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 pul monary artery endothelial cells (BPAEC) treated with 4-hydroxynonenal (4-HNE). Treatment of BPAEC labelled with [P-32] orthophosphate (5 h f or minimal phospholipid labelling) and [H-3] myristic acid (24 h) with 4-HNE in the presence of 0.5% ethanol resulted in the formation of [H -3] phosphatidylethanol (PEt) and [H-3] phosphatidic acid (PA) with ve ry little accumulation of [P-32] PEt. The formation of [H-3] PEt, as o pposed to [P-32] PEt, suggests that PEt synthesis was not through de n ovo pathway but rather through the PLD mechanism. 4-Hydroxynonenal-ind uced PLD activation was dose and time dependent, and was not associate d with cytotoxicity as determined by [H-3] deoxyglucose release. The f ormation of PEt was not affected by chelation of either extracellular Ca2+ with EGTA (5 mM, 30 min) or intracellular Ca2+ with BAPTA-AM (25 muM, 30 min). Treatment of BPAEC with either staurosporine (10 muM, 15 min), a protein kinase C (PKC) inhibitor, or down regulation of PKC b y chronic 12-O-tetradecanoylphorbol-13-acetate (TPA) treatment (100 nM , 18 h) had no effect on 4-HNE-induced PLD activation. These results i ndicate that PLD activation by 4-HNE is independent of PKC activity. W e also examined the specificity of nonylaldehyde derivatives and hydro xyalkenals on PLD activation. In addition to 4-HNE, 4-hydroxyoctenal a nd 4-hydroxyhexenal also stimulated [P-32] PEt formation. Among the va rious nonylaldehydes examined, only trans-2-nonenal and trans-2-cis 6- nonadienal exhibited PLD activation, suggesting the requirement of a t rans double bond at carbon 2 and a hydroxyl group at carbon 4. However , in contrast to 4-HNE-induced PLD activation of BPAEC monolayers, tre atment of 105,000 x g membranes with 4-HNE had no effect on PLD cataly zed hydrolysis of [2-C-14] oleoyl phosphatidylcholine. These data prov ide evidence that 4-HNE, a metabolite of membrane lipid peroxidation, may be involved in endothelial cell signal transduction, through the a ctivation of phospholipase D and the generation of second messengers l ike phosphatidic acid and diacylglycerol.