Pharmacological characterization of the binding of [ '~lbr ine in guinea-pig brain: evidence for multiplicity of the K-opioid receptors

MARIO TIBERI'

Departement & pharmacokogie, Universite' de Montre'al, C.P. 6628, Succ. A, Montrbad (Que'bec), Canada H3C 3J7 AND

JACQUES MAGNAN Centre de recherche, Hdpital Louis-H. LafQntaine, 7406 Hochekaga, Montrbal (Qutbec), Can& HIN 3M5

Received October 6, 1988

TIBERI, M., and MAGNAN , J . 1989 Pharmacological characterization of the binding of [ 3 ~ ] bremazocine in guinea-pig brain: evidence for multiplicity of the K-opioid receptors. Can. J. Physiol. Phmacol. 67: 1336- 1344.

In guinea-pig brain, [3~]bremazocine has a binding capacity of 27.2 pmoVg wet tissue, which is statistically different from that of [3~]ethylketazocine (14.7 pmoyg wet tissue) or the sum of the individual binding capacities of p-, 8-, and K-selective ligands (15.0 pmollg wet tissue). Saturation studies of [3~]bremazocine performed in the presence of unlabelled p-, 8-, and K-blockers still reveal a homogeneous population of binding sites. [3~]~remazocine under suppressed conditions displays at these sites a Kd of 2.5 1 nh.I with a binding capacity of 9.15 pmoVg wet tissue. We have performed the pharmacological characterization of these additional opioid binding sites. Displacement curves measured with a number of opioid substances were all best fitted to a one-site model. The stereoselectivity of these additional sites was demonstrated by using two groups of stereoisomers. Oripavine and benzomorphan opioids were among the most potent drugs at the [3~ ]b remoc ine sites ( p + 8 + K suppressed). Diprenorphine, bremazocine, cyclazocine, and ethylketazocine displayed apparent affinities constants (I/&) of 8.66, 7.57, 21.4, and 38.0 nM, respectively at those sites. The K-selective drugs U5Q488, U69593, PD117302, and tifluadom were inhibitors of the binding of [3~]bremazocine at these sites with apparent affinities of 1 13, 268, 76.9, and 47.9 d. All p- or 8-selective h g s tested in this study have caused weak or no inhibition of the binding. Correlation analyses were done between the different affinities measured at the [3~]bremazocine sites (p + 8 + K suppressed) and those observed at the known p-, 8-, and K-sites of the guinea-pig brain. The results were highly and significantly correlated with the K-binding profile, while no correlation could be established with the p- or 8-binding profiles. Moreover, the results reported here do not correspond to a pharmacological profile that belongs to the a- or e-receptors. Thus, it is likely that in the guinea-pig brain the [3~]bremazocine sites (p + 8 + K suppressed) are a subtype of K-site family, namely K~-sites. Finally, apparent affinities displayed at those sites are correlated with the biological activity of opioids in the guinea-pig ileum and rat vas deferens, suggesting that those sites could be considered as receptors.

Key words: opioid receptors, kappa subtype, guinea-pig brain.

TIBEKI, M., et MAGNAN, J. 1989. Pharmacological characterization of the binding of [3~]bremazocine in guinea-pig brain: evidence for multiplicity of the K-opioid receptors. Can. J. Physiol. Phmacol. 67 : 1336- 1344.

Dans le cerveau de cobaye, la capacitk de liaison de la [3~]brkmazocine (27-2 pmoVg tissu frais) est statistiquement diffkrente de celle mesurCe avec 1'[3~]kthylkktazocine (14,7 pmoUg tissu frais) ou de celle obtenue par la somation des capacitks de liaison des diffkrents ligands p , 8 et K sklectifs (15,O pmoVg eissu frais). Des Ctudes de saturation utilisant la [3~]bh$mazocine en prksence de bloqueurs sClectifs pour les rkcepteurs p , 8 et K a permis de dCteminer l'existence d'une population homoghe de sites de liaison aux opio'ides. Dans ces conditions ex@rimentales, la [3~]bn5mazwine poss&de A ces sites additionels une valeur de Kd de 2,5 1 nh.I et une capacitC de liaison de 9.15 pmollg tissu frais. Nous avons effectuk la caractkrisation phmacologique des ces sites de liaison opioides additionnels. Ha meilleur ajustement des courbes de com#tition obtenues 21 l'aide des diverses substances opioides correspondait dans tous les cas h un msd&le A un site. La stCrkosClectivit6 de ces sites de lidson a CtC d6mont.de grace A l'utilisation de stCrCoisom&res. Les opio'ides de type oripavine ou benzomorphane ont kt6 pami les composCs les plus puissants aux sites marquks par la [3~]brkmazocine ( p + 8 + K bloquts). La diprenorphine, la brkmazocine, la cyelazocine et 1'Cthykktazocine possCdaient B ees sites de liaison des valeurs de constante apparente d'affinitk (lIK,) de 8,66,7,59, 21,4 et 38,O nh.I, respectivement. Les compsQ K-sklectifs U50488, U69593, PD117302 et tifluadom ont tous CtC des inhibiteurs de la liaison de la [3~]brCmazwine B ces sites avec des affinitks de 113,268,76,9 et 47,9 d. Tous les composks p- et 8-sklectifs testks dans eette Ctude ont causk des inhibitions faibles ou inexistantes de la liaison. Des analyses de corrklation ont CtC effecmeks entre les diffkrentes affinitks Ctablies aux sites marquCs par la [3~]brCmazocine ( p + 8 + K bloquks) et celles mesureks aux sites p , 8 et K du cerveau de cobaye. Ces rCsultats ont dCmontrC une corrklation significative avec le profil de liaison des sites K, dors qu'aueune corrklation n'exiseait avec les profils de liaison des sites p ou 8. De plus, les rksultats sugg8rent que le profil phmacologique n9est pas Equivalent h celui qui a Cd dkmontrk pour les rkcepteurs a ou e. Constquement, dans le cerveau de cobaye les sites marquks par la [ ' ~ ] b ~ m z o c i n e (p + 8 + K bloquks) correspondent A un sous-type des sites K

nomdment les sites ~ 2 . Finalement, les affinitks des comgosks mesurkes a ces sites KZ sont en corrklation avec I'activitC biologique des opioi'des dans l'ilkon de eobaye et le vas dkfkrens de rat, sugg8rant que ces sites peuvent &re considkrks c o m e des rkceptews.

Introduction Pen2, D-Pens-eaakephalin (BPDP) has permitted direct binding

The characterization of the p-, 8-, and K-opioid binding sites studies to be perfoked (Kosterlitz and kklterson 198 1 ; Cotton et

has been done using two different approaches. For the and id. 1985). Until the recent synthesis sf kl selective radioligand

8-sites, the availability of relatively selective probes such as for the K-sites (Lahti et al. 19859, indirect studies, in which

[ ~ H I D - A ~ ~ ~ , Mephe4, ~ l ~ - ~ l s - ~ & ~ ~ h ~ l i ~ (DAGO), and ~ 3 ~ 1 ~ - nonselective ligands such as brernazocine and ethylketazocine were used in the presence of unlabelled p- and 8-opisids (to

l~anthor to whom all copkespondenee should be addressed. effectively suppress their binding at these sites), were done to

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MAGNAN 1337

reveal the K-sites (Magnan et al. 1982; Robson et al. 1985). This methodological approach has led to the pharmacological char- acterization of the K-sites and has established their regional distribution in mammalian brain (Goodman and Snyder 1982; Magnan et al. 1982; Robson et al. 1985). However, the use of the nonselective opioid [%]bremazocine has led to conflicting reports about the total number of sites discriminated by this ligand.

For instance, Robson et al. (1985) have measured in the guinea-pig brain, with bremazocine , a total opioid binding capacity of 12.6 pmoYg of wet tissue, whereas Lahti et al. (1985) have obtained 25.0 pmol/g of wet tissue using the same tritiated ligand. These results suggest that [3~]bremazocine might discriminate additional opioid sites (Magnan and Tiberi 1989). However, as for the identity of these additional sites labelled by [3~]bremazocine, this is as yet unknown. Other opioid binding sites such a and E have been postulated to exist and could account for these additional sites discriminated by [3~]bremazocine (Wiister et al. 1979; Z&in and Zukin 1979). Those sites could be also equivalent to other sites that have been described using [3~]diprenorphine or naloxone in rat brain but which were not fully characterized (Grevel and Sadde 1983; Morris and Herz 1986). Furthermore, the additional number of sites probed by [3~]bremazocine might be in accordance with previous studies that have postulated the existence of subtypes of p-, 8-, or K-binding sites in several biological preparations (Wolozin and Pasternak 1981; Attali et al. 1982; Castanas et al. 1984; Rothman et al. 1984; Pasternak 1988; Zukin et al. 1988).

The aim of the present study is to establish the pharmacologi- cal charactefistics, in the guinea-pig brain, of the additional sites labelled by [3~]bremazocine using a series of opioid com- pounds known to be )a-, ti-, or K-selective and also nonselective opioid substances. We also will establish a correlation between the various affinities of the compounds under study at these sites and their respective affinities previously reported at p-, 8-, and K-binding sites in the guinea-pig brain. Finally, correlation analyses will be performed with affinities of the compounds measured at the additional sites labelled by [3~]bremazocine and their biological effects in different isolated preparations to establish the physiological relevancy of these sites. The results will be discussed with regard to the multiplicity of opioid receptors.

Materials and methods Drugs and peptides

(-)Bremazocine hydrochloride (9,9-dimethyl-5-ethyl-2-(I-hy- droxy)-cyclopropylmethyl-2'-hydroxy-6,7-benzomorphan) and tiflua- dom base (l-methyl-2-(3-thienylcarbonyl)-minomethyl-5-(2-fluoro- pheny1)-H-2,3-dihydro-1 ,4-benzodiazepine) were provided by Dr. D. Romer (Sandoz, Basel, Switzerland). (-)Cyclazocine (a-5,9- dimethyl- 2 -cyclopropylmethyl- 2' - hydroxy - 6,7 -benzomorphan) and (-)ethyllcet.zocine methanesulfonate (a-9-methyl-8-0x0-2-cyclopro- pylmethyl-5-ethyl-2'-hydroxy-6,7-benzomorphan) were given by Dr. W. Michne (Sterling-Winthp, Rensselaer, NY). U56488 (tram- 3,4-dichloro-N-methyl-N-(2-(1 -pyrrolidinyl)cyclohexyl)benzeneacet- amide) was provided by Dr. P. F. Von Voigtlander (UpJohn Company, Kalamazoo, MI). U69593 ((5a9 7a, 88)-(-)-N-methyl-N-(7-(1 -pyr- rolidiny1)- l -oxaspiro(4,5)dec-8-y1)bnzeneacetamide) was purchased from Amersham, Oakville, Ont . ( 9 ) and ( -)SKFlMM7 hydrochloride (a-5,9-dimethyl-2-ally1-2'-hydroxy-6,7-benzomorphan) were donated by Dr. R. Quirion (Douglas Hospital, Verdun, Qu6). PD117382 hy- drochloride (trans- N-methyl-N- [2-( l-pyrrolidinyl)-cyclohexyl~nzo- [b] thiophene-4-acetamide) was given by Dr. J . C . Hunter (Parke-Davis Research Unit, England). LevorphanoI tartrate, dextrophan tartrate,

and levallorphan tartrate were obtained from Hoffmann-LaRoche Ltd., Etobicoke, Ont. Diprenorphine hydrochloride was from Reckitt & Colmm, Hull, Yorkshire, U.K. Morphine sulfate was obtained from BDH Pharmaceuticds, Toronto, Ont. D - ~ l a ~ , D-LeuS-enkephalin (DADL), la^, ~ e ~ h e ~ , ~ l ~ - o l ~ e n k e ~ h a l i n (DAGO), B-pen2, D-pen5-edephalin (DPDP), D-~e?, ~eu ' , TI@-enkephalin (DSTLE), and morphiceptin were purchased from Institut h a n d Frappier Bio- chemicals (Laval, Qu6 .) BC30 1 6 (1 4-P-methyl-8-oxacyclorphan) was provided by Dr. B. Belleau (Institut h a n d Frappier Biochemicals, Laval, Qu6). Naloxone hydrochloride and naltrexone hydrochloride were purchased from Sigma (St. Louis, MO).

Labelled Iigands [ 3H ]~ -~ la2 , ~ e P h e ~ , Gly-o15-enkephalin ( [ 3 ~ ] ~ ~ ~ O ; 30 Ci/mmol;

1 Ci = 37 GBq), [ 3 ~ ] ~ ~ e n 2 , D-pen5-enkephalin ( [ 3 ~ ] B ~ ~ ~ ; 54 Ci/mmol), [ 3 ~ ] ( - )ethyket.zocine (27 Ci/mmol) , and ~~H](-)bre- mazocine (2 1.3 C2mmol) were purchased from New England Nuclear (Boston, MA). [ 3 ~ ] ~ 6 9 5 9 3 (58 C2mmol) was obtained from Amer- sham, Oakville, Ont.

Binding m s q s Whole brain homogenates from male Hartley guinea pigs (350-

5QO g) were prepared as described previously (Magnan et al. 1982; Tiberi et al. 1988). The incubations were performed at 25°C for 48 min in a final volume containing 1.9 mE of membranes (10- 19 mg original wet weight) in Tris buffer (50 d; pH 7.4 at 25°C). Bound and free radioactivity were separated by vacuum filtration followed by three washes of the glass-fiber filters (Whatman GF/B) that had been presoaked in 0.3% polyethylenimine for at least 3 h. The filters were then immersed in 10 ~-IJ-. scintillation fluid (Universol Cocktail, ICN Biochemicals, Montrkal, Qu6.) and counted at 45-50% efficiency in a Rackbeta 1219 counter (LKB Instruments).

Saturation experiments were performed by incubating increasing concentrations (routinely 0.61-20 nM) of the various ligands with the cmde membranes. Meanwhile, competition experiments were done in the presence of increasing concentrations of unlabelled drugs against a constant concentration (=2 nM) of ~~Hlbremazocine in crude mem- branes containing 100 nM unlabelled DAGO, 100 nM unlabelled DADL, and 120 nM unlabelled U69593 for the selective blockade of the p-, 6-, and K-sites. Under such experimental conditions, specific binding ranged between 70 and 75%. Total and nonspecific binding were determined in the presence and absence of excess cold opioid. In the case of the selective ligands, 5 pM (-)cyclazocine was used to delineate nonspecific binding, since it has a chemical structure that differs from the structures of DAGO, DPDP, and U69593, and moreover, this opioid displays a high for p-, 6-, and K-binding sites (Magnan et d. 1982). For the benzomorphans E ~ H ] ethylketazo- cine and [3~]brema%scine, 2.5 pM diprenorphine was chosen for the determination of nonspecific binding of these two nonselective ligands. Biprenorphine does not have a bnzomorphan-like structure, and similar to (-)cyclazocine, it has high-affinity binding at p-, 6-, and K-opioid sites ( M a g m et al. 1982).

Data analysis The binding parameters were obtained using the computer-assisted

curve-fitting program EBDA~LIGANB (McPherson 1985; Biosoft, Else- vier). These calculations are done by a weighted nonlinear multiple regression based on the least squares method. First, the raw data are preprocessed by the EBDA program to give slope factors ("pseudo Hill coefficients") and estimates of the binding parameters. It also creates data files used for the final evaluation of the exact fitting model by the LIGAND program. For each model analysed (one site, two sites), LaGaND calculates a residual variance which is indicative of the difference between the theoretical fit and the experimental data obtained. A statistical analysis by a partial F-test is then performed with these residual variances to establish which one of the models best fits the data. Analysis of saturation curves of the radiolabelled drug by the LIGAND program gives the binding capacity (R) and the affinity constant (K,) expressed in molar-'. Thus, the relative equilibrium dis- sociation constant (Kd is obtained by taking the reciprocal of the Ka.

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11338 CAN. J . PHYSHOL. PRARMACQL. VOL. 67, 1989

As for the andysis sf displacement studies, LIGAND program evaluates the binding capacity and the affinity constant (expressed in M-') of the unlabelled drug at the sites probed by the radioligmd under study. The apparent affinity constant of the unlabelled drug is obtained with the reciprocal of Ka. It is worth noting that the apparent affinity constant is not equivalent to the inhibition constant determined by the equation of Cheng and Pmsoff (1973). Indeed, the affinity of the drug is estimated directly fiom the data.

Statistical treatment The total binding capacity of unsuppressed [3~]bremazocine and

unsuppressed [3~]ethylketazmine were compared pair-wise with the m e total opioid binding capacity (RF + R6 + RK) using the test of simple hypothesis. The c o m p ~ s o n of the binding capacity of unsuppressed [3~]brernazocine with those of unsuppressed [3~]ethyl- ketazocine was made using a Student's t-test for unpaired values.

The slope factors were compared with unity using the test of simple h pothesis to establish a possible heterogeneity of the sites labelled by Y [ Hlbremazocine under suppressed conditions. Moreover, we have cornpared the binding capacity value of ['W]bremazocine (under suppressed conditions) obtained by computer-assisted andysis of the saturation curves with those evaluated from displacement curves. To do so, we have made a Scheffk9s andysis of variance that permits all unplanned comparisons (a posteriori comparisons) among pairs of means. Prior to all those statistical tests, we have assessed the nonheterogemeity of the variances for each parameter group using a Bartlett9s test. The level of significance was established at 5%* All statistical tests and correlation analyses performed in the present study have been described elsewhere (Wimer 897 1 ; Sokal and Rohlf 1981).

Results Binding characteristics sf p -, 6-, and K -selective opisid

l iganh For each selective ligand used, the specific binding was best

fitted to a one-site model indicating that those ligands were interacting with a homogeneous class of binding sites. Figure 1 shows representative examples of Scatchard transfornations of tbe saturation curves obtained with p-, ti-, and K-selective opioid ligands. Hw homogenates of guinea-pig brain, the studies performed with ['HIDPDP indicate that this 8-selective opioid has an R value of 3.33 pmol/g wet tissue and its Kd value is 1 .%5 nM. The p-selective ['H]H)AGO binds to its receptors with a & value sf 1.3 1 nM. Its binding capacity is 7.22 pmoVg wet tissue. Finally [ ' ~ ] ~ 6 9 5 9 3 , a K-selective opioid, labels 4.47 pmoUg wet tissue and it has a Kd value of 1.21 nM at those sites. These binding characteristics are summarized in Table 1.

Binding characteristics of nonseBective spisid Bigandts The binding of ['~]eth~lketazocine is best represented by a

curvilinear Scatchard representation (Fig. 1). The two-site model used to evaluate the parameters of the specific binding shows a statistical improvement over the one-site model. Thus, [3~]etlaylketazocine has a & of 0.19 nlki for the high-affinity binding sites and 2.38 nM for the low-affinity binding sites. The R vdues are 5 -32 and 9.34 pmoug wet tissue for the high- and low-affinity binding sites, respectively. The overall labelling by ['~]etlaylketazocine accounts for a total 14.7 pmoVg wet tissue of spioid sites in guinea-pig brain (Table 2). This value is not statistically different from the total binding capacity obtained by adding together the individual binding capacities measured with the p-, 6-, and K-selective ligands (15.0 pmollg wet tissue; Table 1).

The specific binding of [3~]bremazocine was best fitted to a two-site model suggesting an interaction with heterogeneous classes of sites (Fig. 2b). [ '~]~rernazocine displays at the high-affiwity binding sites a Kd of 0.09 nM with an R value of

* EKC 0.360 O DACO 0 L869593 a DPDP

BOUND ( p ~ )

FIG. 1. Representative example of the Scatchard representations of the specific binding obtained in guinea-pig brain homogenates in saturation experiments with a p-selective ligand, [ 3 ~ ] ~ ~ ~ 0 (@); a 8-selective opioid [%]DPDP (A); a K-selective ligand, [ 3 ~ ] ~ 6 9 5 9 3 (0); and a nonselective Bigmd, [3~~ethylketazocine (+). For each of these liganads, specific binding represented 90-5896 of the total binding (not shown) over the range of concentrations used (routinely 0.02-20 M). Each point is the result of triplicate determinations.

TABLE 1. Binding characteristics of p-, 8-, and K-selective opioid ligands in the guinea-pig brain membrane preparations

Kd R Eigand n ( n w (pmollg tissue)

['H]DAGO 4 1.31 28.09 7.22zk0.66 (p-selective)

[ 3 ~ ] ~ ~ ~ ~ 3 1.2520.05 3.33k0.10 (6-selective)

[ ' ~ ] ~ 6 9 5 9 3 9 1.2120.03 4.47k0.20 (K-selective)

To ta lb ind ing=p+6+~= 15.0 -

NOTE: Values are given as the mean * SEM: n is the number of experiments. Saturation c w e s were obtaiwed with 15- 16 points each Qne in triplicate. K,, is the relative equilibrium dissociation constant, and R is the binding capacity.

I 1 .dB pmollg wet tissue, whereas at the low-affinity binding sites, the Kd was 0.90 nAM with an R value of 16.2 pmol/g wet tissue (Table 2). Thus, ['H]bremazocine labels a total of 27.2 pmollg wet tissue of opioid sites in guinea-pig brain, a value that is statistically different from the total binding capacity measured with ['Hlethylketazocine. The total R value for [3wbremazo- cine was also statistically different from the total binding capacity obtained by summing up the binding capacities of the selective ligands. To establish whether this difference in the binding capacities could be related to the use of diprenorpkine as the h g of choice for the determination of the nonspecific binding, we performed two sets of saturation experiments in which 5 p M (-)cyclazocirae (opioid benzomorphm) and 5 pha levallorphan (opioid morphinan) were selected for the delinea- tion of the nonspecific binding of ['~]bremazocine. Neither the use of cyclazocine or levallorphan modified the binding parameters obtained with ['~lbremazocine (data not shown).

To define the binding characteristics of a potential fourth class of opioid binding sites, we have used the same approach that was first described to characterize the K-sites with nonselec- tive ligmds such as ethylketazocine (Magnara et al. 1982). To do so, we used sufficient concentration (1W times their respective Kd) of unlabelled selective p,-, 6-, and K-opioids to suppress the binding of the nonselective ligand at these sites. Consequently,

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TIBEIPI AND MAGNAN

TABLE 2. Binding c.haracteristics of the nonselective opioid ligmds [3~]ethyl- ketazocine and [3~]bremazocine in guinea-pig brain membrane preparations

Kd R Ligand R (nM? (prnol/g tissue)

-

Two-site model [3H]Ethyketazocine

High affinity Low affinity

[3~]~remazocine High affinity Low affinity

Total Pi = 14.721.11*

I H .0+ 1.34 16.2k 1.69

Total Pi = 24.2k0.86

NOTE: Values are given as the mem + SBM; n is the number of experiments. Saturation curves were obtained with 15-17 pints each dome in triplicate. Kd is the relative equilibrium dissociation constant, sand R is the binding capacity.

" S u p s s i m of p,-, 8-, and K-binding was obtained by performing the saturation curves for [ "H ]b~mwine in the presence of 100 nM unlabelled DAGB, 100 nM unlabelled DADL, and 120 nM unlabelled U69593.

*Statistically different from unsuppressed [3H]bremazocine (p @ 00.05).

TOTAL

z 2 rsooo a2

SUPPRESSED

0 TOTAL p + 6 + ~ [Oe3'

FIG. 2. Representative specific saturation curve obtained in the guinea-pig brain homogenates with [3~]bremuocine. The TOTAL c w e represents specific binding obtained in the absence of unlabelled selective opioids (unsuppressed [3~]bremazocine) and the b + 6 + K

SUPPRESSED curve represents the specific binding obtained in the presence of B 00 nM unlabelled DAGB, 188 nM udabelled BABL, and 120 nM unlaklled U69593. FOP the TOTAL curve, specific binding represented 98-65% of the signal (not shown) over the range of concentrations used (0.0 1-20 d) . For the + 6 + K SUPPRESSED curve the specific signal ranged from 80 to 50%. Each point is the result of triplicate determinations. (b ) Scatchard representation of the same saturation curves (shown in a). The TOTAL curve (unsuppressed [ 3 ~ ] b ~ m m a i n e ) is k s t fitted to a two-site model while the F + ~ + K

S U W ~ S S B B c w e is best fitted to a one-site model.

we measured the binding of ['~]bremaaocine and [3~]ethylke- tazocine (as a control) in the presence of 100 nM DAGO (to suppress the psites), 100 nh4 DADL (to suppress the &-sites), and 120 nM U69593 (to suppress the K-sites). Under these conditions, the binding of [3~]ethyketazocine is almost totally suppressed with less than 10% of residual binding left, as compared with the unsuppressed binding. Furthermore, prelimi- nary experiments showed that under these blocking conditions, the binding of [ 3 ~ ] ~ A G 0 , [ 3 ~ ] ~ ~ ~ ~ , and [ 3 ~ ] ~ ~ 9 5 9 3 at a radioligand concentration equaling their respective Kd values was totally inhibited (data not shown). However, under these specific experimental conditions, [3~]bremazocine still hecog- nizes a homogeneous population of sites that was best fitted to a one-site model (Fig. 2b). The Kd value of [%]bremazocine at those residual sites was 2.5 1 nM and the maximum binding capacity was 9.15 pmollg wet tissue (Table 2). Adding this capacity to the capacities obtained with p,-, 8-, and K-selective ligands (15 .O pmollg wet tissue; Table 1) we find a total binding capacity of 24.2 pmol/g wet tissue, which is approximately 90% of the binding capacity measured with h3H]bremazocine under unsuppressed conditions (Table 2).

Phamacs~ogical profile of the binding sf f~]bremmcirae We performed a series of competition studies against the

binding of [3H]bremazocine under p- , ti-, and K-suppressed conditions. The results me summarized in Table 3. Re1imina-y processing of the displacement curves using the EBBA curve- fitting program (McPherson 1985) gives slope factors that are not statistically different from unity except for those obtained with naloxone and ndtrexone. However, using the LIGAND

curve-fitting program (McPherson 19$5), all displacement curves done in the present study were best fitted to a one-site model.

The stereoselectivity of these additional opioid binding sites was demonstrated using two groups sf stereoissmers with different chemical structures. The (-)opioid levorphanol (hy- Qpoxymorphinan) displays an appmnt flmity of 294 nh4 at those additional sites, whereas 8extrorpha.n has a BIK, vdue of 11 525 nM. (-)SKF10047 (benzomorphan) binds to those sites with an

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1340 CAN. J. PHYSIBL. PHAWMACOL. VOL. 67, 1989

TABLE 3. Pharmacological characteristics of unlabelled opioids and drugs at binding sites labelled by [3H]bremz~ine (k-, ti-, md K-sites

suppressed) in guinea-pig brain membranes - -

Unlabelled 1/Ka R drug (a) (~rnol/g tissue) Slope factor

-Peptides DAGO Morphiceptin 6 -Peptides DADE DSTLE DBDP

Msrphinans Levorphanol kxtrorphan Naloxone Nal trexone Levallorphan BC3016 Nalorphine Morphine

r = 0.99 p ( 0.05

nlx

1 / ~ a values at [ '~]brarnozocine sites (nM)

FIG. 3. Correlation analysis between l/K, values of a number of opioids at the sites labelled by [3~]bremazocine under suppressed conditions and Ki values at K-sites in the guinea-pig brain. All Ki values at K-sites me retrieved from Magnan et al. (1982) except for U69593 U50488 and tifluadsm (Tiberi et al. 1988). B R W , bremazocine; DIP, diprenorphe; BC, BC3016; CYC, cyclazocine; EKC, ethylketazo- cine; U50, U50488; U69, U69593; NW, nalorphine; NEX, naloxone; NTX, naltrexone; LAN, levallorphan; LOL, levorphanol; SKF, (-)SKF100$7; BD, PD117302; TW, tifluadom; MOR, morphine.

Oripavines cine under suppressed conditions. Conversely, all re-selective Diprenorphine 8.66iz0. lo .35 a 1420. drugs were able to displace the binding of ['~lbrernazocine Benzomsrphans under su~~ ressed conditions. Indeed. PD117302 was the most ( - ) ~ ~ ~ 1 8 8 4 7 (+)SMF10047 Bremazmine Cyclazocine Ethylke~ocine

Benzeneacetarnides U50488 U69593 PD 1 17302

Benaodiazepines Tifluadom 47.9k4.18 11.420.76 0.85k0.16

NOTE: Values are given as the mean + SEM of thee to six experiments. Competition curves were obtained with 10-15 points each done in triplicate. The displacement studies were performed with = 2 mM [3H]bremazcrcine (suppressed conditions). The binding specificity was approximately 78-758. l/Ka is apparent affinity and R is binding capacity. MI. m t determined.

*Statistically different from unity @ < 0.85).

apparent affinity of 45.4 nM, while (+)SKFlOW7 displays a lower affinity with a 1/K, value of 7227 nM.

Among the antagonists tested, diprenorphine and naltrexone were the most potent drugs with respective apparent affinities at those sites of 8.44 and 45.1 nM. The classical antagonist naloxone was the least potent with an apparent affinity of 240 nM. All benzomorphans used in the present study were good inhibitors of the binding of [3~]bremazocine (under suppressed conditions). Unlabelled bremazocine was the most potent with a 1/K, value of 7.57 nM, which was quite similar to the Kd value measured by saturation studies. Cyclazocine and ethylketazo- cine displayed lower affinity m compared with bremazocine i .e. , 2 1.4 and 38.0 nh/l. The apparent affinity of unlabelled ethyl- ketazocine might explain why we were unable to probe those sites with [3Hlethylketmo@ine using saturation studies, since at this concentration the level of nonspecific binding is very high. Levallorphan was the most potent morphinm with a 1/K, value of 28.9 PnM, whereas nalorphine and morphine displayed respec- tive apparent affinities of 190 and 4409 nM. All p- and 6-pep- tides were very weak inhibitors of the binding of [3H]bremazo-

potent b;:Heneacetamide tested with an apparent affinity of 76.9 nM. U50488 had a 1/K, value of 1 13 nM, while U69593 was the least potent with an apparent affinity of 268 nM. Tifluadom, a benzodiazepine shown to be more selective for re-opioid sites than for benzodiazepine receptors (Romer et al. 1982), gave a 1/K, value of 47.9 nM. Finally, the binding capacities obtained from the analysis of the competition curves were not statistically different from the binding capacity obtained with saturation studies.

Corrcjation analysis with binding assays We performed several correlation analyses to establish the

degree of relationship of the pharmacological profile of opioids at the [%]bremazocine binding sites ( p + 6 + K suppressed) in the guinea-pig brain with the binding profile of the same drugs at the well-known la-, 6-, and K-receptors described previously in the same preparation (Magmn et al. 1982; Cotton et al. 1985; Claxk et al. 1988). No significant correlation could be established with either a k- ( r = -0.85; n = 14) or a 6-phmacological profile ( r = -0.08; n = 14). However, the correlation analysis between the different apparent affinities at the [3~]bremazocine sites (under suppressed conditions) and the Ki values calculated at re-opioid binding sites has resulted in a significant correlation (Fig. 3). This might suggest that the opioid sites discriminated by ['~Ibremazocine under suppressed conditions could be linked to the K-receptor family.

Correlation with bislogica! assays To establish the possible physiological relevancy of the addi-

tional sites labelled by [3H]brernazocine, we made correlation analyses with several biological preparations that were pre- viously used to characterize opioid receptors. Those are the guinea-pig ileum (GPI) , mouse vas deferens (MVB) , and the rat vas deferens (RVB). No significant correlation could be demonstrated between the phiurraacological profile at the ['~lbremazocine sites and the agonist activity in the MVD ( r = 0.68; n = 9). However, apparent affinities displayed at the ['H]bremazocine sites were significantly correlated with the

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TIBERI AND MAGNAN 134 1

‘--+ IOQ identification (Leslie 1987). Firstly, those sites are saturable 5= e

(Fig. 2a) and specific binding for these have been demonstrated

5 with the use of thee chemically unrelated opioids. Secondly,

0 10 the radioligand chosen for the discrimination of opioid sites C .- must exhibit a high affinity and selectivity for those sites, i.e., m Q)

the radioligand must have a Kd lower than 10 nn/Z (Leslie 1987). 3 - 1 [3~]~remazocine under suppressed conditions displays a Kd Q v value of about 2 nM with 70% of specificity at this concentra- 0 a tion. Thirdly, the other criterion should be the stereoselectivity.

0.1 In fact, the binding of a radioligand to receptor sites has to be I 1 o I 00 1000 displaced by a (-)isomer in a dose range that is at least one order

3 1 /Ko values a t [ H]bremozocine sites (nM)

FIG. 4. Correlation analysis between 1 lK , values of a number of opioids at the sites labelled by [3~]bremazocine under suppressed conditions and ICS0 values for inhibition of the contractions in guinea-pig ileum (GPI). All ICm values are retrieved from Magnan et al. (1982) except for tifluadom (R6mer et al. 1982), U69593 (Corbett and Kosterlitz 1986), U50488, and PB 1 17302 (Clark et al. 1988). For abbreviations see legend of Fig. 3.

ntx

1/Ka values a t [ '~]bremozocine sites (nM)

FIG. 5. Correlation analysis between 1 IK, values of a number of ogioids at the sites labelled by [3~]bremmocine under suppressed conditions and K, values for antagonist activity in rat vas deferens (RVB). A%% K, values were retrieved from Magnan et al. (1982) except for tifluadorn and morphine (Carroll et al. 1988). For abbreviations see legend of Fig. 3.

agonist activity in the SPI (Fig. 4). Figure 5 depicts the correlation of the apparent affinities at the [3~]bremazocine sites with the antagonist activity in the RVD. The correlation analysis was statistically significant. Finally, looking at the antagonism produced in the GPI and MVD (Magnm et d . 1982), no correlation could be observed between the K, values measured in GPI (r = 0.15; n = 7) or in MVD (r = -0.81; n = 7) and the apparent affinities displayed at the [3~]brernazocine sites.

Discussion In the last few years, the availability of selective radioactive

probes for p- ,6-, and K-opioid sites has reinforced the dogmatic classification of the opioid receptors as being mostly of the p-, 8-, and K-types (Paterson et al. 1983). Meanwhile, other receptors such as E- and a-type have been postulated to belong also to the opioid receptor family (Wuster et al. 1979; Zukin and Zukin 1979). The results obtained in the present study suggest that ['~~bremazocine labels an additional class of opioid sites that is not recognized with high affinity by [3~]ethylketazocine. We firmly believe that this additional site should be considered as an opioid receptor because it fulfills all criteria for receptor

of -magnitude lower or higher than- the dose range of the corresponding (+)isomer. At these additional sites labelled by ~~~]brernazocine (under suppressed conditions), levorphanol ((-)isomer) is 40 times more potent than dextrorphan ((+)iso- mer), whereas (-)SKF10047 shows an affinity that is 160-fold higher than (+)SKFlW7. Finally, using correlation analyses, the physiological relevancy of these additional sites has been demonstrated, suggesting that those opioid sites could be considered as receptors.

On the basis of the phamacological profile described at the a- or phencyclidine (PCP) receptors in NCB-20 cells (Kushner et al . 1988) or in rat brain (Largent et al. 1987), the nature of the additional sites labelled by [3~]bremazocine cannot be accoun- ted for by the a- or PCP receptor type. First, in our study ( -)SKF10047 is more potent than (+)SKF10847, whereas in NCB-20 cells (f )SKF10847 is 20-fold more potent than (-)SKF10047 at the a-receptors, and these two drugs are equipotent at the PCP receptors. Second, bremazocine displays an affinity of 7.57 nM in our study, whereas its affinity is 120 nM at the a-sites and 5900 nM at the PCP sites. Moreover, the pharmacological profile of these additional sites does not agree with the one reported for e-receptors (Law et al. 1979; Houghten et al. 1984). DADL and morphine are relatively good inhibitors of the binding of [3H]P-endorphin at the E-receptors (Law et al. 1979; Houghten et al. 1984), whereas in the present study those drugs were weak inhibitors of the binding [3~]bremazocine. Additionally, all p- or &selective opioid peptides we tested against the binding of [3~~bremazocine showed very low affinity suggesting that those receptors do not exhibit a p- or 8-profile. The pharmacological profile of the ['HI bremazocine sites (under suppressed conditions) was significantly correlated to those observed with the known K-binding sites in the guinea- pig brain indicating a degree of association with the K-opioid receptor family. At the pment time, we c m o t ascertain whether these results reflect the existence of two affinity forms of the same K-receptor protein or are indicative of two independent K-receptor proteins that are encoded by two distinct genes. Modulation studies using guanine nucleotides or ions could help clarify this point. Attali et al. (1982) have been the first to suggest the existence of multiple K-binding sites. In the guinea- pig lumbosacral spinal cord, these authors have proposed two distinct K-receptor subtypes on the basis of their sensitivity to DADL: DADL-insensitive sites (rel-sites) and DADL-sensitive sites (K~-sites). The K-selective opioid U50488 displayed at these sites in the spinal cord a Ki value of about 1 p M (Souardkres and Cros 1984). Our binding profile does not correspond to the K*-sites reported in spinal cord, since the binding of [3~]bremazocine (under suppressed conditions) was insensitive to DADL and sensitive to U50488. However, when we compare the potencies of the different benzomorphans and selective drugs at the sites of the rat brain (Zukin et al. 19881, they are in good agreement with our results except for

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1 342 CAN. 1. PHYSIOL. PHAWWBACOL. VOL. 67, 1989

U69593. Zufin et al. (1988) have reported that U69593 has no affinity for the rat brain K~-sites, while it displays an affinity of 268 nM at the [3~]bremazocine sites in guinea-pig brain. If we do not consider U69593, there is a significant correlation between the two binding profiles (n = 6; a = 0.90). Although, we cannot explain the discrepancy a b u t U69593, we would like to propose that these additional sites labelled by ["lbrema- zocine are similar to the sites reported by Zukin et d. (1988). Nevertheless, Zukin et al. (1988) have claimed that no sites are detected in the guinea-pig brain; this could be explained by the fact that they have used [3~]ethylketazocine with r$-, ti-, and #-selective blockers to label their sites. Indeed, we have demonstrated that under similar experimental condi- tions, only a very low level of specific binding of 13H]ethylketa- zwine could be measured in the guinea-pig brain. This would make the discrimination of the ~2-sites very difficult in this tissue. We thus propose that the use of ['H]bremazocine under yl- ,6-, and K -suppressed conditions is the adequate experimen- tal approach to probe the sites in guinea-pig brain. There is some physiological evidence to support the existence of a multiplicity of K-sites (Wiister et al. 198 1 ; Iyengx et al. 1986). The physiological relevancy of the si sites in guinea-pig brain is supported by the correlation analyses made with the activity measured in several biological preparations. The appaxnt affinities measured at the sites correlate significantly with the agonism produced in the GPI, which contains both p- and K-receptors (Hutchinson et al. 1975; Chavkin and Goldstein 198 1). Meanwhile, no correlation could be established between those affinities at sites and the agonism in MVD, a tissue that contains predominantly 6- and preceptors (Lord et al. 1977). Those results strengthen the hypothesis that [3~]bremazocine sites (under suppressed conditions) are related to a K-receptor subtype rather than to a p- or &receptor subtype. Reincubation of the GPI with the potent irreversible p-antagonist P- funalagexamine causes a loss of popioid agonist activity without modifying the agonist potency of opioids interacting with the K-receptors in this preparation (Takemori et al. 1981; Ward et d. 1982; Huidobro-Toro et al. 1982). Using GPI pretreated with f&=funaltrexamine, agonist activity of diprenor- phine, bremzocine, U69593, U50488, and morphine has been measured at the remaining K-receptors (Corbett et al. 1985; Corbett and KosterHitz 1986; Traynor et al. 1987). Interestingly, the order of potency of these drugs is significantly correlated with the apparent affinities of those substances at the sites of the guinea-pig brain (n = 5; a = 0.99). Furthermore, the profile of activity of bremazocine, ethylketazocine, tifluadom, and U50488 in the rabbit vas deferens (a preparation containing only K-receptors) measured by Hayes and Kelly (1985) is not significantly correlated with the binding profile of these drugs at the sites despite the fact that the coefficient of correlation was high (n = 4; r = 8.97). This is explained by the few number of compounds tested in the rabbit vas deferens. Collectively, these data confirm the nature of the sites discriminated by [a~]bremazocine (under suppressed conditions) as being of the K-type.

Another striking feature is that the apparent affinities calcu- lated at the K~-receptors of the guinea-pig brain are also correlated (a = 0.78) with the antagonist activity in the rat vas deferens (Fig. 3). It has been postulated that p- and (or) E-

receptors could be discriminated in this particular tissue (WUster et al. 1979; Gillan et al. 198 1). As previously discussed above, the results obtained with [3~]bremazocine (under suppressed

conditions) do not suggest a relationship with either the binding profile of p- or e-receptors. Thus, it is likely that the significant correlation observed in WVD could not be explained by an inter- action with one of these two receptors. We propose that K-type receptors are located on this tissue, and this explains why we measured a significant correlation in the WVB. The proposal that opioids can be agonists or antagonists in different tissues while interacting with the same type of receptors is not new. It has been reported that diprenorphine, for instance, could be an agonist at K-receptors found in the guinea-pig ileum and also an antagonist at K-receptors located on the rabbit vas deferens (Traynor et al. 1987). A more exhaustive study is needed to fully ascertain which K-receptor subtype are located on these three preparations. Finally, taking the total signal measured with unsuppressed [3~]bremazocine as representing 100% of the opioid signal, we have thus the following proportions of receptors in the guinea-pig brain: p-receptors, 30%; 6.- receptors, 14%; sgl-receptors, 18%, and the K~-receptors, 34%. Interestingly, Lahti et al. (1985) using the K~-selective ligand ['H]U69593 has established a proportion of 13% for K,- recep- tors in the guinea-pig brain.

The present study is the first to present phmacological evidence for the existence of subtypes of K-receptors in guinea- pig brain namely KI- and K~-sites. The possibility of such multi- plicity for opioid receptors is not necessarily that farfetched. Indeed, the recent cloning of genes encoding for subtype (or sub-subtype) of different receptors such as in the adrenergic (a l , a 2 9 Pi, and 82) , m u s c ~ n i c (MI, M2, M3, M4, and M5), serotonergic (SPIT1,, 5HTI,), or dopaminergic systems (B2) suggest that the multiplicity of subtype (or sub-subtype) of receptors established by the phmacological method is more realistic than mythical (Bmard 1988, Bunzow et al. 1988; Cotecchia et al. 1988; Fargin et al. 1988; Julius et d. 1988; Lefkowitz and Caron 1988; Wegan et al. 1988).

Acknowledgements This work was supported by a grant from the Medical

Research Council of Canada (MA-81%). Mario Tiberi is the recipient of a studentship from the Fonds de la recherche en santC du QuCbec. We thank Chantal Bigras and Paul Payette for their technical assistance. The authors are also grateful for the generous gifts of the drugs mentioned in the Methods section.

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