Duarte Mota de Freitas

Correlations of Na+-Li+ exchange activity with Na+ and Li+ binding and phospholipid composition in erythrocyte membranes of white hypertensive and normotensive individuals: a nuclear magnetic resonance investigation.

Enhanced Na+-Li+ exchange activity has been reported in red blood cells (RBCs) of white patients with essential hypertension compared with RBCs of normotensive individuals. To understand the factors responsible for this finding, we applied novel and conventional spectroscopic and kinetic methods to blood samples from 10 hypertensive and 10 normotensive individuals. We measured the kinetic parameters (V std, V max, and K m) for RBC Na+-Li+ exchange by atomic absorption spectrophotometry and used 23Na and 7Li nuclear magnetic resonance relaxation methods to measure Na+ and Li+ binding to RBC membranes as well as 31P nuclear magnetic resonance spectroscopy to measure membrane phospholipid compositions. We found significant differences between the two groups for the affinity of Na+ for the RBC membrane (0.202 +/- 0.054 mmol/L-1 for hypertensive patients versus 0.296 +/- 0.071 mmol/L-1 for normotensive subjects, P<.005). The kinetic parameters of RBC Na+-Li+ exchange (V std, V max, and K m) were 0.32 +/- 0.09 and 0.66 +/- 0.17 mmol Li+/L cell.h and 160 +/- 62 mmol/L, respectively, for hypertensive patients versus 0.21 +/- 0.06 and 0.32 +/- 0.14 mmol Li+/L cell.h and 86 +/- 69 mmol/L for normotensive subjects (P<.05). The fractions of phosphatidylserine and phosphatidylethanolamine were 0.153 +/- 0.009 and 0.294 +/- 0.016 for hypertensive patients versus 0.138 +/- 0.013 and 0.325 +/- 0.018 for normotensive subjects (P<.05). The Na+ binding constants were negatively correlated with the Km values for both the hypertensive (r=-.61, P=.01) and normotensive (r=-.43, P=.04) groups. Changes in lipid-protein interactions in the RBC membranes of hypertensive patients appear to be responsible for weaker Na+ binding to the membrane and for the faster rates of RBC Na+-Li+ exchange.

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 ATP in human erythrocytes A 31P NMR and optical spectroscopy study

We have investigated the influence of Li+ on free intracellular Mg2+ concentration in human erythrocytes by 31P NMR and optical absorbance spectroscopies. In red cells loaded with 3 mM intracellular Li+, the chemical shift separation between the α‐ and β‐phosphate resonances of MgATP2− was approx. 0.9 ppm larger than that observed in Li+‐free red cells. By analyzing the interaction of each red cell component with Mg2+ and Li+, we found that Mg2+ is displaced in part from MgATP2− upon addition of Li+ and that the released Mg2+ is bound to the red cell membrane causing an overall decrease in free intracellular Mg2+ concentration.

Competition between Li+ and Mg2+ for ATP and ADP in aqueous solution: A multinuclear NMR study

We used 7Li NMR spin-lattice relaxation times and 31P NMR chemical shifts to study the binding of Li+ and Mg2+ to the phosphate moieties of ATP and ADP. To examine the binding of Li+ and Mg2+ to the base and ribose moieties, we used 1H and 13C NMR chemical shifts. The 7Li NMR relaxation times of Li+/Mg2+ mixtures of ATP or ADP increased with increasing concentrations of Mg2+, suggesting competition between the two ions for adenine nucleotides. No significant binding of Li+ and Mg2+ to the base and ribose moieties occurred. At the pH and ionic strength used, 2:1 and 1:1 species of the Li(+)-ATP and Li+-ADP complexes were present, with the 2:1 species predominating. In contrast, 1:1 species predominated for the Mg(2+)-ADP and Mg(2+)-ATP complexes. We calculated the Li(+)-nucleotide binding constants in the presence and absence of Mg2+ and found them to be somewhat greater in the presence of Mg2+. Although competition between Li+ and Mg2+ for ATP and ADP phosphate binding sites in solution is consistent with the 31P chemical shift data, the possibility that the Li+ and Mg2+ form mixed complexes with the phosphate groups of ATP or ADP cannot be ruled out.

7Li Nuclear Magnetic Resonance Study for the Determination of Li+ Properties in Neuroblastoma SH‐SY5Y Cells

Abstract: Lithium has been used clinically in the treatment of manic depression. However, its pharmacologic mode of action remains unclear. Characteristics of Li+ interactions in red blood cells (RBCs) have been identified. We investigated Li+ interactions on human neuroblastoma SH‐SY5Y cells by developing a novel 7Li NMR method that provided a clear estimation of the intra‐ and extracellular amounts of Li+ in the presence of the shift reagent thulium‐1,4,7,10‐tetrazacyclododecane‐N,N′,N″,N‴‐tetramethylene phosphonate (HTmDOTP4−). The first‐order rate constants of Li+ influx and efflux for perfused, agarose‐embedded SH‐SY5Y cells in the presence of 3 mM HTmDOTP4− were 0.055 ± 0.006 (n = 4) and −0.025 ± 0.006 min−1 (n = 3), respectively. Significant increases in the rate constants of Li+ influx and efflux in the presence of 0.05 mM veratridine indicated the presence of Na+ channel‐mediated Li+ transport in SH‐SY5Y cells. 7Li NMR relaxation measurements showed that Li+ is immobilized more in human neuroblastoma SH‐SY5Y cells than in human RBCs.

Measurement of lithium transport in RBC from psychiatric patients receiving lithium carbonate and normal individuals by 7Li NMR spectroscopy

A reproducible 7Li nuclear magnetic resonance (NMR) method, based on a modified inversion recovery (MIR) pulse sequence, was used to discriminate between intra- and extracellular lithium concentrations in red blood cell (RBC) suspensions. The rates of Na(+)-Li+ countertransport determined by the 7Li NMR method were significantly correlated with the measurements made by atomic absorption (AA) for 14 psychiatric patients receiving lithium carbonate (r = 0.937) and 14 normal individuals (r = 0.931). As expected, the rates of Na(+)-Li+ countertransport measured by MIR were significantly lower for the psychiatric patients receiving lithium carbonate than for normal individuals. The 7Li NMR method provides RBC Li+ countertransport information comparable to AA for psychiatric patients and normal individuals. A description of the advantages of the 7Li NMR method in contrast to the AA method, including the study of Li+ interactions with RBC components such as membrane proteins and anionic phospholipids, is included.

Ionophore-induced Cl− transport in human erythrocyte suspensions: A multinuclear magnetic resonance study

To investigate the effect of ionophores on Cl- distribution in human erythrocyte suspensions, we measured the membrane potential by using 19F and 31P NMR methods. Incubation of human erythrocytes with 0.005 mM of the neutral ionophores valinomycin and nonactin resulted in membrane potentials of -21.2 and -17.8 mV in the presence and absence of DIDS. However, 0.020 mM of the carboxylic ionophores lasalocid, monensin, and nigericin yielded membrane potentials similar to those measured in the absence of ionophore (-9.4 mV). In methanol, the 35Cl- NMR linewidth in the presence of valinomycin was twice as broad as those observed in the presence of carboxylic ionophores, suggesting that neutral ionophores induce Cl- efflux in part via ion pairing.

Competition between Li+ and Mg2+ for red blood cell membrane phospholipids: A 31P, 7Li, and 6Li nuclear magnetic resonance study.

The mode of action of the lithium ion (Li+) in the treatment of manic depression or bipolar illness is still under investigation, although this inorganic drug has been in clinical use for 50 yr. Several research reports have provided evidence for Li+/Mg2+ competition in biomolecules. We carried out this study to characterize the interactions of Li+ and Mg2+ with red blood cell (RBC) membrane components to see whether Li+/Mg2+ competition occurs. 31P nuclear magnetic resonance chemical shift measurements of the phospholipids extracted from the RBC membranes indicated that the anionic phospholipids, phosphatidylserine and phosphatidylinositol, bind Li+ and Mg2+ most strongly. From 6Li relaxation measurements, the Li+ binding constant to the phospholipid extract was found to be 45±5M−1. Thus, these studies showed that the phospholipids play a major role in metal ion binding. 7Li spin-lattice relaxation measurements conducted on unsealed and cytoskeleton-depleted RBC membrane in the presence of magnesium indicated that the removal of the cytoskeleton increases lithium binding to the more exposed anionic phospholipids (357±24 M−1) when compared to lithium binding in the unsealed RBC membrane (221±21 M−1). Therefore, it can be seen that the cytoskeleton does not play a major role in Li+ binding or in Li+/Mg2+ competition.

Nuclear magnetic resonance studies of lithium transport in erythrocyte suspensions of hypertensives

We have applied a nuclear magnetic resonance (NMR) method, based on the 7Li nucleus, to discriminate between intracellular and extracellular lithium ions (Li+) in red blood cell (RBC) suspensions. The NMR method was compared with atomic absorption, a technique that requires physical separation of intra- and extracellular Li+ prior to chemical analysis. The rates and rate constants of RBC Na(+)-Li+ countertransport measured by the 7Li NMR method correlated significantly with the measurements made by atomic absorption for both the hypertensive (r = 0.964) and control (r = 0.961) groups. The rates of RBC Na(+)-Li+ countertransport measured by NMR were significantly higher for hypertensive patients than for normotensive controls. The fact that the NMR method does not require cell membrane lysis, and its potential to reveal structural and mechanistic information on Li+ binding and transport in cellular systems, makes it promising for understanding the basis of Li+ transport variations in RBCs, and possibly other tissues, from hypertensive patients.

Lithium in Medicine: Mechanisms of Action

In this chapter, we review the mechanism of action of lithium salts from a chemical perspective. A description on how lithium salts are used to treat mental illnesses, in particular bipolar disorder, and other disease states is provided. Emphasis is not placed on the genetics and the psychopharmacology of the ailments for which lithium salts have proven to be beneficial. Rather we highlight the application of chemical methodologies for the characterization of the cellular targets of lithium salts and their distribution in tissues.