Cysteine starvation activates the redox-dependent mitochondrial permeability transition in retinal pigment epithelial cells
Cysteine starvation activates the redox-dependent mitochondrial permeability transition in retinal pigment epithelial cells
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DOI：
10.1167/iovs.04-0570
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发表时间：
2004-11-01
影响因子：
4.4
通讯作者：
Sternberg, P
中科院分区：
文献类型：
--
作者：
Armstrong, JS;Whiteman, M;Sternberg, P
目的.谷胱甘肽（GSH）在抗氧化应激中起着关键作用。L-半胱氨酸被认为是谷胱甘肽（GSH）合成的限速剂，因此可能是保护免受氧化应激的关键成分。本研究旨在探讨L-半胱氨酸对人视网膜色素上皮（hRPE）细胞GSH代谢和氧化应激的影响。为探讨半胱氨酸在hRPE细胞GSH代谢中的作用，采用半胱氨酸饥饿的方法，通过测定细胞内活性氧（ROS）的产生，（2）线粒体膜电位，（Deltapsi（m））和线粒体超微结构，（3）细胞活力和凋亡指数，包括含亚二倍体DNA量的细胞分析。半胱氨酸饥饿导致GSH浓度在24小时内下降约95%。GSH能斯特氧化还原电位（E-h）增加约70 mV（对照细胞中E-h=-248+/-2.9 mV，而半胱氨酸饥饿细胞中E-h=-179+/-2.0 mV），表明细胞内氧化显著。半胱氨酸饥饿增加了线粒体呼吸复合物III（细胞色素bc（1））的ROS产生，使用导致Deltapsi（m）损失和细胞死亡的药理学策略确定。Deltapsi（m）的丢失和细胞死亡被bongkrekic酸（腺嘌呤核苷酸转运抑制剂的抑制剂）阻止，表明线粒体通透性转换（MPT）的激活。这一结论得到了电子显微镜研究的进一步支持，该研究显示了显著的线粒体肿胀，这是MPT活化的标志。广谱caspase抑制剂zVADfc 1或caspase 3特异性抑制剂DEVD-CHO均不能阻止细胞死亡，表明细胞色素bc（1）介导的ROS产生导致MPT和坏死。这些结果表明，半胱氨酸是正常GSH代谢和保护hRPE细胞免受氧化应激的必需组分。
Purpose. Glutathione (GSH) plays a key role in protection against oxidative stress. L-Cysteine is thought to be rate-limiting for the synthesis of glutathione (GSH) and therefore may be a critical component in protection against oxidative stress. The purpose of this study was to investigate the role of L-cysteine in GSH metabolism and oxidative stress in human retinal pigment epithelial (hRPE) cells.Methods. To identify the role of cysteine in GSH metabolism in hRPE cells, a strategy of cysteine starvation was used to determine (1) GSH levels and oxidative stress by measuring reactive oxygen species (ROS) production, (2) mitochondrial membrane potential (Deltapsi(m)) and mitochondrial ultrastructure by using conventional electron microscopy (EM), and (3) indices of cell viability and apoptosis including analysis of cells containing hypodiploid amounts of DNA.Results. Cysteine starvation resulted in approximately a 95% decrease in GSH concentrations over 24 hours. The GSH Nernst redox potential (E-h) increased approximately 70 mV (E-h=-248+/-2.9 mV in control cells compared with E-h=-179+/-2.0 mV in cysteine-starved cells) indicating significant intracellular oxidation. Cysteine starvation increased the production of ROS by mitochondrial respiratory complex III (cytochrome bc(1)), determined using a pharmacological strategy that resulted in the loss of Deltapsi(m) and cell death. The loss of Deltapsi(m) and cell death was prevented with bongkrekic acid, an inhibitor of the adenine nucleotide translocator inhibitor, suggesting activation of the mitochondrial permeability transition (MPT). This conclusion was further supported by electron microscopic studies that showed significant mitochondrial swelling, a hallmark of MPT activation. Cell death was not prevented with either the broad-spectrum caspase inhibitor zVADfmk or the caspase 3-specific inhibitor DEVD-CHO, indicating that cytochrome bc(1)-mediated ROS production results in the MPT and necrosis.Conclusions. These results show that cysteine is a required component for normal GSH metabolism and protection against oxidative stress in hRPE cells.