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.