Neuropeptides (1990) 15,213-217 0 Longman Group UK Ltd 1990
0143-4179/90/0015-0213/$10.00
Effect of Thyrotropin Relea,sing Hormone on the Development of Tolerance to the Analgesic and Hyperthermic Actions of Morphine in the Rat P. RAMARAO*
and H. N. BHARGAVA
Department of Pharmacodynamics (m/c 8651, The University of Illinois at Chicago, Health Sciences Center, 833 South Wood Street, Chicago, Illinois, 60672 USA {Correspondence to HNE)
Abstract-The effect of thyrotropin releasing hormone (TRH) on the development of tolerance to the analgesic and hyperthermic actions of morphine was determined in male SpragueDawley rats. The tolerance to morphine was induced by subcutaneous implantation of 6 morphine pellets, each containing 75mg of morphine free base. Rats serving as controls were implanted with 6 placebo pellets. Subcutaneous injections of TRH (I,3 or lOmg/kg) twice a day for 7 days inhibited the development of tolerance to the analgesic, but not to the hyperthermic action of morphine. Doses lower than 1 mg/kg or greater than lOmg/kg administered twice a day for 7 days did not modify tolerance development. The inability of higher doses of TRH to inhibit tolerance to morphine may be related to rapid down-regulation of TRH receptors. The results suggest that in appropriate doses, TRH can selectively inhibit the development of tolerance to the analgesic action of morphine.
Introduction Thyrotropin releasing hormone (TRH), a tripeptide has both endocrine and nonendocrine actions. It releases thyrotropin and prolactin from the anterior pituitary (1). In addition, TRH appears to have direct actions on the CNS since they can be Date received 14 September 1989 Date accepted 30 October 1989 *Present address: Department of Surgery, Beth Israel Hospital, Harvard University Medical School, Boston, Massachusetts, USA
213
observed in hypophysectomized and thyroidectomized animals (see reference 2 for a review). TRH antagonizes the actions of a variety of CNS depressants (3, 4). TRH also appears to interact with endogenous and exogenous opiates. It antagonizes morphineinduced hypothermia and respiratory depression without affecting morphine-induced CNS depression (5) and analgesia (5, 6). It also antagonizes the depressant (7), hypothermic and cataleptic, but not the analgesic effects of P-endorphin (8). Intracerebrally administered TRH inhibits the hypothermic and stereotyped withdrawal
214 jumping syndrome induced by naloxone in morphine-dependent mice (9). In addition, subcutaneous administration of TRH inhibits the development of physical dependence on morphine in mice as evidenced by suppression of hypothermic response observed during naloxone precipitated withdrawal (9). The present studies were undertaken to determine whether TRH affects the development of tolerance to the analgesic and hyperthermic actions of morphine in the rat.
Methods Animals
Male Sprague-Dawley rats weighing 22525Og, obtained from the Sasco-King Animal Co., Oregon, Wisconsin, were acclimated to a room with controlled ambient temperature (23 f l”C), humidity (50 4 10%) and artificial lighting (light 06.00-18.OOhr). The animals were housed under these conditions for at least 4 days prior to being used. The rats were given food and water continuously. Drugs Morphine sulfate (Mallinckrodt Chemical Co., St. Louis, MO), TRH (Hoecht Corporation, Somerville, N.J., courtesy of Mr. Val Wagner) were dissolved in normal saline. Morphine sulfate was injected intraperitoneally whereas TRH was administered subcutaneously in a volume of I ml/kg body weight. Morphine and placebo pellets were obtained from the National Instute on Drug Abuse, Rockville, MD (courtesy of Drs. Rapaka and R.L. Hawks). Induction and assessment of tolerance to the analgesic effect of morphine
Rats were rendered tolerant to, and physically dependent on, morphine by subcutaneous implantation of six morphine pellets during a 7-day period as described previously (10, 11). Each pellet contained 75mg free base. The control rats received pellets containing the excipients without the morphine. The pellets were implanted under light anaesthesia. Briefly, one pellet was implanted on the first day, two on the third day and three on the fifth day. All pellets were removed on the seventh day. Eighteen hours after the removal of the pellets the degree of tolerance to morphine
NEUROPEPTIDES
was estimated by determining the analgesic response to morphine (8mg/kg, i.p.) in placebo and morphine pellet implanted rats. The analgesic effect of morphine was measured by using a tail-flick apparatus as previously described (12,13). Briefly, the apparatus consists of a light source in a wooden box. The light is focused on to a single point by using a convex lens. The opening and closing of the shutter triggers a digital timer. The light beam is focused on the tail of the animal. When the tail is removed by the animal, the circuit is broken and this stops the digital timer. The tail-flick latencies to thermal stimulation were determined before the administration of morphine. The light source was adjusted in such a way that the basal tail-flick reaction time was 1.7 + 0.2 (S.E.M.). The tail-flick latencies to thermal stimulation were determined at various times up to 240 min after an injection of morphine (8mg/kg), i.p.). A cut-off time of 10 set was used to prevent any injury to the tail. The basal response was subtracted from the effect induced by morphine. The area under the time-response curve was calculated for each rat. The data are expressed as mean area under the time-response curve (AUC) + S.E.M. The differences in mean values in various groups were compared by analysis of variance followed by Scheffe’s test. A value of P < 0.05 was considered to be significant. Tolerance to the hyperthermic effect of morphine
The colonic temperature of rats was determined in response to morphine (8mg/kg, i.p.) in placebo and morphine pellet implanted rats. The colonic temperature of each rat was measured using a telethermometer (14). The temperature was recorded when a constant reading was obtained after the rectal insertion of probe to approximately 6cm. The temperature of each rat was measured before and at 30 min intervals after the injection of morphine for a period of 240 min. Eight rats were used for each treatment group. The data analysis was similar to that used in determining the analgesic response to morphine. Administration of TRH
Rats were divided into two groups. One group of rats was injected subcutaneously with vehicle and
215
ANALGESIC AND HYPERTHERMIC ACTIONS OF MORPHINE IN THE RAT
the other with an appropriate dose of TRH. One hour after the TRH injection, the rats were further divided into two subgroups, one being implanted with placebo pellets and the other with morphine pellets as described above. The injections of TRH or its vehicle were given twice a day for 7 days. The pellets were removed on the evening of the 7th day and the test for analgesia and temperature were done on the morning of day 8. Results Effect of TRH on the development of tolerance to the analgesic action of morphine
Chronic administration of morphine by the pellet implantation procedure resulted in the development of tolerance to the analgesic effect of morphine. As shown in Figure lA, the tail-flick latency to thermal stimulatin following a challenge dose (8mg/kg) of morphine was much higher during the 240-min period of observation in placebo pellet implanted rats when compared to morphine pellet implanted rats. The analgesic response, represented as area under the time-response curve, was 14-fold lower in morphine pellet implanted rats than in placebo pellet implanted rats (Fig. 1B). Subcutaneous administration of TRH in doses of 1.0 and 3.0mg/kg given twice a day for 7 days resulted in the inhibition of tolerance to the analgesic action of morphine as evidenced by a greater analgesic response to morphine in TRHtreated than in vehicle-treated morphine-tolerant
0 D
PLACEBO MORPHINE
rh _
Fig. 2 The effect of TRH (0.3-3.0mgIkg) injected S.C.twice a day for 7 days on the development of tolerance to the analgesic effect of morphine in the rat. The asterisks represent P < 0.05 vs. the vehicle-injected morphine tolerant rats.
rats (Fig. 2). The lower dose 0.3mg/kg did not modify morphine tolerance development. In addition, TRH treatment did not modify morphine induced analgesia in rats implanted with placebo pellets. Higher doses of TRH (10 and 3Omg/kg) given twice a day did not modify the development of tolerance to morphine (Fig. 3). Effect of TRH on the development of tolerance to the hyperthermic action of morphine
Chronic administration of morphine by the pellet implantation method resulted in the development of tolerance to the hyperthermic effect of morphine in the rat. As shown in Figure 4, the increase of colonic temperature of placebo pelleted rats given 8mg/kg of morphine was significantly greater than in morphine pellet implanted rats. None of the doses of TRH (0.3 to 30mg/kg) administered twice a day altered the tolerance to the hyperthermic effect of morphine. Since the time-response curve for all doses overlapped for TRH treated rats, only the response to TRH 10 and 30mg doses of TRH is shown (Fig. 4). Discussion
Fig. 1 The time course of the analgesic response to morphine (8mg/kg, i.p.) in morphine-tolerant and non-tolerant rats (panel A). The vertical lines represent S.E.M. The response was transformed into area under the curve (panel B). *P < 0.05 vs. the placebo group.
The present studies clearly indicate that chronic administration of morphine by subcutaneous implantation of six pellets results in the development of a high degree of tolerance to its analgesic and hyperthermic effects. A lo- 15fold tolerance to the analgesic effect of morphine was achieved as
216
NEUROPEPTIDES
0 I
0
PLACEBO MORPHINE
10
DOSE OF TRH (MG/KG;
30 S.C.)
Fig. 3 The effect of TRH (10 and 30mg/kg) injected S.C.twice a day for 7 days on the development of tolerance to the analgesic effect of morphine in the rat.
evidenced by a decrease in the analgesic response to morphine. The results also indicate that in appropriate doses, TRH given twice a day for 7 days inhibited the development of tolerance to the analgesic, but not to the hyperthermic effects of morphine in the rat. Interestingly, multiple injection of TRH did not modify morphine analgesia in morphine-naive rats (placebo pelleted rats). It has been shown earlier that TRH neither possesses analgesic action of its own nor does it modify the analgesic of morphine (6). The inability of higher doses of TRH to modify the development of tolerance to the analgesic action of morphine may be related to down-regulation of TRH receptors at the appropriate loci because of the long term treatment. Since the development of tolerance to the hyperthermic action of morphine was not modified by TRH, it appears that the processes or mechanisms involved in the development of tolerance to the analgesic and hyperthermic responses are not the same. Previously, it has been shown that in the mouse, the tolerance to the hypothermic action of morphine is unaffected by TRH (15). The mechanisms by which TRH inhibits the development of tolerance to the analgesic action of morphine is not evident. TRH does not alter the distribution of morphine in brain and blood (15). Therefore, the possible effect of TRH on dispositional factors can be ruled out. It is possible that TRH and opiates interact at the level of their respective receptors in the CNS. Studies from this laboratory indicate that morphine has no effect on
the binding of [3H]MeTRH to rat brain membranes, however drugs acting at K- and &opiate receptors inhibit the binding of [3H]MeTRH (16-18). However, TRH or its more stable analogs do not affect the binding of tritiated ligands for l.i,6- or K-opiate receptors on brain membranes either at 0°C or 37°C (19). The development of tolerance to morphine is also associated with changes in central receptors, e.g., of dopamine (20-22) and serotonin (23-24). Although there is evidence that in vitro TRH alters central dopamine (25) and serotonin (26) receptors, it is not clear if in vivo administration of TRH reverses morphine-induced changes in these systems. In summary, administration of TRH inhibits the development of tolerance to the analgesic action but not to the hyperthermic action of morphine in the rat. These results further suggest that endogenous neuropeptides like TRH may have modulatory or regulatory role on the chronic actions of opiates. Acknowledgements These studies were supported by a grant from DA-02598 from the National Insitute on Drug Abuse. The authors, thank Mr. J. Johnson for secretarial assistance.
References 1. Bowers, C. Y., Freisen, H. G., Hwang, P., Guyda, H. J. and Folkers, K. (1971). Prolactin and thyrotropin release in man by synthetic pyroglutamyl-histidyl-proiineamide.
6
-1.4
1 0
60 TIME AFlER
Fig. 4
120 ADMINISTRATION
160 OF MORPHINE
240 (min)
The effect of TRH (10 and 30mg/kg) injected S.C.twice a day for 7 days on the development of tolerance to the hyperthermic effect of morphine in the rat.
ANALGESK
AND HYPERTHERMIC ACTIONS OF MORPHINE IN THE RAT
Biochem. Biophys. Res. Commun. 45: 1033-1041. 2. Bhargava, H. N., Yousif, D. J. and Matwyshyn, G. A. (1983). Interactions of thyrotropin releasing hormone, its metabolites and analogues with endogenous and exogenous opiates, Gen. Pharmacol. 14: 565-570. 3. Bhargava, H. N. (1980). The effects of thyrotropin releasing hormone and histidyl-proline diketopiperazine on delta-9-tetra hydrocannabinol induced hypothermia. Life Sci 26: 845850. 4. Bhargava, H. N. and Matwyshyn, G. A. (1980). Influence of thyrotropin releasing hormone and histidyl-proline diketopiperazine on spontaneous motor activity and analgesia induced by delta-9-tetrahydrocannabinol in the mouse. Eur. J. Pharmacol. 68: 147-154. 5. Horita. A., Carino, M. A. and Chestnut, R. M. (1976). Influence of thyrotropin releasing hormone (TRH) on drug induced narcosis and hypothermia in rabbits. Psychopharmacology 49: 57-62. 6. Martin, B. R.. Dewey, W. L., Chau-Pham, T. and Prange, A. J. Jr. (1977). Interactions of thyrotropin releasing hormone and morphine sulfate in rodents. Life Sci. 20: 715-722. 7. Tache, Y., Lis, M. and Collu, R. (1977). Effects of thyrotropin releasing hormone on behavioural and hormonal changes induced by B-endorphin. Life Sci. 21: 841-846. 8. Holaday, J. W., Tseng, L. F., Loh, H. H. and Li, C. H. (1978). Thyrotropin releasing hormone antagonizes 8-endorphin hypothermia and catalepsy. Life Sci. 22: 1537-1544. 9. Bhargava, H. N. (1980). The effects of thyrotropin releasing hormone on the central nervous system responses to chronic morphine administration. Psychopharmacology 68: 185-189. 10. Bhargava, H. N. (1977). Rapid induction and quantitation of morphine dependence in the rat by pellet implantation. Psychopharmacology 52: 55-62. 11. Bhargava, H. N. (1978). Quantitation of morphine tolerance induced by pellet implantation in the rat. J. Pharm. Pharmacol. 30: 133-135. 12. Ramaro, P. and Bhargava, H. N. (1988). Evidence for the involvement of central opiodergic systems in L-tyrosine methyl ester-induced analgesia in the rat. Pharmacology 37: 1-7. 13. Bhargava, H. N. and Ramarao, P. (1989). Comparative effects of Pro-Leu-Gly-NH* and cycle (Leu-Gly) administered orally on the development of tolerance to the analgesic effect of morphine in the rat. Peptides 10:
217
767-771. 14. Bhargava, H. N. (1981). Inhibition to tolerance to the pharmacological effects of human-B-endorphin by prolylleucyl-glycinamide and cycle (leucyl-glycine) in the rat. J. Pharmacol. Exp. Ther. 218: 404-408. 15. Bhargava, H. N. (1981). Dissociation of tolerance to the analgesic and hypothermic effects of morphine by using thyrotropin releasing hormone. Life. Sci. 29: 1015-1020. 16. Bhargava, H. N. and Das, S. (1986). Evidence for opiate action at the brain receptors for thyrotropin releasing hormone. Brain Res. 368: 262-267. 17. Das, S. and Bhargava, H. N. (1987). Inhibition of 3H-(3MeHis’) thyrotropin releasing hormone recognition sites in the brain by tifluadom, a kappa opiate agonist. Neuropharmacology 26: 1141-1146. 18. Bhargava, H. N., Das, S. and Gulati, A. (1988). Stereoselectivity of kappa opiate receptor ligands in inhibiting the binding of ‘H-(3-MeHis*) thyrotropin releasing hormone to brain membranes. J. Pharm. Pharmacol. 40: 70-72. 19. Das, S. and Bhargava, H. N. (1987). Unidirectional interaction between thyrotropin releasing hormone and opiates at the level of their brain receptors. Gen. Pharmacol. 18: 99-102. 20. Bhargava. H. N. (1983). Binding of 3H-spiroperidol to striatal membranes of rats treated chronically with morphine: influence of Pro-Leu-Gly-NH2 and cycle (Leu-Gly). Neuropharmacology 22: 1357-1361. 21. Das, S. and Bhargava, H. N. (1985). The effect of chronic morphine treatment on striatal ‘H-N-propylnorapomorphine binding in the rat: influence of prolyl-leucyl-glycinamide. Pharmacology 31: 241-247. 22. Bhargava. H. N. and Gulati, A. (1989). Modification of brain spinal cord dopamine D1 receptors labeled with “H-SCH 23390 in morphine abstinent rats. J. Pharmacol. Exp. Ther. (in press). 23. Gulati. A. and Bhargava. H. N. (1988). Cerebral cortical 5-HTi and 5-HTz receptors of morphine tolerant-dependent rats. Neuropharmacology 27: 1231-1237. 24. Gulati, A. and Bhargava, H. N. (1989). Brain and spinal cord 5-HTz receptors of morphine tolerant-dependent and abstinent rats. Eur. J. Pharmacol (in press). 25. Funatsu. K. S. and Inanaga, K. (1987). Modulation of dopamine receptors by thryotropin releasing hormone in the rat brain. Peptides 8: 319-325. 26. Funatsu, K., Teshima. S. and Inanaga, K. (1985). Thyrotropin releasing hormone increases 5-hydroxytryptamine, receptors in the limbic brain of the rat. Peptides 6: 563-566.
0143-4179/90/0015-0213/$10.00
Effect of Thyrotropin Relea,sing Hormone on the Development of Tolerance to the Analgesic and Hyperthermic Actions of Morphine in the Rat P. RAMARAO*
and H. N. BHARGAVA
Department of Pharmacodynamics (m/c 8651, The University of Illinois at Chicago, Health Sciences Center, 833 South Wood Street, Chicago, Illinois, 60672 USA {Correspondence to HNE)
Abstract-The effect of thyrotropin releasing hormone (TRH) on the development of tolerance to the analgesic and hyperthermic actions of morphine was determined in male SpragueDawley rats. The tolerance to morphine was induced by subcutaneous implantation of 6 morphine pellets, each containing 75mg of morphine free base. Rats serving as controls were implanted with 6 placebo pellets. Subcutaneous injections of TRH (I,3 or lOmg/kg) twice a day for 7 days inhibited the development of tolerance to the analgesic, but not to the hyperthermic action of morphine. Doses lower than 1 mg/kg or greater than lOmg/kg administered twice a day for 7 days did not modify tolerance development. The inability of higher doses of TRH to inhibit tolerance to morphine may be related to rapid down-regulation of TRH receptors. The results suggest that in appropriate doses, TRH can selectively inhibit the development of tolerance to the analgesic action of morphine.
Introduction Thyrotropin releasing hormone (TRH), a tripeptide has both endocrine and nonendocrine actions. It releases thyrotropin and prolactin from the anterior pituitary (1). In addition, TRH appears to have direct actions on the CNS since they can be Date received 14 September 1989 Date accepted 30 October 1989 *Present address: Department of Surgery, Beth Israel Hospital, Harvard University Medical School, Boston, Massachusetts, USA
213
observed in hypophysectomized and thyroidectomized animals (see reference 2 for a review). TRH antagonizes the actions of a variety of CNS depressants (3, 4). TRH also appears to interact with endogenous and exogenous opiates. It antagonizes morphineinduced hypothermia and respiratory depression without affecting morphine-induced CNS depression (5) and analgesia (5, 6). It also antagonizes the depressant (7), hypothermic and cataleptic, but not the analgesic effects of P-endorphin (8). Intracerebrally administered TRH inhibits the hypothermic and stereotyped withdrawal
214 jumping syndrome induced by naloxone in morphine-dependent mice (9). In addition, subcutaneous administration of TRH inhibits the development of physical dependence on morphine in mice as evidenced by suppression of hypothermic response observed during naloxone precipitated withdrawal (9). The present studies were undertaken to determine whether TRH affects the development of tolerance to the analgesic and hyperthermic actions of morphine in the rat.
Methods Animals
Male Sprague-Dawley rats weighing 22525Og, obtained from the Sasco-King Animal Co., Oregon, Wisconsin, were acclimated to a room with controlled ambient temperature (23 f l”C), humidity (50 4 10%) and artificial lighting (light 06.00-18.OOhr). The animals were housed under these conditions for at least 4 days prior to being used. The rats were given food and water continuously. Drugs Morphine sulfate (Mallinckrodt Chemical Co., St. Louis, MO), TRH (Hoecht Corporation, Somerville, N.J., courtesy of Mr. Val Wagner) were dissolved in normal saline. Morphine sulfate was injected intraperitoneally whereas TRH was administered subcutaneously in a volume of I ml/kg body weight. Morphine and placebo pellets were obtained from the National Instute on Drug Abuse, Rockville, MD (courtesy of Drs. Rapaka and R.L. Hawks). Induction and assessment of tolerance to the analgesic effect of morphine
Rats were rendered tolerant to, and physically dependent on, morphine by subcutaneous implantation of six morphine pellets during a 7-day period as described previously (10, 11). Each pellet contained 75mg free base. The control rats received pellets containing the excipients without the morphine. The pellets were implanted under light anaesthesia. Briefly, one pellet was implanted on the first day, two on the third day and three on the fifth day. All pellets were removed on the seventh day. Eighteen hours after the removal of the pellets the degree of tolerance to morphine
NEUROPEPTIDES
was estimated by determining the analgesic response to morphine (8mg/kg, i.p.) in placebo and morphine pellet implanted rats. The analgesic effect of morphine was measured by using a tail-flick apparatus as previously described (12,13). Briefly, the apparatus consists of a light source in a wooden box. The light is focused on to a single point by using a convex lens. The opening and closing of the shutter triggers a digital timer. The light beam is focused on the tail of the animal. When the tail is removed by the animal, the circuit is broken and this stops the digital timer. The tail-flick latencies to thermal stimulation were determined before the administration of morphine. The light source was adjusted in such a way that the basal tail-flick reaction time was 1.7 + 0.2 (S.E.M.). The tail-flick latencies to thermal stimulation were determined at various times up to 240 min after an injection of morphine (8mg/kg), i.p.). A cut-off time of 10 set was used to prevent any injury to the tail. The basal response was subtracted from the effect induced by morphine. The area under the time-response curve was calculated for each rat. The data are expressed as mean area under the time-response curve (AUC) + S.E.M. The differences in mean values in various groups were compared by analysis of variance followed by Scheffe’s test. A value of P < 0.05 was considered to be significant. Tolerance to the hyperthermic effect of morphine
The colonic temperature of rats was determined in response to morphine (8mg/kg, i.p.) in placebo and morphine pellet implanted rats. The colonic temperature of each rat was measured using a telethermometer (14). The temperature was recorded when a constant reading was obtained after the rectal insertion of probe to approximately 6cm. The temperature of each rat was measured before and at 30 min intervals after the injection of morphine for a period of 240 min. Eight rats were used for each treatment group. The data analysis was similar to that used in determining the analgesic response to morphine. Administration of TRH
Rats were divided into two groups. One group of rats was injected subcutaneously with vehicle and
215
ANALGESIC AND HYPERTHERMIC ACTIONS OF MORPHINE IN THE RAT
the other with an appropriate dose of TRH. One hour after the TRH injection, the rats were further divided into two subgroups, one being implanted with placebo pellets and the other with morphine pellets as described above. The injections of TRH or its vehicle were given twice a day for 7 days. The pellets were removed on the evening of the 7th day and the test for analgesia and temperature were done on the morning of day 8. Results Effect of TRH on the development of tolerance to the analgesic action of morphine
Chronic administration of morphine by the pellet implantation procedure resulted in the development of tolerance to the analgesic effect of morphine. As shown in Figure lA, the tail-flick latency to thermal stimulatin following a challenge dose (8mg/kg) of morphine was much higher during the 240-min period of observation in placebo pellet implanted rats when compared to morphine pellet implanted rats. The analgesic response, represented as area under the time-response curve, was 14-fold lower in morphine pellet implanted rats than in placebo pellet implanted rats (Fig. 1B). Subcutaneous administration of TRH in doses of 1.0 and 3.0mg/kg given twice a day for 7 days resulted in the inhibition of tolerance to the analgesic action of morphine as evidenced by a greater analgesic response to morphine in TRHtreated than in vehicle-treated morphine-tolerant
0 D
PLACEBO MORPHINE
rh _
Fig. 2 The effect of TRH (0.3-3.0mgIkg) injected S.C.twice a day for 7 days on the development of tolerance to the analgesic effect of morphine in the rat. The asterisks represent P < 0.05 vs. the vehicle-injected morphine tolerant rats.
rats (Fig. 2). The lower dose 0.3mg/kg did not modify morphine tolerance development. In addition, TRH treatment did not modify morphine induced analgesia in rats implanted with placebo pellets. Higher doses of TRH (10 and 3Omg/kg) given twice a day did not modify the development of tolerance to morphine (Fig. 3). Effect of TRH on the development of tolerance to the hyperthermic action of morphine
Chronic administration of morphine by the pellet implantation method resulted in the development of tolerance to the hyperthermic effect of morphine in the rat. As shown in Figure 4, the increase of colonic temperature of placebo pelleted rats given 8mg/kg of morphine was significantly greater than in morphine pellet implanted rats. None of the doses of TRH (0.3 to 30mg/kg) administered twice a day altered the tolerance to the hyperthermic effect of morphine. Since the time-response curve for all doses overlapped for TRH treated rats, only the response to TRH 10 and 30mg doses of TRH is shown (Fig. 4). Discussion
Fig. 1 The time course of the analgesic response to morphine (8mg/kg, i.p.) in morphine-tolerant and non-tolerant rats (panel A). The vertical lines represent S.E.M. The response was transformed into area under the curve (panel B). *P < 0.05 vs. the placebo group.
The present studies clearly indicate that chronic administration of morphine by subcutaneous implantation of six pellets results in the development of a high degree of tolerance to its analgesic and hyperthermic effects. A lo- 15fold tolerance to the analgesic effect of morphine was achieved as
216
NEUROPEPTIDES
0 I
0
PLACEBO MORPHINE
10
DOSE OF TRH (MG/KG;
30 S.C.)
Fig. 3 The effect of TRH (10 and 30mg/kg) injected S.C.twice a day for 7 days on the development of tolerance to the analgesic effect of morphine in the rat.
evidenced by a decrease in the analgesic response to morphine. The results also indicate that in appropriate doses, TRH given twice a day for 7 days inhibited the development of tolerance to the analgesic, but not to the hyperthermic effects of morphine in the rat. Interestingly, multiple injection of TRH did not modify morphine analgesia in morphine-naive rats (placebo pelleted rats). It has been shown earlier that TRH neither possesses analgesic action of its own nor does it modify the analgesic of morphine (6). The inability of higher doses of TRH to modify the development of tolerance to the analgesic action of morphine may be related to down-regulation of TRH receptors at the appropriate loci because of the long term treatment. Since the development of tolerance to the hyperthermic action of morphine was not modified by TRH, it appears that the processes or mechanisms involved in the development of tolerance to the analgesic and hyperthermic responses are not the same. Previously, it has been shown that in the mouse, the tolerance to the hypothermic action of morphine is unaffected by TRH (15). The mechanisms by which TRH inhibits the development of tolerance to the analgesic action of morphine is not evident. TRH does not alter the distribution of morphine in brain and blood (15). Therefore, the possible effect of TRH on dispositional factors can be ruled out. It is possible that TRH and opiates interact at the level of their respective receptors in the CNS. Studies from this laboratory indicate that morphine has no effect on
the binding of [3H]MeTRH to rat brain membranes, however drugs acting at K- and &opiate receptors inhibit the binding of [3H]MeTRH (16-18). However, TRH or its more stable analogs do not affect the binding of tritiated ligands for l.i,6- or K-opiate receptors on brain membranes either at 0°C or 37°C (19). The development of tolerance to morphine is also associated with changes in central receptors, e.g., of dopamine (20-22) and serotonin (23-24). Although there is evidence that in vitro TRH alters central dopamine (25) and serotonin (26) receptors, it is not clear if in vivo administration of TRH reverses morphine-induced changes in these systems. In summary, administration of TRH inhibits the development of tolerance to the analgesic action but not to the hyperthermic action of morphine in the rat. These results further suggest that endogenous neuropeptides like TRH may have modulatory or regulatory role on the chronic actions of opiates. Acknowledgements These studies were supported by a grant from DA-02598 from the National Insitute on Drug Abuse. The authors, thank Mr. J. Johnson for secretarial assistance.
References 1. Bowers, C. Y., Freisen, H. G., Hwang, P., Guyda, H. J. and Folkers, K. (1971). Prolactin and thyrotropin release in man by synthetic pyroglutamyl-histidyl-proiineamide.
6
-1.4
1 0
60 TIME AFlER
Fig. 4
120 ADMINISTRATION
160 OF MORPHINE
240 (min)
The effect of TRH (10 and 30mg/kg) injected S.C.twice a day for 7 days on the development of tolerance to the hyperthermic effect of morphine in the rat.
ANALGESK
AND HYPERTHERMIC ACTIONS OF MORPHINE IN THE RAT
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