Abstract

A C6 glioma cell line stably transfected with the humankappa opioid receptor (κOR) was used to characterize receptor binding and G protein activation via the κOR by a comprehensive series of opioid ligands. The ligand-binding affinity for [³H]5α,7α,8β(-)-N-methyl-N-(7-Cl-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzene acetamide (U69593) was similar to that observed in monkey brain membranes and was 10-fold lower in the presence of sodium and GDP. Both peptide and nonpeptide agonists maximally stimulated [³⁵S]GTPγS binding. The stimulation of [³⁵S]GTPγS binding was blocked by pretreatment of cells with pertussis toxin. Partial stimulation of [³⁵S]GTPγS binding via the κOR was observed for several ligands that are antagonists at the mu opioid receptor, suggesting an additional mechanism of drug action. The ability of isomers of tifluadom and levallorphan to stimulate [³⁵S]GTPγS binding indicates that the chiral carbon of levallorphan, a benzomorphan derivative, imparts a greater degree of stereoselectivity than does the chiral carbon in the benzodiazepine derivative tifluadom. In addition, (−)tifluadom, the less potent isomer of tifluadom, which is also a γ-aminobutyric acid_A receptor agonist, stimulated [³⁵S]GTPγS binding. In contrast,d-pentazocine, (+)SKF10047, (+)cyclazocine, andd-ethylketocyclazocine displayed no agonist activity. κOR-selective antagonist norbinaltorphimine competitively inhibited the stimulation of [³⁵S]GTPγS binding by the active isomers of ethylketocyclazocine, cyclazocine, and nalorphine to the same degree, indicating that all three ligands are eliciting an effect via the κOR. The results suggest that these cells express a homogeneous population of κOR, and that their [³⁵S]GTPγS-binding properties make them an excellent means to assess κOR efficacy.