Ethanol induces glutamate secretion by Ca 2+ mobilization and ROS generation in rat hippocampal astrocytes Miguel Salazar, Jose ´ A. Pariente, Gine ´s M. Salido, Antonio Gonza ´lez * Department of Physiology (Cell Physiology Research Group), University of Extremadura, Avenida Universidad s/n, E-10071, Ca ´ceres, Spain Received 29 May 2007; received in revised form 30 October 2007; accepted 7 November 2007 Available online 17 November 2007 Abstract In this study we have investigated the effect of ethanol on [Ca 2+ ] c by microfluorimetry and glutamate secretion using an enzyme-linked system, in rat hippocampal astrocytes in culture. Our results show that ethanol (1–200 mM) evoked a dose-dependent increase in glutamate secretion. 50 mM ethanol, a concentration within the range of blood alcohol levels in intoxicated humans, induced a release of Ca 2+ from intracellular stores in the form of oscillations. Ca 2+ -mobilizing effect of ethanol was not prevented by preincubation of cells in the presence of 2 mM of the antioxidant dithiothreitol. Ethanol-evoked glutamate secretion was reduced when extracellular Ca 2+ was omitted (medium containing 0.5 mM EGTA) and following preincubation of astrocytes in the presence of the intracellular Ca 2+ chelator 1,2-bis-(o-aminophenoxy)-ethane-N,N,N 0 ,N 0 -tetraacetic acid tetraacetoxy-methyl ester (10 mM). Preincubation of astrocytes in the presence of 2 mM of the antioxidant dithiothreitol significantly reduced ethanol-evoked glutamate secretion. Finally, preincubation of astrocytes in the presence of bafilomycin (50 nM) significantly reduced ethanol- induced neurotransmitter release, indicating that exocytosis is involved in glutamate secretion. In conclusion, our results suggest that ethanol mobilizes Ca 2+ from intracellular stores, and stimulates a Ca 2+ -dependent glutamate secretion, probably involving reactive oxygen species production, and therefore creating a situation potentially leading to neurotoxicity in the hippocampus. # 2007 Elsevier Ltd. All rights reserved. Keywords: Ethanol; Calcium; Astrocytes; Hippocampus; Glutamate secretion; Imaging Astrocytes represent the major brain cell population in mammalian central nervous system (CNS). This cell type was initially considered to form a substrate with supportive and metabolic roles in the CNS. However, they provide more than a merely structural and trophic support for the neurons and, in addition, are able to regulate neuronal activity and synaptic neurotransmission (Jourdain et al., 2007; Tritsch and Bergles, 2007). Astrocytes respond with an increase in intracellular free Ca 2+ concentration ([Ca 2+ ] c ) after stimulation with a variety of neurotransmitters, neuromodulators and hormones, and play an important role in the developmental guidance of migrating neurons, in the regulation of neurotransmitter and ion levels, in the nutrition of neurons and in the production of neurotrophic factors (Eysseric et al., 2000). Furthermore, the secretion of neuroactive agents by astrocytes serves to communicate with other surrounding astrocytes and neurons with which they are intimately associated (Malarkey and Parpura, 2007; Perea and Araque, 2007). Glutamate is the principal excitatory neurotransmitter and its interaction with specific membrane receptors is responsible for many neurological functions, including cognition, memory and sensation. It has been postulated that excessive activation of glutamate receptors may mediate neuronal injury or death (Emerit et al., 2004; Hazell, 2007). The CNS is a major target for alcohol and its consumption has long been associated with brain damage. Alcohol may have several targets in astrocytes and other cell types, impairing for example cell growth and differentiation, interfering with the stimulatory effect of trophic factors or altering the expression of www.elsevier.com/locate/neuint Available online at www.sciencedirect.com Neurochemistry International 52 (2008) 1061–1067 Abbreviations: BAPTA-AM, 1,2-bis-(o-aminophenoxy)-ethane-N,N,N 0 ,N 0 - tetraacetic acid tetraacetoxy-methyl ester; CNS, central nervous system; DMEM, Dulbecco’s modified Eagle’s medium; [Ca 2+ ] c , intracellular free Ca 2+ concentration; DTT, dithiothreitol; EAAT, excitatory amino acid trans- porters; EGTA, ethylene glycol-bis(2-aminoethylether)-N,N,N 0 ,N 0 -tetraacetic acid; ER, endoplasmic reticulum; GFAP, glial fibrillary acidic protein; HBSS, Hank’s Balanced Salts; ROS, reactive oxygen species; SERCA, sarcoendo- plasmic reticulum Ca 2+ -ATPase; TPS, thapsigargin. * Corresponding author. Tel.: +34 927 257000x7155; fax: +34 927 257110. E-mail address: agmateos@unex.es (A. Gonza ´lez). 0197-0186/$ – see front matter # 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuint.2007.11.001