Type 2 Diabetes and Vascular BK Channel Regulation

描述（由申请人提供）：我们研究的总体目标是了解心血管系统中调节离子通道的机制。 在Zucker糖尿病肥胖（ZDF）大鼠小冠状动脉平滑肌细胞上发现了钙激活的大电导钾通道（BK通道）对电压和钙激活的反应性改变。 ZDF大鼠是研究2型糖尿病的成熟模型，其冠状动脉血管显示内皮依赖性和非依赖性舒张受损。 由于BK通道是介导血管舒张的主要离子决定因素，并且是内皮源性超极化因子（EDHF）的靶点，因此BK通道调节的改变可能导致2型糖尿病中内皮依赖性和非依赖性血管功能障碍。 将测试以下假设：A）花生四烯酸代谢物（推定的EDHF）对血管舒张的调节在2型糖尿病中是异常的。 B）2型糖尿病中异常的EDHF介导的血管舒张是由于血管BK通道的异常调节。 C）BK通道调节异常是由于通道的结构修饰。 我们提出了以下具体目标：1）为了确定花生四烯酸介导的冠状动脉血管舒张在对照组和ZDF大鼠，我们计划确定细胞色素P450和脂氧合酶途径的花生四烯酸的作用，使用药理学方法和微电极测量膜电位在离体冠状动脉血管。 2)为了确定ZDF大鼠BK通道调节的改变，我们将使用全细胞和单通道电生理方法测定BK通道的特性，包括通道密度、I-V关系、V1/2、Ca 2+ EC 50、对通道激活剂的反应以及通道动力学。 3)为了确定BK通道缺陷的分子机制，我们将探索三种可能的机制，即蛋白激酶C的激活，通道变体的表达，以及使用生物化学和分子方法与通道β亚基的功能解偶联。 本研究有助于我们更好地了解血管BK通道的调节，并阐明EDHF在糖尿病冠状动脉生理调节中的作用。 我们的研究结果可能为2型糖尿病相关血管反应性异常的发病机制提供重要的机制见解，并可能导致2型糖尿病患者心血管并发症预防和治疗的新方法的发展。

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.