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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., University of B r i t i s h Columbia, 1958. A thesis 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 of MASTER OP SCIENCE i n the Department of ' PHYSIOLOGY We accept this thesis as conforming to the required standard. THE UNIVERSITY OP BRITISH COLUMBIA OCTOBER 1961 In presenting t h i s t h e s i s i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the U n i v e r s i t y of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s t h e s i s f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representatives. It i s understood that copying or p u b l i c a t i o n of t h i s t h e s i s f o r f i n a n c i a l gain s h a l l not be allowed without my written permission. Department The U n i v e r s i t y of B r i t i s h Columbia, Vancouver 8, Canada. Date OofoMjLj f . /*76f i i 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 (externa! potassiu* 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 first two hours of incubation, but after this time, intracellular i i i sodium rose and potassium f e l l . Fresh dystrophic mouse muscle had lower potassium and higher sodium content than normal fresh muscle. The cation changes following incuba-tion resembled those found for normal muscle. The changes 42 i n i n t r a c e l l u l a r cations were correlated with the K up-take results, and discussed i n some d e t a i l . i v ACKNOWLEDGEMENT It i s an honour and a pl e a s u r e to acknowledge the help and guidance g i v e n to me over the past three y e a r s hy Dr. Hugh McLennan of the Department of Phys i o l o g y . H i s u n f a i l i n g i n t e r e s t has been a source of constant i n s p i r a t i o n to me. I would a l s o l i k e to thank the other members of the Department of Ph y s i o l o g y 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 i n 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 . I am e s p e c i a l l y g r a t e f u l to Dr. D.H. Copp f o r p e r m i t t i n g me the u n l i m i t e d use of the Departmental f a c i l i t i e s , . a n d to Mr. Kurt Henze f o r the e x c e l l e n t p r e p a r a t i o n of the graphs and t a b l e s . My sinc e r e , t h a n k s go to Dr. 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 of 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 are a l s o extended to Dr. J . R. M i l l e r , Depart-ment of N e u r o l o g i c a l Research, and Miss Ann Cooper, Department of Pathology f o r a constant source of normal and d y s t r o p h i c 129 s t r a i n mice. F i n a l l y , I wish to thank my wife, Margaret, f o r her pa t i e n c e and encouragement, and f o r her help i n p r e p a r i n g the f i n a l d r a f t , and Miss Tanya Dournovo f o r t y p i n g the f i n a l copy. i TABLE OF CONTENTS SECTION I: INTRODUCTION 1 SECTION II: METHODS 25 SECTION III: RESULTS 38 SECTION IV: DISCUSSION 69 SECTION V: CONCLUSIONS AND SUMMARY 85 SECTION VI: BIBLIOGRAPHY 88 SECTION I INTRODUCTION A. Purposes of the study B. General aspects of bioelectricity 1. The composition of tissues 2. Ionic equilibria 3. Maintenance of ionic gradients G # Tissue compartments D. Electrolytes and tissues 1, Nerve tissue 2. Muscle tissue E. Interpretation of results: muscle models F. Muscular dystrophy: human and murine 1. Electrophysiological changes i n dystrophic muscle 2, Histology of dystrophy 3« Electrolyte studies 1 A. PURPOSES OF THE STUDY The investigations reported in this thesis centre around two objectives: to investigate and measure the rates of movement of potassium into mammalian skeletal muscle under varying environmental conditions, with the aid of the radioactive isotope K^2, and to compare the rates of movement of the ion i n normal and dystrophic mouse skeletal muscle. The results were to be expressed as m. equiv. intracellular K moving per (K • t ) 2 unit. Thus, several problems required solvingj the non-inulin space volume had to be determined, involving an e s t i -mation of the extracellular inulin space and a determination of the dry weight of the musclej the normal concentration of K i n fresh muscle tissue also had to be known. As the experiments would involve incuba-tion i n a modified Locke's solution for several hours, a knowledge of the intracellular ionic concentration changes due to incubation 1 was necessary. Net alterations i n K lev e l during incubation could then be appropriately corrected. The concentrations of sodium and potassium i n some of the incubation fluids were altered, to see i f differences in extracellular concentrations of the ions would affect the rate of K entry into the muscle f i b r e . Concomitant observations of muscle resting potentials under similar experimental conditions have been investigated separately by Professor H. McLennan. 2 B. GENERAL ASPECTS OF BIOELECTRICITT 1. The Composition of Tissues It i s a characteristic of the excitable c e l l to exhibit a resting potential difference between i t s environment and the c e l l i n t e r i o r . I t i s also characteristic for protoplasm and tissue f l u i d to contain differing concentrations of certain ionic species, the most obvious examples being the alkal i metal ions sodium and potas-sium (Na and K), which are present in high concentrationsj this i s illu s t r a t e d in Table I. Other substances are also distributed un-equally between the c e l l interior and environment; Table II, from Conway (1950), shows values for some substances found i n frog and rat muscle. To maintain the ionic balance of excised tissue, a r t i f i c i a l fluids were devised to enable the tissue, when incubated i n the f l u i d , to survive and function , lnormally , ,. Sydney Ringer (e.g. (1883)) i n -tensively studied the effect of ions on the frog heart. He observed that small quantities of KC1 and CaClg added to a 0.1% NaCl bathing solution would enable the heart to beat for hoursj without these two salts, i t stopped in a few minutes. Locke (1895) demonstrated that glucose had the same effect as addition of the salts. He also altered the tonicity, and used the f l u i d for perfusing mammalian heart, oxygena-ting and warming i t to body temperature (Locke 1904). Many "physiolo-gical 1* solutions have been devised since then for specific tissuesj Ringer-Tyrode for gut tissue, Ringer-Krebs-Hensleit for brain tissue, and Ringer-Conway for frog muscle are a few common ones. These fluids d i f f e r principally in the concentrations of their constituents, or have special substances added to them; they are a l l supposedly i n osmotic equilibrium with the tissues for 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 in -mV. Calculated values from Nernst Equation. K Na R.P. TISSUE 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 Zierler (1959). TABLE II; DISTRIBUTION OF SUBSTANCES IN FROG AND RAT MUSCLE. Values expressed as m.equiv. per l i t r e fibre or plasma water. H4" expressed as pH. FROG SKELETAL MUSCLE RAT SKELETAL MUSCLE SUBSTANCE FIBRE PLASMA FIBRE PLASMA SODIUM 3.6 104.0 3.0 150.0 POTASSIUM 124.0 2.2 152.0 6.4 CALCIUM 4.9 2.0 1.9 3.1 MAGNESIUM 14.0 1.2 16.1 1.5 CHLORIDE 1.5 74.3 5.0 119.0 BICARBONATE 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 H+ Ca. 6.9 7.4 Ca. 6.2 7.4 5 2. Ionic Equilibria Diffusion i s the movement of particles from a region of higher concentration to one of a lower concentration, due to the random migrations of thermally agitated solute particles. In a fixed structure such as NaCl crystal, this movement does not occurj in the crystal l a t t i c e , each sodium ion i s surrounded by six chlor-ides, fixed by electrostatic forces in a face-centered cube form. In solution, the ions dissociate from tiie c r y s t a l i n equal numbers, and at any point i n a homogenous solution, a situation similar 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 this solution, there w i l l be no potential difference between any two points (Principle of Microscopic Electroneutrality), for the random movements of the ions cancel any transient imbalance. However, i f these ion species are diffusing, the rate of diffusion w i l l be pro-portional to the square root of their hydrated r a d i i j a chloride ion, with a smaller radius than a sodium ion, w i l l diffuse more rapidly; but as i t moves, the rate w i l l tend to be retarded by the attraction of the positive sodium ions l e f t behind, and a small but measurable difference i n distribution of e l e c t r i c a l charges between various parts of the solution w i l l arise. A region of high s a l t concentration w i l l be more positive than a region of lower concentration, and a diffusion potential may be measured; this potential w i l l run down as diffusion proceeds, and w i l l disappear when diffusional equilibrium i s attained. To maintain a potential between two compartments, either there must be no net movement of the ions causing the potential, or there must be an indiffusible (impenetrable) ion in one of them. The Gibbs-Donnan equilibrium rule states that i n a system ifaere an impermeable 6 anion i s on one side of a fixed membrane, and a solution permeable to the membrane (e.g. NaCl) bathes both sides of i t , the ions w i l l become distributed unequally i n the two compartments, with more cation and less diffusible anion i n the indiffusible 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 electric neutrality i n the solutions. The indiffusible anion compart-ment w i l l be sl i g h t l y negative with respect to the other, thus cations w i l l try to move in to i t j this, 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 distribution 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 in ion concentrations of l i v i n g c e l l s and their 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 electric neutrality i s opposed by the concentration potential across the membrane. If an electrode i s placed on each side of the membrane in a Donnan system, a potential of magnitude E = — In l°lb- (Eq. 1) Z F ( c + ) 2 can be measured; R is 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 to be impermeable to Na and Gl; this concept would satisfactorily explain the resting poten-t i a l as arising from passive distribution of K by the Donnan system, and account for the dependence of the magnitude of the potential on the external K concentration (K e); the potential of Eq. 1 adapted for potas-7 slum i s E = RT In ( K + ) i (Eq. 2) F The resting potential is linearly related to the log K e for most values of KQ, but for muscle the slope of plotted experimental observations is 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 e had the greatest influence on the resting potential, i t was also affected by the Na ion ratio; his data were best f i t t e d by the equation 3. Maintenance of Ionic Gradients The distribution of muscle K (or Na) during steady state conditions can be explained electrochemically; the r esting potential 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 potential to a lower one, even though the chemical gradient be opposing the movement; the K ions may move against a 25tl ratio, and s t i l l be mov-ing "passively". This i s dis t i n c t l y different from diffusion, for the l a t t e r occurs down a concentration gradient, not "uphill" against a gradient. The term "passive transport" has been coined to describe this phenomenon of ion movement by electric forces against a diffusion gradient. This passive transport does not require metabolic energy E - M i n K i + 0.005 Nai (Eq. 3) 0.005 Na Q 8 and should not be stopped by metabolic inhibitors. The Nernst electrochemical potential equation consists of two terms, an osmotic, and an electric, which are opposed; for i n transferring an ion against the concentration gradient, osmotic work is done on the system, while electric forces concomitantly try to push the ion back down the gradient. If the two terms are equal, work i s zero, and no transfer occurs; the osmotic term RT n Ki/K e i s equal to the potential term zFE. Sodium ions do not follow this Nernst relationship; how, then, does one account for the Na ion distribution? A mechanism, termed "active transport" was postulated for maintaining the d i s t r i -bution (Dean 1941); a simple definition would be "transport against an electrochemical gradient, using energy derived from cellular meta-bolism"; consequently, -the sodium ion was extruded as fast as i t penetrated the c e l l , keeping the intracellular Na concentration low, and providing a functional impermeability to the ion. In distinction to passive transport, energy was required for this process; a wealth of studies (e.g. references page 60 of Ussing (I960)) showed a depen-dance of the continued extrusion on a continuous metabolic supply of energy; i f Na extrusion could thus be maintained, K ions would tend to equilibrate with the electrochemical gradient of the tissue, i . e . be distributed by a Donnan system. This theory is able to account for most of the phenomena observed i n excitable tissues. However, i t has been suggested (e.g. Ling (1953)) that active transport i s an unnecessary hypothesis, and that a series of fixed sites that bind K and Na (the binding requiring continuous metabolic energy) would exhibit the properties experimen-t a l l y observed. 9 C. TISSUE COMPARTMENTS In Part B, i t was seen that intracellular ion concentra-tions differ from extracellular concentrations. Tissue f l u i d ion concentrations in turn differ from those i n plasma; thus, i t would be misleading to draw off a sample of blood, analyse the plasma, and represent this plasma value as being equivalent to the tissue f l u i d concentration of the ion, for the plasma sample is not a true repres-entation 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 essentially an u l t r a f i l t r a t e of plasma, although i t contains some protein. A Gibbs-Donnan equilibrium i s established between the plasma and tissue fluids; since the plasma protein con-centration i s greater than the tissue f l u i d protein concentration, there w i l l be sl i g h t l y less Na and K, and slig h t l y more Cl 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 ratio tissue f l u i d K/plasma K i s theoretically 0,90-0.95; some experimental values reported were as low as 0,75 (Manery 1954); i f these lat t e r plasma samples were used to determine extracellular K concentrations, they could have been 25% i n error. Caution must also be used when estimating extracellular Ca or Mg ion; these ions may be par t i a l l y bound to plasma proteins, for their distribution ratios are much lower than the K or Na values. Tissue f l u i d i s scant i n muscular tissue; i t i s d i f f i c u l t to sample, for there are no normal accumulations of i t ; i t was estimated that i n a muscle belly with an extracellular space (ECS) of 10$, the tissue f l u i d would be only a 15 micron film covering the surface of the c e l l s . Maurer (1938) punctured frog muscles and drew off small quanti-ties 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 ; his Cl analyses corresponded closely to the calculated values reported by 10 Gamble (1950). Edema f l u i d has been analysed (Manery 1954); the ionic 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 in the two fluids was the same. Accurate intracellular concentrations could not be calculated unless the ECS was known; Na and Cl were considered to be exclusively extracellular ions, and since they appeared not to enter the tissue c e l l s , they were often used as Indicators for ECS estimations. The basis for this was that i f Na and Cl were found only i n the extracellu-lar f l u i d , then the amount of Cl (or Na) i n tissue and plasma samples could be used to calculate the ECS of the tissue. However, muscle ce l l s have been shown to contain both these ions and the po s s i b i l i t y of their cellular uptake was suggested to account for the higher values calcu-lated for these spaces. Cl and Na spaces are now used less often for estimating ECS than in the past, Thiocyanate (SON), a negative ion similar i n mobility to chloride (but not normally found i n the body), has also been used to determine the ECS of tissues; values for this space were 4% too large according to Pappius and E l l i o t t (1956), for this amount was taken up by the tissue c e l l s . And "ideal" indicator for ECS determinations (either tissue or whole-body ECS) would be a substance that was not harmful to the animal or i t s tissues, was distributed evenly in the extracellular f l u i d , did not penetrate into or adsorb onto the c e l l , and was readily assayed. The disaccharides maltose and sucrose (MW 360 and 342) and the polysaccharide inulin (Ml1 991) satisfied these c r i t e r i a ; with the disaccharides, there was the poss i b i l i t y of metabolic u t i l i z a t i o n , whereas inulin 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)); results using this tracer corresponded closely to inulin space estimations. The values for other space estimations varied according to the i n d i -cator used; for amphibian s a r t o r i i , the spaces measured with Na, Cl, CNS, sucrose, inulin, and R1SA decreased i n that order; i t was sugges-ted that the higher values obtained may have been due to the tracer entering the ce l l s (Tasker, et al,l959). The volume of muscle ECS must be known to calculate the muscle fibre or non-ECS volume, and ideally 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 for ions, and assayed the other for ECS. There i s usually a variation between the ECS values of the pair, but the value for the companion muscle i s a better estimation of the ECS than are group averages. Tasker et a l (1959), showed a significant variation of the average ECS in s a r t o r i i from different batches of toads; they also found a seasonal variation i n ECS and tissue ion content. The authors concluded that applying group ECS values to individual muscles was not a v a l i d procedure because of the large variations among and within groups of muscles. Each muscle seems to have a characteristic ECS value, which varies with s i t e , age, weight, and condition. McLennan (1956) reported rat diaphragm ECS to be 25%, gastrocnemius 12%, and extensor digitorum longus to be 15%. Tasker et a l (1959) found a negative correlation between ECS and muscle weight in excised toad s a r t o r i i ; this finding was confirmed by Burr and McLennan (I960) for mouse muscle. The latter authors assayed dystrophic tissue as well, and found the ECS to be greater than i n normal muscle. 12 D. ELECTROLYTES AND TISSUES 1. Nerve Tissue Individual mammalian nerve fibres are too small for con-venient study; consequently, many investigators of cation movements in nerve have used giant nerve fibres - the squid and cuttlefish giant axons that are 0.2-1.0 mm. diameter. These nerve fibres ex-hibited a resting potential of 40-80 mv (inside negative), apparently maintained by an active transport of Na and Kj when studied i n v i t r o the axons were liable to lose K and gain Na. Hodgkin (1957), Shanes (1955) (1958), and Keynes (1951) wrote review articles on the bioelec-t r i c and ionic phenomena observed in nervous tissue. Ion movements associated with nerve activity w i l l not be considered in this thesis. If 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 distribution of K between the fibre and i t s environment seemed to be a Donnan dis-tribution, and i t was proposed that Na was pumped out and K equilibra-ted passively. On the other hand, Hodgkin and Keynes (1955a) showed that there was an apparent active transport of K inwards i n giant axons. The inhibitors of Na active transport also reduced the influx of K from sea-water. This drop in K influx equalled the change i n Na efflux, suggesting a coupled exchange of the two ions; however, some Na extrusion persisted in K-free solution, and the active Na flux exceeded the active K flux. 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 loss. The resting potential did not change when K and Na fluxes decreased after poisoning the nerve (1955a); apparently the active transport per se does not affect the potential. The persisting fluxes 1 3 may have been due to passive diffusion of the ions; this passive movement did not vary as expected when the resting potential was a r t i f i c i a l l y altered; the flux change was much greater than pre-dicted (Hodgkin & Keynes 1955), and always occurred down the K electrochemical gradient. Hodgkin and Keynes (1955) proposed a "long-pore" membrane model that adequately accounted for the observed phenomena. Excess K ions moving out may have diminished the K influx by competing for sites in the membrane. 2. Muscle Tissue Approximately half the weight of the human body i s skeletal muscle; the individual muscles are composed of groups of cells 1 to 40 mm long and 10 to 40 micra i n diameter; simultaneous contraction of the fibres produces a violent shortening of the muscle. Each fibre i s a complete c e l l , multinucleate, with a membrane enclosing i t . In the space between the fibres i s tissue f l u i d , the milieu interier of the body. A potential difference of 60-80 mv (inside negative) exists across the membrane of these c e l l s , due mainly to differing 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 fibres, are high i n K and low in Na (see Table I ) . Recent publications by Harris (i960), Conway (1957), Ussing (i960), and Harris and Sjodin (1961) discuss K in muscle tissue. 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 ce l l s were also permeable to Na, which could replace some of the K i n rat muscle; he confirmed this a year later using radioactive Na (Heppel 1940). 14 Excised frog muscles soaked in ordinary Ringer solution lost K and gained Na; this 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 reversibly depleted the intracellular K (the K^). This was accompanied by a reversible increase in Na^« It was further demon-strated (Creese 1952) that the addition of bicarbonate to the normal medium decreased K loss. In high-KCl solutions, the fibres swelled and gained KC1. These solutions resulted i n the replacement of some of the Naj_ by K; however, the resting potential f e l l due to the lowered Kj/K e ratio. Chloride therefore entered the c e l l more readily, 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 neutrality; consequently, the cells swelled. If non-penetrating anions such as methyl-sulphate were used, the swelling was markedly reduced. Dean (1941) proposed the rtNa pump" theory to explain the maintenance of Na and K gradients; Keynes (1954) suggested that K was the exchange partner for the pump (resting potential must be kept constant), for a reduction of Na efflux occurred i n K-free bathing fluids, and an increase in K-rich f l u i d s . Steinbach (1952) showed an optimum K e for Na extrusion i n Na-loaded muscle; he suggested that K diffusion i n the muscle was the limiting factor i n the exchange of Na for K. The low K. of i n t e r s t i t i a l f l u i d could affect K exchange with muscle fibres, because a small rise i n K„ could lead to an * e appreciable back diffusion into the fibre; this factor could lead to incorrect interpretation of tracer flux calculations. Harris and Burn (1949) found the fibre-interspace exchange to be about equal i n rate to the interspace-soaking solution, i f the muscle fibres impede 15 the movements of the ions. McLennan (1956) (1956a) (1957), working with mammalian muscle discussed the problem at some length, and con-cluded 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./cm2/min, close to McLennan*s value of 1.4 for rat muscle. The latter found that K^2 efflux from equilibrated 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 e would enable a l l K to exchange. 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 extra-cellular space, and less than the total fibre K. The amount of K in this fast portion seemed to vary with the KQ 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 in tissue appeared to differ from "normal" Na: the efflux into non-electrolyte solutions was slower, High Ke solutions increased the efflux more than usual, and the 16 temperature dependance was greater (Conway & Carey 1956). Possibly this Na had replaced K in this "special region"; i f only a limited number of sites to bind cations were available, competition for them might have retarded K or Na movement into or out of the c e l l . E. INTERPRETATION OF RESULTS: MUSCLE MODELS The "classical" concept of a muscle c e l l involved a proto-plasmic mass enclosed by a thin, resistive membrane, one with no capa-c i t y to store ions, and through which ions passed when entering or leaving the c e l l ; the resistance supposedly varied for each ionic species, as some ion species could enter more easily than others; the membrane was considered as the rate-limiting step for ion movement into or out of the c e l l . I t was assumed that extracellular diffusion would maintain the tissue f l u i d ionic composition constant, and that intracellular diffusion was more rapid than the rate of movement through the membrane into the c e l l ; consequently, the intracellular ions became homogenously distributed, and new ions entering the c e l l would readily diffuse and equilibrate within i t . The characteristics of ion uptake therefore depended upon the rate of internal mixing and the resistance of the membrane, assuming that the membrane contained no ions; thus the uptake should have followed first-order kinetic relations, 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 total K i n the fractions did not always equal the total muscle K. If muscles were incubated in 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 did not undergo exchange; 17 however, the amount of exchangeable K increased i f the K e of the bathing solution was above normal. Radioactive K also f a i l e d to become uniformly mixed with frog muscle K after limited exposure to the tracer (Harris & Steinbach 1956)j the specific a c t i v i t i e s of successive extracts of these muscles diminished rapidly; the inhomo-geneity of the K seemed to be intracellular, and not due to a greater degree of exchange of superficial than deep fibres. The membrane theory inferred that the tracer efflux from loaded muscles would be independent of the time of loading, but this result has not been confirmed; muscles exposed for a short time appeared to have more K that would exchange rapidly than others incubated for a longer time (Harris 1953). Apparently this thin resistive membrane model did not adequately interpret the kinetics of ion movements; Ling, Shaw et a l , and Harris have each presented hypotheses i n an attempt to explain their observations more completely. Ling (1952) (1955) (i960) proposed a "fixed-charge" hypo-thesis to explain selective K accumulation by muscle c e l l s . The hypothesis maintained that free moving ions (such as K and Na) tended to approach fixed anions (e.g. protein chains) as closely as their charge would allow; since hydrated K i s smaller than hydrated Na, i t moved closer to the fixed anion; this represented a lower energy state for K than for Na, and the K would be held i n a more stable manner. Since a system w i l l tend to attain 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 ihis 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 radius); but Rb (with a smaller hydrated radius 18 than K) was found to compete for these fixed sites, instead of replacing the already bound K. The anionic sites were maintained by energy from c e l l u l a r metabolism; poisoning the muscle led to K loss and Na gain, but poisoning of an "active transport" system was not necessary to explain this s h i f t ; a lack of ion binding gave the same result. The theory could not adequately explain cation shifts i n cooled muscle; neither did i t account for Na replacing K intra-ce l l u l a r l y , nor the sequence of relative cation permeability of the muscle. - Shaw and his collaborators exhaustively investigated the relations of Na, K and Gl of toad s a r t o r i i (Shaw, Simon & Johnstone 1957) (Simon, Uohnstone, Shankly & Shaw 1959) (Frater, Simon & Shaw 1959) (Simon, Shaw, Bennett & Muller 1957); they found that muscle K did not exchange as expected when K Q was altered, but that Na and Cl movements were independent of K movements or concentrations. The muscles did not swell i n hypertonic KC1, but did i n high NaCl solu-tions; also, there was no replacement of Na by K i n low Na solutions. To account for these findings, the authors postulated a "three-phase theory", consisting of extracellular, free intracellular, and ordered phases. It 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 intracellular phase was i n diffusional equilibrium with the extracellular phase; the apparent cellular concentration gradient was due to ion exclusion from the ordered phase. No energy was thus nec-essary to maintain the c e l l i n this manner. Harris (1953) (1957) postulated a permeation-diffusion model to account for tissue K exchange; this differed i n several respects from 19 the models presented above. Ion accumulation was regarded as a two-stage process: 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 diffusion from this outer region into the c e l l interior. This second step would be bal-anced by diffusion from the internal region to the outer one; conse-quently, there would be no net change i n the K concentration of the c e l l . The model was thus a three compartment one, with the compart-ment between i n t r a - and extra-cellular fluids having ion exchange properties; K movement i n this compartment was slow, due to binding by fixed 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 solution more rapidly than they exchange with internal ions, and (2) when the internal d i f f u -sion i s so rapid that the rate of passage through the outer region, rather than equilibration within i t , determines the rate of exchange. Condition (2) was the "clas s i c a l " resistive membrane that gave rise to first-order kinetics of K movement; condition (1) followed ordinary diffusion laws. This model can satisfactorily account for the kinetic pheno-mena observed i n K exchange experiments. The model can be used to distinguish between K—K exchange and net cellular gain of K, whether the latt e r i s with an anion or in exchange for Na; the K—K exchange seems to rely on thermal agitation and diffusion, while the net move-ment 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 effect on K uptake of adding other cations to the incubation media may be due to a competition for sites i n this interme-20 diate compartment; gain or loss of w i l l also affect the rate of K# uptake, especially i f added ions alter the K^. Metabolic poisons would tend to weaken the binding of the cations i n the special region, and lead to a loss of K and gain of Na by the c e l l s . Temperature would affect the uptake rate, by increasing the thermal agitation of the particles, 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 diffusion laws, Harris and Sjodin could show that K equilibration i s independent of the intracellular d i f f u s i v i t y (D) i f r 2 /D i s less than unity (r i s the radius of a cylinder, and a constant dependant upon K 0); the ratio i s usually ca. 0.5 for muscle c e l l s ; the authors further showed that plotting exchange against •t (or K e°t) w i l l approximate a l l curves to one. Any K—K exchange w i l l f a l l along this curve, but i f the muscle i s gaining or losing K the values w i l l be above or below the exchange curve. If the K 1 uptake i s plotted against ( K e * t ) 2 , much of the exchange wall follow a linear relationship. . The experimental results for K exchange presented i n this thesis w i l l be interpreted with the aid of the Harris permeation-dif-fusion muscle model, for i t is f e l t that this model can best aid i n the explanation and interpretation of the kinetic data recorded. F. MUSCULAR DYSTROPHY: HUMAN AND MURINE Until the nineteenth century, medical practitioners general-l y believed that a l l muscular disorders, weaknesses, and atrophies were of neurogenic origin, 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 alteration i n the nervous system. The modern term progressive muscular dystrophy is 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 strain 129 mice (reported i n Michelson, Russell & Harmon 1955)J i t was subsequently shown (Stevens, Russell & Southard 1957) to be similar to the human muscular dystrophies. 1. Electrophysiological Changes in Dystrophic Mouse Muscle Dystrophic mice have been used to study e l e c t r i c a l altera-tions i n muscle. Conrad and Glaser (1959) reported micro-electrode studies showing no change in resting potentials i n dystrophy, but reported an increased excitability of the dystrophic muscle to e l e c t r i c a l stimulation, as well as an increase in Na conductance. This latter observation was confirmed by McLennan (I96I), who also suggested that the similarity between the potentials from the two types of muscle was because resting potentials could only be recorded from sound fibres, and not from degenerate ones. Sandow and Brust (1958) reported dystrophic muscles to be weaker (l/lO to 1/5 the strength), the "active state" duration to be 1/3 shorter, and the relaxation per-iod three times longer than for the muscles of normal littermates. 2. Histology of Dystrophy Adams et a l (1954) reviewed the histology of dystrophy; great variation i n fibre size and the large proportion of fat and fibre cells were striking features of dystrophic muscle sections. Fibres were found randomly scattered i n their bundles, with swollen 22 and atrophied f i b r e s mixed. The hypertrophy observed c l i n i c a l l y i s evidently due 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 nuclei are swollen and more numerous, and vacuolation and granu-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. Tissue culture (Geiger & Garvin 1957) has shown that the c e l l s develop hypertrophy and begin to degenerate after 3 to 4 weeks of growth. The 129 strain mice show similar microscopic changes. Michel son et_ a l (1955;) could see no pathological a l t e r a t i o n i n any nervous tissue examined, but muscle f i b r e preparations revealed changes similar to those found i n human muscle. 3i E l e c t r o l y t e Studies Pew studies have been done on the electrolyte l e v e l s associated with human muscular dystrophy, Danowski (1955) reported s l i g h t l y elevated calcium and phosphorous l e v e l s and decreased Cl levels i n serum samples of children with dystrophy; other indices such as serum Na, K, protein, and NPN were essentially normal. Dowben and Holley (1959) described erythrocyte electrolyte l e v e l s i n dystrophic patientss Sodium: Normals 13©1 nu equiv./Kg Dystrophics 15©5 " Potassium: Normals 87*2 " Dystrophics 91•O " This elevated K l e v e l i s i n contrast to the usual lowered muscle K levels associated with dystrophy; i t may indicate a reciprocal relationship between muscle and plasma potassium, with the possibi-l i t y of the normal r a t i o being altered by any changes i n muscle membrane permeability. 23 Williams (1957) used neutron activation analysis to assay f o r muscle Na and K, and found that the Na l e v e l was elevated (normal 1 1 3 m. equiv./Kg dry weight, dystrophic 1 9 1 m. equiv.), and that the K l e v e l was lowered (normal 376 m. equiv., dystrophic 181 m. equiv.), confirming the results repor-ted by Horvath, Berg, Cummings & Shy (1955). Studies on dystrophic mice revealed electrolyte variations similar 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 fresh muscle, whereas dystrophic K was 76 m. equiv./Kg fresh muscle. Sodium determina-tions indicated an increase of ca. 43$ i n the sodium values, from a normal average of 46 m. equiv./Kg fresh 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. increase 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 extracellular space, with the resultant increase i n Na and decrease i n K l e v e l s , or to the leak of K from the c e l l , with compensatory gain of Na. Unfortunately the authors did not take into account the problem of extracellular space; i t i s d i f f i c u l t to draw conclusions about i n t r a c e l l u l a r electrolyte l e v e l s from their data. Young, Young, and Bdelman (1959) analysed skeletal and cardiac muscle from dystrophic mice fo r Na, K and l i p i d content. They reported a 19% decrease i n dystrophic skeletal muscle Na, and no difference i n cardiac muscle K between the normal and dystrophic 24 animals; cardiac Na values were not reported. Again, no allowance f o r ex t r a c e l l u l a r space was mentioned. SECTION II METHODS A. Experimental Animals B. Incubation solutions 1. Ext r a c e l l u l a r space experiments a . Inulin solutions b« Thiocyanate solutions 2e Radioactive Isotope experiments C« Dissection technique D. Chemical methods 1. Ext r a c e l l u l a r space estimations 2» Dry weight determinations 3» Non-radioactive incubation experiments E. Radioactive Isotope techniques 25 EXPERIMENTAL ANIMALS The animals used i n t h i s investigation were pure inbred mice of the Bar Harbour 129 s t r a i n , some of which develop an i n -heritable muscle weakness simil a r to human progressive 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 of Neurological Research, University of B r i t i s h Columbia. Purebred normal mice, with no pedigree history of dystrophy, and littermates of a f f l i c t e d animals (those which did not develop muscular weakness) provided control animals. The dystrophic animals were 2-J to 8 weeks old, of vary-ing body weights, and i n varying stages of d i s a b i l i t y due to the dystrophy. The controls were used only i f i n perfect condition, and ranged from about 2 weeks to several months of age. The weight increase with age i s predictable i n the normal animals; the dystrophics, however, do not grow as regularly nor as quickly; they lag behind the normal animals by about 35-50% i n their development; a three-week old normal mouse may weigh 15-20 gm., while a dystrophic littermate may be only 8-9 gm. i n weight. At maturity the normal animals weigh 25-35 gnu, depending on sex, while the dystrophics are only 15-18 gm. i n weight. The animals were kept i n the departmental animal room for several days before being used. During t h i s time the dystro-phics were kept separately from their normal littermates, and were maintained on powdered commercial chow and water, both ad li b i t u m . 26 The chow and water were placed i n special low feeding containers f o r ease of reach by the dystrophics. The normal animals were treated s i m i l a r l y , with the exception that no special containers were provided, and the food was i n pel l e t form. INCUBATION SOLUTIONS Locked solution, as mentioned e a r l i e r , i s an a r t i f i c i a l e xtracellular f l u i d . In the majority of the experiments performed, this solution, or variants of i t , was used for the i n v i t r o incuba-tion of skeletal muscles. For,convenience, stock isotonic solutions (0.154 M. f o r salts dissociating into two ions, 0.11 M. f o r those hielding three) were prepared. Table III l i s t s the stock solutions used. From these constituents the experimental solutions of desired i o n i c composition could readily be prepared, and were freshly made up before each experiment. The f i n a l i o n i c concen-trations i n the various solutions 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 gm/litre KC1 11»47 gm/litre KHCO, j 15.41 gm/litre NaHCOj, j 12.49 gm/litre KH 2P0 4 19.94 gm/litre Na 2HP0 4 21.25 gm/litre CaCl 2 16.18 gm/litre NaH 2P0 4 15.62 gm/litre MgCl 2 22.34 gm/ntre *10 times normal strength. TABLE IV: COMPOSITION OP EXPERIMENTAL BATHING SOLUTIONS. Pinal i o n i c concentrations of constituent substances expressed i n m.equiv. per l i t r e . 3 gm. of glucose per l i t r e was added i n a l l cases, and the solutions equilibrated with 95$ 0 2-5% 00 2 gas mixture. SOLUTION Na K Cl HCO* Oa Mg Choline Phosphate 1 144 6 134 24 2 1 — — 2 150 - 134 24 2 1 - -3 138 12 134 24 2 1 - -4 265 6 254 24 2 1 - -5 18 6 134 24 2 1 126 -6 144 6 7 _ 2 1 _ 151 28 l o E x t r a c e l l u l a r Space Experiments (a) Inulin Space Estimations: The normal Solution 1 was used f o r the i n v i t r o incubation of excised muscles f o r i n u l i n space estimations, with dried B. D. H. Inulin added to 1% (w/v) concentration. Inulin was r e l a t i v e l y insoluble at room tempera-ture, so the appropriate amount was weighed out accurately and dissolved i n hot 0.154 M NaCl solution. After the i n u l i n - s a l i n e had cooled s l i g h t l y , the other constituents of the Solution 1 were added. Por the i n vivo i n j e c t i o n of i n u l i n into nephrecto-mised mice, a concentrated solution was needed. A 25% (w/v) solu-tion was prepared i n hot 0.9% saline. This amount of i n u l i n dissolved readily i n the hot NaCl, but on cooling to room tempera-ture s o l i d i f i e d completely. Gentle reheating resulted f i r s t i n a milky f l u i d , which upon further heating cleared completely. A standard solution f o r the colourimetric estimation of muscle i n u l i n was needed; the Dilute Inulin Standard was 1.0 ml. of the inulin-containing bathing f l u i d diluted i n a volumetric f l a s k to 100 ml. This provided a reference standard of 100 microgm. inulin/ml. The reagents f o r analysis were prepared as follows: Resorcinol: 125 mgm. resorcinol dissolved i n 100 ml. of alcohol; 30% HCl: 80 ml. of concentrated HCl were added to 20 ml. of water; Trichloroacetic Acid: 12 gm. of TCA was dissolved i n 100 ml. of demineralised water. 29 (b) Thiocyanate Space Estimations: Estimations of thio-cyanate space were done in vitro as a check for the inulin space determinations. The thiocyanate bathing solution was prepared with a CNS"" concentration of 48 ra. equiv./l. re-placing chloride in the normal bathing solution. A dilute standard reference solution for the colour-imetric estimation of muscle thiocyanate was prepared by diluting 1,0 ml. of the bathing solution to 100 ml., giving a CMS" concen-tration of 10 microgm. CNS" per ml. The 12$ TCA reagent used in the CNS" estimation i s the same as for the inulin; f e r r i c nitrate reagent was prepared by dissolving 5.0 gm. of Fe^Oj)^ • 9H20 i n 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 specific activity of the stock 0.154 M. K^2 Cl solution was usually not too high, and second, the required 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 else the specific activity of the f l u i d would be too low to be useful. Each shipment of the powdered K 4 2 gCOj arrived by air from Amersham, England, encapsuled i n two nestled screw-top aluminum containers, sealed inside a thick lead castle. The screw-top con-tainers, were opened with tongs and pl i e r s , and the container and it s contents (6.65 gm. of K 4 2 2C0^, powdered) were carefully lowered into a large beaker of water. The powder was washed out of the 30 container, and dissolved i n the water. Indicator was added, and the K4'22CQ5 converted to K 4 2C1 with LOW HCl. This solution was then diluted to 0.154 M and used as a stock solution for making up the experimental incubation f l u i d s . The fluids were prepared with the K^2C1 added as the la s t ingredient; 48 ml. of KCl-free non-active solution of the desired type was placed in a small glass bottle; to this was added 2 . 0 of the isotonic K^2C1, completing the ionic requirements. C. DISSECTION TECHNIQUE The m. gastrocnemius of the 129 strain mice has been used almost exclusively in these experiments. The mice were sacrificed by cervical fracture of the spine. The skin of the thigh was cut completely around with a small pair of sharp scissors and peeled down the leg and over the heel, exposing the muscles, tendons and fasciae. The skinned leg was then gently washed free of hair cuttings, etc., with a cotton pledget soaked with 37°C. Solution 1. A small pair of forceps was carefully inserted between the tendon of Achilles and the t i b i a , and moved back and forth to create a small patent space. Another pair of forceps was then used to strip away the f a s c i a l coverings of the gastrocnemius and the nearby muscles, starting from this space. The popliteal nerves were severed in the popliteal space, close to the body of the gastrocnemius; the muscle was then freed by blunt dissection from those bordering i t , the tendon of Achilles severed near the caleaneous, and the entire muscle l i f t e d upwards by pulling on the freed tendon. The soleus muscle was then separa-ted from the gastrocnemius, and the leg was cut through completely just above the knee; the preparation was placed on the stage of a 31 dissecting 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 37°G. Solution 1 to prevent drying of the tissues. Using fine forceps and a sharp scalpel, excess fat and nervous tissue was stripped and cut from the preparation. The tendon (Achilles) was trimmed free of fat and residual shreds of soleus muscle, and the excess length of tendon was carefully cut off. However, i t was not cut off too short so as to damage any muscle fibres, 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 easily. Now the knee joint was carefully 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 mini-mum amount of damage to the muscle. The completely dissected muscle was then weighed immediately on a Roller-Smith Precision Torsion Balance, and transferred immediately to the test tube containing the bathing fluid. Fow a few of the inulin space studies, m. peroneus longus was similarly excised by loosening and cutting the tendinous ends. Small pieces of thin coloured thread were tied around the tendon of each gastrocnemius for identification purposes while being incubated. It was determined that this thread did not appre-ciably affect the radioactive counting. CHEMICAL METHODS 1. Extracellular space estimations The polysaccharide inulin has been used i n this study to 32 estimate the volume of extracellular space in muscle. Muscles, dissected and weighed as above, -were incubated for 4 hours in a 1% inulin bathing solution. At the end of the incubation per-iod, the muscles were rinsed b r i e f l y in demineralised water, blotted, and weighed rapidly; they were then placed in small clean mortars, 1.0 ml. of 12% TCA and some acid-washed sand added, and ground with a pestle. 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 inulin i n vivo, b i l a t e r a l ligation of the renal pedicle was carried out under ether anaesthesia, and 0.3 ml. of 25% inulin solution injected under the skin of the back. After a variable period of time to 3j hours), the animals were k i l l e d by exsanguination from the heart, and the gastrocnemii and/or peroneii longii routinely excised, rinsed, blotted, weighed, and further treated as described above. The blood sample taken from each animal was placed i n a tube containing a small known amount of heparin solution, centri-fuged, and 0.1 ml. aliquots of the plasma treated with TCA and assayed for inulin. Aliquots of the muscle supernatant solutions were taken and analysed by the method of Hubbard and Loomis (1942). A stan-dard inulin solution was prepared from the incubation solution, and portions of this were further diluted to give varying concen-trations of inulin for use as standards for colourimetric analysis. Five standard tubes were prepared, with 1.0, 0.75, 0.50, 0*25, and 0,0 ml. of dilute inulin 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 into test tubes also. To each tube was added 0 . 2 5 ml. 12% TCA, 1.0 ml. resorcinol, and 3.0 ml 8 of 30% HCL. This mixture was shaken well, heated i n an 80°C. water bath for 8 minutes, and then placed to cool for 30 minutes in ice water. The contents of a l l the tubes were diluted to 10 ml., mixed well by inversion, and colourimetric values read at 510 m i l l i -microns i n a Klett-summerson Photoelectric Colourimeter. As a check on the extracellular spaces estimated by the inulin method, estimations of thiocyanate space were done on some normal mouse muscles, to see i f there was a significant difference between the apparent inulin spaces and the apparent thiocyanate spaces. The m. gastrocnemii were routinely excised, weighed, and incubated in v i t r o in the thiocyanate bathing solution for a per-iod of two hours. The muscles were then ground and centrifuged, and assayed for thiocyanate by the method of Crandall and Anderson (1934). Standards were prepared by making 2.0, 1.0, 0.50, and 0 .25 ml. portions of dilute thiocyanate standard (1.0 ml. bathing solution 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 solution. To every test tube 2.0 ml. of f e r r i c nitrate reagent was added, and the mixture shaken well. The resulting colour was read immed-iately at 510 milljjnicrons i n a Klett Summerson Colourimeter. The thiocyanate content of the muscles could then be calculated with reference to the standard solutions. 2 . Dry Weight Determinations For the determination of percentage water of representa-34 tive muscles, the following procedure was employed: (a) Small metal planchets were washed, dried, heated for 2 hours at 110°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 in the dessicator u n t i l used. (b) Each muscle was excised i n the routine way, placed on the planchet, and rapidly weighed on the same balance. (c) Wiien a number of muscles were ready, they were placed i n a 110°C, constant temperature oven, dried for one hour, cooled i n a dessicator, and weighed carefully. They were then reheated, cooled and weighed to constant weight. 3. Non-Radioactive Incubation Experiments The mice were sacrificed, and the gastrocnemii excised as previously described; the muscles were then immediately weighed, and transferred to the selected bathing solution. The bathing f l u i d was contained in large glass test tubes suspended i n a constant temperature (33°G.) water bath. The bathing solution was oxygenated and circulated i n the test tubes with a 35fd02~^cC02 gas mixture. After a variable time of soaking, the muscles were retrieved from the bottom of the tubes, immersed b r i e f l y (less than 2 seconds) in d i s t i l l e d water to remove any adhering drops of b athing solution, blotted l i g h t l y on dry f i l t e r paper, transferred to acid rinsed boro-silicate test tubes, and digested with 4 drops of concentrated n i t r i c acid. The tubes were kept tightly corked u n t i l ready for analysis. 35 The samples were heated sli g h t l y to ensure the complete digestion of the muscles. The n i t r i c acid solutions were diluted with a few ml. of demineralised water, and transferred into grad-uated "Pyrex" cylinders; an additional few ml. were added to the digestion tube, and these rinsings added to the cylinder. The solution was diluted 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 plastic funnels into test tubes. Two 1.0 ml. aliquots of the f i l t r a t e were placed i n separate test tubes; to one was added demineralised water to give the amount of dilution necessary to bring the concentration of sodium into a range suitable for analysis (usually 2 to 10 ml.); to the other 1.0 ml. aliquot 1.0 ml. of 0.25 M NaCl and the same amount of water for the K determination were added. The amounts of Na and K i n these solutions were determined with the aid of an Evans Electroselenium Flame Photometer. A l l analytical results are expressed in terms of milliequivalents 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 this investigation, with the soaking experiments, extracellular space determinations, and dry weight analyses a l l ancillary to the isotope flux studies. The isotope K^2 was used as a tracer; whenever the radioactive solu-tions were to be handled, gloves were worn to protect the hands from contact, and a l l work was done over an enamel tray lined with t i n f o i l and absorbent paper. After each experiment or each dilu-ting operation involving the radioisotope storage bottles, the 36 entire area was thoroughly monitored with an end-window portable Geiger counter. At a l l times, extreme care was used to limit any contamination by the active solution of equipment used (such as glassware) to a bare minimum. The main disadvantage of using the K*2 was i t s short h a l f - l i f e . Every 24 hours 74$ of i t disintegrated, and after 4 days, the level of activity would be down to 0,40$ of the original value. Thus ike experiments had to be done in the f i r s t three or four days after receipt of a shipment of the K^2. There was no disposal problem, for the radioactive solutions were kept for 3 weeks after 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. This active solution was the medium for the influx studiesj i t was placed in a "Pyrex" test tube in a 33 C. water bath, and mixed and oxygenated with a 95$02-5$CO2 gas mixture. The muscles were routinely excised, rapidly weighed, and placed in the solution to incubate;- after approximately 10, 30, 60, 90, 120, and 150 minutes of incubation the muscles were removed from the solution with a curved glass rod, and placed for 1,0 minutes in a beaker containing 200 ml. of non-active solution of the same composition as the incubation medium. This short soaking period effectively reduced the extracellular K^2 to ca. 50$ of i t s previous le v e l (McLennan 1955), thus lessen-ing the error i n the counting of the fibre K. The muscles were then blotted b r i e f l y on f i l t e r paper, and arranged on a plastic counting 37 tray i n a standard reproducible position. The tray and muscles were then placed under a Geiger-Muller end-window counter and counted for three one-minute intervals. The impulses from the counter were transmitted to an electronic 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 boro-silicate test 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 diluted digest was set i n a sample holder tray, and placed under the Geiger counter to be assayed for radioactivity. After the counting period was finished, the planchet was removed from the sample holder, and the digested muscle mixture poured back into the boro-silicate test tubej the planchet was rinsed twice with 2.0 ml. portions of demineralised water, these added to the test tube, and the tube tightly corked u n t i l used for flame analysis. A 0.1 ml. aliquot of the bathing solution was placed i n a metal planchet, diluted with 1.9 ml. water, and similarly assayed for radioactivity. Thus, a relationship between the amount of K^ "2 i n the muscle and that i n the bathing solution would be established. After 2 to 3 weeks, when the radiation was at a low level, the muscles were assayed for total sodium and potassium by flame photometry. SECTION III RESULTS A. Extracellular Space Estimations 1. Muscle Inulin Space: Normal Strain 129 mice 2. Muscle Inulin Space: Dystrophic Strain 129 Mice 3. Muscle Inulin Space: Swiss Albino Mice 4. Muscle Thiocyanate Space: Normal Strain 129 Mice B. Dry Weight and Non-Inulin Space Water Estimations 1. Dry Weight Determinations 2. Non-Inulin Water Estimations C. Chemical Analyses 1. Fresh Muscle 2. Incubated Muscle: Solution 1 3« Incubated Muscle: Summary of Results of incubation in solutions 2 to 6 4» Conculsions D « Results of K 4 2 Uptake Studies 1. Normal Mouse Skeletal Muscle a) Effects of Varying K e b) Effects of Varying Na^ c) Effects of Varying K e and Na e 2. Dystrophic Mouse Skeletal Muscle a) Effects of Varying E e b) Effects of Varying Na e 3. Summary of K 4 2 Results 38 A. EXTRACELLULAR SPACE ESTIMATIONS Intracellular ion concentrations cannot be estimated without making adequate allowance for the ion content of the extracellular f l u i d . To make this correction, the volume of the extracellular space (ECS) and the concentration of the ion i n the extracellular f l u i d must be known. The calculated amount of ion present in the ECS can then be subtracted from the total muscle content to give the intracellular value. Muscles from normal and dystrophic animals were assayed for ECS as described on page 28 but the averaged results from groups of muscles were found to be variable from group to group; small muscles seemed to have larger ECS values than larger ones, and muscles from the dystrophic ani-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 Fig. I, i.e., there was a negative correlation between muscle weight and magnitude of the ECS. 1 . Muscle Inulin Space: Normal Strain 129 Mice Gastrocnemii from normal strain 129 mice were soaked for a standard time of four hours i n an inulin-containing solution; the muscles were analysed for inulin content, the raw data corrected for muscle swelling during incubation, and the results divided by the muscle weights to give the amount of inulin per gm. of muscle; this was expressed as . mg. inulin per gm. fresh muscle . ————————_____________________ X lOCyo mg. inulin per ml. soaking solution Fig. I i s a graph obtained when the inulin spaces of 34 muscles were plotted against the fresh muscle weights. On inspec-Muscle Fresh Weight, mg 40 tion, there was an obvious relationship between the size of the muscle and i t s inulin space, with the space decreasing with i n -creasing muscle size. The calculated regression line (as drawn ± in Fig. I) i s y - 44,7 - 0.43x (S.E.~ 1.1). However, i t was considered that excised muscles incuba-ted i n an a r t i f i c i a l f l u i d might not have the same inulin space as muscles in s i t u . In order to examine this possibility, normal strain 129 mice were nephrectomised and injected with inulin solu-tion subcutaneouslyj after a variable time, gastrocnemii and/or peronei longi were excised and analysed. (Peroneus longus was a small muscle, with an ECS similar to the gastrocnemius, and i t provided a useful check on the values previously found for the gastrocnemii.) Plasma samples were taken and assayed for inulin content; each plasma value was used as the divisor for muscles from the same animal, i n place of the soaking solution value. Results were calculated as described above, and plotted similarly. F i g . II illustrates the results obtained; values from both the gastrocnemius and the peroneus longus muscles are plotted; the calculated regression line of the 23 points i s y = 54.0 - 0.472x (S.E.* 2.8). The lines i n Figs. I and II do not differ s i g n i f i -cantly. This result indicates that incubated muscles have inulin spaces s l i g h t l y less than but not significantly different from those of fresh muscles; at least part of the swelling noted during incu-bation appears to occur i n a region of the muscle that i s not available to inulin. 2. Muscle Inulin Space: Dystrophic Strain 129 Mice There was only a limited number of dystrophic mice ava i l -able for this investigation, and i n the ligh t of the previous results 41 Fig.JL Inulin Space of normal Mouse Muscle: Injection Method. * Gastrocnemii x Peronei Longi Figures in parentheses show the number of hours between injection and sacrifice of the animals. x(3) (3) X ( | /2) X ( 2 ) (2 1/2) • ( l )»x (2 ) # (3) •(1/2) (3 1/2) • ( 2 1/2) ( 3 ) « 12) — i 1 1 1 1— 20 40 60 Muscle Fresh Weight, mg. 42 with normal mice, i t was decided to pool the results obtained on dystrophic tissue by the incubation and injection methods. Since the peronei longi were too small to excise intact, only the gastro-cnemii of these dystrophic animals were used; Fig. I l l i s a graph of the dystrophic muscle inulin spaces plotted against the muscle weight. The negative correlation described for normal muscles i s also observed in these 17 dystrophic muscles, but there i s a greater dependance of the inulin space on muscle weight. The calculated regression line i s y » 94.6 - l a65x (S.E.~ 2.8). It should be noted that some of the smallest muscles had inulin spaces as high as 30%; this may have been due to a permeability of the muscle membrane to i n u l i n . If this was the case, and the inulin could penetrate into the degenerating muscle cells i t would account for the high inulin space values observed. 3. Muscle Inulin Space: Swiss Albino Mice Inherited muscular dystrophy i s unknown i n Swiss albino mice. Some analyses of gastrocnemii from Swiss mice were done to eliminate the poss i b i l i t y that the previously observed results were a peculiarity of the 129 strain. Twenty-three muscles were excised, soaked, and analysed i n the usual way. A relationship between muscle weight and inulin space similar to that observed i n the strain 129 muscles was noted (see Fig. HT). The calculated regression line i n + Fig. 17 i s y o 39.8- - 0.249x (S.E." 1.2). This line i s not signi-ficantly different from that of either Fig. I or Fig. I I . 4. Muscle Thiocyanate Space; Normal Strain 129 Mice Some thiocyanate (SCN) estimations were done on normal strain 1 2 9 mice, as a check on the inulin space results. Twenty-three muscles were excised and incubated as usual, and analysed for 43 o + 0 i 1 1 1 -i 10 20 30 40 50 Muscle Fresh Weight, mg 0 _ 1 ~JZ r — i 1 1 r~ 1 1 r — 0 2 0 4 0 6 0 80 w M u s c l e F r e s h W e i g h t , m g . 45 SCN. These results are illustrated i n Fig. V; the calculated regression line drawn i s y = 35.7 - 0.196x (S.E.~ 1.2), and tiae slope of the line i s , i n this case, not significantly different from zero, but also i s not significantly different from those of Figures I, II, or I?. However, there appeared to be a trend for 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 intracellular ion per kilogram fresh tissue weight, for which purpose a knowledge of the to t a l water content and the volume of the ECS of the tissue was necessary (see previous section). Muscles were excised, weighed and treated as described on page 33; a series of muscles was also incubated for several hours before the dry weight assay; Table VI i s a tabulation of the average percentage weight loss, expressed as weight loss x 100$ fresh weight None of the averages l i s t e d i n Table VI are significantly different from one another. The fresh and incubated muscles seem to have the same total water content of 76$; also, no significant 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 diffusible inulin, either after injecticaa of inulin into the nephrectomised animal, or following incubation of an excised muscle i n an inulin-containing solution. The available inulin space, usually considered as being identical to the ECS, was determined as described on page 28. The non-inulin space water was Fig. V. Thiocynate Space of normal Mouse Muscle In Vitro Method. 50 n Muscle Fresh Weight , mg TABLE VI: WEIGHT LOSS OP FRESH AND INCUBATED MUSCLE. INCUBATION SOLUTION HOURS INCUB. MUSCLE TYPE * PERCENTAGE WEIGHT LOSS FRESH 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 this 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 this was expressed as a percentage of the fresh muscle weight. Table VII contains the average non-inulin space water values obtained for 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 significatnly different from the soaked muscle value. The dystrophic muscle non-inulin space water was less than half the normal muscle value for fresh tissue; following incubation for 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 reflect 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 in inulin space with incubation, and the constancy of the to t a l tissue water, suggests that during incubation the muscle fibres 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 inulin molecule; inulin could enter the fibre, and the apparent inulin space would be larger than the "true" space. Zierler (1957) reported that normal rat muscle was permeable to the enzyme aldolase (MW147,000); he also reported that dystrophic mouse muscle had a higher efflux of aldolase than did normal muscle (Zierler 1958). It seems possible that i f normal muscle were per-TABLE VII: PERCENTAGE NON-INULIN SPACE WATER. OF FRESH AND INCUBATED MUSCLE*. INCUBATION HOURS MUSCLE PERCENTAGE SOLUTION INCUB. TYPE** NON-INULIN WATER FRESH - N 46.8 4.6 D 20.9 5.9 1 2 N 58.4 8.0 4 N 65.3 1.8 D 21.6 6.5 2 4 N 64.2 3.0 Expressed as percentage fresh tissue weight. N = Normal muscle, D = Drystrophic tissue. 50 meable to aldolase, i t could likewise be permeable to the much smaller i n u l i n molecule (MW991). C. CHEMICAL ANALYSES Na and K analyses of 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 concentrations unless a corr e c t i o n f o r the ion content of the ECS i s made. Also, i f i n -cubated muscles swell, there are l i k e l y to be ions from the incu-bation media i n the water of sw e l l i n g . Correction of muscle analysis data f o r these factors should y i e l d the i n t r a c e l l u l a r i on per u n i t weight of t i s s u e , per l i t r e of i n t r a c e l l u l a r water, per gram of f a t - f r e e d r i e d s o l i d s , or i n other ways, providing the parameters can be c a l c u l a t e d . The a n a l y t i c a l r e s u l t s i n t h i s thesis w i l l be expressed as m. equiv. non-inulin space ion per Kg. f r e s h t i s s u e . This i s probably comparable to the i n t r a c e l l u -l a r i o n content i n fresh muscle, but with incubated muscle one must account f o r the apparent increase i n non-inulin space due to swelling, and the i n t r a c e l l u l a r i o n content and non-inulin space ion content may not be s i m i l a r . Analyses were performed on f r e s h and incubated normal and dystrophic muscle t i s s u e to determine the i n t r a c e l l u l a r (non-inulin space) Na and K concentrations. The muscles were treated as des-c r i b e d on page 34, the raw data corrected f o r i n u l i n space and water of swelling ion content, and t h i s r e s u l t divided by the appropriate t i s s u e weight. The ca l c u l a t e d r e s u l t s are. expressed as m. equiv. ion/Kg fresh t i s s u e ; the standard deviations of the values are al s o given. Tabulated r e s u l t s of the Na and K analyses appear i n Table V I I I . TABLE VIII: Na, K CONTENTS OF FRESH AND INCUBATED MOUSE MUSCLE. Results expressed as m.equiv. non-inulin space ion per Kg. of fresh tissue. INCUBATION SOLUTION HOURS INCTJB. MUSCLE TYPE Na K S.D. Na K FRESH N D 30.0 89.9 5.2 5.2 58.2 68.2 11.4 11.1 N D 31.6 77.4 90.6 60.0 7.8 4.7 2.6 4.2 N D 41.3 77.1 57.6 63.4 19.1 13.3 4.0 8.5 N D 34.6 78.2 72.1 47.3 8.8 11.2 6.6 6.6 N D 69.5 95.1 37.4 39.7 12.0 23.8 8.9 3.5 (CONTINUED OVERLEAF) (CONTINUED PROM PREVIOUS PAGE) INCUBATION SOLUTION HOURS INCUB. MUSCLE TYPE Na K S.D. Na K 2 N 43.0 90.6 10.6 11.9 3 D 73.5 80.4 9.7 7.4 4 N 41.2 62.3 10.9 6.3 D 82.4 91.8 9.2 7.6 4 2 N 110.1 60.8 8.2 6.6 4 N 134.0 41.2 16.4 9.2 D 126.1 62.4 4.5 4.3 5 4 N 14.1 27.2 2.5 3.2 D 17.-7 40.6 2.0 2.5 6 4 N 45.9 35.5 10.2 1.6 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 for the intracellular ions (Na^ and K^) are significantly different from the normal muscle values (p<0.001)j this gain of Na and loss of K i n dystrophic tissue might be caused by a leakage or altered permeability of the muscle membrane, result-ing i n an in a b i l i t y to retain or exclude certain ion species; i f the Na and K ions were to diffuse down their concentration gradients across such a leaky membrane, the cellular K would be expected to f a l l , and the cellular Na to r i s e . This "membrane leak" would be one explanation for the observed alterations in the cellular ion contents. 2. Incubated Muscle; Solution 1. Incubation of excised normal muscle in Locke's solution (Solution 1, Table II) did not significantly alter the non-inulin space Na or K content from that calculated for 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 significant (p<0.01). After 4 hours soaking, normal tissue Na had risen 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 for 2 hours i n Locke's solution, and the intracellular (non-inulin space) ion con-tents w i l l not have changed. The ion levels are not stable for a 54 longer period, however, for after 4 hours soaking there are signi-ficant alterations i n the intracellular Na and K. Apparently the changes in ion content that occur are the same i n both types of tissue, that i s , the Na rises and the K f a l l s . The dystrophic muscle seems to undergo these changes more rapidly than the normal muscle; this may be a reflection of the larger ECS of the dystrophic tissue, or a consequence of an abnormal permeability of the dystrophic fibre membrane. 3. Incubated Muscle: Summary of Results of Incubation i n Solutions  2 to 6. The data for 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 rise and K to f a l l , as mentioned above, was noted as occurring generally in most muscles incubated in the altered media; the change was especially marked following 4 hours' incubation. Muscles incubated i n altered K e solutions gained Na; the gain was less i n 12 mM K e than i n 0 mM K solution; the dystrophic muscles seemed to gain Na more rapidly, and the gain appeared to occur earlier than i n the normal tissue. Incubation of normal muscle in the altered K media led to a decrease i n muscle K; the loss was greater i n the K-free solution, and less i n the 12 mM K e solution, than observed i n the "reference" Solution 1. Dystrophic tissue K rose in the 12 mM. K e solution, but rapidly f e l l i n the K-free medium. During the f i r s t 2 hours of incubation, the K^ value for normal muscle in the 12 mM K was constant, but in the e ' K-free medium i t f e l l somewhat; after 4 hours soaking, normal muscle K was significantly lower than the fresh value. 55 Solution 4 contained 288 mM Nae, and was hypertonicj as expected, both types of muscle gained Na during incubation; normal muscle K had dropped significatnly after 2 hours of soaking, and after 4 hours, both normal and dystrophic tissue K was significantly lower than the fresh tissue value. Soaking excised muscle in 18 mM Na a lowered Na and K i n both types of tissue, a greater drop in Na occurring i n the dystrophic tissue than i n the normal, but less of a decrease in K^. Solution 6 was a phosphate buffered solution; i t was used in 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 aid the cellular 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 levels also changed during the 4 hours incubation, the changes being greater i n this solution than in Solution 1. On the basis of these results, i t was decided not to use this Solution 6, or variants of i t , for incubation purposes. 4. Conculsions It was apparent from the foregoing results -that muscle K levels were relatively stable for at least 2 hours of incubation, at least 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 interpret radioactive K (K#) data i n the l i g h t of the Harris diffusion-permeation hypothesis, for in order to calculate and plot a normal K exchange relationship (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 . It was decided to l i m i t the inter-pretation of K-8- exchange studies to the data obtained during the f i r s t 2 hours of incubation in the radioactive media, for correc-56 tions could not be made for changes that occurred after this time. Consequently, only the uptake of K# could be followed; efflux studies would necessitate a previous loading of the tissue with radioactive K by non-incubation methods, and this was not attempted. If the exchange follows diffusion principles, the efflux graph should be similar i n form to the influx graph. D. RESULTS OF K 4 2 UPTAKE STUDIES AO The radioactive isotope K7 was used as a tracer in the experiments reported i n this section; the studies were done to determine the rate at which radioactive K (K#) i n solution ex-changed with intracellular K, and Aether alterations i n the ionic composition of the incubation solutions would affect the exchange rate. The methods of incubation, radioactive assay, and chemical analysis were described on page 35. In this section, exchange means the movement of an ion occurring i n the absence of a net change in tissue ion content (the exchange of K, as followed by the uptake of K 4 2, w i l l be the only exchange process reported); the Harris diffusion-permeation hypothesis states that the exchange of tissue K should follow a diffusion curve i f the K# uptake i s plotted against the expression (K e x time); i f the square root of this expression is used, the resulting curve should be a single straight line for most of i t s extent. A H K* uptake points should f a l l along this l i n e , and be independant of the external K concen-tration providing the intracellular K level i s constant. It was thought that this method of expressing K* uptake results might pro-vide evidence to support or repudiate the Harris hypothesis; i f the "diffusion-permeation" concept (see Introduction, page 16) i s an accurate representation of the conditions existing i n skeletal 57 muscle tissue, these data may help to elucidate the kinetics of ion movements in muscle. Preliminary experiments showed that tissue K levels remained f a i r l y constant under most conditions for the f i r s t 2 hours of incubation (Table ¥111 and Burr and McLennan (1961)), and were comparable to fresh tissue values. Data from the K^2 exchange experiments were plotted as m. equiv. K# per Kg tissue (ordinate) against (K *t)2 (abscissa), a correction having been applied for the amount of K* remaining i n the ECS, assuming 50% removal during the timed 1.0 minute wash (McLennan 1955). The method of plotting is i l l u s t r a t e d in Fig. VI; the slope of the calculated line i s expressed as m. equiv./Kg/(mM • min)2. Comparison of the slopes of the calculated regression lines enabled an estimation of the relative rates of K exchange under the various experimental condi-tions to be made. 1. Normal Mouse Skeletal Muscle (a) Effects of varying K » The rise 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 radioactivity w i l l be the sum of the exchange and the net gain. F i g . VI shows the results from 19 experiments, with data from muscles incubated i n 2 mM, 6 mM, and 12 mM K e solutions a l l plotted on the same graph; i t i s apparent by inspection that the points cluster around a straight l i n e , and the calculated regression line drawn in the Figure has a slope of 0.98 m. equiv./Kg/(mM • min) 2 (S.E." 0.03). Muscle K exchange observed in these incubation media compares favourably to that predicted by the Harris diffusion-permea-tion model, in which the rate controlling step i s equilibration with-58 Fig yi_ Uptake of by normal Mouse Muscle: Normal and altered K e Incubation Media. 2 mM K e - • 6 mM K e - o 12 mM K e - x ( Ke • t )l/2 (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 led to a net increase i n Kj_. Fig. VII is a graph of the K* uptake of normal muscle tissue in the high-K media; results from 4 experiments were plotted; there were not enough points to calculate a meaningful regression line, so the solid lines i n the Figure are freehand estimations of the slopes of the 24 mM (lower line) and Hie 48 mM solution (upper line) uptake curves; the slopes of the linear portions of the curves are 2.65 and 3.11 m. equiv./Kg/(mM • min)2 respectively. The dashed line i s the regression line from Fig. VI. Both high-K lines are obviously different from the normal l i n e . The net gain in Kj_ can be calculated from the K 4 2 data; Table IX AO compares the chemical analysis data and the calculated TL* data for 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 4 2 data was calculated as 80 m. equiv./Kg; comparable values for 48 mM medium were 120 and 126 m. equiv./Kg. If the K analysis values for each K-* uptake reading were calculated, and the net gain subtracted at each K# point, one would predict that the corrected points would f a l l along a straight line, 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 similar to that observed in the normal K media, i f e ' appropriate correction i s made for the net K gain; the K exchange seems to be a diffusional phenomenon, but i t i s obscured in the 60 F ig . vij_ Uptake of K by normal Mouse Muscle: High - K Incubation M e d i a . 2 4 meg. / L K - • I I I I 1 L_ 0 20 40 60 ( Ke • t ) ! / 2 ( mM min.) l/2 TABLE IX: EXCHANGE AND NET GAIN OP K IN NORMAL MUSCLE : HIGH -Ke INCUBATION MEDIA. Values i n m.equiv./Kg. fresh tissue. K e 1 NET GAIN BY ANALYSIS (1) * 2 K AFTER 2 HOURS (PIG. VII) (2) * 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 for Na^ and as uptake of KC1. (b) Effects of varying Na . The interdependance of Na and K ions i n muscle has been repeatedly demonstrated, and changes i n the external concentration of one ion can affect the movement of the other (see Introduction). Muscles were incubated i n two altered Na solutions, one with 18 mM e ' Na , the other with 288 mM Na . The latter solution was hypertonic e e e i If K# uptake i s plotted against the appropriate (K Q* t ) 2 values, the resultant points f a l l along two lines, as illustrated i n Fig. VIII. The dashed line i s from Fig. VIj the solid line above i t i s the regression line for 8 experiments measuring K uptake i n 288 mM Na e medium; the slope i s 1.02 m. equiv./Kg/(mM•min)2 (S.E.-0.08). The low-Na regression line i s significant at the 1% level i f the uptakes i n normal (144 mM) Na and low (18 mM) Na are com-pared. The tendency for exchange to be slightly increased i n high-Na incubation media i s not significant. The tissues were losing K in both media, but appeared, on the basis of the 4 hour incubation v a l -ues, to be losing i t more rapidly i n the 18 mM Na solution. The unknown interaction between K leaving the muscle and the K# from the ECS attempting to enter and exchange with the might affect the net uptake of K#, and alter the slope of the uptake curve; the K# uptake appears to be reduced in the 18 mM Na g solution, and to be sli g h t l y raised i n the 288 mM solution; the reduction may have been due to this interaction, while the gain i n 288 mM medium could have been influenced by the large rise i n Na i observed even after 2 hours incubation in this f l u i d . (c) Effects of varying K and Na e e A few experiments were performed using combinations of 62 40 r F i g . VjU U p t a k e o f K 4 2 by M o u s e M u s c l e A l t e r e d N a e I n c u b a t i o n M e d i a . 18 m M N a e - o 2 8 8 m M N a e — x D a s h e d L i n e i s f r o m F i g . v i 30 20 > a-a> 0) _» 00 00 10 0 X X X X X X (mM • min) 63 increased and decreased Na_ and K_ levels. 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 illustrated i n Fig. IX w i l l be reported. (Normal K e «= 6 mM, normal Na e = 144 mM.). In 12 mM K-288 mM Na solution, the line of points seems to be s l i g h t l y above the "normal" regression line, and approximates the upper s o l i d line i n Fig. VIII. One experiment i n 2 mM KQ - 18 mM Na Q medium showed the K exchange to be below the "normal" l i n e , and to approximate the lower line in Fig. VIII. Incubation i n 12 mM K e - 18 mM Na Q solution resulted i n the plotted points lying between the normal regression line and the lower line i n Fig. VIII. Chloride-free medium (sulphate replacing Cl) was used for two experiments. With 6 mM K the uptake line was sli g h t l y below the normal regression line, but with 12 mM K_ approximated i t . These results correspond to those reported earlier, that i s , slight alterations of K did not seem to affect the rate of K e exchange significantly; altering Na_ as well as K should have 6 6 produced a curve similar to those seen previously for altered Na Q alone; 2 mM or 12 mM K_ should not affect the uptake appreciably. The experiments with Cl-free media yielded an uptake curve resembling the nontial one, indicating that chloride may not exert a significant effect on K exchange i n muscle. Harris and Sjodin (1961) stated that K exchange was somewhat reduced in Cl-free media; "the reduc-tion 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 Cl-free media. 64 Fig.JX. Uptake of K by normal Mouse Muscle : Altered N a e and K e Incubation Media. x 12 m M K e - 2 8 8 mM N a e o 2 m M K e - 18 mM N a e e 12 m M K e - 18 m M N a e • Chlor ide — f ree medium Dashed line is normal uptake s lope 4 On 30-20 S1 > 3 cr CO CO 10-X / x / y X • x A . • X / o x x O O ~ l — 10 —T— 20 ~40 0 30 (Ke • t ) £ (mM • min.)^ 65 2. Dystrophic Mouse Skeletal Muscle (a) Effects of Varying K Thirteen experiments were performed with muscles from dystrophic animals. Muscles were incubated i n 2 mM, 6 mM and 12 mM K Q incubation media, and the K* uptake plotted; Fig. X shows the experimental values calculated, the dystrophic regression line (solid), and the normal tissue regression lin e (dashed). Again, the points f a l l along a single li n e when plotted against i l . (K e • t ) 2 ; the slope of the line i s 1 033 m. equiv./Kg/(mM • min) 2 (S.E.* 0„06), as compared to 0,98 for the normal tissue. The slopes of the lines are significantly different (p <0 o01). (b) Effects of varying Na Q A few experiments have been performed using altered Naa media; the results for IB mM and 288 mM Na Q media are plotted i n Fig. XI; i t can be seen that K* uptake tends to be somewhat more rapid i n the 288 mM medium, for the slope of the line i s 1.80 m. equiv./Kg/(mM • min) 3 (S.E.~ 0o04) as compared with 1.33 for dystrophic tissue in normal E solutions. The difference between this slope and the normal slope i s significant (p<0,01). The 18 mM Na medium exhibits a definitely slow K# uptake, for the slope of this line i s 0,81 (S.E,- 0,04), considerably less than the 6 mM dystrophic tissue l i n e ; the difference between the two lines i s significant (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 results were plotted as K-a- uptake against (K Q • t ) 2 ! , as described by Harris and Sjodin (1961). The uptake rate for normal muscle in these solutions was 0.98 m. equiv.K*/Kg/(mM • min) 2; the comparable rate for dystrophic 66 F ig . X.: Uptake of K by Dystrophic o Mouse Musc l e : Normal and o x l i i i i i 1 0 i 1 0 2 0 3 0 ( Ke -t ) '* ( mM • min.) 67 Fig._<l_ Uptake of K by Dystrophic Mouse Muscle. Altered Na e Incubation Media. • 18 mM Na e o 288 mM N a e Dashed line is dystrophic regression line from figure JT 0 H 1 1 1 0 10 2 0 3 0 ( Ke • t)^2 (mM • min.) ^2 68 muscle was 1.33, one-third more than for normal tissue. In high-K media, normal muscle accumulated K; this was reflected in a very high uptake rate of 2.65 m. equiv./Kg/ A (mM • min) 8 for 24 mM K g and 3.11 for 48 mM K Q incubation media. Doubling Na led to an increase in the rate of uptake i n both normal and dystrophic tissue, from 0,98 m. equiv./Kg/ (mM • min) to 1,02 for the former, and from 1,33 to 1.80 for the la t t e r . Incuba tion i n 18 mM Na slowed uptake to 0.80 i n normal muscle, and to 0,81 in dystrophic muscle. The effect noted in 18 mM Na medium may be due to interaction between K being lost from the c e l l during incuba-tion (see Table VIII) and the K* entering the c e l l . Harris and Sjodin (I960) found that altering Na e (replacing any d e f i c i t with sucrose) did not appreciably affect the rate of K* uptake; a slight tendency for enhanced or decreased uptake (as compared with normal uptake) might be due to cation interaction in those cases where the muscles are gaining or losing K. This faster rate of Re-uptake in 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, resulting in a rapid uptake of K. There has been a report of increas ed permeability of dystrophic tissue to aldolase (Zierler 1958); possibly the capacitative membrane or region of the dystrophic c e l l i s changed in some way, enabling the K in solution to equilibrate within this resistive layer more rapidly than usual, thus f a c i l i t a t -ing i t s diffusion into the interior of the c e l l . The mechanism by which equilibration within this region occurs does not seem to have been affected by the dystrophic change, for the uptake curve i s s t i l l a straight line, even though the rate of uptake i s increased. SECTION IV DISCUSSION A. Tissue Compartments B. Chemical Analyses: Presh and Incubated Muscle 42 C. K Uptake Studies 42 1. K Uptake of Normal Muscle (a) "Normal" K Solutions © (b) The Effect of High K Media © (c) The Effect of Low K Media © (d) The Effect 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 129 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 sub-clinical muscle defect. However, ECS assay on muscles from Swiss mice yielded results similar to those found for -the strain 129 mice; the same dependency of ECS to muscle size was noted, and it 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 fibres were to expand by growth, they would f i r s t encroach on the ECS surrounding them, and a limited growth of 5 to 10$ might occur without an increase i n the volume of the muscle; this factor may account for a large amount of the variation 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 in the myofibrils of the muscle fibre, for 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 fibre enlarge. This myofibrillar (and subsequent fibre) growth would be grad-ually 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 prob-ably affect the ECS, for the ECS of tendon i s much larger than that of muscle (Manery and Hastings 1939). As the muscle increased in length, the relative amount of tendon might not increase i n pro-portion to muscle size, and the total muscle ECS would be less i f compared to developmentally earlier ECS values. These factors would also be related to the "packing" of the muscle fibres which seems to occur during development; the fibres attempt to occupy the least 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 belly. The muscle inulin space appeared to be less following incubation, for although the slopes of the regression lines for the in vAvo and injection methods were essentially the same, the injec-tion ECS values tended to be greater than the incubation values for the same size of muscle. It also appeared that the non-inulin space increased during incubation; the muscles swelled during incubation 71 and although a correction was deducted for this swelling, a portion of the ECS seems to have been added to the non-inulin (non-ECS) space. It 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 inulin, which penetrates into the sarcoplasm, and occupies the region inside the sarcolemma that i s not occupied by myofibrils. Equi-libration of this region (which apparently contains the sarcoplas-mic reticulum) may take several hours, resulting in the slow increase i n non-inulin space water previously noted. Zierler (1957, 1958) reported a leakage of the enzyme aldolase (MW 147,000) from normal rat and mouse musclej Cotlove (1954) showed that prolonged intravenous administration of inulin or sucrose into rats would eventually lead 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 inulin, although this does not seem to be the case for incubation up to 4 hours* duration (McLennan 1956). Muscles from dystrophic mice seem to have abnormally permeable membranes and Zierler (1958) observed a noticeably greater leakage of aldolase from dystrophic tissue. It is possible that the abnor-mally large inulin space values for dystrophic tissue reported i n this thesis are due to a penetration of the degenerating muscle membrane by the inulin tracer. The ECS of a small normal muscle would be f a i r l y high, but small dystrophic muscles have inulin spaces almost twice as large as their normal counterparts. Either the sarcolemma of the fibres has broken down, leaving the protoplasm and myofibrils exposed to the extracellular f l u i d , or the membrane per-meability has been altered sufficiently to permit entry of inulin 72 into the fibre. The smallest dystrophic muscles had inulin spaces of ca. 90$; this would indicate few functional muscle fibres i f the inulin permeable ones were completely degenerated, yet visible twitches could easily be observed upon e l e c t r i c a l stimulation of the muscle, and resting and action potentials could be recorded without d i f f i c u l t y (Burr and McLennan, unpublished observation). Thus i t seems l i k e l y that the change i n the dystrophic tissue i s in the nature of an alteration i n permeability of the fibre sarco-lemmaj this may be followed i n terminal degenerative stages of •toe fibre by complete degeneration of the membrane, effectively adding the volume of the c e l l to the extra cellular space. Percentage water loss averaged 76$ for normal muscle and 77$ for dystrophic muscle (see Table VI); these two values were not significantly different. The normal muscle water loss was the same whether the muscles were fresh, incubated i n Solution 1, or incubated in Solution 2. It seems that incubation did not affect the total muscle water content; however, when the non-inulin space water was calculated for these muscles (see Table VII), i t was found (for normal muscle) to rise from 47$ of the fresh weight for non-incubated tissue, to 58$ after 2 hours incubation, and to 65$ after 4 hours incubation. One must conclude that the non-inulin space water increases at the expense of the extracellular water. The total muscle water was also constant i n muscles of varying size; i f the ECS progressively decreased with increasing size, the non-inulin space water must have progressively increased along with the muscle size. The ECS results 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 feels that a more thorough investigation of the problem is necessary before any further conclusions regarding the v a r i a b i l i t y of the non-inulin space water with muscle weight or incubation time can be just 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 VIII (page 51); approximately 375 analyses for K and Na were done on both types of muscle. The expected distribution of ions was observed i n fresh normal muscle, for the was high and the Na^ low; 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 for ECS ion content, and thus are not s t r i c t l y comparable to the figures from Table VIII. The ECS correction for Na i s especially important, because the Na e i s a large value. Conway (1950) found that i f one assumed the intracellular sodium to be i n two fractions, one bound firmly to cellular constituents, and the other free to exchange rapidly with the extracellular f l u i d . He assumed further that the "true" intra-cellular 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 kinetic studies of Na* efflux from muscle for a discontin-uity i n the efflux curve indicated two discreet fractions of ion that differed in the time taken to leave the muscle fibre. The correction made in this thesis was the subtraction of the appropriate ion content of the 4 hour inulin space water, with no attempt having 74 been made to correct for the "fast fraction" 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 VIII). These higher Na. and lower K. levels were similar to that observed by Baker et a l (1958), who reported that K± was 76 m. equiv./Kg and Na^ 66,0 m. equiv./Kg. The percentage rise 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 half the normal figure, and Na^ to rise 60% to 60,0 m. equiv./Kg. Young et a l (1959) found similar changes in ion levels i n their normal and dystrophic analyses, with dystrophic K^ 18% greater than the normal muscle values. Analyses of muscle biopsies from human patients with muscular dystrophy (Horyath, Berg, Curamings and Shy 1955) showed Na levels to be increased and K levels to be decreased as compared to cation levels i n biopsy samples from normal i n d i v i -duals. Blaxter (1952) reported similar alteration of electrolytes in muscle tissue from dystrophic calves. It i s apparent that the Na and K differences between nor-mal and dystrophic tissue are not limited to mouse muscle, but are rather a manifestation of the dystrophic change in general. The abnormal permeability of the muscle membrane, noted above for inu-l i n , might i t s e l f account for the lowered Kj_ i n dystrophic tissue, either directly or by leakage of intracellular protein. Since a large part of the K^ i s held i n a Gibbs-Donnan equilibrium, a lower intracellular protein would be expected to reduce this effect, and thus to make K i more nearly equal to KQ. There i s the poss i b i l i t y also that this lowered protein content could be due to an abnormal cellular metabolism. Thus, for example, Milman (1954) reported 75 that glycogen synthesis was abnormal i n dystrophic animals; Rosenkrantz and Laferte (i960) observed that dehydrogenase a c t i v i t y i n dystrophic mouse muscle was greater than normal, and Weinstock, Epstein, and Milhorat (1958) found that cytochrome oxidase a c t i v i t y was increased i n dystrophic mouse t i s s u e . Other enzyme systems appear t o be a f f e c t e d as w e l l ; Kruh, Dreyfus, Schapira and Gey (I960), using C 1 4 - l a b e l l e d glycine, reported a f a s t e r p r o t e i n turnover r a t e f o r dystrophic mouse t i s s u e . Zymaris, S a i f e r , and Volk (I960) found a f a s t e r turnover rate of acid-soluble nucleotides i n dystrophic muscle and Rabinowitz (I960) reported more r a p i d lipogenesis i n dystrophic mice than i n t h e i r normal l i t t e r m a t e s . From the purely 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 not possible to support e i t h e r the increased membrane permeability hypothesis of the a l t e r e d c e l l u l a r metabolism hypothesis as being the cause of the abnormal Naj_ and l e v e l s found i n dystrophic mouse muscle. The i n u l i n space data seems t o support the former view, but i t i s d i f f i c u l t to d i f f e r e n t i a t e adequately between the two conditions. Incubation of muscle t i s s u e generally r e s u l t e d i n K l o s s and Na gain; even the 12 mM K s o l u t i o n d i d not maintain con-stant during 4 hours of incubation. I t i s b e l i e v e d (Harris i960) that c o r r e c t incubation f l u i d i o n i c concentration and corre c t i s o -t o n i c i t y are not the only conditions that must be provided to maintain c e l l u l a r s t a b i l i t y i n v i t r o ; adequate oxygen supply, s u i t a b l e temperature, and adequate substrates f o r metabolism are also needed. These requirements are e s p e c i a l l y important i n mammalian muscle, iiihich has a high rate of c e l l u l a r metabolism, and which r e l i e s h e avily on continuous oxidative metabolism f o r i t s energy supply. Creese, D'Silva, and Northover (1958), observed 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$ 02 - 5$ C02 gas mixture. The C02 that dissolved in the medium formed a buffer pair with HCO^ , and maintained the pH at ca. 7.4; the bicarbonate medium was found to maintain cellular electrolytes better than a phosphate-buffered medium (Solution 6). Creese (i960) showed that rat dia-phragms 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 al 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 Ke solutions. All the muscles incubated for 4 hours lost K; 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 for early Na loss; the continued Na loss 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 at a rate comparable to that of Na ion, but was lost more slowly; Keynes and Swan (1959) observed the same effect when L i was substituted for Na, but L i can substitute for Na in maintenance of the resting potential, whereas choline cannot. The replacement of Na by choline in t r a -c e l l u l a r l y leads to a drop i n the resting potential (Keynes and Swan 1959); this could be one cause of the excessive K loss noted in the choline incubation medium. It i s not known whether mammalian muscle fibres are permeable to choline but frog muscle i s apparently permeable to both L i and choline, and mammalian muscle may be expec-ted to show similar characteristics. 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 for K^ rise or f a l l can be calculated. The Harris permeation-diffusion hypothesis, described b r i e f l y on page 18, is used as a model to aid the interpretation of the kinetic data which have been presented i n this thesis. Under this model exchange of an ion involves two pro-cesses; f i r s t , an exchange with ions adsorbed near the c e l l surface, and second, the diffusion of ions inwards from the adsorbed layer at the same time as an outward diffusion of an equal number of i n -ternal ions takes place. The ions moving outward w i l l compete for sites i n the adsorbed region with those ions entering the region from the extracellular space. Thus the adsorbed region can act as 78 an ion exchanger, with chemically different ions such as K, Na, and Kb being interchanged, or ions of the same species being exchanged, such as the exchange occurring between labelled K ions from the ECS and unlabelled K ions from the c e l l . The equilibra- : tion of the adsorbed layer i s the rate controlling process. If 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 time) 2, a l l experimental results w i l l f a l l along a single curve (at constant temperature). 1. K 4 2 Uptake of Normal Muscle (a) "Normal" K e Solutions McLennan (1955) working with mammalian muscle, and Harris and Steinbach (1956) using frog muscle, could not f i t their kinetic 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 thin resistive membrane separating two ionic reservoirs. The results presented earlier in this thesis do not f i t this single exponential curve either, but when plotted in accordance with the Harris hypothesis, concur with his results for K-K# exchange i n frog muscle. It i s useful to consider the muscle c e l l as possessing "regions" that have a definite K capacity, and a low diff u s i v i t y for ions equilibrating 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 , for each exchange of outer layer tracer ions for non-tracer ions of an inner region w i l l delay the equilibration of the outer adsorption layer. There can be inter-ference to diffusion i n this K region, and a c e l l losing K would maintain a low K# concentration within i t s outer region, and impede 79 equilibration with the rest of the c e l l . Interaction between species of ions could also occur in this region, and influence the flux by competition for adsorption s i t e s . In Fig. VI, the uptake of K^-2 follows a single straight line (slope 0.98 m. equiv./Kg(mM • min) 3) for muscles incubated in 2 mM, 6 mM, and 12 mM Kffl solutions. It was determined (page 51, and Burr and McLennan 1961) that i n these solutions the remained constant for the f i r s t two hours of incubation; measurement of Re-uptake would therefore give a true measure of the normal exchange process, without the necessity of correcting for any net K gain or loss. The uptake curve i s linear for most of i t s length, and even at the beginning, during the i n i t i a l equilibration of the ECS with the bathing solution, which i s unquestionably a diffusion process, there is no difference between the K uptake curves in the various K e media. The K 4 2 uptake characteristics also preclude the necessity of invoking non-exchangeable fractions of muscle K, for the increase in K g does not lead to a greater degree of K being available for exchange or to a faster rate of exchange. This i s in contradistihc— tion to the "classical" interpretation of potassium kinetics (see Introduction pages 16 to 18 for a brief account of the "cla s s i c a l " view). A similar curve would be expected i f K diffusion i n the ECS were the rate-limiting step. The timed 1.0 minute wash given to the muscles after removal from the K* soaking solution reduced the extracellular K* by 50%; this i s presumably regained i n the f i r s t minute following immersion in the incubation f l u i d , for Carey and Conway (1954) observed that the wash does not affect the Re-uptake kinetics. Harris (1957) thought that diffusion in the ECS would be slower than intracellular diffusion; a comparison of his 80 r e s u l t s f o r i n t r a c e l l u l a r d i f f u s i v i t y (1954) and those of McLennan (1955) lead on to the same conclusion. Hodgkin and Horowicz (1959) stu d i e d K uptake of s i n g l e muscle f i b r e s and observed uptake curves s i m i l a r to those found f o r whole muscle. Ha r r i s and Burn (1949) c a l -culated that f r o g s a r t o r i u s would take only 15$ longer t o e q u i l i b r a t e than a s i n g l e f i b r e ; Harris and Sjodin (1961) showed that the Na d i f f u s i v i t y was even closer to the free s o l u t i o n value. The l a t t e r authors also compared data from Hodgkin and Horowicz (1959) with t h e i r f i g u r e s f o r whole muscle K exchange and concluded that turn-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 same as the mean exchange i n a whole muscle. I t appears that the s l i g h t hindrance 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 smaller f i b r e s so as to make the r e s u l t s i m i l a r to that holding f o r a large single f i b r e , (b) The E f f e c t of High K Media Incubation of muscles i n high KC1 media leads to a net gain of K. (Boyle and Conway 1941). The gain occurs f a i r l y r a p i d l y and depends on the amount of K i n the medium. A swelling of the muscle i s associated with the gain from media containing c h l o r i d e , but t h i s i s eliminated i f non-penetrating methyl-sulphate i s used instead. H a r r i s and Sjodin (1961) found that K* uptake was more r a p i d i n C l media than methylsulphate; the C l may f a c i l i t a t e K entry 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 imbalance i s incurred. F i g . VII shows the r e s u l t s of incubation i n 24 mM and 48 mM K e media. The slopes of the l i n e s are markedly steeper than the normal exchange l i n e , f o r net gain of K (as w e l l as exchange) has occurBed. The net gain and the exchange can be separated only i f net gain were known f o r each K* uptake point. Table X showed 81 the differences at the end of 2 hours soaking; the net gain 42 K values as estimated by K data and non-radioactive incubation data are similar. This would indicate that the (K • t ) a method e of plotting s e n s i t i v e l y r e f l e c t s c e l l u l a r ion le v e l s ; upon i n -spection of an uptake curve, and comparison with a normal curve, one could accurately predict whether net K gain had occurred or not© (c) The effect of Low K Media G When muscle i s exposed to a K-free medium, i t loses c e l l u l a r K r a p i d l y . The uptake curve, i n this 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 ionic movement w i l l tend to retard the rate of adsorption 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 . Harris and Sjodin (l96l) reported that the K* uptake from loaded muscles was i n i t i a l l y less than normal, and their graph of K* uptake showed a concavity during the f i r s t hour of soaking, but the remainder of the curve had essentially the same slope as a normal uptake curve. They attributed the long concavity at the beginning to the K loss occurring; when the excess K had l e f t the muscle, the exchange curve approached the usual slope. (d) The Effec t of Na e The movement of muscle K appears to be affected by alterations i n sodium concentration of incubation media (Steinbach 1950). Harris and Sjodin (l96l) found that K uptake was less when Na was lowered, but considered that this was due to a loss e of K from the tissue. Keynes and Swan (1959) showed that there 82 i s not complete independence of the Na and K fluxes, f o r alterations i n K w i l l a f f e c t Na fluxes (see also Keynes 1954)• © McLennan (1957) noted ( i n rat muscle) that incubation i n low-Na e media resulted i n less K uptake as compared to normal Na g solu-tions. The parallelism observed between Na and K movements suggests that their movements are linked i n some way. But the movement i s not always a r i g i d 1:1 exchange of Na for K or vice versa. McLennan (l957) found the ratio to be closer 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 results of K uptake i n altered Na media are shown i n P i g . VIII. The regression l i n e of the 288 mM Na medium points © i s not s i g n i f i c a n t l y different from the normal l i n e , but the 18 mM Na l i n e is- significant at the 1% l e v e l . In 288 mM Na , the e e muscles were rapidly gaining Na, but were also losing some K. This doubling of the incubation medium Na g did not affect K uptake by mouse muscle; the same phenomenon was observed i n frog muscle by Harris and Sjodin ( l 9 6 l ) . The 18 mM Na solution caused a large 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 less than the normal curve, and this can be attributed to the interaction 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 layer, or the exchange from this region to the c e l l i n t e r i o r , may be hampered by the large amount of cation leaving the c e l l . Again, this concurs with the r e s u l t s reported by Harris and Sjodin ( l 9 6 l ) . 83 42 2. K Uptake of Dystrophic Muscle 42 Uptake of K by dystrophic tissue during incuba-tion i n 2 mM, 6 mM, and 12 mM K media i s plotted i n F i g . X. The slope of the l i n e i s much greater f o r the dystrophic tissue 42 than the normal; evidently the entry of K into the muscle f i b r e s occurs more rapidly than usual. This may be a r e f l e c t i o n of an abnormal permeability of the muscle membrane, as postulated by Burr and McLennan (i960). With the Harris model of the muscle membrane, the increased rate of exchange appears to be due to one of two things: a f a c i l i t a t e d increase i n the equilibration of the ion exchange layer, or an increase i n the K d i f f u s i v i t y of the internal c e l l u l a r compartment. These p o s s i b i l i t i e s may be related to the higher rate of metabolism i n dystrophic tissue, to an early dystrophic change i n the sarcoplasmic reticulum (Grant I960) causing a decrease i n the amount of material with ion-exchanger properties, or to an a l t e r a t i o n i n the structure or functioning of the ion exchange layer. Harris and Sjodin (l961a) took electron photomicrographs of normal and incubated frog muscle, and inferred from the results that part of the ion-exchange compart-ment might be situated i n the cytoplasmic reticulum. The e a r l i e s t change occurring during the development of muscular dystrophy i n mice has been found by Grant (i960) to be vacuolation of the r e t i -culum close to the I band; the vacuolation progresses u n t i l i t involves the entire reticulum. This may e f f e c t i v e l y reduce the size of the ion exchange compartment, and lead to a more rapid equilibration of the muscle i n t e r i o r with the external environment. There does not seem to be a qualitative change i n the mode of operation of the ion adsorption region, for the uptake 84 curves from dystrophic muscle show no indications of d i f f e r i n g from those for normal muscle, except that the slope i s steeper. The effects of altering the Na g of the incubation media f o r dystrophic muscles were essentially the same as noted for normal muscles, except the effects were more marked* The high ECS values found e a r l i e r are not s u f f i c i e n t to explain these dystrophic findings, unless the membrane permea-b i l i t y i s grossly d i f f e r e n t . The differences i n metabolism of dystrophic tissue mentioned previously may be of some importance i n interpreting these exaggerated phenomena found i n the dystro-phic muscles. If the ion exchange compartment were smaller than usual, i t might not function 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 leak of K ions from the i n t e r i o r of the muscle into the ECS, thus reducing the to the value reported i n Table VIII. This could also account fo r the minor differences i n resting potentials 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 testing the d i s a b i l i t y of the dystrophic mice, either physiologically or biochemically. If a suitable method for estimating the degree of a f f l i c t i o n would be devised, i t might be easier to interpret the observations re-garding the dystrophic muscle; i f the nature of the dystrophic change were known, or i f one could describe more accurately the properties of the ion exchange compartment, phenomena concerned with uptake of ions by mouse tissue, both normal and dystrophic, could be more accurately and properly described. SECTION V CONCLUSIONS AND SUMMARY A. Conclusions B. Summary 85 CONCLUSIONS 1. There i s a negative correlation between extracellular volume and muscle size i n strain 129 mice. Mice a f f l i c t e d with hereditary muscular dystrophy show the dependency more strongly than normal mice. The i n u l i n molecule may be able to penetrate the membrane. 2. Normal mouse muscle (fresh or incubated) loses 76% of i t s water during heating and dessication. During the incubation, the non-inulin space water appears to increase from 47% to 65% of the muscle volume. This l a t t e r observation needs further study before conclusions can be drawn. 3 . Fresh normal mouse muscle contains 3 0 . 0 m. equiv. Na^/ Kg tissue, and 89.9 m. equiv. K^Kg. Dystrophic muscle has 58.2 m. equiv. Na^/Kg and 68.2 m. equiv. K^/Kg. Intracellular ion concentrations are stable during the f i r s t 2 hours of incu-bation i n a r t i f i c i a l media; prolonged incubation results i n Na i r i s i n g and f a l l i n g . Varying the concentrations of Na and K i n the incubation media influences 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 rapidly i n dystrophic ti s s u e . 42 4. K i s taken up by normal mouse muscle i n much the same way as i t i s i n frog muscle (Harris 1957, Harris and Sjodin 1961). The uptake appears to be an exchange of K* i n solution for unlabelled K i n the muscle f i b r e s , and occurs at the rate of 0.98 m. equiv. K*/Kg tissue per (K g • t ) 3 unit. The process appears to be governed by the laws of diffusion, and the rate of 86 uptake to be independent of the K and of the time of incuba-tion i n the medium. Doubling the Na g does not affect the up-take rate, but reducing Na g to 18 mM slows the uptake noticeably. Dystrophic mouse muscle exchanges K* for K faster than normal muscle; the rate i s 1.33 m. equiv. K*/Kg per (K • t ) ^ uni t . Varying the K g or the length of incubation does not affect the rate of uptake, but a l t e r i n g the Ka g has more of an effe c t on the dystrophic 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 properties. This compart-ment would result i n the K* uptake curve following the general pattern of a d i f f u s i o n curve, with the rate being independent of the time of exposure. The dystrophic change appears to be either a change i n membrane permeability or a reduction of the ion-exchange compartment. These two p o s s i b i l i t i e s cannot be adequately di f f e r e n t i a t e d by the methods used i n t h i s thesis. B. SUMMARY 1. Muscle from 129 strain mice was assayed f o r ECS follow-ing i n vivo administration of i n u l i n or soaking of the muscle i n an i n u l i n solution. The ECS was found to decrease with increasing muscle size, and to be l i n e a r . The results obtained on 129 strain mice were confirmed on Swiss albino 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 strain 129 mice shows a greater dependency on muscle size than muscle from normal animals. The p o s s i b i l i t y of an increased permeability of the 87 muscle i s suggested. 2. Muscles were excised from 129 strain mice and dried. Normal muscle l o s t 16% of the fresh weight, and dystrophic muscle 1 1 % of the fresh weight, either fresh or following i n -cubation i n an a r t i f i c i a l medium. A tendency for the non-inulin space water to increase during incubation was noted. 3. Na and K analyses were done on normal and dystrophic tissue (fresh and a f t e r incubation). Results for fresh normal mouse muscle were Na i = 30.0 m. equiv./Kg tissue and = 89.9 m. equiv./Kg. Fresh dystrophic tissue had Na i = 58.2 m. equiv./ Kg and K^ = 68.2 m. equiv./Kg. Incubation of excised muscles i n a modified Locke's solution resulted i n a rise i n Na^ and a f a l l i n K^ aft e r 4 hours' soaking. During the f i r s t 2 hours of incubation, the ion l e v e l s were comparable to fresh tissue values. 42 4. The radioactive isotope K was used to follow the K exchange of muscles during incubation i n variants of Locke's 42 solution. K uptake followed an unique course when plotted as described by Harris and Sjodin ( l 9 6 l ) . The results are in t e r -preted i n the l i g h t of the Harris diffusion-permeation hypothesis. The effec-t of varying the Na and K concentrations of the soaking 42 solution on the uptake of K i s considered i n some d e t a i l . Results of uptake studies on dystrophic mouse muscle are presen-ted and the differences between normal and dystrophic tissue discussed. SECTION VI BIBLIOGRAPHY 88 Adams, R.D., Denny-Brown, D., and Pearson, CM. (l954). Diseases of muscle; a study i n pathology. Paul B. Hoeber, New York. Baetjer, A.M. (1935). The diffusion of potassium from resting skeletal muscles following a reduction i n the blood supply. Amer. J . Physiol. 112, 1 3 9 . Baker, N., Blahd, W.H., Hart, P. (l958). 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Fig.JJL Inulin Space of normal Mouse Muscle: Injection Method. * Gastrocnemii x Peronei Longi Figures in parentheses show the number of hours between injection and sacrifice of the animals. Oi 1 1 1 1 1 r 0 2 0 4 0 6 0 Muscle Fresh Weight, mg. F ig . |V_ Inulin Space of Swiss Albino Mouse Muscle In V i t ro Method. F i g . V . T h i o c y n a t e S p a c e o f n o r m a l M o u s e M u s c l e In V i t r o M e t h o d . 50 n F ig .v i _ Uptake of K by normal Mouse Musc le : Normal and altered K e Incubation Med ia . 2 m M K e - • 6 m M K e — o 12 m M K e - x F ig . yn_ Uptake of K 4 2 by normal Mouse Muscle: High - K Incubation M e d i a . I 8 C V 1 6 0 1 4 0 > '-3 CT OJ 120 £ 1 0 0 z> CO CO 8 0 * 6 0 4 0 2 0 0 2 4 meg. / L K - • 4 8 m e g . / L K — o Dashed L ine is f rom Fig.VI 2 0 4 0 ( Ke \)l/2 (mM min.)'^ 6 0 40 r F i g . VIH U p t a k e o f K 4 2 by M o u s e M u s c l e A l t e r e d N a e I n c u b a t i o n M e d i a . 18 m M N a e - o 2 8 8 m M N a e - x D a s h e d L i n e i s f r o m F i g . v i . 30 20 \ cr CD CD 3 CO CO 10 X X X X X X ( K e • t ) 2 ( m M • m i n ) 42 Fig.JX.. Uptake of K by normal Mouse Muscle : Altered N a e and K e Incubation Media. x 12 m M K e - 2 8 8 mM N a e o 2 m M K e - 18 mM N a e e 12 m M K e - 18 m M N a e • Chlor ide — f ree medium Dashed line is normal uptake s lope 40-, 30-20-10-0 2 > Cr OJ x / x / / X • x / OJ © A x # © e 3 CO CO / A • / ; o x x o* 10 20 30 40 (Ke - \ % (mM -min.)^ o F i g . X . ' U p t a k e o f K by D y s t r o p h i c ( Ke - t ) ^ (mM • min.) F i g . X _ Uptake of K by Dystrophic Mouse Musc le : Altered N a e Incubation Media. • 18 m M N a e o 2 8 8 m M N a e Dashed line is dystrophic regress ion line f rom f igure JT CH 1 1 1 0 10 20 30 ( Ke -t)^2 (mM • min.) ^ 2 Table I K, N a , and resting potentials of excitable tissues , Ion values expressed as m.equiv ./ l i t re of cell or p lasma water. R.P. values in mV. Ca lcu la ted values f rom Nernst Equation. T i ssue K Na R.P Cell Environ Cel l Environ Ca l c . Actual Frog Skel. Musc le 1 2 4 . 0 2 . 2 3 . 6 , 1 0 4 . 0 9 8 9 2 Rat Skel. Muscle 1 5 2 . 0 4 . 7 3 . 0 1 5 0 . 0 8 7 7 4 Crab - Musc le 1 4 6 . 0 1 2 . 9 5 4 . 0 5 1 3 . 0 61 7 2 Crab - Nerve 1 1 2 . 0 12 .1 5 4 . 0 4 6 8 . 0 6 0 8 2 Squid - Giant Axon 3 6 9 . 0 1 3 . 0 4 4 . 0 4 9 8 . 0 8 3 6 5 *- Values f r o m : S h a n e s ( 1 9 5 8 ) , L i n g ft Gerrard ( 1 9 4 9 ) , Hodgking 8k Keynes ( 1 9 5 5 ) , Conway (1950 ) , and Zier ler (1959) 


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