Allosteric interactions between agonists and antagonists within the adenosine A2A receptor-dopamine D2 receptor heterotetramer.

Adenosine A2A receptor (A2AR)-dopamine D2 receptor (D2R) heteromers are key modulators of striatal neuronal function. It has been suggested that the psychostimulant effects of caffeine depend on its ability to block an allosteric modulation within the A2AR-D2R heteromer, by which adenosine decreases the affinity and intrinsic efficacy of dopamine at the D2R. We describe novel unsuspected allosteric mechanisms within the heteromer by which not only A2AR agonists, but also A2AR antagonists, decrease the affinity and intrinsic efficacy of D2R agonists and the affinity of D2R antagonists. Strikingly, these allosteric modulations disappear on agonist and antagonist coadministration. This can be explained by a model that considers A2AR-D2R heteromers as heterotetramers, constituted by A2AR and D2R homodimers, as demonstrated by experiments with bioluminescence resonance energy transfer and bimolecular fluorescence and bioluminescence complementation. As predicted by the model, high concentrations of A2AR antagonists behaved as A2AR agonists and decreased D2R function in the brain.

Dopamine D2 and adenosine A2A receptors regulate NMDA-mediated excitation in accumbens neurons through A2A-D2 receptor heteromerization.

Bursting activity of striatal medium spiny neurons results from membrane potential oscillations between a down- and an upstate that could be regulated by G-protein-coupled receptors. Among these, dopamine D(2) and adenosine A(2A) receptors are highly enriched in striatal neurons and exhibit strong interactions whose physiological significance and molecular mechanisms remain partially unclear. More particularly, respective involvements of common intracellular signaling cascades and A(2A)-D(2) receptor heteromerization remain unknown. Here we show, by performing perforated-patch-clamp recordings on brain slices and loading competitive peptides, that D(2) and A(2A) receptors regulate the induction by N-methyl-D-aspartate of a depolarized membrane potential plateau through mechanisms relying upon specific protein-protein interactions. Indeed, D(2) receptor activation abolished transitions between a hyperpolarized resting potential and a depolarized plateau potential by regulating the Ca(V)1.3a calcium channel activity through interactions with scaffold proteins Shank1/3. Noticeably, A(2A) receptor activation had no effect per se but fully reversed the effects of D(2) receptor activation through a mechanism in which A(2A)-D(2) receptors heteromerization is strictly mandatory, demonstrating therefore a first direct physiological relevance of these heteromers. Our results show that membrane potential transitions and firing patterns in striatal neurons are tightly controlled by D(2) and A(2A) receptors through specific protein-protein interactions including A(2A)-D(2) receptors heteromerization.

Expression of adenosine A 2A receptors in the rat lumbar spinal cord and implications in the modulation of N-methyl-d-aspartate receptor currents.

BACKGROUND: The presence of A(2A) receptors in the dorsal horn of the spinal cord remains controversial. At this level, activation of N-methyl-d-aspartate (NMDA) receptors induces wind-up, which is clinically expressed as hyperalgesia. Inhibition of NMDA receptor currents after activation of A(2A) receptors has been shown in rat neostriatal neurons. In this study, we sought to establish the presence of adenosine A(2A) receptors in the lamina II of the rat lumbar dorsal horn neurons and investigated whether the activation of A(2A) receptors is able to modulate NMDA receptor currents. METHODS: Experiments were conducted in the rat lumbar spinal cord. The presence of adenosine A(2A) receptor transcripts inside the lumbar spinal cord is assessed with the reverse transcriptase polymerase chain reaction (RT-PCR) technique. Western blot experiments are performed at the same level. The RT-PCR technique is also performed specifically in the lamina II, and the presence of adenosine A(2A) receptor transcripts is assessed in neurons from the lamina II with the single-cell RT-PCR technique. The effect of adenosine A(2A) receptor activation on NMDA receptor currents is studied by the whole-cell configuration of the patch clamp technique. RESULTS: RT-PCR performed on the lumbar spinal cord revealed the presence of adenosine A(2A) receptor transcripts. Western blot experiments revealed the presence of A(2A) receptors in the lumbar spinal cord. RT-PCR performed on the substantia gelatinosa also revealed the presence of adenosine A(2A) receptor transcripts. Finally, single cell RT-PCR revealed the presence of adenosine A(2A) receptor transcripts in a sample of lamina II neurons. Patch clamp recordings showed an inhibition of NMDA currents during the application of a selective A(2A) agonist. CONCLUSIONS: These results demonstrate the presence of A(2A) receptor on neurons from the substantia gelatinosa of the rat lumbar dorsal horn and the inhibition of NMDA-induced currents by the application of a selective A(2A) receptor agonist. Therefore, A(2A) receptor ligands could modulate pain processing at the spinal cord level.

Activation of protein kinase C and inositol 1,4,5-triphosphate receptors antagonistically modulate voltage-gated sodium channels in striatal neurons.

Regulation of voltage-gated sodium channels is crucial to firing patterns that constitute the output of medium spiny neurons (MSN), projecting neurons of the striatum. This modulation is thus critical for the final integration of information processed within the striatum. It has been shown that the adenylate cyclase pathway reduces sodium currents in MSN through channel phosphorylation by cAMP-dependent protein kinase. However, it is unknown whether a phospholipase C (PLC)-mediated signaling cascade could also modulate voltage-gated sodium channels within MSN. Using the whole-cell patch clamp technique, we investigated the effects of activation of two key components in PLC-mediated signaling cascades: protein kinase C (PKC) and inositol-1,4,5-triphosphate (IP(3)) receptors on voltage-dependent sodium current. Cellular dialysis with phorbol 12-myristate 13-acetate, an activator of PKC, significantly reduced peak sodium current amplitude, while adenophostin A, an activator of IP(3) receptors, significantly increased peak sodium current amplitude. This effect of adenophostin was abolished by calcium chelation or by FK506, an inhibitor of calcineurin. These results suggest an antagonistic role of PKC and IP(3) in the modulation of striatal voltage-gated sodium channels, peak current amplitude being decreased through phosphorylation by PKC and increased through dephosphorylation by calcineurin.