Cooke, Nahrwold and Grossman, 1967]. In the present experiments, attempts. Wales, 2033, Australia.

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1 Quarterly Journal of Experimental Phyeiology (1973) 58, BASAL AND POSTPRANDIAL PANCREATIC SECRETION IN RATS. By H. M. SiHw and T. J. HEATH. From the School of Physiology and Pharmacology, The University of New South Wales, Kensington, New South Wales, 2033, Australia. (Received for publication 13th March 1973) When pancreatic juice which was collected separate from bile was diverted from the duodenum of rats for 24 hr, the basal flow rate of juice and its output of protein increased greatly over values recorded immediately after operation. Similar increases were recorded when pancreatic juice was recirculated back into the duodenum for 24 hr. Infusion of bicarbonate or enzyme into the duodenum failed to affect these increases in secretion. Thus the hypersecretion of juice did not appear to be the result of loss of juice from the duodenum. Attempts were then made to estimate rates of pancreatic secretion without diverting bile from the common bile duct. Immediately after operation, rates of pancreatic secretion estimated indirectly from the differences between combined bile-pancreatic juice and bile alone were higher than in those rats in which pancreatic juice was collected by diverting bile from the common bile duct. This suggested that bile in some way assisted the passage of viscous pancreatic juice down the common bile duct. Indirect estimates of pancreatic secretion made 24 hr after operation were greater than the estimates made immediately after operation but were similar to those values obtained after 24 hr in rats from which pure pancreatic juice was collected. This was taken as evidence to support the view that the trauma associated with anaesthesia and surgery may depress the rate of pancreatic secretion and that hypersecretion of juice may indeed be a normal phenomenon. In contrast to the situation reported to exist in dogs, indirect estimates of the effects of feeding on pancreatic secretion were similar irrespective ofwhether secretions were diverted from or returned to the duodenum. At present, there exists very little precise information on either the basal or the postprandial rate of secretion ofpancreatic juice in the rat. It has been shown that when pancreatic juice is diverted from the duodenum of rats and the animals are allowed to recover from the direct effects of anaesthesia and operation for 24 hr, the rate of flow of pancreatic juice and its content of protein increase greatly over those values recorded immediately after operation [Grossman, 1958; Shaw and Heath, 1972]. These rates are much higher than those recorded from anaesthetized rats, and because of this some workers consider that high levels of secretion represent a pathological hypersecretion of the pancreas, and that the volume of juice secreted may have been influenced by diverting pancreatic juice from the intestine [Grossman, 1958]. There are also reports to indicate that pancreatic secretion in response to a meal is altered when pancreatic juice is diverted from the intestine [Annis and Hallenbeck, 1951; Cooke, Nahrwold and Grossman, 1967]. In the present experiments, attempts were made to show that loss of pancreatic juice from the duodenum did not influence either the basal or postprandial level of pancreatic secretion, and that in rats which have recovered from effects of anaesthesia there may exist a high continuous rate of pancreatic secretion. 335

2 336 Shaw and Heath MATERIALS AND METHODS Male albino rats which weighed from g were fasted for 18 hr, then anaesthetized with intraperitoneal pentobarbital sodium (40 mg/kg). Fourteen of the rats each had two cannulas inserted in the common bile duct to collect bile and pancreatic juice separately [Shaw and Heath, 1972]. This method of collecting pancreatic juice entailed the diversion of bile from the common bile duct. Two clear vinyl tubes (i.d mm, o.d mm) were inserted into the duodenum of each rat approximately 15 mm from the pylorus, and were fastened with a purse-string suture. An additional group of 9 rats each had a single cannula inserted in the distal end of the common bile duct to collect combined bile-pancreatic juice. In another group of 6 rats, each had a single cannula placed into the common bile duct at the hilus of the liver to collect pure bile. Each of the rats in the last two groups had a single vinyl tube placed in the duodenum. All rats were allowed to recover from the direct effects of the operation and anaesthetic in Bollman restraining cages for 24 hr. Rats received normal saline either from drinking bottles, or in some cases by infusion into the gut through one of the duodenal cannulas at 1-8 ml./hr with a Harvard infusion pump. Samples of pancreatic juice were collected in plastic vials and the volumes estimated from the net weight. When estimations of bicarbonate concentration were required, pancreatic juice was collected under paraffin oil. The concentration of bicarbonate was estimated by the titrimetric method described by Shaw and Heath [1972], and the concentration of protein by the method of Lowry, Rosebrough, Farr and Randall [1951]. Analyses of variance and Student's t tests were used to estimate the statistical significance of changes that occurred in the various parameters during the experiments. The standard error of the mean (S.E.M.) was estimated from the error mean square (E.M.S.) and the sample number (N): S.E.M. = (E.M.S./N)i RESULTS 1. Basal pancreatic secretion estimated in rats provided with cannulas of the common bile duct to enable collection of pancreatic juice separate from bile The experiments performed on this group of 14 rats were designed to study basal pancreatic secretion in rats in which pure pancreatic juice was collected by diverting bile from the common bile duct. In all rats the flow rate of pancreatic juice and its content of protein were estimated over two 20-min collection periods immediately after cannulation of the common bile duct. No initial estimate was made of the output of bicarbonate, since it would have taken needed for each estimation. several hours to collect the 200,ll. Effects of diversion of pancreatic juice from the duodenum In 6 of the 14 rats, pancreatic juice was diverted from the duodenum, whilst bile was recirculated back through one of the duodenal cannulas. Immediately after cannulation of the common bile duct and while the rats were still under pentobarbital anaesthesia, they secreted pancreatic juice at ,ul./min/kg (Table I). Pancreatic juice was then drained for 24 hr, during which time rats received an infusion of normal saline through the other duodenal cannula. The basal rate of flow of pancreatic juice and its content of bicarbonate and protein

3 Pancreatic Secretion in Rats were then determined during three 40-min collection periods. It was found that the rate of flow of juice and its content of protein had increased significantly (P < 0.001), and had reached values many times greater than those recorded immediately after operation (Table I). TABLE I. Effects on basal pancreatic secretion of diversion or recirculation of pancreatic juice or infu8ion of bicarbonate and trypsin into the duodenum Infusion of Diversion' Recirculation2 HCO, Trypsin4 Number of rats Immediately after operation Flow (l./min/kg) 2-5± ± ±0-2 2*4±O02 Protein output (mg/hr/kg) 9 1± O± ±07 24 hours after operation Flow (,ul./min/kg) 33-4± ± ± ±2-1 Protein output (mg/hr/kg) 37*6±3*1 406± ± ±3-7 Bicarbonate output (,u-equiv/min/kg) 076±0± ± ± ±0*08 1 Pancreatic juice was diverted from the duodenum since cannulation of the common bile duct. 2 Pancreatic juice was recirculated into the duodenum since cannulation of the common bile duct. 3 Pancreatic juice was diverted from the duodenum but sodium bicarbonate solution was infused continuously since cannulation of the common bile duct. 4 Pancreatic juice was diverted from the duodenum for 24 hr and then trypsin-sodium bicarbonate solution was infused into the duodenum for 3 hr. Effects of recirculation of pancreatic juice back into the duodenum In 4 of the 14 rats, bile and all pancreatic juice except one 50,ul. aliquot necessary for protein estimation was recirculated back into the duodenum of the rats for 24 hr. The rate of flow of pancreatic juice and its content of protein were determined immediately after cannulation of the common bile duct, and during three 40-min collection periods 24 hr later. The results obtained from these rats initially and those obtained after 24 hr were similar to those obtained with the first group of 6 rats from which pancreatic juice had been drained for 24 hr (Table I). Effects of diversion of pancreatic juice but infusion of bicarbonate and enzyme to the duodenum In the remaining 4 of the 14 rats, pancreatic juice was diverted from the duodenum, whilst bile was recirculated back through one of the duodenal cannulas. Initial values for flow rate and protein output were estimated, and did not differ significantly from those recorded in the first two groups of rats (Table I). VOL. LVIIII, NO

4 338 Shaw and Heath During the entire 24-hour period after operation, each rat received an infusion of NaHCO m-equiv/hr into the duodenum. This solution contained NaHCO3 dissolved in normal saline to produce a final concentration of 30 m- equiv/l. The ph of this solution was 8-3 and the osmolality was adjusted to 280 m-osmole/kg with distilled water. It was estimated that this amount of bicarbonate was more than three times the amount normally delivered to the duodenum by pancreatic juice [Shaw and Heath, 1972]. The basal rate of flow of pancreatic juice and its content of bicarbonate and protein were determined during three 40-min periods. It was found that the rate of flow of pancreatic juice and its content of bicarbonate and protein reached values similar to those recorded 24 hr after operation in the first group of rats in which pancreatic juice was diverted from the duodenum (Table I). The rats then received an infusion of trypsin (15 mg/hr) into the duodenum for the next five 40-min periods and pancreatic juice was collected during each period. This solution contained pancreatic trypsin (Sigma Chemical Company, St. Louis, Mo.) 8 mg/ml. in sodium bicarbonate 30 m-equiv/l.; the ph of the solution was 8-3 and the osmolality was 280 m-osmole/kg. The trypsin-sodium bicarbonate solution was stored at 0 C, but brought to 37 C just prior to infusion. It was estimated that the amount of enzyme activity infused into the duodenum of these rats was approximately the same as would have been secreted by rats with chronic diversion of pancreatic juice [Shaw and Heath, 1972]. Although this trypsinsodium bicarbonate solution was infused into the duodenum for more than 3 hr, no changes in pancreatic secretion were detected (Table I). 2. Basal and postprandial pancreatic secretion estimated in rats in which pancreatic juice was not separated from bile The experiments performed on rats from this group of 15 rats were designed to study the basal and postprandial levels of pancreatic secretion under more physiological conditions. Pancreatic juice is a viscous substance, and because of this, bile may normally assist the passage of pancreatic juice down the common bile duct. Any errors in estimating pancreatic secretory rate brought about by diverting bile from the common bile duct were therefore obviated by the collection of combined bile-pancreatic juice. In all rats, the flow rate of secretions and their content of protein were estimated directly after cannulation of the duct. Relatively large volumes of juice were needed to estimate bicarbonate concentration and as we were attempting to recirculate as much juice as possible into the duodenum, these estimates were not made. Effects of recirculation of combined bile-pancreatic juice back into the duodenum, and the response of the combined secretion to feeding In 9 of the 15 rats, all bile-pancreatic juice except one 50,ul. aliquot necessary for protein estimation was recirculated back into the duodenum through the duodenal cannula for 24 hr, then the rate of flow of bile-pancreatic juice was estimated during three 40-min collection periods. It was found that the flow rate and protein output of the combined secretion had increased significantly

5 Pancreatic Secretion in Rats over values obtained for these parameters immediately after operation (P < 0-001; Table II). The responses of bile-pancreatic juice to feeding were then determined. Rats were offered 3 g of dried food (Liver Dinner, Kg Pet Foods, Carnation Co. Pty. Ltd., Blacktown, N.S.W.) during the fourth 40-min period. Bile-pancreatic juice was collected for the subsequent three 40-min collection periods, but was re-infused back into the duodenum immediately after flow rate had been estimated. Significant increases over control values were recorded during all four 40-min collection periods in the rate of flow of the combined secretion (P < 0-001; Fig. 1). After the feeding experiments, bile-pancreatic juice was diverted from the duodenum of the rats for a further period of 24 hr. After this period of drainage, the basal flow rate of bile-pancreatic juice fell to 51-0±5*4,ul./min/kg. This was assumed to be due to the interruption of the enterohepatic circulation of bile salts and the consequent fall in the bile fraction of the combined secretion. The rats were then fed again, but the combined secretion was not returned to the duodenum. It was found that the average percentage increase in flow after feeding (41 %) was similar to that obtained on the previous day during continuous replacement of the secretion into the duodenum (Fig. 1). TABLE II. Effects on indirect estimates of basal pancreatic secretion of recirculation of pancreatic juice back into the duodenum Estimated Bile-pancreatic pancreatic juice Bile juice' Number of rats 9 6 Immediately after operation Flow (,ul./min/kg) 51-6±1'5 37.5±i ±2.1 Protein output (mg/hr/kg) 33.0± ± ± hours after operation2 Flow (,ul./min/kg) 66.3 ± ± ±4-1 Protein output (mg/hr/kg) 41.7± ± ± Indirect estimates of flow rate and protein output in pancreatic juice were made by subtracting the values for these parameters in bile from the corresponding values in combined bile-pancreatic juice. 2 Both combined bile-pancreatic juice and bile were recirculated into the duodenum since cannulation of the common bile duct. Effects of recirculation of bile back into the duodenum, and the choleretic response to feeding In the remaining 6 of the 15 rats, a protocol similar to that used in the previous group of 9 rats was followed except that in these rats, bile alone was collected. When the enterohepatic circulation of bile salts was kept intact by recirculating bile back into the duodenum for 24 hr, no significant changes occurred in the flow rate or output of protein in bile (Table II). When these rats were fed and bile was continuously re-infused into the duodenum, small but significant increases over control values were recorded during all four 40-min collection periods in the rate of flow of bile (P < 0-001; Fig. 1). After bile was drained for 24 hr, its flow rate decreased to 17-7 ±0-6

6 Shaw and Heath 4l./min/kg due to interruption of the enterohepatic circulation of bile salts. When these rats were fed, but bile was not returned back into the duodenum, the average percentage increase in flow after feeding (18%) was similar to that obtained on the previous day during continuous replacement of bile into the duodenum (Fig. 1). RETURNED NOT RETURNED 120 COMBINED BILE-PANCREATIC JUICE FOOD FOOD 90 'I I cm 1 o c I E BILE FOOD rihs s * * s ~~ 340 I II I- FOOD frrm I I I " I I I I htff TIME (nin) FIG. 1. Effects of diversion of combined bile-pancreatic juice and bile from the duodenum on the postprandial level ofthese secretions. The top panels show the mean flows of combined bile-pancreatic juice in 9 rats after feeding when the combined secretions were returned and not returned back into the duodenum. The bottom panels show the mean flows of bile in 6 rats after feeding when bile was returned and not returned back into the duodenum. Vertical bars at the left represent ±S.E.M. for a single time interval. It was considered that a more physiological measure for the values of the parameters in pancreatic juice could be made by subtracting the- values for flow rate and protein output in bile from the corresponding values for these parameters in combined bile-pancreatic juice. If it is assumed that the differences represented estimates of pancreatic secretion, it was found that both flow rate and protein output immediately after operation were much higher than

7 Pancreatic Secretion in Rats those estimated immediately after operation from rats in which pure pancreatic juice was collected by diverting bile from the common bile duct (P < 0*001; Tables I and II). It was also found that after 24 hr of recirculation, the values for these parameters tended to increase significantly (P < 0 001) over those obtained immediately after operation, and to approach levels obtained after 24 hr recirculation from rats in which pure pancreatic juice was collected (Tables I and II.) DIsCUSSION The present experiments have indicated that when pancreatic juice is diverted from fasting conscious rats for 24 hr, the rate of flow of pancreatic juice and its content of protein increase greatly over those values recorded immediately after operation. Similar findings have been reported by Grossman [1958]. In other rats in which both bile and pancreatic juice were recirculated for 24 hr, the values for flow of pancreatic juice and content of protein and bicarbonate reached values similar to those obtained in rats when pancreatic juice was diverted from the duodenum. These findings are similar to those of Zucker, Newburger and Berg [1932] and Scott, Graham and McCartney [1940] who showed that uninterrupted introduction of pancreatic juice to the duodenum of conscious dogs failed to diminish the continuous high rate of flow of pancreatic juice. Nevertheless, the situation appears to differ in anaesthetized rats, since Green and Lyman [1972] have shown that return of bile-pancreatic juice prevents any increases in pancreatic enzyme secretion. In rats in which pancreatic juice was diverted from the duodenum, it was found that neither the infusion of bicarbonate solution nor trypsin-bicarbonate solution into the duodenum prevented these increases in pancreatic secretion. Thus it would appear that these increases are probably not due to either excess acidity of the duodenum as suggested by Cooke, Nahrwold and Grossman [1967], or absence of feedback inhibition from proteolytic enzymes in the intestine as suggested by Green and Lyman [1972], both of which could be brought about by diversion of pancreatic juice from the duodenum. Most of the information on rate ofpancreatic secretion in rats has been derived from experiments on anaesthetized animals, in which very small amounts of pancreatic juice are secreted under basal conditions [Debray, de la Tour, Vaille, Roze and Souchard, 1962; Ramirez, Hubel and Clifton, 1966; Heatley, 1968; Dockray, 1972]. Thus the high rates of flow recorded in conscious rats have been regarded as unphysiological by some workers [Dockray, 1972; Green and Lyman, 1972]. The initial slow rate of secretion in anaesthetized rats may be due to two factors. Firstly, it seems likely that anaesthesia and the immediate effects of operation may depress pancreatic secretion [Colwell, 1951; Scott, Graham and McCartney, 1940; Zucker, Newburger and Berg, 1932]. Secondly, in order to collect pure pancreatic juice and estimate flow rate, most workers insert a cannula into the most distal portion of the common bile duct, then either ligate the bile duct at the hilus of the liver [Dockray, 1972], drain bile to the exterior [Debray, de la Tour, Vaille, Roze and Souchard, 1962] or 341

8 342 Shaw and Heath redivert bile to the duodenum [Love, 1957]. In all these cases an abnormal situation may be created, since bile, which is secreted at high pressures even in anaesthetized animals [Mann and Foster, 1918] and may normally assist the passage of viscous pancreatic juice down the common bile duct, is absent, and so cannot perform this function. As a result of this diversion of bile from the common bile duct, coupled with the effects of anaesthesia, abnormally irregular or slow flow of pancreatic juice may occur, and this may make the estimation of the rate of basal pancreatic secretion technically difficult in anaesthetized rats [Heatley, 1968]. When the possible errors brought about by diversion of bile from the common bile duct were obviated, and initial rates of flow of pancreatic juice and output of protein were estimated from the difference between combined bile-pancreatic juice and bile, evidence was obtained to support the view that in fasted anaesthetized rats immediately after operation, there exists a high rate of flow of pancreatic juice and output of protein. When the rats were allowed to recover from the direct effects of operation and anaesthesia for 24 hr, and when both secretions were recirculated to the animals, the values for these parameters in pancreatic juice increased, indicating that pancreatic secretion may have been initially depressed by the trauma of anaesthesia and operation. When rats were fed, it was found that regardless of whether combined bilepancreatic juice was being continuously returned to the animals or not, the average percentage increases in the flow of the combined secretions were similar. Since in another group of rats, the average percentage increases in bile flow were also similar irrespective of whether bile was returned to the duodenum or not, it was considered reasonable to assume that the pancreatic response to feeding might also be the same whether pancreatic juice was returned or not. These findings are in contrast to the situation reported to exist in dogs. Annis and Hallenbeck [1951] showed that returning pancreatic juice to the small intestine during feeding experiments reduced by about 50% the volume of juice and its content of bicarbonate. Similarly, Cooke, Nahrwold and Grossman [1967] found that when pancreatic juice was re-introduced into the duodenum of dogs, peak bicarbonate and volume of juice was about 60% of that obtained when all pancreatic juice was diverted to the exterior. Nevertheless, they could demonstrate no change in the pancreatic enzyme response to feeding, regardless of whether pancreatic juice was diverted from the duodenum or not. Therefore, it is possible that the control of postprandial pancreatic secretion in rats could differ in some respects from that in dogs. The present experiments have indicated that in rats, the rate of flow of pancreatic juice and its output of protein may be initially depressed due to the effects of anaesthesia and operation. Diversion of pancreatic juice from the duodenum does not appear to cause the hypersecretion of juice or influence postprandial levels of secretion. Since indirect estimates of pancreatic secretion have provided evidence in support of the view that rates of pancreatic secretion recorded from anaesthetized rats may be artefactual, it is possible that the hypersecretion of pancreatic juice in this species may represent a normal phenomenon.

9 Pancreatic Secretion in Rats 343 REFERENCES ANNIS, D. and HALLENBECK, G. A. (1951). Effect of excluding pancreatic juice from duodenum on secretory response of pancreas to a meal. Proceedings of the Society of Experimental Biology and Mledicine, 77, COLWELL, A. R. (1951). Collection of pancreatic juice from rats and consequences of its continued loss. American Journal of Physiology, 164, COOKE, A. R., NAHRWOLD, D. L. and GROSSMAN, M. I. (1967). Diversion of pancreatic juice on gastric and pancreatic response to a meal stimulus. American Journal of Physiology, 213, DEBRAY, C., DE LA TOUR, J., VAILLE, C., RoziE, C. and SOUCHARD, M. (1962). Contribution 'a l'etude de la s6cretion biliare et pancreatique externe chez le rat. Journal de Physiologie, 54, DOCKRAY, G. J. (1972). The action of secretin, cholecystokinin-pancreozymin and caerulein on pancreatic secretion in the rat. Journal of Physiology, 225, GREEN, G. M. and LYMAN, R. L. (1972). Feedback regulation of pancreatic enzyme secretion as a mechanism for trypsin inhibitor-induced hypersecretion in rats. Proceedings of the Society of Experimental Biology and Medicine, 140, GROSSMAN, M. I. (1958). Pancreatic secretion in the rat. American Journal of Physiology, 194, HEATLEY, N. G. (1968). The assay of secretin in the rat. Journal of Endocrinology, 42, LOVE, J. W. (1957). A method for the assay of secretin, using rats. Quarterly Journal of Experimental Physiology, 42, LOWRY, 0. H., ROSEBROUGH, N. J., FARR, A. L. and RANDALL, R. J. (1951). Protein measurement with the folin phenol reagent. Journal of Biological Chemistry, 193, MANN, F. C. and FOSTER, J. P. (1918). The secretory pressure of the liver with special reference to the presence or absence of a gallbladder. American Journal of Physiology, 47, RAMIREZ, J., HuBEL, K. A. and CLIFTON, J. A. ( 1966). Intestinal factors affecting pancreatic exocrine secretion in the rat. American Journal of Physiology, 211, SCOTT, V. B., GRAHAM, J. S. and MCCARTTN Y, D. H. (1940). Exocrine pancreatic secretion in the fasting dog. American Journal of Digestive Diseases, 7, SHAW, H. M. and HEATH, T. (1972). The significance of hormones, bile salts, and feeding in the regulation of bile and other digestive secretions in the rat. Australian Journal of Biological Sciences, 25, ZUCKER, T. F., NEWBURGER, P. G. and BERG, B. N. (1932). Continuous pancreatic secretion. American Journal of Physiology, 102,