Studies on the Mechanism for the Gastric Mucosal Protection by Famotidine in Rats

Japan. J. Pharmacol. 55, 211-222 (1991) 211

KeijiMiyata, Takeshi Kamato, Akito Nishida and Kazuo Honda DepartmentofPharmacology, Medicinal Research Laboratories 1, YamanouchiPharmaceutical Co.,Ltd., 21 Miyukigaoka, Tsukuba, Ibaraki 305, Japan ReceivedAugust 10, 1990 AcceptedNovember 15,1990

ABSTRACT-Theeffect of famotidineon gastriclesions induced by the decreasein mucosaldefensive resistance was investigatedin rats and comparedwith those of cimetidine,pirenzepine and cetraxate.Famotidine (0.03, 0.1 and 0.3 mg/kg,p.o.) in- hibiteddose-dependently the developmentof gastriclesions produced by taurocholate- histaminein dosesthat suppressedhistamine-induced acid secretionin pylorus-ligated rats. The H2-antagonistalso prevented gastric mucosal lesions induced by taurocholate-serotonin,iodoacetamide, acidified aspirin and acidifiedethanol. Cimeti- dine, pirenzepineand cetraxateshowed the inhibitoryeffects on almostall types of the gastric lesions,but their inhibitoryeffects were much less potent than those of famotidine.On the other hand, famotidineinhibited the decreasesof gastricmucosal blood flow inducedby acidifiedethanol and the mucosalcontents of glycoprotein inducedby waterimmersion restraint stress. In addition,famotidine increased the trans- gastricpotential difference (PD) and promotedthe recoveryof decreasedtransgastric PD induced by acidifiedethanol in rats. These resultssuggest that the preventive effect of famotidineon gastriclesions is attributablenot only to suppression of acid secretionbut to activationof the gastricmucosal defensive mechanisms.

Histamine H2-receptor antagonists, famoti- ity. dine, cimetidine and ranitidine, have been Famotidine has been reported to be a po- shown to be effective in preventing the forma- tent H2-receptor antagonist in vitro (10-12) tion of gastric mucosal lesions in rats (1-3) and in vivo (4, 13) with greater antisecretory and in promoting the healing in rat (2, 4) and activity than cimetidine and ranitidine (3, 14, human ulcers (5 -7). The mechanisms by 15). Famotidine has also been reported to in- which H2-receptor antagonists suppress ex- hibit the gastric mucosal lesions that were de- perimental lesions are thought to be secondary pendent on gastric acid (2-4). The aims of to their potent gastric acid antisecretory activ- this investigation were to examine the effect of ity (1, 3). However, cimetidine and ranitidine famotidine on the formation of gastric mucosal blocked acidified aspirin-induced gastric le- lesions induced by the decrease in mucosal de- sions in rats in doses that do not suppress acid fensive factors in comparison with cimetidine, secretion (8, 9), suggesting that mechanisms pirenzepine and cetraxate. other than inhibition of acid secretion might be involved in their mucosal protective activ- MATERIALS AND METHODS caused by acidified (150 mM HCI) aspirin in a dose of 150 mg/kg, i.g., or 60% ethanol, i.g., Materials containing 150 mM HCl (acidified ethanol). Famotidine, cimetidine and pirenzepine One hour after i.g.-administration of acidified hydrochloride were synthesized in our labora- aspirin or acidified ethanol in a 1-ml volume tories. Cetraxate hydrochloride was extracted to rats, the animals were killed by chloroform and purified from Neuer R capsule S (Daiichi and the stomach was removed. The length Pharmaceutical). Each drug was suspended (mm) of each lesion was measured macro- with 0.5% methylcellulose solution and admin- scopically, summed per stomach, and used as istered to rats at a volume of 5 ml/kg. Drugs a lesion index. Drugs were given orally 1 hr used for induction of gastric lesions were before ulcerogens administration. sodium taurocholate (Sigma Chemical), hista- In another series of experiments to evaluate mine dihydrochloride (Wako Pure Chemical), the involvement of endogenous prostaglan- serotonin creatinine sulfate (E. Merck), aspi- dins, indomethacin was subcutaneously in- rin (Sanko Pharmaceutical), indomethacin jected to rats 1 hr before drug administration. (Yamanouchi Pharmaceutical) and iodoaceta- Iodoacetamide-induced gastric lesion: Male mide (Tokyokasei). Taurocholate, histamine Wistar rats weighing 200 to 230 g were used and serotonin, and iodoacetamide were dis- for production of gastric lesions by iodoacet- solved in distilled water, physiological saline amide according to the method of Szabo et al. and tap water, respectively. Aspirin was sus- (16). Briefly, rats were placed in individual pended with 150 mM HCI. Indomethacin was mesh cages to prevent coprophagy with free suspended with physiological saline containing access to food and water. Gastric lesion was 0.2% Tween 80. Other drugs used were tryp- caused by 0.1% iodoacetamide in drinking sin (Sigma Chemical), glucose (Nacalai Tes- water given to rats for 7 days; and on the 8th que) and glucosamine (Sigma Chemical). day, rats were sacrificed by chloroform, and the stomach was removed. The length (mm) Gastric lesion production of each lesion was measured macroscopically, Taurocholate-induced gastric lesion: Male summed per stomach, and used as a lesion in- Wistar rats weighing 200 to 230 g were dep- dex. Test compounds were given orally once rived of food but allowed free access to water daily to rats during the 7 days. for 24 hr before the experiments. Acute gastric lesions were induced by taurocholate Gastric acid secretion alone and in the combination with histamine Male Wistar rats weighing 200 to 230 g were or serotonin. Just after 30 mM taurocholate deprived of food, but allowed free access to was given intragastrically in a 1-ml volume, tap water for 24 hr before the experiments. histamine was injected i.p. in a dose of 100 With the rats under ether anesthesia, the mg/kg or serotonin was injected sub- abdomen was incised, and the pylorus was li- cutaneously in a dose of 20 mg/kg. Four hours gated. They were placed in individual mesh later, the animals were killed by chloroform, cages to prevent coprophagy. Four hours lat- and the stomach was removed. The area er, the animals were killed by chloroform, (mm2) of each lesion was measured macro- and the gastric contents were analyzed for scopically, summed per stomach, and used as volume and acidity. Acidity of the gastric juice a lesion index. Drugs were given orally 1 hr was determined by titration with 0.05 N before taurocholate administration. NaOH using an automatic titrator (Hiranuma, Aspirin or ethanol-induced gastric lesion: Comtite-7) to an endpoint pH of 7.0. In Male Sprague-Dawley rats weighing 210 to 230 another series of experiments to examine the g were deprived of food and water for 24 hr influence of histamine against gastric secretion before the experiments. Gastric lesions were in pylorus-ligated rats, histamine at a dose of 100 mg/kg was intraperitoneally injected to was exposed, and two catheters were passed the rats immediately after the abdomen was into the stomach through an incision in the sutured. Test compounds were given orally 1 duodenum and were held in place by a liga- hr before pylorus ligation. ture around the pylorus. One catheter, which was filled with 4% agar in saturated KCI, Measurement of gastric mucosal blood flow served as the intragastric electrode. The circuit Male Sprague-Dawley rats weighing 200 to for the PD recording was coupled with an in- 240 g were deprived of food with free access different electrode placed in the abdominal to water for 24 hr before the experiments. cavity filled with saline. Both the intragastric Under urethane (1.25 g/kg, i.p.) anesthesia, and intraperitoneal electrodes were placed in the trachea was cannulated and the abdomen separate beakers containing saturated KCl was opened. The esophagus was ligated with- solution in which a balanced Ag-AgCI elec- out disturbing the vagus nerves. Gastric trode was positioned. The changes in PD were mucosal blood flow was measured using a la- continuously monitored using a recorder ser Doppler blood flowmeter (TSI, Laserflo; (Rikadenki, R-52) connected to the millivolto- BPM403) according to the method of Saita et meter (Toa Electronics, HM-16S). The second al. (17). The optical flow probe was inserted gastric catheter led to a pH glass electrode of into the stomach through a temporary gastric the flow type (Toa Electronics, GS-80) to de- fistula, which was prepared using a termine the changes in pH of fluids emerging polyethylene tube, in the forestomach and was from the pylorus and which was connected to placed on the gastric mucosa. The probe was a pH meter (Toa Electronics, HM-16S) for mounted in a commercially available balancer simultaneous measurement of the changes in (Physio-Tech). Approximately 30 min after PD. The whole interior of the stomach anesthesia, 60% ethanol containing 150 mM was gently rinsed with warmed saline 3 to HCl in a 1-m] volume was intragastrically 4 times, and then 3 ml of saline was instilled administered through a gastric fistula. The into the stomach. The perfusion of warmed changes in gastric mucosal blood flow was saline (2 ml/min) was controlled by a peristal- continuously monitored. Drugs were given tic pump (Taitec, N-18) located at the middle orally 30 min before urethane anesthesia. of entry or exit tube connected to the fistula or to the pH glass electrode. Measurement of the transgastric potential dif- Approximately 30 min after anesthesia, the ference (PD) perfusion system was interrupted, and the The experimental procedure was essentially solution in the stomach was withdrawn. The the same as that described by Takeuchi and stomach was then exposed for 10 min to 3 ml Okabe (18). Male Sprague-Dawley rats of acidified ethanol. After acidified ethanol weighing 200 to 250 g were derived of food for exposure, the stomach was gently rinsed again 24 hr but water was allowed ad libitum. After several times, another 3 ml of saline was instil- anesthesia with urethane (1.25 g/kg, i.p.), the led, and the perfusion system was resumed. trachea of the rat was cannulated. The abdo- Drugs were given orally 30 min before men was opened and the stomach was ex- urethane anesthesia. posed. The esophagus was ligated without disturbing the vagus nerves. A temporary gastric Measurement of the contents of gastric mucosal fistula was prepared using a polyethylene tube glycoprotein in the forestomach. The fistula that led to a Male Wistar rats weighing 200 to 240 g were three-way tap was used for intragastric instilla- deprived of food but allowed free access to tion and removal of gastric contents and for tap water for 18 hr before the study. The continuous intraluminal perfusion with stress condition was evoked by placing the warmed saline. The pyloroduodenal junction animals in individual compartments of the spe- cial stress cages and then immersing them in a small areas of lesions, with indices of 0.4 ± water bath at 23-'C for 7 hr to the xyphoid 0.2, 2.6 ± 0.4 and 3.1 ± 0.5 mm2, respective- level as described by Takagi and Okabe (19). ly. When taurocholate was administered to After the animals were killed by chloroform, rats in the combination with histamine or sero- the stomachs were removed, placed in ice-cold tonin, however, marked gastric lesions were physiological saline solution, and opened observed with lesion indices of 12.3 ± 1.4 and along the greater curvature. The gastric corpus 7.2 ± 1.0 mm2, respectively. wall was then placed between two glass slides As shown in Figs. 1 and 2, famotidine inhib- and frozen by immersion in hexane cooled in ited taurocholate-histamine and taurocholate- a dry ice-ethanol bath. The gastric mucosa was serotonin-induced gastric lesions at p.o. doses separated from its underlying muscle layer by of 0.03 to 0.3 mg/kg in a dose-dependent pulling the two glass slides apart and then manner with the ED50 values (95% confi- weighed. The frozen mucosa was homogenized dence limits) of 0.04 (0.04-0.05) and 0.08 in 2.5 ml of water with a Polytron homogeniz- (0.02-0.19) mg/kg, p, o. , respectively. Cime- er. The resultant homogenate was digested tidine (3-30 mg/kg, p.o.) and pirenzepine with trypsin (0.5 mg) for 20 hr at 37°C and centrifuged to eliminate non-digestable com- ponents. A portion of the supernatant was hydrolyzed in conc-HCl in a sealed ampoule at 110°C for 16 hr. Hexoses in the hydrolyzate were assayed colorimetrically using the anthrone-H2SO4 method (20). Hexosamine in the hydrolyzate was also assayed colorimetrically after separation through a Dowex 50 col- umn. Glucose and glucosamine were used as the standard for calibration, respectively. Fig. 1. Effects of famotidine, cimetidine, pirenzepine and cetraxate on taurocholate (30 mM)-histamine (100 Statistics mg/kg, i.p.)-induced gastric lesion in rats. Each point All values represent the mean ± standard represents the mean ± S.E.M. for 8- 10 rats. The sta- error of the mean (S.E.M.), and the statistical tistical significance was determined by ANOVA (**: P significance was determined by analysis of < 0.01). variance (ANOVA) or Student's t-test. The differences between treatment groups were compared by the Neuman-Keuls multiple range test. Probabilities of < 5% (P < 0.05) were considered significant. The doses producing 50% inhibition of acid secretion or gastric lesion (ED50 values) were determined by log- probit analysis from data obtained for 3 doses of each compound.

RESULTS

Effect on taurocholate-histamine and taurocho- Fig. 2. Effects of famotidine, cimetidine, pirenzepine and cetraxate on taurocholate (30 mM)-serotonin (20 late-serotonin-induced gastric lesion mg/kg, s.c.)-induced gastric lesion in rats. Each point Administration of taurocholate at 30 mM/ represents the mean ± S.E.M. for 10 rats. The statis- rat, i,g. , histamine at 100 mg/kg, i, p. , and tical significance was determined by ANOVA (*: P < serotonin at 20 mg/kg, s.c., resulted in only 0.05, **: P < 0.01). (10-100 mg/kg, p.o.) also inhibited dose- sion index was 54.0 ± 5.6 mm. Famotidine at dependently these lesions in rats. Cetraxate 0.3 to 1 mg/kg, p.o., inhibited gastric lesions, (100-1000 mg/kg, p.o.) produced dose-de- and the degrees of inhibition were 50.2 and pendent inhibition against taurocholate-hista- 44.4%, respectively. Cimetidine at a dose of mine-induced lesions, whereas cetraxate did 100 mg/kg, p.o., inhibited the lesion forma- not affect taurocholate-serotonin-induced gas- tion by 53.7%. In contrast, pirenzepine and tric lesions at oral doses of 30 and 100 mg/kg cetraxate at 10 to 100 mg/kg, p.o., dose-de- and inhibited them by 68. 1% at 300 mg/kg. pendently inhibited the lesion formation induced by acidified ethanol with ED50 values of Effect on acidified aspirin-induced gastric le- 21.2 (17.6 - 25.5) and 50.6 (30.0 - 85.3) mg/kg, sion p.o. (Fig. 4). Intragastric administration of acidified aspirin produced hemorrhagic damage along the long axis of the stomach, and the lesion index was 32.6 ± 5.4 mm. Oral administration of famotidine (0.3 and 1 mg/kg), cimetidine (3 to 30 mg/kg), pirenzepine (10 to 100 mg/kg) and cetraxate (30 to 300 mg/kg) produced a significant inhibition against acidified aspirin- induced lesion in a dose-dependent manner (Fig. 3). The ED50 values for famotidine, cimetidine, pirenzepine and cetraxate were 0.14 (0.12-0.16), 4.1 (3.5-4.7), 30.8 (21.1- 45.0) and 45.2 (21.4-95.8) mg/kg, p.o., re- Fig. 4. Effects of famotidine, cimetidine, pirenzepinc spectively. and cetraxate on acidified (150 mM HCl) ethanol (60%)-induced gastric lesion in rats. Each point represents the mean ± S.E.M. for 8- 10 rats. The statistical significance was determined by ANOVA (**: P < 0.01).

Effect on iodoacetamide-induced gastric lesion In the control animals, mild erosion was induced in the upper glandular portion (mainly in the corpus) by 0.1% iodoacetamide in drinking water for 7 days with a lesion index of 11.9 ± 1.1 mm. Oral administration of famotidine to rats once daily for 7 days significantly showed a preventive effect on Fig. 3. Effects of famotidine, cimetidine, pirenzepine iodoacetamide-induced gastric lesion. The de- and cetraxate on acidified (150 mM HCI) aspirin (150 mg/kg, i.g.)-induced gastric lesion in rats. Each point grees of inhibition were 52.9 and 56.3% at the represents the mean ± S.E.M. for 9- 10 rats. The dose of 0.3 and 1 mg/kg, p.o., respectively. statistical significance was determined by ANOVA Treatment with orally administered pirenze- (*:P<0.05,**:P<0.01). pine and cetraxate also significantly prevented gastric lesions in response to iodoacetamide, Effect on acidified ethanol-induced gastric le- the degrees of inhibition being 55.2% by sion pirenzepine at 100 mg/kg and 58.0% by cetra- When acidified ethanol was given to the xate at 300 mg/kg. Cimetidine at a dose of control animals, multiple severe lesions were 100 mg/kg once daily for 7 days did not sig- induced in the glandular portion, and the le- nificantly protect the gastric mucosa against iodoacetamide-induced lesion, the degree of p.o.) also inhibited dose-dependently acid inhibiton being 33.6% (Fig. 5). secretion with EDS() values of 23.3 (10.0- 54.2), 21.9 (19.0-25.3) and 143.9 (132.3- 156.6) mg/kg, p.o., respectively. Total acid output was increased by approximately 20% compared with basal acid output by i.p. injection of histamine at a dose of 100 mg/kg in pylorus-ligated rats. Famotidine and cimetidine inhibited gastric acid secretion induced by histamine in a dose-dependent manner with ED50 values of 0.06 (0.04-0.09) and 6.8 (5.5-8.3) mg/kg, p.o., respectively. In contrast, pretreatment with pirenzepine and cetraxate showed a weak antisecretory effect Fig. 5. Effects of famotidine, cimetidine, pirenzepine on histamine-stimulated acid secretion in and cetraxate on 0.1% iodoacetamide-induced gastric pylorus-ligated rats and the degrees of inhibi- lesion in rats. lodoacetamide in drinking water (0.1%) tion were 22.6 ± 4.8% at 30 mg/kg, p.o., of was given to rats for 7 days and on the 8th day, rats pirenzepine and 43.0 ± 9.7% at 100 mg/kg, were sacrificed. Test compounds were given once daily p.o., of cetraxate (Table 1). to rats during the 7 days. Each point represents the mean ± S.E.M. for 10 rats. The statistical significance was determined by ANOVA (*: P < 0.05, **: P < Effect on gastric mucosal blood flow 0.01). The basal blood flow of the gastric mucosa was 112.2 ± 4.4 ml/100 g tissue/min in anes- Effect on basal and histamine-stimulated acid thetized rats. Oral administrations of famoti- secretion in pylorus-ligated rats dine (0.1 to 1 mg/kg, p.o.) and cimetidine (30 Famotidine given orally at doses of 0.1, 0.3 and 100 mg/kg, p.o.) had no significant effect and l mg/kg inhibited basal gastric secretion on the basal blood flow (data not shown). As in a dose-dependent manner with ED50 values shown in Fig. 6, intragastric application of aci- (95% confidence limits) of 0.45 (0.21-0.95) dified ethanol produced a sustained decrease mg/kg in pylorus-ligated rats. Cimetidine (10- in gastric mucosal blood flow. Famotidine, 100 mg/kg, p.o.), pirenzepine (10-100 given orally at doses of 0.1 and 0.3 mg/kg, mg/kg, p.o.) and cetraxate (30-300 mg/kg, p.o., about 1 hr before acidified ethanol, pre-

Table 1. Effects of famotidine, cimetidine, pirenzepine and cetraxate on basal and histamine-stimulated acid secretion in pylorus-ligated rats

"The doses producing 50% inhibition of acid secretion (ED 5() values) were determined by log-probit analysis from data obtained for 3 doses of each compound. h)Figures in parentheses indicate the 95% confidence limits from 5 to 10 animals. Fig. 6. Effects of famotidine and cimetidine on acidified (150 mM HCI) ethanol (60%)-induced decrease in gastric mucosal blood flow in anesthetized rats. Test drugs were orally administered 1 hr before acidified ethanol treatment. The gastric mucosal blood flow (GMBF) immediately before acidified ethanol was desig- nated as 100%. Each point represents the mean ± S.E.M. for 4-5 rats. The statistical significance was determined by Student's t-test (*: P < 0.05, **: P < 0.01). normal control; (-O-) HCI-ethanol control; (-0-) famotidine, 0.1 mg/kg, p.o.; (-D } .amotidine, 0.3 mg/kg, p.o.; (--•-) famotidine, 1 mg/kg, p.o.; (-A-) cimetidine, 30 mg/kg, p.o.. •-) cimetidine, 100 mg/kg, p.o. vented the decrease in blood flow induced by sure (data not shown). acidified ethanol; and rather, at the higher dose of 1 mg/kg, the blood flow increased after acidified ethanol. Oral cimetidine also prevented the acidified ethanol-induced decrease in blood flow at 30 and 100 mg/kg, p.o., and increased blood flow at 100 mg/kg, p.o.

Effect on the transgastric PD The transgastric PD (mucosa negative) was significantly increased by oral administration of famotidine at a dose of 1 mg/kg. The PD was significantly decreased in response to intragastric application of 3 ml acidified ethanol for 10 min, and it decreased after removal of the solution from the stomach and remained lowered for about 1 hr. Famotidine at a dose Fig. 7. Effects of famotidine and cimetidine on of 1 mg/kg promoted the recovery of de- luminal potential difference before and after exposure creased transgastric PD. On the other hand, to acidified (150 mM HCI) ethanol (60%) for 10 min in cimetidine (100 mg/kg, p.o.) did not affect the anesthetized rats. Test drugs were orally administered basal PD or the responses caused by acidified 1 hr before acidified ethanol treatment. Each point ethanol (Fig. 7). Although the basal pH was represents the mean ± S.E.M. for 5 rats. The statistical significance was determined by Student's t-test (*: P significantly higher in the drug-treated groups, < 0.05, **: P < 0.01). (-0-) HCI-ethanol control; the pH changes were not influenced by famo- (-n-) famotidine, 0.3 mg/kg, p.o. ; (-D-) famoti- tidine (0.3 and 1 mg/kg, p.o.) and cimetidine dine, 1 mg/kg, p.o.; (-•-) cimetidine, 100 mg/kg, (100 mg/kg, p.o.) after acidified ethanol expo- p.o. Effect on the contents of gastric mucosal gly- ethanol was significantly reduced by the pre- coprotein treatment with indomethacin (Fig. 8). The contents of gastric mucosal hexosamine and hexose were 372.7 ± 19.3 and 168.7 ± 6.8 DISCUSSION ,ug/100 mg tissue in normal rats, respectively, and were reduced to 76.5 and 79.5% by water Peptic ulcer is well accepted to be caused by immersion restraint stress. Famotidine at 1 an imbalance between the mucosal defensive mg/kg, p.o., inhibited the decreases of hex- factors and the aggressive factors of acid and osamine and hexose induced by stress by 73.7 pepsin (21). The mechanisms by which an H2- and 76.9%, respectively. Cimetidine at 30 receptor antagonist, cimetidine, suppresses ex- mg/kg, p.o., also inhibited the decreases of perimental lesions are thought to be secondary these glycoproteins by 53.9 and 64.7%, re- to its potent gastric acid antisecretory activity spectively. (1). Famotidine has also been reported to inhibit the gastric mucosal lesions induced by Involvement of endogenous prostaglandins water immersion-restraint stress, indomethacin The formation of acidified ethanol-induced and pylorus-ligation that were all dependent gastric mucosal lesions was aggravated from on gastric acid (2-4). A muscarinic M,-recep- 55.6 ± 4.5 to 90.2 ± 3.2 mm by the pretreat- tor antagonist, pirenzepine, has been shown to ment with indomethacin (5 mg/kg, s.c.) in inhibit gastric secretion, improve gastric rats. Oral famotidine at a dose of 1 mg/kg and mucosal microcirculation, and protect gastric cimetidine at a dose of 100 mg/kg showed mucus contents, resulting in the prevention of almost the same inhibitory proportion on gastric lesion formation (22-24). It is re- indomethacin-acidified ethanol-induced lesions ported that the cytoprotection by pirenzepine as that on acidified ethanol induced lesions. might not be due to endogenous prostaglan- On the contrary, the inhibitory effect of 20% dins but be due to a direct action on the gastric mucosa (25). It is also reported that the prevention by cetraxate of experimental gastric lesions may be attributable to its ability to reinforce the gastric mucosal defensive factors such as microcirculation (26) and mucus glycoproteins (27) through a release of endogenous prostaglandins (28), although cetraxate has been shown to exert an inhibitory effect on the gastric secretion in pylorus-ligated rats (29). In the present study, acute gastric lesions were introduced by taurocholate-histamine, taurocholate-serotonin, iodoacetamide, acidified aspirin and acidified ethanol in rats. Fig. 8. Effects of famotidine, cimetidine and 20% Taurocholate is one component of bile acids ethanol on acidified (150 mM HCI) ethanol (60%)-in- that may cause gastric lesion by the retrogres- duced gastric lesion with or without pretreatment of sive influx from the duodenum to the stomach indomethacin (5 mg/kg, s.c.) in rats. Each bar repre- (30), and it has been reported to induce gas- sents the mean ± S.E.M. for 6- 10 rats. The statistical tric erosion by the consecutive administration significance was determined by ANOVA. * denotes statistical significance from the control without in- to rats (31). Histamine develops gastric lesion domethacin treatment (*: P < 0.05, **: P < 0.01). & as a result of a marked vascular disturbance in denotes statistical significance from the control with in- the gastric wall as well as an increase in acid domethacin pretreatment (&: P < 0.05, &&: P < 0.01). secretion (32). Although single administration of taurocholate or histamine to rats showed of prostaglandin biosynthesis (39) and the dis- only a little influence on the gastric mucosa, ruption of the gastric mucosal barrier followed marked lesions were observed after the admin- by back-diffusion of hydrogen ions, stasis in istration of taurocholate in combination with the proximal gastric mucosal microcirculation, histamine. Famotidine and cimetidine inhib- and increased vascular permeability (40). ited taurocholate-histamine-induced gastric Ethanol has been shown to produce gastric le- lesions in rats at doses that suppressed sions with concomitant impairments in such histamine-stimulated acid secretion in pylorus- factors as mucus (41) and mucosal circulation ligated rats. Pirenzepine and cetraxate also in- (42). It was reported that famotidine and hibited these lesions at doses that suppressed cimetidine did not inhibit the gastric lesions basal acid secretion, although these com- induced by such severe conditions as 600 mM pounds did not affect histamine-stimulated HCl and absolute ethanol (43, 44). In the pre- secretion. It is, therefore, suggested that the sent study, aspirin and ethanol were adminis- antisecretory effects of famotidine, cimetidine, tered to rats together with sufficient amounts pirenzepine and cetraxate are mainly respon- of exogenous acid (150 mM HCl) in order to sible for their antiulcer activity in the exclude the possibility that the antisecretory taurocholate-histamine induced lesion model. activity may be responsible for the protective Serotonin causes gastric lesions by platelet effect of test drugs. Famotidine and cimetidine aggregation, followed by fibrous thrombi inhibited acidified aspirin- and acidified formation and vasoconstriction, followed by a ethanol-induced gastric lesions like pirenze- disturbance in the peripheral circulation (33), pine (25) and cetraxate (28), which have been without affecting gastric acid secretion (33, shown to possess cytoprotective activity. The 34). lodoacetamide is a sulfhydryl (SH) block- degree of inhibitory effects of famotidine and ing agent and is thought to cause gastric lesion cimetidine on acidified ethanol-induced gastric by a counteraction on the endogenous prosta- lesion were not so marked (approximately glandins, resulting in the depression of the 50%) but significant. In these gastric lesions mucosal defensive factors (35). Therefore, the induced by acidified aspirin and acidified etha- formation of taurocholate-serotonin- and nol, we used Sprague-Dawley rats instead of iodoacetamide-induced lesions seemed to be Wistar rats because the lesion formation in dependent on defensive factors rather than Sprague-Dawley rats was more marked than gastric acid. Famotidine and other drugs inhib- that in Wistar rats (data not shown). This may ited these gastric lesions in doses suppressing be due to the difference of acid secretion be- basal or histamine-stimulated acid secretion. It tween these rats, that is, Sprague-Dawley rats is known that acid is likely to penetrate the in- used in this study secreted gastric acid much jured gastric mucosa and to make the lesion more than Wistar rats, indicating that endoge- worse after the mucosal epithelium is damaged nous gastric acid plays a role in the by the ulcerogenic agents (36). The control of pathogenesis of even gastric lesions induced by acid secretion, therefore, should help to re- aspirin and ethanol in combination with ex- duce mucosal lesions induced by taurocholate- ogenous acid. It is, therefore, possible that the serotonin and Iodoacetamide. It is reported prevention by famotidine and cimetidine of that the antisecretory effect of cimetidine is acidified aspirin- and acidified ethanol-induced shorter than that of famotidine in dogs (3) and gastric lesions may be partly attributable to humans (37, 38). This could account for the their ability to suppress acid secretion. From observation that cimetidine did not show any these results, in any case, mechanisms other significant influence on Iodoacetamide-induced than suppression of acid secretion might be in- gastric lesions. volved, at least in part, in the mucosal protec- The mechanism by which aspirin induces tion produced by famotidine and cimetidine. gastric damage is thought to be the inhibition Therefore, in the next series of experiments,

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the effect of famotidine on the mucosal defen- iodoacetamide; and it is superior to cimeti- sive factors was examined in rats. Famotidine dine, pirenzepine and cetraxate in rats. The and cimetidine have been shown to inhibit the preventive effect of famotidine is thought to decrease in gastric mucosal blood flow induced be attributable not only to the suppression of by hemorrhagic shock in rats (45, 46) and to acid secretion, but to the activation of the promote the transfer of bicarbonate into the mucosal defensive factors such as mucosal gastric lumen in dogs with gastric pouch (47, blood flow, mucus glycoproteins and bicarbon- 48). In the present study, farnotidine and ate secretion. Moreover, the results of the cimetidine inhibited the decreases in gastric present study indicate that the mucosal protec- mucosal blood flow induced by acidified etha- tion by famotidine is not mediated through en- nol and in mucosal contents of glycoproteins dogenous prostaglandins, but may be due to a (hexose and hexosamine) by water immersion direct action on the gastric mucosa like the ac- restraint stress. Furthermore, famotidine, not tions of cimetidine and pirenzepine. cimetidine, increased the transgastric PD and promoted the recovery of decreased PD in- REFERENCES duced by acidified ethanol in rats. Therefore, it is suggested that there are some differences between the mucosal defensive mechanisms strengthened by famotidine and cimetidine. 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