15-HETE对肺动脉内皮细胞一氧化氮合酶苏氨酸495位点(eNOS Thr495)磷酸化的影响  

Effect of 15(S)-hydroxyeicosate traenoic acid(15-HETE) on eNOS Thr495 phosphorylation in pulmonary artery endothelial cells

作　　者：叶宏[1] 吕昌莲[1] 朱大岭[1] 

机构地区：[1]哈尔滨医科大学

出　　处：《中国病理生理杂志》2006年第10期1896-1900,共5页Chinese Journal of Pathophysiology

基　　金：国家自然科学基金资助项目(No.30370578)

摘　　要：目的:探讨15-羟化二十烷四烯酸(15-HETE)对肺动脉内皮细胞(pu lm anory artery endothelialcells,PAECs)一氧化氮合酶(endothelia n itric oxide synthase,eNOS)活性的影响。方法:通过测定大鼠离体肺动脉环张力,比较去血管内皮、eNOS抑制剂L-NAME对15-HETE收缩肺动脉的影响;通过免疫沉淀和免疫印迹方法测定牛PAECs经2×10-6mol/L 15-HETE作用后,eNOS Thr 495磷酸化情况;用免疫印迹方法测定15-HETE对牛PAECs eNOS总蛋白表达的影响;用G re iss法检测15-HETE及15-脂氧酶(15-LO)抑制剂CDC和NDGA对牛肺动脉内皮细胞一氧化氮(NO)产量的影响。结果:(1)除去肺动脉内皮后,15-HETE收缩血管作用明显增强(P<0.01)。(2)抑制剂L-NAME 10-4mol/L可使15-HETE收缩肺动脉的作用明显增强(P<0.05)。(3)牛PAECs经2×10-6mol/L 15-HETE作用后,eNOS Thr 495位点磷酸化水平增强(P<0.01)。(4)10-6mol/L 15-HETE可抑制亚硝酸盐(NO2-/NO3-)的产生(P<0.05),抑制内源性15-HETE可明显增加NO2-/NO3-的产量,和对照组相比10-5mol/L CDC组P<0.05,10-4mol/L NDGA组P<0.01。结论:肺动脉内皮细胞eNOS/NO通路参与抑制15-HETE收缩大鼠肺动脉,15-HETE抑制肺动脉内皮细胞eNOS活性,使NO产量(NO2-/NO3-)下降。AIM ： To examine the effect of 15 （S） - hydroxyeicosate traenoic acid （ 15 - HETE） on eNOS activity in pulmonary artery endothelial cells （PAECs）. METHODS： 1. Rat pulmonary artery （PA） rings of intrapulmonary arteries were suspended under isometric tension in oxygenated Krebs＇ buffer in order to observe the differences on constriction induced by 15 - HETE using nitro - L - arginine methyl ester （ L - NAME, 10^-4 mol/L, an inhibitor of eNOS） and removal of endothelium. 2. The bovine PAECs was cultured for NO assay by Greiss reaction. 3. Immunoprecipitation （IP） and Western blotting （WB） were used to analyze the cell extracts from bovine PAECs treated with 2 ×10^-6 mol/L 15 - HETE for 30 and 60 min, using pbosphatase - eNOS （Thr495） antibody for IP and eNOS antibody for WB. RESULTS： 1. Inhibition of eNOS with L - NAME and removal of endothelium significantly enhanced 15 - HETE induced contractions in rat PA rings in a concentration - dependent manner （ P 〈 0.05, P 〈 0. 01 ）. 2. 15 - HETE increased phospho - eNOS （Thr495） levels （ P 〈 0. 01 ） in cultured bovine PAECs. 3. 10 -6 mol/L 15 - HETE （ P 〈 0. 05 ） did significantly reduce nitrite production, whereas 10^-5 mol/L cinnamy 1 3,4- dihydroxy- [alpha] -cyanocinnamate （CDC, P 〈0.05 ）, 10^-4 moL/L nordihydroguiairetic acid （NDGA, P 〈 0.01 ） significantly increased nitrite production, as measured by a spectrophotometric assay. CONCLUSION： 15 - HETE inhibits eNOS activity and decreases production of NO （ NO2^-/NO3^- ）. The pathway of eNOS/ NO is involved in 15 - HETE - induced contraction in the rat PA.

关 键 词：羟基花生四烯酸类 一氧化氮 血管收缩 肺动脉 内皮细胞 一氧化氮合酶 

分 类 号：R363[医药卫生—病理学]