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A behavioural pharmacological study on intracerebroventricularly administered CCK-8 related peptides in mice
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Neuropeprides (1989) 13,107-l 13 @ Longman Group UK Ltd 1989
A Behavioural Pharmacological Study on Intracerebroventricularly Administered CCK-8 Related Peptides In Mice Y. HAGINO*,
T. MOROJI*
and R. llZUKA*t
*Department of Psychopharmacology, Psychiatric Research Institute of Tokyo, 2-l-8 Kamikitazawa, Setagaya-ku, Tokyo 156, tDepartment of Psychiatry, Juntendo University, School of Medicine, 2-l-7 Hongo, Bunkyo-ku, Tokyo 113, Japan (reprint requests to T.M.).
Abstract-The sulfated form of cholecystokinin octapeptide (CCK-8) and ceruletide (CER), but not their non-sulfated forms or CCK4, significantly decreased the rates of locomotor activity and rearing during the first 10 min test session 10 min after intracerebroventricular (ICV) administration at doses more than 25 and 3.125mg, respectively. CER-S antagonized methylphenidate-induced hypermotility after ICV administration at a dose of 800ng. Plasma levels of CER-like immunoreactivity (CER-LI) measured at 120 min after subcutaneous injection, when the locomotor suppressive activity induced by 100 and 2OOug was no longer observed, were similar to or much higher than that 30 min after ICV administration at a dose of 800ng, suggesting that the effects of ICV CER-S are not mediated by a peripheral redistribution. These findings indicate that (1) the structural requirement for the locomotor suppressive activity is sulfated tyrosine residue; (2) the behavioural effects of ICVadministered CCK8-S and CER-S are due to their central actions and mediated by the/ inhibition of the central dopamine (DA) function; and (3) CCK-8-S in the brain is functionally associated with the central DA system.
Introduction
Cholecystokinin (CCK)-like peptides have been found to be widely distributed in the mammalian brain. Biochemical studies have demonstrated that the major form of CCK in the brain is the sulfated form of the COOH-terminal octapeptide
Date received 22 August 1988 Date accepted 20 October 1988
(CCK-8-S) (1, 2, 3), followed by the tetrapeptide (CCK4) (1, 2, 3). Other molecular forms of CCK-like peptides are CCK-58, CCK-39, CCK-33 and CCK-12 in mammalian brain (4,5,6,7,8) and a non-sulfated form (CCK-8-NS) in the rat brain /o, [O’* Recently, neuropharmacological studies have indicated that CCK-8 and its related peptides produce behavioural changes resembling those of neuroleptics in rodents when injected centrally as
107
108 well as peripherally (9, 10). However, differences in behavioural effects especially neuroleptic-like properties, have been noted in rodents after intracerebroventricular (ICV) administration of CCK-8 related peptides. These differences in behavioural effects following ICV administration may be due not only to differences in animal the experimental procedures species and employed, but also to CCK heterogeneity. In the present study, we investigated the effects of CCK-8 related peptides on spontaneous locomotor activity, rearing and methylphenidateinduced hyperactivity in mice after ICV administration.
Materials and Methods General
Male ddY strain mice weighing 2533g were housed in groups of 6 mice per cage in a temperature-controlled animal room with a 12:12 hour light/dark cycle (light on at 8:OOa.m.) with free access to commercial food and tap water. CCK-8 related peptides tested were administered to unanesthetized, manually restrained animals ICV in a volume of 10 ~1 by the method of Haley and McCormick ( 11). To ascertain the areas into which the needles were inserted, the brains were sectioned and studied histologically after the experiment.
NEUROPEPTIDES
Behavioural measurements
Evaluation of locomotor activity and rearing rate have been described previously (12). Each test session was 10 min. Both locomotor activity and rearing rate were determined 10 or 30 min after ICV injection of drugs tested. After a 50-min acclimation period in the test cage, methylphenidate was injected intraperitoneally just after ICV administration of CER-S at a dose 800ng in a volume of 10 ~1 and locomotor activity was evaluated every 10 min for 120 min. Measurement plasma
of CER-like
immunoreactivity
in
To evaluate whether the behavioural effects of ICV-administered CER-S were due to central or peripheral action, plasma levels of CER-like immunoreactivity (CER-LI) were determined after ICV and subcutaneous (SC) administration of CER-S. Blood samples were obtained via heart puncture under ether anesthesia using a heparinized syringe. Heparinized blood sample from 3 different animals were collected into a small glass tube containing EDTA (1.5 mg/ml blood) and trasylol (500 IU/ml blood). Then, the plasma was separated by centrifugation at 1600 g for 15 min at 4°C and stored at -80°C until used. CER-LI was measured by radioimmunoassay using antiserum directed towards the N-terminal of CER (13). No cross reactivity for other related peptides such as CCK-8, CCK-33, gastrin-II was observed (0.01%). Statistical analysis
Chemicals CCK-8-S,
its non-sulfated form (CCK-8-NS) and CCK-4 were purchased from Peptide Institute Inc. (Osaka, Japan); ceruletide diethylamine (CER-S) and its non-sulfated (CER-NS) were gifts from Shionogi Company (Osaka, Japan); methylphenidate hydrochloride was a gift from Ciba-Geigy (Basal, Switzerland). CCK4 was dissolved in distilled water containing 0.01% cysteine and 2% sodium bicarbonate. CER-NS was dissolved in 1% sodium bicarbonate and was neutralized with 0.1 N hydrochloric acid. Other peptides were dissolved in physiological saline. All solutions were freshly prepared just before use.
Means and standard errors of the means were calculated using statistical procedures. Student’ t-test (two-tailed) was used comparison against the control group treated with the physiological saline or vehicle.
Results Effects of ICV-administered CCK-8-S motor activity and rearing rate
on loco-
CCK-8-S caused a significant, but not doserelated, reduction in both locomotor activity and rearing rate during the lo-min test session 10 min
INTRACEREBROVENTRICULARLY
ADMINISTERED
after ICV administration at doses ranging from 25 to 800ng (Table 1). The inhibitory effects of CCK-8-S were also observed 30 min after ICV administration at a dose of 800ng but not at a dose of 200ng (Table l), suggesting that its potency is not dose-related, but the duration is prolonged dose-dependently.
Effects of NIV-administered CER-S on locomotor activityand rearing rate Effects of CER-S on locomotor activity and rearing rate 10 min after ICV administration were investigated at doses ranging from 1.0 to 25ng. CER-S at doses ranging from 3.125 to 25 ng caused a significant, but not dose-related, reduction in locomotor activity during the lo-min test session (Table 2). The effect on rearing rate was also observed at doses ranging from 6.25 to 25ng, but was not dose-dependent (Table 2). Effects of CER-S on locomotor activity and rearing rate 30 min after ICV administration were studied at doses ranging from 50 to 800ng. CER-S significantly decreased the rates of both behavioural parameters (Table 2).
Table 1
Effects of ICV-Administered
Effects of ZCV administration of other CCK-8 related peptides on locomotor activityand rearing CCK-&NS,
Efiect of ICV-administered CER-S on methylphenidate-induced hyperactivity Effect of CER-S on methylphenidate-induced hyperactivity was studied at a dose of 8OOng, since its inhibitory effects on both locomotor activity and rearing were still observed 30 min after ICV administration. CER-S (800ng in a volume of 10~1) inhibited methylphenidate-induced hyperactivity from 30 min to 80 min after ICV administration (Fig 1). Plasma levels of CER-LI after ICV and subcutaneous administration of CER-S Plasma CER-LI levels 15 and 30 min after ICV administration of 800ng CER-S in a volume of 10 t.Awere 3.2 + 0.2 and 1.4 + 0.1 @ml, respectively (Fig 2). Plasma levels of CER-LI reached the peak values 15 min after SC injections of 100
CCK-8-S on Locomotor Activity and Rearing Rate in Mice The 10 min test session
The 10 min test session
10 min after ICV administration
30 min after ICV administration
Number
Vehicle CCK-8-S 12sng 25 ng 50 ng 100 ng 200 ng 400 ng 800 ng Vehicle CCK-8-S 200 ng 800 ng
44
344.7 k 21.6
18.5 + 3.1
11 11 6 10 12 12 13 22
286.1 + 44.4 165.4 zb 26.2** 160.8 + 29.6”** 212.1 IL 27.3** 185.2 rt 36.4**’ 155.2 -t 32.2*** 204.2 * 15.8**’
13.7 5.2 2.7 5.9 4.0 2.4 4.8
12 12
and CCK4 had no effect on at doses of up to 1.6 to 1Okg
CER-NS
either parameter (Table 3).
Treatment and dose per rat
of animals
109
CCK-8 RELATED PEPTIDES IN MICE
Locomotor
activity Rearing rate (Mean + S. E. M.)
Locomotor
activity Rearing rate (Mean + S. E. M.)
+ 4.1 + 1.8*** + 0.8*** ?r 1.5*** f 1.3*** +- l.t*** + 1.4*** 597.5 t 45.8
40.3 -I 6.5
524.9 -I 69.1 297.5 + 30.3***
38.6 f 12.1 12.2 z!z2.7***
The data were analysed using Student’s t-test (two-tailed) for comparison against the group treated with vehicle. ** p < 0.01; ***p< 0.001. Abbreviations: CCK-S; sulfated form of cholecystokinin octapeptide, ICV: intracerebroventricular(ly).
110
NEUROPEPTIDES
and 200 p@kg of CER-S and the mean peak values were 18.6 + 1.6 and 33.7 + 2.9ng/ml, respectively. Plamsa CER-LI was still detected 120 min after SC injection of CER-S. Discussion Spontaneous locomotor activity of rats was not affected by CCK-8-S (14,15) but increased by CCK-&NS following administration (14). HowTable 2
Effects of ICV-Administered
Treatment and dose per rat
Vehicle CER-S 1.0 ng 3.125ng 6.25 ng 12.5 ng 25.0 ng Vehicle CER-S 50 ng 100 ng 200 ng 800 ng
ever, van Ree et al. have demonstrated that ICV-administered CCK-8-S but not CCK-8-N& produced significant immobility in rats at a dose level of 2Opg (16). Likewise, conflicting results have been reported that methamphetamineinduced hyperactivity of rats was inhibited by ICV administration of CCK-8-S but not of CCK-8-NS (14), ICV injected CCK-8-S did not alter amphetamine-induced locomotor activity of rats (15). Therefore, we examined the neuropharmaco-
CER-S on Locomotor Activity and Rearing Rate in Mice The 10 min test session 10 min after ICV administration
Number
Locomotor
of animals
Rearing rate activity (Mean + S. E. M.)
41
322.8 f 26.2
16.2 + 2.7
10 9 10 11 11 31
322.2 201.2 186.3 164.9 131.0
14.5 5.8 4.5 5.6 3.0
f f f + ?
The 10 min test session 30 min after ICV administration
39.5 37.9* 27.1** 28.8** 18.6**’
Locomotor
activity Rearing rate (Mean + S. E. M.)
+ 3.9 f 3.3 + 1.8*** IL 2.8* f 1.1***
13 15 15 12
637.0 + 33.0
42.9 f 4.0
558.3 536.1 369.3 310.2
36.0 34.3 13.5 9.3
f f f +
46.5 59.0 30.5*** 33.5***
+ f + zt
3.2 6.5 3.0*** 2.2***
The data were analysed using Student’s t-test (two-tailed) for comparison against the group treated with vehicle. * p < 0.05; **p< 0.01; *** p < 0.001. Abbreviation: CER-S; sulfated form of ceruletide.
Table 3 Effects of ICV-Administration Rate in Mice
of Other CCK-8 Related Peptides on Locomotor Activity and Rearing
The 10 min test session 10 min after ICV administration
Number Treatment
Vehicle CCK-&NS Vehicle CCK-4 Vehicle CER-NS
Dose per rat
0.8ug 1.6ug 1.6kg lO.Oug 1.6tq
Locomotor
of animals
19 10 10 23 11 10 12 11
Abbreviations: CCK-8-NS; non-sulfated form of cholecystokinin non-sulfated form of ceruletide.
253.9 275.6 266.8 243.0 207.3 183.6 282.8 219.4 octapeptide,
+ + f t + f + f
activity (Mean _+ S. E. M.)
Rearing rate 14.8 13.2 19.1 9.4 9.7 2.8 17.2 10.2
30.3 40.5 56.7 34.0 65.5 36.5 37.2 28.0
CCK-4; cholecystokinin
tetrapeptide,
+ f + + f + + f
3.0 4.2 6.9 3.7 5.0
1.0 5.2 2.3
CER-NS;
INTRACEREBROVENTRICULARLY ADMINISTERED CCK-8 RELATED PEPTIDES IN MICE lWUCEE+LLI.+WEllWLll?.
o-o
IllhI
.-.
CBIYII
II41 I III
111
logical action of CCK-8 related peptides on spontaneous locomotor activity, rearing rate and methylphenidate-induced hyperactivity in mice after ICV injection. Effects of ICV-administered CCK-8 related peptideson spontaneous locomotor activityand rearing rate
+l
b
120me.
60
Fig 1 Effect of XV-administered CER-S on methylphenidate-induced hyperactivity Each value is the mean + S.E.M. of the number of mice indicated in parenthesis. The data were analyzed using Student’s t-test (two-tailed) for comparison against the group treated with physiological saline. * p < 0.05; ** p < 0.01. Abbreviations: MPD; methylphenidate, I.C.V.; intracerebroventricularly administration, I.P.; intraperitoneal administration.
O-O
CEI-StWmU.
151
O-O
CL,S INHS.C.
151
CEB-S880 m51C.V. I8 I
0
‘0 1550
00
140ah.
Fig 2CER-LI plasma levels after ICV and subcutaneous administration of CER-S Each value is the mean f S.E.M. of the number of plasma samples indicated in parenthesis. Abbreviations: CER-LI; ceruletide-like immunoreactivity, S.C.; subcutaneous administration.
ICV administration of CCK-8 and CER-S significantly decreased the rates of locomotor activity and rearing at doses ranging from 25 to 8OOng. These findings indicate that CER-S is much more potent in inhibiting locomotor activity and rearing than CCK-8 (12,16). Similar results were obtained in our previous study in which these two peptides were SC administered (12). In addition, the present findings are in good agreement with I& values in competitive inhibition of [propionyl-3H] propionylated CCK-8 ([3H]pCCK-8)-specific binding to the slide mounted tissue sections of the rat brain by CCK-8-S and CER-S (17). This suggests that ICV-administred CCK-8-S or CER-S directly affects the brain function. In contrast to SC administration, however, no dose-related inhibition was observed after ICV administration. At present, the reasons for this discrepancy are unclear. CCK-S-S at dose of SOOng, but not of 2OOng, had an inhibitory effect on the rates of locomotor activity and rearing 30 min after its ICV administration. Such inhibitory effects were also observed after ICV administration of CER-S at doses of 200 and SOOkg, but not of lOOl.~g.These findings indicate that the duration of the inhibitory effect is related to the dose given. In our competitive binding studies, four peptides structurally related to CCK-8, e.g. CER, CCK-8, CCK-S-NS and CCK-4, completely inhibited binding of [3H] pCCK-8 to the tissue sections from the rat brain (17). The order of inhibitory potencies of the peptide tested were CER CCK-8 CCK-S-NS CCK-4. However, no behavioural changes were in mice after ICV administration of CER-NS or CCK-NS or CCK-S-NS at doses ranging from 0.8 to 1.6pg. Thus, the presence of O-sulfate on the tyrosine residue in the CCK-molecule seems to be necessary for the inhibitory effect of CCK-8 related peptides on the locomotor activity and
112 rearing in mice after ICV administration (12). In the present study, furthermore, CCK-4 at doses more than 1.6 pg caused no behavioural changes in mice. These finding, taken together with those on CCK-8-NS and CCK4 in the mammalian brains, indicate that CCK-8-NS and CCK-4 in the brain have different physiological roles from CCK-8-S. ICV-administered [ ‘251]CCK-8 demonstrated to diffuse rapidly into the peripheral blood by a non-carrier-mediated mechanism (18). In our previous study, SC-administered CCK-8-S and CER-S at doses more than 50 and 25l~g/kg, respectively, inhibited locomotor activity and rearing in mice (12). At doses more than 100 l.~g/ kg, these inhibitory effects were observed 60 min after the administration. To clarify whether or not the central effects of CCK-8-S and CER-S are the results of a peripheral redistribution of these peptides, we measured the plasma CER-LI levels after ICV and SC administrations of CER-S. The plasma CER-LI level 30 min after ICV administration of 800ng CER-S was about one-fifth of that at 120 min after SC injections, when the locomotor suppressive activity induced by 200 kg/ kg of CER-S was no longer observed. Thus, the inhibitory effects of CCK-8-S and CER-S after ICV administration may be due to their central actions. Effect of ICV-administered CER-S on methylphenidate-induced hyperactivity was hyperactivity Methylphenidate-induced inhibited by ICV-administered CER-S. Methylphenidate, an indirect dopamine (DA) agonists, causes DA release from nerve terminals of DA neurons in the brain, resulting in behavioural changes, such as hyperactivity and stereotypy, in rodents. Thus, this finding suggests that ICVadministered CER-S affects the neural transmission in central DA systems, antagonizing methylphenidate-induced hyperactivity. Furthermore, it seems likely CCK-8-S in the brain is closely associated with the central DA functions. In conclusion, CCK-8-S and CER-S, but not their non-sulfated forms or CCK-4, caused a reduction in the rates of locomotor activity and rearing in mice when ICV-administered. In addiICV-administered CER-S inhibited tion,
NEUROPEPTIDES
methylphenidate-induced hyperactivity. These findings indicate that (1) the structural requirement for the locomotor suppressive activity is a sulfated tyrosine residue; (2) the behavioural changes induced by ICV-administered CCK-8-S and CER-S are mediated through their inhibitory effects on the central DA functions; consequently (3) CCK-8-S in the brain is functionally associated with the central DA systems; and (4) there are different structure-central nervous system (CNS) action relationships in the CNS-mediated effects of CCK-8 related peptides in the brain.
Acknowledgements We are greatly indebted to Shionogi Company (Osaka, Japan) for the kind gift of ceruletide diethylamine and its non-sulfated form and for the measurement of plasma levels of ceruletide. This study was supported in part by a research grant from the Japanese Ministry of Health and Welfare (to R.I.)
References 1. Muller, J. E., Straus, E. and Yalow, R. S. (1977). Cholecystokinin and its COOH-terminal octapeptide in the pig brain. Proceedings of the National Academy of Sciences of the United States of America 74: 3035-3037. 2. Schneider, B. S., Monahan, J. W. and Hirsch, J. (1979). Brain cholecystokinin and nutritional status in rats and mice. Journal of Clinical Chemistry 64: 13481356. 3. Rehfeld, J. F. and Golterman, R. (1979). Immunochemical evidence of cholecystokinin tetrapeptides in hog brain. Journal of Neurochemistry 32: 1339-1341. 4. Eng, J., Shiina, Y., Pan, Y.-C. E., Blather, R., Chang, M., Stein, S. and Yalow. R. S. (1983). Pig brain contains cholecystokinin octapeptide and several cholecystokinin desoctapeptides. Proceedings of the National Academy of Sciences of the United States of America 80: 6381-6385. 5. Ichihara, K., Eng, J. andYalow, R. S. (1984). Ontogeny of molecular forms of CCK-peptides in rat brain and gut. Life Sciences 34: 93-98. 6. Jansen, J. B. M. J. and Lamer, C. B. H. W. (1983). Immunological evidence of cholecystokinin-39 in porcine brain. Life Sciences 32: 911-913. Rehfeld, J. F. (1978). Immunochemical studies on cholecystokinin. II. Distribution and molecular heterogeneity in the central nervous system and small intestine of man and hog. Journal of Biological Chemistry 253: 40224030. Frey, P. (1983). Cholecystokinin octapeptide (CCK26-33), nonsulfated octapeptide and tetrapeptide (CCK 30-33) in rat brain: analysis by high pressure liquid chromatography (HPLC) and radioimmunoassay (RIA). Neurochemistry International 5: 811-813.
INTRACEREBROVENTRICULARLY
ADMINISTERED
CCK-8 RELATED
9. Zetler, G. (1985). Neuropharmacological profile of cholecystokinin-like peptides. Annals of the New York Academy of Sciences 448: 448-469. 10. Nair, N. P. V., Lal, S. and Bloom, D. M. (1986). Cholecystokinin and Schizophrenia. In: van Ree, J. M. and Matthyse, S. (eds) Progress in Brain Research, Volume 65, Elseveir Science Publishers B. V., Amsterdam, p. 237-258. 11. Haley, T. J. and McCormick, G. (1957). Pharmacological effects of produced by intracerebral injection of drugs in the conscious mouse. British Journal of Pharmacology 12: 12-15. 12. Moroji, T. and Hagino, Y. (1986). A behavioural pharmacological study on CCK-8 related peptides in mice. Neuropeptides 8: 273-286. 13. Kominai, G., Mori, S. and Kono, M. (in press). A highly specific radioimmunoassay of caerulein. Journal of Immunoassay. 14. Katsuura, G. and Itho, S. (1982). Sedative action of cholecystokinin octapeptide on behavioural excitation by
15.
16.
17.
18.
PEPTIDES IN MICE
113
thyrotropin-releasing hormone and methamphetamine. Japanese Journal of Physiology 32: 83-91. Widerlov, E., Kalivas, P. W., Lewis, M. H., Prange, Jr.. A. J. and Breese, G. R. (1983). Influence of cholecystokinin on central monoaminergic pathway. Regulatory Peptides 6: 99-109. van Ree, J. M., Gaffori, 0. and de Wied, D. (1983). In rats, the behavioural profile of CCK-8 related peptides resembles that of antipsychotic agents. European Journal OFPharmacology. 93: 63-78. Sekiguchi, R. and Moroji, T. (1986). Comparative study on characterization and distribution of cholecystokinin binding sites among the rat, mouse and guinea pig brain. Brain Research 399: 271-281. Passaro, Jr., E., Debas, H., Oldendorf, W. and Yamada, T. (1982). Rapid appearance of intraventricularly administered neuropeptides in the peripheral circulation. Brain Research 241: 335-340.
Neuropeprides (1989) 13,107-l 13 @ Longman Group UK Ltd 1989
A Behavioural Pharmacological Study on Intracerebroventricularly Administered CCK-8 Related Peptides In Mice Y. HAGINO*,
T. MOROJI*
and R. llZUKA*t
*Department of Psychopharmacology, Psychiatric Research Institute of Tokyo, 2-l-8 Kamikitazawa, Setagaya-ku, Tokyo 156, tDepartment of Psychiatry, Juntendo University, School of Medicine, 2-l-7 Hongo, Bunkyo-ku, Tokyo 113, Japan (reprint requests to T.M.).
Abstract-The sulfated form of cholecystokinin octapeptide (CCK-8) and ceruletide (CER), but not their non-sulfated forms or CCK4, significantly decreased the rates of locomotor activity and rearing during the first 10 min test session 10 min after intracerebroventricular (ICV) administration at doses more than 25 and 3.125mg, respectively. CER-S antagonized methylphenidate-induced hypermotility after ICV administration at a dose of 800ng. Plasma levels of CER-like immunoreactivity (CER-LI) measured at 120 min after subcutaneous injection, when the locomotor suppressive activity induced by 100 and 2OOug was no longer observed, were similar to or much higher than that 30 min after ICV administration at a dose of 800ng, suggesting that the effects of ICV CER-S are not mediated by a peripheral redistribution. These findings indicate that (1) the structural requirement for the locomotor suppressive activity is sulfated tyrosine residue; (2) the behavioural effects of ICVadministered CCK8-S and CER-S are due to their central actions and mediated by the/ inhibition of the central dopamine (DA) function; and (3) CCK-8-S in the brain is functionally associated with the central DA system.
Introduction
Cholecystokinin (CCK)-like peptides have been found to be widely distributed in the mammalian brain. Biochemical studies have demonstrated that the major form of CCK in the brain is the sulfated form of the COOH-terminal octapeptide
Date received 22 August 1988 Date accepted 20 October 1988
(CCK-8-S) (1, 2, 3), followed by the tetrapeptide (CCK4) (1, 2, 3). Other molecular forms of CCK-like peptides are CCK-58, CCK-39, CCK-33 and CCK-12 in mammalian brain (4,5,6,7,8) and a non-sulfated form (CCK-8-NS) in the rat brain /o, [O’* Recently, neuropharmacological studies have indicated that CCK-8 and its related peptides produce behavioural changes resembling those of neuroleptics in rodents when injected centrally as
107
108 well as peripherally (9, 10). However, differences in behavioural effects especially neuroleptic-like properties, have been noted in rodents after intracerebroventricular (ICV) administration of CCK-8 related peptides. These differences in behavioural effects following ICV administration may be due not only to differences in animal the experimental procedures species and employed, but also to CCK heterogeneity. In the present study, we investigated the effects of CCK-8 related peptides on spontaneous locomotor activity, rearing and methylphenidateinduced hyperactivity in mice after ICV administration.
Materials and Methods General
Male ddY strain mice weighing 2533g were housed in groups of 6 mice per cage in a temperature-controlled animal room with a 12:12 hour light/dark cycle (light on at 8:OOa.m.) with free access to commercial food and tap water. CCK-8 related peptides tested were administered to unanesthetized, manually restrained animals ICV in a volume of 10 ~1 by the method of Haley and McCormick ( 11). To ascertain the areas into which the needles were inserted, the brains were sectioned and studied histologically after the experiment.
NEUROPEPTIDES
Behavioural measurements
Evaluation of locomotor activity and rearing rate have been described previously (12). Each test session was 10 min. Both locomotor activity and rearing rate were determined 10 or 30 min after ICV injection of drugs tested. After a 50-min acclimation period in the test cage, methylphenidate was injected intraperitoneally just after ICV administration of CER-S at a dose 800ng in a volume of 10 ~1 and locomotor activity was evaluated every 10 min for 120 min. Measurement plasma
of CER-like
immunoreactivity
in
To evaluate whether the behavioural effects of ICV-administered CER-S were due to central or peripheral action, plasma levels of CER-like immunoreactivity (CER-LI) were determined after ICV and subcutaneous (SC) administration of CER-S. Blood samples were obtained via heart puncture under ether anesthesia using a heparinized syringe. Heparinized blood sample from 3 different animals were collected into a small glass tube containing EDTA (1.5 mg/ml blood) and trasylol (500 IU/ml blood). Then, the plasma was separated by centrifugation at 1600 g for 15 min at 4°C and stored at -80°C until used. CER-LI was measured by radioimmunoassay using antiserum directed towards the N-terminal of CER (13). No cross reactivity for other related peptides such as CCK-8, CCK-33, gastrin-II was observed (0.01%). Statistical analysis
Chemicals CCK-8-S,
its non-sulfated form (CCK-8-NS) and CCK-4 were purchased from Peptide Institute Inc. (Osaka, Japan); ceruletide diethylamine (CER-S) and its non-sulfated (CER-NS) were gifts from Shionogi Company (Osaka, Japan); methylphenidate hydrochloride was a gift from Ciba-Geigy (Basal, Switzerland). CCK4 was dissolved in distilled water containing 0.01% cysteine and 2% sodium bicarbonate. CER-NS was dissolved in 1% sodium bicarbonate and was neutralized with 0.1 N hydrochloric acid. Other peptides were dissolved in physiological saline. All solutions were freshly prepared just before use.
Means and standard errors of the means were calculated using statistical procedures. Student’ t-test (two-tailed) was used comparison against the control group treated with the physiological saline or vehicle.
Results Effects of ICV-administered CCK-8-S motor activity and rearing rate
on loco-
CCK-8-S caused a significant, but not doserelated, reduction in both locomotor activity and rearing rate during the lo-min test session 10 min
INTRACEREBROVENTRICULARLY
ADMINISTERED
after ICV administration at doses ranging from 25 to 800ng (Table 1). The inhibitory effects of CCK-8-S were also observed 30 min after ICV administration at a dose of 800ng but not at a dose of 200ng (Table l), suggesting that its potency is not dose-related, but the duration is prolonged dose-dependently.
Effects of NIV-administered CER-S on locomotor activityand rearing rate Effects of CER-S on locomotor activity and rearing rate 10 min after ICV administration were investigated at doses ranging from 1.0 to 25ng. CER-S at doses ranging from 3.125 to 25 ng caused a significant, but not dose-related, reduction in locomotor activity during the lo-min test session (Table 2). The effect on rearing rate was also observed at doses ranging from 6.25 to 25ng, but was not dose-dependent (Table 2). Effects of CER-S on locomotor activity and rearing rate 30 min after ICV administration were studied at doses ranging from 50 to 800ng. CER-S significantly decreased the rates of both behavioural parameters (Table 2).
Table 1
Effects of ICV-Administered
Effects of ZCV administration of other CCK-8 related peptides on locomotor activityand rearing CCK-&NS,
Efiect of ICV-administered CER-S on methylphenidate-induced hyperactivity Effect of CER-S on methylphenidate-induced hyperactivity was studied at a dose of 8OOng, since its inhibitory effects on both locomotor activity and rearing were still observed 30 min after ICV administration. CER-S (800ng in a volume of 10~1) inhibited methylphenidate-induced hyperactivity from 30 min to 80 min after ICV administration (Fig 1). Plasma levels of CER-LI after ICV and subcutaneous administration of CER-S Plasma CER-LI levels 15 and 30 min after ICV administration of 800ng CER-S in a volume of 10 t.Awere 3.2 + 0.2 and 1.4 + 0.1 @ml, respectively (Fig 2). Plasma levels of CER-LI reached the peak values 15 min after SC injections of 100
CCK-8-S on Locomotor Activity and Rearing Rate in Mice The 10 min test session
The 10 min test session
10 min after ICV administration
30 min after ICV administration
Number
Vehicle CCK-8-S 12sng 25 ng 50 ng 100 ng 200 ng 400 ng 800 ng Vehicle CCK-8-S 200 ng 800 ng
44
344.7 k 21.6
18.5 + 3.1
11 11 6 10 12 12 13 22
286.1 + 44.4 165.4 zb 26.2** 160.8 + 29.6”** 212.1 IL 27.3** 185.2 rt 36.4**’ 155.2 -t 32.2*** 204.2 * 15.8**’
13.7 5.2 2.7 5.9 4.0 2.4 4.8
12 12
and CCK4 had no effect on at doses of up to 1.6 to 1Okg
CER-NS
either parameter (Table 3).
Treatment and dose per rat
of animals
109
CCK-8 RELATED PEPTIDES IN MICE
Locomotor
activity Rearing rate (Mean + S. E. M.)
Locomotor
activity Rearing rate (Mean + S. E. M.)
+ 4.1 + 1.8*** + 0.8*** ?r 1.5*** f 1.3*** +- l.t*** + 1.4*** 597.5 t 45.8
40.3 -I 6.5
524.9 -I 69.1 297.5 + 30.3***
38.6 f 12.1 12.2 z!z2.7***
The data were analysed using Student’s t-test (two-tailed) for comparison against the group treated with vehicle. ** p < 0.01; ***p< 0.001. Abbreviations: CCK-S; sulfated form of cholecystokinin octapeptide, ICV: intracerebroventricular(ly).
110
NEUROPEPTIDES
and 200 p@kg of CER-S and the mean peak values were 18.6 + 1.6 and 33.7 + 2.9ng/ml, respectively. Plamsa CER-LI was still detected 120 min after SC injection of CER-S. Discussion Spontaneous locomotor activity of rats was not affected by CCK-8-S (14,15) but increased by CCK-&NS following administration (14). HowTable 2
Effects of ICV-Administered
Treatment and dose per rat
Vehicle CER-S 1.0 ng 3.125ng 6.25 ng 12.5 ng 25.0 ng Vehicle CER-S 50 ng 100 ng 200 ng 800 ng
ever, van Ree et al. have demonstrated that ICV-administered CCK-8-S but not CCK-8-N& produced significant immobility in rats at a dose level of 2Opg (16). Likewise, conflicting results have been reported that methamphetamineinduced hyperactivity of rats was inhibited by ICV administration of CCK-8-S but not of CCK-8-NS (14), ICV injected CCK-8-S did not alter amphetamine-induced locomotor activity of rats (15). Therefore, we examined the neuropharmaco-
CER-S on Locomotor Activity and Rearing Rate in Mice The 10 min test session 10 min after ICV administration
Number
Locomotor
of animals
Rearing rate activity (Mean + S. E. M.)
41
322.8 f 26.2
16.2 + 2.7
10 9 10 11 11 31
322.2 201.2 186.3 164.9 131.0
14.5 5.8 4.5 5.6 3.0
f f f + ?
The 10 min test session 30 min after ICV administration
39.5 37.9* 27.1** 28.8** 18.6**’
Locomotor
activity Rearing rate (Mean + S. E. M.)
+ 3.9 f 3.3 + 1.8*** IL 2.8* f 1.1***
13 15 15 12
637.0 + 33.0
42.9 f 4.0
558.3 536.1 369.3 310.2
36.0 34.3 13.5 9.3
f f f +
46.5 59.0 30.5*** 33.5***
+ f + zt
3.2 6.5 3.0*** 2.2***
The data were analysed using Student’s t-test (two-tailed) for comparison against the group treated with vehicle. * p < 0.05; **p< 0.01; *** p < 0.001. Abbreviation: CER-S; sulfated form of ceruletide.
Table 3 Effects of ICV-Administration Rate in Mice
of Other CCK-8 Related Peptides on Locomotor Activity and Rearing
The 10 min test session 10 min after ICV administration
Number Treatment
Vehicle CCK-&NS Vehicle CCK-4 Vehicle CER-NS
Dose per rat
0.8ug 1.6ug 1.6kg lO.Oug 1.6tq
Locomotor
of animals
19 10 10 23 11 10 12 11
Abbreviations: CCK-8-NS; non-sulfated form of cholecystokinin non-sulfated form of ceruletide.
253.9 275.6 266.8 243.0 207.3 183.6 282.8 219.4 octapeptide,
+ + f t + f + f
activity (Mean _+ S. E. M.)
Rearing rate 14.8 13.2 19.1 9.4 9.7 2.8 17.2 10.2
30.3 40.5 56.7 34.0 65.5 36.5 37.2 28.0
CCK-4; cholecystokinin
tetrapeptide,
+ f + + f + + f
3.0 4.2 6.9 3.7 5.0
1.0 5.2 2.3
CER-NS;
INTRACEREBROVENTRICULARLY ADMINISTERED CCK-8 RELATED PEPTIDES IN MICE lWUCEE+LLI.+WEllWLll?.
o-o
IllhI
.-.
CBIYII
II41 I III
111
logical action of CCK-8 related peptides on spontaneous locomotor activity, rearing rate and methylphenidate-induced hyperactivity in mice after ICV injection. Effects of ICV-administered CCK-8 related peptideson spontaneous locomotor activityand rearing rate
+l
b
120me.
60
Fig 1 Effect of XV-administered CER-S on methylphenidate-induced hyperactivity Each value is the mean + S.E.M. of the number of mice indicated in parenthesis. The data were analyzed using Student’s t-test (two-tailed) for comparison against the group treated with physiological saline. * p < 0.05; ** p < 0.01. Abbreviations: MPD; methylphenidate, I.C.V.; intracerebroventricularly administration, I.P.; intraperitoneal administration.
O-O
CEI-StWmU.
151
O-O
CL,S INHS.C.
151
CEB-S880 m51C.V. I8 I
0
‘0 1550
00
140ah.
Fig 2CER-LI plasma levels after ICV and subcutaneous administration of CER-S Each value is the mean f S.E.M. of the number of plasma samples indicated in parenthesis. Abbreviations: CER-LI; ceruletide-like immunoreactivity, S.C.; subcutaneous administration.
ICV administration of CCK-8 and CER-S significantly decreased the rates of locomotor activity and rearing at doses ranging from 25 to 8OOng. These findings indicate that CER-S is much more potent in inhibiting locomotor activity and rearing than CCK-8 (12,16). Similar results were obtained in our previous study in which these two peptides were SC administered (12). In addition, the present findings are in good agreement with I& values in competitive inhibition of [propionyl-3H] propionylated CCK-8 ([3H]pCCK-8)-specific binding to the slide mounted tissue sections of the rat brain by CCK-8-S and CER-S (17). This suggests that ICV-administred CCK-8-S or CER-S directly affects the brain function. In contrast to SC administration, however, no dose-related inhibition was observed after ICV administration. At present, the reasons for this discrepancy are unclear. CCK-S-S at dose of SOOng, but not of 2OOng, had an inhibitory effect on the rates of locomotor activity and rearing 30 min after its ICV administration. Such inhibitory effects were also observed after ICV administration of CER-S at doses of 200 and SOOkg, but not of lOOl.~g.These findings indicate that the duration of the inhibitory effect is related to the dose given. In our competitive binding studies, four peptides structurally related to CCK-8, e.g. CER, CCK-8, CCK-S-NS and CCK-4, completely inhibited binding of [3H] pCCK-8 to the tissue sections from the rat brain (17). The order of inhibitory potencies of the peptide tested were CER CCK-8 CCK-S-NS CCK-4. However, no behavioural changes were in mice after ICV administration of CER-NS or CCK-NS or CCK-S-NS at doses ranging from 0.8 to 1.6pg. Thus, the presence of O-sulfate on the tyrosine residue in the CCK-molecule seems to be necessary for the inhibitory effect of CCK-8 related peptides on the locomotor activity and
112 rearing in mice after ICV administration (12). In the present study, furthermore, CCK-4 at doses more than 1.6 pg caused no behavioural changes in mice. These finding, taken together with those on CCK-8-NS and CCK4 in the mammalian brains, indicate that CCK-8-NS and CCK-4 in the brain have different physiological roles from CCK-8-S. ICV-administered [ ‘251]CCK-8 demonstrated to diffuse rapidly into the peripheral blood by a non-carrier-mediated mechanism (18). In our previous study, SC-administered CCK-8-S and CER-S at doses more than 50 and 25l~g/kg, respectively, inhibited locomotor activity and rearing in mice (12). At doses more than 100 l.~g/ kg, these inhibitory effects were observed 60 min after the administration. To clarify whether or not the central effects of CCK-8-S and CER-S are the results of a peripheral redistribution of these peptides, we measured the plasma CER-LI levels after ICV and SC administrations of CER-S. The plasma CER-LI level 30 min after ICV administration of 800ng CER-S was about one-fifth of that at 120 min after SC injections, when the locomotor suppressive activity induced by 200 kg/ kg of CER-S was no longer observed. Thus, the inhibitory effects of CCK-8-S and CER-S after ICV administration may be due to their central actions. Effect of ICV-administered CER-S on methylphenidate-induced hyperactivity was hyperactivity Methylphenidate-induced inhibited by ICV-administered CER-S. Methylphenidate, an indirect dopamine (DA) agonists, causes DA release from nerve terminals of DA neurons in the brain, resulting in behavioural changes, such as hyperactivity and stereotypy, in rodents. Thus, this finding suggests that ICVadministered CER-S affects the neural transmission in central DA systems, antagonizing methylphenidate-induced hyperactivity. Furthermore, it seems likely CCK-8-S in the brain is closely associated with the central DA functions. In conclusion, CCK-8-S and CER-S, but not their non-sulfated forms or CCK-4, caused a reduction in the rates of locomotor activity and rearing in mice when ICV-administered. In addiICV-administered CER-S inhibited tion,
NEUROPEPTIDES
methylphenidate-induced hyperactivity. These findings indicate that (1) the structural requirement for the locomotor suppressive activity is a sulfated tyrosine residue; (2) the behavioural changes induced by ICV-administered CCK-8-S and CER-S are mediated through their inhibitory effects on the central DA functions; consequently (3) CCK-8-S in the brain is functionally associated with the central DA systems; and (4) there are different structure-central nervous system (CNS) action relationships in the CNS-mediated effects of CCK-8 related peptides in the brain.
Acknowledgements We are greatly indebted to Shionogi Company (Osaka, Japan) for the kind gift of ceruletide diethylamine and its non-sulfated form and for the measurement of plasma levels of ceruletide. This study was supported in part by a research grant from the Japanese Ministry of Health and Welfare (to R.I.)
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INTRACEREBROVENTRICULARLY
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CCK-8 RELATED
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PEPTIDES IN MICE
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