4-Hydroxynonenal, a metabolite of lipid peroxidation, activates phospholipase D in vascular endothelial cells

We have examined the activation of phospholipase D (PLD) in bovine pulmonary artery endothelial cells (BPAEC) treated with 4-hydroxynonenal (4-HNE). Treatment of BPAEC labelled with [32P] orthophosphate (5 h for minimal phospholipid labelling) and [3H] myristic acid (24 h) with 4-HNE in the presence of 0.5% ethanol resulted in the formation of [3H] phosphatidylethanol (PEt) and [3H] phosphatidic acid (PA) with very little accumulation of [32P] PEt. The formation of [3H] PEt, as opposed to [32P] PEt, suggests that PEt synthesis was not through de novo pathway but rather through the PLD mechanism. 4-Hydroxynonenal-induced PLD activation was dose and time dependent, and was not associated with cytotoxicity as determined by [3H] deoxyglucose release. The formation of PEt was not affected by chelation of either extracellular Ca2+ with EGTA (5 mM, 30 min) or intracellular Ca2+ with BAPTA-AM (25 microM, 30 min). Treatment of BPAEC with either staurosporine (10 microM, 15 min), a protein kinase C (PKC) inhibitor, or down regulation of PKC by chronic 12-0-tetradecanoylphorbol-13-acetate (TPA) treatment (100 nM, 18 h) had no effect on 4-HNE-induced PLD activation. These results indicate that PLD activation by 4-HNE is independent of PKC activity. We also examined the specificity of nonylaldehyde derivatives and hydroxyalkenals on PLD activation. In addition to 4-HNE, 4-hydroxyoctenal and 4-hydroxyhexenal also stimulated [32P] PEt formation. Among the various nonylaldehydes examined, only trans-2-nonenal and trans-2-cis 6-nonadienal exhibited PLD activation, suggesting the requirement of a trans double bond at carbon 2 and a hydroxyl group at carbon 4. However, in contrast to 4-HNE-induced PLD activation of BPAEC monolayers, treatment of 105,000 x g membranes with 4-HNE had no effect on PLD catalyzed hydrolysis of [2-14C] oleoyl phosphatidylcholine. These data provide evidence that 4-HNE, a metabolite of membrane lipid peroxidation, may be involved in endothelial cell signal transduction, through the activation of phospholipase D and the generation of second messengers like phosphatidic acid and diacylglycerol.