Membrane depolarization and intracellular Ca2+ increase caused by high external Ca2+ in a rat calcitonin-secreting cell line.
1. 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. 2. 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. 3. 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. 4. An increase in [Ca2+]o depolarized the cell membrane and elevated [Ca2+]i even in the presence of 10 microM-tetrodotoxin. The rise in [Ca2+]i was greatly reduced when action potentials were inhibited by applying hyperpolarizing 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. 5. 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. 6. 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. 7. Under voltage clamp, high [Ca2+]o caused a membrane conductance increase to Na+ ions. 8. 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 sustained 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.(ABSTRACT TRUNCATED AT 400 WORDS)
Yamashita
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Department of Physiology, Jerry Lewis Neuromuscular Research Center, University of California School of Medicine, Los Angeles 90024.
Hagiwara
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D000200:Action Potentials / Q000187:drug effects; D000818:Animals; D002116:Calcitonin / Q000378:metabolism*; D002118:Calcium / Q000493:pharmacokinetics* / Q000502:physiology; D002413:Cations, Divalent / Q000494:pharmacology; D002460:Cell Line; D004095:Dihydropyridines / Q000494:pharmacology; D016257:Fura-2 / Q000031:analogs & derivatives; D015640:Ion Channel Gating / Q000502:physiology*; D007473:Ion Channels / Q000502:physiology*; D011188:Potassium / Q000502:physiology; D051381:Rats; D012684:Sensory Thresholds / Q000502:physiology; D012964:Sodium / Q000502:physiology; D013779:Tetrodotoxin / Q000494:pharmacology; D013997:Time Factors
D002413:Cations, Divalent; D004095:Dihydropyridines; D007473:Ion Channels; C049925:fura-2-am; D013779:Tetrodotoxin; D002116:Calcitonin; D012964:Sodium; D011188:Potassium; D002118:Calcium; D016257:Fura-2