Mecanismos moleculares desencadenados por el deoxicolato de sodio en intestino: implicancias en la absorción intestinal de calcio

Altas concentraciones de deoxicolato de sodio (DXCS) producen efectos toxicos en el intestino. Este estudio explora el efecto de concentraciones fisiologicas altas de DXCS sobre la absorcion intestinal de Ca2+ y los mecanismos moleculares involucrados. Para ello, se usaron pollos de 4 semanas de edad, los cuales se dividieron en dos grupos: controles y tratados con DXCS en la luz intestinal, a diferentes tiempos y concentraciones. La absorcion intestinal de Ca2+ se midio por la tecnica del asa intestinal ligada in situ. Se estudio la expresion de genes y de proteinas involucradas en la via transcelular de la absorcion del cation. El contenido de glutation (GSH) y la actividad de enzimas del sistema antioxidante se evaluaron por espectrofotometria. La produccion de ROS se determino por espectrometria de resonancia de espin y los cambios en la permeabilidad de la membrana interna itocondrial mediante la tecnica de swelling. La apoptosis se estudio a traves de la localizacion subcelular de citocromo c por Western blot y la fragmentacion del ADN por la tecnica de TUNEL. DXCS inhibio la absorcion intestinal de Ca+2, efecto que fue dependiente de la concentracion de la sal biliar. La expresion del ARNm de la Ca+2- ATPasa disminuyo por el tratamiento con la sal biliar y lo mismo ocurrio con la expresion de las proteinas involucradas en el proceso de absorcion del cation: Ca+2- ATPasa, intercambiador Na+/Ca+2 y calbindina D28k. DXCS produjo estres oxidativo, a juzgar por la generacion de ROS, la deplecion de glutation y el swelling mitocondrial. Ademas, la presencia del antioxidante quercetina en el medio de incubacion bloqueo el efecto inhibitorio del DXCS sobre la absorcion intestinal de Ca+2. SUMMARY: High concentrations of sodium deoxycholate (NaDOC) produce toxic effects. This study explores the effect of a single high concentration of NaDOC on the intestinal Ca2+ absorption and the underlying mechanisms. Chicks were divided into two groups: 1) controls, 2) treated with different concentrations of NaDOC in the duodenal loop for variable times. Intestinal Ca2+ absorption was measured as well as the gene and protein expression of molecules involved in the Ca2+ transcellular pathway. Glutathione (GSH) content and the activity of antioxidant enzymes were assessed by spectrophotometry. ROS was determined by spin resonance spectrometry and permeability changes of the internal mitochondrial membrane by the swelling technique. Apoptosis was studied by cytochrome localization through Western blot and DNA fragmentation (TUNEL procedure). NaDOC inhibited the intestinal Ca2+ absorption, which was dose dependent. Ca2+- ATPase mRNA decreased by the ile salt and the same occurred with the protein expression of Ca2+-ATPase, calbindin D28k and Na+/Ca2+ exchanger. NaDOC produced oxidative stress as judged by ROS generation, mitochondrial swelling and glutathione depletion. Furthermore, the antioxidant quercetin blocked the inhibitory effect of NaDOC on the intestinal Ca2+ bsorption. Apoptosis was also triggered by the bile salt, as indicated by the TUNEL staining and the cytochrome crelease from the mitochondria. As a compensatory mechanism, enzyme activities of the antioxidant system were all increased, but the cellular redox state was not normalized. In conclusion, a single high dose of NaDOC inhibits intestinal Ca2+.