Jian Xu1, Zhonglin Xie, Richard Reece, David Pimental, Ming-Hui Zou. 1. Section of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK 73104, USA.
Abstract
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 (> or = 100 micromol/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-zeta. Further, genetic or pharmacological inhibition of either NAD(P)H oxidase-derived O2*- or PKC-zeta 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-zeta-dependent NAD(P)H oxidase.
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 (> or = 100 micromol/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-zeta. Further, genetic or pharmacological inhibition of either NAD(P)H oxidase-derived O2*- or PKC-zeta 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-zeta-dependent NAD(P)H oxidase.
Authors: Richard A Cohen; Colin E Murdoch; Yosuke Watanabe; Victoria M Bolotina; Alicia M Evangelista; Dagmar J Haeussler; Melissa D Smith; Yu Mei; XiaoYong Tong; Jingyan Han; Jessica B Behring; Markus M Bachschmid; Reiko Matsui Journal: J Cardiovasc Pharmacol Date: 2016-06 Impact factor: 3.105