Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice

The endothelium plays an important role in maintaining vascular homeostasis by synthesizing and releasing several endothelium-derived relaxing factors , such as prostacyclin, nitric oxide (NO), and the previously unidentified endothelium-derived hyperpolarizing factor (EDHF). In this study, we examin ed our hypothesis that hydrogen peroxide (H2O2) derived from endothelial NO synthase (eNOS) is an EDHF. EDHF-mediated relaxation and hyperpolarization in response to acetylcholine (ACh) were markedly attenuated in small mesen teric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vaso dilating and hyperpolarizing responses of vascular smooth muscle per se wer e fairly well preserved, as was the increase in intracellular calcium in en dothelial cells in response to ACh, Antihypertensive treatment with hydrala zine failed to improve the EDHF-mediated relaxation. Catalase, which dismut ates H2O2 to form water and oxygen, inhibited EDHF-mediated relaxation and hyperpolarization, but it did not affect endothelium-independent relaxation following treatment with the K+ channel opener levcromakalim. Exogenous H2 O2 elicited similar relaxation and hyperpolarization in endothelium-strippe d arteries. Finally, laser confocal microscopic examination with peroxide-s ensitive fluorescence dye demonstrated that: the endothelium produced H2O2 upon stimulation by ACh acid that the H2O2 production was markedly reduced in eNOS-KO mice. These results indicate that H2O2 is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.