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The effects of peptides on tolerance to the cataleptic and hypothermic effects of morphine in the rat
00?&3902(‘n1 040385.0680?.00/0 Pergamon Press Ltd
THE EFFECTS OF PEPTIDES ON TOLERANCE TO THE CATALEPTIC AND HYPOTHERMIC EFFECTS OF MORPHINE IN THE RAT H. N. BHARGAVA Department of Pharmacognosy and Pharmacology, University of Illinois at the Medical Center, Chicago, IL60680, U.S.A. (Accepted 14 September 1980) Summary-The effects of melanotropin release inhibiting factor (MIF) and a cyclic analog, cycle (LeuGly) derived from MIF on the development of tolerance to the cataleptic and hypothermic effects of morphine in the rat were investigated. The rats were made tolerant to morphine by the subcutaneous implantation of four morphine pellets over a 3-day period. Each pellet contained 75 mg of morphine-free base. Following morphine pellet implantation, tolerance developed to the cataleptic and hypothermic effects of morphine. A dose of morphine (50 mg/kg) which produced hypothermia in placebo-pelleted rats, produced hyperthermia in morphine-pelleted rats. Administration of MIF and cycle (Leu-Gly) prior to and during morphine pellet implantation inhibited the development of tolerance to the cataleptic effect of morphine. Similarly the hyperthermic effect of morphine in morphine-tolerant rats was prevented by both the peptides. Previously, the present authors reported Ithat these peptides also inhibited tolerance to the analgesic effect of morphine. It is concluded that tolerance to analgesic, cataleptic and hypothermic effects of morphine may share common features in its genesis.
In recent years several hypothalamic peptide factors, either inhibiting or stimulating the release of hormones from the pituitary, have been shown to have direct effects on the central nervous system (CNS). These direct effects caused by the hypothalamic factors are independent of their action on the hypophysis. For instance, thyrotropin releasing hormone (TRH) antagonizes the pharmacological effects of CNS depressant drugs, like barbiturates (Breese, Cott, Cooper, Prange, Lipton and Plotnikoff, 1973 alcohol (Breese, Cott, Cooper, Prange and Lipton, 1974), morphine (Horita, Carino and Chestnut, 1976; Bhargava, 1980a) and delta-9-tetrahydrocannabinol (Bhargava, 1980b; Bhargava and Matwyshyn, 1980). Similarly, another hypothalamic hormone, melanotropin release inhibiting factor (Pro-Leu-Gly-NH,, MIF) and several of its analogs have been shown to facilitate the development of tolerance to and physical dependence on morphine in the rat (Van Ree and De Wied, 1976). In earlier studies, a dipeptide, carbobenzyloxy-Pro-D-Leu (Z-Pro-D-Leu) was synthesized in which L-leucine was replaced with the D-k0me.r in the hope that this would create a possible misfit at the receptor site so that these analogs could result in the inhibitory rather than the facilitatory effect on morphine tolerance development. Studies with Z-Pro-DLeu indeed indicated that it could inhibit morphine tolerance and dependence in mice (Walter, Ritzmann, Bhargava, Rainbow, Flexner and Krivoy, 1978). Further studies indicated that MIF and a variety of Key words: peptides, MIF, cycle (Leu-Gly), morphine catalepsy, hypothermia, tolerance.
its di-, tri- and cyclic-analogs also blocked morphine tolerance and/or dependence in mice (Walter, Ritzmann, Bhargava and Flexner, 1979; Bhargava, Walter and Ritzmann, 1980) and rats (Bhargava, 198Oc). In many of these studies, the tolerance to the analgesic effect was studied. However, morphine also causes catalepsy and hypothermia and, on chronic administration, tolerance develops to these effects. Therefore, in order to ascertain if these peptides had effects on the other actions of morphine, the effects of MIF and cycle (Leu-Gly) on tolerance to the cataleptic and hypothermic effects of morphine are described here. METHODS Animals Male Sprague-Dawley rats weighing 200-250 g (King Animal Laboratories, Oregon, WI) were acclimatized to a room with controlled ambient temperature (23 + l°C), humidity (65 f 2%) and a 12 hr dark-light cycle (L 0600-1800 hr). The animals were housed under these conditions for at least 4 days prior to being used. The rats were given food and water ad libitum. Drugs Through the courtesy of Dr A. 0. Geiszler. MIF was obtained as a gift from the Abbott Laboratories, North Chicago, IL. Cycle (Leu-Gly) was synthesized according to the method of Fischer (1906). Thin layer chromatographic analyses on silica Gel plates showed a single spot on two solvent systems (Solvent I, Chloroform: Methanol, 9:l v/v, R, 0.30; Solvent II, Chloroform: Methanol 7: 3 v/v, R, 0.40) when sprayed 385
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H. N. BHARGAVA
with chlorine-tolidine reagent. Cycle (Leu-Gly) and MIF were dissolved in water and injected subcutaneously (s.c.) in a volume of 1 ml/kg body weight. Morphine sulfate (Mallidckrodt Chemical Co., St Louis, MO) was dissolved in saline and injected intraperitoneally (ip.) in a volume of 1 ml/kg body weight. All solutions were prepared fresh on the day of their use. Effect of MIF and cycle (Leu-G/y) on the development of tolerance to catalepsy and hypothermia induced by morphine Induction and assessment of tolerance. Rats were rendered tolerant to and dependent on morphine by subcutaneous implantation of pellets (each containing 75 mg/kg of morphine free base), over a 3-day period as described previously (Bhargava, 1977, 1978, 1980~). Briefly, the back of the rat was shaved. On day 1, under light ether anesthesia at 10a.m. one morphine pellet was implanted after making a small incision in the lower back. The second pellet was implanted at 4p.m. on the same day. On day 2, two more pellets were implanted at 4 p.m. All the pellets were removed 70 hr after the first implantation. Control rats received placebo pellets. At various times thereafter catatonic and hypothermic responses to morphine were determined as described below. To determine the effect of MIF or cycle (Leu-Gly). the peptide was injected subcutaneously in an appropriate dose 2 hr before the first placebo or morphine pellet implantation. The control rats received vehicle (water) instead of the peptide. The injections of water and peptide were given two more times 24 hr apart. Thus, the last injection of the vehicle or the peptide was given 24 hr prior to pellet removal. Quantijcation of the cataleptic action of morphine The degree of cataleptic state was determined by measuring its duration using a bar test for catatonia (Costall and Naylor, 1974). The forepaws of the rat were gently placed across a bar supported by two iron stands 10 cm off the floor. The time for removal of the paws from the bar was measured to the nearest second after treatment with morphine sulfate (50 mg/kg, i.p.). Animals that had not stepped off the bar within 60 set were removed from the bar. If the rat stayed at the bar for 60sec or more it was considered to have 100% catalepsy. If it was less than 60 set then the percentage catalepsy was calculated using 60 set as 100%. The catalepsy was expressed as mean *SEM and was calculated for the following groups viz. (a) vehicle + placebo (b) peptide + placebo, (c) vehicle + morphine and (d) peptide + morphine. The statistical significance between the means of the catalepsy score was determined by using the paired or unpaired Student’s t-test. Measurement of the body temperature The rats were treated with vehicle or the peptide in an appropriate dose and then implanted with placebo
or morphine pellets as described above. At various times after the pellet removal. morphine sulfate (50mg/kg, i.p.) was administered to rats in all the different groups. The recta1 temperature of each rat was measured at fixed times after morphine injection using a telethermometer (Yellow Springs Instrument Co., Yellow Springs, OH) model No. 73 and Probe Type 423. Temperatures were recorded when a constant reading was obtained after rectal insertion of the probe to approx. 6cm. This procedure usually took 30sec for each rat. The data are expressed as mean temperature +SEM. The difference in the means for different groups was analyzed by the Student’s r-test. A value of P > 0.05 was considered nonsignificant. RESULTS
Effect of MIF on tolerance to the cataleptic and hypothermic effect of morphine Multiple injections of MIF inhibited the development of tolerance to the cataleptic action of morphine in the rat. The effect of MIF on morphine-induced catalepsy in placebo and rats with implanted morphine pellets from which the pellets had been removed for 6 hr is shown in Table 1. A dose of 50 mg/kg of morphine produced 71”” catalepsy in placebo pellet-implanted rats which were treated with vehicle. Administration of three injections of MIF to rats implanted with placebo pellets did not alter morphine-induced catalepsy. Implantation of morphine pellets resulted in the development of tolerance as shown by a 4O”h cataleptic response to morphine. In doses of 0.25 and 0.5 mg/kg administered daily for 3 days, MIF did not modify tolerance development; however, a 1.0 mg/kg dose significantly (P < 0.05) inhibited the tolerance to morphine. In another experiment (II). the effect of 2 mg/kg of MIF was studied in rats from which pellets had been removed for 24 hr. Results very similar to those described above were obtained. Thus, treatment with 1 or 2 mg/kg, which did not modify morphine catalepsy in placebopelleted rats, significantly inhibited the development of morphine-induced catalepsy. The effects of MIF on morphine-induced temperature responses in placebo and morphine-pelleted rats from which the pellets had been removed for 6 (Experiment I) or 24 hr (Experiment II) are shown in Table 2. Administration of morphine (50 mg/kg) produced a hypothermic response in placebo pelletimplanted rats and this response was not altered in rats given MIF in any dose used. Rats implanted with morphine pellets, given the vehicle injections, failed to show the hypothermia when injected with morphine, indicating the development of tolerance. The rectal temperatures at 30 min after morphine injection in the MIF (0.25 and 0.5 mg/kg)-treated morphine-tolerant rats, were similar to those of vehicle-injected morphine-tolerant rats. However, the rectal temperatures of rats given MIF (1 mg/kg) were significantly lower than their corresponding vehicle-injected controls.
387
Peptides on chronic morphine actions Table 1. Effect of MlF on the development of tolerance to the cataleptic action of morphine Dose of MIF (mg/kg. s.c.)
Treatment*
Percent catalepsyt (Mean f SEM)
N
Experiment I
(6 hr after the pellet removal) Vehicle: + placebo MIF + placebo Vehicle: + morphine MIF + morphine MIF + morphine MIF + morphine
1.0 0.25 0.50 1.0
4 4 6 8 7 6
71 f 81 f 4O* 31 * 27 k 70 *
11 19 136 5 14 157
8 8 10 10
68 f 60 + 13 * 31 +
14 12 15 147
Experiment II (24 hr after the pellet removal)
Vehicle3 + placebo MIF + placebo Vehicle: + morphine MIF + morphine
2.0 2.0
* Rats were injected with vehicle or MIF and implanted with 4 placebo or 4 morphine pellets over a 3-day period. The injections of the vehicle and MIF were repeated two more times 24 hr apart. The pellets were removed 70 hr after the first implantation. t Thirty min after the morphine sulfate (50 mg/kg, i.p.) injection. $ Water $ P < 0.05 vs the vehicle + placebo group. 7 P < 0.05 vs the vehicle + morphine group.
Effect of cycle (Leu-Gly)
When the above experiment was repeated with MIF in a dose of 2 mg/kg in rats from which the pellets had been removed for 24 hr, a hypothermic response to morphine was observed in placebopelleted rats given the vehicle of MIF. A hyperthermic (P < 0.05) response to morphine was obtained in morphine-tolerant rats. Again as in Experiment I, in the rats treated with 2 mg/kg of MIF, the rectal temperature following morphine injection was lower than in the corresponding vehicle-injected controls.
and hypothermic
on tolerance
to the cataleptic
effect of morphine
Administration of cycle (Leu-Gly) also inhibited the tolerance to morphine-induced catalepsy. The catalepsy produced by morphine (50mg/kg) in placebo or morphine pellet implanted rats given three injections of cycle (Leu-Gly) (2mgfkg) and from which the pellets had been removed for 24 (Experiment I) or 48 hr (Experiment II) is shown in Table 3. As seen with MIF, multiple injections of cycle (Leu-
Table 2. ElTect of MIF on the development of tolerance to the hypothermic effect of morphine
Treatment*
Dose of MIF (mgikg, s.c.)
N
Rectal temperature, “C (Mean k SEM) Time after morphinet injection (min) 0 30
Experiment I (6 hr after the pellet removal)
Vehicle: + placebo MIF + placebo Vehicle: + morphine MIF + morphine MIF + morphine MIF + morphine
1.0 0.25 0.50 1.0
4 4 6 8 I 6
37.9 38.1 37.9 38.0 38.0 31.9
+ k * k + f
0.3 0.2 0.2 0.2 0.2 0.2
35.6 35.7 38.4 38.1 38.7 31.5
If: 0.2 + 0.3 + 0.35 + 0.2 f 0.2 + o.Lq
36.5 + 36.5 5 36.8 f 36.4 f
0.1 0.1 0.2 0.1
35.5 f 35.4 f 38.6 k 38.0 *
Experiment II (24 hr after the pellet removal)
Vehicle MIF + Vehicle MIF +
+ placebo placebo + morphine morphine
2.0 2.0
8 8 10 10
0.1 0.1 0.2$ 0.2q
* Rats were injected with vehicle or MIF and implanted with 4 placebo or 4 morphine pellets over a 3-day period. Two more injections of the vehicle and MIF were made 24 hr apart. The pellets were removed 70 hr after the first implantation. i Morphine sulfate 50 mg/kg (i.p.). $ Water. 5 P < 0.05 vs the vehicle + placebo group. 7 P < 0.05 vs the vehicle + morphine group.
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H. N.
Table 3. Effect of cycle (Leu-Gly) on tolerance to the cataleptic effect of morphine Treatment*
N
Percent catalepsyt (Mean + SEM)
8 8 10 10
68 f 62 f 13 + 43 +
Experiment I (24 hr after the
pellet removal) Vehicle1 + placebo Cycle (Leu-Gly) + placebo Vehicle1 + morphine Cycle (Leu-Gly) + morphine
14 10 l# 117
Experiment II (48 hr after the
pellet removal Vehicle1 + placebo Cycle (Leu-Gly) + placebo Vehicle1 + morphine Cycle (Leu-Gly) + morphine
6 6 6 6
100 + 0 look0 13 f 5s 26 f 7ll
* Rats were injected with the vehicle or cycle (Leu-Gly) (2 mg/kg, s.c.) daily for 3 days and implanted with 4 placebo or 4 morphine pellets as described in the text. The pellets were removed 70 hr after the first implantation. t Thirty min after the morphine sulfate (50 mg/kg, i.p.) injection. $ Water. # P c 0.05 vs the vehicle + placebo group. fi P <: 0.05 vs the vehicle + morphine group.
Gly) did not modify morphine catalepsy in placebopelleted rats. Tolerance developed to the cataleptic effect of morphine as a result of morphine pellet implantation, since the percentage catalepsy decreased from 67.8 in placebo-pelleted rats to 12.6 in morphine-pelleted rats. In contrast to the Iatter, the morphine-tolerant rats given cycle (Leu-Gly) showed 43.2% catalepsy. Similar results were obtained in Experiment II where pellets had been removed for 48 hr. The temperature responses to morphine were also similar to those obtained with MIF. As shown in
BHARGAVA
Table 4, morphine produced hypothermia in placebo hyperthermia was pellet-implanted rats, whereas obtained in morphine pellet-implanted rats. In morphine-tolerant rats treated with cycle (Leu-Gly) significantly (P < 0.05) less hyperthermia was observed (Experiment I, Table 4). In Experiment II where the pellets were removed for 48 hr, cycle (Leu-Gly) treatment completely prevented the morphine-induced hyperthermia. The rectal temperatures in this group of rats were not different from the rats in the two placebo groups. DISCUSSION
The present studies indicate that chronic administration of morphine results in the development of tolerance to the catatonic and hypothermic effects of morphine and that this process is inhibited by daily administration of MIF and cycle (Leu-Gly). Previous studies from these laboratories have indicated that MIF and cycle (Leu-Gly) do not modify analgesia produced by a single administration of morphine; however, these peptides block the development of tolerance to the analgesic effect of morphine (Bhargava, 198Ck, 1981). Blockage of tolerance to the catatonic effect was also demonstrated in the present study. A high correlation was observed between the production of analgesia and of catatonia when the narcotic drug etorphine was injected in the rat either intraperitoneally or in the periaqueductal gray region of the midbrain (Thorn and Levitt, 1980). These studies and studies in this laboratory on tolerance also suggest that a common mechanism may be involved, not only in the production of the two effects of opiates, but also on the development of tolerance. In non-tolerant or placebo-pelleted rats, morphine produced hypothermia. which was unaffected by MIF
Table 4. Effect of cycle (Leu-Gly) on tolerance to the hypothermic effect of morphine
Treatment*
N
Rectal temperature, “C (Mean + SEM) Time after morphine? injection (min) 0 30
Experiment I (24 hr after the
pellet removal) Vehicle3 + placebo Cycle (Leu-Gly) + placebo Vehicle3 + morphine Cycle (Leu-Gly) + morphine
8 8 10 10
36.5 f 0.1 36.4 + 0.1 36.8 -f: 0.2 36.4 + 0.1
35.5 35.4 38.6 38.2
* * k +
0.1 0.1 0.2$ 0.17
36.7 36.8 36.8 36.7
35.8 f 35.7 * 38.0 + 36.1 k
0.4 0.2 0.4$ 0.q
Experiment II (48 hr after the
pellet removal) Vehicle3 + placebo Cycle (Leu-Gly) + placebo Vehicle1 + morphine Cycle (Leu-Gly) + morphine
+ + k f
0.2 0.2 0.2 0.2
* Rats were injected with the vehicle or cycle (Leu-Gly) (2 mg/kg, s.c.) daily for 3 days and implanted with 4 placebo or 4 morphine pellets as described in the text. The pellets were removed 70 hr after the first implantation. t 50 mg/kg (i.p.). $ Water. 5 P < 0.05 vs the vehicle + placebo group. 7 P < 0.05 vs the vehicle + morphine group.
Peptides on chronic morphine actions or cycle (Leu-Gly) treatment. When the same dose of morphine was administered to morphine-tolerant rats, a hyperthermic effect was observed. The morphineinduced hypothermia in non-tolerant rats and the hyperthermia in tolerant rats are consistent with previous studies (Oka, Nozaki and Hosoya, 1972; Warwick, Blake, Miya and Bousquet, 1973). Daily administration of MIF or cycle (Leu-Gly) inhibited the development of the hyperthermic response to morphine in rats implanted with morphine pellets. The thermoregulatory effects of morphine are indeed complex and many neurotransmitter-systems including dopamine have been implicated. It is believed that the hypothermic response to morphine may be related to the hypothalamic serotoninergic system (Lotti, Lomax and George, 1965). However, its involvement in the chronic effect is unlikely (Warwick, et al., 1973). The hyperthermic response may involve cholinergic mechanisms since cholinergic antagonists, like scopolamine and atropine, antagonize morphineinduced hyperthermia (Oka et al., 1972). It is not clear at present if MIF and cycle (Leu-Gly) interact with cholinergic mechanisms. The mechanism by which MIF and cycle (Leu-Gly) inhibit morphine-tolerance development remains to be elucidated. The cataleptic and analgesic actions of narcotic analgesics is accompanied by an increased turnover of dopamine and increased concentration of homovanillic acid in the striatum (Kuschinsky, 1973; Kuschinsky and Hornykiewicz, 1972). On chronic administration of morphine, supersensitivity of brain dopamine receptors develops as a result of chronic depression of dopaminergic neurotransmission (Bhargava, 1980d; Gianutsos, Hynes, Puri, Drawbaugh and Lal, 1974; Iwatsubo and Clouet, 1975; Puri and Lal, 1973; Ritzmann. Walter, Bhargava and Flexner, 1979). It has been shown that MIF and cycle (LeuGly) block the development of morphine-induced dopamine receptor supersensitivity in mice and rats (Bhargava, 198Od, 1981; Ritzmann, et al., 1979). Thus, morphine-induced tolerance to cataleptic and analgesic effects may be related to brain dopamine supersensitivity. Further evidence for the role of dopaminergic systems in the inhibitory action of MIF and cycle (Leu-Gly) on morphine tolerance is provided by the fact that the dopamine receptor supersensitivity induced by long-term administration of haloperidol is inhibited by these peptides (Bhargava and Ritzmann, 1980). In conclusion, it appears that morphineinduced tolerance to its cataleptic, analgesic and hypothermic actions may involve a common mechanism, perhaps changes in dopamine receptor supersensitivity since all these phenomena were blocked by MIF and cycle (Leu-Gly).
Acknowledgements-These studies were supported by a National Institute on Drug Abuse grant DA-02598. The author thanks George Matwyshyn for providing excellent
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technical assistance, Mrs Cheryl King and MS Mary Lou Quinn for their help in the preparation of this manuscript. 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. (1980a). The effects of thyrotropin releasing hormone on the central nervous system responses to chronic morphine administration. Psychopharmacology 6% 185-189. Bhargava, H. N. (1980b). The effects of thyrotropin releasing hormone and histidyl-proline diketopiperazine on delta-9-tetrahydrocannabinol induced hypothermia. Life Sci. 26: 845-850. Bhargava, H. N. (1980~). Cycle (Leucylglycine) inhibits the development of morphine-induced analgesic tolerance and dopamine receptor supersensitivity in rats. Life Sci. 27: 117-123. Bhargava, H. N. (1980d). Inability of cycle (leucylglycine) to facilitate the development of tolerance to and physical dependence on morphine in the rat. L$ .&i 27: 1075-1081. Bhargava, H. N. (1981). The effect of melantropin release inhibiting factor and cycle (Leu-Gly) on the tolerance to morphine induced antinociception in the rat: a doseresponse study. Br. J. Pharmac. In press. Bhargava, H. N. and Matwyshyn, G. A. (1980). Influence of thyrotropin releasing hormone and histidylproline diketopiperazine on spontaneous locomotor activity and analgesia induced by delta-9-tetrahydrocannabinol in the mouse. Eur. J. Pharmac. 68: 147-154. Bhargava, H. N. and Ritzmann, R. F. (1980). Influence of cycle (leyclglycine) on the development of behavioral supersensitivity of dopamine receptors induced by long term administration of haloperidol. The Pharmacologist 22: 259.
Bhargaia, H. N., Walter, R. and Ritzmann, R. F. (1980). Development of narcotic tolerance and physical dependence: effects of Pro-Leu-Gly-NH* and cycle (Leu-Gly). Pharmac. Biochem. Behav. 12: 73-77.
Breese, G. R., Cott, J. M., Cooper, B. R., Prange, A. J. and Lipton, M. A. (1974). Antagonism of ethanol narcosis by thyrotropin-releasing hormone. L$e Sci. 14: 1053-1063. Breeie, G.-R.. Cott, i M. Cooper, B. R., Prange, A. J., Lioton. M. A. and Plotnikoff. N. P. (1975). Effects of thirotropin-releasing hormone on the actions of pentobarbital and other centrally acting drugs. J. Pharmac. exp. Ther. 196: 594604.
Costall, B. and Naylor, R. (1974). A role for the amygdala in the development of the cataleptic and sterotypic actions of the narcotic agonists and antagonists in the rat. Psychopharmacology 35: 203-213. 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 of apomorphine and nigrostriatal lesions on aggression and striatal dopamine turnover during morphine withdrawal. Evidence for dopaminergic supersensitivity in protracted abstinence. Psychopharmacologia (Bed.) 34: 3744.
Horita, A., Carino, M. A. and Chestnut, R. M. (1976). Influence on drug induced narcosis and hypothermia in rabbits. Psychopharmacology 49: 57-62. Iwatsubo, K. and Clouet, D. H. (1975). Dopamine sensitive adenylate cyclase of the caudate nucleus of rats treated with morphine or haloperidol. Biochem. Pharmac. 24: 1499-1503.
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dopamine utilization in corpora striata of rats. Experimentiu 29: 1365-1366. Kuschinsky. K. and Hornykiewicz, 0. (1972). Morphine catalepsy in the rat: relation to striated dopamine metabolism. Eur. J. F~ffr~~. 19: 119-122. Lotti. V. 1.. Lomax. P. and George, R. (l965). Temperature responses in the rat following intracerebral microinjection of morphine. J. Pharmac. exp. Ther. 150: 135-139. Oka. T.. Nozaki, M. and Hosoya. E. (1972). Effects of p-chlorophenylalamine and choiinergic antigonists on body temperature changes induced by the administration of morphine to nontolerant and morphjne-tolerant rats. J. P~urmu~. exn. Ther. 180: 136-143. Puri. S. K. and Lal,’ H. (1973). Effect of dopaminergic stimulation on blockade on morphine withdrawal aggression. Ps,whophurmacoloyiu (Burl.) 32: 113-l 18. Ritzmann. R. F.. Walter. R., Bhargava. H. N. and Flexner, L. B. (1979). Blockage of narcotic induced dopamine receptor supersensitivity by cycle (Leu-Gly). Proc. na~n. Acud. SK.
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Thorn, B. E. and Levitt, R. A. (1980). Etorphine induction of analgesia and catatonia in the rat: systemic or intracranial injection. Neuropharmucology 19: 203-207. Van Ree. J. M. and De Wied. D. (1976). Prolyl-leucylglycinamide (PLG) facilitates morphine dependence. Lifv Sri. 19: 1331-1340. Walter, R. Ritzmann. R. F., Bhargava, H. N. and Flexner. L. B. (1979). Prolyl leucylglycinamide. cycle (leucylglytine) and derivatives block development on morphine in mice. Proc. natn. Acad. Sci.. U.S.A. 76: 518-520. Walter, R., Ritzmann, R. F., Bhargava, H. N.. Rainbow, T. C.. Flexner. L. B. and Krivoy, W. A. (1978). Inhibition by Z-Pro-D-Leu of development of tolerance to and physical dependence on morphine in mice. Prof. natn. Acad. Sri.. U.S.A. 75: 457334576. Warwick. R. 0.. Blake, D. E.. Miya. T. S. and Bousquet, W. F, (1973). Serotonin involvement in thermoregulation following administration of morphine to nontolerant and morphine-tolerant rats. Res. Commun. Chem. Puth. P~ar~e.
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THE EFFECTS OF PEPTIDES ON TOLERANCE TO THE CATALEPTIC AND HYPOTHERMIC EFFECTS OF MORPHINE IN THE RAT H. N. BHARGAVA Department of Pharmacognosy and Pharmacology, University of Illinois at the Medical Center, Chicago, IL60680, U.S.A. (Accepted 14 September 1980) Summary-The effects of melanotropin release inhibiting factor (MIF) and a cyclic analog, cycle (LeuGly) derived from MIF on the development of tolerance to the cataleptic and hypothermic effects of morphine in the rat were investigated. The rats were made tolerant to morphine by the subcutaneous implantation of four morphine pellets over a 3-day period. Each pellet contained 75 mg of morphine-free base. Following morphine pellet implantation, tolerance developed to the cataleptic and hypothermic effects of morphine. A dose of morphine (50 mg/kg) which produced hypothermia in placebo-pelleted rats, produced hyperthermia in morphine-pelleted rats. Administration of MIF and cycle (Leu-Gly) prior to and during morphine pellet implantation inhibited the development of tolerance to the cataleptic effect of morphine. Similarly the hyperthermic effect of morphine in morphine-tolerant rats was prevented by both the peptides. Previously, the present authors reported Ithat these peptides also inhibited tolerance to the analgesic effect of morphine. It is concluded that tolerance to analgesic, cataleptic and hypothermic effects of morphine may share common features in its genesis.
In recent years several hypothalamic peptide factors, either inhibiting or stimulating the release of hormones from the pituitary, have been shown to have direct effects on the central nervous system (CNS). These direct effects caused by the hypothalamic factors are independent of their action on the hypophysis. For instance, thyrotropin releasing hormone (TRH) antagonizes the pharmacological effects of CNS depressant drugs, like barbiturates (Breese, Cott, Cooper, Prange, Lipton and Plotnikoff, 1973 alcohol (Breese, Cott, Cooper, Prange and Lipton, 1974), morphine (Horita, Carino and Chestnut, 1976; Bhargava, 1980a) and delta-9-tetrahydrocannabinol (Bhargava, 1980b; Bhargava and Matwyshyn, 1980). Similarly, another hypothalamic hormone, melanotropin release inhibiting factor (Pro-Leu-Gly-NH,, MIF) and several of its analogs have been shown to facilitate the development of tolerance to and physical dependence on morphine in the rat (Van Ree and De Wied, 1976). In earlier studies, a dipeptide, carbobenzyloxy-Pro-D-Leu (Z-Pro-D-Leu) was synthesized in which L-leucine was replaced with the D-k0me.r in the hope that this would create a possible misfit at the receptor site so that these analogs could result in the inhibitory rather than the facilitatory effect on morphine tolerance development. Studies with Z-Pro-DLeu indeed indicated that it could inhibit morphine tolerance and dependence in mice (Walter, Ritzmann, Bhargava, Rainbow, Flexner and Krivoy, 1978). Further studies indicated that MIF and a variety of Key words: peptides, MIF, cycle (Leu-Gly), morphine catalepsy, hypothermia, tolerance.
its di-, tri- and cyclic-analogs also blocked morphine tolerance and/or dependence in mice (Walter, Ritzmann, Bhargava and Flexner, 1979; Bhargava, Walter and Ritzmann, 1980) and rats (Bhargava, 198Oc). In many of these studies, the tolerance to the analgesic effect was studied. However, morphine also causes catalepsy and hypothermia and, on chronic administration, tolerance develops to these effects. Therefore, in order to ascertain if these peptides had effects on the other actions of morphine, the effects of MIF and cycle (Leu-Gly) on tolerance to the cataleptic and hypothermic effects of morphine are described here. METHODS Animals Male Sprague-Dawley rats weighing 200-250 g (King Animal Laboratories, Oregon, WI) were acclimatized to a room with controlled ambient temperature (23 + l°C), humidity (65 f 2%) and a 12 hr dark-light cycle (L 0600-1800 hr). The animals were housed under these conditions for at least 4 days prior to being used. The rats were given food and water ad libitum. Drugs Through the courtesy of Dr A. 0. Geiszler. MIF was obtained as a gift from the Abbott Laboratories, North Chicago, IL. Cycle (Leu-Gly) was synthesized according to the method of Fischer (1906). Thin layer chromatographic analyses on silica Gel plates showed a single spot on two solvent systems (Solvent I, Chloroform: Methanol, 9:l v/v, R, 0.30; Solvent II, Chloroform: Methanol 7: 3 v/v, R, 0.40) when sprayed 385
386
H. N. BHARGAVA
with chlorine-tolidine reagent. Cycle (Leu-Gly) and MIF were dissolved in water and injected subcutaneously (s.c.) in a volume of 1 ml/kg body weight. Morphine sulfate (Mallidckrodt Chemical Co., St Louis, MO) was dissolved in saline and injected intraperitoneally (ip.) in a volume of 1 ml/kg body weight. All solutions were prepared fresh on the day of their use. Effect of MIF and cycle (Leu-G/y) on the development of tolerance to catalepsy and hypothermia induced by morphine Induction and assessment of tolerance. Rats were rendered tolerant to and dependent on morphine by subcutaneous implantation of pellets (each containing 75 mg/kg of morphine free base), over a 3-day period as described previously (Bhargava, 1977, 1978, 1980~). Briefly, the back of the rat was shaved. On day 1, under light ether anesthesia at 10a.m. one morphine pellet was implanted after making a small incision in the lower back. The second pellet was implanted at 4p.m. on the same day. On day 2, two more pellets were implanted at 4 p.m. All the pellets were removed 70 hr after the first implantation. Control rats received placebo pellets. At various times thereafter catatonic and hypothermic responses to morphine were determined as described below. To determine the effect of MIF or cycle (Leu-Gly). the peptide was injected subcutaneously in an appropriate dose 2 hr before the first placebo or morphine pellet implantation. The control rats received vehicle (water) instead of the peptide. The injections of water and peptide were given two more times 24 hr apart. Thus, the last injection of the vehicle or the peptide was given 24 hr prior to pellet removal. Quantijcation of the cataleptic action of morphine The degree of cataleptic state was determined by measuring its duration using a bar test for catatonia (Costall and Naylor, 1974). The forepaws of the rat were gently placed across a bar supported by two iron stands 10 cm off the floor. The time for removal of the paws from the bar was measured to the nearest second after treatment with morphine sulfate (50 mg/kg, i.p.). Animals that had not stepped off the bar within 60 set were removed from the bar. If the rat stayed at the bar for 60sec or more it was considered to have 100% catalepsy. If it was less than 60 set then the percentage catalepsy was calculated using 60 set as 100%. The catalepsy was expressed as mean *SEM and was calculated for the following groups viz. (a) vehicle + placebo (b) peptide + placebo, (c) vehicle + morphine and (d) peptide + morphine. The statistical significance between the means of the catalepsy score was determined by using the paired or unpaired Student’s t-test. Measurement of the body temperature The rats were treated with vehicle or the peptide in an appropriate dose and then implanted with placebo
or morphine pellets as described above. At various times after the pellet removal. morphine sulfate (50mg/kg, i.p.) was administered to rats in all the different groups. The recta1 temperature of each rat was measured at fixed times after morphine injection using a telethermometer (Yellow Springs Instrument Co., Yellow Springs, OH) model No. 73 and Probe Type 423. Temperatures were recorded when a constant reading was obtained after rectal insertion of the probe to approx. 6cm. This procedure usually took 30sec for each rat. The data are expressed as mean temperature +SEM. The difference in the means for different groups was analyzed by the Student’s r-test. A value of P > 0.05 was considered nonsignificant. RESULTS
Effect of MIF on tolerance to the cataleptic and hypothermic effect of morphine Multiple injections of MIF inhibited the development of tolerance to the cataleptic action of morphine in the rat. The effect of MIF on morphine-induced catalepsy in placebo and rats with implanted morphine pellets from which the pellets had been removed for 6 hr is shown in Table 1. A dose of 50 mg/kg of morphine produced 71”” catalepsy in placebo pellet-implanted rats which were treated with vehicle. Administration of three injections of MIF to rats implanted with placebo pellets did not alter morphine-induced catalepsy. Implantation of morphine pellets resulted in the development of tolerance as shown by a 4O”h cataleptic response to morphine. In doses of 0.25 and 0.5 mg/kg administered daily for 3 days, MIF did not modify tolerance development; however, a 1.0 mg/kg dose significantly (P < 0.05) inhibited the tolerance to morphine. In another experiment (II). the effect of 2 mg/kg of MIF was studied in rats from which pellets had been removed for 24 hr. Results very similar to those described above were obtained. Thus, treatment with 1 or 2 mg/kg, which did not modify morphine catalepsy in placebopelleted rats, significantly inhibited the development of morphine-induced catalepsy. The effects of MIF on morphine-induced temperature responses in placebo and morphine-pelleted rats from which the pellets had been removed for 6 (Experiment I) or 24 hr (Experiment II) are shown in Table 2. Administration of morphine (50 mg/kg) produced a hypothermic response in placebo pelletimplanted rats and this response was not altered in rats given MIF in any dose used. Rats implanted with morphine pellets, given the vehicle injections, failed to show the hypothermia when injected with morphine, indicating the development of tolerance. The rectal temperatures at 30 min after morphine injection in the MIF (0.25 and 0.5 mg/kg)-treated morphine-tolerant rats, were similar to those of vehicle-injected morphine-tolerant rats. However, the rectal temperatures of rats given MIF (1 mg/kg) were significantly lower than their corresponding vehicle-injected controls.
387
Peptides on chronic morphine actions Table 1. Effect of MlF on the development of tolerance to the cataleptic action of morphine Dose of MIF (mg/kg. s.c.)
Treatment*
Percent catalepsyt (Mean f SEM)
N
Experiment I
(6 hr after the pellet removal) Vehicle: + placebo MIF + placebo Vehicle: + morphine MIF + morphine MIF + morphine MIF + morphine
1.0 0.25 0.50 1.0
4 4 6 8 7 6
71 f 81 f 4O* 31 * 27 k 70 *
11 19 136 5 14 157
8 8 10 10
68 f 60 + 13 * 31 +
14 12 15 147
Experiment II (24 hr after the pellet removal)
Vehicle3 + placebo MIF + placebo Vehicle: + morphine MIF + morphine
2.0 2.0
* Rats were injected with vehicle or MIF and implanted with 4 placebo or 4 morphine pellets over a 3-day period. The injections of the vehicle and MIF were repeated two more times 24 hr apart. The pellets were removed 70 hr after the first implantation. t Thirty min after the morphine sulfate (50 mg/kg, i.p.) injection. $ Water $ P < 0.05 vs the vehicle + placebo group. 7 P < 0.05 vs the vehicle + morphine group.
Effect of cycle (Leu-Gly)
When the above experiment was repeated with MIF in a dose of 2 mg/kg in rats from which the pellets had been removed for 24 hr, a hypothermic response to morphine was observed in placebopelleted rats given the vehicle of MIF. A hyperthermic (P < 0.05) response to morphine was obtained in morphine-tolerant rats. Again as in Experiment I, in the rats treated with 2 mg/kg of MIF, the rectal temperature following morphine injection was lower than in the corresponding vehicle-injected controls.
and hypothermic
on tolerance
to the cataleptic
effect of morphine
Administration of cycle (Leu-Gly) also inhibited the tolerance to morphine-induced catalepsy. The catalepsy produced by morphine (50mg/kg) in placebo or morphine pellet implanted rats given three injections of cycle (Leu-Gly) (2mgfkg) and from which the pellets had been removed for 24 (Experiment I) or 48 hr (Experiment II) is shown in Table 3. As seen with MIF, multiple injections of cycle (Leu-
Table 2. ElTect of MIF on the development of tolerance to the hypothermic effect of morphine
Treatment*
Dose of MIF (mgikg, s.c.)
N
Rectal temperature, “C (Mean k SEM) Time after morphinet injection (min) 0 30
Experiment I (6 hr after the pellet removal)
Vehicle: + placebo MIF + placebo Vehicle: + morphine MIF + morphine MIF + morphine MIF + morphine
1.0 0.25 0.50 1.0
4 4 6 8 I 6
37.9 38.1 37.9 38.0 38.0 31.9
+ k * k + f
0.3 0.2 0.2 0.2 0.2 0.2
35.6 35.7 38.4 38.1 38.7 31.5
If: 0.2 + 0.3 + 0.35 + 0.2 f 0.2 + o.Lq
36.5 + 36.5 5 36.8 f 36.4 f
0.1 0.1 0.2 0.1
35.5 f 35.4 f 38.6 k 38.0 *
Experiment II (24 hr after the pellet removal)
Vehicle MIF + Vehicle MIF +
+ placebo placebo + morphine morphine
2.0 2.0
8 8 10 10
0.1 0.1 0.2$ 0.2q
* Rats were injected with vehicle or MIF and implanted with 4 placebo or 4 morphine pellets over a 3-day period. Two more injections of the vehicle and MIF were made 24 hr apart. The pellets were removed 70 hr after the first implantation. i Morphine sulfate 50 mg/kg (i.p.). $ Water. 5 P < 0.05 vs the vehicle + placebo group. 7 P < 0.05 vs the vehicle + morphine group.
388
H. N.
Table 3. Effect of cycle (Leu-Gly) on tolerance to the cataleptic effect of morphine Treatment*
N
Percent catalepsyt (Mean + SEM)
8 8 10 10
68 f 62 f 13 + 43 +
Experiment I (24 hr after the
pellet removal) Vehicle1 + placebo Cycle (Leu-Gly) + placebo Vehicle1 + morphine Cycle (Leu-Gly) + morphine
14 10 l# 117
Experiment II (48 hr after the
pellet removal Vehicle1 + placebo Cycle (Leu-Gly) + placebo Vehicle1 + morphine Cycle (Leu-Gly) + morphine
6 6 6 6
100 + 0 look0 13 f 5s 26 f 7ll
* Rats were injected with the vehicle or cycle (Leu-Gly) (2 mg/kg, s.c.) daily for 3 days and implanted with 4 placebo or 4 morphine pellets as described in the text. The pellets were removed 70 hr after the first implantation. t Thirty min after the morphine sulfate (50 mg/kg, i.p.) injection. $ Water. # P c 0.05 vs the vehicle + placebo group. fi P <: 0.05 vs the vehicle + morphine group.
Gly) did not modify morphine catalepsy in placebopelleted rats. Tolerance developed to the cataleptic effect of morphine as a result of morphine pellet implantation, since the percentage catalepsy decreased from 67.8 in placebo-pelleted rats to 12.6 in morphine-pelleted rats. In contrast to the Iatter, the morphine-tolerant rats given cycle (Leu-Gly) showed 43.2% catalepsy. Similar results were obtained in Experiment II where pellets had been removed for 48 hr. The temperature responses to morphine were also similar to those obtained with MIF. As shown in
BHARGAVA
Table 4, morphine produced hypothermia in placebo hyperthermia was pellet-implanted rats, whereas obtained in morphine pellet-implanted rats. In morphine-tolerant rats treated with cycle (Leu-Gly) significantly (P < 0.05) less hyperthermia was observed (Experiment I, Table 4). In Experiment II where the pellets were removed for 48 hr, cycle (Leu-Gly) treatment completely prevented the morphine-induced hyperthermia. The rectal temperatures in this group of rats were not different from the rats in the two placebo groups. DISCUSSION
The present studies indicate that chronic administration of morphine results in the development of tolerance to the catatonic and hypothermic effects of morphine and that this process is inhibited by daily administration of MIF and cycle (Leu-Gly). Previous studies from these laboratories have indicated that MIF and cycle (Leu-Gly) do not modify analgesia produced by a single administration of morphine; however, these peptides block the development of tolerance to the analgesic effect of morphine (Bhargava, 198Ck, 1981). Blockage of tolerance to the catatonic effect was also demonstrated in the present study. A high correlation was observed between the production of analgesia and of catatonia when the narcotic drug etorphine was injected in the rat either intraperitoneally or in the periaqueductal gray region of the midbrain (Thorn and Levitt, 1980). These studies and studies in this laboratory on tolerance also suggest that a common mechanism may be involved, not only in the production of the two effects of opiates, but also on the development of tolerance. In non-tolerant or placebo-pelleted rats, morphine produced hypothermia. which was unaffected by MIF
Table 4. Effect of cycle (Leu-Gly) on tolerance to the hypothermic effect of morphine
Treatment*
N
Rectal temperature, “C (Mean + SEM) Time after morphine? injection (min) 0 30
Experiment I (24 hr after the
pellet removal) Vehicle3 + placebo Cycle (Leu-Gly) + placebo Vehicle3 + morphine Cycle (Leu-Gly) + morphine
8 8 10 10
36.5 f 0.1 36.4 + 0.1 36.8 -f: 0.2 36.4 + 0.1
35.5 35.4 38.6 38.2
* * k +
0.1 0.1 0.2$ 0.17
36.7 36.8 36.8 36.7
35.8 f 35.7 * 38.0 + 36.1 k
0.4 0.2 0.4$ 0.q
Experiment II (48 hr after the
pellet removal) Vehicle3 + placebo Cycle (Leu-Gly) + placebo Vehicle1 + morphine Cycle (Leu-Gly) + morphine
+ + k f
0.2 0.2 0.2 0.2
* Rats were injected with the vehicle or cycle (Leu-Gly) (2 mg/kg, s.c.) daily for 3 days and implanted with 4 placebo or 4 morphine pellets as described in the text. The pellets were removed 70 hr after the first implantation. t 50 mg/kg (i.p.). $ Water. 5 P < 0.05 vs the vehicle + placebo group. 7 P < 0.05 vs the vehicle + morphine group.
Peptides on chronic morphine actions or cycle (Leu-Gly) treatment. When the same dose of morphine was administered to morphine-tolerant rats, a hyperthermic effect was observed. The morphineinduced hypothermia in non-tolerant rats and the hyperthermia in tolerant rats are consistent with previous studies (Oka, Nozaki and Hosoya, 1972; Warwick, Blake, Miya and Bousquet, 1973). Daily administration of MIF or cycle (Leu-Gly) inhibited the development of the hyperthermic response to morphine in rats implanted with morphine pellets. The thermoregulatory effects of morphine are indeed complex and many neurotransmitter-systems including dopamine have been implicated. It is believed that the hypothermic response to morphine may be related to the hypothalamic serotoninergic system (Lotti, Lomax and George, 1965). However, its involvement in the chronic effect is unlikely (Warwick, et al., 1973). The hyperthermic response may involve cholinergic mechanisms since cholinergic antagonists, like scopolamine and atropine, antagonize morphineinduced hyperthermia (Oka et al., 1972). It is not clear at present if MIF and cycle (Leu-Gly) interact with cholinergic mechanisms. The mechanism by which MIF and cycle (Leu-Gly) inhibit morphine-tolerance development remains to be elucidated. The cataleptic and analgesic actions of narcotic analgesics is accompanied by an increased turnover of dopamine and increased concentration of homovanillic acid in the striatum (Kuschinsky, 1973; Kuschinsky and Hornykiewicz, 1972). On chronic administration of morphine, supersensitivity of brain dopamine receptors develops as a result of chronic depression of dopaminergic neurotransmission (Bhargava, 1980d; Gianutsos, Hynes, Puri, Drawbaugh and Lal, 1974; Iwatsubo and Clouet, 1975; Puri and Lal, 1973; Ritzmann. Walter, Bhargava and Flexner, 1979). It has been shown that MIF and cycle (LeuGly) block the development of morphine-induced dopamine receptor supersensitivity in mice and rats (Bhargava, 198Od, 1981; Ritzmann, et al., 1979). Thus, morphine-induced tolerance to cataleptic and analgesic effects may be related to brain dopamine supersensitivity. Further evidence for the role of dopaminergic systems in the inhibitory action of MIF and cycle (Leu-Gly) on morphine tolerance is provided by the fact that the dopamine receptor supersensitivity induced by long-term administration of haloperidol is inhibited by these peptides (Bhargava and Ritzmann, 1980). In conclusion, it appears that morphineinduced tolerance to its cataleptic, analgesic and hypothermic actions may involve a common mechanism, perhaps changes in dopamine receptor supersensitivity since all these phenomena were blocked by MIF and cycle (Leu-Gly).
Acknowledgements-These studies were supported by a National Institute on Drug Abuse grant DA-02598. The author thanks George Matwyshyn for providing excellent
389
technical assistance, Mrs Cheryl King and MS Mary Lou Quinn for their help in the preparation of this manuscript. REFERENCES
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