García-Ruiz C, Colell A, Morales A, Kaplowitz N, Fernández-Checa J C
Department of Medicine, Hospital Clinic i Provincial, Barcelona, Spain.
Mol Pharmacol. 1995 Nov;48(5):825-34.
Mitochondria are an important source of reactive oxygen intermediates because they are the major consumers of molecular oxygen in cells. Respiration is associated with toxicity, which is related to the activation of oxygen to reactive intermediates. The purpose of the present study was to examine the role of reduced glutathione (GSH) in the maintenance of mitochondrial functions during oxidative stress induced through selective inhibition of the complex III segment of the electron transport chain. Hydrogen peroxide monitored by the fluorescence of dichlorofluorescein increased in a time- and dose-dependent manner on incubation of mitochondria with antimycin A (AA), an inhibitor of complex III. However, blockade of complex I or II with rotenone or thenoyltrifluoroacetone, respectively, did not result in accumulation of hydrogen peroxide. Depletion of mitochondrial GSH to 10-20% of control by preincubation with diethylmaleate (0.8 mM) or ethacrynic acid (250 microM) also increased dichlorofluorescein and malondialdehyde levels and resulted in an additional (2-3-fold) increase after AA. Similar results were obtained when mitochondrial GSH depletion was produced by treatment with buthionine L-sulfoximine before mirochondria isolation. The endogenous oxidative stress induced by AA was accompanied by a moderate loss of activity of ATPase complex (77% of control) and complex IV of respiration (75% of control), which was accentuated after depletion of mitochondrial GSH (51% and 45% of control, respectively). Similar results were observed in isolated hepatocytes in which depletion of mitochondrial GSH and AA led to peroxidation and mitochondrial dysfunction. In addition, with electrophoretic mobility shift assay of the transcription factor nuclear factor-kappa B (NF-kappa B), we detected its activation in response to AA (2-3-fold). Depletion of mitochondrial GSH in hepatocytes (20% of control) led to further enhancement of NF-kappa B activation (2-4-fold), which correlated with generation of hydrogen peroxide. Thus, our results suggest that GSH protects mitochondria against the endogenous oxidative stress produced at the ubiquinone site of the electron transport chain. Mitochondrial GSH depletion potentiates oxidant-induced loss of mitochondrial functions. Oxidant stress in mitochondria can promote extramitochondrial activation of NF-kappa B and therefore may affect nuclear gene expression.
线粒体是活性氧中间体的重要来源，因为它们是细胞中分子氧的主要消耗者。呼吸作用与毒性相关，这与氧被激活为活性中间体有关。本研究的目的是探讨还原型谷胱甘肽（GSH）在通过选择性抑制电子传递链复合体III片段诱导的氧化应激过程中对维持线粒体功能的作用。用复合体III抑制剂抗霉素A（AA）孵育线粒体时，通过二氯荧光素荧光监测的过氧化氢以时间和剂量依赖性方式增加。然而，分别用鱼藤酮或噻吩甲酰三氟丙酮阻断复合体I或II，并未导致过氧化氢的积累。通过与马来酸二乙酯（0.8 mM）或依他尼酸（250 microM）预孵育将线粒体GSH消耗至对照的10 - 20%，也会增加二氯荧光素和丙二醛水平，并在加入AA后导致额外（2 - 3倍）增加。在用丁硫氨酸-L-亚砜亚胺处理分离线粒体之前产生线粒体GSH消耗时，也获得了类似结果。AA诱导的内源性氧化应激伴随着ATP酶复合体活性（对照值的77%）和呼吸复合体IV活性（对照值的75%）的适度丧失，在线粒体GSH消耗后这种丧失更加明显（分别为对照值的51%和45%）。在分离的肝细胞中也观察到类似结果，其中线粒体GSH消耗和AA导致过氧化和线粒体功能障碍。此外通过转录因子核因子-κB（NF-κB）的电泳迁移率变动分析，我们检测到其对AA反应激活（2 - 3倍）。肝细胞中线粒体GSH消耗至对照的20%导致NF-κB激活进一步增强（2 - 4倍），这与过氧化氢的产生相关。因此，我们的结果表明，GSH保护线粒体免受电子传递链泛醌位点产生 的内源性氧化应激。线粒体GSH消耗会增强氧化剂诱导的线粒体功能丧失。线粒体中的氧化应激可促进线粒体外NF-κB的激活，因此可能影响核基因表达。
