j. vet. pharmacol. therap. cloning and pharmacological ... · pharmacological target for equine...

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:

[email protected]

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


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).



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.



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.



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.



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.



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.



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.



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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