- Email: [email protected]
Naltrexone-insensitive facilitation and naltrexone-sensitive inhibition of passive avoidance behavior of the ACTH-(4–9) analog (ORG 2766) are located in two different parts of the molecule
European Journal of Pharmacology, 81 (1982) 441-448 Elsevier Biomedical Press
44 1
NALTREXONE-INSENSITIVE FACILITATION AND NALTREXONE-SENSITIVE INHIBITION O F P A S S I V E A V O I D A N C E B E H A V I O R O F T H E A C T H - ( 4 - 9 ) A N A L O G ( O R G 2766) A R E LOCATED IN TWO DIFFERENT PARTS OF THE MOLECULE MATYAS FEKETE * and DAVID DE WIED ** Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands Received 18 February 1982, revised MS received 1 April 1982, accepted 13 April 1982
M. FEKETE and D. DE WIED, Naltrexone-insensitive facifitation and naltrexone-sensitive inhibition of passive avoidance behavior of the ACTH-(4-9) analog (ORG 2766) are located in two different parts of the molecule, European J. Pharmacol. 81 (1982) 441-448. Subcutaneous injection of the ACTH-(4-9) analog (ORG 2766) in ng amounts prior to the retention test facilitated, while/~g doses attenuated passive avoidance behavior. The inhibitory effect could easily be overcome by treatment with ACTH-(4-10) either before or after ORG 2766 administration. Thus, inhibition of passive avoidance behavior by O R G 2766 probably was not due to competition with ACTH-like peptides or a functional antagonistic influence on brain structures sensitive to ACTH-like peptides. Intracerebroventricular administration of ACTH-(4-10) in a wide dose range (0.5-10.0/zg) and of O R G 2766 in low doses (0.5-1.0 ng) facilitated passive avoidance behavior, whereas 'high' doses of O R G 2766 (5.0 and 10.0 ng) and graded doses of the COOH terminal tripeptide of ORG 2766 (Phe-D-Lys-Phe; PDLP; 0.5-10.0 ng) attenuated passive avoidance behavior. The NH 2 terminal tetrapeptide of ORG 2766 (H-Met/O2/-Glu-His-Phe) facilitated passive avoidance behavior, whereas the NH 2 terminal tripeptide (HM e t / O 2/-Glu-His) was ineffective. Naltrexone pretreatment antagonized the attenuating effect of ORG 2766 and PDLP. Following pretreatment with this opiate antagonist both 'low' and 'high' doses of ORG 2766 and the NH 2 terminal tetrapeptide of ORG 2766 induced facilitation of passive avoidance behavior, while PDLP was ineffective in the presence of naltrexone. Thus, O R G 2766 exerts a dual effect on passive avoidance behavior. The facilitating effect of O R G 2766 resides in the NH 2 terminal part and is unrelated to naltrexone-sensitive brain opiate receptor sites, whereas the inhibiting influence is located in the COOH terminal part of the peptide and depends on naltrexone-sensitive brain opiate receptor sites. Passive avoidance behavior
ACTH-(4-9) analog (ORG 2766)
1. Introduction It h a d b e e n f o u n d that the A C T H - ( 4 - 9 ) a n a l o g (H-Met/OE/-Glu-His-Phe-D-Lys-Phe-OH; ORG 2766), following its s u b c u t a n e o u s a d m i n i s t r a t i o n in 'low' doses (ng) facilitated passive a v o i d a n c e b e h a v i o r a n d in 'high' doses (#g), i n h i b i t e d passive a v o i d a n c e r e t e n t i o n ( F e k e t e a n d D e Wied, in prep a r a t i o n ) . This differential effect of O R G 2766 was n o t o b s e r v e d on extinction o f active a v o i d a n c e * On leave of absence from the Department of Pathophysiology, University Medical School, Szeged, Hungary. ** To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75
© 1982 Elsevier Biomedical Press
Naltrexone
b e h a v i o r a n d was a b s e n t following A C T H - ( 4 - 1 0 ) a n d [D-Phe7]ACTH-(4-10) when tested in 'low' a n d 'high' doses on passive a v o i d a n c e behavior. It was c o n c l u d e d that the i n h i b i t o r y effect of O R G 2766 on passive a v o i d a n c e b e h a v i o r is p r o b a b l y l o c a t e d in the C O O H t e r m i n a l p a r t of the peptide. T h e aim of the p r e s e n t e x p e r i m e n t s was to further analyse this d u a l effect of O R G 2766. Thus, the effect of the N H 2 t e r m i n a l tri- a n d t e t r a p e p t i d e of O R G 2766 on passive a v o i d a n c e r e t e n t i o n was studied. In a d d i t i o n , it was investig a t e d whether the C O O H t e r m i n a l t r i p e p t i d e (PheD - L y s - P h e ; P D L P ) , the m a i n m e t a b o l i c p r o d u c t of O R G 2766, i n d e e d is r e s p o n s i b l e for the i n h i b i t o r y
442
effect of the parent molecule on passive avoidance behavior. It was found that the facilitating effect of O R G 2766 resides in the N H 2 terminal tetrapeptide, [Met(O2)4]ACTH-(4-7) and the inhibitory effect in the C O O H terminal tripeptide, PDLP. In addition, the effect of the latter peptide appeared to depend on naltrexone-sensitive opiate receptor sites.
2. Materials and methods
2.1. Animals
5 min), unavoidable scrambled footshock (0.25 mA, 2 s) was delivered through the grid floor of the dark compartment (learning trial). Rats were removed from the shock box 10 s after the termination of the shock. Passive avoidance latencies were tested 24 h (first retention test) and 48 h (second retention test) after the learning trial. The rat was placed on the platform and the latency to enter the dark compartment was measured up to a maxim u m of 300 s.
2.4. Pep tides
For intracerebroventricular administration of peptides, a plastic cannula was implanted into the lateral cerebral ventricle under anesthesia (0.1 ml H y p n o r m ~ s.c.) as described by De Wied (1976). The rats were allowed to recover for 7 days. The correct positioning of the cannula was checked individually by injection of methylene blue after the experiments had been completed.
For peripheral injection, ACTH-(4-10) (H-MetGlu-His-Phe-Arg-Trp-Gly-OH), the ACTH-(4-9) analog ( H - M e t / O z / - G l u - H i s - P h e - D - L y s - P h e - O H ; O R G 2766) were dissolved in one drop of 1 0 - S N HC1 then diluted with 0.9% saline (pH 6.5-6.7). Peripheral injections were given s.c. in a volume of 0.5 ml. Control animals received the same volume of vehicle. For central injection, ACTH-(4-10), O R G 2766, the N H 2 terminal tripeptide of O R G 2766 (HM e t / O 2 / - G l u - H i s - O H ) , the N H 2 terminal tetrapeptide of O R G 2766 (H-Met/Oz/-Glu-His-PheO H ) and the C O O H terminal tripeptide of O R G 2766 (H-Phe-D-Lys-Phe-OH; PDLP) were dissolved in 0.9% saline containing 0.5% bovine serum albumin (BSA; Sigma Chemical Company, St. Louis, U.S.A., Lot. No.: 41F-9350) preventing adhesion of small amounts of injected peptides. All intracerebroventricular injections were given in a volume of 1/~1 with a Unimetrics microsyringe. Control animals received the same volume of vehicle.
2.3. Passive avoidance behavior
2.5. Naltrexone pretreatment
Animals were trained in a step-through type one-trial learning passive avoidance test (Ader et al., 1972). Training was started between 3 p.m. and 5 p.m. The experimental apparatus consisted of an illuminated platform attached to a large, dark compartment equipped with a grid floor. After habituation to the dark compartment (2 rain), rats were placed on the platform and allowed to enter the dark compartment; since rats prefer dark to light, they normally enter within 15 s. On the next day after three more trials (intertrial interval
Naltrexone HC1 (Endo Laboratories, New York, U.S.A.) was dissolved in 0.9% saline and injected in a volume of 0.5 ml s.c. 2 h before the behavioral testing, i.e. 1 h before the intracerebroventricular peptide or vehicle treatment (De Wied et al., 1978). Control animals received the same volume of 0.9% saline.
Male Wistar rats of an inbred strain (CPB-TNO, Zeist, The Netherlands) weighing 140-160 g, were used. The operated animals were housed in individual cages after operation, while non-operated ones were housed 6 per cage at room temperature (20-21°C). All animals had access to commercial food and tap water ad libitum. The animals were kept under a standard illumination schedule (light on between 6 a.m. and 8 p.m.).
2.2. Surgery
2.6. Statistical analysis Mann-Whitney's non-parametric ranking test was used for statistical analyses.
443
3. Results
median Latency (s) 30C
o
20C
10[
o
- 60rain :
lO
3o
90 ~JgACTH4.10
Fig. 1. Effect of graded doses of ACTH-(4-10) on the retrieval of passive avoidance behavior in rats pretreated with saline or O R G 2766 (1 #g s.c.). O R G 2766 or saline were given 2 h, ACTH-(4-10) or saline 1 h prior to the first retention test. O P<0.05 vs. saline+saline; * P<0.05 vs. O R G 2766+saline. Number in bars indicates the number of rats. [] saline; [] O R G 2766. median totency(s)
30C
]
200
10C
-120rain:
o
30
0
90 ~Jg ACTH4-10
Fig. 2. Effect of O R G 2766 on retrieval of passive avoidance behavior in rats pretreated with saline or graded doses of ACTH-(4-10). ACTH-(4-10) or saline were given 2 h, ORG 2766 or saline I h prior to the first retention test. O P<0.05 vs. saline+saline; * P<0.05 vs. s a l i n e + O R G 2766. [] saline; [] ORG 2766.
preretention treatment (lh) median Latency(s}
Subcutaneous treatment with 30 and 90 gg of ACTH-(4-10) 1 h before the first retention test significantly facilitated passive avoidance behavior in a dose-dependent way (fig. 1). Pretreatment with 1 gg of ORG 2766 1 h prior to the injection with the same doses of ACTH-(4-10) did not affect the response to ACTH-(4-10) in spite of the fact that this dose of ORG 2766 attenuated passive avoidance responding in saline-pretreated rats. Conversely, the same dose of O R G 2766 given 1 h after 30 and 90 gg of ACTH-(4-10) (i.e. 1 h before the first retention test) also failed to affect the dose-dependent facilitation of passive avoidance behavior, although ORG 2766 again attenuated passive avoidance responding in saline-pretreated animals (fig. 2). Intracerebroventricular treatment with 0.5, 1.0, 5.0 and 10.0 #g of ACTH-(4-10) 1 h before the first retention test significantly facilitated the passive avoidance response in the first retention test only (fig. 3). Doses of 5.0 and 10.0 gg ACTH-(4-10) also increased passive avoidance latency in the second retention test. Administration of 0.5 and 1.0 ng of ORG 2766 facilitated passive avoidance response in the first retention test, while 1.0 ng of this peptide also increased it in the second retention test. However, 5.0 and 10.0 ng of ORG 2766 inhibited passive avoidance response in both the first and second retention test.
prere~ntion ~'eotment (lh) median LotencyIs)
300
200
100
0
05
1.0
ACTH4.10 icy.
5,0
1O0~Jg
0
0.5
1.0 5.0 0RG 2766 icy.
10.0ng
Fig. 3. Effect of graded doses of ACTH-(4-10) and ORG 2766 on the retrieval of passive avoidance behavior. Intracerebroventricular treatment 1 h prior to the first retention test. * P<0.05 vs. control. [] 1st retention test (24 h); [] 2nd retention test (48 h).
444
30pg nattrexone sc
saline sc media n Lotency( s )
0RG2766
median tatency(s )
0
1
0
10
90~Jg nottrexone sc median tatency(s )
1
10
o) L 0
1
10 ng icv.
Fig. 4. Effect of two different doses of O R G 2766 in saline- and naltrexone (30 and 90 t.tg s.c.)-pretreated animals on the retrieval of passive avoidance behavior. Naltrexone or saline were given 2 h, peptide or vehicle 1 h prior to the first retention test. * P<0.05 vs. control. [] 1st retention test (24 h); [] 2nd retention test (48 h).
median Latency (s)
median totency(s) 100
30C
200 50 10C
0
0.3
1.0 ~g icy.
O5
30/sg no[trexonesc.
median Latency(s) 300
200 100 Me'~O2)-GLu-His-Phe 0
median Latency(s)
50
100 ng
Fig. 7. Effect of graded doses of Phe-D-Lys-Phe (PDLP) on retrieval of passive avoidance behavior. Intracerebroventricular treatment was given 1 h prior to the first retention test. Abscissa: dose of PDLP (ng).* P<0.05 vs. control. [] 1st retention test (24 h); [] 2nd retention test (48 h).
Fig. 5. Effect of graded doses of M e t / O 2 / G l u - H i s - P h e on retrieval of passive avoidance behavior. Intracerebroventricular treatment was given 1 h prior to the first retention test. * P < 0.05 vs. control. [] 1st retention test (24 h); [] 2nd retention test (48 h).
saline sc.
10
gO)Jg naLtmxonesc. median Latency(s)
]
1
0
0
.ug icy.
Fig. 6. Effect o f M e t / O 2 / G l u - E l i s - P h e in rats pretreated w i t h saline or naltrexone (30 and 90 #g, s.c.) on the retrieval o f passive avoidance behavior. N a l t r e x o n e or saline were given 2 h, peptide or vehicle 1 h p r i o r to the first retention test. * P < 0 . 0 5 vs. control.
[] 1st retention test (24 h); [] 2nd retention test (48 h).
445
median totency(s] 100
50
6
6 naUrexone(sc.) phe- D-tys mp ~ (icVl)
0 0
0 ]0
30 0
30 10
go 0
90 10
JJg ng
Fig. 8. Effect of PDLP in rats pretreated with saline or naltrexone (30 and 90 #g s.c.) on the retrieval of passive avoidance behavior. Naltrexone or saline were given 2 h, peptide or vehicle 1 h prior to the first retention test. * P<0.05 vs. control; [] 1st retention test (24 h); [] 2nd retention test (48 h).
Fig. 4 shows the effect of pretreatment with naltrexone in doses of 30 and 90 #g given s.c. on passive avoidance behavior of rats treated intracerebroventricularly with O R G 2766. Naltrexone pretreatment as such failed to affect passive avoidance behavior. A dose of 1 ng of O R G 2766 facilitated passive avoidance behavior of both the saline- and naltrexone-pretreated groups in the first and second retention test. However, 10 ng of O R G 2766 inhibited passive avoidance behavior of the saline-pretreated group in both retention tests, while it facilitated passive avoidance behavior of the animals pretreated with 90/~g of naltrexone in the first retention test. It was ineffective in rats pretreated with 30 #g of naltrexone. Intracerebroventricular treatment with 0.3 and 1.0 #g of the N H 2 terminal tetrapeptide of O R G 2766 1 h before the first retention test significantly facilitated passive avoidance behavior in the first retention test only (fig. 5). Administration of 1.0, 3.0 and 10.0 #g of the N H 2 terminal tripeptide of O R G 2766 was ineffective. None of these doses increased passive avoidance latencies significantly as compared to saline-treated rats. A dose of 1.0 #g of the N H 2 terminal tetrapeptide of O R G 2766 (H-Met/O2/-Glu-His-PheOH) facilitated passive avoidance behavior of both saline- and naltrexone-pretreated rats in the first retention test (fig. 6). Passive avoidance behavior was dose-depen-
dently attenuated after intracerebroventricular administration of the COOH terminal tripeptide of O R G 2766, PDLP (fig. 7). A dose of 0.5 ng decreased passive avoidance latency in the first retention test, while 1.0, 5.0 and 10.0 ng decreased it in both the first and second retention test. While 30/xg of naltrexone given s.c. partially blocked, 90 #g of the opiate antagonist totally prevented the inhibitory effect of 10 ng of PDLP (fig. 8).
4. Discussion
As found in previous studies, systemic administration of O R G 2766 in ng doses appeared to facilitate passive avoidance behavior (Fekete and De Wied, 1982a), while #g doses had an opposite effect (Fekete and De Wied, 1982b). Since ACTH(4-10) facilitated passive avoidance behavior in a similar fashion in rats which were treated with either attenuating doses of O R G 2766 or with saline, the inhibitory effect of the 'high' dose of O R G 2766 on passive avoidance behavior probably was not due to competitive action with A C T H and related peptides or a functional antagonistic influence on brain structures sensitive to A C T H and related peptides. These effects are apparently exerted on different brain systems involved in the facilitation or in the attenuation of passive avoidance behavior.
446
O R G 2766 as well as ACTH-(4-10) administered systematically or intracerebroventricularly dose dependently facilitated the passive avoidance behavior. When 'high' doses were given, only O R G 2766 had an inhibitory effect whether administered systemically or intracerebroventricularly. The similarity between the influence of peripherally and centrally administered ACTH-(4-10) and O R G 2766 suggests that these peptides interact with central nervous mechanisms independent of the route of administration. In addition, O R G 2766 (in 'low' doses) appeared to be a thousand times more potent than ACTH-(4-10) and this potency ratio also was independent of the route of administration. The N H 2 terminal tetrapeptide of O R G 2766 (sequence 4-7) was as active as ACTH-(4-10) on facilitation of passive avoidance behavior, while the N H 2 terminal tripeptide of O R G 2766 (sequence 4-6) was ineffective in this respect. ACTH(4-7) is the shortest sequence which is as active as ACTH-(4-10) in delaying the extinction of polejumping avoidance behavior (Greven and De Wied, 1973). Loss of activity following the removal of phenylalanine in position 7 again indicates the key role of this amino acid residue in the mediation of the behavioral effect of ACTH-like peptides. This has already been demonstrated with analogs of ACTH-(1-10), ACTH-(4-10) or ACTH-(4-7), in which phenylalanine in position 7 was replaced by the D-enantiomer. Such [D-PheV]ACTH analogs act in an opposite manner and facilitate the extinction of pole-jumping avoidance behavior (Bohus and De Wied, 1966; Greven and De Wied, 1967; De Wied et al., 1975; Wiegant et al., 1978; Fekete and De Wied, 1982a; Fekete et al., 1982b). A C T H competitively displaces radiolabelled dihydromorphine (Terenius, 1975; Terenius et al., 1975; Wiegant et al., 1977) or fl-endorphin (Akil et al., 1980) from opiate receptor sites in the brain and therefore these receptors may represent a site of action of A C T H in the central nervous system. However, it has been suggested that opiate effects are mediated by two receptors: a stereospecific opiate fl-endorphin receptor which mediates the analgesic and cataleptic effects and a non-stereospecific opiate A C T H receptor which mediates the opiate abstinence syndrome and the excitatory el-
fects of the opiates (Jacquet, 1978). A C T H antagonizes morphine inhibition of spinal reflexes (Krivoy et al., 1974; Zimmermann and Krivoy, 1973, 1974), morphine-induced hyperactivity and Straub-tail (Katz, 1979) and counteracts morphine-induced analgesia (Gispen et al., 1976; Wiegant et al., 1977). These results are consistent with the hypothesis that ACTH and related peptides are partial agonist/antagonists of opiate receptors. ACTH-(4-10) has a variety of behavioral effects (De Wied, 1977; Gispen et al., 1977). Most of these effects are probably not related to opiate receptor function (De Wied et al., 1978; Gispen et al., 1977). Although the different parts of the A C T H molecule show similar activities in the pole-jumping or passive avoidance tests, their effect is not identical with respect to other CNS activities, e.g. they differ in affinity for opiate receptor sites (Terenius et al., 1975) and their ability to induce excessive grooming (Gispen et al., 1975; Gispen and Isaacson, 1981). Opiate antagonists (naloxone, naltrexone) suppress ACTH-(124)-induced excessive grooming in rats (Gispen and Wiegant, 1976), but not the effects of ACTHlike neuropeptides on extinction of active avoidance behavior (De Wied et al., 1978). Similarly, the present experiments show that the facilitating effect of O R G 2766 and the N H 2 terminal tetrapeptide of O R G 2766 on passive avoidance behavior is not blocked by the opiate antagonist naltrexone. However, the inhibitory effect of 'high' doses of O R G 2766 on passive avoidance behavior is inhibited in naltrexone-pretreated animals and is even reversed to facilitation. The COOH terminal tripeptide (PDLP) is the main metabolic product of O R G 2766 (Witter et al., 1975; Verhoef and Witter, 1976). This tripeptide is much less active than O R G 2766, but still contains approximately 10% of the activity of ACTH-(4-10) on extinction of pole-jumping active avoidance behavior (De Wied et al., 1975). However, passive avoidance behavior was dose dependently attenuated after intracerebroventricular injection of PDLP. This attenuating effect of the tripeptide was as potent as that of O R G 2766 and was also blocked by pretreatment with naltrexone. PDLP in low doses did not facilitate passive avoidance behavior (Naltrexone pretreatment blocked
447
the attenuating effect but did not induce facilitation as found with O R G 2766). The fact that only 'high' doses of O R G 2766 attenuated passive avoidance behavior suggests that such doses generate sufficient amounts of PDLP from O R G 2766 in the brain to inhibit passive avoidance behavior. Taken together, the results of the present experiments suggest that O R G 2766 has a dual effect on passive avoidance behavior. Facilitation of passive avoidance behavior by 'low' doses of O R G 2766 may be an intrinsic activity of the peptide exerted via a naltrexone-insensitive mechanism as was also found in pole-jumping active avoidance behavior (De Wied et al., 1978). This effect of O R G 2766 resides in the N H 2 terminal part of the molecule. The suppression of the passive avoidance response by 'high' doses of O R G 2766 is possibly mediated by naltrexone-sensitive opiate receptors and this inhibitory influence is located in the COOH terminal part of the peptide. This inhibitory effect of high dose of O R G 2766 or of the tripeptide, PDLP, on retention of passive avoidance behavior may be explained in different ways. For example by the affinity of these peptides for naltrexone-sensitive opiate receptors. O R G 2766 does not displace dihydromorphine (Terenius et al., 1975) or naloxone (Walker et al., 1981) in vitro. However, Plomp and Van Ree (1978) demonstrated opiate-like effects of N H 2 terminal ACTH-fragments as measured on electrically evoked contractions of the mouse vas deferens and the displacement of radiolabelled morphine from morphine antiserum. Structure-activity studies in both assay systems showed that the active core resided in the sequence ACTH-(7-10). It remains possible therefore that PDLP has an affinity for opiate receptor sites. Other explanations for the attenuating effect of PDLP on passive avoidance behavior are, a change in the sensitivity of naltrexone-sensitive receptors, the release of neuropeptide(s) with amnestic properties, the release of opiate-like peptides with an inhibitory effect on passive avoidance behavior (Kovacs and De Wied, 1981; Beluzzi and Stein, 1981; Messing et al., 1981; Garzon et al., 1981) or inhibition of enzymes which degrade opiate-like peptides in the brain. Further experiments are needed to unravel the question of the attenuating influence of the tripeptide PDLP on passive avoidance behavior.
Acknowledgements The peptides were donated by Dr. H.M. Greven of Organon International B.V., Oss, The Netherlands. Endo Laboratories provided us with naltrexone. This study was supported in part by Stichting Pharmacologisch Studiefonds.
References Ader, R., J.A.W.M. Weijnen and P. Moleman, 1972, Retention of a passive avoidance response as a function of the intensity and duration of electric shock, Psychon. Sci. 26, 125. Akil, H., W. Hewlett, J.D. Barchas and C.H. Li, 1980, Binding of 3H-fl-endorphin to rat brain membranes: Characterization and opiate properties, European J. Pharmacol. 64, 1. Beluzzi, J.D. and L. Stein, 1981, Facilitation of long-term memory by brain endorphins, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr., R.A. Jensen, R.B. Messing, H. Rigter and J.L. McGaugh (Academic Press, New YOrk) p. 291. Bohus, B. and D. De Wied, 1966, Inhibitory and facilitatory effect of two related peptides on extinction of avoidance behavior, Science 153, 318. De Wied, D., 1976, Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments, Life Sci. 19, 685. De Wied, D., 1977, Peptides and behavior, Life Sci. 20, 195. De Wied, D., B. Bohus, J.M. Van Ree and I. Urban, 1978, Behavioral and electrophysiological effects of peptides related to lipotropin (fl-LPH), J. Pharmacol. Exp. Ther. 204, 570. De Wied, D., A. Witter and H.M. Greven, 1975, Behaviourally active ACTH analogues, Biochem. Pharmacol. 24, 1463. Fekete, M. and D. De Wied, 1982a, Potency and duration of action of the ACTH 4-9 analog (ORG 2766) as compared to ACTH 4-10 and [D-PheT]ACTH 4-10 on active and passive avoidance behavior of rats, Pharmac. Biochem. Behav. 16, 387. Fekete, M. and D. De Wied, 1982b, Dose-related facilitation and inhibition of passive avoidance behavior by the ACTH(4-9) analog (ORG 2766), Pharmac. Biochem. Behav. (in press). Garzon, J., J. Rubio and J. Del Rio, 1981, Naloxone blocks the effect of 5-hydroxy-tryptophan on passive avoidance learning in rats: implication of endogenous opioid peptides, Life Sci. 29, 17. Gispen, W.H., J. Buitelaar, V.M. Wiegant, L. Terenius and D. De Wied, 1976, Interaction between ACTH fragments, brain opiate receptors and morphine-induced analgesia, European J. Pharmacol. 39, 393. Gispen, W.H. and R.L. Isaacson, 1981, ACTH-induced excessive grooming in the rat, Pharmacol. Ther. 12, 209. Gispen, W.H., J.M. Van Ree and D. De Wied, 1977, Lipotropin and the central nervous system, Int. Rev. Neurobiol. 20, 209.
448
Gispen, W.H. and V.M. Wiegant, 1976, Opiate antagonists suppress ACTHi.24-induced excessive grooming in the rat, 1976, Neurosci. Lett. 2, 159. Gispen, W.H., V.M. Wiegant, H.M. Greven and D. De Wied, 1975, The induction of excessive grooming in the rat by intraventricular application of peptides derived from ACTH: Structure-activity studies, Life Sci. 17, 645. Greven, H.M. and D. De Wied, 1976, The active sequence in the ACTH molecule responsible for inhibition of the extinction of conditioned avoidance behavior in rats, European J. Pharmacol. 2, 14. Greven, H.M. and D. De Wied, 1973, The influence of peptides derived from corticotrophin (ACTH) on performance. Structure activity studies, in: Drug Effects on Neuroendocrine Regulation, eds. E. Zimmermann, W.H. Gispen, B.H. Marks and D. De Wied (Elsevier, Amsterdam), Progress in Brain Research, Vol. 39, p. 429. Jacquet, Y.F., 1978, Opiate effects after adrenocorticotropin or fl-endorphin injection in the periaqueductal gray matter of rats, Science 201, 1032. Katz, R.J., 1979, ACTH4.10 antagonism of morphine-induced behavioural activation in the mouse, European J. Pharmacol. 53, 383. Kovacs, G.L. and D. De Wied, 1981, Endorphin influences on learning and memory, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martines, Jr., R.A. Jensen, R.B. Messing, H. Rigter and J.L. McGaugh (Academic Press, New York) p. 231. Krivoy, W.A., D. Kroeger, A.N. Taylor and E. Zimmermann, 1974, Antagonism of morphine by fl-melanocyte stimulating hormone and by tetracosactin, Europrean J. Pharmacol. 27, 339. Messing, R.B., R.A. Jensen, B.J. Vasquez, J.L. Martinez, Jr., V.R. Spiehler and J.L. McGaugh, 1981, Opiate modulation of memory, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr., R.A. Jensen, R.B. Messing, H. Rigter and J.L. McGaugh (Academic Press, New York) p. 431.
Plomp, G.J.J. and J.M. Van Ree, 1978, Adrenocorticotrophic hormone fragments mimic the effect of morphine in vitro, Br. J. Pharmacol. 64, 223. Terenius, L., 1975, Effects of peptides and amino acids on dihydromorphine binding to the opiate receptor, J. Pharm. Pharmacol. 27, 450. Terenius, L., W.H. Gispen and D. De Wied, 1975, ACTH-Iike peptides and opiate receptors in the brain: Structure activity studies, European J. Pharmacol. 33, 395. Verhoef, J. and A. Witter, 1976, In vivo fate of a behaviorally active ACTH 4-9 analog in rats after systemic administration, Pharmacol. Biochem. Behav. 4, 583. Walker, J.M., G.G. Berntson, C.A. Sandman, A.J. Kastin and H. Akil, 1981, Induction of analgesia by central administration of ORG 2766, an analog of ACTH4. 9, European J. Pharmacol. 69, 71. Wiegant, V.M., D. Colbern, Tj.B. Van Wimersma Greidanus and W.H. Gispen, 1978, Differential behavioral effect of ACTH 4-10 and (D-PheT)ACTH 4-10, Brain Res. Bull. 3, 167. Wiegant, V.M., W.H. Gispen, L. Terenius and D. De Wied, 1977, ACTH-like peptides and morphine: Interaction at the level of the CNS, Psychoneuroendocrinology 2, 63. Witter, A., H.M. Greven and D. De Wied, 1975, Correlation between structure, behavioral activity and rate of biotransformation of some ACTH 4-9 analogs, J. Pharmacol. Exp. Ther. 193, 853. Zimmermann, E. and W.A. Krivoy, 1973, Antagonism between morphine and the polypeptides ACTH, ACTHI.24 and flMSH in the nervous system, in: Drug Effects on Neuroendocrine Regulation, Progr. Brain Res. Vol. 39, eds. E. Zimmermann, W.H. Gispen, B.H. Marks and D. De Wied (Elsevier, Amsterdam) p. 383. Zimmermann, E. and W.A. Krivoy, 1974, Depression of frog isolated spinal cord by morphine and antagonism by tetracosactin, Proc. Soc. Exp, Biol. 146, 575.
44 1
NALTREXONE-INSENSITIVE FACILITATION AND NALTREXONE-SENSITIVE INHIBITION O F P A S S I V E A V O I D A N C E B E H A V I O R O F T H E A C T H - ( 4 - 9 ) A N A L O G ( O R G 2766) A R E LOCATED IN TWO DIFFERENT PARTS OF THE MOLECULE MATYAS FEKETE * and DAVID DE WIED ** Rudolf Magnus Institute for Pharmacology, Medical Faculty, University of Utrecht, Vondellaan 6, 3521 GD Utrecht, The Netherlands Received 18 February 1982, revised MS received 1 April 1982, accepted 13 April 1982
M. FEKETE and D. DE WIED, Naltrexone-insensitive facifitation and naltrexone-sensitive inhibition of passive avoidance behavior of the ACTH-(4-9) analog (ORG 2766) are located in two different parts of the molecule, European J. Pharmacol. 81 (1982) 441-448. Subcutaneous injection of the ACTH-(4-9) analog (ORG 2766) in ng amounts prior to the retention test facilitated, while/~g doses attenuated passive avoidance behavior. The inhibitory effect could easily be overcome by treatment with ACTH-(4-10) either before or after ORG 2766 administration. Thus, inhibition of passive avoidance behavior by O R G 2766 probably was not due to competition with ACTH-like peptides or a functional antagonistic influence on brain structures sensitive to ACTH-like peptides. Intracerebroventricular administration of ACTH-(4-10) in a wide dose range (0.5-10.0/zg) and of O R G 2766 in low doses (0.5-1.0 ng) facilitated passive avoidance behavior, whereas 'high' doses of O R G 2766 (5.0 and 10.0 ng) and graded doses of the COOH terminal tripeptide of ORG 2766 (Phe-D-Lys-Phe; PDLP; 0.5-10.0 ng) attenuated passive avoidance behavior. The NH 2 terminal tetrapeptide of ORG 2766 (H-Met/O2/-Glu-His-Phe) facilitated passive avoidance behavior, whereas the NH 2 terminal tripeptide (HM e t / O 2/-Glu-His) was ineffective. Naltrexone pretreatment antagonized the attenuating effect of ORG 2766 and PDLP. Following pretreatment with this opiate antagonist both 'low' and 'high' doses of ORG 2766 and the NH 2 terminal tetrapeptide of ORG 2766 induced facilitation of passive avoidance behavior, while PDLP was ineffective in the presence of naltrexone. Thus, O R G 2766 exerts a dual effect on passive avoidance behavior. The facilitating effect of O R G 2766 resides in the NH 2 terminal part and is unrelated to naltrexone-sensitive brain opiate receptor sites, whereas the inhibiting influence is located in the COOH terminal part of the peptide and depends on naltrexone-sensitive brain opiate receptor sites. Passive avoidance behavior
ACTH-(4-9) analog (ORG 2766)
1. Introduction It h a d b e e n f o u n d that the A C T H - ( 4 - 9 ) a n a l o g (H-Met/OE/-Glu-His-Phe-D-Lys-Phe-OH; ORG 2766), following its s u b c u t a n e o u s a d m i n i s t r a t i o n in 'low' doses (ng) facilitated passive a v o i d a n c e b e h a v i o r a n d in 'high' doses (#g), i n h i b i t e d passive a v o i d a n c e r e t e n t i o n ( F e k e t e a n d D e Wied, in prep a r a t i o n ) . This differential effect of O R G 2766 was n o t o b s e r v e d on extinction o f active a v o i d a n c e * On leave of absence from the Department of Pathophysiology, University Medical School, Szeged, Hungary. ** To whom all correspondence should be addressed. 0014-2999/82/0000-0000/$02.75
© 1982 Elsevier Biomedical Press
Naltrexone
b e h a v i o r a n d was a b s e n t following A C T H - ( 4 - 1 0 ) a n d [D-Phe7]ACTH-(4-10) when tested in 'low' a n d 'high' doses on passive a v o i d a n c e behavior. It was c o n c l u d e d that the i n h i b i t o r y effect of O R G 2766 on passive a v o i d a n c e b e h a v i o r is p r o b a b l y l o c a t e d in the C O O H t e r m i n a l p a r t of the peptide. T h e aim of the p r e s e n t e x p e r i m e n t s was to further analyse this d u a l effect of O R G 2766. Thus, the effect of the N H 2 t e r m i n a l tri- a n d t e t r a p e p t i d e of O R G 2766 on passive a v o i d a n c e r e t e n t i o n was studied. In a d d i t i o n , it was investig a t e d whether the C O O H t e r m i n a l t r i p e p t i d e (PheD - L y s - P h e ; P D L P ) , the m a i n m e t a b o l i c p r o d u c t of O R G 2766, i n d e e d is r e s p o n s i b l e for the i n h i b i t o r y
442
effect of the parent molecule on passive avoidance behavior. It was found that the facilitating effect of O R G 2766 resides in the N H 2 terminal tetrapeptide, [Met(O2)4]ACTH-(4-7) and the inhibitory effect in the C O O H terminal tripeptide, PDLP. In addition, the effect of the latter peptide appeared to depend on naltrexone-sensitive opiate receptor sites.
2. Materials and methods
2.1. Animals
5 min), unavoidable scrambled footshock (0.25 mA, 2 s) was delivered through the grid floor of the dark compartment (learning trial). Rats were removed from the shock box 10 s after the termination of the shock. Passive avoidance latencies were tested 24 h (first retention test) and 48 h (second retention test) after the learning trial. The rat was placed on the platform and the latency to enter the dark compartment was measured up to a maxim u m of 300 s.
2.4. Pep tides
For intracerebroventricular administration of peptides, a plastic cannula was implanted into the lateral cerebral ventricle under anesthesia (0.1 ml H y p n o r m ~ s.c.) as described by De Wied (1976). The rats were allowed to recover for 7 days. The correct positioning of the cannula was checked individually by injection of methylene blue after the experiments had been completed.
For peripheral injection, ACTH-(4-10) (H-MetGlu-His-Phe-Arg-Trp-Gly-OH), the ACTH-(4-9) analog ( H - M e t / O z / - G l u - H i s - P h e - D - L y s - P h e - O H ; O R G 2766) were dissolved in one drop of 1 0 - S N HC1 then diluted with 0.9% saline (pH 6.5-6.7). Peripheral injections were given s.c. in a volume of 0.5 ml. Control animals received the same volume of vehicle. For central injection, ACTH-(4-10), O R G 2766, the N H 2 terminal tripeptide of O R G 2766 (HM e t / O 2 / - G l u - H i s - O H ) , the N H 2 terminal tetrapeptide of O R G 2766 (H-Met/Oz/-Glu-His-PheO H ) and the C O O H terminal tripeptide of O R G 2766 (H-Phe-D-Lys-Phe-OH; PDLP) were dissolved in 0.9% saline containing 0.5% bovine serum albumin (BSA; Sigma Chemical Company, St. Louis, U.S.A., Lot. No.: 41F-9350) preventing adhesion of small amounts of injected peptides. All intracerebroventricular injections were given in a volume of 1/~1 with a Unimetrics microsyringe. Control animals received the same volume of vehicle.
2.3. Passive avoidance behavior
2.5. Naltrexone pretreatment
Animals were trained in a step-through type one-trial learning passive avoidance test (Ader et al., 1972). Training was started between 3 p.m. and 5 p.m. The experimental apparatus consisted of an illuminated platform attached to a large, dark compartment equipped with a grid floor. After habituation to the dark compartment (2 rain), rats were placed on the platform and allowed to enter the dark compartment; since rats prefer dark to light, they normally enter within 15 s. On the next day after three more trials (intertrial interval
Naltrexone HC1 (Endo Laboratories, New York, U.S.A.) was dissolved in 0.9% saline and injected in a volume of 0.5 ml s.c. 2 h before the behavioral testing, i.e. 1 h before the intracerebroventricular peptide or vehicle treatment (De Wied et al., 1978). Control animals received the same volume of 0.9% saline.
Male Wistar rats of an inbred strain (CPB-TNO, Zeist, The Netherlands) weighing 140-160 g, were used. The operated animals were housed in individual cages after operation, while non-operated ones were housed 6 per cage at room temperature (20-21°C). All animals had access to commercial food and tap water ad libitum. The animals were kept under a standard illumination schedule (light on between 6 a.m. and 8 p.m.).
2.2. Surgery
2.6. Statistical analysis Mann-Whitney's non-parametric ranking test was used for statistical analyses.
443
3. Results
median Latency (s) 30C
o
20C
10[
o
- 60rain :
lO
3o
90 ~JgACTH4.10
Fig. 1. Effect of graded doses of ACTH-(4-10) on the retrieval of passive avoidance behavior in rats pretreated with saline or O R G 2766 (1 #g s.c.). O R G 2766 or saline were given 2 h, ACTH-(4-10) or saline 1 h prior to the first retention test. O P<0.05 vs. saline+saline; * P<0.05 vs. O R G 2766+saline. Number in bars indicates the number of rats. [] saline; [] O R G 2766. median totency(s)
30C
]
200
10C
-120rain:
o
30
0
90 ~Jg ACTH4-10
Fig. 2. Effect of O R G 2766 on retrieval of passive avoidance behavior in rats pretreated with saline or graded doses of ACTH-(4-10). ACTH-(4-10) or saline were given 2 h, ORG 2766 or saline I h prior to the first retention test. O P<0.05 vs. saline+saline; * P<0.05 vs. s a l i n e + O R G 2766. [] saline; [] ORG 2766.
preretention treatment (lh) median Latency(s}
Subcutaneous treatment with 30 and 90 gg of ACTH-(4-10) 1 h before the first retention test significantly facilitated passive avoidance behavior in a dose-dependent way (fig. 1). Pretreatment with 1 gg of ORG 2766 1 h prior to the injection with the same doses of ACTH-(4-10) did not affect the response to ACTH-(4-10) in spite of the fact that this dose of ORG 2766 attenuated passive avoidance responding in saline-pretreated rats. Conversely, the same dose of O R G 2766 given 1 h after 30 and 90 gg of ACTH-(4-10) (i.e. 1 h before the first retention test) also failed to affect the dose-dependent facilitation of passive avoidance behavior, although ORG 2766 again attenuated passive avoidance responding in saline-pretreated animals (fig. 2). Intracerebroventricular treatment with 0.5, 1.0, 5.0 and 10.0 #g of ACTH-(4-10) 1 h before the first retention test significantly facilitated the passive avoidance response in the first retention test only (fig. 3). Doses of 5.0 and 10.0 gg ACTH-(4-10) also increased passive avoidance latency in the second retention test. Administration of 0.5 and 1.0 ng of ORG 2766 facilitated passive avoidance response in the first retention test, while 1.0 ng of this peptide also increased it in the second retention test. However, 5.0 and 10.0 ng of ORG 2766 inhibited passive avoidance response in both the first and second retention test.
prere~ntion ~'eotment (lh) median LotencyIs)
300
200
100
0
05
1.0
ACTH4.10 icy.
5,0
1O0~Jg
0
0.5
1.0 5.0 0RG 2766 icy.
10.0ng
Fig. 3. Effect of graded doses of ACTH-(4-10) and ORG 2766 on the retrieval of passive avoidance behavior. Intracerebroventricular treatment 1 h prior to the first retention test. * P<0.05 vs. control. [] 1st retention test (24 h); [] 2nd retention test (48 h).
444
30pg nattrexone sc
saline sc media n Lotency( s )
0RG2766
median tatency(s )
0
1
0
10
90~Jg nottrexone sc median tatency(s )
1
10
o) L 0
1
10 ng icv.
Fig. 4. Effect of two different doses of O R G 2766 in saline- and naltrexone (30 and 90 t.tg s.c.)-pretreated animals on the retrieval of passive avoidance behavior. Naltrexone or saline were given 2 h, peptide or vehicle 1 h prior to the first retention test. * P<0.05 vs. control. [] 1st retention test (24 h); [] 2nd retention test (48 h).
median Latency (s)
median totency(s) 100
30C
200 50 10C
0
0.3
1.0 ~g icy.
O5
30/sg no[trexonesc.
median Latency(s) 300
200 100 Me'~O2)-GLu-His-Phe 0
median Latency(s)
50
100 ng
Fig. 7. Effect of graded doses of Phe-D-Lys-Phe (PDLP) on retrieval of passive avoidance behavior. Intracerebroventricular treatment was given 1 h prior to the first retention test. Abscissa: dose of PDLP (ng).* P<0.05 vs. control. [] 1st retention test (24 h); [] 2nd retention test (48 h).
Fig. 5. Effect of graded doses of M e t / O 2 / G l u - H i s - P h e on retrieval of passive avoidance behavior. Intracerebroventricular treatment was given 1 h prior to the first retention test. * P < 0.05 vs. control. [] 1st retention test (24 h); [] 2nd retention test (48 h).
saline sc.
10
gO)Jg naLtmxonesc. median Latency(s)
]
1
0
0
.ug icy.
Fig. 6. Effect o f M e t / O 2 / G l u - E l i s - P h e in rats pretreated w i t h saline or naltrexone (30 and 90 #g, s.c.) on the retrieval o f passive avoidance behavior. N a l t r e x o n e or saline were given 2 h, peptide or vehicle 1 h p r i o r to the first retention test. * P < 0 . 0 5 vs. control.
[] 1st retention test (24 h); [] 2nd retention test (48 h).
445
median totency(s] 100
50
6
6 naUrexone(sc.) phe- D-tys mp ~ (icVl)
0 0
0 ]0
30 0
30 10
go 0
90 10
JJg ng
Fig. 8. Effect of PDLP in rats pretreated with saline or naltrexone (30 and 90 #g s.c.) on the retrieval of passive avoidance behavior. Naltrexone or saline were given 2 h, peptide or vehicle 1 h prior to the first retention test. * P<0.05 vs. control; [] 1st retention test (24 h); [] 2nd retention test (48 h).
Fig. 4 shows the effect of pretreatment with naltrexone in doses of 30 and 90 #g given s.c. on passive avoidance behavior of rats treated intracerebroventricularly with O R G 2766. Naltrexone pretreatment as such failed to affect passive avoidance behavior. A dose of 1 ng of O R G 2766 facilitated passive avoidance behavior of both the saline- and naltrexone-pretreated groups in the first and second retention test. However, 10 ng of O R G 2766 inhibited passive avoidance behavior of the saline-pretreated group in both retention tests, while it facilitated passive avoidance behavior of the animals pretreated with 90/~g of naltrexone in the first retention test. It was ineffective in rats pretreated with 30 #g of naltrexone. Intracerebroventricular treatment with 0.3 and 1.0 #g of the N H 2 terminal tetrapeptide of O R G 2766 1 h before the first retention test significantly facilitated passive avoidance behavior in the first retention test only (fig. 5). Administration of 1.0, 3.0 and 10.0 #g of the N H 2 terminal tripeptide of O R G 2766 was ineffective. None of these doses increased passive avoidance latencies significantly as compared to saline-treated rats. A dose of 1.0 #g of the N H 2 terminal tetrapeptide of O R G 2766 (H-Met/O2/-Glu-His-PheOH) facilitated passive avoidance behavior of both saline- and naltrexone-pretreated rats in the first retention test (fig. 6). Passive avoidance behavior was dose-depen-
dently attenuated after intracerebroventricular administration of the COOH terminal tripeptide of O R G 2766, PDLP (fig. 7). A dose of 0.5 ng decreased passive avoidance latency in the first retention test, while 1.0, 5.0 and 10.0 ng decreased it in both the first and second retention test. While 30/xg of naltrexone given s.c. partially blocked, 90 #g of the opiate antagonist totally prevented the inhibitory effect of 10 ng of PDLP (fig. 8).
4. Discussion
As found in previous studies, systemic administration of O R G 2766 in ng doses appeared to facilitate passive avoidance behavior (Fekete and De Wied, 1982a), while #g doses had an opposite effect (Fekete and De Wied, 1982b). Since ACTH(4-10) facilitated passive avoidance behavior in a similar fashion in rats which were treated with either attenuating doses of O R G 2766 or with saline, the inhibitory effect of the 'high' dose of O R G 2766 on passive avoidance behavior probably was not due to competitive action with A C T H and related peptides or a functional antagonistic influence on brain structures sensitive to A C T H and related peptides. These effects are apparently exerted on different brain systems involved in the facilitation or in the attenuation of passive avoidance behavior.
446
O R G 2766 as well as ACTH-(4-10) administered systematically or intracerebroventricularly dose dependently facilitated the passive avoidance behavior. When 'high' doses were given, only O R G 2766 had an inhibitory effect whether administered systemically or intracerebroventricularly. The similarity between the influence of peripherally and centrally administered ACTH-(4-10) and O R G 2766 suggests that these peptides interact with central nervous mechanisms independent of the route of administration. In addition, O R G 2766 (in 'low' doses) appeared to be a thousand times more potent than ACTH-(4-10) and this potency ratio also was independent of the route of administration. The N H 2 terminal tetrapeptide of O R G 2766 (sequence 4-7) was as active as ACTH-(4-10) on facilitation of passive avoidance behavior, while the N H 2 terminal tripeptide of O R G 2766 (sequence 4-6) was ineffective in this respect. ACTH(4-7) is the shortest sequence which is as active as ACTH-(4-10) in delaying the extinction of polejumping avoidance behavior (Greven and De Wied, 1973). Loss of activity following the removal of phenylalanine in position 7 again indicates the key role of this amino acid residue in the mediation of the behavioral effect of ACTH-like peptides. This has already been demonstrated with analogs of ACTH-(1-10), ACTH-(4-10) or ACTH-(4-7), in which phenylalanine in position 7 was replaced by the D-enantiomer. Such [D-PheV]ACTH analogs act in an opposite manner and facilitate the extinction of pole-jumping avoidance behavior (Bohus and De Wied, 1966; Greven and De Wied, 1967; De Wied et al., 1975; Wiegant et al., 1978; Fekete and De Wied, 1982a; Fekete et al., 1982b). A C T H competitively displaces radiolabelled dihydromorphine (Terenius, 1975; Terenius et al., 1975; Wiegant et al., 1977) or fl-endorphin (Akil et al., 1980) from opiate receptor sites in the brain and therefore these receptors may represent a site of action of A C T H in the central nervous system. However, it has been suggested that opiate effects are mediated by two receptors: a stereospecific opiate fl-endorphin receptor which mediates the analgesic and cataleptic effects and a non-stereospecific opiate A C T H receptor which mediates the opiate abstinence syndrome and the excitatory el-
fects of the opiates (Jacquet, 1978). A C T H antagonizes morphine inhibition of spinal reflexes (Krivoy et al., 1974; Zimmermann and Krivoy, 1973, 1974), morphine-induced hyperactivity and Straub-tail (Katz, 1979) and counteracts morphine-induced analgesia (Gispen et al., 1976; Wiegant et al., 1977). These results are consistent with the hypothesis that ACTH and related peptides are partial agonist/antagonists of opiate receptors. ACTH-(4-10) has a variety of behavioral effects (De Wied, 1977; Gispen et al., 1977). Most of these effects are probably not related to opiate receptor function (De Wied et al., 1978; Gispen et al., 1977). Although the different parts of the A C T H molecule show similar activities in the pole-jumping or passive avoidance tests, their effect is not identical with respect to other CNS activities, e.g. they differ in affinity for opiate receptor sites (Terenius et al., 1975) and their ability to induce excessive grooming (Gispen et al., 1975; Gispen and Isaacson, 1981). Opiate antagonists (naloxone, naltrexone) suppress ACTH-(124)-induced excessive grooming in rats (Gispen and Wiegant, 1976), but not the effects of ACTHlike neuropeptides on extinction of active avoidance behavior (De Wied et al., 1978). Similarly, the present experiments show that the facilitating effect of O R G 2766 and the N H 2 terminal tetrapeptide of O R G 2766 on passive avoidance behavior is not blocked by the opiate antagonist naltrexone. However, the inhibitory effect of 'high' doses of O R G 2766 on passive avoidance behavior is inhibited in naltrexone-pretreated animals and is even reversed to facilitation. The COOH terminal tripeptide (PDLP) is the main metabolic product of O R G 2766 (Witter et al., 1975; Verhoef and Witter, 1976). This tripeptide is much less active than O R G 2766, but still contains approximately 10% of the activity of ACTH-(4-10) on extinction of pole-jumping active avoidance behavior (De Wied et al., 1975). However, passive avoidance behavior was dose dependently attenuated after intracerebroventricular injection of PDLP. This attenuating effect of the tripeptide was as potent as that of O R G 2766 and was also blocked by pretreatment with naltrexone. PDLP in low doses did not facilitate passive avoidance behavior (Naltrexone pretreatment blocked
447
the attenuating effect but did not induce facilitation as found with O R G 2766). The fact that only 'high' doses of O R G 2766 attenuated passive avoidance behavior suggests that such doses generate sufficient amounts of PDLP from O R G 2766 in the brain to inhibit passive avoidance behavior. Taken together, the results of the present experiments suggest that O R G 2766 has a dual effect on passive avoidance behavior. Facilitation of passive avoidance behavior by 'low' doses of O R G 2766 may be an intrinsic activity of the peptide exerted via a naltrexone-insensitive mechanism as was also found in pole-jumping active avoidance behavior (De Wied et al., 1978). This effect of O R G 2766 resides in the N H 2 terminal part of the molecule. The suppression of the passive avoidance response by 'high' doses of O R G 2766 is possibly mediated by naltrexone-sensitive opiate receptors and this inhibitory influence is located in the COOH terminal part of the peptide. This inhibitory effect of high dose of O R G 2766 or of the tripeptide, PDLP, on retention of passive avoidance behavior may be explained in different ways. For example by the affinity of these peptides for naltrexone-sensitive opiate receptors. O R G 2766 does not displace dihydromorphine (Terenius et al., 1975) or naloxone (Walker et al., 1981) in vitro. However, Plomp and Van Ree (1978) demonstrated opiate-like effects of N H 2 terminal ACTH-fragments as measured on electrically evoked contractions of the mouse vas deferens and the displacement of radiolabelled morphine from morphine antiserum. Structure-activity studies in both assay systems showed that the active core resided in the sequence ACTH-(7-10). It remains possible therefore that PDLP has an affinity for opiate receptor sites. Other explanations for the attenuating effect of PDLP on passive avoidance behavior are, a change in the sensitivity of naltrexone-sensitive receptors, the release of neuropeptide(s) with amnestic properties, the release of opiate-like peptides with an inhibitory effect on passive avoidance behavior (Kovacs and De Wied, 1981; Beluzzi and Stein, 1981; Messing et al., 1981; Garzon et al., 1981) or inhibition of enzymes which degrade opiate-like peptides in the brain. Further experiments are needed to unravel the question of the attenuating influence of the tripeptide PDLP on passive avoidance behavior.
Acknowledgements The peptides were donated by Dr. H.M. Greven of Organon International B.V., Oss, The Netherlands. Endo Laboratories provided us with naltrexone. This study was supported in part by Stichting Pharmacologisch Studiefonds.
References Ader, R., J.A.W.M. Weijnen and P. Moleman, 1972, Retention of a passive avoidance response as a function of the intensity and duration of electric shock, Psychon. Sci. 26, 125. Akil, H., W. Hewlett, J.D. Barchas and C.H. Li, 1980, Binding of 3H-fl-endorphin to rat brain membranes: Characterization and opiate properties, European J. Pharmacol. 64, 1. Beluzzi, J.D. and L. Stein, 1981, Facilitation of long-term memory by brain endorphins, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr., R.A. Jensen, R.B. Messing, H. Rigter and J.L. McGaugh (Academic Press, New YOrk) p. 291. Bohus, B. and D. De Wied, 1966, Inhibitory and facilitatory effect of two related peptides on extinction of avoidance behavior, Science 153, 318. De Wied, D., 1976, Behavioral effects of intraventricularly administered vasopressin and vasopressin fragments, Life Sci. 19, 685. De Wied, D., 1977, Peptides and behavior, Life Sci. 20, 195. De Wied, D., B. Bohus, J.M. Van Ree and I. Urban, 1978, Behavioral and electrophysiological effects of peptides related to lipotropin (fl-LPH), J. Pharmacol. Exp. Ther. 204, 570. De Wied, D., A. Witter and H.M. Greven, 1975, Behaviourally active ACTH analogues, Biochem. Pharmacol. 24, 1463. Fekete, M. and D. De Wied, 1982a, Potency and duration of action of the ACTH 4-9 analog (ORG 2766) as compared to ACTH 4-10 and [D-PheT]ACTH 4-10 on active and passive avoidance behavior of rats, Pharmac. Biochem. Behav. 16, 387. Fekete, M. and D. De Wied, 1982b, Dose-related facilitation and inhibition of passive avoidance behavior by the ACTH(4-9) analog (ORG 2766), Pharmac. Biochem. Behav. (in press). Garzon, J., J. Rubio and J. Del Rio, 1981, Naloxone blocks the effect of 5-hydroxy-tryptophan on passive avoidance learning in rats: implication of endogenous opioid peptides, Life Sci. 29, 17. Gispen, W.H., J. Buitelaar, V.M. Wiegant, L. Terenius and D. De Wied, 1976, Interaction between ACTH fragments, brain opiate receptors and morphine-induced analgesia, European J. Pharmacol. 39, 393. Gispen, W.H. and R.L. Isaacson, 1981, ACTH-induced excessive grooming in the rat, Pharmacol. Ther. 12, 209. Gispen, W.H., J.M. Van Ree and D. De Wied, 1977, Lipotropin and the central nervous system, Int. Rev. Neurobiol. 20, 209.
448
Gispen, W.H. and V.M. Wiegant, 1976, Opiate antagonists suppress ACTHi.24-induced excessive grooming in the rat, 1976, Neurosci. Lett. 2, 159. Gispen, W.H., V.M. Wiegant, H.M. Greven and D. De Wied, 1975, The induction of excessive grooming in the rat by intraventricular application of peptides derived from ACTH: Structure-activity studies, Life Sci. 17, 645. Greven, H.M. and D. De Wied, 1976, The active sequence in the ACTH molecule responsible for inhibition of the extinction of conditioned avoidance behavior in rats, European J. Pharmacol. 2, 14. Greven, H.M. and D. De Wied, 1973, The influence of peptides derived from corticotrophin (ACTH) on performance. Structure activity studies, in: Drug Effects on Neuroendocrine Regulation, eds. E. Zimmermann, W.H. Gispen, B.H. Marks and D. De Wied (Elsevier, Amsterdam), Progress in Brain Research, Vol. 39, p. 429. Jacquet, Y.F., 1978, Opiate effects after adrenocorticotropin or fl-endorphin injection in the periaqueductal gray matter of rats, Science 201, 1032. Katz, R.J., 1979, ACTH4.10 antagonism of morphine-induced behavioural activation in the mouse, European J. Pharmacol. 53, 383. Kovacs, G.L. and D. De Wied, 1981, Endorphin influences on learning and memory, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martines, Jr., R.A. Jensen, R.B. Messing, H. Rigter and J.L. McGaugh (Academic Press, New York) p. 231. Krivoy, W.A., D. Kroeger, A.N. Taylor and E. Zimmermann, 1974, Antagonism of morphine by fl-melanocyte stimulating hormone and by tetracosactin, Europrean J. Pharmacol. 27, 339. Messing, R.B., R.A. Jensen, B.J. Vasquez, J.L. Martinez, Jr., V.R. Spiehler and J.L. McGaugh, 1981, Opiate modulation of memory, in: Endogenous Peptides and Learning and Memory Processes, eds. J.L. Martinez, Jr., R.A. Jensen, R.B. Messing, H. Rigter and J.L. McGaugh (Academic Press, New York) p. 431.
Plomp, G.J.J. and J.M. Van Ree, 1978, Adrenocorticotrophic hormone fragments mimic the effect of morphine in vitro, Br. J. Pharmacol. 64, 223. Terenius, L., 1975, Effects of peptides and amino acids on dihydromorphine binding to the opiate receptor, J. Pharm. Pharmacol. 27, 450. Terenius, L., W.H. Gispen and D. De Wied, 1975, ACTH-Iike peptides and opiate receptors in the brain: Structure activity studies, European J. Pharmacol. 33, 395. Verhoef, J. and A. Witter, 1976, In vivo fate of a behaviorally active ACTH 4-9 analog in rats after systemic administration, Pharmacol. Biochem. Behav. 4, 583. Walker, J.M., G.G. Berntson, C.A. Sandman, A.J. Kastin and H. Akil, 1981, Induction of analgesia by central administration of ORG 2766, an analog of ACTH4. 9, European J. Pharmacol. 69, 71. Wiegant, V.M., D. Colbern, Tj.B. Van Wimersma Greidanus and W.H. Gispen, 1978, Differential behavioral effect of ACTH 4-10 and (D-PheT)ACTH 4-10, Brain Res. Bull. 3, 167. Wiegant, V.M., W.H. Gispen, L. Terenius and D. De Wied, 1977, ACTH-like peptides and morphine: Interaction at the level of the CNS, Psychoneuroendocrinology 2, 63. Witter, A., H.M. Greven and D. De Wied, 1975, Correlation between structure, behavioral activity and rate of biotransformation of some ACTH 4-9 analogs, J. Pharmacol. Exp. Ther. 193, 853. Zimmermann, E. and W.A. Krivoy, 1973, Antagonism between morphine and the polypeptides ACTH, ACTHI.24 and flMSH in the nervous system, in: Drug Effects on Neuroendocrine Regulation, Progr. Brain Res. Vol. 39, eds. E. Zimmermann, W.H. Gispen, B.H. Marks and D. De Wied (Elsevier, Amsterdam) p. 383. Zimmermann, E. and W.A. Krivoy, 1974, Depression of frog isolated spinal cord by morphine and antagonism by tetracosactin, Proc. Soc. Exp, Biol. 146, 575.