Analysis of [H-3] bremazocine binding in single and combinatorial opioid receptor knockout mice

Despite ample pharmacological evidence for the existence of multiple mu-. d elta- and kappa -opioid receptor subtypes. only three genes encoding mu-(MO R), delta-(DOR) and kappa-(KOR) opioid receptor have been cloned. The KOR g ene encodes kappa (1)-sites. which specifically bind arylacetamide compound s, and the possible existence of kappa -opioid receptor subtypes derived fr om another kappa -opioid-receptor gene, yet to be characterized, remains a very contentious issue. kappa (2)-Opioid receptors are described as binding sites typically labelled by the non-selective benzomorphan ligand [H-3]bre mazocine in the presence of mu-, delta- and kappa (1)-opioid receptor block ing ligands. To investigate the genetic origin of kappa (2)-opioid receptor s, we have carried out homogenate binding experiments with [H-3]bremazocine in brains of single MOR-, DOR-, KOR- and double MOR/DOR-deficient mice. Sc atchard analysis showed that 68 +/- 12% of the binding sites arise from the MOR gene, 27 +/- 1% from the DOR gene and 14.5 +/- 0.2% from the KOR gene, indicating that the three known genes account for total [H-3]bremazocine b inding. Experiments in the presence of mu-. delta- and kappa (1)-opioid rec eptor suppressor ligands further showed that non-kappa (1)-opioid receptor labelling can be accounted for by binding to both the mu- and delta -opioid receptors. Finally, [H-3]bremazocine binding experiments performed on brai n membranes from the triple MOR/DOR/KOR-deficient mice revealed a complete absence of binding sites, confirming definitively that no additional gent: is required to explain the total population of [H-3]bremazocine binding sit es. Altogether the data show that the putative kappa (2)-opioid receptors a re in fact a mixed population of KOR, DOR and predominantly MOR gene produc ts. (C) 2001 Elsevier Science B.V. All rights reserved.