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(Arteriosclerosis, Thrombosis, and Vascular Biology. 2006;26:2688.)
© 2006 American Heart Association, Inc.

Atherosclerosis and Lipoproteins

Uncoupling of Endothelial Nitric Oxidase Synthase by Hypochlorous Acid

Role of NAD(P)H Oxidase–Derived Superoxide and Peroxynitrite

Jian Xu; Zhonglin Xie; Richard Reece; David Pimental; Ming-Hui Zou

From the Section of Endocrinology and Diabetes, Department of Medicine (J.X., Z.X., R.R., M.-H.Z.), University of Oklahoma Health Sciences Center, Oklahoma City; and the Division of Cardiology, Department of Medicine (D.P.), Boston University School of Medicine, Boston, Mass.

Correspondence to Ming-Hui Zou, MD, PhD, Division of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, BSEB 325, 941 Stanton L. Young Blvd, Oklahoma City, OK 73104. E-mail ming-hui-zou{at}ouhsc.edu

Objective— The aim of the present study is to determine whether hypochlorous acid (HOCl), the major oxidant of leukocyte-derived myeloperoxidase (MPO), oxidizes the zinc-thiolate center of endothelial nitric oxide synthase (eNOS) and uncouples the enzyme.

Methods and Results— Exposure of purified recombinant eNOS to HOCl (100 µmol/L) released zinc and disrupted the enzyme-active eNOS dimers. In parallel with increased detections of both O₂^·– and ONOO^–, clinically relevant concentrations of HOCl disrupted eNOS dimers in cultured human umbilical vein endothelial cells (HUVEC) at concentration 10- to 100-fold lower than those required for recombinant eNOS. In HUVEC, HOCl increased the translocation of both p67^phox and p47^phox of NAD(P)H oxidase and the phosphorylation of atypical protein kinase C-. Further, genetic or pharmacological inhibition of either NAD(P)H oxidase–derived O₂^·– or PKC- or NOS abolished the effects of HOCl on eNOS dimers. Consistently, HOCl increased both O₂^·– and ONOO^– and eNOS dimer oxidation in isolated mouse aortas from C57BL/6 but less in those of gp91^phox knock-out mice. Finally, in human carotid atherosclerotic arteries, eNOS predominantly existed as monomers in parallel with increased staining of both MPO and 3-nitrotyrosine.

Conclusions— We conclude that HOCl uncouples eNOS by ONOO^– generated from PKC-–dependent NAD(P)H oxidase.

Endothelial nitric oxide synthase (eNOS) uncoupling is observed in cardiovascular diseases, but its mechanisms remain elusive. Here we reported that hypochlorous acid activated PKC-–dependent NAD(P)H oxidase and generated kindling radicals (O₂^·–) and oxidants (ONOO^–), which further oxidizes the zinc-thiolate center, dissociates the enzyme-active dimers, and uncouples the enzyme.

Key Words: hypochlorite • endothelial dysfunction • NAD(P)H oxidase • peroxynitrite

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