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. 2000 Dec;106(12):1521-30.
doi: 10.1172/JCI10506.

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice

T Matoba  1 , H Shimokawa, M Nakashima, Y Hirakawa, Y Mukai, K Hirano, H Kanaide, A Takeshita
Affiliations

Hydrogen peroxide is an endothelium-derived hyperpolarizing factor in mice

T Matoba et al. J Clin Invest. 2000 Dec.

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 (H(2)O(2)) 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 H(2)O(2) 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 H(2)O(2) elicited similar relaxation and hyperpolarization in endothelium-stripped arteries. Finally, laser confocal microscopic examination with peroxide-sensitive fluorescence dye demonstrated that the endothelium produced H(2)O(2) upon stimulation by ACh and that the H(2)O(2) production was markedly reduced in eNOS-KO mice. These results indicate that H(2)O(2) is an EDHF in mouse small mesenteric arteries and that eNOS is a major source of the reactive oxygen species.

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Figures

Figure 1
Figure 1
Reduced EDHF-mediated endothelium-dependent relaxation and hyperpolarization in eNOS-KO mice. (a) Endothelium-dependent relaxation to ACh of the aorta (left, n = 11) and of small mesenteric arteries (right, n = 9) in control mice. NO plays a primary role in the aorta, whereas EDHF plays a primary role in small mesenteric arteries. AP < 0.01, BP < 0.05. (b) Endothelium-dependent relaxation to ACh of the aorta (left) and of small mesenteric arteries (right, n = 10 each) in eNOS-KO mice. The relaxation was absent in the aorta, whereas in small mesenteric arteries those responses were partially compensated by vasodilator prostaglandins with a marked reduction of EDHF-mediated responses. AP < 0.01, BP < 0.05. (c) Endothelium-dependent hyperpolarization to ACh was also markedly reduced in small mesenteric arteries of eNOS-KO mice (n = 5–6). AP < 0.01, BP < 0.05 vs. control.
Figure 2
Figure 2
Preserved endothelium-independent relaxation and increase in intracellular Ca2+ levels upon stimulation by agonists, and a failure of antihypertensive therapy to improve the reduced EDHF-mediated responses in eNOS-KO mice. (a) Endothelium-independent relaxation in small mesenteric arteries to levcromakalim was preserved (left, n = 6 each) while those to SNP were enhanced (right, n = 5–6) in eNOS-KO mice. AP < 0.01. (b) Direct measurement of the changes in intracellular Ca2+ levels in endothelial cells. ACh (1 nM–10 μM), CPA (3 μM), and ionomycin (25 μM) caused an increase, while EGTA (2 mM) caused a decrease in intracellular Ca2+ levels as shown by F348/F380 (left). The increase in intracellular Ca2+ levels, when normalized to ionomycin-induced maximal F348/F380, were comparable between the two strains (right, n = 5 each). (c) Although the antihypertensive therapy with hydralazine for 6 weeks normalized blood pressure in eNOS-KO mice (left), the treatment failed to improve the reduced EDHF-mediated responses (KCl-sensitive component after the blockade of cyclooxygenase with 10 μM indomethacin and the blockade of eNOS with 100 μM L-NNA) in eNOS-KO mice (right, n = 8–10). AP < 0.01, BP < 0.05 vs. control. sBP, systolic blood pressure.
Figure 3
Figure 3
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).
Figure 4
Figure 4
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).
Figure 5
Figure 5
ACh-induced production of H2O2 by the endothelium. DCF-dependent fluorescence intensities (determined 3 minutes after application of ACh) are expressed as fold increase from that under basal conditions. (a) ACh (10 μM) caused a significant increase in fluorescence intensity. Pretreatment with indomethacin (10 μM), L-NNA (100 μM), or both did not affect the responses (n = 5–7). NS, not significant. (b) Inhibitory effect of catalase on the ACh-induced increase in fluorescence intensity (n = 4–7). (c) The ACh-induced increase in fluorescence intensity was significantly reduced in eNOS-KO mice (n = 4). Experiments were performed in the presence of indomethacin (10 μM) and L-NNA (100 μM). (d) Inhibitory effect of calmodulin antagonists calmidazolium (10 μM) and fendiline (100 μM) on the ACh-induced increase in fluorescence intensity (n = 5–6). Indo, indomethacin (10 μM); Cat, catalase (1250 U/ml). AP < 0.05 by one-way ANOVA; BP < 0.05 vs. basal condition by paired Student’s t test.
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