The mechanism of action responsible for the motor depressant effects of cannabinoids, which operate through centrally expressed cannabinoid CB1 receptors, is still a matter of debate. In the present study, we report that CB1 and adenosine A2A receptors form heteromeric complexes in co-transfected HEK-293T cells and rat striatum, where they colocalize in fibrilar structures. In a human neuroblastoma cell line, CB1 receptor signaling was found to be completely dependent on A2A receptor activation. Accordingly, blockade of A2A receptors counteracted the motor depressant effects produced by the intrastriatal administration of a cannabinoid CB1 receptor agonist. These biochemical and behavioral findings demonstrate that the profound motor effects of cannabinoids depend on physical and functional interactions between striatal A2A and CB1 receptors.
Activation of cannabinoid CB1 receptors mediates most central effects of cannabinoids, such as Δ9-tetrahydrocannabinol (THC), the main psychoactive ingredient of marihuana. One of the most salient effects of CB1 receptor activation is motor depression, which is related to the significant modulatory role played by endocannabinoids in the basal ganglia (Gough and Olley, 1978; Ledent et al, 1999; Sanudo-Pena et al, 1999; van der Stelt and Di Marzo, 2003). CB1 receptors are abundantly expressed in different structures of the basal ganglia, including the striatum (Herkenham et al, 1991). In the striatum, CB1 receptors are localized in both types of GABAergic efferent neurons, enkephalinergic and dynorphinergic (Hohmann and Herkenham, 2000; Fusco et al, 2004), which constitute more than 90% of the striatal neuronal population (Gerfen, 2004). Furthermore, striatal CB1 receptors are localized in parvalbumin-expressing GABAergic interneurons (Hohmann and Herkenham, 2000; Fusco et al, 2004) and presynaptically in glutamatergic and GABAergic terminals (Rodriguez et al, 2001; Kofalvi et al, 2005).
Similar to endocannabinoids, the neuromodulator adenosine plays a very important integrative role in striatal function (Ferré et al, 1997, 2005). Adenosine A2A receptors are more concentrated in the striatum than anywhere else in the brain and they are strategically located, both pre- and postsynaptically, to modulate glutamatergic neurotransmission in GABAergic enkephalinergic neurons (Hettinger et al, 2001; Ferré et al, 2005; Ciruela et al, 2006). In the present study, we found that A2A and CB1 receptors co-immunoprecipitate from extracts of rat striatum, where they colocalize in fibrilar structures. In co-transfected mammalian cells we demonstrated that both receptors form direct physical interactions, that is A2A–CB1 receptor heteromers. At a functional level, we also demonstrated that CB1 receptor function is dependent on A2A receptor activation both in vitro and in vivo. Thus, activation of A2A receptors was necessary for CB1 receptor signaling in a human neuroblastoma cell line and blockade of A2A receptors significantly decreased the motor depressant effects of the central administration of the synthetic cannabinoid receptor agonist WIN 55 212-2 into the rat striatum.
MATERIALS AND METHODS
Adult male C57BL/6 wild type, A2A receptor KO and CB1 receptor KO mice, weighing 25–30 g, and adult male Wistar rats, weighing 250–300 g (Instituto Cajal, CSIC, Madrid, Spain) were used. Mice were only used to validate the antibodies to be applied in the immunohistochemical confocal experiments in the rat brain. The rat was the target animal species, as it was the most suitable to study the behavioral effects of intrastriatal administration of cannabinoid agonists. A2A receptor KO and CB1 receptor KO mice were generated as described elsewhere (Ledent et al, 1999; Chen et al, 1999). All animals used in a given experiment originated from the same breeding series, and were matched for age and weight. Mice were housed in groups of 4–5 per cage in clear plastic cages and maintained in a temperature- (22°C) and humidity-controlled room on a 12 h light–dark schedule with food and water provided ad libitum. The maintenance of the animals, as well as the experimental procedures, followed the guidelines from European Union Council Directive 86/609/EEC. All efforts were made to minimize the number of animals used and their suffering. The experimental protocols involving animals were approved by the local (CSIC) ethic committee. Animals were anesthetized by an intraperitoneal (i.p.) administration of pentobarbital (Lab Normon, Madrid, Spain) and perfused by means of a cannula introduced into the ascending aorta through the left ventricle. The vascular network was first washed of blood with saline solution (0.9% NaCl), followed by fixation with 4% paraformaldehyde in phosphate buffer (PB, 0.1 M, pH 7.4). The brains were then extracted and postfixed by immersion in the same fixative for 12–24 h at 4°C. Thereafter they were washed in two to three changes of PB 0.1 M and then cut with a vibrating blade microtome into 30-μm-thick coronal serial sections. Single- and double-labeling immunocytochemical techniques were employed for the detection of the antigens in free-floating sections. Adjacent serial sections of the same brains where the primary antibodies were omitted were analyzed to discriminate nonspecific staining. The absence of signal in sections from single CB1 and A2A KO mice showed the specificity of the primary antibodies used (see Figure 1). Sections were washed with three changes of PBT (PB 0.1 M, pH 7.4, containing 0.3% of Triton X-100), followed by a blocking solution (PBT containing 5% normal donkey serum). The primary antibodies were diluted in this blocking solution (1 : 250 for the CB1 receptor and 1 : 500 for the A2A). CB1 receptor antibody was a rabbit polyclonal antiserum raised against a synthetic peptide corresponding to the first 14 amino acids on the amino terminus of the sequence for the rat receptor (Sigma, Saint Luis, MO, USA), and has been described and characterized elsewhere (Howlett et al, 1998). A2A receptor antibody was a mouse monoclonal antibody (05-717, Upstate, Lake placid, NY, USA). Sections were incubated for 3 and 1 days with the CB1 receptor and the A2A antibody, respectively, under continuous shaking at 4°C. Then, sections were washed several times in PBT and incubated for 90 min with a donkey biotinylated anti-rabbit secondary antibody (1 : 500) for the CB1 receptor (RPN1004V1, Amersham Biosciences, Buckinghamshire, UK) and with a donkey anti-mouse secondary antibody conjugated to Alexa Fluor 594 (red fluorescence) (1 : 500) (Molecular Probes, Leiden, The Netherlands) for the A2A receptor. In order to determine the presence of the CB1 receptor, sections were washed several times in PBT and incubated for 1 h with Streptavidine conjugated to Alexa Fluor 488 (green fluorescence) (1 : 3000) (Molecular Probes). Finally, sections were washed in PB repeatedly and mounted using Polyvinyl alcohol mounting medium plus 1,4-diazobicyclo[2,2,2]-octane (antifading) (Sigma). Images of the sections were obtained by confocal microscopy.
Male Sprague–Dawley rats (Charles River Laboratory, Wilmington, MA, USA) weighing 300–350 g were used in co-immunoprecipitation experiments. Animals were maintained in accordance with guidelines of the Institutional Care and Use Committee of the Intramural Research Program, National Institute on Drug Abuse, NIH. Rats were killed with an overdose of Equithesin (NIDA Pharmacy, Baltimore, MD, USA). The striatal tissue was dissected on ice and the tissue was homogenized in 50 mM Tris-HCl, 5 mM EDTA, and Complete Mini peptidase inhibitors (Roche Applied Sciences, Basel, Switzerland), and centrifuged for 30 min at 20 000g at 4°C. Pellets were resuspended in solubilization buffer (10 mM Tris-HCl, 1 mM EDTA, 1% CHAPS, and the peptidase inhibitors) and centrifuged for 1 h at 150 000g at 4°C. Protein concentration in the supernatant was assayed using the bichinconinic acid assay (Pierce Biotechnology, Rockford, IL, USA). The specific receptor antibodies used were anti-A2A receptor rabbit polyclonal IgG (VCR1) (Hillion et al, 2001) and anti-CB1 receptor rabbit polyclonal IgG (PA1-745; Affinity BioReagents, Golden, CO, USA). The selectivity of both antibodies has been previously characterized (Hillion et al, 2001; Twitchell et al, 1997). The antibody against the cAMP response element binding protein (Cell Signaling Technologies, Beverly, MA, USA) was used as a control antibody. All three antibodies were immobilized on AminoLink Plus Coupling Gel using the Seize Primary Mammalian Immunoprecipitation Kit from Pierce Biotechnology. The solubilized striatal tissue was incubated with the immobilized antibody support and the receptors were co-immunoprecipitated using the immobilized antibodies. The beads were washed repeatedly with buffers containing varying concentrations of sodium chloride and Tris to wash away any nonspecifically bound proteins to the beads. The immunoprecipitated receptors were eluted off the immobilized antibody support using 2% SDS solution and the antibody support was regenerated for reuse. The immunoprecipitates were mixed with the loading buffer and resolved by SDS-PAGE. Western blots were performed with anti-CB1 receptor antibodies.
Cell Cultures and Transfections
HEK-293T cells were used to demonstrate heteromerization of co-transfected constructs of A2A and CB1 receptors (A2A-Rluc and CB1-YFP) with bioluminescence resonance energy transfer (BRET) experiments. The human neuroblastoma cell line SH-SY5Y was used to investigate whether there is functional A2A–CB1 receptor cross-talk, as this cell line has been reported to constitutively express functional A2A and CB1 receptors (Salim et al, 2000; Hillion et al, 2001; Klegeris et al, 2003). HEK-293T cells were grown in Dulbecco's modified Eagle's medium (DMEM) (Gibco, Paisley, Scotland, UK) supplemented with 2 mM L-glutamine, 100 UI/ml penicillin/streptomycin, and 5% (v/v) heat-inactivated fetal bovine serum (FBS) (all supplements were from Invitrogen, Paisley, Scotland, UK). The SH-SY5Y neuroblastoma cell line was grown in DMEM (Gibco) supplemented with 2 mM L-glutamine, 100 UI/ml penicillin/streptomycin, 1 mM sodium pyruvate, and 10% (v/v) heat-inactivated FBS. Cells were maintained at 37°C in an atmosphere of 5% CO2, and were passaged when they were 80–90% confluent, twice a week. Human A2A receptor cDNA without its stop codon was amplified using sense and antisense primers harboring unique EcoRI and BamHI sites. The fragment was then subcloned to be in-frame with Rluc into the EcoRI and BamHI restriction sites of a Renilla luciferase expressing vector (pcDNA 3.1-Rluc) yielding the A2A-Rluc construct. Human CB1 receptor cDNA without its stop codon was amplified using sense and antisense primers harboring unique BamHI and EcoRI sites. The fragment was then subcloned to be in-frame with EYFP into the BamHI and EcoRI restrictions sites of a multiple cloning site of pEYFP-N1 (enhanced yellow variant of GFP; Clontech, Heidelberg, Germany) yielding the CB1-YFP construct. Both constructs express Rluc or EYFP on the C-terminal ends of the receptor. Functionality of the constructs transiently transfected in HEK 239T cells was tested by ERK1/2 phosphorilation assay (data not show). HEK-293T cells growing in six-well dishes were transiently transfected with 12 μg of DNA by PolyEthylenImine (PEI; Sigma, Steinheim, Germany) method. Various amounts of DNA for the construct CB1-YFP were used with 2 μg of A2A-Rluc. To maintain the ratio of DNA in co-transfections, the empty vector, pcDNA 3.1, was used to equilibrate the amount of total DNA transfected. For transient transfections, cells were incubated with a mix containing constructs DNA, 5.47 mM nitrogen residues of PEI, and 150 mM NaCl in a serum-starved medium. After 4 h, medium was changed to a fresh complete medium. BRET immunolabeling and BRET experiments were performed 48 h after transfection.
SH-SY5Y cells were grown on glass coverslips coated with poly-L-lysine (Sigma). At 60% confluence, cells were rinsed with PBS, fixed in 4% paraformaldehyde for 15 min, and washed with PBS containing 20 mM glycine. Cells were permeabilized with PBS containing 20 mM glycine, 1% bovine serum albumin (BSA) (buffer A), and 0.05% Triton X-100 during 5 min, and were blocked with buffer A for 1 h at room temperature. Cells were labeled for 1 h with mouse monoclonal anti-A2A receptor antibody (05-717, Upstate, Lake Placid, NY, USA). Then, were washed and stained for 1 h with cyanine 3-conjugated affinity purified donkey anti-mouse IgG (Jackson ImmunoResearch, West Grove, USA) and with cyanine 5-conjugated rabbit anti-CB1 receptor antibody (Affinity Bioreagents), labelled in our laboratory using FluoroLink Cy5 reactive dye pack (Amersham Biosciences). The coverslips were rinsed for 30 min in buffer A and mounted with Vectashield Mounting Medium for Fluorescence (Vector Laboratories Inc. Burlingame, CA, USA). Microscopic observations were made in Olympus FV 300 confocal scanning laser microscope (Leica Lasertechnik, Leica Microsystems, Mannheim, Germany). Expression of the A2A-Rluc and CB1-YFP constructs were also tested by confocal microscopy. HEK-293T cells transiently transfected with the cDNA of fusion proteins were grown on glass coverslips coated with poly-L-lysine (Sigma), 48 h after transfections, rinsed with PBS, and fixed in 4% paraformaldehyde for 15 min. To detect the expression of the A2A-Rluc construct the same protocol as described above was used, whereas to detect the CB1-YFP construct its fluorescent properties were used. Coverslips were washed with buffer A and mounted as describe above.
At 48 h after transfection, cells were rapidly washed twice in HBSS with 10 mM glucose, detached, and resuspended in the same buffer containing 1 mM EDTA. To control the cell number, sample protein concentration was determined using a Bradford assay kit (Bio-Rad, Munich, Germany) using BSA dilutions as standards. To quantify fluorescence of CB1-YFP, cells (20 μg protein) were distributed in duplicated 96-well microplates (black plates with a transparent bottom) and read in a Fluostar Optima Fluorimeter (BMG Labtechnologies, Offenburg, Germany) equipped with a high-energy xenon flash lamp, using a 10-nm bandwidth excitation filter at 485 nm. YFP fluorescence was the fluorescence of the sample minus the fluorescence of cells not expressing CB1-YFP. For BRET measurement, the equivalent of 20 μg of cell suspension were distributed in triplicates in 96-well microplates (Corning 3600, white plates with white bottom) and 5 μM coelenterazine H (Molecular Probes, Eugene, OR, USA) was added. After 1 min, the readings were collected using a Mithras LB 940 (Berthold Technologies, DLReady, Germany) that allows the integration of the signals detected in the filter at 485 nm (440–500 nm) and the 530 nm (510–590 nm). To quantify luminescence of Rluc, readings were taken after 10 min of adding 5 μm coelenterazine H. The BRET signal was determined by calculating the ratio of the light emitted by YFP (510–590 nm) over the light emitted by the Rluc (440–500 nm). The net BRET values were obtained by subtracting the BRET background signal detected when Rluc-tagged construct was expressed alone. Curves were fitted using a nonlinear regression and one-phase exponential association fit equation (GraphPad Prism, San Diego, CA, USA).
Total cellular RNA was isolated from confluent cultures of SH-SY5Y cells using QuickPrep Total RNA Extraction Kit (Amersham Biosciences) following the manufacturer's instructions. For the RT-PCR assay, 1 μg of total RNA was reverse transcribed by random priming using M-MLV Reverse Transcriptase, RNase H Minus, and Point Mutant, following the protocol of two-step RT-PCR provide by data sheet of Promega (Promega, Madison, WI, USA). The resulting single-stranded cDNA was used to perform PCR amplification for CB1 receptor, A2A receptor, and tubulin as an internal control of PCR technique. Samples, composed by master mix, that includes Taq DNA Polymerase, dNTPs, MgCl2, and reaction buffers at optimal concentrations for efficient amplification of DNA templates (Promega), primers and cDNA, were denatured at 95°C for 2 min, and then subjected to 35 cycles of 95°C for 1 min, 58°C to annealing CB1 receptor primers, and 60°C to annealing A2A receptor primers during 1 min and extensions of 2 min at 72°C, with a 10 min extension at 72°C during the last cycle on a Techne thermal cycler. The primers used to amplify the human CB1 receptor gene were 5′-IndexTermTGGGCAGCCTGTTCCTCAC-3′ (forward) and 5′-IndexTermCATGCGGGCTTGGTC-3′ (reverse). To amplify the human A2A receptor, the primers used were 5′-IndexTermCATCCCCTTTGCCATCACCATCAG-3′ (forward) and 5′-IndexTermGTAGGGGCAGCCAGCAGAGG-3′ (reverse). To amplify tubulin, the primers used were 5′-IndexTermCATGATGGCCGCCTGCGACC-3′ (forward) and 5′-IndexTermCCTGGATGGCCGTGCTGTTGC-3′ (reverse). The expected size of the amplicons was 400 bp for the CB1 receptor, 571 bp for A2A receptor, and 232 bp for tubulin. The PCR products were electrophoresed on a 1% agarose gel. RNA without reverse transcriptions did not yield any amplicons, indicating that there was no genomic DNA contamination.
The accumulation of cAMP was measured with Cyclic AMP (3H) Assay System (Amersham Biosciences) as described in the manual from the manufacturer. About 80% confluent SH-SY5Y cells were serum-starved during 12–16 h supplemented or not with 2 UI/ml of adenosine deaminase (ADA; Roche, Basel, Switzerland) and the medium was replaced for the same fresh medium immediately before 50 μM Zardaverine addition as phosphodiesterase inhibitor. After 15 min at 37°C, the A2A receptor antagonist 4-(2-[7-amino-2-(2-furyl[1,2,4]-triazolo[2,3-a[1,3,5]triazin-5-yl-aminoethyl)phenol (ZM241385; 1 μM) (Cunha et al, 1997) or the CB1 receptor antagonist N-(piperidin)-1-yl)-5-(4-iodophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboxamide (AM251; 1 μM) (Lan et al, 1999) were added and incubated 5 min before agonist addition. The A2A receptor agonist 2-p-(2-carboxyethyl)phenethylamino-5′-N-ethylcarboxamidoadenosine (CGS 21680, 200 nM) (Karcz-Kubicha et al, 2003) and the selective CB1 receptor agonist arachidonyl-2-chloroethylamide (ACEA, 50 nM) (Hillard et al, 1999) were added with or without 10 μM forskolin and incubated for 30 min at 37°C. CGS 21680 and forskolin were from Sigma, the rest of reagents were from Tocris, Bristol, UK. CGS 21680 and ZM241385 were initially dissolved in DMSO (concentration of DMSO in the final dilution was <0.01%). ACEA and AM251 were initially diluted in ethanol (concentration of ethanol in the final dilution was <0.01%). One-way ANOVA followed by Newman–Keuls post hoc test was used for statistical comparisons.
Male Sprague–Dawley rats (Charles River Laboratory) weighing 300–350 g were used in the motor activity experiments. The animals were stereotaxically implanted with stainless-steel guide cannulae (22 G, Plastic ONE, Roanoke, VA, USA) in the right and left dorsal striatum under Equithesin (NIDA Pharmacy) anesthesia (coordinates respect to bregma: A 0.0, L±3.5, V −5.0). Guide cannulae were fixed with dental acrylic and stainless-steel screws to the skull surface. Stainless-steel stylets were inserted into the cannulae to prevent occlusion. A recovery period of 3 or 4 days was allowed before testing. For intrastriatal administration, injection needles (28 G) extending 0.5 mm below the guide were inserted into the cannulae. The cannabinoid receptor agonist R-(+)-[2,3-dihydro-5-methyl-3-(4-morpholinylmethyl) pyrrolo-[1,2,3-d,e]-1,4-benzoxazin-6-yl]-1-naphthalenyl-methanone mesylate (WIN 55 212–2; 40 μg dissolved in 5% Tween 80-saline) (Felder et al, 1995; Hillard et al, 1999) or vehicle (5% Tween 80-saline) was administered intrastriatally at a rate of 0.5 μl/min by means of a microdrive pump (final injection volume: 1 μl). The dose and rate of administration of WIN 55 212–2 was chosen according to pilot experiments and previously published studies on motor depressant effects induced by intrastriatal administration of THC (Gough and Olley, 1978). The needle was then left in place for an additional 2 min before being replaced by the stylet. About 20 min before the intrastriatal administration, saline, the CB1 receptor antagonists AM251 (3 mg/kg), and N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1H-pyrazole-3-carboximide hydrochloride (SR141716A; 3 mg/kg) (Felder et al, 1995) or the A2A receptor antagonist 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-7-methyl-1-propargylxanthine phosphate disodium (MSX-3; 3 mg/kg) (Karcz-Kubicha et al, 2003; Sauer et al, 2000) were administered i.p. (1 ml/kg for SR141716A and MSX-3 and 2 ml/kg for AM251). SR141716A and AM251 were dissolved in a vehicle of 2% ethanol, 2% Tween 80, and saline and MSX-3 was dissolved in saline with a few drops of 0.1 N NaOH (final pH: 7.4). SR141716A and AM251 were from Sigma and MSX-3 was synthesized at the Pharmaceutical Institute, University of Bonn, Germany. About 20 min after the intrastriatal administration, the animals were placed in a Columbus Instruments Auto-Track system (Coulbourn Instruments, Lehigh Valley, PA) to quantify motor activity (ambulatory distance and stereotypies). Motor activity was recorded during the first 20 min (maximal period of exploratory activity) in 5-min intervals. The average of the motor activity recordings obtained during the first four 5-min intervals was used to analyze differences between the differently treated groups of animals. One-way ANOVA with Dunnett's multiple comparison tests were used for statistical analysis. At the end of the experiment, rats were killed with an overdose of Equithesin, the brain was removed and placed in a 10% formaldehyde solution, and coronal sections were cut to verify cannulae location.
Competition Binding Assays
Frozen rat brains obtained from Pel Freez, Rogers, AR, USA, were dissected to obtain cortical and striatal membrane preparations as described elsewhere (Sauer et al, 2000). ZM241385 and MSX-2 and its phosphate pro-drug MSX-3 were investigated in competition experiments versus the CB1 receptor agonist [3H]CP55 940 (158 Ci/mmol; Amersham, Rossendaal, The Netherlands). MSX-2 was tested as it is the dephosphorylated active compound of MSX-3, which cannot be converted to MSX-2 in in vitro experiments. Stock solutions of the compounds were prepared in DMSO. Final DMSO concentrations in the assays did not exceed 2.5%. Competition experiments were performed using 0.1 nM [3H]CP55 940 at room temperature in plastic tubes with rat brain cortical or striatal membranes (50 μg of protein per tube) in 1 ml (final volume) of buffer solution (50 mM Tris-HCl, 3 mM MgCl2, 0.1% BSA, pH 7.4). Nonspecific binding was determined in the presence of 10 μM of CP55 940. After 2 h the incubation was stopped, solutions were rapidly filtered through GF/C glass fiber filters on a 24-Brandell cell harvester, and washed three times with 3 ml each of ice-cold washing buffer solution (50 mM Tris-HCl, 0.1% BSA, pH 7.4). Filters were dried, scintillation cocktail (Ultima Gold, Perkin-Elmer) was added, and radioactivity was measured using a liquid scintillation counter (Tri-Carb 2900TR, Packard). Three independent experiments were performed, each in triplicate. Protein concentrations were determined by the method of Lowry.
Colocalization and Co-immunoprecipitation of A2A and CB1 Receptors in the Rat Striatum
Immunohistochemical experiments were carried out to test whether A2A and CB1 receptors are coexpressed in striatal neurons. Immunological staining of coronal sections of mouse striatopallidal complex with the anti-CB1 or the anti-A2A receptor antibodies showed a predominant labeling of A2A and CB1 receptors in the striatum and globus pallidus (Figures 1a and b). CB1 receptor immunostaining was moderate in the striatum and strongest in the pallidum, whereas A2A receptors were mainly localized in the striatum. The labeling profile of the two antibodies was very similar, tagging primarily the neuropil and avoiding the neuronal cell bodies. Similar results were obtained using coronal sections of rat brain (results not shown), which agrees with previous reports (Herkenham et al, 1991; Hettinger et al, 2001). No immunostaining was found in samples from genetically modified CB1 or A2A knockout mice (Figure 1c and d), which demonstrates the specificity of the primary antibodies.
Double immunofluorescence staining of coronal sections of rat striatum revealed a green fluorescent signal corresponding to CB1 receptors and a red fluorescent signal corresponding to A2A receptors, in fibrilar structures (Figure 2). Both labeled profiles had a dispersed appearance with clear contrast between the unlabeled cell bodies or fiber bundles (with no fluorescence signal) and the surrounding labeled neuropil. Thus, CB1 or A2A receptor-immunoreactive fibers had the appearance of a meshwork, indicative of striatal neuropil, perforated by unlabeled cell bodies (Figure 2a–f). This was clearly evident at a higher magnification, where sharply delineated cells bodies devoid of any of the two labels were observed, whereas cell membrane profiles were clearly labeled with both CB1 and A2A receptor antibodies (Figure 2g and h, respectively). Occasionally, primary dendrites were also labeled with the two antibodies (Figure 2g and 2h). Merging of the red and green images showed a strong colocalization (yellow signal) of CB1 and A2A receptors in the same striatal neurons (Figure 2c, f and i). A yellow fluorescent signal showed colocalization of CB1 and A2A receptors in approximately half of the total fibers expressing CB1 receptors, and the presence of green (CB1 receptor immunoreactivity) and absence of red (A2A receptor immunoreactivity) signal in the merged images indicates that most A2A receptors are colocalized with CB1 receptors, but that there is a proportion of CB1 receptors that does not colocalize with A2A receptors (Figure 2c, f and i). This agrees with the more widespread localization of striatal CB1 receptors compared to A2A receptors (see Introduction). Overall, results shown in Figure 2 demonstrate that CB1 and A2A receptors are present in the same striatal neurons. It remains to be determined if striatal A2A and CB1 receptors are preferentially colocalized postsynaptically, in GABAergic enkephalinergic dendrites, or also presynaptically, in glutamatergic terminals, as positive immunoreactive fibrilar structures were compatible with both dentritic processes and nerve terminals.
To test for the existence of physical interactions between A2A and CB1 receptors in the striatum, co-immunoprecipitation experiments were performed. As shown in Figure 3, a predominant band at about 60 kDa, corresponding to the CB1 receptor, could be observed in the lysate and in the immunoprecipitate obtained using either the CB1 receptor antibody or the A2A receptor antibody, but not when another antibody (anti-CREB) was used. These results indicate that heteromeric A2A–CB1 receptor complexes exist in the striatum. However, co-immunoprecipitation does not demonstrate the existence of true heteromers, as they do not discard the existence of intermediate proteins indirectly linking A2A and CB1 receptors.
A2A–CB1 Receptor Heteromerization in Living Cells
To demonstrate the existence of a direct physical interaction between A2A and CB1 receptors, BRET was carried out in living HEK293 cells transfected with cDNAs encoding the fusion proteins A2A-Rluc (human A2A receptor-Renilla luciferase) and CB1-YFP (human CB1 receptor-yellow fluorescent protein). After transfection, the receptors expression was high at the membrane level (Figure 4a). As energy transfer between two specifically interacting proteins has to reach a plateau, a saturable BRET curve was obtained for the A2A-Rluc/CB1-YFP pair when constant amounts of the cDNA for the Rluc construct were co-transfeted with increasing amounts of the plasmid cDNA for the YFP construct (Figure 4b). Maximum net BRET was 0.061±0.004 (see Materials and methods) and BRET50 was attained at a relatively low CB1-YFP/A2A-Rluc ratio (0.023±0.003). As negative controls, no significant BRET was obtained in a mixture of cells transfected with A2A-Rluc and cells transfected with CB1-YFP or in cells co-transfected with A2A-Rluc and with CD4-YFP (Figure 4b). These results indicate that the BRET signal obtained using A2A-Rluc/CB1-YFP was specifically due to A2A–CB1 receptor heteromerization. Treatment with either the A2A receptor agonist CGS 21680 (200 nM), the CB1 receptor agonist ACEA (100 nM), or both for 15 and 45 min did not induce any significant changes in the BRET signal, indicating that acute agonist treatment does not modify A2A–CB1 receptor heteromerization (data not shown).
Functional Cross-Talk between CB1 and A2A Receptors
The human neuroblastoma cell line SH-SY5Y with constitutive expression of A2A and CB1 receptors was used to investigate A2A–CB1 receptor cross-talk. The presence of A2A and CB1 receptors was confirmed by RT-PCR (Figure 5a), immunocytochemistry, and confocal laser microscopy (Figure 5b). Confocal analysis revealed high colocalization of both receptors (white color in Figure 2b, right panel). By coupling to Gs-olf proteins, A2A receptors stimulate adenylyl-cyclase and induce cAMP accumulation (Kull et al, 1999). On the other hand, CB1 receptors couple to Gi-o proteins, and inhibit adenylyl cyclase (Bidaut-Russell et al, 1990; Felder et al, 1995; Hillard et al, 1999). Functional interaction between A2A and CB1 receptors were then assessed in cAMP accumulation experiments. Treatment of SH-SY5Y cells with the selective CB1 receptor agonist ACEA counteracted the increase in cAMP levels induced by forskolin, but the effect of ACEA was not significant in the presence of the A2A receptor antagonist ZM241385 (Figure 6). This demonstrates that under basal conditions, CB1 receptors are negatively coupled to adenylate cyclase and suggests that coupling of CB1 receptors to Gi requires previous or simultaneous activation of A2A receptors. Previous studies have shown that SH-SY5Y cells release significant amounts of adenosine, which could provide the sufficient tonic activation of A2A receptors required to enable CB1 receptor function (Salim et al, 2000). In fact, when cAMP levels were determined in the presence of the enzyme ADA (2 UI/ml), which rapidly metabolizes released adenosine and prevents tonic activation of adenosine receptors (Salim et al, 2000), ACEA was unable to affect forskolin-induced increases in cAMP levels (Figure 7). The A2A receptor agonist CGS 21680 substantially increased cAMP levels when SH-SY5Y cells where preincubated with ADA and this was prevented by treatment with the A2A receptor antagonist ZM241385 or by ACEA (Figure 8). The reversal of the CGS21680-induced increase of cAMP by ACEA was counteracted by the CB1 receptor antagonist AM251 (Figure 8). Altogether, these results indicate that in human neuroblastoma SH-SY5Y cells, activation of A2A receptors is necessary for CB1 receptor signaling.
Counteraction of Striatal CB1 Receptor-Mediated Motor Depression by A2A Receptor Antagonist
The in vitro biochemical studies described above predicted that A2A receptor antagonists would reduce in vivo behavioral effects of CB1 activation involving striatal function. To test this hypothesis, we studied the effects of blockade of A2A receptors by a previous systemic administration of the potent and selective A2A receptor antagonist MSX-3 on the motor effects induced by the bilateral striatal administration of the cannabinoid receptor agonist WIN 55 212-2. Here we show that the striatal administration of WIN 55 212-2 produces a significant motor depressant effect, both on locomotion and stereotypied behavior in nonhabituated rats (Figure 9). Although WIN 55 212-2 is a nonselective CB1–CB2 receptor agonist (Felder et al, 1995; Hillard et al, 1999), previous studies have shown that the motor depressant effects produced by systemic administration of WIN 55 212-2 are mediated by CB1 receptors (Gifford et al, 1999; Darmani, 2001). Nevertheless, in the present behavioral model, the involvement of CB1 receptors was demonstrated by the ability of two selective CB1 receptor antagonists, SR141716A and AM251, to counteract the motor depression induced by the intrastriatal administration of WIN 55 212-2 (Figure 9). Importantly, previous systemic administration of MSX-3 did not by itself significantly modify motor activity in vehicle-treated animals, but it completely counteracted the motor depression produced by WIN 55 212-2, indicating that in vivo CB1 receptor signaling that controls motor activity depends on A2A receptor activation (Figure 9).
Binding of A2A Receptor Antagonists to CB1 Receptors
Although A2A receptor antagonists behaved as CB1 receptor antagonists in both in vitro and in vivo models, their ability to bind to the CB1 receptor was not supported by radioligand binding experiments. No significant inhibition of the binding of a low concentration (0.1 nM) of [3H]CP55 940 to rat striatal or cortical membranes was observed with 1 μM of ZM241385 (2±3 and 4±3, respectively), MSX-2 (6±3 and 1±1, respectively), or MSX-3 (9±2 and 3±6, respectively). In the same assay, CP55 940 displayed a Ki value of 0.83±0.06 nM in striatal membranes.
It is becoming clear that the concept of heptaspanning G protein-coupled receptors (GPCRs) as single functional units has to be changed to a new concept that considers GPCRs as components of supramolecular aggregates, which include the same or other GPCRs, forming homomers or heteromers (Agnati et al, 2003, 2005; Franco et al, 2003, 2005). Heteromerization considerably increases the possible functional responses of GPCRs and its potential in drug discovery is just beginning to be considered (George et al, 2002; Maggio et al, 2005). By means of in vitro and in vivo approaches, we now demonstrate that both physical and functional interactions between striatal A2A and CB1 receptors exist and that these interactions play a significant role in the motor depressant effects of CB1 receptor agonists.
Adenosine A2A receptors are most abundant in the striatum, where they are preferentially localized in the dendritic spines of the striatopallidal GABAergic enkephalinergic neurons and also presynaptically in glutamatergic terminals (Hettinger et al, 2001; Ciruela et al, 2006). Striatal CB1 receptors are located in the dendritic spines of GABAergic neurons, including the striatopallidal neurons, and they are also located on nerve terminals (Rodriguez et al, 2001; Kofalvi et al, 2005; Julian et al, 2003). By using immunofluorescent histochemical techniques, we demonstrated that A2A and CB1 receptors are in fact colocalized in the same striatal elements, in fibrilar structures, which represent either dendritic processes or nerve terminals. A yellow fluorescent signal showed colocalization of CB1 and A2A receptors in approximately half of the total fibers expressing CB1 receptors, and the absence of a red immunoreactive signal in the merged images indicates that most A2A receptors are colocalized with CB1 receptors (Figure 2c, f, and i). This agrees with the more widespread localization of striatal CB1 receptors (see Introduction). These results provide the anatomical basis needed to sustain A2A–CB1 receptor interactions in the striatum. We first demonstrate the existence of A2A–CB1 heteromeric receptor complexes in rat striatal membranes by co-immunoprecipitation. We then show that A2A and CB1 receptors can form ‘true heteromers’ using BRET in HEK-293T living cells. These physical interactions suggested a functional interdependence between A2A and CB1 receptors. Such a functional interdependence is demonstrated by our findings that both CB1 receptor signaling in a human neuroblastoma cell line and the motor depressant effects of CB1 receptor agonists in rats are dependent on A2A receptor activation.
In recent studies, some biochemical effects of CB1 receptor agonists have been reported to depend on A2A receptor function, although it was suggested that these effects might be due to indirect interactions involving dopamine D2 receptors (Yao et al, 2003; Andersson et al, 2005). In the present study, we found a functional A2A–CB1 receptor interdependence in cells (human neuroblastoma SH-SY5Y cell line) that do not express D2 receptors. In those cells, CB1 receptor stimulation could only produce a decrease in cAMP levels if A2A receptors were simultaneously co-activated. This indicates that activation of A2A receptors in the CB1–A2A receptor heteromer allows the effective coupling of CB1 receptor to Gi proteins. It must however be pointed out that Soria et al (2004) found that the genetic activation of A2A receptors did not impair the ability of cannabinoid agonists to activate Gi proteins. Similarly, previous studies in transfected cells have also shown that CB1 receptors couple and activate Gi proteins (CB1 receptor agonist-induced inhibition of forskolin-induced cAMP accumulation) in the absence of A2A receptors (Felder et al, 1995; Hillard et al, 1999). Therefore, our results strongly suggest that it is in the presence of A2A receptors (when forming A2A–CB1 receptor heteromers) that CB1 receptor function depends on A2A receptor activation. The results, however, do not discard the existence of Gi-independent signal-transduction pathways that do not depend on A2A receptor function. In fact, several studies suggest that CB1 receptors can also couple to Gs under some conditions (for review, see Demuth and Molleman, 2006). Furthermore, we cannot discard the possibility that functional A2A–CB1 receptor interactions could take place with the receptors being close enough but not physically (directly or indirectly) connected.
It is generally accepted that the basal ganglia are the main brain areas involved in the motor depressant effects of cannabinoids and CB1 receptor agonists (Gough and Olley, 1978; Sanudo-Pena et al, 1999; van der Stelt and Di Marzo, 2003). However, there is no consensus about the role played by the different structures of the basal ganglia, with some authors giving more relevance to the striatum and others to projecting striatal areas (globus pallidus and substantia nigra pars reticulata) (Gough and Olley, 1978; Sanudo-Pena et al, 1999; van der Stelt and Di Marzo, 2003). The present study indicates an important role for the striatum, as a pronounced depression of exploratory activity was observed with the intrastriatal administration of the cannabinoid receptor agonist WIN 55 212-2, which was counteracted by the systemic administration of CB1 receptor antagonists. Furthermore, the motor depressant effect of WIN 55 212-3 was completely counteracted by previous systemic administration of the selective A2A receptor antagonist MSX-3. Importantly, these results provide an in vivo behavioral correlate for the biochemical results obtained with the neuroblastoma cell line, indicating that some functional effects of striatal CB1 receptors effects depend on A2A receptor function. The inability of A2A receptor antagonists to bind to the CB1 receptor shown in the present radioligand binding experiment indicates that the CB1-receptor-antagonist-like behavior of A2A receptor antagonists is due to a functional A2A–CB1 receptor interdependence. It has recently been shown that genetic and also pharmacological blockade of A2A receptors significantly, but only partially, reduces cataleptogenic effects induced by systemic administration of the CB1 receptor agonist CP55 940 (Andersson et al, 2005). On the other hand, Soria et al (2004) reported a lack of changes in the motor depressant effects induced by the systemic administration of THC in A2A receptor knockout mice. Our results demonstrate that a selective A2A receptor antagonist can completely counteract the motor depression produced by the striatal activation of CB1 receptors, suggesting that motor-depressant effects of systemically administered CB1 receptor agonists depend on both striatal (A2A receptor-dependent) and nonstriatal (A2A receptor-independent) CB1 receptors. Soria et al (2004) also reported a decreased place preference to THC in mice with genetic blockade of A2A receptors. This further suggests that the rewarding effects of cannabinoids might also depend on striatal A2A–CB1 receptor heteromeric complexes, although the main anatomical target for those effects is still a matter of debate.
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This research was supported in part by grants from Spanish Ministerio de Ciencia y Tecnología (SAF2005-00903 to FC and SAF2005-00170 to EIC and SAF2003-04864 and PNSD to RM) and in part by the Intramural Research Program of the National Institutes of Health.
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Carriba, P., Ortiz, O., Patkar, K. et al. Striatal Adenosine A2A and Cannabinoid CB1 Receptors Form Functional Heteromeric Complexes that Mediate the Motor Effects of Cannabinoids. Neuropsychopharmacol 32, 2249–2259 (2007). https://doi.org/10.1038/sj.npp.1301375
- adenosine A2A receptor
- cannabinoid CB1 receptor
- receptor heteromerization
- cyclic AMP
- motor activity
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