Department of Cell & Developmental Biology, University of North Carolina at Chapel Hill, 27599, USA.
Arch Biochem Biophys. 2010 Mar 15;495(2):174-81. doi: 10.1016/j.abb.2010.01.008. Epub 2010 Jan 25.
The electron transport chain of mitochondria is a major source of reactive oxygen species (ROS), which play a critical role in augmenting the Ca(2+)-induced mitochondrial permeability transition (MPT). Mitochondrial release of superoxide anions (O(2)(-)) from the intermembrane space (IMS) to the cytosol is mediated by voltage dependent anion channels (VDAC) in the outer membrane. Here, we examined whether closure of VDAC increases intramitochondrial oxidative stress by blocking efflux of O(2)(-) from the IMS and sensitizing to the Ca(2+)-induced MPT. Treatment of isolated rat liver mitochondria with 5microM G3139, an 18-mer phosphorothioate blocker of VDAC, accelerated onset of the MPT by 6.8+/-1.4min within a range of 100-250microM Ca(2+). G3139-mediated acceleration of the MPT was reversed by 20microM butylated hydroxytoluene, a water soluble antioxidant. Pre-treatment of mitochondria with G3139 also increased accumulation of O(2)(-) in mitochondria, as monitored by dihydroethidium fluorescence, and permeabilization of the mitochondrial outer membrane with digitonin reversed the effect of G3139 on O(2)(-) accumulation. Mathematical modeling of generation and turnover of O(2)(-) within the IMS indicated that closure of VDAC produces a 1.55-fold increase in the steady-state level of mitochondrial O(2)(-). In conclusion, closure of VDAC appears to impede the efflux of superoxide anions from the IMS, resulting in an increased steady-state level of O(2)(-), which causes an internal oxidative stress and sensitizes mitochondria toward the Ca(2+)-induced MPT.
线粒体的电子传递链是活性氧（ROS）的主要来源，ROS 在增强 Ca(2+)诱导的线粒体通透性转变（MPT）中起着关键作用。来自线粒体外膜的电压依赖性阴离子通道（VDAC）介导了超氧阴离子（O(2)(-)）从膜间空间（IMS）向细胞质的释放。在这里，我们研究了 VDAC 的闭合是否通过阻断 O(2)(-)从 IMS 的流出以及对 Ca(2+)诱导的 MPT 敏感来增加线粒体内部的氧化应激。用 5μM G3139（一种 18 聚体 VDAC 的硫代磷酸酯阻断剂）处理分离的大鼠肝线粒体，在 100-250μM Ca(2+)范围内将 MPT 的起始时间提前了 6.8+/-1.4 分钟。G3139 介导的 MPT 加速可被 20μM 水溶性抗氧化剂丁羟甲苯逆转。G3139 预处理还增加了线粒体中二氢乙啶荧光监测的 O(2)(-)的积累，并且用胆钙化醇处理线粒体导致线粒体外膜的通透性增加，逆转了 G3139 对 O(2)(-)积累的影响。IMS 内 O(2)(-)产生和周转的数学模型表明，VDAC 的闭合导致线粒体中 O(2)(-)的稳态水平增加了 1.55 倍。总之，VDAC 的闭合似乎阻碍了超氧阴离子从 IMS 的流出，导致 O(2)(-)的稳态水平增加，从而引起内部氧化应激，并使线粒体对 Ca(2+)诱导的 MPT 敏感。
