CONTROL OF CA2-KINASE-C AND CALCINEURIN( CHANNEL CURRENT AND EXOCYTOSIS IN RAT LACTOTROPHS BY BASALLY ACTIVE PROTEIN)

Modulation of voltage-activated Ca2+ channel activity by phosphorylati on was studied in metabolically intact voltage-clamped rat lactotrophs . Experiments using Ba2+ as a charge carrier indicated that a phorbol ester protein kinase C activator stimulates high-voltage-activated Ca2 + channel currents, but has no effect on low-voltage-activated current s. Extracellular application of structurally and mechanistically disti nct protein kinase C inhibitors (staurosporin, H7, calphostin C, chele rythrine and Po 31-8220) preferentially inhibited the high-voltage-act ivated Ba2+ current. This suggests that protein kinase C is required f or maintainance of Ca2+ channel activity even in the absence of modula tors. Cyclosporin A, an inhibitor of the Ca2+/calmodulin-dependent pro tein phosphatase calcineurin, increased the high-voltage-activated Ca2 + channel current, and staurosporin reversed this effect. Thus, dephos phosphorylation by calcineurin may limit basal Ca2+ channel activity. Time-domain monitoring of cellular capacitance changes demonstrated th at cyclosporin A and 12-O-tetradecanoyl-phorbol-13-acetate do not affe ct exocytosis at a hyperpolarized potential, but each enhances depolar ization-induced exocytosis. Facilitation of exocytosis by cyclosporin A differed from 12-O-tetradecanoyl-phorbol-13-acetate in that it was b iphasic. The delayed facilitation induced by cyclosporin A could be ac counted for by stimulation of the voltage-gated Ca2+ current. These re sults suggest that the high-voltage activated Ca2+ channel current in rat lactotrophs is determined by the opposing basal activities of prot ein kinase C and calcineurin. Furthermore, it is concluded that the re gulation of Ca2+ channels by protein kinase C and calcineurin affects depolarization-induced exocytosis.