Signaling Pathways Involved in Adenosine Triphosphate-Induced Endothelial Cell Barrier Enhancement

Abstract

Abstract-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 Ca 2ϩ and Erk activation caused by ATP were irrelevant to barrier enhancement. Experiments using biochemical inhibitors or siRNA indicated that G proteins (specifically G ␣q and G ␣i2 ), 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 ␣q with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca 2ϩ , but involve activation of myosin phosphatase via a novel G-protein-coupled mechanism and PKA. Key Words: endothelial barrier Ⅲ extracellular adenosine triphosphate Ⅲ G protein Ⅲ myosin phosphatase I nflammatory agonist-induced endothelial cell (EC) barrier dysfunction is associated with cytoskeletal remodeling, disruption of cell-cell contacts, and the formation of paracellular gaps. 1 Less is known about the mechanisms of EC barrier maintenance and protection. Some naturally occurring substances such as sphingosine 1-phosphate, angiotensin 1, and the second messenger cAMP are known to enhance the EC barrier. Recently, much attention has been given to the therapeutic potential of purinergic agonists and antagonists for the treatment of cardiovascular and pulmonary diseases. 2 Accumulated experimental data suggest that adenosine triphosphate (ATP) and other purines are promising as physiologically-relevant barrier-protective agents as they are readily present in the surrounding EC microenvironment in vivo, and they decrease transendothelial permeability in vitro. ATP can be released into the bloodstream from platelets 3 and red blood cells. 4 Extracellular ATP concentrations may temporarily exceed 100 mol/L in blood. 5 Furthermore, the endothelium provides a source of ATP locally within vascular beds. ATP is released constitutively across the apical membrane of EC under basal conditions. 6 Enhanced release of ATP was observed from EC in response to various stimuli including hypotonic challenge, 6 calcium agonists, 6 shear stress, 7 thrombin, 7 ATP itself, 8 and lipopolysaccharide. 9 Once released, ATP is degraded rapidly and its metabolites, adenosine diphosphate (ADP) and adenosine, have also been characterized as signaling molecules, able to regulate various cellular functions. 10

TIMAP is a positive regulator of pulmonary endothelial barrier function

“…Although TIMAP depletion did not have a significant effect on basal TER (Fig. 4B), it markedly attenuated the effects of barrierprotective agents (S1P and ATP) (18,22) (Fig. 4D).…”

“…The effects of both S1P and ATP, two barrier-protective agents (18,22), were attenuated, whereas the effects of thrombin and nocodazole, both evoking barrier dysfunction (11), were enhanced by TIMAP depletion (Fig. 4D).…”

Signaling pathways involved in OxPAPC-induced pulmonary endothelial barrier protection

“…A number of agonists including phospholipid products (Zhang, Magnusson et al 1997;Huang, Subbaiah et al 2005) and bioactive peptides (Garcia, Siflinger-Birnboim et al 1986;van Nieuw Amerongen, Draijer et al 1998;Petrache, Verin et al 2001;Moy, Blackwell et al 2002;Birukova, Adyshev et al 2005) increase vascular endothelial permeability, which my lead to alveolar flooding and pulmonary edema. However, much smaller number of bioactive molecules capable of enhancing endothelial barrier properties have been reported so far (Garcia, Liu et al 2001;Liu, Schaphorst et al 2002;VouretCraviari, Bourcier et al 2002;Birukov, Bochkov et al 2004;Kolosova, Mirzapoiazova et al 2005), and signaling mechanisms underlying these barrier protective effects have been a major focus of ongoing studies (Lee, Lee et al 2000;Garcia, Liu et al 2001;Vouret-Craviari, Bourcier et al 2002;Birukov, Leitinger et al 2004;Dudek, Jacobson et al 2004;Kolosova, Mirzapoiazova et al 2005;Mehta, Konstantoulaki et al 2005;Singleton, Dudek et al 2005). …”

Protective effect of purinergic agonist ATPγS against acute lung injury

“…Little is known about processes that determine barrier enhancement or protection. Recently, it has been shown that extracellular ATP stabilizes barrier function of endothelial monolayers (15,18,21) and attenuates barrier disruption caused by thrombin (12) and reperfusion (13). Extracellular purines (adenosine, ADP, and ATP) are important signaling molecules that mediate diverse biological effects via cellsurface receptors.…”