Characterization of kappa opioid binding using dynorphin A1-13 and U69,593 in the rat brain

Previous studies of kappa opioid binding sites have suggested heterogeneous binding to this class of opioid receptors. To further investigate kappa receptor heterogeneity, we analyzed the binding properties of various "kappa-selective" ligands in rat brain homogenates. Displacement assays were carried out using [3H]bremazocine in the presence of various displacing ligands under mu and delta receptor-blocked conditions. Homologous displacement of [3H]bremazocine produced "shallow" displacement which best fit a two-site model of drug-receptor interaction. Dynorphin A1-13 and U69,593 exhibited similar biphasic displacement of [3H]bremazocine. Maximal displacement by these ligands, however, represented only approximately 55% of total [3H]bremazocine binding, which suggests the existence of a third component of [3H]bremazocine binding. Biphasic displacement by dynorphin A1-13 was detected in tissue throughout the brain and the spinal cord, whereas the dynorphin-resistant component of [3H]bremazocine binding was uniquely absent in the spinal cord. U50,488H, tifluadom and ethylketocyclazocine appeared to displace from additional, dynorphin-insensitive sites, as their maximal displacement exceeded that seen with either dynorphin A1-13 or U69,593. These results strongly suggest the existence of at least three components of non-mu, non-delta [3H]bremazocine binding in the rat brain: two with differential affinity for dynorphin A1-13 and U69-593 (kappa-1 and kappa-2 sites), and a third (termed here R1) that was further resolved into two binding sites by bremazocine. Preliminary analysis of the R1 component using naloxone revealed one high-affinity site, which may be opiate in nature, and a second site whose binding properties closely resemble those of the sigma receptor described by others.