Psychoneuroendocrinology, 1977, Vol. 2, pp. 35-41. Pergamon Press. Printed in Great Britain
EFFECT OF NEUROHYPOPHYSEAL HORMONES ON MORPHINE DEPENDENCE JAN M. VAN REE and DAVID DE WIED Rudolf Magnus Institute for Pharmacology,Universityof Utrecht--Medical Faculty, Vondellaan 6, Utrecht, The Netherlands (Received21 July 1976) SUMMARY (1) The developmentof physicaldependenceon morphine is facilitatedin rats treated with [desglycinamideg-arginineg]-vasopressin(DG-AVP) or with oxytocin.(2) Oxytocinappeared to be more potent than DG-AVP. (3) The essential elements of vasopressin and oxytocin required for facilitatingmorphine tolerance and physicaldependence, are located in the Cterminal part of these hormones. (4) It was found that the C-terminaltripeptide of oxytocin, prolyl-leucyl-glycinamide,was the most potent oligopeptidein this respect. Key Words--morphine; tolerance;physicaldependence;oxytocin;vasopressin;Pro-Leu-Gly. INTRODUCTION ABLATIONof the posterior lobe of the pituitary interferes with the maintenance of avoidance behavior. Extinction of shuttle-box avoidance behavior which is facilitated in posterior lobectomized rats, can be normalized by the administration of vasopressin (De Wied, 1969). Extinction of active avoidance behavior is also rapid in Brattleboro rats with hereditary diabetes insipidus, which lack the ability to synthesize vasopressin (De Wied, Van Wimersma Greidanus & Bohus, 1975a). The influence of posterior pituitary substances on conditioned behavior can also be demonstrated in intact rats. Vasopressin and related peptides facilitate active avoidance behavior in hypophysectomized rats (Bohus, Gispen & De Wied, 1973) and increase resistance to extinction of active and passive avoidance behavior in intact rats (De Wied & Bohus, 1966; De Wied, 1971 ; Ader & De Wied, 1972; King & De Wied, 1974). Conversely, the intraventricular administration of vasopressin antiserum prevents passive avoidance behavior (Van Wimersma Greidanus, Dogterom & De Wied, 1975). Finally, vasopressin and congeners protect against CO2-induced amnesia in rats (Rigter, Van Riezen & De Wied, 1974) and puromycine-induced amnesia in mice (Walter, Hoffman, Flexner & Flexner, 1975). These observations suggest that vasopressin is involved in learning and memory plocesses. The development of tolerance to morphine can be regarded as a form of learning or memory: morphine sensitive cells "learn" to tolerate and remember the effect of morphine (Cohen, Keats, Krivoy & Ungar, 1965; Caldwell & Sever, 1974; Clouet & Iwatsubo, 1975). This reasoning was used by Krivoy, Zimmermann & Lande (1974) to study the influence of vasopressin on the development of morphine tolerance. These authors reported that high doses of [desglycinamide 9, lysinea]-vasopressin (DG-LVP) facilitate the development of resistance to the analgesic action of morphine in mice. The physiological involvement 35
36
JAN M. VANRr~ and DAVIDDE WIED
of vasopressin in the development of morphine tolerance was subsequently demonstrated in rats with hereditary diabetes insipidus. It was shown that these animals develop tolerance to the analgesic action of morphine at a much slower rate (De Wied & Gispen, 1976). Because the development of tolerance to moxphine seems to run paxallel with the development of physical dependence (Clouet & Iwatsubo, 1975; Takemori, 1975), it might be possible that the development of physical dependence would also be facilitated by vasopressin. The present experiments, which were designed to test this hypothesis, show that indeed the development of physical dependence to morphine is facilitated by vasopressin and related peptides. Subsequently, the effect of various neurohypophyseal hormones and fragments on tolerance and dependence to morphine was determined in order to establish the essential elements required for this effect. MATERIALS AND METHODS The degree of physical dependence was measured by the loss of body weight following an injection of naloxone in rats chronically treated with morphine. Morphine-HCl was administered intraperitoneally twice daily (at 10.00 and 17.00 hr) in a dose of 40 mg/kg. One hr prior to each morphine injection, animals received subcutaneously saline (0.2 ml) or peptide (1 vg/animal, except when otherwise indicated). On subsequent days of morphine treatment groups of animals were injected intraperitoneallywith naloxone at a dose of 4 mg/kg I hr after the first daily injection of morphine. At various time intervals before and after the challenge with naloxone, the body weights of the animals were measured. In addition, body temperatures were determined to measure the antagonistic action of naloxone on morphine induced hyperthermia in tolerant animals. Body temperature was recorded with a telethermometer by inserting the thermister probe approx 4 cm into the rectum. The median differences between the values obtained at half an hour (body temperature) or 3 hr (body weight) after the challenge with naloxone and the values recorded at 1 hr before morphine administration were calculated. At this time of the day body weight and temperature remain relatively constant. In another set of experiments a test procedure comparable to that described by Takemori (1975) was used. Before and half an hour after treatment with morphine (80 mg/kg i.p.) and naloxone (1 rng/kg, i.p.) the response latency of animals to heat was measured on a hot plate (54.2 4- 0.1°C) according to Eddy & Leimbach (1953). The difference in response latency before and after the test dose of morphine/naloxone was used as the parameter of the effect of the drugs. On the day before testing took place the rats were injected twice with morphine (10.00 hr: 20 mg/kg, 18.00 hr: 40 mg/kg). One hr before morphine administration rats were treated either with saline or peptide. Statistical analysis of the data was performed using Mann-Whitney U-test. The following drugs were used: morphine (Morphine Hydrochloride, O.P.G., Utrecht, The Netherlands); naloxone (Naloxone Hydrochlodde, Endo Laboratories, Brussels, Belgium); DG-AVP (Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-OH); oxytocin (C~s-Tyr-Ile-Gln-Asn-C~-Pro-Leu-Gly-NH2); I
!
tocinamide (Cys-Tyr-Ile-Gln-Asn-Cys-NHz); PLG (Pro-Leu-GIy-NH2); I
I
pressinamide (Cys-Tyr-Phe-Gln-Asn-Cys-NHz); PAG (Pro-Arg-Gly-NHz); I
I
AVT (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NHa); Pro-His-Phe-Arg-Gly-NHz ; cycio[Len-Gly] (Dr. Walter, Chicago, USA). Peptides were obtained from Organon InternationalB.V. (Oss, The Netherlands).
EFFECT OF ~EUROHYPOPHYSEALHORMONESON MORPHINE DEPENDENCE
37
RESULTS Morphine administration hardly affected body weight on the first day of treatment (Fig. 1). However, an increase in body weight was found on subsequent days of testing. This increase was higher in animals treated with DG-AVP on the 3rd day of testing than in saline t~eated controls (p < 0.03). Naloxone administretion on the 3rd day of morphine txeatment precipitated a marked fall in body weight in animals treated with DG-AVP but not in placebo treated animals. Loss of body weight at that time in peptide treated animals was of the same magnitude as that found in the placebo treated rats on the 5th day.
E
-2 -3 t,)
-4 -5 -6
I
I 2
I 3
4
I
I 6
Doysof morphine "freofment
FIG. 1. Median change in body weight (g) without or after injection with naloxone in rats treated with morphine twice daily for subsequent days. • • DG-AVP. O ~ O placebo. [] [] DG-AVP and naloxone. O O placebo and naloxone. Body temperature changed biphasically as a result of morphine treatment: hypothermia was followed by hyperthermia. The hyperthermic response appeared to occur sooner after the morphine injection on subsequent days of testing. It was found that naloxone completely counteracted the morphine induced hyperthermia on the 5th day of morphine treatment. However, in the DG-AVP treated animals naloxone antagonized the morphine hyperthermia on the 4th day. Similar results were obtained in a subsequent experiment in which the last peptide injection--on the test day--was omitted. In order to ascertain the potency of oxytocin in facilitating morphine dependence, the effect of graded doses of oxytocin was compared with that of graded amounts of DG-AVP. In this particular experiment morphine was administered at a dose of 20 mg/kg on the first day only because the rats were somewhat more sensitive to the acute action of morphine due, perhaps, to seasonal variations, and on the test day no peptide was injected. It appeared that oxytocin was approx 5 times more potent than DG-AVP in naloxoneinduced body weight loss (Table I). In the same test procedure the covalent ring of oxytocin (tocinamide) was ineffective, but the C-terminal tripeptide of oxytocin (PLG) appeared to be more potent than the parent hormone oxytocin.
JA_~M. VANREEand DAV~I3DE TIED
38 TABLE L EFFECT OF
DG-AVP AND OXYTOCIN ON BODY WEIGHT CHANGE
FOLLOWING NALOXONE
IN RATS TREATED FOR 3 DAYS WITH MORPHINE
Mean body weight change (n = 11-12)
Amount of peptide/rat
(g)
DG-AVP
Oxytocin
0.04 t~g
--0.7
--1.7
0.2 t~g 1.0 /~g
--1.5 --3.0
--4.0 --4.0
Comparable results were obtained with another test procedure. In this system, the response on the hot plate was measured after treatment with morphine and naloxone (see Material and Methods). The response latency was decreased in animals pretreated with oxytocin, DG-AVP, AVT, PLG, PAG, cyclo (Leu-Gly) but not in rats treated with tocinamide, pressinamide or Pro-His-Phe-Arg-Gly (Table II). Oxytocin and PLG were approx 5 times more potent than DG-AVP, AVT and PAG. TABLEII. Tr~ Err~CT OF OXYTOCIN, VASOPRESSINAND FRAGMENTS OF THESE POLYPEPTIDF~
ON THE ANALGF~IC
RESPONSE OF RATS AFTER MORPHINE/NALOXONE ADMINISTRATION
The animals were pretreated with morphine and with saline or peptide Amount of peptide/rat (/~g) Saline Oxytocin Tocinamide
Median increase in response latency (sec) 48.1"
1 1
Number of rats 22
11.3.+
21
1I
50.0 ~
18
II
Pro-Leu-Gly
1
11.7.* ~ ' - ~
17
Cyclo (Leu-Gly)
1
DG-AVP
1
50.0
DG-AVP
5
23.9 ~ . _
Pressinamide
5
50.0 ~
Pro-Arg-Gly
5
18.0t /
AVT
1
40.0 ~
7
AVT
5
15.9.* "~-
9
Pro-His-Phe-Arg-Gly
5
50.0
§
8.5** ~
12 9 ~
9 21
~
**
11
12
* The trial was terminated if the response latency exceeded 60 sec. Response latency prior to treatment was approx I0 sec. t Different from saline treated animals (level of significance: t P < 0.1, .+p < 0.05), § Significant difference between groups (§p < 0.05, UP < 0.02, **p < 0.001).
EFFECT OF NEUROHYPOPHYSEALHORMONESON MORPHINE DEPENDENCE
39
DISCUSSION In the present experiment physical dependence on morphine was measured by the loss of body weight following treatment with naloxone. Body weight loss has been shown to be a reliable index of physical dependence in rats (Nozaki, Akera, Lee & Brody, 1974; Stolerman, Johnson, Bunker & Jarvik, 1975). The results show that DG-AVP treated animals aremore sensitive to naloxone. Physical dependence refers to the fact that the organism becomes more sensitive to the morphine antagonist (Clouet & Iwatsubo, 1975; Takemori, 1975). Thus, it can be concluded that DG-AVP facilitates the development of physical dependence. This is supported by the observation that the morphine-induced hyperthermia was already antagonized by naloxone in the peptide treated animals before this occurred in the saline treated rats. Omitting the last injection with DG-AVP--on the test day--did not affect the results. From electrophysiological studies with vasopressin analogues we know that the effect does not last for more than 8 hr (Urban & De Wied, unpublished data). It is therefore unlikely that the influence of peptides is due to a direct interaction with naloxone. Thus, DG-AVP does not modify the expression of withdrawal signs, but indeed facilitates the development of physical dependence. This was substantiated in the test described by Takemori (1975). It has been argued that this procedure is a sensitive indicator of the development of tolelance and physical dependence. Also in this test DG-AVP facilitated the development of tolerance and dependence. The present experiments were designed in view of the involvement of vasopressin and related analogues in learning and memory processes. Vasopressin increases resistance to extinction of avoidance behavior (De Wied, 1971 ; Ader & De Wied, 1972) and protects against various forms of amnesia (Lande, Flexner & Flexner, 1972; Rigter et al., 1974; Walter et al., 1975). Oxytocin was also active in this respect, but the potency of this peptide is much lower than that ofvasopressin (De Wied, Bohus, Urban, Van Wimersma Greidanus & Gispen, 1975b). The covalent ring structure of vasopressin is the most important part of this molecule for effects on avoidance behavior. Surprisingly, in the present experiments oxytocin is much more potent than DG-AVP in facilitating tolerance to, and physical dependence on, morphine. For this effect the Cterminal part of oxytocin (PLG) is essential. The same holds for vasopressin, although PLG was more active than PAG. AVT was as active as DG-AVP and PAG. The C-terminal tripeptide of AVT is identical to that of [argininea]-vasopressin (AVP). This explains why it has the same potency as the vasopressin analogues. As mentioned before, neurohypophyseal hormones and their fragments protect against puromycin-induced amnesia in mice (Lande et al., 1972; Walter et aL, 1975). Although vasopressin and related compounds had marked activity, PLG and shorter C-terminal peptides of oxytocin were as active in this respect, but the covalent ring of oxytocin was not. This is in contrast to results obtained in the avoidance test. The present data, therefore, compare rather well with the results obtained in the puromycin amnesia test. Accordingly, neurohypophyseal hormones and their fragments are involved in learning and memory processes. However, it might be postulated that various learning processes are specifically controlled by different peptides. The physiological role of vasopressin in memory processes has been demonstrated in
40
JAN M. VANREEand DAVIDDE WIED
Brattleboro rats with hereditary diabetes insipidus in which active and passive avoidance behavior is markedly disturbed (De Wied, Bohus & Van Wimersma Greidanus, 1974). In addition, intraventricular injections of specific antisera against vasopressin which prevents passive avoidance behavior, support the physiological role of vasopressin in the consolidation of avoidance behavior (Van Wimersma Greidanus et al., 1975). P L G and the dipeptide Leu-Gly might be generated in vivo from oxytocin by hypothalamic enzymes (Walter, Gritfiths & Hooper, 1973) and P L G has been isolated as such from hypothalamic tissue (Nair, Kastin & Schally, 1971). It is tempting to speculate that P L G and related peptides are essentially involved in the development of morphine tolerance and dependence. This is in accord with previous findings that the development of resistance to the analgesic action of morphine is reduced in Brattleboro rats (De Wied & Gispen, 1976). These animals however do not lack the ability to synthesize oxytocin. Although they have a lower than normal amount of oxytocin in the posterior pituitary (Van Wimersma Greidanus, personal communication), the level of this hormone in the brain has to be determined, before we might suggest that a disturbance in oxytocin production might be the underlying cause of the decrease in tolerance development in homozygous diabetes insipidus rats. The authors wish to acknowledge the skilful assistance of Willy Spaapen-Kok and Yvonne SchuchardFerric. They also express their thanks to Organon International B.V. (Oss, The Netherlands) for supplying the various peptides, to Dr. R. Walter (Chicago, U.S.A.) for cycle (Leu-Gly), and to Endo Laboratories (Brussels, Belgium) for naloxone. REFERENCES AD~,~, R. & DE Wxr=D,D. (1972) Effects of lysine vasopressin on passive avoidance learning. Psychon. Scl. 29, 46-48. Bom.Ts, B., GIsPEN, W. H. & DE WIFx~,D. (1973) Effect of lysine vasopressin and ACTH 4-10 on conditioned avoidance behavior of hypophysectomized rats. Neuroendocrinology 11, 137-143. CAtDWELL,J. & SEVER,P. S. (1974) The biochemical pharmacology of abused drugs. Clin. Pharm. Ther. 16, 989-1013. O.OtnET, D. H. & IWAxstmo, K. (1975) Mechanisms of tolerance to and dependence on narcotic analgesic drugs. Ann. Rev. Pharmae. 15, 49-71. Co~N, M., KEATS,A. S., gauvov, W. A. & UNGAR,G. (1965) Effect of actinomycin on morphine tolerance Prec. Soc. exp. Biol. 119, 381-384. DE WIED, D. (1969) Effects of peptide hormones on behavior. In Frontiers in Neuroendocrinology, W. F. Ganong and L. Martini (Eds.), pp. 97-140. Oxford University Press, New York. DE WIED,D. (1971) Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature, Lend. 232, 58-60. DE WIED,D. & Bonus, B. (1966) Long term and short term effect on retention of a conditioned avoidance response in rats by treatment respectively with long acting pitressin or a-MSH. Nature, Lend. 212, 14841486. DE WtED, D. & GXSPEN,W. H. (1976) Impaired development of tolerance to morphine analgesia in rats with hereditary diabetes insipidus. Psychopharmacologia, Berlin 46, 27-29. DE WIED, D., Bonus, B. & VAN WIMERSMAGR~DANOS, Tj. B. (1974) The hypothalamo-hypophyseal system and the preservation of conditioned avoidance behavior in rats. In Integrative Hypothalamic Activity, D. F. Swaab and J. P. Schad6 (Eds.), Progr. Brain Res. 41,417-428. Elsevier, Amsterdam. D~ WIED, D., VAN WIMERSMAGn~ZDA~'US,Tj. B. & Bonus, B. (1975a), Pituitary peptides and behavior: influence on motivational learning and memory processes. Neuropsychopharmacology, Prec. IX Congr. of the Collegium Intern. Neuropsychopharmacologicum, pp. 653-658. Excerpta Medica, Amsterdam. D~ WIED, D., Bonus, B., URBAN,I., VANWIMERSMAGREIDANUS,Tj. B. & GISPEN, W. H. (1975b) Pituitary peptides and memory. In Peptides: Chemistry, Structure and Biology, R. Walter and J. Meienhofer (Eds.), pp. 635-643. Ann Arbor Science, Ann Arbor. EDDY, N. B. & LEIMBACH,D. (1953) Synthetic analgesics. II. Dithienylbutenyl and dithienylbutylamines. J. Pharm. exp. Ther. 107, 385-393.
EFFECTOF NEUROHYPOPHYSEALHORMONESON MORPHINEDEPENDENCE
41
KING, A. R. & DE WIED,D. (1974) Localized behavioral effects of vasopressin on maintenance of an active avoidance response in rats. J. comp. physiol. PsychoL 86, 1008-1018. KmvoY, W. A., ZI~ra~ANN, E. & LAr,rOE,S. (1974) Facilitation of development of resistance to morphine analgesia by desglycinamideg-lysine-vasopressin. Proc. hath. Acad. Sci. U.S.A. 71, 1852-1856. LANDE, S., FLEXNER,J. B. & FLEXNER, L. B. (1972) Effect of corticotropin and desglycinamideg-lysinevasopressin on suppression of memory by puromycin. Proc. natn. Acad. Sci, U.S.A. 69, 558-560. NAm, R. M. G., KASTIN,A. J. & SCHALLY,A. V. (1971) Isolation and structure of hypothalamic MSH release-inhibiting hormone. Biochem. biophys. Res. Communs. 43, 1376-1381. NOZAKI,M., AKERA,T., LEE,C. Y. & BRODY,T. M. (1974) The effects of age on the development of tolerance to and physical dependence on morphine in rats..L Pharm. exp. Ther. 192, 506--512. RIGTER, H., VAN R/EZEN, H. & DE WIED, D. (1974) The effects of ACTH- and vasopressin-analogues on CO2-induced retrograde amnesia in rats. PhysioL Behao. 13, 381-388. STOLERMAN, I. P., JOHNSON, C. A., BUNKER,P. & JARVIK, M. E. (1975) Weight loss and shock-elicited aggression as indices of morphine abstinence in rats. Psychopharmacologia, Berlin 45, 157-161. TAKEMORI,A. E. (1975) Neurochemical bases for narcotic tolerance and dependence. Biochem. Pharmac. 24, 2121-2126. VAN WIMERSMAGREIDANUS,Tj. B., DOGTEROM,J. & DE WIED, D. (1975) Intraventricular administration of anti-vasopressin serum inhibits memory consolidation in rats. Life Sci. 16, 637-644. WALTER,R., GmFFITHS,E. C. & HOOPFa~,K. C. (1973) Production of MSH-release-inhibiting hormone by a particulate preparation of hypothalami: mechanisms of oxytocin inactivation. Brain Res. 60, 449-457. WALTER,R., HOFFMAN,P. L. FLEXNER,J. B. & FLEXr,mR, L. B. (1975) Neurohypophyseal hormones, analogs, and fragments: their effect on puromycin-induced amnesia. Proc. hath. Acad. Sci. U.S.A.. 72, 4180--4184.
EFFECT OF NEUROHYPOPHYSEAL HORMONES ON MORPHINE DEPENDENCE JAN M. VAN REE and DAVID DE WIED Rudolf Magnus Institute for Pharmacology,Universityof Utrecht--Medical Faculty, Vondellaan 6, Utrecht, The Netherlands (Received21 July 1976) SUMMARY (1) The developmentof physicaldependenceon morphine is facilitatedin rats treated with [desglycinamideg-arginineg]-vasopressin(DG-AVP) or with oxytocin.(2) Oxytocinappeared to be more potent than DG-AVP. (3) The essential elements of vasopressin and oxytocin required for facilitatingmorphine tolerance and physicaldependence, are located in the Cterminal part of these hormones. (4) It was found that the C-terminaltripeptide of oxytocin, prolyl-leucyl-glycinamide,was the most potent oligopeptidein this respect. Key Words--morphine; tolerance;physicaldependence;oxytocin;vasopressin;Pro-Leu-Gly. INTRODUCTION ABLATIONof the posterior lobe of the pituitary interferes with the maintenance of avoidance behavior. Extinction of shuttle-box avoidance behavior which is facilitated in posterior lobectomized rats, can be normalized by the administration of vasopressin (De Wied, 1969). Extinction of active avoidance behavior is also rapid in Brattleboro rats with hereditary diabetes insipidus, which lack the ability to synthesize vasopressin (De Wied, Van Wimersma Greidanus & Bohus, 1975a). The influence of posterior pituitary substances on conditioned behavior can also be demonstrated in intact rats. Vasopressin and related peptides facilitate active avoidance behavior in hypophysectomized rats (Bohus, Gispen & De Wied, 1973) and increase resistance to extinction of active and passive avoidance behavior in intact rats (De Wied & Bohus, 1966; De Wied, 1971 ; Ader & De Wied, 1972; King & De Wied, 1974). Conversely, the intraventricular administration of vasopressin antiserum prevents passive avoidance behavior (Van Wimersma Greidanus, Dogterom & De Wied, 1975). Finally, vasopressin and congeners protect against CO2-induced amnesia in rats (Rigter, Van Riezen & De Wied, 1974) and puromycine-induced amnesia in mice (Walter, Hoffman, Flexner & Flexner, 1975). These observations suggest that vasopressin is involved in learning and memory plocesses. The development of tolerance to morphine can be regarded as a form of learning or memory: morphine sensitive cells "learn" to tolerate and remember the effect of morphine (Cohen, Keats, Krivoy & Ungar, 1965; Caldwell & Sever, 1974; Clouet & Iwatsubo, 1975). This reasoning was used by Krivoy, Zimmermann & Lande (1974) to study the influence of vasopressin on the development of morphine tolerance. These authors reported that high doses of [desglycinamide 9, lysinea]-vasopressin (DG-LVP) facilitate the development of resistance to the analgesic action of morphine in mice. The physiological involvement 35
36
JAN M. VANRr~ and DAVIDDE WIED
of vasopressin in the development of morphine tolerance was subsequently demonstrated in rats with hereditary diabetes insipidus. It was shown that these animals develop tolerance to the analgesic action of morphine at a much slower rate (De Wied & Gispen, 1976). Because the development of tolerance to moxphine seems to run paxallel with the development of physical dependence (Clouet & Iwatsubo, 1975; Takemori, 1975), it might be possible that the development of physical dependence would also be facilitated by vasopressin. The present experiments, which were designed to test this hypothesis, show that indeed the development of physical dependence to morphine is facilitated by vasopressin and related peptides. Subsequently, the effect of various neurohypophyseal hormones and fragments on tolerance and dependence to morphine was determined in order to establish the essential elements required for this effect. MATERIALS AND METHODS The degree of physical dependence was measured by the loss of body weight following an injection of naloxone in rats chronically treated with morphine. Morphine-HCl was administered intraperitoneally twice daily (at 10.00 and 17.00 hr) in a dose of 40 mg/kg. One hr prior to each morphine injection, animals received subcutaneously saline (0.2 ml) or peptide (1 vg/animal, except when otherwise indicated). On subsequent days of morphine treatment groups of animals were injected intraperitoneallywith naloxone at a dose of 4 mg/kg I hr after the first daily injection of morphine. At various time intervals before and after the challenge with naloxone, the body weights of the animals were measured. In addition, body temperatures were determined to measure the antagonistic action of naloxone on morphine induced hyperthermia in tolerant animals. Body temperature was recorded with a telethermometer by inserting the thermister probe approx 4 cm into the rectum. The median differences between the values obtained at half an hour (body temperature) or 3 hr (body weight) after the challenge with naloxone and the values recorded at 1 hr before morphine administration were calculated. At this time of the day body weight and temperature remain relatively constant. In another set of experiments a test procedure comparable to that described by Takemori (1975) was used. Before and half an hour after treatment with morphine (80 mg/kg i.p.) and naloxone (1 rng/kg, i.p.) the response latency of animals to heat was measured on a hot plate (54.2 4- 0.1°C) according to Eddy & Leimbach (1953). The difference in response latency before and after the test dose of morphine/naloxone was used as the parameter of the effect of the drugs. On the day before testing took place the rats were injected twice with morphine (10.00 hr: 20 mg/kg, 18.00 hr: 40 mg/kg). One hr before morphine administration rats were treated either with saline or peptide. Statistical analysis of the data was performed using Mann-Whitney U-test. The following drugs were used: morphine (Morphine Hydrochloride, O.P.G., Utrecht, The Netherlands); naloxone (Naloxone Hydrochlodde, Endo Laboratories, Brussels, Belgium); DG-AVP (Cys-Tyr-Phe-Gln-Asn-Cys-Pro-Arg-OH); oxytocin (C~s-Tyr-Ile-Gln-Asn-C~-Pro-Leu-Gly-NH2); I
!
tocinamide (Cys-Tyr-Ile-Gln-Asn-Cys-NHz); PLG (Pro-Leu-GIy-NH2); I
I
pressinamide (Cys-Tyr-Phe-Gln-Asn-Cys-NHz); PAG (Pro-Arg-Gly-NHz); I
I
AVT (Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Arg-Gly-NHa); Pro-His-Phe-Arg-Gly-NHz ; cycio[Len-Gly] (Dr. Walter, Chicago, USA). Peptides were obtained from Organon InternationalB.V. (Oss, The Netherlands).
EFFECT OF ~EUROHYPOPHYSEALHORMONESON MORPHINE DEPENDENCE
37
RESULTS Morphine administration hardly affected body weight on the first day of treatment (Fig. 1). However, an increase in body weight was found on subsequent days of testing. This increase was higher in animals treated with DG-AVP on the 3rd day of testing than in saline t~eated controls (p < 0.03). Naloxone administretion on the 3rd day of morphine txeatment precipitated a marked fall in body weight in animals treated with DG-AVP but not in placebo treated animals. Loss of body weight at that time in peptide treated animals was of the same magnitude as that found in the placebo treated rats on the 5th day.
E
-2 -3 t,)
-4 -5 -6
I
I 2
I 3
4
I
I 6
Doysof morphine "freofment
FIG. 1. Median change in body weight (g) without or after injection with naloxone in rats treated with morphine twice daily for subsequent days. • • DG-AVP. O ~ O placebo. [] [] DG-AVP and naloxone. O O placebo and naloxone. Body temperature changed biphasically as a result of morphine treatment: hypothermia was followed by hyperthermia. The hyperthermic response appeared to occur sooner after the morphine injection on subsequent days of testing. It was found that naloxone completely counteracted the morphine induced hyperthermia on the 5th day of morphine treatment. However, in the DG-AVP treated animals naloxone antagonized the morphine hyperthermia on the 4th day. Similar results were obtained in a subsequent experiment in which the last peptide injection--on the test day--was omitted. In order to ascertain the potency of oxytocin in facilitating morphine dependence, the effect of graded doses of oxytocin was compared with that of graded amounts of DG-AVP. In this particular experiment morphine was administered at a dose of 20 mg/kg on the first day only because the rats were somewhat more sensitive to the acute action of morphine due, perhaps, to seasonal variations, and on the test day no peptide was injected. It appeared that oxytocin was approx 5 times more potent than DG-AVP in naloxoneinduced body weight loss (Table I). In the same test procedure the covalent ring of oxytocin (tocinamide) was ineffective, but the C-terminal tripeptide of oxytocin (PLG) appeared to be more potent than the parent hormone oxytocin.
JA_~M. VANREEand DAV~I3DE TIED
38 TABLE L EFFECT OF
DG-AVP AND OXYTOCIN ON BODY WEIGHT CHANGE
FOLLOWING NALOXONE
IN RATS TREATED FOR 3 DAYS WITH MORPHINE
Mean body weight change (n = 11-12)
Amount of peptide/rat
(g)
DG-AVP
Oxytocin
0.04 t~g
--0.7
--1.7
0.2 t~g 1.0 /~g
--1.5 --3.0
--4.0 --4.0
Comparable results were obtained with another test procedure. In this system, the response on the hot plate was measured after treatment with morphine and naloxone (see Material and Methods). The response latency was decreased in animals pretreated with oxytocin, DG-AVP, AVT, PLG, PAG, cyclo (Leu-Gly) but not in rats treated with tocinamide, pressinamide or Pro-His-Phe-Arg-Gly (Table II). Oxytocin and PLG were approx 5 times more potent than DG-AVP, AVT and PAG. TABLEII. Tr~ Err~CT OF OXYTOCIN, VASOPRESSINAND FRAGMENTS OF THESE POLYPEPTIDF~
ON THE ANALGF~IC
RESPONSE OF RATS AFTER MORPHINE/NALOXONE ADMINISTRATION
The animals were pretreated with morphine and with saline or peptide Amount of peptide/rat (/~g) Saline Oxytocin Tocinamide
Median increase in response latency (sec) 48.1"
1 1
Number of rats 22
11.3.+
21
1I
50.0 ~
18
II
Pro-Leu-Gly
1
11.7.* ~ ' - ~
17
Cyclo (Leu-Gly)
1
DG-AVP
1
50.0
DG-AVP
5
23.9 ~ . _
Pressinamide
5
50.0 ~
Pro-Arg-Gly
5
18.0t /
AVT
1
40.0 ~
7
AVT
5
15.9.* "~-
9
Pro-His-Phe-Arg-Gly
5
50.0
§
8.5** ~
12 9 ~
9 21
~
**
11
12
* The trial was terminated if the response latency exceeded 60 sec. Response latency prior to treatment was approx I0 sec. t Different from saline treated animals (level of significance: t P < 0.1, .+p < 0.05), § Significant difference between groups (§p < 0.05, UP < 0.02, **p < 0.001).
EFFECT OF NEUROHYPOPHYSEALHORMONESON MORPHINE DEPENDENCE
39
DISCUSSION In the present experiment physical dependence on morphine was measured by the loss of body weight following treatment with naloxone. Body weight loss has been shown to be a reliable index of physical dependence in rats (Nozaki, Akera, Lee & Brody, 1974; Stolerman, Johnson, Bunker & Jarvik, 1975). The results show that DG-AVP treated animals aremore sensitive to naloxone. Physical dependence refers to the fact that the organism becomes more sensitive to the morphine antagonist (Clouet & Iwatsubo, 1975; Takemori, 1975). Thus, it can be concluded that DG-AVP facilitates the development of physical dependence. This is supported by the observation that the morphine-induced hyperthermia was already antagonized by naloxone in the peptide treated animals before this occurred in the saline treated rats. Omitting the last injection with DG-AVP--on the test day--did not affect the results. From electrophysiological studies with vasopressin analogues we know that the effect does not last for more than 8 hr (Urban & De Wied, unpublished data). It is therefore unlikely that the influence of peptides is due to a direct interaction with naloxone. Thus, DG-AVP does not modify the expression of withdrawal signs, but indeed facilitates the development of physical dependence. This was substantiated in the test described by Takemori (1975). It has been argued that this procedure is a sensitive indicator of the development of tolelance and physical dependence. Also in this test DG-AVP facilitated the development of tolerance and dependence. The present experiments were designed in view of the involvement of vasopressin and related analogues in learning and memory processes. Vasopressin increases resistance to extinction of avoidance behavior (De Wied, 1971 ; Ader & De Wied, 1972) and protects against various forms of amnesia (Lande, Flexner & Flexner, 1972; Rigter et al., 1974; Walter et al., 1975). Oxytocin was also active in this respect, but the potency of this peptide is much lower than that ofvasopressin (De Wied, Bohus, Urban, Van Wimersma Greidanus & Gispen, 1975b). The covalent ring structure of vasopressin is the most important part of this molecule for effects on avoidance behavior. Surprisingly, in the present experiments oxytocin is much more potent than DG-AVP in facilitating tolerance to, and physical dependence on, morphine. For this effect the Cterminal part of oxytocin (PLG) is essential. The same holds for vasopressin, although PLG was more active than PAG. AVT was as active as DG-AVP and PAG. The C-terminal tripeptide of AVT is identical to that of [argininea]-vasopressin (AVP). This explains why it has the same potency as the vasopressin analogues. As mentioned before, neurohypophyseal hormones and their fragments protect against puromycin-induced amnesia in mice (Lande et al., 1972; Walter et aL, 1975). Although vasopressin and related compounds had marked activity, PLG and shorter C-terminal peptides of oxytocin were as active in this respect, but the covalent ring of oxytocin was not. This is in contrast to results obtained in the avoidance test. The present data, therefore, compare rather well with the results obtained in the puromycin amnesia test. Accordingly, neurohypophyseal hormones and their fragments are involved in learning and memory processes. However, it might be postulated that various learning processes are specifically controlled by different peptides. The physiological role of vasopressin in memory processes has been demonstrated in
40
JAN M. VANREEand DAVIDDE WIED
Brattleboro rats with hereditary diabetes insipidus in which active and passive avoidance behavior is markedly disturbed (De Wied, Bohus & Van Wimersma Greidanus, 1974). In addition, intraventricular injections of specific antisera against vasopressin which prevents passive avoidance behavior, support the physiological role of vasopressin in the consolidation of avoidance behavior (Van Wimersma Greidanus et al., 1975). P L G and the dipeptide Leu-Gly might be generated in vivo from oxytocin by hypothalamic enzymes (Walter, Gritfiths & Hooper, 1973) and P L G has been isolated as such from hypothalamic tissue (Nair, Kastin & Schally, 1971). It is tempting to speculate that P L G and related peptides are essentially involved in the development of morphine tolerance and dependence. This is in accord with previous findings that the development of resistance to the analgesic action of morphine is reduced in Brattleboro rats (De Wied & Gispen, 1976). These animals however do not lack the ability to synthesize oxytocin. Although they have a lower than normal amount of oxytocin in the posterior pituitary (Van Wimersma Greidanus, personal communication), the level of this hormone in the brain has to be determined, before we might suggest that a disturbance in oxytocin production might be the underlying cause of the decrease in tolerance development in homozygous diabetes insipidus rats. The authors wish to acknowledge the skilful assistance of Willy Spaapen-Kok and Yvonne SchuchardFerric. They also express their thanks to Organon International B.V. (Oss, The Netherlands) for supplying the various peptides, to Dr. R. Walter (Chicago, U.S.A.) for cycle (Leu-Gly), and to Endo Laboratories (Brussels, Belgium) for naloxone. REFERENCES AD~,~, R. & DE Wxr=D,D. (1972) Effects of lysine vasopressin on passive avoidance learning. Psychon. Scl. 29, 46-48. Bom.Ts, B., GIsPEN, W. H. & DE WIFx~,D. (1973) Effect of lysine vasopressin and ACTH 4-10 on conditioned avoidance behavior of hypophysectomized rats. Neuroendocrinology 11, 137-143. CAtDWELL,J. & SEVER,P. S. (1974) The biochemical pharmacology of abused drugs. Clin. Pharm. Ther. 16, 989-1013. O.OtnET, D. H. & IWAxstmo, K. (1975) Mechanisms of tolerance to and dependence on narcotic analgesic drugs. Ann. Rev. Pharmae. 15, 49-71. Co~N, M., KEATS,A. S., gauvov, W. A. & UNGAR,G. (1965) Effect of actinomycin on morphine tolerance Prec. Soc. exp. Biol. 119, 381-384. DE WIED, D. (1969) Effects of peptide hormones on behavior. In Frontiers in Neuroendocrinology, W. F. Ganong and L. Martini (Eds.), pp. 97-140. Oxford University Press, New York. DE WIED,D. (1971) Long term effect of vasopressin on the maintenance of a conditioned avoidance response in rats. Nature, Lend. 232, 58-60. DE WIED,D. & Bonus, B. (1966) Long term and short term effect on retention of a conditioned avoidance response in rats by treatment respectively with long acting pitressin or a-MSH. Nature, Lend. 212, 14841486. DE WtED, D. & GXSPEN,W. H. (1976) Impaired development of tolerance to morphine analgesia in rats with hereditary diabetes insipidus. Psychopharmacologia, Berlin 46, 27-29. DE WIED, D., Bonus, B. & VAN WIMERSMAGR~DANOS, Tj. B. (1974) The hypothalamo-hypophyseal system and the preservation of conditioned avoidance behavior in rats. In Integrative Hypothalamic Activity, D. F. Swaab and J. P. Schad6 (Eds.), Progr. Brain Res. 41,417-428. Elsevier, Amsterdam. D~ WIED, D., VAN WIMERSMAGn~ZDA~'US,Tj. B. & Bonus, B. (1975a), Pituitary peptides and behavior: influence on motivational learning and memory processes. Neuropsychopharmacology, Prec. IX Congr. of the Collegium Intern. Neuropsychopharmacologicum, pp. 653-658. Excerpta Medica, Amsterdam. D~ WIED, D., Bonus, B., URBAN,I., VANWIMERSMAGREIDANUS,Tj. B. & GISPEN, W. H. (1975b) Pituitary peptides and memory. In Peptides: Chemistry, Structure and Biology, R. Walter and J. Meienhofer (Eds.), pp. 635-643. Ann Arbor Science, Ann Arbor. EDDY, N. B. & LEIMBACH,D. (1953) Synthetic analgesics. II. Dithienylbutenyl and dithienylbutylamines. J. Pharm. exp. Ther. 107, 385-393.
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41
KING, A. R. & DE WIED,D. (1974) Localized behavioral effects of vasopressin on maintenance of an active avoidance response in rats. J. comp. physiol. PsychoL 86, 1008-1018. KmvoY, W. A., ZI~ra~ANN, E. & LAr,rOE,S. (1974) Facilitation of development of resistance to morphine analgesia by desglycinamideg-lysine-vasopressin. Proc. hath. Acad. Sci. U.S.A. 71, 1852-1856. LANDE, S., FLEXNER,J. B. & FLEXNER, L. B. (1972) Effect of corticotropin and desglycinamideg-lysinevasopressin on suppression of memory by puromycin. Proc. natn. Acad. Sci, U.S.A. 69, 558-560. NAm, R. M. G., KASTIN,A. J. & SCHALLY,A. V. (1971) Isolation and structure of hypothalamic MSH release-inhibiting hormone. Biochem. biophys. Res. Communs. 43, 1376-1381. NOZAKI,M., AKERA,T., LEE,C. Y. & BRODY,T. M. (1974) The effects of age on the development of tolerance to and physical dependence on morphine in rats..L Pharm. exp. Ther. 192, 506--512. RIGTER, H., VAN R/EZEN, H. & DE WIED, D. (1974) The effects of ACTH- and vasopressin-analogues on CO2-induced retrograde amnesia in rats. PhysioL Behao. 13, 381-388. STOLERMAN, I. P., JOHNSON, C. A., BUNKER,P. & JARVIK, M. E. (1975) Weight loss and shock-elicited aggression as indices of morphine abstinence in rats. Psychopharmacologia, Berlin 45, 157-161. TAKEMORI,A. E. (1975) Neurochemical bases for narcotic tolerance and dependence. Biochem. Pharmac. 24, 2121-2126. VAN WIMERSMAGREIDANUS,Tj. B., DOGTEROM,J. & DE WIED, D. (1975) Intraventricular administration of anti-vasopressin serum inhibits memory consolidation in rats. Life Sci. 16, 637-644. WALTER,R., GmFFITHS,E. C. & HOOPFa~,K. C. (1973) Production of MSH-release-inhibiting hormone by a particulate preparation of hypothalami: mechanisms of oxytocin inactivation. Brain Res. 60, 449-457. WALTER,R., HOFFMAN,P. L. FLEXNER,J. B. & FLEXr,mR, L. B. (1975) Neurohypophyseal hormones, analogs, and fragments: their effect on puromycin-induced amnesia. Proc. hath. Acad. Sci. U.S.A.. 72, 4180--4184.
