Effect of Li+ upon the Mg2+-dependent activation of recombinant Gialpha1.

Although lithium salts have been used in the treatment and prophylaxis of manic-depressive or bipolar patients for 50 years, the mechanism of the pharmacologic action of Li+ is unknown. Based on activity studies of inhibitory and stimulatory guanine-binding (G) proteins in rat cortical membranes, it was proposed that Li+ inhibition of G-proteins may account for its pharmacologic action. We used the purified alpha subunit of the recombinant inhibitory G-protein, rGialpha1, and found that Li+ at therapeutic levels significantly inhibited the formation of the GDP.AlF4-.rGialpha1 complex. Because our studies were conducted with a purified, metal-reconstituted G-protein rather than with cell membrane suspensions, our Li+ inhibition results lend additional support to the G-protein hypothesis for Li+ action.

Comparison of fluorescence, (31)P NMR, and (7)Li NMR spectroscopic methods for investigating Li(+)/Mg(2+) competition for biomolecules.

The biochemical action of lithium in the treatment of manic-depressive illness is still unknown. One hypothesis is that Li(+) competes for Mg(2+)-binding sites in biomolecules. We report here our studies on metal ion competition by three distinct methods: fluorescence, (31)P NMR, and (7)Li NMR spectroscopy, using ATP as a model ligand. By fluorescence spectroscopy, we used the dye, furaptra, by measuring the increases in Mg(2+) levels in an ATP solution as Li(+) levels were increased in the solution. This increase in Mg(2+) levels was indicated by increases in the fluorescence intensity ratio (335/370) of furaptra. By (31)P NMR spectroscopy, this competition was demonstrated by changes in the (31)P NMR spectrum of ATP. The Li(+)/Mg(2+) competition was indicated by predictable changes in the separation between the alpha and beta resonances of the phosphates of ATP. For (7)Li NMR spectroscopy, spin-lattice relaxation measurements were used, which provided free Li(+) concentrations that could be used for determining the free Mg(2+) values in ATP solutions. The values of the free Mg(2+) concentrations obtained by all three methods were in good agreement. The fluorescence and (7)Li NMR methods, however, proved to be more sensitive to low concentrations of Li(+) than the (31)P NMR method.

Competition between Li+ and Mg2+ in neuroblastoma SH-SY5Y cells: a fluorescence and 31P NMR study.

Because Mg2+ and Li+ ions have similar chemical properties, we have hypothesized that Li+/Mg2+ competition for Mg2+ binding sites is the molecular basis for the therapeutic action of lithium in manic-depressive illness. By fluorescence spectroscopy with furaptra-loaded cells, the free intracellular Mg2+ concentration within the intact neuroblastoma cells was found to increase from 0. 39 +/- 0.04 mM to 0.60 +/- 0.04 mM during a 40-min Li+ incubation in which the total intracellular Li+ concentration increased from 0 to 5.5 mM. Our fluorescence microscopy observations of Li+-free and Li+-loaded cells also indicate an increase in free Mg2+ concentration upon Li+ incubation. By 31P NMR, the free intracellular Mg2+ concentrations for Li+-free cells was 0.35 +/- 0. 03 mM and 0.80 +/- 0.04 mM for Li+-loaded cells (final total intracellular Li+ concentration of 16 mM). If a Li+/Mg2+ competition mechanism is present in neuroblastoma cells, an increase in the total intracellular Li+ concentration is expected to result in an increase in the free intracellular Mg2+ concentration, because Li+ displaces Mg2+ from its binding sites within the nerve cell. The fluorescence spectroscopy, fluorescence microscopy, and 31P NMR spectroscopy studies presented here have shown this to be the case.

Competition between Li+ and Mg2+ for the phosphate groups in the human erythrocyte membrane and ATP: an NMR and fluorescence study.

We investigated the mechanism of competition between Li+ and Mg2+ in Li(+)-loaded human red blood cells (RBCs) by making 7Li and 31P NMR and fluorescence measurements. We used 7Li NMR relaxation times to probe Li+ binding to the human RBC membrane and ATP; an increase in Mg2+ concentration caused an increase in both 7Li T1 and T2 values in packed Li(+)-loaded RBCs, in suspensions of Li(+)-loaded RBC ghosts, in suspensions of Li(+)-containing RBC membrane, and in aqueous solutions of ATP, indicating competition between Li+ and Mg2+ for binding sites in the membrane and ATP. We found that increasing concentrations of either Li+ or Mg2+ in the presence of human RBC membrane caused an increase in the 31P NMR chemical shift anisotropy parameter, which describes the observed axially symmetric powder pattern, indicating metal ion binding to the phosphate groups in the membrane. Competition between Li+ and Mg2+ for phosphate groups in ATP and in the RBC membrane was also observed by both fluorescence measurements and 31P NMR spectroscopy at low temperature. The ratio of the stoichiometric binding constants of Mg2+ to Li+ to the RBC membrane was approximately 20; the ratio of the conditional binding constants in the presence of a free intracellular ATP concentration of 0.2 mM was approximately 4, indicating that Li+ competes for approximately 20% of the Mg(2+)-binding sites in the RBC membrane. Our results indicate that, regardless of the spectroscopic method used, Li+ competes with Mg2+ for phosphate groups in both ATP and the RBC membrane; the extent of metal ion competition for the phosphate head groups of the phospholipids in the RBC membrane is enhanced by the presence of ATP. Competition between Li+ and Mg2+ for anionic phospholipids or Mg(2+)-activated proteins present in cell membranes may constitute the basis of a general molecular mechanism for Li+ action in human tissues.