Xu Jian, Xie Zhonglin, Reece Richard, Pimental David, Zou Ming-Hui
Section of Endocrinology and Diabetes, Department of Medicine, University of Oklahoma Health Sciences Center, 941 Stanton L. Young Blvd, Oklahoma City, OK 73104, USA.
Arterioscler Thromb Vasc Biol. 2006 Dec;26(12):2688-95. doi: 10.1161/01.ATV.0000249394.94588.82. Epub 2006 Oct 5.
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.
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.
We conclude that HOCl uncouples eNOS by ONOO- generated from PKC-zeta-dependent NAD(P)H oxidase.
本研究旨在确定白细胞衍生的髓过氧化物酶（MPO）的主要氧化剂次氯酸（HOCl）是否会氧化内皮型一氧化氮合酶（eNOS）的锌硫醇盐中心并使该酶解偶联。
将纯化的重组eNOS暴露于HOCl（≥100 μmol/L）会释放锌并破坏具有酶活性的eNOS二聚体。与超氧阴离子（O2*-）和过氧亚硝酸盐（ONOO-）检测增加同时发生的是，临床相关浓度的HOCl在培养的人脐静脉内皮细胞（HUVEC）中破坏eNOS二聚体，其浓度比重组eNOS所需浓度低10至100倍。在HUVEC中，HOCl增加了NAD（P）H氧化酶的p67（phox）和p47（phox）的转位以及非典型蛋白激酶C-zeta的磷酸化。此外，对NAD（P）H氧化酶衍生的O2*-或PKC-zeta或NOS的基因或药理学抑制消除了HOCl对eNOS二聚体的影响。同样，HOCl增加了来自C57BL/6的分离小鼠主动脉中的O2*-和ONOO-以及eNOS二聚体氧化，但在gp91（phox）基因敲除小鼠的主动脉中增加较少。最后，在人颈动脉粥样硬化动脉中，eNOS主要以单体形式存在，同时MPO和3-硝基酪氨酸的染色增加。
我们得出结论，HOCl通过PKC-zeta依赖性NAD（P）H氧化酶产生的ONOO-使eNOS解偶联。
