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Binding of [3H]spiroperidol to striatal membranes of rats treated chronically with morphine
Neuropharrnacology Vol. 22, No. 12A, pp. 1357-1361, 1983 Printed in Great Britain
0028-3908/83$3.00+0.00 Pergamon Press Ltd
BINDING OF [3H]SPIROPERIDOL TO STRIATAL MEMBRANES OF RATS TREATED CHRONICALLY WITH MORPHINE INFLUENCE OF PRO-LEU-GLY-NH2 AND CYCLO(LEU-GLY) H. N. BHARGAVA Department of Pharmacodynamics, University of Illinois at Chicago, Health Sciences Center, Chicago, IL 60612, U.S.A.
(Accepted 1 May 1983) Summary--The effect of the chronic administration of morphine to Sprague Dawley rats on striatal dopamine receptors labelled with [3H]spiroperidol was determined. For chronic administration of morphine, rats were implanted subcutaneously with four morphine pellets (each containing 75 mg of morphine free base) during a 3-day period. Placebo pellet-implanted rats served as controls. Twenty-four hours after the removal of the pellet binding of [3H]spiroperidol to striatal dopamine receptors was determined. Rats receivingmorphine showed no differencein the total number of binding sites (Bmax value) but there was a 60~ decrease in the apparent dissociation constant (Kd value) compared with placebo controls. The effect of two peptides, Pro-Leu-Gly-NH2 and cyclo(Leu-Gly) (2 mg/kg), which have been shown to inhibit tolerance to and dependence on morphine, on the changes in the binding of [3H]spiroperidol induced by the implantation of morphine pellets was also determined. Both peptides antagonized the decreases in K0 values induced by the administration of morphine. However, when administered chronically by themselves, the peptides had no effect on either the Bmax o r Kd values of [3H]spiroperidol. It is concluded that the behavioral supersensitivityof dopamine receptors and the genesis of tolerance-dependence to opiates may be related to the affinity and not to the density of striatal dopamine receptors. Key words: morphine, tolerance-dependence, dopamine receptor, supersensitivity, [3H]spiroperidol binding, Pro-Leu-Gly-NH2, cyclo(Leu-Gly), striatum.
Considerable evidence suggests that the chronic administration of morphine to rodents is associated with aberations in dopaminergic neurons in the central nervous system. Both behavioural and biochemical studies indicate the appearance of a supersensitive dopamine receptor system following chronic treatment (Puri and Lal, 1973; Gianutosos, Hynes, Puri, Drawbaugh and Lal, 1974; Iwatsubo and Clouet, 1975; Ritzmann, Walter, Bhargava and Flexner, 1979; Bhargava, 1980, 1981a). Similar findings have been arrived at after chronic administration of human fl-endorphin to rats (Bhargava, 1981b). However, several studies have either been unable to show supersensitivity or, in some cases, have provided evidence for the presence of the subsensitivity of dopamine receptors after chronic treatment with morphine (Kuschinsky, 1975; Merali, Singhal, Hrdina and Ling, 1975; Iversen and Joyce, 1978). More recently, the function of dopamine receptors following the chronic administration or morphine have been studied using [3H]spiroperidol as the ligand. Puri, Spaulding and Mantione (1978) demonstrated that acute treatment of rats with 30 mg/kg did not alter the binding of [3H]spiroperidol. The implantation of two morphine pellets (50 mg morphine
in each) caused an increase in Kd values, whereas the implantation of 4 morphine pellets resulted in decreases in the Bm~x value (fewer binding sites) with a lower Kd value (enhanced affinity). In both cases the pellets were removed 24 or 48 hr earlier. Christie and Overstereet (1979) on the other hand, found decreased affinity (higher Kd value) for [3H]spiroperidol in the striatum of rats withdrawn from morphine and concluded that morphine-tolerant rats were supersensitive to dopamine agonists and rats withdrawn from morphine were subsensitive. More recent studies suggest that chronic treatment with morphine is associated with an increase in affinity without a change in the number of binding sites for [3H]spiroperidol (Ritzmann, Lee and Fields, 1982). In view of these conflicting data, studies were undertaken to examine the binding parameters of [3H]spiroperidol in the striata of rats treated chronically with morphine. In addition, the effects of two peptides, Pro-Leu-Gly-NH2 (MIF) and cyclo(LeuGly) (CLG), which have been shown to inhibit tolerance to and dependence on opiates (Bhargava, 1980, 1981 a, c, d; Bhargava, Walter and Ritzmann, 1980), on the binding changes of [3H]spiroperidol induced by chronic treatment with morphine has been determined.
1357
1358
H.N. BHARGAVA METHODS
Animals Male Sprague-Dawley rats weighing 200-250g (King Animal Laboratories, Oregon, Wisconsin) were acclimatized to a room with controlled ambient / temperature (23 + 1o,oC), humidity (50 _+ 10%) and a 12hr dark-light cycle (L 0600-1800hr). The rats were given food and water ad libitum. The animals were housed under these conditions for at least 4 days before being used.
Drugs Pro-Leu-Gly-NH2 was a gift from the Abbott Laboratories, North Chicago, Illinois through the courtesy of Dr E. L. Woroch. Cyclo (Leu-Gly) (CLG) was synthesized in the laboratory according to the method of Fischer (1906). The peptides were found to be pure as judged by thin layer chromatographic analysis on two solvent systems as described previously (Bhargava, 1981c). The peptides were dissolved in distilled deionized water and injected subcutaneously, such that each rat received the drug in 1 ml of the solution per kg of body weight. [~H]spiroperidol (sp. act. 26.0 Ci/mmol) was obtained from New England Nuclear, Boston, Massachusetts. d-Butaclamol was a gift from Ayerst Laboratories, Montreal, Canada.
Schedule of the administration of peptide and morphine The rats were treated chronically with morphine by the subcutaneous implantation of four morphine pellets (each containing 75 mg of morphine free base) during a 3-day period as described previously (Bhargava, 1977, 1978). Rats serving as controls were implanted with four placebo pellets. The schedule of treatment with peptide and morphine was similar to that described previously (Bhargava 1981a, c). Briefly, the rats were injected with either vehicle (water), Pro-Leu-Gly-NH2 (2 mg/kg) or cyclo(LeuGly) (2 mg/kg), respectively. Each group of rats was further divided into two subgroups. One hour after the injection of water or the peptide, rats in one subgroup were implanted with a placebo pellet, whereas those in the other subgroup were implanted with a morphine pellet. This initial implantation procedure was followed by two more injections of vehicle or the peptide and implantation of three more placebo or morphine pellets, in their respective groups as described previously (Bhargava, 1981 a, c). All pellets were removed from the rats, under light ether anesthesia, 70 hr after the first implantation of pellets. Twenty four hours after the removal of pellets the rats were sacrificed, their striata removed and frozen at - 7 0 C until assayed for the binding of [3H]spiroperidol. The untilization of the above procedure for the implantation of pellets in the rat has been shown to produce a high deffree of tolerance to and physical dependence on morphine (Bhargava, 1977, 1978). In
addition, the levels of morphine could not be detected in brain or plasma at the time the animals were sacrificed (Bhargava, 1978), although acute treatment with morphine has been shown not to alter the binding of [3H]spiroperidol to the striatal tissue of rats (Puri et al., 1978).
The binding of [3H]spiroperidol to dopamine receptors in the striatal membranes o[ the rat The binding of [3H]spiroperidol was carried out essentially as described previously (Creese and Snyder, 1979) with a slight modification. The striata were homogenized in 30vol of 50mM Tris HCI buffer (pH 7.7 at 25"C) using a Brinkman Polytron homogenizer (setting 6 for 20 sec). The tissue homogenate was twice centrifuged at 49,000g for 15 min in a refrigerated Sorvall RC-5B centrifuge after resuspending in fresh Tris-buffer. The final pellet was suspended in 50 mM, with the Tris buffer (pH 7.4 at 25~C) containing 0.1% ascorbic acid, 120 mM NaCI, 5mM KC1, 2 m M CaCI 2 and 10/tM pargyline to get a concentration of 10 mg wet tissue per ml of incubation buffer. The standard assay mixture contained 0.2 ml of the homogenate containing approx. 200 ~g of protein and 0.1 ml of [3H]spiroperidol and buffer to make up the total volume to 1 ml. Incubation was carried out in triplicate in a shaking water bath maintained at 3 7 C for 15min. At the end of the incubation period, the contents of the incubation tubes were rapidly filtered under partial vacuum using a Millipore manifold filtration unit and Whatman G F / F glass fiber filters. This was followed by two 5ml washes of ice-cold 50raM Tris-buffer (pH 7.4). The filters were transferred to liquid scintillation vials containing 10 ml of 3 a 70 scintillation cocktail (Research Products International Corp. Arlington Heights, Illinois). After an overnight period of equilibration, the radioactivity in the samples was determined in a Packard Tricarb liquid scintillation spectrometer. The stereospecific binding of [3H]spiroperidol was defined as the difference in
c-o 3
•
Morphine - water • Placebo + water
~oOoB
I IpM
Kd=49+-
2
max= 1 4 2 + - [ 6 f m o l / m g
Kd =111_+ I I ~ M t . " . I B too. =,45 :t 9 frnol/rng 0 pro e 13 I
20
I
,
40
60
l
80
protein
--~ I
100
~ ~]
120
"%
140
I
160
B
Fig. 1. Scatchard plots of the binding of [~H]spiroperidol to striatal membranes of nontolerant (placebo pellets) and morphine tolerant-dependent (morphine pellets) rats. The lines are drawn by linear regression analyses.
Peptides, morphine tolerance-dependence and DA receptors binding in the absence and presence of 1/~M dbutaclamol. The concentration of protein in the homogenates was determined according to the method of Lowry, Rosebrough, Farr and Randall (1951). The apparent dissociation constant (Kd) and the maximal binding capacity (Bmax)were calculated from saturation curves generated by using six to seven concentrations of [3H]spiroperidol. The Scatchard plots were generated by least square regression analyses. Four rats were used for each treatment group. The means of Bm.x and Kd values in different groups were analyzed by one-way analysis of variance followed by Schaffe's S-test. RESULTS
O i1 Placebo ,Morphine
Bmox(fmol / mg protein )
~f- 120 ;~
+1
g E
30 0
J Iil K~(pM)
zS S "o
,2o 9O
~- 6o
o & CO
As indicated previously, a high degree of tolerance to and physical dependence on morphine developed as a result of the implantation of four pellets of morphine during a 3-day period (Bhargava, 1977, 1978). Chronic administration of morphine resulted in an increase in the affinity of the binding of [3H]spiroperidol to dopamine receptors. However, the number of binding sites remained unchanged. The Scatchard plot for the specific binding of [3H]spiroperidol to striatal membranes from rats implanted with placebo and morphine pellets is shown in Fig. 1. The maximal number of binding sites (Bmax value) in the rats treated chronically with morphine and rats implanted with placebo pellets were 141.5_+15.6 and 143.2_+9.0fmol of [3H]spiroperidol bound per milligram of protein, respectively (Fig. 1). These values were not significantly different from each other. Furthermore, these values are in agreement with those reported for high affinity, [3H]spiroperidol-binding sites in the rat striatum (Briley and Langer, 1978). The chronic administration of morphine, however, decreased the apparent dissociation constant (Ko). The Kd values in rats implanted with placebo and morphine pellets were 111.3 + 11.2 and 49.3 +_ 10.7 pM, respectively. Analysis of variance indicated a significant interaction (F(I, 12)= 19.33, P <0.001 (Fig. 1).
m
Studies from these laboratories have indicated that both Pro-Leu-Gly-NH2 and cyclo (Leu-Gly) inhibit the development of tolerance to and physical dependence on opiates. To seek further for a relationship between tolerance to opiates and the functions of dopamine receptors, the effect of peptides, in doses that inhibit tolerance to and physical dependence on opiates, on the function of dopamine receptors altered by chronic treatment with morphine
i
90
LIJ CO 6O
Effect of chronic administration of morphine on the binding of [3H]spiroperidol in the rat striatum
Eff~cts o[peptides on the changes in binding parameters qf striatal [3H]spiroperidol indueed by chronic treatment with morphine to rats
1359
3O
0
i i1 Vehicle
MIF
CLG
Fig. 2. Effect of Pro-Leu-Gly-NH 2 (MW) and cyclo(Leu-
Gly) (CLG) on morphine-induced changes in the binding constants of [3H]spiroperidol. Rats were injected with vehicle (water) or the appropriate peptide and implanted subcutaneously with either four morphine or four placebo pellets as described in the text. The pellets were removed 70 hr after the first implantation. Twenty four hours after the removal of the pellet the animals were sacrificed, their striata removed and frozen at - 70°C until receptor binding studies were undertaken. *P < 0.05 vs vehicle + placebo group, tP < 0.05 vs vehicle + morphine group. was studied. Chronic treatment with either Pro-LeuGly-NH 2 or cyclo (Leu-Gly) in rats implanted with placebo pellets did not alter either the Bm,x or Kd values of [3H]spiroperidol in the striatum (Fig. 2). Both the peptides did not affect the Bin,x values in rats implanted with morphine pellets. However, the Ko values of [3H]spiroperidol, which were decreased as a result of the implantation of morphine pellets, increased significantly. In comparison to the Ko of 49.3pM in the control group, the Kd in the Pro-Leu-Gly-NH2 morphine and cyclo (Leu-Gly) morphine-treated groups were 88.2 _+ 9.6 and 87.4 _+ 8.8 _+ pM, respectively (Fig. 2). DISCUSSION
Previous studies have indicated that the schedule for the implantation of morphine pellets utilized in the present study produced a high degree of tolerance to and physical dependance on morphine (Bhargava, 1977, 1978, 1980, 1981a,c). Earlier behavioral and neurochemical findings suggested the development of enhanced sensitivity to dopaminergic drugs in rodents treated chronically with morphine (Puri and Lal, 1973; Gianutsos et al., 1974; Ritzmann et al., 1979; Bhargava, 1980). Increased sensitivity to dopaminergic drugs results with drugs which decrease dopaminergic transmission on acute administration.
1360
H.N. BHARGAVA
Puri et al. (1978) have reported that a single injection of haloperidol decreased the binding of [3H]spiroperidol but morphine, even at a large concentration, was without any effect on the binding of [3H]spiroperidol. This suggests that morphine decreases dopaminergic transmission by mechanisms other than the direct blockade of dopamine receptors. Similar conclusions were arrived at by Iwatsubo and CIouet (1975) who showed that administration of morphine in vivo produced a dose-dependent increase in the activity of adenylate cyclase without changes in dopamine sensitivity. The changes in the function of dopamine receptors on chronic treatment with morphine resulting in behavioral supersensitivity have been observed by several investigators, as mentioned above. However, considerable disparity exists in the literature for the binding characteristics of [3H]spiroperidol on chronic treatment with morphine. Puri et al. (1978) reported that, in the rat, a small degree of dependence induced by the implantation of two morphine pellets caused increases in both the Kd and Bin,x values for [3H]spiroperidol in striatal membranes, whereas a large degree of dependence induced by four morphine pellets resulted in decreases in both the Kd and Bm,x values. Christie and Overstreet (1979) on the other hand, reported that in the morphine-tolerant rat, the Ko or B..... values of the binding of [3H]spiroperidol did not differ; however, in the morphine-withdrawn group the K~ value increased without any change in B..... values. These studies thus suggest that subsensitivity of striatal dopamine receptors occurs. Chronic treatment with morphine results in decreases in Ka values without a change in the Bm,x value for the binding or [3H]spiroperidol (Ritzmann et al., 1982). The present studies agree with those of Ritzmann et al. (1982) and partially, with those of Purl et al. (1978) in the sense that the latter group also reported decreases in Ka values for the binding of [3H]spiroperidol but found decreases in Bin,Xvalues as well, and are in contrast with those of Christie and Overstreet (1979). Thus, the present studies show that chronic treatment with morphine by the implantation of morphine pellets in the rat caused an increase in the affinity of [3H]spiroperidol to dopamine receptors without changing the maximum number of binding sites. So, the supersensitivity following chronic treatment with morphine, observed in various behavioral measures, may be related to the affinity of ligand to dopamine receptors. This is consistent with the report by Cross, Longden, Owen, Poulter and Waddington (1978) who found a direct relationship between affinity for [~H]spiroperidol and behavioral supersensitivity to apomorphine. In order to seek a relationship between the functions of dopamine receptors and the tolerancedependence process induced by morphine, the effects of two peptides Pro-Leu-Gly-NH2 and cyclo (LeuGly), which are knwon to inhibit tolerance to and dependence on morphine, on the changes in binding
of [3H]spiroperidol following the chronic administration of morphine, were evaluated. The chronic administration of either Pro-Leu-Gly-NH2 or cyclo (Leu-Gly) in rats implanted with placebo pellets did not alter either the apparent dissociation constant or the maximum number of binding sites for [3H]spiroperidol in the striatum. However, both peptides, when administered prior to and during the implantation of morphine pellets, antagonized the enhanced affinity (decreases in Kd values) observed after chronic treatment with morphine. It is, therefore, tempting to speculate that tolerance to and physical dependence on morphine involves changes in the affinity and not the density of striatal dopamine receptors. To summarize, the chronic administration of morphine is associated with an increased affinity of ligands to striatal dopamine receptors and it may be one of the several mechanisms by which tolerance to and physical dependence on morphine is produced. Acknowledgements--This study was supported in part by grant USPHS and grant DA-02598 from the National Institute on Drug Abuse. The author thanks Mr George A. Matwyshyn for providing excellent technical assistance and Mr Pathrose Paulose for secretarial assistance.
REFERENCES
Bhargava H. N. (1977) Rapid induction and quantitation of morphine dependence in the rat by pellet implantation. Psychopharmacology 52:55 62. Bhargava H. N. (1978) Quantitation of morphine tolerance induced by pellet implantation in the rat. J. Pharm. Pharmac. 30: 133-135. Bhargava H. N. (1980) Cyclo(leucylglycine)inhibits the development of morphine-induced analgesic tolerance and dopamine receptor supersensitivity in rats. L(/e Sci. 27:117-123.
Bhargava H. N. (1981a) The effect of melanotropin release inhibiting factor (MIF) and cyclo(Leu-GLy) on the tolerance to morphine-induced antinociception in the rat: a dose-response study. Br. J. Pharmac. 72: 707-714. Bhargava H. N. (1981b) Enhanced response to apomorphine in rats treated with multiple injections of human beta endorphin and its blockade by Pro-Leu-GlyNH 2 and cyclo(Leu-Gly). Life Sci. 29: 1945-1949. Bhargava H. N. (1981c) The effects of peptides on tolerance to the cataleptic and hypothermic effects of morphine in the rat. Neuropharmacology 20: 385-390. Bhargava H. N. (1981d) Inhibition of tolerance to the pharmacological effects of human beta endorphin in the rat by prolyl leucyl glycinamideand cyclo(leucylglycine). J. Pharmac. exp. Ther. 218:404 408. Bhargava H. N., Walter R. and Ritzmann R. F. (1980) Development of narcotic tolerance and physical dependence: effects of Pro-Leu-Gly-NH2 and cyclo(LeuGly). Pharmac. Biochem. Behav. 12: 73-77. Briley M. and Langer S. Z. (1978) Two binding sites for 3H-spiroperidol on rat striatal membranes. Eur. J. Pharmac. 50:283 284. Christie M. J. and Overstreet D. H. (1979) Sensitivity of morphine-tolerant rats to muscarinic and dopaminergic agonists: relation to tolerance and withdrawal. P.~vchopharmacology 65: 27-34. Creese I. and Snyder S. H. (1979) Nigrostriatal lesions enhance striatal 3H-apomorphine and 3Hbspiroperidol binding. Eur. J. Pharmac. 56: 277-281.
Peptides, morphine tolerance-dependence and DA receptors Cross A. J., Longden A., Owen F., Poutter M. and Waddington J. L. (1978) Interrelationship between behavioural and neurochemical indices of dopaminergic neurons. Br. J. Pharmac. 63" 383. Fischer E. (1906) Synthese von Polypeptiden XV. Chem. Ber. 39: 2893-2931. Gianutsos G., Hynes M. D., Puri S. K., Drawbaugh R. B. and Lal H. (1974) Effect on apomorphine and nigrostriatal lesions on agression and striatal dopamine turnover during morphine withdrawal: evidence for dopaminergic supersensitivity during protracted abstinence. P.~vchopharmacologia 34:37 44. Iverson S. D. and Joyce E. M. (1978) Effects in the rat of chronic morphine treatment on the behavioral responses to apomorphine. Br. J. Pharmac. 62: 390. lwatsubo K. and Clouet D. H. (1975) Dopamine-sensitive adenylate cyclase of the caudate nucleus or rats treated with morphine or haloperidol. Biochem. Pharmac. 24" 1499-1505. Kuschinsky K. (1975) Does chronic morphine induce a supersensitivity of dopamine receptors in rat brain? Psychopharmacologia 42: 225-229.
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Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with Folin Phenol reagent. J. biol. Chem. 139: 265-275. Merali Z., Singhal R. L., Hrdina P. D. and Ling G. M. (1975) Changes in brain cyclic AMP metabolism and acetylcholine and dopamine during narcotic dependence and withdrawal. Life Sci. 16: 1889-1894. Puri S. K. and Lal H. (1973) Effect of dopaminergic stimulation of blockade on morphine withdrawal aggression. Psychopharmacologia 32:113-120. Puri S. K., Spaulding T. C. and Mantione C. R. (1978) Dopamine antagonist binding: a significant decrease with morphine dependence in the rat striatum. Life Sci. 23: 637-642. Ritzmann R. F., Lee J. M. and Fields J. Z. (1982) Modification of morphine-induced changes in striatal 3H-spiroperidol binding and stereotype behavior by cyclo (Leu-Gly). Life Sci. 30" 1573-1580. Ritzmann R. F., Walter R., Bhargava H. N. and Flexner L. B. (1979) Blockage of narcotic induced dopamine receptor supersensitivity by cyclo(Leu-Gly). Proc. natln. Acad. Sci. U.S.A. 76:5997 5998.
0028-3908/83$3.00+0.00 Pergamon Press Ltd
BINDING OF [3H]SPIROPERIDOL TO STRIATAL MEMBRANES OF RATS TREATED CHRONICALLY WITH MORPHINE INFLUENCE OF PRO-LEU-GLY-NH2 AND CYCLO(LEU-GLY) H. N. BHARGAVA Department of Pharmacodynamics, University of Illinois at Chicago, Health Sciences Center, Chicago, IL 60612, U.S.A.
(Accepted 1 May 1983) Summary--The effect of the chronic administration of morphine to Sprague Dawley rats on striatal dopamine receptors labelled with [3H]spiroperidol was determined. For chronic administration of morphine, rats were implanted subcutaneously with four morphine pellets (each containing 75 mg of morphine free base) during a 3-day period. Placebo pellet-implanted rats served as controls. Twenty-four hours after the removal of the pellet binding of [3H]spiroperidol to striatal dopamine receptors was determined. Rats receivingmorphine showed no differencein the total number of binding sites (Bmax value) but there was a 60~ decrease in the apparent dissociation constant (Kd value) compared with placebo controls. The effect of two peptides, Pro-Leu-Gly-NH2 and cyclo(Leu-Gly) (2 mg/kg), which have been shown to inhibit tolerance to and dependence on morphine, on the changes in the binding of [3H]spiroperidol induced by the implantation of morphine pellets was also determined. Both peptides antagonized the decreases in K0 values induced by the administration of morphine. However, when administered chronically by themselves, the peptides had no effect on either the Bmax o r Kd values of [3H]spiroperidol. It is concluded that the behavioral supersensitivityof dopamine receptors and the genesis of tolerance-dependence to opiates may be related to the affinity and not to the density of striatal dopamine receptors. Key words: morphine, tolerance-dependence, dopamine receptor, supersensitivity, [3H]spiroperidol binding, Pro-Leu-Gly-NH2, cyclo(Leu-Gly), striatum.
Considerable evidence suggests that the chronic administration of morphine to rodents is associated with aberations in dopaminergic neurons in the central nervous system. Both behavioural and biochemical studies indicate the appearance of a supersensitive dopamine receptor system following chronic treatment (Puri and Lal, 1973; Gianutosos, Hynes, Puri, Drawbaugh and Lal, 1974; Iwatsubo and Clouet, 1975; Ritzmann, Walter, Bhargava and Flexner, 1979; Bhargava, 1980, 1981a). Similar findings have been arrived at after chronic administration of human fl-endorphin to rats (Bhargava, 1981b). However, several studies have either been unable to show supersensitivity or, in some cases, have provided evidence for the presence of the subsensitivity of dopamine receptors after chronic treatment with morphine (Kuschinsky, 1975; Merali, Singhal, Hrdina and Ling, 1975; Iversen and Joyce, 1978). More recently, the function of dopamine receptors following the chronic administration or morphine have been studied using [3H]spiroperidol as the ligand. Puri, Spaulding and Mantione (1978) demonstrated that acute treatment of rats with 30 mg/kg did not alter the binding of [3H]spiroperidol. The implantation of two morphine pellets (50 mg morphine
in each) caused an increase in Kd values, whereas the implantation of 4 morphine pellets resulted in decreases in the Bm~x value (fewer binding sites) with a lower Kd value (enhanced affinity). In both cases the pellets were removed 24 or 48 hr earlier. Christie and Overstereet (1979) on the other hand, found decreased affinity (higher Kd value) for [3H]spiroperidol in the striatum of rats withdrawn from morphine and concluded that morphine-tolerant rats were supersensitive to dopamine agonists and rats withdrawn from morphine were subsensitive. More recent studies suggest that chronic treatment with morphine is associated with an increase in affinity without a change in the number of binding sites for [3H]spiroperidol (Ritzmann, Lee and Fields, 1982). In view of these conflicting data, studies were undertaken to examine the binding parameters of [3H]spiroperidol in the striata of rats treated chronically with morphine. In addition, the effects of two peptides, Pro-Leu-Gly-NH2 (MIF) and cyclo(LeuGly) (CLG), which have been shown to inhibit tolerance to and dependence on opiates (Bhargava, 1980, 1981 a, c, d; Bhargava, Walter and Ritzmann, 1980), on the binding changes of [3H]spiroperidol induced by chronic treatment with morphine has been determined.
1357
1358
H.N. BHARGAVA METHODS
Animals Male Sprague-Dawley rats weighing 200-250g (King Animal Laboratories, Oregon, Wisconsin) were acclimatized to a room with controlled ambient / temperature (23 + 1o,oC), humidity (50 _+ 10%) and a 12hr dark-light cycle (L 0600-1800hr). The rats were given food and water ad libitum. The animals were housed under these conditions for at least 4 days before being used.
Drugs Pro-Leu-Gly-NH2 was a gift from the Abbott Laboratories, North Chicago, Illinois through the courtesy of Dr E. L. Woroch. Cyclo (Leu-Gly) (CLG) was synthesized in the laboratory according to the method of Fischer (1906). The peptides were found to be pure as judged by thin layer chromatographic analysis on two solvent systems as described previously (Bhargava, 1981c). The peptides were dissolved in distilled deionized water and injected subcutaneously, such that each rat received the drug in 1 ml of the solution per kg of body weight. [~H]spiroperidol (sp. act. 26.0 Ci/mmol) was obtained from New England Nuclear, Boston, Massachusetts. d-Butaclamol was a gift from Ayerst Laboratories, Montreal, Canada.
Schedule of the administration of peptide and morphine The rats were treated chronically with morphine by the subcutaneous implantation of four morphine pellets (each containing 75 mg of morphine free base) during a 3-day period as described previously (Bhargava, 1977, 1978). Rats serving as controls were implanted with four placebo pellets. The schedule of treatment with peptide and morphine was similar to that described previously (Bhargava 1981a, c). Briefly, the rats were injected with either vehicle (water), Pro-Leu-Gly-NH2 (2 mg/kg) or cyclo(LeuGly) (2 mg/kg), respectively. Each group of rats was further divided into two subgroups. One hour after the injection of water or the peptide, rats in one subgroup were implanted with a placebo pellet, whereas those in the other subgroup were implanted with a morphine pellet. This initial implantation procedure was followed by two more injections of vehicle or the peptide and implantation of three more placebo or morphine pellets, in their respective groups as described previously (Bhargava, 1981 a, c). All pellets were removed from the rats, under light ether anesthesia, 70 hr after the first implantation of pellets. Twenty four hours after the removal of pellets the rats were sacrificed, their striata removed and frozen at - 7 0 C until assayed for the binding of [3H]spiroperidol. The untilization of the above procedure for the implantation of pellets in the rat has been shown to produce a high deffree of tolerance to and physical dependence on morphine (Bhargava, 1977, 1978). In
addition, the levels of morphine could not be detected in brain or plasma at the time the animals were sacrificed (Bhargava, 1978), although acute treatment with morphine has been shown not to alter the binding of [3H]spiroperidol to the striatal tissue of rats (Puri et al., 1978).
The binding of [3H]spiroperidol to dopamine receptors in the striatal membranes o[ the rat The binding of [3H]spiroperidol was carried out essentially as described previously (Creese and Snyder, 1979) with a slight modification. The striata were homogenized in 30vol of 50mM Tris HCI buffer (pH 7.7 at 25"C) using a Brinkman Polytron homogenizer (setting 6 for 20 sec). The tissue homogenate was twice centrifuged at 49,000g for 15 min in a refrigerated Sorvall RC-5B centrifuge after resuspending in fresh Tris-buffer. The final pellet was suspended in 50 mM, with the Tris buffer (pH 7.4 at 25~C) containing 0.1% ascorbic acid, 120 mM NaCI, 5mM KC1, 2 m M CaCI 2 and 10/tM pargyline to get a concentration of 10 mg wet tissue per ml of incubation buffer. The standard assay mixture contained 0.2 ml of the homogenate containing approx. 200 ~g of protein and 0.1 ml of [3H]spiroperidol and buffer to make up the total volume to 1 ml. Incubation was carried out in triplicate in a shaking water bath maintained at 3 7 C for 15min. At the end of the incubation period, the contents of the incubation tubes were rapidly filtered under partial vacuum using a Millipore manifold filtration unit and Whatman G F / F glass fiber filters. This was followed by two 5ml washes of ice-cold 50raM Tris-buffer (pH 7.4). The filters were transferred to liquid scintillation vials containing 10 ml of 3 a 70 scintillation cocktail (Research Products International Corp. Arlington Heights, Illinois). After an overnight period of equilibration, the radioactivity in the samples was determined in a Packard Tricarb liquid scintillation spectrometer. The stereospecific binding of [3H]spiroperidol was defined as the difference in
c-o 3
•
Morphine - water • Placebo + water
~oOoB
I IpM
Kd=49+-
2
max= 1 4 2 + - [ 6 f m o l / m g
Kd =111_+ I I ~ M t . " . I B too. =,45 :t 9 frnol/rng 0 pro e 13 I
20
I
,
40
60
l
80
protein
--~ I
100
~ ~]
120
"%
140
I
160
B
Fig. 1. Scatchard plots of the binding of [~H]spiroperidol to striatal membranes of nontolerant (placebo pellets) and morphine tolerant-dependent (morphine pellets) rats. The lines are drawn by linear regression analyses.
Peptides, morphine tolerance-dependence and DA receptors binding in the absence and presence of 1/~M dbutaclamol. The concentration of protein in the homogenates was determined according to the method of Lowry, Rosebrough, Farr and Randall (1951). The apparent dissociation constant (Kd) and the maximal binding capacity (Bmax)were calculated from saturation curves generated by using six to seven concentrations of [3H]spiroperidol. The Scatchard plots were generated by least square regression analyses. Four rats were used for each treatment group. The means of Bm.x and Kd values in different groups were analyzed by one-way analysis of variance followed by Schaffe's S-test. RESULTS
O i1 Placebo ,Morphine
Bmox(fmol / mg protein )
~f- 120 ;~
+1
g E
30 0
J Iil K~(pM)
zS S "o
,2o 9O
~- 6o
o & CO
As indicated previously, a high degree of tolerance to and physical dependence on morphine developed as a result of the implantation of four pellets of morphine during a 3-day period (Bhargava, 1977, 1978). Chronic administration of morphine resulted in an increase in the affinity of the binding of [3H]spiroperidol to dopamine receptors. However, the number of binding sites remained unchanged. The Scatchard plot for the specific binding of [3H]spiroperidol to striatal membranes from rats implanted with placebo and morphine pellets is shown in Fig. 1. The maximal number of binding sites (Bmax value) in the rats treated chronically with morphine and rats implanted with placebo pellets were 141.5_+15.6 and 143.2_+9.0fmol of [3H]spiroperidol bound per milligram of protein, respectively (Fig. 1). These values were not significantly different from each other. Furthermore, these values are in agreement with those reported for high affinity, [3H]spiroperidol-binding sites in the rat striatum (Briley and Langer, 1978). The chronic administration of morphine, however, decreased the apparent dissociation constant (Ko). The Kd values in rats implanted with placebo and morphine pellets were 111.3 + 11.2 and 49.3 +_ 10.7 pM, respectively. Analysis of variance indicated a significant interaction (F(I, 12)= 19.33, P <0.001 (Fig. 1).
m
Studies from these laboratories have indicated that both Pro-Leu-Gly-NH2 and cyclo (Leu-Gly) inhibit the development of tolerance to and physical dependence on opiates. To seek further for a relationship between tolerance to opiates and the functions of dopamine receptors, the effect of peptides, in doses that inhibit tolerance to and physical dependence on opiates, on the function of dopamine receptors altered by chronic treatment with morphine
i
90
LIJ CO 6O
Effect of chronic administration of morphine on the binding of [3H]spiroperidol in the rat striatum
Eff~cts o[peptides on the changes in binding parameters qf striatal [3H]spiroperidol indueed by chronic treatment with morphine to rats
1359
3O
0
i i1 Vehicle
MIF
CLG
Fig. 2. Effect of Pro-Leu-Gly-NH 2 (MW) and cyclo(Leu-
Gly) (CLG) on morphine-induced changes in the binding constants of [3H]spiroperidol. Rats were injected with vehicle (water) or the appropriate peptide and implanted subcutaneously with either four morphine or four placebo pellets as described in the text. The pellets were removed 70 hr after the first implantation. Twenty four hours after the removal of the pellet the animals were sacrificed, their striata removed and frozen at - 70°C until receptor binding studies were undertaken. *P < 0.05 vs vehicle + placebo group, tP < 0.05 vs vehicle + morphine group. was studied. Chronic treatment with either Pro-LeuGly-NH 2 or cyclo (Leu-Gly) in rats implanted with placebo pellets did not alter either the Bm,x or Kd values of [3H]spiroperidol in the striatum (Fig. 2). Both the peptides did not affect the Bin,x values in rats implanted with morphine pellets. However, the Ko values of [3H]spiroperidol, which were decreased as a result of the implantation of morphine pellets, increased significantly. In comparison to the Ko of 49.3pM in the control group, the Kd in the Pro-Leu-Gly-NH2 morphine and cyclo (Leu-Gly) morphine-treated groups were 88.2 _+ 9.6 and 87.4 _+ 8.8 _+ pM, respectively (Fig. 2). DISCUSSION
Previous studies have indicated that the schedule for the implantation of morphine pellets utilized in the present study produced a high degree of tolerance to and physical dependance on morphine (Bhargava, 1977, 1978, 1980, 1981a,c). Earlier behavioral and neurochemical findings suggested the development of enhanced sensitivity to dopaminergic drugs in rodents treated chronically with morphine (Puri and Lal, 1973; Gianutsos et al., 1974; Ritzmann et al., 1979; Bhargava, 1980). Increased sensitivity to dopaminergic drugs results with drugs which decrease dopaminergic transmission on acute administration.
1360
H.N. BHARGAVA
Puri et al. (1978) have reported that a single injection of haloperidol decreased the binding of [3H]spiroperidol but morphine, even at a large concentration, was without any effect on the binding of [3H]spiroperidol. This suggests that morphine decreases dopaminergic transmission by mechanisms other than the direct blockade of dopamine receptors. Similar conclusions were arrived at by Iwatsubo and CIouet (1975) who showed that administration of morphine in vivo produced a dose-dependent increase in the activity of adenylate cyclase without changes in dopamine sensitivity. The changes in the function of dopamine receptors on chronic treatment with morphine resulting in behavioral supersensitivity have been observed by several investigators, as mentioned above. However, considerable disparity exists in the literature for the binding characteristics of [3H]spiroperidol on chronic treatment with morphine. Puri et al. (1978) reported that, in the rat, a small degree of dependence induced by the implantation of two morphine pellets caused increases in both the Kd and Bin,x values for [3H]spiroperidol in striatal membranes, whereas a large degree of dependence induced by four morphine pellets resulted in decreases in both the Kd and Bm,x values. Christie and Overstreet (1979) on the other hand, reported that in the morphine-tolerant rat, the Ko or B..... values of the binding of [3H]spiroperidol did not differ; however, in the morphine-withdrawn group the K~ value increased without any change in B..... values. These studies thus suggest that subsensitivity of striatal dopamine receptors occurs. Chronic treatment with morphine results in decreases in Ka values without a change in the Bm,x value for the binding or [3H]spiroperidol (Ritzmann et al., 1982). The present studies agree with those of Ritzmann et al. (1982) and partially, with those of Purl et al. (1978) in the sense that the latter group also reported decreases in Ka values for the binding of [3H]spiroperidol but found decreases in Bin,Xvalues as well, and are in contrast with those of Christie and Overstreet (1979). Thus, the present studies show that chronic treatment with morphine by the implantation of morphine pellets in the rat caused an increase in the affinity of [3H]spiroperidol to dopamine receptors without changing the maximum number of binding sites. So, the supersensitivity following chronic treatment with morphine, observed in various behavioral measures, may be related to the affinity of ligand to dopamine receptors. This is consistent with the report by Cross, Longden, Owen, Poulter and Waddington (1978) who found a direct relationship between affinity for [~H]spiroperidol and behavioral supersensitivity to apomorphine. In order to seek a relationship between the functions of dopamine receptors and the tolerancedependence process induced by morphine, the effects of two peptides Pro-Leu-Gly-NH2 and cyclo (LeuGly), which are knwon to inhibit tolerance to and dependence on morphine, on the changes in binding
of [3H]spiroperidol following the chronic administration of morphine, were evaluated. The chronic administration of either Pro-Leu-Gly-NH2 or cyclo (Leu-Gly) in rats implanted with placebo pellets did not alter either the apparent dissociation constant or the maximum number of binding sites for [3H]spiroperidol in the striatum. However, both peptides, when administered prior to and during the implantation of morphine pellets, antagonized the enhanced affinity (decreases in Kd values) observed after chronic treatment with morphine. It is, therefore, tempting to speculate that tolerance to and physical dependence on morphine involves changes in the affinity and not the density of striatal dopamine receptors. To summarize, the chronic administration of morphine is associated with an increased affinity of ligands to striatal dopamine receptors and it may be one of the several mechanisms by which tolerance to and physical dependence on morphine is produced. Acknowledgements--This study was supported in part by grant USPHS and grant DA-02598 from the National Institute on Drug Abuse. The author thanks Mr George A. Matwyshyn for providing excellent technical assistance and Mr Pathrose Paulose for secretarial assistance.
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Lowry O. H., Rosebrough N. J., Farr A. L. and Randall R. J. (1951) Protein measurement with Folin Phenol reagent. J. biol. Chem. 139: 265-275. Merali Z., Singhal R. L., Hrdina P. D. and Ling G. M. (1975) Changes in brain cyclic AMP metabolism and acetylcholine and dopamine during narcotic dependence and withdrawal. Life Sci. 16: 1889-1894. Puri S. K. and Lal H. (1973) Effect of dopaminergic stimulation of blockade on morphine withdrawal aggression. Psychopharmacologia 32:113-120. Puri S. K., Spaulding T. C. and Mantione C. R. (1978) Dopamine antagonist binding: a significant decrease with morphine dependence in the rat striatum. Life Sci. 23: 637-642. Ritzmann R. F., Lee J. M. and Fields J. Z. (1982) Modification of morphine-induced changes in striatal 3H-spiroperidol binding and stereotype behavior by cyclo (Leu-Gly). Life Sci. 30" 1573-1580. Ritzmann R. F., Walter R., Bhargava H. N. and Flexner L. B. (1979) Blockage of narcotic induced dopamine receptor supersensitivity by cyclo(Leu-Gly). Proc. natln. Acad. Sci. U.S.A. 76:5997 5998.