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Departments of Gastroenterology (K. Shim., K. Shir., S.W., N.H.) and Pathology (Y.K., M.K.), Tokyo Women’s Medical College, Tokyo 162, Japan; and Departments of Medicine (Y.S., T.-M.C., W.Y.C.) and Pediatrics (Y.D., R.F.), University of Rochester Medical Center, Rochester, New York 14642
Address all correspondence and requests for reprints to: Dr. Kyoko Shimizu, Tokyo Women’s Medical College, Department of Gastroenterology, 8-1 Kawada-cho, Shinjuku-Ku, Tokyo 162, Japan.
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Introduction |
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Materials and Methods |
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Isolation of intact islets
Intact islets of Langerhans were isolated from adult male Wistar
rats weighing 200–300 g according to the method of Lacy et
al. (24). After anesthetizing the animals with ip and im
injections of urethane (50% wt/vol, 0.5 ml/100 g BW), the distal end
of the pancreatic duct adjacent to the duodenum was clamped and a
polyethylene tube (PE-50, 0.58 mm ID) was inserted into the common bile
duct near the hilus of the liver. The abdominal aorta was cannulated
with a polyethylene tube (PE-50, 0.58 mm ID) below the renal arteries
and the portal vein was sectioned. A 0.15 M NaCl solution
was infused into the aorta to flush the blood out of the pancreas.
Collagenase solution (50 mg of collagenase type V (Worthington
Biochemical Corporation, Freehold, NJ) dissolved in 5 ml of HBSS was
injected into the common bile duct. Approximately 10 min after the
injection of collagenase, the whole pancreas was carefully removed and
placed in a test tube containing the collagenase solution described
above. The tube was shaken vigorously by a hand in a 37°C water bath
for 5 min until no tissue pieces were visible on the wall of the tube,
and the digested pancreas was then washed three times with HBSS. The
sediment containing the islets was suspended in 30% (wt/wt) Ficoll
(Sigma Chemical Co., St. Louis, MO). This was layered over a
discontinuous Ficoll gradient consisting of 4 ml each of 20%, 23%,
and 27% Ficoll in HBSS (wt/wt). After centrifugation of the
discontinuous gradients at 700 x g for 10 min, the
islets were withdrawn from the interface between the 20% and 23%
Ficoll layer, fixed in 4% paraformaldehyde overnight, and embedded in
paraffin for in situ hybridization.
Immunohistochemical study
Adjacent pairs of the sections were mounted on separate slides
as the mirror images for colocalization studies of CCK and insulin,
glucagon, somatostatin, and pancreatic polypeptide (PP). The
immunohistochemical protocol used was basically the avidin-biotin
complex (ABC) method with reagents provided by Vector Laboratories,
Inc. (Burlingame, CA). Anti-CCK-8 antiserum was raised with BSA-CCK-8
conjugate and had a final titer of 1:1,000,000. The antiserum has the
following relative reactivity toward various CCK/gastrin family
peptides: CCK-8, 100%; unsulfated CCK-8, 50%; porcine CCK-33, 33%;
canine CCK-58, 20%; CCK-27-33 (CCK-7), 50%; CCK-28-33 (CCK-6), 2.5%;
CCK-4, <0.1%; human gastrin-17-I, 0.3%. The antiserum does not
cross-react with other islet peptides including insulin, glucagon, PP,
and somatostatin at concentrations as high as 10 µM. The
sharp decrease in cross-reaction with unsulfated CCK-8, CCK-6, and
CCK-4 indicated that the antibody reacts predominantly with an epitope
involving the N-terminal region of CCK-8 that contains the sulfated
tyrosine residue. Anti-CCK 1–22 was raised against the N-terminal
1–22 fragment of porcine CCK-33 to a titer of 1:250,000 and reacts
exclusively toward porcine CCK-33. Primary antisera included rabbit
anti-CCK-8 and anti-N-terminal CCK-33 (CCK 1–22) at 1:1000 dilution;
rabbit antihuman gastrin-17, rabbit antiporcine insulin, rabbit
antihuman gastrin-17, rabbit antiporcine insulin, rabbit antiporcine
glucagon, rabbit antihuman somatostatin, and rabbit antihuman PP at
1:100 dilution (Vector). After incubation with the antiserum at 4 C
overnight, a biotinated goat antirabbit serum IgG (Vector) was used as
the second antibody. Peroxidase staining was performed with
3,3'-diaminobenzidine (DAB).
In order to clarify the localization of CCK in the islets, double immunohistochemical staining was performed with ABC method using CCK antiserum followed by horseradish peroxidase-antiperoxidase (PAP) method using insulin, glucagon, somatostatin, PP, or gastrin antisera. The primary antibodies used were described above. Immunoperoxidase staining for CCK was detected by ABC method using aminoethylcarbazole (AEC). After the observation, these slides were treated in 0.01 M citrate buffer (pH 6.0) at 90–95 C for 10 min before the second immunoperoxidase staining for insulin, glucagon, somatostatin, PP, and gastrin. The localization of these endocrine hormones were detected by PAP method using DAB.
The absorption studies used CCK-8 antiserum absorbed with 10 µM CCK-8 or 100 µM gastrin (Sigma Chemical Company). After preincubation of the CCK-8 antiserum with CCK-8 or gastrin overnight, the supernatant was used as the primary antiserum.
HPLC of rat pancreas extracts
Pancreata were quickly removed from eight anesthetized rats and
immediately boiled for 10 min. They were then homogenized in a Polytron
tissue homogenizer, and the pH of the homogenate was adjusted to 9.2
with aqueous ammonia. 2-mercapto-ethanol was added to a final
concentration of 0.05%, and stirring was performed for 2 hours at 4 C.
After a lipid layer had been separated out by centrifugation at
12,000 x g at 4 C and removed, the supernatant was
lyophilized and extracted with a C18 Sep-Pak cartridge. The molecular
heterogeneity of the CCK-LI in the C18 Sep-Pak extract was analyzed by
reverse-phase HPLC on a Varian MCH10 C18 analytical column (4.6 x
300 mm), as described previously (25).
RT-PCR
Total RNA was extracted from the isolated islets and the
duodenum by the method of Chomczynski (26) and quantified by measuring
the absorbance at 260 nm. The RT reaction was carried out in a 20 µl
volume, containing 4 µl of 5 x RT buffer, 1 µl of
deoxyribonucleotide triphosphate (dNTP, 25 mM each), 100
pmol of random hexamer, 1 U/µg of RNase inhibitor, 1 µg of total
RNA, and 200 U Moloney murine leukemia virus (M-MuLV) reverse
transcriptase. The reaction mixture was incubated at 37 C for 60 min,
heated to 95 C for 10 min and then quickly chilled on ice. One tenth of
the resulting complementary DNA (cDNA) was used as a template for the
PCR. PCR was done in a final volume of 20 µl, containing 3
mM MgCl2, 50 µM of each dNTP, 50
pmol each of the up and downstream primers, and 2 U Thermus Aquatics
(Taq) DNA polymerase. Oligonucleotide primers were designed
on the basis of the DNA sequence of rat CCK. Amplification of cDNA with
primers located in exon 2 and exon 3 of the gene expected to yield a
specific PCR product of 350 bp. The sequences of the upstream primers
was 5'-CAAGATCTATGAAGTGCGGCGTGT-3', and the downstream primer was
5'-GGCGGATCCACTACGATGGGTA-3'. A thermal cycle program of 40 cycles of
45 sec at 94 C, 45 sec at 60 C, and 2 min at 72 C was employed. The
products were separated by electrophoresis on a 2% agarose gel and
visualized with ethidium bromide.
Restriction digestion by endonuclease
The amplified PCR products from islet and duodenum were purified
by Magic PCR Prep (Promega, Madison, MI) to remove the excess primers.
The purified PCR products were digested by HAE III at 37 C for 1
h. The digested PCR products were electrophoresed on a 2% agarose gel
containing ethidium bromide.
Southern blot analysis
PCR amplified products from islets and duodenum were
electrophoresed on a 2% agarose gel containing ethidium bromide. The
agarose gel was denatured in 0.5 N NaOH and 1.5
M NaCl, and was neutralization in 1.0 M
Tris-HCl, pH 8.0, 1.5 M NaCl for 60 min at room temperature
with gentle agitation. Then the DNA was transferred to a nylon
membrane. The membrane was prehybridized for 2 h at 65 C in
standard prehybridization solution. A CCK specific cDNA was labeled
with digoxigenin (DIG)-11-dUTP (Boehringer Mannheim
Corporation, Indianapolis, IN) using the random primer method.
Hybridization with DIG-labeled CCK cDNA probe was carried out in
the standard prehybridization solution at 65 C overnight. The membrane
was washed twice with 2 x SSC containing 0.1% SDS at 65 C. These
hybridized probe was immunodetected with an alkaline
phosphatase-conjugated antidigoxigenin antibody and visualized
colorimetrically using NTB and X-phosphate substrate.
In situ hybridization
An EcoR1-Hind III restriction fragment of
the plasmid containing the cDNA encoding rat preprocholecystokinin was
labeled by random primed DNA labeling with digoxigenin dUTP using a
Genius DNA labeling kit (Boehringer Mannheim). The slides were immersed
in 0.2 N HCl at room temperature for 15 min and transferred
into 2 x SSC at 70 C for 30 min. The sections were then digested
in proteinase K solution at a concentration of 10 µg/ml in 20
mM Tris-HCl (pH 7.4) at 37 C for 60 min. After digestion,
the sections were washed in PBS and fixed with 4% paraformaldehyde in
PBS at room temperature for 5 min. Following fixation, the sections
were washed in PBS and dehydrated in 100% ethanol and air dried, and
then immersed in 0.25% acetic anhydride containing 0.1 M
triethanolamine at room temperature for 10 min. As a negative control,
RNase (1 mg/ml) pretreatment was carried out at this stage. The
sections were washed in 2 x SSC and then in diethylpyrocarbonate
(DEPC)-treated water, and after dehydration in 100% ethanol and
air-drying, they were incubated at 42 C for 2 h in hybridization
buffer consisting of 50% formamide, 5 x SSPE (900 mM
NaCl, 50 mM NaH2PO4, 5
mM EDTA, pH 7.4), 5 x Denhardt’s solution, 0.1%
SDS, and sonicated salmon sperm DNA. Plasmid containing the cDNAs
encoding rat preprocholecystokinin (Dr. J. Dixon, University of
Michigan) (27), preprogastrin (28), and preproinsulin were labeled with
digoxigenin (DIG)-11-dUTP by the PCR method. For hybridization, each
digoxigenin-labeled cDNA probe was added to hybridization buffer
containing 50% dextran sulfate, and the sections were then incubated
at 42 C overnight. As a negative control, unlabeled CCK cDNA was
hybridized overnight before the hybridization with labeled CCK probe.
Following hybridization, the sections were washed in 2 x SSC at
room temperature for 60 min, then in 50% formamide containing 1
x SSC at 45 C for 20 min, and briefly in 2 x SSC at room
temperature. Color detection was performed according to the standard
procedure for the nonradioactive DNA detection kit (Boehringer
Mannheim). Briefly, the sections were immersed in 100 mM
Tris-HCl (pH 7.5) and 15 mM NaCl, and incubated with
blocking solution consisting of 2% normal sheep serum, 0.3% Triton
X-100, 100 mM Tris-HCl (pH 7.5), and 15 mM NaCl
at room temperature for 30 min. They were then incubated with the
antidigoxigenin Fab fragment conjugated to alkaline phosphatase at 4 C
overnight. After incubation, the sections were washed in 100
mM Tris-HCl (pH 7.5), 150 mM NaCl followed by
100 mM Tris-HCl (pH 9.5), 100 mM NaCl, and 50
mM MgCl2. The antidigoxigenin alkaline
phosphatase activity was detected with X-phosphate and NTB.
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). Neither CCK-containing nerve fibers
nor nerve cell bodies were observed in the rat pancreas.
CCK-LI-positive cells had round nuclei and cytoplasm filled with
granules of uniform size. Based on the number and pattern of
distribution of these cells, they appeared most likely to be
insulin-containing B cells. Glucagon-LI cells (A cells) were situated
in the periphery (mantle area) of the islets. Somatostatin cells (D
cells) were seldom encountered and were interposed between the
insulin and glucagon cells. PP-containing cells were distinct from
the A, B, and D cells and were located in the periphery of the islets
and scattered among the exocrine cells. The mirror sections of rat
pancreas were processed with CCK-8 and the other endocrine hormone
antisera to confirm the identity of CCK-LI and the other endocrine
hormones. Sections immunostained for insulin revealed positive cells
with characteristics identical to those that stained positive for CCK-8
(Fig. 1A-INS, 1A-CCK). The other cells containing glucagon (Fig. 1B-GLU, 1B-CCK), somatostatin (Fig. 1C-SOM, 1C-CCK), and PP (Fig. 1D-PP, 1D-CCK) were completely different from the CCK-cells. No
positive staining was observed in the islets with either antiserum to
N-terminal CCK-33 (CCK 1–22) or gastrin-17 antiserum. The double
staining studies also confirmed that CCK-LI was colocalized with
insulin (Fig. 2A-INS, 2A-CCK) but not
with other endocrine peptides including glucagon (Fig. 2B-GLU, 2B-CCK),
somatostatin (Fig. 2C-SOM, 2C-CCK), PP (Fig. 2D-PP, 2D-CCK), and
gastrin (Fig. 2E-GAS, 2E-CCK). The CCK-LI in the islets was completely
abolished by preabsorption of the CCK antiserum with CCK-8 but not with
gastrin (Fig. 3).
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). The mean CCK-8
concentration in the whole pancreas was 463 ± 59 pmol/g wet
tissue (n = 6).
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). Restriction digestion pattern of PCR
fragments from these two sources were identical (Fig. 5B). To further
confirm that the PCR product was derived from CCK messenger RNA (mRNA),
the amplified cDNA was transferred to nylon membrane and hybridized
with a digoxigenin labeled CCK-specific cDNA probe. The CCK cDNA probe
identified a single DNA of 350 bp in PCR products from islets and
duodenum by Southern blot analysis (Fig. 5C).
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, A and B). As a positive control,
insulin mRNA was seen diffusely in the center of the islet (Fig. 6, C
and D). There was no gastrin mRNA signals in the islet (Fig. 6E). As a
negative control, the prehybridization with unlabeled CCK cDNA before
hybridization with the CCK probe completely abolished the hybridization
signal (Fig. 6F).
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Discussion |
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In the present study, CCK-LI was detected in the central portion of the islets by immunohistochemical studies. Analysis of mirror section and double staining clearly demonstrated that CCK and insulin immunoreactivity coexisted in the same cells. Glucagon, somatostatin, and PP cells, on the other hand, were located in the periphery of the islets. There was no gastrin immunoreactivity in the whole islets. CCK antiserum saturated with CCK showed no immunoreactivity in the immunohistochemical study, and CCK antiserum incubated with rat gastrin failed to abolish CCK-LI in the islets. Cross-reactivity of CCK antiserum with the related peptide gastrin makes it difficult to segregate the CCK-LI from gastrin-LI. CCK-8 is partially homologous with gastrin, with the two sharing a C-terminal pentapeptide (Gly-Trp-Met-Asp-Phe-NH2). To distinguish the CCK-LI in the islets as CCK-8 rather than gastrin, we used three different rabbit antisera: antiserum against CCK-8, N-terminal CCK-33 (CCK1–22), and gastrin-17. Strongly positive staining was observed only with the anti-CCK-8 serum, indicating that the CCK-LI in the islets most likely represented CCK-8. It has been previously reported that gastrin is maximally expressed in the islets during late fetal gestation and rapidly disappears during the first week after birth (2, 3). This also supports our observations. Due to the low cross-reactivity toward CCK-4, we were unable to detect neurons or nerve fibers in the pancreas containing this form of CCK (13) using our CCK-8 antiserum.
Moreover, HPLC analysis of the pancreatic extracts failed to detect gastrin. In the HPLC analysis of pancreatic extracts, standard CCK-8 (5 ng), pCCK-33 (10 ng) and cCCK-58 (20 ng) were run though the same column individually and in combination under the same chromatographic conditions and then detected by RIA to obtain their retention times. Both CCK-7 (20 ng) and CCK-6 (100 ng) exhibited a retention time of 28 min when run individually. Therefore, except for the peak eluted at 10 min, which had the same retention time as that of oxidized CCK-8 (prepared by oxidation of CCK-8 with dithionate), we were unable to identify the other forms of CCK-LI in the pancreatic extract that were eluted at 24 min or earlier. When applied at 100 ng, CCK-4 exhibited a very small immunoreactive peak eluted at 34 min and thus could represent the small peak eluted after but not well resolved from CCK-8 that was occasionally found in the pancreatic extract. Because CCK-4 was found in the islet endocrine cells only in feline pancreas (13), it was possible this peak represent CCK-4 in the pancreatic neurons of rat pancreas. Due to the low yield of isolated islet, we did not study the molecular form of CCK-LI in the endocrine pancreas. However, the results of immunohistochemical study indicated that only the islet endocrine cells contained CCK-LI recognizable with our antiserum, it is logical to assume that the form of CCK-LI detected in the pancreatic extract represented those detected in the endocrine cells. However, we cannot rule out the presence of CCK-4 containing nerve fibers in the islet as observed by Rehfeld et al. (13) in other species.
Examination of the rat pancreas by in situ hybridization and RT-PCR revealed that the CCK gene was expressed in the islets of adult rats. The CCK gene was expressed in the islets, corresponding to those observed with mRNA from the duodenal mucosa by RT-PCR. The restriction digestion pattern and Southern blot analysis of the PCR amplified cDNA from the islet and the duodenum strongly suggest that this is the product of authentic CCK mRNA.
The CCK mRNA was observed diffusely in the islet by in situ hybridization as well as CCK-LI detected by immunohistochemical studies. Insulin mRNA was also clearly demonstrated in the center of the islet; on the other hand, there was no gastrin mRNA in the whole islet by in situ hybridization. The preabsorption study with unlabeled CCK cDNA before the hybridization with labeled CCK cDNA probe confirmed the existence of CCK mRNA in the islet. These results suggest that the cells expressing the CCK gene are also able to translate CCK mRNA.
Madsen et al. (22) reported a cloned cell line from a transplantable radiation-induced islet cell tumor that coexpressed CCK and glucagon, and CCK mRNA was revealed by Northern blot analysis in a human glucagonoma but not in insulinoma or normal pancreatic tissue. Grube (21) et al. also demonstrated colocalization of CCK-LI with glucagon in A-cells in adult rat and human pancreatic islets. These observations suggest that CCK is synthesized in A cells, but that is inconsistent with our own findings. Although CCK-LI was found in the intrapancreatic nerve fibers or A cells in the pancreas, it has never been reported the presence of CCK in B cells. The discrepancy between our observation and the previous other studies might depend on the species, age, and antibody used. In the present study, neither CCK-containing nerve fiber nor ganglia was found in the rat pancreas. Also, the characteristics of islet cell tumors may be different from that of normal tissue.
Pancreatic endocrine cells appear to arise from the multipotential stem cells. Several gastrointestinal hormones, such as gastrin (1, 2, 3), secretin (4), PYY (5, 6), and TRH (7), have been reported to be present in the pancreatic islets during the developmental period. These gastrointestinal hormones, including glucagon, are maximally expressed during late gestational period and rapidly disappear after birth. This suggests that gene expression of the gastrointestinal hormones in pancreatic islets are developmentally regulated and may influence pancreatic differentiation and growth. On the other hand, a large portion of the islets expressed both CCK mRNA and CCK immunoreactivity as well as insulin in the adult, suggesting that CCK may contribute to pancreatic growth or physiological regulation. In single-pass, isolated vascularly perfused rat pancreas with the duodenum excluded, the CCK-A antagonist L-364718 was shown to inhibit the flow of pancreatic juice and amylase output in response to vagal stimulation (15). This suggested that the inhibition is attributable to local release of CCK in the pancreas. CCK/gastrin immunoreactive nerves innervated both ganglionic cells and glucagon cells in the islets of the hamster, porcine, and feline pancreas (13, 16, 17). These nerve were found to be extrinsic (13) and thus could not be the source of CCK mRNA. Further, more recent study suggested that CCK immunoreactive ganglion cells were found in the feline pancreas (18). These observation indicated that CCK may act as a neurotransmitter in the pancreas. On the other hand, we could identify CCK-8 immunoreactivity and its transcript in B cells but neither in the nerve terminals nor in the ganglia in the rat pancreas. This observation suggests CCK is expressed and synthesized in the islet B cells that may have a distinct function from the extrinsic CCK-containing nerves found in other species.
Morphological studies indicate that numerous efferent vessels from the islets reach the acinar and duct cells, and thus that the exocrine pancreas receives significant blood flow from the islet-acinar portal system in the horse (29), rat (30), human (31), and monkey (32, 33), and local delivery of islet hormones via islet-acinar portal vessels may stimulate pancreatic exocrine secretion. The previous studies reported that a peptide corresponding to the C-terminal tetrapeptide amide (CCK-4) of CCK/gastrin was present in the nerve terminals in the hamster, pig, and cat (13, 16, 17, 18) and camel (19), but not in the snake (20). While CCK-4 in the nerve is thought to be an insulin releaser in isolated perfused porcine pancreas (13), others have reported that CCK-8 or CCK with longer amino acid residues are more potent stimulant of insulin release in the perfused rat pancreas (12). It is possible that CCK-8 in the islets may be involved not only in the release of insulin but also in the stimulation of exocrine pancreatic secretion in the rat.
In conclusion, we have presented evidence that the CCK gene is expressed in rat pancreatic islets and that CCK-LI is colocalized with insulin, indicating that CCK is synthesized in the islets of Langerhans. The main molecular form of CCK-LI in the pancreas is CCK-8. While the physiological role of CCK in the islets has not yet been characterized, CCK in the islets may play an important role in pancreatic exocrine and endocrine secretion.
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Acknowledgments |
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Footnotes |
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Received April 25, 1997.
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