Nicotera P, McConkey D, Svensson S A, Bellomo G, Orrenius S
Department of Toxicology, Karolinska Institutet, Stockholm, Sweden.
Toxicology. 1988 Nov 14;52(1-2):55-63. doi: 10.1016/0300-483x(88)90196-5.
To investigate the relationship between alterations of cytosolic Ca2+ concentration and development of cytotoxicity, isolated rat hepatocytes were loaded with the fluorescent indicator Quin-2 AM and then incubated with non-toxic or toxic levels of menadione (2-methyl-1,4-naphthoquinone) or tert-butyl hydroperoxide (t-BH). The resulting changes in cytosolic Ca2+ concentration were compared to those seen upon exposure of the hepatocytes to an alpha 1-adrenergic agonist, phenylephrine, as well as to those induced by menadione and t-BH in hepatocytes pretreated with agents that modify their toxicity. Exposure of hepatocytes to phenylephrine or non-toxic levels of menadione caused a moderate and transient increase in cytosolic Ca2+ (less than or equal to 0.7 microM), whereas a toxic concentration of menadione produced a marked, sustained increase in Ca2+ which fully saturated the binding capacity of Quin-2 (greater than 1.5 microM). Treatment of the hepatocytes with the protective agent, dithiothreitol, prevented both the increase in cytosolic Ca2+ and the cytotoxicity induced by menadione. On the other hand, pretreatment of cells with diethylmaleate to deplete intracellular glutathione made otherwise non-toxic concentrations of menadione cause both a sustained increase in cytosolic Ca2+ and cytotoxicity. Similarly, toxic concentrations of t-BH also caused a sustained increase in cytosolic Ca2+. The iron chelator, desferrioxamine, and dithiothreitol (DTT), which protected the cells from t-BH toxicity, also prevented the sustained elevation of cytosolic Ca2+. Our findings provide further support for the hypothesis that a perturbation of intracellular Ca2+ homeostasis is an early and critical event in the development of toxicity in hepatocytes exposed to oxidative stress.
为了研究胞质Ca2+浓度变化与细胞毒性发展之间的关系，将分离的大鼠肝细胞用荧光指示剂喹啉-2 AM加载，然后与无毒或有毒水平的甲萘醌（2-甲基-1,4-萘醌）或叔丁基过氧化氢（t-BH）孵育。将由此产生的胞质Ca2+浓度变化与肝细胞暴露于α1-肾上腺素能激动剂去氧肾上腺素时的变化进行比较，同时也与用改变其毒性的药物预处理的肝细胞中甲萘醌和t-BH诱导的变化进行比较。肝细胞暴露于去氧肾上腺素或无毒水平的甲萘醌会导致胞质Ca2+适度且短暂升高（小于或等于0.7微摩尔），而有毒浓度的甲萘醌会使Ca2+显著且持续升高，使喹啉-2的结合能力完全饱和（大于1.5微摩尔）。用保护剂二硫苏糖醇处理肝细胞可防止胞质Ca2+升高以及甲萘醌诱导的细胞毒性。另一方面，用马来酸二乙酯预处理细胞以耗尽细胞内谷胱甘肽会使原本无毒浓度的甲萘醌导致胞质Ca2+持续升高和细胞毒性。同样，有毒浓度的t-BH也会导致胞质Ca2+持续升高。铁螯合剂去铁胺和二硫苏糖醇（DTT）可保护细胞免受t-BH毒性，同时也可防止胞质Ca2+持续升高。我们的研究结果为以下假设提供了进一步支持，即细胞内Ca2+稳态的扰动是暴露于氧化应激的肝细胞毒性发展过程中的一个早期关键事件。
