Effect of preinduction of heat-shock proteins on acetic acid-induced small intestinal lesions in rats

Bowel dysfunction such as irritable bowel syndrome caused by stress is well described. Previous reports suggest that stress is known to cause the release of endogenous substances such as catecholamine, beta-endorphine, 5-hydroxytryptamine, corticotropin-releasing factor, and thyrotropin-releasing hormone (TRH). However, the role played by these neurohormonal mediators in bowel dysfunction under stress conditions is not well known. We investigated the influence of water-immersion stress or TRH administration on the expression of 60-kDa, 72-kDa, and 90-kDa heat-shock proteins (HSP60, HSP72, and HSP90, respectively) in rat small intestinal mucosa by Western blot and immunohistochemical analyses. The cytoprotective function of preinduced HSPs on experimentally induced mucosal damage also was studied. In order to investigate the influence of preinduction of HSP60 on small intestinal damage, the small intestinal lumen was perfused with 1.5% acetic acid 1 ml/min for 15 min with or without pretreatment with water-immersion stress or TRH administration. Expression of HSP60 was significantly increased by water-immersion stress or TRH administration in the small intestinal mucosa, whereas HSP72 and HSP90 did not increase. Interestingly, expression of this protein showed the biphasic peak pattern after water-immersion stress or TRH administration. Each peak was observed 3-6 hr and 21-24 hr after the initiation of water-immersion stress or TRH administration. Immunohistochemical study also showed a significant increment of HSP60 in both the cytoplasm and nuclei of the small intestinal mucosal cells. No histopathologic alteration was observed in rat small intestinal mucosa after each treatment. Small intestinal damage caused by 1.5% acetic acid perfusion was not influenced by preinduction of HSP60. We demonstrated that water-immersion stress or TRH administration specifically induced HSP60, although preinduction of this protein did not show a cytoprotective function in the small intestinal mucosa.     

Effects of intravenous lansoprazole on acute gastric mucosal lesions and acid secretion

The effects of lansoprazole given intravenously on gastric mucosal lesions, gastric bleeding and acid secretion were investigated in rats in comparison with those of omeprazole, famotidine and ranitidine. Lansoprazole inhibited the formation of gastric mucosal lesions in rats induced by water-immersion stress or aspirin with ID50 values of 0.26 and 0.99 mg/kg, respectively, and also inhibited gastric bleeding induced by hemorrhagic shock or water-immersion stress with ID50 values of 0.46 and 1.22 mg/kg, respectively. Lansoprazole was more potent than omeprazole, famotidine and ranitidine in inhibiting gastric mucosal lesions and hemorrhagic shock- or stress-induced bleeding. Famotidine and ranitidine showed negligible inhibition of water-immersion stress-induced gastric bleeding. Lansoprazole strongly inhibited water-immersion stress-stimulated acid secretion in rats, whereas famotidine and ranitidine did not show a potent inhibitory effect. These results indicate that lansoprazole exerts prominent inhibitory actions against the formation of gastric mucosal lesions and gastric bleeding by inhibiting acid secretion, and they show that it is superior to histamine H2-receptor antagonists in inhibiting stress-induced gastric bleeding.     

Effect of marzulene on the restitution of rat gastric mucosa after NaOH-induced injury

BACKGROUND/AIMS: The purpose of this study was to assess the effect of marzulene (L-glutamine plus azulene) on the repair of NaOH-induced gastric mucosal injury in rats. METHODOLOGY: Gastric mucosal injury was induced with intragastric instillation of 3.0 ml of 5% NaOH for 1 minute. From 2 days after the operation, the rats were orally given chow pellets containing 0%, 0.25%, or 0.5% of marzulene for 25 weeks. RESULTS: Oral administration of marzulene at both dosages significantly increased the mucosal heights of the fundic and antral mucosa at week 25. Marzulene also increased the labeling indices of the fundic and antral epithelial cells, but not the mucosal blood flow. CONCLUSIONS: These findings indicate that marzulene stimulates repair mechanisms of rat gastric mucosa after NaOH injury. This effect of marzulene may be associated with a stimulation of gastric epithelial cell proliferation.     

The cytoprotective effect of leminoprazole on indomethacin-induced damage to rabbit gastric mucosal cells

Leminoprazole (an acid pump inhibitor) has a mucosal protective effect against various experimental gastric lesions, but the underlying mechanism remains unknown. We examined whether leminoprazole prevents indomethacin-induced damage to cultured gastric mucosal cells. The viability of rabbit gastric mucosal cells was assessed by the 3-(4,5-dimethyl-2-thiazoyl)-2,5-diphenyl-2H-tetrazolium bromide and dye exclusion methods. [35S]Methionine-labeled proteins were detected by autoradiography after sodium dodecylsulfate-polyacrylamide gel electrophoresis. Western blot analysis was carried out using anti-heat shock protein (HSP)-70 and anti-HSP-72 antibodies. Exposure of gastric mucosal cells to indomethacin for 4 hr apparently reduced their viability in a dose-related manner. Pretreatment with leminoprazole for 4 hr significantly prevented the reduction in cell viability caused by 50 microM indomethacin, although omeprazole was not effective. However, such pretreatment did not prevent the severe damage induced by 500 microM indomethacin. 16,16-Dimethyl prostaglandin E2 significantly prevented the cell damage induced by indomethacin at both 50 and 500 microM. Leminoprazole alone did not affect cell viability. The cytoprotection by leminoprazole was expressed after a 2-hr lag period. Leminoprazole did not promote prostaglandin E2 synthesis by cells, but it apparently induced the synthesis of 83-kDa, 72-kDa, 52-kDa and 35-kDa proteins. Both the cytoprotection and the induction of such protein synthesis were abolished by cycloheximide and actinomycin D. The leminoprazole-induced 72-kDa protein did not react with the antibodies against HSP-70 and HSP-72. These results indicate that leminoprazole directly protects gastric mucosal cells against mild damage caused by indomethacin and that its cytoprotective effect might be mediated through de novo synthesized proteins. In addition, it is suggested that the leminoprazole-induced proteins might be unknown proteins related to cytoprotection, although the exact characters of the proteins are unclear.     

Effects of tofisopam on gastric functions in rats (author's transl)

The effects of tofisopam on gastric functions were examined in rats. Intracerebroventricular (i.c.v.) injection of tofisopam (50 ot 100 microgram) increased both basal gastric acid output and mucosal blood flow (MBF) in rats anesthetized with urethane, while intravenous injection of tofisopam (10 mg/kg) did not change the basal gastric acid output. Ten micrograms of tofisopam, i.c.v., a dose which did not show any effect on the basal gastric acid output, significantly inhibited the decrease in gastric acid output induced by noradrenaline (5 microgram, i.c.v.). Tofisopam (10 mg/kg, i.v. or 100 microgram, i.c.v.) showed no effect on the increase in gastric acid output induced by electrical stimulation of the lateral hypothalamic area (LHA). These results, together with the previous findings, suggest the tofisopam (i.c.v.) acts on the nucleus dorsalis n. vagi and/or LHA and competes with noradrenaline. The gastric acid output was increased remarkably under water-immersion stress, and this increase lasted during the stress-loading, but the MBF did not show a corresponding increase. Pretreatment of rats with tofisopam (100 mg/kg, intraduodenal) significantly increased the MBF and inhibited the ulcer formation caused by the stress. From these results, tofisopam may restore the unbalance between sympathetic and parasympathetic nervous tones induced by stress-loading.     

Endoscopic observation of the gastric mucus in vivo stained with azure A

Gastric mucus was stained with Azure A, a cationic dye, which had the highest affinity with macromolecular constituents of the mucus, under such conditions as 0.2% Azure A-0.5% NaHCO3 solution (pH 8.1) in dye concentration, staining for ten minutes, 37 degrees C in reaction temperature and the salt concentration and ionic strength below 6.0 x 10(-2). In rat and resected human stomachs, gastric mucus was clearly stained under these conditions. In human subjects, the in vivo stained muscu was observed endoscopically. The pyloric gland region. The difference was seen in the pattern of the gastric area between the fundic and pyloric gland region. Histological examination revealed that only the mucous layer was stained with Azure A. The stained macromolecules in the mucus and factors affecting the staining were discussed.     

Gastric mucosal lesions after burn injury: relationship to H+ back-diffusion and the microcirculation

Rats were subjected to a 30% body surface area full-thickness burn. Two hours after injury, 93% of animals had gastric mucosal erosions. At 5 hours this increased to 100%, but at 24 and 72 hours, lesions were fewer and less severe. Histologic study suggested that lesions noted at 24 and 72 hours represented erosions formed earlier. No mucosal abnormalities were noted in control rats. A causal relationship between mucosal ischemia and the development of erosions is suggested by the presence of A-V shunts at 2 and 5 hours only. Significant increases in H+ back-diffusion and protein leakage into the gastric lumen at 2 and 5 hours also implicated changed mucosal permeability in the etiology of erosions. The return of H+ back-diffusion to control values at 24 and 72 hours, when lesions were still present, appears to contradict the theory that permeability changes are secondary to erosion formation.     

Transient kinetics of polyamine oxidase from Zea mays L

Restraint water-immersion stress-induced expression of heat shock protein (HSP)70 mRNA in the cerebral cortex and stomach of rats was evaluated by Northern blotting. Cerebral and gastric HSP70 mRNA significantly increased in the 6 h-stressed rats and the amount of mRNA measured as optical densities was highest in the 12 h-stressed rats. These data confirmed our previous observations and suggest that families of HSPs play a salient cytoprotective role in stress-vulnerable organs.     

The dynamics of pAL2-1 homologous linear plasmids in Podospora anserina

We have investigated the properties of the newly synthesized proton-pump inhibitor, 3-butyryl-8-methoxy-4-[(2-thiophenyl)amino]quinoline (YJA20379-6), on gastric mucosal proton-pump (H+/K+-ATPase) activity, gastric acid secretion and gastroduodenal lesions in experimental rats. YJA20379-6 markedly inhibited H+/K+-ATPase activity in rabbit isolated gastric mucosal microsomes, confirming its classification as a proton-pump inhibitor. The inhibitory efficacy of YJA20379-6 on the proton pump was approximately 14-times higher than that of omeprazole at pH 7.4. YJA20379-6 given intraduodenally had a potent inhibitory effect on gastric secretion in pylorus-ligated rats (ED50 22.9 mg kg(-1)) but was less active than omeprazole. Pretreatment of rats with YJA20379-6 dose-dependently protected the gastric mucosa from damage induced by water-immersion stress, indomethacin and absolute ethanol, and the duodenal mucosa from damage induced by mepirizole. Repeated administration of YJA20379-6 also dose-dependently accelerated the spontaneous healing of acetic acid-induced gastric ulcers. These results suggest that YJA20379-6 has potent anti-secretory and anti-ulcer effects which are exerted by suppression of H+/K+-ATPase activity in gastric parietal cells. YJA20379-6 might be useful for the clinical treatment of peptic ulcer diseases.     

The vascular effects of topical and intravenous alpha2-adrenoceptor agonist clonidine on canine pial microcirculation

Helicobacter pylori infection of the gastric mucosal surface was investigated in patients with hamartomatous fundic polyps or hyperplastic polyps and in patients without endoscopic evidence of disease (healthy subjects). Presence of H. pylori infection was determined by culture, histologic examination, and the endoscopic phenol red test. Adherence of H. pylori was evaluated with scanning electron microscopic examination of antral biopsy specimens. Both prevalence of H. pylori infection (P < 0.001) and H. pylori adherence (P < 0.05) were less in patients with hamartomatous fundic polyps than in healthy subjects and patients with hyperplastic polyps. However, the percentages of plasma cells in gastric mucosa that contained IgA and of gastric epithelial cells that expressed Lewis b did not differ significantly among the three groups. These findings suggest that defense mechanisms against the attachment of H. pylori other than IgA or Lewis b antigen are present in patients with hamartomarous fundic polyps.     

Evidence that GABA transmission mediates context-specific extinction of learned fear

Recent evidence has suggested that molecular chaperones participate in the conformational change between the normal cellular prion protein (PrPC) and its scrapie isoform (PrPSc). To study a role of PrPC in the regulation of expression of heat shock proteins (HSPs), a group of molecular chaperones, heat-induced expression of major HSPs (HSP105, HSP90alpha, HSP72, HSC70, HSP60, and HSP25) was investigated in cultured skin fibroblasts isolated from the mice homogeneous for a disrupted PrP gene (PrP-/- mice) by Western blot analysis and immunocytochemistry. Two lines of fibroblasts were established and designated SFK derived from the PrP-/- mice and SFH derived from the PrP+/+ mice, respectively. In both SFK and SFH cells, HSP105, HSP72, and HSP25 were expressed at low levels under unstressed conditions but they were induced markedly following exposure to heat stress (43 degreesC/20 min) at 3-72 h postrecovery. In both cell types, HSC70 and HSP60 were expressed at high levels under unstressed conditions and their levels remained unchanged after heat shock treatment. HSP90alpha was undetectable in both cell types under any conditions examined. The pattern of expression, induction, and subcellular location of HSP105, HSP72, HSC70, HSP60, and HSP25 was not significantly different between SFK and SFH cells under unstressed and heat-stressed conditions. Furthermore, the levels of constitutive expression of HSP105, HSC70, HSP60, and HSP25 were similar between the brain tissues isolated from the PrP-/- and PrP+/+ mice. These results indicate that HSP induction is not affected by either the existence or the absence of PrPC in the cells.     

Transplantation. Abstract from 1970

We previously suggested that hydroxyl free radical (-OH) production may play a role in carcinogenesis induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). MNNG-induced gastric cancer in rats and human gastric carcinoma occur most often in the antral mucosa and rarely in the normal fundic mucosa. We hypothesized that regional differences in anti-oxidant activity may be responsible. In the present study, we examined anti-oxidant activity by comparing the relative rates of reduction of a nitroxide free radical, 4-hydroxy-2,2,6,6-tetramethyl-1-piperidinyloxy (Tempol), in the antral and fundic mucosa of male Wistar rats using ESR. The relative rate of Tempol reduction was significantly slower in the antral portion of the wall than in the fundic portion when Tempol [4 x 10(-6) mole/mg wet weight of gastric wall] in HEPES buffer (pH 7.4) was spread over the mucosal surface of a section of the gastric wall. Addition of a sulfhydryl group modulator, N-ethylmaleimide, to the mucosal surface before treatment with Tempol removed the significant difference observed in the rates of reduction in the antral and fundic portions of the gastric wall. No signals were detected in the muscle layer. Our results indicate that the relative rate of free radical reduction by sulfhydryl groups was significantly slower in the antral mucosa than in the fundic mucosa. We therefore conclude that a regional difference in the rates of reduction of free radicals by sulfhydryl groups may result in the site susceptible to development of MNNG-induced gastric cancer.     

Histone H3 messenger RNA in situ hybridization for identifying proliferating cells in formalin-fixed rat gastric mucosa

To devise a more sensitive method for identifying proliferative cells in routinely formalin-fixed, paraffin-embedded tissues, we applied an in situ hybridization (ISH) technique for the detection of histone H3 mRNA in rat gastric mucosa and amplified the signal by a silver intensification method. ISH was performed using a Fluorescein-labelled, single-stranded DNA probe for the human histone H3 gene. To determine the optimal conditions for detecting H3 mRNA in rat gastric mucosa, we tested the effect of changing conditions, such as fixation time and digestion time, by a proteinase before hybridization. Next, the proliferation indices obtained using H3 ISH were compared with those obtained using bromodeoxyuridine (BrdU) immunohistochemistry. In normal rat gastric mucosa, H3 ISH- and BrdU-positive cells were confined to the neck region of both fundic and pyloric mucosa. The two labelling indices were almost the same. In all the serial sections studied, H3 ISH-positive cells were almost always BrdU-positive too. Taken together, these results indicate that the H3 ISH technique is useful for the evaluation of proliferative activity in gastric epithelial cells by virtue of its detection of S-phase cells.     

Childhood derivatives of high and low reactivity in infancy

BACKGROUND & AIMS: Regenerating gene (Reg) has been isolated from rat regenerating pancreatic islets, and Reg protein is mitogenic to islet cells. We have recently shown that Reg gene and Reg protein are expressed in gastric enterochromaffin-like (ECL) cells. This study aimed to clarify whether gastrin enhances Reg protein production in ECL cells and whether Reg protein is mitogenic to gastric mucosal cells. METHODS: Reg gene expression in response to acute and chronic hypergastrinemia was investigated in rats. Immunohistochemical studies, Northern blotting, and in situ hybridization were performed to investigate the expression of Reg protein and Reg gene. The direct effect of gastrin on Reg gene expression was investigated using isolated ECL cells, and the trophic effect of Reg protein on cultured gastric epithelial cells was assessed by [3H]thymidine uptake. RESULTS: Both chronic hypergastrinemia and short-term gastrin administration stimulated Reg gene expression and Reg protein production in fundic mucosa. Reg gene expression was also augmented in isolated ECL cells after incubation with rat gastrin. Reg protein was mitogenic to cultured rat gastric epithelial cells. CONCLUSIONS: Gastrin stimulates the production of Reg protein in gastric ECL cells, which may be involved in the gastrin-induced gastric mucosal cell growth.     

The fundamentals of pediatric patient and disease management

We have reported that increases in lipid peroxide (LPO) formation, nonprotein sulfhydryl (NP-SH) oxidation, and inducible NO synthase (iNOS) activity and a decrease in constitutive NO synthase (cNOS) activity in the gastric mucosa of rats with water immersion restraint (WIR) stress are closely related to gastric mucosal lesion development. Peroxynitrite, which is produced by the reaction of nitric oxide (NO) with superoxide anion, can initiate intracellular LPO formation and NP-SH oxidation, resulting in producing an extreme cellular membrane damage. In this study, the relation of changes in cNOS and iNOS activities to LPO formation and NP-SH oxidation was examined in the gastric mucosa of rats with WIR stress. An increase in iNOS activity, but not a decrease in cNOS activity, correlated well with an increase in LPO concentration (r = 0.750) and NP-SH concentration (r = -0.808) in the gastric mucosa of rats with WIR stress. In addition, the above-mentioned changes in iNOS activity and LPO and NP-SH concentrations with lesion development in the gastric mucosa of rats with WIR stress were attenuated with both prevention of the lesion development and an increase in the concentration of gastric mucosal nitrite/nitrate, the breakdown products of NO, by pretreatment with aminoguanidine, a selective iNOS inhibitor. These results suggest that in the gastric mucosa of WIR-stressed rats, NO produced by increased iNOS could contribute to enhanced LPO formation and NP-SH oxidation, resulting in lesion development.     

The effects of hange-shashin-to on gastric function in comparison with sho-saiko-to

The effects of "Hange-shashin-to (TJ-14)" on gastric function were examined in comparison with "Sho-saiko-to (TJ-9)". Oral treatment with TJ-14 (125-500 mg/kg) caused dose-dependent suppression of ethanol-induced gastric injury, while it did not suppress gastric lesions induced by water-immersion stress. TJ-9 (125-500 mg/kg, p.o.) suppressed both water-immersion stress-induced gastric lesions and ethanol-induced gastric injury in a dose-dependent manner. Intraduodenal administration of TJ-14 even at 500 mg/kg did not affect gastric juice secretion, while TJ-9 at 125 to 500 mg/kg dose-dependently suppressed gastric juice secretion. TJ-14 (125-500 mg/kg, p.o.) accelerated gastric emptying in normal rats and improved the delayed gastric emptying induced by BaCl2 in a dose-dependent manner, whereas such effect was not noted with TJ-9. Oral treatment with TJ-14 at 500 mg/kg significantly suppressed apomorphine-induced vomiting, but it did not affect copper sulfate-induced vomiting. These results suggest that TJ-14 exhibits an anti-ulcer action (probably based on its ability to protect the gastric mucosa), improvement of gastric emptying and an anti-emetic action. TJ-9 also showed anti-ulcer effects, probably based on its ability to suppress gastric secretion and to protect the gastric mucosa. Thus, the present study demonstrated the effectiveness of TJ-14 and TJ-9 against gastric disease, and provided basic data which explain the differences in clinical application between these two kampo medicines.     

A laser ablation method for the spatial segregation of enzyme and redox sites on carbon fiber microelectrodes

Glucagon-like polypeptide 1 (GLP-1) may be a major enterogastrone, slowing gastric emptying when released by intestinal nutrients. In six conscious dogs, we studied the effects of GLP-1, on antropyloric motility, gastric emptying, and transpyloric flow after instillation of 500 ml of saline into the stomach. The meal was given and recordings were started 15 min after intravenous bolus and infusion of either saline or three different doses of GLP-1. Intravenous GLP-1 produced a dose-related retardation of gastric emptying associated with a decrease in the number and volume of flow pulses in comparison to saline. This change in transpyloric flow was associated with an inhibition of antropyloric pressure waves, a stimulation of isolated pyloric pressure waves, and an increase in basal pyloric tone induced by intravenous GLP-1 infusion. Our findings show that GLP-1 has a potent dose-dependent inhibitory effect on transpyloric flow and gastric emptying. This effect is temporally associated with inhibition of antral "pumping" and stimulation of pyloric "braking" mechanisms.     

Direct effect of endotoxin on the gastric mucosal microcirculation and electrical gradient

The effects of intra-arterial infusion of E. coli endotoxin at 1.0 mg. per minute on the gastric total and mucosal blood flows, electrical potential difference, and ionic fluxes across the gastric mucosa were studied in an exteriorized, chambered preparation of canine fundic stomach. Gamma-labelled microsphere technique was used in addition to venous drainage and plasma aminopyrine clearance for the measurement of total and mucosal blood flow, respectively. In spite of normal systemic blood pressure throughout the experiment, E. coli endotoxin infusion caused a significant decrease in total gastric blood flow and in the fractional distribution of flow to the mucosae. There was no significant arteriovenous shunting of microspheres. Significant reduction in potential difference and hydrogen-ion back diffusion also was noted after endotoxin infusion, possibly as a consequence of reduced mucosal blood flow. The results indicate that significant gastric mucosal ischemia can occur and may represent a mechanism in the development of gastric erosions in endotoxemia, even in the absence of systemic hypotension.     

Heat shock protein 72 modulates pathways of stress-induced apoptosis

The resistance to stress-induced apoptosis conferred by the thermotolerant state or by exogenous expression of HSP72 was measured in mouse embryo fibroblasts. The induction of thermotolerance protects cells from heat, tumor necrosis factor alpha (TNFalpha), and ceramide-induced apoptosis but not from ionizing radiation. Because the development of thermotolerance is associated with increased levels of heat shock proteins, we determined whether constitutive expression of one of the major inducible heat shock proteins, HSP72, could also protect cells from stress-induced apoptosis. Cells expressing constitutive HSP72 were shown to have significantly reduced levels of apoptosis after heat, TNFalpha, and ceramide but not after ionizing radiation. Activation of stress-activated protein kinase/c-Jun N-terminal kinase (SAPK/JNK) was found to be strongly inhibited in thermotolerant cells after heat shock but not after other stresses. Cells that constitutively express HSP72 did not demonstrate decreased SAPK/JNK activation after any of these stresses. Thus, factors other than HSP72 that are induced in the thermotolerant state are able to reduce activation of SAPK/JNK after heat stress. Notably, the level of activation of SAPK/JNK did not correlate with the amount of apoptosis detected after different stresses. Constitutive HSP72 expression inhibited poly(ADP-ribose) polymerase cleavage in cells after heat shock and TNFalpha but not after ceramide or ionizing radiation. The results suggest either that SAPK/JNK activation is not required for apoptosis in mouse embryo fibroblasts or that HSP72 acts downstream of SAPK/JNK. Furthermore, the data support the concept that caspase activity, which can be down-regulated by HSP72, is a crucial step in stress-induced apoptosis. Based on data presented here and elsewhere, we propose that the heat shock protein family can be classified as a class of anti-apoptotic genes, in addition to the Bcl-2 and inhibitor of apoptosis protein families of genes.