Enhanced mitochondrial superoxide in hyperglycemic endothelial cells: direct measurements and formation of hydrogen peroxide and peroxynitrite
Enhanced mitochondrial superoxide in hyperglycemic endothelial cells: direct measurements and formation of hydrogen peroxide and peroxynitrite
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DOI：
10.1152/ajpheart.00761.2007
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发表时间：
2007-12-01
影响因子：
4.8
通讯作者：
Radi, Rafael
中科院分区：
文献类型：
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作者：
Quijano, Celia;Castro, Laura;Radi, Rafael
对牛主动脉内皮细胞 (BAEC) 的高血糖挑战会增加氧化剂的形成和细胞损伤，而这些损伤可通过 MnSOD 过度表达而消除，这意味着线粒体超氧化物 (O-2(中心点-)) 作为中心介质。然而，线粒体 O-2（中心点-）及其稳态浓度尚未直接测量。因此，我们的目标是通过不同的技术检测和量化 O-2（中心点-）以及从中衍生的氧化剂。与在 5 mM 葡萄糖（正常血糖条件）中培养的细胞相比，在 30 mM 葡萄糖（高血糖条件）中培养的 BAEC 中，O-2（中心点-）的敏感靶点线粒体乌头酸酶失活 60%。在高血糖条件下，可以特异性地检测到线粒体靶向氢乙啶衍生物 (MitoSOX) 向羟基乙锭（与 O-2（中心点-）反应的产物）的氧化增加。据估计，线粒体 O-2（中心点）稳态浓度（至 250 pM）和形成速率（至 6 mu M/s）增加了 8.8 倍。超氧化物的形成增加了细胞内过氧化氢（通过 3-氨基-2,4,5-三唑介导的过氧化氢酶失活来测量）和一氧化氮衍生的氧化剂（即过氧亚硝酸盐）的浓度，通过 3-硝基酪氨酸的免疫化学检测来证明。通过氯甲基二氯二氢荧光素 (CM-H2DCF) 氧化进一步评估氧化剂的形成。暴露于高血糖条件下会引发 CM-H2DCF 的氧化，并被降低线粒体膜电位、抑制电子传递（即 myxothiazol）和清除线粒体氧化剂（即 MitoQ）的药物显着减少。在缺乏线粒体的 BAEC（rho(0) 细胞）中，高血糖条件不会增加 CM-H2DCF 氧化。高血糖条件下线粒体 O-2（中心点-）的形成与克雷布斯循环中葡萄糖代谢的增加和线粒体膜的超极化有关。
Hyperglycemic challenge to bovine aortic endothelial cells (BAECs) increases oxidant formation and cell damage that are abolished by MnSOD overexpression, implying mitochondrial superoxide (O-2(center dot-)) as a central mediator. However, mitochondrial O-2(center dot-) and its steady-state concentrations have not been measured directly yet. Therefore, we aimed to detect and quantify O-2(center dot-) through different techniques, along with the oxidants derived from it. Mitochondrial aconitase, a sensitive target of O-2(center dot-), was inactivated 60% in BAECs incubated in 30 mM glucose (hyperglycemic condition) with respect to cells incubated in 5 mM glucose (normoglycemic condition). Under hyperglycemic conditions, increased oxidation of the mitochondrially targeted hydroethidine derivative (MitoSOX) to hydroxyethidium, the product of the reaction with O-2(center dot-), could be specifically detected. An 8.8-fold increase in mitochondrial O-2(center dot-) steady-state concentration (to 250 pM) and formation rate (to 6 mu M/s) was estimated. Superoxide formation increased the intracellular concentration of both hydrogen peroxide, measured as 3-amino-2,4,5-triazole-mediated inactivation of catalase, and nitric oxide-derived oxidants (i.e., peroxynitrite), evidenced by immunochemical detection of 3-nitrotyrosine. Oxidant formation was further evaluated by chloromethyl dichlorodihydrofluorescein (CM-H2DCF) oxidation. Exposure to hyperglycemic conditions triggered the oxidation of CM-H2DCF and was significantly reduced by pharmacological agents that lower the mitochondrial membrane potential, inhibit electron transport (i.e., myxothiazol), and scavenge mitochondrial oxidants (i.e., MitoQ). In BAECs devoid of mitochondria (rho(0) cells), hyperglycemic conditions did not increase CM-H2DCF oxidation. Mitochondrial O-2(center dot-) formation in hyperglycemic conditions was associated with increased glucose metabolization in the Krebs cycle and hyperpolarization of the mitochondrial membrane.