Synergistic depletion of astrocytic glutathione by glucose deprivation and peroxynitrite: Correlation with mitochondrial dysfunction and subsequent cell death

Previously we reported that immunostimulated astrocytes were highly vulnerable to glucose deprivation. The augmented death was mimicked by the peroxynitrite (ONOO^-)-producing reagent 3-morpholinosydnonimine (SIN-1). Here we show that glucose deprivation and ONOO^- synergistically deplete intracellular reduced glutathione (GSH) and augment the death of astrocytes via formation of cyclosporin A-sensitive mitochondrial permeability transition (MPT) pore. Astrocytic GSH levels were only slightly decreased by glucose deprivation or SIN-1 (200 μM) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/SIN-1 -treated astrocytes. The depletion of GSH occurred before a significant release of lactate dehydrogenase (a marker of cell death). Superoxide dismutase and ONOO^- scavengers completely blocked the augmented death, indicating that the reaction of nitric oxide with superoxide to form ONOO^- was implicated. Furthermore, nitrotyrosine immunoreactivity (a marker Of ONOO^-) was markedly enhanced in glucose-deprived/SIN-1-treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose- deprived/SIN-1-treated astrocytes. The glutathione synthase inhibitor L- buthionine-(S,R)-sulfoximine markedly decreased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1-treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevented the MTP depolarization as well as the enhanced LDH releases in glucose-deprived/SIN-1-treated astrocytes.