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We have previously reported that endothelial cell phospholipase D (PLD), activated by 4-hydroxynonenal (4-HNE), was independent of protein kinase C activation. To determine whether PLD stimulation by 4-HNE is related to protein tyrosine phosphorylation, the effects of tyrosine kinase (Tyrk) and protein tyrosine phosphatase (PTPase) inhibitors on PLD activation were investigated. Pretreatment of bovine pulmonary artery endothelial cells (BPAEC) with Tyrk inhibitors, such as genistein, erbstatin, and herbimycin attenuated 4-HNE-induced PLD activation. Furthermore, vanadate, phenylarsine oxide, and diamide, inhibitors of PTPases, markedly increased the 4-HNE-induced PLD activation. The effects of Tyrk and PTPase inhibitors were specific towards the 4-HNE, as these agents had no effect on the agonist- or TPA- induced PLD activation. In addition to PLD activation, treatment of BPAEC with 4-HNE increased tyrosine phosphorylation of proteins including bands of molecular weights 40,000-60,000, 70,000-90,000, and 110,000-130,000. The 4-HNE-mediated increase in protein tyrosine phosphorylation was partly inhibited by genistein (100 μM). Vanadate (10 μM) pretreatment also potentiated 4-HNE-induced protein tyrosine phosphorylation. These data suggest that 4-HNE-mediated stimulation of PLD may occur as a result of activation of tyrosine kinases.