Acta Physiologica Sinica, October 25, 2005, 57 (5): 612-618

Acta Physiologica Sinica, October 25, 2005, 57 (5): 612-618

Received 2005-02-16 Accepted 2005-08-04
This work was supported by the National Natural Science Foundation of China (No. 30370578).
^*Corresponding author. Tel: +86-451-86614075; Fax: +86-451-86665559; E-mail: dalingz@yahoo.com

Research Paper

15-hydroxyeicosatetraenoic acid depressed endothelial nitric oxide synthase activity in pulmonary artery

YE Hong¹, BI Hai-Rong¹, LÜ Chang-Lian¹, TANG Xiao-Bo¹, ZHU Da-Ling^1,2,3,*

¹College of Pharmacy, Harbin Medical University, Harbin 150086, China; ²Department of Pharmacy, the Second Affiliated Hospitalof Harbin Medical University, Harbin 150086, China;³Key Laboratory of Biopharmaceutical Engineering of Heilongjiang Province, Harbin 150086, China

Abstract: 15-hydroxyeicosatetraenoic acid (15-HETE) plays an important role in hypoxia-induced pulmonary vasoconstriction. Release of nitric oxide (NO) is apparently decreased and activity of endothelial nitric oxide synthase (eNOS) is impaired in chronic hypoxia. However, little is known whether 15-HETE contributes to eNOS/NO pathway in the constriction induced by 15-HETE. We examined the response of rat pulmonary artery (PA) rings to 15-HETE, the production of NO, total eNOS expression and the phosphorylation of eNOS in bovine pulmonary artery endothelial cells (BPAECs) stimulated by 15-HETE. Rat PA rings were divided into three groups: endothelium intact group, endothelium denuded group, and nitro-L-arginine methyl ester (L-NAME, 0.1 mmol/L, an inhibitor of eNOS) group. Constrictions to 15-HETE were significantly enhanced in endothelium denuded group and L-NAME group (both P< 0.05 vs endothelium intact group, n= 9); BPAECs were incubated in different conditions to test nitrite production by Greiss method. Nitrite production was significantly reduced by 1 mmol/L 15-HETE (P< 0.05), and increased by the lipoxygenase inhibitors, 10 μmol/L cinnamyl 3,4- dihydroxy-[alpha] -cyanocinnamate (CDC, P< 0.05) and 0.1 mmol/L nordihydroguiairetic acid (NDGA, P< 0.01 ); Western blot analysis of extracts from BPAECs incubated with 15-HETE in different time was carried out to test total eNOS expression, and the expression was changed unobviously. Immunoprecipitation (IP) and Western blot analysis of cell extracts from BPAECs treated with 2 μmol/L 15-HETE in different length of time were accomplished, using phospo-eNOS-threonine 495 (Thr495, an inhibitory site) antibody for IP, and eNOS or 15-lipoxygenase (15-LO) antibodies for Western blot. 15-HETE depressed eNOS activity by increasing the levels of phospho-eNOS-Thr 495. The data suggest that eNOS/NO pathway is involved in PA constrictions induced by 15-HETE and that 15-HETE depresses eNOS activity by phosphorylation in Thr495 site. The protein interaction between phospho-eNOS (Thr495) and 15-LO is discovered for the first time.

Key words: 15-hydroxyeicosatetraenoic acid; 15-lipoxygenase; pulmonary artery vasoconstriction; endothelium cells; endothelial nitric oxide synthase

15-羟二十碳四烯酸下调肺动脉内皮型一氧化氮合酶的活性

叶宏¹，毕海荣¹，吕昌莲¹，唐晓波¹，朱大岭^1,2,3,*

1哈尔滨医科大学药学院; ²哈尔滨医科大学附属二院药学部; ³黑龙江省生物医药工程重点实验室省部共建国家重点实验培育基地，哈尔滨150086

摘要: 15-羟二十碳四烯酸(15-hydroxyeicosatetraenoic acid，15-HETE)在低氧性肺血管收缩中起着重要作用，低氧肺动脉高压下调内皮型一氧化氮合酶(endothelial nitric oxide synthase，eNOS)，使一氧化氮(nitric oxide，NO)的产量下降，但目前尚无关于15-HETE与eNOS/NO相互作用研究的报道。我们通过Wistar大鼠肺动脉环张力、牛肺动脉内皮细胞NO 产量、总eNOS表达及eNOS磷酸化测定等方法对15-HETE与eNOS/NO的相互作用进行研究。首先分离大鼠肺动脉，分为eNOS抑制剂 L-NAME组(0.1mmol/L)、去血管内皮组与内皮完整组，用15-HETE作用大鼠离体肺动脉环，测定肺动脉张力。结果表明，L-NAME组、去除内皮组与内皮完整组分别比较，15-HETE对血管的收缩作用增强，且都有统计学意义(P< 0.05)。培养牛肺动脉内皮细胞，分别用15-HETE、15-脂氧酶(15-lipoxygenase，15-LO)抑制剂[(cinnamyl 3,4-dihydroxy-[alpha] -cyanocinnamate，CDC)和(nordihydroguiairetic acid，NDGA)]处理细胞，通过Greiss方法检测亚硝酸盐含量，间接测定NO产量，与对照组比较，1 mmol/L 15-HETE明显降低肺动脉内皮细胞NO水平( P< 0.05 )， 10 μmol/L CDC和0.1 mmol/L NDGA显著增加NO水平(分别是P< 0.05, P< 0.01); 通过Western blot检测不同时间(5，10，15，20，30，60 min) eNOS的表达情况，结果显示，15-HETE的不同作用时间，没有引起eNOS表达的明显不同；用苏氨酸495位点磷酸化eNOS (Thr495)抗体进行免疫沉淀，再用总eNOS抗体和15-LO抗体通过Western blot检测磷酸化型含量，间接测定eNOS活性，结果表明15-HETE增强Thr495磷酸化型eNOS含量。由于Thr495为eNOS抑制性磷酸化位点，因此15-HETE降低eNOS活性。这些数据表明：15-HETE的缩血管作用有eNOS/NO参与，15-HETE可以通过磷酸化Thr495位点降低eNOS活性，并且首次发现磷酸化eNOS (Thr495)和15-LO之间存在蛋白质相互作用。

关键词: 15-羟二十碳四烯酸；15-脂氧酶；肺动脉收缩；内皮细胞；内皮型一氧化氮合酶

中图分类号: R363