RESUMO
Folic acid (folate) is a vitamin of the B-complex group crucial for neurological function. Considering that excitotoxicity and cell death induced by glutamate are involved in many disorders, the potential protective effect of folic acid on glutamate-induced cell damage in rat hippocampal slices and the possible intracellular signaling pathway involved in such effect were investigated. The treatment of hippocampal slices with folic acid (100 µM) significantly abrogated glutamate (1 mM)-induced reduction of cell viability measured by MTT reduction assay and inhibited glutamate-induced D-[3H]-aspartate release. To investigate the putative intracellular signaling pathways implicated in the protective effect of folic acid, we used a PI3K inhibitor, LY294002, which abolished the protective effects of folic acid against glutamate-induced cell damage and D-[3H] aspartate release. Moreover, hippocampal slices incubated with folic acid alone for 30 min presented increased phosphorylation of GSK-3ß at Ser9, indicating an inhibition of the activity of this enzyme. Furthermore, folic acid in the presence of glutamate insult in hippocampal slices maintained for an additional period of 6 h in fresh culture medium without glutamate and/or folic acid induced phosphorylation of GSK-3ß and ß-catenin expression. In addition, glutamate-treated hippocampal slices showed increased iNOS expression that was reversed by folic acid. In conclusion, the results of this study show that the protective effect of folic acid against glutamate-induced excitotoxicity may involve the modulation of PI3K/GSK-3ß/ß-catenin pathway and iNOS inhibition.
Assuntos
Ácido Fólico/farmacologia , Ácido Glutâmico/farmacologia , Glicogênio Sintase Quinase 3 beta/metabolismo , Hipocampo/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , Óxido Nítrico Sintase Tipo II/metabolismo , Animais , Sobrevivência Celular/efeitos dos fármacos , Hipocampo/metabolismo , Masculino , Fosfatidilinositol 3-Quinases/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Wistar , Transdução de Sinais/efeitos dos fármacosRESUMO
This study was designed to determine whether calreticulin (CRT), a chaperone protein, is present in in vitro-matured (IVM) pig oocytes and to study its potential role in the block to polyspermy. Western blot analysis, using an anti-CRT antibody, of oocyte lysate showed an immunoreactive band of â¼60 âkDa. Simultaneous labeling of IVM oocytes with anti-CRT antibody and peanut agglutinin lectin (PNA lectin, a porcine cortical granules (CG)-specific binding lectin) revealed localization of CRT in the subplasmalemmal region with a 27.7% colocalization with PNA staining. After IVF, PNA labeling was not observed and anti-CRT labeling decreased significantly in zygotes and disappeared in two-cell embryos. Western blot analysis of oocyte exudate obtained from zona pellucida (ZP)-free oocytes activated with calcium ionophore confirmed the presence of a band that reacted with an anti-CRT antibody. Anti-CRT antibody and PNA labeling were not observed in activated oocytes despite being detectable in non-activated oocytes. The presence of CRT in vesicles located under the oolemma was demonstrated using immunogold cytochemistry at the ultrastructural level. To study the role of CRT in fertilization, ZP-enclosed and ZP-free oocytes were incubated with exogenous CRT and then inseminated. Whereas ZP-free oocytes showed fewer penetrating sperm and lower polyspermy rates than untreated oocytes, the opposite effect was observed in ZP-enclosed oocytes. In conclusion, CRT is confined to subplasmalemmal vesicles partially overlapping with CG contents. Its exocytosis after the oocyte activation seems to participate in the membrane block to polyspermy in pigs but is not involved in the ZP block.
Assuntos
Calreticulina/fisiologia , Membrana Celular/fisiologia , Grânulos Citoplasmáticos/metabolismo , Interações Espermatozoide-Óvulo , Suínos , Animais , Calreticulina/metabolismo , Células Cultivadas , Técnicas de Cultura Embrionária , Exocitose , Fertilização , Fertilização in vitro/veterinária , Técnicas de Maturação in Vitro de Oócitos/veterinária , Masculino , Oócitos/citologia , Oócitos/metabolismo , Interações Espermatozoide-Óvulo/fisiologia , Suínos/metabolismo , Distribuição Tecidual , Zona Pelúcida/metabolismoRESUMO
Preclinical and clinical studies indicate that deficiency in folic acid plays a role in the pathophysiology of depression. Considering that alterations in the signaling pathways that regulate neuroplasticity and cellular survival are implicated in depressive disorders, the present study investigated the involvement of the phosphoinositide 3-kinase (PI3K), glycogen synthase kinase-3 (GSK-3ß), and peroxisome proliferator-activated receptor-γ (PPARγ) in the antidepressant-like effect of folic acid in the forced swimming test (FST). The intracerebroventricular (i.c.v.) pre-treatment of mice with LY294002 (10 nmol/site, a PI3K inhibitor) or GW-9662 (1 µg/site, a PPARγ antagonist) prevented the antidepressant-like effect of folic acid (50 mg/kg, p.o.) in the FST. In addition, the administration of subeffective doses of the selective GSK-3ß inhibitor, AR-A014418 (3 mg/kg, i.p.), a non-selective GSK-3ß inhibitor, lithium chloride (10 mg/kg, p.o) or a PPARγ agonist, rosiglitazone (1 µg/site, i.c.v.) in combination with a subeffective dose of folic acid (10 mg/kg, p.o.) significantly reduced the immobility time in the FST as compared with either drug alone, without altering the locomotor activity. These results indicate that the antidepressant-like effect of folic acid in the FST might be dependent on inhibition of GSK-3ß and activation of PPARγ, reinforcing the notion that these are important targets for antidepressant activity.