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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2009;29:739.)
© 2009 American Heart Association, Inc.

Cell Biology/Signaling

Bradykinin-Induced Dilation of Human Coronary Arterioles Requires NADPH Oxidase–Derived Reactive Oxygen Species

Brandon T. Larsen; Aaron H. Bubolz; Suelhem A. Mendoza; Kirkwood A. Pritchard, Jr; David D. Gutterman

From the Cardiovascular Center (B.T.L., A.H.B., S.A.M., K.A.P., D.D.G.), the Departments of Surgery (K.A.P.) and Medicine (D.D.G.), Medical College of Wisconsin, and Veterans Administration Medical Center (D.D.G.), Milwaukee, Wisconsin.

Correspondence to David D. Gutterman, MD, Senior Associate Dean for Research, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail dgutt{at}mcw.edu

Objective— Hydrogen peroxide (H₂O₂) is an endothelium-derived hyperpolarizing factor in human coronary arterioles (HCAs). H₂O₂ mediates bradykinin (BK)-induced vasodilation and reduces bioavailability of epoxyeicosatrienoic acids (EETs); however, the cellular and enzymatic source of H₂O₂ is unknown.

Methods and Results— NADPH oxidase expression was determined by immunohistochemistry. Superoxide and H₂O₂ production was assayed in HCAs and human coronary artery endothelial cells (HCAECs) using dihydroethidium and dichlorodihydrofluorescein histofluorescence, respectively. Superoxide was quantified by HPLC separation of dihydroethidium products. Diameter changes of HCAs were measured by videomicroscopy. NADPH oxidase subunits Nox1, Nox2, Nox4, p22, p47, and p67 were each expressed in HCA endothelium. In HCAs or HCAECs incubated with dihydroethidium and dichlorodihydrofluorescein, BK induced superoxide and H₂O₂ formation, which was inhibited by gp91ds-tat or apocynin but not by gp91scram-tat or rotenone. HPLC analysis confirmed that BK specifically induced superoxide production. Gp91ds-tat reduced vasodilation to BK but not to papaverine. 14,15-EEZE (an EET antagonist) further reduced the residual dilation to BK in the presence of gp91ds-tat, but had no effect in the presence of gp91scram-tat, suggesting that NADPH oxidase–derived ROS modulate EET bioavailability.

Conclusion— We conclude that endothelial NADPH oxidase is a functionally relevant source of H₂O₂ that mediates agonist-induced dilation in the human heart.

H₂O₂ mediates bradykinin-induced vasodilation and reduces bioavailability of epoxyeicosatrienoic acids in human coronary arterioles, but the source of H₂O₂ was previously unknown. We identified NADPH oxidase as a functionally relevant source of H₂O₂ in this vascular bed. This enzyme complex may therefore modulate human coronary vascular resistance and myocardial perfusion.

Key Words: oxidant stress • endothelium • coronary circulation • neurohumoral control of circulation • hypertension–basic studies