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 chromaff in cells, suggesting that voltage-sensitive Ca2+ channels (VSCCs) are inhib ited by protein kinase C-mediated phosphorylation. We now address the possi bility that L- and P/Q-type Ca2+ channel subtypes might be differentially i nvolved 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 f luorescence in a dose-dependent manner (IC50: 18 and 7 nM; maximal inhibiti ons: 45 and 48 %, respectively). The protein kinase C inhibitor staurospori ne (100 nM) reverted the inhibitory action of PMA. PMA (0.1-1 mu M) inhibit ed the early and late phases of the ionomycin (2 mu M)-evoked [Ca2+](i) tra nsients 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 (IC5 0 = 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 Ca2influx persisted in the combined presence of omega-agatoxin IVA (100 nM) an d nitrendipine (1 mu M). Simultaneous exposure to omega-agatoxin IVA and PM A inhibited both the early [Ca2+](i) transients and Mn2+ quenching to a muc h greater extent than each drug separately. Inhibition of the [Ca2+](i) tra nsients by nitrendipine and PMA did not significantly exceed that produced by PMA alone. It is concluded that phorbol ester-mediated activation of pro tein kinase C inhibits preferentially L-type VSCCs over P/Q type channels i n adrenal chromaffin cells. However, the possibility cannot be ruled out th at dihydropyridine-resistant, non-P/Q type channels might also be negativel y regulated by protein kinase C. This may represent an important pathway fo r the specific control of VSCCs by protein kinase C-linked receptors, not o nly in paraneurones but presumably also in neurones and other excitable cel ls. (C) 1999 Elsevier Science B.V. All rights reserved.