RESUMO
Atrial natriuretic peptide (ANP) can regulate aqueous humor production in the eye and has recently been suggested to play some functional roles in the retina. It has also been reported that ANP increases tyrosine hydroxylase (TH) mRNA levels and intracellular dopamine levels in PC12 cells. The effect of ANP on TH levels and the role of ANP in retinal excitotoxicity remain unknown. In this study, we investigated the effects of ANP on TH expression and dopamine levels in rat retina after intravitreal injection of NMDA. Immunohistochemistry localized natriuretic peptide receptor-A (NPRA) in the ganglion cell layer (GCL), the inner nuclear layer (INL) and the outer nuclear layer (ONL) in the rat retina. Quantitative real-time PCR and Western blot analysis showed a dramatic reduction in retinal TH levels 5 days after NMDA injection, while ANP, at a concentration of 10(-4) M, ameliorated this reduction in TH mRNA and TH protein levels. High-performance liquid chromatography (HPLC) analysis showed that NMDA reduced dopamine levels in the retina, and that ANP attenuated this reduction. Moreover, morphological analysis showed that ANP ameliorated NMDA-induced neurotoxicity through NPRA. The ameliorative effect of ANP was inhibited by a dopamine D(1) receptor antagonist. These results suggest that ANP may have a neuroprotective effect through possible involvement of dopamine induction.
Assuntos
Fator Natriurético Atrial/administração & dosagem , Agonistas de Aminoácidos Excitatórios/toxicidade , N-Metilaspartato/toxicidade , Fármacos Neuroprotetores/administração & dosagem , Síndromes Neurotóxicas/etiologia , Degeneração Retiniana/prevenção & controle , Análise de Variância , Animais , Anticorpos/farmacologia , Fator Natriurético Atrial/imunologia , Benzazepinas/farmacologia , Western Blotting/métodos , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Domperidona/farmacologia , Dopamina/metabolismo , Antagonistas de Dopamina/farmacologia , Relação Dose-Resposta a Droga , Interações Medicamentosas , Eletroquímica/métodos , Imuno-Histoquímica/métodos , Espaço Intracelular/efeitos dos fármacos , Masculino , Fármacos Neuroprotetores/imunologia , Síndromes Neurotóxicas/complicações , Síndromes Neurotóxicas/patologia , Células PC12 , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Degeneração Retiniana/etiologia , Degeneração Retiniana/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Tirosina 3-Mono-Oxigenase/metabolismoRESUMO
Transcription factors of the nuclear factor-kappa B (NF-kappaB)/Rel family may be involved in neuronal cell death or survival. We examined the role of NF-kappaB p65 in N-methyl-D-aspartate (NMDA)-induced neurotoxicity in the rat retina. Western blot analysis showed that elevated levels of retinal NF-kappaB p65 protein at days 1 and 5 after intravitreal NMDA injection. Immunohistochemistry localized increased NF-kappaB p65 immunoreactivity in the ganglion cell layer (GCL) and the inner nuclear layer (INL) after NMDA injection especially in retinal ganglion cells (RGCs), displaced amacrine cells, and amacrine cells. Concomitant with the early increase in NF-kappaB p65 protein levels, there was an increase in NF-kappaB DNA binding activity after NMDA injection as shown by electrophoretic mobility shift assay (EMSA). These increases in NF-kappaB p65 protein levels and NF-kappaB DNA binding activity were totally abolished by simultaneous injection of NF-kappaB p65 antisense oligodeoxynucleotide (AS ODN). A partial but significant protective effect on the inner retina was noted when the AS ODN was given together with NMDA as shown by morphological analysis, morphometry of cells in the GCL and morphometry of inner plexiform layer thickness as well as quantitative real-time PCR of Thy-1 mRNA levels. These results suggest that activated NF-kappaB p65 may participate in NMDA-induced retinal neuronal cell death and that inhibition of NF-kappaB activation such as the use of AS ODN may be a viable neuroprotective strategy for protective RGCs and other inner retinal neurons.
Assuntos
Agonistas de Aminoácidos Excitatórios/toxicidade , N-Metilaspartato/toxicidade , NF-kappa B/genética , Oligodesoxirribonucleotídeos Antissenso/farmacologia , Doenças Retinianas/patologia , Animais , Morte Celular/efeitos dos fármacos , Masculino , Microinjeções , NF-kappa B/metabolismo , Fármacos Neuroprotetores/farmacologia , Ratos , Ratos Wistar , Retina/efeitos dos fármacos , Retina/patologia , Doenças Retinianas/induzido quimicamente , Doenças Retinianas/terapia , Fator de Transcrição RelARESUMO
N-methyl-D-aspartate (NMDA) may affect dopaminergic cells, which contain tyrosine hydroxylase (TH), the rate-limiting enzyme in dopamine synthesis. To clarify the involvement of TH in the neuroprotective effects of nitric oxide (NO), we investigated whether NMDA alters TH mRNA and TH protein levels and whether NO inhibits NMDA-induced changes in the rat retina. Dopamine levels in the retina were measured by high-performance liquid chromatography (HPLC). Reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR showed that intravitreal injection of NMDA caused a significant reduction in TH mRNA levels in the retina. Similarly, Western blot analysis showed that NMDA decreased the production of TH protein. These reductions in TH mRNA and TH protein levels were attenuated by concomitant injection of NOC 18, an NO donor. HPLC analysis showed that NMDA reduced dopamine levels in the retina and that NO attenuated this reduction. Furthermore, morphological analysis showed that NO prevents NMDA-induced neurotoxicity through dopamine D(1) receptors. These results suggest that the neuroprotective effect of NO may be associated with the induction of TH expression and increased levels of dopamine.
Assuntos
Sequestradores de Radicais Livres/uso terapêutico , Regulação da Expressão Gênica/efeitos dos fármacos , Síndromes Neurotóxicas/prevenção & controle , Óxido Nítrico/uso terapêutico , Retina/efeitos dos fármacos , Tirosina 3-Mono-Oxigenase/metabolismo , Animais , Benzazepinas/farmacologia , Western Blotting , Contagem de Células , Cromatografia Líquida de Alta Pressão/métodos , Domperidona/farmacologia , Dopamina/análise , Antagonistas de Dopamina/farmacologia , Interações Medicamentosas , Agonistas de Aminoácidos Excitatórios/toxicidade , Masculino , N-Metilaspartato/toxicidade , Síndromes Neurotóxicas/etiologia , Doadores de Óxido Nítrico/uso terapêutico , Compostos Nitrosos/uso terapêutico , Ratos , Ratos Wistar , Retina/metabolismo , Retina/patologia , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Fatores de Tempo , Tirosina 3-Mono-Oxigenase/genéticaRESUMO
RhoA, a key protein involved in cytoskeleton regulation modulating neurogenesis and neural plasticity, has been implicated in a variety of cellular functions including the modulation of N-methyl-D-aspartate (NMDA) receptor activity. We examined its possible involvement in NMDA-induced excitotoxicity in the retina, and evaluated the neuroprotective effect of fasudil, a Rho kinase inhibitor, in this model of neurotoxicity. RhoA protein levels in NMDA-treated retinas were assessed by Western blot analysis and localized by immunohistochemistry. Fasudil (10(-6)-10(-4) M together with 4 x 10(-2) M NMDA) was given intravitreally and its effect was evaluated by counting the number of cells in the ganglion cell layer (GCL), measuring the thickness of the inner plexiform layer (IPL), and measuring retinal Thy-1 mRNA levels at 5 days after injection. Western blot analysis showed a transient increase in the level of retinal RhoA and ROCKII proteins at 1 day after NMDA injection, and that this increment was significantly prevented by simultaneous injection of fasudil. Immunohistochemistry showed that NMDA induced a substantial increase in RhoA immunoreactivity in the GCL and the IPL. Fasudil injection reduced cell loss in the GCL and the reduction in IPL thickness after NMDA injection. The reduction in Thy-1 mRNA levels by NMDA was also significantly attenuated by concomitant injection of fasudil. These results suggest that RhoA and ROCKII are upregulated and may be involved in NMDA-induced retinal neurotoxicity, and that fasudil is neuroprotective against glutamate-related excitotoxicity.
Assuntos
1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/análogos & derivados , 1-(5-Isoquinolinasulfonil)-2-Metilpiperazina/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Retina/enzimologia , Degeneração Retiniana/enzimologia , Proteína rhoA de Ligação ao GTP/metabolismo , Animais , Contagem de Células , Modelos Animais de Doenças , Interações Medicamentosas/fisiologia , Inibidores Enzimáticos/farmacologia , Ácido Glutâmico/metabolismo , Peptídeos e Proteínas de Sinalização Intracelular , Masculino , N-Metilaspartato/farmacologia , Degeneração Neural/tratamento farmacológico , Degeneração Neural/enzimologia , Degeneração Neural/genética , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Neurotoxinas/metabolismo , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/efeitos dos fármacos , RNA Mensageiro/efeitos dos fármacos , RNA Mensageiro/metabolismo , Ratos , Ratos Wistar , Receptores de N-Metil-D-Aspartato/efeitos dos fármacos , Retina/efeitos dos fármacos , Retina/fisiopatologia , Degeneração Retiniana/tratamento farmacológico , Degeneração Retiniana/genética , Células Ganglionares da Retina/citologia , Células Ganglionares da Retina/efeitos dos fármacos , Células Ganglionares da Retina/enzimologia , Antígenos Thy-1/genética , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genética , Quinases Associadas a rhoRESUMO
PURPOSE: To investigate the effects of antiglaucoma eyedrops and vehicles on the proliferation of human corneal epithelial cells. MATERIAL AND METHODS: Seven eyedrops[prostaglandin F2 alpha analogs(2), beta blockers(40), topical carbonic anhydrase inhibitor(1)], and six of the eyedrop vehicles, excluding that of Xalatan, were used. Anti-glaucoma eyedrops and vehicles were serially diluted 2-fold with culture medium(10-2,560 fold). The mixture was added to human corneal epithelial cells and incubated for 48 hrs. Cell proliferation was measured by commercial assay kit. Dye-reagents were added to the wells and incubated for 1 h at 37 degrees C. Optical density were measured at 490 nm. The dilution rate for 50% inhibition was calculated as the dilution rate of drugs or vehicles necessary to produce 50% inhibition of cell proliferation. RESULT: All drugs completely inhibited cell proliferation when the dilution rate was low. At 40-fold dilution, Trusopt and Timoptol showed a significant decrease in cell growth inhibition. On the other hand, Rescula showed almost 100% inhibition at 160-fold dilution. Above 640-fold dilution, the inhibition rate of all drugs became 50% or less and there was no significant difference between drugs. Vehicles also inhibited cell growth. The dilution rates for growth inhibition by vehicles were different from those of drugs. The dilution rate at 50% inhibition of anti-glaucoma eyedrops decreased in the following order: Rescula > Xalatan > Betoptic > Hypadil > Mikelan > Timoptol > Trusopt. The dilution rate for 50% inhibition of vehicles decreased in the following order: Rescula vehicle > Hypadil vehicle > Betoptic vehicle > Mikelan vehicle > Timoptol vehicle > Trusopt vehicle. CONCLUSION: All anti-glaucoma eyedrops inhibited cell proliferation. These effects were stronger in prostaglandin F2 alpha analogs and weakest in Trusopt. Furthermore, the inhibition of cell proliferation was caused also by the vehicle of eyedrops, and the influence of the vehicle varied in each type of eyedrops.
Assuntos
Antagonistas Adrenérgicos beta/farmacologia , Dinoprosta/farmacologia , Epitélio Corneano/efeitos dos fármacos , Glaucoma/tratamento farmacológico , Inibidores do Crescimento/farmacologia , Inibidores da Anidrase Carbônica/farmacologia , Divisão Celular/efeitos dos fármacos , Epitélio Corneano/citologia , Humanos , Soluções Oftálmicas , Sulfonamidas/farmacologia , Tiofenos/farmacologiaRESUMO
The aim of this study was to investigate whether an excitotoxic concentration of N-methyl-D-aspartate (NMDA) increases the expression of the phosphorylated cAMP response element-binding protein (p-CREB) and the DNA-binding activity of the cAMP response element (CRE) in rat retina. Intravitreal injection of NMDA was performed in adult male Wistar rats. p-CREB protein levels in the retina were examined by Western blot analysis. DNA-binding activity of CRE in the retina was evaluated by an electrophoretic mobility gel shift assay (EMSA). We confirmed that NMDA induced the reduction of ganglion cells and the inner plexiform layer of the retina. Western blot analysis showed increases in the expression of p-CREB in the retina 12 and 24 h after intravitreal NMDA injection and dimer formation of CREB in the nuclear fraction at 24 h. Increases of DNA-binding activity were observed in the retina 24 h after NMDA injection by EMSA. Our results suggest that phosphorylation of CREB may involved in NMDA-induced excitotoxicity in rat retina. Phosphorylated CREB seems to be the active form and the one that is transcribed.