Vol. 282, Issue 2, 676-684, 1997

Activation of the Cloned Human Kappa Opioid Receptor by Agonists Enhances [³⁵S]GTPS Binding to Membranes: Determination of Potencies and Efficacies of Ligands¹

Jinmin Zhu, Lai-Yi Luo, Jian-Guo Li, Chongguang Chen and Lee-Yuan Liu-Chen

Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania

Activation of kappa receptors inhibits adenylate cyclase, enhances K⁺ conductance and reduces Ca⁺⁺ conductance via pertussis toxin-sensitive G proteins. We recently cloned a human kappa opioid receptor and stably expressed it in Chinese hamster ovary (CHO) cells. In this study, the effects of activation of the human kappa receptor by agonists on [³⁵S]GTPS binding to CHO cell membranes were examined. The presence of GDP and Mg⁺⁺ was essential for the kappa agonist ()-U50,488H-induced increase in [³⁵S]GTPS binding to be observed and the optimal concentration was 3 µM and 5 mM, respectively. The presence of 100 mM Na⁺ was necessary to produce the maximal signal-to-background ratio. ()U50,488H-induced increase in [³⁵S]GTPS binding was time- and tissue concentration-dependent. ()U50,488H increased [³⁵S]GTPS binding in a dose-dependent manner with an EC₅₀ of 3.1 nM. (+)-U50,488H had no effect, which indicates that this effect is stereospecific. Naloxone (1 µM) or norbinaltorphimine (10 nM) shifted the dose-response curve of ()-U50,488H to the right by 100-fold. These results indicate that enhancement of [³⁵S]GTPS binding by ()-U50,488H is a kappa receptor-mediated event. Pretreatment of the cells with pertussis toxin, but not cholera toxin, abolished the ()-U50,488H-induced increase in [³⁵S]GTPS binding, which indicates the involvement of G_i and/or G_o proteins. [³⁵S]GTPS binding induced by ()-U50,488H had a K_d value of 0.34 ± 0.08 nM and a B_max value of 431 ± 29 fmol/mg protein. The rank order of potencies of opioid ligands tested in stimulating [³⁵S]GTPS binding was dynorphin A 1-17 > (±)-ethylketocyclazocine > -funaltrexamine, ()-U50,488H, tifluadom > nalorphine > pentazocine, nalbuphine > buprenorphine. Dynorphin A 1-17, (±)-ethylketocyclazocine, ()-U50,488H, tifluadom and -funaltrexamine were full agonists, but nalorphine and pentazocine were partial agonists producing maximal responses of 68% and 23% of those of full agonists, respectively. Nalbuphine and buprenorphine had low levels of agonist activities. Norbinaltorphimine and naloxone were antagonists devoid of activities. Enhancement of [³⁵S]GTPS binding by kappa agonists provides a simple functional measure for receptor activation and can be used for determination of potencies and efficacies of opioid ligands at the kappa receptor.