Neuroscience Letters, 144 (1992) 161-164 © 1992 Elsevier Scientific Publishers Ireland Ltd. All rights reserved 0304-3940/92/$ 05.00

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Calcium channels in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker Masaakira K a n o , Ryohei Satoh and Yumiko N a k a b a y a s h i Department of Physiology, Kitasato University School of Medicine, Kanagawa (Japan) (Received 25 May 1992; Revised version received 17 June 1992; Accepted 17 June 1992)

Key words." Ca channel; Development; Embryonic chick skeletal muscle; Culture; D600; Nifedipine The effects of chronic treatment with calcium channel blockers were studied on the expression of voltage-dependent calcium channels (VDCCs) in chick skeletal muscle cells developing in culture. Myotubes were treated after 2 days in culture with either 20/aM D600 or 10/aM nifedipine, and measurements were made of the maximum rate of rise (M.R.R.) of the two components of action potential, operated by T- and L-type VDCCs, respectively. Treatment with either blocker reduced the M.R.R. of the action potential component operated by the L-type VDCC throughout the culture period examined. The M.R.R. of the T-type VDCC component, on the other hand, was unaffected by either treatment. The reduction in the M.R.R. of the L-type component in blocker-treated cells is thought to be due to the down-regulation of the expression of L-type VDCC. Thus, it appears that the expression of L-type VDCC in the chick skeletal muscle cells can be regulated by a function of L-type VDCC, which mediate the entry of Ca 2÷ into the cells. The physiological significance of the L-type VDCC, which expressed prominently early in the development of skeletal muscle cells, for the differentiation of excitability is discussed.

Voltage-dependent calcium channels (VDCCs) mediate the entry of Ca R+into the cells, where this ion plays an important role in the regulation of a wide variety of cellular processes in mature cells [7]. It has been shown that VDCCs are more prominent in the less differentiated states of developing nerve and muscle cells [16], and these channels therefore may have central roles also in the differentiation of developing cells. In embryonic chick skeletal muscle cells in culture two different types of VDCCs (referred to as T- and L-type) have been shown to be expressed prominently at the early state of differentiation [8, 9, 10], but the regulatory mechanism and physiological significance for this developmentally transient expression of VDCCs remain to be determined. In recent work on the effects of chronic exposure of this preparation to Ca channel blocker, we have demonstrated that the L-type channel of the two classes of VDCC can down-regulate tetrodotoxin (TTX)-sensitive Na channel expression via transmembrane influx of Ca 2÷ through the L-type VDCC [14]. Thus it has been suggested that the density of L-type VDCC is one of important determinant of the electrical differentiation of this preparation. Correspondence." M. Kano, Department of Physiology, Kitasato University School of Medicine, 1-15-1 Kitasato, Sagamihara, Kanagawa 228, Japan,

Continuing our studies regarding the role of L-type VDCC in the differentiation of developing muscle cells in culture, we examined the effect of chronic treatment with L-type VDCC blockers on the expression of VDCCs itself in the present investigation. Muscle cell cultures were prepared from pectoral muscles of 11-day-old chick embryos as previously described [9]. Chronic treatment with L-type VDCC blockers was begun at 2 days in culture, at which time numerous multinucleated myotubes formed. To each culture dish was added either D600 (Knoll) or nifedipine (Sigma) as a concentrated solution. The final concentration of these blockers was 20/IM for D600 and 10/IM for nifedipine. These concentrations were used because the L-type VDCC in this preparation was found to be largely blocked at these concentrations [13]. Care was taken not to expose the light-sensitive nifedipine to light. Electrophysiological experiments were performed 3-14 days after plating. Before the experiment, myotubes were extensively washed with a recording solution to remove blocker; in addition, nifedipine was inactivated by exposure to bright light for 10 min during washing. The recording solution was the same as described previously [10], which was designed to record specific components of action potential operated by VDCCs without contamination by other parallel components operated by volt-

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Fig. l Effect of chronic treatment with 20 #M D600 (A) and 10 pM nifedipine (B) on the M.R.R. of Ba action potentials in cultured chick skeletal muscle ceils. Treatment with D600 or nifedipine was begun at 2 days in culture, and measurements of the M.R.R. were made after 3 days in culture. The M.R.R. of Ba action potential components operated by T-type (©,O) and L-type ([],11) VDCCs are plotted against the age of culture. The open symbols refer to untreated control cells and the filled symbols to blocker-treated cells. Points with error bars (± S.D.) represent averages derived from measurements made on 20 cells. Insets in A show Ba action potentials (upper traces) and their rates of rise (lower traces) recorded from 6-day-old, control (a) and D600-treated (b) myotubes: filled and open arrows in the lower traces point to the M.R.R. of the T- and L-type VDCC components. respectively; arrowheads indicate the beginning and the end of depolarizing pulse current: short dashed lines are the reference potential.

age-depenent Na, C1 and K channels. Thus, it contained (in raM): barium acetate 50.0, tetraethylammonium acetate 50.0, cesium acetate 2.7, magnesium acetate 10.0, glucose 12.0; the pH was buffered at 7.4 by adding 10 mM Tris-maleate. Ba action potentials obtained from myotube immersed in this recording solution often consist of two components when elicited by depolarization from a hyperpolarized level o f - 8 0 mV, which are operated by T-type and L-type VDCCs, respectively [9, 10]. An example of such a two-component action potential is shown in the inset (a) of Fig. 1A (upper trace), The lower trace in the inset is the rate of rise of the Ba action potential; the two spikes in this rate trace correspond to the components operated by T- and L-type VDCCs, respectively. The maximum value for the rate of rise of each component was taken as a quantitative measure of the expression of VDCC involved, since the maximum rate of rise (M.R.R.) should be proportional to the density of the ionic channel involved. Intracellular recording of Ba action potential and measurement of the M.R.R. of the action potential have been described [9]. The Ba action potentials and their rates of rise were displayed on a storage oscilloscope and stored digitally (at 2 kHz) on floppy disk using a laboratory computer. From a result of the measurement of the M.R.R. of Ba action potential, we have previously demonstrated that embryonic chick skeletal muscle cells developing in culture sequentially express two types of VDCC (T- and

L-type) with distinct developmental properties during cell differentiation [8 10]. Almost all cells examined at 3 days in culture are found to express the T-type VDCC alone. The average value of the M.R.R. ofT-type VDCC action potential component peaks at 4 days in culture, and then rapidly decreases to zero before 10 days in culture. Unlike the T-type VDCC, the L-type VDCC are not expressed in almost all cells examined at 3 days in culture. The average value of the M.R.R. of L-type VDCC action potential component subsequently increases, reaches a maximum by 8 days in culture, after which it tends to decrease. Effect of chronic treatment of cells with L-type VDCC blocker on the expression of these two types of VDCC was examined in this experiment. Two blockers (D600 and nifedipine) which act at different sites on L-type VDCC were used, and treatment with either blocker was performed from the second day of culture onward. Blocker-treated cells showed no apparent changes in morphology as compared to control, untreated cells. Fig. 1 shows the effect of chronic treatment with D600 (A) and nifedipine (B) on the M.R.R. of two components of action potential. As clearly shown, the M.R.R. of T-type VDCC component of action potential (circles) was not significantly altered with either treatment throughout the culture period examined. However, the M.R.R. of Ltype VDCC component (squares) was drastically decreased in cells treated either with D600 or with nifedip-

163 ine. An example of a Ba action potential and its rate of rise recorded from a 6-day-old, D600-treated cell is shown in the inset (b) of Fig. 1A. At all ages of culture examined with the exception of that at day 3, the decrease in the M.R.R. of L-type VDCC component by treatment with blocker was evident. Before the measurements, blocker-containing culture medium was completely replaced with a recording solution by extensive wash in the recording solution for at least 15 min; in addition, the cultures treated with nifedipine were exposed to bright light for 10 min during washing. In the preliminary experiment, it has been corfirmed that the reducing effect of acutely applied D600 or nifedipine on the M.R.R. of L-type VDCC component was reversed upon 15 min of wash or 10 min exposure to bright light during washing, respectively (not shown). Hence, the decrease in the M.R.R. of L-type VDCC component in blocker-treated cells cannot be explained by the direct effect of blockers persisting after wash or exposure to light during washing. Rather it is plausible that chronic treatment of cells with L-type VDCC blocker down-regulated the expression of L-type VDCC. It has become increasingly evident that L-type is subject to regulatory influence, being altered in density by a variety of factors, including chronic drug treatment, hormone influence, disease state [5], or developmental stage [16]. Ca 2÷ may be an important intracellular mediator of the regulation of expression of different receptor or channel proteins, protein synthesis showing a requirement for intracellular Ca > [2]. In the present experiment we showed that chronic treatment of developing skeletal muscle ceils with L-type VDCC blocker can produce a down-regulation of the expression of L-type VDCCs, suggesting that Ca 2+ influx through the L-type VDCC is required for the normal expression of L-type VDCC. Chronic blockade of L-type VDCC would be predicted to decrease the time-averaged intracellular levels of Ca 2+ by decreasing the Ca 2÷ influx. The decreased intracellular levels of Ca 2+ may be responsible for the observed down-regulation of expression of L-type VDCC with chronic exposure to L-type VDCC blocker, consistent with Ca > being the intracellular mediator of the regulation of expression of L-type VDCC. This interpretation is consistent with prior reports that the expression of binding sites for the dihydropyridine L-type VDCC blocker is down-regulated in chick skeletal muscle cells grown in low-Ca > media [11, 12]. However, in chick neural retina cells [6] PCI2 cells [3], the expression of dihydropyridine binding sites has been shown to be downregulated by chronic depolarization, which induces Ca 2+ influx through the L-type VDCC. Taken together these observations indicate that intracellular levels of Ca 2+ play a crucial role in regulation of the expression of L-

type VDCC, although the regulation differs according to tissue; the rise in intracellular Ca 2+ up-regulates in some types of cell and down-regulates in other types of cell. In skeletal muscle cells the L-type VDCC was not expressed up to 3 days in culture (Fig. 1). At this very early stage the expression of L-type VDCC might be triggered by Ca 2+ influx through T-type VDCC, which have prominently been expressed already at this stage. Furthermore, since the T-type VDCC is activated by relatively small depolarization, its role may be to activate the highthreshold L-type VDCC, and thereby to facilitate Ca > influx via L-type VDCC at the early state of differentiation up to 6 days in culture. Subsequently, the T-type VDCC disappeared and the intracellular levels of Ca 2÷ is expected to be lowered in these stages of differentiation (after 6 days in culture). In addition, around these stages the development of sarcoplasmic reticulum has been shown to occur in chick skeletal muscle cells in culture [4]. This event is also expected to lower intracellular of Ca 2+ by Ca 2+ pump in the sarcoplasmic reticulum. The present experiment also demonstrated that the expression of L-type VDCC down-regulated after 8 days in culture. This is likely to be due to the decrease in intracellular Ca 2+, caused probably by disappearance of T-type VDCC and development of sarcoplasmic reticulum. In the previous studies we have demonstrated that the expression of voltage-dependent Na channel (VDNC) begins late and progresses slowly in comparison with the expression of VDCCs [8] and up-regulates by treatment with L-type VDCC blocker [14]. Thus, reduced intracellular Ca 2+ has been shown to up-regulate the expression of VDNC, as is the case with rat skeletal muscle cells in culture [1, 15]. Taken together these results suggest that in the developing chick skeletal muscle cells, one of the developmental roles for L-type VDCC, which is prominently expressed during a particular time in development, may be in regulation of the expression of the voltage-dependent cationic channels, up-regulation of the L-type VDCC itself and down-regulation of the VDNC. We thank Mr. A. Tanakadate for computer programing, Mr. H. Ishikawa and Mr. M. Souma for technical help. This study was supported in part by a Scientific Research Grant from the Mininstry of Education of Japan (No. 03770050). 1 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. 2 Brostrom, C.O. and Brostrom, M.A., Calcium-dependent regulation of protein synthesis in intact mammalian cells, Annu. Rev Physiol., 52 (1990) 577-590.

3 DeL_orme, E.M., Rabe. C.S. and McGee .h-., R.. Regulation of the number of functional voltagc-scnsiti~c ( ' a channels ~m P('I2 ceils by chronic changes in membrane potential. ,I. F'harnlacol. Exp. Ther., 244 (1988) 838 843. 4 Ezerman, E.B. and lshikawa. H., Differentiation of lhe sarcoplasmic reticulum and F system in developing chick skeletal muscle in vitro, J. Cell Biol.. 35 (1967)405 420. 5 Ferrante, J. and Triggle, D.J., Drug- and disease-induced regulation of voltage-dependent calcium channels, Pharmacol. Rev.. 42 (1990) 29 44. 6 Ferraute, J., Triggle, D.J. and Rutledge, A., The effects of chronic depolarization on L-type 1,4-dihydropyridine-sensitive, voltage-dependent Ca > channels in chick neural retina and rat cardiac cells, Can. J. Physiol. Pharmacol., 69 (1991) 914 920. 7 Hagiwara, S. and Byerly, L., Calcium channel, Annu. Rev. Neurosci..4(1981)69 125. 8 Kano, M., Satoh, R. and Nakabayashi, Y., Developmental changes in voltage-dependent calcium and sodium channels during differentiation of embryonic chick skeletal muscle cells in culture, Biomed. Res,, 12 (Supp12) (1991)197 198. 9 Kano, M., Wakuta, K. and Satoh, R.. Calcium channel components of action potential in chick skeletal muscle cells developing in culture, Dev. Brain Res., 32 (1987} 233 240.

IO Kano. M., Wakuta, K. and Satoh. R., Two components ol calciun" channel current in embryonic chick skeletal muscle cells developing incuhure, Dev. Brain Res.,47(I989) 101 112. 1I Navarro, J., Dihydropyridine [mcthyl-*H}PN 2(10 l I{1 binding anti myogenesis in intact muscle cells in vitro, ,I. Neurochen~., 46 (198~ 1166 1169. 12 Pauwels, P.J., Van Assouw, H.P. and Leysen. J.E., Depolarization of chick myotubes triggers the appearance of I+)-[~H]PN200-110binding sites, Mol. Pharmacol., 32 (1987)785 791. 13 Satoh, R., Nakabayashi, Y. and Kano, M.. Pharmacological properties of two types of calcium channel in embryonic chick skeletal muscle cells in culture, Neurosci. Lett,, 122 (1991) 233 236. 14 Satoh, R., Nakabayashi, Y. and Kano, M., Chronic treatment with D600 enhances development of sodium channels in cultured chick skeletal muscle cells, Neurosci. Lett., 138 (1992) 249 252. 15 Sherman, S.J. and Catterall, W.A., Electrical activity and cytosolic calcium regulate levels of tetrodotoxin-sensitive sodium channels in cultured rat muscle cells, Proc. Natl, Acad. Sci. USA, 81 {1984} 262 266. 16 Spitzer. N.C., Ion channels in development, Annu. Rev. Neurosci., 2 (1979) 363 397.

Calcium channels in embryonic chick skeletal muscle cells after cultivation with calcium channel blocker.

The effects of chronic treatment with calcium channel blockers were studied on the expression of voltage-dependent calcium channels (VDCCs) in chick s...
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