Intracellular lithium and cyclic AMP levels are mutually regulated in neuronal cells.

In this work, we studied the effect of intracellular 3',5'-cyclic adenosine monophosphate (cAMP) on Li+ transport in SH-SY5Y cells. The cells were stimulated with forskolin, an adenylate cyclase activator, or with the cAMP analogue, dibutyryl-cAMP. It was observed that under forskolin stimulation both the Li+ influx rate constant and the Li+ accumulation in these cells were increased. Dibutyryl-cAMP also increased Li+ uptake and identical results were obtained with cortical and hippocampal neurons. The inhibitor of the Na+/Ca2+ exchanger, KB-R7943, reduced the influx of Li+ under resting conditions, and completely inhibited the effect of forskolin on the accumulation of the cation. Intracellular Ca2+ chelation, or inhibition of N-type voltage-sensitive Ca2+ channels, or inhibition of cAMP-dependent protein kinase (PKA) also abolished the effect of forskolin on Li+ uptake. The involvement of Ca2+ on forskolin-induced Li+ uptake was confirmed by intracellular free Ca2+ measurements using fluorescence spectroscopy. Exposure of SH-SY5Y cells to 1 mm Li+ for 24 h increased basal cAMP levels, but preincubation with Li+, at the same concentration, decreased cAMP production in response to forskolin. To summarize, these results demonstrate that intracellular cAMP levels regulate the uptake of Li+ in a Ca(2+)-dependent manner, and indicate that Li+ plays an important role in the homeostasis of this second messenger in neuronal cells.

Ion pairing between Cl- or ClO4- and alkali metal complexes of ionophore antibiotics in organic solvents: a multinuclear NMR and FT-IR study.

The extent of ion pairing in chloride and perchlorate salts was studied by measurement of the Cl- and ClO4- resonances and the observation of the perchlorate stretching frequency by use of nuclear magnetic resonance (NMR) spectroscopy and Fourier transform infrared spectroscopy (FT-IR), respectively, for a variety of ionophores in various solutions and in large unilaminar vesicles (LUVs). The NMR line widths of chloride and perchlorate were larger in solutions containing the neutral ionophores valinomycin (Val) and nonactin (Non) than in solutions containing the negatively charged ionophores nigericin (Nig), lasalocid (Las), and monensin (Mon). The viscosity-corrected perchlorate NMR line widths in solutions containing Val and Las were significantly negatively correlated (r2 > or = 0.99) with the dielectric constant of the solvent. Solvents with low dielectric constants favored ion pair formation. From methanolic solutions containing the Li+, Na+, K+, and Cs+ salts of Cl- and ClO4-, it was determined that the cation with the highest selectivity for the ionophore affords the most ion pairing. A decrease in pH from 7 to 3 had no significant effect on the NMR line widths of chloride and perchlorate in methanolic solutions containing Val, whereas a similar decrease in pH in a methanolic solution containing Mon caused a 2-fold increase in the line widths. The FT-IR difference spectrum of KClO4 in a methanolic solution containing Val showed splitting at the perchlorate stretching frequency. No band splitting was observed in the FT-IR difference spectrum of KClO(4) in methanolic solutions containing Las. The efflux of 35Cl in LUVs containing the neutral ionophore Val followed first-order kinetics with an efflux constant of 1.70 x 10(-3) x min(-1), as determined by 35Cl NMR spectroscopy. The induction of increased membrane permeability in LUVs by the ionophore was determined to be negligible for Val and Nig by fluorescence spectroscopy.

Li+/Mg2+ competition at therapeutic intracellular Li+ levels in human neuroblastoma SH-SY5Y cells.

OBJECTIVES: One proposed mechanism of lithium action in the treatment of bipolar disorder is that Li+ competes with Mg2+ for Mg2+ binding sites within the cell. In this study, we investigated this competition at therapeutic intracellular Li+ levels in human neuroblastoma SH-SY5Y cells. METHODS: We used fluorescence spectroscopy and a Mg2+ indicator, furaptra, to investigate this competition in human neuroblastoma SH-SY5Y cells. Atomic absorption spectrophotometry was used for determination of the intracellular Li+ levels. RESULTS: The neuroblastoma cells, incubated in 15 mM or 30 mM Li+-containing buffer, showed a significant increase in free intracellular Mg2+ levels [using a positive linear within-groups contrast t-test, the 15 mM condition produced t(2) = 5.0, one-tailed p < 0.02, and the 30 mM Li+-incubation conditions gave t(2) = 9.2, one-tailed p < 0.006] but did not significantly increase over time in the Li+-free condition [t(2) = 0.1, one-tailed p > 0.96]. At the earlier times during the incubation (1 or 10 min for the 15 mM or 30 mM Li+-containing buffers), the intracellular Li+ concentrations were 0.6-2.5 mM, values which are comparable to those reached in the brain of Li+-treated patients. CONCLUSION: We demonstrated that competition between Li+ and Mg2+ can occur at therapeutic intracellular Li+ levels.

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 +/- 5 M(-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.

Competition between Na(+) and Li(+) for unsealed and cytoskeleton-depleted human red blood cell membrane: a (23)Na multiple quantum filtered and (7)Li NMR relaxation study.

Evidence for competition between Li(+) and Na(+) for binding sites of human unsealed and cytoskeleton-depleted human red blood cell (csdRBC) membranes was obtained from the effect of added Li(+) upon the (23)Na double quantum filtered (DQF) and triple quantum filtered (TQF) NMR signals of Na(+)-containing red blood cell (RBC) membrane suspensions. We found that, at low ionic strength, the observed quenching effect of Li(+) on the (23)Na TQF and DQF signal intensity probed Li(+)/Na(+) competition for isotropic binding sites only. Membrane cytoskeleton depletion significantly decreased the isotropic signal intensity, strongly affecting the binding of Na(+) to isotropic membrane sites, but had no effect on Li(+)/Na(+) competition for those sites. Through the observed (23)Na DQF NMR spectra, which allow probing of both isotropic and anisotropic Na(+) motion, we found anisotropic membrane binding sites for Na(+) when the total ionic strength was higher than 40 mM. This is a consequence of ionic strength effects on the conformation of the cytoskeleton, in particular on the dimer-tetramer equilibrium of spectrin. The determinant involvement of the cytoskeleton in the anisotropy of Na(+) motion at the membrane surface was demonstrated by the isotropy of the DQF spectra of csdRBC membranes even at high ionic strength. Li(+) addition initially quenched the isotropic signal the most, indicating preferential Li(+)/Na(+) competition for the isotropic membrane sites. High ionic strength also increased the intensity of the anisotropic signal, due to its effect on the restructuring of the membrane cytoskeleton. Further Li(+) addition competed with Na(+) for those sites, quenching the anisotropic signal. (7)Li T(1) relaxation data for Li(+)-containing suspensions of unsealed and csdRBC membranes, in the absence and presence of Na(+) at low ionic strength, showed that cytoskeleton depletion does not affect the affinity of Na(+) for the RBC membrane, but increases the affinity of Li(+) by 50%. This clearly indicates that cytoskeleton depletion favors Li(+) relative to Na(+) binding, and thus Li(+)/Na(+) competition for its isotropic sites. Thus, this relaxation technique proves to be very sensitive to alkali metal binding to the membrane, detecting a more pronounced steric hindrance effect of the cytoskeleton network to binding of the larger hydrated Li(+) ion to the membrane phosphate groups.

Nuclear magnetic resonance and oxygen affinity study of cesium binding in human erythrocytes.

We investigated the interaction of the cesium ion (Cs(+)) with the anionic intracellular components of human red blood cells (RBCs); the components studied included 2,3-bisphosphoglycerate (BPG), ADP, ATP, inorganic phosphate (P(i)), carbonmonoxy hemoglobin (COHb), and RBC membranes. We used spin-lattice (T(1)) and spin-spin (T(2)) (133)Cs NMR relaxation measurements to probe Cs(+) binding, and we found that Cs(+) bound more strongly to binding sites in BPG and in RBC membranes than in any other intracellular component in RBCs at physiologic concentrations. By using James-Noggle plots, we obtained Cs(+) binding constants per binding site in BPG (66 +/- 8 M(-1)), ADP (19 +/- 1 M(-1)), ATP (25 +/- 3 M(-1)), and RBC membranes (55 +/- 2 M(-1)) from the observed T(1) values. We also studied the effect of Cs(+) on the oxygen (O(2)) affinity of purified Hb and of Hb in intact RBCs in the absence and in the presence of BPG. In the absence of BPG, the O(2) affinity of Hb decreased upon addition of Cs(+). However, in the presence of BPG, the O(2) affinity of Hb increased upon addition of Cs(+). The O(2) affinity of Cs(+)-loaded human RBCs was larger than that of Cs(+)-free cells at the same BPG level. (31)P NMR studies on the pH dependence of the interaction between BPG and Hb indicated that the presence of Cs(+) resulted in a smaller fraction of BPG available to bind to the cleft of deoxyHb. Our NMR and O(2) affinity data indicate that a strong binding site for Cs(+) in human RBCs is BPG. A partial mechanism for Cs(+) toxicity might arise from competition between Cs(+) and deoxyHb for BPG, thereby increasing oxygenation of Hb in RBCs, and thus decreasing the ability of RBCs to give up oxygen in tissues. The presence of Cs(+) at 12.5 mM in intact human RBCs containing BPG at normal concentrations did not, however, alter significantly the O(2) affinity of Hb, thus ruling out the possibility of Cs(+)-BPG interactions accounting for Cs(+) toxicity in this cell type.

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.

7Li nuclear magnetic resonance study for the determination of Li+ properties in neuroblastoma SH-SY5Y cells.

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.

Na(+)-H+ and Na(+)-Li+ exchange are mediated by the same membrane transport protein in human red blood cells: an NMR investigation.

Na(+)-H+ exchange is a transport system present in erythrocytes which plays an important role in the regulation of intracellular pH, cellular volume, and transmembrane ion transport. Na(+)-Li+ exchange has received much attention and has been investigated in more detail than have any of the other ion transport systems, because of its high reproducibility. Both red blood cell (RBC) Na(+)-H+ and Na(+)-Li+ exchange are elevated in essential hypertensive patients relative to normotensive individuals. RBC Na(+)-Li+ exchange may be a mode of operation of Na(+)-H+ exchange. Amiloride and its analogue, 5-(N,N-hexamethylene)amiloride (HMA), are well-known inhibitors of Na(+)-H+ exchange, whereas phloretin strongly inhibits Na(+)-Li+ exchange. In this study, we tested the effects of amiloride, HMA, and phloretin on Na(+)-Li+ exchange activity in intact RBCs by using atomic absorption. We investigated by using 7Li nuclear magnetic resonance (NMR) spectroscopy the effects of HMA and phloretin inhibition on Li+ efflux across resealed H(+)- and Li(+)-loaded RBC ghosts in the absence and presence of pH gradients. Amiloride inhibitory activities on both Na+ and Li+ binding to exposed RBC membranes under different pH conditions were also studied by 23Na and 7Li NMR relaxation time measurements. We found that Na(+)-Li+ exchange activity was inhibited by amiloride, HMA, and phloretin in suspensions of intact RBCs and of resealed RBC ghosts. Li+ efflux rates across resealed H(+)- and Li(+)-loaded RBC ghosts were significantly lower when a pH gradient was present, presumably because of the competition between Li+ and H+ for transport by the same transport protein. Amiloride had similar inhibitory constants on both Na+ and Li+ binding to RBC membranes (1021 +/- 48 M-1 vs 964 +/- 40 M-1 at pH 8.0; 731 +/- 147 M-1 vs 716 +/- 27 M-1 at pH 7.0). These results suggest that Na(+)-H+ exchange and Na(+)-Li+ exchange are mediated by the same RBC membrane transport protein.

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+ 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.

7Li NMR relaxation study of Li+ binding in human erythrocytes.

We used 7Li NMR spin-lattice (T1) and spin-spin (T2) relaxation time measurements to investigate the binding of Li+ in human red blood cell (RBC) suspensions. In RBCs containing 1.4 mM Li+, the intracellular 7Li NMR T2 relaxation value (0.30 +/- 0.03 s) was much smaller than the corresponding T1 value (6.0 +/- 0.1 s), yielding a ratio of T1 to T2 of 20. For 1.5 mM LiCl solutions whose viscosities were adjusted to 5 cP with glycerol, the values of the T1/T2 ratios were as follows: 49 for unsealed RBC membrane (2.0 mg of protein/mL); 4.4 for spectrin (1.9 mg/mL); 1.5 for 5.4 mM 2,3-bisphosphoglycerate (BPG); 2.2 for 2.7 mM carbonmonoxyhemoglobin (COHb); 1.6 for 2.0 mM ATP; and 1.2 for a 50/50% (v/v) glycerol-water mixture. Intracellular viscosity and the electric field gradients experienced by Li+ when traversing the spectrin-actin network therefore are not responsible for the large values of the T1/T2 ratios observed in Li(+)-loaded RBCs. We conclude that the RBC membrane is the major Li+ binding site in Li(+)-loaded RBCs (Kb = 215 +/- 36 M-1) and that the binding of Li+ to COHb, BPG, spectrin-actin, or ATP is weak. Partially relaxed 7Li NMR spectra of Li(+)-containing RBC membrane suspensions indicated the presence of two relaxation components, one broad and one narrow.(ABSTRACT TRUNCATED AT 250 WORDS)

Reinvestigation of the transmembrane difference in 7Li NMR T1 values in Li(+)-loaded human erythrocyte suspensions.

In contrast to the findings in a recent study (R. P. Gullapalli, R. M. Hawk, R. A. Komoroski, Magn. Reson. Med. 20, 240 (1991)), we found that, even at high hematocrits, the T1 values for extracellular 7Li were at least three times longer than those for intracellular Li+. We conclude that a transmembrane difference in T1 values can be used for separate observation of intracellular and extracellular Li+ in human red blood cell suspensions.

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.

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.

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.

Phosphate is an inhibitor of copper-zinc superoxide dismutase.

The superoxide dismutase (SOD) activity of bovine copper-zinc superoxide dismutase (Cu,Zn-SOD) in 50 mM Hepes [4-(2-hydroxyethyl)-1- piperazineethanesulfonic acid], pH 7.4, was decreased by approximately 50% when the solution was made 10 mM in phosphate, in spite of the fact that the ionic strength of both solutions was adjusted to be equal. A similar experiment was carried out with bovine Cu,Zn-SOD chemically modified at Arg-141 with phenylglyoxal, which consequently had approximately 20% of the activity of the unmodified protein. (This activity was shown not to be due to residual unmodified protein.) Addition of 10 mM phosphate to solutions of the modified protein caused only a small decrease (less than 5%) in the SOD activity. The presence of phosphate also caused the affinity of Cu,Zn-SOD for binding azide or cyanide anions to be reduced; this effect of phosphate was also much less for the arginine-modified protein. We conclude that the inhibitory effect of phosphate on bovine Cu,Zn-SOD is due primarily to the neutralization of the positive charge on the side chain of Arg-141. The effect of increasing ionic strength on the activities of the native and arginine-modified proteins was also investigated. We found that at high concentrations of phosphate (greater than or equal to 10 mM), the SOD activities of native and arginine-modified Cu,Zn-SOD were inhibited comparably when the ionic strength was increased. This effect is presumably due to the lysine residues near the active site.(ABSTRACT TRUNCATED AT 250 WORDS)