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

Previously we reported that immunostimulated astrocytes were highly vulnera ble to glucose deprivation. The augmented death was mimicked by the peroxyn itrite (ONOO-)-producing reagent 3-morpholinosydnonimine (SIN-1). Here we s how that glucose deprivation and ONOO- synergistically deplete intracellula r reduced glutathione (GSH) and augment the death of astrocytes via formati on of cyclosporin A-sensitive mitochondrial permeability transition (MPT) p ore. Astrocytic GSH levels were only slightly decreased by glucose deprivat ion or SIN-1 (200 mu M) alone. In contrast, a rapid and large depletion of GSH was observed in glucose-deprived/SIN-1-treated astrocytes. The depletio n 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 im munoreactivity (a marker of ONOO-) was markedly enhanced in glucose-deprive d/SIN-1-treated astrocytes. Mitochondrial transmembrane potential (MTP) was synergistically decreased in glucose-deprived/SIN-1 treated astrocytes. Th e glutathione synthase inhibitor L-buthionine-(S,R)-sulfoximine markedly de creased the MTP and increased lactate dehydrogenase (LDH) releases in SIN-1 -treated astrocytes. Cyclosporin A, an MPT pore blocker, completely prevent ed the MTP depolarization as well as the enhanced LDH releases in glucose-d eprived/SIN-1-treated astrocytes.