The effects of high glucose on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations of isolated rat mesenteric artery and the possible involvement of reactive oxygen species in these responses were investigated, After precontraction with phenylephrine (3 x 10 (8) - 10(-7) M), acetylcholine (10(-8)-3 x 10(-6) M) and A 23187 (10(-8)-3 x 10 (6) M), a calcium ionophore, induced concentration-dependent relaxations in the presence of N-W-nitro-L-arginine methyl ester (L-NAME) (10(-4) M) and indomethacin (10 (5) M). These relaxations were ;abolished in the presence of charybdotoxin (2 x 10(-7) M) plus aparnin ( 10 (7) M) and were assumed to be mediated by EDHF. Effects of elevated glucose were examined by incubating the arterial rings for 6 h in Krebs-Henseleit solution containing 212 MM glucose. Under these conditions relaxation to acetylcholine was significantly attenuated but was unchanged when the tissues were incubated for 6 It in solution containing 11.1 mM mannitol used as hyperosmotic control. Addition of superoxide dismutase (SOD) (75 U/ml) and combination of SOD with catalase (200 U/ml) during incubation with high glucose significantly preserved the impairment of EDHF-mediated relaxations to acetylcholine. A 23187-induced endothelium-dependent relaxation was not affected by high glucose, Similarly, relaxations to pinacidil (10(-10) 10(-5) M) and to sodium nitroprusside (SNP) (10(-10)-3 x 10(-7) M) were also unchanged in the rings exposed to high glucose. These results suggest that in rat mesenteric arteries exposed to elevated glucose receptor-dependent EDHF-mediated relaxations (acetylcholine-induced) are impaired whereas receptor-independent ones (A 23187-induced) and responses to smooth muscle relaxants that exert their effects through mechanisms independent of endothelium are unaffected. Our findings lead us to propose that reactive oxygen species like superoxide (-O-2,) and hydrogen peroxide (H2O2,) do seem to play a role in the impairment of EDHF-mediated relaxations in the presence of elevated glucose, (c) 2005 Elsevier Inc. All rights reserved.