Coordinate regulation of glutathione metabolism in astrocytes by Nrf2

The tripeptide glutathione (GSH) represents the major brain thiol and is essential for prevention of oxidative stress. Using monochlorobimane to label intracellular GSH in a glutathione S-transferase catalyzed reaction we have examined the kinetics of GSH metabolism including its rate of conjugation, total GSH content, synthesis, and efflux in astrocyte cultures under basal conditions and after induction of antioxidant response element (ARE)-mediated gene expression by the transcription factor Nrf2. In the presence of a cerebral spinal fluid-like salt solution astrocytes could not synthesize detectable levels of GSH. Addition of GSH precursors, cystine, glutamate, and glycine, rapidly restored GSH synthesis. Astrocytes were able to use either glutamate or glutamine as precursors equally for GSH synthesis. Using the small molecule chemical inducer tert-butylhydroqunione (tBHQ) we report that induction of ARE-mediated gene expression is associated with a coordinated increase in GSH content and synthesis rate with little effect on the rate of GSH conjugation or efflux. Consistent with the effect of the inducer, adenovirus-mediated overexpression of the transcription factor Nrf2 that mediates tBHQ's effects also increased GSH content, confirming that GSH metabolism can be regulated by the Nrf2 pathway.