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.
| Umapathy, Siddaramappa Nagavedi; Siddaramappa Umapathy, Nagavedi; Kaczmarek, Elzbieta et al. (2013) Adenosine A1 receptors promote vasa vasorum endothelial cell barrier integrity via Gi and Akt-dependent actin cytoskeleton remodeling. PLoS One 8:e59733 |
| Kim, Kyung-Mi; Csortos, Csilla; Czikora, Istvan et al. (2012) Molecular characterization of myosin phosphatase in endothelium. J Cell Physiol 227:1701-8 |
| Gorshkov, Boris A; Zemskova, Marina A; Verin, Alexander D et al. (2012) Taxol alleviates 2-methoxyestradiol-induced endothelial permeability. Vascul Pharmacol 56:56-63 |
| Umapathy, Nagavedi Siddaramappa; Gonzales, Joyce; Fulzele, Sadanand et al. (2012) ?-Nicotinamide adenine dinucleotide attenuates lipopolysaccharide-induced inflammatory effects in a murine model of acute lung injury. Exp Lung Res 38:223-32 |
| Poirier, Christophe; Berdyshev, Evgeny V; Dimitropoulou, Christiana et al. (2012) Neutral sphingomyelinase 2 deficiency is associated with lung anomalies similar to emphysema. Mamm Genome 23:758-63 |
| 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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