Advertisement
OtherNEUROPHARMACOLOGY

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

Jinmin Zhu, Lai-Yi Luo, Jian-Guo Li, Chongguang Chen and Lee-Yuan Liu-Chen
Journal of Pharmacology and Experimental Therapeutics August 1997, 282 (2) 676-684;

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.

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 Voigtlanderet 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). (−)-U50,488H is the prototype of selectivekappa 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 kappaopioid receptor belongs to the superfamily of GPCRs. Activation ofkappa 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 [32P]azidoanilido-GTP into Gα subunits (Prather et al., 1995), increase in inward-rectifying K+ conductance (Maet al., 1995; Henry et al., 1995; Grudt and Williams, 1993) and decrease in Ca++ conductance through N-type channels (Gross and MacDonald, 1987). Kappaagonist-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 receptors.

G proteins undergo activation/inactivation cycle (for reviews, Gilman, 1987; Birnbaumer et al., 1990). At resting state, the guanine nucleotide binding site of the alpha subunits of G proteins is occupied by GDP. When the GPCRs are activated by agonists, GDP bound to Gα is displaced by GTP. Binding by GTP causes dissociation of G proteins into alpha andbeta/gamma subunits. GTP-bound alphasubunit and beta/gamma subunits activate downstream effectors. Hydrolysis of GTP bound to alphasubunits by a GTPase intrinsic to Gα to GDP terminates agonist effects. Gα-GDP is then reassociated with beta/gamma subunits to re-enter the cycle. When the poorly hydrolyzed GTP analog GTPγS is used instead of GTP, the half-lives of GTPγS-bound alphasubunits are prolonged and α subunits are persistently activated.

Agonist-stimulated binding of [35S]GTPγS to G proteins has been used as a sensitive assay of activation of many GPCRs. It was first used in a reconstitution system of purified G protein and purified beta adrenergic receptors (Asanoet al., 1984). Since then, this method has been applied to many GPCRs in cell membranes, including muscarinic (Hilf et al., 1989; Lazareno et al., 1993), A1 adenosine (Lorenzen et al., 1993), alpha-2D adrenergic (Tian et al., 1994) and mu opioid (Traynor and Nahorski, 1995) receptors. It was recently used to localize G proteins activated by agonists for mu opioid, cannabinoid and γ-aminobutyric acidB receptors in rat brain sections by in vitro autoradiography (Sim et al., 1995).

In this study, we characterized effects of activation of the hkor stably expressed in CHO cells (CHO-hkor cells) on [35S]GTPγS binding to membranes. In addition, we determined whether this assay could be used to determine efficacy and potency of various opioid ligands on the kappa receptor.

Methods and Materials

Materials.

[3H]Diprenorphine (35 Ci/mmol) and [35S]GTPγS (1000–1300 Ci/mmol) were obtained from Amersham Corp. (Arlington Heights, IL) and NEN Life Sciences Co. (Boston, MA), respectively. Naloxone was a gift from DuPont/Merck (Wilmington, DE), and (−)-U50,488H was provided by Upjohn (Kalamazoo, MI). Dynorphin, DPDPE and DAMGO were purchased from Peninsula Laboratories (Belmont, CA). GDP and GTPγS were purchased from Sigma Chemical Co. (St. Louis, MO). (±)-Ethylketocyclazocine, tifluadom, nalorphine, nalbuphine, pentazocine, β-funaltrexamine, norbinaltorphimine and buprenorphine were provided by the National Institute on Drug Abuse.

Stable expression of hkor in CHO cells.

CHO cells were transfected with the hkor cDNA in the vector pBK-CMV (Zhu et al., 1995) and clonal cell lines stably expressing hkor (CHO-hkor) were established with Geneticin selection as described (Sambrooket al., 1989).

[35S]GTPγS binding.

Determination of [35S]GTPγS binding to G proteins was carried out with a modified procedure of Traynor and Nahorski (1995).

After removal of the growth medium, CHO-hkor cells were washed twice with 100 mM phosphate-buffered saline, harvested in Versene solution (0.54 mM EDTA, 0.14 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.46 mM KH2PO4, 1 mM glucose), centrifuged at 500 × g for 3 min and washed once with phosphate-buffered saline. The cell pellet was suspended in a buffer of 50 mM TEL/0.1 mM phenylmethylsulfonyl fluoride, sonicated and centrifuged at 1000 × g for 10 min. The pellet was sonicated and centrifuged again. Combined supernatant was centrifuged at 46,000 × g for 30 min. The pellet was resuspended in 50 mM Tris, pH 7.0, and centrifuged again. The membrane pellet was resuspended in 50 mM Tris, 0.32 M sucrose, pH 7.0, aliquoted at ∼600 μg protein/ml, frozen in dry ice/ethanol and stored in −70°C until use. All procedures were performed at 4°C.

Immediately before [35S]GTPγS binding assay, membranes was thawed at 37°C, chilled on ice, passed through a 22-gauge needle and diluted with 50 mM HEPES, pH 7.4, 100 mM NaCl, 5 mM MgCl2 and 1 mM EDTA with 1 mM dithiothreitol and 0.1% bovine serum albumin freshly added (buffer A). Membranes (∼7.5 μg protein) were incubated in buffer A containing [35S]GTPγS (100,000–150,000 dpm, ∼80 pM) and GDP with or without an opioid ligand (10−11 to 10−5M) in a total volume of 0.5 ml for 60 min at 30°C. Nonspecific binding was defined by incubation in the presence of 10 μM GTPγS. Nonspecific binding was found to be similar in the presence and absence of (−)-U50,488H and was subtracted from total stimulated and total basal binding. Bound and free [35S]GTPγS were separated by filtration with GF/B filters under reduced pressure. Radioactivity on filters was determined by liquid scintillation counting. The routine assay conditions, which included 100 mM NaCl, 5 mM MgCl2 and 3 μM GDP in the binding buffer, and performed with 80 to 120 pM [35S]GTPγS and 7.5 to 10 μg membrane protein for 60 min at 30°C, yielded 3,500 to 5,500 dpm and 1,000 to 1,500 dpm for maximal stimulated and basal [35S]GTPγS binding, respectively. EC50 values of drugs were determined by curve fitting to the equation for a sigmoidal curve E =E max·[D]/([D]n+ EC50 n), where E is effect produced by a certain concentration of the drug, [D], E max is the maximal response elicited by the drug and n is a fitting parameter.

Opioid receptor binding.

Membranes were prepared from CHO-hkor cells as described previously (Zhu et al., 1996). Opioid receptor binding was conducted with [3H]diprenorphine according to our published procedure (Zhu et al., 1995). Binding was carried out in 50 mM Tris-HCl buffer containing 1 mM EGTA and 10 μM leupeptin (pH 7.4 at room temperature) (TEL buffer) or in [35S]GTPγS binding buffer at 25°C for 1 h in duplicate in a volume of 1 ml with 30 to 60 μg protein. Binding data were analyzed with the EBDA program (McPherson, 1983).Ki values were determined by use of the equation of Cheng and Prusoff (1973). Comparison of Ki values of each drug in the two buffers were performed with Student’s t test, with P <.05 being considered significantly different.

Determination of protein content.

Protein contents of membranes were determined by the BCA method of Smith et al.(1985) with bovine serum albumin as the standard.

Results

Stable expression of the human kappa opioid receptor cDNA in CHO cells.

The human kappa opioid receptor cDNA was expressed stably in CHO cells. Saturation binding of [3H]diprenorphine to membranes of CHO-hkor cells was carried out in 50 mM TEL buffer. Kd and B max were determined to be 0.12 ± 0.02 nM and 1292 ± 52 fmol/mg protein (mean ± S.E.M., n = 3), respectively. [3H]Diprenorphine binding to the membranes was potently inhibited by kappa selective ligands, but not bymu or delta ligands (see table 1).

View this table:
Table 1

EC50 values and maximal effects of opioid ligands in stimulating [35S]GTPγS binding to CHO-hkor cell membranes and their K i values in inhibiting [3H]diprenorphine binding to the human kappareceptors expressed in CHO cells

Effects of GDP on [35S]GTPγS binding.

Experiments were performed with 5 mM Mg++, 100 mM Na+ and various concentrations of GDP in the presence and absence of 3 μM (−)-U50,488H (fig. 1). Without GDP, no increase in [35S]GTPγS binding by thekappa receptor agonist (−)-U50,488H was observed. GDP decreased [35S]GTPγS binding in a dose-dependent manner both in the presence and absence of (−)-U50,488H (fig. 1). With ≥0.3 μM GDP, the magnitude of reduction in [35S]GTPγS binding was larger in the absence than the presence of (−)-U50,488H and, thus, the increase in [35S]GTPγS binding caused by the agonist could be observed. The presence of 3 μM GDP produced the maximal stimulatory response. At concentrations of GDP ≥ 100 μM, effects of (−)-U50,488H were greatly diminished or obliterated. In this study, 3 μM GDP was used in all other experiments.

Figure 1
Figure 1

Effect of GDP on [35S]GTPγS binding to CHO-hkor membranes in the absence (open triangle) or presence (dark triangle) of 3 μM (−)-U50,488H. [35S]GTPγS binding was performed in the absence or presence of various concentrations of GDP, 5 mM Mg++ and 100 mM Na+with ∼10 μg membrane proteins at 30°C for 60 min as described under “Materials and Methods.” Each value represents the mean ± S.E.M. of three independent experiments in duplicate. Control represents [35S]GTPγS binding without GDP in the absence of 3 μM (−)-U50,488H, which yielded 4 to 5.5 fmol/10 μg protein.

Effects of Mg++ concentration on [35S]GTPγS binding.

The importance of Mg++ in [35S]GTPγS binding was determined in the absence and presence of 3 μM (−)-U50,488H with 3 μM GDP and 100 mM Na+(fig. 2). At concentrations between 5 μM and 0.5 mM, there was no significant difference in [35S]GTPγS binding between with and without (−)-U50,488H. At 5 or 15 mM, the basal level of [35S]GTPγS binding was doubled. From 1.5 to 15 mM, binding in the presence of (−)-U50,488H was greatly increased and a plateau was reached at 5 and 15 mM. At the plateau, agonist-induced binding was 2.5- to 3.5-fold of basal binding. At 50 mM, both basal level and agonist-induced binding decreased. Based on these results, 5 mM Mg++ in the form of MgCl2 was used in all other experiments.

Figure 2
Figure 2

Effect of Mg++ on [35S]GTPγS binding to CHO-hkor membranes in the absence (open triangle) or presence (dark triangle) of 3 μM (−)-U50,488H. [35S]GTPγS binding was performed in the presence of various concentrations of Mg++, 3 μM GDP and 100 mM Na+ with ∼7.5 μg membrane proteins at 30°C for 60 min as described under “Materials and Methods.” Each value represents the mean ± S.E.M. of three independent experiments in duplicate.

Effects of Na+ on [35S]GTPγS binding.

Effects of Na+ on [35S]GTPγS binding were investigated in the presence of 3 μM GDP and 5 mM Mg++ with or without 3 μM (−)-U50,488H (fig.3). Without Na+, (−)-U50,488H-stimulated increase in [35S]GTPγS binding was evident, although the magnitude of the increase was relatively small (not shown, but similar to 0.1 mM NaCl of fig. 3). At ≤1 mM, Na+ did not affect basal or agonist-induced [35S]GTPγS binding. Basal [35S]GTPγS binding was decreased by [Na+] ≥ 10 mM, whereas binding in the presence of (−)-U50,488H was reduced at [Na+] ≥ 100 mM, both in a concentration-dependent manner. Binding in the presence of 100 mM Na+ provides the best signal-to-background difference. At this concentration, the basal binding amounted to approximately 28% of the maximal binding achieved by (−)-U50,488H. In all other experiments, binding was performed in the presence of 100 mM NaCl.

Figure 3
Figure 3

Effect of Na+ on [35S]GTPγS binding to CHO-hkor membranes in the absence (open triangle) or presence (dark triangle) of 3 μM (−)-U50,488H. [35S]GTPγS binding was performed in the presence of various concentrations of Na+, 3 μM GDP and 5 mM Mg++ with ∼ 7.5 μg membrane proteins at 30°C for 60 min as described under “Materials and Methods.” Each value represents the mean ± S.E.M. of three independent experiments in duplicate.

Time course of basal and (−)-U50,488H-stimulated [35S]GTPγS binding.

Time courses of [35S]GTPγS binding were conducted in the presence and absence of 3 μM (−)-U50,488H at 30°C. Basal [35S]GTPγS binding increased with time, whereas (−)-U50,488H-stimulated [35S]GTPγS binding (difference between with and without (−)-U50,488H) reached a plateau at ∼90 min.

Relationship between [35S]GTPγS binding and the amount of CHO-hkor membrane.

[35S]GTPγS binding increased linearly with or without 3 μM (−)-U50,488H up to 40 μg of membrane proteins in each assay tube. Routinely, 7.5 to 10 μg of membrane proteins were used, which gave 3,500 to 5,500 dpm and 1,000 to 1,500 dpm for stimulated and basal [35S]GTPγS binding, respectively.

Effect of (−)-U50,488H on [35S]GTPγS binding to CHO-hkor membranes.

[35S]GTPγS binding to CHO-hkor membranes with various concentrations of (−)-U50,488H was examined in the presence of 3 μM GDP, 5 mM Mg++ and 100 mM Na+ (fig. 4). Binding of [35S]GTPγS was increased by (−)-U50,488H in a concentration-dependent manner with an EC50 of 3.1 nM. The maximal response, which represented ∼3.5-fold of the basal level, was reached at 0.3 μM (−)-U50,488H (fig. 4). Ten micromolar (−)-U50,488H failed to stimulate [35S]GTPγS binding to membranes of untransfected CHO cells (not shown). (+)-U50,488H up to 1 μM did not increase [35S]GTPγS binding, compared with the basal level (not shown), which indicated stereospecificity of receptor activation. Naloxone (1 μM) or norbinaltorphimine (10 nM) shifted the concentration-effect curve of (−)-U50,488H to the right by about 100-fold (fig. 4). Ke values of naloxone and norbinaltorphimine calculated from these data were 10 nM and 0.1 nM, respectively, indicating that their potencies in the CHO-hkor system are similar to those in other kappa opioid receptor assays (Portoghese et al., 1987). The muagonist DAMGO and the delta agonist DPDPE had no effect (fig. 4). Taken together, these results indicate that (−)-U50,488H-induced increase in [35S]GTPγS binding is mediated by specific activation of the kappaopioid receptor.

Figure 4
Figure 4

Dose-response relationship of [35S]GTPγS binding to CHO-hkor membranes induced by (−)-U50,488H, (−)-U50,488H + 1 μM naloxone, (−)-U50,488H + 10 nM norbinaltorphimine, DAMGO and DPDPE. [35S]GTPγS binding was performed in the presence of 3 μM GDP, 5 mM Mg++ and 100 mM Na+ with ∼7.5 μg membrane proteins at 30°C for 60 min as described under “Materials and Methods.” Basal [35S]GTPγS binding was 0.5 to 0.7 fmol/10 μg protein, whereas stimulated levels were 1.8 to 2.7 fmol/10 μg protein. Basal binding was subtracted from each datum. Data were expressed as % of maximal binding produced by (−)-U50,488H. Each value represents the mean ± S.E.M. of three independent experiments in duplicate.

Effects of pretreatment with pertussis toxin or cholera toxin on [35S]GTPγS binding.

To assess the G proteins that were involved in the action of (−)-U50,488H, CHO-hkor cells were treated with pertussis toxin (100 ng/ml) or cholera toxin (20 μg/ml) for 24 h before membrane preparation. Pertussis toxin pretreatment completely abolished (−)-U50,488H-induced increase in [35S]GTPγS binding and reduced basal binding of [35S]GTPγS by 60% (fig.5A). In contrast, cholera toxin pretreatment did not affect basal or (−)-U50,488H-induced [35S]GTPγS binding (fig. 5B). Thus, (−)-U50,488H-induced [35S]GTPγS binding was most likely caused by binding to pertussis toxin-sensitive G proteins,i.e., Gi and/or Go.

Figure 5
Figure 5

Effect of pretreatment with pertussis toxin (A) or cholera toxin (B) on (−)-U50,488H-induced [35S]GTPγS binding. CHO-hkor cells were treated with pertussis toxin (PTX; 100 ng/ml) or cholera toxin (CTX; 20 μg/ml) for 24 h and [35S]GTPγS binding to membranes was performed with or without (−)-U50,488H. Binding to membranes of untreated cells served as control. [35S]GTPγS binding was performed in the presence of 3 μM GDP, 5 mM Mg++ and 100 mM Na+ with ∼7.5 μg membrane proteins at 30°C for 60 min as described under “Materials and Methods.” Each value represents the mean ± S.E.M. of three independent experiments in duplicate. Data are expressed as % of the control basal level. All pertussis toxin-treated binding values were significantly lower than the control basal level (P <.05, Student’s t test).

Determination of Kd andBmax of (−)-U50,488H-induced [35S]GTPγS binding.

Displacement of [35S]GTPγS binding with unlabeled GTPγS was performed in the absence and presence of 10 μM (−)-U50,488H for 180 min (fig. 6) to determineKd and B max of [35S]GTPγS binding that could be maximally stimulated by (-),U50,488H. Scatchard analysis of the difference between the two curves revealed a Kd of 0.34 ± 0.08 nM and a B max of 431 ± 29 fmol/mg protein for [35S]GTPγS binding (mean ± S.E.M., n = 3).

Figure 6
Figure 6

Displacement curves of [35S]GTPγS binding by GTPγS in the presence (open circle) and absence (dark circle) of 3 μM (−)-U50,488H. [35S]GTPγS binding was performed in the presence of 3 μM GDP, 5 mM Mg++ and 100 mM Na+ with ∼7.5 μg membrane proteins at 30°C for 60 min as described under “Materials and Methods.” Each value represents the mean ± S.E.M. of three independent experiments in duplicate. Differences between two curves were analyzed with Scatchard analysis (inset), which yielded Kd andB max values of 0.34 ± 0.08 nM and 431 ± 29 fmol/mg protein.

Determination of potencies and efficacies of opioid ligands on [35S]GTPγS binding.

Several opioid receptor ligands were examined for their potencies and efficacies in stimulating [35S]GTPγS binding to membranes. EC50 values and maximal responses were determined and compared with those of (−)-U50,488H (fig.7, table1). Dynorphin A 1–17, (±)-ethylketocyclazocine, tifluadom and β-funaltrexamine produced maximal responses similar to (−)-U50,488H and were full agonists. Dynorphin A 1–17 and (±)-ethylketocyclazocine had higher potencies than (−)-U50,488H with EC50 values of 0.18 and 0.57 nM, respectively, whereas β-funaltrexamine and tifluadom were potent similar to (−)-U50,488H with EC50 values of 1.7 nM and 3.9 nM, respectively. Nalorphine and pentazocine acted as partial agonists with the maximal stimulation of [35S]GTPγS binding at 68% and 23% of that of (−)-U50,488H, respectively. EC50 values of nalorphine and pentazocine, determined as the dose that elicited 50% of the maximal response of each drug, were 17.9 nM and 103 nM, respectively. Nalbuphine and buprenorphine had some stimulatory effects at ≥100 nM, but no plateau in maximal response was detected at up to 30 μM. The rank order of potencies of ligands tested for stimulation of [35S]GTPγS binding was dynorphin A 1–17 > (±)-ethylketocyclazocine > β-funaltrexamine, tifluadom, (−)-U50,488H > nalorphine > pentazocine, nalbuphine > buprenorphine. Although norbinaltorphimine and naloxone had high affinity for the kappa receptor, they did not increase [35S]GTPγS binding. In addition, naloxone and norbinaltorphimine shifted the dose-response curve of (−)-U50,488H to the right (fig. 4), indicating that they are antagonists.

Figure 7
Figure 7

Dose-response curves of opioid ligands on [35S]GTPγS binding to CHO-hkor membranes. [35S]GTPγS binding was performed in the presence 3 μM GDP, 5 mM Mg++ and 100 mM Na+ with ∼7.5 μg membrane proteins at 30°C for 60 min as described under “Materials and Methods.” A dose-response curve of (−)-U50,488H was included in each experiment and data are expressed as % of the maximal response induced by (−)-U50,488H. Basal [35S]GTPγS binding was 0.5 to 0.7 fmol/10 μg protein, whereas stimulated levels were 1.8 to 2.5 fmol/10 μg protein. Basal binding was subtracted from each datum. Each value represents the mean ± S.E.M. of three independent experiments in duplicate.

Binding affinities of opioid ligands to the humankappa opioid receptor.

Competitive inhibition of [3H]diprenorphine binding by opioid ligands to CHO-hkor membranes was conducted to determine binding affinities of opioid ligands tested in [35S]GTPγS binding. Binding was performed in [35S]GTPγS binding buffer as well as in TEL buffer, because Tris-HCl buffer is the most commonly used buffer for binding studies.Ki values of these ligands were listed in table 1. There was no significant difference inKi values of each drug in these two buffers. These values determined in membranes of CHO-hkor cells were similar to those determined in membranes of COS-1 cells transiently expressing the kappa receptor (Zhu et al., 1995). Ligands selective for kappa receptor ((−)-U50,488H, norbinaltorphimine, dynorphin A 1–17) had much higher affinity than the mu selective ligand DAMGO or the deltaselective ligand DPDPE. For the five full agonists (dynorphin A 1–17, (±)-ethylketocyclazocine, tifluadom, β-funaltrexamine and (−)-U50,488H), the Ki values were very similar to the EC50 values for stimulation of [35S]GTPγS binding (table 1).

Discussion

Opioid receptors mediate functional effects of agonistsvia activation of G proteins. In the present study, we demonstrated the ability of kappa agonists to enhance binding of [35S]GTPγS to membranes of CHO-hkor cells. The presence of GDP and Mg++ was essential for agonist-stimulated [35S]GTPγS binding. Na+ was necessary for maximal signal-to-background ratios. The extent of (−)-U50,488H-induced increase in [35S]GTPγS binding was dependent on agonist concentration, incubation time and tissue concentration. The effect of (−)-U50,488H was stereospecific and reversed by naloxone or norbinaltorphimine, which indicates that this effect is mediated by thekappa receptor. Pertussis toxin-, but not cholera toxin-sensitive G proteins were involved. In this system, [35S]GTPγS binding assay allowed classification of high-affinity kappa opioid ligands into full agonists, partial agonists and antagonists. Although full agonists elicit maximal effects, partial agonists and antagonists produce submaximal and no effects, respectively. This biochemical assay thus permits determination of efficacies of kappa opioid ligands. In addition, the CHO-hkor cell, being devoid of other receptors, is an excellent system for this purpose. EC50 values of five full agonists tested in stimulating [35S]GTPγS binding were in the nanomolar range, very similar to their Ki values in inhibiting [3H]diprenorphine binding, which strongly suggests that there is no or little spare receptors in this system.

In addition to its relatively large stimulated signals and its allowing determination of ligand efficacy, [35S]GTPγS binding assay has some practical advantages. Membranes can be prepared and frozen for convenience. The assay itself is easy, quick and reproducible. Recently, Befort et al. (1996) reported use of [35S]GTPγS binding to evaluate activity ofdelta opioid receptors transiently expressed COS cells. The utility of this assay for transiently expressed receptors will facilitate examination of effects of mutations on receptor functional activity.

Stimulation of [35S]GTPγS binding by agonists allows one to examine G protein activation by ligand-occupied receptors regardless of the types of G proteins and effector systems involved. In this regard, it is similar to kappa agonist-induced enhancement of GTPase activity of G proteins (Clark et al., 1986; Clark and Medzihradsky, 1987; Lawrence et al., 1995) and increase in labeling of Gα subunits by [32P]azidoanilido-GTP (Prather et al., 1995). (−)-U50,488H and dynorphin peptides stimulated low-Km GTPase activity by 10 to 20% with EC50 values of 3 to 23 μM in guinea pig cerebellum membranes (Clark et al., 1986) and in rat and monkey brain membranes pretreated with β-funaltrexamine andcis-(±)-3-methylfentanyl isothiocyanate to alkylate themu and delta receptors, respectively (Clark and Medzihradsky, 1987). These EC50 values are more than 100-fold greater than their Ki values of binding to the kappa receptor. In three mouse thymoma cell lines, stimulation of low-Km GTPase activity by (−)-U50,488H was observed at ≥ 10 nM and had a 21 to 53% increase over the basal level at 30 μM (−)-U50,488H (Lawrenceet al., 1995). Activation of a cloned rat kappaopioid receptor stably expressed in CHO cells increased the incorporation of [32P]azidoanilido-GTP into four Gα subunits with maximal increases of 20 to 44%, and EC50 values of (−)-U50,488H and dynorphin A 1–17 were in the nanomolar range, similar to theirKi values in binding to thekappa receptor (Prather et al., 1995). In these studies, (−)-U50,488H and dynorphin peptides, but no partial agonists, were examined.

In addition to activation of GTPase, inhibition of forskolin-stimulated adenylate cyclase activity has been used as a biochemical measure of activation of the kappa opioid receptor (Attali et al., 1989; Konkoy and Childers, 1989, 1993; Lawrence and Bidlack, 1993; Lawrence et al., 1995; Prather et al., 1995). In CHO cells stably transfected with a cloned ratkappa receptor, dynorphin A 1–17 and (−)-U50,488H were very potent in inhibiting forskolin-stimulated adenylate cyclase with EC50 values in the nanomolar range (Pratheret al., 1995). In contrast, in many other systems, EC50 values of (−)-U50,488H, U69,593 and dynorphin A 1–17 for the inhibition of forskolin-stimulated adenylate cyclase activity were in the micromolar range, 100- to 1000-fold of their Ki values of binding to thekappa opioid receptor. These included the rat spinal cord and spinal cord-dorsal root ganglion culture (Attali et al., 1989), guinea pig cerebellar membranes (Konkoy and Childers, 1989), guinea pig brain membranes (Konkoy and Childers, 1993) and mouse thymoma cells (Lawrence et al., 1995; Lawrence and Bidlack, 1993). Maximal inhibition that could be achieved by (−)-U50,488H or dynorphin A 1–17 was 40% in guinea pig brain membranes (Konkoy and Childers, 1993), 37 to 66% in mouse thymoma cell lines (Lawrenceet al., 1995; Lawrence and Bidlack, 1993) and 56 to 75% in CHO cells expressing a rat kappa receptor (Prather et al., 1995). Only (−)-U50,488H, U69,593 and dynorphin peptides were examined in the studies on inhibition of adenylate cyclase. Whether partial agonists were active in these systems was not determined.

The presence of GDP is essential for (−)-U50,488H-induced increase in [35S]GTPγS binding. Optimal signals were obtained with 1 to 10 μM GDP. The requirement of GDP for the agonist-induced increase in binding of [35S]GTPγS has also been demonstrated for many other receptors including muscarinic, A1 adenosine andmu opioid receptors (Hilf et al., 1989; Lorenzenet al., 1993; Traynor and Nahorski, 1995). For thealpha-2D adrenergic receptor, GDP is not required for agonist-induced increase in [35S]GTPγS binding, but it increased the magnitude of stimulation caused by an agonist (Tian et al., 1994). The mechanism of action of GDP, however, is not fully understood. From the data in figure 1, we propose the following hypothesis. In the absence of an agonist, GDP inhibits [35S]GTPγS binding in a dose-dependent manner with an IC50 value of ∼0.8 μM. With no or a low concentration of GDP, [35S]GTPγS competes favorably over GDP for binding to guanine nucleotide binding sites of Gα subunits. As a result, the agonist-induced increase in [35S]GTPγS binding is obscured. Addition of GDP in the micromolar range results in the binding of GDP to most guanine nucleotide binding sites of Gαsubunits. Binding of an agonist to the receptor increases dissociation of GDP from the interacting G proteins and their association of [35S]GTPγS (Gilman, 1987) and, therefore, allows the increase in [35S]GTPγS binding to be observed.

Kappa agonist-stimulated binding of [35S]GTPγS depends on the presence of Mg++, and the maximal signal-to-background ratio was observed between 5 and 15 mM. Mg++ has multiple effects on signal transduction of G protein-coupled receptors (for reviews, Gilman, 1987; Birnbaumer et al., 1990). The effects that require Mg++ are as follows: 1) formation of agonist-receptor-G protein ternary complex; 2) activation of G proteins by agonist-occupied receptors; 3) stimulation of GTP binding to G proteins and GDP dissociation by an agonist-receptor complex; 4) reduction of dissociation of GTPγS from G proteins to near zero; 5) βγ-stimulated GDP dissociation and GTPγS-induced subunit dissociation; 6) GTPase activity. The net results are that Mg++ promotes dissociation of oligomeric G proteins and the formation of an “activated” state of Gα. Effects of Mg++ on [35S]GTPγS binding are likely caused by a combination of effects 1 through 5.

Na+ is not required for kappa agonist stimulation, because (−)-U50,488H can increase [35S]GTPγS binding even without Na+. However, better stimulated-to-basal difference was obtained in the presence of >30 mM [Na+]. Similar observations have also been reported in studies of G protein activation by muscarinic (Hilfet al., 1989), formyl peptide (Gierschik et al., 1989), A1 adenosine (Lorenzen et al., 1993) andalpha-2D adrenergic (Tian et al., 1994) receptors. It appears that Na+ prevents activation of G proteins by unoccupied receptors (Gierschik et al., 1989). This effect of Na+ is in accord with the findings that Na+ was required forkappa agonist-induced increase in low-Km GTPase activity (Clark et al., 1986; Clark and Medzihradsky, 1987; Lawrence et al., 1995), inhibition of forskolin-stimulated adenylate cyclase (Attali et al., 1989; Konkoy and Childers, 1989, 1993;Lawrence and Bidlack, 1993; Lawrence et al., 1995) and increase in [32P]azidoanilido-GTP into Gα subunits (Prather et al., 1995).

Enhancement of [35S]GTPγS binding by thekappa opioid agonist (−)-U50,488H was completely blocked by pretreatment of the cells with pertussis toxin, but not with cholera toxin, which confirms that the event is mediated entirely through pertussis toxin-sensitive G proteins. This result agrees with the findings that actions of kappa opioid receptors are mediated through pertussis toxin-sensitive Gi and/or Go proteins (Lawrence and Bidlack, 1993; Pratheret al., 1995; Ma et al., 1995). Inhibition of adenylate cyclase by kappa receptor activation was blocked by pertussis toxin pretreatment (Lawrence and Bidlack, 1993). Activation of a cloned rat kappa opioid receptor increased the incorporation of [32P]azidoanilido-GTP into four Gα subunits, three of which were identified as Gi3α, Gi2α and Go2α(Prather et al., 1995). Pertussis toxin treatment abolishedkappa agonist-induced increase in K+conductance through an inward rectifying K+channel (Ma et al., 1995). Basal [35S]GTPγS binding was also lowered by pertussis toxin treatment (see fig. 5A). A similar reduction of basal [35S]GTPγS binding by pertussis toxin was observed in SHSY-5Y cells (Traynor and Nahorski, 1995). This may be caused by active coupling of unoccupied receptors to pertussis toxin-sensitive G proteins and, thus, stimulation of [35S]GTPγS binding to G proteins at resting condition, similar to alpha-2D adrenergic receptor (Tianet al., 1994). In this CHO-hkor system, kappaopioid receptors are not coupled to cholera toxin-sensitive G proteins. Coupling of the kappa opioid receptor to cholera toxin-sensitive G proteins was reported in the dorsal root ganglion-spinal dorsal horn culture (Crain and Shen, 1990) and in myenteric plexuses (Gintzler and Xu, 1991).

Potencies and efficacies of opioid ligands in stimulating muopioid receptor-mediated [35S]GTPγS binding to SHSY-5Y cell membranes correlated very well with those in other functional assays, such as an in vivo antinociceptive test and inhibition of contraction in guinea pig ileum in vitro(Traynor and Nahorski, 1995). In the present study, opioid agonists had different potencies in kappa receptor-mediated enhancement of [35S]GTPγS binding and produced varying maximal responses, indicative of their efficacies. The rabbit vas deferens contained kappa opioid receptor, but notmu and delta receptors (Oka et al., 1981). Inhibition of field-stimulated contraction of the rabbit vas deferens was used to determine efficacies and potencies of ligands onkappa opioid receptors (Hayes and Kelly, 1985; Milleret al., 1986; Verlinde and De Ranter, 1988). Our finding that ethylketocyclazocine, tifluadom and (−)-U50,488H were full agonists in stimulating [35S]GTPγS binding is consistent with the observation that these three compounds were full agonists in the rabbit vas deferens (Romer et al., 1982;Hayes and Kelly, 1985; Verlinde and De Ranter, 1988). (−)-U50,488H and tifluadom were equally potent in stimulating [35S]GTPγS binding, whereas tifluadom was 7 to 400 times more potent than (−)-U50,488H in the rabbit vas deferens (Hayes and Kelly, 1985; Miller et al., 1986; Verlinde and De Ranter, 1988). Although ethylketocyclazocine was about 10 times more potent than (−)-U50,488H, in stimulating [35S]GTPγS binding it is 6 or 60 times more potent than (−)-U50,488H in the rabbit vas deferens (Hayes and Kelly, 1985; Miller et al., 1986). The observation that β-funaltrexamine enhances [35S]GTPγS binding in CHO-hkor cells is consistent with the original findings that it is a kappa agonist, in addition to having irreversiblemu antagonist activities (Portoghese et al., 1980).

In the present study, nalorphine and pentazocine were partial agonists at the kappa receptor in stimulating [35S]GTPγS binding and nalbuphine had low level of agonist activity. Our findings are consistent with those ofMiller et al. (1986) that pentazocine and nalorphine inhibited electrically induced contraction of guinea pig ileum by acting on the kappa opioid receptor with aKe value of naloxone of ∼20 nM. In contrast, pentazocine, nalbuphine and nalorphine were inactive in vas deferens preparations of the rat, mouse and rabbit, but they could antagonize the action of ethylketocyclazocine in these preparations (Hayes and Kelly, 1985; Miller et al., 1986; Verlinde and De Ranter, 1988). The variation in potencies of these compounds in thesein vitro preparations was hypothesized to be caused by the difference in the number of spare receptors in these models (Hayes and Kelly, 1985; Miller et al., 1986).

Our observations that (−)-U50,488H, tifluadom and ethylketocyclazocine are full agonists and nalorphine and nalbuphine are partial agonists are in accord with results from in vivo pharmacological studies. (−)-U50,488H, tifluadom and ethylketocyclazocine were found to be highly efficacious kappa agonists in kappareceptor-mediated antinociception (Von Voigtlander et al., 1983; Piercey et al., 1982; France et al., 1994;Dykstra et al., 1987) and diuresis (Von Voigtlander et al., 1983; Leander, 1983a; Dykstra et al., 1987;Takemori et al., 1988). In drug discrimination procedures, these drugs substitute completely for kappa agonists ethylketocyclazocine, bremazocine and spiradoline (Holtzman et al., 1991; France et al., 1994; Dykstra et al., 1987; Picker, 1994b; Smith and Picker, 1995). On the contrary, maximal effects produced by nalorphine were less than those of full agonists in kappa receptor-mediated diuresis (Leander, 1983a, b). Nalorphine and nalbuphine substituted partially or failed to substitute for bremazocine in drug discrimination tests (Smith and Picker, 1995; Picker, 1994a). In addition, nalorphine and nalbuphine antagonized the stimulus effect of bremazocine (Picker, 1994a; Smith and Picker, 1995). Nalorphine antagonized the diuretic effect of bremazocine (Leander, 1983b) as well as the antinociceptive effect of U50,488H (Dykstra, 1990).

Naloxone and norbinaltorphimine shifted the dose-response curve of (−)-U50,488H to the right, but neither had any effect on the basal [35S]GTPγS binding. These results indicate that both compounds are pure antagonists without positive or negative intrinsic activity in this system.

For five full agonists, the EC50 values in stimulating [35S]GTPγS binding were similar to their Ki values in inhibiting [3H]diprenorphine binding. This finding indicates that there are few or no spare receptors in this CHO-hkor system.

The binding affinities of ligands examined, including full agonists, partial agonists and antagonists, for the kappareceptor were similar in [35S]GTPγS binding buffer and in commonly used Tris-based receptor binding buffer. [35S]GTPγS binding buffer contains 100 mM Na+, 5 mM Mg++ and 3 μM GDP, whereas the Tris-based receptor binding buffer does not. We found that the addition of 100 mM Na+, 5 mM Mg++, 3 μM GDP or all three to Tris-based receptor binding buffer did not change the apparent Ki value of (−)-U50,488H (Li, J.-G. and Liu-Chen, L.-Y., unpublished observation). Based on results in table 1, it is likely that Na+, Mg++, GDP or all three at the concentrations used did not affect binding affinities of other ligands examined. Contrary to our observations, Lawrence and Bidlack (1992) showed that in R1.1 thymoma cell membranes, 100 mM Na+ and 10 mM Mg++inhibited 50 pM (−)-[3H]bremazocine binding by 45% and 37%, respectively. Although 100 μM GDP did not have an effect on (−)-[3H]bremazocine binding, a combination of 100 μM GDP and 30 mM Na+inhibited binding by 54%. Thus, effects of Na+, Mg++ and GDP on kappa agonist binding may also depend on the ligand examined. This issue needs further investigation.

In conclusion, stimulation of [35S]GTPγS binding to membranes of CHO cells stably expressing the humankappa opioid receptor by kappa agonists provides a useful functional measure for interaction between kappaopioid receptors and pertussis toxin-sensitive G proteins. In addition, this assay distinguishes ligands of full agonists, partial agonists and antagonists, which, in general, agrees with results obtained fromin vitro tissue preparations and in vivopharmacology.

Acknowledgments

We thank Drs. Alan Cowan and Ronald J. Tallarida for helpful discussions.

Footnotes

  • Send reprint requests to: Dr. Lee-Yuan Liu-Chen, Department of Pharmacology, Temple University School of Medicine, 3420 N. Broad St., Philadelphia, PA 19140.

  • ↵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.

References

  1. ↵
    1. Asano T.,
    2. Pedersen S. E.,
    3. Scott C. W.,
    4. Ross E. M.
    (1984) Reconstitution of catecholamine-stimulated binding of guanosine 5′-O-(3-thiotriphosphate) to the stimulatory GTP-binding protein of adenylate cyclase. Biochemistry 23:5460–5467.
    OpenUrlCrossRefPubMed
  2. ↵
    1. Attali B.,
    2. Saya D.,
    3. Vogel Z.
    (1989) Kappa-opiate agonists inhibit adenylate cyclase and produce heterologous desensitization in rat spinal cord. J. Neurochem. 52:360–369.
    OpenUrlCrossRefPubMed
  3. ↵
    1. Befort K.,
    2. Tabbara L.,
    3. Kieffer B. L.
    (1996) [35S]GTPγS binding: A tool to evaluate functional activity of a cloned opioid receptor transiently expressed in COS cells. Neurochem. Res. 21:1301–1307.
    OpenUrlCrossRefPubMed
  4. ↵
    1. Birnbaumer L.,
    2. Abramowitz J.,
    3. Brown A. M.
    (1990) Receptor-effector coupling by G proteins. Biochim. Biophys. Acta 1031:163–224.
    OpenUrlPubMed
  5. ↵
    1. Conn P. M.
    1. Chang K. -J.
    (1984) Opioid receptors: multiplicity and sequelae of ligand-receptor interactions. in The Receptors, ed Conn P. M. (Academic Press, Inc. New York, NY), 1:1–81.
  6. ↵
    1. Chavkin C.,
    2. Goldstein A.
    (1981) Specific receptor for the opioid peptide dynorphin: Structure-activity relationships. Proc. Natl. Acad. Sci. U.S.A. 78:6543–6547.
    OpenUrlAbstract/FREE Full Text
  7. ↵
    1. Cheng Y.-C.,
    2. Prusoff W. H.
    (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22:3099–3108.
    OpenUrlCrossRefPubMed
  8. ↵
    1. Clark M. J.,
    2. Levenson S. D.,
    3. Medzihradsky F.
    (1986) Evidence for coupling of the kappa opioid receptor to brain GTPase. Life Sci. 39:1721–1727.
    OpenUrlCrossRefPubMed
  9. ↵
    1. Clark M. J.,
    2. Medzihradsky F.
    (1987) Coupling of multiple opioid receptors to GTPase following selective receptor alkylation in brain membranes. Neuropharmacology 26:1763–1770.
    OpenUrlCrossRefPubMed
  10. ↵
    1. Crain S. M.,
    2. Shen K. F.
    (1990) Opioids can evoke direct receptor-mediated excitatory effects on sensory neurons. [review]. Trends Pharmacol. Sci. 11:77–81.
  11. ↵
    1. Dykstra L. A.,
    2. Gmerek D. E.,
    3. Winger G.,
    4. Woods J. H.
    (1987) Kappa opioids in Rhesus monkeys. I. diuresis, sedation, analgesia and discriminative stimulus effects. J. Pharmacol. Exp. Ther. 242:413–420.
    OpenUrlAbstract/FREE Full Text
  12. ↵
    1. Dykstra L. A.
    (1990) Butorphanol, levallorphan, nalbuphine and nalorphine as antagonists in the squirrel monkey. J. Pharmacol. Exp. Ther. 254:245–252.
    OpenUrlAbstract/FREE Full Text
  13. ↵
    1. Evans C.,
    2. Keith D. E., Jr.,
    3. Morrison H.,
    4. Magendzo K.,
    5. Edwards R. H.
    (1992) Cloning of a delta opioid receptor by functional expression. Science 258:1952–1955.
    OpenUrlAbstract/FREE Full Text
  14. ↵
    1. France C. P.,
    2. Medzihradsky F.,
    3. Woods J. H.
    (1994) Comparison of kappa opioids in rhesus monkeys: behavioral effects and receptor binding affinities. J. Pharmacol. Exp. Ther. 268:47–58.
    OpenUrlAbstract/FREE Full Text
  15. ↵
    1. Gierschik P.,
    2. Sidiropoulos D.,
    3. Steisslinger M.,
    4. Jakobs K. H.
    (1989) Na+ regulation of formyl peptide receptor-mediated signal transduction in HL 60 cells: evidence that the cation prevents activation of the G protein by unoccupied receptors. Eur. J. Pharmacol. 172:481–492.
    OpenUrlCrossRefPubMed
  16. ↵
    1. Gilman A. G.
    (1987) G proteins: Transducers of receptor-generated signals. Annu. Rev. Biochem. 56:615–649.
    OpenUrlCrossRefPubMed
  17. ↵
    1. Gintzler A. R.,
    2. Xu H.
    (1991) Different G proteins mediate the opioid inhibition or enhancement of evoked [5-methionine]enkephalin release. Proc. Natl. Acad. Sci. U.S.A. 88:4741–4745.
    OpenUrlAbstract/FREE Full Text
  18. ↵
    1. Gross R. A,
    2. MacDonald R. L.
    (1987) Dynorphin A selectively reduces a large transient (N-type) calcium current of mouse dorsal root ganglion neurons in cell culture. Proc. Natl. Acad. Sci. U.S.A. 84:5469–5473.
    OpenUrlAbstract/FREE Full Text
  19. ↵
    1. Grudt T. J.,
    2. Williams J. T.
    (1993) Kappa-opioid receptors also increase potassium conductance. Proc. Natl. Acad. Sci. U.S.A. 90:11429–11432.
    OpenUrlAbstract/FREE Full Text
  20. ↵
    1. Hayes A.,
    2. Kelly A.
    (1985) Profile of activity of kappa receptor agonists in the rabbit vas deferens. Eur. J. Pharmacol. 110:317–322.
    OpenUrlCrossRefPubMed
  21. ↵
    1. Henry D. J.,
    2. Grandy D. K.,
    3. Lester H. A.,
    4. Davidson N.,
    5. Chavkin C.
    (1995) Kappa-opioid receptors couple to inwardly rectifying potassium channels when coexpressed by Xenopus oocytes. Mol. Pharmacol. 47:551–557.
    OpenUrlAbstract
  22. ↵
    1. Hilf G.,
    2. Gierschik P.,
    3. Jakobs K. H.
    (1989) Muscarinic acetylcholine receptor-stimulated binding of guanosine 5′-O-(3-thiotriphosphate) to guanine-nucleotide-binding proteins in cardiac membranes. Eur. J. Biochem. 186:725–731.
    OpenUrlPubMed
  23. ↵
    1. Holtzman S. G.,
    2. Cook L.,
    3. Steinfels G. F.
    (1991) Discriminative stimulus effects of spiradoline, a kappa-opioid agonist. Psychopharmacology 105:447–452.
    OpenUrlCrossRefPubMed
  24. ↵
    1. Kieffer B.,
    2. Befort K.,
    3. Gaveriaux-Ruff C.,
    4. Hirth C. G.
    (1992) The δ-opioid receptor: Isolation of a cDNA by expression cloning and pharmacological characterization [published erratum appears in Proc. Natl. Acad. Sci. U.S.A. 91: 1193,1994]. Proc. Natl. Acad. Sci. U.S.A. 89:12048–12052.
    OpenUrlAbstract/FREE Full Text
  25. ↵
    1. Konkoy C. S.,
    2. Childers S. R.
    (1989) Dynorphin-selective inhibition of adenylyl cyclase in guinea pig cerebellum membranes. Mol. Pharmacol. 36:627–633.
    OpenUrlAbstract
  26. ↵
    1. Konkoy C. S.,
    2. Childers S. R.
    (1993) Relationship between kappa1 opioid receptor binding and inhibition of adenylyl cyclase in guinea pig brain membranes. Biochem. Pharmacol. 45:207–216.
    OpenUrlCrossRefPubMed
  27. ↵
    1. Lawrence D. M. P.,
    2. Bidlack J. M.
    (1992) Kappa opioid binding sites on the R1.1 murine lymphoma cell line: Sensitivity to cations and guanine nucleotides. J. Neuroimmunol. 41:223–230.
    OpenUrlCrossRefPubMed
  28. ↵
    1. Lawrence D. M.,
    2. Bidlack J. M.
    (1993) The kappa opioid receptor expressed on the mouse R1.1 thymoma cell line is coupled to adenylyl cyclase through a pertussis toxin-sensitive guanine nucleotide-binding regulatory protein. J. Pharmacol. Exp. Ther. 266:1678–1683.
    OpenUrlAbstract/FREE Full Text
  29. ↵
    1. Lawrence D. M.,
    2. Joseph D. B.,
    3. Bidlack J. M.
    (1995) Kappa opioid receptors expressed on three related thymoma cell lines. Differences in receptor-effector coupling. Biochem. Pharmacol. 49:81–89.
    OpenUrlCrossRefPubMed
  30. ↵
    1. Lazareno S.,
    2. Farries T.,
    3. Birdsall N. J.
    (1993) Pharmacological characterization of guanine nucleotide exchange reactions in membranes from CHO cells stably transfected with human muscarinic receptors m1–m4. Life Sci. 52:449–456.
    OpenUrlCrossRefPubMed
  31. ↵
    1. Leander J. D.
    (1983a) A kappa opioid effect: increased urination in the rat. J. Pharmacol. Exp. Ther. 224:89–94.
    OpenUrlAbstract/FREE Full Text
  32. ↵
    1. Leander J. D.
    (1983b) Evidence that nalorphine, butorphanol and oxilorphan are partial agonists at a kappa-opioid receptor. Eur. J. Pharmacol. 86:467–470.
    OpenUrlCrossRefPubMed
  33. ↵
    1. Lorenzen A.,
    2. Fuss M.,
    3. Vogt H.,
    4. Schwabe U.
    (1993) Measurement of guanine nucleotide-binding protein activation by A1 adenosine receptor agonists in bovine brain membranes: stimulation of guanosine-5′-O-(3-[35S]thio)triphosphate binding. Mol. Pharmacol. 44:115–123.
    OpenUrlAbstract
  34. ↵
    1. Ma G. H.,
    2. Miller R. J.,
    3. Kuznetsov A.,
    4. Philipson L. H.
    (1995) Kappa-opioid receptor activates an inwardly rectifying K+ channel by a G protein-linked mechanism: coexpression in Xenopus oocytes. Mol. Pharmacol. 47:1035–1040.
    OpenUrlAbstract
  35. ↵
    1. Mansson E.,
    2. Bare L.,
    3. Yang D.
    (1994) Isolation of a human κ opioid receptor cDNA from placenta. Biochem. Biophys. Res. Commun. 202:1431–1437.
    OpenUrlCrossRefPubMed
  36. ↵
    1. McPherson G. A.
    (1983) A practical computer based approach to the analysis of radioligand binding experiments. Comput. Prog. Biomed. 17:107–114.
    OpenUrlCrossRefPubMed
  37. ↵
    1. Miller L.,
    2. Shaw J. S.,
    3. Whiting E. M.
    (1986) The contribution of intrinsic activity to the action of opioids in vitro. Br. J. Pharmacol. 87:595–601.
    OpenUrlPubMed
  38. ↵
    1. Oka T.,
    2. Negishi K.,
    3. Suda M.,
    4. Matsumiya T.,
    5. Inazu T.,
    6. Ueki M.
    (1981) Rabbit vas deferens: a specific bioassay for opioid kappa-receptor agonists. Eur. J. Pharmacol. 73:235–236.
    OpenUrlCrossRefPubMed
  39. ↵
    1. Pfeiffer A.,
    2. Brantl V.,
    3. Herz A.,
    4. Emrich H. M.
    (1986) Psychotomimesis mediated by kappa opiate receptors. Science 233:774–776.
    OpenUrlAbstract/FREE Full Text
  40. ↵
    1. Picker M. J.
    (1994a) Kappa agonist and antagonist properties of mixed action opioids in a pigeon drug discrimination procedure. J. Pharmacol. Exp. Ther. 268:1190–1198.
    OpenUrlAbstract/FREE Full Text
  41. ↵
    1. Picker M. J.
    (1994b) Discriminative stimulus and response rate-decreasing effects of kappa opioids: antagonism by naloxone. Life Sci. 54:1767–1775.
    OpenUrlCrossRefPubMed
  42. ↵
    1. Piercey M. F.,
    2. Lahti R. A.,
    3. Schroeder L. A.,
    4. Einspaher F. J.,
    5. Brasuhn C.
    (1982) U50,488H, a pure kappa receptor agonist with spinal analgesia loci in the mouse. Life Sci. 31:1197–1200.
    OpenUrlCrossRefPubMed
  43. ↵
    1. Portoghese P. S.,
    2. Larson D. L.,
    3. Sayre L. M.,
    4. Fries D. S.,
    5. Takemori A. E.
    (1980) A novel opiate receptor site directed alkylating agent with irreversible narcotic antagonistic and reversible agonistic activities. J. Med. Chem. 23:233–234.
    OpenUrlCrossRefPubMed
  44. ↵
    1. Portoghese P. S.,
    2. Lipkowski A. W.,
    3. Takemori A. E.
    (1987) Binaltorphimine and nor-binaltorphimine, potent and selectivity κ-opioid receptor antagonists. Life Sci. 4:1287–1292.
  45. ↵
    1. Prather P. L.,
    2. McGinn T. M.,
    3. Claude P. A.,
    4. Liu-Chen L. Y.,
    5. Loh H. H.,
    6. Law P.-Y.
    (1995) Properties of a kappa-opioid receptor expressed in CHO cells: interaction with multiple G-proteins is not specific for any individual Gα subunit and is similar to that of other opioid receptors. Mol. Brain Res. 29:336–346.
    OpenUrlCrossRefPubMed
  46. ↵
    1. Romer D.,
    2. Buscher H. H.,
    3. Hill R. C.,
    4. Maurer R.,
    5. Petcher T. J.,
    6. Zeugner H.,
    7. Benson W.,
    8. Finner E.,
    9. Milkowski W.,
    10. Thies P. W.
    (1982) An opioid benzodiazepine. Nature 298:759–760.
    OpenUrlCrossRefPubMed
  47. ↵
    1. Sambrook J.,
    2. Fritsch E. F.,
    3. Maniatis T.
    (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY).
  48. ↵
    1. Sim L. J.,
    2. Selley D. E.,
    3. Childers S. R.
    (1995) In vitro autoradiography of receptor-activated G proteins in rat brain by agonist-stimulated guanylyl 5′-[γ-[35S]thio]triphosphate binding. Proc. Natl. Acad. Sci. U.S.A. 92:7242–7246.
    OpenUrlAbstract/FREE Full Text
  49. ↵
    1. Smith M. A.,
    2. Picker M. J.
    (1995) Examination of the kappa agonist and antagonist properties of opioids in the rat drug discrimination procedure: influence of training dose and intrinsic efficacy. Behav. Pharmacol. 6:703–717.
    OpenUrlPubMed
  50. ↵
    1. Smith P. K.,
    2. Krohn R. I.,
    3. Hermanson G. T.,
    4. Mallia A. K.,
    5. Gartner F. H.,
    6. Provenzano M. D.,
    7. Fujimoto E. K.,
    8. Goeke N. M.,
    9. Olson B. J.,
    10. Klenk D. C.
    (1985) Measurement of protein using bicinchoninic acid. Anal. Biochem. 150:76–85.
    OpenUrlCrossRefPubMed
  51. ↵
    1. Takemori A. E.,
    2. Schwartz M. M.,
    3. Portoghese P. S.
    (1988) Suppression by nor-binaltorphimine of kappa opioid-mediated diuresis in rats. J. Pharmacol. Exp. Ther. 247:971–974.
    OpenUrlAbstract/FREE Full Text
  52. ↵
    1. Tian W. N.,
    2. Duzic E.,
    3. Lanier S. M.,
    4. Deth R. C.
    (1994) Determinants of α2-adrenergic receptor activation of G proteins: evidence for a precoupled receptor/G protein state. Mol. Pharmacol. 45:524–531.
    OpenUrlAbstract
  53. ↵
    1. Traynor J. R.,
    2. Nahorski S. R.
    (1995) Modulation by mu-opioid agonists of guanosine-5′-O-(3-[35S]thio)triphosphate binding to membranes from human neuroblastoma SH-SY5Y cells. Mol. Pharmacol. 47:848–854.
    OpenUrlAbstract
  54. ↵
    1. Verlinde C.,
    2. De Ranter C.
    (1988) Assessment of the kappa-opioid activity of a series of 6,7-benzomorphans in the rabbit vas deferens. Eur. J. Pharmacol. 153:83–87.
    OpenUrlCrossRefPubMed
  55. ↵
    1. Von Voigtlander P. F.,
    2. Lahti R. A.,
    3. Ludens J. H.
    (1983) U-50,488: a selective and structurally novel non-μ (κ) opioid agonist. J. Pharmacol. Exp. Ther. 224:7–12.
    OpenUrlAbstract/FREE Full Text
  56. ↵
    1. Zhu J.,
    2. Chen C.,
    3. Xue J.-C.,
    4. Kunapuli S.,
    5. de Riel J. K.,
    6. Liu-Chen L. Y.
    (1995) Cloning of a human κ opioid receptor from the brain. Life Sci. 56:PL201–PL207.
    OpenUrlCrossRefPubMed
  57. ↵
    1. Zhu J.,
    2. Yin J.,
    3. Law P.-Y.,
    4. Claude P. A.,
    5. Rice K. C.,
    6. Evans C. J.,
    7. Chen C.,
    8. Yu L.,
    9. Liu-Chen L.-Y.
    (1996) Irreversible binding of SUPERFIT to the cloned δ receptor and determination of its binding domain. J. Biol. Chem. 271:1430–1434.
    OpenUrlAbstract/FREE Full Text
Back to top

In this issue

Download PDF
Article Alerts
Email Article
Citation Tools

OtherNEUROPHARMACOLOGY

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

Jinmin Zhu, Lai-Yi Luo, Jian-Guo Li, Chongguang Chen and Lee-Yuan Liu-Chen
Journal of Pharmacology and Experimental Therapeutics August 1, 1997, 282 (2) 676-684;
Reddit logo Twitter logo Facebook logo Mendeley logo

Jump to section

Advertisement