Regulation of Ca 2+ influx by a protein kinase C activator in chromaffin cells: differential role of P/Q- and L-type Ca 2+ channels

Abstract

Phorbol esters reduce depolarization-evoked Ca 2+ influx in adrenal chromaffin cells, suggesting that voltage-sensitive Ca 2+ channels (VSCCs) are inhibited by protein kinase C-mediated phosphorylation. We now address the possibility that L- and P/Q-type Ca 2+ 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 Ca 2+ concentration ([Ca 2+] i) and the early component of the depolarization-evoked Mn 2+ quenching of fura-2 fluorescence in a dose-dependent manner (IC 50: 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 μM) inhibited the early and late phases of the ionomycin (2 μM)-evoked [Ca 2+] i transients by 14–23%. ω-Agatoxin IVA, a blocker of P/Q-type Ca 2+ channels, inhibited high K +-evoked [Ca 2+] i rises in a dose-dependent fashion (IC 50=50 nM). In contrast, 0.1 μM ω-conotoxin GVIA, a blocker of N-type channels, was without effect. A sizeable (<45%) component of early Ca 2+ influx persisted in the combined presence of ω-agatoxin IVA (100 nM) and nitrendipine (1 μM). Simultaneous exposure to ω-agatoxin IVA and PMA inhibited both the early [Ca 2+] i transients and Mn 2+ quenching to a much greater extent than each drug separately. Inhibition of the [Ca 2+] 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 but presumably also in neurones and other excitable cells.