Glutathione in the brain. Studies on efflux and possible implications in neurotoxicity

Neuronal cell death in acute insults such as stroke, trauma and epilepsy involves overactivation of glutamate receptors, calcium influx and increased formation of potentially toxic reactive oxygen species. Glutathione is a key component in the antioxidant defense system against reactive oxygen species. In stroke models the levels of glutathione decrease in the tissue and increases extracellularly. This suggests that efflux of glutathione may lead to a loss of cellular glutathione and hence decreased intracellular antioxidant capacity. The main focus of the work in this thesis was to characterize efflux and extracellular metabolism of glutathione in situations involving glutamate receptor activation and/or oxygen deprivation. Glutathione levels in the tissue and in a mitochondrial enriched fraction were determined after hypoxia-ischemia in the neonatal rat, a model that involves overactivation of glutamate receptors and neurodegeneration.Efflux of glutathione was characterized by incubation of tissue slices, primary cultures and organotypic cultures of the rat hippocampus. Glutathione and amino acid concentrations were determined by the use of precolumn derivatization followed by liquid chromatography separation and fluorescence detection. Hypoxia-ischemia in 7-day-old rats was induced by unilateral carotid artery ligation followed by 65 min of hypoxia.Depolarization, anoxia-aglycemia and stimulation of glutamate receptors induced a delayed increase in efflux of glutathione from hippocampal slices. The glutamate receptor mediated glutathione efflux was dependent on extracellular calcium but unrelated to dantrolene-sensitive intracellular calcium release and independent of nitric oxide synthesis. The delayed increase in efflux of glutathione was attenuated by treatment with a calmodulin antagonist and an anion transport inhibitor. Extracellular glutathione was mainly recovered in the reduced form. Glutamate receptor mediated glutathione efflux was observed in mixed neuronal-astroglial cultures but not in neuron-free astroglial cultures. Extracellular catabolism of glutathione by g-glutamyl transpeptidase was enhanced by depolarization, anoxia-aglycemia and glutamate receptor stimulation. The concentration of glutathione was transiently decreased in the mitochondrial fraction immediately after hypoxia-ischemia. At 14-72 h after hypoxia-ischemia glutathione concentrations were decreased both in the mitochondrial fraction and in the tissue. Oxidized glutathione increased selectively in the mitochondrial fraction directly after hypoxia-ischemia. The studies show that glutamate receptor stimulated efflux of the reduced form of glutathione is delayed and likely to be of neuronal origin. The possible neurotoxic and/or modulatory implications are: i) decreased intracellular capacity to inactivate reactive oxygen species, ii) altered intra- and extracellular redox state, which can change the activity of enzymes, transporters and transcription factors, iii) increased extracellular concentrations of the breakdown products cysteine and glycine, both neurotoxic at high concentrations, iv) increased NMDA-receptor activity via reduction of disulfide bonds. The decreased glutathione levels after hypoxia-ischemia may be of pathophysiological relevance by rendering the cells more vulnerable to oxidative stress and/or by affecting redox regulated factors