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 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 (Km 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 microM. 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.
Schweigel
Monika
M
Department of Veterinary Physiology, Free University of Berlin, Germany. mschweigel@fbn-dummerstorf.de
Park
Hi-Sung
HS
Etschmann
Benjamin
B
Martens
Holger
H
D000818:Animals; D017920:Antiporters / Q000378:metabolism; D001693:Biological Transport, Active / Q000187:drug effects; D002478:Cells, Cultured; D003035:Cobalt / Q000494:pharmacology; D004847:Epithelial Cells / Q000187:drug effects* / Q000378:metabolism*; D007099:Imipramine / Q000494:pharmacology; D008274:Magnesium / Q000378:metabolism*; D009942:Organometallic Compounds / Q000494:pharmacology; D012417:Rumen / Q000166:cytology*; D012756:Sheep; D012964:Sodium / Q000378:metabolism / Q000494:pharmacology*; D013997:Time Factors
D017920:Antiporters; D009942:Organometallic Compounds; C046250:sodium-magnesium antiporter; D003035:Cobalt; D012964:Sodium; D008274:Magnesium; D007099:Imipramine