ROVIDED. Endothelial cell (EC) barrier dysfunction, a prominent feature of acute lung injury (ALT), is tightly linked to cytoskeletal remodeling, which leads to the disruption of cell-cell contacts and includes activation of contraction initiated by myosin light chain (MLC)phosphorylation followed by F-actin stress fiber formation and formation of paracellular gaps. Little is known about processes which determine barrier enhancement or protection; however, our published data implicate a critical role for cytoskeletal dynamics in this response. Extracellular ATP is an important vascular mediator, which elicits cellular effects on EC mainly through P2Y family receptors coupled to specific trimeric G- proteins. Our novel findings indicate that ATP at physiologically relevant concentrations produces rapid, sustained and dose-dependent increases in transendothelial electrical resistance (TER), indicating profound barrier enhancement and potently reversed barrier dysfunction elicited by the edemagenic agent, thrombin. Specific depletion of a subunits of Gq and Gi2 significantly attenuated ATP-induced increase in TER indicating the involvement of these G-proteins inATP- induced EC barrier enhancement. The ATP-induced increase in TER is tightly linked to an increase in myosin-associated phosphatase (PPase) 1 (MLCP) activity. Inhibition of PPase 1 abolished the ATP-induced increase in TER and lead to phosphorylation of several cytoskeletal targets includingMLC, ezrin/radixin/moezin (ERM) and caldesmon suggesting that dephosphorylation of these proteins may be involved in the barrier-enhancing effect of ATP. In addition, protein kinase A (PKA) inhibition attenuates both ATP-induced increases in TER and phosphorylation of vasolidator- stimulated protein (VASP), which in the phosphorylated form inhibits stress fiber formation supporting the involvement of the PKA/VASP pathway in ATP-induced EC barrier enhancement. Our working hypothesis is that ATP-induced EC barrier enhancement and cytoskeletal remodeling is dependent, at least in part, upon activation of specific P2Y/G protein complexes followed by coordinated activation of MLCP and PKA signaling. SA#1will define the role of specific P2Y/G-protein complexes in the activation of MLCP- and PKA-dependent signaling. SA#2 will define the involvement of MLCP and its cytoskeletal targets in ATP-induced EC barrier enhancement. SA #3 will explore the molecular mechanisms by which PKA activity is involved in ATP-induced EC barrier enhancement focusing on VASP and MLCP as potential PKA targets. SA#4 will characterize the potential barrier-protective effects of ATP in murine models of ALL These studies will provide an understanding of the novel signaling pathways involved in ATP-induced lung EC barrier enhancement and promise new directions and targets for treatment of lung disorders.

Agency
National Institute of Health (NIH)
Institute
National Heart, Lung, and Blood Institute (NHLBI)
Type
Research Project (R01)
Project #
5R01HL083327-04
Application #
7330349
Study Section
Lung Injury, Repair, and Remodeling Study Section (LIRR)
Program Officer
Moore, Timothy M
Project Start
2006-01-01
Project End
2009-12-31
Budget Start
2008-01-01
Budget End
2008-12-31
Support Year
4
Fiscal Year
2008
Total Cost
$356,843
Indirect Cost
Name
Georgia Regents University
Department
Internal Medicine/Medicine
Type
Schools of Medicine
DUNS #
966668691
City
Augusta
State
GA
Country
United States
Zip Code
30912
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Poirier, Christophe; Gorshkov, Boris A; Zemskova, Marina A et al. (2011) TIMAP protects endothelial barrier from LPS-induced vascular leakage and is down-regulated by LPS. Respir Physiol Neurobiol 179:334-7
Bogatcheva, Natalia V; Zemskova, Marina A; Gorshkov, Boris A et al. (2011) Ezrin, radixin, and moesin are phosphorylated in response to 2-methoxyestradiol and modulate endothelial hyperpermeability. Am J Respir Cell Mol Biol 45:1185-94
Jezierska, Agnieszka; Kolosova, Irina A; Verin, Alexander D (2011) Toll Like Receptors Signaling Pathways as a Target for Therapeutic Interventions. Curr Signal Transduct Ther 6:428-440
Zemskov, Evgeny; Lucas, Rudolf; Verin, Alexander D et al. (2011) P2Y receptors as regulators of lung endothelial barrier integrity. J Cardiovasc Dis Res 2:14-22
Bogatcheva, Natalia V; Zemskova, Marina A; Poirier, Christophe et al. (2011) The suppression of myosin light chain (MLC) phosphorylation during the response to lipopolysaccharide (LPS): beneficial or detrimental to endothelial barrier? J Cell Physiol 226:3132-46

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