Chronic treatment with D600 enhances development of sodium channels in cultured chick skeletal muscle cells

Neuroscience Letters, 138 (1992) 249-252 ~ 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Chronic treatment with D600 enhances development of sodium channels in cultured chick skeletal muscle cells R y o h e i Satoh, Y u m i k o N a k a b a y a s h i a n d M a s a a k i r a K a n o Department of Physiology, Kitasato University School of Medicine, Kanagawa (Japan) (Received 16 December 1991; Revised version received 23 January 1992; Accepted 24 January 1992)

K~:v words: Embryonic chick skeletal muscle; Na channel; Ca channel: Development; D600: Nifedipine: BAY K 8644 We have studied the long-term effects of D600, a blocker of L-type voltage-dependent Ca channels (VDCC), on the development of voltagedependent Na channels (VDNC) that are sensitive to tetrodotoxin (TTX), by electrophysiological measurements of the maximum rate of rise of the YTX-sensitive Na spike in cultured chick skeletal muscle cells. Chronic treatment with D600 (2-20/~M) caused a dose-dependent increase in the density of VDNC. The density of VDNC was increased by 150 250% when D600 was added to the cultures at 20,uM from the second day of culture onward. Co-treatment with an inhibitor of the transcription of RNA from DNA, c~-amanitin, or with cycloheximide, an inhibitor of protein synthesis, prevented the up-regulation by D600. Nifedipine, a different type of blocker of L-type VDCC, was also effective in increasing the density of VDNC, and BAY K 8644, an agonist of L-type VDCC, had the opposite effect. It is suggested that the effect of D600 was mediated via a mechanism specific for L-type VDCC that involves regulation of cytosolic levels of Ca -'+ and protein synthesis de novo.

In skeletal muscle cells, the developmental changes [4, 13, 15, 20, 21] and the electrophysiological properties [1, 3, 6, 14] of the various types of voltage-dependent cationic channel, such as the tetrodotoxin (TTX)-sensitive Na channel (VDNC) or the T- and L-type Ca channels (VDCCs), have been well characterized, but the mechanisms that control the development of these channels are still unknown. Our previous studies have demonstrated that the VDCCs are more prominent than the V D N C in cultured chick skeletal muscle cells at less differentiated stages and there is a gradual switching from the VDCCs to the VDNC with age in such cultures [9]. Because of the unique time course of the appearance of VDCCs and VDNC, our attention has been focused on the interrelationships between VDCCs and VDNC in differentiating chick skeletal muscle cells. It has been demonstrated that the chronic blockade of VDNC by T T X causes an increase in the density of VDNC in cultured rat skeletal myotubes, and it has been suggested that the mechanism for regulating the density of VDNC in cultured myotubes involves the regulation of levels of cytosolic Ca 2+ by electrical activity [2, 19]. It is generally agreed that L-type VDCC currents constitute Corre.q~ondence." R. Satoh, Department of Physiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, Japan.

a major pathway for the voltage-gated influx of Ca 2+ [17, 18] and, hence, L-type VDCC play an essential role in the control of cytosolic levels of Ca 2+, especially at the early stage of differentiation of skeletal muscle cells [5]. With these considerations in mind, we might expect that the development of VDNC is related to some function of the L-type VDCC. To examine this possibility, the effect of chronic treatment with modulators of L-type VDCC on the development of VDNC was investigated in the present study. Some of these results have been reported elsewhere in abstract form [8]. Cultures of muscle cells were prepared from pectoral muscles of 12-day-old chick embryos as described elsewhere [13]. D600, nifedipine or BAY K 8644 was added to the cultures from the second day of culture onward, except in the experiment for which results are shown in Fig. 3, where treatment with D600 was begun at 6 days in culture. The electrophysiological techniques used have also been described elsewhere [15]. In brief, the cells were bathed in Tyrode's solution buffered with Tris-HC1 (pH 7.4) and maintained at 36°C during experiments. A conventional technique with two intracellular glass microelectrodes was employed. Both intracellular electrodes, one for passing a transmembrane current and the other for recording the transmembrane potential, were filled with 3 M KC1 and had resistances of 2 0 4 0 MI2. To ensure the maximum generation of Na spike, the cell was

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Fig. 1. Effect of chronic treatment with D600 on the M.R.R. of TTXsensitive Na spikes in cultured chick skeletal muscle cells. Treatment with 2 0 # M D600 was begun at 2 days in culture, and measurements of the M.R.R. were made from the third day of culture. The M.R.R. in treated cultures (e) and untreated control cultures (©) is plotted against the age of the culture. Points with error bars (_+ S.D.) represent averages derived from measurements made on 20 cells. Inset shows a typical Na spike potential (upper trace) and the corresponding, electronically differentiated trace (lower trace) from a muscle cell in an untreated 14day-old culture; arrowhead in the lower trace indicates the beginning o f a stimulatory current pulse; the short dashed line is the reference potential.

hyperpolarized locally to -80 mV with a constant current before a stimulatory current pulse was applied. In this experiment, the development of TTX-sensitive VDNC was assessed quantitatively in terms of the maximum rate of rise (M.R.R.) of the Na spike, which has been shown to be TTX-sensitive [15], since the M.R.R. should be proportional to the density of the VDNC. The M.R.R. of the Na spike was estimated by measuring the height of the differential of the Na spike obtained with a RC circuit (the time constant 200 #s). Drugs used in the present study were: D600 (Knoll), nifedipine (Sigma), BAY K 8644 (Sigma), ~-amanitin (Sigma), cycloheximide (Sigma) and TTX (Sankyo). Statistical significance of data was examined by Student's t-test. As is illustrated in Fig. 1, exposure to 20 #M D600 from the second day of culture onward resulted in an increase in the M.R.R. of the Na spike throughout the culture period examined. The dose-dependence of this effect was also evaluated (Fig. 2). This result suggests that D600 caused an increase in the density of TTXsensitive VDNC in cultured chick skeletal muscle cells. Similar findings have been reported recently with verapamil, a phenylalkylamine, in an experiment that involved binding of [3H]saxitoxin [2]. In order to clarify the relationship between influx of Ca 2+ through L-type VDCC and increases in the density of VDNC, we exam-

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Fig. 2. Dose-response relationship for the effect of treatment with D600 on the increase in the M.R.R. of Na spikes from muscle cells in 10-day-old cultures (treatment with D600 for 8 days). Values are the means _+ S.D. of results from 10 samples. *Difference between the values for experimental and control cultures is significant (P < 0.0001).

ined the effects of the chronic exposure to two other modulators of L-type VDCC, nifedipine and BAY K 8644, on the M.R.R. of the Na spike. Nifedipine and BAY K 8644 have antagonistic and agonistic effects on L-type VDCC, respectively [7, 17]. An increase in the M.R.R.'s by chronic treatment with nifedipine (10 #M) was of the same extent as by chronic treatment with D600 (20 #M). Conversely, the M.R.R.'s measured in 8-day-old cultures decreased by approximately 20% upon chronic treatment with BAY K 8644 (20 #M) as compared with untreated control cultures (not illustrated). Although modulators of L-type VDCC including D600, do not always interact exclusively with L-type VDCC [22], these data support the hypothesis that the chronic effects of modulators of L-type VDCC on the density of VDNC are, at least in part, mediated by alterations in the rate of entry of Ca 2+ via L-type VDCC. The D600-induced up-regulation of VDNC may be explained in several ways: (1) by inhibition of the degradation of VDNC; (2) by activation of the inactivated form of the VDNC; (3) by increased biosynthesis of the VDNC. To examine the third possibility, we tested the effects of a-amanitin [12, 16] and cycloheximide, inhibitors of the transcription of RNA from DNA and of protein synthesis, respectively. Simultaneous treatment with these inhibitors (a-amanitin at 1 #g/ml of culture medium; cycloheximide at 3 BM) with D600 abolished the D600-induced up-regulation (Fig. 3). From these results it seems likely that the D600-induced development of VDNC could be explained by increased biosynthesis of the VDNC. Furthermore, a-amanitin caused an increase in the density of the VDNC to a level higher than that found in the cultures treated with D600 alone,

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Fig. 3. Effects of c~-amanitin (A) and cycloheximide (B) on the D600induced increase in the M.R.R. of N a spikes of cultured chick skeletal muscle cells. In this experiment, treatment with D600 (20 t M ) was begun at 6 days in culture, at which time the M.R.R. reached almost the m a x i m u m value measured in untreated control cultures (o). The treatment with D600 induced further increases in the M.R.R. (e). The other symbols are as follows: I , simultaneous treatment with D600 and an appropriate inhibitor (~-amanitin at 1 t g / m l of culture medium; cycloheximide at 3 tiM); A, addition o f inhibitor after a 24-h treatment with D600; D, treatment with inhibitor alone. Each point is the average of results from 10 cells.

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V when treatment with ~-amanitin was initiated 24 h after the start of treatment with D600. Further studies are needed to explain this phenomenon. The present study extends and confirms previous findings by Sherman and Catterall [19] and by Brodie et al. [2] that chronic treatment of skeletal myotubes with TTX up-regulates the VDNC through decreases in cytosolic levels of Ca 2+ in association with a decrease in electrical activity. However, such findings provide no appropriate explanation for the relationship between changes in cytosolic levels of Ca 2+ and L-type V D C C . It has been shown that VDNC, as well as V D C C , are present in cultured chick and rat skeletal myotubes [1, 10, 13]. In the chick myotubes, many of the action potentials consist of, at least, two components, one being single or repetitive spikes mediated by the VDNC and the other being represented by a plateau and mediated by the L-type VDCC, as shown in Figs. 4A-1 and 4B-1. In good agreement with our previous results [11], TTX abolished both the Na spikes and the Ca plateau (Fig. 4A-2), although the plateau could be elicited with a slight increase in intensity o f the stimulatory current pulse (Fig. 4A-3). This observation indicates that the plateau was triggered by the Na spikes. The Ca plateau, by contrast, was abolished by application of D600, but the Na spike was not affected by this drug (Fig. 4B-2). These findings demonstrate that TTX has similar effects to those of D600 with respect to blocking the Ca plateau in skeletal muscle cells that exhibit both spike and plateau responses. Thus,

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Fig. 4. Acute effects o f T T X (A) and D600 (B) on two-component action potentials of cultured chick skeletal muscle cells. Records A and B were obtained from different myotubes in 13-day-old untreated control cultures. A-l, Control before exposure to TTX; A-2 and A-3, after exposure to 0.3 t M TTX. The intensity of the stimulatory current pulse was increased slightly in A-3. B-l, control before exposure to D600; B-2, after a 10-min exposure to 20 t M D600. The upper trace in each panel shows the stimulatory current pulse recorded with fast-sweepspeed: the middle and lower traces are fast- and slow-sweep-speed recordings of the action potentials, respectively. The base lines of the upper traces represent reference potential level to the middle traces. Note that both T T X and D600 exhibit similar effects in terms of the complete blockade of the plateau, as can be seen in A-2 and B-2.

chronic treatment with D600 or with TTX may cause a decrease in cytosolic levels of Ca 2+. Although we did not directly determine the cytosolic levels of Ca 2+ in our preparation, it seems likely that blockers of L-type VDCC decrease the level of Ca 2+ in the cytosolic pool and thereby up-regulate the VDNC, as occurs in experiments with TTX [2, 19]. Taking all these data together, we suggest that the upregulation of VDNC by chronic treatment with L-type VDCC blockers is mediated via a function of L-type VDCC which regulate the cytosolic levels of Ca 2+. Hence, the VDNC could be influenced by L-type VDCC that are expressed predominantly at an early stage of ontogenesis ofmyotubes [9, 13, 14]. However, the molecular mechanism(s) of the up-regulation that involves intracellular Ca2+-signal transduction and subsequent control of gene expression is, as yet, unclear.

252

From results of the experiments described above, we propose that the TTX-sensitive VDNC is down-regulated by the increase in the level of cytosolic free Ca 2+ that enter via the L-type VDCC at the early stage of development of cultured chick skeletal muscle cells. This finding may be relevant to the physiological role of Ltype VDCCs which are highly developed at this stage. This hypothesis is further supported by our previous observations that the increase in the density of TTXsensitive VDNC is, to some extent, inversely proportional to the decrease in the density of L-type VDCC [9]. It will be interesting to pursue further these investigations of the mechanisms that control voltage-dependent ion channels via cytosolic levels of free Ca 2+. We thank Mr. A. Tanakadate for computer programming and Mr. H. Ishikawa for technical help. This study was supported in part by a Scientific Research Grant from the Ministry of Education of Japan (No. 03770050). 1 Beam, K.G. and Knudson, C,M., Calcium currents in embryonic and neonatal mammalian skeletal muscle, J. Gen. Physiol.. 91 (1988) 781-798. 2 Brodie, C., Brody, M. and Sampson, S.R., Characterization of the relation between sodium channels and electrical activity in cultured rat skeletal myotubes: regulatory aspects, Brain Res., 488 (1989) 186-194. 3 Cognard, C., Romey, G., Galizzi, J.-P., Fosset, M. and Lazdunski, M., Dihydropyridine-sensitive Ca > channels in mammalian skeletal muscle cells in culture: electrophysiological properties and interactions with Ca 2+ channel activator (Bay K8644) and inhibitor (PN 200-110), Proc, Natl. Acad. Sci. USA, 83 (1986) 1518-1522, 4 Gonoi, T. and Hasegawa, S., Post-natal disappearance of transient calcium channels in mouse skeletal muscle: effects of denervation and culture, J. Physiol., 401 (1988) 617-637. 5 Grouselle, M., Koenig, J., Lascombe, M.-L,, Chapron, J., M616ard, P. and Georgescauld, D., Fura-2 imaging of spontaneous and electrically induced oscillations of intracellular free Ca > in rat myotubes, Pfltigers Arch., 418 (1991) 40-50. 6 Hagiwara, S. and Byerly, L., Calcium channel, Annu. Rev. Neurosci., 4 (1981) 69-125. 7 Hosey, M.M. and Lazdunski, M., Calcium channels: molecular pharmacology, structure and regulation, J. Membrane Biol., 104 (1988) 81-105.

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