Outils chimiques et méthodologies d'analyse des radicaux superoxyde et monoxyde d'azote (étude par RPE de la régulation du stress oxydant dans la paroi artérielle)

TOULOUSE3-BU Sciences (315552104) / SudocSudocFranceF

Electron spin resonance detection of oxygen radicals released by UVA-irradiated human fibroblasts

This work reports the electron spin resonance (ESR) detection of oxygenated radicals (OR) released by cultured human fibroblasts after UVA (365 nm) exposure. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as spin trap. After a UVA irradiation of one hour, followed by a latent period of at least 45 min., and an incubation time of 30 min. in a trapping medium containing DMPO, glucose, Na+, K+ and Ca2+ an ESR signal was recorded. By contrast, an ESR signal was produced after only 15 min. incubation when calcium ionophore A23187 was used. Although the ESR signal was characteristic of the hydroxyl adduct DMPO-OH, the use of catalase and superoxide dismutase (SOD) revealed that UVA stimulated fibroblasts released the superoxide anion O$_2^-$ in the medium. SOD, vitamin C and (+)-catechin inhibited the release of superoxide generated by human fibroblasts stimulated with A23187 calcium ionophore at 5 units/ml, 10-5 M and $2\times 10^{-4}$ M, respectively

Superoxide release by confluent endothelial cells, an electron spin resonance (ESR) study

In the present study we used ESR to detect the release of oxygen radicals by endothelial cells stimulated with calcium ionophore A23187. Dimethyl-1-pyrroline-N-oxide (DMPO) was used as a spin trap. Although the observed adduct (DMPO-OH) suggested the presence of the hydroxyl radical, the use of superoxide dismutase and catalase revealed that superoxide anion was released in the medium. Superoxide production was more efficient when the cells were post-confluent for a few days. The release of superoxide was 3-fold greater in growth arrested cells (D6-D9) than in proliferating cells (D0). Although two inhibitors of the mitochondrial respiratory chain carbanyl cyanide m-chlorophenylhydrazone (CCCP), antimycine decreased the ESR signal by 35%, the use of superoxide dismutase (SOD) and tumor necrosis factor (TNF) suggested that the release of O$_2^-$ occurred in the cell membrane. The physiological significance of this extracellular superoxide release by post-confluent cells deserves further study