Ethics Statement

All animals used in the study were handled in accordance with the National Institutes of Health Animal care guidelines. The animal research conducted to perform this study was approved by the NIDA IRP Animal Care and Use Committee (under the auspices of protocol 09-BNRB-73) on 12/7/2009.

Animals

Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) weighting between 300–350 g were used in these experiments. Rats were housed 2 per cage and they maintained at a temperature of 22±2°C on a regular 12-h light–dark cycle. Food and water were available ad libitum.

Adenosine A_2AR antagonists

The following A_2AR antagonists were used: 2-(2-Furanyl)-7-[3-(4-methoxyphenyl)propyl]-7H-pyrazolo[4,3-e][1], [2], [4]triazolo[1,5-c]pyrimidin-5-amine (SCH-442416), 2-(2-Furanyl)-7-(2-phenylethyl)-7H-pyrazolo[4,3-e][1], [2], [4]triazolo[1,5-c]pyrimidin-5-amine (SCH-58261), 2-(2-furanyl)-7-[2-[4-[4-(2-methoxyethoxy)phenyl]-1-piperazinyl]ethyl]-7H-pyrazolo[4,3-e][1], [2], [4]-triazolo[1,5-c]pyrimidin-5-amine (SCH-420814), 4-(2-[7-Amino-2-(2-furyl)[1], [2], [4]triazolo[2,3-a][1], [3], [5]triazin-5-ylamino]ethyl)phenol (ZM-241385), (E)-1, 3-diethyl-8-(3,4-dimethoxystyryl)-7-methyl-3,7-dihydro-1H-purine-2,6-dione (KW-6002), (E)-3-(3-hydroxypropyl)-8-[2-(3-methoxyphenyl)vinyl]-7-methyl-1-prop-2-ynyl-3,7-dihydropurine-2,6-dione (MSX-2) and its water-soluble phosphate prodrug (E)-phosphoric acid mono-(3-{8-[2-(3-methoxyphenyl)vinyl]-7-methyl-2,6-dioxo-1-prop-2-ynyl-1,2,6,7-tetrahydropurin-3-yl}propyl) ester disodium salt (MSX-3). MSX-3 is a water-soluble phosphate pro-drug of MSX-2; in vivo MSX-3 is readily converted to the A_2AR antagonist MSX-2 (Sauer et al., 2002). For their systemic administration, the compounds were prepared as follows:

SCH-442416 and SCH 58261 were suspended in a solution of 5% dimethyl- sulfoxide (DMSO) (Sigma-Aldrich, St. Louis,MI), 5% TWEEN80 (Sigma-Aldrich, St. Louis, MI) and 90% ddH₂O; SCH-420814 was suspended in a solution of 20% PEG400, 40% β-cyclodextrin and 40% Lutrol 1% (in ddH₂O); ZM-241385 was suspended in a solution of 15% DMSO, 10% TWEEN80 and 75% ddH₂O; KW-6002 was suspended in a solution of 8% TWEEN80 and 92% ddH₂O; MSX-3 was dissolved in sterile saline (with 3 µl/ml saline of 1 M NaOH solution, final pH 7.4). All drugs but MSX-3 (Sigma-Aldrich, St. Louis, MI) were provided by CHDI Foundation Inc. (Los Angeles, CA, US). SCH-420814 was administered subcutaneously (s.c.) at 1 ml/kg and the other drugs were administered via intraperitonal (i.p.) injection at volume of 2 ml/kg.

Locomotor Activity

Locomotor activity was measured by placing the animals individually in motility soundproof chambers (50×50 centimeters; Med Associates Inc., VT). Locomotion was measured by counting the number of breaks in the infrared beams of the chambers. The animals were placed in individual acrylic chambers at noon on the day of testing. A lamp inside each chamber remained lit during this period. Following 90 min of habituation, the rats were injected i.p. with different doses of each compound or vehicle and locomotor activity was recorded for 90 min after the drug or vehicle administration. All the animals were tested only once. The effect of different doses of the A_2AR antagonists on locomotor activity were analyzed using a one-way analysis of variance (ANOVA), followed by Newman-Keuls' post-hoc test.

Surgical procedures

Rats were anesthetized with 3 ml/kg of Equithesin (4.44 g of chloral hydrate, 0.972 g of Na pentobarbital, 2.124 g of MgSO₄, 44.4 ml of propylene glycol, 12 ml of ethanol and distilled H₂O up to 100 ml of final solution; NIDA Pharmacy, Baltimore, MD) and implanted unilaterally with bipolar stainless steel electrodes, 0.15 mm in diameter (Plastics One, Roanoke, VA), into the orofacial area of the lateral agranular motor cortex (3 mm anterior, 3 and 4 mm lateral, and 4.2 mm below bregma). The electrodes and a head holder (connected to a swivel during stimulation) were fixed on the skull with stainless steel screws and dental acrylic resin. For the experiments with electromyographic (EMG) recording, electrodes were also implanted in mastication muscles (during the same surgical procedure). Two 5 mm-long incisions were made in the skin on the upper and lower jaw areas to expose the masseter and the lateral pterygoid muscles. Two silicon rubber-coated coiled stainless steel recording electrodes (Plastics One, Roanoke, VA) were slipped below the skin from the incision in the skull until the tips showed up from the incisions in the jaw. The bare tips of the electrodes were then held in contact with the masseter and the lateral pterygoid muscles and the skin was closed with surgical staples. The other end of the recording electrodes was encased in a molded plastic pedestal with a round threaded post which was attached to an electrical swivel and then to a differential amplifier (Grass LP511, Grass Instruments, Warwick, RI). The pedestal was secured to the skull with dental cement together with the stimulation electrodes. For the in vivo microdialysis experiments, concentric microdialysis probes with 2-mm long dialysis membranes (Eicom Corp., Tokio, Japan) were implanted respectively into the striatum ipsilateral to the stimulation electrodes (0.0 mm AP, 4.5 ML and 7.0 mm DV).

EMG recording and power correlation analysis

Rats were placed in individual bowl chambers. Both stimulation electrodes and recording electrodes were attached using flexible shielded cabling to a four channel electrical swivel. Stimulation electrodes were connected to two-coupled constant current isolation units (PSIU6X, Grass Instruments West Warwick, RI) driven by an electrical stimulator (Grass S88X; Grass Instruments). The recording electrodes were connected to a differential amplifier (Grass LP511, West Warwick, RI). This configuration allows the rat to move freely while the stimulation and EMG recordings are taking place. After 60 min of habituation, biphasic current pulse trains (pulse of 0.1 ms at 120–200 µA; 100 Hz, 160 ms trains repeating once per 2 seconds) were delivered. The current intensity was adjusted to the threshold level, defined as the minimal level of current intensity allowing at least 95% of the stimulation pulses to elicit a positive EMG response. Positive EMG response was defined as at least 100% increase of the peak to peak amplitude respect to the background tonic EMG activity lasting more than 100 ms or at least 70% increase in the power of the EMG signal respect to the baseline. Positive EMG responses always matched observable small jaw movements. The threshold level was different for each animal but it was very stable and reproducible once established. The threshold level was in the 100 to 150 µA range for most cases and it reached 200 µA in a few (6) animals. Animals that failed to show a positive EMG response with electrical cortical stimulation intensities of 200 µA were discarded from the experimental procedure (less than 10%). Both stimulator monitoring and the amplified and filtered EMG signal (20,000 times gain, bandwidth from 10 to 1,000 Hz with a notch filter set at 60 Hz) were directed to analog-to-digital converter for recording (Lab-Trax-4, World Precision Instruments, Sarasota, FL) and backup (NI 9215, National Instruments, Austin, TX) and digitized at a sampling rate of 10,000 samples/second. Recordings of the digitized data were made using the software Data Trax2 software (World Precision Instruments) and LabVIEW SignalExpress (National Instruments). A power correlation analysis was used to quantify the correlation between the stimulation pulses of current delivered into the orofacial motor cortex (input signal; µA) and the elicited EMG response in the jaw muscles (output signal; µV). Decrease in the power correlation coefficient (PCC) between these two signals is meant to describe a decrease in the efficacy of the transmission in the neural circuit. Off-line, both signals were rectified and the root mean square (RMS) over each period of the stimulation pulses was calculated in the recorded signals using Data Trax2 software. The transformed data (RMS) from the stimulator monitor and the EMG were then exported with a time resolution of 100 samples/second to a spreadsheet file. The stimulation signal values were used as a reference to select data in a time window of 320 ms starting at the beginning of each train of pulses. This time window was chosen to ensure the analysis of any EMG response whose occurrence or length was delayed from the onset of the stimulation trains and to maximize the exclusion from the analysis of spontaneous jaw movements not associated with the stimulation. Pearson's correlation between the RMS values from the stimulation and EMG signals was then calculated for each experimental subject. PCC was calculated using the data recorded 40 min after the administration of the dose of any compound or vehicle. The effects of the different doses of A_2AR antagonists on PCC were analyzed by a one-way ANOVA, followed by Dunnett's post-hoc test.

In vivo microdialysis

The experiments were performed on freely moving rats 24 h after probe implantation. An artificial cerebrospinal solution of (in mM) 144 NaCl, 4.8 KCl, 1.7 CaCl₂, and 1.2 MgCl₂ was pumped through the microdialysis probe at a constant rate of 1 µl/min. After a washout period of 90 min, dialysate samples were collected at 20-min intervals. After 60 min of collecting samples for baseline, the rats were injected either with the A_2AR antagonists KW-6002 or SCH-442416. Both compounds were compared to vehicle controls (5% DMSO, 5% of TWEEN80 and 90% of ddH₂O). After 20 min from drug or vehicle injection, electrical stimulation pulses were applied through the electrodes implanted in the orofacial motor cortex for 20 min (pulse of 0.1 ms at 50–150 µA; 100 Hz, 160 ms trains repeating once × second) and samples were collected for 2 additional hours. Glutamate content was measured by reverse-phase HPLC coupled to a flourimetric detector (Shimadzu Inc., Tokio, Japan) [15]. Glutamate values were transformed as percentage of the mean of the three values before the drug or vehicle injection and transformed values were statistically analyzed. The effect of KW-6002, SCH-442416 and vehicle were analyzed using a one-way ANOVA for repeated measures followed by a Tukey's post-hoc test.

Cell clones

To obtain CHO cells expressing single receptors or co-expressing A_2AR and A₁R or A_2AR and D₂R, the human cDNAs for A₁R or D₂R cloned in pcDNA3.1 vector (containing a geneticin resistance gene) were used. The human A_2AR was cloned into a pcDNA3.1/Hygro vector with a hygromycin resistance gene. For single transfections, CHO cells were transfected with the cDNA corresponding to A_2AR, A₁R or D₂R using lipofectamine (Invitrogen, Carlsbad, USA) method following the instructions of the supplier. 24 h after transfection the selection antibiotic was added at a concentration that was previously determined by a selection antibiotic test. Antibiotic resistant clones were isolated in the presence of the selection antibiotic (1200 µg/ml geneticin or 1000 µg/ml hygromycin). After an appropriate number of days/passes, several stable lines were selected and cultured in the presence of the selection antibiotic (600 µg/ml geneticin or 300 µg/ml hygromycin). To obtain clones co-expressing A_2AR and A₁R or A_2AR and D₂R, CHO cells expressing high affinity A_2AR (obtained as above described) were transfected with the human cDNAs for A₁R or D₂R cloned in pcDNA3.1 vector using lipofectamine. After an appropriate number of days/passes stable lines were selected and cultured in the presence of the selection antibiotic. The receptor(s) expression in the cell clones was first detected by dot-blot of cell lysates using commercial available antibodies and wild-type CHO cells lysates as negative basal staining. Positively moderated stained clones were grown to obtain membranes in which the receptor expression was quantified by radioligand-binding experiments (see Results).

Bioluminescence Resonance Energy Transfer (BRET) assays

The fusion proteins A_2AR-Renilla Luciferase (A_2AR-RLuc), A₁R-Yellow Fluorescence Protein (A₁R-YFP) and D₂R-YFP were prepared and characterized as described elsewhere [16]. The cDNA encoding serotonin 5HT_2B-YFP receptor was kindly provided by Dr. Irma Nardi (University of Pisa, Italy). CHO cells were transiently transfected with the corresponding fusion protein cDNA (see Figure legends) using lipofectamine. Cells were incubated (4 h) with the corresponding cDNA together with lipofectamine and Opti-MEM medium (Invitrogen). After 4 hours, the medium was changed to a fresh complete culture medium. Twenty-four hours after transfection, cells were washed twice in quick succession in HBSS with 10 mM glucose and scraped in 0.5 ml of the same buffer. To control the cell number, sample protein concentration was determined using a Bradford assay kit (Bio-Rad, Munich, Germany) using bovine serum albumin dilutions as standards. To quantify fluorescence proteins, cells (20 µg protein) were distributed in 96-well microplates (black plates with a transparent bottom) and fluorescence was read at 400 nm in a Fluo Star Optima Fluorimeter (BMG Labtechnologies, Offenburg, Germany) equipped with a high-energy xenon flash lamp, using a 10 nm bandwidth excitation filter. Receptor-fluorescence expression was determined as fluorescence of the sample minus the fluorescence of cells expressing protein-Rluc alone. For BRET measurements, the equivalent of 20 µg of cell protein were distributed in 96-well microplates (Corning 3600, white plates; Sigma) and 5 µM coelenterazine H (Molecular Probes, Eugene, OR) was added. After 1 minute of adding coelenterazine H, the readings were collected using a Mithras LB 940, which allows the integration of the signals detected in the 485 nm-short- (440–500 nm) and the 530 nm-long-(510–590 nm) wavelength filters. To quantify receptor-Rluc expression luminescence readings were performed after 10 minutes of adding 5 µM coelenterazine H. The net BRET is defined as [(long-wavelength emission)/(short-wavelength emission)]-Cf where Cf corresponds to [(long-wavelength emission)/(short-wavelength emission)] for the Rluc construct expressed alone in the same experiment.

Radioligand binding experiments

Cells were disrupted with a Polytron homogenizer (PTA 20 TS rotor, setting 3; Kinematica, Basel, Switzerland) for two 5 s-periods in 10 volumes of 50 mM Tris-HCl buffer, pH 7.4 containing a proteinase inhibitor cocktail (Sigma, St. Louis, MO, USA). Cell debris was removed by centrifugation at 1,500 g for 5 min at 4°C and membranes were obtained by centrifugation at 105,000 g (40 min, 4°C). Membranes were resuspended and centrifuged under the same conditions. The pellet was stored at −20°C, washed once more as described above and resuspended in 50 mM Tris-HCl buffer for immediate use. Membrane protein was quantified by the bicinchoninic acid method (Pierce Chemical Co., Rockford, IL, USA) using bovine serum albumin dilutions as standard. For competition experiments, membrane suspensions (0.2 mg of protein/ml) were incubated for 2 h at 25°C in 50 mM Tris-HCl buffer, pH 7.4, containing 10 mM MgCl₂ and 0.2 U/ml of adenosine deaminase (ADA, EC 3.5.4.4; Roche, Basel, Switzerland) with the indicated free concentration of the A₁R, A_2AR, or D₂R antagonist [³H]DPCPX (GE Healthcare, UK), [³H]ZM-241385, or [³H]YM-09151-2, respectively (NEN Perkin Elmer, Wellesley, MA, USA) or the A₁Ragonist [³H](R)-PIA (Moravek Biochemicals Inc., Brea, CA, USA) and increasing concentrations of DPCPX, ZM-241385, YM-09151-2, the A_2AR agonist CGS-21680 or the tested A_2AR antagonist (all provided by CHDI Foundation Inc.). Non-specific binding was determined in the presence of 11 µM of the corresponding non-radiolabelled ligand. Free and membrane-bound ligand were separated by rapid filtration of 500 µl aliquots in a cell harvester (Brandel, Gaithersburg, MD, USA) through Whatman GF/C filters embedded in 0.3% polyethylenimine that were subsequently washed for 5 s with 5 ml of ice-cold 50 mM Tris-HCl buffer. The filters were incubated with 10 ml of Ecoscint H scintillation cocktail (National Diagnostics, Atlanta, GA, USA) overnight at room temperature and radioactivity counts were determined using a Tri-Carb 1600 scintillation counter (PerkinElmer, Boston, MA, USA) with an efficiency of 62% [17]. All displacers were dissolved in DMSO and diluted in the binding medium. The DMSO concentration in the binding incubates was less than 0.5% and, at this concentration, it did not affect agonist or antagonist affinity for their respective receptors.

Binding data analysis

Radioligand competition curves were analyzed by nonlinear regression using the commercial Grafit curve-fitting software (Erithacus Software, Surrey, UK), by fitting the binding data to the mechanistic two-state dimer receptor model [18], [19]. Since there is now abundant evidence for GPCR oligomerization, including A₁R, A_2AR and D₂R [20]–[23] and the minimal functional unit of GPCRs in biological tissues seems to imply dimerization [23], this model considers a homodimer as the minimal structural unit of the receptor. Here, we also consider the possibility of a homodimer as the minimal structural unit of a receptor forming homomers or forming heteromers with another receptor. To calculate the macroscopic equilibrium dissociation constants the following equation for a competition binding experiment deduced previously [19], [24] was considered: (1)

where A represents free radioligand (the adenosine A₁R or A_2AR or dopamine D₂R antagonist [³H]DPCPX, [³H]ZM-241385 or [³H]YM-09151-2, respectively or the A₁R agonist [³H](R)-PIA) concentration, R_T is the total amount of receptor dimers and K_DA1 and K_DA2 are the macroscopic equilibrium dissociation constants describing the binding of the first and the second radioligand molecule (A) to the dimeric receptor; B represents the assayed competing compound concentration, and K_DB1 and K_DB2 are, respectively, the macroscopic equilibrium dissociation constants for the binding of the first ligand molecule (B) to a dimer and for the binding of the second ligand molecule (B) to the semi-occupied dimer; K_DAB is the hybrid equilibrium radioligand/competitor dissociation constant, which is the dissociation constant of B binding to a receptor dimer semi-occupied by A.

When the radioligand A shows non-cooperative behaviour, eq. (1) can be simplified to eq. (2) due to the fact that K_DA2  =  4K_DA1 [19], [25] and, therefore, K_DA1 is enough to characterize the binding of the radioligand A:(2)

Binding to GPCRs quite often displays negative cooperativity. Under these circumstances K_D2/K_D1>4 and then K_D1 and K_D2 represent the “high-affinity” and the “low-affinity” binding sites, respectively. On the other hand, for positive cooperativity, K_D2/K_D1<4 and then K_D2 represents the “high-affinity” and K_D1 represents the “low-affinity”binding sites [25]. The two-state dimer model also introduces a cooperativity index (D_CB). The dimer cooperativity index for the competing ligand B is calculated as [19], [25]:

The way the index is defined is such that its value is “0” for non-cooperative binding, positive values of D_C indicate positive cooperativity, whereas negative values imply negative cooperativity [14], [19].

In experimental conditions when both the radioligand A and the competitor B (i.e., most adenosine A_2A receptor antagonist tested in the present study) show non-cooperativity, it results that K_DA2  =  4K_DA1 and K_DB2  =  4K_DB1, and eq. (1) can be simplified to: (3)

When both the radioligand A and the competitor B (DPCPX, ZM241385, SCH 23390 or YM-09151-2) are the same compound and the binding is non-cooperative, eq. (3) simplifies to:(4)

Goodness of fit was tested according to reduced χ² value given by the nonlinear regression program. The test of significance for two different population variances was based upon the F-distribution (see ref. [25] for details). Using this F test, a probability greater than 95% (p<0.05) was considered the criterion to select a more complex equation to fit binding data over the simplest one. In all cases, a probability of less than 70% (p>0.30) resulted when one equation to fit binding data was not significantly better than the other. Results are given as parameter values ± S.E.M. of three-four independent experiments.

Striatal pre- versus postsynaptic profile of A_2A receptor antagonists

Dose-response experiments with the six A_2AR antagonists indicated that four compounds (SCH-420814, SCH-58261, MSX-3 and ZM-241385) had a similar potency (similar minimal significant effective doses) at inducing locomotor activation (Fig. 1) and at reducing PCC (Fig. 2). The other two compounds had a very different profile: KW-6002 produced a strong locomotor activation already at the dose of 0.3 mg/kg i.p., while it did not reduce PCC at the highest tested dose (10 mg/kg i.p.). On the other hand, SCH-442416 produced a very weak locomotor activation, only significant at doses higher than 3 mg/kg i.p., while it significantly decreased PCC already at the dose of 0.1 mg/kg i.p.

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Figure 1. Locomotor activation in rats induced by A_2AR antagonists.

Data represent means ± S.E.M. of the locomotor activity (distance traveled, in cm, of total accumulated counts) in habituated rats (90 min) during 90 min following the drug administration (n = 6–8 per group). * and **: p<0.05 and p<0.01, respectively in comparison to vehicle-treated animals (0 mg/kg); ANOVA with post-hoc Newman–Keuls' comparisons, p<0.5 and p<0.01, respectively).

https://doi.org/10.1371/journal.pone.0016088.g001

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Figure 2. Blockade by A_2AR antagonists of the motor output induced by cortical electrical stimulation.

Dose-dependent decrease in the Power Correlation Coefficient (PCC) induced by the administration of different A_2AR antagonists. Results represent means ± S.E.M. (n = 5–6 per group). * and **: p<0.05 and p<0.01, respectively in comparison to vehicle-treated animals (0 mg/kg); ANOVA with post-hoc Dunnett' comparisons, p<0.5 and p<0.01, respectively).

https://doi.org/10.1371/journal.pone.0016088.g002

In vivo microdialysis with cortical electrical stimulation was used as an additional in vivo evaluation of the preferential pre- and postsynaptic activity of SCH-442416 and KW-6002, respectively. SCH-442416 significantly counteracted striatal glutamate release induced by cortical stimulation at a dose that strongly reduced PCC but did not induce locomotor activation (1 mg/kg i.p.; Fig. 3). On the other hand, KW-6002 did not modify striatal glutamate release induced by cortical stimulation at a dose that produced a pronounced locomotor activation but did not reduce PCC (1 mg/kg i.p.; Fig. 3).

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Figure 3. Blockade by A_2AR antagonists of striatal glutamate release induced by cortical electrical stimulation.

(a) Representative coronal sections of a rat brain, stained with cresyl violet, showing the tracks left by the bipolar stimulation electrode in the orofacial area of the lateral agranular motor cortex (top) and by the microdialysis probe in the lateral striatum (bottom). (b) Effect of systemic administration of the A_2AR antagonists SCH-442416 and KW-6002 (1 mg/kg, i.p., in both cases) on the increase in glutamate extracellular levels in the lateral striatum induced by cortical electrical stimulation. Results are expressed as means ± S.E.M. of percentage of the average of the three values before the stimulation (n = 5–7 per group). Time ‘0’ represents the values of the samples previous to the stimulation. The arrow indicates the time of systemic administration. The train of vertical lines represents the period of cortical stimulation. *: p<0.05 compared to value of the last sample before the stimulation (repeated-measures ANOVA followed by Tukey's test).

https://doi.org/10.1371/journal.pone.0016088.g003

Development of CHO cell-lines expressing A₁-A_2A or A_2A-D₂ receptor heteromers

Cell clones expressing A_2AR, A₁R-A_2AR heteromers or A_2AR-D₂R heteromers and control clones expressing A₁R or D₂R were generated (see Materials and Methods). First of all, the ability of A_2AR to form heteromers with A₁R or D₂R in CHO cells was demonstrated by BRET experiments in cells transiently co-expressing A_2AR-Rluc and A₁R-YFP or A_2AR-Rluc and D₂R-YFP. A positive BRET signal for energy transfer was obtained (Fig. 4). The BRET signal increased as a hyperbolic function of the concentration of the YFP-fusion construct added reaching an asymptote. As a negative control the BRET pair formed by A_2AR-Rluc and 5-HT_2BR-YFP was used. As shown in Figure 4, the negative control gave a linear non-specific BRET signal. The significant and hyperbolic BRET signal found for these fusion proteins indicates that the intermolecular interaction between A_2AR and A₁R or A_2AR and D₂R in CHO cells is specific.

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Figure 4. Identification of receptor heteromers in CHO cells by BRET saturation curve.

BRET experiments were performed with CHO cells co-expressing A_2AR-RLuc and A₁R-YFP (A) or A_2AR-RLuc and D₂R-YFP (B). Co-transfections were performed with increasing amounts of plasmid–YFP (0.25 to 4 µg cDNA corresponding to A₁R-YFP and 0.5 to 8 µg corresponding to D₂R-YFP) whereas the A_2AR-RLuc construct was maintained constant (0.5 µg cDNA). Both fluorescence and luminiscence of each sample were measured before every experiment to confirm similar donor expressions (about 100,000 luminescent units) while monitoring the increase acceptor expression (10,000–25,000 fluorescent units). As a negative control, linear BRET was obtained in cells expressing equivalent luminescence and fluorescence amounts corresponding to A_2AR-RLuc, (0.5 µg transfected cDNA) and serotonin 5HT_2B-YFP (0.5 to 8 µg transfected cDNA) receptors. The relative amount of acceptor is given as the ratio between the fluorescence of the acceptor minus the fluorescence value of cells expressing the donor alone (YFP) and the luciferase activity of the donor (Rluc). BRET data are expressed as means ± S.D. of 4–6 different experiments grouped as a function of the amount of BRET acceptor.

https://doi.org/10.1371/journal.pone.0016088.g004

A_2AR-D₂R and A₁R-A_2AR heteromerization in stably transfected CHO cells was shown by ligand binding experiments. This is an indirect approach for the identification of a receptor heteromer in native tissues or cells [13]. In the A_2AR-D₂R heteromer, an allosteric interaction between both receptors in the heteromer has been described, in which the dopamine D₂R agonist affinity decreases in the presence of an A_2AR agonist [14]. In CHO cells stably expressing A_2AR and D₂R, the affinity of the D₂R for dopamine was determined by competition experiments of the D₂R antagonist [³H]YM-09151-2 versus dopamine in the presence (Fig. 5a) or in the absence (Fig. 5b) of the A_2AR agonist CGS-21680 (200 nM). By fitting data obtained in the absence of CGS-21680 to eq. 3 (Methods; considering K_DA1 = 2.9 nM see below) the calculated K_DB1 was 9±2 µM. In the presence of CGS-21680, 5 µM of dopamine was unable to decrease the radioligand bound and more than 50% of radioligand bound was found in the presence of 100 µM of dopamine (Fig. 5b). A K_DB1 >30 µM was estimated and it was shown that CGS-21680 induced a decrease in the dopamine affinity for D₂R. An allosteric interaction in the A₁R-A_2AR heteromer has also been described, in which the A₁R agonist affinity decreases in the presence of an A_2AR agonist [11]. As shown in Figure 6a, the displacement of the A₁R agonist [³H]R-PIA by CGS21680 was significantly (p<0.001) better fitted by a biphasic than by a monophasic curve. At low CGS-21680 concentrations, when it binds preferentially to A_2AR (at concentrations of CGS-21680 <500 nM, the direct binding of CGS-21680 to A₁R is <1%, according to the calculated affinity of A₁R for CGS-21680), CGS-21680 decreased the binding of [³H]R-PIA to the A₁R with an IC₅₀ value of 386±35 nM (n = 3). At high CGS-21680 concentrations (>10 µM), the [³H]R-PIA binding displacement reflects the binding of CGS-21680 directly to the A₁R and the competition between CGS-21680 and R-PIA for the binding to the A₁R. In fact, in the control clone expressing only A₁R, the displacement by CGS-21680 of [³H]R-PIA only occurred at CGS-21680 concentrations higher than 10 µM (Fig. 6b).

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Figure 5. Allosteric interaction between A_2AR and D₂R in A_2AR-D₂R CHO cells.

Competition experiments were performed in membrane preparations from CHO cells expressing A_2AR and D₂R with 0.5 nM [³H]YM-09151-2 and increasing concentrations of dopamine (from 0.1 nM to 30 µM) in the absence (a) or in the presence (b) of 200 nM CGS-21680 as indicated in Methods. Data represent means ± S.E.M. of a representative experiment performed with triplicates.

https://doi.org/10.1371/journal.pone.0016088.g005

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Figure 6. Allosteric interaction between A₁R and A_2AR in A₁R-A_2AR CHO cells.

Competition experiments were performed in membrane preparations from CHO cells expressing A₁R or A₁R and A_2AR with 12 nM [³H]R-PIA versus increasing concentrations of the A_2AR agonist CGS-21680 as indicated in Methods. Data represent means ± S.E.M. of a representative experiment performed with triplicates.

https://doi.org/10.1371/journal.pone.0016088.g006

A pharmacological characterization of selected cell clones was performed with competition experiments of radio-labeled antagonists of A₁, A_2A and D₂ receptors versus selective agonists or antagonists. In all cases, the competition curves of the A_2AR antagonist [³H]ZM-241385 (2 nM) versus ZM-241385 (0.1 nM to 11 µM), the D₂R antagonist [³H]YM-09151-2 (0.2 nM) versus YM-09151-2 (0.01 nM to 11 µM) or the A₁R antagonist [³H]DPCPX (2 nM) versus DPCPX (0.1 nM to 11 µM), were monophasic, indicating the absence of cooperativity (see Materials and Methods). By fitting the binding data to eq. 4 (Materials and Methods), the K_D (K_D1) values obtained for the antagonists ZM-241385 or YM-09151-2 were 8±3 nM and 2.9±0.3 nM, respectively, for the chosen A_2AR-D₂R clone, the K_D values obtained for the A₁R and A_2AR antagonists were 8±2 nM (DPCPX) and 1.8±0.4 nM (ZM-241385), respectively, for the chosen A₁R-A_2AR cell clone and the K_D value obtained for A_2AR antagonist (ZM-241385) was 0.9±0.3 nM for the chosen A_2AR cell clone. Also by fitting the binding data to eq. 4 (Materials and Methods), the K_D value obtained for the A₁R antagonist (DPCPX) was 8.6±0.9 nM for the A₁R cell clone and the K_D value obtained for the D₂R antagonist (YM-09151-2) was 0.23±0.08 nM for the D₂R cell clone. These values were then used to determine the affinity constants showed in Tables 1 and 2. The agonists affinity in each selected clone was determined by competition experiments using the A_2AR antagonist [³H]ZM-241385 (2 nM) versus the agonist CGS-21680 (1 nM to 50 µM), the D₂R antagonist [³H]YM-09151-2 (0.2 nM), versus the agonist quinpirole (0.1 nM to 30 µM), or the A₁R antagonist [³H]DPCPX (2 nM), versus the agonist R-PIA (1 nM to 50 µM). As it is shown in Tables 1 and 2, the agonist affinity for A_2AR in A_2AR, A_2AR-D₂R or in A_2AR-A₁R cells is in the same range as that reported for brain striatum or for cells expressing human A_2AR (between 30 and 250 nM) [7]. Nevertheless, the affinity of the A_2AR for the selective agonist CGS-21680 was slightly but significantly lower when co-expressed with D₂R (see Table 2). A₁R (but not A_2AR or D₂R) agonist binding showed negative cooperativity (negative D_CB values, see Materials and Methods), both in cells expressing A₁R and in cells co-expressing A₁R and A_2AR (Tables 1 and 2).

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Table 1. Pharmacological parameters for agonist binding to A₁R, A_2AR and D₂R in A₁R, A_2AR and D₂R CHO cells.

https://doi.org/10.1371/journal.pone.0016088.t001

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Table 2. Pharmacological parameters for agonist binding to A₁R-A_2AR and A_2AR-D₂R CHO cells.

https://doi.org/10.1371/journal.pone.0016088.t002

Screening of A_2AR antagonists on cells expressing A₁-A_2A or A_2A-D₂ receptor heteromers

To test if selected A_2AR antagonists display different selectivity for A₁R-A_2AR or A_2AR-D₂R heteromers, competition experiments with these ligands were performed using CHO cells expressing A_2AR, A₁R-A_2AR or A_2AR-D₂R. We found that none of the six A_2AR antagonists first tested in the in vivo models were able to bind with moderate affinity to A₁R or to D₂R in CHO cells expressing A₁R or D₂R (data not shown), indicating that these compounds are specific ligands for A_2AR. Competition experiments of [³H]ZM-241385 (2 nM) binding versus increasing concentrations of each A_2AR antagonist (1 nM to 100 µM) were performed as indicated in Methods and binding data from competition experiments were fitted assuming that receptors are dimers and statistically (F test, see Materials and Methods) testing whether the competitor (A_2AR antagonists) binding was cooperative (biphasic competition curves; fitting to eq. 2) or non-cooperative (monophasic competition curves; fitting to eq. 3). Since the screened compounds are A_2AR antagonists, competition curves were expected to be monophasic, assuming that antagonist binding is not cooperative. In fact, in all cell clones, MSX-2, KW-6002, SCH-420814, ZM-241385 and SCH-58261 gave monophasic competition curves (fitting binding data to eq. 2 was not better than fitting to eq. 3; see Methods and Fig. 7 a–c as an example). Accordingly, the pharmacological characterization for these compounds gave D_CB = 0 and K_DB2 = 4K_DB1 (see Table 3). For all compounds, co-transfection with A₁R did not significantly modify their affinity for A_2AR. On the other hand, co-transfection with D₂R significantly reduced the affinity of A_2AR for MSX-2, SCH-420814, SCH-58261 and ZM-241385, from two to about nine times, and did not significantly modify the affinity of A_2AR for KW-6002 (Table 3).

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Figure 7. Binding of the A_2AR antagonists KW-6002 and SCH-442416 to A₁R-A_2AR and A_2AR-D₂R CHO cells.

Competition experiments of [³H]ZM-241385 (2 nM) versus increasing concentrations of KW-6002 (a and c) or SCH-442416 (b and d) were performed as indicated in Methods in membrane preparations from CHO cells expressing A₁R and A_2AR (a and b) or A_2AR and D₂R (c and d). Data are means ± S.E.M. of a representative experiment performed with triplicates.

https://doi.org/10.1371/journal.pone.0016088.g007

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Table 3. Pharmacological parameters for A_2AR antagonist binding to A_2AR, A₁R-A_2AR and A_2AR-D₂R CHO cells.

https://doi.org/10.1371/journal.pone.0016088.t003

For SCH-442416, a careful statistically-based analysis of the monophasic or biphasic nature of the competition curves led to an unexpected finding: in A_2AR-D₂R cells, competition curves of [³H]ZM-241385 (2 nM) binding versus increasing concentrations of SCH-442416 were biphasic (fitting to eq. 2 improves the fitting to eq. 3; see Methods) (Fig. 7d). Table 4 shows the deduced pharmacological parameters from competition experiments of [³H]ZM-241385 versus SCH-442416 in cells expressing A_2AR, A₁R-A_2AR and A_2AR-D₂R. In A_2AR and A₁R-A_2AR cells the curves were monophasic. Accordingly, the pharmacological characterization gave a D_CB values of 0 and a K_DB2 = 4K_DB1. In contrast, as mentioned above, in cells expressing A_2AR-D₂R, competition curves were biphasic, and binding data were then fitted to eq. 2 (Methods) and robust parameters were obtained (Table 4). Thus, in A_2AR-D₂R cells, SCH-442416 binding showed a strong negative cooperativity and, consequently, with a marked loss of affinity (an increase of 600 times in K_DB2) respect to cells expressing A_2AR. This is reflected by the B₅₀ value (concentration competing 50% of radioligand binding), which was more than 40 times higher in A_2AR-D₂R cells than in A₁R-A_2AR cells or A_2AR cells.

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Table 4. Pharmacological parameters for SCH-442416 binding to A_2AR, A₁R-A_2AR and A_2AR-D₂R CHO cells.

https://doi.org/10.1371/journal.pone.0016088.t004