He L, Poblenz A T, Medrano C J, Fox D A
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-6052, USA.
J Biol Chem. 2000 Apr 21;275(16):12175-84. doi: 10.1074/jbc.275.16.12175.
Calcium overload is suggested to play a fundamental role in the process of rod apoptosis in chemical-induced and inherited retinal degenerations. However, this hypothesis has not been tested directly. We developed an in vitro model utilizing isolated rat retinas to determine the mechanisms underlying Ca(2+)- and/or Pb(2+)-induced retinal degeneration. Confocal microscopy, histological, and biochemical studies established that the elevated [Ca(2+)] and/or [Pb(2+)] were localized to photoreceptors and produced rod-selective apoptosis. Ca(2+) and/or Pb(2+) induced mitochondrial depolarization, swelling, and cytochrome c release. Subsequently caspase-9 and caspase-3 were sequentially activated. Caspase-7 and caspase-8 were not activated. The effects of Ca(2+) and Pb(2+) were additive and blocked completely by the mitochondrial permeability transition pore (PTP) inhibitor cyclosporin A, whereas the calcineurin inhibitor FK506 had no effect. The caspase inhibitors carbobenzoxy-Leu-Glu-His-Asp-CH(2)F and carbobenzoxy-Asp-Glu-Val-Asp-CH(2)F, but not carbobenzoxy-Ile-Glu-Thr-Asp-CH(2)F, differentially blocked post-mitochondrial events. The levels of reduced and oxidized glutathione and pyridine nucleotides in rods were unchanged. Our results demonstrate that rod mitochondria are the target site for Ca(2+) and Pb(2+). Moreover, they suggest that Ca(2+) and Pb(2+) bind to the internal metal (Me(2+)) binding site of the PTP and subsequently open the PTP, which initiates the cytochrome c-caspase cascade of apoptosis in rods.
钙超载被认为在化学诱导和遗传性视网膜变性中视杆细胞凋亡过程中起关键作用。然而，这一假说尚未得到直接验证。我们利用分离的大鼠视网膜建立了一个体外模型，以确定Ca(2+)和/或Pb(2+)诱导视网膜变性的潜在机制。共聚焦显微镜、组织学和生化研究表明，升高的[Ca(2+)]和/或[Pb(2+)]定位于光感受器，并导致视杆细胞选择性凋亡。Ca(2+)和/或Pb(2+)诱导线粒体去极化、肿胀和细胞色素c释放。随后，caspase-9和caspase-3被依次激活。caspase-7和caspase-8未被激活。Ca(2+)和Pb(2+)的作用具有相加性，并被线粒体通透性转换孔（PTP）抑制剂环孢素A完全阻断，而钙调神经磷酸酶抑制剂FK506则无作用。caspase抑制剂苄氧羰基-Leu-Glu-His-Asp-CH(2)F和苄氧羰基-Asp-Glu-Val-Asp-CH(2)F，但不是苄氧羰基-Ile-Glu-Thr-Asp-CH(2)F，分别阻断线粒体后事件。视杆细胞中还原型和氧化型谷胱甘肽以及吡啶核苷酸的水平未发生变化。我们的结果表明，视杆线粒体是Ca(2+)和Pb(2+)的靶位点。此外，这些结果提示Ca(2+)和Pb(2+)与PTP的内部金属（Me(2+)）结合位点结合，随后打开PTP，从而启动视杆细胞中细胞色素c-caspase凋亡级联反应。
