Evaluation of magnesium fluxes in rat erythrocytes using a stable isotope of magnesium

The mechanisms that maintain intracellular Mg concentration at physiologic levels are not fully understood. In this work, we described for the first time, a new method using Mg-25 stable isotopes, that allows simultaneous determination of Mg2+ efflux and Mg2+ influx in non- loaded cells at physiological levels of extracellular Mg. Erythrocytes from rats were suspended as a 10% suspension in NaCl medium or choline medium. The erythrocyte suspension was incubated at 37 degrees C, and aliquots of the cell suspension were centrifuged at the beginning of the incubation and after 60 and 120 min. The quantification of Mg-24, Mg-25 and Mg-26 in supernatants and in erythrocytes were determined by ICP/ MS. Simultaneous Mg2+ efflux and Mg2+ influx were calculated from the intra- extracellular distribution of the three isotopes. By this new approach we characterized Mg2+ influx and Mg2+ efflux at 0.4 mM extracellular Mg in both NaCl and choline Cl medium. Mg2+ efflux and Mg2+ influx were largely inhibited by amiloride in NaCl medium and by cinchonine in choline Cl medium. Apparent velocity and LineWeaver- Burk kinetics showed that Mg2+ influx is different from Mg2+ efflux suggesting the involvement of two distinct transport mechanisms. Moreover, modifying extracellular Mg concentrations, to mimic hypo- or hyper-magnesaemia, we showed that Mg2+ efflux and Mg2+ influx increased with extracellular Mg up to 0.8 mM, the physiologic concentration of total extracellular Mg. Our data demonstrate that Mg2+ fluxes are directly related to the levels of extracellular Mg and that in physiological conditions, Na-dependent and Na- independent Mg2+ efflux counterbalance Mg influx to maintain constant intracellular Mg level.