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Aims Atrial fibrillation (AF) is associated with abnormal sarcoplasmic reticulum (SR) calcium release, which is promoted by adenosine A(2A) receptor (A(2A)R) activation. Here, we tested the hypothesis that abnormal calcium release in AF is linked to A(2A)R remodelling. Methods and results Western blotting and quantitative real-time PCR were used to determine A(2A)R mRNA and protein levels in right atrial samples from patients with and without AF. Effects of A(2A)R activation on calcium handling were assessed with patch-clamp technique and confocal calcium imaging. A(2A)R mRNA levels and functional A(2A)Rs were moderately up-regulated in patients with atrial dilation and markedly up-regulated in those with AF. Accordingly, A(2A)R stimulation significantly increased ryanodine receptor phosphorylation in AF patients, and spontaneous calcium waves increased moderately in myocytes from patients with atrial dilation and strongly in patients with AF (2.2 +/- 2.1 to 14.3 +/- 8.8 min(-1), n = 6, P = 0.01). Moreover, the high baseline level of calcium waves in AF was reduced by A(2A)R antagonists (3.5 +/- 2.0 to 1.3 +/- 1.3 min(-1), n = 6, P = 0.007) or adenosine deaminase (1.7 +/- 1.5 to 0.5 +/- 0.6 min(-1), n = 10, P = 0.02) suggesting that A(2A)Rs are activated by endogenous adenosine. Indeed, intracellular perfusion with adenosine significantly increased the calcium wave frequency (1.1 +/- 0.8 to 8.2 +/- 3.3 min(-1), n = 8), whereas adenosine removal from the cytosol decreased it (2.1 +/- 0.9 to 0.3 +/- 0.3 min(-1), n = 8, P = 0.04). Conclusions Atrial fibrillation patients show increased A(2A)R expression that may account for the high baseline level of spontaneous SR calcium release seen in myocytes from these patients, and the ability of A(2A)R antagonists to reduce this abnormal calcium release points to the A(2A)R as a novel molecular target in AF.

Functional interactions between the G protein-coupled dopamine D1 and histamine H3 receptors have been described in the brain. In the present study we investigated the existence of D1-H3 receptor heteromers and their biochemical characteristics.D1-H3 receptor heteromerization was studied in mammalian transfected cells with Bioluminescence Resonance Energy Transfer and binding assays. Furthermore, signalling through mitogen-activated protein kinase (MAPK) and adenylyl cyclase pathways was studied in co-transfected cells and compared with cells transfected with either D1 or H3 receptors.Bioluminescence Resonance Energy Transfer and binding assays confirmed that D1 and H3 receptors can heteromerize. Activation of histamine H3 receptors did not lead to signalling towards the MAPK pathway unless dopamine D1 receptors were co-expressed. Also, dopamine D1 receptors, usually coupled to G(s) proteins and leading to increases in cAMP, did not couple to G(s) but to G(i) in co-transfected cells. Furthermore, signalling via each receptor was blocked not only by a selective antagonist but also by an antagonist of the partner receptor.D1-H3 receptor heteromers constitute unique devices that can direct dopaminergic and histaminergic signalling towards the MAPK pathway in a G(s)-independent and G(i)-dependent manner. An antagonist of one of the receptor units in the D1-H3 receptor heteromer can induce conformational changes in the other receptor unit and block specific signals originating in the heteromer. This gives rise to unsuspected therapeutic potentials for G protein-coupled receptor antagonists.

The results presented in this paper show that adenosine A2A receptor (A2AR) form homodimers and that homodimers but not monomers are the functional species at the cell surface. Fluorescence resonance energy transfer (FRET) and bioluminescence resonance energy transfer (BRET) techniques have been used to demonstrate in transfected HEK293 cells homodimerization of A2AR, which are heptaspanning membrane receptors with enriched expression in striatum. The existence of homodimers at the cell surface was demonstrated by time-resolved FRET. Although agonist activation of the receptor leads to the formation of receptor clusters, it did not affect the degree of A2AR-A2AR dimerization. Both monomers and dimers were detected by immunoblotting in cell extracts. However, cell surface biotinylation of proteins has made evident that more than 90% of the cell surface receptor is in its dimeric form. Thus, it seems that homodimers are the functional form of the receptor present on the plasma membrane. A deletion mutant version of the A2A receptor, lacking its C-terminal domain, was also able to form both monomeric and dimeric species when cell extracts from transfected cells were analyzed by immunoblotting. This suggests that the C-terminal tail does not participate in the dimerization. This is relevant as the C-terminal tail of A2AR is involved in heteromers formed by A2AR and dopamine D2 receptors. BRET ratios corresponding to A2AR-A2AR homodimers were higher than those encountered for heterodimers formed by A2AR and dopamine D2 receptors. As A2AR and dopamine D2 receptors do indeed interact, these results indicate that A2AR homodimers are the functional species at the cell surface and that they coexist with A2AR/D2 receptor heterodimers.