Intracellular potassium activities in muscles of normal and dystrophic mice: An in vivo electrometric study

EXPERIMENTAL

NEUROLOGY

71, 203-219 (1981)

Intracellular Potassium Activities in Muscles of Normal and Dystrophic Mice: An in Vivo Electrometric Study MILTON Department

P. CHARLTON, of Zoology, Received

HAROLD University April

SILVERMAN,

AND HAROLD

of Toronto,

Toronto,

21, 1980; revision

received

Ontario June

M5S

L. ATWOOD’ IAI,

Canada

10, 1980

Intracellular potassium ion activity (aK:) was measured in vivo in gastrocnemius muscle fibers of normal and dystrophic (dy-2J/dy-2J) C57BY6J mice. Rapid measurements were made by means of a double-barrel ion selective microelectrode. The aK: in muscles of dy-2J mice was about 8% lower than in muscles of normal mice. This reduction in aK: was not sufficient to account for the low resting potential of the dy-2J fibers. The fibers measured were from the superficial region of the muscle, a region previously shown to be composed of fast-twitch fibers. This region shows only minor morphologic changes in young dy-2J mice. The reduction of aK: in these fibers is consistent with the hypothesis that there is a general membrane defect associated with muscular dystrophy. It is also suggested, however, that the neurally derived pseudomyotonia associated with the dy-2J mutation and the concomitant increase in the oxidative capacity in the measured muscle fibers might also affect the concentration of intracellular K+.

INTRODUCTION Murine muscular dystrophy caused by the inheritance of a mutation at a single locus (33) shows many of the abnormal features also found in the human muscular dystrophies. Associated with this neuromuscular disorder are the degeneration of muscle fibers, lower than normal resting potentials in the fibers (12, 13, 22, 24, 25, 27, 37, 44), and apparent intracellular ion imbalances (3, 15, 16,46). In particular, the potassium ion concentration is estimated to be lower than normal in dystrophic skeletal muscle fibers. Abbreviation: aK:-intracellular potassium ion activity. ’ Dr. Charlton’s present address is College of Medicine and Department of Zoology and Microbiology, Ohio University, Athens, Ohio 45701. This work was supported by operating grants from the National Research Council of Canada and the Muscular Dystrophy Association of Canada awarded to Dr. Atwood. Dr. Silverman is a postdoctoral fellow of the MDAC. Reprint requests should be addressed to Dr. Atwood. 203 00144886/81/010203-17$02.00/O Copyright All rights

0 1981 by Academic Press, Inc. of reproduction in any form reserved

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In those studies the total muscle ion content was measured by flame photometry or atomic absorption spectrophotometry and the intracellular ion concentration was calculated by the application of a correction factor based on an estimate of extracellular ion content. Such determinations of ion content, although useful, have several major drawbacks. First, most of the muscles studied are mixed.muscles [e.g., the soleus; see Dribin and Simpson (8); Silverman and Atwood, (45)], and there may well be differences between fast and slow muscle fiber potassium concentrations. Second, the amount of connective tissue present in dystrophic muscles varies from muscle to muscle and this could affect the estimation of extracellular space (15). Third, fibers showing severe degeneration might have very low potassium values whereas the more normal fibers in the same muscle could have normal potassium concentrations. Fourth, the previous methods reported only the total ion content and gave no information about the concentration of ions actually in solution in the cell water. The latter parameter is expected to be of physiological significance. To answer more clearly some of these objections to the previous studies, we made direct measurements of the potassium ion activity within individual fibers of normal and dystrophic mice. The measurements were made using a double-barrel potassium-sensitive electrode. This electrode has been used to make rapid measurements on an in viva preparation using a muscle which has been demonstrated to have a region completely homogeneous for fast-twitch fibers (45). We confirmed that the potassium ion activity of dystrophic fast-twitch fibers is somewhat less than that of fibers from the same muscle in normal animals. MATERIALS

AND METHODS

Animal Preparation. Female mice of the C57BL6J strain (2 to 6 months of age) were used in all experiments. Control (+/+ or i/?) animals and animals suffering from the dy2J/dy 2J defect were anesthetized with pentobarbital (80 mg/kg, i.p.) and kept anesthetized throughout the duration of the experiment by a further 40 mg/kg of pentobarbital administered subcutaneously as needed. The right hind leg of the animal was shaved, and the mouse placed in a chamber kept warm (32°C) containing Liley’s solution modified by the addition of 5 mM Na glutamate and 10 mM BES (N,N-bis[2-hydroxyethyll2-aminoethane sulfonic acid). The Liley’s solution (137 mM NaCl, 5 mM KCl, 2 mM CaC12, 1 mM MgC12, 24 mM NaH,CO,, 1 mM NaH,PO,) was continuously bubbled with 95% 02-5% CO,. The Liley’s solution covered the lower two-thirds of the animal, whose head was elevated on a slanted platform rising from the bottom of the chamber. The leg was pinned and

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clamped in an extended position, and the skin removed from the leg. The thin biceps femoris was gently teased and removed from the surface of the gastrocnemius muscle. The gastrocnemius was thus exposed with its nerve and blood supply intact. Immediately after dissection, the Liley’s solution in the chamber was completely replaced, and was also changed at appropriate intervals throughout the experiment. Exposure of the muscle was rapid and initial measurements could be made within 30 min of the initial anesthetic dose. Mice were capable of surviving as long as 6 h in the preparation chamber, and microelectrodes were used to make measurements during this time. Penetration of dystrophic fibers was complicated by the movement of the muscle associated with the pseudomyotonia which occurs in these animals. Pentobarbital does not inhibit the pseudomyotonia, and it was necessary to administer phenytoin (20 mg/kg, i.p.) to effect quiescence of the hind limb muscles (46). Rationale for Use of the Double-Barrel Electrode. Double-barrel K+sensitive electrodes offer several advantages over two single-barrel electrodes. In general, measurements proceed much faster with doublebarrel electrodes because only one penetration of a fiber is required, and visual identification of individual muscle fibers is not necessary. When two electrodes are used, many attempts are sometimes required to get both electrodes into the same cell [compare Fig. 6 of Aicken and Thomas (1) with Fig. 1 here]. In addition, it is likely that the double-barrel electrode technique provides more accurate data than those obtainable with two independent electrodes because each barrel of the former electrode senses the same membrane potential (a value which must be subtracted from the potential measured by the K+-sensitive electrode). This might not be the case if independent electrodes were widely separated and caused different amounts of damage on entry. Attempts to place two independent electrodes close together in the same muscle fiber often lead to lower resting potentials and fiber damage, especially in dystrophic muscle. Preparation of Electrodes. Double-barrel electrodes were pulled from theta tubing with a thick partition (3-mm diameter, Brown and Flaming style, R+D Optical, Bethesda, Maryland). The tubing was soaked in a chromic-sulfuric acid cleaning solution, washed several times in distilled water, and dried in acetone before pulling. Waterproofing of the ion-selective barrel was accomplished by coating the glass with silicone. Two techniques were used. The first involved placing a 33% mixture of trimethylchlorosilane in xylene into one barrel of the pulled pipet. After being at room temperature for about 3 min, the pipet was placed in an oven with the tip down and allowed to bake for an hour at 200°C. After cooling, the treated barrel was back-filled with a small drop of

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K+ exchanger (Corning 477317, Medfield, Massachusetts) and the tip coaxed to fill by the application of heat from a small soldering iron. The reference barrel was then filled with Na formate (3 M containing 65 mM NaCl) (2 1) and the treated barrel was flushed vigorously and filled with 200 mM KCl. In the second technique the ends of the theta tube blanks were lightly firepolished and covered tightly with paraffin film. The film over one barrel was punctured at one end and this end inserted 0.5 to 1 min into a vial containing a few drops of trimethylchlorosilane. One barrel of the blank was thus filled with vapor of the trimethylchlorosilane. The tube was immediately placed in the microelectrode puller and pulled into pipets which were filled as described above. The vapor method provided a quick easy method of coating the glass with silicone which resulted in few plugged pipets. Good results were also obtained with the vapor of light dimethylpolysiloxane (Dow Corning DC 200). Analysis of K+-Sensitive Electrode Characteristics. Three different tests indicated that Na+ leakage from the reference barrel had no effect on the K+-sensitive barrel. First, vigorous stirring applied while the electrode was immersed in 100 mM KC1 failed to change the potential measured by the electrode. Second, strong vacuum applied to the reference barrel, sufficient to raise its resistance, caused no change in the K+ barrel potential. Finally an independent, single-barrel K+ electrode was inserted into a crustacean muscle fiber (about 100 pm in diameter) simultaneously with a double-barrel K+ electrode. The measurements of internal K+ by the two electrodes were within 5 mM of each other. Also, Na formate (3 M, 65 mM NaCl) was compared with the more standard 3 M KC1 to determine whether any differences could be found between the performance of these two solutions in the reference barrel. There was no appreciable difference either in the slope of the electrode calibration curve or in the selectivity of the electrodes. The Na formate solution was used as our reference solution to avoid any possibility of artifactual introduction of K+ to our system of measurement. In several experiments one barrel of a double-barrel electrode was filled with Na formate and the other barrel filled with 3 M KCl. Membrane potentials recorded by the two barrels were within 1 mV. The resistance of the reference barrels was 7 to 15 Ma. Electrodes were used if the tip potential of the reference barrel was less than 5 mV. Some drugs affect the sensitivity of the potassium electrode (10,23,34). Phenytoin and pentobarbital were tested by adding to a 100 mM K+ calibration solution concentrations of these drugs which exceeded the maximum concentration used in an animal. Neither drug had any effect on the K+-sensitive electrode or on the tip potential of the reference barrel.

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1

K+ Activity and Activity Coefficients (y) in Calibration Solutions at 32”C, Calculated by the Extended Debye-Huckel Equation Activity coefficient Solution 100 mM 115 mM 125 mM 130 mM 150 mM

KCl, KCI, KCl, KCl, KCI, 200mM KCl, 100 mM KCl, 200 mM KCl,

50 50 50 50 50 50 -

K+ activity (aK+) (meq/liter)

(YK+)

mM mM mM mM mM mM

NaCl NaCl NaCl NaCl NaCl NaCl

0.7313 0.7241 0.71% 0.7174 0.7094 0.6925 0.7617 0.7094

73.1 83.3 89.9 93.3 106.4 138.5 76.2 141.9

Selectivity of the double-barrel K+ electrodes for K+ over Na+ was 50 to 80: 1 as determined by the separate solution technique [i.e., the electrode response to pure Na+ solutions (to 5 M) was evaluated]. The electrodes had a typical slope of 55 to 58 mV/decade [K+] when tested in solutions of KC1 (100, 115, 125, 150, and 200 mM) containing 50 mM NaCl at 32°C. The calibration concentrations were chosen to be close to the expected intracellular potassium concentration [K+],. The 50 mM NaCl was included in the calibration solutions in order to mimic the intracellular [Na] and serve as a severe control for the slight Na+ sensitivity of the potassium electrodes (constant interference method). The K+ activity coefficient (yK+) in the calibration solutions was calculated by the extended Debye-Huckel equation (3 1, 32, 42): logy =

AGl+Bafl

+cz

’

where A, B, C, and a are constants with the following valuesA = -0.5186 [Robinson and Stokes (41), Table 11, B = 0.3306 [Robinson and Stokes (41), Table 11, a = 3.5 [Meier ef al. (32)], and C = 0.0187 [Parsons (38), Table 24]-and Z is the total ionic strength [= 0.5 C,, c, z:, where cn is the concentration of each ion (including NaCl) and z,, is the charge of each ion]. The total ionic strength (I) includes the 50 mM NaCI. The presence of Na+ should increase the activity coefficient of K+ by less than 0.1% at 32°C [an effect different from that on ionic strength (1 l)]. This small effect was ignored. The results of these calculations are shown in Table 1. The voltage response of the K+ electrode in each calibration solution was used to construct a calibration curve, the equation of which was used to

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calculate the intracellular [K+] and aK:. Calibration solutions were contained in small vials (15 ml) mounted beside the mouse in the warm preparation chamber. All calibrations were thus performed at the same temperature as the intracellular measurements. Measurement oflntraceflular K+. Contact with the calibration solutions was made through a bridge containing 3 M KC1 in agar and a heavily chlorided silver wire. During experiments the same bridge was used, but it contacted an additional bridge consisting of Liley’s solution in agar (2%) which in turn contacted the Liley’s solution in the preparation chamber. This was done to avoid any K+ leakage to the preparation solution. The electrodes were connected to an electrometer headstage (WPI F223) by a fine chlorided silver wire. Upon penetration of a fiber, the reference barrel registers the membrane potential and the K+-sensitive barrel registers the membrane potential plus a potential proportional to the aK:. The membrane potential component must therefore be subtracted from the K+ electrode signal. The reference electrode potential was noted before and after penetration and the difference was calculated to give the membrane potential. This figure was then subtracted from the K+ electrode signal so that the potential was proportional to the K+ activity (potassium potential). Voltages were read from a digital meter with O.l-mV resolution. The reference signal was recorded on one channel of a chart recorder (Brush 2200) and the potassium potential was recorded on the second channel. Electrodes were calibrated immediately before and after 5 to 10 fibers had been penetrated. If the slope or intercept of the calibration curve changed, the data were rejected and the experiment was terminated, or the electrode was replaced with a new one. RESULTS Typical records of cell potentials during electrode penetrations are presented in Fig. 1. The ease of penetration of both normal and dy-2J fibers is indicated by the rapid deflection of the membrane potential record in most fibers (Fig. l), but penetration of dy-2J fibers was slightly more difficult to effect than penetration of normal fibers, perhaps due to a thicker connective tissue layer. In most fibers the resting potential (E,) stabilized rapidly after penetration and the double-barrel electrode could be left in these fibers for several minutes without any reduction in the E,. Other fibers (Fig. 1) showed slight damage of the membrane as indicated by a drop of a few millivolts in the initial E, before stabilization. All fibers eventually showed a stabilization of the E,. The potassium potential remained constant (after the initial response time) regardless of the changes which occurred in the E, of slowly stabilizing fibers. Thus, fibers which appeared to show some membrane

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b

FIG. 1. Records of penetrations of gastrocnemius muscle fibers in normal (a) and dystrophic (b) mice. Membrane potentials (downward deflections) recorded on channel B; difference between the membrane potential and the signal from the K+-selective barrel (upward deflection) displayed as A-B. The arrows show the initial recordings outside the fiber. Note that A-B remains steady even in fibers showing some drift of membrane potential after the penetration.

damage did not appear to be leaking potassium through the electrode penetration site. It was then possible to record the potassium potential any time after fiber penetration with reference to the E, recorded at that time. When concentrated KC1 was applied to the bath surrounding a penetrated fiber there was a rapid reduction in the E, but no immediate change in the K+ signal (Fig. 2). All these observations confirmed the idea that measurements of aK: were not affected by changes in the membrane potential. Histograms of the E,, [K]i, and aK: of normal and dy-2J fibers are presented in Figs. 3-5. The dy-2J fibers had average E, (initial measurement on penetration), [K]i, and aK: values significantly lower than those of normal fibers (Table 2). The histograms indicate that the fibers sampled in the surface of the gastrocnemius were drawn from fiber populations with values normally distributed about a mean in both normal and dy-2J animals.

210

CHARLTON

15 mMK*

, SILVERMAN,

AND ATWOOD

200 mMK’

10mV%ic

FIG. 2. The addition of extracellular K+ depolarizes the muscle fiber(B) but has hardly any effect on the aK: reading (A-B).

Although the E, and aK, are both lower than normal in dy-2J muscle fibers, the correlation coefficient of these two parameters was not significantly different from zero in either dy-2J or normal muscle. Reduction of the [K]i has been reported after immersion of isolated NORMAL

Em (m”) 30

5 0 c

i

DYSTROPHIC

20

100 LA

40

50

60 E,

70

80

90

(mV)

FIG. 3. Histograms to compare the resting membrane potentials (E,) in normal and dystrophic mouse gastrocnemius fibers.

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NORMAL

(mM CK+li

K+)

lIK+li(mM

Kt)

30

20

10

0!

50

FIG. 4. Histograms to compare sarcoplasmic gastrocnemius fibers.

K+ in normal and dystrophic

mouse

muscle in saline for in vitro experiments (7,30). In our in viva preparation, however, a plot of penetration number (a function of time) against [K]i revealed no reduction of the [K]i during the course of an experiment. Reduction of the [K]i in fibers did occur in experiments in which measurements were made after the death of the animal. The predicted Nemstian E, in our muscles at 32°C was determined by E, = 60.4 log(aKT/aQ). Assuming that the extracellular activity coefficient for potassium (yK,f) is 0.76 (41), then aK,+ is 3.8. The average predicted E, in normal fibers was 81.2 mV and the average E, measured was 71.8 mV. In the dy-2J fibers the average predicted E, was 79.1 mV and the average measured E, was 67.1 mV. Because the difference in the predicted Em between normal and dy-2J fibers was 2.1 mV and the difference in the measured E, was 3.9 mV, the lower E, in the dy-2J fibers cannot be entirely explained by the lower aK: in the latter fibers. The average aK: in normal and dy-2J fibers was 85.4 and 78.8 mM, respectively. The activity coefficient of intracellular K+ (K:) can be determined by comparing the total [Kli measurements of Silverman et al.

212

CHARLTON,

SILVERMAN,

AND ATWOOD NORMAL

aK+

(mM K’)

OYSTROPHIC

40

60

80 aK+

100

120

(mM K+)

FIG. 5. Histograms to compare sarcoplasmic potassium activity (aK+) in normal and dystrophic mouse gastrocnemius fibers.

(46) with the present aK: values. The activity coefficient for the normal fibers was YK: = aK:/[K]i = 85.4/143.9 = 0.59 and the YK~ for the dy-2J fibers was yKr = aK:/[K]i = 78.81134.7 = 0.58. The age of the mice used in this study was 2 to 6 months. To test whether age differences were a factor in our results, 2- and 6-month-old fibers were compared separately between normal and dy-2J animals. There appeared to be a slightly lower aK: in the older fibers compared with the younger fibers (Table 3). The average aK: of the 2-month-old dy-2J fibers was significantly lower than that of the 2-month-old normal fibers, and the same was also true when fibers from 6-month-old animals were compared. The average difference between normal and dy-2J fibers was essentially the same in both 2- and 6-month-old fibers.

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TABLE

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MUSCLE

2

Average Membrane Potential (E,), Intracellular Potassium Concentration ([K+],), and Activity (aK:) in Gastrocnemius Muscle Fibers of Normal (+/+, +/?) and Dystrophic (dy-2J/dy-2J) Mice

Age Genotype

(months)

Fiber N

WI,

-&II (mV 2

2.6 1.3 1.7 1.1 1.3 0.6

112.9 118.1 115.3 112.6 129.6 115.8

7 28 12 25 23 29

Total N and x k SE Elemental estimate of Silverman et al. (47) Average predicted E,

124

2 2 4 6 6

20 19 20 10 20

72.0 61.3 62.5 68.8 71.9

Total N and 8 f SE Elemental estimate of Silverman et al. (47) Average predicted E,

89

67.1 2 0.9*

dy-2J/dy-2J dy-2Jldy-2J dy-2J/dy-2J dy-2J/dy-2J dy-2J/dy-2J

k 2 + + ” k

(mM

2 2 2 6 2 2

+I+

i/+ +/+ -+I? ii? Cl?

73.4 69.4 73.5 72.7 68.7 74.9

SE)

71.8 Z!I0.6

k SE)

? 2 2 2 f +

3.7 5.3 4.4 2.2 2.4 2.7

118.2 2 1.3

aK, (mM + 81.3 85.3 82.8 81.7 92.8 83.5

SE)

+ 2.3 2 2.0 2 3.0 2 1.4 zk 1.6 + 1.7

85.4 t 0.9

143.9 2 2.6 81.2 2 k k k k

1.3 1.6 1.5 1.6 1.4

117.1 107.7 101.0 111.8 104.6

2 2.4 rf: 4.2 2 5.6 ? 1.8 +- 1.9

107.9 ” 1.7*

84.2 78.6 74.1 81.3 76.5

k k k * k

1.6 2.8 3.6 1.1 1.2

78.8 2 l.l*

134.7 f 2.7 79.1

* Significantly different from normal, P 5 0.01 (one-way analysis of variance).

DISCUSSION To our knowledge, this is the first in vivo study of intracellular potassium ion activity in mammalian muscle, and the first attempt to compare intracellular potassium activities in normal and dystrophic muscle fibers using ion-selective microelectrodes. The in vivo approach minimizes the risk of anoxia-induced changes in ion activity which could occur (and were observed by us) during in vitro experiments. Rapid measurement of ion activity was facilitated by the use of a double-barrel electrode. This further reduced the risk of postdissection degeneration. The present results indicate that in mouse gastrocnemius muscles, myoplasmic potassium ion activity, aK:, is about 8% lower in muscle fibers which are homozygous for the dy-2J (dystrophic) allele than in normal fibers. These results are in qualitative agreement with previous

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SILVERMAN, TABLE

[Kfli of 2- and 6-Month-Old

AND ATWOOD 3

Normal and dy-2.1 Fibers from C57BLi6J Mouse Gastrocnemius Muscle

N Fibers

Age (months)

Normal

99 25

2 6

119.6 t 1.6 112.6 t 2.1

Dystrophic

39 30

2 6

112.2 t 0.4* 107.0 k 1.5*

[K+]i 2

SE

* Significantly different from age-matched normal, P 5 0.05 (I test)

studies which indicated that areduction in intracellular muscle potassium is associated with muscular dystrophy in several species including man (4,5, 13,15,16,19,29,46,49) and contradict, at least in the dy-2J dystrophy, the idea that aK: is not different in dystrophic muscle fibers as was reported in the dy mouse dystrophy (44). The studies noted above all used chemical analysis techniques which measure total elemental concentrations. To gain an estimate of the intracellular ion concentration, those techniques require some estimate of the extracellular space in order to correct for extracellular elemental concentrations. As pointed out by Hoh and Salafsky (16), differences in the extracellular space component between normal and dystrophic muscles can cause errors in comparison of the [K+]i based on whole muscle analysis. Furthermore, measurements of extracellular space appear to vary “widely even in the same tissue under similar conditions” (28) and are known to depend to a certain extent on the condition of the particular muscle being evaluated (35, 36). Our data are a measure of the concentration of free potassium ions in the myoplasm and are not subject to the errors inherent in estimating the extracellular space or its ionic content. The studies on the total elemental concentrations in whole muscles were generally done on muscles containing mixed fiber populations [i.e., soleus of the mouse, which contains approximately 50% slow-twitch muscle fibers (8)]. Different fiber types are known to differ in [K+]i (47, 48). The interpretation of these data is further complicated by the unknown contribution to the average [K+]i value made by highly degenerate fibers in dystrophic muscles. In contrast, our data were obtained from a region of the gastrocnemius muscle which contains almost exclusively fast-twitch fibers in both normal and dy-2J animals (45). Although the dy-2J fibers in

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this region of muscle are more oxidative than their normal counterparts (8, 45), they show only minor mophologic changes (both grossly and ultrastructurally) at the age used in this study (45). Thus the decrease in the average aK: observed in this study is not the result of a large decrease limited to highly degenerated fibers, but represents a general decline found in fibers of a muscle region which is nearly normal morphologically. Had there been a population of highly degenerate fibers, a bimodal rather than unimodal histogram of aK: values for the dy-2J muscles would have been expected together with a larger variance in aK: in a dy-2J mice in comparison with normal mice. Our direct measurements of aK: in the dy-2J mice contrast with the inferential measurements of Sellin and Speralakis (44), who found that in both normal and dystrophic mice (dy allele), the same [K+], was required to reduce the E, of extensor digitorum longus fibers to 0 mV. Although the response of the E, to changes in the [K+], was not Nernstian and other results showed differences in PNa:PK and in electrogenic ion pumping, it was concluded that the [K+]i was identical in normal and dystrophic mice of the dy allele. It is evident that both in C57B16J (results reported here) and in ReJ129 (44) mouse strains, membrane potential is not well predicted by the Nernst equation, and other factors must be considered [for example, possible effects of [Cl],; see Dulhunty (9)]. The average YK: (0.59) of normal fibers is near the value of yK+ = 0.612 in rabbit ventricular papillary muscle found by Lee and Fozzard (26) but is lower than that found in many nonmammalian tissues (28). The potassium activity coefficient is considerably lower than that of the saline (0.76) or calibration solutions (-0.72) which were chosen to mimic the K+ and Na+ contents of the myoplasm. The elemental [K]i estimates of Silverman et al. (46) reflect an average value for the whole muscle including the more oxidative underlying region. The measurements for the aK: were obtained only from the superficial glycolytic region. Thus -yK+ might be slightly overestimated because oxidative muscle may contain less potassium than glycolytic muscle (48). The activity coefficient comparisons are based on the assumption that all the fiber water is a solvent for all the intracellular K+ [cf. (6, 17, 18)]. The results can be explained if there is significant binding or compartmentalization of intracellular K+ or if the activity coefficient of the myoplasm is lower than that of simple water solutions due to electrostatic interactions in the polyelectrolyte solution (6, 26, 28). It is tempting to suggest that, because YK: is the same in normal and dy-2J fibers, the above interactions are similar in both fiber populations. Enthusiasm for this conclusion must be tempered by the realization that YK: is no more accurate than the

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determination of total fiber [K] and the latter figure is prone to the many errors discussed above. The finding that aK: is lower in dy-2J fibers than in normal fibers is in general agreement with the hypothesis that there is a general membrane disorder associated with the genetic defects causing dystrophy. Evidence for this membrane hypothesis has been reviewed recently by a number of authors (42-44). The finding that potassium is decreased in morphologically intact fibers suggests a general phenomenon, but does not offer in itself strong evidence for a membrane deficiency. In addition to, or as a consequence of, the possible membrane defect, the dy-2J animals exhibit two abnormalities which could contribute to an altered Na+/K+ ratio. First, high muscle activity is known to result in K+ loss (2, 14,47), and these mice display a neurally derived pseudomyotonia which results in a high degree of muscle activity and extension of hind limbs (20, 40, 45, 46). Future experiments with ion-selective electrodes will determine whether or not aK: is likely to be affected by the amount of overactivity found in the hindlimb muscles of the dy-2J mice. However, the present data indicate that when the pseudomyotonia is reduced by the chronic administration of phenytoin to dy-2J mice, there is no change in the total muscle potassium (46). Second, superficial gastrocnemius fibers which are predominantly fast-twitch glycolytic in the normal animals are gradually converted in the dy-2J muscle to a more oxidative type[(46); Silverman, unpublished data] which may contain less K+ (even in normal animals) (48). Further observations will show whether it is the dystrophy per se or the secondary conversion of muscle fibers to a more oxidative type which is more important in lowering intracellular aK+. In this regard, it will be useful to compare ion activities in hind limb muscles with those of the forelimb as forelimb muscle fibers in dy-2J animals contain the defective allele but do not suffer from the pseudomyotonia present in the hind limbs. Whatever the etiology of the decreased aK;t in dy-2J fibers, there is evidence that aK: and aNa: can exert an influence on several cell activities including protein synthesis [see reviews by Pestka (39) and Civan (6)]. Whether the reduction in aK: in dy-2J fibers is sufficient to cause further alteration in cell metabolic processes is unknown at this time. REFERENCES 1. AICKEN, C. C., AND R. C. THOMAS. 1977. Micro-electrode measurements of the intracellular pH and buffering power of mouse soleus fibers. J. Physiol. (London) 267: 791-810. 2. AKAIKE, N. 1978. Resting and action potentials in white muscle of potassium deficient rats. Comp. Biochem. Physiol. 61: 629-633.

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3. ATWOOD, H. L., AND I. KWAN. 1978. Dystrophic and normal mice show age-dependent divergence of muscle sodium concentrations. Exp. Neural. 60: 386-392. 4. BAKER, N., W. H. BLAHD, AND P. HART. 1958. Concentrations of K and Na in skeletal muscle of mice with a hereditary myopathy (Dystrophia muscularis). Am. J. Physiol. 193: 530-533.

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