C007 Redox-sensitive activation of endothelial nitric oxide synthase by catechins: role of hydroxyl moieties

Abstract

Objectives Several rich sources of polyphenols have been shown to strongly increase the endothelial formation of nitric oxide (NO), a potent vasoprotecting factor, via the redox-sensitive activation of the PI3-kinase/Akt pathway leading to the phosphorylation of endothelial NO synthase. The purpose of the present study was to investigate the molecular mechanisms underlying the stimulatory effect of catechins on the endothelial formation of NO using different catechins (flavan-3-ols). Methods Vascular reactivity studies were performed using porcine coronary artery rings, which were suspended in organ chambers for the measurement of changes in isometric tension. All experiments were performed in the presence of indomethacin (an inhibitor of cyclooxygenases), and the combination of apamin and charybdotoxin (two inhibitors of endothelium-derived hyperpolarizing factor-mediated effects) to assess only the NO component of the relaxation. Cultures of porcine coronary artery endothelial cells (P1) were used to determine the phosphorylation level of Akt and endothelial NO synthase by Western blot analysis. Both natural and synthetic catechins were evaluated. Results (-)-Epigallocatechin-3-O-gallate (EGCg) induced potent endothelium-dependent relaxations in porcine coronary artery rings. The EGCg-induced relaxation was inhibited by MnTMPyP (a membrane permeant analogue of superoxide dismutase, SOD) whereas extracellular SOD had no effect, indicating a major role of the intracellular formation of ROS. Relaxations to EGCg were minimally affected by rotenone (an inhibitory of the mitochondrial respiratory chain), sulphenazol (an inhibitor of cytochrome P450), apocynin (an inhibitor of NADPH oxidase) or allopurinol (an inhibitor of xanthine oxidase). The replacement of all hydroxyl groups of EGCg by O-methyl groups resulted in the total loss of the relaxing activity whereas partial replacement decreased the relaxing activity. Conclusions EGCg caused endothelium-dependent relaxations of coronary arteries via the redox-sensitive formation of NO in endothelial cells. The stimulatory effect does not involve major intracellular sources of ROS including the mitochondrial respiratory chain, xanthine oxidase, NADPH oxidase and cytochrome P450 but is critically dependent on the presence of hydroxyl groups possibly leading to auto-oxidation of the polyphenol.