Cyclic nucleotide gradients regulate the balance of mechanical forces underlying pulmonary endothelial barrier integrity

环核苷酸梯度调节肺内皮屏障完整性的机械力平衡

基本信息

批准号：
10198006
负责人：
THOMAS C RICH
金额：
$ 35.63万
依托单位：
UNIVERSITY OF SOUTH ALABAMA
依托单位国家：
美国
项目类别：
财政年份：
2001
资助国家：
美国
起止时间：
2001-12-01 至 2023-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10198006
关键词：
3-Dimensional A kinase anchoring protein Adenylate Cyclase Adhesions Adult Respiratory Distress Syndrome Affect Agonist Alprostadil Anisotropy Apical Bacterial Infections Bicarbonates Blood Blood Circulation Cell membrane Cell-Matrix Junction Cells Cyclic AMP Cyclic AMP-Dependent Protein Kinases Cyclic GMP Cyclic Nucleotides Data Development Dimensions Distant Endothelial Cells Endothelium Equilibrium Extracellular Matrix Face Feedback Filtration Fingerprint Forskolin Frequencies Future G Protein-Coupled Receptor Signaling G-Protein-Coupled Receptors Generations Goals Heterogeneity Imaging technology Individual Inflammation Inflammation Mediators Intercellular Junctions Investigation Isometric Exercise Isoproterenol Kinetics Liquid substance Location Lung Maps Measurement Measures Mechanics Mediating Membrane Modulus Molecular Monolayer Stress Microscopy Movement Oxygen Permeability Phenotype Physiological Prostaglandins Proteins Pulmonary Edema Regulation Second Messenger Systems Signal Pathway Signal Transduction Site Stabilizing Agents Syndrome Testing Time Work base cell motility experimental study imaging approach insight interstitial mechanical force mechanical properties monolayer novel receptor response spatiotemporal temporal measurement

项目摘要

PROJECT SUMMARY Pulmonary microvascular endothelial cells (PMVECs) form contiguous, semi-permeable barriers between the bloodstream and the interstitial space. cAMP generated by plasma membrane-localized adenylyl cyclases (ACs) enhances PMVEC barriers. In contrast, cytosolic cAMP, cGMP, and cUMP generated by exogenous and endogenous soluble cyclases disrupt PMVEC barriers. These observations suggest that cyclic nucleotide signals are highly localized, or compartmentalized, and that near-membrane and cytosolic cAMP, cGMP, and perhaps cUMP signals have opposing effects on endothelial function in the lung microvasculature. The concept of compartmentalized signals implies that feedback networks localized to specific subcellular domains control the kinetics of second messenger signals. However, our understanding of the physiological and pathophysiological implications of localized feedback networks within pulmonary endothelial cells is at best rudimentary. Thus, the overall goal of this project is to determine the spatial and temporal relationships between compartmentalized cAMP signals, PKA-mediated feedback networks, and regulation of mechanical forces in pulmonary endothelial cells. Experiments described in this proposal will for the first time identify where cAMP signals occur in the 3D space of PMVECs, identify important temporal components of cAMP signals, and chart feedback mechanisms contributing to signal localization and kinetics of these signals. In other words, we will provide roadmaps identifying the spatial locations of cAMP signals that are critical for controlling the dynamics of cellular forces. We will then overlay these responses onto PKA activity maps and underlying distributions of A kinase anchoring proteins (AKAPs). As such, successful completion of the studies proposed in this application will identify the spatial and temporal fingerprints of specific cAMP signalosomes that regulate mechanical forces within pulmonary endothelial cells, and thus control endothelial barrier integrity. The spatial and temporal fingerprints will direct future studies aimed at identifying target proteins within these signalosomes, leading to both a better understanding of the molecular mechanisms underlying localized signal transduction and identifying translational targets within signalosomes.

项目概要肺微血管内皮细胞 (PMVEC) 在肺微血管之间形成连续的半透性屏障血液和间隙空间。由质膜定位的腺苷酸环化酶产生的 cAMP (AC) 增强 PMVEC 屏障。相反，胞质 cAMP、cGMP 和 cUMP 是由外源性和内源性可溶性环化酶破坏 PMVEC 屏障。这些观察结果表明环核苷酸信号高度局部化或区室化，并且近膜和胞质 cAMP、cGMP 和也许 cUMP 信号对肺微血管系统的内皮功能具有相反的影响。概念区室化信号意味着反馈网络局限于特定的亚细胞域控制第二信使信号的动力学。然而，我们对生理和肺内皮细胞内局部反馈网络的病理生理学影响充其量是简陋的。因此，该项目的总体目标是确定空间和时间关系区室化的 cAMP 信号、PKA 介导的反馈网络和机械调节之间的关系肺内皮细胞中的力。该提案中描述的实验将首次确定 cAMP 信号出现在 PMVEC 的 3D 空间中，识别 cAMP 的重要时间成分信号，以及有助于信号定位和这些信号动力学的图表反馈机制。在换句话说，我们将提供路线图来识别 cAMP 信号的空间位置，这对于控制细胞力的动态。然后，我们将这些响应叠加到 PKA 活动图上， A 激酶锚定蛋白 (AKAP) 的基本分布。就这样顺利完成了学业本申请中提出的方法将识别特定 cAMP 信号体的空间和时间指纹调节肺内皮细胞内的机械力，从而控制内皮屏障的完整性。空间和时间指纹将指导未来的研究，旨在识别这些区域中的目标蛋白。信号体，使人们更好地理解局部信号背后的分子机制转导和识别信号体内的翻译靶标。