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. 2011 Jan 11;6(1):e16088.
doi: 10.1371/journal.pone.0016088.

Striatal pre- and postsynaptic profile of adenosine A(2A) receptor antagonists

Marco Orru  1 , Jana Bakešová, Marc Brugarolas, César Quiroz, Vahri Beaumont, Steven R Goldberg, Carme Lluís, Antoni Cortés, Rafael Franco, Vicent Casadó, Enric I Canela, Sergi Ferré
Affiliations

Striatal pre- and postsynaptic profile of adenosine A(2A) receptor antagonists

Marco Orru et al. PLoS One. .

Abstract

Striatal adenosine A(2A) receptors (A(2A)Rs) are highly expressed in medium spiny neurons (MSNs) of the indirect efferent pathway, where they heteromerize with dopamine D(2) receptors (D(2)Rs). A(2A)Rs are also localized presynaptically in cortico-striatal glutamatergic terminals contacting MSNs of the direct efferent pathway, where they heteromerize with adenosine A(1) receptors (A(1)Rs). It has been hypothesized that postsynaptic A(2A)R antagonists should be useful in Parkinson's disease, while presynaptic A(2A)R antagonists could be beneficial in dyskinetic disorders, such as Huntington's disease, obsessive-compulsive disorders and drug addiction. The aim or this work was to determine whether selective A(2A)R antagonists may be subdivided according to a preferential pre- versus postsynaptic mechanism of action. The potency at blocking the motor output and striatal glutamate release induced by cortical electrical stimulation and the potency at inducing locomotor activation were used as in vivo measures of pre- and postsynaptic activities, respectively. SCH-442416 and KW-6002 showed a significant preferential pre- and postsynaptic profile, respectively, while the other tested compounds (MSX-2, SCH-420814, ZM-241385 and SCH-58261) showed no clear preference. Radioligand-binding experiments were performed in cells expressing A(2A)R-D(2)R and A(1)R-A(2A)R heteromers to determine possible differences in the affinity of these compounds for different A(2A)R heteromers. Heteromerization played a key role in the presynaptic profile of SCH-442416, since it bound with much less affinity to A(2A)R when co-expressed with D(2)R than with A(1)R. KW-6002 showed the best relative affinity for A(2A)R co-expressed with D(2)R than co-expressed with A(1)R, which can at least partially explain the postsynaptic profile of this compound. Also, the in vitro pharmacological profile of MSX-2, SCH-420814, ZM-241385 and SCH-58261 was is in accordance with their mixed pre- and postsynaptic profile. On the basis of their preferential pre- versus postsynaptic actions, SCH-442416 and KW-6002 may be used as lead compounds to obtain more effective antidyskinetic and antiparkinsonian compounds, respectively.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Locomotor activation in rats induced by A2AR 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).
Figure 2
Figure 2. Blockade by A2AR 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 A2AR 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).
Figure 3
Figure 3. Blockade by A2AR 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 A2AR 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).
Figure 4
Figure 4. Identification of receptor heteromers in CHO cells by BRET saturation curve.
BRET experiments were performed with CHO cells co-expressing A2AR-RLuc and A1R-YFP (A) or A2AR-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 A2AR-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 A2AR-RLuc, (0.5 µg transfected cDNA) and serotonin 5HT2B-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.
Figure 5
Figure 5. Allosteric interaction between A2AR and D2R in A2AR-D2R CHO cells.
Competition experiments were performed in membrane preparations from CHO cells expressing A2AR 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.
Figure 6
Figure 6. Allosteric interaction between A1R and A2AR in A1R-A2AR CHO cells.
Competition experiments were performed in membrane preparations from CHO cells expressing A1R or A1R and A2AR with 12 nM [3H]R-PIA versus increasing concentrations of the A2AR agonist CGS-21680 as indicated in Methods. Data represent means ± S.E.M. of a representative experiment performed with triplicates.
Figure 7
Figure 7. Binding of the A2AR antagonists KW-6002 and SCH-442416 to A1R-A2AR and A2AR-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 A2AR (a and b) or A2AR and D2R (c and d). Data are means ± S.E.M. of a representative experiment performed with triplicates.
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