Regulation of hydrogen peroxide generation in cultured endothelial cells.
Regulation of hydrogen peroxide generation in cultured endothelial cells.
复制标题
培养内皮细胞中过氧化氢生成的调节。
DOI：
10.1165/ajrcmb/6.2.175
复制
发表时间：
1992
影响因子：
6.4
通讯作者：
Crapo,JD
中科院分区：
文献类型：
--
作者：
Kinnula,VL;Whorton,AR;Chang,LY;Crapo,JD
研究了内源性过氧化氢（h2o）在抗氧化酶抑制剂、线粒体抑制剂、微粒体细胞色素P-450抑制剂存在下以及h2o或甲萘醌诱导氧化应激后从主动脉内皮细胞释放的情况。用3-甲氧基-4-羟基苯基乙酸测定细胞外H20的生成，用氨三唑间接测定细胞内H202的产生（在过氧化物酶体内或附近），氨三唑在H20存在下使过氧化氢酶失活。在汉克斯平衡盐溶液中，细胞外H20的释放量为0.079±0.005 nmol/min/mg蛋白，在120分钟的孵化期内保持不变，不受细胞传代数的影响。氨基三唑灭活过氧化氢酶的半衰期为23 min。抑制过氧化氢酶、谷胱甘肽还原酶或“)”-谷氨酰半胱氨酸合成酶不会改变细胞外H20释放的速率。此外，抑制线粒体呼吸链（鱼tenone，抗霉素A）或微粒体细胞色素P-450（8-甲氧基补骨脂素）不会改变内皮细胞或谷胱甘肽还原酶失活的细胞的细胞外H20释放或细胞内H20产生（在过氧化物酶体上）。当细胞暴露于外源性H20 （30 j. tM）时，细胞外的H20主要通过谷胱甘肽氧化还原途径清除。只有当谷胱甘肽氧化还原循环失活时，外源添加的H20 （100 j. tM）才会改变细胞内（过氧化物酶体内或附近）的H20产量。Menadione （20 j. tM）经过细胞内氧化还原循环，使细胞外h2o释放增加近4倍，达到0.3 nmol/min/mg蛋白。此外，甲萘醌增加过氧化物酶体H20 2水平，并将氨基三唑存在下过氧化氢酶失活的半衰期缩短至13分钟。过氧化氢酶抑制增加了甲萘醌处理期间细胞外H20 2的释放，表明在氧化应激期间H20 2可以通过质膜扩散。结果表明，在培养的内皮细胞中，线粒体或微粒体水平产生的活性氧可以在没有过氧化物酶过氧化氢酶参与的情况下局部清除，并且在细胞外释放的活性氧是由这些细胞在过氧化氢酶或谷胱甘肽还原酶无法进入的部位产生的。组织中活性氧的形成和降解的调控不仅对我们理解正常的氧化代谢很重要，而且对我们理解活性氧过量产生的病理条件也很重要。活性氧在呼吸过程中由多种细胞类型产生和释放。血管内皮细胞对氧化损伤非常敏感（1-4）。内皮细胞的损伤程度取决于氧化应激的程度和抗氧化防御系统的状态(5)。虽然内皮细胞含有过氧化氢酶和su-
Endogenous hydrogen peroxide (H20 2) release from aortic endothelial cells was studied in the presence of antioxidant enzyme inhibitors, mitochondrial inhibitors, a microsomal cytochrome P-450 inhibitor, and after oxidative stress induced with H20 2 or menadione. Extracellular H20 2 generation was determined spectrofluorometrically using 3-methoxy-4-hydroxy phenylacetic acid, and intracellular H202 production (in or near peroxisomes) was measured indirectly using aminotriazole, which inactivates catalase in the presence of H20 2• Extracellular H20 2 release was 0.079±0.005 nmol/min/mg protein in Hanks' balanced salt solution, was constant during a 120-min incubation period, and was not affected by the cell passage number. The half-life for catalase inactivation with aminotriazole was 23 min. Inhibition of catalase, glutathione reductase, or')'-glutamylcysteine synthetase did not change the rate of extracellular release of H20 2• Furthermore, inhibition of the mitochondrial respiratory chain (rotenone, antimycin A) or microsomal cytochrome P-450 (8-methoxypsoralen) did not change extracellular H20 2 release or intracellular H20 2 production (at peroxisomes) by endothelial cells or cells in which glutathione reductase was inactivated. When the cells were exposed to exogenous H20 2 (30 j. tM), extracellular H20 2 was scavenged primarily by the glutathione redox pathway. Exogenously added H20 2 (100 j. tM) changed intracellular H20 2 production (in or near peroxisomes) only when the glutathione redox cycle was inactivated. Menadione (20 j. tM), which undergoes intracellular redox cycling, increased extracellular H20 2 release almost 4-fold to 0.3 nmol/min/mg protein. Furthermore, menadione increased peroxisomal H20 2 levels and decreased the half-life for catalase inactivation in the presence of aminotriazole to 13 min. Catalase inhibition increased extracellular H20 2 release during menadione treatment, indicating that H20 2 can diffuse across the plasma membrane during oxidant stress. The results suggest that in cultured endothelial cells, reactive oxygen species generated at the mitochondrial or microsomal levels can be scavenged locally without the involvement of peroxisomal catalase and that reactive oxygen species that are released extracellularly are generated by these cells at a site that is inaccessible to catalase or glutathione reductase.Regulation of reactive oxygen species formation and degradation in tissues is important not only to our understanding of normal oxidative metabolism but also to our understanding of pathologic conditions in which reactive oxygen species are overproduced. Reactive oxygen species are generated and released during respiration by a variety of cell types. Vascular endothelial cells are well recognized as being extremely sensitive to oxidative injury (1-4). The extent of injury in endothelial cells is dependent on the degree of oxidative stress and the status of antioxidant defense systems (5). Although endothelial cells contain both catalase and su-