Elevation in intracellular cAMP level has been associated with increased endothelial barrier integrity and linked to the activation of protein kinase A (PKA). Recent studies have shown a novel mechanism of cAMP-mediated endothelial barrier regulation via cAMP-dependent nucleotide exchange factor Epac1 and Rap1 GTPase. This study examined a contribution of PKA-dependent and PKA-independent pathways in the human pulmonary endothelial (EC) barrier protection by cAMP. Synthetic cAMP analog, 8-bromoadenosine-3',5'-cyclic monophosphate (Br-cAMP), induced dose-dependent increase in EC transendothelial electrical resistance which was associated with activation of PKA, Epac/Rap1, and Tiam/Vav/Rac cascades and significantly attenuated thrombin-induced EC barrier disruption. Both specific Epac/Rap1 activator 8CPT-2Me-cAMP (8CPT) and specific PKA activator N(6)-benzoyl-adenosine-3',5'-cyclic monophosphate (6Bnz) enhanced EC barrier, suppressed thrombin-induced EC permeability, and independently activated small GTPase Rac. SiRNA-induced Rac knockdown suppressed barrier protective effects of both PKA and Epac signaling in pulmonary EC. Intravenous administration of either 6Bnz, or 8CPT, significantly reduced lung vascular leak in the murine model of lung injury induced by high tidal volume mechanical ventilation (HTV, 30 ml/kg, 4 h), whereas combined treatment with 6Bnz and 8CPT showed no further additive effects. This study dissected for the first time PKA and Epac pathways of lung EC barrier protection caused by cAMP elevation and identified Rac GTPase as a hub for PKA and Epac signaling leading to enhancement of lung vascular barrier.
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