Tirosh O, Sen C K, Roy S, Packer L
Molecular and Cell Biology, University of California, Berkeley, CA 94720-3200, USA.
Neuroscience. 2000;97(3):531-41. doi: 10.1016/s0306-4522(00)00028-2.
Elevated levels of extracellular glutamate are neurotoxic. The cytotoxic property of extracellular glutamate is known to mediate two primary mechanisms, excitotoxicity and excitotoxicity-independent processes. The excitotoxicity-independent pathway was investigated in the current study in a mouse hippocampal-derived HT4 cell line. Exposure of HT4 cells to glutamate for 12h induced loss of cell viability preceded by rapid loss of intracellular reduced glutathione followed by accumulation of intracellular reactive oxygen species, elevation of intracellular Ca(2+), progressive loss of mitochondrial membrane potential swelling and loss of mitochondrial outer membrane integrity. Glutamate-induced loss of DNA integrity has been detected. The antioxidants alpha-tocopherol and trolox, mitochondrial calcium uniporter inhibitor Ruthenium Red and protein synthesis inhibitor cycloheximide all showed protection against glutamate-induced toxicity. None of the protective agents except for alpha-tocopherol controlled the glutamate-induced reactive oxygen species build-up. However, these cell death regulators prevented the glutamate-induced mitochondrial damage and regulated glutamate-induced increase in intracellular Ca(2+). Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone, a mitochondrial uncoupler, partially protected against glutamate-induced cell death and mitochondrial damage, while the mitochondrial ribosomal inhibitor chloramphenicol and extracellular Ca(2+) chelator ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid did not protect the cells against glutamate treatment. The results of this study demonstrated that mitochondrial dysfunction was a key event in the excitotoxicity-independent component of neuronal cell death. Reactive oxygen species accumulation and glutathione depletion were prominent in glutamate-treated cells; however, these events were not direct mediators of cell death.
细胞外谷氨酸水平升高具有神经毒性。已知细胞外谷氨酸的细胞毒性作用介导两种主要机制，即兴奋性毒性和非兴奋性毒性过程。在本研究中，我们在小鼠海马来源的HT4细胞系中研究了非兴奋性毒性途径。将HT4细胞暴露于谷氨酸12小时会导致细胞活力丧失，在此之前细胞内还原型谷胱甘肽迅速减少，随后细胞内活性氧积累、细胞内Ca(2+)升高、线粒体膜电位逐渐丧失、肿胀以及线粒体外膜完整性丧失。已检测到谷氨酸诱导的DNA完整性丧失。抗氧化剂α-生育酚和曲洛司坦、线粒体钙单向转运体抑制剂钌红以及蛋白质合成抑制剂环己酰亚胺均显示出对谷氨酸诱导的毒性具有保护作用。除α-生育酚外，没有一种保护剂能控制谷氨酸诱导的活性氧生成增加。然而，这些细胞死亡调节因子可防止谷氨酸诱导的线粒体损伤，并调节谷氨酸诱导的细胞内Ca(2+)增加。线粒体解偶联剂羰基氰化物对三氟甲氧基苯腙可部分保护细胞免受谷氨酸诱导的细胞死亡和线粒体损伤，而线粒体核糖体抑制剂氯霉素和细胞外Ca(2+)螯合剂乙二醇双(β-氨基乙基醚)-N,N,N',N'-四乙酸不能保护细胞免受谷氨酸处理。本研究结果表明，线粒体功能障碍是神经元细胞死亡非兴奋性毒性成分中的关键事件。在谷氨酸处理的细胞中，活性氧积累和谷胱甘肽耗竭较为突出；然而，这些事件并非细胞死亡的直接介导因素。
