1 Service de Gastro-entérologie, Département de Médecine, Université de Sherbrooke, Sherbrooke, Québec, Canada J1H 5N4; and 2 CURE Digestive Diseases Research Center, UCLA Medical Center, Los Angeles, California 90073-1792
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ABSTRACT |
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In rodents,
cholecystokinin (CCK) induces pancreatic enzyme secretion and pancreas
growth through its CCKA receptors. It is unknown whether
occupation of the CCKB receptors present in pig and human
pancreas can cause the same effects. This study evaluates
CCKB receptor expression in rat, mouse, pig, and fetal human pancreata using Northern blot, Western blot, and
immunofluorescence techniques. The reported 2.7-kb CCKB
receptor mRNA transcript in the rat brain and gastric fundus is absent
in pancreas; the message was, however, detected by RT-PCR and by a
CCKB receptor antibody as an 80-kDa protein present
uniquely in islet -cells. Proteins of 50 and 80 kDa appear in mouse
pancreas, and proteins of 50 and 115 kDa appear in pig and human
pancreas, respectively, all localized in islet
-cells. Gastrin mRNAs
are strongly present in fetal rat pancreas, and the hormone is
localized in islets; both are repressed 10 days after birth. In
conclusion, the CCKB receptors are present in pancreas of
four species with exclusive location in islet
-cells. In such a
location, they could be indirectly involved in the control of enzyme secretion.
rat; mouse; pig; human; cholecystokinin
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INTRODUCTION |
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THE GASTRIN FAMILY OF PEPTIDES and their receptors are widely distributed throughout the central nervous system (11) and gastrointestinal organs (13). The receptors for gastrin and cholecystokinin (CCK) represent a population of two different subtypes: the CCKA subgroup with high affinity for sulfated CCK-8 and the CCKB subtype with almost equal affinity for sulfated and nonsulfated CCK analogs (43, 46).
The CCKA receptors are primarily found in the periphery, where they regulate pancreatic enzyme secretion and growth and gallbladder contraction (31, 40, 49). The CCKB subtype was originally found in the central nervous system and seems to control anxiety and panic attacks (2); in periphery, it is associated with gastric acid secretion and histamine release (28).
The physiology and pharmacology of the CCKB/gastrin receptor seem to be dependent on the species, and, so far, no clear biological functions have been assigned to its occupation. This receptor subtype has been identified in human (49), calf (26), dog (8, 16), guinea pig (56), and most recently in pig pancreas (36). Zhou et al. (57) indicated that the CCKB receptor could not be detected in the rat pancreas by Northern blot analysis, a finding confirmed later by RT-PCR (34).
No clear biological functions have been assigned to the pancreatic CCKB/gastrin receptors, although gastric enterochromaffin-like (ECL) cell receptors do have clear functions. Their occupation was associated with ECL cell growth (7), histamine release (20), and mucosal growth (53). In CCKB/gastrin receptor-deficient mice, parietal and ECL cells were decreased, along with a reduction in somatostatin cell density and an increase in antral gastrin cell number (18). Overexpression of this CCKB/gastrin receptor in the stomach of naturally occurring CCKA receptor gene knockout rats led to thickening of the fundic mucosa and hyperplasia and hypertrophy of the parietal cells (32).
Although the presence of the CCKB/gastrin receptors in the pancreas of different species is not contested, the physiological responses resulting from their occupation raise questions and present controversial results. In CCKB receptor gene knockout mice, Miyasaka et al. (33) recently indicated that the CCKB receptor has no role in pancreatic growth, exocrine secretion, or bile secretion. These results are in total opposition to those of Saillan-Barreau et al. (42), who demonstrated in a transgenic mouse strain expressing the human CCKB/gastrin receptor that CCK and sulfated gastrin stimulated enzyme secretion with identical potencies and efficacies, and with those of Detjen et al. (6), who showed in CCKB receptor-transfected human pancreatic cancer cells that their occupation by CCK led to inhibition of their growth. In the pig, whose pancreas possesses predominantly CCKB receptors rather than CCKA receptors (36), CCK is a rather weak stimulant of pancreatic enzyme secretion in live animals (5) and in freshly isolated acini (36). Such insensitivity to CCK was also observed in newborn rat pancreata (55), suggesting a paracrine effect of gastrin found in large quantity in pancreata of fetal and newborn rats (30). Such a hypothesis is supported by our recent findings that treatment of pregnant female rats during their entire pregnancy with either gastrin or the CCKB receptor antagonist L-365260 resulted in fetal pancreas hypertrophy and atrophy, respectively (35), suggesting a role for the CCKB receptors and gastrin at least in pancreas differentiation early in life.
In view of the great uncertainty regarding the physiological roles of CCKB receptor occupation in pancreatic physiology, this study was undertaken 1) to document the ontogeny of expression of the rat pancreatic CCKB receptor and pancreatic gastrin mRNA, its specific agonist; 2) to confirm the CCKB receptor mRNA expression by evaluating the CCKB receptor protein by Western blot and immunofluorescence; and 3) to establish the presence and localization of the CCKB receptor protein in the pancreas of three different species other than the rat: the mouse, the pig, and the human. We expect that these new data will clarify the importance and the role of the CCKB receptors in pancreatic physiology.
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MATERIALS AND METHODS |
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Animals. These studies were performed on male (180-200 g) and female (250-300 g) Sprague-Dawley rats from Charles River (St-Constant, QC, Canada) and their pups after birth. Fetal rat pancreata were excised from 21-day-old fetuses, neonatal pancreata came from newborn rats, and other pancreata came from rats 3-35 days after birth and adult animals (60 days). Pieces of pancreas from suckling and adult mice and pig and from 18-wk human fetuses were taken for evaluation. Human fetal pancreatic tissues were obtained from normal elective pregnancy terminations. No tissue was collected from cases associated with known fetal abnormality or fetal death. These studies were approved by the Institutional Human Subject Review Board.
Tissue preparation. Once excised, tissues were 1) quickly frozen in liquid nitrogen and processed later for RNA extraction and Northern blot analysis; 2) embedded in Tissue-Tek optimum cutting temperature (OCT) 4583 compound (Sakuta Fine Tek, Torrance, CA) and frozen in liquid nitrogen for indirect immunofluorescence studies; or 3) homogenized in a specific buffer for Western blot analysis.
Membrane preparation.
All procedures were carried out at 4°C. Freshly removed tissues were
minced and disrupted in 10 mM HEPES, pH 7.5, 250 mM sucrose, 1 mM EGTA,
1 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 21 µM leupeptin, and
1.5 µM aprotinin with the use of five passes of a Potter-Elvehjem
homogenizer. Unbroken cells and nuclei were removed by centrifugation
at 500 g for 5 min. Membranes were collected by
centrifugation at 30,000 g for 30 min using a TLS-55 rotor. The supernatants were removed, and membranes were suspended to a
concentration of 15-30 mg/ml in homogenization buffer and stored at 80°C until used. Protein concentrations were determined using a
bicinchoninic acid protein assay reagent (Pierce Chemical, Rockford, IL).
Gel electrophoresis and immunoblotting.
Membrane proteins (30 µg) were boiled 5 min in 62.5 mM Tris · HCl, pH 6.8, 10% glycerol, 2% SDS, and 5% -mercaptoethanol and
separated by SDS-PAGE using 10% polyacrylamide gels. After transfer to
polyvinylidene difluoride membranes (Bio-Rad Laboratories, Mississauga,
ON, Canada), the membranes were blocked for 2 h with 6% nonfat
dry milk in TBST (20 mM Tris · HCl, pH 7.4, 0.15 M NaCl, and
0.1% Tween 20) and incubated overnight at 4°C with polyclonal antibodies corresponding to different areas of the canine or rat CCKB/gastrin receptor molecule (10). These
antibodies were raised in rabbit using the following sequences:
antibody 9262 (amino acids 42-55) from the
extracellular part of the canine receptor and antibody 9252 (amino acids 253-264) from the intracellular part of the canine
receptor. We also used antibody 9413 (amino acids
252-266) from the intracellular part of the rat receptor. Characterization of these antibodies has been previously described (10). The blots were then washed 5 times for 5 min in TBST
and incubated for 1 h with horseradish peroxidase-conjugated
anti-rabbit IgG (Amersham Pharmacia Biotech, Baie d'Urfé, QC,
Canada) at 1:8,000 in TBST-3% nonfat dry milk. After membranes were
washed six times for 5 min, they were developed with Lumi-Light Plus chemiluminescence substrate (Roche Diagnostics, Montreal, QC, Canada).
Immunoneutralization was performed by preincubating the primary
antibody with its specific peptide antigen (10 µg/ml) for 1 h.
Indirect immunofluorescence. Tissues embedded in Tissue-Tek OCT 4583 compound were handled as previously described (1). For this specific technique, the CCKB/gastrin antibody 9262 (canine) was used at a 1:500 dilution. The gastrin antibody 8007, a generous gift from Dr. Rehfeld (Copenhagen, Denmark) was used at a 1:100 dilution. The antibody raised against insulin, used at a 1:50 dilution, was a gift from Dr. Bendayan (University of Montreal, Montreal, QC, Canada). The somatostatin antibody used at a 1:2,000 dilution, glucagon (1:50), and pancreatic polypeptide (1:2,000) were generous gifts from Dr. Lebel (Department of Biology, Université de Sherbrooke).
RNA extraction and probe preparation. Total RNAs from rat pancreata and gastric fundus were isolated according to a modification of the procedure of Chirgwin et al. (4) as previously described (3). The gastrin open reading frame with 5'- and 3'-flanking sequences (nucleotides 96-376) was cloned by PCR using the following sense and antisense primers, respectively, composed with Kpn I and Sac I restriction sites: 5'-TGCTGGCTCTAGGTACCTTCTCGG-3' (nucleotides 80-103) and 5'-GATGGCTGAGCTCTGGAAGAGC-3' (complementary to nucleotides 365-386). Single-stranded cDNA reverse transcribed from rat brain mRNA served as the DNA template, and this amplification product was used for sequencing and transcription of cRNA probe with a specific RNA polymerase, as described by the Riboprobe System (Promega, Madison, WI). A fragment of 490 bp (nucleotides 207-697) subcloned from the rat CCKB receptor cDNA (54) was also used.
Northern blot analysis. Total RNA (20 µg, quantified by measuring absorbance at 260 nm) was size fractionated on a 1% agarose gel containing 2.2 M formaldehyde and transferred to nylon membranes (Nytran Plus; Schleicher & Schuell, Keene, NH) as described by Sambrook et al. (33). The remainder of the technique was as described earlier (3). The autoradiograms were quantified by scanning densitometry, and values were normalized against the corresponding 18S RNA levels.
DNA amplification. For amplification starting from 5 µg of RNA as template, single-stranded cDNA was synthesized from total RNA using an oligo(dt) primer and first-strand cDNA kit (Pharmacia Biotech). For direct amplification, 0.5 µg of total RNA that had been reverse transcribed was used for each reaction. Reactions were performed using 2 units of Taq polymerase (Pharmacia Biotech) and the CCKB receptor primers: sense 5'-CTTCATCCCGGGTGTGGTTATTGCG-3' (corresponding to nucleotides 816-840) and antisense: 5'-CCCCAGTGTGCTGATGGTGGTATAGC-3' (nucleotides 1460-1485) at 2.5 mM each in 50 µl of 67 mM Tris · HCl, pH 8.8, 16 mM (NH4)2SO4, 1.5 mM MgCl2, deoxyribonucleotides at 0.25 mM each, and Tween 20 (0.01% vol/vol). Parameters for DNA amplification were 94°C for 30 s, annealing temperature (57°C) for 30 s, and 72°C for 30 s. Oligonucleotide primers used for DNA amplification were synthesized by GIBCO (Life Technologies, Burlington, ON, Canada). DNA amplification products were analyzed by gel electrophoresis on a polyacrylamide gel stained with ethidium bromide.
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RESULTS |
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Ontogeny of CCKB/gastrin receptor mRNA in rat pancreas.
As shown in Fig. 1A, we were
unable to detect any mRNA coding for the CCKB receptor in
the rat pancreas; this absence of receptor messenger was evident at all
ages, including the fetus. This lack of detection did not result from a
probe problem, since a strong message of 2.7 kb was detected in the rat
brain and gastric fundus, our positive controls (54). With
the use of RT-PCR, the CCKB receptor mRNAs were, however,
detected in rat fetal and adult pancreata and in rat brain and gastric
fundus, but the messenger was not amplified in the gastric antrum
sample, our negative control (12) (Fig. 1B).
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Ontogeny of CCKB/gastrin receptor protein in rat
pancreas.
Using a specific antibody raised against the intracellular part of the
canine CCKB receptor, we were able to detect the
CCKB receptor protein in the rat pancreatic membranes, as
shown in Fig. 2A. We were
surprised to see that its concentration remained unaffected with age.
The receptor protein was absent in the gastric antrum but present in
the gastric fundus, our respective negative (12) and
positive (54) controls. The receptor protein was visualized as an 80-kDa protein in adult pancreas, and its specificity was ensured by the observation that the 80-kDa band disappeared after
preabsorption of the antibody with the corresponding antigen, as shown
in Fig. 2B.
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Ontogeny of the pancreatic rat CCKB/gastrin receptor
visualized by indirect immunofluorescence.
By immunofluorescence (Fig. 3), the
presence of the CCKB receptor protein can be ascertained in
the rat pancreas at all ages examined. Even if labeling is less intense
in the fetal and newborn organs, it is surprisingly located at the
level of the islets of Langerhans at all ages, from fetal life up to
adulthood, with no labeling at all on the acinar and ductal cells.
Specificity of the labeling was confirmed with tissue exposition to
preimmune serum.
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Pancreatic and gastric fundus cellular localization of the
CCKB/gastrin receptor by indirect immunofluorescence.
Because of the exclusive location of the CCKB receptor
protein in the pancreatic islet of Langerhans anatomic structure, the specific cellular location within the islet was investigated. As shown
in Fig. 4, serial sections of pancreatic
tissue clearly indicate that the CCKB/gastrin receptor
protein colocalizes with somatostatin in the islet's -cells;
indeed, there is no match with the insulin, glucagon, or pancreatic
polypeptide cells. In the gastric fundus, the CCKB receptor
protein also clearly colocalizes with the somatostatin
-cells. The
receptor was also detected on the parietal cells with much less
intensity. It is interesting to visualize the somatostatin
-cells
closely associated with the parietal cells.
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Identification and localization of the CCKB/gastrin
receptor and somatostatin in mouse, pig, and human fetal pancreas.
As shown in Fig. 5A, the adult
mouse pancreas exhibits two CCKB/gastrin receptor proteins
with a major band of ~80 kDa and a minor one around 50 kDa. In the 8- and 16-day suckling mice, the 80-kDa protein is present, whereas we
were unable to detect the 50 kDa protein. Preabsorption of
antibody 9262 with its corresponding antigen prevented
recognition of both proteins in the samples (only the adult was
presented), thus confirming the specificity of the antibody. As in the
rat pancreas, the mouse CCKB/gastrin receptor observed in
the adult pancreas was also localized in the islets of Langerhans, more
specifically in the -cells surrounding the islets, as shown by its
colocalization with somatostatin. The pig pancreas also expresses the
CCKB/gastrin receptor as a 50-kDa protein recognized by
antibody 9252 (Fig. 5B). This binding was
impaired by the antibody's preabsorption with its corresponding antigen. As observed in the rat and mouse pancreas, the pig
CCKB/gastrin receptor was also exclusively detected in the
islets of Langerhans, in the
-cells surrounding the islets where it
colocalizes with somatostatin. The human fetal pancreas is no
exception, with the CCKB/gastrin receptor protein also
specifically located in the
-cells of the islets of Langerhans in
colocalization with somatostatin, as observed in Fig. 5C.
Contrary, however, to what was observed in adult rat, mouse, and pig
pancreas, the fetal human pancreas is richer in islet cells containing
somatostatin and CCKB/gastrin receptor protein. The human
CCKB/gastrin receptor protein is bigger than that of the
three other species studied, with an estimated molecular mass of 115 kDa. Recognition of this 115-kDa receptor protein by its specific
antibody was also impaired by preabsorption of the antibody by its
antigen.
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Ontogeny of the pancreatic gastrin mRNA.
Since gastrin has been reported in human fetal and neonatal rat
pancreas (19, 30) and the hormone binds
specifically to its CCKB/gastrin receptor, we evaluated
whether there was a correlation between pancreatic gastrin expression
and its receptor in the rat. As shown in Fig.
6, the gastrin mRNA is strongly expressed in fetal and newborn rat pancreas. This expression, although still quite intense in the early days of life, exhibited a strong decrease at
day 10, with the message almost absent later on. With a
specific gastrin antibody, we were able to localize the rat gastrin
protein in the G cells of the gastric antrum (Fig.
7A), our positive control. In
support of the data obtained with the gastrin mRNA in the rat pancreas,
the gastrin protein is intensely visible in the fetal and newborn rat
pancreas and undetectable in 10-day-old and adult rat pancreas (Fig.
7B). These data suggest that expression of the
CCKB/gastrin receptor protein is not under the direct
control of pancreatic gastrin, at least after 10 days of life.
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DISCUSSION |
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The major objective of this study was to ascertain the status of
the pancreatic CCKB/gastrin receptor with regard to its
presence and cellular localization in different species. Our data
clearly indicate that 1) the CCKB/gastrin
receptor mRNA could not be identified in the rat pancreas by Northern
blot but was detectable by RT-PCR; 2) the
CCKB/gastrin receptor was present in rat pancreas at all ages studied and in equal proportions as an 80-kDa protein and was
localized exclusively in the somatostatin -cells of the islets of
Langerhans; 3) the CCKB/gastrin receptor protein
was also identified in mouse, pig, and human fetal pancreas, with its
specific location also in the somatostatin
-cells of the islets of
Langerhans; and 4) the presence of the
CCKB/gastrin receptor in the rat pancreas does not
correlate with that of gastrin throughout the gland's development.
Identification of the CCKB/gastrin receptor in the rat pancreas has been a difficult task in recent years. Indeed, the receptor mRNA remained undetectable by means of Northern blot (54, 57) and RT-PCR techniques (34), whereas its message has been detected in fetal pancreas by Northern blot analysis and in adult pancreas only by RT-PCR (9). Our data confirm results from investigators unsuccessful in showing CCKB/gastrin mRNAs by Northern blots (54, 57) and those who were successful by RT-PCR (9). We cannot explain, however, why Monstein et al. (34) and Zhou et al. (57) failed to detect this receptor mRNA by RT-PCR, although we and Funakoshi et al. (9) succeeded. The detection of the CCKB/gastrin receptor protein with a specific antibody for the first time in the rat pancreas indicates undoubtedly that the message has been translated.
Demonstration of the CCKB/gastrin receptor protein in pig and human pancreas also confirms for the first time that the CCKB/gastrin receptor mRNAs previously detected in these two species (24, 36) are indeed translated. The mouse CCKA receptor gene was recently cloned (17). From the sequence of the CCKB receptor gene present in GenBank but not yet published, we established that the 14-amino acid sequence of the dog CCKB receptor chosen to develop the antibody has 78.6% homology with that of the rat and mouse and 92.9% homology with that of the human CCKB receptor. Similarities are of 87.9%, 82.9%, and 97.1% with those of the rat, mouse, and human CCKB receptor, respectively. Our data indicate that the receptor protein is indeed also present in the pancreas of suckling and adult mice.
By the different techniques of SDS-PAGE, affinity labeling, and radiolabeling, studies agreed on the various molecular masses of the rat CCK receptors between 26 and 200 kDa or greater (37); a predicted relative molecular mass of 48,954 (which can reach 90 kDa when the protein is glycosylated) was established by molecular biology (54). The estimated relative molecular mass of 80,000 determined with our specific CCKB/gastrin receptor antibody agrees with the previously published data. In the mouse pancreas, nothing is available for comparison with the size of its CCKB/gastrin receptor. Our results, however, present two proteins of ~50 and ~80 kDa recognized by our specific antibody; the smaller faint protein band could represent the core protein and the larger band the glycosylated receptor. This pancreatic mouse CCKB/gastrin receptor protein is quite comparable to that present in the rat pancreas and in the isolated ECL cells from the African mastomys (50), two other rodents. By photoaffinity labeling, the pig pancreatic CCKB receptor was recently described as a 48- to 52-kDa protein (38), which corresponds quite accurately with the 50-kDa protein identified with our specific antibody. The size of the human CCKB/gastrin receptor core protein was estimated at 48.5 kDa (25, 39); the ~115 kDa protein identified with our specific antibody probably represents the glycosylated form of the receptor, which was recently recognized with a specific antibody as a 105-kDa protein following transfection of the CCKB/gastrin gene in mouse (42).
Localization of the CCKB/gastrin receptor has been
performed by storage phosphor autoradiography in human
(49) and in transgenic mouse (42) pancreata;
in both species, binding of 125I-labeled Bolton-Hunter
(BH)-CCK-8 and 125I-BH-sulfated gastrin, respectively, was
diffusely and homogeneously distributed in the exocrine component of
the gland. In the dog and guinea pig gastric mucosae (10),
staining with the same CCKB/gastrin receptor antibodies
used in our study revealed the receptor exclusively on the gastric
somatostatin -cells. Our data confirm this location on the
-cells, but, contrary to Helander et al. (10), we were
able to demonstrate the presence of the CCKB receptor
protein also on the parietal cells, although with much less intensity
than on the
-cells. The difference in the two studies may reside in
the tissue preparation. Helander et al. (10) fixed their
tissues in 4% formaldehyde and embedded them in paraffin, whereas ours
were embedded in OCT. Our technical approach seems more sensitive,
because we used the somatostatin antibody at a 1:2,000 dilution,
whereas Helander et al. used theirs at a 1:30 dilution; their
CCKB receptor antibody was diluted 1:100, whereas ours, the
same antibody, was at 1:500.
Even though it was previously stated that no CCKB/gastrin
mRNA transcripts nor receptors were present on pancreatic islets (15, 52), our data clearly and convincingly
show the contrary in the rat, mouse, pig, and human pancreas. Indeed,
serial sections stained with specific CCKB/gastrin and
somatostatin antibodies reveal the presence of the
CCKB/gastrin receptor in the somatostatin -cells, in
agreement with a recent study performed in rabbit fundic mucosa with
specific CCK receptor agonists (41). This finding was
quite unexpected because the receptors were anticipated mostly on
acinar cells of the human and pig pancreas for secretion of their
enzyme content in response to CCK, whose receptors were described on
acinar cells either by autoradiography (49) or by
physiological secretory studies (36), respectively. As
indicated by Helander et al. (10), this absence of
immunofluorescence on pancreatic acinar cells does not exclude the
presence of the CCKB/gastrin receptors on these cells; it
has been postulated that the receptor density could be too low to allow
detection by the antibodies used. This hypothesis is plausible and is
supported by our data in the gastric fundus, in which labeling of the
CCKB receptor on the
-cells was much more intense than
on the parietal cells.
A constant low level of CCKB/gastrin receptor mRNA
undetected by Northern blot but present under RT-PCR may reflect a
transcription rate leading to the synthesis of a certain amount of
receptors that accumulate at the cell surface in the pancreas. When
this receptor type development is finished, a lower level of receptor mRNA would be required because it serves uniquely to maintain the
receptor number. Our data on failure to detect the
CCKB/gastrin receptor mRNA by Northern blot and on our
success by RT-PCR, combined with the constant concentration of
CCKB/gastrin receptor protein observed by Western blot
during pancreas development, support this hypothesis. The few data
indicating that the half-life for receptor proteins is much longer than
that of the corresponding mRNA also support this hypothesis
(29). Moreover, a recent study by Sanchez et al.
(45) indicated no progressive increase or decrease in the
pattern of gene expression of the four endocrine pancreatic hormones
during human fetal pancreas development. Furthermore, no increase in
immunostaining of the four hormones was observed and a dispersion of
endocrine cells within the exocrine tissue was noticed. It is highly
plausible that a similar phenomenon proceeds after birth involving the
CCKB/gastrin receptors present on the rat pancreatic
-cells.
If, as suggested by Helander et al. (10), the
CCKB/gastrin receptor present on pig and human pancreatic
acinar cells are undetectable by immunofluorescence because of their
low density, it would indicate in these two species that CCK via the
occupation of its CCKB/gastrin receptor can control the
secretion of the pancreatic enzymes from the acinar cells and that of
somatostatin from the endocrine -cells. In the rat and mouse
pancreas, however, the occupation of the CCKB/gastrin
receptors would be uniquely involved in somatostatin release because
the CCKA receptors were located on acinar and endocrine
-cells in these two species (1).
Assuming that the CCKB/gastrin receptors are absent from
the pig and human pancreatic acinar cells but present only on -cells as shown in this study, how can we then envisage the control of the
secretory effects of CCK on pancreatic enzyme secretion from pig
(36) and human (49) acinar cells? It was
recently reported that endogenous insulin is a major player regulating
the postprandial pancreatic enzyme secretion in rats (24)
and dogs (22). Indeed, immunoneutralization of circulating
insulin following a meal or the infusion of secretin-CCK totally
inhibited pancreatic enzyme secretion in response to these two stimuli,
accompanied by a significant increase in somatostatin in portal venous
effluent (23).
The following mechanism may operate to explain the control of
pancreatic enzyme secretion in pig and human pancreas in the absence of
any CCK receptor subtypes on their acinar cells. In response to a meal,
the CCK-immunoreactive nerve fibers demonstrated in the pancreas of
several species (20) would release CCK early, which in
turn can induce insulin secretion from the -cells through occupation
of the CCKA receptor subtype present on these cells (1, 14). Since this physiological step
involves the CCKA receptors with high affinity for CCK
within the picomolar range, CCK could come either from the gut and/or
the pancreatic nerve terminals. It is also expected that
intrapancreatic local concentrations of CCK released from the nerves
are much higher than in the plasma and reach the nanomolar range,
sufficient to occupy the less-sensitive CCKB/gastrin
receptors present on the somatostatin
-cells and release their
hormonal content (51). Levels of pancreatic nanomolar concentrations of CCK could be reached late in response to meal consumption, causing a delayed release of somatostatin involved in the
control of insulin (27) and enzyme (47) secretions.
Finally, in addition to its potential control of insulin and
somatostatin secretion via occupation of the CCKA and
CCKB receptors, respectively, CCK may also be important in
maintaining the pancreatic -cell population at a normal density.
Indeed, in CCKB/gastrin-deficient mice, the
-cell
population was reduced by 43% in the gastric antrum (18);
unfortunately, the pancreatic
-cell population was not looked at in
these animals.
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ACKNOWLEDGEMENTS |
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We acknowledge Christiane Ducharme for secretarial assistance.
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FOOTNOTES |
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This research was supported by grants from the Natural Sciences and Engineering Research Council of Canada (Grant GP6369) and le Ministère de l'Éducation du Québec (Grant ER1092).
Address for reprint requests and other correspondence: J. Morisset, Service de gastro-entérologie, Dép. de médecine, Faculté de Médecine-Université de Sherbrooke, Sherbrooke, QC, Canada J1H 5N4 (E-mail: jmori7{at}courrier.usherb.ca).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.
Received 15 November 1999; accepted in final form 24 February 2000.
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