Effects of ceruletide and haloperidol on the hypothalamo-pituitary β-endorphin system and brain β-endorphin contents in the rat: With special reference to effects of ceruletide in chronically haloperidol-treated rats

Neuropeptides (1991) 18, 1-14 0 Longman Group UK Ltd 1991

Effects of Ceruletide and Haloperidol On The Hypothalamo-pituitary P-Endorphin System And Brain P-Endorphin Contents In The Rat: With Special Reference To Effects Of Ceruletide In Chronically Haloperidol-Treated Rats Y. HAGINO”,

M. OKUWA*t,

AND T. MOROJI”

*Department of Psychopharmacology, Psychiatric Research Institute of Tokyo, 2-l-8 Kamikitazawa, Setagaya-ku, Tokyo 156, Japan tNew Product Development Department, Shionogi & Company Ltd, Osaka, Japan (Reprint requests to TM)

Abstract-Subcutaneous (SC) administration of 200kg/kg ceruletide (CER), a decapeptide chemically related CCK-8, and 5mg/kg haloperidol (HLP) to rats increased the plasma immunoreactive p-endorphin (ir-P-END) level. The combined injection of CER and haloperidol caused higher plasma ir-P-END levels than either drug alone. High plasma ir-p-END levels returned to control levels on the 2nd day. Prior intraperitoneal (ip) administration of a CCK receptor antagonist, L-364,718 (3mg/kg), but not proglumide (400mg/kg, ip), inhibited CERinduced, but not HLP-induced, elevation in plasma it--P-END levels. The dopamine agonist, bromocriptine (1 mg/kg, ip) decreased plasma ir-p-END levels, but had not effect on CERinduced elevation in plasma ir-p-END levels, whereas bromocriptine-induced reduction in plasma ir+-END levels was antagonised by HLP. CER injection to chronically HLP-treated rats caused a greater elevation of plasma ir+-END levels compared to saline-injected rats. In contrast to the acute experiment, plasma ir-p-END levels remained elevated over a period of 24h. In the acute experiment, CER, HLP or the combined treatment with these two drugs had no effect on ir-P-END contents in the pituitary gland and brain. In the chronic experiment, HLP increased the adenohypophyseal and septal ir-P-END contents and decreased the hippocampal it--P-END contents 24h after the final HLP injection. CER caused a small reduction only in the hippocampal ir-p-END contents of CER-injected rats 15min after injection. When determined on the 2nd day, however, the increases in the adenohypophyseal and septal ir+END contents and the decrease in the hippocampal ir+-END contents observed in CER-injected rats were of the same magnitude as those of rats not given the CER injection. These findings indicate that CER stimulates the release of ir-P-END from the Date received 5 June 1990 Date accepted 20 July 1990

2

~EUROPE~~ES

adenohypophysis through CCK-A receptors and that elevated plasma ir-@END levels is partly involved in some behavioural effects induced by CER. Furthermore, sustained elevation of plasma ir-P-END levels after a single injection of CER to chronically HLP-treated rats may explain its long-lasting therapeutic and behavioural effects.

Cholecystokinin octapeptide (CCK-8) and ceruletide (CER), a decapeptide chemically related to CCK-8, have various central effects, i.e. satiation, behavioural sedation, analgesia and neurolepticlike effects when administered intracerebroventricularly (ICV) or systemically. Zetler and M~rsdolf (1) reported that an enkephalin analogue, (D-Ala)* (MePhe)4(Met(0)-ol)5-enkephalin (FK 33-824), antagonises the inhibitory effect of CER on food intake and that the opioid antagonist, naloxone, enhances it. Wilson et al (2) also demonstrated that the pre-treatment with morphine antagonises the suppress~t action of 0X-8 on food consumption in the rat and naloxone potentiates the CCK-8 effect on feeding. Similarly, ICV and systemic administration of CCK-8 and CER potentially antagonised opiate-mediated analgesia produced by foot shock, morphine and B-endorphin, but not opiate-independent analgesia (3,4). In contrast, CCK-8 and CER have been demonstrated to possess analgesic properties in the hot-plate test and in the writhing test in the rat. The analgesic effect of CCK-like peptides is antagonised by very low doses of naloxone. CCK-8 and CER alter pituitary hormone release in rats, which suggests that CCK related peptides act as a neurotransmitter or modulator of neuronal activity controlling the release of pituitary hormones (S-7). Basso et al (8) demonstrated that intravenously injected CER significantly increases P-endorphin (P-END) levels in both plasma and cerebrospinal fluid (CSF) and has a psychotonic effect in particular on the anxiety state and mood in patients with psychogenic headache. Thus, they speculated that the effect of CER on mood and pain is mediated by an increase in P-END release. CCK immunoreactive nerves have been demonstrated to display a distribution similar to that of enkephalin, which suggests a functional interaction between these two neurotransmitter systems

in the brain (9). In addition, Kiraly and van Ree (10) showed that endogenous opioids are concerned in certain interaction between CCK-8 and dopamine (DA) systems. Matsubara and Matsushita (11-12) suggested that when given together with haloperidol (HLP), the long-lasting antagonistic effect of CER on amphet~ine-induced h~eractivity in rats is mediated by the endogenous P-END. Previously, we reported that when administered ICV and systemically, neurolepticlike properties of CCK-8 and CER are mediated by their inhibitory effects on the central DA functions (13-14). The purposes of this study were: a) to investigate the effect of CER on plasma levels of immunoreactive ~-endorphin (ir-~-END) in the rat, including effects of CCK-A type receptor antagonist, L-364,718, and the D2 agonist, bromocriptine on CER-induced ir-P-END release; b) to study the effects of CER on ir$-END in both rat brain and pituitary gland following the acute and chronic HLP treatment.

Materials and Methods Male Wistar rats weighing 290-3108 were used throughout the experiments. The animals were housed two to a cage with free access to food and tap water in a temperature controlled room (22 St 2OC) under a 12:12h ligh~dark cycle (light on at 8.00a.m.) In the acute experiments, the animals were sacrificed by decapitation 15min and 60min after the subcutaneous (SC)injection of CER at a dose of 200 pg/kg. Pre-treatment with vehicle, L-364,718 (3 mg/kg intraperitoneally (ip)) or proglumide (4~mg/kg ip) was performed 45 min prior to CER administration. Bromocriptine (1 m&kg ip) was administered 75min prior to CER treatment. In addition, effects of L-364,718 (3mg/kg ip) and bromocriptine (1 mg/kg ip) on HLP-induced ir+-END release were studied. L-364,718 was injected concurrently with HLP (5 mg/kg SC). Bro-

EFFECTS OF CERULETtDE AND HALOPERIDOL ON THE HYPOTHALAMO-PITU~ARY

mocriptine was ip injected 30min prior to the sc administration of HLP. The animals were sacrificed by decapitation 6Omin following the SCinjection of 5 mg/kg HLP. Finally, the effects of combined treatment with CER and HLP on ir+-END in the plasma, pituitary gland and various discrete regions of the brain were investigated. The pre-treatment with HLP or vehicle was performed 45min prior to the SCinjection of CER. The animals were sacrificed by decapitation 1 or 24 h after the pre-treatment with HLP or vehicle. In the the chronic experiments, HLP (5m~kg SC)and vehicle were SCadministered once a day for 3 weeks. CER or physiological saline was SC injected 45min after the final HLP injection. The animals were sacrificed by decapitation 60min or 24h after given the final injection. CER and proglumide were dissolved in physiological saline. HLP and bromocriptine were dissolved in 1% lactic acid and neutralised with 0.05 N sodium hydrogen carbonate. L-364,718 was suspended in 0.5% methylcellose. CER and HLP were SCinjected in a volume of O.lm~l~g body weight. L-364,718, proglumide and bromocriptine were ip injected in a volume of 0.2ml/lOOg body weight. Preparations of plasma and tissues

Trunk blood was collected into test tubes containing EDTA (1.5 mg/ml blood) and Trasylol(5OOIU/ ml blood), and plasma was separated be centrifugation at 16OOgfor 20min at 4°C. The brain was quickly removed from the skull after decapitation and dissected into six discrete regions according to the method described by Glowinski and Iversen (15). The pituitary was divided into the adenohypophysis and neurointermediate lobe. Tissue and plasma samples were stored frozen at -80°C until use. The dissected brain tissues and pituitary lobes were weighed and homogenised by sonication in 0.1 N acetic acid. After the homogenates were boiled for 1Omin and centrifuged at 25,OOOg for 20min at 4”C, the supernatant was lyophilised and stored at -80°C until use. The protein was measured by the method of Lowry et al (16). Plasma n-B-END was extracted according to the method described by Matsumura et al (17). Plasma (2.5 ml) was mixed with 5 ml of acid acetone (acetic

~-ENDORPHIN SYSTEM IN THE RAT

3

acid: acetone, 3: 100, v/v) and centri~ged for 5 min at 16OOg. The supernatant was washed twice with 1Oml of petroleum ether and lyophilised. Enzyme immunoassay procedure

Our enzyme immunoassay (EIA) method was described in detail elsewhere (18). A 96 well flat-bottom polystyrene microplate (Dynatech, West Germany) was coated with 1.28u.g of goat antiserum against rabbit IgG per well in 250~~1in 0.1 M carbonated buffer (pH 9.6) by incubation at 4°C overnight. After incubation each well was rinsed five times with 0.02M phosphate-buffered saline (pH 7.4), containing 0.05% Tween-20 (washing buffer). Then, 1OOpJ assay buffer (0.14M phosphate buffer pH 7.4 containing 25 mM EDTA, 0.5% bovine serum albumin (BSA) and 0.02% Tween 20), 50~1 standard or unknown sample prepared in assay buffer and 50~1 B-END antiserum were added to each well. After overnight incubation at 4°C 50~1 of horseradish peroxidase (HRP~-~-END conjugate (1:2000 diluted with assay buffer, the HRP conjugated with P-END by a periodate oxidation method (19) was added to each well and allowed to react at 4°C for 4 h. Each well was washed five times with washing buffer. 250~1 of substrate (44mg of o-phenylenediamine and 100(~1 of 3% H201 dissolved in 100ml of 0.1 M phosphatecitrate buffer pH 5.0) was added in each well and incubated at room temperature for 20min. The reaction was stopped by the addition of 50 ~1 of 5 N of the resulting HZSO+ The absorbance chromogen was obtained by a Microplate photometer MTP-22 (Corona Electric Co, Japan) at 492nm. The intraassay variation is 5.5% and sensitivity is 5pg/well. HPLC separation of B-END immunoreactive peptides. P-END immunoreactive peptides were separated by reverse-phase high performance liquid chromatography (HPLC) using a Shimadzu Model LC-6A liquid chromatograph equipped with a Nucleosil column (300-7C4, 50 x 4.6mm; Chemco Scientific Co, Ltd, Japan). The lyophilised extract was reconstituted in lOOir_lof phosphate buffer (pH 7.4) and 80~1 was layered onto the HPLC column after centrifugation. A 30mi non-linear gradient from 12.5-36% acetonitril in 0.1% tri-Auoroacetic acid with lmllmin flow rate

4

NEUROPEPTLDES

was used routinely. Fractions were collected at 1 min intervals and processed for EIA after lyophilisation. Drugs and antisera

Ceruletide diethylamine and L-364,718 were gifts Shionogi Research Laboratories (Osaka, Japan). Haloperidol was a gift from Yoshitomi Pharm. Co (Osaka, Japan). Rat l3-END1-31 and other B-END related peptides were purchased from Peninsula Lab Inc (San Carlos, USA). Human B-lipotropin was a gift from the National Hormone and Pituitary Program (USA). Bromocriptine mesylate was purchased from Sigma Chem Co (Chicago, USA). The P-END antiserum, which is raised in rabbits against porcine B-END, was purchased from Amersham International plc (Buckinghamshire, UK). This antiserum recognises B-END1_31 and its a-N-acetylated form on an approximately equimolar basis. The cross-reactions between human @lipotropin, camel 13-END1-27,camel l3-ENDI-26, camel acetyl B-ENDi-3i’ camel acetyl P-ENDi-27 and camel acetyl P-END~_x in the assay were 55, 94, 71, 78, 57 and 52%, respectively. The cross-

0

0

I

15

00 Hil.

Effects of SC injection of CER on plasma levels of rmmunoreactive P-END. Each value represents the mean + SE of plasma samples indicated in parenthesis. The data were analysed using Student’s t-test (two-tailed) for comparison against the saline-treated group. * p < 0.05. Abbreviations: CER; ceruletide, SAL; saline, HLP; haloperidol, VEH: Vehicle, ir+-END; immunoreactive g-endorphin.

reactions with OL-and y-endorphin, leucine- and methionine- enkephalins were less than 1%. The following were purchased: goat antiserum against rabbit IgG (Miles-Yeda Ltd, Israel); horseradish peroxidase (Toyobo Co Ltd, Japan); o-phenylenediamine dihydrochroride (Sigma Chem Co, Chicago, USA).

Stastistical analysis

All data are shown as the arithmetic mean (f SE). The statistical significance of differences between values in different groups was analysed by Student’s t-test (two-tailed). P values less than 0.05 were considered to be statistically significant. Results

Effects of SCinjection of CER on plasma levels of P-END. Plasma levels of immunoreactive ir-B-END were increased by approximately 170% 15 min after SCadministration of 200 pg/kg CER as compared to those of saline-injected rats (p < 0.05). Plasma ir-P-END levels returned to control levels 60min after SCinjection of CER (Fig. 1). Effects of SC injected CER on the molecular forms of plasma B-END immunoreactive peptides. The effect of CER on the individual molecular forms of B-END immunoreactive peptides was determined by separating ii--B-END using a reverse-phase HPLC equipped a Nucleosil column. The reverse-phase HPLC revealed three major immunoreactive peptides in the rat plasma 15 min after the injection of 2OO&kg CER or saline. The first and second major immunoreactive peptides eluted in the same positions as authentic ir-P-END peptides, such as camel B-END1-3i, rat B-END1-31, camel B-END1-27, human B-END1_3i and camel 13-END1-z6, and human B-lipotropin, respectively. Thus, the first major immunoreactive component was tentatively identified as ir-P-END consisting of 13-END1-26, l3-ENDr-27 and l3END1_3i. The second major peak was considered to correspond to B-lipotropin. However, the third major immunoreactive component could not be identified. The SCadministration of 200&kg CER caused a parallel elevation of all three major immunoreactive components in the rat plasma (Fig. 2).

EFFECTS OF CERULETIDE AND HALOPERIDOLON THE HYPOTHALAMO-PITUITARY

b-0 ScllitlO! 0-0

CER I ZOOylik# l.c. 1I.5 I

100

Fig. 2 Effects of sc injected CER on the molecular forms of plasma P-END immunoreactive peptides. Animals were treated with CER (200pg/kg) or saline 15min before decapitation. Plasma extracts were separated by reverse-phase HPLC. Fractions were collected at lmin intervals and were analysed for ir-p-END by enzyme immunoassay. The arrows (left to right) correspond to the elution positions of the reference peptides: camel P-endorphinr_3r, rat @-endorphinr3f, camel ~-endo~hin~-~~, human ~-endorphin,~~~, camel P-endorphinr..a6, camel acetyl P-endorphint_31, camel acetyi f3-endorphinl_z7, camel acetyl S-endorphinr_z6, human f3-lipotropin. Each value represents the mean + SE of plasma samples indicated in parenthesis. The data wee an~ysed using Student’s t-test (two-tailed) for comparison against the saline-treated group. ** p c 0.01. Abbreviations: CER; ceruletide, SAL; saline, HLP; haloperidol, VEH: Vehicle, ir-S-END; immunoreactive ~-endorphin.

3

&ENDORPHIN SYSTEM IN THE RAT

Thus, ir+-END in the adenohypophysis originated mainly from B-END and B-lipotropin, whereas ir-B-END in the neurointermediate lobe from P-END and its acetylated derivatives. Effects of the pre-treatment with two different types of pe~pheral CCK receptor blockers and dopamine agonist on CER-induced ir-B-END release. Plasma ir+-END levels were markedly increased 1.5min after SC injection of 2OOFg/kg CER. The two different types of peripheral CCK receptor blockers, L-364,718 (3mg/kg ip) and proglumide (4OOmg/kg ip), did not cause any changes in plasma is-@-END levels. However, the pre-treatment with 3mg/kg of L-364,718 completely inhibited CER-induced elevation in plasma ir-B-END levels, but not proglumide when given ip at a dose of 400mglkg (Fig. 4A). The pre-treatment with the DA agonist, bromocriptine (lmg/kg ip), had no effect on CER-induced elevation in ir-P-END levels, plasma whereas plasma ir+-END levels were significantly reduced by bromocriptine alone as compared to those of the rats treated with saline (Fig. 4B). Effects of L-364,718 and bromocriptine on HLP-induced ir+-END release. The SC administration of .5mg/kg HLP produced a marked elevation of plasma ir-B-END levels. Bromocriptine at a dose of lmg/kg (ip) but not L-364,718 this HLP-induced (3m~kg ip), antagonised ir+-END release (Figs 5A and 5B).

_ j

Aloncbypophyrir

I

sir

H t =:

25’

3

The molecular forms of ir-B-END in the pituitary gland. In the adenohypophysis (Fig. 3A), B-ENDiand B-lipotropin were major immunoreactive peptides. Acetylated derivatives of P-ENDr-s1 and l3-ENDi-27 were also present in this tissue but in relatively small quantities. The major immunoreactive peptide in the neurointermediate lobe comigrated with acetylated derivatives of B-END1-27 and B-END1-26 (Fig. 3B). In addition, the neurointermediate lobe contained the acetylated form of B-ENDi-3i and B-ENDI_31.

0

Frlrtitr

II.

Fig. 3 The molecuiar forms of immunorea~tive ~-endo~hin in the pituitary gland. Pituitary extracts were separated by reverse-phase HPLC. Fractions were collected at 1 m intervals and were analysed for ir-@-END by enzyme immunoassay. The arrows (left to right) correspond to the eIution positions of the reference peptides: camel f3-endorphinI_B1rat f3-endorphinr_31, camel f%endorphinI_z7, human S-endorphin,_31, camel P-endorphinl_r6, camel acetyl f3-endorphinr_3r, camel acetyl P-endorphinr_ar, camel acetyl ~-endo~hin~_~~, human ~-~i~tropin. Abbreviation: ir-P-END; immunoreactive p-endorphin.

6

7mimmlZ

NEUROPEPTIDES

Vulll SAL CER

Pdmun SAL CEI

L.J(I4.718 S11 CEI

YYdl SAL CER

ML CEI

Fig. 4 Effects of the pre-treatment with two different types of peripheral CCK receptor antagonists and dopamine agonist on CER-induced ir-B-END release. The animals were sacrificed by decapitation 15min after SCinjection of CER (2OOpg/kg). The pre-treatment with vehicle, L-364,718 (3mg/kg ip) or proglumide (4OOmg/kgip) were performed 45min prior to CER administration. Bromocriptine (1 mg/kg ip) was administered 75min prior to CER treatment. Each bar and vertical line represent the mean + SE. The number in the column represents the number of plasma samples. Statistical differences were analysed by Student’s t-test (two-tailed). * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: SAL; saline, CER; ceruletide, ir-B-END; immunoreactive B-endorphin.

Effects of the combined treatment with CER and HLP on plasma ir+-END levels. As mentioned above, CER (200&kg SC) and HLP (5mg/kg SC) caused an elevation in plasma ir+-END levels, respectively. The combined injection of CER and HLP produced significantly higher plasma ir-P-END levels than CER alone, but not HLP (Fig. 6). High plasma ir+-END levels caused by CER, HLP or the combined treatment #withthese two drugs returned to control levels on the 2nd day after the treatment (Fig. 6). Effects of chronic HLP administration on CERinduced elevation in plasma ir-P-END levels. Plasma ir+-END levels in chronically HLPtreated rats 1 h after the final drug injection were significantly higher than those in vehicle-treated rats. When 200 kg/kg of CER was SCadministered 45min after the final injection of HLP, plasma ir-P-END levels were higher, but not significantly different from those in rats without CER treatment (Fig. 7). In contrast to the acute experiments, plasma ir-P-END levels in chronically HLP-treated rats remained significantly elevated over a period of 24h (Fig. 7).

Effects of CER or HLP administration and the combined treatment with these two drugs on ir+-END contents in the pituitary gland. In the acute experiments, the SCadministration of 200 kg/ kg CER or 5mg/kg HLP and the combined treatment with these two drugs had no effect on ir-P-END contents in both adenohypophysis and neurointermediate lobe (Table 1). Furthermore, no changes in ir-P-END contents in both pituitary lobes were observed 24 h after each drug treatment. Regardless of the treatment with CER, ir-P-END contents in the adenohypophysis of chronically HLP-treated rats were significantly higher 24h after the final HLP injection than at 60min. Ir-P-END contents in the neurointermediate lobe also showed a tendency to be increased, but not statistically significant (Table 2). The molecular forms of ir-P-END in various discrete brain regions of the rat. The reverse-phase HPLC revealed two major ir+-END, e.g., I3-END1_31 and /3-ENDi_27, in all the various discrete brain regions studied, except for the hypothalamus (Fig. 8). In addition, the acetylated form of l3-END1-27 was present in the striatum

a 0.6

b r ** 1

r’**7

-

6.6

7

I

*** --

-iv-

? 0.6 = J R4 c: 0.3 c i] 0.3 Z 0.1

7r~almanlL VEH

IllP

L-364.716 VEH HLP

Mm VEH HLP

VEII

HLP

Fig. 5 Effects of L-364,718 and bromocriptine on HLPinduced ir+END release. The animals were sacrificed by decapitation 60min after SC injection of HLP (5mg/kg). L364,718 (3mgIkg ip) was injected concurrently with HLP. Bromocriptine (1 mg/kg ip) was injected 30min prior to HLP treatment. Each bar and vertical line represent the mean + SE. The number in the column represents the number of plasma samples. Statistical differences were analysed by Student’s t-test (two-tailed). * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviations: VEH; vehicle, HLP; haloperidol, ir-B-END; immunoreactive B-endorphin.

EFFECTS OF CERULETIDE AND HALOPERIDOL ON THE HYPOTHALAMO-PITUITARY

B-ENDORPHIN SYSTEM IN THE RAT

7

regions. In the acute experiments, saline or CER was SCinjected 45 min after the pre-treatment with vehicle or HLP (5mg/kg) and then sacrificed by decapitation 15min after SCinjection of saline or CER. In all the various discrete brain regions studied (Table 3), there were no differences in

Id-

5 0.6: y O.B-

-?

(2

.L

Fig. 6 Effects of the combined treatment with CER and HLP on plasma h-P-END levels. The animals were sacrificed by decapitation 1 h (1st day) or 24 h (2nd day) after the pre-treatment with HLP or vehicle. The pre-treatment with HLP (5mg/kg SC) or vehicle was performed 45min prior to SC injection of CER (2OOpg/kg) or saline. Each bar and vertical line represent the mean + SE. The number in the column represents the number of plasma samples. Statistical differences were analysed by Student’s t-test (two-tailed). *p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviation: ir-P-END; immunoreactive @endorphin.

(STR), add P-lipotropin in the frontal cortex (FC), STR, nucleus accumbens (NAc) and hippocampus (HIPP) but in relatively minor quantities. Effects of CER or HLP administration and the combined treatment with these two drugs on ir-P-END contents in various discrete brain

z RI-

z =

L

I i

Fig. 7 Effects of chronic HLP administration on CERinduced elevation in plasma ir+-END levels. The animals were repeatedly treated with daily injections of HLP (Smg/kg SC)or vehicle for 3 weeks. CER (200 &kg SC)or saline was SCinjected 45min after the final HLP injection. The animals were sacrificed by decapitation 1 h (1st day) or 24 h (2nd day) after given the final HLP injection. Each bar and vertical line represent the mean t SE. The number in the column represents the number of plasma samples. The data were analysed using Student’s t-test (two-tailed) for comparison against the group treated with vehicle and saline. * p < 0.05, ** p < 0.01, *** p < 0.001. Abbreviation: ir+-END; immunoreactive P-endorphin.

Table 1 Effects of CER or HLP administration and the combined treatment with these two drugs on pituitary ir+-END contents in the rats. The animals were sacrificed by decapitation 1 h (1st day) or 24h (2nd day) after the pre-treatment with HLP or vehicle. The pre-treatment with HLP (5mg/kg SC)or vehicle was performed 45min prior to SCinjection of CER (200 &kg) or saline. Each value represents the mean k SE of the number of pituitary samples indicated in parenthesis. Abbreviations: CER; ceruletide, SAL; saline, HLP; haloperidol, VEH; vehicle, ir-P-END; immunoreactive P-endorphin.

Vehicle + saline

immunoreactive P-endorphin (kg-mgprotein) Vehicle Haloperidol + Ceruletide + Vehicle

Haloperidol + Ceruletide

Adenohypophysis

1st day 2nd day

1.3482 + 0.1067 (10) 1.3804 + 0.1691 (14)

1.1082 f 0.0854 (8) 1.2220 f 0.1802 (8)

1.0620 f 0.0899 (8) 1.2198 + 0.1261 (8)

1.1289 ?I 0.0987 (8) 1.6070 f 0.2805 (8)

Neurointermediate lobe

1st day 2nd day

17.6117 + 1.2228 (10) 23.0368 + 2.9299 (14)

14.6693 + 1.1293 (8) 18.6130 f 2.3435 (8)

15.6339 + 2.3413 (8) 15.0923 + 2.1468 (8)

18.1245 f 1.7991 (8) 13.5392 f 3.0584 (8)

8

NEUROPEPTIDES

final HLP injection. In addition, ir-P-END contents in the NAc tended to be reduced. CER at 200pglkg caused a decrease in the hippocampal ir-P-END contents in chronically HLP-treated rats 15 min after the SCinjection. However, ir-P-END contents in the other regions studied were not affected by the SCinjection of CER. On the other hand, the septal ir+-END contents were significantly increased at about 23 h after the injection of CER, whereas the hippocampal ir-P-END contents were decreased. There were no differences in the magnitude of increment in septal ir+-END contents and reduction in the hippocampal ir-P-END contents between CER- and saline-

Discussion

ll~ralb~lmur

4iziiTl ;;

,I,,,,

.E

I,

I

5 I.0

50

-= :

y 0.5 & .= o

26

5 IO I5 20 25 30 55 Fr1cli.n I@.

O

Fig. 8 The molecular forms of immunoreactive B-endorphin in various discrete brain regions of the rat. Brain extracts were separated by reverse-phase HPLC. Fractions were collected at lmin intervals and were analysed for ir+-END by enzyme immunoassay. The arrows (left to right) correspond to the elution positions of the reference peptides: camel B-endorphinr-sr, rat B-endorphinr-sr, camel f3-endorphinr_27, human B-endorphinr_sr, camel f3-endorphinI_26, camel acetyl B-endorphinr-sr, camel acetyl f3-endorphinr_27, camel acetyl B-endorphinr_a6, human B-lipotropin. Abbreviation: ir-B-END; immunoreactive B-endorphin.

ir-P-END contents between the groups 1 h and 24 h after the above-mentioned treatments. In the chronic experiments, the SC administration of 200t&kg CER caused no changes in ir-P-END contents in any brain regions of chronically vehicle-treated rats when determined 1 h and 24 h after the final injection. No changes in ir-P-END contents were found 1 h after the final HLP injection in any brain regions of chronically HLPtreated rats. However, the septal ir-P-END contents were increased and the hippocampal ir+END contents were decreased 24h after the

CCK-8 is present in the hypothalamus at high concentrations (20). In addition, CCK-containing axons and nerve endings have been found in the external layers of the median eminence (21). As mentioned in the Introduction section, these findings suggest that CCK acts as a neurohormone or neurotransmitter of hypothalamo-pituitary neuronal activity controlling the release of pituitary hormones. In in vivo and in vitro animal studies, CCK-8 stimulated the release of growth hormone and prolactin (5, 22). CCK-8 stimulates the release of P-END from the rat anterior pituitary as well (23-24). Furthermore, the finding that CCK-8, but not desulfated CCK-8 and CCK-4, causes dose-dependent stimulation of P-END release from dispersed cells of the rat anterior pituitary suggests that intravenously administered CCK-8 acts directly and specifically on anterior pituitary cells to stimulate P-END release. CER has been also reported to elevate P-END levels in both plasma and CSF in human after the intravenous administration of CER 1 or 2ng/kg/min for 15min (8). In the present study, CER at a dose of 200tqg/kg also caused an increment of plasma P-END levels 15min after SC injection without altering contents of ir-P-END in the adenohypophysis and neurointermediate lobe. The reverseplate HPLC revealed that CER produces a parallel elevation of three major P-END immunoreactive peptides, e.g., P-END size peptides, @lipotropinsize immunoreactive peptide and unidentified

EFFECTS OF CERULETIDE AND HALOPERIDOL ON THE HYPOTHALAMO-PITUITARY

9

f3-ENDORPHIN SYSTEM IN THE RAT

Table 2 Effects of CER or chronic HLP administration and the combined treatment with these two drugs on pituitary ir+-END contents in the rats. The animals were repeatedly treated with daily injections of HLP (5mg/kg SC)or vehicle for 3 weeks. CER (200 &kg SC)or saline was injected SC45min after the final HLP injection. The animals were sacrificed by decapitation 1 h (1st day) or 24h (2nd day) after given the final HLP injection. Each value represents the mean + SE of the number of pituitary samples indicated in parenthesis. The data were analysed by Student’s t-test (two-tailed) for comparison against the group treated with vehicle and saline. * p c 0.05. Abbreviations: CER; ceruletide, SAL; saline, HLP; haloperidol, VEH; vehicle, ir-P-END; immunoreactive P-endorphin. immunoreactive

P-endorphin (pglmg protein) Chronic haloperidol Ceruletide + Saline

Control

Adenohypophysis

1st day

2.1879 + 0.2218 (5)

2nd day Neurointermediate lobe

1st day 2nd day

35.7234

+ 8.6094 (5)

Chronic haloperidol + Ceruletide

2.0354 f

0.2192 (5)

1.8622 + 0.2094 (6)

1.8483 + 0.0565 (6)

2.1901 +

0.3197 (6)

3.3561 I!Z0.3671* (6)

3.1533 zk 0.2909*

45.4877 37.6407

immunoreactive component with higher molecular size, in the rat plasma. In addition, no material with the same molecular size as P-lipotropin was found in the neurointermediate lobe. Therefore, we assume that the plasma ir-P-END determined here is mainly of adenohypophyseal origin. Furthermore, the present results provide evidence

?I 15.7494 (5) f 8.7943 (6)

50.1325 63.3704

f 7.2584 f 9.3661

(6) (6)

53.4839 63.8194

+ 9.5449 f 8.8545

(6) (6) (6)

that CER stimulates the secretion of ir+END from the adenohypophysis and supports the assumption that the effect of CER on mood and pain may be mediated by an increase in P-END release (8). In the present study, both non-peptide CCK A receptor blocker, L-364,718, and CCK antagonist,

Table 3 Effects of CER or HLP administration and the combined treatment with these two drugs on ir+-END contents in various discrete brain regions. The animals were sacrificed by decapitation 1 h (1st day) or 24 h (2nd day) after the pre-treatment with HLP or vehicle. The pre-treatment with HLP (5mg/kg SC)or vehicle was performed 45min prior to SCinjection of CER (2OOt.@kg) or saline. Each value represents the mean + SE of the number of brain samples indicated in parenthesis. Abbreviations: CER; ceruletide, SAL; saline, HLP; haloperidol, VEH; Vehicle, ir+END; immunoreactive P-endorphin

Vehicle + Saline

immunoreactive P-endorphin (nglmg protein) Vehicle Haloperidol + Ceruletide + Saline

Haloperidol + Ceruletide

F. Cortex

1st day 2nd day

0.1142 0.1427

f 0.0235 (10) f 0.0233 (14)

0.0954 + 0.0204 (8) 0.1115 + 0.0224 (8)

0.0865 f 0.0254 (8) 0.1342 + 0.0328 (8)

0.1861 f 0.0839 (8) 0.0872 + 0.0229 (8)

N. Accumbens

1st day 2nd day

0.2424 + 0.0261 (10) 0.2698 + 0.0455 (14)

0.2020 + 0.0294 (8) 0.2694 f 0.0540 (8)

0.2453 + 0.0354 (8) 0.2024 f 0.0238 (8)

0.3194 0.2028

Hypothalamus

1st day 2nd day

1.9823 f 0.1570 (10) 2.3681 + 0.1241 (14)

2.1030 + 0.2338 (8) 2.2268 k 0.1687 (8)

1.8520 + 0.2083 (8) 2.1500 f 0.1885 (8)

1.9512 + 0.2321 (8) 2.2749 + 0.0758 (8)

Striatum

1st day 2nd day

0.6894 f 0.1004 (10) 0.6893 + 0.0627 (14)

0.6319 + 0.0588 (8) 0.6271 f 0.0671 (8)

0.6247 + 0.0775 (8) 0.6740 + 0.0740 (8)

0.6147 + 0.0706 (8) 0.5581 + 0.0731 (8)

Septum

1st day 2nd day

0.7363 + 0.1108 (10) 0.5533 + 0.0671 (14)

0.6567 + 0.0908 (8) 0.5662 f 0.0643 (8)

0.6295 f 0.0635 (8) 0.4775 + 0.0777 (8)

0.5435 + 0.0626 (8) 0.4445 f. 0.0449 (8)

Hippocampus

1st day 2nd day

0.4298 0.4107

+ 0.0409 (10) f 0.0247 (14)

0.4640 f 0.0472 (8) 0.4299 f 0.0512 (8)

0.4044 + 0.0224 (8) 0.4254 + 0.1658 (8)

0.4152 + 0.0258 (8) 0.4162 + 0.0291 (8)

+ 0.0536 (8) f 0.0174 (8)

10

NEUROPEF’TIDES

Table 4 Effects of CER or chronic HLP administration and the combined treatment with these two drugs on ir+-END contents in various discrete brain regions. The animals were repeatedly treated with daily injections of HLP (5mg/kg SC)or vehicle for 3 weeks. CER (2OO&kg SC)or saline was injected SC45min after the final HLP injection, The animals were sacrificed by decapitation 1 h (1st day) or 24 h (2nd day) after given the final HLP injection. Each value represents the mean + SE of the number of brain samples indicated in parenthesis. The data were analysed by Student’s t-test (two/tailed) for comparison against the group treated with vehicle and saline. * p c 0.05. Abbreviations: CER; ceruletide, SAL; saline, HLP; haloperidol, VEH; Vehicle, ir+END; immunoreactive P-endorphin. immunoreactive

F. Cortex N. Accumbens

Striatum

Chronic haloperidol + Ceruletide

Control

Ceruletide

1st day 2nd day

0.0858 + 0.0110 (5)

0.1004 + 0.0189 (5) 0.0811 + 0.0157 (6)

0.1233 + 0.0167 0.1041 + 0.0152

(6) (6)

0.1080 + 0.0060 0.0888 + 0.0008

(6) (6)

1st day

0.2242 rt 0.0496 (5)

0.2014 f 0.0492 (5) 0.2237 f 0.0491 (6)

0.2480 z!z0.0862 0.0979 f 0.0157

(6) (6)

0.1454 + 0.0222 0.1105 f 0.0067

(6) (6)

1st day 2nd day

3.9738 + 0.3370 (5)

4.2504 + 0.5254 (5) 4.3849 + 0.2887 (6)

3.7892 + 0.3889 4.1579 f 0.4202

(6) (6)

3.4439 + 0.3468 4.0140 + 0.2597

(6) (6)

1st day

0.9799 + 0.1918 (5)

0.8797 + 0.1101 (5)

0.7763 f 0.1266

(6)

0.5964 + 0.0758

(6)

0.9673 + 0.1647 (6)

0.6329 rk 0.0982

(6)

0.7542 + 0.1356

(6)

h 2nd day Hypothalamus

P-endorphin (nglmg protein) Chronic haloperidol + Saline

2nd day Septum

1st day 2nd day

1.2715 + 0.2133 (5)

1.6759 + 0.2279 (5) 1.2045 f 0.1263 (6)

1.5635 + 0.2531 (6) 2.3807 + 0.2107** (6)

1.4262 f 0.3068 (6) 2.5350 + 0.2858** (6)

Hippocampus

1st day 2nd day

0.9009 f 0.1718 (5)

0.6469 f 0.1042 (5) 0.6937 f 0.1051 (6)

0.6010 f 0.0234 0.4499 + 0.0615:

0.5099 f 0.0614* (6) 0.4463 f 0.0726* (6)

proglumide, had no effects plasma ir-P-END. However,

on basal

levels

of

L-364,718, but not completely CERproglumide , antagonised induced increment of plasma ir-P-END levels. L-364,718 is an extremely potent, competitive and specific antagonist of CCK-A receptor in both in vitro radioligand binding and isolated tissue assays compared to proglumide (25). The acute HLP treatment has been reported to increase the plasma concentrations of ir-P-END in a dosedependent manner (26) with little or no changes in pituitary contents of ir+-END. In the present study, HLP increased plasma ir-P-END levels 60 min after SCadministration at a dose of 5 mg/kg. On the other hand, the Dz agonist, bromocriptine, reduced the basal levels of plasma ir$-END. The treatment of rats with 5mg/kg HLP (SC) antagonised bromocriptine-induced reduction in plasma P-END levels, whereas L-364,718 had no effect on HLP-induced increase of plasma ir+-END levels. These observations are consistent with the previous study showing that acute administration of dopamine agonists or antagonists in vivo changes

(6) (6)

peripheral blood ir+-END (27). The dopaminergic receptor antagonist has been suggested to stimulate the secretion of POMC-derived peptides by interrupting the tonic inhibitory control of intermediate lobe melanotrophs exerted by dopaminergic neurons projecting from the arcuate nucleus. Dopaminergic receptor agonists have the opposite effect on the neurointermediate pituitary (28-30). The Dzselective agonist, LY-141865, also has a stimulatory effect on secretion of anterior pituitary ir-P-END, which indicates that its effect is mediated by its actions on hypothalamic corticotropin-releasing hormone (CRH) (31). In fact, CRH has been reported to be a potent stimulator of ACTH/P-END-L1 secretion on cultured adenohypophyseal cells (32). In the present study, the combined administration of CER and HLP caused a greater increase in plasma ir-P-END levels than either drug alone, e.g., the effect of CER on plasma ir-P-END levels was additional to that of HLP. Therefore, our findings indicate that the stimulatory effect of CER on ir-P-END release may be mediated by CCK-A receptors which

EFFECTS OF CERULETIDE AND HALOPERIDOL ON THE HYPOTHALAMO-PITUITARY

possibly exist in the adenohypophysis, but not by the tuberoin-findibular dopaminergic mechanism and by HLP-stimulated CRH release. As mentioned above, CER or HLP and the combined treatment with these two drugs caused a pronounced elevation in plasma ir-P-END 1 h after each drug treatment. The plasma ir-P-END levels returned to the basal levels 24h later. However, ir+END contents in the adenohypophysis and neurointermediate lobe did not change signi~cantly under any experimental condition. The earlier studies demonstrated that chronic HLP treatment resulted in an elevation of plasma ir-P-END levels with an increase in the content of ir-P-END in the neurointermediate lobe, but not in the adenohypophysis and also found that plasma ir-P-END levels continued to be elevated after even though the animals were sacrificed 24 h after the final drug treatment (27,30,33). In the present study, chronic HLP administration produced an increment of plasma ir-P-END levels 1 h after the final drug treatment as well. Elevated plasma ir+-END levels returned to the basal levels over a period of 24h. On the other hand, CER injection to chronically HLP-treated rats caused a greater, but not significant, elevation of plasma ir+-END levels as compared to those in saline-injected rats. Furthermore, plasma ir-P-END levels remained significantly elevated over a period of 24h. Previously, we found that CER was clinically effective against some types of chronic neuroleptic-resistant schizophrenia on the medication of neuroleptics and that the symptomatic improvement persisted for about 2 weeks after a single injection (34-36). Matsubara and Matsushita (37) have reported a long-lasting antagonistic effect of CER as mentioned above. In contrast to the earlier reports, ir-P-END contents in the adenohypophysis and neurointermediate lobe of rats with or without CER injection were increased at 24h, but not at 1 h, after the final HLP treatment. The increment of ir+-END contents in both pituitary lobes of CER-injected rats was of the same magnitude as that of rats without CER injection. This indicates that the increment of ir-p-END contents may be due to chronic HLP treatment. Zakarian and Smyth (38) have shown that the principal peptides in the rat brain are 13-END1_31 AND &END1_,6, and that the acetylated forms

~-ENDORPHIN SYSTEM IN THE RAT

11

are relatively minor. Ion-exchange chromatography of the g-END-related peptides, however, extracted from dissected regions of the rat brain reveals two different processing patterns, one for the hypoth~amus, midbrain and amygdala, and the other for the hippocampus, dorsal colliculae and brain stem. The hypothalamus contains predominantly @END1_3i and the midbrain and amygdala also contains 13-END1-3i but it is accompanied by 13-END1-26; in these regions there are negligible acetylated peptides. On the other hand, the hippocampus, dorsal colliculae and brain stem contains the (Y,N-acetylated forms of P-ENDi-27 and 13-END1-2+ However, in the present study the reverse-phase HPLC of acid extracts from dissected rat brain regions indicates that the principal peptides in all the brain regions studied were l3-ENDI_31 and P-ENDi-27; the acetylated forms of P-END related peptides were negligible in these regions except for the STR. l3-LPH was also found in the FC, NAc, STR and hippocampus, but was less signi~cnt. The distribution of P-END within the central nervous system is not clear. The factors responsible for conflicting results probably include varying cross-reactivities and sensitivities of antisera as well as differing extraction procedures and methods of animal sacrifice (39). As Zakarian and Smyth pointed out, however, our observations also point to the existence of differential processing mechanisms of the precursor in various regions of the rat brain. There are many reports that endogenous opioid may be involved in the action of 0X-8 and CER (l-4, S-12). In the present study, CER injection produced an elevation of plasma ir-P-END levels, but it did not affect the content of ir+-END in any brain region studied. Zetler et al have suggested the existence of allosteric interactions between the receptor for opioids and that for CCK-8 and CER. In addition, CCK-8 treatment has been reported to result in a significant decrease in levels of b- and b-sites in the FC of the neonatal rat (40). Kiraly and van Ree (10) have indicated that y-END may be a candidate to mediate the influence of UK-8 on presumably postsynapic located dopaminergic receptor systems, since y-END, but not e-END, P-END, Met-enkephalin and Leu-enkephalin, antagonises the apomorphine-induced hyperactivity following the injection of CCK-8 into the NAc

12

(41). Thus, CER may act on other opioid systems in the brain, consequently producing behavioural effects. However, further studies will be required to confirm this assumption. The treatment with neuroleptic drugs has been well known to produce alterations in contents of several brain neuropeptides. Several earlier studies have reported that chronic treatment to the rat with HLP produces alterations in ir-B-END contents in some regions of both brain and pituitary gland (26-28, 30, 33, 4244). Hollt and Bergman (26) have reported that chronic treatment with lmg/kg HLP over a period of 3 weeks resulted in an increase in ir-B-END contents of the hypothalamus and septum. Ham and Smyth (42) found a marked increase in the brain stem, but not in the hypothalamus, after chronic treatment with lmg/kg HLP over a lo-day period. In addition, they have reported that the increase in B-END under HLP treatment was confined to the acetylated derivatives. There are some reports that chronic treatment with HLP led to a significant reduction in ir+-END contents in the STR (43) and NAc (44). By contrast, chronic treatment to the rat with HLP has been reported to have no effect on contents of B-END related-peptides not only in the hypothalamus (33, 43-44) and septum (43-44) but also in the FC (43-44), midbrain (33), STR (44) and NAc (43). As mentioned above, the findings concerning the effect of chronic HLP treatment on brain B-END immunoreactivity are conflicting. In the present study, acute treatment with HLP had no effect on ir-B-END contents in any brain region studied. On the other hand, chronic treatment produced an elevation in septal contents of ir-P-END at 24h after the final HLP injection. A small reduction in ir-B-END contents was also found in the hippocampus and NAc; however, the reduction in the latter region was not statistically significant (p < 0.1). Because we found no change in brain ir-P-END contents at 24h after a single injection of HLP, the alterations in ir+-END contents in brain regions mentioned above appear to be due to chronic treatment with HLP. However, the discrepancy between the findings cited above and our results cannot be explained, but it may be due to factors similar to those responsible for the discrepancy between the findings concerning pituitary ir-P-END contents

NEUROPEPTIDES

discussed above. Taken together with the findings cited above, however, the present results suggest a functional interaction between B-END and dopaminergic systems in the central nervous system. Moreover, this functional interaction appears partly to be involved in therapeutic efficacy for schizophrenia. Previously, we reported that although CER did not suppress the discriminated avoidance behaviour (DAR) over a broad range of doses, the combined administration of CER and neuroleptics at critical doses that suppress the DAR caused a greater reduction in the avoidance rate when compared to neuroleptics alone (45). These findings suggest that CER influences the central dopaminergic systems, potentiating the central effects of neuroleptics. Furthermore, Matsubara and Matsushita (11-12) have reported that a single injection of CER has a long-lasting antagonistic effect on amphetamine-induced hyperactivity observed in chronically HLP-treated rats and suggested that dopamine release by amphetamine in the nucleus accumbens is presynaptically inhibited by opiate receptors activated by B-END. In fact, opiate receptors are located presynaptically on dopaminergic terminals in the limbic areas (46). Thus, it is of interest to ascertain whether a single injection of CER has any effect on ir-B-END contents in the brain of chronically HLP-treated rats. In the present study, CER at 200ug/kg caused a small reduction only hippocampal ir-P-END contents shortly after the SCinjection. The septal ir-B-END contents were increased and ir-P-END contents were the hippocampal decreased when determined on the 2nd day after SC injection of CER. In addition, ir+-END contents in the NAc showed a tendency to reduce. alterations in ir-B-END contents However, observed in the brain regions of CER-injected rats were of the same magnitude as those of rats not given the CER injection. The intra-accumbal pre-treatment with CCK-8 has been reported to inhibit the hyperactivity induced by injection of apomorphine into the NAc in a dose dependent manner (10). In addition, the local treatment with the opioid antagonist, naloxone, antagonised this inhibitory action of CCK-8, but did not change the blocking effect of HLP on apomorphine-induced hyperactivity. These findings suggest that endoge-

EFTE(JTSOFCERULE?1DEANDHALOPERIDOLONTHEHYPOTHALAMO-PITUITARY8-ENDORPHINSYSTEMINTHERAT

nous opioids are concerned in certain interactions between CCK-8 and dopaminergic systems. Conflicting results have been reported concerning effects of chronic HLP administration of CCK-like immunoreactivity in the NAc of the rat brain (47-50). Considering the findings by Kiraly and van Ree and a small reduction in accumbal ir-P-END contents mentioned above, thus, sustained elevation in plasma ir-P-END levels after the combined treatment with HLP and CER may be relevant to long-lasting therapeutic effect of CER in schizophrenia treated with neuroleptics and longlasting antagonising effect on amphetamine-induced hyperactivity in rats when given together with HLP. In conclusion, CER stimulates the release of ir-P-END from the adenohypophysis through CCK-A receptors which presumably exist in the adenohypophysis, and elevated plasma ir+-END levels is partly involved in some behavioural effects induced by CER. Furthermore, sustained elevation of plasma ir-P-END levels after a single injection of CER to chronically HLP-treated rats may explain its long-lasting therapeutic and behavioural effects. References 1. Zetler. G. and Morsdorf, K.-H. (1984). Effects of ceruletide and cholecystokinin octapeptide on eating in mice: Interaction with naloxone and the enkephalin analogue. FK 33-824. Naunyn-Schmidberg’s Archives of Pharmacology 325: 20%213. 2. Wilson, M. C., Denson, D., Bedford, A. and Hunsinger, R. N. (1983). Pharmacological manipulation of sincalide (CCK-Q-induced suppression of feeding. Peptides 4: 351357. 3. Faris, P. L., Komisaruk, B. R., Watkins, L. R. and Mayer, D. J. (1983). Evidence for the neuropeptide cholecystokinin as antagonist of opiate analgesia. Science 219: 310-312. 4. Itoh. S., Katsuura, G. and Maeda, Y. (1982). Caerulein and cholecystokinin suppress 8-endorphin-induced analgesia in the rat. European Journal of Pharmacology 80: 421-425. 5. Vijayan, E., Samson, W. K. and McCann, S. M. (1979). In vivo and in vitro effects of cholecystokinin on gonadotropin, prolactin, growth hormone and thyrotropin release in the rat. Brain Research 172: 295-302. 6. Porter, J. R. and Sander, L. D. (1981). The effect of cholecystokinin octapeptide on pituitary adrenal hormone secretion. Regulatory Peptides 2: 245-252. 7 Itho, S., Katsuura, G., Hirota, R. and Odaguchi. K. (1980). Effect of caerulein on plasma corticosterone concentration in the rat. Life Science 27: 2205-2210.

13

8. Basso, N., Materia, A., D’Intinosante, V., Ginaldi, A., Pona. V., Reilly, P., Ruggeri, S. and Fioravanti, M. (1981). Effect of ceruletide on pituitary-hypothalamic peptides and on emotion in man. Peptides 2, Supp. 2: 71-75. 9. Stengaard-pedersen, K. and Larsson, L.-I. (1981). Localisation and opiate receptor binding of enkephalin, CCK and ACTH@endorphin in the rat central nervous system. Peptides 2: 3-19. 10. Kiraly, I. andVan Ree. J. M. (1987). Behaviouralevidence for the involvement of endogenous opioids in the interaction between cholecystokinin and brain dopamine systems. Neuroscience Letters 74: 343-347. Il. Matsubara, K. and Matsushita, A. (1986). B-endorphin involvement in the antidopaminergic effect of caerulein. Japan Journal of Pharmacology 40: 417-422. 12. Matsubara, K. and Matsushita, A. (1986). Further evidence for B-endorphin involvement in the long-lasting antagonistic effect of caerulein on amphetamine hyperactivity in rats. European Journal of Pharmacology 121: 297-301. 13. Moroji, T. and Hagino, Y. (1986). A behaviouralpharmacological study on CCK-8 related peptides in mice. Neuropeptides 8: 273-286. 14. Hagino, Y. and Moroji, T. (1989). A behavioural pharmacological study on CCK-8 related peptides in mice. Neuropeptides 8: 273-286. 15. Glowinski. J. and Iversen L. L. (1966). Regional studies of catecholamines in rat brain. Journal of Neurochemistry 13: 655-669. 16. Lowry, 0. H.. Rosebrough, N. J., Farr, A. L. and Randall, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry 193: 265-275. 17. Matsumura. M., Fukunda, N., Saito, S. and Mori, H. (1982). Effect of a test meal, duodenal acidification and tetragastrin on the plasma concentration of P-endorphinlike immunoreactivity in man. Regulatory Peptides 4: 173-181. 18. Arai, H., Moroji, T., Kosaka, K. and Iizuka, R. (1986). Extrahypophyseal distribution of a-Melanocyte stimulating hormone (a-MSH)-like immunoreactivity in postmortem brains from normal subjects and Alzheimer-type dementia patients. Brain Research 377: 305-310. 19. Kato, N., Honda, Y., Ebihara, S.. Naruse. H. and Takahashi, Y. (1984). Development of an enzyme immunoassay for delta sleep-inducing peptide (DISP) and its use in the determination of the metabolic clearance rate of DSIP administration to dog. Neuroendocrinology 39: 39-44. 20. Beinfeld, M. C., Meyer, D. K.. Eskay, R. L., Jensen, R. T. and Brownstein, M. J. (1981). The distribution ofcholecystokinin immunoreactivity in the central nervous system of the rat as determined by radioimmunoassay. Brain Research 212: 51-57. 21. Vanderhaeghen. J. J., Lotstra, F., DeMay. J. and Gilles, C. (1980). Immunohistochemical localisation of cholecystokinin- and gastrin- like peptides in the brain and hypophysis of the rat. Proceedings of the National Academy of Sciences of the United States of America. 77: 1190-1194.

14 22. Morley, J. E., Melmed S., Briggs, J., Carlson, H. E., Hershman, J. M., Solomon, T. E., Lamers, C. and Damassa, D. A. (1979). Cholecystokinin octapeptide refeases growth hormone from the pituitary in vitro. Life Sciences 25: 1201-1206. 23. Matsumura, M., Yamanoi, A., Yamamoto, S. and Saito, S. (1983). In vivo and in vitro effects of cholecystokinin octapeptide on the release of B-endorphin-like immunoreactivity. Neuroendocrinology 36: 443-338. 24. Meyer, D. K., Anhut, H., Nutto, D., Beinfeld, M. C. and Knepel, W. (1982). Choiecystokin release ~-endo~hin from the anterior pituitary gland. Neuropeptides 2: 371375. 25. Chang, R. S. L. and Lotti, V. J. (1986). Biochemical and pharmacological characterisation of an extremely potent and selective nonpeptide cholecystokin antagonist. Proceedings of the National Academy of Sciences of the United States of America X3: 4923-4926. 26. Hollt, V. and Bergmann, M. (1982). Effects of acute and chronic haloperidol treatment on the concentrations immunoreactive 8-endorphin in plasma, pituitary and brain of rats. Neuropharmacology 21: 147-154. 27. Millington, W. R., O’Donohue, T. L. and Mullet, G. P. (1987). Dopaminergi~ agents selectively alter the posttranslational processing of ~-endo~hin in the intermediate pituitary of the rats. The Journal of Pharmacology and Experimental Therapeutics. 243: 160-170. 28. Chen, C. L. C., Dionne, F. T. and Roberts, J. L. (1983). Regulation of the pro-opiomelanocortin mRNA levels in rat pituitary by dopaminergic compounds. Proceedings of the National Academy of Sciences of the United States of America. 80: 2211-2215. 29. Beaulieu, M., Goldman, M. E., Miyazaki, K.,Fray, E. A., Eskay, R. L., Kebabian, J. W. and Cote, T. E. (1984). Bromocriptine-induced changes in the biochemistry, physiology, and histology of the intermediate lobe of the rat pituitary gland. Endocrinology 114: 1871-1184. 30. Millington, W. R. O’Donohue, T. L., Chappell, M. C, Roberts, J. L. and Mueller, G. P. (1986). Coordinate regulation of peptide acetyltransferase activity and proopiomelanocortin gene expression in the intermediate lobe of the rat pituitary. Endocrinology 118: 2024-2033. 31. Farah, J. M., Jr. and Mueller, G. P. (1989). A D-2 dopaminergic agonist stimulates secretion of anterior pituitary immunoreactive ~-endorphin in rats. Neuroendocrinology 50: 26-32. 32. Vale, W., Vaughan, J., Smith, M., Yamamoto, G., River, J. and River, C. (1983). Effects of synthetic ovine corticotropin-releasing factor, glucocorticoids, catecholamines, neurohypophysial peptides, and other substances on cultured corticotropic cells. Endocrinology 113: 1121-1131. 33. Millington, W. R., Maiewski, S., O’Donohue, T. L. and Mueller, G. P. (1985). Long-term haloperidol treatment elevates B-endorphin levels in the intermediate pituitary but not in rat brain. Neuropeptides 6: 365-372. 34. Moroji, T., Wantanabe, N., Aoki, N. and Itho, S. (1982). Antipsychotic effects of ceruletide (caerulein) on chronic schizophrenia. Archives of General Psychiatry 39: 48% 486. 35. Moroji, T., Wantanabe, N., Aoki, N. and Itho, S. (1982). a decapeptide Antipsychotic effects of caerulein,

NEUROPEFTIDES

36.

37.

38.

39.

40.

41.

42.

43.

44.

45.

46.

47.

48.

49.

50.

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