Receptor crosstalk: haloperidol treatment enhances A(2A) adenosine receptor functioning in a transfected cell model

Abstract

A(2A) adenosine receptors are considered an excellent target for drug development in several neurological and psychiatric disorders. It is noteworthy that the responses evoked by A(2A) adenosine receptors are regulated by D(2) dopamine receptor ligands. These two receptors are co-expressed at the level of the basal ganglia and interact to form functional heterodimers. In this context, possible changes in A(2A) adenosine receptor functional responses caused by the chronic blockade/activation of D(2) dopamine receptors should be considered to optimise the therapeutic effectiveness of dopaminergic agents and to reduce any possible side effects. In the present paper, we investigated the regulation of A(2A) adenosine receptors induced by antipsychotic drugs, commonly acting as D(2) dopamine receptor antagonists, in a cellular model co-expressing both A(2A) and D(2) receptors. Our data suggest that the treatment of cells with the classical antipsychotic haloperidol increased both the affinity and responsiveness of the A(2A) receptor and also affected the degree of A(2A)-D(2) receptor heterodimerisation. In contrast, an atypical antipsychotic, clozapine, had no effect on A(2A) adenosine receptor parameters, suggesting that the two classes of drugs have different effects on adenosine-dopamine receptor interaction. Modifications to A(2A) adenosine receptors may play a significant role in determining cerebral adenosine effects during the chronic administration of antipsychotics in psychiatric diseases and may account for the efficacy of A(2A) adenosine receptor ligands in pathologies associated with dopaminergic system dysfunction.

Electronic supplementary material: The online version of this article (doi:10.1007/s11302-010-9201-z) contains supplementary material, which is available to authorized users.

Keywords: A2A adenosine receptors; A2A binding parameters; D2 dopamine receptor antagonists; Receptor crosstalk; Receptor functioning.

Fig. 1

Co-immunoprecipitation (IP) and immunoblotting (IB) of A_2AAR. CHO cells were treated with medium alone (control) or with 1 μM haloperidol or 1 μM clozapine for 24 h. One milligram of protein was immunoprecipitated using a polyclonal antibody against D₂DR and resolved by SDS electrophoresis. Proteins were transferred to nitrocellulose membranes and A_2AAR IB was performed using a polyclonal antibody against A_2AAR. In control A_2AAR-transfected cells, but not in wild-type cells, A_2AARs were detected following immunoprecipitation with the A_2A AR antibody. Immunoreactive proteins were visualised by chemioluminescence (a). Densitometric analysis of A_2A AR immunoreactive bands (b)

Fig. 2

Effect of different haloperidol or clozapine concentrations on [³H] NECA-specific binding of A_2AAR. CHO cells were treated for 24 h with medium alone or haloperidol or clozapine (1 nM–10 μM) and then [³H]NECA binding assays were performed. Data are expressed as the percentage of [³H]NECA-specific binding with respect to untreated control cells (100%) and represent the mean ± SEM of three different experiments

Fig. 3

Effect of antipsychotic treatment on [³H] NECA binding parameters of A_2AAR. CHO cells were treated for 1–24 h with medium alone (a), 1 μM haloperidol (b) or 1 μM clozapine (c), and then [³H]NECA competitive binding experiments were performed. Graphs show a representative Scatchard plot of [³H] NECA competitive binding data obtained from three experiments yielding similar results

Fig. 4

Effect of antipsychotic treatment on A_2AAR functional responsiveness. CHO cells were treated for 24 h with medium alone (a), with 1 μM haloperidol (b) or with 1 μM clozapine (c), and then the ability of different NECA concentrations to stimulate cAMP accumulation was evaluated. Data are expressed as picomoles per milligram proteins per minute and represent the mean ± SEM of four different experiments