Caveolae and mitochondria: A structural interface functionally linking calcium an

描述（由申请人提供）：动脉壁主要由平滑肌细胞组成。通过收缩或放松，这些细胞决定动脉直径，进而调节血流量和血压。动脉平滑肌中钙的浓度部分决定了收缩的程度。钙进入这些细胞的一个主要来源是通过电压依赖性的l型钙通道。本研究的总体目标是研究尚不清楚的动脉平滑肌钙通道功能的控制机制。更具体地说，本研究探讨了一种新的调节机制，即局部氧化和钙信号微域在动脉平滑肌细胞中功能收敛。这促进了l型钙通道活性的增加，平滑肌细胞内钙的增加，最终导致动脉收缩。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，我们提议测试一个模型，其中氧化还原和钙微域信号的收敛需要与外周线粒体密切相反的质乳小泡（含有NADPH氧化酶和l型钙通道）。我们还将研究由此产生的氧化还原/钙信号耦合是否有助于正常动脉功能和肥胖的动脉功能障碍。特异性目的1验证了NADPH氧化酶和l型钙通道在动脉平滑肌小窝中共定位产生功能偶联的氧化还原和钙微域的假设。特异性目标2测试了外周线粒体亚群调节氧化还原和钙微域的功能偶联的假设。特异性目的3验证了功能偶联氧化还原和钙微域增加导致肥胖动脉功能障碍的假设。这些特定目标的实验将使用电压钳电生理学，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加压动脉的组合来检查健康和肥胖动物动脉平滑肌中氧化还原和钙微域信号传导的小窝和线粒体的结构和功能作用。这些实验的结果将为肥胖症中潜在的动脉功能障碍事件提供机制见解，并可能导致开发新的合理治疗方法来管理和预防心血管疾病。

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.