Membrane depolarization and intracellular Ca2+ increase caused by high external Ca2+ in a rat calcitonin‐secreting cell line.

Publisher Website
Google Scholar
Abstract
Calcitonin secretion is regulated by the external Ca2+ concentration ([Ca2+]o) via a rise in intracellular Ca2+ concentration ([Ca2+]i). The mechanism which couples an increase in [Ca2+]o to an increase in [Ca2+i) was explored in a rat calcitonin-secreting cell line (rMTC 44-2). [Ca2+]i was monitored using Fura-2 AM, and the membrane potential or current was simultaneously measured. Using the conventional whole-cell clamp, tetrodotoxin-sensitive voltage-gated Na+ channels, T- and L-type Ca2+ channels, and three types of K+ channels, the delayed K+ channel, the A-channel and the inward-rectifying channel were observed. Using the nystatin-perforated whole-cell clamp technique, the resting potential measured under current clamp in standard extracellular medium was -59.0 .+-. 5.0 mV (mean .+-. S.D., n = 25), and the input resistance was 3.9 .+-. 1.9 G .OMEGA. (n = 10). In 0.5 mM [Ca2+]o most cells (22/25) showed spontaneous action potentials. An increase in [Ca2+]o depolarized the cell membrane and elevated [Ca2+]i even in the presence of 10 .mu.M-tetrodotoxin. The rise in [Ca2+]i was greatly reduced when action potentials were inhibited by applying hyperdepolarizing current. The increase in [Ca2+]i saturated with 3-4 mM [Ca2+]o. In 3 mM [Ca2+]o, [Ca2+]i was 188.9 .+-. 40.5% (n = 12) of that in 0.5 mM [Ca2+]o. In high [Ca2+]o the duration of action potentials was prolonged, but the action potential frequency did not always increase. In some cases it even decreased in high [Ca2+]o. Two types of action potential were observed in high [Ca2+]o, one with a shorter duration and the other with a longer duration. [Ca2+]i transiently increased in association with the long-duration action potentials. These long-duration action potentials were also accompanied by a larger after hyperpolarization. Under voltage clamp, high [Ca2+]o caused a membrane conductance increase to Na+ ions. Even when the membrane potential was clamped at a level below the threshold for Ca2+ channel activation, high [Ca2+]o provoked an increase of [Ca2+]i which was composed of an initial transient increase followed by a sustained increase, indicating an involvement of mechanisms other than Ca2+ influx through voltage-gated channels. The sustainerd increase was more frequently observed than the initial transient increase. The amplitude of the sustained phase was dependent on [Ca2+]o, and in 5 mM [Ca2+]o it was 120.9 .+-. 18.9% (103-194%) (n = 58) of that in 0.5 mM [Ca2+]o. Under voltage clamp, a brief application (500 ms) of high [Ca2+]o caused an initial transient increase which reached the maximum 10-15 s after high [Ca2+]o application, indicating that Ca2+ ions mobilized from the internal store are involved in the initial transient increase. Sr2+ substituted for Ca2+ in increasing the membrane conductance and [Ca2+]i under voltage clamp whereas Mg2+ and Co2+ did not. Nitrendipine (1 .mu.M) and Bay K 8644 (1 .mu.M) had little effect on membrane conductance and on [Ca2+]i under voltage clamp. In rat GH3 cells, high [Ca2+]o neither depolarized the cell membrane nor changed [Ca2+]i. Three modes of [Ca2+]i increase are discussed. One mode is an influx through voltage-gated Ca2+ channels. The second one, which manifested as the initial transient increase of [Ca2+]i under voltage clamp, may be ascribed to a mobilization from the internal Ca2+ store. The third one, which constituted the sustained increase, is undetermined.