Effects of Aging on Duodenal Bicarbonate Secretion SUN W. KIM, M.D., DILIPKUMAR PAREKH, M.D., COURTNEY M. TOWNSEND, JR., M.D., and JAMES C. THOMPSON, M.D. From the Department of Surgery, The University of Texas Medical Branch, Galveston, Texas
The incidence of duodenal ulcer increases with age, but acid secretion does not. We have investigated the effects of aging on a mechanism of duodenal mucosal defense. Basal and acid-stimulated bicarbonate secretions were measured in the proximal duodenum in anesthetized rats of three different age groups (3 months, 1 year, and 2 years). The proximal duodenum was cannulated in situ between two plastic tubes that extend downward from a titrating chamber, and bicarbonate secretion was measured by the method of Flemstrom et al. Although there was no significant difference in basal secretion among three groups, bicarbonate secretion in response to luminal acid (100 mmol/L Imillimolarl HCI) was diminished in 1-year-old and 2-year-old rats (1-hour integrated bicarbonate secretion; 3 months = 5.8 ± 0.7; 1 year = 3.1 ± 1.0*; 2 years = 2.0 + 0.7*). We also studied the effects of two mediators for acid-stimulated duodenal bicarbonate secretion, vasoactive intestinal polypeptide (VIP), and prostaglandin E2 (PGE2). Intravenous infusion of VIP (0.4,4,40 nmol/ kg/hr) and intraluminal administration of PGE2 (10-5 mol/L [molar] and 10-4 mol/L) induced duodenal bicarbonate secretion in a dose-dependent manner in all three groups, without significant difference between groups. These findings suggest that the release of mediator(s) in response to acid is decreased in the duodenum of the aging rats. The progressive breakdown in mucosal defense mechanisms with increasing age may explain, at least in part, the age-related increase of incidence of duodenal ulcer disease.
duodenum is as low as 1.5 to 2.o.4-7 Hydrogen ions within the lumen are removed from the proximal duodenum by neutralization by bicarbonate that originates from the duodenal surface epithelium (as well as by pancreaticobiliary bicarbonate secretion).8'9 Recent studies suggest that impaired duodenal mucosal secretion of bicarbonate is an important factor in the pathogenesis of duodenal ulcer. 10-13 The mechanisms that govern mucosal bicarbonate secretion include neurohumoral factors9 14-16 and luminal acid. 17-19 The ability of the mucosa to respond to acid seems very important in the maintenance of the surface pH gradient and in the protection of mucosa. Acid-stimulated bicarbonate secretion from the duodenal mucosa appears to be mediated by endogenous prostaglandins, vasoactive intestinal polypeptide (VIP), and possibly by nervous mechanisms.19-26 Aging is known to be associated with secretory and morphologic changes in the gastrointestinal tract,27-3 i but the effect of aging on mucosal bicarbonate secretion has not been investigated previously. In the present study we measured basal and acid-stimulated bicarbonate secretion from the proximal duodenal mucosa in rats of three different age groups. The effects of mediators of acid-stimulated bicarbonate secretion, prostaglandin E2 (PGE2) and VIP, were examined in rats ofthe same different ages.
T n HE INCIDENCE OF duodenal ulcer increases with age. 1-3 Because there is no evidence of increased
aggressiveness of acid peptic digestion with aging, we have investigated age-related changes in bicarbonate secretion from the duodenal mucosa, which is an important mucosal mechanism for defense against acid and pepsin.4 Mucosal bicarbonate secretion maintains the pH within the mucus gel on the mucosal surface of the duodenum at neutrality, even when the intraluminal pH of Presented at the I 1 0th Annual Meeting of the American Surgical Association, Washington, D.C., April 5-8, 1990. Supported by grants from the National Institutes of Health (5R37 DK 15241, PO1 DK 35608), from the American Cancer Society (PDT-220), and in conjunction with the Walls Medical Research Foundation. Dr. Kim is Visiting Scientist from the Department of Surgery, College of Medicine, Seoul National University, Seoul, Korea. Dr. Parekh is Visiting Scientist from the University of Witwatersrand, Johannesburg, South Africa and is supported by scholarships from the Medical Research Council and the Michael and Janie Miller Foundation. Address reprint requestst to James C. Thompson, M.D., Department of Surgery, The University of Texas Medical Branch, Galveston, TX 77550. Accepted for publication April 12, 1990. *p < 0.05 versus 3 months.
Materials and Methods Male Fischer 344 rats (obtained from the National Institute of Aging, Bethesda, Maryland) of three different age groups (3 month, 1 year, and 2 years) were used in this study. Rats were fasted overnight in wire-bottom cages with free access to drinking water. Rats were anesthetized with an intraperitoneal injection of 1.0 to 1.2 g/kg urethane, and a tracheostomy was performed. Cannulas were placed into a jugular vein and a femoral artery for infusion of test agents, and for monitoring blood pressure. Normal saline was infused intravenously at 4 mL/kg/hr to prevent dehydration during the procedure. The abdomen was opened through an upper midline incision. The two plastic cannulas, which extend downward from a titrating cham-
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ber, were inserted into the first portion of the duodenum, which was then ligated around flanges on the tips of the proximal and distal to the tubes in such a manner as to provide an isolated segment of duodenum for study that was excluded from the presence of gastric juice and pancreaticobiliary secretions (Fig. 1). The intraluminal distance between the ends of the two tubes was 12 mm. The abdomen was closed with the titrating chamber clamped 5 cm above the anterior abdominal wall of the rats. The titrating chamber was filled with a luminal perfusate (10 mL of 154 mEq NaCl), which had been bubbled with 100% oxygen for at least 30 minutes before the experiment. The perfusate was circulated by constant-pressure gas lift (0.4 kg/cm2) of 100% oxygen, and was changed two or three times until it was clear. The temperature of the perfusate was maintained at 37°C by means of a warmwater jacket, and body temperature was maintained at 37 to 38 C by a heat lamp. Bicarbonate secretion was measured every 15 minutes by titration of the perfusate to an end point of pH 7.6 with 25 mmol/L HCI, as measured by an autotitration pH stat system (Radiometer, Denmark). After a period of stabilization (usually 30 minutes to 1 hour), two consecutive basal values were obtained, and test agents were then administered. Rats were excluded from the study if the mean arterial pressure was not maintained above 80 mmHg. Arterial blood samples (0.1 mL) were taken through arterial cannula at the beginning and at the end of experiment to check blood pH and HCO3-. If these values were not in In vivo Measurement of Duodenal Bicarbonate Secretion in Rat H"t lamp
333
TABLE 1. Basal and Acid-stimulated Bicarbonate Secretion in Three Age Groups Bicarbonate secretion in three age groups
(AEq/cm/hr) Time (minutes) Basal 1 Basal 2 15 30 45 60
3 month (n = 8)
10.18 10.55 18.91 17.26 14.44 13.93
±0.73 ± 1.04 ± 1.40 1.84 ± 1.33 ± 1.52
1 year (n= 8) 11.60 12.30 ± 15.51 ± 15.96 ± 15.25 ± 13.50
1.56 1.33 1.86 1.98 2.59 1.94
2 years (n = 8) 11.27 11.15 ± 14.31 ± 14.32 ± 12.30 ± 11.73 ±
1.47 1.48 1.53 1.56 1.42 1.12
the normal range (pH: 7.34 to 7.46; HCO3-: 18 to 27 mmol/L), the rats were excluded. To study the effect of luminal acid, the perfusate was replaced by 10 mL of 100 mmol/L HCI (with addition of 54 mmol/L NaCl to achieve isotonicity of the perfusate) for 5 minutes. At the end of this period, the acid was removed and the reservoir and the cannulated duodenal segment were washed with 15 mL of circulating perfusate five times in 10 minutes. After this, bicarbonate secretion was measured for another hour. In the second experiment we studied the effect of VIP and PGE2 on duodenal bicarbonate secretion. Vasoactive intestinal polypeptide in saline that contained 0. 1% bovine serum albumin was infused intravenously at the rate of 0.4, 4, and 40 nmol/kg/hr (4 mL/kg/hr) for 15 minutes in a sequence of increasing molarity. Because topical (intraluminal) administration of PGE2 is more effective than systemic administration,26 we studied the effect of intraluminal infusion of PGE2 on the secretion of duodenal bicarbonate. We compared two dosages of PGE2 (10-5 mol/L and 10-4 mol/L) that were added individually to the perfusate. Vasoactive intestinal polypeptide was purchased from Bachem Inc. (Torrance, CA) and all other chemicals from Sigma Chemical Co. (St. Louis, MO). Vasoactive intestinal polypeptide was dissolved in distilled water and PGE2 was stored at -20°C and dissolved in 95% ethanol just before use. Statistical analysis was performed with Student's t test. Results are expressed as microequivalents of HCO3- per centimeter of duodenum per hour (,uEq/cm/ hr), and percentage of basal bicarbonate secretion. Results
FIG. 1. Diagram of in vivo system for measuring duodenal bicarbonate secretion in the rat (modified from Flemstrom et al., 1982'4).
Twenty-two (of eighty-five) rats were excluded because of hypotension or because of failure to meet acid-base criteria. Basal and acid-stimulated bicarbonate secretion in the three groups are shown in Table 1. There was no significant difference in basal bicarbonate secretion among the three groups. Perfusion of the proximal duodenum with 100 mmol/L HCI significantly stimulated duodenal
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bicarbonate secretion above basal for the entire hour in the 3-month-old rats. The peak bicarbonate output was increased by almost two times over basal. However bicarbonate secretion was weakly stimulated by luminal acid in the 1- and 2-year-old rats; the 1-hour integrated bicarbonate secretions after exposure to luminal acid were calculated and are shown in Figure 2. The response to acid was significantly less in 1-year and 2-year-old rats when compared to the 3-month-old rats. There was no significant difference between the HC03- outputs of the 1- and 2-year-old rats. Although the intraluminal distance between the ends of the two tubes of the reservoir and the length of test segment of duodenum was fixed at 12 mm in all rats, different-sized rats may have different diameter or surface area of duodenal mucosa. Therefore we examined our data expressed as percentage ofbasal secretion to compare the data from rats of different ages and sizes. The mean of two basal values of each rat was expressed as 100% basal output and the percentage bicarbonate output was calculated and shown at different times over the course of the experiment in Figure 3. Exposure of duodenum to luminal acid produced an immediate significant increase in bicarbonate output in all three groups. In young rats bicarbonate output was increased by 85.8% and 66.1% during the first and second 15 minutes after acidification, whereas in 1- and 2-year-old rats, it was increased by 33.6% to 39.2% during the same periods. Furthermore, in the young rats, acid-stimulated bicarbonate output remained
N=8
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Time (min.) FIG. 3. Effect of luminal HCI (100 mmol/L) on bicarbonate secretion from proximal duodenum in three age groups (3 months, open circle; 1 year, open triangle; 2 years, open square). The results are expressed as percentage of basal secretion. Number of rats of each group is eight and mean ± SEM are shown.
above basal for at least 1 hour, whereas in 1- and 2-yearold rats bicarbonate returned to near-basal levels after only 30 minutes. Intravenous infusion of VIP increased duodenal bicarbonate secretion in a dose-dependent manner in all three groups (Fig. 4). All three groups responded well to two higher doses of peptide without difference between groups. Bicarbonate secretion was increased three times during infusion with VIP 40 nmol/kg/hr, and this was greater than that achieved by 100 mmol/L HCl stimulation. Similarly the response to both doses of PGE2 was similar in the three age groups (Fig. 5). In the 3-month-old rat, the bicarbonate output during l0-4 mol/L PGE2 stimulation was similar to the peak output in response to 100 mmol/ L HCI.
-
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______\ Age group
FIG. 2. Effect of luminal acid (100 mmol/L HC1) on duodenal bicarbonate secretion in rats of three age groups. Results in this figure are 1-hour integrated values and expressed as mean ± SEM. * = p < 0.05 vs. 3month-old rats.
Although many consider peptic ulcers a disease ofmiddle age, this concept is based on the number rather than on the rate of ulcer patients. Most epidemiologic studies have not used strict criteria in determining the ulcer-incidence rate, which is defined as the number of new cases arising during a given time interval per specified unit of population.32'33 The well-designed studies of ulcer incidence by Bonnevie" 2 in Denmark and by Kurata and colleagues3 in the United States have demonstrated that the age-specific incidences of gastric ulcer and duodenal ulcer increase in an almost linear manner in men and women from the age of 15 to 80. On the other hand, basal
..,....,...
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EFFECTS OF AGING ON DU(OE)ENAL BICARBONATE SECRETION
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FIG. 4. Duodenal bicarbonate secretion in response to VIP. Vasoactive intestinal polypeptide was infused intravenously at a rate of 0.4, 4, 40 nmol/kg/hr serially. All three groups responded in a similar manner. Mean ± SEM are shown and number of rats for each group is six (open circle, 3 months; open triangle, 1 year; open square, 2-year-old group).
and stimulated gastric acid secretions is decreased progressively with aging in humans27-29 and in rats.30 Therefore the higher incidence of peptic ulcer disease in the aging population, despite a decreased secretion of acid, suggests that factors concerned with mucosal defense may be relatively more important in the pathogenesis of duodenal ulcer in the elderly. The secretion of bicarbonate and mucus in the duodenum is the main defense mechanism against acid.4 Mucus adherent to the luminal surface of the mucosa provides a zone of low turbulence (so-called unstirred layer), allowing the development of a gradient for HC03- from the mucosal side and H+ from the luminal side.34 Small amounts of bicarbonate protect the mucosa against large amounts of acid by neutralizing H+ ions that diffuse back into the mucus layer. Impaired duodenal bicarbonate secretion is thought to play an important role in the pathogenesis of experimental'0-'2 and clinical'3 duodenal ulcer disease. Isenberg and colleagues'3 have reported recently that patients with inactive duodenal ulcers have decreased production of bicarbonate, both basal and acid-stimulated, in the mucosa of the proximal duodenum. Acid is the major physiologic stimulant of bicarbonate secretion.'7-'9 Vasoactive intestinal polypeptide and endogenous prostaglandins are involved in mediating acidstimulated bicarbonate secretion, i',17-'9 but a role for mediation via other neurohumoral pathways has not been
335
clearly demonstrated.35'36 Vasoactive intestinal polypeptide is the most likely humoral factor mediating the bicarbonate secretary response to acid because it is a potent stimulant of duodenal bicarbonate response20 and is released from nerve endings during the exposure of duodenal mucosa to acid.37'38 Algazi and colleagues22 found that the VIP antagonist ([4C1-D-Phe6, Leu'7] VIP) inhibited duodenal bicarbonate secretion stimulated by luminal acidification. Stimulation of bicarbonate secretion is an important action of PGE2.39 Exposure of the duodenum to acid stimulates release of PGE2 from duodenal mucosa,14"9 and acid-induced bicarbonate secretion is blocked by indomethacin.25 Our study indicates that there is an age-related decrease in bicarbonate secretion stimulated by exposure of the duodenal mucosa to acid. We do not know whether this finding can be applied to humans. 'Old' animals are defined as having achieved the age at which one half of the population ordinarily dies (median survival time).404' That age in humans is about the eighth decade of life and in Fischer 344 rats the age is 23 to 26 months.42 Although the exact corresponding age of humans cannot be matched with that of rats used in the present study, our results obtained from three different age groups clearly reflect an age-related change and suggest a progressive breakdown in mucosal defense mechanisms in the proximal duodenum with increasing age. If similar changes are found PGE2 0-5M- pAde
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Time (min.) FIG. 5. Duodenal bicarbonate secretion in response to luminal PGE2. Two different concentrations of PGE2 (10-5 mol/L and 10-4 mol/L) in the perfusate were tested by adding PGE2 solution to circulating perfusate. Mean ± SEM are shown. No difference between groups is noted. Open circle, 3 months (n = 6); open triangle, 1 year (n = 9); open square, 2-
year-old group (n = 6).
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KIM AND OTHERS
in humans, this breakdown may explain, at least in part, the age-related increase in the clinical incidence of duodenal ulcer. Age-related changes in other mucosal defense mechanisms in the gastroduodenal region, including mucus secretion, mucosal blood flow, and rapid epithelization after mucosal injury also require investigation. Hemodynamic and acid-base status influence duodenal mucosal bicarbonate secretion.4344 Metabolic alkalosis, expansion of systemic blood volume, and increased mucosal blood flow may lead to a significant increase in bicarbonate secretion, whereas metabolic acidosis, shock, and decreased mucosal blood flow may reduce secretion. The network of microscopic blood vessels in the mucosa is deficient in the aged rat stomach.45 Because aged rats may be more sensitive to systemic changes induced by our experimental procedures (including anesthesia and operation), pH and blood pressure were monitored during the experiment; 22 of 85 rats were excluded at the time of the experiment because of hypotension or abnormal acid-base balance. Twenty per cent of young rats (5 of 25), 23% (7 of 30) of 1-year-old rats and 33% (10 of 30) of 2-year-old rats were excluded. We have demonstrated that duodenal bicarbonate secretion in response to VIP and PGE2 is intact in 1- and 2-year-old rats, at least in the range of doses tested here. Because VIP and PGE2 appear to be important physiologic mediators of acid-induced bicarbonate secretion, the results suggest that secretory potential of aged duodenal mucosa for bicarbonate is preserved and that the decrease in acid-stimulated bicarbonate secretion found with aging is due to decreased sensitivity of the mucosa to luminal acid or to decreased release of mediators in response to acid. It is unlikely that differences in blood flow or general condition of the rats in different age groups influenced the results as the diminution in bicarbonate secretion was in response to acid only, while the secretion in response to VIP and PGE2 was preserved. A decrease in the number of neuropeptide-containing nerve fibers (substance P, VIP, somatostatin) in the small intestine has been reported in senescent rats (RLEF/LATI female rats, 36 months old).46 We plan to determine whether acid-stimulated VIP release and endogenous PGE2 production and release by the duodenal mucosa are disturbed with aging. Basal and stimulated bicarbonate secretion from the proximal duodenal mucosa were measured in anesthetized rats of different ages. Although there was no difference in basal secretion, acid-stimulated secretion was decreased with increasing age. Vasoactive intestinal polypeptide and PGE2 increased duodenal bicarbonate secretion in all three groups in a similar manner. The present study provides support for the concept that the increasing incidence of duodenal ulcer disease with aging may be caused, at least in part, by a progressive breakdown of the mucosal defensive response to acid in the proximal duodenum.
References 0. The incidence of gastric ulcer in Copenhagen County. Scand J Gastroent 1975; 10:231-239. 2. Bonnevie 0. The incidence of duodenal ulcer in Copenhagen County. Scand J Gastroent 1975; 10:385-393. 3. Kurata JH, Honda GD, Frankl H. The incidence of duodenal and gastric ulcers in a large Health Maintenance Organization. Am J Public Health 1985; 75:625-629. 4. Flemstrom G, Turnberg LA. Gastroduodenal defence mechanisms. Clinics in Gastroenterology 1984; 13:327-354. 5. Dorricott NJ, Fiddian-Green RG, Silen W. Mechanisms of acid disposal in canine duodenum. Am J Physiol 1975; 228:269-275. 6. Harmon JW, Woods M, Gurll NJ. Different mechanisms of hydrogen ion removal in stomach and duodenum. Am J Physiol 1978; 235:E692-E698. 7. Flemstrom G, Kivilaakso E. Demonstration of a pH gradient at the luminal surface of rat duodenum in vivo and its dependence on mucosal alkaline secretion. Gastroenterology 1983; 84:787-794. 8. Simson JNL, Merhav A, Silen W. Alkaline secretion by amphibian duodenum. I. General characteristics. Am J Physiol 1981; 240 (Gastrointest Liver Physiol 3):G401-408. 9. Flemstrom G, Garner A. Gastroduodenal HCO3 transport: characteristics and proposed role in acidity regulation and mucosal protection. Am J Physiol 1982; 242 (Gastrointest Liver Physiol 5):G183-193. 10. Okabe, S, Ishihare Y, Inoo H, Tanaka H. Mepirizole-induced duodenal ulcers in rats and their pathogenesis. Dig Dis Sci 1982; 27: 242-249. 11. Briden S, Flemstrom G, Kivilaakso E. Cysteamine and propionitrile inhibit the rise of duodenal mucosal alkaline secretion in response to luminal acid in rats. Gastroenterology 1985; 88:295-302. 12. Takeuchi K, Furukawa 0, Tanaka H, Okabe S. A new model of duodenal ulcers induced in rats by indomethacin plus histamine. Gastroenterology 1986; 90:636-645. 13. Isenberg JI, Selling JA, Hogan DL, Koss MA. Impaired proximal duodenal mucosal bicarbonate secretion in patients with duodenal ulcer. N Engl J Med 1987; 316:374-379. 14. Flemstrom G, Garner A, Nylander 1, et al. Surface epithelial HCO3 transport by mammalian duodenum in vivo. Am J Physiol 1982; 243 (Gastrointest Liver Physiol 6):G348-358. 15. Flemstrom G, Heylings JR, Garner A. Gastric and duodenal HCO3 transport in vitro: effects of hormones and local transmitters. Am J Physiol 1982; 242 (Gastrointest Liver Physiol 5):GI00-G 110. 16. Hogan DL, Isenberg JI. Gastroduodenal bicarbonate production. Adv Intern Med 1988; 33:385-408. 17. Konturek SJ, Bilski J, Tasler J, Laskiewicz J. Gastroduodenal alkaline response to acid and taurocholate in conscious dog. Am J Physiol 1984; 247 (Gastrointest Liver Physiol 10):G 149-G 154. 18. Heylings JR, Garner A, Flemstrom G. Regulation of gastroduodenal HCO3- transport by luminal acid in the frog in vitro. Am J Physiol 1984; 246 (Gastrointest Liver Physiol 9):G235-G242. 19. Isenberg JI, Smedfors B, Johansson C. Effect of graded doses of intraluminal H', prostaglandin E2, and inhibition of endogenous prostaglandin synthesis on proximal duodenal bicarbonate secretion in unanesthetized rat. Gastroenterology 1985; 88:303307. 20. Flemstrom G, Kivilaakso E, Briden S, et al. Gastroduodenal bicarbonate secretion in mucosal protection. Dig Dis Sci 1985; 30(Suppl 11):63S-68S. 21. Wolosin JD, Thomas FJ, Hogan DL, et al. The effect of vasoactive intestinal peptide, secretin, and glucagon on human duodenal bicarbonate secretion. Scand J Gastroenterol 1989; 24:151-157. 22. Algazi, MC, Chen H-S, Koss, MA, et al. Effect of VIP antagonist on VIP-, PGE2, and acid- stimulated duodenal bicarbonate secretion. Am J Physiol 1989; 256 (Gastrointest Liver Physiol 19): G833-G836. 23. Flemstrom G. Stimulation of HCO3- transport in isolated proximal bullfrog duodenum by prostaglandins. Am J Physiol 1980; 239 (Gastrointest Liver Physiol 2):G 198-G203. 24. Flemstrom G. Gastroduodenal mucosal secretion of bicarbonate and mucus. Am J Med 1986; 81(Suppl 2A):18-22.
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25. Selling JA, Hogan DL, Aly A, et al. Indomethacin inhibits duodenal mucosal bicarbonate secretion and endogenous prostaglandin E2 output in human subjects. Ann Intern Med 1987; 106:368-37 1. 26. Heylings JR, Feldman M. Basal and PGE2 stimulated duodenal bicarbonate secretion in the rat in vivo. Am J Physiol 1988; 255 (Gastrointest Liver Physiol 1 8):G470-G475. 27. Grossman MI, Kirsner JB, Gillespie IE. Basal and histalog-stimulated gastric secretion in control subjects and in patients with peptic ulcer or gastric cancer. Gastroenterology 1963; 45:14-26. 28. Baron JH. Studies of basal and peak acid output with an augmented histamine test. Gut 1963; 4:136-144. 29. Kekki M, Samloff IM, Ihamaki T, et al. Age- and sex-related behaviour of gastric acid secretion at the population level. Scand J Gastroenterol 1982; 17:737-743. 30. Khalil T, Singh P, Fujimura M, et al. Effect of aging on gastric acid secretion, serum gastrin, and antral gastrin content in rats. Dig Dis Sci 1988; 33:1544-1548. 31. Singh P, Rae-Venter B, Townsend CM Jr, et al. Gastrin receptors in normal and malignant gastrointestinal mucosa: age-associated changes. Am J Physiol 1985; 249 (Gastrointest Liver Physiol 12): G76 I -G769. 32. Kurata JH, Haile BM. Epidemiology of peptic ulcer disease. Clinics in Gastroenterology 1984; 13:289-307. 33. Kurata JH. Ulcer epidemiology: an overview and proposed research framework. Gastroenterology 1989; 96:569-580. 34. Heatley NG. Mucosubstance as a barrier to diffusion. Gastroenterology 1959; 37:313-317. 35. Smedfors B, Johansson C. Cholinergic influence on duodenal bicarbonate response to hydrochloric acid perfusion in the conscious rat. Scand J Gastroentrol 1986; 21:809-815.
36. Jonson C, Tunback-Hanson P, Fandriks L. Splanchnic nerve activation inhibits the increase in duodenal HCO3 secretion induced by luminal acidification in the rat. Gastroenterology 1989; 96: 45-49. 37. Fahrenkrug J, Haglund U, Jodal M, et al. Nervous release of vasoactive intestinal polypeptide in the gastrointestinal tract of cats: possible physiological implications. J Physiol 1978; 284:291-305. 38. Bloom SR, Mitchell SJ, Greenberg GR, et al. Release of VIP, secretion and motilin after duodenal acidification in man. Acta Hepato-Gastroenterol 1978; 25:365-368. 39. Silen W. What is cytoprotection of the gastric mucosa? Gastroenterology 1988; 94:232-235. 40. Walford RL. When is a mouse "old"? J Immunol 1976; 117:352353. 41. Khalil T, Thompson JC. Aging and gut peptides. In Thompson JC, Greeley GH Jr, Rayford PL, eds. Gastrointestinal Endocrinology. New York: McGraw Hill, pp 147-157. 42. Chesky J, Rockstein M. Life span characteristics in the male Fischer rat. Experimental Aging Research 1976; 2:399-407. 43. Vattay P, Wenzl E, Feil W, et al. Role of acid base balance and mucosal blood flow in alkaline secretion of rabbit duodenum. Acta Physiologic Hungarica 1989; 73:81-87. 44. Jonson C, Fandriks L, Pettersson A. Increased duodenal HCO3 output after blood volume expansion in the rat: an effect mediated by atrial natriuretic peptide (ANP)? Acta Physiol Scand 1989; 136:263-269. 45. Ohno T, Uramoto H, Masuda Y, et al. Influence of aging on stress ulcer formation in rats. Gastroenterology 1989; 96:A374. 46. Feher E, Penzes L. Density of substance P, vasoactive intestinal polypeptide and somatostatin-containing nerve fibers in the ageing small intestine of the rats. Gerontology 1987; 33:341-348.
DISCUSSION
would need to eliminate that as part of this response that they have shown.
DR. WILLIAM SILEN (Boston, Massachusetts): It was only about 10 years ago that it was recognized almost simultaneously in two laboratories, one in Sweden by Gunnar Flemstrom and in our own laboratory, that the duodenal mucosa alone, quite independent of bile and pancreatic juice, is capable of secreting large quantities of alkaline juice. In some systems stimulated alkaline secretion by the duodenal mucosa comes close to approximating maximal acid output by the stomach. This alkaline secretion is most profound in the first 2 or 3 cm in the duodenum, decreases rapidly as one progresses distally and is not produced by Brunner's glands. It has been demonstrated to occur in the human duodenum, and it has been shown to be reduced in at least one study in humans with duodenal ulcer. Duodenal alkaline secretion probably is the most important mechanism in the duodenal mucosa as a protective mechanism aside from the inherent capability of that mucosa to rapidly reconstitute its integrity when
injured. Whereas for years we have focused our attention on the injurious properties of gastric acid, now we must direct our attention to the protective mechanisms. We and others have shown that nonsteroidal antiinflammatory drugs can reduce this alkaline secretion, and we are indebted to Dr. Kim and his colleagues for calling to our attention the possibility that another factor such as age might also interfere with this important mechanism. As surgeons who manipulate this area to a fairly considerable degree, we need to look forward to recognizing other factors that influence duodenal alkaline secretion. I have one question of Dr. Kim and that relates to whether the acid that was instilled into the duodenum causes an injury. In some species 100 mmol/L acid will indeed do that, and I think Kim and associates need to exclude that possibility here because the appearance of alkaline secretion in the duodenum, while it is mainly an active transport, may occur under injurious circumstances by passive diffusion. The authors
DR. FRANK G. MOODY (Houston, Texas): The experiments of Dr. Kim and his associates were well conceived and presented and relate to what is being found in very carefully performed epidemiologic studies of ulcer disease. For example, in Sheffield in the United Kingdom, there has been a population-corrected, increased incidence of admission of older people to their hospital district with ulcer disease complicated by perforation and hemorrhage. I do not know how old a 2-year rat is. Possibly the author can tell us, but it is known that as people get older, they have less acid secretion. Is it possible also in the rat that there is less acid secretion, leading to either stimulation or inhibition of the generation of prostanoids or VIP? Have you measured the tissue level of either of these agonists of bicarbonate secretion? DR. DAVID FROMM (Detroit, Michigan): A component of alkali secretion is related to capillary filtration of bicarbonate. This then raises the question: Is there a difference in hemodynamic response of a 3month-old as opposed to a 1-year-old rat? Do the authors have any blood flow data in terms of age? DR. JOSEF E. FISCHER (Cincinnati, Ohio): This is an elegantly simple and beautifully designed experiment, which tends to address the paradox of increased ulcer occurrence in the face of declining acid. What they find briefly is a decreased not only volume of response but also duration of response and with duodenal installation of acid; in the face of a preserved response to PGE2 and, at least to my reading, in the upper two dosage range of VIP. I don't know whether I am reading too much into the flat response in the 1-year-old and the 2-year-old rats to the lowest dose of VIP for micromoles per kilogram per hour, but it seemed to me that there was
The incidence of duodenal ulcer increases with age, but acid secretion does not. We have investigated the effects of aging on a mechanism of duodenal mucosal defense. Basal and acid-stimulated bicarbonate secretions were measured in the proximal duodenum in anesthetized rats of three different age groups (3 months, 1 year, and 2 years). The proximal duodenum was cannulated in situ between two plastic tubes that extend downward from a titrating chamber, and bicarbonate secretion was measured by the method of Flemstrom et al. Although there was no significant difference in basal secretion among three groups, bicarbonate secretion in response to luminal acid (100 mmol/L Imillimolarl HCI) was diminished in 1-year-old and 2-year-old rats (1-hour integrated bicarbonate secretion; 3 months = 5.8 ± 0.7; 1 year = 3.1 ± 1.0*; 2 years = 2.0 + 0.7*). We also studied the effects of two mediators for acid-stimulated duodenal bicarbonate secretion, vasoactive intestinal polypeptide (VIP), and prostaglandin E2 (PGE2). Intravenous infusion of VIP (0.4,4,40 nmol/ kg/hr) and intraluminal administration of PGE2 (10-5 mol/L [molar] and 10-4 mol/L) induced duodenal bicarbonate secretion in a dose-dependent manner in all three groups, without significant difference between groups. These findings suggest that the release of mediator(s) in response to acid is decreased in the duodenum of the aging rats. The progressive breakdown in mucosal defense mechanisms with increasing age may explain, at least in part, the age-related increase of incidence of duodenal ulcer disease.
duodenum is as low as 1.5 to 2.o.4-7 Hydrogen ions within the lumen are removed from the proximal duodenum by neutralization by bicarbonate that originates from the duodenal surface epithelium (as well as by pancreaticobiliary bicarbonate secretion).8'9 Recent studies suggest that impaired duodenal mucosal secretion of bicarbonate is an important factor in the pathogenesis of duodenal ulcer. 10-13 The mechanisms that govern mucosal bicarbonate secretion include neurohumoral factors9 14-16 and luminal acid. 17-19 The ability of the mucosa to respond to acid seems very important in the maintenance of the surface pH gradient and in the protection of mucosa. Acid-stimulated bicarbonate secretion from the duodenal mucosa appears to be mediated by endogenous prostaglandins, vasoactive intestinal polypeptide (VIP), and possibly by nervous mechanisms.19-26 Aging is known to be associated with secretory and morphologic changes in the gastrointestinal tract,27-3 i but the effect of aging on mucosal bicarbonate secretion has not been investigated previously. In the present study we measured basal and acid-stimulated bicarbonate secretion from the proximal duodenal mucosa in rats of three different age groups. The effects of mediators of acid-stimulated bicarbonate secretion, prostaglandin E2 (PGE2) and VIP, were examined in rats ofthe same different ages.
T n HE INCIDENCE OF duodenal ulcer increases with age. 1-3 Because there is no evidence of increased
aggressiveness of acid peptic digestion with aging, we have investigated age-related changes in bicarbonate secretion from the duodenal mucosa, which is an important mucosal mechanism for defense against acid and pepsin.4 Mucosal bicarbonate secretion maintains the pH within the mucus gel on the mucosal surface of the duodenum at neutrality, even when the intraluminal pH of Presented at the I 1 0th Annual Meeting of the American Surgical Association, Washington, D.C., April 5-8, 1990. Supported by grants from the National Institutes of Health (5R37 DK 15241, PO1 DK 35608), from the American Cancer Society (PDT-220), and in conjunction with the Walls Medical Research Foundation. Dr. Kim is Visiting Scientist from the Department of Surgery, College of Medicine, Seoul National University, Seoul, Korea. Dr. Parekh is Visiting Scientist from the University of Witwatersrand, Johannesburg, South Africa and is supported by scholarships from the Medical Research Council and the Michael and Janie Miller Foundation. Address reprint requestst to James C. Thompson, M.D., Department of Surgery, The University of Texas Medical Branch, Galveston, TX 77550. Accepted for publication April 12, 1990. *p < 0.05 versus 3 months.
Materials and Methods Male Fischer 344 rats (obtained from the National Institute of Aging, Bethesda, Maryland) of three different age groups (3 month, 1 year, and 2 years) were used in this study. Rats were fasted overnight in wire-bottom cages with free access to drinking water. Rats were anesthetized with an intraperitoneal injection of 1.0 to 1.2 g/kg urethane, and a tracheostomy was performed. Cannulas were placed into a jugular vein and a femoral artery for infusion of test agents, and for monitoring blood pressure. Normal saline was infused intravenously at 4 mL/kg/hr to prevent dehydration during the procedure. The abdomen was opened through an upper midline incision. The two plastic cannulas, which extend downward from a titrating cham-
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ber, were inserted into the first portion of the duodenum, which was then ligated around flanges on the tips of the proximal and distal to the tubes in such a manner as to provide an isolated segment of duodenum for study that was excluded from the presence of gastric juice and pancreaticobiliary secretions (Fig. 1). The intraluminal distance between the ends of the two tubes was 12 mm. The abdomen was closed with the titrating chamber clamped 5 cm above the anterior abdominal wall of the rats. The titrating chamber was filled with a luminal perfusate (10 mL of 154 mEq NaCl), which had been bubbled with 100% oxygen for at least 30 minutes before the experiment. The perfusate was circulated by constant-pressure gas lift (0.4 kg/cm2) of 100% oxygen, and was changed two or three times until it was clear. The temperature of the perfusate was maintained at 37°C by means of a warmwater jacket, and body temperature was maintained at 37 to 38 C by a heat lamp. Bicarbonate secretion was measured every 15 minutes by titration of the perfusate to an end point of pH 7.6 with 25 mmol/L HCI, as measured by an autotitration pH stat system (Radiometer, Denmark). After a period of stabilization (usually 30 minutes to 1 hour), two consecutive basal values were obtained, and test agents were then administered. Rats were excluded from the study if the mean arterial pressure was not maintained above 80 mmHg. Arterial blood samples (0.1 mL) were taken through arterial cannula at the beginning and at the end of experiment to check blood pH and HCO3-. If these values were not in In vivo Measurement of Duodenal Bicarbonate Secretion in Rat H"t lamp
333
TABLE 1. Basal and Acid-stimulated Bicarbonate Secretion in Three Age Groups Bicarbonate secretion in three age groups
(AEq/cm/hr) Time (minutes) Basal 1 Basal 2 15 30 45 60
3 month (n = 8)
10.18 10.55 18.91 17.26 14.44 13.93
±0.73 ± 1.04 ± 1.40 1.84 ± 1.33 ± 1.52
1 year (n= 8) 11.60 12.30 ± 15.51 ± 15.96 ± 15.25 ± 13.50
1.56 1.33 1.86 1.98 2.59 1.94
2 years (n = 8) 11.27 11.15 ± 14.31 ± 14.32 ± 12.30 ± 11.73 ±
1.47 1.48 1.53 1.56 1.42 1.12
the normal range (pH: 7.34 to 7.46; HCO3-: 18 to 27 mmol/L), the rats were excluded. To study the effect of luminal acid, the perfusate was replaced by 10 mL of 100 mmol/L HCI (with addition of 54 mmol/L NaCl to achieve isotonicity of the perfusate) for 5 minutes. At the end of this period, the acid was removed and the reservoir and the cannulated duodenal segment were washed with 15 mL of circulating perfusate five times in 10 minutes. After this, bicarbonate secretion was measured for another hour. In the second experiment we studied the effect of VIP and PGE2 on duodenal bicarbonate secretion. Vasoactive intestinal polypeptide in saline that contained 0. 1% bovine serum albumin was infused intravenously at the rate of 0.4, 4, and 40 nmol/kg/hr (4 mL/kg/hr) for 15 minutes in a sequence of increasing molarity. Because topical (intraluminal) administration of PGE2 is more effective than systemic administration,26 we studied the effect of intraluminal infusion of PGE2 on the secretion of duodenal bicarbonate. We compared two dosages of PGE2 (10-5 mol/L and 10-4 mol/L) that were added individually to the perfusate. Vasoactive intestinal polypeptide was purchased from Bachem Inc. (Torrance, CA) and all other chemicals from Sigma Chemical Co. (St. Louis, MO). Vasoactive intestinal polypeptide was dissolved in distilled water and PGE2 was stored at -20°C and dissolved in 95% ethanol just before use. Statistical analysis was performed with Student's t test. Results are expressed as microequivalents of HCO3- per centimeter of duodenum per hour (,uEq/cm/ hr), and percentage of basal bicarbonate secretion. Results
FIG. 1. Diagram of in vivo system for measuring duodenal bicarbonate secretion in the rat (modified from Flemstrom et al., 1982'4).
Twenty-two (of eighty-five) rats were excluded because of hypotension or because of failure to meet acid-base criteria. Basal and acid-stimulated bicarbonate secretion in the three groups are shown in Table 1. There was no significant difference in basal bicarbonate secretion among the three groups. Perfusion of the proximal duodenum with 100 mmol/L HCI significantly stimulated duodenal
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334
bicarbonate secretion above basal for the entire hour in the 3-month-old rats. The peak bicarbonate output was increased by almost two times over basal. However bicarbonate secretion was weakly stimulated by luminal acid in the 1- and 2-year-old rats; the 1-hour integrated bicarbonate secretions after exposure to luminal acid were calculated and are shown in Figure 2. The response to acid was significantly less in 1-year and 2-year-old rats when compared to the 3-month-old rats. There was no significant difference between the HC03- outputs of the 1- and 2-year-old rats. Although the intraluminal distance between the ends of the two tubes of the reservoir and the length of test segment of duodenum was fixed at 12 mm in all rats, different-sized rats may have different diameter or surface area of duodenal mucosa. Therefore we examined our data expressed as percentage ofbasal secretion to compare the data from rats of different ages and sizes. The mean of two basal values of each rat was expressed as 100% basal output and the percentage bicarbonate output was calculated and shown at different times over the course of the experiment in Figure 3. Exposure of duodenum to luminal acid produced an immediate significant increase in bicarbonate output in all three groups. In young rats bicarbonate output was increased by 85.8% and 66.1% during the first and second 15 minutes after acidification, whereas in 1- and 2-year-old rats, it was increased by 33.6% to 39.2% during the same periods. Furthermore, in the young rats, acid-stimulated bicarbonate output remained
N=8
7
*p < 0.05 vs. 3mo
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Time (min.) FIG. 3. Effect of luminal HCI (100 mmol/L) on bicarbonate secretion from proximal duodenum in three age groups (3 months, open circle; 1 year, open triangle; 2 years, open square). The results are expressed as percentage of basal secretion. Number of rats of each group is eight and mean ± SEM are shown.
above basal for at least 1 hour, whereas in 1- and 2-yearold rats bicarbonate returned to near-basal levels after only 30 minutes. Intravenous infusion of VIP increased duodenal bicarbonate secretion in a dose-dependent manner in all three groups (Fig. 4). All three groups responded well to two higher doses of peptide without difference between groups. Bicarbonate secretion was increased three times during infusion with VIP 40 nmol/kg/hr, and this was greater than that achieved by 100 mmol/L HCl stimulation. Similarly the response to both doses of PGE2 was similar in the three age groups (Fig. 5). In the 3-month-old rat, the bicarbonate output during l0-4 mol/L PGE2 stimulation was similar to the peak output in response to 100 mmol/ L HCI.
-
h.
Discussion 2 01~~
E 0
.
______\ Age group
FIG. 2. Effect of luminal acid (100 mmol/L HC1) on duodenal bicarbonate secretion in rats of three age groups. Results in this figure are 1-hour integrated values and expressed as mean ± SEM. * = p < 0.05 vs. 3month-old rats.
Although many consider peptic ulcers a disease ofmiddle age, this concept is based on the number rather than on the rate of ulcer patients. Most epidemiologic studies have not used strict criteria in determining the ulcer-incidence rate, which is defined as the number of new cases arising during a given time interval per specified unit of population.32'33 The well-designed studies of ulcer incidence by Bonnevie" 2 in Denmark and by Kurata and colleagues3 in the United States have demonstrated that the age-specific incidences of gastric ulcer and duodenal ulcer increase in an almost linear manner in men and women from the age of 15 to 80. On the other hand, basal
..,....,...
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EFFECTS OF AGING ON DU(OE)ENAL BICARBONATE SECRETION
-
VIP (nmolelkg/hr)
400-
-*- 40 -
300-
-
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45
FIG. 4. Duodenal bicarbonate secretion in response to VIP. Vasoactive intestinal polypeptide was infused intravenously at a rate of 0.4, 4, 40 nmol/kg/hr serially. All three groups responded in a similar manner. Mean ± SEM are shown and number of rats for each group is six (open circle, 3 months; open triangle, 1 year; open square, 2-year-old group).
and stimulated gastric acid secretions is decreased progressively with aging in humans27-29 and in rats.30 Therefore the higher incidence of peptic ulcer disease in the aging population, despite a decreased secretion of acid, suggests that factors concerned with mucosal defense may be relatively more important in the pathogenesis of duodenal ulcer in the elderly. The secretion of bicarbonate and mucus in the duodenum is the main defense mechanism against acid.4 Mucus adherent to the luminal surface of the mucosa provides a zone of low turbulence (so-called unstirred layer), allowing the development of a gradient for HC03- from the mucosal side and H+ from the luminal side.34 Small amounts of bicarbonate protect the mucosa against large amounts of acid by neutralizing H+ ions that diffuse back into the mucus layer. Impaired duodenal bicarbonate secretion is thought to play an important role in the pathogenesis of experimental'0-'2 and clinical'3 duodenal ulcer disease. Isenberg and colleagues'3 have reported recently that patients with inactive duodenal ulcers have decreased production of bicarbonate, both basal and acid-stimulated, in the mucosa of the proximal duodenum. Acid is the major physiologic stimulant of bicarbonate secretion.'7-'9 Vasoactive intestinal polypeptide and endogenous prostaglandins are involved in mediating acidstimulated bicarbonate secretion, i',17-'9 but a role for mediation via other neurohumoral pathways has not been
335
clearly demonstrated.35'36 Vasoactive intestinal polypeptide is the most likely humoral factor mediating the bicarbonate secretary response to acid because it is a potent stimulant of duodenal bicarbonate response20 and is released from nerve endings during the exposure of duodenal mucosa to acid.37'38 Algazi and colleagues22 found that the VIP antagonist ([4C1-D-Phe6, Leu'7] VIP) inhibited duodenal bicarbonate secretion stimulated by luminal acidification. Stimulation of bicarbonate secretion is an important action of PGE2.39 Exposure of the duodenum to acid stimulates release of PGE2 from duodenal mucosa,14"9 and acid-induced bicarbonate secretion is blocked by indomethacin.25 Our study indicates that there is an age-related decrease in bicarbonate secretion stimulated by exposure of the duodenal mucosa to acid. We do not know whether this finding can be applied to humans. 'Old' animals are defined as having achieved the age at which one half of the population ordinarily dies (median survival time).404' That age in humans is about the eighth decade of life and in Fischer 344 rats the age is 23 to 26 months.42 Although the exact corresponding age of humans cannot be matched with that of rats used in the present study, our results obtained from three different age groups clearly reflect an age-related change and suggest a progressive breakdown in mucosal defense mechanisms in the proximal duodenum with increasing age. If similar changes are found PGE2 0-5M- pAde
300-
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>
250-
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00 2000
3mo
0" 1500
100-
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15
30
45
60
Time (min.) FIG. 5. Duodenal bicarbonate secretion in response to luminal PGE2. Two different concentrations of PGE2 (10-5 mol/L and 10-4 mol/L) in the perfusate were tested by adding PGE2 solution to circulating perfusate. Mean ± SEM are shown. No difference between groups is noted. Open circle, 3 months (n = 6); open triangle, 1 year (n = 9); open square, 2-
year-old group (n = 6).
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KIM AND OTHERS
in humans, this breakdown may explain, at least in part, the age-related increase in the clinical incidence of duodenal ulcer. Age-related changes in other mucosal defense mechanisms in the gastroduodenal region, including mucus secretion, mucosal blood flow, and rapid epithelization after mucosal injury also require investigation. Hemodynamic and acid-base status influence duodenal mucosal bicarbonate secretion.4344 Metabolic alkalosis, expansion of systemic blood volume, and increased mucosal blood flow may lead to a significant increase in bicarbonate secretion, whereas metabolic acidosis, shock, and decreased mucosal blood flow may reduce secretion. The network of microscopic blood vessels in the mucosa is deficient in the aged rat stomach.45 Because aged rats may be more sensitive to systemic changes induced by our experimental procedures (including anesthesia and operation), pH and blood pressure were monitored during the experiment; 22 of 85 rats were excluded at the time of the experiment because of hypotension or abnormal acid-base balance. Twenty per cent of young rats (5 of 25), 23% (7 of 30) of 1-year-old rats and 33% (10 of 30) of 2-year-old rats were excluded. We have demonstrated that duodenal bicarbonate secretion in response to VIP and PGE2 is intact in 1- and 2-year-old rats, at least in the range of doses tested here. Because VIP and PGE2 appear to be important physiologic mediators of acid-induced bicarbonate secretion, the results suggest that secretory potential of aged duodenal mucosa for bicarbonate is preserved and that the decrease in acid-stimulated bicarbonate secretion found with aging is due to decreased sensitivity of the mucosa to luminal acid or to decreased release of mediators in response to acid. It is unlikely that differences in blood flow or general condition of the rats in different age groups influenced the results as the diminution in bicarbonate secretion was in response to acid only, while the secretion in response to VIP and PGE2 was preserved. A decrease in the number of neuropeptide-containing nerve fibers (substance P, VIP, somatostatin) in the small intestine has been reported in senescent rats (RLEF/LATI female rats, 36 months old).46 We plan to determine whether acid-stimulated VIP release and endogenous PGE2 production and release by the duodenal mucosa are disturbed with aging. Basal and stimulated bicarbonate secretion from the proximal duodenal mucosa were measured in anesthetized rats of different ages. Although there was no difference in basal secretion, acid-stimulated secretion was decreased with increasing age. Vasoactive intestinal polypeptide and PGE2 increased duodenal bicarbonate secretion in all three groups in a similar manner. The present study provides support for the concept that the increasing incidence of duodenal ulcer disease with aging may be caused, at least in part, by a progressive breakdown of the mucosal defensive response to acid in the proximal duodenum.
References 0. The incidence of gastric ulcer in Copenhagen County. Scand J Gastroent 1975; 10:231-239. 2. Bonnevie 0. The incidence of duodenal ulcer in Copenhagen County. Scand J Gastroent 1975; 10:385-393. 3. Kurata JH, Honda GD, Frankl H. The incidence of duodenal and gastric ulcers in a large Health Maintenance Organization. Am J Public Health 1985; 75:625-629. 4. Flemstrom G, Turnberg LA. Gastroduodenal defence mechanisms. Clinics in Gastroenterology 1984; 13:327-354. 5. Dorricott NJ, Fiddian-Green RG, Silen W. Mechanisms of acid disposal in canine duodenum. Am J Physiol 1975; 228:269-275. 6. Harmon JW, Woods M, Gurll NJ. Different mechanisms of hydrogen ion removal in stomach and duodenum. Am J Physiol 1978; 235:E692-E698. 7. Flemstrom G, Kivilaakso E. Demonstration of a pH gradient at the luminal surface of rat duodenum in vivo and its dependence on mucosal alkaline secretion. Gastroenterology 1983; 84:787-794. 8. Simson JNL, Merhav A, Silen W. Alkaline secretion by amphibian duodenum. I. General characteristics. Am J Physiol 1981; 240 (Gastrointest Liver Physiol 3):G401-408. 9. Flemstrom G, Garner A. Gastroduodenal HCO3 transport: characteristics and proposed role in acidity regulation and mucosal protection. Am J Physiol 1982; 242 (Gastrointest Liver Physiol 5):G183-193. 10. Okabe, S, Ishihare Y, Inoo H, Tanaka H. Mepirizole-induced duodenal ulcers in rats and their pathogenesis. Dig Dis Sci 1982; 27: 242-249. 11. Briden S, Flemstrom G, Kivilaakso E. Cysteamine and propionitrile inhibit the rise of duodenal mucosal alkaline secretion in response to luminal acid in rats. Gastroenterology 1985; 88:295-302. 12. Takeuchi K, Furukawa 0, Tanaka H, Okabe S. A new model of duodenal ulcers induced in rats by indomethacin plus histamine. Gastroenterology 1986; 90:636-645. 13. Isenberg JI, Selling JA, Hogan DL, Koss MA. Impaired proximal duodenal mucosal bicarbonate secretion in patients with duodenal ulcer. N Engl J Med 1987; 316:374-379. 14. Flemstrom G, Garner A, Nylander 1, et al. Surface epithelial HCO3 transport by mammalian duodenum in vivo. Am J Physiol 1982; 243 (Gastrointest Liver Physiol 6):G348-358. 15. Flemstrom G, Heylings JR, Garner A. Gastric and duodenal HCO3 transport in vitro: effects of hormones and local transmitters. Am J Physiol 1982; 242 (Gastrointest Liver Physiol 5):GI00-G 110. 16. Hogan DL, Isenberg JI. Gastroduodenal bicarbonate production. Adv Intern Med 1988; 33:385-408. 17. Konturek SJ, Bilski J, Tasler J, Laskiewicz J. Gastroduodenal alkaline response to acid and taurocholate in conscious dog. Am J Physiol 1984; 247 (Gastrointest Liver Physiol 10):G 149-G 154. 18. Heylings JR, Garner A, Flemstrom G. Regulation of gastroduodenal HCO3- transport by luminal acid in the frog in vitro. Am J Physiol 1984; 246 (Gastrointest Liver Physiol 9):G235-G242. 19. Isenberg JI, Smedfors B, Johansson C. Effect of graded doses of intraluminal H', prostaglandin E2, and inhibition of endogenous prostaglandin synthesis on proximal duodenal bicarbonate secretion in unanesthetized rat. Gastroenterology 1985; 88:303307. 20. Flemstrom G, Kivilaakso E, Briden S, et al. Gastroduodenal bicarbonate secretion in mucosal protection. Dig Dis Sci 1985; 30(Suppl 11):63S-68S. 21. Wolosin JD, Thomas FJ, Hogan DL, et al. The effect of vasoactive intestinal peptide, secretin, and glucagon on human duodenal bicarbonate secretion. Scand J Gastroenterol 1989; 24:151-157. 22. Algazi, MC, Chen H-S, Koss, MA, et al. Effect of VIP antagonist on VIP-, PGE2, and acid- stimulated duodenal bicarbonate secretion. Am J Physiol 1989; 256 (Gastrointest Liver Physiol 19): G833-G836. 23. Flemstrom G. Stimulation of HCO3- transport in isolated proximal bullfrog duodenum by prostaglandins. Am J Physiol 1980; 239 (Gastrointest Liver Physiol 2):G 198-G203. 24. Flemstrom G. Gastroduodenal mucosal secretion of bicarbonate and mucus. Am J Med 1986; 81(Suppl 2A):18-22.
1. Bonnevie
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25. Selling JA, Hogan DL, Aly A, et al. Indomethacin inhibits duodenal mucosal bicarbonate secretion and endogenous prostaglandin E2 output in human subjects. Ann Intern Med 1987; 106:368-37 1. 26. Heylings JR, Feldman M. Basal and PGE2 stimulated duodenal bicarbonate secretion in the rat in vivo. Am J Physiol 1988; 255 (Gastrointest Liver Physiol 1 8):G470-G475. 27. Grossman MI, Kirsner JB, Gillespie IE. Basal and histalog-stimulated gastric secretion in control subjects and in patients with peptic ulcer or gastric cancer. Gastroenterology 1963; 45:14-26. 28. Baron JH. Studies of basal and peak acid output with an augmented histamine test. Gut 1963; 4:136-144. 29. Kekki M, Samloff IM, Ihamaki T, et al. Age- and sex-related behaviour of gastric acid secretion at the population level. Scand J Gastroenterol 1982; 17:737-743. 30. Khalil T, Singh P, Fujimura M, et al. Effect of aging on gastric acid secretion, serum gastrin, and antral gastrin content in rats. Dig Dis Sci 1988; 33:1544-1548. 31. Singh P, Rae-Venter B, Townsend CM Jr, et al. Gastrin receptors in normal and malignant gastrointestinal mucosa: age-associated changes. Am J Physiol 1985; 249 (Gastrointest Liver Physiol 12): G76 I -G769. 32. Kurata JH, Haile BM. Epidemiology of peptic ulcer disease. Clinics in Gastroenterology 1984; 13:289-307. 33. Kurata JH. Ulcer epidemiology: an overview and proposed research framework. Gastroenterology 1989; 96:569-580. 34. Heatley NG. Mucosubstance as a barrier to diffusion. Gastroenterology 1959; 37:313-317. 35. Smedfors B, Johansson C. Cholinergic influence on duodenal bicarbonate response to hydrochloric acid perfusion in the conscious rat. Scand J Gastroentrol 1986; 21:809-815.
36. Jonson C, Tunback-Hanson P, Fandriks L. Splanchnic nerve activation inhibits the increase in duodenal HCO3 secretion induced by luminal acidification in the rat. Gastroenterology 1989; 96: 45-49. 37. Fahrenkrug J, Haglund U, Jodal M, et al. Nervous release of vasoactive intestinal polypeptide in the gastrointestinal tract of cats: possible physiological implications. J Physiol 1978; 284:291-305. 38. Bloom SR, Mitchell SJ, Greenberg GR, et al. Release of VIP, secretion and motilin after duodenal acidification in man. Acta Hepato-Gastroenterol 1978; 25:365-368. 39. Silen W. What is cytoprotection of the gastric mucosa? Gastroenterology 1988; 94:232-235. 40. Walford RL. When is a mouse "old"? J Immunol 1976; 117:352353. 41. Khalil T, Thompson JC. Aging and gut peptides. In Thompson JC, Greeley GH Jr, Rayford PL, eds. Gastrointestinal Endocrinology. New York: McGraw Hill, pp 147-157. 42. Chesky J, Rockstein M. Life span characteristics in the male Fischer rat. Experimental Aging Research 1976; 2:399-407. 43. Vattay P, Wenzl E, Feil W, et al. Role of acid base balance and mucosal blood flow in alkaline secretion of rabbit duodenum. Acta Physiologic Hungarica 1989; 73:81-87. 44. Jonson C, Fandriks L, Pettersson A. Increased duodenal HCO3 output after blood volume expansion in the rat: an effect mediated by atrial natriuretic peptide (ANP)? Acta Physiol Scand 1989; 136:263-269. 45. Ohno T, Uramoto H, Masuda Y, et al. Influence of aging on stress ulcer formation in rats. Gastroenterology 1989; 96:A374. 46. Feher E, Penzes L. Density of substance P, vasoactive intestinal polypeptide and somatostatin-containing nerve fibers in the ageing small intestine of the rats. Gerontology 1987; 33:341-348.
DISCUSSION
would need to eliminate that as part of this response that they have shown.
DR. WILLIAM SILEN (Boston, Massachusetts): It was only about 10 years ago that it was recognized almost simultaneously in two laboratories, one in Sweden by Gunnar Flemstrom and in our own laboratory, that the duodenal mucosa alone, quite independent of bile and pancreatic juice, is capable of secreting large quantities of alkaline juice. In some systems stimulated alkaline secretion by the duodenal mucosa comes close to approximating maximal acid output by the stomach. This alkaline secretion is most profound in the first 2 or 3 cm in the duodenum, decreases rapidly as one progresses distally and is not produced by Brunner's glands. It has been demonstrated to occur in the human duodenum, and it has been shown to be reduced in at least one study in humans with duodenal ulcer. Duodenal alkaline secretion probably is the most important mechanism in the duodenal mucosa as a protective mechanism aside from the inherent capability of that mucosa to rapidly reconstitute its integrity when
injured. Whereas for years we have focused our attention on the injurious properties of gastric acid, now we must direct our attention to the protective mechanisms. We and others have shown that nonsteroidal antiinflammatory drugs can reduce this alkaline secretion, and we are indebted to Dr. Kim and his colleagues for calling to our attention the possibility that another factor such as age might also interfere with this important mechanism. As surgeons who manipulate this area to a fairly considerable degree, we need to look forward to recognizing other factors that influence duodenal alkaline secretion. I have one question of Dr. Kim and that relates to whether the acid that was instilled into the duodenum causes an injury. In some species 100 mmol/L acid will indeed do that, and I think Kim and associates need to exclude that possibility here because the appearance of alkaline secretion in the duodenum, while it is mainly an active transport, may occur under injurious circumstances by passive diffusion. The authors
DR. FRANK G. MOODY (Houston, Texas): The experiments of Dr. Kim and his associates were well conceived and presented and relate to what is being found in very carefully performed epidemiologic studies of ulcer disease. For example, in Sheffield in the United Kingdom, there has been a population-corrected, increased incidence of admission of older people to their hospital district with ulcer disease complicated by perforation and hemorrhage. I do not know how old a 2-year rat is. Possibly the author can tell us, but it is known that as people get older, they have less acid secretion. Is it possible also in the rat that there is less acid secretion, leading to either stimulation or inhibition of the generation of prostanoids or VIP? Have you measured the tissue level of either of these agonists of bicarbonate secretion? DR. DAVID FROMM (Detroit, Michigan): A component of alkali secretion is related to capillary filtration of bicarbonate. This then raises the question: Is there a difference in hemodynamic response of a 3month-old as opposed to a 1-year-old rat? Do the authors have any blood flow data in terms of age? DR. JOSEF E. FISCHER (Cincinnati, Ohio): This is an elegantly simple and beautifully designed experiment, which tends to address the paradox of increased ulcer occurrence in the face of declining acid. What they find briefly is a decreased not only volume of response but also duration of response and with duodenal installation of acid; in the face of a preserved response to PGE2 and, at least to my reading, in the upper two dosage range of VIP. I don't know whether I am reading too much into the flat response in the 1-year-old and the 2-year-old rats to the lowest dose of VIP for micromoles per kilogram per hour, but it seemed to me that there was
