Labelling of A1 and A3 adenosine receptors with the new selective A1 agonist 2'-Me-CCPA

Brain is rich of adenosine receptors. Radiolabelled adenosine receptor agonists such as [3H]N6-cyclohexyladenosine ([3H]CHA) and [3H]phenylisopropyladenosine were largely used for labelling adenosine A1 receptor sites. Unfortunately, these radioligands label both A1 and A3 receptors. Hence, the majority of studies performed so far did not discriminate between these adenosine receptor subtypes. Some studies have introduced non labelled adenosine A3 receptor antagonists to discriminate between the 2 sites, whereas no highly selective agonists allowing such a discrimination are available. In this study we propose the use of 2'-C-methyl-2-chloro-N6-cyclopentyladenosine (2'-Me-CCPA) to discriminate A1 and A3 adenosine receptors in sections of rat cerebral cortex and hippocampus. This compound synthesized by us proved to be a potent and selective agonist at A1 receptors active in the nanomolar range. It has affinity for A2 and A3 receptors in the micromolar range only. In sections of rat frontal and occipital cortex and hippocampus exposed to [3H]CHA in the presence or in the absence of different adenosine receptor agonists, [3H]CHA was bound with high affinity, in a manner consistent with the labelling of adenosine receptors. The highest density of binding sites was found in the occipital cortex, followed by the frontal cortex and hippocampus. Concentrations of 2'-Me-CCPA between 0.1 and 10 nM labelled almost all adenosine A1 receptors. The compound reduced by about 70%, 50% and 25% [3H]CHA binding to frontal cortex, occipital cortex and hippocampus, respectively. These findings indicate that in the frontal cortex the majority of [3H]CHA binding belongs to the A1 receptor subtype, in the occipital cortex A1 and A3 receptors are expressed in similar percentages, whereas in the hippocampus the A3 receptor predominates. Based on these results we suggest that 2'-Me-CCPA may represent an adenosine receptor agonist suitable for discriminating A1 and A3 receptors.