Exploring Vasomotor Mechanisms using New PKG Inhibitors and cGMP Biosensors

使用新型 PKG 抑制剂和 cGMP 生物传感器探索血管舒缩机制

基本信息

批准号：
8038300
负责人：
WOLFGANG R DOSTMANN
金额：
$ 37.63万
依托单位：
UNIVERSITY OF VERMONT & ST AGRIC COLLEGE
依托单位国家：
美国
项目类别：
财政年份：
2002
资助国家：
美国
起止时间：
2002-01-01 至 2013-03-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8038300
关键词：
Acute Address Adenoviruses Animals Arteries Biochemistry Biosensor Blood Pressure Blood Vessels Blood flow C-Type Natriuretic Peptide Calcium Calcium Signaling Cardiovascular system Cell Culture Techniques Cell membrane Cell physiology Cellular biology Coronary Arteriosclerosis Cyclic GMP Data Development Diffuse Disease Energy Transfer Enzyme Kinetics Enzymes Event Explosion Feedback Fluorescence Fluorescence Microscopy Fluorescence Resonance Energy Transfer Frequencies Guanylate Cyclase Health Heart Atrium Hydrolysis Hypertension Image Imaging Techniques In Vitro Insecta Ion Channel Isoenzymes Label Lead Link Measurement Measures Mediating Membrane Modeling Molecular Molecular Biology Muscle function Mutagenesis Myography Natriuretic Peptides Nitric Oxide Particulate Patch-Clamp Techniques Pathway interactions Pattern Phosphorylation Physiology Play Potassium Channel Prevention Protein Engineering Protein Kinase Proteins Regional Blood Flow Regulation Resistance Resolution Role Ryanodine Receptors Sarcoplasmic Reticulum Signal Pathway Signal Transduction Smooth Muscle Smooth Muscle Myocytes Soluble Guanylate Cyclase Speed Stroke System Techniques Testing Therapeutic Agents Vascular Diseases Vascular Smooth Muscle Vascular resistance Vasodilation Vasomotor Work atrial natriuretic factor receptor A base blood flow measurement falls in vitro activity in vivo inhibitor/antagonist multidisciplinary novel phosphodiesterase V phospholamban ratiometric receptor response theories tool

项目摘要

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.

描述（由申请人提供）：细胞内CGMP和CA2+调节健康和疾病中的血管平滑肌（VSM）功能。新兴的观点是细胞内Ca2+信号是高度动态的，而Ca2+信号的模式决定了VSM函数。了解细胞内CGMP信号传导动力学，随后的CGMP依赖性蛋白激酶（PKG）的激活及其与Ca2+信号的关系滞后。在这里，使用新型的CGMP生物传感器和PKG抑制剂，我们将提供证据表明CGMP（例如Ca2+）在空间和时间上是动态的，并且取决于多个相互关联的控制机制。我们的方法将涉及CA2+和CGMP的高分辨率测量结果，并提供了这些信符分子对VSM功能的交互式控制的无与伦比的视图。在特定目的1中，我们将测试膜（PGC）和胞质（SGC）Guanylyl循环酶具有从根本上不同的CGMP形成模式的理论。我们建议NO和ANP分别激活SGC和PGC，创建了细胞内CGMP的离散池，因此为CGMP信号的功能分隔提供了结构基础。我们还将测试PKG通过作为CGMP池的负反馈调节器来控制血管张力的机制。通过我们最近开发的新型CGMP生物传感器，这些评估已成为可能，这些传感器可以直接测量具有高时间和空间分辨率的细胞CGMP。在特定目标2中，我们将阐明CGMP/PKG和钙信号传导之间的相互作用。我们建议CGMP和PKG部分通过调节Ca2+火花，BKCA通道和全球钙来调节血管功能。在特定目标3中，我们将通过研究PKG抑制剂增加血压和完整动物血管耐药性的功效来确定PKG对体内血管控制的贡献。我们测试该提案的三个目标的方法将是多学科的，该方法采用生理学的最先进技术（高速钙成像，斑块夹技术，耐药性动脉my术，血压和血液流量测量），细胞生物学，共焦荧光显微镜（共核显微镜，反射量荧光显微镜，平滑的细胞培养），滋生培养物培养物，滋生培养群，滋生培养物，促进群体群体群体群体群体群体群体群体群体群体群体群体群体群体群体群体群体群体群体群体，腺病毒）和生物化学（酶动力学技术，荧光能量转移，细胞蛋白递送系统）。这项工作将提供对调节血管平滑肌中PKG活性的因素的综合观点，从而显着增强我们对健康和疾病动脉功能的理解。公共卫生相关性CGMP依赖性蛋白激酶（PKG）是整个体内血管中细胞功能的重要调节剂。该建议旨在确定涉及PKG及其信号伴侣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旨在预防和治疗血管疾病（例如高血压，中风和冠状动脉疾病）的新策略和治疗剂至关重要。