Metabolic inhibition increases glutamate susceptibility on a PC12 cell line.

The effect of energetic metabolism compromise, obtained by chemical induction of hypoglycaemia (glucose deprivation), hypoxia (mitochondrial respiratory chain inhibition), and ischaemia (hypoglycaemia plus hypoxia), on glutamate toxicity was analyzed on PC12 cells. The respiratory status of these cells, measured by the MTT [3-(4,5-dimethylthiazol-2-yl)2,5-diphenyltetrazolium bromide] assay, was significantly decreased after metabolic inhibition induced by ischaemia, but it was not affected by both hypoglycaemia and hypoxia. Under hypoglycaemia, but not under hypoxia, ATP levels were significantly reduced (from 12.67+/-0.48 to 5.38+/-1.41 nmol/mg protein). However, ischaemic-like conditions greatly potentiated the decline of ATP levels (95% decrease) observed after hypoglycaemia. The influence of metabolic inhibition on glutamate-induced cytotoxicity was also analyzed. When the cells were preincubated under conditions that deplete ATP (hypoglycaemia and ischaemia), the inhibition of MTT reduction, measured after glutamate incubation, was potentiated. This effect could be reverted when vitamin E and idebenone were present during the induction of metabolic inhibition. The ATP levels above which glutamate susceptibility was enhanced were also determined. These results indicate that glutamate toxicity on PC12 cells, which occurs by a mechanism independent of N-methyl-D-aspartate (NMDA) receptor activation, can be enhanced by the depletion of intracellular ATP upon metabolic stress; it is dependent on the extent of ATP depletion and seems to involve the generation of free radicals. It can be concluded that under ischaemic conditions, the deleterious effects of glutamate can be potentiated by the energetic compromise associated with this pathologic situation.