Effects of indomethacin on the rat small intestinal mucosa: immunohistochemical and biochemical studies using anti-mucin monoclonal antibodies.

BACKGROUND: The luminal surface of the gastrointestinal tract is covered by a viscoelastic gel layer that acts as a protective barrier against the intraluminal environment. Because the situation of the small intestine has not been elucidated to the same degree as other sections, in this study, we investigated the effects of indomethacin on the rat small intestinal mucosa. METHODS: Male Wistar rats were given indomethacin 10 mg/kg s-c and sacrificed 1, 3, 7, or 14 days later. The small intestine was opened along the anti-mesenteric side, and examined macroscopically. Total mucin content in the small intestinal epithelium was measured and immunoreactivity was examined using anti-mucin monoclonal antibodies HCM31 and PGM34. RESULTS: Indomethacin caused punched out and linear ulcers located mostly along the mesenteric margin of the distal jejunum with sparing of the ileum. Histological examination showed sialomucin recognized by HCM31 increased on day 3 especially in the regenerating epithelium around the ulcer edge. Furthermore, the surface mucous gel layer displayed a multilaminated pattern, consisting of non-sulfated sialomucin-rich layers and sulfated mucin-rich layers, where both mucins had the common core protein, MUC2. Biochemical measurements also showed the total mucin content of the jejunum increased transiently and HCM31-positive mucin increased approximately 4 times greater than baseline on day 3, but no marked changes were observed in the ileum, with few ulcers observed. CONCLUSIONS: Indomethacin administration causes quantitative and qualitative change in jejunal mucin. In particular, sialomucin plays an important role in regenerating epithelium during the healing process following indomethacin-induced mucosal damage.

Effects of combination treatment with famotidine and methylmethionine sulfonium chloride on the mucus barrier of rat gastric mucosa.

BACKGROUND AND AIM: In Japan, peptic ulcer disease (PUD) is treated clinically with a combination of a mucosal protectant and acid suppressants, but there is scant information regarding the effects of these drugs on normal gastric mucus cells. In the present study, the effects of co-administration of methylmethionine sulfonium chloride (MMSC) and famotidine on rat gastric mucus cells were investigated using both biochemical and histological methods. METHODS: Rats were divided into four groups: controls were given carboxymethylcellulose orally once daily for 7 days and the second, third and fourth groups were treated similarly with famotidine (famotidine group), MMSC (MMSC group) or famotidine plus MMSC (combination group). After killing the rats on the 8th day, the stomachs were removed and the biosynthesis and amount of mucin in different areas of the gastric mucosa were compared among groups. Using anti-mucin monoclonal antibodies, the mucin content and immunoreactivity were also compared. RESULTS: Both the biosynthesis and accumulation of mucin were significantly decreased in the famotidine group, but increased in the MMSC and combination groups. The amount and immunoreactivity of surface mucus cell-derived mucin were both reduced in the famotidine group, and increased in the MMSC and combination groups. There was no difference among the groups in the content and immunoreactivity of gland mucus cell-derived mucin. CONCLUSION: Famotidine-induced suppression of gastric surface mucus cell function is prevented by combined treatment with MMSC, raising the possibility of a more effective cure of PUD.

Effects of tea catechins on the gastrointestinal mucosa in rats.

Although tea catechins are known to exert a potent antiulcer effect on the alimentary tract, there is scant information concerning their effects on normal mucus cell functions. Using original anti-mucin monoclonal antibodies, we studied the influences of long-term administration of catechins on the quantity and quality of mucin in rat gastrointestinal mucosa. Administration of 0.5% tea catechins significantly increased the mucin content of the ileum, but not the stomach. An enzyme-linked immunosorbent assay (ELISA) showed no remarkable qualitative changes in gastric mucin, but a selective increase and decrease in sulfo- and sialomucins, respectively, in the ileum of rats administered catechins. The ELISA results were consistent with both the immunohistochemical findings and the high-iron diamine-alcian blue staining pattern. These findings indicate that tea catechins modulate ileal mucin metabolism in the ileal mucosa, suggesting that further studies focusing on the ileal epithelium will assist in further elucidation of the mechanism of catechin effects.

Changes in the mucus barrier of the rat during 5-fluorouracil-induced gastrointestinal mucositis.

OBJECTIVE: A frequent complication of antineoplastic chemotherapy (CT) is gastrointestinal (GI) mucositis. Although clinically this mucositis can be treated, data on the effect of CTon the mucosal defense mechanisms are scant, so the effects of 5-fluorouracil (5-FU) on mucin, one of the principal defense factors of the GI mucosa, were investigated. MATERIAL AND METHODS: 5-FU was administered orally to rats at a dose of 50 mg/kg once daily for 5 days. Using anti-mucin monoclonal antibodies, the immunoreactivity in different areas of the rats' GI tracts was compared, as well as the mucin content. Changes in the GI mucin during the process of recovery from the injury were also investigated. Immunohistochemical analysis of proliferating cell nuclear antigen (PCNA) was used to determine whether or not the effects of 5-FU on cell proliferation contributed to the changes in mucin. RESULTS: 5-FU caused significant alterations of the immunoreactivity and content of mucin in the rat GI mucosa, especially in the jejunum. The jejunal mucin content was most markedly reduced on day 1 after drug withdrawal, and increased thereafter. By day 7, the content had transiently but significantly increased approximately 1.5-fold, and returned to the basal level by day 13. The number of PCNA-positive cells strikingly decreased at day 1, but by day 7 had increased approximately 2-fold, compared with the control. CONCLUSION: The activation of mucus cells in the jejunum, if appropriately manipulated, could lead to more effective prevention of CT-induced GI mucositis.

Effects of acid antisecretory drugs on mucus barrier of the rat against 5-fluorouracil-induced gastrointestinal mucositis.

OBJECTIVE: Acid antisecretory agents are used for the prophylaxis of cancer chemotherapy (CT)-induced gastrointestinal (GI) mucositis. Although these drugs seem to be clinically beneficial, data on their effects on the GI mucosal defense during CT treatment are scant. The objective of this study was to compare the effects of omeprazole, lansoprazole, and lafutidine on mucin, a major mucus component, during 5-fluorouracil (5-FU) treatment, as a CT regimen. MATERIAL AND METHODS: Rats, weighing approximately 230 g, were divided into five groups. The control group was administered 0.5% carboxymethylcellulose orally once daily for 5 days. The second, third, fourth, and fifth groups were treated with 5-FU (50 mg/kg), 5-FU plus omeprazole (10 mg/kg), 5-FU plus lansoprazole (10 mg/kg), and 5-FU plus lafutidine (30 mg/kg) in the same way, respectively. The rats were sacrificed on the sixth day, and their stomachs and small intestines were removed. Using anti-mucin monoclonal antibodies, we compared the immunoreactivity in different areas of the rats' GI tracts as well as the mucin content. RESULTS: Body-weight decreased in rats in the 5-FU group. Lafutidine, but neither omeprazole nor lansoprazole, inhibited the 5-FU-induced weight loss. Mucosal damage and reduced mucin content in stomach and small intestine were observed in rats receiving 5-FU alone. In the stomach, all antisecretory drugs caused the protective effects against 5-FU-induced mucosal injury and alleviation of the decreased mucin accumulation. In the jejunum and ileum, lafutidine, but neither omeprazole nor lansoprazole, ameliorated the 5-FU-induced mucosal damage and decreased mucin accumulation. CONCLUSION: Lafutidine could offer the possibility of more effective prevention of CT-induced mucositis through the activation of GI mucus cells.

Effects of a novel histamine H2-receptor antagonist, lafutidine, on the mucus barrier of human gastric mucosa.

BACKGROUND AND AIM: Lafutidine is a novel histamine H(2)-receptor antagonist used primarily as an antisecretory agent in Japan. Previous human studies have not assessed its gastroprotective effects. The purpose of the present study was to determine the effects of lafutidine on the human gastric mucus layer using both histological and biochemical methods. METHODS: Of the 14 patients scheduled for gastrectomy who consented to participate, seven were given 14 days of lafutidine 20 mg/day (lafutidine group) and the others received no medication (control group). The surface mucus gel layer in Carnoy-fixed tissue sections was examined immunohistochemically. Both the thickness of the mucus layer and its mucin content were measured in gastric corpus mucosa. RESULTS: There was no detectable difference between the groups in the grade of gastritis or the immunohistochemical staining characteristics. The laminated structure of the surface mucus gel layer was retained after administration of lafutidine and it was thicker than the layer in the control group. The surface layer in the lafutidine group had three-fold more mucin than that in the control group. There was no difference between the two groups in the mucin content of the deep mucosa. CONCLUSION: Lafutidine, given at clinical dosages, not only inhibits acid secretion but also strengthens the mucus barrier of the human gastric mucosa.

Analysis of mucin composition in gastric juices of chronic rheumatic patients with upper gastrointestinal damage.

Assessment of the mucin subclasses in the gastric juices of severe chronic rheumatoid arthritis (RA) patients was compared with non-RA cases which received the eradication treatment of Helicobacter pylori (H. pylori). Gastric juice samples were obtained from 8 RA patients (5 for H. pylori-negative and 3 for H. pylori-positive) and 5 control subjects in which we confirmed the successful eradication of H. pylori. The gastric luminal mucins were extracted and isolated by the ethanol precipitation method. These mucin solutions were digested with chymotrypsin, dialyzed, lyophilized, and redissolved. The obtained specimen was applied to an ion exchange column containing DEAE-Sepharose CL-6B and eluted with a discontinuous salt gradient in three salt steps. The gastric luminal mucins were divided into three fractions based on the distinctive sialic acid content. The proportion of acidic mucin rich in sialic acid from the gastric juice of RA patients without the H. pylori infection was significantly lower than those RA patients with H. pylori or the control subjects. A decrease in the acidic mucin content after eradication of H. pylori was commonly observed in all the control subjects. Our investigation raises the possibility that the gastric mucosae of RA patients have resistance against H. pylori infection. And the analysis of the composition in the gastric luminal mucin may be a very useful tool for the evaluation of gastric homeostasis in RA patients.

Appearance of specific mucins recognized by monoclonal antibodies in rat gastric mucosa healing from HCl-induced gastric mucosal damage.

BACKGROUND: Mucus is an important factor in the physiological defense mechanism of the gastrointestinal tract. We have reported that two distinct antigenicities reacting with anti-mucin monoclonal antibodies (mAbs), HCM31 and RGM26, emerged in epithelial cells regenerating from acetic acid-induced gastric damage in the rat. Here, we examined whether the expression of specific mucins occurred during the healing stage of acute gastric mucosal lesions, and what was the principal alteration of the mucus in the regenerating process of gastric epithelia from slight mucosal lesions. METHODS: Eight-week-old male Wistar rats were used. The animals were administered 0.6 N hydrochloric acid, or 0.5% carboxymethyl cellulose sodium salt into their stomachs. Twenty-four, 48, and 72 h after the HCl administration, their stomachs were removed. Immunohistochemical observation was performed after staining with the mAbs, RGM21, RGM26, HIK1083, or HCM31. RESULTS: Twenty-four hours after the administration of HCl, mucous cells stained with RGM26 emerged in the deeper area of the surface epithelial cells in the damaged corpus mucosa. After 48 h, HCM31-positive cells were noted in the epithelial cells where the mucosal damage reached more deeply. CONCLUSIONS: The appearance of specific mucin species was observed in the regenerating epithelia of the rat during the healing process from acute gastric mucosal damage.

Characterization of rat gastric mucins using a monoclonal antibody, RGM23, recognizing surface mucous cell-type mucins.

A novel anti-mucin monoclonal antibody (mAb), designated RGM23, was developed against mucin purified from rat gastric mucosa. RGM23 reacted with the mucin attached to the ELISA well. The reactivity was lost by trypsin treatment, but not by periodate oxidation, indicating that RGM23 recognizes the peptide moiety of the mucin molecule. Histochemical study showed that RGM23 stained the corpus and antral surface mucosa of rat stomach, but not their glandular mucosa, nor duodenal, small intestinal or large intestinal mucosa. The area stained with RGM23 was coincident with that stained with 45M1, a mAb reacting with MUC5AC mucin. Examination of the mucin subunits extracted from rat stomach by Sepharose CL-4B and Q-Sepharose chromatography and CsTFA equilibrium centrifugation showed that RGM23 reacted with the surface mucous cell-type mucins that were stained with periodate-Schiff (PAS) and reacted with mAb RGM21. The gastric gland-type mucin, which reacted with mAb HIK1083, did not react with RGM23. On Q-Sepharose chromatography, a part of the RGM21-reactive mucins was only faintly stained with PAS and did not react with RGM23. The results together indicated that RGM23 probably reacted with the rat MUC5AC (rMuc5AC) mucin present in the surface mucosa of the stomach, and that the surface mucosal cells in rat stomach may contain mucin bearing non-rMuc5AC core protein in addition to rMuc5AC mucins.

Cyclooxygenase-2 inhibitors attenuate increased blood pressure in renovascular hypertensive models, but not in deoxycorticosterone-salt hypertension.

COX-2 is an inducible cyclooxygenase (COX) that has been reported to be expressed in the macula densa and surrounding cortical thick ascending limb in normotensive rats. The present study assessed the contribution of COX-2 in three different rat models of hypertension, each characterized by a different activation of the renal renin-angiotensin system. Mean blood pressure (MBP) in the rat 2 kidney-1 clip (2K1C) model was significantly reduced with a COX-2 selective inhibitor, NS-398 (10 mg/kg, p.o., twice a day) (vehicle-administered rats (n = 8): 154 +/- 6 mmHg; NS-398-administered rats (n = 5): 128 +/- 10 mmHg). By contrast, a COX-1 selective inhibitor, mofezolac, did not lower MBP. Increased plasma renin activity (23 +/- 8 ng/kg/h (n = 6) vs. sham operation, 2.4 +/- 0.9 ng/kg/h (n = 4)) was markedly reduced to 6.8 +/- 2.7 ng/ml/h (n = 5) by NS-398, but not by mofezolac. The development of 1 kidney-1 clip (1K1C) hypertension was also inhibited by NS-398 (vehicle (n = 12): 133 +/- 1 mmHg; NS-398 (n = 7): 122 +/- 3 mmHg) accompanied by a reduction in plasma renin activity (3.0 +/- 0.3 ng/ml/h, n = 4) to 1.0 +/- 0.2 ng/ml/h (n = 5). The COX-2 inhibitor increased urinary excretions in the 1K1C model, but not in the 2K1C model. In a deoxycorticosterone acetate (DOCA)-salt model, plasma renin activity was markedly suppressed to less than 0.3 ng/ml/h. The COX-2 inhibitor caused no significant changes in MBP, plasma renin activity, or urinary excretion, suggesting that COX-2 made a lesser contribution in this model. Increased expression of COX-2 mRNA and protein was observed in the kidneys of 1K1C and 2K1C rats, but not in DOCA-salt rats. These results suggest that COX-2 plays a significant role in the development of 2K1C and 1K1C renovascular hypertension, in addition to making a substantial contribution to the diuretic effect in the 1K1C model.