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Cloning and pharmacological characterization of the equine adenosine A2A
receptor: a potential therapeutic target for the treatment of equine
endotoxemia
C. I. BRANDON*
M. VANDENPLAS�
H. DOOKWAH�
J. LINDEN– &
T. F. MURRAY*
Departments of *Physiology and
Pharmacology, �Large Animal Medicine and�Anatomy and Radiology, College of
Veterinary Medicine, University of Georgia,
Athens, GA; –Department of Physiology,
Health Sciences Center, University of
Virginia, Charlottesville, VA, USA
Brandon, C. I., Vandenplas, M., Dookwah, H., Linden, J., Murray, T. F. Cloning
and pharmacological characterization of the equine adenosine A2A receptor: a
potential therapeutic target for the treatment of equine endotoxemia. J. vet.
Pharmacol. Therap. 29, 243–253.
The aim of the current study was to clone the equine adenosine A2A receptor
gene and to establish a heterologous expression system to ascertain its
pharmacologic profile via radioligand binding and functional assays. An eA2A-
R expression construct was generated by ligation of the eA2A cDNA into the
pcDNA3.1 expression vector, and stably transfected into human embryonic
kidney cells (HEK). Binding assays identified those clones expressing the eA2A-
R, and equilibrium saturation isotherm experiments were utilized to determine
dissociation constants (KD), and receptor densities (Bmax) of selected clones.
Equilibrium competition binding revealed a rank order of agonist potency of
ATL > CV-1808 > NECA > 2-CADO > CGS21680, and a rank order of ant-
agonist potency as ZM241385 > 8-phenyltheophylline > p-sulfophenylthe-
ophylline > caffeine. Furthermore, adenylate cyclase assays using selective
A2A-R agonists revealed that the eA2A-R functionally coupled to Gas as
indicated by an increase in intracellular [3H]cAMP upon receptor activation.
Finally, NF-jB reporter gene assays revealed a CGS21680 concentration-
dependent inhibition of NF-jB activity. These results indicate that the
heterologously expressed eA2A-R has a pharmacological profile similar to that
of other mammalian A2A receptors and thus can be utilized for further
characterization of the eA2A-R to ascertain whether it can serve as a suitable
pharmacological target for equine inflammatory disease.
(Paper received 21 March 2006; accepted for publication 5 April 2006)
Thomas F. Murray, Editor, Critical Reviews in Neurobiology, Distinguished Research
Professor and Head, Department of Physiology and Pharmacology, College of
Veterinary Medicine, University of Georgia, Athens, GA 30602, USA. E-mail:
tmurray@vet.uga.edu
INTRODUCTION
Adenosine is an endogenous purine nucleoside that has evolved
to modulate many physiologic processes. Cellular signaling by
adenosine occurs through four known adenosine receptor
subtypes (A1, A2A, A2B, and A3), all of which are seven
transmembrane spanning G-protein coupled receptors (Hettinger
et al., 1998; Olah & Stiles, 2000). These four receptor subtypes
are further classified based on their ability to either stimulate or
inhibit adenylate cyclase activity. The A2A and A2B receptors
couple to Gas and mediate the stimulation of adenylate cyclase,
while the A1 and A3 adenosine receptors couple to Gai which
inhibits adenylate cyclase activity (Cronstein, 1994; Linden,
2001). Additionally, A1 receptors couple to Gao, which has been
reported to mediate adenosine inhibition of Ca2+ conductance,
whereas A2B and A3 receptors also couple to Gaq and stimulate
phospholipase activity.
Extracellular adenosine concentrations from normal cells are
approximately 300 nM (Hirschhorn et al., 1981); however, in
response to cellular damage (e.g. in inflammatory or ischemic
Abbreviations: NECA, 5¢-N-ethylcarboxamidoadenosine; CGS-21680, 2-
p-(2-carboxyethyl)phenethylamino-5¢-N-ethylcarboxamidoadenosine; 2-
chloroadenosine, 6-amino-2-chloropurine riboside; CV-1808, 2-phenyl-
aminoadenosine; [3H]ZM241385, [2-3H]-4-(2-[7-Amino-2-(2-furyl)-
[1,2,4]-triazolo-[2,3-a]-[1,3,5]-triazin-5-ylamino]ethyl)phenol; ATL303,
Adenosine Therapeutics LLC 2-propynylcyclohexyl-5¢-N: ethylcarb-
oxamidoadenosine analog.
J. vet. Pharmacol. Therap. 29, 243–253, 2006.
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd 243
tissue), these concentrations are quickly elevated (600–
1200 nM) (Cronstein et al., 1995). Thus, in regard to stress or
injury, the function of adenosine is primarily that of cytoprotec-
tion preventing tissue damage during instances of hypoxia,
ischemia, and seizure activity (Ibayashi et al., 1988). Activation
of A2A receptors produces a constellation of responses that in
general can be classified as anti-inflammatory (Sullivan &
Linden, 1998; Salvatore et al., 2000).
Inflammation represents a host defense response to a variety
of harmful stimuli and is crucial to the survival of an organism
(Lukashev et al., 2004). The inflammatory cascade is mediated
by proinflammatory cytokines such as tumor necrosis factor-
alpha (TNF-a), lymphotoxin-a, and interferon-gamma as well as
many others; the interplay among these cytokines and cytokine-
induced chemokines results in the activation of macrophages,
lymphocytes, and neutrophils (Rosenberg & Gallin, 2003).
Whereas the primary function of these proinflammatory
molecules is to remove the invading pathogen, it is not without
its consequences as local tissue damage and prolonged
inflammation can also occur. One of the most potent of these
cytokines is TNF-a, and in some instances of inflammatory
disease its expression can become dysregulated via activation of
the NF-jB pathway. NF-jB is a nuclear transcription factor that
activates the transcription of a host of inflammatory genes –
including that of TNF-a. Remarkably, NF-jB activation is self-
limiting; while it transcribes proinflammatory genes, it also
drives the transcription of its own inhibitory protein – inhibitory-
jB (I-jB). Thus, NF-jB is active for approximately 30 min; after
which I-jB is translated and binds NF-jB forming an inactive
complex in the cytoplasm. However, the expressed TNF-a can
also bind to and activate its own receptor on certain cell types
and this also activates the NF-jB pathway in some instances of
inflammatory disease. Therefore, in this situation, NF-jB activity
is no longer self-limiting; thus TNF-a transcription continues
constitutively.
It has been shown that activation of adenosine A2A receptors
strongly inhibits the NF-jB pathway, as well as the production of
TNF-a (Bshesh et al., 2002). With this in mind, it seems
reasonable to assume that administration of adenosine may be
of therapeutic value. However, due to the relatively short
biological half-life of adenosine and potential adverse side effects
(e.g. hypotension, bradycardia, and hypothermia), its therapeu-
tic usefulness may be limited. To that end, research into the
activation of adenosine receptor subtypes has primarily focused
on the use of adenosine analogs that may not have these adverse
effects due to their specificity for individual adenosine receptor
subtypes.
Thus, the long-term goal stemming from our laboratory is to
characterize fully the equine adenosine A2A receptor (A2A-R) to
ascertain if it can be utilized as a therapeutic target for the
treatment of equine endotoxemia. Thus, the present study was
designed to characterize the heterologously expressed equine
A2A receptor via radioligand binding and functional assays using
selective receptor antagonists and an array of adenosine analogs.
It is our belief that results from this study will provide the
foundation for future research to determine the effects of these
agonists on cytokine production by equine monocytes chal-
lenged with LPS. To this end, the objectives of this study were (i)
to heterologously express equine A2A receptor in human
embryonic kidney 293 (HEK) cells; (ii) to characterize the
pharmacological signature of this receptor utilizing radioligand
binding assays; (iii) to determine the ability of adenosine analogs
to alter adenylate cyclase activity and intracellular concentra-
tions of cAMP; and (iv) to determine what effect eA2A-R
activation plays on signaling components of the inflammatory
cascade.
MATERIALS AND METHODS
Cloning and sequencing of the equine A2A receptor cDNA
An equine monocyte cDNA library was constructed as described
in the Uni-ZAP II cDNA library construction kit (Stratagene,
LaJolla, CA, USA), and subsequently screened for the equine A2A
receptor cDNA by hybridization with a 32P end-labeled rat A2A
oligonucleotide as a probe. A single hybridization positive plasmid,
isolated by alkali lysis over Qiagen columns (Valencia, CA, USA),
contained an EcoRI/XhoI insert of the estimated size for the full-
length equine A2A receptor, and was selected for sequencing. The
plasmid was sequenced in either direction with an ABI Prism
BigDyeTM Terminator Cycle Sequencing Ready Reaction Kit (ver.
3.0) in both 96- and 384-well format. Sequencing reactions
included 4 lL of plasmid DNA (100 ng), 44 pmol of primer,
0.70 lL of BigDyeTM, 0.58 lL ddH2O, 0.34 lL DMSO, 1.4 lL 5x
reaction buffer (5x ¼ 10 mM MgCl2, 400 mM Tris-Cl pH 9.0),
resulting in a total reaction volume of 7.2 lL. Thermal cycling
reactions were performed on a 96-well GeneAmp� PCR System
(Applied Biosystems, Foster City, CA, USA) and an Autolid Dual
384-well GeneAmp� PCR System 9700 (Applied Biosystems). The
purified extension products were separated on an ABI Prism�
3700 DNA Analyzer (Applied Biosystems).
The eA2A-R-containing plasmid was then amplified by trans-
formation into bacterial cells (Top10 cells; Invitrogen, Carlsbad,
CA, USA), which were grown overnight at 37 �C on LB agar
plates containing 50 lg/mL ampicillin (Sigma, St Louis, MO,
USA). Ampicillin-resistant colonies were then inoculated in
300 mL of LB broth (Sigma) containing 50 lg/mL ampicillin
and shaken overnight at 37 �C, 5% CO2. Plasmid purification of
the resulting culture was achieved using an endotoxin-free
plasmid purification kit (Endo-Free Maxi Kit; Qiagen) as des-
cribed previously (Meyer, 1990). A BamHI/DraI restriction
fragment of the eA2A-R cDNA was then directionally subcloned
into a BamHI/NotI restriction site of the pcDNA3.1 expression
vector (Invitrogen) using a Takara DNA ligation kit (Fisher
Scientific, Suwannee, GA, USA). Specifically, the eA2A-R cDNA
was digested with BamHI and DraI, while the pcDNA3.1
expression vector was digested with BamHI and NotI; the
digestion reactions were carried out in separate tubes at 37 �C
for 3 h. Prior to ligation, the pcDNA3.1 vector was treated
further with calf intestinal alkaline phosphatase to prevent
re-circularization of the plasmid DNA; after which both
244 C. I. Brandon et al.
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd
the eA2A-R cDNA and pcDNA3.1 were extracted in phenol-
chloroform to remove extraneous salts and proteins. The
resulting eA2A-R BamHI/DraI restriction fragment and BamHI/
NotI-digested pcDNA3.1 were resolved on an agarose gel,
purified and ligated together in a reaction using T4 DNA ligase
at 16 �C for 24 h. The eA2A-R cDNA was sequenced fully from
either end on an ABI 3700 automated sequencer (Amersham
Biosciences, Piscataway, NJ, USA) using the Transposon based
EZ::TN insertion kit (Epicentre, Madison, WI, USA) with the Kan-
2 FP-1 and Kan-2 RP-1 primers supplied by the manufacturer.
Sequence fragments were then assembled into a contig using
AssemblyLIGN software (Accelrys, Norwalk, CT, USA), and
translation start and stop codons identified via sequence analysis
utilizing MACVECTOR 6.5.3 software (Accelrys). The resulting
coding region of the eA2A-R cDNA was then translated into its
corresponding amino acid sequence and aligned with other
mammalian A2A-R proteins (MACVECTOR). Conserved amino acid
residues were then divided by the total alignment score to
determine sequence homology.
Heterologous expression system
For stable transfection, HEK293 were seeded in six-well microt-
iter plates (Becton-Dickinson, Bedford, MA, USA) at a density of
6 · 104 cells/well, and cultured in complete medium containing
Minimal Essential Media (MEM; Sigma), 10% fetal calf serum
(FCS), and 1% penicillin–streptomycin to approximately 75%
confluence. Transfection of the eA2A-R cDNA was performed
using Polyfect transfection reagent (Qiagen). Initially, 2 lg of the
eA2A-pcDNA plasmid were added to Eppendorf tubes, and
brought to a final volume of 100 lL with TE buffer (Tris–EDTA),
pH 7.5. Polyfect transfection reagent (40 lg) was then added
and allowed to incubate at room temperature for 10 min. HEK
cell growth media was removed and replaced with 1.5 mL
complete media in the absence of antibiotics, followed by the
addition of the eA2A-R cDNA/Polyfect reaction complex. The
transfection reaction was incubated for 6 h at 37 �C, 5% CO2,
and 95% relative humidity, after which the transfection media
was replaced with MEM supplemented with 10% FCS and 1%
penicillin–streptomycin. At 24 h post-transfection, the selection
media, comprised the complete media that included 0.25 mg/mL
of the antibiotic geneticin (G418, Invitrogen), was added. To
isolate individual eA2A-R clones, plaques were isolated using
6.4 · 8 mm cloning cylinders (Fisher Scientific), trypsinized, and
transferred to separate culture flasks. Individual eA2A-R-expres-
sing HEK cell clones were then maintained at 37 �C, 5% CO2,
95% relative humidity until pharmacological characterization
experiments were undertaken.
[3H]ZM241385 Radioligand Binding Assay
To screen individual clones for membrane receptor expression,
binding assays were performed on eA2A-R/HEK cell membrane
preparations. Initially, eA2A-R-expressing HEK cells were grown
to confluence in 150 · 15 mm Nunc culture plates (Fisher),
after which cells were harvested via cell scraping in ice cold
25 mM Tris, pH 7.5. Equine A2A/HEK cell membrane prepara-
tions were then obtained by Dounce homogenization (20
strokes), and the resulting membranes centrifuged at 40 000 g
for 20 min. The membranes were then resuspended in Tris buffer
and washed two additional times prior to the binding assays.
For the binding assays, 100 lg of eA2A/HEK cell membrane
were incubated in the presence of 1.0 nM of the selective A2A
antagonist [3H]ZM241385 (Tocris Cookson, Ellisville, MO, USA)
to define total binding and with the addition of 10 lM 2-
chloroadenosine (2-CADO) to define nonspecific binding. Prior to
use in binding assays, cell membrane preparations were
incubated with 2.0 U/mL adenosine deaminase for 30 min at
22 �C. The final reaction volume was 500 lL. The binding
reaction was incubated for 45 min at room temperature, after
which the membranes were harvested via rapid filtration onto
Brandell GF/B filters using a 48-well cell harvester (Brandell,
Gaithersburg, MD, USA). Four milliliters of scintillation cocktail
was added to each filter, and counts determined on a Beckman
LS6000 scintillation counter (Beckman Coulter, Fullerton, CA,
USA). Specific binding was determined by subtracting nonspe-
cific binding from that of the total. Data were analyzed using
Graphpad Prism Software (Prism 4.0; Graphpad Software Inc.,
San Diego, CA, USA).
Equilibrium saturation isotherms
To determine the antagonist affinity (KD) and receptor density
(Bmax) of selected clones, equilibrium saturation isotherms were
performed. Briefly, 100 lg of eA2A-HEK cell membranes were
incubated with increasing concentrations of [3H]ZM241385
(0.0625–2.0 nM) in the presence or absence of 10 lM 2-CADO to
define nonspecific and total binding, respectively. Data were
analyzed utilizing nonlinear regression analysis with Graphpad
Prism Software (Prism 4.0).
Adenylate cyclase assays
Adenylate cyclase assays were performed to determine whether
the heterologously expressed eA2A-R functionally coupled to
intracellular heterotrimeric G-proteins. Briefly, eA2A-HEK cells
were plated in six-well plates at a density of 7.5 · 104 cells/well
and cultured for 24 h. The cells were then washed three times in
serum-free media, and preloaded with [3H]-adenine at a final
concentration of 1.2 lCi/mL for 5 h. Cells were then washed three
times in serum-free media followed by the addition of 10 lL of
individual A2A agonists (CGS21680, ATL303, NECA, and
2-CADO) in MEM containing 50 lM of the phosphodiesterase
inhibitor rolipram (Sigma) and incubated for 40 min at 37 �C, and
5% CO2. The cells were then lysed, and the supernatant
transferred to 1.5 mL microcentrifuge tubes and centrifuged at
10 000 g for 15 min at 4 �C. The resulting supernatant was
washed over Dowex (50W-X4 Resin; Bio-Rad, Hercules, CA, USA)
followed by alumina columns, and [3H]cAMP was eluted from the
alumina columns by the addition of 0.1 M imidazole buffer and
counts determined. Data were analyzed utilizing nonlinear
regression analysis with Graphpad Prism Software (Prism 4.0).
Equine adenosine A2A receptor 245
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd
Equilibrium competition binding
To determine the pharmacological signature of the heterolo-
gously expressed e-A2A-Rs, equilibrium competition experiments
were performed. Briefly, 100 lg of e-A2A/HEK cell membranes
were incubated with 0.25 nM [3H]ZM241385 and increasing
concentrations of either A2A agonists or antagonists. Agonists
utilized in these experiments were ATL303, NECA, 2-CADO,
CGS21680, and CV-1808, and the antagonists utilized were
caffeine, 8-phenyltheophylline, p-sulfophenyltheophylline and
unlabeled ZM241385. Data were analyzed utilizing nonlinear
regression analysis with Graphpad Prism Software (Prism 4.0).
Reporter gene and electrophoretic mobility shift assays
To determine the effect of receptor activation on downstream
signal transduction, NF-jB reporter gene assays were performed.
The eA2A-F receptor clone was seeded in a 96-well microtiter
plate at a density of 2.5 · 104 cells per well at 37 �C, 5% CO2,
and 95% relative humidity until approximately 75–80% conflu-
ence was achieved. The cells were then transiently transfected
with 50 ng/well of the Endothelial Leukocyte Adhesion Molecule
(ELAM) NF-jB-dependent firefly luciferase reporter construct (a
generous gift from Dr Douglas Golenbock; University of Massa-
chusetts Medical School, Worcester, MA, USA), and 5 ng/well of
the synthetic Renilla luciferase reporter plasmid (Promega,
Madison, WI, USA) with the Polyfect transfection reagent
(Qiagen). Initially, 100 ng total DNA (50 ng ELAM, 5 ng Renilla,
45 ng empty vector [pcDNA]) was added to individual tubes,
followed by the addition of serum-free media to a final volume of
20 lL DNA/well. The transfection reagent Polyfect was then
added (3 lg/well), and incubated at room temperature for
10 min, after which MEM + 10% FCS was added to a final
volume of 100 lL/well. After the DNA-Polyfect incubation
period, media were removed and replaced with 100 lL complete
media containing 10% FCS, 1% penicillin–streptomcyin, fol-
lowed by the addition of 100 lL/well of the ELAM/Renilla/
pcDNA-Polyfect reaction mixture. The transfection reaction was
allowed to proceed for 6 h, after which the cells were washed in
serum-free media. The media was then replaced with MEM
containing 10% FCS, 1% penicillin–streptomycin, and 0.25 mg/
mL G418. The cells were allowed to recover for 48 h and then
stimulated with human recombinant TNF-a at a final concen-
tration of 10 ng/mL for 4 h in the presence of the selective A2A
agonist CGS21680 at a concentration range from 10 nM to
100 lM. Following the stimulation reaction, cells were lysed
with 50 lL passive lysis buffer and assayed for firefly and Renilla
luciferase activities, respectively using the Dual-Luciferase
Reporter Assay kit (Promega). Luciferase activity was normalized
relative to the Renilla luciferase activity to control for differences
in transfection efficiencies.
To determine the effect of eA2A-F receptor activation on NF-jB
nuclear translocation, electrophoretic mobility shift assays were
performed. Briefly, cells were plated in 15 mm culture plates
(Becton-Dickinson) and allowed to reach 75–80% confluency.
The cells were then stimulated as in the reporter gene assays for
50 min. Growth media utilized were either MEM + 10% FCS or
MEM + 10% FCS including 50 lM of the phosphodiesterase
inhibitor rolipram. Following stimulation, cells were trypsinized,
washed 2x in ice cold PBS, and the nuclear extracts obtained by
incubation in a hypotonic HEPES buffer (10 mM HEPES, 10 mM
KCl, 0.1 mM EDTA, 0.1 mM EGTA, 1 mM DTT, and 0.5 mM
PMSF, pH 7.9), followed by the addition of 10% NP-40. Cells
were then centrifuged, supernatants discarded, and the nuclear
pellet suspended in a hypertonic HEPES buffer (20 mM HEPES,
400 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM DTT, 0.5 mM
PMSF, pH 7.9), extracted at 4 �C for 30 min with mixing,
followed by centrifugation at 1.4 · 104 g, 4 �C for 5 min.
Equivalent amounts of nuclear extract were then incubated
with a [c32P] end-labeled NF-jB oligonucleotide for 20 min at
room temperature. A one-tenth volume of 10x gel loading buffer
consisting of 250 mM Tris/HCl, pH 7, 0.2% bromophenol blue,
and 40% glycerol was then added to each reaction tube, and the
protein/oligonucleotide complexes were resolved on a 4%
nondenaturing polyacrylamide gel. After protein migration, the
gel was dried and opposed to a phosphoimager for at least 24 h;
the resulting bands were then analyzed utilizing a 170–7856
Molecular Imager FX Pro (Bio-Rad).
RESULTS
Equine A2A-R cloning and sequencing
The cloning and sequencing of the eA2A-R cDNA revealed a full-
length transcript complete with start and stop codons, 5¢ and 3¢untranslated regions (UTR), as well as a poly-adenylation signal
in the 3¢ UTR. ClustalW alignment of the eA2A-R cDNA indicated
this receptor cDNA had a high degree of sequence similarity with
that of other mammalian A2A receptor transcripts. The position
of the start codon was conserved in the sequences for the equine
and human A2A-R cDNAs, while the position of the translation
start sequence for the mouse, rat, and guinea pig was 10 bp
downstream from that of the equine receptor. The termination
signal was conserved for the equine, human, and guinea pig,
while that of the rat and mouse were located 27 bp downstream.
Furthermore, BLAST homology screening of the deduced amino
acid sequence of the eA2A-R with that of other mammalian A2A-
R proteins revealed a similar degree of homology (Fig. 1;
Table 1).
Evaluation of eA2A receptor expression
Initially, eA2A-R/HEK clones were screened for membrane
expression utilizing radioligand binding assays to document
displaceable binding of 1 nM [3H]ZM241385 in the presence of
10 lM 2-CADO. Ten individual G418-resistant plaque-isolated
clones were screened and compared with membranes from rat
striatum which served as a positive control. From these 10
G418-resistant clones, four were identified that expressed the
eA2A-R at levels greater than untransfected HEK293 cells as
indicated by the specific binding of [3H]ZM241385 (Fig. 2a).
246 C. I. Brandon et al.
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd
Based on these levels of receptor expression, four clones (A2A-D,
A2A-E, A2A-F, and A2A-G) were selected from the original pool of
10 to utilize in further characterization experiments (Fig. 2b).
Equilibrium saturation isotherms
To further characterize the four selected eA2A-HEK clones,
equilibrium saturation isotherm experiments were performed.
Fig. 1. ClustalW protein alignments of the equine adenosine A2A receptor with that of other mammalian species.
Table 1. Comparison of the equine adenosine A2A receptor homology
with that of other mammalian species
A2A protein sequence % Overall homology GenBank accesssion no.
Human 90.0 NM00675
Guinea pig 81.8 D63674
Mouse 77.9 NM009630
Rat 77.4 NM053294
Fig. 2. (a) One point-binding assay of eA2A-R transfected HEK293 cells to screen for receptor membrane expression. Total binding was defined with
1.0 nM [3H]ZM241385, and nonspecific binding with [3H]ZM241385 in the presence of 10 lM 2-chloroadenosine (arrows denote those clones selected
for further characterization). (b) Replot of the data in histogram 2A showing specific membrane receptor densities of the four selected clones; data are
expressed in pmol/mg protein bound. Membranes from rat striatum (Str., in both panels) served as the positive control.
Equine adenosine A2A receptor 247
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd
Results from these studies revealed that [3H]ZM241385 bound
saturably and with high affinity to membranes derived from the
stably transfected HEK cells (Fig. 3a). All four clones had
dissociation constants that were within the published range of
0.79–0.92 nM for rat striatal membranes (Alexander & Millns,
2001). Isotherm analysis of nontransfected HEK cells revealed no
displaceable binding, indicating that binding occurring in the
four selected clones was due to the presence of the eA2A-R.
Additionally, a Scatchard replot of these data resulted in a linear
transformation indicating specific binding to a homogeneous
population of binding sites (Fig. 3b). Based on a similar affinity
for [3H]ZM241385 between clone eA2A-F and that of rat
striatum (0.76 nM vs. 0.74 nM, respectively) coupled with that
clone’s high level of receptor expression (1.575 pmol/mg vs.
0.7620 pmol/mg), the eA2A-F clone was utilized in all subse-
quent characterization experiments (Fig. 3b).
Equilibrium competition binding
To determine the rank order of agonist potency of clone eA2A-F,
equilibrium competition binding assays were performed. The
specific A2A agonists used in these experiments were ATL303,
NECA, CV-1808, 2-CADO, and CGS21680. Nonlinear regression
analysis of these data revealed that ATL303 and CV-1808
displacement data were better fit (P < 0.05) with a two-site
competition model. The remaining three compounds (2-CADO,
CGS21680, and NECA) were adequately fit by a one-site
competition model (Fig. 4). The rank order of high-affinity binding
of these agonists at the equine A2A receptor was ATL303 > CV-
1808 > NECA > 2-CADO > CGS21680 (Table 2).
To determine the rank order of potency for antagonists,
competition experiments were performed using the antagonists
caffeine, 8-phenyltheophylline, p-sulfophenyltheophylline, and
unlabeled ZM241385 in competition with [3H]ZM241385.
Additionally, the selective A3 antagonist MRS1220 was inclu-
ded to document further the pharmacologic profile of the
equine A2A receptor. The deduced rank order of antagonist
potency was ZM241385 > 8-phenyltheophylline > p-sulfo-
phenyltheophylline > caffeine (Fig. 5); the selective A3 antag-
onist MRS1220 did not inhibit the binding of [3H]ZM241385.
To demonstrate that the expressed equine A2A receptor was
sensitive to GTP, competition binding experiments were per-
formed in the presence and absence of the nonhydrolysable GTP
analog GTPcS. In these experiments, the ATL303 compound was
used inasmuch as it was the most potent agonist and distin-
guished two eA2A-R agonist affinity states. When 100 lM of
GTPcS was added, the high affinity state of the receptor was
eliminated, producing a rightward shift in the displacement
curve characterized by one low-affinity component (Fig. 6).
Adenylate cyclase assays
To document the functionality of the heterologously expressed
equine A2A receptors, adenylate cyclase assays were performed.
Fig. 3. (a) Equilibrium saturation isotherm analysis of the four selected equine A2A receptors stably transfected in HEK293 cells. To determine the
antagonist affinity (KD) and receptor densities (Bmax), 100 lg of eA2A-R membrane fractions were incubated with an increasing concentration of
[3H]ZM241385 (0.0625–2.0 nM), and 10 lM 2-CADO to define total and nonspecific binding, respectively. Rat striatum and nontransfected HEK293
cells served as positive and negative controls, respectively. (b) Replot of clone eA2A-F when compared with rat striatum and nontransfected HEK cells;
inset shows a scatchard replot of the data indicating homogeneity of binding.
Fig. 4. Equilibrium competition binding experiments to determine the
rank order of agonist potency of selective A2A receptor agonists. One
hundred micrograms of eA2A-R/HEK cell membranes were incubated
with a fixed concentration of [3H]ZM241385 (0.25 nM) and increasing
concentration of agonist (10 pM–10 lM). Results are normalized data
from four replicate experiments revealing an agonist rank order of
potency of ATL303 > CV-1808 > NECA > 2-CADO > CGS21680.
248 C. I. Brandon et al.
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd
The selected A2A agonists used in these experiments were
ATL303, NECA, CGS21680, and 2-CADO at concentrations
ranging from 1 nM to 300 lM. The rank order of potency of these
agonists for production of [3H]cyclic AMP accumulation was
ATL303 > CGS21680 > NECA > 2-CADO (Fig. 7).
Adenylate cyclase assays were then performed in the presence
of the A2A antagonist ZM241385 (1 lM) to determine whether
the A2A agonist stimulation of adenylate cyclase was due to
eA2A-R activation. In these experiments, CGS21680 was incu-
bated with the HEK293 cells expressing the eA2A-R in the
presence or absence of 1 lM ZM241385. Addition of ZM241385
resulted in a rightward shift in the concentration–response curve
by approximately one order of magnitude (Fig. 8).
Reporter gene and electrophoretic mobility shift assays
In these experiments, eA2A-R activation by CGS21680 resulted
in a concentration-dependent inhibition of TNF-a-stimulated
NF-jB activity, with maximal inhibition (74%) occurring at
100 lM CGS21680. A return to baseline (e.g. cells stimulated
with 10 ng/mL TNF-a alone) was observed in cells co-
incubated with 10 nM CGS21680 and TNF-a. Furthermore,
the inhibitory effect of CGS21680 (1 lM) was largely reversed
with the addition of 10 lM of the A2A selective antagonist
ZM241385. Inhibition of the effects of TNF-a by CGS21680
was apparently mediated via cyclic AMP as this inhibitory
effect was enhanced by the addition of the phosphodiesterase
inhibitor rolipram. Incubation of the cells with 10 lM forskolin,
a direct activator of adenylate cyclase, in the presence of TNF-a
Table 2. EC50 and fractional occupancy values for the five A2A receptor agonists used in the equilibrium competition binding experiments. ATL303 and
CV-1808 both fit a two-site binding model whereby there were two affinity sites identified; fractional occupancy values are the percentage binding at the
high affinity state. NECA, 2-CADO, and CGS21680 all fit a one-site binding model
ATL303 (nM) CV-1808 (nM) NECA (nM) 2-CADO (nM) CGS21680 (nM)
EC50-1 0.833 (0.15–4.47) 53.8 (39.8–72.7) 74.79 (63.6–87.9) 208.06 (147.2–294.0) 243.62 (167.7–354.0)
EC50-2 18.1 (10.7–30.6) 110.48 (76.5–159.5) – – –
Fraction RH 0.28 (0.05–0.50) 0.17 (0.11–0.23) 1.0 1.0 1.0
95% confidence intervals are denoted in parentheses.
ZM241385(nM)
Phenyl Theo. (nM)
p-sulfophenyltheophylline(nM)
Caffeine(nM)
EC50 0.22 1770 19200 3541095% CI 0.09-0.54 1045-2986 10410-35412 12989-96538
Fig. 5. Equilibrium competition binding
experiments to determine the rank order of
antagonist potency at the equine A2A recep-
tor. One hundred micrograms of eA2A-R/HEK
cell membranes were incubated with a fixed
concentration of [3H]ZM241385 (0.25 nM)
and increasing concentration of antagonist
(1 nM–1 mM). 95% confidence intervals are
denoted in parentheses. Results indicate a
rank order of antagonist potency of
ZM241385 > phenyltheophylline > p-sulfo-
phenyltheophylline > caffeine > MRS1220.
ATL303 (nM) ATL303+GTPγγS (nM)
EC50-15.67 -
EC50-2 164.44 45.08Fraction RH 0.27 0
Fig. 6. GTP shift of agonist competition curves for antagonist radioligand
binding. The plot illustrates specific binding from a representative
competition experiment of the agonist ATL303 with the antagonist
radioligand [3H]ZM241385 in the presence and absence of 100 lM
GTPcs.
Equine adenosine A2A receptor 249
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd
also resulted in almost complete inhibition of NF-jB activity
(Fig. 9).
Data from the electrophoretic mobility shift assays indicated
that the inhibitory effect of CGS21680 on TNF-a-stimulated
NF-jB activity was not due to an inhibition of nuclear
translocation as NF-jB subunits were identified in nuclear
extracts from TNF-a-stimulated cells at all concentrations of
CGS21680 (Fig. 10; Table 3). Furthermore, to show that the
bands visualized in the electrophoretic mobility shift assays in
the TNF-a-treated cells were activated NF-jB subunits, nuclear
extracts were incubated with a polyclonal antibody to the p65
subunit of NF-jB. Addition of this antibody shifted the band to a
higher molecular weight, providing evidence that the TNF-a-
activated complex consisted of the p65 (RelA) subunit. Addi-
tionally, the elimination of the band by incubating nuclear
extracts with excess cold oligonucleotide lends further support
for the presence of NF-jB subunits in the nuclear extracts of
TNF-a-stimulated cells.
DISCUSSION
Tumor necrosis factor-a plays an important role in the response
of mammals to proinflammatory substances such as the
lipopolysaccharide (LPS) component of the outer cell wall of
gram-negative bacteria. Interactions among LPS and its recep-
tors on the surface of mononuclear phagocytes result in
activation of the NF-jB pathway leading to the transcription of
a host of inflammatory genes, including TNF-a. The TNF-aprotein is then secreted to the extracellular space where it can
bind to TNF-a receptors on target cells, and in turn cause
activation of the NF-jB pathway. In this situation, TNF-a
CGS21680(nM)
CGS21680+ZM241385 (nM)
EC50 674.83 581295%CI
355.11-1282 4351-7764
Fig. 8. Adenylate cyclase assays: effect of A2A-R antagonism on
adenylate cyclase activity. Equine A2A/HEK cells were pre-loaded with
[3H]adenine (1.2 lCi/mL) for 5 h, followed by incubation with
CGS21680 (10 nM–300 lM) in the presence or absence of 1 lM
ZM241385. Cells were then lysed and the resulting supernatant filtered
over Dowex followed by alumina columns. Table inset depicts respective
EC50 values and 95% confidence intervals (nM) for CGS21680 in the
presence and absence of the A2A-selective antagonist ZM241385.
Fig. 9. NF-jB reporter gene assays: equine A2A receptor-expressing
HEK293 cells were transiently transfected with the ELAM and Renilla
luciferase reporter plasmids after which they were treated with the
indicated concentrations of CGS21680 and stimulated with TNF-a(10 ng/mL) for 4 h either in complete media (CM), or complete media in
the presence of the phosphodiesterase rolipram (CM + RO). Following
stimulation, the cells were lysed and the resulting supernatant assayed
for TNF-a-stimulated NF-jB-dependent luciferase activity. TNF, tumor
necrosis factor; DMSO, dimethyl sulfoxide; RO, rolipram; Forsk., forskolin;
CGS, CGS21680; ZM, ZM241385.
ATL303(nM)
CGS21680(nM)
NECA(nM)
2-CADO(nM)
EC50 0.07 10.77 12.72 53.4195%CI
(0.006-0.076) 6.07-19.12 6.64-24.37 34.46-82.80
Fig. 7. Adenylate cyclase assays: equine A2A-R/HEK cells were pre-
loaded with [3H]adenine (1.2 lCi/mL) for 5 h, followed by incubation
with the selected A2A agonist (1.0 nM–100 lM) in the presence of
phosphodiesterase inhibitors. Cells were then lysed and the resulting
supernatant filtered over Dowex followed by alumina columns. The
[3H]cAMP was then eluted into scintillation vials by the addition of 0.1 M
imidazole buffer. Data are normalized from three replicate experiments.
Table inset denotes respective EC50 values and 95% confidence intervals
for agonists utilized in these experiments.
250 C. I. Brandon et al.
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd
expression becomes dysregulated and can result in dire conse-
quences to the host, including septic shock and death. Thus,
agents capable of inhibiting the response to TNF-a may have
enormous therapeutic potential for the treatment of inflamma-
tory disease.
The results obtained in this study indicate that activation of
the eA2A-R with the A2A-selective agonist CGS21680 reduces
TNF-a-induced NF-jB-driven reporter gene expression in a
concentration-dependent manner without altering nuclear
translocation of NF-jB (Figs 9 & 10; Table 3). Specifically, we
observed a 74% inhibition of NF-jB activity at the highest
concentration of CGS21680 (100 lM) (Fig. 9), and this effect
was potentiated by the addition of the phosphodiesterase
inhibitor rolipram. These findings are consistent with a cyclic
AMP-mediated inhibitory effect on NF-jB activation. The strong
inhibition of NF-jB activity by forskolin alone also supports a
role for cyclic AMP. Increased concentrations of cyclic AMP have
been reported to inhibit NF-jB-mediated transcription of TNF-ain human monocytic and endothelial cells (Ollivier et al., 1996).
In this earlier report, forskolin and dibutyryl cyclic AMP
inhibited LPS-stimulated TNF-a gene expression and NF-jB
activity without altering nuclear translocation of NF-jB het-
erodimers. We demonstrate herein an eA2A-R-mediated concen-
tration-dependent inhibition of TNF-a activation of NF-jB
reporter gene activity, that was potentiated by inhibition of
phosphodiesterase and occurred in the absence of changes in NF-
jB nuclear translocation. These results strongly suggest that the
inhibition of TNF-a-stimulated NF-jB activity via eA2A-R
activation is a cyclic AMP-mediated effect. This hypothesis is
further supported by the results of a recent report summarizing
the effects of A2A-R activation in equine articular chondrocytes
(Tesch et al., 2002). In that study adenosine, the A2A-selective
agonist N(6)-[(dimethoxyphenyl)-ethyl]adenosine (DMPA), and
forskolin alone, significantly increased intracellular concentra-
tions of cyclic AMP, and suppressed LPS-stimulated nitric oxide
production by articular chondrocytes. Moreover, similar to our
results, inhibition of phosphodiesterase potentiated actions of
DMPA and forskolin.
Using the electrophoretic mobility shift assays, we identified
NF-jB subunits in nuclear extracts of TNF-a-stimulated cells in
both the presence and absence of an A2A receptor agonist
(Fig. 10; Table 3). Additionally, supershift assays using a p65
polyclonal antibody (Santa Cruz Biotech, Inc., Santa Cruz, CA,
USA) clearly indicated that the DNA binding complex induced by
TNF-a contained the active p65 NF-jB subunit. Thus, the
inhibitory effect on NF-jB is occurring through a route other
than that of nuclear translocation; this effect has been reported
previously for forskolin-mediated modulation of LPS-stimulated
NF-jB activity (Majumdar & Aggarwal, 2003). Additional work
will be required to determine the precise mechanism by which
eA2A-R activation inhibits NF-jB transcriptional activity. How-
ever, the ability of A2A-R agonists to inhibit both the early
induction of NF-jB activity by LPS as reported by Ollivier et al.
(1996), as well as later induction of NF-jB activity by TNF-a that
we report here, would seem to imply great potential in the
treatment of endotoxemia.
Additionally, the results obtained from the reporter gene
assays performed in the present study suggest possible species
and/or cell-type-dependent activation of the NF-jB pathway.
Fig. 10. Electrophoretic mobility shift assays (EMSA) of TNF-a-stimula-
ted eA2A-HEK cells. Cells were treated with four concentrations of
CGS21680 (10 lM–0.1 lM) in the presence or absence of the phospho-
diesterase inhibitor rolipram (RO), then stimulated with TNF-a (10 ng/
mL) for 50 min, and nuclear extracts collected to analyze for NF-jB
translocation. Lane assignments are: 1, media control; 2, TNF-a control;
3, CGS21680 control; 4, RO control; 5, TNF-a + 10 lM CGS21680; 6,
TNF-a + 10 lM CGS21680 + RO; 7, cold oligo; 8, anti-p65 supershift.
Table 3. Densitometry analysis of electrophoretic mobility shift assays
(EMSA) data. Band densities were measured and converted to percentage
NF-jB translocation
Lane no. Cell treatment Scan analysis % Translocation
1 Media control 171 (0) 0
2 2 ng/mL TNF 2153 (1982) 100
3 10 lM CGS21680 in
0.5% DMSO
152 ()19) 0
4 50 lM Rolipram 193 (22) 1
5 TNF + 10 lM
CGS21680
1954 (1783) 99
6 TNF + 10 lM
CGS21680 + RO
2243 (2072) 105
Numbers in parentheses are relative optical densities with background
subtracted.
Equine adenosine A2A receptor 251
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd
For example, the results of a recent study using LPS-stimulated
RAW 246.7 cells reported that adenosine regulates macroph-
age function independently of NF-jB activity (Nemeth et al.,
2003). In that study, neither adenosine nor adenosine receptor
agonists inhibited NF-jB binding in mobility shift assays;
furthermore, adenosine treatment of LPS-stimulated cells failed
to inhibit NF-jB-specific activity as assessed by reporter gene
assays. A possible explanation for the discrepancy in NF-jB
activity between that study and the results reported herein may
be that RAW 246.7 cells express A2B receptors but not the A2A
receptor subtype. The fact that A2B receptors are relatively low
affinity receptors when compared with A2A may account for
the lack of an inhibitory effect on TNF-a-stimulated NF-jB
activity. In contrast, the inhibitory effect of adenosine analogs
in our eA2A heterologous expression system was marked. In
agreement with our findings are the results of a study by
Bshesh et al. (2002) in which CGS21680 inhibited NF-jB
activity in LPS-stimulated human monocytic leukemia cells
(THP-1). The results of that study also indicated that inhibition
of NF-jB activity occurred in the absence of an alteration in
nuclear translocation of NF-jB. These authors concluded that
adenosine receptor activation resulted in activation of PKA,
phosphorylation of CREB, and a reduction in TNF-a production,
most likely via inhibition of NF-jB-dependent transcription of
the TNF-a gene. Additionally, the results of a recent study
performed in human myeloid KBM-5 cells indicate that
adenosine suppresses TNF-a-stimulated NF-jB-driven reporter
gene expression (Majumdar & Aggarwal, 2003). To define
which adenosine receptor subtypes were mediating these
effects, these authors incubated KBM-5 cells with adenosine
in the presence of either the A1-selective (DPCPX) or
A2-selective (DMPX) antagonists. Results from these experi-
ments revealed that the A2-selective antagonist DMPX reversed
the adenosine-mediated inhibition of TNF-a-stimulated NF-jB
activity in a concentration-dependent manner, while the A1
antagonist had no effect. Thus, the inhibitory effect on NF-jB
activity was mediated through the A2-receptor subtype. In
contrast to our results, adenosine treatment of TNF-a-stimula-
ted KBM-5 cells significantly reduced NF-jB nuclear binding as
evidenced by mobility shift assays. This may imply that the
cellular context is the primary determinant for the mechanism
of adenosine receptor-mediated regulation of TNF-a production.
It has been suggested by Majumdar and Aggarwal (2003) that
the effects of adenosine are selective as it had no effect on
NF-jB activity induced by other inflammatory agents (phorbol-
12-myristate-13-acetate [PMA], LPS, H2O2, and ceramide), all of
which activate the NF-jB pathway. This finding suggests that
the mechanism by which these agents activate NF-jB differs
from the pathway used by TNF-a. Taken together, the contra-
dictory findings regarding adenosine regulation of the NF-jB
pathway suggest that this inhibitory signal transduction path-
way may be species and/or cell-type specific, as well as
dependent on the subtype of adenosine receptor involved. By
using cells heterologously expressing equine A2A-R, we demon-
strated that adenosine A2A receptor-specific agonists modulate
TNF-a-induced activation of NF-jB.
The results of equilibrium saturation isotherm experiments
revealed that [3H]ZM241385 bound saturably and with high
affinity to the eA2A-F clone. The affinity of clone eA2A-F for
[3H]ZM241385 (0.74 nM) was comparable with that for rat
striatum (0.84 nM) (Alexander & Millns, 2001). Similarly, the
affinity of [125I]ZM241385 for bovine striatal A2A-R is 1.4 nM,
and 1.6 nM for canine A2A-R expressed in CHO cells (Palmer
et al., 1995). In a more recent study, [3H]ZM241385 had a
binding affinity of 1.2 nM in the equine striatum (Chou &
Vickroy, 2003). Thus, the A2A-selective antagonist
[3H]ZM241385 binds to the equine A2A receptor with an affinity
comparable with that of other mammalian species.
Equilibrium competition binding assays revealed an agonist
rank order of potency to be ATL303 > CV-1808 > NECA > 2-
CADO > CGS21680 (Fig. 4, Table 2), and the antagonist rank
order as ZM241385 > 8-phenyltheophylline > p-sulfophenyl-
theophylline > caffeine (Fig. 5). Results from adenylate cyclase
assays revealed a rank order of agonist potency to be
ATL303 > CGS21680 > NECA > 2-CADO (Fig. 7). Overall, the
pharmacological characteristics of the equine A2A receptor are
similar to those of other species, with the exception of a
somewhat higher affinity for CV-1808. The pharmacological
signature of the eA2A-R is most similar to the human A2A-R
(Table 4), which is to be expected inasmuch as the eA2A-R
shares 90% amino acid sequence homology with the human
A2A-R (Fig. 1; Tables 1 & 4).
In summary, the cloned equine adenosine A2A receptor stably
expressed in HEK293 cells provides a unique heterologous
expression system to characterize the receptor. In these
experiments we have demonstrated that: (i) the eA2A-R is
expressed in HEK293 cell membranes and binds the selective
A2A antagonist [3H]ZM241385 with both high affinity and
Table 4. Equilibrium competition binding
rank order of potency of the eA2A-R when
compared with that of the rat and human
A2A-R, respectively; the expression cell utilized
is noted in parenthesis
eA2A-R (HEK293) (nM) rA2A-R (PC12) (nM) hA2A-R (CHO) (nM)
ATL303 0.83 (0.15–4.47) NA NA
CV-1808 53.8 (39.78–72.66) 949 (589–1,530) 76 (62–93)
NECA 74.8 (63.62–87.92) 160 (110–234) 66 (40–110)
2-CADO 208.1 (147.24–294.00) 879 (722–1,070) 164 (92–293)
CGS21680 243.6 (167.66–353.99) 298 (216–412) 221 (156–311)
Rank order values for the rat and human A2A-R are taken from Kull et al. (1999). 95% confidence
intervals for each mean value are denoted in parentheses.
252 C. I. Brandon et al.
� 2006 The Authors. Journal compilation � 2006 Blackwell Publishing Ltd
selectivity, as is characteristic of other mammalian A2A
receptors; (ii) equilibrium competition binding and cyclase data
demonstrate a rank order of potency that is similar to that of
other mammalian adenosine A2A receptors; (iii) that the
expressed receptor is functional as indicated by the activation
of adenylate cyclase; and (iv) adenosine receptor activation
results in a concentration-dependent inhibition of TNF-a-stimu-
lated NF-jB activity. These results are encouraging for the
further characterization of the equine A2A receptor as a
molecular target for the treatment of equine endotoxemia.
ACKNOWLEDGMENTS
The authors gratefully acknowledge Morris Animal Foundation
for providing financial assistance for a portion of this study
(DO2EQ-03). Additionally, we thank the laboratories of Dr Lee H.
Pratt (Department of Plant Science, University of Georgia) for
performing the initial sequencing reactions of the eA2A-R/
pBluescript plasmid.
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