Published online before print June 30, 2005, doi: 10.1161/01.RES.0000175561.55761.69
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© 2005 American Heart Association, Inc.
Molecular Medicine |
Signaling Pathways Involved in Adenosine Triphosphate-Induced Endothelial Cell Barrier Enhancement
From the Department of Medicine (I.A.K., T.M., D.A., P.U., J.R.J., V.N., D.B.P., J.G.N.G., A.D.V.), Division of Pulmonary and Critical Care Medicine, and Departments of Anesthesiology (L.H.R.), Johns Hopkins University School of Medicine, Baltimore, Md.
Correspondence to Dr Alexander D. Verin, 5200 Eastern Ave, MFL Bldg, Center Tower, Room 660, Baltimore, MD 21224. E-mail averin1{at}jhmi.edu
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Abstract |
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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 Ca2+ 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 Gi2), 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 Gq with siRNA prevented ATP-induced activation of myosin phosphatase. We conclude that the mechanisms of ATP-induced barrier enhancement are independent of intracellular Ca2+, but involve activation of myosin phosphatase via a novel G-protein–coupled mechanism and PKA.
Key Words: endothelial barrier • extracellular adenosine triphosphate • G protein • myosin phosphatase
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Introduction |
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Inflammatory 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 platelets3 and red blood cells.4 Extracellular ATP concentrations may temporarily exceed 100 µmol/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
Extracellular nucleotides and adenosine act via purinoreceptors, which are divided into 2 classes: P1, or adenosine receptors, and P2 receptors, that recognize extracellular ATP, ADP, uridine 5'-triphosphate (UTP), and uridine 5'-diphosphate (UDP).10 Four different P1 receptors have been identified and pharmacologically characterized: A1, A2A, A2B, and A3.11 The A2A and A2B receptors preferably interact with members of the Gs family of G proteins and the A1 and A3 receptors with Gi/o proteins.11 The P2 receptors are divided into 2 subclasses, X and Y. P2X receptors are ATP-gated nonselective cation channels.12 The P2Y receptors are G-protein coupled. P2Y1, 2, 4, 6, and 11 are coupled to Gq and activate PLCß. P2Y12, 13, and 14 are coupled to Gi and inhibit adenylate cyclase.10
The expression of purinoreceptors in human EC is variable and dependent on the specific EC type. Among P2 receptors, P2X46,13,14 and P2Y213–15 are the most abundant and widely expressed in different EC. Wang et al14 also demonstrated that human umbilical vein endothelial cells (HUVEC) express a high level of P2Y1 and P2Y11. The P2Y expression profile suggests that nucleotide signaling in EC is likely mediated specifically via Gq pathway. Recently P2Y12 has been characterized in rat EC,16 implicating Gi-mediated nucleotide signaling. Adenosine receptors also have been found in human EC.17
Recently the barrier-protective properties of ATP have been reported in HUVEC, bovine, and porcine EC.18,19 The exact nature of ATP-induced barrier augmentation is not well defined. In this study we examined the mechanisms of EC barrier enhancement caused by extracellular ATP using a combination of pharmacological and molecular approaches. We sought to define the molecular components coupling receptor activation with barrier enhancement.
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Materials and Methods |
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Sources of reagents and details of procedures are provided in the expanded Materials and Methods section in the online data supplement available at http://circres.ahajournals.org. Human and bovine pulmonary artery EC (HPAEC; Clonetics, Walkersville, Md and BPAEC; American Type Tissue Culture Collection, Rockville, Md, respectively), and human lung microvascular EC (HLMVEC; Clonetics) were used in the study. siRNA-based protein depletion of small GTPases were performed as described elsewhere.20 The barrier properties of EC monolayers were characterized using electrical cell impedance sensor system.21 Described immunostaining protocol was used.22 The percentage of total cell surface area occupied by VE-cadherin labeled cell–cell junctions was quantitatively determined using Openlab (4.0) software (Improvision). Concentrations of cytosolic Ca2+ were measured as described previously.23 cAMP concentration in EC lysates was determined with TRK 432 radioassay system (Amersham). PKA activity was measured using a nonradioactive PKA Kinase Activity assay Kit (Stressgen). Myosin-enriched fraction of HPAEC was prepared as described previously.24 Ser/Thr Phosphatase Assay Kit (Upstate) was used to determine myosin light chain phosphotase (MLCP) activity. MLC phosphorylation was analyzed by either phosphospecific antibody or urea polyacrylamide gel electrophoresis as previously described.24 For basic statistical analysis a GraphPad Prism Program was used. Data were compared by a Student t test. Probability values <0.05 were considered to be significant. Values are expressed as mean±SE.
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Results |
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ATP Increases Transendothelial Electrical Resistance
ATP increased the transendothelial electrical resistance (TER) of HPAEC monolayers in a concentration-dependent manner (Figure 1A). ADP, another nonselective P2 receptor agonist, and the stable ATP analogs ATP-
-S and 2-MeS-ATP also increased TER. AMP-CCP, which is more specific for the P2X1 and P2X3, receptors was completely inactive (Figure 1B). Other types of EC (HPAEC, HLMVEC, and BPAEC) demonstrated similar responses to ATP stimulation characterized by increased TER (online Table I).
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ATP Affects Cell–Cell Junctions
Immunofluorescence studies revealed changes in distributions of cell–cell junctional proteins after ATP treatment. VE-cadherin, a major component of endothelial adherens junctions, was more pronounced at the cellular periphery, presumably at cell–cell contacts (Figure 2A). The calculated percentage of total cell surface area occupied by VE-cadherin–labeled cell–cell junctions confirmed that ATP induced a significant increase in the surface area of cell–cell interfaces as a percentage of total cell surface area (Figure 2B). Furthermore, whereas the tight junctional component zonula occludens-1 (ZO-1) had a thin and somewhat discontinuous pattern at cell–cell borders of unstimulated monolayers, a thicker, more regular and continuous ZO-1 distribution was observed after ATP treatment (Figure 2C). This rearrangement of ZO-1 is consistent with a tightening of permeability barrier.
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ATP-Induced Increases in EC Barrier Function Are Independent of Changes in Cytosolic Ca2+
Purenergic stimulation is known to increase intracellular Ca2+ concentrations.10 However, inflammatory agonists that increase Ca2+ compromise EC barrier function.25 It has been previously reported that ATP decreases transendothelial albumin permeability despite increase on intracellular Ca2+.18 In our experiments, ATP increased Ca2+ in HPAEC (Figure 3A) in a dose-dependent fashion. 10 µmol/L 1,2-bis(0-Aminophenoxy)ethane-N,N,N',N'-tetraacetic Acid Tetra(acetoxymethyl) Ester (BAPTA) completely inhibited the ATP-induced Ca2+ increase (Figure 3B). BAPTA itself caused a decrease in TER, followed by recovery, but did not affect the TER response to ATP (Figure 3C). Enhancement of VE-cadherin–mediated intercellular junctions by ATP was also unaffected (Figure 3D). Therefore ATP-induced enhancement of endothelial barrier and adherens junctions is independent of intracellular Ca2+.
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Inhibition of Erk Phosphorylation Does not Prevent ATP-Induced Barrier Enhancement
The mitogen-activated protein kinase (MAPK) cascade is a signal transduction system, which is known to participate in multiple cellular functions.26 It has been previously shown that extracellular ATP induces Erk MAPK phosphorylation in EC.27 In our system time-dependent Erk phosphorylation occurred after ATP stimulation (Figure 4A). To investigate the involvement of Erk in the ATP-induced barrier response, the upstream kinase (MEK) was inhibited with U0126. Pretreatment of HPAEC with U0126 completely abolished ATP-induced Erk phosphorylation (Figure 4B), but had no effect on ATP-induced TER increase (Figure 4C). These data suggest the absence of a functional relationship between Erk phosphorylation and increased EC barrier function after ATP stimulation.
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ATP Response Is Mediated Via G Proteins
To determine the role of G proteins in ATP-induced barrier enhancement, HPAEC were treated either with a G protein–specific silencing RNA or with pertussis toxin (PTX). Depletion of either Gi (Figure 5A) or Gq (Figure 5B) with specific siRNAs significantly attenuated the increased TER induced by ATP, which confirms the involvement of both Gi and Gq subunits. Depletion of G12 had an opposite effect as it potentiated the response to ATP (Figure 5C), whereas depletion of G13 had no effect (Figure 5D). Based on the sensitivity to PTX, G proteins are grouped into 2 families. The Gi/Go family is sensitive to PTX whereas the Gq family is insensitive to this toxin. Pretreatment of HPAEC with PTX blocked the ATP response (Figure 5E), suggesting the exclusive role of Gi/Go proteins in ATP-induced barrier enhancement. The apparent discrepancy between PTX and siRNA data regarding the contribution of Gi and Gq into ATP response may be caused by additional effects of PTX, different from ADP ribosylation of Gi/o.28–30
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ATP Induces PKA Activation
In the P2Y family of purine receptors only P2Y11 has been reported to activate adenylate cyclase. However, adenylate cyclase activation and cAMP production are established steps in signal transduction via adenosine receptors. ATP did not significantly raise the intracellular cAMP level in HPAEC as opposed to adenosine receptor agonist NECA (Figure 6A), suggesting that the ATP effect is not mediated by adenosine receptors. A known activator of cAMP/PKA forskolin was used as a positive control in these experiments. ATP challenge did, however, lead to a transient increase in PKA activity, that could be inhibited by PKA inhibitor H89 (Figure 6B). H89 and another PKA inhibitor KT5720 also significantly attenuated the ATP-induced increase of TER (Figure 6C). These data implied that activation of PKA via a cAMP-independent mechanism was a necessary component of ATP-induced barrier enhancement.
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Vasodilator-stimulated phosphoprotein (VASP) is a known PKA effector protein which has been recently shown to localize to cell–cell junctions and participate in EC cytoskeletal rearrangement leading to permeability changes.31 ATP induced PKA-specific phosphorylation of VASP on Ser157 (Figure 6D, left) simultaneously with PKA activation (5 minutes). It should be noted that phosphorylation of VASP persisted later, when PKA was no more activated. This may occur because dephosphorylation requires increased phosphatase activity, which may be inhibited or unchanged after ATP treatment. VASP phosphorylation was insensitive to Ca2+ chelation with BAPTA, which correlates with the Ca2+-insensitivity of ATP-induced increase in TER (Figure 6D, middle). ATP-induced VASP phosphorylation was completely inhibited by H89 (Figure 6, right).
To directly examine the role of VASP in ATP-induced barrier enhancement, we used specific silencing RNA. VASP-depleted HPAEC exhibited an appreciably more robust response to ATP (Figure 6E). Taken together, these results indicated that VASP may serve as a negative regulator of barrier function, and that its phosphorylation after ATP exposure eliminated this property, resulting in enhanced EC barrier.
Myosin-Associated Phosphatase Is Involved in ATP-Induced Barrier Enhancement
It has been previously reported that ATP-induced decreases in transendothelial albumin flux correlates with decreased phosphorylation of MLC.32 In our experimental system ATP treatment had a biphasic effect with initial stimulation of MLC phosphorylation (at 5 minutes) followed by inhibition (at 30 minutes) and a return to baseline values by 1 hour (Figure 7A). These data differ from those of Noll et al,18 who demonstrated that ATP caused a fast and sustained MLC dephosphorylation in porcine aortic EC. Pretreatment of cells with BAPTA prevented the early transient increase in MLC phosphorylation but did not affect decreased MLC phosphorylation at later time points (Figure 7A). These results suggested that early MLC phosphorylation is associated with intracellular Ca2+ elevation but is not causally related to barrier enhancement. It also suggested that the later Ca2+-independent reduction of MLC phosphorylation may be functionally related to the observed barrier response. Data shown on Figure 7B confirms the decreased amount of phosphorylated form of MLC after 30 minutes of ATP treatment using urea gel electrophoresis.
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In the next set of experiments we attempted to clarify the role of MLCP in the ATP-induced reduction of MLC phosphorylation. First, the effect of ATP on TER was dramatically suppressed by microcystin, an inhibitor of phosphatase type 1 (PP1) and type 2 (PP2A), but not by fostriecin, an inhibitor of PP2A, implicating the involvement of PP1 (online Table II). Second, treatment of HPAEC with ATP led to increased myosin-associated phosphatase activity (Figure 8A) in a time-dependent manner, which agreed with the time course of ATP-induced barrier enhancement (Figure 8B). Increased association of the PP1
isoform with the myosin fraction was also observed after ATP challenge (Figure 8C and 8D). Furthermore, the increase of myosin-associated phosphatase activity was completely abolished by calyculin (inhibitor of PP1 and PP2A), but not by fostriecin (online Table III). Importantly, phosphatase activation (Figure 8A), increase in TER (Figure 8B), PP1 association (Figure 8C and 8D), and MLC dephosphorylation (Figure 7A) reached their maximum at approximately the same time (30 minutes), suggesting a strong correlation between these processes. Taken together, these results indicate that MLCP plays an important role in barrier enhancement induced by ATP. Furthermore, there is a clear association between ATP-induced activation of MLCP and G proteins. Depletion of Gq, but not Gi2 with siRNA abolished ATP-induced increase in phosphatase activity (0.34±0.04 pmoles of phosphate per mg protein compared with 1.22±0.04 pmoles of phosphate per mg protein in control cells.)
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Discussion |
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The inflammatory response of lung endothelium includes increased transendothelial permeability, leading to extravasation of fluid and blood cells and resulting in lung edema. Inflammatory mediators acting via G protein–coupled receptors trigger increased endothelial permeability by increasing intracellular Ca2+ concentrations which in turn activate signaling pathways leading to cytoskeletal reorganization and disassembly of VE-cadherin at adheren junctions.1 Extracellular nucleotides activate ion-channel P2X receptors and G protein–coupled P2Y receptors inducing apoptotic, proinflammatory, and thrombotic changes in many tissues and cell types.10 However, unlike other inflammatory stimuli, ATP and its analogues do not compromise endothelial barrier function.
In this study we show that extracellular ATP, despite inducing increases in intracellular Ca2+, acts as a potent barrier-protective agonist on EC derived from different types of lung blood vessels. ATP induced an increase in TER (Figure 1A) and rearrangement of VE-cadherin and ZO-1, suggesting a tightening of cell–cell contacts (Figure 2). Because ATP undergoes hydrolysis within minutes on the surface of EC, producing ADP and adenosine,33 it is possible that both purinergic systems (P1 and P2) are involved in ATP-induced endothelial barrier enhancement. As nonhydrolyzed ATP analogues also enhanced endothelial barrier function (Figure 1B) and, as has been previously published, the adenosine receptor antagonist 8-phenyltheophylline does not inhibit the barrier-protective effects of ATP,18 it is evident that ATP directly triggers barrier-protective mechanisms via P2 receptors.
Many effects of ATP as an extracellular mediator have been attributed to an increase in intracellular Ca2+ via P2Y receptors (see reference 10 for review). Intracellular Ca2+, however, is not important for the ATP-induced decrease in albumin flux across porcine endothelial monolayers.18 Our study confirms that the barrier-protective property of ATP is unrelated to intracellular Ca2+ concentrations. Although ATP caused a dose-dependent rise of intracellular Ca2+, the chelation of this Ca2+ with BAPTA did not affect either increased barrier function or enhanced VE-cadherin staining at the cell periphery (Figure 3).
ATP has been shown to activate Erk in different cell types, including endothelium.34,35 However, Erk activation appears to be involved in endothelial barrier dysfunction, rather than protection.36 We demonstrate that ATP-induced Erk activation does not play a functional role in barrier enhancement (Figure 4).
ATP-induced EC barrier enhancement occurs via a G protein–coupled mechanism because treatment of EC with siRNA designed to target Gq and Gi2 markedly decreased ATP effect (Figure 5A and 5B). PTX also prevented ATP effects on TER (Figure 5E), suggesting the involvement of Gi. Interestingly, depletion of G12 protein potentiated effects of ATP on TER (Figure 5C). Previously published data suggest that G12 may contribute to a procontractile phenotype of EC. Specifically, G12 activates the small GTPase Rho.37 Rho activation ultimately leads to MLC phosphorylation, cell contraction, and barrier disruption.1 Activation of G12 increased paracellular permeability38 and disrupted tight and adherens junctions39 in epithelial cells. The introduction of mutationally activated G12 protein into K562 cells blocked cadherin-mediated cell adhesion.40 Considering these data we speculate that activation of G12 by ATP may negatively contribute to TER measurements in control cells, whereas G12 depletion eliminated this negative effect (Figure 5C), thereby potentiating the effect of ATP.
Elevation of cAMP levels and activation of PKA are known to be associated with barrier enhancement.41 Previous data, however, suggest that ATP-induced barrier enhancement is cAMP-independent.18 In our experiments ATP challenge did not produce an elevation of cAMP (Figure 6A) but did increase PKA activity (Figure 6B). PKA activation independent of cAMP has recently been described. This signaling pathway uses activation of PKA via anchoring proteins such as AKAP11042 and NF-
B.43 Despite an established role for PKA in barrier protection, PKA targets involved in endothelial barrier function remain largely unknown. The focal adhesion- and microfilament-associated protein VASP is a known PKA target. Because VASP has been implicated in many actin-based processes,44 its involvement in barrier regulation seems plausible. It has been demonstrated that PKA-dependent phosphorylation of VASP acts as a negative regulator of actin dynamics45 and occurs on cell spreading.46 VASP is abundantly expressed in EC, however its role in endothelial physiology is only starting to be explored. VASP might participate in maintaining an open paracellular pathway, acting as a negative regulator of barrier function, whereas phosphorylation on Ser157 may be associated with relaxation of the actin cytoskeleton and increased barrier function.31 Our data support this idea. First, extracellular ATP triggered PKA-dependent VASP phosphorylation, which occurred in parallel with an increase in TER and cell spreading (Figure 6D) suggesting that nonphosphorylated VASP is a negative regulator of barrier function, and its phosphorylation diminishes that negative effect. Second, depletion of total VASP also eliminates the negative effect and strengthens the barrier (Figure 6E), even if the amount of the phosphorylated form should be reduced accordingly. Therefore, both VASP phosphorylation and depletion are associated with EC barrier enhancement, implicating a role for unphosphorylated VASP as a negative regulator.
Another molecular mechanism of ATP effects on endothelial barrier function defined in the current work is MLC dephosphorylation. EC contraction driven by MLC phosphorylation is a key event in several models of agonist-induced barrier dysfunction.1 It is unclear, however, if the opposite is true: the role of MLC dephosphorylation in endothelial barrier protection has not been confirmed. Noticeable dephosphorylation of MLC occurs at 30 minutes after ATP challenge and coincides with the peak of barrier enhancement (Figure 7A and 8
A). However, the activity of MLCP starts increasing shortly after ATP treatment and completely correlates with the time course of barrier enhancement (Figure 8A and 8B). Early lack of MLC dephosphorylation may be explained by intracellular Ca2+ elevation resulting in the increased activity of MLC kinase, which in turn, leads to an increased level of phosphorylated MLC. But it neither overcomes the effect of ATP nor is it causally related to ATP-induced barrier enhancement.
Because the catalytic subunit of MLCP was identified as a PP1 isoform,47 we specifically studied the association of PP1 with the myosin fraction (Figure 8C) and found it to be increased (Figure 8C and 8D). This confirms the involvement of MLCP in ATP-induced enhancement of endothelial barrier. Furthermore, we found that ATP causes activation of MLCP via a G protein–coupled mechanism. Interestingly, although both Gq and Gi2 are involved in barrier-enhancing ATP signaling (Figure 5A and 5B), only Gq appears to be important for phosphatase activation. MLCP is regulated via its regulatory subunit myosin-binding phosphatase targeting. Phosphorylation of myosin-binding phosphatase targeting by Rho kinase leads to inhibition of its activity.48 Our data provide novel evidence of a positive regulation for MLCP via a G protein–coupled mechanism. To our knowledge, a positive regulatory mechanism for MLCP has only been shown in a study of cell division.49
Our study presents an attempt to clarify some of the signaling pathways involved in ATP-induced endothelial cell barrier enhancement. Lack of experimental data leaves room for many speculations regarding the similarity of ATP signaling to other known barrier-protective mechanisms. For instance, action of potent barrier protector sphingosine 1-phosphate has been long investigated and involves activation of small GTPase Rac followed by strong enhancement of cortical actin.50 We have not studied these particular mechanisms in our work. Further studies are needed to fully characterize complex signaling machinery involved in ATP-induced enhancement of endothelial barrier. Based on our data, however, several signaling elements can be distinguished. These include a G protein–coupled receptor (most likely P2Y type), Gq and Gi2, PKA, and MLCP. PKA activation, however, occurs via a cAMP-independent mechanism, possibly involving protein kinase A–anchoring proteins (AKAPs). Ca2+ signaling and Erk activation are not involved in the effect of ATP on endothelial barrier.
Beneficial effects of ATP on barrier function suggest that endothelium is a potential therapeutic target for purine-based agonists. Further studies, using isolated lung and animal models, should clarify the possible use of such agonists in the treatment of acute lung injury.
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Acknowledgments |
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This work was supported by grants from National Heart, Lung, and Blood Institutes (HL67307, HL68062, and HL58064), and American Lung Association of Maryland Research Grant.
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Footnotes |
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This manuscript was sent to Donald Heistad, Consulting Editor, for review by expert referees, editorial decision, and final disposition.
Original received January 5, 2005; revision received June 9, 2005; accepted June 20, 2005.
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