Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709; Instituto de Inmunología, Escuela de Medicina, Universidad Austral de Chile, Valdivia 511-0566, Chile.
Department of Pharmacology and Physiology, University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07101-1709.
J Biol Chem. 2011 Sep 2;286(35):30409-30414. doi: 10.1074/jbc.M111.234294. Epub 2011 Jul 13.
Endothelial NOS (eNOS)-derived NO is a key factor in regulating microvascular permeability. We demonstrated previously that eNOS translocation from the plasma membrane to the cytosol is required for hyperpermeability. Herein, we tested the hypothesis that eNOS activation in the cytosol is necessary for agonist-induced hyperpermeability. To study the fundamental properties of endothelial cell monolayer permeability, we generated ECV-304 cells that stably express cDNA constructs targeting eNOS to the cytosol or plasma membrane. eNOS-transfected ECV-304 cells recapitulate the eNOS translocation and permeability properties of postcapillary venular endothelial cells (Sánchez, F. A., Rana, R., Kim, D. D., Iwahashi, T., Zheng, R., Lal, B. K., Gordon, D. M., Meininger, C. J., and Durán, W. N. (2009) Proc. Natl. Acad. Sci. U.S.A. 106, 6849-6853). We used platelet-activating factor (PAF) as a proinflammatory agonist. PAF activated eNOS by increasing phosphorylation of Ser-1177 and inducing dephosphorylation of Thr-495, increasing NO production, and elevating permeability to FITC-dextran 70 in monolayers of cells expressing wild-type and cytosolic eNOS. PAF failed to increase permeability to FITC-dextran 70 in monolayers of cells transfected with eNOS targeted to the plasma membrane. Interestingly, this occurred despite eNOS Ser-1177 phosphorylation and production of comparable amounts of NO. Our results demonstrate that the presence of eNOS in the cytosol is necessary for PAF-induced hyperpermeability. Our data provide new insights into the dynamics of eNOS and eNOS-derived NO in the process of inflammation.
内皮型一氧化氮合酶（eNOS）衍生的 NO 是调节微血管通透性的关键因素。我们之前已经证明，eNOS 从质膜向细胞质易位是通透性增加所必需的。在此，我们测试了这样一个假设，即细胞质中 eNOS 的激活对于激动剂诱导的通透性增加是必需的。为了研究内皮细胞单层通透性的基本特性，我们生成了稳定表达靶向质膜或细胞质中 eNOS 的 cDNA 构建体的 ECV-304 细胞。eNOS 转染的 ECV-304 细胞再现了后微静脉内皮细胞（Sánchez，F. A.，Rana，R.，Kim，D. D.，Iwahashi，T.，Zheng，R.，Lal，B. K.，Gordon，D. M.，Meininger，C. J.，and Durán，W. N.（2009）Proc. Natl. Acad. Sci. U.S.A. 106, 6849-6853）中 eNOS 易位和通透性特性。我们使用血小板激活因子（PAF）作为促炎激动剂。PAF 通过增加 Ser-1177 的磷酸化和诱导 Thr-495 的去磷酸化来激活 eNOS，增加 NO 的产生，并增加表达野生型和细胞质 eNOS 的细胞单层对 FITC-葡聚糖 70 的通透性。PAF 未能增加靶向质膜的 eNOS 转染细胞单层对 FITC-葡聚糖 70 的通透性。有趣的是，尽管 eNOS Ser-1177 磷酸化和产生相当数量的 NO，但仍发生这种情况。我们的结果表明，细胞质中存在 eNOS 是 PAF 诱导的通透性增加所必需的。我们的数据为炎症过程中 eNOS 和 eNOS 衍生的 NO 的动态提供了新的见解。
