1 Department of Clinical
Physiology, The changes in levels of the newly discovered
luminal CCK-releasing factor (LCRF) in the small intestinal lumen
before and after bile-pancreatic juice diversion in conscious rats were
examined by a specific RIA. Moreover, we also examined
whether LCRF secretion was under cholinergic control. Anti-LCRF
antiserum was raised in rabbits, and a sensitive RIA was established.
The localization of LCRF was examined by immunohistochemistry. The
luminal content of LCRF was significantly increased by bile-pancreatic
juice diversion, during which luminal trypsin activity was eliminated.
The increase in luminal LCRF content was not inhibited by intravenous
infusion of atropine. The changes in plasma levels of CCK and
pancreatic secretion were similar to those in luminal LCRF contents.
LCRF immunostaining was observed in villus tip enterocytes of the small intestine and was most prominent in the duodenal portion. These results
support our original hypothesis that LCRF may be released spontaneously
into the small intestinal lumen from the villus tip enterocytes and its
intraluminal degradation by proteases regulates CCK release.
Furthermore, LCRF release was not subject to cholinergic regulation.
luminal CCK-releasing factor; CCK-releasing peptide; luminal
feedback
PANCREATIC EXOCRINE secretion in conscious rats is
mainly regulated by protease activities in the proximal intestine
(luminal feedback regulation) (3); intraduodenal proteases are
inhibitory, while ingestion of protease inhibitors or diversion of
bile-pancreatic juice stimulates pancreatic enzyme secretion. This
increase is mediated by release of CCK (1, 10, 20). Moreover, we
reported (20) that exclusion of both bile and pancreatic juice is a
strong stimulant of CCK release and pancreatic protein secretion in
conscious rats.
To explain the mechanism by which intestinal trypsin inhibits
pancreatic secretion and CCK release, we hypothesized the presence of a
trypsin-sensitive polypeptide [CCK-releasing peptide
(CCK-RP)], which is considered to be spontaneously secreted by
the small intestine into the intestinal lumen. This CCK-RP was
hypothesized to stimulate CCK release and pancreatic secretion
subsequent to its secretion into the lumen (14). This hypothesis was
supported by our subsequent study (16) and a report by Lu et al. (11). Recently, Spannagel et al. (22) purified CCK-RP from rat intestinal perfusate and named it luminal CCK-releasing factor (LCRF). This peptide is composed of 70- to 75-amino-acid residues and has a molecular mass of 8,136 Da, and the
NH2-terminal 41 residues were determined.
We synthesized the LCRF
NH2-terminal
fragment-(1 LCRF Antiserum
ABSTRACT
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
35) and examined its stimulatory effect on
pancreatic exocrine secretion in conscious rats (12). We
confirmed that intraduodenal injection of this LCRF-fragment rapidly
increased pancreatic fluid and protein secretion (12). In the present
study, we established a specific RIA for LCRF to clarify its
physiological role. Using this method, we examined whether LCRF was
secreted into the lumen. LCRF immunoreactivity in the duodenal mucosa
was also measured, and the localization of LCRF was determined by
immunohistochemistry. Whether a cholinergic mechanism is involved in
luminal feedback regulation is controversial. Guan et al. (4, 5)
observed no contribution of a cholinergic mechanism to pancreatic
hypersecretion produced by bile-pancreatic juice diversion, whereas Lu
et al. (11) reported that the release of CCK-RP was inhibited by
atropine. Therefore, we examined whether the secretion of LCRF into the
lumen was inhibited by atropine.
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials and methods
Results
Discussion
References
35) was conjugated with keyhole-limpet
hemocyanin using 3-maleimidobenzoic acid
N-hydroxy-succinimide ester, according
to the previously described procedure (12). Rabbits were
immunized with conjugate (100 µg as peptide) emulsified in complete
Freund's adjuvant (6, 25). Booster injections of the same dose of
conjugate in incomplete Freund's adjuvant were administered at 2-wk
intervals beginning 3 wk after the first immunization. The antiserum
used for this study was obtained 10 days after the eighth immunization.
Animal Preparation
Male Wistar rats (330-346 g body wt) were obtained from Shizuoka Jikkenn Dobutsu (Shizuoka, Japan). Animals were fed commercial rat chow (CRF-1, Oriental, Tokyo, Japan) before surgery and during recovery.The operating procedures have been described in detail previously (17, 19). Briefly, a midline abdominal incision was made under enflurane anesthesia (Abbott, North Chicago, IL) delivered through a plastic face mask by means of a vaporizer. The cannulas used in this study were Silastic medical grade tubing (Dow-Corning, Midland, MI; ID, 0.025 in.; OD, 0.037 in.). Separate cannulas were introduced for draining bile and pancreatic juice along with a duodenal cannula and a right external jugular vein cannula. After the operation, the rats were placed in modified Bollman-type restraint cages and had free access to food and water in a room at 24°C with filtered air and light from 0500 through 1700. Bile and pancreatic juice were returned continuously to the intestine via the duodenal cannula.
Experimental Design
Bile and pancreatic juice were collected separately for a 30-min period, and the volume of pancreatic juice was measured with a Hamilton syringe. Samples of 20 µl of pancreatic juice were used to determine protein concentration, and the rest was mixed with the bile and infused into the duodenum with a syringe pump (compact infusion pump, Harvard Apparatus, Southnatick, MA) over the next 30 min. Bile and pancreatic juice were diverted from the intestine after a basal collection period of 90 min with return of bile and pancreatic juice, during which 0.05 M NaHCO3 solution was infused at a rate of 1 ml/h for 4 h. The animals were killed before bile-pancreatic juice diversion and 2 or 4 h after the diversion. Samples of 5-6 ml of blood were withdrawn through the venous cannula, and the animals were killed. The proximal quarter of the small intestine was removed and washed with 10 ml of ice-chilled distilled water. The intestinal contents were immediately frozen and lyophilized for subsequent LCRF RIA. Blood samples were collected in ice-chilled EDTA-containing tubes and immediately centrifuged at 4°C at 3,000 rpm for 15 min. The plasma samples were stored atTo examine the effects of atropine, we started intravenous infusion of
100 µg · kg1 · h
1
of atropine 60 min before bile-pancreatic juice diversion, as previously reported (13). The changes in protein secretion, plasma CCK
levels, and LCRF contents were examined.
Protein concentration in pancreatic juice was determined by measuring the optical density at 280 nm (9) of samples diluted 200-fold with 0.04 M Tris buffer, pH 7.8.
Immunohistochemistry
Three segments of small intestinal tissues (2 cm length) at 10, 45, and 70 cm from the pylorus were removed, fixed in 10% Formalin, and embedded in paraffin. Sections 5-µm thick were incubated with antiserum to LCRF-(1Assays
RIA of LCRF.
125I-labeled LCRF-(135) was
prepared by the chloramine-T method and purified by Sephadex G-10
chromatography and diethylaminoethyl-ion exchange chromatography
(eluted with a gradient of 0-1 M NaCl in 0.01 M imidazole buffer,
pH 7.5). Standard LCRF-(1
35) and samples were incubated with
antiserum (R601; final dilution, 1:40,000) for 48 h at 4°C in a
total volume of 500 µl of 0.01 M phosphate buffer (pH 7.4) containing
0.5% BSA, 0.025 M EDTA, 0.14 M NaCl, 0.05% (vol/vol) Tween 20, and
0.01% sodium azide. Next, 0.1 µl of tracer (
6,000 cpm) was
added, and incubation was continued for 48 h at 4°C. The bound and
unbound peptides were separated by adding goat-anti-rabbit IgG.
Synthetic LCRF-(1
35) for use as a standard was prepared according to
the net peptide content determined by amino acid analysis.
|
Preparation and extraction of basal intestinal wash, intestinal mucosal tissues, and plasma samples. The samples of intestinal wash obtained before bile-pancreatic juice diversion (basal) contained high protease activities. As proteases digested antibody in the RIA system resulting in high values, proteases had to be excluded immediately. The intestinal wash (10 ml of ice cold water) was boiled at 96-100°C for 15 min, followed by overnight dialysis (using the membrane, cutoff level mol wt 1,000) at 4°C, centrifuged at 3,000 rpm at 4°C, and then lyophilized. We examined whether this extraction procedure affected the LCRF radioimmunoreactivity itself by comparing the results of samples obtained after 2 h of bile-pancreatic juice diversion with or without extraction. This extraction procedure did not significantly affect LCRF radioimmunoactivity (t = 1.52, P > 0.15, n = 5 and 7 for with and without extraction, respectively). Therefore, samples obtained 2 and 4 h after bile-pancreatic juice diversion were directly assayed without extraction.
To measure tissue content of LCRF, we killed an additional four rats by decapitation, and the proximal small intestine (20 cm) was removed, washed with 10 ml of cold distilled water, and opened longitudinally and the intestinal mucosa was removed by gently scraping the surface with a spatula. LCRF was extracted from the intestinal mucosa. Briefly, samples were homogenized with ice-cold distilled water for 1 min and centrifuged at 12,000 rpm at 4°C for 10 min. The supernatants were used for LCRF measurement. Plasma samples of 2-3 ml were obtained from each rat, and two 3-ml or three 2-ml plasma samples from different rats were combined into one 6-ml sample for LCRF measurement. LCRF was extracted from plasma samples by adsorption onto C-18 (Mega Bond Elut C18 Varian Sample Preparation Products) (24). The recovery of LCRF-(1RIA of CCK. Plasma CCK extracted by adsorption on Sep-Pak cartridges (Waters, MA) was measured by RIA as described previously (2, 6, 19, 24, 25), using antiserum OAL-656 and CCK-8 as a standard.
Statistics
Values are expressed as means ± SE. To analyze the results of pancreatic secretion, we performed multiple ANOVA followed by Newman-Keuls multiple comparison test with respect to time and treatment. Increases in protein secretion with or without atropine treatment were compared by Student's t-test. P < 0.05 was considered to be significant. ![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Changes in Protein Secretion, Plasma LCRF and CCK Levels, and LCRF Contents in Rats Without Atropine
The exclusion of bile-pancreatic juice significantly increased pancreatic fluid and protein secretion as previously reported (19, 20). The results of protein secretion are shown in Fig. 2. The protein secretion peaked at 1-2 h after diversion and decreased thereafter, then remained at a level twofold higher than the basal value.
|
Plasma CCK levels also significantly increased and peaked 2 h after
bile-pancreatic juice diversion, decreasing slightly thereafter (Fig.
3A). The
luminal LCRF contents also increased significantly after
bile-pancreatic juice diversion, and the changes were similar to those
in CCK levels (Fig. 3B). The plasma
LCRF levels were very low (4.0-5.4 fmol/ml) and were not affected
by bile-pancreatic juice diversion.
|
Effects of Atropine
Intravenous infusion of atropine significantly decreased basal (bile-pancreatic juice return) protein secretion. However, the secretion in response to bile-pancreatic juice diversion was markedly increased, as previously reported (13) (Fig. 2). The integrated increase in protein secretion during the 4 h of bile-pancreatic juice diversion [(the value of protein secretion during 4 h of bile-pancreatic juice diversion minus protein secretion during 0.5 h before bile-pancreatic juice diversion) × 8] was 68.3 ± 7.5 mg (n = 8) in rats without atropine vs. 82.8 ± 17.5 mg (n = 5) in rats with atropine. The difference was not significant (t = 1.27, P > 0.2).The plasma CCK levels and luminal LCRF contents were also increased significantly by bile-pancreatic juice diversion in rats treated with atropine (Fig. 3). There was no significant difference between rats with and without atropine treatment.
Immunohistochemistry and Tissue Contents of LCRF
LCRF immunoreactivity was strongest in enterocytes at the tips of villi of the proximal small intestine (Fig. 4A). Although luminal mucus strands contained LCRF immunoreactivity, proximal enterocytes, goblet cells, and enteroendocrine cells were negative for LCRF immunoreactivity (Fig. 4B).
|
Tissue contents of LCRF in the small intestine were 418.4 ± 78.8 fmol/g wet wt (n = 4).
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
We raised antiserum specific to LCRF, established a specific RIA, and measured luminal LCRF content before and after bile-pancreatic juice diversion in rats. Furthermore, we examined the localization of LCRF in the small intestine by immunostaining and quantified its immunoreactivity in the small intestine. LCRF immunoreactivity in the small intestinal lumen increased after bile-pancreatic juice diversion in conscious rats. LCRF content peaked during the 2 h after bile-pancreatic juice diversion, and these changes paralleled those in plasma CCK levels. These changes also paralleled the protein output (19, 20), although the plasma LCRF levels were very low and did not change during bile-pancreatic juice diversion.
LCRF immunostaining was observed in the villus tips of the small intestinal mucosa but not in enteroendocrine cells, although we could not verify the origin of this LCRF. Moreover, the staining was stronger in the proximal than the distal intestine. This evidence is in accordance with the previous report (21) that luminal feedback regulation was only operative in the proximal small intestine. Taken together, these observations suggested that LCRF is secreted from the proximal small intestine to the lumen, but not to the circulation, and has a physiological role in CCK release and pancreatic protein secretion during bile-pancreatic juice diversion. Although immunohistochemical staining was recently observed in the myenteric plexus of the duodenum (23), neurons were not stained in our study. As our antiserum was raised against part of the NH2-terminal fragment, these observations might not be necessarily specific to LCRF, and further examinations are therefore necessary.
Intravenous infusion of atropine did not diminish luminal LCRF content, CCK release, or protein secretion produced by bile-pancreatic juice diversion but tended to enhance the responses to bile-pancreatic juice diversion. Thus we clearly showed that the release of LCRF is atropine resistant. Moreover, the changes in plasma CCK levels and the increases in protein output during 4 h of bile-pancreatic juice diversion were not different with or without atropine. The present results support those of the previous studies by Guan et al. (4, 5) in which the luminal feedback regulation in conscious rats was shown to be cholinergic independent. On the other hand, Lu et al. (11) reported that the CCK-releasing activity of intestinal perfusate obtained from donor rats treated with atropine was eliminated and that its secretion was cholinergic dependent. These different observations might be due to differences in experimental conditions between the studies. We (17) and Guan et al. (4, 5) analyzed animals after complete recovery from abdominal surgery, whereas Lu et al. (11) examined acutely anesthetized animals. Because bile-pancreatic juice diversion is a strong and long-lasting stimulator of CCK release and pancreatic protein secretion (20), anesthetized animals might be more susceptible to severe experimental conditions and/or to atropine treatment. Alternatively, because the CCK-RP proposed by Lu et al. (11) has not been purified, it is not known whether this peptide is the same as LCRF. As our RIA system did not show cross-reactivity with another candidate of CCK-RP, DBI (7), interaction of LCRF with this protein is unknown.
The maximal protein response was obtained by intraduodenal injection of
30 pmol of LCRF fragment (12). However, the luminal content of LCRF
measured by RIA was <100 fmol. There are two possible explanations
for this discrepancy: 1) LCRF
fragment-(135) may be less potent than natural LCRF (22) or
2) when the bioactivity of LCRF was
examined, a physiological dose of bile acid (taurocholate) was infused
into the duodenum to reproduce physiological conditions, although
pancreatic enzymes were eliminated (12). Thus, because luminal bile
acid prevents CCK release directly and indirectly (19), larger doses of
LCRF might have been required for the bioassay.
Feedback regulation of CCK release manifested by dietary protease
inhibitors or intact protein was proposed to be mediated by both
PSTI-61 (monitor peptide) in the pancreatic juice and LCRF (8, 12, 18,
22). The essential mechanism of action of these CCK-RPs is the same;
when the luminal protease activity decreases below the threshold (15)
by ingestion of trypsin inhibitors or bile-pancreatic
juice diversion, CCK-RPs survive and elicit CCK release (Fig.
5). Exclusion of bile-pancreatic juice from the intestine is a strong stimulator of pancreatic secretion associated with a decrease in luminal trypsin activity in conscious rats (20).
Nevertheless, PSTI-61 (monitor peptide) cannot be responsible for CCK
release produced by bile-pancreatic juice diversion, because PSTI-61
was excluded from the intestinal lumen and PSTI-61 is not present in
intestinal secretions (4). Therefore, LCRF may be more effective as a
CCK-RP than PSTI-61.
|
![]() |
ACKNOWLEDGEMENTS |
---|
We gratefully acknowledge the assistance of Dr. G. M. Green in reviewing the manuscript. We thank S. Kanai for technical assistance.
![]() |
FOOTNOTES |
---|
This study was supported in part by grants from the Ministry of Education, Science, and Culture, the Life Science Foundation of Japan, the Sandoz Foundation for Gerontological Research, the Sasagawa Medical Research Foundation, and the Research Foundation of Pancreatic Disease of Japan.
Address for reprint requests: K. Miyasaka, Dept. of Clinical Physiology, Tokyo Metropolitan Institute of Gerontology, 35-2 Sakaecho, Itabashiku, Tokyo 173, Japan.
Received 11 September 1997; accepted in final form 7 October 1998.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Fölsch, U. R.,
P. Cantor,
H. M. Wilms,
A. Schafmayer,
H. D. Becker,
and
W. Creutzfeldt.
Role of cholecystokinin in the negative feedback control of pancreatic enzyme secretion in conscious rats.
Gastroenterology
92:
449-458,
1987[Medline].
2.
Funakoshi, A.,
I. Nakano,
H. Shinozaki,
K. Tateishi,
T. Hamaoka,
and
H. Ibayashi.
High plasma cholecystokinin levels in patients with chronic pancreatitis having abdominal pain.
Am. J. Gastroenterol.
81:
1174-1178,
1986[Medline].
3.
Green, G. M.,
and
R. L. Lyman.
Feedback regulation of pancreatic enzyme secretion as a mechanism for trypsin inhibitor-induced hypersecretion in rats.
Proc. Soc. Exp. Biol. Med.
140:
6-12,
1972.
4.
Guan, D.,
H. Ohta,
T. Tawil,
R. A. Liddle,
and
G. M. Green.
CCK-releasing activity of rat intestinal secretion: effect of atropine and comparison with monitor peptide.
Pancreas
5:
677-684,
1990[Medline].
5.
Guan, D.,
H. Ohta,
T. Tawil,
A. W. Spannagel,
R. A. Liddle,
and
G. M. Green.
Lack of cholinergic control in feedback regulation of pancreatic secretion in the rat.
Gastroenterology
98:
437-443,
1990[Medline].
6.
Hashimura, E.,
F. Shimizu,
T. Ishino,
K. Imagawa,
K. Tateishi,
and
T. Hamaoka.
Production of rabbit antibody specific for amino-terminal residues of cholecystokinin octapeptide (CCK-8) by selective suppression of cross-reactive antibody response.
J. Immunol. Methods
55:
375-387,
1982[Medline].
7.
Herzig, K. H.,
I. Shön,
K. Tatemoto,
Y. Ohe,
Y. Li,
U. R. Fölsch,
and
C. Owyang.
Diazepam binding inhibitor is a potent cholecystokinin-releasing peptide in the intestine.
Proc. Natl. Acad. Sci. USA
93:
7927-7932,
1996
8.
Iwai, K.,
S. Fukuoka,
T. Fushiki,
M. Tsujikawa,
M. Hirose,
S. Tsunasawa,
and
F. Sakiyama.
Purification and sequencing of a trypsin-sensitive cholecystokinin-releasing peptide from rat pancreatic juice.
J. Biol. Chem.
262:
8956-8959,
1987
9.
Keller, P. J.,
E. Cohen,
and
H. Neurath.
The proteins of bovine pancreatic juice.
J. Biol. Chem.
233:
344-349,
1958
10.
Louie, D. S.,
D. May,
P. Miller,
and
C. Owyang.
Cholecystokinin mediates feedback regulation of pancreatic enzyme secretion in rats.
Am. J. Physiol.
250 (Gastrointest. Liver Physiol. 13):
G252-G259,
1986[Medline].
11.
Lu, L.,
D. S. Louie,
and
C. Owyang.
A cholecystokinin-releasing peptide mediates feedback regulation of pancreatic secretion.
Am. J. Physiol.
256 (Gastrointest. Liver Physiol. 19):
G430-G435,
1989
12.
Miyasaka, K.,
and
A. Funakoshi.
Stimulatory effect of synthetic luminal cholecystokinin releasing factor (LCRF) fragment-(135) on pancreatic exocrine secretion in conscious rats.
Pancreas.
15:
310-313,
1997[Medline].
13.
Miyasaka, K.,
and
G. M. Green.
The effect of atropine on rat basal pancreatic secretion during return or diversion of bile pancreatic juice.
Proc. Soc. Exp. Biol. Med.
l74:
187-192,
1983.
14.
Miyasaka, K.,
and
G. M. Green.
Effect of rapid washout of proximal small intestine on pancreatic secretion in conscious rat (Abstract).
Gastroenterology
84:
1251,
1983.
15.
Miyasaka, K.,
and
G. M. Green.
Effect of exclusion of pancreatic juice on rat basal pancreatic secretion.
Gastroenterology
86:
114-119,
1984[Medline].
16.
Miyasaka, K.,
D. Guan,
R. A. Liddle,
and
G. M. Green.
Feedback regulation by trypsin: evidence for intraluminal CCK-releasing peptide.
Am. J. Physiol.
257 (Gastrointest. Liver Physiol. 20):
G175-G181,
1989
17.
Miyasaka, K.,
K. Kitani,
and
G. Green.
The sequential changes in pancreatic exocrine function after abdominal surgery.
Pancreas
1:
347-353,
1986[Medline].
18.
Miyasaka, K.,
R. Nakamura,
A. Funakoshi,
and
K. Kitani.
Stimulatory effect of monitor peptide and human pancreatic secretory trypsin inhibitor on pancreatic secretion and cholecystokinin release in conscious rat.
Pancreas
4:
139-144,
1989[Medline].
19.
Miyasaka, K.,
N. Sazaki,
A. Funakoshi,
M. Matsumoto,
and
K. Kitani.
Two mechanisms of inhibition by bile on luminal feedback regulation of rat pancreas.
Gastroenterology
104:
1780-1785,
1993[Medline].
20.
Nakamura, R.,
K. Miyasaka,
A. Funakoshi,
and
K. Kitani.
Interactions between bile and pancreatic juice diversions on cholecystokinin release and pancreas in conscious rats.
Proc. Soc. Exp. Biol. Med.
192:
182-186,
1989[Abstract].
21.
Schneeman, B. O.,
and
L. Lyman.
Factors involved in the intestinal feedback regulation of pancreatic enzyme secretion in the rats.
Proc. Soc. Exp. Biol. Med.
148:
897-903,
1975[Abstract].
22.
Spannagel, A. W.,
G. M. Green,
D. Guan,
R. A. Liddle,
K. Faull,
and
U. R. Reeve.
Purification and characterization of a luminal cholecystokinin-releasing factor from rat intestinal secretion.
Proc. Natl. Acad. Sci. USA
93:
4415-4420,
1996
23.
Tarasova, N.,
A. W. Spannagel,
G. M. Green,
G. Gomez,
J. T. Reed,
J. C. Thompson,
M. R. Hellmich,
J. R. Reeve,
R. A. Liddle,
and
G. H. Greeley.
Distribution and localization of a novel cholecystokinin-releasing factor in the rat gastrointestinal tract.
Endocrinology
138:
5550-5554,
1997
24.
Tateishi, K.,
A. Funakoshi,
A. Jimi,
S. Funakoshi,
H. Tamamura,
H. Yajima,
and
Y. Matsuoka.
High plasma pancreastatin like immunoreactivity in a patient with malignant insulinoma.
Gastroenterology
97:
1313-1318,
1989[Medline].
25.
Tateishi, K.,
T. Hamaoka,
N. Sugiura,
C. Yanaihara,
and
N. Yanaihara.
A novel immunization procedure for production of anti-cholecystokinin-specific antiserum of low cross-reactivity.
J. Immunol. Methods
47:
249-258,
1981[Medline].