PHORBOL ESTER ENHANCES ACTIVATION OF ADENYLATE-CYCLASE IN BOVINE AORTIC ENDOTHELIAL-CELLS

Endothelial cells possess beta-adrenoceptors linked to adenylate cycla se which may regulate several aspects of endothelial cell function. Th e potential for this second messenger system to be modulated by protei n kinase C activity was investigated. Bovine aortic endothelial cells (BAECs) were cultured in the absence or presence of phorbol 12-myrista te 13-acetate (PMA), an activator of protein kinase C. Basal and forsk olin-, sodium fluoride (NaF)-, and isoproterenol-stimulated adenylate cyclase activity was measured in homogenates from BAECs. Beta-adrenoce ptor density on membranes from BAECs was measured by I-125-iodocyanopi ndolol binding. Sodium dodecylsulfate-polyacrylamide gel electrophores is of immunoprecipitated proteins was used to identify phosphorylated proteins. Pretreatment of BAECs with 100 nM PMA for 30 min increased b asal adenylate cyclase activity above control levels, and also increas ed enzyme activity stimulated by forskolin, NaF, or isoproterenol. Pre treatment of BAECs for 60 min with 100 nM staurosporine, an inhibitor of protein kinase C, prevented the enhancement of adenylate cyclase ac tivity caused by PMA. Treatment of BAECs with PMA did not trigger phos phorylation of the inhibitory guanine nucleotide-binding protein, and there was no change in BAEC beta-adrenoceptor density following PMA pr etreatment. Exposure of BAECs to ATP or bradykinin did not mimic the e ffects 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 t o phospholipase C did not mimic this effect.