0.196x (S.E.~ 1.2), and tiae slope of the l i n e i s , i n t h i s case, not s i g n i f i c a n t l y d i f f e r e n t from zero, but also i s not s i g n i f i c a n t l y d i f f e r e n t from those o f Figures I, I I , or I ? . However, there appeared t o be a trend f o r the SCN space to decrease with increasing muscle weight. B.  DRY WEIGHT AND NON-INULIN SPACE WATER ESTIMATIONS 1.  Dry Weight Determinations Results of the Na and K analyses were to be expressed  as i n t r a c e l l u l a r i o n per kilogram f r e s h tissue weight, f o r which purpose a knowledge o f the t o t a l water content and the volume of the ECS of the tissue was necessary (see previous s e c t i o n ) .  Muscles  were excised, weighed and treated as described on page 33; a series of muscles was also incubated f o r several hours before the dry weight assay;  Table VI i s a tabulation of the average percentage  weight l o s s , expressed as weight loss  x 100$  fresh weight None of the averages l i s t e d i n Table VI are s i g n i f i c a n t l y d i f f e r e n t from one another.  The fresh and incubated muscles seem to have the  same t o t a l water content of 76$;  also, no s i g n i f i c a n t difference  i n water content between normal and dystrophic tissues was apparent. 2.  Non-inulin Space Water Estimations The non-inulin space water volumeof a tissue i s that volume  of water not accessible to d i f f u s i b l e i n u l i n , either a f t e r injecticaa of i n u l i n i n t o the nephrectomised animal, or following incubation of an excised muscle i n an inulin-containing s o l u t i o n .  The available  i n u l i n space, usually considered as being i d e n t i c a l t o the ECS, was determined as described on page 28.  The non-inulin space water was  Fig. V.  Thiocynate Space In  of normal  Mouse  Vitro Method.  50 n  Muscle  Fresh  Weight , mg  Muscle  TABLE VI:  INCUBATION SOLUTION  WEIGHT LOSS OP FRESH AND INCUBATED MUSCLE.  HOURS INCUB.  FRESH  MUSCLE TYPE *  PERCENTAGE WEIGHT LOSS  N  75.6  D  77.2  N  75.9  N  75.4 76.8  4  N  76.8  N = Normal muscle, D = Dystrophic muscle. A l l standard deviations are less than 1.1, except t h i s average, which i s 3.8,  48  calculated from a knowledge of this ECS value, the dry muscle weight, and the fresh weight, and was estimated as being fresh muscle weight - ECS - dry weight  X  IOQ%  fresh -weight t h i s was expressed as a percentage of the fresh muscle weight. Table VII contains the average non-inulin space water values obtained f o r fresh and incubated muscles.  The normal muscle  values increased greatly with incubation, i n both normal and K-free incubation solutions;  however, the fresh dystrophic  muscle value was not s i g n i f i c a t n l y d i f f e r e n t from the soaked muscle value.  The dystrophic muscle non-inulin space water was  l e s s than h a l f the normal muscle value f o r fresh tissue;  following  incubation f o r 4 hours, the normal value rose from 47?2 (fresh) to 65% of the fresh tissue weight, while the dystrophic value remained unchanged at 21%.  This increase i n non-inulin space water i n  normal tissue may r e f l e c t a swelling or increase of volume" i n an area of the muscle not available to i n u l i n .  The decrease observed  i n i n u l i n space with incubation, and the constancy of the t o t a l tissue water, suggests that during incubation the muscle f i b r e s swell by taking up water from the ECS.  The increase i n c e l l u l a r  water might be more than has been measured here;  i f the muscle  membrane were distended by this extra water, i t might be more permeable to the i n u l i n molecule; i n u l i n could enter the f i b r e , and the apparent i n u l i n space would be larger than the "true" space. Z i e r l e r (1957) reported that normal r a t muscle was permeable to the enzyme aldolase (MW147,000);  he also reported that dystrophic mouse  muscle had a higher e f f l u x of aldolase than d i d normal muscle ( Z i e r l e r 1958).  I t seems possible that i f normal muscle were per-  TABLE VII:  INCUBATION SOLUTION FRESH  1  2  PERCENTAGE NON-INULIN SPACE WATER. OF FRESH AND INCUBATED MUSCLE*.  HOURS INCUB. -  MUSCLE TYPE**  PERCENTAGE NON-INULIN WATER  N  46.8  4.6  D  20.9  5.9  2  N  58.4  8.0  4  N  65.3  1.8  D  21.6  6.5  N  64.2  3.0  4  Expressed as percentage fresh tissue weight. N = Normal muscle, D = Drystrophic tissue.  50  meable t o a l d o l a s e , i t c o u l d l i k e w i s e be permeable to the much s m a l l e r i n u l i n molecule C.  (MW991).  CHEMICAL ANALYSES Na and K a n a l y s e s o f s k e l e t a l muscle w i l l not y i e l d u s e f u l data about the i n t r a c e l l u l a r i o n c o n c e n t r a t i o n s u n l e s s a c o r r e c t i o n f o r the i o n c o n t e n t o f t h e ECS i s made.  Also, i f i n -  cubated muscles s w e l l , t h e r e a r e l i k e l y t o be i o n s from t h e i n c u b a t i o n media i n the water o f s w e l l i n g .  C o r r e c t i o n o f muscle  a n a l y s i s d a t a f o r these f a c t o r s s h o u l d y i e l d t h e i n t r a c e l l u l a r i o n p e r u n i t weight o f t i s s u e , p e r l i t r e p e r gram o f f a t - f r e e d r i e d s o l i d s , parameters c a n be c a l c u l a t e d .  o f i n t r a c e l l u l a r water,  o r i n o t h e r ways, p r o v i d i n g the  The a n a l y t i c a l r e s u l t s i n t h i s  t h e s i s w i l l be e x p r e s s e d as m. e q u i v . n o n - i n u l i n space i o n p e r Kg. f r e s h t i s s u e .  T h i s i s p r o b a b l y comparable t o the i n t r a c e l l u -  l a r i o n c o n t e n t i n f r e s h muscle, b u t w i t h i n c u b a t e d muscle one must account  f o r t h e apparent  i n c r e a s e i n n o n - i n u l i n space due t o  s w e l l i n g , and the i n t r a c e l l u l a r i o n c o n t e n t and n o n - i n u l i n space i o n c o n t e n t may n o t be s i m i l a r . A n a l y s e s were performed  on f r e s h and i n c u b a t e d normal and  d y s t r o p h i c muscle t i s s u e t o determine space) Na and K c o n c e n t r a t i o n s .  the i n t r a c e l l u l a r ( n o n - i n u l i n  The muscles were t r e a t e d as des-  c r i b e d on page 34, the raw d a t a c o r r e c t e d f o r i n u l i n space and water o f s w e l l i n g i o n c o n t e n t , and t h i s r e s u l t d i v i d e d b y the a p p r o p r i a t e t i s s u e weight.  The c a l c u l a t e d r e s u l t s are. expressed  as m. e q u i v . ion/Kg f r e s h t i s s u e ;  the s t a n d a r d d e v i a t i o n s o f the  values are a l s o given. T a b u l a t e d r e s u l t s o f the Na and K a n a l y s e s appear i n Table V I I I .  TABLE V I I I :  Na, K CONTENTS OF FRESH AND INCUBATED MOUSE MUSCLE. Results expressed as m.equiv. n o n - i n u l i n space i o n per Kg. o f f r e s h t i s s u e .  INCUBATION SOLUTION  FRESH  HOURS INCTJB.  MUSCLE TYPE  Na  K  S.D. Na  K  N  30.0  89.9  5.2  5.2  D  58.2  68.2  11.4  11.1  N  31.6  7.8  2.6  D  77.4  90.6 60.0  4.7  4.2  N D  41.3 77.1  63.4  N  34.6  D N D  19.1 13.3  4.0  72.1  8.8  6.6  78.2  47.3  11.2  6.6  69.5 95.1  37.4 39.7  12.0 23.8  8.9 3.5  (CONTINUED OVERLEAF)  57.6  8.5  (CONTINUED PROM PREVIOUS PAGE)  INCUBATION SOLUTION  HOURS INCUB.  S.D. Na  K  90.6  10.6  D  73.5  80.4  9.7  11.9 7.4  N  41.2  D  82.4  62.3 91.8  10.9 9.2  6.3 7.6  2  N  110.1  60.8  8.2  6.6  4  N D  134.0 126.1  41.2  9.2  62.4  16.4 4.5  N  27.2 40.6  2.5 2.0  3.2  D  14.1 17.-7  N  45.9  35.5  10.2  1.6  4  6  K  43.0  3  5  Na  N  2  4  MUSCLE TYPE  4  4  4.3  2.5  53  1.  Fresh Muscle Normal mouse muscle contained 30.0 m. equiv. Na/Kg, while  the dystrophic muscle had 58.2 m. equiv. Na/Kg.  The normal muscle  K was 89.9 m. equiv./Kg, but the dystrophic K was only 68.2 m. equiv./ Kg.  Both dystrophic values f o r the i n t r a c e l l u l a r ions (Na^ and K^)  are s i g n i f i c a n t l y d i f f e r e n t from the normal muscle values (p<0.001)j t h i s gain of Na and l o s s of K i n dystrophic tissue might be caused by a leakage or a l t e r e d permeability of the muscle membrane, r e s u l t ing i n an i n a b i l i t y to r e t a i n or exclude c e r t a i n ion species; i f the Na and K ions were to diffuse down their concentration gradients across such a leaky membrane, the c e l l u l a r K would be expected to f a l l , and the c e l l u l a r Na to r i s e .  This "membrane leak" would be  one explanation f o r the observed a l t e r a t i o n s i n the c e l l u l a r i o n contents. 2.  Incubated Muscle;  Solution 1.  Incubation of excised normal muscle i n Locke's s o l u t i o n (Solution 1, Table II) did not s i g n i f i c a n t l y a l t e r the non-inulin space Na or K content from that calculated f o r fresh muscle.  How-  ever, the Na content of dystrophic tissue rose 33/£ to 77.4 m. equiv. Na/Kg, and the K dropped 12$ to 60.0 m. equiv./Kg;  both these  changes are s i g n i f i c a n t (p<0.01). After 4 hours soaking, normal tissue Na had r i s e n to 41.3 m. equiv./Kg, and K had dropped to 57.6 m. equiv./Kg;  the dystro-  phic tissue contents were 77.1 m, equiv. Na/Kg and 63.4 m. equiv. K/Kg. The excised muscle tissue can be incubated f o r 2 hours i n Locke's s o l u t i o n , and the i n t r a c e l l u l a r (non-inulin space) i o n contents w i l l not have changed.  The ion l e v e l s are not stable f o r a  54  longer period, however, f o r a f t e r 4 hours soaking there are s i g n i f i c a n t alterations i n the i n t r a c e l l u l a r Na and K.  Apparently the  changes i n ion content that occur are the same i n both types of t i s s u e , that i s , the Na r i s e s and the K f a l l s .  The dystrophic  muscle seems t o undergo these changes more r a p i d l y than the normal muscle;  t h i s may be a r e f l e c t i o n of the larger ECS of the dystrophic  tissue, or a consequence of an abnormal permeability of the dystrophic f i b r e membrane. 3.  Incubated Muscle:  Summary of Results of Incubation i n Solutions  2 t o 6. The data f o r ion content following 2 hours and 4 hours of incubation i n the altered media are to be found i n Table VIII; these data w i l l be summarised and b r i e f l y compared i n this section. The tendency of Na to r i s e and K to f a l l , as mentioned above, was noted as occurring generally i n most muscles incubated i n the a l t e r e d media;  the change was e s p e c i a l l y marked following  4 hours' incubation. Muscles incubated i n altered K gain was l e s s i n 12 mM K  e  than i n 0 mM K  e  solutions gained Na; solution;  the  the dystrophic  muscles seemed to gain Na more rapidly, and the gain appeared to occur e a r l i e r than i n the normal t i s s u e .  Incubation of normal  muscle i n the altered K media l e d to a decrease i n muscle K; loss was greater i n the K-free solution, and l e s s i n the 12 mM solution, than observed i n the "reference" Solution 1. tissue K rose i n the 12 mM. K-free medium.  K  e  the K  Dystrophic  solution, but r a p i d l y f e l l i n the  During the f i r s t  2 hours of incubation, the K^  value f o r normal muscle i n the 12 mM K e K-free medium i t f e l l somewhat;  was constant, but i n the '  a f t e r 4 hours soaking, normal  muscle K was s i g n i f i c a n t l y lower than the fresh value.  e  55  Solution 4 contained 288 mM  Na , e  and was hypertonicj as  expected, both types of muscle gained Na during incubation;  normal  muscle K had dropped s i g n i f i c a t n l y a f t e r 2 hours of soaking,  and  a f t e r 4 hours, both normal and dystrophic tissue K was lower than the f r e s h tissue value. Na  a  significantly  Soaking excised muscle i n 18  mM  lowered Na and K i n both types of tissue, a greater drop i n Na  occurring i n the dystrophic tissue than i n the normal, but l e s s of a decrease i n  K^.  Solution 6 was  a phosphate buffered solution;  i t was  used  i n an attempt to reduce the swelling that occurred during incubation i n the bicarbonated-buffered media;  perhaps a reduction i n the  amount of swelling would a i d the c e l l u l a r retention of Na and K. However, the swelling during incubation s t i l l occurred, and to almost the same degree as i n Solution 1.  The tissue Na and K  l e v e l s a l s o changed during the 4 hours incubation, the changes being greater i n t h i s s o l u t i o n than i n Solution 1.  On the basis  of these r e s u l t s , i t was decided not to use t h i s Solution 6, or variants of i t , f o r incubation purposes. 4.  Conculsions I t was apparent from the foregoing results -that muscle K  l e v e l s were r e l a t i v e l y stable f o r at l e a s t 2 hours of incubation, at l e a s t i n most soaking media.  This s t a b i l i t y of  i s a necessary  condition i f one wishes to i n t e r p r e t radioactive K (K#)  data i n  the l i g h t of the Harris diffusion-permeation hypothesis, for i n order to calculate and p l o t a normal K exchange r e l a t i o n s h i p (K# uptake against time) the Kj_ must be constant, or the data w i l l not f a l l along the predicted l i n e .  I t was decided to l i m i t the i n t e r -  pretation of K-8- exchange studies to the data obtained during the f i r s t 2 hours of incubation i n the radioactive media, f o r correc-  56  tions could not be made for time.  changes that occurred a f t e r t h i s  Consequently, only the uptake of K# could be  followed;  e f f l u x studies would necessitate a previous loading of the t i s s u e with radioactive K by non-incubation methods, and t h i s was attempted.  not  I f the exchange follows d i f f u s i o n p r i n c i p l e s , the e f f l u x  graph should be s i m i l a r i n form to the i n f l u x graph. D.  RESULTS OF K  4 2  UPTAKE STUDIES  AO The radioactive isotope K7 experiments reported i n t h i s section;  was used as a t r a c e r i n the the studies were done to  determine the rate a t which radioactive K (K#) i n s o l u t i o n exchanged with i n t r a c e l l u l a r K, and A e t h e r a l t e r a t i o n s i n the i o n i c composition rate.  of the incubation solutions would a f f e c t the exchange  The methods of incubation, radioactive assay, and  analysis were described on page 35.  chemical  In t h i s section, exchange  means the movement of an ion occurring i n the absence of a net change i n tissue ion content (the exchange of K, as followed by the uptake of K , 42  w i l l be the only exchange process  the Harris diffusion-permeation hypothesis  reported);  states that the exchange  of tissue K should follow a d i f f u s i o n curve i f the K# uptake i s p l o t t e d against the expression  (K  e  x time);  i f the square root  of t h i s expression i s used, the r e s u l t i n g curve should be a s i n g l e s t r a i g h t l i n e f o r most of i t s extent.  AH  K* uptake points should  f a l l along this l i n e , and be independant of the external K concent r a t i o n providing the i n t r a c e l l u l a r K l e v e l i s constant.  I t was  thought that this method of expressing K* uptake r e s u l t s might provide evidence to support or repudiate the Harris hypothesis;  if  the "diffusion-permeation" concept (see Introduction, page 16) i s an accurate representation of the conditions e x i s t i n g i n s k e l e t a l  57  muscle tissue, these data may help to elucidate the k i n e t i c s of ion movements i n muscle. Preliminary experiments showed that tissue K l e v e l s remained f a i r l y constant under most conditions f o r the f i r s t 2 hours of incubation (Table ¥111 and Burr and McLennan (1961)), and were comparable to f r e s h tissue values.  Data from the K^  2  exchange  experiments were p l o t t e d as m. equiv. K# per Kg tissue (ordinate) against (K *t)2  (abscissa), a correction having been applied f o r  the amount of K* remaining i n the ECS, assuming 50% removal during the timed 1.0 minute wash (McLennan 1955). is i l l u s t r a t e d i n F i g . VI;  The method of p l o t t i n g  the slope of the calculated l i n e i s  expressed as m. equiv./Kg/(mM • min)2.  Comparison of the slopes  of the calculated regression l i n e s enabled an estimation of the r e l a t i v e rates of K exchange under the various experimental conditions to be made. 1.  Normal Mouse S k e l e t a l Muscle (a)  E f f e c t s of varying K » The r i s e i n K* w i l l represent exchange only i f the  remains constant;  i f the tissue i s gaining K, the net increase i n  tissue r a d i o a c t i v i t y w i l l be the sum of the exchange and the net gain.  F i g . VI shows the r e s u l t s from 19 experiments, with data from  muscles incubated i n 2 mM, on the same graph;  6 mM,  and 12 mM K  e  solutions a l l plotted  i t i s apparent by inspection that the points  cluster around a straight l i n e , and the calculated regression l i n e drawn i n the Figure has a slope of 0.98 m. equiv./Kg/(mM • m i n )  2  (S.E." 0.03). Muscle K exchange observed i n these incubation media compares favourably to that predicted by the Harris diffusion-permeation model, i n which the rate c o n t r o l l i n g step i s e q u i l i b r a t i o n with-  58  Fig yi_ Uptake of by normal Mouse Muscle: Normal and altered K Incubation Media. e  2  mM K  6  mM K  12  mM K  ( Ke • t )  2  l/  e  e  e  -  •  - o - x  (mM • min.)  ^2  59  i n an adsorbed layer of K.  With this "normal" exchangeability  established, i t was possible to measure K* uptake under conditions leading to a net increase of tissue K. Incubation of muscle i n high-K media (24 mM,  48 mM  K) Q  resulted i n rapid uptake of K*- by the tissue, and l e d to a net increase i n Kj_.  F i g . VII i s a graph of the K* uptake of normal  muscle tissue i n the high-K media; plotted;  r e s u l t s from 4 experiments were  there were not enough points to calculate a meaningful  regression l i n e , so the s o l i d l i n e s i n the Figure are freehand estimations of the slopes of the 24 mM solution (upper l i n e ) uptake curves;  (lower l i n e ) and Hie 48 mM the slopes of the l i n e a r  portions of the curves are 2.65 and 3.11 m. equiv./Kg/(mM • min)2 respectively.  The dashed l i n e i s the regression l i n e from F i g . VI.  Both high-K l i n e s are obviously d i f f e r e n t from the normal l i n e . The net gain i n Kj_ can be calculated from the K  4 2  data;  Table IX  AO compares the chemical analysis data and the calculated TL* data f o r the net K gain.  K gain by analysis i n 24 mM K  e  was 85 m. equiv./Kg  fresh weight;  the K gain from the K  m. equiv./Kg;  comparable values f o r 48 mM medium were 120 and  126 m. equiv./Kg.  4 2  data was calculated as 80  I f the K analysis values f o r each K-* uptake  reading were calculated, and the net gain subtracted a t each K# point, one would predict that the corrected points would f a l l along a s t r a i g h t l i n e , one which would correspond to the normal solution K# uptake l i n e . From the foregoing data i t seems that K exchange i n high-K solutions i s s i m i l a r to that observed i n the normal K  media, i f  e appropriate correction i s made f o r the net K gain;  '  the K exchange  seems t o be a d i f f u s i o n a l phenomenon, but i t i s obscured i n the  60  F i g . vij_  Uptake High  2 4 meg. / L  I 0  of  K  - K K  I 20  ( Ke • t  by n o r m a l  Incubation -  I  ) 2 ! /  Mouse  Muscle:  Media.  •  I 40  1  ( mM min.) 2 l/  L_ 60  TABLE IX:  EXCHANGE AND NET GAIN OP K IN NORMAL MUSCLE : HIGH -K  e  INCUBATION MEDIA. Values i n m.equiv./Kg. f r e s h  K  e  1 NET GAIN BY ANALYSIS (1)  *  2  K AFTER 2 HOURS (PIG. V I I ) (2)  tissue.  * 3 K EXCHANGE AT 2 HOURS (PIG. VI) (5)  4 DIFFERENCE COLUMN (2)  12)  24  85  127  47  80  48  120  194  68  126  61  high-K media by the large uptake of K-* due to net tissue K gain, both i n exchange f o r Na^ and as uptake of KC1. (b)  E f f e c t s of varying Na . The interdependance of Na and K ions i n muscle has been  repeatedly demonstrated, and changes i n the e x t e r n a l concentration of one ion can a f f e c t the movement of the other (see Introduction). Muscles were incubated i n two altered Na Na , the other with 288 mM Na . e e  solutions, one with 18 mM e ' The l a t t e r s o l u t i o n was hypertonic  e  i  I f K# uptake i s p l o t t e d against the appropriate (K * t ) values, 2  Q  the r e s u l t a n t points f a l l along two l i n e s , as i l l u s t r a t e d i n Fig. VIII.  The dashed l i n e i s from F i g . VIj  the s o l i d l i n e above  i t i s the regression l i n e f o r 8 experiments measuring K uptake i n 288 mM Na 0.08).  e  medium;  the slope i s 1.02 m. equiv./Kg/(mM•min)  2  (S.E.-  The low-Na regression l i n e i s s i g n i f i c a n t at the 1% l e v e l  i f the uptakes i n normal (144 mM) Na and low (18 mM) Na are compared.  The tendency f o r exchange to be s l i g h t l y increased i n high-Na  incubation media i s not s i g n i f i c a n t .  The tissues were l o s i n g K i n  both media, but appeared, on the basis of the 4 hour incubation v a l ues, to be l o s i n g i t more r a p i d l y i n the 18 mM Na  solution.  unknown i n t e r a c t i o n between K leaving the muscle and the K# the ECS attempting  to enter and exchange with the  The from  might a f f e c t  the net uptake of K#, and a l t e r the slope of the uptake curve; K# uptake appears to be reduced i n the 18 mM Na be s l i g h t l y r a i s e d i n the 288 mM s o l u t i o n ;  g  the  s o l u t i o n , and t o  the reduction may have  been due to this i n t e r a c t i o n , while the gain i n 288 mM medium could have been influenced by the large r i s e i n Na  i  observed even a f t e r  2 hours incubation i n t h i s f l u i d . (c)  E f f e c t s of varying K  and Na e e A few experiments were performed using combinations of  62  F i g . VjU  Uptake Altered  40  of N a  K  4  2  by  Mouse  Incubation  e  Muscle Media.  r  18  mM  Na  288  mM  Na  Dashed  e  -  o  — x e Line is f r o m Fig.vi X X X X  30  X X  >  aa>  0) _»  20  00 00  10  0  (mM  • min)  63  increased and decreased Na_ and K_ l e v e l s .  The number of experi-  ments was such that s t a t i s t i c a l evaluation would be inconclusive; only the general tendencies i l l u s t r a t e d i n F i g . IX w i l l be reported. (Normal K  e  «= 6 mM,  normal Na  e  = 144  mM.).  In 12 mM K-288 mM Na s o l u t i o n , the l i n e of points seems to be s l i g h t l y above the "normal" regression l i n e , and approximates the upper s o l i d l i n e i n F i g . V I I I . One experiment i n 2 mM K  Q  - 18 mM Na  Q  medium showed the  K exchange to be below the "normal" l i n e , and to approximate the lower l i n e i n F i g . V I I I . Incubation i n 12 mM K  e  - 18 mM Na  Q  solution r e s u l t e d i n  the p l o t t e d points l y i n g between the normal regression l i n e and the lower l i n e i n F i g . VIII. Chloride-free medium (sulphate replacing Cl) was used f o r two experiments.  With 6 mM K  the uptake l i n e was s l i g h t l y below  the normal regression l i n e , but with 12 mM K_ approximated i t . These r e s u l t s correspond to those reported e a r l i e r , that i s , s l i g h t a l t e r a t i o n s of K exchange s i g n i f i c a n t l y ;  d i d not seem to a f f e c t the rate of K e a l t e r i n g Na_ as well as K should have 6  6  produced a curve s i m i l a r to those seen previously f o r altered Na alone;  2 mM  Q  or 12 mM K_ should not a f f e c t the uptake appreciably.  The experiments with Cl-free media yielded an uptake curve resembling the nontial one, i n d i c a t i n g that chloride may not exert a s i g n i f i c a n t e f f e c t on K exchange i n muscle.  Harris and Sjodin (1961) stated  that K exchange was somewhat reduced i n Cl-free media;  "the reduc-  t i o n noted here corresponds to this finding, and may have been due to a decreased net uptake of K due to a lack of swelling by the muscle i n the C l - f r e e media.  64  Fig.JX.  Uptake  of K  Altered  Na  by and  e  x  12 m M  K  o  2 mM  K  e  12 m M  K  •  e  e  e  normal K  line  is  Incubation  e  288  mM  Na  -  18  mM  Na  -  18  mM  Na  free  normal  Muscle : Media.  -  Chloride —  Dashed  Mouse  e  e  e  medium uptake  slope  4 On X x  S 30-  /  1  y  > 3  X  cr  20  /  •  CO CO  A  x  .  • X  10/ xx  0  ~l— 10  o OO  (Ke • t ) £  —T— 20  30  (mM • min.)^  ~40  65 2.  Dystrophic Mouse S k e l e t a l Muscle (a)  E f f e c t s of Varying K Thirteen experiments were performed with muscles from  dystrophic animals. Muscles were incubated i n 2 mM, mM K  incubation media, and the K* uptake plotted;  Q  6 mM and 12 F i g . X shows  the experimental values calculated, the dystrophic regression l i n e ( s o l i d ) , and the normal tissue regression l i n e (dashed). Again, the points f a l l along a single l i n e when p l o t t e d against (K  i • t) ; 2  e  l. the slope of the l i n e i s 1 33 m. equiv./Kg/(mM • m i n ) 2  0  (S.E.* 0„06), as compared to 0,98 f o r the normal t i s s u e .  The slopes  of the l i n e s are s i g n i f i c a n t l y d i f f e r e n t (p <0 01). o  (b)  E f f e c t s of varying  Na  Q  A few experiments have been performed using altered Na media; F i g . XI;  the r e s u l t s f o r IB mM and 288 mM Na  Q  a  media are p l o t t e d i n  i t can be seen that K* uptake tends to be somewhat more  rapid i n the 288 mM medium, f o r the slope of the l i n e i s 1.80 m. equiv./Kg/(mM • m i n )  3  ( .E.~ 0 04) as compared with 1.33 f o r S  o  dystrophic tissue i n normal E solutions.  The difference between  t h i s slope and the normal slope i s s i g n i f i c a n t (p<0,01).  The  18 mM Na medium exhibits a d e f i n i t e l y slow K# uptake, f o r the slope of this l i n e i s 0,81 (S.E,- 0,04), considerably l e s s than the 6 mM dystrophic tissue l i n e ;  the difference between the two  l i n e s i s s i g n i f i c a n t (p <0,01). 3.  Summary of K  4 2  Results  Uptake of K* from incubation media with 2 to 12 mM  K  6  seemed to follow a unique course when the r e s u l t s were plotted as K-a- uptake against (K  • t ) , as described by Harris and Sjodin (1961). 2 !  Q  The uptake rate f o r normal muscle i n these solutions was m. equiv.K*/Kg/(mM • m i n ) ; 2  0.98  the comparable rate f o r dystrophic  66  F i g . X.:  Uptake Mouse  l  0  i  i  i  ( Ke -t ) '*  of  K  by  Muscle:  Dystrophic  Normal  i  1 0  ( mM • min.)  o o x  and  i 2  0  i  1 3  0  67  Fig._<l_  Uptake of  K  by  Altered N a  Dashed  e  Incubation  •  18  mM  Na  o  288  mM  Na  line  Dystrophic  is dystrophic  Mouse  Muscle.  Media.  e  e  regression  line  from  figure JT  0 H 0  1  1  1  10  20  30  ( Ke • t)^2  (mM • min.) ^2  68  muscle was 1.33,  one-third more than f o r normal t i s s u e .  In high-K  media, normal muscle accumulated K;  this  was r e f l e c t e d i n a very high uptake rate of 2.65 m. equiv./Kg/ A (mM  • min)  8  f o r 24 mM K  g  and 3.11 f o r 48 mM K  Q  incubation media.  Doubling Na l e d to an increase i n the rate of uptake i n both normal and dystrophic t i s s u e , from 0,98 m. equiv./Kg/ (mM  • min)  to 1,02 f o r the former, and from 1,33  Incuba  t i o n i n 18 mM Na  to 1.80 f o r the l a t t e r .  slowed uptake to 0.80 i n normal muscle, and t o 0,81  i n dystrophic muscle.  The e f f e c t noted i n 18 mM Na  medium may be  due to i n t e r a c t i o n between K being l o s t from the c e l l during tion (see Table VIII) and the K* entering the c e l l . Sjodin (I960) found that a l t e r i n g Na  e  incuba-  Harris and  (replacing any d e f i c i t with  sucrose) d i d not appreciably a f f e c t the rate of K* uptake;  a  s l i g h t tendency f o r enhanced or decreased uptake (as compared with normal uptake) might be due to cation i n t e r a c t i o n i n those cases where the muscles are gaining or l o s i n g K.  This f a s t e r rate of Re-  uptake i n dystrophic tissue i s not due to any net gain of KC1 by the c e l l s , but may be an abnormal permeability of the c e l l membrane, r e s u l t i n g i n a r a p i d uptake of K.  There has been a report of increas  ed permeability of dystrophic tissue to aldolase ( Z i e r l e r 1958); possibly the capacitative membrane or region of the dystrophic  cell  i s changed i n some way, enabling the K i n s o l u t i o n to e q u i l i b r a t e within t h i s r e s i s t i v e layer more r a p i d l y than usual, thus f a c i l i t a t ing i t s d i f f u s i o n i n t o the i n t e r i o r of the c e l l .  The mechanism by  which e q u i l i b r a t i o n within t h i s region occurs does not seem to have been affected by the dystrophic change, f o r the uptake curve i s s t i l l a s t r a i g h t l i n e , even though the rate of uptake i s increased.  SECTION IV DISCUSSION  A.  Tissue  Compartments  B.  Chemical Analyses:  C.  K  Presh and Incubated Muscle  42  1.  Uptake Studies 42 K Uptake of Normal Muscle (a)  "Normal" K  Solutions ©  (b)  The E f f e c t  of High K  Media ©  (c)  The E f f e c t  of Low K  Media ©  (d)  The E f f e c t of Na ©  42 2,  K  Uptake of Dystrophic Muscle  69  A. TISSUE COMPARTMENTS The ECS volume of muscle appears to depend on the size of the muscle, and to vary according to its condition and possibly its location in the body. This latter factor may be only a reflection of muscle size, for whole muscles vary in size depending on their location and function. On the basis of the inulin space results, one would predict that the extensor digitorum longus muscle, a naturally small muscle, would have a larger ECS value than the gastrocnemius, and this has been reported (McLennan 1956). The size dependence of ECS on muscle size observed in the strain 1 2 9 mice could have been a peculiarity of the strain;  since the dys-  trophics exhibited the dependency to a more marked degree than the normal animals, the normal animals might have carried a mild subclinical muscle defect. However, ECS assay on muscles from Swiss mice yielded results similar to those found for -the strain 1 2 9 mice; the same dependency of ECS to muscle size was noted, and i t was concluded that this relation would probably be found for most muscle tissue;  Tasker 6t al (1959) reported finding a similar  negative correlation between size and extracellular volume for toad skeletal muscle. This dependency may be a function of the development of the muscle; i t is believed that the number of fibres in a skeletal muscle is fixed at birth (Ham 1957) and that growth of a muscle in size is due to an increased volume of individual fibres, and not to the generation of nex^r fibres. Hines (1952) showed that there was a decrease in the relative vascular volume of a muscle with age, but the effect of this change on the ECS seems small compared to the differences observed in the present study. If the muscle  70  f i b r e s were to expand by growth, they would f i r s t encroach on the  ECS surrounding them, and a l i m i t e d growth of 5 to 10$  might occur without an increase i n the volume of the muscle; this f a c t o r may account f o r a large amount of the v a r i a t i o n found i n muscles of the same weight (see Figures I and I I ) .  The  expansion might i n i t i a l l y occur i n the myofibrils of the muscle f i b r e , f o r these structures could hypertrophy at the expense of the  sarcoplasm; only when the hypertrophy was such that the  sarcoplasm volume was minimal would the whole f i b r e enlarge. This m y o f i b r i l l a r (and subsequent f i b r e ) growth would be gradu a l l y occurring, as the muscle i s developing i n s i t u .  The  amount of tendon or tough fibrous tissue i n the muscle would probably a f f e c t the ECS, f o r the ECS of tendon i s much l a r g e r than that of muscle (Manery and Hastings 1939).  As the muscle increased i n  length, the r e l a t i v e amount of tendon might not increase i n proportion to muscle s i z e , and the t o t a l muscle ECS would be less i f compared to developmentally e a r l i e r ECS values.  These factors  would also be r e l a t e d to the "packing" of the muscle f i b r e s which seems to occur during development; the  the f i b r e s attempt to occupy  l e a s t volume compatible with maximum s trength and performance,  and only the minimum amount of connective tissue, blood vessels, et cetera i s present i n the muscle b e l l y . The muscle i n u l i n space appeared to be l e s s following incubation, f o r although the slopes of the regression l i n e s f o r the i n vAvo and i n j e c t i o n methods were e s s e n t i a l l y the same, the i n j e c t i o n ECS values tended to be greater than the incubation values f o r the  same size of muscle.  I t also appeared that the non-inulin space  increased during incubation;  the muscles swelled during incubation  71  and although a correction was  deducted f o r t h i s swelling, a  portion of the ECS seems to have been added to the non-inulin (non-ECS) space.  I t may  be that during incubation i n an a r t i -  f i c i a l medium, the muscle sarcolemma becomes permeable to i n u l i n , which penetrates  i n t o the sarcoplasm, and occupies the region  inside the sarcolemma that i s not occupied by m y o f i b r i l s .  Equi-  l i b r a t i o n of t h i s region (which apparently contains the sarcoplasmic reticulum) may  take several hours, r e s u l t i n g i n the slow  increase i n non-inulin space water previously noted. 1958)  reported a leakage of the enzyme aldolase (MW  normal r a t and mouse musclej intravenous  Z i e r l e r (1957,  147,000) from  Cotlove (1954) showed that prolonged  administration of i n u l i n or sucrose i n t o rats would  eventually l e a d to the muscle ECS indicated by these substances equalling the space occupied by chloride ion, an ion that i s largely extracellular.  These findings suggest that the muscle  membrane may be permeable to i n u l i n , although this does not seem to be the case f o r incubation up to 4 hours* duration (McLennan 1956). Muscles from dystrophic mice seem to have abnormally permeable membranes and Z i e r l e r (1958) observed a noticeably greater leakage of aldolase from dystrophic t i s s u e .  I t i s possible that the abnor-  mally large i n u l i n space values f o r dystrophic tissue reported i n t h i s thesis are due to a penetration of the degenerating muscle membrane by the i n u l i n t r a c e r .  The ECS  of a small normal muscle  would be f a i r l y high, but small dystrophic muscles have i n u l i n spaces almost twice as large as t h e i r normal counterparts.  E i t h e r the  sarcolemma of the f i b r e s has broken down, leaving the protoplasm and myofibrils exposed to the e x t r a c e l l u l a r f l u i d , or the membrane permeability has been altered s u f f i c i e n t l y to permit entry of i n u l i n  72  into the f i b r e . of c a . 90$;  The smallest dystrophic muscles had i n u l i n spaces  this would indicate few functional muscle f i b r e s i f  the i n u l i n permeable ones were completely degenerated, yet v i s i b l e twitches could e a s i l y be observed upon e l e c t r i c a l stimulation of the muscle, and resting and action potentials could be recorded without d i f f i c u l t y (Burr and McLennan, unpublished observation). Thus i t seems l i k e l y that the change i n the dystrophic tissue i s i n the nature of an a l t e r a t i o n i n permeability of the f i b r e sarcolemmaj  t h i s may be followed i n terminal degenerative stages of  •toe f i b r e by complete degeneration of the membrane, e f f e c t i v e l y adding the volume of the c e l l to the extra c e l l u l a r space. Percentage water loss averaged 76$ for normal muscle and 77$ f o r dystrophic muscle (see Table V I ) ; not s i g n i f i c a n t l y d i f f e r e n t .  these two values were  The normal muscle water l o s s was the  same whether the muscles were fresh, incubated i n Solution 1, or incubated i n Solution 2.  I t seems that incubation d i d not a f f e c t  the t o t a l muscle water content;  however, when the non-inulin space  water was calculated f o r these muscles (see Table V I I ) , i t was found (for normal muscle) to r i s e from 47$ of the fresh weight f o r nonincubated t i s s u e , to 58$ a f t e r 2 hours incubation, and t o 65$ a f t e r 4 hours incubation.  One must conclude that the non-inulin space  water increases at the expense of the e x t r a c e l l u l a r water.  The  t o t a l muscle water was also constant i n muscles of varying s i z e ; i f the ECS progressively decreased with increasing s i z e , the noni n u l i n space water must have progressively increased along with the muscle s i z e . The ECS r e s u l t s were obtained from a large number of muscles;  the dry weight and calculated non-inulin space, water  73  figures were from a small number of muscles;  one f e e l s that a  more thorough investigation of the problem i s necessary before any further conclusions regarding the v a r i a b i l i t y of the noni n u l i n space water with muscle weight or incubation time can be j u s t i f i a b l y made.  B.  CHEMICAL ANALYSES:  FRESH AND INCUBATED MUSCLE  A summary of the K and Na analyses i s found i n Table V I I I (page 51);  approximately 375 analyses f o r K and Na were  done on both types of muscle.  The expected d i s t r i b u t i o n of ions  was observed i n f r e s h normal muscle, f o r the Na^ low;  was high and the  the figures of 89.9 m. equiv./Kg and 30.0 m. equiv./Kg  respectively compare with 100 m. equiv./Kg and 46 m. equiv./Kg as reported by Baker, Blahd and Hart (1958), and 121.0 m. equiv./Kg and 36.5 m. equiv./Kg as reported by Williams et a l (1957).  These  reported values have not been corrected f o r ECS ion content, and thus are not s t r i c t l y comparable t o the figures from Table V I I I . The ECS correction f o r Na i s e s p e c i a l l y important, because the Na  e  i s a large value.  Conway (1950) found that i f one assumed the  i n t r a c e l l u l a r sodium to be i n two f r a c t i o n s , one bound f i r m l y to c e l l u l a r constituents, and the other free to exchange r a p i d l y with the e x t r a c e l l u l a r f l u i d .  He assumed further that the "true" i n t r a -  c e l l u l a r Na was the bound portion and was only 2-3 m. equiv./Kg instead of 15-20 m. equiv./Kg as usually reported. His data were based on k i n e t i c studies of Na* e f f l u x from muscle f o r a discontinu i t y i n the e f f l u x curve indicated two discreet fractions of ion that d i f f e r e d i n the time taken to leave the muscle f i b r e .  The  correction made i n this thesis was the subtraction of the appropriate ion content of the 4 hour i n u l i n space water, with no attempt having  74  been made to correct for the "fast f r a c t i o n " of non-inulin space Na, Muscle tissue from dystrophic mice was found to contain 68.2 m. equiv.K/Kg and 58.2 m. equiv. Na/Kg (Table V I I I ) .  These  higher Na. and lower K. l e v e l s were s i m i l a r to that observed by Baker et a l (1958), who reported that K  ±  Na^ 66,0 m. equiv./Kg.  was  76 m. equiv./Kg and  The percentage r i s e i n Na^ or f a l l i n K^  i s the same i n both these instances.  Williams et a l (1957) found  the dystrophic Kj_ to drop to 56.7 m. equiv./Kg, nearly h a l f the normal figure, and Na^ to r i s e 60% to 60,0 m. equiv./Kg.  Young  et a l (1959) found similar changes i n ion l e v e l s i n t h e i r normal and dystrophic analyses, with dystrophic K^ 18% greater than the normal muscle values.  Analyses  patients with muscular dystrophy 1955)  of muscle biopsies from human (Horyath, Berg, Curamings and Shy  showed Na l e v e l s to be increased and K l e v e l s to be  decreased  as compared to cation l e v e l s i n biopsy samples from normal i n d i v i duals.  Blaxter (1952) reported s i m i l a r a l t e r a t i o n of e l e c t r o l y t e s  i n muscle tissue from dystrophic calves. I t i s apparent that the Na and K differences between normal and dystrophic tissue are not l i m i t e d to mouse muscle, but are rather a manifestation of the dystrophic change i n general.  The  abnormal permeability of the muscle membrane, noted above f o r inul i n , might i t s e l f account f o r the lowered Kj_ i n dystrophic t i s s u e , either d i r e c t l y or by leakage of i n t r a c e l l u l a r p r o t e i n .  Since a  large part of the K^ i s held i n a Gibbs-Donnan equilibrium, a lower i n t r a c e l l u l a r protein would be expected to reduce this e f f e c t , and thus to make K  i  more nearly equal to K . Q  There i s the p o s s i b i l i t y  also that t h i s lowered protein content could be due to an abnormal c e l l u l a r metabolism.  Thus, f o r example, Milman (1954) reported  75  t h a t glycogen  s y n t h e s i s was abnormal i n d y s t r o p h i c  animals;  Rosenkrantz and L a f e r t e (i960) observed t h a t dehydrogenase a c t i v i t y i n d y s t r o p h i c mouse muscle was g r e a t e r than normal, and Weinstock, E p s t e i n , and M i l h o r a t (1958) found t h a t cytochrome oxidase  a c t i v i t y was i n c r e a s e d i n d y s t r o p h i c mouse t i s s u e .  enzyme systems appear t o be a f f e c t e d as w e l l ; S c h a p i r a and Gey ( I 9 6 0 ) , u s i n g C  1 4  Kruh,  Dreyfus,  - l a b e l l e d glycine, reported a  f a s t e r p r o t e i n t u r n o v e r r a t e f o r d y s t r o p h i c mouse Zymaris, S a i f e r , and V o l k  Other  tissue.  (I960) f o u n d a f a s t e r t u r n o v e r  rate  o f a c i d - s o l u b l e n u c l e o t i d e s i n d y s t r o p h i c muscle and Rabinowitz (I960) r e p o r t e d more r a p i d l i p o g e n e s i s i n d y s t r o p h i c mice than i n t h e i r normal l i t t e r m a t e s .  From t h e p u r e l y i o n i c  analyses  performed on mouse muscle f o r t h i s t h e s i s , i t i s n o t p o s s i b l e t o support  e i t h e r the i n c r e a s e d membrane p e r m e a b i l i t y  o f the a l t e r e d c e l l u l a r metabolism h y p o t h e s i s o f t h e abnormal Naj_ and The  d i f f i c u l t t o d i f f e r e n t i a t e adequately  and Na g a i n ;  as b e i n g t h e c a u s e  l e v e l s f o u n d i n d y s t r o p h i c mouse muscle.  i n u l i n space d a t a seems t o s u p p o r t  Incubation  hypothesis  the former view, b u t i t i s between the two c o n d i t i o n s .  o f muscle t i s s u e g e n e r a l l y r e s u l t e d i n K l o s s  even t h e 12 mM K s o l u t i o n d i d n o t m a i n t a i n  s t a n t d u r i n g 4 hours o f i n c u b a t i o n .  con-  I t i s b e l i e v e d ( H a r r i s i960)  t h a t c o r r e c t i n c u b a t i o n f l u i d i o n i c c o n c e n t r a t i o n and c o r r e c t i s o t o n i c i t y a r e n o t t h e o n l y c o n d i t i o n s t h a t must be p r o v i d e d t o maintain  cellular stability i n vitro;  adequate oxygen  supply,  s u i t a b l e temperature, and adequate s u b s t r a t e s f o r metabolism are a l s o needed.  These requirements a r e e s p e c i a l l y important i n  mammalian muscle, iiihich has a h i g h r a t e o f c e l l u l a r metabolism, and which r e l i e s h e a v i l y on continuous energy s u p p l y .  o x i d a t i v e metabolism f o r i t s  Creese, D ' S i l v a , and Northover (1958), o b s e r v e d  76  that the addition of colloids to the incubation media aided K and Na retention in excised mammalian muscles; recently Creese and Northover (1961) reported that crude human serum globulin would maintain rat diaphragm cations at the normal level during 2 hours of incubation. Baetjer (1935) showed that a 20$ reduction of blood supply to muscle in situ would cause a marked K loss and Na gain. The solutions that were used for incubation media in the present study were constantly oxygenated by a 95$ 0 - 5$ C0 gas mixture. 2  2  The C0 that dissolved in the medium formed a buffer pair with 2  HCO^,  and maintained the pH at ca. 7.4;  the bicarbonate medium  was found to maintain cellular electrolytes better than a phosphatebuffered medium (Solution 6). Creese (i960) showed that rat diaphragms lost K rapidly i f incubated in hypoxic media; Hill (1928) showed that thick tissues (more than 1.95 mm.)  would become anoxic  during incubation because of the limitation of the rate of oxygen diffusion.  If any anoxia occurred in the incubated muscles in the  present study, the rapid K loss and Na gain from the anoxic fibres could not be differentiated from a slight K loss in a l l fibres; however, for -the purpose of measuring  uptake, the media used  appeared to be adequate, for the intracellular K was stable for 2 hours in the normal and 12 mM K incubated for 4 hours lost K;  e  solutions. A l l the muscles  those incubated in 12 mM K did not  lose as much as tissue in 6 mM K. Muscles in the K-free solution lost the most K, although in the low Na medium, much K as well as Na was lost.  Choline had been used to replace the deficit of Na  in the latter solution; one would expect a loss of Na to occur under these conditions, accompanied by an initial gain of K to  77  compensate f o r early Na l o s s ;  the continued Na l o s s may have  altered the metabolism or the ionic balance of the c e l l to the extent that there was interference with K binding.  Renkin (1961)  showed that choline entered frog s a r t o r i i a t a rate comparable to that of Na ion, but was l o s t more slowly;  Keynes and Swan (1959)  observed the same e f f e c t when L i was substituted f o r Na, but L i can substitute f o r Na i n maintenance of the r e s t i n g p o t e n t i a l , whereas choline cannot.  The replacement of Na by choline i n t r a -  c e l l u l a r l y leads to a drop i n the r e s t i n g p o t e n t i a l (Keynes and Swan 1959);  t h i s could be one cause of the excessive K loss noted  i n the choline incubation medium.  I t i s not known whether mammalian  muscle f i b r e s are permeable to choline but frog muscle i s apparently permeable to both L i and choline, and mammalian muscle may be expected t o show s i m i l a r c h a r a c t e r i s t i c s .  C.  K  4 2  UPTAKE "Exchange" of K has been defined on page 56. Measurement  of the rate of exchange by following K  4 2  uptake, w i l l only give a  true picture i f the K^ i s constant, or i f a known correction f o r K^ r i s e or f a l l can be c a l c u l a t e d . hypothesis,  The Harris  permeation-diffusion  described b r i e f l y on page 18, i s used as a model to a i d  the i n t e r p r e t a t i o n of the k i n e t i c data which have been presented i n this thesis. cesses;  Under t h i s model exchange of an i o n involves two pro-  f i r s t , an exchange with ions adsorbed near the c e l l surface,  and second, the d i f f u s i o n of ions inwards from the adsorbed layer at the same time as an outward d i f f u s i o n of an equal number of i n t e r n a l ions takes place.  The ions moving outward w i l l compete f o r  s i t e s i n the adsorbed region with those ions entering the region from the e x t r a c e l l u l a r space.  Thus the adsorbed region can act as  78  an i o n exchanger, with chemically d i f f e r e n t ions such as K, Na, and Kb being interchanged, or ions o f the same species being exchanged, such as the exchange occurring between l a b e l l e d K ions from the ECS and unlabelled K ions from the c e l l .  The e q u i l i b r a - :  t i o n of the adsorbed layer i s the r a t e c o n t r o l l i n g process. I f the adsorption region of the c e l l accumulates ions at a rate that i s proportional to the applied concentration, i t can be shown (Harris and Sjodin 1961) that when the amount of exchange i  i s plotted against (applied concentration x t i m e ) , a l l experimental 2  r e s u l t s w i l l f a l l along a single curve (at constant 1.  K  4 2  (a)  temperature).  Uptake of Normal Muscle "Normal" K  e  Solutions  McLennan (1955) working with mammalian muscle, and Harris and Steinbach (1956) using frog muscle, could not f i t t h e i r k i n e t i c data to a single exponential function of time, as i s required by the " c l a s s i c a l " concept of a t h i n r e s i s t i v e membrane separating two i o n i c r e s e r v o i r s . The r e s u l t s presented e a r l i e r i n this thesis do not f i t this single exponential curve e i t h e r , but when plotted i n accordance with the Harris hypothesis, concur with h i s results f o r K-K# exchange i n frog muscle.  I t i s u s e f u l to consider the  muscle c e l l as possessing "regions" that have a definite K capacity, and a low d i f f u s i v i t y f o r ions e q u i l i b r a t i n g within the c e l l .  Ion  movement through the adsorbed ion region w i l l involve a series of exponential terms to describe i t , f o r each exchange of outer layer tracer ions f o r non-tracer ions of an inner region w i l l delay the e q u i l i b r a t i o n of the outer adsorption l a y e r .  There can be i n t e r -  ference to d i f f u s i o n i n t h i s K region, and a c e l l l o s i n g K would maintain a low K# concentration within i t s outer region, and impede  79  e q u i l i b r a t i o n with the r e s t of the c e l l .  Interaction between  species of ions could also occur i n this region, and influence the f l u x by competition f o r adsorption s i t e s . In F i g . VI, the uptake of K^  follows a single s t r a i g h t  -2  l i n e (slope 0.98 m. equiv./Kg(mM • min) ) f o r muscles incubated i n 3  2 mM,  6 mM,  and 12 mM K  ffl  Burr and McLennan 1961)  solutions.  I t was determined (page 51,  that i n these solutions the  constant f o r the f i r s t two hours of incubation;  and  remained  measurement of Re-  uptake would therefore give a true measure of the normal exchange process, without the necessity of correcting f o r any net K gain or loss.  The uptake curve i s l i n e a r f o r most of i t s length, and even  at the beginning, during the i n i t i a l e q u i l i b r a t i o n of the ECS the bathing s o l u t i o n , which i s unquestionably  with  a d i f f u s i o n process,  there i s no difference between the K uptake curves i n the various media.  The K  4 2  K  e  uptake c h a r a c t e r i s t i c s also preclude the necessity  of invoking non-exchangeable f r a c t i o n s of muscle K, f o r the increase in K  g  does not lead to a greater degree of K being available f o r  exchange or to a faster rate of exchange.  This i s i n c o n t r a d i s t i h c —  t i o n to the " c l a s s i c a l " i n t e r p r e t a t i o n of potassium kinetics (see Introduction pages 16 to 18  f o r a b r i e f account of the " c l a s s i c a l "  view). A similar curve would be expected i f K d i f f u s i o n i n the ECS were the r a t e - l i m i t i n g step.  The timed 1.0 minute wash given  to the muscles a f t e r removal from the K* soaking solution reduced the e x t r a c e l l u l a r K* by 50%;  this i s presumably regained i n the  f i r s t minute following immersion i n the incubation f l u i d , for Carey and Conway (1954) observed that the wash does not a f f e c t the Reuptake k i n e t i c s .  Harris (1957) thought that d i f f u s i o n i n the  would be slower than i n t r a c e l l u l a r d i f f u s i o n ;  ECS  a comparison of h i s  80  results for intracellular diffusivity  (1954) and those of McLennan  (1955) l e a d on t o the same c o n c l u s i o n .  Hodgkin and Horowicz (1959)  s t u d i e d K uptake o f s i n g l e muscle f i b r e s and s i m i l a r t o those f o u n d f o r whole muscle.  observed uptake c u r v e s  H a r r i s and Burn (1949) c a l -  c u l a t e d t h a t f r o g s a r t o r i u s would take o n l y 15$ l o n g e r than a s i n g l e f i b r e ; d i f f u s i v i t y was  H a r r i s and  to equilibrate  S j o d i n (1961) showed t h a t the  even c l o s e r t o the f r e e s o l u t i o n v a l u e .  The  Na  latter  a u t h o r s a l s o compared d a t a from Hodgkin and Horowicz (1959) w i t h t h e i r f i g u r e s f o r whole muscle K exchange and c o n c l u d e d t h a t over i n the l a r g e r s i n g l e f i b r e s i s about the exchange i n a whole muscle.  turn-  same as the mean  I t appears t h a t the s l i g h t h i n d r a n c e  imposed by e x t r a c e l l u l a r d i f f u s i o n i s compensated by  the  presence  of the s m a l l e r f i b r e s so as t o make the r e s u l t s i m i l a r t o t h a t holding (b)  for a large single f i b r e ,  The  E f f e c t o f High K Incubation  g a i n o f K.  (Boyle  and  Media  of muscles i n h i g h KC1 media l e a d s t o a Conway 1941).  The  net  g a i n occurs f a i r l y r a p i d l y  and depends on the amount o f K i n the medium.  A s w e l l i n g of  the  muscle i s a s s o c i a t e d w i t h the g a i n from media c o n t a i n i n g c h l o r i d e , but  t h i s i s e l i m i n a t e d i f n o n - p e n e t r a t i n g m e t h y l - s u l p h a t e i s used  instead.  H a r r i s and S j o d i n  (1961) found t h a t K* uptake was  r a p i d i n C l media than m e t h y l s u l p h a t e ;  the C l may  e n t r y by accompanying i t i n t o the c e l l ,  and no e l e c t r i c a l  is  more  facilitate  K  imbalance  incurred. F i g . V I I shows the r e s u l t s o f i n c u b a t i o n i n 24 mM  48 mM  K  e  media.  The  slopes  and  o f the l i n e s are markedly s t e e p e r  than  the normal exchange l i n e , f o r n e t g a i n o f K (as w e l l as exchange) has  occurBed.  The  n e t g a i n and the exchange can be  separated  only  i f n e t g a i n were known f o r each K* uptake p o i n t . Table X showed  81  the d i f f e r e n c e s at the end of 2 hours soaking;  the net g a i n  42 K values as estimated by K data and non-radioactive i n c u b a t i o n data are s i m i l a r . of  This would i n d i c a t e that the (K • t ) method e a  plotting sensitively reflects cellular ion levels;  upon i n -  s p e c t i o n of an uptake curve, and comparison with a normal one  curve,  could a c c u r a t e l y p r e d i c t whether net K g a i n had occurred or  not© (c)  The e f f e c t of Low K  Media G  When muscle i s exposed t o a K-free medium, i t l o s e s cellular K rapidly.  The uptake curve, i n t h i s case, would not  be as steep as the normal curve, f o r K i s being l o s t , a.nd the outward i o n i c movement w i l l tend to r e t a r d the r a t e of a d s o r p t i o n of  K* onto the c e l l .  Experiments i n low K  media are best done on e  K-loaded c e l l s .  H a r r i s and S j o d i n ( l 9 6 l ) r e p o r t e d that the K*  uptake from loaded muscles was i n i t i a l l y l e s s than normal, and t h e i r graph of K* uptake showed a concavity during the f i r s t hour of  soaking, but the remainder of the curve had e s s e n t i a l l y the  same slope as a normal uptake curve. concavity at the beginning excess K had l e f t  They a t t r i b u t e d the long  to the K l o s s o c c u r r i n g ;  when the  the muscle, the exchange curve approached the  usual slope. (d)  The E f f e c t of Na e The  movement of muscle K appears to be a f f e c t e d by  a l t e r a t i o n s i n sodium c o n c e n t r a t i o n of i n c u b a t i o n media (Steinbach 1950). H a r r i s and S j o d i n ( l 9 6 l ) found that K uptake was l e s s when Na was lowered, but considered that t h i s was due to a l o s s e of K from the t i s s u e . Keynes and Swan (1959) showed that there  82  i s not complete independence of the Na and K f l u x e s , f o r alterations i n K  w i l l a f f e c t Na f l u x e s (see a l s o Keynes 1954)• ©  McLennan (1957) noted ( i n r a t muscle) that i n c u b a t i o n i n low-Na e media r e s u l t e d i n l e s s K uptake as compared to normal Na tions.  solu-  The p a r a l l e l i s m observed between Na and K movements  suggests that t h e i r movements are l i n k e d i n some way. movement i s not always a r i g i d 1:1 exchange versa.  g  But the  of Na f o r K or v i c e  McLennan (l957) found the r a t i o to be c l o s e r to 2:1  (Na:K);  however, Hodgkin and Keynes (1955) found the r a t i o i n  squid axon to be  1:1. 42  The r e s u l t s of K in P i g . VIII.  uptake i n a l t e r e d Na media a r e shown  The r e g r e s s i o n l i n e of the 288 mM Na  medium p o i n t s ©  i s not s i g n i f i c a n t l y d i f f e r e n t from the normal l i n e , but the 18 mM Na l i n e i s - s i g n i f i c a n t at the 1% l e v e l . In 288 mM Na , the e e muscles were r a p i d l y g a i n i n g Na, but were a l s o l o s i n g some K. This doubling  of the i n c u b a t i o n medium Na  uptake by mouse muscle;  d i d not a f f e c t K  the same phenomenon was observed i n  f r o g muscle by H a r r i s and S j o d i n The 18 mM Na  g  (l96l).  s o l u t i o n caused a l a r g e f a l l i n i n t r a c e l ©  l u l a r K.  The slope  of the uptake curve i s l e s s than the normal  curve, and t h i s can be a t t r i b u t e d to the i n t e r a c t i o n between the K and Na being l o s t from the muscle, and the K* attempting to enter.  The rate of buildup  of K* on the surface l a y e r , or the  exchange from t h i s r e g i o n to the c e l l i n t e r i o r , may be hampered by the large amount of c a t i o n l e a v i n g the c e l l .  Again, t h i s  concurs with the r e s u l t s reported by H a r r i s and S j o d i n  (l96l).  83 2.  K  42  Uptake of Dystrophic  Muscle  42 Uptake of K t i o n i n 2 mM, The  6 mM,  by dystrophic  and 12 mM  K  t i s s u e during  incuba-  media i s p l o t t e d i n F i g . X.  slope of the l i n e i s much g r e a t e r f o r the dystrophic t i s s u e 42  than the normal;  e v i d e n t l y the entry of K  f i b r e s occurs more r a p i d l y than u s u a l .  i n t o the muscle  T h i s may  be a r e f l e c t i o n  of an abnormal p e r m e a b i l i t y of the muscle membrane, as by Burr and McLennan ( i 9 6 0 ) .  postulated  With the H a r r i s model of the muscle  membrane, the i n c r e a s e d r a t e of exchange appears to be due of two  things:  to  one  a f a c i l i t a t e d i n c r e a s e i n the e q u i l i b r a t i o n of  the i o n exchange l a y e r , or an increase i n the K d i f f u s i v i t y  of  the i n t e r n a l c e l l u l a r compartment.  be  These p o s s i b i l i t i e s may  r e l a t e d to the higher r a t e of metabolism i n dystrophic an e a r l y dystrophic change i n the sarcoplasmic  t i s s u e , to  reticulum  (Grant  I960) causing a decrease i n the amount of m a t e r i a l with i o n exchanger p r o p e r t i e s , or to an a l t e r a t i o n i n the s t r u c t u r e or f u n c t i o n i n g of the i o n exchange l a y e r .  H a r r i s and S j o d i n (l961a)  took e l e c t r o n photomicrographs of normal and incubated  f r o g muscle,  and i n f e r r e d from the r e s u l t s that p a r t of the ion-exchange compartment might be  s i t u a t e d i n the cytoplasmic  change o c c u r r i n g during  reticulum.  The  earliest  the development of muscular dystrophy i n  mice has been found by Grant (i960) to be v a c u o l a t i o n of the culum c l o s e to the I band;  the v a c u o l a t i o n progresses  i n v o l v e s the e n t i r e r e t i c u l u m .  T h i s may  reti-  until i t  e f f e c t i v e l y reduce the  s i z e of the i o n exchange compartment, and l e a d to a more r a p i d e q u i l i b r a t i o n of the muscle i n t e r i o r with the e x t e r n a l environment. There does not  seem to be a q u a l i t a t i v e change i n the  mode of operation of the i o n a d s o r p t i o n  region, f o r the uptake  84  curves from dystrophic muscle show no i n d i c a t i o n s of d i f f e r i n g from those f o r normal muscle, except that the slope i s steeper. The  e f f e c t s of a l t e r i n g the Na  g  of the i n c u b a t i o n  media f o r dystrophic muscles were e s s e n t i a l l y the same as noted f o r normal muscles, except the e f f e c t s were more marked* The high ECS to e x p l a i n these  values found e a r l i e r are not  d y s t r o p h i c f i n d i n g s , u n l e s s the membrane permea-  b i l i t y i s grossly different.  The  d i f f e r e n c e s i n metabolism of  d y s t r o p h i c t i s s u e mentioned p r e v i o u s l y may i n i n t e r p r e t i n g these p h i c muscles.  sufficient  be of some importance  exaggerated phenomena found i n the d y s t r o -  I f the i o n exchange compartment were smaller than  u s u a l , i t might not f u n c t i o n as e f f e c t i v e l y as i t does i n the normal muscle;  there could be a l e a k of K i o n s from the  of the muscle i n t o the ECS, r e p o r t e d i n Table V I I I .  thus reducing  the  to the  interior value  This could a l s o account f o r the minor  d i f f e r e n c e s i n r e s t i n g p o t e n t i a l s observed by Conrad and  Glaser  (1959) and McLennan ( l 9 6 l ) . There i s no r e l i a b l e method of t e s t i n g the  disability  of the dystrophic mice, e i t h e r p h y s i o l o g i c a l l y or b i o c h e m i c a l l y . I f a s u i t a b l e method f o r estimating the degree of a f f l i c t i o n would be devised, i t might be e a s i e r to i n t e r p r e t the observations r e garding  the d y s t r o p h i c muscle;  change were known, or i f one  i f the nature  of the  dystrophic  could describe more a c c u r a t e l y the  p r o p e r t i e s of the i o n exchange compartment, phenomena concerned with uptake of ions by mouse t i s s u e , both normal and c o u l d be more a c c u r a t e l y and p r o p e r l y  described.  dystrophic,  SECTION V  CONCLUSIONS AND SUMMARY  A.  Conclusions  B.  Summary  85  CONCLUSIONS 1.  There i s a negative c o r r e l a t i o n between e x t r a c e l l u l a r  volume and muscle s i z e i n s t r a i n 129 mice.  Mice a f f l i c t e d  with h e r e d i t a r y muscular dystrophy show the dependency more s t r o n g l y than normal mice. to  The i n u l i n molecule may be able  penetrate the membrane.  2.  Normal mouse muscle ( f r e s h or incubated) l o s e s 76% of  i t s water during h e a t i n g and d e s s i c a t i o n . the  During the i n c u b a t i o n ,  n o n - i n u l i n space water appears to increase from 47% to 65%  of  the muscle volume.  This l a t t e r observation needs f u r t h e r  study before c o n c l u s i o n s can be drawn. 3.  Fresh normal mouse muscle contains 3 0 . 0 m. equiv. Na^/  Kg t i s s u e , and 89.9 m. equiv. K ^ K g .  Dystrophic muscle has  58.2 m. equiv. Na^/Kg and 68.2 m. equiv. K^/Kg. ion  concentrations are stable during the f i r s t  bation i n a r t i f i c i a l Na  i  r i s i n g and  media;  falling.  Intracellular  2 hours of i n c u -  prolonged i n c u b a t i o n r e s u l t s i n V a r y i n g the concentrations of Na and  K i n the i n c u b a t i o n media i n f l u e n c e s the changes i n i n t r a c e l l u l a r Na and K.  The changes appear to occur more r a p i d l y i n dystrophic  tissue. 42 4.  K  i s taken up by normal mouse muscle i n much the  same way as i t i s i n f r o g muscle ( H a r r i s 1957, H a r r i s and S j o d i n 1961). for  The uptake appears to be an exchange of K* i n s o l u t i o n  u n l a b e l l e d K i n the muscle f i b r e s , and occurs at the rate of  0.98 m. equiv. K*/Kg t i s s u e per ( K  • t ) unit. 3  g  The process  appears to be governed by the laws of d i f f u s i o n , and the r a t e of  86  uptake to be independent of the K t i o n i n the medium.  and of the time of incuba-  Doubling the N a  take r a t e , but reducing Na  g  to 18 mM  g  does not a f f e c t  the up-  slows the uptake n o t i c e a b l y .  Dystrophic mouse muscle exchanges K* f o r K f a s t e r than normal muscle;  the r a t e i s 1.33  m. equiv. K*/Kg per (K  unit.  Varying the K  affect  the rate of uptake, but a l t e r i n g the K a  • t)^  or the l e n g t h of i n c u b a t i o n does not  g  g  has more of an  e f f e c t on the d y s t r o p h i c muscle than on the normal muscle. 5.  K exchange  i n muscle may be mediated by a compartment  i n the f i b r e s that has ion-exchange p r o p e r t i e s .  This  compart-  ment would r e s u l t i n the K* uptake curve f o l l o w i n g the general p a t t e r n of a d i f f u s i o n curve, with the rate being independent of the time of exposure.  The d y s t r o p h i c change appears to be  e i t h e r a change i n membrane p e r m e a b i l i t y or a r e d u c t i o n of the ion-exchange compartment.  These two p o s s i b i l i t i e s cannot be  adequately d i f f e r e n t i a t e d by the methods used i n t h i s B.  thesis.  SUMMARY 1.  Muscle from 129 s t r a i n mice was assayed f o r ECS f o l l o w -  i n g i n vivo a d m i n i s t r a t i o n of i n u l i n or soaking of the muscle i n an i n u l i n s o l u t i o n .  The ECS was found to decrease with  i n c r e a s i n g muscle s i z e , and to be l i n e a r .  The r e s u l t s obtained  on 129 s t r a i n mice were confirmed on Swiss a l b i n o mice, so the tendency i s not a p e c u l i a r i t y of the 129 s t r a i n . The ECS of muscle from dystrophic s t r a i n 129 mice shows a g r e a t e r dependency animals.  on muscle s i z e than muscle from normal  The p o s s i b i l i t y of an i n c r e a s e d p e r m e a b i l i t y of the  87  muscle i s suggested. 2.  Muscles were excised from 129 s t r a i n mice and d r i e d .  Normal muscle l o s t 16% of the f r e s h weight, and dystrophic muscle 1 1 % of the f r e s h weight, e i t h e r f r e s h or f o l l o w i n g i n cubation i n an a r t i f i c i a l  medium.  A tendency f o r the n o n - i n u l i n  space water to i n c r e a s e during i n c u b a t i o n was noted. 3.  Na and K analyses were done on normal and dystrophic  tissue  ( f r e s h and a f t e r i n c u b a t i o n ) .  mouse muscle were N a m. equiv./Kg.  i  Results f o r f r e s h normal  = 30.0 m. equiv./Kg t i s s u e and  F r e s h dystrophic t i s s u e had N a  i  = 89.9  = 58.2 m.  equiv./  Kg and K^ = 68.2 m. equiv./Kg. Incubation  of excised muscles i n a modified Locke's  s o l u t i o n r e s u l t e d i n a r i s e i n Na^ and a f a l l i n K^ a f t e r 4 hours' soaking. ion  During the f i r s t  l e v e l s were comparable  2 hours of i n c u b a t i o n , the  to f r e s h t i s s u e v a l u e s .  42 The r a d i o a c t i v e isotope K was used to f o l l o w the K  4.  exchange of muscles during i n c u b a t i o n i n v a r i a n t s of Locke's 42 solution.  K  uptake followed an unique course when p l o t t e d as  d e s c r i b e d by H a r r i s and S j o d i n ( l 9 6 l ) .  The r e s u l t s are i n t e r -  p r e t e d i n the l i g h t of the H a r r i s d i f f u s i o n - p e r m e a t i o n  hypothesis.  The effec-t of v a r y i n g the Na and K concentrations of the soaking 42 s o l u t i o n on the uptake of K  i s considered i n some d e t a i l .  R e s u l t s of uptake s t u d i e s on d y s t r o p h i c mouse muscle are presented  and the d i f f e r e n c e s between normal and dystrophic t i s s u e  discussed.  SECTION V I  BIBLIOGRAPHY  88  Adams, R.D., Denny-Brown, D., and Pearson, CM. ( l 9 5 4 ) . Diseases of muscle; a study i n pathology. Paul B. Hoeber, New York. B a e t j e r , A.M. (1935). The d i f f u s i o n of potassium from r e s t i n g s k e l e t a l muscles f o l l o w i n g a r e d u c t i o n i n the blood supply. Amer. J . P h y s i o l . 112, 1 3 9 . Baker, N., Blahd, W.H., Hart, P. 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P h y s i o l . 125,, 232. Conrad, J.T., & Glaser, G.H. (1959). A study of membrane r e s t i n g and a c t i o n p o t e n t i a l s of dystrophic mammalian muscle. Trans. Am. N u r . Assoc. 1959, 170. e  Conway, E . J . (1950). C a l c u l a t i o n of the i d i o m o l a r value and i t s e l e c t r o s t a t i c equivalent i n normal mammalian s k e l e t a l muscle. I r i s h J . Med. S c i . S e r i e s 6. 295,, 216. Conway, E . J . (1957). Nature and s i g n i f i c a n c e of concentration r e l a t i o n s of potassium and sodium ions i n s k e l e t a l muscle. P h y s i o l . Rev. _1» 84. Conway, E . J . & Carey, M.J. (1956). A c t i v e s e c r e t i o n of sodium from i s o l a t e d N a - r i c h s k e l e t a l muscle. Nature, London 178, 644. Cosmos, E . & H a r r i s , E . J . ( l 9 6 l ) . In v i t r o s t u d i e s of the g a i n and exchange of calcium i n f r o g s k e l e t a l muscle. J . Gen. P h y s i o l , i i , 1121. Cotlove, E. (1954). Mechanism and extent of d i s t r i b u t i o n of i n u l i n and sucrose i n c h l o r i d e space of t i s s u e . Amer. J . P h y s i o l . 176, 396.  89  C r a n d a l l , L.A. J r . , & Anderson, M.X. (1934). E s t i m a t i o n of the state of h y d r a t i o n of the body by the amount of water a v a i l a b l e f o r the s o l u t i o n of sodium t h i o c y a n a t e . Am. J . D i g e s t . D i s . Nutr. 1, 126. Creese, R. (1952). Bicarbonate i o n and muscle potassium. Biochem. J . JjO, XVIII. Creese, R. ( i 9 6 0 ) . Potassium i n d i f f e r e n t l a y e r s of i s o l a t e d diaphragm. J . P h y s i o l . 154> 133. Creese, R., D * S i l v a , J.L., Northover, J . ( l 9 5 8 ) . The e f f e c t of i n s u l i n on-sodium i n mammalian muscle. Nature 181, 1278. Creese, R., N e i l , M., and Stephenson, G. (1956). E f f e c t of c e l l v a r i a t i o n on potassium exchange of muscle. Trans. Paraday Soc. J52, 1022. Danowski, T.S. (l955)« E l e c t r o l y t e and endocrine studies i n muscular dystrophy. Amer. J . Phys. Med. .3_4_, 281. Dean, R.B. ( l 9 4 l ) . Theories of e l e c t r o l y t e e q u i l i b r i u m i n muscle. B i o l . Symp. _3_, 341. Duchenne, G.B. ( l 8 4 8 ) . De l B a l l i S r e et P i l s , P a r i s .  1  e l e c t r i s a t i o n localise*e.  J.B.  P r a t e r , R., Simon, S.E. & Shaw, P.H. ( l 9 5 9 ) . Muscle: a three phase system. The p a r t i t i o n of d i v a l e n t i o n s across the membrane. J . Gen. P h y s i o l . _43_, 81. Gamble, J.L. (1954). Chemical anatomy, p h y s i o l o g y , and pathology of e x t r a c e l l u l a r f l u i d . Harvard U n i v e r s i t y P r e s s . Cambridge, Mass. Geiger, R.S., & Garvin, J.S. ( l 9 5 7 ) . P a t t e r n of regeneration of muscle from p r o g r e s s i v e muscular dystrophy p a t i e n t s c u l t u r e d i n v i t r o as compared with normal human s k e l e t a l muscle. J . Neuropath. Exp. Neurol. 16, 532. Grant, A.E. 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( l 9 5 7 ) • Ionic movements and e l e c t r i c l i n g i a n t nerve f i b r e s . Proc. Roy. Soc. B. 148, 1.  activity  Hodgkin, A.L., & Horowicz, P. (1959). Movements of Na and K i n s i n g l e muscle f i b r e s . J . P h y s i o l . 145, 4 3 2 . Hodgkin, A.L. & Keynes, R.D. ( l 9 5 5 ) . The K p e r m e a b i l i t y g i a n t nerve f i b r e . J . P h y s i o l . 128, 61.  of a  Hodgkin, A.L. & Keynes, R.D. (l955a). A c t i v e transport of c a t i o n s i n g i a n t axons from S e p i a and L p l i g o . J . P h y s i o l . 128, 28^ Horvath, B., Berg, L., Cummings, D.J., & Shy, G.M. ( l 9 5 5 ) . Musc u l a r Dystrophy. Cation c o n c e n t r a t i o n s i n r e s i d u a l muscle. J . A p p l . P h y s i o l . 8, 22.  91  Hubbard, R.S. & Loomis, T.A. (1942). i n u l i n . J . B i o l . Chem. ,145., 641.  The determination of  J e n e r i c k , H.P. (1956). The e f f e c t s of calcium on s e v e r a l e l e c t r i c a l p r o p e r t i e s of muscle membrane. J . Gen. 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An index of membrane p e r m e a b i l i t y . Amer. J . P h y s i o l . 190, 201. Z i e r l e r , K.L. (1958). A l d o l a s e l e a k from muscle of mice with h e r e d i t a r y muscular dystrophy. B u l l . Johns Hopkins Hosp. 102, 17. Z i e r l e r , K.L. (1959). E f f e c t of i n s u l i n on membrane p o t e n t i a l and potassium content of r a t muscle. Amer. J . P h y s i o l . 197. 515. Zymaris, M.C., S a i f e r , A., V o l k , B.W. ( i 9 6 0 ) . S p e c i f i c a c t i v i ty of a c i d - s o l u b l e n u c l e o t i d e s i n hindleg muscles of mice with dystrophia m u s c u l a r i s . Nature 188, 323.  Muscle  Fresh  Weight,  mg.  Fig.JJL  Inulin Space of normal Mouse Muscle: Injection Method. * Gastrocnemii x Peronei Longi  Figures in parentheses between injection and  Oi 0  1  1  20  show the number of hours sacrifice of the animals.  1  1  40  Muscle Fresh Weight, mg.  1  r 60  Fig. |V_  Inulin  Space  of  In  Vitro  Swiss  Albino  Method.  Mouse  Muscle  Fig. V.  Thiocynate  Space In  50  n  of  Vitro  normal Method.  Mouse  Muscle  Fig.vi_  Uptake  of  Normal  and  altered  2  mM  K  -  •  6  mM  K  —  o  12  mM  K  -  x  e  e  e  K  by normal K  e  Mouse  Incubation  Muscle: Media.  F i g . yn_  Uptake High  I8CV  of  - K  K  4  2  Incubation  2 4 meg. / L  K  -  4 8 meg./ L  K  — o  Dashed  Line  is  by n o r m a l  Mouse  Muscle:  Media.  •  from  Fig.VI  160  140  >  120  '-3  CT OJ £ 100  z>  CO CO  *  80  60  40  20  0  20  ( Ke  60  40  \) 2 (mM l/  min.)'^  F i g . VIH  Uptake Altered  40  of Na  K e  4  2  by  Mouse  Incubation  Muscle Media.  r 18  mM  Na  e  288  mM  Na  e  Dashed  Line  -  o  -  x  is f r o m  Fig.vi. X X X X  30  X  \  cr CD  20  X  CD 3 CO CO  10  ( Ke  • t )  2  (mM  • min)  42 Fig.JX..  Uptake  of K  Altered  Na  by and  e  x  12 m M  K  o  2 mM  K  e  12 m M  K  •  e  e  e  normal K  line  Media.  288  mM  Na  -  18  mM  Na  -  18  mM  Na  is  Muscle :  Incubation  e  -  Chloride —  Dashed  Mouse  free  normal  e  e  e  medium uptake  slope  40-, x x  /  /  2 30-  20-  X  •  /  OJ / A  x  ©  3  CO CO  •  A  x #  e  10-  /  > Cr OJ  ©  /  o x x o* ;  0  10 (Ke - \ %  20 (mM  30 -min.)^  40  o Fig.  X.'  Uptake  ( Ke - t ) ^  of  (mM  K  by  •  min.)  Dystrophic  Fig.X_  Uptake  of  Altered  •  Dashed  18  o  288  line  is  K  by  Na  e  0  Incubation  mM  Na  mM  Na  Mouse  Muscle:  Media.  e  e  dystrophic figure  CH  Dystrophic  regression  line  from  JT  1  1  10  20  ( Ke -t)^2  (mM  • min.) ^2  1  30  Table I  K,  N a , a n d resting  as  m.equiv./litre  potentials of  cell  Calculated  of  or  values  excitable  plasma from  water. Nernst  K Tissue  Cell  Environ  124.0  2.2  3.6  Rat  Skel. Muscle  152.0  4.7  Crab  -  Muscle  146.0  Crab  -  Nerve  Values  from:  Ion  values  values  in  expressed  mV.  Equation.  R.P  Cell  Skel. M u s c l e  *-  R.P.  ,  Na  Frog  Squid - Giant A x o n  tissues  Environ  Actual  104.0  98  92  3.0  150.0  87  74  12.9  54.0  513.0  61  72  1 12.0  12 .1  54.0  468.0  60  82  369.0  13.0  44.0  498.0  83  65  Shanes  (1958),  8k K e y n e s  Ling  (1955),  ft  ,  Calc.  Gerrard  (1949),  C o n w a y ( 1 9 5 0 ) , and  Hodgking Z i e r l e r (1959)  

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