Opioid ligand binding sites in the spinal cord of the guinea-pig

Newopharmacology Vol. 25, No. 5, pp. 471-480, 1986 Printed in Great Britain

0028-3908186 $3.00 t 0.00 Pergamon Press Ltd

OPIOID LIGAND BINDING SITES IN THE SPINAL CORD OF THE GUINEA-PIG G. D. ZARR, LINDA L. WERLING, S. R. BROWN and B. M. Cox Department of Pharmacology, Uniformed Services University of the Health Sciences, Bethesda, MD 20814, U.S.A. (Accepted 4 fury 1985)

Summary-The properties of opioid binding sites in membranes from the spinal cord of the guinea-pig were analyzed in experiments employing radiolabeled opioid ligands, selective or partially selective for p, 6 and K-type binding sites. Incubation was conducted at 37°C in a quasi-physiological modified Krebs medium, containing sodium and magnesium. The types of binding sites were discriminated on the basis of their tinities for [3H-~-Ala~-MePhe~-Giys-ol]enkepha~n (r3H]DAGO), [3H-5Ala*-D-~us~nkephalin, and [jH]ethyket~yclaz~ne and the relative potencies of the displacing Iigands, DAGO, [D-Se&Leu’]enkephalyi-Thr and trans-3,4-dichloro-N-methyl-N-[2-(l-pyrrolidinyl)-cyclohexyl]~nzeneacetamide methanesulfonate hydrate (U50488H), which are selective for p, 6 and K type binding sites respectively. In membranes from whole spinal cord, K type sites comprised about 60%, /Labout 30% and S about 10% of the total of p, S and K binding sites. Binding sites of the p type were also found in the lumbo-sacral region of guinea-pig spinal cord, in contrast to earlier reports of their absence from this tissue. Morphine showed a better than SOO-fold selectivity for Jo over K sites in spinal cord, while n~buphine and (-)1-cyc1openty1-5-(1,2,3,4,5,~hexahydro-8-hydroxy-3,6,1 I-trimethyl-2,6-methane-3benzazocin-l l-yl)3-pentanone methanesulfonate (WIN 44441-3) showed about a IO-fold selectivity for g sites, The drug U50488H had about a 150-fold greater affinity for K than p-type binding sites. Key words: binding sites, opioid delta receptors, spinal cord kappa receptors, spinal cord mu receptors, spinal cord, opioid binding.

The ability of morphine and related drugs to induce a selective analgesia limited to the lower part of the body after direct application to the spinal cord (Yaksh and Rudy, 1976; Yaksh, 1978) has led to the dinical use of opiates, administered in~athe~ally, to produce localized analgesia (Behar, Olshwang, Magora and Davidson, 1979; Cousins, Mather, Glynn, Wilson and Graham, 1979; Magora, Olshwang, Eimerl, Shorr, Katzenelsson, Cotev and Davidson, 1980). These studies indicate that the spinal cord is an important site for the analgesic action of morphine, an opiate with preferential affinity for p-type opioid receptors. Other workers have shown that opioids with preferential affinity for K-type receptors, such as ethylketocyclazocine, also exert significant analgesic effects when applied to the spinal cord (Wood, Rackham and Richard, 1981; Schmauss and Yaksh, 1984). There is some controversy regarding the nature of high affinity opioid binding sites in spinal cord. Several groups have reported the presence of p, S and x-type sites in homogenates of the spinal cord of the rat (Fields, Emson, Leigh, Gilbert and Iversen, 1980; Traynor, Kelly and Rance, 1982; Czlonkowski, Costa, Przewlocki, Pasi and Herz, 1983; Mack, Killian and Weyhenmeyer, 1984). However, Gouarderes, Audigier and Cros (1982), and Attali, Gouarderes, Mazarquil, Rudigier and Cros (1982) have reported that p-type receptors are not detectable in homogenates of the lumbo-sacral cord of

rats and guinea-pigs. Since morphine has been shown to produce analgesia when administered to the lumbo-sacral cord of rats (Yaksh and Rudy, 1976; Wood et al., 1981), the failure to detect p receptors in this region of the cord is surprising. The properties of opioid binding sites in neural tissue of the rat and guinea-pig, determined in a modified Krebs buffer at 37°C have recently been described (Werling, Zarr, Brown and Cox, 1985). Reported here is the characterization of opioid binding sites in the spinal cord of the guinea-pig, using the same experimental conditions. Since the ionic composition of the incubation medium affects the properties of p, S and K-type binding sites in different ways (Werling, Brown and Cox, 1984), it is considered that these incubation conditions permit the determination of the ligand binding properties of receptors in the spinal cord opioid under more physiologically-relevant conditions than those employed in previous studies. The present study reports the characteristics of ,u, 6 and K opioid binding sites in homogenates of the spinal cord of the guinea-pig in a modified Krebs buffer, and demonstrates that sites with fl receptor characteristics are present in lumbo-sacral regions of the spinal cord of the guinea-pig. METHODS Materials

[3H]Ethylketocyclazocine (13H]EKC) mmol, was purchased from New England

19.9 Ci/ Nuclear

412

G. D. ZARRet al.

Corporation (Boston, Massachusetts, U.S.A.) and [‘HTyr’-D-Ala2-MePhe4-Glyo15]enkephalin (PHIDAGO), 60 Ci/mmol and [3H-Tyr’-o-Ala2-n-Leu5]enkephalin ([‘HIDADLE), 36.5 Ci/mmol, from Amersham Corporation (Arlington Heights, Illinois, U.S.A.). [D-Ala’-MePhe4-Gly’-ol]Enkephalin (DAGO) was purchased from Cambridge Research Biochemicals Ltd (Beach, New York, U.S.A.) and [D-Ser’Leu5]enkephalyl-Thr (DSLET) and HEPES from Sigma Chemical Company (St Louis, Missouri, U.S.A.). Ethylketocyclazocine methane sulfonate (EKC), (+)l-cyclopentyl-5-(1,2,3,4,5,6-hexahydro-8 hydroxy-3,6,1l-trimethyl-2,6-methano-3-benzazocin11.yl)3pentanone methanesulfonate (WIN 44441-2) and WIN 44441-3 were supplied by Sterling Winthrop Labs (Rensselaer, New York, U.S.A.), naloxone hydrochloride by Endo Labs (Garden City, New York, U.S.A.), and morphine sulfate by Merck (Rahway, New Jersey, U.S.A.); U50488H was a gift from Dr Robert Lahti (Upjohn) and nalbuphine hydrochloride, a gift from Dr S. Muldoon. Fresh dilutions of drugs were prepared for each experiment in modified Krebs buffer (see below). Preparation of spinal cord membranes

Male Hartley guinea-pigs (400-500 g, CAMM, Wayne, New Jersey, U.S.A.) were killed by decapitation, the vertebral column was dissected in block, divided into four segments, and placed on ice. Segments of spinal cord were rapidly removed from each vertebral column segment by application of a high pressure air line to the distal end. The spinal cord segments were rapidly placed in ice-cold modified Krebs buffer (25 mM HEPES, 118 mM NaCl, 4.8 mM KCl, 1.2 mM MgCl,, 2.4 mM CaCl,, pH adjusted to 7.4). Tissue from 4 to 6 animals was pooled and homogenized using a teflon-glass homogenizer (ten strokes). The crude membrane homogenate was centrifuged at 27,000g for 15 min at 4°C the supernatant discarded and the pellet resuspended in 20 vol of modified Krebs buffer with a Vortex mixer and incubated at 0°C for 60 min to facilitate the removal of any endogenous opioids. At the end of 60 min, the suspension of membrane was centrifuged at 27,000g for 15 min at 4”C, the supernatant discarded, and the pellet was resuspended in 10 vol of modified Krebs buffer. After two additional washes, the final pellet was resuspended to a concentration of 2% w/v in modified Krebs buffer and frozen at -70°C for not more than 3 weeks prior to use in receptor binding assays. Protein concentrations were determined by a modification of the Lowry procedure (Peterson, 1977). The protein content in the 2% suspensions of membranes of spinal cord ranged from 290 to 32Opg (mean value 3OOpg) protein/250 ~1 membrane suspension. Measurement of radioligand binding

Labelled opioid ligand binding was determined as described previously (Werling et al., 1985). Briefly,

aliquots of spinal cord membrane suspension, in modified Krebs buffer, were incubated at a l%(w/v) final concentration in a final volume of 500 ~1 at 37’C for 20min with [‘HIDAGO (for p receptors), [3H]DADLE in the presence of 10 nM unlabelled DAGO (for 6 receptors), or [3H]EKC in the presence of 1 PM unlabelled DAGO (for K receptors). Nonspecific binding was determined by conducting parallel incubations with a large excess of unlabelled competing ligand. A 1 PM concentration of DAGO was used for g receptors, 1 PM DSLET for 6 receptors and 1 PM EKC for ICreceptors. The stability of the labelled and unlabelled competing ligands under these incubation conditions was determined as described previously (Werling et al., 1985). Approximately 300 pg of membrane protein were present in each assay tube. Control experiments indicated that binding was linear with the concentration of the protein at least to 6OOpg protein per tube. The incubation was terminated by rapid dilution in icecold modified Krebs buffer and filtration through glass fiber filters (No. 32, Schleicher and Schuell Inc, Keene, New Hampshire, U.S.A.) under vacuum. After two additional washes with 4 ml of cold buffer, the radioactivity retained on the filter was determined by liquid scintillation spectrometry. Control experiments revealed that there was insignificant loss of specifically bound radioactivity during these washing procedures (the half time for dissociation at 4°C was greater than 10 min for all radioligands). Binding parameters for each labelled ligand were estimated with the assistance of LIGAND (Munson and Rodbard, 1980), a non-linear curve fitting program which assumes the simultaneous contribution to total binding of one or more independent sites. An F-statistic on the mean square residual error, and a runs test examining the occurrence of serial positive or negative deviations of the observed data from the computed binding isotherm, were used to select between different models for each set of data. RESULTS

Equilibrium binding

The rate at which the specific binding of all three labelled ligands reached equilibrium under the incubation conditions was determined. For each ligand, specific binding approached a plateau within the first 5 min of incubation at 37°C and reached equilibrium within 20 min. All subsequent incubations were conducted for 20mir1, at which time there was no evidence of any degradation of labelled or unlabelled ligands. Characterization of specijc binding of [‘HIDAGO

A relatively high level of non-specific binding of [3H]DAG0 was observed in the membranes from spinal cord. Nevertheless, a specific component of high affinity [‘HIDAGO binding, declining from 40% at 1 nM to 5% of total binding at 50 nM free ligand concentration, was always detectable (Fig. 1A). Com-

413

Opioid binding in spinal cord (A) Whole

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Fig. 1. Analysis of high affinity saturable binding of [3H]DAG0, l-50 nM, by membranes of the spinal cord of the guinea-pig. Aliquots of homogenates of (A) whole spinal cord, (B) lumbo-sacral region of spinal cord, or (C) cervicalthoracic region of spinal cord, were incubated with various concentrations of [‘HIDAGO, as indicated in Methods. Specific binding estimates, determined in two independent experiments (open and closed circles) in each condition, are presented as Scatchard (1949) plots. The binding parameters were estimated by non-linear curve fitting (LIGAND; Munson and Rodbard, 1980) for each saturation curve individually, in pooled sets of two curves for each type of

homogenate, and as a group of six saturation curves. In each case the data were best fitted by a single site model. Since no significant differenceswere observed at a 5% probability level between binding by each part of the cord and binding by whole cord, the lines drawn indicate the parameters estimated from the pooled group of six saturation curves, with a K, of 8 nM, and a B,, of 12pM corresponding to a tissue concentration of binding sites of 18fmol/mg protein. puter analysis of the binding [3H]DAG0 in homogenates of whole spinal cord indicated that a single site model was most consistent with the experimental data, with a dissociation constant (K,) of 6 nM and a &,, of 18 fmol/mg protein. This binding site was further characterized by displacement studies. Specific binding of [3H]DAG0 was readily displaced by morphine and nalbuphine. In contrast, the analog of enkephalin DSLET, which has modest selectivity for the 6 type receptor (David, Moisand, Meunier,

Table

Morgat, Gacel and Roques, 1982; Werling et al., 1985) and U50488H, which has preferential affinity for K type receptors (James and Goldstein, 1984), were much less potent (Table 1). These results suggest that the specific binding of [3H]DAG0 in spinal cord membranes from the guinea-pig was to a binding site with the characteristics of p-type receptors, as has been observed with this ligand in the brain of the rat (Gillan and Kosterlitz, 1982) and guinea-pig (James and Goldstein, 1984; Werling et al., 1985). Measurements of the binding of [‘HIDAGO in the separated cervical-thoracic and lumbo-sacral regions of the spinal cord of the guinea-pig showed binding to sites with p characteristics in both regions. Scatchard plots for both tissue preparations were linear (Fig. 1B and C). Computer analysis indicated that a one site model with a K, of 7 nM and a B,,,,, of 18 fmol/mg protein for lumbo-sacral membranes and a one site model with a K, of 9nM and a B,,,,, of 20 fmol/mg protein in cervical-thoracic membranes, provided the best fit to the experimental observations. These derived parameters were not significantly different from one another or from binding in membranes from the whole cord (Fig. 1) at the P < 0.05 level. Re-analysis of the pooled data yielded a K, of 8 nM with B,,,,, of 18 fmol/mg protein. Displacement of 5 nM rH]DAGO binding in lumbo-sacral spinal cord membranes with morphine sulfate showed a K, of 12nM (Fig. 2A), while U50488H had an I&, > 10 PM (data not shown). Thus, the site labelled by [3H]DAG0 in lumbo-sacral spinal cord had similar K, and B,,,,, values to the site labelled by [3H]DAG0 in membrane from whole spinal cord in saturation binding experiments. In addition, the K, for morphine in whole spinal cord and lumbo-sacral spinal cord were similar. The present results for binding studies in the lumbo-sacral spinal cord of the guinea-pig with [)H]DAGO indicate that the population of receptors labelled had characteristics consistent with a p opioid receptor site binding. An interesting feature of the displacement of [3H]DAG0 binding in membrane preparations from

I. Affinities of opioid ligands for p and K binding sites in membranes from the spinal cord of the guinea-pig p binding site*

K binding site?

Ligand

K&M) + SEM

Maximum inhibition (%)

Morphine DAGG DSLET WIN 44441-3 WIN 44441-2 Nalbuphine U50488H

15*4(2) 10 f 2 (3) 54+ L?(3) 0.058 * 0.025 (2) not determined I I * 3 (3) 1900 f 380 (2)

100 100 100 100 100 100

K,(nM) f SEM z 1000 (3) not determined > 10,000 (2) 0.7 f 0.2 (2) 1900(l) 118*54(3) 14 + 2 (3)

Maximum inhibition (%) 20(10pM) O(lOpM) 100 100 100 100

K, ratio <0.015
*Affinity for P sites was estimated by displacement of specific binding of 5 t&i [‘HIDAGO from spinal cord membranes of the guinea-pig. tAtiinity for K sites was estimated by displacement of the specific binding of 2 nM [‘H]EKC in the presence of 1 pM DAGO (for details, see Methods). The data from two or three complete displacement curves were modelled together by a non-linear curve-fitting procedure (LIGAND; Munson and Rodbard, 1980) to obtain the pooled K, values and estimates of error. Figures in parentheses after the K, values indicate the number of separate full displacement curves which were analyzed in each case. When complete displacement of the labelled ligand was not obtained at a displacing concentration of ligand of 10 PM, a K, value was not calculated. The maximum displacement (as a percentage) at this concentration is indicated in the Table.

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Fig. 2. Displacement of specific binding of t3H]DAG0, 5 nM, in spinal cord membranes by increasing ~on~ntrations of displacing hgands. Panel A shows displacement curves for ligands which appeared to show a shoulder in the displacement curve; (0) morphine in whole cord membranes; (a) morphine in lumbo-sacral cord membranes; (A) DSLET in whole cord membranes. Panel B shows the displacement curves in whole cord membranes for ligands which did not display any obvious shoulder in their displacement curves; (II) unlabelled DAGO; (A) nalbuphine; (0) U50488H.

spinal cord was that the log. logit slopes (pseudo Hill plots) of displacement curves for most ligands had slopes of less than the value of unity anticipated for simple mass action binding to a single site. Thus, displacement slopes in the range 0.5-0.7 were observed with DAGO, DSLET, morphine and nalbuphine. For some ligands, a shoulder in the displacement curves was apparent (Fig. 2A). These results might indicate the binding of [3H]DAG0 to two types of binding site which could be discriminated by some displacing ligands. However, the apparent discrepancy from a single site model was not sufficiently large for a two site model to fit the data significantly better at a 5% probability level. The possible heterogeneity of specific binding of [3H]DAG0 is considered further in the Discussion section. Characterization

of speci$c binding of [3HH]EKC

Specific binding was found to be saturable when the suspension of membrane was incubated with [‘H]EKC at concentrations of O.l-50nM. A curvilinear Scatchard plot was obtained from saturation curves for [3H]EKC (Fig. 3A). A model of two independent binding sites fitted the data better than a one-site model or a three-site model. Displacement of 2.0 nM [3H]EKC by increasing concentrations of DAGO revealed a high affinity site for the displacement of DAGO with a K, of 5SnM, and a low affinity site with a K, greater than 10 PM. The high affinity displacement of 30% of the 2nM [3H]EKC specific binding by DAGO is consistent with displacement of t3H]EKC from p-opioid receptor sites. Therefore a 1 PM concentration of DAGO was employed for the selective blockade of /1 receptor sites in this system. Displacement of 2.0 nM [3H]EKC with U50488H revealed a high affinity site with a Kr of 2.3 nM, and

a low affinity site with a K, of 1.9HM (Fig. 4A). Inspection of the displacement curves for U50488H in spinal cord and in cortex of the guinea-pig (Werling et al., 1985) indicated that U50488H did not displace ligands from /J binding sites until the concentration exceeded I PM. A final concentration of 1 ,LLMof U5~88H was therefore selected to block rc binding without affecting p binding. To be certain that [3H]EKC was not binding to S receptors in the present system, DSLET was used to displace 2.0 nM [jH]EKC in the presence and absence of 1 PM DAGO. However, DSLET had a K, of 45 nM when no DAGO was included and was unable to produce complete displacement of 13H]EKC at concentrations up to 10 PM. When I PM DAGO was added to block the binding of [3H]EKC to g sites, DSLET was unable to displace any specific binding even at a final concentration of !‘iOpM (Fig. 4A and B). Saturation analysis of 0.12-50 nM [3H]EKC binding was conducted under three conditions in parallel In the absence of both U50488H and DAGO, Scatchard transformation of the saturation curve for [jH]EKC showed a curvilinear plot (Fig.3A). Computer analysis indicated that a model assuming two independent binding sites provided the best fit of the experimental data. A high affinity site (presumably a rc site) with Ku of 0.6 nM and S,,,,, of 42 fmol/mg protein and a low affinity site with Kn of 44 nM and B,,, of 153 fmol/mg protein were identified. When a 1 PM DAGO block was used in the [3H]EKC saturation assay, Scatchard (1949) transformation of the data still produced a curvilinear plot (Fig. 3B). A model assuming two independent binding sites continued to fit the data better than a one-site model, with a high affinity site with a K, of 0.8 nM and a B,,, of 31 fmol/mg protein and a very low affinity site with K, of 350 nM and B,,,,, of 660 fmol/mg protein (there are considerable errors in the estimates of the

Opioid binding in spinal cord

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DAGO. Under these conditions, binding of [‘H]EKC was readily displaced by U50488H, but morphine had a very low affinity, only producing significant displacement at concentrations greater than 1 PM (Fig. 4A and B). At concentrations up to 20pM, DSLET did not produce significant displacement of [3H]EKC from this binding site. Studies with enantiomers of WIN 44,441 clearly showed the stereoselectivity of the binding site. The (-)enantiomer, WIN 44441-3, a potent analgesic antagonist (Michne, Lewis, Michalec, Pierson, Gillan, Paterson, Robson and Kosterlitz, 1978), with high affinity at several types of opioid binding site (Wood, Pilapil, Thakur and Richard, 1984), and the (+)enantiomer, WIN 44,441-2 and nalbuphine HCI were used to displace 2 nM [‘H]EKC. The enantiomers WIN 44441-3 had a K, of 0.7 nM while WIN 44441-2 had a K, of 1.9 p M. In the absence of blockade by DAGO of p-binding of [3H]EKC, nalbuphine gave an IC,, of lOOnM, but when the binding of [3H]EKC was restricted to ICsites in the presence of DAGO, the I& for nalbuphine was 400 nM (K,, 118 nM). The potencies of ligands in displacing the binding of [3H]EKC at the K site are summarized in Table 1. The log. logit slopes (pseudo Hill plot) for the displacing ligands at the K site were less than unity, varying from about 0.5 for U50488H and WIN 44441-3 to 0.9 for nalbuphine. Characterization of specific binding of [-‘H]DADLE

Bound (PM)

Fig. 3. Analysis of high affinity saturable binding of [3H]EKC, 0.1-50 nM, by spinal cord membranes of the guinea-pig. The results of single experiments, in which the specific binding of [)H]EKC was measured in the absence of any displacing ligand (A, triangles), in the presence of 1 PM DAGO (B, circles) and in the presence of 1 PM U50488H (C, squares) are represented as Scatchard plots. Non-specific bindine determined in the nresence of I PM EKC, has been subtracted. Repeat experiments yielded similar results. The dashed lines indicate the properties of the binding sites that best fitted the experimental data, determined by non-linear curve-fitting (LIGAND; Munson and Rodbard, 1980). (A) A two-site model fitted the experimental data best with K,, of 0.6 nM, B_, of 42 fmol/mg protein, K,, of 44 nM and Bmnx2of 153 fmol/mg protein. In the presence of 1 FM DAGO (B), a two-site model also provided the best fit, with K,, of 0.8 nM, B,,, of 31 fmol/mg protein, K,, of 350 mM, Bmar2of 660 fmol/mg protein. In the presence of 1 PM U50488H, the binding was best represented by a single-site model, with K, of 12 nM, E,,,,, of 90 fmol/mg protein.

low affinity site parameters). Finally, when a 1 PM U50488H block was used in the [3H]EKC saturation assay, Scatchard transformation yielded a linear plot and computer modelling was only able to fit a one-site model with a K, of 12nM and a B,,,,, of

90fmol/mg protein (Fig. 3C). Additional displacement studies were performed to analyze ligand binding selectivity at the high affinity site in membranes from the spinal cord of the guinea pig labelled with [3H]EKC in the presence of 1 PM

Specific binding was found to be saturable when suspension of membrane was incubated with increasing concentrations of [3H]DADLE from 0.3 to 30nM. At greater concentrations of [3H]DADLE, non-specific binding was 70-80% of total binding. A linear Scatchard plot was obtained from transformation of saturation curves (Fig. 5). Computer analysis of the saturation curves for [3H]DADLE indicated that a one-site model, with a Kn of 8.0 nM and a B,,,,, of 28 fmol/mg protein provided the best fit of the data. Previous work has shown that [3H]DADLE discriminates poorly between p and 6 opioid receptors (Gillan and Kosterlitz, 1982; James and Goldstein, 1984; Werling et al., 1985) and labels 6 receptors with only a 2-5-fold preference over p receptors. Displacement of 5 nM [3H]DADLE with DSLET was determined in order to identify the binding of [)H]DADLE to p and 6 subtypes (Fig. 6). The specific binding of [3H]DADLE was displaced by

DSLET with an IC,, of 15 nM and displacement of [3H]DADLE was complete within a concentration range covering two orders of magnitude (Fig. 6). Displacement of 5 nM [3H]DADLE with DAGO showed a high affinity displacement with an IC,, of less than 5 nM producing about 60% inhibition of control binding at 10 nM DAGO, but DAGO was unable to displace the remaining binding of [3H]DADLE except at concentrations exceeding 1 PM (Fig. 6). Based on these experiments, a concentration of 10nM DAGO was selected to block the

G. D. ZARR et al.

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Fig. 4. Displa~ment of specific binding of [3H]EKC, 2 nM, in the absence (A) or presence (3) of I PM DAGO to block p-type binding sites, in spinal cord membranes of the guinea pig by increasing concentrations of displacing ligands. Results from single experiments are shown. Repeat experiments yielded similar results. (0) U50488H; (0) morphine; (A) DSLET.

binding of 13H]DADLE to p sites and allow binding studies of isolated 6 sites. Saturation analysis of OS-25 nM [‘HJDADLE binding was repeated in the presence of 10 nM DAGO (Fig. 5), yielding a linear plot. A one-site model provided the best fit to the experimental observations (K, of 1.9 nM and BmaXof &Ofmol/mg protein). Blocking p sites with 10 nM DAGO resulted in an approx. Cfold increase in the affinity of [‘HIDADLE for the remaining sites and an 80% reduction in the number of receptor sites. Displacement studies of 5 nM [‘HIDADLE binding in the presence of a 10 nM DAGO block were not possible due to the very small amount of residual s~cifically bound labelled ligand.

2.5

DISCUSSION

The results presented above confirm earlier reports that specific opioid binding sites can be demonstrated in spinal cord membranes by the use of conventional radiolabelled ligand binding techniques. Even when binding is measured in the presence of sodium and other cations, suf3icient specific binding was observed with the ligands employed in this study for reliable quantification of the parameters of the high affinity binding site to be made. High affinity binding sites with characteristics similar to p receptors have been examined with 13H]DAG0. These sites had a K, of 8 nM, a value very similar to that observed under the same experimental conditions in membranes from the cerebral cortex of the guinea-pig (Werling et al., 1985). It was found that [3H]DAG0 was readily displaced from the binding sites in spinal cord by morphine, nalbuphine

I

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Fig. 5. Analysis of the specific binding of rH]DADLE, 0.5-25nM, by spinal cord membranes of the guinea pig. Specific binding is represented as a Scatchard plot. The results of four inde~ndent experiments, in the absence of any competing iigand (open and solid circles) and in the presence of 1OnM DAGO to block p-type binding sites (open and solid triangles), are show. The lines indicate the best fit (LIGAND, Munson and Rodbard, 1980) to the pooled experimental data in each case. In the absence of DAGO, Ko was 8 nM, B,, 28 fmol/mg protein; in the presence of DAGO; K, was 2 nM, B_ 6 fmol/mg protein.

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Fig. 6. Displacement of specific binding of [‘HIDADLE, 5nM, by increasing concentration of DAGO (m) and DSLET (A). Results are the mean values from two independent experiments with each ligand.

Opioid binding in spinal cord

and WIN 44,441-3, but was relatively resistant to displacement by DSLET, a modestly selective ligand for 6 type binding sites (Werling et al., 1985) or by U50488H, a very selective ligand for K binding sites (James and Goldstein, 1984; Werling et al., 1985). Binding at g-type sites comprised about 30% of the total binding at p, S and K sites in membranes from the guinea pig spinal cord. High affinity specific binding of [-‘HIDAGO was observed in homogenates of the lumbo-sacral region of the spinal cord of the guinea pig, in addition to homogenates of whole cord or homogenates of the cervical-thoracic region of the cord. The affinity of the [‘HIDAGO binding site, the density of sites relative to tissue protein, and sensitivity to displacement by unlabelled ligands, were similar in the lumbo-sacral and cervical-thoracic regions of the spinal cord of the guinea-pig. These results thus confirm the previous reports of Traynor et al. (1982), Czlonkowski et al. (1983) and Mack et al. (1984) that p opioid receptors are present in lumbo-sacral spinal cord of the guinea-pig, and they are inconsistent with the reported failure of Attali et al. (1982) and Gouarderes et al. (1982) to detect of [3H]dihydromorphine or specific binding [‘HIDADLE, both ligands with good affinity for p-type opioid binding sites, in the lumbo-sacral cord of rats and guinea-pigs. The reasons for the discrepancy are not clear, but may be related to the high level of non-specific binding relative to the total binding of these ligands in spinal cord tissue. Some features of the studies of binding of the [)H]DAGO in the spinal cord of the guinea-pig suggest that the binding sites for this ligand might not be homogeneous. When small concentrations of [3H]DAG0 were used, displacement curves obtained with unlabelled morphine and DSLET showed an apparent shoulder at around l&25% displacement and the slopes of log. logit displacement plots for most ligands were significantly less than unity. It has been suggested that in receptor systems coupled to adenylate cyclase, agonists show low slopes in displacement curves because of the existence of multiple affinity states of the receptor, regulated by guanine nucleotides, while predicted displacement slopes are observed with antagonists, and intermediate slopes with partial agonists (Tsai and Lefkowitz, 1979; Childers and Snyder, 1980). However, in the present studies the partial agonist, nalbuphine had a displacement slope as small as that of the full agonists morphine and DAGO, in the range 0.5-0.7. Small slopes for the displacement curve were not observed in studies of the binding of the [3H]DAG0 in homogenates of the cortex of the guinea pig under similar conditions (Werling et al., 1985). Thus, the alternative explanation, that there is heterogeneity in the binding sites for [3H]DAG0 in spinal cord unrelated to the regulation by guanine nucleotide appears more likely. However, the experimental data did not fit better to a two-site model than a one-site model by computer analysis of the binding of

477

rH]DAGO in homogenates of spinal cord, either when analyzed as separate experiments or when the data from several experiments were pooled. It is possible that the fairly high variance of binding estimates in this tissue, which derives in part from the high level of non-specific ligand binding, precludes the reliable detection in membranes from spinal cord of a low capacity site for [3H]DAG0 with very high affinity for some ligands. The possibility that p-type opioid receptors are heterogeneous in some tissues has been raised by Schulz and Wuster (1981) and Pastemak, 1982). Wolozin and Pastemak (1981) and Lutz, Cruciani, Costa, Munson and Rodbard (1984) have proposed that there is a low capacity site, designated p1, with a high affinity not only for conventional p ligands, but also for some peptide analogues of enkephalin such as DADLE. Thus, the present observation that DSLET might also have a high affinity for a postulated very high affinity for binding site DAGO would be compatible with this hypothesis. The proposed p, site has an estimated K, for DAGO of 0.14.3 nM (Lutz et al.,1984). Accurate quantitative analysis of this site therefore requires small concentrations of very high specific activity [3H]DAG0 and also an adequately large binding site concentration. In spinal cord membranes of the guinea pig, the amount of specifically-bound radioactivity at concentrations of [‘HIDAGO of less than 1 nM was too small for reliable estimates of very high affinity binding site parameters to be made, either in displacement or saturation binding experiments. Full characterization of this site in spinal cord will require the availability of a selective ligand with very specific radioactivity. Since no evidence for the heterogeneity of the [3H]DAG0 binding site was apparent in earlier studies in the cortex of the guinea pig (Werling et al., 1985), the present results suggest that the postulated very high affinity for binding site [‘HIDAGO is present in a greater concentration, compared to the remaining p-type binding sites, in spinal cord, than in cerebral cortex. Saturation analysis of specific binding of [‘HIDADLE suggested that there was a single class of sites for this ligand, but displacement studies with DAGO revealed that about 30-40% of binding sites for [3H]DADLE at 2 nM were very resistant to displacement by this p-selective ligand. In contrast, DSLET produced complete displacement of specific binding to [3H]DADLE over a relatively narrow concentration range. These results indicate that most of the specific binding of [‘HIDADLE in the homogenates of spinal cord in the guinea pig was to sites with the characteristics of p binding site, but about 20% of the binding of this ligand was to sites that were very sensitive to DSLET but not to DAGO. At these sites, the K, for [3H]DADLE was about 2 nM, a value very similar to the K, observed for this ligand at d-sites in the cerebral cortex of the guinea-pig (Werling et al., 1985). These sites thus have the characteristics of d-tvne binding sites observed in

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other tissues (Gillan and Kosterlitz, 1982; James and Goldstein, 1984; Werling et nl., 1985). The very low density of the 6 binding sites in the spinal cord of the guinea-pig and the fairly high non-specific binding of [3H]DADLE in this tissue precluded a quantitative characterization of the displacement potencies of several ligands at this site. Binding sites of the d-type comprised only about 10% of the total of p, 6 and IC binding sites in this tissue. Analysis of the specific binding of [3H]EKC in the spinal cord of the guinea pig indicated that the data was best represented by a model with more than one class of binding sites. A component of this binding was readily displaced by U50488H, a selective K ligand (James and Goldstein, 1984), and WIN 44441-3 [the (-)enantiomer] but not by DAGO, WIN 44441-2 [the (+)enantiomer] or DSLET. This binding site could be studied by the use of small concentrations of [3H]EKC after binding sites with intermediate affinity were occluded with unlabelled DAGO. Under these conditions, almost all of the binding of t3H]EKC appeared to be at the high affinity site, although a residual very low affinity binding site which was insensitive to DAGO could be revealed by saturation binding analysis. The high affinity site had an affinity for [3H]EKC of around 1 nM, a value very similar to the affinity for EKC of the K binding sites in the cortex of the guinea-pig measured under similar conditions, and comprised about 60% of the total of p, 6 and K binding sites in guinea-pig spinal cord. Examination of the displacement of [3H]EKC from this high affinity binding site by unlabelled EKC, U50488H and WIN 44441-3 after blockade of p sites with DAGO revealed that the slopes of the log. logit displacement plots were significantly less than unity. In contrast, nalbuphine showed a displacement slope very close to unity, and in the cortex of the guineapig, U50488H also displaced [3H]EKC from K binding sites with a slope very close to unity (Werling et al., 1985). Thus, there appears to be a significant difference between the properties of high affinity binding sites for [)H]EKC in cortex and spinal cord. Heterogeneity of K opioid receptors has been proposed in other studies (Schulz and Wuster, 1981), and the present results might indicate that there is heterogeneity in the high affinity binding sites for EKC in the spinal cord of the guinea-pig, even though computer analysis of the binding of [3H]EKC to this high affinity site was best characterized by a single site model. In this connection, it is noteworthy that Traynor and Rance (1985) have very recently reported that displacement studies of high affinity binding of [3H]bremazocine to presumed K type binding sites in the lumbo-sacral spinal cord of the rat have revealed some anomalies, inconsistent with a single site model of binding. Possible explanations of the anomalies apparent in this and the present study include the occurrence in spinal cord of sub-types of the K opioid binding site, or the regulation of a

subfraction of a single population of K binding sites by a guanine nucleotide. Studies of the effects or GTP on the binding of EKC to K binding sites in homogenates of the guinea pig cortex under the same conditions as were employed here have demonstrated that GTP produces a modest but significant reduction in the maximum binding of EKC (Werling et al., 1984). The low affinity binding sites for [3H]EKC which are insensitive to concentrations of U50488H of less than 1 PM are also probably heterogeneous. Part of this low affinity binding is sensitive to displacement by DAGO, with a K, of about 6nM and thus probably represents EKC binding to p binding sites, as has been observed by many other groups (e.g. Gillan and Kosterlitz, 1982; James and Goldstein, 1984; Werling et al., 1985). However, the number of sites insensitive to 1 PM U50488H was too high to be accounted for entirely by p binding sites. It seems probable that there are additional classes of sites which bind [3H]EKC with relatively low affinities. These might include the benzomorphan/epsilon binding sites described by Chang, Blanchard and Cuatrecasas (1984) and perhaps sigma opioid binding sites (Zukin and Zukin, 1981). Since DSLET produced no inhibition of the binding of [‘H]EKC in the presence of DAGO, it seems probable that there was very little binding of EKC to 6 binding sites under the experimental conditions used. The present results therefore indicate that binding sites with characteristics similar to those of p, 6 and K binding sites observed in other tissues are also present in the spinal cord of the guinea-pig. However, displacement studies with selective synthetic opioids appear to indicate that the p and ~-types of binding site in the spinal cord of the guinea-pig might not be homogeneous, in contrast to previous studies in cerebral cortex of guinea-pig under similar conditions, where no evidence for heterogeneity of the p and K binding sites was obtained (Werling et al., 1985). Further studies are required to confirm that the apparent heterogeneity is not artifactual and to demonstrate its functional significance in relation to the pharmacological actions of the ligands exposing the apparent heterogeneity of the binding site. These studies confirm that DAGO and U50488H should be useful opioid agonists capable of discriminating between p and K binding sites in the spinal cord. However, the partial agonist, nalbuphine and the very potent opioid antagonist, WIN 44441-3 had significant affinity at both p, and K binding sites. Both showed approx. IO-fold preference for p sites under the experimental conditions used, an affinity difference which is not sufficient to make them useful discriminators between actions mediated at p and ~-type receptors. The present estimates of the properties of binding sites are derived from studies employing a modified Krebs medium since it is suggested that the heterogeneity of the binding site is best evaluated under conditions in which concentrations of critical ions

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opinions or assertions contained herein are the private ones approach those found in intact neurons. In the few of the authors and are not to be construed as official or cases where comparisons between the present estireflecting the view of the Department of Defense or the mates of affinities of ligands can be compared with Uniformed Services University of the Health Sciences. The published estimates of their pharmacological potenexperiments reported herein were conducted according to cies in actions mediated in the spinal cord, there is the principles set forth in the “Guide for the Care and Use of Laboratory Animals,” Institute of Laboratory Animal generally reasonable agreement between the in vitro Resources, National Research Council (DHEW Publication estimates of affinity and the in vivo estimates of No. [NIH]78-23, 1978). analgesic potency. Yaksh (1981) has published EC&, estimates of the analgesic potencies of several drugs after intrathecal administration in rats. In his experiREFERENCES ments, morphine had an EC,, of 13 nM and in the Attali B., Gouarderes C., Mazarquil H., Rudigier Y. and present studies morphine displaced the specific bindCros J. (1982) Evidence for multiple “kappa” binding ing of [3H]DAG0 with a K, of 15 nM; DADLE had sites by the use of opioid peptides in the guinea pig an analgesic EC,, of I1 nM and in the present studies lumbo-sacral spinal cord. Neuropeptides 3: 53-64. the K, for [3H]DADLE was 8 nM in the absence of Behar M., Olshwang D., Magora- F. and Davidson J. T. (1979) Epidural morphine in the treatment of pain. Lancef DAGO, when most of the binding of DADLE was i: 527. probably to p-type sites. Yaksh (1981) found the Chang K.-J., Blanchard S. G. and Cuatrecasas P. (1984) ECso for EKC was 53 nM. This value is in reasonable Benzomorphan sites are ligand recognition sites of putaaccord with the present authors’ estimate of an tive. I e-receptors. Molec. Pharmac. 26: 484488. Childers S. R. and Snyder S. H. (1980) Differential reguaffinity for EKC at p binding sites of about 40 nM. lation by guanine nucleotides of opiate agonist and The analgesic activity of EKC in spinal cord of the antagonist receptor interactions. J. Neurochem. 34: rat as measured by the tail flick method is very 583-593. probably mediated, at least in part, by /* receptors Cousins M. J., Mather L. E., Glynn C. J., Wilson P. R. and Graham J. R. (1979) Selective spinal analgesia. Lancet i: (Schmauss and Yaksh, 1984). In view of the sug1141-1142. . gestion by Schmauss and Yaksh that responses to Czlonkowski A., Costa T., Przewlocki R., Pasi A. and Herz some types of chemically-mediated pain are particuA. (1983) Opiate receptor binding sites in human spinal larly sensitive to inhibition by ICopioids, comparisons cord. Brain Rex 267: 392-396. of EC,, values obtained in this kind of test with David M., Moisand C., Meunier J-C., Morgat J-L., Gacel G. and Roques B. P. (1982) [3H]Tyr-o-Ser-Gly-Pheestimates of affinity to ICbinding site in spinal cord I_eu-Thr: a specific probe for the h-opiate receptor subwould be of particular interest, but such estimates are type in brain membranes. Eur. J. Pharmac. 78: 385-387. not at present available. Fields H. L., Emson P. C., Leigh B. K., Gilbert R. F. T. and These results suggest a remarkable similarity beIversen L. L. (1980) Multiple opiate receptor sites on primary afferent fibers. Nature 284: 351-353. tween estimates of analgesic potency after intrathecal administration and estimates of ligand affinity at p Gillan M. C. G. and Kosterlitz H. W. (1982) Spectrum of the p-, a-, and k-binding sites in homogenates of rat binding sites. However, the similarity should be brain. Br. J. Pharmac. 77: 461469. treated with caution. The estimates of opioid potency Gouarderes C., Audigier Y. and Cros J. (1982) Benzomorphan binding sites in rat lumbo-sacral spinal cord. are based on assumptions regarding the volume of Eur. J. Pharmac. 78: 483486. distribution of intrathecally-administered opioids James I. F. and Goldstein A. (1984) Site-directed alkylation which may not be correct. Secondly, some potential of multiple opiate receptors: 1. Binding selectivity. Molec. intracellular regulators of opioid binding were not Pharmac. 25: 337-342. included in the present assays. It is quite possible Lutz R. A., Cruciani R. A., Costa T., Munson P. and Rodbard D. (1984) A very high affinity opioid binding site therefore that the similarity in values is fortuitous. in rat brain: demonstration by computer modeling. BioThe main contribution of the present study is the them. biophys. Res. Commun. 122: 265-269. observation that the types of opioid binding site Mack K. J.. Killian A. and Weyhenmeyer J. A. (1984) initially demonstrated in buffers such as Tris-HCl Comparison of mu, delta, and kappa opiate binding sites (which do not support many cell functions and do not in rat brain and spinal cord. Life Sci. 34: 281-285. contain essential regulatory cations such as Nat and Magora T., Olshwang D., Eimerl D., Shorr J., Katzenelson R.. Cotev S. and Davidson J. T. (1980) Observations on Mg’+) can be demonstrated in homogenates of the extradural morphine a analgesia in various pain condispinal cord of the guinea pig in the presence of these tions. Br. J. Anaesth. 52: 247-252. ions. Further studies will be needed to clarify the Michne W. F.. Lewis T. R., Michalec J. T., Pierson A. K., Gilan M. G. C., Paterson S. J., Robson L. E. and complex relationship between in vitro estimates of Kosterlitz H. W. (1978) Novel developments of Nreceptor affinity and in vivo estimates of potency. narcotic antagonists. In: Characteristics and Functions of Opioids (Van Ree J. and Tere-

methylbenzomorphan

Acknowledgements-We

thank Dr Robert Lahti of The Upjohn Company for the gift of U50488H, Sterling Winthrop Laboratories for ethylketocyclazocine and WIN 44441 isomers, Dr Sheila Muldoon for nalbuphine HCI, and Dr P. J. Munson and Dr D. Rodbard for making the DEC-10 version of their non-linear curve fitting program LIGAND available to us. This work was supported by a grant from the National Institute for Drug Abuse. The

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