Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability.
Internalization of eNOS and NO delivery to subcellular targets determine agonist-induced hyperpermeability.
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eNOS 的内化和 NO 向亚细胞靶标的传递决定了激动剂诱导的通透性过高。
DOI：
10.1073/pnas.0812694106
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发表时间：
2009
影响因子：
11.1
通讯作者：
Duran,WalterN
中科院分区：
文献类型：
--
作者：
Sanchez,FabiolaA;Rana,Roshniben;Kim,DavidD;Iwahashi,Toru;Zheng,Ruifang;Lal,BrajeshK;Gordon,DonnaM;Meininger,CynthiaJ;Duran,WalterN
内皮型一氧化氮合酶(ENOS)调节微血管通透性的分子机制尚不清楚。激动剂诱导的内化可能在这一过程中起作用。我们在这里证明了eNOS的内化需要将NO传递到亚细胞位置，从而增加内皮单层对大分子的通透性。利用动力蛋白-2(Dyn2K44A)和小窝蛋白-1(Av1Y14F)的显性-负性突变体，我们发现将eNOS锚定在质膜上可以抑制由血小板激活因子(PAF)、血管内皮生长因子(VEGF)诱导的ECV-CD8eNOSGFP(ECV-304转基因细胞)和毛细血管后静脉内皮细胞(CVEC)的高通透性。我们还观察到，将空泡eNOS锚定在质膜上会使Ser-1177处的eNOS磷酸化与NO的产生脱钩。这种解离是以突变和细胞依赖的方式发生的。PAF可诱导转染dyn2K44A的ECV-CD8eNOSGFP和CVEC中Ser-1177-eNOS的磷酸化，而对转CAV1Y14F的CVEC中Ser-1177处eNOS的去磷酸化。有趣的是，dyn2K44A消除了NO的产生，而CAV1Y14F则导致CVEC中NO的产生减少。转CAV1Y14F基因的血管内皮细胞在结合质膜的小窝中产生NO，并且不能引起通透性的增加。我们的研究表明，eNOS内化是激动剂诱导的高通透性所必需的，并提示eNOS通过质膜上的磷酸化及其内吞作用激活eNOS的机制需要将NO输送到亚细胞靶点，从而导致高通透性。
The molecular mechanisms of endothelial nitric oxide synthase (eNOS) regulation of microvascular permeability remain unresolved. Agonist-induced internalization may have a role in this process. We demonstrate here that internalization of eNOS is required to deliver NO to subcellular locations to increase endothelial monolayer permeability to macromolecules. Using dominant-negative mutants of dynamin-2 (dyn2K44A) and caveolin-1 (cav1Y14F), we show that anchoring eNOS-containing caveolae to plasma membrane inhibits hyperpermeability induced by platelet-activating factor (PAF), VEGF in ECV-CD8eNOSGFP (ECV-304 transfected cells) and postcapillary venular endothelial cells (CVEC). We also observed that anchoring caveolar eNOS to the plasma membrane uncouples eNOS phosphorylation at Ser-1177 from NO production. This dissociation occurred in a mutant- and cell-dependent way. PAF induced Ser-1177-eNOS phosphorylation in ECV-CD8eNOSGFP and CVEC transfected with dyn2K44A, but it dephosphorylated eNOS at Ser-1177 in CVEC transfected with cav1Y14F. Interestingly, dyn2K44A eliminated NO production, whereas cav1Y14F caused reduction in NO production in CVEC. NO production by cav1Y14F-transfected CVEC occurred in caveolae bound to the plasma membrane, and was ineffective in causing an increase in permeability. Our study demonstrates that eNOS internalization is required for agonist-induced hyperpermeability, and suggests that a mechanism by which eNOS is activated by phosphorylation at the plasma membrane and its endocytosis is required to deliver NO to subcellular targets to cause hyperpermeability.