title: Glutamate-induced free radical formation in rat brain synaptosomes is not dependent on intrasynaptosomal mitochondria membrane potential.
abstract: Glutamate induces reactive oxygen species formation (ROS) in neurons. Free radicals can potentially be synthesized by NADPH oxidase or mitochondria. The primary source of ROS origin has yet to be identified. In addition, pro-oxidant action of glutamate receptors on neuronal presynaptic terminals is still not characterized. We investigated the influence of glutamate and agonists of its ionotropic receptors on ROS formation detected by fluorescent dye DCFDA in rat brain synaptosomes. Glutamate in concentration 10 and 100μM led to an increase of probe fluorescence pointing to free radical accumulation. This effect was mimicked by 100μM of NMDA or 100μM of kainate. Glutamate-induced ROS formation was sensitive to NMDA inhibitors MK-801 (10μM), NO synthase (NOS) inhibitor l-NAME (100μM) and NADPH oxidase inhibitors DPI (30μM) and not affected by mitochondrial uncoupler CCCP (10μM) and mitochondrial toxins rotenone (10μM)+oligomycin (5μg/ml). We also showed that 100μM of glutamate leads to a decrease of intrasynaptosomal mitochondrial potential monitored by fluorescent dye Rhodamine-123. Hence, the depolarization of intrasynaptosomal mitochondria is not a primary cause of glutamate-induced ROS formation in neuronal presynaptic terminals. Activation of NMDA receptors might be responsible for a certain part of glutamate pro-oxidant action. Most likely, sources of glutamate-induced ROS formation in neuronal presynaptic terminals are NADPH oxidase and NOS activation.
authors.affiliation: Laboratory of Biophysics and Engineering of Cell, Institute of Biophysics and Cell Engineering, Akademicheskaya St., 27, Minsk 220072, Belarus.
authors.forename: Aleksandra V
authors.initials: AV
authors.lastname: Alekseenko
authors.forename: Valeriya V
authors.initials: VV
authors.lastname: Lemeshchenko
authors.forename: Tatyana G
authors.initials: TG
authors.lastname: Pekun
authors.forename: Tatyana V
authors.initials: TV
authors.lastname: Waseem
authors.forename: Sergei V
authors.initials: SV
authors.lastname: Fedorovich
mesh_terms: D000818:Animals; D001921:Brain / Q000187:drug effects / Q000378:metabolism*; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone / Q000494:pharmacology; D005609:Free Radicals / Q000378:metabolism*; D018698:Glutamic Acid / Q000494:pharmacology*; D008297:Male; D053078:Membrane Potential, Mitochondrial / Q000187:drug effects / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D061209:Proton Ionophores / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D017382:Reactive Oxygen Species / Q000378:metabolism; D012402:Rotenone / Q000494:pharmacology; D013574:Synaptosomes / Q000187:drug effects / Q000378:metabolism*; D014475:Uncoupling Agents / Q000494:pharmacology
chemical_list: D005609:Free Radicals; D061209:Proton Ionophores; D017382:Reactive Oxygen Species; D014475:Uncoupling Agents; D012402:Rotenone; D018698:Glutamic Acid; D002258:Carbonyl Cyanide m-Chlorophenyl Hydrazone