J. Phyeiol. (1977), 270, pp. 29-36 29 With 5 text-ftgure8 Printed in Great Britain THE SECRETION OF PEPSIN BY T. KONDO* AND D. F. MAGEEt From the Creighton University School of Medicine, Department of Physiology, 2500 California Street, Omaha, Nebraska 68178 (Received 16 July 1976) SUMMARY 1. Ligation and division of the vessels supplying the stomach, except those from the spleen, result in greatly increased pepsin secretion in response to pentagastrin, pilocarpine or 2-deoxy-D-glucose. 2. This effect was still present 6 months after initial ligation. 3. Analysis of dose-response curves shows that for pepsin secretion the Vmax values were raised by this procedure. 4. Right-sided vessel ligation and division does not alter acid secretion in response to the stimuli used. 5. It is suspected that the procedure of right-sided vessel ligation removes a non-competitive inhibitor mechanism to gastric pepsin secretion. INTRODUCTION In the Heidenhain pouch, acid and pepsin secretion is stimulated by cholinomimetic agents and by histamine, but pentagastrin and endogenous gastrin will stimulate only acid from the vagally denervated pouch mucosa (Dutt & Magee, 1972). Heidenhain pouches differ from the main stomach in their innervation, blood supply and in the absence of muscular and mucosal continuity with the main stomach. We have attempted to evaluate these factors in the secretion of pepsin. METHODS In six dogs with simple gastric fistulae, dose-response relationships were studied by plotting pentagastrin or pilocarpine dose against acid and pepsin secretion before and after (3-4 weeks) truncal vagotomy. Following vagotomy, the main arteries supplying the stomach, except the short gastrics (which are the only supply to Heidenhain pouches), were cut between ligatures and the dose-response relationship reinvestigated 3-4 weeks later. In this operation the left gastric artery and surrounding tissue, including visible nerves, was ligated and divided as it left the coeliac axis. * Visiting Instructor from Nagoya University, Nagoya, Japan. t Present address: Department of Physiology, Creighton University School of Medicine, 2500 California Street, Omaha, Nebraska 68178.
30 T. KONDO AND D. F. MAGEE The right gastro-epiploic was ligated and divided at the gastroduodenal junction and any remaining pulsating vessels entering the stomach from the oesophagus were divided. Hereafter this procedure is referred to as vessel ligation. A few weeks after the last experiment the completeness of the vessel ligation and division was confirmed by inspection at laparotomy in each of the dogs. In six dogs (three with lesser curvature innervated pouches) the order was reversed in that vessel ligation preceded vagotomy. The innervated pouches were made by the method of Fausnaugh, De Vito & Chapman (1960) except that a mucosal septum (1 cm) was left. These are called Pavlov pouches hereafter. In these the response to intravenous pentagastrin and to pilocarpine was studied as before, but at 1, 3 and 6 months after vessel ligation. The effect of vagal stimulation was examined using 2-deoxy-D-glucose at a dose of 50 mglkg. min intravenously for 20 min. The resulting dose-response curves were compared with those obtained before the vessel ligation and division procedure. In the experiments in which truncal vagotomy was the initial procedure, pentagastrin was given in doses of 0.5, 1 and 2 #ug/min, in ascending order. In the later experiments, in which blood vessel ligation was the initial procedure, it was given in doses of 0 05, 0-125, 0-25 and 0 5 Itg/min. Pilocarpine was given always in doses of 20, 40 and 80,ug/min. All were given continuously and intravenously. Acid was titrated using a Radiometer automatic machine. Pepsin was estimated using the method of Anson (1938). Pepsin units are mg of tyrosine liberated from Hb during 5 min at 370 C. In the figs. the group mean and S.E. of mean were plotted, but significance was determined by paired difference using Student's t. Kinetic analysis of group means of the fistula and pouch pepsin secretion in response to pentagastrin was calculated from the data in Figs. 3 and 4. In this dose/response S/V is plotted against dose S. This analytical procedure was used also on the pilocarpine data. This permits calculation of V.x. and Ki,, since the slopes of these lines are 1/ J'.. and the x intercepts - Km (Segal, 1968). In order to determine significance of the difference between V.. and K., the values for each dog were calculated and subjected to paired analysis with the control values using Student's t. The results of this analysis are given in Table 1. RESULTS In Figs. 1 and 2 it will be seen that vagotomy reduces the acid and pepsin secretion of the stomach in response to pentagastrin at each dose level used, but acid and pepsin still increase with increasing pentagastrin dose. The basal secretion is noticeably depressed. After transaction of the major vessels in addition, the basal secretion of pepsin is higher than after vagotomy alone, but basal acid secretion is unchanged. Increasing doses of gastrin significantly increased acid secretion alone, when both the vagi and the major arteries were interrupted. In both the pilocarpine and pentagastrin experiments in which the major blood vessels were ligated prior to vagotomy, both resting pepsin and stimulated pepsin secretions were higher than preligation control levels of secretion from the fistula (Figs. 2, 3). When the data were plotted as shown, for fistula secretion, in Fig. 3, it was found, using either group or paired comparison, that the Vmax for pentagastrin-stimulated pepsin secretion was significantly increased by vessel ligation, 3 and 6 months
THE SECRETION OF PEPSIN 31 TABLE 1. Regression data calculated from Figs. 3 and 4 (+ S.E. of mean) Pentagastrin Pilocarpine Pepsin Before 1 2 3 Before 3 VmaS (u./10 min) Pouch 41P47 +9*1 100-39 +34-43 52.7* +9.42 67.33* +9.44 - - Fistula 213-36 +72*59 142-42 ±495 54 395.25* + 78*38 734-34* +218-81 560-0 +110.0 1140.0* +470 0 Km (/tg/min) Pouch -0-0173 + 0-0278 0-0989 ±00549 0-0097 +0-0106 0-0242 +0*0254 Fistula -0 0535 0-1824 0.004* 0.1945* + 0 0057 +0-1761 + 0-0022 + 0-1215 Note: Correlation coefficients significant (P < 0 005), except for lines 1 which are insignificant throughout. * Difference from before significant at the 95 % level. 7-7 - 6-6 1-5 2 - D- 40' - 2 1 2 20 40 80 PG (pag/min) Pilo (pg/min) 270~~~~2 2 PlY Fig. 1. Left: the effect of pentagastrin (PG) on acid secretion from a simple gastric fistula. B, before truncal vagotomy; 1, after truncalvagotomyalone; 2, after truncal vagotomy and vessel ligation (n = 6). Right: the effect of pilocarpine (Pilo) on acid secretion from a gastric fistula; B, before; 1, after vessel ligation; 2, after vessel ligation and bilateral truncal vagotomy (n = 6). 2 PHY 270
32 T. KONDO AND D. F. MAGEE later. With pilocarpine, the Vmax for pepsin from the fistula only was increased (Table 1). Paired comparison showed that the Km for pentagastrin-stimulated pepsin secretion from the fistula had significantly increased 3 and 6 months following vessel ligation (Table 1). This is not obvious from Fig. 3. In the three animals with Pavlov pouches, basal 1100 1000 900 800 i 700 600 I-: c 500.C cl 400 300 200 100 NB 1.--- --2 0 1 1 2 PG (pg/min) 1 2 B 0 l20 40 80 Pilo (pg/min) Fig. 2. Left: the effect of pentagastrin (PG) on pepsin secretion in the same experiment as Fig. 1; B, before; 1, after vagotomy; 2, after vagotomy and vessel ligation. All points on 1 and 2 significantly different from B. Zero point line 2 significantly greater than 0 line 1 (n = 6). Right: the effect of pilocarpine (Pilo) on pepsin secretion in the same experiment as Fig. 1; B, before; 1, after vessel ligation; 2, after vessel ligation and bilateral truncal vagotomy. * Paired difference from B significant. pepsin levels were too erratic before operation to show significant elevation of resting pepsin secretion after vessel ligation. Pentagastrin did, however, show a significantly elevated Vmax for pouch pepsin (Fig. 4). Vessel ligation without vagotomy left pouch- and fistula-stimulated acid (with pentagastrin or pilocarpine) unchanged (Fig. 5), however. In the pilocarpine experiments (Fig. 2), added vagotomy brought about a depression in pepsin secretion in response to small doses, but Vmax andkm and the response to 80,ug/min in this case were not different from the control.
THE SECRETION OF PEPSIN 33 Direct vagal stimulation with 2-deoxy-D-glucose showed that, after vessel ligation, acid secretion was no greater than preligation secretion, but pepsin secretion from the fistula was significantly greater (A = 5206 + 151 pepsin units, or 49-2 % increase). The average increase from the pouch did not reach significance. B 1001-3 in 0 1-1 n 50 A 005 0125 025 0*5 S (g/min) Fig. 3. Response of fistula pepsin secretion to pentagastrin (PG) dose. S = PG dose. S/V = PG dose/pepsin activity per 10 min collection, expressed as tyrosine (mg). B, before; 1, 2 and 3 = 1, 3 and 6 months after vessel ligation. Since slope = l/v.,,, the steeper the slope the smaller the Vmax. dogs. See Table 1. Each point, mean + S.E. of mean of experiments in three I DISCUSSION The experiments described make it clear that division of the main left gastric and the right gastroepiploic arteries, plus an occasional oesophageal artery, increased the ability of the stomach to secrete pepsin in response to pentagastrin, 2-deoxy-D-glucose, or pilocarpine. Acid seemed to be unaffected. This augmentation was not seen in the absence of the vagi, but a suggestion that it is there was seen in the slightly elevated basal pepsin secretion when vessel division was superimposed on vagotomy. Bilateral vagotomy in these and previous experiments lowered pepsin secretion, basal or pentagastrin-stimulated, whether or not the 2-2
34 T. KONDO AND D. F. MAGEE stomach had been subjected to vessel ligation. This was true also of pilocarpine stimulation, except at the highest dose level. The proposition posed by these results is that, in ligating the vessels to the right side of the stomach, an inhibitory influence has been removed which apparently depressed the sensitivity of the chief cells and decreased 30 B Lo 0 20 x ~~~~~~~~~~~~~ 0 0 05 0 125 0-25 0.5 S (pg/min) Fig. 4. Response of pepsin secretion from Pavlov pouches to pentagastrin (PG) dose. S = PG dose. S/V = PG dose/pepsin activity per 10 min collection, expressed as tyrosine (mg). B, before; 1, 2 and 3 = 1, 3 and 6 months after vessel ligation. See Table 1. Each point mean + s.e. of mean of experiments in three dogs. their Vmax value. It is, therefore, not a competitive inhibitory mechanism. Since removal augmented pilocarpine (a direct cholinergic stimulant), pentagastrin (which we believe to be an indirect cholinergic stimulant) and 2-deoxy-D-glucose (another indirect cholinergic stimulant), such an inhibitory mechanism seemed to act non-competitively on cholinergic sites on the chief cell itself. What is the source of such inhibition? Were it vascular, one would expect alteration of acid as well, and a depression in secretion would fit expectation better than augmentation. An acute reaction to partial ischaemia should be over by 6 months but the observed augmented pepsin secretion was not.
THE SECRETION OF PEPSIN 35 The network of nerve fibres around the left gastric artery is so obvious that an inhibitory mechanism mediated by it seems reasonable. The nature of these nerves is assumed to be sympathetic. The presence of,8-adrenergic agents has been shown to depress acid and pepsin secretion (Magee, 1976). Preliminary calculations, though inconclusive, favour a non-competitive mechanism for this. 7 6 I : 1-3 0*9 - -c 5' C E0S 4._ + I 2 0 8-071, 0-7 - E+.7 04 03 0 jil 0 005 0-125 0-25 0-5 PG (pg/min) 0-2 - 0 I I 0 0-05 0-125 0-25 0-5 PG (,ug/min) Fig. 5. Acid secretion in response to pentagastrin (PG) (left) from simple gastric fistula (right) from Pavlov pouch in the same animals. B, before; 1, 2 and 3 = 1, 3, and 6 months respectively after vessel ligation. n = 3. A difficulty with this explanation is that sympathetic fibres in profusion are known to reach the stomach from the spleen, and these were intact in our experiments. Additionally, fl-adrenergic antagonists have not been shown to augment pepsin from Heidenhain pouches or gastric fistulae under pentagastrin stimulation (Magee, 1976). These earlier findings with f-blockade and the lack of any effect of vessel ligation on acid secretion (which is influenced by fl-blockade) argue against the interrup- as the explanation for the phenomenon observed. tion of fl-adrenergics The work was supported by N.I.H. grant no. 5 ROI AM 17125-03, 'The Secretion of Pepsin'.
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