Peroxynitrite (PN), generated from the reaction of nitric oxide (NO) and superoxide, is implicated in the pathogenesis of ischemic and neurodegenerative brain injuries. Mitochondria produce NO from mitochondrial NO synthases and superoxide by the electron transport chain. Our objective was to detect the generation of PN of mitochondrial origin and characterize its effects on mitochondrial respiratory function. Methods. Freshly isolated brain non-synaptosomal mitochondria from C57Bl/6 (wild type, WT) and endothelial NO synthase knock out (eNOS-KO) mice were treated with exogenous PN (0.1, 1, 5 µmol/L) or a PN donor (SIN-1; 50 µmol/L), or PN scavenger (FeTMPyP; 2.5 µmol/L). Oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer and mitochondrial respiratory parameters were calculated. Mitochondrial membrane potential, superoxide, and PN were determined from rhodamine 123, dihydroethidium, and DAX-J2 PON green fluorescence measurements, respectively. Mitochondrial protein nitrotyrosination was determined by western blots. Results. Both exogenous PN and SIN-1 decreased respiratory function in WT isolated brain mitochondria. FeTMPyP enhanced state III and state IVo mitochondrial respiration in both WT and eNOS-KO mitochondria. FeTMPyP also elevated state IIIu respiration in eNOS-KO mitochondria. Unlike PN, neither SIN-1 nor FeTMPyP depolarized the mitochondria. Although mitochondrial protein nitrotyrosination was unaffected by SIN-1 or FeTMPyP, FeTMPyP reduced mitochondrial PN levels. Mitochondrial superoxide levels were increased by FeTMPyP but unaffected by PN or SIN-1. Conclusions. Thus, we present the evidence of functionally significant PN generation in isolated brain mitochondria. Mitochondrial PN activity was physiologically relevant in WT mice and pathologically significant under conditions with eNOS deficiency.

中文翻译：

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过氧亚硝酸盐分解催化剂增强离体脑线粒体的呼吸功能


过氧亚硝酸盐 (PN) 由一氧化氮 (NO) 和超氧化物反应生成，与缺血性和神经退行性脑损伤的发病机制有关。线粒体通过电子传递链从线粒体 NO 合酶和超氧化物中产生 NO。我们的目标是检测线粒体来源的 PN 的产生并表征其对线粒体呼吸功能的影响。方法。用外源 PN (0.1, 1, 5 µmol/L) 或 PN 供体 (SIN) 处理来自 C57Bl/6（野生型，WT）和内皮 NO 合酶敲除 (eNOS-KO) 小鼠的新鲜分离的脑非突触体线粒体。 -1; 50 µmol/L) 或 PN 清除剂 (FeTMPyP; 2.5 µmol/L)。使用安捷伦 Seahorse XFe24 分析仪测量耗氧率 (OCR)，并计算线粒体呼吸参数。线粒体膜电位、超氧化物和 PN 分别通过罗丹明 123、二氢乙锭和 DAX-J2 PON 绿色荧光测量来确定。通过蛋白质印迹法测定线粒体蛋白硝基酪氨酸化。结果。外源性 PN 和 SIN-1 均降低 WT 分离脑线粒体的呼吸功能。 FeTMPyP 增强 WT 和 eNOS-KO 线粒体中的 III 态和 IVo 态线粒体呼吸。 FeTMPyP 还升高 eNOS-KO 线粒体中的 IIIu 态呼吸。与 PN 不同，SIN-1 和 FeTMPyP 都不会使线粒体去极化。尽管线粒体蛋白硝基酪氨酸化不受 SIN-1 或 FeTMPyP 的影响，但 FeTMPyP 降低了线粒体 PN 水平。 FeTMPyP 增加了线粒体超氧化物水平，但不受 PN 或 SIN-1 的影响。结论。因此，我们提出了在分离的脑线粒体中产生具有功能性意义的 PN 的证据。 线粒体 PN 活性在 WT 小鼠中具有生理相关性，在 eNOS 缺乏的情况下具有病理学意义。