Hydrogen peroxide induces vasorelaxation by enhancing 4-aminopyridine-sensitive Kv currents through S-glutathionylation

Hydrogen peroxide (H 2 O 2 ) is an endothelium-derived hyperpolarizing factor. Since opposing vasoactive effects have been reported for H 2 O 2 depending on the vascular bed and experimental conditions, this study was performed to assess whether H 2 O 2 acts as a vasodilator in the rat mesenteric artery and, if so, to determine the underlying mechanisms. H 2 O 2 elicited concentration-dependent relaxation in mesenteric arteries precontracted with norepinephrine. The vasodilatory effect of H 2 O 2 was reversed by treatment with dithiothreitol. H 2 O 2 -elicited vasodilation was significantly reduced by blocking 4-aminopyridine (4-AP)-sensitive Kv channels, but it was resistant to blockers of big-conductance Ca 2+ -activated K + channels and inward rectifier K + channels. A patch-clamp study in mesenteric arterial smooth muscle cells (MASMCs) showed that H 2 O 2 increased Kv currents in a concentration-dependent manner. H 2 O 2 speeded up Kv channel activation and shifted steady state activation to hyperpolarizing potentials. Similar channel activation was seen with oxidized glutathione (GSSG). The H 2 O 2 -mediated channel activation was prevented by glutathione reductase. Consistent with S -glutathionylation, streptavidin pull-down assays with biotinylated glutathione ethyl ester showed incorporation of glutathione (GSH) in the Kv channel proteins in the presence of H 2 O 2 . Interestingly, conditions of increased oxidative stress within MASMCs impaired the capacity of H 2 O 2 to stimulate Kv channels. Not only was the H 2 O 2 stimulatory effect much weaker, but the inhibitory effect of H 2 O 2 was unmasked. These data suggest that H 2 O 2 activates 4-AP-sensitive Kv channels, possibly through S -glutathionylation, which elicits smooth muscle relaxation in rat mesenteric arteries. Furthermore, our results support the idea that the basal redox status of MASMCs determines the response of Kv currents to H 2 O 2 .