Duan Shili, Hajek Petr, Lin Catherine, Shin Soo Kyung, Attardi Giuseppe, Chomyn Anne
Division of Biology, California Institute of Technology, Pasadena 91125, USA.
J Biol Chem. 2003 Jan 10;278(2):1346-53. doi: 10.1074/jbc.M209269200. Epub 2002 Oct 25.
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.
我们在此表明,凋亡诱导剂星形孢菌素会使完整的143B.TK(-)细胞的内源性呼吸速率早期下降。另一方面,通过对完整细胞耗氧量的测量确定,星形孢菌素处理后的前8小时,细胞色素c氧化酶的活性未发生变化。内源性呼吸速率的下降先于细胞色素c从线粒体的释放。此外,我们排除了半胱天冬酶、通透性转换和蛋白激酶C抑制是呼吸速率下降的原因。此外,Bcl-2基因的过表达并不能阻止呼吸速率的下降。最后这一发现表明,Bcl-2在呼吸扰动的下游起作用。在经星形孢菌素处理的143B.TK(-)骨肉瘤细胞中,复合体I和复合体III的酶活性正常,这一证据表明呼吸速率下降的原因可能是线粒体外膜通透性的改变。据推测,电压依赖性阴离子通道关闭,从而阻止ADP和可氧化底物进入线粒体。这一解释得到了另一个惊人发现的证实,即在经星形孢菌素处理的143B.TK(-)细胞中,用不影响未处理细胞线粒体膜的浓度的洋地黄皂苷使其透化后,线粒体外膜失去完整性;这导致其对外源性底物的不透性逆转。外膜完整性的丧失还导致细胞色素c从线粒体大量过早释放。最重要的是,Bcl-2过表达可防止星形孢菌素诱导的外膜对洋地黄皂苷的超敏感性。因此,我们的实验揭示了线粒体外膜的早期变化,这些变化早在完整细胞中细胞色素c从线粒体释放之前就已发生。