Endocrinology Vol. 138, No. 12 5550-5554
Copyright © 1997 by The Endocrine Society
Distribution and Localization of a Novel Cholecystokinin-Releasing Factor in the Rat Gastrointestinal Tract1
Nadya Tarasova,
Alan W. Spannagel,
Gary M. Green,
Guillermo Gomez,
Jason T. Reed,
James C. Thompson,
Mark R. Hellmich,
Joseph R. Reeve, Jr.,
Rodger A. Liddle and
George H. Greeley, Jr.
Advanced Bioscience Laboratories-Basic Research Program (N.T.),
National Cancer Institute-Frederick Cancer Research and Development
Center, Frederick, Maryland 21702; Department of Physiology (A.W.S.,
G.M.G.), University of Texas Health Science Center, San Antonio, Texas
78284; Department of Surgery (G.G., J.T.R., J.C.T., M.R.H., G.H.G.),
The University of Texas Medical Branch, Galveston, Texas 77555; Center
for Ulcer Research Education: Center for Digestive Disease Research
(J.R.R.), Veterans Administration-West Los Angeles, Los Angeles,
California 90073; and Department of Medicine (R.A.L.), Duke University
Medical Center, Durham, North Carolina 27710
Address all correspondence and requests for reprints to: George H. Greeley, Jr., Ph.D., Department of Surgery, The University of Texas Medical Branch, Galveston, Texas 77555-0725. E-mail:
ggreeley{at}mspo2.med.utmb.edu
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Abstract
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The purpose of this study was to examine the distribution and
localization of an intestinal cholecystokinin (CCK)-releasing factor,
called luminal CCK-releasing factor (LCRF), in the gastrointestinal
tract and pancreas of the rat. RIA analysis indicates that LCRF
immunoreactivity is found throughout the gut including the pancreas,
stomach, duodenum, jejunum, ileum, and colon with the highest levels in
the small intestine. Immunohistochemistry analysis shows LCRF
immunoreactivity staining in intestinal villi, Brunner’s glands of the
duodenum, the duodenal myenteric plexus, gastric pits, pancreatic
ductules, and pancreatic islets. These results indicate potential
sources for secretagogue-stimulated release of luminal LCRF and support
the hypothesis that LCRF is secreted into the intestinal lumen to
stimulate CCK release from mucosal CCK cells.
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Introduction
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CHOLECYSTOKININ (CCK) is a major gut
hormone involved in the regulation of gallbladder contraction,
pancreatic secretion, gastric emptying, bowel motility, satiety, and
release of peptide YY from the lower intestine (1, 2). CCK is found in
mucosal enteroendocrine cells of the upper small intestine and is
secreted into the systemic circulation primarily by ingestion of fats
and proteins (1, 3, 4). The exact mechanisms controlling release of CCK
into the bloodstream are not clear.
In the rat and chicken, chronic dietary intake of raw soybean-based
meals results in an enlarged pancreas (5, 6). Additionally, oral
ingestion (7) or direct intraduodenal (ID) infusion of soybean trypsin
inhibitor elevates pancreatic exocrine secretion and plasma CCK levels
in rats (8). Dietary soybean trypsin inhibitor is thought to prevent
degradation of a luminal CCK-releasing factor by pancreatic proteases
(i.e. trypsin, chymotrypsin) (9, 10, 11). The partial structure
of this novel CCK-releasing factor has been described recently, and the
peptide is called luminal CCK-releasing factor (LCRF) (12). Amino acid
analysis and mass spectral analysis show that the purified peptide is
composed of 70–75 residues and has a mass of 8136 kDa. The N-terminal
41 residues have been sequenced. The N-terminal fragment of LCRF,
LCRF1–35, is biologically active (13). The purpose of this report is
to describe the distribution and localization of LCRF in the gut of the
rat using RIA and immunohistochemical techniques.
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Materials and Methods
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Animal tissues
Animal use for these studies was approved by The University of
Texas Medical Branch Institutional Animal Care and Use Committee. For
RIA analysis of LCRF-immunoreactivity (LCRF-IR) distribution, adult
(250 g) male Sprague-Dawley rats (n = 24) were killed after being
fed ad libitum. The stomach, duodenum, ileum, jejunum, and
colon were removed, the mucosal layer was separated from the muscle
layer, and both were homogenized immediately in water (1:10). The
pancreas was harvested and homogenized in 2 N
acetic acid. The kidney, abdominal muscle, spleen, and liver were also
extirpated and homogenized in water. Homogenates were boiled for 20 min
and then cooled on ice. Supernatants were prepared by centrifugation
and lyophilized. Dried extracts were resuspended in RIA buffer and
assayed at various dilutions.
LCRF antisera
These studies made use of polyclonal antisera generated against
synthetic fragments: LCRF1–10 (STFWAYQPDG [called
STFW]); Cys-LCRF5–23 (CAYQPDGDNDPTDYQKEHT [called
CAYQ]); LCRF7–23 (QPDGDNDPTDYQKEHT [called QPDG]); and
LCRF22–37 (HTSSPSQLLAPGDYPC [called HTSS]. LCRF
polyclonal antisera were raised using an immunization protocol
previously described with modifications (14). STFW was conjugated to
BSA; AYQP, QPDG, and HTSS were conjugated to keyhole limpet hemocyanin.
As expected, none of these LCRF antisera recognize other
gastrointestinal peptides or monitor peptide, another CCK-releasing
peptide (15, 16).
To demonstrate the neutralization properties of our LCRF antisera,
adult male rats were prepared with self-emptying blind loops of the
jejunum and a biliary pancreatic fistula (10, 17), which permitted
infusion of test secretagogues in the absence of bile-pancreatic juice
in the perfused segment (17). Bile-pancreatic juice is returned to the
duodenum; aliquots are collected to monitor pancreatic secretion.
Antisera were given intraluminally. In this model, peptone is a potent
stimulant for pancreatic exocrine secretion. Five percent peptone
solution (5% lactalbumin hydrolysate, pH 7.0), containing 23 µg/ml
of either anti-LCRF IgG (QPDG) or normal rabbit Ig (Quality Controlled
Biochemicals, Inc., Hopkinton, MA) was infused at 4 ml/h for 2 h
into the jejunum (n = 6–9 rats). The anti-LCRF IgG completely
abolished the pancreatic secretory response to intrajejunal infusion of
peptone, whereas normal rabbit IgG antiserum was ineffective (Fig. 1
).
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Figure 1. Anti-LCRF7–23 IgG abolishes the
pancreatic secretory response to intraduodenal peptone in rats.
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LCRF RIA
A RIA for LCRF-IR was developed and validated using a polyclonal
antiserum generated against a synthetic peptide, CAYQPDGDND PTDYQKYEHT
[Cys-LCRF5–23]; residues 5–23 of the LCRF molecule
conjugated to keyhole limpet hemocyanin through a cysteine residue
introduced at the zero position of the synthetic peptide. The
ID50 (50% inhibition of 125I-labeled ligand)
and sensitivity of the antiserum for synthetic LCRF1–35
were 0.20 ± 0.005 and 0.015 ± 0.005 ng/tube, respectively.
The antigen (Cys-LCRF5–23) was radioiodinated by the
chloramine T method and purified by reverse phase HPLC using an
ASAHIPAK ODP-C8P column (Keystone Scientific Inc., Belle
Fonte, PA). The LCRF antiserum detected, in a dose-dependent fashion,
LCRF-IR contained in a diluted extract of the rat duodenum and a
perfusate collected from an isolated rat jejunal Thiry-Vella fistula
(Fig. 2). The displacement curves for the
LCRF-IR contained in the extracts were parallel to the
LCRF1–35 standard line. In addition, LCRF-IR detected in
the luminal perfusate eluted on HPLC in the same position as the
original LCRF isolated and sequenced (Ref. 12 and data not shown). The
intra- and interassay variances are 5–8 and 10–12%,
respectively.
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Figure 2. LCRF RIA: Displacement curves of
LCRF1–35 standard and duodenal and intestine luminal
extracts containing LCRF-IR.
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LCRF-IR immunohistochemistry
For analysis of LCRF-IR localization by immunohistochemistry,
the Sprague-Dawley rat stomach, duodenum, pancreas, liver, and spleen
were cut in 0.2 x 0.5 x 0.5 mm pieces, fixed in 4%
formaldehyde in PBS for 6 h, dehydrated gradually in 70% and
100% ethanol, and paraffin embedded. Tissue blocks were cut into
4-µm sections. Deparaffinized, rehydrated sections were treated with
3% hydrogen peroxide for 5 min, blocked with 20% swine serum, and
incubated with respective antibodies at 1:500 dilution overnight at
room temperature, followed by biotinylated secondary antibodies (20
min), streptavidin conjugated to peroxidase (20 min) and aminoethyl
carbazole-H2O2 or diamino benzidine [all
reagents were from LSAB-2 kit (DAKO Corp., Carpinteria, CA)]. Three
different LCRF antisera were used in these studies; they were STFW,
QPDG, and HTSS. Washes were done with 0.02 M
Tris-HCl, pH 7.2, containing 0.1% Tween-20 and 0.3
M NaCl. Finally, the slides were counterstained
with Mayer’s hematoxylin and observed under Nikon Optiphot II light
microscope. For specificity studies, the various LCRF antisera were
immunoabsorbed with the corresponding peptides used for immunization.
LCRF fragments were added to the appropriate prediluted antisera to
give a final concentration of the peptide (50 µg/ml). Antisera
reacted with the LCRF fragments for 8 h before being used for
staining.
Perfusion of isolated jejunal Thiry-Vella loops and collection of
luminal perfusate containing LCRF-IR
Animal use for these studies was approved by The University of
Texas Health Science Center at San Antonio Institutional Animal Care
and Use Committee. Adult male Wistar rats were prepared with a 20- to
25-cm jejunal Thiry-Vella fistulas. During recovery from surgery (4
days) and between experimental collections, rats were allowed to eat
ad libitum, and the intestinal fistulas were perfused
(
2.5 ml/h) with a liquid diet [2.5% Vital (Ross Laboratories,
Columbus, OH), 2.5% peptone, 1.0% L-glutamine,
0.5% taurocholic acid]. Collection of intestinal perfusates for assay
of LCRF-IR was done 4 days after surgery. Rats were fasted for 4–5
h, and the intestinal fistulas were perfused with saline at 0.5 ml/min
for 30 min to remove debris. The intestinal fistulas were then perfused
with saline at 1.0 ml/min for 4 h, and the outflow was immediately
placed on ice. The collected perfusates were boiled, cooled on ice,
filtered (Whatman no. 4 filter paper), and concentrated using Waters
Classic C18 Sep-Paks (Millipore Corporation, Milford, MA).
Sep-Paks were activated with 100% ethanol (5 ml) and then washed with
0.1% acetic acid (10 ml). Intestinal perfusates (240 ml, pH adjusted
to 3.2) were loaded onto each Sep-Pak, followed by 0.1% acetic acid
(5 ml). LCRF-IR was eluted from each Sep-Pak with 75% ethanol in 0.1%
acetic acid (1.2 ml). Ethanol was evaporated from the eluates by means
of a Speed-Vac. The final volumes were then adjusted to 1 ml with
saline, and the pH of the material was adjusted to approximately pH
6–8 and stored at 4 C until assayed. Samples stored in this manner are
stable and give similar LCRF-IR values with repeat RIA
determinations.
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Results and Discussion
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RIA and immunohistochemistry findings demonstrate that LCRF-IR is
found in the pancreas, stomach, and intestine of the rat (Table 1 and Figs. 3, 4, and 5). LCRF-IR is detected in extracts of
the muscle and mucosal layers. LCRF-IR is also detected in the
intestinal lumen. By RIA, the highest concentrations of LCRF-IR were
found in the small intestine. Extracts of the duodenum and an
intestinal lumen perfusate gave dose-dependent inhibition curves that
were parallel to the synthetic LCRF standard, suggesting that the
immunoreactive substances detected in the extracts are similar or same
in identity to the LCRF standard (Fig. 2
). Extracts of other
gastrointestinal tissues also gave dose-dependent and parallel
displacement curves in the LCRF RIA (data not shown). LCRF-IR was below
the detection limit of the LCRF assay (0.015 ng/tube) in the liver,
kidney, spleen, and abdominal muscle extracts. Western blotting
analysis of a pancreatic extract with our various LCRF antisera has
identified an approximately 20-kDa immunoreactive protein (E. Kraig, K.
Clarkin, G. Green, D. Whitcomb, and G. Greeley, manuscript in
preparation).
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Figure 3. LCRF-IR staining in rat duodenum. A, HTSS
antibodies. Strong staining in the microvilli, Brunner’s glands, and
myenteric plexus and faint staining in the smooth muscle can be
observed. B, Staining with HTSS antibodies, preabsorbed with the
immunization peptide. C, Brunner’s glands, staining with QPDG
antibodies. D, The Meissner’s plexus with strongly positive ganglion
cells, QPDG antibodies. Original magnification: A and B, x50; C,
x200; and D, x400.
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Figure 4. LCRF-IR staining of jejunum. A, Villi, STFW
antibodies; B, smooth muscle and Meissner myenteric plexus, QPDG
antibodies. Original magnification: A, x200; B, x100.
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Figure 5. LCRF-IR staining in rat pancreas, QPDG antibodies.
A, Ductules; B, an islet. Original magnification, x100.
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Immunostaining for LCRF-IR localization in the pancreas and
gastrointestinal tract was performed with three different LCRF antisera
to different parts of the LCRF molecule (
Figs. 3–5). All the
antibodies showed the same pattern of staining. Preabsorption of the
antisera with immunization peptides abolished the staining, confirming
the specificity of detection (Fig. 3B). Immunohistochemistry revealed
LCRF-IR positive cells in the intestine, duodenum, and pancreas.
Gastric pits also showed the presence of LCRF-IR, but staining was a
lot less intense than in the intestine, which agrees with the RIA
findings. In the intestine, intense staining of villi and in the
myenteric plexus was observed (Figs. 3 and 4). Brunner’s glands of
duodenum were also strongly positive. The immunohistochemical technique
is exceptionally sensitive; therefore, it is conceivable, but unlikely,
that the localized LCRF-IR staining represents LCRF-IR sequestered by
LCRF receptors and LCRF-IR not produced in situ. The
abundant LCRF-IR localization in the Brunner’s glands of the duodenum
and intestinal villi suggests that these regions of the gut secrete
LCRF directly into the intestinal lumen to stimulate CCK release from
intestinal CCK-containing cells in a paracrine manner. Furthermore, the
detection of LCRF-IR by RIA (Fig. 2) in the lumen perfusate of an
isolated jejunal Thiry-Vella fistula indicates that the proximal small
intestine is a potential source of LCRF secretion. RIA analysis also
indicates the presence of LCRF-IR in the jejunum tissue. In the
pancreas, islets and ductules were positive for LCRF-IR, while acinar
cells were negative. By immunohistochemical examination, LCRF-IR was
also not detected in the liver and kidneys (data not shown). The
exocrine pancreas may also secrete LCRF into pancreatic juice that
eventually reaches the intestinal lumen, much like monitor peptide,
another CCK-releasing peptide (11, 15, 16). LCRF-IR contained in
pancreatic islets, such as pancreatic insulin and glucagon, may be
released into the general circulation.
It is worth pointing out that our LCRF antisera given intraluminally
abolishes the increase in pancreatic exocrine secretion seen in
response to infusion of peptone, a secretagogue for pancreatic and CCK
secretion (18). These results, when considered along with the
localization findings of LCRF in the gut and the measurement of LCRF-IR
in the intestine, indicate that our antisera are generated against an
authentic, endogenous luminal regulatory peptide that participates in
the physiologic stimulation of CCK secretion.
Interestingly, an earlier immunohistochemical report (19) indicates
that LCRF-IR was detected in the myenteric plexus and submucosal plexus
of the stomach and duodenum, and in extraintestinal nerve bundles,
particularly in the nodose ganglion. LCRF-IR staining was also
identified in nerve fibers throughout the pancreas. In this earlier
report, LCRF-IR was not detected in the Brunner’s gland or in
pancreatic ductules. This earlier report suggested that LCRF is
primarily a gut neuropeptide; however, the authors of this report did
not suggest a secretory pathway of neurally produced LCRF into the
intestinal lumen. The present studies failed to detect LCRF-IR in the
submucosal plexus or in pancreatic nerve fibers and found abundant
LCRF-IR in the Brunner’s glands and lesser amounts in the pancreatic
ductules. The differences in the results of the two studies may be a
result of a more sensitive technique that was used in the present
study.
There is evidence in man that the pancreatic proteases, trypsin and
chymotrypsin, may exert a negative feedback effect on their own
secretion (20, 21, 22). However, whether this negative feedback action on
the exocrine pancreas in man involves a protease-sensitive releasing
factor is not clear presently. Interestingly, multiple gut peptides
found in the gastrointestinal lumen have been described earlier (23).
We have termed these luminal gut peptide hormones "lumones." In
this report, RIA detection of LCRF-IR in the rat intestinal lumen is
the first example of a physiologic relevance for a luminal peptide
possibly derived from the intestinal mucosa, which stimulates CCK
secretion from mucosal CCK cells.
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Acknowledgments
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The authors wish to thank Karen Martin, Krista Lutz, Liz Cook,
Mary Lou Mraz, and Eileen Figueroa for their help with preparation of
the manuscript.
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Footnotes
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1 This research was sponsored in part by National Cancer Institute,
DHHS, by a contract with Advanced Bioscience Laboratories. The authors
were supported by funding from the NIH (DK-15241, DK-37482) and
National Science Foundation (IBN 9506305) to G.H.G. and J.C.T.; Texas
Higher Education (003659–069) and DK-37482 (to G.M.G.); and DK-38626
(to R.A.L.). 
Received May 30, 1997.
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Abstract
Introduction
