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 microM (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+]i mobilization, resulting in the inhibition of NA release from PC12 cells.
Oda
H
H
Department of Pharmacology, Faculty of Pharmaceutical Sciences, Hokkaido University, Sapporo, Japan.
Murayama
T
T
Nomura
Y
Y
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000730:Androstadienes / Q000494:pharmacology*; D000818:Animals; D002118:Calcium / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D036341:Intercellular Signaling Peptides and Proteins; D009281:Naphthalenes / Q000494:pharmacology; D009638:Norepinephrine / Q000502:physiology*; D016716:PC12 Cells; D010455:Peptides; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins / Q000378:metabolism; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors*; D011188:Potassium / Q000494:pharmacology; D011493:Protein Kinase C / Q000037:antagonists & inhibitors*; D051381:Rats; D012636:Secretory Rate / Q000187:drug effects; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D014862:Wasp Venoms / Q000494:pharmacology; D000077191:Wortmannin
D000255:Adenosine Triphosphate; D000730:Androstadienes; D002118:Calcium; D004791:Enzyme Inhibitors; D036341:Intercellular Signaling Peptides and Proteins; D009281:Naphthalenes; D009638:Norepinephrine; D010455:Peptides; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017853:Phosphotransferases (Alcohol Group Acceptor); D011188:Potassium; D011493:Protein Kinase C; D013755:Tetradecanoylphorbol Acetate; D014862:Wasp Venoms; D000077191:Wortmannin; C037500:mastoparan; C058819:calphostin C