Actions of Estrogen on Uterine Artery Endothelium

项目摘要 我们在上一个资金周期中由这个RO1资助的工作已经确定了内生氢 硫化物(H_2S)是由选择性上调的H_2S合成酶--胱硫醚β合成酶(CBS)产生的。 一种新的子宫动脉(UA)扩张器系统有助于雌激素诱导和妊娠相关的升高 子宫血流。这一新的途径重塑了人们对子宫血流动力学如何在 正常怀孕。最近，我们报道了硫化氢通过激活Smooth来刺激人类尿酸松弛 肌肉(SM)大电导钙激活的电压门控钾(BKCa)通道；然而，硫化氢是如何 在正常和复杂妊娠中，调节雌激素诱导的UA扩张的作用在很大程度上仍不清楚。 蛋白质中反应性半胱氨酸(S)上-SSH基团的形成，称为硫水化或过硫化。 已成为硫化氢发挥其生物学功能的主要信号途径。硫水化反应可自由转化 硫醇(-SH)转变为过硫化物(-SSH)导致修饰半胱氨酸的反应性增加 SSH与SH的亲核性比较。在这一竞争性续订RO1应用程序中，我们提供了新数据 显示雌激素和怀孕可以显著刺激人类尿酸中的蛋白质硫水化；以及更多 有趣的是，BKCa的总水合蛋白和硫水化β1和γ1亚基水平的升高 正常妊娠时，子痫前期患者尿酸通道明显减少。因此，我们建议 检验一个新的假设，即增强的CBS/H_2S硫水化增加的β1和γ1 BKCa(通过 雌激素受体依赖的转录)导致SM BKCa激活以介导雌激素诱导 UA在正常妊娠时扩张，这一机制在先兆子痫中受损。这个猜想将会是 通过两个特定的目标进行测试，以确定雌激素反应的BKCa通道以及如何 硫酸水合导致这些BKCa通道的激活，这些通道与妊娠期尿酸扩张和 子痫前期，具有全面的生化、细胞、分子、药理、生理和 UA细胞体外原代培养模型的电生理学研究 子宫切除术后不同雌激素状态对人体主要尿酸影响的研究 正常妊娠和子痫前期妊娠的子宫肌层尿酸样本，以及在活体大鼠模型中的作用 体内的外源性和内源性雌激素。拟议的研究将建立一个新的机制，以 通过硫酸水合调节β1和γ1亚基激活BKCa通道，从而广泛影响离子通道 生物学。这些研究将理解雌激素反应性SM激活的具体机制 BKCa通道与雌激素诱导的妊娠和子痫前期尿酸扩张有关。所获得的数据将 促进我们对雌激素和子宫血流生物学的理解，为帮助 制定防治先兆子痫的替代战略。获得的新数据也会让人大吃一惊 对雌激素心血管保护作用的认识。 1

Project Summary Our work funded by this RO1 during this last funding cycle has established that endogenous hydrogen sulfide (H2S) produced by selective upregulated H2S synthesizing enzyme cystathionine β-synthase (CBS) is a new uterine artery (UA) dilator system contributing to estrogen-induced and pregnancy-associated rises in uterine blood flow. This novel pathway has reshaped the view how uterine hemodynamics is regulated during normal pregnancy. More recently, we reported that H2S stimulates human UA relaxation via activating smooth muscle (SM) large conductance Ca2+-activated voltage-gated potassium (BKCa) channels; yet, how H2S mediates estrogen-induced UA dilation in normal and complicated pregnancies remains largely unknown. Formation of -SSH groups on reactive cysteine(s) in proteins, referred to as sulfhydration or persulfidation, has emerged as the main signaling route for H2S to exert its biological function. Sulfhydration converts free thiols (-SH) to persulfide (-SSH) resulting in increased reactivity of modified cysteines due to increased nucleophilicity of SSH compared with SH. In this competitive renewal RO1 application, we present new data showing that estrogen and pregnancy can significantly stimulate protein sulfhydration in human UA; and more interestingly, the elevated levels of total sulfhydrated proteins and sulfhydrated β1 and γ1 subunits of BKCa channels in human UA in normal pregnancy is significantly reduced in preeclampsia. Thus, we propose to test a novel hypothesis herein that augmented CBS/H2S sulfhydrates the increased β1 and γ1 BKCa (via estrogen receptor-dependent transcription) resulting in activation of SM BKCa to mediate estrogen-induced UA dilation in normal pregnancy and this mechanism is impaired in preeclampsia. This conjecture will be tested by two specific aims targeting on determining the estrogen-responsive BKCa channels and how sulfhydration results in activation of these BKCa channels pertaining to UA dilation in pregnancy and preeclampsia, with comprehensive biochemical, cellular, molecular, pharmacological, and physiological and electrophysiology approaches using in vitro primary cell culture models of UA smooth muscle cells, ex vivo studies of human main UA samples associated with different estrogens status from hysterectomy and myometrial UA samples from normal and preeclamptic pregnancies, and in vivo rat models to study the role of exogenous and endogenous estrogens in vivo. The proposed studies will establish a novel mechanism for BKCa channel activation via sulfhydrating its regulatory β1 and γ1 subunits to broadly impact on ion channel biology. These studies will comprehend specific mechanisms for activation of the estrogen-responsive SM BKCa channels pertaining to estrogen-induced UA dilation in pregnancy and preeclampsia. Data obtained will advance our understanding of estrogens and uterine blood flow biology, informing new pathways to assist the development of alternative strategies for combating preeclampsia. New data obtained will also shed lights on the understanding of the cardiovascular protective effects of estrogens. 1