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

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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.