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

The fluorescent Ca2+ indicator FURA-2 was used to characterize the depolarization-related intracellular Ca2+ signalling process in bovine adrenal chromaffin cells. Depolarization with high K+ (10-65 mM) gave rise to a very rapid increase in intracellular free Ca2+ 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, 1 microM] raised intracellular free Ca2+ concentration further in the presence of 30 mM K+, and it enhanced the initial intracellular Ca2+ 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 microM], known to be active on L-type channels, caused only modest inhibition of K+ -induced increase in intracellular free Ca2+ concentration: c. 50% (at 30 mM K+) and 25% (at 40-70 mM K+). In addition, omega-conotoxin GVIA (1-10 microM), a blocker of neuronal N- and L-type calcium channels, reduced the initial increase in intracellular free Ca2+ concentration only slightly at 55 mM K+. Further, the dihydropyridine-insensitive component of the intracellular Ca2+ signal was also insensitive to omega-conotoxin, which was otherwise quite active in a central nervous rat in vivo preparation Gd3+ (40 microM), a potent calcium antagonist in the chromaffin cell, blocked the intracellular Ca2+ response to depolarization. When added at different times after K+ stimulation, however, Gd3+ reduced intracellular free Ca2+ concentration to control levels along a slow time course of several minutes. Similar results were obtained when EGTA was added to reduce extracellular Ca2+ 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 Ca2+ influx, may also lead to enhancement of Ca2+ release from an intracellular store.