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Chronic, but not acute, administration of morphine alters antiopiate (Tyr-MIF-1) binding sites in rat brain
Life Sciences, Vol. 44, pp. 555-561 Printed in the U.S.A.
Pergamon Press
CHRONIC, BUT NOT ACUTE, ADMINISTRATION OF MORPHINE ALTERS ANTIOPIATE (TYR-MIF-1) BINDING SITES IN RAT BRAIN James E. Zadina, Abba J. Kastin, Lin-Jun Ge, Heath Gulden and Kimberly J. Bungart Veterans Administration Medical Center and Tulane University School of Medicine New Orleans, LA 70146 (Received in final form December 28, 1988) SUMMARY
Opiate addiction could involve a change in the binding of endogenous antiopiates. A candidate for such a role is Tyr-MIF-1 (Tyr-Pro-LeuGly-NH2), a brain peptide that can antagonize exogenous and endogenous opiates and bind to opiate receptors. Its primary action, however, may be through its own binding site in brain, which we now report is altered by chronic administration of morphine. Rats given morphine pellets had reduced binding of both iodinated and tritiated Tyr-MIF-1 on day 5, when substantial tolerance is evident. In contrast, mu and delta opiate receptors were increased. Acute injection of an analgesic dose of morphine did not reduce Tyr-MIF-1 binding, indicating that chronic administration is required for the change. These findings open new approaches to the study of addiction by focusing on antiopiate activity.
The discovery of opiate receptors and their endogenous peptide ligands in the mid- 1970's led to the hope for a rapid increase in understanding of the mechanisms underlying addiction. One reasonable mechanism postulated to be involved in the processes of tolerance and dependence was regulation of the opiate receptor by chronic administration of opiates (1,2). Early binding studies, however, indicated that opiate receptors either did not change (384) or increased in a manner that did not correlate with the time course of symptoms of tolerance and dependence; the effect could be observed within a few minutes after injection (5,6) and antagonists were at least as effective as agonists in producing the change (5). Recent studies (7,8) have shown up-regulation of delta opiate receptors after chronic administration of morphine. However, the role, if any, of these changes in the processes of tolerance and dependence remains enigmatic (9). Another possible mechanism underlying tolerance and dependence after chronic administration of opiates would be a change in the activity or binding of endogenous antiopiates. An up-regulation of binding sites for an antiopiate might be expected in addiction as a reflection of increased sensitivity to the antiopiate. However, it is also possible that down-regulation could occur; increased activity of the antiopiate could result in down-regulation of its own receptor, or decreased receptor numbers could result in greater sensitivity by a reduction in the concentration of antiopiate required to occupy a sufficient Such changes in an proportion of receptors to induce a response (1,2). antiopiate system could be involved in the hallmark of tolerance, an increase in the dose required to achieve a given biological effect of an opiate, such as analgesia. 0024-3205/89 $3.00 + .OO
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A candidate for such a role is Tyr-MIF-1, a peptide isolated from bovine brain (10) that reduces the effects of morphine in tests of thermal and chemical pain (11,12) and antagonizes endogenous opiates in stress-induced analgesia (13,14). The long-term nociceptive changes observed after administration of beta-endorphin or morphiceptin to neonatal rats (15,16) can also be blocked by Tyr-MIF-1 (16). Tyr-MIF-1 inhibits binding of mu opiate ligands to their receptors more potently and selectively than other putative antiopiate peptides (17,18). However, except for high affinity inhibition of casomorphin-like peptides, the IC5O's of Tyr-MIF-1 against opiate ligands are in the micromolar range, and it may primarily act at its own binding site at nanomolar concentrations (17,19) We now report that chronic, but not acute, administration of morphine to rats significantly decreases the binding of Tyr-MIF-1 to its binding site in rat brain.
MATERIALS AND METHODS
Chemicals Tyr-MIF-1 was obtained from Bachem Switzerland, iodinated by the chloramine T method, and purified by high performance liquid chromatography (HPLC) to isolate the monoiodinated fraction (1900-2100 Ci/mmol). 3H-DAG0 (Tyr-D-Ala-Gly-NMe-Phe-Gly-01, 60 Ci/mmol) and 3H-DPDPE (D-Pen-Gly-Gly-PheD-Pen-OH, 26 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). 3H-DADLE (Tyr-D-Ala-Gly-Phe-D-Leu-OH, 46.9 Ci/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). 3H-Tyr-MIF-1 (6-12 Ci/mmol) was custom labeled by DuPont-New England Nuclear. All radioactive peptides were purified in our laboratory by HPLC. Animals Male Sprague-Dawley-derived adult rats were obtained from Zivic-Miller (Allison Park, PA) and housed in a 12:12 dark:light cycle at 21 + L'C. Chronic implantation of morphine pellets Animals were randomly divided into groups of 6-8 rats, anesthetized with methoxyflurane, and implanted subcutaneously with pellets containing 75 mg morphine or placebo. The implantation schedule was that of Rothman et al.(g), a modification of that of Wei, Loh and Way (20): Two pellets were implanted on day 1, and four pellets on day 2. Binding was measured on day 5, when substantial tolerance is evident (8). Acute morphine treatment Rats were injected IP with either 7.5 mg/kg morphine sulfate dissolved in Thirty minutes diluent (0.9% NaCl, O.OlM acetic acid) or with diluent alone. later, the animals were decapitated. This method has been shown to produce significant analgesia 15-30 min after injection and significantly increased mu (3H-naloxone-labeled) and delta (3H-DADLE-labeled) opiate receptors (6). Tissue preparation Crude synaptic plasma membranes (SPM) were prepared according to previously published methods (17) with minor modifications. Rat brains were rapidly removed and placed in 0.32 M sucrose at 4OC. Three morphine-treated and three control animals were individually processed in each set, and two sets were separately processed in each assay for a total of six animals, or two sets, comprising each group. Brains minus cerebelli were weighed, minced, and homogenized in 20 volumes of ice-cold 0.32M sucrose using a Brinkman polytron
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at setting 6 for 20 sec. Homogenates were centrifuged at 1000 x g for 15 min at 4OC to separate the crude nuclear pellet. Centrifugation of the supernatant and all subsequent centrifugations were at 30,000 x g for 15 min. The resulting pellet was reconstituted in 50 mM Tris (pH 7.5 at 23OC) and incubated for one hour in a water bath at room temperature to remove endogenous ligands. The preincubation increases specific binding of Tyr-MIF-1 (17). Homogenates were again centrifuged and pellets reconstituted in STEM buffer (0.25 M sucrose, 5 mM Tris, 0.5 mM EDTA, and 1 mM MgS04) and centrifuged. The STEM buffer was originally developed for freezing and storing membrane vesicles in a manner maintaining activity of amino acid transport (21). Tyr-MIF-1 binding after freezing in STEM buffer was significantly higher than that after freezing in Tris or phosphate (25 mM Na2P04, pH 7.4) buffer, and not significantly different from that of fresh tissue. Similarly, opiate receptor binding was nearly identical in fresh tissue preparations and in those frozen in STEM buffer. Binding was stable for at least one month. Two protocols were used to provide comparably washed fresh and frozen tissue: pellets of tissue to be frozen were reconstituted in 5 vol STEM, frozen in isopentane at -30°C, and stored at -7O'C for a maximum of 3 weeks. For assay, this tissue was thawed with 15 vol of the appropriate assay buffer, recentrifuged, and reconstituted in 20 vol of the appropriate assay buffer. Pellets for assays with unfrozen tissue were reconstituted with a 5:15 STEM:phosphate or STEM:Tris mixture, recentrifuged, and reconstituted in 20 vol of assay buffer. All comparisons between morphine-treated and control animals involved tissues that were processed, stored, and assayed at the same time with identical procedures. Protein concentrations (500-600 pg/ml final incubation concentration) were determined for individual animals as well as pooled group tissues using the Lowry method with BSA as the standard. 1251-Tyr-MIF-1 and 3H-Tyr-MIF-1 binding Methods of binding assays were similar to those previously published (17, 19). Briefly, 200 ~1 of tissue homogenate was incubated at 4'C for 18-24 hours equilibrium conditions (19)) with 0.1 - 0.5 nM 1251-Tyr-MIF-1 or with 20-30 nM 4H-Tyr-MIF-1 and varying concentrations of unlabeled peptide as described below. Nonspecific binding (NSB) was defined as that observed in the presence of 10 PM Tyr-MIF-1. Methods for separation of bound and free peptide with a Skatron Cell Harvester and for liquid scintillation counting were previously decribed (22). For determination of specific binding in individual animals, "single point" assays were used; tissue from each animal was separately processed, and 200 ~1 tissue was incubated with radioligand in the presence (NSB) or absence (TB) of 10 PM Tyr-MIF-1. For determination of apparent dissociation constants (Kd) and maximum number of binding sites (Bmax), tissue from three animals was pooled and inhibition curves were analyzed by the LIGAND program (23) or by Scatchard analysis. Opiate receptor binding assay Opiate receptor binding assays were similar to "single point" assays for Tyr-MIF-1 except that 3H-DAG0 and 'H-DPDPE were incubated in 50 mM Tris and 3H-DADLE in 10 mM Tris (8) for one hour at 23OC. 1.1 nM 3H-DAG0 and 1.5 nM 3H-DPDPE were used to selectively label the mu and delta opiate receptors. Since 3H-DADLE binding has been shown to increase after both acute (6) and chronic (7,8) administration of morphine, incubations were also conducted in 10 mM Tris with 1.5 nM 3H-DADLE. This ligand labels delta opiate receptors and has also been reported to label mu receptors (24). Nonspecific binding for all opiate ligands was that measured in the presence of 10 pM levallorphan.
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RESULTS Binding in individual animals Specific binding of several ligands was determined in "single point" assays. Raw data from 2 sets of 3 animals per group were expressed per mg protein and analyzed by analysis of variance (ANOVA). Figure 1 shows the data expressed as mean percentage of control to facilitate comparisons across ligands.
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FIG. 1. Effects of acute and chronic exposure to morphine on specific binding of 1251-Tyr-MIF-l, 3H-Tyr-MIF-1, and opiate ligands. Bars represent SEM. (* - p < 0.05; *** - p < 0.001).
As shown in the left panel of Figure 1 acute injections of morphine failed to produce a significant change in 125I-Tyr-MIF-1 binding. Chronic pellet implantation, however, resulted in a substantial decrease in specific binding relative to control for both 1251-Tyr-MIF-1 (20% decrease; F(1,8) - 25.66, p 0.001) and 3H-Tyr-MIF-1 (19% decrease; F(1,8) = 6.37, p < 0.05). With chronic exposure to morphine, opiate binding increased, which agrees with the findings of several studies (5,7,8). Tritiated DAGO, DPDPE, and DADLE binding increased significantly by 18% (F(1,8) - 12.04, p < 0.01)‘ 23% (F(1,8) - 11.84, p < 0.01) and 13% (F(1,8) - 6.32, p < 0.05) of control, indicating that both mu and delta opiate receptors increase under these conditions. Determination of apparent Kd and Bmax Figure 2 shows binding inhibition curves in Scatchard coordinates generated by incubation of 12'1-Tyr-MIF-1 with varying concentrations (0 - 60 nM) of unlabeled Tyr-MIF-1. Since iodination did not appear to affect binding of Tyr-MIF-1 in preliminary studies, and in this study both 3H-Tyr-MIF-1 and 12SI-Tyr-MIF-1 produced similar results, bound and bound/free ratios were Inhibition calculated assuming identity of labeled and unlabeled compound. curves were generated with pooled tissue (three animals per tissue pool) in
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experiments on three separate days, with two tissue-processing sessions on each day. A one-site model best described the data, and each tissue preparation yielded one estimate of the Kd and Bmax. These values were analyzed by an ANOVA of the following design: 3 (day of experiment) x 2 (processing set) x 2 (morphine or placebo). For greater accuracy, each of the twelve tissue preparations yielded two data points for each parameter, generated by inhibition curves at each of two concentrations of labeled ligand that varied by 3-fold. Figure 2, showing a Scatchard plot of one pair of inhibition curves, is representative of the overall results in approximating the parameter estimates averaged across all twelve such pairs.
0-0 l-
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FIG. 2. of 1251-Tyr-MIF-1 Representative Scatchard plot of inhibition binding by unlabeled Tyr-MIF-1 in animals treated with morphine or placebo pellets.
Chronic exposure to morphine resulted in a 15% decrease in the number of 1251-Tyr-MIF-1 binding sites relative to controls (2.59 + 0.34 vs 3.04 + 0.31 pmoles/mg protein), and ANOVA showed that this change was highly significant (F(1,12) - 12.36, p < 0.01). Although some individual Scatchard analyses indicated increases in affinity, ANOVA of values from all 24 inhibition curves showed no statistically significant change in this parameter (Kd - 51.9 + 6.9 vs 54.8 * 5.5 nM). In one experiment, a lower dose of morphine was administered; 1 pellet was implanted on day one, and two pellets on day 2 instead of the 2 + 4 pellets used in the other experiments. Tyr-MIF-1 binding was 10% lower in morphine animals relative to placebo-implanted controls (Bmax - 2.08 pmoles/mg protein difference was not statistically vs 2.31 for placebo); however, the significant. Similarly, the Kd was not significantly altered.
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DISCUSSION
Chronic (5 day) administration of morphine produced significant decreases in the number of high-affinity binding sites for the antiopiate Tyr-MIF-1. Although the changes were not large, they were observed with both tritiated and iodinated Tyr-MIF-1 and in both "single point" assays with individual animals and in inhibition experiments estimating the Bmax from pooled tissue with 24 inhibition curves. Although there was some indication of an increase in affinity, analysis of the 24 inhibition curves did not reveal a significant change in this parameter. Opiate receptors increased in the same tissue preparation that showed a decrease in Tyr-MIF-1 binding. This makes it highly unlikely that the change in Tyr-MIF-1 binding is part of a non-specific reduction in protein synthesis or receptors in general. Furthermore, it indicates that regulation of binding sites for this antiopiate peptide is different from that of classically defined opiate receptor sites. Our results are in good agreement with previous studies (5,7,8) which have indicated that the change in opiate receptors is due primarily to a change in Bmax, particularly at the "mu non-competitive delta" binding site (7,8). Although the changes in its own binding site demonstrated in this study are likely to be a major mechanism of any role that Tyr-MIF-1 may play in addiction, we cannot preclude binding site interactions such as allosteric effects or conformational changes that could alter the ability of Tyr-MIF-1 to bind to opiate receptors (17,18). Acute injection of an analgesic dose of morphine did not produce the decreases in binding of Tyr-MIF-1 observed after chronic morphine, indicating that chronic administration is required for the effect. This is consistent with the possibility that the changes correspond to the development of tolerance or dependence rather than acute processes associated with the short-term presence of morphine. In contrast, the increase in opiate receptors observed after chronic administration of morphine can also be seen only minutes after injection of the opiate (5,6). Holaday et al. (7) and Rothman et al. (8) have reported increases in delta opiate receptors after chronic administration of morphine. The "mu-noncompetitive" component of this binding increases after However, the chronic, but not after acute injection of morphine (8). well-documented increases in opiate receptors after chronic administration of opiate antagonists (25) create some difficulty for theories of opiate receptor up-regulation as a mechanism of addiction since the increase in receptors after antagonists is correlated with increased sensitivity to morphine, rather than the decreased sensitivity observed in the tolerant state. Nevertheless, it is unlikely that a single mechanism is involved in the addictive process, and models combining changes in opiate and antiopiate receptors may provide a promising approach to the problem. At least two hypotheses concerning the morphine-induced reductions in Tyr-MIF-1 binding in rat brain are consistent with a role for the peptide or its binding site in addiction: (a) the activity of the peptide increases during chronic administration of morphine producing a down-regulation of its own binding site, or (b) the reduced number of sites could reflect greater sensitivity to the antiopiate if lower concentrations are required to occupy Regardless, the proportion of receptor necessary to induce a response (1,2). this report provides the first evidence that changes in binding sites for an antiopiate peptide could play a role in opiate tolerance and dependence.
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ACKNOWLEDGEMENT
These studies were supported by the Veterans Administration
REFERENCES 1. H.O.J. COLLIER, Nature -:181-182 (1965). 2. A. GOLDSTEIN and D.B.GOLDSTEIN, Res. Publ. Mental Dis. s:265-267 (1968). 3. W.A. KLEE and R.A. STEATY, Nature 248: 61-63 (1974). 4. v. HOLLT, J. DUM, J. BLASIG, P. SCHUBERT and A. HERZ, Life Sci. j.&: 1823-1828 (1975). 5. C.B. PERT and S.H. SNYDER, Biochem. Pharmacol. 5:847-853 (1976). A.J. KASTIN, D.H. COY, and B.A. ADINOFF, Psychoneuro6. J.E. ZADINA, endocrinology, u:367-383 (1985). 7. J.W. HOLADAY, R.J. HITZEMAN, J. CURELL, F.C. TORTELLA, and G.L. BELENKY, Life Sci. =:2359-2362 (1982). 8. R.B. ROTHMAN, J.A. DANKS, A.E. JACOBSON, T.R. BURKE, Jr., K.C. RICE, F.C. TORTELLA and J.W. HOLADAY, Eur. J. Pharmacol. u:113-119 (1986). 9. A.P. SMITH, P.-Y. LAW, and H.H. LOH, In The Ouiate Recenters (G.W. Pasternak, ea.). The Humana Press, Clifton, NJ, 441-485 (1988). 10. A. HORVATH and A.J. KASTIN, J. Biol. Chem. In Press. 11. A.J. KASTIN, E. STEPHENS, R.H. EHRENSING, and A.J. FISCHMAN, Pharmacol. Biochem. Behav. =:937-941 (1984). 12. A.J. KASTIN, E. STEPHENS, J.E. ZADINA, D.H. COY, and A.J. FISCHMAN, Pharmacol. Biochem. Behav. =:1045-1049 (1985). 13. Z.H. GALINA and A.J. KASTIN, Life Sci. x:2153-2159 (1986). 14. Z.H. GALINA and A.J. KASTIN, Br.J. Pharm. a:669-674 (1987). 15. J.E. ZADINA and A.J. KASTIN, Dev. Brain Research 2:21-29 (1986). 16. J.E. ZADINA, A.J. KASTIN, P.K. MANASCO, M.F. PIGNATIELLO, and K.L. NASTIUK, Brain Research 409:10-18 (1987). 17. J.E. ZADINA and A.J. KASTIN, Peptides &:965-970, (1985). 18. J.E. ZADINA and A.J. KASTIN, Pharmacol. Biochem. Behav. 25:1303-1305 (1986). 19. J.E. ZADINA, A.J. KASTIN, E.F. KRIEG, Jr., and D.H. COY, Pharmacol. Biochem. Behav. =:1193-1198 (1982). 20. E. WEI, H.H. LOH and E.L. WAY, J. Pharm. Exp. Ther. 184:398-403 (1973). 21. R. ROSKOSKI Jr., N. RAUCH, and L.M. ROSKOSKI, Archiv. Biochem. Biophys. 207:407-415 (1981). 22. J.E. ZADINA and A.J. KASTIN, Pharmacol. Biochem. Behav. a:947-952 (1984). 23. P.J. MUNSON and D. RODBARD, Anal. Biochem. m:220-239 (1980). 24. R.R. GOODMAN, B. ADLER, and G.W. PASTERNAK, Neurosci Lett. 59.155-158 (1985). 25. R.S. ZUKIN, A. TEMPLE, and E. L. GARDNER, In Mechanisms of Tolerance and Dependence (C.W. Sharp, ed.) NIDA Research Monograph 54, U.S. Government Printing Office, Washington, D.C., 146-161 (1984).
Pergamon Press
CHRONIC, BUT NOT ACUTE, ADMINISTRATION OF MORPHINE ALTERS ANTIOPIATE (TYR-MIF-1) BINDING SITES IN RAT BRAIN James E. Zadina, Abba J. Kastin, Lin-Jun Ge, Heath Gulden and Kimberly J. Bungart Veterans Administration Medical Center and Tulane University School of Medicine New Orleans, LA 70146 (Received in final form December 28, 1988) SUMMARY
Opiate addiction could involve a change in the binding of endogenous antiopiates. A candidate for such a role is Tyr-MIF-1 (Tyr-Pro-LeuGly-NH2), a brain peptide that can antagonize exogenous and endogenous opiates and bind to opiate receptors. Its primary action, however, may be through its own binding site in brain, which we now report is altered by chronic administration of morphine. Rats given morphine pellets had reduced binding of both iodinated and tritiated Tyr-MIF-1 on day 5, when substantial tolerance is evident. In contrast, mu and delta opiate receptors were increased. Acute injection of an analgesic dose of morphine did not reduce Tyr-MIF-1 binding, indicating that chronic administration is required for the change. These findings open new approaches to the study of addiction by focusing on antiopiate activity.
The discovery of opiate receptors and their endogenous peptide ligands in the mid- 1970's led to the hope for a rapid increase in understanding of the mechanisms underlying addiction. One reasonable mechanism postulated to be involved in the processes of tolerance and dependence was regulation of the opiate receptor by chronic administration of opiates (1,2). Early binding studies, however, indicated that opiate receptors either did not change (384) or increased in a manner that did not correlate with the time course of symptoms of tolerance and dependence; the effect could be observed within a few minutes after injection (5,6) and antagonists were at least as effective as agonists in producing the change (5). Recent studies (7,8) have shown up-regulation of delta opiate receptors after chronic administration of morphine. However, the role, if any, of these changes in the processes of tolerance and dependence remains enigmatic (9). Another possible mechanism underlying tolerance and dependence after chronic administration of opiates would be a change in the activity or binding of endogenous antiopiates. An up-regulation of binding sites for an antiopiate might be expected in addiction as a reflection of increased sensitivity to the antiopiate. However, it is also possible that down-regulation could occur; increased activity of the antiopiate could result in down-regulation of its own receptor, or decreased receptor numbers could result in greater sensitivity by a reduction in the concentration of antiopiate required to occupy a sufficient Such changes in an proportion of receptors to induce a response (1,2). antiopiate system could be involved in the hallmark of tolerance, an increase in the dose required to achieve a given biological effect of an opiate, such as analgesia. 0024-3205/89 $3.00 + .OO
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A candidate for such a role is Tyr-MIF-1, a peptide isolated from bovine brain (10) that reduces the effects of morphine in tests of thermal and chemical pain (11,12) and antagonizes endogenous opiates in stress-induced analgesia (13,14). The long-term nociceptive changes observed after administration of beta-endorphin or morphiceptin to neonatal rats (15,16) can also be blocked by Tyr-MIF-1 (16). Tyr-MIF-1 inhibits binding of mu opiate ligands to their receptors more potently and selectively than other putative antiopiate peptides (17,18). However, except for high affinity inhibition of casomorphin-like peptides, the IC5O's of Tyr-MIF-1 against opiate ligands are in the micromolar range, and it may primarily act at its own binding site at nanomolar concentrations (17,19) We now report that chronic, but not acute, administration of morphine to rats significantly decreases the binding of Tyr-MIF-1 to its binding site in rat brain.
MATERIALS AND METHODS
Chemicals Tyr-MIF-1 was obtained from Bachem Switzerland, iodinated by the chloramine T method, and purified by high performance liquid chromatography (HPLC) to isolate the monoiodinated fraction (1900-2100 Ci/mmol). 3H-DAG0 (Tyr-D-Ala-Gly-NMe-Phe-Gly-01, 60 Ci/mmol) and 3H-DPDPE (D-Pen-Gly-Gly-PheD-Pen-OH, 26 Ci/mmol) were obtained from Amersham (Arlington Heights, IL). 3H-DADLE (Tyr-D-Ala-Gly-Phe-D-Leu-OH, 46.9 Ci/mmol) was obtained from DuPont-New England Nuclear (Boston, MA). 3H-Tyr-MIF-1 (6-12 Ci/mmol) was custom labeled by DuPont-New England Nuclear. All radioactive peptides were purified in our laboratory by HPLC. Animals Male Sprague-Dawley-derived adult rats were obtained from Zivic-Miller (Allison Park, PA) and housed in a 12:12 dark:light cycle at 21 + L'C. Chronic implantation of morphine pellets Animals were randomly divided into groups of 6-8 rats, anesthetized with methoxyflurane, and implanted subcutaneously with pellets containing 75 mg morphine or placebo. The implantation schedule was that of Rothman et al.(g), a modification of that of Wei, Loh and Way (20): Two pellets were implanted on day 1, and four pellets on day 2. Binding was measured on day 5, when substantial tolerance is evident (8). Acute morphine treatment Rats were injected IP with either 7.5 mg/kg morphine sulfate dissolved in Thirty minutes diluent (0.9% NaCl, O.OlM acetic acid) or with diluent alone. later, the animals were decapitated. This method has been shown to produce significant analgesia 15-30 min after injection and significantly increased mu (3H-naloxone-labeled) and delta (3H-DADLE-labeled) opiate receptors (6). Tissue preparation Crude synaptic plasma membranes (SPM) were prepared according to previously published methods (17) with minor modifications. Rat brains were rapidly removed and placed in 0.32 M sucrose at 4OC. Three morphine-treated and three control animals were individually processed in each set, and two sets were separately processed in each assay for a total of six animals, or two sets, comprising each group. Brains minus cerebelli were weighed, minced, and homogenized in 20 volumes of ice-cold 0.32M sucrose using a Brinkman polytron
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at setting 6 for 20 sec. Homogenates were centrifuged at 1000 x g for 15 min at 4OC to separate the crude nuclear pellet. Centrifugation of the supernatant and all subsequent centrifugations were at 30,000 x g for 15 min. The resulting pellet was reconstituted in 50 mM Tris (pH 7.5 at 23OC) and incubated for one hour in a water bath at room temperature to remove endogenous ligands. The preincubation increases specific binding of Tyr-MIF-1 (17). Homogenates were again centrifuged and pellets reconstituted in STEM buffer (0.25 M sucrose, 5 mM Tris, 0.5 mM EDTA, and 1 mM MgS04) and centrifuged. The STEM buffer was originally developed for freezing and storing membrane vesicles in a manner maintaining activity of amino acid transport (21). Tyr-MIF-1 binding after freezing in STEM buffer was significantly higher than that after freezing in Tris or phosphate (25 mM Na2P04, pH 7.4) buffer, and not significantly different from that of fresh tissue. Similarly, opiate receptor binding was nearly identical in fresh tissue preparations and in those frozen in STEM buffer. Binding was stable for at least one month. Two protocols were used to provide comparably washed fresh and frozen tissue: pellets of tissue to be frozen were reconstituted in 5 vol STEM, frozen in isopentane at -30°C, and stored at -7O'C for a maximum of 3 weeks. For assay, this tissue was thawed with 15 vol of the appropriate assay buffer, recentrifuged, and reconstituted in 20 vol of the appropriate assay buffer. Pellets for assays with unfrozen tissue were reconstituted with a 5:15 STEM:phosphate or STEM:Tris mixture, recentrifuged, and reconstituted in 20 vol of assay buffer. All comparisons between morphine-treated and control animals involved tissues that were processed, stored, and assayed at the same time with identical procedures. Protein concentrations (500-600 pg/ml final incubation concentration) were determined for individual animals as well as pooled group tissues using the Lowry method with BSA as the standard. 1251-Tyr-MIF-1 and 3H-Tyr-MIF-1 binding Methods of binding assays were similar to those previously published (17, 19). Briefly, 200 ~1 of tissue homogenate was incubated at 4'C for 18-24 hours equilibrium conditions (19)) with 0.1 - 0.5 nM 1251-Tyr-MIF-1 or with 20-30 nM 4H-Tyr-MIF-1 and varying concentrations of unlabeled peptide as described below. Nonspecific binding (NSB) was defined as that observed in the presence of 10 PM Tyr-MIF-1. Methods for separation of bound and free peptide with a Skatron Cell Harvester and for liquid scintillation counting were previously decribed (22). For determination of specific binding in individual animals, "single point" assays were used; tissue from each animal was separately processed, and 200 ~1 tissue was incubated with radioligand in the presence (NSB) or absence (TB) of 10 PM Tyr-MIF-1. For determination of apparent dissociation constants (Kd) and maximum number of binding sites (Bmax), tissue from three animals was pooled and inhibition curves were analyzed by the LIGAND program (23) or by Scatchard analysis. Opiate receptor binding assay Opiate receptor binding assays were similar to "single point" assays for Tyr-MIF-1 except that 3H-DAG0 and 'H-DPDPE were incubated in 50 mM Tris and 3H-DADLE in 10 mM Tris (8) for one hour at 23OC. 1.1 nM 3H-DAG0 and 1.5 nM 3H-DPDPE were used to selectively label the mu and delta opiate receptors. Since 3H-DADLE binding has been shown to increase after both acute (6) and chronic (7,8) administration of morphine, incubations were also conducted in 10 mM Tris with 1.5 nM 3H-DADLE. This ligand labels delta opiate receptors and has also been reported to label mu receptors (24). Nonspecific binding for all opiate ligands was that measured in the presence of 10 pM levallorphan.
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RESULTS Binding in individual animals Specific binding of several ligands was determined in "single point" assays. Raw data from 2 sets of 3 animals per group were expressed per mg protein and analyzed by analysis of variance (ANOVA). Figure 1 shows the data expressed as mean percentage of control to facilitate comparisons across ligands.
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1
I
I
88
I
fi
1 3
H-Tyr-MIF-1
I
“““’ ‘W-OAGO
%-OPOPE
_1
‘,,-OAOLE
FIG. 1. Effects of acute and chronic exposure to morphine on specific binding of 1251-Tyr-MIF-l, 3H-Tyr-MIF-1, and opiate ligands. Bars represent SEM. (* - p < 0.05; *** - p < 0.001).
As shown in the left panel of Figure 1 acute injections of morphine failed to produce a significant change in 125I-Tyr-MIF-1 binding. Chronic pellet implantation, however, resulted in a substantial decrease in specific binding relative to control for both 1251-Tyr-MIF-1 (20% decrease; F(1,8) - 25.66, p 0.001) and 3H-Tyr-MIF-1 (19% decrease; F(1,8) = 6.37, p < 0.05). With chronic exposure to morphine, opiate binding increased, which agrees with the findings of several studies (5,7,8). Tritiated DAGO, DPDPE, and DADLE binding increased significantly by 18% (F(1,8) - 12.04, p < 0.01)‘ 23% (F(1,8) - 11.84, p < 0.01) and 13% (F(1,8) - 6.32, p < 0.05) of control, indicating that both mu and delta opiate receptors increase under these conditions. Determination of apparent Kd and Bmax Figure 2 shows binding inhibition curves in Scatchard coordinates generated by incubation of 12'1-Tyr-MIF-1 with varying concentrations (0 - 60 nM) of unlabeled Tyr-MIF-1. Since iodination did not appear to affect binding of Tyr-MIF-1 in preliminary studies, and in this study both 3H-Tyr-MIF-1 and 12SI-Tyr-MIF-1 produced similar results, bound and bound/free ratios were Inhibition calculated assuming identity of labeled and unlabeled compound. curves were generated with pooled tissue (three animals per tissue pool) in
Tyr-MIF-1 Binding After Chronic Morphine
vol. 44, No. 8, 1989
559
experiments on three separate days, with two tissue-processing sessions on each day. A one-site model best described the data, and each tissue preparation yielded one estimate of the Kd and Bmax. These values were analyzed by an ANOVA of the following design: 3 (day of experiment) x 2 (processing set) x 2 (morphine or placebo). For greater accuracy, each of the twelve tissue preparations yielded two data points for each parameter, generated by inhibition curves at each of two concentrations of labeled ligand that varied by 3-fold. Figure 2, showing a Scatchard plot of one pair of inhibition curves, is representative of the overall results in approximating the parameter estimates averaged across all twelve such pairs.
0-0 l-
Placebo -
-0
Morphine
C
m
0.004-
0.002
I
0‘ 0
I 0.2
I
1
I
\I
0.4 Bound
0.6
I
I
0.8
1
(nM)
FIG. 2. of 1251-Tyr-MIF-1 Representative Scatchard plot of inhibition binding by unlabeled Tyr-MIF-1 in animals treated with morphine or placebo pellets.
Chronic exposure to morphine resulted in a 15% decrease in the number of 1251-Tyr-MIF-1 binding sites relative to controls (2.59 + 0.34 vs 3.04 + 0.31 pmoles/mg protein), and ANOVA showed that this change was highly significant (F(1,12) - 12.36, p < 0.01). Although some individual Scatchard analyses indicated increases in affinity, ANOVA of values from all 24 inhibition curves showed no statistically significant change in this parameter (Kd - 51.9 + 6.9 vs 54.8 * 5.5 nM). In one experiment, a lower dose of morphine was administered; 1 pellet was implanted on day one, and two pellets on day 2 instead of the 2 + 4 pellets used in the other experiments. Tyr-MIF-1 binding was 10% lower in morphine animals relative to placebo-implanted controls (Bmax - 2.08 pmoles/mg protein difference was not statistically vs 2.31 for placebo); however, the significant. Similarly, the Kd was not significantly altered.
560
Tyr-MIF-1 Binding After Chronic Morphine
Vol. 44, No. 8, 1989
DISCUSSION
Chronic (5 day) administration of morphine produced significant decreases in the number of high-affinity binding sites for the antiopiate Tyr-MIF-1. Although the changes were not large, they were observed with both tritiated and iodinated Tyr-MIF-1 and in both "single point" assays with individual animals and in inhibition experiments estimating the Bmax from pooled tissue with 24 inhibition curves. Although there was some indication of an increase in affinity, analysis of the 24 inhibition curves did not reveal a significant change in this parameter. Opiate receptors increased in the same tissue preparation that showed a decrease in Tyr-MIF-1 binding. This makes it highly unlikely that the change in Tyr-MIF-1 binding is part of a non-specific reduction in protein synthesis or receptors in general. Furthermore, it indicates that regulation of binding sites for this antiopiate peptide is different from that of classically defined opiate receptor sites. Our results are in good agreement with previous studies (5,7,8) which have indicated that the change in opiate receptors is due primarily to a change in Bmax, particularly at the "mu non-competitive delta" binding site (7,8). Although the changes in its own binding site demonstrated in this study are likely to be a major mechanism of any role that Tyr-MIF-1 may play in addiction, we cannot preclude binding site interactions such as allosteric effects or conformational changes that could alter the ability of Tyr-MIF-1 to bind to opiate receptors (17,18). Acute injection of an analgesic dose of morphine did not produce the decreases in binding of Tyr-MIF-1 observed after chronic morphine, indicating that chronic administration is required for the effect. This is consistent with the possibility that the changes correspond to the development of tolerance or dependence rather than acute processes associated with the short-term presence of morphine. In contrast, the increase in opiate receptors observed after chronic administration of morphine can also be seen only minutes after injection of the opiate (5,6). Holaday et al. (7) and Rothman et al. (8) have reported increases in delta opiate receptors after chronic administration of morphine. The "mu-noncompetitive" component of this binding increases after However, the chronic, but not after acute injection of morphine (8). well-documented increases in opiate receptors after chronic administration of opiate antagonists (25) create some difficulty for theories of opiate receptor up-regulation as a mechanism of addiction since the increase in receptors after antagonists is correlated with increased sensitivity to morphine, rather than the decreased sensitivity observed in the tolerant state. Nevertheless, it is unlikely that a single mechanism is involved in the addictive process, and models combining changes in opiate and antiopiate receptors may provide a promising approach to the problem. At least two hypotheses concerning the morphine-induced reductions in Tyr-MIF-1 binding in rat brain are consistent with a role for the peptide or its binding site in addiction: (a) the activity of the peptide increases during chronic administration of morphine producing a down-regulation of its own binding site, or (b) the reduced number of sites could reflect greater sensitivity to the antiopiate if lower concentrations are required to occupy Regardless, the proportion of receptor necessary to induce a response (1,2). this report provides the first evidence that changes in binding sites for an antiopiate peptide could play a role in opiate tolerance and dependence.
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ACKNOWLEDGEMENT
These studies were supported by the Veterans Administration
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