Long-term phorbol ester treatment dissociates phospholipase D activation from phosphoinositide hydrolysis and prostacyclin synthesis in endothelial cells stimulated with bradykinin

Bovine pulmonary artery endothelial cells (BPAEC) were prelabeled with [³H]choline or [³H]myristic acid to selectively label endogenous phosphatidylcholine. BPAEC were stimulated with ATP and bradykinin (BK), and phospholipase D (PLD) activation was detected as a 4-fold increase in [³H]choline in cells prelabeled with [³H]choline or as a 2- to 3-fold increase in [³H]phosphatidylethanol in cells prelabeled with [³H]myristic acid and stimulated in the presence of ethanol. Pretreatment of BPAEC with 0.1 μM phorbol 12-myristate 13-acetate (PMA) for 22 hr completely inhibited agonist-induced PLD activation, whereas prostacyclin synthesis and [³H]phosphoinositide ([³H]PIns) hydrolysis were enhanced in pretreated cells. Long-term PMA treatment thus dissociates agonist-induced PLD activation from [³H]PIns hydrolysis, and agonist-induced prostacyclin synthesis is not dependent upon PLD activation.