Issue Archive

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

Ye Hong, Bi Hairong, Lu Changlian, Tang Xiaobo, Zhu Daling

College of Pharmacy, Harbin Medical University.Harbin 150086,Heilongjiang

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 #mu#mol/L 15-HETE (P< 0.05), and increased by the lipoxygenase inhibitors, 10 #mu#mol/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 #mu#mol/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

Received: 　　Accepted:

Corresponding author: 　　E-mail:

Citing This Article：

Ye Hong, Bi Hairong, Lu Changlian, Tang Xiaobo, Zhu Daling. 15--hydroxyeicosatetraenoic acid depressed endothelial nitric oxide synthase activity in pulmonary artery. Acta Physiol Sin 2005; 57 (5): (in Chinese with English abstract).