ATP in Lung Endothelial Barrier Enhancement

Project: Research project

Project Details

Description

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.
StatusNot started

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