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

项目总结 肺微血管内皮细胞(PMVECs)形成连续的、半透明的屏障 血流和间质间隙。质膜定位的腺酰环化酶产生cAMP (ACS)增强了PMVEC的壁垒。相反，胞浆cAMP、cGMP和CUMP由外源和 内源性可溶性环化酶可破坏PMVEC屏障。这些观察结果表明，环核苷酸 信号高度局部化或区分化，近膜和胞质cAMP、cGMP和 可能CUMP信号对肺微血管内皮细胞功能有相反的影响。这一概念 分区化的信号意味着反馈网络局限于特定的亚细胞区域控制 第二信使信号的动力学。然而，我们对生理和心理的理解 肺内皮细胞内局部反馈网络的病理生理学意义最好 很初级的。因此，本项目的总体目标是确定空间和时间关系 CAMP信号、PKA介导的反馈网络和机械调节之间的关系 对肺内皮细胞的作用力。该提案中描述的实验将首次识别出 当cAMP信号出现在PMVECs的3D空间时，识别cAMP的重要时间成分 信号，以及有助于这些信号的信号局部化和动力学的图表反馈机制。在……里面 换句话说，我们将提供路线图，确定营地信号的空间位置，这对 控制细胞力的动态。然后，我们将把这些响应叠加到PKA活动地图上，并 A激酶锚定蛋白(AKAP)的潜在分布。因此，研究工作已圆满完成 在本应用中提出的将识别特定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旨在识别这些蛋白质中的目标蛋白质的研究 信号小体，使我们更好地理解局部信号的分子机制 信号小体内的转导和识别翻译靶标。

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.