title: A kinetic analysis of kappa-opioid agonist binding using the selective radioligand [3H]U69593.
abstract: The interaction of the nonselective opioid ligand [3H]bremazocine and of the kappa-opioid [3H]U69593 with the kappa-receptor was investigated in guinea-pig cortical membranes. Each radioligand bound to a single population of high-affinity sites, although [3H]U69593 apparently recognised only 70% of those sites labelled by [3H]bremazocine. Naloxone and the kappa-selective ligands U69593 and PD117302 exhibited full inhibition of the binding of both radioligands. Kinetic analysis demonstrated biphasic rates of association and dissociation for both [3H]bremazocine and [3H]U69593. Detailed analysis of the binding of [3H]U69593 revealed that the fast rate of association was dependent on radioligand concentration, in contrast to the slow rate, which was independent of ligand concentration. Guanylyl-5'-imidodiphosphate (GppNHp) inhibited binding of [3H]U69593; saturation analysis demonstrated that the inhibitory effects of GppNHp resulted in a decrease in affinity without any significant change in binding capacity. GppNHp attenuated the formation of the slow component of [3H]U69593 binding, while accelerating the fast component. The data are consistent with the formation of a high-affinity complex between the kappa-receptor and a guanine nucleotide binding protein. Guanine nucleotides promote the dissociation of this ternary complex and the stabilisation of a lower-affinity state of the receptor.
authors.affiliation: Parke-Davis Research Unit, Addenbrookes Hospital Site, Cambridge, England.
authors.forename: J A
authors.initials: JA
authors.lastname: Smith
authors.forename: J C
authors.initials: JC
authors.lastname: Hunter
authors.forename: R G
authors.initials: RG
authors.lastname: Hill
authors.forename: J
authors.initials: J
authors.lastname: Hughes
mesh_terms: D000818:Animals; D044482:Benzeneacetamides; D001575:Benzomorphans / Q000378:metabolism*; D001667:Binding, Competitive; D006168:Guinea Pigs; D007700:Kinetics; D008297:Male; D009019:Morphinans / Q000378:metabolism*; D011759:Pyrrolidines / Q000378:metabolism*; D011869:Radioligand Assay; D014316:Tritium
chemical_list: D044482:Benzeneacetamides; D001575:Benzomorphans; D009019:Morphinans; D011759:Pyrrolidines; D014316:Tritium; C027248:bremazocine; C045444:U 69593