Digestive Diseases and Sciences, Vol. 37, No. i0 (October 1992), pp. 1600-1605

Effect of Luminal Acidification on Guinea Pig Gastric Mucosa R A M A N A T H A N V I S V A N A T H A N , BM, BCH, FRCS (Irel, Eng, Ed), F W A C S , FICS

H + secretion was studied in guinea pig fundic mucosa incubated in (A) bicarbonateRinger's gassed with 95% 02-5% C02, or (B) HEPES gassed with 100% 02 before and after luminal p H was lowered to 2.0for periods up to 90 min, At p H 2.0for 60 min, H + secretion in group A tissues fell by 35 +- 4% (P = O.02)from a control rate o f l . 3 5 +- 0.09 txeq/cme/hr and in group B tissues by 50 +- 11% (P = O.01) from a control rate o f l . 5 9 +0.08 txeq/cm2/hr. After 90 min at p H 2.0, H § secretion in group A fell by 53 +- 8% (P = O.02) from a control rate o f l . 4 7 +- 0.07 peq/cm2/hr and in group B fell by 44 +- 6% (P = 0.01) from a control rate of 1.38 +- 0.07 tzeq/cm2/hr. Histamine 1 • 1 0 - 4 M stimulation following exposure to p H 2.0 for 90 min increased secretion in group A tissues from 0.80 +- 0.14 to 1.06 +- 0.13 txeq/cm2/hr (P < 0.05), compared with an increase in nonacidified controls from 1.15 +- 0.22 to 1.80 +- 0.20 tzeq/cm2/hr (P < 0.05) and in group B tissues from 1.27 +- 0.10 to 1.56 +- 0.19 peq/cm2/hr ~P < 0.05) compared with nonacidified controls from 1.43 +- 0.22 to 2.23 +- 0.41 peq/cm /hr (P < 0.05). Secretory function and electrical characteristics were adversely affected by luminal acidification to p H 2.0 and suggested a breach in the mucosal barrier with damage to parietal cells. KEY WORDS: acid secretory rate; luminal acidification;parietal cell injury; histamine stimulation.

Injury to the gastric mucosa by luminal acid has been assumed to play a dominant role in the causation of mucosal ulceration in man. The exact mechanism by which luminal acid injures the gastric mucosa is not known. In particular, whether the injurious action of H § is mainly on the surface, chief cells, parietal ceils, or all of these cell types is unclear. The prevailing opinion of this problem has been that surface cells are the first line of defense and hence the tissue that is first injured by luminal H +. Recent studies, however, indicate that both Manuscript received May 23, 1991; accepted February 12, 1992. From the Department of Surgery, Harvard Medical School and the Harvard Digestive Diseases Center, The Beth Israel Hospital, Boston, Massachusetts 02215. This study was funded by the National Institute of Health Grant No. AM 15681. Address for reprint requests: Dr. R. Visvanathan, Department of Surgery, University of Manchester, Clinical Sciences Building, Hope Hospital, Eccles Old Road, Salford M6 8HD, United Kingdom.

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surface cells and chief cells are remarkably impermient to H + on the luminal surface. The present study was carried out to assess specifically the effect of luminal acidification on the function of the oxyntic cells as well as to examine the effects of luminal acid on the electrical characteristics of the entire mucosa. MATERIALS AND METHODS

Nonfasted female guinea pigs (Hartley strain, Elm Hill, Massachusetts) weighing 266 --- 7 g were killed by cervical dislocation and the stomachs removed. The antrum was discarded and the fundus was rinsed in bicarbonateRinger's solution at 37~ C and divided along the greater and lesser curvatures into dorsal and ventral halves. The seromuscular layer was stripped away from each mucosal half according to the methods of Rutten and Ito (1) and each tissue stretched and mounted separately in a Lucite chamber (internal surface area 1.96 cm2). Mounting of the tissue was accomplished within 15 min of sacrificing the animal. During the period of preparation the tissues were Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)

0163-2116/92/1000-1600506.50/0 9 1992 Plenum Publishing Corporation

LUMINAL ACID ON GUINEA PIG GASTRIC MUCOSA kept submerged in bicarbonate--Ringer's solution at 37~ C, gassed with 95% 02-5% CO2 and moistened with this solution during stripping. The assembled chambers were connected to water-jacketed gas-lift circulators maintained at 37~ C. The nutrient surface was bathed in either (A) 15 ml of a bicarbonate-Ringer's solution at pH 7.49 - 0.02 containing 122 mM NaC1, 25 mM NaHCO3, 5 mM KC1, 1.3 mM MgSO4, 2 mM CaC12, 1 mM KH2PO4, and 20 mM glucose and gassed with a 95% 02-5% CO2 mixture bubbled through distilled water at pH 7.0, or (B) 15 ml of HEPES solution at pH 7.41 - 0.01 containing 122 mM NaC1, 25 mM HEPES buffer, 5 mM KC1, 1.3 mM MgSO4, 2 mM CaC12, 1 mM KH2PO4, and 20 mM glucose gassed with 100% 02. The luminal surface was bathed in 15 ml of unbuffered saline (150 mM NaC1) gassed with 100% 02 in both groups. The osmolarities of nutrient and luminal bathing solutions were 300 and 286 mosmol/kg water, respectively, as measured with an Advanced Instruments Osmometer 3WlI (Needham Heights, Massachusetts). Group A tissues were incubated for 90-120 min before acidifying the luminal surface. Group B tissues were initially mounted and perfused for 60 min under identical conditions to group A tissues before changing the nutrient solution to HEPES 100% 02 for 60-90 min after which luminal acidification was instituted. The luminal surface was acidified to pH 3.0, 2.5, or 2.0 with hydrochloric acid (HC1) in both groups of tissues after these initial baseline observations. At the end of the period of acidification (30, 60, or 90 min), the pH of the luminal solution was rapidly titrated back to the control pH of 5.0 with sodium hydroxide solution (NaOH). For electrophysiological measurements, each chamber was equipped with four 3 M KC1 bridges immobilized in 4% agar with calomel reference electrodes (Dow Coming Corp., Midland, Michigan) and was recorded on Kipp and Zonen BD 41 chart recorders (Kipp and Zonen, Rotterdam, The Netherlands). Two bridges were connected to calomel reference electrodes for measurement of transepithelial potential difference (PD), and the other two bridges were connected to Ag-AgC1 electrodes for passing current across tissue for measurement of transepithelial resistance (R). The calomel and Ag-AgC1 electrodes were connected to a preamplifier and then to an automatic voltage clamp (World Pricision Instruments, New Haven, Connecticut) that monitored transepithelial PD and clamped the PD to zero for recording transepithelial short-circuit current (Isr R was determined by passing across the tissue a 100-~A current of 0.05 sec duration from a 45-V battery connected to a microammeter. The immediate change in PD was recorded, and R was calculated from Ohm's law. Since guinea pig gastric mucosa behaves in an ohmic manner to the passage of small current pulses, Is~ was calculated from the open-circuit PD and R. All R values were corrected for solution resistance by the automatic voltage clamp in the Ussing chamber without any tissue. H § secreted into the luminal solution was titrated continuously by the pH stat method. Fluid in the chamber bathing the luminal surface of the gastric mucosa was titrated with 10 mM NaOH to a constant pH of 5.0 except during periods of luminal acidification (Auto Burette ABU 80, Standard pH Meter Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)

PHM82, and Autotirator 80; Radiometer, Copenhagen, Denmark). The base released was monitored on a chart recorder and the corresponding half-hourly acid secretory rates calculated. All experiments were performed simultaneously on paired tissues from the same animal, onehalf being subjected to luminal acidification while the other served as a nonacidified control. Stimulation of acid secretion with 1 • l0 -4 M histamine dihydrochloride (Sigma Chemical Co., St. Louis, Missouri) was also conducted on paired tissues in groups A and B after luminal acidification of one in each pair to pH 2.0 for 90 min. All results are expressed as mean values - SEM (standard error of mean) for each subgroup of tissues in groups A and B. Tissue responses were compared by the t test for paired samples or by analysis of covariance with P < 0.05 being considered significant.

RESULTS Luminal acidification to p H 3.0 and 2.5 had no effect on PD, R and Isr in either group. H o w e v e r , small but significant decreases of 8 --- 1% (P < 0.02) and 16 --- 2% (P < 0.05) in H § secretions were o b s e r v e d in group A ( N = 15) and B (N = 15) tissues, respectively (Figure 1). In tissues e x p o s e d to a luminal p H of 2.0 for 60 min, a significant rise in PD of 4.1 --- 1.1 m V was o b s e r v e d in group A but not in group B tissues during the period of acidification (Figure 2). One h o u r after return of these tissues to control conditions, the PD, R, and Isc were not different from controls in group A tissues (N = 15) e v e n though H + secretion had d e c r e a s e d significantly b y 35 - 4% (P < 0.02) (Figure 3). In the group B tissues ( N = 9), the PD was reduced significantly by 20 --- 5% (P < 0.05), the R was raised significantly b y 11 --- 3% (P < 0.05), the I~c significantly reduced by 26 --- 6% (P < 0.02), and the H + secretion significantly reduced b y 50 --- 11% (P < 0.01) in c o m p a r i s o n with paired controls one hour after return f r o m luminal acidification to control conditions (Figures 2 and 3). Luminal acidification to a p H o f 2.0 for 90 min in group A tissues ( N = 7) did not c a u s e the rise in PD noted after lesser degrees o f acidification or for s h o r t e r p e r i o d s o f time. T h e only significant changes in group A tissues after return to control conditions was a 53 -+ 8% reduction in H + secretion as c o m p a r e d with controls (P = 0.02) (Figure 4). In group B tissues ( N = 11) there w e r e highly significant decreases of 6.2 - 1.4 m V in PD (P = 0.01) (Figure 5) and 44 --- 6% in H § secretion (P = 0.01) (Figure 4). The data do not include those o f four tissues that perforated during or soon after exposure to luminal acidification. E a c h o f these tissues showed a profound fall in PD before perforation.

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VISVANATHAN Luminal acidification of Guinea-pig gastric mucosa Effect on H+secretion

n-15

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30

60

il 60 min.Post acidification

90

Fig 1. Acid secretory rates of HCO 3- Ringer (group A) tissues (N = 15) and HEPES (group

B) tissues (N = 15) before and after exposure to pH 2.5-3.0 for 90 min. In a separate group of experiments designed to define further the degree of injury to the oxyntic cells induced by luminal acidification, H + secretion was stimulated with histamine 1 x 10-4 M beginning 1 hr after removal of luminal acidification and return to control conditions. The tissues had been exposed to a luminal p H of 2.0 for 90 min. Control tissues in groups

A (N = 6) and B (N = 6) showed an increase in H + secretion of 80 +- 27% and 56 --- 21%, respectively (P < 0.05), significantly greater than the stimulated tissues previously exposed to luminal acidification, viz, increase of 57 --- 29%, in group A (P < 0.05) and increase of 21 --- 7% in group B (P < 0.05) (Figure 6). One tissue in group B perforated during exposure to

Luminal acidification of Guinea-pig gastric mucosa Effect on P. D. & tissue resistance

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Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)

LUMINAL ACID ON GUINEA PIG GASTRIC MUCOSA Luminal acidification of Guinea-pig gastric mucosa

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the luminal acid, and the secretory data of this tissue were not included in the results. DISCUSSION Luminal acidification has been implicated in the etiology of peptic ulcer disease, and its damaging effects were presumed to be mainly upon surface ceils where ulceration began (2). This view has been challenged (3), and the injurious circumstances may be more damaging to the much more metabolically active

oxyntic ceils than to other cells in the gastric mucosa (4, 5). The present study showed that a luminal pH of 2.0 was the critical pH at which the mucosal barrier to luminal acid was overcome, producing irreversible falls in transepithelial PD and H + secretion. The results obtained correspond closely to the barrier breaching pH of carmine chief cell monolayers suspended in Ussing chambers across H + gradients (6). They support the contension that oxyntic cells are injured by luminal acid, a surprising finding in view of

Luminal acidification of Guinea-pig gastric mucosa Effect on H+secretion T

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Fig 4. Acid secretory rates of HCO 3- Ringer (group A) tissues (N = 7) and HEPES (group B) tissues (N = 11) before and after exposure to pH 2.0 for 90 min. Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)

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VISVANATHAN Luminal acidification of guinea-pig gastric mucosa Effect on P.D and tissue resistance

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and HEPES (group B) tissues (N = 11) prior to, during, and after exposure to pH 2.0 for 90 min. the normal physiological process of secretion o f H + at the luminal plasma membrane of these cells (4, 5). Tissues bathed in HEPES 100% 02 are more adversely affected by luminal acid than tissues bathed in HCO3- Ringer's 95% 02-5% CO2 in view of perforation of tissues only in HEPES 100% 02 and of rapid decays in PDs in these tissues. These observations are

consistent with reports of enhanced susceptibility of amphibian gastric mucosa to ulceration under similar conditions (absence of HCO3- and CO2) (6, 7). It is known that maximal acid secretion has a protective effect on gastric mucosa against ulceration caused by luminal acid (8, 9). This is presumably due to the alkaline tide of bicarbonate that appears on the

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Fig 6. Acid secretory rates of HCO3- Ringer (group A) tissues and HEPES (group B) tissues following luminal acidification (test tissues, T, only) and after histamine stimulation (both control, C, and test tissues) compared to basal rates.

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Digestive Diseases and Sciences, Vol. 37, No. 10 (October 1992)

LUMINAL ACID ON GUINEA PIG GASTRIC MUCOSA serosal surface in an amount equal to that of acid secreted (2). Physiological injury during absence of overt ulceration therefore could conceivably begin in the oxyntic rather than the surface cells. Present studies do not shed light on the all important question of mechanism of cell death in the gastric mucosa. Further studies (unpublished) suggest that a transient alkalinization of the intracellular p H of oxyntic cells occurs when conditions are changed from H C O 3Ringer's 95% 0 2 - 5 % CO 2 to H E P E S 100% 02. It might be expected that acidification would be better tolerated by such a change, but the reverse is clearly true. It is possible, however, that cellular buffering capacity is enhanced by the H C O 3 - - C O 2 system. Whether luminal acidification injures the mucosa (including oxyntic cells) by gaining access to the cell cytoplasm from the nutrient side has not been determined, but the highly impermeant nature of the apical membrane o f both surface and chief cells suggests that the nutrient or basal side of the cell might be more vulnerable (6). ACKNOWLEDGMENTS I thank Dr. W. Silen for his support and helpful advice and Ms. Rudi Jansons for technical assistance.

Digestive Diseases and Sciences, Vot. 37, No. t0 (October 1992)

REFERENCES 1. Rutten MJ, Ito S: Morphology and electrophysiology of guinea pig gastric mucosal repair in vivo. Am J Physiol 244:6171-6182, 1983 2. Kivilaakso E, Silen W: Pathogenesis of experimental gastric mucosal injury. N Engl J Med 301:364-369, 1979 3. Ashley SW, Soybel DI, Moore D, Cheung LY: Intercellular pH (pHi) in gastric surface epithelium is more susceptible to serosal than mucosal acidification. Surgery 102:371-379, 1987 4. Silen W: New concepts of the gastric mucosal barrier. Am J Surg 133:8-12, 1977 5. Fromm D: Gastric mucosal barrier. In Physiology of the Gastrointestinal Tract. LR Johnson (ed). New York, Raven Press, 1981, pp 733-748 6. Sanders MJ, Ayaion A, Roll M, Soil AH: The apical surface of canine chief cell monolayers resist H § back-diffusion. Nature 313:52-54, 1985 7. Schiessel R, Merhav A, Matthews JB, Fleisher LA, Barzilai A, Silen W: Role of nutrient HCO3- in protection of amphibian gastric mucosa. Am J Physiol 239:6536-6542, 1980 8. Smith P, O'Brien P, Fromm D, Silen W: Secretary state in gastric mucosa and resistance to injury by exogenous acid. Am J Surg 133:81-85, 1977 9. O'Brien P, Silen W: Influence of acid secretary state on the gastric mucosal tolerance to back diffusion of H § Gastroenterology 71:760-765, 1976

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