Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells

Monika Schweigel, Hi-Sung Park, Benjamin Etschmann, Holger Martens
2006 American Journal of Physiology - Gastrointestinal and Liver Physiology  
This study examines the routes by which Mg 2ϩ leaves cultured ovine ruminal epithelial cells (REC). Mg 2ϩ -loaded (6 mM) REC were incubated in completely Mg 2ϩ -free solutions with varying Na ϩ concentrations, and the Mg 2ϩ extrusion rate was calculated from the increase of the Mg 2ϩ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na ϩ salt). In other experiments, REC were also studied for the intracellular free Mg 2ϩ concentration ([Mg 2ϩ ]i;
more » ... tration ([Mg 2ϩ ]i; using magfura 2), the intracellular Na ϩ concentration (using Na ϩ -binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na ϩ /Mg 2ϩ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na ϩ /Mg 2ϩ exchanger]. Mg 2ϩ -loaded REC show a Mg 2ϩ efflux that was strictly dependent on extracellular Na ϩ . The Mg 2ϩ extrusion rate increased from 0.018 Ϯ 0.009 in a Na ϩ -free medium to 0.73 Ϯ 0.3 mM ⅐ l cells Ϫ1 ⅐ min Ϫ1 in a 145 mM Na ϩ medium and relates to extracellular Na ϩ concentration ([Na ϩ ]e) according to a typical saturation kinetic (Km value for [Na ϩ ]e ϭ 24 mM; maximal velocity ϭ 11 mM ⅐ l cells Ϫ1 ⅐ min Ϫ1 ). Mg 2ϩ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na ϩ -free media. Furthermore, an elevation of [cAMP]i led to an [Mg 2ϩ ]i decrease that amounted to 375 Ϯ 105 M. The anti-Na ϩ /Mg 2ϩ exchanger mAb inhibits Mg 2ϩ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na ϩ /Mg 2ϩ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg 2ϩ extrusion and can be assumed to play a considerable role in the process of Mg 2ϩ absorption as well as the maintenance of the cellular Mg 2ϩ homeodynamics. sheep rumen; magnesium efflux; sodium/magnesium exchanger; cobalt(III)hexaammine; mag-fura 2
doi:10.1152/ajpgi.00014.2005 pmid:16109844 fatcat:xdxx6ouda5drlmbl2spoo5chpi