Voltage-dependent increase in ionized cytoplasmic calcium in the L6H9 muscle cell line detected with quin2.

The concentration of free Ca2+ ions in the cytoplasm ([Ca2+]i) is a key parameter in the function of muscle cells. This study describes the effect of membrane depolarization on [Ca2+]i in differentiating cells of the L6H9 line of rat skeletal muscle. [Ca2+]i was assessed using the fluorescent indicator quin2. In the presence of 1 mM extracellular Ca2+, [Ca2+]i averaged 250 nM. Replacement of extracellular Na+ with K+ resulted in cellular depolarization from -64 to -20 mV, measured with a fluorescent oxonol indicator. Depolarized cells showed a significant increase in [Ca2+]i, from 250 to 390 nM. The increase was prevented by nifedipine (5 microM) and was in great part dependent on the presence of extracellular Ca2+. A residual significant increase in [Ca2+]i was observed upon depolarization in Ca2+-free medium; this rise may be attributed to Ca2+ release from intracellular organelles. In the presence of extracellular Ca2+, replacement of extracellular Na+ by N-methylglucamine+ did not depolarize the cells, yet resulted in a significant increase in [Ca2+]i. This rise may be ascribed to inhibition or reversal of Na+/Ca2+ exchange activity due to the absence of extracellular Na+. The data are consistent with the presence of voltage-sensitive Ca2+ channels and Na+/Ca2+ antiporters at the cell surface, and of mechanisms of voltage-sensitive Ca2+ release from intracellular organelles.