Petrosillo Giuseppe, Ruggiero Francesca M, Pistolese Marilva, Paradies Giuseppe
Department of Biochemistry and Molecular Biology and Consiglio Nazionale delle Ricerche Institute of Biomembranes and Bioenergetics, University of Bari, Via E. Orabona, 4, 70126 Bari, Italy.
J Biol Chem. 2004 Dec 17;279(51):53103-8. doi: 10.1074/jbc.M407500200. Epub 2004 Oct 8.
Release of cytochrome c from mitochondria is considered a critical, early event in the induction of an apoptosis cascade that ultimately leads to programmed cell death. Mitochondrial Ca(2+) loading is a trigger for the release of cytochrome c, although the molecular mechanism underlying this effect is not fully clarified. This study tested the hypothesis that distinct Ca(2+) thresholds may induce cytochrome c release from rat liver mitochondria by membrane permeability transition (MPT)-dependent and independent mechanisms. The involvement of reactive oxygen species (ROS) and cardiolipin in the Ca(2+)-induced cytochrome c release was also investigated. Cytochrome c was quantitated by a new, very sensitive, and rapid reverse-phase high performance liquid chromatography method with a detection limit of 0.1 pmol/sample. We found that a low extramitochondrial Ca(2+) level (2 microM) promoted the release of approximately 13% of the total alamethicin releasable pool of cytochrome c from mitochondria. This release was not depending of MPT; it was mediated by Ca(2+)-induced ROS production and cardiolipin peroxidation and appears to involve the voltage-dependent anion channel. High extramitochondrial Ca(2+) level (20 microM) promoted approximately 45% of the total releasable pool of cytochrome c. This process was MPT-dependent and was also mediated by ROS and cardiolipin. It is suggested that distinct Ca(2+) levels may determine the mode and the amount of cytochrome c release from rat liver mitochondria. The data may help to clarify the molecular mechanism underlying the Ca(2+)-induced release of cytochrome c from rat liver mitochondria and the role played by ROS and cardiolipin in this process.
线粒体细胞色素c的释放被认为是诱导凋亡级联反应的关键早期事件，最终导致程序性细胞死亡。线粒体Ca(2+) 负载是细胞色素c释放的触发因素，尽管这种效应背后的分子机制尚未完全阐明。本研究检验了以下假设：不同的Ca(2+) 阈值可能通过膜通透性转换 (MPT) 依赖性和非依赖性机制诱导大鼠肝脏线粒体释放细胞色素c。还研究了活性氧 (ROS) 和心磷脂在Ca(2+) 诱导的细胞色素c释放中的作用。细胞色素c通过一种新的、非常灵敏且快速的反相高效液相色谱法进行定量，检测限为0.1 pmol/样品。我们发现，低细胞外Ca(2+) 水平 (2 microM) 促进了线粒体中约13% 的总阿拉霉素可释放细胞色素c池的释放。这种释放不依赖于MPT；它是由Ca(2+) 诱导的ROS产生和心磷脂过氧化介导的，似乎涉及电压依赖性阴离子通道。高细胞外Ca(2+) 水平 (20 microM) 促进了约45% 的细胞色素c总可释放池的释放。这个过程是MPT依赖性的，也是由ROS和心磷脂介导的。提示不同的Ca(2+) 水平可能决定大鼠肝脏线粒体释放细胞色素c的方式和量。这些数据可能有助于阐明Ca(2+) 诱导大鼠肝脏线粒体释放细胞色素c的分子机制以及ROS和心磷脂在此过程中所起的作用。
