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 (H₂O₂) is an EDHF in porcine coronary microvessels with use of an electron spin resonance (ESR) method to directly detect H₂O₂ 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 H₂O₂ to the EDHF-mediated responses was demonstrated by the inhibitory effect of catalase and by the relaxing and hyperpolarizing effects of exogenous H₂O₂. Endothelial production of H₂O₂ was quantified in bradykinin- or substance P-stimulated intact blood vessels by ESR spectroscopy. Tiron, a superoxide scavenger that facilitates H₂O₂ formation, enhanced bradykinin-induced production of H₂O₂, 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 Ba²⁺ or exogenous K⁺. Conclusion - These results provide ESR evidence that H₂O₂ is an EDHF in porcine coronary microvessels.