Release of hydrogen peroxide in response to hypoxia-reoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane.
Release of hydrogen peroxide in response to hypoxia-reoxygenation: role of an NAD(P)H oxidase-like enzyme in endothelial cell plasma membrane.
复制标题
DOI：
10.1165/ajrcmb.12.1.7529030
复制
发表时间：
1995
影响因子：
6.4
通讯作者：
J. Zulueta;F. Yu;I. Hertig;V. Thannickal;P. Hassoun
中科院分区：
文献类型：
--
作者：
J. Zulueta;F. Yu;I. Hertig;V. Thannickal;P. Hassoun
应用多种酶系统抑制剂和直接测定分离的牛肺动脉内皮细胞质膜产生过氧化氢(H_2O_2)的方法，研究了缺氧、低氧和低氧后复氧条件下牛肺动脉内皮细胞(EC)细胞外H_2O_2释放的动力学和机制。用荧光分光光度法测细胞外过氧化氢含量。缺氧(3%O2)和缺氧(0%O2)的EC释放的H_2O_2较常氧(20%O_2)低(分别为29.6+/-1.3%和4.2+/-0.7%)。缺氧后即刻(20%O2)内皮细胞胞外H_2O_2释放量增加至272+/-48%，而常氧持续暴露(100%)后，细胞外H_2O_2释放量增加。用别嘌醇抑制黄嘌呤氧化酶(XO)并不能减少常氧或低氧后复氧的细胞释放H_2O_2。此外，环氧合酶(吲哚美辛)、磷脂酶A2(奎纳克林和氯丙嗪)、一氧化氮合酶(L-精氨酸类似物)、线粒体电子传递链(鱼藤酮和氰化物)和细胞色素P-450(甲氧补骨脂素)的抑制剂对这种释放没有或几乎没有影响。另一方面，蛋白激酶C的抑制剂(钙磷蛋白和星形孢子素)和NADPH氧化酶(二苯基碘)以剂量依赖的方式减少了EC中的H_2O_2的释放。在单独的实验中，从EC分离的质膜被发现在NADH或NADPH作为电子供体的存在下产生H_2O_2。这一作用可被二苯基碘抑制，但不能被别嘌醇抑制。
The dynamics and mechanisms of extracellular release of hydrogen peroxide (H2O2) from bovine pulmonary artery endothelial cells (EC) subjected to anoxia, hypoxia, and hypoxia followed by reoxygenation were examined using various inhibitors of enzymatic systems in intact cells and by direct measurement of H2O2 production from isolated EC plasma membranes. Extracellular H2O2 was measured with a fluorometric assay. EC exposed to hypoxia (3% O2) and anoxia (0% O2) released less H2O2 (29.6 +/- 1.3% and 4.2 +/- 0.7%, respectively) compared with EC exposed to normoxia (20% O2). The extracellular release of H2O2 from EC previously exposed to hypoxia for 24 h increased immediately after reoxygenation (20% O2) to 272 +/- 48%, as compared with EC exposed continuously to normoxia (100% release). Inhibition of xanthine oxidase (XO) by allopurinol did not reduce the release of H2O2 from cells exposed to normoxia or hypoxia followed by reoxygenation. Furthermore, inhibitors of cyclooxygenase (indomethacin), phospholipase A2 (quinacrine and chlorpromazine), nitric oxide synthase (L-arginine analogs), the mitochondrial electron transport chain (rotenone and cyanide), and cytochrome P-450 (methoxypsoralen) had no or minimal effect on this release. On the other hand, inhibitors of protein kinase C (calphostin and staurosporine) and NADPH oxidase (diphenyliodonium) reduced the release of H2O2 from EC in a dose-dependent manner in both exposure groups. In separate experiments, plasma membranes isolated from EC were found to produce H2O2 in the presence of NADH or NADPH as electron donors. This was inhibited by diphenyliodonium but not by allopurinol.(ABSTRACT TRUNCATED AT 250 WORDS)