Differential mechanism and site of action of CCK on the pancreatic secretion and growth in rats

Mitsuyoshi Yamamoto, Munenori Otani, Dong-Mei Jia, Ken-Ichiro Fukumitsu, Hiroyuki Yoshikawa, Toshiharu Akiyama, and Makoto Otsuki

Third Department of Internal Medicine, University of Occupational and Environmental Health, Japan, School of Medicine, Kitakyushu 807-8555, Japan

Submitted 29 July 2002 ; accepted in final form 3 June 2003


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Recent studies demonstrated that cholecystokinin (CCK) at physiological levels stimulates pancreatic enzyme secretion via a capsaicin-sensitive afferent vagal pathway. This study examined whether chemical ablation of afferent vagal fibers influences pancreatic growth and secretion in rats. Bilateral subdiaphragmatic vagal trunks were exposed, and capsaicin solution was applied. Pancreatic wet weight and pancreatic secretion and growth in response to endogenous and exogenous CCK were examined 7 days after capsaicin treatment. Perivagal application of capsaicin increased plasma CCK levels and significantly increased pancreatic wet weight compared with those in the control rats. Oral administration of CCK-1 receptor antagonist loxiglumide prevented the increase in pancreatic wet weight after capsaicin treatment. In addition, continuous intraduodenal infusion of trypsin prevented the increase in plasma CCK levels and pancreatic wet weight after capsaicin treatment. There were no significant differences in the expression levels of CCK-1 receptor mRNA and protein in the pancreas in capsaicin-treated and control rats. Intraduodenal administration of camostat or intravenous infusion of CCK-8 stimulated pancreatic secretion in control rats but not in capsaicin-treated rats. In contrast, repeated oral administrations of camostat or intraperitoneal injections of CCK-8 significantly increased pancreatic wet weight in both capsaicin-treated and control rats. Present results suggest that perivagal application of capsaicin stimulates pancreatic growth via an increase in endogenous CCK and that exogenous and endogenous CCK stimulate pancreatic growth not via vagal afferent fibers but directly in rats.

cholecystokinin; pancreatic growth; capsaicin


RECENT STUDIES HAVE DEMONSTRATED that the vagal nerve plays an important role in digestive physiology, including the control of pancreatic functions (7, 22). In addition, both cholecystokinin (CCK)-1 and CCK-2 receptors are revealed to be present on rat and rabbit vagal fibers (25, 45). Although CCK has been shown to play an important physiological role both in the meal-induced release of pancreatic enzymes and in the regulation of pancreatic growth (10, 30, 33, 37), the influence of vagotomy on the pancreatic growth is still controversial (2, 5, 16, 26, 27, 39, 41).

Capsaicin, the pungent ingredient in red peppers, is a selective neurotoxin for unmyelinated primary afferent sensory neurons (36). It is therefore widely used as a pharmacological tool to assess the involvement of sensory neurons in biological functions. In addition, immunohistochemistry and retrograde tracing demonstrated the presence of capsaicin-sensitive afferent nerve fibers in the gastrointestinal mucosa and the pancreas (3). Although the mechanism and site of action of CCK on the pancreatic secretion is still controversial, Li and Owyang (20, 21) have revealed that exogenous and endogenous CCK at physiological concentrations stimulates pancreatic enzyme secretion via a capsaicin-sensitive afferent vagal pathway in anesthetized rats. A recent study also showed that vagal hyperactivity itself stimulates cell proliferation of pancreatic {beta} and acinar cells primarily through a cholinergic receptor in rats (15). Along this line, we hypothesized that the ablation of afferent vagal fibers interrupts the effect of CCK on the pancreatic growth and results in atrophy or hypoplasia of the pancreas. In the present study, we examined our hypothesis and determined the site of action of CCK on pancreatic secretion and growth in rats.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials

The synthetic trypsin inhibitor camostat (FOY-305) was supplied by Ono Pharmaceutical (Osaka, Japan). The following were also purchased: capsaicin, calf thymus DNA, enterokinase, and trypsin (type I; 10,100 U/mg solid) (Sigma, St. Louis, MO); octapeptide of CCK (CCK-8) (Peptide Institute, Protein Research Foundation, Osaka, Japan); CCK-8 NH2-terminal-specific rabbit antiserum OAL-656 (Otsuka Assay Laboratory, Tokushima, Japan); fluorescent dye H-33258 (Hoechst, Frankfurt, Germany); Phadebas amylase test (amylase test A) (Shionogi Pharmaceutical, Osaka, Japan); guanidine thiocyanate (Fluka Biochemika, Buchs, Switzerland); nylon membranes (Hybond-N), polyvinylidene difluoride (PVDF) membranes (Hybond-P), and [{alpha}-32P]deoxycytidine triphosphate (Amersham Pharmacia Biotech, London, UK); and a random primer DNA labeling kit (version 2) (Takara Shuzo, Shiga, Japan).

Animals

Male Wistar rats weighing 200–220 g were used in the present study and maintained in a temperature (23 ± 2°C)- and humidity (55 ± 5%)-controlled room with a 12:12-h light-dark cycle (lights on at 7:00 AM). Rats received humane care according to the guidelines of our institution, and the experimental protocol was approved by our institutional animal welfare committee.

Animal Preparation

For capsaicin treatment, rats were anesthetized with intraperitoneal injection of pentobarbital sodium at 50 mg/kg body wt. After a midline laparotomy, the abdominal vagal trunks were exposed, and a piece of gauze soaked in 0.1 ml capsaicin solution (10 mg/ml dissolved in Tween 80 and olive oil) was left on the vagal trunks for 30 min. Vehicle alone was applied to control rats. Experiments were performed on the seventh day after the treatment.

For continuous administration of trypsin (2 mg/h) into the duodenum after capsaicin treatment, rats were fasted overnight and anesthetized with pentobarbital sodium (50 mg/kg body wt ip), and a cannula was inserted into the duodenum. Rats were placed in Bollman-type restraint cages and were allowed to access food and water ad libitum after the surgery.

For pancreatic secretory study, rats were fasted overnight and anesthetized with pentobarbital sodium (50 mg/kg body wt ip). Two cannulas were inserted into the biliopancreatic duct and bile duct to drain pure pancreatic juice and pure bile separately. In addition, two cannulas were inserted into the duodenum to return bile pancreatic juice and infuse camostat. A jugular vein cannula was inserted for infusion of CCK-8.

Experimental Protocols

Food intake study. Since capsaicin application to central and peripheral vagal fibers attenuates CCK-induced satiety (38), food intake after infusion of CCK-8 was determined to demonstrate that perivagal capsaicin application successfully ablated vagal afferent fibers. On postoperative day 3, rats were fasted from 9:00 AM to 5:00 PM and then injected with CCK-8 (200 pmol/kg body wt ip). Five minutes later, rats were allowed to eat for 30 min, and food intake during this period was measured.

Effect of capsaicin treatment on pancreatic growth. To examine the effect of chemical ablation of afferent vagal fibers by capsaicin on pancreatic growth, the whole pancreas was removed and weighed without fasting (9:00–10:00 AM) on day 7 after the treatment. A portion of the pancreas was then homogenized in a 0.15 N NaCl solution by using a motor-driven, Teflon-coated glass homogenizer. The homogenates were filtered through three layers of gauze and then sonicated for 1 min. The aqueous phase was used for DNA, protein, amylase, lipase, and trypsinogen assay. Blood was taken for the determination of plasma CCK levels.

To examine the role of CCK-1 receptor on pancreatic growth in capsaicin-treated rats, the specific CCK-1 receptor antagonist loxiglumide at 50 mg/kg body wt was administered orally twice daily (9:00 AM and 9:00 PM) for 7 days. The whole pancreas was removed and weighed without fasting on day 7 after capsaicin treatment.

To investigate the mechanism of the increases in plasma CCK levels and pancreatic wet weight after capsaicin treatment, trypsin (2 mg · 0.5 ml1 · h1) was continuously infused into the duodenum for 7 days. Previous studies have shown that trypsin and chymotrypsin in the duodenum exert a negative feedback regulation on the CCK secretion in rats (12, 31). Blood was taken for the determination of plasma CCK levels, and the whole pancreas was removed and weighed without fasting on day 7 after the treatment.

Northern blot analysis for CCK-1 receptor mRNA. To examine the effect of perivagal application of capsaicin on the expression level of CCK-1 receptor mRNA in the pancreas, Northern blot analysis was performed in capsaicin-treated and control rats. Total RNA was extracted from the frozen pancreatic tissue by the acid guanidium thiocyanate/phenol/chloroform method (17). Purified rat CCK-1 receptor and the mouse 7S cDNA probes were labeled with [{alpha}-32P]deoxycytidine triphosphate with a random-primer DNA labeling kit (version 2). For Northern blot analysis, 20 µg of total RNA was size-fractionated on a 1.2% agarose-1.8 M formaldehyde gel, and RNAs were transferred onto nylon membranes (Hybond-N) followed by cross-linking by ultraviolet irradiation. The filters were incubated in prehybridization solution containing 50 mM phosphate buffer (pH 7.4), 0.75 M NaCl, 5 mM EDTA, 50% formamide (vol/vol), 1% SDS (vol/vol), 10% dextran sulfate (vol/vol), 5x Denhardt's solution, and 10 mg/ml salmon sperm DNA and then were hybridized overnight with a labeled cDNA probe (1 x 106 cpm/ml) at 42°C. After being washed with SSC solution containing 0.1% SDS, images were scanned from the Northern blot filters with the FUJIX Bio-Image analyzing system (BAS 2000) (Fuji Film, Tokyo, Japan).

Immunoblot analysis for CCK-1 receptor protein. To examine the effect of perivagal application of capsaicin on the expression level of CCK-1 receptor protein in the pancreas, immunoblot analysis was performed in capsaicin-treated and control rats. Frozen pancreatic tissue was homogenized with a Polytron homogenizer in ice-cold lysis buffer (pH 7.4) containing (in mM) 25 Tris · HCl, 25 NaCl, 0.5 EGTA, 10 NaF, 1 Na3VO4, 10 sodium pyrophosphate, and 1 phenylmethylsulfonyl fluoride, with 10 mg/ml aprotinin, 10 mg/ml leupeptin, and 0.1 mg/ml soybean trypsin inhibitor. Samples were then centrifuged at 15,000 rpm for 10 min at 4°C. Protein concentration was determined by the Bradford method (1) using bovine serum albumin as a standard. The supernatant was prepared for one-dimensional SDS-PAGE. Proteins (10 µg/lane) were then separated by 10% SDS-PAGE. After SDS-PAGE, proteins were transferred to PVDF membranes. Membranes were blocked with blocking solution [10% nonfat dry milk in PBS (pH 7.4) and 0.05% Triton X-100 (PBS-T)] overnight at 4°C, washed in PBS-T, and then incubated overnight with CCK-1 receptor antibody at a 1:6,000 dilution in PBS-T containing 3% nonfat dry milk at 4°C. After being washed, membranes were incubated with anti-rabbit IgG antibody conjugated to horseradish peroxidase at a 1:10,000 dilution in PBS-T for 1 h at room temperature. Antibody binding was detected by a chemiluminescence detection system (ECL High Plus; Amersham Pharmacia Biotech) and exposed to X-ray films (Scientific Bio-Imaging film; Kodak, Rochester, NY).

Site of action of endogenous CCK on pancreatic secretion and pancreatic growth. To examine the effect of endogenous CCK on pancreatic exocrine secretion and growth in capsaicin-treated and control rats, a synthetic trypsin inhibitor, camostat, at 100 mg · kg body wt1 · h1 was infused into the duodenum for 1 h after a 16-h fast. This dose of camostat was shown to cause significant elevation of plasma CCK levels in anesthetized rats (42). Pancreatic secretion was collected every 10 min, and fluid volume and protein concentration were determined. Blood was taken to determine the plasma CCK level at fasting and immediately after the end of camostat administration.

To examine the effect of endogenous CCK on pancreatic growth, camostat at a dose of 100 mg/kg body wt was given orally once daily for 7 days, and the whole pancreas was removed and weighed in the capsaicin-treated and control rats. Vehicle alone was administered orally once daily for 7 days in control rats.

Site of action of exogenous CCK on pancreatic secretion and growth. To examine the effect of exogenous CCK on pancreatic exocrine secretion and pancreatic growth in capsaicin-treated and control rats, a synthetic CCK-8 at 40 pmol · kg body wt1 · h1 was infused via the jugular vein for 1 h. Pancreatic secretion was collected every 10 min and was analyzed for volume and protein. Blood was taken at the end of CCK-8 infusion to determine plasma CCK levels.

To examine the effect of exogenous CCK on pancreatic growth in capsaicin-treated and control rats, CCK-8 at 40 pmol/kg body wt was injected intraperitoneally twice daily (9:00 AM and 9:00 PM) for 7 days, and the whole pancreas was removed and weighed.

Assays

Protein concentrations in pancreatic homogenate and pancreatic juice were measured by the method of Lowry et al. (23) with bovine plasma albumin as a standard. Pancreatic DNA was measured fluorometrically by the reaction between 3, 5-diaminobenzoic acid and deoxyribose sugar by using calf thymus DNA as a standard (18). CCK concentrations in plasma were measured by a sensitive and specific radioimmunoassay using the antiserum OAL-656 with CCK-8 as a standard (40). Amylase activity was determined by a chromogenic method with Phadebas amylase test and expressed as a Somogyi unit. Trypsinogen was determined as tryptic activity after activation with enterokinase (9). Lipase activity was determined by the method of Whitaker (43) and expressed as international units.

Statistical Analysis

Each experiment was performed in 7–9 rats, and results were expressed as means ± SE. Statistical analysis was performed by unpaired Student's t-test using a commercial software, StatView (Abacus Concepts/Brain Power, Berkeley, CA). Differences of P < 0.05 were considered statistically significant.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Effect of Capsaicin Treatment on CCK-8-Induced Inhibition of Food Intake

CCK-8 had no influence on food intake in capsaicin-treated rats, whereas it significantly inhibited food intake in control rats (Table 1). These results indicate that the vagal afferent fibers mediating the satiety effect of CCK are functionally ablated after capsaicin treatment.


View this table:
[in this window]
[in a new window]
 
Table 1. Effect of capsaicin treatment on CCK-8-induced inhibition of food intake; body and pancreatic weight; pancreatic contents of protein, DNA, and enzymes; and pancreatic growth in response to camostat or CCK-8

 

Effect of Capsaicin Treatment on Pancreatic Growth

Chemical ablation of the afferent vagal fibers by capsaicin had no influence on body weight gain, but it caused a significant increase in pancreatic wet weight compared with that in the control rats (Table 1). Pancreatic protein and DNA contents were also significantly increased after perivagal application of capsaicin, whereas the ratio of protein to DNA, an indication of cell size, remained unchanged in both groups (Table 1). These observations indicate that the increase in pancreatic wet weight after capsaicin treatment is due to hyperplasia.

Effect of Capsaicin Treatment on Pancreatic Enzyme Contents

Pancreatic amylase, lipase, and trypsinogen contents were increased in capsaicin-treated rats compared with those in control rats, although a statistically significant difference was found only in the trypsinogen content (Table 1). When pancreatic enzyme contents were related to pancreatic DNA, pancreatic amylase concentration showed no significant change, lipase concentration slightly decreased, and trypsinogen concentration significantly increased after capsaicin treatment (Table 1).

Plasma CCK Levels and the Effect of Loxiglumide on Pancreatic Growth after Capsaicin Treatment

Plasma CCK levels in fasting and nonfasting states in capsaicin-treated rats were higher than those in the control rats (Table 2). Oral administration of the CCK-1 receptor antagonist loxiglumide prevented the increase in pancreatic wet weight in capsaicin-treated rats (Fig. 1). These results suggest that endogenous CCK plays an important role in the pancreatic growth in capsaicin-treated rats.


View this table:
[in this window]
[in a new window]
 
Table 2. Plasma CCK levels in control and capsaicin-treated rats

 


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 1. Effect of oral administration of cholecystokinin (CCK)-1 receptor antagonist loxiglumide and intraduodenal infusion of trypsin on pancreatic weight in capsaicin-treated rats. Values are expressed as means ± SE of 7–9 rats. *Significant difference vs. control rats.

 

Effect of Intraduodenal Infusion of Trypsin on Plasma CCK Levels and Pancreatic Growth after Capsaicin Treatment

Continuous intraduodenal infusion of trypsin completely inhibited the elevation of plasma CCK levels in both fasting and nonfasting states in capsaicin-treated rats and in the nonfasting state in control rats (Table 2). The increase in pancreatic wet weight was also completely prevented by intraduodenal infusion of trypsin in capsaicin-treated rats (Fig. 1). These results suggest that the increase in plasma CCK levels after capsaicin treatment is due to the decrease of proteolytic activity in the duodenum.

Expression Levels of CCK-1 Receptor mRNA and Protein in the Capsaicin-Treated and Control Rats

There were no significant differences in the CCK-1 receptor mRNA and protein levels between capsaicin-treated and control rats (Fig. 2).



View larger version (77K):
[in this window]
[in a new window]
 
Fig. 2. The expression levels of CCK-1 receptor mRNA (A) and protein (B) in the pancreas. Figure shows representative blots from control and capsaicin-treated rats.

 

Effect of Capsaicin Treatment on Pancreatic Exocrine Secretion and Growth in Response to Endogenous CCK Releaser Camostat

Intraduodenal administration of camostat at a dose of 100 mg · kg body wt1 · h1 produced a great increase in pancreatic protein and fluid secretion in control rats but not in capsaicin-treated rats (Fig. 3A), although pancreatic fluid in basal secretion was significantly higher in capsaicin-treated rats compared with that in control rats (Fig. 3B). Intraduodenal infusion of camostat elicited significant increases in plasma CCK levels above the basal values in both control and capsaicin-treated rats. There were no significant differences in plasma CCK levels between control and capsaicin-treated rats after camostat infusion (Table 2). Once-daily oral administration of 100 mg · kg body wt camostat for 7 days significantly increased pancreatic weight compared with that in vehicle-treated control rats, irrespective of whether vagal nerves were treated with capsaicin or not (Table 1).



View larger version (19K):
[in this window]
[in a new window]
 
Fig. 3. Effect of intraduodenal administration of camostat at 100 mg · kg body wt1 · h1 on pancreatic protein (A) and fluid secretion (B) in control and capsaicin-treated rats. Values are expressed as means ± SE of 7–9 rats. aSignificant difference vs. control. bSignificant difference vs. respective basal value before camostat administration (at time 0).

 

Effect of Capsaicin Treatment on Pancreatic Exocrine Secretion and Growth in Response to Exogenous CCK

Intravenous infusion of CCK-8 for 1 h caused a marked increase in protein secretion and a tendency to increase in fluid secretion in control rats, whereas it had no effects in capsaicin-treated rats (Fig. 4). Plasma CCK levels in capsaicin-treated rats at the end of CCK-8 infusion were not significantly different from those in control rats (Table 1).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Effect of intravenous administration of CCK-8 at 40 pmol · kg body wt1 · h1 on pancreatic protein (A) and fluid secretion (B) in control and capsaicin-treated rats. Values are expressed as means ± SE of 7–9 rats. aSignificant difference vs. control. bSignificant difference vs. respective basal value before CCK-8 administration (at time 0).

 

Twice-daily intraperitoneal injection of 40 pmol/kg body wt CCK-8 for 7 days significantly increased pancreatic wet weight in both groups of rats compared with that in vehicle-treated control rats (Table 1). These results suggest that the vagal pathway is the primary site of action of CCK on the pancreatic secretion but not on pancreatic growth.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
We found in the present study that pancreatic growth is promoted after chemical ablation of vagal afferent fibers in rats and that this growth is accompanied by increases in pancreatic protein, trypsinogen, and DNA content. Similar trophic effects on the pancreas have been demonstrated after repeated injections of exogenous CCK (30) or caerulein (8) and endogenous CCK stimulation by a high-protein diet (11) or trypsin inhibitor (28). In addition, several results also suggest that CCK plays an important role in the pancreatic growth after perivagal application of capsaicin. First, plasma CCK levels were higher in capsaicin-treated rats than those in control rats (Table 2). Second, intraduodenal infusion of trypsin prevented the increase in plasma CCK levels and pancreatic weight in capsaicin-treated rats (Table 1, Fig. 1). Third, the CCK-1 receptor antagonist loxiglumide prevented the pancreatic growth after chemical ablation of vagus afferent fibers (Fig. 1). Therefore, it is likely that increased endogenous CCK stimulates pancreatic growth after perivagal application of capsaicin and that elevated plasma CCK levels in capsaicin-treated rats is attributable to the decreased pancreatic protein, especially trypsin secretion into the duodenum.

The expression of pancreatic CCK-1 receptor mRNA and protein in the capsaicin-treated rats were not significantly different from that in control rats (Fig. 2). However, previous studies demonstrated that pancreatic acinar cells possess both high- and low-affinity CCK-1 receptors (14, 34) and that the high-affinity receptors mediate CCK-stimulated enzyme secretion, whereas the low-affinity receptors mediate the inhibition of enzyme secretion observed at a high concentration of CCK (4, 35). Although CCK-1 receptor subtypes were not examined in the present study, the ablation of vagal afferent fibers might increase the ratio of high-affinity receptors to compensate for the decreased pancreatic exocrine secretion. Since the trophic effect of CCK is believed to be mediated by high-affinity CCK-1 receptors (6), an increased ratio of high-affinity receptors could be responsible for the pancreatic growth after chemical ablation of vagal afferent fibers.

We demonstrated in the present study that pancreatic growth is stimulated after chemical ablation of afferent vagal fibers by capsaicin and that CCK-1 receptor antagonist prevents the pancreatic growth. These results suggest that CCK exerts a trophic effect on the pancreas directly, not via capsaicin-sensitive vagal afferent fibers in rats. To confirm this hypothesis, we examined the effect of endogenous and exogenous CCK on pancreatic secretion and growth. As previously shown by Li and Owyang (20, 21), chemical ablation of vagal afferent fibers abolished the effect of exogenous and endogenous CCK on pancreatic secretion (Figs. 3 and 4). However, perivagal application of capsaicin showed no influence on pancreatic growth stimulated by endogenous and exogenous CCK (Table 1). These results further suggest that CCK exerts a trophic effect not via a vagal afferent pathway but directly on the pancreas in rats. In addition, previous studies demonstrated that CCK receptors present on the afferent neurons are lost after treatment with capsaicin (24, 25). Since loss of CCK receptors on the afferent nerves interrupts the effect of CCK via afferent fibers, these findings also support our view that CCK stimulates pancreatic growth and secretion by a different pathway in rats. However, the question of whether CCK stimulates pancreatic secretion by a capsaicin-sensitive vagal afferent pathway is not settled, and conflicting findings have also been reported (13, 19). Further studies seem to be needed to settle this issue.

Pancreatic fluid secretion was significantly increased after perivagal application of capsaicin (Figs. 3B and 4B). Although the precise mechanism of the hypersecretion remained unclear, we previously observed pancreatic fluid hypersecretion in the proliferative process of acinar cells after oral administration of the protease inhibitor camostat (44). Since the pancreatic growth is stimulated in the capsaicin-treated rats (Table 1), it is conceivable that acinar cell proliferation might influence pancreatic fluid hypersecretion in capsaicin-treated rats. On the other hand, intraduodenal administration of camostat, but not intravenous infusion of CCK-8, significantly stimulated pancreatic fluid secretion in control rats (Figs. 3B and 4B). The difference seems to arise from the effect of secretin (29) and different molecular forms of CCK released after camostat administration. A very recent study demonstrated that CCK-58, not CCK-8, is the only detectable endocrine form of CCK in rat (32).

In conclusion, we have demonstrated in the present study that pancreatic growth is promoted after chemical ablation of afferent vagal fibers by capsaicin and that CCK largely participates in the pancreatic growth. These results suggest that CCK exerts a trophic effect not via a vagal afferent pathway but directly on the pancreas in rats.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. Otsuki, Third Dept. of Internal Medicine, Univ. of Occupational and Environmental Health, Japan, School of Medicine, 1-1 Iseigaoka, Yahatanishiku, Kitakyushu 807-8555, Japan (E-mail: mac-otsk{at}med.uoeh-u.ac.jp)

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. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976.[ISI][Medline]
  2. Buchler M, Malfertheiner P, Glasbrenner B, and Beger HG. Pancreatic trophism after truncal vagotomy in rats. Am J Surg 154: 300–304, 1987.[ISI][Medline]
  3. Buck SH and Burks TF. The neuropharmacology of capsaicin: review of some recent observations. Pharmacol Rev 38: 179–226, 1986.[ISI][Medline]
  4. Burnham DB, McChesney DJ, Thurston KC, and Williams JA. Interaction of cholecystokinin and vasoactive intestinal polypeptide on function of mouse pancreatic acini in vitro. J Physiol 349: 475–482, 1984.[Abstract]
  5. Chen D, Nylander AG, Rehfeld JF, Axelson J, Ihse I, and Hakanson R. Does vagotomy affect the growth of the pancreas in the rat? Scand J Gastroenterol 27: 606–608, 1992.[ISI][Medline]
  6. Dawra R, Saluja A, Lerch MM, Saluja M, Logsdon C, and Steer M. Stimulation of pancreatic growth by cholecystokinin is mediated by high affinity receptors on rat pancreatic acinar cells. Biochem Biophys Res Commun 193: 814–820, 1993.[ISI][Medline]
  7. Debas HT, Konturek SJ, and Grossman MI. Effect of extra-gastric and truncal vagotomy on pancreatic secretion in the dog. Am J Physiol 228: 1172–1177, 1975.[Abstract/Free Full Text]
  8. Dembinski AB and Johnson LR. Stimulation of pancreatic growth by secretin, caerulein, and pentagastrin. Endocrinology 106: 323–328, 1980.[ISI][Medline]
  9. Erlanger BE, Kokowasky N, and Cohen W. The preparation and properties of two new chromogenic substrates of trypsin. Arch Biochem Biophys 95: 271–278, 1961.[ISI]
  10. Fölsch UR. Regulation of pancreatic growth. In: Clinics in Gastroenterology. The Exocrine Pancreas, edited by Creutzfeld W. Philadelphia, PA: Saunders, 1984, vol. 13, p. 679–699.
  11. Green GM, Levan VH, and Liddle RA. Plasma cholecystokinin and pancreatic growth during adaptation to dietary protein. Am J Physiol Gastrointest Liver Physiol 251: G70–G74, 1986.[Abstract/Free Full Text]
  12. Green GM and Lyman RL. 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.
  13. Guan D, Phillips WT, and Green GM. Pancreatic secretion stimulated by CCK is not mediated by capsaicin-sensitive vagal afferent pathway in awake rats. Am J Physiol Gastrointest Liver Physiol 270: G881–G886, 1996.[Abstract/Free Full Text]
  14. Jensen RT, Lemp GF, and Gardner JD. Interaction of cholecystokinin with specific membrane receptors on pancreatic acinar cells. Proc Natl Acad Sci USA 77: 2079–2083, 1980.[Abstract]
  15. Kiba T, Tanaka K, Numata K, Hoshino M, Misugi K, and Inoue S. Ventromedial hypothalamic lesion-induced vagal hyperactivity stimulates rat pancreatic cell proliferation. Gastroenterology 110: 885–893, 1996.[ISI][Medline]
  16. Koop H, Schwarting H, Trautmann M, Borger HW, Lankisch PG, Arnold R, and Creutzfeldt W. Trophic effect of truncal vagotomy on the rat pancreas. Digestion 33: 198–205, 1986.[ISI][Medline]
  17. Korc M, Chandrasekar B, Yamanaka Y, Friess H, Bücher M, and Beger HG. Overexpression of the epidermal growth factor receptor in human pancreatic cancer is associated with concomitant increases in the levels of epidermal growth factor and transforming growth factor alpha. J Clin Invest 90: 1352–1360, 1992.[ISI][Medline]
  18. Labarca C and Paigen K. A simple, rapid and sensitive DNA assay procedure. Anal Biochem 102: 344–352, 1980.[ISI][Medline]
  19. Levan VH and Green GM. Effect of atropine on rat pancreatic secretory response to trypsin inhibitors and protein. Am J Physiol Gastrointest Liver Physiol 251: G64–G69, 1986.[Abstract/Free Full Text]
  20. Li Y and Owyang C. Vagal afferent pathway mediates physiological action of cholecystokinin on pancreatic enzyme secretion. J Clin Invest 92: 418–424, 1993.[ISI][Medline]
  21. Li Y and Owyang C. Endogenous cholecystokinin stimulates pancreatic enzyme secretion via vagal afferent pathway in rats. Gastroenterology 107: 525–531, 1994.[ISI][Medline]
  22. Lindskov J, Amtorp O, and Larsen HR. The effects of highly selective vagotomy on exocrine pancreatic function in man. Gastroenterology 70: 545–549, 1976.[ISI][Medline]
  23. Lowry OH, Rosebrough NJ, Farr AL, and Randall RJ. Protein measurement with the folin phenol reagent. J Biol Chem 193: 265–275, 1951.[Free Full Text]
  24. Moran TH, Norgren R, Crosby RJ, and McHugh PR. Central and peripheral vagal transport of cholecystokinin binding sites occurs in afferent fibers. Brain Res 526: 95–102, 1990.[ISI][Medline]
  25. Moriarty P, Dimaline R, Thompson DG, and Dockray GJ. Characterization of cholecystokininA and cholecystokininB receptors expressed by vagal afferent neurons. Neuroscience 79: 905–913, 1997.[ISI][Medline]
  26. Nylander AG, Chen D, Ding XQ, Norlen P, and Hakanson R. The trophic response of rat pancreas to sulfated cholecystokinin-8 is dose- and time-dependent and not affected by vagotomy or atropine. Pharmacol Toxicol 80: 142–146, 1997.[ISI][Medline]
  27. Oscarson J, Hakanson R, Liedberg G, Lundqvist G, Sundler F, and Thorell J. Variated serum gastrin concentration: trophic effects on the gastrointestinal tract of the rat. Acta Physiol Scand Suppl 475: 2–27, 1979.
  28. Otsuki M, Ohki A, Okabayashi Y, Suehiro I, and Baba S. Effect of synthetic protease inhibitor camostate on pancreatic exocrine function in rats. Pancreas 2: 164–169, 1987.[ISI][Medline]
  29. Otsuki M, Sakamoto C, Ohki A, Yuu H, Maeda M, and Baba S. Pancreatic exocrine secretion and immunoreactive secretin release after intraduodenal instillation of 1-phenyl-1-hydroxy-npentane and HCl in rats. Dig Dis Sci 26: 538–44, 1981.[ISI][Medline]
  30. Otsuki M and Williams JA. Amylase secretion by isolated pancreatic acini after chronic cholecystokinin treatment in vivo. Am J Physiol Gastrointest Liver Physiol 244: G683–688, 1983.[Abstract/Free Full Text]
  31. Owyang C, Louie DS, and Tatum D. Feedback regulation of pancreatic enzyme secretion. Suppression of cholecystokinin release by trypsin. J Clin Invest 77: 2042–2047, 1986.[ISI][Medline]
  32. Reeve JR Jr, Green GM, Chew P, Eysselein VE, and Keire DA. CCK-58 is the only detectable endocrine form of cholecystokinin in rat. Am J Physiol Gastrointest Liver Physiol. In press.
  33. Rehfeld JF. Cholecystokinin. In: Handbook of Physiology. The Gastrointestinal System. Neural and Endocrine Biology. Bethesda, MD: Am. Physiol. Soc., 1989, sect. 6, vol. II, chapt. 16, p. 337–358.
  34. Sankaran H, Goldfine ID, Bailey A, Licko V, and Williams JA. Relationship of cholecystokinin receptor binding to regulation of biological functions in pancreatic acini. Am J Physiol Gastrointest Liver Physiol 242: G250–G257, 1982.[Abstract/Free Full Text]
  35. Sankaran H, Goldfine ID, Deveney CW, Wong KY, and Williams JA. Binding of cholecystokinin to high affinity receptors on isolated rat pancreatic acini. J Biol Chem 255: 1849–1853, 1980.[Abstract/Free Full Text]
  36. Sharkey KA, Williams RG, and Dockray GJ. Sensory substance P innervation of the stomach and pancreas. Demonstration of capsaicin-sensitive sensory neurons in the rat by combined immunohistochemistry and retrograde tracing. Gastroenterology 87: 914–921, 1984.[ISI][Medline]
  37. Solomon TE. Regulation of pancreatic secretion. In: Clinics in Gastroenterology. The Exocrine Pancreas, edited by Creutzfeld W. Philadelphia, PA: Saunders, 1984, vol. 13, p. 657–678.
  38. South EH and Ritter RC. Capsaicin application to central or peripheral vagal fibers attenuates CCK satiety. Peptides 9: 601–612, 1988.[ISI][Medline]
  39. Stock-Damge C, Aprahamian M, Lhoste E, Pousse A, Humbert W, Noriega R, and Grenier JF. Pancreatic hyperplasia after small bowel resection in the rat: dissociation from endogenous gastrin levels. Digestion 29: 223–230, 1984.[ISI][Medline]
  40. Tachibana I, Watanabe N, Shirohara H, Akiyama T, Nanano S, and Otsuki M. Effects of tetraprenylacetone on pancreatic exocrine secretion and acute pancreatitis in two experimental models in rats. Int J Pancreatol 17: 147–154, 1995.[ISI][Medline]
  41. Tiscornia OM, Perec CJ, Celener D, De Lehmann ES, Caro L, de Paula J, Baratti C, Martinez JL, and Dreiling DA. Chronic truncal vagotomy: its effects on the weight and function of the rat's pancreas. Mt Sinai J Med 48: 295–304, 1981.[ISI][Medline]
  42. Watanabe S, Takeuchi T, and Chey WY. Mediation of trypsin inhibitor-induced pancreatic hypersecretion by secretin and cholecystokinin in rats. Gastroenterology 102: 621–628, 1992.[ISI][Medline]
  43. Whitaker JF. A rapid and specific method for the determination of pancreatic lipase in serum and urine. Clin Chim Acta 44: 133–138, 1973.[ISI][Medline]
  44. Yamamoto M, Shirohara H, and Otsuki M. CCK-, secretin-, and cholinergic-independent pancreatic fluid hypersecretion in protease inhibitor-treated rats. Am J Physiol Gastrointest Liver Physiol 274: G406–G412, 1998.[Abstract/Free Full Text]
  45. Zarbin MA, Wamsley JK, Innis RB, and Kuhar MJ. Cholecystokinin receptors: presence and axonal flow in the rat vagus nerve. Life Sci 29: 697–705, 1981.[ISI][Medline]