PEPSIN SECRETION DURING DAMAGE BY ETHANOL AND SALICYLIC ACID

GASTROENTEROLOGY Copyriht 1972 by The Williams & Wilkins Co. Vol. 62. No. 3 Printed in U.S. A. PEPSIN SECRETION DURING DAMAGE BY ETHANOL AND SALICYLIC ACID LEONARD R. JOHNSON, PH.D. Department of Physiology and Biophysics, University of Oklahoma Medical Center, Oklahoma City, Oklahoma Previous reports from this laboratory have demonstrated that pepsin secretion increases when hydrogen ions back diffuse through the gastric mucosa of the canine Heidenhain pouch. Pepsin output was measured in the effluent from the pouches of 4 dogs during irrigation with 15% ethanol plus or minus HCI and during irrigation with 20 mn salicylic acid. Ethanol by itself damaged the pouches as evidenced by large net fluxes of Na+ and K + into the pouch and the appearance of protein in the fluid. Pepsin secretion, however, was not stimulated. The combination of ethanol and 100 mn HCI produced the same effects on Na+, K+, and protein and also increased pepsin secretion 4-fold. Small amounts of HCI (10 mn) in combination with ethanol also increased pepsin secretion 2- or 3-fold. Salicylic acid (20 mn) plus 100 mn HCI caused severe damage and increased pepsin output from 3 mg per 15 min during control periods to 26 mg per 15 min. It is concluded that: (1) damage alone does not stimulate pepsin secretion; acid must be present; (2) pepsin secretion during damage is independent of the appearance of total protein in the irrigation fluid; (3) small amounts of acid are effective stimulators of pepsin secretion in the presence of a damaged barrier; and (4) damage by ethanol and salicylic acid in the presence of H+ results in a significant stimulation of pepsin secretion. The gastric mucosa contains a barrier to the free diffusion of H +. If, however, the barrier is weakened, back diffusion of acid occurs and damage results. Numerous studies from Davenport's laboratory have described the physiological consequences of disrupting this barrier. 1-4 During injury H+ moves out of the lumen toward the serosal side and the Na+ flux in- Received August 9, 1971. Accepted September 24, 1971. Address requests for reprints to: Leonard R. Johnson, Ph.D., Department of Physiology and Biophysics, 800 Northeast 13th Street, Oklahoma City, Oklahoma 73104. This study was supported by a grant from the G. A. Manahan Trust Fund and by National Institutes of Health Grant AM-14392. The. expert technical assistance of Louise H. Wyss is gratefully acknowledged. 412 creases in the opposite direction. Cells are disrupted and K + moves into the fluid bathing the mucosa. In addition, fluid accumulates in the pouch along with plasma protein. Recent studies from this laboratory have shown that pepsin output from the canine Heidenhain (vagally denervated) pouch increases when the pouch mucosa is damaged with hydrochloric or acetic acid. 5, 6 The amount of pepsin appearing in the pouch contents is directly related to the next flux of H + out of the pouch. 6 Since the increased output of pepsin can be blocked by atropine, this output is probably due to secretion triggered at some point by a cholinergic reflex. 6 This study investigates the following questions: (1) does pepsin secretion increase when the mucosa is damaged by alcohol which

March 1972 DAMAGE AND PEPSIN SECRETION 413 does not need acid to cause injury; (2) can the increased pepsin output be separated from the general appearance of protein which occurs during damage; (3) are low concentrations of H+ effective stimulators of pepsin secretion when the barrier is damaged; (4) what is the effect of salicylic acid and Hel on pepsin secretion. Methods Four dogs were surgically prepared with a gastric fistula drained by a Thomas 7 cannula and a vagally denervated pouch with a Gregory' cannula. These particular experiments were begun about 3 years after surgery. The animals were fasted for a least 18 hr prior to an experiment. At the start of an experiment the dogs were placed on tables where they stood quietly supported, in part, by slings. The gastric fistula was opened and the stomach rinsed with saline. The fistula was left open for the duration of the experiment to prevent gastric juice from entering the duodenum. The metal cannula of the Heidenhain pouch was connected by rubber tubing to a reservoir with 50 ml of a control solution which, depending on the experiment, contained either 0.03 M phosphate buffer ph 7.5 or 50 mn NaCI plus 100 mn HCI. The solution was allowed to enter and leave the pouch influenced only by gravity and pouch contractions. The level of fluid in the reservoir was about 20 cm above the pouch. At the end of each 15-min period the reservoir and the pouch were drained and the solution replaced. Volumes of recovered fluid ranged from 48 to 51 ml, and after the first two 15-min periods, which were not included in the experiment, were usually 50 ml. The control solution used during the first series of experiments with ethanol was phosphate buffer, ph 7.5. Collections of the control solution continued for four 15-min periods after the beginning of an experiment. The pouches were then irrigated (periods 5 to 8) with either 100 mn HCI plus 50 mn NaCI, 15% ethanol plus phosphate buffer, or 15% ethanol plus 100 mn HCI and 50 mn NaC!. A final hour of collections with the control solution ended the experiment. Some control experiments consisted entirely of irrigating the pouches with phosphate buffer. In the other series of alcohol experiments periods 1 to 4 and 9 to 12 consisted of irrigations with 15% ethanol in phosphate buffer. In some studies this was continued through periods 5 to 8. In the other tests either phosphate buffer alone or 15% ethanol plus 10 mn HCI and 50 mn NaCI was substituted for the buffer and alcohol. During the experiments with salicylic acid the pouches were irrigated with 50 mn NaCI plus 100 mn HCI throughout. On some days the irrigation fluid during periods 5 to 8 contained 20 mn salicylic acid as well. Following each 15-min collection the volume of the sample was measured and an aliquot taken for pepsin determination. This aliquot was either diluted or had HCI added to it so that it contained 50 mn HC!. This was done immediately after collecting the fluid from the dog. The pepsin concentration was determined using a modification 9 of the Anson lo hemoglobin method and expressed as milligrams of pepsin per milliliter by reading the trichloroacetic acid supernate at 280 mil and comparing it with solutions incubated with different pepsin standards (Hog pepsin, three times crystallized, Pentex Biochemicals, Kankakee, II!,). Pepsin outputs were calculated, taking into consideration the dilution of the assay, and expressed as milligrams of pepsin per 15 min. The Na+ and K+ concentrations of each sample were determined by flame photometry (IL, Model 143). Total amounts ofna+ and K + in the recovered fluid were calculated for each period. The net flux of each ion was determined by subtracting the amount put in the pouch from the amount recovered and expressed as microequivalents per 15 min. Positive numbers indicate a net gain of an ion by the fluid in the pouch. The total protein content of some samples was determined by direct reading on the spectrophotometer at 280 mil and comparison with standards of bovine serum albumin. Protein was expressed as milligrams per 15 min. Experiments were carried out on alternate days and no more than three times a week. The order of studies within each series of experiments was randomly assigned. Except for the salicylate series all the results are expressed as the means and standard errors of the means of three observations in each of 4 dogs. Salicylic acid damaged the pouches severely and since the results were clear cut the experiment was repeated only once (means ± SEM of two observations in each of 4 dogs). P values were determined by t-test for unpaired data. Results Irrigation of the pouches with 15% ethanol in combination with 100 mn Hel caused a 6- or 7-fold increase in pepsin secretion (fig. 1). Ethanol by itself had no significant effect or pepsin output. Hel by itself caused a significant stimulation of

414 JOHNSON Vol. 62, No.3 8.0 J 100 15% EtOH. '" E z iii w 6.0 4.0, 2.0 1,, z.e iii E 1-'" E ;;:; E <no!!, 80 60 40 20 4 6 8 10 12 FIG. 1. Pepsin secretion in response to 15% ethanol and 100 mn Hel. Pouches were irrigated with phosphate buffer, ph 7.5, periods 1 to 4 and periods 9 to 12. The pepsin responses to ethanol and Hel during periods 5, 6, and 7 are significantly higher (P < 0.001) than those to either Hel or ethanol alone. Means and standard errors of the means of three observations in each of 4 dogs. IS"*' PEFOOOS FIG. 4. Large increase in protein output during damage with ethanol alone with no significant effect on pepsin secretion. Means and standard errors of the means of three observations in each of 4 dogs. '.0 400 300 '" w "- 200,. ::> i5 s: l "I 100. _. 4 EIOH+HCI L 1 EtOH 1 -'"I 6 8 10 12! 4.0 } 3.0 2.0 EtOH+ tomn Hel 1.0 ""' 1!5 min PERIOOS 10 12 E'OH+ Pa. Butt. FIG. 5. Pepsin secretion in response to 15% ethanol plus 10 mn Hel. Pepsin outputs during periods 5, 6, 7, and 8 are significantly higher than comparable control periods with buffer alone or buffer plus ethanol. Means and standard errors of the means of three observations in each of 4 dogs. FIG. 2. Net fluxes of Na+ into the pouches during the experiment described in figure 1. 24 40. 30 on "-,. 20 ::> iii <n ;! 0 10 6 10 12 FIG. 3. Net fluxes of K + into the pouches during the experiments described in figure 1. i 20 l 16 :. i: 12 8 4 4 6 8 10 12 IS min PERIODS FIG. 6. Pepsin secretion in response to 20 mn salicylic acid in combination with 100 mn Hel. During periods 5 to 8, pepsin secretion was significantly higher (P < 0.001) when the pouches were irrigated with the combination than when they were exposed to Hel alone. Means and standard errors of the means of two observations in each of 4 dogs.

March 1972 DAMAGE AND PEPSIN SECRETION 415 pepsin secretion in periods 6 and 8 when compared with control periods 3 and 4 (P < 0.05). Although it did not stimulate pepsin secretion, ethanol alone caused as much damage as ethanol plus acid for the Na + and K + fluxes were essentially the same in both experiments (figs. 2 and 3). There was an exchange of Na + for H + during the first period the pouches were exposed to 100 mn Hel (fig. 2), but this concentration of acid failed to damage the mucosa as evidenced by the normal Na+ fluxes in the following periods and the failure of the intracellular K + to appear in the irrigation fluid (fig. 3). Figure 4 presents results demonstrating that even though alcohol alone caused no stimulation of pepsin output the increased appearance of protein, an integral result of damaging the barrier, still occurs. Ethanol in combination. with as Iowa concentration of Hel as 10 mn resulted in a significant stimulation of pepsin secretion (fig. 5). It, therefore, is readily a pparent why buffering was necessary during the control periods to observe this stimulation. Salicylic acid in amounts equivalent to a normal dose of aspirin is one of the most injurious substances to the gastric mucosal barrier. In the current set of experiments the combination of salicylic acid with 100 mn Hel injured the pouches severely as evidenced by bleeding and a large increase in the volume of fluid collected. As might be predicted pepsin secretion increased significantly in the presence of both agents (fig. 6). Pepsin secretion remained higher than control levels during the periods following the removal of salicylic acid from the 100 mn Hel solution, indicating that H+ was still moving across the damaged barrier. Discussion Earlier work by Davenport 11 showed that ethanol in concentrations of 8% or less did not break the gastric mucosal barrier, while 14 and 27% solutions disrupted the barrier as evidenced by large increases in the net fluxes of H+, Na +, and K +. Davenport 11 also showed that alcohol was no more damaging when given in 100 mn Hel than it was when the solution was buffered to ph 7.5. The current experiments reproduced this finding for the fluxes of Na + and K + (figs. 2 and 3) were essentially identical whether the 15% ethanol was given in 100 mn Hel or phosphate buffer. Therefore, the large increase in pepsin output which was observed only in the presence of acid (fig. 1) cannot be thought of as a general consequence of damaging the mucosa. Hydrogen ion must be present to back diffuse through the mucosa and stimulate pepsin secretion. A previous report has indicated that this is indeed a secretor:y phenomenon for the appearance of pepsin is blocked by atropine. 6 The conclusions mentioned in the preceding paragraph, namely, that the increased pepsin output is due to secretion and not a part of damage in general, are reinforced by the finding (fig. 4) that ethanol alone causes a large output of protein into the pouch without any increase in pepsin output. This indicates that pepsin does not appear in the irrigation fluid to any appreciable extent as part of the protein which usually appears when the barrier is damaged. Figure 1 shows that 100 mn Hel in the presence of 15% ethanol stimulates pepsin secretion significantly. While 100 mn Hel must be considered a physiological concentration of acid, the question arises as to the sensitivity of the mechanism which when triggered by H+ results in increased pepsin production. The results depicted in figure 5 show that once the barrier to diffusion of H+ is broken a low concentration of acid is sufficient to stimulate pepsin secretion. Salicylic acid in combination with 100 mn Hel damages the barrier severely. The experiments in figure 6 were terminated earlier than expected, for it was evident that the mucosa had not been fully repaired between experiments. This is probably the explanation for the relatively high levels of pepsin output during the control periods when the mucosa was exposed to Hel alone. These experiments were done primarily for the sake of completeness for salicylic acid is routinely used in experiments with the barrier. We

416 JOHNSON Vol. 62, No. 3 have now demonstrated increased pepsin secretion when the mucosa is exposed to acetic acid 6 and Hel in combination with salicylic acid and ethanol. High (300 mn) concentrations of Hel break the barrier when administered alone with similar effects on pepsin secretion. 5, 6 From the data presented here it can be concluded that (1) increased pepsin secretion is not a product of breaking the barrier per se but that H+ must be present to produce the effect; (2) the increased pepsin output does not come from pepsinogen released by damaged chief cells; (3) low concentrations of H+ are sufficient to stimulate pepsin secretion if the barrier has been damaged; and (4) pepsin secretion increases in the presence of Hel and either salicylic acid or ethanol. Earlier work has shown that pepsin secretion is stimulated by exposing the mucosa to concentrations of Hel (100,200, 250 mn) below those necessary to damage the barrier and that the increased pepsin secretion can be blocked by atropine, 6 This indicates the possible presence of a mechanism for the regulation of pepsin secretion under physiological conditions. In light of the results reported here with a low concentration of Hel (10 mn) it will be interesting to note the effects of small amounts of acid in the intact mucosa. REFERENCES 1. Davenport HW: Gastric mucosal injury by fatty and acetylsalicylic acids. Gastroenterology 46: 245-253, 1964 2. Davenport HW: Damage to the gastric mucosa: effects of salicylates and stimulation. Gastroenterology 49: 189-196, 1965 3. Davenport HW: Potassium fluxes across the resting and stimulated gastric mucosa: injury by salicylic and acetic acids. Gastroenterology 49: 238-245, 1965 4. Davenport HW: Fluid produced by the gastric mucosa during damage by acetic and salicylic acids. Gastroenterology 50:487-499, 1966 5. Johnson LR: Pepsin output from the damaged canine Heidenhain pouch. Am J Dig Dis 16:403-407, 1971 6. Johnson LR: Pepsin stimulated by topical hydrochloric and acetic acids. Gastroenterology 62: 33-38, 1972 7. Thomas JE: An improved cannula for gastric and intestinal fistulas. Proc Soc Exp Bioi Med 46: 260-261, 1941 8. Gregory RA: Gastric secretory responses after portal venous ligation. J Physiol (Lond) 144:123-127, 1958 9. Northrup JH, Kunitz M, Herriot RM: Crystalline Enzymes. Second edition. New York, Columbia University Press, 1948, p 303-307 10. Anson ML: The estimation of pepsin, trypsin, papain and cathepepsin with hemoglobin. J Gen Physiol 22:79-89, 1938 11. Davenport HW: Ethanol damage to canine oxyntic glandular mucosa. Proc Soc Exp Bioi Med 126:657-662, 1967