European Journal of Pharmacolo,~,, 229 (1992) 211-216
211
Cc~1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.011
EJP 52813
ACTH: a structure-activity study on pilocarpine-induced epilepsy G e r d a Croiset a n d David D e W i e d Rudolf Magnus Institute, Department of Pharmacology, Medical Faculty, Unicersi~ of Utrecht, Vondeflaan 6, Utrecht, Netherlands Received 27 February 1992, revised MS received 8 September 1992, accepted 22 September 1992
Intracerebroventricularly applied pilocarpine (2.4 mg/2 gl) immediately produced symptoms of epilepsy, ranging from akinesia to motor seizures, in rats. Whereas ACTH-(1-39), ACTH-(I-24), ACTH-(1-18), ACTH-(1-16) and ACTH-(18-39) were not active, subcutaneous prctreatment with smaller ACTH-Iike fragments, such as ACTH-(4-9), ACTH-(4-10), ACTH-(410X7D-Phe), ACTH-(7-16), and Org2766, reduced the severity of the epilepsy. Moreover, fewer rats developed motor seizures. Thus, ACTH fragments devoid of peripheral endocrine activity reduce pilocarpine-induced epileptiform activity in rats. A narrow bell-shaped dose-response relationship was found. Except for ACTH-(7-16), which was active in a dose of 1 and 10 p~g/rat s.c., the other fragments were only active at one dose (10 p,g/rat). The anti-epileptic properties appeared to reside in the sequence 1-16, and more specifically in the sequences 4-7 and 7-16, of the ACTH molecule. Seizures (chemically induced); Pilocarpine; Epilepsy; Anticonvulsants; ACTH (adrenocorticotrophic hormone)
1. Introduction
Besides classical anti-epileptic drugs, neuropeptides also exert anti-epileptic effects in some epileptogenic encephalopathies. A C T H is currently used to treat infantile spasms (West syndrome), an advantage being the higher incidence of spasm-free states compared to other treatments (Klein, 1970). However, an adverse effect is an ACTH-(1-24)-induced Cushing-like phenomenon. In a limited number of animal studies, fragments of ACTH devoid of peripheral endocrine activity were found to possess anti-epileptic properties. Org2766, an ACTH-(4-9) analogue, effectively reduced the course and severity of convulsive seizures in amygdaloid-kindled rats (Goldman et al., 1987), and a dorsal hippocampal kindling-induced epileptic syndrome was attenuated by ACTH-like neuropeptides (Cottrell et al., 1983). The N-terminal portion appeared to possess anti-epileptic properties: A C T H - ( 1 16), ACTH-(7-16) and ACTH-(7-16)(7D-Phe) inhibited after-discharges, whereas ACTH-(1-16), ACTH(4-16) and ACTH-(4-10)(7D-Phe) effectively inhibited behavioral depression. In a previous study we found that ACTH-(4-10) and ACTH-(7-16) have beneficial effects on pilocarpine-induced seizures (Wamil et al.,
Correspondence to: G. Croiset, Rudolf Magnus Institute, Department of Pharmacology, Medical Faculty, University of Utrecht, Vondellaan 6, Utrecht, Netherlands.
1989). In this study we investigated the moiety of the A C T H molecule possessing anti-epileptic properties in the pilocarpine (a muscarinic acetylcholine receptor agonist) model of epilepsy.
2. Materials and methods
2.1. Animals Male Wistar rats of an inbred stain (CPB-TNO, Zeist, The Netherlands) weighing 200-250 g were used. They were kept under standard conditions (room temperature 22 + 1 ° C, lights on from 6:00 a.m. until 7:00 p.m.), housed individually and received pellet food and water ad libitum. The experiments were performed between 10:00 a.m. and 5:00 p.m.
2.2. Peptides, drugs and injection procedures ACTH, its fragments and Org2766 were gifts from Organon Int. B.V. (Oss, The Netherlands). Peptides were freshly dissolved in 0.9% saline. Systemic injections were given s.c. in a volume of 0.5 ml 1 h prior to pilocarpine administration. The amino acid sequence of ACTH-(1-39) is H-Ser-Tyr-Ser-Met-Glu-His-PheArg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-ProVal-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-SerA l a - G l u - A l a - P h e - P r o - L e u - G l u - P h e - O H , that of Org2766 (ACTH-(4-9) analogue) is H-Met-(O2)-Glu-
H i s - P h e - L y s - P h e - O H a n d that of 7 2 - M S H is H-TyrVal-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-GIy-OH. P i l o c a r p i n e h y d r o c h l o r i d e (Sigma, St. Louis, M O , U S A ) was dissolved in 0.9% saline and injected i.c.v. (2.4 r a g / 2 /,tl p e r rat). F o r i.c.v, injections, rats were e q u i p p e d with a p o l y e t h y l e n e c a n n u l a in the lateral c e r e b r a l ventricle, the c a n n u l a b e i n g fixed to the skull with d e n t a l c e m e n t ( D e W i e d , 1976), Rats were used for the e x p e r i m e n t 1 w e e k after surgery. Injections (2 /,1) were given with a H a m i l t o n syringe. T r e a t m e n t s were given blind.
status epilepticus, stage 5: d e a t h . T h e behavioral stage was d e t e r m i n e d every, min and scored as follows: stage 1, 1; stage 2, 2; etc. T h e m a x i m u m score was 75. T h e n u m b e r of rats that d e v e l o p e d g e n e r a l i z e d convulsions d u r i n g the o b s e r v a t i o n p e r i o d is e x p r e s s e d as a perc e n t a g e of the total n u m b e r of rats.
2.4. N u m b e r o[-"animals A C T H - ( 4 - 1 0 ) : control n - 2 7 , 1 # g n = 8, 10 /ag n = 10, 100 # g n = 9; A C T H - ( 7 - 1 6 ) : control n 21, 1 /xg n = 6, 10 /xg n = 9, 100 /.tg n = 6: Org2766: control n-37, 1 p,g n = 9 , 3 p,g n = 9 , 10 / , g n 9, 30 /,tg n = 10; A C T H - ( 1 - 1 6 ) : control n = 19, 0.1 # g n = 4 , 1 fig n = 4, 10 # g n - 11: A C T H - ( 1 - 3 9 ) : control n = 16, 10 # g n = 7: A C T H - ( I - 2 4 ) : control n - 1 1 , 10 /.~g n=ll; A C T H - ( I - 1 8 ) : control n = 8 , 10 fig n = 8 ; A C T H - ( 1 8 - 3 9 ) : control n - 10, 10 # g n = 13: A C T H (11-24): control n - 8, 10 / , g n = 1(1; A C T H - ( 1 1 - 1 6 ) : c o n t r o l n = 10, 10/xg n = 10; T 2 - M S H : control n = 10, 10 # g n = 1(1; A C T H - ( 4 - 1 0 ) ( 7 D - P h e ) : control n = 13, 10 # g n - 13; A C T H - ( 4 - 9 ) : control n = 8, 1 0 / , g n = 8;
2.3. Behavioral measurements I m m e d i a t e l y a f t e r p i l o c a r p i n e a d m i n i s t r a t i o n , the rat was p l a c e d in a t r a n s p a r e n t box (30 × 30 cm) a n d its b e h a v i o u r was o b s e r v e d for 15 rain. Five successive b e h a v i o r a l stages w e r e d i s t i n g u i s h e d ( m o d i f i e d from R a c i n e (1972)). Stage 1: akinesia, staring and salivation; stage 2: h e a d clonus, f o r e l i m b clonus a n d w e t - d o g shakes; stage 3: r e a r i n g with f o r e l i m b clonus; stage 4: r e a r i n g a n d falling with g e n e r a l i z e d convulsions and
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Fig. 1. D o s e - r e s p o n s e effect of A C T H - ( 4 - t 0 ) , Org2766, A C T H - ( I 16) and A C T H - ( 7 - 1 6 ) on epilepsy. On the left side the total score ( + S.E.M.) is depicted and on the right side tile '7~- of rats that developed convulsions.
213
ACTH-(4-7): control n = 8, 10 /xg n = 8; Phe-D-LysPhe: control n = 11, 10 p~g n = 11.
2.5. Statistics The Fisher test (as a form of the Chi-square analysis) was used for statistical analysis of the effect of peptide treatment on the number of rats that developed convulsions after pilocarpine administration. The Mann-Whitney U test was used for statistical analysis of the effect of peptide treatment on the total epilepsy score during the observation period. P ~< 0.05 was taken as the level of significance. Results are expressed as means _+ S.E.M.
3. Results The effect of i.c.v, pilocarpine was direct. Two minutes after its i.c.v, administration the rats became motionless (stage 1). During the following minute head clonus and forelimb clonus were observed (stage 21, and within 4 rain the majority of the rats had developed generalized convulsions (stage 4). The occurrence of seizures gradually decreased 15 rain after the injection. No difference was found in the time of onset of convulsions between the groups of rats.
3.1. Dose-response study (fig. 11 In a first series of experiments ACTH-(1-16), ACTH-(4-10), A C T H - ( 7 - 1 6 ) and ORG2766 were tested in several doses. These peptides attenuated the effect of pilocarpine. However, either a narrow or no dose-response relationship was found, but the fragments were active at a dose of 10/xg. Only A C T H - ( 7 161 was effective at a dose of 1 p.g (total score 1 p.g: U = 2 1 (21,6), P = 0 . 0 1 ; 10 /xg: U = 3 3 . 5 (21,9) P = 0.005). A significant decrease in the total epilepsy score and in the number of animals that developed general convulsions (stage 4) was observed after pretreatment with a dose of 10 # g ACTH-(4-10) or Org2766 (total score: Org2766: U = 28 (37,91, P = 0.0001; ACTH-(4-10): U = 46 (27,10), P = 0.002). A significant reduction in the total epilepsy score was also observed after 10 /xg ACTH-(1-16) (U = 40 (19,111, P = 0.02). A dose of 10 /xg was chosen for further experiments. No difference was found in the time of onset of convulsions.
3.2. Effect of large A C T H fragments on pilocarpine-induced epilepsy (fig. 1 and table 1) ACTH-(1-391, ACTH-(1-241, A C T H - ( I - 1 8 ) and ACTH-(18-39) did not affect the number of animals
TABLE 1 Effect of A C T H f r a g m e n t s on p i l o c a r p i n e - i n d u c e d epilepsy. Total score ACTH-(1-39) Control 42 ± 3 10 ,ag 46 + 2 A C T I t - ( 1 24) Control 33 _+ 6 10/xg 32+6 A C T H - ( 1- 18) Control 39 ± 5 10 ,ag 33+5 ACTH-(18-39) Control 42 _+4 10 # g 42+_4 ACTH-(11-24) Control 49 + 1 10 ,ag 27+4 * A C T H - ( I 1 161 Control 40 +_3 10 # g 37 + 4 y2-MSlt Control 47 + 4 10/xg 49 ± 3 ACTH-(4-10)(7D-Phe) Control 51 + 2 10 g g 37+_4 * A C T H - ( 4 9) Control 47 + 3 1(I/xg 29 +_ 7 ACTH-(4-7) Control 411 ± 4 10 # g 26+6 Phe-D-Lys-Phe Control 45 + 1 10/xg 44+3 A C T | t-(6-9) Control 51 + 1 10 # g 44 +_ 5 ACTH-(7-10) Control 39 + 3 10 # g 33+5
Onset (rain)
% convulsions
3.5 ± (/.5 3.8 + 0.4
75 100
3.0 ± (1.6 3.2+_0.8
54 73
6.2 ± 1.8 5 . 5 + 1.0
75 5(1
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80 100
5.5 + 0.3 6 . 2 + 1.6
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9(} 90
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100 69
4.2 ± (1.7 5 . 0 + 1.8
100 5(}
5.6 + 1.4 5 . 3 + 1.8
87 37
4.6 +_0.6 4.4+0.7
100 91
2.8 + 0.3 3.8 ± ().5
100 78
4.7 ± 0.7 3.4+0.9
83 58
that developed tonic/clonic seizures, nor the total epilepsy score. ACTH-(11-24) reduced both measures (total score: U = 8.5 (8,10), P = 0.02). No difference was found in the time of onset of convulsions.
3.3. Effect of intermediate ACTH fragments on pilocarp±he-induced epilepsy (fig. 1 and table 11 As mentioned above, ACTH-(1-16) and A C T H - ( 7 161 attenuated the severity of the epilepsy, but did not decrease the number of animals that developed t o n i c / clonic seizures. Other fragments, such as A C T H - ( l l 16) and y2-MSH, did not affect the severity of the epilepsy. No difference was found in the time of onset of convulsions.
214
3.4. Effect of small A C T H fragments on pilocarpine-mduced epilepsy (fig. 1 and table 1) Treatment with A C T H - ( 4 - 1 0 ) or ACTH-(4-10)(7DPhe) significantly reduced the total epilepsy score (U = 24 (13,13), P = 0.001). The smaller fragments A C T H (4-9) and A C T H - ( 4 - 7 ) tended to reduce the severity of the epilepsy. A significant reduction in the number of rats with t o n i c / c l o n i c convulsions was found after treatment with A C T H - ( 4 - 1 0 ) or A C T H - ( 4 - 9 ) , and a modest but not significant reduction after treatment with A C T H - ( 4 - 7 ) . Org2766 effectively reduced both the severity and the number of animals with t o n i c / c l o n i c convulsions. The Org2766 C-terminal fragment Phe-D-Lys-Phe was not active in this respect. No difference was found in the time of onset of convulsions.
4. Discussion
Torda and Wolff (1952), who used pentymethylenetetrazol, were the first to find anticonvulsive effects of chronic treatment with a crude A C T H preparation. In the present study we used a muscarinic acetylcholine receptor agonist (pilocarpine) to produce epileptiform seizures. Pilocarpine injection into the lateral ventricle results in immediate epileptiform activity (Turski et al., 1989; Wamil et at., 1989). Pretreatment with A C T H neuropeptides protected rats from pilocarpine-induced seizures, and either no or a narrow bell-shaped doseresponse relationship was found, There is no explanation yet for this phenomenon, although it has been reported in several studies (De Wied et al., 1987; Wolterink et al., 1991), The anti-epileptic activity appears to reside mainly in fragments of the A C T H molecule; neither the whole molecule (1-39), nor large fragments such as (1-24) and (18-39) were active. The anti-epileptic activity was found predominantly in the N-terminal fragments of ACTH. Sequences as small as five amino acids, such as A C T H - ( 4 - 9 ) and its analogue Org2766, appeared to possess anti-epileptic activity. However, Org2766, which is a thousandfold more active on active and passive avoidance behaviour (De Wied and Jolles, 1982), was not more potent than A C T H - ( 4 - 1 0 ) in protecting against pilocarpine-induced epilepsy. The active moiety resides in the (4-7) sequence, the (7-10) sequence not being effective. In this active sequence the fifth amino acid, Glu, is probably of more importance than the seventh amino acid, Phe, as the structurally related neuropeptide, y2-melanocyte-stimulating h o r m o n e (y2-MSH), which contains an A C T H - ( 4 - 9 ) sequence with Gly instead of Glu in position 5, did not have anti-epileptic effects. The D-enantiomer A C T H - ( 4 10)(7D-Phe) appeared to be almost as effective as
ACTH-(4-10). Interestingly, the D-enantiomer has an effect opposite to that of ACTH-(4-10)(7L-Phe) on the extinction of active and passive avoidance behaviour (De Wied and Jolles, 1982). Another sequence, A C T H - ( 7 - 1 6 ) also protected against pilocarpine-induced epilepsy. The entire 10amino acid sequence is necessary for activity as neither (7-10) nor (11-16) had anti-epileptic activity. Cottrell et al. (1983) found that A C T H - ( 1 - 1 6 ) and A C T H - ( 7 16) were equally effective on after-discharges elicited by hippocampal kindling. They also found the D-enantiomer of A C T H - ( 7 - 1 6 ) was more active in that it reduced after-discharges as well as behavioral depression. In this study, A C T H - ( I - 1 6 ) , which contains two anti-epileptic sites ((4-7) and (7-16)), had only minor protective effects. This may point to the importance of the three-dimensional structure of these two fragments. It may well be that when these two sites are combined, as in the (1-16) fragment, the three-dimensional effective structures of both fragments are concealed. These fragments might be generated from A C T H - ( 1 - 1 6 ) in the brain, as brain synaptosomes havc been shown to convert the (1-16) fragment to A C T H (7-16) after incubation with brain peptidases (Burbach, 1989). A behaviorally potent analogue of ACTH-(4-9), Org2766, also possesses anti-epileptic properties. In other models of epilepsy, A C T H is beneficial in reducing the course and duration of seizures whereas Org2766 markedly reduces seizures after amygdaloid kindling (Goldman et al., 1987) but is not very, active in hippocampal kindling (Cottrell et a[., 1983). Thus the site in the brain where the syndrome is generated also determines the sensitivity to peptides. Apparently, the attenuating effect of a compound, in this case an ACTH-Iike neuropeptide, on kindling cannot be generalized to related substances. Pilocarpine is a cholinomimetic agent. Thus the cholinergic system and structures innervated by this system may be the site of action of A C T H fragments. It is also possible that ACTH-Iike fragments exert their effects in the pathways which ultimately result in motor seizures. Cholinomimetics (Olney et al., 1983; Turski, et al., 1984), kainic acid (Bcn-Ari ct al., 1980), folates (Olney et al., 1981) and G A B A antagonists (Turkski et al., 1984) produce very similar brain abnormalities. Although initiation of abnormal elcctrographic activity within limbic circuits is linked to divergent receptor mechanisms, it seems evident that epileptic brain damage in experimental animals involves the same mechanism, and that one neurotransmitter system cannot be considered pivotal in the genesis of seizures and their pathological sequelae (Turski et al., 1989). Many areas in the central nervous system are involved in epilepsy. Basal ganglia, such as the substantia nigra and the
215 globus pallidus, serve as relay stations in the propagation of seizures elicited from the motor cortex or the limbic system. Excitation in the substantia nigra is probably mediated by L-glutamate or L-aspartate. Microinjection of a N M D A receptor antagonist and of the G A B A agonist muscimol into the substantia nigra or entopeduncular nucleus protects against the convulsant action of pilocarpine (Hosford and McNamara, 1988; Patel et al., 1988; Turski et al., 1983; 1986). Dopamine is also a neurotransmitter involved in pilocarpine-induced epilepsy (Turski et al., 1988). /3-MSH given i.v. facilitates ventral root responses after dorsal root stimulation in the cat, and antagonizes the depressive action of chlorpromazine on dorsal root potentials. Thus /3-MSH could influence dopaminergic, cholinergic and histaminergic transmission (Krivoy, 1970). A C T H neuropeptides are reported to affect excitatory and inhibitory neurotransmission. Calvet et al. (1992) studied the effect of an A C T H - ( 4 - 1 0 ) analogue (BIM 22015) (D-Ala-Gln-Tyr-Phe-Arg-Trp-Gly-NH 2) on the neuronal firing pattern of rat spinal cord cultures. The BIM peptide decreased the spontaneous firing rate after a single dose. These changes were enhanced in a low Ca 2+ medium. Chronic exposure to the peptide increased phasic bursting patterns, resembling strychnine-induced effects. These findings suggest that these A C T H - r e l a t e d peptides induce changes in the m e m b r a n e properties of spinal cord neurons to either decrease (acute) or increase (chronic) the frequency of action potentials. As suggested by the authors, BIM 22015 may inhibit synaptic transmission by reducing presynaptic calcium entry and impairing excitatory transmitter release. The effect of chronic treatment was interpreted as an attenuating effect on transmitter release at inhibitory presynaptic terminals. The anticonvulsive effects of A C T H neuropeptides may also be explained by an attenuating influence on excitatory neurotransmission. A C T H - ( 1 - 2 4 ) and A C T H - ( 4 - 1 0 ) reduce the binding of [3H]glutamate, although with rather low affinity (Ito et al., 1988). Recently, Trifiletti and Pranzatelli (1992) found that micromolar concentrations of ACTH-(1-39), ACTH-(1-24), and A C T H (1-17) displaced the binding of [3H]MK 801 in rat hippocampus homogenates; however, A C T H - ( 4 - 1 0 ) was not active. The affinity was of the same order of magnitude as that found for A C T H fragments at other receptor sites. Cholinergic agents applied in the vicinity of CA~ or CA 3 pyramidal neurons enhance firing induced by synaptic activation by acting presynaptically to decrease GABAergic inhibition and by acting postsynaptically to decrease K + conductance (Meldrum, 1985). A C T H - ( 4 - 1 0 ) may interfere with one or more of the above-mentioned processes, as A C T H fragments bind to muscarinic receptors (Tonnaer et al., 1986) and A C T H - ( 1 - 2 4 ) has been shown to inhibit the in vitro
binding of the muscarinic antagonist [~H]quinuclidinylbenzilate (QNB) in the rat. However, A C T H - ( 4 10) is not active in this respect. A C T H and related peptides have been shown to affect cholinergic transmission. Geiger et al. (1987) reported that A C T H fragments decreased acetylcholine concentrations in the rat striatum, with Org2766 being a thousandfold more active than A C T H - ( 4 - 9 ) . Since these two fragments were equally active in protecting against pilocarpine-induced epilepsy, it does not seem likely that their primary effect is on the cholinergic system. It has been hypothesized that endogenous opioids may play a role in seizure activity (Cain and Corcoran, 1985). /3-Endorphin as well as Met-enkephalin facilitates electrical kindling of the amygdala. /3-Endorphin itself can produce after-discharge activity in limbic structures, an effect which can be reversed by naloxone (Henriksen et al., 1978). It is possible that the anticonvulsive effect of A C T H is mediated through the opioid system. A C T H - ( 4 - 1 0 ) and A C T H - ( 7 - 1 6 ) have been shown to inhibit [3H]dihydromorphine binding to synaptic plasma m e m b r a n e fractions of rat brain (Terenius et al., 1975) and to counteract the antinociceptive action of morphine (Gispen et al., 1976). The finding that high doses of Org2766 in contrast to low doses have a naloxone-reversible effect on avoidance behaviour is not in favour of an ACTH-like opiate antagonistic effect on epilepsy as opiate activity appears to be located in the (7-10) sequence (Fekete and de Wied, 1982), a sequence which was inactive in the present experiment. Nevertheless, in other studies the involvement of the opioid system in seizure activity has been demonstrated. Cottrell et al. (1983)showed that y 2 - M S H can reduce the seizures induced by hippocampal kindling, but is ineffective in pilocarpine-induced epilepsy. It is not possible to determine the mechanism of action of A C T H neuropeptides on pilocarpine-induced epilepsy from the data in the literature and the present results. Studies on the influence of neuropeptides on glutamatergic (GABAergic) or on dopamine transmission may be of importance in revealing the underlying mechanism of action of these neuropeptides.
Acknowledgement The authors wisk to thank Leo van Halewijn for his excellent technical assistance.
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Klein, R., 197/), Effects of A C T H and corticosteroids on epileptifl~rm disorders, Progr. Brain Res. 32, 263. Krivoy, W.A., 1970, Effects of A C T H and related polypeptides on spinal cord, Progr. Brain Res. 32, 108. Meldrum, B., 1985, Anticonvulsants and the Limbic System, Psychopharmacology of the Limbic System, ed. Trimble (()xfl~rd University Press) p. 78. Olney, J.W., T.A. Fuller and T. DeGubareff, 1981, Kainate-likc neurotoxicity of folates, Nature 292, 165. Olney, ,I.W., T. DeGabureff and J. Labruyere, 1983, Seizure-relatcd brain damage induced by cholinergic agents, Nature 301,520. Putel, S., 1988, Pathways involved in the spread of seizure activity and the effects of excitato~' aminoacid antagonists, in: Frontiers in Excitatory Aminoacid Research, eds. E.A. Cavalheiro, J. L e h m a n n and L. Turski (Alan R. Liss, New York) p. 255. Racine, R.J., 1972, Modification of seizure activity by electrical stimulation: II Motor seizure, Electroencephalogr. Clin. Neuro physiol. 32, 281. Terenius, 1.... W.H. Gispen and I5). De Wied, 1975, A(q'tl-like peptides and opiate receptors in the rat brain: strncture-activily studies, Eur. J. Pharmacol. 33, 395. Tonnaer, J.A.D.M.. M. Van Vugt and S.J. De Graaf, 1986, In vitro interaction of A C T H with ral brain muscarinic receptors, Peptides 7, 425. Torda, C. and I I.G. Wolff. 1952, Effects of various concentrations of adrenocorticotrophic hormone on electrical activity of brain and on sensitivity to convulsion-inducing agent, Am. J. Physiol. 168, 4116. Trifi]etti. R.R. and M.R, Pranzatelli, 1992. A ( ' T ] I binds to [~H]MKS()I labelled rat hippocampal N M D A receptors, Eur. J. Pharmacol. 226, 377. Turski, W.A., E.A. Cavalheiro, M. Schwarz, S.J. ('zuczwar, Z. Klein rok and L. Tnrski, 1983. Limnbic seizures produced by pilocarpinc in rats: behavioral, electronencephalographic and neuropathological study, Behav. Brain Res. 9, 315. Turksi, W.A.. E.A. Cava]heiro, Z.A. Borlololto, L.M. Mello, M. Schwarz and L. Turski, 1984. Seizures produced by pilocarpinc in mice: a behavioral, electrocncephalographic and morphological analysis, Brain Res. 321, 237. Turksi, L., E.A. Cawdheiro and W.A. Turski, 19~6, ExcitatoLw neurotransmission v, ithin substantia nigra pars reticulata regulates threshold for seizures produced by pilocarpine in rats: Effects of intranigral 2-amino-7-phosphonoheptanoatc and N-methyl-l) aspartate, Neuroscience IB, 61. Turski, L., E.A. Cavalheiro, Z.A. Bortolotto. C. Ikonomidou-Turski, Z. Kleinrok and W.A. Turski, 1988, Dopamine-sensitivc anticon vulsam site in tile rat striatum, J. Neurosci. 8, 3837. Turski, 1,., C. Ikonomidou, W.A. Turski, Z.A. Bortolott and E.A. Cawdheiro, 1980, Review: cholinergic mechanisms and cpileptogenesis, the seizures induced by pilocarpine: A novel experimental model of intractable epilepsy, Synapse 3, 154. Wamil, A., G. Croiset. Z. Kleinrok and D. de Wied, 1980, Beneficial effects of ACTtt-(4 101 on pilocarpine induced seizures, Neurosci. Res. Commun. 4(21, 109. Wolterink, G., J.M. Van Ree, J.W. Van Nispen and D. l)e Wicd, 1991, Structural modifications of the ACTtt-(4 9) analog Org2766 yields peptides with high biological activity. Life Sci. 48, 155.
211
Cc~1992 Elsevier Science Publishers B.V. All rights reserved 0014-2999/92/$05.011
EJP 52813
ACTH: a structure-activity study on pilocarpine-induced epilepsy G e r d a Croiset a n d David D e W i e d Rudolf Magnus Institute, Department of Pharmacology, Medical Faculty, Unicersi~ of Utrecht, Vondeflaan 6, Utrecht, Netherlands Received 27 February 1992, revised MS received 8 September 1992, accepted 22 September 1992
Intracerebroventricularly applied pilocarpine (2.4 mg/2 gl) immediately produced symptoms of epilepsy, ranging from akinesia to motor seizures, in rats. Whereas ACTH-(1-39), ACTH-(I-24), ACTH-(1-18), ACTH-(1-16) and ACTH-(18-39) were not active, subcutaneous prctreatment with smaller ACTH-Iike fragments, such as ACTH-(4-9), ACTH-(4-10), ACTH-(410X7D-Phe), ACTH-(7-16), and Org2766, reduced the severity of the epilepsy. Moreover, fewer rats developed motor seizures. Thus, ACTH fragments devoid of peripheral endocrine activity reduce pilocarpine-induced epileptiform activity in rats. A narrow bell-shaped dose-response relationship was found. Except for ACTH-(7-16), which was active in a dose of 1 and 10 p~g/rat s.c., the other fragments were only active at one dose (10 p,g/rat). The anti-epileptic properties appeared to reside in the sequence 1-16, and more specifically in the sequences 4-7 and 7-16, of the ACTH molecule. Seizures (chemically induced); Pilocarpine; Epilepsy; Anticonvulsants; ACTH (adrenocorticotrophic hormone)
1. Introduction
Besides classical anti-epileptic drugs, neuropeptides also exert anti-epileptic effects in some epileptogenic encephalopathies. A C T H is currently used to treat infantile spasms (West syndrome), an advantage being the higher incidence of spasm-free states compared to other treatments (Klein, 1970). However, an adverse effect is an ACTH-(1-24)-induced Cushing-like phenomenon. In a limited number of animal studies, fragments of ACTH devoid of peripheral endocrine activity were found to possess anti-epileptic properties. Org2766, an ACTH-(4-9) analogue, effectively reduced the course and severity of convulsive seizures in amygdaloid-kindled rats (Goldman et al., 1987), and a dorsal hippocampal kindling-induced epileptic syndrome was attenuated by ACTH-like neuropeptides (Cottrell et al., 1983). The N-terminal portion appeared to possess anti-epileptic properties: A C T H - ( 1 16), ACTH-(7-16) and ACTH-(7-16)(7D-Phe) inhibited after-discharges, whereas ACTH-(1-16), ACTH(4-16) and ACTH-(4-10)(7D-Phe) effectively inhibited behavioral depression. In a previous study we found that ACTH-(4-10) and ACTH-(7-16) have beneficial effects on pilocarpine-induced seizures (Wamil et al.,
Correspondence to: G. Croiset, Rudolf Magnus Institute, Department of Pharmacology, Medical Faculty, University of Utrecht, Vondellaan 6, Utrecht, Netherlands.
1989). In this study we investigated the moiety of the A C T H molecule possessing anti-epileptic properties in the pilocarpine (a muscarinic acetylcholine receptor agonist) model of epilepsy.
2. Materials and methods
2.1. Animals Male Wistar rats of an inbred stain (CPB-TNO, Zeist, The Netherlands) weighing 200-250 g were used. They were kept under standard conditions (room temperature 22 + 1 ° C, lights on from 6:00 a.m. until 7:00 p.m.), housed individually and received pellet food and water ad libitum. The experiments were performed between 10:00 a.m. and 5:00 p.m.
2.2. Peptides, drugs and injection procedures ACTH, its fragments and Org2766 were gifts from Organon Int. B.V. (Oss, The Netherlands). Peptides were freshly dissolved in 0.9% saline. Systemic injections were given s.c. in a volume of 0.5 ml 1 h prior to pilocarpine administration. The amino acid sequence of ACTH-(1-39) is H-Ser-Tyr-Ser-Met-Glu-His-PheArg-Trp-Gly-Lys-Pro-Val-Gly-Lys-Lys-Arg-Arg-ProVal-Lys-Val-Tyr-Pro-Asn-Gly-Ala-Glu-Asp-Glu-SerA l a - G l u - A l a - P h e - P r o - L e u - G l u - P h e - O H , that of Org2766 (ACTH-(4-9) analogue) is H-Met-(O2)-Glu-
H i s - P h e - L y s - P h e - O H a n d that of 7 2 - M S H is H-TyrVal-Met-Gly-His-Phe-Arg-Trp-Asp-Arg-Phe-GIy-OH. P i l o c a r p i n e h y d r o c h l o r i d e (Sigma, St. Louis, M O , U S A ) was dissolved in 0.9% saline and injected i.c.v. (2.4 r a g / 2 /,tl p e r rat). F o r i.c.v, injections, rats were e q u i p p e d with a p o l y e t h y l e n e c a n n u l a in the lateral c e r e b r a l ventricle, the c a n n u l a b e i n g fixed to the skull with d e n t a l c e m e n t ( D e W i e d , 1976), Rats were used for the e x p e r i m e n t 1 w e e k after surgery. Injections (2 /,1) were given with a H a m i l t o n syringe. T r e a t m e n t s were given blind.
status epilepticus, stage 5: d e a t h . T h e behavioral stage was d e t e r m i n e d every, min and scored as follows: stage 1, 1; stage 2, 2; etc. T h e m a x i m u m score was 75. T h e n u m b e r of rats that d e v e l o p e d g e n e r a l i z e d convulsions d u r i n g the o b s e r v a t i o n p e r i o d is e x p r e s s e d as a perc e n t a g e of the total n u m b e r of rats.
2.4. N u m b e r o[-"animals A C T H - ( 4 - 1 0 ) : control n - 2 7 , 1 # g n = 8, 10 /ag n = 10, 100 # g n = 9; A C T H - ( 7 - 1 6 ) : control n 21, 1 /xg n = 6, 10 /xg n = 9, 100 /.tg n = 6: Org2766: control n-37, 1 p,g n = 9 , 3 p,g n = 9 , 10 / , g n 9, 30 /,tg n = 10; A C T H - ( 1 - 1 6 ) : control n = 19, 0.1 # g n = 4 , 1 fig n = 4, 10 # g n - 11: A C T H - ( 1 - 3 9 ) : control n = 16, 10 # g n = 7: A C T H - ( I - 2 4 ) : control n - 1 1 , 10 /.~g n=ll; A C T H - ( I - 1 8 ) : control n = 8 , 10 fig n = 8 ; A C T H - ( 1 8 - 3 9 ) : control n - 10, 10 # g n = 13: A C T H (11-24): control n - 8, 10 / , g n = 1(1; A C T H - ( 1 1 - 1 6 ) : c o n t r o l n = 10, 10/xg n = 10; T 2 - M S H : control n = 10, 10 # g n = 1(1; A C T H - ( 4 - 1 0 ) ( 7 D - P h e ) : control n = 13, 10 # g n - 13; A C T H - ( 4 - 9 ) : control n = 8, 1 0 / , g n = 8;
2.3. Behavioral measurements I m m e d i a t e l y a f t e r p i l o c a r p i n e a d m i n i s t r a t i o n , the rat was p l a c e d in a t r a n s p a r e n t box (30 × 30 cm) a n d its b e h a v i o u r was o b s e r v e d for 15 rain. Five successive b e h a v i o r a l stages w e r e d i s t i n g u i s h e d ( m o d i f i e d from R a c i n e (1972)). Stage 1: akinesia, staring and salivation; stage 2: h e a d clonus, f o r e l i m b clonus a n d w e t - d o g shakes; stage 3: r e a r i n g with f o r e l i m b clonus; stage 4: r e a r i n g a n d falling with g e n e r a l i z e d convulsions and
ACTH-(4-
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Fig. 1. D o s e - r e s p o n s e effect of A C T H - ( 4 - t 0 ) , Org2766, A C T H - ( I 16) and A C T H - ( 7 - 1 6 ) on epilepsy. On the left side the total score ( + S.E.M.) is depicted and on the right side tile '7~- of rats that developed convulsions.
213
ACTH-(4-7): control n = 8, 10 /xg n = 8; Phe-D-LysPhe: control n = 11, 10 p~g n = 11.
2.5. Statistics The Fisher test (as a form of the Chi-square analysis) was used for statistical analysis of the effect of peptide treatment on the number of rats that developed convulsions after pilocarpine administration. The Mann-Whitney U test was used for statistical analysis of the effect of peptide treatment on the total epilepsy score during the observation period. P ~< 0.05 was taken as the level of significance. Results are expressed as means _+ S.E.M.
3. Results The effect of i.c.v, pilocarpine was direct. Two minutes after its i.c.v, administration the rats became motionless (stage 1). During the following minute head clonus and forelimb clonus were observed (stage 21, and within 4 rain the majority of the rats had developed generalized convulsions (stage 4). The occurrence of seizures gradually decreased 15 rain after the injection. No difference was found in the time of onset of convulsions between the groups of rats.
3.1. Dose-response study (fig. 11 In a first series of experiments ACTH-(1-16), ACTH-(4-10), A C T H - ( 7 - 1 6 ) and ORG2766 were tested in several doses. These peptides attenuated the effect of pilocarpine. However, either a narrow or no dose-response relationship was found, but the fragments were active at a dose of 10/xg. Only A C T H - ( 7 161 was effective at a dose of 1 p.g (total score 1 p.g: U = 2 1 (21,6), P = 0 . 0 1 ; 10 /xg: U = 3 3 . 5 (21,9) P = 0.005). A significant decrease in the total epilepsy score and in the number of animals that developed general convulsions (stage 4) was observed after pretreatment with a dose of 10 # g ACTH-(4-10) or Org2766 (total score: Org2766: U = 28 (37,91, P = 0.0001; ACTH-(4-10): U = 46 (27,10), P = 0.002). A significant reduction in the total epilepsy score was also observed after 10 /xg ACTH-(1-16) (U = 40 (19,111, P = 0.02). A dose of 10 /xg was chosen for further experiments. No difference was found in the time of onset of convulsions.
3.2. Effect of large A C T H fragments on pilocarpine-induced epilepsy (fig. 1 and table 1) ACTH-(1-391, ACTH-(1-241, A C T H - ( I - 1 8 ) and ACTH-(18-39) did not affect the number of animals
TABLE 1 Effect of A C T H f r a g m e n t s on p i l o c a r p i n e - i n d u c e d epilepsy. Total score ACTH-(1-39) Control 42 ± 3 10 ,ag 46 + 2 A C T I t - ( 1 24) Control 33 _+ 6 10/xg 32+6 A C T H - ( 1- 18) Control 39 ± 5 10 ,ag 33+5 ACTH-(18-39) Control 42 _+4 10 # g 42+_4 ACTH-(11-24) Control 49 + 1 10 ,ag 27+4 * A C T H - ( I 1 161 Control 40 +_3 10 # g 37 + 4 y2-MSlt Control 47 + 4 10/xg 49 ± 3 ACTH-(4-10)(7D-Phe) Control 51 + 2 10 g g 37+_4 * A C T H - ( 4 9) Control 47 + 3 1(I/xg 29 +_ 7 ACTH-(4-7) Control 411 ± 4 10 # g 26+6 Phe-D-Lys-Phe Control 45 + 1 10/xg 44+3 A C T | t-(6-9) Control 51 + 1 10 # g 44 +_ 5 ACTH-(7-10) Control 39 + 3 10 # g 33+5
Onset (rain)
% convulsions
3.5 ± (/.5 3.8 + 0.4
75 100
3.0 ± (1.6 3.2+_0.8
54 73
6.2 ± 1.8 5 . 5 + 1.0
75 5(1
4.3 + (I.6 5 . 0 + 1.1
80 100
5.5 + 0.3 6 . 2 + 1.6
10(} 4(}
5.6 + 1.1 5.4 + 0.6
9{} 811
4.2 + 0.9 3.5 + 0.6
9(} 90
3.7 ± 0.6 5.1 +11.7
100 69
4.2 ± (1.7 5 . 0 + 1.8
100 5(}
5.6 + 1.4 5 . 3 + 1.8
87 37
4.6 +_0.6 4.4+0.7
100 91
2.8 + 0.3 3.8 ± ().5
100 78
4.7 ± 0.7 3.4+0.9
83 58
that developed tonic/clonic seizures, nor the total epilepsy score. ACTH-(11-24) reduced both measures (total score: U = 8.5 (8,10), P = 0.02). No difference was found in the time of onset of convulsions.
3.3. Effect of intermediate ACTH fragments on pilocarp±he-induced epilepsy (fig. 1 and table 11 As mentioned above, ACTH-(1-16) and A C T H - ( 7 161 attenuated the severity of the epilepsy, but did not decrease the number of animals that developed t o n i c / clonic seizures. Other fragments, such as A C T H - ( l l 16) and y2-MSH, did not affect the severity of the epilepsy. No difference was found in the time of onset of convulsions.
214
3.4. Effect of small A C T H fragments on pilocarpine-mduced epilepsy (fig. 1 and table 1) Treatment with A C T H - ( 4 - 1 0 ) or ACTH-(4-10)(7DPhe) significantly reduced the total epilepsy score (U = 24 (13,13), P = 0.001). The smaller fragments A C T H (4-9) and A C T H - ( 4 - 7 ) tended to reduce the severity of the epilepsy. A significant reduction in the number of rats with t o n i c / c l o n i c convulsions was found after treatment with A C T H - ( 4 - 1 0 ) or A C T H - ( 4 - 9 ) , and a modest but not significant reduction after treatment with A C T H - ( 4 - 7 ) . Org2766 effectively reduced both the severity and the number of animals with t o n i c / c l o n i c convulsions. The Org2766 C-terminal fragment Phe-D-Lys-Phe was not active in this respect. No difference was found in the time of onset of convulsions.
4. Discussion
Torda and Wolff (1952), who used pentymethylenetetrazol, were the first to find anticonvulsive effects of chronic treatment with a crude A C T H preparation. In the present study we used a muscarinic acetylcholine receptor agonist (pilocarpine) to produce epileptiform seizures. Pilocarpine injection into the lateral ventricle results in immediate epileptiform activity (Turski et al., 1989; Wamil et at., 1989). Pretreatment with A C T H neuropeptides protected rats from pilocarpine-induced seizures, and either no or a narrow bell-shaped doseresponse relationship was found, There is no explanation yet for this phenomenon, although it has been reported in several studies (De Wied et al., 1987; Wolterink et al., 1991), The anti-epileptic activity appears to reside mainly in fragments of the A C T H molecule; neither the whole molecule (1-39), nor large fragments such as (1-24) and (18-39) were active. The anti-epileptic activity was found predominantly in the N-terminal fragments of ACTH. Sequences as small as five amino acids, such as A C T H - ( 4 - 9 ) and its analogue Org2766, appeared to possess anti-epileptic activity. However, Org2766, which is a thousandfold more active on active and passive avoidance behaviour (De Wied and Jolles, 1982), was not more potent than A C T H - ( 4 - 1 0 ) in protecting against pilocarpine-induced epilepsy. The active moiety resides in the (4-7) sequence, the (7-10) sequence not being effective. In this active sequence the fifth amino acid, Glu, is probably of more importance than the seventh amino acid, Phe, as the structurally related neuropeptide, y2-melanocyte-stimulating h o r m o n e (y2-MSH), which contains an A C T H - ( 4 - 9 ) sequence with Gly instead of Glu in position 5, did not have anti-epileptic effects. The D-enantiomer A C T H - ( 4 10)(7D-Phe) appeared to be almost as effective as
ACTH-(4-10). Interestingly, the D-enantiomer has an effect opposite to that of ACTH-(4-10)(7L-Phe) on the extinction of active and passive avoidance behaviour (De Wied and Jolles, 1982). Another sequence, A C T H - ( 7 - 1 6 ) also protected against pilocarpine-induced epilepsy. The entire 10amino acid sequence is necessary for activity as neither (7-10) nor (11-16) had anti-epileptic activity. Cottrell et al. (1983) found that A C T H - ( 1 - 1 6 ) and A C T H - ( 7 16) were equally effective on after-discharges elicited by hippocampal kindling. They also found the D-enantiomer of A C T H - ( 7 - 1 6 ) was more active in that it reduced after-discharges as well as behavioral depression. In this study, A C T H - ( I - 1 6 ) , which contains two anti-epileptic sites ((4-7) and (7-16)), had only minor protective effects. This may point to the importance of the three-dimensional structure of these two fragments. It may well be that when these two sites are combined, as in the (1-16) fragment, the three-dimensional effective structures of both fragments are concealed. These fragments might be generated from A C T H - ( 1 - 1 6 ) in the brain, as brain synaptosomes havc been shown to convert the (1-16) fragment to A C T H (7-16) after incubation with brain peptidases (Burbach, 1989). A behaviorally potent analogue of ACTH-(4-9), Org2766, also possesses anti-epileptic properties. In other models of epilepsy, A C T H is beneficial in reducing the course and duration of seizures whereas Org2766 markedly reduces seizures after amygdaloid kindling (Goldman et al., 1987) but is not very, active in hippocampal kindling (Cottrell et a[., 1983). Thus the site in the brain where the syndrome is generated also determines the sensitivity to peptides. Apparently, the attenuating effect of a compound, in this case an ACTH-Iike neuropeptide, on kindling cannot be generalized to related substances. Pilocarpine is a cholinomimetic agent. Thus the cholinergic system and structures innervated by this system may be the site of action of A C T H fragments. It is also possible that ACTH-Iike fragments exert their effects in the pathways which ultimately result in motor seizures. Cholinomimetics (Olney et al., 1983; Turski, et al., 1984), kainic acid (Bcn-Ari ct al., 1980), folates (Olney et al., 1981) and G A B A antagonists (Turkski et al., 1984) produce very similar brain abnormalities. Although initiation of abnormal elcctrographic activity within limbic circuits is linked to divergent receptor mechanisms, it seems evident that epileptic brain damage in experimental animals involves the same mechanism, and that one neurotransmitter system cannot be considered pivotal in the genesis of seizures and their pathological sequelae (Turski et al., 1989). Many areas in the central nervous system are involved in epilepsy. Basal ganglia, such as the substantia nigra and the
215 globus pallidus, serve as relay stations in the propagation of seizures elicited from the motor cortex or the limbic system. Excitation in the substantia nigra is probably mediated by L-glutamate or L-aspartate. Microinjection of a N M D A receptor antagonist and of the G A B A agonist muscimol into the substantia nigra or entopeduncular nucleus protects against the convulsant action of pilocarpine (Hosford and McNamara, 1988; Patel et al., 1988; Turski et al., 1983; 1986). Dopamine is also a neurotransmitter involved in pilocarpine-induced epilepsy (Turski et al., 1988). /3-MSH given i.v. facilitates ventral root responses after dorsal root stimulation in the cat, and antagonizes the depressive action of chlorpromazine on dorsal root potentials. Thus /3-MSH could influence dopaminergic, cholinergic and histaminergic transmission (Krivoy, 1970). A C T H neuropeptides are reported to affect excitatory and inhibitory neurotransmission. Calvet et al. (1992) studied the effect of an A C T H - ( 4 - 1 0 ) analogue (BIM 22015) (D-Ala-Gln-Tyr-Phe-Arg-Trp-Gly-NH 2) on the neuronal firing pattern of rat spinal cord cultures. The BIM peptide decreased the spontaneous firing rate after a single dose. These changes were enhanced in a low Ca 2+ medium. Chronic exposure to the peptide increased phasic bursting patterns, resembling strychnine-induced effects. These findings suggest that these A C T H - r e l a t e d peptides induce changes in the m e m b r a n e properties of spinal cord neurons to either decrease (acute) or increase (chronic) the frequency of action potentials. As suggested by the authors, BIM 22015 may inhibit synaptic transmission by reducing presynaptic calcium entry and impairing excitatory transmitter release. The effect of chronic treatment was interpreted as an attenuating effect on transmitter release at inhibitory presynaptic terminals. The anticonvulsive effects of A C T H neuropeptides may also be explained by an attenuating influence on excitatory neurotransmission. A C T H - ( 1 - 2 4 ) and A C T H - ( 4 - 1 0 ) reduce the binding of [3H]glutamate, although with rather low affinity (Ito et al., 1988). Recently, Trifiletti and Pranzatelli (1992) found that micromolar concentrations of ACTH-(1-39), ACTH-(1-24), and A C T H (1-17) displaced the binding of [3H]MK 801 in rat hippocampus homogenates; however, A C T H - ( 4 - 1 0 ) was not active. The affinity was of the same order of magnitude as that found for A C T H fragments at other receptor sites. Cholinergic agents applied in the vicinity of CA~ or CA 3 pyramidal neurons enhance firing induced by synaptic activation by acting presynaptically to decrease GABAergic inhibition and by acting postsynaptically to decrease K + conductance (Meldrum, 1985). A C T H - ( 4 - 1 0 ) may interfere with one or more of the above-mentioned processes, as A C T H fragments bind to muscarinic receptors (Tonnaer et al., 1986) and A C T H - ( 1 - 2 4 ) has been shown to inhibit the in vitro
binding of the muscarinic antagonist [~H]quinuclidinylbenzilate (QNB) in the rat. However, A C T H - ( 4 10) is not active in this respect. A C T H and related peptides have been shown to affect cholinergic transmission. Geiger et al. (1987) reported that A C T H fragments decreased acetylcholine concentrations in the rat striatum, with Org2766 being a thousandfold more active than A C T H - ( 4 - 9 ) . Since these two fragments were equally active in protecting against pilocarpine-induced epilepsy, it does not seem likely that their primary effect is on the cholinergic system. It has been hypothesized that endogenous opioids may play a role in seizure activity (Cain and Corcoran, 1985). /3-Endorphin as well as Met-enkephalin facilitates electrical kindling of the amygdala. /3-Endorphin itself can produce after-discharge activity in limbic structures, an effect which can be reversed by naloxone (Henriksen et al., 1978). It is possible that the anticonvulsive effect of A C T H is mediated through the opioid system. A C T H - ( 4 - 1 0 ) and A C T H - ( 7 - 1 6 ) have been shown to inhibit [3H]dihydromorphine binding to synaptic plasma m e m b r a n e fractions of rat brain (Terenius et al., 1975) and to counteract the antinociceptive action of morphine (Gispen et al., 1976). The finding that high doses of Org2766 in contrast to low doses have a naloxone-reversible effect on avoidance behaviour is not in favour of an ACTH-like opiate antagonistic effect on epilepsy as opiate activity appears to be located in the (7-10) sequence (Fekete and de Wied, 1982), a sequence which was inactive in the present experiment. Nevertheless, in other studies the involvement of the opioid system in seizure activity has been demonstrated. Cottrell et al. (1983)showed that y 2 - M S H can reduce the seizures induced by hippocampal kindling, but is ineffective in pilocarpine-induced epilepsy. It is not possible to determine the mechanism of action of A C T H neuropeptides on pilocarpine-induced epilepsy from the data in the literature and the present results. Studies on the influence of neuropeptides on glutamatergic (GABAergic) or on dopamine transmission may be of importance in revealing the underlying mechanism of action of these neuropeptides.
Acknowledgement The authors wisk to thank Leo van Halewijn for his excellent technical assistance.
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