RESUMO
Cartilage was isolated from pig articular joints, and the production of reactive oxygen species (ROS) by chondrocytes embedded within the cartilage was assessed by two methods: the reduction of nitro blue tetrazolium and by the use of diaminobenzidine in the presence of manganese ions. Little constitutive generation of ROS was seen, but it could be detected after the addition of the calcium ionophore ionomycin. Further, the response seen was extremely heterogeneous; some cells showed a far greater release of ROS than others. Cells arranged in the columnar arrays of the deep zone were the most active, while those furthest from the cartilage/bone interface (i.e. nearer to the outer face of the cartilage) were unresponsive. Chondrocytes cultured in alginate beads also showed a similar heterogeneity in their response, suggesting that the isolation of these cells and the measurement of ROS production in a population is not representative of the true situation.
Assuntos
Cartilagem Articular/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Animais , Técnicas de Cultura de Células , Condrócitos/metabolismo , SuínosRESUMO
The immortalized human chondrocyte cell line C-20/A4 has the ability to produce superoxide constitutively at low levels of 5.4 x 10(-2) nmol/min/10(6) cells (S.E.M. = +/-0.5, n = 30) and at raised levels upon stimulation with ionomycin and phorbol 12-myristate 13-acetate. Priming and anti-priming effects of interleukin (IL)-1 beta and IL-4, respectively, are also demonstrated. Reverse transcriptase polymerase chain reaction (RT-PCR) amplification using oligonucleotide primers to components of the NADPH oxidase enzyme complex showed mRNA expression of p22-phox, p40-phox and p47-phox. Western blot analysis using polyclonal antisera indicated the presence of the p47-phox p67-phox polypeptide components. These results show that the C-20/A4 cells contain an NADPH oxidase-like complex, similar to that found in other cell types, which produces superoxide anions.
Assuntos
Cartilagem/química , Cartilagem/citologia , NADPH Oxidases/análise , NADPH Oxidases/genética , RNA Mensageiro/análise , Sequência de Bases , Western Blotting , Carcinógenos/farmacologia , Cartilagem/metabolismo , Linhagem Celular , DNA Complementar/análise , DNA Complementar/química , DNA Complementar/genética , Eletroforese em Gel de Poliacrilamida , Regulação Enzimológica da Expressão Gênica , Humanos , Interleucina-1/farmacologia , Interleucina-4/farmacologia , Ionomicina/farmacologia , Ionóforos/farmacologia , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/química , RNA Mensageiro/genética , Espécies Reativas de Oxigênio/metabolismo , Superóxidos/metabolismo , Acetato de Tetradecanoilforbol/farmacologiaRESUMO
Porcine articular chondrocytes have the capacity to release superoxide in response to the addition of the calcium ionophore ionomycin in a concentration-dependent manner. This activity was not stimulated by the addition of fMetLeuPhe or the kinase activator phorbol myristate acetate (PMA). However, this release of superoxide was inhibited by iodonium diphenyl (IDP), suggesting the involvement of NADPH oxidase. Reverse transcriptase polymerase chain reaction (RT-PCR) using oligonucleotides designed against the known sequences for the human phagocyte NADPH oxidase showed the expression of p22-phox, p40-phox, and p47-phox mRNA, while Western blot analysis of chondrocyte extracts using polyclonal antisera raised against the human phagocyte NADPH oxidase suggested the presence of the p67-phox polypeptide. These results suggest that porcine articular chondrocytes can release reactive oxygen species using a NADPH oxidase-like complex.
