Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat
Impaired endothelial nitric oxide synthase activity associated with enhanced caveolin binding in experimental cirrhosis in the rat
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DOI：
10.1016/s0016-5085(99)70408-7
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发表时间：
1999-11-01
期刊：
影响因子：
29.4
通讯作者：
Groszmann, RJ
中科院分区：
文献类型：
--
作者：
Shah, V;Toruner, M;Groszmann, RJ
背景与目的：一氧化氮（NO）的减少被认为是肝硬化肝内血管收缩的一个原因，但其调节机制尚不明确。本研究的目的是研究caveolin-1（一种被认为是内皮NO合酶的负调节因子）在介导完整肝硬化肝内NO生成不足中的作用。方法：采用吸入四氯化碳诱导肝硬化。观察灌注后大鼠肝脏NO生成及灌注压力的血流调节。Western blotting和免疫组织化学检测内皮细胞NO合成酶（eNOS）、小窝蛋白和钙调蛋白的表达。采用瓜氨酸生成法和化学发光法分别测定NOS活性和NO生成量。采用全组织蛋白免疫沉淀法检测蛋白-蛋白相互作用。结果：随着流量的增加，肝硬化动物产生的氮氧化物明显少于对照组动物。与对照组动物相比，在eNOS蛋白水平相似的情况下，肝硬化动物肝组织中NOS活性显著降低。eNOS活性不足与eNOS与小窝蛋白结合的数倍增加有关。肝硬化患者小窝蛋白-1蛋白水平明显升高。结论：这些研究提供了证据，证明小窝蛋白与eNOS的表达增强和相互作用有助于损害完整肝硬化肝脏的NO生成，降低NOS活性和血管收缩。
Background & Aims: A reduction in nitric oxide (NO) has been implicated as a cause of intrahepatic vasoconstriction in cirrhosis, but the regulatory mechanisms remain undefined. The aim of this study was to examine a contributory role for caveolin-1, a putative negative regulator of endothelial NO synthase, in mediating deficient intrahepatic NO production in the intact cirrhotic liver. Methods: Cirrhosis was induced by carbon tetrachloride inhalation. Flow regulation of NO production and perfusion pressure was examined in the perfused rat liver. Protein expression of endothelial NO synthase (eNOS), caveolin, and calmodulin was examined by Western blotting and immunohistochemistry. NOS activity and NO production were assessed by citrulline generation and chemiluminescence, respectively. Protein-protein interactions were examined using whole tissue protein immunoprecipitation. Results: In response to incremental increases in flow, cirrhotic animals produced significantly less NOx than control animals. NOS activity was significantly reduced in liver tissue from cirrhotic animals compared with control animals in the presence of similar eNOS protein levels. Deficient eNOS activity was associated with a severalfold increase in binding of eNOS with caveolin. Protein levels of caveolin-1 were markedly increased in the cirrhotic liver. Conclusions: These studies provide evidence that enhanced expression and interaction of caveolin with eNOS contribute to impaired NO production, reduced NOS activity, and vasoconstriction in the intact cirrhotic liver.