High affinity [3H]ethylketazocine binding: Evidence for specific κ receptors

Neuropharmacolooy Vol. 21, pp. 215 to 219, 1982

0028-3908/82/030215-05503.00/0 Pergamon Press Ltd

Printed in Great Britain

HIGH AFFINITY [3H]ETHYLKETAZOCINE BINDING: EVIDENCE FOR SPECIFIC x RECEPTORS P. L. WOOD* and S. CHARLE$ON Merck Frosst Laboratories, Department of Pharmacology, P.O. Box 1005, Pointe-Claire-Dorval, Quebec, Canada H9R 4P8 (Accepted 23 September 1981) Summary--A study has been made of the inactivation of /4mu) ([3H]-dihydromorphine),

6(delta) ([3H](D-ala2-D-leu~)enkephalin) and x(kappa) ([3H]ethylketazocine) opiate receptor binding sites by N-ethylmaleimide (NEM) and it was observed that in contrast to/~ and 6 sites, the x sites of rat brain membrane preparations were resistant to low concentrations of N-ethylmaleimide. Furthermore, this x site was selectively protected, from inactivation with high concentrations of N-ethylmaleimide, by the h agonists ethylketazocine and (-)-~t-(1R,5R,9R)-5,9-dimethyl-2-(L-tetrahydrofurfuryl)-2'-hydroxy-6,7benzomorphan (MR-2034) but not by morphine or (D-ala2-D-leuS)-enkephalin.These studies suggest that a unique x receptor is present in the rat CNS.

Early studies have clearly delineated the stereospecific actions of narcotic analgesic agents (Becket and Casy, 1954). These data laid the foundation for subsequent radioligand binding studies (Pert and Snyder, 1973; Simon, Hiller and Edelman, 1973) which indicated the presence of specific opiate receptors in the CNS. With the discovery of the enkephalins (Hughes, 1975), putative endogenous ligands for these receptors, the existence of multiple opiate receptors was suggested (Martin, Eades, Thompson, Huppler and Gilbert, 1976; Lord, Waterfield, Hughes and Kosterlitz, 1977). These proposed receptor subpopulations include #, 6, x and a (sigma) sites. The presence of distinct/~ and 6 receptors in the CNS has been clearly demonstrated by the selective protection by/~ and 6 agonists against the inactivation of their respective receptors by alkylating (Robson and Kosterlitz, 1979) and sulfhydryl reagents (Smith and Simon, 1980). However, despite an abundance of behavioral (Martin et al., 1976; Gilbert and Martin, 1976; Pickworth and Sharpe, 1979; Ward, Metcalf and Rees, 1978; Metcalf, Rees and Ward, 1979; Woods, Gly and Swain, 1978) and neurochemical (Wood and Stotland, 1980; Wood, Stotland, Richard and Rackham, 1980; Wood and Rackham, 1981) evidence suggesting that a separate x site is responsible for the CNS actions of the ketazocines and N-furylbenzomorphans, several binding studies (Hiller and Simon, 1979, 1980; Pasternak, 1980; Snyder and Goodman, 1980; Harris and Sethy, 1980) have not revealed a unique binding site. In contrast, several studies have described (Kosterlitz and Paterson, 1980; Wood, Charleson, Lane and Hudgin,

unpublished) a binding site for [3H]ethylketazocine which is insensitive to /~, 6, cr and E(epsilon) receptor agonists but sensitive to x, partial/~ and agonist/antagonist analgesics. Similarly, Roemer, Buscher, Hill, Maurer, Petcher, Welle, Bakel and Akkerman (1980) have reported a distinctive [3H]bremazocine binding site in the rodent CNS. In this report such studies are extended and evidence is presented for selective protection of [3H]ethylketazocine binding by the x agonists ethylketazocine and MR 2034 and for lack of protection by morphine or o-ala2-t)-leu-enkephalin (DADLE) in the case of receptor inactivation by N-ethylmaleimide. METHODS

Materials

Tritiated ligands were purchased from New England Nuclear Corporation (Boston, MA); [3H]dihydromorphine (DHM, 76 Ci/mmol); [3H]ethylketazocine (EKC, 15 Ci/mmol) and Amersham [-3H](Dala2-D-leuS)-enkephalin (DADLE, 52 Ci/mmol). Morphine sulfate (Allen & Hanburys), ethylketazocine (Sterling Winthrop), (-)-~-(1R,5R,9R)-5,9-dimethyl2-(L-tetrahydrofur furyl)-2'-hydr oxy-6,7-benzomorphan (MR-2034) (Dr Merz, Boehringer Ingelheim) and DADLE (Peninsula) were dissolved in distilled water. N-Ethylmaleimide (NEM) and reduced glutathione were purchased from Sigma.

* To whom correspondence should be addressed at: Douglas Hospital Research Centre, 6875 Boul LaSalle Verdun, Quebec H4H 1R3. Key words: x receptor, ethylketazocine, MR-2034, selective protection. 215

Procedure

Membrane preparations (rat whole brain minus cerebellum) and subsequent opiate binding assays were performed as reported elsewhere (Wood et al., unpublished). Briefly, the brain mince was homogenized with a Polytron PT-10 at a setting of 4 for 30 sec and subsequently diluted 100-fold with cold buffer. This buffer was 0.05 M Tris-HC1 (pH 7.7) for the

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P.L. WOODand S. CHARLESON

[aH]dihydromorphine and [aH]DADLE binding assays and 0.05 M K2HPO,~" HCI (pH 7.4) + 1 mM E D T A ' K 2 for the [aH]ethylketazocine assay. The diluted homogenates were centrifuged at 49,000 g for 15 rain (4°C) and the pellets homogenized again in buffer and diluted 100-fold. These suspensions were incubated for 40 min at 37°C after which the membranes were again isolated at 49,000 g. These final pellets were either stored frozen for further [3H]ethylketazocine binding assays or homogenized in buffer and diluted for [3H]DADLE or [3H]dihydromorphine binding. For incubations, 1 ml of brain membrane suspension (0.54).7mg protein) and radioactive label 75,000-100,000 cpm were made to a volume of 2 ml with buffer and the incubations carried to equilibrium which was 30 min at 4°C for [3H]ethylketazocine and 60rain at 25°C for [3H]DADLE and [3H]dihydromorphine. The assays were terminated by vacuum filtration (Whatman GF/B filters) with 2 washes of 5 ml cold buffer and the filters counted in 3 ml of biofluor (NEN Canada) after a 10-18 hr extraction period. Non-specific blanks were carried out in the presence of 10-SM cold ligand; hot ligands were 2 n M ([aH]EKC), 0.5nM ([aH]DHM) and l nM ([3H] DADLE). For the analysis of receptor inactivation by N-ethylmaleimide, the following protocols were adopted. (A) Inactivation time-course. The time course of receptor inactivation was followed with 0.5 and 5.0mM concentrations of N-ethylmaleimide. Membranes were incubated at 37°C with NEM for 0-60 min after which 2.5 or 25 mM GSH was added to the membranes which were then isolated by centrifugation (28,000 o, 15 min) at 4°C and washed twice with buffer. These treated membranes were then diluted to the required protein concentration and tested in the standard manner for [3H]ethylketazocine, [3H]dihydromorphine and [3H]DADLE binding. (B) Protection experiments. Membranes were incubated at 37°C for 10 rain with varying concentrations (5-5000 nM) of either cold MR 2034, ethylketazocine, morphine or DADLE after which 0.5mM ([~H]DHM and [3H]DADLE assays) or 5raM ([aH]EKC assay) N-ethylmaleimide was added for a further 12 and 15 min incubation at 37°C respectively. Next, 2.5 mM ([3H]DHM and [3H]DADLE assays) or 25 mM GSH ([3H]EKC assay) was added and the tubes centrifuged (28,000 0, 15 min) at 4°C. The pellets were resuspended in fresh buffer and incubated at 37°C for 5 min to dissociate any cold ligand. These membranes were again isolated and washed twice. The final membrane preparation was then diluted to the required protein concentration and used for estimation of [3H]dihydromorphine, [3H]DADLE or [aH]ethylketazocine binding. Control experiments with non-alkylated membranes established that the described washing steps were sufficient to remove all the reduced GSH and cold ligand and not interfere with subsequent 3H-ligand binding.

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Fig. 1. (A) Time-course of inactivation of opiate receptor binding by 0.5 mM N-ethylmaleimide (NEM) The tritiated ligands are ethylketazocine (EKC) (r), dihydromorphine (DHM) (/d and DADLE (~). (B) Receptor inactivation with 5.0 mM NEM.

RESULTS Receptor inactivation Initial studies with 0.5 mM N-ethylmaleimide (Fig. 1A) demonstrated that the [3H]ethylketazocine site was partially resistant to inactivation while the [3H]dihydromorphine and [aH]DADLE were rapidly destroyed by alkylation of sulfhydryl groups with N-ethylmaleimide. A 10-fold increase in the concentration of N-ethylmaleimide resulted in a more rapid rate of destruction of the ~ site but the degree of inactivation was less than that observed for the p and 6 sites.

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Fig. 2. Selective protection of I-3H]dihydromorphine ([3H]DHM) binding from 0.5mM N-ethylmaleimide (NEM) inactivation by morphine but not by DADLE, ethylketazocine or MR-2034. Inactivation time was 12 min. Results are the means of 2 experiments; control binding was approximately 2000 cpm/mg protein.

Fig, 4, Selective protection of [3H]ethylketazocine binding from 5 m M N-ethylmaleimide inactivation by ethy]ket~ocine and MR-2034 but not by morphine or D A D L E . Inactivation period was 15 min. Results are means N SE of 4 experiments; control binding was approximately 3 ~ cpmlmg protein,

Receptor protection

the rat brain. However, to control for different assay conditions with [3H]ethylketazocine binding, an experiment in Tris buffer at 25°C for 60 min was also performed. In this case, treatment with 5 mM of N-ethylamleimide resulted in a 65~o decrease in [3H]ethylketazocine binding. Using cold ligands for protection experiments the following data were obtained (~o of control binding): (a) morphine (10 nM, 36%; 1000 nM, 45%) and (b) MRo2034 (10nM, 410/o; 1000nM, 73~o). These data are in agreement with those in Figure 4.

Using inactivation of membrane binding sites by N-ethylmaleimide at 37°C, the/~ receptor site (Fig. 2) was selectively protected by morphine and the ~ site (Fig. 3) by DADLE, supporting previous observations (Smith and Simon, 1980; Robson and Kosterlitz, 1979). In addition, ethylketazocine, but not MR-2034, was found to protect the 6 site from inactivation by N-ethylmaleimide indicating some cross-reactivity of ethylketazocine with this receptor. In marked contrast, the /~ site (Fig. 2) was only protected by the y agonist morphine and the [3H]ethylketazocine site (Fig. 4) by the purported x agonists ethylketazocine and MR-2034. This marked pharmacological specificity cannot be explained by differences in the final binding assay conditions since the membrane inactivation and protection by cold ligand were all performed under identical conditions at 37°C. Therefore, the selective protection data presented in Figures 2-4 suggest the presence of unique #, ~ and x receptors in 70 60

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DISCUSSION

The selective protection experiments of Robson and Kosterlitz (1979) and those of Smith and Simon (1980) have convincingly demonstrated the presence of distinct p (alkaloid) and 6 (peptide) receptors in the CNS. The present data, using a similar experimental paradigm, further suggest that a ~ site, which has been labelled with [-3H]ethylketazocine (Kosterlitz and Paterson, 1980; Wood et al., unpublished), appears to be resistant to inactivation by low concentrations of N-ethylmaleimide. Furthermore, this site can be protected from N-ethylmaleimide inactivation (5.0mM) by the x agonists ethylketazocine and MR-2034, but not by the/~ agonist, morphine or the 6 agonist, DADLE. The specificity, of the site is further supported by the selective protection of [3H]dihydromorphine binding sites by morphine but not by ethylketazocine, MR-2034 or DADLE. These observations, as well as a wealth of behavioral data, do not therefore agree with the proposal that ethylketazocine-induced analgesia is dependent upon /~ receptors (Pasternak, 1980). While Roemer et al. (1980) have also described a unique binding site for the putative r¢ agonist bremazocine, several studies (Snyder and Goodman, 1980; Harris and Sethy, 1980) have not been able to detect a unique [3H]ethylketazocine binding site. Hiller and Simon (1979, 1980) also

218

P.L. Wooo and S. CHARLESON

deny the presence of a separate [3H]ethylketazocine binding site; however, they have reported a binding site 32 times more sensitive to ethylketazocine than morphine in displacing this binding. Similarly, ethylketazocine was 7 times more potent than leu-enkephalin in displacing [3H]ethylketazocine. These data, while not of the same magnitude, agree with previous reports of a [~H]ethylketazocine binding site sensitive to x, agonist/antagonist and partial ,u analgesics, but insensitive to 6, e, # and ¢r receptor agonists (Kosterlitz and Paterson, 1980; Wood, Charleson, Lane and Hudgin, 1982). Pasternak (1980) also has reported a [aH]ethylketazocine site with characteristics similar to [3H]morphine binding except for a much greater resistance to digestion by proteolytic enzymes. Therefore, while not in total agreement, these studies indicate one cannot exclude the possible existence of ~ receptors in the CNS. While it is clear from these protection experiments that the #, 6 and x receptor sites are unique separate entities, it also appears that the x agonist ethylketazocine, but not MR-2034, has significant 6 receptor affinity. While competition studies (Wood et al., 1982) also indicated that ethylketazocine and, MR-2034 inhibited [3H]dihydromorphine binding, no protection of the [-3H]dihydromorphine site from inactivation by Noethylmaleimide was noted with these agents. It therefore appears that protection against receptor inactivation by N-ethylmaleimide may be a useful in vitro test for examining the receptor specificity of opiate analgesics. The interaction between ethylketazocine and 6 receptors may also be of significance in vivo since depression of hippocampal acetylcholine turnover (TRAch) has been noted with ethylketazocine at 16 32 times the analgesic EDso (Wood and Rackham, 1981). This decrease in ACh turnover was naloxone-reversible and thought to be/~ or 6 receptor-mediated. The present receptor protection experiments would suggest that delta receptor activation may account for these actions of ethylketazocine in large doses. In summary, the present protection experiments demonstrate that a unique ~: receptor is present in the rodent C N S and they support other reports of ~ binding sites (Roemer et al., 1980; Kosterlitz and Paterson, 1980; Wood et al., unpublished). Furthermore, it appears that MR-2034 is a selective h- agonist while ethylketazocine may also interact with ,5 receptors at high dosage levels. Acknowledoements--The authors wish to thank Dr H.

Merz (Boehringer) for the supply of MR-2034 Winthrop Lab for the EKC.

and

REFERENCES

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Kappa receptors Woods, J. H., Gly, C. L. and Swain, H. E. (1978). Behavioral actions of some N-furyl benzomorphans and ketazocines in rhesus monkeys and mice. In: Characteristics and Function of Opioids (Van Ree J. M. and Terenius L. Eds), pp. 403-411. Elsevier/North Holland, Amsterdam.

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Wood, P. L., Stotland, L. M., Richard, J. W. and Rackham, A. (1980). Actions of mu, kappa, sigma, delta and agonist/antagonist opiates on striatal dopaminergic function. J. Pharmac. exp. Ther. 215: 697-703.