Abstract
Adenosine and its analogs exert negative inotropic, chronotropic and dromotropic effects in a variety of cardiac preparations. The cardioinhibitory effects of adenosine are mediated through an interaction with adenosine receptors of the A_1 subtype. The pharmacological profile of the adenosine receptor present in porcine atrial membranes, as labeled by (-)-N^6 -[^^125 I]-p-hydroxyphenylisopropyl-adenosine ([^^125 I]HPIA), is of the A_1 subtype (Leid et al., Mol. Pharmacol. 34, 334, 1988). To further define the molecular properties of the cardiac A_1 adenosine receptor and as an obligatory first step toward purification, it is necessary to solubilize the receptors from myocyte membranes in an active form. We have characterized the solubilized A_1 adenosine receptor from porcine atria with respect to its interactions with adenosine analogs and modulation by guanine nucleotides. The A_1 adenosine receptor was solubilized from a porcine atrial P3 preparation in 0.8% w/v digitonin and 0.16% w/v cholate buffer. Using a dual extraction procedure the cardiac A_1 adenosine receptors were solubilized in approximately 50% yield (in [^^125 I]HPIA sites). Adenosine deaminase-treated extracts were used directly in radioligand binding experiments. [^^125 I]HPIA binding experiments were carried out at 37℃ in a volume of 95 ul. Bound [^^125 I]HPIA was separated from free radioligand by rapid filtration over glass fiber filters that had been presoaked in a 0.5%, w/v, polyethyleneimine. [^^125 I]HPIA labeled an apparently homogenous population of solubilized recognition sites with a K_D of 1.4±0.1 nM and a Bmax of 88±4 fmol/mg protein. The use of this mixed-detergent system resulted in a 2.5 fold enrichment of A_1 adenosine receptor specific activity. The rank order potency of adenosine receptor agonists as inhibitors of [^^125 I]HPIA binding was consistent with the labeling of an A_1 receptor, demonstrating that the pharmacological signature of the recognition site was retained in the solubilized preparation. Kinetic studies suggested that the association of [^^125 I]HPIA with the solubilized porcine atrial A_1 receptor was consistent with that of a simple bimolecular reaction. The interaction of the cardiac A_1 adenosine receptor with guanine nucleotide binding proteins was preserved in this detergent extract. Addition of guanosine-5'-0-(3-thio)triphosphate to an equilibrated mixture of solubilized porcine atrial A_1 adenosine receptors and [^^125 I]HPIA effected a rapid and complete dissociation of the radioligand. This dissociation was resolved into two kinetic phases which appear to arise from two populations of independent, noninterconvertible receptor-G protein complexes with distinct sensitivities to guanine nucleotides. This interpretation is consistent with the observation of Linden et al. (in Purines in Cellular Signaling： Targets for New Drugs, Springer, in press) that multiple G-protein speices including α_o , α_i and α_i3 copurify with A_1 adenosine receptors on an agonist affinity gel.