Glutamate induces release of glutathione from cultured rat astrocytes – a possible neuroprotective mechanism? Joa ˜o Frade,* Simon Pope, Maike Schmidt,à Ralf Dringen,à Rui Barbosa,* , § Jennifer Pocock,¶ Joa ˜o Laranjinha* , ** and Simon Heales  ,    *Centre for Neurosciences and Cell Biology, University of Coimbra, Coimbra, Portugal  Department of Clinical Biochemistry (Neurometabolic Unit), National Hospital of Neurology and Neurosurgery, London, UK àCenter of Biomolecular Interactions, University of Bremen, Faculty 2 (Biology/Chemistry), Bremen, Germany §Biochemistry Laboratory, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal ¶Department of Neuroinflammation, Institute of Neurology, University College London, London, UK **Laboratory of Instrumental Analysis, University of Coimbra, Coimbra, Portugal   Department of Molecular Neuroscience, Institute of Neurology, University College London, London, UK Glutamate is the major excitatory amino acid of the mammalian brain (Danbolt 2001). It acts through a variety of ionotropic and metabotropic receptors: the first exert their effects via ligand-gated ion channels, whereas the second act through coupling to G proteins and activation of intracellular secondary messengers (Greenamyre and Porter 1994; Mel- drum 2000). Although glutamate is an important excitatory neurotransmitter it can be toxic if its extracellular levels are not tightly controlled. In conditions where release and/or uptake of glutamate are altered, extracellular glutamate can accumulate causing a persistent or excessive activation of glutamate-gated ion channels (excitotoxicity) (Mark et al. 2001; Coyle and Puttfarcken 1993). A number of pathways have been implicated in glutamate excitotoxicity, namely calcium deregulation, loss of membrane potential, mito- chondrial impairment and production of reactive nitrogen/ oxygen species (RNOS), which can lead to oxidative/ nitrosative stress and ultimately cell death (Coyle and Received September 14, 2007; revised manuscript received December 1, 2007; accepted December 19, 2007. Address correspondence and reprint requests to Simon Pope, Department of Clinical Biochemistry (Neurometabolic Unit), National Hospital of Neurology and Neurosurgery, London WC1N 3BG, UK. E-mail: spope@ion.ucl.ac.uk Abbreviations used: cGT, c-glutamyl transferase; AMPA, a-amino-3- hydroxy-5-methyl-4-isoxazole propionic acid; BSO, buthionine sul- phoxime; DIV, day in vitro; GSH, glutathione; GSx, total glutathione (amount of GSH + twice the amount of GSSG); LDH, lactate dehy- drogenase; MM, minimal medium; NO, nitric oxide; RNOS, reactive nitrogen/oxygen species. Abstract Glutamate is the major excitatory amino acid of the mamma- lian brain but can be toxic to neurones if its extracellular levels are not tightly controlled. Astrocytes have a key role in the protection of neurones from glutamate toxicity, through regu- lation of extracellular glutamate levels via glutamate trans- porters and metabolic and antioxidant support. In this study, we report that cultures of rat astrocytes incubated with high extracellular glutamate (5 mM) exhibit a twofold increase in the extracellular concentration of the tripeptide antioxidant glutathione (GSH) over 4 h. Incubation with glutamate did not result in an increased release of lactate dehydrogenase, indicating that the rise in GSH was not because of membrane damage and leakage of intracellular pools. Glutamate-induced increase in extracellular GSH was also independent of de novo GSH synthesis, activation of NMDA and non-NMDA glutamate receptors or inhibition of extracellular GSH break- down. Dose–response curves indicate that GSH release from rat astrocytes is significantly stimulated even at 0.1 mM glu- tamate. The ability of astrocytes to increase GSH release in the presence of extracellular glutamate could be an important neuroprotective mechanism enabling neurones to maintain levels of the key antioxidant, GSH, under conditions of glu- tamate toxicity. Keywords: antioxidant, astrocytes, cell culture, glutamate, glutathione, oxidative stress. J. Neurochem. (2008) 105, 1144–1152. d JOURNAL OF NEUROCHEMISTRY | 2008 | 105 | 1144–1152 doi: 10.1111/j.1471-4159.2008.05216.x 1144 Journal Compilation Ó 2008 International Society for Neurochemistry, J. Neurochem. (2008) 105, 1144–1152 Ó 2008 The Authors