Apparent heterogeneity of cardiac A1 adenosine receptors as revealed by radioligand binding experiments on N-ethylmaleimide-treated membranes

Abstract

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 KL and KH 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: Gi/Go 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 Gi, and perhaps to Go, we have performed A1 adenosine receptor radioligand "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 KL 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.(ABSTRACT TRUNCATED AT 250 WORDS)

Fig. 1

A–C. Inhibition curves for antagonist radioligand binding by R-RIA (circles) and NECA (triangles) to sites in cortical (open symbols) and cardiac (closed symbols) membranes. Panel A [³H]XAC, control membranes. Panel B [³H]XAC, NEM-pretreated membranes in presence of 0.1 mM Gpp(NH)p. Panel C [³H]CPX: NEM-pretreated membranes in presence of 0.1 mM Gpp(NH)p. Points shown are averages of duplicate determinations. Nonspecific binding as defined by theophylline (5 mM) is substracted from all data. The two site fits are shown as solid tines. One site fits are shown as – – – (heart) or ⋯ (Brain)