CONTRIBUTION OF THE MITOCHONDRIAL PERMEABILITY TRANSITION TO LETHAL INJURY AFTER EXPOSURE OF HEPATOCYTES TO T-BUTYLHYDROPEROXIDE
CONTRIBUTION OF THE MITOCHONDRIAL PERMEABILITY TRANSITION TO LETHAL INJURY AFTER EXPOSURE OF HEPATOCYTES TO T-BUTYLHYDROPEROXIDE
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DOI：
10.1042/bj3070099
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发表时间：
1995-04-01
影响因子：
4.1
通讯作者：
LEMASTERS, JJ
中科院分区：
文献类型：
--
作者：
NIEMINEN, AL;SAYLOR, AK;LEMASTERS, JJ
我们已经开发了一种新的方法来监测线粒体通透性转换在单个完整的肝细胞损伤与叔丁基过氧化氢（t-BuOOH）。用荧光探针钙黄绿素和四甲基罗丹明甲酯（TMRM）加载培养的肝细胞。根据加载条件，钙黄绿素仅标记胞质空间并且不进入线粒体或其染色胞质和线粒体。TMRM标记线粒体作为线粒体极化的指标。使用激光扫描共聚焦显微镜同时成像两个探针的荧光。在正常的孵育过程中，TMRM标记的线粒体无限期（超过63分钟），钙黄绿素没有重新分配之间的细胞质和线粒体。这些发现表明，线粒体渗透性转换孔（“megachannel”）保持连续关闭。加入100 μ M t-BuOOH后，线粒体迅速充满钙黄绿素，表明线粒体渗透性转换的开始。该事件伴有线粒体去极化，如TMRM丧失所示。随后，ATP浓度下降，细胞失去活力。三氟拉嗪是一种磷脂酶抑制剂，可抑制分离线粒体的通透性转换，防止钙黄绿素重新分布到线粒体中，线粒体去极化，ATP耗竭和细胞死亡。羰基氰化物间氯苯腙（CCCP），线粒体解偶联剂，也迅速去极化完整的肝细胞的线粒体，但并没有单独诱导渗透性转变。三氟拉嗪没有阻止ATP耗竭和细胞死亡后，添加CCCP。总之，在肝细胞正常孵育期间，渗透性转换孔不会“闪烁”打开，而是保持持续关闭。此外，线粒体去极化本身不会引起完整细胞的通透性转变。然而，在氧化应激过程中，渗透性转变迅速发生，导致线粒体去极化和细胞死亡。
We have developed a novel method for monitoring the mitochondrial permeability transition in single intact hepatocytes during injury with t-butylhydroperoxide (t-BuOOH). Cultured hepatocytes were loaded with the fluorescence probes, calcein and tetramethylrhodamine methyl ester (TMRM), Depending on loading conditions, calcein labelled the cytosolic space exclusively and did not enter mitochondria or it stained both cytosol and mitochondria. TMRM labelled mitochondria as an indicator of mitochondrial polarization. Fluorescence of two probes was imaged simultaneously using laser-scanning confocal microscopy. During normal incubations, TMRM labelled mitochondria indefinitely (longer than 63 min), and calcein did not redistribute between cytosol and mitochondria. These findings indicate that the mitochondrial permeability transition pore ('megachannel') remained closed continuously, After addition of 100 mu M t-BuOOH, mitochondria filled quickly with calcein, indicating the onset of mitochondrial permeability transition. This event was accompanied by mitochondrial depolarization, as shown by loss of TMRM. Subsequently, the concentration of ATP declined and cells lost viability. Trifluoperazine, a phospholipase inhibitor that inhibits the permeability transition in isolated mitochondria, prevented calcein redistribution into mitochondria, mitochondrial depolarization, ATP depletion and cell death. Carbonyl cyanide m-chlorophenylhydrazone (CCCP), a mitochondrial uncoupler, also rapidly depolarized mitochondria of intact hepatocytes but did not alone induce a permeability transition. Trifluoperazine did not prevent ATP depletion and cell death after the addition of CCCP. In conclusion, the permeability transition pore does not 'flicker' open during normal incubation of hepatocytes but remains continuously closed. Moreover, mitochondrial depolarization per se does not cause the permeability transition in intact cells. During oxidative stress, however, a permeability transition occurs quickly which leads to mitochondrial depolarization and cell death.