The role of external and matrix pH in mitochondrial reactive oxygen species generation.
The role of external and matrix pH in mitochondrial reactive oxygen species generation.
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DOI：
10.1074/jbc.m801019200
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发表时间：
2008-10-24
期刊：
影响因子：
--
通讯作者：
Votyakova TV
中科院分区：
文献类型：
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作者：
Selivanov VA;Zeak JA;Roca J;Cascante M;Trucco M;Votyakova TV
作为电子和质子通过内膜运输的副产物，线粒体中产生的活性氧（ROS）对于正常细胞运作以及病理学的发展非常重要。基质和细胞质碱化稳定半醌自由基，一个潜在的超氧化物生产商，我们假设质子不足下过量的电子供体增强活性氧的产生。我们通过测量线粒体释放的活性氧的pH依赖性来验证这一假设。 在复合物II底物琥珀酸盐存在下或在具有复合物I底物谷氨酸盐和苹果酸盐的更生理条件下，在pH从6至8变化的培养基中进行实验。基质pH值由无机磷酸盐、尼日利亚碱和低浓度解偶联剂或缬氨霉素操纵。我们发现，在所有研究的条件下，高pH值强烈增加了自由基产生的速率，即使在尼日利亚菌素存在下ΔpH = 0时也是如此。在无机磷酸盐的情况下，当基质是最碱性的，在介质中的pH值高于7引起的渗透性转变伴随着ROS产生的减少。观察到与完整的呼吸线粒体，以及在复合物I抑制剂鱼藤酮，增强活性氧释放的存在下，由培养基的碱化诱导的ROS生产增加。本报告中揭示的现象对于理解线粒体产生活性氧的机制非常重要，特别是与解偶联蛋白相关的机制。
Reactive oxygen species (ROS) generation in mitochondria as a side product of electron and proton transport through the inner membrane is important for normal cell operation as well as development of pathology. Matrix and cytosol alkalization stabilizes semiquinone radical, a potential superoxide producer, and we hypothesized that proton deficiency under the excess of electron donors enhances reactive oxygen species generation. We tested this hypothesis by measuring pH dependence of reactive oxygen species released by mitochondria. The experiments were performed in the media with pH varying from 6 to 8 in the presence of complex II substrate succinate or under more physiological conditions with complex I substrates glutamate and malate. Matrix pH was manipulated by inorganic phosphate, nigericine, and low concentrations of uncoupler or valinomycin. We found that high pH strongly increased the rate of free radical generation in all of the conditions studied, even when ΔpH = 0 in the presence of nigericin. In the absence of inorganic phosphate, when the matrix was the most alkaline, pH shift in the medium above 7 induced permeability transition accompanied by the decrease of ROS production. ROS production increase induced by the alkalization of medium was observed with intact respiring mitochondria as well as in the presence of complex I inhibitor rotenone, which enhanced reactive oxygen species release. The phenomena revealed in this report are important for understanding mechanisms governing mitochondrial production of reactive oxygen species, in particular that related with uncoupling proteins.