EVIDENCE THAT DRUG-BINDING TO NONADRENERGIC [H-3] IDAZOXAN BINDING-SITES (I-RECEPTORS) OCCURS TO INTERACTING OR INTERCONVERTIBLE AFFINITY FORMS OF THE RECEPTOR

Characterization of [H-3]idazoxan binding to guinea pig kidney membranes showed that approximately 90% bound to non-adrenergic I-receptors and approximately 10% to alpha(2)-adrenoceptors. I-Receptors could be studied separately by including 3-mu-M rauwolscine to the assay. During these conditions 22 different compounds out of 29 (including imidazoline and guanidinium compounds) generated biphasic or shallow competion curves (Hill coefficients down to 0.57), for which computer modelling suggested that drugs bound to two sites with different affinities. However, the proportion of sites varied considerably depending on which drug was used as competitor; the variation being from approximately 50/50% to approximately 8/92% and analysis of variance clearly indicated that the variation of proportions of sites could be attributed to an effect induced by the drugs which indicated that the sites were dynamically formed or modulated by the presence of the drug. A few drugs (guanabenz, (-)-medetomidine, phentolamine, clemastin, prazosin and idazoxan itself) yielded steep uniphasic curves (Hill coefficients near unity) which were resolved only into one site fits. One drug (detomidine) yielded supersteep competition curves (Hill coefficient 1.29), the data being reminiscent of positive cooperativity. In another set of experiments attempts were made to block one of the affinity forms of the I-receptor with histamine, a compound which had grossly different affinities for the two I-receptor sites, and competition curves were then obtained using other drugs which also seemed to distinguish between the two sites. Computer analysis of experimentally obtained as well as simulated, computer generated, competition curves clearly showed that the kidney I-receptors did not exist in two independent non-interacting forms. Instead the data suggested that the I-receptor was composed of interacting or interconverting receptor entities with different affinities for drugs. Monovalent cations such as Cs+ or NH4+ were found to be powerful inhibitors of [H-3]idazoxan binding to the kidney I-receptors, the ions decreasing both the apparent B(max) and affinity of the ligand for the receptor. The cations also decreased the affinities of UK-14,304 for both the high and low affinity forms of the I-receptor, but the apparent proportions of sites were not at all affected by the ions. Moreover, ligand binding to kidney I-receptor were not at all affected by non-hydrolyzable guanine nucleotides. It is suggested that the binding data reflects functional properties of the I-receptor protein.