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Abstract: 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 [...]

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 (~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. KEY WORDS: * aquaporins * water channel * small interfering RNA * colon

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. aspirin; chloride channel blockers; cyclooxygenases; indomethacin; NS-398

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 [[sup.14]C]-guanidine were studied using Caco-2 cells grown on polycarbonate permeable membranes. The basolateral-to-apical flux of [[sup.14]C]-guanidine (i.e., its secretion) was quantitatively higher than the apical-to-basolateral transport (i.e., its absorption). When [Na.sup.+] was replaced by [K.sup.+] or [Li.sup.+], 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 [[sup.14]C]-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.sup.+] gradient. Influx and efflux of [[sup.14]C]-guanidine were unaffected by the presence of tetraethyl-ammonium, cimetidine or decynium-22 in the donor compartment. Only quinine significantly reduced [[sup.14]C]-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. KEY WORDS: * guanidine * transepithelial transport * Caco-2 cells * apical and basolateral membranes * efflux

Human gastric epithelial cells in culture were used to investigate the uptake, localization and fate of the Helicobacter pylori vacuolating toxin A (VacA) and the time of persistence of toxin inside the cell. Findings showed that VacA is transported to cells in both free and membrane-bound form, localizes inside the endosomal-lysosomal compartment, persists within the cell for at least 72 hours and does not degrade into smaller fragments. These suggests that a large amount of VacA avoids the main lysosomal degradative processes and retains its molecular integrity.