Oxidative glutamate toxicity involves mitochondrial dysfunction and perturbation of intracellular Ca2+ homeostasis.
Glutamate toxicity on PC12 cells is mediated by oxidative stress as a consequence of the inhibition of a cystine uptake system with depletion of GSH. In this study we report that glutamate decreases PC12 cell viability, inhibiting the reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). This decrease was prevented by the antioxidants vitamin E, idebenone and L-deprenyl, which were also shown to be effective in reducing the accumulation of reactive oxygen species (ROS) in cells exposed to glutamate, decreasing the fluorescence of 2',7'-dichlorofluorescein (DCF). Incubation of PC12 cells with high glutamate concentrations induced mitochondrial dysfunction, leading to the loss of mitochondrial transmembrane potential, evaluated as a decrease in rhodamine 123 (Rh123) retention by mitochondria, and to the decrease of intracellular ATP levels. The mitochondrial dysfunction, induced by glutamate, can be involved in the observed increase of [Ca2+]i. The elevation of [Ca2+]i occurred after GSH depletion, suggesting that oxidative stress is involved in the disturbances of intracellular calcium homeostasis. In conclusion, our data indicate that glutamate, at concentrations which block cystine uptake in PC12 cells leading to GSH depletion and inducing oxidative stress, increases ROS accumulation and decreases cell survival by a mechanism involving mitochondrial dysfunction and impairment of Ca2+ homeostasis.
Faculty of Medicine and Center for Neuroscience of Coimbra, University of Coimbra, Portugal. cpereira@cnc.uc.pt
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Oliveira
D000818:Animals; D000975:Antioxidants / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism*; D018698:Glutamic Acid / Q000187:drug effects / Q000378:metabolism / Q000506:poisoning*; D005978:Glutathione / Q000378:metabolism; D006706:Homeostasis / Q000187:drug effects*; D007425:Intracellular Membranes / Q000378:metabolism*; D008928:Mitochondria / Q000187:drug effects* / Q000502:physiology*; D009994:Osmolar Concentration; D010084:Oxidation-Reduction; D018384:Oxidative Stress / Q000502:physiology; D016716:PC12 Cells; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism
D000975:Antioxidants; D017382:Reactive Oxygen Species; D018698:Glutamic Acid; D005978:Glutathione; D002118:Calcium