三氧化二砷诱导线粒体通透性转变孔道开放的机制研究

背景与目的:线粒体通透性转变孔道(permeabilitytransitionpore,PTP)是线粒体释放细胞色素c和凋亡诱导因子的主要途径,亚砷酸盐可能通过PTP影响细胞凋亡,为了揭示其诱导PTP开放的机制,本实验研究了Ca2 介导的线粒体Ca2 释放(Ca2 -inducedCa2 releasefrommitochondria,mCICR)在As2O3介导的PTP开放及细胞色素C释放中的作用。方法:提取大鼠肝线粒体。通过紫外分光光度仪检测As2O3作用下线粒体的膨胀,测定线粒体PTP的开放状态;采用双波长双光束紫外分光光度仪检测As2O3作用下测试体系内Ca2 浓度的变化引起的吸光度值的变化,以反映线粒体Ca2 的转运(即mCICR)情况;Westernblot检测线粒体上清液中细胞色素c的含量,反映线粒体释放细胞色素c的情况。结果:10μmol/LAs2O3和低浓度Ca2 不能引起PTP开放和细胞色素c释放,10μmol/LAs2O3和高浓度Ca2 诱导PTP开放和细胞色素c释放;当抑制mCICR时,As2O3和Ca2 对PTP开放和细胞色素c释放的作用完全抑制。结论:As2O3介导的PTP开放和细胞色素c释放依赖于mCICR。

Mechanism of opening of mitochondrial permeability transition pore induced by arsenic trioxide

BACKGROUND & OBJECTIVE: Permeability transition pore (PTP) is central for apoptosis by acting as a good candidate pathway for the release of cytochrome c and apoptosis-induction factors from mitochondria. Arsenite may induce apoptosis via a direct effect on PTP. To characterize the exact mechanism for arsenite to induce PTP opening, the correlations of calcium-induced calcium release from mitochondria (mCICR) to As2O3-induced PTP opening and cytochrome c release from mitochondria were studied. METHODS: Mitochondria were prepared from Wistar rat livers. The effect of As2O3 on mitochondrial PTP opening was measured with ultraviolet (UV) spectrophotometer. The changes of Ca(2+) concentration were detected with UV spectrophotometer to monitor Ca(2+) -induced Ca(2+) release from mitochondria. Cytochrome c release mediated by Ca(2+) was measured with Western blot. RESULTS: As2O3 (10 micromol/L) combined with low concentration of Ca(2+) didn't induce PTP opening and cytochrome c release from mitochondria; while As2O3 (10 micromol/L) combined with high concentration of Ca(2+) induced PTP opening and cytochrome c release. When mCICR was inhibited, the effect of As2O3 and Ca(2+) on PTP opening and cytochrome c release was completely inhibited. CONCLUSION: As2O3-induced PTP opening and cytochrome c release depend on mCICR.