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Attenuation of morphine tolerance and dependence by α-melanocyte stimulating hormone (α-MSH)
Life Sciences, Vol . 24, pp . 1931-1938 Printed in the U .S .A .
Pergamon Press
ATTENUATION OF MORPHINE TOLERANCE AND DEPENDENCE BY a-MÉLANOCYTE STIMULATING HORMONE(a-MSH) Jbzsef I .Szbkely, Erzsbbet Miglécz, Zsuzsa Duaai-Rov~ca, Istvdn Tarnawa, Andr~s Z . Rbaai, Llszlb Grlf and S~ador Bajuez Research Institute for Pharmaceutical Chemistry, H-1325 Budapest, P .O . Box 82 ., Hungary (Received in final form April 5, 1979) Summary Repeated administration of morphine resulted in significant reduction of its analgesic potency . If 0 .1 mg/kg a-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance . Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma a-MSH level at the time of morphine administration . Of the morphine abstinence symptoms the naloxoneinduced jumping in morphine pretreated mice could not be modified either by a-MSH coadministration or by MIF pretreament, but the withdrawal body weight lose was found to be diminished by the former and increased by the latter peptide . The possible role of a-MSH is preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed . One of the most intriguing problems in the pharmacology of endorphins is their ability to induce morphine-like tolerance and dependence (1-3) . Cross tolerance between opiates and opioidahas been reported (2,4-6) . Thus, the question arises, how it is possible, that in certain circumstances the organism becomes tolerant to and dependent on its own morphine-like aeuropeptides . Hypothetically, some endogenous factors might inhibit the tolerance-dependence development under physiological conditions . If such substances really existed they should probably be released concomitantly with the opioid peptides . Recent studies on an ACTH secreting mouse pituitary tumor cell line have shown that these cells synthetize a large glycoprotein molecule (pro - ACTH/endorphin), which serves as a precursor for at least three smaller molecular forms of ACTH and also for ß - lipotropia (7,8) . Further cleavage of ACTH to a -melanotropin (a-MSH) and corticotropin-like intermediate lobe peptide (CLIP), as suggested previously by Lowry's group (9), has also been demonstrated in cell suspensions of rat intermediate lobe (10) . Thus, the above mentioned peptides i .e . ß-lipotropin, and ACTH and 0024-3205/79/211931-0802 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1932
Inhibition of Morphine Tolerance by MSH
Vol . 24, No . 21, 1979
their natural fragments (ß - endorphin, y-lipotropin, ß-MSH, CLIP and a-MSH) appear to be jointly released by the pituitary . Otherwise of the known peptides of the brain it was vasopreasin, oxytocin and its C-terminal tripeptide that were reported to facilitate the morphine tolerance and dependence by van Ree and de Wied (11) . The latter fragment is called MIF (MSH release inhibiting factor) because of its plasma a-MSH level decreasing activity (12) . If the decreased a-MSH level is connected with the reported facilitation of morphine tolerance, it was of interest to teat the effect of a-MSH itself i .e . to examine whether or not the above mentioned findings (11) are connected with a possible role of physiological regulation . All the more since being a natural fragment of the ACTH chain, it is released into the plasma together with ß-endorphin (13) . Therefore experiments were designed to teat whether morphine tolerance and dependence can be influenced by the biosynthetic products of ACTH (a-MSH and CLIP) and also by MIF . Methods Subjects were male rata of CFY strain weighing 150-200 g at the beginning of the experiments and male CFLP mice weighing 20-25 g . The animals were purchased from LATI (Breeding Farm of Labora tory Animals, Gödöllö, Hungary) . The rats were caged by 5, the mice by 10 in plastic boxes with free access to standard semieynthetic laboratory diet and tap water . The analgesic effect of substances was tested in rats by the tail-flick teat (14) . The radiant heat source was adjusted so, that the reaction time measured automatically to the nearest 0 .1 sec varied between 3 and 5 sec . A cut-off time of 10 sec was used to prevent blistering . Tolerance was induced as described previously (15) . On day 1 at 9 :00 A .M . after having read the control reaction time, the rats were treated with 4 .35 mg/kg of morphine s .c . (4 .35 mg/kg is 300 x of the analgesic ED50 determined in previous experiments) . The readings were repeated 15, 30, 60 min later and upon conclusion of the control session an additional injection was given to bring the total dose to 14 .5 mg/kg (i .e . 10 times the ED50) . At 6 :00 P . M. on the same day then at 9 :00 A .M . and 6 :00 P .M . on the following 3 days the same amount was given . On day 5 at 9 :00 A . M . the animals were challenged with 4 .35 mg/kg again and the analgesic effect was tested (test session) . In the first experiment, with the exception of the first and last treatments, i .e . when the animals were tested for analgesia (on day 1 and 5), 0 .1 mg/kg a-MSH was given s .c . immediately prior to each morphine injection . In the second experiment instead of a-MSH 1 .0 mg/kg CLIP was given in the same way . In the third experiment the rats received 1 .0 mg/kg MIF s .c . either 1 hour prior to morphine treatment or immediately prior to it . Thus the possible direct effect of MIF could probably be separated from the consequences of inhibition of MSH release from the pituitary, which could be detected 20 min after treatment (16) . For dependence studies mice were used . Morphine was
injected
Vol . 24, No . 21, 1979
Inhibition of Morphine Tolerance by MSH
1933
s .c . in increasing amounts until the done of 100 mg/kg was reached . Using the "4 day test" schedule as described by Saeleas et al . (17) the animals received 15 injections, i .e . altogether 1470 mg/kg . On the fourth day 2 hours after the last treatment the abstinence syndrome was precipitated with naloaone . Immediately after the injection the animals were weighed then placed in separate glass cylinders and the jumps (all 4 paws off the bottom) were counted for 15 min . The proportion of animals jumping in response to varying doses to naloxone was determined (18) and the median effective doses were estimated (19) . Moreover the severity of abstinence syndrome was characterized by the weight loan recorded 1,2,3 hours after challenge with naloaone . The body weight was measured to the nearest 0 .01 g on a Mettler balance . Separate groups of animals were treated s .c . with 0 .1 mg/kg MSH or 0 .1 mg/kg MIF before each morphine treatment . The former was given immediately prior to morphine injections, the latter 1 hour earlier . Drugs purchased were morphine hydrochloride (Alkaloids, Hungary) and naloxone hydrochloride (Endo Labs .) . a-MSH was kindly donated by Prof . Medzihradszky of EötvBs University, Budapest, Hungary, MIF (H-Pro-Leu-Gly-NH2 ) and CLIP was syathetised by one of us (S .B .) . Results As shown by the saline pretreated (control) groups, repeated morphine administration induced tolerance, i .e . comparing the tailflick latencies measured in the test session (on day 5) to those recorded in the control session (on day 1) a significant reduction of morphine's analgesic potency could be observed (Table 1) . The development of tolerance was influenced by the different pretreamenta is different ways . In the teat session, the level of analgesia displayed by rats chronically pretreated with 0 .1 mg/kg a-MSH prior to morphine was higher than that of the saline pretreated ones . In the control session, however, both groups showed similar analgesic responsiveness . Thus the data indicate the inhibition of development of morphine tolerance in a-MSH pretreated animals (Table 1 .) . However, no differences were observed in tolerance development, if the rata were pretreated with 10 times higher doses of CLIP . It means that similar tail-flick latencies were found in the control and test session both in the saline and CLIP pretreated groups (Table 1) . Pretreating the animals with 1 .0 mg/kg MIF one hour prior to the morphine administration reduced analgesic activity was observed on day 5 if comparing them to rata pretreated with the same dose of MIF, but immediately prior to morphine . That is, MIF accelerated the tolerance development if applying it at a certain interval before the alkaloid (Table 2) . Otherwise, comparing the saline pretreated group (Table 1,A) to the MIF pretreated one (Table 2,A) no statistically significant differences were found in the test session, i .e . MIF injected immediately prior to morphine did not alter tolerance development .
1934
Vol . 24, No . 21, 1979
Inhibition of Morphine Tolerance by MSH
TABLE 1 Effect of a-MSH and CLIP on Development of Morphine Tolerance
Tail-Flick Reaction Time in Sec (Mean t S .E .) Group Session
Time Elapsed After Morphine Injections Control
15 min
30 min
60 min
Tolerance induced by saline A n~39
90 min
120 min
morphine b
Control
3 " 53 0 .09
7 .54 0 .37
8 .96 0 .25
7 .93 0,31
6 .81 0 .46
5 .23 0 .34
Teat
3 .62 0 .09
5 .90 0 .30
6 .23 0 .36
4 .82 0 .32
4 .24 0 .26
3 .61 0 .14
Tolerance induced by a-MSHc + morphineb B n~31
Control
344 " 0 .10
740 . 0 .40
847 . 0 .34
837 . 0 .35
611 0,40
509 0 .38
Test
3 .65 0 .11
6 .84d 0 .35
7 .94 e 0,34
6 .25e 0,43
5 .03 0,43
4 .304 0 .27
Tolerance induced by saline + morphineb C n~39
Control
3 " 68 0 .09
8 .63 0 .28
9 .58 0 .17
9,14 0 .27
7 .70 0 .33
5,01 0 .25
Teat
3 .59 0 .09
5 .99 0,32
6 .48 0 .30
4 .50 0 .25
3 .87 0 .22
3 .70 0 .15
Control
3'80 0 .10
8 .84 0 .28
9 .24 0,21
8 .97 0 .32
7 .18 0 .37
4 .88 0 .34
Test
3 .53 0 .08
5,68 0 .28
6 .36 0 .33
4 .32 0 .27
3 .55 0 .16
3 .35 0 .17
Tolerance induced by CLIP f + morphineb D n~33
84 .35 mg/kg s .c,
b 2x14 .5 mg/kg/day s .c .
c 0 .1 mg/kg s .c .
d Sigaificantly different from group A, p<0 .05, Student's t-test . e Significantly different from group A, p<0 .01, Student's t-test . f1 .0 mg/kg s .c .
Inhibition of Morphine Tolerance by MSH
Vol, 24, No, 21, 1979
1935
TABLE 2 Effect of MIF on Development of Morphine Tolerance Tail-Flick Reaction Time in Sec (Mean t S .E .) Time Elapsed After Morphine Injections
Group Session
Control
15 min
30 min
60 min
90 min
120 min
During tolerance induction MIFb given immediately prior to morphine A a~49
Control
3 " 63 0 .14
8 .16 0 .32
9 .09 0 .35
9 .04 0 .35
7 .01 0 .28
5 .49 0 .22
Teat
3 .62 0 .14
5 .51 0 .22
6 .30 0 .25
4 .56 0 .18
3 .70 0 .15
3 .34 0 .13
During tolerance induction MIFb given 1 hour prior to morphine ° B n~44
Control
3 " 71 0 .18
8 .55 0 .41
9 .55 0 .46
9 .42 0 .45
7 .64 0 .38
5 .61 0 .28
Test
3 .67 0 .17
4 .97 0 .24
5 .484 0 .27
3 .44e 0 .17
3 .244 0 .15
3 .16 0 .15
84 .35 mg/kg s .c .
b l mg/kg s .c .
° 2x14 .5 mg/kg/day s .c . d Significaatly different from group A, p<0 .05, Student's t-test . e Significantly different from group A, p<0 .01, Student's t-test . In morphine dependent mice neither a-MSH nor MIF pretreatment modified significantly the naloaone-induced jumping, i .e . similar amounts of aaloxone were necessary to precipitate the withdrawal jumping in the case of either pretreatment (Table 3) . Measuring, however, the withdrawal body weight loan, significant differences were found . Mice pretreated with a-MSH prior to morphine administration showed reduced weight lose upon naloxone treatment . The MIF pretreated ones, however, loot more weight than the saline pretreated animals, i .e . MIF seemed to accelerate the dependence development (Table 4) . Discussion Considering the treatment schedule applied it may be supposed that in the test session not the peptides' analgesia modifying effect was recorded but their influence on the tolerance development . Since the last treatment with a-MSH, CLIP of MIF was given 15-16 hours prior to the second analgesia testing, the direct effect of these substances on morphine-induced analgesia was unlikely . Our data on facilitation by MIF of morphine dependence
1936
Inhibition of Morphine Tolerance by MSH
Vol . 24, No . 21, 1979
confirm and extend van Ree's and de Wied's previous similar findings (11) . Furthermore, the data concerning the attenuation by a-MSH of morphine tolerance and its facilitation by MIF are in good agreement if considering that the latter lowers the plasma level of a-MSH . Pretreatments with CLIP had no significant effect emphasizing the specificity of the tolerance inhibiting action of a-MSH . TABLE 3 Effect of Repeated a-MSH and MIF Treatments on the Naloxone-Precipitated Jumping in Morphine Dependent Mice ED 50 of Naloxone and 95z Confidence Limite
Group
Treatment
A n~38
Saline + Morphine b
0 .12 (0 .08- 0 .19)
B n~36
a-MSHc + Morphine b
0 .10 (0 .07-0 .16)
(mg/kg s .c .) a
C Saline + 0,14 n~77 Morphine b (0 .10-0 .19) D n~74
MIFd + Morphine b
0 .13 (0 .09-0 .20)
a Naloxone was given in doses of 0 .03 ; 0 .10 ; 0 .30 mg/kg to separate groupa of animale and the ED 50 . was calculated by determining the percentage of mice dumping .
bGiving 15 s .c . injections during 4 consecutive days as described in part Method . c 0 .1 mg/kg s .c . immediately prior to each morphine treatment . d1 .0 mg/kg s .c . 1 hour prior to each morphine treatment .
As to the effect on opiate dependence a-MSH pretreatment reduced, while pretreatment with MIF increased the abstinence weight lose, whereas withdrawal jumping had not been affected . These data may suggest but by no means prove the physiological role of a-MSH and/or MIF in regulating the development of tolerance to the endogenous opioida provided that such a process would occur in physiological circumstances . In this context it may be recalled that the mechanisms of the conversion of ACTH to a-MSH (9) and ß-LPH to ß-endorphin (20) appear to be related to each other, the intermediate pituitary being the richest in both a-MSH (9,10) and ß-endorphin (10,21) .
Inhibition of Morphine Tolerance by MSH
Vol . 24, No . 21, 1979
1937
TABLE 4 Effect of Repeated a-MSH and MIF Treatments on the Naloxone-Precipitated Body Weight Loss in Morphine Dependent Mice Group Treatment
Dose of Naloxone mg/kg s .c .
Weight Loss a (Mean t S .E .) Time Elapsed After Naloxone Injection 1 2 3 hours 1 .13±0 .16 1 .6210 .17 2 .13±0 .10
No . of mice used
A
Saline + Morphine b
0 .03 0 .10 0 .30
0 .5210 .13 1 .2210 .12 1 .5710 .11
B
a-MSHc + Morphine b
0 .03 0 .10 0 .30
0 .24±0 .13 0 .72t0 .17 1 .1910 .19 E 0 .7210 .13E 0 .9710 .16 E 1 .3910 .14 h 0 .7810 .06h 0 .9710 .07 h 1 .3710 .11 h
10 14 12
C
Saline d + Morphine b
0 .03 0 .10 0 .30
0 .6110 .08 0 .7210 .05 1 .1210 .09
27 25 25
D
MIF e + b Morphine
0 .03 0 .10 0 .30
1 .0510 .13 8 1 .6710 .14 E 1 .9910 .12 8 1 .0910 .108 1 .43±0 .13 1 .8510 .12 1 .12±0 .06 1 .7510 .108 2 .1010 .12 E
1 .23±0 .14 1 .1710 .09 1 .2710 .11
1 .6810 .10 2 .10±0 .12 2 .4710 .09
1 .4210 .15 1 .6710 .11 1 .7210 .14
14 12 12
24 23 27
aMeaeured to the nearest 0 .01 g .
b Giving 15 s .c . injections during 4 consecutive days as described in part Method . c0 .1 mg/kg s .c . immediately before each morphine treatment . d0 .1 ml/10 g b .wt . s .c . 1 hour prior to each morphine treatment . e 1 .0 mg/kg s .c . 1 hour prior to each morphine treatment . f,g,h Significantly different from controls, Group B was compared to A and group D to C . p<0 .05,<0 .01 and <0 .001, respectively, Student's t-test .
References 1. 2. 3. 4. 5. 6. 7.
E .WEI and H .H .LOH, Science _193, 1262-1263 (1976) . J .M . VAN REE, D .DE WIED, A .F .BRADBURY, E .C .HULME, D .G .SMYTH, and C .R .SNELL, Nature 264, 792-794 (1976) . J .BLÄSIG and A .HERZ, Naunyn-Schmiedeberg's Arch . Pharmacol . 294, 297-300 (1976) . .A .WATERFIELD, J .HUGHES, and H .W .ROSTERLITZ, Nature 260, A 624-625 (1976) . L .-F .TSENG, H .H .LOH, and C .H .LI, Proc . Nat .Acad . Sci .U .S .A . 7~ 4187-4189 (1976) . J .I . $Z~ICELY, A .Z .RÔNAI, Z .DUNAI-ROVÂCS, E .MIGLÉCZ, S .BAJUSZ, L .GRAF, Life Sci . _20, 1259-1264 (1977) . R .E .MAINS, B .A .EIPPER, and N .LING, Proc .Nat .Acad .Sci .II .S .A . 74, 3014-3018 (1977) .
1938
8, 9. 10, 11 . 12 . 13 . 14 . 15 : 16 . 17 . 18 . 19 . 20 . 21 .
Inhibition of Morphine Tolerance by MSH
Vol, 24, No . 21, 1979
B .A .EIPPER and R .E .MAINS, J .Biol,Chem . 253, 5732 - 5744 (1978) . A .P .SCOTT, J .G .RATCLIFFE, L .H .REES, J .LANDON, H,P .J .BENETT, P,J .LOWRY, and C .McMARTIN, Nature New Biol . _244, 65-67 (1973) B .A .EIPPER and R .E,MAINS, J .Supramol, Struct . (in press) . J .M,VAN REE and D .DE WIED, Life Sci . 19, 1331 -1340 (1976), M .E .CELIS, S .TALEISNIK, and R .WALTER, Proc, Nat .Acad . Sci . U .S .A . 68, 1428-1433 (1971) . J .ROSSIÉR, E .D .FRENCH, C .RIVIER, N .LING, R .GIIILLEMIN, and F,E .BLOOM, Nature _270, 618-620 (1977) . F .E .D'AMOUR and D .L .SMITH, J .Pharmacol . Exp .Ther . _72, 74 - 79 (1941) . Z .DUNAI-KOVÂCS and J,I .SZÉKELY,Psyhopharmacology _53, 65-72 (1977), M,E .CELIS, S,TALEISNIR, and R .WALTER, Proc .Nat .Acad .Sci, U,S .A, _68, 1428-1433 (1971) . J .R .SAELENS, F .R .GRANAT, and W.R .SAWYER, Arch . Int . Pharmacodyn, _190, 213-218 (1971) . E .L . WAY, H .H,LOH, and F .-H . SHEN, J .Pharmacol . Exp,Ther . 167, 1-8 (1969) . .T J . LITCHFIELD, Jr ., and F .WILCORON, J .Pharmacol . Exp .Ther . 96, 9-113 (1949) . L .GR~F, A .RENESSEY, I .BERZETEI, and A .Z .RÔNAI, Biochem . Biophys . Res,Commun . 78,1114-1123 (1977) . J .ROSSIER, T .M .VARGO, S .MINICK, N,LING, F,E .BLOOM, and R . GUILLEMIN, Proc . Nat .Acad,Sci .U .S .A, 74, 5162 -5165 (1977) .
Pergamon Press
ATTENUATION OF MORPHINE TOLERANCE AND DEPENDENCE BY a-MÉLANOCYTE STIMULATING HORMONE(a-MSH) Jbzsef I .Szbkely, Erzsbbet Miglécz, Zsuzsa Duaai-Rov~ca, Istvdn Tarnawa, Andr~s Z . Rbaai, Llszlb Grlf and S~ador Bajuez Research Institute for Pharmaceutical Chemistry, H-1325 Budapest, P .O . Box 82 ., Hungary (Received in final form April 5, 1979) Summary Repeated administration of morphine resulted in significant reduction of its analgesic potency . If 0 .1 mg/kg a-MSH was coadministered, the tolerance development was attenuated, 1 mg/kg MIF (MSH release inhibiting factor), given simultaneously with morphine, did not affect tolerance . Injecting, however, MIF 1 hour prior to the daily opiate treatment resulted in accelerated development of tolerance supposedly by lowering the plasma a-MSH level at the time of morphine administration . Of the morphine abstinence symptoms the naloxoneinduced jumping in morphine pretreated mice could not be modified either by a-MSH coadministration or by MIF pretreament, but the withdrawal body weight lose was found to be diminished by the former and increased by the latter peptide . The possible role of a-MSH is preventing the development of tolerance to the analgesic effect of endogenous opioid peptides is discussed . One of the most intriguing problems in the pharmacology of endorphins is their ability to induce morphine-like tolerance and dependence (1-3) . Cross tolerance between opiates and opioidahas been reported (2,4-6) . Thus, the question arises, how it is possible, that in certain circumstances the organism becomes tolerant to and dependent on its own morphine-like aeuropeptides . Hypothetically, some endogenous factors might inhibit the tolerance-dependence development under physiological conditions . If such substances really existed they should probably be released concomitantly with the opioid peptides . Recent studies on an ACTH secreting mouse pituitary tumor cell line have shown that these cells synthetize a large glycoprotein molecule (pro - ACTH/endorphin), which serves as a precursor for at least three smaller molecular forms of ACTH and also for ß - lipotropia (7,8) . Further cleavage of ACTH to a -melanotropin (a-MSH) and corticotropin-like intermediate lobe peptide (CLIP), as suggested previously by Lowry's group (9), has also been demonstrated in cell suspensions of rat intermediate lobe (10) . Thus, the above mentioned peptides i .e . ß-lipotropin, and ACTH and 0024-3205/79/211931-0802 .00/0 Copyright (c) 1979 Pergamon Press Ltd
1932
Inhibition of Morphine Tolerance by MSH
Vol . 24, No . 21, 1979
their natural fragments (ß - endorphin, y-lipotropin, ß-MSH, CLIP and a-MSH) appear to be jointly released by the pituitary . Otherwise of the known peptides of the brain it was vasopreasin, oxytocin and its C-terminal tripeptide that were reported to facilitate the morphine tolerance and dependence by van Ree and de Wied (11) . The latter fragment is called MIF (MSH release inhibiting factor) because of its plasma a-MSH level decreasing activity (12) . If the decreased a-MSH level is connected with the reported facilitation of morphine tolerance, it was of interest to teat the effect of a-MSH itself i .e . to examine whether or not the above mentioned findings (11) are connected with a possible role of physiological regulation . All the more since being a natural fragment of the ACTH chain, it is released into the plasma together with ß-endorphin (13) . Therefore experiments were designed to teat whether morphine tolerance and dependence can be influenced by the biosynthetic products of ACTH (a-MSH and CLIP) and also by MIF . Methods Subjects were male rata of CFY strain weighing 150-200 g at the beginning of the experiments and male CFLP mice weighing 20-25 g . The animals were purchased from LATI (Breeding Farm of Labora tory Animals, Gödöllö, Hungary) . The rats were caged by 5, the mice by 10 in plastic boxes with free access to standard semieynthetic laboratory diet and tap water . The analgesic effect of substances was tested in rats by the tail-flick teat (14) . The radiant heat source was adjusted so, that the reaction time measured automatically to the nearest 0 .1 sec varied between 3 and 5 sec . A cut-off time of 10 sec was used to prevent blistering . Tolerance was induced as described previously (15) . On day 1 at 9 :00 A .M . after having read the control reaction time, the rats were treated with 4 .35 mg/kg of morphine s .c . (4 .35 mg/kg is 300 x of the analgesic ED50 determined in previous experiments) . The readings were repeated 15, 30, 60 min later and upon conclusion of the control session an additional injection was given to bring the total dose to 14 .5 mg/kg (i .e . 10 times the ED50) . At 6 :00 P . M. on the same day then at 9 :00 A .M . and 6 :00 P .M . on the following 3 days the same amount was given . On day 5 at 9 :00 A . M . the animals were challenged with 4 .35 mg/kg again and the analgesic effect was tested (test session) . In the first experiment, with the exception of the first and last treatments, i .e . when the animals were tested for analgesia (on day 1 and 5), 0 .1 mg/kg a-MSH was given s .c . immediately prior to each morphine injection . In the second experiment instead of a-MSH 1 .0 mg/kg CLIP was given in the same way . In the third experiment the rats received 1 .0 mg/kg MIF s .c . either 1 hour prior to morphine treatment or immediately prior to it . Thus the possible direct effect of MIF could probably be separated from the consequences of inhibition of MSH release from the pituitary, which could be detected 20 min after treatment (16) . For dependence studies mice were used . Morphine was
injected
Vol . 24, No . 21, 1979
Inhibition of Morphine Tolerance by MSH
1933
s .c . in increasing amounts until the done of 100 mg/kg was reached . Using the "4 day test" schedule as described by Saeleas et al . (17) the animals received 15 injections, i .e . altogether 1470 mg/kg . On the fourth day 2 hours after the last treatment the abstinence syndrome was precipitated with naloaone . Immediately after the injection the animals were weighed then placed in separate glass cylinders and the jumps (all 4 paws off the bottom) were counted for 15 min . The proportion of animals jumping in response to varying doses to naloxone was determined (18) and the median effective doses were estimated (19) . Moreover the severity of abstinence syndrome was characterized by the weight loan recorded 1,2,3 hours after challenge with naloaone . The body weight was measured to the nearest 0 .01 g on a Mettler balance . Separate groups of animals were treated s .c . with 0 .1 mg/kg MSH or 0 .1 mg/kg MIF before each morphine treatment . The former was given immediately prior to morphine injections, the latter 1 hour earlier . Drugs purchased were morphine hydrochloride (Alkaloids, Hungary) and naloxone hydrochloride (Endo Labs .) . a-MSH was kindly donated by Prof . Medzihradszky of EötvBs University, Budapest, Hungary, MIF (H-Pro-Leu-Gly-NH2 ) and CLIP was syathetised by one of us (S .B .) . Results As shown by the saline pretreated (control) groups, repeated morphine administration induced tolerance, i .e . comparing the tailflick latencies measured in the test session (on day 5) to those recorded in the control session (on day 1) a significant reduction of morphine's analgesic potency could be observed (Table 1) . The development of tolerance was influenced by the different pretreamenta is different ways . In the teat session, the level of analgesia displayed by rats chronically pretreated with 0 .1 mg/kg a-MSH prior to morphine was higher than that of the saline pretreated ones . In the control session, however, both groups showed similar analgesic responsiveness . Thus the data indicate the inhibition of development of morphine tolerance in a-MSH pretreated animals (Table 1 .) . However, no differences were observed in tolerance development, if the rata were pretreated with 10 times higher doses of CLIP . It means that similar tail-flick latencies were found in the control and test session both in the saline and CLIP pretreated groups (Table 1) . Pretreating the animals with 1 .0 mg/kg MIF one hour prior to the morphine administration reduced analgesic activity was observed on day 5 if comparing them to rata pretreated with the same dose of MIF, but immediately prior to morphine . That is, MIF accelerated the tolerance development if applying it at a certain interval before the alkaloid (Table 2) . Otherwise, comparing the saline pretreated group (Table 1,A) to the MIF pretreated one (Table 2,A) no statistically significant differences were found in the test session, i .e . MIF injected immediately prior to morphine did not alter tolerance development .
1934
Vol . 24, No . 21, 1979
Inhibition of Morphine Tolerance by MSH
TABLE 1 Effect of a-MSH and CLIP on Development of Morphine Tolerance
Tail-Flick Reaction Time in Sec (Mean t S .E .) Group Session
Time Elapsed After Morphine Injections Control
15 min
30 min
60 min
Tolerance induced by saline A n~39
90 min
120 min
morphine b
Control
3 " 53 0 .09
7 .54 0 .37
8 .96 0 .25
7 .93 0,31
6 .81 0 .46
5 .23 0 .34
Teat
3 .62 0 .09
5 .90 0 .30
6 .23 0 .36
4 .82 0 .32
4 .24 0 .26
3 .61 0 .14
Tolerance induced by a-MSHc + morphineb B n~31
Control
344 " 0 .10
740 . 0 .40
847 . 0 .34
837 . 0 .35
611 0,40
509 0 .38
Test
3 .65 0 .11
6 .84d 0 .35
7 .94 e 0,34
6 .25e 0,43
5 .03 0,43
4 .304 0 .27
Tolerance induced by saline + morphineb C n~39
Control
3 " 68 0 .09
8 .63 0 .28
9 .58 0 .17
9,14 0 .27
7 .70 0 .33
5,01 0 .25
Teat
3 .59 0 .09
5 .99 0,32
6 .48 0 .30
4 .50 0 .25
3 .87 0 .22
3 .70 0 .15
Control
3'80 0 .10
8 .84 0 .28
9 .24 0,21
8 .97 0 .32
7 .18 0 .37
4 .88 0 .34
Test
3 .53 0 .08
5,68 0 .28
6 .36 0 .33
4 .32 0 .27
3 .55 0 .16
3 .35 0 .17
Tolerance induced by CLIP f + morphineb D n~33
84 .35 mg/kg s .c,
b 2x14 .5 mg/kg/day s .c .
c 0 .1 mg/kg s .c .
d Sigaificantly different from group A, p<0 .05, Student's t-test . e Significantly different from group A, p<0 .01, Student's t-test . f1 .0 mg/kg s .c .
Inhibition of Morphine Tolerance by MSH
Vol, 24, No, 21, 1979
1935
TABLE 2 Effect of MIF on Development of Morphine Tolerance Tail-Flick Reaction Time in Sec (Mean t S .E .) Time Elapsed After Morphine Injections
Group Session
Control
15 min
30 min
60 min
90 min
120 min
During tolerance induction MIFb given immediately prior to morphine A a~49
Control
3 " 63 0 .14
8 .16 0 .32
9 .09 0 .35
9 .04 0 .35
7 .01 0 .28
5 .49 0 .22
Teat
3 .62 0 .14
5 .51 0 .22
6 .30 0 .25
4 .56 0 .18
3 .70 0 .15
3 .34 0 .13
During tolerance induction MIFb given 1 hour prior to morphine ° B n~44
Control
3 " 71 0 .18
8 .55 0 .41
9 .55 0 .46
9 .42 0 .45
7 .64 0 .38
5 .61 0 .28
Test
3 .67 0 .17
4 .97 0 .24
5 .484 0 .27
3 .44e 0 .17
3 .244 0 .15
3 .16 0 .15
84 .35 mg/kg s .c .
b l mg/kg s .c .
° 2x14 .5 mg/kg/day s .c . d Significaatly different from group A, p<0 .05, Student's t-test . e Significantly different from group A, p<0 .01, Student's t-test . In morphine dependent mice neither a-MSH nor MIF pretreatment modified significantly the naloaone-induced jumping, i .e . similar amounts of aaloxone were necessary to precipitate the withdrawal jumping in the case of either pretreatment (Table 3) . Measuring, however, the withdrawal body weight loan, significant differences were found . Mice pretreated with a-MSH prior to morphine administration showed reduced weight lose upon naloxone treatment . The MIF pretreated ones, however, loot more weight than the saline pretreated animals, i .e . MIF seemed to accelerate the dependence development (Table 4) . Discussion Considering the treatment schedule applied it may be supposed that in the test session not the peptides' analgesia modifying effect was recorded but their influence on the tolerance development . Since the last treatment with a-MSH, CLIP of MIF was given 15-16 hours prior to the second analgesia testing, the direct effect of these substances on morphine-induced analgesia was unlikely . Our data on facilitation by MIF of morphine dependence
1936
Inhibition of Morphine Tolerance by MSH
Vol . 24, No . 21, 1979
confirm and extend van Ree's and de Wied's previous similar findings (11) . Furthermore, the data concerning the attenuation by a-MSH of morphine tolerance and its facilitation by MIF are in good agreement if considering that the latter lowers the plasma level of a-MSH . Pretreatments with CLIP had no significant effect emphasizing the specificity of the tolerance inhibiting action of a-MSH . TABLE 3 Effect of Repeated a-MSH and MIF Treatments on the Naloxone-Precipitated Jumping in Morphine Dependent Mice ED 50 of Naloxone and 95z Confidence Limite
Group
Treatment
A n~38
Saline + Morphine b
0 .12 (0 .08- 0 .19)
B n~36
a-MSHc + Morphine b
0 .10 (0 .07-0 .16)
(mg/kg s .c .) a
C Saline + 0,14 n~77 Morphine b (0 .10-0 .19) D n~74
MIFd + Morphine b
0 .13 (0 .09-0 .20)
a Naloxone was given in doses of 0 .03 ; 0 .10 ; 0 .30 mg/kg to separate groupa of animale and the ED 50 . was calculated by determining the percentage of mice dumping .
bGiving 15 s .c . injections during 4 consecutive days as described in part Method . c 0 .1 mg/kg s .c . immediately prior to each morphine treatment . d1 .0 mg/kg s .c . 1 hour prior to each morphine treatment .
As to the effect on opiate dependence a-MSH pretreatment reduced, while pretreatment with MIF increased the abstinence weight lose, whereas withdrawal jumping had not been affected . These data may suggest but by no means prove the physiological role of a-MSH and/or MIF in regulating the development of tolerance to the endogenous opioida provided that such a process would occur in physiological circumstances . In this context it may be recalled that the mechanisms of the conversion of ACTH to a-MSH (9) and ß-LPH to ß-endorphin (20) appear to be related to each other, the intermediate pituitary being the richest in both a-MSH (9,10) and ß-endorphin (10,21) .
Inhibition of Morphine Tolerance by MSH
Vol . 24, No . 21, 1979
1937
TABLE 4 Effect of Repeated a-MSH and MIF Treatments on the Naloxone-Precipitated Body Weight Loss in Morphine Dependent Mice Group Treatment
Dose of Naloxone mg/kg s .c .
Weight Loss a (Mean t S .E .) Time Elapsed After Naloxone Injection 1 2 3 hours 1 .13±0 .16 1 .6210 .17 2 .13±0 .10
No . of mice used
A
Saline + Morphine b
0 .03 0 .10 0 .30
0 .5210 .13 1 .2210 .12 1 .5710 .11
B
a-MSHc + Morphine b
0 .03 0 .10 0 .30
0 .24±0 .13 0 .72t0 .17 1 .1910 .19 E 0 .7210 .13E 0 .9710 .16 E 1 .3910 .14 h 0 .7810 .06h 0 .9710 .07 h 1 .3710 .11 h
10 14 12
C
Saline d + Morphine b
0 .03 0 .10 0 .30
0 .6110 .08 0 .7210 .05 1 .1210 .09
27 25 25
D
MIF e + b Morphine
0 .03 0 .10 0 .30
1 .0510 .13 8 1 .6710 .14 E 1 .9910 .12 8 1 .0910 .108 1 .43±0 .13 1 .8510 .12 1 .12±0 .06 1 .7510 .108 2 .1010 .12 E
1 .23±0 .14 1 .1710 .09 1 .2710 .11
1 .6810 .10 2 .10±0 .12 2 .4710 .09
1 .4210 .15 1 .6710 .11 1 .7210 .14
14 12 12
24 23 27
aMeaeured to the nearest 0 .01 g .
b Giving 15 s .c . injections during 4 consecutive days as described in part Method . c0 .1 mg/kg s .c . immediately before each morphine treatment . d0 .1 ml/10 g b .wt . s .c . 1 hour prior to each morphine treatment . e 1 .0 mg/kg s .c . 1 hour prior to each morphine treatment . f,g,h Significantly different from controls, Group B was compared to A and group D to C . p<0 .05,<0 .01 and <0 .001, respectively, Student's t-test .
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Inhibition of Morphine Tolerance by MSH
Vol, 24, No . 21, 1979
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