Activation of the cloned human kappa opioid receptor by agonists enhances [35S]GTPgammaS 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 [35S]GTPgammaS 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]GTPgammaS binding to be observed and the optimal concentration was 3 microM 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]GTPgammaS binding was time- and tissue concentration-dependent. (-)U50,488H increased [35S]GTPgammaS 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 microM) 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]GTPgammaS 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]GTPgammaS binding, which indicates the involvement of Gi and/or Go proteins. [35S]GTPgammaS binding induced by (-)-U50,488H had a Kd value of 0.34 +/- 0.08 nM and a Bmax value of 431 +/- 29 fmol/mg protein. The rank order of potencies of opioid ligands tested in stimulating [35S]GTPgammaS binding was dynorphin A 1-17 > (+/-)-ethylketocyclazocine > beta-funaltrexamine, (-)-U50,488H, tifluadom > nalorphine > pentazocine, nalbuphine > buprenorphine. Dynorphin A 1-17, (+/-)-ethylketocyclazocine, (-)-U50,488H, tifluadom and beta-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]GTPgammaS 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.
Zhu
J
J
Department of Pharmacology, Temple University School of Medicine, Philadelphia, Pennsylvania 19140, USA.
Luo
L Y
LY
Li
J G
JG
Chen
C
C
Liu-Chen
L Y
LY
D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D037361:Adenylate Cyclase Toxin; D000818:Animals; D001665:Binding Sites; D016466:CHO Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002772:Cholera Toxin / Q000494:pharmacology; D003001:Cloning, Molecular; D006224:Cricetinae; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D020881:Enkephalin, D-Penicillamine (2,5)-; D004745:Enkephalins / Q000378:metabolism; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006153:Guanosine Diphosphate / Q000494:pharmacology; D006801:Humans; D007700:Kinetics; D008274:Magnesium / Q000378:metabolism / Q000494:pharmacology; D037342:Pertussis Toxin; D011759:Pyrrolidines / Q000494:pharmacology; D017473:Receptors, Opioid, kappa / Q000819:agonists* / Q000235:genetics / Q000378:metabolism; D011994:Recombinant Proteins / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology; D013462:Sulfur Radioisotopes; D010566:Virulence Factors, Bordetella / Q000494:pharmacology
D037361:Adenylate Cyclase Toxin; D004745:Enkephalins; D011759:Pyrrolidines; D017473:Receptors, Opioid, kappa; D011994:Recombinant Proteins; D013462:Sulfur Radioisotopes; D010566:Virulence Factors, Bordetella; D020875:Enkephalin, Ala(2)-MePhe(4)-Gly(5)-; D006153:Guanosine Diphosphate; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D019900:3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cyclohexyl)-benzeneacetamide, (trans)-Isomer; D020881:Enkephalin, D-Penicillamine (2,5)-; D002772:Cholera Toxin; D012964:Sodium; D037342:Pertussis Toxin; D008274:Magnesium