Differential binding of opioid peptides and other drugs to two subtypes of opioid δncx binding sites in mouse brain: Further evidence for δ receptor heterogeneity

Peptides,Vol. 14, pp. 893-907, 1993

0196-9781/93 $6.00 + .00 Copyright © 1993 PergamonPress Ltd.

Printed in the USA.

Differential Binding of Opioid Peptides and Other Drugs to Two Subtypes of Opioid 6nc x Binding Sites in Mouse Brain: Further Evidence for 6 Receptor Heterogeneity H E N G X U , * J O H N S. P A R T I L L A , * B R I A N R. DE COSTA,J" K E N N E R C. RICE~" A N D R I C H A R D B. R O T H M A N *1

*Clinical Psychopharmacology Section, NIDA Addiction Research Center, PO Box 5180, Baltimore, M D 21224 and /Laboratory of Medicinal Chemistry, NIDDK, NIH, Bethesda, M D 20892 R e c e i v e d 25 F e b r u a r y 1993 XU, H., J. S. PARTILLA, B. R. DE COSTA, K. C. RICE AND R. B. ROTHMAN. Differential binding ofopioidpeptides and other drugs to two subtypes of opioid 6nc~binding sites in mouse brain: Further evfdencefor 6 receptorheterogeneity. PEPTIDES 14(5) 893-907, 1993.--Research into the functional role of the opioid 6 receptor has intensified with the recent in vivo identification of 6 receptor subtypes, termed 6~and 62, which mediate antinociception in the mouse. A variety of data also support the hypothesis of an opioid receptor complex composed of distinct, yet interacting, u, 6, and perhaps r binding sites. This model postulates two classes of 6 binding sites: a 6 binding site not associated with the opioid receptor complex, termed the 6ncxsite, and a 6 site associated with the receptor complex, termed the 6¢x site. A major purpose of this study was to clarify the relationship between the 6,¢xbinding sites and the 6~ and 62 receptors. Mouse brain membranes were depleted of u sites by pretreatment with the sitedirected acylating agent, BIT, and the 6,~ binding sites were labeled with [3H][D-Ala2,D-LeuS]enkephalin.Binding surface analysis readily resolved two binding sites (6.~x.~ and 6.~x_:)in the absence and presence of 100 mM NaCI. Control experiments with guanine nucleotides and the ligand-selectivity analysis indicated that the two sites were not two states of a single receptor. Pretreatment of membranes with DALCE, but not [Cys4]deltorphin, decreased [3H][D-Ala2,D-LeuS]enkephalinand [3H][D-SerZ,Thrr]enkephalin binding. Ligand-selectivity analysis of the two binding sites suggested that neither 6,¢xbinding site had the characteristics expected of the 62 receptor, and that the 6,~,.~ site, but not the 6,¢~.2site, was synonymous with the 6~ receptor. Moreover, our finding that the racemic nonpeptide 6 agonist, BW373U86, had high affinity at and selectivity for the 6,c~.2site suggests that this site may be a novel 6 receptor that mediates some of the effects of BW373U86. Opioid peptides

Opioid binding sites

Mouse brain

6 receptor heterogeneity

R E S E A R C H into the functional role of the opioid 6 receptor, first described by Lord et al. (33), has intensified with the recent in vivo identification of 6 receptor subtypes, termed b j a n d ~2, which mediate antinociception in the mouse (24,35,67,68). This work was facilitated by the d e v e l o p m e n t of selective agonist a n d antagonist drugs for these two receptors. Thus, [D-Pen2,D-PenS]enkephalin (DPDPE) a n d Tyr-DAla-Phe-GIu-Val-Val-GIy-NH2 (deltorphin-II, DELT-II) are agonists, whereas 7-benzylidene-7-dehydronaltrexone (BNTX) a n d naltriben (NTB) are reversible antagonists, for the 6, a n d ~2 receptors, respectively (24,46,67,70). The enkephalin analog, [D-Ala2,LeuS,Cys6]enkephalin (DALCE), is a selective irreversible i n h i b i t o r of the ~1 receptor, a n d 5'naltrindole isothiocyanate (5'-NTII) a n d Tyr-D-Ala-Phe-Cys-

Val-Val-Gly-NH2 ([Cys4]DELT) are selective irreversible inhibitors at the 62 receptor (21,24). Other lines of data also support the existence of 6 receptor subtypes. For example, much data support the hypothesis of an opioid receptor complex composed of distinct, yet interacting, u, b, and perhaps r binding sites ( 19,58,59). This model postulates two classes of 6 binding sites: a ~ binding site not associated with the opioid receptor complex, termed the 6,c~ site (the ncx stands for not in the complex), and a 6 site associated with the receptor complex, termed the 6 , site (the cx stands for in the complex). The 6ncx site has high affinity for ligands such as D P D P E (38), low affinity for morphine, and is synonymous with what is commonly identified as the 6 binding site. Both u and 6 fig,ands appear to be competitive inhibitors of [3H][I~Ala/,D-LeuS]enkephalin

Requests for reprints should be addressed lo Richard B. Rothman.

893

894

XU ET AL.

binding to the 6ncxsite (53,56,59). The 6cx site has high affinity for tz ligands, low affinity for DPDPE, and is optimally labeled with [3H][D-Ala2,LeuS]enkephalin. Mu ligands are noncompetitive inhibitors at the 6¢x site (60). Under appropriate assay conditions, recent work demonstrated that the ligand-selectivity pattern of the 6cx site is markedly different from that of the # receptor (60). The 6ncxsite is thought to mediate the direct antinociceptive effects of DPDPE, an effect that is selectively reversed by the 6selective irreversible antagonist, DALCE (22). In contrast, the 6c~ site is thought to mediate the modulatory effects of subantinociceptive doses of enkephalin-related peptides on morphineinduced antinociception, an effect that is blocked by the irreversible antagonist, 5'-NTII (58). Recent data that 5'-NTII blocks 62 receptor-mediated modulation of morphine antinociception supports the hypothesis that the ~2 receptor and the 6¢~ receptor may be synonymous (44). It is apparent that two different schemes of 6 receptor subtypes have developed: an earlier model, based on studies of the opioid receptor complex, and a more recent model, which evolved with the use of recently available agonists and antagonists. A point of some interest is the relationship between the 6nc x and 6cx binding sites on the one hand, and the 6~ and 62 receptors on the other. We decided to test the hypothesis that the 6n~ and 6~ receptor are the same by comparing detailed ligand-selectivity studies of the 6,~x binding site with the known pharmacology of the 6~ receptors. An unforeseen result of our first study, which was conducted using rat brain membranes, was that the 6ncx binding site could be resolved into two binding sites (77). Whereas site 2 had high affinity for all peptide ligands tested {Tyr-D-Ala-Gly-Phe-D-Leu (DADL), [D-Ser2,Thr6]enkephalin (DSTLE), DPDPE, Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2 (DELT-I), and DELT-I1}, site 1 had high affinity only for DADL, DSTLE, and DELT-II. This pattern of results, along with the observation that DALCE irreversibly binds to the 6,c~ site (55), suggested that we had resolved two subtypes of the 6,~x site, not 6~ and 62 receptors. Rather than invest additional time characterizing the multiple 6,cx sites of the rat, we decided to extend these studies to mouse brain, since 6~ and 62 receptors have been pharmacologically characterized in the mouse, not the rat. The primary question addressed by this study is the relationship between the 6,cx binding sites and the 6~ and 62 receptors. The major finding is that, as observed in the rat, the 6,c~ binding site can be resolved into two sites, which are not 62-like. METHOD

Preparation of Membranes Membrane preparations were prepared with minor modifications of published procedures (59). Briefly, 25 frozen mouse brains (Pel Freeze) were placed into ice-cold 10 m M Tris-HC1, pH 7.4 (5 ml/brain), and homogenized while still frozen with a polytron for 20 s at setting number 5. The homogenates were pooled and centrifuged at 30,000 )< g for 10 min. The pellets were resuspended in an equal volume of ice-cold l0 m M TrisHCI, pH 7.4, and recentrifuged. The pellets were then resuspended in 10 m M Tris-HC1, pH 7.4, containing 100 m M NaC1, 3 m M MnCI2, and 2 # M GTP and incubated for 60 min at 25°C. After the membranes were washed three times by repeated centrifugation and resuspension with ice-cold 10 m M Tris-HCl, pH 7.4, the membrane pellets were resuspended with 10 m M 3-[N-morpholino]propanesulfonic acid (MOPS) buffer, pH 7.4, containing 3 m M MnC12. Following a 60-min incubation at 25 °C with 1 #M of the irreversible # ligand, 2-(p-ethoxybenzyl)-l-

diethylaminoethyl-5-isothiocyanatobenzimidazole-HCl(BIT) or no drug, the membranes were washed three times by repeated centrifugation and resuspension with ice-cold 10 m M Tris-HC1, pH 7.4. The final membrane pellets were kept at -70°C. According to previously established nomenclature, these membranes are termed CNT/BIT membranes if the incubation occurred with BIT, or CNT/CNT membranes if the incubation occurred without BIT (59). For the preparation of DALCE- or [Cysa]DELT-II (DELT4C)-pretreated membranes, standard mouse CNT/BIT membranes were resuspended with 10 m M MOPS, pH 7.4, containing 3 m M MnC12 and 10 m M 2-mercaptoethanol. Following a 60min incubation at 25°C with DALCE or DELT-4C, the membranes were washed three times by repeated centrifugation and resuspension in ice-cold 10 m M Tris-HC1, pH 7.4. After a 60min incubation at 37°C in 50 m M Tris, pH 7.4, containing 200 m M NaC1 and 50 u M GppNHp, the membranes were washed three times with ice-cold 10 m M Tris, pH 7.4, and the final membrane pellets were kept at - 7 0 ° C until assayed.

Ligand Binding Assay, Data Analysis, Statistics, and Chemicals As in earlier studies (59), incubations with [3H][D-AlaZ,DLeuS]enkephalin were conducted for 4-6 h at 25°C in 10 m M Tris-HC1, pH 7.4, containing 100 m M choline chloride, 3 m M MnCI2, and a protease inhibitor cocktail (bacitracin 100 t~g/ml, bestatin 10 ug/ml, leupeptin 4 ug/ml, and chymostatin 2 ug/ ml). In a series of experiments, 100 m M NaC1 was used instead of choline and MnC12, and in other experiments 100 n M LY164929 was used to block [3H][D-Ala2,D-LeuS]enkephalin binding to the 6cx site (57).

TABLE l INHIBITION OF [3HIDADL BINDINGTO THE ~,~ BINDING SITE OF MOUSE BRAIN MEMBRANES BY VARIOUSPEPTIDES IN THE TRIS/CHOLINECONDITION IC~o(nM _+SD) (SlopeFactor + SD) Drug DPDPE DPLPE DELT-I DELT-II

CNT/CNT Membranes

CNT/BIT Membranes

6.87 ± 0.57 (0.87 ± 0.06) 6.94 _+0.96 (0.70 _+0.07) 6.99 _+0.79 (0.63 _+0.04) 9.80 ± 0.86 (0.92 _+0.07)

13.7 ± 2.2* (0.84 + 0.10) 9.61 _+ 1.72 (0.67 ± 0.07) 18.6 _+2.5* (0.65 -+ 0.06) 9.47 ± 1.57 (0.82 _+0.10)

Each value is mean _+SD. Usingthe Tris]choline assay condition, [3H][D-AlaZ,D-LeuS]enkephalin (2.5 nM) was displaced by nine concentrations of the indicated peptides. The data of two experiments were combined ( 18 data points) and fit to the twoparameter logistic equation for the best-fitparameter estimatesreported above.When assayingCNT/ CNT membranes, 100 nMLYl64929 was included to block [3H][D-AIa2,D-Leu5]enkephalinbinding to the 6cxbinding site. * When compared to control, p < 0.01.

BINDING OF OPIOID PEPTIDES IN MOUSE BRAIN

TABLE 2 EFFECTS OF GppNHp ON DPLPE INHIBITION CURVES GppNHp (~M)

IC.~o (riM+_SD)

Slope Factor (_+SD)

0 10 50

10.7 _ 1.2 18.7 _+2.6* 16.6 _ 2.7*

0.77 _ 0.06

0.69 +-0.06 0.62 _+0.06*

Using the Tris/choline assay condition and mouse CNT/BIT membranes, [3H]D-Ala2,DLeuS]enkephalin (3 nM) was displaced by nine concentration of DPLPE in the absence and presence of GppNHp. The data of two experiments were combined ( 18 data points) and fit to the twoparameter logisticequation for the best-fitparameter estimates (_+SD)reported above. * When compared to control, p < 0.01.

As described in other papers in greater detail (54), single inhibition curves were fit to the two-parameter logistic equation (50) for the best-fit estimates of the IC50 and slope factor using MLAB-PC (Civilized Software, Bethesda, MD). Binding surfaces (36,51,64) were fit to one- and two-site binding models for the best-fit parameter estimates using MLAB-PC, which is a true implementation of MLAB as described by Knott and Reece for the DEC-10 computer system (28). Statistical differences between one- and two-site binding models, and between binding parameters, used the F-test (39), as previously described (56). DALCE, [D-Ala2,LeuS,Serr]enkephalin (DALES), DELT-4C, Tyr-D-Ala-Phe-Ser-Val-Val-Gly-NH2([Ser4]DELT, DELT-4S), and 3-lodo-Tyr-o-Ala-Gly Phe-D-Leu ([t27I]DADL) were synthesized and purified at the Brown University Macromolecular Synthetic Facility (Providence, RI). [3H][D-AlaZ,D-LeuS]Enkephalin (sp.act. = 34.68 Ci/mmol) and [3H]DSTLE (sp.act. = 24.74 Ci/mmol) were obtained from Multiple Peptide Systems (San Diego, CA), by arrangement with Robert Walsh, Research Technology Branch, NIDA. When necessary, the [3H]ligands were repurified by high pressure liquid chromotography. 5'Guanylyimidodiphosphate (GppNHp) was purchased as the lithium salt from Sigma Chemical Co. (St. Louis, MO). Naltriben and BNTX were provided by Philip S. Portoghese, Ph.D., Professor of Medicinal Chemistry, Department of Medicinal Chemistry, University of Minnesota, College of Pharmacy, Health Sciences, Rm. 8-114, Unit F, 308 Harvard Street SE, Minneapolis, MN 55455. The sources of the other reagents have been described (54). Interested investigators can obtain complimentary samples of BIT and FIT by writing to Kenner C. Rice, Ph.D., Chief, LMC, Bldg. 8-B120, NIDDK, NIH, Bethesda, MD 20892. RESULTS

Effect of BIT and GppNHp on Peptide Inhibition Curves The first series of experiments assessed the effect of BIT on [3H][D-Ala2,D-LeuS]enkephalin binding to the ~¢x binding site. As reported in Table 1, slope factors less than 0.7 were observed for [D-Pen2,L-PenS]enkephalin (DPLPE) and DELT-I, whether or not the membranes were pretreated with BIT. Pretreatment with BIT increased the IC5o values of DPDPE and DELT-I by about 2-fold. Thus, as observed in rat brain (77), pretreatment of mouse brain membranes with BIT does not create the low

895

slope factors, which are a primary indicator of the possible occurrence of multiple binding sites. To determine if the low slope factors resulted from the occurrence of two states of the 6ncxbinding site, DPLPE inhibition curves were generated in the absence and presence of GppNHp. As reported in Table 2, GppNHp increased the ICs0 value by almost 2-fold, but also decreased the slope factor, changes that are not consistent with the presence of two states of a G-proteinlinked receptor (74).

Binding Surface Analysis of [3H][D-Ala2,D-LeuS]Enkephalin Binding The experimental design use to characterize [3H][D-Ala2,DLeuS]enkephalin binding to 8,¢xbinding sites is described in Table 3. In these experiments, two concentrations of [3H][D-Ala2,DLeuS]enkephalin (0.5 and 2.5 nM) were each displaced by eight concentrations of primary displacers, in the absence or presence of the indicating blocking agents. For the Tris/choline assay condition, the entire data set, consisting of 324 data points, was fit to one- and two-site binding models. The two-site model fit the data significantly better than did the one-site model (p < 0.001). Representative binding surfaces are shown in Fig. 1. The best-fit parameter estimates, reported in Table 4, indicated that [3H][D-Ala2,D-LeuS]enkephalin labeled two binding sites present at densities of 104 fmol/mg protein (6.... l) and 54 fmol/mg protein (~ncx-2). Whereas [DAlaE,o-LeuS]enkephalin, naltrindole, and DELT-II were essentially nonselective between the two sites, DPDPE was about 10fold selective for the 6.... 1 binding site.

TABLE 3 BINDING SURFACEANALYSISOF [3H][D-Ala2,D-LeuS]ENKEPHALIN BINDING TO MOUSE BRAIN ~.¢~BINDING SITES Surface

PrimaryDisplacer

Blocker

Panel

1 2 3 4 5 6 7 8 9 10 11 12 13

DADL DADL DADL DADL DPDPE DPDPE DPDPE DELT-II DELT-I1 DELT-II Naltrindole Naltrindole Naltrindole

None DPDPE (1 nM) DELT-II (2 riM) Naltrindole* None DELT-1I (2 riM) Naltrindole* None DPDPE (1 nM) Naltrindole* None DPDPE (1 nM) DELT-II ( 1 nM)

A A B B C C C D D D E E E

The experimental design used for binding surface analysis of [3H][DAla2,D-Leu5]enkephalin binding to mouse brain 6ncxbinding sites is described. In these experiments, two concentrations of [3H][D-AIa2,DLeu5]enkephalin (0.5 and 2.5 nM) were each displacedby eight concentrations of primary displacers,in the absenceor presenceof the indicating blocking agents. Unblocked surfaces generated 18 data points each, whereas the blocked surfaces generated 20 points each. The entire data set was generated by combining the data of several independent experiments. Representative data are shown in Fig. 1 for data generated using the TRIS/choline condition, and Fig. 2 for data generated using the TRIS/sodium condition. The best-fit parameter estimates are reported in Table 4 (TRIS/cholinecondition)and Table 5 (Tris/sodiumcondition). * The concentration of naltrindole was 0.2 nM (TRIS/choline condition) and 0.5 nM (TRIS/sodium condition).

896

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FIG. 1. [~H][D-Ala2,D-LeuS]Enkephalin binding surfaces in the Tris/choline condition. The experimental design used for these experiments is described in Table 3. For each panel of this figure, two concentrations of [3H][D-Ala2,D-LeuS]enkephalin (0.5 nM, solid line; 2.5 nM, dashed line) were each displaced by eight concentrations of test drug. When a blocker was present, the data are presented as a percent of the control binding in the absence of any drug. (A) [3H][D-AlaE,D-LeuS]enkephalin (0.5 and 2.5 riM) was displaced by [D-AIa2,DLeuS]enkephalin in the absence (X) or presence of 1 riM DPDPE ([]). (B) [3H][D-Ala2,D-LeuS]Enkephalin (0.5 and 2.5 nM) was displaced by [D-Ala2,D-LeuS]enkephalin in the presence of 2 nM DELT-II (X) or 0.2 nM naltrindole (n). (C) [3H][D-Ala2,D-LeuS]Enkephalin (0.5 and 2.5 nM) was displaced by DPDPE in the absence (X) or presence of 2 nM DELT-II (D) or 0.2 nM naltrindole (+). (D) [3H][D-Ala2,DLeuSlEnkephalin (0.5 and 2.5 nM) was displaced by DELT-II in the absence (X) or presence of 1 nM DPDPE ([2) or 0.2 nM naltrindole (+). (E) [3H][D-Ala2,D-LeuS]Enkephalin (0.5 and 2.5 nM) was displaced by naltrindole in the absence (X) or presence of 1 a M DPDPE ([3) or 1 aM DELT-II (+).

BINDING OF OPIOID PEPTIDES IN MOUSE BRAIN

897

Binding surfaces were similarly generated in the presence of 100 m M sodium chloride (Fig. 2). As reported in Table 5, two binding sites were also readily resolved. The major effect of sodium was to decrease the Bma x of the 6,cx-2 site to 31 fmol/mg protein and to substantially increase the K~ values of DPDPE, DELT-II, and naltrindole for the 6.¢x.2binding site so that these drugs were 1546-fold, 1194-fold, and 492-fold selective for the 6,c~_~ binding site, respectively.

In contrast to the selectivity of DPDPE for the ~.cx-~site, the novel nonpeptide 6 agonist, BW373U86 (9), was 20-fold selective for the 6.cx.2site. In fact, this racemic drug was the most selective agent for the 6,cx-2 binding site. We recognize that studies with racemic compounds can only provide preliminary data, and it is hoped that preparation of the enantiomers of BW373U86 may provide tools that may reveal even greater degrees of selectivity. Similar studies were conducted using the Tris/choline assay condition (Table 7). For most test drugs, the presence or absence of sodium had little effect on their K~values for the 6ncx. 1 binding site. In contrast, with the exception of DADL, DSTLE, [pCI]DPDPE, BW373U86, and NTB, sodium substantially (greater than 10-fold) increased the K~ values of most test drugs for the 6ncx.2binding site. Interestingly, sodium decreased the Ki values of BW373U86 and NTB for the 6.c~.2 site by more than 10-fold. Comparison of the selectivity of the test drugs for the 6.~x.j and 6,¢x_2binding sites (Fig. 4) showed that whereas sodium had little effect on the selectivity of DADL, DSTLE, [~27I]DADL, [pC1]DPDPE, and BW373U86 for the 6,¢x_~ site, it markedly increased the selectivity of DPDPE, DPLPE, DELT-I, DELTII, naltrindole, and oxymorphindole for the fi.... ~ binding site. In contrast, sodium actually reversed the selectivity of NTB and BNTX. Thus, in the absence of sodium, NTB and BNTX are selective for the 6ncx-t and 6,cx-2 sites, respectively, but in the presence of sodium they are selective for the ~,~-2 and 6.... binding sites. These changes in selectivity result from the different degrees of sodium shift observed for each drug at the two binding sites (Fig. 5).

Ligand-Selectivity Analysis

Effect of DALCE and [Cys4]DELT on 6,ex Binding Sites

TABLE 4 BEST-FIT PARAMETER ESTIMATESOF THE TWO-SITE MODEL: TRIS/CHOLINE ASSAYCONDITION Parameter value +_SD Parameter Bmax

[3H]D-AIa2,D-Leu5]Enkephalin (Kd, nM) DPDPE (K~,nM) DELT-II (K~,nM) Naltrindole (K~,nM)

Site I 6.~,_~

Site 26~¢~_2

104 _+46

54 + 46

0.67 _+0.14 0.35 +_0.24 1.15 + 0.42 0.20 _+0.08

0,36 -+ 0.17 3,18 -+ 1.73 2,41 _ 0.80 0.52 ___0.15

As described in the legend to Table 3, the data of several independent experiments, conducted using the Tris/choline assay condition, were combined, generating a total of 324 data points. Fitting the data to a one-site model resulted in a sum of square (SS) of 6306. Fitting to a twosite model resulted in a highly significant (F = 5.24, p < 0.001) decrease in the SS to 5820. The best-fit parameter estimates (+SD) are reported.

As described in the legend to Table 6, the ligand selectivity of the 6~x., and 6,cx.2binding sites was determined by generating, for each test drug, binding surfaces in the absence of a blocker, in the presence of 2 nM DPDPE, and in the presence of 4 nM DELT-II. The data of these three independent experiments were pooled, and fit to the two-site model with the Bm~ values, the Kd values of [3H][D-Ala2,D-LeuS]enkephalin, and Ki values of DPDPE and DELT-II fixed to the values determined by analysis of the primary data sets (Tables 4 and 5) for the best-fit parameter estimates of the two-site model. An example of such an experiment is shown in Fig. 3 for BW373U86, except that only the data of the inhibition curves generated against 2.5 nM [3H][DAla2,D-LeuS]enkephalin are shown. The ligand-selectivity pattern obtained for the Tris/sodium assay condition is reported in Table 6. The enkephalin-related peptides, [D-Ala2,D-LeuS]enkephalin, [~27I]DADL, and DSTLE were relatively nonselective between the two 6.¢x binding sites: whereas [D-Ala2,D-LeuS]enkephalin and [127I]DADL were 4.7fold and 3. l-fold selective for the ~.... ~binding site, DSTLE was 2-fold selective for the 6n~x.2site. In contrast, the cyclic enkephalin analogs, DPDPE and DPLPE, were highly selective for the 6.... ~ site. Both DELT-I and DELT-II were also highly selective for the 6~_~ binding site. Unlike DPDPE, its halogenated analog [pCl-Phe4,D-PenS]enkephalin ([pCI]DPDPE) had about the same affinity for both binding sites. Naltrindole, and the 6~-selective antagonist BNTX (46), were moderately selective for the 6,~.~ binding site. In contrast, the 62-selective antagonist NTB (67,70) was moderately selective for the 6,~_2 binding site. The 6 agonist, oxymorphindole (23), like naltrindole, was also highly selective for the 6~cx.~binding site. Morphine had much lower affinity for the 6,cx_1binding site than the 6 agonists.

DALCE is a cystinine-containing analog of [LeuS]enkephalin that acts as an irreversible inhibitor at 61 receptors in vivo (24), probably via disulfide bond formation (6). [Cys4]DELT acts as an irreversible antagonist at 62 receptors in vivo (21). However, this is not due to covalent bond formation, since [Ser4]DELT, which cannot form a covalent bond, also acts as an apparent irreversible inhibitor of 62 receptors in vivo. The next series of experiments was conducted to determine if [Cys4]DELT acts as an apparent irreversible inhibitor of the 6,cx binding sites. As an additional control, the Ser4 analogs were tested, since these do not act as irreversible inhibitors (21,34). In a reversible binding assay, three of four peptides had high affinity for the 6,cx binding sites. [Ser4]DELT had the lowest affinity, with an IC~o value of 48 nM. DALCE, DALES, and [Cys4]DELT had slope factors close to unity, indicating that they probably had equal affinity for the two 6,cx binding sites (Table 8). When tested as irreversible inhibitors, both DALES (1 #M) and [Ser4]DELT (1 uM) failed to produce any wash-resistant inhibition of either [3H][D-AlaZ,D-LeuS]enkephalin or [3H]DSTLE binding (data not shown). As reported in Table 9, DALCE (2 uM), but not [Cys4]DELT (2 •M), acted as an apparent irreversible i n h i b i t o r of both [3H][D-Ala2,D LeuS]enkephalin or [3H]DSTLE binding. Moreover, 1 # M [Cysa]DELT did not alter the effect of 1 ~zM DALCE.

Prediction Experiments A direct prediction of this selectivity of BW373U86 for the 6n~x.2 site is that partial blockade of [3H][D-AlaZ,DLeuS]enkephalin binding to the 6.... 1 binding site with either DPDPE or DELT-II should shift the BW373U86 inhibition curve to the left, not to the fight, as one might otherwise expect.

898

XU ET AL

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-10

-~

LOG [DADL] M C. DPDPE BINDING SURFACES 100

J rm rr

6O

I.L 0

5O

I-Z W

-7

-6

-5

-

-3

[DADL] M

D. DELTORPHIN-II

_... ".

~

I-Z 0 U

8

LOG

BINDING SURFACES

100 [ ~ < . ~ , .

3

7O

.

+

~

&%.

~0

,01

.

,

,',+

W

0

-11

-10

-9

-8

-7

-6

-5

-3

-~

0

-10

-11

LOG [DPDPE] M

-9

-8

-7

-6

-5

-~'

-3

LOG [ D E L T O R P H I N - l ] ] M E. NALTRINDOLE

BINDING SURFACES

100

90 0 n," ~Z E3 (j

E

8O 70 60

~o

W W n

20

10

-11

-10

-9

-8

-7

-6

)5

_l

-3

LOG [NALTRINDOLE] M

FIG. 2. [3H][D-AlaZ,D-LeuS]Enkephalin binding surfaces in the Tris/sodium condition. The experimental design used for these experiments is described in Table 3. For each panel of this figure, two concentrations of [3H][D-Ala2,D-LeuS]enkephalin (0.5 nM, solid line; 2.5 nM, dashed line) were each displaced by eight concentrations of test drug. When a blocker was present, the data are presented as a percent of the control binding in the absence of any drug. (A) [3H][D-Ala2,o-LeuS]Enkephalin (0.5 and 2.5 nM) was displaced by [DAla2,D-LeuS]enkephalin in the absence (X) or presence of 1 nM DPDPE (F]). (B) [3H][D-Ala2,D-LeuS]Enkephalin (0.5 and 2.5 nM) was displaced by [D-Ala2,D-LeuS]enkephalin in the presence of 2 nM DELT-II (X) or 0.5 nM naltrindole (O). (C) [3H][D-Ala2,DLeuS]Enkephalin (0.5 and 2.5 nM) was displaced by DPDPE in the absence (X) or presence of 2 nM DELT-II (n) or 0.5 nM naltrindole (+). (D) [3H][D-Ala2,o-LeuS]Enkephalin (0.5 and 2.5 riM) was displaced by DELT-II in the absence (X) or presence of 1 nM DPDPE ([3) or 0.5 nM naltrindole (+). (E) [3H][D-Ala2,D-LeuS]Enkephalin (0.5 and 2.5 nM) was displaced by naltrindole in the absence (X) or presence of 1 nM DPDPE (O) or l nM DELT-11 (+).

B I N D I N G O F O P I O I D P E P T I D E S IN M O U S E B R A I N

TABLE 5 BEST-FIT PARAMETER ESTIMATESOF THE TWO-SITE MODEL: TRIS/SODIUM ASSAY CONDITION Parameter Value _+SD Site I bncx_~

Parameter

Bmax

132 +- 9

[D-AIa2,D-Leu5]Enkephalin (K~, nM) DPDPE (Ki, nM) DELT-II (K~, riM) Naltrindole (K~, nM)

0.53 0.52 0.69 0.10

_+ 0.07 +_ 0.07 _+ 0.13 _+ 0.02

Site 2 ~-2 31 +- 13 2.49 804 824 49.2

+_ 1.35 _+ 1095 _+ 1652 _+ 47

As described in the legend to Table 3, the data of several independent experiments, conducted using the Tris/sodium assay condition, were combined, generating a total of 330 data points. Fitting the data to a one-site model resulted in a sum of square (SS) of 16,610. Fitting to a two-site model resulted in a highly significant (F = 9.01, p - 5E-8) decrease in the SS to 14,560. The best-fit parameter estimates (+_SD) are reported.

In accord with this prediction, blockade with D P D P E and DELTII produced a highly statistically significant decrease in the IC50 values of BW373U86 from 3.3 n M to 1.4 n M and 0.7 nM, respectively (Fig. 3). Another direct prediction of the binding parameters reported in Table 6 for the Tris/sodium condition is that, using [3H]DSTLE, it should be possible to construct selective labeling conditions of the two ~ binding sites. Thus, it can be calculated

899

that, using 1 n M [3H]DSTLE, the use of 50 n M D P D P E as a blocker results in greater than 90% labeling of the 5ncx-2site, and that the use of 5 n M BW373U86 as a blocker results in greater than 90% labeling of the ~.... 1 site. A direct prediction of the two-site model is that DELT-II should be considerably more potent under BW373U86-blocked conditions than DPDPEblocked conditions. In accord with the prediction, the observed IC50 values were 320 +-- 44 n M (DPDPE-blocked) and 2.7 + 0.1 n M (BW373U86-blocked) (data not shown). DISCUSSION Previous studies from this laboratory presented evidence for subtypes of the 6~c~binding site (76,77). The present study considerably extends these earlier reports in two important ways: 1) mouse brain membranes, rather than rat brain membranes are used, and 2) a more detailed ligand-selectivity analysis of the two ~,~x binding sites is presented, including a n u m b e r of antagonists (NTB, B N T X , DALCE, [Cys4]DELT, DALCE) that have been used to characterize ~ and 62 receptors (23,24,67,70). Since ligands that distinguish between two binding sites have inhibition curves characterized by low slope factors (5), we first determined the IC50 values and slope factors of several ligands, which we anticipated might have low slope factors, at the b,~x binding site. As reported in Table 1 and observed in our previous studies (76,77), DPDPE, DPLPE, DELT-I, and DELT-II all had slope factors significantly less than 1.0, both in membranes depleted of ~t binding sites by pretreatment with BIT and in control membranes in which [3H][D-Ala2,D-LeuS]enkephalin binding to the ~ site was blocked by inclusion of the t~-selective peptide, LY164929 (57). The possibility that subtypes of opioid receptors detected via ligand binding methods are in fact pools of opioid receptors

TABLE 6 LIGAND-SELECTIVITY STUDY OF MULTIPLE 6,~ BINDING SITES IN MOUSE CNT/BIT MEMBRANES: TRIS/SODIUM CONDITION Drug

K~(b.c~-,)(nM _+SEM) [B~ = 132 fmol/mg protein]

[D-AIa2,D-Leus ]Enkephalin DPDPE DELT-II Naltrindole Oxymorphindole DSTLE DPLPE DELT-I [t271]DADL [pCI]DPDPE Morphine BW373U86 BNTX NTB

0.53 + 0.004 0.52 ___0.004 0.69 + 0.04 0.10 _+ 0.001 0.82 _+ 0.01 1.07 + 0.01 0.97 + 0.02 0.36 ___0.01 4.74 _ 0.10 0.89 _+0.01 88.6 +__1.6 1.52 + 0.03 2.83 _+ 0.06 0.15 + 0.002

K~(6n¢~-2)(nM _+SEM) [B,~, = 31 fmol/mg protein] 2.49 804 824 49,2 140 0.54 118 227 14.5 0.66 1859 0.074 81.4 0.046

_+ 0.07 +_ 60 + 91 _+ 2.6 +__13 _+0.05 _+ 22 _+ 25 + 2.1 _+ 0.07 +_ 208 + 0.01 -2-_13.3 _+0.004

Ki (b~,.2)/Ki (b,¢~-0 4.69 1546 1194 492 170 0.50 121 630 3.06 0.74 20.9 0.048 28.7 0.31

Using mouse CNT/BIT membranes, initial experiments established the dose range over which test compounds inhibited [3H][D-Ala2,D-Leu5]enkephalin binding from 90% to 10% of control. In subsequent experiments, two concentrations of [3H][D-AIa2,D-Leu5]enkephalin (0.5 and 2.5 riM) were each displaced by eight concentrations of oxymorphindole, DSTLE, DPLPE, DELT-I, [~27I]DADL, [pC1]DPDPE, morphine, BW373U86, BNTX, and NTB. In a second experiment, two concentrations of [3H][D-Ala:,D-Leu5]enkephalin (0.5 and 2.5 nil/) were each displaced by above 10 drugs in the presence of 4 nM of DELT-II. In a third series of experiments, two concentrations of [3H][D-Ala2,D-Leus ]enkephalin were each displaced by above 10 drugs in the presence of 2 nM of DPDPE. The combined surface were fit to the two-site binding model for the best fit Ki values, with the Bm~, and Ka values of [3H][D-AIa2,D-Leu5]enkephalin, DPDPE, and DELT-II fixed to the values reported in Table 5. The best-fit parameter estimates (+SEM) are reported.

900

X U ET AL. 100 .... .

~

80

UNBLOCKED

~, " ' . ~ " . , •

BLOCKED WITH 2 nM DPDpE

~%

60

BLOCKEDWITH • 4 nM DELTORPHIN-II

o i

........

_,. ~I

i .... -11

-10

i -9

.

• •

.

.

.

.

.

-8

.

.

-7

-6

LOG [BW373U86] M

FIG. 3. Ligand-selectivity study: BW373U86. The ligand selectivity analysis was carried out as described in the legend to Table 6. This figure shows the data obtained for displacement of 0.5 nM [3H][D-Ala2,DLeuS]enkephalin binding by BW373U86 in the absence of a blocker (O), the presence of 4 nM DELT-II (m), and the presence of 2 aM DPDPE (A). The data are presented as a percent of the bindng observed in the presence of the blocker. The ICso values (±SD) were 3.3 ± 0.9 nM, 0.70 ± 0.21 aM, and 1.35 _+ 0.26 nM, for the unblocked, DELT-ll-blocked, and DPDPE-blocked curves, respectively. The decreases in the ICso values were highly significant for both DELT-II (F = 14.4, p = 0.0026) and DPDPE (F = 10.1, p = 0.0078). coupled and uncoupled from G-proteins has long been recognized as creating the appearance of subtypes where none actually exist (3,4,10,74). In particular, opioid receptors, as with other receptors that interact with G-proteins, can exist in two confor-

mations: a high-affinity state (coupled to G-protein), which is characterized by high-affinity interactions with agonists, and a low affinity state (uncoupled to G-proteins), which is characterized by low-affinity interactions with agonists. Antagonists bind with high affinity to both states of the receptor. According to this two-state model, GTP, or its nonhydrolyzable analog, G p p N H p , converts the high-affinity agonist state to the lowaffinity agonist state. Thus, using a radiolabeled antagonist to label the receptor, G T P alters the characteristics of an agonist inhibition curve, which in the absence of G T P has low IC5o values and low slope factors, reflecting the presence of both highand low-affinity agonist states, by increasing the IC50 and increasing the slope factor to 1, reflecting the conversion of highaffinity states to low-affinity states (26,74). This model gives rise to certain testable predictions. One, agonist inhibition curves characterized by low slope factors in the absence of G p p N H p should be characterized by slope factors of 1 in the presence of of G p p N H p . Two, since the ligand-selectivity analysis of the two sites would hypothetically reflect the presence of high- and low-affinity agonist states, the observed ligand-selectivity analysis should be consistent with the known pharmacology of the test drugs. Three, drugs with antagonist activity should have equal affinity for both of the binding sites. To examine the two-state hypothesis, DPLPE inhibition curves were generated in the absence and presence of the guanyl nucleotide G p p N H p . The data (Table 2) indicated that although G p p N H p (10 and 50 #M) modestly increased the ICs0 value, it decreased the slope factor, which is not consistent with the predictions of the two-state hypothesis (see above). Moreover, the ligand-selectivity pattern of the two 6ncx sites follows a pattern that is not consistent with the two-state hypothesis (Table 6). For example, although the 6 agonists DELT-II, DPDPE, DPLPE,

TABLE 7 LIGAND-SELECT1VITY STUDY OF MULTIPLE ~,:x BINDING SITES IN MOUSE CNT/BIT MEMBRANES: TRIS/CHOLINE CONDITION Drug [D-ala2,D-Leu5]enkephalin DPDPE DELT-II Naltrindole Oxymorphindole DSTLE DPLPE DELT-I [~27I]DADL [pCI]DPDPE Morphine BW373U86 BNTX NTB

K~(6,cx.0(nM _+SEM) [B~ = 104 fmol/mg protein] 0.67 + 0.14 0.35 ± 0.24 1.15 ± 0.42 0.20 ± 0.08 0.38 ± 0.06 0.81 + 0.17 0.38 _+ 0.07 0.90 ± 0.15 1.36 ± 0.20 0.28 ± 0.05 58.3 ± 10.2 5.84 _+ 1.33 15.9 ± 5.4 0.06 ± 0.01

K~(rnex.2)(aM ~ SEM) 54 fmol/mg protein]

[Bm~ =

0.36 + 0.17 3.18 ± 1.73 2.41 ± 0.80 0.52 ± 0.15 1.67 ± 0.35 0.91 ± 0.23 3.98 ± 1.05 4.16 _+0.86 3.14 _+0.59 1.16 _+0.24 125 ± 24 0.34 ± 0.09 5.26 ± 2.15 0.67 ± 0.14

Ki (6.~,_z)/K, (~,~.0

0.53731 9.0857 2.0957 2.6000 4.3947 1.1235 10.474 4.6222 2.3088 4.1429 2.1441 0.058219 0.33082 11.167

Using mouse CNT/BIT membranes, initial experiments established the dose range over which test compounds inhibited [3H]D-AIa2,D-Leu5]enkephalin binding from 90% to 10% of control. In subsequent experiments, two concentrations of [3H][D-Ala2,D-Leus ]enkephalin (0.5 and 2.5 nM) were each displaced by eight concentrations of oxymorphindole, DSTLE, DPLPE, DELT-I, [~27I]DADL, [pCI]DPDPE, morphine, BW373U86, BNTX, and NTB. In a second experiment, two concentrations of [3H][D-AIa2,D-Leu5]enkephalin (0.5 and 2.5 riM) were each displaced by above 10 drugs in the presence of 2 aM of DELT-II. In a third series of experiments, two concentrations of [3H][D-AIa2,D-Leu5]enkephalin were each displaced by above 10 drugs in the presence of 2 aM of DPDPE. The combined surface were fit to the two-site binding model for the best fit K~ values, with the Bm~ and Kd values of [3H][D-Ala2,D-LeuS]enkephalin, DPDPE, and DELT-II fixed to the values reported in Table 4. The best-fit parameter estimates (+SEM) are reported.

BINDING OF OPIOID PEPTIDES IN MOUSE BRAIN LOG(Ki L

DADL

. . . . .

.

.

L ....

m

.........................................~

DPDPE

.

.

DELT-I!

JKit"

l ~

~

.

i

1) .

....

.

'

::

....

I ....

i

.................................................................................... ................... i ............................ i .............................

Naltrlndole ............................... o.morph,n.,.

..........................

DSTLE ........................ ir .........

.

. . . .

............................[

................................

i ............................ ..........

! ............

DELT-| [1271]DADL

-

Morphine~

i . . . . . . . . . . . . . . . . . . . . .i . . . . . . . . . . . . . . . . . . . . . . . . . . .

i. . . .

~

[]

•

~

:0:0

.r. Y

FIG. 4. Selectivity of drugs for the 6,~x_~and 8,~x-2sites as a function of the assay condition. The selectivity index is the log of the ratio of the K~ of site 2 to the K, of site 1.

and DELT-I are highly selective for the 6ncx-~site, other 6 agonists such as DADL, DSTLE, and [pC1]DPDPE are not. In addition, not only does the nonpeptide 6 agonist BW373U86 have the opposite selectivity as DPDPE, but the antagonists naltrindole, BNTX, and NTB are not nonselective between the two sites, as predicted by the two-state model. In light of these findings, it is important to comment on a recent paper, which concluded that "much of the previous characterization of opioid receptor subtypes reflects.., a significant pool of G-protein-uncoupled opioid receptors" (49). The primary evidence for this conclusion can be summarized by their studies with human placental K receptors, which showed that [3H]U69,593, [3H]etorphine, and [3H]ethylketocyclazocine each labeled about 50% of the binding sites labeled by [3H]bremazocine. The authors hypothesized that whereas [3H]bremazocine labeled the total population of uncoupled and coupled receptors, the other three radioligands labeled only the coupled pool (high-affinity agonist state) of the K receptor. Although data obtained with various opioid receptors clearly demonstrate that agonist inhibition curves of [3H]antagonist binding are characterized by low slope factors in the absence of guanine nucelotides (10,29,74), these investigators observed agonist-inhibition curves of [3H]bremazocine binding characterized by slope factors approaching unity, indicating that ethylketocyclazocine, for example, has high affinity for all the Kreceptors labeled by [3H]bremazocine. Viewed collectively with studies that clearly demonstrate that bremazocine is a full K agonist in the guinea pig ileum bioassay (12), the rat diuresis model (30,31), and antinociceptive models (20), the data presented by Richardson et al. (49) are inconsistent with a two-state model. Moreover, their data are inconsistent with data of other investigators who showed that [3H]ethylketocyclazocine and [3H]naloxone labeled the same number of binding sites in human placenta (1), and that [3H]etorphine and [3H]diprenorphine labeled a similar number of binding sites in human placenta both in the absence and presence of G p p N H p and NaCI (45). An alternative explanation for their observation that two agonist [3H]ligands apparently label the same site with Bmax values

901 that differ by a factor of 2 is the phenomenon of half-of-thesites reactivity, which refers to the situation where one class of ligand has access to all ligand binding sites, and others do not (32). This phenomenon has been documented to occur for the binding of [3H]dihydromorphine and [3H]naloxone to # receptors (8), for [3H][LeuS]enkephalin and [3H][MetS]enkephalin binding (63), for [D-Pen2'au25I-Phe4,D-PenS]enkephalin and [3H][pC1]-DPDPE binding to 8 receptors (27,72), and for the binding of the ¢Ladrenerigc antagonists [~251]cyanopindolol and p2~l]hydroxybenzylpindolol to platelet ~ receptors (69). We applied the method of binding surface analysis (51) to test the hypothesis that [3H][D-Ala2,D-LeuS]enkephalin labeled more than one binding site. Various studies have established that this is a robust experimental design that facilities the resolution of multiple binding sites (36,41,61,64). The primary data set was generated by combining the binding surfaces of several independent experiments, as described in Table 3. These experiments were conducted using our standard assay condition of 10 rn_MTris-HCl, pH 7.4, containing 100 m M choline chloride and 3 m M MnC12 (the Tris/choline condition) (52,77), and in the presence of 100 mJk/NaC1 (the Tris/sodium condition), since we anticipated that the ligand-selectivity analysis (see below) might demonstrate considerable differences between the two assay conditions. Representative data sets are shown in Fig. 1 for the Tris/choline condition, and in Fig. 2 for the Tris/sodium condition. For both sets of data, nonlinear least squares curve fitting demonstrated that the two-site model fit the data considerably better (p < 0.001) than did a one-site model (Tables 4 and 5). Two binding sites, designated 6.... ~ and ~ncx-2, were resolved. Relative to the Tris/choline condition, the Tris/sodium condition resulted in a modest increase in the Bm~x of the 6,cx-~ site from 104 fmol/mg protein to 132 fmol/mg protein, and a modest decrease in the Bmax of the 6,cx-2 site from 54 fmol/mg protein to 31 fmol/mg protein. Whereas in the Tris/choline condition DPDPE, DELT-II, and naltrindole were only slightly selective for the ~.... ~ site, the presence of NaC1 in the assay medium substantially increased their Ki values for the 6.cx-2binding site.

LOG ( K i o d i u / K i ..... .} -

DADL

°

I ........~J l

.................... . . . .

DPDPE

o~,~.,, .....

Oxymorphindole - -

DELT-I

=

.... i , , .......... , ,-l-.i

........................

=

Naltrindole

DSTLE

,,,

i ........

7

~i

--

!

~

~

•

...........

~

a. ~"

~

..........

0(~

~.

. . . . . . . . . . . . i................................

[1271]DADL [pCI]DPDPE

Morphine

BB m~EI .......

•

[]

=-" ~ =

~

/

BW373U86

NTB

i

..........................

....

~

i

| ........

F~'~' _~

~

I

1

FIG. 5. Sodium shift ratio of 6.¢~ binding sites. The log of the sodium shift ratios at each binding site is presented.

902

X U ET AL. TABLE 8 INHIBITION OF [3H][D-AIa2,D-LeuS]ENKEPHALIN BINDING TO THE di.~ BINDING SITES BY PUTATIVE IRREVERSIBLE LIGANDS Peptide

K~ (nM+ SD)

Slope Factor (_+SD)

DALCE DALES [Cys4]DELT-II [Ser4]DELT-II

7.21 --- 0.79 6.88 +__1.08 12.6 ___2.1 47.5 _+ 2.1

1.14 _+0.12 1.08 _+ 0.16 1.23 _+0.22 0.83 _+ 0.27

[3H][D-Ala2,D-LeuS]Enkephalin (2.2 riM)was displaced by nine concentrations of the indicated peptides, using the Tris/sodium system. The data of two experiments were combined (18 data points) and fit to the two-parameter logistic equation for the best-fit parameter estimates reported above.

Thus, the Tris/sodium condition appeared to permit a robust discrimination of the two [3H][D-Ala2,D-LeuS]enkephalin binding sites. A limitation of purely quantitative approaches to analyzing binding data is often the lack of traditional qualitative indices of receptor heterogeneity, such as biphasic Scatchard plots or inhibition curves characterized by prominent plateaus. Fortunately, examination of the BW373U86 inhibition curves provides compelling evidence of the presence of two binding sites (Fig. 3). The binding parameters obtained for BW373U86 indicate that it is about 20-fold selective for the 6,~.2 site, and that DPDPE and DELT-II are highly selective for the 6.... ~site. These parameters predict that, in the presence of concentrations of DPDPE and DELT-II that partially block [3H][D-Ala2,D-LeuS]enkephalin binding to the 6.... 1 site, the BW373U86 displacement curves should shift to the left, not to the right, as would be expected if [3H][D-Ala2,D-LeuS]enkephalin labeled, in fact, a single binding site. As shown in Fig. 3, the BW373U86 inhibition curves did shift to the left in the presence of partial blocking concentration of DPDPE and DELT-II, providing visual evidence for a twosite model. The data obtained here are also consistent with other studies that generally report a single class of 6 binding sites. For example, in the Tris/choline condition, naltrindole has about the same affinity for both sites (Ki = 0.2 and 0.6 nM). These data predict that in the absence of NaCI, [3H]naltrindole should label an

apparent single class of binding sites with high affinity for DPDPE, DELT-II, and [pC1]DPDPE. Published data validate this prediction (78). Furthermore, given the low affinity of naltrindole for the 6ncx.2site in the Tris/sodium condition, our data also predict that, in the presence of sodium, [3H]naltrindole should label an apparent single class of binding sites (the 6,cx.~ site) with high affinity for DPDPE, DELT-II, and [pC1]DPDPE. It is unlikely that the two binding sites resolved in the Tris/ choline condition are 6 and u sites. Besides the fact that the u sites are depleted by pretreatment with BIT (52), the high affinity of DELT-II, which is highly selective for fi sites relative to u sites (14), essentially rules out significant labeling o f # sites by [3H][DAlaZ,D-LeuS]enkephalin in the Tris/choline condition. Similarly, the high affinity of the moderately f-selective peptide, DSTLE (17), and the highly 6-selective peptide, [pC1]DPDPE (71), for both 6,cx binding sites also rules out significant labeling o f u sites by [3H][D-Ala2,D-LeuS]enkephalin in the Tris]sodium condition. Finally, the essentially equal affinity of [3H][D-AIa2,D LeuS]enkephalin for the two ~,~ binding sites validates the fitting of [D-AlaZ,D-LeuS]enkephalin self-displacement inhibition curves to one-site binding models (59). Ligand-selectivity studies were conducted as described for the K opioid (54), a (61), and PCP receptor systems (48,62). In the Tris/sodium condition, the selectivity profiles of the test drugs readily fell into four groups: 1. drugs greater than 100-fold selective for the 6ncx-~site (DPDPE, DPLPE, DELT-I, DELT-II, oxymorphindole, naltrindole); 2. drugs greater than 20-fold selective for the 6ncx_, site (morphine); 3. drugs less than 5-fold selective for the ~.... ~ site (DADL, DSTLE, [127I]DADL, [pC1]DPDPE); 4. drugs greater than 15-fold selective for the 6ncx-2 site (BW373U86). In the Tris/choline condition, however, most drugs were less than 10-fold selective for the ~n¢~-~site. Interestingly, BW373U86 was about 20-fold selective for the 6°cx-2 site under both assay conditions. As illustrated by Fig. 4, it is clear that whereas NaC1 markedly increased the selectivity of DPDPE, DPLPE, DELT-I, DELTII, naltrindole, and oxymorphindole for the 6.... , binding site, it had little effect on the selectivity of DADL, DSTLE, [~27I]DADL, [pC1]DPDPE, and BW373U86 for the 6.... , site. Interestingly, in the absence of sodium, NTB and BNTX are moderately selective for the 6,cx-, and 6,cx.2 sites, respectively,

TABLE 9 WASH-RESISTANT INHIBITION OF 6,c~ BINDING SITES BY DALCE AND [Cys4]DELTORPHIN [3H]DADL Binding Condition Control DALCE (2 uM) [Cys4]Deltorphin (2 uM) DALCE (1 #M) + [Cys4]deltorphin (1 #M)

Specific Binding (fmol/mg protein) 62.7 29.3 60.2 36.8

+ + + +

4.4 4.7* 2.0 0.7*

Percent Inhibition 0 53,4* 4,2 41,3"

[3H]DSLETBinding SpecificBinding (fmol/mgprotein) 60.9 34.1 63.3 39.5

_+2.8 _+ 1.6" + 1.6 _+2.1"

Percent Inhibition 0 44.1" 0 35.1"

As described in the Method section, CNT/BIT membranes were incubated for 60 min at 25 °C with either DALCE, [Cys4]deltorphin, or no drug (control), extensively washed, and then assayed for residual [3H][D-Ala2,D-Leu5]enkephalin binding. DALES and [Ser4]DELTII did not produce wash-resistant inhibition at a 1 uM concentration. Each value is the mean + SEM of the results obtained with three membrane preparations.

BINDING OF OPIOID PEPTIDES IN MOUSE BRAIN but in the presence of sodium they are selective for the 6~x.2 (3.2-fold) and 6.... l (29-fold) binding sites, respectively. Studies of the mechanisms by which NaC1 affects the opioid receptors have a long and venerable history (43,66). More recent work, conducted in membranes of guinea pig cortex, showed that NaC1 decreased the Bmax of 6 sites labeled by a 6 agonist. The proposed mechanism of this effect was an increase in the proportion of the low-affinity state of the 6 receptor (73). Similar results were obtained in the NG 108-15 cell line (37). The results obtained here differ from these earlier reports. In this study, NaC1 did not alter the total Bmax of 6 binding sites labeled by [3H][D-Ala2,D-LeuS]enkephalin: 158 fmol/mg protein in the Tris/ choline condition and 163 fmol/mg protein in the Tris/sodium condition. Moreover, although a two-state model would predict that NaCI should increase the Bm~ of a low-affinity component at the expense of the high-affinity component, the data obtained here suggest that NaCI decreases the Bmax of the low-affinity component (Tables 4 and 5). In addition, the pattern of K~shifts is not consistent with a two-state model, since some peptides (e.g., DPDPE, D E L T - I , and DELT-II) have very low affinity for the presumed low-affinity component (rn~x_2 site), and others do not (e.g., [D-Ala2,D-LeuS]enkephalin, DSTLE, and [pC1]DPDPE). Viewed collectively, the simplest explanation of the data is, as shown in Fig. 5, that the primary effect of NaC1 is to markedly increase the Ki values of certain drugs for the 6~cx-2binding site. As will be noted below, the effect of irreversible agents on the 6~x binding sites is an important criteria for determining whether or not they are 6~ or 62 receptors. For these experiments, we used DALCE (24) and [Cysa]DELT (21), which have been shown to block 6~ and 62 receptors, respectively. The irreversible action of DALCE peptides is thought to result from disulfide bond formation between the Cys and the receptor drug recognition site (6). Since [Cys4]DELT acts as an apparent irreversible inhibitor in vivo, we thought it would also be of interest to examine its in vitro effects. DALES and [Ser4]DELT were included as controls, since, due to the substitution of Ser for Cys, they would not be expected to act as irreversible ligands. Initial experiments with [Cys4]DELT indicated that with 20 aM, but not 2 lzM, a considerable amount of this peptide was not removed by the superwash procedure (see the Method section). Therefore, in the experiments reported here, we used much lower concentrations of putative irreversible peptides. Using 2 ~tM DALCE, we were able to demonstrate a selective effect of DALCE on the 6,cx binding sites (Table 9). Although the magnitude of the effect was about 50%, previous studies showed that pretreatment of membranes with 20 ~tM DALCE, followed by extensive washing of the membranes, almost completely inhibited [3H]DPDPE binding (6) and [3H][D-Ala2,D-Leu~]enkephalin binding to the 6,~x site by about 90% (55), indicating that the total population of 6,~ binding sites is sensitive to DALCE. As mentioned in the Introduction, a primary goal of this study was to determine the relationship between the 6~x binding sites and the 6t and 62 receptors by comparing detailed ligandselectivity studies of the 6~x binding site with the known pharmacology of the 61 receptors. Ligand-selectivity studies do not provide information on the intrinsic activity or efficacy of test drugs. Thus, comparisons of in vitro binding data and in vivo pharmacological data must be interpreted cautiously. Nevertheless, to faciliate this comparison, we first developed working definitions of what the in vitro characteristics of the 6~ and 62 receptors should be. As described in Table 10, the 6~ receptor can be defined as being sensitive to the irreversible effects of DALCE, but not the apparent irreversible effects of

903 [Cys4]DELT or 5'-NTII. In contrast, the 62 receptor should be sensitive to the apparent irreversible effects of [Cys4]DELT and 5'-NTII, not DALCE. The 6~ receptor should have high affinity for DPDPE, and if the deltorphins and DSTLE have high affinity at the 61 binding site, then they should act as antagonists at the 6t receptor. Similarly, the 62 receptor would be expected to have high affinity for the deltorphins and DSTLE, and lower affinity for DPDPE. If DPDPE has high affinity for the 62 receptor, then it would be expected to act as an antagonist at that site. Furthermore, one would expect that the selective 62 antagonist, NTB (67,70), should have higher affinity for the 62 site than for the 6t site, and the selective 6t antagonist, BNTX (46), to have lower affinity for the 62 site than for the 6t site. Additionally, one might expect that NTB would have higher affinity than BNTX for the 62 site, and lower affinity than BNTX at the 6~ site. Comparison of the results using the Tris/sodium condition and the criteria outlined in Table l0 suggest that neither the 6ncx.t nor the 6ncx.2sites are related to the 62 receptor: l) [3H][DAla2,D-LeuS]enkephalin binding to the 6ncxbinding sites is sensitive to the irreversible effects of DALCE, not that of [Cys4]DELT (Table 9). 2) Although the 29-fold selectivity of BNTX for the 6ncx.~site might support the notion that the 6ncx-2 is the 62 receptor, the low degree of selectivity of NTB for the 6~cx.2 site (3.3-fold) suggests otherwise. 3) The very low affinity of both DELT-I and DELT-II for the 6ncx-2site. Comparison of the results using the Tris/sodium condition and the criteria outlined in Table l0 suggest that the 6.... ~ site, not the 6,cx_2 site, may be related to the 61 receptor: l) [3H][DAla2,D-LeuS]enkephalin binding to the 6~cxbinding sites is sensitive to the irreversible effects of DALCE, but not [Cys4]DELT. 2) The relatively selective interaction of both naltrindole and BNTX, which are known to block the effects of DPDPE (67), with the 6.... ~binding site (relative to the 6,c~.2 site). 3) The high affinity of DPDPE for the 6.cx.t site. Importantly, the very low affinity of DPDPE for the 0ncx-: site strongly suggests that it is unlikely to be the 6~ receptor. Given the relatively low degree of selectivity of most drugs between the 6.... ~ and the 6~.2 binding sites in the Tris/choline condition, comparisons of these data with the criteria outlined in Table 10 is problematic. Nevertheless, as pointed out above, the insensitivity of the Gcx sites to apparent irreversible effects of [Cys4]DELT, coupled with their sensitivity to DALCE (55), strongly suggests that the 6,¢~ binding sites are not 62-like. Although the data discussed above suggest that neither 6,cx binding site is the 62 receptor, the situation would certainly be clarified if a binding site with the characteristics of a 62 receptor were described. However, the data presented here also indicate that most commonly used 6 receptor ligands should label the 6.... ~ site, which, as described above, is probably synonymous with the 6t receptor. Indeed, there are no studies, which we are aware of, that have demonstrated substantial differences between the 6 site labeled by [3H]DPDPE and that labeled by other ligands, such as [3H]DSTLE (17), [3H]deltorphin (7), [12sI][DAla2]deltorphin-I (13), and [3H]naltrindole (78) in mouse or rat brain. In fact, the data presented here show that DALCE pretreatment affects [3H]DSTLE binding and [3H][D-AIa2,DLeuS]enkephalin binding equally (Table 9), indicating that it too is probably labeling 6t receptors in mouse brain membranes. An unexpected finding was the 20-fold selectivity of the nonpeptide 6 agonist, BW373U86, for the 6~.2 binding site, both in the absence and presence of NaC1. In fact, in the Tris/sodium condition, BW373U86 was the most potent of the 6 agonists tested, where it was 10,000-fold and 16,000-fold more potent than DPDPE and DELT-II, respectively. Given observations

904

XU ET AL. TABLE l0 WORKING DEFINITIONS OF THE |N VITRO CHARACTERISTICS OF 6~ AND 6z RECEPTORS Characteristic

6~

Irreversible inhibition produced by DALCE Irreversible inhibition produced by [Cys4]DELT Irreversible inhibition produced by 5'-NTII Affinity for DPDPE

Yes No No Higher

Affinity for DELT-I, DSTLE, DELT-II

Higher (assuming that they are antagonists at the 61 receptor) Lower Higher

Affinity for NTB Affinity for BNTX

62 No

Yes Yes Lower (assuming it is not an antagonist) Higher Higher Lower

As noted in the text, the high affinity of the deltorphins and DSTLE for the [3H][D-AIa2,D-Leu5]enkephalin binding sites predicts that they should be antagonists at the ~ receptor. Similarly, DPDPE would be expected to have low affinity for the 62 receptor, except in the case if it acted as an antagonist at that site.

that BW373U86 possesses a somewhat different pharmacological profile than DPDPE (F. Porreca and J. H. Woods, personal communication), it is tempting to speculate that the 6ncx.2 site may be a novel 6 receptor mediating certain effects of BW373U86. The nomenclature of 6 sites associated with a ~t-6 opioid receptor complex (the 6cx site), and a 6 site not associated with the opioid receptor complex (the 6ncxsite), arose from a variety of binding and in vivo pharmacological studies, which were recently reviewed (58). The recent cloning of a 6 receptor by two research groups strongly supports the commonly held assumption that the opioid receptors are members of the seven transmembrane G-protein-linked receptor family (15,25). These recent results emphasize the need to reconcile the concept of a # 6 opioid receptor complex with what is now known about the structural features of seven transmembrane G-protein-linked receptors. These receptors are single polypeptide proteins that most often exist as monomers. Thus, it is clear that if the ~t-6 opioid receptor complex exists as a physical entity, then it must express, in a single polypeptide chain, two distinct recognition sites: a ~z-like site and a 6cx-like site. Since a ~t-6 opioid receptor complex has not yet been cloned, we can't directly test this hypothesis. However, we can test the hypothesis by seeing if other seven transmembrane G-protein-linked receptors express multiple recognition sites for drugs. If they do, then we can conclude that the hypothesis of a single opioid receptor expressing distinct recognition sites for ~t and 6cx ligands is a reasonable idea. Indeed, data from several receptor systems support this idea. For example, replacement of the Asn 385 residue of the human 5-HTIa receptor with Val markedly decreased the affinity ofpindolol and other aryloxyalkylamines for the mutated receptor without altering the binding of other classes of drugs, including 5-HT agonists and antagonists (18). Work with chimeric 5-HT2/ 5-HT~c receptors strongly suggested that "structurally diverse 5HT2 antagonists utilize distinct regions of the 5-HT2 receptor for high affinity binding," (l 1), a finding consistent with the model of Evardson et al. (16), who suggested that 5-HT and 5HT2 antagonists bind to different domains of the 5-HT2 receptor. Similarly, site-directed mutagenesis studies of the ~2-adrenergic receptor showed that selected amino acid substitutions increased agonist affinity without changing antagonist affinity (42). With the endothelian receptor, opposite results were obtained: substitution of Asp for Lys 181 decreased antagonist affinity to a much greater extent than agonist affinity (79). Data also indicate that

the cloned dopamine D2 receptor has multiple domains for drug recognition sites. For example, using the cloned D2 receptor, the antagonist ligand [3H]emonapride consistently detects more D2 receptors than does [3H]spiperone (65), and replacement of Asp 8° increases the Ko of epidepride 25-fold, while increasing the Ko of spiperone by 2-fold (40). The studies reviewed above clearly show that several wellcharacterized members of the seven transmembrane G-proteinlinked receptors express multiple recognition sites for drugs. In the context of receptors for the biogenic amines, which have only one endogenous ligand, the multiple recognition sites are detected as different binding domains for drugs. Extension of these findings to opioid receptors, which bind many endogenous ligands, merely requires that there be separate binding domains for different endogenous ligands. In the case of the postulated #-6 opioid receptor complex, there may be separate domains for # and 6~x ligands. Indeed, it is interesting to note that the recent molecular biological studies of receptors have provided new insights into the physical nature of binding domains, which have been postulated for some time for the opioid receptors on the basis of classic pharmacological investigations (2,47,75). Unfortunately, the clarification of the issues discussed above must await the cloning of additional opioid receptors. In regard to the present study, it is fortunate that its major conclusions give rise to testable hypotheses. In the case of the 6nex.l binding site, the high affinity of the deltorphins for this site, coupled with their apparent lack of activity at 6~ receptors, clearly predicts that the deltorphins should be antagonists at the 61 receptor. This may be difficult to experimentally verify, since 61 and 62 receptors appear to mediate the same effects. However, it may be possible to demonstrate blockade of DPDPE antinociception in mice pretreated with 62-selective irreversible ligands, such as 5'-NTII or [Cys4]DELT. Alternatively, given the lack of crosstolerance between DPDPE and DELT-II/DSTLE antinociception (35,68), it is possible that coinfusion of DPDPE and DELTII will prevent the development of tolerance to DPDPE, but not to DELT-II. In the case of the hypothesis that the 6,¢~.2 site might mediate some of the effects of BW373U86, our data predict that BW373U86 should produce some effects that are not produced by DPDPE or DELT-II. Moreover, since BNTX is about 1000fold more potent at the 6n¢x-2 site than is naltrindole, the data predict that whereas effects of DPDPE should be readily reversed by both naltrindole and BNTX, certain effects of

B I N D I N G O F O P I O I D P E P T I D E S IN M O U S E B R A I N

BW373U86 should be readily reversed by B N T X , but not naltrindole. In summary, the data presented here provide: additional evidence for heterogeneity of 6 receptors; preliminary evidence for

905

a novel subtype of the/~ receptor for which the nonpeptide t5 agonist, BW373U86, has high affinity; and preliminary data that support the hypothesis that the tS~receptor and the fi.... ~binding site may be synonymous.

REFERENCES 1. Agbas, A.; Simon, J.; Avni Oktem, H.; Varga, E.; Borsodi, A. Characterization of human placental opioid receptors by 3H-ethylketocyclazocine and 3H-naloxone binding. Neuropeptides 12:171-176; 1988. 2. Band, L.; Xu, H.; Bykov, V,; et at. The potent opiate agonist, (+)cis-3-methylfentanyl binds pseudoirreversibly to the opioid receptor complex in vitro and in vivo: Evidence for a novel mechanism of action. Life Sci. 47:2231-2240; 1990. 3. Blume, A. J. Interaction ofligands with the opiate receptors of brain membranes: Regulation by ions and nucleotides. Proc. Natl. Acad. Sci. USA 75:1713-1717; 1978. 4. Blume, A. J.; Boone, G.; Lichtshtein, D. Regulation of the neuroblastoma × glioma hybrid opiate receptors by Na ÷ and guanine nucleotides. Adv. Exp. Med. Biol. 116:163-174; 1979. 5. Boeynaems, J. M.; Dumont, J. E. Quantitative analysis of the binding of ligands to their receptors. J. Cyclic Nucl. Res. 1:123-142; 1975. 6. Bowen, W. D.; Hellewell, S. B.; Kelemen, M.; Huey, R.; Stewart, D. Affinity labeling of delta-opiate receptors using [D-Ala2,Leu 5, Cys6]enkephalin. Covalent attachment via thiol-disulfide exchange. J. Biol. Chem. 262:13434-13439; 1987. 7. Buzas, R.; Toth, G.; Cavagnero, S,; Hruby, V. J.; Borsodi, A. Synthesis and binding characteristics of the highly specific delta-selective new tritiated opioid peptide, [3H][D-Ala2]deltorphin I1. Life Sci. 50: PL75-PL78; 1992. 8. Byrne, W. L.; Codd, E. E. Half-of-the-sites reactivity in the binding of opiate agonists and antagonists to the opiate receptor. In: Way, E. L., ed. Exogenous opiate agonists and antagonists. New York: Pergamon Press; 1980:67-80. 9. Chang, K. J.; Rigdon, G. C.; Howard, J. L.; McMutt, R. W. A novel potent and selective nonpeptidic delta-opioid receptor agonist, BW373U86. J. Pharmacol. Exp. Ther. (in press). 10. Childers, S. R,; Snyder, S. H. Guanine nucleotides differentiate agonist and antagonist interactions with opiate receptors. Life Sci. 23: 759-761; 1978. I I. Choudhary, M. S.; Craigo, S.; Roth, B. L. Identification of receptor domains that modify ligand binding to 5-hydroxytryptamine2 and 5-hydroxytryptamine~c serotonin receptors. Mol. Pharmacol. 42:627633; 1992. 12. Corbett, A. D.; Kosterlitz, H. W. Bremazocine is an agonist at kappaopioid receptors and an antagonist at mu-opioid receptors in the guinea-pig myenteric plexus. Br. J. Pharmacol. 89:245-249; 1986. 13. Dupin, S.; Tafani, J.-A. M.; Mazarguil, H.; Zajac, J.-M. [~2sI][DAla2]deltorphin-I: A high affinity, delta-selective opioid receptor ligand. Peptides 12:825-830; 1991. 14. Erspamer, V.; Melchiorri, P.; Falconieri Erspamer, G.; et al. Deltorphins: A family of naturally occurring peptides with high affinity and selectivity for delta opioid binding sites. Proc. Natl. Acad. Sci. USA 86:5188-5192; 1989. 15. Evans, C. J.; Keith, D.E., Jr.; Morrison, H.; Magendzo, K.; Edwards, R. H. Cloning of a delta opioid receptor by functional expression. Science 258:1952-1955; 1992. 16. Evardsen, O.; Sylte, l.; Dahl, S. G. Molecular dynamics ofserotonin and ritanserin interacting with the 5-HT2 receptor. Mol. Brain Res. 14:166-178; 1992. 17. Gacel, G.; Fournie-Zaluski, M. C.; Roques, B. P. Tyr-D-Ser-Gly Phe-Leu-Thr, a highly preferential ligand for delta-opiate receptors. FEBS Lett. 118:245-247; 1980. 18. Guan, X.-M.; Peroutka, S. J.; Kobilka, B. K. Identification of a single amino acid residue responsible for the binding of a class of ~-adrenerglc receptor antagonists to 5-hydroxytryptamineta receptors. Mol. Pharmacol. 41:695-698; 1992. 19. Holaday, J. W.; Porreca, F.; Rothman, R. B. Functional coupling among opioid receptor types: Implications for anesthesiology. In:

20.

21.

22.

23. 24.

25.

26.

27.

28. 29.

30. 31. 32. 33. 34.

35.

36.

Estafanous, F. G., ed. Opioids in anesthesia II. Boston: ButterworthHeineman Publishers; 1990:50-60. Horan, P.; de Costa, B. R.; Rice, K. C.; Porreca, F. Differential antagonism ofU69,593- and bremazocine-induced antinociception by (-)-UPHIT: Evidence of kappa opioid receptor multiplicity in mice. J. Pharmacol. Exp. Ther. 257:1154-1161; 1991. Horan, P. J.; Wild, K. D.; Misicka, A.; Lipowski, A.; Haaseth, R. C.; Hruby, V.; Weber, S. J.; Davis, T. P.; Yamamura, H. I.; Porreca, F. Agonist and antagonist profiles of [D-Ala2,Glu4]deltorphin and its [Cys4]- and [SerA]-substituted derivatives: Further evidence of opioid delta receptor multiplicity. J. Pharmacol. Exp. Ther. (in press) Jiang, Q.; Bowen, W. D.; Mosberg, H. I.; Rothman, R. B.; Porreca, F. Opioid agonist and antagonist antinociceptive properties of [DAla2,LeuS,Cys6]enkephalin: Selective actions at the delta.onoomp~e~ site. J. Pharmacol. Exp. Ther. 255:636-641; 1990. Jiang, Q.; Rice, K. C.; Decosta, B.; Porreca, F. Effects of oxymorphindole on morphine-induced antinociception in mice and rats. NIDA Res. Monogr. 105:384-385; 1991. Jiang, Q.; Takemori, A. E.; Sultana, M.; et al. Differential antagonism of opioid delta antinociception by enkephalin and naltrindole 5'isothiocyanate: Evidence for delta receptor subtypes. J. Pharmacol. Exp. Ther. 257:1069-1075; 1991. Keiffer, B. L.; Befort, K.; Gaveriaux-Ruff, C.; Hirth, C. G. The/~opioid receptor: Isolation of a cDNA by expression cloning and pharmacological characterization. Proc. Natl. Acad. Sci. USA 89: 12048-12052; 1992. Kent, R. S.; De Lean, A.; Lefkowitz, R. J. A quantitative analysis ofbeta-adrenergic receptor interactions: Resolution of high and low affinity states of the receptor by computer modeling ofligand binding data. Mol. Pharmacol. 17:14-23; 1980. Knapp, R. J.; Sharma, S, D.; Toth, G.; et al. [D-Pen2'4'-125I-Phea,DPenS]enkephalin: A selective high affinity radioligand for delta opioid receptors with exceptional specific activity. J. Pharmacol. Exp. Ther. 258:1077-1083; 1991. Knott, G. D.; Reece, D. K. MLAB: A civilized curve fitting system. Proc. Online 1972 Int. Conf. 1:497-526; 1972. Law, P. Y.; Horn, D. S.; Loh, H. H. Multiple affinity states of opiate receptor in neuroblastoma × glioma NG 108-15 hybrid cells. Opiate agonist association rate is a function of receptor occupancy. J. Biol. Chem. 260:3561-3569; 1985. Leander, J. D. Further study of kappa opioids on increased urination. J. Pharmacol. Exp. Ther. 227:35-41; 1983. Leander, J. D.; Zerbe, R. L.; Hart, J. C. Diuresis and suppression of vasopressin by kappa opioids: Comparison with mu and delta opioids and clonidine. J. Pharmacol. Exp. Ther. 234:463-469; 1985. Levitzki, A.; Stallcup, W. B.; Koshland, D. E.; Jr. Half-of-the-sites reactivity and the conformational states of cytidine triphosphate synthetase. Biochemistry 10:3371-3378; 1971. Lord, J. A.; Waterfield, A.; Hughes, J.; Kosteditz, H. W. Endogenous opioid peptides: Multiple agonists and receptors. Nature 267:495499; 1977. Mattia, A.; Vanderah, T.; Mosberg, H. I.; Omnaas, J. R.; Bowen, W. D.; Porreca, F. Pharmacological characterization of [DAlaZ,LeuS,Ser6]enkephalin (DALES) antinociceptive actions at the deltanon~omp~exeaopioid receptor. Eur. J. Pharmacol. 192:371-376; 1991. Mattia, A.; Vanderah, T.; Mosberg, H. I.; Porreca, F. Lack of antinociceptive cross-tolerance between [D-Pen2,D-PenS]enkephalin and [D-Ala2]deltorphin II in mice: Evidence for delta receptor subtypes. J. Pharmacol. Exp. Ther. 258:583-587; 1991. McGonigle, P.; Neve, K. A.; Molinoff, P. B. A quantitative method of analyzing the interaction of slightly selective radioligands with multiple receptor subtypes. Mol. Pharmacol. 30:329-337; 1986.

906 37. McLean, S.; Rice, K. C.; Lessor, R. A.; Rothman, R. B. [3H]CycloFOXY, a ligand suitable for positron emission tomography, labels mu and kappa opioid receptors. Neuropeptides 10:235-239; 1987. 38. Mosberg, H. I.; Hurst, R.; huby, V. J.; Gee, K.; Yamamura, H. I.; Galligan, J. J.; Burkes, T. F. Bis-penicillamine enkephalins possess highly improved specificity toward delta opioid receptors. Proc. Natl. Acad. Sci USA 80:5871-5874; 1983. 39. Munson, P. J.; Rodbard, D. LIGAND: A versatile computerized approach for characterization of ligand-binding systems. Anal. Biochem. 107:220-239; 1980. 40. Neve, K. A.; Cox, B. A.; Henningsen, R. A.; Spanoyannis, A.; Neve, R. L. Pivotal role for aspartate-80 in the regulation ofdopamine D2 receptor affinity for drugs and inhibition of adenyl cyclase. Mol. Pharmacol. 39:733; 1991. 41. Neve, K. A.; McGonigle, P.; Molinoff, P. B. Quantitative analysis of the selectivity of radioligands for subtypes of beta adrenergic receptors. J. Pharmacol. Exp. Ther. 238:46-53; 1986. 42. O'Dowd, B. F.; Hantowich, M.; Regan, J. W.; Leader, W. M.; Caron, M. G. Site-directed mutagenesis of the cytoplasmic domains of the human beta-2-adrenergic receptor localization of regions involved in G protein-receptor coupling. J. Biol. Chem. 263:15985-15992; 1988. 43. Pert, C. B.; Snyder, S. H. Opiate receptor binding of agonists and antagonists affected differentially by sodium. Mol. Pharmacol. 10: 868-879; 1974. 44. Porreca, F.; Takemori, A. E.; Sultana, M.; Portoghese, P. S.; Bowen, W. D.; Mosberg, H. I. Modulation of mu-mediated antinociception in the mouse involves opioid delta-2 receptors. J. Pharmacol. Exp. Ther. 263:147-152; 1992. 45. Porthe, G.; Frances, B.; Verrier, B.; Cros, J.; Meunier, J. C. The kappa-opioid receptor from human placenta: Hydrodynamic characteristics and evidence for its association with a G protein. Life Sci. 43:559-567; 1988. 46. Portoghese, P. S.; Sultana, M.; Nagase, H.; Takemori, A. E. A highly selective 6ropioid receptor antagonist: 7-benzylidenenaltrexone. Eur. J. Pharmacol. 218:195-196; 1992. 47. Portoghese, P. S.; Takemori, A. E. Different receptor sites mediate opioid agonism and antagonism. J. Med. Chem. 26:1341-1343; 1983. 48. Reid, A. A.; Mattson, M. V.; de Costa, B. R.; et al. Specificity of phencyclidine-like drugs and benzomorphan opiates for NMDAcoupled and dopamine uptake carrier associated phencyclidine binding sites in guinea pig brain. Neuropharmacology 29:811-817; 1990. 49. Richardson, A.; Demoliou-Mason, C.; Barnard, E. A. Guanine nucleotide-binding protein-coupled and -uncoupled states of opioid receptors and their relevance to the determination of subtypes. Proc. Natl. Acad. Sci. USA 89:10198-10202; 1992. 50. Rodbard, D.; Lenox, R. H.; Wray, H. L; Ramseth, D. Statistical characterization of the random errors in the radioimmunoassay doseresponse variable. Clin. Chem. 22:350-358; 1976. 51. Rothman, R. B. Binding surface analysis: An intuitive yet quantitative method for the design and analysis of ligand binding studies. Alcohol Drug Res. 6:309-325; 1986. 52. Rothman, R. B.; Bowen, W. D.; Bykov, V.; et al. Preparation of rat brain membranes greatly enriched with either type-I-delta or typell-delta opiate binding sites using site directed alkylating agents. Neuropeptides 4:201-215; 1984. 53. Rothman, R. B.; Bowen, W. D.; Herkenham, M.; Jacobson, A. E.; Rice, K. C.; Pert, C. B. A quantitative study of [3H]D-Ala2-D-LeuSenkephalin binding to rat brain membranes. Evidence that oxymorphone is a noncompetitive inhibitor of the lower affinity deltabinding site. Mol. Pharmacol. 27:399-409; 1985. 54. Rothman, R. B.; Bykov, V.; de Costa, B. R.; Jacobson, A. E.; Rice, K. C.; Brady, L. S. Interaction of endogenous opioid peptides and other drugs with four kappa opioid binding sites in guinea pig brain. Peptides 11:311-331; 1990. 55. Rothman, R. B.; Bykov, V.; Jacobson, A. E.; Rice, K. C.; Long, J. B.; Bowen, W. D. A study of the effect of the irreversible delta receptor antagonist [D-Ala2,LeuS,Cys6]enkephatin on 6cx and bncx opioid binding sites in vitro and in vivo. Peptides 13:691-694; 1992.

X U ET AL. 56. Rothman, R. B.; Bykov, V.; Mahboubi, A.; et al. Interaction ofbetafunaltrexamine with [3H]cycloFOXY binding in rat brain: Further evidence that beta-FNA alkylates the opioid receptor complex. Synapse 8:86-99; 1991. 57. Rothman, R. B.; Bykov, V.; Ofri, D.; Rice, K. C. LY164929: A highly selective ligand for the lower affinity [3H]D-ala2-D-leuS-enkephalin binding site. Neuropeptides 11:13-16; 1988. 58. Rothman, R. B.; Holaday, J. W.; Porreca, F. Allosteric coupling among opioid receptors: Evidence for an opioid receptor complex. In: Herz, A., ed. Handbook of experimental pharmacology, vol. 104, "Opioids." Berlin: Springer-Verlag; 1992:217-237. 59. Rothman, R. B.; Long, J. B.; Bykov, V.; Jacobson, A. E.; Rice, K. C.; Holaday, J. W. Beta-FNA binds irreversibly to the opiate receptor complex: In vivo and in vitro evidence. J. Pharmacol. Exp. Ther. 247:405-416; 1988. 60. Rothman, R. B.; Mahboubi, A.; Bykov, V.; et al. Probing the opioid receptor complex with (+)-trans-superfit. I1. Evidence that u ligands are noncompetitive inhibitors of the 6~xopioid peptide binding site. Peptides 13:1137-1143: 1992. 61. Rothman, R. B.; Reid, A. A.; Mahboubi, A.; et al. Labeling by [3H]l,3-Di(2-tolyl)guanidine of two high affinity binding sites in guinea pig brain: Evidence for allosteric regulation by calcium channel antagonists and pseudoallosteric modulation by s ligands. Mol. Pharmacol. 39:222-232; 1991, 62. Rothman, R. B.; Reid, A. A.; Silverthorn, M.; et al. Structure-activity studies on the interaction of biogenic amine reuptake inhibitors and potassium channel blockers with MK-801-sensitive (PCP site 1) and -insensitive (PCP site 2) [3H]TCP binding sites in guinea pig brain. In: Domino, E. F.; Kamenka, J.-M., eds. Multiple sigma and PCP receptor ligands: Mechanisms for neuromodulation and protection. Ann Arbor, MI: NPP Books; 1992:137-146. 63. Rothman, R. B.; Westfall, T. C. Further evidence for an opioid receptor complex. J. Neurobiol. 14:341-351; 1983. 64. Rovati, G. E.; Rodbard, D.; Munson, P. J. DESIGN: Computerized optimization of experimental design for estimating Kd and Bmax in ligand binding experiments. II Simultaneous analysis of homologous and heterologous competition curves and analysis blocking and of "multiligand" dose-response surfaces. Anal. Biochem. 184: 172-183; 1990. 65. Seeman, P.; Guan, H.-C.; Civelli, O.; van Tol, H. H. M.; Sunahara, R.K.; Niznik, H. B. The cloned dopamine D2 receptor reveals different densities for dopamine receptor antagonist ligands. Implications for human brain positron emission tomography. Eur. J. Pharmacol. 227:139-146; 1992. 66. Simon, E. J.; Hiller, J. M.; Groth, J.; Edelman, I. Further properties of stereospecific opiate binding sites in rat brain: On the nature of the sodium effect. J. Pharmacol. Exp. Ther. 192:531-537; 1975. 67. Sofuoglu, M.; Portoghese, P. S.; Takemori, A. E. Differential antagonism of delta opioid agonists by naltrindole and its benzofuran analog (NTB) in mice: Evidence for delta opioid receptor subtypes. J. Pharmacol. Exp. Ther. 257:676-680; 199 I. 68. Sofuoglu, M.; Portoghese, P. S.; Takemori, A. E. Cross-tolerance studies in the spinal cord ofbeta-FNA-treated mice provides further evidence for delta opioid receptor subtypes. Life Sci. 49:PLI53PL156; 1991. 69. Steer, M, L.; Atlas, D. Demonstration of human platelet beta-adrenergic receptors using ~25I-labeledcyanopindolol and ~25I-labeled hydroxybenzylpindolol. Biochim. Biophys. Acta 686:240-244; 1982. 70. Takemori, A. E.; Sultana, M.; Nagase, H.; Portoghese, P. S. Agonist and antagonist activities of ligands derived from naltrexone and oxymorphone. Life Sci. 50:1491-1495; 1992. 71. Vaughn, L. K.; Knapp, R. J.; Toth, G.; Wan, Y. P.; huby, V. J.; Yamamura, H. 1. A high affinity, highly selective ligand for the delta opioid receptor: [3HJ-[D-Pen2,pC1-Phe4,D-PenS]enkephalin. Life Sci. 45:1001-1008; 1989. 72. Vaughn, L. K.; Knapp, R. J.; Toth, G.; Wan, Y. P.; huby, V. J.; Yamamura, H. I. A high affinity, highly selective ligand for the delta opioid receptor: [3H]-[D-Pen2,pCI-Phe4,D-PenS]enkephalin. Life Sci. 45:1001-1008; 1989. 73. Werling, L. L.; Brown, S. R.; Puttfarcken, P.; Cox, B. M. Sodium regulation of agonist binding at opioid receptors. II Effects of sodium

BINDING O F OPIOID PEPTIDES IN MOUSE BRAIN replacement on opioid binding in guinea pig cortical membranes. Mol. Pharmacol. 30:90-95; 1986. 74. Werling, L. L.; Puttfarcken, P. S.; Cox, B. M. Multiple agonist-affinity states of opioid receptors: Regulation of bindingby guanyl nucleotides in guinea pig cortical, NG 108-15, and 7315c cell membranes. Mol. Pharmacol. 33:423-431; 1988. 75. Xu, H.; Kim, C.-H.; Zhu, Y. C.; Weber, R. J.; Rice, K. C.; Rothman, R. B. (+)-cis-Methylfentanyland its analogs bind pseudoirreversibly to the mu opioid binding site: Evidence for pseudoallosteric modulation. Neuropharrnacology 30:455-462; 1991. 76. Xu, H.; Ni, Q.; Jacobson, A. E.; Rice, K. C.; Rothman, R. B. Preliminary ligand binding data for subtypes of the delta opioid receptor in rat brain membranes. Life Sci. 49:PLI41-PLI46; 1991.

907 77. Xu, H.; Partilla, J. S.; de Costa, B. R.; Rice, K. C.; Rothman, R, B. Interaction of opioid peptides and other drugs with multiple ~in~x binding sites in rat brain: Further evidence for heterogenity. Peptides 13:1207-1213; 1992. 78. Yamamura, M. S.; Horvath, R.; Toth, G.; Otvos, F.; Malatynska, E.; Knapp, R. J.; Porreca, F.; Hruby, V. J.; Yamamura, H. I. Characterization of naltrindole binding to delta opioid receptors in rat brain. Life Sci. 50:PL119-PL124; 1992. 79. Zhu, G.; Wu, L.H.; Mauzy, C.; Egloff, A. M.; Mirzadegan, T.; Chung, F. Z. Replacement of lysine-181 by aspartic acid in the third transmembrane region of endothelin type b receptor reduces its affinity to endothelin peptides and sarafotoxin 6c without affecting G protein coupling. J. Cell. Biochem. 50:159-164; 1992.