Uncoupling of Endothelial Nitric Oxidase Synthase by Hypochlorous Acid

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 (≥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 p67phox and p47phox of NAD(P)H oxidase and the phosphorylation of atypical protein kinase C-ζ. Further, genetic or pharmacological inhibition of either NAD(P)H oxidase–derived O2·− or PKC-ζ or NOS abolished the effects of HOCl on eNOS dimers. Consistently, HOCl increased both O2·− and ONOO− and eNOS dimer...