Avaliação da síntese de óxido nítrico em defeitos da cadeia respiratória

• Ano de defesa: 2006
• Autor(a) principal: Gamba, Juliana [UNIFESP]
• Tipo de documento: Dissertação
• Tipo de acesso: Acesso aberto
• Idioma: por
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Óxido nítrico; Mitocôndria; Fosforilação oxidativa; DNA mitocondrial
• Link de acesso: http://repositorio.unifesp.br/handle/11600/9048

A mitochondrial production of nitric oxide (NO) hás been recently identified. Besides being a free radical, NO also participates in several physiological processes. NO itself is relatively non-reactive and exhibits an inhibitory effect on the respiratory chain, specially, cytochrome c oxidase (COX). NO can also be converted into a number of more reactive derivatives, which can lead to oxidative damage of several mitochondrial components, such as mitochondrial DNA (mtDNA), mitochondrial proteins and membranes. The existence of a mitochondrial nitric oxide synthase suggests that NO has an important regulatory role on mitochondrial function. The aim of this study was to evaluate nitric oxide synthesis in cell lines carrying different respiratory chain defects and to verify if there was protein nitration, which would indicate the presence of oxidative damage by nitrogen radicals. NO synthesis was verified through nitrite quantitation and using a fluorescent NO indicator, DAF-FM diacetate. We analyzed cybrid cell lines carrying mtDNA mutations 3243 A>G, 8344 A>G and mtDNA deletion, and fibroblasts from patients with confirmed and suspected mitochondrial disease. Control samples were: the cell line originated from osteosarcoma 143B and fibroblasts from a normal individual. We observed increased nitrite production in all cybrid cell lines and a decrement in all fibroblasts from patients. All cell lines showed intracellular fluorescence with DAF-FM diacetate. The quantitative analysis showed a significantly increase of the fluorescence obtained for the 8344 A>G cell line and a decrease in most of the fibroblasts. Protein nitration was verified through Western blotting method using the anti-nitrotyrosine antibody, in cybrid cells, fibroblasts and skeletal muscle. There was no detection of proteins containing nitrotyrosines, suggesting that these samples were not nitrated. Our results suggest a variation of nitric oxide synthesis in certain respiratory chain defects, without indication of oxidative damage as a consequence of nitrogen radicals. This variation can suggest NO involvement on mitochondrial regulatory process of respiratory chain defects. The knowledge of this regulatory process may have implications on treatment strategies of mitochondrial diseases and neurodegenerative diseases with mitochondrial involvement.