0013-7227/91/1296-2867$03.00/0 Endocrinology Copyright © 1991 by The Endocrine Society
Vol. 129, No. 6 Printed in U.S.A.
Endogenous Cholecystokinin Release Responsible for Pancreatic Growth Observed after Pancreatic Juice Diversion* N. RIVARD, D. GUAN, D. MAOUYO, G. GRONDIN, F. L. BERUBE, AND J. MORISSET Centre de Recherche sur les Mecanismes de Secretion, Departement de Biologie, Faculte des Sciences, Uniuersite de Sherbrooke, Sherbrooke, Quebec, Canada J1K 2R1
ABSTRACT. This study was undertaken to determine whether intermittent pancreatic juice diversion (PJD) from the intestine can induce pancreatic and duodenal growth. Concomitant infusions of SMS 201-995, a somatostatin analog, and L364,718, a cholecystokinin (CCK) receptor antagonist, were used to establish the involvement of endogenous CCK. Fed rats equipped with biliary, duodenal, and pancreatic cannulae had their pancreatic juice diverted 8 h/day for 4 days and were infused or not with either SMS 201-995 (5 /xg/kg- h) or L-364,718 (0.5 mg/kg-h) during diversion. After 4 days, rats were killed, and their pancreas and duodenum were excised for measurements of parameters indicative of growth. In normally fed rats with pancreatic juice returned, SMS 201-995 inhibited daily
/ C H O L E C Y S T O K I N I N (CCK) is recognized as one V y of the major regulators of pancreatic enzyme secretion, gall bladder contraction, and gastric emptying (1). With its analogs, CCK is also known for its trophic effects on the pancreas (2-4) and intestinal mucosa (5-7). These initial observations on the growth-promoting effects of CCK and its analogs were obtained from studies in which the trophic agents were administered exogenously. Therefore, the role of endogenous CCK in the regulation of pancreatic and intestinal growth has not yet been fully characterized, although some evidence exists for its involvement. Indeed, chronic feeding of a trypsin inhibitor has been reported to stimulate CCK release and induce pancreatic growth (8-10). Bile-pancreatic juice diversion (BPJD) in the rat caused hypercholecystokinemia and enlargement of the pancreas and small intestine (11-13). The observation that L-364,718, Received May 3,1991. Address all correspondence and requests for reprints to: Dr. Jean Morisset, Departement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1K 2R1. * This work was supported by the Natural Sciences and Engineering Research Council of Canada and le Ministere de l'Education du Quebec (Grants A-6369 and 733).
pancreatic secretions of volume and protein, whereas L-364,718 inhibited only protein output. These two inhibitors had no effect on normal pancreatic and duodenal growth. PJD was associated with increased volume and protein output, increased plasma CCK level, and pancreatic growth. All of these effects were completely blocked by SMS 201-995 and L-364,718, with the exception of plasma CCK level by the CCK antagonist. None of these treatments affected duodenal growth. These results suggest that intermittent infusions of these two inhibitors had no effect on normal pancreatic and duodenal growth, but were successful in preventing pancreatic growth induced by PJD. They also indicate that endogenous CCK is involved in PJD-induced pancreatic growth. (Endocrinology 129: 2867-2874,1991)
a specific CCK receptor antagonist, can totally prevent the trophic effect of BPJD on the pancreas, but not on the intestine (11), raises the importance of endogenous CCK as a direct trophic factor for the intestine. Furthermore, in that latter study, the weight of the pancreas and the thickness of the intestinal mucosa were the only parameters used to characterize growth. To further evaluate the role of endogenous CCK release on the growth of gastrointestinal organs, inhibitors of CCK release and specific CCK receptor antagonists become important tools. Indeed, somatostatin is a known inhibitor of pancreatic exocrine secretion (14,15) as well as the release of secretin (16) and CCK (17,18). It is also recognized as an antitrophic factor for the pancreas (3) and a potent inhibitor of the growth-promoting effect of gastrin on the fundic and antral mucosae (19). Recently, long term infusion of SMS 201-995, a potent long-lasting analog of somatostatin, inhibited normal pancreatic and duodenal growth and significantly reduced the plasma CCK level (20). Similarly, the CCK receptor antagonist L-364,718 prevented caerulein-induced pancreatic growth (21) as well as that induced by camostate administration (22) or BPJD (11, 12). However, the effect of this CCK antagonist on normal pancreatic development
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remains controversial, with some inhibition (11, 23) or no effect (10, 21, 24) being reported. This study was, therefore, undertaken 1) to determine whether intermittent PJD induces pancreatic and duodenal growth in association with increased CCK plasma levels, 2) to correlate such growth with changes in pancreatic volume and protein outputs, and 3) to establish the ability of L-364,718 and SMS 201-995 to prevent normal pancreatic and duodenal development as well as the effects of PJD on these two organs.
Materials and Methods Surgical procedures Male Wistar rats (300-350 g), purchased from Charles River (Montreal, Quebec, Canada), were used in this study. They were anesthetized with methoxyflurane and prepared with Silastic cannula (0.020 in. id x 0.037 in. od). Surgical procedures consisted of cannulation of the pancreatic and biliary ducts to collect pure pancreatic and hepatic secretions. A cannula was inserted into the duodenum to return bile and pancreatic juice; another cannula was placed into the abdominal cavity for ip infusion. Animals were also fixed with a jugular cannula for the infusion of L-364,718 or SMS 201-995. Each cannula was routed under the skin to exit on the back of the animal. After surgery, rats were kept in modified Bollmann-type restraint cages. Rats were fed ad libitum Purina rat chow and water before surgery, during recovery, and during experiments. During the recovery period and between experiments, bile and pancreatic juice were diverted, but continuously returned to the intestine by a servo-system consisting of a collection tube in a liquid level photodetector coupled to a peristaltic pump, as previously described (25).
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Effect of iv infusion of SMS 201-995 or L-364,718 on pancreas and duodenum In this experiment all rats had their pancreatic juice and bile constantly returned to the small intestine. Control rats received either the SMS 201-995 or the L-364,718 solvent. Data from these two control groups were subsequently pooled, since no significant difference was observed between them for all of the parameters studied. SMS 201-995 was given iv at a dose of 5 Mg/kg-h, 8 h/day for 4 days, whereas L-364,718, given through the same route, was infused at a dose of 0.5 mg/kg- h, also 8 h/ day for 4 days. Both inhibitors were prepared daily and infused at a rate of 1 ml/h. Effect of intermittent PJD on pancreas and duodenum In this experiment the pancreatic juice of the control rats was collected and immediately returned with the bile into the intestine. The pancreatic juice of the diverted rats was collected, but not returned to the intestine with the bile. Therefore, pancreatic juice was diverted 8 h/day (0800-1600 h) for 4 days. Effect of iv infusion of SMS 201-995 or L-364,718 on the effects of intermittent PJD In the first experiment, one group of diverted rats was infused iv with a solution of 0.05% BSA (vehicle), whereas the other group of diverted rats received SMS 201-995 in BSA daily at a dose of 5 Mg/kg h during 8 h. In the second experiment one group of diverted rats was infused iv with the vehicle of L364,718, and the other group received daily L-364,718 at 0.5 mg/kg-h during 8 h. Control rats had their pancreatic juice constantly returned to the duodenum. Infusions of both inhibitors were started 15 min before PJD. Data from diverted rats infused with the vehicle of SMS 201-995 or L-364,718 were pooled with those of the diverted rats, since no significant difference was found between these groups.
Infusates
Assays
SMS 201-995 (Sandoz, Canada, Inc., Montreal, Quebec, Canada) was provided as a stock solution of 0.5 mg/ml in acetate buffer, pH 4.2. Dilutions of the stock solution were made in saline containing 0.05% BSA. Syringes and cannula used for SMS 201-995 infusion were rinsed with 1% BSA. L-364,718 (Merck, Sharp, and Dohme Research Laboratory, Rahway, NJ) was provided as a powder. For these studies, L364,718 was dissolved in dimethylsulfoxide-Tween-80 (1:1). This preparation was diluted in saline to a final concentration of 2% dimethylsulfoxide and Tween-80.
In all of these experiments rats were killed by CO2 inhalation at the end of the fourth day of each experiment. Their abdomen was cut open, and blood samples were collected in heparinized syringes from the inferior vena cava. Samples were immediately put on ice and centrifuged at 4 C. The plasma was quickly frozen at -70 C for CCK assays. The pancreas was dissected out and trimmed free of fat, mesentery, and lymph nodes. A 200-mg piece was homogenized in ice-cold buffer, as previously described, for protein, amylase and chymotrypsin assays (4). A second 100- to 140-mg piece was homogenized in 0.6 N perchloric acid for RNA and DNA extraction and determination. The duodenum (defined as the segment of small intestine between the pylorus and the entrance of the common bile pancreatic duct) was excised, opened longitudinally, washed with saline, blotted on filter paper, and weighed. The tissue was then homogenized in 0.6 N perchloric acid for DNA and RNA extraction and protein determination. Protein was determined by the method of Lowry et al. (26), with BSA as a standard. Amylase was assayed using a chromogenic assay according to the method of Bernfeld (27), and
Experimental procedures All experiments were performed from the fourth to the eighth day postoperatively. During these 4 days, bile and pancreatic juice were collected separately every hour (0800-1600 h). The volume of pancreatic juice was measured, and 10 ^1 were taken and diluted 1:300 in 0.02 M phosphate buffer for protein assay (absorbance, 280 nm). The remainder of the pancreatic juice was mixed (or not) with bile and returned (or not) into the intestine depending on the experiment.
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PJD-INDUCED PANCREATIC GROWTH chymotrypsin was assayed using iV-benzoyl-L-tyrosine ethyl ester as a substrate according to the method of Hummel (28). RNA and DNA were extracted as described by Mainz et al. (2). DNA was determined by the method of Volkin and Cohn (29). RNA was measured from the final 0.1 N perchloric acid extract according to the procedure of Munro and Fleck (30). All data on the pancreatic and duodenal components represent total amount per pancreas or the duodenal fragment. Plasma CCK was determined according to the method of Turkelson et al. (31). In this assay [125I]CCK-39 was prepared by the Iodogen method. Recoveries from the Sep-Pak cartridges of CCK-8 or CCK-39 added to charcoal-stripped plasma were 85 ± 6% for CCK-8 and 81 ± 11% for CCK-39. Binding of the tracer to the antibody was inhibited by 50% in the presence of 11.5 pM sulfated CCK-8. This assay detects only CCK molecular forms and shows less than 1% cross-reactivity with sulfated and unsulfated gastrins. Microscopy
Immediately after death, five pieces of tissue were taken from different regions of the pancreas of each animal and fixed in 2% glutaraldehyde-0.1 M cacodylate buffer (pH 7.4) for 1 h at room temperature. The tissues were processed as previously described (32). Semithin sections (1 /xm) stained with toluidine blue were used for light microscopic examination. Representative micrographs from each treatment were taken with a standard Universel Zeiss microscope (Zeiss, New York, NY) equipped with a green filter on Kodak 32ASA Panatomic film (Eastman Kodak, Rochester, NY). Statistical analysis All results were expressed as the mean ± SE. Statistical analyses were performed using Student's t test, and differences were considered significant at P < 0.05.
Results Effects of SMS 201-995 and L-364,718 on pancreatic secretion of control fed rats In all of these experiments food intake was measured in each group on a daily basis during the 4-day experiment. The rats ate an average of 20-25 g food daily, and no difference was observed among groups. As shown in Table 1, over the 4-day study period, control rats secreted between 4-5 ml pancreatic juice/day, with no significant difference between the daily collections. SMS 201-995 significantly reduced the daily volume output by 45%. On the contrary, infusion of the CCK receptor antagonist L-364,718 had no significant effect on secretion volume, although a slight daily reduction was observed. Control rats released daily between 125-140 mg protein. The somatostatin analog significantly reduced that protein secretion by 67%. The CCK receptor antagonist also exhibited a significant inhibition of 47%.
TABLE 1. Effects of SMS 201-995 or L-364,718 on pancreatic secretion and pancreatic parameters in control-fed rats Control (n = 15) Vol secreted (ml) 4.7 ± 132 ± Proteins secreted (mg) Pancreatic wt (mg) 1,115 ± Total DNA (mg) 7.61 ± Total RNA (mg) 25 ± Total protein (mg) 200.3 ± Total amylase (U) 15,960 ± Total chymotrypsin 1,156 ± (U)
0.4 7.3
SMS 201-995 (n = 5)
L-364,718 (n = 6)
2.6 ± 0.6° 44 ± 7.8°
3.5 ± 0.5 70 ± 6.8"
23.5 1,433 ± 57° 934 ± 43 0.51 8.3 ± 0.68 7.92 ± 0.48 31.6 ± 1.5° 24.1 ± 1.26 1.1 13.7 329 ± 13.1° 185.5 ± 5.3 1,790 36,984 ± 1,996" 23,798 ± 3,038° 125 2,043 ±117° 1,142 ± 106
Rats equipped with pancreatic, biliary, and duodenal cannulae had their bile and pancreatic juice constantly returned into the duodenum. The volume of pancreatic juice was measured hourly, and 10 nl were saved for protein measurement. SMS 201-005 was infused 8 h/day from 0800-1600 h during 4 consecutive days at a dose of 5 jig/kg-h. L364,718 was infused as SMS 201-995 at a dose of 0.5 mg/kg- h. Control rats received either the SMS 201-995 or the L-364,718 solvent. Volume and protein outputs represent the daily means for the 4 days. For the pancreatic components, rats were killed at the end of the fourth day of treatment. Values are the mean ± SE. ° Significantly different from control at P < 0.05.
Effects of SMS 201-995 or L-364,718 on pancreatic and duodenal components of control fed rats In control rats with pancreatic juice constantly returned to the duodenum, the 8-h daily infusion of SMS 201-995 resulted in significant increases in pancreatic weight (29%), RNA (26%), protein (64%), amylase (131%), and chymotrypsin (77%; Table 1). The only significant effect of L-364,718 was a 46% increase in total amylase content. Furthermore, none of these inhibitors modified the total pancreatic DNA content. These modifications observed at the biochemical level are supported by the histological study. Indeed, pancreases from control rats (Fig. la) present a normal histology, with nuclei located in the basal region of the cells and the zymogen granules filling a third of each cell. The somatostatin analog treatment resulted in a tremendous increase in zymogen granules (Fig. lb), which confirms the increases in all pancreatic components and the inhibition of secretion. Although L-364,718 inhibited pancreatic protein secretion, the tissue components were not modified; these results were corroborated from the histology (Fig. Id), which presents pancreatic acinar cells comparable to those of the control group (Fig. la). As shown in Fig. lc, the daily infusion of the CCK receptor antagonist solvent had no effect on pancreatic morphology. As shown in Table 2, the daily intermittent infusion of SMS 201-995 or L-364,718 had no effect on duodenal weight or its total protein, RNA, and DNA contents. Finally, at the end of the fourth day of infusion, SMS
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rD FIG. 1. Effects of SMS 201-995 or L-364,718 on the morphology of the pancreatic tissue of control-fed rats. Treatments are explained in Table 1. a, Control rats; b, SMS 201-995-infused rats; c, control rats infused with the vehicle of L-364,718; d, L-364,718-infused rats. Magnification, X525. Arrow, Zymogen granules. TABLE 2. Effects of SMS 201-995 or L-364,718 on the duodenal components of control fed rats
Control (n = 15) SMS 201-995 (n = 5) L 364,718 (n = 6)
Duodenal wt (nig)
Total DNA (mg)
Total RNA (mg)
362 ± 25 321 ± 24 358 ± 31
2.8 ± 0.18 2.7 ± 0.14 2.5 ± 0.27
2.9 ± 0.44 2.9 ± 0.55 3.1 ± 0.27
Total protein (mg) 42.6 ± 3.7 45.3 ± 6.9 41.3 ± 2.8
Values are the mean ± SE. Treatments are described in Table 1.
201-995 significantly reduced the plasma CCK concentration by 52%, while L-364,718 had no effect (Fig. 2).
On the contrary, L-364,718 maintained protein secretion at daily control levels during the 4 days of treatment.
Effects of SMS 201-995 or L-364,718 on pancreatic secretion of pancreatic juice-diverted rats
Effects of SMS 201-995 or L-364,718 on pancreatic and duodenal components of pancreatic juice-diverted rats
As shown in Table 3, daily intermittent PJD over 4 days significantly increased the daily volume outputs above control values by an average of 127%. The infusion of SMS 201-995 or L-364,718 prevented these daily increases in volume and kept them at control levels. Similarly, PJD over 4 days significantly increased the daily protein secretion above control values by an average of 59%. SMS 201-995 not only prevented these increases in protein outputs stimulated by PJD, but significantly reduced them below control values by an average of 32%.
As shown in Table 3, daily PJD for 4 days significantly increased pancreatic weight by 52% and total DNA and RNA contents by 30% and 50%, respectively. However, under this condition, total protein remained at the control level, whereas total amylase and chymotrypsin were significantly reduced by 93% and 77%, respectively. The daily infusion of SMS 201-995 prevented all of the modifications induced by PJD on pancreatic weight and total contents of DNA, RNA, protein, amylase, and chymotrypsin. The CCK receptor antagonist also pre-
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PJD-INDUCED PANCREATIC GROWTH
U
5 CD
a
CS
CL
FIG. 2. Effects of SMS 201-995 or L-364,718 on plasma CCK concentrations in control-fed rats. Treatment are explained in Table 1. Blood samples were taken when animals were killed. Values are the mean ± SE. *, Significantly different from controls at P < 0.05. C, Control-fed rats; CS, fed rats infused with SMS 201-995; CL, fed rats infused with L-364,718.
vented the trophic effects of PJD, but failed to return pancreatic weight and total protein, amylase, and chymotrypsin contents to control values. After 4 days of PJD, the pancreases of these animals showed normal morphology, with acinar cells grouped in acini and forming lobules; no inflammation or inflammatory cells were present. However, the acinar cells were emptied of zymogen granules and contained some lipid vesicules (see Fig. 3a). The diverted rats infused with the CCK antagonist solvent also presented pancreatic tissue comparable to that of the PJD rats without lipid vesicules (Fig. 3c). However, SMS 201-995 (Fig. 3b) prevented the acinar cell degranulation caused by PJD and presented a morphological image comparable to that of the controls (Fig. la). L-364,718 (Fig. 3d) partially prevented the acinar cell degranulation caused by PJD; this supports the biochemical data indicating incomplete recovery to
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control values of total protein, amylase, and chymotrypsin contents. While PJD induced pancreatic growth, as indicated by significant increases in pancreatic weight and total contents of DNA and RNA, it failed to affect duodenal growth, as shown in Table 4. Indeed, duodenal weight and its contents of protein, DNA, and RNA were comparable to control values. Furthermore, the somatostatin analog and the CCK receptor antagonist had no effect on the duodenum of these diverted rats. Finally, PJD was associated with a significant 212% increase in plasma CCK levels, which was totally blocked by SMS 201-995 infusion, but not at all by L-364,718 (Fig. 4). Discussion The major findings from this study are that 1) intermittent infusions of SMS 201-995, an analog of somatostatin, or L-364,718, a CCK receptor antagonist, had no effect on pancreatic and duodenal growth of normally fed rats, although both inhibitors reduced basal protein output; 2) intermittent PJD induced increases in pancreatic volume and protein output in association with pancreatic growth; 3) infusion of SMS 201-995 or L364,718 reduced to or below the control level volume and protein secretion stimulated by PJD and prevented pancreatic growth stimulated by PJD; 4) SMS 201-995 and L-364,718 seem to control pancreatic secretion and growth by two different mechanisms; and finally, 5) PJD and both inhibitors had no effect on duodenal growth.
The observation that SMS 201-995 is a potent inhibitor of basal volume and protein output from the exocrine pancreas of fed rats may partially result from the reduced plasma CCK level and from reduced release of secretin (16) and acetylcholine from pancreatic nerve terminals (33) or a direct action on acinar (34) and ductal cells (35).
TABLE 3. Effects of SMS 201-995 or L-364,718 on pancreatic secretion and pancreatic parameters in pancreatic juice-diverted rats Control (n = 15) Vol secreted (ml) Proteins secreted (mg) Pancreatic wt (mg) Total DNA (mg) Total RNA (mg) Total protein (mg) Total amylase (U) Total chymotrypsin (U)
4.7 ± 0.4 132 ± 7.3 1,115 ± 23.5 7.61 ± 0.51 25 ± 1.1 200.3 ± 13.7 15,960 ± 1,790 1,156 ± 125
Diverted (n = 16)
SMS 201-995 (n = 6)
L-364,718 (n = 6)
10.7 ± 1.3° 210 ± 30.3° 1,687 ± 47° 9.83 ± 0.4° 37.5 ± 1.16° 242 ± 12.3 1,187 ± 98° 267 ± 63°
3.5 ± 0.4 82.5 ± 16° 1,166 ± 49.6 6.6 ± 0.4 28.6 ± 1.9 206 ± 12.4 15,380 ± 2,715 1,244 ± 257
4.2 ± 0.5 137 ± 10.6 908.5 ± 47° 6.4 ± 0.3 22.2 ± 2.2 141 ± 8.1° 9,373 ± l,358a'5 502 ± 103o>fc
Rats, prepared as described in Table 1, had their pancreatic juice diverted daily for 4 days from 0800-1600 h. The control and diverted rats received either the SMS 201-995 or the L-364,718 solvent 8 h a day. SMS 201-995 was infused at a dose of 5 jug/kg-h, and L-364,718 at 0.5 mg/ kg-h. Control rats had their pancreatic juice constantly returned. Volume and protein outputs as well as the pancreatic components are expressed as indicated in Table 1. Values are the mean ± SE. 0 Significantly different from control at P < 0.05. 6 Significantly different from diverted rats at P < 0.05.
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r,B
7
\D ; FIG. 3. Effects of SMS 201-995 or L-364,718 on the morphology of the pancreatic tissue of pancreatic juice-diverted rats. Treatments are explained in Table 3. a, Diverted rats; acini contain lipid vesicules (arrow); b, diverted rats infused with SMS 201-995; c, diverted rats infused with the vehicle of L-364,718; d, diverted rats infused with L-364,718. Magnification, X525. Arrow, Zymogen granules. Arrowhead, Lipid granules. TABLE 4. Effects SMS 201-995 or L-364,718 on the duodenal components of pancreatic juice-diverted rats Duodenal wt (nig) Control (n = 15) Diverted (n = 16) SMS 201-995 (n = 6) L-364,718 (n = 6)
342 ± 25 321 ± 11 311 ± 3 1 326 ± 26
Total DNA (mg) 2.8 ± 2.3 ± 2.4 ± 2.5 ±
0.18 0.23 0.20 0.08
Total RNA (mg) 2.9 ± 2.9 ± 2.2 ± 2.3 ±
0.44 0.19 0.31 0.17
Total protein (mg) 42.6 ± 34.6 ± 41.3 ± 43.8 ±
3.7 4.6 3.1 2.4
Values are the mean ± SE. Treatments are described in Table 3.
The daily inhibition of pancreatic protein secretion caused by the infusion of SMS 201-995 resulted in pancreatic hypertrophy at the end of 4 days, as indicated by increases in pancreatic weight and total RNA, protein, amylase, and chymotrypsin, and is supported by the tremendous increment in zymogen granules of the acinar cells. However, this intermittent somatostatin analog treatment had no effect on total DNA content, while its constant infusion over 7 days caused pancreatic aplasia (20). This observation stresses the importance of the continuous presence of the inhibitor to exert its antitrophic potential. Although L-364,718 and SMS 201-995 had comparable
inhibitory effects on daily protein secretion, the CCK antagonist failed to modify the basal serum CCK level, pancreatic morphology, and pancreatic components, with the exception of increased total amylase content. Such an increase in amylase content has previously been observed with L-364,718, given orally twice daily for 14 days (36). The observed increases in volume and protein output in response to PJD support the important role of CCK in the negative feedback regulation of enzyme secretion (37-39), as increments in plasma CCK concentration are comparable to those obtained after long term feeding of trypsin inhibitor (8, 22) or chronic BPJD (12).
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PJD-INDUCED PANCREATIC GROWTH 30
20
U
oE tn
o
w
10"
DL FIG. 4. Effects of SMS 201-995 or L-364,718 on plasma CCK in pancreatic juice-diverted rats. Treatments are explained in Table 3. Blood samples were taken when animals were killed. Values are the mean ± SE. *, Significantly different from controls at P < 0.05. C, Control rats; D, rats with their pancreatic juice diverted; DS, same as D with the infusion of SMS 201-995; DL, same as D with the infusion of L-364,718.
The concomitant infusion of SMS 201-995 or L364,718 during PJD resulted in a complete inhibition of the stimulatory effect of PJD on volume and protein output. Furthermore, the somatostatin analog reduced the secretion of protein below control values. The action of SMS 201-995 seems to be mediated by an inhibition of CCK release, and the specific reduction of protein output below control values may result from a direct effect on the acinar cells via the somatostatin receptor (34). On the contrary, L-364,718 did not reduce the plasma CCK concentration, and thus, its action is presumably at the CCK receptor level. The observation that infusion of proglumide or immunoneutralization of CCK abolished the increase in pancreatic secretion in response to PDJ supports the above explanation (39). The increases in pancreatic weight and total DNA and RNA observed in response to PJD are characteristic of pancreatic hyperplasia caused by exogenous CCK and secretin (3, 4) or serine protease inhibitor feeding (8, 9). The reduced contents of amylase and chymotrypsin under these conditions at the time of death are indicative of the active secretion during the 8 h preceding death. The increased plasma CCK concentration in response to PJD supports the involvement of endogenous CCK as one of the trophic agents. This study reports for the first time a complete prevention of PJD-induced pancreatic growth by the infusion of 5 jig/kg-h SMS 201-995. This treatment is more efficient than 25 ^g/kg SMS 201-995 given twice daily, which failed to alter the growth effect of camostate feeding (22), or than somatostatin given sc and is reported to reduce caerulein-induced pancreatic growth (3). This antitrophic effect of SMS 201-995 may result from
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its inhibition of endogenous CCK release (18) or that of CCK-releasing peptide (17). A direct action on the pancreatic cells cannot be excluded at this point (34, 35). The implication of endogenous CCK as the trophic factor released by PJD is further supported by the potent inhibitory effect of the CCK receptor antagonist L364,718. Contrary to SMS 201-995, the CCK receptor antagonist failed to return protein, amylase, and chymotrypsin contents to control levels. This observation further supports the suggestion that the two inhibitors act differently and may indicate that PJD could release other factors involved in the control of pancreatic enzyme secretion and synthesis. Recently, we have demonstrated that PJD increased peptide-YY release, which was inhibited by SMS 201-995 (40). Pancreatic peptide release was also observed in response to BPJD (41). These factors are known inhibitors of pancreatic enzyme secretion, but their effects on enzyme synthesis are still unknown. The lack of effect of L-364,718 on duodenal growth of control-fed rats confirms similar data previously obtained by Axelson et al. (11) with this same antagonist and by Niederau et al. (10) with another CCK receptor antagonist, CR 1409. These data seem to indicate that endogenous CCK is not an important factor in maintaining the growth of this organ. However, the intermittent infusion of SMS 201-995 was ineffective on duodenal growth, while its constant infusion at the same dose for 7 days resulted in an impressive inhibition (20). These data indicate that the constant presence of SMS 201995 is needed to suppress duodenal growth. Intermittent PJD is definitely not an important inducer of duodenal growth. This finding contrasts with data indicating that chronic BPJD is a potent stimulus for small intestine growth, an effect not inhibited by L364,718 (11). Our data indicate that the induction of intestinal growth needs more than an intermittent diversion of the pancreatic juice and suggest that bile diversion might also be necessary.
Acknowledgments The authors thank Micheline Vanier for technical assistance, and Anne Rousseau for secretarial assistance. We also thank Drs. Turkelson and Solomon for the RIA of CCK.
References 1. Dunbar Lewis L, Williams JA 1990 Cholecystokinin: a key integrator of nutrients assimilation. News Physiol Sci 5:163-175 2. Mainz DL, Black O, Webster PD 1973 Hormonal control of pancreatic growth. J Clin Invest 52:2300-2304 3. Morisset J 1984 Somatostatin: a potential antigrowth factor for the exocrine pancreas. Regul Peptides 10:11-22 4. Solomon TE, Petersen H, Elashoff I, Grossman MI 1978 Interaction of caerulein and secretin on pancreatic size and composition in rat. Am J Physiol 235:E714-E719
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5. Fine H, Levine GM, Shiau YF 1983 Effect of cholecystokinin and secretin on intestinal structure and function. Am J Physiol 245:G358-G363 6. Morisset J, Genik P 1983 Effects of acute and chronic administration of secretin and caerulein on rat duodenal and gastric growth. Regul Peptides 5:111-123 7. Weser E, Bell D, Tawil T 1981 Effects of octapeptide cholecystokinin, secretin and glucagon on intestinal mucosal growth in parenterally nourished rats. Dig Dis Sci 26:409-416 8. Goke B, Printz H, Koop I, Rausch U, Ritcher G, Arnold R, Adler G 1986 Endogenous CCK release and pancreatic growth in rats after feeding a proteinase inhibitor (camostate). Pancreas 1:505515 9. Miyasaka K, Nakamura R, Kitani K 1989 Effects of trypsin inhibitor (camostate) in pancreas and CCK release in young and old female rats. J Gerontol Med Sci 44:M136-M14O 10. Niederau C, Liithen R, Niederau M, Strohmeyer G, Ferrell LD, Grendel JH 1990 Effects of long-term CCK stimulation and CCK blockade on pancreatic and intestinal growth, morphology and function. Digestion [Suppl 2] 46:217-225 11. Axelson J, Hakanson I, Ihse I, Lilja I, Rehfeld JF, Sundler F 1990
12.
13.
14. 15. 16. 17.
18. 19. 20.
21.
22.
Effects of endogenous and exogenous cholecystokinin and of infusion with the cholecystokinin antagonist L-364,718 on pancreatic and gastrointestinal growth. Scand J Gastroenterol 25:471-480 Gasslander T, Axelson J, Hakanson I, Ihse I, Lilja I, Rehfeld JF 1990 Cholecystokinin is responsible for growth of the pancreas after pancreaticobiliary diversion in rats. Scand J Gastroenterol 25:1060-1065 Miazza BM, Turberg Y, Guillaume P, Hahne W, Chayvialle JA, Loizeau E 1985 Mechanism of pancreatic growth induced by pancreatico-biliary diversion in the rat: inhibition by proglumide, benzotript and ranitidine. Scand J Gastroenterol 20:75-83 Folsch UR, Lankisch PG, Creutzfeldt W 1978 Effect of somatostatin on basal and stimulated pancreatic secretion in the rat. Digestion 17:194-203 Guan D, Maouyo D, Sarfati P, Morisset J 1990 Effects of SMS 201-995 on basal and stimulated pancreatic secretion in rats. Endocrinology 127:298-304 Boden G, Sivitz MC, Owen OT, Ersa-Koumar N, Landor JH 1975 Somatostatin suppresses secretin and pancreatic exocrine secretion. Science 190:163-165 Herzig KH, Lee L, Maj D, Owyang C 1989 Somatostatin inhibits feedback regulation of pancreatic enzyme secretion: activation of the inhibitory regulatory protein (Gi) to inhibit secretion of CCKreleasing peptide. Gastroenterology 96:A207 (Abstract) Schlegel W, Raptis S, Harvey KF, Olivier JM, Pfeiffer EF 1977 Inhibition of cholecystokinin-pancreozymin release by somatostatin. Lancet 2:166-168 Lehy T, Dubrasquet M, Bonfils S 1979 Effect of somatostatin on normal and gastrin-stimulated cell proliferation in the gastric and intestinal mucosae in the rat. Digestion 19:99-109 Rivard N, Guan D, Solomon TE, Turkelson CM, Petitclerc D, Morisset J 1991 Negative control by sandostatin on pancreatic and duodenal growth: a possible implication of insulin-like growth factor I. Regul Peptides 34:13-23 Schmidt WE, Choudhury AR, Siegel EG, Loser C, Conlon JM, Folsch UR, Creutzfeldt W 1989 CCK-antagonist L364.718: influence on rat pancreatic growth induced by caerulein and bombesinlike peptides. Regul Peptides 24:67-79 Schmidt WE, Stockmann F, Choudhury AR, Wilms HM, Siegel EG, Nustede R, Folsch UR, Creutzfeldt W 1989 Influence of CCK antagonist L-364,718, pancreastatin(33-49) and a somatostatin
23.
24. 25.
26. 27. 28.
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analogue on camostat-induced rat pancreatic hypertrophy. Digestion 44:105-116 Wisner JR, McLaughlin RE, Rich KA, Zawa SO, Renner IG 1988 Effect of L-364,718, a new cholecystokinin receptor antagonist on camostat-induced growth of the rat pancreas. Gastroenterology 94:109-113 Zucker KA, Adrian TE, Bilchik AJ, Modlin IM 1989 Effects of the CCK receptor antagonist L-364,718 on pancreatic growth in adult and developping animals. Am J Physiol 257:G511-G516 Sarfati P, Green GM, Morisset J 1988 Secretion of protein, fluid and immunoreactive somatostatin in rat pure pancreatic juice: Adaptation to chronic caerulein and secretin treatment. Pancreas 3:375-382 Lowry OH, Rosebrough NJ, Farr AL, Randall RJ 1951 Protein measurement with the Folin phenol reagent. J Biol Chem 193:265275 Bernfeld P 1955 Amylases a and 0. In: Colowick P, Kaplan NO (eds) Methods in Enzymology. Academic Press, New York, pp 149158 Hummel BC 1959 A modified spectrophotometric determination of chymotrypsin, trypsin and thrombin. Can J Biochem Physiol
37:1393-1399 29. Volkin E, Cohn WE 1954 Estimation of nucleic acids. Biochem Anal 1:287-303 30. Munro HN, Fleck A 1966 Recent developments in the measuring of nucleic acids in biological materials. Analyst 91:78-88 31. Turkelson CM, Dale WE, Reidelberger R, Solomon TE 1986 Development of cholecystokinin radioimmunoassay using synthetic CCK-10 as immunogen. Regul Peptides 15:205-218 32. Morisset J, Grondin G 1987 Implication of ornithine decarboxylase and polyamines in pancreatic growth of neonatal rats. Pancreas 2:303-311 33. Hoist JJ, Schaffalitzky Muckadell OB, Fahrenkrug J 1990 Somatostatin inhibits neurally stimulated pancreatic secretion indirectly. Pancreas 5:611-614 34. Esteve JP, Susini C, Vaysse N, Antonietti H, Wunsch E, Berthon G, Ribet A 1984 Binding of somatostatin to pancreatic acinar cells. Am J Physiol 247:G62-G69 35. Vaysse N, Susini C, Esteve JP, Pradayrol L, Balas D, Ribet A 1979 Action of somatostatin, big somatostatin and secretin on rat pancreatic duct cells. In: Rosselin G, Fromageot P, Bonfils S (eds) Hormone Receptors in Digestion and Nutrition. Elsevier/NorthHolland, New York, p 215 36. Wisner JR, Ozawa S, Xue BG, Renner IG 1990 Chronic administration of a potent cholecystokinin receptor antagonist, L-364,718, fails to inhibit pancreas growth in preweanling rats. Pancreas 5:434-438 37. Green GM, Lyman RL 1972 Feedback regulation of pancreatic enzyme secretion as a mechanism for trypsin inhibitor-induced hypersecretion in rats. Proc Soc Exp Biol Med 140:6-13 38. Louie DS, May D, Miller P, Owyang C 1986 Cholecystokinin mediates feedback regulation of pancreatic enzyme secretion in rats. Am J Physiol 250:G252-G259 39. Shiratori K, Chen YF, Chey WY, Lee KY, Chang TM 1986 Mechanism of increased exocrine pancreatic secretion in pancreatic juice-diverted rats. Gastroenterology 91:1171-1178 40. Guan D, Maouyo D, Taylor IL, Gettys TW, Greely Jr GH, Morisset J 1991 Peptide-YY, a new partner in the negative feedback control of pancreatic secretion. Endocrinology 128:911-916 41. Myasaka K, Funakoshi A, Myasaki K, Kitani K 1989 Release of pancreatic polypeptide and its mechanism in luminal feedback regulation in the conscious rat. Digestion 43:73-80
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Vol. 129, No. 6 Printed in U.S.A.
Endogenous Cholecystokinin Release Responsible for Pancreatic Growth Observed after Pancreatic Juice Diversion* N. RIVARD, D. GUAN, D. MAOUYO, G. GRONDIN, F. L. BERUBE, AND J. MORISSET Centre de Recherche sur les Mecanismes de Secretion, Departement de Biologie, Faculte des Sciences, Uniuersite de Sherbrooke, Sherbrooke, Quebec, Canada J1K 2R1
ABSTRACT. This study was undertaken to determine whether intermittent pancreatic juice diversion (PJD) from the intestine can induce pancreatic and duodenal growth. Concomitant infusions of SMS 201-995, a somatostatin analog, and L364,718, a cholecystokinin (CCK) receptor antagonist, were used to establish the involvement of endogenous CCK. Fed rats equipped with biliary, duodenal, and pancreatic cannulae had their pancreatic juice diverted 8 h/day for 4 days and were infused or not with either SMS 201-995 (5 /xg/kg- h) or L-364,718 (0.5 mg/kg-h) during diversion. After 4 days, rats were killed, and their pancreas and duodenum were excised for measurements of parameters indicative of growth. In normally fed rats with pancreatic juice returned, SMS 201-995 inhibited daily
/ C H O L E C Y S T O K I N I N (CCK) is recognized as one V y of the major regulators of pancreatic enzyme secretion, gall bladder contraction, and gastric emptying (1). With its analogs, CCK is also known for its trophic effects on the pancreas (2-4) and intestinal mucosa (5-7). These initial observations on the growth-promoting effects of CCK and its analogs were obtained from studies in which the trophic agents were administered exogenously. Therefore, the role of endogenous CCK in the regulation of pancreatic and intestinal growth has not yet been fully characterized, although some evidence exists for its involvement. Indeed, chronic feeding of a trypsin inhibitor has been reported to stimulate CCK release and induce pancreatic growth (8-10). Bile-pancreatic juice diversion (BPJD) in the rat caused hypercholecystokinemia and enlargement of the pancreas and small intestine (11-13). The observation that L-364,718, Received May 3,1991. Address all correspondence and requests for reprints to: Dr. Jean Morisset, Departement de Biologie, Faculte des Sciences, Universite de Sherbrooke, Sherbrooke, Quebec, Canada J1K 2R1. * This work was supported by the Natural Sciences and Engineering Research Council of Canada and le Ministere de l'Education du Quebec (Grants A-6369 and 733).
pancreatic secretions of volume and protein, whereas L-364,718 inhibited only protein output. These two inhibitors had no effect on normal pancreatic and duodenal growth. PJD was associated with increased volume and protein output, increased plasma CCK level, and pancreatic growth. All of these effects were completely blocked by SMS 201-995 and L-364,718, with the exception of plasma CCK level by the CCK antagonist. None of these treatments affected duodenal growth. These results suggest that intermittent infusions of these two inhibitors had no effect on normal pancreatic and duodenal growth, but were successful in preventing pancreatic growth induced by PJD. They also indicate that endogenous CCK is involved in PJD-induced pancreatic growth. (Endocrinology 129: 2867-2874,1991)
a specific CCK receptor antagonist, can totally prevent the trophic effect of BPJD on the pancreas, but not on the intestine (11), raises the importance of endogenous CCK as a direct trophic factor for the intestine. Furthermore, in that latter study, the weight of the pancreas and the thickness of the intestinal mucosa were the only parameters used to characterize growth. To further evaluate the role of endogenous CCK release on the growth of gastrointestinal organs, inhibitors of CCK release and specific CCK receptor antagonists become important tools. Indeed, somatostatin is a known inhibitor of pancreatic exocrine secretion (14,15) as well as the release of secretin (16) and CCK (17,18). It is also recognized as an antitrophic factor for the pancreas (3) and a potent inhibitor of the growth-promoting effect of gastrin on the fundic and antral mucosae (19). Recently, long term infusion of SMS 201-995, a potent long-lasting analog of somatostatin, inhibited normal pancreatic and duodenal growth and significantly reduced the plasma CCK level (20). Similarly, the CCK receptor antagonist L-364,718 prevented caerulein-induced pancreatic growth (21) as well as that induced by camostate administration (22) or BPJD (11, 12). However, the effect of this CCK antagonist on normal pancreatic development
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remains controversial, with some inhibition (11, 23) or no effect (10, 21, 24) being reported. This study was, therefore, undertaken 1) to determine whether intermittent PJD induces pancreatic and duodenal growth in association with increased CCK plasma levels, 2) to correlate such growth with changes in pancreatic volume and protein outputs, and 3) to establish the ability of L-364,718 and SMS 201-995 to prevent normal pancreatic and duodenal development as well as the effects of PJD on these two organs.
Materials and Methods Surgical procedures Male Wistar rats (300-350 g), purchased from Charles River (Montreal, Quebec, Canada), were used in this study. They were anesthetized with methoxyflurane and prepared with Silastic cannula (0.020 in. id x 0.037 in. od). Surgical procedures consisted of cannulation of the pancreatic and biliary ducts to collect pure pancreatic and hepatic secretions. A cannula was inserted into the duodenum to return bile and pancreatic juice; another cannula was placed into the abdominal cavity for ip infusion. Animals were also fixed with a jugular cannula for the infusion of L-364,718 or SMS 201-995. Each cannula was routed under the skin to exit on the back of the animal. After surgery, rats were kept in modified Bollmann-type restraint cages. Rats were fed ad libitum Purina rat chow and water before surgery, during recovery, and during experiments. During the recovery period and between experiments, bile and pancreatic juice were diverted, but continuously returned to the intestine by a servo-system consisting of a collection tube in a liquid level photodetector coupled to a peristaltic pump, as previously described (25).
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Effect of iv infusion of SMS 201-995 or L-364,718 on pancreas and duodenum In this experiment all rats had their pancreatic juice and bile constantly returned to the small intestine. Control rats received either the SMS 201-995 or the L-364,718 solvent. Data from these two control groups were subsequently pooled, since no significant difference was observed between them for all of the parameters studied. SMS 201-995 was given iv at a dose of 5 Mg/kg-h, 8 h/day for 4 days, whereas L-364,718, given through the same route, was infused at a dose of 0.5 mg/kg- h, also 8 h/ day for 4 days. Both inhibitors were prepared daily and infused at a rate of 1 ml/h. Effect of intermittent PJD on pancreas and duodenum In this experiment the pancreatic juice of the control rats was collected and immediately returned with the bile into the intestine. The pancreatic juice of the diverted rats was collected, but not returned to the intestine with the bile. Therefore, pancreatic juice was diverted 8 h/day (0800-1600 h) for 4 days. Effect of iv infusion of SMS 201-995 or L-364,718 on the effects of intermittent PJD In the first experiment, one group of diverted rats was infused iv with a solution of 0.05% BSA (vehicle), whereas the other group of diverted rats received SMS 201-995 in BSA daily at a dose of 5 Mg/kg h during 8 h. In the second experiment one group of diverted rats was infused iv with the vehicle of L364,718, and the other group received daily L-364,718 at 0.5 mg/kg-h during 8 h. Control rats had their pancreatic juice constantly returned to the duodenum. Infusions of both inhibitors were started 15 min before PJD. Data from diverted rats infused with the vehicle of SMS 201-995 or L-364,718 were pooled with those of the diverted rats, since no significant difference was found between these groups.
Infusates
Assays
SMS 201-995 (Sandoz, Canada, Inc., Montreal, Quebec, Canada) was provided as a stock solution of 0.5 mg/ml in acetate buffer, pH 4.2. Dilutions of the stock solution were made in saline containing 0.05% BSA. Syringes and cannula used for SMS 201-995 infusion were rinsed with 1% BSA. L-364,718 (Merck, Sharp, and Dohme Research Laboratory, Rahway, NJ) was provided as a powder. For these studies, L364,718 was dissolved in dimethylsulfoxide-Tween-80 (1:1). This preparation was diluted in saline to a final concentration of 2% dimethylsulfoxide and Tween-80.
In all of these experiments rats were killed by CO2 inhalation at the end of the fourth day of each experiment. Their abdomen was cut open, and blood samples were collected in heparinized syringes from the inferior vena cava. Samples were immediately put on ice and centrifuged at 4 C. The plasma was quickly frozen at -70 C for CCK assays. The pancreas was dissected out and trimmed free of fat, mesentery, and lymph nodes. A 200-mg piece was homogenized in ice-cold buffer, as previously described, for protein, amylase and chymotrypsin assays (4). A second 100- to 140-mg piece was homogenized in 0.6 N perchloric acid for RNA and DNA extraction and determination. The duodenum (defined as the segment of small intestine between the pylorus and the entrance of the common bile pancreatic duct) was excised, opened longitudinally, washed with saline, blotted on filter paper, and weighed. The tissue was then homogenized in 0.6 N perchloric acid for DNA and RNA extraction and protein determination. Protein was determined by the method of Lowry et al. (26), with BSA as a standard. Amylase was assayed using a chromogenic assay according to the method of Bernfeld (27), and
Experimental procedures All experiments were performed from the fourth to the eighth day postoperatively. During these 4 days, bile and pancreatic juice were collected separately every hour (0800-1600 h). The volume of pancreatic juice was measured, and 10 ^1 were taken and diluted 1:300 in 0.02 M phosphate buffer for protein assay (absorbance, 280 nm). The remainder of the pancreatic juice was mixed (or not) with bile and returned (or not) into the intestine depending on the experiment.
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PJD-INDUCED PANCREATIC GROWTH chymotrypsin was assayed using iV-benzoyl-L-tyrosine ethyl ester as a substrate according to the method of Hummel (28). RNA and DNA were extracted as described by Mainz et al. (2). DNA was determined by the method of Volkin and Cohn (29). RNA was measured from the final 0.1 N perchloric acid extract according to the procedure of Munro and Fleck (30). All data on the pancreatic and duodenal components represent total amount per pancreas or the duodenal fragment. Plasma CCK was determined according to the method of Turkelson et al. (31). In this assay [125I]CCK-39 was prepared by the Iodogen method. Recoveries from the Sep-Pak cartridges of CCK-8 or CCK-39 added to charcoal-stripped plasma were 85 ± 6% for CCK-8 and 81 ± 11% for CCK-39. Binding of the tracer to the antibody was inhibited by 50% in the presence of 11.5 pM sulfated CCK-8. This assay detects only CCK molecular forms and shows less than 1% cross-reactivity with sulfated and unsulfated gastrins. Microscopy
Immediately after death, five pieces of tissue were taken from different regions of the pancreas of each animal and fixed in 2% glutaraldehyde-0.1 M cacodylate buffer (pH 7.4) for 1 h at room temperature. The tissues were processed as previously described (32). Semithin sections (1 /xm) stained with toluidine blue were used for light microscopic examination. Representative micrographs from each treatment were taken with a standard Universel Zeiss microscope (Zeiss, New York, NY) equipped with a green filter on Kodak 32ASA Panatomic film (Eastman Kodak, Rochester, NY). Statistical analysis All results were expressed as the mean ± SE. Statistical analyses were performed using Student's t test, and differences were considered significant at P < 0.05.
Results Effects of SMS 201-995 and L-364,718 on pancreatic secretion of control fed rats In all of these experiments food intake was measured in each group on a daily basis during the 4-day experiment. The rats ate an average of 20-25 g food daily, and no difference was observed among groups. As shown in Table 1, over the 4-day study period, control rats secreted between 4-5 ml pancreatic juice/day, with no significant difference between the daily collections. SMS 201-995 significantly reduced the daily volume output by 45%. On the contrary, infusion of the CCK receptor antagonist L-364,718 had no significant effect on secretion volume, although a slight daily reduction was observed. Control rats released daily between 125-140 mg protein. The somatostatin analog significantly reduced that protein secretion by 67%. The CCK receptor antagonist also exhibited a significant inhibition of 47%.
TABLE 1. Effects of SMS 201-995 or L-364,718 on pancreatic secretion and pancreatic parameters in control-fed rats Control (n = 15) Vol secreted (ml) 4.7 ± 132 ± Proteins secreted (mg) Pancreatic wt (mg) 1,115 ± Total DNA (mg) 7.61 ± Total RNA (mg) 25 ± Total protein (mg) 200.3 ± Total amylase (U) 15,960 ± Total chymotrypsin 1,156 ± (U)
0.4 7.3
SMS 201-995 (n = 5)
L-364,718 (n = 6)
2.6 ± 0.6° 44 ± 7.8°
3.5 ± 0.5 70 ± 6.8"
23.5 1,433 ± 57° 934 ± 43 0.51 8.3 ± 0.68 7.92 ± 0.48 31.6 ± 1.5° 24.1 ± 1.26 1.1 13.7 329 ± 13.1° 185.5 ± 5.3 1,790 36,984 ± 1,996" 23,798 ± 3,038° 125 2,043 ±117° 1,142 ± 106
Rats equipped with pancreatic, biliary, and duodenal cannulae had their bile and pancreatic juice constantly returned into the duodenum. The volume of pancreatic juice was measured hourly, and 10 nl were saved for protein measurement. SMS 201-005 was infused 8 h/day from 0800-1600 h during 4 consecutive days at a dose of 5 jig/kg-h. L364,718 was infused as SMS 201-995 at a dose of 0.5 mg/kg- h. Control rats received either the SMS 201-995 or the L-364,718 solvent. Volume and protein outputs represent the daily means for the 4 days. For the pancreatic components, rats were killed at the end of the fourth day of treatment. Values are the mean ± SE. ° Significantly different from control at P < 0.05.
Effects of SMS 201-995 or L-364,718 on pancreatic and duodenal components of control fed rats In control rats with pancreatic juice constantly returned to the duodenum, the 8-h daily infusion of SMS 201-995 resulted in significant increases in pancreatic weight (29%), RNA (26%), protein (64%), amylase (131%), and chymotrypsin (77%; Table 1). The only significant effect of L-364,718 was a 46% increase in total amylase content. Furthermore, none of these inhibitors modified the total pancreatic DNA content. These modifications observed at the biochemical level are supported by the histological study. Indeed, pancreases from control rats (Fig. la) present a normal histology, with nuclei located in the basal region of the cells and the zymogen granules filling a third of each cell. The somatostatin analog treatment resulted in a tremendous increase in zymogen granules (Fig. lb), which confirms the increases in all pancreatic components and the inhibition of secretion. Although L-364,718 inhibited pancreatic protein secretion, the tissue components were not modified; these results were corroborated from the histology (Fig. Id), which presents pancreatic acinar cells comparable to those of the control group (Fig. la). As shown in Fig. lc, the daily infusion of the CCK receptor antagonist solvent had no effect on pancreatic morphology. As shown in Table 2, the daily intermittent infusion of SMS 201-995 or L-364,718 had no effect on duodenal weight or its total protein, RNA, and DNA contents. Finally, at the end of the fourth day of infusion, SMS
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rD FIG. 1. Effects of SMS 201-995 or L-364,718 on the morphology of the pancreatic tissue of control-fed rats. Treatments are explained in Table 1. a, Control rats; b, SMS 201-995-infused rats; c, control rats infused with the vehicle of L-364,718; d, L-364,718-infused rats. Magnification, X525. Arrow, Zymogen granules. TABLE 2. Effects of SMS 201-995 or L-364,718 on the duodenal components of control fed rats
Control (n = 15) SMS 201-995 (n = 5) L 364,718 (n = 6)
Duodenal wt (nig)
Total DNA (mg)
Total RNA (mg)
362 ± 25 321 ± 24 358 ± 31
2.8 ± 0.18 2.7 ± 0.14 2.5 ± 0.27
2.9 ± 0.44 2.9 ± 0.55 3.1 ± 0.27
Total protein (mg) 42.6 ± 3.7 45.3 ± 6.9 41.3 ± 2.8
Values are the mean ± SE. Treatments are described in Table 1.
201-995 significantly reduced the plasma CCK concentration by 52%, while L-364,718 had no effect (Fig. 2).
On the contrary, L-364,718 maintained protein secretion at daily control levels during the 4 days of treatment.
Effects of SMS 201-995 or L-364,718 on pancreatic secretion of pancreatic juice-diverted rats
Effects of SMS 201-995 or L-364,718 on pancreatic and duodenal components of pancreatic juice-diverted rats
As shown in Table 3, daily intermittent PJD over 4 days significantly increased the daily volume outputs above control values by an average of 127%. The infusion of SMS 201-995 or L-364,718 prevented these daily increases in volume and kept them at control levels. Similarly, PJD over 4 days significantly increased the daily protein secretion above control values by an average of 59%. SMS 201-995 not only prevented these increases in protein outputs stimulated by PJD, but significantly reduced them below control values by an average of 32%.
As shown in Table 3, daily PJD for 4 days significantly increased pancreatic weight by 52% and total DNA and RNA contents by 30% and 50%, respectively. However, under this condition, total protein remained at the control level, whereas total amylase and chymotrypsin were significantly reduced by 93% and 77%, respectively. The daily infusion of SMS 201-995 prevented all of the modifications induced by PJD on pancreatic weight and total contents of DNA, RNA, protein, amylase, and chymotrypsin. The CCK receptor antagonist also pre-
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PJD-INDUCED PANCREATIC GROWTH
U
5 CD
a
CS
CL
FIG. 2. Effects of SMS 201-995 or L-364,718 on plasma CCK concentrations in control-fed rats. Treatment are explained in Table 1. Blood samples were taken when animals were killed. Values are the mean ± SE. *, Significantly different from controls at P < 0.05. C, Control-fed rats; CS, fed rats infused with SMS 201-995; CL, fed rats infused with L-364,718.
vented the trophic effects of PJD, but failed to return pancreatic weight and total protein, amylase, and chymotrypsin contents to control values. After 4 days of PJD, the pancreases of these animals showed normal morphology, with acinar cells grouped in acini and forming lobules; no inflammation or inflammatory cells were present. However, the acinar cells were emptied of zymogen granules and contained some lipid vesicules (see Fig. 3a). The diverted rats infused with the CCK antagonist solvent also presented pancreatic tissue comparable to that of the PJD rats without lipid vesicules (Fig. 3c). However, SMS 201-995 (Fig. 3b) prevented the acinar cell degranulation caused by PJD and presented a morphological image comparable to that of the controls (Fig. la). L-364,718 (Fig. 3d) partially prevented the acinar cell degranulation caused by PJD; this supports the biochemical data indicating incomplete recovery to
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control values of total protein, amylase, and chymotrypsin contents. While PJD induced pancreatic growth, as indicated by significant increases in pancreatic weight and total contents of DNA and RNA, it failed to affect duodenal growth, as shown in Table 4. Indeed, duodenal weight and its contents of protein, DNA, and RNA were comparable to control values. Furthermore, the somatostatin analog and the CCK receptor antagonist had no effect on the duodenum of these diverted rats. Finally, PJD was associated with a significant 212% increase in plasma CCK levels, which was totally blocked by SMS 201-995 infusion, but not at all by L-364,718 (Fig. 4). Discussion The major findings from this study are that 1) intermittent infusions of SMS 201-995, an analog of somatostatin, or L-364,718, a CCK receptor antagonist, had no effect on pancreatic and duodenal growth of normally fed rats, although both inhibitors reduced basal protein output; 2) intermittent PJD induced increases in pancreatic volume and protein output in association with pancreatic growth; 3) infusion of SMS 201-995 or L364,718 reduced to or below the control level volume and protein secretion stimulated by PJD and prevented pancreatic growth stimulated by PJD; 4) SMS 201-995 and L-364,718 seem to control pancreatic secretion and growth by two different mechanisms; and finally, 5) PJD and both inhibitors had no effect on duodenal growth.
The observation that SMS 201-995 is a potent inhibitor of basal volume and protein output from the exocrine pancreas of fed rats may partially result from the reduced plasma CCK level and from reduced release of secretin (16) and acetylcholine from pancreatic nerve terminals (33) or a direct action on acinar (34) and ductal cells (35).
TABLE 3. Effects of SMS 201-995 or L-364,718 on pancreatic secretion and pancreatic parameters in pancreatic juice-diverted rats Control (n = 15) Vol secreted (ml) Proteins secreted (mg) Pancreatic wt (mg) Total DNA (mg) Total RNA (mg) Total protein (mg) Total amylase (U) Total chymotrypsin (U)
4.7 ± 0.4 132 ± 7.3 1,115 ± 23.5 7.61 ± 0.51 25 ± 1.1 200.3 ± 13.7 15,960 ± 1,790 1,156 ± 125
Diverted (n = 16)
SMS 201-995 (n = 6)
L-364,718 (n = 6)
10.7 ± 1.3° 210 ± 30.3° 1,687 ± 47° 9.83 ± 0.4° 37.5 ± 1.16° 242 ± 12.3 1,187 ± 98° 267 ± 63°
3.5 ± 0.4 82.5 ± 16° 1,166 ± 49.6 6.6 ± 0.4 28.6 ± 1.9 206 ± 12.4 15,380 ± 2,715 1,244 ± 257
4.2 ± 0.5 137 ± 10.6 908.5 ± 47° 6.4 ± 0.3 22.2 ± 2.2 141 ± 8.1° 9,373 ± l,358a'5 502 ± 103o>fc
Rats, prepared as described in Table 1, had their pancreatic juice diverted daily for 4 days from 0800-1600 h. The control and diverted rats received either the SMS 201-995 or the L-364,718 solvent 8 h a day. SMS 201-995 was infused at a dose of 5 jug/kg-h, and L-364,718 at 0.5 mg/ kg-h. Control rats had their pancreatic juice constantly returned. Volume and protein outputs as well as the pancreatic components are expressed as indicated in Table 1. Values are the mean ± SE. 0 Significantly different from control at P < 0.05. 6 Significantly different from diverted rats at P < 0.05.
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Endo • 1991 Vol 129 • No 6
r,B
7
\D ; FIG. 3. Effects of SMS 201-995 or L-364,718 on the morphology of the pancreatic tissue of pancreatic juice-diverted rats. Treatments are explained in Table 3. a, Diverted rats; acini contain lipid vesicules (arrow); b, diverted rats infused with SMS 201-995; c, diverted rats infused with the vehicle of L-364,718; d, diverted rats infused with L-364,718. Magnification, X525. Arrow, Zymogen granules. Arrowhead, Lipid granules. TABLE 4. Effects SMS 201-995 or L-364,718 on the duodenal components of pancreatic juice-diverted rats Duodenal wt (nig) Control (n = 15) Diverted (n = 16) SMS 201-995 (n = 6) L-364,718 (n = 6)
342 ± 25 321 ± 11 311 ± 3 1 326 ± 26
Total DNA (mg) 2.8 ± 2.3 ± 2.4 ± 2.5 ±
0.18 0.23 0.20 0.08
Total RNA (mg) 2.9 ± 2.9 ± 2.2 ± 2.3 ±
0.44 0.19 0.31 0.17
Total protein (mg) 42.6 ± 34.6 ± 41.3 ± 43.8 ±
3.7 4.6 3.1 2.4
Values are the mean ± SE. Treatments are described in Table 3.
The daily inhibition of pancreatic protein secretion caused by the infusion of SMS 201-995 resulted in pancreatic hypertrophy at the end of 4 days, as indicated by increases in pancreatic weight and total RNA, protein, amylase, and chymotrypsin, and is supported by the tremendous increment in zymogen granules of the acinar cells. However, this intermittent somatostatin analog treatment had no effect on total DNA content, while its constant infusion over 7 days caused pancreatic aplasia (20). This observation stresses the importance of the continuous presence of the inhibitor to exert its antitrophic potential. Although L-364,718 and SMS 201-995 had comparable
inhibitory effects on daily protein secretion, the CCK antagonist failed to modify the basal serum CCK level, pancreatic morphology, and pancreatic components, with the exception of increased total amylase content. Such an increase in amylase content has previously been observed with L-364,718, given orally twice daily for 14 days (36). The observed increases in volume and protein output in response to PJD support the important role of CCK in the negative feedback regulation of enzyme secretion (37-39), as increments in plasma CCK concentration are comparable to those obtained after long term feeding of trypsin inhibitor (8, 22) or chronic BPJD (12).
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PJD-INDUCED PANCREATIC GROWTH 30
20
U
oE tn
o
w
10"
DL FIG. 4. Effects of SMS 201-995 or L-364,718 on plasma CCK in pancreatic juice-diverted rats. Treatments are explained in Table 3. Blood samples were taken when animals were killed. Values are the mean ± SE. *, Significantly different from controls at P < 0.05. C, Control rats; D, rats with their pancreatic juice diverted; DS, same as D with the infusion of SMS 201-995; DL, same as D with the infusion of L-364,718.
The concomitant infusion of SMS 201-995 or L364,718 during PJD resulted in a complete inhibition of the stimulatory effect of PJD on volume and protein output. Furthermore, the somatostatin analog reduced the secretion of protein below control values. The action of SMS 201-995 seems to be mediated by an inhibition of CCK release, and the specific reduction of protein output below control values may result from a direct effect on the acinar cells via the somatostatin receptor (34). On the contrary, L-364,718 did not reduce the plasma CCK concentration, and thus, its action is presumably at the CCK receptor level. The observation that infusion of proglumide or immunoneutralization of CCK abolished the increase in pancreatic secretion in response to PDJ supports the above explanation (39). The increases in pancreatic weight and total DNA and RNA observed in response to PJD are characteristic of pancreatic hyperplasia caused by exogenous CCK and secretin (3, 4) or serine protease inhibitor feeding (8, 9). The reduced contents of amylase and chymotrypsin under these conditions at the time of death are indicative of the active secretion during the 8 h preceding death. The increased plasma CCK concentration in response to PJD supports the involvement of endogenous CCK as one of the trophic agents. This study reports for the first time a complete prevention of PJD-induced pancreatic growth by the infusion of 5 jig/kg-h SMS 201-995. This treatment is more efficient than 25 ^g/kg SMS 201-995 given twice daily, which failed to alter the growth effect of camostate feeding (22), or than somatostatin given sc and is reported to reduce caerulein-induced pancreatic growth (3). This antitrophic effect of SMS 201-995 may result from
2873
its inhibition of endogenous CCK release (18) or that of CCK-releasing peptide (17). A direct action on the pancreatic cells cannot be excluded at this point (34, 35). The implication of endogenous CCK as the trophic factor released by PJD is further supported by the potent inhibitory effect of the CCK receptor antagonist L364,718. Contrary to SMS 201-995, the CCK receptor antagonist failed to return protein, amylase, and chymotrypsin contents to control levels. This observation further supports the suggestion that the two inhibitors act differently and may indicate that PJD could release other factors involved in the control of pancreatic enzyme secretion and synthesis. Recently, we have demonstrated that PJD increased peptide-YY release, which was inhibited by SMS 201-995 (40). Pancreatic peptide release was also observed in response to BPJD (41). These factors are known inhibitors of pancreatic enzyme secretion, but their effects on enzyme synthesis are still unknown. The lack of effect of L-364,718 on duodenal growth of control-fed rats confirms similar data previously obtained by Axelson et al. (11) with this same antagonist and by Niederau et al. (10) with another CCK receptor antagonist, CR 1409. These data seem to indicate that endogenous CCK is not an important factor in maintaining the growth of this organ. However, the intermittent infusion of SMS 201-995 was ineffective on duodenal growth, while its constant infusion at the same dose for 7 days resulted in an impressive inhibition (20). These data indicate that the constant presence of SMS 201995 is needed to suppress duodenal growth. Intermittent PJD is definitely not an important inducer of duodenal growth. This finding contrasts with data indicating that chronic BPJD is a potent stimulus for small intestine growth, an effect not inhibited by L364,718 (11). Our data indicate that the induction of intestinal growth needs more than an intermittent diversion of the pancreatic juice and suggest that bile diversion might also be necessary.
Acknowledgments The authors thank Micheline Vanier for technical assistance, and Anne Rousseau for secretarial assistance. We also thank Drs. Turkelson and Solomon for the RIA of CCK.
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