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

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 [S]GTPgS binding to CHO cell membranes were examined. The presence of GDP and Mg was essential for the kappa agonist (2)-U50,488H-induced increase in [S]GTPgS binding to be observed and the optimal concentration was 3 mM and 5 mM, respectively. The presence of 100 mM Na was necessary to produce the maximal signal-tobackground ratio. (2)U50,488H-induced increase in [S]GTPgS binding was timeand tissue concentration-dependent. (2)U50,488H increased [S]GTPgS binding in a dose-dependent manner with an EC50 of 3.1 nM. (1)-U50,488H had no effect, which indicates that this effect is stereospecific. Naloxone (1 mM) or norbinaltorphimine (10 nM) shifted the dose-response curve of (2)-U50,488H to the right by 100-fold. These results indicate that enhancement of [S]GTPgS binding by (2)-U50,488H is a kappa receptor-mediated event. Pretreatment of the cells with pertussis toxin, but not cholera toxin, abolished the (2)-U50,488H-induced increase in [S]GTPgS binding, which indicates the involvement of Gi and/or Go proteins. [ S]GTPgS binding induced by (2)U50,488H had a Kd value of 0.34 6 0.08 nM and a Bmax value of 431 6 29 fmol/mg protein. The rank order of potencies of opioid ligands tested in stimulating [S]GTPgS binding was dynorphin A 1–17 . (6)-ethylketocyclazocine . b-funaltrexamine, (2)U50,488H, tifluadom . nalorphine . pentazocine, nalbuphine . buprenorphine. Dynorphin A 1–17, (6)-ethylketocyclazocine, (2)U50,488H, tifluadom and b-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 [S]GTPgS 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. Opioid receptors play important roles in many physiological functions. The presence of multiple types of opioid receptors, at least mu, delta, kappa and epsilon, in the nervous system has been established by pharmacological and binding studies as well as differential anatomical localization in the brain (for a review, Chang, 1984). Activation of kappa opioid receptors produces many effects including analgesia (Von Voigtlander et al., 1983; Dykstra et al., 1987), dysphoria (Pfeiffer et al., 1986) and water diuresis (Leander, 1983a; Dykstra et al., 1987). Dynorphins are thought to be endogenous ligands for kappa receptors (Chavkin and Goldstein, 1981). (2)-U50,488H is the prototype of selective kappa agonists (Von Voigtlander et al., 1983), whereas norbinaltorphimine is a selective kappa antagonist (Portoghese et al., 1987). There is ample evidence supporting the notion that the kappa opioid receptor belongs to the superfamily of GPCRs. Activation of kappa opioid receptors leads to inhibition of adenylate cyclase (Attali et al., 1989; Konkoy and Childers, 1989, 1993; Lawrence and Bidlack, 1993; Lawrence et al., 1995; Prather et al., 1995), activation of low-Km GTPase (Clark et al., 1986; Clark and Medzihradsky, 1987; Lawrence et al., 1995), enhancement of incorporation of [P]azidoanilido-GTP into Ga subunits (Prather et al., 1995), increase in inward-rectifying K conductance (Ma et al., 1995; Henry et Received for publication January 7, 1997. 1 This work was supported in part by a grant from National Institute on Drug Abuse (DA 04745). J.Z. was supported by a training grant from National Institute on Drug Abuse (T32 DA07237). ABBREVIATIONS: CHO cells, Chinese hamster ovary cells; CHO-hkor cells, Chinese hamster ovary cells stably expressing human k opioid receptor; DAMGO, Try-D-Ala-Gly-(Me)Phe-Gly-ol; DPDPE, Tyr-D-Pen-Gly-Phe-D-Pen-OH; EDTA, ethylenediaminetetraacetic acid; G protein, guanosine triphosphate-binding regulatory protein; EGTA, ethylene glycol-bis(b-aminoethyl ether)-N,N,N9,N9-tetraacetic acid; GDP, guanosine diphosphate; GPCRs, G protein-coupled receptors; GTPgS, guanosine-59-O-(3-thio)triphosphate; (2)-U50,488H, (trans)-3,4-dichloro-N-methylN-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide; hkor, human k opioid receptor; HEPES, N-2-hydroxyethylpiperazine-N9-2-ethanesulfonic acid; TEL buffer, 50 mM Tris-HCl buffer, 1 mM EGTA and 10 mM leupeptin, pH 7.5. 0022-3565/97/2822-0676$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 282, No. 2 Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 282:676–684, 1997 676 at A PE T Jornals on A uust 9, 2017 jpet.asjournals.org D ow nladed from al., 1995; Grudt and Williams, 1993) and decrease in Ca conductance through N-type channels (Gross and MacDonald, 1987). Kappa agonist-induced inhibition of adenylate cyclase and increase in K conductance were shown to be mediated through pertussis toxin-sensitive G proteins (Lawrence and Bidlack, 1993; Prather et al., 1995; Ma et al., 1995). After the cloning of the mouse delta opioid receptor (Kieffer et al., 1992; Evans et al., 1992), we (Zhu et al., 1995) and Mansson et al. (1994) reported cloning of the human kappa opioid receptor. Hydropathy analysis of the amino acid sequence shows the presence of seven putative transmembrane domains, a structural motif common to all G protein-coupled