κ Opioid receptor stimulation of [35S] GTPγS binding in guinea pig brain

Abstract Although only one gene for κ opioid receptors has been cloned to date, κ 1 and κ 2 receptors have been defined pharmacologically, with drugs such as bremazocine binding to both putative κ receptor subtypes. To examine whether κ receptor subtypes can be distinguished at the level of the G-protein, the ability of the κ 1 agonist ( trans -(dl)-3,4-dichloro- N -methyl- N -[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide) methane sulfonate (U-50488H) to stimulate [ 35 S]guanosine-5′-O-(γ-thio)-triphosphate ([ 35 S]GTPγS) binding in guinea pig brain was compared with that of bremazocine and dynorphin. In membranes prepared from guinea pig striatum, both bremazocine and U-50488H stimulated [ 35 S]GTPγS binding with the same relative efficacy, while dynorphin produced at least two-fold greater efficacy than the other two agonists. In vitro autoradiography of agonist-stimulated [ 35 S]GTPγS binding revealed similar regional distributions of bremazocine- and U-50488H-activated G-proteins. In striatal membranes, the κ antagonist nor-binaltorphimine (nor-BNI) blocked both bremazocine- and U-50488H-stimulated [ 35 S]GTPγS binding with similar K e values. In agonist additivity experiments, the stimulation of [ 35 S]GTPγS binding by the δ agonist [ d -pen 2,5 , p -Cl-Phe 4 ]enkephalin (p-Cl-DPDPE) was approximately additive with the two κ agonists. Stimulation of [ 35 S]GTPγS binding by the μ agonist [ d -Ala 2 , N -Me 4 , Gly 5 -ol]-enkephalin (DAMGO) was additive with U-50488H, but not with bremazocine, reflecting the μ antagonist properties of this compound. The combination of bremazocine and U-50488H together produced no greater stimulation of binding than either agonist alone, indicating that they were binding to the same site. These results demonstrate that bremazocine and U-50488H activate G-proteins in guinea pig brain through the same receptor, and suggest that κ 2 receptors are not coupled through the same signal transduction mechanisms as κ 1 receptors.