Caveolae and mitochondria: A structural interface functionally linking calcium an

描述(申请人提供)：动脉壁主要由平滑肌细胞组成。通过收缩或松弛，这些细胞决定动脉直径，进而调节血流和血压。动脉平滑肌中钙的浓度在一定程度上决定了收缩的程度。钙离子进入这些细胞的一个主要来源是通过电压依赖的L型钙通道。这项建议的总体目标是研究目前知之甚少的控制动脉平滑肌钙通道功能的机制。更具体地说，这项研究探索了一种新的调控机制，在这种机制中，局部氧化剂和钙信号微域在功能上聚合在动脉平滑肌细胞中。这促进了L型钙通道活性的增加，增加了平滑肌细胞内的钙离子，最终导致了动脉收缩。重要的是，氧化应激增加和钙通道活性增加被认为与肥胖相关心血管疾病(如高血压和中风)的血管功能障碍有关。在这个应用中，我们建议测试一个模型，在这个模型中，氧化还原和钙微域信号的融合需要质膜小窝(包含NADPH氧化酶和L型钙通道)，而质膜小窝与外周线粒体密切相反。我们还将调查由此产生的氧化还原/钙信号耦合是否有助于正常的动脉功能和肥胖患者的动脉功能障碍。特异性目的1验证NADPH氧化酶和L类钙通道共存于血管小窝产生氧化还原功能偶联微域和钙微域的假说。特定目的2验证了外周线粒体亚群调节氧化还原微域和钙微域功能耦合的假设。特定目标3测试了功能偶联氧化还原和钙微域增加导致肥胖患者动脉功能障碍的假设。这些特定目标的实验将结合电压钳电生理学、全内反射荧光(TIRF)显微镜、分子生物学、透射电子显微镜和完整的加压动脉来研究健康和肥胖动物动脉平滑肌中小窝和线粒体在氧化还原和钙微域信号中的结构和功能作用。这些实验的结果将为肥胖的动脉功能障碍的潜在事件提供机械性的见解，并可能导致管理和预防心血管疾病的新的合理疗法的开发。

DESCRIPTION (provided by applicant): The walls of arteries are largely composed of smooth muscle cells. By contracting or relaxing, these cells determine arterial diameter, which in turn regulates blood flow and blood pressure. The concentration of calcium in arterial smooth muscle determines in part the degree of contraction. A major source of calcium entry into these cells is through voltage-dependent L-type calcium channels. The general goal of this proposal is to investigate the poorly understood mechanisms controlling calcium channel function in arterial smooth muscle. More specifically, this research investigates a novel regulatory mechanism where localized oxidant and calcium signaling microdomains functionally converge in arterial smooth muscle cells. This promotes increased L-type calcium channel activity, increased calcium within the smooth muscle cells, and ultimately arterial contraction. Importantly, increased oxidative stress and increased calcium channel activity are thought to be related to vascular dysfunction in obesity-related cardiovascular diseases such as hypertension and stroke. In this application we propose to test a model where the convergence of redox and calcium microdomain signaling requires plasmalemmal caveolae (containing NADPH oxidase and L-type calcium channels) that are closely opposed to peripheral mitochondria. We will also investigate if the resulting coupled redox/calcium signaling contributes to normal arterial functio and to arterial dysfunction in obesity. Specific Aim 1 tests the hypothesis that NADPH oxidase and L-type calcium channels colocalize in caveolae to produce functionally coupled redox and calcium microdomains in arterial smooth muscle. Specific Aim 2 tests the hypothesis that a subpopulation of peripheral mitochondria modulate functional coupling of redox and calcium microdomains. Specific Aim 3 tests the hypothesis that increased functionally coupled redox and calcium microdomains contribute to arterial dysfunction in obesity. The experiments in these Specific Aims will use a combination of voltage-clamp electrophysiology, total internal reflection fluorescence (TIRF) microscopy, molecular biology, transmission electron microscopy, and intact pressurized arteries to examine the structural and functional role of caveolae and mitochondria in redox and calcium microdomain signaling in arterial smooth muscle from healthy and obese animals. The outcome of these experiments will provide mechanistic insights into events underlying arterial dysfunction in obesity and may lead to the development of new rational therapies for managing and preventing cardiovascular disease.