Voltage-sensitive calcium flux into bovine chromaffin cells occurs through dihydropyridine-sensitive and dihydropyridine- and ω-conotoxin-insensitive pathways

Abstract

The fluorescent Ca 2+ indicator FURA-2 was used to characterize the depolarization-related intracellular Ca 2+ signalling process in bovine adrenal chromaffin cells. Depolarization with high K + (10–65 mM) gave rise to a very rapid increase in intracellular free Ca 2+ concentration, which subsequently decayed slowly towards a “plateau”. The size of this initial increase varied sigmoidally with the calculated membrane potential, the relationship being described well by a Boltzmann distribution function for a transition between two states (transition potential, −23 mV). A dihydropyridine calcium channel agonist [(+)202-791, 1μM] raised intracellular free Ca 2+ concentration further in the presence of 30 mM K +, and it enhanced the initial intracellular Ca 2+ response to depolarization. Voltage-sensitive calcium channels in chromaffin cells are believed to include the l-type. Several dihydropyridine calcium channel antagonists [(−)202-791, nifedipine, nitrendipine; 1–5 μM], known to be active on l-type channels, caused only modest inhibition of K + -induced increase in intracellular free Ca 2+ concentration: c. 50% (at 30 mM K +) and 25% (at 40–70 mM K +). In addition, ω-conotoxin GVIA (1–10 μM), a blocker of neuronal N- and l-type calcium channels, reduced the initial increase in intracellular free Ca 2+ concentration only slightly at 55mM K +. Further, the dihydropyridine-insensitive component of the intracellular Ca 2+ signal was also insensitive to ω-conotoxin, which was otherwise quite active in a central nervous rat in vivo preparation. Gd 3+ (40 μM), a potent calcium antagonist in the chromaffin cell, blocked the intracellular Ca 2+ response to depolarization. When added at different times after K +. stimulation, however, Gd 3+ reduced intracellular free Ca 2+ concentration to control levels along a slow time course of several minutes. Similar results were obtained when EGTA was added to reduce extracellular Ca 2+ concentration to sub-nanomolar levels, in the presence of high K +. We conclude that bovine chromaffin cells are equipped with at least two different classes of voltage-dependent calcium channels, only one of which is likely to be the l-type channel. We also propose that depolarization, in addition to stimulating Ca 2+ influx, may also lead to enhancement of Ca 2+ release from an intracellular store.