Rat Gastroduodenal Research Articles

Translational research of a novel humanized epidermal growth factor receptor-related protein: a putative inhibitor of pan-ErbB

The ErbB family members are protein tyrosine kinases, which play a crucial role in the signal transduction pathways that regulate key cellular functions. Overexpression of the ErbB family members is associated with oncogenicity, metastatic potential, cell proliferation, apoptosis, angiogenesis, and prognosis in cancer. Molecular-targeted therapies centered on the ErbB signaling pathway are the currently promising anti-cancer therapies. We reviewed the literature to summarize the current knowledge of epidermal growth factor receptor (EGFR)-related protein (ERRP) and determine the potential of this protein to be translated into a molecular-targeting treatment for cancer. ERRP isolated from rat gastroduodenal mucosa is a pan-ErbB inhibitor that targets multiple members of the ErbB family both in vitro and in vivo. Sequestration of ErbB ligands by ERRP results in the formation of inactive ErbB heterodimers and subsequent attenuation of signaling pathways activated by ErbB. We suggest a strategy to develop a humanized ERRP protein based on activity of rat EERP in vitro. As rat ERRP protein is expected to generate an immune response in humans, we propose a hypothesis that a humanized version of ERRP has potential therapeutic value for cancer patients.

Histological Examination of the Relationship between Respiratory Disorders and Repetitive Microaspiration Using a Rat Gastro-Duodenal Contents Reflux Model

Microaspiration due to gastroesophageal reflux (GER) has been suggested as a factor contributing to the development and exacerbation of several respiratory disorders. To explore the relationship between GER and respiratory disorders, we histologically examined the bilateral lungs of a rat gastroduodenal contents reflux model, which was previously used to investigate the histogenesis of Barrett's esophagus and esophageal carcinoma. GER was surgically induced in male Wistar rats. The bilateral lungs of the reflux rats were examined with hematoxylin and eosin (HE), PAS-Alcian blue, and Azan staining at 10 and 20 weeks after surgery. Immunohistochemical staining of CD68 and α-SMA was also performed. Aspiration pneumonia with severe peribronchiolar neutrophilic and lymphocytic infiltrates, goblet cell hyperplasia, prominence of blood vessels, and increased thickness of the smooth muscle layer were detected. Bronchiolitis obliterans (BO)-like lesions comprising granulation tissue with macrophages, spindle cells, and multinucleated giant cells in the lumen of respiratory bronchioles were observed in the bilateral lungs of the reflux animals. These findings suggest that the severe inflammation and the BO-like lesions may play a role in exacerbation of the forced expiratory volume in 1 second (FEV 1) in human cases. In conclusion, we speculate that repetitive microaspiration due to GER may contribute to the exacerbation of various respiratory diseases, particularly asthma and chronic obstructive pulmonary disease (COPD), and the development of BO syndrome following lung transplantation. The reflux model is a good tool for examining the causal relationships between GER and respiratory disorders.

Gastroprotective effect of nonstarch polysaccharide calcium pectate under experimental conditions

Course prophylactic administration and single therapeutic treatment with calcium pectate improved resistance of rat gastroduodenal mucosa to ethanol-induced ulcers, prednisolone-induced ulcers, and H. Shay ulceration of the gastric mucosa.

W1724 Hydrogen Sulfide Stimulates Hco3- Secretion in Rat Stomach and Duodenum

Background/Aim: Hydrogen sulfide (H2S), generated endogenously from L-cysteine by cystationine-β-synthase (CBS) and cystathionine-g-lyase (CSE) in mammalian cells, has recently been recognized as an important mediator that affects various functions under pathophysiological conditions. However, the functional role H2S plays in the gastrointestinal tract remains largely unknown. In the present study, we examined the effects of NaHS, a H2S donor, on HCO3secretion in the rat stomach and duodenum and investigated the mechanism involved in these responses. Methods: Male SD rats were used after 18 h fasting. Under urethane anesthesia, an ex-vivo chambered stomach or a duodenal loop was perfused with saline, and HCO3secretion was measured at pH 7.0 using a pH stat-method. NaHS (0.1~10 μmol/ ml) or HCl (mucosal acidification; 200 mM) was applied topically to the mucosa for 5 min or 10 min, respectively, while PGE2 (0.3 mg/kg) was given IV as a single injection. Indomethacin (5 mg/kg, SC), glibenclamide (KATP channel blocker; 10 mg/kg, IP) was given 30 min before NaHS, while L-NAME (nitric oxide (NO) synthase inhibitor; 20 mg/kg, SC) was given 3 h before. Propargylglycine (CSE antagonist; 10 mg/kg/h) was infused IV starting 1 h before mucosal acidification. Results: Intraluminal application of NaHS significantly and dosedependently increased the secretion of HCO3in both the stomach and duodenum. The HCO3stimulatory action of NaHS in these tissues were significantly attenuated by indomethacin and L-NAME as well as sensory deafferentation. Gastric but not duodenal HCO3 response was also attenuated by gibenclamide. PGE2 stimulated gastric and duodenal HCO3secretions, and these effects were both partially mitigated by glibenclamide but not indometahcin, L-NAME or sensory deafferentation. The acid-induced HCO3secretion was significantly mitigated by propargylglycine, the inhibitor of H2S production, in the duodenum but not the stomach. In addition, the duodenal lesions induced by acid perfusion (150 mM HCl for 4 h) was significantly aggravated by pretreatment with propargylglycine. Conclusions: These results suggest that H2S increases HCO3secretion in the rat gastroduodenal mucosa, and this action is partly mediated by PG, NO and sensory neurons in these tissue and partly associated with the activation of ATP-sensitive K+ channels in the stomach. In addition, it is assumed that endogenous H2S is involved in the regulatory mechanism of HCO3secretion and mucosal protection in the duodenum but not the stomach, because the acid-induced HCO3secretion was attenuated only in the duodenum by the inhibitor of H2S production.

Cloning of a novel EGFR-related peptide: a putative negative regulator of EGFR.

Although epidermal growth factor receptor (EGFR) plays a key role in regulating cell proliferation, differentiation, and transformation in many tissues, little is known about the factor(s) that may modulate its function. We have isolated a cDNA clone from the rat gastroduodenal mucosa whose full length revealed 1,958 bp that contained 227 bp of 5'-untranslated region (UTR) and an open-reading frame encoding 479 amino acids, followed by 290 bp of 3'-UTR. It showed ~85% nucleotide homology to the external domain of the rat EGFR. We refer to the product of the newly isolated cDNA as EGFR-related protein (ERRP). In Northern blot analysis with poly(A)(+) RNA from different rat tissues, ERRP cDNA hybridized to several mRNA transcripts with the strongest reaction noted with a transcript of approximately 2 kb. Maximal expression of the 2-kb mRNA transcript was observed in the small intestine, followed by colon, liver, gastric mucosa, and other tissues. Transfection of ERRP cDNA into a colon cancer cell line, HCT116, resulted in a marked reduction in proliferation in monolayer and colony formation in soft agar compared with the vector-transfected controls. In another colon cancer cell line, Caco-2, with a tetracycline-regulated promoter system, induction of ERRP expression in the absence of doxycycline was associated with a marked reduction in EGFR activation and proliferation. We conclude that the ERRP cDNA may represent a new member of the EGFR gene family and that ERRP plays a role in regulating cell proliferation by modulating the function of EGFR.

Immunolocalization of transforming growth factor-alpha and epidermal growth factor receptor in the rat gastroduodenal area.

The maintenance of gastrointestinal epithelium integrity requires a fine balance between proliferation and differentiation as well as protection against gastric acid secretion. Transforming growth factor-alpha (TGF-alpha) regulates these functions by binding to epidermal growth factor receptor (EGF-R). This study was designed to identify the localization of TGF-alpha and EGF-R in the rat gastroduodenal region. In the stomach, the surface and gastric pit cells showed staining for TGF-alpha antibodies in the cytoplasm and basolateral and apical membranes. TGF-alpha and EGF-R were observed in the supranuclear region of the cells lining the gland. In the duodenum, the enterocytes coexpressed both TGF-alpha and EGF-R in the supranuclear area. The EGF-R was also observed in the apical membrane. Brunner's glands were positive for both TGF-alpha and EGF-R antibodies. Our results demonstrate the coexpression of TGF-alpha and EGF-R in the rat gastroduodenal area, which suggests a functional role for them in the establishment and maintenance of the epithelial renewal.

Rat gastroduodenal motility in vivo: involvement of NO and ATP in spontaneous motor activity.

Our studies of fasted anesthetized rats have shown that all spontaneous relaxations of the antrum are nitric oxide (NO) dependent. Duodenal motility is patterned into propagating "grouped" motor activity interposed with "intergroup" periods of nonpropagating motor activity; in the duodenum, only intergroup relaxations are NO dependent. We examined the involvement of NO and ATP in spontaneous motor activities of the gastroduodenum in vivo: contractions and relaxations were recorded and analyzed simultaneously from the antrum (S1) and proximal duodenum (D1) of anesthetized Sprague-Dawley rats (n = 10/group), using extraluminal foil strain gauges. Treatment with the NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg iv) attenuated (P < 0.05) antral and intergroup relaxations, whereas grouped relaxations were enhanced (P < 0.05). These effects were reversed with L-arginine (300 mg/kg iv). L-NAME also increased (P < 0.05) the amplitude of duodenal contractions. ATP (8 mg. kg-1. min-1 iv) stimulated relaxations at S1 and D1 that were blocked by the P2-purinoceptor antagonist suramin (60 mg/kg iv). This treatment did not affect spontaneous antral relaxations; however, duodenal grouped relaxations were attenuated. Desensitization to the P2x-purinoceptor agonist alpha,beta-methylene ATP (300 micrograms/kg iv) gave results similar to suramin. In contrast, the P2y-purinoceptor agonist 2-methylthio-ATP (2-MeS-ATP; 360 micrograms/kg iv) evoked duodenal relaxations that were attenuated by L-NAME, and desensitization to 2-MeS-ATP attenuated intergroup relaxations. Spontaneous relaxations of the rat antrum and duodenal intergroup relaxations are NO dependent. Both gut regions relax in response to systemically administered ATP; this response is sensitive to suramin. Grouped duodenal relaxations display functional sensitivity to suramin and P2x- purinoceptor desensitization, indicative of the involvement of ATP and P2x purinoceptors. P2y purinoceptors must also be present; however, these occur on elements releasing NO. Although NO does not mediate grouped relaxations or duodenal contractions, the sensitivity of these responses to L-NAME indicates that the pathway(s) controlling these responses is modulated by NO.

Rat gastroduodenal motility in vivo: interaction of GABA and VIP in control of spontaneous relaxations.

Spontaneous relaxations occurring within motor activity in the rat gastroduodenum in vivo can be distinguished by their dependence on either nitric oxide (NO) or ATP. We examined the interaction of gamma-aminobutyric acid (GABA) and vasoactive intestinal peptide (VIP) within pathways controlling this activity in the antrum (S) and duodenum (D) of anesthetized Sprague-Dawley rats, using miniaturized extraluminal foil strain gauges oriented perpendicular to (S1, D1) or in the axis of (S2) the circular smooth muscle. The NO synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg iv) attenuated (P < 0.05) antral relaxations and, in the duodenum, nonpropagating "intergroup" relaxations. The GABAA receptor antagonist bicuculline (350 micrograms/kg sc) had similar effects. The GABAA agonist 3-amino-1-propanesulfonic acid stimulated L-NAME-sensitive relaxations at S1 and D1. Propagating "grouped" responses were unchanged. VIP (6 micrograms/kg iv) always induced a relaxation of the duodenum, which was attenuated by bicuculline and L-NAME. VIP caused simultaneous responses at S1 and S2; however, the antrum displayed either contraction or relaxation in response to VIP. All antral relaxations in response to VIP were attenuated (P < 0. 05) by L-NAME; however, only VIP-induced relaxations at S1 were sensitive to bicuculline. VIP-induced contractions were also unaffected. GABAA receptors mediate the pathway(s) controlling NO-related spontaneous relaxations of the antrum and duodenal circular muscle. All VIP-induced relaxations are mediated by NO. Spontaneous relaxations of the rat gastroduodenum include responses that involve a GABAAergic NO-related pathway, which is targeted by VIP. In addition, VIP can target NO relaxations of the antrum via other pathways.

Distinct effects of tetragastrin in rat gastroduodenal mucosa on mucin content and mucosal protective action against histamine-induced injury.

We examined the effects of tetragastrin on mucin (mucus glycoprotein) content and mucosal damage in the rat stomach and duodenum. Following an injection of tetragastrin (12, 120, or 400 microg/kg subcutaneously), no macroscopic damage was found to the gastric mucosa but an increase in corpus mucin content was noted, whereas mucosal lesions appeared and the mucin content decreased in the duodenum in a dose-related manner. In the groups with histamine (0.8, 8, or 80 mg/kg intraperitoneally) administration, the extent of mucosal damage and the decrease in mucin content were dose-related in both these regions. For assessment of the effect of tetragastrin on the protective action in gastroduodenal mucosa, changes in mucin content and mucosal damage with histamine (80 mg/kg) -induced injury were examined. Coadministration of tetragastrin prevented the gastric mucosal damage and inhibited the decrease in corpus mucin content. In the duodenum, tetragastrin aggravated the histamine-induced mucosal damage and did not inhibit the reduction of the mucin content. From the present results, the increase in gastric mucins induced by tetragastrin might be related to the protective effect of gastric mucosa against injury. Tetragastrin did not protect the duodenal mucosa, and histamine-induced injury occurring in this region would be aggravated by the increase in HCl secretion and the decrease in mucin content induced by tetragastrin.

Deranged hydrophobic barrier of the rat gastroduodenal mucosa after parenteral nonsteroidal anti-inflammatory drugs

BACKGROUND & AIMS: Parenteral administration of nonsteroidal anti- inflammatory drugs (NSAIDs) may cause gastrointestinal mucosal lesions. The aim of this study was to investigate whether parenteral NSAIDs alter surface hydrophobicity of the gastroduodenal mucosa. METHODS: Conscious rats received indomethacin or diclofenac subcutaneously at different doses (0.5-10 mg/kg). Surface hydrophobicity of gastric and duodenal mucosa was determined by contact angle measurement at various time points; mucosal prostaglandin synthesis and mucus phospholipid content were measured. Also, the effects of NSAIDs were studied in bile duct-ligated rats. RESULTS: A single 1-2-mg/kg dose significantly decreased hydrophobicity in the stomach and duodenum. The decrease was associated with a reduction in mucus phosphatidylcholine. In the duodenum, mucosal prostaglandin synthesis was restored 24 hours after NSAID dosing, but hydrophobicity was still decreased. There was no adaptation to long-term treatment. In bile duct-ligated rats, NSAIDs did not decrease gastric or duodenal hydrophobicity. Moreover, oral administration of bile from rats pretreated with parenteral NSAIDs significantly decreased mucosal hydrophobicity in untreated rats. CONCLUSIONS: Low-dose NSAIDs by parenteral route impair the physicochemical barrier against luminal acidity and render the mucosa susceptible to injury. Excretion of NSAIDs in bile seems to play a key role in this effect. (Gastroenterology 1997 Jun;112(6):1931-9)

Establishment of monoclonal antibodies against carbohydrate moiety of gastric mucins distributed in the different sites and layers of rat gastric mucosa

Eight monoclonal antibodies (MAbs), designated RGM21 approximately RGM42, were generated against mucin purified from the rat gastric mucosa. By applying ELISA, all of these MAbs were proved to react not only with the purified mucin, but also with the oligosaccharide mixture obtained from the antigenic mucin by alkaline borohydride treatment. Treatment of the mucin-attached ELISA well with trypsin, sodium periodate or galactose oxidase prior to the addition of the MAb was applied to characterize these MAbs. Histochemical observation indicated that all these MAbs were able to stain the formalin fixed-paraffin embedded sections of the rat gastroduodenal mucosa. Although each of these MAbs reacted with distinct mucus-producing cells localized in particular regions of the gastroduodenal mucosa, their staining specificity could generally be classified into four groups. These MAbs might be useful for estimating the physiological and pathological changes of mucins in the gastric mucosa.

A new method for measurement of blood flow, pH, and transmucosal potential difference in rat gastroduodenal mucosa by endoscopy

A method was developed to measure the mucosal blood flow (BF), mucosal pH (pH), and transmucosal potential difference (PD) in various sites from the oral cavity to the duodenum without surgical operation or damage to the subject rats. These measurements were carried out by using three indicator electrodes, which were attached to the various sites through the suction channel of an endoscope. The hydrogen gas clearance method was used for the measurement of BF. BF values obtained at the fundic, pyloric, and duodenal regions were 119 +/- 17, 69.9 +/- 8.8, and 114 +/- 18 ml/min/100 g (mean +/- SE), respectively. The pH values were lowest at the cardiac portion and the forestomach and highest at the duodenum. PD showed higher values at the stomach and lower values at the pharynx and duodenum. Using this technique, it was possible to measure the BF, pH, and PD repeatedly and safely at various sites in the same rat. Therefore, it was suggested that this method is useful in studying the physiological functions of the stomach and duodenum and the pathogenesis of gastroduodenal ulceration and that this method is applicable to measure the change of the above parameters in the healing process of gastric ulcer in rats.

Phospholipid- and neutral lipid-containing organelles of rat gastroduodenal mucous cells: Possible origin of the hydrophobic mucosal lining

In this study, an attempt was made to localize the cell types and subcellular organelles that synthesize and secrete surface-active lipids from the rat gastroduodenal mucosa. Two fluorescent hydrophobic probes—Nile Red and 1-anilinonaphthalene-8-sulfonic acid—and a phospholipid-selective cytochemical stain—iodoplatinate—were used for fluorescence and electron microscopic studies, respectively. The results showed the presence of phospholipid and neutral lipid fluorescent hot spots in the mucous cells of both the gastric mucosa and the submucosal Brunner's glands of the duodenum. In contrast, other cell types of the stomach (parietal, chief, and endocrine cells) or the duodenum (goblet cells and villous and crypt enterocytes) were either unreactive or weakly stained with the dyes. Ultrastructurally, two classes of large infranuclear inclusion bodies were observed in these two types of mucous cells. The location and size of these inclusion bodies appeared to be in agreement with the position and size of the large hydrophobic fluorescent hot spots detected at the light microscopic level. The ultrastructural appearance, localization, and histochemical staining pattern of these lipid-containing organelles suggest that they are specific for these cell types and not a general feature of degenerative epithelium undergoing lysosome-induced autolysis. It was concluded that the mucous cells of both the stomach and the Brunner's glands contain a subcellular organelle that stores hydrophobic material constituting both neutral lipids and phospholipids. These lipidic substances may be secreted into the gastroduodenal mucous gel and provide its surface with a nonwettable lining to repel luminal acid.

Prostaglandin E 2 formation by rat gastroduodenal tissue following intragastric acid perfusion

Rat gastroduodenal mucosa forms prostaglandin (PG) E 2. However, little is known about regional differences in PGE 2 formation or the effect of gastric hydrochloric acid (HCl) perfusion on regional PGE 2 formation. In this study, the rats were divided into 3 groups. Group 1 received intravenous (i.v.), 1 ml/h, and intragastric (i.g.), 8 ml/h, perfusions of saline simultaneously for 3 h. Group 2 received saline i.v. and 0.15 N HCl i.g., 8 ml/h. Group 3 was injected with a bolus of aspirin (ASA), 60 mg/kg, followed by ASA, 40 mg/kg/h i.v., and 0.15 N HCl i.g.. The gastric aspirates were analyzed for volume and pH. Segments of gastroduodenal tissue from the fundus, corpus, antrum, and duodenum were minced and then incubated in 1 ml of 50 mM Tris buffer, pH 8.4, for 30 sec with mixing; the incubate was assayed for PGE 2 by radioimmunoassay. Intragastric HCl decreased the pH of aspirate without producing gastric mucosal lesions. However, when combined with i.v. ASA, ulcer formation was present in all animals (p < 0.05). PGE 2 was formed by isolated tissue from four different gastroduodenal regions. The duodenum formed significantly greater amounts than the fundus, antrum, or corpus, which were similar. Intragastric HC1 produced a trend toward increased PGE 2 formation (pmol PGE 2/mg tissue) in the fundus, 143 ± 36 to 237 ± 57; corpus, 87 ± 13 to 200 ± 57; antrum, 157 ± 28 to 224 ± 65; and duodenum, 235 ± 56 to 338 ± 51. However, statistical significance was not reached. Intragastric HC1 combined with i.v. ASA reduced PGE 2 formation by tissue from all four regions below detectable levels. The data indicate that PGE 2α formation by rat gastroduodenal tissue differs regionally, and i.g. HCl perfusion did not significantly increase PGE 2 formation. However, when i.g. HCl perfusion combined with i.v. ASA, the PGE 2 formation was reduced below detectable levels.

Cytoprotective effect of S-adenosylmethionine compared with that of misoprostol against ethanol-, aspirin-, and stress-induced gastric damage

The administration of oral S-adenosylmethionine (SAMe) (100 mg/kg body weight) was well tolerated by the rat gastroduodenal tract. Moreover, rats given SAMe exhibited a significant increase in non-protein sulfhydryl groups of gastroduodenal mucosa as compared with control animals. The abilities of SAMe and misoprotol, a protaglandin E 1 analogue, to protect the gastric mucosa against necrosis induced by various noxious stimuli (ethanol, aspirin, stress) were also compared in standardized, experimental rat models. Pretreatment with SAMe or misoprostol significantly and to the same extent reduced gastric mucosal injury. The experiments described herein indicate that SAMe, a molecule used for the treatment of osteoarthritis, can exert a gastric cytoprotective effect in animals. As preliminary data have shown that this effect may also be reproduced in humans, SAMe seems to provide a therapeutic advantage, in contrast to currently available nonsteroidal anti-inflammatory agents.