Phorbol ester enhances activation of adenylate cyclase in bovine aortic endothelial cells.
Endothelial cells possess beta-adrenoceptors linked to adenylate cyclase which may regulate several aspects of endothelial cell function. The potential for this second messenger system to be modulated by protein kinase C activity was investigated. Bovine aortic endothelial cells (BAECs) were cultured in the absence or presence of phorbol 12-myristate 13-acetate (PMA), an activator of protein kinase C. Basal and forskolin-, sodium fluoride (NaF)-, and isoproterenol-stimulated adenylate cyclase activity was measured in homogenates from BAECs. beta-adrenoceptor density on membranes from BAECs was measured by 125I-iodocyanopindolol binding. Sodium dodecylsulfate-polyacrylamide gel electrophoresis of immunoprecipitated proteins was used to identify phosphorylated proteins. Pretreatment of BAECs with 100 nM PMA for 30 min increased basal adenylate cyclase activity above control levels, and also increased enzyme activity stimulated by forskolin, NaF, or isoproterenol. Pretreatment of BAECs for 60 min with 100 nM staurosporine, an inhibitor of protein kinase C, prevented the enhancement of adenylate cyclase activity caused by PMA. Treatment of BAECs with PMA did not trigger phosphorylation of the inhibitory guanine nucleotide-binding protein, and there was no change in BAEC beta-adrenoceptor density following PMA pretreatment. Exposure of BAECs to ATP or bradykinin did not mimic the effects of phorbol ester. In conclusion, activation of protein kinase C by PMA enhanced adenylate cyclase activity in BAECs. However, ATP and bradykinin which activate endothelial cell surface receptors linked to phospholipase C did not mimic this effect.
Lefroy
D C
DC
Department of Clinical Pharmacology, Royal Postgraduate Medical School, London, UK.
Donnelly
L E
LE
McEwan
J R
JR
MacDermot
J
J
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000262:Adenylyl Cyclases / Q000187:drug effects*; D000470:Alkaloids / Q000494:pharmacology; D000704:Analysis of Variance; D000818:Animals; D001011:Aorta; D001920:Bradykinin / Q000494:pharmacology; D002417:Cattle; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology / Q000201:enzymology*; D004789:Enzyme Activation / Q000187:drug effects; D019204:GTP-Binding Proteins / Q000187:drug effects; D010766:Phosphorylation / Q000187:drug effects; D011493:Protein Kinase C / Q000187:drug effects*; D011943:Receptors, Adrenergic, beta / Q000187:drug effects; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology*
D000470:Alkaloids; D011943:Receptors, Adrenergic, beta; D000255:Adenosine Triphosphate; D011493:Protein Kinase C; D019204:GTP-Binding Proteins; D000262:Adenylyl Cyclases; D019311:Staurosporine; D013755:Tetradecanoylphorbol Acetate; D001920:Bradykinin