Effect of centrally administered losartan on gastric and duodenal ulcers in rats.

The effect of centrally administered losartan, an AT(1) receptor antagonist, on gastric acid secretion and gastric cytoprotection was studied using different models of gastric ulcers, such as acetic acid-induced chronic gastric ulcers, pylorus ligation, ethanol-induced and stress-induced acute gastric ulcers and cysteamine hydrochloride-induced duodenal ulcer. Losartan was administered intracerebroventrically (i.c.v.) at 2 different doses (125 and 250 microg/kg). Both doses of losartan increased the healing of acetic acid-induced chronic gastric ulcers. In pylorus-ligated rats, a significant reduction in free acidity, total acidity and ulcer index was observed with high dose (250 microg/kg, i.c.v.), while low dose (125 microg/kg, i.c.v.) produced reduction only in free acidity and ulcer index. Both doses also produced a significant antiulcer effect in ethanol-induced and stress-induced gastric ulcers. Losartan also reduced ulcer area in cysteamine-induced duodenal ulcer. We conclude that AT(1) receptor antagonism in the brain increases healing of gastric ulcers and reduces gastric acid secretion and increases gastric mucin content.

The protease-activated receptor-2 agonist induces gastric mucus secretion and mucosal cytoprotection.

Protease-activated receptor-2 (PAR-2), a receptor activated by trypsin/tryptase, modulates smooth muscle tone and exocrine secretion in the salivary glands and pancreas. Given that PAR-2 is expressed throughout the gastrointestinal tract, we investigated effects of PAR-2 agonists on mucus secretion and gastric mucosal injury in the rat. PAR-2-activating peptides triggered secretion of mucus in the stomach, but not in the duodenum. This mucus secretion was abolished by pretreatment with capsaicin, which stimulates and ablates specific sensory neurons, but it was resistant to cyclo-oxygenase inhibition. In contrast, capsaicin treatment failed to block PAR-2-mediated secretion from the salivary glands. Intravenous calcitonin gene-related peptide (CGRP) and neurokinin A markedly elicited gastric mucus secretion, as did substance P to a lesser extent. Specific antagonists of the CGRP1 and NK2, but not the NK1, receptors inhibited PAR-2-mediated mucus secretion. Pretreatment with the PAR-2 agonist strongly prevented gastric injury caused by HCl-ethanol or indomethacin. Thus, PAR-2 activation triggers the cytoprotective secretion of gastric mucus by stimulating the release of CGRP and tachykinins from sensory neurons. In contrast, the PAR-2-mediated salivary exocrine secretion appears to be independent of capsaicin-sensitive sensory neurons.

Mechanisms of gastroduodenal protection by sucralfate.

Over the past 5-10 years, a number of studies have shown that topical sucralfate enhances a number of gastric and duodenal mechanisms, e.g., the "mucus-bicarbonate barrier," mucosal hydrophobicity, mucosal blood flow, cell viability, and local production of prostaglandins, as well as endogenous mediators of tissue injury and repair. It seems likely that the complex actions of sucralfate are in part related to direct interaction between the drug or its components (aluminum, sucrose, and sulfate) and gastric mucosal tissues, and in part related to effects of the drug on the various mucosal mediators of tissue injury and repair. Local actions may play a role in accelerating healing of ulcer-damaged mucosa, but this does not explain the protective actions of sucralfate on normal mucosa. Thus sucralfate appears to enhance the protective function of the "mucus-bicarbonate" barrier by actions on both components. This may depend in part on an interaction with the unstirred layer overlying gastric epithelium. Sucralfate has also been shown to increase the hydrophobicity of mucus gel. There is little doubt that sucralfate increases local production and release of protective prostaglandins (PGs), but the precise role played by these agents in mediating mucosal protection and in chronic ulcer healing remains uncertain. Currently, the mechanism of action of sucralfate on vascular integrity remains unknown and the role of PGs in this protective function is unclear. There is little evidence that epidermal growth factor plays any role in mediating mucosal protection by sucralfate, but it may be important in its ulcer-healing action. Sucralfate has been shown to be truly "cytoprotective" in that it protects isolated epithelial cells from damage by noxious agents. In animals treated with sucralfate, the surface epithelial cells were disrupted, but necrotic lesions in the deep proliferative zone were virtually absent. It seems likely that investigations of the actions of sucralfate and its components will move ever closer to defining the target cells, the intracellular events, and the mediators that bring about its protective and ulcer-healing activity.

Administration of sucralfate prolongs survival of animals with experimental peptic ulceration.

Ligation of the pig bile duct (BDL) results in 100% incidence of pars esophageal ulceration within 48 hours of the procedure. Usually such ulceration is uniformly fatal unless a highly selective vagotomy is performed simultaneously with the BDL. The administration of sucralfate to pigs with BDL prolonged their survival for up to 7 days, with evidence of healing of the ulcer on macroscopic and histologic observations. An increase in cell proliferation in the squamous epithelium of the ulcerated area was also seen in this sucralfate group. These features were not seen in controls, pigs with BDL only, or pigs with BDL and with magaldrate (Riopone), colloidal bismuth subcitrate (DeNol), or carbenoxolone. Analysis by Sepharose 2B gel filtration showed that there was no significant difference in the amounts of polymeric mucin in any group, with a wide scatter of the data seen especially for pigs in the untreated BDL-only group. This study suggests that sucralfate may enhance healing in this experimental pig ulcer model via a mechanism independent of the stimulation of mucus secretion. We propose that coating the mucosa with sucralfate provides a temporary substitute barrier that creates a microenvironment conducive to wound repair by mucosal proliferation.

Mucins (MUC1 and MUC3) of gastrointestinal and breast epithelia reveal different and heterogeneous tumor-associated aberrations in glycosylation.

In a comprehensive study, we examined the expression of the membrane and secretory mucins MUC1 and MUC3, respectively, in normal and neoplastic gastrointestinal and breast epithelia before and after specific alterations of their glycan structures by neuraminidase, alpha-fucosidase, or carbohydrate-specific periodate oxidation. MUC1 mRNA was also identified in normal colorectal tissues by in situ hybridization. The data revealed that normal colorectal epithelia express both MUC1 mRNA and protein, which were detectable after periodate oxidation with all tested MUC1-specific antibodies. During tumorigenesis in the colon, MUC1 became recognizable without periodate treatment concomitantly with highly dysplastic lesions and the malignant state. In the breast, in which MUC1 is detectable with most antibodies in normal epithelium as well as in carcinomas, staining could be enhanced by pretreatment with periodate and casually by enzyme treatments. MUC3 was detectable in normal and neoplastic colorectal tissues and was more intensely stained after periodate oxidation. It was absent in normal breast even after pretreatment but was expressed in seven of 20 breast carcinomas. Therefore, incomplete glycosylation, abnormal distribution, and ectopic expression of mucins are characteristics of malignancy. Periodate oxidation may be widely applicable to immunohistochemistry for examining changes in glycosylation and for detecting antigens masked by glycans.

Age-related stimulation by tetragastrin of gastric mucin biosynthesis in rat.

The effects of tetragastrin on gastric mucin biosynthesis in middle-aged rats were compared with those in young rats. The incorporation of [3H]glucosamine and [35S]sulfate into mucin was stimulated by tetragastrin in cultured corpus mucosa from 7-week-old rats. In contrast, tetragastrin could not enhance mucin biosynthesis in stomachs from 52-week-old rats. The isosorbide dinitrate-induced stimulation of corpus mucin biosynthesis observed in middle-aged rats was essentially the same as that seen in young rats. Nitric oxide (NO) synthase activity of the corpus was significantly reduced in the middle-aged rats compared to the young rats. NO synthase-immunoreactivity was observed at surface mucous cells in the corpus mucosa of young, but not of middle-aged, rats. These results suggest that aging decreases the effect of gastrin on gastric mucin biosynthesis through the age-related loss of NO synthase function in the surface mucous cell layer of rat stomach.

Impact of ethanol on innate protection of gastric mucosal epithelial surfaces and the risk of injury.

Earlier investigations on the effect of ethanol on synthesis and posttranlational glycosylation of gastric mucus glycoprotein (mucin) revealed quantitative changes in the apoprotein assembly, glycosylation, and mucin retention on the mucosal surface (Slomiany et al.., Alcoholism: Clin. Exp. Res. 21, 417-423, 1998). To assess whether metabolic consequences of ethanol ingestion, documented in the in vitro system are also occurring in vivo the rats were subjected to 8 weeks of ethanol containing liquid diet. The retention of mucin on the surface of gastric mucosa was quantitated by measuring the binding of gastric mucin to Mucin Binding Protein (MBP) of gastric mucosa. The results were compared with those obtained with the rats subjected to pair-feeding the isocaloric-control diet. Before alcohol administration, and in two weeks' intervals thereafter, the gastric contents from the animals was collected and mucin purified. After 8 weeks of the respective diet, the animals were sacrificed and their gastric mucosa used for MBP preparation. The binding of mucin to MBP before ethanol, and after 2, 4, 6, and 8 weeks of ethanol diet was quantitated with Enzyme Linked Lectin Assay (ELLA). The study with standard mucin revealed that binding of mucin to MBP differs substantially between individual animals. The same variability in binding was observed with the individual mucin preparations collected at the onset of the experiment. However, with the progression of ethanol feeding, the mucin samples besides displaying the variable and animal-specific binding to MBP at the initiation of the experiment, also showed a dramatic decrease in binding. In five animals, after two weeks of ethanol diet, mucin binding to MBP decreased by 50%; in two animals, the drastic decrease in binding was observed in mucin collected after four weeks of alcohol feeding; and in one animal a 20% decrease in binding persisted for six weeks, and then decreased to 50% in the last collection. Also, in two animals, the mucin collected after 8 weeks of ethanol feeding retained only 6-9% of the initial binding capacity. In contrast, in pair-fed controls, the mucin binding to MBP remained the same or increased up to 20%. Results of the studies, performed on mucin of the individual animals and matching preparations of MBP, showed that each animal expresses different degree of mucin binding. Moreover, in chronic ethanol ingestion, the individual variations are accompanied by a decrease in mucin binding to MBP. Since the observed decrease in binding occurred in samples containing the same preparation of MBP, the component affected by alcohol resides on mucin. Thus, considering the in vitro impact of ethanol on generation of carbohydrate chains in Golgi, and the finding on mucin oligosaccharides-dependent mucin-MBP complex formation, we conclude that ethanol impairs the synthesis of mucin oligosaccharide structures required for binding with MBP, and the retention on gastric mucosal surfaces.

The effects of verapamil on stress- and histamine-induced gastric lesions in rats.

The influence of verapamil on stress-induced and histamine-induced gastric ulcers was investigated in rats. The influence of verapamil was also examined on various biochemical parameters that affect the development of these ulcer models. The animals were pretreated with intraperitoneal verapamil (1, 5, 25 mg/kg) by injection 1 h before the induction of experimental ulceration. The gastric lesions were induced by cold-restraint stress or intraperitoneal injection of histamine (300 mg/kg). The gastroprotective effects of verapamil were evaluated by determining the ulcer index, gastric mucus content, free and total acidity, lipid peroxidation and non-protein sulfhydryl content. Verapamil pretreatment at a dose of 25 mg/kg significantly reduced stress-induced ulcers. Verapamil enhanced mucus secretion, reduced total acidity and lipid peroxidation and decreased non-protein sulfhydryl content in a dose-dependent fashion. On the other hand, pretreatment with verapamil at any dose had no significant effect on histamine-induced ulcers. L-Arginine (L-A) (100 mg/kg) or L-nitroarginine (L-NNA) (100 mg/kg) were also injected i.p. to the animals 1 h before stress to test the role of nitric oxide (NO) in the mechanism of the gastroprotective activity of verapamil (25 mg/kg). The results suggested that verapamil stimulates gastric NO production, but the overproduction of NO worsens gastric ulcers. The effects of verapamil on experimentally induced ulcers may be related to its ability to induce biochemical alterations in the parameters measured in gastric tissue.

Sucralfate counteracts the inhibition of gastric mucosal mucin receptor by Helicobacter pylori lipopolysaccharide.

BACKGROUND: Among the disturbances in gastric mucosal defense associated with Helicobacter pylori infection is the loss of mucus coat continuity, which results in a severe disturbance in the ability of mucus coat to maintain its functions as the pre-epithelial element of gastric mucosal defense. Here, we show that H. pylori, through its cell-wall lipopolysaccharide, disrupts the interaction between gastric mucin and its mucosal receptor, and that sucralfate is capable of counteracting this untoward effect of the bacterium. METHODS: The receptor was isolated from octylglucoside-solubilized gastric mucosal epithelial cell membranes by affinity chromatography on Sepharose-bound wheat germ agglutinin and following iodination with 125I, used in the binding assays for mucin in the presence of H. pylori lipopolysaccharide and sucralfate. RESULTS: Preincubation of the receptor protein with H. pylori lipopolysaccharide led to a decrease in mucin binding. The inhibitory effect was proportional to the concentration of lipopolysaccharide and reached a maximum of 91% at 30 micrograms/ml. The effect of H. pylori lipopolysaccharide was countered by sucralfate, which caused a dose-dependent relief of the inhibitory effect. The maximum (75%) restoration in mucin-receptor binding occurred at 60 micrograms/ml sucralfate. CONCLUSIONS: The results provide strong evidence for the effectiveness of sucralfate in preventing the loss of gastric mucus coat continuity caused by H. pylori.

[Digestion of human gastric mucous by extracellular Helicobacter pylori enzyme].

Helicobacter pylori (Hp) is a causative pathogen of chronic gastritis and strongly associated with recurrence of duodenal ulcers, but the mechanism is not known. We studied the possible digestion of gastric mucus by Hp extracellular enzyme in human stomach. Hp was isolated from biopsy specimens taken from peptic ulcer patients or normal individuals and its extracellular enzyme was collected. Incubation of human gastric mucosa with Hp extracellular enzyme resulted in a significant decrease of PAS-positive staining, indicating digestion of gastric mucus. Analysis of protein profile of extracellular enzyme revealed no difference among Hp strains. Subsequent zymography showed 2 bands; 97 K and 20 K, indicating existence glycosyl-hydrase. These results suggest that Hp weakens gastric mucosal defense by digesting carbohydrates in the protective mucus layer.

[A method for determining the strength of the disulphide bonds of glycoproteins from gastric mucus]. / Sposob opredeleniia prochnosti disul'fidnykh sviazei glikoproteinov zheludochnoi slizi.

The author describes a simple and highly informative method for the assessment of disulfide bonds of gastric mucus glycoproteins, based on studies of the mucus rheology before and after incubation with 5% unithiol for an hour at 37 degrees C. Such an exposure was found to induce in normal subjects just a slight reduction of viscous elastic characteristics of the mucus (under 30% of the initial values), whereas in patients with duodenal ulcer this reduction was marked: in 83 +/- 7% of patients the rheologic characteristics dropped by more than 30%, in 1/3 of the patients the mucous gel was found completely dissolved. Such an effect was observed both in the patients with the initially reduced and with normal rheologic parameters of gastric mucus.

In vitro measurements of mucoadhesive properties of six types of pectin.

The objective of this study was to measure and compare the specific- and general mucin interaction of six pectin types from three manufacturers, differing mainly in the degree of methoxylation and degree of amidation. Mucoadhesive properties were measured using a texture analyzer. It was found that an intermediate degree of methoxylation (35 and 36%) improved the specific mucin interaction. Amidation did not increase mucin interaction. Samples from different manufacturers did not alter these conclusions. This study indicates that the general classification of pectin as a poor mucoadhesive, without differentiating between the amount and type of substituents, probably is an oversimplification.

Up-regulation in endothelin-1 by Helicobacter pylori lipopolysaccharide interferes with gastric mucin synthesis via epidermal growth factor receptor transactivation.

OBJECTIVE: Endothelin-1 (ET-1), a key mediator of inflammatory processes associated with bacterial infection, is a 21-amino acid peptide produced from a biologically inactive big ET-1 by the action of endothelin-converting enzyme-1 (ECE-1) that acts through G protein-coupled ET(A) and ET(B) receptors. Here we report on the role of ET-1 in the mediation of the detrimental influence of Helicobacter pylori on the synthesis of gastric mucin. MATERIAL AND METHODS: Rat gastric mucosal cells were exposed to H. pylori key virulence factor, lipopolysaccharide (LPS). RESULTS: The LPS inhibitory effect on gastric mucin synthesis was accompanied by a marked increase in ET-1 generation and enhancement in ECE-1 activity. Inhibition of ECE-1 with phosphoramidon not only led to the impedance of LPS-induced ET-1 generation, but also countered the detrimental effect of LPS on mucin synthesis. Moreover, the LPS inhibitory effect on mucin synthesis was blocked by ET(A) receptor antagonist BQ610, but not by ET(B) receptor antagonist BQ788. Furthermore, the LPS-induced suppression in gastric mucin synthesis was countered in a concentration-dependent fashion by PD153035 (81.7%), a specific inhibitor of epidermal growth factor receptor (EGFR) kinase as well as PP2 (69.8%), a selective inhibitor of tyrosine kinase Src responsible for ligand-independent EGFR transactivation. CONCLUSIONS: Our findings are the first to show that the detrimental effect of H. pylori on gastric mucin synthesis is intimately linked to the events associated with ECE-1 up-regulation, enhancement in ET-1 production, and G protein-coupled ET(A) receptor activation that triggers the EGFR transactivation.

Gastrointestinal tolerability of metamizol, acetaminophen, and diclofenac in subchronic treatment in rats.

The gastrointestinal tolerability of metamizol and acetaminophen [weak cyclooxygenase (COX) inhibitors] in comparison with diclofenac (nonselective cyclooxygenase inhibitor) was evaluated in subchronic treatments in rats. Wistar rats received 60 mg/kg body weight of metamizol and acetaminophen, and 3 mg/kg body weight of diclofenac by oral route twice daily for 14 days. Myeloperoxidase activity, an index of neutrophil infiltration, COX expression and the effects on blood parameters used as indicators of liver and renal functions were also studied. Metamizol and acetaminophen did not cause apparent gastrointestinal lesions; in contrast diclofenac showed swelling and an increased thickness on the distal intestinal mucosa. Myeloperoxidase activity was significantly increased in the small bowel with diclofenac treatment. In gastric mucosa the expression of the cyclooxygenase-1 was not affected and the expression of cyclooxygenase-2 was not observed. Diclofenac treatment significantly diminished hematocrit, hemoglobin, and corpuscular volume and increased the number of platelets. Aspartate aminotransferase and gamma-glutamyltransferase activity were also altered and, regarding the renal biochemical parameters, the animals treated with diclofenac had increased urea values. In contrast, acetaminophen treatment did not affect either of these parameters and metamizol increased only the alanine aminotransferase activity. Under our experimental conditions, metamizol and acetaminophen seem to be safe drugs. In contrast, with diclofenac treatment blood loss and anemia are observed which could stem from the small intestinal injury. Moreover, this drug could to impair kidney function.

Helicobacter pylori aggregating activity of gastric mucin with ulcer healing by ebrotidine.

The mucin isolated from gastric secretion of duodenal ulcer patients before and after therapy with a new antiulcer agent, ebrotidine, was assessed for H. pylori aggregating activity and macromolecular organization. Analyses of mucin molecular forms revealed that successful therapy with ebrotidine was accompanied by a 2.6-2.9-fold increase in the high molecular weight mucin form. The H. pylori aggregation inhibition assays showed that therapy with ebrotidine evoked a 4-fold increase in mucin anti-H. pylori titer. The changes in the functional properties of mucin following ebrotidine therapy were also accompanied by a 36% increase in the content of sulfomucin. The results demonstrate that ulcer therapy with ebrotidine lead to a marked enhancement in mucin's qualities associated with maintenance of gastric mucosal integrity and strengthening the indigenous defenses against H. pylori.

Use of fluorescent hydrophobic dyes in establishing the presence of lipids in the gastric mucus gel layer.

The hydrophobic property of crude samples of canine and porcine mucus was demonstrated by its binding and reactivity with two fluorescent dyes, 1-anilinonaphthalene-8-sulfonic acid (ANS) and 1,6-diphenyl-1,3,5-hexatriene (DPH). The contribution of lipids to these hydrophobic binding sites was indicated by our observations that the DPH-induced fluorescence of both porcine and canine gastric mucus was reduced greater than 75% after lipid extraction. Fluorescence microscopy revealed an extracellular band of intense reactivity in association with the mucus gel layer overlying the rat gastric mucosa that was abolished if the frozen sections were pretreated with lipid solvents. When rats pretreated with indomethacin were injected with a cytoprotective dose of 16,16-dimethyl-PGE2, there was an increase in fluorescence of the gastric perfusate treated with ANS, suggesting that hydrophobic factors, perhaps lipids, were being secreted in association with mucus. These extracellular lipids may play an important role in conferring protective barrier properties to the mucus gel layer.

Effects of tetragastrin on mucus glycoprotein in rat gastric mucosal protection.

The effects of tetragastrin on mucus glycoprotein (mucin) metabolism and mucosal protection in rat gastric mucosa were investigated. Rats were administered with various doses of tetragastrin (12, 120, or 400 micrograms/kg body weight; s.c.), followed by 50% ethanol-induced gastric injury. Tetragastrin caused a significant increase in mucin content in the corpus mucosa and prevented 50% ethanol-induced gastric mucosal damage in a dose-dependent manner. For assessment of the effects of tetragastrin on the metabolism of gastric mucin in detail, changes in mucin distribution in the three different layers of rat gastric mucosa were examined one hour after single administration of tetragastrin. A significant increase in the mucin content was noted in the mucus gel and surface mucosal layer. Mucin content in the deep mucosa corresponding mainly to the mucus neck cell mucin underwent virtually no change by this treatment. An increase in mucin in the mucus gel and surface mucosa would thus appear due to the administration of tetragastrin and may possibly be related to the protective action of the gastric mucosa against injury. The data demonstrate a possibility that gastrin may have potential for enhancing gastric mucosal protection associated with mucus secretion and/or mucus synthesis on the surface mucosa of rat gastric mucosa.

Gastric mucosal damage accompanying changes in mucin induced by histamine in rats.

The present study was carried out to clarify the mechanism of histamine-induced gastric mucosal damage in rats. Following an injection of histamine (80 mg/kg), pH dropped within 30 min. and then recovered control value (pH = 4-5) 4 hr later. The decrease in mucosal mucin content and the appearance of haemorrhagic erosions followed the drop in pH. The recovery of mucosal mucin content preceded the healing of haemorrhagic erosions and pH recovery to the control level. Histamine also caused qualitative changes in corpus mucins. These qualitative changes induced by histamine were eliminated at 7 hr following the administration of histamine. It appears from the present results that increase in HCl and decrease in mucins induced by histamine bring about gastric damage.

Reversal of the gastric effects of nicotine by nitric oxide donor treatment.

Nicotine intensifies experimental gastric ulceration by reducing gastric mucosal blood flow (GMBF) and mucus. As both these parameters can be improved by nitric oxide (NO), we evaluated the impact of a NO donor in ethanol-induced gastric mucosal injury in rats administered nicotine. A nicotine solution or water was administered for 20 days to Sprague-Dawley rats. NO donor (isosorbide dinitrate) was given 60 and 10 min before preparation of ex vivo gastric chambers and exposure to ethanol. Chronic nicotine intake significantly reduced GMBF and gastric mucus content. Nicotine intensifies ethanol-induced gastric injury and short-term administration of NO donor failed to antagonize the ulcerogenic action from either nicotine or alcohol. In another study, rats drank nicotine solution for 20 days, after which the nicotine was withdrawn and replaced by water for 10 additional days. NO donor was provided during these last 10 days. The gastric effects of nicotine persisted for at least 10 days after nicotine was withdrawn but then these effects could be abolished by prolonged NO treatment. Nicotine reduces plasma nitrite level, but gastric mucosal MPO activity remained unchanged. Our data suggest that nicotine cessation plus a longer period of NO donor administration can completely abolish the gastric effects of nicotine.

Assessment of UDP-galactosyl-transferase activity in gastric mucosa of patients with chronic liver disease using an enzyme-linked peanut agglutinin binding assay.

Gastric mucin plays an important role in protecting mucosa from irritants such as acids and pepsin, and UDP-galactosyltransferase is a key enzyme in mucin synthesis. In order to study the synthesis of gastric mucin in patients with chronic liver disease, we developed a new assay using a peanut agglutinin lectin to measure this enzyme in human gastric mucosa obtained by endoscopic biopsy. Enzyme activity correlated well with that determined with a previous method using radiolabeled galactose. The enzyme activity in gastric mucosa of cirrhotic patients was significantly lower than in patients with chronic hepatitis or in normal controls and correlated with the amount of mucin in surface epithelial cells. Our findings suggest that the synthesis of gastric mucin is impaired in patients with liver cirrhosis.