Influence of caveolin on constitutively activated recombinant eNOS: insights into eNOS dysfunction in BDL rat liver
Influence of caveolin on constitutively activated recombinant eNOS: insights into eNOS dysfunction in BDL rat liver
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DOI：
10.1152/ajpgi.00143.2003
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发表时间：
2003-09-01
影响因子：
4.5
通讯作者：
Shah, V
中科院分区：
文献类型：
--
作者：
Hendrickson, H;Chatterjee, S;Shah, V
减少内皮型一氧化氮合酶（eNOS）衍生的NO从肝血管内皮细胞的生产有助于肝血管收缩门静脉高压症。本研究的目的是研究这一过程的机制，通过测试的影响的eNOS（S1179 DeNOS）在原代和繁殖的肝细胞在体外和在假手术和胆管结扎（BDL）大鼠肝脏在体内，使用腺病毒载体编码绿色荧光蛋白（AdGFP）和S1179 DeNOS（AdS1179 DeNOS）。AdS1179DeNOS转导增强了非实质肝细胞中基础和激动剂刺激的NO生成。与AdGFP转导的假手术大鼠相比，AdS1179DeNOS体内转导的假手术大鼠对递增剂量的血管收缩剂甲氧胺的升压反应降低。然而，BDL大鼠转导AdS1179DeNOS并没有表现出改善的血管舒张反应所证明的类似的流量依赖性的压力增加中观察到的BDL大鼠转导AdGFP，尽管类似的病毒转基因表达水平。我们接下来检查了eNOS抑制蛋白小窝蛋白对肝硬化中S1179DeNOS功能障碍的影响。免疫金电镜分析表明，小窝蛋白在BDL肝不仅在肝内皮细胞，而且在肝星状细胞显着表达。在LX2肝星状细胞系中的体外研究表明，小窝蛋白沉淀LX2细胞中的重组S1179DeNOS，重组S1179DeNOS共沉淀小窝蛋白，并且在小窝蛋白过表达的存在下结合增强。此外，小窝蛋白过表达抑制重组S1179DeNOS活性。这些研究表明，重组S1179DeNOS蛋白功能正常的肝细胞和组织，但证据功能障碍，在肝硬化大鼠肝脏和小窝蛋白的表达和抑制BDL非实质细胞，包括肝星状细胞，可能会导致这种功能障碍。
Diminished endothelial nitric oxide (NO) synthase (eNOS)-derived NO production from the hepatic vascular endothelium contributes to hepatic vasoconstriction in portal hypertension. The aim of this study was to examine the mechanism of this process by testing the influence of a constitutively active form of eNOS (S1179DeNOS) in both primary and propagated liver cells in vitro and in the sham and bile duct ligated (BDL) rat liver in vivo, using an adenoviral vector encoding green fluorescent protein (AdGFP) and S1179DeNOS (AdS1179DeNOS). AdS1179DeNOS transduction augmented basal and agonist-stimulated NO generation in nonparenchymal liver cells. Sham rats transduced in vivo with AdS1179DeNOS evidenced a decreased pressor response to incremental doses of the vasoconstrictor methoxamine compared with sham rats transduced with AdGFP. However, BDL rats transduced with AdS1179DeNOS did not display improved vasodilatory responses as evidenced by similar flow-dependent pressure increases to that observed in BDL rats transduced with AdGFP, despite similar levels of viral transgene expression. We next examined the influence of the eNOS inhibitory protein caveolin on S1179DeNOS dysfunction in cirrhotic liver. Immunogold electron microscopic analysis of caveolin in BDL liver demonstrated prominent expression not only in liver endothelial cells, but also in hepatic stellate cells. In vitro studies in the LX2 hepatic stellate cell line demonstrate that caveolin precipitates recombinant S1179DeNOS in LX2 cells, that recombinant S1179DeNOS coprecipitates caveolin, and that binding is enhanced in the presence of overexpression of caveolin. Furthermore, caveolin overexpression inhibits recombinant S1179DeNOS activity. These studies indicate that recombinant S1179DeNOS protein functions appropriately in normal liver cells and tissue but evidences dysfunction in the cirrhotic rat liver and that caveolin expression and inhibition in BDL nonparenchymal cells, including hepatic stellate cells, may account for this dysfunction.