Proc. Natl Acad. Sci. USAVol. 79, pp. 7570-7574, December 1982Neurobiology

Preparation of brain membranes containing a single type of opioidreceptor highly selective for dynorphin

(neuropeptide/multiple receptors/K opioid/,3chlornaltrexamine)

LAIN F. JAMES, CHARLES CHAVKIN*, AND AVRAM GOLDSTEINAddiction Research Foundation and Stanford University, Palo Alto, California 94304

Contributed by Avram Goldstein, August 30, 1982

'ABSTRACT Opioid receptors-on guinea pig brain membraneswere alkylated by the naltrexone analogue (3-chiornaltrexamine.Binding oftheprototypical .i and Kcligands, [3H]dihydromorphineand [3H]ethylketocyclazocine, was more readily affected by thereagent than was binding of the 8 ligand, 3H-labeled [D-Ala2, D-Leu5]enkephalin. Treatment of membranes with 3-chlornaltrex-'amine in the presence of dynorphin resulted in significant pro-.tection of [3H]ethylketocyclazocine'binding sites, without protec-tion-of[H]dihydromorphine or3H-labeled [DwAla, D-Leu5lenkeph-alin sites. Similarly, [D-Ala2, D-Leu5]enkephalin and sufentanilselectively protected binding sites for 3H-labeled [D-Ala2, D-Leu5]enkephalin and [3H]dihydromorphine, respectively. Scat-chard analysis of [3H]ethylketocyclazocine' binding to untreatedmembranes suggested two types of binding site with 40-fold dif-ference in affinities. Membranes treated with (-chlornaltrex-amine in the presence of dynorphin retained about 40% of thehigh-affinity sites and lost the low-affinity sites. Selective protec-tion of sites with high affinity for dynorphin and ethylketocyclazo-cine was confirmed in competition binding assays. These resultsstrongly suggest that the three types of opioid receptor are notinterconvertible and provide -further evidence that the endoge-nous peptide dynorphin is a highly selective ligand of the K opioidreceptor.

The pharmacologic effects of opiate alkaloids and endogenousopioid peptides on mammalian nervous systems are mediatedby specific receptors on target cell membranes. Opioid recep-tors have been divided into at least three types-p, 8, and K-on the basis of different physiologic and pharmacologic effectsproduced by different types of opioids (1-5), 'differences inbinding ofvarious opioids to brain tissue (3, 6-9), and selectiveprotection ofreceptors from alkylation (6, 10-12). The physical,chemical, and functional differences between the receptortypes have not yetobeen defined. Indeed, because opioid li-gands are often only partially selective for receptor type, it hasbeen difficult to study a single type of opioid receptor withoutinterference from the other types.Our aim has been to produce opioid receptor systems in vitro

that contain only one type of binding site. Our approach hasebeen to inactivate receptors by alkylation with the irreversibleopioid antagonist /3chlornaltrexamine (CNA) (13, 14) and toprotect a specific type of receptor by treatment with CNA inthe presence of a ligand'maximally selective for that type (15).In the guinea pig ileum myenteric plexus-longitudinal musclepreparation we showed that dynorphin-(1-13) protected .K re-ceptors but not A receptors from alkylation by CNA (6). In thesame tissue we were able to protect A receptors but not K re-ceptors with [D-Ala2, D-Leu5]enkephalin (DADLE). DADLEis normally a.8 agonist, but because 8 receptors do not function

in control of guinea pig ileum longitudinal muscle contraction,the enkephalin analogue interacts with A receptors in this assay(3). Thus, by selective protection, we could produce prepara-tions with functional K receptors only, or functional u receptorsonly.

These same experiments showed that dynorphin is selectivefor K receptors. Similar conclusions about dynorphin selectivityhave been drawn from selective tolerance experiments (16) andfrom measurements of the sensitivity of dynorphin to antago-nism by naloxone in several tissues (6, 17).We now report that CNA destroys 1L, 8, and Kopioid binding

sites on guinea pig brain membranes. Each of the three typesof binding site was selectively protected from alkylation by re-action of membranes with CNA in the presence of an appro-priate selective ligand-dynorphin for K sites, DADLE for 8sites, and sufentanil for ,.a sites. Dynorphin protected a singletype of high-affinity site with properties typical of K receptorsin binding assays.

'MATERIALS AND METHODSReagents.''[3H]Dihydromorphine ([3H]DHM, 65 Ci/mmol)

and [3HIDADLE (40 Ci/mmol) were obtainedfrom Amersham,and [3H]ethylketocyclazocine ([3H]EKC, 15 Ci/mmol) wasfrom New England Nuclear (1 Ci = 3.7 X 1010 becquerels).Dynorphin, dynorphin-(1-13) amide, and'DADLE were fromPeninsula Laboratories (San Carlos, CA), normorphine hemi-hydrate from Applied Science (Gardena, CA), EKC methanesulfonate from Sterling Winthrop (Rensselaer, NY), and sufen-tanil citrate from Janssen Pharmaceutical (New Brunswick, NJ).CNA was a generous gift from P. S. Portoghese and A. E. Tak-emori.

Purity of radioligands was confirmed by thin-layer chroma-tography, purity of peptides by reverse-phase high-perfor-mance liquid chromatography on a Waters Associates (Milford,MA) system with a piBondapak C18 column. Elution was by alinear gradient'from 15% to 35% acetonitrile .in'6 mM trifluo-roacetic acid over 30 min at a flow rate of 1.5 ml/min. EKC,normorphine, and sufentanil were used without'further puri-fication.

Preparation of Membranes. A male guinea pig (Simonsen,400-500 g) was killed by decapitation. Brain tissue without cer-ebellum was homogenized, using a Tissumizer (Tekmar, Cin-cinnati, OH) for 30 sec in 10 vol of 370C Krebs-Ringer solution(KR solution) of the following composition (mM): NaCl, 118;KCI, 4.75; CaCl2, 2.54; KH2PO4, 1.19; MgSO4, 1.20; NaHCO3,25; glucose, 11; choline chloride, 0.02; and mepyraminemaleate (125 nM). The tissue was prepared and treated in KR

Abbreviations: CNA, 1-chlornaltrexamine; DADLE, [D-Ala2, D-Leu5]enkephalin; DHM, dihydromorphine;.EKC, ethylketocyclazo-cine; KR solution, Krebs-Ringer solution.* Present address: Arthur V. Davis Center for Behavioral Neurobiology,The Salk Institute, P. 0. Box 85800, San Diego, CA 92138.

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The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact.

Proc. Natl. Acad. Sci. USA 79 (1982) 7571

solution in order to keep conditions as close as possible to thoseused in our previous experiments with the guinea pig ileumpreparation (6). Throughout the experiment, the KR stock so-lution was maintained at 37C and bubbled with 95% 02/5%CO2. Membranes were incubated in capped tubes without gas-sing; under these conditions pH 7.4 was maintained for at least30 min. The homogenate was centrifuged (14,500 x g, 370C,20 min), resuspended in KR solution, and incubated at 370C for20 min to remove endogenous opioids. Membranes were againcentrifuged as above, then resuspended in KR solution to a con-centration of 16 mg (original tissue) per ml, for reaction withCNA. Protecting ligands, when present, were added 1 min be-fore addition of CNA. Reaction was for 20 min at 370C. Sus-pensions were then diluted at least 1:4 with KR solution andcentrifuged (here, and below, 14,500 X g, 370C, 5 min) andwashed once in KR solution at 370C by resuspension and re-centrifugation then four times by resuspension in KR solution,incubation for 20 min at 370C, and centrifugation. Finally, mem-branes were washed twice in 50 mM Tris HCl (pH 7.4) by sus-pension and immediate centrifugation and then resuspendedin the Tris buffer at room temperature to a concentration of 20mg (original tissue) per ml. Two controls were included in eachprotection experiment: (i) untreated membranes, which weresubjected to the washing procedure but were not treated withprotecting ligand or CNA; (ii) membranes treated with pro-tecting ligand but not CNA, as a control for removal of the pro-tecting ligand from the binding sites. For the experiments re-ported here, recovery ofbinding capacity in membranes treatedwith protecting ligands but not CNA was greater than 75%.

Radioreceptor Binding Assays. Binding of the prototypical,u, 8, and K ligands-[3H]DHM, [3H]DADLE, and [3H]EKC,respectively-was assayed as reported (6), with the modifica-tions described in figure and table legends.

Dissociation constants (Kd) and concentrations of bindingsites (Bma) describing the binding of [3H]EKC to brain mem-branes were estimated by nonlinear least-squares regression byusing the Statistical Analysis System program NLIN (18). Themodel and derivative statements for this program were kindlyprovided by D. B. Bylund. The program fits data to equationsfor the Langmuir isotherm, using both one-site and two-sitemodels. For presentation of results, parameters were estimatedfor each separate experiment, then data were normalized bydividing each measurement of bound ligand by the sum of thesaturable high-affinity and low-affinity binding sites obtainedfrom the parameter estimation in that experiment. Such nor-malized data from three separate experiments were combinedand presented as a Scatchard plot. Finally, parameter estimateswere obtained from the combined normalized data by using theNLIN program. To avoid statistical errors inherent in Scatchardanalysis (errors on the x and y axes are highly correlated), non-linear regression was performed directly on the binding iso-therm data (19).

For estimation of free dynorphin-(1-13) amide at the end ofthe binding assay, samples were centrifuged (12,300 X g, 4°C,5 min) and 1.5 ml of supernatant solution was removed and ly-ophilized. The dried material was dissolved in 100 ,Al of 0.1%Triton X-100 in 0.1 M HCl, and dynorphin was determined byradioimmunoassay as described (20). Standards were assayedin 100 ,ul of0. 1% Triton X-100 in 0.1 M HC1 containing the samehigh concentration of Tris as the samples. Binding of '"I-la-beled dynorphin-(1-13) was reduced by about 30% but IC50values for the standards were not significantly affected.

RESULTSTreatment of guinea pig brain membranes with CNA causeddestruction of high-affinity binding sites for [3H]DHM, [3H]EKC,

and [3H]DADLE (Fig. 1). The radioligand concentrations werechosen for selective binding of each ligand at its high-affinitysite (6). Sites for [3H]DADLE were somewhat less sensitive toCNA than were [3H]EKC or [3H]DHM sites. Binding of allthree ligands was reduced by at least 85% when membraneswere treated with 10 nM CNA.

Treatment of membranes with 10 nM CNA for 20 min in thepresence of increasing concentrations of dynorphin resulted inincreasing protection of [3H]EKC binding with no protectionof [3H]DHM or [3H]DADLE binding (Fig. 2A). Treatmentwith CNA, as above, in the presence of DADLE resulted in se-lective protection of [3H]DADLE binding sites (Fig. 2B), andtreatment in the presence of sufentanil resulted in selectiveprotection of [3H]DHM binding sites (Fig. 2C). Dynorphin andsufentanil could not be used at higher concentrations thanshown because the control experiments without CNA showedincomplete recovery of binding capacity even after repeatedwashing of the membranes as described in Materials and Meth-ods.

Scatchard analysis of binding to untreated membranes sug-gested more than one type of binding site for [3H]EKC (Fig.3). Nonlinear regression analysis gave estimates of 0.18 and 7.7nM for Kd and 3.3 and 10 pmol/g of tissue, respectively, forBmax assuming a two-site model. Membranes treated with 10nM CNA in the presence of 100 nM dynorphin retained about40% of the higher-affinity binding sites (Kd = 0.13 nM, Bm.= 1.3 pmol/g of tissue, by nonlinear regression analysis) andlost most or all of the lower-affinity sites. In the absence of dy-norphin (Fig. 3 Inset), the high-affinity sites were also de-stroyed, leaving a saturable binding site of very low affinity andhigh capacity (represented by the horizontal part of the Scat-chard plot). It appears, therefore, that there are three types ofbinding site for [3H]EKC, a high-affinity site and a lower-affinitysite, both of which are destroyed by CNA, and a very-low-af-finity site, which is resistant to CNA. Dynorphin protects onlythe high-affinity site.

Sites protected by dynorphin were compared to sites on un-treated membranes in competition binding assays. Because

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CNA, nM

FIG. 1. Inactivation of guinea pig brain opioid binding sites byCNA. Guinea pig brain membranes were treated with CNA, and bind-ing of [3H]DHM (1.3 nM; A), [3H]DADLE (1.8 nM; *), and [3H]EKC(0.6 nM; o) was then assayed as described previously (6) except thatthe assay volume was 1 ml. Saturable binding is the difference betweenbinding in the absence and presence of 1 gM EKC. Though EKC issomewhat selective for K receptors, at a concentration of 1 AM it dis-places as much [3H]DADLE or [3HIDHM as do 1 uM concentrationsof DADLE or levallorphan, respectively. Data are means + SEM fromthree independent experiments on different membrane preparations.In each experiment, measurements were made in triplicate.

Neurobiology: James et al.

7572 Neurobiology: James et al.

50

a02525

1 10 100 100 1000 10,000Protecting ligand, nM

FIG. 2. Selective protection of brain opioid binding sites. Guinea pig brain membranes were treated with 10 nM CNA in the presence of variousconcentrations of protecting ligands. Then binding of radioligands was assayed as for Fig. 1: *, [3H]DADLE; 0, [3HIEKC; and A, [3H]DHM. A,Protection by dynorphin B. protection by DADLE; C, protection by sufentanil. ForA and B, concentrations of radioligands were: ['HIEKC, 0.6 nM;[3H]DHM, 1.3 nM; and [3H]DADLE, 1.8 nM, as in Fig. 1. For C, concentrations were ['H]EKC, 0.1 nM; [3H]DHM, 0.2 nM; and [3H]DADLE, 0.3 nM.For explanation of these lower radioligand concentrations, see the text. Percent protection is the percentage of sites normally destroyed by CNA(compare Fig. 1) that were not destroyed in the presence of protecting ligand. When necessary, a correction was made for incomplete removal ofprotecting ligand from the binding sites by taking binding to membranes treated with protecting ligand but not CNA as 100%. Data are means± SEM from three or four independent experiments on different membrane preparations. All ligands were tested on each membrane preparationin triplicate.

high-affinity binding of both [3H]DHM and [3H]DADLE wasdestroyed by treatment of membranes with CNA in the pres-ence of dynorphin, the only radioligand to be considered here

2.00~~~~~.

CNA

0

1.0n , \ ~ ~~~~00.5 1.0

t \ Untreated+Dy

0 L0 0.5 1.0

FIG. 3. Scatchard plots for binding of [3H]EKC to untreated mem-branes and to membranes treated with CNA in the presence of dy-norphin. Saturable binding of [3HIEKC (at concentrationsbetween 0.1and 20 nM) to untreated guinea pig brain membranes (e), membranestreated with 10 nM CNA and 100 nM dynorphin (A), or membranestreated with 10 nM CNA alone (o, Inset) was measured as describedfor Fig. 1 except 10 ,M EKC was used for estimation of nonsaturablebindingbecause of the higher radioligand concentrations used in theseexperiments. Results from three experiments were combined (afternormalization to the total number of binding sites) and are presentedas Scatchard plots, in which B is the fractional occupancy of all thesaturable binding sites and F is the concentration of free ligand atequilibrium, calculated by subtracting the measured value for totalligand bound from that for total ligand added to the incubation mix-ture. When free ligand was measured separately, the value for totalbound ligand plus free ligand accounted for at least 90% of the totalligand in the assay. The solid curves are fitted to the parameter esti-mates reported in the text. The broken line is from the Scatchard plotfor binding of [3H]EKC to membranes treated with 10 nM CNA alone(see Inset).

is [3H]EKC. As found previously (6), dynorphin-(1-13) amidewas the most potent competer, with EKC next and normor-phine and DADLE least potent (Table 1). Dynorphin-(1-13)amide was used for these experiments because it is stable underthe conditions of the binding assay (22). The IC50 value foundhere for the competition of DADLE for [3H]EKC binding sitesis about 40 times that reported previously. We could show di-rectly that a part of this difference was due to the different so-lutions used in preparation of the membranes. Thus, mem-branes prepared in KR solution (as described here) yielded anIC50 value 5 times that for a parallel preparation of membranesin Tris [as in the previous study (6)]. In addition, the presentmore thorough washing procedures may remove cofactors suchas ions or nucleotides, which can alter affinities for thereceptors.

After destruction ofA and 8 receptors with CNA in the pres-ence of dynorphin, dynorphin-(1-13) amide was even morepotent than before as a competer, whereas normorphine andDADLE were less potent. The potency of EKC did not change.After correction for the loss of free dynorphin-(1-13) amide bynonspecific binding to the membranes, the affinity of dynor-phin-(1-13) amide for the receptors in treated membranes wasfound to be more than 20 million times that of DADLE, morethan 250,000 times that of normorphine, and more than 500times that of the putative K-selective ligand EKC (Table 1).

DISCUSSION

We have shown that treatment of guinea pig brain membraneswith the site-directed alkylating agent CNA results in inacti-vation of ,A 8, and K opioid binding sites. Each type of site canbe selectively protected from alkylation by the presence of itsselective ligand-dynorphin for K sites, DADLE for 8 sites, andsufentanil for ,u sites. Dynorphin protects a single type of high-affinity site for [3H]EKC, which has the properties ascribed tothe K opioid receptor in competition binding assays. Normor-phine and DADLE (,u and 8 receptor ligands) have higher IC50values in competition with [3H]EKC for sites protected by dy-norphin than for sites on untreated membranes, whereas dy-norphin-(1-13) amide has a lower IC50 value in competition forprotected sites than for untreated sites. Estimates of dissocia-tion constants (Ks, Table 1) show that dynorphin-(1-13) amide

Proc. Natl. Acad. Sci. USA 79 (1982)

Proc. NatL Acad. Sci. USA 79 (1982) 7573

Table 1. Characterization of [3H]EKC binding sites on untreated membranes and membranestreated with CNA in the presence of dynorphin

IC50, nMCompeting Untreated Treated

ligand membranes membranes R Ki, M pKiNormorphine 170 ± 27 750 ± 280 4.4 1.30 X 10-7 6.9DADLE 8,000 ± 2,000 54,000 ± 25,000 6.7 9.60 x 10-6 5.0EKC 1.3 ± 0.2 1.4 ± 0.5 1.1 2.50 x 10-1o 9.6Dynorphin-(1-13)amide 0.28 ± 0.08 0.13 ± 0.04 0.46 <5 x 10-13* 12.3

Competition for [3H]EKC binding sites was measured as described (6). Saturable binding was takenas the difference between binding in the absence of unlabeled drug and that in the presence of 1 ,uM EKC.IC50, the nominal concentration of competing ligand (i.e., the amount of ligand added, divided by thevolume of incubation mixture) required to reduce the saturable binding of [3H]EKC by 50%, was esti-mated from log-logit plots and is reported as mean + SEM from four independent experiments. Treatedmembranes were allowed to react with 10 nM CNA in the presence of 100 nM dynorphin then washedas described in Materials and Methods. Untreated membranes were washed similarly but were not al-lowed to react with CNA or dynorphin. R is the ratio of IC50 values for treated and untreated membranes.Ki is an estimate of dissociation constant forbinding of unlabeled ligand to treated membranes, calculatedfrom the equation, Ki = IC50/[1 + (L/Kd)] (21), in which L is the concentration of radioligand and Kdis its dissociation constant. Because of the wide range of binding constants, it is convenient to use pKi,the negative logarithm of Ki.* For these competition experiments, concentration of [3H]EKC free in solution at equilibrium variedbetween 98% of total added radioligand at high concentrations of competing ligand and about 83% whenno competing ligand was present. It was not possible, however, to measure free dynorphin-(1-13) amideremaining in solution at equilibrium because, at the low concentrations used in competition experi-ments, the amount of free ligand was below the detection limit of our radioimmunoassay. At higherconcentrations (between 1 and 10 nM) the free concentration at the end of the binding assay incubationwas only 1-2% of the nominal concentration, and this loss occurred within the first 10 min. The estimateof an upper limit for Ki reported in the table is based on a free concentration 2% of the nominal con-centration. For normorphine and DADLE, nonspecific binding is negligible at the high concentrationsemployed, so that the free concentration is virtually equal to the nominal concentration.

has much higher affinity for dynorphin-protected sites thandoes EKC, normorphine, or DADLE. The data presented hereshow clearly that dynorphin, at the concentrations tested, pro-tects only K receptors.The observation that most of the added dynorphin-(1-13)

amide is adsorbed nonspecifically to membranes is in agree-ment with data already published (23). The resulting high con-centration on membrane surfaces and the exceptionally low Kisuggest that the nonspecific binding may play a role in receptorinteraction. For example, if the adsorbed peptide were stillavailable to the receptor, nonspecific binding might act as amechanism for concentration of the ligand in the vicinity of thereceptor. In that case, the actual affinity of ligand for receptormight not be nearly so great as the apparent affinity measuredhere.The observed differences in IC50 values for competition with

[3H]EKC for sites on untreated and dynorphin-protected mem-branes (Table 1) would be expected if [3H]EKC were bindingto two types of site on untreated membranes (a major site towhich the radioligand binds preferentially, and a minor site oflower affinity) but only to one type (the major site) on treatedmembranes. The predicted effect of CNA treatment with se-lective protection of the major site can be expressed by an em-pirical ratio R, the IC50 in treated membranes divided by theIC50 in untreated membranes. When competing ligand has thesame selectivity profile as radioligand (e.g., when it is the non-radioactive form ofradioligand), R = 1. When competing ligandis more selective than radioligand for the major site, R is lessthan 1; and when it is less selective for the major site, R is greaterthan 1.

Initial attempts to demonstrate selective protection of the ,receptor with a range of ligands including sufentanil, morphi-ceptin (24), naloxone, and normorphine were unsuccessful.Although binding sites for [3H]DHM were protected, so weresites for [3H]DADLE and [3H]EKC. In these experiments la-

beled ligands were used at concentrations of 0.75 times theirKd for high-affinity sites as estimated previously (6), a conditionchosen so that the high-affinity sites for each radioligand wouldbe labeled predominantly. This was sufficient for demonstrationof selective protection of K and 8 receptors. However, onlywhen the concentration of labeled ligands was reduced to 0.125times the previously estimated Kd could we show selective pro-tection of a receptors by sufentanil. Evidently, at the higherconcentrations both [3H]EKC and [3H]DADLE crossreact sig-nificantly with ,u receptors, and those are the sites protectedby sufentanil. At the lower concentrations the two ligands aremore selective for K and 8 receptors, respectively, which arenot protected by sufentanil.The unavailability of radioligands that are completely selec-

tive for a single receptor type is a major problem in using com-petition binding assays for assessing receptor selectivity. Evenat concentrations below the dissociation constant for high-affin-ity sites there can be significant crossreaction of a supposedlyselective ligand with other receptor types. If no correction ismade for this crossreaction, estimates of Ki or comparisons ofIC50 values from competition experiments can be misleading.For example, initial reports ofcompetition binding experimentswith [3H]EKC and rat brain membranes provided no evidencefor K receptors (25, 26). Subsequently, K receptor-like bindingsites were detected in rat brain (27, 28). Binding at minor sitescan be blocked by the simultaneous presence of high concen-trations of appropriate unlabeled ligands (26), provided suffi-ciently selective ones are available. However, the use of block-ing ligands is restricted to binding assays and cannot be ex-tended to the study ofa single receptor type in a bioassay, unlessthe tissue is first made tolerant to the blocking ligands, a con-dition that may alter the properties of the receptors. Destruc-tion of minor sites and selective protection of the major site, asdescribed here, allows direct measurements of affinities foreach receptor type. We have illustrated this approach for K re-

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7574 Neurobiology: James et al.

ceptors, but of course it can be extended to u and 8 receptorsby protecting with sufentanil and DADLE, respectively.

With membrane preparations that each contain a single typeof opioid receptor, as described here, it should be possible todescribe the selectivity of any ligand in a nonarbitrary, ther-modynamically meaningful way, as ratios ofequilibrium bindingconstants, or (on a logarithmic scale) as differences in pKi values.We have already demonstrated the use ofselective protection

in a bioassay of opioid activity with the guinea pig ileum prep-aration (6). In principle the technique could be applied to in-vestigate the involvement of a given type of receptor in any invitro assay for opioids. For example, opioids inhibit release ofvasopressin from isolated pituitary (29), inhibit adenylate cy-clase in rat striatal membranes (30), and modulate the firing ofneurons in rat hippocampal slices (31). In all such cases selectiveprotection may indicate which receptor types are involved inthe response to opioids.The anatomical distribution of multiple opioid receptors has

been studied by autoradiography, using either "selective" li-gands or manipulation of the incubation conditions to distin-guish receptor types (32, 33). The same objections to partiallyselective ligands that apply to binding assays apply with evengreater force to autoradiography, in which it may be difficultto distinguish between a high density of low-affinity receptorsand a low density of high-affinity receptors. Selective protec-tion of high-affinity sites and destruction of low-affinity siteswould distinguish these possibilities. Hence it may be possibleto map specific dynorphin pathways in the central nervous sys-tem by comparing the distribution of immunoreactive dynor-phin, measured by immunohistochemistry (34, 36), to that ofspecific dynorphin (K) receptors, measured by selective pro-tection and autoradiography. The same procedure could, inprinciple, be applied to the other types of opioid receptor.

Our results provide further evidence that dynorphin is a Kreceptor ligand in brain, in agreement with pharmacologic as-sessments of dynorphin selectivity in peripheral tissues (6, 16,17). Selective protection of the three types of opioid receptorsuggests strongly that these are physically distinct entities andnot simply interconvertible forms of the same receptor, as hasbeen proposed (33). A preliminary account of the present in-vestigation has been presented elsewhere (37).We thank Madeline Rado for skillful technical assistance, Dr. W. T.

Nelson for setting up the computer programs, and especially Drs. P.S. Portoghese and A. E. Takemori for the gift ofCNA. This investigationwas supported by Grant DA-1199 from the National Institute on DrugAbuse.

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