Apparent heterogeneity of cardiac A 1 adenosine receptors as revealed by radioligand binding experiments on N-ethylmaleimide-treated membranes

Summary While G protein-coupled receptors are often studied by analyzing antagonist radioligand: cold agonist inhibition curves using an independent site model, it is now clear that K_L and K_H values determined in these analyses are not reliable estimates of the affinities of the agonists for free and G protein-coupled forms of the receptor. Thus, such experiments cannot be used to contrast the characteristics of a given type of receptor in different tissues, i.e., to probe for the existence of receptor subtypes. Since treatment with N-ethylmaleimide treatment blocks receptor: G_i/G₀ protein interactions, such analyses on N-ethylmaleimide-pretreated membranes should allow direct assessment of the affinities of competing ligands for the free receptor or for multiple receptor subtypes.As A₁ adenosine receptors couple to G_i, and perhaps to G_o, we have performed A₁ adenosine receptor radio-ligand competition studies first on control, then on N-ethylmaleimide-pretreated bovine cardiac and cerebral cortical membranes. Results of experiments with the antagonist radioligand ³H]xanthine amine congener appeared to be confounded by ligand binding to A₂ adenosine receptors present in the cardiac membrane preparations. Further experiments utilized the A₁-specific radioligand ³H] 1,3-dipropyl-8-cyclopentylxanthine. These experiments confirmed once more that the K_L values determined by computer analysis of competition curves performed on control membranes are not reliable estimates of the affinities of the competing ligand for free receptors. Furthermore the results supported the hypothesis that similar analyses on NEM-treated membranes provide reliable estimates of the affinity(s) of competing ligands for free receptors. Lastly, the results suggest that cardiac membranes contain two subtypes of A₁ adenosine receptors that are differentiated by 5-modified but not N⁶-modified adenosine analogs. One of these receptor subtypes appears to be the same as the A₁ receptor detected in cortical membranes.Abbreviations ¹²⁵I]ABA 1251](N⁶-p-aminobenzyl)adenosine - ³H]CPX ³H]8-cyclopentyl-1,3-dipropylxanthine - ³H]R-PIA ³H]N⁶-R-phenylisopropyladenosine - ⁶H]XAC ⁶H]xanthine amine congener - CHA N⁶-cyclohexyladenosine - EDTA ethylenediaminetetraacetic acid - ¹²⁵I]BW-A844U ¹²⁵I]3-(4-amino)phenethyl-l-propyl-8-cyclopentylxanthine - NCCA 5-N-cyclopropylcarboxamide adenosine - NECA 5-N-ethylcarboxamide adenosine - PMSF phenylmethylsulphonyl fluoride - NEM N-ethylmaleimide Recipient of a Postdoctoral Fellowship from the Chicago Heart Association