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Vol. 51, Issue 4, 745-781, December 1999
Institut National de la Santé et de la Recherche Médicale U266, Centre National de la Recherche Scientifique UMR 8600, Université René Descartes, Paris, France (B.P.R., F.N.); Royal Ottawa Hospital, University of Ottawa, Ottawa, Ontario, Canada (J.B.); Section on Behavioral Neuropharmacology, Experimental Therapeutics Branch, National Institute of Mental Health, Bethesda, Maryland (J.N.C.); National Institutes of Health, Digestive Diseases Branch, Bethesda, Maryland (S.A.W.); Department of Anatomy and Neurobiology, Chandler Medical Center, University of Kentucky, Lexington, Kentucky (K.B.S.); and Institut National de la Santé et de la Recherche Médicale U288, Faculté de Médecine Pitié-Salpêtrière, Paris, France (M.H.)
I. Introduction
II. Characterization of Cholecystokinin (CCK) Receptors
A. CCK1 (CCK-A) Receptors
1. CCK1 Receptor Clones.
2. Antagonists of CCK1 Receptors.
3. Agonists of CCK1 Receptors.
B. CCK2 (CCK-B) Receptors
1. CCK2 Receptor Clones.
2. Gastrin Receptors Are CCK2 Receptors.
3. Antagonists of CCK2 Receptors.
4. Agonists of CCK2 Receptors.
III. Molecular Biology of CCK Receptors
A. CCK Receptor Gene Structure
B. Chromosomal Localization of CCK Receptor Genes
C. Animal Models without Detectable Levels of CCK Receptors
IV. Receptor Structure/Function Studies
A. Signal Transduction
1. CCK1 Receptors.
2. CCK2 Receptors.
B. Ligand-Receptor Interaction
1. Agonists.
2. Antagonists.
C. Receptor Regulation.
V. Radioligands and Binding Assays: Heterogeneity of CCK1 and CCK2 Receptors
A. Radioligands at CCK1 Receptors
B. Radioligands at CCK2 Receptors
C. Heterogeneity of CCK2 Receptor-Binding Sites
VI. Distribution of CCK Receptors
A. Distribution in Central Nervous System
1. CCK1 Receptors.
2. CCK2 Receptors.
3. Regulation of CCK Receptors.
B. Distribution in Gastrointestinal and Other Systems
VII. Physiological Implications of CCK Receptors
A. Peripheral Functions
B. Central Functions
1. CCK in Panic Attacks and Anxiety.
2. CCK and Schizophrenia.
3. CCK and Depression.
4. CCK and Memory Processes.
5. Interactions between CCK and Enkephalin
Systems.
VIII. Conclusion
Acknowledgments
References
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I. Introduction |
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The peptide cholecystokinin
(CCK)2
was originally discovered in the gastrointestinal
tract (Ivy and Oldberg, 1928
) and has been shown to mediate pancreatic
secretion and contraction of gallbladder. Then, CCK was described in
the mammalian central nervous system (CNS) as a gastrin-like
immunoreactive material (Vanderhaeghen et al., 1975), and it is now
generally believed to be the most widespread and abundant neuropeptide
in the CNS. This peptide, initially characterized as a 33-amino-acid
sequence, is present in a variety of biologically active molecular
forms derived from a 115-amino-acid precursor molecule (prepro-CCK; Deschenes et al., 1984), such as CCK-58, CCK-39, CCK-33, CCK-22, sulfated CCK-8
[Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2]
and CCK-7, unsulfated CCK-8 and CCK-7, CCK-5, and CCK-4
(Trp-Met-Asp-Phe-NH2; Fig.
1; Rehfeld and Nielsen, 1995). The
presence of CCK in both gut and brain raises the intriguing issue of
the evolutionary significance of separate pools of a peptide in two
systems originating from different embryonic zones (i.e., endoderm and
ectoderm, respectively).
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Receptors for CCK have been pharmacologically classified on the basis
of their affinity for the endogenous peptide agonists CCK and gastrin,
which share the same COOH-terminal pentapeptide amide sequence but
differ in sulfation at the sixth (gastrin) or seventh (CCK) tyrosyl
residue. Two types of CCK receptors (type A, "alimentary", and type
B, "brain") have thus been distinguished. The CCK-A receptor was
first characterized using pancreatic acinar cells (Sankaran et al.,
1980), whereas the CCK-B receptor, with a different pharmacological
profile, was discovered in the brain (CCK-B; Innis and Snyder, 1980b).
The gastrin receptor mediating acid secretion in the stomach was
initially thought to constitute a third type of high-affinity receptor
on the basis of its location and small differences in affinity for CCK
and gastrin-like peptides (Song et al., 1993). However, subsequent
cloning of gastrin and CCK-B receptors revealed their molecular
identity (see later). CCK-A and CCK-B receptor types have been shown to
differ by their relative affinity for the natural ligands, their
differential distribution, and their molecular structure. The CCK-A
receptor binds sulfated CCK with a 500- to 1000-fold higher affinity
than sulfated gastrin or nonsulfated CCK (Silvente-Poirot et al.,
1993a). The CCK-B/gastrin receptor binds gastrin and CCK with almost
the same affinity and discriminates poorly between the sulfated and nonsulfated CCK analogs (Saito et al., 1980). The distribution of CCK-A
and CCK-B/gastrin receptors is tissue dependent (see below).
Based on pharmacological and biochemical studies, the existence of
subtypes of CCK-A and CCK-B receptors has been postulated. Nevertheless, only two genes have been cloned. The initial nomenclature of the receptors as CCK-A and CCK-B receptors is generally accepted by
pharmacologists and molecular biologists. Based on the guidelines defined by the International Union of Pharmacology (IUPHAR) Committee on Receptor Nomenclature and Drug Classification, receptors should be
named after their endogenous ligands and identified by a numerical subscript corresponding to the chronological order of the formal demonstration of their existence by cloning and sequencing (Vanhoutte et al., 1996). Because the CCK-A receptor was the first to be cloned,
it should be renamed CCK1, and the CCK-B receptor
should become CCK2. According to these
guidelines, new splice variants, if pharmacologically relevant, should
be indicated by subscript lowercase letters, in parentheses, such as
CCK1(a), CCK1(b),
CCK2(a), and CCK2(b)
receptors. This new nomenclature would allow any newly discovered CCK
receptor to be logically named according to the same informative
guidelines (see Vanhoutte et al., 1996).
This rational nomenclature has been adopted in the present review, which is devoted to the two CCK receptors whose existence has been firmly established through cloning.
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II. Characterization of Cholecystokinin (CCK) Receptors |
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A. CCK1 (CCK-A) Receptors
1. CCK1 Receptor Clones.
The size of the
CCK1 receptor demonstrated by ligand affinity
crosslinking studies varied depending on the ligand, the crosslinking reagent, the species, and the tissue expressing the CCK receptor (Svoboda et al., 1982; Rosenzweig et al., 1983; Miller, 1984; Fourmy et
al., 1987; Pearson and Miller, 1987; Pearson et al., 1987a,b; Shaw et
al., 1987; Schjoldager et al., 1988; Powers et al., 1991). In rat
pancreatic acinar cells, the CCK1 receptor was
found to be an 85- to 95-kDa, N-linked glycoprotein with a 42- to 44-kDa protein core.). Microsequencing of five peptide products derived from either
enzymatic digestion or chemical cleavage of the protein receptor
allowed the design of degenerate oligonucleotide primers for cloning
the cDNA of the CCK1 receptor from a rat
pancreatic cDNA library. The deduced sequence of the rat
CCK1 receptor corresponds to a 429-amino-acid
protein with a calculated molecular mass of 48 kDa. Hydropathy analysis
predicts seven transmembrane-spanning domains (TM) as expected for a
member of the G protein-coupled receptor (GPCR) superfamily (Dohlman et
al., 1991; Fig. 2). The sequence contains
at least three consensus sites for N-linked glycosylation
(Asn-X-Ser/Thr), consistent with the heavy and variable degree of
glycosylation reported using ligand-affinity crosslinking techniques
(de Weerth et al., 1993b). The CCK1 receptor has
three consensus sequence sites for protein kinase C (PKC)
phosphorylation in the third intracellular loop (Graff et al., 1989),
consistent with previous data showing that CCK-8- and
12-O-tetradecanoylphorbol-13-acetate-stimulated phosphorylation of serine and threonine residues involves predominantly the third intracellular loop and to a minor extent the cytoplasmic tail
of the rat pancreatic CCK1 receptor (Kawano et
al., 1992; Ozcelebi and Miller, 1995). In addition, there are conserved
cysteines in the first and second extracellular loops (ECLs) of both
CCK1 and CCK2 receptors
(Figs. 2 and 3), which may form a
disulfide bridge required for stabilization of their tertiary structure (Silvente-Poirot et al., 1998), and another cysteine in the C terminus
may serve as a membrane-anchoring palmitoylation site (O'Dowd et al.,
1988; Ovchinikov et al., 1988).
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), human gallbladder (de Weerth et al., 1993a;
Ulrich et al., 1993), and rabbit gastric (Reuben et al., 1994) cDNA
libraries using either low-stringency hybridization or polymerase chain reaction methods. The CCK1 receptor is highly
conserved among these species with an overall amino acid homology of
80% and a pairwise amino acid sequence identity of 87 to 92% in
humans, guinea pig, rat, and rabbit (Table
1).
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2. Antagonists of CCK1 Receptors. Several structurally different CCK1 receptor antagonists have been synthesized. They belong to various series of chemicals, including dipeptoid, benzodiazepine, pyrazolidinine, and amino acid derivatives, and have both excellent selectivity and high affinity for CCK1 receptors.
The first CCK antagonists were derived from a naturally occurring benzodiazepine, asperlicin (Table 2), which has been isolated from the fungus Aspergillus alliaceus (Chang et al., 1985). The demonstrated high in vitro and
in vivo potency of asperlicin at CCK1 receptors
conferred clear advantages over previously reported CCK antagonists as
a tool for investigation of the physiological and pharmacological
actions of CCK. The first analogs of asperlicin were designed to assess
which structural features of asperlicin could be modified to further
enhance its CCK inhibitory potency without compromising its
CCK1 selectivity. Unfortunately, this approach
failed to overcome the key defects of asperlicin (Bock et al., 1986).
Interestingly, asperlicin contains elements of the 1,4-benzodiazepine
ring system found in antianxiety agents such as diazepam. On the other
hand, several studies support the concept that the natural ligand for
the antianxiety benzodiazepine receptor is a peptide (Guidotti et al.,
1983; Alho et al., 1985), suggesting that the
5-phenyl-1,4-benzodiazepine ring is in fact a chemical structure that
recognizes a peptide receptor. This explains why the
5-phenyl-1,4-benzodiazepine ring was proposed as the basis for the
design of improved CCK receptor antagonists (Evans et al., 1986).
Indeed, the 3-amino-5-phenyl-1,4-benzodiazepin-2-one derivatives,
typified by L-364,718 (MK-329, devazepide; Tables 2 and
3), remained for several years the most
potent CCK antagonists described with a good selectivity for
CCK1 receptors (IC50
CCK2/CCK1 = 3750).
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; Tables 2 and 3) was selected as a
candidate for further evaluation. The results obtained showed that
FK-480 is a highly selective and potent CCK1
receptor antagonist (Akiyama and Otsuki, 1994; Ito et al., 1994a).
Several other potent and selective antagonists of the
CCK1 receptor have been described, including
glutamic acid derivatives such as loxiglumide (CR-1505) or
lorglumide (CR-1409; Makovec et al., 1985; Table 2), and partial
sequences of the C-terminal region of CCK. The dipeptide,
N-tert-butyloxycarbonyl-aspartyl-phenylalaninamide (Boc-Asp-Phe-NH2), representing the
two-amino-acid C-terminal fragment common to both CCK and gastrin, is a
low-affinity partial agonist at CCK2 receptors
but has no activity at CCK1 receptors. This
selectivity is abolished by removal of the C-terminal amide. Replacement of the N-tert-butyloxycarbonyl group
in this dipeptide with an analog, the 2-naphthalene sulfonyl group,
gave 2-naphthalenesulfonyl 1-aspartyl-(2-phenethyl)amide (2-NAP; Tables
2 and 3), which behaves as a competitive antagonist at
CCK1 receptors. Interestingly, this compound has
a 300-fold greater affinity for CCK1 than
CCK2 receptors (Hull et al., 1993).
On the other hand, further development of "dipeptoids", initially
characterized as CCK2 receptor antagonists (see
below), led to a molecule that has a 100-fold selectivity for the
CCK1 receptor, where it acts as a potent
competitive antagonist (PD-140,548; Boden et al., 1993).
Several years ago, synthetic peptides with
CCK1 receptor antagonist properties were
described (Lignon et al., 1987). One of these compounds, designated
JMV-179
[Tyr(SO3H)-Ahx-Gly-D-Trp-Ahx-Asp-phenylethylester], corresponds to the C-terminal heptapeptide of CCK in which the phenylalamide and the L-tryptophan residues were
substituted by a phenylethyl ester and a D-tryptophan,
respectively. In addition, to protect the peptide against oxidation,
the two methionines were replaced by a 6-aminohexanoic acid (Ahx)
residue. The pharmacological results obtained demonstrated that JMV-179
is a full CCK1 receptor antagonist. In contrast,
JMV-180
[Boc-Tyr(SO3H)-Nle-Gly-Trp-Nle-Asp-phenylethylester] appeared to be an agonist of the stimulatory phase of the amylase release by pancreatic acini (low concentration range) and an antagonist of the inhibitory phase (high concentrations; Galas et al., 1988).
A new serine derivative,
(R)-1-[3-(3-carboxypyridine-2-yl)-thio-2-(indol-2-yl)carbonylamino]propionyl-4-diphenylmethylpiperazine (TP-680)] has been recently developed (Akiyama et al., 1996; Tables 2
and 3). This compound showed approximately 2 and 22 times greater
selectivity for CCK1 receptors relative to
CCK2 receptors than L-364,718 and loxiglumide,
respectively. Pharmacological data showed that TP-680 is a selective
and irreversible antagonist of CCK1 receptors
(Akiyama et al., 1996).
Other CCK1 receptor antagonists have been
developed, such as T-0632 (Tables 2 and 3), which is a novel nonpeptide
and water-soluble compound that inhibits the specific binding of
125I-CCK-8 to rat CCK1
receptor in a concentration-dependent and competitive manner. The
Ki value of T-0632 for the
CCK1 receptor, 0.24 nM, is 23,000-fold less than
its Ki value (5,600 nM) for the
CCK2 receptor (Taniguchi et al., 1996).
Interest in nonpeptide CCK receptor-selective ligands has directed
efforts toward the incorporation of conformationally restricted structures as spacers between Trp and Phe residues in the sequence of
the CCK2 receptor endogenous ligand CCK-4
(Trp-Met-Asp-Phe-NH2). Thus, recently, a new
series of CCK-4 restricted analogs with a 3-oxoindolizidine ring were
synthesized. The most remarkable results were obtained with IQM-95,333
(Tables 2 and 3), which displays a CCK1 receptor
affinity (Ki = 0.62 nM) similar to
that of L-364,718, but with a much higher selectivity
(Ki
CCK2/Ki
CCK1 > 8000; Martin-Martinez et al., 1997).
Another CCK1 receptor antagonist, SR-27,897
(Tables 2 and 3), which is chemically unrelated to peptoids,
benzodiazepines, or glutamic acid derivatives, has been developed. This
compound was obtained by optimization of a lead compound discovered
through the random screening of a large chemical library. SR-27,897 is a highly potent (Ki = 0.2 nM) and
selective (CCK2/CCK1
IC50 = 800) antagonist of
CCK1 receptors (Gully et al., 1993).
3. Agonists of CCK1 Receptors. Only a few compounds have been reported to be CCK1-selective agonists; most of them are tetrapeptides, hexapeptides, and benzodiazepine derivatives.
Two series of CCK analogs have been developed. One series, exemplified by A-71378 [des-NH2-Tyr(SO3H)-Nle-Gly-Trp-Nle-(NMe)Asp-Phe-NH2], contains an (NMe)Asp residue that is critical for CCK1 receptor selectivity (Holladay et al., 1992). The other series derived by replacement of the methionine
residue of Boc-CCK-4 (Boc-Trp-Met-Asp-Phe-NH2) with side chain-substituted Lys derivatives:
Boc-Trp-Lys(X)-Asp-(NMe)Phe-NH2, such as A-71623
(X = o-toluylaminocarbonyl [Tac]) and A-70874 (X = p-hydroxycinnamoyl [Hyc]; Lin et al., 1991; Tables
4 and 5).
Exploration of this tetrapeptide series continued through the
examination of the effects of N-methylation at the Asp
residue. The results obtained showed that analogs containing either
(NMe)Asp or (NMe)Asp-(NMe)Phe are highly potent
(IC50 values in the nanomolar range) and
selective CCK1 receptor agonists (Holladay et
al., 1992).
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).
In the hexapeptide series, it has also been reported that replacement
of Asp-Tyr(SO3H) of CCK-8 with
Hpa(SO3H) (Hpa is 4-hydroxyphenylacetyl) and
N-methylation of Phe do not diminish the affinity for
CCK1 or CCK2 receptors
(Pierson et al., 1997). Inversion of the chirality of Asp7 in
conjunction with N-methylation of Phe8 produces a compound [Hpa(SO3H)-Met-Gly-Trp-Met-D-Asp-MePhe-NH2]
that exhibits high affinity and 2100-fold selectivity for
CCK1 receptors. Moreover, moving the
N-methyl group from Phe to Asp decreased the affinity for
CCK2 receptors without affecting that for
CCK1 receptors, giving a compound
Hpa(SO3H)-Nle-Gly-Trp-Nle-MeAsp-Phe-NH2
(ARL-15849; Tables 4 and 5) with a 6600-fold higher selectivity for the latter receptors (Pierson et al., 1997).
Recently, a series of 1,5-benzodiazepines acting as
CCK1 receptor agonists in vitro and in vivo were
discovered. Potency within this series was modulated by substituents on
the N1-anilinoacetamide moiety (Aquino et al., 1996), with
substitution and/or replacement of the C3-position phenylurea moiety
(GW5823, GW7854; Hirst et al., 1996; Willson et al., 1996; Henke et
al., 1997; Tables 4 and 5).
B. CCK2 (CCK-B) Receptors
1. CCK2 Receptor Clones.
Affinity crosslinking
studies of the CCK2 receptor using
125I-[Leu or
NLeu15]-gastrin-2-17,disuccinimidyl suberate and
either a 60 to 70% pure canine gastric parietal cell preparation or a
solubilized porcine gastric mucosal extract identified two
glycoproteins of 78 and 74 kDa, respectively (Svoboda et al., 1982;
Baldwin et al., 1986; Chiba et al., 1988; Baldwin, 1993).). At the same
time, the gastrin receptor cDNA was also cloned from a canine parietal
cell cDNA library using a COS cell plasmid expression approach (Kopin
et al., 1992). The rat and canine CCK2 receptors are 452 and 453 amino acids long, respectively, and share an 84% amino
acid identity. This degree of homology is consistent with interspecies
variations of the same receptor and has been considered as an early
indication that the gastrin receptor is simply the CCK2 expressed in the stomach (see below).
Similar to the CCK1 receptor, hydropathy analysis
predicts seven TM domains as expected of a member of the GPCR
superfamily (Dohlman et al., 1991). The sequence contains at least
three consensus sites for N-linked glycosylation
(Asn-X-Ser/Thr), consistent with the heavy and variable degree of
glycosylation reported using ligand affinity crosslinking techniques
(Baldwin et al., 1986; Chiba et al., 1988; Baldwin, 1993). Similar to
the CCK1 receptor, there are conserved cysteines in the first and second ECLs that may form a disulfide bridge required
for stabilization of the tertiary structure (Silvente-Poirot et al.,
1998), and a cysteine in the C terminus of the receptor may serve as a
membrane-anchoring palmitoylation site (O'Dowd et al., 1988;
Ovchinikov et al., 1988; Fig. 3).
), human brain (Pisegna et al., 1992; Ito et
al., 1993; Lee et al., 1993; Denyer et al., 1994) and stomach (Pisegna et al., 1992), and guinea pig gallbladder and stomach (de Weerth et
al., 1993b). In addition, CCK2 receptor cloning
has been achieved from gastric enterochromaffin and parietal cells and
brain of Mastomys natalensis (Nakata et al., 1992), calf
pancreas (Dufresne et al., 1996), and a rabbit genomic library
(Blandizzi et al., 1994; Table 1). The CCK2
receptor is highly conserved in humans, canine, guinea pig, calf,
rabbit, M. natalensis, and rat, with an overall amino acid
identity of 72% and pairwise amino acid sequence identities of 84 to
93%.
2. Gastrin Receptors Are CCK2 Receptors.
Gastrin
receptors in the stomach and CCK2 receptors in
the brain were historically viewed as distinct types of CCK receptors on the basis of their different relative affinities for CCK and gastrin-like peptides (Menozzi et al., 1989). However, the canine parietal cell gastrin receptor expressed in COS cells exhibits the same
relative affinities for CCK-8 and gastrin as those of native
human and guinea pig CCK2 receptors. The canine
parietal gastrin receptor was also considered to be a distinct receptor because of a reversal in affinity for L-364,718 versus L-365,260 in
comparison with CCK2 receptors in the brain of
other species (Lotti and Chang, 1989). The basis for this reversal has
subsequently been ascribed to a species-specific change of a single
nucleotide resulting in a single amino acid substitution (Leu355 in
canine receptor versus Val319 in the human receptor) in TMVI (Beinborn et al., 1993). Similar to the human, guinea pig, and rat
CCK2 receptors (Pisegna et al., 1992; Wank et
al., 1992b), cloning of the CCK2 receptor from
canine brain (Wank, 1995) resulted in a single cDNA identical to that
for the canine parietal cell gastrin receptor (Kopin et al., 1992).
Clearly, the identification of a single CCK2
receptor-encoding gene through low- and high-stringency hybridization
of cDNA and genomic libraries and Northern and Southern blot analyses
in numerous species indicates that gastrin receptors do correspond to
CCK2 receptors located in the gastrointestinal tract and do not constitute a third type of CCK receptor (Wank, 1995).
3. Antagonists of CCK2 Receptors.
Many attempts
have been made to develop potent and specific nonpeptide antagonists of
CCK2/gastrin receptor. As a result, several new
chemical entities appeared, exhibiting high selectivity for specific
populations of CCK2/gastrin receptors. The
various compounds under development belong to the following main
chemical classes: amino acid, benzodiazepine, dipeptoid,
pyrazolidinone, and ureidoacetamides derivatives (for a review, see
Makovec and D'Amato, 1997).
). A combination of
the key fragments of the Lilly and Merck series led to the development
of novel nonpeptide CCK2 receptor antagonists
with substitution on the quinazolinone and phenyl rings. Binding data
for this class of compounds suggest that the linker between these rings
is a critical determinant for CCK2 receptor-binding affinity. However, these new compounds have a low
selectivity for CCK2 receptor (Padia et al.,
1997). Indeed, the spatial arrangement of the two moieties appears to
be critical for both potency and selectivity. The introduction of
---NH--- as a linker significantly enhanced CCK2
receptor-binding affinity and selectivity, providing compounds with
nanomolar binding affinity and good selectivity
(Ki
CCK1/Ki
CCK2 > 500). Moreover, these compounds are
active when administered per os (Padia et al., 1998).
On the other hand, the moderate affinity of L-364,718 for
CCK2 receptors suggested that the benzodiazepine
nucleus might also hold a key to selective ligands for these receptors.
The first compound of interest developed using this strategy was
L-365,260 (Tables 6 and
7), which revealed to be the first potent
and selective non- peptide CCK2 receptor
antagonist (Bock et al., 1989). One factor that determined CCK receptor
selectivity in this series was the C3-stereochemistry of the
benzodiazepine ring system, with the (3R)-enantiomer
generally providing CCK2 receptor selectivity.
Moreover, recent studies have shown that the C5-phenyl moiety of the
core benzodiazepine structure could be replaced by C5-cycloalkyl
groups, a modification that retained CCK2
receptor affinity and selectivity. In particular, the C5-cyclohexyl
analog displayed subnanomolar affinity for CCK2
receptors (IC50 = 0.28 nM), with improved
selectivity (Ki
CCK1/Ki
CCK2 = 6500) compared with L-365,260 (Chambers et
al., 1993).
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) or a 2-pyridyl group at the
5-position (Semple et al., 1996b) of the parent benzodiazepine
structure provides a significant increase in absorption. Similar
results have been achieved through the incorporation of an amine-based
cationic solubilizing group within the benzodiazepine framework, with a cyclic amine to form an amidino functionality in the 5-position (L-740,093; Showell et al., 1994; Tables 6 and 7). Other attempts to
improve aqueous solubility included the introduction of acidic groups
(L-368,935 and L-369,466; Bock et al., 1994) or lipophilic surrogates
(Chambers et al., 1995) into the 3-position of the aryl urea component
of either the 1,4-benzodiazepin-2-one parent system or closely related
structures (CP-212,454; Lowe et al., 1995; Tables 6 and 7). The
opposite strategy has also been used with the introduction of basic
amino substituents into the same region. YM022 is the optimal structure
of this new series, with subnanomolar affinity for
CCK2 receptors (Nishida et al., 1994). Moreover,
when these modifications are combined within the same molecule, the
resulting improvements in the in vivo effects appear to be essentially
additive, as shown by the compound YF476 (Tables 6 and 7), which has a
good oral bioavailability in dogs (Semple et al., 1997).
Other nonpeptide CCK2 receptor antagonists have
been developed, derived through rational design from the CCK
tetrapeptide (Hughes et al., 1990). This led to tryptophan dipeptoid
derivatives such as PD-134,308 (CI-988; Tables 6 and 7) with nanomolar
affinity for CCK2 receptors (Horwell, 1991;
Horwell et al., 1991). PD-134,308 exhibits a 1600-fold selectivity for
CCK2 over CCK1 receptors. C-terminal modifications of this compound led to molecules with subnanomolar affinity for CCK2 receptors. For
example, further attempts to optimize the substitution on the phenyl
ring led to a compound 19, which has an extraordinarily high affinity
for the CCK2 receptor (IC50 = 0.08 nM) and a high degree of selectivity (Ki
CCK1/Ki
CCK2 = 940; Augelli-Szafran et al., 1996). A
direct comparison of the structure of the dipeptoid derivatives showed that the size of these molecules could be reduced to increase their
lipophilicity. Such compounds have been synthesized, and some of them
have been found to be potent and selective CCK2
receptor antagonists. Moreover, as expected, one of them (RB 211) was
shown to be more efficient in crossing the blood-brain barrier than the
parent compounds (Blommaert et al., 1993) and devoid of the weak
CCK1 receptor agonist properties of dipeptoids
(Höcker 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., 1992b), macrocyclization
(Didier et al., 1992; Bolton et al., 1993), nor rigidification of the amide bond (Fincham et al., 1992a) led to any positive result. Only a
C-terminal cyclization of PD-134,308 derivatives, by means of a
tetrahydronaphtyl group, has been reported to increase the affinity for
CCK2 receptors (Higginbottom et al., 1993). This approach has also been used for compounds such as RB 210 (Tables 6 and
7), in which C-terminal constraints can be easily introduced. Thus, the
-carbon of the phenethyl side chain of RB 210 was linked to the
-carbon bearing the carbonyl function, by means of a methylene bridge. This resulted in the formation of a proline ring (Bellier et
al., 1997). The most potent compounds of this new series had similar
affinities for CCK2 receptors as RB 210. Structure-affinity relationships of this series indicated that
lengthening of the distance between the amide nitrogen atom and the
phenyl ring was of little importance, whereas the position of the
carboxylate group could not be modified. Therefore, the pyrrolidine
ring was replaced by piperidine to slightly modify the possible
orientation of the aromatic moiety toward the carboxylate without
violating any of the requirements previously established in both linear and constrained series for the recognition of
CCK2 receptors. However, the resulting compounds
behave as moderately potent CCK2 receptor
antagonists (Bellier et al., 1998).
As previously mentioned, the clinical development of PD-134,308
(CI-988) was limited due to its poor bioavailability, which was
attributed to poor absorption and efficient hepatic extraction. Scientists at Parke-Davis also envisaged that reducing the molecular weight of the parent compound would lead to better absorption. Thus,
they synthesized a series of analogs in which the key
-methyltryptophan and adamantyloxycarbonyl moieties, required for
receptor binding, were kept intact and the C terminus was extensively
modified. These modifications led to compounds such as CI-1015 (Tables
6 and 7) for which the oral bioavailability in rat was improved nearly
10-fold and the blood-brain barrier permeability was also enhanced
relative to CI-988 (Trivedi et al., 1998).
Two other series have been described, leading to the synthesis of
derivatives that have both excellent selectivity and high affinity for
CCK2 receptors: the ureidoacetamide class of
CCK2 receptor antagonists (RP-73,870; Pendley et
al., 1995) and the pyrazolidinones (LY-288,513; Howbert et al., 1992;
Tables 6 and 7). Development of the latter series has been discontinued
because of adverse effects in preclinical toxicological studies. The
nonpeptide ureidoacetamides are potent and selective ligands with
nanomolar or subnanomolar affinities for CCK2
receptors and a 100- to 1000-fold selectivity for these receptors over
CCK1 receptors. Despite its relatively poor oral
bioavailability, RP-73,870 was as potent as other antiulcer compounds
after oral administration in a duodenal ulceration model (Pendley et
al., 1995).
4. Agonists of CCK2 Receptors.
Different
strategies have been followed to design potent and selective
agonists of CCK2 receptors. One of these was
to protect CCK-8
[Asp-Tyr(SO3H)-Met-Gly-Trp-Met-Asp-Phe-NH2]
from degrading enzymes such as aminopeptidase A (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). The biologically active
Boc[Nle28,31]CCK27-33 (BDNL; Ruiz-Gayo et al.,
1985) was used as the parent compound to design enzyme-resistant
analogs. In this compound, the major sites of cleavage are at the
Trp30/Nle31 and Nle28/Gly29 bonds. BDNL is potentially resistant to
aminopeptidase cleavage due to its tert-butyloxycarbonyl
N-terminal-protecting group (Ruiz-Gayo et al., 1985; Durieux et al.,
1986a). Thus, several enzyme-resistant BDNL analogs containing either a
retro-inversion of the Nle28-Gly amide bond, an (NMe)Nle31
residue, or a combination of these two modifications have been
synthesized (Charpentier et al., 1988a). This led to BC 264 (Tables
8 and 9), a
highly potent CCK2 receptor agonist that exhibits
about the same affinity (Ki = 0.1-0.5
nM) in all species (guinea pig, rat, mouse, monkey, humans) and was at
that time the only systemically active CCK2
receptor agonist (Charpentier et al., 1988a; Durieux et al., 1991). The
peptidase-resistant bioactive analog [3H]pBC264
was also developed (Durieux et al., 1989) by replacing the Boc group
with a tritiated propionyl residue. The radioactivity present in the
mouse brain 15 min after 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, because more than 85% of the radioactivity present in
the brain corresponded to the intact molecule (Ruiz-Gayo et al., 1990).
On the other hand, despite its intrinsic flexibility, CCK-8 was found
through NMR to exist preferentially under a folded form in aqueous
solution (Fournié-Zaluski 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 between - or
-carboxyl group of Glu1 and Lys4 side chains, such as BC 254 and BC 197 (Tables 8 and 9), which were found highly potent and
selective CCK2 receptor agonists (Charpentier et
al., 1988b, 1989). Another nonsulfated CCK-8 analog, [N-methyl-Nle28,31]CCK26-33
(SNF-8702; Tables 8 and 9), has also been described, which has about
4000-fold greater affinity for CCK2 than for
CCK1 receptors (Knapp et al., 1990).
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).
Because nonpeptide ligands have historically offered greater
opportunity for manipulation of both pharmacodynamic (selectivity and
efficacy) and pharmacokinetic (oral bioavailability, duration) parameters, the development of nonpeptidic CCK2
receptor selective agonists endowed with good stability and
bioavailability should provide useful pharmacological tools and
possibly therapeutic agents. To design such derivatives, the C-terminal
tetrapeptide CCK-4 appeared to be a good molecule to start with,
because of its significant CCK2 receptor affinity
and selectivity, although it has been shown to trigger panic attacks in
humans (de Montigny, 1989; Bradwejn et al., 1991b). Several
modifications were made to CCK-4, such as the N-terminal protection of
the tetrapeptide in Boc-CCK4 (Harhammer et al.,
1991) or modifications of the different amino acids such as the
replacement of Met by Nle or (NMe)Nle (Corringer et al., 1993). Recent
NMR and molecular dynamics studies indicated that the
CCK2 receptor-selective CCK-4 analogs adopt an
S-shaped conformation with a relatively well-defined orientation of the
side chains (Goudreau et al., 1994). The same type of folded structures
has been reported for several potent agonists derived from CCK-4 and
containing a
[trans-3-propyl-L-proline] (Nadzan et al., 1991), a diketopiperazine skeleton (Shiosaki et al., 1990), or
a [(alkylthio)proline] residue (Kolodziej et al., 1995). With this
template, other cyclic CCK-4 analogs have been synthesized in which the
Trp-Met dipeptide was changed to 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). Moreover,
the side chain of Nle in the compound Boc-Trp-(NMe)Nle-Asp-Phe-NH2 together with the N
terminus of Trp appeared to be good candidates for another possible
cyclization. Thus, cyclic compounds were designed through molecular
modeling to mimic the proposed biologically active conformation of
these CCK-4 analogs. The goal of this study was to stabilize the
bioactive conformation of CCK2 receptor agonists
to aid in the design of nonpeptide ligands. This led to the development
of macrocyclic constrained CCK-4 analogs that are endowed with agonist
properties and able to cross the blood-brain barrier (Blommaert et al.,
1997).
Selective and peptidase-resistant CCK2 receptor
ligands that derive from
Boc-[Nle31]CCK30-33 through the
incorporation of non-natural hydrophobic amino acids have also been
developed (Weng et al., 1996b). Among these compounds,
Boc-[Phg31,Nal33]CCK30-33
proved to be a full agonist at rat hippocampal
CCK2 receptors. Moreover, it appeared that
modifications of the hydrophobic and steric character of either the C-
or N-terminal amino acid substituents of CCK-4 derivatives could affect
the agonist or antagonist profile of these peptides. This was shown by
the fact that the agonist
Boc-[Phg31,Nal33]CCK30-33
could be chemically converted to an antagonist through the addition of
two alkyl groups on the terminal CONH2 (Weng et al., 1996b).
Very recently, a new series of highly potent and selective
CCK2 receptor agonists were 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 affinity
and to behave as a specific agonist at CCK2
receptors and to adopt the S-shaped preferential conformation. To
determine the essential structural components of specific
CCK2 receptor agonists, a step-by-step
lengthening of the C-terminal tetrapeptide of BC 264 was carried out.
Various diacidic moieties, such as malonate or succinate residues, were
coupled to the N-terminal portion of the tetrapeptide, leading to RB
400 [HOOC-CH2-CO-Trp-(NMe)Nle-Asp-Phe-NH2] and RB 403 (Tables 8 and 9). RB 400 was also derivatized under its
benzylamide and methyl ester forms. Compounds that belong to the RB 400 series possess high affinities for the CCK2
receptor, with a subnanomolar affinity
(Ki = 0.42 nM) being obtained in case
of RB 400 itself (Million et al., 1997).
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III. Molecular Biology of CCK Receptors |
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A. CCK Receptor Gene Structure
The genes encoding the CCK1 receptor (Miller
et al., 1995; Wank, 1995; Inoue et al., 1997) and the
CCK2 receptor (Song et al., 1993) in humans are
organized in a similar manner consisting of five exons and four
introns. The receptor genes have homologous exon/intron splice sites
with exon 1 coding for the extracellular N-terminal sequence, exon 2 coding for the sequence from the beginning of TMI to the first part of
TMIII, exon 3 coding for the sequence from TMIII to the beginning of
TMV, exon 4 coding for the sequence from TMV to the first fourth of the
third intracellular loop, and exon 5 coding for the remainder of the
receptor (Fig. 4). The genes for the rat
(Takata et al., 1995) and mouse (Lacourse et al., 1997)
CCK1 receptors and rabbit (Blandizzi et al.,
1994) and mouse (Nagata et al., 1996) CCK2
receptors are organized similarly to those for humans. This high degree
of conservation of the sequence and organization between
CCK1 and CCK2 receptor
genes and the fact that the brain and pancreas of the bullfrog
Rana catesbeiana and Xenopus laevis express only
one CCK receptor (Vigna et al., 1984, 1986) suggest that the
CCK1 and CCK2 receptor
genes evolved sometime after amphibia from duplication of a common
ancestral gene (as for the gene encoding the receptor ligands, CCK and
gastrin). This concept is further supported by the cloning of a gene
encoding a CCK receptor from a X. laevis brain cDNA library.
This receptor is expressed in brain and stomach but is undetectable in
pancreas. The deduced amino acid sequence from this gene has 55 and
56% amino acid identity with the human CCK1 and
CCK2 receptors, respectively. This receptor
expressed in COS-7 cells has a CCK1 receptor type pharmacological profile (sulfated CCK > gastrin-17 > nonsulfated CCK-8 > CCK-4) like that of the native receptor in
X. laevis brain and pancreas (Vigna et al., 1986; Schmitz et
al., 1996) but with a relatively high affinity for sulfated gastrin, as
expected for a CCK2 receptor. Nevertheless, like
typical CCK1 receptors, the CCK receptor obtained
from the X. laevis brain cDNA library has a higher affinity
for L-364,718 than for L-365,260, and it is not recognized by CAM 1714 or CAM 1028 (Schmitz et al., 1996).
|
Alternative splicing of exon 4 of the human CCK2
receptor gene results in two CCK2 receptor
transcripts that differ by a block of five amino acids within the third
intracellular loop (Song et al., 1993; Fig. 4). The shorter transcript
is largely predominant in stomach, although its relative distribution
in individual cell types has not been examined. To date, the
physiological relevance of the two isoforms of the human
CCK2 receptor is not known. A comparison of the
shorter and longer isoforms revealed no significant differences in
agonist affinity and signal transduction (Ito et al., 1993, 1994b; Wank
et al., 1994b).
Another splice variant of the human CCK2 receptor
transcript, designated 
CCK2 receptor, which
differs at the 5' end from the CCK2 receptor
transcript described earlier, was discovered using a polymerase chain
reaction-based cloning strategy (Miyake, 1995).
CCK2 receptor encodes an N-terminally
truncated receptor that starts with the methionine Met67 in TMI and is
otherwise identical in the remaining sequence. The gene structure is
similar to that previously reported for the human
CCK2 receptor (Song et al., 1993) except that the
first intron was of ~10 kb (compared with 1.177 kb) and contained the
sequence for the alternative first exon that makes up the 5'
untranslated region of CCK2 receptor (Fig. 4).
The first methionine of exon 2, which is common to both CCK2 and CCK2 receptors,
serves as the translational initiation site for the
CCK2 receptor. CCK2
receptor transiently expressed in COS-7 cells has a ~3-fold lower
affinity for CCK-8 and a ~30-fold lower affinity for gastrin compared
with the CCK2 receptor, but its affinity for the
antagonists L-365,260 and L-364,718 is unchanged. Both
CCK2 and CCK2 receptor
transcripts have been detected in brain, stomach, and pancreas through
the use of reverse transcription-polymerase chain reaction (Miyake,
1995). According to the guidelines defined by the IUPHAR committee,
because these splice variants do not appear to be major variants, they
are not indicated by subscript lowercase letters.
On the other hand, Jagerschmidt et al. (1994) isolated several
CCK2 receptor mRNA isoforms from rat brain
tissue, including a truncated mRNA species. Unspliced precursor mRNA
and the mature form were identified in the cerebral cortex,
hypothalamus, and hippocampus in apparently differing proportions
according to the region examined, suggesting that the expression of the
CCK2 receptor could be modulated at a
post-transcriptional level. Thus, although five precursor mRNAs were
found in the cerebral cortex and the hypothalamus, only one fully
processed messenger was detected in the hippocampus. In the case of the
cerebellum, only a completely unspliced mRNA form was found, which is
in agreement with previous studies showing that
CCK2 receptor-binding sites are not expressed in
this structure in the rat (Pélaprat et al., 1987).
B. Chromosomal Localization of CCK Receptor Genes
The human CCK1 receptor gene has been
localized to chromosome 4 using a panel of human/hamster hybrid DNAs
(Huppi et al., 1995). The mouse CCK1 receptor
gene has been mapped to a syntenic region on chromosome 5 using a wild × inbred backcross panel of mice [(BALB/cAN × Mus spretus)
F1 × BALB/cAN] (Huppi et al., 1995). This
region of mouse chromosome 5 is syntenic with human chromosome 4p16.2-p15.1 (Huppi et al., 1995). The human CCK1
receptor was further mapped to 4p15.1-p15.2 using fluorescence in situ
hybridization and physically mapped between the markers AFMb355ya5 and
AFMa283yh5 (Inoue et al., 1997). The rat CCK1
receptor gene has been localized to a syntenic region on chromosome 14 by fluorescence in situ hybridization (Takiguchi et al., 1997).
The human CCK2 receptor has been localized to
chromosome 11 in humans and a syntenic region on chromosome 7 in the
mouse using a panel of human/hamster hybrid DNAs (Huppi et al., 1995).
Fluorescence in situ hybridization of human metaphase chromosomal
spreads has further localized the human CCK2
receptor gene to the distal short arm of chromosome 11 (11p15.4; Song
et al., 1993; Zimonjic et al., 1994). The colocalization of the
CCK1 receptor gene with the dopamine
D5 receptor gene at 4p15.1-p15.3 (Sherrington et al., 1993) and of the CCK2 receptor gene with the
gene encoding the dopamine D4 receptor at
11p15.4-p15.5 (Gelernter et al., 1992; Pisegna et al., 1992) is
especially interesting in view of the coexistence of CCK and dopamine
in midbrain neurons and the regulation of mesolimbic dopaminergic
pathways by both CCK1 and
CCK2 receptors (Crawley and Corwin, 1994).
C. Animal Models without Detectable Levels of CCK Receptors
An inbred strain of Long Evans rats, the Otsuka Long-Evans
Tokushima Fatty rats, that is considered to be a model for late-onset non-insulin-dependent diabetes mellitus, was discovered to have no
detectable levels of CCK1 receptor gene
expression. Subsequent cloning of their CCK1
receptor gene revealed a deletion of 6847 bp encompassing the promoter
region and first and second exons (Takiguchi et al., 1997). Although
these rats are known to have polygenic abnormalities, the presence of
several metabolic and behavioral abnormalities has been attributed to
the loss of CCK1 receptor expression.
Targeted disruption of the CCK2 receptor gene has
been achieved in mice (Nagata et al., 1996). Homozygous mutant mice
were viable and fertile and appeared to be grossly normal into
adulthood (Langhans et al., 1997).
CCK2
/ mutant mice have much
fewer gastric parietal and ECL cells than so wild-type animals, which
is in line with the growth-promoting effects of gastrin at the
CCK2 receptor previously seen in patients with
hypergastrinemia due to the Zollinger-Ellison syndrome. Also, as
expected, these mice were hypochlorhydric and hypergastrinemic (Nagata
et al., 1996). Together, these results demonstrate the importance of
the CCK2 receptor in maintaining the normal
cellular composition and function of the gastric mucosa.
Moreover, the physiological implication of CCK2
receptor can now be further investigated in CCK2
receptor-deficient mice obtained through gene targeting. The first
experiments reported with this interesting model show a critical role
of CCK2 receptors in memory process.
CCK2 receptor-deficient mice have an impairment
of performance in the memory task (Sebret et al., 1999; for more
details, see VIIB4. CCK and Memory Processes).
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IV. Receptor Structure/Function Studies |
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A. Signal Transduction
1. CCK1 Receptors.
The modulation of
CCK1 receptor affinity by guanine nucleotides in
early studies suggested that they belong to the GPCR superfamily. This
has been confirmed through the cloning of CCK1
receptors (Wank et al., 1992a), which revealed their
seven-transmembrane receptor structure.). The insensitivity of CCK1 receptor inositol phosphate
signaling to pertussis toxin suggests that its couples through the
Gq family of G proteins (Pang and Sternweiss,
1990). Recently, a study using both phospholipase C (PLC) and G
protein -subunit-specific antibodies indicated that both
Gq and G11
are present
in pancreas and that the CCK1 receptor couples to
Gq or G11 to activate
PLC-1 in pancreatic cell membranes (Piiper et al., 1997).
; Yoshida et al., 1997). Nevertheless, other studies have
shown that there are at least two pathways responsible for the
increased production of arachidonic acid in response to
CCK1 receptor stimulation. One is the sequential
effects of phospholipase C (PLC) and diglyceride lipase on
phosphatidylinositol, and the other involves the action of the
PLA2 effect on phosphatidylcholine. Both pathways
cause stimulation of amylase release (Pandol et al., 1991). In addition to the activation of the PLC and PLA2
signal-transduction pathways, CCK1 receptor
stimulation can lead to an increase in the adenylyl cyclase
signal-transduction cascade (Marino et al., 1993).
Thus, CCK1 receptor is capable of coupling to
both PLC and adenylyl cyclase at physiological concentrations in native
cells. It is not clear whether this is a result of the independent
coupling of CCK1 receptor to
Gs and Gq or simply the
result of G protein 
-subunit activation of an isotope of adenylyl
cyclase. A study using a chimeric CCK receptor in which the first
intracellular loops between CCK1 and
CCK2 receptors were exchanged showed that Arg68
and Asn69 belonging to the loop of CCK1 receptor
are important for the stimulatory coupling of this receptor with
adenylyl cyclase but are not involved in its coupling with
Gq. These results support the idea that the
CCK1 receptor is directly coupled with both Gs and Gq (Wu et al.,
1997).
Recent studies (for reviews, see Müller and Lohse, 1995; Daaka et
al., 1997) have shown that some GPCRs use the same effectors as those
of the tyrosine kinase receptor pathway [e.g., Shc (adapter protein)/growth factor receptor-bound protein 2/product of son of
sevenless (SOS)], resulting in Ras and mitogen-activated protein kinase (MAPK) activation and leading to expression of transcriptional factors, such as c-myc, c-jun, and
c-fos. It was recently shown that MAPKs and c-Jun
NH2-terminal kinases (JNKs, which phosphorylate serine residues of c-Jun) are rapidly activated by CCK-8 in rat pancreas both in vitro and in vivo (Dabrowski et al., 1996a,b; Tateishi
et al., 1998). These results suggest that CCK might stimulate cell
proliferation via its action at CCK1 receptors.
Moreover, the activation of both MAPKs and JNKs may be of importance in the early pathogenesis of acute pancreatitis (Dabrowski et al., 1996a).
The mechanism by which the Gq protein-coupled CCK
receptor activates Ras is not well understood. Results obtained by
Dabrowski et al. (1996b) suggest that formation of Shc/growth factor
receptor-bound protein 2/SOS complex via a PKC-dependent mechanism may
provide the link between Gq protein-coupled CCK
receptor stimulation and Ras activation.
A case report of a woman with gallstones and obesity was ascribed to
abnormal processing of transcripts from a normal
CCK1 receptor gene that resulted in the
predominance of mRNA with a 262-bp deletion corresponding to the third
exon. Although this mutation could negatively affect expression or
coupling to G proteins, neither in vivo nor in vitro data were obtained
in support of such inferences. Unfortunately, other affected family
members were not examined and expected splicing abnormalities in
transcripts from other genes were not studied, so only an association
could be established between the common phenotype of gallstones and obesity and the putative RNA processing abnormality in the affected patient (Miller et al., 1995).
2. CCK2 Receptors.
Molecular cloning of
CCK2 receptors has shown that this receptor is a
member of the seven-transmembrane domain GPCR superfamily (Wank et al.,
1992b). This confirmed previous results showing that nonhydrolyzable
GTP analogs reduced the binding of selective CCK2
receptor agonists, as expected of the coupling of these receptors with
G proteins (Knapp et al., 1990; Durieux et al., 1992).
) and in the embryonic pituitary cell line (Lo and Hughes,
1988). More recently, Zhang et al. (1992) showed that CCK-8 increased
the turnover of phosphoinositides and IP3
labeling in dissociated neonatal rat brain cells, in which both
CCK1 and CCK2 receptors
were expressed. One study of CCK2 receptors,
using synaptoneurosomes from guinea pig cortex, failed to provide
support to their possible coupling with adenylyl cyclase or PLC,
although Ca2+ release from intracellular stores,
possibly via a G protein-independent mechanism, could be triggered by a
CCK analog (Galas et al., 1992).
Expression of receptor cDNAs in a mammalian expression system allows
for a readily available source of receptor for functional studies. In
transfected cells (Cos, Chinese hamster ovary), it has been shown that
like the CCK1 receptor, the
CCK2 receptor couples to a pertussis
toxin-insensitive G protein (Roche et al., 1990) that is probably
related to the Gq/11 family, thereby causing activation of PLC (Tsunoda et al., 1988a,b, 1989; Delvalle et al.,
1992). The region of the CCK2 receptor
interacting with Gq was determined in
CCK2 receptor with Lys333 Met, Lys334Thr, and Arg335Leu mutations transiently expressed in COS-7 cells and X. laevis oocytes. Indeed, these mutations resulted in the loss of Gq activation without affecting receptor affinity
(Wang, 1997).
Site-directed mutagenic replacement of Asp100 in the rat
CCK2 receptor, a highly conserved residue in TMII
of most GPCRs, results in a 50% reduction in CCK-8-stimulated
phophoinositide turnover with no change in CCK-8 affinity and only a
small (<6-fold) decrease in antagonist affinity (Jagerschmidt et al.,
1995). These data led to the hypothesis that Asp100 points in the
direction of the cluster of basic amino acids (Lys333/Lys334/Arg335),
located in the third intracellular loop of the receptor at the bottom of the TMVI, that plays a critical role in
CCK2 receptor activation of
Gq proteins (Wang, 1997).
Another residue, Phe347, which belongs to the TMVI domain, was
identified as essential for the signal transduction process. Thus, the
exchange of Phe347 for alanine disrupts the
phosphatidylinositol-signaling pathway without affecting the binding of
CCK receptor agonists (Jagerschmidt et al., 1998). This amino acid
could be a residue implicated in transduction processes through its
possible role in agonist-induced changes in receptor conformation and
subsequent triggering of G protein activation. Indeed, the exchange of
Phe347 for Ala could produce a conformational change in the sequence containing the basic triplet, located just beneath TMVI.
On the other hand, by analogy with CCK1
receptors, it has been shown that CCK2 receptors
are coupled to a phospholipase pathway leading to the release of
arachidonic acid via a PTX-sensitive G protein (Pommier et al., 1999)
and to an MAPK pathway (Taniguchi et al., 1994).
B. Ligand-Receptor Interaction
1. Agonists.
The examination of a 42-amino-acid
N-terminal truncation of the human CCK1 receptor
and site-directed mutants in the region near the top of TMI suggested
the interaction of amino acid residues Trp39 and Gln40 with CCK.
Further binding data for the interaction between wild-type and Trp39Phe
and Gln40Asn mutant CCK1 receptors and a series
of N-terminally modified CCK analogs that were applied to a model of
the CCK1 receptor (based on data from
bacteriorhodopsin, rhodopsin, and the -adrenergic receptors)
suggested that the N-terminal moiety of CCK-8 interacts via hydrogen
bonding with Trp39 and Gln40 (Kennedy et al., 1997). However,
photoaffinity labeling with
125I-desaminotyrosyl-Gly-[Nle28,31,pNO2-Phe33]CCK-(26-33)
of rat CCK1 receptors overexpressed in Chinese
hamster ovary cells demonstrated just the opposite result: the
placement of Trp39 proximate to the C-terminal
pNO2-Phe33 residue of the probe (Ji et al.,
1997). The interaction of CCK with the CCK1 receptor was further modeled using separate single amino acid mutations, Lys105Val and Arg337Val, that resulted in a loss in CCK-8-stimulated calcium release. These data suggest that Lys105 and
Arg337 in the CCK1 receptor interact with
Tyr(SO3H) and Asp of CCK-8, respectively (Tsunoda
et al., 1997).). Chimeric and
site-directed mutagenesis studies of the rat CCK2
receptor containing CCK1 receptor segmental
substitutions suggested that a block of five amino acids (residues
204-208, including Cys205, which putatively forms a disulfide bridge
with Cys127 at the top of TMIII) is important for gastrin selectivity (Silvente-Poirot and Wank, 1996) and that His207 is also important for
CCK-8 affinity (Silvente-Poirot et al., 1998). Studies of human
chimeric CCK1/CCK2
receptors made through exon shuffling of the respective receptor genes
also demonstrated the importance of this area near the top of TMIII for
conferring high gastrin affinity (Wu et al., 1997). Chimeric studies
replacing the X. laevis CCK receptor with variable-length
N-terminal segments of the human CCK2 receptor
revealed the need for multiple contact points in the N-terminal
two-thirds (through TMV) of the CCK2 receptor for
conferring gastrin selectivity (Schmitz et al., 1996). Studies of Ala
scanning mutagenesis in the N terminus near the top of TMI and the
first ECL (ECL1) of the rat CCK2 receptor
identified one nonconserved (Arg57Ala) and four conserved amino acids
(Asn115Ala, Leu116Ala, Phe120Ala, and Phe122Ala) that adversely
affected CCK-8 affinity when mutated to Ala. Reciprocal mutations of
these amino acids at equivalent positions in the rat
CCK1 receptor revealed only two mutations,
Leu103Ala and Phe107Ala, that decreased CCK-8 affinity (Silvente-Poirot
et al., 1998). These studies suggest that CCK peptide agonists interact
with multiple amino acids in the extracellular domain of CCK receptors
and that CCK1 and CCK2 receptors have distinct binding sites despite their shared high affinity for CCK-8. With the use of site-directed mutagenesis, the
roles of three aromatic residues located in TMV (Phe227) and TMVI
(Phe347 and Trp351) of the rat CCK2 receptor were
also evaluated in binding experiments. The results demonstrated that
the highly conserved residues in GPCRs, Phe227 and Phe347, do not play
an important role in the recognition of the agonists. In contrast, Trp351 appeared to be in the agonist-binding site of the receptor, where it probably interacts with the C-terminal sequence of CCK-8, as
illustrated by the similar reduction in affinity for both CCK-8 and
CCK-4 (Jagerschmidt et al., 1998).
2. Antagonists.
Data from CCK receptor chimeric and
site-directed mutagenesis studies suggest that the outer third of TMVI
and TMVIII interacts with the benzodiazepine-based antagonists,
L-364,718 and L-365,260. A survey of all TM amino acids of the human
CCK2 receptor in which one to four amino acids
were replaced with the corresponding CCK1 receptor amino acids identified two single-point mutations, Thr111Asn and His376Leu, that cause a 23-fold decrease in L-365,260 affinity and
a 63-fold increase in L-364,718 affinity, respectively (Kopin et al.,
1995). The importance of the TMVII domain for antagonist affinity was
confirmed by a rat CCK2 receptor TMVII chimera
with a 13-fold decrease in L-364,718 affinity (Mantamadiotis and
Baldwin, 1994) that could be explained by the single-point mutation
His381Leu (Jagerschmidt et al., 1996). The reversal of the relative
affinity for L-364,718 and L-365,260 between canine gastrin receptor
and both the rat and human CCK2 receptors noted
earlier has been explained by an interspecies variation of a single
amino acid in TMVI (Leu355 in dog versus the corresponding Val349 in
humans; Marino et al., 1993). The lack of effect of these TMVI and
TMVII mutations on agonist affinity suggests that agonist- and
antagonist-binding sites are, at best, only partially overlapping.
C. Receptor Regulation.
GPCR function is significantly
regulated by the mechanisms that determine receptor trafficking within
the cell. The molecular and cellular mechanisms involved in regulation
of translocation, sequestration, recycling, and degradation of GPCRs
are not well understood, and the available data are largely
controversial. Fusion of the C terminus of GPCR to the N terminus of
the green fluorescent protein is a valuable tool in the study of
receptor localization and trafficking. CCK1-green
fluorescent protein allowed for the direct observation of spontaneous
and ligand-induced internalization of the receptor (Tarasova et al.,
1997).
; Go et al., 1998). In contrast, internalization
of the CCK2 receptor is at least in part
dependent on the phosphorylation of Ser/Thr residues in its C terminus
(Pohl et al., 1997). In the phosphorylation-deficient
CCK1 receptor mutant with PKC consensus site
mutations Ser260Ala and Ser264Ala, desensitization of the CCK-stimulated inositol 1,4,5-triphosphate response is delayed until
the occurrence of receptor internalization (Rao et al., 1997).
Desensitization of CCK2 receptor stably expressed
in Chinese hamster ovary cells does not require the C terminus and is
independent of internalization, unlike the CCK1
receptor (Choi et al., 1998).
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V. Radioligands and Binding Assays: Heterogeneity of CCK1 and CCK2 Receptors |
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Initial studies describing the distribution and the binding
characteristics of CCK1 and
CCK2 receptors have used nonselective CCK
receptor radioligands. Because CCK-8 is the physiological ligand of CCK
receptors, it was first considered to be the most suitable probe for
the characterization of CCK receptors in radioligand-binding studies.
Preparation of stable, high-specific-activity radioiodinated CCK
through conjugation to 125I-Bolton Hunter reagent
(125I-BH) has been described using several CCK
fragments, such as CCK-8 or CCK-33 (Sankaran et al., 1979; Lin and
Miller, 1985). Specific binding sites for CCK have also been
characterized using a 125I-CCK-8 probe made
resistant to degradation through reaction with the iodinated form of
the imidoester, methyl-p-hydroxybenzimidate (Praissman et
al., 1983). Characterization of CCK1 and
CCK2 receptors was performed in the presence of
selective nonradiolabeled ligands to saturate only one of the CCK
receptors (Hill and Woodruff, 1990). Now, selective radioligands are
available for the specific labeling of CCK1 or
CCK2 receptors.
A. Radioligands at CCK1 Receptors
[3H]-(±)-L-364,718 is a potent and
selective CCK1 receptor antagonist that binds saturably and
reversibly to rat pancreatic membranes. The radioligand recognizes a
single class of binding sites with a high affinity
(Kd = 0.23 nM), and the potency of various CCK receptor agonists and antagonists to inhibit its binding correlates with both their ability to inhibit
125I-CCK-8-specific binding and the known
pharmacological properties of these compounds in peripheral tissues
(Chang et al., 1986). Nevertheless, in a more recent study, Talkad et
al. (1994) showed that 125I-CCK-8 binds to two
different states of the CCK1 receptor in rat
pancreatic acini (a high-affinity state and a low-affinity state),
whereas [3H]L-374,718 binds to a low-affinity
state and to a previously unrecognized very low-affinity state. Similar
measurements using transfected COS cells also identified three
different states of the CCK1 receptor, suggesting
that this feature is an intrinsic property of the
CCK1 receptor molecule itself (Huang et al.,
1994)
The peptide antagonist of the CCK1 receptor
JMV-179 was modified at its N terminus through the incorporation of
p-hydroxyphenylpropionate (BH reagent) and was subsequently
radioiodinated (Silvente-Poirot et al., 1993b). The results obtained
with this first antagonist radioligand,
125I-BH-JMV-179, demonstrated that
CCK1 receptors exist under two interconvertible
affinity states regulated by G proteins in rat pancreatic plasma membranes.
B. Radioligands at CCK2 Receptors
Several peptide ligands have been used to characterize
CCK2-binding sites, such as
[3H]pentagastrin,
[3H]gastrin or
125I-gastrin, and
[3H]CCK-4 (Gaudreau et al., 1985; Clark et al.,
1986; Durieux et al., 1988).
The highly potent agonist [3H]pBC264 (Durieux
et al., 1989) has a subnanomolar affinity for
CCK2 receptors
(Kd = 0.15-0.20 nM) in brain
membranes from mouse, cat, rat, guinea pig, and humans (Durieux et al.,
1992). [3H]pBC264 binds to membranes in a
time-dependent, reversible, and saturable manner. Moreover, even in the
rat brain, a tissue with high levels of nonspecific binding and low
density of CCK receptors (Williams et al., 1986), the specific binding
of [3H]pBC264 reached 80% of total binding at
a radioligand concentration close to the
Kd value (Durieux et al., 1992). In
guinea pig and mouse brain, specific [3H]pBC264
binding was almost not affected by NaCl and/or
guanyl-5'-yl-imidodiphosphate. In contrast, in rat brain, the affinity
of [3H]pBC264 was decreased and the maximal
number of binding sites was increased by NaCl and the guanyl
nucleotide, suggesting that a proportion of CCK2
receptors are constitutively coupled to G proteins (Durieux et al.,
1992).
The high selectivity of [3H]SNF8702 also
permits the characterization of CCK2 receptors in
brain tissues without interference from the population of
CCK1 receptors present (Knapp et al., 1990). The
results obtained in guinea pig brain cortex demonstrated that [3H]SNF8702 binds to a larger population of
CCK2 sites than
[3H]pBC264, which is not the case in mouse
brain. These results could reflect the presence of several CCK-binding
states with different sensitivities to ions and nucleotides. Thus, a
part of the receptors labeled by [3H]pBC264 in
guinea pig brain may be insensitive to these reagents, unlike the
additional sites bound by [3H]SNF8702 (Knapp et
al., 1990; Durieux et al., 1992).
Selective nonpeptide antagonist radioligands have been developed.
[3H]L-365,260 binds saturably and reversibly to
brain membranes, and Scatchard analysis indicated a single class of
high-affinity (Kd = 2 nM) binding
sites (Chang et al., 1989). Recently, a new series of nonpeptide
CCK2 receptor antagonists has been described by
Horwell et al. (1991). Some of these compounds have been radioiodinated (125I-PD-142,308; Horwell et al., 1995) or
tritiated ([3H]PD-140,376; Hill et al., 1993).
The latter radioligand has advantages over the alternative radioligand
[3H]L-365,260 because it has a greater
selectivity and affinity for the CCK2 receptors
and yields a higher ratio of specific to nonspecific binding in both
cerebral cortex and gastric mucosa (Hunter et al., 1993).
Interestingly, in addition to the high-affinity population of
CCK2 receptors,
[3H]PD-140,376 labeled a low-affinity state.
C. Heterogeneity of CCK2 Receptor-Binding Sites
Binding studies using linear or cyclic CCK-8 analogs allowed the
discovery of a heterogeneity of CCK2-binding
sites in guinea pig brain (Durieux et al., 1986b; Knapp et al., 1990;
Rodriguez et al., 1990). Thus, CCK2 receptors
have been shown to exist in three different affinity states (Huang et
al., 1994). This heterogeneity has been confirmed in saturation and
competition binding studies. Thus, the Hill coefficient was in general
significantly lower than unity in different tissues (Hunter et al.,
1993; Huang et al., 1994; Harper et al., 1996).
The existence of CCK2 receptor heterogeneity has
also been proposed from experiments performed in the presence of
guanosine-5'-(,-imido)diphosphate or
guanosine-5'-O-(3-thio)triphosphate. The results obtained
clearly showed that these nonhydrolyzable GTP analogs reduced the
binding of selective CCK2 receptor ligands
(Wennogle et al., 1988). However, different sensitivities to guanyl
nucleotides were observed depending on the structures of the ligands
used (Knapp et al., 1990; Durieux et al., 1992; Lallement et al., 1995;
Suman-Chauhan et al., 1996).
Several authors have described CCK2 receptor
agonists apparently capable of discriminating two (Durieux et al.,
1986b; Derrien et al., 1994b; Million et al., 1997) or even three
(Huang et al., 1994) different affinity states. More recently, similar
results have been obtained with antagonists (Hunter et al., 1993;
Harper et al., 1996; Bellier et al., 1997).
Several hypotheses could be proposed to explain this apparent
heterogeneity of CCK2 receptor-binding sites. It
is possible that the coupling of CCK2 receptors
to different G proteins (see IVA2. CCK2
Receptors) induces different receptor conformation with different
affinities for the ligands (for a review, see Kenakin, 1995). Another
explanation would be that depending on the molecular interaction of a
ligand with its binding site, preferential or differential coupling
with a G protein can occur (Spengler et al., 1993).
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VI. Distribution of CCK Receptors |
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A. Distribution in Central Nervous System
Specific CCK-binding sites were demonstrated in membranes from
brain homogenates almost two decades ago (Hays et al., 1980; Innis and
Snyder, 1980a,b; Saito et al., 1980; Praissman et al., 1983). Since
then, numerous studies using autoradiography and, more recently, in
situ hybridization and immunocytochemistry have investigated the
regional distribution and specific cellular localization of CCK
receptors throughout the neuraxis. Early studies used radioligands such
as 125I-CCK-33, 125I-CCK-8,
[3H]pentagastrin,
[3H]CCK-8, [3H]CCK-4 or
[3H]Boc[Nle28,31]CCK27-33
(Gaudreau et al., 1983, 1985; Zarbin et al., 1983; Van Dijk et al.,
1984; Dietl et al., 1987; Pélaprat et al., 1987; Durieux et al.,
1988; Niehoff, 1989) that do not distinguish between the two CCK
receptors. In general, these studies performed in several species
(e.g., rat, guinea pig, monkey, humans) showed high densities of
CCK-binding sites in several areas, including the cerebral cortex,
striatum, olfactory bulb and tubercle, and certain amygdaloid nuclei.
Moderate levels were observed in the hippocampus, claustrum, substantia
nigra, superior colliculus, periaqueductal gray matter, and pontine
nuclei. Low densities were reported in several thalamic and
hypothalamic nuclei and in the spinal cord (Fig.
5).
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Initial evidence for species differences in the distribution of CCK
receptors was also provided by these studies. For example, in the
cerebellum, high densities of CCK-binding sites were present in guinea
pig, whereas only low levels were detected in rat (Zarbin et al., 1983;
Gaudreau et al., 1985; Mantyh and Mantyh, 1985). CCK-binding sites have
now been identified and visualized in the nervous system of numerous
species ranging from goldfish to humans (e.g., Dietl et al., 1987;
Kritzer et al., 1988, 1990; Hyde and Peroutka, 1989; Miceli and
Steiner, 1989; Hill et al., 1990; Ghilardi et al., 1992; Moons et al.,
1992; Schiffmann et al., 1992; Kuehl-Kovarik et al., 1993; Madtes and
King, 1994; Morency et al., 1994; Himick et al., 1996; Mercer et al.,
1996; Oliver and Vigna, 1996). These studies showed both similarities
and sometimes striking differences in the comparative distribution of
CCK receptors from one species to another. More comprehensive analyses
and discussion about CCK receptor distribution differences in several
brain regions among multiple species can be found elsewhere (Gaudreau
et al., 1985; Sekiguchi and Moroji, 1986; Williams et al., 1986; Dietl
and Palacios, 1989).
With the advent of specific radioligands that could differentiate
between the two types of CCK receptors, it has become apparent that
CCK1 and CCK2 receptors
exhibit a sometimes overlapping, yet distinct, distribution throughout
the CNS. The vast majority of CCK receptors in the CNS are of the
CCK2 type, with CCK1
receptors restricted to rather discrete regions. The precise anatomical localization of the two CCK receptor types, as detailed later, serves
to provide morphological substrates for many of the diverse functions
attributed to neural CCK, including involvement in feeding, satiety,
cardiovascular regulation, anxiety, pain, analgesia, memory,
neuroendocrine control, osmotic stress, dopamine-related behaviors, and
neurodegenerative and neuropsychiatric disorders (see Crawley and
Corwin, 1994).
1. CCK1 Receptors.
Radioligand studies,
initially conducted in the rat, showed CCK1
receptors to be mainly located in the interpeduncular nucleus, area
postrema, and medial nucleus tractus solitarius, with additional areas
of binding found in the habenular nuclei, dorsomedial nucleus of the
hypothalamus, and central amygdala (Moran et al., 1986; Hill et al.,
1987, 1988a; Moran and McHugh, 1988; Woodruff et al., 1991; Carlberg et
al., 1992; Zajac et al., 1996; Qian et al., 1997). Studies in
primates have revealed dramatic species differences, demonstrating a
much higher prevalence and broader distribution of
CCK1 receptors in the monkey and humans than that in rodents (Hill et al., 1988b, 1990; Graham et al., 1991). Thus, in
the monkey, CCK1 receptor-binding sites are
located not only in the area postrema, nucleus, tractus solitarius, and
hypothalamic dorsomedial nucleus, but also in the supraoptic nucleus,
paraventricular nucleus, mammillary bodies, supramammillary region,
infundibular region, dorsal motor nucleus of the vagus, and the
neurohypophysis. In addition, the mesostriatal dopaminergic system
exhibits CCK1 receptor binding in both its origin
(substantia nigra pars compacta and adjacent ventral tegmental area)
and forebrain targets (caudate and putamen). CCK1
receptors are also found in the dorsal horn of monkey and human spinal
cord. Peripherally, the nodose ganglion and vagus nerve contain and
transport CCK1 receptors (Corp et al., 1993;
Widdop et al., 1994).
). Moreover, additional areas
containing CCK1 receptor mRNA were revealed. In
the forebrain, moderate to light mRNA expression is localized in the
olfactory bulb, anterior olfactory nuclei, olfactory tubercle, piriform
cortex, neocortex, claustrum, taenia tecta, all principal cell layers
of the hippocampal formation, medial nucleus of the amygdala, and
nucleus of the lateral olfactory tract. Moderate expression is also
present in the lateral septal nucleus, bed nucleus of the stria
terminalis, preoptic nucleus, thalamic reticular nucleus, and several
hypothalamic regions, including the arcuate nucleus and lateral and
posterior hypothalamic areas. Limited labeling for
CCK1 mRNA has been observed in the brainstem,
with expression found only in the dorsal motor nucleus of the vagus
nerve and the interpeduncular, caudal linear raphe, and hypoglossal nuclei.
Finally, it should be noted that a recent report on the
immunohistochemical distribution of the CCK1
receptor in rat CNS, using a newly developed and partially
characterized antiserum, described numerous brain regions displaying
CCK1 receptor-like immunoreactivity (Mercer and
Beart, 1997). In addition to being present within most of the areas
shown above to contain CCK1 receptor-binding sites or mRNA, other regions with either perikaryal or axonal/dendritic immunolabeling included the nucleus accumbens, anteroventral thalamic nucleus, medial mammillary nucleus, superior colliculus, periaqueductal gray matter, nuclei raphe obscurus and dorsalis, and parabrachial, trigeminal, vestibular, and inferior olivary nuclei, as well as layers
2 to 6 of the spinal cord. Further studies are necessary to confirm
these results.
2. CCK2 Receptors.
In the telencephalon,
autoradiographic binding studies (Moran et al., 1986; Pélaprat et
al., 1987; Durieux et al., 1988; Woodruff et al., 1991; Carlberg et
al., 1992; Qian et al., 1997) showed that high densities of
CCK2 receptors are localized in the external
plexiform layer of the main olfactory bulb, middle layers of the
neocortex (with particularly high levels in the retrosplenial and
cingulate cortices), piriform cortex, nucleus accumbens, and
parasubiculum (Table 10). Moderate
levels are found in the olfactory bulb glomerular layer, deep layers of
neocortex, olfactory tubercle, islands of Calleja, fundus striata,
ventral pallidum, caudate-putamen, hippocampus, dentate gyrus,
presubiculum, and some amygdaloid nuclei. Only low densities are
present in other telencephalic areas such as the taenia tecta, septum,
bed nucleus of the stria terminalis, diagonal band of Broca, globus pallidus, superficial layers of neocortex, and most amygdaloid nuclei.
In the diencephalon, moderate levels of CCK2
receptors are distributed within several hypothalamic nuclei, including the suprachiasmatic, supraoptic and ventromedial nuclei, and within the
thalamic reticular nucleus. Low binding densities are found in other
diencephalic regions such as the medial preoptic, arcuate, and
dorsomedial hypothalamic nuclei; paraventricular, mediodorsal and
reuniens thalamic nuclei; and zona incerta and lateral habenular nucleus. In the mesencephalon, moderate densities of
CCK2 receptor binding are localized in the
parabigeminal nucleus, substantia nigra, and superior colliculus, with
low levels present in the inferior colliculus, parabrachial nucleus,
dorsal raphe nucleus, and periaqueductal gray matter. Relatively few
CCK2 receptor-binding sites are found in the
myelencephalon, with low to moderate levels distributed within the
pontine and superior olivary nuclei, and nucleus tractus solitarius. As
noted, CCK2 receptor binding in the cerebellum is
species dependent. Indeed, with autoradiographic studies,
CCK2 receptors have been detected in the guinea
pig, human, and mouse cerebellum, but not in rat cerebellum (Sekiguchi and Moroji, 1986; Williams et al., 1986, Dietl et al., 1987;
Jagerschmidt et al., 1994). Finally, low levels of binding are observed
in the dorsal and ventral horns of the spinal cord. In the periphery, CCK2 receptor-binding sites are located in the
trigeminal and dorsal root ganglia (DRG; Ghilardi et al., 1992) and in
the vagus nerve (Corp et al., 1993).
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) is in good agreement with that of CCK2
receptor-binding sites (see also Shigeyoshi et al., 1994; Hansson et
al., 1998). Although some discrepancies were observed, virtually all of
the nuclei and regions described earlier were shown to exhibit
hybridization for CCK2 receptor mRNA, with
particularly strong signals found in the neocortex, piriform cortex,
anterior olfactory nuclei, and several amygdaloid nuclei. Some areas
with moderate to weak expression included the olfactory bulb and
tubercle, hippocampal formation, claustrum, other amygdaloid nuclei,
septum, nucleus accumbens, caudate-putamen, substantia nigra,
thalamic reticular nucleus, paraventricular, supraoptic and
ventromedial hypothalamic nuclei, interpeduncular nucleus, red nucleus,
vestibular nuclei, dorsal column nuclei, reticular formation, and
lateral cerebellar nucleus. Diffuse labeling was also reported
throughout the spinal cord. In peripheral sensory ganglia,
CCK2 receptor mRNA has been localized to a small
population of DRG neurons (Zhang et al., 1993).
3. Regulation of CCK Receptors.
It has become apparent that
expression of CCK receptor-binding sites and mRNAs in the nervous
system is not static but rather is malleable on different kind of
perturbations. This is particularly evident in the hypothalamus where
the levels of binding sites and/or mRNA for CCK2
and/or CCK1 receptors have been shown to increase
in response to various physiological or pharmacological stimuli such as
osmotic stress, hypophysectomy, food and water deprivation, and chronic
morphine treatment (Day et al., 1989; Meister et al., 1994; Hinks et
al., 1995; O'Shea and Gundlach, 1995; Munro et al., 1998). In primary
sensory neurons, the expression of CCK2 receptor
mRNA is dramatically up-regulated after peripheral axotomy from the
normal low percentage of in situ hybridization-labeled cells to
encompass about two-thirds of all DRG neurons across all size
categories on peripheral axotomy (Zhang et al., 1993). In contrast,
mild cortical infarction results in decreased levels of
CCK2 receptor mRNA and binding sites in the
entire ipsilateral cerebral hemisphere (Van Bree et al., 1995). These
data on CCK receptor alterations are in line with previous
demonstrations of changes in CCK mRNA and peptide levels after certain
perturbations, thereby providing further evidence that neural CCK
ligand-receptor systems are capable of plastic responses to various stimuli.
B. Distribution in Gastrointestinal and Other Systems
In the gastrointestinal tract and other peripheral systems,
CCK1 receptors are present in pancreatic acinar
cells, chief cells and D cells of the gastric mucosa, smooth muscle
cells of the gallbladder, pyloric sphincter, sphincter of Oddi, some
gastrointestinal smooth muscle and enteric neuronal cells, and anterior
pituitary corticotrophs (for reviews, see Jensen et al., 1994; Wank et
al., 1994a; Wank, 1995). CCK1 receptors can also
be expressed in several tumors, including pancreatic adenocarcinomas,
meningiomas, and some neuroblastomas (Reubi et al., 1997a; Weinberg et
al., 1997), as well as in certain pancreatic carcinoma, neuroblastoma,
and lung cancer cell lines (Logsdon, 1986; Klueppelberg et al., 1990; Sethi et al., 1993). Furthermore, CCK1 receptor
mRNA has been found in esophageal, gastric, and colon cancers (Clerc et
al., 1997). On the other hand, peripheral CCK2
receptors are located in smooth muscle cells throughout the
gastrointestinal tract (including the gallbladder), parietal,
enterochromaffin-like, D cells and chief cells of the gastric mucosa,
myenteric plexus neurons, pancreatic acinar cells, monocytes, and T
lymphocytes (Sacerdote et al., 1991; Jensen et al., 1994; Mantyh
et al., 1994; Wank et al., 1994; Wank, 1995; Song et al., 1996;
Tarasova et al., 1996; Helander et al., 1997; Reubi et al., 1997b).
Tumors and tumor cell lines expressing CCK2
receptors include medullary thyroid, gastric, colon, ovarian and small
cell lung carcinomas, astrocytomas, and certain pancreatic and lung
cancer cell lines (Sethi et al., 1993; Wank, 1995; Reubi and Waser,
1996; Clerc et al., 1997; Reubi et al., 1997a).
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VII. Physiological Implications of CCK Receptors |
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A. Peripheral Functions
As described in detail in VIB. Distribution in
Gastrointestinal and Other Systems, CCK1
receptors in the periphery are primarily localized in the pancreas,
gallbladder, pylorus, intestine, and vagus nerve (Sankaran et al.,
1980; Smith et al., 1984; Moran et al., 1987, 1990; Szecowka et al.,
1989; Hill et al., 1990; Wank et al., 1992a). In the pancreas, CCK acts
at CCK1 receptors on acinar cells to stimulate
the secretion of the digestive enzyme pancreatic amylase (Liddle et
al., 1984; Freidinger, 1989; Jensen et al., 1989). In the gallbladder,
CCK acts at CCK1 receptors to stimulate
gallbladder contraction (Chang and Lotti, 1986; Gully et al., 1993).
Commercial preparations of CCK are used clinically to evaluate
gallbladder contraction in human gallbladder disease (Ondetti et al.,
1970).
The role of peripheral CCK1 receptors in the
regulation of feeding behavior is an area of intense investigations.
CCK1 receptors appear to mediate the transmission
of sensory information from the gut to the brain. Peripherally
administered CCK inhibits food consumption, even after fasting, in many
species, including humans (Gibbs et al., 1973; Pi-Sunyer et al., 1982;
Stacher et al., 1982; for reviews, see Smith and Gibbs, 1992; Crawley
and Corwin, 1994). Furthermore, CCK1 receptor
antagonists increase food consumption and postpone satiety in several
species, supporting the idea that endogenous CCK participates in the
physiological regulation of feeding behavior (Dourish et al., 1989;
Wolkowitz et al., 1990; Corwin et al., 1991; Reidelberger et al., 1991;
Moran et al., 1992, 1993; for a review, see Crawley and Corwin, 1994).
The entry of food into the intestine triggers the release of endogenous CCK by the intestinal mucosa, thereby activating
CCK1 receptors in the periphery. In particular,
CCK1 receptors on the vagus nerve (Moran et al.,
1987) appear to be critical for the satiety-inducing action of CCK.
Thus, lesions of the vagus nerve completely block the CCK-induced
satiety syndrome (Crawley et al., 1981; Smith et al., 1981; South and
Ritter, 1988). These findings have led to the hypothesis that CCK
released from the intestine after a meal activates
CCK1 receptors on the vagus nerve to transmit
sensations of fullness to the brain, which subsequently terminates
feeding behaviors and initiates the sequence of behaviors associated
with satiety (Smith and Gibbs, 1992; Fig.
6). CCK1 receptor
agonists have been proposed as anorectics for the treatment of obesity (Simmons et al., 1994; Wettstein et al., 1994). Conversely,
CCK1 receptor antagonists have been proposed for
the treatment of anorexia disorders (Wolkowitz et al., 1990).
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CCK2 receptors in the periphery are primarily
localized in the stomach (Kopin et al., 1992) and on the vagus nerve in
some species (Mercer and Lawrence, 1992). As previously demonstrated, gastrin acts at CCK2 receptors to stimulate
gastric acid secretion (Schubert and Shamburek, 1990). Similarly, CCK
stimulates gastric acid secretion (Sandvik and Waldum, 1991), and this
effect can be blocked by CCK2 receptor
antagonists (Bado et al., 1991; Pendley et al., 1995). To further
explore the peptidergic pharmacology of the pyloric sphincter, it is
desirable to have a preparation that would allow the examination of
contraction independent of basal motor activity and could exclude
contribution from the enteric nervous system. Such a preparation of
isolated antral cells has been obtained through enzymatic
disaggregation of tissue strips from different species, as well as
disaggregated isolated cell preparations from the pyloric sphincter.
Results obtained from these assays show that pyloric smooth muscle
contractions are stimulated by low doses of CCK and that gastric
emptying induced by a lipid-enriched meal is inhibited by
CCK2 receptor antagonists (Debas et al., 1975;
Lopez et al., 1991). The latter compounds have been proposed for the
treatment of gastric ulcers (Pendley et al., 1995).
Another relatively simple functional assay for CCK receptors is the
guinea pig ileum longitudinal muscle myenteric plexus, which contains
both CCK1 and CCK2
receptors. It has been demonstrated that CCK-8 elicits contraction
through both receptors. Moreover, it has been shown that activation of
CCK2 receptor released only acetylcholine,
whereas activation of CCK1 receptor is
responsible for the release of both substance P and acetylcholine (Dal
Forno et al., 1992; Corsi et al., 1994).
B. Central Functions
In line with its wide distribution in brain, CCK is involved in the modulation/control of multiple central functions. In particular, numerous experimental and clinical studies have clearly shown that CCK, through its action at CCK1 and CCK2 receptors, participates in the neurobiology of anxiety, depression, psychosis, cognition, and nociception.
1. CCK in Panic Attacks and Anxiety.
The initial suggestion
that the CCK system might be involved in anxiety came from experiments
of Bradwejn and de Montigny (1984, 1985a,b) that showed that
benzodiazepine receptor agonists could attenuate CCK-induced excitation
of rat hippocampal neurons. Subsequent clinical studies demonstrated
that bolus injections of the CCK2 receptor
agonist CCK-4 or pentagastrin provoke panic attacks in patients with
panic disorders (Bradwejn et al., 1990, 1991b, 1992a,b). The induced
symptoms are comparable to those produced by a standard panic-provoking
agent (35% CO2; Bradwejn and Koszycki, 1991) and
can be attenuated by antipanic pharmacological agents such as
antidepressants (Bradwejn and Koszycki, 1994; Shlik et al., 1997a; van
Megen et al., 1997). CCK-4 also provokes panic attacks in healthy human
subjects (de Montigny, 1989; Bradwejn et al., 1991a; McCann et al.,
1994); however, sensitivity to the peptide is enhanced in panic
disorder patients relative to healthy volunteers (Bradwejn et al.,
1991b; van Megen et al., 1994), suggesting that endogenous CCK system
may be altered in panic disorder and contributes to pathological
anxiety. Recent investigations have revealed that the panicogenic
effects of CCK2 receptor agonists are not limited
to panic disorder, because individuals with social phobia, generalized
anxiety disorder, obsessive compulsive disorder, and premenstrual
dysphoric disorder also exhibit an augmented behavioral response to
these ligands (Le Melledo et al., 1995; De Leeuw et al., 1996; van
Vliet et al., 1997; Brawman-Mintzer et al., 1997; Katzman et al.,
1997). Although these data suggest that CCK sensitivity is not peculiar
to panic disorder, the threshold of vulnerability to
CCK2 receptor agonists appears to be lower in
panic disorder relative to other psychopathologies in which anxiety is
a significant component (Katzman et al., 1997). In parallel, a number
of investigators have reported that CCK peptides (Boc-CCK-4, BC 197)
administered systemically or intracerebrally produce anxiogenic-like
effects in different animal species, including mouse, rat, guinea pig,
cat, and monkey (Blommaert et al., 1993; Harro et al., 1993; for a
review, see Daugé and Roques, 1995). However, the anxiogenic
effects of CCK peptides in animals have not been observed by all
investigators, and the relevant negative findings should not be ignored
(Shlik et al., 1997b). The conflicting data reported in the animal
literature are attributable in part to the failure to address the
various factors that potentially influence susceptibility to the
anxiogenic effects of CCK (Bradwejn and Vasar, 1995). For instance,
rats with low exploratory behavior (i.e., "anxious" rats) have been
reported to exhibit a higher density of CCK receptor-binding sites in
the frontal cortex and hippocampus relative to that in rats with high
exploratory behavior (i.e., "nonanxious" rats; Harro et al., 1990;
Koks et al., 1997). Thus, the effects of CCK compounds could vary
considerably because of existing differences in the distribution and
binding characteristics of CCK receptor types and/or affinity states
among species. Recently, the effects of the selective
CCK2 receptor agonists BC 264 and BC 197 and of
the nonselective CCK receptor agonist BDNL were investigated in rats
subjected to the elevated plus-maze. Surprisingly, BDNL and BC 197 did induce anxiogenic-like effects, but BC 264 was devoid of any
effect (Fig. 7). The behavioral responses
to BDNL and BC 197 could be suppressed by CI-988, as expected from the
involvement of CCK2 receptors (Derrien et al.,
1994b). On the other hand, Palmour et al. (1993) studied the anxiogenic
effects of CCK receptor agonists in a nonhuman primate model. CCK-4
administered i.v. to African green monkeys has strong and dose-related
effects on behaviors thought to reflect anxiety and panic.
Interestingly, BC 264 also produces these behavioral responses, but the
profile of behavior is somewhat different because at low doses,
hypervigilance and stereotypy are prominent.
|
, 1998), increased
hypothalamic-pituitary-adrenal axis activity (de Montigny, 1989;
Abelson et al., 1991; Kellner et al., 1997; Shlik et al., 1997a), and
elevated blood levels of dopamine, epinephrine, norepinephrine, and
neuropeptide Y (Boulenger et al., 1996). The extent to which the
biological alterations due to CCK2 receptor
agonist administration are comparable to those underlying naturally
occurring panic attacks remains to be determined. Functional imaging
studies in healthy volunteers have shown that CCK-4-induced anxiety is
associated with cerebral blood flow activation in the anterior
cingulate gyrus, the claustrum-insular-amygdala region, and the
cerebellar vermis (Benkelfat et al., 1995). Although these studies
indicate that brain mechanisms are activated after CCK-4
administration, they do not elucidate the precise neuronal circuitry
subserving CCK-4-induced panic. It has been proposed that brainstem
nuclei, including nucleus tractus solitarius, medulla, and parabrachial
nucleus, are important sites of action of exogenous CCK-4 (Shlik et
al., 1997b). These structures contribute to the regulation of
respiration and cardiopulmonary function and have close anatomical and
functional links with the locus ceruleus, a brain region involved in
the expression of fear and anxiety. Studies in animals have shown that
CCK interacts with brainstem structures to modulate respiration, heart
rate, and blood pressure (Denavit-Saubié et al., 1985), and it is
likely that the prominent cardiorespiratory symptoms elicited by
exogenous CCK-4 in humans result from direct or indirect stimulation of
CCK receptors in brainstem nuclei. The emotional symptoms evoked by
CCK-4 may rise from an action of this peptide on brainstem structures
and a subsequent activation or inhibition of higher CNS regions
mediated through neuronal projections.
The neurobiological mechanisms by which CCK2
receptor agonists provoke panic and concomitant biological changes have
been the subject of considerable research activity. Animal studies suggest that anxious behavior induced by various CCK fragments is
associated with selective CCK2 receptor
stimulation (Harro et al., 1993). CCK2 receptors
also appear to participate in the expression of anxiety in humans after
systemic administration of CCK-4 and pentagastrin. Thus, acute
treatment with the selective CCK2 receptor
antagonist L-365,260 was reported to block CCK-4-induced panic attacks
in panic disorder patients (Bradwejn et al., 1994) and
pentagastrin-induced panic symptoms in healthy volunteers (Lines et
al., 1995). Although CCK2 receptors appear to be
the key component from which CCK-4 triggers panic symptoms, there is
growing evidence that the peptide produces its effects through interactions with other neurotransmitter systems. Animal studies have
demonstrated that serotonin, norepinephrine, dopamine, opioids, corticotropin-releasing factor, and the benzodiazepine/-aminobutyric acid complex play salient roles in the induction of anxiety with CCK (Crawley, 1995; Zacharko et al., 1995). Similarly, clinical studies
have revealed important interactions between CCK and serotonin (Shlik
et al., 1997a; van Megen et al., 1997), norepinephrine (Le Melledo et
al., 1998), and the benzodiazepine/-aminobutyric acid complex (de
Montigny, 1989) in the induction of panic-like behavioral and
physiological symptoms.
Interestingly, single-strand conformational polymorphism analysis
showed that a significant association exists between panic disorder and
polymorphism of the CCK2 receptor gene (Kennedy
et al., 1999). The CA repeat polymorphism in the upstream promoter region appears to be different in patients versus control subjects, suggesting that CCK2 receptor gene variations may
be a relevant factor in the neurobiology of panic disorder. In
addition, a polymorphism, also revealed by single-strand conformational
polymorphism analysis, has been found in the promoter region of the
gene encoding the CCK precursor (Wang et al., 1998).
Recent attempts to evaluate the therapeutic effects of
CCK2 receptor antagonists in panic disorder have
produced disappointing results (Adams et al., 1995; Kramer et al.,
1995), mainly because the two compounds available for human use,
L-365,260 and CI-988, have unfavorable pharmacokinetic properties.
Fortunately, several pharmaceutical compagnies have developed
CCK2 receptor antagonists with superior
pharmacokinetic profiles. These compounds are currently under
evaluation for their potential interest in the treatment of
anxiety and other psychopathologies.
2. CCK and Schizophrenia.
To date, modifications in
functioning of the dopamine system are generally accepted as a key
component in the hypothetical pathophysiological mechanisms of
schizophrenia. The existence of interactions between dopaminergic and
CCKergic systems has been demonstrated by a large body of
electrophysiological, behavioral, and neurochemical data (for a review,
see Crawley, 1991; Derrien et al., 1993a; Ladurelle et al., 1993).
Moreover, dopamine has been shown to be colocalized with CCK in the
posterior part of the nucleus accumbens (Hökfelt et al., 1980).
This observation can have clinical relevance because the A-10
dopaminergic neurons that project to the nucleus accumbens, much more
than the other dopaminergic systems, are probably concerned by the
pathophysiological mechanisms of schizophrenia (Crawley and Corwin,
1994). Numerous experiments have shown that CCK modulates the release
of dopamine and that dopaminergic agents modulate the release of CCK
(Crawley and Corwin, 1994). The interactions between CCK and dopamine
are complex and often bidirectional, with CCK potentiating or
inhibiting the action of dopamine, depending on the brain region
examined. Thus, local administration of the CCK2
receptor agonists BC 264 or CCK-8 reduced dopamine release in the
nucleus accumbens of microdialysed rats, whereas via the i.p. route,
the former agonist produced a large increase in dopamine release in the
same area (Ladurelle et al., 1993, 1997). One hypothesis to account for the i.p. effects of BC 264 could be that this agonist, acting on the
CCK2 receptors located in the dorsal
subiculum/CA1 of the hippocampus, stimulates the glutamatergic
projections to the anterior nucleus accumbens, resulting in dopamine
release (Sebret et al., 1999).
; Bachus et al., 1997). In
addition, significant reductions in CCK-like immunoreactivity have been
reported in several brain regions of schizophrenic patients (Ferrier et
al., 1983, 1985; Carruthers et al., 1984), especially those with
predominantly negative symptoms. On the other hand, a lower density of
CCK receptor-binding sites has been found in the hippocampus and
frontal cortex of schizophrenic patients compared with controls
(Farmery et al., 1985). However, it should be noted that not all
studies confirmed the decrease in CCK mRNA levels in schizophrenia.
Indeed, in the postmortem study of Schalling et al. (1990),
schizophrenic patients had even higher CCK mRNA levels in the ventral
tegmental area and substantia nigra than control subjects. Such a
finding should suggest that elevated CCK synthesis in regions rich in
dopaminergic neurons may be associated with schizophrenia.
Methodological problems, study groups of patients that were too small,
and patient heterogeneity might have contributed to these inconsistent
results. Nevertheless, on the whole, the available data suggest that
schizophrenia may be associated with reduced CCK activity. This
reduction may be attributed to either a decreased processing of
preproCCK in neurons or a reduction in synaptic levels of CCK due to
activations in catabolic or putative reuptake processes (Migaud et al.,
1995) or some neurodegeneration of CCKergic neurons in schizophrenia.
The inference that schizophrenia may be associated with hypoactive
CCKergic transmission along with reports that CCK analogs have
neuroleptic-like activity in animal paradigms relevant to schizophrenia
spurred a great deal of interest in the potential antipsychotic
activity of CCK peptides. Several open studies reported that
administration of nonselective CCK receptor agonists (CCK-8; CCK-33,
cerulein) improved psychotic symptoms in schizophrenic patients when
added to ongoing neuroleptic treatment (for a review, see Montgomery
and Green, 1988; Payeur et al., 1993). These findings were encouraging
and suggested that CCK receptor agonists in combination with typical
neuroleptics may be useful for the treatment of schizophrenia. However,
subsequent placebo-controlled studies indicated that nonselective CCK
receptor agonists or antagonists are ineffective in the treatment of
schizophrenia (Innis et al., 1986; Whiteford et al., 1992). New
generations of agonists and antagonists acting with selectivity at
CCK1 or CCK2 receptors are
available, and clinical trials with these new compounds, alone or in
combination with dopaminergic agents, are eagerly expected.
3. CCK and Depression.
One of the physiological actions of
the neuropeptide CCK seems to involve modulation of the nigrostriatal
and mesolimbic dopaminergic pathways. Taking into consideration that
the mesolimbic dopaminergic pathways play a crucial role in motivation
and rewarding processes, which are likely to be altered in depression
(for a review, see Willner, 1990), a role of CCK in mood disorders
cannot be excluded.
). These results suggest that CCK2
receptor antagonists have antidepressant-like properties in mice.
The involvement of CCK in behavioral responses associated with
anticipatory stress has already been demonstrated, and the importance
of external stimuli, such as a novel environment, in revealing the
behavioral effects of CCK receptor agonists or antagonists has been
emphasized in several studies (Crawley, 1984; Daugé et al., 1989;
O'Neill et al., 1991; Lavigne et al., 1992). In the conditioned
immobility test, anticipatory stress on the day of the test might
increase the sensitivity of the CCK system, allowing the effects of
CCK2 receptor agonists and antagonists to be
detected. The antidepressant-like effects observed with CCK2 receptor antagonists could result from an
increase in extracellular dopamine, because they were preventable by
both D1 and D2 receptor antagonists in the forced-swim test (Hernando et al., 1994; Fig. 8). Taken together, these data suggest
that depression is associated with a hyperactive
CCK2 receptor system and that
CCK2 receptor antagonists may be useful in the
treatment of depressive syndromes (Daugé and Roques, 1995).
|
;
Geracioti et al., 1993). However, there is some evidence that an
increase in cerebrospinal fluid CCK levels can occur in
particularly severe depression (Löfberg et al., 1998). On the
other hand, postmortem studies have revealed that compared with healthy
controls and patients with schizophrenia, suicide victims have elevated
prepro-CCK mRNA levels and an increased density of CCK-containing
neurons in the dorsolateral prefrontal cortex and a higher density of
CCK receptors in the frontal cortex (Ferrier et al., 1985).
4. CCK and Memory Processes.
There is increasing preclinical
evidence that the CCK system may play a role in memory processes. The
presence of CCK is conspicuous in brain regions suspected to underlie
memory functions, including the hippocampal formation, amygdaloid
nuclei, and cerebral cortex. It has been suggested that
CCK1 and CCK2 receptors
have different roles in learning and memory functions (Harro and
Oreland, 1993). In particular, a balance between
CCK1 receptor-mediated facilitatory effects and
CCK2 receptor-mediated inhibitory effects on
memory retention has been postulated (Lemaire et al., 1992, 1994).
However, there are conflicting reports on the effects of
CCK2 receptor agonists in animal models of
memory. For instance, although some groups have reported that selective
CCK2 receptor agonists (e.g., CCK-4, BC 264)
impair memory (Katsuura and Itoh, 1986; Daugé et al., 1992;
Lemaire et al., 1992; Derrien et al., 1994a), others have found that
these peptides enhance memory (Gerhardt et al., 1994). Treatment with
BC 264 has also been described to elicit prominent hypervigilance in
monkeys and to increase behavioral arousal in rats (Daugé and
Roques, 1995). The latter findings suggest a possible role for
CCK2 receptor in attentional activation that can
facilitate learning.
).
Ceruletide has also been reported to improve cognitive processing in
young, but not in elderly, healthy subjects (Dodt et al., 1996).
Recently, Shlik et al. (1998) found that the continuous administration
of the selective CCK2 receptor agonist, CCK-4, had no effect on psychomotor performance, although it produced impairments in cognitive tests of free recall and recognition. The
results of this study suggest that CCK-4 may exert a negative influence
on memory consolidation and retrieval.
Factors that potentially contribute to discrepant findings
include differences in experimental paradigms, dosage, and mode of drug
administration. Another possible explanation of the discrepant findings
on the role of CCK receptors in memory function might be due to the
heterogeneity of CCK receptors (discussed earlier). In the two-trial
memory task based on exploration of novelty, it has been shown that BC
264 enhanced spatial working memory, supporting the cognitive-enhancing
properties of this agonist, whereas BC 197 was found to induce an
amnesic effect (Fig. 9), a result in good
agreement with the memory deficit obtained with CCK-4 (for a review,
see Daugé and Léna, 1998). Interestingly, similar
observations were made with a propionyl analog of BC 264, pBC 264, in
both young and aged rats (Taghzouti et al., 1999). Thus, the latter
CCK2 receptor agonist enhanced consolidation and
retrieval processes in young and aged rats but did not affect acquisition. Moreover, it has been shown through microdialysis that BC
264, injected i.p. at pharmacologically active doses, increased the
extracellular levels of dopamine and its metabolites (dihydroxyphenyl
acetic acid and homovanillic acid) in the anterior part of the nucleus
accumbens (Ladurelle et al., 1997). 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 effect on attention and/or
memory. On the other hand, the effects due to BC 197 might be
nonspecific. Indeed, BC 197 can exert anxiogenic-like effects (Derrien
et al., 1994b), and the response observed after peripheral
administration of this CCK2 receptor agonist in
the two-trial memory task could reflect more such effects than a true
disruption of memory processes (review in Daugé and Léna,
1998).
|
5. Interactions between CCK and Enkephalin
Systems.
a. In the Control of Pain.
Anatomical studies have shown that
the distribution of CCK-8 and CCK receptors parallels that of
endogenous opioids and opioid receptors in the pain-processing regions
in both the brain and the spinal cord (Gall et al., 1987; Pohl et al.,
1990). This overlapping distribution triggered numerous investigations
on the role of CCK in nociception. Thus, several groups described a
naloxone-reversible antinociceptive effect of CCK-8 or its analogs in
relevant antinociceptive tests, such as the hot-plate, writhing, and
tail-flick tests (for a review, see Baber et al., 1989). However, it
has also been reported that CCK-8 has antiopioid properties. Thus,
Faris et al. (1983) found that CCK reduced the antinociceptive effects
produced by the release of endogenous opioids but did not modify
opioid-independent analgesia induced by hind paw foot shock. In
addition, numerous studies have shown that peripherally administered
CCK receptor antagonists potentiate opioid antinociceptive responses,
confirming the existence of a functional antagonism by endogenous CCK
and opioid systems (for a review, see Roques and Noble, 1996). It has
been hypothesized that CCK down-regulates opioid effects through activation of CCK2 receptors. This hypothesis is
supported by the data obtained with selective
CCK2 receptor antagonists. Indeed, these ligands
strongly potentiate (+200-800%) the antinociceptive effects of
endogenous enkephalins in rodents treated with RB 101, a mixed
inhibitor of enkephalin-metabolizing enzymes (Fournié-Zaluski et
al., 1992; Valverde et al., 1994). Interestingly, the combination of
opioids with selective CCK2 receptor antagonists
enhanced the antiallodynic effects of morphine, suppressed the
development of autotomy behavior in a model of neuropathic pain in rat,
and efficiently relieved the allodynia-like symptoms in spinally
injured rats (review in Roques and Noble, 1996).
; Fig. 10). Schematically, the
potentiation of the effects of exogenous or endogenous opioids by BDNL,
a nonselective CCK1/CCK2
receptor agonist (Ruiz-Gayo et al., 1985), could be related to an
increase in the release of enkephalins due to
CCK1 receptor activation (like that occurring by
combined treatment with CCK-8 and a cocktail of peptidase inhibitors,
Hendrie et al., 1989) and/or a direct improvement in the efficacy of
transduction processes of the OP3 (µ) opioid
receptors, which might be allosterically evoked by
CCK1 receptor occupation (Magnuson et al., 1990).
On the other hand, CCK2 receptor activation could
in turn negatively modulate the opioidergic system; this explains why
the selective CCK2 receptor agonist BC 264 produced a decrease in the lick latency in the hot-plate test in mice
(Derrien et al., 1993b). If stimulation of CCK receptors is capable of
modulating the opioid system, this sytem can in turn regulate the
release of CCK peptides. Thus, the stimulation of
OP3 (µ) opioid receptors has an inhibitory influence on the K+-evoked release of CCK-like
material 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
OP1 (
) opioid receptor agonists enhance the
K+-evoked release of CCK-like material from
slices of rat substantia nigra and spinal cord (Benoliel et al., 1991,
1992). Also, the in vivo binding of the CCK2
receptor selective agonist [3H]pBC 264 in the
mouse brain was found to be reduced by the administration of RB 101, a
mixed inhibitor of enkephalin-degrading peptidases or BUBU
[Tyr-D-Ser(O-tert-butyl)-Gly-Phe-Leu-Thr(O-tert-butyl)], an OP1 () receptor-selective agonist,
supporting the idea that endogenous enkephalins increase the
extracellular levels of CCK (competing with
[3H]pBC 264 at CCK2
receptors) through the activation of OP1 () opioid receptors (Ruiz-Gayo et al., 1992).
|
;
for a review, see Roques and Noble, 1996). A dysfunction in the balance
between the two peptidergic systems involved in reward in the case of
opioids and in attention and anxiety in the case of CCK could
participate in the neurobiological mechanisms underlying vulnerability
in drug addiction. Furthermore, it has been suggested that endogenous opioid peptides, especially enkephalins, might be involved in the cause
of depression (for a review, see Roques et al., 1993) and that
CCK-mediated processes might possibly counteract the antidepressant-like effects of opioids. In line with these hypotheses, increasing the levels of endogenous enkephalins by RB 101 was shown to
induce antidepressant-like effects in relevant paradigms, such as the
forced swimming, conditioned suppression of motility, and learned
helplessness tests (for a review, see Roques and Noble, 1996). In all
these models of depression, rodents treated with RB 101 react to an
adverse situation in the same way as after the administration of
"classic" antidepressants, such as imipramine, desipramine, and amitriptyline.
Behavioral studies showed that blockade of
CCK1 and CCK2 receptors
produces opposite effects on the opioid-induced reduction of
conditioned suppression motility due to endogenous enkephalins protected from peptidase inactivation by RB 101. Thus, the
antidepressant-like effects of RB 101 were suppresed by the
CCK1 receptor antagonist L-364,718 and enhanced
by the CCK2 receptor antagonist L-365,260 (Smadja
et al., 1995; Fig. 11). Given the
reliable and strong facilitatory effects of CCK2
receptor antagonists on the behavioral responses to RB 101, it was of
interest to investigate the regions involved in the endogenous
interactions between CCK and opioid systems. Because the mesolimbic
system is known to be widely involved in the control of motivational
and affective responses, two mesolimbic structures were studied: the
anterior nucleus accumbens and the central amygdala. Moreover, the
nucleus accumbens has been implicated in the interaction between CCK
and opioid systems in the control of other pharmacological responses
(Kiraly and Van Ree, 1987; Mueller and Whiteside, 1990). The results
obtained showed that the antidepressant-like effects of RB 101 were
potentiated by microinjection of the CCK2
receptor antagonist PD-134,308 in the anterior nucleus accumbens and
the central amygdala, but not in the caudate nucleus, suggesting that
the mesolimbic system plays an important role in the interaction
between CCK and opioid systems in the control of these behavioral
responses (Smadja et al., 1997).
|
). The recent demonstration that activation of
CCK2 receptors could negatively modulate the opioid system (see earlier) suggests that in contrast, the selective blockade of these receptors should increase the ability of mixed inhibitors to decrease the withdrawal signs. Indeed, this has recently
been confirmed using RB 101 in association with the
CCK2 receptor antagonist PD-134,308 (Maldonado et
al., 1995). Moreover, the protracted abstinence syndrome could be
improved due the antidepressant-like properties of mixed inhibitors
administered alone or in combination with the selective
CCK2 receptor antagonists. Thus, the possibility of relapse, the most important problem in the management of opioid addiction, should be minimized.
Interestingly, all of these behavioral studies showed that
CCK2 receptor antagonists do not apparently
potentiate the subjective effects of opioids (for a review, see Roques
and Noble, 1996). This finding should have important clinical
implications in the management of pain, taking into account the strong
antinociceptive responses to opioids in association with the
CCK2 receptor antagonists.
| |
VIII. Conclusion |
|---|
|
TopPreviousNextReferences
|
|---|
Since the original characterization of CCK by Ivy and Oldberg in
1928, followed by the isolation and sequencing of this hormone (Jorpes
and Mutt, 1966), and its detection in the CNS (Vanderhaeghen et al.,
1975), considerable advances have been made in the knowledge of the
roles of this neuropeptide. The actions of CCK and related peptides
have been extended to include endocrine secretion; motility and growth
in the gastrointestinal system; and regulation of satiety, anxiety,
pain, and dopamine-mediated behavior in the central and peripheral
nervous systems. These actions are mediated by at least two distinct
receptors, which have been pharmacologically characterized. The
existence of these CCK receptors (CCK1 and
CCK2) has subsequently been confirmed by their
molecular cloning. Nevertheless, the large variety of functions
mediated by CCK receptors, as well as pharmacological studies, suggests
that some heterogeneity exists in CCK1 and
CCK2 receptors. However, such a heterogeneity has
not been confirmed in molecular biology studies, which have so far
identified only two members of the CCK receptor family. The
physiological and pathophysiological implications of these receptors
can now be further investigated in CCK2
receptor-deficient mice obtained through gene targeting (Nagata et al.,
1996) and in Otsuka Long-Evans Tokushima Fatty rats, which have no
functional CCK1 receptors (Kobayashi et al.,
1996). Several potential clinical applications concern the treatment of
brain disorders and/or pain with CCK2 receptor
agonists or antagonists and of diseases involving food consumption with
CCK1 receptor ligands.
| |
Acknowledgments |
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TopPreviousNextReferences
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We 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 gratefully acknowledged.
| |
Footnotes |
|---|
1 Address for correspondence: Prof. Bernard P. Roques, INSERM U266, Centre National de la Recherche Scientifique UMR 8600, Université René Descartes, 4, Avenue de l'Observatoire, 75270 Paris Cedex 06, France. E-mail: roques{at}pharmacie.univ-paris5.fr
| |
Abbreviations |
|---|
CCK, cholecystokinin; IUPHAR, International Union of Pharmacology; CNS, central nervous system; PKC, protein kinase C; Hpa, 4-hydroxyphenylacetyl; DRG, dorsal root ganglia; PLC, phospholipase C; IP3, inositol triphosphate; GPCR, G protein-coupled receptor; PLA2, phospholipase A2; MAPK, mitogen-activated protein kinase; BH, Bolton-Hunter; TM, transmembrane domain; ECL, extracellular loop; SOS, the product of son of sevenless.
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References |
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TopPrevious |
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 B. J. Wang and Z. J. Cui How does cholecystokinin stimulate exocrine pancreatic secretion? From birds, rodents, to humans Am J Physiol Regulatory Integrative Comp Physiol, February 1, 2007; 292(2): R666 - R678. [Abstract] [Full Text] [PDF]
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P. Giacobini and S. Wray Cholecystokinin Directly Inhibits Neuronal Activity of Primary Gonadotropin-Releasing Hormone Cells through Cholecystokinin-1 Receptor Endocrinology, January 1, 2007; 148(1): 63 - 71. [Abstract] [Full Text] [PDF]
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T. J. Weiland, N. J. Voudouris, and S. Kent CCK2 receptor nullification attenuates lipopolysaccharide-induced sickness behavior Am J Physiol Regulatory Integrative Comp Physiol, January 1, 2007; 292(1): R112 - R123. [Abstract] [Full Text] [PDF]
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 F. Benedetti, M. Amanzio, S. Vighetti, and G. Asteggiano The Biochemical and Neuroendocrine Bases of the Hyperalgesic Nocebo Effect. J. Neurosci., November 15, 2006; 26(46): 12014 - 12022. [Abstract] [Full Text] [PDF]
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K. G. Harikumar, D. I. Pinon, and L. J. Miller Fluorescent Indicators Distributed throughout the Pharmacophore of Cholecystokinin Provide Insights into Distinct Modes of Binding and Activation of Type A and B Cholecystokinin Receptors J. Biol. Chem., September 15, 2006; 281(37): 27072 - 27080. [Abstract] [Full Text] [PDF]
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 M. Dufresne, C. Seva, and D. Fourmy Cholecystokinin and gastrin receptors. Physiol Rev, July 1, 2006; 86(3): 805 - 847. [Abstract] [Full Text] [PDF]
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M. Foucaud, I. G. Tikhonova, I. Langer, C. Escrieut, M. Dufresne, C. Seva, B. Maigret, and D. Fourmy Partial Agonism, Neutral Antagonism, and Inverse Agonism at the Human Wild-Type and Constitutively Active Cholecystokinin-2 Receptors Mol. Pharmacol., March 1, 2006; 69(3): 680 - 690. [Abstract] [Full Text] [PDF]
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 S. Schulz, C. Rocken, C. Mawrin, and S. Schulz Immunohistochemical Localization of CCK1 Cholecystokinin Receptors in Normal and Neoplastic Human Tissues J. Clin. Endocrinol. Metab., November 1, 2005; 90(11): 6149 - 6155. [Abstract] [Full Text] [PDF]
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N. Tsujino, A. Yamanaka, K. Ichiki, Y. Muraki, T. S. Kilduff, K.-i. Yagami, S. Takahashi, K. Goto, and T. Sakurai Cholecystokinin Activates Orexin/Hypocretin Neurons through the Cholecystokinin A Receptor J. Neurosci., August 10, 2005; 25(32): 7459 - 7469. [Abstract] [Full Text] [PDF]
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I. Langer, I. G. Tikhonova, M.-A. Travers, E. Archer-Lahlou, C. Escrieut, B. Maigret, and D. Fourmy Evidence That Interspecies Polymorphism in the Human and Rat Cholecystokinin Receptor-2 Affects Structure of the Binding Site for the Endogenous Agonist Cholecystokinin J. Biol. Chem., June 10, 2005; 280(23): 22198 - 22204. [Abstract] [Full Text] [PDF]
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C. Morel, G. Ibarz, C. Oiry, E. Carnazzi, G. Berge, D. Gagne, J.-C. Galleyrand, and J. Martinez Cross-interactions of Two p38 Mitogen-activated Protein (MAP) Kinase Inhibitors and Two Cholecystokinin (CCK) Receptor Antagonists with the CCK1 Receptor and P38 MAP Kinase J. Biol. Chem., June 3, 2005; 280(22): 21384 - 21393. [Abstract] [Full Text] [PDF]
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M. Dong, E. M. Hadac, D. I. Pinon, and L. J. Miller Differential Spatial Approximation between Cholecystokinin Residue 30 and Receptor Residues in Active and Inactive Conformations Mol. Pharmacol., June 1, 2005; 67(6): 1892 - 1900. [Abstract] [Full Text] [PDF]
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Z. Zheng, M. W. Lewis, and R. A. Travagli In vitro analysis of the effects of cholecystokinin on rat brain stem motoneurons Am J Physiol Gastrointest Liver Physiol, May 1, 2005; 288(5): G1066 - G1073. [Abstract] [Full Text] [PDF]
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E. Archer-Lahlou, C. Escrieut, P. Clerc, J. Martinez, L. Moroder, C. Logsdon, A. Kopin, C. Seva, M. Dufresne, L. Pradayrol, et al. Molecular Mechanism Underlying Partial and Full Agonism Mediated by the Human Cholecystokinin-1 Receptor J. Biol. Chem., March 18, 2005; 280(11): 10664 - 10674. [Abstract] [Full Text] [PDF]
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R. D. Reidelberger, J. Hernandez, B. Fritzsch, and M. Hulce Abdominal vagal mediation of the satiety effects of CCK in rats Am J Physiol Regulatory Integrative Comp Physiol, June 1, 2004; 286(6): R1005 - R1012. [Abstract] [Full Text] [PDF]
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P. Giacobini, A. S. Kopin, P. M. Beart, L. D. Mercer, A. Fasolo, and S. Wray Cholecystokinin Modulates Migration of Gonadotropin-Releasing Hormone-1 Neurons J. Neurosci., May 19, 2004; 24(20): 4737 - 4748. [Abstract] [Full Text] [PDF]
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G. Mazzocchi, L. K. Malendowicz, F. Aragona, R. Spinazzi, and G. G. Nussdorfer Cholecystokinin (CCK) Stimulates Aldosterone Secretion from Human Adrenocortical Cells via CCK2 Receptors Coupled to the Adenylate Cyclase/Protein Kinase A Signaling Cascade J. Clin. Endocrinol. Metab., March 1, 2004; 89(3): 1277 - 1284. [Abstract] [Full Text] [PDF]
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M. Beinborn, Y. Ren, M. Blaker, C. Chen, and A. S. Kopin Ligand Function at Constitutively Active Receptor Mutants Is Affected by Two Distinct Yet Interacting Mechanisms Mol. Pharmacol., March 1, 2004; 65(3): 753 - 760. [Abstract] [Full Text] [PDF]
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 J. C. Reubi Peptide Receptors as Molecular Targets for Cancer Diagnosis and Therapy Endocr. Rev., August 1, 2003; 24(4): 389 - 427. [Abstract] [Full Text] [PDF]
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C. Gales, M. Poirot, J. Taillefer, B. Maigret, J. Martinez, L. Moroder, C. Escrieut, L. Pradayrol, D. Fourmy, and S. Silvente-Poirot Identification of Tyrosine 189 and Asparagine 358 of the Cholecystokinin 2 Receptor in Direct Interaction with the Crucial C-Terminal Amide of Cholecystokinin by Molecular Modeling, Site-Directed Mutagenesis, and Structure/Affinity Studies Mol. Pharmacol., May 1, 2003; 63(5): 973 - 982. [Abstract] [Full Text] [PDF]
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 A. S. Kopin, E. W. McBride, C. Chen, R. M. Freidinger, D. Chen, C.-M. Zhao, and M. Beinborn Identification of a series of CCK-2 receptor nonpeptide agonists: Sensitivity to stereochemistry and a receptor point mutation PNAS, April 29, 2003; 100(9): 5525 - 5530. [Abstract] [Full Text] [PDF]
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J. Pannequin, C. Oiry, C. Morel, J. Kucharczak, I. Camby, R. Kiss, D. Gagne, J.-C. Galleyrand, and J. Martinez C-Terminal Heptapeptide of Gastrin Inhibits Astrocytomas Motility by Interacting with a New Gastrin Binding Site J. Pharmacol. Exp. Ther., July 1, 2002; 302(1): 274 - 282. [Abstract] [Full Text] [PDF]
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B. Pommier, F. Beslot, A. Simon, M. Pophillat, T. Matsui, V. Dauge, B. P. Roques, and F. Noble Deletion of CCK2 Receptor in Mice Results in an Upregulation of the Endogenous Opioid System J. Neurosci., March 1, 2002; 22(5): 2005 - 2011. [Abstract] [Full Text] [PDF]
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S. Lal, A. J. Kirkup, A. M. Brunsden, D. G. Thompson, and D. Grundy Vagal afferent responses to fatty acids of different chain length in the rat Am J Physiol Gastrointest Liver Physiol, October 1, 2001; 281(4): G907 - G915. [Abstract] [Full Text] [PDF]
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L. K. Malendowicz, M. Nowak, L. Gottardo, C. Tortorella, M. Majchrzak, and G. G. Nussdorfer Cholecystokinin Stimulates Aldosterone Secretion from Dispersed Rat Zona Glomerulosa Cells, Acting Through Cholecystokinin Receptors 1 and 2 Coupled with the Adenylate Cyclase-Dependent Cascade Endocrinology, October 1, 2001; 142(10): 4251 - 4255. [Abstract] [Full Text] [PDF]
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 G. Szalmay, G. Varga, F. Kajiyama, X.-S. Yang, T. F Lang, R M. Case, and M. C Steward Bicarbonate and fluid secretion evoked by cholecystokinin, bombesin and acetylcholine in isolated guinea-pig pancreatic ducts J. Physiol., September 15, 2001; 535(3): 795 - 807. [Abstract] [Full Text] [PDF]
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C. Becker, M.-H. Thiebot, Y. Touitou, M. Hamon, F. Cesselin, and J.-J. Benoliel Enhanced Cortical Extracellular Levels of Cholecystokinin-Like Material in a Model of Anticipation of Social Defeat in the Rat J. Neurosci., January 1, 2001; 21(1): 262 - 269. [Abstract] [Full Text] [PDF]
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20 to
P < .05 and 
P < .05 compared with the
same dose of L-365,260 without antagonists (Newmann-Keuls test).
