title: Excitotoxic death induced by released glutamate in depolarized primary cultures of mouse cerebellar granule cells is dependent on GABAA receptors and niflumic acid-sensitive chloride channels.
abstract: Excitotoxic neuronal death has been linked to neurological and neurodegenerative diseases. Several studies have sought to clarify the involvement of Cl(-) channels in neuronal excitotoxicity using either N-methyl-D-aspartic acid (NMDA) or alpha-amino-3-hydroxy-5-methylisoxazole-4-propionate/kainic acid agonists. In this work we induced excitotoxic death in primary cultures of cerebellar granule cells by means of endogenously released glutamate. Excitotoxicity was provoked by exposure to high extracellular K(+) concentrations ([K(+)](o)) for 5 min. Under these conditions, a Ca(2+)-dependent release of glutamate was evoked. When extracellular glutamate concentration rose to between 2 and 4 microM, cell viability was significantly reduced by 30-40%. The NMDA receptor antagonists (MK-801 and D-2-amino-5-phosphonopentanoic acid) prevented cell death. Exposure to high [K(+)](o) produced a (36)Cl(-) influx which was significantly reduced by picrotoxinin. In addition, the GABA(A) receptor antagonists (bicuculline, picrotoxinin and SR 95531) protected cells from high [K(+)](o)-triggered excitotoxicity and reduced extracellular glutamate concentration. The Cl(-) channel blockers niflumic acid and 5-nitro-2-(3-phenylpropylamino)benzoic acid also exerted a neuroprotective effect and reduced extracellular glutamate concentration, even though they did not reduce high [K(+)](o)-induced (36)Cl(-) influx. Primary cultures of cerebellar granule cells also contain a population of GABAergic neurons that released GABA in response to high [K(+)](o). Chronic treatment of primary cultures with kainic acid abolished GABA release and rendered granule cells insensitive to high [K(+)](o) exposure, even though NMDA receptors were functional. Altogether, these results demonstrate that, under conditions of membrane depolarization, low micromolar concentrations of extracellular glutamate might induce an excitotoxic process through both NMDA and GABA(A) receptors and niflumic acid-sensitive Cl(-) channels.
authors.affiliation: Department of Neurochemistry, Institut d'Investigacions Biomediques de Barcelona, Consejo Superior de Investigaciones Cientificas, CSIC, IDIBAPS, Rossello 161, 08036 Barcelona, Spain.
authors.forename: Zoila
authors.initials: Z
authors.lastname: Babot
authors.forename: Rosa
authors.initials: R
authors.lastname: Cristòfol
authors.forename: Cristina
authors.initials: C
authors.lastname: Suñol
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D000831:Animals, Newborn; D001224:Aspartic Acid / Q000493:pharmacokinetics / Q000494:pharmacology; D016923:Cell Death / Q000187:drug effects; D002470:Cell Survival / Q000187:drug effects; D002478:Cells, Cultured; D002531:Cerebellum / Q000166:cytology*; D018118:Chloride Channels / Q000037:antagonists & inhibitors / Q000502:physiology*; D002712:Chlorides / Q000378:metabolism; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D016291:Dizocilpine Maleate / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D018691:Excitatory Amino Acid Antagonists / Q000494:pharmacology; D018756:GABA Antagonists / Q000494:pharmacology; D005968:Glutamate Decarboxylase / Q000378:metabolism; D018698:Glutamic Acid / Q000032:analysis*; D007150:Immunohistochemistry / Q000379:methods; D051379:Mice; D009281:Naphthalenes; D009474:Neurons / Q000187:drug effects*; D009498:Neurotoxins / Q000633:toxicity; D009544:Niflumic Acid / Q000494:pharmacology; D010082:Oxepins; D011189:Potassium Chloride / Q000633:toxicity; D011419:Propidium; D011963:Receptors, GABA-A / Q000502:physiology*; D016194:Receptors, N-Methyl-D-Aspartate / Q000502:physiology; D019782:Riluzole / Q000494:pharmacology; D014316:Tritium / Q000493:pharmacokinetics; D005680:gamma-Aminobutyric Acid / Q000032:analysis
chemical_list: C461941:1-phenyl-1,4-epoxy-1H,4H-naphtho(1,8-de)(1,2)dioxepin; D018118:Chloride Channels; D002712:Chlorides; D016861:Cyclooxygenase Inhibitors; D018691:Excitatory Amino Acid Antagonists; D018756:GABA Antagonists; D009281:Naphthalenes; D009498:Neurotoxins; D010082:Oxepins; D011963:Receptors, GABA-A; D016194:Receptors, N-Methyl-D-Aspartate; C120673:benzyloxyaspartate; D014316:Tritium; D001224:Aspartic Acid; D011419:Propidium; D018698:Glutamic Acid; D009544:Niflumic Acid; D005680:gamma-Aminobutyric Acid; D011189:Potassium Chloride; D016291:Dizocilpine Maleate; D019782:Riluzole; D005968:Glutamate Decarboxylase