Characterization of agonist radioligand interactions with porcine atrial A₁ adenosine receptors

The agonist radioligand (-)-N⁶-[¹²⁵I]-p-hydroxyphenylisopropyladenosine ([¹²⁵I]HPIA) was used to characterize adenosine recognition sites in porcine atrial membranes. [¹²⁵I]HPIA showed saturable binding to an apparently homogeneous population of sites with a maximum binding capacity of 35 ± 3 fmol/mg of protein and an equilibrium dissociation constant of 2.5 ± 0.4 nM. Kinetic experiments were performed to address the molecular mechanism of [¹²⁵I]HPIA binding in porcine atrial membranes. [¹²⁵I]HPIA apparently interacts with the cardiac adenosine receptor in a simple bimolecular reaction. A kinetically derived [¹²⁵I]HPIA dissociation constant (2.4 nM) was in good agreement with that parameter measured at equilibrium. Guanyl nucleotides negatively modulated [¹²⁵I]HPIA binding by increasing its rate of dissociation. This finding is consonant with the formation of a ternary complex in porcine atrial membranes, consisting of ligand, receptor, and guanyl nucleotide-binding protein. Prototypic adenosine receptor agonists and antagonists inhibited specific binding in a manner consistent with the labeling of an A₁ adenosine receptor. The results of these experiments suggest that the adenosine receptor present in porcine atrial membranes, as labeled by [¹²⁵I]HPIA, is of the A₁ subtype.