HISTAMINE EFFECTS ON H+ PERMEABILITY BY ISOLATED GASTRIC MUCOSA

Transcription

1 GASTROENTEROLOGY 70: ,1976 Copyright 1976, by The Williams & Wilkins Co. Vol. 70, No.6 Printed in U.S.A. HISTAMINE EFFECTS ON H+ PERMEABILITY BY ISOLATED GASTRIC MUCOSA DAVID FROMM, M.D., MARK SILEN, AND RUSSELL ROBERTSON, B.S. Department of Surgery, Beth Israel Hospital, 330 Brookline Avenue, Boston, Massachusetts This study examines the effects of histamine on mucosal permeability to acid by isolated stomach of rabbits. Histamine (9 x 10-5 M) decreased antral luminal acid loss which could not be accounted for by active H+ secretion or appearance of organic acid. Histamine also increased antral electrical resistance and decreased the unidirectional luminal to serosal flux ( J I He-erythritol. ~ o f Histamine, theophylline, and N 6,02-dibutyryl adenosine 3',5'-monophosphate did not significantly alter secretion by fundus in the presence of salicylate (5 x 10-3 M). However, after removal of salicylate, these agents stimulated acid secretion. Histamine decreased luminal acid loss by both antrum and fundus treated with salicylate, increased the electrical resistance of these tissues, and decreased Jis erythritol of fundic mucosa. Burimamide (1 x 10-3 M) inhibited the permeability effects of histamine on antrum and fundus. The data indicate that histamine decreases spontaneous antral mucosal permeability as well as salicylateinduced increase in mucosal permeability of both antrum and fundus. The effects of burimamide suggest that antrum contains H2 receptor sites unrelated to acid secretion and that fundus contains H2 receptor sites which also govern permeability but are independent of those related to acid secretion. The effects of histamine on gastric mucosal permeability to acid in vivo are controversial. For normal gastric mucosa, histamine has been reported to decrease,2, 3 increase,4 or have no effects, 6 on the back diffusion of acid, For stomach with enhanced back diffusion of acid, little is known about the effects of histamine on gastric mucosal permeability and the active secretion of H+, Increased quantities of histamine have been shown to be present in the lumen 7 and venous effluent 8 of stomach exposed to agents which increase mucosal permeability. It has been postulated that this increased availability of histamine results in stimulation of acid secretion, thereby potentiating existing tissue damage. 9 However, data for these effects are incomplete. It is also unclear whether the reported -effects of histamine are the result of a direct action of the hormone on the mucosa, or an indirect one, perhaps related to an increase' in blood flow. This study examines the effects of histamine on the rates of luminal acid loss and acid secretion by isolated antral and fundic mucosa of the rabbit in the absence and presence of salicylate. Methods New Zealand white male rabbits (2 to 3 kg), previously allowed free access to food and water, were killed by an Received September 25, Accepted November 26, A portion of this work was presented at the Annual Meeting of the American Gastroenterological Association Research Forum, San Antonio, Texas, May 1975, and a preliminary report appeared in abstract form.' This investigation was supported by Research Grant AM from the National Institute of Arthritis, Metabolism a d Digestive Disease, and by Research Career Development Award AM to Dr. Fromm. intravenous injection of sodium pentobarbital. Either a segment of fundus immediately adjacent to the medial tip of the spleen or the antrum was excised and washed with Ringer's solution and mounted on a lucite half-chamber. By blunt and sharp dissection the serosa and muscularis were stripped from the underlying mucosa, which was continuously bathed in oxygenated Ringer's solution. The mucosa, with its thin supporting muscularis mucosa, was then fixed between two identical half-chambers which were perfused with gas lift circulating reservoirs kept at 37.5 C. The luminal and serosal surfaces were bathed with identical Ringer's solution bubbled with 100% O2 and containil1g the following in millimoles per liter: Na+, 136; Ca++, 3.5; K+, 5; Mg++, 1.2; CI-, 150.4; glucose, 10. Steady state rates of acid secretion, measured at ph 7, using NaOH, 0.01 N, as the titrant, and luminal acid Joss, measured at ph 4, using HCI, 0.01 N, were determined by a ph stat technique as described previously. '0 Each tissue served as its own control, so that after an initial 30- to 40-min flux measurement at a given ph, one or two more 30- to 45-min flux determinations were made after the addition of a given compound at the same ph. Uniform concentrations of given substances were used throughout the experiments. Histamine (Eli Lilly Company, Indianapolis, Ind.), 9 x 10-5 M, was added to either the serosal or luminal bathing solution; burimamide (the generous gift of John G. Paul, Smith, Klein and French Laboratories, Philadelphia, Pa.), 1.3 x 10-3 M, theophylline (Nutritional Biochemical Co., Cleveland, Ohio), 1 -x 1 02 ~ M, or N6,02-dibutyryl adenosine 3 ~, 5 " - m o n o p(db h o s p h a t AMP) (Sigma Chemical Co., St. Louis, Mo.), 2 x 10-3 M, was added only to serosal bathing solution. Sodium salicylate (Fisher Scientific Co., Fair Lawn, N. J.), 5 X 10-3 M, was added to the luminal bathing solution, and 60 to 90 min were allowed to pass in order for the electrical resistance to stabilize at its decreased value. The electrical resistance of each tissue was determined by 1076

2 June 1976 HISTAMINE EFFECTS ON GASTRIC PERMEABILITY 1077 measuring the transmural electrical potential difference response to a pulse of current. Luminal and serosal accumulations of lactate and pyruvate were measured before and after the addition of histamine to antral mucosa. Once steady state conditions were achieved, the luminal and serosal bathing solutions were replaced with fresh solution. Thirty to 45 min later, these solutions were replaced and histamine was added to the serosal side. Lactate and pyruvate concentrations of these solutions were assayed by a lactic dehydrogenase method. 11 Steady state unidirectional luminal to serosal fluxes of 14C-erythritol (New England Nuclear Corp., Boston, Mass.) were measured over two 30-min intervals. The luminal bathing solution contained 5 mm erythritol (Sigma Chemical Co.). The technique of flux measurements and assay of 14C were those described previously for small intestine. 12 Statistical analyses were performed using Student's t-test for paired varieties. Differences were considered significant if the P value was less than Results Antral Mucosa The rate of luminal acid appearance,titrated at ph 7, for 10 antral mucosal segments was less than 0.3 IlM per hr cm 2. The addition of histamine did not significantly alter the rate of luminal acid appearance (P > 0.5) for these tissues. Furthermore, the rate of acid appearance was not diminished by bubbling both sides of five tissues with 100% N 2. The appearance of acid in the luminal bathing solution appears to be predominantly lactic acid (table 2). The effects of histamine on luminal acid loss and electrical resistance of antral mucosa are shown in table 1. In the absence of addition of a compound to the serosal or luminal bathing solutions, the rate of luminal acid loss and electrical resistance did not change significantly during two intervals of measurement. However, after the addition of histamine to the serosal side, a 31 % decrease (P < 0.(01) in rate of luminal acid loss occurred and was associated with a 35% increase (P < 0.001) in electrical resistance. These parameters were not significantly altered by the addition of histamine to the luminal side of the mucosa. The luminal appearance of lactate (table 2) before the addition of histamine did not differ significantly from that observed in the presence of this agent. Similarly, TABLE 1. Effects of histamine on luminal acid loss and electrical resistance of antral mueosa Agent JU+ C Period" No. rabbits net ~ J d added' I'Eq/hr em' (ohms em') I ± ± ± ± ± 7.0 I ± ± ± ± 0.04' ± 9.0 I ± ± 8.5 Histamine/L 0.82 ± ± ± 9.1 ~ a R 1.1 ± ± 5.9' 4.2 ± 3.5 a Fluxes and resistances for periods I and were measured over 30- to 45-min intervals. b Agent IS indicates addition to serosal bathing solution; agent /L indicates addition to luminal bathing solution. C J ~ e = ~ rate of luminal acid loss. Flux and resistance values are given as mean ± 1 SEM. d a = Mean difference ± 1 SEM between periods I and for J ~ e (J) ~ and resistance (R). 'P < TABLE 2. Effects of histamine on lactate and pyruvate appearance and erythritol flux of antral mucosa Period" Agent Luminal No. rabbits {).LC Serosal added' (I'M/hrem') (I'M/hr em') A"I.' Lactate I ± ± ± ± ± 0.06 I ± ± ± ± ± 0.07 Pyruvate I ± ± ± Jl8d (I'M/hrem') A'ld (ohms em') Erythritol I ± ± ± ± ± 13.2 I ± ± ± ± 0.001' ± ± ± 0.04 ~ R a -3.3 ± ± 6.2' a Fluxes (mean ± 1 SEM) and resistances (mean ± 1 SEM) for periods I and were measured over 30-min intervals. b Agent IS indicates addition to serosal bathing solution. cal indicates mean ± 1 SEM difference between periods I and for luminal appearance and as for serosal appearance. d JI. = unidirectional luminal to serosal flux. aj = mean ±1 SEM difference between periods I and for JIB and ar for resistance. 'P <

3 1078 FROMM ETAL. Vol. 70, No.6 histamine did not significantly alter the serosal appearance of lactate. The luminal appearance of pyruvate (table 2) before and after the addition of histamine did not differ significantly and was negligible with respect to the rate of luminal acid loss. The unidirectional flux of erythritol (table 2) did not change significantly over the two intervals of measurement in the absence of histamine. However, the flux of erythritol decreased by 31 % after the addition of histamine (P < 0.001). Table 3 shows the effects of histamine on permeability of antral mucosa treated with burimamide and/or salicylate. The H2 receptor antagonist, burimamide, did not significantly alter the rate of luminal acid loss or electrical resistance in the absence of histamine. Subsequent addition of histamine had no discernible effect on the rate of luminal acid loss or electrical resistance. Sodium salicylate has been previously shown to increase the rate of luminal acid loss by isolated antral ' mucosa of the rabbit. 13 This increase in rate does not change significantly over two intervals of measurement begun after the transmural electrical potential difference and short circuit current decrease and obtain new steady state values. After the addition of histamine to salicylate-treated antral mucosa, a 24 % decrease (P < 0.001) in the rate of luminal acid loss occurs. This effect of histamine is blocked by pretreatment with burimamide. Fundic Mucosa The effects of histamine on acid secretion by fundic mucosa exposed to salicylate are shown in table 4. It has been previously shown that relatively low concentrations of salicylate do not inhibit acid secretion.14 In the presence of salicylate in the luminal bathing solution, the rate of acid secretion does not change significantly over the two intervals of flux measurement. Although histamine stimulates acid secretion by isolated fundic mucosa of the rabbit, 10 histamine is ineffective when the mucosa is exposed to salicylate. This was observed when histamine was added to the serosal side only or to the serosal and luminal sides of the mucosa. It has also been shown previously that the effect of histamine on acid secretion by rabbit fundic mucosa is greater in the presence of theophylline and that DB-AMP is a stimulant of acid secretion. 15 Neither subsequent addition of theophylline to mucosa exposed to salicylate and then histamine nor addition of DB-AMP to salicylate-treated mucosa had a significant effect on the rate of acid secretion. However, when salicylate is removed from the luminal bathing solution, subsequent addition of histamine resulted in a significant increase (P < 0.001) in the rate of acid secretion. Theophylline and DB-AMP were also observed to increase acid secretion after removal of salicylate (n = 3 for each). Because histamine stimulates acid secretion in the absence of salicylate, it is difficult to determine whether or not histamine causes a true decrease in the rate of luminal acid loss. Abolition of the rate of luminal acid loss occurs after the addition of histamine. This is predominantly due to stimulation of acid secretion. 10 However, the effect of histamine on the luminal acid loss presumably can be determined in the presence of salicylate, since this prevents the stimulatory effect of histamine on acid secretion. The effects of histamine on luminal acid loss by fundic mucosa exposed to salicylate are shown in table 5. Salicylate has been previously shown to increase the rate of luminal acid loss by isolated fundic mucosa of the rabbit. 14 The enhanced rate of acid loss and decrease in electrical resistance did not change significantly over the two intervals of measurement. Addition of histamine to the serosal side caused a 13% decrease (P < 0.001) in luminal acid loss and a 16% increase (P < 0.001) in electrical resistance. Subsequent addition of histamine to the luminal side had no significant additional effect on either parameter. In a separate series of experiments, histamine added initially to the luminal side also caused TABLE 3. Effects of histamine on luminal acid loss and electrical resistance of antral mucosa treated with burimamide and/or salicylate Agent JH+ (' Period" No. rabbits ne.t t,.j. added' (I'Eq/hr em' ) (ohms em') t,.r lu 1.10 ± ± 12.9 burimamide/s 1.07 ± ± ± ± 15.3 I histamine/s 1.03 ± ± ± ± 10.6 I salicylate/l ± ± ± ± ± ± 4.1 I salicylate/l ± ± 5.1 histamine/s 1.17 ± ± 0.04' 44.3 ± ± 2.1' I salicylate/l ± ± 6.5 burimamide/s 1.69 ± ± ± ± 3.7 I histamine/s 1.61 ± ± ± ± 2.9 Fluxes and resistances for periods I, and I were measured over 30- to 45-min intervals except for the last group of experiments (salicylate, burimamide, histamine), which were measured over 20-min intervals. Agent /S indicates addition to serosal bathing solution; agent /L indicates addition to luminal bathing solution. C J ne, = rate of luminal acid loss. Flux and resistance values are given as mean ± 1 SEM. d ~ = mean difference ± 1 SEM between periods I and, and I for J ne, (J) and resistance (R). ' P <

4 June 1976 HISTAMINE EFFECTS ON GASTRIC PERMEABILITY TABLE 4. Effects of histamine on acid secretion by fundic mucosa exposed to salicylate IOn Period JH +r Agent - net No. rabbits added' (j1eq/hr em' ).a.,1 d I salicylatell ± 0.43 salicylatell 3.00 ± 0.42 I salicylatell ± 0.42 histamine/s 1.89 ± 0.37 I salicylatell ± 0.41 histamine IS 2.43 ± 0.44 ill histaminell 2.44 ± 0.40 I salicylatell ± 0.46 histamine/s 2.24 ± 0.44 I theophylline/s 2.41 ± 0.47 I salicylatell ± 0.38 DB-AMP/S 2.10 ± 0.40 I salicylatell ± 0.53 salicylatell removed 2.93 ± 0.54 I histamine/s 4.04 ± ± ± ± ± ± ± ± ± ± 0.26 e a Fluxes for periods I, and I were measured over 30- to 45-min intervals. Agent IS indicates addition to serosal bathing solution; agent IL indicates addition to luminal bathing solution., - J : ~ =. rate of acid secretion (mean ± 1 SEM). d D. J = mean ± 1 SEM difference between flux periods I and I, periods and I. e P < TABLE 5. Effect of histamine on luminal acid loss and erythritol flux by fundic mucosa exposed to salicylate Period Agent added' No. rabbits JH + C ne' (j1eq/hr em').a.,1 d (ohms em'} ~ l t d I I I I HistaminelL HistaminelL Burimamide/S ± ± ± ± ± ± ± ± ± ± ± 0.34 JI. erythritol (/lm/hr em') 46.6 ± ± ± ± ± O.l1e 57.4 ± ± ± ± ± O.l1e 50.9 ± ± ± ± ± ± '± 1.g e ± ± 2.1' -2.1 ± 1.8 I ± ± ± ± ± ± ± ± ± 0.002' 46.3 ± ± ± 1.7' a Fluxes for periods I, and I were measured over 20- to 30-min invervals. Agent/S indicates addition to serosal bathing solution; agentll indicates addition to luminal bathing solution. ' J,:'.: = Rate of luminal acid loss. Flux and resistance values are given as mean ± 1 SEM. d D. = mean difference ± 1 SEM between flux periods I and and periods and I for J ~ :(J). and resistance (R). e P < 'P < g P < a decrease in acid loss (P < 0.001) and an increase in electrical resistance (P < 0.025). Subsequent addition of histamine to the serosal side of these tissues had no significant additional effect. The unidirectional flux of erythritol (table 5) did not change significantly over two 20-min intervals of measurement in the presence of salicylate. After the addition of histamine, the erythritol flux decreased by 9% (P < 0.01). Burimamide (table 5) had no effect on acid loss or electrical resistance for salicylate-treated mucosae. However, burimamide prevented the effects of histamine observed in the absence of the H2 receptor antagonist. Theoretically, one might expect an increase in luminal

5 1080 FROMM ETAL. Vol. 70, No.6 acid loss to occur in the presence of an H2 receptor antagonist, since such agents are known to decrease acid secretion. The latter would result in an apparent but not true increase in acid loss. However, the H2 receptor antagonists do not depress basal acid secretion by isolated fundic mucosa of the rabbit (the H2 receptor antagonists do depress histamine-stimulated acid secretion by this tissue). 15 Discussion Histamine added to the serosal, but not mucosal, side of isolated antral mucosa of the rabbit decreases the rate of luminal acid loss. This change cannot be accounted for by stimulation of H+ secretion or by an increase in organic acid appearance. Further evidence that histamine decreases permeability is provided by the observation of an increase in electrical resistance 16 and unidirectional flux of a water- but not lipid-soluble neutral molecule, erythritol. 17 The response of antral mucosal permeability to histamine persists even when permeability to acid is increased by salicylate. It is possible that the effect of histamine on antral mucosa was influenced by the release of gastrin, since the ph of the luminal bathing solution was above that required for inhibition of gastrin release (at least in vivo). However, isolated antrum of the rat has been reported not to spontaneously release gastrin 18; the authors are unaware of histamine's having a direct effect on antral mucosal gastrin release; and exogenous addition of gastrin to isolated antral mucosa of the rabbit has been found to have no effect on 1 uminal acid loss or electrical resistance. 19 Although histamine stimulates H+ secretion by fundic mucosa in the absence of salicylate, histamine is ineffective in the presence of salicylate. Theophylline, which both stimulates and augments the effect of histamine on H+ secretion by rabbit fundus in the absence of salicylate, IS does not alter H+ secretion by mucosa exposed to both salicylate and histamine. Since histamine is believed to stimulate acid secretion by affecting adenylate cyclase activity and theophylline stimulates acid secretion by affecting phosphodiesterase activity, IS this suggests that salicylate may either alter adenylate cyclase and phosphodiesterase activity or affect the metabolic pathway beyond cyclic AMP. DB-AMP, which stimulates H+ secretion in the absence of salicylate, IS also is ineffective in the presence of salicylate, suggesting the latter alternative as a site altered by salicylate. The effect of salicylate in preventing stimulation of acid secretion, however, is readily reversible after removal of salicylate from the bathing solution. In the presence of salicylate the lack of an effect of histamine on H+ secretion does not imply that mucosal permeability also is unaltered by histamine. Although histamine added to the serosal or luminal bathing solution has no discernible effect on H+ secretion at ph 7 in the presence of salicylate, it cannot be strictly assumed that this is so at ph 4. At this ph, histamine causes a decrease in the rate of luminal acid loss. Because mucosal permeability l4 is markedly increased by salicylate at ph 4, but not at ph 7, the effect of histamine conceivably could be due to enhanced access of this agent to H2 receptor sites governing acid secretion. Accessibility of histamine to the sites governing permeability presumably is increased at ph 4, since either serosal or luminal addition increases electrical resistance and decreases the apparent luminal loss of acid. However, the change in electrical resistance and associated decrease in unidirectional erythritol flux suggest that the decrease in H+ flux is at least in part due to a decrease in permeability of salicylate-treated fundic mucosa, just as observed for antral mucosa. The effect of histamine on antral mucosal permeability implies that this portion of stomach contains an H2 receptor which is not associated with acid secretion. Isolated antral mucosa of the rabbit does not actively secrete acid, and the permeability responses to histamine are blocked by an H2 receptor antagonist, burimamide. H2 receptor antagonists added to fundic mucosa block acid secretion stimulated by histamine 15 and block the decrease in permeability caused by histamine under conditions in which acid secretion presumably is resistant to stimulation. These observations suggest that fundic mucosa contains two H2 receptors. One is related to acid secretion, and the other, like that of antrum, is related to mucosal permeability. REFERENCES 1. Fromm D, Silen M, Robertson R: Histamine effects on H+ permeability by isolated gastric mucosa (abstr). Gastroenterology 68:895, Wlodek GK, Leach RK: The effect of histamine stimulation on the net ionic fluxes in Heidenhain and Pavlov fundic pouches. Can J Surg 10:47-52, Rudick J, Chapman ML, Werther JL, et al: Ionic fluxes across canine antral mucosa: effects of pentagastrin and histamine (abstr). Clin Res 18:388, Moody FG, Davis WL: Hydrogen and sodium permeation of canine gastric mucosa during histamine and sodium thiocyanate administration. Gastroenterology 59: , Altamirano M: Backdiffusion of H+ during gastric secretion. Am J Physiol 218:1-6, Altamirano M, Cruz L: Ionic permeability of canine antrum. Am J Physiol 227: , Johnson LR: Histamine liberation by gastric mucosa of pylorusligated rats damaged by acetic or salicylic acids. Proc Soc Exp Bioi Med 121: , Johnson LR, Overholt BF: Release of histamine into gastric venous blood following injury by acetic or salicylic acid. Gastroenterology 52: , Davenport HW: Fluid produced by the gastric mucosa during damage by acetic and salicyclic acids. Gastroenterology 50: , Fromm D, Schwartz JH, Quijano R: Transport of H+ and other electrolytes across isolated gastric mucosa of the rabbit. Am J Physiol 228: , Hohorst H: L-lactate determination with lactic dehydrogenase and D.P.N. In Methods of Enzymatic Analysis. Edited by HV Bergmeyer. New York, Academic, 1963, p Fromm D, Field M, Silen W: Effect of insulin on sugar and amino acid transport across isolated small intestine. Am J Physiol 217:53-57, 1969

6 June 1976 HISTAMINE EFFECTS ON GASTRIC PERMEABILITY Fromm D, Schwartz JR: Ion transport and effects of salicylate and bile salt on isolated antral mucosa (abstr). Gastroenterology 66:695, Fromm D, Schwartz JR, Quijano R: The effects of salicylate and bile salt on ion transport by isolated gastric mucosa of the rabbit. Am J Physiol (in press) 15. Fromm D, Schwartz JH, Quijano R: Effects of cyclic adenosine 3',5'monophosphate and related agents on acid secretion by isolated rabbit gastric mucosa Gastroenterology 69: , Linderholm H: Theoretical considerations regarding the active transport of ions. Acta Physiol Scand 27 (Suppl 97) :1-33, Birkett D, Silen W: Alteration of the physical pathways through the gastric mucosa by sodium taurocholate. Gastroenterology 67: , Dretler R, Wesdorp RIC, Fischer JE: Release of gastrin from isolated perfused gastric antra (abstr). Gastroenterology 66:686, Fromm D, Robertson R: Effects of alcohol on ion transport by isolated gastric and esophageal mucosa. Gastroenterology (in press)