Coronary ion channels, gender and vasoreactivity

描述（由申请人提供）：最近的前瞻性临床试验强调了我们缺乏关于性别和性激素对冠状动脉血管的影响的知识，特别是在细胞/分子水平上。性激素对血管平滑肌的增殖、离子通道活性和通道表达等生理机能有着深远的影响。冠状动脉平滑肌（CSM）离子通道的活性对冠状动脉血流的调节和血管疾病的进展至关重要。我们已经证明CSM钙和钾通道活性受性别的强烈影响。本研究将确定CSM电压门控钙（VGCC）和K通道活性的性别差异机制，以及随之对细胞内钙（Cai）调节和血管反应性的影响。总的假设是，男性冠状动脉反应性更强，是由于睾酮（TST）依赖性VGCC合成和pkc依赖性活性的增加，导致冠状血管痉挛（CVS）的倾向增强。体内（完整的、去性腺的和激素替代的猪）和体外技术将用于确定性别、TST和雌激素（E2）在调节CSM离子通道活性和冠状动脉反应性中的作用。目的1将利用电生理、免疫印迹和RT-PCR确定性别和性激素诱导VGCC活性和表达变化的机制。由于血管反应活性和VGCC活性都受PKC调节，性别和性激素对PKC调节VGCC的影响也将被研究。由于VGCC活性对Cai和收缩的影响是通过耦合到K通道和细胞钙缓冲来调节的，Aim 2将使用同步电压钳和微荧光法确定VGCC活性的性别差异对ca激活（BK）和电压依赖的K电流（Kv）和Cai的影响。生理和病理生理条件下，VGCC活性的性别差异对大血管和微血管反应性的影响将在体外和体内进行评估。CVS模型将用于评估VGCC活性的性别特异性差异在血管疾病进展和严重程度中的作用。本研究的目的是确定CSM中与性别相关的细胞和分子差异，因为它们与冠状动脉疾病倾向的性别差异有关。

DESCRIPTION (provided by applicant): Recent prospective clinical trials have underscored our lack of knowledge with regard to the effect of gender and sex hormones on the coronary vasculature, especially at the cellular/molecular level. Sex hormones exert profound influence on vascular smooth muscle physiology, including proliferation, ion channel activity and channel expression. Coronary smooth muscle (CSM) ion channel activity is central to both regulation of coronary blood flow and progression of vascular disease. We have demonstrated that CSM calcium and potassium channel activities are strongly influenced by gender. This study will determine the mechanism for gender-specific differences in CSM voltage-gated calcium (VGCC) and K channel activity and the consequent effects on intracellular calcium (Cai) regulation and vasoreactivity. The overall hypothesis is that coronary arterial reactivity in males is greater due to a testosterone (TST)-dependent increase in VGCC synthesis and PKC-dependent activity resulting in an enhanced propensity for coronary vasospasm (CVS). Both in vivo (intact, gonadectomized and hormone-replaced swine) and in vitro techniques will be used to determine the role of gender, TST and estrogen (E2) in regulating CSM ion channel activity and coronary arterial reactivity. Aim 1 will determine the mechanism of gender and sex hormone induced changes in VGCC activity and expression using electrophysiology, immunoblot and RT-PCR. As both vasoreactivity and VGCC activity are modulated by PKC, gender and sex hormone effects on PKC regulation of VGCC will also be examined. As the effect of VGCC activity on Cai and contraction is modulated via coupling to K channels and cellular calcium buffering, Aim 2 will determine the effect of sex-specific differences in VGCC activity on Ca-activated (BK) and voltage-dependent K current (Kv) and Cai using simultaneous voltage clamp and microfluorometry. The effect of sex-specific differences in VGCC activity on macro- and microvascular reactivity under both physiological and pathophysiological conditions will be assessed in vitro and in vivo in Aim 3. A model of CVS will be used to assess the role of sex-specific differences in VGCC activity on the progression and severity of vascular disease. The goal of this research is to determine gender-related cellular and molecular differences in CSM as they relate to gender differences in propensity for coronary disease.