Signaling Mechanisms Underlying Myogenic Tone in Arterioles of Skeletal Muscle: R

描述(申请人提供)：压力诱导的(肌源性)血管收缩是设定外周阻力和自动调节血流的关键。肌源性收缩依赖于平滑肌(SM)膜去极化和电压依赖性钙内流。尽管肌源性反应是小动脉SM的固有特性，但它受到超极化和血管扩张剂影响的调节。钙敏感大电导钾离子通道(BKCa)是SM中许多血管扩张剂刺激的重要中介。BKCa通过去极化和细胞内[Ca~(2+)]升高来激活，被认为是一种‘反馈’抑制物，防止随着腔内压的增加而过度的小动脉肌源性收缩。这一假说在很大程度上是基于在大脑小动脉上进行的研究，然而我们的工作并不支持BKCa在调节骨骼肌小动脉的肌源性张力方面发挥相同的作用，骨骼肌小动脉是外周阻力的主要贡献者。有趣的是，我们的研究表明，骨骼肌小动脉和大脑小动脉在SM膜电位和压力引起的收缩之间的关系上存在根本的差异，这表明在骨骼肌血管中，BKCa的激活可能被最小化，以允许在静息条件下持续的血管收缩。这项研究的总体目标是明确BKCa在骨骼肌小动脉SM中的生理作用和调节机制。BKCa的调控是复杂的，在分子水平和翻译后水平上都有调控，同时也受到BKCa相对于其他信号相关分子的细胞位置的调控。这种调节的复杂性为组织特异性异质性提供了广泛的机会，其中通道行为与局部组织要求相匹配。因此，我们的总体假设是，BKCa在脑肌和骨骼肌的血管SM中受到不同的调节。此外，BKCa调节的这种区域差异允许局部血流动力学控制与组织功能适当匹配。结合分子、细胞和电生理方法，在骨骼肌和脑血管制备中，本研究旨在：1.检测BKCa亚基的组成和表达；2.利用siRNA敲除方法测试BKCa亚基在调节肌源性收缩中的作用；3.确定BKCa在分离的SM细胞中的调节机制，重点是钙离子和电压敏感性；cGMP/cGMP依赖的蛋白激酶的调节；4.测试BKCa在调节分离血管的肌张力中的作用，重点是张力的反馈调节和BKCa通道对cGMP/cGMP依赖的蛋白激酶和血管扩张机制激活第二信使系统的敏感性。了解BKCa功能调节的组织特异性机制将影响作为血管疾病治疗措施开发的通道调节剂的设计和使用。公共卫生相关性：这项研究将提高我们对小动脉如何改变其直径从而控制局部血流的了解。重要的是，获得的数据还将有助于识别在病理生理情况下可能改变的生化部位。因此，有可能识别细胞信号中的步骤，这些步骤最终可能用于药物治疗的靶向。

DESCRIPTION (provided by applicant): Pressure-induced (myogenic) vasoconstriction is key to setting of peripheral resistance and autoregulation of blood flow. Myogenic constriction depends on smooth muscle (SM) membrane depolarization and voltage- dependent Ca2+-influx. Although myogenic responsiveness is an inherent property of arteriolar SM, it is modulated by hyperpolarizing and vasodilator influences. An important mediator of a number of vasodilator stimuli in SM is the Ca2+-sensitive large-conductance K+ ion channel (BKCa). Activation of BKCa by depolarization and increased intracellular [Ca2+] has been proposed to serve as a 'feedback' inhibitor, preventing excessive myogenic constriction of arterioles as intralumenal pressure increases. This hypothesis is largely based on studies performed in small cerebral arteries, however our work does not support an identical role for BKCa in regulating myogenic tone of skeletal muscle arterioles, which are a major contributor to peripheral resistance. Interestingly, our studies suggest a fundamental difference between skeletal muscle arterioles and small cerebral arteries in the relationship between SM membrane potential and pressure-induced constriction suggesting that in skeletal muscle vessels BKCa activation may be minimized to allow sustained vasoconstriction under resting conditions. The overall goals of the studies in this proposal are to define the physiological role and mechanisms of regulation for BKCa in arteriolar SM of skeletal muscle. Control of BKCa is complex with regulation occurring at the molecular and post-translational levels as well as by its cellular location with respect to other signaling-related molecules. This complexity of regulation provides extensive opportunity for tissue-specific heterogeneity where channel behavior is matched to local tissue requirements. Thus, our overall hypothesis is that BKCa is differentially regulated in vascular SM from cerebral and skeletal muscle. Further, such regional differences in regulation of BKCa allow for local control of hemodynamics to be appropriately matched to tissue function. Using a combination of molecular, cellular and electrophysiological approaches, in skeletal muscle and cerebral vascular preparations, the proposed studies aim to: 1. examine subunit composition and expression of BKCa; 2. test the role of BKCa subunits in modulating myogenic constriction using an siRNA knockdown approach; 3. define mechanisms of BKCa regulation in isolated SM cells with emphasis on Ca2+ and voltage sensitivity; modulation by cGMP/cGMP-dependent protein kinase; 4. test the role of BKCa in modulating myogenic tone of isolated vessels, focusing on both feedback-modulation of tone and the sensitivity of the BKCa channel to cGMP/cGMP- dependent protein kinase and vasodilator mechanisms activating this second messenger system. Understanding tissue-specific mechanisms by which BKCa function is regulated will impact on design and utilization of channel modulators developed as therapeutic measures for treatment of vascular diseases. PUBLIC HEALTH RELEVANCE: This study will improve our knowledge of how small arteries can vary their diameters and thus control local blood flow. Importantly, the data obtained will also contribute to identifying biochemical sites that may be altered in pathophysiological situations. Thus the potential exists for the identification of steps in cellular signaling that may ultimately be used for the targeting of pharmacological therapies.