Neuroscience Letters, 111 (1990) 157-163 Elsevier Scientific Publishers Ireland Ltd.
157
NSL 06754
The effect of Ba ions on human muscle cultured in monolayer and innervated with fetal rat spinal cord Takayoshi Kobayashi, Makoto Michikawa, Hiroshi Miyazaki and Hiroshi Tsukagoshi Department of Neurology, Tokyo Medical and Dental University, Tokyo (Japan) (Received 26 September 1989; Revised version received 1 December 1989; Accepted 1 December 1989) Key words." Ba ion; Ca component; Innervation; Human muscle; Tissue culture In human muscle culture, 7% of aneurally (AMs) and 5.9 % of innervated contracting muscle cells (ICMs) showed slow repolarization components (SRCs: duration, less than I0 ms) of action potentials. After an application of 10 mM Ba ion, prolonged SRCs, which were blocked by nifedipine, appeared in 96% of the AMs and 70% of the ICMs. The duration of SRCs under the application of 10 mM Ba solution were not significantly different between AM and ICM (2.8 and 2.0 s, respectively). The resting membrane potentials (RMPs) of AMs and ICMs decreased to 78% and 74% in the medium with I0 mM Ba solution, respectively, and the input resistance (Rin) of AM increased to 161% in the medium with 10 mMBa. Slow hyperpolarizing afterpotentials were observed both in AMs and ICMs, and completely disappeared during the application of Ba.
There have been few d e v e l o p m e n t a l e l e c t r o p h y s i o l o g i c a l studies o f the h u m a n muscle because o f the difficulty in culturing h u m a n muscle a n e u r a l l y which is innervated with m o t o n e u r o n e s [19]. W e recently established a c o - c u l t u r e system with hum a n muscle cells in m o n o l a y e r a n d fetal rat spinal c o r d with d o r s a l r o o t ganglia att a c h e d [3, 12]. In this c o - c u l t u r e system, i n n e r v a t e d muscle cells begin to c o n t r a c t , a n d have g o o d cross-striations. T h e y have chemical transmissions, a n d b e c o m e m o r e m a t u r e e l e c t r o p h y s i o l o g i c a l l y [19], b i o c h e m i c a l l y [13-15], a n d m o r p h o l o g i c a l l y [3, 12]. T h e slow C a - c o m p o n e n t was usually o b s e r v e d in the i m m a t u r e a n e u r a l l y c u l t u r e d chick a n d rat muscle cells a n d d i s a p p e a r e d in m a t u r a t i o n with time [6-11, 18]. Little has been k n o w n a b o u t Ca c o m p o n e n t s o f h u m a n muscles, this study was c o n d u c t e d to clarify the d e v e l o p m e n t a l c h a n g e o f C a c o m p o n e n t by using Ba, the net i n w a r d c u r r e n t o f which is k n o w n to be like the slow C a i n w a r d c u r r e n t [17]. W e were also
Correspondence." T. Kobayashi, Department of Neurology, Tokyo Medical and Dental University, 1-5-45 Ushima, Bunkyo-ku, Tokyo 113, Japan. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
158
interested to know the effects of the Ba ion on human muscles to clarify the cause of periodic paralysis by Ba poisoning [4, 16]. Muscle biopsies of II patients (16-78 years old) were used for this experiment. Muscle biopsies and clinical EMG mainly showed neurogenic changes, and these patients were considered not to have intrinsic muscle disorders. Aneural human muscle cultures were established according to an explant re-explantation technique [l]. Aneural muscle cells were cultured in 67% Dulbecco's modified Eagle's medium (DMEM), 23% medium 199, 10% fetal bovine serum (FBS); and supplemented with 25 ng/ml fibroblast growth factor (FGF), 10 ng/ml epidermal growth factor (EGF), and 10/lg/ml insulin. Cultures were fed twice weekly and examined every day or every other day by phase-contrast inverted microscopy. For innervation experiments, explants of spinal cords with dorsal root ganglia from 13-day-old fetal rats (Spragu~Dawley) were placed on the top of the muscle monolayer shortly after myoblast fusion. From the moment of co-culture, innervation muscle cells were cultured in mixed Dulbecco's modified Eagle medium (DMEM) and medium 199 supplemented only with 10% FBS and 10/tg/ml insulin. For electrophysiological studies, glass microelectrodes filled with 3 M KCI and with the tip resistance of 15 30 ME2 were used for recording the membrane potential and passing current. A culture dish was perfused with a medium containing: (in mM) Na 150, K 4, Ca 2.2, Mg 1.1, CI 160.6, glucose 5.6, and HEPES 10 (pH 7.2); as well as 0.1% bovine serum albumin. The medium in the dish was kept at 35"C. One/~M nifedipine was added to the perfusing medium as needed. Ten mM BaCI2 was also added to the perfusing medium with or without an addition of CaCl> Data were transferred and stored in a personal computer (PC-9801) through an A-D converter. Statistical analysis was done according to the Student t-test. In this culture system, AM cells are fiat and irregular in diameter. They neither contract nor have cross-striations. After about 10 days of co-culture, innervated musTABLE I ELECTRICAL PARAMETERS OF C U L T U R E D H U M A N MUSCLE CELLS Values are mean _+_ S.E.M. and number of cells tested are in parentheses. Amplitude of action potential was measured from resting membrane potential to the peak of off-response after hyperpolarization of membrane to about - 100 mV. Resting membrane potential (mV)
Amplitude of action potential (mV)
Aneurally
55.1 -+ 1.3
71.7+1.3
163.1 ± 10.1
13.9±1.1
cultured cells
(n = 71)
(n =47)
(n = 45)
(n =48)
Innervated contracting
62.0+_1.4" (n = 34)
83.4_+2.6* (n = 26)
223.7_+ 19.8" (n = 15)
9.1 _+ 1.0" (n = 21 )
cells * P < 0.01 compared with aneurally cultured cells.
Maximum rate of rise of action potential (V/s)
Input resistance (M.Q)
159 cle cells began to contract and to develop cross-striations [12]. In co-culture, miniature endplate potentials (MEPPs) were recorded from muscle cells in 3 days of coculture with the spinal cord. Spontaneous endplate potentials (EPPs) and action potentials were observed in 6 days of co-culture. Both MEPPs and EPPs were blocked by 2/~M ( + ) - t u b o c u r a r i n e [2, 19]. Table I shows the summary of electrical parameters of cultured human muscle cells in the present study. Resting membrane potential (RMP), amplitude of action potential and the maximum rate of rise of action potential of I C M fibers were significantly larger than those of A M cells. And the input membrane resistance (Rin) of 1CM fibers was significantly smaller than that of AM cells. Usually no notches of SRCs during the repolarizing phase, which was preceded by the fast rising phase of action potentials, were observed either in human A M or 1CM cells with faster sweep speed (2 ms/div). A few human A M (7%, n = 7 1 ) and I C M cells (5.9%, n = 3 4 ) , however, had these components, the durations of which were only 10.4+0.7 ms (S.E.M., n = 5 ) and 9.5 ms (n=2), respectively (Fig. 1). Fig. 2-I shows the effects of Ba ion on AM. In this cultured A M cell, no SRC is observed. Just after the application of 10 m M Ba ion without Ca ion, the Rin becomes larger and the R M P begins to depolarize, and SRC preceded by the fast rising phase of action potential appears. This component disappears after washing out of 10 m M Ba. Fig. 2-1I shows that this SRC is blocked by 1 ~ M nifedipine. This suggests that the SRC is evoked by a Ca inward current. Fig. 3 shows the effects of Ba ion on ICM. After the application of Ba ion, the R M P begins to depolarize, and the frequency of spontaneous action potentials increases. SRCs appear between 30 s and 1 min after the application of Ba ion. During Ba application, SRCs appear without muscle contractions. During the washing out of Ba ion, the duration and the amplitude of these components decrease, and finally only fast action potentials can be observed with muscle contractions. In a prolonged
AM mV"L
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Fig. 1. Appearance of a slow repolarization component (SRC) elicited by anodal break excitation in the regular medium. AM, aneurally cultured human muscle; ICM, innervated contracting human muscle. Each lower record shows the same action potential of the upper record with faster sweep speed. 1 in AM and ICM: no SRC is preceded by the fast rising phase of action potentials. 2 in AM and ICM: SRCs with about 10 ms duration are detected in the lower records. Hyperpolarizingcurrents are applied in each experiment (AM-I: 500 ms, 4 nA; AM-2:150 ms, 7 hA; ICM-I: 350 ms, 4 nA; ICM-2:150 ms, 8 nA). AM-1 in 16 days of culture, AM-2 in 15 days of culture; ICM-1 and 2 in 36 days of co-culture.
160
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Fig. 2. Effect of Ba ion on aneurally cultured human muscle. 1: A, control; B, just after the application of 10 mM Ba ion without Ca ion; C, during the application of Ba ion without Ca ion; D, after washing out of Ba ion without Ca ion. Hyperpolarizing current (500 ms, 4 nA) is applied during this experiment. II: A, SRC appears in the application of 10 mM Ba ion without Ca ion; B, SRC disappears after the application of 1 pM nifedipine. Hyperpolarizing current (1 s, 4 nA) is applied during this experiment. I and II are at 16 days of culture. III: Change in membrane conductance during SRC of an AM cell. Hyperpolarizing current (500 ms, 8 nA) is applied in this experiment. In 11 days of culture. The upper trace records current intensity, and the lower trace represents membrane potential change.
A
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Fig. 3. Effect of Ba ion on innervated contracting human muscle. A: control. Spontaneous firing with muscle contractions. B: just after the application of 10 mM Ba ion without Ca ion. C: during the application of Ba ion without Ca ion. D, E: during washing out of 10 m M Ba ion without Ca ion. B-E show spontaneous firings with prolonged SRCs without muscle contractions. F: after washing out of Ba ion without Ca ion. Disappearance of these SRCs (in 23 days of co-culture).
Aneurally cultured cells Innervated contracting cells
95.7% (n = 23) 70 % (n = 10)
Percentage of cells with SRC in the medium with 10 mM Ba (%) 56.4±3.4 (n = 7) 66±1.3 (n = 8)
(n = 8)
78.0±5.2 74.0±2.7
~.1±3.9 48.8±1.6
18.7+4.5 (n = 6)
33.7+ 10.8
(D) 161.1 ___+15.0
Control (Mr2) (C) 10 mM Ba (MI2) D/C x 100 (%)
B/Axl00
(%)
10mMBa(mV)
Control (mV) (A)
(B)
Input resistance
Resting membrane potential
2.8±0.4 (n = 22) 2.0±0.8
Max. duration of SRC (s)
SRC, slow repolarization component. The duration of SRC is measured from the inflexion point of the repolarizing fast action potential to the point where SRC reached the resting membrane potential. The means of B/A × 100(%) and D/C × 100(%) are figured out from the decreased or increased percentages of the individual experiments. Values are mean ± S.E.M. and number of cells tested are in the parentheses.
EFFECT OF Ba ION ON CULTURED HUMAN MUSCLE CELLS
TABLE II
162 SRC, the conductance becomes larger at the early phase of the slow repolarizing potential than in the resting stage (Fig. 2-III). In AM and ICM, the slow hyperpolarizing afterpotentials appear; however, these are suppressed by the application of B~ ion (Figs. 1 3) probably because Ba has an inhibitory action on delayed rectification. After the application of 10 m M BaCI2 with 2.2 m M CaCI2, the durations of these slow components (0.9 _+0.7 s, n = 5) became significantly shorter (P < 0.05) than those (4.2+_ 1.4s, n = 5 ) in the medium of 10 m M B a ion without Ca ion. Jmari et al. reported that the same phenomenon was observed in rat uterine smooth muscle under different concentrations of external Ca when Ba was the charger carrier. And their results suggest that Ba is highly permanent when Ca was absent, but only sparsely permanent when Ca was present [5]. Prolonged SRC which appears in 10 m M Ba application without Ca ion, is due mainly to Ba inward current through Ca channels, and Ca dependant repolarizing K current is possibly negligible, because this prolonged SRC is completely blocked by nifedipine. Table II shows the summary of the effect of Ba ion on cultured human muscle. The SRC appeared in both AM and ICM. The appearance rate of this component was less in the innervated culture (70%) than in the aneural culture (96%). It is presumed that, during development, SRC becomes less activated after innervation. The duration of these components, however, was not significantly different between the aneural and innervated cultures (2.8 +_0.4 and 2.0 + 0.8 s, respectively). The resting membrane potentials were decreased to 78% and 74% in the aneural and innervated cultures, respectively, and Rin increased to 161% in the aneural culture. These may be due to a decreased resting potassium conductance [20]. Regarding the Ca components, cultured human muscle seems more 'mature' in its early stage compared to cultured chick and rat muscles, because cultured chick and rat muscle cells usually have prolonged Ca components in their early stages of aneural culture [6-1 l, 18] whereas cultured human muscle cells rarely had these components even in their early stage. The significance of the role of this Ca component in the development of skeletal muscle cells is still unknown. From the clinical point of view, muscle paralysis induced by Ba poisoning might be not due to hypokalemia but to the decrease of the resting membrane potential.
! Askanas, V. and Engel, W.K., New program for investigating adult human skeletal muscle grown aneurally in tissue culture, Neurology, 25 (1975) 58~7. 2 Askanas, V., Engel, W.K. and Kobayashi, T., Thyrotrophin-releasinghormone enhances motor neuron-evoked contractions of cultured human muscle, Ann. Neurol., 18 (1985) 716-719. 3 Askanas, V., Kwan, H., Alvarez, P.B., Engel, W.K., Kobayashi, T., Martinuzzi, A. and Hawkins, E.F., De novo neuromuscularjunction formation on human muscle fibers cultured in monolayer and innervated by fetal rat spinal cord: Ultrastructural and ultrastructural-cytochemicalstudies, J. Neurocytol., 16 (1987) 523 527. 4 Huang, K.W., Pa ping (transient paralysis simulating family periodic paralysis), Chin. Med. J., 61 (1943) 305 312. 5 Jmari, K., Mironneau, C. and Mironneau, J., Selectivity of calcium channels in rat uterine smooth muscle: interactions between sodium, calcium and barium ions, J. Physiol. (Lond.), 384 (1987) 247--261.
163 6 Kano, M. and Shimada, Y., Tetrodotoxin resistant electrical activity in chick skeletal muscle cells differentiated in vitro, J. Cell. Physiol., 81 (1973) 85 90. 7 Kano, M., Development of excitability in embryonic chick skeletal muscle cells, J. Cell. Physiol., 86 (1975) 503 510. 8 Kano, M. and Yamamoto, M., Development of spike potentials in skeletal muscle cells differentiated in vitro from chick embryo, J. Cell. Physiol., 90 (1977) 439~t44. 9 Kidokoro, Y., Development of action potentials in a clonal rat skeletal muscle cell line, Nature New Biol., 241 (1973) 158-159. 10 Kidokoro, Y., Developmental changes of membrane electrical properties in a rat skeletal muscle cell line, J. Physiol. (Lond.), 244 (1975) 129-143. 11 Kidokoro. Y., Sodium and calcium components of the action potential in a developing skeletal muscle cell line, J. Physiol. (Lond.), 244 (1975) 145 159. 12 Kobayashi, T., Askanas, V. and Engel, W.K., Innervation of human muscle cultured in monolayer by rat spinal cord: Importance of dorsal root ganglia for achieving successful functional innervation, J. Neurosci., 7 (1987) 3131-1341. 13 Martinuzzi, A., Askanas, V., Kobayashi, T., Engel, W.E. and DiMauro, S., Expression of muscle gene specific isoenzyme of phosphorylase and creatine kinase in innervated cultured human muscle, J. Cell Biol., 103 (1986) 1423 1429. 14 Martinuzzi, A., Askanas, V., Kobayashi, T., Engel, W.E. and Gorsky, J.E., Developmental expression of the muscle-specific isoenzyme of phosphoglycerate mutase in human muscle cultured in monolayer and innervated by fetal rat spinal cord, Exp. Neurol., 96 (1987) 365-375. 15 Martinuzzi, A., Askanas, V., Kobayashi, T. and Engel, W.E., Asynchronous regulation of muscle specific isoenzymes of creatinine kinase, glycogen phosphorylase, lactic dehydrogenase and phosphoglycerate mutase in innervated and noninnervated cultured human muscle, Neurosci. Lett., 89 (1988) 216-222. 16 Phelan, D.M., Hagley, S.R. and Guerin, M.D., Is hypokalemia the cause of paralysis in barium poisoning?, Br. Med. J., 289 (1984) 882. 17 Raymond, G. and Potreau, D., Barium ions and excitation-contraction coupling of frog single muscle fibers under controlled current and voltage. J. Physiol. (Paris), 73 (1977) 617 631. 18 Saito, K. and Ozawa, E., Contraction of cultured chick myotubes induced by electrical stimulation, Biomed. Res., 7 (1986) 69-77. 19 Saito, K., Kobayashi, T., Askanas, V., Engel, W.K. and Ishikawa, K., Electrical parameters of human muscle cultured in monolayer, aneurally and innervated by rat spinal cord, Muscle Nerve, 9, Suppl. 5S (1986) 162. 20 Sperelakis, N., Schneider, M.F. and Harris, E.J., Decreased K conductance produced by Ba in frog sartorius fibers, J. Gen. Physiol., 50 (1967) 1565-1583.
157
NSL 06754
The effect of Ba ions on human muscle cultured in monolayer and innervated with fetal rat spinal cord Takayoshi Kobayashi, Makoto Michikawa, Hiroshi Miyazaki and Hiroshi Tsukagoshi Department of Neurology, Tokyo Medical and Dental University, Tokyo (Japan) (Received 26 September 1989; Revised version received 1 December 1989; Accepted 1 December 1989) Key words." Ba ion; Ca component; Innervation; Human muscle; Tissue culture In human muscle culture, 7% of aneurally (AMs) and 5.9 % of innervated contracting muscle cells (ICMs) showed slow repolarization components (SRCs: duration, less than I0 ms) of action potentials. After an application of 10 mM Ba ion, prolonged SRCs, which were blocked by nifedipine, appeared in 96% of the AMs and 70% of the ICMs. The duration of SRCs under the application of 10 mM Ba solution were not significantly different between AM and ICM (2.8 and 2.0 s, respectively). The resting membrane potentials (RMPs) of AMs and ICMs decreased to 78% and 74% in the medium with I0 mM Ba solution, respectively, and the input resistance (Rin) of AM increased to 161% in the medium with 10 mMBa. Slow hyperpolarizing afterpotentials were observed both in AMs and ICMs, and completely disappeared during the application of Ba.
There have been few d e v e l o p m e n t a l e l e c t r o p h y s i o l o g i c a l studies o f the h u m a n muscle because o f the difficulty in culturing h u m a n muscle a n e u r a l l y which is innervated with m o t o n e u r o n e s [19]. W e recently established a c o - c u l t u r e system with hum a n muscle cells in m o n o l a y e r a n d fetal rat spinal c o r d with d o r s a l r o o t ganglia att a c h e d [3, 12]. In this c o - c u l t u r e system, i n n e r v a t e d muscle cells begin to c o n t r a c t , a n d have g o o d cross-striations. T h e y have chemical transmissions, a n d b e c o m e m o r e m a t u r e e l e c t r o p h y s i o l o g i c a l l y [19], b i o c h e m i c a l l y [13-15], a n d m o r p h o l o g i c a l l y [3, 12]. T h e slow C a - c o m p o n e n t was usually o b s e r v e d in the i m m a t u r e a n e u r a l l y c u l t u r e d chick a n d rat muscle cells a n d d i s a p p e a r e d in m a t u r a t i o n with time [6-11, 18]. Little has been k n o w n a b o u t Ca c o m p o n e n t s o f h u m a n muscles, this study was c o n d u c t e d to clarify the d e v e l o p m e n t a l c h a n g e o f C a c o m p o n e n t by using Ba, the net i n w a r d c u r r e n t o f which is k n o w n to be like the slow C a i n w a r d c u r r e n t [17]. W e were also
Correspondence." T. Kobayashi, Department of Neurology, Tokyo Medical and Dental University, 1-5-45 Ushima, Bunkyo-ku, Tokyo 113, Japan. 0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
158
interested to know the effects of the Ba ion on human muscles to clarify the cause of periodic paralysis by Ba poisoning [4, 16]. Muscle biopsies of II patients (16-78 years old) were used for this experiment. Muscle biopsies and clinical EMG mainly showed neurogenic changes, and these patients were considered not to have intrinsic muscle disorders. Aneural human muscle cultures were established according to an explant re-explantation technique [l]. Aneural muscle cells were cultured in 67% Dulbecco's modified Eagle's medium (DMEM), 23% medium 199, 10% fetal bovine serum (FBS); and supplemented with 25 ng/ml fibroblast growth factor (FGF), 10 ng/ml epidermal growth factor (EGF), and 10/lg/ml insulin. Cultures were fed twice weekly and examined every day or every other day by phase-contrast inverted microscopy. For innervation experiments, explants of spinal cords with dorsal root ganglia from 13-day-old fetal rats (Spragu~Dawley) were placed on the top of the muscle monolayer shortly after myoblast fusion. From the moment of co-culture, innervation muscle cells were cultured in mixed Dulbecco's modified Eagle medium (DMEM) and medium 199 supplemented only with 10% FBS and 10/tg/ml insulin. For electrophysiological studies, glass microelectrodes filled with 3 M KCI and with the tip resistance of 15 30 ME2 were used for recording the membrane potential and passing current. A culture dish was perfused with a medium containing: (in mM) Na 150, K 4, Ca 2.2, Mg 1.1, CI 160.6, glucose 5.6, and HEPES 10 (pH 7.2); as well as 0.1% bovine serum albumin. The medium in the dish was kept at 35"C. One/~M nifedipine was added to the perfusing medium as needed. Ten mM BaCI2 was also added to the perfusing medium with or without an addition of CaCl> Data were transferred and stored in a personal computer (PC-9801) through an A-D converter. Statistical analysis was done according to the Student t-test. In this culture system, AM cells are fiat and irregular in diameter. They neither contract nor have cross-striations. After about 10 days of co-culture, innervated musTABLE I ELECTRICAL PARAMETERS OF C U L T U R E D H U M A N MUSCLE CELLS Values are mean _+_ S.E.M. and number of cells tested are in parentheses. Amplitude of action potential was measured from resting membrane potential to the peak of off-response after hyperpolarization of membrane to about - 100 mV. Resting membrane potential (mV)
Amplitude of action potential (mV)
Aneurally
55.1 -+ 1.3
71.7+1.3
163.1 ± 10.1
13.9±1.1
cultured cells
(n = 71)
(n =47)
(n = 45)
(n =48)
Innervated contracting
62.0+_1.4" (n = 34)
83.4_+2.6* (n = 26)
223.7_+ 19.8" (n = 15)
9.1 _+ 1.0" (n = 21 )
cells * P < 0.01 compared with aneurally cultured cells.
Maximum rate of rise of action potential (V/s)
Input resistance (M.Q)
159 cle cells began to contract and to develop cross-striations [12]. In co-culture, miniature endplate potentials (MEPPs) were recorded from muscle cells in 3 days of coculture with the spinal cord. Spontaneous endplate potentials (EPPs) and action potentials were observed in 6 days of co-culture. Both MEPPs and EPPs were blocked by 2/~M ( + ) - t u b o c u r a r i n e [2, 19]. Table I shows the summary of electrical parameters of cultured human muscle cells in the present study. Resting membrane potential (RMP), amplitude of action potential and the maximum rate of rise of action potential of I C M fibers were significantly larger than those of A M cells. And the input membrane resistance (Rin) of 1CM fibers was significantly smaller than that of AM cells. Usually no notches of SRCs during the repolarizing phase, which was preceded by the fast rising phase of action potentials, were observed either in human A M or 1CM cells with faster sweep speed (2 ms/div). A few human A M (7%, n = 7 1 ) and I C M cells (5.9%, n = 3 4 ) , however, had these components, the durations of which were only 10.4+0.7 ms (S.E.M., n = 5 ) and 9.5 ms (n=2), respectively (Fig. 1). Fig. 2-I shows the effects of Ba ion on AM. In this cultured A M cell, no SRC is observed. Just after the application of 10 m M Ba ion without Ca ion, the Rin becomes larger and the R M P begins to depolarize, and SRC preceded by the fast rising phase of action potential appears. This component disappears after washing out of 10 m M Ba. Fig. 2-1I shows that this SRC is blocked by 1 ~ M nifedipine. This suggests that the SRC is evoked by a Ca inward current. Fig. 3 shows the effects of Ba ion on ICM. After the application of Ba ion, the R M P begins to depolarize, and the frequency of spontaneous action potentials increases. SRCs appear between 30 s and 1 min after the application of Ba ion. During Ba application, SRCs appear without muscle contractions. During the washing out of Ba ion, the duration and the amplitude of these components decrease, and finally only fast action potentials can be observed with muscle contractions. In a prolonged
AM mV"L
1 i
I.C.M
2 I5~A
mY ]
j
\,
O0
0
my 1
1
2 ISnA
,,vl
I
I
J, ~
40
120
15hA
___j/'
1
60n~ 0
:~
120
~mJc mY 01
n, / ~\\
21,a~
Fig. 1. Appearance of a slow repolarization component (SRC) elicited by anodal break excitation in the regular medium. AM, aneurally cultured human muscle; ICM, innervated contracting human muscle. Each lower record shows the same action potential of the upper record with faster sweep speed. 1 in AM and ICM: no SRC is preceded by the fast rising phase of action potentials. 2 in AM and ICM: SRCs with about 10 ms duration are detected in the lower records. Hyperpolarizingcurrents are applied in each experiment (AM-I: 500 ms, 4 nA; AM-2:150 ms, 7 hA; ICM-I: 350 ms, 4 nA; ICM-2:150 ms, 8 nA). AM-1 in 16 days of culture, AM-2 in 15 days of culture; ICM-1 and 2 in 36 days of co-culture.
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Fig. 2. Effect of Ba ion on aneurally cultured human muscle. 1: A, control; B, just after the application of 10 mM Ba ion without Ca ion; C, during the application of Ba ion without Ca ion; D, after washing out of Ba ion without Ca ion. Hyperpolarizing current (500 ms, 4 nA) is applied during this experiment. II: A, SRC appears in the application of 10 mM Ba ion without Ca ion; B, SRC disappears after the application of 1 pM nifedipine. Hyperpolarizing current (1 s, 4 nA) is applied during this experiment. I and II are at 16 days of culture. III: Change in membrane conductance during SRC of an AM cell. Hyperpolarizing current (500 ms, 8 nA) is applied in this experiment. In 11 days of culture. The upper trace records current intensity, and the lower trace represents membrane potential change.
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Fig. 3. Effect of Ba ion on innervated contracting human muscle. A: control. Spontaneous firing with muscle contractions. B: just after the application of 10 mM Ba ion without Ca ion. C: during the application of Ba ion without Ca ion. D, E: during washing out of 10 m M Ba ion without Ca ion. B-E show spontaneous firings with prolonged SRCs without muscle contractions. F: after washing out of Ba ion without Ca ion. Disappearance of these SRCs (in 23 days of co-culture).
Aneurally cultured cells Innervated contracting cells
95.7% (n = 23) 70 % (n = 10)
Percentage of cells with SRC in the medium with 10 mM Ba (%) 56.4±3.4 (n = 7) 66±1.3 (n = 8)
(n = 8)
78.0±5.2 74.0±2.7
~.1±3.9 48.8±1.6
18.7+4.5 (n = 6)
33.7+ 10.8
(D) 161.1 ___+15.0
Control (Mr2) (C) 10 mM Ba (MI2) D/C x 100 (%)
B/Axl00
(%)
10mMBa(mV)
Control (mV) (A)
(B)
Input resistance
Resting membrane potential
2.8±0.4 (n = 22) 2.0±0.8
Max. duration of SRC (s)
SRC, slow repolarization component. The duration of SRC is measured from the inflexion point of the repolarizing fast action potential to the point where SRC reached the resting membrane potential. The means of B/A × 100(%) and D/C × 100(%) are figured out from the decreased or increased percentages of the individual experiments. Values are mean ± S.E.M. and number of cells tested are in the parentheses.
EFFECT OF Ba ION ON CULTURED HUMAN MUSCLE CELLS
TABLE II
162 SRC, the conductance becomes larger at the early phase of the slow repolarizing potential than in the resting stage (Fig. 2-III). In AM and ICM, the slow hyperpolarizing afterpotentials appear; however, these are suppressed by the application of B~ ion (Figs. 1 3) probably because Ba has an inhibitory action on delayed rectification. After the application of 10 m M BaCI2 with 2.2 m M CaCI2, the durations of these slow components (0.9 _+0.7 s, n = 5) became significantly shorter (P < 0.05) than those (4.2+_ 1.4s, n = 5 ) in the medium of 10 m M B a ion without Ca ion. Jmari et al. reported that the same phenomenon was observed in rat uterine smooth muscle under different concentrations of external Ca when Ba was the charger carrier. And their results suggest that Ba is highly permanent when Ca was absent, but only sparsely permanent when Ca was present [5]. Prolonged SRC which appears in 10 m M Ba application without Ca ion, is due mainly to Ba inward current through Ca channels, and Ca dependant repolarizing K current is possibly negligible, because this prolonged SRC is completely blocked by nifedipine. Table II shows the summary of the effect of Ba ion on cultured human muscle. The SRC appeared in both AM and ICM. The appearance rate of this component was less in the innervated culture (70%) than in the aneural culture (96%). It is presumed that, during development, SRC becomes less activated after innervation. The duration of these components, however, was not significantly different between the aneural and innervated cultures (2.8 +_0.4 and 2.0 + 0.8 s, respectively). The resting membrane potentials were decreased to 78% and 74% in the aneural and innervated cultures, respectively, and Rin increased to 161% in the aneural culture. These may be due to a decreased resting potassium conductance [20]. Regarding the Ca components, cultured human muscle seems more 'mature' in its early stage compared to cultured chick and rat muscles, because cultured chick and rat muscle cells usually have prolonged Ca components in their early stages of aneural culture [6-1 l, 18] whereas cultured human muscle cells rarely had these components even in their early stage. The significance of the role of this Ca component in the development of skeletal muscle cells is still unknown. From the clinical point of view, muscle paralysis induced by Ba poisoning might be not due to hypokalemia but to the decrease of the resting membrane potential.
! Askanas, V. and Engel, W.K., New program for investigating adult human skeletal muscle grown aneurally in tissue culture, Neurology, 25 (1975) 58~7. 2 Askanas, V., Engel, W.K. and Kobayashi, T., Thyrotrophin-releasinghormone enhances motor neuron-evoked contractions of cultured human muscle, Ann. Neurol., 18 (1985) 716-719. 3 Askanas, V., Kwan, H., Alvarez, P.B., Engel, W.K., Kobayashi, T., Martinuzzi, A. and Hawkins, E.F., De novo neuromuscularjunction formation on human muscle fibers cultured in monolayer and innervated by fetal rat spinal cord: Ultrastructural and ultrastructural-cytochemicalstudies, J. Neurocytol., 16 (1987) 523 527. 4 Huang, K.W., Pa ping (transient paralysis simulating family periodic paralysis), Chin. Med. J., 61 (1943) 305 312. 5 Jmari, K., Mironneau, C. and Mironneau, J., Selectivity of calcium channels in rat uterine smooth muscle: interactions between sodium, calcium and barium ions, J. Physiol. (Lond.), 384 (1987) 247--261.
163 6 Kano, M. and Shimada, Y., Tetrodotoxin resistant electrical activity in chick skeletal muscle cells differentiated in vitro, J. Cell. Physiol., 81 (1973) 85 90. 7 Kano, M., Development of excitability in embryonic chick skeletal muscle cells, J. Cell. Physiol., 86 (1975) 503 510. 8 Kano, M. and Yamamoto, M., Development of spike potentials in skeletal muscle cells differentiated in vitro from chick embryo, J. Cell. Physiol., 90 (1977) 439~t44. 9 Kidokoro, Y., Development of action potentials in a clonal rat skeletal muscle cell line, Nature New Biol., 241 (1973) 158-159. 10 Kidokoro, Y., Developmental changes of membrane electrical properties in a rat skeletal muscle cell line, J. Physiol. (Lond.), 244 (1975) 129-143. 11 Kidokoro. Y., Sodium and calcium components of the action potential in a developing skeletal muscle cell line, J. Physiol. (Lond.), 244 (1975) 145 159. 12 Kobayashi, T., Askanas, V. and Engel, W.K., Innervation of human muscle cultured in monolayer by rat spinal cord: Importance of dorsal root ganglia for achieving successful functional innervation, J. Neurosci., 7 (1987) 3131-1341. 13 Martinuzzi, A., Askanas, V., Kobayashi, T., Engel, W.E. and DiMauro, S., Expression of muscle gene specific isoenzyme of phosphorylase and creatine kinase in innervated cultured human muscle, J. Cell Biol., 103 (1986) 1423 1429. 14 Martinuzzi, A., Askanas, V., Kobayashi, T., Engel, W.E. and Gorsky, J.E., Developmental expression of the muscle-specific isoenzyme of phosphoglycerate mutase in human muscle cultured in monolayer and innervated by fetal rat spinal cord, Exp. Neurol., 96 (1987) 365-375. 15 Martinuzzi, A., Askanas, V., Kobayashi, T. and Engel, W.E., Asynchronous regulation of muscle specific isoenzymes of creatinine kinase, glycogen phosphorylase, lactic dehydrogenase and phosphoglycerate mutase in innervated and noninnervated cultured human muscle, Neurosci. Lett., 89 (1988) 216-222. 16 Phelan, D.M., Hagley, S.R. and Guerin, M.D., Is hypokalemia the cause of paralysis in barium poisoning?, Br. Med. J., 289 (1984) 882. 17 Raymond, G. and Potreau, D., Barium ions and excitation-contraction coupling of frog single muscle fibers under controlled current and voltage. J. Physiol. (Paris), 73 (1977) 617 631. 18 Saito, K. and Ozawa, E., Contraction of cultured chick myotubes induced by electrical stimulation, Biomed. Res., 7 (1986) 69-77. 19 Saito, K., Kobayashi, T., Askanas, V., Engel, W.K. and Ishikawa, K., Electrical parameters of human muscle cultured in monolayer, aneurally and innervated by rat spinal cord, Muscle Nerve, 9, Suppl. 5S (1986) 162. 20 Sperelakis, N., Schneider, M.F. and Harris, E.J., Decreased K conductance produced by Ba in frog sartorius fibers, J. Gen. Physiol., 50 (1967) 1565-1583.
