RESUMO
Brain ischemia is accompanied by lowering of intra- and extracellular pH. Stroke often leads to irreversible damage of synaptic transmission by unknown mechanism. We investigated an influence of lowering of pH(i) and pH(o) on free radical formation in synaptosomes. Three models of acidosis were used: (1) pH(o) 6.0 corresponding to pH(i) decrease down to 6.04; (2) pH(o) 7.0 corresponding to the lowering of pH(i) down to 6.92: (3) 1 mM amiloride corresponding to pH(i) decrease down to 6.65. We have shown that both types of extracellular acidification, but not intracellular acidification, increase 2',7'-dichlorodihydrofluorescein diacetate fluorescence that reflects free radical formation. These three treatments induce the rise of the dihydroethidium fluorescence that reports synthesis of superoxide anion. However, the impact of amiloride on superoxide anion synthesis was less than that induced by moderate extracellular acidification. Superoxide anion synthesis at pH(o) 7.0 was almost completely eliminated by mitochondrial uncoupler carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone. Furthermore, using fluorescent dyes JC-1 and rhodamine-123, we confirmed that pH(o) lowering, but not intracellular acidification, led to depolarization of intrasynaptosomal mitochondria. We have shown that pH(o) but not pH(i) lowering led to oxidative stress in neuronal presynaptic endings that might underlie the long-term irreversible changing in synaptic transmission.
Assuntos
Encéfalo/metabolismo , Espaço Extracelular/metabolismo , Espaço Intracelular/metabolismo , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Superóxidos/metabolismo , Sinaptossomos/metabolismo , Acidose/metabolismo , Amilorida/farmacologia , Animais , Corantes Fluorescentes , Concentração de Íons de Hidrogênio , Masculino , Mitocôndrias/metabolismo , Estresse Oxidativo , Ratos , Ratos Wistar , Desacopladores/farmacologia , Imagens com Corantes Sensíveis à VoltagemRESUMO
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.