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MUCOSAL BIOLOGY

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

Department of Veterinary Physiology, Free University of Berlin, Berlin, Germany

Submitted 12 January 2005 ; accepted in final form 12 August 2005

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 mag-fura 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·l cells^–1·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·l cells^–1·min^–1). Mg²⁺ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE₂ (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 µM. 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.

sheep rumen; magnesium efflux; sodium/magnesium exchanger; cobalt(III)hexaammine; mag-fura 2