Instituto de Inmunología, Universidad Austral de Chile, Los Laureles s/n, 511-0566, Valdivia, Chile.
Circ Res. 2012 Aug 17;111(5):553-63. doi: 10.1161/CIRCRESAHA.112.274548. Epub 2012 Jul 9.
Endothelial adherens junction proteins constitute an important element in the control of microvascular permeability. Platelet-activating factor (PAF) increases permeability to macromolecules via translocation of endothelial nitric oxide synthase (eNOS) to cytosol and stimulation of eNOS-derived nitric oxide signaling cascade. The mechanisms by which nitric oxide signaling regulates permeability at adherens junctions are still incompletely understood.
We explored the hypothesis that PAF stimulates hyperpermeability via S-nitrosation (SNO) of adherens junction proteins.
We measured PAF-stimulated SNO of β-catenin and p120-catenin (p120) in 3 cell lines: ECV-eNOSGFP, EAhy926 (derived from human umbilical vein), and postcapillary venular endothelial cells (derived from bovine heart endothelium) and in the mouse cremaster muscle in vivo. SNO correlated with diminished abundance of β-catenin and p120 at the adherens junction and with hyperpermeability. Tumor necrosis factor-α increased nitric oxide production and caused similar increase in SNO as PAF. To ascertain the importance of eNOS subcellular location in this process, we used ECV-304 cells transfected with cytosolic eNOS (GFPeNOSG2A) and plasma membrane eNOS (GFPeNOSCAAX). PAF induced SNO of β-catenin and p120 and significantly diminished association between these proteins in cells with cytosolic eNOS but not in cells wherein eNOS is anchored to the cell membrane. Inhibitors of nitric oxide production and of SNO blocked PAF-induced SNO and hyperpermeability, whereas inhibition of the cGMP pathway had no effect. Mass spectrometry analysis of purified p120 identified cysteine 579 as the main S-nitrosated residue in the region that putatively interacts with vascular endothelial-cadherin.
Our results demonstrate that agonist-induced SNO contributes to junctional membrane protein changes that enhance endothelial permeability.
内皮细胞黏附连接蛋白是控制微血管通透性的重要组成部分。血小板激活因子（PAF）通过将内皮型一氧化氮合酶（eNOS）易位到细胞质并刺激 eNOS 衍生的一氧化氮信号级联反应来增加大分子的通透性。一氧化氮信号调节黏附连接通透性的机制尚不完全清楚。
我们探讨了 PAF 通过黏附连接蛋白的 S-亚硝基化（SNO）刺激超渗透性的假说。
我们测量了 3 种细胞系（ECV-eNOSGFP、EAhy926（源自人脐静脉）和后微静脉内皮细胞（源自牛心内皮））和体内小鼠提睾肌中 PAF 刺激的β-连环蛋白和 p120-连环蛋白（p120）的 SNO。SNO 与黏附连接处β-连环蛋白和 p120 的丰度减少以及通透性增加相关。肿瘤坏死因子-α增加了一氧化氮的产生，并引起与 PAF 相似的 SNO 增加。为了确定 eNOS 亚细胞定位在该过程中的重要性，我们使用转染了细胞质 eNOS（GFPeNOSG2A）和质膜 eNOS（GFPeNOSCAAX）的 ECV-304 细胞。PAF 诱导了β-连环蛋白和 p120 的 SNO，并显著减少了细胞质 eNOS 细胞中这些蛋白之间的结合，但在质膜锚定 eNOS 的细胞中没有。一氧化氮产生和 SNO 的抑制剂阻断了 PAF 诱导的 SNO 和通透性增加，而 cGMP 途径的抑制剂则没有影响。对纯化的 p120 进行质谱分析鉴定出半胱氨酸 579 为该区域中与血管内皮钙黏蛋白相互作用的主要 S-亚硝基化残基。
我们的结果表明，激动剂诱导的 SNO 有助于增强内皮通透性的连接膜蛋白变化。
