Introduction

Abstract Introduction Materials & Methods Results Discussion References

There is considerable interest in the development of delta opioid-selective, nonpeptide compounds as analgesic agents that may show an improved side effect profile over morphine and other currently available mu agonist therapies (Porreca et al., 1995). To expedite this process, a full understanding of the action of delta opioid agonists at the cellular level is essential, particularly because compounds active at this receptor may show agonist, antagonist and inverse agonist properties (Costa and Herz, 1989). Opioid receptors are members of the family of seven-transmembrane domain receptors and couple to inhibitory G proteins, such that opioid agonists stimulate the exchange of bound GDP for GTP, which can be measured as an increase in the binding of the stable GTP analog [³⁵S]GTPS to cell membranes (Sim et al., 1995; Traynor and Nahorski, 1995). Such opioid agonist-mediated stimulation of [³⁵S]GTPS binding has an absolute requirement for GDP (Traynor and Nahorski, 1995). In addition, µM levels of GDP are needed for optimum agonist-stimulation of [³⁵S]GTPS binding at several receptor systems (Gierschik et al., 1991; Lazareno et al., 1993; Lorenzen et al., 1993; Offermanns et al., 1994). However, the level of GDP required varies across systems. Thus, to observe agonist responses at m1 and m3 receptors expressed in CHO or human embryonic kidney cells, only low levels (0.1 µM) of GDP are required, whereas for m2- and m4-mediated increases in the same cells, 10-fold higher levels of GDP are needed (Lazareno et al., 1993; Offermanns et al., 1994). In contrast, the alpha-2 adrenergic-mediated stimulation of [³⁵S]GTPS binding in PC-12 cells requires no GDP (Tian et al., 1994).

The mu opioid receptors in SH-SY5Y cells couple to G_i and G_o proteins, including G_alphai3, G_alphai2, G_alphai1, G_alphao1 and G_alphao2, but have a preference for G_alphai3 (Laugwitz et al., 1993). In contrast, delta opioid receptors in NG108-15 cells couple especially to G_alphai2 (McKenzie and Milligan, 1990). However, when expressed in CHO cells, both mu and delta receptors couple to similar populations of G protein subtypes (Chakrabarti et al., 1995). Such differences or similarities in the coupling of opioid receptors to G protein subtypes may define the specific requirements for GDP and certain ions to obtain an optimum agonist-stimulated [³⁵S]GTPS response and contribute to the properties of the receptor.

We report on the characterization of delta opioid modulation of [³⁵S]GTPS binding response in NG108-15 cells. A complex relationship exists between the concentration of GDP and its effect on [³⁵S]GTPS binding, but opioid agonist-mediated stimulation of this response follows a simple relationship, increasing with increasing GDP. Changes in [³⁵S]GTPS binding provide a functional method for investigating delta opioid activity and allow for study of the action of agonists and inverse agonists, as exemplified by ICI 174864. A preliminary account of some of the results has been presented (Szekeres and Traynor, 1995).

	Materials and Methods

Abstract Introduction Materials & Methods Results Discussion References

Chemicals and drugs. [³H]Diprenorphine (1.11 TBq/mmol) was purchased from Amersham (Aylesbury, UK),and [³⁵S]GTPS (46.1 TBq/mmol) was purchased from Dupont New England Nuclear Research Products (Boston, MA). DAMGO, DPDPE, DSLET, pertussis toxin and all other chemicals were of analytical grade and purchased from Sigma Chemical (St. Louis, MO). [D-Ala²,Glu⁴]Deltorphin II (Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH₂) was obtained from Peninsula (St. Helens, UK), and BNTX, NTB and ICI 174864 [N,N-diallyl-Tyr-Aib-Phe-Leu-OH (Aib = -aminoisobutyric acid)] were purchased from RBI (St. Albans, UK). The following compounds were kindly donated as indicated: CI977 [5R-(5,7,8)-N-methyl-N-[7-(1-pyrrolidinyl)-1-oxaspiro[4,5]dec-8-yl]-4-benzofuranacetamide-HCl; Parke-Davis, Cambridge, UK], etorphine and diprenorphine (Reckitt & Colman, Hull, UK); nalorphine and levallorphan (Zeneca Pharmaceuticals, Alderley Park, UK) and TIPP (Tyr-Tic-Phe-Phe; Tic = tetrahydroisoquinoline-3-carboxylic acid) (Dr. S. J. Paterson, St. Thomas's Hospital, London, UK). Ecoscint scintillation fluid was purchased from National Diagnostics. DMEM (without sodium pyruvate; with 4500 mg/l glucose), DMEM/Nutrient Mix F-12, fetal calf serum, new born calf serum, fungizone (amphotericin B), L-glutamine, penicillin/streptomycin solution and hypoxanthine (13.6 µg/ml)/aminopterin (0.176 µg/ml)/thymidine (3.88 µg/ml) supplement were all purchased from GIBCO Laboratories (Paisley, UK).

Cell culture. NG108-15 cells (kindly provided by Dr. M. Keen, Department of Pharmacology, University of Birmingham, UK) were grown in 175-cm² tissue culture flasks in DMEM supplemented with 5% fetal calf serum and hypoxanthine (13.6 µg/ml)/aminopterin (0.176 µg/ml)/thymidine (3.88 µg/ml) at 37°C in a 5% CO₂ atmosphere. CHO cells transfected with the mouse delta opioid receptor were a kind gift from Dr. C. J. Evans (Department of Psychiatry, University of California at Los Angeles). Cells were grown in 175-cm² tissue culture flasks in DMEM/Nutrient Mix F-12 supplemented with 5% fetal calf serum, 2.5 µg/ml amphotericin B, 50 units/ml penicillin, 50 µg/ml streptomycin and 258 µg/ml L-glutamine at 37°C in a 5% CO₂ atmosphere.

Membrane preparation. Confluent monolayers of NG108-15 cells or CHO cells were rinsed once and harvested by gentle agitation in DMEM, followed by centrifugation (250 × g, 2 min). The cell pellet was resuspended in a buffer of 20 mM HEPES, pH 7.4, 100 mM NaCl and 4 mM MgCl₂ (buffer A) and homogenized using a tissue tearer (twice at 5 sec, 30,000 rpm). The resultant crude membrane fraction was collected by centrifugation (50,000 × g, 15 min), washed in buffer A and recentrifuged as before. The pellet was finally resuspended in buffer A to give a protein concentration of 0.25 mg/ml (Lowry et al., 1951). All procedures were performed at 0° to 4°C. In experiments to determine opioid binding, buffer A was replaced with 50 mM Tris·HCl, pH 7.4, in all procedures.

Opioid binding assays. Binding was performed in Tris·HCl buffer to afford parameters for high-affinity binding of the ligands (Rodriguez et al., 1992) as follows: membrane protein (250 µg) was incubated in Tris·HCl, pH 7.4, with [³H]diprenorphine (0.04-10 nM) or [³H]DPDPE (0.04-20 nM) in a final volume of 1 ml. After allowing the system to reach equilibrium for 1 hr at 25°C (Cotton et al., 1985; Ho et al., 1997), the mixture was rapidly vacuum-filtered through GF/B filters to separate bound from free ligand, and the filters were rinsed three times in 3 ml of ice-cold buffer (Tris·HCl, pH 7.4). Radioactivity retained on the filters was determined by liquid scintillation counting. Nonspecific binding was defined as the binding remaining in the presence of 10 µM naloxone. Specific binding was typically >80% of total binding at the radioligand K_d.

[³⁵S]GTPS binding assay. Unless otherwise stated, cell membranes (250 µg of protein) were incubated in buffer A containing [³⁵S]GTPS (100 pM) and GDP (100 µM) for 1 hr at 30°C in a total volume of 1 ml and in the presence of varying concentrations of opioids. Samples were then rapidly vacuum-filtered through GF/B filters and washed three times with 3 ml of ice-cold buffer A. Bound radioactivity was quantified by liquid scintillation counting. Nonspecific binding was defined using 10 µM unlabeled GTPS. Specific binding was typically 90% to 95% of total binding. To examine the stability of [³⁵S]GTPS at the end of the incubation assay, 100 µl samples of the incubation mixture were taken, centrifuged and applied to cellulose-PEI thin-layer liquid chromatography plates that were developed using a solvent system of 0.5 M K₂HPO₄, containing 0.01 M -mercaptoethanol. After drying, the plates were cut into 1-cm strips and counted for radioactivity as above. The purity of the [³⁵S]GTPS used in the assays, as determined by thin-layer liquid chromatography, was > 90%.

Data analysis. Binding data were analyzed with the program LIGAND (Munson and Rodbard, 1980) to provided estimates for the total receptor number (B_max), binding affinity (K_d or K_i) and slope of the binding isotherms. The data was fitted to both one- and two-site models. To determine whether the two-site model produced a statistically better fit of the data than the one-site model, the variance ratio test (F test) facility of LIGAND was used, where P < .05 was considered significant. The EC₅₀ for stimulation of [³⁵S]GTPS binding obtained at various drug concentrations was determined from nonlinear curve fitting of the data fitted, using GraphPAD Prism (GraphPAD, San Diego, CA). Apparent antagonist affinity constants (K_e) were calculated from [³⁵S]GTPS binding assay concentration-effect curves in the absence or presence of a single concentration of antagonist using the equation K_e = [antagonist]/(DR  1), where DR = (EC₅₀ in the presence of antagonist)/(EC₅₀ in the absence of antagonist) (Kosterlitz and Watt, 1968). Statistical comparisons between groups of data were made by Student's t test, where P < .05 was considered significant.

	Results

Abstract Introduction Materials & Methods Results Discussion References

Characterization of basal and agonist-stimulated [³⁵S]GTPS binding. The NG108-15 cells used in this study expressed a homogeneous delta opioid receptor population. This was demonstrated by the ability of the selective delta opioid agonist DPDPE (K_i = 1.64 ± 0.07 nM; Hill coefficient, 0.95 ± 0.06; n = 3) but not the mu and kappa opioids DAMGO and CI977 (K_i = >1000 nM) to displace binding of the nonselective antagonist [³H]diprenorphine. In addition, the number of receptors labeled by [³H]diprenorphine (559 ± 61 fmol/mg/protein, K_d = 0.32 ± 0.01 nM, n = 3) was the same as the number labeled by [³H]DPDPE (501 ± 94 fmol/mg/protein, K_d = 1.68 ± 0.51 nM). The CHO cells used expressed delta receptors at a density of 404 ± 23 fmol/mg of protein (n = 3) determined with [³H]diprenorphine (K_d = 0.2 ± 0.01 nM).

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Effect of (a) GDP and (b) GDPS on the binding of [35S]GTPS (100 pM) to membranes of NG108-15 cells in the absence () and presence () of DPDPE (1 µM), where 100% represents the level of [35S]GTPS binding in the absence of guanine dinucleotide, which was 75.7 ± 3.3 fmol/mg of protein (a) and 123.8 ± 15.3fmol/mg of protein (b). Insets, degree of stimulation of [35S]GTPS binding by 1 µM DPDPE. Assays were performed as described in Materials and Methods for 60 min at 30°C. Points represent mean ± S.E.M. from three separate determinations performed in duplicate. **P < .01 and *P < .05 compared with control. P < .05 compared with no dinucleotide.

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Effect of the nucleotides ADP (), GMP () and UDP () on [35S]GTPS (100 pM) binding to membranes from NG108-15 cells. Assays were performed as described in Materials and Methods for 60 min at 30°C. Points represent mean ± S.E.M. from three separate determinations performed in duplicate.

View larger version (20K): [in this window] [in a new window]   Fig. 3.   Effect of varying the concentration of GDP in the absence () or presence () of DPDPE (1 µM) on [35S]GTPS (100 pM) binding to membranes from CHO cells expressing the delta receptor. Assays were performed as described in Materials and Methods for 60 min at 30°C. Points represent mean ± S.E.M. from three separate determinations performed in duplicate. *P < .05. Inset, degree of stimulation by 1 µM DPDPE.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of Na+ concentration on basal () and DPDPE (1 µM)-stimulated () [35S]GTPS (100 pM) binding to NG108-15 membranes. Assays were performed in the presence of 4 mM MgCl2 as described in Materials and Methods for 60 min at 30°C. Points represent mean ± S.E.M. from three separate determinations performed in duplicate.

View larger version (20K): [in this window] [in a new window]   Fig. 5.   Effect of Mg++ concentration on basal () and DPDPE (1 µM)-stimulated () [35S]GTPS (100 pM) binding to NG108-15 membranes. Assays were performed in the presence of 100 mM NaCl as described in Materials and Methods for 60 min at 30°C. Points represent mean ± S.E.M. from three separate determinations performed in duplicate.

Effects of agonists and antagonists on [³⁵S]GTPS binding. DPDPE caused a concentration-dependent increase in the binding of [³⁵S]GTPS over control, affording an EC₅₀ value of 31.2 ± 3.1 nM (n = 12) and reaching maximal stimulation at 1 µM of the opioid (figs. 6 and 7). This stimulation was completely blocked after incubation of the cells with pertussis toxin (100 ng/ml) for 24 hr before preparation of the membranes (fig. 7), confirming the involvement of G proteins of the G_o/G_i class. Pertussis toxin treatment also lowered basal binding of [³⁵S]GTPS in these cells by 56.0 ± 2.2%, to 18.7 ± 0.8 fmol/mg protein from a basal level of 41.2 ± 1.2 fmol/mg protein (P < .01; n = 3).

View larger version (26K): [in this window] [in a new window]   Fig. 6.   Stimulation of [35S]GTPS (100 pM) binding to membranes of NG108-15 cells by DPDPE alone () or in the presence of 300 nM naloxone (), 10 nM BNTX () or 0.2 nM NTB (). Assays were performed as described in Materials and Methods for 60 min at 30°C, and values represent mean ± S.E.M. from three separate determinations performed in duplicate. Binding of [35S]GTPS was 39.1 ± 5.3 fmol/mg protein in the absence of DPDPE and 107.9 ± 9.2 fmol/mg of protein in the presence of DPDPE (10 µM).

View larger version (18K): [in this window] [in a new window]   Fig. 7.   DPDPE-mediated stimulation of [35S]GTPS (100 pM) binding to NG108-15 membranes prepared from control () and pertussis toxin (100 ng/ml, 24 hr)-treated () cells. Assays were performed as described in Materials and Methods for 60 min at 30°C. Points represent mean ± S.E.M. from three separate determinations performed in duplicate. All pertussis toxin-treated values were significantly (P < .01) lower than the basal value.

View larger version (31K): [in this window] [in a new window]   Fig. 8.   Relative activity of various ligands to stimulate [35S]GTPS (100 pM) binding to NG108-15 cell membranes. Concentrations used were DSLET, [D-Ala2,Glu4]deltorphin II (DELT II) and DPDPE (3 µM); etorphine, levallorphan, diprenorphine, nalorphine, naltrindole, NTB and ICI 174864 (10 µM); BNTX (1 µM); and TIPP (5 µM). Points represent mean ± S.E.M. from at least three separate determinations performed in duplicate and are normalized to the maximal effect produced by DSLET that represented 37.6 ± 2.9 fmol [35S]GTPS bound/mg of protein. Assays were performed as described in Materials and Methods for 60 min at 30°C. Significantly greater than the DPDPE response (P < .05). *Significantly below basal value (P < .05).

View larger version (36K): [in this window] [in a new window]   Fig. 9.   Effect of ICI 174864 on [35S]GTPS (100 pM) binding to NG108-15 membranes in Na+ (100 mM; open columns)- or K+ (100 mM; hatched columns)-containing buffer. The cross-hatched column represents the effect of 1 µM naloxone on the ICI 174864 (3 µM)-mediated response in K+-containing buffer. Basal [35S]GTPS binding was 76.2 ± 3.2 fmol/mg in Na+-containing buffer and 102.4 ± 9.8 fmol/mg in K+-containing buffer. Assays were performed as described in Materials and Methods, using the appropriate buffer, for 60 min at 30°C. Points represent mean ± S.E.M. from three separate determinations performed in duplicate. *P < .01 compared with effect caused by 3000 nM ICI 174864 alone.

View larger version (19K): [in this window] [in a new window]   Fig. 10.   Diprenorphine stimulation of [35S]GTPS (100 pM) binding to NG108-15 membranes relative to that afforded by DSLET (3 µM) in Na+ (100 mM; )- or K+ (100 mM; )-containing buffer. Basal [35S]GTPS binding was 53.2 ± 6.8 fmol/mg in Na+-containing buffer and 78.5 ± 12.0 fmol/mg in K+-containing buffer. [35S]GTPS binding in the presence of DSLET (3 µM) was 102.8 ± 1.1 fmol/mg in Na+-containing buffer and 112.6 ± 13.6 fmol/mg in K+-containing buffer. Assays were performed as described in Materials and Methods, using the appropriate buffer, for 60 min at 30°C. Points represent mean ± S.E.M. from three separate determinations performed in duplicate.

View larger version (20K): [in this window] [in a new window]   Fig. 11.   Displacement of the specific binding of [3H]diprenorphine (0.5 nM) to membranes from NG108-15 cells by NTB (), BNTX () and DPDPE (). Experiments were performed in Tris·HCl buffer (50 mM, pH 7.4) for 60 min at 25°C as described in Materials and Methods. Points represent mean ± S.E.M. for four experiments performed in duplicate.

View this table: [in this window] [in a new window]   TABLE 1 Binding affinities of BNTX and NTB at the delta opioid receptor in NG108-15 cell membranes Data were obtained from the displacement of specific binding of [3H]diprenorphine (0.5 nM) to membranes from NG108-15 cells by BNTX and NTB (see fig. 11) and fitted using the LIGAND program (Munson and Rodbard, 1980). The two-site model gave a significantly better fit of the data than a single-site model (P < .01).

	Discussion

Abstract Introduction Materials & Methods Results Discussion References

The NG108-15 cell membranes used in this study were shown to express the same number of delta opioid receptors measured by either the delta agonist [³H]DPDPE or the delta antagonist [³H]diprenorphine. Because this cell line expresses only opioid receptors of the delta type, this demonstrates that in Tris buffer the receptors are all (within the limits of measurement) in a conformational state recognized with high affinity by both agonists and antagonists. According to the ternary complex model of receptor/G protein interaction (Birnbaumer et al., 1990), this would indicate that the receptors are in a form tightly coupled to G protein.

To demonstrate delta opioid agonist stimulation of [³⁵S]GTPS binding in membranes from NG108-15 and CHO cells, the presence of GDP, or its hydrolysis resistant analog GDPS, was necessary. This dependence on GDP has also been observed in studies on the stimulation of [³⁵S]GTPS binding by mu opioid agonists in SH-SY5Y cell membranes (Traynor and Nahorski, 1995) and in C6 cells expressing the cloned µ receptor (Emmerson et al., 1996), adenosine A₁ agonists in bovine brain (Lorenzen et al., 1993), muscarinic agonists in porcine cardiac membranes and CHO cells (Hilf et al., 1989; Lazareno et al., 1993) and chemotactic peptide fMet-Leu-Phe in membranes from HL-60 cells (Gierschik et al., 1991). GDP is thought to bind to empty guanine nucleotide binding sites on G proteins and hence reduce the basal level of [³⁵S]GTPS binding (Wieland et al., 1992), such that agonist-induced exchange of GDP for [³⁵S]GTPS can be observed. However, GDP and GDPS affected both basal and agonist-stimulated [³⁵S]GTPS binding in the NG108-15 cell membranes in a complex manner. Thus, GDP and GTPS reduced binding of [³⁵S]GTPS at concentrations below 3 µM, but above this concentration an increase in [³⁵S]GTPS binding was observed, and this reverted to an inhibition at higher concentrations.

The delta opioid-mediated stimulation of [³⁵S]GTPS binding was not observed if GDP or GDPS was replaced by GMP, ADP or UDP. These latter nucleotides did not inhibit basal [³⁵S]GTPS binding but did stimulate the binding of [³⁵S]GTPS in the absence of opioid agonist. Indeed, for ADP, UDP and GMP, the level of stimulation was far in excess of that seen with GDP or GDPS. A number of explanations are possible for this finding. The nucleotides may be stimulating the binding of [³⁵S]GTPS via a G protein-coupled "nucleotide" receptor, although no nucleotide receptor with such a profile is known. However, a preliminary metabolism study suggests the presence of the second unlabeled nucleotide protects [³⁵S]GTPS from breakdown, probably by various nucleotidases and phosphatases present in the membrane preparation (Wieland et al., 1992), thereby increasing the level of [³⁵S]GTPS available to bind to G protein. This is supported by the finding that a reduced level of agonist stimulated [³⁵S]GTPS binding was seen at 37°C, whereas stimulation was increased at 20°C. The decrease in binding at higher concentrations of GDP or GDPS (>30 µM) presumably results from a competition between the guanine dinucleotides and [³⁵S]GTPS for the nucleotide binding site on the G protein(s). As a consequence, this effect is not seen with ADP, UDP or GMP due to the specificity of the nucleotide binding site on the G protein (Rodbell et al., 1971, 1980).

These complex effects of GDP on [³⁵S]GTPS binding in NG108-15 cell membranes are not a property of the delta opioid receptor system because a similar relationship is not seen in membranes from CHO cells expressing the mouse delta receptor. Thus, the effect is cell, not receptor, specific and is independent of the ability of the agonist bound form of the delta receptor to stimulate [³⁵S]GTPS binding. The difference in GDP requirements for expression of delta agonism at the same receptor expressed in different cells highlights considerations that must be examined when looking at receptor-mediated responses outside endogenous systems (Kenakin, 1996).

The ionic environment is also a very important parameter in modulating [³⁵S]GTPS binding. To observe agonist stimulation of [³⁵S]GTPS binding in NG108-15 cell membranes, Mg⁺⁺ ions were required. It is difficult to conclude the mechanism by which Mg⁺⁺ is causing its effects because this cation has many actions on G protein events (Birnbaumer et al., 1990). However, the increase in both basal and agonist-stimulated [³⁵S]GTPS binding may be accounted for by the increased rate of dissociation of GDP (Higashijima et al., 1987) and an increased rate of association of [³⁵S]GTPS (Bokoch et al., 1984; Sternweis et al., 1981) with a decreased rate of dissociation (Higashijima et al., 1987). In addition, delta opioid binding is likely to be enhanced by Mg⁺⁺ (Rodriguez et al., 1992; Standifer et al., 1993), resulting in a differential increase in agonist-stimulated over basal [³⁵S]GTPS binding. Above 10 mM, Mg⁺⁺ had a strong inhibitory effect on both basal and agonist-stimulated [³⁵S]GTPS binding.

Na⁺ ions were not essential to produce an agonist-mediated increase in [³⁵S]GTPS binding. The effect of Na⁺ ions on [³⁵S]GTPS binding was to decrease both basal and agonist-stimulated [³⁵S]GTPS binding. However, in the presence of DPDPE (1 µM), the effect of Na⁺ was shifted, leading to an increased stimulation window at Na⁺ concentrations of 10 to 100 mM. This inhibitory effect of Na⁺ on G protein activity has been interpreted as reflecting a modulation of the affinity of "empty" opioid delta receptors for G protein, that is, reducing the activity of constitutively active receptors (Costa et al., 1990, 1992; Lefkowitz et al., 1993). As expected, because delta opioid receptors are known to couple to G proteins of the G_i/G_o family (Laugwitz et al., 1993; Offermanns et al., 1991; Prather et al., 1994; Roerig et al., 1992), treatment of NG108-15 cells with pertussis toxin (100 ng/ml; 24 hr) completely abolished delta opioid receptor-mediated stimulation of [³⁵S]GTPS binding. Furthermore, pertussis toxin pretreatment reduced control [³⁵S]GTPS binding. Because pertussis toxin only uncouples G proteins from receptors and does not alter other properties of G proteins, such as their ability to bind GTP and interact functionally with adenylyl cyclase (Katada et al., 1986), this provides evidence for the presence of constitutively active receptors in membrane preparations of NG108-15 cells.

Only a single delta opioid receptor has been cloned from NG108-15 cells (Evans et al. 1992; Kieffer et al., 1992). However, the agonist effect of DPDPE was antagonized by the nonselective opioid antagonist naloxone and by the putative delta-1 antagonist BNTX and putative delta-2 antagonist NTB. The higher sensitivity of DPDPE to blockade by NTB, rather than BNTX, suggests the homogeneous delta receptor in NG108-15 cells is of the delta-2 subtype, a conclusion also reached by studying the cloned delta receptor from NG108-15 cells expressed in CHO cells (Law et al., 1994). The K_e values reported in the CHO cells for the two antagonists (Law et al., 1994) suggests they have lower affinities for the transfected receptor in CHO cells than for the endogenous receptor in NG108-15 cells. Moreover, the displacement of binding of [³H]diprenorphine by BNTX and NTB in delta receptor containing CHO membranes is approximately linear, whereas the displacement of [³H]diprenorphine binding by these compounds in the NG108-15 membranes was biphasic. This biphasic recognition by both BNTX and NTB may depend on the environment, such as the type of associated G proteins, which allows differentiation of the receptor into two apparent binding affinities for these antagonists. However, the agonist DPDPE recognizes only a single site. Because agonists are highly susceptible to the presence of Na⁺ ions and guanine nucleotides (Childers and Snyder, 1980; Kelly et al., 1982) and therefore to the state of coupling of the receptor, this suggests that the biphasic activity of BNTX and NTB results from a combination of the chemical nature of the compounds themselves and the membrane environment.

Several opioid agonist alkaloids and peptides were examined for their ability to stimulate [³⁵S]GTPS binding by using maximally effective concentrations as reported for their activity against adenylyl cyclase in these cells (Childers et al., 1993; Law et al., 1983; Roerig et al., 1996). The most efficacious were the peptides, although DSLET was more efficacious than either DPDPE or [D-Ala²,Glu⁴]deltorphin II. Etorphine was equally efficacious with DPDPE and [D-Ala²,Glu⁴]deltorphin II, but other alkaloid derivatives were partial agonists. The relative ability to stimulate [³⁵S]GTPS binding among these compounds was similar to their ability to inhibit adenylyl cyclase. Of the antagonists examined, diprenorphine had some activity, as previously seen at the delta receptor in NG108-15 cells (Law et al., 1983), as did naltrindole, which is reported to have agonist effects at supraspinal delta receptors in the mouse (Stapelfeld et al., 1992). BNTX, NTB and TIPP had no efficacy in this system. In contrast, the antagonist ICI 174864, a delta opioid ligand with negative intrinsic activity (Costa and Herz, 1989), reduced the basal binding of [³⁵S]GTPS in membranes of NG108-15 cells, confirming the presence of constitutively active receptors of the delta type.

The effect of ICI 174864 in lowering basal [³⁵S]GTPS was not as pronounced as that of pertussis toxin. This is in contrast with the findings of Mullaney et al. (1996), using the cloned mouse delta receptor expressed in Rat 1 fibroblasts, in which pertussis toxin and ICI 174864 inhibited basal [³⁵S]GTPS binding to the same extent and suggested ICI 174864 was an inverse agonist with high intrinsic activity. The observed difference could be explained by the fact that in the NG108-15 cells ICI 174864 may not be a full inverse agonist or that other receptors present in NG108-15 cells and also linked to G_o/G_i proteins are active in the agonist-unoccupied state. Such receptors include muscarinic M₄ receptors (Graeser and Neubig, 1993; Lazareno et al., 1990; Michel et al., 1989), alpha-2B adrenergic (Graeser and Neubig, 1993; McClue and Milligan, 1990) and cannabinoid (Caulfield and Brown, 1992; Mackie et al., 1993).

The inverse agonist effect of ICI 174864 was much more pronounced in buffer in which the Na⁺ ions had been replaced with K⁺ ions, an effect previously reported for the inverse agonist activity of ICI 174864 when determined by measures of GTPase activity (Costa and Herz, 1989). Seven-transmembrane domain G protein-linked receptors exist in equilibrium between an inactive (R) and active conformation (R*). In the presence of agonist (A), the R* form is greatly favored and interacts with G protein to form the active ternary complex (AR*G) (Birnbaumer et al., 1990). Constitutive activity occurs when receptors exist in the R* state and are able to spontaneously couple to G proteins in the absence of agonist. Ligands with negative intrinsic activity shift the equilibrium in favor of R and thereby lead to a reduction in constitutive activity, manifest in the present experiments as a reduction in basal [³⁵S]GTPS binding. Thus, despite the fact that Na⁺ ions and ICI 174864 are acting through different mechanisms, the presence of Na⁺ will mask the inverse agonist effect of ICI 174864 because both bring about the same result, namely, a reduction in R* with a concomitant rise in R. When Na⁺ ions in the medium are replaced with K⁺ ions, the basal binding of [³⁵S]GTPS is considerably higher, so the inverse agonist effect of ICI 174864 is apparently greater.

The increase in the basal level of binding of [³⁵S]GTPS afforded by removal of Na⁺ ions should also have an effect on agonist responses. In the presence of 100 mM Na⁺, diprenorphine only produced 30% of the maximal stimulation of [³⁵S]GTPS binding produced by the full agonist DSLET. However, when Na⁺ ions were replaced by K⁺ ions in the reaction buffer, diprenorphine produced 70% of the DSLET response. Thus, because there is a finite number of G proteins that can be labeled by [³⁵S]GTPS, the relative efficacies of compounds can be varied by alteration of the basal level of coupling through changes in the concentration of Na⁺ ions.

In conclusion, delta opioid agonists have been shown to stimulate [³⁵S]GTPS binding to pertussis toxin-sensitive G proteins in membranes from NG108-15 cells in a naloxone-reversible manner. Optimal conditions for this assay have been established to provide a rapid, sensitive and reproducible method to measure the potency and intrinsic activity of G protein activation at an endogenously expressed delta opioid receptor. The relative efficacy of a series of compounds is governed by the level of G protein, which places a ceiling on the maximum effect that can be obtained. Therefore, through judicious manipulation of assay conditions, it should be possible to match the relative delta opioid efficacies in this cell system with those seen in different tissues and thus design screening conditions suitable for the detection of different pharmacological and side effect profiles.