Evaluation of the binding of the A-1 selective adenosine radioligand, cyclopentyladenosine (CPA), to rat brain tissue

Abstract

Summary The binding of [³H]-Cyclopentyladenosine (CPA), an N⁶-substituted analog of adenosine, was examined in vitro. CPA bound with high affinity (K _d=0.48 nmol/l) to rat brain membranes. Specific binding, which represented 90–97% of the total counts bound at a ligand concentration of 1 nmol/l, was saturable, reversible and sensitive to protein denaturation. The pharmacology of binding was consistent with the labeling of an A-1 receptor, the R- and S-diastereomers of N⁶-phenylisopropyladenosine (PIA) showing a sixteenfold difference in their ability to displace CPA. The prototypic A-1 selective adenosine agonist, N⁶-cyclohexyladenosine (CHA) was twofold less active than CPA in displacing radiolabeled CPA. Comparison of the ability of cold CHA and CPA to displace [³H]-CPA gave rat dissociation constants of 1.88 and 1.80×10⁴ s⁻¹, respectively suggesting that both CHA and CPA bound to the same recognition site. In contrast however, comparison of the binding of [³H]-CPA with that of [³H]-CHA showed distinct differences. The K _d for CHA was approximately twice that of CPA while the apparent B _max was 60% greater. In comparing the pharmacology of CPA binding with that of CHA, it was found that CHA, S-PIA and the antagonist, PACPX were more active in displacing CHA than CPA. In general however, CPA has a binding profile very similar to that observed with CHA.