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Opiates and enkephalins: A common binding site mediates their analgesic actions in rats
Life Sciences, Vol. 29, pp. 843-851 Printed in the U.S.A.
Pergamon Press
OPIATES AND ENKEPHALINS: A COMMONBINDING SITE MEDIATES TIIEIR ANALGESIC ACTIONS IN RATS An Zhong Zhang and Gavril W. Pasternak George C. Cotzias Laboratory of Neuro-Oncology Memorial Sloan-Kettcrinq Cancer Center and Departments of PharmacolocLv and Neurology, Cornell University Medical College, New York, NY 10021 (Received in final form June 22, 1981)
SUMMARY 3H-Labelled opiate and enkephalin ligands appear to bind with highest a f f i n i t y to a single site responsible for t h e i r analgesic properties. Administered in vivo, naloxazone, an irreversible opiate, selectively i n h i b i t s for over 24 hours the high a f f i n i t y binding of aH-labelled mu, and kappa opiates and enkephalins. This i n h i b i t i o n of binding gradually resolves over 3 days, perhaps correlating with receptor t~rnover. Naloxazone treatment also abolishes morphine, D-ala~-metD-enkephalinamide and betahendorphin analgesia. Although morphine and D-ala2-met5-enkepha linamide bind with similar potencies to the high a f f i n i t y s i t e , morphine's potency for the low a f f i n i t y D-ala2-met5-enkephalinamide site is f~r less than the enkephalin analog. These results imply that all ~H-ligands examined bind with hiqhest a f f i n i t y to a mu-like receptor while low a f f i n i t y D-ala2-met5-enkephalinamide binding, with a KD of 6 nM, represents a d e l t a - l i k e receptor. The concept of multiple populations of opiate receptors has been proposed on the basis of pharmacological (1,2) and biochemical (3,4) c r i t e r i a . Pharmacologically, opiates have been assigned to one of three major groups named after prototypic drugs: mu (morphine), kappa (ketocyclazocine) and sigma (SKF 10,047). The discovery of the enkephalins (5) led to a fourth c l a s s i f i c a t i o n for the peptides: delta (enkephalin) (2). Riochemically defined subpopulations of receptors have been suggested from binding studies. Despite the i n a b i l i t y of Scatchard analyses to d i f f e r e n t i a t e subpopulations of opiate receptors in early studies, enzymatic treatments raised the p o s s i b i l i t y of t h e i r existence (3). As techniques evolved, nonlinear Scatchards identifying novel, higher a f f i n i t y binding were demonstrated for opiate agonists and antagonists (4) and l a t e r for enkephalin (2) and other opiate drugs. Previous studies have established the ab#lity of naloxazone, an i r r e v e r s i b l e opiate antagonist, to block high a f f i n i t y jH-naloxone binding and morphine analgesia in mice (6,7). Using naloxazone, we now describe in rats i t s actions on the binding of a variety of enkephalins, mu, kappa and antagonist opiates and on opiate and opioid peptide analgesia. Address correspondence to Dr. G.W. Pasternak, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 0024-3205/81/080843-09502.00/0 Copyright (e) 1981 Pergamon Press Ltd.
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Fi9.2 Effect of naloxazone on mu and kappa agonists. Saturation studies on brain membranes treated with either naloxone or n~loxazone 20-24 hours e a r l i e r were performed with a) the mu agonist ~H-dihydromorphine (0.4-37.6 nM) or b) the kappa agonist ~H-ethylketocyclazocine (0.1-14 nM). All points represent only specific binding from one experiment. Similar results were obtained in 3 separate experiments.
Vol. 29, No. 8, 1981
High Affinity Opiate & Enkephalin Binding
845
METHODS Naloxazone was synthesized as previously described (6). Rats were treated in vivo by either the subcutaneous administration of naloxazone or naloxone (300 mg/kg; 50 mg/ml in H20-dilute acetic acid). Twenty to twenty-four hours following the in vivo administration of drug, animals were sacrificed and brain membranes prepared as previously described (8). This membrane preparation includes extensive washing to remove both endogenous and exogenous ligands. Rinding assays were performed (8) using one or two ml aliquots of homogenate (20 mg wet weight tissue/ml). All incubations were performed for 60 minutes at 25°C in t r i p l i c a t e and all results expressed as specific binding, the d i f f e r ence in binding between t r i p l i c a t e s assayed in the presence and absence of i ~M levallorphan. Saturation analyses were performed over a wide concentration range of 3H-labelled ligands. To insure accuracy in the Scatchard analysis, free concentrations of ligand were determined d i r e c t l y by centrifuging additional samples and counting t h e i r supernatants. All binding experiments were performed at least three times each. Analgesia was deter~ined with a radiant lamp technique (7) as o r i g i n a l l y described by D'Amour and Smith (91 . Morphine sulphate was injected subcutaneously and beta-endorphin or O-alaL-metD-enkephalinamide intracerebrovent r i c u l a r l y . Latencies were established prior to and following drug administration and analgesia was defincd as the doubling of the pre-injection latency. S t a t i s t i c a l significance was established using the Fisher Exact Test. RESULTS: We investigated the effects of naloxazone treatment on the binding of a variety of 3H-labelled opiates and enkephalins. 3H-Naltrexone binding yields a c u r v i l i n e a r Scatchard which can be broken ~nto high and low a f f i n i t y binding (Fig. l a ) . The high a f f i n i t y component of ~H-naltrex~ne binding is selectively inhibited, in a manner similar to that seen with JH-naloxone (data not shown). This i n h i b i t i o n of binding is not decreased despite extensive washing which eliminates the inhibStion of reversible opiates. Thus, in vivo naloxazone i n h i b i t s the high a f f i n i t y binding of two separate narcotic antagonists. I t had been proposed that high a f f i n i t y agonist binding might correspend to low a f f i n i t y antagonist binding and vice versa (4). However, the r e l a t i v e l y selective i n h i b i t i o n of high a f f i n i t y mu agonist ~H-dihydromorphine binding by naloxazone (Fig. 2a) suggests that the high a f f i n i t y binding of both agonists and antagonists are the same. As with the mu agonist 3H-dihydromorphine, high a f f i n i t y binding of 3H-ethylketocyclazocine, a member of the kappa class of opiates ( i ) , is quite sensitive to the prolonged i n h i b i t i o n of naloxazone (Fig. 2b), as is the high a f f i n i t y binding of 3H-SKF 10,047, a sigma agonist. The discovery of the enkephalins (5) and the description of t h e i r pharma, cology (2) suggested that they might represent an additional class of receptors. However, naloxazone lowers high a f f i n i t y 3H-met5-enkephalin (Fig. 3a), 3H-leu5-enkephalin (Fig. 3b) and 3H-D-ala2-metS-enkephalinamide binding (Fig. 3c) in a manner identical to the mu and kappa opiates and antagonists. In mice, naloxazone's i n h i b i t i o n lasts approximately three days, with the gradual reappearance of high a f f i n i t y binding and s e n s i t i v i t y to morphine analgesia ( i 0 ) . A similar reappearance of opiate receptor binding after three days is found in rats using 3H-D-ala2-met5-enkephalinamide (Fig. 3d). In an e f f o r t to determine whether the high a f f i n i t y binding components of enkephalins and opiates blocked by naloxazone corresponded to the same s i t e ,
846
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Vol. 29, No. 8, 198
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Fio.3 Effect of naloxazone on enkephalin binding. Saturation studies on brain membranes treated with either naloxone or naloxazone 20-24 hours earlier were performed with a) 3H-met5-enkephalin (0.825 nM) in the presence of bacitracin (50 ~g/ml), b) 3H-leuBenk~phalin ~2-61 nM) in the presence of bacitracin (50 ~g/ml), or c) JH-D-alaZ-metb-enkephalinamide (0.3-13 riM), d) additional studies with 3H-ala2-met5-enkephalinamide (0.4-15 nM) were performed after 72 hours. All points represent only specific binding from one experiment. Similar results were obtained in 3 separate experiments.
direct competition experiments were performed with 3H-D-ala2-met5-enkephalinamide and morphine (Fig. 4). Morphine's inhibition of binding in washed brain membranes from rats treated the previous day with naloxazone is biphasic, almost identical to results previously reported (11,12). The i n i t i a l displacement, whose IC50 is less than 1 nM, corresponds to a morphine, or mu, site since morphine binds so potently; whereas the second displacement, whose IC50 is greater than 100 nM, reflects binding to an enkephalin-selective, or delta, site (11,12). Naloxazone treatment eliminates morphine's inhibition at low concentrations, suggesting that naloxazone blocks a morphine-sensitive recep-
Vol. 29, No. 8, 1981
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Fi9.4 Morphine Displacement of 3H-D-ala2-met5-enkephalinamide Binding in Tissue from Naloxone- and Naloxazone-Treated Rats. Rats were t r e a t ed with naloxone or naloxazone and 18-20 hours l a t e r s a c r i f i c e d and homogenates prepared as described in Methods. Tissue was assayed in t r i p l i c a t e with 3H-D-ala2-met5-enkephalinamide (i nM) and increasing concentrations of morphine. Points represent s p e c i f i c binding in one experiment. S i m i l a r results were obtained in 3 separate experiments.
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Fig.5 Effects of Naloxazone on Opiate Receptor Binding on Rat Spinal Cord. Rats treated with e i t h e r naloxone or naloxazone were s a c r i f i c e d 20 hours l a t e r , spinal cords removed, tissue prepared and assayed with 3H-naloxone (0.2-19 nM). A l l points represent s p e c i f i c binding from one experiment. Similar results were obtained in 3 separate experiments.
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Time (min) Fi9.6 Effects of Naloxazone on Morphine Analgesia. Groups of rats (n=3) were treated with saline (A), naloxone (e) or naloxazone (o) (250 mg/kg SC) and tested for analgesia (A) 18-22 hrs. or (B) 70-72 hrs. l a t e r with morphine sulfate (12 mg/kg). Results are the percentage of animals which were analgesic.
t o r . A similar loss of the i n i t i a l displacement by low concentrations of unlabelled ligand is found for ketocyclazocine, naloxone, levorphanol and levallorphan. Scatchard analysis of binding reveals a loss of the high a f f i n Z i t y component for those ligands tested (Figs. 1,2,3). Together, these studies suggest that all the ligands tested bind with highest a f f i n i t y to the same s i t e , corresponding to a morphine receptor. T a i l f l i c k analgesia is mediated primarily through spinal mechanisms (13). Since opiate receptors have been described in the spinal cord (14), we i n v e s t i gated the effects of naloxazone on spinal cord binding. In vivo administration of naloxazone produces a persistent lowering of H-naTox%n-e binding despite extensive washing. Saturation studies using 3H-naloxone demonstrate a biphasic Scatchard plot similar to those in brain (Fig. 5). Like brain, naloxazone s e l e c t i v e l y i n h i b i t s high a f f i n i t y binding with l i t t l e e f f e c t on low a f f i n i t y binding. Since naloxazone treatment blocked morphine analgesia in mice for longer than 24 hours (6,7,10), we also investigated its effects on analgesia in rats (Fig. 6). Morphine sulphate at 12 mg/kg, is analgesic in all rats treated the day before with naloxone or saline. In contrast, naloxazone treatment 24 hours e a r l i e r t o t a l l y blocks morphine's analgesic actions at this dose. Since Scatchard analysis has demonstrated the return of the vast majority of high a f f i n i t y binding three days a f t e r naloxazone treatment (Fig. 3d), morphine sulphate was administered to animals treated 3 days e a r l i e r with naloxazone, naloxone or saline (Fig. 6b). As expected, the naloxone and untreated groups
Vol. 29, No. 8, 1981
High Affinity Opiate & Enkephalin Binding
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demonstrate a prolonged analgesia lasting over two and one half hours. The naloxazone treated group is also analgesic, but the duration is decreased. These results are not unexpected since the levels of high a f f i n i t y binding had not yet reached completely normal levels by three days. Ue next investigated naloxazone's actions on opio#d peptide analgesia. As with morphine, no analgesic response from e i t h e r O-alaL-met~-enkephalinamide and betah-endorphin is detectable in naloxazone-treated animals in contrast to the marked analgesia in naloxone-treated controls.
DISCUSSION Numerous reports have suggested a variety of types of opiate receptors (1,2,4,11,12). Our results imply that all the 3H-ligands tested bind with highest a f f i n i t y to a single s i t e responsible for opiate and opioid peptide analgesia. This proposal rests on the combination of both Scatchard analysis and displacement experiments. Naloxazone blocks the high a f f i n i t y component in rats of mu (3H-dihydromorphine), kappa (3H-eth~Iketocylazocine) ~nd sigma (JH-SKF 10,047) opiate agonists, antaqonists (~H-naltrexone and ~H-naloxone) and enkephalins (3H-met5-enkephalin, 3H-leu5-enkephalin and 3H-D-ala2-met 5enkephalinamide). Additional studies with the most specific delta compound, 125 - . l - D - a l a 2-D-leu -enkephalln, also show a loss of high a f f i n i t y binding (15).
850
High Affinity Opiate & Enkephalin Binding
Vol. 29, No. 8, 198]
To ensure that naloxazone does not merely have similar effects on d i f f e r e n t receptors, d i r e c t competition experiments were performed. As i l l u s t r a t e d in Figure 4, a portion of 33H-D-ala2-met5-enkephalinamide binding is easily displaced by low morphine concentration (
Vol. 29, No. 8, 1981
High Affinity Opiate and Enkephalin Binding
85
ACKNOWLEDGEMENTS We thank Mary Buatti for her excellent technical assistance and Drs. Posner, Foley, I n t u r r i s i and Ahern for t h e i r help, and Dr. W i l l e t t e for his generous g i f t of 3H-naltrexone. This work has been supported in part by a grant from the American Cancer Society (PDTI69) and NIDA (DA02615). G.W. Pasternak is supported by NINCDS I-KO7NS415. BIBLIOGRAPHY 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
W.R. MARTIN, C.G. EADES, J.A. THOMPSON, R.A. HUPPLER, and P.E. G i l b e r t , J. Pharmacol. Exp. Therap. 197 517 (1976). J. LORD, A. WATERFIELD, J. HUGHES, and H.W. KOSTERLITZ, Nature 267 495 (1977). G.W. PASTERNAK and S.H. SNYDER, Molec. Pharmacol. I0 183-193 (1974). G.W. PASTERNAK and S.H. SNYDER, Nature 253 563-565 (1975). J. HUGHES, R.W. SMITH, H. KOSTERLITZ, L. FOTHERGILL, B.A. MORGAN, and H.R. MORRIS, Nature 258 577-599 (1975). G.W. PASTERNAKand E.F. HAHN, J. Med. Chem., 23 674-676 (1980). G.W. PASTERNAK, S.R. CHILDERS, and S.H. SNYDER, Science 208 514-516 (1980). G.W. PASTERNAK, H.A. WILSON, and S.H. SNYDER, Molec. Pharmacol 11 340-351 (1975). F.E. D'AMOUR and D.L. SMITH, J. Pharmacol. Exp. Therap. 72 74-79 (1941). G.W. PASTERNAK, S.R. CHILDERS, and S.H. SNYDER, J. Pharmacol. Exp. Therap. 214 455-462 (1980). K.J. CHANG, B.R. COOPER, E. HAZUM, and P. CUATRECASAS, Molec. Pharmacol. 16 91-104 (1979) K.J. CHANGand P. CUATRECASAS, J. Biol. Chem. 254 2610-2618 (1979). S. IRWIN, R.W. HOUDE, D.R. BENNETT, L.C. HENDERSHOT, and M.H. Seevers, J. Pharmacol. Exp. Therap. i01 132-143 (1951). C. LaMOTTE, C.B. PERT, and S.H. Snyder, Brain Res. 112 407-412 (1976). E. HAZUM, K.J. CHANG, P. CUATRECASAS, and G.W.PASTERNAK, Life Sci. (in press). P.S. PORTOGHESE, D.L. LARSON, J.B. LIANG, A.E. TAKEMORI, and T.P. CARUSO, J. Med. Chem. 21 598-599 (1978). C.B. PERT, G.W. PASTERNAK, and S.H. SNYDER, Science 182 1359-1361 (1973). H.A. WILSON, G.W. PASTERNAK, and S.H. SNYDER, Nature 253 448-450 (1975). G.W. PASTERNAK, A.M. SNOWMAN, and S.H. SNYDER,'Molec. Pharmacol. i i 735-744 (1975). B.L. WOLOZIN and G.W. PAST[RNAK, Proc. Nat. Acad. Sci. USA (in press). A-Z ZHANG, J.K. CHANG, and G.W. PASTERNAK, L i f e Sci. (in press). G.W. PASTERNAK and S.H. SNYDER, Molec. Pharmacol. LI 478-484 (1975). 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. (in press).
Pergamon Press
OPIATES AND ENKEPHALINS: A COMMONBINDING SITE MEDIATES TIIEIR ANALGESIC ACTIONS IN RATS An Zhong Zhang and Gavril W. Pasternak George C. Cotzias Laboratory of Neuro-Oncology Memorial Sloan-Kettcrinq Cancer Center and Departments of PharmacolocLv and Neurology, Cornell University Medical College, New York, NY 10021 (Received in final form June 22, 1981)
SUMMARY 3H-Labelled opiate and enkephalin ligands appear to bind with highest a f f i n i t y to a single site responsible for t h e i r analgesic properties. Administered in vivo, naloxazone, an irreversible opiate, selectively i n h i b i t s for over 24 hours the high a f f i n i t y binding of aH-labelled mu, and kappa opiates and enkephalins. This i n h i b i t i o n of binding gradually resolves over 3 days, perhaps correlating with receptor t~rnover. Naloxazone treatment also abolishes morphine, D-ala~-metD-enkephalinamide and betahendorphin analgesia. Although morphine and D-ala2-met5-enkepha linamide bind with similar potencies to the high a f f i n i t y s i t e , morphine's potency for the low a f f i n i t y D-ala2-met5-enkephalinamide site is f~r less than the enkephalin analog. These results imply that all ~H-ligands examined bind with hiqhest a f f i n i t y to a mu-like receptor while low a f f i n i t y D-ala2-met5-enkephalinamide binding, with a KD of 6 nM, represents a d e l t a - l i k e receptor. The concept of multiple populations of opiate receptors has been proposed on the basis of pharmacological (1,2) and biochemical (3,4) c r i t e r i a . Pharmacologically, opiates have been assigned to one of three major groups named after prototypic drugs: mu (morphine), kappa (ketocyclazocine) and sigma (SKF 10,047). The discovery of the enkephalins (5) led to a fourth c l a s s i f i c a t i o n for the peptides: delta (enkephalin) (2). Riochemically defined subpopulations of receptors have been suggested from binding studies. Despite the i n a b i l i t y of Scatchard analyses to d i f f e r e n t i a t e subpopulations of opiate receptors in early studies, enzymatic treatments raised the p o s s i b i l i t y of t h e i r existence (3). As techniques evolved, nonlinear Scatchards identifying novel, higher a f f i n i t y binding were demonstrated for opiate agonists and antagonists (4) and l a t e r for enkephalin (2) and other opiate drugs. Previous studies have established the ab#lity of naloxazone, an i r r e v e r s i b l e opiate antagonist, to block high a f f i n i t y jH-naloxone binding and morphine analgesia in mice (6,7). Using naloxazone, we now describe in rats i t s actions on the binding of a variety of enkephalins, mu, kappa and antagonist opiates and on opiate and opioid peptide analgesia. Address correspondence to Dr. G.W. Pasternak, Memorial Sloan-Kettering Cancer Center, 1275 York Avenue, New York, NY 10021 0024-3205/81/080843-09502.00/0 Copyright (e) 1981 Pergamon Press Ltd.
844
High Affinity Opiate & Enkephalin Binding
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Vol. 29, No. 8, 198
-n
Treotment • o
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125
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Vol. 29, No. 8, 1981
High Affinity Opiate & Enkephalin Binding
845
METHODS Naloxazone was synthesized as previously described (6). Rats were treated in vivo by either the subcutaneous administration of naloxazone or naloxone (300 mg/kg; 50 mg/ml in H20-dilute acetic acid). Twenty to twenty-four hours following the in vivo administration of drug, animals were sacrificed and brain membranes prepared as previously described (8). This membrane preparation includes extensive washing to remove both endogenous and exogenous ligands. Rinding assays were performed (8) using one or two ml aliquots of homogenate (20 mg wet weight tissue/ml). All incubations were performed for 60 minutes at 25°C in t r i p l i c a t e and all results expressed as specific binding, the d i f f e r ence in binding between t r i p l i c a t e s assayed in the presence and absence of i ~M levallorphan. Saturation analyses were performed over a wide concentration range of 3H-labelled ligands. To insure accuracy in the Scatchard analysis, free concentrations of ligand were determined d i r e c t l y by centrifuging additional samples and counting t h e i r supernatants. All binding experiments were performed at least three times each. Analgesia was deter~ined with a radiant lamp technique (7) as o r i g i n a l l y described by D'Amour and Smith (91 . Morphine sulphate was injected subcutaneously and beta-endorphin or O-alaL-metD-enkephalinamide intracerebrovent r i c u l a r l y . Latencies were established prior to and following drug administration and analgesia was defincd as the doubling of the pre-injection latency. S t a t i s t i c a l significance was established using the Fisher Exact Test. RESULTS: We investigated the effects of naloxazone treatment on the binding of a variety of 3H-labelled opiates and enkephalins. 3H-Naltrexone binding yields a c u r v i l i n e a r Scatchard which can be broken ~nto high and low a f f i n i t y binding (Fig. l a ) . The high a f f i n i t y component of ~H-naltrex~ne binding is selectively inhibited, in a manner similar to that seen with JH-naloxone (data not shown). This i n h i b i t i o n of binding is not decreased despite extensive washing which eliminates the inhibStion of reversible opiates. Thus, in vivo naloxazone i n h i b i t s the high a f f i n i t y binding of two separate narcotic antagonists. I t had been proposed that high a f f i n i t y agonist binding might correspend to low a f f i n i t y antagonist binding and vice versa (4). However, the r e l a t i v e l y selective i n h i b i t i o n of high a f f i n i t y mu agonist ~H-dihydromorphine binding by naloxazone (Fig. 2a) suggests that the high a f f i n i t y binding of both agonists and antagonists are the same. As with the mu agonist 3H-dihydromorphine, high a f f i n i t y binding of 3H-ethylketocyclazocine, a member of the kappa class of opiates ( i ) , is quite sensitive to the prolonged i n h i b i t i o n of naloxazone (Fig. 2b), as is the high a f f i n i t y binding of 3H-SKF 10,047, a sigma agonist. The discovery of the enkephalins (5) and the description of t h e i r pharma, cology (2) suggested that they might represent an additional class of receptors. However, naloxazone lowers high a f f i n i t y 3H-met5-enkephalin (Fig. 3a), 3H-leu5-enkephalin (Fig. 3b) and 3H-D-ala2-metS-enkephalinamide binding (Fig. 3c) in a manner identical to the mu and kappa opiates and antagonists. In mice, naloxazone's i n h i b i t i o n lasts approximately three days, with the gradual reappearance of high a f f i n i t y binding and s e n s i t i v i t y to morphine analgesia ( i 0 ) . A similar reappearance of opiate receptor binding after three days is found in rats using 3H-D-ala2-met5-enkephalinamide (Fig. 3d). In an e f f o r t to determine whether the high a f f i n i t y binding components of enkephalins and opiates blocked by naloxazone corresponded to the same s i t e ,
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Fio.3 Effect of naloxazone on enkephalin binding. Saturation studies on brain membranes treated with either naloxone or naloxazone 20-24 hours earlier were performed with a) 3H-met5-enkephalin (0.825 nM) in the presence of bacitracin (50 ~g/ml), b) 3H-leuBenk~phalin ~2-61 nM) in the presence of bacitracin (50 ~g/ml), or c) JH-D-alaZ-metb-enkephalinamide (0.3-13 riM), d) additional studies with 3H-ala2-met5-enkephalinamide (0.4-15 nM) were performed after 72 hours. All points represent only specific binding from one experiment. Similar results were obtained in 3 separate experiments.
direct competition experiments were performed with 3H-D-ala2-met5-enkephalinamide and morphine (Fig. 4). Morphine's inhibition of binding in washed brain membranes from rats treated the previous day with naloxazone is biphasic, almost identical to results previously reported (11,12). The i n i t i a l displacement, whose IC50 is less than 1 nM, corresponds to a morphine, or mu, site since morphine binds so potently; whereas the second displacement, whose IC50 is greater than 100 nM, reflects binding to an enkephalin-selective, or delta, site (11,12). Naloxazone treatment eliminates morphine's inhibition at low concentrations, suggesting that naloxazone blocks a morphine-sensitive recep-
Vol. 29, No. 8, 1981
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Time (min) Fi9.6 Effects of Naloxazone on Morphine Analgesia. Groups of rats (n=3) were treated with saline (A), naloxone (e) or naloxazone (o) (250 mg/kg SC) and tested for analgesia (A) 18-22 hrs. or (B) 70-72 hrs. l a t e r with morphine sulfate (12 mg/kg). Results are the percentage of animals which were analgesic.
t o r . A similar loss of the i n i t i a l displacement by low concentrations of unlabelled ligand is found for ketocyclazocine, naloxone, levorphanol and levallorphan. Scatchard analysis of binding reveals a loss of the high a f f i n Z i t y component for those ligands tested (Figs. 1,2,3). Together, these studies suggest that all the ligands tested bind with highest a f f i n i t y to the same s i t e , corresponding to a morphine receptor. T a i l f l i c k analgesia is mediated primarily through spinal mechanisms (13). Since opiate receptors have been described in the spinal cord (14), we i n v e s t i gated the effects of naloxazone on spinal cord binding. In vivo administration of naloxazone produces a persistent lowering of H-naTox%n-e binding despite extensive washing. Saturation studies using 3H-naloxone demonstrate a biphasic Scatchard plot similar to those in brain (Fig. 5). Like brain, naloxazone s e l e c t i v e l y i n h i b i t s high a f f i n i t y binding with l i t t l e e f f e c t on low a f f i n i t y binding. Since naloxazone treatment blocked morphine analgesia in mice for longer than 24 hours (6,7,10), we also investigated its effects on analgesia in rats (Fig. 6). Morphine sulphate at 12 mg/kg, is analgesic in all rats treated the day before with naloxone or saline. In contrast, naloxazone treatment 24 hours e a r l i e r t o t a l l y blocks morphine's analgesic actions at this dose. Since Scatchard analysis has demonstrated the return of the vast majority of high a f f i n i t y binding three days a f t e r naloxazone treatment (Fig. 3d), morphine sulphate was administered to animals treated 3 days e a r l i e r with naloxazone, naloxone or saline (Fig. 6b). As expected, the naloxone and untreated groups
Vol. 29, No. 8, 1981
High Affinity Opiate & Enkephalin Binding
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demonstrate a prolonged analgesia lasting over two and one half hours. The naloxazone treated group is also analgesic, but the duration is decreased. These results are not unexpected since the levels of high a f f i n i t y binding had not yet reached completely normal levels by three days. Ue next investigated naloxazone's actions on opio#d peptide analgesia. As with morphine, no analgesic response from e i t h e r O-alaL-met~-enkephalinamide and betah-endorphin is detectable in naloxazone-treated animals in contrast to the marked analgesia in naloxone-treated controls.
DISCUSSION Numerous reports have suggested a variety of types of opiate receptors (1,2,4,11,12). Our results imply that all the 3H-ligands tested bind with highest a f f i n i t y to a single s i t e responsible for opiate and opioid peptide analgesia. This proposal rests on the combination of both Scatchard analysis and displacement experiments. Naloxazone blocks the high a f f i n i t y component in rats of mu (3H-dihydromorphine), kappa (3H-eth~Iketocylazocine) ~nd sigma (JH-SKF 10,047) opiate agonists, antaqonists (~H-naltrexone and ~H-naloxone) and enkephalins (3H-met5-enkephalin, 3H-leu5-enkephalin and 3H-D-ala2-met 5enkephalinamide). Additional studies with the most specific delta compound, 125 - . l - D - a l a 2-D-leu -enkephalln, also show a loss of high a f f i n i t y binding (15).
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High Affinity Opiate & Enkephalin Binding
Vol. 29, No. 8, 198]
To ensure that naloxazone does not merely have similar effects on d i f f e r e n t receptors, d i r e c t competition experiments were performed. As i l l u s t r a t e d in Figure 4, a portion of 33H-D-ala2-met5-enkephalinamide binding is easily displaced by low morphine concentration (
Vol. 29, No. 8, 1981
High Affinity Opiate and Enkephalin Binding
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ACKNOWLEDGEMENTS We thank Mary Buatti for her excellent technical assistance and Drs. Posner, Foley, I n t u r r i s i and Ahern for t h e i r help, and Dr. W i l l e t t e for his generous g i f t of 3H-naltrexone. This work has been supported in part by a grant from the American Cancer Society (PDTI69) and NIDA (DA02615). G.W. Pasternak is supported by NINCDS I-KO7NS415. BIBLIOGRAPHY 1. 2. 3. 4. 5. 6. 7. 8. 9. I0. ii. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23. 24.
W.R. MARTIN, C.G. EADES, J.A. THOMPSON, R.A. HUPPLER, and P.E. G i l b e r t , J. Pharmacol. Exp. Therap. 197 517 (1976). J. LORD, A. WATERFIELD, J. HUGHES, and H.W. KOSTERLITZ, Nature 267 495 (1977). G.W. PASTERNAK and S.H. SNYDER, Molec. Pharmacol. I0 183-193 (1974). G.W. PASTERNAK and S.H. SNYDER, Nature 253 563-565 (1975). J. HUGHES, R.W. SMITH, H. KOSTERLITZ, L. FOTHERGILL, B.A. MORGAN, and H.R. MORRIS, Nature 258 577-599 (1975). G.W. PASTERNAKand E.F. HAHN, J. Med. Chem., 23 674-676 (1980). G.W. PASTERNAK, S.R. CHILDERS, and S.H. SNYDER, Science 208 514-516 (1980). G.W. PASTERNAK, H.A. WILSON, and S.H. SNYDER, Molec. Pharmacol 11 340-351 (1975). F.E. D'AMOUR and D.L. SMITH, J. Pharmacol. Exp. Therap. 72 74-79 (1941). G.W. PASTERNAK, S.R. CHILDERS, and S.H. SNYDER, J. Pharmacol. Exp. Therap. 214 455-462 (1980). K.J. CHANG, B.R. COOPER, E. HAZUM, and P. CUATRECASAS, Molec. Pharmacol. 16 91-104 (1979) K.J. CHANGand P. CUATRECASAS, J. Biol. Chem. 254 2610-2618 (1979). S. IRWIN, R.W. HOUDE, D.R. BENNETT, L.C. HENDERSHOT, and M.H. Seevers, J. Pharmacol. Exp. Therap. i01 132-143 (1951). C. LaMOTTE, C.B. PERT, and S.H. Snyder, Brain Res. 112 407-412 (1976). E. HAZUM, K.J. CHANG, P. CUATRECASAS, and G.W.PASTERNAK, Life Sci. (in press). P.S. PORTOGHESE, D.L. LARSON, J.B. LIANG, A.E. TAKEMORI, and T.P. CARUSO, J. Med. Chem. 21 598-599 (1978). C.B. PERT, G.W. PASTERNAK, and S.H. SNYDER, Science 182 1359-1361 (1973). H.A. WILSON, G.W. PASTERNAK, and S.H. SNYDER, Nature 253 448-450 (1975). G.W. PASTERNAK, A.M. SNOWMAN, and S.H. SNYDER,'Molec. Pharmacol. i i 735-744 (1975). B.L. WOLOZIN and G.W. PAST[RNAK, Proc. Nat. Acad. Sci. USA (in press). A-Z ZHANG, J.K. CHANG, and G.W. PASTERNAK, L i f e Sci. (in press). G.W. PASTERNAK and S.H. SNYDER, Molec. Pharmacol. LI 478-484 (1975). 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. (in press).