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

This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+](i); using magfura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([ cAMP] i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [ using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018 +/- 0.009 in a Na+-free medium to 0.73 +/- 0.3 mM center dot l cells(-1) center dot min(-1) in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+](e)) according to a typical saturation kinetic (K-m value for [Na+](e) = 24 mM; maximal velocity = 11 mM center dot l cells(-1) center dot min(-1)). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE(2) (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP](i) led to an [Mg2+](i) decrease that amounted to 375 +/- 105 mu M. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.