The role of subtypes of voltage-gated K+ channels in pulmonary vasoconstriction induced by 15-hydroeicosatetraenoic acid / 药学学报

<p><b>AIM</b>To observe the effect of subtypes of Kv channels in rat pulmonary artery smooth muscle cells (PASMCs) on the process of pulmonary vasoconstriction induced by 15-HETE.</p><p><b>METHODS</b>In the present study, ring of rabbit PA with specific Kv channel blockers were employed to functionally identify certain channel subtypes that took part in the process of 15-HETE induced pulmonary vasoconstriction; RT-PCR and Western blotting analysis were also used to measure the expression of subtypes of Kv in PASMCs exposed to 15-HETE,chronic hypoxia.</p><p><b>RESULTS</b>Blocking of Kv1. 1, Kv1. 2, Kv1. 3 and Kv1. 6 channels did not affect 15-HETE induced vasoconstriction in normoxic rats; 15-HETE did not affect expression of Kv1. 1 and Kv1. 2 channels; 15-HETE significantly downregulated the expression of mRNA and protein of Kv1. 5 and Kv2. 1 in rat PASMCs.</p><p><b>CONCLUSION</b>The results suggested that hypoxia may block Kv1. 5 and Kv2. 1 channels via 15-HETE mediated mechanism, leading to decrease numbers of functional Kv1. 5 and Kv2. 1 channels in PASMCs, leading to PA vasoconstriction.</p>

ERK1/2 signaling pathway is involved in 15-hydroxyeicosatetraenoic acid-induced hypoxic pulmonary vasoconstriction / 生理学报

Hypoxia-induced 15-hydroxyeicosatetraenoic acid (15-HETE) is an essential mediator to constrict pulmonary arteries (PA). The signaling pathway involved in 15-HETE-induced PA vasoconstriction remains obscure. The aim of the present study was to test the hypothesis that hypoxic PA constriction induced by 15-HETE was possibly regulated by the extracellular signal-regulated kinase-1/2 (ERK1/2) pathway. PA ring tension measurement, Western blot and immunocytochemistry were used in the study to determine the possible role of ERK1/2 in 15-HETE-induced PA vasoconstriction. The organ bath for PA rings tension study was employed. Adult male Wistar rats were raised in hypoxic environment with fractional inspired oxygen (FIO2, 0.12) for 9 d. PA 1~1.5 mm in diameter were dissected and cut into 3 mm long rings for tension study. ERK1/2 up-stream kinase (MEK) inhibitor PD98059, which blocks the activation of ERK1/2, was used. The results showed that pretreatment of PD98059 significantly blunted 15-HETE-induced PA vasoconstrictions in the rings from hypoxic rat. Moreover, in endothelium-denuded rings, PD98059 also significantly attenuated 15-HETE-induced vasoconstriction. Phosphorylation of ERK1/2 in pulmonary arterial smooth muscle cells (PASMCs) of rat was enhanced evidently when stimulated by 15-HETE. Thus, the data suggest that ERK1/2 signaling pathway is involved in 15-HETE-induced hypoxic pulmonary vasoconstriction.

15-hydroxyeicosatetraenoic acid depressed endothelial nitric oxide synthase activity in pulmonary artery / 生理学报

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 mumol/L 15-HETE (P<0.05), and increased by the lipoxygenase inhibitors, 10 mumol/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 mumol/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.