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Biochemical and Molecular Actions of Nutrients

Aquaporin-8 Is Involved in Water Transport in Isolated Superficial Colonocytes from Rat Proximal Colon^1,2

Umberto Laforenza³, Emanuela Cova, Giulia Gastaldi, Simona Tritto, Monica Grazioli, Nicholas F. LaRusso^*, Patrick L. Splinter^*, Patrizia D’Adamo, Marisa Tosco^** and Ulderico Ventura

Dipartimento di Medicina Sperimentale, Sezione di Fisiologia Umana, Università di Pavia, Italy; ^* Center for Basic Research in Digestive Diseases, Division of Gastroenterology and Hepathology, Mayo Medical School, Clinic and Foundation, Rochester, MN; DIBIT, San Raffaele Scientific Institute, Milan, Italy; and ^** Dipartimento di Scienze Biomolecolari e Biotecnologie, Università di Milano, Italy

³To whom correspondence should be addressed. E-mail: lumberto{at}unipv.it.

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 ß-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

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