Role of Gi/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery
Role of Gi/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery
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DOI：
10.1016/j.cellsig.2011.02.008
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发表时间：
2011-07-01
影响因子：
4.8
通讯作者：
Leblais, Veronique
中科院分区：
文献类型：
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作者：
Banquet, Sebastien;Delannoy, Estelle;Leblais, Veronique
β(2)-肾上腺素能受体（β (2)-AR）的激活引发小鼠肺动脉内皮型一氧化氮合酶（eNOS）依赖性松弛，与毒碱受体依赖性松弛相反，这种松弛在缺氧肺动脉高压中得以保留。因此，我们表征了β (2)- ar介导的eNOS激活的信号通路，特别关注G（i/o）蛋白、蛋白激酶和小泡。在野生型或小窝蛋白-1敲除小鼠的肺动脉中进行了功能研究（用于评估血管松弛反应）、Western blotting（用于评估eNOS和小窝蛋白-1磷酸化）和透射电镜（用于可视化小窝）。在野生型离体动脉中，G（i/o）蛋白（百日毒，PTX）、磷脂酰肌醇3-激酶（PI3K、wortmannin或LY 294002）、Akt （Akt抑制剂X）和src -激酶（PP2）的抑制剂以及胆固醇消耗（使用甲基- β -环dextrin）降低了选择性β (2)-AR激动剂procaterol的松弛。Procaterol诱导eNOS Ser（1177）磷酸化，而PTX、PP2或Akt抑制剂可阻止这一磷酸化。Procaterol还促进了Tyr位点的caveolin-1磷酸化（14），PTX或PP2降低了该磷酸化。小泡蛋白-1基因缺失导致小鼠肺动脉内皮小泡破裂，procaterol诱导的舒张增强。与procaterol不同，乙酰胆碱诱导的松弛不受PTX、甲基- β -环糊精或caveolin-1基因缺失的影响。综上所述，小鼠肺内皮β (2)-AR与G(i/o)-Src激酶- pi3k /Akt通路偶联，促进eNOS Ser（1177）位点磷酸化，导致no依赖性血管松弛。Caveolin-1通过G(i/o)-Src激酶途径在Tyr位点磷酸化，对这种反应施加负性控制（14）。由于肺β (2)- ar和毒碱受体介导的松弛在各自的信号通路中分化，导致缺氧诱导的肺动脉高压中eNOS的激活和敏感性，eNOS激活的机制可能是肺内皮功能障碍的关键决定因素。(C) 2011爱思唯尔公司版权所有。
Activation of the beta(2)-adrenoceptor (beta(2)-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the beta(2)-AR-mediated eNOS activation, with special focus on G(i/o) proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective beta(2)-AR agonist procaterol was reduced by inhibitors of G(i/o) proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-beta-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser(1177), which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr(14), which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-beta-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial beta(2)-AR is coupled to a G(i/o)-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser(1177) leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr(14), through a G(i/o)-Src kinase pathway. Since pulmonary beta(2)-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction. (C) 2011 Elsevier Inc. All rights reserved.