Exploring Vasomotor Mechanisms using New PKG Inhibitors and cGMP Biosensors

描述（由申请人提供）：细胞内cGMP和Ca2+调节血管平滑肌（VSM）在健康和疾病中的功能。新兴的观点是细胞内Ca2+信号是高度动态的，Ca2+信号的模式决定了VSM的功能。对细胞内cGMP信号动力学、cGMP依赖性蛋白激酶（PKG）的后续激活及其与Ca2+信号的关系的理解滞后。在这里，使用新的cGMP生物传感器和PKG抑制剂，我们将提供证据，cGMP，像Ca2+，是空间和时间动态的，并依赖于多个相互关联的控制机制。我们的方法将涉及Ca2+和cGMP的高分辨率测量，并提供这些信使分子对VSM功能的交互控制的无与伦比的视图。在具体目标1中，我们将测试膜（pGC）和细胞质（sGC）观基环化酶参与cGMP形成的根本不同模式的理论。我们认为，分别激活sGC和pGC的NO和ANP在细胞内形成了离散的cGMP池，从而为cGMP信号的功能分区化提供了结构基础。我们还将测试PKG通过作为cGMP池的负反馈和正反馈调节器来控制血管张力的机制。我们最近开发的新型cGMP生物传感器使这些评估成为可能，该传感器允许以高时间和空间分辨率直接测量细胞cGMP。在Specific Aim 2中，我们将阐明cGMP/PKG与钙信号传导之间的相互作用。我们认为cGMP和PKG部分通过Ca2+火花、BKCa通道和全局钙的调节来调节血管功能。在Specific Aim 3中，我们将通过研究PKG抑制剂在完整动物体内增加血压和血管阻力的功效来确定PKG对体内血管控制的贡献。我们测试这三个目标的方法将是多学科的，采用最先进的技术，从生理学（高速钙成像，膜片钳技术，阻力动脉肌图，血压和血流测量），细胞生物学（共聚焦荧光显微镜，比例荧光显微镜，平滑肌细胞培养），分子生物学（昆虫细胞培养，诱变，腺病毒）和生物化学(酶动力学技术，荧光能量传递，细胞蛋白质传递系统)。这项工作将为调节血管平滑肌PKG活动的因素提供一个综合的观点，从而显著提高我们对健康和疾病中动脉功能的理解。cgmp依赖性蛋白激酶（PKG）是全身血管细胞功能的重要调节因子。本研究旨在确定涉及PKG及其信号伙伴的血管控制的分子机制。了解血管如何收缩和扩张对于开发新的策略和治疗药物至关重要，这些药物旨在预防和治疗血管疾病，如高血压、中风和冠状动脉疾病。

DESCRIPTION (provided by applicant): Intracellular cGMP and Ca2+ regulate vascular smooth muscle (VSM) function in health and disease. The emerging view is that intracellular Ca2+ signals are highly dynamic, and that patterning of Ca2+ signals determines VSM function. Understanding of intracellular cGMP signaling dynamics, subsequent activation of cGMP-dependent protein kinase (PKG), and its relationship to Ca2+ signals has lagged. Here, using novel cGMP biosensors and PKG inhibitors, we will provide evidence that cGMP, like Ca2+, is spatially and temporally dynamic, and dependent on multiple interrelated control mechanisms. Our approach will involve high resolution measurements of Ca2+ and cGMP, and provide an unparalleled view of the interactive control of VSM function by these messenger molecules. In Specific Aim 1 we will test the theory that membrane (pGC) and cytosolic (sGC) guanylyl cyclases engage fundamentally different patterns of cGMP formation. We propose that NO and ANP, which primarily activate sGC and pGC, respectively, create discrete pools of intracellular cGMP, and thus provide the structural basis for the functional compartmentalization of cGMP signals. We will also test the mechanisms by which PKG controls vascular tone by serving as both a negative and a positive feedback regulator for cGMP pools. These assessments have been made possible by our recent development of novel cGMP-biosensors, which allow direct measurement of cellular cGMP with high temporal and spatial resolution. In Specific Aim 2 we will elucidate the interplay between cGMP/PKG and calcium signaling. We propose that cGMP and PKG act in part through modulation of Ca2+ sparks, BKCa channels, and global calcium to regulate vascular function. In Specific Aim 3 we will determine the contributions of PKG to vascular control in vivo by studying the efficacy of PKG inhibitors to increase blood pressure and vascular resistance in the intact animal. Our approach to test the three aims of this proposal will be multidisciplinary, employing state-of-the-art techniques from physiology (high speed calcium imaging, patch clamp techniques, resistance artery myography, blood pressure and blood flow measurements), cell biology (confocal fluorescence microscopy, ratiometric fluorescence microscopy, smooth muscle cell culture), molecular biology (insect cell culture, mutagenesis, adenovirus) and biochemistry (enzyme kinetic techniques, fluorescence energy transfer, cellular protein delivery systems). This work will provide an integrated view of the factors that modulate PKG activity in vascular smooth muscle and thereby significantly enhance our understanding of arterial functions in health and disease. PUBLIC HEALTH RELEVANCE The cGMP-dependent protein kinase (PKG) is an essential regulator of cellular function in blood vessels throughout the body. This proposal seeks to ascertain the molecular mechanisms of vascular control involving PKG and its signaling partners. Understanding how blood vessels constrict and dilate is critical for the development of new strategies and therapeutic agents aimed at prevention and treatment of vascular disorders such as hypertension, stroke and coronary artery disease.