Choleresis Induced by Stimulation of the Gastric Antrum

Vol. 52, No. 1 Printed in U.S.A

GASTROENTEROLOGY

Copyright © 1967 b y The Williams & Wilkins Co.

CHOLERESIS INDUCED BY STIMULATION OF THE GASTRIC ANTRUM DAVID

L.

MORTON

N AHRWOLD , M.D., ALLAN R. COOKE, M.R.A.C.P., AND

I.

GROSSMA N, M.D., PH.D.

Research, Veterans Administration Center , and Departments of Physiology and Medicine, UCLA School of Medicine, Los Angeles, California

190,2 mm external diameter) was inserted about 5 cm into the common bile duct through the duodenal fistula. Simultaneously, antral pouch perfusion was begun through a polyethylene tube which was threaded through a Foley catheter positioned in the antral pouch of the dog with mucocutaneous fistula and through a rubber tube attached to the antral cannula of the other 2 dogs. The other end of the rubber tube was connected to a vertical glass reservoir into which the effluent perfusant was allowed to collect before being emptied every 15 min. The a ntral pouch was perfused at a rate of 90 ml per hour using a p eristaltic pump (Harvard Apparatus Company, Dover, Mass.). The solution used throughout was 250 mM glycine, as a buffer, with acetylcholine chloride (0.5 g per 100 ml). For the first 150 min, the pH of the perfusate was adjusted to 1.0 with 1 N HCI. This was followed by perfusion for 120 min with the solution adjusted to pH 7.0 with 1 N NaOH. For the final period of 120 min, the solution was again adjusted to pH 1.0. Gastric juice was collected at 30-min intervals, the volume recorded, and the acid concentration determined by titration to pH 7.0 with 0.2 N NaOH using an automatic titrator and pH meter (Autoburet, Radiometer, Copenhagen, Denmark). Bile was collected by gravity drainage in graduated tubes at 30-min intervals. The bicarbonate concentration was determined by adding 0.5 ml of bile to 1.0 ml of 0.1 N HCI, boiling the mixture, and back-titrating to pH 7.0 with 0.2 N N aOH using t he automatic t itrator and pH meter. Sodium and potassium determinations were done using a flame photometer (Zeiss, Western Germany). Chloride was estimated with a hloric dometer (Buchler Instrument Company, New Jersey) . Bile salt concentration was calculated by the method of Wheeler and Ramos4 as the difference between the sum of the sodium and potassium concentrations and the sum of the chloride and bicarbonate concentrations. The volume rate of bile flow reached a constant

Intravenously administered porcine gastrin extract and the pure polypeptide, porcine gastrin II, were found to be stimuIan ts of bile flow in a previous study from this laboratory.! This is the report of a study of changes in the flow and composition of bile associated with the release of gastrin from the gastric antrum.

Methods Three mongrel dogs, weighing between 16 and 18 kg, were used. Two were prepared with a separated antral pouch drained by a Gregory cannula,2 gastrojejunostomy, and cholecystectomy. The duodenal stump was oversewn, the lesser pancreatic duct ligated, and a Thomas cannula3 placed in the duodenum opposite the opening of the common bile duct. Two weeks later, the gastrohepatic ligament was divided at its attachment to the antral pouch, and a gastric fistula was made using a Thomas cannula. The third dog was prepared with a Heidenhain pouch drained by a Gregory cannula and a separated antral pouch drained by a mucocutaneous fistula at its pyloric end. The remainder of the stomach was excised, the duodenal stump oversewn, and gastrointestinal continuity restored by esophagojejunostomy. Two weeks later, cholecystectomy, lesser pancreatic duct ligat ion, division of the gastrohepatic ligament, and cannulation of the duodenum opposite the common bile duct opening were carried out. Beginning 3 weeks after the second operation, each dog had four identical tests. Following an 18-hr fast, a polyethylene tube (Intramedic PE Received June 27, 1966. Accepted August 16, 1966. Address requests for reprints to: Dr. Morton I. Grossman, Veterans Administration Center, Los Angeles, California 90073 . This work was supported in part by Grant AM 08354 from the United States Public Health Service. 18

level after 90 min of perfusion with acidified acetylcholine solution. The control periods used for statistical analysis were the two 30-min periods before and the first two 30-min periods after perfusion of the antral pouch with acetylcholine solution at pH 7.0. The volume and composition of bile during these control periods were compared with those during antral pouch stimulation with neutral acetylcholine solution. The acid output from t he Heidenhain pouch and t he gastric fistulas was used as the index of antral stimulation. The U-test of Mann-Whitney· was applied to each experiment to determine the significance of the difference between control and test values. These individual probabilities were combined by the method of Fisher.6

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Results

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19

GASTRIN-I N DUCED CHOLERESIS

J anua1'y 1967

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FIG. 1. Volume flow response of hepatic bile in a dog with Heidenhain pouch (open bars) and dogs with gastric fistulas (shaded bars) and gastric acid secretory responses duri ng antral perfusion with acetylcholine chloride. Responses from 1 dog with Heidenhain pouch represent mean of four experiments, and from 2 dogs with gastric fistulas, the mean of eight experiments. In th is and subsequent figures, vertical lines indicate one standard error of the mean.

Acid secretion and bile volume. Acid secretion from both the Heidenhain pouch and the gastric fistulas increased during perfusion of the antral pouch with neutral acetylcholine solution (P < 0.01). During the same period of antral stimulation, there was a significant increase (P < 0.01) in bile volume in both the dog with no gastric tissue in continuity with the gastrointestinal tract and in those with t he remaining stomach drained by a gastric fistula (fig. 1). B icarbonate. There was a significant increase in biliary bicarbonate output in all dogs during the period of antral stimulation (P < 0.01) . Bicarbonate concentration also increased (P < 0.05) but showed little tendency to fall during the second period of antral acidification (fig. 2).

20

NAHRWOLD ET AL.

Chloride, sodium, and potassium. None of the changes in the concentrations of these ions was significant. Chloride concentration showed a tendency to increase throughout the experiment, and this trend was unaltered by antral stimulation. The concentrations of potassium and sodium fell throughout the experiment, and, likewise, these trends were unaltered by antral stimulation. Responses from the totally gastrectomized dog and the dogs with gastric fistulas were similar (fig. 3). Bile salts. Bile salt output declined steadily during the control period of antral perfusion with acidified acetylcholine solution. During the subsequent perfusion with neutral acetylcholine solution, bile salt output remained relatively constant but fell precipitously when the antral pouch was again perfused with acidified acetylcholine solution. Bile salt concentration fell during the ~

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first period of perfusion with acidified acetylcholine solution but remained relatively constant throughout the remainder of the experiment. Differences for both bile salt output and bile salt concentration between the control and test periods were not significant. The totally gastrectomized dog and the dogs with gastric fistulas showed similar responses (fig. 4).

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FIG. 3. Biliary sodium, potassium, and chloride concentration in response to antral perfusion with acetylcholine chloride. Mean of 12 experiments.

Division of the antrum from the body of the stomach and the duodenum and transection of the gastrohepatic ligament left the right gastroepiploic artery as the only attachment of the antral pouch. It is unknown whether neural connections between antrum and liver exist along this vessel. Since choleresis occurred even after the truncal vagotomy incident to total gastrectomy, a neural mechanism is unlikely. Interruption of the enterohepatic circula-

Januw·y 1967

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GASTRIN-INDUCED CHOLERESIS

tion of bile by its collection causes a progressive decrease in the secretion of bile salts. 4 This makes demonstration of a weak stimulant of bile salt secretion diffi cult, and we cannot exclude this possibility. The choleresis in these experiments was similar to that known to occur with secretin, which increases the rate of bile flow as well as the biliary bicarbonate concentration and output. Since secretin is a potent choleretic, studies of bile flow must b e designed to prevent the entry of gastric acid into the small intestine and the consequent liberation of secretin. That secretin did not cause the choleresis induced by antral stimulation in these studies is shown by the fact that choleresis occurred even when no acidsecreting tissue was in continuity with the gastrointestinal tract. Furthermore, in another study/ even during maximal histamine stimulation, drainage of gastric acid by a gastric fistula in animals with antral pouches and gastroenterostomy prevented secretin release as indicated by failure of pancreatic secretion to increase above basal levels. E arlier investigators who found a pancreatic stimulant in antral mucosal extracts assumed that secretin was the agent responsible for this action. Now that gastrin h as been shown to be both a pancreatic7 and a biliaryl stimulant, the question has arisen as to whether gastrin is the sole pancreatic and biliary stimulant in antral mucosa, or whether additional agents such as secretin may also be present. Extracts of gastric antral mucosa made by a method known to be effective in extracting secretin from duodenal mucosa were ineffective in stimulating pancreatic secretion.8 The choleresis in our experiments was observed only during antral stimulation with a neutral solution. If the effect were due to secretin released from the antral mucosa, choleresis would be expected during antral acidification. Jones and Brooks9 showed that the choleresis induced by insulin hypoglycemia was abolished by antrectomy, indicating that the antrum releases a choleretic. Choleresis occurred with irrigation of innervated antral pouches with peptone, liver extract,

and saline in the studies of Jones et al.,10 but those authors did not interpret their findings as indicating that the choleresis was due to gastrin. Irrigation of the gastric antrum with acetylcholine causes release of gastrin. ll This effect is blocked by acidification.l2 That gastrin was released in our experiments is shown by the secretory response from the Heidenhain pouch and the gastric fistulas. Since exogenous gastrin is a choleretic and produces changes in bile composition identical to those in our experiments,l and since choleresis occurred during a period of gastrin release as indicated by gastric acid secretion, we conclude that endogenously released gastrin is a choleretic. SUllllllary

The volume and composition of bile were studied by cannulation of the common bile duct through a duodenal fistula in dogs with separated antral pouches, cholecystectomy, and either total gastrectomy with H eidenhain pouch or gastrojejunostomy with gastric fistulas. Perfusion of the antral pouch with buffered acetylcholine solution at pH 7.0 caused a choleresis characterized by increased bicarbonate concentration and output, as well as increased gastric acid secretion. These effects were not seen during antral perfusion with acetylcholine chloride solution acidified to pH 1.0. These studies indicate that the antral mucosa is the source of a choleretic substance, and they are consistent with the hypothesis that this substance is gastrin. REFERENCES 1. Zaterka, S., and M. 1. Grossman. 1966. The effect of gastrin a nd histamine on the secretion of bile. Gastroenterology 50: 500-505. 2. Gregory, R. A. 1958. Gastric secretory responses after portal venous ligation . J. Physiol. (London) 1#: 123-137. 3. Thomas, J . E. 1941. An improved cannula for gastric an d intestinal fistul as . Proc. Soc . Exp. BioI. Med. 46: 260--261. 4. Wheeler, H. 0., and O. L. Ramos. 1960 . Determinants of the flow and composition of bile in the unanesthetized dog during constant infusions of sodium taurocholate. J. Clin. Invest . 39: 161-170.

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5. Siegel, S. 1956. N onparametric statistics for the behavioral sciences. McGraw-Hill Book Company, Inc., New York. 6. Fisher, R. A. 1948. Statistical methods for research workers. Oliver and Boyd, Ltd., Edinburgh. 7. Preshaw, R. M., A. R. Cooke, and M. I. Grossman. 1965. Stimulation of pancreatic secretion by a humoral agent from the pyloric gland area of the stomach. Gastroenterology 49: 617-622. 8. Preshaw, R. M., and M. 1. Grossman. 1965. Comparative effectiveness of subcutaneous and intravenous routes of administration of pancreatic stimulants. Amer. J. Physiol. 209: 803-810.

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9. Jones, R. S., and F. P. Brooks. 19()5. The pyloric antrum as a mediator of insulin induced choleresis. Physiologist 8: 202. 10. Jones, R. S., K. C. Powell, and F. P. Brooks. 1965. The role of the gastric antrum in the control of bile flow. Surg. Forum. 16: 386387. 11. Robertson, C. R., K. Langlois, C. G. Martin, G. Slezak, and M. I. Grossman. 1950. Release of gastrin in response to bathing the pyloric mucosa with acetylcholine. ArneI'. J. Physiol. 163: 27-33. 12. Woodward, E. R., C. Robertson, B. S. Fried, and H. Shapiro. 1957. Further studies on the isolated gastric antrum. Gastroenterology 32: 868-877.