摘要
目的研究CYP4A/20-HETE和eNOS活化有关的调节蛋白间的相互作用。方法培养新生牛肺动脉内皮细胞(PAECs),用免疫沉淀(IP)和免疫印迹(WB)对下列4组的CYP4A,eNOS及eNOS调节蛋白进行测定:对照组:PAECs培养皿中加入与20-HETE处理组体积相同的乙醇溶剂,作用10min;20-HETE处理5min组:PAECs培养皿中20-HETE的浓度为1×10-6mol·L-1,作用5min;20-HET处理10min组:PAECs培养皿中20-HETE的浓度为1×10-6mol·L-1,作用10min;VEGF处理10min组:PAECs培养皿中VEGF的浓度为1×10-8mol·L-1,作用10min。结果经20-HETE处理后:肺动脉内皮细胞eNOS蛋白表达增加、phos-pho-eNOS(Ser1177)合成增加;用VEGF进行的上述实验结果与20-HETE相似;20-HETE促使Hsp、蛋白激酶B(Akt)与eNOS结合。结论免疫沉淀和免疫印迹实验结果表明:CYP4A与eNOS在肺动脉内皮细胞相互结合;20-HETE通过促使Hsp、蛋白激酶B(Akt)与eNOS结合,使eNOS(Ser1177)磷酸化,增加NO释放,舒张血管;VEGF和20-HETE的作用相似。
Aim To investigate the interaction between regulatory proteins related to the activation of eNOS and CYP4A/20-HETE.Methods The endothelia of pulmonary artery of new born bovine were cultured and divided into four groups:controlling group:ethanol solvent as vehicle(the volume is the same with 1×10^-6 mol·L^-120-HETE)was added to culture dish and incubated for 10 min;20-HETE 5 min treatment group:1×10^-6 mol·L^-120-HETE was added to culture dish and incubated for 5 min;20-HETE 10 min treatment group:1×10^-6 mol·L^-120-HETE was added to culture dish and incubated for 10 min;VEGF 10 rain treatment group:1×10^-8 mol·L^-1 VEGF was added to culture dish and incubated for 10 min.CYP4A,eNOS and proteins related to the activation of eNOS were measured by Immunoprecipitating and immunobloting.Results Expressions of eNOS and phospho-eNOS(Serl177)in PAECs were upregulated after the cells were treated with 20-HETE,and similar phenomena were observed when 20-HETE was replaced by VEGF;20-HETE increased the binding of Hsp,Akt and eNOS.Conclusions(1)20-HETE phosphorylates eNOS at Ser1177 site which activates the eNOS to catalyze L-aginine to produce nitric oxide.(2)eNOS binds with Hsp90 and Akt in endothelial cell of pulmonary artery and 20-HETE increase the binding.(3)eNOS binds with CYP4A in endothelial cell of pulmonary artery.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2007年第1期51-54,共4页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助课题(No30370578
30470752)
