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Naloxazone irreversibly inhibits the high affinity binding of [125I]D-ala2-D-leu5-enkephalin
Life Sciences, Vol. 28, pp. 2973-2979 Printed in the U.S.A.
Pergamon Press
NALOXAZONE IRREVERSIBLY INHIBITS THE HIGH AFFINITY BINDING OF [1251]D-ala2-D-Ieu5-ENKEPHALIN Eli Hazum, Kwen-Jen Chang, Pedro Cuatrecasas and Gavril W. Pasternak* The Wellcome Research Laboratories, Research Triangle Park, NC 27709 *George C. Cotzias Laboratory of Neuro-Oncology Memorial Sloan-Kettering Cancer Center, New York 10021 (Received in final form April 27, 1981)
SUMMARY Scatchard analyses of [1251]D-ala2-D-leu5-enkephalin binding in rat brain membranes are c u r v i l i n e a r , suggesting low and high a f f i n i t y sites. Treating the membranes with naloxazone abolishes the high a f f i n i t y bindinq with slight effect on low a f f i n i t y binding. Displacement of [1251]-D-alaC-D-leuD-enkephalin binding by merphine in untreated membranes is biphasic. Displacement by morphine in naloxazone-treated tissue is monophasic, with no i n h i b i t i o n by low concentrations of morphine. Naloxazon~ treatment has l i t t l e effect on displacements by unlabeled D-ala -D-leub-enkephalin. Binding in N4TGI neuroblastoma c e l l s , which demonstrates a linear Scatchard plot with single a f f i n i t y constant similar to that of the low a f f i n i t y binding in brain, is less sensitive to naloxazone's actions. Naloxazone treatment in vivo inhibits D-ala2-D-leu5-enkephalin analgesia. INTRODUCTION Studies of enkephalin binding to brain membranes have demonstrated c u r v i l i n ear Scatchard plots ( I ) similar to those described e a r l i e r for opiate agonists and antagonists (2). These reports raised the p o s s i b i l i t y that both opiates and enkephalins might bind to multiple receptor sub-populations. Detailed binding studies soon showed marked differences in the binding of enkephalin peptides and opiates such as naloxone and morphine (3-5), suggesting the existence of a sub-population of opiate receptors in the central nervous system with higher a f f i n i t y for enkephalin-like peptides than for morphine-like alkaloids. This sub-population has been clearly demonstrated in regional studies of opiate receptor binding (5). In marked contrast to other regions, the receptors in the frontal cortex bound enkephalins far more extensively and with higher a f f i n i t y than opiate alkaloids such as morphine. Naloxazone (6-9) has proven useful in the investigation of high and low a f f i n i t y opiate receptor binding. Whereas naloxazone had no demonstrable effect on a variety of non-opioid receptor systems, i t selectively blocked the high a f f i n i t y binding of a number of [JH]enkephalin analogs (met-enkephalin, l~u-enkephalin, D-ala2-met5-enkephalinamide ( i 0 ) ; as well as a variety of [JH] opiates including the mu agonists, dihydromorphine and morphine (7,8); the kappa agonist, ethylketocyclazocine(9); the sigma agonist, SKF 10,047 (Pasternak, Buatti and Spiegel, in preparation) and the antagonists naloxone and naltrexone ( i 0 ) . We now report the effects of naloxazone on the binding of the potent delta ligand, [1251] D-ala2-D-leu5-enkephalin. 0024-3205/81/262973-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
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Vol. 28, No. 26, 1981
MATERIALS AND METHODS Enkephalin analogs were synthesized by Dr. S. Wilkinson, the Wellcome Research Laboratories, Beckenham, England. Morphine sulfate was obtained from M a l l i n c r o t Chemical Works, and naloxone from Endo Laboratories. Naloxazone was synthesized as previously described (6). [1251] Labeled D-ala2-D-leu 5enkephalin was prepared as previously described using chloramine-T (4,5). Brain membranes were prepared as previously described (4,5). B r i e f l y , whole rat brains were homogenized with a Polytron homogenizer at 4 ° C in 0.32 M sucrose in 5 mM tris-HC1 buffer, pH 7.4. The nuclei and most of the mitochondria were removed by c e n t r i f u g a t i o n f o r 15 minutes at 5,000 g and supernatant was f u r t h e r centrifuged for 30 minutes at 40,000 g and f i n a l l y suspended in 50 mM t r i s - H C l b u f f e r , pH 7.4, at 4°C. N4TGI neuroblastoma cells were grown on DMEM with 5% f e t a l bovine serum. The cells were grown to confluence and removed from the flask by incubation with 1 mM EDTA f o r 5 minutes at 37°C. The detached cells were washed 3 times with 10 mM t r i s buffer, pH 7.7, containing 0.25 M sucrose, 2 mM MgCI2 and 5 mM glucose and f i n a l l y suspended in the same buffer. Membranes were treated with naloxazone (2 ~M) at room temperature for 40 minutes followed by four washes. Each wash consisted of a 15 minute incubation at 37°C and c e n t r i f u g a t i o n . This procedure is s u f f i c i e n t to remove reversible ligands ( i i ) . Binding assays were performed as previously described (3,4). B r i e f l y , the radioactive enkephalin (0.07 nM, specific a c t i v i t y 2,000 Ci/mmole) was incubated at 25°C for 60 minutes with a final volume of 0.25 ml containing N4TGI c e l l s (4 x 106 c e l l s / m l ) or brain membranes (0.7 mg/ml p r o t e i n ) , and the binding was measured by f i l t r a t i o n with vacuum through Whatman GF/C f i l t e r s . Specific binding represents the bound r a d i o a c t i v i t y displaced by unlabeled D-ala2-D-leu5-enkephalin (I ~M). Each value is the mean of duplicate incubations varying less than 7%. Scatchard analyses were f i t by computer using a nonlinear least square program and a f f i n i t i e s and sites determined (Pasternak and Reiman, in preparation). Analgesia was tested 10 min following the i n j e c t i o n i . c . v , of D-ala2-D-leu5-enkephalin (i ~I dissolved in I0 ~I E l l i o t ' s B solution, an a r t i f i c i a l CSF) in mice treated 20 hrs e a r l i e r with e i t h e r naloxone or naloxazone (200 mg/kg s.c.) as previously described (l-g). RESULTS
Saturation curves of [1251]D-ala2-D-leu5-enkephalin to rat brain y i e l d c u r v i l i n e a r Scatchard plots (Fig. I ) . Such plots can be segmented into high a f f i n i t y (KD 0.52 nM; 0.89 fmoles) and a low a f f i n i t y (KD 5.23nM; 11.04 fmoles) components. Treatment of membranes with naloxazone in v i t r o markedly lowers the high a f f i n i t y binding, transforming the c u r v i l i n e a r Scatchard plots into linear ones (KD 5.9 nM; 9.47 fmoles). Whereas the high a f f i n i t y component is v i r t u a l l y abolished, the low a f f i n i t y one is decreased by only 15%, with no s i g n i f i c a n t change in a f f i n i t y . The persistence of this i n h i b i t i o n , despite extensive washing of the brain membranes which does eliminate the i n h i b i t i o n of reversible drugs, strongly implies that naloxazone's actions are irreversible. To f u r t h e r explore the properties of the low a f f i n i t y binding remaining a f t e r naloxazone treatment, displacement experiments were performed on [1251]D-ala2-D-leu5-enkephalin binding with both morphine and unlabeled D-ala2-D-leu5-enkephalin (Fig.2). Control tissue demonstrates a monophasic displacement of binding by unlabeled D-ala2-D-leu5-enkephalin with an IC50 of approximately 2.5 nM. Naloxazone treatment does not change the displacement by unlabeled D-ala2-D-leu5-enkephalin s i g n i f i c a n t l y (IC50 4 nM).
Vol. 28, No. 26, 1981
Interactions of Naloxazone and DADL-enk
I
2975
I
I
KD(nM) Bmax(fmOles)
4 1 ,,p o • -
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Fig.l Scatchard plots of [1251]D-Ala2-D-Leu5-enkephalin binding to brain membranes (1) and to brain membranes pretreated with 2 ~M naloxazone (o) and then washed. The data were determined from saturation experiments over a wide concentration range (0.06 nM to 13 nM). All binding presented is s p e c i f i c . This experiment has been repeated 3 times. Morphine displacement of control tissue is biphasic (Fig. 2 bottom), as previously described (3,4), with an i n i t i a l displacement at concentrations less than 0.3 nM and a f u r t h e r displacement occurring with an approximate "IC50" of 50 nM. The total apparent IC50, which is actually a mixture of binding sites is about 15 nM. Abolishing the high a f f i n i t y binding with naloxazone correspondingly changes the morphine displacement curves. Morphine displaces binding in the treated tissue in a monophasic fashion with an approximate IC50 of 63 nM, a value similar to the IC50 of the lower portion of the control curve. Thus, blockade of high a f f i n i t y binding by naloxazone eliminates the displacement of [1251] D-ala-D-leu-enkephalin binding by morphine at low concentrations. Opiate binding in N4TGI neuroblastoma c e l l s is primarily delta or enkephalin in nature (3,4). Scatchard analysis of [1251] D-ala2-D-leu5-enkephalin binding in these c e l l s (Fig.3) is l i n e a r , suggesting a single population of s i t e s ; t h e i r a f f i n i t y (3.3 nM) is similar to that of the low a f f i n i t y sites found in brain membranes (5.23 nM). Treatment of these cells with naloxazone results in a r e l a t i v e l y small reduction in the number of binding sites (about 35%) with no change in a f f i n i t y . This reduction, which is similar to the decrease in low a f f i n i t y sites normally seen with brain membranes (15-20%; Fig. I ) , can be contrasted to the v i r t u a l elimination of the high a f f i n i t y binding observed upon treatment of brain membranes.
2976
Interactions of Naloxazone and DADL-enk
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Vol. 28, No. 26,
,
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Competition of [1251] labeled D-Ala~-l)-Leu5-enkephalin binding to brain Membranes (o) and naloxazone-treated membrane (o) by D-AIa2-DLeu5-~nkephalin (top) and by morphine (bottom). Radioactive D-Ala2D-Leu -enkephalin (0.07 nM) was incubated with increasing concentrations of unlabeled morphine or peptide, and specific binding was determined and expressed as percent of specific control binding. This experiment has been replicated three times. TABLE 1.
D-Ala2-D-Leu5-Enkephalin Analgesia in NaloxazoneTreated Mice Treatment
Analgesia (n)
Control Naloxone Naloxazone
100% (5) 93% (14) 42% (7)*
p < 0.01
Mice were treated with naloxone or naloxazone (200 mg/kg sc) and administered D-ala2-D-leu5-enkephalin. Analgesia, defined as latency twice the baseline value, was tested i0 min a f t e r administrat i o n of drug. S i g n i f i c a n c e values determined by the Fisher Exact Test show no d i f f e r e n c e between the control and naloxone group. The naloxazone group is s i g n i f i c a n t l y d i f f e r e n t from the naloxone group (p < 0.025) and the pooled naloxone and control group (p < 0.01).
198]
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Interactions of Naloxazone and DADL-enk
2977
Naloxazone treatment in vivo markedly attenuates the analgesic actions of morphine (7,8), ketocyclazo~ine (9), SKF 10,047 (Pasternak, Buatti and Spiegel, in preparation), enkephalins and beta-endorphin (30). The similar blockade of the high a f f i n i t y binding of these drugs ~nd [125D]-D-ala2-b-leu5-enkephalin led us to test naloxazone s actions on D-ala~-D-leuU-enkephalin analgesia (Table 1). At a dose of i ~g, D-ala2-D-leu5-enkephalin is analgesic in all untreated and in 13 of 14 naloxone-treated mice. However, only 42% (p < 0.01) of the naloxazone animals were analgesic.
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0 0.5 1.0 1.5 2.0 [ "'1] D-ala'-D-leu'-Enkephalin Binding (f m01es/lO' cells) Scatchard plots of [1251] D-AlaL-D-Leu5-enkephalin binding to N4TG1 neuroblastoma cells (e) and to N4TGI cells pretreated with 2 pM naloxazone (o) and then washed. The data were determined from saturation experiments over a wide concentration range (0.06 nM to 13 nM). 0.25 ml of N4TGI cells (4 X I0 ° cells/ml) were incubated with various concentrations of [1251]-labeled D-Ala2-D-Leu 5 enkephafin and assayed as described in METHODS. All binding is specific, and the experiment has been replicated three times. DISCUSSION Both the curvilinear Scatchard analysis and the biphasic displacement of [1251]D-ala2-D-leu5-enkephalin by morphine suggest that [1251]D-ala2-D-leu 5enkephalin binds to two populations of receptors, presumably mu and delta typeS. Interpretation of complex displacement curves is extremely d i f f i c u l t and correlations between mu and delta receptors and high and low a f f i n i t y sites have only been tentative. Naloxazone provides a unique method of simplifying such analyses. These studies clearly show that naloxazone's i r r e v e r s i b l e i n h i b i t i o n is r e l a t i v e l y selective for the high a f f i n i t y component of [1251]Dala2-D-leu5-enkephalin binding and the high a f f i n i t y morphine displacement of [1251]D-ala2-D-leu5-enkephalin, suggesting they might be the same receptor.
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Interactions of Naloxazone and DADL-enk
Vo].. 28, No. 26, 1981
Although the assignment of mu or delta c h a r a c t e r i s t i c s remains t e n t a t i v e , these studies suggest that high a f f i n i t y [1251]D-ala2-D-leu5-enkephalin binding is mu-like and low a f f i n i t y binding d e l t a - l i k e . Naloxazone treatment blocks high a f f i n i t y binding of the mu agonists [3H]morphine and [3H]dihydromorphine with the same potency and s e l e c t i v i t y that i t blocks high a f f i n i t y [1251]Dala2-D-leu5-enkephalin binding (7,8,10-12) (Fig. 2) and s i q n i f i c a n t l y lessens morphine's potency in competitively displacing [1251]D-ala2-D-leu5-enkephalin binding (Fig. 2). Low a f f i n i t y [1251]D-ala2-D-leu5-enkephalin binding appears to be quite d i f f e r e n t from the hiqh a f f i n i t y s i t e . The term "low a f f i n i t y " s i t e is a r e l a t i v e one, since [1251]D-ala2-D-leu5-enkephalin s t i l l binds very potently with a KD of about 5 nM. Unlike the high a f f i n i t y s i t e , the low a f f i n i t y one is not very sensitive to naloxazone and binds D-ala2-D-leu5-enkephalin (IC50 4 riM) f a r more potently than morphine (IC50 63 nM), f u l f i l l i n g the major c r i t e r i a of a delta receptor. In neuroblastoma c e l l s , which are predominantly delta and which bind [1251]D-ala2-D-leu5-enkephalin with an a f f i n i t y (KD 3.3 nM) similar to the low a f f i n i t y s i t e in brain (KD 5 nM), naloxazone reduces the number of sites to a far lesser extent than in the brain, which has a higher proportion of mu receptors. This t e n t a t i v e assignment of high a f f i n i t y sites as mu-like and low a f f i n i t y sites as d e l t a - l i k e is consistent with the regional l o c a l i z a t i o n of these receptor types (5,13). Chang et al (5) clearly showed that the receptors in the frontal cortex strongly preferred enkephalins, whereas the hypothalamus binding sites were predominantly opiate, or mu-like. When brain regions were tested for t h e i r s e n s i t i v i t y to naloxazone (13), binding in the frontal cortex was v i r t u a l l y unchanged, whereas binding in the hypothalamus was decreased over 40% (p < 0.005). Naloxazone i n h i b i t s morphine, ketocyclazocine, B-endorphin, D-ala 2met5-enkephalinamide, D-ala2-met5-enkephalin (6-10,12) and SKF 10,047 analgesia (Pasternak, Buatti and Spiegel, in preparation). The s i m i l a r reduction of D-ala2-D-leu5-enkephalin analgesia, coupled with the evidence suggesting that they all bind with highest a f f i n i t y to the same s i t e , suggests that a single s i t e mediates opiate (mu, kappa and sigma), enkephalin and B-endorphin analgesia seen with standard drug doses. Why analgesia remains a f t e r naloxazone treatment at very high drug doses remains unknown. Most probably a small number of high a f f i n i t y sites are not blocked by naloxazone. Although high a f f i n i t y binding following naloxazone treatment cannot be discerned by Scatchard analgesia, this technique might not detect very low levels of high a f f i n i t y sites (10-15% of normal levels) in view of the large number of low a f f i n i t y s i t e s . In addition, naloxazone administered subcutaneously probably has s i g n i f i c a n t v a r i a b i l i t y in i t s absorption and entry into the brain, resulting in less than complete blockade of all the high a f f i n i t y sites in all animals. However, an additional analgesic system mediated by d i f f e r e n t receptors requiring larger drug dosages cannot be excluded. ACKNOWLEDGEMENTS GWP is a r e c i p i e n t of NINCDS Teacher Investigator Award (1 KO7NS415-02). This work was supported in part by a grant from NIDA (DA 02615) and the American Cancer Society (PDT 169). We thank Dr. R. Reiman for his help with the computer programs and M. Buatti for her technical assistance.
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REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Lord, J . , Waterfield, A., Hughes, J. and K o s t e r l i t z , H.W. Nature 267, 495 (1977). Pasternak, G.W. and Snyder, S.H. Nature 253, 563-564 (1975). Chang, K-J. , M i l l e r , R.J. and Cuatrecasas, P. Molec.Pharmacol. 14, 961-970 (1978). Chang, K-J and Cuatrecasas, P. J. Biol. Chem. 254:2610-2618 (1979). Chang, K-J, Cooper, B.R., Hazum, E. and Cuatrecasas, P. Molec. Pharm. 16: 91- 104 (1979). Pasternak, G.W. and Hahn, E.F. J. Med. Chem. 23:674-676 (1980). Pasternak, G.W., Childers, S.R. and Snyder, S.H. Science 208:514-516 (1980). Pasternak, G.W., Childers, S.R. and Snyder, S.H. J. Pharmacol. Exp. Therap. 214:455-462 (1980). Pasternak, G.W. Proc. Nat. Acad. Sci., USA 77:3691-3694 (1980). Pasternak, G.W. Neurology , in press. Childers, S.R. and Pasternak, G.W. Europ. J. Pharmacol., in press. Zhang, A-Z. and Pasternak, G.W. L i f e Sci., in press. Zhang, A-Z. and Pasternak, G.W. Europ. J. Pharmacol. 6__77:323-324 (1980). Chang, K-J, Hazum, E. and Cuatrecasas, P. Proc. Nat. Acad. Sci., USA 77:4469-4473 (1980).
Pergamon Press
NALOXAZONE IRREVERSIBLY INHIBITS THE HIGH AFFINITY BINDING OF [1251]D-ala2-D-Ieu5-ENKEPHALIN Eli Hazum, Kwen-Jen Chang, Pedro Cuatrecasas and Gavril W. Pasternak* The Wellcome Research Laboratories, Research Triangle Park, NC 27709 *George C. Cotzias Laboratory of Neuro-Oncology Memorial Sloan-Kettering Cancer Center, New York 10021 (Received in final form April 27, 1981)
SUMMARY Scatchard analyses of [1251]D-ala2-D-leu5-enkephalin binding in rat brain membranes are c u r v i l i n e a r , suggesting low and high a f f i n i t y sites. Treating the membranes with naloxazone abolishes the high a f f i n i t y bindinq with slight effect on low a f f i n i t y binding. Displacement of [1251]-D-alaC-D-leuD-enkephalin binding by merphine in untreated membranes is biphasic. Displacement by morphine in naloxazone-treated tissue is monophasic, with no i n h i b i t i o n by low concentrations of morphine. Naloxazon~ treatment has l i t t l e effect on displacements by unlabeled D-ala -D-leub-enkephalin. Binding in N4TGI neuroblastoma c e l l s , which demonstrates a linear Scatchard plot with single a f f i n i t y constant similar to that of the low a f f i n i t y binding in brain, is less sensitive to naloxazone's actions. Naloxazone treatment in vivo inhibits D-ala2-D-leu5-enkephalin analgesia. INTRODUCTION Studies of enkephalin binding to brain membranes have demonstrated c u r v i l i n ear Scatchard plots ( I ) similar to those described e a r l i e r for opiate agonists and antagonists (2). These reports raised the p o s s i b i l i t y that both opiates and enkephalins might bind to multiple receptor sub-populations. Detailed binding studies soon showed marked differences in the binding of enkephalin peptides and opiates such as naloxone and morphine (3-5), suggesting the existence of a sub-population of opiate receptors in the central nervous system with higher a f f i n i t y for enkephalin-like peptides than for morphine-like alkaloids. This sub-population has been clearly demonstrated in regional studies of opiate receptor binding (5). In marked contrast to other regions, the receptors in the frontal cortex bound enkephalins far more extensively and with higher a f f i n i t y than opiate alkaloids such as morphine. Naloxazone (6-9) has proven useful in the investigation of high and low a f f i n i t y opiate receptor binding. Whereas naloxazone had no demonstrable effect on a variety of non-opioid receptor systems, i t selectively blocked the high a f f i n i t y binding of a number of [JH]enkephalin analogs (met-enkephalin, l~u-enkephalin, D-ala2-met5-enkephalinamide ( i 0 ) ; as well as a variety of [JH] opiates including the mu agonists, dihydromorphine and morphine (7,8); the kappa agonist, ethylketocyclazocine(9); the sigma agonist, SKF 10,047 (Pasternak, Buatti and Spiegel, in preparation) and the antagonists naloxone and naltrexone ( i 0 ) . We now report the effects of naloxazone on the binding of the potent delta ligand, [1251] D-ala2-D-leu5-enkephalin. 0024-3205/81/262973-07502.00/0 Copyright (c) 1981 Pergamon Press Ltd.
2974
Interactions
of Naloxazone and DADL-enk
Vol. 28, No. 26, 1981
MATERIALS AND METHODS Enkephalin analogs were synthesized by Dr. S. Wilkinson, the Wellcome Research Laboratories, Beckenham, England. Morphine sulfate was obtained from M a l l i n c r o t Chemical Works, and naloxone from Endo Laboratories. Naloxazone was synthesized as previously described (6). [1251] Labeled D-ala2-D-leu 5enkephalin was prepared as previously described using chloramine-T (4,5). Brain membranes were prepared as previously described (4,5). B r i e f l y , whole rat brains were homogenized with a Polytron homogenizer at 4 ° C in 0.32 M sucrose in 5 mM tris-HC1 buffer, pH 7.4. The nuclei and most of the mitochondria were removed by c e n t r i f u g a t i o n f o r 15 minutes at 5,000 g and supernatant was f u r t h e r centrifuged for 30 minutes at 40,000 g and f i n a l l y suspended in 50 mM t r i s - H C l b u f f e r , pH 7.4, at 4°C. N4TGI neuroblastoma cells were grown on DMEM with 5% f e t a l bovine serum. The cells were grown to confluence and removed from the flask by incubation with 1 mM EDTA f o r 5 minutes at 37°C. The detached cells were washed 3 times with 10 mM t r i s buffer, pH 7.7, containing 0.25 M sucrose, 2 mM MgCI2 and 5 mM glucose and f i n a l l y suspended in the same buffer. Membranes were treated with naloxazone (2 ~M) at room temperature for 40 minutes followed by four washes. Each wash consisted of a 15 minute incubation at 37°C and c e n t r i f u g a t i o n . This procedure is s u f f i c i e n t to remove reversible ligands ( i i ) . Binding assays were performed as previously described (3,4). B r i e f l y , the radioactive enkephalin (0.07 nM, specific a c t i v i t y 2,000 Ci/mmole) was incubated at 25°C for 60 minutes with a final volume of 0.25 ml containing N4TGI c e l l s (4 x 106 c e l l s / m l ) or brain membranes (0.7 mg/ml p r o t e i n ) , and the binding was measured by f i l t r a t i o n with vacuum through Whatman GF/C f i l t e r s . Specific binding represents the bound r a d i o a c t i v i t y displaced by unlabeled D-ala2-D-leu5-enkephalin (I ~M). Each value is the mean of duplicate incubations varying less than 7%. Scatchard analyses were f i t by computer using a nonlinear least square program and a f f i n i t i e s and sites determined (Pasternak and Reiman, in preparation). Analgesia was tested 10 min following the i n j e c t i o n i . c . v , of D-ala2-D-leu5-enkephalin (i ~I dissolved in I0 ~I E l l i o t ' s B solution, an a r t i f i c i a l CSF) in mice treated 20 hrs e a r l i e r with e i t h e r naloxone or naloxazone (200 mg/kg s.c.) as previously described (l-g). RESULTS
Saturation curves of [1251]D-ala2-D-leu5-enkephalin to rat brain y i e l d c u r v i l i n e a r Scatchard plots (Fig. I ) . Such plots can be segmented into high a f f i n i t y (KD 0.52 nM; 0.89 fmoles) and a low a f f i n i t y (KD 5.23nM; 11.04 fmoles) components. Treatment of membranes with naloxazone in v i t r o markedly lowers the high a f f i n i t y binding, transforming the c u r v i l i n e a r Scatchard plots into linear ones (KD 5.9 nM; 9.47 fmoles). Whereas the high a f f i n i t y component is v i r t u a l l y abolished, the low a f f i n i t y one is decreased by only 15%, with no s i g n i f i c a n t change in a f f i n i t y . The persistence of this i n h i b i t i o n , despite extensive washing of the brain membranes which does eliminate the i n h i b i t i o n of reversible drugs, strongly implies that naloxazone's actions are irreversible. To f u r t h e r explore the properties of the low a f f i n i t y binding remaining a f t e r naloxazone treatment, displacement experiments were performed on [1251]D-ala2-D-leu5-enkephalin binding with both morphine and unlabeled D-ala2-D-leu5-enkephalin (Fig.2). Control tissue demonstrates a monophasic displacement of binding by unlabeled D-ala2-D-leu5-enkephalin with an IC50 of approximately 2.5 nM. Naloxazone treatment does not change the displacement by unlabeled D-ala2-D-leu5-enkephalin s i g n i f i c a n t l y (IC50 4 nM).
Vol. 28, No. 26, 1981
Interactions of Naloxazone and DADL-enk
I
2975
I
I
KD(nM) Bmax(fmOles)
4 1 ,,p o • -
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Fig.l Scatchard plots of [1251]D-Ala2-D-Leu5-enkephalin binding to brain membranes (1) and to brain membranes pretreated with 2 ~M naloxazone (o) and then washed. The data were determined from saturation experiments over a wide concentration range (0.06 nM to 13 nM). All binding presented is s p e c i f i c . This experiment has been repeated 3 times. Morphine displacement of control tissue is biphasic (Fig. 2 bottom), as previously described (3,4), with an i n i t i a l displacement at concentrations less than 0.3 nM and a f u r t h e r displacement occurring with an approximate "IC50" of 50 nM. The total apparent IC50, which is actually a mixture of binding sites is about 15 nM. Abolishing the high a f f i n i t y binding with naloxazone correspondingly changes the morphine displacement curves. Morphine displaces binding in the treated tissue in a monophasic fashion with an approximate IC50 of 63 nM, a value similar to the IC50 of the lower portion of the control curve. Thus, blockade of high a f f i n i t y binding by naloxazone eliminates the displacement of [1251] D-ala-D-leu-enkephalin binding by morphine at low concentrations. Opiate binding in N4TGI neuroblastoma c e l l s is primarily delta or enkephalin in nature (3,4). Scatchard analysis of [1251] D-ala2-D-leu5-enkephalin binding in these c e l l s (Fig.3) is l i n e a r , suggesting a single population of s i t e s ; t h e i r a f f i n i t y (3.3 nM) is similar to that of the low a f f i n i t y sites found in brain membranes (5.23 nM). Treatment of these cells with naloxazone results in a r e l a t i v e l y small reduction in the number of binding sites (about 35%) with no change in a f f i n i t y . This reduction, which is similar to the decrease in low a f f i n i t y sites normally seen with brain membranes (15-20%; Fig. I ) , can be contrasted to the v i r t u a l elimination of the high a f f i n i t y binding observed upon treatment of brain membranes.
2976
Interactions of Naloxazone and DADL-enk
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Vol. 28, No. 26,
,
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Competition of [1251] labeled D-Ala~-l)-Leu5-enkephalin binding to brain Membranes (o) and naloxazone-treated membrane (o) by D-AIa2-DLeu5-~nkephalin (top) and by morphine (bottom). Radioactive D-Ala2D-Leu -enkephalin (0.07 nM) was incubated with increasing concentrations of unlabeled morphine or peptide, and specific binding was determined and expressed as percent of specific control binding. This experiment has been replicated three times. TABLE 1.
D-Ala2-D-Leu5-Enkephalin Analgesia in NaloxazoneTreated Mice Treatment
Analgesia (n)
Control Naloxone Naloxazone
100% (5) 93% (14) 42% (7)*
p < 0.01
Mice were treated with naloxone or naloxazone (200 mg/kg sc) and administered D-ala2-D-leu5-enkephalin. Analgesia, defined as latency twice the baseline value, was tested i0 min a f t e r administrat i o n of drug. S i g n i f i c a n c e values determined by the Fisher Exact Test show no d i f f e r e n c e between the control and naloxone group. The naloxazone group is s i g n i f i c a n t l y d i f f e r e n t from the naloxone group (p < 0.025) and the pooled naloxone and control group (p < 0.01).
198]
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Interactions of Naloxazone and DADL-enk
2977
Naloxazone treatment in vivo markedly attenuates the analgesic actions of morphine (7,8), ketocyclazo~ine (9), SKF 10,047 (Pasternak, Buatti and Spiegel, in preparation), enkephalins and beta-endorphin (30). The similar blockade of the high a f f i n i t y binding of these drugs ~nd [125D]-D-ala2-b-leu5-enkephalin led us to test naloxazone s actions on D-ala~-D-leuU-enkephalin analgesia (Table 1). At a dose of i ~g, D-ala2-D-leu5-enkephalin is analgesic in all untreated and in 13 of 14 naloxone-treated mice. However, only 42% (p < 0.01) of the naloxazone animals were analgesic.
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0 0.5 1.0 1.5 2.0 [ "'1] D-ala'-D-leu'-Enkephalin Binding (f m01es/lO' cells) Scatchard plots of [1251] D-AlaL-D-Leu5-enkephalin binding to N4TG1 neuroblastoma cells (e) and to N4TGI cells pretreated with 2 pM naloxazone (o) and then washed. The data were determined from saturation experiments over a wide concentration range (0.06 nM to 13 nM). 0.25 ml of N4TGI cells (4 X I0 ° cells/ml) were incubated with various concentrations of [1251]-labeled D-Ala2-D-Leu 5 enkephafin and assayed as described in METHODS. All binding is specific, and the experiment has been replicated three times. DISCUSSION Both the curvilinear Scatchard analysis and the biphasic displacement of [1251]D-ala2-D-leu5-enkephalin by morphine suggest that [1251]D-ala2-D-leu 5enkephalin binds to two populations of receptors, presumably mu and delta typeS. Interpretation of complex displacement curves is extremely d i f f i c u l t and correlations between mu and delta receptors and high and low a f f i n i t y sites have only been tentative. Naloxazone provides a unique method of simplifying such analyses. These studies clearly show that naloxazone's i r r e v e r s i b l e i n h i b i t i o n is r e l a t i v e l y selective for the high a f f i n i t y component of [1251]Dala2-D-leu5-enkephalin binding and the high a f f i n i t y morphine displacement of [1251]D-ala2-D-leu5-enkephalin, suggesting they might be the same receptor.
2978
Interactions of Naloxazone and DADL-enk
Vo].. 28, No. 26, 1981
Although the assignment of mu or delta c h a r a c t e r i s t i c s remains t e n t a t i v e , these studies suggest that high a f f i n i t y [1251]D-ala2-D-leu5-enkephalin binding is mu-like and low a f f i n i t y binding d e l t a - l i k e . Naloxazone treatment blocks high a f f i n i t y binding of the mu agonists [3H]morphine and [3H]dihydromorphine with the same potency and s e l e c t i v i t y that i t blocks high a f f i n i t y [1251]Dala2-D-leu5-enkephalin binding (7,8,10-12) (Fig. 2) and s i q n i f i c a n t l y lessens morphine's potency in competitively displacing [1251]D-ala2-D-leu5-enkephalin binding (Fig. 2). Low a f f i n i t y [1251]D-ala2-D-leu5-enkephalin binding appears to be quite d i f f e r e n t from the hiqh a f f i n i t y s i t e . The term "low a f f i n i t y " s i t e is a r e l a t i v e one, since [1251]D-ala2-D-leu5-enkephalin s t i l l binds very potently with a KD of about 5 nM. Unlike the high a f f i n i t y s i t e , the low a f f i n i t y one is not very sensitive to naloxazone and binds D-ala2-D-leu5-enkephalin (IC50 4 riM) f a r more potently than morphine (IC50 63 nM), f u l f i l l i n g the major c r i t e r i a of a delta receptor. In neuroblastoma c e l l s , which are predominantly delta and which bind [1251]D-ala2-D-leu5-enkephalin with an a f f i n i t y (KD 3.3 nM) similar to the low a f f i n i t y s i t e in brain (KD 5 nM), naloxazone reduces the number of sites to a far lesser extent than in the brain, which has a higher proportion of mu receptors. This t e n t a t i v e assignment of high a f f i n i t y sites as mu-like and low a f f i n i t y sites as d e l t a - l i k e is consistent with the regional l o c a l i z a t i o n of these receptor types (5,13). Chang et al (5) clearly showed that the receptors in the frontal cortex strongly preferred enkephalins, whereas the hypothalamus binding sites were predominantly opiate, or mu-like. When brain regions were tested for t h e i r s e n s i t i v i t y to naloxazone (13), binding in the frontal cortex was v i r t u a l l y unchanged, whereas binding in the hypothalamus was decreased over 40% (p < 0.005). Naloxazone i n h i b i t s morphine, ketocyclazocine, B-endorphin, D-ala 2met5-enkephalinamide, D-ala2-met5-enkephalin (6-10,12) and SKF 10,047 analgesia (Pasternak, Buatti and Spiegel, in preparation). The s i m i l a r reduction of D-ala2-D-leu5-enkephalin analgesia, coupled with the evidence suggesting that they all bind with highest a f f i n i t y to the same s i t e , suggests that a single s i t e mediates opiate (mu, kappa and sigma), enkephalin and B-endorphin analgesia seen with standard drug doses. Why analgesia remains a f t e r naloxazone treatment at very high drug doses remains unknown. Most probably a small number of high a f f i n i t y sites are not blocked by naloxazone. Although high a f f i n i t y binding following naloxazone treatment cannot be discerned by Scatchard analgesia, this technique might not detect very low levels of high a f f i n i t y sites (10-15% of normal levels) in view of the large number of low a f f i n i t y s i t e s . In addition, naloxazone administered subcutaneously probably has s i g n i f i c a n t v a r i a b i l i t y in i t s absorption and entry into the brain, resulting in less than complete blockade of all the high a f f i n i t y sites in all animals. However, an additional analgesic system mediated by d i f f e r e n t receptors requiring larger drug dosages cannot be excluded. ACKNOWLEDGEMENTS GWP is a r e c i p i e n t of NINCDS Teacher Investigator Award (1 KO7NS415-02). This work was supported in part by a grant from NIDA (DA 02615) and the American Cancer Society (PDT 169). We thank Dr. R. Reiman for his help with the computer programs and M. Buatti for her technical assistance.
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Interactions
of Naloxazone and DADL-enk
2979
REFERENCES 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14.
Lord, J . , Waterfield, A., Hughes, J. and K o s t e r l i t z , H.W. Nature 267, 495 (1977). Pasternak, G.W. and Snyder, S.H. Nature 253, 563-564 (1975). Chang, K-J. , M i l l e r , R.J. and Cuatrecasas, P. Molec.Pharmacol. 14, 961-970 (1978). Chang, K-J and Cuatrecasas, P. J. Biol. Chem. 254:2610-2618 (1979). Chang, K-J, Cooper, B.R., Hazum, E. and Cuatrecasas, P. Molec. Pharm. 16: 91- 104 (1979). Pasternak, G.W. and Hahn, E.F. J. Med. Chem. 23:674-676 (1980). Pasternak, G.W., Childers, S.R. and Snyder, S.H. Science 208:514-516 (1980). Pasternak, G.W., Childers, S.R. and Snyder, S.H. J. Pharmacol. Exp. Therap. 214:455-462 (1980). Pasternak, G.W. Proc. Nat. Acad. Sci., USA 77:3691-3694 (1980). Pasternak, G.W. Neurology , in press. Childers, S.R. and Pasternak, G.W. Europ. J. Pharmacol., in press. Zhang, A-Z. and Pasternak, G.W. L i f e Sci., in press. Zhang, A-Z. and Pasternak, G.W. Europ. J. Pharmacol. 6__77:323-324 (1980). Chang, K-J, Hazum, E. and Cuatrecasas, P. Proc. Nat. Acad. Sci., USA 77:4469-4473 (1980).