Electron spin resonance detection of hydrogen peroxide as an endothelium-derived hyperpolarizing factor in porcine coronary microvessels

Objective— Endothelium-derived hyperpolarizing factor (EDHF) plays an important role in modulating vascular tone, especially in microvessels, although its nature has yet to be elucidated. This study was designed to examine whether hydrogen peroxide (H 2 O 2 ) is an EDHF in porcine coronary microvessels with use of an electron spin resonance (ESR) method to directly detect H 2 O 2 production from the endothelium. Methods and Results— Isometric tension and membrane-potential recordings demonstrated that bradykinin and substance P caused EDHF-mediated relaxations and hyperpolarizations of porcine coronary microvessels in the presence of indomethacin and N ω -nitro-l-arginine. The contribution of H 2 O 2 to the EDHF-mediated responses was demonstrated by the inhibitory effect of catalase and by the relaxing and hyperpolarizing effects of exogenous H 2 O 2 . Endothelial production of H 2 O 2 was quantified in bradykinin- or substance P–stimulated intact blood vessels by ESR spectroscopy. Tiron, a superoxide scavenger that facilitates H 2 O 2 formation, enhanced bradykinin-induced production of H 2 O 2 , as well as the EDHF-mediated relaxations and hyperpolarizations. By contrast, cytochrome P-450 inhibitors (sulfaphenazole or 17-octadecynoic acid) or a gap junction inhibitor (18α-glycyrrhetinic acid) failed to inhibit the EDHF-mediated relaxations. Involvement of endothelium-derived K + was not evident in experiments with ouabain plus Ba 2+ or exogenous K + . Conclusion— These results provide ESR evidence that H 2 O 2 is an EDHF in porcine coronary microvessels.