Inhibition of Protein Kinase C-Dependent Noradrenaline Release by Wortmannin in PC12 Cells

Wortmannin is a specific phosphatidylinositol 3-kinase inhibitor, and it inhibits secretion in many cell types. Previously we reported that high K+and external ATP stimulated a Ca2+influx and [3H]noradrenaline ([3H]NA) release from rat pheochromocytoma cell line 12 (PC12) cells in the presence of extracellular CaCl2. Addition of phorbol 12-myristate 13-acetate (PMA) stimulated [3H]NA release by itself and enhanced the maximal responses of high K+and ATP. In this study, we investigated the effects of wortmannin on NA release from PC12 cells. Wortmannin inhibited the [3H]NA release induced by high K+and ATP, and the stimulatory effects of PMA, in a dose-dependent manner. Wortmannin caused 50% inhibition of high K+-induced [3H]NA release at a concentration of 2.78 ± 0.68 μM(n= 5). The increased cytosolic free Ca2+concentrations ([Ca2+]i), induced by ATP, were not inhibited by wortmannin. Wortmannin inhibited PMA-induced phosphorylation of a 80-kDa protein in the cytosol fraction of PC12 cells. Calphostin C, a specific protein kinase C inhibitor, also inhibited high K+-, ATP-, and PMA-induced NA release, and the phosphorylation of the 80-kDa protein induced by PMA. Mastoparan, an amphiphilic tetradecapeptide from wasp venom, stimulated NA release in the presence or absence of extracellular CaCl2. Neither wortmannin nor calphostin C inhibited the NA release induced by mastoparan. These findings suggest that wortmannin inhibits the PKC-dependent pathway, not [Ca2+]imobilization, resulting in the inhibition of NA release from PC12 cells.