RESUMO
The endocannabinoid system (ECS) regulates several physiological processes in the Central Nervous System, including the modulation of neuronal excitability via activation of cannabinoid receptors (CBr). Both glutaric acid (GA) and quinolinic acid (QUIN) are endogenous metabolites that, under pathological conditions, recruit common toxic mechanisms. A synergistic effect between them has already been demonstrated, supporting potential implications for glutaric acidemia type I (GA I). Here we investigated the possible involvement of a cannabinoid component in the toxic model exerted by QUINâ¯+â¯GA in rat cortical slices and primary neuronal cell cultures. The effects of the CB1 receptor agonist anandamide (AEA), and the fatty acid amide hydrolase inhibitor URB597, were tested on cell viability in cortical brain slices and primary neuronal cultures exposed to QUIN, GA, or QUINâ¯+â¯GA. As a pre-treatment to the QUINâ¯+â¯GA condition, AEA prevented the loss of cell viability in both preparations. URB597 only protected in a moderate manner the cultured neuronal cells against the QUINâ¯+â¯GA-induced damage. The use of the CB1 receptor reverse agonist AM251 in both biological preparations prevented partially the protective effects exerted by AEA, thus suggesting a partial role of CB1 receptors in this toxic model. AEA also prevented the cell damage and apoptotic death induced by the synergic model in cell cultures. Altogether, these findings demonstrate a modulatory role of the ECS on the synergic toxic actions exerted by QUINâ¯+â¯GA, thus providing key information for the understanding of the pathophysiological events occurring in GA I.
Assuntos
Ácidos Araquidônicos/farmacologia , Córtex Cerebral/efeitos dos fármacos , Endocanabinoides/farmacologia , Glutaratos/toxicidade , Neurônios/efeitos dos fármacos , Alcamidas Poli-Insaturadas/farmacologia , Ácido Quinolínico/toxicidade , Animais , Benzamidas/farmacologia , Agonistas de Receptores de Canabinoides/farmacologia , Carbamatos/farmacologia , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Sinergismo Farmacológico , Endocanabinoides/metabolismo , Feminino , Masculino , Neurônios/metabolismo , Piperidinas/farmacologia , Gravidez , Pirazóis/farmacologia , Ratos , Ratos Endogâmicos WF , Receptores de Canabinoides/metabolismoRESUMO
It has been shown that synergistic toxic effects of quinolinic acid (QUIN) and glutaric acid (GA), both in isolated nerve endings and in vivo conditions, suggest the contribution of these metabolites to neurodegeneration. However, this synergism still requires a detailed characterization of the mechanisms involved in cell damage during its occurrence. In this study, the effects of subtoxic concentrations of QUIN and/or GA were tested in neuronal cultures, co-cultures (neuronal cells + astrocytes), and mixed cultures (neuronal cells + astrocytes + microglia) from rat cortex and striatum. The exposure of different cortical and striatal cell cultures to QUIN + GA resulted in cell death and stimulated different markers of oxidative stress, including reactive oxygen species (ROS) formation; changes in the activity of antioxidant enzymes such as superoxide dismutase, catalase, and glutathione peroxidase; and depletion of endogenous antioxidants such as -SH groups and glutathione. The co-incubation of neuronal cultures with QUIN + GA plus the N-methyl-D-aspartate antagonist MK-801 prevented cell death but not ROS formation, whereas the antioxidant melatonin reduced both parameters. Our results demonstrated that QUIN and GA can create synergistic scenarios, inducing toxic effects on some parameters of cell viability via the stimulation of oxidative damage. Therefore, it is likely that oxidative stress may play a major causative role in the synergistic actions exerted by QUIN + GA in a variety of cell culture conditions involving the interaction of different neural types.
Assuntos
Glutaratos/toxicidade , Modelos Biológicos , Neurônios/metabolismo , Estresse Oxidativo , Ácido Quinolínico/toxicidade , Animais , Antioxidantes/metabolismo , Catalase/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Córtex Cerebral/patologia , Técnicas de Cocultura , Maleato de Dizocilpina/farmacologia , Feminino , Gliose/metabolismo , Gliose/patologia , Glutaratos/administração & dosagem , Glutationa/metabolismo , Melatonina/farmacologia , Neostriado/patologia , Neuritos/efeitos dos fármacos , Neuritos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/patologia , Estresse Oxidativo/efeitos dos fármacos , Ácido Quinolínico/administração & dosagem , Ratos Wistar , Espécies Reativas de Oxigênio/metabolismo , Superóxido Dismutase/metabolismoRESUMO
The brain of children affected by organic acidemias develop acute neurodegeneration linked to accumulation of endogenous toxic metabolites like glutaric (GA), 3-hydroxyglutaric (3-OHGA), methylmalonic (MMA) and propionic (PA) acids. Excitotoxic and oxidative events are involved in the toxic patterns elicited by these organic acids, although their single actions cannot explain the extent of brain damage observed in organic acidemias. The characterization of co-adjuvant factors involved in the magnification of early toxic processes evoked by these metabolites is essential to infer their actions in the human brain. Alterations in the kynurenine pathway (KP) - a metabolic route devoted to degrade tryptophan to form NAD(+) - produce increased levels of the excitotoxic metabolite quinolinic acid (QUIN), which has been involved in neurodegenerative disorders. Herein we investigated the effects of subtoxic concentrations of GA, 3-OHGA, MMA and PA, either alone or in combination with QUIN, on early toxic endpoints in rat brain synaptosomes. To establish specific mechanisms, we pre-incubated synaptosomes with different protective agents, including the endogenous N-methyl-d-aspartate (NMDA) receptor antagonist kynurenic acid (KA), the antioxidant S-allylcysteine (SAC) and the nitric oxide synthase (NOS) inhibitor nitro-l-arginine methyl ester (l-NAME). While the incubation of synaptosomes with toxic metabolites at subtoxic concentrations produced no effects, their co-incubation (QUIN+GA, +3-OHGA, +MMA or +PA) decreased the mitochondrial function and increased reactive oxygen species (ROS) formation and lipid peroxidation. For all cases, this effect was partially prevented by KA and l-NAME, and completely avoided by SAC. These findings suggest that early damaging events elicited by organic acids involved in metabolic acidemias can be magnified by toxic synergism with QUIN, and this process is mostly mediated by oxidative stress, and in a lesser extent by excitotoxicity and nitrosative stress. Therefore, QUIN can be hypothesized to contribute to the pathophysiology of brain degeneration in children with metabolic acidemias.