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High and low affinity opioid binding sites: Relationship to mu and delta sites
Life Sciences, Vol. 31, pp. 1303-1306 Printed in the U.S.A.
Pergamon Press
HIGH AND LOW AFFINITY OPIOID BINDING SITES: RELATIONSHIP TO MU AND DELTA SITES Gavril W. Pasternak The George C. Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, and Departments of Neurology and Pharmacology Cornell University Medical College New York, NY 10021 (Received in fln~l form June 14, 1982)
Summary Binding and pharmacological studies suggest a common opiate and enkephalin binding site in addition to their previously reported selective sites. This common high a f f i n i t y site has tentatively been named mu1, distinguishing i t from the morphineselective site (mu2) and enkephalin-selective site (delta). The existence of t~is additional common high a f f i n i t y site and its association with opiate and opioid peptide analgesia may help explain some pharmacological observations, such as the cross tolerance between morphine and enkephalin analgesia and the lack of cross tolerance between them in the guinea pig ileum and mouse vas deferens bioassays. Pharmacological comparisons between the opiates and enkephalins have revealed a number of significant differences as well as important similarities. The selectivity of the guinea pig ileum preparation for morphine-like drugs and the mouse vas deferens for enkephalins was one basis for classifying potential receptor subtypes (1). This concept was supported by the lack of cross tolerance between drug classes in these two bioassays (2,3). Analgesia, on the other hand, showed significant cross tolerance between morphine and the enkephalins (4-6). Similarly, enkephalins can suppress much of the withdrawal syndrome in morphine-dependent animals (4). Thus, the peripheral bioassay systems suggest receptors selective for either the enkephalins or morphine while those receptors mediating analgesia may not discriminate between the two classes of drugs. Selective binding sites for enkephalins and morphine within the CNS which may correspond to the receptors in the peripheral bioassays have been described (7). We now present further evidence for a common site for morphine and the enkephalins which appears to be important in opiate and opioid peptide analgesia. Methods Binding assays were performed and data analyzed as previously described (8). In brief, rat brain homogenates were prepared, including a preincubation at 37°C, and t r i p l i c a t e tubes assayed in the absence and presence of levallorphan (1 ~M). The difference between these sets of t r i p l e t s was considered specific binding. Only specific binding is reported. All experiments were performed at least 3 times. 0024-3205/82/121303-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1304
Opiate Receptor Classification
Vol. 31, No.s 12 & 13, 1982
Results In 1975 a novel binding site for 3H-dihydromorphine and 3H-naloxone was described (9) whose a f f i n i t y was lO-fold greater than those sites originally reported (10-12). Soon afterwards, nonlinear Scatchard plots were reported for the enkephalins (1,13) and most other ligands. The selective, irreversible inhibition by naloxazone of the high a f f i n i t y binding component for a large series of radiolabeled opiates and opioid peptides suggested the possibility of a common binding site (13-18). To examine this possibility further, we have examined the direct competitive interactions between a series of opiates and opioid peptides. Previous studies reporting selective enkephalin and morphine sites demonstrated multiphasic displacement curves for morphine's inhibition of radiolabeled enkephalin binding (7). A portion of the enkephalin binding was quite sensitive to low morphine concentrations (<1 nM) while the remainder required far greater morphine concentrations. After confirming these results, we examined morphine's inhibition of 3H-D-ala2-D-leu5-enkephalin in tissue treated with naloxazone. Naloxazonetreatment eliminated the i n i t i a l , sensitive displacement of 3H-D-ala2-D-leu5-enkeph~lin binding by morphine (<1 nM). Since naloxazone also biocked high a f f i n i t y aH-D-ala~-D-leu~-enkephalin and morphine binding, these results were most consistent with a common high a f f i n i t y site. Similar results were seen with the displacement of 3H-dihydromorpiline binding with unlabeled 3H-D-ala2-D-leu5-enkephalin and using unlabeled ketocyclazocine, SKF 10,047 and naloxone. These findings further support the concept of a common high a f f i n i t y site for opiates and enkephalins. In the multiphasic displacements of radiolabeled enkephalins by morphine, the portion of binding sensitive to morphine represented less than 35% of the total. IC50 values of total binding~ therefore, were very similar to the second, less sensitive displacement. Since the reports of selective sites used IC50 values for total binding (7), we examined the a b i l i t y of opiates and enkephalins to inhibit 3H-dihydromorphine and 3H-D-ala2-D-leu5-enkephalin binding in naloxazone-treated tissue (Table 1). By eliminating the sensitive i n i t i a l displacement we were able to examine the second displacement more directly. Overall, mu drugs were far more potent displacing H-dihydromorphine binding and delta drugs more potent displacing 3H-D-ala2-D-leu5enkephalin binding. These results indicate that the sites remaining in naloxazone-treated tissue selectively bind either morphine or enkephalins and were responsible for the differences between the opiate and enkephalin binding noted in previous studies (7). TABLE 1. INHIBITIONOF 3H-OPIOID BINDING IN NALOXAZONE-TREATED TISSUES. IC50 (nM) 3H-D-ala2-D-leu5-enkephalin Morphine 89 D-ala2-D-leu5-enkephalin 10 D-ala2-D-met5-enkephalin 8 Naloxazone 42 FK 33,824 51
3H-dihydromorphine 11 64 97 19 17
Results are the mean of three separate IC50 determinations performed in naloxazone-treated tissues (18).
Vol. 31, No.s 12 & 13, 1982
TABLE 2.
Opiate Receptor Classification
1305
SELECTIVITYMU1, MU2 AND DELTA SITES Approximate KD values (nM)
Morphine D-ala2-D-leu5-enkephalin
mul
mu2
0.4 0.5
10 50
delta 70 6
Discussion The results presented above suggest three subtypes of morphine and enkephalin binding sites (Table 2): i) a common site (mul) to which both morphine and the enkephalins bind with highest a f f i n i t y , 2) a site (mu~) preferentially binding morphine rather than enkephalins such as 3H-D-a~a2D-leub-enkephalin, and 3) a site (delta) selectively binding enkephalins such as 3H-D-alaLD-leub-enkephalin. In addition to explaining the binding data, this model is consistent with much of the pharmacological evidence. Selective receptors for morphine in the guinea pig ileum and the enkephalins in the mouse vas deferens were important in the original proposal of delta receptors (1). This concept has been supported by recent studies showing lack of cross tolerance between the two classes of drugs in these bioassays (2,3). These receptors might correspond to the mu2 and delta sites. Analgesia, on the other hand, does not f i t well as either a morphineselective or an enkephalin-selective action, although Martin has suggested i t is mediated through mu receptors (19). Both the enkephalins and morphine are potent analgesics. More important, both classes of agents demonstrate cross tolerance with each other, suggesting that a single site may mediate their analgesic actions. Binding studies have implied that the muI site binds both classes quite well and might correspond to this receptor. This conclusion is further supported by analgesic studies using naloxazone. Under conditions in which muI sites are blocked in vivo, the analgesic potency of morphine (13), 3H-D-ala2-D-leu5-enkephalin--(14--~-20) and B-endorphin (14,20) was markedly reduced. Although analgesia is a complex response and can be influenced by a variety of factors, these results were consistent with a role for the muI site in analgesia. This does not mean that analgesia is mediated solely by muI sites. Evidence suggesting delta or kappa analgesia has been reported (21, 22). However, our results would suggest that these additional sites require higher doses of drug for activity than the mu1 sites since muI blockade shifts the dose-response curves of kappa and delta drugs to the right. The existence of distinct muI sites is supported by a wide variety of evidence. These sites have a unique regional localization based upon naloxazone sensitivity (23) and autoradiographic studies (Goodman, Kuhar, Snyder and Pasternak, in preparation)..They show a different developmental appearance (24) and phylogenetic distribution (25). Pharmacologically, the muI sites also appear to be involved in prolactin release and catalepsy but not growth TABLE 3.
TENTATIVE CLASSIFICATION OF OPIOID ACTIONS MEDIATED THROUGHMU1 SITES MuI Mediated
Not muI mediated
Analgesia CatalepsF Prolactin Release
Sedation Lethality Growth hormone release
1306
Opiate Receptor Classification
Vol. 31, No.s 12 & 13, 1982
homone release, sedation or lethality (Table 3) (26). confirmed an earlier report (27).
The lethality results
Acknowledgements These studies were supported in part by grants from NIDA (DA 02615) and The American Cancer Society (PDT 169). GWP is the recipient of a TeacherInvestigator Award from the NINCDS (NS 0415). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
J.A.H. LORD, A.A. WATERFIELD, J. HUGHESand H.W. KOSTERLITZ, Nature 267 495-499 (1977). R. SCHULZ, M. WUSTER, H. KRENSS, and A. HERZ, Nature 285 242-243 (1980). R. SCHULZ, M. WUSTER, P. RUBINI, and A. HERZ, J. P h a ~ o l . Exp. Ther. 219 547-550 (1981). J. BLASIG and A. HERZ, Nauyn-Schmiedeberg's Arch Pharmacol 294 297-300 (1976). H.W. KOSTERLITZ and J. HUGHES, Alcohol Intoxication and Withdrawal~ M. GROSS, ed (pp 141-154) Plenum Press, New York (1977). J.M. VAN REE, D. DEWIED, A.F. BRODBURY,E.C. HUME, O.G. SMYTHand C.R. SNELL, Nature 264 792-794 (1976). K-J CHANGand P. CUATRECASAS, J. Biol. Chem. 254 2610-2618 (1978). G.W. PASTERNAK, H.A. WILSON and S.H. SNYDER, Molec. Pharmacol. 11 340-351 (1975). G.W. PASTERNAKand S.H. SNYDER, Nature 253 563-565 (1975). C.B. PERT and S.H. SNYDER, Science 179~1-1014 (1973). E.J. SIMON, J.M. HILLER and I. EDELMAN, Proc. Nat. Acad. Sci. USA 70 1947-1949 (1973). L. TERENIUS, Acta Pharmacol. Toxicol. 33 377-384 (1973). G.W. PASTERNAK, S.R. CHILDERS and S.H. SNYDER, J. Pharmacol. Exp. Ther. 214 455-462 (1980). A.Z. ZHANGand G.W. PASTERNAK, Life Sci. 29 843-851 (1981). G.W. PASTERNAK, Proc. Nat. Acad. Sci. USA 7---7 3691-3694 (1980). G.W. PASTERNAK, M. CARROLL-BUATTI and K. SPIEGEL, J. Pharmacol. Exp. Ther. 219 192-198 (1981). B.L. WOLOZIN, S. NISHIMURA and G.W. PASTERNAK, J. Neurosci., in press. B.L. WOLOZIN and G.W. PASTERNAK, Proc. Nat. Acad. Sci. USA 78 6181-6185 (1981). W.R. MARTIN, Br. J. Clin. Pharmacol. 7 2735-2795 (1979). G.W. PASTERNAK, Neurology 31 1311-1315 (1981). P.L. WOOD, A. RACKHAMand J. RICHARD, Life Sci. 28 2119-2125 (1981). R.C.A. FREDERICKSON, E.L. SMITHWICK, R. SHUMANand K.G. BEMIS, Science 211 603-605 (1981). A.Z. ZHANGand G.W. PASTERNAK, Europ. J. Pharmacol. 67 323-324 (1980). A.Z. ZHANGand G.W. PASTERNAK, Europ. J. Pharmacol. 7~ 29-40 (1981). M.C. BUATTI and G.W. PASTERNAK, Brain Res. 281 400-40~ (1981). K. SPIEGEL, I. KOURIDES and G.W. PASTERNAK, Science, in press. R. DINGLEDINE and A. GOLDSTEIN, Br. J. Pharmacol. 48 718-719 (1973).
Pergamon Press
HIGH AND LOW AFFINITY OPIOID BINDING SITES: RELATIONSHIP TO MU AND DELTA SITES Gavril W. Pasternak The George C. Cotzias Laboratory of Neuro-Oncology, Memorial Sloan-Kettering Cancer Center, and Departments of Neurology and Pharmacology Cornell University Medical College New York, NY 10021 (Received in fln~l form June 14, 1982)
Summary Binding and pharmacological studies suggest a common opiate and enkephalin binding site in addition to their previously reported selective sites. This common high a f f i n i t y site has tentatively been named mu1, distinguishing i t from the morphineselective site (mu2) and enkephalin-selective site (delta). The existence of t~is additional common high a f f i n i t y site and its association with opiate and opioid peptide analgesia may help explain some pharmacological observations, such as the cross tolerance between morphine and enkephalin analgesia and the lack of cross tolerance between them in the guinea pig ileum and mouse vas deferens bioassays. Pharmacological comparisons between the opiates and enkephalins have revealed a number of significant differences as well as important similarities. The selectivity of the guinea pig ileum preparation for morphine-like drugs and the mouse vas deferens for enkephalins was one basis for classifying potential receptor subtypes (1). This concept was supported by the lack of cross tolerance between drug classes in these two bioassays (2,3). Analgesia, on the other hand, showed significant cross tolerance between morphine and the enkephalins (4-6). Similarly, enkephalins can suppress much of the withdrawal syndrome in morphine-dependent animals (4). Thus, the peripheral bioassay systems suggest receptors selective for either the enkephalins or morphine while those receptors mediating analgesia may not discriminate between the two classes of drugs. Selective binding sites for enkephalins and morphine within the CNS which may correspond to the receptors in the peripheral bioassays have been described (7). We now present further evidence for a common site for morphine and the enkephalins which appears to be important in opiate and opioid peptide analgesia. Methods Binding assays were performed and data analyzed as previously described (8). In brief, rat brain homogenates were prepared, including a preincubation at 37°C, and t r i p l i c a t e tubes assayed in the absence and presence of levallorphan (1 ~M). The difference between these sets of t r i p l e t s was considered specific binding. Only specific binding is reported. All experiments were performed at least 3 times. 0024-3205/82/121303-04503.00/0 Copyright (c) 1982 Pergamon Press Ltd.
1304
Opiate Receptor Classification
Vol. 31, No.s 12 & 13, 1982
Results In 1975 a novel binding site for 3H-dihydromorphine and 3H-naloxone was described (9) whose a f f i n i t y was lO-fold greater than those sites originally reported (10-12). Soon afterwards, nonlinear Scatchard plots were reported for the enkephalins (1,13) and most other ligands. The selective, irreversible inhibition by naloxazone of the high a f f i n i t y binding component for a large series of radiolabeled opiates and opioid peptides suggested the possibility of a common binding site (13-18). To examine this possibility further, we have examined the direct competitive interactions between a series of opiates and opioid peptides. Previous studies reporting selective enkephalin and morphine sites demonstrated multiphasic displacement curves for morphine's inhibition of radiolabeled enkephalin binding (7). A portion of the enkephalin binding was quite sensitive to low morphine concentrations (<1 nM) while the remainder required far greater morphine concentrations. After confirming these results, we examined morphine's inhibition of 3H-D-ala2-D-leu5-enkephalin in tissue treated with naloxazone. Naloxazonetreatment eliminated the i n i t i a l , sensitive displacement of 3H-D-ala2-D-leu5-enkeph~lin binding by morphine (<1 nM). Since naloxazone also biocked high a f f i n i t y aH-D-ala~-D-leu~-enkephalin and morphine binding, these results were most consistent with a common high a f f i n i t y site. Similar results were seen with the displacement of 3H-dihydromorpiline binding with unlabeled 3H-D-ala2-D-leu5-enkephalin and using unlabeled ketocyclazocine, SKF 10,047 and naloxone. These findings further support the concept of a common high a f f i n i t y site for opiates and enkephalins. In the multiphasic displacements of radiolabeled enkephalins by morphine, the portion of binding sensitive to morphine represented less than 35% of the total. IC50 values of total binding~ therefore, were very similar to the second, less sensitive displacement. Since the reports of selective sites used IC50 values for total binding (7), we examined the a b i l i t y of opiates and enkephalins to inhibit 3H-dihydromorphine and 3H-D-ala2-D-leu5-enkephalin binding in naloxazone-treated tissue (Table 1). By eliminating the sensitive i n i t i a l displacement we were able to examine the second displacement more directly. Overall, mu drugs were far more potent displacing H-dihydromorphine binding and delta drugs more potent displacing 3H-D-ala2-D-leu5enkephalin binding. These results indicate that the sites remaining in naloxazone-treated tissue selectively bind either morphine or enkephalins and were responsible for the differences between the opiate and enkephalin binding noted in previous studies (7). TABLE 1. INHIBITIONOF 3H-OPIOID BINDING IN NALOXAZONE-TREATED TISSUES. IC50 (nM) 3H-D-ala2-D-leu5-enkephalin Morphine 89 D-ala2-D-leu5-enkephalin 10 D-ala2-D-met5-enkephalin 8 Naloxazone 42 FK 33,824 51
3H-dihydromorphine 11 64 97 19 17
Results are the mean of three separate IC50 determinations performed in naloxazone-treated tissues (18).
Vol. 31, No.s 12 & 13, 1982
TABLE 2.
Opiate Receptor Classification
1305
SELECTIVITYMU1, MU2 AND DELTA SITES Approximate KD values (nM)
Morphine D-ala2-D-leu5-enkephalin
mul
mu2
0.4 0.5
10 50
delta 70 6
Discussion The results presented above suggest three subtypes of morphine and enkephalin binding sites (Table 2): i) a common site (mul) to which both morphine and the enkephalins bind with highest a f f i n i t y , 2) a site (mu~) preferentially binding morphine rather than enkephalins such as 3H-D-a~a2D-leub-enkephalin, and 3) a site (delta) selectively binding enkephalins such as 3H-D-alaLD-leub-enkephalin. In addition to explaining the binding data, this model is consistent with much of the pharmacological evidence. Selective receptors for morphine in the guinea pig ileum and the enkephalins in the mouse vas deferens were important in the original proposal of delta receptors (1). This concept has been supported by recent studies showing lack of cross tolerance between the two classes of drugs in these bioassays (2,3). These receptors might correspond to the mu2 and delta sites. Analgesia, on the other hand, does not f i t well as either a morphineselective or an enkephalin-selective action, although Martin has suggested i t is mediated through mu receptors (19). Both the enkephalins and morphine are potent analgesics. More important, both classes of agents demonstrate cross tolerance with each other, suggesting that a single site may mediate their analgesic actions. Binding studies have implied that the muI site binds both classes quite well and might correspond to this receptor. This conclusion is further supported by analgesic studies using naloxazone. Under conditions in which muI sites are blocked in vivo, the analgesic potency of morphine (13), 3H-D-ala2-D-leu5-enkephalin--(14--~-20) and B-endorphin (14,20) was markedly reduced. Although analgesia is a complex response and can be influenced by a variety of factors, these results were consistent with a role for the muI site in analgesia. This does not mean that analgesia is mediated solely by muI sites. Evidence suggesting delta or kappa analgesia has been reported (21, 22). However, our results would suggest that these additional sites require higher doses of drug for activity than the mu1 sites since muI blockade shifts the dose-response curves of kappa and delta drugs to the right. The existence of distinct muI sites is supported by a wide variety of evidence. These sites have a unique regional localization based upon naloxazone sensitivity (23) and autoradiographic studies (Goodman, Kuhar, Snyder and Pasternak, in preparation)..They show a different developmental appearance (24) and phylogenetic distribution (25). Pharmacologically, the muI sites also appear to be involved in prolactin release and catalepsy but not growth TABLE 3.
TENTATIVE CLASSIFICATION OF OPIOID ACTIONS MEDIATED THROUGHMU1 SITES MuI Mediated
Not muI mediated
Analgesia CatalepsF Prolactin Release
Sedation Lethality Growth hormone release
1306
Opiate Receptor Classification
Vol. 31, No.s 12 & 13, 1982
homone release, sedation or lethality (Table 3) (26). confirmed an earlier report (27).
The lethality results
Acknowledgements These studies were supported in part by grants from NIDA (DA 02615) and The American Cancer Society (PDT 169). GWP is the recipient of a TeacherInvestigator Award from the NINCDS (NS 0415). References 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24. 25. 26. 27.
J.A.H. LORD, A.A. WATERFIELD, J. HUGHESand H.W. KOSTERLITZ, Nature 267 495-499 (1977). R. SCHULZ, M. WUSTER, H. KRENSS, and A. HERZ, Nature 285 242-243 (1980). R. SCHULZ, M. WUSTER, P. RUBINI, and A. HERZ, J. P h a ~ o l . Exp. Ther. 219 547-550 (1981). J. BLASIG and A. HERZ, Nauyn-Schmiedeberg's Arch Pharmacol 294 297-300 (1976). H.W. KOSTERLITZ and J. HUGHES, Alcohol Intoxication and Withdrawal~ M. GROSS, ed (pp 141-154) Plenum Press, New York (1977). J.M. VAN REE, D. DEWIED, A.F. BRODBURY,E.C. HUME, O.G. SMYTHand C.R. SNELL, Nature 264 792-794 (1976). K-J CHANGand P. CUATRECASAS, J. Biol. Chem. 254 2610-2618 (1978). G.W. PASTERNAK, H.A. WILSON and S.H. SNYDER, Molec. Pharmacol. 11 340-351 (1975). G.W. PASTERNAKand S.H. SNYDER, Nature 253 563-565 (1975). C.B. PERT and S.H. SNYDER, Science 179~1-1014 (1973). E.J. SIMON, J.M. HILLER and I. EDELMAN, Proc. Nat. Acad. Sci. USA 70 1947-1949 (1973). L. TERENIUS, Acta Pharmacol. Toxicol. 33 377-384 (1973). G.W. PASTERNAK, S.R. CHILDERS and S.H. SNYDER, J. Pharmacol. Exp. Ther. 214 455-462 (1980). A.Z. ZHANGand G.W. PASTERNAK, Life Sci. 29 843-851 (1981). G.W. PASTERNAK, Proc. Nat. Acad. Sci. USA 7---7 3691-3694 (1980). G.W. PASTERNAK, M. CARROLL-BUATTI and K. SPIEGEL, J. Pharmacol. Exp. Ther. 219 192-198 (1981). B.L. WOLOZIN, S. NISHIMURA and G.W. PASTERNAK, J. Neurosci., in press. B.L. WOLOZIN and G.W. PASTERNAK, Proc. Nat. Acad. Sci. USA 78 6181-6185 (1981). W.R. MARTIN, Br. J. Clin. Pharmacol. 7 2735-2795 (1979). G.W. PASTERNAK, Neurology 31 1311-1315 (1981). P.L. WOOD, A. RACKHAMand J. RICHARD, Life Sci. 28 2119-2125 (1981). R.C.A. FREDERICKSON, E.L. SMITHWICK, R. SHUMANand K.G. BEMIS, Science 211 603-605 (1981). A.Z. ZHANGand G.W. PASTERNAK, Europ. J. Pharmacol. 67 323-324 (1980). A.Z. ZHANGand G.W. PASTERNAK, Europ. J. Pharmacol. 7~ 29-40 (1981). M.C. BUATTI and G.W. PASTERNAK, Brain Res. 281 400-40~ (1981). K. SPIEGEL, I. KOURIDES and G.W. PASTERNAK, Science, in press. R. DINGLEDINE and A. GOLDSTEIN, Br. J. Pharmacol. 48 718-719 (1973).