Autoradiographic evidence for multiple CNS binding sites for adenosine derivatives.

The binding of the adenosine analogues, 5'-N-ethylcarboxamido[3H]adenosine and N6-cyclohexyl[3H]adenosine, to the rat brain was examined utilizing light-microscopic autoradiographic techniques. While associated with many of the same structures, binding sites for these compounds showed distinct differences in both their patterns of distribution and their capacities to be inhibited by the adenosine analogue, R-phenylisopropyladenosine. Previous studies have shown that, at nanomolar concentrations, cyclohexyladenosine and R-phenylisopropyladenosine bind rather exclusively to the A1 type of adenosine receptors. In contrast, 5'-N-ethylcarboxamidoadenosine has almost equal affinity for A1 and A2 sites. Taking advantage of these characteristics, non-A1 binding sites were resolved by examining 5'-N-ethylcarboxamidoadenosine binding in the presence of micromolar concentrations of unlabeled R-phenylisopropyladenosine. The autoradiographically demonstrated distribution of R-phenylisopropyladenosine-insensitive 5'-N-ethylcarboxamidoadenosine binding sites differed significantly from that of the cyclohexyladenosine binding sites. Such non-A1 binding sites were concentrated in the striatum, nucleus accumbens, medial geniculate, olfactory tubercle, amygdala and certain thalamic nuclei. In contrast to the distribution of A1 adenosine receptor sites, R-phenylisopropyladenosine-insensitive 5'-N-ethylcarboxamidoadenosine binding was only low to moderate in the hippocampus, cerebellum and superior colliculus, regions which are strongly positive for cyclohexyladenosine binding. The present study provides the first autoradiographic evidence for multiple adenosine binding sites in the brain by demonstrating that the adenosine analogue 5'-N-ethylcarboxamidoadenosine can bind to a site or sites distinct from the A1 adenosine receptor site. The 5'-N-ethylcarboxamidoadenosine binding site which is not displaced by low concentrations of R-phenylisopropyladenosine may correspond to an A2 adenosine receptor site and/or an as yet uncharacterized type of adenosine receptor.