Introduction

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Activation of the delta opioid receptor³ is suggested to play a role in multiple behavioral and physiological effects ranging from analgesia and mood-driven behaviors to olfaction and gastrointestinal motility (for review, see Dhawan et al., 1996). Delta opioid receptors are members of the seven-transmembrane G protein-coupled receptor superfamily (for review, see Reisine and Bell, 1993). Delta opioids, acting at the delta opioid receptor, have mediated the inhibition of forskolin-stimulated adenylyl cyclase (Evans et al., 1992), an increase in the production of inositol phosphates (Tsu et al., 1995) as well as modulation of ion channel opening (Taussig et al., 1992; Ikeda, 1996) in a pertussis toxin-sensitive manner.

The delta opioid receptor transduces its signal through inhibitory G proteins, G_i2, G_i3, and G_o in tumor cell lines (Taussig et al., 1992; Prather et al., 1994a) and in vivo (Hescheler et al., 1987; Sanchez-Blazquez and Garzon, 1993). The G proteins G_o, G_i1,2 and G_i3 have also been found to mediate inhibition of adenylyl cyclase by delta opioids (McKenzie and Milligan, 1990; Carter and Medzihradsky, 1993). There have also been reports of coupling of the delta opioid receptor to the stimulatory G protein (Shen and Crain, 1990; Gintzler and Xu, 1991). In addition, intrathecal administration in mice of antisense oligodeoxynucleotides directed against G_s blocked DPDPE-induced spinal analgesia (Standifer et al., 1996). Thus, we were interested in determining if the delta opioid receptor expressed in C6 cells would interact with the stimulatory G protein.

Although definitive proof of delta opioid receptor subtypes has yet to be provided, behavioral and biochemical data suggest the presence of two receptor subtypes (Sofuoglu et al., 1991; Jiang et al., 1991; Buzas et al., 1994; for review see Traynor and Elliott, 1993). Study of a homogeneous delta receptor population was mostly limited to the NG108-15 neuroblastoma cell line until the cloning of the delta opioid receptor (Evans et al., 1992; Kieffer et al., 1992). The pharmacological properties of the three cloned opioid receptors have also been investigated in COS monkey fibroblast cells and the CHO cell line (Raynor et al., 1994). They proposed that the cloned delta opioid receptor has a pharmacological profile similar to that of the "delta-2" opioid receptor subtype. In addition to examining the pharmacology in non-neural cell lines, the ligand binding was determined with use of conditions to maximize agonist interactions. We were interested in examining the pharmacological profile of the cloned rat delta opioid receptor expressed in C6 glioma cells.

Although there is a fair amount of data describing ligand affinities at the delta opioid receptor, there is less information regarding ligand efficacy. Drugs with similar receptor binding affinities are not necessarily equally efficacious in evoking behavioral or biochemical responses. Efficacy is of importance for potential analgesic drugs; maximally efficacious ligands may relieve more severe pain than weaker agonists. In a continuation of our interest in studying the opioid receptor in its native milieu, we examined ligand pharmacology and efficacy at the cloned rat delta opioid receptor stably expressed in a rat C6 glioma cell line under somewhat more physiological conditions (100 mM sodium chloride and 50 µM GDP). Recent results from our laboratory (Yabaluri and Medzihradsky, 1997) support a role for extracellular sodium in regulating not only the ligand interactions with the receptor, but also signal transduction through the mu opioid receptor. We estimated the efficacy of coupling to the G protein by measuring the agonist-stimulated binding of the nonhydrolyzable GTP analog, [³⁵S]GTPS, which has been an effective method of assessing mu opioid efficacy in SH-SY5Y neuroblastoma cell membranes (Traynor and Nahorski, 1995), C6 glioma cells transfected with the cloned rat mu opioid receptor (Emmerson et al., 1996), as well as in COS cells transiently expressing the mouse delta opioid receptor (Befort et al., 1996). Under conditions of physiological extracellular sodium concentration and in the presence of GDP, we determined the efficacies for stimulation of [³⁵S]GTPS binding and the ligand affinities for the delta opioid receptor, and found no evidence for the cloned receptor favoring delta-1- or delta-2-selective ligands. Given the robust signal, we were also able to determine efficacies for relatively weak agonists at the delta opioid receptor. Given the essential role of receptor-G protein coupling in delta opioid receptor signal transduction, the efficacy of opioid ligands at the delta opioid receptor to activate G proteins in vitro may be an important indicator of their in vivo efficacy.

	Materials and Methods

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Materials. [³⁵S]GTPS (1300 Ci/mmol), [³H]naltrindole (32 Ci/mmol) and [³H]pCl-DPDPE (41 Ci/mmol) were purchased from DuPont NEN (Boston, MA). The cAMP assay kit was purchased from Diagnostic Products (Los Angeles, CA). BW373U86 was obtained from Burroughs Wellcome Co. (Research Triangle Park, NC). DPDPE and pCl-DPDPE were generous gifts from H. Mosberg (University of Michigan). Naltrindole, NTB, BNTX, ICI 174864, SINTX, SIOM, SNC80, DSLET, deltorphin II, etorphine, U69,593, oxymorphindole and diprenorphine were obtained through the Opioid Basic Research Center at the University of Michigan (Ann Arbor, MI). Fetal bovine serum and Geneticin were purchased from Gibco Life Sciences (Gaithersberg, MD). Pertussis toxin and cholera toxin were purchased from List Biochemicals (Campbell, CA). DAMGO, DMEM, Trizma and other biochemicals were purchased from Sigma Chemical (St. Louis, MO).

Cell culture. The cDNA encoding the rat delta opioid receptor was cloned by Meng et al. (1995), and the coding region is identical with the sequence reported by Fukuda et al. (1993). A pCMV-neo expression vector, courtesy of Dr. Mike Uhler (University of Michigan) (Huggenvik et al., 1991), was used to express the receptors in C6 glioma cells. Twenty micrograms of plasmid DNA were transfected into a 100-mm dish of cells by 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. Individual colonies were screened for opioid receptor binding, and a single clone (C613) 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 two times with ice-cold phosphate-buffered saline (0.9% NaCl, 0.61 mM Na₂HPO₄, 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, 1 mM ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, pH 7.4) at 37°C and pelleted by centrifugation at 200 × g for 3 min. The cell pellet was resuspended in 10 volumes of ice-cold 0.32 M sucrose, 1 mM Tris HCl (pH 7.4) with a Teflon-glass dounce mounted to a Tri-R Stir-R motor at 1000 rpm. The suspension was then centrifuged for 10 min at 1000 × g at 4°C, and the supernatant was removed and kept on ice. The resuspension and centrifugation was 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 15,000 × g 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 20,000 × g 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) with a 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). The assay medium for determination of [³H]pCl-DPDPE binding contained membrane protein (8.3 µg, 41 fmol receptor) diluted in Tris-Mg buffer (50 mM Tris HCl, 5 mM MgCl₂, pH 7.4), 50 µl water or unlabeled ligand (1 µM pCl-DPDPE final concentration for maximum specific displacement) and 25 µl [³H]pCl-DPDPE (0.09-10 nM) in a final volume of 525 µl. The assay medium for [³H]naltrindole contained membrane protein (4.8-5.6 µg, 28 fmol receptor), water or unlabeled ligand (1 µM naltrindole final concentration for maximum specific displacement) and [³H]naltrindole (final concentration of 0.04 nM for the competition experiment or 0.01-1.4 nM for saturation curve) in 50 mM Tris HCl, 100 mM NaCl, 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol, 50 µM GDP in a total volume of 2 ml. After the membranes were preincubated for 15 min at 25°C 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 (Schleicher and Schuell no. 32, Keene, NH) 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 ethanol and 4 ml scintillation cocktail and subjected to liquid scintillation counting. For the determination of K_i values (0.04 nM [³H]naltrindole), seven concentrations of competing ligand in duplicate were included in the binding assay. K_i values were calculated from the EC₅₀ for inhibition of the specific binding of 0.04 nM [³H]naltrindole in [³⁵S]GTPS binding assay buffer obtained from two to three experiments and analyzed by the one-site competition curve fit with Graph Pad Prism (San Diego, CA).

[³⁵S]GTPS binding assay. Agonist stimulation of [³⁵S]GTPS binding was measured as described by Tian et al. (1994). Membranes (5-7 µg/tube) were mixed with ligand and preincubated for 10 min at 25°C. The experiment was initiated by the addition of assay buffer 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, 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 and Schuell no. 32). The filters were then washed an additional three times with 2 ml of buffer. Filters were placed in vials containing 400 µl ethanol and 4 ml 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 percent of increase in [³⁵S]GTPS binding over basal, 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. Cells grown to confluence were washed twice with phosphate-buffered saline (as above) and lifted off the surface by incubation with lifting buffer as described above. The cell suspension was then centrifuged for 3 min at 200 × g and the pellet was resuspended in A2 buffer (128 mM NaCl, 2.4 mM KCl, 1.3 mM CaCl₂, 2.0 mM NaHCO₃, 3.0 mM MgSO₄, 10 mM Na₂HPO₄, 10 mM glucose, 8 mM theophylline, pH 7.4). After 10 min of preincubation at 37°C, inhibition of adenylyl cyclase activity was initiated by the addition of 50 µl of cells (5-10 µg protein) to 50 µl of A2 buffer with forskolin (final concentration, 10 µM) and opioid. The assay was terminated after 15 min (37°C) by the addition of 50 µl of ice-cold 0.15 M HCl. The samples were heated at 80°C for 3 to 4 min, and then frozen at 80°C overnight. After thawing, the samples were neutralized with 0.5 M Tris and the cAMP content was determined with a radioligand binding assay kit from Diagnostic Products (Los Angeles, CA). The experiment was performed four to five times in duplicate.

Data analysis. [³⁵S]GTPS binding and adenylyl cyclase data from three to five experiments were combined and fit to a sigmoidal curve with a variable slope with use of GraphPad Prism, and the 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) with 0.027 nM for the naltrindole K_d value. Saturation binding data for [³H]pCl-DPDPE and [³H]naltrindole were fit to a one-site binding hyperbola. Efficacy was calculated as the fraction of the maximum stimulation of [³⁵S]GTPS binding by BW373U86. For adenylyl cyclase inhibition, efficacy was calculated as the fraction of the maximum inhibition of adenylyl cyclase by BW373U86. Unpaired, two-tailed t tests comparing control with drug addition (fig. 4) or toxin treatment (fig. 5) were performed with the GraphPad Prism.

View larger version (25K): [in this window] [in a new window]   Fig. 4.   Stimulation of [35S]GTPS binding by BW373U86 or DPDPE in the presence of other agonists or antagonist. The percent of stimulation of [35S]GTPS binding over basal by 10 nM BW373U86 or 3 µM DPDPE in the absence (control) or presence of 10 µM deltorphin II, 10 µM DPDPE, 10 µM oxymorphindole or 10 µM ICI 174,864 was measured as described under "Materials and Methods." All ligands were added to membranes 10 min before the [35S]GTPS binding assay was initiated. Shown are the mean and standard error for three experiments, each measured in duplicate, with the basal amounts measured in triplicate. Asterisks indicate a significant (P < .05) difference compared with control values.

View larger version (26K): [in this window] [in a new window]   Fig. 5.   [35S]GTPS binding in membranes from cells treated with pertussis toxin or cholera toxin. Cells were treated for 24 hr with pertussis toxin (PTX), cholera toxin (CTX) or without treatment (control) and membranes were prepared as described under "Materials and Methods." Shown is the mean and standard error of [35S]GTPS bound (pmol/mg) from three experiments in the presence of no ligand (basal), 1 µM BW373U86, 10 µM DSLET or 10 µM DPDPE for each treatment, measured as described under "Materials and Methods." Each experiment was carried out in triplicate. Asterisks indicate a significant (P < .05) difference compared with control values.

	Results

Top Abstract Introduction Materials & Methods Results Discussion References

Radioligand binding to the delta opioid receptor in C6 membranes. Equilibrium binding of antagonist [³H]naltrindole revealed a single population of saturable binding sites on membranes prepared from C6 glioma cells stably expressing the rat delta opioid receptor (fig. 1). Membrane preparations from these cells routinely expressed 4 to 6 pmol receptor/mg membrane protein. The binding affinity (0.03 nM) was similar to that found for [³H]naltrindole binding in monkey brain membrane preparations (0.04 nM, Emmerson et al., 1994). Saturation binding of [³H]pCl-DPDPE in 50 mM Tris, pH 7.4, containing 5 mM MgCl₂ (Tris-Mg buffer), conditions which typically favor high-affinity binding, was also best described by a single saturable binding site. The K_d was 1.5 nM, which is similar to the K_d of 1.2 nM found for [³H]pCl-DPDPE in monkey cortex membranes (Emmerson et al., 1994). The B_max was 3.3 pmol/mg protein. (data not shown). In contrast, approximately 35 fmol/mg [³H]DPDPE binding sites were detected in monkey brain membranes (Emmerson et al., 1994). K_i values for several ligands were determined by displacement of [³H]pCl-DPDPE in Tris-Mg buffer (table 1).

View larger version (25K): [in this window] [in a new window]   Fig. 1.   [3H]Naltrindole equilibrium binding in GTPS binding assay buffer. Plasma membranes were preincubated for 15 min at 25°C in the same buffer used for GTPS binding assays (except for [35S]GTPS) as described under "Materials and Methods." [3H]Naltrindole was added and the samples were incubated an additional 90 min at 25°C. Specific binding at each concentration of [3H]naltrindole is shown in (a) and the Scatchard plot of these data is shown in the inset (b). Shown are the combined data from two assays, with each point measured in duplicate.

Stimulation of [³⁵S]GTPS binding by delta opioid agonists. Stimulation of [³⁵S]GTPS binding by agonists, dependent on the presence of GDP and NaCl, has been described as a useful measurement of G protein activation by agonists (Wieland and Jakobs, 1994). This method was used as a measurement of efficacy for several agonists in membranes from C6 glioma cells stably expressing the mu opioid receptor (C6µ cells) (Emmerson et al., 1996). The stimulation of [³⁵S]GTPS binding by opioid agonist depended on the presence of GDP. Agonist (BW373U86) stimulation of 0.05 nM [³⁵S]GTPS binding was not observed in the absence of GDP. Maximal stimulation (% of basal) was observed in the presence of 50 µM GDP (data not shown). [³⁵S]GTPS binding increased in a linear fashion for 30 min. Basal binding of [³⁵S]GTPS was 0.01 to 0.02 pmol [³⁵S]GTPS/mg protein/30 min and was similar to the basal level found in the C6µ membrane preparation (Emmerson et al., 1996). The nonpeptide delta opioid agonists BW373U86 and SNC80 stimulated basal [³⁵S]GTPS binding by 640 ± 20% (EC₅₀, 1.3 nM) and 650 ± 20% (EC₅₀, 57 nM), respectively, over basal [³⁵S]GTPS binding (fig. 2a, table 1). The maximum increase in [³⁵S]GTPS binding of 0.10 pmol/mg was similar to the increase observed by mu agonists in C6µ cells, despite the higher level of receptor expression in the C6µ cells (Emmerson et al., 1996). The peptide delta opioid agonists DSLET and pCl-DPDPE were also highly efficacious with a maximum stimulation of [³⁵S]GTPS binding of 576 ± 20% (EC₅₀, 72 nM) and 510 ± 20% (EC₅₀, 69 nM).

View larger version (23K): [in this window] [in a new window]   Fig. 2.   Stimulation of GTPS binding. Plasma membranes were preincubated with opioids for 10 min at 25°C. [35S]GTPS and the GTPS binding assay buffer were added, and the samples were incubated for an additional 30 min at 25°C. The reactions were terminated by rapid filtration as described under "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 standard error for three to four experiments, each carried out in duplicate. The efficacy values are listed in table 1.

Evaluation of the delta opioid receptor subtype. To ensure that the difference in efficacies between the presumably full delta opioid agonists were not caused by the presence of both delta-1 and delta-2 receptors, and to assess the delta receptor subtype in this preparation, we antagonized [³H]naltrindole binding as well as the stimulation of [³⁵S]GTPS binding with selective and nonselective antagonists. If there were more than one subtype present, we would expect an antagonist selective for one subtype to be less potent in antagonizing the effect of a different receptor subtype. The antagonists naltrindole (both delta-1 and delta-2), NTB (delta-2 selective) and BNTX (delta-1 selective) displayed similar relative potencies for inhibition of [³H]naltrindole binding (table 1, fig. 3c). However, the relative potencies of the selective antagonists, NTB and BNTX, did not change whether they were antagonizing a delta-1 selective agonist or a nonselective (delta-1 and delta-2) agonist for delta opioid receptor subtypes. Both selective and nonselective antagonists were able to completely inhibit stimulation of [³⁵S]GTPS binding by maximally efficacious concentrations of BW373U86 (delta-1 and delta-2) or DPDPE (delta-1 selective) (fig. 3, a and b). The pK_B values for inhibition of [³⁵S]GTPS binding stimulation by 10 nM BW373U86 were 8.53, 8.56 and 8.14 for naltrindole, NTB and BNTX, respectively. The pK_B values for inhibition of [³⁵S]GTPS binding stimulation by 3 µM DPDPE were similar at 8.74, 8.76 and 8.54 for naltrindole, NTB and BNTX, respectively. In contrast, BNTX possessed a 100-fold greater affinity for [³H]DPDPE binding sites (delta-1) relative to those of [³H]DSLET (delta-2) in guinea pig brain membranes (Portoghese et al., 1992).

View larger version (22K): [in this window] [in a new window]   Fig. 3.   Antagonism of stimulated GTPS and [3H]naltrindole binding. The antagonists naltrindole (), NTB () and BNTX () were added with 10 nM BW373U86 (a) or 3 µM DPDPE (b) for measurement of GTPS binding, or with 0.04 nM [3H] naltrindole (c) for measurement of binding as described under "Materials and Methods." The data are expressed as the percent of stimulation in the presence of the agonist only for [35S]GTPS binding or as percent of specific binding in the presence of [3H]naltrindole only. Shown are the mean and standard error bars for three experiments, each carried out in duplicate.

Verification of partial agonism. To verify partial agonist characteristics of oxymorphindole, deltorphin II and DPDPE in this assay, the following experiments were performed. We measured the effect of 10 µM oxymorphindole on the stimulation of [³⁵S]GTPS binding by 10 nM BW373U86 or 3 µM DPDPE, concentrations which induced nearly maximum stimulation. The presence of 10 µM oxymorphindole reduced the stimulation of [³⁵S]GTPS binding by BW373U86 and DPDPE to 9 ± 1% and 17 ± 5% that of full agonist alone, respectively (fig. 4). Deltorphin II and DPDPE, which appear to be partial agonists compared to BW373U86, also antagonized the stimulation of [³⁵S]GTPS binding by BW373U86 to the level stimulation observed with 10 µM deltorphin II or 10 µM DPDPE alone (fig. 4). Antagonist ICI 174,864 (10 µM) was able to completely inhibit stimulation of [³⁵S]GTPS binding by DPDPE (fig. 4), but not BW373U86 (not shown). However, 90 µM ICI 174,864 did completely inhibit stimulation of [³⁵S]GTPS binding by BW373U86 (fig. 4). The decreased inhibition of BW373U86 by ICI 174,864 was possibly caused by a slower ligand dissociation rate observed with BW373U86 (Childers et al., 1993). Furthermore, addition of both 10 µM deltorphin II and 3 µM DPDPE did not significantly increase the stimulation of [³⁵S]GTPS binding compared with 3 µM DPDPE alone (fig. 4). If DPDPE and deltorphin II acted through separate receptor types in this system, we would expect their effects to be at least partially additive. However, there was no additive effect on stimulation by the deltorphin II and DPDPE, providing further evidence for partial agonist actions by these ligands, as well as for a single delta opioid receptor subtype present in these cells.

Treatment of C6 cells with pertussis toxin and cholera toxin. To determine whether the stimulation of [³⁵S]GTPS binding in the membranes is caused by coupling of the delta opioid receptor to the pertussis toxin-sensitive G proteins, G_i and G_o, or whether there is some stimulation of the cholera toxin-sensitive stimulatory G protein, G_s, cells were pretreated with pertussis or cholera toxin. Pertussis toxin pretreatment completely inhibited the stimulation of [³⁵S]GTPS binding by 1 µM BW373U86, 10 µM DSLET and 10 µM DPDPE (fig. 5), which indicates that the stimulation of [³⁵S]GTPS binding by both full and partial agonists is mediated by pertussis toxin-sensitive G proteins. Treatment with cholera toxin reduced the basal and stimulated [³⁵S]GTPS binding by BW373U86 and DSLET (fig. 5). After cholera toxin pretreatment, the apparent percent stimulation (expressed as percent of basal binding) actually increased because the basal [³⁵S]GTPS binding decreased. Although there is basal [³⁵S]GTPS binding to G_s, pertussis toxin pretreatment completely eliminated agonist-stimulated [³⁵S]GTPS binding, which indicates that opioid receptor activates only pertussis toxin inhibitory (G_o/G_i) G proteins.

Inhibition of adenylyl cyclase activity in whole cells. To determine whether the greater stimulation of [³⁵S]GTPS binding by BW373U86 and SNC80 in membranes resulted in greater inhibition of adenylyl cyclase activity in intact cells than by DPDPE, cAMP accumulation in whole cells in the presence of BW373U86, SNC80 or DPDPE was measured. BW373U86 inhibited forskolin-stimulated adenylyl cyclase activity by 79 ± 9% with an EC₅₀ of 0.9 nM (fig. 6 and table 1). As with [³⁵S]GTPS binding, SNC80 inhibited adenylyl cyclase activity to nearly the same extent (71 ± 5%), but was less potent (EC₅₀, 15 nM). Surprisingly, DPDPE similarly inhibited adenylyl cyclase activity by 72 ± 6% with an EC₅₀ of 80 nM (fig. 6). Therefore, the greater stimulation of [³⁵S]GTPS binding by BW373U86 than with DPDPE did not result in greater inhibition adenylyl cyclase activity.

View larger version (19K): [in this window] [in a new window]   Fig. 6.   Inhibition of forskolin-stimulated adenylyl cyclase by opioid agonist in whole cells. Whole cells were incubated 15 min with or without BW373U86 (), SNC80 () or DPDPE () in the presence of 1 µM forskolin. The cAMP content was determined as described under "Materials and Methods." The cAMP levels in the presence of agonist were expressed as percent of cAMP accumulation in the absence of added agonist. Forskolin-stimulated cAMP was 0.13 pmol cAMP/µg protein. Shown is the mean and standard error bars from four to five experiments, each carried out in duplicate.

	Discussion

Top Abstract Introduction Materials & Methods Results Discussion References

The purpose of this study was to characterize the efficacy of G protein coupling of the cloned delta opioid receptor stably expressed in a C6 glioma cell line. The C6 cells highly express a homogeneous population of delta opioid receptor and thus provide an excellent means to assess efficacy of ligands at the delta opioid receptor. The magnitude of receptor-stimulated [³⁵S]GTPS binding in the C6 membrane preparation enables one to evaluate efficacy at the delta opioid receptor for opioid ligands including very weak partial agonists. We found that both peptide and nonpeptide ligands were maximally efficacious in the stimulation of [³⁵S]GTPS binding and inhibition of adenylyl cyclase. Previously, our laboratory has shown that C6 cells provide a suitable environment for the mu opioid receptor, which was similarly transfected into the C6 cells at a high receptor density (Emmerson et al., 1996). Recently, the stimulation of [³⁵S]GTPS binding by opioid agonist was also characterized for the mouse delta receptor transiently expressed in COS cells (Befort et al., 1996).

Ligand binding affinities for naltrindole and pCl-DPDPE measured in the membranes from C6 cells transfected with the cloned delta opioid receptor were similar to those found in monkey membranes. For example, the K_d of [³H]pCl-DPDPE in the absence of sodium was 1.5 nM in membranes from C6 and 1.2 nM in monkey brain membranes (Emmerson et al., 1994). However, the binding affinities of other ligands measured here did not correlate with the binding affinity found in monkey membranes for the delta receptor, which indicates that the cloned delta receptor differs pharmacologically from those characterized in the monkey membrane preparation, despite their characterization in a cell line of glial origin. In addition, little correlation was found between the literature K_i values for delta receptor ligands measured in the membranes from CHO cells transfected with the mouse delta opioid receptor and those measured in other membrane preparations (Raynor et al., 1994). The differences in receptor pharmacology may be caused by the heterogeneous population of delta receptor subtypes in other systems, different post-translational modifications or unknown proteins associated with the receptor.

Raynor et al. (1994) proposed that the cloned mouse delta receptor is of the delta-2 subtype. Although we saw a similar trend in the binding data regarding receptor subtype (i.e., deltorphin II and NTB having higher affinities than DPDPE and BNTX), the efficacies of deltorphin II and DPDPE to stimulate [³⁵S]GTPS binding were identical. Because of these equal efficacies and the greater efficacy of ligands that bind to both subtypes, i.e., BW373U86 and SNC80 (Bilsky et al., 1995), we were concerned about the possibility of the presence of both delta receptor subtypes, perhaps because of a posttranslational modification. However, naltrindole, NTB and BNTX antagonized stimulation of [³⁵S]GTPS binding by BW373U86 or DPDPE with equal potencies. Furthermore, the intrinsic activities of DPDPE and deltorphin II were not additive, which indicates that their action was through the same receptor. Based on these data, we are unable to conclude that the cloned delta receptor is of the delta-2 or delta-1 subtype. Although substantial evidence from rat and mouse both in in vivo (Mattia et al., 1991; Sofuoglu et al., 1991) and in vitro (Buzas et al., 1994) studies indicates the existence of delta opioid receptor subtypes, this clone does not exhibit the characteristics of one subtype, which suggests that perhaps neuroanatomical and subcellular localization of the receptor may contribute to the observation of receptor subtypes (Zaki et al., 1996).

Although the [³⁵S]GTPS binding data in membranes from the C6 cells suggest that the receptor-G protein interaction was functional, the data do not discriminate which population of G proteins were interacting with the delta opioid receptor. There is evidence that the delta opioid receptor may couple to G_s (Shen and Crain, 1990; Gintzler and Xu, 1991; Standifer et al., 1996). Cruciani et al. (1993) found that opioid receptors couple to both cholera toxin- and pertussis toxin-sensitive G proteins in F-11 (neuroblastoma-dorsal root ganglion neuron) hybrid cells. Specificity of G protein coupling did not appear to depend on the level of delta opioid receptor expression. Prather et al. (1994b) showed that overexpression of the delta opioid receptor in neuroblastoma and neuroblastoma × glioma cell lines does not alter the population of G proteins that couples to the delta opioid receptor. In addition, Befort et al. (1996) demonstrated that transiently expressed mouse delta opioid receptor (25 pmol receptor/mg protein) showed similar agonist efficacy and maximal stimulation of [³⁵S]GTPS binding compared with the receptor stably expressed in CHO cells (4 pmol receptor/mg protein). In C6 cells transfected with high levels of the mu opioid receptor, the receptor retained its specificity for inhibitory G proteins (Emmerson et al., 1996); however, cells expressing high levels of alpha-2 adrenergic receptor did not (Eason et al., 1992). In this study, pertussis toxin treatment completely abolished agonist stimulation of [³⁵S]GTPS binding, which demonstrated that the delta opioid receptor is only coupled to inhibitory (G_o/G_i) G proteins.

Despite the high levels of receptor expression, we found no evidence of spare receptors. As receptor occupancy increased, stimulation of [³⁵S]GTPS binding also increased with a linear correlation where maximal receptor occupation was necessary for maximal stimulation of [³⁵S]GTPS binding. Given the differential sensitivity of ligands to sodium and guanine nucleotides (Childers et al., 1993; Emmerson et al., 1994), K_i values were determined by displacement of [³H]naltrindole under conditions identical with the [³⁵S]GTPS binding assay. The K_i values were similar to the EC₅₀ values for stimulation of [³⁵S]GTPS binding. Thus, receptor occupancy {([L]/(K_i + [L]) where [L] is ligand concentration and K_i is binding affinity} correlated closely with stimulation of [³⁵S]GTPS binding. In addition, there appears to be a relationship between the potency of agonist to inhibit adenylyl cyclase and the potency for activation of G proteins.

Although we observed a wide range of responses for [³⁵S]GTPS binding stimulation by agonists, the differences did not carry over to inhibition of adenylyl cyclase activity. We found no significant difference between the maximum inhibition of adenylyl cyclase by BW373U86, SNC80 or DPDPE. Knapp et al. (1995) also found similar maximum inhibition of adenylyl cyclase by SNC80 and pCl-DPDPE in CHO cells stably transfected with the human delta opioid receptor. SNC80 and DPDPE were also fully efficacious in bioassays in mouse vas deferens and guinea pig ileum preparations (Bilsky et al., 1995). In addition, the relative efficacies of several peptide ligands, including deltorphin II and DPDPE, were indistinguishable in the mouse vas deferens bioassay (Kramer et al., 1993). The full agonist properties of DPDPE could possibly be caused by an excess number of G proteins relative to adenylyl cyclase, thus an apparent partial agonist for stimulation of [³⁵S]GTPS binding could maximally inhibit adenylyl cyclase. We did not determine the K_i values for the ligands in the adenylyl cyclase assay buffer so we could not construct an occupancy-response curve to determine whether spare receptors were present in the inhibition of adenylyl cyclase.

By the characterization of the binding affinity and efficacy at the delta opioid receptor 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 increased understanding of the mechanism of delta opioid receptor-mediated signal transduction at the cellular level will promote the use of delta opioid ligands as pharmacological tools and potential therapeutic agents.