In vivo binding affinities of cholecystokinin agonists and antagonists determined using the selective CCKB agonist [3H]pBC 264

European Journal o f Pharmacology, 209 (1991) 185-193 © 1991 Elsevier Science Publishers B.V. All rights reserved 0014-2999/91/$03.50

185

EJP 52179

In vivo binding affinities of cholecystokinin agonists and antagonists determined using the selective CCK B agonist [ 3H] pBC 264 C h r i s t i a n e D u r i e u x , M a r i a n o R u i z - G a y o a n d B e r n a r d P. R o q u e s UFR des Sciences Pharmaceutiques et Biologiques, Ddpartement de Chimie Organique, U266 I N S E R M - UA498 CNRS, 4 Az~enue de l'Obsert~atoire, 75270 Paris Cedex 06, France Received 10 May 1991, revised MS received 28 August 1991, accepted 17 September 1991

The respective role of central vs. peripheral CCK-B receptors in the recently reported anxiolytic effects of CCK-B antagonists remains to be firmly established. We therefore investigated the in vivo binding properties of cerebral CCK receptors after i.c.v. injection into mice of [3H]pBC 264 ([3H]propionyl-Tyr(SO3H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH2), a highly potent, peptidase-resistant and selective CCK-B agonist. The specific binding of [3H]pBC 264 was reversible and saturable. The dose producing 50% receptor occupancy was 25 pmol and the Bma x w a s 0.9 pmol/brain 15 min after injection. I.c.v. administered CCKs (IDs0 8500 pmol) was 200-fold less potent than pBC 264 (IDs0 43 pmol) in inhibiting specific [3H]pBC 264 binding; CCKsNS, CCK 5 and CCK 4 being slightly less potent than CCK8. Aminopeptidases play a major role in degrading CCKs since the protected analog pCCKs or CCKs in the presence of an aminopeptidase inhibitor exhibited higher affinities than CCK 8. I.v. administration of pBC 264 (20 mg/kg) inhibited [3H]pBC 264 specific binding by about 72%, confirming its ability to enter the brain. In contrast, C C K 4 w a s unable to modify [3H]pBC 264 binding. As expected, the CCK-A antagonist (L364,718) did not inhibit [3H]pBC 264 binding, while at the highest dose used (40 mg/kg i.p.) the CCK-B antagonist (L365,260) inhibited binding by 20%. Several hypotheses are discussed to account for the very low i.v. doses of C C K 4 and L365,260 needed to produce anxiogenic and anxiolytic responses, respectively.

CCK-8 (cholecystokinin octapeptide); CCK B receptors; Binding in vivo; Peptidase inhibitors; CCK receptor agonists; CCK receptor antagonists; [3H]pBC 264; (Mouse)

1. Introduction

The octapeptide cholecystokinin (CCK 8) is abundant in both the brain and the gastrointestinal system, where it interacts with nanomolar affinities with at least two types of binding sites designated C C K - A and CCK-B receptors, although subtypes of these receptors may also exist (Durieux et al., 1986a; Yu et al., 1990; Knapp et al., 1990). These two types of receptors were initially characterized by their s i m i l a r affinities for CCK~, contrasting with the 100-1000 times lower affinity of the endogenous CCK fragments, unsulfated CCKs and CCK 4 (Innis and Snyder, 1980) for CCK-A

Correspondence to: B.P. Roques, U F R des Sciences Pharmaceutiques et Biologiques, D ~ p a r t e m e n t de Chimie Organique, U266 I N S E R M - UA498 CNRS, 4 Avenue de l'Observatoire, 75270 Paris Cedex 06, France.

sites. CCK-A type receptors are predominant in the periphery but have also been reported to be present in restricted brain areas (Moran et al., 1986; D.R. Hill et al., 1987) whereas CCK-B receptors have a wide distribution in the CNS (Zarbin et al., 1983; Van Dijk et al., 1984; P61aprat et al., 1987) Selective tritiated antagonists and agonists for each type of receptor have recently been synthesized and their in vitro binding properties characterized (Chang et al., 1986; Chang et al., 1989; Durieux et al., 1989; K n a p p et al., 1990). Most of the pharmacological properties of CCK s have been studied after intracerebral (i.c.v.) administration of the peptide as it does not readily penetrate the b l o o d - b r a i n barrier. CCKs-related peptides have been shown to be implicated in satiety (Gibbs et al., 1973; Schick et al., 1986), nociception (review in Baber et al., 1989), memory (review in Itoh and Lal, 1990), spontaneous or dopamine-dependent locomotor activity (Van Ree et al., 1983; Weiss et al., 1988; Hagino et al., 1989) and anxiety (Singh et al., 1991). However, the effective

186

doses of CCK8 or its fragments varied over a wide range in the different studies and the involvement of central o r / a n d peripheral CCK-A or CCK-B receptors in these effects has not yet been clearly demonstrated. For example, we have recently shown that the plasma levels of a CCK s analog, administered i.c.v, to rats, were sufficient to decrease food intake through stimulation of peripheral receptors (Crawley et aI., 1991). Interestingly, although it is difficult to observe clear anxiogenic responses after selective stimulation of CCK-B receptors (Ravard and Dourish, 1990; Daug6 et al., in press; Singh et al., 1991), peripheral administration of very low doses of CCK-B antagonists has been shown to produce anxiolytic effects (Hughes et al., 1990), increase food intake (Dourish et al., 1989) and potentiate morphine-induced analgesia (Dourish et al., 1990; Wiesenfeld-Hallin et al., 1990). The anxiolytic effects could be related to the panic attacks or severe anxiogenic syndrome induced by peripheral administration of low doses of C C K 4 t o healthy volunteers (De Montigny, 1989). However, the respective role of central a n d / o r peripheral CCK-B receptors in these responses remains to be firmly established. An answer to these questions could be found by evaluating the brain concentrations of CCK agonists and antagonists after their peripheral or i.c.v, administration and the related occupancy of CCK-B receptors. This requires a preliminary determination of the in vivo binding properties of these molecules for brain binding sites, with the aim of studying the relationships between pharmacological responses and receptor activation. For this purpose we used [3H]pBC 264, the first reported highly potent and selective radiolabelled CCK-B analog (Durieux et al., 1989). This CCKs-related peptide, which has the formula propionylTyr(SO3 H)gNIe-mGIy-Trp-(NMe)NIe-Asp-Phe-NH 2, has been shown to be very resistant to peptidases (Charpentier et al., 1988) and to behave as a highly potent and selective CCK-B agonist in various pharmacological assays and electrophysiological experiments (Daug6 et al., 1990, 1991; Crawley et al., 1991; Bado et al., 1991). The in vivo binding properties of [3H]pBC 264 were determined after i.c.v, co-injections of the radioligand with CCK-related peptides or antagonists, and also after i.v. administration of pBC 264, which has been shown to cross the blood-brain barrier (Ruiz-Gayo et al., 1990). Moreover, as different peptidases, such as aminopeptidases (Deschodt-Lanckman et al., 1983), and neutral endopeptidase 24.11 (NEP) (Matsas et al., 1984), have been reported to participate in the in vitro degradation of CCK 8 and analogs, the efficiency of CCKs in displacing [3H]pBC 264 binding was measured after i.c.v, coinjection of the sulfated peptide with the aminopeptidase inhibitor bestatin and the NEP inhibitor thiorphan (Roques et al., 1980).

2. Materials and methods

2.1. Animals" Male Swiss mice (Depr6, France) weighing 20-22 g were used in all experiments. All procedures were carried out between 13:00 and 18:00 h. 2.2. Drugs CCK8, nonsulfated CCK s (CCKsNS), and the fragments CCK 5 and C C K 4 w e r e synthesized in the laboratory according to previously described methods (Charpentier et al., 1988). [3H]pBC 264 ([3H]propionylTyr(SO 3H)-gNle-mGly-Trp-(NMe)Nle-Asp-Phe-NH 2, 98-100 C i / m m o l ) was prepared as reported previously (Durieux et al., 1989). Thiorphan, L 364,718 and L 365,260 were synthesized according to previously reported methods (Roques et al., 1980; Evans et al., 1986; Bock et al., 1989). Bestatin was purchased from Roger Bellon Laboratories, France. The drugs were dissolved in saline, except for CCKsNS, CCK 4 and CCK 5, which were dissolved in phosphate buffer, pH 7.4. For i.c.v, injections L 364,718 and I 365,260 were prepared as stock solutions in ethanol-propylene glycol (1:1) and diluted 20-fold with saline, and for i.p. injections as suspensions in 0.5% carboxymethylcellulose. I.c.v. injections were given in a volume of 10 ~1 by the method of Haley and McCormick (1957). I.v. injections were given in a volume of 200 #1 of saline or pBC 264 and CCK 4 in saline. The vehicle for L365,260 was 5% E t O H - 3 0 % propylene glycol-65% saline. 2.3. In t:il,o binding assay The experiments were performed as described by Meucci et al. (1989) for opioid agonists, with minor modifications. Mice were killed by cervical dislocation 15 min after i.c.v, injection of the tracer and their brains were quickly removed. The total brain (minus cerebellum) was homogenized for 14 s in 9.8 ml of ice-cold 50 mM Tris-HC1, pH 7.4 containing 0.2 m g / m l bacitracin with a Brinkman Polytron (PT-10/35, setting 4). Twelve aliquots of 0.15 ml were immediately filtered through Whatman G F / B glass filters precoated with 50 mM Tris-HC1, pH 7.4, containing 0.1% (w/v) bovine serum albumin. The filters were rinsed twice with 5 ml of ice-cold buffer. Four filters were placed together in a scintillation vial in 15 ml of Biofluor (NEN) and then counted. Total radioactivity of tritiated ligand in the homogenate was determined by counting 0.6 ml aliquots in 15 ml of Biofluor. Free radioactivity (F) was calculated as the difference between total radioactivity and radioactivity retained in the filters (bound radioactivity). Isotopic dilution with

187

2.4. In t~it,o metabolism

2

so

E'

F g

After i.c.v, injection of 10 pmol [3H]pBC 264, the mouse brain was homogenized in 1 ml of water and rapidly heated at 95 °C for 5 min to inactivate peptidases released from the tissue. Four mice were used for each determination. A fraction of the homogenate was filtered through a SEP-PAK cartridge to extract [3H]pBC 264 and its metabolites. Elution was carried out with 1.5 ml methanol. The resulting eluate was concentrated (Speed Vac Concentrator, Savant) to 0.15 ml, centrifuged at 9000 × g for 15 min and diluted with 0.15 ml H 2 O before HPLC analysis. Controls were treated in a same way, with [3H]pBC 264 added directly to mouse brain preparations. The overall recovery of radioactivity was about 96%. The samples were co-injected with 10 -4 M pBC 264 onto a Shimadzu HPLC apparatus equipped with a Cls #Bondapak column (4.6 × 250 mm). Elution was carried out with a linear gradient (20 min) of 25 mM triethylammonium phosphate buffer pH 6.5/acetonitrile rising from 28 to 38% acetonitrile, at a flow rate of 1.2 ml/min. Fractions of 0.6 ml were collected and counted after the addition of 5 ml scintillation liquid.

2

10o

unlabelled pBC 264 was used for the highest concentration (75 pmol) in saturation experiments. Competition experiments were carried out using 10 pmol of [3H]pBC 264 co-injected with or without various quantities of CCKs analogs, antagonists or peptidase inhibitors.

D

2O

o

I

!

I

I

~10

40

60

80

o 1oo

Time. r~ln

Fig. 1. Time course of total radioactivity [] and specific binding • of [3H]pBC 264 in mouse brain after i.c.v, injection of 10 pmol. Values are expressed as a percentage of total cpm injected (9.4+_0.8 105). Each point is the mean of six determinations (+S.E.M.). Specific binding was derived by subtracting nonspecific binding, defined as the binding measured after co-injection of 10 nmol cold ligand.

pmol. Only 15% of the total radioactivity injected was still present in the brain 15 min after injection, and 10% remained 90 min later (fig. 1). This relatively low recovery is usually obtained with neuropeptides after i.c.v, injection (Meucci et al., 1989) and could result from rapid elimination of the intact molecule from the brain, as has been shown for pCCK 8 (Crawley et al., 1991). Only 15 to 20% of [3H]pBC 264 was found to be metabolized 15 min after injection (fig. 2) and less than 55% at 60 rain. 3000

2.5. Data analysis Data from saturation experiments were analyzed by using Scatchard plots. Nonspecific binding was calculated by linear regression analysis of nonspecific binding as a function of the total radioactivity present in the brains of mice that had received 10 nmol of pBC 264. For each concentration, specific binding was determined by subtracting the calculated nonspecific binding from the total binding. In competition experiments, the levels of radioactivity in the brain are expressed as the ratio between bound and free radioactivity. Binding data were analyzed with the Hill plot log [(B 0 - B ) / B ] = log (I). Student's t-test was used for statistical comparisons, and differences were considered to be significant if P < 0.05.

cpm

2000

1000

........

,

I]11

..,,,,

. . . . . . . . . . . . . . . . . .

25

3. Results

3.1. Metabolism of [L~H/pBC 264 The agonist [3H]pBC 264 was rapidly eliminated from mouse brain tissue after i.c.v, administration of 10

50

Fraction number Fig. 2. A chromatogram of the radioactivity extracted from the brain 15 min after i.c.v, injection of 10 pmol [3H]pBC 264 in mice. The quantity of radioactivity co-eluting with a standard of pBC 264 (10 4 M) was measured after H P L C separation under the conditions described in Materials and methods. Fraction n u m b e r s 26 and 27 correspond to authentic [3H]pBC 264.

188

B/Bo

0.8 0.6

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0.4 0.2 0

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0

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1

40 60 pmol injected

80

100

0.05

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1

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LOG {dose), pmol Fig. 4. Inhibition of [3H]pBC 264 (10 pmol) specific binding to mouse brain after i.c.v, co-injection with • pBC 264, zx CCKs, • C C K s + bestatin. Each point is the mean of four determinations, each done in triplicate.

$ 0.02 {3.

-6 E 0.01 -d c

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m

0

0

J

0.2

0.4 0.6 0.8 Bound, pmol/brain Fig. 3. Saturation curve (A) and corresponding Scatchard analysis (B) for the specific binding of [3H]pBC 264 to mouse brain after i.c.v. injection. The saturation plot represents the specific binding of [3H]pBC 264 in the brain vs. the dose injected. Each point represents the mean (_+ S.E.M.) of 10 individual determinations. Specific binding was derived by subtracting from each experimentally determined total binding a value for nonspecific binding, which was calculated by linear regression analysis of nonspecific binding (in the presence of 10 nmol cold ligand) as a function of total cpm measured in the brain.

3.2. Binding parameters Fifteen minutes after i.c.v, injection of 10 pmol of [3H]pBC 264, the total bound radioactivity (fraction retained on G F / B filters) represented 24% of the total radioactivity recovered from the brain. The fraction of bound radioactivity which disappeared after co-injection of 10 nmol of cold pBC 264 was defined as specific binding. The level of nonspecific binding increased linearly as a function of the dose of [3H]pBC 264 and represented 7.3% of the total radioactivity present in the brain 15 min after i.c.v, administration and 30% of the bound radioactivity. The specific binding reached a maximum 15 min after injection and was found to be reversible, as illustrated by its slow decrease between 20 and 90 min (fig. 1). Binding parameters were therefore determined 15 min after i.c.v, injection. The specific binding of [3H]pBC 264 was saturable, as shown by the binding isotherm as a function of the doses injected (fig. 3A). Scatchard transformation of the data indicated that 50% receptor occupancy was produced by injecting

25 4- 3 pmol, and that the maximal number of sites was 0.89 + 0.13 pmol per brain (fig. 3B).

3.3. Pharmacological profile of in ciuo [3H]pBC 264 binding Inhibition of the specific binding of 10 pmol [3H]pBC 264 by CCK s analogs and CCK 8 fragments was studied 15 min after i.c.v, co-injections. At 10 pmol of [3H]pBC 264, the ratio of bound radioactivity to free radioactivity was 0.278 ± 0.008 for the total binding and 0.108 ± 0.002 for the nonspecific binding (mean _+ S.E.M. of 52 measurements). The ID50 values and the Hill coefficients, evaluated from competition curves (fig. 4), are reported in table 1. The IDs0 value for pBC 264 (43 pmol) was of the same order as the apparent affinity calculated from saturation experiments (25 pmol). CCK s was approximately 200-fold less potent than pBC 264 (ID50 8500 pmol). As expected, p e C K s , a CCK s analog protected from aminopeptidases by a N-terminal propionyl group, TABLE 1 Inhibition of [3HlpBC 264 (10 pmol) binding in vivo by i.c.v, injected C C K s and related compounds. Competition experiments were carried out with five different concentrations of analogs, using four mice for each dose, and each determination was in triplicate; ± represents the 95% confidence limits on IDso values. Compounds

ID50 (pmol)

nH

pBC 264 CCK8 pCCK~ CCK s + 50 nmol bestatin CCK s + 50 nmol thiorphan

43± 9 8500 ± 416 8 6 + 11 797 _+ 125 9500 ± 890

0.68 0.49 0.56 0.69 0.48

189 TABLE 2

100,

"i

"1-

60

--[-

Effect of i.p. injections of C C K antagonists on the in vivo binding of [3H]pBC 264. Values are means_+S.E.M, of percentage of specific binding. In parentheses are the n u m b e r of mice in each experiment.

:i:i:!:i

Treatment mg/kg

O

Saline

i:i:!:i:

20-

L 364

L 365

CCX 5

I nmol

has a greater affinity, with only a twofold lower potency (IDs0 86 pmol) than pBC 264. The CCK 8 fragments CCKsNS, CCK 5 and CCK 4 were also co-injected i.c.v, with [3H]pBC 264. However, due to their limited solubility in physiological vehicles, the highest possible dose was 10 nmol. Although slightly lower than the inhibition observed with CCKs, [3H]pBC 264 binding was inhibited by about 48, 35 and 40% by CCKsNS, CCK 5 and CCK4, respectively (fig. 5). The CCK-A, L 364,718 and CCK-B, L 365,260 antagonists, which are even less soluble, were used at 1 nmol. Under these conditions no inhibition was observed with L 364,718 while L 365,260 yielded about 16% inhibition. 3.4. Effect of peptidase inhibitors on the in t.,ivo binding

of [3HlpBC 264 The influence of peptidase inhibitors was studied by i.c.v, co-administration of 10 pmol [3H]pBC 264 with ..,...

100

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80

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0.25 1/.5 1.0 2.0

89.1 + 83.3_+ 81.8+ 78.9+

L 364,718

40

1.96

99.3-+ 11.3 (7)

10 n m o l

Fig. 5. Inhibition of in vivo binding of l(I pmol [3H]pBC 264 by i.c.v. administered CCK-related peptides and antagonists. Values are means-+S.E.M, of 7 - 1 2 determinations, each done in triplicate, * P <0.05, ** P <0.001 as compared to controls in the Student's t-test.

, i / i / ,

¢////~

V///z 5#/././.,

0 Veh

0.05

iiiiiiiiii

CCK4

20

20

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Fig. 6. Comparison of the inhibition of in vivo specific binding of 10 pmol [~H]pBC 264 by pBC 264 or C C K 4 administered i.v. Values (_+S.E.M.) are m e a n s of six individual determinations, each performed in triplicate. ** P < 0.001 as compared to controls in the Student's t-test.

lll0.lJ+ 7.9 (14) 5 10 20 40

CC~8N S

Cg~ 4

% of specific binding

L 365,260 _ _ - ~ Veh

/xmol/mouse

12.3 8.6 5.4 7.1

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various doses of CCKs and either the aminopeptidase inhibitor bestatin (50 nmol) or the endopeptidase 24.11 inhibitor thiorphan (50 nmol). Neither inhibitor alone changed the specific binding of [3H]pBC 264, but bestatin increased the affinity of CCK~ by an order of magnitude (IDs0 797 pmol), and also the Hill coefficient (Hill = 0.69). In contrast, thiorphan was unable to improve the binding efficiency of CCK~ (table 1).

3. 5. Effect of systemic administration of pBC 264, CCK 4 and CCK-B (L365,260) or CCK-A (L364,718) antagonists on brain [3H]pBC 264 binding A large inhibition (72%, P < 0.01) of [3H]pBC 264 binding was observed after i.v. injection of 20 mg/kg pBC 264 15 min prior to its i.c.v, administration (fig. 6). CCK a at the same dose did not modify the specific binding of [3H]pBC 264. The selective CCK-B antagonist L 365,260 when injected i.v. 15 min prior to i.c.v. administration of [3H]pBC did not produce any effect at the highest possible dose used, 3 mg/kg (data not shown). At doses ranging from 5 to 40 mg/kg i.p. 30 min before i.c.v, injection of [3H]pBC 264, L 365,260 decreased the specific binding of [3H]pBC 264 from 5 to 21%, but even at the highest dose this effect was just at the limit of statistical significance. As expected, the selective CCK-A antagonist L 364,718 injected i.p. at 40 mg/kg under the same conditions was unable to modify the specific binding of [3H]pBC 264 (table 2).

4. Discussion

In spite of problems inherent to in vivo binding studies, such as the problem that radioligand binding will not achieve equilibrium and the possibility that some receptor sites may be inaccessible in intact tissue, systemic injection of radioligands into the brain of an intact animal could provide information about binding parameters under physiological conditions.

190

The results of this study show that the in vivo binding of the selective CCK-B agonist [3H]pBC 264 to mouse brain is specific, reversible and saturable. Thus 15 min after i.c.v, administration of 10 pmol [-~H]pBC 264, which corresponds to the time of maximum binding, the specific binding was high and represented about 70% of the bound radioactivity. Under these conditions, the dose of [3H]pBC 264 resulting in 50% occupation of receptors was 25 pmol and its maximal binding capacity 0.89 pmol/brain. These results obtained with whole brain minus cerebellum may be influenced by brain regions close to the ventricle, since the ligand would have limited access to areas distant from this injection site. As already observed under in vitro conditions (Charpentier et al., 1988) and after i.v. injection (Ruiz-Gayo et al., 1990), [3H]pBC 264 was highly resistant to peptidases as at least 80% of the radioactivity present in the brain 15 rain after i.c.v. injection corresponded to the intact molecule, and only 55% of the ligand was metabolized after 1 h. In competition experiments, various CCK fragments showed the same rank order of potency for CCK-B receptors as that found in vitro with mouse brain homogenates (Roques et al., in press), namely pBC 264 > CCK 8 > CCKsNS > CCK 4 ~ CCK 5. Nevertheless, the absolute potencies of CCKs (IDs0 8500 pmol) and its fragments were notably weaker than the potency of pBC 264 (IDs0 43 pmol) in inhibiting in vivo [3H]pBC 264 binding, a rather unexpected result considering the results of in vitro studies, where pBC 264 and CCK~ exhibit similar high binding affinities, with KDS of 0.2 and 0.5 nM, respectively (Roques et al., in press). This could be due either to different pharmacokinetic properties of the synthetic and natural peptides a n d / o r to various degrees of susceptibility to peptidases. The potency of CCK 8 in the presence of the aminopeptidase inhibitor bestatin increased by an order of magnitude, but did not change with the NEP inhibitor thiorphan, suggesting a major role of aminopeptidases in the degradation of exogenously administered CCK 8. In agreement with this, the CCK 8 analog pCCK 8, which is protected from aminopeptidase action by a propionyl group, exhibited a 100-fold higher apparent affinity than CCK 8 and was only twofold less potent than pBC 264. The possible additional action of thiol or serine peptidases shown to be involved in vitro in CCK s degradation (McDermott et al., 1983; Durieux et al., 1986b; Rose et al., 1988) was not tested due to the lack of potent specific inhibitors. The results are also in agreement with the enhanced potency observed in various tests of the decapeptide analog of CCK 8 caerulein, which is also protected from aminopeptidase action by a N-terminal pyroGlu residue (Jurna and Zetler, 1981; Griesbacher et al., 1989). Likewise, i.c.v, co-injection of CCK 8 with bestatin, thiorphan and captopril, an inhibitor of angiotensin converting enzyme, has been reported to

potentiate the feeding (Griesbacher et al., 1989) or the antinociceptive (R.G. Hill et al., 1987) effects induced by the sulfated peptide. The latter response was reversed by prior administration of the opioid antagonist naloxone, thereby supporting the involvement of endogenous enkephalins in this effect. This could explain why bestatin alone or administered together with CCK 8 was not able to potentiate the antinociceptive effect (R.G. Hill et al., 1987), since inhibition of both aminopeptidases and NEP has been shown to be necessary to obtain a significant physiological analgesia triggered by enkephalins (review in Roques, 1991). Taken together, the present results indicate that high doses (>~ 1 nmol) of i.c.v, administered CCK s are necessary to produce central pharmacological effects and are in agreement with previous reports (Barbaz et al., 1986; Schick et al., 1986; R.G. Hill et al., 1987; Weiss et al., 1988; Griesbacher et al., 1989). However, i.v. administration of 20 m g / k g (0.36 /xmol) of pBC 264 15 min prior to i.c.v, injection of [3H]pBC 264 inhibited the specific binding of the tritiated ligand by about 72%. In a previous study, we have shown that the amount of intact [3H]pBC 264 present in mouse brain 15 min after i.v. administration is about 0.014% (Ruiz-Gayo et al., 1990), leading to an estimated concentration of about 50 pmol pBC 264 in the brain after i.v. injection of 20 mg/kg. This result is in good agreement with the 75% inhibition of [3H]pBC 264 specific binding obtained 15 min after an i.c.v. injection of 100 pmol pBC 264, which corresponded to 15 pmol in the brain, since 15% of the intact ligand was found in the brain at this time. I.v. administration of C C K 4 at 50 txg/kg or 20 m g / k g did not modify the binding of [3H]pBC 264. Low doses of C C K 4 ( 5 0 / x g / k g s.c.) have been reported to decrease the exploratory activity of mice in the elevated plus maze (Harro and Vasar, 1991), and this could be explained by prior activation of peripherally located CCK-B receptors relaying to the nucleus tractus solitarius via vagus terminals (Zarbin et al., 1981; Morin-Surun et al., 1983; Mailleux and Vanderhaeghen, 1990; Branchereau et al., submitted). However, in this experiment the similar potencies of CCK-A antagonists such as lorglumide and devazepide suggest that CCK-A receptors could also be implicated in the behavioral response. In addition, the existence of the previously proposed high-affinity specific binding sites for C C K 4 (Durieux et al., 1988) could also account for this response. As expected from its low in vitro affinity for CCK-B sites, the selective CCK-A antagonist L 364,718 (Chang et al., 1986) did not modify the specific binding of [3H]pBC 264 after i.c.v, or i.p. administration. In contrast, L 365,260, a selective CCK-B antagonist (Chang et al., 1989), inhibited [3H]pBC 264 binding after both i.c.v, and i.p. injections, although the 20% inhibition obtained with the highest dose used (40 m g / k g i.p.)

191

was at the limit of statistical significance. At 10 m g / k g i.p., corresponding to 0.5 /xmol of L 365,260, 16% inhibition was observed and, interestingly, a similar inhibition was obtained after i.c.v, injection of 1 nmol of L 365,260. This is in good agreement with a recent pharmacokinetic study (Hargreaves and Lin, in press) showing that 0.1% of an i.v. administered dose of L 365,260 penetrates into rat brain 1 min after injection. The in vitro affinity in mouse of L 365,260, using [3H]pBC 264 as ligand, was 5.2 nM as compared to 0.17 nM for pBC 264 (Roques et al., in press). Assuming that there are no drastic differences between the pharmacokinetic properties of these molecules, the apparent affinity of the CCK-B antagonist should therefore be about 1.5 nmol. Unfortunately, due to the limited solubility of this CCK-B antagonist, it was not possible to inject a dose greater than 1 nmol to determine the precise IDs0 value. The weak inhibition of in vivo binding of the CCK-B agonist [3H]pBC 264 by L 365,260 at a concentration as high as 40 m g / k g i.p. appears striking, inasmuch as low doses (/~g/kg range) of L 365,260 have been reported to give anxiolytic responses in the rat, probably as a result of blockade of brain CCK-B receptors (Ravard et al., 1990). One possible explanation is that low doses of antagonist are sufficient to reverse the effects of the low levels of extracellularly released endogenous CCKs, whereas higher doses of the same antagonist are necessary to have an effect on central responses induced by exogenously administered CCKs. Accordingly, Daug6 et al. (1991) have found that antagonism of the modification of alternation behavior induced by intra accumbens injection of BC 264 is obtained at dose of L365,260 as high as 5 m g / k g i.p. At this concentration, the inhibition of [3H]pBC 264 binding can be estimated to be about 11%, suggesting that a low occupation of receptors is sufficient to block the pharmacological responses induced by CCKs. Moreover, it is possible that only a small proportion of the binding sites labelled by [3H]pBC 264 in vivo are implicated in the behavioral response, and that L365,260 might only recognize this type of receptor. However, the participation of peripheral CCK-B receptors in the anxiolytic properties of L365,260 cannot be completely excluded. The response to CCK 4 could involve an initial stimulation of peripheral CCK-B binding sites. In conclusion, we have characterized the in vivo binding of a selective CCK-B agonist [3H]pBC 264 in mouse brain. This ligand behaves as a highly selective probe for CCK-B receptors in vivo and is very resistant to peptidases. The characterization of the in vivo binding of [3H]pBC 264 should permit the apparent affinities of various CCK agonists and antagonists in vivo to be determined, and allow their concentration in the brain to be estimated after i.c.v, or systemic administration. A correlation between the in vivo binding

characteristics of a ligand and the pharmacological responses induced by stimulation of CCK-B receptors can therefore be established.

Acknowledgements We wish to thank Dr. A. Beaumont for critical reading of the manuscript C. Dupuis for typing it, P.J. Corringer for synthesizing the molecules and M. Derrien for fruitful discussion. This study was supported by Rh6ne Poulenc Rorer. M. Ruiz-Gayo is a recipient of CEE fellowship.

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