title: Activation of endothelial nitric oxide synthase is dependent on its interaction with globular actin in human umbilical vein endothelial cells.
abstract: 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.
authors: Key Laboratory of Cardiovascular Disease and Molecular Intervention, Key Laboratory of Human Functional Genomics, Nanjing Medical University, Nanjing, China.
authors: Qiongyu
authors: Q
authors: Mi
authors: Nan
authors: N
authors: Chen
authors: Yasin
authors: Y
authors: Shaifta
authors: Liping
authors: L
authors: Xie
authors: Hui
authors: H
authors: Lu
authors: Zhen
authors: Z
authors: Liu
authors: Qi
authors: Q
authors: Chen
authors: Colleen
authors: C
authors: Hamid
authors: Silke
authors: S
authors: Becker
authors: Yong
authors: Y
authors: Ji
authors: Albert
authors: A
authors: Ferro
mesh_terms: D000199:Actins / Q000378:metabolism*; D000241:Adenosine / Q000494:pharmacology; D002478:Cells, Cultured; D015638:Cytochalasin D / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D057809:HEK293 Cells; D006632:Histamine / Q000494:pharmacology; D061307:Human Umbilical Vein Endothelial Cells / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism; D006801:Humans; D052250:Nitric Oxide Synthase Type III / Q000378:metabolism*; D010590:Phalloidine / Q000494:pharmacology; D010766:Phosphorylation / Q000187:drug effects; D011485:Protein Binding / Q000187:drug effects; D055503:Protein Multimerization / Q000502:physiology; D051057:Proto-Oncogene Proteins c-akt / Q000378:metabolism; D012694:Serine / Q000378:metabolism; D013917:Thrombin / Q000494:pharmacology
chemical_list: D000199:Actins; D010590:Phalloidine; D015638:Cytochalasin D; D012694:Serine; D006632:Histamine; D052250:Nitric Oxide Synthase Type III; D051057:Proto-Oncogene Proteins c-akt; D013917:Thrombin; D000241:Adenosine