Key Laboratory of Cardiovascular Disease and Molecular Intervention, Key Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing, China.
J Mol Cell Cardiol. 2011 Sep;51(3):419-27. doi: 10.1016/j.yjmcc.2011.06.017. Epub 2011 Jun 29.
Endothelial nitric oxide synthase (eNOS) has been reported to associate with globular actin, and this association increases eNOS activity. Adenosine, histamine, salbutamol and thrombin cause activation of eNOS through widely different mechanisms. Whether these eNOS agonists can regulate eNOS activity through affecting its association with actin is unknown. As previously reported, we confirmed in cultured human umbilical vein endothelial cells (HUVEC) that histamine and thrombin increased intracellular Ca(2+) whereas adenosine and salbutamol did not, and that these four agonists caused different effects on actin filament structure. Nevertheless, despite their divergent effects on intracellular Ca(2+) and on actin filament structure, we found by immunoprecipitation that adenosine, histamine, salbutamol and thrombin all caused an increase in association between eNOS and globular actin. This increase of association was inhibited by pre-treatment with phalloidin, an actin filament stabilizer. All of these agonists also increased phosphorylation of eNOS on serine residue 1177, eNOS activity, and cyclic guanosine-3', 5'-monophosphate, and these increases were all attenuated by phalloidin. Agonist-induced phosphorylation of eNOS on serine 1177 was attenuated by Akt inhibition, whereas association of eNOS with actin was not. We also found, in HEK-293 cells transfected with the eNOS mutants eNOS-S1177A or eNOS-S1177D, that the association between eNOS and globular actin was decreased as compared to cells transfected with wild-type eNOS. We conclude that association of globular actin with eNOS plays an essential and necessary role in agonist-induced eNOS activation, through enabling its phosphorylation by Akt at serine residue 1177.
内皮型一氧化氮合酶（eNOS）已被报道与球状肌动蛋白结合，这种结合增加了 eNOS 的活性。腺苷、组氨酸、沙丁胺醇和凝血酶通过广泛不同的机制引起 eNOS 的激活。这些 eNOS 激动剂是否可以通过影响其与肌动蛋白的结合来调节 eNOS 活性尚不清楚。正如之前所报道的，我们在培养的人脐静脉内皮细胞（HUVEC）中证实，组氨酸和凝血酶增加了细胞内 Ca(2+)，而腺苷和沙丁胺醇则没有，这四种激动剂对肌动蛋白丝结构有不同的影响。然而，尽管它们对细胞内 Ca(2+)和肌动蛋白丝结构有不同的影响，我们通过免疫沉淀发现，腺苷、组氨酸、沙丁胺醇和凝血酶都导致 eNOS 与球状肌动蛋白的结合增加。这种结合的增加被肌动蛋白丝稳定剂鬼笔环肽预处理所抑制。所有这些激动剂都增加了 eNOS 丝氨酸 1177 位点的磷酸化、eNOS 活性和环鸟苷酸-3',5'-单磷酸，这些增加都被鬼笔环肽所减弱。激动剂诱导的 eNOS 丝氨酸 1177 磷酸化被 Akt 抑制所减弱，而 eNOS 与肌动蛋白的结合则不受影响。我们还发现，在转染了 eNOS 突变体 eNOS-S1177A 或 eNOS-S1177D 的 HEK-293 细胞中，与转染野生型 eNOS 的细胞相比，eNOS 与球状肌动蛋白的结合减少。我们得出结论，球状肌动蛋白与 eNOS 的结合在激动剂诱导的 eNOS 激活中起着至关重要和必要的作用，通过使其丝氨酸 1177 位点被 Akt 磷酸化。
