Uncoupling of endothelial nitric oxidase synthase by hypochlorous acid: role of NAD(P)H oxidase-derived superoxide and peroxynitrite.
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.


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.
Xu
Jian
J
Section of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK 73104, USA.
Xie
Zhonglin
Z
Reece
Richard
R
Pimental
David
D
Zou
Ming-Hui
MH
D000818:Animals; D050197:Atherosclerosis / Q000378:metabolism / Q000473:pathology; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects / Q000378:metabolism / Q000473:pathology; D005260:Female; D006801:Humans; D006997:Hypochlorous Acid / Q000494:pharmacology*; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases / Q000235:genetics / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism; D052250:Nitric Oxide Synthase Type III / Q000235:genetics / Q000378:metabolism*; D016877:Oxidants / Q000494:pharmacology*; D010100:Oxygen / Q000378:metabolism; D009195:Peroxidase / Q000235:genetics / Q000378:metabolism; D030421:Peroxynitrous Acid / Q000378:metabolism*; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D014443:Tyrosine / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology*
D008562:Membrane Glycoproteins; D016877:Oxidants; D010750:Phosphoproteins; D014475:Uncoupling Agents; C079789:neutrophil cytosol factor 67K; D013481:Superoxides; D030421:Peroxynitrous Acid; D009569:Nitric Oxide; C002744:3-nitrotyrosine; D014443:Tyrosine; D006997:Hypochlorous Acid; D009195:Peroxidase; D052250:Nitric Oxide Synthase Type III; C488842:Cybb protein, mouse; D000074662:NADPH Oxidase 2; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D010100:Oxygen