Regulation of Ca2+ influx by a protein kinase C activator in chromaffin cells: differential role of P/Q- and L-type Ca2+ channels

Phorbol esters reduce depolarization-evoked Ca2+ influx in adrenal chromaffin cells, suggesting that voltage-sensitive Ca2+ channels (VSCCs) are inhibited by protein kinase C-mediated phosphorylation. We now address the possibility that L- and P/Q-type Ca2+ channel subtypes might be differentially involved in phorbol ester action. In bovine chromaffin cells, short-term (10 min) incubations with phorbol 12-myristate 13-acetate (PMA) inhibited early high K+-evoked rises in cytosolic free Ca2+ concentration ([Ca2+]i) and the early component of the depolarization-evoked Mn2+ quenching of fura-2 fluorescence in a dose-dependent manner (IC50: 18 and 7 nM; maximal inhibitions: 45 and 48%, respectively). The protein kinase C inhibitor staurosporine (100 nM) reverted the inhibitory action of PMA. PMA (0.1-1 [mu]M) inhibited the early and late phases of the ionomycin (2 [mu]M)-evoked [Ca2+]i transients by 14-23%. [omega]-Agatoxin IVA, a blocker of P/Q-type Ca2+ channels, inhibited high K+-evoked [Ca2+]i rises in a dose-dependent fashion (IC50=50 nM). In contrast, 0.1 [mu]M [omega]-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (<45%) component of early Ca2+ influx persisted in the combined presence of [omega]-agatoxin IVA (100 nM) and nitrendipine (1 [mu]M). Simultaneous exposure to [omega]-agatoxin IVA and PMA inhibited both the early [Ca2+]i transients and Mn2+ quenching to a much greater extent than each drug separately. Inhibition of the [Ca2+]i transients by nitrendipine and PMA did not significantly exceed that produced by PMA alone. It is concluded that phorbol ester-mediated activation of protein kinase C inhibits preferentially L-type VSCCs over P/Q type channels in adrenal chromaffin cells. However, the possibility cannot be ruled out that dihydropyridine-resistant, non-P/Q type channels might also be negatively regulated by protein kinase C. This may represent an important pathway for the specific control of VSCCs by protein kinase C-linked receptors, not only in paraneurones ...