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Meal-stimulated exocrine pancreatic secretion and release of GI peptides in normal and nicotine-treated rats
Regulatory Peptides, 33 (1991) 11-20
I1
Elsevier
REGPEP 01013
Meal-stimulated exocrine pancreatic secretion and release of GI peptides in normal and nicotine-treated rats Parimal C h o w d h u r y , M a s a a k i Ami*, Ryo H o s o t a n i * and Phillip L. Rayford Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR (U.S.A.) Received 10 September 1990; revised version received 17 December 1990; accepted 18 December 1990)
Key words: Effect of nicotine; Food stimulated; Plasma gastrin; Plasma CCK; Exocrine pancreatic secretion
Summary In rats, treated chronically with saline and nicotine, we studied the postprandial release of gastrin and cholecystokinin by specific radioimmunoassays and simultaneously measured secretory outputs of the exocrine pancreas. Rats were prepared surgically with gastric and pancreatic fistulas. Meal-stimulated release of peptides and exocrine secretory outputs were measured 24 h postoperatively in conscious rats. Infusion of food via intragastric cannula significantly stimulated plasma gastrin levels in both control and nicotine treated rats. Postprandial gastrin levels in nicotine treated rats were significantly higher compared to gastrin levels obtained after food in untreated control rats. Plasma CCK levels were increased in both groups after food. These levels remained significantly elevated from the basal values only for a transient period following infusion of the liquid meal. There were no differences in postprandial plasma CCK levels between the two groups. Outputs of exocrine pancreatic volume, protein and trypsin increased significantly after food in both control and nicotine treated groups of rats. The differences in outputs of volume and protein between the two groups of rats were not significant; however, the trypsin outputs in the nicotine rats were decreased significantly when compared to control rats. * Present address: First Department of Surgery, Kyoto University, Kyoto, Japan. Correspondence: P. Chowdhury, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205, U.S.A. 0167-0115/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
12
The data indicate that in rats, administration of food stimulated the release of immunoreactive gastrin and CCK with concomitant increase in exocrine pancreatic secretions of volume, protein and trypsin. Chronic nicotine treatment and its effect on food, however, appeared to have induced hyperfunction of G-cells that resulted in increased gastrin secretion and a decrease in trypsin secretion by exocrine pancreas. These data may have important implications in the etiology of the development of exocrine pancreatic dysfunction in chronic smokers.
Introduction
Nicotine, a component found in substantial amounts in tobacco, cigarettes and smokeless tobacco, has been linked to the development of various pathophysiological disorders [1-3]. Previous studies in rats exposed to nicotine via different routes of administration showed the development of gastrointestinal and pancreatic lesions and it was suggested that these lesions might have been mediated through alteration of hormonal and metabolic factors [4]. The precise mechanism by which nicotine potentiates the development of gastrointestinal mucosal injury has not been clearly delineated. In earlier studies, we have reported that animals exposed chronically to low levels of nicotine via gavage feeding had significantly increased basal circulating levels of gastrin (92 pM vs. 47 pM) and cholecystokinin (106 pM vs. 81 pM) when compared to the animals exposed to saline by the same routes [5]. The results suggest that intragastric topical administration of nicotine stimulates the endocrine cells that specifically secrete gastrin and CCK. The observation that chronic ingestion of nicotine induced higher basal plasma levels of gastrin and CCK, prompted us to study the effect of a physiologic stimulus on postprandial release and actions ofgastrin and CCK. In this study, we report the effect of intragastric administration of food on exocrine pancreatic secretions and plasma levels of gastrin and CCK in both control and chronic nicotine treated rats.
Materials and Methods
Male Sprague-Dawley rats, 8 weeks old and approx. 150 g body weight, were divided into two groups of 12 rats each and were used in the study. The animals were fed daily at 9:00 a.m. for 120 days with either saline or nicotine (20 mg/kg/day (0.12 mmol)) via a gavage tube. Animals from each group were housed individually in galvanized steel metabolic cages (7 in x 10 in x 7 in) equipped with an automatic on-line water intake system in a room with 12 h light-dark cycle. Weight and food intake of each rat was monitored at regular time intervals. At 120 days, each rat was fasted for 24 h, anesthetized and an external jugular vein cannulated with PE-90 tubing. An abdominal incision was made and a gastric cannula (PE-160) was placed in the avascular part of the stomach (fundic portion) to introduce the liquid meal. A pancreatic cannula (PE-50)
13 was then placed and secured in the pancreatic duct to collect pancreatic juice continuously. Prior to the placement of the pancreatic cannula the bile duct was ligated near the liver beyond the point where it is surrounded by pancreatic tissue. Bile duct was cannulated with a PE-50 tubing of about 3 cm long and the bile was diverted to the duodenum at 1.0 cm from the pylorus ring. All PE tubings (cannulae) were supported and secured by purse string nylon sutures with 6.0 prolene (Ethicon, Sommervile, N J) and were exteriorized through the subcutaneous tissues. The incision was then closed and rats were transferred to Bollman type restraining cages (Fisher Scientific, Fairlawn, N J) for recovery. During recovery, the animals received water but no food for an additional 24 h. The i.v. cannula was kept open through infusion of saline at a flow rate of 0.4 ml/h by infusion pump (Sage Instruments). Composition of liquid meal: The liquid meal consists of a packet of sustacal nutritional powder (Mead Johnson, Nutritional Division, Evansville, IN, containing 20.5 g protein, 9.2 g fat, 48 g carbohydrate, vitamins, minerals and salts and dissolved in 100 ml of whole milk).
Sample collection During the experiment two basal blood samples were taken at 9 : 00 a.m. which were followed successively by two 20 rain basal pancreatic juice samples. 5 ml of liquid meal, as described above, were then infused through the gastric cannula at a flow rate of 3.94 ml/min. Following the infusion of liquid meal, blood samples (2 ml) were collected at 10 min intervals through juglar vein catheter for 40 min while pancreatic juice samples were collected continuously for 100 min at 20 min intervals. The volume of blood withdrawn was replaced with equal volume of 4~o bovine serum albumin made up in saline. Blood was centrifuged and plasma stored at - 80 ° C. Pancreatic juice collected in the pancreatic cannula (PE tubing) was measured for volume and diluted to a fixed volume with 200 #1 of saline and stored in the freezer for measurements of protein and trypsin. Measurements Gastrin and CCK levels in plasma were measured using specific radioimmunoassays [6,7]. These assays have been validated for measuring gastrin and CCK in tissues and body fluid of dogs [8] and rats [9,10]. Protein concentration in the pancreatic juice was measured by Bradford's method [ 11 ] using bovine serum albumin as standard. Outputs of total protein were calculated by multiplying the protein concentration times the total volume of pancreatic juice obtained at that time interval. Trypsin concentration in the pancreatic juice was measured using the method of Erlanger et al. [ 12] employing N-~-benzoyl-arginine-p-nitroanilide (BAPNA) as substrate. Prior to trypsin assay, pancreatic juice was incubated with enterokinase (25 #g). Outputs of trypsin were calculated in the same manner as described above for protein. Calculations Hormone concentrations were calculated as pM and expressed as mean + S.E. of the mean. Basal and stimulated values for both plasma and exocrine pancreatic outputs
14
were divided by its corresponding basal value and multiplied by 100. All values were reported as percent increments over the basal. ANOVA and Student's unpaired t-tests were used to test the significant differences between the means. Differences with a P-value of less than 0.05 were considered statistically significant.
Results
Food intakes were not different between the two groups throughout the entire treatment period. At killing, body weights of rats treated with saline were 572 + 16 g and that of nicotine were 499 + 7 g, respectively. The differences in weight gain between the two groups were statistically significant, P < 0.05. Basal plasma levels of gastrin in normal and nicotine treated rats were 39 _+ 11 pM and 60 + 12 pM, respectively. Plasma levels of gastrin, measured after intragastric infusion of liquid meal are shown in Fig. 1 and are represented as percent increments over the basal. Infusion of liquid meal significantly stimulated the release of plasma gastrin levels (as shown by significant increase over basal) in both saline and nicotine treated rats at all time points. The increments in gastrin levels after food in nicotine
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Fig. I. Postprandial levels ofimmunoreactive plasma gastrin (shown as percent increments over the basal value) in rats. The basal value was considered as 100% (see text for details). O , control; IN, nicotine treated. *, P < 0.05, significantly different from basal; ~ , P < 0.05, significantly different between two groups.
15
treated rats, however, was significantly higher when compared to the gastrin levels in the saline treated group (Fig. 1). Basal plasma levels of CCK in normal and nicotine treated rats were 68 + 4 and 86 + 11 pM, respectively. Plasma CCK levels measured after intragastric infusion of food are shown in Fig. 2. Plasma levels of CCK increased significantly but transiently after intragastric infusion of food in both groups. The levels of CCK measured at 20, 30 and 40 min time periods were not significantly different from their respective basal. The differences in postprandial plasma CCK levels between the two groups were not significant (Fig. 2). Outputs of pancreatic juice volume, protein and trypsin measured before and after the infusion of liquid meal were calculated as percent of basal and are shown in Figs. 3 and 4. Pancreatic juice volumes measured at basal (prior to food administration) in control and nicotine treated rats were 119 + 12 and 114 + 11 #1, respectively. These values increased to 158 + 17 and 149 + 18/~1 at 40 min after food. 10 min after food infusion, the outputs of pancreatic juice volume in the saline treated group were 17 ~o over the basal, P < 0.05; in comparison, the volume outputs in nicotine treated group were 40% above the basal, P < 0.05 (Fig. 3, upper panel). Maximal volume outputs after food were obtained at 40 rain in both groups and at 100 min, the volume outputs in each of the groups were not different from their own basal. The protein concentrations in the pancreatic juice samples obtained at basal in control and nicotine treated rats were of 1.2 + 0.3 and 1.3 + 0.3 mg/ml, respectively. These values 200
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Fig. 2. Postprandial levels ofimmunoreactive plasma CCK (shown as percent increments over the basal) in rats. The basal was considered as 100% (see text for details), r-l, control; P3, nicotine treated. *, P < 0.05. significantly different from basal.
16
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increased to 2.3 + 0.4 and 2.8 + 0.4 mg/ml at 40 min after food. The protein outputs for both groups were maximal at 40 min after food (Fig. 3, lower panel). The outputs of protein measured beyond 60 min after food were not different from the basal in any of the two groups. No significant differences in postprandial protein outputs were found between the control and nicotine treated groups. Outputs of trypsin measured (percent of basal) in the pancreatic juice are shown in Fig. 4. Trypsin concentrations measured in pancreatic juice samples obtained at basal in control and nicotine treated rats were 900 + 199 and 949 + 128 #g/ml, respectively. These values increased to 1121 + 186 and 1412 _+ 169#g/ml at 40min after food. Trypsin outputs increased significantly in both groups after food and were maximal at 40 rain. The integrated 100 min outputs of trypsin (° o basal) between control (1031 + 220) vs. nicotine (678 + 101) were significantly different, P < 0.05. Trypsin outputs in nicotine treated groups after food were significantly lower when compared to saline treated group.
17
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Fig. 4. Postprandial exocrine secretory outputs of trypsin in rats. ©, control; @, nicotine treated. *, P < 0.05, significantly different from control.
Discussion Food intake was not different between control and nicotine treated rats; however, body weights were significantly greater in control rats when compared to nicotine rats. This suggests that food intake was not a major factor in body weight loss in nicotine treated rats. The results of the current study show that in rats, intragastric administration of food stimulated the release of gastrin and CCK. The data further demonstrate that release of these G I peptides are associated with concomitant stimulation of pancreatic secretory outputs of protein and trypsin. These data are consistent with the postprandial release of G I peptides and associated exocrine pancreatic secretory outputs shown in other species [ 13-17]. The postprandial release of C C K in rats found in this study is transient when compared to other species. These results are consistent with the reported studies [18,19]; however, results from other laboratories showed that several types of luminal nutrients were capable of producing prolonged increase of plasma C C K [20]. In rats, two forms of circulating C C K have been described (by gel filtration in combination with bioassay): one peak of intermediate size between CCK-33 and CCK-8 and another peak with the same size as CCK-8 [ 18]. In a more recent study, two types of C C K are detected, one corresponding to CCK-8 and one to CCK-33, 39 [21]. The antibody used in this study for C C K radioimmunoassay has its greatest affinity for molecular forms of CCK-33 and other larger forms [7]. The transient rise of C C K as found in our study represents CCK-33, but does not rule out the possibility of circulating
18 CCK-8 which was not measured in this study. The prolonged response of pancreatic enzyme secretion after food may be due to the circulating levels of CCK-8; however, other humoral and neural factors may also be implicated in this phenomenon. The data obtained in this study are consistent with the feedback regulation of pancreatic enzyme secretion described in rats [22-24]. It has been shown that bile is important in controlling the rate of disappearance of trypsin and chymotrypsin activities from the small intestines [25]. In this study, although the pancreatic juice was diverted during the experiment, bile was constantly infused in the small intestine, thus maintaining the requirements of luminal trypsin and chymotrypsin activities necessary for feedback regulation of exocrine pancreatic secretion. The current study, thus, lends additional support to CCK-mediated feedback regulatory mechanism of pancreatic secretion. Absence of any significant differences in protein outputs and plasma CCK levels after food in control and nicotine treated rats indicate that nicotine treatment did not impair the CCK mediated feedback regulatory mechanism of pancreatic secretion. Administration of a low dose of nicotine for a prolonged period of time did not alter the physiologic response of CCK release but it affected gastrin release. Gastrin release in response to feeding has been reported in rats [26] and our observation is consistent with these findings. The gastrin release in nicotine treated animals was consistently higher compared to control suggesting the hyperfunction of antral G-cells induced presumably by nicotine. The increased response of gastrin release after food in nicotine treated rats may be due to increased catecholamine levels in the circulation. In studies conducted by other investigators, it has been reported that nicotine may indirectly enhance gastrin release through the release of catecholamines [27-30]. In earlier studies, we have shown that nicotine given to rats via drinking water produced major changes in gastric mucosa and pancreatic acinar cell [4]. We have also reported that acinar cells isolated from rats treated chronically with nicotine show significant inhibition in their ability to secrete amylase in response to CCK-8 and carbachol [31]. These studies indicate that functional impairments are induced by nicotine in stomach and exocrine pancreas of rats. The fact that the trypsin outputs after food were significantly decreased in nicotine treated animals when compared to control is indicative of the functional impairments of exocrine pancreas. These findings are consistent with our earlier published report [31]. Thus, the results from our current study indicate that food stimulated exocrine pancreatic secretion in normal and nicotine treated rats may have been influenced by the release of larger and perhaps smaller forms of CCK. The data lend support to the existence of a feedback regulatory system in rats and nicotine ingestion does not appear to impair or alter that regulatory mechanism.
Acknowledgements The authors wish to thank Mr. Danny McKay for his technical assistance in conducting radioimmunoassays of CCK and gastrin. Thanks are also due to Mrs. Mae Agnew for her excellent typing of this manuscript. This work was presented, in part,
19 at the 71st A n n u a l M e e t i n g o f F e d e r a t i o n o f A m e r i c a n Societies o f E x p e r i m e n t a l Biology a n d M e d i c i n e . T h i s s t u d y w a s s u p p o r t e d in p a r t by N I H R01-30415.
References 1 Surgeon General. In The Health Consequences of Smoking: Cancer and chronic lung disease in the workplace. Washington, D.C., U.S. Dept. of Health and Human Services, U.S. Government Printing Office, 1985. 2 Surgeon General. In Smoking and Health, Peptic Ulcer Disease. DHEW Publication No. PHS 79-50066, 1979. 3 Weir, J.M. and Dunn, J.E., Smoking and mortality: a perspective, Cancer, 25 (1970) 105-112. 4 Chowdhury, P., Hosotani, R., Chang, L.W. and Rayford, P.L., Metabolic and pathologic effects of nicotine on gastrointestinal tract and pancreas of rats, Pancreas, 5 (1990) 222-229. 5 Chowdhury, P., Hosotani, R. and Rayford, P. L., Weight loss and altered circulating GI peptide levels of rats exposed chronically to nicotine, Pharmacol. Biochem. Behav., 33 (1989) 591-594. 6 Jackson, B.M., Reeder, D.D. and Thompson, J.C., Dynamic characteristics of gastrin release, Am. J. Surg., 123 (1972) 137-142. 7 Rayford, P. L. and McKay, D. W., Cholecystokinin radioimmunoassay, J. Clin. Immunoassay, 10 (1987) 74-78. 8 Fried, G.M., Ogden, D.W., Swierczek, J., Greeley, G.H., Rayford, P.L. and Thompson, J. C., Release of CCK in conscious dogs: correlation with simultaneous measurements of gall bladder pressure and pancreatic protein secretion, Gastroenterology, 85 (1983) 1113-1119. 9 Baba, N., Suzuki, T., Tobe, T. et al., Influence of obstructive jaundice on pancreatic growth and on basal plasma levels of cholecystokinin and gastrin in rats, Dig. Dis. Sci., 31 (1986) 1233-1241. 10 Baba, N., Chowdhury, P., Inoue, K., Ami, M. and Rayford, P. L., Ileocaecal resection induced pancreatic growth in rats, Peptides, 6 (1985) 211-215. 11 Bradford, M.M., A rapid and sensitive method for quantitation of microgram quantities of protein utilizing the principle dye binding, Anal. Biochem., 72 (1976) 248-254. 12 Erlanger, B., Kokowsky, F. and Cohen, W., The preparation and properties of two new chromogenic substrates of trypsin, Arch. Biochem. Biophys., 95 (1961) 271-278. 13 Lilja, P., Weiner, I., Inoue, K., Fried, G. M., Greeley, G.H. and Thompson, J. C., Release of cholecystokinin in response to food and intraduodenal fat in pigs, dogs and man, Surg. Gynecol. Obstet., 159 (1984) 557-561. 14 Hayakawa, T., Katada, N., Kondo, T., Shibata, T., Kodaira, T., Ikei, N. and Hamoka, T., Meal stimulated cholecystokinin release and exocrine pancreatic secretion in dogs, Res. Exp. Med., 188 (1988) 19-25. 15 Himeno, S., Tarui, S., Kanayama, S., Kuroshima, T., Shinomura, Y., Hayashi, C., Tateishi, K., Imagawa, K., Hashimura, E. and Hamoka, T., Plasma cholecystokinin responses after ingestion of liquid meal and intraduodenal infusions of fat, amino acids, or hydrochloric acids in man: analysis with region specific radioimmunoassay, Am. J. Gastroenterol., 78 (1983) 703-707. 16 Solomon, T.E., Regulation of pancreatic secretion, Clin. Gastroenterol., 13 (1984) 657-678. 17 Katada, N., Noda, A. and Hayakawa, T., Postprandial pancreatic secretion and plasma hormones in dogs with pancreatic fistula, Am. J. Gastroenterol., 78 (1983) 740-744. 18 Liddle, R. A., Goldfine, I. D. and Williams, J. A., Bioassay of plasma CCK in rats: effects of food, trypsin inhibitor and alcohol, Gastroenterology, 87 (1984) 542-549. 19 Liddle, R.A., Green, G.M., Conrad, C.K. and Williams J.A., Proteins but not amino acids, carbohydrates, or fats stimulate cholecystokinin secretion in rat, Am. J. Physiol.~ 251 (1986) G243-G248. 20 Douglas, B. R., Woutersen, R. A., Jansen, J. M. M. J., DeJong, A.J.L. and Lamers, C. B. H., The influence of different nutrients on plasma choleeystokinin levels in the rat, Experientia Basel, 44 (1988) 12-23. 21 Linden, A., Carlquist, M., Hansen, S. and Uvnas-Moberg, K., Plasma concentrations ofcholecystokinin, CCK-8 and CCK-33, 39 in rats, determined by a method based on enzyme digestion of gastrin before HPLC and RIA detection of CCK, Gut 30 (1989) 213-222.
20 22 Louie, D.S., May, D., Miller, P. and Owyang, C., Cholecystokinin mediates feedback regulation of pancreatic enzyme secretion in rats, Am. J. Physiol., 250 (1986) G252-G259. 23 Fushiki, T. and Iwai, K., Two hypotheses on the feedback regulation of pancreatic enzyme secretion, FASEB J., 122 (1989) 121-126. 24 Folsch, U.R., Cantor, P., Wilms, H.M., Schafmayer, A., Becker, H.D. and Creutzfeldt, W., Role of cholecystokinin in the negative feedback control of pancreatic enzyme secretion in conscious rats, Gastroenterology, 92 (1987) 449-458. 25 Green, G.M. and Nasset, E.S., Importance of bile in regulation of intraluminal proteolytic enzyme activities in the rat, Gastroenterology, 79 (1980) 695-702. 26 Lichtenberger, L.M., Importance of food in the regulation of gastrin release and formation, Am. J. Physiol., 243 (1982) 429-441. 27 Lefholtz, K., Release induced by nicotinic agonists, In Paton, D. M., (Ed.), The release ofcatecholamines for adrenergic neurons. Oxford, Pergamon Press, 1979, pp. 275-301. 28 Starke, K., Regulation ofnoradrenaline release by presynaptic receptor systems, Rev. Physiol. Biochem. Pharmacol., 77 (1977) 2-124. 29 Christensen, K. C. and Stadil, F., Effect of epinephrine and norepinephrine on gastrin release and gastric secretion of acid in man, Scand. J. Gastroenterol., 11 (Suppl. 37) (1976) 87-92. 30 Hayes, J. R., Ardill, J., Shanks, R.G. and Buchanan, K. D., Effect of catecholamines on gastrin release, Metabolism, 27 (1978) 385-391. 31 Chowdhury, P., Hosotani, R. and Rayford, P.L., Inhibition of CCK or carbachol-stimulated amylase release by nicotine, Life Sci., 45 (1989) 2163-2168.
I1
Elsevier
REGPEP 01013
Meal-stimulated exocrine pancreatic secretion and release of GI peptides in normal and nicotine-treated rats Parimal C h o w d h u r y , M a s a a k i Ami*, Ryo H o s o t a n i * and Phillip L. Rayford Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, Little Rock, AR (U.S.A.) Received 10 September 1990; revised version received 17 December 1990; accepted 18 December 1990)
Key words: Effect of nicotine; Food stimulated; Plasma gastrin; Plasma CCK; Exocrine pancreatic secretion
Summary In rats, treated chronically with saline and nicotine, we studied the postprandial release of gastrin and cholecystokinin by specific radioimmunoassays and simultaneously measured secretory outputs of the exocrine pancreas. Rats were prepared surgically with gastric and pancreatic fistulas. Meal-stimulated release of peptides and exocrine secretory outputs were measured 24 h postoperatively in conscious rats. Infusion of food via intragastric cannula significantly stimulated plasma gastrin levels in both control and nicotine treated rats. Postprandial gastrin levels in nicotine treated rats were significantly higher compared to gastrin levels obtained after food in untreated control rats. Plasma CCK levels were increased in both groups after food. These levels remained significantly elevated from the basal values only for a transient period following infusion of the liquid meal. There were no differences in postprandial plasma CCK levels between the two groups. Outputs of exocrine pancreatic volume, protein and trypsin increased significantly after food in both control and nicotine treated groups of rats. The differences in outputs of volume and protein between the two groups of rats were not significant; however, the trypsin outputs in the nicotine rats were decreased significantly when compared to control rats. * Present address: First Department of Surgery, Kyoto University, Kyoto, Japan. Correspondence: P. Chowdhury, Department of Physiology and Biophysics, University of Arkansas for Medical Sciences, 4301 W. Markham, Little Rock, AR 72205, U.S.A. 0167-0115/91/$03.50 © 1991 Elsevier Science Publishers B.V. (Biomedical Division)
12
The data indicate that in rats, administration of food stimulated the release of immunoreactive gastrin and CCK with concomitant increase in exocrine pancreatic secretions of volume, protein and trypsin. Chronic nicotine treatment and its effect on food, however, appeared to have induced hyperfunction of G-cells that resulted in increased gastrin secretion and a decrease in trypsin secretion by exocrine pancreas. These data may have important implications in the etiology of the development of exocrine pancreatic dysfunction in chronic smokers.
Introduction
Nicotine, a component found in substantial amounts in tobacco, cigarettes and smokeless tobacco, has been linked to the development of various pathophysiological disorders [1-3]. Previous studies in rats exposed to nicotine via different routes of administration showed the development of gastrointestinal and pancreatic lesions and it was suggested that these lesions might have been mediated through alteration of hormonal and metabolic factors [4]. The precise mechanism by which nicotine potentiates the development of gastrointestinal mucosal injury has not been clearly delineated. In earlier studies, we have reported that animals exposed chronically to low levels of nicotine via gavage feeding had significantly increased basal circulating levels of gastrin (92 pM vs. 47 pM) and cholecystokinin (106 pM vs. 81 pM) when compared to the animals exposed to saline by the same routes [5]. The results suggest that intragastric topical administration of nicotine stimulates the endocrine cells that specifically secrete gastrin and CCK. The observation that chronic ingestion of nicotine induced higher basal plasma levels of gastrin and CCK, prompted us to study the effect of a physiologic stimulus on postprandial release and actions ofgastrin and CCK. In this study, we report the effect of intragastric administration of food on exocrine pancreatic secretions and plasma levels of gastrin and CCK in both control and chronic nicotine treated rats.
Materials and Methods
Male Sprague-Dawley rats, 8 weeks old and approx. 150 g body weight, were divided into two groups of 12 rats each and were used in the study. The animals were fed daily at 9:00 a.m. for 120 days with either saline or nicotine (20 mg/kg/day (0.12 mmol)) via a gavage tube. Animals from each group were housed individually in galvanized steel metabolic cages (7 in x 10 in x 7 in) equipped with an automatic on-line water intake system in a room with 12 h light-dark cycle. Weight and food intake of each rat was monitored at regular time intervals. At 120 days, each rat was fasted for 24 h, anesthetized and an external jugular vein cannulated with PE-90 tubing. An abdominal incision was made and a gastric cannula (PE-160) was placed in the avascular part of the stomach (fundic portion) to introduce the liquid meal. A pancreatic cannula (PE-50)
13 was then placed and secured in the pancreatic duct to collect pancreatic juice continuously. Prior to the placement of the pancreatic cannula the bile duct was ligated near the liver beyond the point where it is surrounded by pancreatic tissue. Bile duct was cannulated with a PE-50 tubing of about 3 cm long and the bile was diverted to the duodenum at 1.0 cm from the pylorus ring. All PE tubings (cannulae) were supported and secured by purse string nylon sutures with 6.0 prolene (Ethicon, Sommervile, N J) and were exteriorized through the subcutaneous tissues. The incision was then closed and rats were transferred to Bollman type restraining cages (Fisher Scientific, Fairlawn, N J) for recovery. During recovery, the animals received water but no food for an additional 24 h. The i.v. cannula was kept open through infusion of saline at a flow rate of 0.4 ml/h by infusion pump (Sage Instruments). Composition of liquid meal: The liquid meal consists of a packet of sustacal nutritional powder (Mead Johnson, Nutritional Division, Evansville, IN, containing 20.5 g protein, 9.2 g fat, 48 g carbohydrate, vitamins, minerals and salts and dissolved in 100 ml of whole milk).
Sample collection During the experiment two basal blood samples were taken at 9 : 00 a.m. which were followed successively by two 20 rain basal pancreatic juice samples. 5 ml of liquid meal, as described above, were then infused through the gastric cannula at a flow rate of 3.94 ml/min. Following the infusion of liquid meal, blood samples (2 ml) were collected at 10 min intervals through juglar vein catheter for 40 min while pancreatic juice samples were collected continuously for 100 min at 20 min intervals. The volume of blood withdrawn was replaced with equal volume of 4~o bovine serum albumin made up in saline. Blood was centrifuged and plasma stored at - 80 ° C. Pancreatic juice collected in the pancreatic cannula (PE tubing) was measured for volume and diluted to a fixed volume with 200 #1 of saline and stored in the freezer for measurements of protein and trypsin. Measurements Gastrin and CCK levels in plasma were measured using specific radioimmunoassays [6,7]. These assays have been validated for measuring gastrin and CCK in tissues and body fluid of dogs [8] and rats [9,10]. Protein concentration in the pancreatic juice was measured by Bradford's method [ 11 ] using bovine serum albumin as standard. Outputs of total protein were calculated by multiplying the protein concentration times the total volume of pancreatic juice obtained at that time interval. Trypsin concentration in the pancreatic juice was measured using the method of Erlanger et al. [ 12] employing N-~-benzoyl-arginine-p-nitroanilide (BAPNA) as substrate. Prior to trypsin assay, pancreatic juice was incubated with enterokinase (25 #g). Outputs of trypsin were calculated in the same manner as described above for protein. Calculations Hormone concentrations were calculated as pM and expressed as mean + S.E. of the mean. Basal and stimulated values for both plasma and exocrine pancreatic outputs
14
were divided by its corresponding basal value and multiplied by 100. All values were reported as percent increments over the basal. ANOVA and Student's unpaired t-tests were used to test the significant differences between the means. Differences with a P-value of less than 0.05 were considered statistically significant.
Results
Food intakes were not different between the two groups throughout the entire treatment period. At killing, body weights of rats treated with saline were 572 + 16 g and that of nicotine were 499 + 7 g, respectively. The differences in weight gain between the two groups were statistically significant, P < 0.05. Basal plasma levels of gastrin in normal and nicotine treated rats were 39 _+ 11 pM and 60 + 12 pM, respectively. Plasma levels of gastrin, measured after intragastric infusion of liquid meal are shown in Fig. 1 and are represented as percent increments over the basal. Infusion of liquid meal significantly stimulated the release of plasma gastrin levels (as shown by significant increase over basal) in both saline and nicotine treated rats at all time points. The increments in gastrin levels after food in nicotine
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40
TIME (MIN)
Fig. I. Postprandial levels ofimmunoreactive plasma gastrin (shown as percent increments over the basal value) in rats. The basal value was considered as 100% (see text for details). O , control; IN, nicotine treated. *, P < 0.05, significantly different from basal; ~ , P < 0.05, significantly different between two groups.
15
treated rats, however, was significantly higher when compared to the gastrin levels in the saline treated group (Fig. 1). Basal plasma levels of CCK in normal and nicotine treated rats were 68 + 4 and 86 + 11 pM, respectively. Plasma CCK levels measured after intragastric infusion of food are shown in Fig. 2. Plasma levels of CCK increased significantly but transiently after intragastric infusion of food in both groups. The levels of CCK measured at 20, 30 and 40 min time periods were not significantly different from their respective basal. The differences in postprandial plasma CCK levels between the two groups were not significant (Fig. 2). Outputs of pancreatic juice volume, protein and trypsin measured before and after the infusion of liquid meal were calculated as percent of basal and are shown in Figs. 3 and 4. Pancreatic juice volumes measured at basal (prior to food administration) in control and nicotine treated rats were 119 + 12 and 114 + 11 #1, respectively. These values increased to 158 + 17 and 149 + 18/~1 at 40 min after food. 10 min after food infusion, the outputs of pancreatic juice volume in the saline treated group were 17 ~o over the basal, P < 0.05; in comparison, the volume outputs in nicotine treated group were 40% above the basal, P < 0.05 (Fig. 3, upper panel). Maximal volume outputs after food were obtained at 40 rain in both groups and at 100 min, the volume outputs in each of the groups were not different from their own basal. The protein concentrations in the pancreatic juice samples obtained at basal in control and nicotine treated rats were of 1.2 + 0.3 and 1.3 + 0.3 mg/ml, respectively. These values 200
-
175 ' 'k 150 ' A .-I < 03 ,'n
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125,
100 '
[]
NICOTINE
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CONTROL
75-
.-I n SO,
25,
BASAL
10
20
TIME
30
40
(MIN)
Fig. 2. Postprandial levels ofimmunoreactive plasma CCK (shown as percent increments over the basal) in rats. The basal was considered as 100% (see text for details), r-l, control; P3, nicotine treated. *, P < 0.05. significantly different from basal.
16
160
o
Food
RATS
P
-
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= >-5
/ I00-
_~
550-
~
140-
v
120-
Nicotine
~ n t r o I
300c~
) 250-
~/Confrol
._C.
o 200" (3_
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ao,o,
2'o
2o
go
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MINUTES Fig. 3. Postprandial exocrine secretory outputs in rats. Volume outputs in upper panel. Protein outputs in lower panel. O, control; @, nicotine treated. *, P < 0.05, significantly different from control.
increased to 2.3 + 0.4 and 2.8 + 0.4 mg/ml at 40 min after food. The protein outputs for both groups were maximal at 40 min after food (Fig. 3, lower panel). The outputs of protein measured beyond 60 min after food were not different from the basal in any of the two groups. No significant differences in postprandial protein outputs were found between the control and nicotine treated groups. Outputs of trypsin measured (percent of basal) in the pancreatic juice are shown in Fig. 4. Trypsin concentrations measured in pancreatic juice samples obtained at basal in control and nicotine treated rats were 900 + 199 and 949 + 128 #g/ml, respectively. These values increased to 1121 + 186 and 1412 _+ 169#g/ml at 40min after food. Trypsin outputs increased significantly in both groups after food and were maximal at 40 rain. The integrated 100 min outputs of trypsin (° o basal) between control (1031 + 220) vs. nicotine (678 + 101) were significantly different, P < 0.05. Trypsin outputs in nicotine treated groups after food were significantly lower when compared to saline treated group.
17
RATS
300" ¢J w
m 2502 200-
F°°d /~T~ X
~~Contro,
c
,-
150"
I00" Basal
20
4'0 6'0 MINUTES
8'0
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Fig. 4. Postprandial exocrine secretory outputs of trypsin in rats. ©, control; @, nicotine treated. *, P < 0.05, significantly different from control.
Discussion Food intake was not different between control and nicotine treated rats; however, body weights were significantly greater in control rats when compared to nicotine rats. This suggests that food intake was not a major factor in body weight loss in nicotine treated rats. The results of the current study show that in rats, intragastric administration of food stimulated the release of gastrin and CCK. The data further demonstrate that release of these G I peptides are associated with concomitant stimulation of pancreatic secretory outputs of protein and trypsin. These data are consistent with the postprandial release of G I peptides and associated exocrine pancreatic secretory outputs shown in other species [ 13-17]. The postprandial release of C C K in rats found in this study is transient when compared to other species. These results are consistent with the reported studies [18,19]; however, results from other laboratories showed that several types of luminal nutrients were capable of producing prolonged increase of plasma C C K [20]. In rats, two forms of circulating C C K have been described (by gel filtration in combination with bioassay): one peak of intermediate size between CCK-33 and CCK-8 and another peak with the same size as CCK-8 [ 18]. In a more recent study, two types of C C K are detected, one corresponding to CCK-8 and one to CCK-33, 39 [21]. The antibody used in this study for C C K radioimmunoassay has its greatest affinity for molecular forms of CCK-33 and other larger forms [7]. The transient rise of C C K as found in our study represents CCK-33, but does not rule out the possibility of circulating
18 CCK-8 which was not measured in this study. The prolonged response of pancreatic enzyme secretion after food may be due to the circulating levels of CCK-8; however, other humoral and neural factors may also be implicated in this phenomenon. The data obtained in this study are consistent with the feedback regulation of pancreatic enzyme secretion described in rats [22-24]. It has been shown that bile is important in controlling the rate of disappearance of trypsin and chymotrypsin activities from the small intestines [25]. In this study, although the pancreatic juice was diverted during the experiment, bile was constantly infused in the small intestine, thus maintaining the requirements of luminal trypsin and chymotrypsin activities necessary for feedback regulation of exocrine pancreatic secretion. The current study, thus, lends additional support to CCK-mediated feedback regulatory mechanism of pancreatic secretion. Absence of any significant differences in protein outputs and plasma CCK levels after food in control and nicotine treated rats indicate that nicotine treatment did not impair the CCK mediated feedback regulatory mechanism of pancreatic secretion. Administration of a low dose of nicotine for a prolonged period of time did not alter the physiologic response of CCK release but it affected gastrin release. Gastrin release in response to feeding has been reported in rats [26] and our observation is consistent with these findings. The gastrin release in nicotine treated animals was consistently higher compared to control suggesting the hyperfunction of antral G-cells induced presumably by nicotine. The increased response of gastrin release after food in nicotine treated rats may be due to increased catecholamine levels in the circulation. In studies conducted by other investigators, it has been reported that nicotine may indirectly enhance gastrin release through the release of catecholamines [27-30]. In earlier studies, we have shown that nicotine given to rats via drinking water produced major changes in gastric mucosa and pancreatic acinar cell [4]. We have also reported that acinar cells isolated from rats treated chronically with nicotine show significant inhibition in their ability to secrete amylase in response to CCK-8 and carbachol [31]. These studies indicate that functional impairments are induced by nicotine in stomach and exocrine pancreas of rats. The fact that the trypsin outputs after food were significantly decreased in nicotine treated animals when compared to control is indicative of the functional impairments of exocrine pancreas. These findings are consistent with our earlier published report [31]. Thus, the results from our current study indicate that food stimulated exocrine pancreatic secretion in normal and nicotine treated rats may have been influenced by the release of larger and perhaps smaller forms of CCK. The data lend support to the existence of a feedback regulatory system in rats and nicotine ingestion does not appear to impair or alter that regulatory mechanism.
Acknowledgements The authors wish to thank Mr. Danny McKay for his technical assistance in conducting radioimmunoassays of CCK and gastrin. Thanks are also due to Mrs. Mae Agnew for her excellent typing of this manuscript. This work was presented, in part,
19 at the 71st A n n u a l M e e t i n g o f F e d e r a t i o n o f A m e r i c a n Societies o f E x p e r i m e n t a l Biology a n d M e d i c i n e . T h i s s t u d y w a s s u p p o r t e d in p a r t by N I H R01-30415.
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20 22 Louie, D.S., May, D., Miller, P. and Owyang, C., Cholecystokinin mediates feedback regulation of pancreatic enzyme secretion in rats, Am. J. Physiol., 250 (1986) G252-G259. 23 Fushiki, T. and Iwai, K., Two hypotheses on the feedback regulation of pancreatic enzyme secretion, FASEB J., 122 (1989) 121-126. 24 Folsch, U.R., Cantor, P., Wilms, H.M., Schafmayer, A., Becker, H.D. and Creutzfeldt, W., Role of cholecystokinin in the negative feedback control of pancreatic enzyme secretion in conscious rats, Gastroenterology, 92 (1987) 449-458. 25 Green, G.M. and Nasset, E.S., Importance of bile in regulation of intraluminal proteolytic enzyme activities in the rat, Gastroenterology, 79 (1980) 695-702. 26 Lichtenberger, L.M., Importance of food in the regulation of gastrin release and formation, Am. J. Physiol., 243 (1982) 429-441. 27 Lefholtz, K., Release induced by nicotinic agonists, In Paton, D. M., (Ed.), The release ofcatecholamines for adrenergic neurons. Oxford, Pergamon Press, 1979, pp. 275-301. 28 Starke, K., Regulation ofnoradrenaline release by presynaptic receptor systems, Rev. Physiol. Biochem. Pharmacol., 77 (1977) 2-124. 29 Christensen, K. C. and Stadil, F., Effect of epinephrine and norepinephrine on gastrin release and gastric secretion of acid in man, Scand. J. Gastroenterol., 11 (Suppl. 37) (1976) 87-92. 30 Hayes, J. R., Ardill, J., Shanks, R.G. and Buchanan, K. D., Effect of catecholamines on gastrin release, Metabolism, 27 (1978) 385-391. 31 Chowdhury, P., Hosotani, R. and Rayford, P.L., Inhibition of CCK or carbachol-stimulated amylase release by nicotine, Life Sci., 45 (1989) 2163-2168.