CCK-B receptor: chemistry, molecular biology, biochemistry and pharmacology

Progress in Neurobiology Vol. 58, pp. 349 to 379, 1999 # 1999 Elsevier Science Ltd. All rights reserved Printed in Great Britain 0301-0082/99/$ - see front matter

PII: S0301-0082(98)00090-2

CCK-B RECEPTOR: CHEMISTRY, MOLECULAR BIOLOGY, BIOCHEMISTRY AND PHARMACOLOGY FLORENCE NOBLE and BERNARD P. ROQUES* DeÂpartement de Pharmacochimie MoleÂculaire et Structurale, INSERM U266-CNRS UMR 8600, Universite Rene Descartes, UFR des Sciences Pharmaceutiques et Biologiques, 4, Avenue de l'Observatoire, 75270 Paris Cedex 06, France AbstractÐCholecystokinin (CCK) is a peptide originally discovered in the gastrointestinal tract but also found in high density in the mammalian brain. The C-terminal sulphated octapeptide fragment of cholecystokinin (CCK8) constitutes one of the major neuropeptides in the brain; CCK8 has been shown to be involved in numerous physiological functions such as feeding behavior, central respiratory control and cardiovascular tonus, vigilance states, memory processes, nociception, emotional and motivational responses. CCK8 interacts with nanomolar anities with two di€erent receptors designated CCK-A and CCK-B. The functional role of CCK and its binding sites in the brain and periphery has been investigated thanks to the development of potent and selective CCK receptor antagonists and agonists. In this review, the strategies followed to design these probes, and their use to study the anatomy of CCK pathways, the neurochemical and pharmacological properties of this peptide and the clinical perspectives o€ered by manipulation of the CCK system will be reported. The physiological and pathological implication of CCK-B receptor will be con®rmed in CCK-B receptor de®cient mice obtained by gene targeting (Nagata et al., 1996. Proc. Natl. Acad. Sci. USA 93, 11825±11830). Moreover, CCK receptor gene structure, deletion and mutagenesis experiments, and signal transduction mechanisms will be discussed. # 1999 Elsevier Science Ltd. All rights reserved

CONTENTS 1. Introduction 2. Development of peptide, nonpeptide and peptidomimetic CCK-B agonists and antagonists 2.1. Design of selective agonists for CCK-B receptors 2.2. Design of selective antagonists for CCK-B receptors 3. Cloning and characterization of the CCK-B receptor 4. CCK-B receptor gene structure 5. CCK-B receptor localization 6. Signal-transduction cascade for CCK-B receptors 7. Site directed mutagenesis of the CCK-B receptor: characterization of residues involved in binding of ligands and functional coupling 8. CCK-B receptor heterogeneity 9. Pharmacology and therapeutic interests of CCK-B ligands 9.1. Gastric acid secretion 9.2. Interactions between endogenous CCK and opioid systems 9.2.1. Evidence of regulatory mechanisms between endogenous CCK and enkephalin systems in the control of pain 9.2.2. Evidence of antidepressant-like e€ects induced by CCK-B antagonists: interaction with the enkephalinergic system 9.2.3. Interest of enkephalin-degrading enzyme inhibitors in combination with CCK-B antagonists in the treatment of opioid addiction 9.3. CCK-B receptors and anxiety 9.4. CCK-receptors and memory 10. Conclusion Acknowledgements References

ABBREVIATIONS AP CCK CCK4 CCK8 DAG IP3

Aminopyrine Cholecystokinin Cholecystokinin-4 Sulphated cholecystokinin-8 Diacylglycerol Inositol triphosphate

i.c.v. PKC PKA PLA2 PLC TM

Intracerebroventricular Protein kinase C Protein kinase A Phospholipase A2 Phospholipase C Transmembrane

* Corresponding author. Tel.: (33)-1-43.25.50.45; Fax: (33)-1-43.26.69.18; e-mail: [email protected]. 349

351 351 351 352 354 355 355 357 358 361 362 362 363 363 366 366 369 371 372 372 372

Table 1. Selective CCK-B agonists 350 F. Noble and B. P. Roques

CCK-B Receptor

1. INTRODUCTION Cholecystokinin (CCK) is a gut±brain peptide that exerts a variety of physiological actions in the gastrointestinal tract and central nervous system through cell surface CCK receptors. CCK was initially isolated from the porcine duodenum as a 33 amino acid peptide (Mutt and Jorpes, 1968). A number of biologically-active molecular variants were subsequently described (Rehfeld et al., 1982) and the most abundant peptide present in the brain was shown to be CCK8: Asp±Tyr(SO3H)±Met±Gly± Trp±Met±Asp±Phe±NH2. On the basis of their pharmacological properties and speci®cities for ligand binding, CCK receptors have been divided into two subtypes, namely, the CCK-A and CCK-B receptors both belonging to the class of G proteincoupled receptors characterized by seven transmembrane (TM) domains. CCK-A receptors are located mainly in the periphery but are also found in some regions of the brain (Hill et al., 1987a,b). The major population of central CCK receptors are of CCK-B subtype (Hill et al., 1987a), which is also found in the stomach and vagus nerve. The gastrin receptor was found to be identical to the CCK-B receptor. Considerable interest is devoted to the pharmacology of CCK-B receptors, since administration of selective agonists produces behavioral changes such as anxiety, perturbation of memory and hyperalgesia, and dysfunctioning of CCK-B related neural pathways could be involved in neuropsychiatric disorders. Accordingly, CCK-B antagonists have been shown to block panic attacks induced in humans by systemic administration of low doses of CCK4 (Bradwejn et al., 1991), Trp±Met±Asp±Phe±NH2, which has a 300-fold higher anity for the CCK-B receptor than for the peripheral CCK-A receptor (Dauge et al., 1990).

2. DEVELOPMENT OF PEPTIDE, NONPEPTIDE AND PEPTIDOMIMETIC CCKB AGONISTS AND ANTAGONISTS 2.1. Design of Selective Agonists for CCK-B Receptors At CCK-A receptors, sulphated CCK8 [Asp± Tyr(SO3H)±Met±Gly±Trp±Met±Asp±Phe±NH2]was the minimal sequence for high anity binding, whereas at central binding sites, CCK4, gastrin and unsulphated CCK8 can be bound, albeit they have somewhat lower potency compared to sulphated CCK8. Di€erent strategies have been followed to design potent and selective agonists and antagonists of CCK-B receptors. In spite of its intrinsic ¯exibility, CCK8 was found by NMR to exist preferentially under folded form in aqueous solution (FournieÂZaluski et al., 1986) with a proximity between Asp1 and Gly4. This property was used to synthesize cyclic peptides through amide bond formation between Asp1 or a-, b-Glu1 and Lys4 side chains. Compounds such as BC 254 and BC 197 were found highly potent and selective CCK-B agonists (Charpentier et al., 1988a, 1989) (Table 1).

351

Another approach towards CCK-B agonists was to protect CCK8 from degrading enzymes such as aminopeptidase A (APA) (Migaud et al., 1996) and a thiol/serine protease cleaving this peptide at the Met±Gly bond (Camus et al., 1989; Rose et al., 1996). In attempting to design enzyme-resistant analogs, the biologically active Boc[Nle28,31]CCK27±33 (referred as BDNL in the text) (Ruiz-Gayo et al., 1985) was used as the parent compound (Table 1). In this compound the major sites of cleavage were at the Trp30±Nle31 and Nle28±Gly29 bonds. BDNL is potentially resistant to aminopeptidases cleavage owing to its t-butyloxycarbonyl N-terminal-protecting group (Ruiz-Gayo et al., 1985; Durieux et al., 1986a). Consequently, several enzyme-resistant BDNL analogs containing either a retro-inverso 28± 29 amide bond, or a (NMe)Nle31 residue, or a combination of these two modi®cations have been synthesized (Charpentier et al., 1988b). This led to BC 264, a highly potent CCK-B agonist which exhibits about the same anity (KD=0.1±0.5 nM) in all species (guinea pig, rat, mouse, monkey, human, transfected cells) and was at that time the only systemically active CCK-B agonist (Charpentier et al., 1988b; Durieux et al., 1991) (Table 1). A quantitative study of the passage of CCK-like peptides across the blood±brain barrier is possible thanks to the development of the radiolabelled, peptidase-resistant bioactive analogue [3H]pBC264 (Durieux et al., 1989), obtained by replacing the Boc group by a tritiated propionyl residue. Preliminary studies on the bioavailability of [3H]pBC264 have been carried out in the mouse. The radioactivity present in the brain 15 min after intravenous (i.v.) injection of the tritiated compound represented 1.6/10 000 of the total radioactivity injected. Moreover, as shown by HPLC, [3H]pBC264 was very resistant to metabolism, since >85% of the radioactivity present in the brain corresponded to the intact molecule (RuizGayo et al., 1990). The behavioral results obtained with BC 264 suggest that the development of nonpeptidic CCK-B selective agonists endowed with good stabilities and bioavailability should provide useful pharmacological tools and possibly therapeutic agents. In order to design such derivatives, the C-terminal tetrapeptide CCK4 appeared to be the shortest peptide showing signi®cant CCK-B anity and selectivity, although it has been shown to trigger panic attacks in humans (de Montigny, 1989; Bradwejn et al., 1991). Several modi®cations were made to CCK4 which increased its CCK-B selectivity, such as the N-terminal protection of the tetrapeptide in BocCCK4 (Harhammer et al., 1991) and modi®cations of the di€erent amino acids such as the replacement of Met by Nle and (NMe)Nle (Corringer et al., 1993). Recent NMR and molecular dynamics studies indicated that the potent and CCK-B selective CCK4 analogues adopt an S-shaped conformation with a relatively well de®ned orientation of the side chains (Goudreau et al., 1994). The same type of folded structures have been reported for several potent agonists derived from CCK4 and containing a [trans-3-propyl-L-proline] (Nadzan et al., 1991), a diketopiperazine skeleton (Shiosaki et al., 1990) or a [(alkylthio)proline] (Kolodziej et al., 1995). Using

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this template the synthesis of cyclic CCK-4 analogues has been reported which contain in place of the Trp±Met dipeptide, a diketopiperazine moiety resulting from a cyclization between Nle and N-substituted (D)Trp residues and coupled with a small linker to Asp±Phe±NH2 (Weng et al., 1996a) (Table 1). Moreover, the side chain of Nle in the compound Boc±Trp±(NMe)Nle±Asp±Phe±NH2 together with the N-terminus of Trp appeared as good candidates for another possible cyclization. Thus, cyclic compounds were designed by molecular modeling in order to mimic the proposed biologically active conformation of this CCK4 analogues. The goal of this study was to stabilize the bioactive conformation of CCK-B agonists in order to aid the design of nonpeptide ligands. This led to the development of macrocyclic constrained CCK4 analogs endowed with agonist properties, able to cross the blood±brain barrier (Blommaert et al., 1997) (Table 1). It is noteworthy that all these CCK-B agonists have a common C-terminal dipeptide: Asp±Phe± NH2. Selective and peptidase-resistant CCK-B ligands which derive from Boc-[Nle31]CCK30±33, by incorporation of nonnatural hydrophobic amino acids have also been developed (Weng et al., 1996b). Among these compounds Boc±[Phg31, 33 Nal ]CCK30±33 proved to be a full agonist in an electrophysiological assay on rat hippocampal CCK-B receptors. Moreover, it appears that modi®cations of the hydrophobic and steric character of either the C- or N-terminal amino acid substituents of CCK-4 derivatives seem to be able to change the agonist or antagonist pro®le of these peptides. This was shown by the fact that the agonist Boc-[Phg31, Nal33]CCK30±33 can be chemically modi®ed to become an antagonist, by addition of two alkyl groups on the terminal CONH2 (Weng et al., 1996b). Very recently a new series of highly potent and selective CCK-B agonists has been developed (Million et al., 1997). Boc±Trp±(NMe)Nle±Asp± Phe±NH2, the C-terminal tetrapeptide of BC 264 was shown to have a high anity and to behave as a speci®c agonist of the CCK-B receptor, and to adopt the S-shaped preferential conformation. In order to determine the essential structural components of speci®c CCK-B agonists, a step by step lengthening of the C-terminal tetrapeptide of BC 264 was carried out. Various di-acidic moieties, such as malonate or succinate residues, were coupled to the N-terminal part of the tetrapeptide leading to RB 400 (HOOC±CH2±CO±Trp±(NMe)Nle±Asp± Phe±NH2) and RB 403. RB 400 was also derivatized under its benzylamide and methyl ester forms. The compounds which belong to this RB 400 series possess high anities for the CCK-B receptor, a subnanomolar anity being obtained in the case of RB 400 (Ki=0.42 nM) (Million et al., 1997) (Table 1). 2.2. Design of Selective Antagonists for CCK-B Receptors Much of the early research regarding the physiological e€ects of CCK was hindered by the lack of selective antagonists. There now are representative

CCK antagonists of various chemical structures, including dipeptoid, benzodiazepine, pyrazolidinone and amino acid derivatives that have both excellent selectivity and high anity for either CCK-A or CCK-B receptors. For a long time, the most potent and selective CCK-B antagonists reported were the benzodiazepine derivative L-365 260 (Bock et al., 1989), the peptoid PD-134 308 (or CI-988) (Horwell et al., 1991), the ureidoacetamide RP-69758 (BoÈhme et al., 1992), the diphenylpyrazolidinone LY-288 513 (Rasmussen et al., 1992), and the asperlicin-related quinazolinones (Yu et al., 1991). However, one of the main problems encountered with most of these compounds is their low bioavailability (Dethlo€ and De La Iglesia, 1992; Chen et al., 1992) (Table 2). CCK-B receptor antagonist properties can be introduced in CCK peptides by reducing the sequence, as in Boc±Trp±Orn(Z)±Asp±NH2 (Gonzalez-Muniz et al., 1990), or by introducing large and hindering residues as illustrated with Boc± Trp±Phg±Asp±1NalN(CH3)2 (Corringer et al., 1993) which behaves as a relatively potent (Ki=38 nM), peptidase resistant and selective CCK-B antagonist. A direct comparison of the structure of some of the CCK-B ligands designed by Horwell et al. (1991), such as PD-134 308, showed that the size of these latter molecules could be reduced to increase their lipophilicity. These compounds have been synthesized and some of them found to be potent and selective of CCK-B receptors. Moreover, as expected, one of them (RB 211) was shown more ecient in crossing the blood±brain barrier that the parent compounds (Blommaert et al., 1993), and devoid of the weak CCK-A agonist properties of dipeptoids (HoÈcker et al., 1993; Ding et al., 1995). On the other hand, to improve the properties of PD-134 308, numerous conformational restrictions were introduced in its structure. Unfortunately, neither N-terminal cyclization (Fincham et al., 1992a), nor macrocyclization (Didier et al., 1992; Bolton et al., 1993), or rigidi®cation of the amide bond (Fincham et al., 1992b), led to any signi®cant positive result. Only a C-terminal cyclization of PD134 308 derivatives, by means of a tetrahydronaphtyl group, has been reported to increase the anity of CCK-B receptors (Higginbottom et al., 1993). This approach has also been used for compounds such as RB 210 (Table 2), in which C-terminal constraints can be easily introduced. Thus, the b carbon of the phenethyl side chain of RB 210 was linked to the a carbon bearing the carbonyl function, by means of a methylene bridge (Table 2). This resulted in the formation of a proline ring (Bellier et al., 1997). The most potent compounds of this new series had similar anities for the CCK-B receptor as RB 210. Structure±anity relationships of this series indicated that lengthening the distance between the amide nitrogen atom and the phenyl ring was of little importance, while the position of the carboxylate could not be modi®ed. Therefore, the pyrrolidine ring was replaced by piperidine, in order to slightly modify the possible orientation of the aromatic moiety towards the carboxylate, without violating any of the requirements previously established in both linear and constrained series for

CCK-B Receptor

353

Table 2. Selective CCK-B antagonists Compound

Name

Ki (nM) [3H]pCCK8 CCK-A

Reference

CCK-B

L-365 260

800

PD-134 308

1440

LY-288 513

11 600

RP-73 870

1634

0.5

Pendley et al. (1995)

YM-022

150

0.1

Nishida et al. (1994)

RB-210

1518

14

Blommaert et al. (1993)

1560

24

Bellier et al. (1997)

CCK-B binding. Nevertheless, the resulting compounds behave as moderately potent CCK-B antagonists (Bellier et al., 1998). As previously mentioned, the clinical development of PD-134 308 (CI-988) was limited due to its weak bioavailability, which was attributed to poor absorption and ecient hepatic extraction. Thus, Parke±Davis scientists also envisaged that reducing

7

0.5

31

Lotti and Chang (1989)

Horwell et al. (1991)

Howbert et al. (1992)

the molecular weight of the parent compound would lead to better absorption. Accordingly, they synthesized a series of analogues in which the key amethyltryptophan and adamantyloxycarbonyl moieties, required for receptor binding, were kept intact and the C-terminus was extensively modi®ed. These modi®cations led to compounds such as CI-1015 for which the oral bioavailability in rat was improved

354

F. Noble and B. P. Roques

nearly ten times, and the blood±brain permeability was also enhanced relative to CI-988 (Trivedi et al., 1998). Most of the high anity nonpeptide receptor antagonists for CCK-B receptors were identi®ed directly from natural products screening programs (Lam et al., 1993; Ohtsuka et al., 1993), or from structural leads derived from natural products. Merck researchers were the ®rst to identify selective high anity nonpeptide antagonists of CCK-B receptors [L-365 260; Lotti and Chang (1989)], using a benzodiazepine pharmacophore for the natural product asperlicin (Chang et al., 1985; Bock et al., 1986). A major drawback associated with these early benzodiazepine-based CCK-B antagonists was their lack of oral ecacy. Nevertheless, incorporation of a (t-butylcarbonyl)methyl group at the 1position (Semple et al., 1996a) or a 2-pyridyl group at the 5-position (Semple et al., 1996b) of the parent benzodiazepine structure provides a signi®cant increase in absorption. Other attempts to improve aqueous solubility have been to introduce acidic groups (Bock et al., 1994) or lipophilic surrogates (Chambers et al., 1995), into the three-position of the aryl urea position of either the 1,4-benzodiazepine-2-one parent system. The opposite strategy has also been used with the introduction of basic amino substituents into the same region. YM 022 is the optimal structure in this new series, with sub-nanomolar anity for the CCK-B receptor (Nishida et al., 1994) (Table 2). Moreover, when combinations of the above modi®cations are incorporated into the same molecule, the improvements in the in vivo e€ects appears to be essentially additive, resulting in compounds such as YF 476, which has a good oral bioavailability in dogs (Semple et al., 1997). On the other hand, since the asperlicin structure is comprised of several heterocyclic systems, it was hypothesized that alternative substructures embedded within the molecular framework of this natural product may provide a rational starting point for e€orts to design structurally novel series of nonpeptide CCK ligands. On these observations, Lilly scientists developed a series of quinazoline derivatives by a bond disconnection approach (Yu et al., 1991). Combination of the key fragments of the Lilly and Merck series led to the development of novel nonpeptide CCK-B receptor antagonists with substitution on the quinazolinone and phenyl rings. Binding data of this class of compounds suggest that the linker between these both rings is a critical determinant for CCK-B receptor binding anity. Nevertheless these new compounds have a low selectivity for CCK-B receptor (Padia et al., 1997). Two other series have been described, leading to the synthesis of derivatives that have both excellent selectivity and high anity for CCK-B receptors: the ureidoacetamides class of CCK-B antagonists [RP-73870; Pendley et al. (1995)] and the pyrazolidinones [LY-288 513; Howbert et al. (1992)] (Table 2).

3. CLONING AND CHARACTERIZATION OF THE CCK-B RECEPTOR Although there is general agreement that the CCK-A receptor has a distinct agonist and antagonist binding pro®le from the gastrin and CCK-B receptors, controversy remains regarding the existence of distinct CCK-B and gastrin receptor subtypes. Gastrin, CCK and CCK-related peptides comprise a hormone family, characterized by the identical carboxyl-terminal pentapeptide amide structure, a domain critical for receptor binding. Agonist binding studies on brain membranes and parietal cells show a six- to ten-fold and a one- to two-fold higher anity for CCK than for gastrin, respectively (Jensen et al., 1990). These small di€erences in agonist binding have created controversy regarding the existence of subtypes within this receptor class. Whether the CCK-B/gastrin receptor class is heterogenous was awaiting isolation and direct comparison of putative subtypes. Several strategies have been followed to puri®ed the CCK-B/gastrin receptor and cloned its cDNA. Assuming CCK-A and CCK-B receptors are closely related members of the same receptor family on the basis of their shared high anity for CCK, to obtain the CCK-B receptor cDNA, the 32 P-labeled, randomly primed full-length coding region of the CCK-A receptor cDNA isolated from rat pancreas (Wank et al., 1992a) was used to screen a cDNA library constructed from AR42-J cells, a rat pancreatic acinar carcinoma cell line known to express predominantly (80%) CCK-B subtype CCK receptors, pharmacologically identical to brain CCK-B receptors (Lambert et al., 1991). Several clones were isolated only under low-stringency conditions. Two of them were sequenced and found to contain identical long open reading frames highly homologous to the CCK-A receptor cDNA. Using this new CCK-B open reading frame sequence as a 32 P-labeled prime probe, high-stringency hybridizing clones were isolated from the rat brain cortex cDNA library. One of this clone was a 2243-base pair (bp) clone which had identical cDNA sequence to those of the two clones isolated from the AR42-J cell cDNA library (Wank et al., 1992b). At the same time, the gastrin receptor cDNA was cloned from a canine parietal cell cDNA library using a Cos cell plasmid expression approach relying on a single-subunit receptor-binding protein suggested by anity cross-linking studies (Kopin et al., 1992). The CCK-B receptor cDNA has subsequently been cloned from rat stomach, human brain and stomach, and guinea pig gallbaldder and stomach [review in Wank (1995)]. The deduced amino acid sequences of the di€erent CCK-B receptors cloned show a high degree of sequence homology, in the range expected for interspecies variation of the same receptor. Moreover, the high homology observed between the rat brain CCK-B receptor and the canine parietal cell gastrin receptor was the ®rst indication that the gastrin receptor is the same as the CCK-B receptor (Wank et al., 1992b; Kopin et al., 1992). This has been recently con®rmed. Thus, analysis of human genomic DNA indicates that a single gene encodes both the brain

CCK-B Receptor

and the stomach CCK-B/gastrin receptors (Lee et al., 1993). In rat, the ®rst in frame ATG consistent with a consensus translation initiation site, initiates a single long open reading frame encoding a unique 452 amino acid protein with a predicted Mr of 48 954 (Wank et al., 1992b) (Fig. 1). Hydropathy analysis predicts that their amino acid sequence contains seven TM-spanning domains, suggesting that CCKB receptor belongs to the G protein-coupled superfamily of receptors, in good agreement with its reported modulation by guanine nucleotides (Merrit et al., 1985; Roche et al., 1990). The sequence contains four potential N-linked glycosylation sites, three in the amino terminus and one in the third intracellular loop, which could be consistent with the heavy and variable degree of glycosylation reported using ligand anity cross-linking techniques (Pearson et al., 1987; Lambert et al., 1991). There is multiple potential serine and threonine phosphorylation sites, one potential site for protein kinase C (PKC) phosphorylation (serine 82, in the ®rst intracellular loop) and two potential site for protein kinase A (PKA) phosphorylation (serine 154 in the second intracellular loop, and serine 442 in the cytoplasmic tail). Moreover, as in CCK-A receptor, there are two cysteins in the ®rst and second extracellular loops, which may form a disul®de bridge required for stabilization of the tertiary structure as demonstrated for other receptors (Karnik et al., 1988; Dixon et al., 1987). A cysteine in the C-terminal region conserved in many of the G proteincoupled receptors may be a membrane-anchoring palmitoylation site as demonstrated for rhodopsin and the b2-adrenergic receptors (O'Dowd et al., 1988; Ovchinikov et al., 1988). The CCK-B receptors cloned from other species have the same structural features described above for the rat CCK-B receptor (Kopin et al., 1992; Lee et al., 1993; Ito et al., 1993).

4. CCK-B RECEPTOR GENE STRUCTURE The gene for the CCK-B receptor has been cloned in humans (Song et al., 1993). This gene exceeded 8 kb in length and contained a 1356-bp open reading frame which was interrupted by four introns of 164± 1177 bp. Exon 1 encodes the putative extracellular amino terminus of the receptor. Exons 2 and 3 encode TM regions I±IV, and exon 4 encodes the ®fth TM region and an initial portion of the third intracellular loop. Exon 5 encodes the remainder of this intracellular loop, the remaining TM regions VI and VII, and the intracellular carbonyl terminus (Song et al., 1993). An isoform cDNA of the CCK-B receptor was isolated from human stomach using PCR-based cloning strategies (Miyake, 1995). This cDNA differed from initially cloned cDNA only in the 5'-end region and encoded a truncated isoform (DCCK-B) in which the putative N-terminal extracellular domain of the CCK-B receptor was completely lost, and was otherwise identical in the remaining sequence. Isolation of genomic CCK-B receptor DNA revealed that the gene structure was similar to

355

what was previously reported except that the ®rst intron was 010 kb compared with 1.177 kb and contained the sequence for the alternative ®rst exon, 1b. The alternative usage of this exon and promoter causes no change in translated receptor protein, because this exon corresponds to the 5'-noncoding region (Miyake, 1995). Using PCR, Song et al. (1993) determined that the human CCK-B receptor gene was alternatively spliced in exon 4, resulting in the presence of two CCK-B receptor transcripts di€ering by a block of ®ve amino acids (GGAGP) within the third intracellular loop. Alternative splicing occurs in the third intracellular loop of certain other G protein-coupled receptors, such as the human D2 and D3 dopamine receptors (Giros et al., 1989; Monsma et al., 1989; Fishburn et al., 1993). In the case of the D2 dopamine receptor, alternative splicing may a€ect the coupling of the receptor to second messenger pathways (Montmayeur and Borrelli, 1991; Liu et al., 1992). Thus, the pentapeptide cassette in the CCK-B receptor may be of potential functional importance. Nevertheless, despite the location of the variably spliced amino acid sequence, no signi®cant di€erence in agonist anity or signal transduction was measured between the shorter and longer isoforms (Wank, 1995). The shorter transcript is predominant in the stomach. Several CCK-B receptor mRNA forms from rat brain tissues and fundus glands have also been isolated, allowing the detection of truncated mRNA species and the determination of the CCK-B receptor gene structure (Jagerschmidt et al., 1994; Luciani et al., in preparation). In the cerebral cortex and the hypothalamus, three precursor CCK-B receptor mRNA forms that contain introns were detected. These three unspliced CCK-B receptor mRNA forms probably represent maturation intermediates of the CCK-B receptor transcript. The precursor CCK-B receptor RNA forms observed were di€erentially distributed in the rat brain. Indeed, although several precursor RNAs were detected in the cerebral cortex and the hypothalamus, only a completely unspliced mRNA form was detected in the cerebellum, and no unprocessed messengers in the hippocampus (Jagerschmidt et al., 1994). To identify chromosomal location of CCK-B receptor, DNA from a clone of the CCK-B receptor gene was labeled with biotin and hybridized to human or mouse metaphase chromosomes. Thus, CCK-B receptor has been localized on the terminal short arm of chromosome 11 in humans (Song et al., 1993) and on a syntenic region on mouse chromosome 7 (Huppi et al., 1995).

5. CCK-B RECEPTOR LOCALIZATION CCK-A receptors are found principally in the gastrointestinal tract and select areas of the CNS, while CCK-B/gastrin receptors are found principally in the CNS and select areas of the gastrointestinal tract, on pancreatic acinar cells and parietal cells. Autoradiographic studies using CCK-related peptide-binding sites in the rat brain, such as [125I]CCK8 (Nieho€, 1989; PeÂlaprat et al., 1987),

Fig. 1. Schematic representation of the rat CCK-B receptor showing the postulated transmembrane topology, sites for putative NH2-linked glycosylation (tridents), serine and threonine phosphorylation by protein kinases C and A (PO3), and conserved cysteines in the ®rst and second extracellular loops, possibly forming a disul®de bridge, and possible a palmitoylated conserved cysteine in the cytoplasmic tail. NH2-, amino terminus; COOH-, carboxy terminus.

356 F. Noble and B. P. Roques

CCK-B Receptor

[3H]BDNL (PeÂlaprat et al., 1987), [3H]CCK8 (Dijk et al., 1984), [3H]pentagastrin (Gaudreau et al., 1983) or selective CCK antagonists (Woodru€ et al., 1991; Hill and Woodru€, 1990), were generally in good agreement with the distribution of the CCK receptors mRNA. Nevertheless, some discrepancies were found between the distribution of mRNA for the CCK-B receptors and that of the CCK binding sites reported (Honda et al., 1993; Jagerschmidt et al., 1994). These discrepancies between the distribution of CCK receptors demonstrated by autoradiographic studies and that of CCK receptor mRNAs identi®ed by in situ hybridization could be explained in part by the fact that receptor mRNAs are located only in cell bodies, however, the proteins translated from their mRNAs are transported to terminal ®elds where binding sites for ligand are expressed. Reciprocally, mRNAs may not always be translated eciently into receptor proteins, thus the presence of receptor mRNAs does not necessarily imply the presence of functional receptor proteins in the same location. In the rat, CCK-B mRNA was shown to be widely distributed in areas such as the cerebral cortex, the olfactory regions, the hippocampal formation, the septum, the amygdala, the basal ganglia and related regions (nucleus accumbens, caudate putamen, substantia nigra), the interpeduncular nucleus and the cerebellum (Honda et al., 1993). This mRNA localization is largely consistent with previous reported histochemical binding studies (Gaudreau et al., 1983; PeÂlaprat et al., 1987; Nieho€, 1989), except for regions such as the cerebellum where neither CCK-B receptors (PeÂlaprat et al., 1987) nor prepro-CCK mRNA (Lanaud et al., 1989) were previously detected. Receptors in the posterior cortical amygdaloid nucleus and in the pyramidal cell layer of the hippocampus correlate with the presence of immunohistochemically de®ned CCK nerve terminals in these regions (Hendry and Jones, 1985). Other regions of good CCK receptor± peptide correspondence include the area postrema, nucleus of the solitary tract, dorsal tegmental nucleus, medial and cortical nuclei of the amygdala, caudate-putamen, nucleus accumbens and supraoptic nucleus (Nieho€, 1989; HoÈkfelt et al., 1991). However, areas of noncorrespondence also occur. For example, the amount of CCK found in the central nucleus of the amygdala is one of the highest in the brain, and includes CCK-containing terminals, but this region contains levels of CCK receptor close to background (Nieho€, 1989; Fallon et al., 1983; Roberts et al., 1982). Other regions with CCK-positive cells and/or terminals, but low levels of receptors include many hypothalamic nuclei, ventral tegmental area, substantia nigra, dorsal raphe nucleus and periaqueductal grey area. Species-speci®c heterogeneity in tissue expression is apparent in di€erent structures, and indicates that the results of studies performed in one species may not necessarily be generalized to other species (Nieho€, 1989; Mercer et al., 1996). For example, by autoradiographic studies CCK-B receptors have been detected in the guinea pig, human and mouse cerebellum (Sekiguchi and Moroji, 1986; Williams et al., 1986; Dietl et al., 1987).

357

CCK-B receptors are also present on immune cells such as monocytes and T lymphocytes, where their function is unknown. Moreover, like CCK-A receptors, CCK-B receptors are also present on tumors and tumor-derived cell lines such as a human leiomyosarcoma and the AR42J cell line where they may stimulate growth (Wank, 1995). Several studies of vagal CCK receptors have reported an accumulation of binding sites proximal to the site of ligation of the nerve, indicating the axonal transport of receptors towards the periphery (Moran et al., 1987; Zarbin et al., 1981). Evidence that these receptors are present on a€erent neurons has been provided by the observation that they are present on the cell soma of a€erent neurons in the nodose ganglion and are lost after treatment with the a€erent neurotoxin, capsaicin. Moreover, a very recent study provided direct evidence for the both CCK-A and CCK-B receptors are expressed by rat vagal a€erent neurons, and reported that both receptors are expressed in human nodose ganglia (Moriarty et al., 1997).

6. SIGNAL-TRANSDUCTION CASCADE FOR CCK-B RECEPTORS The signal transduction mechanism of CCK-A receptors has been best characterized in pancreatic acinar cells, where CCK stimulates digestive enzyme release, usually assayed as amylase activity. One physiologically important signaling pathway is the hydrolysis of polyphosphoinositides (PPI) by phospholipase C and the subsequent formation of the second messengers, inositol 1,4,5-triphosphate (1,4,5-IP3) and 1,2-diacylglycerol (DAG), leading to the release of intracellular Ca2+ and the activation of PKC, respectively (Berridge and Irvine, 1989). However, recent results suggest that depending on the agonists used, CCK-A receptor activation may result in di€erential stimulation of the phospholipase A2 (PLA2) and PLC pathways (Tsunoda and Owyang, 1993; Yoshida et al., 1997). Thus, it has been shown that some agonists utilize PLA2-arachidonic acid cascades to mediate Ca2+ oscillations and amylase secretion, while others utilize the PLCphosphoinositol pathways to produce IP3 and DAG. IP3 and DAG mediate Ca2+ release from intracellular pools and activation of PKC, respectively, and these act in concert to stimulate amylase secretion (Berridge and Irvine, 1989). Moreover, it has been shown that high concentrations of CCK8 increases the formation of other intracellular messengers such as adenosine 3',5'-cyclic monophosphate (cAMP) through CCK-A receptor activation (Yule et al., 1993). The signal transduction cascade for CCK-B receptors has been less well characterized, largely because of the diculty of working with isolated neurons expressing CCK-B receptors or isolated gastric mucosal cells expressing CCK-B/gastrin receptors. Thus, numerous studies have investigated the signal transduction pathways by using the expression of CCK-B receptors in di€erent cell lines. It has been demonstrated that the CCK-A receptor when transfected into the CHO cell line couples to signal trans-

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duction pathways with characteristics similar to those reported for the receptor expressed in the native pancreatic acinar cell (Yule et al., 1993). In isolated canine, porcine, or rabbit parietal cells, CCK-B receptors, like CCK-A receptors, couple to a pertussis toxin-insensitive G protein (Roche et al., 1991a,b), subsequently causing activation of PLC, formation of IP3 and DAG, release of intracellular Ca2+, and translocation and activation of PKC (Tsunoda et al., 1988; DelValle et al., 1992). To our knowledge, central CCK-B receptors have not yet been proved to link to any second-messenger system in the brain, including the phosphoinositide system, although phosphoinositide metabolism has been shown to be a€ected by CCK in cells expressing endogenous CCK-B receptors (Shinohara and Kawasaki, 1994; Barrett et al., 1989), and an embryonic pituitary cell line (Lo and Hughes, 1988). Enzymatically dissociated neonatal-rat brain cells were also used to study the in¯uence of CCK8 on the phosphoinositide turnover in brain. The results obtained provided evidence that CCK8 stimulated the turnover of phosphoinositide and increase IP3 labelling in this model, in which both CCK-A and CCK-B receptors seem to be involved (Zhang et al., 1992). One study of CCK-B receptors, using synaptoneurosomes from guinea pig cortex, did not demonstrate CCK analogues-stimulated increase in PLC, although Ca2+ was released from intracellular stires, possibly via a G protein-independent mechanism (Galas et al., 1992). Expression of receptor cDNAs in a mammalian expression system that closely approximates the native cell allows a readily available source of receptor for study [see Yule et al. (1993)]. Moreover, as there is still some possibility for the existence of multiple gastrin receptors other than CCK-B, it might be necessary to analyze the signal transduction using a system in which a single class of CCKB receptor is present. Studies of second messenger signaling in these models have demonstrated functional coupling to phospholipase C via a pertussis toxin-insensitive G protein (Gq and/or G11), using inositol phosphate generation and Ca2+ mobilization (Lignon et al., 1993; Wank et al., 1994;

Jagerschmidt et al., 1995). Moreover, Akagi et al. (1997) have recently shown that the [Ca2+]i oscillation induced by the weak activation of CCK-B receptor requires Ca2+ in¯ux to support the activity of the small amount of IP3. PKC and PLA2 activation and subsequent arachidonic acid production might be involved in this Ca2+ in¯ux into CHO cells mediated by CCK-B receptors (Akagi et al., 1997). Pommier et al. (submitted) have demonstrated that CCK-B receptor activation led to arachidonic acid through activation of both DAG lipase and another phospholipase which is a PLA2. Moreover, the results obtained indicate that CCK-B receptor may couple to two G proteins: a PTXinsensitive G protein (Gq/G11)-PLC pathway and a PTX-sensitive G protein±phospholipase A2 pathway leading to the release of arachidonic acid (Fig. 2). In order to con®rm the physiological signi®cance of these observations, other experiments are required using native cells, such as isolated gastric glands.

7. SITE DIRECTED MUTAGENESIS OF THE CCK-B RECEPTOR: CHARACTERIZATION OF RESIDUES INVOLVED IN BINDING OF LIGANDS AND FUNCTIONAL COUPLING In order to develop more speci®c and selective compounds, it is important to elucidate the molecular interactions involved in CCK receptor ligand binding. In contrast to the endogenous peptide ligands, nonpeptide antagonists often show substantial di€erences in anity among species. These species-related di€erences in binding anity often re¯ect di€erences in the primary structure of the receptors. Thus, although canine and human CCKB receptors share 090% amino acid identity and have similar agonist binding anity, they exhibit opposite rank orders of anity for two nonpeptide CCK antagonists: L-365 260 shows selectivity for the human receptor, whereas L-364 718 displays a higher anity for the dog CCK receptor. Mutational analysis of the CCK-B receptor demonstrated that antagonist anities can be altered dra-

Fig. 2. CCK8 stimulation of inositol phosphate (IP) production and arachidonic acid release in CHO cells expressing the rat CCK-B receptor in absence or in presence of the pertussis toxin (PTX). To characterize the e€ect of PTX on CCK8-stimulated IP production or arachidonic acid release, cells were treated with PTX for 16 hr at 378C before treatment with the CCK-B agonist.

CCK-B Receptor

matically by a single amino acid substitution which in turn explain species-related di€erences (Beinborn et al., 1993). The replacement of Val349 in the sixth TM region of the human receptor with the corresponding amino acid in the canine receptor, leucine, decreases the anity of L-365 260 and increases the anity of L-364 718 to the values seen in the canine receptor. Conversely, substitution of Leu355 to Val in the canine receptor results in antagonist anities that resemble those observed in the human receptor. Similarly, whereas the endogenous agonist, CCK-8, triggers comparable signaling with each of the CCK-B receptor homologs, marked species di€erences are observed in the ability of the synthetic agonists to trigger receptor-mediated inositol phosphate production. Further analysis revealed that this observed di€erences in drug ecacy are in large part explained by single or double aliphatic amino acid substitutions between respective species homologs in TM domain VI (Kopin et al., 1997). These studies emphasize the need for species-appropriate models for the screening of agonists and antagonists in drug development. The CCK-B receptor is a member of the G protein-coupled receptor superfamily. Within this broad group, it is well established that the TM domain residues of biogenic amine receptors are the primary determinants of ligand anity (Schwartz and Rosenkilde, 1996). In contrast, relatively little is known about the molecular basis for ligand anity to peptide receptors. Several peptide ligands were reported to interact with both TM and extracellular domains of these receptors. Thus, for instance, it has been shown by point mutagenesis and chimeric studies that opioid receptors interact with their ligands at multiple sites, both extracellular and intramembranous (Wang et al., 1994; Befort et al., 1996; Pepin et al., 1997). Likewise, numerous studies have provided evidence for an important role of residues in the N-terminal domain, the extracellular loops and the TM helices for the ability of several receptors to bind their ligands, such as neurokinin receptor (Fong et al., 1992), V1a vasopresin receptor (Chini et al., 1995), oxytocin receptor (Chini et al., 1996), somatostatin receptor (Kaupman et al., 1995) and vasoactive intestinal peptide (VIP) receptor (Du et al., 1997). A few studies have been devoted to the CCK-B receptor. Several strategies have been followed to analyze the role of CCK-B receptor amino acids in determining high anity binding of subtype selective agonists and antagonists. The ®rst one which has been utilized to identify potentially important residues as a possible determinant for CCK-A vs CCKB receptor selectivity, was based on di€erences between the primary sequences of CCK-A and CCK-B receptors. On the other hand, several aromatic residues were shown to be highly conserved within the TM domains of G protein-coupled receptors in particular in helices IV, V and VI (Befort et al., 1996; Underwood et al., 1994; Probst et al., 1992). These conserved aromatic residues were proposed to play an important role in the spatial organization of the binding site (Underwood et al., 1994). Moreover, these amino acids could be involved in the signal transduction mechanism occuring after

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agonist-induced receptor activation as described in the case of the neurokinin type 1 receptor (Huang et al., 1994a), the 5-HT2 receptor (Choudhary et al., 1993), and the angiotensin II type AT1 receptor (Marie et al., 1994). Finally, site-directed mutagenesis can be proposed from a rhodopsin-derived model of G protein-coupled receptor extended to the CCK-B receptor (Jagerschmidt et al., 1996; Baldwin, 1993). Several studies have been performed using these di€erent strategies (Fig. 3). The cloning and sequencing of both CCK-A and CCK-B receptor types from di€erent species have shown a marked amino acid sequence homology. Thus, in rat, in the seventh TM domain, there are only ®ve amino acids di€ering between both receptor types. Among these residues, His381 was selected because based on a rhodopsin-derived model of G protein-coupled receptor extended to the CCK-B receptor, His381 was found to be embedded within the hydrophobic core formed by the seven helices and located near the position in which a lysine residue was shown to be involved in the retinal Schi€ base formation (Bownds, 1967). Moreover, site directed mutagenesis experiments have shown that histidines present in TM domains often play a key role in binding various agonists and/or antagonists to G protein-coupled receptors (Olah et al., 1992; Fong et al., 1993, 1994; Huang et al., 1994a). The mutation of the His381 residue to leucine or phenylalanine had no apparent e€ect on the structural integrity of the receptor. Moreover, the results obtained show that this residue is not involved in the agonist binding site and its mutation had also no e€ects on the binding of CCK-B selective antagonists. In contrast, antagonists highly selective for CCK-A receptors had increased anities for H381L and H381F mutants. Thus, as suggested His381 residue of the rat CCK-B receptor is essential for CCK-B vs CCK-A antagonist selectivity (Jagerschmidt et al., 1996). This result is in good agreement with a similar independent work (Kopin et al., 1995) in which the authors predicted the relative position of residues among the TM helices of the human CCK receptors, which are involved in A/B selectivity. The results obtained show that no single residue substitution is sucient to completely reverse subtype selectivity, but that multiple residues should act in concert. Eight residues appeared pharmacologically important. Moreover, the rhodopsin-based three-dimensional model of G protein-coupled receptor structure predicts that these residues project into a putative ligand pocket, similar to the one which is well established for biogenic amine receptors (Caron and Lefkowitz, 1993; Strader et al., 1989). In addition, a segment of ®ve amino acids in the second extracellular loop of the CCK-B receptor was shown to be essential for the high anity of the natural peptide agonits, gastrin, suggesting that determinants of the binding site of the CCK-B receptor are also situated within the extracellular domains (Silvente-Poirot and Wank, 1996). On the other hand, several aromatic residues located in the TM helices are conserved among most of the G protein-coupled receptors (Probst et al., 1992). This is the case of the Ar±X2±Pro±X7±Ar

Fig. 3. Schematic representation of the important amino acids involved in the binding of CCK-B agonists and antagonists, and in the G protein coupling determined by sitedirected mutagenesis.

360 F. Noble and B. P. Roques

CCK-B Receptor

motif (where Ar is aromatic and X is any residue) which is very often found in the TM-V domain, and Ar±X3±Ar±X±Pro, found in the TM-VI helix of most of the G protein-coupled receptors (Underwood et al., 1994). These motifs were also found in the CCK-B receptor and the high conservation of these constituting amino acids suggests that they could have a critical role in the structural organization and/or in the functioning of G proteincoupled receptors. Using site-directed mutagenesis (Jagerschmidt et al., 1998) have demonstrated that the highly conserved aromatic acid residues between G protein-coupled receptors, Phe227 and Phe347, do not play an important role in the recognition of the agonists, while a loss in the anity of the antagonists to the mutated receptor was observed. Moreover, these authors have also identi®ed one amino acid Trp351 of the agonist binding site of the receptor involved in CCK4 binding. Studies on the functionality of the CCK-B receptor showed a di€erent pattern of response in the case of F347A and F227A mutants. Thus, mutation of Phe347 disrupts phosphatidylinositol pathway, indicating a complete loss of the transduction process associated with agonists binding. In contrast, both ecacy and potency values were found very close for the wild-type and F227A receptors indicating that the Phe227 residue of the rat CCK-B receptor is not involved in the signal transduction pathway. Another residue, Asp100 has also been shown to be involved in signal transduction (Jagerschmidt et al., 1995). It was hypothesized that Asp100 points in a direction of a cluster of basic amino acid (Lys333/Lys334/Arg335) located in the third intracellular loop of the receptor at the bottom of the TM-VI domain. This was con®rmed by results reported by Wang (1997) showing that these three basic amino acids play a critical role in CCKB receptor activation of Gq proteins. Thus, although not directly involved in the binding of CCK-B ligands as shown by the lack of change in binding anities following replacement of Phe347 by Ala, Phe347 which belongs to the TM-VI domain, could be a residue implicated in transduction processes by playing a key role in agonist-induced changes in receptor conformation triggering Gq/11 protein stimulation. As described above (see Section 6), CCK-B receptors can activate multiple e€ector pathways via coupling to distinct G proteins (Pommier et al., submitted). Further experiments performed with the F347A mutant receptor seem to emphasize this result. Thus, the replacement of Phe347 by Ala was shown to disrupt the phosphatidylinositol pathway, while activation of this mutant receptor by CCK8 resulted in release of arachidonic acid (Noble et al., in preparation). These results suggest that the exchange of Phe347 for Ala induces alteration of intracellular loop structure a€ecting the Gq/11 protein coupling following binding of a ligand, while no modi®cation with another G protein leading to the release of arachidonic acid can be observed. The importance of the external N-terminal region of several G protein-coupled receptors for the binding of peptide ligands has been reported (Hjorth et al., 1994). Since the N-terminal extracellular domain

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of rat CCK-B receptor includes three putative Nglycosylation sites, N-terminal truncation changes molecular size and perhaps structural conformation. Nevertheless, Miyake (1995) has shown that a splice variant of the CCK-B receptor, in which the Nterminal extracellular domain and almost all the residues of the ®rst TM domain helix were absent, only displayed moderate altered binding and functional properties. These results indicate that this domain is not essential for ligand binding, in good agreement with a recent study, showing that deletion of this domain did not modify the binding of di€erent ligands (Jagerschmidt et al., 1998).

8. CCK-B RECEPTOR HETEROGENEITY On the basis of the pharmacological data obtained receptor subtypes has been proposed to exist in the CCK family. However, despite extensive searching by hybridization screening of cDNA libraries from di€erent tissues [reviewed in Wank (1995)], only two CCK receptors have been identi®ed. Southern blot hybridization using human, guinea pig and rat DNA with either CCK-A or CCK-B receptor species-speci®c, full-length coding sequence probes under both high- and low-stringency conditions has also failed to identi®ed additional members of the CCK receptor family. Radiolabelled ligands derived from nonpeptide antagonists represent useful tools to characterize the di€erent anity states of neuropeptide receptors. By comparing results obtained from binding of agonist and antagonist radioligands, pancreatic CCK-A receptors were shown to exist in three di€erent anity states for the agonist CCK8; the agonist ligand [125I]CCK8 identi®ed the high- and low-anity states, while the antagonist ligand [3H]L-364 718 bound to the low-anity state and to a previously unidenti®ed very-low-anity state which represents 60±80% of the receptor (Talkad et al., 1994; Huang et al., 1994b). This result is in good agreement with the values of Bmax. Thus, the antagonist [3H]L364 718 recognized a signi®cantly higher number of receptors than the agonist [125I]CCK8. A heterogeneity of CCK-B binding sites has been reported in guinea pig brain from binding studies using linear or cyclic CCK8-analogues (Durieux et al., 1986b; Knapp et al., 1990; Rodriguez et al., 1990). Similarly, CCK-B receptor has been shown to exist in three di€erent anity states. Nevertheless, in contrast to CCK-A receptors, the very-low-anity states represents only 10±15% of the CCK-B receptors (Huang et al., 1994b), in good agreement with the lack of di€erence observed between the maximal number of binding sites for the antagonist [3H]L-365 260 and the natural agonist [125I]CCK8 (Chang et al., 1989). The heterogenity of the CCK-B receptor has been con®rmed in saturation and competition binding studies. Thus, the slope values were in general signi®cantly lower than unity in di€erent tissues (Hunter et al., 1993; Harper et al., 1996; Huang et al., 1994b). The presence of CCK-B receptor heterogeneity has also been proposed from the experiments performed in the presence of Gpp(NH)p or GTPgS.

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The results obtained clearly showed that these nonhydrolyzable GTP analogues, reduced the binding of selective CCK-B ligands. Nevertheless, the anity of these compounds was di€erently a€ected and sometimes almost una€ected (Lallement et al., 1995; Suman-Chauhan et al., 1996; Knapp et al., 1990; Durieux et al., 1992). The fact that the anity of CCK related peptide was almost una€ected could indicate that there is only a partial coupling of CCK-B receptors to G proteins with these peptides (Wennogle et al., 1988). On the other hand, as reported in Section 6 (signal-transduction cascade for CCK-B receptors), it appears that CCK-B receptor may couple to two G proteins: a PTX-insensitive G protein (Gq/11)-PLC pathway and a PTX-sensitive G protein phospholipase pathway. Thus, there is a di€erence in the pool of G proteins coupled to the CCK-B receptor when it is activated by ligands. This could explain the weak modi®cations observed in presence of GTPgS or Gpp(NH)p, as it has been shown that G protein aq/11 subunits are relatively insensitive to GTP analogues (Pang and Sternweiss, 1990). Thus, several authors have described CCK-B agonists, apparently capable of discriminating two (Durieux et al., 1986b; Derrien et al., 1994b; Million et al., 1997) or even three (Huang et al., 1994b) binding states. More recently, similar results have been obtained with antagonists (Harper et al., 1996; Bellier et al., 1997; Hunter et al., 1993). The most interesting point is that several compounds bind to the receptor in a way that is signi®cantly better described by a two-site binding model in CHO cells which have been stably transfected with rat CCK-B receptors (Million et al., 1997; Bellier et al., 1997) (Fig. 4). In contrast to the results obtained with guinea pig and rat brain membranes, and Cos cells, stable transfection of CHO cells ensured that the receptor panel is homogenous, indicating that the two-site binding must result from the coexistence of two anity states of a single receptor, in terms of amino acid sequence.

Several explanations could be proposed. It is possible that the coupling of CCK-B receptors to di€erent G proteins induces a receptor conformation with a di€erent anity for the ligands which recognize two binding sites. Another explanation would be that depending on the molecular interaction of a ligand within its binding sites, preferential or di€erential coupling with a G protein can occur (Spengler et al., 1993). Our understanding of CCK-B receptor heterogeneity could be enhanced by site-directed mutagenesis studies in which a di€erence in important amino acid residues involved in the binding of ligands which recognized two binding sites could be evidenced.

9. PHARMACOLOGY AND THERAPEUTIC INTERESTS OF CCK-B LIGANDS 9.1. Gastric Acid Secretion Gastrin and CCK, which are two related peptides that share homology at their biologically active pentapeptidic C-terminal sequence, have been shown to stimulate gastric acid secretion in vitro. Thus, both peptides bind to receptor sites located on parietal (Magous et al., 1989) and induce an increase in phosphoinositide turnover (Roche and Magous, 1989; Chew and Brown, 1986) and an uptake in [14C]aminopyrine ([14C]AP) (an index of acid secretion in vitro) with the same ecacy and potency (Roche and Magous, 1989). The use of selective antagonists allowed speci®cation of what type of receptor is involved in the regulation of gastric acid secretion. Thus, when comparing the e€ects of L-365 260 and L-364 718, it appeared that, in all cases, the CCK-B antagonist was 70±100 times more potent that the CCK-A antagonist (Roche et al., 1991a). Moreover, the abilities of these antagonists to inhibit gastrin- or CCK8-induced [14C]AP uptake in parietal cells are related to their e€ect on in vivo gastrin-stimulated

Fig. 4. Inhibition of [3H]pCCK8 speci®c binding to the rat CCK-B receptor transfected in CHO cells by the selective CCK-B agonist RB 400.

CCK-B Receptor

acid secretion in various animal models; L-365 260 antagonizes the in vivo response mediated by gastrin with a 400-fold higher potency than L-364 718 (Lotti and Chang, 1989). Gastrin and CCK have numerous e€ects on enterochroman-like (ECL) cells, D cells and parietal cells. Two hypotheses for the action of these peptides have been proposed: 1. they could act directly on the parietal cells to stimulate acid secretion (Soll et al., 1984); 2. they could act by stimulating the release of histamine from ECL cells, which then acts on parietal cells via histamine H2 receptor (Sandvik et al., 1987; Lloyd et al., 1992). Today, evidence regarding the regulation of acid secretion appears to support the theory that histamine release from ECL cells and direct e€ects on parietal cells are both physiologically relevant e€ects of gastrin and CCK (Chuang et al., 1991; Oiry et al., 1995). The therapeutic potential of the high anity CCK-B antagonists has not yet been ®rmly established. In view of the clinical application, the healing of peptic ulcers is greatly assisted by antisecretory drugs such as histamine H2 blockers and proton pump inhibitors. However, long-term treatment with antisecretory drugs leads to hypergastrinemia, which is also found in the Zollinger±Ellison syndrome (Isenberg et al., 1973). Because gastrin is not only a strong stimulant of gastric acid secretion (Sandvik and Waldum, 1991), but also a growth factor of gastric mucosal (Nagata et al., 1996), CCK-B antagonists would be expected to block both the hypersecretion and the gastric mucosal growth that characterizes this syndrome. Moreover, it has been reported that treatment with a proton pump inhibitor induced gastric lesions and hyperplasia of ECL cells (Lamberts et al., 1988; Larsson et al., 1988) and that these e€ects were inhibited by the CCK-B antagonist PD-136 540 in rats (Eissele et al., 1992). Thus, these observations suggest that CCK-B antagonists may prevent the relapse of peptic ulcers during therapy with the above antisecretory drugs (Koizumi et al., 1996) or may be of value in the treatment of refractory peptic ulcer disease (Pendley et al., 1995). 9.2. Interactions Between Endogenous CCK and Opioid Systems A large body of evidence has now been accumulated supporting physiological interaction between CCK8 and enkephalins. The opioid and CCK systems are critically involved, generally in an opposite manner, in various physiological processes, including respiratory functions and cardiovascular tonus [review in Roques et al. (1993); Crawley and Corwin (1994); Olson et al. (1995)]. The overlapping distribution of the neuropeptides enkephalin and CCK and their respective receptors in pain-processing regions of the brain and spinal cord (Gall et al., 1987; Pohl et al., 1990) has focused attention on the role of CCK in nociception. Few studies have been done to investigate the functional relationships between CCK and opioid peptides in other beha-

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vioral responses. It has been suggested that a dysfunctioning of the endogenous opioid system might be involved in the aetiology of depression and addiction. It was therefore of interest to investigate the possible existence of CCK-mediated process which would counteract the euphorogenic e€ects of opioids. These studies could open interesting therapeutical possibilities [review in Roques and Noble (1996)]. 9.2.1. Evidence of Regulatory Mechanisms Between Endogenous CCK and Enkephalin Systems in the Control of Pain It has been suggested that CCK8 has an antiopioid activity. Thus, Faris et al. (1983) found that CCK reduced the antinociceptive e€ects produced by the stress-induced release of endogenous opioids, and did not modify nonopiate responses induced by hind paw foot shock. In addition, numerous studies have shown that peripherally administered CCK antagonists or active immunization against CCK potentiate exogenous opiate-produced antinociception (Faris et al., 1984; Baber et al., 1989). However, few studies have been performed on the possible physiological interactions between endogenous CCK and endogenous opioid systems. It is now well established that endogenous opioid peptides, enkephalins are cleaved into inactive fragments by means of ectopeptidases [review in Roques et al. (1993)]. The development of ecient inhibitors of these metabolizing enzymes allow the extracellular levels of enkephalins to be monitored. The joint use of these inhibitors and CCK antagonists allowed physiological responses of the two neuropeptide systems to be studied. The existence of regulatory mechanisms between CCK and enkephalin systems in the control of pain have been proposed. Thus, activation of CCK-A receptors potentiates the analgesic e€ects induced by the complete inhibitor of enkephalin-degrading enzymes able to cross the blood±brain barrier, RB 101 (FournieÂ-Zaluski et al., 1992; Noble et al., 1992), while activation of CCK-B receptors reduces them (Derrien et al., 1993a; Noble et al., 1993a). Schematically, stimulation of CCK-A receptors could enhance opioid release, and/or directly improve the ecacy of transduction processes occuring at the m-sites, which might be allosterically evoked by CCK-A site occupation (Magnuson et al., 1990). In contrast, CCK-B receptor activation could negatively modulate the opioidergic system. Furthermore, the opioid system appears also able to regulate the release of CCK peptides (Fig. 5). Thus, the stimulation of m opioid receptors has an inhibitory in¯uence on the K+-evoked release of CCKlike material (CCKLM) at spinal and supraspinal levels (Rattray and De Belleroche, 1987; Rodriguez and Sacristan, 1989; Benoliel et al., 1991, 1992). On the other hand, in vitro studies have shown that d opioid agonists enhance the K+-evoked release of CCKLM from slices of rat substantia nigra (Benoliel et al., 1991, 1992). This result has been con®rmed by in vivo binding studies. Thus, the binding of the CCK-B selective agonist [3H]pBC 264 was found to be reduced by administration of the d

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Fig. 5. Hypothetical model of the supraspinal interactions between CCK, via CCK-A, CCK-B receptors and the opioid system via d-opioid and m-opioid receptors. CCK agonists, endogenous or exogenous, stimulate CCK-B and/or CCK-A receptors, which can modulate the opioidergic systems either directly (via binding of opioid agonists or via C-®ber evoked activity) or indirectly (via the release of endogenous enkephalins). In addition, activation of m-opioid receptors, which leads to antinociceptive responses, could negatively modulate the release of endogenous CCK, while d-opioid receptors may enhance it.

selective agonist BUBU, as by RB 101, through activation of d opioid receptors by endogenous enkephalins (Ruiz-Gayo et al., 1992). Consequently, activation of d opioid receptors potentiates the release of CCK8, which could bind to CCK-A and CCK-B receptors. This increase is supported by the blockade of CCK-B binding sites by selective antagonists (blocking the negative feedback control achieved by CCK8 via CCK-B receptor activation) which strongly potentiates (200±800%) the antinociceptive e€ects induced by RB 101 in the rat tail ¯ick test and the mouse hot plate test (Valverde et al., 1994) and prolonged the action of the inhibitor (Valverde et al., 1995) (Fig. 6). CCK may function as a powerful functional opioid antagonist. Thus, it has been suggested that endogenous CCK is released to counteract the opioid e€ect and to serve as one of the mechanisms of opioid tolerance. In good agreement with this hypothesis, Zhou et al. (1992) and Pu et al. (1994) have shown that the development of morphine tolerance in the rat was associated with increased hybridization signals for the CCK mRNA in the brain. Nevertheless, these results are not consistent with another study indicating that morphine addiction was not associated with any change in tissue levels of CCKLM and CCK mRNA. These ®ndings suggest that opioid-CCK interactions may occur only at the synaptic level, by modulation of peptide release, without necessarily implicating variations in peptide levels or synthesis (Pohl et al., 1992). Previous studies reported that CCK-B antagonists could prevent and reverse tolerance to analgesic e€ects of morphine without a€ecting morphineinduced physical dependence [review in Roques and Noble (1996); IdaÈnpaÈaÈn-HeikkilaÈ et al. (1997a)]. Thus, it was also important to investigate the possible development of side-e€ects after chronic treatment with RB 101 and CCK-B antagonists to evaluate the therapeutic interest of this association in the management of pain. The results showed that chronic administration of RB 101 plus PD-134 308 did not induce development of tolerance to antinociception at the peak e€ect time, and a longer duration of analgesia was observed than that reported with RB 101 alone (Valverde et al., 1995). On the

other hand, the endogenous CCK has been proposed to modulate opioid-induced the rewarding properties. Thus, the selective CCK-A antagonist L364 718, prevented the acquisition of a morphine place preference, while the selective CCK-B antagonist L-365 260, appeared to potentiate it (Higgins et al., 1991, 1992). In previous studies, it has been shown that repeated systemic administration of the mixed inhibitor RB 101 failed to establish a conditioned place preference in mice (Noble et al., 1993b) and induce weak rewarding e€ects in rats (Valverde et al., 1996). In rats, the selective CCK-B antagonist PD-134 308 facilitated the conditioned place preference induced by a sube€ective dose of RB 101. Nevertheless, only signi®cant e€ect for the association of the mixed enkephalin-degrading enzyme inhibitor and CCK-B antagonist was observed vs the control group, whereas no e€ects were observed when compared with RB 101 alone (Valverde et al., 1996). This result suggests that the physiological role played by CCK in modulating the rewarding responses induced by the activation of the endogenous opioid system is weaker than the regulatory mechanism between both systems in the control of nociceptive transmission. This hypothesis is in good agreement with a recent study, in which Higgins et al. (1994) have demonstrated that CCKA antagonists, as CCK-B antagonists, were ine€ective in a morphine drug discrimination paradigm and did not alter the pattern of heroin responding in a group of rats trained to self-administer heroin. In conclusion, these results suggest that CCK-B antagonists may not potentiate the subjective e€ects of opioids. This observation has some important clinical implications in the management of pain, taking into account the strong antinociceptive responses induced by RB 101 given alone or in association with CCK-B antagonists [review in Roques and Noble (1995)]. Nevertheless, the facilitatory e€ects of opiate-induced analgesia by CCK-B antagonists seem to be restricted to m-opioid receptor-mediated responses. Thus, in contrast to the results obtained in the tail ¯ick and hot plate tests, in the formalin assay the antinociceptive e€ects of RB 101 (mainly due to d-opioid receptor stimulation) are not potentiated by administration of

CCK-B Receptor

365

Fig. 6. Antinociceptive e€ects of RB 101 (i.v.) in the tail ¯ick test in rats and potentiation by the selective CCK-B antagonist PD-134 308. (A) PD-134 308 was administered at time ÿ20 min (30 min before the tail ¯ick test), and RB 101 at time 0 min (10 min before the tail ¯ick test). (B) PD-134 308 was administered at time ÿ20 min and RB 101 at time 0 min. Tail ¯ick latencies were tested at di€erent times. The analgesic responses were expressed as percentage analgesia using the following equation: % analgesia = (test latency ÿ control latency)/(cut-o€ time ÿ control latency)  100 (cut-o€ time = 15 sec). PP < 0.05 and PPP < 0.01 as compared to control group; pP < 0.05 and ppP < 0.01 as compared to same dose of RB 101 without PD-134 308.

selective CCK-B antagonists (Noble et al., 1995). The results obtained with animal models of in¯ammatory pain are still sparse and sometimes contradictory. Thus, it has been demonstrated that the carrageenin-injected rats, CCK is able to attenuate the antinociceptive e€ect of intrathecal morphine, while the CCK-B antagonist L-365 260, which enhanced the ability of low dose of systemic morphine or RB 101 to reduce c-Fos expression in the spinal cord of carrageenin-injected rats (Chapman et al., 1995; Honore et al., 1997), was ine€ective in enhancing the potency of this opioid (Stanfa and Dickenson, 1993). It has been proposed that morphine-mediated CCK release may be abolished in in-

¯ammatory states. In a recent study, Perrot et al. (1998) have shown a di€erential and limited e€ect of CCK-B antagonist on opioid antinociception in this model depending on the dose of morphine, the phase of in¯ammation and the intensity of hyperalgesia. It has been suggested that CCK may have a physiological role in neuropathic pain, since an increase in primary sensory neurons of endogenous CCK was observed after peripheral axotomy in rat (Verge et al., 1993; Stanfa et al., 1994). It could be suggested that a possible increased release of CCK from terminals of primary a€erents will antagonize the actions of opioid analgesics either released en-

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dogenously or applied exogenously, resulting in the development of neuropathic pain syndrome and the relative ine€ectiveness of opioids (Xu et al., 1993). Thus, it has been demonstrated that combination of opioids and selective CCK-B antagonists enhanced morphine antiallodynic ecacy (Nichols et al., 1995) and suppressed the development of autotomy behavior in a model of neuropathic pain in rat (Xu et al., 1993, 1994a), and e€ectively relieved the allodynialike symptom in spinally injured rats (Xu et al., 1994b). The e€ects of the CCK-B antagonists appeared to be dual: the peak e€ect of morphine was increased, and the duration of the e€ect of the alkaloid was also expanded (IdaÈnpaÈaÈn-HeikkilaÈ et al., 1997b). The systemic administration of drugs represents a common route of administration and delivers drugs to tissues naturally via the circulation. However, the site of drug actions is not precisely determined. Previous studies have provided evidence for a spinal (Stanfa and Dickenson, 1993; Zhou et al., 1993; Wiesenfeld-Hallin et al., 1990; Xu et al., 1997) and supraspinal (Miller and Lupica, 1994; Pu et al., 1994) site for the potentiation between morphine or endogenous enkephalins and CCK-B receptor antagonists. Moreover, in a recent study, SchaÈfer et al. (1998) have shown that CCK into in¯amed paws of the rat attenuated peripheral antinociceptive e€ects induced by m-opioid agonists, suggesting a possible CCK±opioid interaction at the single cell level in sensory nerve terminals of primary e€erent neurons. 9.2.2. Evidence of Antidepressant-Like E€ects Induced by CCK-B Antagonists: Interaction with the Enkephalinergic System One of the physiological actions of the neuropeptide CCK seems to involve modulation of the nigrostriatal and mesolimbic dopaminergic pathways [review in Crawley (1991)]. The evidence for interactions between dopaminergic and CCKergic systems includes a large body of electrophysiological, behavioral and neurochemical data (Ladurelle et al., 1993; Derrien et al., 1993b; Dauge et al., 1990). Moreover, dopamine has been shown to be co-localized with CCK in the posterior part of the nucleus accumbens (HoÈkfelt et al., 1980). Mesolimbic dopaminergic pathways play a crucial role in incentive motivation and rewarding processes which are likely to be altered during depression [review in Willner (1990)], and therefore the possibility that CCK-mediated regulation of these neurons could be altered in this emotional disorders, cannot be excluded. Thus, the selective CCK-B agonists BC 264 and BC 197 accentuated the suppression of motility test in mice, an animal model used to select antidepressant drugs. Moreover, this e€ect was inhibited by L-365 260, demonstrating the selective involvement of the CCK-B receptors (Derrien et al., 1994a). However, the most interesting results were obtained with the CCK-B antagonist, which alone decreased motor inhibition in shocked mice and induced antidepressant-type e€ect in the forced swim test in mice (Fig. 7). This could result from an increase of extracellular dopamine contents since this e€ect was suppressed both by D1 or D2 selective

antagonists. Moreover, it has been shown that the association of ine€ective doses of nomifensine (a blocker of dopamine re-uptake) and L-365 260 leads to a signi®cant decrease in the duration of immobility, suggesting that both drugs could act by a related mechanism (Hernando et al., 1994). Moreover, it has been suggested that the endogenous enkephalins might be involved in the aetiology of depression. Accordingly, the `anxious' behavioral responses triggered by forced swimming, conditioned suppression of motility and learned helplessness were attenuated by treatment with the enkephalin-degrading enzyme inhibitors (Ben Natan et al., 1984; Lecomte et al., 1986; Gibert-Rahola et al., 1990; Tejedor-Real et al., 1993, 1995; Baamonde et al., 1992; Smadja et al., 1995) or with antidepressant drugs, suggesting a potential role of endogenous enkephalins in depressive syndroms. It has been demonstrated in these tests, that the inhibitors modulated the functioning of the mesocorticolimbic and nigrostriatal dopaminergic systems, which are known to be implicated in mood control and shown to be connected with enkephalin pathways [review in Roques et al. (1993)]. In line with this, increasing the level of endogenous enkephalins with peptidase inhibitors induced antidepressant-like e€ects, which were suppressed by both d opioid antagonist, naltrindole and the dopamine D1 antagonist, SCH 23390 (Baamonde et al., 1992) (Fig. 8). It was therefore of interest to investigate the possible modulation of the RB 101 induced behavioral responses by CCK ligands. The results obtained showed that the antidepressant-like e€ects induced by the CCK-B antagonist, L-365 260 was suppressed by the selective antagonist for d opioid receptors (Fig. 7), suggesting the occurrence of physiological adverse interactions between CCK and opioid systems (Derrien et al., 1994a; Hernando et al., 1996), and indicate that CCK-B antagonists could block centrally located CCK-B receptors, thus reinforcing the antidepressant-like e€ects induced by d opioid receptor stimulation. Accordingly, the antidepressant-like e€ect of RB 101 was potentiated by L365 260 and suppressed by BC 264. As expected, the facilitation induced by L-365 260 on RB 101 responses was blocked by naltrindole (Smadja et al., 1995). In a recent study, Smadja et al. (1997) have shown that the endogenous CCK system, through CCK-B receptors, could modulate opioid behavioral responses by a mechanism directly involving two di€erent mesolimbic structures, the anterior nucleus accumbens and the central amygdala. Taken together these data suggest that the clinical use of CCK-B antagonists, administered alone, or in association with classical treatments or inhibitors of enkephalin catabolism, could be extended to the treatment of depressive syndromes. 9.2.3. Interest of Enkephalin-Degrading Enzyme Inhibitors in Combination with CCK-B Antagonists in the Treatment of Opioid Addiction It has been proposed that the craving and self-administration of opioid drugs could be explained either by a pre-existing de®cit in the endogenous opioid system or by a de®cit that could occur after

Fig. 7. E€ects of the CCK-B agonist, BC 264 and the CCK-B antagonist, L-365 260 in shocked and non-shocked mice in the conditioned suppression of motility test. (A) Antidepressant-like e€ects of L-365 260 and reversal by naltrindole, a selective d-opioid receptor antagonist. PP < 0.05 as compared to control group; pP < 0.05 as compared to the same dose of L-365 260 without naltrindole. (B) Depressant-like e€ects of the CCK-B agonist BC 264. PP < 0.05 and PPP < 0.01 as compared to control group.

CCK-B Receptor 367

Fig. 8. Conditioned suppression of motility test in mice. (A) E€ects of naltrindole (d antagonist) and SCH 23390 (D1 antagonist) on the antidepressant-like e€ects induced by i.v. injected RB 101. PPP < 0.01 as compared to control group; ppP < 0.01 as compared to the same dose of RB 101 without antagonist. (B) E€ects of L-365 260 on the antidepressant-like e€ects induced by i.v. injected RB 101. PP < 0.05 and PPP < 0.01 as compared to control group; ppP < 0.01 as compared to the same dose of RB 101 without L-365 260.

368 F. Noble and B. P. Roques

CCK-B Receptor

chronic administration of opiates. Thus, the use of a treatment increasing the level of endogenous opioid peptides could be an interesting new approach in the treatment of drug abuse. Indeed, it has been shown that mixed inhibitors such as RB 101 reduced the severity of the withdrawal syndrome in morphine-dependent rats after administration of naloxone (Maldonado et al., 1995). Moreover, as indicated above, several studies demonstrated that activation of CCK-B receptors could modulate the opioid system negatively, suggesting that selective blockade of these receptors may increase the ability of mixed enkephalin-degrading enzyme inhibitors to reduce the opioid withdrawal syndrome precipitated by naloxone. This recently has been con®rmed using RB 101 in association with the CCK-B antagonist PD-134 308 (Maldonado et al., 1995). Although early abstinence syndrome may be an important clinical problem, in the treatment of addiction, the most dicult aspect is the protracted abstinence syndrome, one of the main factors contributing to relapse. Indeed, the ®rst days after cessation of prolonged drug use leads to acute withdrawal syndrome, which consists of physiological changes (i.e. agitation, hyperalgesia, tachycardia, hypertension, diarrhea and vomiting) and a variety of phenomena (i.e. cardiovascular, visceral, thermoregulatory and subjective changes) or depressive states may persist for months or more after the last dose of opiate. Thus, the main challenge in the management of opioid addiction is to develop a pharmacotherapy to minimize the short-term withdrawal syndrome and protracted opiate abstinence syndrome. The complete inhibitors of enkephalin degrading enzymes could be administered alone or in combination with the selective CCK-B antagonists to increase the endogenous opioid peptide levels, thus reducing the discomfort of the short-term withdrawal syndrome, as previously described (Maldonado et al., 1995). Moreover, the protracted abstinence syndrome also could be ameliorated owing to the antidepressant-like properties of these compounds, and thus, the possibility of relapse, the most important problem in the management of opioid addiction should be minimized [review in Roques and Noble (1995)]. 9.3. CCK-B Receptors and Anxiety Co-localization of CCK and dopamine in the ventral tegmental area and the ascending mesolimbic pathways suggests that CCK could act as a neuromodulator of dopaminergic neurotransmission (HoÈkfelt et al., 1980). These dopaminergic pathways have been closely related to motivational mechanisms and reward (Phillips and Le Piane, 1982; Bozarth, 1986; Cador et al., 1991), and, thus, CCK would have a place in the regulation of motivated behaviors and anxiogenic-like e€ects which are almost certainly mediated by these mechanisms (Derrien et al., 1993b). There is now substantial evidence in the literature suggesting that CCK, could be involved in mechanisms related to anxiety and panic attacks [review in Bourin et al. (1996)]. Clinical studies have shown that the CCK-B receptor agonist CCK4 elicits panic

369

attacks in patients with panic disorders and in healthy volunteers; these e€ects are antagonized by the nonpeptide CCK-B antagonist L-365 260 (Bradwejn et al., 1994). Furthermore, clinical tests performed with the selective CCK-B receptor antagonist PD-134 308 revealed a decrease in sum intensity scores in panic attack frequency (Bradwejn, 1995). These data support the role of CCK-B receptors in the mediation of panicogenic-like action of CCK4. Nevertheless, a placebo-controlled trial of L365 260 did not result in any clinically signi®cant improvement in patients with generalized anxiety disorder (Kramer et al., 1995). It is possible that this lack of e€ect is related to the limited solubility and brain penetrability of these ®rst generation CCK-B antagonists. Although, there is no data in rodents corresponding to panic attacks in humans due to the lack of a speci®c animal model of this disease, several studies indicate that CCK may act as an endogenous anxiogenic peptide. Thus, various CCK-B antagonists were able to produce anxiolytic e€ects in several models of fear and/or anxiety in rodents, such as the black and white box in mice, or the elevated plus maze in mice and rats [review in Dauge and Roques (1995)]. Even the selective CCK-A antagonists lorglumide and L-364 718 show similar properties, but at doses probably active also at CCK-B receptors (Woodru€ and Hughes, 1991; Harro and Vasar, 1991). The involvement of CCK-B receptors in these tests was emphasized by the anxiogenic-like e€ects produced by the selective CCK-B agonist BC 197, which decreases the exploratory behavior of rats in the elevated plus maze test, and their suppression by the CCK-B antagonist PD-134 308. Furthermore, CCK agonists decrease the time spent and locomotor activity in the light compartment of the black and white box, and support acquisition and retention in fear-motivated tests. Pharmacological heterogeneity of CCK-B receptors has been proposed from the results obtained in animal models of anxiety. Recently, the e€ects of selective CCK-B agonists, BC 264 and BC 197 and the nonselective CCK agonist BDNL were investigated in the elevated plus maze in rats. As expected, BDNL and BC 197 induced anxiogenic-like e€ects, while BC 264 was devoid of e€ect (Fig. 9). Furthermore, the behavioral responses induced by BDNL and BC 197 were suppressed by PD-134 308, but not by L-365 260 (Derrien et al., 1994b). In the black and white box test in mice, BC 197 was found to induce anxiogenic-like e€ects at doses as low as 1 mg kgÿ1 i.p., while an approximately 300 times higher dose of BC 264 was required to observe the same responses. As previously mentioned, binding competition experiments performed with BC 197 were signi®cantly better when results were analyzed by a two-site than by a one-site model. Thus, BC 197 could interact with two di€erent states of anity of the CCK-B receptor, whereas BC 264 could have the same anity for the two states. The hypothesis that BC 197 interacts di€erently with two CCK-B receptor subtypes, could explain the bellshaped dose±response curves produced by BC 197 in the elevated plus maze. It is possible that BC 197 at a dose of 0.3 mg kgÿ1 stimulates only one of the

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Fig. 9. E€ects of intraperitoneal injection of BDNL, BC 197 and BC 264 administered 30 min before the experiment in the elevated plus-maze. The behavioral responses of rats were measured in the elevated plus-maze for 5 min and are expressed as the percentage of time spent in open arms. PP < 0.05 and PPP < 0.01 as compared to control group.

CCK-B binding sites, producing a decrease in the percentage of time spent in the open arms, and that the stimulation of the other CCK-B site by BC 197 at a higher dose compensates for the e€ects produced by activation of the ®rst site. In contrast, BC 264 may simultaneously activate both binding sites,

resulting in the lack of behavioral change in this particular test. There is some evidence for the participation of particular brain structures in the behavioral expression of the anxiogenic-like e€ects. The nucleus tractus solitarius (NTS) was postulated to directly or indirectly participate in some components of panic attacks (Branchereau et al., 1992) triggered by stimulation of CCK-B binding sites. Indeed, CCK-B receptor stimulation by BC 264 induces postsynaptic excitation of neurons involved in cardiorespiratory control and in emotional responses through activation of direct or indirect ascending pathways projecting to the nucleus accumbens, amygdala, periaqueductal grey matter and locus coeruleus. Other brain regions linked or not to the NTS seem to be involved in CCK-induced anxiogenic e€ects. Thus, CCK8 has been shown to activate rat hippocampal neurons (Bradwejn and de Montigny, 1984; BoÈhme et al., 1989; Dauge et al., 1990), an e€ect suppressed by activation of the neuronal type of benzodiazepine receptor (Bradwejn and de Montigny, 1984). Moreover, this study was the starting point leading to the hypothesis that alterations of CCK-ergic mechanisms contribute to the pathophysiology of panic disorder. The authors provided evidence that anxiolytic benzodiazepines could antagonize the central action of a neuropeptide and proposed that benzodiazepine-mediated antagonism of CCK-induced excitation might be an important mechanism by which benzodiazepines exert their clinically relevant action. More complex results have been described after local injection of CCK agonists. Thus, data obtained using microdialysis technique in awake rats showed that the stimulation of CCK-B receptors by BC 264, locally perfused in the rostral part of the nucleus accumbens, decreased the extracellular level of dopamine, whereas CCK8 in the posterior part of this structure increased it in agreement with experiments performed on nucleus accumbens slices (Ladurelle et al., 1993; Marshall et al., 1991). These data suggest that the CCK modulation of dopamine involves two di€erent mechanisms in the rostral and the caudal regions of the nucleus accumbens which could be related to the heterogeneity of responses obtained in CCK behavioral studies. Moreover, the results obtained with the selective CCK-B agonists BC 197 and BC 264 support further the existence of distinct CCK-B subsites and suggest that, in the anterior nucleus accumbens, their stimulation mediate opposite e€ects on K+evoked stimulated dopamine release via a presynaptic mechanism (LeÂna et al., 1997), in good agreement with the U-shaped dose±response curve obtained by Marshall et al. (1991) with the octapeptide CCK8 on dopamine release. Moreover, after local injection of CCK8 in the posterior part of nucleus accumbens, hypoexploration and anxiogenic-like responses were obtained only when the rats were stimulated by a novel environment (Dauge et al., 1989; Ladurelle et al., 1995). The transfer of animals from their home cages to a novel environment induced a transient increase in dopamine e‚ux in the posterior nucleus accumbens. Microadministration of CCK8 in these

CCK-B Receptor

conditions immediately enhanced dopamine release and produced a longer e€ect than that observed with rats placed in a familiar environment. These data indicate that the intensity of the CCK8 e€ects on extracellular dopamine levels and on anxiety-like response seems to depend on the activity of these neurons previous to CCK8 treatment (Ladurelle et al., 1995). This observation that particular experimental conditions are requested to reveal CCK8 e€ects has been con®rmed by using African green monkeys and rodents submitted to preinjection stress [review in Harro et al. (1993)] or novel environment (Lavigne et al., 1992).

9.4. CCK-Receptors and Memory On the basis of anatomical data, studies of CCK on memory processes have constituted an important ®eld of investigation, as this neuropeptide is present in regions such as limbic structures and cortical areas, which are implicated in the control of cognitive processes, motivational and emotional behaviors. Present results from rodent experiments indicate that CCK or CCK analogues tend to enhance performance and retention in memory-related tasks [review in Itoh and Lal (1990)]. Thus, Flood and Morley (1989) have demonstrated that exogenous or endogenous CCK released during a meal, improve memory retention in mice trained to avoid footshocks in a T maze. Furthermore, peripheral injection of CCK8 or its analogues, ceruletide, produced memory facilitation in a passive avoidance test and accelerated extinction in rats trained to avoid electric footshocks in the active avoidance paradigm (Itoh et al., 1989). In addition, blockade of CCK-A receptors by intracerebroventricular (i.c.v.) or peripheral injection of selective CCK-A antagonists produced memory de®cits in passive and active avoidance tests, and impaired spatial memory in the Morris maze (Itoh et al., 1992). In contrast to CCK8, central injection of the tetrapeptide CCK4 induced amnesia in rats (Katsuura and Itoh, 1986). Taken together, these results suggest that CCK-A receptor activation could improve learning and memory, whereas CCK-B receptor stimulation could produce memory de®ciency. In support of this hypothesis, Dauge et al. (1992) have shown that the selective CCK-B agonist BC 264 decreases spontaneous alternation in the Y maze following microinjection into the nucleus accumbens, a result interpreted in part, by a memory de®ciency. This result has been con®rmed in the three-panel runway test in rats. Thus, Derrien et al. (1994c) have shown that BC 264, peripherally administered or infused into the nucleus accumbens impaired socially reinforced memory in this test. This e€ect was suppressed by the selective CCK-B antagonist L-365 260, supporting the involvement of CCK-B receptors and the nucleus accumbens in memory processes. Although the CCK-B antagonist has no intrinsic action under these experimental conditions, these molecules could re-established normal learning and memory functions in the situation of increased release of CCK8 and subsequent overstimulation of CCK-B receptors.

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Moreover, CCK-A receptor agonists and CCK-B antagonists both enhance memory in an olfactory recognition test via distinct neuronal pathways. Memory enhancement induced by CCK-A agonists appears to involve ®rst vagal relay from the periphery to the brain. Conversely, memory enhancement induced by CCK-B antagonists is directly central and involves, at least in part, the hippocampal system (Lemaire et al., 1992, 1994). In addition, our recent results indicate that the stimulation of CCK-B receptors by BC 264 peripherally administered at low doses could enhance vigilance and/or attention of rats in the Y maze (Ladurelle et al., 1997). Moreover, an intrinsic e€ect of L-365 260 was observed with perturbation of the spontaneous alternation of rats, suggesting the existence of a tonic release of CCK in certain cerebral structures probably involved in attention or memory. However, as mentioned above, in the same dose of range, L-365 260 was also reported to improve memory in rats using an olfactory recognition test (Lemaire et al., 1992). The apparent discrepancies observed with BC 264 and L-365 260 indicate that CCK-B receptors could have di€erent functions involving probably di€erent neuronal pathways, according to the task carried out by the animal. Heterogeneity of CCK-B receptor has also been suggested from results obtained with BC 264 and BC 197 in a two-trial memory task based on exploration of novelty [review in Dauge and LeÂna (1998)]. This task is based on the innate tendency of animals to explore new environments (Dellu et al., 1992, 1997). It has been assumed that detection of spatial novelty or a change introduced into a familiar environment implies a comparison between the previously stored information of the familiar environment and the currently perceived arrangement of the novel situation.This test o€ers the advantage of avoiding the use of appetitive or aversive reinforcers unlike many memory tasks and therefore minimizes the in¯uence of these factors on memory. This is especially important given the well known e€ects of CCK peptides in satiety and anxiety related behaviors [review in Crawley and Corwin (1994)]. In this test, BC 264 enhanced spatial working memory, supporting the cognitive-enhancing properties of this agonist. A similar result was obtained with a propionyl analogue of BC 264, pBC 264 in young and aged rats (Taghzouti et al., 1998). This CCK-B agonist enhanced consolidation and retrieval processes of young and aged rats and did not a€ect acquisition. Moreover, it has been shown by microdialysis that BC 264, injected i.p. at pharmacologically active doses increased the extracellular levels of dopamine, DOPAC and HVA in the anterior part of the nucleus accumbens (Ladurelle et al., 1997). The involvement of dopamine receptors in memory ®elds has also been con®rmed in other brain regions such as the prefrontal cortex (Williams and Goldman-Rakic, 1995). Thus, it could be hypothesized that activation of dopaminergic transmission in the nucleus accumbens, which has been involved in some components of memory processes (Taghzouti et al., 1985; Ploeger et al., 1994; Floresco et al., 1996), could be the mechanism by which BC 264 produces its e€ect on attention and/

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or memory. In contrast, BC 197 was found to induce an amnesic e€ects, a result in good agreement with the de®cit in memory described with CCK4. However, it has been shown that BC 197 induced anxiogenic-like e€ects (Derrien et al., 1994b), and the response observed after peripheral administration of this CCK-B agonist in the twotrial memory task could be more related to an e€ect on anxiety rather to a true disruption of memory processes [review in Dauge and LeÂna (1998)]. These results provide further evidence for the heterogeneity of CCK-B receptors, and show that the stimulation of CCK-B receptors in rats, depending on the CCK-B agonists used, can mediate distinct behavioral responses. The modulation of memory processes by BC 264 or analogues could o€er new perspective in the treatment of attention/memory disorders with ageing or with neurodegenerative diseases.

10. CONCLUSION The peptide CCK exists in numerous brain and peripheral regions where it serves as a neurotransmitter and neuromodulator or hormone. The development of selective and highly potent agonists and antagonists has constituted a major breakthrough in the ®eld of CCK research. Numerous data support the existence of physiological interactions between endogenous CCK system and other systems, as opioid or dopaminergic systems. The obvious neuroanatomical association between dopamine and CCK continues to stimulate research directed towards the development of new antipsychotic drugs. It is only recently that a possible antipsychotic e€ect of CCK-B antagonists has been associated with a known mechanism of action of conventional neuroleptic drugs, that is, modulation of the activity of dopamine neurons in the midbrain [review in Crawley (1991)]. Nevertheless, in spite of considerable e€ort made in this area, it is rather doubtful that CCK agonists or antagonists can be potent antipsychotic drugs. The substantial changes in the content of CCK and density of CCK receptors occuring in schizophrenia are most likely secondary to the disease-induced changes in the brain [review in Bourin et al. (1996)]. The two peptidergic systems using enkephalins or CCK as endogenous e€ectors seem to be activated in several stressful and con¯icting situations. The phasic release of endogenous CCK8 could lead to various behavioral responses such as nociception and anxiety through interaction with CCK-B receptors, whereas the enkephalinergic system appears to produce opposite e€ects through activation of m and d opioid receptors, respectively. CCK compounds, especially the selective CCK-B antagonists may be interesting drugs in the management of pain. Indeed, even if they do not induce antinociceptive response alone, they are able to strongly potentiate the antinociceptive e€ects of the opioids. The clinical implications of this potentiation are very interesting. Indeed, CCK-B antagonists may be useful in potentiating the antinociceptive properties of exogenous and of en-

dogenous opioids, consequently further reducing the eventual side e€ects, which may occur after chronic treatment. Furthermore, CCK-B antagonists could be co-administered with inhibitors of enkephalin catabolism, to increase the endogenous opioid peptide levels in the treatment of drug abuse, reducing the discomfort of the short-term withdrawal syndrome. The protracted abstinence syndrome also could be ameliorated owing to the antidepressantlike properties of the CCK-B antagonists. Thus, the possibility of relapse, the most important problem in the management of opioid addiction, should be minimized [review in Roques and Noble (1995)]. One of the main characteristics of the e€ects of CCK compounds, are the bell shaped dose±response curves which are observed with the same timecourse in behavioral tests. The mechanisms sustaining these dose±response curves remain to be clari®ed, but similar ®ndings have frequently been observed with peptides, particularly with CCK8 and related compounds in various behavioral and neurophysiological studies. One hypothesis to explain the bell-shaped curves could be a recruitment of diverse neuronal pathways and/or cerebral structures generating various e€ects as a function of the doses. Another hypothesis could also be the interaction with two subsites of CCK-B receptors leading to di€erent and/or opposite responses. This hypothesis seems to be supported by the observed e€ects of CCK-B agonists, Boc-CCK4, BC 264 and BC 197 (LeÂna et al., 1997; Derrien et al., 1994b; Ladurelle et al., 1998). Activation of one of these subsites could o€er new perspective in the treatment of attention/ memory disorders associated with ageing or with neurodegenerative diseases. AcknowledgementsÐThe authors would like to thank C. Dupuis for typing the tables. All members of the laboratory and colleagues whose names appear in the references cited in this review are acknowledged. They thank RhonePoulenc-Rorer for their ®nancial supports.

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