睾酮对TNF-α作用于内皮细胞表达HO-1的影响

目的 观察不同浓度睾酮对肿瘤坏死因子-α(TNF-α)作用于人脐静脉内皮细胞 (human umbilical vein endothelial cells)ECV-304表达血红素加氧酶-1(HO-1)的影响,初步探讨其作用机制.方法 用ELISA法观察不同浓度睾酮及雄激素受体拮抗剂Flutimide、雌激素受体拮抗剂ICI182,780对ECV-304表达HO-1干预作用.结果 睾酮在3×10-6,3×10-7,3×10-8,3×10-9mol/L几种浓度下均具有促进TNF-α诱导的ECV-304细胞表达HO-1的量.与3.0×10-8mol/L睾酮干预组相比,予1.0×10-6mol/L ICI182,780预处理,ECV-304细胞释放的HO-1的量明显减少,并诱导HO-1的表达呈时间(当t=24 h达到最理想的分泌量)、浓度依赖关系.结论 睾酮对TNF-α诱导的ECV-304细胞表达HO-1的影响与其浓度有关,生理浓度睾酮对其具有一定的促进作用,而且这种生物学效应是部分通过转化为雌激素后,作用于雌激素受体实现.     

睾酮对血管内皮细胞衰老的干预作用

目的 观察睾酮对过氧化氢(H2O2)诱导衰老的人脐静脉内皮细胞(HUVECs)的影响,初步探讨其作用机制.方法 分别用MTT还原法和SA-β-gal细胞化学检测法观察不同浓度睾酮及雄激素受体拮抗剂Flutimide、雌激素受体拮抗剂ICI182,780对HUVECs衰老的干预作用.结果 睾酮在3.0×10-9、3.0×10-8和3.0×10-7 mol/L 3种浓度下均具有延缓H2O2诱导的HUVECs的细胞增殖率降低和SA-β-gal阳性率明显增加的趋势.与3.0×10-8 mol/L睾酮干预组相比,予1.0×10-6 mol/L ICI182,780预处理, HUVECs的细胞增殖率明显降低,SA-β-gal阳性率明显增加.结论 睾酮对H2O2诱导的HUVECs衰老的影响与其剂量有关,生理浓度睾酮对其具有一定的延缓作用,而且这种生物学效应是部分通过转化为雌激素后,作用于雌激素受体实现的.     

性激素水平及平衡对大鼠血管内皮细胞增殖的调节作用及意义

目的　探讨雌、雄及孕激素分别对雌、雄性大鼠肺血管内皮细胞 (VEC)增殖的影响。方法　采用组织贴块法培养大鼠肺血管内皮细胞 ,应用噻唑蓝 (MTT)比色法检测大鼠VEC的增殖情况。结果　 (1) 3× 10 -8mol/L、3×10 -7mol/L 17 β雌二醇 (E2 )可促进雌鼠肺VEC的增殖 (P <0 .0 1) ;3× 10 -9mol/L、3× 10 -8mol/L、3× 10 -7mol/L 17 βE2 均能促进雄鼠VEC的增殖 (P<0 .0 5 ) ,各组间无明显差异。雌激素受体拮抗剂他莫昔芬可阻断 17 βE2 上述促增殖作用。 (2 ) 3× 10 -8mol/L、3× 10 -7mol/L睾酮 (T)均可明显促进雄鼠VEC的增殖(P <0 .0 5 ) ,但对雌鼠VEC增殖无促进作用。 (3)E2 /孕激素 (P) =3/ 10时能促进雌鼠VEC的增殖 (P<0 .0 5 )。(4)E2 /T =1亦可促进雌鼠VEC增殖 (P<0 .0 5 )。结论　雌激素可促进雌、雄性大鼠VEC的增殖 ,其促增殖作用无性别差异 ,且通过雌激素受体 (ER)介导。雄激素仅可促进雄性大鼠VEC增殖 ,单纯孕激素对雌鼠VEC增殖无明显影响。E2 /T、E2 /P比值的平衡对雌鼠VEC的增殖也起重要作用     

HSP27对雌激素诱导的内皮保护作用的影响

目的观察雌二醇(E2)及雌激素受体拮抗剂他莫昔芬对HUVEC HSP27表达的影响,以及HSP27 siRNA对雌激素内皮保护作用的影响。方法分别用不同浓度(10-9、10-8和10-7mol/L)E2处理HUVEC 24 h;10-7mol/L雌二醇及10-6mol/L他莫昔芬处理HUVEC 24 h。以空白组为阴性对照,RT-PCR和Western blot检测HSP27的表达。分别转染HSP27 siRNA和mockRNA48 h后,再与10-7mol/L E2孵育24 h。空白组做为阴性对照,RT-PCR和Western blot检测HSP27的表达;分别用硝酸还原酶法和ELISA检测培养基中NO和内皮素1的含量;ELISA检测细胞表面细胞间黏附分子1(ICAM-1)的含量。结果 E2处理HUVEC可上调HSP27的mRNA和蛋白表达水平,并且具有剂量依赖性,表达的峰值在10-7mol/L。10-6mol/L他莫昔芬可阻断10-7mol/L E2的作用。RT-PCR、Western blot检测HSP27 siRNA序列能明显下调HSP27mRNA及蛋白的表达。HU-VEC转染HSP27 siRNA4...     

雌激素受体介导Genistein对内皮型一氧化氮合酶表达的诱导作用

目的 研究雌激素受体介导的基因组效应在Genistein促进内皮型一氧化氮合酶表达过程中的作用.方法 体外培养人脐静脉内皮细胞,在氧化型低密度脂蛋白(100 mg/L)干预内皮细胞的基础上,经雌激素受体拮抗剂ICI182780(1 μmol/L)或经基因转录阻断剂放线菌素D(5 mg/L)作用30 min后,再加入Genistein(100 nmol/L)作用24 h,实时定量PCR检测内皮型一氧化氮合酶mRNA表达,Western Blotting检测内皮型一氧化氮合酶蛋白的表达.结果 与空白对照组相比,氧化型低密度脂蛋白(100 mg/L)明显下调内皮型一氧化氮合酶 mRNA和蛋白表达(P<0.05);Genistein明显上调内皮型一氧化氮合酶 mRNA和蛋白表达(P<0.05),而且Genistein对内皮型一氧化氮合酶的诱导作用被雌激素受体拮抗剂ICI182780和基因转录阻断剂放线菌素D明显抑制(P<0.05 ).结论 Genistein促进内皮型一氧化氮合酶的表达与雌激素受体介导的基因组效应密切相关.     

睾酮通过减少氧化应激干预血管内皮细胞衰老

目的观察睾酮对过氧化氢(H_2O_2)诱导衰老的人脐静脉内皮细胞(HUVECs)的影响。方法用SA-βgal细胞化学检测法观察不同浓度睾酮及雄激素受体拮抗剂、雌激素受体拮抗剂对H_2O_2诱导的HUVECs衰老的干预作用,用2,7二氯氢化荧光素二酯(H2DCF-DA)染色检测细胞内活性氧簇(ROS)水平,用免疫印迹分析(Western印迹)法检测细胞中去磷酸化Rb蛋白水平。结果睾酮在3.0×10~(-9)、3.0×10~(-8)、3.0×10~(-7)mol/L等3种浓度下均具有延缓H_2O_2诱导的HUVECs的SA-βgal阳性率明显增加的趋势;与3.0×10~(-8)mol/L睾酮干预组相比,予10~(-6)mol/L ICI182,780预处理,HUVECs的SA-βgal阳性率明显增加,ROS水平增加,去磷酸化Rb蛋白水平明显增加。结论睾酮对H_2O_2诱导的HUVECs衰老的影响与其剂量有关;生理浓度睾酮具有延缓H_2O_2诱导的HUVECs衰老的趋势,其作用可能是通过转化为雌激素,作用于雌激素受体,减少氧化应激和调节去磷酸化Rb蛋白产生。     

不同浓度雌二醇对去卵巢小鼠T淋巴细胞凋亡的影响及作用机制

目的 研究不同浓度雌二醇(E2)对去卵巢小鼠脾脏T淋巴细胞凋亡的影响并探讨其作用机制.方法 分离出小鼠脾脏T淋巴细胞,分为10组,分别为青年组、假手术组、去卵巢对照组、去卵巢+E2(10-11mol/L)组、去卵巢+E2(10-10mol/L)组、去卵巢+E2(10-8mol/L)组、去卵巢+E2(10-6mol/L)组、去卵巢+E2(10-10mol/L)+雌激素受体拮抗剂IC1182 780(10-7mol/L)组、去卵巢+E2(10-10mol/L)+1-甲基-4-苯基-四氢吡啶(MPP,10-6mol/L)组、去卵巢+E2(10-10mol/L)+四氢化吡咯二硫代氨基甲酸酯(PDTC,10-6mol/L)组.流式细胞仪检测各组细胞凋亡率,Westernblot方法检测各组细胞雌激素受体(ER)α、ERβ、Bax、Bcl-2蛋白表达及细胞核中P65蛋白水平.结果 去卵巢对照组细胞凋亡率为(19.4±2.5)%,高于青年组(14.6±2.4)%和假手术组(14.5±2.3)%,Bcl-2、核内P65蛋白水平分别为0.25±0.05、0.09±0.01,低于青年组(0.40±0.07、0.15±0.02)和假手术组(0.38±0.05、0.15±0.02),均P<0.01;去卵巢+E2(10-10mol/L)组凋亡率为(16.6±1.8)%,低于去卵巢对照组(P<0.05),Bel-2及核内P65蛋白水平高于去卵巢对照组(P<0.01),而去卵巢+E2(10-8mol/L,10-6mol/L)组凋亡率高于去卵巢对照组(分别为P<0.05、P<0.01).去卵巢对照组ERα、ERβ蛋白水平分别为0.23±0.01、0.22±0.03,低于青年组(0.27±0.02、0.29±0.04)和假手术组(0.28±0.03、0.29±0.02),去卵巢+E2(10-11mol/L、10-10mol/L、10-8mol/L)组ERα、ERβ蛋白水平均高于去卵巢对照组,而去卵巢+E2(10-mol/L)组(0.09±0.01、0.14±0.02)低于去卵巢对照组(均P<0.01).去卵巢+E2(10-10mol/L)+IC1182 780组及去卵巢+E2(10-10mol/L)+PDTC组凋亡率与去卵巢对照组差异无统计学意义,去卵巢+E2(10-10mol/L)+MPP组凋亡率与去卵巢+E2(10-10mol/L)组比较,差异无统计学意义.结论 去卵巢小鼠T淋巴细胞凋亡增多,E2对去卵巢小鼠T淋巴细胞的凋亡呈剂量依赖的双相调节作用,生理范围内可部分逆转去卵巢小鼠T淋巴细胞凋亡,而超过生理范围则增加其凋亡.生理剂量E2可能通过ERβ及核因子-kB途径抑制去卵巢小鼠的T淋巴细胞凋亡.     

D1类多巴胺受体对肾上腺素α受体介导的促动脉平滑肌细胞增殖作用的影响

目的 观察D1类多巴胺受体对肾上腺素α受体介导的血管平滑肌(VSM)细胞增殖的影响.方法 以去甲肾上腺素(NE)10-6～10-9 mol/L刺激Sprague-Dawley(SD)大鼠主动脉分离的VSM细胞,观察在D1类多巴胺受体激动剂(Fenoldopam)10-5～10-8 mol/L存在的情况下,NE促细胞增殖作用的变化,细胞增殖用3H-TdR掺入量表示.结果 NE呈浓度依赖性的促进SD大鼠VSM细胞的增殖,该作用由肾上腺素α受体介导,酚妥拉明存在的情况下可消除NE的促增殖作用.Fenoldopam本身对VSM细胞增殖无影响,但Fenoldopam可通过D1类多巴胺受体减弱NE(10-6 mol/L)介导的VSM细胞增殖;该实验结果得到了人VSM细胞实验结果的印证.结论 D1类多巴胺受体对NE介导的促VSM细胞增殖具有抑制作用,该作用可能在高血压的发生、发展中发挥一定的作用.     

D3多巴胺受体对α肾上腺素能受体介导的促动脉平滑肌细胞增殖作用的影响

目的 观察D3多巴胺受体对肾上腺素α受体介导的血管平滑肌细胞(VSMC)增殖的影响.方法 用去甲肾上腺素(NE)刺激SD大鼠的VSMC,观察在D3受体激动剂(PD128907)存在的情况下,NE促增殖作用的变化,其中细胞增殖用3H-TdR掺入量表示.结果 NE通过肾上腺素α受体促进SD大鼠VSMC增殖,该作用呈现浓度依赖性关系.PD128907低浓度(10(-8)、10(-7) mol/L)对VSMC增殖无影响,但高浓度(10(-6)、10(-5) mol/L)却促进VSMC的增殖[PD128907 10(-6) mol/L=(4982±529)计数/min、PD128907 10(-5) mol/L=(5782±483)计数/min与对照=(3798±438)计数/min相比,P＜0.05],此作用可被α受体阻断剂酚妥拉明阻断.低浓度的PD128907(10(-7) mol/L)可通过D3受体减弱NE 10(-6) mol/L引起的VSMC增殖[NE 10(-6) mol/L=(6315±245)计数/min与NE 10(-6) mol/L PD128907 10(-7) mol/L=(4898±286)计数/min相比,P＜0.05.结论 D3受体对NE所致的VSMC增殖具有抑制作用,该作用可能在高血压的发生、发展中发挥作用.     

雌激素抑制内质网应激引起的血管内皮细胞凋亡及机制

目的通过建立人脐静脉内皮细胞(HUVEC)内质网应激(ERS)的细胞模型,研究雌激素抑制内质网应激引起的凋亡的信号传导机制,以探讨雌激素对心血管的保护机制。方法分别用10μmol/L的衣霉素(TM)或2 mmol/L的二硫苏糖醇(DTT)诱导HUVEC,建立内质网应激细胞模型,提前给予10-8mol/L的17-β雌二醇(E2)预处理1 h,用Western blot检测葡萄糖调节蛋白78(GRP78)判断模型是否建立成功,并探索E2对内质网应激的作用。检测内质网应激的三条主要信号通路蛋白的变化,上调最显著的为内质网应激最主要的信号通路。Western blot检测内质网应激凋亡蛋白C/EBP-同源蛋白(CHOP),Hochest染色检测细胞凋亡率,探索E2对内质网应激凋亡的作用。添加E2受体拮抗剂ICI182780(ERα、ERβ拮抗剂及GPER激动剂)和G15(GPER拮抗剂)后检测内质网应激最主要通路蛋白表达量的变化,探索雌激素受体在其抑制内质网应激中的作用。添加E2受体后信号通路阻断剂,检测雌激素抑制内质网应激的过程中活化其受体后激活的最主要受体后信号通路。结果 TM/DTT组GRP78的表达量显著上调,内质网应激三条信号通路中蛋白激酶R样内质网激酶(PERK)信号通路上调最明显,而TM/DTT+E2组上调显著回复。TM/DTT组CHOP的表达量显著上调且细胞凋亡率显著增加,而TM/DTT+E2组上调明显回复,凋亡细胞减少。E2有显著抑制p-PERK/PERK上调的作用,而E2的保护作用可分别被ICI182780和G15阻断,同时添加ICI182780和G15时阻断作用最显著。分别添加信号通路阻断剂后,E2抑制pPERK/PERK上调的作用均减弱,其中以磷脂酰肌醇-3羟基激酶(PI3K)通路阻断剂的作用最显著。结论 E2可抑制TM/DTT诱导的HUVEC内质网应激。p-PERK/PERK通路可能为TM/DTT诱导的HUVEC内质网应激最主要的信号通路。E2可抑制过度内质网应激引起的细胞凋亡。E2受体在E2抑制内质网应激凋亡的作用中起重要作用。E2受体激活包括PI3K-蛋白激酶B(PKB/Akt)、细胞外信号调节激酶1/2(ERK1/2)、c-Jun氨基末端激酶(JNK)和p38-丝裂原活化蛋白激酶(p38-MAPK)在内的信号通路快速起到抑制内质网应激的作用,其中PI3K-Akt通路可能为最主要的通路。雌激素通过抑制PERK信号通路引起的内质网应激凋亡,保护血管内皮细胞,其抑制内质网应激的机制主要为活化的雌激素受体激活PI3K/Akt通路。     

Estrogen-mediated endothelial progenitor cell biology and kinetics for physiological postnatal vasculogenesis

Estrogen has been demonstrated to promote therapeutic reendothelialization after vascular injury by bone marrow (BM)-derived endothelial progenitor cell (EPC) mobilization and phenotypic modulation. We investigated the primary hypothesis that estrogen regulates physiological postnatal vasculogenesis by modulating bioactivity of BM-derived EPCs through the estrogen receptor (ER), in cyclic hormonally regulated endometrial neovascularization. Cultured human EPCs from peripheral blood mononuclear cells (PB-MNCs) disclosed consistent gene expression of ER alpha as well as downregulated gene expressions of ER beta. Under the physiological concentrations of estrogen (17beta-estradiol, E2), proliferation and migration were stimulated, whereas apoptosis was inhibited on day 7 cultured EPCs. These estrogen-induced activities were blocked by the receptor antagonist, ICI182,780 (ICI). In BM transplanted (BMT) mice with ovariectomy (OVX) from transgenic mice overexpressing beta-galactosidase (lacZ) regulated by an endothelial specific Tie-2 promoter (Tie-2/lacZ/BM), the uterus demonstrated a significant increase in BM-derived EPCs (lacZ expressing cells) incorporated into neovasculatures detected by CD31 immunohistochemistry after E2 administration. The BM-derived EPCs that were incorporated into the uterus dominantly expressed ER alpha, rather than ER beta in BMT mice from BM of transgenic mice overexpressing EGFP regulated by Tie-2 promoter with OVX (Tie-2/EGFP/BMT/OVX) by ERs fluorescence immunohistochemistry. An in vitro assay for colony forming activity as well as flow cytometry for CD133, CD34, KDR, and VE-cadherin, using human PB-MNCs at 5 stages of the female menstrual-cycle (early-proliferative, pre-ovulatory, post-ovulatory, mid-luteal, late-luteal), revealed cycle-specific regulation of EPC kinetics. These findings demonstrate that physiological postnatal vasculogenesis involves cyclic, E2-regulated bioactivity of BM-derived EPCs, predominantly through the ER alpha.     

Locally produced estrogen promotes fetal rat metatarsal bone growth; an effect mediated through increased chondrocyte proliferation and decreased apoptosis

The importance of estrogens for the regulation of longitudinal bone growth is unequivocal. However, any local effect of estrogens in growth plate cartilage has been debated. Recently, several enzymes essential for estrogen synthesis were shown to be expressed in rat growth plate chondrocytes. Local production of 17beta-estradiol (E2) has also been demonstrated in rat costal chondrocytes. We aimed to determine the functional role of locally produced estrogen in growth plate cartilage. The human chondrocyte-like cell line HCS-2/8 was used to study estrogen effects on cell proliferation (3H-labeled thymidine uptake) and apoptosis (cell death detection ELISA kit). Chondrocyte production of E2 was measured by RIA and organ cultures of fetal rat metatarsal bones were used to study the effects of estrogen on longitudinal growth rate. We found that significant amounts of E2 were produced by HCS-2/8 chondrocytes (64.1 +/- 5.3 fmol/3 days/10(6) cells). The aromatase inhibitor letrozole (1 microM) and the pure estrogen receptor antagonist ICI 182,780 (10 microM) inhibited proliferation of HCS-2/8 chondrocytes by 20% (P < 0.01) and almost 50% (P < 0.001), respectively. Treatment with ICI 182,780 (10 microM) increased apoptosis by 228% (P < 0.05). Co-treatment with either caspase-3 or pan-caspase inhibitors completely blocked ICI 182,780-induced apoptosis (P < 0.001 vs ICI 182,780 only). Moreover, both ICI 182,780 (10 microM) and letrozole (1 microM) decreased longitudinal growth of fetal rat metatarsal bones after 7 days of culture (P < 0.01). In conclusion, our data clearly show that chondrocytes endogenously produce E2 and that locally produced estrogen stimulates chondrocyte proliferation and protects from spontaneous apoptosis. In addition, longitudinal growth is promoted by estrogens locally produced within the epiphyseal growth plate.     

Clinically used estrogens differentially inhibit human aortic smooth muscle cell growth and mitogen-activated protein kinase activity

Some estrogenic compounds modify vascular smooth muscle cell (SMC) biology; however, whether such effects are mediated in part by estrogen receptors is unknown. The purpose of this study was to evaluate whether the actions of clinically used estrogens on human aortic SMC biology are mediated by estrogen receptors. We examined the effects of various clinically used estrogens in the presence and absence of ICI 182,780, an estrogen receptor antagonist, on cultured human aortic SMC DNA synthesis ([(3)H]thymidine incorporation), cellular proliferation (cell counting), cell migration (modified Boyden chamber), collagen synthesis ([(3)H]proline incorporation), and mitogen-activated protein kinase activity. FCS-induced DNA synthesis, cell proliferation, collagen synthesis, platelet-derived growth factor-induced SMC migration, and mitogen-activated protein kinase activity were significantly inhibited by physiological (10(-9) mol/L) concentrations of 17beta-estradiol and low concentrations (10(-8) to 10(-7) mol/L) of 17beta-estradiol, estradiol valerate, estradiol cypionate, and estradiol benzoate but not by estrone, estriol, 17alpha-estradiol, or estrone sulfate. The inhibitory effects of 17beta-estradiol and other inhibitory estrogens were completely reversed by 100 micromol/L ICI 182,780, and the rank-order potency of various estrogens to inhibit SMC biology matched their rank-order affinity for estrogen receptors. The inhibitory effects of estrogens on SMC biology are in part receptor-mediated. Because the cardioprotective effects of hormone replacement therapy are most likely mediated by modification of SMC biology, whether hormone replacement therapy protects a given postmenopausal woman against cardiovascular disease will depend partially on the affinity of the estrogen for estrogen receptors in vascular SMCs.     

Astrocyte-derived transforming growth factor-{beta} mediates the neuroprotective effects of 17{beta}-estradiol: involvement of nonclassical genomic signaling pathways

17beta-Estradiol (E2) and selective estrogen receptor modulators (SERMs), such as tamoxifen, mediate numerous effects in the brain, including neurosecretion, neuroprotection, and the induction of synaptic plasticity. Astrocytes, the most abundant cell type in the brain, influence many of these same functions and thus may represent a mediator of estrogen action. The present study examined the regulatory effect and underlying cell signaling mechanisms of E2-induced release of neurotropic growth factors from primary rat cortical astrocyte cultures. The results revealed that E2 (0.5, 1, and 10 nm) and tamoxifen (1 mum) increased both the expression and release of the neuroprotective cytokines, TGF-beta1 and TGF-beta2 (TGF-beta), from cortical astrocytes. The stimulatory effect of E2 was attenuated by the estrogen receptor (ER) antagonist, ICI182,780, suggesting ER dependency. The effect of E2 also appeared to involve mediation by the phosphotidylinositol 3-kinase (PI3K)/Akt signaling pathway, because E2 rapidly induced Akt phosphorylation, and pharmacological or molecular inhibition of the PI3K/Akt pathway prevented E2-induced release of TGF-beta. Additionally, the membrane-impermeant conjugate, E2-BSA, stimulated the release of TGF-beta, suggesting the potential involvement of a membrane-bound ER. Finally, E2, tamoxifen, and E2-BSA were shown to protect neuronal-astrocyte cocultures from camptothecin-induced neuronal cell death, effects that were attenuated by ICI182,780, Akt inhibition, or TGF-beta immunoneutralization. As a whole, these studies suggest that E2 induction of TGF-beta release from cortical astrocytes could provide a mechanism of neuroprotection, and that E2 stimulation of TGF-beta expression and release from astrocytes occurs via an ER-dependent mechanism involving mediation by the PI3K/Akt signaling pathway.     

Increased testicular Sertoli cell population induced by an estrogen receptor antagonist

Sertoli cell proliferation is prolonged in neonatal boars treated with the aromatase inhibitor letrozole, but porcine testicular aromatase synthesizes a potent, non-aromatizable androgen, 1-hydroxytestosterone, as well as estradiol. Therefore, experiments were conducted to determine whether the Sertoli cell proliferative response to letrozole is due to a loss of estrogen or a loss of androgen signaling. Littermate boars were treated with letrozole, the estrogen receptor blocker ICI 182,780, or vehicle, from 1 week of age and testes collected at 6.5 weeks. Sertoli cell number was increased 30% by letrozole or ICI 182,780 compared with vehicle. Neither treatment affected testosterone, gonadotropins or prolactin. We conclude that Sertoli cell proliferation in neonatal boars is restricted by the local activation of estrogen receptors. The response to letrozole is apparently not mediated by the novel capacity of the porcine gonadal aromatase for 1-hydroxytestosterone but by estradiol synthesis; therefore, aromatase inhibition may have similar effects on Sertoli cell proliferation in other species.     

Membrane estrogen receptor-dependent extracellular signal-regulated kinase pathway mediates acute activation of endothelial nitric oxide synthase by estrogen in uterine artery endothelial cells

Rapid uterine vasodilatation after estrogen administration is believed to be mediated by endothelial production of nitric oxide (NO) via endothelial NO synthase (eNOS). However, the mechanism(s) by which estrogen activates eNOS in uterine artery endothelial cells (UAEC) is unknown. In this study, we observed that estradiol-17beta (E2) and E2-BSA rapidly (<2 min) increased total NOx production in UAEC in vitro. This was associated with rapid eNOS phosphorylation and activation but was unaltered by pretreatment with actinomycin-D. Estrogen receptor-alpha protein was detectable in isolated plasma membrane proteins by immunoblotting, and E2-BSA-fluorescein isothiocyanate binding was evident on the plasma membrane of UAEC. E2 did not mobilize intracellular Ca2+, but E2 and ionomycin in combination induced greater eNOS phosphorylation than either E2 or ionomycin alone. E2 did not stimulate rapid Akt phosphorylation. E2 stimulated rapid ERK2/1 activation in a time- and dose-dependent manner, with maximal responses observed at 5-10 min with E2 (10 nm to 1 microm) treatment. Acute activation of eNOS and NOx production by E2 could be inhibited by PD98059 but not by LY294002. When E2-BSA was applied, similar responses in NOx production, eNOS, and ERK2/1 activation to those of E2 were achieved. In addition, E2 and E2-BSA-induced ERK2/1 activation and ICI 182,780 could inhibit NOx production by E2. Thus, acute activation of eNOS to produce NO in UAEC by estrogen is at least partially through an ERK pathway, possibly via estrogen receptor localized on the plasma membrane. This pathway may provide a novel mechanism for NO-mediated rapid uterine vasodilatation by estrogen.