Solute transporters and aquaporins are impaired in celiac disease.

BACKGROUND INFORMATION: Celiac disease is a chronic inflammatory disorder of the small bowel induced in genetically susceptible subjects by gluten ingestion. Diarrhoea, weight loss and malabsorption represent the major clinical presentation of the disease. Here we examined the possible alteration in the expression and localization of water channels [AQPs (aquaporins)] and some solute transporters in duodenal mucosa of celiac disease patients. Duodenal biopsies from untreated celiacs, treated celiacs, healthy controls and disease controls were considered in the present study. The expressions of some AQPs and transporter mRNAs in human duodenal biopsies were determined by semi-quantitative RT-PCR (reverse transcription PCR) and real-time RT-PCR. The localization of AQPs 3, 7 and 10 and of SGLT1 (Na+/glucose co-transporter 1), PEPT1 (H+/oligopeptide transporter 1) and NHE3 (Na+/H+ exchanger 3) was evaluated by immunohistochemistry. RESULTS: AQPs 3, 7, 10 and 11, SGLT, PEPT and NHE, CFTR (cystic fibrosis transmembrane conductance regulator) and NKCC (Na-K-2Cl co-transporter) mRNAs were expressed in duodenal biopsies of healthy controls, treated celiac patients and disease controls. The expression of transcripts was virtually absent in duodenal biopsies of untreated celiac disease patients except for CFTR and NKCC. In healthy controls, immunohistochemistry revealed a labelling in the apical membrane of surface epithelial cells of the duodenum. The immunolabelling was heavily reduced or absent in untreated celiac patients, while it was normal in patients consuming a gluten-free diet for at least 12 months. CONCLUSIONS: Our results indicate that the main routes for water and solute absorption are deficient in celiac disease and may play a role in the onset of malabsorption symptoms.

Aquaporin-6 is expressed along the rat gastrointestinal tract and upregulated by feeding in the small intestine.

BACKGROUND: Several aquaporins (a family of integral membrane proteins) have been recently identified in the mammalian gastrointestinal tract, and their involvement in the movement of fluid and small solutes has been suggested. In this direction we investigated, in some regions of the rat gastrointestinal tract, the presence and localization of aquaporin-6, given its peculiar function as an ion selective channel. RESULTS: RT-PCR and immunoblotting experiments showed that aquaporin-6 was expressed in all the investigated portions of the rat gastrointestinal tract. The RT-PCR experiments showed that aquaporin-6 transcript was highly expressed in small intestine and rectum, and less in stomach, caecum and colon. In addition, jejunal mRNA expression was specifically stimulated by feeding. Immunoblotting analysis showed a major band with a molecular weight of about 55 kDa corresponding to the aquaporin-6 protein dimer; this band was stronger in the stomach and large intestine than in the small intestine. Immunoblotting analysis of brush border membrane vesicle preparations showed an intense signal for aquaporin-6 protein. The results of in situ hybridization experiments demonstrate that aquaporin-6 transcript is present in the isthmus, neck and basal regions of the stomach lining, and throughout the crypt-villus axis in both small and large intestine. In the latter regions, immunohistochemistry revealed strong aquaporin-6 labelling in the apical membrane of the surface epithelial cells, while weak or no labelling was observed in the crypt cells. In the stomach, an intense staining was observed in mucous neck cells and lower signal in principal cells and some parietal cells. CONCLUSION: The results indicate that aquaporin-6 is distributed throughout the gastrointestinal tract. Aquaporin-6 localization at the apical pole of the superficial epithelial cells and its upregulation by feeding suggest that it may be involved in movements of water and anions through the epithelium of the villi.

Osmotic water permeability of rat intestinal brush border membrane vesicles: involvement of aquaporin-7 and aquaporin-8 and effect of metal ions.

Water channels AQP7 and AQP8 may be involved in transcellular water movement in the small intestine. We show that both AQP7 and AQP8 mRNA are expressed in rat small intestine. Immunoblot and immunohistochemistry experiments demonstrate that AQP7 and AQP8 proteins are present in the apical brush border membrane of intestinal epithelial cells. We investigated the effect of several metals and pH on the osmotic water permeability (Pf) of brush border membrane vesicles (BBMVs) and of AQP7 and AQP8 expressed in a cell line. Hg2+, Cu2+, and Zn2+ caused a significant decrease in the BBMV Pf, whereas Ni2+ and Li+ had no effect. AQP8-transfected cells showed a reduction in Pf in the presence of Hg2+ and Cu2+, whereas AQP7-transfected cells were insensitive to all tested metals. The Pf of both BBMVs and cells transfected with AQP7 and AQP8 was not affected by pH changes within the physiological range, and the Pf of BBMVs alone was not affected by phlorizin or amiloride. Our results indicate that AQP7 and AQP8 may play a role in water movement via the apical domain of small intestine epithelial cells. AQP8 may contribute to the water-imbalance-related clinical symptoms apparent after ingestion of high doses of Hg2+ and Cu2+.

Intracellular, intercellular, and stromal invasion of gastric mucosa, preneoplastic lesions, and cancer by Helicobacter pylori.

BACKGROUND AND AIMS: It is not clear how Helicobacter pylori, an apparently extracellular pathogen colonizing the luminal side of the gastric epithelium, invariably causes an immune-inflammatory response on the stromal side of the mucosa. Penetration of H pylori into epithelial cell lines and its interaction with immune-inflammatory cells have been documented in vitro. Several investigations also showed in vivo bacterial penetration into the epithelium up to the lamina propria; however, the identification as H pylori of the bacteria-like bodies observed in unchanged, metaplastic, or neoplastic mucosa remained sometimes questionable. METHODS: To search for bacteria-like organisms, we used transmission electron microscopy on endoscopic biopsy specimens from 20 dyspeptic subjects and surgical specimens of neoplastic and nonneoplastic mucosa from 20 cancerous stomachs. To ascertain the H pylori nature of the organisms found, we used 6 different antibodies directed against bacterial lysates, purified vacuolating cytotoxin A, or purified cytotoxin-associated antigen A in immunogold tests. The results were compared with those of H pylori strains cultivated in vitro. RESULTS: In nonmetaplastic gastric epithelium, cytochemically proven H pylori were detected, in the majority of cases, inside cytoplasm of epithelial cells, in intraepithelial intercellular spaces, and in underlying lamina propria, often in direct contact with immune-inflammatory cells and sometimes inside small blood vessels. Cytochemically proven H pylori were also observed inside 6 of 8 intestinal metaplasias and 9 of 20 cancers. CONCLUSIONS: H pylori penetrates normal, metaplastic, and neoplastic gastric epithelium in vivo, intracellularly, or interstitially to cause a strong immune-inflammatory response and promote gastric carcinogenesis.

Free-soluble and outer membrane vesicle-associated VacA from Helicobacter pylori: Two forms of release, a different activity.

Helicobacter pylori releases VacA both as free-soluble and as outer membrane vesicle (OMV)-associated toxin. In this study, we investigated the amount of VacA released in each of the two forms and the role of each form in VacA-induced cell vacuolation in vitro. We found that: (1) free-soluble toxin accounted for about 75% of released VacA, while the remaining 25% was OMV-associated; (2) although OMV-associated VacA caused a statistically significant vacuolation, virtually all the vacuolating activity of a H. pylori broth culture filtrate was due to free-soluble VacA. While it is widely accepted that OMVs may represent an important vehicle for delivering virulence factors to the gastric mucosa, our results suggest that OMV-associated VacA could play a pathobiological role different from that of free-soluble toxin. This conclusion fits with mounting evidence that VacA exerts a large pattern of pathobiological effects among which cell vacuolation might not be the main one.

Expression and immunolocalization of aquaporin-7 in rat gastrointestinal tract.

BACKGROUND INFORMATION: In the gastrointestinal tract of mammals, water can either be secreted with digestive juices or absorbed by the small and large intestine. Transcellular water movement can be mediated by the transmembrane protein family of AQPs (aquaporins), as has also been recently identified in the gastrointestinal tract. However, the localization, expression and functioning of AQPs in the gastrointestinal tract have not been completely characterized. For the present study, we investigated: (1) the expression of AQP7 in some portions of rat gastrointestinal tract by semiquantitative reverse transcriptase-PCR and by immunoblotting and (2) the cellular and subcellular localization of AQP7 by immunohistochemistry. RESULTS: AQP7 mRNA and proteins were highly expressed in the small intestine, weakly in the caecum, colon and rectum and were absent in the stomach. Immunoblotting analysis using rat gastrointestinal tract membrane fractions showed two major bands corresponding to a molecular mass of approx. 34 and 40 kDa for the AQP7 protein. No bands were observed when the anti-AQP7 antibody was preadsorbed with the immunizing peptide. Immunohistochemistry revealed strong AQP7 labelling in the surface epithelial cells of duodenum, jejunum, ileum, caecum, colon and rectum, whereas weak or no labelling was observed in the crypt cells. The labelling was manifest particularly in the apical membrane but intracellular staining was also observed. CONCLUSIONS: The results indicate that AQP7 is present in the small and large intestine. The higher expression of AQP7 protein at the apical pole of the superficial epithelial cells suggests its involvement in rapid fluid movement through the villus epithelium.

Aquaporin-8 is involved in water transport in isolated superficial colonocytes from rat proximal colon.

Water is an essential nutrient because it must be introduced from exogenous sources to satisfy metabolic demand. Under physiologic conditions, the colon can absorb and secrete considerable amounts of water even against osmotic gradients, thus helping to maintain the body fluid balance. Here we describe studies on both aquaporin (AQP) expression and function using cells isolated from the superficial and lower crypt regions of the rat proximal colon. The expression of AQP-3, -4, and -8 in isolated colonocytes was determined by semiquantitative RT-PCR and by immunoblotting. The localization of AQP-8 in the colon was evaluated by immunohistochemistry. A stopped-flow light scattering method was used to examine osmotic water movement in isolated colonocytes. Moreover, the contribution of AQP-8 to overall water movement through isolated colonocytes was studied using RNA interference technology. Colonocytes from the proximal colon express AQP-3, -4, and -8 with increasing concentrations from the lower crypt cells toward those on the surface. Osmotic water permeability was higher in surface than in crypt colonocytes (P < 0.05); it was significantly inhibited by the water channel blocker dimethyl sulfoxide, and reversed by beta-mercaptoethanol (P < 0.05). Immunohistochemistry revealed a strong AQP-8 labeling in the apical membrane of the superficial colonocytes. Inhibition of aquaporin-8 expression by small interfering RNA significantly decreased osmotic water permeability (approximately 38%; P < 0.05). Current results indicate that aquaporin-8 may play a major role in water movement through the colon by acting on the apical side of the superficial cells.

Guanidine transport across the apical and basolateral membranes of human intestinal Caco-2 cells is mediated by two different mechanisms.

The functional characteristics of the intestinal absorption and secretion of guanidine as a model of a nutritionally and metabolically essential organic cation were examined in the Caco-2 human intestinal cell line. Both apical and basolateral transport of [14C]-guanidine were studied using Caco-2 cells grown on polycarbonate permeable membranes. The basolateral-to-apical flux of [14C]-guanidine (i.e., its secretion) was quantitatively higher than the apical-to-basolateral transport (i.e., its absorption). When Na+ was replaced by K+ or Li+, both apical and basolateral accumulation were significantly inhibited. Studies using the cell monolayers and apical membrane vesicles obtained from Caco-2 cells showed a potential-independent mechanism of guanidine apical uptake and efflux. Conversely, basolateral uptake and efflux were membrane potential dependent. Kinetic analysis revealed that both saturable and nonsaturable mechanisms accounted for the apical and basolateral accumulations. The [14C]-guanidine efflux from cells through the apical and basolateral membranes was significantly reduced at 4 degrees C, suggesting carrier-mediated mechanisms. Moreover, the apical efflux was stimulated by an inwardly directed H+ gradient. Influx and efflux of [14C]-guanidine were unaffected by the presence of tetraethylammonium, cimetidine or decynium-22 in the donor compartment. Only quinine significantly reduced [14C]-guanidine entrance through apical and basolateral membranes and its exit through the basolateral membrane. In conclusion, our results suggest that the influx and the efflux through the apical membrane is mediated by different transporters, whereas transport across the basolateral membrane is mediated by a member of the organic cation transporter family with high affinity for guanidine.

NSAIDs counteract H. pylori VacA toxin-induced cell vacuolation in MKN 28 gastric mucosal cells.

The relationship between nonsteroidal anti-inflammatory drugs (NSAIDs) and Helicobacter pylori-induced gastric mucosal injury is still under debate. VacA toxin is an important H. pylori virulence factor that causes cytoplasmic vacuolation in cultured cells. Whether and how NSAIDs affect VacA-induced cytotoxicity is unclear. This study was designed to evaluate the effect of NSAIDs on H. pylori VacA toxin-induced cell vacuolation in human gastric mucosal cells in culture (MKN 28 cell line). Our data show that 1) NSAIDs (indomethacin, aspirin, and NS-398) inhibit VacA-induced cell vacuolation independently of inhibition of cell proliferation and prostaglandin synthesis; 2) NSAIDs impair vacuole development/maintenance without affecting cell binding and internalization of VacA; and 3) NSAIDs, as well as the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid, also inhibit cell vacuolation induced by ammonia. We thus hypothesize that NSAIDs might protect MKN 28 cells against VacA-induced cytotoxicity by inhibiting VacA channel activity required for vacuole genesis.

PYY-Tag transgenic mice displaying abnormal (H+-K+)ATPase activity and gastric mucosal barrier impairment.

The mechanism by which the gastrointestinal hormones peptide YY and glucagon inhibit gastric acid secretion is largely unknown. PYY-Tag transgenic mice develop endocrine tumors in the colon that are composed mainly of peptide YY/enteroglucagon-producing L type cells. Therefore we studied the functional activity of such tumors and the gastric functions of PYY-Tag mice. Fasting and fed PYY-Tag transgenic mice and CD1 controls were assayed for circulating levels of peptide YY, glucagon, insulin, and gastrin. The gastric pH was determined and gastric samples were examined for (a) histologic appearance; (b) K(+)-stimulated p-nitrophenylphosphatase activity and [(14)C]aminopyrine accumulation of apical and tubulovesicle membranes; (c) adherent mucus determination by Alcian blue recovery; and (d) DNA/RNA/protein epithelial content and in vivo incorporation of [(3)H]thymidine into DNA. Transgenic mice showed high serum levels of peptide YY and glucagon, increased gastric pH, and a high incidence of gastric ulcers after fasting. p-Nitrophenylphosphatase activity, [(14)C] aminopyrine accumulation, and proton pump redistribution from cytoplasmic tubulovesicles to apical membranes were significantly lower in the gastric mucosa of transgenic mice compared with the controls. In addition, the adherent mucus was thinner, and [(3)H]thymidine incorporation into the DNA was decreased. The abnormal and unregulated levels of circulating peptide YY and glucagon led to gastric acid inhibition and an impairment of gastric barrier function as a result of a striking reduction in epithelial proliferation.