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系统影响的知识，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有着重要的影响。冠状动脉平滑肌（CSM）离子通道活性是调节冠状动脉血流和血管疾病进展的中心。我们已经证明，CSM钙和钾通道的活动受到性别的强烈影响。本研究将确定CSM电压门控钙（VGCC）和K通道活性的性别特异性差异的机制，以及对细胞内钙（Cai）调节和血管反应性的影响。总体假设是，由于VGCC合成和PKC依赖性活性的睾酮（TST）依赖性增加导致冠状动脉痉挛（CVS）倾向增强，因此男性冠状动脉反应性更大。将使用体内（完整、性腺切除和去势替代猪）和体外技术来确定性别、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通过电生理、免疫印迹和RT-PCR等方法，探讨性别和性激素诱导VGCC活性和表达变化的机制。由于血管反应性和VGCC活性均受PKC调节，因此还将检查性别和性激素对VGCC的PKC调节的影响。由于VGCC活性对Cai和收缩的影响是通过与K通道和细胞钙缓冲的偶联来调节的，因此目标2将使用同步电压钳和显微荧光测定法确定VGCC活性的性别特异性差异对Ca激活（BK）和电压依赖性K电流（Kv）和Cai的影响。在目的3中，将在体外和体内评估生理和病理生理条件下VGCC活性的性别特异性差异对大血管和微血管反应性的影响。CVS模型将用于评估VGCC活性的性别特异性差异对血管疾病进展和严重程度的作用。本研究的目的是确定CSM中与性别相关的细胞和分子差异，因为它们与冠心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.