Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide

We have investigated oxidant-mediated stimulation of phospholipase D (PLD) activity in bovine pulmonary artery endothelial cells (BPAEC), prelabeled with [³²P] orthophosphate or [³²P]lysophospholipids. Treatment of cells incubated in Hanks' balanced salt solution (HBSS) containing 0.5% ethanol with hydrogen peroxide (H₂O₂) or linoleic acid hydroperoxide (18:2-OOH) enhanced the formation of ³²P-labeled phosphatidylethanol (PEt) and phosphatidic acid (PA) in a dose-and time-dependent manner, indicating the activation of PLD. The H₂O₂- and 18:2-OOH-mediated PLD activation was not associated with cytotoxicity as determined by [³H]deoxyglucose release. The addition of ferrous chloride (50 μM) augmented H₂O₂-induced formation of [³²P]PEt and [³²P]PA about 2-fold, whereas the addition of the iron chelator desferoxamine blocked the potentiating effect of ferrous chloride. Replacement of the HBSS medium with Medium 199 containing 20% calf serum also potentiated the effect of H₂O₂-induced PLD activation. In addition to phosphatidylcholine (PC), phosphatidylethanolamine (PE), and phosphatidylinositol (PI) were readily hydrolyzed by PLD in response to H₂O₂ and 18:2-OOH treatment. The substrate specificity for oxidant-stimulated PLD activity differed from that observed in the presence of bradykinin or exhibited by agonist stimulation with 12-O-tetradecanoylphorbol 13-acetate (TPA) where PC was the major phospholipid hydrolyzed by PLD. The formation of PEt in the presence of H₂O₂ and 18:2-OOH was not abolished by chelation of either extracellular Ca²⁺ with EGTA (5 mM) or intracellular Ca²⁺ with 1,2-bis-(2-aminophenoxy)ethane-N,N,N,N-tetraacetic acid-acetoxymethyl ester (BAPTA-AM) (25 μM, 30 min). Furthermore, pretreatment of BPAEC with the protein kinase C (PKC) inhibitor staurosporine and down-regulation of PKC by chronic TPA treatment (100 nM, 18 hr) had no effect on H₂O₂-induced PLD activation, suggesting that PLD activation by H₂O₂ is independent of PKC activity. It is possible that H₂O₂- and 18:2-OOH-induced activation of PLD represents an important mechanism to produce PA and diacylglycerol in endothelial cells.