Boyer C S, Bannenberg G L, Neve E P, Ryrfeldt A, Moldéus P
Division of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Biochem Pharmacol. 1995 Sep 7;50(6):753-61. doi: 10.1016/0006-2952(95)00195-6.
The intracellular events that lead to arachidonic acid release from bovine endothelial cells in culture treated with hydrogen peroxide were characterized. The hydrogen peroxide-stimulated release of arachidonic acid was time- and dose-dependent, with maximal release achieved at 15 minutes after the addition of 100 microM hydrogen peroxide. Hydrogen peroxide-stimulated release of arachidonic acid was blocked with the phospholipase A2 inhibitor quinacrine. Treatment of the cells with hydrogen peroxide did not result in liberation of oleic acid, indicating that hydrogen peroxide exercised its effect on an arachidonate-specific phospholipase. Pretreatment of the cells with antioxidants, transition metal chelators, and hydroxyl radical scavengers did not affect the hydrogen peroxide-stimulated arachidonic acid release, indicating that the response to hydrogen peroxide is not oxygen radical-mediated. The response to hydrogen peroxide does not appear to be calcium-dependent, due to the following two observations: (a) No increase in intracellular calcium was seen upon exposure of the FURA2-loaded cells to hydrogen peroxide at concentrations sufficient to release arachidonic acid, and (b) no change in the release response was detected in cells loaded with the intracellular calcium chelator BAPTA. Significant inhibition of arachidonic acid release was seen when the cells were pretreated with inhibitors of protein kinase C, but not with inhibitors of tyrosine kinase. The results of these studies indicate that hydrogen peroxide-stimulated arachidonic acid release is mediated by a specific signal-responsive phospholipase A2, and that this process is not mediated via the actions of either lipid peroxidation or calcium but, rather, that a stimulation of intracellular kinase activity is necessary for this response.
对培养的牛内皮细胞在用过氧化氢处理后导致花生四烯酸释放的细胞内事件进行了表征。过氧化氢刺激的花生四烯酸释放具有时间和剂量依赖性，在加入100微摩尔过氧化氢后15分钟达到最大释放量。过氧化氢刺激的花生四烯酸释放被磷脂酶A2抑制剂奎纳克林阻断。用过氧化氢处理细胞不会导致油酸释放，这表明过氧化氢对花生四烯酸特异性磷脂酶发挥作用。用抗氧化剂、过渡金属螯合剂和羟自由基清除剂对细胞进行预处理不会影响过氧化氢刺激的花生四烯酸释放，这表明对过氧化氢的反应不是由氧自由基介导的。对过氧化氢的反应似乎不依赖于钙，这基于以下两个观察结果：(a) 用FURA2负载的细胞在暴露于足以释放花生四烯酸的浓度的过氧化氢时，细胞内钙没有增加；(b) 在负载细胞内钙螯合剂BAPTA的细胞中未检测到释放反应的变化。当细胞用蛋白激酶C抑制剂预处理时，花生四烯酸释放受到显著抑制，但用酪氨酸激酶抑制剂预处理时则没有。这些研究结果表明，过氧化氢刺激的花生四烯酸释放是由一种特定的信号响应性磷脂酶A2介导的，并且这个过程不是通过脂质过氧化或钙的作用介导的，而是对细胞内激酶活性的刺激对于这种反应是必要的。
