生理学报Acta Physiologica Sinica, April

研究论文

膜雌激素受体介导一氧化氮合酶活性增高的快速非基因效应

王庭槐*, 付晓东, 杨丹, 谈智, 潘敬运

中山大学医学院生理学教研室, 广州 510080

摘要: 实验利用新生小牛胸主动脉内皮细胞(BAECs)作为模型, 观察17β-雌二醇(E2)、 E2BSA对BAECs中内皮型一氧化氮合酶(eNOS)的快速激活作用, 并探讨了丝裂素活化蛋白激酶(MAPK)信号通路在其中的作用。结果显示, 不同浓度的E2 (0.001-1 μmol/L)作用于BAECs 15 min均能快速激活eNOS; 0.01 μmol/L浓度的E2作用于BAECs, 5 min即能激活eNOS, 15 min达到最大效应, 随后eNOS快速失活; E2BSA (17.5 ng/ml)作用于BAECs, 15 min同样可激活eNOS。E2、 E2BSA激活eNOS的作用均能被雌激素受体(ER)拮抗剂tamoxifen (0.1 μmol/L)或MAPK激酶特异抑制剂PD98059 (50 μmol/L)所阻断。放线菌素D (25 μg/ml)不能阻断E2、 E2BSA对eNOS的激活作用。E2 (0.01 μmol/L)、 E2BSA (17.5 ng/ml)作用于BAECs 15 min后可明显促进p42/p44磷酸化MAPK蛋白表达, 而对p42/p44 MAPK总蛋白表达无影响。Tamoxifen可部分阻断E2、 E2BSA激活p42/p44磷酸化MAPK的作用。这些结果提示, BAECs膜上可能存在膜雌激素受体(membrane estrogen receptor, mER), E2、 E2BSA作用于mER后可通过MAPK信号途径快速激活eNOS。

关键词: 雌激素; 膜雌激素受体; 一氧化氮合酶; 丝裂素活化蛋白激酶; 血管内皮细胞

中图分类号: Q463; R331.36

Membrane estrogen receptor mediates the rapid nongenomic activation of endothelial nitric oxide synthase by estrogen

WANG Ting-Huai*, FU Xiao-Dong, YANG Dan, TAN Zhi, PAN Jing-Yun

Department of Physiology, Sun Yat-sen University of Medical Sciences, Guangzhou 510080

Abstract: In the present study, confluent bovine aortic endothelial cells (BAECs) were used to study the rapid nongenomic effects of 17β-estradiol and the membrane impermeable conjugated 17β-estradiol (E2BSA) on the activation of endothelial nitric oxide synthase (eNOS) and mitogen activated protein kinase (MAPK). eNOS activation was assessed in whole cells by measuring [3H]L-arginine conversion to [3H]L-citrulline. MAPK activity was determined by Western blotting. The results obtained show that the addition of various concentrations of E2 (0.001-1 μmol/L) resulted in 122±29, 186±17, 83±20 and 157±29% increases in eNOS activity, respectively, in BAECs within 15 min of exposure to the hormone. E2 (0.01 μmol/L)-stimulated eNOS activity was detectable during 5-, 15- and 30- min incubation which yielded increases of 37±6, 56±9 and 38±8%, respectively. The increase reached a plateau from 15 through 30 min and rapidly declined thereafter. E2BSA （17.5 ng/ml） also enhanced eNOS activity by an increase of 35±9% above the basal activity. The effect of E2 and E2BSA on eNOS activation was unaffected by actinomycin D （25 μg/ml） but was obviously inhibited by tamoxifen (0.1 μmol/L) and PD98059 (50 μmol/L). Compared with control， E2 and E2BSA stimulation of BAECs for 15 min caused an increase in MAPK activity by 428±17 and 360±14% respectively. This effect was blocked by tamoxifen. These results suggest that there might be the membrane estrogen receptor localized on BAECs, which mediates the rapid nongenomic effect of estrogen on eNOS activation through MAPK pathways.

Key words: estrogen; membrane estrogen receptor; nitric oxide synthase; mitogen-activated protein kinase; vascular endothelial cell