Functional interaction between pre-synaptic α6β2-containing nicotinic and adenosine A2A receptors in the control of dopamine release in the rat striatum

Abstract

Background and purpose: Pre-synaptic nicotinic ACh receptors (nAChRs) and adenosine A2A receptors (A2A Rs) are involved in the control of dopamine release and are putative therapeutic targets in Parkinson's disease and addiction. Since A2A Rs have been reported to interact with nAChRs, here we aimed at mapping the possible functional interaction between A2A Rs and nAChRs in rat striatal dopaminergic terminals.

Experimental approach: We pharmacologically characterized the release of dopamine and defined the localization of nAChR subunits in rat striatal nerve terminals in vitro and carried out locomotor behavioural sensitization in rats in vivo.

Key results: In striatal nerve terminals, the selective A2A R agonist CGS21680 inhibited, while the A2A R antagonist ZM241385 potentiated the nicotine-stimulated [(3) H]dopamine ([(3) H]DA) release. Upon blockade of the α6 subunit-containing nAChRs, the remaining nicotine-stimulated [(3) H]DA release was no longer modulated by A2A R ligands. In the locomotor sensitization experiments, nicotine enhanced the locomotor activity on day 7 of repeated nicotine injection, an effect that no longer persisted after 1 week of drug withdrawal. Notably, ZM241385-injected rats developed locomotor sensitization to nicotine already on day 2, which remained persistent upon nicotine withdrawal.

Conclusions and implications: These results provide the first evidence for a functional interaction between nicotinic and adenosine A2A R in striatal dopaminergic terminals, with likely therapeutic consequences for smoking, Parkinson's disease and other dopaminergic disorders.

Keywords: adenosine; adenosine A2A receptor; adenosine A2B receptor; dopamine; locomotor sensitization; nAChRs; nicotine; rat; striatum.

Figure 1

Nicotine stimulates [³H]dopamine ([³H]DA) release from rat striatal synaptosomes. (A) Time course of the averaged release of [³H]DA. Synaptosomes were treated with various concentrations of nicotine (1, 30, 300 nM or 3 μM) for 8 min, as indicated by the horizontal bar. (B) Concentration-response curve for nicotine to trigger the release of [³H]DA. *P < 0.001 versus non-stimulated control; n = 10–21 independent observations in duplicate.

Figure 2

The adenosine A_2AR agonist CGS (30 nM), but neither the A_2AR antagonist ZM (100 nM) nor the A_2BR antagonist MRS (200 nM), stimulates [³H]DA release from rat striatal synaptosomes. The non-selective adenosine receptor antagonist, caffeine (50 μM), also stimulates [³H]DA release, which may be explained by the observation that the simultaneous blockade of A_2ARs and A_2BRs by ZM and MRS also facilitated [³H]DA release. Data are mean ± SEM of 7–38 experiments performed in duplicate. *P < 0.05, ***P < 0.01 versus 0 FR% (i.e. no change in baseline); n.s., not significant.

Figure 3

Adenosine A_2AR activation inhibits the nicotine-induced [³H]DA release from rat striatal synaptosomes. (A) Time course and (B) bar graph displaying the averaged release of [³H]DA induced by various concentrations of nicotine alone or in the presence of A_2AR agonist, CGS (10–100 nM). The co-administration of CGS and nicotine occurred as indicated by the horizontal bar in (A). Data are mean ± SEM of 6–18 experiments performed in duplicate. *P < 0.05, **P < 0.01 and ***P < 0.001 versus 0 FR% (i.e. no change in baseline); ^$P < 0.05 and ^$$P < 0.01 between nicotine alone (green bar) and nicotine with CGS; n.s., not significant.

Figure 4

Adenosine A_2AR blockade increases the nicotine-induced [³H]DA release from rat striatal synaptosomes. (A) Time course and (B) bar graph displaying the averaged release of [³H]DA induced by nicotine alone or in the presence of the A_2AR antagonist, ZM (100 nM). The co-administration of ZM and nicotine occurred as indicated by the horizontal bar in (A). (C) In a similar experimental paradigm, the non-selective adenosine receptor antagonist, caffeine (50 μM), failed to mimic the action of ZM, that is, to facilitate the effect of nicotine. (D) This lack of caffeine effect may be due to the involvement of A_2BRs because the selective A_2BR antagonist MRS (200 nM) inhibited the effect of nicotine and prevented the facilitatory action of ZM when the two antagonists were combined. Data are mean ± SEM of 6–13 experiments performed in duplicate. *P < 0.05 and ***P < 0.001 versus 0 FR% (i.e. no change in baseline); ^$P < 0.05 and ^$$P < 0.01 between the indicated bars in (B) and (C) and when compared to control (nicotine in the absence of any adenosine receptor antagonist, displayed with the leftmost bar) in (D); n.s., not significant.

Figure 5

HPLC analysis reveals that striatal synaptosomes release adenosine. (A) Mean chromatograms and (B) mean ± SEM values for the quantified extracellular levels of adenosine and its metabolites in the presence of the adenosine deaminase inhibitor, EHNA (20 μM), upon incubation of synaptosomes (∼1.2 mg protein × mL⁻¹) in the absence or in the presence of nicotine (1 μM); similar results were obtained with 30 nM nicotine (not shown). n.s., not significant.

Figure 6

The adenosine A_2AR-mediated inhibition of the nicotine-induced [³H]DA release mainly depended on α6β2-containing nicotinic ACh receptors (nAChRs). (A) Western blot analysis revealed specific bands for the different major nAChR subunits in pre-synaptic membrane preparations, with the molecular weight indicated on the right of each excerpt. (B) Quantification of the density of the different subunits obtained from three (α4, α7 and β2) and four (α6) rats under three different protein loads to ensure that the signal is not saturated (maximum = 100% = saturated signal). (C) Time course displaying the averaged [³H]DA release induced by nicotine (30 nM) in the absence or in the presence of either the α6 subunit antagonist α-CTX (30 nM), the α7 subunit antagonist α-BTX (100 nM) or the β2 subunit antagonist DHβE (100 nM). (D) Time course displaying the averaged [³H]DA release, induced by nicotine (30 nM) in the presence of α-CTX (30 nM), when combined with either the A_2AR agonist CGS (30 nM) or the A_2AR antagonist ZM (100 nM). (E) Bar graph summarizing the sensitivity of 30 nM nicotine-stimulated [³H]DA release under the difference conditions tested in (C) and (D). Data are mean ± SEM of nine experiments performed in duplicate. *P < 0.05, **P < 0.01 and ***P < 0.001 versus 0 FR% (i.e. no change in baseline); ^$$$P < 0.001 between nicotine alone (blue bar) and nicotine with antagonists of nicotinic acetylcholine receptors; n.s., not significant. Note that neither CGS nor ZM affected the non-α6 subunit-containing nAChR-induced release of [³H]DA, suggesting that it is these α6 subunit-containing nAChRs that are modulated by A_2ARs to control striatal dopamine release.

Figure 7

Adenosine A_2AR blockade in vivo facilitates the sensitization to nicotine-stimulated hyperlocomotion. Sixteen rats were used to create factorial × groups for vehicle–vehicle, ZM–vehicle, vehicle–nicotine and ZM–nicotine injections. The A_2AR antagonist ZM (1 mg·kg⁻¹) or its vehicle was injected 30 min before the test, while nicotine (0.5 mg·kg⁻¹) or its vehicle was injected immediately before the test. After the first 2 days of 30 min habituation to the open-field arena each day, the rats received daily injections for 8 days. Significant sensitization to nicotine developed on day 7, which was blunted by the subsequent week of abstinence, as determined by a single injection of nicotine on the day 8 of abstinence (challenge), resulting in no statistically significant difference between the vehicle–vehicle group (to which all data points were normalized, represented by the dashed line) and the nicotine–vehicle group, represented by the red circles at day 18 of the experiment. ZM had no or a minimal hypolocomotor effect throughout the duration of the experiment (green upside-down triangles). However, ZM-injected rats developed sensitization to nicotine already on day 2 (blue diamonds), which was not blunted by the drug-free period, as indicated by a statistically significant difference between the ZM–vehicle group (green upside-down triangles) and the ZM–nicotine group (blue diamonds) at day 18 of the experiment. Data are mean ± SEM of distance travelled during each 30 min of open-field observation. A *P < 0.05 representing statistical differences between the nicotine-injected (red circles) or ZM-injected (green triangles) and the vehicle-injected (dashed line) rats; and ^$P < 0.05 representing statistical differences between the ZM-injected and the ZM + nicotine-injected rats (blue diamonds), as determined with anova of repeated measures and Dunett's post hoc analysis.