Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina.
Post-translational regulation of endothelial nitric oxide synthase (eNOS) by estrogens in the rat vagina.
复制标题
大鼠阴道雌激素对内皮一氧化氮合酶（eNOS）的翻译后调节。
DOI：
10.1111/j.1743-6109.2010.01750.x
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发表时间：
2010
期刊：
影响因子：
--
通讯作者：
Burnett,ArthurL
中科院分区：
文献类型：
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作者：
Musicki,Biljana;Liu,Tongyun;Strong,TravisD;Lagoda,GwenA;Bivalacqua,TrinityJ;Burnett,ArthurL
雌激素在女性性唤醒过程中主要通过一氧化氮(NO)调控阴道血流。虽然雌激素的血管效应归因于增加内皮细胞NO的产生，但雌激素调节阴道内皮细胞一氧化氮合酶(ENOS)的机制尚不清楚。目的我们的假设是雌激素在翻译后调节阴道内的eNOS，提供了一种机制，在不改变eNOS蛋白表达的情况下影响NO的生物利用度。方法我们检测了动情间期雌性大鼠阴道远端和近端eNOS的磷酸化及其与小窝蛋白-1和热休克蛋白90(HSP90)的相互作用。大鼠去卵巢后7天和去卵巢大鼠去卵巢后2天用雌二醇17β(15µg)替代。主要结果雌激素调节大鼠阴道eNOS的分子机制。结果用免疫组织化学方法将磷酸化eNOS(Ser1177)定位于血管内皮细胞和阴道窦。停用雌激素后，eNOS正调控位点(Ser-1177)的磷酸化水平降低，eNOS与其负调控基因Caveolin-1的结合增加(不影响eNOS/HSP90的相互作用)，雌激素替代后两者均恢复正常。雌激素对eNOS(Ser-1177)的磷酸化Akt和细胞外信号调节蛋白激酶1/2(ERK1/2)的蛋白表达无明显影响，提示雌激素对eNOS(Ser-1177)磷酸化的影响不是由激活的AKT或ERK1/2介导的。eNOS负性调节位点(Ser-114)的磷酸化水平随雌激素水平的降低而增加，经雌激素替代后恢复正常，提示雌激素维持eNOS在该部位的低磷酸化水平可能限制eNOS与小窝蛋白-1的相互作用并保持酶的活性。卵巢切除或雌二醇替代对总eNOS、诱导型一氧化氮合酶、小窝蛋白-1和HSP90蛋白表达无明显影响。结论这些结果明确了新的雌激素信号传导机制，涉及eNOS的磷酸化和eNOS-小窝蛋白-1的相互作用。
IntroductionEstrogens control vaginal blood flow during female sexual arousal mostly through nitric oxide (NO). Although vascular effects of estrogens are attributed to an increase in endothelial NO production, the mechanisms of endothelial NO synthase (eNOS) regulation by estrogens in the vagina are largely unknown.AimsOur hypothesis was that estrogens regulate eNOS post-translationally in the vagina, providing a mechanism to affect NO bioavailability without changes in eNOS protein expression.MethodsWe measured eNOS phosphorylation and eNOS interaction with caveolin-1 and heat shock protein 90 (HSP90) in the distal and proximal vagina of female rats at diestrus, 7 days after ovariectomy and 2 days after replacement of ovariectomized rats with estradiol-17β (15 µg).Main Outcome MeasuresMolecular mechanisms of eNOS regulation by estrogen in the rat vagina.ResultsWe localized phospho-eNOS (Ser-1177) immunohistochemically to the endothelium lining blood vessels and vaginal sinusoids. Estrogen withdrawal decreased phosphorylation of eNOS on its positive regulatory site (Ser-1177) and increased eNOS binding to its negative regulator caveolin-1 (without affecting eNOS/HSP90 interaction), and they were both normalized by estradiol replacement. Protein expressions of phosphorylated Akt (protein kinase B) and extracellular signal-regulated protein kinase 1/2 (ERK1/2) were not affected by estrogen status, suggesting that the effect of estrogens on eNOS (Ser-1177) phosphorylation was not mediated by activated AKT or ERK1/2. eNOS phosphorylation on its negative regulatory site (Ser-114) was increased in the vagina by estrogen withdrawal and normalized by estradiol replacement, implying that the maintenance of low phosphorylation of eNOS on this site by estradiol may limit eNOS interaction with caveolin-1 and preserve the enzyme’s activity. Total eNOS, inducible NOS, caveolin-1, and HSP90 protein expressions were not affected by ovariectomy or estradiol replacement in the distal or proximal vagina.ConclusionsThese results define novel estrogen signaling mechanisms in the vagina which involve eNOS phosphorylation and eNOS–caveolin-1 interaction.