Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species.
Mitochondrial permeability transition in hepatocytes induced by t-BuOOH: NAD(P)H and reactive oxygen species.
复制标题
DOI：
10.1152/ajpcell.1997.272.4.c1286
复制
发表时间：
1997-04
期刊：
影响因子：
--
通讯作者：
A. Nieminen;Aaeon M. Byrne;B. Herman;J. Lemasters
中科院分区：
文献类型：
--
作者：
A. Nieminen;Aaeon M. Byrne;B. Herman;J. Lemasters
叔丁基过氧化氢（t-BuOOH）诱导肝细胞线粒体通透性转换（MPT），导致细胞死亡。使用共聚焦显微镜，我们可视化的吡啶核苷酸氧化和活性氧（ROS）的形成诱导t-BuOOH。还原的线粒体吡啶核苷酸（NADH和NADPH）通过自体荧光成像。线粒体膜电位，活性氧，MPT的发病，和细胞死亡进行了监测与四甲基罗丹明甲酯（TMRM），二氯荧光素，钙黄绿素，碘化丙啶，分别。t-BuOOH迅速氧化线粒体NAD（P）H。氧化是双相的，第二个较慢的阶段发生在线粒体ROS的产生。随后，发生MPT，线粒体去极化，细胞死亡。减少线粒体NAD+的β-羟基丁酸盐延迟了细胞杀伤，但减少胞质NAD+的乳酸盐却没有。抑制MPT的三氟拉嗪不能阻断NAD（P）H的初始氧化，但能阻止氧化的第二阶段，部分阻断ROS的形成，并保持细胞活力。抗氧化剂，去铁胺和二苯基苯二胺，也阻止了NAD（P）H氧化的第二阶段。它们还几乎完全阻断了ROS的形成，并停止了细胞杀伤。这两种抗氧化剂也阻止了线粒体通透性转换和随后的线粒体去极化。总之，NAD（P）H氧化和ROS形成是促进MPT的关键事件，在肝细胞的氧化损伤和死亡。
Tert-butyl hydroperoxide (t-BuOOH) induces the mitochondrial permeability transition (MPT) in hepatocytes, leading to cell death. Using confocal microscopy, we visualized pyridine nucleotide oxidation and reactive oxygen species (ROS) formation induced by t-BuOOH. Reduced mitochondrial pyridine nucleotides (NADH and NADPH) were imaged by autofluorescence. Mitochondrial membrane potential, ROS, onset of MPT, and cell death were monitored with tetramethylrhodamine methyl ester (TMRM), dichlorofluorescin, calcein, and propidium iodide, respectively. t-BuOOH rapidly oxidized mitochondrial NAD(P)H. Oxidation was biphasic, and the second slower phase occurred during mitochondrial ROS generation. Subsequently, MPT took place, mitochondria depolarized, and cells died. beta-Hydroxybutyrate, which reduces mitochondrial NAD+, delayed cell killing, but lactate, which reduces cytosolic NAD+, did not. Trifluoperazine, which inhibits MPT, did not block the initial oxidation of NAD(P)H but prevented the second phase of oxidation, partially blocked ROS formation, and preserved cell viability. The antioxidants, deferoxamine and diphenylphenylenediamine, also prevented the second phase of NAD(P)H oxidation. They also blocked ROS formation nearly completely and stopped cell killing. Both antioxidants also prevented the mitochondrial permeability transition and subsequent mitochondrial depolarization. In conclusion, NAD(P)H oxidation and ROS formation are critical events promoting MPT in oxidative injury and death of hepatocytes.