Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice
Tetsuya Matoba, … , Hideo Kanaide, Akira Takeshita
Tetsuya Matoba, … , Hideo Kanaide, Akira Takeshita
Published December 15, 2000
Citation Information: J Clin Invest. 2000;106(12):1521-1530. https://doi.org/10.1172/JCI10506.
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Article

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice

Abstract

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 examined 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 mesenteric arteries from eNOS knockout (eNOS-KO) mice. In the eNOS-KO mice, vasodilating and hyperpolarizing responses of vascular smooth muscle per se were fairly well preserved, as was the increase in intracellular calcium in endothelial cells in response to ACh. Antihypertensive treatment with hydralazine failed to improve the EDHF-mediated relaxation. Catalase, which dismutates 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 H2O2 elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H2O2 upon stimulation by ACh and 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.

Authors

Tetsuya Matoba, Hiroaki Shimokawa, Mikio Nakashima, Yoji Hirakawa, Yasushi Mukai, Katsuya Hirano, Hideo Kanaide, Akira Takeshita

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ACh-induced production of H2O2 by the endothelium detected as an increas...
ACh-induced production of H2O2 by the endothelium detected as an increase in fluorescence intensity in the DCF-loaded endothelial cells in small mesenteric arteries of mice. Fluorescence images of the endothelium in small mesenteric artery of a control mouse were obtained before (a) and 3 minutes after (b, d, and e) the application of ACh (10 μM). (c) Fluorescence image of smooth muscle layer of a control mouse obtained at the same visual field as a and b. The direction and depth of the smooth muscle layer were apparently different from those of the endothelial layer. (d) ACh-induced increase in fluorescence intensity was almost abolished by pretreatment with catalase (1250 U/ml) in a control mouse. (e) ACh-induced increase in the fluorescence intensity was markedly reduced in an eNOS-KO mouse. All experiments were performed in the presence of indomethacin (10 μM) and L-NNA (100 μM).