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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Identification of the nature of EDHF in mouse small mesenteric arteries....
Identification of the nature of EDHF in mouse small mesenteric arteries. (a) Catalase (1250 U/ml) markedly inhibited the endothelium-dependent relaxation to ACh in control mice (after the blockade of cyclooxygenase and eNOS) (left, n = 5) and in eNOS-KO mice (after the blockade of cyclooxygenase) (right, n = 4). AP < 0.01. (b) Catalase also inhibited the ACh-induced endothelium-dependent hyperpolarization in small mesenteric arteries of control mice (left, n = 5), whereas it did not affect the levcromakalim-induced (10 μM) hyperpolarization (right, n = 3). AP < 0.01 vs. control. (c) H2O2, when exogenously applied, caused endothelium-independent relaxation (left, n = 5) as well as hyperpolarization (right, n = 4) in small mesenteric arteries of control mice without endothelium. AP < 0.01 vs. control, BP < 0.05 vs. resting membrane potentials. Experiments were performed in the presence of indomethacin (10 μM) and L-NNA (100 μM) (b and c).