【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。更多还原

【Abstract】 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~ -1 20-HETE) was added to culture dish and incubated for 10 min; 20-HETE 5 min treatment group:1×10~ -6 mol·L~ -1 20-HETE was added to culture dish and incubated for 5 min; 20-HETE 10 min treatment group:1×10~ -6 mol·L~ -1 20-HETE was added to culture dish and incubated for 10 min; VEGF 10 min 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(Ser1177)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 ①20-HETE phosphorylates eNOS at Ser1177 site which activates the eNOS to catalyze L-aginine to produce nitric oxide.②eNOS binds with Hsp90 and Akt in endothelial cell of pulmonary artery and 20-HETE increase the binding. ③eNOS binds with CYP4A in endothelial cell of pulmonary artery.更多还原