THE ENHANCEMENT BY WORTMANNIN OF PROTEIN-KINASE C-DEPENDENT ACTIVATION OF PHOSPHOLIPASE-D IN VASCULAR ENDOTHELIAL-CELLS

Phosphatidic acid generation by phospholipase D (PLD) activation has b een implicated in agonist- and oxidant-mediated endothelial cell signa l transduction. We examined the effect of wortmannin on PLD activation in pulmonary artery endothelial and smooth muscle cells in culture. P retreatment of bovine pulmonary artery endothelial cells (BPAECs) with wortmannin potentiated TPA- (100 nM), ATP- (100 mu M), and bradykinin - (1 mu M) induced [P-32]PEt formation, an index of PLD activation. Ho wever, wortmannin by itself had no effect on PLD activity. The potenti ating effect of wortmannin on TPA-induced PLD activation was dose- (1- 10 mu M) and time-dependent (5-30 min) and was inhibited by bisindoylm alemide, an inhibitor of protein kinase C (PKC). Furthermore, down-reg ulation of PKC by prolonged treatment with TPA (100 nM, 18 h) attenuat ed the wortmannin effect. This effect of wortmannin was specific for T PA- or agonist-induced PLD activation as no potentiation of [P-32]PEt formation was observed with H2O2 (1 mM) or ionomycin (1 mu M). The eff ect of wortmannin was not due to activation of PKC alpha as determined by western blot analysis of PKC alpha in the cytosol and membrane fra ctions. Also, genistein, an inhibitor of tyrosine kinases, did not att enuate the wortmannin-mediated potentiation of PLD thereby suggesting non-involvement of protein tyrosine phosphorylation. These results ind icate that wortmannin potentiates PKC-dependent stimulation of PLD in vascular endothelial cells. (C) 1997 Elsevier Science Ireland Ltd.