Am J Physiol Lung Cell Mol Physiol 291: L289 –L295, 2006;
doi:10.1152/ajplung.00343.2005.
Endothelial cell barrier enhancement by ATP is mediated by the small
GTPase Rac and cortactin
Jeffrey R. Jacobson, Steven M. Dudek, Patrick A. Singleton,
Irina A. Kolosova, Alexander D. Verin, and Joe G. N. Garcia
Department of Medicine, Pritzker School of Medicine, University of Chicago, Chicago, Illinois
Submitted 4 August 2005; accepted in final form 20 February 2006
vascular permeability; cytoskeleton; actin
EXTRACELLULAR NUCLEOTIDES,
including ATP, are known to
mediate a number of physiologically relevant vascular functions via effects on both smooth muscle cells and endothelial
cells (EC). The effects of ATP on the endothelium are of
particular interest as large amounts of these nucleotides are
released by activated platelets (as well as EC) in response to
shear stress and in the setting of acute inflammation (2, 3, 19,
34). Accordingly, local effects of ATP on the endothelium may
be relevant to a variety of clinical settings characterized by
derangements in endothelial function and the increased vascular permeability observed in inflammatory lung syndromes
such as acute lung injury (ALI), a condition with significant
morbidity and mortality and for which there are currently few
treatment options. Although it is known that ATP-induced
increases in EC intracellular Ca2⫹ with the subsequent release
Address for reprint requests and other correspondence: J. G. N. Garcia,
Pritzker School of Medicine, Univ. of Chicago, 5841 S. Maryland Ave.,
MC6092 AMBW604, Chicago, IL 60637 (e-mail: jgarcia@medicine
.bsd.uchicago.edu).
http://www.ajplung.org
of nitric oxide and prostacyclin promote vasodilation (5, 15,
23), the direct effects of ATP on EC with respect to activation
of the actin cytoskeleton and barrier function remain poorly
characterized.
ATP affects intracellular EC signaling events through activation of specific P2 purinergic receptors (20). This family of
receptors is divided into two groups, ion-coupled P2X receptors, composed of seven cloned subtypes, and G proteincoupled P2Y receptors, composed of six subtypes. Of these,
P2X4, P2X5, P2Y11, P2Y1, and P2Y2 are the most highly
expressed subtypes in EC (28, 31). Whereas the activation of
P2X receptors promotes the influx of extracellular Ca2⫹, binding of adenosine nucleotides to P2Y receptors leads to increased inositol 1,4,5-trisphosphate (IP3) via G protein-mediated phospholipase C (PLC) activation culminating in the
release of Ca2⫹ from intracellular stores (1).
Our lab has characterized (7, 8) several agonists that also
induce IP3-mediated increases in cytosolic calcium, including
thrombin, a serine protease, and the phospholipid sphingosine
1-phosphate (S1P), which is also released by activated platelets. Notably, although both of these agonists are potent activators of the EC actin cytoskeleton, they exert diametrically
opposite effects on EC barrier function as thrombin induces
rapid and pronounced barrier disruption and S1P is a robust EC
barrier enhancer (7, 8). Both thrombin and S1P induce EC
myosin light chain (MLC) phosphorylation and actomyosin
contraction via the coordinate activities of the Ca2⫹/calmodulin-dependent MLC kinase (MLCK) and Rho kinase (6, 7, 8).
However, beyond these effects there are several discernible
differences. For example, unlike thrombin, S1P activates the
small GTPase Rac in association with translocation of the
80-/85-kDa actin-binding protein cortactin to the cell periphery
and augmentation of the cortical actin ring, events that may
account for its barrier-enhancing properties (6, 13). Hence,
although ATP also induces IP3-mediated increases in cytosolic
Ca2⫹, its effects with respect to EC cytoskeletal rearrangement
and barrier function cannot be predicted on this basis alone.
Accordingly, we sought to characterize and examine specific
mechanisms underlying EC cytoskeletal activation and barrier
enhancement by ATP.
MATERIALS AND METHODS
Measurement of transendothelial electrical resistance. Human pulmonary artery EC (HPAEC) were grown to confluence in complete
medium over evaporated gold microelectrodes connected to a phasesensitive lock-in amplifier. Experiments relying on small interfering
The costs of publication of this article were defrayed in part by the payment
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1040-0605/06 $8.00 Copyright © 2006 the American Physiological Society
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Jacobson, Jeffrey R., Steven M. Dudek, Patrick A. Singleton,
Irina A. Kolosova, Alexander D. Verin, and Joe G. N. Garcia.
Endothelial cell barrier enhancement by ATP is mediated by the small
GTPase Rac and cortactin. Am J Physiol Lung Cell Mol Physiol 291:
L289 –L295, 2006; doi:10.1152/ajplung.00343.2005.—ATP is a
physiologically relevant agonist released by various sources, including activated platelets, with complex effects mediated via activation of
P2 purinergic receptors. ATP-induced endothelial cell (EC) production of prostacyclin and nitric oxide is recognized, and EC barrier
enhancement evoked by ATP has been described. ATP effects on EC
barrier function and vascular permeability, however, remain poorly
characterized. Although the mechanisms involved are unclear, we
previously identified activation of the small GTPase Rac and translocation of cortactin, an actin-binding protein, as key to EC barrier
augmentation induced by simvastatin and sphingosine 1-phosphate
and therefore examined the role of these molecules in ATP-induced
EC barrier enhancement. ATP induced rapid, dose-dependent barrier
enhancement in human pulmonary artery EC as measured by transendothelial electrical resistance, with a peak effect appreciable at 25
min (39% increase, 10 ␮M) and persisting at 2 h. These effects were
associated with rearrangement of the EC actin cytoskeleton, early
myosin light chain phosphorylation, and spatially defined (cell periphery) translocation of both Rac and cortactin. ATP (10 ␮M)-treated EC
demonstrated a significant increase in Rac activation relative to
controls, with a maximal effect (⬃4-fold increase) at 10 min. Finally,
ATP-induced barrier enhancement was markedly attenuated by reductions of either Rac or cortactin (small interfering RNA) relative to
controls. Our results suggest for the first time that ATP-mediated
barrier protection is associated with cytoskeletal activation and is
dependent on both Rac activation and cortactin.
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ENDOTHELIAL BARRIER ENHANCEMENT BY ATP
RNA (siRNA) transfection were performed in 1% serum medium as
described below. Transendothelial electrical resistance (TER) was
measured in response to agonists applied directly to the medium with
an electrical cell-substrate impedance sensing system (ECIS) (Applied
BioPhysics, Troy, NY) as previously described (9). As cells adhere
and spread out on the microelectrode TER increases, whereas cell
retraction, rounding, or loss of adhesion is reflected by a decrease in
TER. For each experiment TER values were plotted at 5-min intervals
and were derived from the average values measured over the surrounding 3-min period. Data points from each 5-min interval were
then averaged across all experiments of a given condition and plotted.
MLC phosphorylation in intact endothelium. HPAEC were analyzed for MLC phosphorylation by SDS-PAGE followed by Western
immunoblotting with antibody specific for diphosphorylated MLC as
previously described (25). Blots were scanned and quantitatively
Fig. 2. Actin cytoskeletal rearrangement and cortactin translocation in EC treated with ATP. Immunofluorescent imaging of confluent HPAEC monolayers
treated with ATP (10 ␮M) demonstrate enhanced cortical actin (A, arrows). Although early cytoskeletal rearrangement is apparent at earlier time points, these
effects are fully realized at 30 min in association with decreased central stress fibers and decreased paracellular gaps relative to vehicle controls. Evidence of
the peripheral translocation of cortactin (B, arrowheads) is seen at 5 min and is even more pronounced at 30 min.
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Fig. 1. Effect of ATP on endothelial cell (EC) barrier function. Human
pulmonary artery EC (HPAEC) were grown to confluence on gold microelectrodes before treatment with ATP and measurement of transendothelial electrical resistance (TER). Relative to vehicle controls, ATP (1–100 ␮M) induced
a rapid, dose-dependent increase in barrier function as measured by TER (n ⫽
3 for each group). A maximal effect was observed at 20 –30 min after ATP at
all concentrations, whereas higher doses of ATP (10 or 100 ␮M) were
associated with persistent barrier enhancement (⬎2 h).
analyzed with ImageQuant software (version 5.2, Amersham Biosciences, Piscataway, NJ).
Immunofluorescent microscopy. Confluent HPAEC grown on coverslips were exposed to experimental conditions, fixed with 3.7%
formaldehyde, and permeabilized with 0.25% Triton X-100. After
blocking with 2% BSA, cells were exposed to primary antibodies for
60 min. Fluorescently tagged secondary antibodies were applied for
60 min in the dark. F-actin was detected by staining with Texas
red-conjugated phalloidin. Cells were imaged with a Nikon videoimaging system.
Rac activation assay. Determination of Rac activation (Rac-GTP)
was performed with a commercially available kit (Upstate, Lake
Placid, NY). Rac-GTP was pulled down with a p21-activated kinase
(PAK)1-glutathione S-transferase fusion protein, and samples were
subjected to Western blotting and detection with anti-Rac1 antibody.
Silencing RNA. For construction of the siRNA, a transcriptionbased kit from Ambion (Austin, TX) was used (Silencer siRNA
construction kit). The siRNA sequences targeting human cortactin and
Rac1 were generated with mRNA sequences from GenBank (gi:
20357555, 29792301). Potential target sequences were aligned to the
human genome database in a BLAST search to eliminate sequences
with significant homology to other human genes. For each mRNA (or
scramble), two targets were identified. Specifically, cortactin target
sequence 1 (5⬘-AATGCCTGGAAATTCCTCATT-3⬘), cortactin target sequence 2 (5⬘-AAACAGAATTTCGTGAACAGC-3⬘), Rac1 target sequence 1 (5⬘-AAAACTTGCCTACTGATCAGT-3⬘), Rac1 target sequence 2 (5⬘-AACTTGCCTACTGATCAGTTA-3⬘), scramble
sequence 1 (5⬘-AAGAGAAATCGAAACCGAAAA-3⬘) and scramble sequence 2 (5⬘-AAGAACCCAATTAAGCGCAAG-3⬘) were
used. EC cells were then transfected with siRNA, using siPORTamine
as transfection reagent. Cells (⬃40% confluent) were serum starved
for 2 h, followed by incubation with 3 ␮M (1.5 ␮M of each siRNA)
target siRNA (or scramble siRNA or no siRNA) for 6 h in serum-free
medium. Medium with serum was then added (1% serum final
concentration) for 42 h before Western blotting or functional assays
were conducted.
Statistical analysis. One-way ANOVA was used to compare the
means of data from two or more different experimental groups. If a
significant difference was present by ANOVA (P ⬍ 0.05), a least
significant difference test was performed post hoc. Subsequently,
differences between groups were considered statistically significant
when P ⬍ 0.05. Results are expressed as means ⫾ SE.
ENDOTHELIAL BARRIER ENHANCEMENT BY ATP
RESULTS
monolayers treated with ATP (10 ␮M) demonstrated dramatic
cytoskeletal rearrangement characterized by decreased central
actin stress fibers and enhanced cortical actin that was fully
realized at 30 min (Fig. 2A). These changes were associated
with reductions in the number of paracellular gaps consistent
with augmentation of barrier integrity. Additionally, evidence
of the peripheral translocation of cortactin was noted at 5 min
and was even more pronounced at 30 min, consistent with the
time course of actin cytoskeletal rearrangement (Fig. 2B).
ATP induces EC MLC phosphorylation. EC cytoskeletal
rearrangement is driven by actomyosin contraction, which is
dependent on the phosphorylation of MLC. We measured
MLC diphosphorylation in HPAEC treated with ATP (10 ␮M)
by Western blotting, using an antibody specific for diphosphorylated MLC (Fig. 3A). MLC phosphorylation levels were
dramatically increased early (⬃7-fold increase at 1 and 5 min),
consistent with the observed time course of cytoskeletal rearrangement. Levels were decreased at 10 min and were back to
Fig. 3. EC myosin light chain (MLC) phosphorylation by ATP. Confluent HPAEC were treated with ATP (10 ␮M) before Western blotting and probing with
antibody specific for diphosphorylated MLC (MLC-pp). Relative to vehicle controls, ATP-induced a rapid and dramatic increase in MLC diphosphorylation that
was significantly attenuated at 10 min (A; n ⫽ 3, *P ⬍ 0.05 relative to vehicle). Separately, HPAEC monolayers treated with ATP (10 ␮M) and then probed
for diphosphorylated MLC (B) demonstrate a predominant localization at the periphery of EC (arrows) that is evident at 5 min and no longer appreciable at 20
min, consistent with the observed time course of actin cytoskeletal rearrangement. In contrast, thrombin (1 U/ml, 5 min), a barrier-disruptive agonist, induces
increased MLC phosphorylation throughout EC that corresponds to transcellular actin stress fiber formation.
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EC barrier function is enhanced by ATP. TER was used as
a sensitive measure of EC monolayer barrier integrity and
barrier function. HPAEC grown to confluence in complete
medium and then treated with 100 ␮M ATP exhibited a rapid
and significant increase in TER (45% increase at 25 min),
indicative of enhanced barrier function, with a persistent effect
after ⬎2 h (Fig. 1). A dose-dependent response was observed,
as 1 and 10 ␮M ATP affected a significant but lesser increase
in TER (20% and 39% increase at peak effects, respectively).
On the basis of these results, we chose to use 10 ␮M ATP for
subsequent experiments, as a significant difference between the
maximal effects of 10 ␮M and 100 ␮M was not appreciable.
Moreover, these concentrations of ATP are most consistent
with concentrations used in other reports (10, 22).
EC cytoskeletal rearrangement and cortactin translocation
by ATP. Immunofluorescent images were obtained to link the
effects of ATP on EC TER with cytoskeletal changes. EC
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ENDOTHELIAL BARRIER ENHANCEMENT BY ATP
ences in MLC phosphorylation that correspond to the barrier
effects of thrombin and ATP.
ATP induces endothelial cell Rac activation. As we had
previously identified EC barrier augmentation in association
with enhanced cortical actin as associated with activation of the
small GTPase Rac, we measured levels of Rac activation in EC
challenged with ATP (10 ␮M). Relative to vehicle, a significant increase in activated Rac was evident at 10 min (⬃4-fold
increase; Fig. 4A). Moreover, Rac activation was increased as
early as 1 min, although these results did not achieve significance.
Activation of the small GTPases is dependent of isoprenylation and ultimately translocation to the cell periphery, where
they can bind to GTP. Immunofluorescent images of EC
treated with ATP (10 ␮M) followed by an antibody specific for
Rac1 confirmed increased translocation of Rac to the cell
Fig. 4. Rac activation in ATP-treated EC. HPAEC were grown to ⬃70% confluence before stimulation with vehicle, sphingosine 1-phosphate (S1P; 1 ␮M, 1
min), or ATP (10 ␮M, 1–20 min). Activated Rac (Rac-GTP) was pulled down with a p21-activated kinase 1-glutathione S-transferase fusion protein. Samples
were then subjected to Western blotting and detection with anti-Rac1 antibody (A). A trend toward increased Rac activation is suggested early (1 and 5 min),
and a significant increase is evident at 10 min. (n ⫽ 3, *P ⬍ 0.05 relative to vehicle). Separately, immunofluorescent imaging of HPAEC monolayers treated
with ATP (10 ␮M) demonstrate early (within 5 min) peripheral translocation of Rac (B, arrows), consistent with the observed time course of actin rearrangement.
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baseline within 20 min. We next obtained immunofluorescent
images to determine the site of ATP-induced MLC phosphorylation. Relative to vehicle, HPAEC grown to confluence and
then treated with ATP (10 ␮M) demonstrated increased MLC
phosphorylation at the cell periphery, the site of cortical actin
enhancement, at 5 min (Fig. 3B). This increased MLC phosphorylation was no longer evident at 20 min, consistent with
our Western blotting data and the observed time course of
active cytoskeletal rearrangement. The peripheral distribution
of MLC phosphorylation in response to ATP stands in stark
contrast to the colocalization of MLC phosphorylation with
actin stress fibers in response to thrombin, an agonist known to
induce MLC phosphorylation in the setting of EC contraction
and barrier disruption. Although quantitative differences corresponding to our Western blotting densitometry are not appreciable, these images clearly demonstrate qualitative differ-
ENDOTHELIAL BARRIER ENHANCEMENT BY ATP
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DISCUSSION
As ATP is a potentially prevalent constituent of the endothelial microenvironment under a variety of clinically relevant
conditions, an understanding of its effect on the endothelium is
of significant interest. The literature is highly inconsistent with
respect to ATP-induced EC barrier regulation, with both barrier disruption, characterized by increased permeability, and
barrier enhancement described (10, 29). We now extend the
initial report of ATP-induced EC barrier enhancement (10) and
demonstrate for the first time the dynamic EC cytoskeletal
rearrangement by ATP in association with specific biochemical
events associated with cytoskeletal activation including rapid
MLC phosphorylation, activation of the small GTPase Rac,
and cortactin translocation. In addition, we have identified
these latter two events as necessary to achieve maximal EC
barrier enhancement by ATP.
Early EC MLC dephosphorylation by ATP has also been
reported (10, 22). Although our findings should not necessarily
supersede the work of previous investigators, our data from
several complementary experiments are notable as ATP-induced EC barrier enhancement, measured by TER, was associated with decreased paracellular gaps and enhanced cortical
actin histologically, and dynamic actin cytoskeletal rearrangement in this setting was associated with increased MLC phosphorylation, a requirement for actomyosin contraction and
cytoskeletal activation. Indeed, we have also described EC
MLC dephosphorylation by ATP (14); however, these findings
were evident only at time points (30 min) later than those of the
present study and are compatible with rapid cytoskeletal rearrangement by ATP that is fully realized by 30 min. Finally, the
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Fig. 5. Effect of Rac and cortactin small interfering RNA (siRNA) on
ATP-induced EC barrier enhancement. HPAEC were transfected with siRNA
and grown to confluence on gold microelectrodes. Rac1 and cortactin siRNA
efficacy and specificity were confirmed by Western blotting (A). Measurements
of TER demonstrate a significant attenuation of ATP-induced (10 ␮M) barrier
enhancement in EC transfected with siRNA specific for either Rac (B) or
cortactin (C) relative to both nontransfected controls and EC transfected with
scramble siRNA (n ⫽ 5 for each group).
barrier-enhancing effects of ATP were dependent on both
activation of the small GTPase Rac and translocation of the
actin-binding protein cortactin, events associated with EC
barrier enhancement and consistent with the effects of other
agonists and stimuli our lab has studied that also promote EC
barrier function (6, 12).
EC Rac activation by ATP is a novel finding; however, the
mechanisms responsible for this event remain speculative.
Consistent with the known effects of ATP, recent evidence
suggests that Rac activation is dependent on intracellular Ca2⫹
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periphery within 5 min (Fig. 4B) and no longer appreciable at
20 min. The time course of these events was similar to that of
Rac activation measured by Western blotting.
ATP-induced EC barrier enhancement is dependent on both
Rac activation and cortactin translocation. To establish the
functional significance of both Rac activation and cortactin
translocation in ATP-induced EC barrier enhancement we used
specific siRNAs designed to reduce either Rac or cortactin
expression and then measured TER. EC were transfected with
Rac siRNA, cortactin siRNA, or scramble siRNA and grown to
confluence overlying ECIS gold microelectrodes. Separately,
untransfected cells were used as a control. EC were then
treated with ATP (10 ␮M), and TER was measured. Notably,
although Rac activity has been shown to be necessary for
maintenance of EC monolayer integrity (32), there was no
appreciable difference in baseline electrical resistances measured among untransfected EC and any of the siRNA-transfected EC. We speculate that this may be due to sufficient
residual Rac activity in our Rac-silenced EC as well as differences attributable to the specific techniques used to measure
barrier integrity. Although scramble siRNA attenuated ATPinduced barrier enhancement relative to untransfected controls,
Rac silencing elicited a further and highly significant attenuation (60% reduction relative to scramble siRNA at peak effect
of ATP; Fig. 5B). In separate experiments, silencing of cortactin resulted in a significant reduction in peak ATP effects (47%
reduction relative to scramble; Fig. 5C). These data confirm an
important functional role for both Rac and cortactin in ATPmediated EC barrier enhancement.
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ENDOTHELIAL BARRIER ENHANCEMENT BY ATP
ACKNOWLEDGMENTS
The authors express special thanks to Lakshmi Natarajan for expert cell
culture preparation.
GRANTS
The authors acknowledge the support of the Center for Translational
Respiratory Medicine, support from the National Heart, Lung, and Blood
Institute (Grants HL-71411, HL-58064, HL-69340, and HL-66583), and support from the Parker B. Francis Foundation (J. R. Jacobson) and the Dr. Lowell
T Coggeshall Endowment (J. G. N. Garcia).
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and activation of protein kinase C (PKC) (17, 26). At the same
time, however, there is evidence that ATP-induced EC barrier
enhancement occurs independent of effects on Ca2⫹ or PKC
(21). Unfortunately, the authors who reported these findings
did not assess Rac activation but were able to abolish ATPinduced EC barrier enhancement by inhibition of PLC, an
effector of IP3-mediated release of Ca2⫹ from intracellular
stores. Although the possibility exists that the rapid activation
of Rac by ATP is mediated by events that are PLC dependent
but Ca2⫹ independent, this finding would be highly unexpected. Separately, specific effectors of Rac activation have
now been identified including several guanine nucleotide exchange factors (GEFs) as well as the disruption of Rac from
guanine dissociation inhibitor, which enables Rac activation
via GEF-mediated GTP exchange (4, 18). Further investigation
of the potential role of these mediators in ATP-induced Rac
activation is required.
We previously characterized EC cortactin translocation in
association with Rac activation, and a linkage between these
two events is supported by the colocalization of cortactin with
Rac on EC stimulation at sites of polymerized actin peripherally as well as evidence of cortactin phosphorylation and
translocation by PAKs, downstream effectors of Rac whose
activation can be inhibited by the inhibition of Rac (6, 7, 12).
Our findings now are consistent with the growing body of
evidence (30, 33), implicating an important association of these
two molecules with each other in the setting of EC barrier
enhancement.
Ultimately, characterization of the EC barrier-enhancing
properties of ATP may have profound clinical implications.
Already, our lab has begun using a murine model of ALI to
investigate the attenuation of inflammation via effects on lung
vascular permeability by intravenously administered ATP. Furthermore, elucidation of the precise mechanisms involved in
these effects, particularly as they are relevant to other barrierenhancing agonists that we have previously investigated as
potentially novel treatment strategies in ALI (11, 16, 24), may
lead to highly specific and effective therapies for a variety of
clinical conditions characterized by increased vascular permeability.
ENDOTHELIAL BARRIER ENHANCEMENT BY ATP
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