名古屋学芸大学健康･栄養研究所年報第３号

２００９年《Original Article》

Distribution of CCK receptors in guinea-pig gastrointestinal tract
Kotoyo Fujiki＊, Hiroshi Ishiguro＊＊ and Satoru Naruse＊＊＊[Abstract]
Cholecystokinin (CCK) is a regulatory peptide in the gastrointestinal tract. The cell/tissue distribution of
CCK receptors is variable among species. Guinea-pig is a useful animal model to study the physiology ofgastrointestinal tract. However, its structure and distribution are not known. In the present study, we have cloned
guinea-pig CCK-BR and examined the distribution of CCK receptors in guinea-pig gastrointestinal tract andpancreas. A large amount of CCK-AR mRNA was detected in pancreatic acinar cells, gallbladder, and stomachmucosal layer. CCK-BR was expressed in pancreatic acinar and duct cells and mucosal and muscle layers ofduodenum and stomach. However, only a truncated isoform of CCK-BR with a deletion of exon 1 (Δexon 1)
was detected in pancreatic duct cells.
Key words; CCK-1 receptor, CCK-2 receptor, gastrin, truncated isoform

with an extracellular N-terminal and intracellular[Introduction]

C-terminal.

Cholecystokinin (CCK) belongs to a family of gas-CCK-AR has been identified in various tissues

trointestinal peptides that regulate various functions

such as gallbladder, sphincter of oddi, and pancreaticin the gastrointestinal tract including stimulation ofacinar cells5, 6, 7, 8). CCK-BR is typically found in

gastric acid production, induction of gastric smooth

gastric parietal cells, gastrointestinal smooth muscle

muscle contraction, and promotion of growth andcells and in the central nervous system2). However,differentiation of the stomach mucosa1). CCK also

the cell/tissue distribution of CCK-AR and CCK-BR

mediates enzyme secretion from pancreatic acinar

is variable among species. For example, human pan-cells and functions as a neurotransmitter in the cen-creatic acinar cells lack CCK-AR and only express

tral nervous system2, 3)

CCK-BR, while mouse and rat acinar cells possess.

Receptors for CCK have been pharmacologically

CCK-AR. CCK-AR and CCK-BR have several iso-classified into two subtypes, CCK-1 receptor (CCK-forms. Among them N-terminally truncated isoform

A receptor; CCK-AR) and CCK-2 receptor (CCK-B

shows low affinity with CCK9, 10).

receptor; CCK-BR)4). CCK receptors belong to theGuinea-pig is a useful animal model to study thesuperfamily of G protein-coupled receptors which

physiology of gastrointestinal tract because of someare characterized by seven transmembrane domains

similarities to humans. Guinea-pig has gallbladderconnected by intracellular and extracellular loops

and its pancreatic juice contains high concentrations＊ School of Nutritional Sciences, Nagoya University of Arts and Sciences, Nisshin, Japan＊＊ Nagoya University Graduate School of Medicine, Nagoya, Japan＊＊＊ Miyoshi Hospital, Aichi, Japan11

of HCO3–. Guinea-pig CCK-AR cDNA was cloned14)

in exon5 (NM007627); for second PCR were;

and the mRNA was expressed in gallbladder, pan-sense 5’-ATGTGGTGGGCGGGAGCCTGAGC-3’2)

creas, stomach . However, guinea-pig CCK-BR has

in 5’ untranslated reasion (AF264177), antisense

not been cloned and the distribution of CCK-BR5’-GATTGGGCAGGAGGGTGAA-3’ in exon 2in gastrointestinal tract are not known. In the pres-(NM176875), sense 5’- CGCAGTGATCTTCCT-

ent study, we have cloned guinea-pig CCK-BR and

GATGAGCG-3’ in exon 2 (NM176875), antisense

examined the distribution of CCK receptors includ-5’-TGCAGTGGTCGGCAGATGGCG-3’ in exon

ing the truncated isoform of CCK-BR in guinea-pig

3 (NM176875), sense 5’-GTGTCTGTGAGCGT-gastrointestinal tract and pancreas.

GTCCACG-3’ in exon 3 (this paper), antisense5’-CTGCTCTGGCTCTCACTGTCG-3’ in exon
4 (this paper), sense 5’-TATGGACTCATCTC-[Material and Methods]

CCGCGAA-3’ in exon 4 (NM007627), antisense

Animals and tissue isolation

5’-TGTGCTGATGGTGGTATAGCT-3’ in exon5

Female guinea pigs were obtained from Japan

NM013165). The primer selection was based on

SLC (Hamamatsu, Japan). All protocols were ap-the region found in each GenBank accession no.

proved by the Animal Use Committees of Nagoya

that has high homology in human, mouse, and rat

University and the National Institute for Physiologi-sequences.

cal Sciences. Guinea-pigs were killed by cervical

PCR products were purified using the High Puredislocation and the pancreas, duodenum, stomach,

PCR Product Purification Kit (Roche Diagnostics

and gallbladder were removed. The pancreatic acini

GmbH Mannheim, Germany) and sequenced directly

and ducts were isolated as described previously15, 16).

using automated sequencer (BECKMAN COULTER,

The duodenum and stomach were separated to mu-

Inc Fullerton, CA) after terminator reaction using
DNA Sequencing Kit CEQTM DTCS-Quick Start Kit17)

cosal and muscle layers as described previously .

(BECKMAN COULTER, Inc Fullerton, CA).
Total RNA extraction
RT-PCR

Total RNA was extracted using RNeasy Protect
Mini Kit (QIAGEN GmbH, Germany). Reverse tran-Polymerase chain reactions (PCR) were car-scription was performed using an oligo-dT primer

ried out in a 50 mL reaction mixture containing

and TaqMan Reverse Transcription Reagents (Roch,50 ng cDNA, 5 mL of 10×Ex Taq buffer, 4 mL of

Branchburg NJ, U.S.A.).

2.5 mM dNTP mix, 50 pmol of each primer and1.5 U Ex Taq DNA polymerase (TaKaRa, Shiga,

Direct sequencing

Otsu Japan). The conditions were; denaturation

Polymerase chain reactions (PCR) were carriedfor 3 min at 94°C, then 35 cycles of denaturationout using the Ex Taq (TaKaRa, Japan). The PCR

for 30 sec at 94°C, annealing for 30 sec at 62°C,protocol was: 94°C, 1 min; 60°C, 1 min; 72°C, 2extension for 30 sec at 72°C, followed by 7 minmin; 30 cycles for first PCR and 94°C, 30 s; 60°C,at 72°C using GeneAmp PCR System 9700 (Ap-60 s; 72°C, 30 s; 25 cycles for second PCR using

plied Biosystems, Foster CA). The oligonucleotide

GeneAmp PCR System 9700 (Applied Biosystems,primers used for CCK-AR were; sense 5’- ATG-Foster, CA ).

GACGTGGTAGACAGCCTT-3’ in exon 1, anti-

The oligonucleotide primers used for first

sense 5’-ACATCGTTTGGCAGTAGGAAG -3’ in

PCR were; sense 5’-GGGCGGAAACTAGGAG-exon 3 (S68242, 576 bp), for CCK-BR were; sense

GGGC-3’ in 5’ untranslated reasion (AF264177),

5’- CAGGCAACGTCAGCTGCGAAA-3’ in exon

antisense 5'-TGTGCTGATGGTGGTATAGCT-3’1 (this paper), antisense 5’-GATTGGGCAGGAG-12

Distribution of CCK receptors in guinea-pig gastrointestinal tract

GGTGAA-3’ in exon 2 (NM176875, 246 bp). sense[Result]

5’- TTCACCCTCCTGCCCAATC -3’ in exon 2, antisense 5’-AGTGGCTAAAATCACGCGAGC-3’ inAlignment of guinea pig CCK-B receptor andexon 3 (this paper, 207 bp). The primers for GAPDH

human CCK-B receptor

were; sense 5’- ACCACAGTCCATGCCATCAC-3’,

Alignment of guinea pig CCK-B receptor andantisense 5’-TCCACCACCCTGTTGCTGTA-3’.

human CCK-B receptor are shown in Fig. 1 fornucleotide sequences and in Fig. 2 for deduced protein sequences. They have 88% and 91% homology
respectively. The homology to mouse and rat CCKBR is 83% for nucleotide sequence and 87% fordeduced protein sequence.
Expression of CCK-AR and CCK-BR mRNA in
guinea-pig gastrointestinal tract and pancreas
RT-PCR analysis of the tissue distribution of CCKAR and CCK-BR is shown in Fig. 3. The fragments
amplified with primers for exon 1 (sense) and exon
2 (antisense) of CCK-BR indicate mRNA expression of a complete isoform (wild type) of CCK-BR.
The fragments amplified with primers for exon 2(sense) and exon 3 (antisense) of CCK-BR indicate
mRNA expression of both complete isoform (wildtype) and truncated isoform (Δexon 1) of CCK-BR.
A large amount of CCK-AR mRNA was detectedin pancreatic acinar cells, gallbladder, and stomachmucosal layer while its amounts in pancreatic ductcells, duodenum mucosal and muscle layers, andstomach muscle layer were small. CCK-BR wasFig. 1 Alignment of nucleotide sequences for guinea pig
CCK-BR (upper) and human CCK-BR (lower).

Fig. 2 Alignment of deduced protein sequences for guinea
pig CCK-BR (upper) and human CCK-BR (lower).

13Fig. 3

Distribution of CCK-AR and CCK-BR mRNA in guinea-pig gastrointestinal tract and pancreas.

expressed in pancreatic acinar and duct cells andshows about 10 fold lower affinity for CCK-8 andmucosal and muscle layers of duodenum and stom-

100 fold lower affinity for gastrin than the complete

ach. However, only a truncated isoform of CCK-BR

form of CCK-BR9). The CCK-AR which lacked thewith a deletion of exon 1 (Dexon 1) was detected infirst 42 amino acids did not directly bind CCK-910).

pancreatic duct cells [Fig. 3].

Truncated isoforms of CCK-BR are identified in thetumor cells11) and so their function may be related toproliferation and growth of the cells.

[Discussion]

Rat duodenum mucosa expresses a small amountIn the present study we have successfully obtainedof CCK-AR and does not express CCK-B recep-

cDNA sequence of guinea-pig CCK-BR. The de-tor12). Human duodenum mucosa and muscle layer

duced amino acid sequence of CCK-B receptor isexpress CCK-AR and does not express CCK-BR13).

50% identical to the CCK-A receptor. Wank et al. re-The present study demonstrated that guinea-pig

ported that CCK-A receptors have 1000 fold higher

duodenum predominantly expresses CCK-BR while

affinity for CCK than for gastrin. CCK-B receptor

stomach mucosa expresses both CCK-AR and CCK-18)

have similar affinity for CCK and gastrin .

BR. CCK- BR in the duodenum may play an important role in the intestinal and neural phase of theIn guinea-pig the expression of CCK-A recep-

regulation of pancreatic exocrine secretion.

tors were detected in pancreatic acini, but not inpancreatic ducts. Mice pancreatic ducts express
CCK-A receptors but not secretin receptors19). Only

[Reference]a truncated isoform of CCK-BR with a deletion of1

exon1 was detected in guinea-pig pancreatic ducts.

Jonson, L. R (1994) in Physiolpgy of the Gastrointestinal Tract, 1st ed., pp. 49-67, Raven Press, New York.

The exon1 codes initial 50 amino acids which is the2N-terminal extracellular domain of CCK-BR. TheWank SA (1995) Cholecystokinin receptors. Am J
Physiol 32:G628–G646

N-terminally truncated form of the human CCK-BR14

Distribution of CCK receptors in guinea-pig gastrointestinal tract

3Shulkes A, Baldwin GS. (1997) Biology of gut chole-secretion from rat pancreatic acini. Biochem Biophys

cystokinin and gastrin receptors. Clin Exp Pharmacol

Res Commun. 24;269:792-7.
16 Ishiguro H, Steward MC, Case RM. et al. (1996) Bi-Physiol. 24; 209-216
45Jensen, R. T., Wank, S. A., Gardner, J. D. et al. (1989)

carbonate secretion in interlobular ducts from guinea-Trends Pharmacol Sci 10, 418-423.

pig pancreas. J Phy 495; 179-191

Steigerwalt RW, Goldfine ID, Williams JA. (1984)

17 Ishikawa K, Ozaki T. (1997) Distribution of several

Characterization of cholecystokinin receptors on bo-gut neuropeptides and their effects on motor activity

vine gallbladder membranes. Am J Physiol. 247:G709-in muscularis mucosae of guinea-pig proximal colon. JAut Ner Sys 64; 91-100714.
678Schrenck T von, Moran TH, Jensen RT. et al. (1988)

18 Wank SA, Pisegna JR, de Weerth A. (1994）receptor

Cholecystokinin receptors on gallbladder muscle andfamily. Molecular cloning, structure, and functional ex-pancreatic acinar cells: a comparative study. Am J

pression in rat, guinea pig, and human. Ann N Y AcadPhysiol. 255:G512-521

Sci. 23;713:49-66.
19

Grider JR, Makhlouf GM. (1990) Distinct receptors for

CCK-A receptor for pancreatic function in mice: aand gallbladder. Am J Physiol. 259:G184-190

study in CCK-A receptor knockout mice. Pancreas.

Cox KL, Schrenck T von, Jensen RT et al. (1990)

24:276-83.

Characterization of cholecystokinin receptors on thesphincter of Oddi. Am J Physiol. 257:G873–G881
9Miyake A. (1995) A truncated isoform of human CCKB/gastrin receptor generated by alternative usage of anovel exon. Biochem Biophys Res Commun. 208:230237

10Kennedy K, Escrieut C, Fourmy D. et al. (1995) Identification of a region of the N-terminal of the human
CCKA receptor essential for the high affinity interaction with agonist CCK. Biochem Biophys Res Commun. 213:845-852

11McWilliams DF, Watson SA, Seth R. et al. (1998) Coexpression of gastrin and gastrin receptors (CCK-B anddelta CCK-B) in gastrointestinal tumour cell lines. Gut
42:795-798

12

Monstein HJ, Nylander AG, Hakanson R. et al. (1996)
Cholecystokinin-A and cholecystokinin-B/gastrin receptor mRNA expression in the gastrointestinal tract
and pancreas of the rat and man. A polymerase chain
reaction study. Scand J Gastroenterol. 31:383-390

13

Funakoshi A, Fukamizu Y, Miyasaka K. (1999) Mechanism of cholecystokinin-A- receptor antagonist on
human pancreatic exocrine secretion. Localization ofCCK-A receptor in the human duodenum. Digestion.
60:75-80

14De Weerth A, Pisegna JR, Wank SA. (1993) Guinea pig
gallbladder and pancreas possess identical CCK-A receptor subtypes: receptor cloning and expression. Am JPhysiol. 265:G1116-1121

15

Takiguchi S, Suzuki S, Noda T. et al. (2002) Role ofcholecystokinin and gastrin on muscle cells of stomach

Mizuno N, Naruse S, Kitagawa M, et al. (2000) Effects
of an inhibitor of myosin light chain kinase on amylase

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和文抄録

モルモット消化管における CCK 受容体の分布
藤木＊

理代

石黒洋

＊＊

成瀬＊＊＊

達［要旨］
CCK（Cholecystokinin）は消化管ホルモンの一つで、その受容体の分布は動物種によって異なる。
モルモットは消化管研究における実験モデルとして有用だが、CCK 受容体の構造や分布については明らかではない。
本研究において、我々は CCK-B 受容体の cDNA をクローニングすることに成功した。CCK-A受容体と共にそれらの消化管における分布を mRNA レベルで調べた。その結果、CCK-A 受容体は膵腺房細胞、胆嚢、胃粘膜層に多く発現しており、CCK-B 受容体は膵腺房細胞、膵導管細胞、十二指腸および胃の粘膜層と筋層に発現していた。しかし、膵導管細胞における CCK-B 受容体はエクソン １ が欠損したアイソフォームであった。
キーワード；CCK-1レセプター，CCK-2 レセプター，ガストリン，選択的スプライシング＊名古屋学芸大学管理栄養学部＊＊名古屋大学大学院医学系研究科健康栄養医学＊＊＊三好町民病院

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