Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide.
Activation of endothelial cell phospholipase D by hydrogen peroxide and fatty acid hydroperoxide.
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DOI：
10.1016/s0021-9258(18)54023-0
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发表时间：
1993-01
期刊：
影响因子：
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通讯作者：
V. Natarajan;M. Taher;B. Roehm;N. Parinandi;H. Schmid;Z. Kiss;J. Garcia
中科院分区：
文献类型：
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作者：
V. Natarajan;M. Taher;B. Roehm;N. Parinandi;H. Schmid;Z. Kiss;J. Garcia
我们研究了氧化剂介导的牛肺动脉内皮细胞 (BPAEC) 中磷脂酶 D (PLD) 活性的刺激，预先用 [32P] 正磷酸盐或 [32P] 溶血磷脂标记。用过氧化氢 (H2O2) 或亚油酸过氧化氢 (18:2-OOH) 处理在含有 0.5% 乙醇的 Hanks 平衡盐溶液 (HBSS) 中孵育的细胞，以剂量和时间依赖性方式增强 32P 标记的磷脂酰乙醇 (PEt) 和磷脂酸 (PA) 的形成，表明 PLD 的激活。 H2O2 和 18:2-OOH 介导的 PLD 激活与 [3H] 脱氧葡萄糖释放测定的细胞毒性无关。添加氯化亚铁 (50 microM) 使 H2O2 诱导的 [32P]PEt 和 [32P]PA 形成增加约 2 倍，而添加铁螯合剂去铁胺则阻断了氯化亚铁的增强作用。用含有 20% 小牛血清的培养基 199 替代 HBSS 培养基也增强了 H2O2 诱导的 PLD 激活的效果。除了磷脂酰胆碱 (PC) 之外，磷脂酰乙醇胺 (PE) 和磷脂酰肌醇 (PI) 在 H2O2 和 18:2-OOH 处理下很容易被 PLD 水解。氧化剂刺激的 PLD 活性的底物特异性不同于在缓激肽存在下观察到的底物特异性或通过 12-O-十四烷酰佛波醇 13-乙酸酯 (TPA) 激动剂刺激所表现出的底物特异性，其中 PC 是 PLD 水解的主要磷脂。在 H2O2 和 18:2-OOH 存在下，PEt 的形成不会因细胞外 Ca2+ 与 EGTA (5 mM) 或细胞内 Ca2+ 与 1,2-双-(2-氨基苯氧基)乙烷-N,N,N,N-四乙酸-乙酰氧基甲酯 (BAPTA-AM)（25 µM，30 分钟）的螯合而消除。此外，用蛋白激酶 C (PKC) 抑制剂十字孢菌素预处理 BPAEC 以及通过长期 TPA 处理（100 nM，18 小时）下调 PKC 对 H2O2 诱导的 PLD 活化没有影响，表明 H2O2 引起的 PLD 活化与 PKC 活性无关。 H2O2-和18:2-OOH诱导的PLD激活可能是内皮细胞中产生PA和二酰基甘油的重要机制。
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 microM) 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 microM, 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.