Chronic Administration of Morphine and Naltrexone Up-Regulate Ft-Opioid

European Journal of Pharmacology, 160 (1989) 71-82 71 Elsevier

EJP 50622

Chronic administration of morphine and naltrexone up-regulate ft-opioid binding sites labeled by [3H][D-Ala2,MePhe4,Gly-olS]enkephalin: further evidence for two p-binding sites

Richard B. Rothman 1,. Victor Bykov 1 Joseph B. Long 2, Linda S. Brady 3, Arthur E. Jacobson 4, Kenner C. Rice 4 and John W. Holaday 2 / Unit on Receptor Studies, Laboratory of Clinical Science, N1MH, Bldg. 10-3D41, Bethesda, MD 20892, 2 Division of Neuropsyehiatry, Walter Reed Army Institute of Research, Washington, DC 20307-5100, ~ Unit on Functional Neuroanatomy, N1MH, Bethesda, MD 20892, and 4 Section on Drug Design and Synthesis', LN, NIDDK, Bethesda, MD 20892, U.S.A.

Received 31 March 1988, revised MS received 25 October 1988, accepted 1 November 1988

A variety of data support the hypothesis of an opiate receptor complex composed of distinct, yet interacting/Z and 6 binding sites (termed /zcx and 6cx to indicate binding sites 'in the complex'), in addition to independent /Z and 6 binding sites, termed /znc~ and 6no~, to indicate binding sites 'not in the complex'. Ligand binding studies using membranes and slide-mounted sections of rat brain support the hypothesis that the irreversible /z-antagonist fl-funaltrexamine (FNA) selectively alkylates the opiate receptor complex, altering the binding of ~ agonists to the/zcx binding site and the binding of [3 H][D.Ala2 D_Leu 5]enkephalin to the 8~x site. Previous studies demonstrated that the chronic administration of morphine to rats selectively 'upregulates' the opiate receptor complex. In contrast, the chronic administration of naltrexone upregulates several types of opioid receptors, including x, the 6noX binding site, and multiple binding sites labeled by ~ agonists. A prediction based upon these observations is that, using [3H][D-Alae,MePhe4,Gly-olS]enkephalin to label /Z binding sites, chronic morphine should upregulate only the ~cx binding site, whereas chronic naltrexone should additionally up-regulate the /Ln~ binding site. In this study we test and confirm this hypothesis, using sensitivity to FNA to define the ~ binding site. The implications of these data for models of the opioid receptors and the mechanism(s) of tolerance and dependence are discussed.

/~ Opioid receptors; [3H][D-Ala2,MePhe4,Gly-olS]enkephalin; Morphine; Naltrexone; fl-Funaltrexamine; x Opioid receptors; (Receptor upregulation)

1. Introduction rive inhibitors at the higher affinity [3H][D- Ala2,D-LeuS]enkephalin binding site (commonly Chang and Cuatrecasas (1979) first demon- identified as 6), and potent noncompetitive inhibi- strated that [3H][D-Ala2,D-LeuS]enkephalin tors at the lower affinity [3H][D-Ala2,D-LeuS]en- labeled two binding sites which are distinguished kephalin binding site (commonly identified as #). on the basis of differential displacement by This suggested that the lower affinity [3H][D- morphine. Rothman et al. (1985a) extended this AlaZ,D-LeuS]enkephalin binding site was the 6 work by showing that/~ ligands are weak competi- binding site of an opiate receptor complex and that the non-competitive inhibition was mediated through an adjacent ~ binding site. These two binding sites were named the 8cx and /zcx binding * To whom all correspondence should be addressed. sites, the 'cx' indicating 'in the complex' (Roth- 0014-2999/89/$03.50 © 1989 Elsevier Science Publishers B.V. (Biomedical Division) 72 man et al., 1987a). The higher affinity [3H][D- TABLE 1 Ala2,D-LeuS]enkephalin binding site is termed Selected characteristics of opioid binding sites. A partial tabu- 8.... for the 8 binding site 'not in the complex'. lation of the properties of selected populations of opioid Other studies demonstrated that leucine en- receptors is provided. Citations: ~ (Rothman et al., 1987a). b (Rothman et al, 1985a). ~ (Rothman et al., 1987b). d (Danks kephalin is a non-competitive inhibitor of a et al., 1988); this study. ~ (Rothman et al., 1987a; Rothman et binding site labeled by the opioid antagonists, al., 1988c). [3H]naloxone (Rothman and Westfall, 1981; Rothman et al., 1985c) or [3H]17-cyclopropyl- Opiate receptor complex methyl-3,14-dihydroxy-4,5a-epoxy-6fl-fluoro- /~ ..... ~ - 8~ 8n,. ~ morphinan ([3H]cycloFOXY) (Rothman et al., Labeled by 1987b), leading to the hypothesis that the/~ bind- [3 HJcycloFOXY ~ No Yes No No Labeled by ing site labeled by these opioid antagonists is the [ 3H][D_AIa2 D_Leu 5 ] t~x binding site. To test this hypothesis, Rothman enkephalin b No No Yes Yes et al. (1987b) examined the effect of chronic Labeled by morphine administration, which upregulates the [3H]DAGO ~ Yes Yes No No 8~ binding site (Rothman et al., 1986), on the /~ Upregulated by chronic morphine d No Yes Yes No binding site labeled by [3H]cycloFOXY. As pre- Upregulated by dicted, chronic morphine upregulated the/~ bind- chronic naltrexone d Yes Yes Yes Yes ing site at which leucine enkephalin is a non-com- Sensitive to FNA ~ No Yes Yes No petitive inhibitor, supporting the assignment of Commonly accepted the name/~ to this site. (We use the term 'upreg- nomenclature /~ /~ ~ 8 ulate' to indicate an increase in the B.... .) In its simplest formulation, the model of an opiate receptor complex, consisting of distinct yet interacting /~ and 8~x binding sites, predicts a one-to-one ratio of /~ agonist binding sites to 6~.~ (lower affinity [3H][D-AlaZ,D-LeuS]enkephalin) Studies using the irreversible /~ antagonist /3- binding sites. However, in certain regions of the funaltrexamine (FNA) support the latter interpre- rat brain, such as the thalamus, the number of t~ tation (Rothman et al., 1988c). Pretreatment of agonist sites exceeds the number of 8~ binding slide ounted sections of rat brain with FNA pro- sites (Rothman et al., 1985a), consistent with the duces a 60% decrease in the Bm~~ of [3H]DAGO existence of a t~ binding site not part of the opiate binding sites without altering the K d (Rothman et receptor complex, termed the /~× binding site al., 1987a), while pretreatment of membranes with (Rothman et al., 1987a). FNA decreases the B...... of the 6~x binding site The hypothesis of two /~ binding sites, sum- without altering the 6ocx binding site (Rothman et marized in table 1, leads to the prediction that two al., 1988c). These data support the hypothesis that binding sites should be apparent from ligand bind- FNA alkylates the opiate receptor complex, pro- ing studies conducted using tritiated t~ agonists. ducing a conformational change resulting in such However, computer assisted analysis of the bind- a large increase in the K~ of [3H][D-AlaZ,D - ing of [3H][D-Ala2,MePhe4,Gly-olS]enkephalin LeuS]enkephalin for the 8~x binding site, and ([3H]DAGO) and the /~ agonist [3H]3,14-dihy- [3H]DAGO for the /~x binding site, that these droxy-4,5 a-epoxy-6fl- fluoro-17-methylmorphinan sites can no longer be labeled (observed as a ([3H]FOXY) (Spain et al., 1985; Zajac and Roques, decrease in the Bmax). Consistent with the hy- 1985; Rothman et al., 1988b) does not resolve two pothesis that the residual FNA-insensitive binding sites. This observation suggests either (1) [3H]DAGO binding sites are /~ncx binding sites, is the hypothesis of two/~ sites is wrong, or (2) the the different anatomical distribution of FNA-sen- Kd'S of /~ ligands for the two sites are almost sitive and FNA insensitive/~ binding sites (Roth- identical. man et al., 1987a). 73

Although pretreatment of slide mounted sec- 2. Materials and methods tions with FNA produces a decrease in the Bma x of [3H]DAGO binding sites, pretreatment of mem- 2.1. Drug treatments branes with FNA causes a 2-fold increase in the Male Sprague-Dawley rats (200-250 g), four K d without altering the Bm~X (data of this study rats per experimental group, were implanted un- and Rothman et al., 1988c). The simplest explana- der light halothane anesthesia as previously de- tion of this observation is that the FNA-induced scribed (Rothrnan et al., 1986) with two 75 mg increase in the K d of [3H]DAGO for the /~x morphine pellets (day 1). Four more pellets were binding site is small (about 2-fold) using mem- implanted on day 2 and membranes were pre- branes (thus [3H]DAGO continues to label both pared on day 5. Control rats received placebo the /~cx and /1no× binding sites), but very large pellets under an identical schedule. This protocol using slide mounted sections (thus [3H]DAGO produces a high degree of tolerance to and depen- labels only the /~ .... binding site). Taken collec- dence on morphine (Danks et al., 1988). tively, these observations support the definition of For treatment with naltrexone, rats were im- the t~. X binding site as the FNA-sensitive compo- planted s.c. on day 1 with two naltrexone pellets nent of/~ agonist binding. (30 mg naltrexone with 105 mg cholesterol and 15 Our studies of the effect of chronic morphine mg tristearin) under light halothane anesthesia administration on opioid receptors demonstrated (Yoburn et al., 1985). The pellets were wrapped in a selective upregulation of the opiate receptor nylon mesh and removed intact on day 8. Mem- complex (6cx and /~cx binding sites) (Rothman et branes were prepared on day 9. Control rats re- al., 1986; Danks et al., 1986; 1988). In contrast, ceived placebo pellets. chronic administration of naltrexone causes a more To ascertain the effect of acute drug adminis- generalized upregulation of opioid receptor sub- tration, groups of rats were administered either types, including the 6~.~ and 6 ..... binding sites saline, morphine sulfate (10 mg/kg) or naltrexone (Danks et al., 1986; 1988), ~¢ binding sites and /x (1 mg/kg) i.p. binding site(s) (Tempel et al., 1982; 1985; Paden et al., 1987). These observations led to the hy- 2.2. Preparation of membranes pothesis that, using [3H]DAGO to label /z binding sites, chronic morphine would upregulate only the For [3H]DAGO binding assays (see section FNA-sensitive, bt~× binding site, whereas chronic 2.3,), control and FNA-treated membranes were naltrexone would upregulate, in addition, the prepared as described previously (Rothman et al., FNA-insensitive btn~× binding site. This result 1983). Briefly, lysed-P2 membranes were prepared would represent a fourth line of evidence support- from a single brain and split into two aliquots for ing the existence of two/z binding sites. a 60 rain incubation at 25°C in 10 mM Tris-HCl, These considerations led us to formulate the pH 7.4, 100 mM NaCI, 3 mM MnC12 and 2 ktM following predictions: (1) chronic administration GTP in the absence (CNT-P2) and presence of 1 of morphine and naltrexone will upregulate /~M FNA (FNA-P2). The homogenates were then [3H]DAGO binding sites, (2) # binding sites up- washed three times by centrifugation and the pel- regulated by chronic morphine will be completely lets stored at -70°C until assayed. For acute sensitive to FNA, and (3) t~ binding sites upregu- injection experiments, lysed-P2 membranes were lated by chronic naltrexone will be partially sensi- prepared as described elsewhere (Rothman et al., tive to FNA, reflecting upregulation of /x~x and 1985a). /xn~ binding sites. The experiments reported in For assay of ~ binding sites (see section 2.3.), this paper test and confirm these predictions. Ad- CNT-P2 was prepared as described above. The ditionally, data are presented that chronic mor- pellets were then resuspended with 3 ml/g wet phine does not, and chronic naltrexone does, up- weight 10 mM 3-[N-morpholino]propanesulfonic regulate ~ binding sites. acid (MOPS), pH 7.4 containing 3 mM MnC12, 74

and incubated 1 /~M 2-(4-ethoxybenzyl)-l-diethyl- the experimental conditions described in section aminoethyl-5-isothiocyanatobenzimidazole • HC1 2,3., are best fit by a one site model. The equa- (BIT) and 1 ~M N-phenyl-N-[1-(2-(4-isothiocya- tions used in these fits were:

nato)phenethyl)-4-piperidinyl]propanamide - HC1 (B..... xL) (FIT), respectively, as previously described (Roth- G1S(L,LL) - (1) man et al., 1985b). Pellets were kept frozen at

- 70 ° C until assayed. GIS(L,LL) GDI(LLL) (2) 2.3. Ligand binding assays G1S(LO) The corresponding equations used to weight the [3H]DAGO binding assays were conducted at points were (Rothman et al.. 1985a): 25°C as previously described (Rothman et al., 1 1988b) in 50 mM Tris-HC1, pH 7.4, containing G1SW(L,LL) - (1') (0.0001 KGIS(L,LL) xG1S(L,LL)) protease inhibitors bacitracin (0.1 mg/ml), be- 1 statin (0.01 mg/ml), leupeptin (0.004 mg/ml) and GDIW(L,LL) = (2') chymostatin (0.002 mg/ml). [3H]D-AlaZ,D - (0.0001 + 0.0003 × GD1 (L,LL)) LeuS]enkephalin binding assays were conducted as In these equations, L refers to the concentration described (Rothman et al., 1986). [3H]Bremazo- of radiolabeled ligand, LL to the concentration of cine binding assays were conducted at 0 °C in 50 non-radioactive ligand. Equation (1) is a standard mM potassium phosphate buffer, pH 7.4, contain- one-site competitive binding model, except that L ing 0.4 M NaC1, as previously described (Roth- and LL are constrained to have the same dissoci- man et al., 1985b). Each assay condition was ation constant. Equation (2) computes a "fraction filtered after a 4-6 h incubation ([3H]DAGO) or of control' by dividing the binding observed in the 2-4 b incubation ([3H]bremazocine) (steady state presence of drugs by the binding in the presence conditions) in triplicate using a Brandell Cell of L alone. Harvester with less than 5% variation between To illustrate the application of these equations triplicates. Filters were washed twice with 5 ml to the experimental design used in this study, aliquots of ice-cold 10 mM Tris-HC1, pH 7.4 consider the displacement of two concentrations ([3H]DAGO) or ice-cold 50 mM phosphate buffer, of L, each by eight concentrations LL. This surface pH 7.4 ([3H]bremazocine). The non-specific bind- requires two measurements of total binding, two ing was determined by incubations in the presence measurements of non-specific binding, and mea- of 20 /~M levallorphan. Protein concentrations surements of binding in the presence of the 16 (Lowry et al., 1951) ranged between 0.5 and 1.0 different concentrations of LL. These data gener- mg/ml. ate two displacement curves, each associated with a level of specific binding and eight displacement 2.4. Experimental design and data analysis points. The two specific binding points are fit using equation (1) and weighted using equation Binding surfaces (Rothman, 1986) were gener- (1'), while the displacement curves are fit using ated by displacing two concentrations of 3H-ligand equation (2) and weighted using equation (2'). by eight concentrations of cold ligand, generating Both data sets (18 data points) are fit simulta- 18 data points per surface. The data were fit to a neously, after the 'specific binding' points are one site binding model for the best-fit estimates of divided by a 'scale factor' (equal to the estimated the Bn .... and K d using MLAB (Knott and Reece, B...... ) so that their units range between 0 and 1, 1972), which utilizes a weighted nonlinear least the same as the displacement curve points. Statis- squares curve fitting algorithm. Previously pub- tical significance between parameter estimates was lished data has demonstrated that [3H]DAGO assessed using a modified Student's t-test as de- (Rothman et al., 1988b) and [3H]bremazocine scribed by Brown and Hollander (1977), which (Rothman et al., 1985b) binding surfaces, using does not assume normality. 75

2.5. Chemicals 3. Results

[3H]Bremazocine (SA = 30 Ci/mmol) and [3H][D-Ala2,D-LeuS]enkephalin (SA = 36.6 3.1. Acute administration of morphine and naltre- Ci/mmol) were purchased from New England xone Nuclear Corp. [3H]DAGO (SA = 59.9 Ci/mmol) was purchased from Amersham Corp. (-)- Pert and Snyder (1976) reported that the acute Bremazocine was kindly supplied by Dr. Roemer, administration of opiate agonists or antagonists Sandoz Pharmaceuticals. DAGO and [D-Pen2,L- increased [3H]naloxone binding to rat brain mem- PenS]enkephalin were purchased from Peninsula branes. Our previous work demonstrated that the Laboratories. Peptidase inhibitors were purchased acute injection of morphine sulfate (10 mg/kg from Sigma Chemical Corp. BIT and FIT were i.p.) had no effect on [3H][D-Ala2,D-LeuS]en- synthesized as described in Dr. Rice's laboratory kephalin binding (Rothman et al., 1986). In this (Burke et al., 1986). Dr. Rapaka of NIDA kindly study, rats were administered either saline, mor- supplied the morphine, naltrexone and placebo phine sulfate (10 mg/kg) or naltrexone (1 mg/kg) pellets used in this study. B-FNA and MeTyr-D- i.p. Lysed-P2 membranes were prepared (30 min Ala-Gly-N(Et)-CH(CH 2-Ph)CH 2-N(CH 3) 2(LY- after drug administration) for assay with [3H] 164929) was generously provided by Dr. Zimmer- DAGO and [3H][D-AlaZ,D-LeuS]enkephalin. man, Eli Lilly Co., Indianapolis, IN. Aliquots of these membranes were pretreated with BIT and FIT, as described in Materials and methods, prior to assay with [3H]bremazocine. The TABLE 2 results (table 2) demonstrated no significant effect Effect of acute administration of morphine and naltrexone on (P < 0.01) on [3H]bremazocine or [3H][D-AlaZ,D- [3H]DAGO, [3H][D-Ala2,D-LeuS]enkephalin and [3H]brema- Leu 5]enkephalin binding sites. However, the acute zocine binding. As described in the text, rats received i.p. administration of naltrexone produced a small injections of saline, morphine sulfate (10 mg/kg), or naltrexone (1 mg/kg). Lysed-P2 membranes were prepared 30 rain (22%) but significant decrease in [3H]DAGO later. [3H]DAGO (2.0 nM), [3H]bremazocine (2.0 nM), and binding. [3H][D-Ala 2, LeuS]enkephalin (3.0 nM) binding sites were labeled as described in the text. The higher and lower affinity [3H][D-Ala2,D-LeuS]enkephalin binding sites were selectively 3.2. Effect of chronic morphine and naltrexone on K labeled using 50 nM [D-Pen2,L-PenS]enkephalin and 100 nM binding sites MeTyr-D-Ala-GIy-N(Et)-CH(CH 2-Ph)CH 2-N(CH 3 ) 2( LY 164929) to block binding to the higher and lower affinity binding sites, respectively (Rothman et al., 1988a). Each point As reported in table 3, chronic morphine failed is the mean_+S.D. (n = 3). a p < 0.01 when compared to con- to alter the binding of [3H]bremazocine to ~ bind- trol. ing sites, whereas chronic naltrexone produced a Assay condition Specific binding small, but significant 25% increase in the B.... . (fmol/mg protein) Saline Morphine Naltrexone Higher affinity 3.3. Effect of chronic morphine and naltrexone on i~ [3 HI[D-Ala2,D-LeuS]- opioid binding sites enkephalin binding site (Sn~ ~ binding site) 18.1_+1.8 20.2_+4.2 23.6_+ 3.0 Lower affinity Table 4 reports the effect of chronic morphine [ 3 H][D_AIa 2, D_Leu 5 ]_ and chronic naltrexone on /a binding sites labeled enkephalin binding site with [3H]DAGO. Since the data of the placebo (6~ binding site) 18.4_+0.7 21.2_+2.6 22.6_+ 2.3 groups did not significantly differ, these data were [3H]DAGO binding 16.3_+0.6 14.7_+2.2 12.7_+ 1.0 ~ combined. Using the methods described in this [ 3 H]Bremazocine study, pretreatment of rat brain membranes in binding 125 _+7.3 137 _+9.6 144 _+11.7 vitro with FNA increases the K d of [3H]DAGO 76

BREMAZOCINE BINDING SURFACE TABLE 3 Effect of chronic nahrexone and chronic morphine on ~ binding sites. Morphine or nahrexone were administered to rats gO,00 and membranes pretreated with BIT and FIT prepared as 8O described in Materials and methods. [3H]Bremazocine binding surfaces were generated by displacing 0.47 and 2.0 nM [3H]bremazocine by 16 concentrations of ( )-bremazocine e()'°L between 0.2 and 51.2 nM. Each experimental group consisted \ 60 0\ of four separate membrane preparations, each prepared from a single rat. The combined data of each group (72 data points, 40 0 \ n 4) were fit to a one site binding model for the best-fit 30 parameter estimates reported below. Figure 1 illustrates the \ binding surfaces obtained with placebo membranes. Each value is the mean 4- S.E. ,l p < 0.01 when compared to placebo. I 111 0 I l J 1 J _ i Bmax K d -11 -10.6 -10 -11.6 -8 -8.6 -8 -7.5 -7 4.8 -e (fmol/mg protein) (nM) Chronic naltrexone LOG [BR~'~.ZGCiNEIM Placebo 368 4-16 1.97 _+ 0.1 Fig. 1. [3 H]Bremazocine binding surfaces (placebo membranes). Naltrexone 463 4__ 8.0 ~ 1.41 4-0.03 '' Membranes were prepared from rats pelleted with placebo Chronic morphine pellets. Binding surfaces were generated by displacing each of Placebo 451 _+ 8.0 1.38 + 0.04 two concentrations of [~H]bremazocine (0.47 and 2.0 nM) by Morphine 472 _+ 15 1.47 _+ 0.04 eight concentrations of bremazocine (0.2-51.2 nM). The control specific binding was 98.4 ± 5.26 and 272 4-12 fmol/mg protein (mean 4_ S.D., n = 4) for the 0.47 and 2.0 nM conditions. The combined data of the four membrane preparations were fit to the one site binding model as described in table 3. The best fit parameter estimates (B~ = 368 fmol/mg protein, K d = 1.97 nM) generated the 'predicted' curves depicted in the figure. TABLE 4 The solid and dashed lines are the 0.47 and 2.0 nM displace- Effect of chronic morphine and chronic naltrexone on /~ opioid ment curves, respectively. Open circles and squares depict data binding sites. Morphine or naltrexone were administered to points derived from the 0.47 and 2.0 nM displacement curves. rats and CNT-P2 (control membranes) and FNA-P2 (FNA- respectively. pretreated membranes) were prepared as described in Materi- als and methods. [3H]DAGO binding surfaces were generated by displacing 0.5 and 2.2 nM (chronic nahrexone experiment) and 0.7 and 3.4 nM [3H]DAGO (chronic morphine experiment) by 16 concentrations of DAGO between 0.5 and 128 nM. Each experimental group consisted of four separate mem- binding sites without altering the B .... (Rothman brane preparatios, each prepared from a single rat. The com- et al., 1988c). bined data of each group (72 data points, n - 4) were fit to a one site binding model for the best-fit parameter estimates. Using CNT-P2, chronic administration of The data of the two placebo groups did not significantly differ morphine increased the Bmax of [3H]DAGO bind- and were therefore combined (144 data points, n = 8 mem- ing site(s) by 200% and increased the K d by 210%. brane preps), Each value is the mean4_S.E. ~'P < 0.01 when However, using membranes pretreated with FNA, compared to control. no increase in the BmaX was observed. Different CNT-P2 FNA-P2 results were obtained in the chronic naltrexone B .... K d B~ X K d experiment. Using CNT-P2, chronic naltrexone (fmol/mg (nM) (fmol/mg (nM) increased the Bma× by 176%, but did not increase protein) protein) the K d. Pretreating these same membranes with Placebo 74.14_2.5 2.94_0.04 69.7_+4.3 7.5_+0.1 FNA resulted in a significant increase in the K d Chronic (from 2.7 to 5.8 nM), and an increase in the B .... morphine 150 4_5.0" 6.1_+0.12" 75.0_+6.0 8.0_+0.2 of [-~H]DAGO binding sites (from 69.7 to 94 Chronic naltrexone 130 4_2.2 ~' 2.7_+0.05 94.0+3.7 ~ 5.8_+0.12 ~ fmol/mg protein). 77

4. Discussion alteration in the B...... However, previously published reports (Rothman et al., 1983; Tam and 4.1. Effect of chronic morphine and chronic naltre- Liu-Chen, 1986) led to the expectation that/~-FNA xone on tz opioid binding site(s) would produce a partial decrease in the Bma× of ~-binding sites. Our previous study (Rothman et The major goal of this study was to examine al., 1983) compared FNA-P2 to lysed-P2 mem- the hypothesis that [3H]DAGO labels two distinct branes and used [3H]oxymorphone to label t~ populations of binding sites, one being the/~ bind- binding sites. In this study we compared CNT-P2 ing site of the opiate receptor complex (#~), and to FNA-P2. The results obtained here with the other being a t~ binding site not associated [3H]DAGO demonstrate that lysed-P2 membranes with the opiate receptor complex (~,cx). The fact are not the proper control for FNA-P2, a fact not that [3H]DAGO labels an apparently homoge- appreciated at the time our previous study was neous population of ~ binding sites prompted us conducted. to examine this hypothesis using chronic drug The differences between our data and those of treatments and a site directed alkylating agent. We Tam and Liu-Chen (1986) probably reflect differ- hoped that these independent approaches would ent experimental procedures. They showed that in provide the means to test the hypothesis. As ex- vitro exposure of membranes to/~-FNA decreased plained in the Introduction, previously published the Bma x of [3H]DAGO binding site(s). However, data supported defining the/~ binding site as the they utilized membranes derived from guinea pig FNA-sensitive binding component of [3H]DAGO brain, and did not include either MnC12 or GTP binding. Other studies indicated that whereas when exposing membranes to fl-FNA. It is possi- chronic morphine selectively increased the Bmax of ble that apparently minor changes like these alter the opiate receptor complex, chronic naltrexone the exact site of alkylation, producing different produced a more generalized increase in the B.... s conformational changes in the receptor. For exam- of the various opioid receptor subtypes. This led ple, incubating slide mounted sections of molded to the prediction that whereas [3 H]DAGO binding minced rat brain with fl-FNA using a protocol sites upregulated by chronic morphine would be similar to that used in this study for membranes, completely sensitive to FNA (reflecting the selec- produced a decrease in the [3H]DAGO BmaX tive upregulation of the ~× binding site), [~H] without a change in the K d (Rothman et al., DAGO binding sites upregulated by chronic nal- 1987a). trexone would be partially sensitive to FNA (re- The observed differences are probably quanti- flecting the FNA-insensitivity of the/~.~x binding tative rather than qualitative. For example, a de- site). crease in the Bma× can be viewed as a very large The results obtained with CNT-P2 confirm increase in the K d, such that the binding site is no these predictions. As found when [3H][D-Ala2,D - longer measurable. Thus the effect of FNA can be LeuS]enkephalin was used to label the 6~ binding viewed as increasing the K a of [3H]DAGO for the site, chronic morphine, but not chronic naltre- /~x binding site. If the increase is very large, then xone, increased the K d of binding sites labeled by a decrease in the B.... is observed, since the bind- [3H]DAGO. The mechanism(s) responsible for this ing site will no longer be measurable. If the in- difference, as well as the fact that both chronic crease is small, then an increase in the K d is agonist and antagonist increased the Bm~x remains observed. Thus the alteration of binding parame- unexplored. However, it does suggest that the ters by FNA might depend not only on the ligand mechanism(s) underlying agonist- and antagonist- used to label /1 binding site(s), but also on the induced upregulation might differ, a hypothesis preparation of brain used. supported by the work of Danks et al. (1986, 1988). 4.2. Implications of these findings for models of tolerance and dependence As reported previously (Rothman et al., 1988c), pretreatment of membranes with FNA increased As originally defined by the lessons of 'classic' the K d of [3H]DAGO binding site(s) without an adrenergic pharmacology, chronic administration 78 of antagonists upregulate receptor binding sites, xone, synergizes with endogenous anti-opiates to and, as a consequence of blocking the actions of produce the withdrawal syndrome. Thus, the more endogenous agonists, also produce supersensitiv- tolerant the animal, the more anti-opiate is re- ity. Indeed, rats chronically treated with the opiate leased, resulting in a physiological context where antagonist naltrexone are more sensitive to the less naloxone is required to precipitate withdrawal. antinociceptive actions of morphine (Yoburn et This speculative, but testable, model postulates al., 1985; Tempel et al., 1985). Unlike the tonically an integrated role for the anti-opiate peptides, a active adrenergic systems, the opioid systems in potential mechanism for the development of toler- the central nervous system appear to be tonically ance and dependence, an explanation for why inactive, and are subject to activation by a variety chronic administration of agonist and antagonist of stresses including stress, endotoxic shock and both upregulate receptors, and is consistent with electroconvulsive shock (Holaday, 1985). growing evidence for parasynaptic transmission Why chronic administration of opiate antago- via the CSF as a major conduit of information nists upregulates opioid receptors of tonically in- flow in the CNS (Herkenham, 1987). Further- active systems remains to be explained. Perhaps more, because this model identifies anti-opiates as the level of tonic activity is too low to be detected the mediators of tolerance and dependence, a di- by acute challenge with an antagonist, but is de- rect prediction is that the acute and chronic ef- tectable as an upregulation with chronic challenge. fects of morphine need not be coupled. Pharmaco- Alternatively, the fact that chronic administration logical blockade of the anti-opiate(s) should pre- of both agonists and antagonists upregulate opioid vent the development of tolerance and depen- binding sites suggests a more complex explana- dence, while not blocking the acute effects of tion. morphine. Finally, the model leads to such clini- It is possible that the chronic administration of cally testable predictions that heroin addicts might morphine and naltrexone releases endogenous suffer from an abnormally high 'anti-opiatergic' peptides such as cholecystokinin-8 (Faris et al., tone, producing a state of dysphoria, most effec- 1983), c~-MSH (Contreras and Takemori, 1984), tively relieved by the self administration of an dynorphin (Huidobro-Toro et al., 1981), fl-en- opiate agonist. dorphin-(1-27) (Hammonds et al., 1984), endogenous Phe-Met-Arg-Phe-NH 2 like peptides (Yang 4,3. Effect of chronic' morphine and naltrexone on et al., 1985) and [MetS]enkephalin (Lee et al., binding sites 1980: Vaught and Takemori, 1979; Vaught et al., 1982), which have been shown to antagonize Due to a lack of selective ligands, ~ binding opioid-mediated responses, and that one or a com- sites are typically assayed with a non-selective bination of these 'anti-opiates' participate in the ligand such as [3 H]bremazocine or [ ~H]ethylketo- development of tolerance, dependence, and the cyclazocine in the presence of drugs which block observed upregulation. binding of the ligand to /, and 3 binding sites This model potentially provides a mechanism (Kosterlitz et al., 1981). Thus ~ binding sites are to explain the concurrent development of toler- operationally defined as the residual non-/*, non-6 ance and dependence, that is the increasing sensi- binding sites. tivity of the organism to challenge with an In this study we pretreated membranes with antagonist as tolerance develops (Way et al., 1969; irreversible ligands BIT (/,-selective) and FIT (3- Wei et al., 1973). Possible, the continued adminis- selective), and conducted the assay at 0 °C in the tration of morphine causes increasing release of presence of 0.4 M NaCI. Previously published anti-opiates into the cerebrospinal fluid, which ligand binding and autoradiographic data support distribute throughout the CNS, upregulating the the hypothesis that using these assay conditions opiate receptor complex, attenuating the effects of [3H]bremazocine labels ~ binding sites (Rothman morphine, thereby producing tolerance. The ad- et al., 1985b; McLean et al., 1987). The density of ministration of exogenous antagonist, i,e. nalo- the residual non-/,, non-3 binding sites is an order 79

of magnitude higher than previously reported observed by Pasternak and associates (for exam- (Robson et al., 1985). The reason for this awaits ple, see Buatti and Pasternak, 1981). (2) Computer clarification. One possibility is that x binding sites simulation studies (Rothman, 1986) demonstrate are optimally assayed at 0°C in the presence of that the three site model proposed by Pasternak is 0.4 M NaC1. A second possibility (Weyhenmeyer inconsistent with the data from which it is derived. and Mack, 1985) is that there are multiple popula- (3) The model predicts that the ICs0 for morphine tions of K binding sites, and the particular popula- displacing [3H][D-Ala 2,D_Leu 5]enkephalin bind- tion assayed depends not only on the method used ing should increase as the concentration of [3H] to prevent labeling of /~ and 6 binding sites, but [D-Ala2,D-LeuS]enkephalin is increased. How- on the ligand used to label the residual non-t~, ever, the ICs0 of morphine (Barrett and Vaught, non-8 binding sites, a concept supported by the 1983) and the highly/z-selective peptide, LY164929 findings of Zukin et al. (1988) and DeCosta et al. (Rothman et al., 1988a) required to displace (in press). [3H][D-Ala2,D-LeuS]enkephalin binding decreases Our results obtained by comparison of placebo as the concentration of [3H][D-AlaZ,D-Leu 5] membranes prepared identically to, and on the enkephalin is increased. This leftward shift in the same day, as membranes of the experimental morphine or LY 164929 displacement curve oc- group, confirm the results of Tempel et al. (1982) curs because higher concentrations of [3H][D- and Paden et al. (1987), that the chronic adminis- Ala2,D-LeuS]enkephalin label more of the lower tration of naltrexone upregulates K binding sites affinity binding sites at which the/~ ligands inter- by about 25%. The lack of effect of chronic act more potently. (4) As described in the Intro- morphine on [3H]bremazocine binding sites is evi- duction, quantitative ligand binding studies of /x dence that they are not identical to the lower agonist binding fails to resolve more than one affinity [3H][D-Ala 2,D_Leu 5]enkephalin binding binding site (Spain et al., 1985; Zajac and Roques, site or to/x binding sites labeled with [3H]DAGO. 1985; Rothman et al., 1988b). The Pasternak model predicts that three binding sites should be re- 4.4. Relationship of Ix~ and txz binding sites to the solved. (5) Similarly, quantitative binding studies present study using [3H][D-AlaZ,D-LeuS]enkephalin resolves only two binding sites (Chang and Cuatrecasas, The hypothesis of Pasternak (1982) of a com- 1979; Rothman et al., 1985a), not the three predic- mon high affinity binding site for/~ and 6 ligands ted by the Pasternak model. (6) Additional data resembles in some ways, particularly semantic, the published by Sarne and Kenner (1987) support the presently described model of two /~ and two data cited above that the hypothesis of Pasternak binding sites: those in the receptor complex, and (1982) is inconsistent with published binding data. those not associated with the receptor complex. Thus there exists only a semantic resemblance The model proposed by Pasternak is an integra- between the model of two /~ binding sites devel- tion of in vitro binding studies, as well as in vitro oped in this and previous publications (Rothman and in vivo observations made with the irreversi- et al., 1987a), and the model of Pasternak and ble ligand, naloxonazine. associates. This model is a three site binding model, which postulates that ligands such as [3H][D-Ala2,D - Leu 5]enkephalin and [3 H]DAGO.:will label three 4.5. Evidence for two classes of I1 binding sites sites termed /z 1, 8 and ~2- The /~1 site is the common high affinity binding site for /~ and 6 The three predictions of the hypothesis ad- ligands. However, several lines of evidence argue dressed by this study have been confirmed: (1) against this model. (1) The published binding chronic administration of morphine and naltre- parameters, which are purported to describe the xone upregulate [3H]DAGO binding sites, (2) ~t model (Pasternak, 1982), generate displacement binding sites upregulated by chronic morphine are curves without the prominent plateaus actually completely sensitive to FNA, and (3) t~ binding 80 sites upregulated by chronic naltrexone are par- that 125I-,8-endorphin labels several t~-type bands tially sensitive to FNA. using crude membrane preparations (Howard et These data provide additional evidence for the al., 1985), and (5) the observation reported in this existence of two classes of tt binding sites, one site paper of the differential upregulation of the two in the receptor complex, the/zc~ binding site, and [3H]DAGO binding sites by two different chronic one not associated with the receptor complex, the drug treatments. Further examination of this hy- /z .... binding site. According to this hypothesis, pothesis will depend upon the ability to selectively illustrated in table 1, FNA selectively alkylates the assay the/z,, x and tLn~x binding sites. receptor complex, leading to a very large increase in the K d of [3H][D-Ala2,D-LeuS]enkephalin for the 6c~ binding site (observed as a decrease in the Note added in proof B ..... ) and an increase in the K d of the/~ binding site (Rothman et al., 1988c), Using membranes, While this paper was in review, a quantitative autoradio- the increase in the K d of the /z~ site is small, so graphic study demonstrating upregulation of /z opioid recep- that the overall K d, which is a combination of the tors by chronic morphine was accepted for publication in Brain Research (Brady, L.S., M. Herkenham, J.B. Long and Kj of [3H]DAGO for the /zc~ and /z .... binding R.B. Rothman, Chronic morphine increases t~-opiate receptor sites, increases about 2-fold without a decrease in binding in rat brain: a quantitative autoradiographic study, the B.... . Chronic morphine selectively upregu- Brain Res., in press). lates the/zc× binding site, while chronic naltrexone upregulates both the tt~,~ and /znc~ binding sites. Upregulated tt~.~ binding sites differ from normal References /z~ binding sites in that alkylation with FNA results in a large increase in the K d of [3H]DAGO Barrett, R.R. and J.L. Vaught, 1983. Evaluation of the interac- for these sites, which is observed as a decrease in tions of mu and delta selective ligands with ~H-D-ala2-D- leu%enkephalin binding to mouse brain membanes, Life the B ...... Thus in the chronic morphine experi- Sci. 33, 2439. ment, all the upregulated [3H]DAGO binding sites Brown, B.M.J. and M. Hollander, 1977, Statistics, a Bio- were eliminated by FNA, while in the chronic medical Introduction (John Wiley & Sons) p. 118. naltrexone experiment only a portion of upregu- BuattL M.C. and G.W. Pasternak, 1981, Multiple opiate recep- lated t~ binding sites were eliminated. The hy- tors: phylogenic differences, Brain Res. 218, 400. Burke, T.R., Jr., A.E. Jacobson, K.C. Rice, J.V. Silverton, W.F. pothesis that the tLc~ binding sites upregulated by Simonds. R.A. Streaty and W.A. Klee, 1986. Probes for chronic morphine and naltrexone must differ in narcotic receptor mediated phenomena. 12. cis-( + )-3-meth- some way from binding sites present in placebo ylfentanyl isothiocyanate, a potent site-directed acylating membranes is supported by the observation that agent for delta opioid receptors. Synthesis, absolute config- upregulated lower affinity [3HI[D-Ala2,D- uration, and receptor enantioselectivity, J. Med. Chem. 29, 1087. LeuS]enkephalin binding sites are labile to prein- Chang, K.-J. and P. Cuatrecasas, 1979, Multiple opiate recep- cubation of membranes in high concentrations of tors: enkephalins and morphine bind to receptors of differ- NaC1 (Danks et al., 1986: 1988). ent specificity, J. Biol. Chem. 254, 2610. The hypothesized existence of two populations Contreras, P.C. and A.E. Takemori, 1984, Antagonism of of tL opioid receptors is consistent with (1) the morphine-induced analgesia, tolerance, and dependence by alpha-melanocyte-stimulating hormone. J. Pharmaco[. Exp. varying ratio of # and ~ binding sites across Ther. 22% 21. regions of the brain (Rothman et al., 1985a), (2) Danks, J.A., F.C. Tortella, V. Bykov, A.E. Jacobson, K.C. Rice the observation that FNA-sensitive and FNA-in- and R.B. Rothman, 1986, Up-regulation of the mu-non- sensitive [-~H]DAGO binding sites possess differ- competitive delta binding site by chronic morphine admin- ent anatomical distributions (Rothman et al., istration: effect of preincubating membranes in 400 mM sodium chloride, in: NIDA Research Monograph 75, 'Pro- 1987a), (3) physiological data (Sayre et al., 1983; gress in Opioid Research - Proceedings of the 1986 Inter- McGilliard and Takemori, 1978: Schulz and Wus- national Narcotics Research Conference', eds. J.W. Hola- ter, 1981; Ward et al., 1986; Porreca and Tortella, day, P.-Y. Law and A. Herz (Department of Health and 1987; Sheldon et al., 1987), (4) biochemical data Human Services, Washington DC) p. 93. 81

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