Natarajan V, Scribner W M, Taher M M
Department of Medicine, Indiana University School of Medicine, Indianapolis 46202.
Free Radic Biol Med. 1993 Oct;15(4):365-75. doi: 10.1016/0891-5849(93)90036-t.
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.
我们检测了用4-羟基壬烯醛（4-HNE）处理的牛肺动脉内皮细胞（BPAEC）中磷脂酶D（PLD）的激活情况。在0.5%乙醇存在下，用4-HNE处理预先用[32P]正磷酸盐（5小时以实现最小程度的磷脂标记）和[3H]肉豆蔻酸（24小时）标记的BPAEC，导致[3H]磷脂酰乙醇（PEt）和[3H]磷脂酸（PA）的形成，而[32P] PEt的积累很少。与[32P] PEt不同，[3H] PEt的形成表明PEt的合成不是通过从头合成途径，而是通过PLD机制。4-羟基壬烯醛诱导的PLD激活呈剂量和时间依赖性，并且如通过[3H]脱氧葡萄糖释放所确定的，与细胞毒性无关。用乙二醇双四乙酸（EGTA，5 mM，30分钟）螯合细胞外Ca2+或用1,2-双（2-氨基苯氧基）乙烷-N,N,N',N'-四乙酸四乙酰甲酯（BAPTA-AM，25 microM，30分钟）螯合细胞内Ca2+，均不影响PEt的形成。用蛋白激酶C（PKC）抑制剂星形孢菌素（10 microM，15分钟）处理BPAEC，或通过长期用12-O-十四酰佛波醇-13-乙酸酯（TPA，100 nM，18小时）处理下调PKC，对4-HNE诱导的PLD激活均无影响。这些结果表明，4-HNE诱导的PLD激活与PKC活性无关。我们还检测了壬醛衍生物和羟基烯醛对PLD激活的特异性。除了4-HNE外，4-羟基辛烯醛和4-羟基己烯醛也刺激了[32P] PEt的形成。在检测的各种壬醛中，只有反式-2-壬烯醛和顺式-2,反式-6-壬二烯醛表现出PLD激活，这表明在碳2处需要一个反式双键，在碳4处需要一个羟基。然而，与4-HNE诱导的BPAEC单层的PLD激活相反，用4-HNE处理105,000×g的膜对PLD催化的[2-14C]油酰磷脂酰胆碱的水解没有影响。这些数据提供了证据，即膜脂质过氧化的代谢产物4-HNE可能通过激活磷脂酶D以及产生磷脂酸和二酰甘油等第二信使参与内皮细胞信号转导。
