ORIGINAL PAPER H 2 O 2 Mobilizes Ca 2+ from Agonist- and Thapsigargin-sensitive and Insensitive Intracellular Stores and Stimulates Glutamate Secretion in Rat Hippocampal Astrocytes Antonio Gonza ´lez Æ Marı ´a P. Granados Æ Jose ´ A. Pariente Æ Gine ´s M. Salido Accepted: 20 April 2006 / Published online: 23 June 2006 Ó Springer Science+Business Media, Inc. 2006 Abstract The effect of hydrogen peroxide (H 2 O 2 ) on cytosolic free calcium concentration ([Ca 2+ ] c ) as well as its effect on glutamate secretion in rat hippocampal astrocytes have been the aim of the present research. Our results show that 100 lMH 2 O 2 induces an increase in [Ca 2+ ] c , that remains at an elevated level while the oxidant is present in the perfusion medium, due to its release from intracellular stores as it was observed in the absence of extracellular Ca 2+ , followed by a significant increase in glutamate secretion. Ca 2+ -mobilization in response to the oxidant could only be reduced by thapsigargin plus FCCP, indi- cating that the Ca 2+ -mobilizable pool by H 2 O 2 includes both endoplasmic reticulum and mitochondria. We con- clude that ROS in hippocampal astrocytes might contribute to an elevation of resting [Ca 2+ ] c which, in turn, could lead to a maintained secretion of the excitatory neurotransmitter glutamate, which has been considered a situation poten- tially leading to neurotoxicity in the hippocampus. Keywords Reactive oxygen species Æ Calcium Æ Fluorescence Æ Astrocytes Æ Hippocampus Æ Glutamate secretion Introduction Astrocytes form the largest population of non-excitable cells in mammalian central nervous system (CNS) and are intimately associated with neurons. This cell type was ini- tially considered to form a substrate with supportive and metabolic roles in the CNS. However, nowadays it is known that astrocytes provide more than a merely structural and trophic support for the neurons and, in addition, are able to regulate neuronal activity and synaptic neurotransmission [1–3]. They respond with an increase in intracellular free Ca 2+ concentration ([Ca 2+ ] c ) after stimulation with a variety of neurotransmitters, neuromodulators and hormones. Many of these stimuli mediate their effect through the activation of membrane metabotropic receptors which, coupled via G-proteins to phospholipase C, lead to the generation of the intracellular messenger inositol 1,4,5- trisphosphate (IP 3 ) that, in turn, releases Ca 2+ from the endoplasmic reticulum (ER). Astrocytes can as well increase [Ca 2+ ] c via ionotropic receptors, which are ligand- gated cation channels which mediate Ca 2+ entry into the cell [4]. Rise in [Ca 2+ ] c regulates intracellular events such as metabolic responses, ion transport systems, secretion of neuroactive substances, differentiation and gene expression. Due to its critical importance for the cellular functions, resting [Ca 2+ ] c and the recovery from increases in [Ca 2+ ] c produced by receptor/channel activation is provided by the plasma membrane Ca 2+ -ATPase (PMCA), the electrochemically driven Na + /Ca 2+ exchanger and the sarcoendoplasmic reticulum Ca 2+ -ATPase (SERCA) [5]. Astrocytes contain an elaborate ER that serves as a major source for rapid release of Ca 2+ into the cytosol. Together with the ER another capacious Ca 2+ pool is represented by mitochondria, and it has been proposed that they play an active role in Ca 2+ signaling both in physiological and in A. Gonza ´lez (&) Æ M. P. Granados Æ J. A. Pariente Æ G. M. Salido Department of Physiology, University of Extremadura, Avenida Universidad s/n, E-10071 Ca ´ceres, Spain e-mail: agmateos@unex.es Neurochem Res (2006) 31:741–750 DOI 10.1007/s11064-006-9078-y 123