Biochemical and pharmacological evidence for opioid receptor multiplicity in the central nervous system

Life Sciences, Vol. 33, Sup. I, 1983, pp. 167-173 Printed in the U.S.A.

Pergamon Press

BIOCHEMICAL AND PHARMACOLOGICAL EVIDENCE FOR OPIOID RECEPTOR MULTIPLICITY IN THE CENTRAL NERVOUS SYSTEM G.W. Pasternak*, A. R. G i n t z l e r * * , R.A. Houghten***, G.S.F. Ling*, R.R. Goodman*, K. Spiegel*, S. Nishimura*, N. Johnson*, L. D. Recht* *Laboratory of Neuro-Oncology Memorial Sloan-Kettering Cancer Center, and Departments of Neurology and Pharmacology Cornell University Medical College New York, NY 10021

**Departments of Biochemistry and Psychiatry Downstate Medical Center Brooklyn, NY ***Department of Microbiology Scripps Clinic and Research Foundation La J o l l a , CA (Received in final form June 26, 1983)

Summary Evidence from a variety of experimental models has suggested the existence of muI , mu2 and delta binding sites for morphine and the enkephalins in the central nervous system. Additional biochemical experiments now support t h i s concept of a common high a f f i n i t y s i t e for opiates and opioid peptides. Mu sites have now been implicated in a number of pharmacological actions, including supraspinal analgesia, prolactin release, and catalepsy, but not in others (spinal analgesia, respiratory depression, and the guinea pig ileum). The hypothesis of muI sites was supported by the unique opioid meptazinol, which s e l e c t i v e l y bound to muI s i t e s . As expected from i t s muI binding s e l e c t i v i t y , its analgesic actions in the mouse, localized supraspinally, were antagonized by the selective muI antagonist naloxonazine and i t had no respiratory depressant actions. Other binding studies suggested the presence of discrete SKFlO,O47-selective (KD approximately 5 nM) binding sites in rat brain which d i f f e r e d from both kappa sites and the previously reported PCP-binding sigma sites. Additional binding and autoradiograpical studies have also implied the presence of B-endorphin, or epsilon, sites in the CNS.

A major goal of opioid research has been the correlation of opioid binding sites with pharmacological function. Although in vivo studies had suggested opioid receptor m u l t i p l i c i t y (1,2), multiple classes of binding sites were not observed in the early binding studies. The identification of a new, higher a f f i n i t y opiate binding site (3) provided the f i r s t evidence of m u l t i p l i c i t y using binding technology. Subsequently, the enkephalins (4) provided strong phramacological evidence for receptor multiplicity. In addition to pharmacological differences between morphine and the enkephalins in peripheral bioassays, binding (15) and autoradiographical (6,7) studies clearly 0024-3205/83 $3.00 + .00 Copyright (c) 1983 Pergamon Press Ltd.

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implied different classes of binding sites for morphine and the enkephalins. However~ the interpretation of binding experiments was complicated by binding of the H-ligands to more than one class of sate. Fo~ example, i t was noted in these early studies that enkephalins l i k e OH-O-alaZ-D-leub-enkephalin bound to morphine, or mu, sites as well as enkephalin, or delta, s i t e s ( 5 ) . This was clearly seen with the multiphasic displacement of radiolabeled enkephalins by morphine. A small portion of the radiolabeled enkephalin binding, usually 25 to ~5% of the t o t a l , was easily and potently displaced by low morphine concentrations while the remainder was far less sensitive. The interpretation of these findings has been controversial. Although these results might be explained by just two types of sites, mu and delta, evidence from our laboratory led us to propose three types of morphine and enkephalin binding sites: mu1, a common, very high a f f i n i t y (KD< 1 nM) site for both opiates and enkephalins corresponding to the novel high a f f i n i t y binding site described in 1975 (3); mu2, a site which p r e f e r e n t i a l l y bound morphine far more potently than enkephalin; and delta, ~ site which selectively bound enkephalins (8). In this hypothesis, the ~H-labeled enkephalin binding which was easily displaced by morphine represented muI binding. A variety of developmental (13-16) phylogenetic ( I i ) , regional ~12), biochemical and pharmacological studies (9,10) have supported this proposal. We now present additional biochemical and pharmacological evidence supporting the concept of muI sites, as well as results suggesting the existence of discrete B-endorphin (epsilon), kappa and SKFIO,047 binding sites in rat brain. METHODS Binding assays were performed on brain homogenates of Sprague-Dawley rats as previously described (8). Analgesia was determined using the t a i l f l i c k or benzoquinone writhing assays (14,15). Respiratory depression was assessed by measuring pO2, pCn2 and pH in arterial blood gases (17). The guinea pig ileum bioassays was performed as previously described (18). Naloxonazine and naloxazone were synthesized as previously reported (19). RESULTS AND DISCUSSION Biochemical

evidence for muI sites

The multiphasic displacement of radiolabeled enkephalins by morphine strongly suggested that the enkephalins were labeling a morphine, or mu site (5). Since saturation studies had demonstrated high and low a f f i n i t y binding components for a number of radiolabeled enkephalins (4,8-14) i t had been suggested that the enkephalins bound with higher a f f i n i t y to the delta site and with lower a f f i n i t y to the mu s i t e . We investigated this p o s s i b i l i t y by performing saturation studies with 3H-D-ala2-D-leu5-enkephalin in the presence and absence of low morphine concentrations (I nM). We found that t h i s low concentration of morphine appeared to competitively i n h i b i t the higher a f f i n i t y component of the °H-enkephalin binding far more extensively than ~he lower a f f i n i t y component, implying that the morphine-sensitive portion of H-enkephalin binding corresponded to the high a f f i n i t y 3H2enkephalinb binding component. Similarly, low concentrations of D-ala -D-leu'-enkephalin (I nM)~selectively inhibited the higher a f f i n i t y binding component of the mu agonist JH-dihydromorphine and the antagonist ~H-naloxone, supporting the concept of a common high a f f i n i t y binding site for morphine and the enkephalin. Early binding studies examining the effects of N-ethylmaleimide suggested that the high a f f i n i t y binding component of 3H-dihydromorphine was extremely sensitive to the reagent (20). We therefore examined the effects of N-ethylmaleimide on the binding of the delta 3H-ligand D-ala2-D-leu --

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enkephalin. Treating the tissue with N-ethylmaleimide at a very low #oncentrat i o n (25 uM) eliminated the higher affinitY3binding component of the °Hlabeled enkephalin, H-dihydromorphine^and -Hmnaloxone. In addition, the morphine-~ensiti~e portion of °H-D-alaZ-D-leub-enk~phalin binding and the D-ala -D-leu~-enkephalin-sensitive portion of ~H-dihydromorphine binding was lost following the treatment of the tissue with N-ethylmaleimide at 25 ~M. Thus, N-et~ylmaleimide treatment abolished the high a f f i n i t y binding component of JH-labeled mu and delta ligands in saturation ~xperiments, as well as the high a f f i n i t y disnlacement of °H-D-ala -D-leu ~enkephalin by morphine and 3H-dihydromorphine by D-ala2-D-leu 5enkephalin. I f both morphine and the enkephalins were binding to a common very high a f f i n i t y binding s i t e , both classes of compounds should be able to protect the binding of a ~H-labeled ligand such as dihydromorphine from N-ethylmaleimide, a concept termed cross-protection. Previous studies f a i l e d to demonstrate cross-protection for enkephalins and morphine against N-ethylmaleimide (21). However, the concentration of reagent used (0.5 mM) was 20-fold greater than the level which we found r e l a t i v e l y selective for the high a f f i n i t y component (25 ~M). We therefore performed cross-protection experiments using therlow (25 uM) N-ethylmaleimide concentration. ~o~h morphine and D-ala~-D-leu ~enkephalin at 1 nM s i g n i f i c a n t l y protected ~H-dihydromorphine binding from N-ethylmaleimide (p
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Pharmacological role of muI sites in respiratory depression The relationship between morphine's analgesic and respiratory depressant actions has remained a major question in opioid pharmacology. We examined t h i s problem by comparing the effects of muI blockade with naloxonazine on morphine analgesia and respiratory depression, assessed by serial a r t e r i a l blood gases (17). At 3.5 mg/kg ( i . v . ) , morphine produced both a s i g n i f i c a n t increase in t a i l f l i c k latency and respiratory depression in control animals. Pretreating rats with naloxonazine (i0 mg/kg, i . v . ) 24 hours e a r l i e r eliminated the analgesic response without affecting the drop in pO2 and pH or the rise of pCO2. Full dose-response curves demonstrated a s i g n i f i c a n t 2.5-fold s h i f t in morphine's analgesic response. Dose-response curves examining morphine's depression of PO2 and pH and elevation of pCO2 were v i r t u a l l y the same in the two treatment groups. Although the receptor mechanisms involved with morphine's respiratory depression remain unknown, muI sites appear not to be involved• Guinea pig ileum bioassay: mediation by mu2 sites The marked s e l e c t i v i t y of the guinea pig ileum bioassay for morphine over the enkephalins was one of the major findings suggesting the existence of discrete mu and delta receptors (4) . This s e l e c t i v i t y raised the p o s s i b i l i t y }~ t h i s response might be mediated through mu2, as opposed to muI , sites • We examined t h i s p o s s i b i l i t y with both binding and contraction assays. n binding assays, naloxonazine i r r e v e r s i b l y i n h i b i t e d the binding of H-dihydromorphine to both rat and guinea pig brain homogenates approximately 70%. However, binding to guinea pig longitudinal muscle-myenteric plexus homogenates was lowered only 5% (p
~

Table 1: Tentative Receptor C l a s s i f i c a t i o n of Opioid Actions MuI Mediated

Not MuI Mediated

Supraspinal Analgesia (in the mouse)

Spinal Analgesia (in the mouse; possibly delta)

Prol actin release

Growth hormone release

Hypothermia

Bradycardia (mu2)

Catalepsy

Sedation (kappa)

Acetylcholine turnover in hippocampus and parietal cortex

Respiratory depression (possible mu2 and/or delta) Lethality Dopamine turnover in striatum (mu2 and delta) Reversal of endotoxic shock (delta) I n h i b i t i o n of guinea pig ileum contractions (mu2)

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Other pharmacological roles for muI sites A number of other pharmacological properties of opioids have been studied regarding t h e i r relationship to muI sites (26-30) and are summarized i n Table I. Meptazinol: a muI selective opioid Meptazinol has proven to be a unique opioid. Although i t s analgesic actions were reversed by naloxone, i t d i f f e r e d in a number of respects from other opioids, such as its lack of respiratory depression and cardiovascular effects and its poor a b i l i t y to displace JH-opioid binding (31,32). When we ~xamined the a b i l i t y of meptazinol to i n h i b i t the binding of a series of H-labeled opioids, we found that She compound at concentations under I0 nM displaced approximately 20-30% of ~H-opioid while the remainder of the binding required far greater concentrations. The simil~r a b i l i t y of meptazinol to displace a portion of the binding from a number of "H-opioids of various classes raised the p o s s i b i l i t y that the compound was interacting with the high a f f i n i t y (muI) ~ites. To examine t h i s p o s s i b i l i t y , saturation studies were performed with ~H-dihydromorphine in the presence of meptazinol (5 nM). The meptazi~ol competitively inhibited the high a f f i n i t y (KD 0.2 nM) component of the H-dihydromorphine binding far more extensively than the lower a f f i n i t y (KD 3 nM) component. Together with other binding studies, our results implied that meptazinol s e l e c t i v e l y bound to the muI class of opioid binding s i t e . This binding s e l e c t i v e l y of meptazinol for muI sites permitted us to reexamine a number of opioid actions previously studied with naloxazone and naloxonazine. As anticipated, meptazinol analgesia was potently reduced by the p r i o r blockade of muI sites with naloxonazine. Since e a r l i e r studies had implied that muI analgesia in the mouse was supraspinal, we tested meptazinol analgesia in spinalized mice. No analgesic response in the t a i l f l i c k asssay could be demonstrated in spinalized mice at meptazinol doses which dramatically elevated t a i l f l i c k latencies in control animals. Since our previous studies had indicated that muI sites did not play a role in respiratory depression, we next compared meptazinol's effects on respiration to those of morphine at equianalgesic doses. Unlike morphine, which potently decreased the pO2 and pH and raised the pcO2, meptazinol did not depress respiratory function. Furthermore, meptazinol did not reverse morphine's respiratory depressant actions when both drugs were given together, implying that meptazinol was not acting as e i t h e r an agonist or an antagonist in t h i s system. Thus, the pharmacological properties of meptazinol agreed exceedingly well with our predictions based upon its muI s e l e c t i v i t y in binding assays. Characterization of kappa and sigma opiate and B-endorphin binding The above studies strongly supported the concept of multiple opioid binding sites, s p e c i f i c a l l y muI sites which bound both opiates and enkephalins with very high a f f i n i t y , mu2 sites which p r e f e r e n t i a l l y bound morphine and delta sites which s e l e c t i v e l y bound enkephalins. Additional evidence from our laboratory also supports the existence of d i s t i n c t SKF10,047- selective and B-endorphin-selective sites in the central nervous system in addition to mu, kappa and delta sites (33,34). Using 3H-B-endorphin of high specific a c t i v i t y in rat brain homogenate binding assays, we found that H-B-endorphin s e l e c t i v e l y labeled a s i t e to which opiates such as morphine and enkephalins bound with far less affinity. In addition, the 3H-B-endorphin also bound to muI sites with

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high a f f i n i t y , consistent with the a b i l i t y of selective muI blockade by naloxonazine to antagonize B-endorphin analgesia and catalepsy (16). Additional evidence for selective central epsilon sit~s was obtained from the comparison of the autoradiographical l o c a l i z a t i o n of ~H-~-endorphin binding ~ites in2rat br~in with the regional d i s t r i b u t i o n of JH-dihydromorphine and ~H-D-ala -D-leu -enkephalin. ~n b r i e f , a number of areas clearly showed differences between the three JH-ligands, p a r t i c u l a r l y the cortex, hippocampus, olefactory bulb, striatum and claustrum. In addition, we have found that 3H-SKFIO,047 s e l e c t i v e l y labeled a site which differed from mu, kappa or delta ~ites. Most of the compound~ we examined did not d i f f e r e n t i a t e between "H-ethylketocyclazocine and HSKFIO,047 binding. However, bo~h ketocyclazocine and metkephamid were far more potent displacers against ~H-ethylketocyclazocine than against ~H" SKFIO,047. Further studies have shown that this site was highly stereoselect i v e for (-)-isomers of opiates, i~cluding SKFIO,047, did not bind phencyclidine (PCP) derivatives, and bound ~H-SKFIO,047 with an a f f i n i t y constant of approximately 5-10 nM. Thus, t h i s SKFIO,047 site differed dramatically from the PCP-binding sigma site previously reported (35). Acknowledgements This work was supported by grants from Ives Laboratories, Inc, NIDA (002615) and the American Cancer Society (PDTI69) to GWP, NINCDS (NS18113) to RAH, and NIDA (DA03893 to ARG. GWP is a recipient of a Teacher-lnvestigator Award from NINCDS (NS00415) and GSFL is supported by Fellowship DRG-564 of the Damon Runyon-Walter Winchell Cancer Fund. REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. 11. 12. 13. 14. 15. 16. 17. I~. 19.

W.R. Martin, Pharmacological Reviews 19:463-521 (1967). W.R. Martin, C.G. Eades, J.A. Thompson, R.E. Huppler, and P.E. G i l b e r t , J. Pharmacol. Exp. Ther. 197:517-532 (1976). G.W. Pasternak and S.H. Snyder, Nature 253:563-565 (1975). J.H. Lord, A.A. Waterfield, J. Hughes and H.W. K o s t e r l i t z , Nature 267:495-499 (1977). K-J Chang, and P. Cuatrecasas, J. Biol. Chem. 254:2610-2618 (1979). R.R. Goodman, S.H. Snyder, M.J. Kuhar, and W.S. Young, Proc. Nat. Acad. Sci, USA 77:6239-6243 (1980). T. Ouka, P. Schubert, M. Wuster, R. Stoiber, and A. Hertz, Neurosci. Letters 21:119-124 (1981). ~.L. Wolozin and G.W. Pasternak, Proc. Nat. Acad. Sci., USA 78:6181-6185 (1981). G.W. Pasternak, A-Z Zhang and L. Tecott, Life Sci. 27:1185-1190 (1980). A-Z Zhang and G.W. Pasternak, Europ. J. Pharmacol. 73:29-40 (1981). M.C. 8uatti and G.W. Pasternak, Rrain Res. 218:400-405 (1981). A-Z Zhang and G.W. Pasternak, Europ. J. Pharmacol. 67:323-324 (1980). A-Z Zhang and G.W. Pasternak, L i f e Sci. 29:843-851 (1981). G.W. Pasternak, S.R. Childers, and S.H. Snyder, Science 208:514-516 (1980) G.W. Pasternak, S.R. Childers, and S.H. Snyder, J. Pharmacol. Exp, Ther. 214:455-462 (1980). G.W. Pasternak, Neurology 31:1311-1315 (1981). G.S.F. Ling, K. Spiegel, S. Nishimura, and G.W. Pasternak, Europ J. Pharmacol. 86:487-488 (1983). A.R. Gintzler and G.W. Pasternak, Neurosci. Letters, in press. E.F. Hahn, M. Carroll-Buatti and G.W. Pasternak, J. Neurosci. 2:572-576 (1982).

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2Q. 21. 22. 23. 24. . 25. 26. 27. 28. 29. 30. 31. 32. 33. 34. 35.

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G.W. Pasternak, H.A. Wilson and S.H. Snyder, Molec. Pharmacol. 11:340-351 (1975). J.R. Smith and E.J. Simon, Proc. Nat. Sci, USA 77:281-284 (1980). G.S.F. Ling and G.W. Pasternak, Brain Res., in press. G.W. Pasternak, Proc. Nat. Acad. Sci., lISA 77:~691-3694 (1980). G.W. Pasternak, M. C a r r o l l - B u a t t i , and K. Spiegel, J. Pharmacol. Exp. Ther. ~19:192-198 (1981). C. Burkhardt, R.C.A. Frederikson, and G.W. Pasternak, Peptides 3:869-~71 (1982). A.L. Kirchgessner, R.J. Bodnar and G.W. Pasternak, Pharmacol, Biochem. Behav. 17:1175-1179 (1982). P.L. Wood and G.W. Pasternak, Neurosci. Letters, in press. J.W. Holaday, G.W. Pasternak, R.J. D'Amato, B.A. Ruvio and A.I. Faden, Europ. J. Pharmacol., in Dress. J.W. Holaday, G.W. Pasternak, and A . I . Faden, Neurosci. Letters, in press P.L. Wood, A. Rackham, and J. Richard, Life Sci. 28:2119-2125 (1982). R.J. Stephens, J.F. Waterfall, and R.A. Franklin, Gen. Oharmac. 9:73-78 (1978). D. B i l l , I.S. Cowlrick, J. Fox, M.H. Todd, P.J. Ward, M.D. and M.G. Wyllie, Br. J. Pharmacol. 74:866P (1981). B.L. Wolozin, S. Nishimura and G.W. Pasternak, J. Neurosci. 2:708-713 (1982). N. Johnson, R.A. Houghten and G.W. Pasternak, Life Sci. 31:1381-1384 (1982). R.S. Zukin and S.R. Zukin, L i f e Sci. 29:~681-2590 (1981).