The aim of this proposal is to determine the receptor signaling pathways by which the natriuretic peptides (NP) modulate pulmonary microvascular endothelial cell (PMVEC) barrier function and pulmonary edema formation. Pulmonary edema is the primary cause of lung dysfunction in most cases of acute lung injury. Transudation of vascular fluid and protein from the pulmonary capillary lumen to the interstitial and alveolar space impairs gas exchange and initiates a cascade of inflammatory events that lead to acute respiratory distress syndrome. The NPs (atrial, ANP, brain, BNP, and C-type, CNP) play important physiologic roles in modulating vascular endothelial cell permeability. Numerous studies have documented their ability to reduce permeability of proximal pulmonary artery endothelial cells, however, the receptors and signal pathways responsible for this effect are not well defined and few studies have examined the effect of NPs on PMVECs, the cells that are responsible for regulating trans-pulmonary fluid flux. Recent studies in the applicant's lab have shown that the NPs differentially affect PMVEC barrier function and that the guanylyl cyclase- and non- guanylyl cyclase-linked receptors (NPR-A, NPR-C, respectively) have differing effects. The overall goal of this proposal is to identify the receptor-signaling pathways that mediate the inhibitory effects of the NPs on PMVEC barrier function. This proposal will test the hypotheses that NPs blunt thrombin-induced increases in PMVEC permeability via NPR-A by a cGMP/PKG pathway and facilitate restoration of PMVECs barrier dysfunction via NPR-C. In addition, the proposal hypothesizes that the inhibitory effects of the NPs on thrombin-induced increases in PMVEC permeability are mediated via downstream inhibition of the small GTPases RhoA, Rac1 and cdc42. The proposal aims to test these hypotheses by 1) examining the effect of NPs, phosphodiesterase inhibitors and PKG inhibitors on the permeability of PMVECs, 2) examining the effect of altered expression of NPR-A, NPR-C, PKG and the small GTPases on the ability of NPs to protect against thrombin-induced barrier dysfunction in vitro, and 3) examine the effect of NPs on pulmonary edema formation in mice with targeted disruption of NPR-A and NPR-C. Findings from these studies will further our understanding of cellular mechanism that modulate pulmonary edema formation and determine if the NPs and their receptors are potential therapeutic targets for acute lung injury.
