ANNALS O F CLINICAL AND LABORATORY SCIEN CE, Vol. 16, No. 6Copyright © 1986, Institu te for Clinical Science, Inc.

Effect o f Cadmium and Bombesin on Exocrine Pancreatic Secretions and Plasma Levels of Gastrin and Cholecystokinin (CCK) in RatsPARIMAL CHOWDHURY, Ph .D ., MASAAKI AMI, M .D .,

and PHILLIPL. RAYFORD, Ph .D .

Department o f Physiology and Biophysics, University o f Arkansas fo r Medical Sciences,

Little Rock, AR 72205

ABSTRACTThe effects of cadmium and bombesin on exocrine pancreatic secretions

and plasma levels of gastrin and cholecystokinin (CCK) were studied in anesthetized rats with pancreatic and gastric fistulas. Rats treated only with saline were used as controls. Both control and cadmium (0.1 mg per kg) treated rats were infused with saline, secretin, and bombesin (BBS). Blood and pancreatic juice samples were collected at regular time inter­vals. Plasma levels of gastrin and CCK were measured in blood samples by specific radioimmunoassay. Pancreatic juice samples were measured for volume, protein, and trypsin outputs. Compared to saline treated rats, outputs of volume, protein, and trypsin were significantly greater in cad­mium treated rats. Plasma levels of gastrin were suppressed with secretin but significantly elevated with BBS. Plasma CCK levels were not different from basal after secretin or BBS in rats treated with either cadmium or saline. The results suggest that the administration of cadmium stimulated exocrine pancreatic secretion by a mechanism that does not involve gas­trin or CCK. Bombesin may have a direct influence on the stimulation of exocrine pancreatic secretion in rats.

IntroductionStudies in man and experimental ani­

mals have shown that digestive processes are regulated, at least in part, by actions and interactions of gut hormones re­leased postprandially.15,16 In addition, plasma levels of secretin, cholecysto­kinin (CCK) and pancreatic polypeptide (PP) have been associated with exocrine

pancreatic secretions.16,27,30 Bombesin (BBS), a gut peptide isolated from the amphibian skin,31 is known to influence the release of several gastrointestinal hormones (including CCK, gastrin, and PP) and the release of pancreatic exo­crine secretion.11,28 Secretin and CCK have been shown to have dose depen­dent effects on volume, bicarbonate, and protein secretions of pancreatic juice in

0091-7370/86/1100-0479 $01.50 © Institute for Clinical Science, Inc.

4 8 0 CHOWDHURY, AMI, AND RAYFORD

man and dogs.9,17,20 To our knowledge, the effects of cadmium on the biological actions of these peptides in rats have not been studied.

Cadm ium is a naturally occurring environmental toxic trace elem ent and is in gested through various dietary sou rces13,14,22 and through cigarette sm oking.40 In dogs, intravenous infu­sions of cadmium alone and cadmium with secretin had no significant effect on outputs of exocrine pancreatic secretions or plasma levels of gastrin, CCK, and PP.4 It has been demonstrated in dogs that intravenous administration of cad­mium potentiated bombesin stimulated release of plasma levels of CCK and pan­creatic polypeptide (PP)3 but not gastrin. The current study was conducted in rats to determine the effects of orally admin­istered cadmium on exocrine pancreatic secretions and plasma levels of gastrin and cholecystokinin during infusions of saline (basal), secretin, and bombesin.Materials and MethodsA n im a l s

Twenty male Sprague Dawley rats, approxim ately 400 g of body w eight, were used in the study. The animals were divided into two groups of 10 rats each. Ten rats received saline and ten received cadmium. For 14 days daily, the animals w ere fed either saline or cadmium (0.1 mg per kg body weight) as CdCl2 solution in a total volume of 0.2 ml via intragastric tube. The dose of cad­mium selected was based on the 5 to 10 percent absorption of cadmium by GI tract as shown by us5,6 and o thers.22 Based on this estimate, the oral dose of cadmium used in this study should pro­duce an equivalent blood cadmium level which could come from dietary or envi­ronmental exposure. After 14 days of continuous treatment, the animals were fasted for 24 hours prior to experimental procedure.

S u r g e r y

Rats were anesthetized with a mixture o f k etam ine-H C l and aceprom azine maleate (1:10). The animals were surgi­cally prepared with pancreatic and gas­tric fistulas by use of the following proce­dures. An abdominal incision was made to locate the bile duct. The bile duct was cannulated with PE-50 tubing near the liver and bile was diverted to the duode­num. A second PE tubing about 10cm long was inserted into the bile pancreatic duct and was led out to the exterior and secured through the wall of the abdomi­nal muscle to collect pancreatic juice. In order to avoid the contributions of gastric juice in the stimulation of exo­crine pancreatic secretions, pylorus was lig a te d , and a gastric cannula was inserted and secured to the avascular part of the stomach. The gastric cannula was left open throughout the study period.

E x p e r im e n t a l P r o t o c o l

Saline was continuously infused dur­ing and after the surgical procedures were completed. After surgery, secretin (5U per kg-hr)* was infused throughout the experim ent. This dose of secretin was found optimal to attain a background volume output in rats, as found in our preliminary experiments.38 After 45 min of secretin infusion, BBS (1 (xg per kg- hr)f was infused for 45 min. After BBS infusion was stopped, secretin infusion was continued for another 45 min. The experiment was terminated after a final infusion of saline for 15 min.

Pancreatic ju ice sam ples w ere col­lected continuously for 15 min intervals for measurements of volume, protein, and trypsin. Blood samples were col­lected before and after infusions of test substances. Blood was kept cold after

* Kabi Industries, Stockholm, Sweden, t Peninsula Laboratories, CA.

GUT PEPTID ES AND PANCREATIC SECRETION IN RATS 48 1

co llec tio n , and cen tr ifu g ed at 4°C; plasma was separated and kept frozen at— 20°C for future m easu rem en ts of im m unoreactive gastrin and CCK by specific radioimmunoassays.

M e a s u r e m e n t

Volume was measured in the PE tub­ing with known internal diameters and was converted to microliters. Pancreatic juice was diluted to a fixed volume of 200 fxl of saline. Protein concentration in the pancreatic juice was measured by Brad­ford’s method1 with bovine plasma albu­min as a standard. Total protein outputs were calculated by multiplying the pro­tein concentration with the volume mea­sured during that time interval. Trypsin concentration in pancreatic juice was measured using the method of Erlanger et al12 em ploying N -a -b en zo y l-D L - arginine-p-nitroanilide (BAPNA) as sub­strate. Enterokinase (25 (Jig) was added to 0.1 ml of diluted pancreatic juice and incubated for 30 min before being sub­jected to trypsin assay. Trypsin outputs were calculated by similar procedures as described under protein measurements.RadioimmunoassayG a s t r in

Plasma gastrin was measured26 by a specific radioimmunoassay using anti­serum (UT.55).* The gastrin radioimmu­noassay was developed utilizing anti­serum U T -55 that r e c o g n iz e d the C-terminal amino acid part of the gastrin molecule. The antiserum measures all molecular forms of gastrin. Physiologic changes in gastrin levels are detected with this antiserum with accuracy after food and after various stimuli. In pre­vious studies, it has been shown that

* University of Texas Medical Branch, Galveston, TX.

the antiserum measures gastrin in rat plasma.2,38

C h o l e c y s t o k in in

C oncentrations o f CCK in plasm a were measured by a specific radioimmu­noassay. Validation of the assay has been reported;15,37 however, a brief descrip­tion of the assay procedure is given for clarity. An antibody was generated in New Zealand white rabbits by repeated inoculation with 16 percent pure CCK over a six month period, followed by one injection of 50 |jug of 99 percent pure CCK. The CCK variant (CCK-39)t was labeled with 125I by the modified chlora- mine-T method.15 The labeled hormone was separated from contaminants on a column (0.86 X 16 cm) containing Seph- adex G-25 fine and purified further on a 4 cm colu m n co n ta in in g c e llu lo se C F -llt immediately before being used in the assay.

Antibody (1:2,500 initial dilution), sam­ple, and buffer were added to each tube, incubated for 24 hours; labeled CCK in 200 jjoI (7500 to 8000 cpm) was then added to each tube and incubated for an additional 48 hrs. Bound hormone was separated from free horm one by the addition of goat anti-rabbit gamma glob­ulin and incubation continued overnight at 4°C. The CCK antibody bound 25 to 35 percent of labeled CCK-39 at a final dilution of 1:25,000; more than 80 per­cent of the tracer was bound with excess antibody.

CCK-33 and CCK-39t were equally potent when measured in radioimmuno­assay, whereas CCK fragments and gas­trin varied in potency from 0.3 percent to 0 .0 5 p ercen t w hen com pared to

t Kindly supplied by Professor Viktor M utt Gas­trointestinal Hormone Laboratory, Karolinska Insti- tutet, Stockholm.

$ Whatman Chemical Corporation, Clifton, NJ.

482 CHOWDHURY, AMI, AND RAYFORD

CCK-33 and CCK-39.15 Little or no dis­placement of 125I CCK-39 from combina­tion with antibody was measured with gastrin inhibitory peptide, vasoactive intestinal p eptide, secretin , m otilin, bombesin, gastrin releasing peptide, and other peptides of pancreatic, gastrointes­tinal or pituitary origin .15,37 D ose re­sp o n se cu rv es o f 99 p er c e n t pure CCK-33, our reference standard, and rat plasma in serial dilutions were generated in the assay. The slopes of these dose response curves did not differ signifi­cantly from each other, suggesting that the antibody measures substances in rat plasma that are immunologieally similar to CCK -33 and C C K -39.2 The assay measures the release of CCK by food, and plasma levels of CCK in rats have been correlated with exocrine pancreatic secretions (unpublished observations).

C a l c u l a t io n s

Hormone concentrations were calcu­lated as pg per ml and expressed as mean ± standard error of the mean. Students unpaired “t” test was used to test the sig­nificance of differences between means. Differences with a p-value of less than 0.05 were considered statistically signifi­cant.ResultsE f f e c t o f S e c r e t in a n d B B S o n B a sa l O u t p u t s o f V o l u m e in C o n t r o l a n d C a d m iu m T r e a t e d R ats

Volume outputs (|jl1 per 45 min) during the initial saline infusion were 7.90 ± 2.30 and 6.03 ± 0.50 in control and cad­mium treated rats, respectively (figure 1). A significant stimulation of volume to 75.60 ± 6.05 for control and 101.90 ±9.00 for Cd treated rats occurred after infusion of secretin . W hen BBS was administered during secretin infusion, the volume outputs were enhanced sig­nificantly from both saline and secretin

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Control * *V

Rats

Cadmium

Saline Secretin Secretin Secretin Saline +BBS

F ig u r e 1. Exocrine pancreatic secretion in rats: Volume output (|xl to 45 min) during infusion of saline, secretin, secretin + BBS, secretin and saline* Significantly different from initial saline; JSignifi- cantly different from initial secretin infusion. Clear bar = control; Dark bar = Cd-treated.

phase to 237.40 ± 51.80 for control and390.00 ± 47.00 for cadmium treated rats. Withdrawal of BBS infusion re­sulted in volume outputs of 126.50 ±25.00 for control and 199.40 ± 11.00 for Cd treated rats, leve ls sign ificantly higher than the pre-BBS phase. During the last infusion with saline, the volume outputs were reduced; however, these levels were found significantly higher than those found during the initial infu­sion of saline (figure 1).

Pancreatic protein outputs (mg per 45 min) during the initial saline infusion were not different between control and Cd treated rats (figure 2). Secretin infu­sion resulted in moderate but significant increase in protein outputs to 0.49 ± 0.06 for control and 0.54 ± 0.13 for Cd treated rats. With BBS infusion during secretin, protein outputs were signifi­cantly increased to 2.34 ± 0.57 for con­trol and 4.41 ± 0.60 for Cd-treated rats (P < 0.05). Secretin infusion without

GUT PEPTID ES AND PANCREATIC SECRETION IN RATS 4 8 3

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Saline Secretin Secretin Secretin Saline +BBS

F i g u r e 2 . Exocrine pancreatic secretion in rats: Protein output (mg to 45 min) during infusions of saline, secretin, secretin + BBS, secretin and saline. * Significantly different from the initial saline infu­sion. * Significantly different from the initial secretin infusion. C lear bar = contro l; D ark bar = Cd- treated.

BBS reduced the protein output to 0.98 ± 0.25 for control and 1.50 ± 0.13 for Cd treated rats, values significantly higher than the secretin phase before BBS infusion. W hen secretin infusion was stopped, the protein outputs were reduced to levels not different from those obtained during the initial saline infusion.

Trypsin outputs ( | J i g per 45 min) dur­ing the initial saline infusion were 2.06 ± 0.44 for control and 3.34 ± 0.59 for Cd treated rats, respectively (figure 3). With secretin infusion, the trypsin out­puts w ere sign ificantly increased to 53.60 ± 8.30 and 68.00 ± 12.80 for both control and Cd-treated rats. Infusion of BBS during secretin resulted in further en h a n cem en t o f trypsin outputs to 102.80 ± 22.30 and 131.00 ± 20.50 for

control and Cd-treated rats. After secre­tin plus BBS, secretin infused alone resulted in the decrease in trypsin out­puts to 54.40 ± 12.60 for control and90 .00 ± 12.30 for C d-treated rats, values not significantly different from the initial secretin phase. Outputs of trypsin during the final saline infusion were sig­nificantly higher than those found during the initial saline infusion.

E f f e c t o f S e c r e t in a n d BBS o n P la sm a GI P e p t id e L e v e l s in C o n t r o l a n d C d - t r e a t e d R ats

Basal levels of gastrin (pg per ml) dur­ing saline were 134.90 ± 23.90 for con­trol and 94.30 ± 20.30 for Cd-treated rats (figure 4). With secretin infusion, gastrin levels were decreased to 72.30 ± 12.90 and 53.30 ± 9.80 for control and Cd-treated rats. The reduction in gastrin levels in control rats during secretin was significant when compared to saline, but they were not different from Cd-treated

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F i g u r e 3. Exocrine pancreatic secretion in rats: Trypsin output (fxg to 45 min), during infusion of saline, secretin, secretin + BBS, secretin and saline. * Significantly different from initial saline infusion. ^Significantly different from initial secretin infusion. Clear bar = control; Dark bar = Cd-treated.

484 CHOWDHURY, AMI, AND RAYFORD

180

150

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Solme Secretin Secretin Secretin Saline +BBS

F ig u r e 4. Plasma GI peptide levels in rats: Gas­trin (pg per ml) levels as measured by radioimmuno­assay during infusions of saline, secretin, secretin + BBS, secretin and saline. * Significantly different from initial saline infusion. ^Significantly different from initial secretion infusion. Clear bar = control; Dark bar = Cd-treated.

rats. Infusion of BBS during secretin resulted in gastrin levels of 135.80 ± 23.70 and 159.00 ± 39.70 for control and Cd rats, values significantly different from the secretin phase. No further changes in gastrin levels were found with or without the continuation of secretin or saline infusion.

Basal levels of CCK (pg per ml) during saline infusion were 145.20 ± 9.00 for control and 132.20 ± 16.00 for Cd- treated rats (data not show n). W ith secretin, the CCK levels were 101.80 ±11.00 and 97.20 ± 15.00 for control and Cd rats. With BBS during secretin infu­sion, the CCK levels w ere 91.70 ±15.00 for control and 94.50 ± 6.00 for Cd treated rats. No significant changes in plasma CCK levels were found before

and after infusions of either secretin or BBS.Discussion

Gastrointestinal peptides, in general, play a major role in the process of diges­tion .36 Release and interactions of GI peptides during the digestive phase after a meal have also been considered impor­tant regulating factors for stimulation of exocrine pancreatic secretion.36 Gastro­intestinal tract, in addition to liver and kidney, has been shown to be a primary target site for accumulation of cadmium when cadmium is injected through var­ious route of adm inistration.5’6,22 The effects of chronic cadmium administra­tion on GI peptides and exocrine pancre­atic secretions in rats have not been reported; however, acute studies of cad­mium from our laboratory indicated that it influenced exocrine pancreatic secre­tions in dogs.7 Our major interest in this study was to determine the effect of cad­mium on normal biological actions of peptides.

The basal (saline infusion) volume out­puts in both contro l and cadm ium treated animals found in this study are not different from each other (figure 1); however, they are low compared to the volume outputs reported in conscious and anesthetized rats.18,34 Differences in basal outputs of volume could have been due to the differences in species and the anesthetics that were used in the two studies. Secretin stimulated volume out­puts significantly in both saline and cad­mium treated rats, and this observation is consistent with the earlier reports by Petersen and Grossman34 and others.17,20 The outputs of volum e were substan­tially enhanced in control rats when bombesin was infused with secretin. In C d-treated animals, the potentiating effect of bombesin is more pronounced compared to the control group (figure 1). These in vivo data in rats confirm the in

GUT PEPTID ES AND PANCREATIC SECRETION IN RATS 4 8 5

vitro observations of Iwatsuki and Peter­son25 who showed that BBS acts directly on mouse pancreatic acinar cells to stim­ulate exocrine secretion. The enhanced response of exocrine pancreatic secre­tory outputs, as observed in this study, appeared to be the direct effect of BBS on pancreatic acinar and duct cells of rats treated with cadmium.

Protein and trypsin outputs were sub­stantially higher in the Cd group when compared to control group (figures 2 and 3). In a previous study in dogs, found by some of us that cadmium, when infused alone and with secretin, did not increase outputs of exocrine pancreatic secretions or plasma levels of GI peptides.4 In this study in rats, it was found that cadmium potentiates the effects of BBS on protein and volume outputs which suggests that cadmium synergizes the effects of bom­besin on acinar and duct cells of the pan­creas. These results provide evidence that BBS and cadmium act on different receptors of the exocrine secreting cells of the pancreas. Henrikson21 and Way42 described the potentiation or synergism to occur only when two agonists stimu­late different receptors resulting in the activation of the same effector mecha­nism.

Divalent cations such as C aff, M gtt, and Z n ft have b een show n to have direct effect on the release of GI pep­tides (gastrin, CCK, and PP) and on exo­crine pancreatic secretions.23 Effects of cadmium on release of GI peptides and stimulation of exocrine pancreatic secre­tin have not been studied extensively and may be different since this metal exhibited some unique characteristics in regard to its affinity to certain phase reactant proteins.8 In addition, C d ff and Z n ft have been shown to influence the release of C aff from its intracytoplasmic stores,35 and the released calcium may act as an important mediator for endo­crine and exocrine function. 10’19>29’33>39>43 This may be the mechanism by which

cadmium potentiates the effects of BBS on exocrine pancreatic secretion , as found in this study.

Our stu d y in d ica tes that gastrin release is significantly in h ib ited by secretin, an observation that is consis­tent with the earlier reports in dogs.41 In the current study, BBS stimulated the release of gastrin in rats. The dose of bombesin used and pattern of release of gastrin in this study in rats is similar to the dose of bombesin and the pattern of release of gastrin observed in other spe­cies with BBS.11,28 Thus, BBS at the dose levels used did not release measurable im m u n oreactive CCK in rats. This observation is consistent with studies reported earlier by this group.38 Exoge­nous adm inistrations of CCK-8 have been shown to stimulate pancreatic pro­tein outputs by Izzo, Brugge, and Praiss- man;24 gastrin is a weak stimulant for exocrine pancreatic secretion.24,32 Cho- lecystokinin octapeptide (CCK-8), a cir­culating form of CCK, and gastrin are not detectable by our CCK radioimmu­noassay unless present at a very high concentration. Since in this study e le ­vated plasma levels of gastrin were found but not CCK, increased pancreatic enzyme output in both control and cad­mium treated rats may be due to either a direct response to BBS, release of forms of CCK that are not detected by our radioimmunoassay, or release of other stimulatory substances. The current study, however, does suggest that in addition to the stim ulatory effect of secretin and bombesin, the presence of Cd appears to provide an additional stimulus to gastrin cells of the gut and exocrine cells of the pancreas.

AcknowledgmentsThanks are extended to Mr. Danny McKay, Mrs.

Rena Humphrey, and Miss Peggy Roshell for techni­cal assistance and Mrs. Patricia Johnson and Ms. Yvonne R. Studer for typing this manuscript. This work was supported by NIH Grant # AM30415.

4 8 6 CHOWDHURY, AMI, AND RAYFORD

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