Abstract
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 kappaopioid receptor and stably expressed it in Chinese hamster ovary (CHO) cells. In this study, the effects of activation of the humankappa receptor by agonists on [35S]GTPγS 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]GTPγS 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]GTPγS binding was time- and tissue concentration-dependent. (−)U50,488H increased [35S]GTPγS 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]GTPγS 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]GTPγS binding, which indicates the involvement of Gi and/or Goproteins. [35S]GTPγS binding induced by (−)-U50,488H had a Kd 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]GTPγS binding was dynorphin A 1–17 > (±)-ethylketocyclazocine > β-funaltrexamine, (−)-U50,488H, tifluadom > nalorphine > pentazocine, nalbuphine > buprenorphine. Dynorphin A 1–17, (±)-ethylketocyclazocine, (−)-U50,488H, tifluadom and β-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]GTPγS binding by kappaagonists 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.
Footnotes
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Send reprint requests to: Dr. Lee-Yuan Liu-Chen, Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140.
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↵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 κ 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(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid
- GDP
- guanosine diphosphate
- GPCRs
- G protein-coupled receptors
- GTPγS
- guanosine-5′-O-(3-thio)triphosphate
- (−)-U50
- 488H, (trans)-3,4-dichloro-N-methyl-N-[2-(1-pyrrolidiny)-cyclohexyl]benzeneacetamide
- hkor
- human κ opioid receptor
- HEPES
- N-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid
- TEL buffer
- 50 mM Tris-HCl buffer, 1 mM EGTA and 10 μM leupeptin, pH 7.5
- Received January 7, 1997.
- Accepted April 21, 1997.
- The American Society for Pharmacology and Experimental Therapeutics
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