EFFECTS OF THE NON-STEROIDAL ANTIPHLOGISTICS ON THE GASTRIC MUCOSAL BARRIER AND HEXOSAMINE CONTENT IN RATS. Shigehiko NARUMI and Morio KANNO

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1 EFFECTS OF THE NON-STEROIDAL ANTIPHLOGISTICS ON THE GASTRIC MUCOSAL BARRIER AND HEXOSAMINE CONTENT IN RATS Shigehiko NARUMI and Morio KANNO Biological Research Laboratories, Central Research Division, Takeda Chemical Industries, Ltd., Higashiyodogawa-ku, Osaka, Japan Received for publication April 15, 1972 The gastric mucosal barrier consisting of mucous and mucosal barriers in the rat is known to protect the gastric mucosa from the digestive activity of the gastric juice. The postulation by Plessis (1) that the permeability change of the gastric mucosal barrier is the main trigger in generating the gastric ulcer is originates from the observation that bile juice frequently present in the stomach of patients with gastric and duodenal ulcers induces the decreased secretion of the gastric mucus, by which the gastric mucosa is made more sensitive to the gastric acid there by activating the development of ulcers. Intragastric administration of bile juice in the dog was reported by Davenport (2) to result in the destruction of the mucosal barrier and the back diffusion of hydrogen ion to the mucosal layer. Similar results were recently obtained by the same procedure in humans (3). Decreased response of the gastric acid secretion to alcohol, caffein, or histamine in patients with gastric ulcers was also due to the back diffusion of hydrogen ion caused by the destruction of the gastric mucosal barrier (4, 5).<BR> Correlation between the gastric mucosal barrier and the development of the gastric ulcers was studied in animals treated with non-steroidal antiphlogistics and stress stimuli induced by a combination of restraint and immersion of rats into cold water. METHODS 1. Procedures in formation of the gastric ulcers Male SD rats 7 to 8 weeks in age and fasted for approx. 18 hr prior to the experiment were divided into groups of 5. Acetylsalicylic acid (ASA), phenylbutazone (PBZ), ibufenac, indomethacin, or 6-chloro-5-cyclohexylindan-l-carboxylic acid (TAI 284) suspended in 5 % gumi arabicum was administered orally by use of a gastric tube, and 6 hr after the animals were sacrificed under deep ether anesthesia for extirpation of whole stomach. The control group was treated with the vehicle alone. Animals of one of the groups were restrained on the metal net in supine position and were immersed in water at 23 C at the vertical direction untill the xiphoid for 0, 3, 8 and 22 hr (6). The ulcers formed on the gastric mucosa were scored by grading the longest size (diameter) of the ulcers by use of stereoscope; 0: no change, l : mucosal and submucosal bleeding or mucosal erosion,

2 2: ulcer below 1 mm in size and below 10 in number, 3: ulcers 1 to 3 mm in size and less than 10 in number or below 1 mm in size and more than 10 in number, 4: ulcers 3 to 5 mm in size and below 10 in number or I to 3 mm in size and above 10 in number and 5: ulcers above 5 mm in size. 2. Measurement of hexosamine in the gastric mucosa The glandular part of the stomach after the observation of the ulcers was dried at 70 to 76'C overnight and then weighed. The part of the stomach sampled was sometimes divided into corpus and antrum. After hydrolysing the dried stomach overnight in 2 N HCI(7), the gastric hexosamine was measured following the method of Boas (8). Hexosamine in the hydrolyzates was adsorbed by passing through the Dowex-50 column ( mesh) and the adsorbed hexosamine was eluted with 2 H NCI. The eluates in volume of 1 to 3 ml to which 1 ml of acetylaceton reagent had been added were boiled for 45 min at 89 to 92 C. The boiled eluates after being cooled at room temperature were mixed after addition of 2.5 ml of ethylalcohol. One to two hr after the addition of 1 ml of Ehrlich's reagent, hexosamine was determined at a wavelength of 530 mp using glucosamine as standard. 3. Evaluation of the function of the gastric mucosal barrier The function of the gastric mucosal barrier was evaluated in groups of male rats 7 to 8 weeks in age and fasted previously for 24 hr following the method of Overholts et al. (9). Surgical laparotomy in ether anesthetized rats was followed by exposure of the stomach and ligation of the cardiac and pyloric rings and a simultaneous bilateral section of the cardiac branch of the vagus nerve. The gastric lumen was washed with warm deionized water until the return was clear, and the infusion of 5 ml of 100 mm HCI solution into the lumen was followed by suturing of the incised wounds. The antiphlogistic agents were administered intragastrically 3 hr before surgery. The animals were sacrificed under deep ether anesthesia 2 hr after the termination of the surgical operation for the extirpation of the whole stomach. The concentration of hydrogen, sodium, potassium and chloride ions in the gastric content was measured. Sodium and potassium were measured by use of a flame photometer, chloride by use a chloridmeter and hydrogen by titration with 0.01 N NaOH using phenolphthalein as an indicator. The estimated values expressed are the differences 'from those before infusion of 100 mm HCI solution. RESULTS 1. Production of gastric ulcers by antiphlogistics Gross gastric lesions in rats treated with ASA (260 mg/kg) were irregularly circular to oval with diameters ranging from 1 to 3 mm in glandular stomach. Slight hemorrhage was occasionally present. Treatment of the animals with PBZ (200 mg/kg), indomethacin (30 mg/kg), ibufenac (500 mg/kg), or TAI 284 (30 mg/kg) also produced the gastric ulcers at the glandular stomach and the shape or diameter was nearly equal of that of ASA treat ment. 2. Effects of the antiphlogistics on the gastric hexosamine content Intragastric administration of the antiphlogistics produced gastric ulcers at the

3 TABLE 1. Effects of antiphlogistics on gastric hexosamine content in rats. Antiphlogistics in dose described were orally administered, and 6 hr thereafter, rats were sacrificed. Each number represents mean of 5 rats and standard error of mean. TABLE 2. Effects of antiphlogistics on the hexosamine contents of the antrum and corpus in rats. glandular stomach and the score of ulcer ranging from 2 to 3 (Table 1). The animals treated with ASA or PBZ produced a decrease of gastric hexosamine which was remarkable in the former and slight in the latter. On the other hand, treatment of the animals with indomethacin, TAI 284 or ibufenac did not affect the gastric hexosamine. As shown in Table 2, the level of hexosamine was found to be higher in the antrum than in the corpus of non-treated animals. Decrease of hexosamine by ASA was more obvious in the

4 FIG. 1. Effect of ASA on the gastric hexosamine content in rats. Numbers in upper part are score of ulcer. ASA suspended in 5% gumi arabicum was administered orally, and 6 hr there after, rats were sacrificed. antrum than that in the corpus. The hexosamine level in the antrum and corpus of the rat treated with PBZ, indomethacin, or TAI 284 was rather elevated. The oral dose of 32.5 mg/kg of ASA did not affect the gastric hexosamine content, though it did produce mucosal ulcers in the glandular stomach. The same procedure with 65 and 130 mg/kg of ASA produced a decrease of the gastric hexosamine dose-dependently (Fig. 1). 3. Effects of the antiphlogistics on the function of the gastric mucosal barrier The secretion of hydrogen and chloride ions beyond the back diffusion of hydrogen ion in the stomach of the vagally intact rats was found to limit the estimation of the back diffu sion (10). In order to remove such limiting factor, the cardiac branch of the vagus nerve was sectioned bilaterally prior to experiment. As shown in Fig. 2, the intragastric administration of 32.5, 65 and 130 mg/kg of ASA 3 hr previously increased dose-dependently the back diffusion of hydrogen ion and the efflux of sodium ion at the equivalent rate. The same procedure of 200 and 400 mg/kg of PBZ produced the back diffusion of hydrogen ion and the efflux of sodium ion almost to the same extent in non-treated vagotomized rats (Fig. 3). As shown in Fig. 4, the oral administration of 30 mg/kg of indomethacin and TAI 284 did not affect the gastric mucosal barrier, though the glandular stomach exhibited the mucosal ulcers. Ibufenac in dose of 500 mg/kg behaved similarly to ASA but to a less extent with ASA (Fig. 5).

5 Fic. 2. Dose-response relationship of ASA in back diffusion of hydrogen ion and the intraluminal efflux of sodium ion to/from the gastric content. Ordinate indicates net flux of sodium and hydrogen ions concentration (meq/1). Abscissa indicates doses of ASA, (mg!kg, p.o.). FIG. 3. Effects of ASA and PBZ on gastric mucosal barrier in vagotomized rats. The pattern of net transfer of electrolytes and volume across the gastric walls during 2 hr. ASA and PBZ in doses described there suspended in 5% gumi arabicum were administered orally 3 hr before pyloric ligation.

6 FIG. 4. Effects of ASA, indomethacin and TAI 284 on gastric mucosal barrier in vagotomized rats. FIG. 5. Effects of ASA and ibufenac on gastric mucosal barrier in vagotomized rats.

7 4. Effect of the stress stimuli on the gastric mucosal barrier and hexosamine content Immersion of the restraint animals in cold water for 3 and 8 hr resulted in the formation of the mucosal ulcers in the glandular stomach with grades of and 5.0+0, respectively. As shown in Fig. 6, the back diffusion of hydrogen ion and the efflux of sodium ion were not affected by 3 and 8 hr cold restraint, however, the same procedure for 22 hr increased both parameters slightly. As shown in Table 3, the gastric hexosamine content FIG. 6. Effects of cold and restraint on gastric mucosal barrier in vagotomized rats. Intact rats were restrained on a metal net and were immersed in water for 0, 3, 8 and 22 hr. Thereafter, the animals were used to evaluate the function of the gastric mucosal barrier under vagotomy for 2 hr. TABLE 3. Effects of stress stimuli with a combination of cold and restraint on the hexosamine contents of the antrum and corpus in rats. " Hours after cold and restraint " in this table means " hours " indicated in Fig. 6 plus 2 hr.

8 was not affected by 5 and 10 hr stress stimuli, though the same procedure for 24 hr decreased the hexosamine content. DISCUSSION During the healing processes of the gastric ulcers caused by acetic acid in the rat, the elevation of the hexosamine level in the gastric mucosa at the margin of the ulcers as well as the lowering of its level by depression of the healing process due to treatment of the animals with ASA or glucocorticoid were reported by Takagi et al. (11). The electron-microscopical observation of the gastric mucosa sampled from the patients with gastric ulcers demonstrated the healing processes that monolayer of undifferentiated and regenerating epithelical covering developed into the mucous cells and the foliated epithels to form matured gastric gland (12). This evidence indicates that the mucous substances present in the mucous cells of the gastric mucosa participated to the protection of the gastric mucosa from injury and also to the healing mechanism of the gastric ulcer. Since intragastric administration of ASA was reported to destroy the gastric mucosal barrier in the rat (13), correlation between the formation of the gastric ulcers by anti phlogistics or stress stimuli and the destruction of the gastric mucosal barrier in reference to the hexosamine level was studied in the present experiment. Gastric hexosamine was obviously lowered by ASA, slightly by PBZ but not by indomethacin, TAI 284, ibufenac and the stress stimuli (5 and 10 hr). The activity of L-glutamine-D-fructose-6-phosphate transaminase present in the gastric mucosa was reported to be inhibited by ASA and PBZ in vitro and in vivo (14). Kent and Allen (15) showed that salicylate depressed the synthesis of acetyl-coa. hibition of the enzyme activity or that of acetyl-coa synthesis was likely to result in the depressed formation of intermediary aminosugars and hexosamine. Treatment of the dog with an oral administration In of 5 mg/kg of indomethacin for 15 days produced gastric ulcers in association with a decrease in the mucosal levels of sialic acid and L-fucose, both of which were incorporated into the mucosal polysaccharide after the synthesis of the intermediary aminosugars (16). Decrease in the mucosal level of mucopolysaccharide in the rat (17) in response to the ulcerogenic restraint stress (24 hr) and that in mucosal level of mucous substances with positive Schiff reaction in the guinea-pig (18) treated with the similar process for 18 hr were reported. In our results, indomethacin and stress stimuli for 5 and 10 hr did not affect the hex osamine level in the gastric mucosa, but the same process for 24 hr decreased the hexosamine content. Only ASA among the antiphologistics used impaired the gastric mucosal barrier as indicated by increase in the back diffusion of hydrogen ion and the efflux of sodium ion, however, the ineffective agents in this respect also produced the gastric ulcers. The results clearly show therefore that the experimentally induced gastric ulcers were produced not only by interference with the gastric mucosal barrier. This conclusion was further sup ported by previous results (19) with thiocyanate which was demonstrated to destroy the

9 gastric mucosal barrier but not to produce the gastric ulcer. A similar conclusion was presented by Takagi et al. (20) from the results of treatment of the rat with ASA but not with indomethacin. PBZ was observed to increase the mucosal efflux of pontamine sky blue injected intravenously. Such an effect of ASA was likely to be derived from decrease in the gastric hexosamine level, which regulates the efflux or inff7ux of the electrolyte and water through the gastric mucosa. Correlation SUMMARY between the gastric mucosal barrier and the development of the gastric ulcers was studied in the rat treated with non-steroidal antiphlogistics or restraint and water immersion stress. 1. All of the oral treatment of rats with acetylsalicylic acid (ASA, 200 mg/kg), phenyl butazone (PBZ, 200 mg/kg), indomethacin (30 mg/kg), 6-chloro-5-cyclohexylindan-l-carbox ylic acid (TAI 284, 30 mg/kg) or ibufenac (500 mg/kg) produced the gastric ulcers similar in shape or diameter at the glandular stomach (score: 2-3). The immersion of the restraint rats in cold water for 3 and 8 hr resulted in the formation of mucosal ulcers in the glandular stomach with grades of and 5.0+0, respectively. 2. Among the ulcerogenic agents tested under the same experimental conditions, ASA (above 65 mg/ kg) and also PBZ (200 mg/kg) to a lesser extent decreased the gastric hexosa mine content. The remaining compounds were ineffective in this respect. The cold re straint for 5 and 10 hr proved to be ineffective, but the same procedure for 24 hr decreased the hexosamine content. 3. ASA in oral doses of 32.5, 65 and 130 mg/kg 3 hr prior to experiments dose-de pendently increased the back diltusion of hydrogen ion and the efflux of sodium ion at the equivalent rate, while the remaining drugs mentioned above did not. The cold restraint for 3 and 8 hr did not affect the movement of both ions, though the same procedure for 22 hr slightly increased both parameters. From these results, it is concluded that the destruction of the gastric mucosal barrier is correlated with the decrease of the gastric hexosamine content, but a causal relationship between the development the gastric ulcers and either parameter is not always found. Acknowledgments: The authors thank Drs. K. Shimamoto and K. Kikuchi for valuable advice and criticism. maibashi for technical assistance. Gratitude is also due to Mr. T. Hirata and Mr. K. Go 1) PLESSIS, D.J.: Lancet 1, 974 (1965) REFERENCES 2) DAVENPORT, H.W.: Gastroenterology 54, 175 (1968) 3) IVEY, K.J., DENBESTEN, L. AND CLIFTON, A.: Gastroenterology 59, 683 (1970) 4) DAVENPORT, H.W.: Gut 6, 513 (1965) 5) OVERHOLT, B.F. AND POLLARD, M.H.: Gastroenterology 54, 182 (1968) 6) TAKAGI, K. AND OKABE, S.: Jap. J. Pharmac. 18, 9 (1968)

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