Maintenance of sodium and potassium ions in frog sartorius muscle fibers by aspartate salts

Vol . 7, Part I, pp. 811-823, 1988 . Life Sciences Printed in Great Britain .

Pergamon Press

MAINTENANCE OF 30DIUM AND POTASSIUM IONS IN FROG SARTORIUS MUSCLE FIBERS BY ASPARTATE SALTS M . Sato, T . Kiyohara and Nobuko Kobayashi Department oß Physiology Kumamoto University Medical School Kumamoto, Japan

(Received 11 March 1968; in final form 24 May 1988) FROG muscle Bibers, when immersed in Ringer's solution at room temperature for a long time, gain Na and lose K (1, 2), because Na and K ions in- and outside the plasma membrane move according to the electrochemical gradient while in isolated muscle fibers enough metabolic energy is not supplied to extrude Na ions actively and to absorb K ions, thereby balancing the intracellular Na and K concéntracions . In the present experiments an investigation was made whether or not the intracellular Na and K concentrations were maintained at a more normal value, when frog sartorius muscles were immersed ßor 24 hr in Ringer's solution containing various kinds oß aspartate salts, than that obtained with normal Ringer's solution .

In addi-

tion, to see whether the resting potential oß muscle fibers is hyperpolarized by a solution with K- and Mg-aspartates as reported by Henderson and üalkenstein (3), resting potentials oß muscle ßibers, stored either ßor a short time or ßor 24 hr in Ringer's solution containing K- and Mg-aspartatea, were measured and compared with those in muscle ßibers soaked in normal Ringer's solution . Materials and Methods One oß a pair oß sartorius muscles, dissected Brom a common Japanese ßrog, was immersed in normal Ringer's solution (composition in

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mMs NaCl 113 .0, %C1 2 .5, CaC1 2 1 .8, MgC1 2 2 .0, Na2HP04 2 .15 and NaH2P0 4 0 .85) ßor 24 hr, while the other oß the pair in a test solution containing various aspartate salts ßor the same period .

Usu-

ally several pairs of sartorii were immersed in a teat solution and in Ringer's solution at the same time .

Temperature oß the immersion

solution was maintained at 20 ° , 8 ° or 2° C .

Muscles were removed

ßrom the solution 24 hr aßter immersion and subjected to chemical analysis ßor Na and R, using a Blame spectrophotometer (UNICAM SP 900) .

Intracellular Na and S concentrations were calculated ßrom

the Na and S content in muscles according to a formula by Boyle and Conway (1) and Boyle et sl . (4), assuming 12 .5

as a volume oß the

extracelluler space (4, 5) and 0 .185 as a dry-to-wet-weight ratio (5) . Measurements of resting potentials oß sartorius muscle ßibers immersed either ßor a short time or 24 hr in an aspartate-Ringer's solution or in normal Ringer's solution were carried out with a glass capillary microelectrode Billed with 3 M gCl, using conventional electrophyaiological equipment (6) . For preparing teat solutions, KC1, Mg~C1 2 and CaC1 2 in Ringer's solution were replaced by equimolar g-aspartate, Mg-aspartate and Ca-aspartate, reapective-ly.

In other kinds oß experiments 5 mM-NaCl

were substituted ßor equimolar Na-aspartate . Results Sodium and Potassium Concentrations Tßßecta oß replacement oß both gCl and M¢C1 2 or %C1 alone in Ringer's solution b~ % end M¢-eapsrtates on intraçellule~r Na and S concentrations .

One oß a pair oß sartorius muscles was stored in

Ringer's solution ßor 24 hr, while the other oß the pair in a solution, in which gCl and MgC1 2 in Ringer's solution had been replaced

Vol . 7, No. 15 by K- and Mg-aspartates . 2° , 8° or 20 °C .

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Temperature oß solutions was maintained at

The results are shown in Table 1 .

Comparing mus-

cles, which were~stored in Ringer's solution containing K- and Mgaspartates at 2 °C with those immersed in normal Ringer's solution, no signißicant difße.rence is found in ~Na~i and ~K~ i (the intracellular Na and K concentration, respectively) between the two groups of muscles .

TABLE 1 Intracellular Na and K concentrations in sartorius muscle ßibers stored ßor 24 hr àt various temperatures in normal

Ringer's solution, Ringer's solution with 2 .5 mM K-aspartate plus 2 .0 mM Mg-aspartate and that with 1 .25 mM K2S0 4 plus 2 .0 mM MgS0 4.

Immersion solutions Ringer solution (10) Ringer solution with K-, Mg-aspartate (10)

T&m . ( C~

Mean muscle weight (mg)

20

55 .9

23 .1±2 .0

137 .3±2 .4

20

56 .5

16 .7±1 .0

144 .0±2 .6

t test

Ringer solution (12)

Ringer solution with K-, Mg-aspartate (12) t test Ringer solution (7) Ringer solution with K-, Mg-aspartate (7) t test Ringer solution (8)

Ringer solution with K2S0 4 and Mg50 4 (8) t test

~Na~ i (K] i (mmole~kg.H 20)

0 .01
60 .9

11 .7±1 .9

139 .8±2 .1

8

61 .8

8 .9+1 .8

141 .9±1 .7

0 .01
58 .9

13 .8±1 .7

150 .8±1 .2

2

59 .3

15 .7±1 .3

151 .3+1 .0

0 .25
P>0 .5

20

53 .9

20 .4±3 .3

133 .4±3 .8

20

53 .7

18 .6+3 .3

134 .0+3_9

P70 .5

P>0 .5

Values listed are the means ± S .E ., and numerals inside Lhe parenthesis indicate number of muscles measured .

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However, ~Na~ i and ~K~i in muscles, having been soaked in Ringer's solution with K- and Mg-aspartates at 8° or 20 °C, are signißicantly different Brom those in muscles stored in normal Ringer's solution ; in the former muscles ~Na~ i is lower and [K]i is higher than those in the latter .

Since muscle ßibers lose K and gain Na gradually

aßter their immersion in Ringer's solution, the above observation indicates the eßßect oß K- and Mg-aspartates in maintaining the cell K and Na close to a normal value . In order to check whether or not such an eßßect of maintaining the cell K and Na close to a normal value is characteristic of K, Mg-aspartate solution, one of a pair oß sartorü was stored for 24 hr at 20 °C in Ringer's solution, in which KC1 and MgC1 2 had been replaced by K2S0 4 and MgS0 4 , and the intracellular Na and K concentrations oß the muscles were compared with those stored in normal Ringer's solution . Table 1 .

The results are shown in the lowest column oß

The table indicates the absence oß significant difference

in [Na~ i and [K]i between the two groups oß muscles, indicating that only the presence oß K- and Mg-aspartates is capable oß maintaining the cell K and Na close to a normal value . Eßßects oß replacement oß KC1 . M¢C1 2 or CaC1 2 alone by K- . M¢or Ca-aspartate on Na and K concentrations .

To see whether or not

K-, Mg- or Ca-aspartate alone shows such an action to maintain the cell K and Na .a s observed by K-, Mg-aspartate solution, sartorii were immersed for 24 hr in Ringer's solution at 20 °C, in which KC1, MgC1 2 or Cs,C1 2 had been replaced by K-, Mg- or Ca-aspartate, and then their ~Na~i and ~K]i were compared with those in muscles which had been stored in normal Ringer's solution under similar conditions . The results are shown in Table 2 .

As shown in this table, no signi-

ßicant difference is seen in tNa]i and ~K]i between the muscles stored in Ringer's solution with K-aspartate and those in normal

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Ringer's solution, indicating that 2 .5 mM K-aspartate alone is not capable of maintaining the cell K and Na at a normal level .

A sim-

ilar result was obtained when eight pairs of sartorii were immersed for 24 hr at 20 °C in normal Ringer's solution and in Ringer's solution, in which KC1 had been replaced by K-glutamate, and their cation concentrations were compared with each other .

Muscles stored

in K-glutamate solution showed ~Na]i of 12 .8 ± 1 .0 mmole/kg .H 20 and

(x1i

of 157 .9 ± 0.4 mmole/kg .H 20 (mean ± S .E .), while the control

muscles showed 13 .5 ± 1 .6 and 148 .9 ± 1 .7 mmole/kg .H 20, respectively . No significant difference was found in (Na]i, though (K]i in muscles stored in K-glutamate solution was significantly higher than that in control muscles (P ~ 0 .001) . Muscles stored in Ringer's solution containing only Mg- or Caaspartate showed a greater value of ~Na~i and a significantly smaller value of (K]i than those stored in normal Ringer's solution .

On

the other hand, muscles stored in Ringer's solution, in which 5 mM NaCl had been replaced by equimolar Na-aspartate, showed a smaller value of tNa~i and a higher value of (K~i than those in muscles stored in normal Ringer's solution (Table 2) . One of a pair of sartorii was immersed for 24 hr at 20 °C in Ringer's solution, to which 2 mM Mg-aspartate had been added, and was compared with the other of the pair stored in normal Ringer's solution for their ~Na]i and (K~i .

Although muscles in the former

group showed a higher (K]i (0 .025

P ~ 0 .05) than that in the latter

muscles, no significant difference was found in ~Na~i between the two groups of muscles . Effects of replacement of KC1, MQC1 2 and CaC1 2 in Ringer's solution by K-,

MA- and

Ca-as partates on Na and K çoncentrations .

Muscles stored for 24 hr at 20 ° C in Ringer's solution, in which KC1, . MgC1 2 and CaCi 2 had been replaced by K-, Mg- and Ca-aspartates,

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TABLE 2 Intracellular Na and K concentrations in sartoriua muscle fibers stored at 20 °C ßor 24 hr in Ringer's solution, in which KC1, MgC1 2 , CaC1 2 or 5 mM NaCl was replaced by equimolar R-, Mg-, Ca- or Na-aspartate .

Immersion solutions

Tim . ( C~

Mean muscle weight (mg)

(Na)i (K]i Immole /kg .H20)

Ringer solution (9) Ringer solution with K-aspartate (9)

20

62 .9

23 .5±2 .3

137 .4±4 .5

20

61 .1

23 .8+2 .2

137 .2+2 .9

Ringer solution (8) Ringer solution with Mg-aspartate (8) t test

20

45 .6

21 .0±1 .9

138 .6±1 .7

20

46 .6

24 .6±3 .2

124 .4±3 .5

Ringer solution (7) Ringer solution with Ca-aspartate (7)

20

57 .2

21 .4±2 .2

136.E±1 .7

20

59 .3

34 .7+4 .2

108 .4+5 .0

Ringer solution (9) Ringer solution with Na-aspartate (9) t test

20

49 .8

19 :912 .4

137 .113 .0

20

49 .3

14 .5+0 .9

139 .7+2 .5

t test

~0 . 5

0 .25
t test

0 .025
0 .025
P~0 .5

0 . 001( PC 0 . 00 5

P<0 .001

P~0 .5

Values listed are the means ± S .E ., and numerals inside the parenthesis indicate number oß muscles measured . showed ~Na]i oß 17 .3 ± 1 .4 mmole/kg .H 20 (mean oß 15 muscles ± S .E .) and (K~i oß 139.0 1 1 .4 mmole/kg .H 20, while the control muscles showed 19 .3 ± 2.2 and 136 .4 ± 1 .4 mmole/kg .H 20, respectively . in the ßormer group of muscles is smaller (0 .05

(Na~i

P ~ 0 .1) and EK~i in

these muscles greater (0 .025 ~ P ~ 0 .05) than those in control muscles . The low ability oß K-, Mg-, Ca-e.spartate solution in maintaining (Na]i and (K)i compared with that oß K-, Mg-solution may be explained by an antagonistic eßfect oß Ca-aspartate solution to that oß K-, Mg-aspartate .

Vol. 7,

5 mM K-asp. 2 mM Mg-asp.

5 mM KCl

-70

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No . 15

E c o~ ..

I I I r r r i I 1 r

-8 0

0

a v~ c

~d

-90

~- J

Normal Ringer ( 2 .5 mM KCl ) 0

20 10 Time

30 40 (min )

50

60

70

FIG . 1 Resting potentials of sartorius muscle fibers in normal Ringer's solution, Ringer's solution with 5 mM KC1 and that containing 5 mM K-aspartate and 2 mM Mg-aspartate . Each point represents the average value of resting potentials in about 30 fibers from four muscles, measured successively in 10 min, and the vertical bar indicates ± S .E . of the mean . Resti~Potent ials Restin¢ po tentials of sartorius muscle fibers in K-, M¢-asnartate solution

Recently Henderson and Walkenstein (3) reported that

frog sartorius muscle fibers, which had been depolarized by addition of 5-10 mM KC1 to Ringer's solution, was hyperpolarized by 5-10 mV after their immersion in a solution containing 2 mM Mg-aspartate and 5-10 mM K-aspartate .

Fig . 1 represents changes in the resting po-

tential of muscle fibers, which were immersed in Ringer's solution containing 5 mM K-aspartate and 2 mM Mg-aspartate after they had been depolarized by Ringer's solution with 5 mM KC1 .

As shown in

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818

-70

~ -

10 mM KCl

E 10 mM K-asp.

-° -80 r c Y r O d

a c y -90 Y

1 1 l

~_-J

T

Normal Ringer ( 2 .5 mM KCl ) 0

10

Time

20

(min)

30

40

50

FIG . 2 Resting potentials oß sartorius muscle ßibers in normal Ringer's solution, Ringer's solution with 10 mM KC1 and that containing 10 mM K-aspartate . Each point represents the average value oß resting potentials in about 30 ßibers ßrom ßour muscles, measured successively in 10 min, and the vertical bar indicates ± S .E . oß the mean . this ßigure, no hyperpolarization of muscle ßibers such as reported by Henderson and Walkenstein (3) was observed by replacing KC1 with K-aspartate in Ringer's solution and by adding Mg-aspartate to the solution .Similarly muscle ßibers, depolarized by 10 mM KC1, was not hyperpolarized by replacement oß KC1 with 10 mM K-aspartate (Fig .2) . On the contrary, a slight depolarization was observed immediately aßter replacement oß Ringer's solution with 10 mM KC1 by the solution with 10 mM K-aspartate ; this depolarization may be attributed to a decrease in C1 concentration in the bathing medium (7) . Resting potentials of _muscle fibers stored for 24 hr in K-,

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-90

2.5 mM K-asp. 2 .OmM Mg-asp . ( 20~ ) ->

2.5 mM K-asp. 2 mMMg-asp ~-100

(20 °C .24hr)

~-110 ... 0a

Normal _ Ring¢r(20 °~ )

o~ c

~ -90

819

2.5 mM K-asp ( 20 °C ) 20 mM Mg-asp

Normal Ring¢r ( 20 °C , 24 hr )

-100 0

10 Time

20 (min)

30

40

FIG. 3 Resting potentials of sartorius muscle fibers, stored in Ringer's solution with 2~5 mM K-aspartate and 2 .0 mM Mg-aspartate at 20 ° C for 24 hr (upper figure) and in normal Ringer's solution (lower figure) Each point represents the average of resting potentials in about 20 fibers from three muscles and the vertical bar represents + S .E . of the mean . M~-aspartate solution at 20 °C .

Muscle fibers, which had been immer-

sed in normal Ringer's solution at 20 ° C for 24 hr, showed resting potentials of 96-97 mV, while those stored in Ringer's solution, in which FCI. and P1gCl~ had been replaced by K- a.nd Mg-aspartate, showed resting potentials of 99-100 mV ; the latter is greater by about 3 mV thcLn the former (Fig . 3) .

The difference may be attributed to the

difference in the amount of [K~i between the two kinds of muscle fibers

(see Table 1) .

As shown in Fig

3, muscle fibers having been

immersed in normal Ringer's solution at 20 ° C for 24 hr, did not show any hyperpolarization after their re-immersion in K-, Mg-aspartate

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820 solution .

Discussion Muscle fibers, isolated from the living body and immersed in Ringer's solution for a long time, gain Na and lose K because these ions move according to the electrochemical gradient .

However, in

muscle fibers in vivo Na ions are extruded from the cell and K ions taken up by the active Na-K pump mechanism utilizing the metabolic energy, and consequently internal Na and K concentrations are maintained constant .

In the present experiments it has been shown that

muscle fibers, stored in K-, Mg-aspartate solution at 8° or 20 °C, show a low ~Na~i and a high ~KJi value compared with those in fibers stored in normal Ringer's solution, indicating that in K-, Mg-aspartate solution muscle fibers maintain Na and K concentrations at a more normal value than in Ringer's solution .

The fact that little

difference in ~Na~i and ~K~i was seen between muscles stored in K-, Mg-aspartate solution at cold and those in Ringer's solution at the same temperature may be explained by a possibility that in Ringer's solution at low temperature muscle fibers are capable of maintaining ~Na~i and ~K~i nearly at a normal value because of small efflux rate of K ions and small influx rate of Na ions . Maintainance of ~Na~i and ~K~i in muscle fibers at a normal value was observed when 5 mM NaCl in Ringer's solution had been replaced by isomolar Na-aspartate, though its effect was not as prominent as that by K-, Mg-aspartate solution .

On the contrary,

muscles stored in Ringer's solution, in which either MgC1 2 or CaC1 2 had been replaced by Mg-aspartate or Ca-aspartate, showed an increased ~Na~i and a decreased ~K~i value as compared with those stored in normal Ringer's solution .

This would explain why K-, Mg-,

Ca-aspartate solution produced a less significant effect of

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821

maintaining ~Na~i low and ~K~i high than K-, Mg- aspartate solution . The fact mentioned above suggests a possibility that aspartate ions enter into the cell interior, though the rate of their entrance being probably very slow, and that they are utilized as a source for the cell metabolism which supplies energy for the active transport of Na and K ions .

Since the presence of Mg-aspartate in addition

to that of K-aspartate is necessary for producing a prominent effect, a possible role of Mg ions in the metabolic process should also be considered . When the external medium contained either Mg-aspartate or Caaspartate alone, a significant rise in ~Na~i and a significant decrease in ~K~i were observed,

This probably results from the fact

that aspartate ions form a chelete with Mg or Ca (8) so that they would not diffuse easily into the cell interior, and consequently aspartate ions would not be utilized as a energy source for the active Na-K pump mechanism .

The possibility that aspartate salts

diffuse into the cell interior and are utilized as a energy source only when they are dissociated as ions is supported by the fact that muscles stored in Ringer's solution containing 5 mM Na-aspartate showed a lower value of ~Na~i and a higher value of ~K~i than those in the control muscles . Nakahara et al . (9) measured Na and K content in gastrocnemius muscles of rats after their electrical stimulations with and without preliminary injection of various salts of amino acids into animals, and obtained results indicating that muscles with preliminary injection of K- and ~lg-aspartates or K-aspartate alone showed a low \a and a high K content as compared with those in control muscles . Their results are therefore in essential agreement with those in the present experiments, and may be explained by a facilitation of the Na-K pump mechan=sm by the presence of aspartate ions, though a

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Vol. 7, No . 15

different kind of explanations for this phenomenon was supplied by Nakahara et al ., who s~ipposed that the ions which were utilized for the-metabolism are K and Mg ions transported into the cell interior as a form of complexes with K or Mg and consequently that aspartate ions were not the source for the metabolism but had a role in transporting K and Mg ions into the cell . In the present experiments we could not confirm Henderson and Walkenstein's results (3) that isolated frog sar~.orius muscle fibers were hyperpolarized by several mV when they had been immersed in Ringer's solution containing K and Mg-aspartates after they being depolarized by Ringer's solution with 5-10 mh KC1 .

The reason for

the discrepancy between these two experiments is not clear .

How-

ever, it has been shown in the present experiments that muscle fibers stored in Ringer°s solution containing K- and Mg-aspartates at 20 °C for 24 hr showed a high resting potential value compared with those stored in normal Ringer's solution under the same condition .

The

difference in the resting potentials between the two kinds of muscle fibers may reflect a difference in [K~i .

Summary Frog sartorius muscle fibers, stored in Ringer°s solution containing K- and Mg-aspartates at 8 °C or 20 °C for 24 hr, show a low intracellular Na and a high K concentration compared with those in control muscles, which were stored in normal Rüiger°s solution under the same conditions .

However, no such effect as maintaining the

intracellular Na concentration low and the K concentration high was observed when either KC1, MgC1 2 or CaC1 2 in Rüiger's solution was replaced by isomolar K-, Mg- or Ca- aspartate . Muscle fibers stared in Ringer's solution with F- and Digaspartate at 20 °C for 24 hr showed a greater resting potential value

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than that in fibers immersed in normal Ringer's solution under similar conditions .

The difference may be a consequence of a differ-

ence in the intracellular K concentration between the two groups of muscle fibers .

Acknowledgements The authors express their gratitute to Tanabe Seiyaku Co ., Ltd . for supplying them aspartate salts .

References 1.

P .J . Boyle and E .J . Conway,

J . Phvsiol . 100, 1 (1941) .

2.

M . Sato and S . Kiyosuke,

3.

E .G . Henderson and S .S . Walkenstein,

Kumamoto Med . J . 13, 272 (1960) . J . Cell . Phvsiol . 62, 231

(1967) . 4.

P .J . Boyle, E.J . Conway, F . Kané and H .L . O'Reilly,

J . Phvsiol .

99, 401 (1941) . 5.

J .E . Desmedt,

6.

W . Nastuk and A.L . Hodgkin,

J . Phvsiol . 121, 191 (1953) . J . Cell . COmp . Phvsiol . 35, 39

(1950) . 7.

A .L . Hodgkin and P . Horowicz,

8.

H . Yoshimura and K . Higaki,

J . Phvsiol . 1~, 405 (1959) . Reports of the Study Group on

Aspartate Salts 3, 88 (1965) . (in Japanese) 9.

M. Nakahara, S . Yamada, S . Sakahashi, Y . Nakanishi and T . Toits, Shiryo 11, 115 (1963) . (in Japanese)