Voltage-sensitive calcium flux into bovine chromaffin cells occurs through dihydropyridine-sensitive and dihydropyridine- and ω-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, 1μM] 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 μM], 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, ω-conotoxin GVIA (1-10 μM), a blocker of neuronal N- and l-type calcium channels, reduced the initial increase in intracellular free Ca²⁺ concentration only slightly at 55mM K⁺. Further, the dihydropyridine-insensitive component of the intracellular Ca²⁺ signal was also insensitive to ω-conotoxin, which was otherwise quite active in a central nervous rat in vivo preparation. Gd³⁺ (40 μM), 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.