δ-Opioid receptor binding in mouse brain: Evidence for heterogeneous binding sites

European Journal of Pharmacology, 216 (1992) 273-277

273

© 1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.00

EJP 52483

6-Opioid receptor binding in mouse brain: evidence for heterogeneous binding sites M. Sofuoglu a, P.S. Portoghese b and A.E. Takemori

a

Department of Pharmacology, Medical School and h Department of Medicinal Chemistry, College of Pharmacy, University of Minnesota, Minneapolis, MN 55455, USA Received 17 January 1992, accepted 17 March 1992

In this study we investigated the characteristics of binding sites with which t~ opioid receptor agonists interact in homogenates of mouse brain using Krebs-HEPES medium. [3H][D-Ser2,LeuS,Thr6]enkephalin (DSLET), [3H][D-Ala2,D-LeuS]enkephalin (DADLE) and [3H][D-Pen2,D-PenS]enkephalin (DPDPE) were used to label 3 opioid binding sites. The analyses of the saturation binding data of these ligands (Scatchard plots) gave best fits to single rather than multiple site models. The binding capacity (Bma x) labelled by [3H]DSLET was found to be significantly greater than those of [3H]DADLE and [3H]DPDPE in brains of mice. Naltriben (the benzofuran analogue of naltrindole) was equally effective in competing for [3H]DSLET, [3H]DPDPE and [3H]DADLE binding sites. On the other hand, DADLE was significantly more potent in competing for [3H]DADLE and [3H]DPDPE binding sites than for [3H]DSLET binding sites. Also, DPDPE was more potent in competing for the binding sites of [3H]DADLE and [3H]DPDPE than for those of [3H]DSLET. DSLET was found to be equipotent in competing for [3H]DSLET, [3H]DPDPE and [3H]DADLE binding sites. These results suggest a heterogeneity of 6 opioid receptors which may be explained possibly by the existence of 6 opioid receptor subtypes. Naltriben; 6-Opioid receptor subtypes; DSLET ([D-Ser 2,LeuS,Thr6]enkephalin); DPDPE ([D-Pen2,D-Pen 5]enkephalin); DADLE (D-Ala2,D-Leu5 ]enkephalin)

I. Introduction Recently, we have observed that, the benzofuran analog of naltrindole, naltriben, a highly selective 6 opioid receptor antagonist, is more effective in antagonizing the antinociceptive action of [D-SerZ,Leu 5, Thr6]enkephalin (DSLET) than that of [D-Pen2,D PenS]enkephalin ( D P D P E ) or [D-AlaZ,D-LeuS]en kephalin ( D A D L E ) in mice (Sofuoglu et al., 1991a). In addition to the differential antagonism by naltriben, lack of development of cross-tolerance between D S L E T and D P D P E provided further evidence for ~ opioid receptor heterogeneity (Sofuoglu et al., 1991a,b). Based

Correspondence to: A.E. Takemori, Department of Pharmacology, 3-249 Millard Hall, University of Minnesota, 435 Delaware Street S.E., Minneapolis, MN 55455, U.S.A. Tel. 1.612.625 3248, fax 1.612.625 8408. This investigation was supported by U.S. Public Health Service Grants from the National Institute on Drug Abuse. Studies in this report were carried out in accordance with the Declaration of Helsinki a n d / o r with the guide for the care and use of laboratory animals as adopted and promulgated by the National Institutes of Health.

on these and other observations in vivo, we have suggested that the antinociceptive action of D S L E T and D P D P E may be mediated by different receptors, possibly ~ opioid receptor subtypes (Sofuoglu et al., 1991a). Based on the differential antagonism of 3 opioid receptor agonists by the irreversible ~ opioid receptor antagonists, [D-Ala2,LeuS,Cys6]enkephalin and naltrindole-5'-isothiocyanate, a collaborative study has also revealed the possibility of ~ opioid receptor subtypes (Jiang et al., 1991). In order to gain more insight into the heterogeneity of 6 opioid receptor sites, we performed opioid receptor binding experiments using selective ~ opioid receptor agonists. The binding properties of various radiolabelled opioid ligands that have different degrees of selectivities for the 3 type of opioid receptor have been studied in various species using different incubation mediums (Corbett et al., 1984; Cotton et al., 1985; Werling et al., 1985; Zarr et al., 1986). In this study, we investigated the binding characteristics of three different radiolabelled 6 opioid receptor agonists, D A D L E , D S L E T and D P D P E , in brain homogenates of mice. Additionally, competition among various 6 opioid receptor agonists and antagonists for 6 opioid receptor binding was studied to characterize further these binding sites.

274

2. Materials and methods

/z opioid binding sites. Nonspecific binding was determined in the presence of 1 /xM unlabelled DSLET, D A D L E and D P D P E for [3H]DSLET, [3H]DADLE and [3H]DPDPE binding, respectively. Incubations were performed in duplicates in a final volume of 1 ml at 37°C for 20 min. The incubation was terminated by transferring the incubation tubes to a rack bathed in ice-cold water and rapid filtration through Whatman G F / C filters (soaked in water saturated with t-amyl alcohol) using a Brandell cell harvester. The filters were washed twice with 4 ml buffer and transferred to scintillation vials. Absolute ethanol (0.5 ml) and 4.5 ml Ecolume were added to the vials and the radioactivity was determined by liquid scintillation spectrometry. Protein concentrations were determined by the method of Lowry et al. (1951) using bovine serum albumin (Sigma Chemical Co.) as the standard.

2.1. Animals Random bred male Swiss-Webster mice (Biolab, White Bear Lake, MN) weighing 20-25 g were used in all experiments. All animals were supplied food and water ad libitum and housed in a temperature controlled (23°C) animal room for at least 1 day before experimentation.

2.2. Preparation of neural membranes Assays were performed in modified Krebs-HEPES buffer (mM: NaCI 118; KC1 4.8; CaCl 2 2.5; MgC12 1.2; and H E P E S 25, pH adjusted to 7.4) according to a slight modification of the method of Werling et al. (1985). Briefly, mice were decapitated and their brains minus cerebella were removed for brain preparation. The brain tissue was homogenized in 10 volumes of Krebs-HEPES buffer. The homogenate was centrifuged at 27 0 0 0 × g for 15 min at 4°C. Then the pellet was suspended in 20 volumes of buffer and held on ice for 1 h to remove any endogenous opioid ligands. The suspended pellet was then washed 3 times by centrifugation at 27 000 × g for 15 min at 4°C and resuspended in ice-cold buffer. Fresh membrane preparations were used for all binding experiments.

2.4. Statistical analyses The binding assays were analyzed with the McPherson program which is a modification of the L I G A N D program for the IBM computer (McPherson, 1983). Computer software was used to conduct A N O V A with a post test of Fisher for estimating the significant differences among groups.

2.5. Drugs

2.3. Opioid receptor binding assays

DAMGO, D A D L E and D P D P E were bought from Bachem Inc. (Torrance, CA). D S L E T was bought from Serva Biochemicals Westbury, NY. [3H]DADLE and [3H]DPDPE were purchased from Amersham Corp. (Des Plaines, IL). [3H]DSLET was purchased from Du Pont Co. (Boston, MA). Naltriben was synthesized as described previously (Portoghese et al., 1988).

The radioligands used were [3H]DSLET, [3H]DAD L E and [3H]DPDPE. The homogenate was preincubated with 1 /xM [D-Ala2,MePhe4,GlyS-ol]enkephalin ( D A M G O ) for 20 min at 37°C when [3H]DSLET or [3H]DADLE binding was performed in order to block

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Bound (pM) Fig. 1. Scatchard plots of (a) [3H]DPDPE, (b) [3H]DSLET and (c) [3H]DADLE binding in brain homogenates of mice. The ordinate and abscissa represent bound/free × 103 and bound ligand (pM) (uncorrected for protein concentration mg/ml), respectively. Labelled ligand concentrations between 0.1-40 nM were used in these saturation experiments. The figures shown are data from representative experiments.

275 TABLE 1

TABLE 2

Binding characteristics of 6 opioid receptor agonists in brain membranes of mice.

Competition o f / z and 6 opioid agonist binding in mouse brain ~

Agonist

K d +_S.E. ~ (nM)

[3H]DPDPE [3H]DADLE [3H]DSLET

4.6 + 0,5 (6) 5.6 _+0.3 (3) 1.9_+0.1 (3)

Competitor

Bmax + S.E. ( f m o l / m g protein) DSLET DPDPE DADLE Naltriben

32.5 _+3.0 34.0 + 4.0 46.4+3.5 b

a N u m b e r of experiments are indicated in the parentheses. Binding assays were conducted with 11 increasing concentrations of agonists between 0.1-40 nM. [ 3 H ] D A D L E and [3H]DSLET bindings were assayed in the presence of 1 /zM D A M G O . b Value is significantly different from those of the other two ~ opioid receptor agonists.

3. Results

K i (95% confidence limits) (nM) b [3H]DPDPE

[3H]DADLE

[3H]DSLET

2.1 7.4 2.8 0.6

3.0(1.3- 4.9) 8.2 (3.1-13.5) 4.1 (1.7- 6.9) 0.3 (0.1- 0.5)

3.6 19.2 14.3 0.5

(1.7- 2.5) (2.6-12.6) (1.7- 3.0) (0.1- 1.2)

(0.6- 6.8) (17.4-21.2) c (9.7-19.3) c (0.4- 0.6)

" Competition experiments were performed with 12 different concentrations of cold ligand to compete with 5 nM [3H]DPDPE and 2 nM of [ 3 H ] D S L E T and [ 3 H ] D A D L E binding. C o m p e t i t i o n of [3H]DSLET and [3H]DADLE binding was performed in the presence of 1 p.M D A M G O . b The values given are the geometric m e a n and 95% confidence limits determined from at least three different experiments, c The K i values that show significant difference from those of other 6 opioid receptor agonists.

3.1. Binding characteristics of ~ opioid receptor agonists in brain In saturation experiments using [3H]DPDPE, the Scatchard plots were linear with a Hill coefficient around 1 and were fitted best to a single site model (fig. 1). The Scatchard plot with [3H]DADLE binding (with ~ sites blocked with DAMGO) was similar to that of [3H]DPDPE in that the data fitted best to single site models and gave a Hill coefficient of 1. The Bmax values of [3H]DADLE binding was similar to that of [3H]DPDPE binding (table 1). On the other hand, saturation experiments with [3H]DSLET (with blocked sites) revealed a significantly greater Bmax value than those of [3H]DADLE or [3H]DPDPE. The data for [3H]DSLET binding fitted best to a single site model and revealed a Hill coefficient of 1.

3.2. Competition studies of ~ opioid receptor agonist binding in brain membranes. Naltriben was equally effective in competing with the binding sites of [3H]DSLET, [3H]DPDPE and [3H]DADLE (fig. 2 and table 2). On the other hand, DADLE was more potent in competing with [3H]DADLE and [3H]DPDPE binding sites compared to its

competition for [3H]DSLET binding sites. Similarly, DPDPE was more potent in competing for the binding sites of [3H]DADLE and [3H]DPDPE compared to those of [3H]DSLET. DSLET was found to be equipotent in competing for the binding sites of [3H]DSLET, [3H]DPDPE and [3H]DADLE. The analyses of the competition curves revealed log.logit slopes around 1 for [3H]DPDPE and [3H]DADLE competition while [3H]DSLET competition gave log.logit slopes slightly less than one but LIGAND analysis still considered a one site model the best fit for the experimental data.

4. Discussion

The findings in this study demonstrated that the binding capacities determined with different 6 opioid receptor ligands were different in the brain of the mouse. Previously Cotton et al. (1985) have shown that in the guinea pig brain, differences in the binding capacities of [3H]DSLET, [3H]DPDPE and [3H]DADLE were not evident after blockade of Iz sites with DAMGO. The difference between our results and those of Cotton et al. (1985) may be due to the different

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0 - 1 4 -1'2 -1'0 ~ -'6 -'4 Log concentration of competitor

Fig. 2. Competition of specific binding of (a) 2 nM of [3H]DADLE, (b) 5 nM of [3H]DPDPE and (c) 2 nM of [3H]DSLET by increasing concentrations of naltriben ( × ) , D P D P E (©) and D S L E T (zx). T h e ordinate and abscissa represent percent of specifically bound ligand and log concentration of competitor, respectively. These figures shown are data from representative experiments.

276 species used, the effect of different buffers used in the incubation medium (Zajac et al., 1990) and the concentration of D A M G O used to suppress the tx sites. We used the same concentration of D A M G O to suppress the tx sites in both [3H]DSLET and [ 3 H ] D A D L E binding and this concentration of D A M G O did not interfere with D P D P E binding (data not shown). Competition studies among various ~ receptor agonists in brain homogenates showed that D A D L E was more potent in competing for [ 3 H ] D A D L E and [3H]DPDPE binding sites than for [3H]DSLET binding sites. D P D P E also was more potent in competing for the binding sites of [ 3 H ] D A D L E and [3H]DPDPE than for those of [3H]DSLET. In contrast, D S L E T was found to be equipotent in competing for [3H]DSLET, [3H]DPDPE and [ 3 H ] D A D L E binding sites. These resuits suggest a heterogeneous population of 6 receptors in the brain in which the binding sites for D A D L E and D P D P E a p p e a r e d to be different from those for DSLET. This suggestion was also substantiated by the differences in the binding capacity labelled by [3H]DSLET compared to those labelled by [3H]DAD L E and [3H]DPDPE. Recently, Negri et al. (1991), using two ~ opioid agonists, [3H]DPDPE and [3H]deltorphin I, reported evidence for 8 opioid subtypes in rat brain. Their results suggested that [3H]DPDPE binds to a single ~ opioid site but [3H]deltorphin I binds to two different ~ opioid sites. These results complement those in vivo where subtypes of 6 receptors were postulated in which deltorphin and D S L E T interact with similar receptors whereas D P D P E apparently interacts with different ones (Jiang et al., 1991). In this study, analyses of data from saturation binding experiments did not revealed any evidence for more than one site, although binding capacity labelled by [3H]DSLET was greater than those obtained for [3H]DPDPE or [3H]DADLE. These results may suggest the possibility that [3H]DSLET could label more than one ~ opioid sites which possess similar affinities so that the two different sites could not be discerned on Scatchard plots. In addition as mentioned above, competition experiments also suggest that D A D L E and D P D P E prefer one similar 6 opioid site whereas it is possible that D S L E T may have similar affinities to more than one fi opioid sites. The analyses of our competition experiments also did not revealed a twosite model for [3H]DSLET which may be due to similar reasons as in the saturation experiments, i.e. similar affinities of these two ~ opioid sites. Previous experiments in vivo suggested that naltriben antagonizes preferentially the antinociceptive activity of D S L E T over D A D L E and D P D P E (Sofuoglu et al., 1991a). In the present study, naltriben appeared to c o m p e t e equally well against [ 3 H ] D P D P E , [3H]DSLET and [ 3 H ] D A D L E binding in brain homogenates. The question of why the in vivo selectivity

of naltriben for D S L E T was not observed in whole brain homogenates may need to be answered by performing binding experiments using different regions of the brain especially in those sites that may be involved in the antinociceptive effects of opioids. In this regard, it is relevant that we have recently synthesized an antagonist, 7-benzylidene-7-dehydronaltrexone which competes for [3H]DPDPE 100 times more potently than for [3H]DSLET in binding assays. Also in vivo, this new antagonist inhibits the antinociceptive action of D P D P E in mice at doses that do not affect the antinociceptive activity of D S L E T (Sultana et al., in press). These findings give futher proof and fortify the postulate of ~ opioid receptor subtypes. In summary, our saturation experiments revealed that the binding capacity labelled by D S L E T was found to be different from those of D A D L E and D P D P E in brain membranes of mice. Also in competition studies, D P D P E and D A D L E displayed a similar profile that was different from that of DSLET. These results complement previously reported findings in vivo (Sofuoglu et al., 1991a,b; Jiang et al., 1991) and in vitro (Negri et al., 1991). The compilation of the data in vitro and in vivo suggest a heterogeneous ~ opioid receptor population that may represent 6 opioid receptor subtypes in which D P D P E and D A D L E interact with one subtype and D S L E T and deltorphin interact additionally with a different one.

References Corbett, A.D., M.G.C. Gilian, H.W. Kosterlitz, A.T. McKnight, S.J. Paterson and L.E. Robson, 1984, Selectivities of opioid peptide analogues as agonists and antagonists at the 6-receptor, Br. J. Pharmacol. 83, 271. Cotton, R., H.W. Kosterlitz, S.J. Paterson, M.J. Rance and J.R. Treynor, 1985, The use of [3H]-(D-Pen2,D-PenS)enkephalin as a highly selective ligand for the d binding site, Br. J. Pharmacol. 84, 927. Jiang, Q., A.E. Takemori, M. Sultana, P.S. Portoghese, W.D. Bowen, H.I. Mosberg and F. Porreca, 1991, Differential antagonism of opioid 6 antinociception by [D-Ala2,LeuS,Cys6]enkephalin and naltrindole-5'-isothiocyanate: Evidence for d receptor subtypes, J. Pharmacol. Exp. Ther. 257, 1069. Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall, 1951, Protein measurement with the Folin phenol reagent, J. Biol. Chem. 193, 265. McPherson, G.A., 1983, A practical computer-based approach to the analysis of radioligand binding experiments, Comput. Prog. Biomed. 17, 107. Negri, L., R.L. Potenza, R. Corsi and P. Melchiorri, 1991, Evidence for two subtypes of ~ opioid receptors in rat brain, Eur. J. Pharmacol. 196, 335. Portoghese, P.S., M. Sultana, H. Nagase and A.E. Takemori, 1988, Application of the message-address concept in the design of highly potent and selective non-peptide 6 opioid receptor antagonists, J. Med. Chem. 31, 281. Sofuoglu, M., P.S. Portoghese and A.E. Takemori, 1991a, Differential antagonism of 8 opioid agonists by naltrindole (NTI) and its

277 benzofuran analog (NTB) in mice: Evidence for ~ opioid receptor subtypes, J. Pharmacol. Exp. Ther. 257, 676 Sofuoglu, M., P.S. Portoghese and A.E. Takemori, 1991b, Crosstolerance studies in the spinal cord of /3-FNA-treated mice provides further evidence for delta receptor subtypes, Life Sci. 49, PL153. Sultana, M., A.E. Takemori and P.S. Portoghese, 7-Benzylidene-7dehydronaltrexone (BNTX), a highly selective ~l antagonist. The first clear evidence for ~ receptor subtypes based on binding, Abstr. Coll. Prob. Drug Dep. (in press).

Werling, L.L., G.D. Zarr, S.R. Brown and B.M. Cox, 1985, Opioid binding to rat and guinea-pig neural membranes in the presence of physiological cations at 37°C, J. Pharmacol. Exp. Ther. 233, 723. Zajac, J.M., A. Bigeard, P. Delay-Goyet and B.P. Roques, 1990, Affinity states of rat brain opioid receptors in different tissue preparation, J. Neurochem. 54, 992. Zarr, G.D., L.L. Werling, S.R. Brown and B.M. Cox, 1986, Opioid ligand binding sites in the spinal cord of the guinea-pig, Neuropharmacology 25, 471.