Anales RANF

S7-03 DISSECTING THE MECHANISMS OF VASCULAR BARRIER STRENGTHENING IN ACUTE LUNG INJURY: NOVEL MECHANISMS FOR EXTRACELLULAR PURINES A. Verin Augusta University, Augusta USA Endothelial cells (EC) form a semi-permeable barrier between the interior space of blood vessels and the underlying tissues. In acute lung injury the EC barrier is weakened leading to increased permeability. The mechanisms involved in the preservation of EC barrier integrity are largely unknown. We have demonstrated that extracellular purines, ATP (stable analog, ATPγS) and adenosine, strengthen EC barrier in vitro and in vivo . However, the mechanisms underlying the barrier protective effects of these purinergic agonists are different. Adenosine-induced EC barrier enhancement involves activation of P1 A2 receptors coupling to Gs trimeric G protein, followed by cAMP-dependent protein kinase A (PKA) activation, activation of myosin light chain (MLC) phosphatase (MLCP), and decrease in MLC phosphorylation. In parallel, adenosine strengthens EC barrier through Gs-mediated activation of small GTPase, Rac1. Unlike adenosine, ATPγS activates EC -barrier-protecting signaling via P2Y receptors coupled to Gi and involved unconventional cAMP-independent PKA, MLCP and Rac1 activations. ATPγS, Gi -mediated PKA and MLCP activations may be coordinated through the actions of adapter protein GAB1, Shp2 (a non-receptor Tyr phosphatase), and AKAP2 (PKA anchoring protein 2)-mediated signaling. AKAP2 was found in immune complexes with PKA and Gi and directly interacts with MLCP suggesting that the AKAP2/MLCP axis is a novel regulator of P2Y/Gi-mediated EC barrier enhancement. Further, our data show the involvement of a regulatory molecule ELMO1 (Engulfment and cell motility protein 1) in P2Y/Gi-mediated EC barrier strengthening, which together with the adapter protein Dock180 formed a bipartite GEF for Rac1. Collectively, our data suggested that while adenosine-induced EC barrier enhancement involves activation of Gs/cAMP- mediated signaling, ATPγS -induced P2Y-mediated EC barrier strengthening requires Gi-mediated, coordinated activation of GAB1/Shp2 and Dock180/ELMO1 leading to activation of PKA and Rac1 pathways, respectively.