Introduction

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Opioids can differ in their ability to provoke a response once they have bound to their receptor, and these differences in efficacy have been characterized by a number of procedures. Opioid-sensitive in vivo assays, in which different intensities of a particular stimulus are used, have proven very helpful in ordering opioids with respect to their efficacies. In thermal analgesia assays, for example, low-efficacy mu agonists such as nalbuphine or buprenorphine are less able to produce analgesia at warmer temperatures than are high-efficacy agonists such as fentanyl. At warmer temperatures, where the low-efficacy agonists are ineffective, these low-efficacy mu agonists are able to antagonize the analgesia produced by higher efficacy agonists (Walker et al., 1993, 1995). Fewer experiments of this type have been undertaken with kappa agonists. McClane and Martin (1967) showed that the ability of the putative high-efficacy kappa agonist, cyclazocine, or the putative low-efficacy kappa agonist, nalorphine, to depress the extensor reflex in the spinal dog depended in part on the strength of the stimulus that elicited the reflex. A sufficiently large dose of cyclazocine depressed the reflex elicited by the strongest stimulus, but the most effective dose of nalorphine was less able to depress this most intense reaction.

In vitro assay procedures have utilized measures of the maximum membrane binding of [³⁵S]GTPS, a nonhydrolyzable form of GTP, as an indicator of opioid efficacy at their G protein-coupled receptors. In parallel with in vivo assay systems, most in vitro measures have measured efficacy differences with the mu opioid system. In SH-SY5Y cells, in which mu receptors predominate, differences in maximum [³⁵S]GTPS binding produced by a series of mu and kappa agonists were proportional to their differences in presumed efficacy (Traynor and Nahorski, 1995). Subsequently, this finding has been replicated and extended in more selective mu and delta clones (Emmerson et al., 1996; Clark et al., 1997). Liu-Chen and colleagues (Zhu et al., 1997) recently reported kappa efficacy-related differences in maximum [³⁵S]GTPS binding by a number of kappa opioids in a human kappa receptor clone expressed in Chinese hamster ovary (CHO) cells. Dynorphin A 1-17, (±) ethylketocyclazocine, U50488H and -funaltrexamine were among the drugs that exhibited high-efficacy profiles, whereas nalorphine and pentazocine produced decreasing maximum binding levels indicative of reduced efficacy. In the present study, these observations have been extended to the human kappa receptor expressed in the C6 glioma cell line. A large series of opioids that varied in affinity at the kappa receptor as well as having potential differences in efficacy at this site were evaluated. These included a set of stereoisomers with distinct positions of chirality. The aim of this study was to support and extend in the C6 cells the data provided by Zhu et al. (1997) in CHO cells as well as to determine whether different positions of chirality produced distinct changes in efficacy.

	Materials and Methods

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Materials. [³⁵S]GTPS (1300 Ci/mmol) and [³H]naloxone (53 Ci/mmol) were purchased from DuPont-NEN (Boston, MA), and [³H]5,7,8(-)-N-methyl-N-(7-Cl-pyrrolidinyl)-1-oxaspiro(4,5)dec-8-yl)benzene acetamide (U69593; 60 Ci/mmol) was obtained from Amersham (Arlington Heights, IL). The cyclic AMP assay kit was purchased from Diagnostic Products Corp. (Los Angeles, CA). -chlornaltrexamine (-CNA) was obtained from Research Biochemicals International (Natick, MA). All other opioids and their isomers used in this study were obtained through the Opioid Basic Research Center at the University of Michigan (Ann Arbor, MI). Geneticin was purchased from Mediatech, Inc. (Herndon, VA). Pertussis toxin was purchased from List Biological Laboratories, Inc. (Campbell, CA). Dulbecco's modified Eagle's medium (DMEM), fetal bovine serum, Trizma, and other biochemicals were purchased from Sigma (St. Louis, MO).

Cell Culture. The cDNA encoding the human kappa opioid receptor (OR) in a pCDNA3 expression vector (Mansson et al., 1994) was used to stably express the receptors in C6 glioma cells. Twenty micrograms of plasmid DNA was transfected into a 100-mm dish of cells using the method of Chen and Okayama (1987). Two days after transfection, cells were maintained in tissue culture medium (DMEM and 10% fetal bovine serum) with 1 mg/ml geneticin for 14 days. After this selection period, individual cells were removed and plated in 24-well plates, maintaining antibiotic selection pressure. Stably transfected cells were able to grow in the presence of geneticin. Individual colonies were screened for opioid receptor binding and opioid-stimulated [³⁵S]GTPS binding. A single clone (C62a) was used for this study.

Membrane Preparation. Plasma membranes were prepared by lysis of cells in isotonic sucrose (Emmerson et al., 1996). Cells were washed twice with ice-cold phosphate-buffered saline (0.9% NaCl, 0.61 mM Na₂HPO₄, and 0.38 mM KH₂PO₄, pH 7.4). Cells were detached from flasks by incubation in lifting buffer (5.6 mM glucose, 5 mM KCl, 5 mM HEPES, 137 mM NaCl, and 1 mM EGTA, pH 7.4) at 37°C and pelleted by centrifugation at 200g for 3 min. The cell pellet was resuspended in 10 volumes of ice-cold 0.32 M sucrose and 1 mM Tris-HCl (pH 7.4) using a Teflon-glass dounce mounted to a Tri-R Stir-R motor at 1000 rpm. The suspension was then centrifuged for 10 min at 1000g at 4°C, and the supernatant was removed and kept on ice. The resuspension and centrifugation were repeated with the remaining pellet an additional three times, saving the supernatant from each spin in tubes kept on ice, to further break up the membranes and increase the yield. The combined supernatants were then centrifuged at 15,000g for 20 min at 4°C. After the centrifugation, the upper pellet was removed from the lower pellet by gently washing with ice-cold 0.32 M sucrose. The upper pellet was resuspended in 50 mM Tris-HCl buffer (pH 7.4) and centrifuged 20 min at 15,000g and 4°C. The final pellet was resuspended in 50 mM Tris buffer and frozen at 80°C in 0.5-ml aliquots (0.6-1.0 mg/ml).

Protein Determination. Protein concentration was determined by the method of Lowry et al. (1951) using bovine serum albumin standard. Samples were dissolved with 1 N NaOH for 30 min at room temperature before protein determination.

Receptor-Binding Assay. Ligand binding was carried out as described previously (Fischel and Medzihradsky, 1981). In brief, the assay medium for determination of [³H]U69593 binding contained membrane protein (20 µg) diluted in Tris-Mg buffer (50 mM Tris-HCl and 5 mM MgCl₂, pH 7.4), 50 µl of water or unlabeled ligand (1 µM naloxone final concentration for maximum specific displacement), and 25 µl of [³H]U69593 (0.09-7 nM) in a final volume of 525 µl. After the membranes were preincubated for 15 min at 25^oC in the assay buffer, the binding was initiated by addition of unlabeled and radiolabeled ligands. After incubation for 90 min at 25°C to reach equilibrium, the samples were quickly filtered through glass fiber filters (No. 32, Schleicher & Schuell, Keene, NH) and mounted in a Brandel cell harvester (Biomedical Research and Development Laboratories, Gaithersburg, MD). Each filter was removed and placed in a 5-ml polypropylene scintillation vial with 0.4 ml of ethanol and 4 ml of Ultima Gold (Packard Instrument Co., Meriden, CT) scintillation cocktail and subjected to liquid scintillation counting.

[³⁵S]GTPS-Binding Assay. Agonist stimulation of [³⁵S]GTPS binding was measured as described by Tian et al. (1994). Membranes (5-20 µg/tube) were mixed with ligand and preincubated for 10 min at 25°C. The experiment was initiated by the addition of [³⁵S]GTPS to yield a final concentration in 100 µl of 50 mM Tris-HCl, 100 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol (added fresh), 50 µM GDP, and 50 pM [³⁵S]GTPS (pH 7.4). Tubes were incubated for 30 min at 25°C and the reaction was terminated by diluting the sample with 2 ml of ice-cold 50 mM Tris-HCl buffer containing 5 mM MgCl₂ and 100 mM NaCl and rapidly filtering the tube contents through glass fiber filters (Schleicher & Schuell 32). The filters were then washed an additional three times with 2 ml of buffer. Filters were placed in vials containing 400 µl of ethanol and 4 ml of Econo-Safe scintillation cocktail for liquid scintillation counting. Basal activity was defined by the difference between the [³⁵S]GTPS binding in the absence or presence of 50 µM unlabeled GTPS. To determine the percentage of increase in [³⁵S]GTPS binding over basal, binding in the presence of 50 µM unlabeled GTPS and the basal binding was subtracted from each point, and each value was divided by the basal value and then multiplied by 100%. The experiment was performed three to four times in duplicate.

Whole-Cell Adenylyl Cyclase Assay. Inhibition of forskolin-stimulated adenylyl cyclase was performed as described previously (Clark et al., 1997).

Data Analysis. Saturation binding data for [³H]U69593 and [³H]naloxone were fit to a one-site binding hyperbola using GraphPad Prism. [³⁵S]GTPS-binding data from three to five experiments were combined and fit to a sigmoidal curve with a variable slope using GraphPad Prism and radioligand-binding displacement curves were best fit to one-site competition curves. K_i values were calculated as IC₅₀/(1 + [³HL]/K_d) (Cheng and Prusoff, 1973) using 13.7 nM for the naloxone K_d value and 0.6 nM for the U69593 K_d value. Efficacy was calculated as the fraction of the maximum stimulation of [³⁵S]GTPS binding by [N-methyl-Tyr¹,N--methyl-Arg⁷-D-Leu⁸]dynorphin A-(1-8)ethylamide (E2078).

	Results

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OR Expression in C6 Glioma Cells. The level of OR expression was significantly lower in six different C6 glioma cells stably expressing the human OR (C6) clones as compared with clones expressing either the mu opioid receptor (Emmerson et al., 1996) or the delta opioid receptor (Clark et al., 1997). Membrane preparations from these cells expressed approximately 0.1 to 1 pmol of receptor/mg membrane protein (Fig. 1). The agonist-stimulated [³⁵S]GTPS binding was dependent on receptor density. The binding data in Fig. 2 as well as all subsequent experiments were performed in the clone expressing the greatest amount of receptor. However, the expression level in a single clone was dependent on the lot of fetal bovine serum in the cell culture media. Because opioid-stimulated GTPS binding is dependent on receptor expression levels, all [³⁵S]GTPS-binding assays examining agonist efficacy were performed in membranes with approximately 2.8 pmol receptor/mg membrane protein. In control experiments, we found that the EC₅₀ values for E2078 stimulation of [³⁵S]GTPS binding are similar in membranes expressing 101 to 4433 fmol receptor/mg membrane protein (data not shown), suggesting that agonist potency is independent of receptor number under the conditions of the experiments performed here.

View larger version (22K): [in this window] [in a new window]   Fig. 1.   Receptor expression and [35S]GTPS binding in six different C6 clones. Membranes from six different clones were prepared (crude membrane prep) as described in Materials and Methods. Receptor density was estimated using the specific binding of 7.7 nM [3H]naloxone and a Kd value of 13.7 nM. Agonist (1 µM EKC)-stimulated GTPS binding (pmol/mg membrane protein) was determined as described in Materials and Methods.

View larger version (24K): [in this window] [in a new window]   Fig. 2.   [ 3H]naloxone and [ 3H]U69593 equilibrium binding to C6 membranes. A, plasma membranes were preincubated for 15 min at 25°C in 50 mM Tris, pH 7.4, and 5 mM MgCl2 as described in Materials and Methods. [3H]U69593 was added and samples were incubated an additional 90 min at 25°C. Nonspecific binding was determined using 10 µM U69593. Shown is the combined data from two assays, with each point measured in duplicate. B, [3H]naloxone was incubated with C6 membranes as described above except in the same buffer used for GTPS-binding assays (except omitting [35S]GTPS). Specific binding at each concentration of [3H]naloxone was determined by complete displacement with 10 µM naloxone. Shown is the combined data from two assays, with each point measured in duplicate.

Evaluation of Ligand-Binding Affinities. Equilibrium binding of agonist [³H]U69593 and antagonist [³H]naloxone revealed a single population of saturable binding sites on membranes prepared from C6 (Fig. 2). K_i values for several agonists and antagonists were determined by displacement of [³H]naloxone or [³H]U69593 (Table 1). The [³H]naloxone-binding assay was performed under the same conditions as the measurements of [³⁵S]GTPS binding in buffer containing 100 mM NaCl and 50 µM GDP. The [³H]U69593 displacement assay was performed in the presence of 5 mM MgCl₂. Seven ligands ([(trans)-3,4-dichloro-N-methyl-N-[2-(2-pyrrolidinyl)-cyclohexyl]benzeneacetamide (U50488H), dynorphin 1-17, ethylketocyclazocine (EKC), nalorphine, nalbuphine, naloxone, and norBNI) had very similar affinities for the human OR expressed in C6 glioma cells as compared with human OR expressed in CHO cells (Table 1; Zhu et al., 1997). We extended the evaluation of opioid affinity and efficacy at the OR by examining several other pharmacologically interesting opioids. Inhibition of agonist binding by 100 mM sodium and 50 µM GDP was evident by an approximate 10-fold shift in binding affinity (Table 1). Several agonists (oxilorphan, nalmefene, levallorphan, cyclazocine, and -CNA) displayed subnanomolar affinities for the human OR, whereas agonist nalorphine had nanomolar affinity.

View this table: [in this window] [in a new window]   TABLE 1 Binding affinity and efficacy of stimulation of [35S]GTPS binding in membranes from C6 cells stably transfected with OR

Determination of Potencies and Efficacies of Opioid Ligands to Stimulate [³⁵S]GTPS Binding. Identical conditions were used here to evaluate OR pharmacology as were used to examine mu (Emmerson et al., 1996) and delta (Clark et al., 1997) efficacy. The kappa agonist-stimulated [³⁵S]GTPS-binding dependence on the GDP concentration was identical with that observed for mu- (Emmerson et al., 1996) and delta- (Clark et al., 1997) stimulated binding (data not shown). The presence of 50 µM GDP markedly reduced basal [³⁵S]GTPS binding, which yielded the highest possible sensitivity to agonist stimulation. Maximal stimulation of [³⁵S]GTPS binding was significantly lower here (188 ± 6%) (Fig. 3 and Table 1) compared with the mu and delta opioid receptors expressed in C6 glioma which increased [³⁵S]GTPS binding by 300 and 600%, respectively (Emmerson et al., 1996; Clark et al., 1997). As observed in C6µ and C6 cell membranes, agonist potency, as measured by EC₅₀ to stimulate [³⁵S]GTPS binding, did not correlate with efficacy. Efficacy was measured by the ratio of maximal stimulation of [³⁵S]GTPS binding for the ligand in question to the maximal stimulation produced by E2078. E2078, bremazocine, EKC, U50488H, and U69593 are all full agonists at the OR with E2078, a hydrolysis-resistant dynorphin derivative, slightly more efficacious than all of the other compounds (Fig. 3, A and B). Both dynorphin 1-17 and 1-13 and both isomers of tifluadom stimulated [³⁵S]GTPS binding by approximately 150%, although the pairs had greatly differing potencies (Fig. 3B). Cyclazocine, -CNA, levallorphan, oxilorphan, nalorphine, and nalmefene were all partial agonists whereas norBNI and naloxone possessed no agonist properties (Fig. 3C; Table 1). In control experiments, relative agonist (E2078, EKC, and cyclazocine) efficacy as well as EC₅₀ values were independent of receptor number (femtomoles receptor per milligram of membrane protein). In preparations containing 1540, 2850, and 4433 fmol receptor/mg membrane protein, EC₅₀ values for these three agonists did not differ and the rank order efficacy was identical where E2078  EKC > cyclazocine (data not shown).

View larger version (31K): [in this window] [in a new window]   Fig. 3.   Stimulation of GTPS binding. Plasma membranes were preincubated with opioids for 10 min at 25°C in the GTPS binding assay buffer. [35S]GTPS was added and the samples were incubated for an additional 30 min at 25°C. The reaction was terminated by rapid filtration and the samples processed as described in Materials and Methods. The data are expressed as stimulation relative to GTPS binding in the absence of added ligand (0.1-0.2 pmol/mg membrane protein). Shown are the mean and S.E. for three to four experiments, each carried out in duplicate. The data were fit to a four parameter logistic equation and the curve shown. The efficacy and EC50 values are listed in Table 1.

The Relationship between Stereoselectivity and Efficacy. The () isomer of tifluadom displayed agonist properties, albeit at 32-fold lower potency than (+)-tifluadom (Fig. 3B; Table 1). The inactive isomers of EKC (10 µM d-EKC), cyclazocine (10 µM (+)-cyclazocine), and pentazocine (10 µM d-pentazocine) did not stimulate [³⁵S]GTPS binding (Fig. 4). In addition, neither 10 nor 100 µM (+)SKF10047 was able to stimulate [³⁵S]GTPS binding. Although 10 µM dextrallorphan stimulated [³⁵S]GTPS binding by 15 ± 7%, the increase was not blocked by 1 µM norBNI.

View larger version (27K): [in this window] [in a new window]   Fig. 4.   Stimulation of [35S]GTPS binding by isomers of opioid ligands. Ten micromolar ligand was preincubated for 10 min with C6 membranes before addition of [35S]GTPS. The binding assay was performed as described in Materials and Methods. The effect of dextrallorphan was not blocked by 1 µM norBNI.

Antagonism of the Stimulation of [³⁵S]GTPS Binding by NorBNI. NorBNI is a OR-selective potent antagonist (Portoghese et al., 1987). When 1 nM norBNI was added at the same time as agonist, it inhibited the ability of EKC, nalorphine, and cyclazocine to stimulate [³⁵S]GTPS binding by 16- to 18-fold (Fig. 5). Very similar K_e values (0.06-0.07 nM) for inhibition of the full and two partial agonists response indicate that all three agonists are activating the same receptor.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Stimulation of [35S]GTPS binding in the presence of norBNI. Indicated concentrations of agonist were preincubated with C6 membranes in the absence (filled symbols) or presence (open symbols) of 1 nM norBNI for 10 min. The binding assay and data analysis were performed as described in Materials and Methods. The shift in EC50 caused by 1 nM norBNI for EKC, nalorphine, and cyclazocine was 18.9-fold, 17.5-fold, and 16.0-fold, respectively. The calculated KI values for norBNI inhibition were 0.07 nM, 0.06 nM, and 0.06 nM, respectively.

	Discussion

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The purpose of this study was to examine the efficacy of several ligands at the human OR by evaluating the magnitude of OR-mediated stimulation of [³⁵S]GTPS binding. After the cloning of the human OR (Mansson et al., 1994; Simonin et al., 1995), receptor pharmacology and function has been examined after stable (Zhu et al., 1997; Blake et al., 1997) and transient (Simonin et al., 1995) expression in a variety of cell lines and compared with the pharmacology of the mouse and rat clones (Simonin et al., 1995). Given the widespread distribution of the OR in the human brain and spinal cord (Simonin et al., 1995), the efficacy of ligands at the human OR is of great interest in the evaluation of compounds for potential clinical use. We have extended an initial characterization (Zhu et al., 1997) and have evaluated the efficacy of several compounds at the human OR by examination of ligand-stimulated [³⁵S]GTPS binding. There appear to be no significant spare receptors in this assay in that the EC₅₀ values for ligand-stimulated [³⁵S]GTPS binding are higher than the ligand affinities determined under identical assay conditions. The relatively high OR expression level in these cells compared with brain tissue, and the lack of spare receptors, provide a robust agonist stimulation of [³⁵S]GTPS binding for full agonists. The observation that receptor expression roughly correlates with agonist-stimulated GTPS binding also suggests that there are no spare receptors.

The affinity of agonist [³H]U69593 for the C6 receptor (0.6 nM) is similar to that observed in monkey membranes (0.95 nM; Emmerson et al., 1994) as well as in membranes prepared from PC12 cells stably expressing the cloned mouse OR (2.8 nM; Raynor et al., 1994). The naloxone affinity for the OR is approximately 7-fold weaker than that observed in monkey brain membranes (Emmerson et al., 1994), yet it is comparable with the affinity observed for naloxone in CHO cells stably expressing the human OR (4.5 ± 1.1 nM; Zhu et al., 1997). Similar values were obtained in cell membrane preparations from COS-1 cells transiently expressing the rat and human OR (Meng et al., 1995; Simonin et al., 1995).

In a general comparison of our and Liu-Chen's data (Zhu et al., 1997), ligand efficacy at the human kappa receptor is apparently independent of the cell line in which the receptor is expressed. E2078, a potentially clinically useful stable derivative of dynorphin that is able to penetrate the blood-brain barrier (Terasaki et al., 1989, 1991), was most efficacious in increasing [³⁵S]GTPS binding by 188 ± 6%, whereas several peptide and nonpeptide kappa agonists (dynorphin 1-17, dynorphin 1-13, bremazocine, EKC, U50488H, U69593, and tifluadom isomers) displayed high efficacy (~ 150% stimulation of [³⁵S]GTPS binding). Of these agonists, bremazocine, EKC, U69593, and U50488H were full agonists when tested for analgesic activity by measuring the latency for monkeys to remove their tails from a thermos containing 55° C water (France et al., 1994). In addition, the binding K_i values for bremazocine, EKC, U50488, and nalorphine in the C6 cells correlate well with the dose required to produce a half-maximal EKC-discriminative stimulus effects in monkeys (France et al., 1994). Surprisingly, both dynorphin 1-17 and 1-13 had similar efficacies in the stimulation of [³⁵S]GTPS binding although dynorphin 1-13 was approximately 30-fold less potent.

Based on the limited ligands selective for putative OR subtypes, the human clone appears to be similar to the kappa₁ subtype. The C6 cells are U69593 sensitive; U69593 binds selectively, and with high affinity, to the kappa₁ but not kappa₂ site (Zukin et al., 1988). Nalorphine has been characterized as a kappa₃ analgesic. Animals tolerant to U50488H were not tolerant to nalorphine (Paul et al., 1991). In the C6 cells, we found that norBNI was equally efficacious to block EKC, cyclazocine, and nalorphine-stimulated [³⁵S]GTPS binding, suggesting that although nalorphine may interact with the kappa₃ opioid receptor, in this cell line, it is functional at the kappa₁ opioid receptor subtype.

By suppression of basal [³⁵S]GTPS binding by excess GDP, we were able to evaluate partial agonist efficacy. Efficacy differences at the mu opioid receptor were magnified by increasing GDP concentrations, indicating that the activity state of G proteins can affect agonist efficacy (Selley et al., 1997). Several of the partial agonists have been previously characterized as mu opioid receptor antagonists. For example, nalmefene is marketed as a long-acting mu-selective antagonist with no efficacy. However, monkey discriminative effects at the OR have been observed (Woods et al., 1986) in addition to high affinity for both the monkey mu and kappa opioid receptor (0.13 and 0.28 nM, respectively (Emmerson et al., 1994)). The data presented here suggest that nalmefene is a potent yet weak partial agonist and may exhibit its discriminative effects via the OR.

Natural opioids are levorotatory, whereas several of the synthetic opioids are racemic. The benzomorphan dextrorotatory (+)-enantiomers do not possess opioid activity (for review, see Musacchio, 1990). The ability of isomers of tifluadom and levallorphan to stimulate [³⁵S]GTPS binding indicates that the chiral carbon of levallorphan, a benzomorphan derivative, imparts a greater degree of stereoselectivity than does the chiral carbon in the benzodiazepine derivative tifluadom. In addition ()-tifluadom, the less potent isomer of tifluadom, which is also a GABA_A agonist, stimulated [³⁵S]GTPS binding. In contrast, d-pentazocine, (+)SKF10047, (+)cyclazocine, and (d)EKC displayed no agonist activity. The (+) isomer of SKF10047 is a putative sigma receptor ligand and an N-methyl-D-aspartate receptor noncompetitive antagonist.

One striking difference between the results obtained here and those in the CHO cells is the sensitivity to sodium and GDP observed here. Despite the observation that several ligands had similar efficacies and affinities (in the presence of GDP and sodium) for the human OR expressed in C6 glioma cells as compared to human OR expressed in CHO cells, we found the C6 receptor to be sensitive to sodium and GDP whereas the CHO cell-expressed human OR was not (Zhu et al., 1997). Relative lack of OR sensitivity to sodium compared to the mu opioid receptor was also described in monkey brain membrane preparations (Emmerson et al., 1994); however, ligand binding in guinea pig cerebellar membranes was strongly inhibited by NaCl and a nonhydrolyzable guanine nucleotide analog (Gairin et al., 1989). In addition, agonist binding to the OR (kappa₁ subtype) expressed in the mouse R1.1 lymphoma cell line was reduced to 30% of control binding by 30 mM NaCl and 100 µM GTP (Lawrence and Bidlack, 1992).

We observed minimal OR-mediated inhibition of adenylyl cyclase in contrast to the results of Liu-Chen and colleagues (Zhu et al., 1997) and in contrast to stably expressed mu and delta opioid receptors in C6 glioma cells. Differences in adenylyl cyclase inhibition in the two clonal cell lines (CHO and C6 glioma cells) may be due to a different complement of G proteins in CHO and C6 glioma cells or to the presence of an yet undescribed factor which modulates opioid receptor sodium and/or guanine nucleotide sensitivity. The isoform of adenylyl cyclase as well as the G protein subunit (alpha or beta-gamma) that mediates inhibition is unknown in the C6 glioma cells and the mechanisms of inhibition may vary between receptor types (mu, delta, and kappa). In these same clones, Gutstein et al. (1997) found that mu and delta receptor stimulation activated extracellular signal-related kinase but kappa stimulation did not.

Prather et al. (1995) found that rat OR stably transfected in CHO cells was able to interact with multiple G proteins (Go, Gi₂, and Gi₃) as measured by agonist-stimulated incorporation of azidoanilido-GTP. This pattern was not unlike that observed for both delta and mu opioid receptors, indicating that receptors did not selectively couple to a single isoform of G protein. The coupling of OR to G protein was apparently weaker, as measured by an agonist-stimulated increase in GTPS binding over basal binding, than that observed in C6 cells expressing the mu or delta opioid receptor (Yabaluri and Medzihradsky, 1996, Clark et al., 1997). These differences may be due to the relatively lower expression levels observed in the OR clones. Indeed, when additional experiments were performed to evaluate agonist EC₅₀ values over a range of receptor expression levels, the extent of kappa agonist-stimulated GTPS-binding via the OR was similar to that of delta agonists acting via the OR with similar receptor expression levels (data not shown). Lower expression levels of the OR were also observed when all three opioid receptors were individually expressed in baculovirus-infected insect cells (Massotte et al., 1997).

By the characterization of the binding affinity and efficacy at the OR of a wide range of opioids, the results of this study contribute to the assessment of opioid efficacy in stimulating G protein, a first step in the signal transduction cascade. An assessment of ligands that vary in efficacy at the OR at the cellular level will promote the use of kappa opioid ligands as pharmacological tools and perhaps as potential therapeutic agents.