Signaling pathways involved in adenosine triphosphate-induced endothelial cell barrier enhancement

Endothelial barrier dysfunction caused by inflammatory agonists is a frequent underlying cause of vascular leak and edema. Novel strategies to preserve barrier integrity could have profound clinical impact. Adenosine triphosphate (ATP) released from endothelial cells by shear stress and injury has been shown to protect the endothelial barrier in some settings. We have demonstrated that ATP and its nonhydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers and caused remodeling of cell-cell junctions. Increases in cytosolic Ca²⁺ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically G _αq and G_αi2), protein kinase A (PKA), and the PKA substrate vasodilator-stimulated phosphoprotein were involved in ATP-induced barrier enhancement. ATP treatment decreased phosphorylation of myosin light chain and specifically activated myosin-associated phosphatase. Depletion of G_αq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca²⁺, but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA.