Introduction

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Opioid analgesics are used extensively in the management of pain. However, a limitation to their effectiveness is the development of dependence, a condition for which an effective treatment is unavailable. Buprenorphine is a synthetic oripavine analgesic structurally related to the potent opioid agonist etorphine, and to diprenorphine, an antagonist. It exhibits potent analgesic and antinociceptive actions (Cowan, 1995; Lewis, 1995) with a limited capacity for producing physical dependence and is less reinforcing than other opiates (Negus and Woods, 1995). Although used primarily in clinical pain management (Foley, 1993), buprenorphine is under development as a treatment for opioid dependence because it has been reported to attenuate opiate self-administration in humans and nonhuman primates (Jasinski and Preston, 1995; Fudala and Johnson, 1995; Mello and Mendelson, 1995).

The therapeutic actions of buprenorphine have been mostly ascribed to actions on the mu receptor. Binding studies have shown that buprenorphine binds with high affinity at the mu receptor (Raynor et al., 1994; Blake et al., 1997) and causes a functional desensitization with regard to cAMP inhibition, but no receptor internalization, after pretreatment of the mouse mu receptor expressed in HEK 293 cells (Blake et al., 1997). The pharmacological profile of buprenorphine has been suggested to reflect a mu agonist at low doses, and mu and kappa antagonists at high doses. Some authors have suggested that these mixed agonist/antagonist properties of buprenorphine contribute to its clinical effectiveness (Rothman et al., 1995; Dykstra and Negus, 1995).

Currently, little is known about the functional consequence of buprenorphine on delta receptor function. Binding studies have shown that buprenorphine binds with high affinity to the delta receptor (Kong et al., 1993; Rothman et al., 1995); peripheral administration of buprenorphine up-regulated delta receptors in the forebrain region of the rat brain (Belcheva et al., 1993), which suggests an antagonistic action at the delta receptor for buprenorphine. Other studies, however, have reported inhibition of cAMP accumulation in COS cells expressing the cloned mouse delta opioid receptor by buprenorphine (Kong et al., 1993). These studies suggested that buprenorphine may also display an agonist/antagonist characteristic at the delta receptor.

Recent mutagenesis studies have indicated that agonists and antagonists interact differently with opioid receptors and have distinct binding determinants. Mutation of aspartic acid residue 95 (Asp⁹⁵) and 128 (Asp¹²⁸) of the delta receptor to an asparagine created mutant receptors, D95N and D128N, respectively, which exhibited reduced affinity for delta selective agonists such as DSLET and DPDPE but not for antagonists such as naltrindole, BNTX and NTB (Kong et al., 1993; Befort et al., 1996a). Hence the D95N and D128N mutants discriminated between agonist and antagonist binding, and the Asp⁹⁵ and Asp¹²⁸ residues were essential for the binding of delta selective agonists.

In the present study we demonstrated that buprenorphine binding affinity to the delta receptor was not decreased by mutation of either Asp⁹⁵ or Asp¹²⁸ to an asparagine. However, most importantly, we showed that the Asp⁹⁵ mutation in the delta receptor converted the partial agonistic character of buprenorphine to a pure antagonist. We also showed that full delta selective agonists were much less potent in activating the D95N mutant than the wild-type delta receptor, which indicates that Asp⁹⁵ is essential for full-agonist action at the delta receptor.

	Materials and Methods

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Cell culture. HEK 293 cells were grown and maintained in minimal essential medium with Earle's salts (Life Technologies, Inc., Grand Island, NY) containing 10% fetal calf serum, 100 units ml¹ penicillin and streptomycin sulfate in 10% CO₂ at 37°C. The mouse delta opioid receptor cDNA in pcDNA3 (Invitrogen, San Diego, CA) modified with the FLAG epitope (DYKDDDDK) at the amino terminus was a generous gift from Dr. Mark von Zastrow, University of California, San Francisco. The mouse delta opioid receptor, the D95N and D128N mutant cDNA were stably transfected into HEK 293 cells by a modification of the calcium phosphate protocol (Chen and Okayama, 1988). HEK 293 cell monolayers at approximately 70% confluence were transfected with 30 µg of plasmid. After an overnight incubation at 37°C, the medium was removed and the cells were treated with 5 ml of phosphate-buffered saline containing 10% glycerol for 10 min at room temperature. Cells were then washed twice with phosphate-buffered saline and incubated for 48 h at 37°C in growth medium. Stable transformants were selected in growth medium containing 1.0 mg ml¹ Geneticin (Life Technologies, Inc.) for the D95N mutant and D128N mutant, and maintained in T 75 cm² tissue culture flasks in 10% CO₂ at 37°C.

Mutagenesis of the cloned mouse delta opioid receptor. The mouse delta opioid receptor cDNA was mutated by use of the Altered Site in vitro Mutagenesis system (Promega Corp. Madison WI). Sequences of the oligonucleotides for the D95N and D128N mutants were 5-GCTTTGGCTAATGCGCTGGCC-3 (GAT to AAT) and 5-CTCTCCATTAACTACTACAAC-3 (GAC to AAC), respectively. The delta receptor cDNA was subcloned into pALTER, and a single-stranded template was produced. The 21-mer oligonucleotide containing the desired mutation was annealed to the single-stranded template, elongated with T4 DNA polymerase and transformed into Escherichia coli strain BMH 71-18 mut S. Transformants were selected by growth on LB plates containing 125 µg/ml ampicillin. The mutations were confirmed by dideoxyDNA sequencing, and the cDNA was excised and subcloned into EcoRI-EcoRV site in the expression vector pcDNA3.

Radioligand binding studies. Receptor binding studies were performed with membranes from stably transfected HEK 293 cells expressing the delta WT, D95N or D128N mutant cDNA. Membranes were prepared and receptor binding studies conducted as described (Raynor et al., 1994). Cell monolayers were harvested in 6 ml of buffer containing 50 mM Tris-HCl (pH 7.8) containing 1 mM ethyleneglycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid, 5 mM MgCl₂, 10 µg ml¹ leupeptin, 10 µg ml¹ pepstatin, 200 µg ml¹ bacitracin and 0.5 µg ml¹ aprotinin and placed on ice. A cell pellet was prepared by centrifugation at 24,000 × g for 7 min at 4°C and was homogenized in the same buffer by a Polytron (Brinkmann Instruments, Westbury, NY) at setting 2.5, 30 sec. The cell homogenate was centrifuged at 48,000 × g for 20 min at 4°C, and the resulting cell pellet was homogenized and placed on ice for the binding assays. Binding assays were carried out at 25°C for 40 min in a final volume of 200 µl with 1 nM [³H]naltrindole as radioligand and 1 µM naltrindole to define nonspecific binding. The binding reaction was terminated by the addition of ice-cold 50 mM Tris-HCl (pH 7.8) and rapid filtration over FP-100 Whatman GF/B glass fiber filters that were pretreated with 0.5% polyethyleneimine and 0.1% bovine serum albumin. The filters were rinsed with 12 ml of ice-cold buffer, and the bound radioactivity was determined by use of a liquid scintillation counter. Total binding and nonspecific binding for the wild-type delta receptor were typically 2560 ± 400 cpm and 380 ± 30 cpm, respectively, and for the D95N mutant 1520 ± 210 cpm and 230 ± 40 cpm, respectively.

cAMP accumulation studies. Stably transfected HEK 293 cells were subcultured in 12-well culture plates and allowed to recover for 72 h before the experiments. For agonist pretreatment studies, a 10-fold concentrated stock of agonist was diluted into growth medium and added to individual culture wells for the times indicated in the table and figure legends. The final concentration of all agonists used in regulation studies was 1 µM. After treatment, the medium was removed and replaced with 1 ml of growth medium containing 0.5 mM IBMX and the cells were incubated for 30 min at 37°C. The medium was then replaced with fresh medium containing 10 µM forskolin with or without opioid agonist in the concentration range 10¹² to 10⁶ and the cells transferred to 37°C. After 5 min the medium was removed, 1.0 ml of 0.1 N HCl was added and the monolayers frozen at 20°C. For determination of the cAMP content of each well, the monolayers were thawed, placed on ice, sonicated and the intracellular cAMP levels measured by radioimmunoassay (Amersham plc, Buckinghamshire, UK). Data obtained from the dose-response curves were analyzed by nonlinear regression analysis with GraphPad Prism 2 (GraphPad Software, Inc. San Diego, CA)

	Results

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To investigate buprenorphine regulation of the cloned delta opioid receptor, the wild-type cDNA and mutant form of the delta receptor that contained an aspartate to asparagine substitution at amino acids 95 (D95N) and 128 (D128N) were stably expressed in HEK 293 cells. Pharmacological characterization of the stably transformed cells was carried out by radioligand binding and the functional inhibition of forskolin-stimulated cAMP accumulation, as described previously (Kong et al., 1993; Raynor et al., 1994; Befort et al., 1996a). Saturation binding with the delta selective radioligand, [³H]naltrindole, demonstrated that the wild-type delta receptor was expressed in HEK 293 cells at the level of 9.4 ± 3.0 pmol mg¹ of membrane protein (B_max) with a dissociation constant of (K_D) of 0.3 ± 0.06 nM (n = 3). Saturation analysis of [³H]naltrindole binding revealed a K_D of 1.3 ± 0.5 nM (n = 3) and B_max of 59.0 ± 9.0 fmol mg¹ protein for the D95N mutant and a K_D of 1.5 ± 0.2 nM (n = 3) and B_max of 236.0 ± 3.0 fmol mg¹ protein for the D128N mutant. These results indicate that the mutant delta receptors were expressed at a lower density than the wild-type. No specific [³H]naltrindole binding was detected in untransfected HEK 293 cells (data not shown).

A series of opioids were tested for their binding affinity to the wild-type and D95N mutant delta receptor (table 1). The analysis of competitive radioligand binding data with [³H]naltrindole showed that the expressed wild-type delta receptor had specific, high-affinity binding for the delta selective opiates DSLET, DPDPE, DADLE and SIOM as well as the nonselective ligands bremazocine and ()-buprenorphine. The binding affinities were similar to those previously reported in rat brain membranes (Rothman et al., 1995), in HEK 293 cells (Wang et al., 1995; Bot et al., 1997) and in other surrogate cell lines (Kong et al., 1993; Raynor et al., 1994; Befort et al., 1996a; Meng et al., 1996). Consistent with previous results (Kong et al., 1993), delta selective nonpeptide, SIOM, and peptide agonists, such as DSLET and DPDPE, exhibited reduced affinities for the D95N mutant, whereas bremazocine exhibited a small reduction (table 1). DSLET did not bind to the D95N mutant, whereas SIOM and DPDPE bound with affinities of approximately 0.5 µM. Similar to the results of Kong et al. (1993), the antagonist naltrindole displayed similar affinities at the wild-type and D95N mutant delta receptor (table 1). In agreement with the antagonist binding data, ()-buprenorphine exhibited similar binding affinities at both receptors (table 1).

Studies on opioid receptors expressed in HEK 293 cells have shown that these receptors are coupled to the inhibition of adenylyl cyclase and to G proteins of the G_i or G_o family (Arden et al., 1995; Tsu et al., 1995; Pei et al., 1995). The cloned delta receptor expressed in HEK 293 cells was functionally active and mediated agonist inhibition of forskolin-stimulated cAMP accumulation (fig. 1). The selective delta agonist DSLET and the nonselective agonists bremazocine and buprenorphine inhibited cAMP accumulation (table 1). Their potencies are similar to previously published potencies for the delta selective and nonselective agonists acting at the delta receptor to inhibit cAMP accumulation in the mouse NG108-15 hybrid cells (Cai et al., 1996; Pei et al., 1995) and in delta receptor transfected CHO cell line (Evans et al., 1992; Law et al., 1994; Malatynska et al., 1996), COS-7 cell line (Kong et al., 1993) and HEK 293 cells (Keith et al., 1996; Bot et al., 1997). The potencies of most agonists to inhibit cAMP accumulation were greater than their binding affinities. We have no direct explanation for this difference but it is possible that this could be caused by the presence of spare receptors as suggested from the studies of Law et al. (1994). The large spare-receptor pool may consist of high-affinity and G protein-coupled receptors only a small proportion of which needs to be stimulated to inhibit cAMP accumulation, whereas the binding studies detect both coupled and uncoupled low-affinity delta receptors. Hence the binding studies would detect a mixture of high- and low-affinity receptors. Although the agonist binding displacement studies with [³H]naltrindole conformed best to a one-site binding model, as determined by analysis of the best-curve fit of the dose-response curve, the Hill coefficients of the agonists buprenorphine (0.64 ± 0.02), DSLET (0.55 ± 0.02), SIOM (0.54 ± 0.03) and bremazocine (0.77 ± 0.03) to displace [³H]naltrindole in the wild-type delta receptor were significantly less than 1.0, which suggests the existence of binding sites with different affinities for these compounds. Hence cAMP inhibition may not necessarily be related to opioid occupancy in a linear manner. The extent of maximal inhibition of buprenorphine (maximal inhibition %, n) (66.3 ± 4.4, n = 3), morphine (54.3 ± 4.9, n = 3) and SIOM (54.7 ± 7.2, n = 3) compared with the maximal inhibition of the full agonist DSLET (82.8 ± 4.0, n = 4; Student's t test to buprenorphine; P < .05) suggests that buprenorphine, morphine and SIOM may have partial agonist activity at the delta receptor.

View larger version (16K): [in this window] [in a new window]   Fig. 1.   Dose-dependent opioid inhibition of cAMP accumulation for delta wild-type expressing HEK 293 cells. cAMP accumulation was determined as described under "Materials and Methods." The inhibition of forskolin-stimulated cAMP accumulation is expressed as a percentage of the forskolin control. Intracellular cAMP levels of the cells incubated with forskolin alone served as controls (100%). Forskolin-stimulated cAMP levels were typically 5- to 20-fold higher than basal values. Basal levels were subtracted from the forskolin levels obtained. The data presented are the mean ± S.E. of three or more separate experiments, each performed in duplicate.

Inhibition of maximal cAMP accumulation was blocked by the delta selective antagonist naltrindole. Naltrindole (1 µM) significantly decreased (Student's t test; P < .05) the maximal inhibitory effects of the delta selective agonist DSLET (table 2, fig. 2) and of buprenorphine. The effect on cAMP accumulation of 1 µM DSLET alone or in the presence of 1 µM naltrindole was 82.8 ± 4.0% and 28.8 ± 3.9% inhibition, respectively, and on 1 µM buprenorphine was 66.3 ± 4.4% and 17.8 ± 2.7%, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Effects of naltrindole and ()-buprenorphine on DSLET inhibition of cAMP accumulation for wild-type delta receptors. HEK 293 cell-monolayers were treated with growth medium containing DSLET in the concentration range 1012 to 106 M, together with 10 µM forskolin and 1 µM of either naltrindole or buprenorphine for 5 min at 37°C and then assayed for intracellular cAMP levels as described under "Materials and Methods." The dose-response curves were determined by computer analysis by use of GraphPad Prism 2. The data presented are the mean ± S.E. of three or more separate experiments, each performed in duplicate.

To further investigate whether differences in agonist interaction with D95N mutant are caused by variations in agonist binding, a series of opiates were tested for stimulation of the same mutant receptor as described by Kong et al. (1993). Consistent with their reduced binding affinities reported here and in previous studies (Kong et al., 1993), the delta selective agonists DSLET, DADLE, DPDPE and SIOM (table 1) and the nonselective agonist bremazocine (table 1, fig. 3) were much less potent in inhibiting cAMP accumulation in cells expressing the D95N mutant. This was reflected in a rightward shift of the dose-response curve (fig. 3, table 1). The D95N mutant also was no longer able to be desensitized after 3 h pretreatment with DSLET and levorphanol, agonists which have previously been reported to desensitize the delta receptor expressed in HEK 293 cells (table 3) (Bot et al., 1997). The inability of agonists to desensitize the D95N mutant may be caused by the agonists being not effective in activating cellular processes normally involved in desensitizing the wild-type delta receptor.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Bremazocine inhibition of cAMP accumulation for delta wild-type opioid receptor and the D95N mutant receptor expressed in HEK 293 cells. Intracellular cAMP accumulation was assayed as described under "Materials and Methods." The data presented are the mean ± S.E. of three or more separate experiments, each performed in duplicate.

View this table: [in this window] [in a new window]   TABLE 3 Agonist (106 M) pretreatment (3 h) effects on opioid inhibition of cAMP accumulation for the delta WT and D95N mutant delta receptors expressed in HEK 293 cells

Although ()-buprenorphine, and to a lesser extent the structurally related ligand diprenorphine, inhibited cAMP accumulation in HEK 293 cells expressing the wild-type delta receptor (table 1), they were incapable of inhibiting cAMP accumulation via the D95N mutant despite binding to the receptor with subnanomolar affinity (table 1, fig. 4). The loss of effectiveness of buprenorphine is unlikely to be caused by its low efficacy because SIOM, which was less efficacious than buprenorphine in inhibiting cAMP accumulation in the wild-type delta receptor (table 1), was still able to inhibit cAMP accumulation via the D95N mutant. Likewise morphine, which has also been reported as being a partial agonist at the delta receptor (Bot et al., 1997), was also still able to inhibit cAMP accumulation but with a reduced efficacy in the D95N mutant (% maximal inhibition: 28.0 ± 2.5, n = 3, Student's t test to wild-type, P < .05) compared with the wild-type delta receptor (54.3 ± 4.9, n = 3). The high affinity of buprenorphine for the D95N mutant and the lack of function suggests that buprenorphine was acting as an antagonist at the D95N mutant receptor. To test this possibility, buprenorphine was examined for its ability to block DSLET and bremazocine inhibition of cAMP accumulation. DSLET and bremazocine inhibited cAMP accumulation to the same maximal extent (table 1) and when added together, produced the same maximal inhibition of cAMP accumulation as DSLET did alone (not shown) and did not exhibit a potency less than each agonist individually, which suggests that both are full agonists at the wild-type delta receptor.

View larger version (16K): [in this window] [in a new window]   Fig. 4.   ()-Buprenorphine inhibition of cAMP accumulation for the wild-type delta receptor, the D95N and the D128N mutant receptors expressed in HEK 293 cells. Intracellular cAMP accumulation was assayed as described under "Materials and Methods." The data presented are the mean ± S.E. of three or more separate experiments, each performed in duplicate.

In contrast to its action at the wild-type delta receptor (table 1), buprenorphine did not inhibit cAMP accumulation via the D95N mutant. When added to increasing concentrations of DSLET (0.1-1000 nM), buprenorphine (1 µM) reduced the ability of DSLET to inhibit cAMP via the D95N mutant (table 2, fig. 5). A similar antagonism of the effects of the agonist bremazocine was found when buprenorphine was used (table 4, fig. 6). The ability of buprenorphine to block agonist inhibition of cAMP accumulation via the D95N mutant was similar to the actions of the classical delta antagonist naltrindole (tables 2 and 4, figs. 5 and 6). Both naltrindole and buprenorphine reduced the maximal effectiveness of agonists to inhibit cAMP accumulation via the D95N mutant. This is likely caused by the high concentrations of buprenorphine and naltrindole used in this study, because naltrindole is known to be a competitive antagonist. However, it can not be ruled out that because both compounds may have slow dissociation rates, they may act in a semi-noncompetitive manner to block the actions of DSLET and bremazocine. Buprenorphine (1 µM) did not reduce the maximal ability of DSLET to inhibit cAMP accumulation via the wild-type delta receptor (table 2, fig. 2), which suggests that its binding to the wild-type delta receptor was competitive and that its actions at the wild-type delta receptor are consistent with it being a partial agonist, which would be expected to have agonist property per se but would diminish the potency of full agonists when combined with them (Jasper and Insel, 1992).

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Effects of naltrindole and ()-buprenorphine on DSLET inhibition of cAMP accumulation for D95N mutant delta receptors. HEK 293 cell-monolayers, expressing the D95N mutant delta receptors were treated with growth medium containing DSLET in the concentration range 1011 to 106 M, together with 10 µM forskolin and 1 µM of either naltrindole or buprenorphine for 5 min at 37°C, and then assayed for intracellular cAMP levels as described under "Materials and Methods." The data presented are the mean ± S.E. of three or more separate experiments, each performed in duplicate.

View larger version (14K): [in this window] [in a new window]   Fig. 6.   Effects of naltrindole and ()-buprenorphine on bremazocine inhibition of cAMP accumulation for D95N mutant delta receptors expressed in HEK 293 cells. The cell monolayers were treated with growth medium containing bremazocine in the concentration range 1011 to 106 M, together with 10 µM forskolin and 1 µM of either naltrindole or buprenorphine for 5 min at 37°C and then assayed for intracellular cAMP levels as described under "Materials and Methods." The data presented are the mean ± S.E. of three or more separate experiments, each performed in duplicate.

In addition to aspartic acid 95, recent studies have shown that another conserved aspartic acid at residue 128 (Asp¹²⁸) is also critical for high-affinity agonist binding to the delta receptor. Mutations of Asp¹²⁸ to asparagine (D128N) have reduced the binding of agonists such as DADLE, DPDPE and bremazocine, which suggests that Asp¹²⁸ of the delta receptor has an important role in ligand recognition (Befort et al., 1996b).

In our study, the affinity of buprenorphine for the D128N mutant was increased 15-fold compared with the binding of buprenorphine to the wild-type delta receptor (K_i: wild-type = 2.4 ± 0.6 nM; D128N mutant = 0.6 ± 0.06 nM). Furthermore, buprenorphine was still active as an agonist at the D128N mutant with an EC₅₀ value for inhibition of cAMP accumulation of 1.3 ± 0.5 nM and a maximal inhibitory capacity of 52 ± 4% compared with an EC₅₀ of 1.4 ± 1.3 nM and maximal inhibitory capacity of 66. ± 4.4% for the wild-type delta receptor (fig. 4). The similar potency and efficacy exhibited by buprenorphine acting on the D128N mutant and wild-type delta receptors occurred despite the D128N mutant being expressed at less than 10% the density of the wild-type receptor, which indicates that receptor expression was not a critical factor in agonist potency and efficacy under these expression conditions. Similarly, other agonists, such as DSLET, DADLE, etorphine and levorphanol, were also just as effective in inhibiting cAMP accumulation via the D128N mutant as the wild-type delta receptor (Bot et al., 1997). Thus, unlike the D95N mutant, mutation of Asp¹²⁸ to an asparagine in the delta receptor did not affect the functional properties of buprenorphine, which indicates that Asp⁹⁵ is selectively involved in mediating the functional properties of this opioid.

	Discussion

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In the current study the cloned mouse delta opioid receptor (Evans et al., 1992; Kieffer et al., 1992) and a mutant with substituted asparagine for aspartate residue at position 95 (D95N) (Kong et al., 1993) were stably expressed in HEK 293 cells and the functional activity of opioids was examined. The mouse receptor bound the selective delta agonist DSLET with high affinity, as well as the nonselective opioids buprenorphine and bremazocine.

Mutagenesis studies have revealed the involvement of unique receptor domains that are specific for agonist and antagonist interaction; the agonist binding domains in the opioid delta receptor encompass transmembranes II, III, VI and VII and the extracellular loop III (Kong et al., 1993; Befort et al., 1996a; Fukuda et al., 1995; Valiquette et al., 1996). Furthermore, aromatic amino acid residues in the transmembrane spanning regions have also contributed to agonist binding to the delta receptor (Befort et al., 1996b). Our results indicate that the charged amino acid, Asp⁹⁵, is also involved in agonist binding, but more importantly, is critical for agonist activation and desensitization of the delta receptor.

Our previous results showed that mutation of Asp⁹⁵ to asparagine greatly reduced affinity of the delta receptor for selective agonists (Kong et al., 1993). In the present study we show that the potency and efficacy of delta selective agonists such as DPDPE, DSLET, DADLE and SIOM to activate the D95N mutant and hence to inhibit cAMP accumulation was reduced, even though the receptor was coupled to G proteins (Kong et al., 1993). However, more importantly, buprenorphine was unable to activate the D95N mutant expressed in HEK 293 cells, even though its affinity to bind to the mutant receptor was not reduced by the mutation. Buprenorphine acted as an antagonist at the D95N mutant blocking the effects of the agonists DSLET and bremazocine to inhibit cAMP accumulation. This is similar to the result of Mollereau et al. (1997) who reported that a replacement of Gln²⁸⁰ by His in TM6 of the human ORL1 (opioid receptor-like) increased the binding affinity of lofentanil and etorphine but reduced their intrinsic activity to inhibit cAMP accumulation. Consequently, they no longer acted as pure agonists, as they do at the native ORL1 receptor, but exhibited clear antagonistic properties.

Behavioral studies in humans and nonhuman primates have suggested that buprenorphine is a mixed agonist/antagonist at opioid receptors (see the introduction). This was also suggested by our results in that its effectiveness in inhibiting cAMP accumulation via the wild-type delta receptor was less than that exhibited by the full agonists such as DSLET; it also reduced the potency of DSLET and bremazocine to inhibit cAMP accumulation without altering their maximal inhibitory capacity. This is consistent with the concept of partial agonism as proposed by Jasper and Insel (1992). It is conceivable that mutation of the Asp⁹⁵ residue removed the agonist component of buprenorphine and hence removed the ability of buprenorphine to activate intracellular G protein-associated effector systems, while retaining the ability of the compound to bind to the delta receptor. In support of this it has been suggested recently that opioids exhibit marked differences in efficacy and/or potency in the activation of different G proteins (Garzon et al., 1997). Different receptor-G protein associations for DSLET and buprenorphine may contribute to the presence and absence, respectively, of inhibition of cAMP accumulation in the D95N mutant.

Mutagenesis studies have indicated that antagonists do not bind to the same domains in delta opioid receptors as agonists (Kong et al., 1993; Befort et al., 1996a, b). This may explain why antagonists such as naltrindole and naloxone bound to the delta wild-type and the D95N mutant receptors with similar affinities whereas full agonists exhibited greatly reduced affinities at the D95N mutant. The similar affinities of buprenorphine for the wild-type and D95N mutant delta receptors suggests that buprenorphine may bind to the receptor in a manner similar to antagonists. In fact, ()-buprenorphine has a chemical structure very similar to ()-diprenorphine (fig. 7) which may provide a basis for its similar binding affinity at the wild-type and D95N mutant receptor.

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Chemical structure of agonist ()-buprenorphine and structurally related antagonist ()-diprenorphine.

Both ()-buprenorphine and ()-diprenorphine are N-cyclopropylmethyl-nordihydroorvinol derivatives originally prepared from thebaine-methyl vinyl ketone adducts in the search for analgesics which may be superior to morphine but with fewer side effects. (For a full review of the chemistry of these compounds see Bentley, 1971.) They differ structurally only in the length of the alkyl chain group R at position C₁₉ with diprenorphine having a methyl whereas buprenorphine has a t-butyl substitution. Structure-activity relationships, based on rodent antinociceptive and morphine antagonism tests, established that increasing the length of the alkyl chain group R in the structure at position C₁₉ from methyl to t-butyl had little effect on mu/kappa selectivity but resulted in higher intrinsic activity (Lewis, 1974). Hence a primary alcohol (and diastereoisomeric methyl secondary alcohols) substitutes on C₁₉, as present in diprenorphine, convert the compound to an antagonist with low intrinsic activity, whereas propyl and butyl tertiary alcohol substitutes, as present in buprenorphine, impart a powerful analgesic character on the structure as determined in the rodent tail-pressure test (Lewis, 1974). Mutation of the Asp⁹⁵ residue may have removed the ability of the t-butyl and methyl groups to impart an agonistic character to buprenorphine and diprenorphine, respectively, perhaps by not allowing receptor association with intracellular-effector G protein systems which usually occur after agonist binding to the wild-type delta receptor. In support of this, recent reports have demonstrated dynamic changes in G protein association with the delta receptor after agonist binding (Law and Reisine, 1997). Furthermore, agonist activation has been reported to promote association of G_i2 with the receptor, a G protein proposed to mediate the coupling of the delta receptor to adenylyl cyclase (McKenzie and Milligan, 1990). Hence this specific association of particular G proteins with the wild-type delta receptor may have been altered in the D95N mutant. This structural separation of inherent agonist/antagonist character by the D95N mutant was only evident for the N-cyclopropylmethyl-nordihydroorvinol derivatives diprenorphine and buprenorphine, and not for the other partial agonists SIOM and morphine, which still retained activity via the D95N mutant, which suggests that they might bind differently to the delta receptor than diprenorphine and buprenorphine.

Another conserved aspartic acid residue in transmembrane III (Asp¹²⁸) has also been proposed to be involved in agonist binding and activation of G-protein-linked receptors, including the delta receptor (Befort et al., 1996b). Mutation of Asp¹²⁸ to asparagine resulted in a receptor with greatly reduced affinity for DADLE and DPDPE as well as selective nonpeptide agonists. These findings showed that the Asp¹²⁸ of the delta receptor has an important role in ligand recognition. Consistent with the results of Befort et al. (1996a) that the D128N mutant had lower affinity for delta selective agonists, we have found that Asp¹²⁸ also influences agonist activation of the delta receptor. Selective agonists such as DADLE, DPDPE and DSLET where less potent at inhibiting cAMP accumulation in D128N mutant delta receptor expressing HEK 293 cells than cells expressing the wild-type delta receptor (Bot et al., 1997). Most of the selective agonists had similar maximal inhibitory effects on cAMP accumulation via the D128N and wild-type delta receptors, but exhibited decreased potency, which suggests that Asp¹²⁸ is critical for the affinity of the receptor for these agonists. In contrast, the effect of buprenorphine on the D128N mutant and wild-type delta receptor were indistinguishable, which indicates that Asp¹²⁸ is not essential for this mixed agonist/antagonist to bind to and activate the delta receptor.

The results of this study suggest that full agonists and mixed agonist/antagonists may act via some common mechanisms to stimulate the delta receptor. The differences between these classes of compounds, however, may be in how they bind to the delta receptor and hence which intracellular effector system(s) they activate. Because buprenorphine is an analgesic with limited abuse potential, identification of its ligand binding determinants may be useful in the development of novel opioids with limited abuse potential and limited tolerance after continued use.