Type 2 Diabetes and Vascular BK Channel Regulation

描述(申请人提供)：我们研究的总体目标是了解心血管系统中离子通道的调节机制。我们观察到Zucker糖尿病肥胖(ZDF)大鼠冠状动脉平滑肌细胞的大电导钙激活钾(BK)通道对电压和钙激活的反应发生改变。ZDF大鼠是一种已建立的研究2型糖尿病的模型，其冠脉血管表现为内皮依赖性和非依赖性的松弛受损。由于BK通道是介导血管松弛的主要离子决定因素，也是内皮衍生超极化因子(EDHFs)的靶点，BK通道调节的改变可能在2型糖尿病血管内皮依赖性和非内皮依赖性血管功能障碍中起作用。将检验以下假设：a)花生四烯酸代谢产物对血管松弛的调节在2型糖尿病中是异常的，花生四烯酸代谢物可能是EDHs。2)2型糖尿病患者EDHF介导的血管松弛功能异常是由于血管BK通道调节异常所致。C)BK通道调节异常是由于通道结构改变所致。1)为了研究花生四烯酸对正常大鼠和ZDF大鼠冠脉血管的松弛作用，我们计划用药理学方法和微电极测量冠脉血管膜电位来确定细胞色素P450和脂氧合酶途径在花生四烯酸效应中的作用。2)用全细胞和单通道电生理方法测定BK通道特性，包括通道密度、I-V关系、V1/2、钙EC50、对通道激动剂的反应和通道动力学，以确定ZDF大鼠BK通道调节的变化。3)为了确定BK通道缺陷的分子机制，我们将用生化和分子方法探索三种可能的机制，即蛋白激酶C的激活，通道变异体的表达，以及与通道β亚基的功能解偶联。这一研究有助于我们更好地了解血管BK通道的调节，揭示EDHF在糖尿病冠脉生理调节中的作用。我们的结果可能为2型糖尿病相关的血管反应性异常的发病机制提供重要的机制认识，并可能导致预防和治疗2型糖尿病患者心血管并发症的新方法的发展。

DESCRIPTION (provided by applicant): The overall objective of our research is to understand the mechanisms that regulate ion channels in the cardiovascular system. We made a novel observation that the large conductance Ca2+-activated K+ (BK) channels in smooth muscle cells from small coronary arteries of the Zucker diabetic fatty (ZDF) rats showed altered response to voltage and Ca 2+ activation. ZDF rats are an established model for the study of type 2 diabetes and their coronary vessels showed impaired endothelium-dependent and -independent relaxation. Since BK channels are the major ionic determinant in mediating vasorelaxation and are the target of endothelium-derived hyperpolarizing factors (EDHFs), altered BK channel regulation may contribute to both endothelium-dependent and -independent vascular dysfunction in type 2 diabetes. The following hypotheses will be tested: A) Regulation of vasorelaxation by arachidonic acid metabolites, which are putative EDHFs, is abnormal in type 2 diabetes. B) Abnormal EDHF-mediated vasorelaxation in type 2 diabetes is due to abnormal regulation of vascular BK channels. C) Abnormal BK channel regulation is due to structural modification of the channel. We propose the following specific aims: 1) To determine the arachidonic acid-mediated coronary vasorelaxation in control and ZDF rats, we plan to determine the contribution of the cytochrome P450 and the lipoxygenase pathways to the arachidonic acid effects using pharmacological approaches and microelectrode measurements of membrane potential in isolated coronary vessels. 2) To determine the alterations in the regulation of BK channels in ZDF rats, we will determine the BK channel properties including channel density, I-V relationships, V1/2, Ca 2+ EC50, response to channel activators, and channel kinetics using the whole-cell and single-channel electrophysiological approaches. 3) To determine the molecular mechanisms of the BK channel defects, we will explore three plausible mechanisms, namely activation of protein kinase C, expression of channel variants, and functional uncoupling with the channel beta subunit using biochemical and molecular approaches. This study may help us to better understand the regulation of vascular BK channels and to delineate the role of EDHF in the regulation of coronary physiology in diabetes. Our results may provide important mechanistic insight into the pathogenesis of abnormal vasoreactivity associated with type 2 diabetes and may lead to the development of new approaches in the prevention and treatment of cardiovascular complications in patients with type 2 diabetes.