EFFECT OF VAGOTOMY ON PANCREATIC SECRETION STIMULATED BY ENDOGENOUS AND EXOGENOUS SECRETIN

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1 GASTROENTEROLOGY Copyright, by The Williams & Wilkins Co. Vol. 60, No. 3 P>-inted in U. S. A. EFFECT OF VAGOTOMY ON PANCREATIC SECRETION STIMULATED BY ENDOGENOUS AND EXOGENOUS SECRETIN HARRIS J. MORELAND, M.D., AND LEONARD R. JOH'NSON, PH.D. Department of Surgery and Department of Physiology and Biophysics, University of Oklahoma School of Medicine, Oklahoma City, Oklahoma The role of the vagus nerve in the control of pancreatic secretion was studied in 3 dogs having chronic pancreatic, gastric, and duodenal fistulae. Pancreatic volume, bicarbonate, and protein outputs were determined before and after vagotomy in response to various doses of exogenous and endogenous secretin. Endogenous secretin was released by infusion at constant volume rate of different concentrations of Hel into the duodenal cannula. Vagotomy decreased basal protein output and the protein response to low levels of duodenal acidification. Basal volume and bicarbonate outputs were unaffected by vagotomy. Significant decreases in volume and bicarbonate response to duodenal acidification (1.5 and 6.0 meq of Hel per hr) occurred following vagotomy, but these disappeared at high rates of acid infusion (24.0 meq of Hel per hr). Vagotomy caused a significant increase in volume and bicarbonate output in response to low doses of secretin (0.5 and 1.0 U per kg-hr). With higher doses of secretin (2.0 and 4.0 U per kg-hr) there was no difference following vagotomy. From these results we have tentatively concluded that: (I) the vagus facilitates the release of secretin in response to low levels of duodenal acidification; (2) vagotomy results in an increased sensitivity of the pancreas to low doses of secretin; and (3) vagotomy decreases basal protein output, indicating that the vagus may playa direct role in the stimulation of enzyme secretion. Many studies have been conducted to establish the relationship between the neural and hormonal factors influencing pancreatic secretion. Most of these experiments compared pancreatic secretion be- Received August 7, Accepted October 14, This work was presented at the meeting of the American Gastroenterological Association, May 1970, Boston. Address requests for reprints to: Dr. Leonard R. Johnson, Department of Physiology and Biophysics, 800 Northeast 13th Street, Oklahoma City, Oklahoma This study was supported by a General Research Grant from the University of Oklahoma and by the G. A. Manahan Trust Fund. The authors are grateful to Dr. G. Rainey Williams for providing space in the Department of Surgery and some of the equipment for this study. Dr. Eugene D. Jacobson also provided support for this study. 425 fore and after vagotomy, anticholinergics, parasympathetic drugs, or local anesthetics. In 1967, after reviewing the evidence obtained from these studies, Preshaw 1 concluded that the results were contradictory and offered no firm support for a hypothesis of integrated neural and humoral control of pancreatic secretion. These conflicting results can probably be explained in part by the methods employed, i.e., anesthetized animals, impure hormone preparations, failure to exclude endogenous acid from the duodenum, single injections rather than continuous intravenous infusions, and failure to establish adequate dose-response curves. Most of the above-mentioned studies have dealt with the influence of the vagus on pancreatic secretion or the possible relationship between cholinergic stimulation and the hormones secretin and cholecysto-

2 426 MORELAND AND JOHNSON Vol. 60, No.3 kinin. Two studies demonstrated that topical anesthetics block the release of secretin by duodenal acidification. 2, 3 Until recently, however, few studies have been concerned with the possible involvement of the vagus in the secretin release mechanism. Henriksen and Rune 4 examined the pancreatic response to duodenal acidification (4.0 meq of Hel per 30 min) and, although each of their animals showed a decrease in both volume and bicarbonate output following vagotomy, the difference was not statistically significant. In the present investigation we have studied the effects of vagotomy over the entire physiological range of pancreatic secretion in response to both endogenously released and exogenously administered secretin. In so doing we have found evidence that vagal innervation facilitates the release of secretin in response to low levels of duodenal acidification. Methods Three dogs, weighing from 13 to 15 kg were surgically prepared with a chronic pancreatic fistula according to the method described by Preshaw and Grossman 5 and subsequently modified by Steni:,g.6 At the same time a Thomas cannula 7 was placed in the most dependent portion of the stomach just proximal to the antrum and a Gregory cannulas was inserted into the duodenum 2 cm distal to the entrance of the pancreatic fistula. At least 2 weeks were allowed for recovery from surgery before experiments were begun. Construction of the pancreatic fistula in this manner results in transection of the duodenum at the entrance of the major pancreatic duct. This no doubt interrupts some vagal fibers. There is some question as to the source of vagal innervation of the distal duodenum, that is, whether the nerve fibers are the ones from the stomach which cross the pylorus to the proximal duodenum or whether the fibers reach the gut along with the blood vessels. The actual case is probably a mixture of both. In any event, since all the experiments were conducted after this procedure, its effects do not enter into the interpretation of the results due to truncal vagotomy. The dogs were fasted for 18 hr before each experiment. The stomach was rinsed by irrigation through the gastric fistula which was kept open throughout the experiment. A continuous intravenous infusion of 0.15 M NaCl (60 ml per hr) was delivered with a peristaltic pump (Harvard Apparatus Company) by way of a polyethylene catheter inserted into a leg vein. On other days 0.15 M NaCl was infused into the duodenal cannula at a rate of 150 ml per hr. This rate gave excellent perfusion of the duodenal bulb as evidenced by a 10 to 15% reflux through the gastric fistula. Reflux was essentially constant for all doses of acid and saline both before and after vagotomy. Studies with exogenous secretin or duodenal acidification were not begun until two consecutive 15-min basal collections from the pancreatic fistula were 2.0 ml per 15 min or less. If basal secretion remained higher, the study was cancelled for the day. Exogenous secretin ( , 2.0, or 4.0 U per kg-hr) was then substituted for the pure saline intravenous infusion, and pancreatic secretion was collected for 2 hr at 15-min intervals. In some experiments, endogenous secretin was released by substituting HCl for saline in the duodenal perfusion system. HCl was given in concentrations of 10, 40, or 160 meq per liter which, at the rate of infusion used, corresponded to 1.5, 6.0, and 24.0 meq per hr. In these experiments secretion was also collected for an additional 2 hr. Pancreatic juice was collected every 15 min and the volume recorded to the nearest 0.1 ml. Bicarbonate concentration was measured by adding 0.5 ml of pancreatic juice to 1.0 ml of 0.1 N HCl, boiling the solution briefly, cooling, and back titrating the residual HCl with 0.2 N NaOH. Titration was to ph 7.0 using an automatic titrator (Radiometer, Copenhagen). Outputs of bicarbonate are expressed as meq per 15 min. Protein concentration of the samples was measured with a Zeiss spectrophotometer as absorbance at 280 mm and compared with a standard solution of bovine serum albumin. Protein outputs are expressed as mg per 15 min. After completing the series of experiments in duplicate, bilateral thoracic vagotomies were performed. Two centimeters of each vagus and all visible ramifications were resected. Completeness of vagotomy was verified with the Hollander insulin test. 9 Insulin (0.25 U per kg) caused less than 0.4 meq of H + per 15-min secretion after vagotomy. Pyloroplasty was not performed, but instead of one meal a day the dogs were fed an equal amount of food as mush in two daily feedings. The weights of the animals remained nearly constant throughout the study. Three weeks after vagotomy, the identical series of dose-response experiments were repeated. No experiments were conducted after 3 months postvagotomy and there was no visible change in any dog's pancreatic response during

3 March 1971 EFFECT OF VAGOTOMY ON PANCREATIC SECRETION 427 this period. There was no evidence of vagal regeneration detected by a repeat Hollander test or by autopsy. The order of experiments as to dose and whether secretin or acid was infused was randomly determined. Each experiment was repeated so that both before and after vagotomy observations are expressed as means and standard error of the means of two observations in each of 3 dogs. Results were calculated as the mean of the two highest consecutive 15-min periods for each individual dog. The washout phenomenon was evident for protein secretion during the first two periods of stimulation but these values were omitted from the calculations. Only one dose of secretin or acid was administered on a given day and experiments were conducted on alternate days no more than three times per week. Secretin (batch 17021) was obtained from the Gastrointestinal Hormone Research Unit, Karolinska Institutet, Stockholm, Sweden. Results Vagotomy resulted in a significant decrease in pancreatic volume (fig. 1) and bicarbonate output (fig. 2) in response to duodenal acidification with 1.5 and 6.0 meq of Hel per hr. The effect was most pronounced during irrigation with 1.5 meq of Hel per hr and its statistical significance disappeared when 24.0 meq of Hel per hr were infused. The decrease in the pancreatic response to low levels of duodenal acidification was pronounced, and figure E 12 8 BEFORE 4 t AFTER S J'- ~ "p<o.05 B meq H hr FIG. 1. Pancreatic volume output before and after vagotomy in ml per 15 min in response to different rates of duodenal acidification. B represents basal secretion. Two observations were made in each of 3 dogs. Means and SE: P values were determined by t test for unpaired data using the means and SE for a single dose before and after vagotomy c 'E!Q 2.0 'do C" '" ~ 1.5 E BEFORE Y;,.; 'I",," AFTER! " p<o.ool... p<o.05 B meq H hr FIG. 2. Bicarbonate output in meq per 15 min under the same conditions as figure B.O c ~ 6.0 "- E meq HClhr BEFORE L ~ ~ I -~ 15 min PERIODS -f.., 1 AFTER 't i FIG. 3. Volume response before and after vagotomy during the experiments, with 1.5 meq of H e l per hr infused into the duodenum. depicts the volumes of juice collected during the entire course of the experiment with 1.5 meq of Hel per hr. Vagotomy did not alter either basal volume (fig. 1) or bicarbonate output (fig. 2). Vagotomy did, however, decrease the basal output of pancreatic protein (fig. 4), and this marks the only aspect of basal secretion that was decreased following vagotomy. The pancreatic protein response to duodenal acidification remained less after vagotomy during infusion of the two lower concentrations of acid but, like volume and bicarbonate output, recovered when 24.0 meq of Hel per hr were infused (fig. 4). The decreased volume and bicarbonate outputs following vagotomy in response to duodenal acidification could theoretically be explained by either the removal of vagal facilitation of endogenous secretin release or the abolition of a vagal secretin interaction at the level of the pancreatic secretory cell. Figures 5 and 6 demonstrate that the

4 428 MORELAND AND JOHNSON Vol. 60, No c 200 'E '! z iii f- e 120 If E '{ '! B meq H'hr * p(o.ooi ~ p<o FIG. 4. Protein output in mg per 15 min under the same conditions as figure c: 16 'E ~ 12 '- E 8 4 B AFTER *" --- BEFORE 'p<o.ooi SECRETIN (unitskg-hr) FIG. 5. Same as figure I, except that secretion was stimulated by different doses of exogenous secretin ~ 2.5 It), ~ 2.0 u I 0" 1.5 w E AFTER ;t BEFORE.p(O.OOI B SECRETIN (unitskg-hr) FIG. 6. Same as figure 2, except that secretion was stimulated by different doses of exogenous secretin. second mechanism cannot explain the results observed, for there was actually an increased response to low doses of exogenous secretin following vagotomy. Significant increases in both volume and bicarbonate output occurred after vagotomy in response to 0.5 and 1.0 U of secretin per kg-hr. There was no change in the re ~ 200 E '! z iii f- e 120 0:: a. ~ 'I * - -I" 'I ! '!j- - - AFTER B SECRETIN (unitskg-hr), p(o.ooi... p<o.05 FIG. 7. Same as figure 4, except that secretion was stimulated by different doses of exogenous secretin. sponses at the upper end of the dose-response curve (figs. 5 and 6). Basal secretion (figs. 5 and 6) during the days in which exogenous secretin was given was, as in the previous figures (1 and 2), unaffected by vagotomy. The decrease in basal protein output following vagotomy was also noted on the days exogenous secretin was administered (fig. 7). The decline in protein secretion following vagotomy was observed at each dose of exogenous secretin (fig. 7). DiscuSSIOn The relationship between the neural and hormonal control of gastric secretion has been explored in detail and has shown that the vagus nerve influences gastric secretion in three ways. First, cholinergic activity stimulates the parietal cell directly.1o Second, vagal activity releases gastrin. ll Third, the vagus sensitizes the parietal cell to respond to gastrin. 12 It is impossible now, as it was in 1967 to draw comparable conclusions about the integrative control of pancreatic secretion. The current study investigates the effects of truncal vagotomy over the entire physi.ological range of pancreatic secretion in response to duodenal acidification. Vagotomy reduced the pancreatic response during low levels of duodenal acidification (figs. 1 and 2), but not during high rates of H + infusion. The effect of vagotomy was most apparent at the lowest rate of acid infusion, 1.5 meq of H+ per hr (fig. 3). The pancreatic response to duodenal acidifica- 4.0

5 March 1971 EFFECT OF VAGOTOMY ON PANCREATIC SECRETION 429 tion was mimicked by exogenous secretin. From the data of others who have examined this question, there is no evidence to suggest the major involvement of any hormone other than secretin in the response to physiological amounts of acid There is, however, some evidence that at high rates of duodenal acidification another hormone may be released. 16 The differences in secretion after vagotomy noted during this study can probably be attributed solely to effects on secretin release or action. Since the pancreatic response to low doses of secretin was actually increased after vagotomy, the only explanation of the decreased response to duodenal acidification is that the vagus facilitates the release of secretin from the duodenum. Crider and Thomas 17 in 1944 found that vagotomy decreased the pancreatic response to both HCI and peptone infused into the duodenum. Henriksen and Rune 4 observed a uniform decrease in the pancreatic response of all their dogs to duodenal acidification following vagotomy. However, they were infusing acid at a rate of 8 meq per hr and the decrease was not found to be significant. Interpolating from the results of our study, the postvagotomy decrease would probably not be significant at this dose of acid. Results such as these point out the necessity for studying a phenomenon over the full range of physiological doses. Other investigators using cholinergics, anticholinergics, and anesthetics have also obtained evidence that cholinergic stimulation facilitates secretin release. Urecholine increased the rate of fluid and bicarbonate secretion in response to duodenal acidification in vagotomized dogs. 4 Application of local anesthetics to the intestine inhibited the pancreatic response to acid. 2, 3, 18 Results with topical anesthetics, however, are open to criticism, since these agents often damage mucosa as well as block neural reflexes. Thomas 19 observed that anticholinergics decreased the pancreatic response to secretin less than they decreased the response to duodenal acidification. An unexpected finding in our study was the large increase in the pancreatic response to low doses of exogenous secretin following vagotomy. Again this was a doserelated phenomenon, for the responses to 2.0 and 4.0 U of secretin per kg-hr were identical before and after vagotomy. This observation is not unique with our study, for several groups have observed similar increases after vagotomy. Routley et al. 20 measured pancreatic responses to various stimuli in dogs. The rate of secretion in response to secretin was found to be increased after vagotomy. In a study similar to ours, Tankel and Hollander21 compared the responses to secretin before and after vagotomy in 4 dogs. The basal rate of pancreatic secretion was unchanged by vagotomy, but the responses to secretin were increased significantly in 2 dogs and slightly increased in the remaining animals. Henriksen,22 on the other hand, found no change in volume or bicarbonate output following vagotomy in response to exogenous secretin. In these experiments a doseresponse relationship was obtained and vagotomy had no effect on any part of the curve; however, secretin was administered only as a rapid intravenous injection and not as a continuous infusion. Magee et al. 23 found that vagotomy produced a significant decrease in the rate of pancreatic secretion in response to Vitrum secretin. Vagotomy had no effect on the response to Boots secretin. It can be concluded from our studies and the others cited that vagotomy does not appear to reduce the pancreatic response to exogenous secretin as it does the gastric response to exogenous gastrin. Therefore, the vagus does not interact with secretin at the level of the pancreatic secretory cell to increase the over-all response. On the contrary, our results indicate that the pancreas becomes more sensitive to secretin after vagotomy. That is, the dose-response curve is shifted to the left without a change in the maximal response. Whether this increase in sensitivity is due to withdrawal of cholinergic stimulation or to some other mechanism needs to be established. The observation that basal protein secretion decreased after vagotomy was the only evidence that we obtained for an effect of the vagus nerve on the unstimulated pan-

6 430 MORELAND AND JOHNSON Vol. 60, No. 3 creas. Protein output increased in response to the lowest dose of acid (fig. 4) and the lowest dose of secretin (fig. 7) in both the pre- and postvagotomy states. Throughout the remaining dose range of secretin and acid infusion, protein output remained essentially constant. Infusion of 24.0 meq of HCI per hr into the duodenum resulted in a significant increase in the response of the vagotomized dogs when compared with protein secretion stimulated by 6.0 meq of HCI per hr (fig. 4). This is the only instance noted in either the pre- or postvagotomized animals when secretin could fully account for the pancreatic response to duodenal acidification. Preshaw et al. 16 also noted that at high rates of duodenal acid infusion pancreatic enzyme output was increased over that stimulated by exogenous secretin and, concluded that, under these conditions, acid released some cholecystokinin. The same explanation would account for the increased enzyme secretion we observed at the highest rate of acidification. The physiological significance of this observation, however, is open to question, since during fasting the dog duodenal ph ranges between 4 and 5 and drops only to about 3 during the response to a meal. Furthermore, these lower ph values occur only in the duodenal bulb,24 and during our experiments the entire duodenum was acidified. Since truncal vagotomy abolishes parasympathetic innervation in the pancreas as well as the duodenum, it is difficult to draw meaningful conclusions concerning neural hormonal integration and the control of pancreatic secretion. This problem can be circumvented in the stomach by using innervated pouches of the gastrinproducing antrum and the acid-secreting fundus and then denervating them one at a time. In light of the surgery required for the collection of pancreatic juice, it is more difficult to denervate selectively pouches of the duodenum. Recognizing the above limitations, the dose-response design and the control experiments in the current study permit several tentative conclusions from the results concerning integrative control of pancreatic secretion. Vagal innervation facilitates the release of secretin in response to low levels of duodenal acidification. The sensitivity of the pancreas itself, however, to low doses of exogenous secretin increases after vagotomy. These two findings appear to be physiologically significant, since they are most prominent at the lower end of the dose-response curve. Finally, vagal innervation appears to provide direct stimulation of pancreatic protein secretion. REFERENCES 1. Preshaw RM: Integration of nervous and hormonal mechanisms for external pancreatic secretion, Handbook of Physiology, sect 6, vol 2. Edited by CF Code. Washington, American Physiological Society, 1967, p Shapiro H, Woodward ER: Inhibition of the secretin mechanism by local anesthetics. Amer Surg 31 : , Thomas JE, Swena EM: The effect of local anesthetics applied to the intestinal mucosa on the response of the pancreas to intestinal stimuli (abstr). Fed Proc 22:664, Henriksen FW, Rune SJ: Cholinergic effect on the canine pancreatic response to acidification of the duodenum. Scand J Gastroent 4: , Preshaw RM, Grossman MI: Stimulation of pancreatic secretion by extracts of the pyloric gland area of the stomach. Gastroenterology 48:36-44, Stening GF: A modification of a chronic pancreatic fistula in the dog. Brit J Surg 56: , Thomas JE: An improved cannula for gastric and intestinal fistulas. Proc Soc Exp BioI Med 46: , Gregory RA: Secretory responses after portal venous ligation. J Physiol (London) 144: , Hollander F: The insulin test for the presence of intact nerve fibers after vagal operations for peptic ulcer. Gastroenterology 7: , Pevsner L, Grossman MI: The mechanism of vagal stimulation of gastric acid secretion. Gastroenterology 28: , Pe Thein M, Schofield B: Release of gastrin from the pyloric antrum following vagal stimulation by sham feeding in dogs. J Physiol (London) 148: , Andersson S, Grossman MI: Effect of vagal denervation of pouches on gastric secretion in dogs with intact or resected antrums. Gastroenterology 49: , Pascal JP, Vaysse N, Augier D, et al: Response of

7 March 1971 EFFECT OF VAGOTOMY ON PANCREATIC SECRETION 431 the canine pancreas to duodenal acidification and secretin. Scand J Gastroent 3: , Johnson LR, Grossman MI: Secretin: the enterogastrone released by acid in the duodenum. Arner J Physiol 215: , Preshaw RM: Duodenal acidification and the release of secretin, Exocrine Glands. Edited by SY Botheho, FP Brooks, WB Shelley. University of Pennsylvania Press, 1969, p Preshaw RM, Cooke AR, Grossman MI: Quantitative aspects of response of canine pancreas to duodenal acidification. Arner J Physiol 210: , Crider JO, Thomas JE: Secretion of pancreatic juice after cutting the extrinsic nerves. Amer J Physiol 141: , Slayback JB, Swena EM, Thomas JE, et al: The pancreatic secretory response to topical anaesthetic block of the small bowel. Surgery 61: , Thomas JE: Mechanism of the action of pancreatic stimuli studied by means of atropine like drugs. Amer J Physiol 206: , RoutIey EF, Mann FC, Bollman JL, et al: Effects of vagotomy on pancreatic secretion in dogs with chronic pancreatic fistula. Surg Gynec Obstet 95 : , Tankel HI, Hollander F: Effect of vagotomy on pancreatic secretion. Amer J Physiol 193: , Henriksen FW: Effect of vagotomy and atropine on the canine pancreatic response to secretin and pancreozymin. Scand J Gastroent 4: , Magee DF, Fragola LA, White TT: Influence of parasympathetic innervation on the volume of pancreatic juice. Ann Surg 161:15-20, Brooks AM, Grossman MI: Postprandial ph and neutralizing capacity of the proximal duodenum in dogs. Gastroenterology 59:85-89, 1970