Mitochondrial outer membrane permeability change and hypersensitivity to digitonin early in staurosporine-induced apoptosis
Mitochondrial outer membrane permeability change and hypersensitivity to digitonin early in staurosporine-induced apoptosis
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DOI：
10.1074/jbc.m209269200
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发表时间：
2003-01-10
影响因子：
4.8
通讯作者：
Chomyn, A
中科院分区：
文献类型：
--
作者：
Duan, S;Hájek, P;Chomyn, A
我们在这里已经证明，凋亡诱导剂星形孢子素在完整的143B.TK-细胞中引起内源性呼吸频率的早期下降。另一方面，细胞色素c氧化酶的活性在星状孢子素处理后的前8小时内没有变化，这是通过测量完整细胞的氧耗来确定的。内源呼吸速率的降低先于细胞色素c从线粒体中释放。此外，我们已经排除了半胱氨酸酶、通透性转换和蛋白激酶C抑制是导致呼吸频率下降的原因。此外，过度表达Bcl2基因并不能阻止呼吸频率的下降。最后的发现表明，在呼吸过程中，Bcl2在微扰的下游起作用。星形孢菌素处理的143B.TK-骨肉瘤细胞中，复合物I和复合物III的酶活性正常，提示呼吸减弱的原因可能是线粒体膜外膜通透性的改变。据推测，电压依赖的阴离子通道关闭，从而阻止ADP和可氧化底物被线粒体摄取。另一个令人惊讶的发现证实了这一解释，即在星状孢子素处理的143B.TK-细胞中，洋地黄素在不影响幼稚细胞线粒体膜的浓度下通透，线粒体膜外壁失去完整性；这导致其对外源底物的不通透性发生逆转。外膜完整性的丧失也会导致细胞色素c从线粒体大量提前释放。最重要的是，bc l-2的过表达可以阻止星状孢子素诱导的外膜对洋地黄素的过敏。因此，我们的实验揭示了线粒体外膜的早期变化，这种变化发生在完整细胞的线粒体释放细胞色素c之前很久。
We have shown here that the apoptosis inducer staurosporine causes an early decrease in the endogenous respiration rate in intact 143B.TK- cells. On the other hand, the activity of cytochrome c oxidase is unchanged for the first 8 h after staurosporine treatment, as determined by oxygen consumption measurements in intact cells. The decrease in the endogenous respiration rate precedes the release of cytochrome c from mitochondria. Moreover, we have ruled out caspases, permeability transition, and protein kinase C inhibition as being responsible for the decrease in respiration rate. Furthermore, overexpression of the gene for Bcl-2 does not prevent the decrease in respiration rate. The last finding suggests that Bcl-2 acts downstream of the perturbation in respiration. The evidence of normal enzymatic activities of complex I and complex III in staurosporine-treated 143B.TK- osteosarcoma cells indicates that the cause of the respiration decrease is probably an alteration in the permeability of the outer mitochondrial membrane. Presumably, the voltage-dependent anion channel closes, thereby preventing ADP and oxidizable substrates from being taken up into mitochondria. This interpretation was confirmed by another surprising finding, namely that, in staurosporine-treated 143B.TK- cells permeabilized with digitonin at a concentration not affecting the mitochondrial membranes in naive cells, the outer mitochondrial membrane loses its integrity; this leads to a reversal of its impermeability to exogenous substrates. The loss of outer membrane integrity leads also to a massive premature release of cytochrome c from mitochondria. Most significantly, Bcl-2 overexpression prevents the staurosporine-induced hypersensitivity of the outer membrane to digitonin. Our experiments have thus revealed early changes in the outer mitochondrial membrane, which take place long before cytochrome c is released from mitochondria in intact cells.