9 MECHANISMS OF ENDOTHELIAL BARRIER ENHANCEMENT INDUCED BY ADENOSINE TRIPHOSPHATE

Purpose of Study Endothelial barrier dysfunction is often the underlying cause of vascular leakage and edema. It is important therefore to find ways to preserve barrier properties. Extracellular adenosine triphosphate (ATP) has been known to protect endothelial barrier. In this study we defined the mechanisms of endothelial barrier enhancement caused by extracellular ATP. Methods Combination of pharmacological and molecular approaches is used in this study. Summary of Results ATP and its non-hydrolyzed analogues enhanced barrier properties of cultured endothelial cell monolayers, caused remodeling of cell-cell junctions, and significantly attenuated thrombin-induced barrier disruption. Intracellular Ca2+ increase and Erk activation caused by ATP were irrelevant to barrier enhancement. Inhibitory analysis and silencing RNA revealed the involvement of G proteins (specifically Gαq and Gαi2) as well as protein kinase A and its substrate VASP in ATP-induced barrier enhancement. Contractile state of endothelial cells governed by myosin light chain (MLC) phosphorylation underlies barrier properties. ATP treatment decreased MLC phosphorylation and specifically activated myosin-associated phosphatase. Depletion of Gαq with siRNA prevented ATP-induced activation of myosin phosphatase. Conclusions We conclude that ATP-induced barrier-improving mechanism is independent from intracellular Ca2+, but involves activation of myosin phosphatase via novel G protein coupled mechanism and PKA.