Multiple Opiate Receptors - Journal of Biological Chemistry · Multiple Opiate Receptors 2611 mouse...

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 254, No. 8, Issue of April 25, pp. 2610-2618.1979 Printed in U.S. A.

Multiple Opiate Receptors ENKEPHALINS AND MORPHINE BIND TO RECEPTORS OF DIFFERENT SPECIFICITY*

(Received for publication, May 25, 1978, and in revised form, October 18, 1978)

Kwen-Jen Chang and Pedro Cuatrecasas From the Department of Molecular Biology, The Wellcome Research Laboratories, Research Triangle Park, North Carolina 27709

The use of very low concentrations of 1251-[D-Ala2,D- Leu5]enkephalin, [3H]naloxone, and [3H]dihydromor- phine under similar conditions enables the measure- ment of different opiate binding sites in a rat brain membrane preparation. In the absence of sodium ions, one such site binds enkephalins and their D-Ala2-sub- stituted analogs with higher affinity than morphine and naloxone (enkephalin receptor), while another site exhibits better affinity for naloxone and morphine (morphine receptor).

Morphine, naloxone, and enkephalin do not facilitate the dissociation of bound 12?-[D-Ala2,D-Leu5]enkepha- lin, suggesting that heterogenous or homogenous coop- erativity does not exist for opiate receptors. Sodium ion decreases the high affinity binding of morphine and enkephalins to both receptor sites. Scatchard plots of saturation binding isotherms of ‘251-[D-Ala2,D-Leu5]en- kephalin indicate two binding sites with dissociation constants of 0.8 no and 6 11~ and capacities of 43 and 86 fmol/mg of membrane protein.

Many neuroblastoma cell lines bear only the enkeph- alin receptors. So far, cell lines containing only the morphine receptor have not been found. Homogeniza- tion or exposure to morphine for 24 h does not affect the binding characteristics of neuroblastoma cells.

N- Cyclopropylmethylnoretorphine, an analgesic about 100 times more potent than morphine with less propensity to induce physical dependence, competes for the binding of ‘251-[D-Ala2,D-Leu5]enkephalin and [3H]naloxone with ICsO values of 2.5 and 1 no, respec- tively, and shows no “sodium shift” in the C3H]nalox- one-binding assay. Partial agonists, pentazocine, bu- torphanol, and oxilorphan, also have a weak “sodium shift” and a low I&O ratio in competing with [3H]nalox- one compared to ‘251-[D-Ala2,D-Leu5]enkephalin bind- ing.

Enkephalin and its stable analogs exhibit high affin- ity for the enkephalin receptors. The analog, Try-Gly- Gly-Phe, which retains significant intrinsic activity, is still able to bind to the enkephalin receptor better than to the morphine receptor. Tyr-ethyl ester binds to both receptor sites equally well, showing an I& value of about 0.1111~. These data may suggest that the hydro- phobic group of the phenylalanine residue of enkeph- alin could be responsible for the major structural dif- ference between enkephalin and morphine for receptor recognition. This may also explain why N-cyclopropyl- methylnoretorphine, which contains a hydrophobic group at the C-19 position of oripavine, binds to the

* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accordance with 18 USC. Section 1734 solely to indicate this fact.

enkephalin receptor better than naloxone and mor- phine.

In 1975, several laboratories simultaneously reported that a morphine-like material could be extracted from brain and pituitary. Hughes et al. (1) and Hughes (2) first reported that their opiate-like material consisted of two similar pentapep- tides with sequences of H-Tyr-Gly-Gly-Phe-Met-OH ([Met’lenkephalin) and H-Tyr-Gly-Gly-Phe-Leu-OH ([Leu5]- enkephalin). The existence of these two peptides was rapidly confiied by Pasternak et al. (3) and Simantov and Snyder (4). The structure of [Met’lenkephalin is contained within the sequence (residues 61 to 65) of 91 amino acids of ,&lipotropin, a peptide isolated from the pituitary gland of several verte- brate species (5, 6). Bradbury et al. (7) and Cox et al. (8) reported that the 31 amino acid COOH-terminal fragment of /3-lipotropin was as potent as [Met’lenkephalin. Various COOH-terminal fragments of ,&lipotropin known as a-, fl-, , and y-endorphins have now also been isolated from the pitui- tw (6, 9).

[Met’lEnkephalin and [Leu5]enkephalin are rapidly metab- olized in uiuo and in vitro (10-12). Many analogs of enkephalin have now been synthesized and tested for morphine-like ac- tivity and for stability. Replacement of glycine at position 2 by D-Ala together with substitution of leucine at position 5 by D-h% has yielded a very stable and more potent pentapeptide, [o-Ala*,&Leu5]enkephalin (13-15).

Utilizing labeled narcotic agonists and antagonists of high specific radioactivity, specific opiate binding sites (receptors) have been identified and characterized (16-18). “H-labeled [Leu’lenkephalin has recently been employed to study en- kephalin-receptor interactions in brain membranes (19, 20) and cultured cells (21). ‘““I-labeled [n-Ala”,&Leu”]enkeph- alin has been prepared successfully and is found to bind to opiate receptors stereospecifically and with high affinity (22- 24).

Simultaneous comparison of the potency of narcotic ago- nists, antagonists, and enkephalin analogs in inhibiting the binding of 3H-labeled narcotics and enkephalin, or of lz51- labeled enkephalin, reveal that narcotics are more potent in inhibiting labeled narcotics than enkephalins (19,20,24). The contrary is true for enkephalins and their analogs. Good correlations exist between the relative potency of enkephalin analogs (13, 14) in the mouse vas deferens bioassay and in the opiate binding assay. However, the correlation of binding is not as good with activity in the guinea pig ileum assay (13, 14). AII enkephalin analogs tested so far show considerably greater activity relative to morphine in the mouse vas deferens compared to the guinea pig ileum (13, 14). Certain narcotics are relatively more potent in the guinea pig ileum than on the

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Multiple Opiate Receptors 2611

mouse vas deferens (19). These data have been used to support the view that there are multiple opiate receptors. Martin and colleagues (25, 26) have postulated that there are at least three different opiate receptors.

By using ‘251-labeled [D-Ala’,D-Leu’lenkephahn of high spe- cific radioactivity, direct evidence has been obtained for at least two distinct types of opiate binding sites in the brain. One of these binds enkephalins with higher affinity than narcotics, while the other binds narcotics with higher affinity. Independent measurement of both sites (receptors) with mi- nor interference due to cross-reactivity can be accomplished by using very low concentrations of ‘251-[D-&i2,D-LeU5]f?rF

kephalin and 3H-labeled naloxone or dihydromorphine. The hydrophobic moiety of the benzene ring of enkephalin appears to be one of the major factors differentiating these two binding sites. The binding sites which bind enkephalin with higher affinity probably contain a hydrophobic recognition area for the benzene side chain of enkephalin or the C-19 hydrophobic group of oripavine derivatives.

EXPERIMENTAL PROCEDURES Materials-Enkephalins and their analogs were synthesized by Dr.

S. Wilkinson, The Wellcome Research Laboratories, Beckenham, England (14). Naloxone was obtained from Endo Laboratories, Inc. Morphine sulfate was obtained from Mallinckrodt Chemical Works. Pentazocine and meperidine were obtained from Sterling-Winthrop Research Institute. Butorphanol and oxilorphan were obtained from Bristol Laboratories. N-Cyclopropylmethylnoretorphine was ob- tained from Reckitt and Colman Products, London, England. [“HI- Naloxone (specific activity, 23 Ci/mmol) was purchased from New England Nuclear. [“H]Etorphine (specific activity, 32 Ci/mmol) and [3H]dihydromorphine (specific activity, 70 Ci/mmol) were purchased from Amersham/Searle.

Methods-Crude brain membranes are prepared by the method of differential centrifugation in isotonic sucrose solution. Whole brain without cerebellum from Sprague-Dawley rats (150 to 200 g) is homogenized with a Polytron PT-20 for 1 min at a setting of 3 in 10 volumes (v/w) of 0.32 M sucrose. The homogenates are centrifuged at 6,ooO x g for 15 min to remove the nuclei and mitochondria. The supernatants obtained from two such centrifugations are combined and centrifuged at 40,000 x g for 30 min. The pellets are resuspended in 5 volumes (original wet weight) of 5 mM Tris. HCl, pH 7.7, and allowed to swell for 30 min. The synaptosomes thus obtained are disrupted with a Polytron homogenizer and centrifuged at 6,000 X g for 15 min. The supernatants are centrifuged at 40,000 x g for 30 min. The tight, brownish pellets (which contain most of the mitochondria) are discarded while the top, loose pellets are separated and resus- pended in 5 mM Tris . HCl. These steps of swelling, disruption, and centrifugation are repeated and the final loose membrane pellet is suspended in 2 volumes (original wet weight) of 50 mM Tris . HCl and stored at -20°C.

N4TGl cells were supplied by Dr. A. Gilman, University of Virginia, Charlottesville, Virginia; these cells are routinely cultured in Dul- becco’s modified Eagle’s medium supplemented with 10% fetal calf serum at 37°C in an atmosphere of 90% air and 10% COZ. The cells, at confluency, are scraped from the tissue culture dishes and washed twice with Krebs-Ringer phosphate buffer containing sodium chloride (128 mM), calcium chloride (1.4 mu), magnesium chloride (1.4 mM), potassium chloride (5 mM), and sodium phosphate (5 mu, pH 7.4). When cell membranes are prepared, the cells are allowed to swell in 5 mM Tris . HCl buffer for 30 min before disruption with a Polytron PT-20. The whole particulate fraction is recovered by centrifugation at 40,ooO X g for 30 min. The pellets are then resuspended in 50 mM Tris . HCI buffer, pH 7.7.

lz51-labeled derivatives of [D-Ala*,n-Leu”]enkephalin are prepared as described previously (22). Briefly, to 0.1 ml of 0.25 M sodium phosphate (pH 7.4) 1.25 pg of enkephalin and 4 PCi of carrier-free NaY (Union Carbide) are added. Twenty microliters of chloramine T (0.5 mg/ml) are added, and after 20 s 20 ~1 of sodium metabisulfate (1 mg/ml) are added to stop the reaction. Monoiodinated enkephalin is then purified by Bio-Gel P2 and DEAE-Sephadex chromatography (22). In most studies of competition of binding, the step of DEAE chromatography is omitted. The specific activity of 1251-[D-Ala2,D- Leu5]enkephalin is about 2 Ci/pmol.

Binding assays are performed essentially as described previously,

using a Ntration method (GF/C filter) (22). Final volumes are 0.25 ml and 2 ml for ‘*“I-[D-Ala*,n-Leu”]enkephalin and 3H-labeled nar- cotics. For the competition curves, the concentrations of ‘““I-[D- Ala’,n-Leu”]enkephalin, [3H]naloxone, and C”H]dihydromorphine used are about 0.25 nM, 0.38 nM, and 0.2 nM, respectively. The total incubation time is 60 min at 24’C. The nonspecific binding is deter- mined in the presence of a large excess (1 pM) of[n-Ala’&-Leu5]- enkephalin or naloxone. All assays are performed in duplicate, and variability of the duplicates is usually less than 10% of the mean. The protein concentration is determined by the method of Lowry et al. (27) using crystalline bovine serum albumin as standard.

RESULTS

Direct Evidence for Two Opiate Binding Sites with Differ- ing Specificity-The potency of an inhibitor in competing for binding of a radioactive ligand is dependent upon the concen- tration of the labeled ligand and receptor concentration used in the assay (28,29). When the concentrations of receptor and labeled ligand are lower than one-tenth the dissociation con- stant for labeled ligand, I&, value (the concentration which can inhibit 50% of the labeled ligand binding) is nearly equal to its dissociation constant (28, 29). In a multiple receptor system, selection of the labeled ligand concentration becomes an especially important factor. A high affinity site can be measured without significant interference from a low affinity site provided certain conditions are met. The comparison of the potency is always done in the same membrane prepara- tion, the same conditions, and at the same time. The results are qualitatively reproducible, and the key experiments are repeated at least three times. The variation between individ- ual experiment gives no more than 2-fold difference in ICW value.

[D-Ala’,L-Leu5]EnkephaIin competes for the binding of “‘I- [D-Ala*@-Leu’]enkephaIin and [3H]naloxone and [3H]dihy- dromorphine with different potencies (Fig. 1). The concentra-

-101’ ’ ’ I I I 10-g 10-e

[D-AIO 2 ,L-Leu’“~Lqholin, tY7 5

FIG. 1. Upper panel, comparison of the potency of [D-Ala’&- Leu5]enkephalin in competing for the binding of ‘*51-[D-Ala2,D- Leu’lenkephalin (0), [3H]naloxone (O), and [3H]dihy&omorphine (A). The total specific radioactivity bound was 1180 cpm (2.4% of the total added), 870 cpm (4.6%), and 2430 cpm (8%) for ‘251-[D-&a2,D- Leu5]enkephalin (0.25 nM), [3H]naloxone (0.38 DM), and [“Hldihydro- morphine (0.2 nM), respectively. Values represent the mean of dupli- cate samples which are +5% of the mean. Lowerpanel, Hill plot. Hill coefficient, n = 1, 1, and 0.8 are found for ?-labeled enkephalin, [3H]dihydromorphine, and [3H]naloxone, respectively.

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2612 Multiple Opiate Receptors

TABLE I

ICY, values of enkephalins, [D-Ala’&-Leu’jenkephalin, morphine sulfate, and naloxone when studied by competition of binding of [3HJdihydromorphine, ‘251-[~.Ala2,~-Leu5]enkephalin, and (3H]naloxone in the absence andpresence of Na+ or Mn” ions

The I&, values are estimated from competition curves (Figs. 1 and 2) using 0.2 nM of [3H]dihydromorphine, 0.38 nM of [3H]naloxone, and 0.25 nM of ‘ZSI-[n-Ala2,n-Leu5]enkephahn. The concentration of Na’ and Mn*+ ions is 0.1 M and 1 mu, respectively. The two portions of the competition curve of naloxone and morphine sulfate against ‘251-[n-fiaZ,n-Leu5]enkephabn are separately estimated. Binding is carried out at 24°C. Values are expressed as nM + S.E. The number in parentheses is the number of separate experiments.

Labeled ligand

Inhibitor “‘I-[D-Ala’,o-Leu”]enkephalin

[n-Ala*&-Leu’]En- kephalin

[Lei?]Enkephalin [Met’lEnkephalin Morphine

Naloxone

Tris Tti Nat Ml?+ Tti Nat MI?’

6.0 f 0.5 (3) 1.3 f 0.1 (3) 1.5 + 0.2 (3) 1.5 f 0.1 (3) 7.25 f 0.96 (4) 58 f 38 (3) 4.8 f 0.8 (3)

23 -+ 6 (3) 3.1 * 0.5 (3) 3.0 2% 0.3 (2) 1.3 + 0.8 (3) 28.3 f 10.4 (3) 283 f 104 (3) 3.4 f 1.5 (3) 8.2 f 1.8 (3) 4.4 f 0.9 (4) 2.3 + 0.2 (3) 1.6 f 0.4 (4) 9.3 + 1.2 (2) 60 f 17 (3) 3.5 f 0.7 (3) 0.4 f 0.1 (3) 0.31 * 0.14 (4) 0.30 zt 0.10 (3) 0.50 f 0.26 (3) 16 f 12 (3) 0.28 f 0.13 (3)

47 * 12 (4) 40 f 10 (2) 42 f 5 (3) 1.4 + 0.6 (3) 0.43 f 0.08 (4) 0.8 + 0.3 (3) 1.06 * 0.51 (3) 1.10 * 0.35 (3) 1.10 f 0.30 (3)

22 f 6 (4) 20 + 5 (2) 30 f 10 (3)

tion of [n-Ala*&-Leu5]enkephahn required to inhibit 50% (It&) of the specific binding of 0.25 no ‘251-[D-Lila2,D-

Leu’lenkephalin is about 1.5 nM (Table I). In contrast, much greater concentrations (ICm about 7 no) of [n-Ala’+Leu5]- enkephalm are required to compete with the binding of [3H]naloxone and [3H]dihydromorphine. The concentrations of labeled narcotics used in the experiments are 0.38 nM an 0.2 no for [3H]naloxone and [3H]dihydromorphine, respec- tively, which are below the KD of the high affinity site. Therefore, the decreased potency for competition with the narcotics is not due to the labeled ligands used (28, 29) and is a correct reflection of different binding sites for labeled en- kephalin and narcotics. Hill plots in both cases show linear relationships with a Hill coefficient (n) of about 1 (Fig. 1).

Similar results are obtained for natural [Leu’]- and [Met5]enkephalins (Table I). Although they are less potent than [n-Ala2,L-Leu5]enkephahn in inhibiting the binding of both 3H-labeled narcotics and ‘251-[n-Ala2,n-Leu5]enkephahn, the difference in these two binding assays is still apparent. This difference is smaller for [Met’lenkephalin (Table I).

However, the potency relationship is reversed when mor- phine is used to compete for the binding of ‘251-[D-i%la2,D-

Leu’lenkephalin, [3H]naloxone, and [3H]dihydromorphine (Fig. 2). Morphine competes with [3H]dihydrohomorphine and [3H]naloxone with an I& about 0.5 no (Table I). A biphasic curve is found for narcotics in competing with rz51- [n-Ala*,&Leu5]enkephahn. The approximate I&O values for these two portions of the competition curve are estimated to be about 0.5 nM and 40 I-IM, respectively (Table I). Naloxone competes with the binding of ‘251-[n-Ala2,D-Leu5]enkephahn, E”H]naloxone, and [3H]dihydromorphine in a fashion very similar to morphine (data not shown); the I& values are shown in Table I. The Hill coefficient (n) is 0.8 for both enkephalin and morphine when measured against [3H]nalox- one binding (Fig. 2). This may be due to the slightly different binding of agonist and antagonist such as suggested by Fein- berg et al. (30).

When narcotics are used to compete with the binding of low concentrations of 3H-labeled narcotics, a linear Hill plot with a Hill coefficient (n) of about 1 is obtained (Fig. 2B). This evidence suggests that the 3H-labeled narcotics bind primarily to a homogeneous receptor population under these conditions. Thus, the bimolecular mass action equation

is applied to estimate the kinetic parameters, where Kd is the

-I 01 I 10-9 10-a 0' 10-G

[MORPHINE] ,M

FIG. 2. Upper panel, comparison of the potency of morphine sul- fate in competing for the binding of ‘251-[n-Ala2,n-Leu5]enkephabn (a), [3H]naloxone (O), and [3H]dihyclromorphine (0). The specific binding for ‘251-[n-Ala*,n-Leu5]enkephalin (0.25 nM), [3H]naloxone (0.38 nM), and [3H]dibydromorphine (0.2 11~) are 1220 cpm (2.5%), 1950 cpm (lo%), and 3800 cpm (15%), respectively. Values represent the mean of duplicate samples that are rt_5% of the mean. Lower panel, Hill plot of competition curve of morphine sulfate. Note the Hill coefficients (n), 1 and 0.8 for [3H]dihydromorphine and [3H]- naloxone. Two linear slopes with n = 0.4 and 1 are found for lz51-[~- Ala*,n-Leu’]enkephalin.

apparent dissociation constant, [ Rf] and [ I$] are the concen- trations of unbound receptor and ligand, and [RH] is the receptor .ligand complex. The total concentration of receptor sites (Rf + RH) are estimated to be 85 and 83 nM for [3H]- naloxone and [3H]dihydromorphine, respectively. This nearly equal content of both 3H-labeled narcotics is consistent with their binding to the same receptor population under the conditions described. This conclusion is further supported by their nearly identical ICm values regardless of whether [3H]- naloxone or [3H]dihydromorphine is used (Table I). If the same receptor population were responsible for the majority of the binding of ‘251-[n-Ala2,n-Leu5]enkephahn with an affinity of 0.8 nM (22), 6 to 8% of ‘251-[n-Ala2,n-Leu5]enkephahn should have been bound. However, only about 2.5% of ‘251-[D-Ala2,D-

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Multiple Opiate Receptors 2613

Leu’lenkephalin is bound, making it very unlikely that the binding site with high affinity for narcotics can also bind enkephalin with an affinity of 0.8 nM.

Competition curves of morphine sulfate and naloxone against ‘251-[D-Ala2,D-Leu5]enkephalin show biphasic curves and suggest that ‘251-[D-Ala2,D-Leu5]enkephalin at 0.25 nM is bound to two receptor populations at a ratio of about 3:7 (Fig. 2). Thirty per cent of the total bound ‘251-[D-Ala2,D-Leu6]- enkephalin can be inhibited by very low concentrations of morphine and naloxone, and the remaining 70% can only be inhibited by high concentrations of narcotics. Direct satura- tion curves of ‘251-[D-Ala2,D-Leu5]enkephahn (Fig. 3) show nonlinear Scatchard plots with parameters of 0.8 nM and 6 nM for the apparent KD, and 35 pM (or 44 fmol per mg of membrane protein) and 70 pM (or 88 fmol per mg of membrane protein) for receptor concentration. The receptor concentra- tion calculated for the latter site is very similar to that which binds [3H]naloxone and [3H]dihydromorphine, as described above. Na+ decreases the binding of both the high and the low affinity sites (Fig. 3). Furthermore, a biphasic rate of dissociation with half-times (t& of 5 and 22 min is observed for the ‘251-[D-Ala2,D-Leu5]enkephalin-receptor complexes (Fig. 4) which is consistent with the hypothesis of two opiate receptors of differing affinities.

The data described above clearly indicate that 1251-[D- Ala2,D-Leu’lenkephalin binds to two separate receptor sites which can bind narcotics with a 20- to W-fold, and enkephalin with a 3- to 5-fold difference in affinity (Table I). The use of low concentrations of 3H-labeled narcotics as reported here should allow detection of only the high affinity sites for narcotics. Indeed, morphine and naloxone compete with the binding of “H-labeled narcotics with affinities close to that of the high affinity site (0.5 no and 0.8 no). [D-Na2,L-Lf?U5] enkephalin competes with [3H]naloxone and [3H]dihy- dromorphine with an I& of about 6 no. Therefore, this site is called the “morphine receptor” since it binds morphine more strongly than enkephalin. The other site, which binds enkephalin better than morphine and naloxone, is referred to

E 8

0 -NoCI 0 a- . +NoCI 5 3 6- z

s! - ?i 0.04

0

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0.03 4 3

“s

NZ 2

0.02

z 0.01

23 ,:

Ic 2 Bfmol 8 -

5 IO 15

FREE lz51-[ D-Ala’, D-Leu’]- ENKEPHALIN, nM

FIG. 3. Saturation binding isotherms and Scatchard plots (inset) of the binding of ‘251-[D-Aa2,D-Leu5]enkephahn to brain membranes. 0.1 ml of brain membranes (0.8 mg/ml of protein concentration) is incubated with various concentrations of 1281-[D-AlaZ,D-Leu6]enkeph- alin (specific activity, 2 Ci/pmol) in the absence (0) and presence (0) of 0.1 M NaCl at 24°C for 60 min. The nonspecific binding is determined in the presence of 10 PM of [D-Ala’&-Leu5]enkephalin. Values represent the mean of duplicate determinations which are +5% of the mean. Note that the high affinity binding for “‘I-[D- Ala*,D-Leu’lenkephalin has an apparent dissociation constant of 0.8 nM and a capacity of 3.5 fmol/tube. The low affinity binding exhibited an apparent dissociation constant of about 6 DM and a capacity of 7 fmol/tube.

IO

. DUion Only 0 Dilutwx + I pM Nabxcme 0 Dilution +I IrM[~-~~oTL-~e”5]enkephalln

10 20 30 40 50 60

TIME (min)

FIG. 4. Rate of dissociation of ‘*“I-[D-Ala’,D-Let?]enkephalin-re- ceptor complex and the effect of naloxone and [D-Ala’&-Leu’]en- kephalin. The brain membranes were prelabeled with ‘251-[D-Ala2,D- Leu”]enkephalin (0.5 nM) for 60 min at 24°C and washed twice with cold buffer to remove unbound ‘2”I-[D-Ala2,D-LeuS]enkephalin. The labeled membrane pellets were then suspended in buffer (24°C) containing 1 PM of naloxone (0) or [D-Ala’&-Leu’lenkephalin (A) or dilution only (0). At various times, 2-ml aliquots were filtered, and the remaining specific bound ““I-[D-Ala,D-Leu’lenkephalin was de- termined. Values represent the mean of duplicates which are less than 10% of the mean.

as the “enkephalin receptor.” These two sites are not the same as those described by Pasternak and Snyder (31) in their “two state conformation” hypothesis since in our studies the behavior of the agonist, dihydromorphine, is very similar to that of the antagonist, naloxone. In the model of “agonist- antagonist two state conformation” (31), agonists should have less potency in inhibiting the binding of [3H]naloxone than that of [3H]dihydromorphine, and vice uersa for antagonist. In the present studies the potency of naloxone and morphine and enkephalins does not vary significantly whether [“HI- naloxone or [3H]dihydromorphine is used.

Low concentrations of labeled enkephahn and narcotics are used subsequently in the evaluation of potency of narcotics and enkephalin on the two binding sites since low concentra- tions of labeled narcotics bind preferentially to the morphine- binding sites. However, since the affinities of enkephalin for both sites are quite similar (6fold difference), and since the quantity of morphine receptor is much higher in the brain preparation, lz51-[D-Ala2,D-Leu”]enkephahn at a concentration of 0.25 nM wiIl bind to both sites with a ratio (morphine site to enkephalin site) of 3:7, which is readily detected by binding competition curves of naloxone and morphine. This may also explain the slight deviation of the Hill plot from a linear slope (Fig. 1). Since most of the ‘251-[D-Ala2,D-Leu5]enkephalin is bound to the enkephalin site, the apparent I& value should be closer to the Kd for the enkephalin site. Thus, ‘251-[D- Ala2,D-Leu’lenkephahn at concentrations below 0.25 nM can be used as a marker for the enkephalin receptors.

Absence of Negative Cooperatiuity-The differences in po- tency of enkephalin and narcotics in competing for the binding sites of labeled enkephalin and narcotics could also be due to heterogenous, negatively cooperative interactions between en- kephalin and morphine receptor sites. To test this possibility, the rate of dissociation of the enkephalin.receptor complex was measured by diluting membranes previously labeled with 1251-[D-Ala2,D-Leu5]enkephalin in the absence and presence of excess, unlabeled naloxone, morphine, and enkephalin (Fig. 4). The rate of dissociation of ‘251-[D-Ala2,D-Leu5]enkephalin is not significantly affected by any of these conditions. This indicates that negative, heterogenous, or homogenous coop- erativity does not exist. This conclusion is further supported

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2614 Multiple Opiate Receptors

by the data described in Figs. 1 and 2, where the Hill coeffi- cient (n) is very close to 1 when [D-Ala’&-Leu’]-, [Let?]- and [Met’lenkephahn compete with the binding of any of the labeled ligands and when morphine and naloxone compete with the binding of 3H-labeled naloxone and dihydromor- phine. Linear plots with two different slopes are obtained when naloxone and morphine compete with the binding of ‘251-[D-Ala2,D-Leu5]enkephahn. These data are explained sim- ply by two independent binding sites with widely different affinities for narcotics.

Differential Effects of Cations on Enkephalin and Mor- phine- binding Sites-In the absence and presence of Na+ or Mn’+, the IC% value for [D-Ala’,D-Leulenkephahn is un- changed (Fig. 5, inset). But Mnzf increases significantly the potency of [Leu5]- and [Met’lenkephalin in inhibiting the binding (Table I). When morphine (Fig. 5, lower panel) or naloxone (not shown) are used to compete for binding, the portion inhibited by low concentrations of narcotics (mor- phine-binding sites) disappears completely in the presence of Na+. However, in the presence of 1 mM MnC12, a large increase is noted (about 2-fold) in the portion (the enkephalin-binding site) sensitive to high concentrations of the narcotic.

When [3H]naloxone (0.38 nM) is used as the labeled marker, the potency of enkephalin and morphine is reduced in the presence of 0.1 M NaCl. A 40-fold and a 5- to lo-fold reduction in affinity is observed or morphine and enkephalin analogues, respectively, while the affinity for naloxone is not changed

[O-Ad, D-LW’] enkpholin,M

25, I

[MORPHINE] ,M

FIG. 5. Effect of cations on the binding of ‘*sI-[D-Ala”,D-LeuS]en- kephalin and the potency of enkephalin and morphine in competing for binding. 0.25 nM ‘*51-[n-AlaZ,o-LeuS]enkephalin (about 50,000 cpm) are incubated with brain membrane (0.5 mg/ml) in the absence and presence of various concentrations of [n-Ala’&Let?]enkephalin (up- per panel) and morphine ( lower panel) for 60 min at 24°C without (0) and with 0.1 M NaCl(0) or 1 mM MnCl2 (A). The inset shows the percentage of inhibition with or without Na’ or Mnzc ions. Note that a result similar to that of morphine sulfate is obtained for naloxone. Values represent the mean of duplicate determinations which are +5% of the mean.

10-g 10-S 0’

[INHIBITOR], M

FIG. 6. Comparison of the potency of [n-Ala*&-Ler?]enkephalin and naloxone in competing for the binding of ‘251-[n-Ala2,n-Leu5]- enkephalin (upper panel) and [3H]naloxone ( lower panel) in neuro- blastoma cells (N4TGl). 0.25 ml of neuroblastoma cells (5 x lo6 cells/ ml) are incubated with ‘251-[n-Ala2,n-Leu5]enkephalin (0.25 nM) in the presence of various concentrations of [n-Ala*+-Leu5]enkephalin (0) or naloxone (0) at 24°C for 60 min. 2 ml of neuroblastoma cells are used in the experiment of [3H]naloxone (0.38 nM) binding. Very similar results are also observed for other neuroblastoma cells such as NIE 115, N18TG2, and NG108-15 hybrids. Values represent the mean of duplicates that are &IO% of the mean. Note that in the experiment of [“Hlnaloxone binding, the stereospecific binding is very low (<I50 cpm) and suggests that the affinity for [3H]naloxone in neuroblastoma cells is very low.

significantly (Table I). This reduction suggests that the mor- phine-binding sites may be converted by Na+ to sites which bind morphine and enkephalin only with low affinity but which retain the high affinity for naloxone (31).

Neuroblastoma Cells Exhibit Only Enkephalin-binding Sites-The potency of a series of narcotic agonists and antag- onists in competing with the binding of ‘251-[D-Ala”,D-Leu5]- enkephalin to intact cultured cells is similar to that seen with rat brain membranes (24). This result suggests that the opiate receptor in neuroblastoma cells is similar to the enkephalin receptor in brain membranes. To examine the possible effects of homogenization procedures, cultured cells were homoge- nixed by the same methods used for the preparation of brain membranes, and the membranes were then analyzed for their affinity for enkephalin and narcotics. Naloxone and morphine compete with ‘251-[D-Ala2,D-Leu5]enkephahn in a manner very similar to that seen in intact cells. Notably, neuroblastoma cells or cell membranes bind very little [3H]naloxone or di- hydromorphine (~200 cpm) even though very substantial binding (several thousand counts per min) of ‘251-[D-Ala2,D-

Leu5Jenkephalin occurs under conditions very similar to those used in the brain membrane experiments. Furthermore, brain membranes bind 2 to 3 times more radioactivity of 3H-labeled narcotics than ‘251-[D-Ala2,D-Leu5]enkephahn. The morphine type of receptor which can bind narcotics with high and enkephalin with low affinity is not found in cultured cells. The fact that only the enkephalin type of receptor is detected in neuroblastoma cells even when 3H-labeled naloxone or dihy- dromorphine are used to measure binding (Fig. 6) further supports the hypothesis that there are two different opiate receptors in brain and only one such receptor in neuroblas- toma cells.

Etorphine Binding and its Inhibition by Naloxone and- Enkephalin-Previously, we (24) and other laboratories (19, 20) have shown that etorphine is approximately equipotent in

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Multiple Opiate Receptors 2615

competing with the binding of labeled enkephalin or naloxone. It can, therefore, be anticipated that C3H]etorphine should bind to both sites equally well. Since the affinities of enkeph- alin and naloxone for both sites are different, the competition curve of enkephalin and naloxone against C3H]etorphine should behave heterogenously, and the apparent I& value for enkephalin and naloxone should be greater than those values against ‘251-[n-Ala2,D-Leu5]enkephahn and [3H]nalox- one, respectively. That this is indeed the case is shown in Fig. 7. The apparent I&, values are 4 nM and 10 nM for naloxone and [rr-Ala’,u-Leu5]enkephahn, respectively, and the Hill plots are not linear.

Effect of Enkephalin Analogs and Various Narcotics on the Binding of Enkephalin and Naloxone-The apparent ICm values of various narcotic agonists, antagonists, and par- tial agonists in competing for the binding of the two labeled ligands are given in Table II. The ratios of the I& values for both receptor sites range from 70 to 1.4. Meperidine, a narcotic with a structure quite different from morphine, binds better to the morphine receptor than to the enkephalin receptor (ratio of 10). The mixed agonist-antagonists, pentazocine, butorphanol, and oxilorphan have a similar affinity for both systems. N-Cyclopropylmethylnoretorphine, another mixed agonist-antagonist which is 100 to 300 times more potent than morphine and has antagonist activity about one-fifth that of nalorphine (32, 33), binds to both receptor sites with even more nearly equal affinity. These drugs have been reported to contain less potential for inducing physical dependence. The “sodium shift” is smaller for pentazacine, butophanol, and oxilorphan than for morphine (Table II). Sodium ions do not affect the potency of N-cyclopropylmethylnoretorphine (Fig. 8) in competing for the binding of [3H]naloxone (Table II). In these terms, N-cyclopropylmethylnoretorphine behaves as a pure antagonist in the [3H]naloxone-binding assay.

The increase in affinity for the enkephalin receptor and the decrease in affinity for the morphine receptor resulting from

[‘HI Etorphm

d (

10-s 10-e [ INHIl3ITOR]

FIG. 7. Upperpanel, competition curve of [n-Ala’,L-Leu’lenkeph- alin and naloxone against [3H]etorphine binding. 0.13 nM of etorphine (8600 cpm) is incubated with 2 ml of brain membrane (0.27 mg/ml of membrane protein) in the absence and presence of various concentra- tions of [n-Ala’,L-Leu5]enkeph&n (0) or naloxone (0) for 60 min at room temperature. Lowerpanel, Hill plot of the binding of enkephalin and naloxone against C3H]etorphine. Note the nonlinear biphasic curve for both ligands. Each point is the mean of duplicate determi- nation.

TABLE II Comparison of the potency of narcotics and enkephalin analogs in competing with the binding of ‘251-fu-Ala2,~-LeuS]enkephalin and

[3H]nalorone The apparent IC, values are estimated from competition curves.

The K&o ratios of potency using these two labels are shown in the third column. The last column shows the ICm ratios in inhibiting the binding of [3H]naloxone in the presence and absence of sodium ion (0.1 M). The concentrations are 0.25 nM and 0.38 nM for ‘*“I-[D-Ala’,D- Leu’lenkephalin and [3H]naloxone, respectively. The assays are car- ried at 24°C for 60 min.

Compounds

Morphine Naloxone Meperidine Pentazocine N-Cyclopropylmeth-

ylnoretorphine Butorphanol Oxilorphan

[Met’lEnkephalin [n-Ala’,Met’]En-

kephalin [Leu’lEnkephalin [n-Ala’,Leu’]Enkeph

alin Tyr-Gly-Gly-Phe Tyr-ethyl ester

I ICW “1-[~-Ala’,~- ,eu”]Enkeph-

alin

1

- -

1:

L

[

I I

1% F

‘51-~~-AIft’,~. ,eu ]Enkeph

alin

3H]Naloxon~

70 19 10

3 2.5

io

+Na’”

35 nM 15 nM

3W 30 nM

2.5 nM

0.5 nM 0.8 nM 0.3 pLM

10 nM 1nM

-Nit+

41 1

20 3 1

1.4 DM 1.0 nM

1.0 nM 0.7 niv

1.4 1.4

4 3

4.0 nM 10 nM 0.4 5 1.5 nM 6nM 0.25 8

3.2 nM 25 nM 0.14 1.5 nM 6nM 0.25

10 6

0.3 PM 100 PM ! 1

1.4 PM

WlLM

5 3

a Using [jH]naloxone.

HO CH t “Pf

FIG. 8. Structure of N-cyclopropylmethylnoretorphine.

introduction of a hydrophobic group at C-19 of oripavine suggest that a hydrophobic group in this position is more important for the enkephalin receptor. This hydrophobic area could in principle be occupied by the benzene ring of the phenylalanine residue of enkephalin. This possibility is sup- ported by the fact that the 5-fold difference in enkephalin binding to its receptor compared to the morphine receptor is maintained even with the simple analog, Tyr-Gly-Gly-Phe (Table II, lower part). This difference disappears with Tyr- ethyl ester, even though this amino acid analog still retains some affinity (albeit low) for the receptor. The major differ- ence between the two receptor sites appears to be the region interacting with Gly-Phe of enkephalin and the hydrophobic group of C-19 of oripavine.

Table II also shows that the IC% ratios of the enkephalins and their analogs, determined from inhibiting the binding of the [3H]naloxone in the presence and absence of Na+, range between 3 to 10.

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2616 Multiple Opiate Receptors

DISCUSSION

Morphine inhibits the binding of [3H]dihydromorphine with a ICU value of about 0.5 nM. However, its potency against the binding of ‘251-[D-Ala”,D-Leu5]enkephahn is about 50 times lower, with a I& value of about 35 no (Table II). Creese et al. (34) shows that the binding of 3H-labeled narcotic agonist and antagonist to brain membrane preparation was discrimi- natively affected by temperature. At 25”C, sodium ions in- crease the number of binding sites for opiate antagonist and decrease the number of agonist with no change in affinity. However, at O’C, sodium ions increase the binding of opiate antagonist by enhancing its affinity for binding site without changing the number of binding sites. These effects are about 2- to 3-fold. The differences of morphine and naloxone in inhibiting the binding of 3H-labeled narcotics and lz51-[D- Ala’,D-Leu’lenkephahn are about 50- to lOO-fold, and thus these differences could not be accounted for by the tempera- ture effect. In addition, the comparison of the potency are carefully carried out at the same time and with the same temperature and the same membrane preparations. Similar to the binding of 3H-labeled narcotic agonists (31,35,36,3&J), the binding of ‘251-[D-Ala2,D-Leu5]enkephahn is decreased by Na+ (Fig. 3) and increased by Mn2+ (Fig. 5), and also extremely sensitive to the enzymatic digestion (24). 1251-[D-tia2,D- Leu’lenkephalin probably binds to agonistic conformation as postulated by Pasternak and Snyder (31) under the present experimental condition (low concentration). Because of the wide difference of narcotics and enkephalin in inhibiting the binding of [3H]dihydromorphine and ‘251-[D-Ala2,D-Leu5]en- kephalin, and these differences are not due to the cooperativ- ity and agonist-antagonist conformation (31),‘it has to be concluded that at least two opiate receptors exist in rat brain.

By using the radioactive ligands, ‘251-[D-Ala2,D-Leu5]en- kephalin, [3H]naloxone, [3H]dihydromorphine, and [3H]etor- phine, it is possible to demonstrate that at least two types of opiate binding sites (receptors) exist in rat brain membrane preparations. Table III summarizes the basic features and parameters of these two binding sites. Enkephalin binds to both sites with affinities (Kc& apparent dissociation constant) of about 2 nM and 10 nM. However, narcotic agonists such as morphine, and antagonists such as naloxone, bind to these two sites with affinities which differ by as much as 20- to lOO- fold. Both naloxone and morphine bind to the enkephalin sites with affinities of about 30 no, while their affinities for the morphine sites are about 0.4 no. In conclusion, the relative order of potency for the enkephalin receptor in the absence of sodium ions is [D-Ala’&-Leu”]enkephalin > [Leulenkephalin or [Met5]enkephalin Z N-cyclopropylmethylnoretorphine > naloxone > morphine, and for the morphine receptor it is morphine > naloxone > N-cyclopropylmethylnoretorphine > [D-Ala2,L-Leu’lenkephahn 2 [Met5]enkephalin > [Leu’len- kephalin.

On the basis of pharmacological studies in the guinea pig ileum and the mouse vas deferens it has been suggested that the receptors in the two models are heterogenous and not identical (14, 19). In the mouse vas deferens, stable enkepha- lins are about 200 times more potent than morphine (14), while in the guinea pig ileum enkephalins are equal or slightly less potent than morphine. The concentration of antagonist required to inhibit the enkephalin effects is 10 times higher in the vas deferens than in the guinea pig ileum. In the present studies the apparent affinity of naloxone for enkephalin-bind- ing sites is much lower than that for morphine-binding sites. Morphine and enkephalin bind to the morphine-binding sites with about equal potency in the presence of Na’. Naloxone is much more potent than either enkephalin or morphine in binding to the morphine-binding sites in the presence of Na+.

TABLE III Summary of the distinguishing characteristics of morphine and

enkephalin receptors The apparent dissociation constants for enkephshn, morphine, and

naloxone are calculated from their ICSO values by using Cheng and Prusoffs equation (37),

K’d=-%- 1+[L3

Kd

where [L] is the concentration of labeled ligand and Kd and K’d are the apparent dissociation constants for labeled and unlabeled ligands, respectively. Kd values of enkephalm receptors for ‘?-[D-Ala’,D- Leu’]enkephalin and morphine receptors for [3H]dihydromorphine are ‘0.8 nM and 0.3 nM, respectively. Using [3H]naloxone binding data, and a Kd of 0.5 no, similar values are obtained. The binding capacity is estimated from the Scatchard plots of the binding of ‘251-[D-AaZ,D- Le&]enkephalin.

Affinity, (nM) [n-Ala*&-Leu’]En-

kephalin [L&]Enkephalin [Me@]Enkephalin Morphine Naloxone N-Cyclopropyl-

methylnoretor- phine

Capacity (fmol/mg of protein)

Na+ effect

Mn2+ effect

Enkephalin receptors Morphine receptors

1.2

2.7 15 3.2 6.2

32 0.3 13 0.5

2 0.8

43

1 Enkephalin bind- ing

f Enkephalin bind- ing

5.0

88

f Naloxone binding 4 Morphine binding J Enkephalin binding Slight t affinity of en-

kephalin and mor- phine

Thus, the enkephalin sites seem to resemble the pharmaco- logic receptor of the mouse vas deferens, and the morphine receptor resembles the receptor in guinea pig ileum. In the guinea pig ileum, enkephalin presumably interacts mainly with the putative p receptors which mediate the action of classical morphine-like compounds. In the mouse vas deferens, enkephalin interacts mainly with the putative 8 receptor (19). Martin et al. (25) previously proposed the existence of multi- ple opiate receptor, (I, IL, and k receptors.

The monovalent ion, NaC, and the divalent cation, Mn2+, affect the binding to morphine and enkephalin receptors dif- ferentially. Na+ increases the binding of naloxone without any significant effect on its affinity. In contrast, the affinity for enkephalins is reduced 5- to lo-fold and that for morphine 40- fold. These results are consistent with previous reports by Pert and Snyder (38) and Miller et al. (22). Mn2+ increases slightly the affinity of enkephalin and morphine for the mor- phine-specific sites. In contrast to the effects on the binding of [3H]naloxone, Na’ completely inhibits the binding of lz51- [D-Ala2,D-Leu’lenkephahn to the morphine sites but only partially inhibits its binding to the enkephalin sites. Satura- tion binding isotherms and Scatchard plots indicate that Na+ decreases the binding of enkephalin to both sites, by decreas- ing the amount of binding without altering the affinity. Mn2+ increases the binding of the enkephalin receptor without significantly affecting the affinity of morphine, naloxone, or [D-Ala2,L-Leu’lenkephalin (Table I). The differential effect of cations described here further supports the existence of two opiate receptors in rat brain tissues. Since Mn2+ increases the binding capacity of enkephalin receptors nearly 2-fold (Fig. 5), the relative binding of ‘251-[D-Ala2,D-Leu5]enkephahn to en- kephalin sites is increased in the presence of 1 mu MnC12 (Fig.

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Multiple Opiate Receptors 2617

5). Thus, it is possible to measure more accurately the affinity mally with the enkephalin peptides. Our data suggest that the of compounds forenkephalin receptors in the presence of 1 endogenous substances which normally interact with the mor- mM MnC12. phine receptors may not yet be known.

The ratio of the I& value of a drug in the presence of sodium ion to that in its absence is known as the “sodium Acknowledgments-The excellent technical assistance of Mark

shift” (38), which has been used as an indication of the agonist, Collins is greatly acknowledged. We are grateful to Nancy Davis for

antagonist, and partial agonist properties of compounds. The typing the manuscript.

partial agonists, pentazocine, butorphanol, and oxilorphan which have low “sodium shift” ratios also bind to both recep- tor sites with similar affinity. The most interesting compound is N-cyclopropylmethylnoretorphine. This compound shows no “sodium shift” in the [3H]naloxone-binding assay, and it binds to the enkephalin sites with an affinity higher than morphine and naloxone and binds to morphine sites with an affinity lower than that of morphine. At present, it is not clear what these data mean. However, partial agonists or mixed agonist-antagonists are also known to have less potential in inducing physical dependence. It is conceivable that the rela- tive affinity to these two opiate receptor sites may play some role in the ability of a compound to cause physical dependence or addiction. The ability of opioid peptides to induce physical dependence has recently been demonstrated by Wei and Loh (39) with constant infusion into the periaqueductal gray fourth ventricular space of rat brain. Since the relative affinities of opiates and opioid peptides for these two opiate receptor sites vary, a careful comparison of the analgesic potency and the relative ability to cause physical dependence may provide new insights. Recently, Frenk et al. (40) and Urea et al. (41) showed that intracerebroventricular injection of low doses of [Met]- and [Leulenkephalins or of a high dose of morphine caused epileptic seizures and that all such seizures could be blocked by high doses of naloxone. However, morphine at low dose or enkephalin at high dose induced analgesia. Our data show that enkephalin receptors bind enkephalins with an affinity higher than that of opiates. In contrast, morphine receptors bind enkephalins and opiates with the reverse af- finty. Thus, the data suggest that morphine receptors may play a role in analgesic effects, and enkephalin receptors mediate behavioral epileptic seizures.

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We have screened many cultured neuroblastoma cells for opiate receptor binding. Although many of these bear enkeph- alin receptors, no cells have yet been found to have the morphine receptor sites. Since opiate receptors in situ are continually exposed to enkephalins, it is theoretically possible that exposure of receptors to enkephalins causes some kind of transition which leads to new binding characteristics. This possibility was tested by treating neuroblastoma cells with morphine (10d6 M) for 24 h; however, the binding properties did not change with this treatment.

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K J Chang and P Cuatrecasasdifferent specificity.

Multiple opiate receptors. Enkephalins and morphine bind to receptors of

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