Yang J C, Kahn A, Cortopassi G
Department of Urology, University of California, 95616, Davis, CA, USA.
Toxicology. 2000 Oct 26;151(1-3):65-72. doi: 10.1016/s0300-483x(00)00298-5.
The mechanism by which the mitochondrially-localized Bcl-2 protein inhibits apoptosis is still unclear. Some authors have proposed that apoptosis is dependent on induction of the mitochondrial permeability transition pore (PTP), and that activators of apoptosis such as Bax work through activation of PTP, whereas inhibitors of apoptosis such as Bcl-2 work through inhibition of PTP, and the consequent activation or inhibition of PTP-dependent release of mitochondrial apoptotic factors, including cytochrome c. PTP opening is classically measured by a light-scattering assay of large-amplitude swelling of rodent liver mitochondria in sucrose media. Thus to test the hypothesis that Bcl-2 inhibits either the PTP or the PTP-dependent release of cytochrome c, the rate and extent of PTP, and PTP-dependent release of cytochrome c were compared in liver mitochondria from control and Bcl-2 transgenic mice. We demonstrated that Bcl-2 protein was expressed to high levels in mitochondria of transgenics versus controls. We confirmed that while control mice undergo massive hepatic cell death upon exposure to anti-Fas antibody, the Bcl-2 transgenic livers were resistant, by the criteria of gross morphology, serum enzyme release, and caspase 3 activity. We purified mitochondria from livers of the Bcl-2 transgenics and measured PTP directly by the mitochondrial swelling assay. Purified mitochondria from both transgenics and controls were induced to undergo large-amplitude swelling that was dependent on the classical PTP inducers calcium ion (Ca(2+)), t-butyl hydroperoxide (tBOOH) and atractyloside (Atr); and as expected, pretreatment of mitochondria with cyclosporin A (CsA) completely abolished mitochondrial swelling. However, there was no difference in the rate or final extent of PTP induction in Bcl-2 overexpressors versus control mitochondria. Furthermore, there was no difference in the PTP dependent release of cytochrome c from Bcl-2 overexpressors versus control mitochondria. Therefore, while we observe a strong inhibition of Fas-dependent apoptosis by Bcl-2 overexpression in mouse liver, we observe no effect of Bcl-2 overexpression on either the rate or extent of mitochondrial PTP, or upon the release of cytochrome c from mitochondria in which the PTP has been induced. The simplest explanation of these results is that Bcl-2 inhibits neither PTP nor PTP-dependent release of cytochrome c, however, other possibilities are discussed.
线粒体定位的Bcl-2蛋白抑制细胞凋亡的机制仍不清楚。一些作者提出,细胞凋亡依赖于线粒体通透性转换孔(PTP)的诱导,凋亡激活剂如Bax通过激活PTP发挥作用,而凋亡抑制剂如Bcl-2则通过抑制PTP以及随后激活或抑制PTP依赖的线粒体凋亡因子(包括细胞色素c)的释放来发挥作用。经典的PTP开放检测方法是通过对蔗糖培养基中啮齿动物肝线粒体的大幅度肿胀进行光散射测定。因此,为了验证Bcl-2抑制PTP或PTP依赖的细胞色素c释放这一假设,我们比较了对照小鼠和Bcl-2转基因小鼠肝线粒体中PTP的速率和程度以及PTP依赖的细胞色素c释放情况。我们证明,与对照相比,Bcl-2蛋白在转基因小鼠的线粒体中高表达。我们证实,当对照小鼠暴露于抗Fas抗体后会发生大量肝细胞死亡时,根据大体形态、血清酶释放和半胱天冬酶3活性的标准,Bcl-2转基因肝脏具有抗性。我们从Bcl-2转基因小鼠的肝脏中纯化出线粒体,并通过线粒体肿胀试验直接测量PTP。来自转基因小鼠和对照小鼠的纯化线粒体均被诱导发生大幅度肿胀,这种肿胀依赖于经典的PTP诱导剂钙离子(Ca(2+))、叔丁基过氧化氢(tBOOH)和苍术苷(Atr);正如预期的那样,用环孢素A(CsA)预处理线粒体完全消除了线粒体肿胀。然而,Bcl-2过表达小鼠的线粒体与对照线粒体在PTP诱导的速率或最终程度上没有差异。此外,Bcl-2过表达小鼠的线粒体与对照线粒体在PTP依赖的细胞色素c释放方面也没有差异。因此,虽然我们观察到Bcl-2在小鼠肝脏中过表达可强烈抑制Fas依赖的细胞凋亡,但我们并未观察到Bcl-2过表达对线粒体PTP的速率或程度以及PTP诱导后线粒体中细胞色素c的释放有任何影响。这些结果最简单的解释是,Bcl-2既不抑制PTP也不抑制PTP依赖的细胞色素c释放,不过,我们也讨论了其他可能性。