Lack of evidence of κ2-selective activation of G-proteins -: κ opioid receptor stimulation of [35S]GTPγS binding in guinea pig brain

Although only one gene for kappa opioid receptors has been cloned to date, kappa(1) and kappa(2) receptors have been defined pharmacologically, with drugs such as bremazocine binding to both putative kappa receptor subtypes. To examine whether kappa receptor subtypes can be distinguished at the level of the G-protein, the ability of the kappa(1) agonist (trans-(dl)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]-benzeneacetamide) methane sulfonate (U-50488H) to stimulate [S-35]guanosine-5'-O-(gamma-thio)-triphosphate ([S-35]GTP gamma S) 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 [S-35]GTP gamma S 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 [S-35]GTP gamma S binding revealed similar regional distributions of bremazocine- and U-50488H-activated G-proteins. In striatal membranes, the kappa antagonist nor-binaltorphimine (nor-BNI) blocked both bremazocine- and U-50488H-stimulated [S-35]GTP gamma S binding with similar K-e values. In agonist additivity experiments, the stimulation of [S-35]GTP gamma S binding by the delta agonist [D-pen(2,5), p-Cl-Phe(4)]enkephalin (p-Cl-DPDPE) was approximately additive with the two kappa agonists. Stimulation of [S-35]GTP gamma S binding by the mu agonist [D-Ala(2), N-Me-4, Gly(5)-ol]-enkephalin (DAMGO) was additive with U-50488H, but not with bremazocine, reflecting the mu 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 kappa(2) receptors are not coupled through the same signal transduction mechanisms as kappa(1) receptors. BIOCHEM PHARMACOL 56;1:113-120, 1998. (C) 1998 Elsevier Science Inc.