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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 25Mg 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 37C, and aliquots of the cell suspension were centrifuged at the beginning of the incubation and after 60 and 120 min. The quantification of 24Mg, 25Mg and 26Mg 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.

To study the role of Magnesium in the regulation of cell proliferation we characterized the proliferation behaviour of HC-11 mammary epithelial cells that were grown in media containing low to high Mg concentrations. Cells grown under control conditions (0.5 mM Mg in the medium) or in the presence of high (H) Mg (45 mM) displayed similar log-phases and reached confluence in 72h. In the presence of low (L) Mg (0.025 mM) the cells exhibited a reduced growth rate and did not reach confluence at 72h. Intra cellular total Mg increased from 12 to 36h of culture in all cells examined but returned to basal levels in those cells which reached confluence (i.e., control and H-Mg cells). Intra cellular Mg increased independent of mitosis-induced changes of volume and adenine nucleotides pools but correlated with an increased percentage of cells in the S phase and with total nucleic acid contents. These bell-shaped changes of intra cellular Mg were less evident in L-Mg cells, likely due to a combination of low Mg levels in the medium and decreased growth rate. Changes in membrane potential and pH were important factors that contributed to maintaining intra cellular Mg at physiologic levels in the face of increased or decreased availability of extra cellular Mg. H-Mg cells were depolarised and more acidic than control cells; conversely, L-Mg cells showed a pattern of hyperpolarization and alkalinization. These results lend support to the concept that Mg may be involved in regulating cell proliferation, and show that cells maintain adequate levels of intra cellular Mg, and hence their proliferation potential, even under conditions of extreme changes of extra cellular Mg.

Compelling evidence shows that magnesium (Mg) content is directly correlated to proliferation in normal cells as Mg stimulates DNA and protein synthesis. Some data have demonstrated that upon mitogenic stimuli normal cell are able to increase their intracellular Mg content, likely by activating Mg influx. Mg deprivation, in turn, induces inhibition of DNA and protein synthesis thus promoting growth arrest. From a mechanistic viewpoint, Mg deprivation may influence cell cycle control by upregulating the cyclin inhibitor p27Kip1 thus influencing cyclin E-dependent kinases. In many neoplastic cells, Mg is higher than in normal counterparts and this high Mg is maintained also against concentration gradient. Moderate vs. severe and acute vs chronic effect of Mg deprivation must be distinguished: severe Mg deprivation causes growth arrest also in tumor cells, while chronic Mg deprivation leads to an "adaptation" of tumor cells both to growth rate and Mg content. In tumor cells deranged Mg content and distribution is likely due to an inhibition of Mg efflux via the Na-Mg antiport. When differentiation process is induced by receptor mediated stimuli such as IFN-alpha and ATP, decrease of cell Mg content accompanies with activation of Mg efflux. Transformed cells may thus display high growth rate also because they retain a large amount of Mg. On their whole, these data strongly suggest that regulation of intracellular Mg availability parallels the molecular control of cell proliferation, and maybe also cell differentiation and death.