GSK-3β inhibition by lithium confers resistance to chemotherapy-induced apoptosis through the repression of CD95 (Fas/APO-1) expression

Lithium exerts neuroprotective actions that involve the inhibition of glycogen synthase kinase-3β (GSK-3β). Otherwise, recent studies suggest that sustained GSK-3β inhibition is a hallmark of tumorigenesis. In this context, the present study was undertaken to examine whether lithium modulated cancer cell sensitivity to apoptosis induced by chemotherapy agents. We observed that, in different human cancer cell lines, lithium significantly reduced etoposide- and camptothecin-induced apoptosis. In HepG2 cells, lithium repressed drug induction of CD95 expression and clustering at the cell surface as well as caspase-8 activation. Lithium acted through deregulation of GSK-3β signaling since (1) it provoked a rapid and sustained phosphorylation of GSK-3β on the inhibitory serine 9 residue; (2) the GSK-3β inhibitor SB-415286 mimicked lithium effects by repressing drug-induced apoptosis and CD95 membrane expression; and (3) lithium promoted the disruption of nuclear GSK-3β/p53 complexes. Moreover, the overexpression of an inactivated GSK-3β mutant counteracted the stimulatory effects of etoposide and camptothecin on a luciferase reporter plasmid driven by a p53-responsive sequence from the CD95 gene. In conclusion, we provide the first evidence that lithium confers resistance to apoptosis in cancer cells through GSK-3β inhibition and subsequent repression of CD95 gene expression. Our study also highlights the concerted action of GSK-3β and p53 on CD95 gene expression.     

In Vitro and In Vivo Inhibition of Inositol Monophosphatase by the Bisphosphonate L-690,330

Abstract: We have previously described the synthesis of bis-phosphonate-containing inhibitors of inositol monophosphatase. In the present study, a more detailed examination of the in vitro and in vivo properties of one of these compounds, L-690,330, is described. L-690,330 is a competitive inhibitor of inositol monophosphatase with a K ₁, depending on the source of IMPase, of between 0.2 and 2 μM. Although ∼1,000-fold more potent in vitro than lithium, in muscarinic m1 receptor-transfected Chinese hamster ovary cells prelabelled with [³H]inositol, L-690,330 only produced 40% of the accumulation of [³H]inositol monophosphates achieved by lithium at the same concentration (10 m M ), suggesting that the ability of L-690,330 to cross the cell membrane is limited. Nevertheless, under conditions of cholinergic stimulation (100 mg/kg of pilocarpine s.c.), high doses of L-690,330 were able to increase brain inositol(1)phosphate levels in vivo to three- to fourfold control levels. This effect was dose dependent (ED₅₀= 0.3 mmol/kg s.c.) and was maximal after 1 h. In peripheral tissues, the effects of L-690,330 on inositol(1)phosphate levels mimicked those of lithium both qualitatively and quantitatively. However, in the brain, the effects of L-690,330 were much less than seen with lithium, consistent with the blood-brain barrier restricting access of the polar L-690,330 into the CNS, thereby further limiting entry of compound into cells in the brain. In the future, it may be possible to develop prodrugs of this compound, which circumvent many of the cell permeability problems inherent in bisphosphonate compounds.     

Comparative Effects of Lithium on the Phosphoinositide Cycle in Rat Cerebral Cortex, Hippocampus, and Striatum

Abstract: The effects of lithium on muscarinic cholinoceptor-stimulated phosphoinositide turnover have been investigated in rat hippocampal, striatal, and cerebral cortical slices using [³H]inositol or [³H]cytidine prelabelling and inositol 1,4,5-trisphosphate [lns(1,4,5)P₃] and inositol 1,3,4,5-tetrakisphosphate [lns(1,3,4,5)P₄] mass determination methods. Carbachol addition resulted in maintained increases in lns(1,4,5)P₃ and lns(1,3,4,5)P₄ mass levels in hippocampus and cerebral cortex, whereas in striatal slices these responses declined significantly over a 30-min incubation period. Carbachol-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations were inhibited by lithium in all brain regions studied in a time-and concentration-dependent manner. For example, in hippocampal slices significant inhibitory effects of LiCl were observed at times > 10 min after agonist challenge; IC₅₀ values for inhibition of agonist-stimulated lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations by lithium were 0.22 ± 0.09 and 0.33 ± 0.13 mM, respectively. [³H]CMP-phosphatidate accumulation increased in all brain regions when slices were stimulated by agonist and lithium. The ability of myo-inositol to reverse these effects, as well as lithium-suppressed lns(1,4,5)P₃ accumulation, implicates myo-inositol depletion in the action of lithium in the hippocampus and cortex at least. The results of this study suggest that although significant differences in the magnitude and time courses of changes in inositol (poly)phosphate metabolites occur in different brain regions, lithium evokes qualitatively similar enhancements of [³H]inositol monophosphate and [³H]CMP-phosphatidate levels and inhibitions of lns(1,4,5)P₃ and lns(1,3,4,5)P₄ accumulations. However, the inability of striatal slices to sustain carbachol-stimulated inositol polyphosphate accumulation in the absence of lithium and the inability to reverse effects with myo-inositol may indicate differences in phosphoinositide signalling in this brain region.     

Lithium Enhances Muscarinic Receptor–Stimulated CDP-Diacylglycerol Formation in Inositol-Depleted SK-N-SH Neuroblastoma Cells

Abstract: The psychotherapeutic action of Li⁺ in brain has been proposed to result from the depletion of cellular inositol secondary to its block of inositol monophosphatase. This action is thought to slow phosphoinositide resynthesis, thereby attenuating stimulated phosphoinositidase-mediated signal transduction in affected cells. In the present study, the effect of Li⁺ on muscarinic receptor–stimulated formation of the immediate precursor of phosphatidylinositol, CDP-diacylglycerol (CDP-DAG), has been examined in human SK-N-SH neuroblastoma cells that have been cultured under conditions that alter the cellular content of myo-inositol. Resting neuroblastoma cells, like brain cells in vivo, were found to concentrate inositol from the culture medium, achieving an intracellular level of 60.0 ± 4 nmol/mg of protein. The addition of carbachol to [³H]cytidine-prelabeled cells elicited a four- to fivefold increase in the accumulation of labeled CDP-DAG. This stimulated formation of [³H]CDP-DAG was completely blocked by the addition of 10 μM atropine, was not dependent on the presence of Li⁺, nor was it affected by co-incubation with myo-inositol. This result was in sharp contrast to findings in rat brain slices, in which carbachol-stimulated formation of [³H]CDP-DAG was potentiated ～ 10-fold by Li⁺ and substantially reduced by coincubation with inositol. The formation of [³H]CDP-DAG in labeled SK-N-SH cells by carbachol was both concentration and time dependent. The order of efficacy of muscarinic ligands in stimulating [³H]-CDP-DAG accumulation paralleled that established in these cells for inositol phosphate accumulation, i.e., carbachol ≥ oxotremorine-M > bethanecol ≥ arecoline > oxotremorine > pilocarpine. Extended culture of the SK-N-SH cells in an inositol-free chemically defined growth medium progressively reduced the intracellular inositol content to <5 nmol/mg of protein, a level comparable with that seen in cortical slices. In these inositol-depleted cells, Li⁺ potentiated carbachol-stimulated [³H]CDP-DAG formation, and this effect was completely reversed by coincubation with inositol (EC₅₀ 0.2 mM). The present study thus demonstrates, in the same cultured cell line, the effects of normal and reduced intracellular inositol levels on the ability of Li⁺ to attenuate phosphoinositide resynthesis, as inferred from [³H]CDP-DAG accumulation. The results indicate that Li⁺ can lead to a slowing of stimulated phosphoinositide turnover in neuroblastoma cells, provided that the intracellular inositol content has been significantly reduced.     

Lithium treatment of Nicotiana tabacum microspores blocks polar nuclear migration,disrupts the partitioning of membrane-associated Ca2+, and induces symmetrical mitosis

We have found that the normal developmental pathway of Nicotiana tabacum microspores is blocked or switched when microspores are exposed to lithium, and these effects are reversible with Ca²⁺ and myo-inositol. Normal development was defined by the following characteristics: changes in microspore shape from spherical to oval and then ellipsoid; two nuclear displacements, first from a central location to the cell periphery, and then from the periphery to the generative pole; a localization of membrane-associated Ca²⁺ at the generative pole preceding nuclear division; and, finally, an asymmetrical mitosis that results in a two-celled pollen grain with well-differentiated generative and vegetative nuclei. Lithium treatment blocked the localization of membrane-associated Ca²⁺ at the generative pole, and instead it was evenly distributed at both poles. Lithium treatment also blocked the asymmetrical positioning of the microspore nucleus at the generative pole and resulted in an approximately four-fold increase in the frequency of symmetrical mitosis. When Ca²⁺ and myo-inositol were added along with lithium, the effects were substantially decreased, and there was only a small increase in the frequency of symmetrical mitosis compared with controls. The timing of treatment was important; microspores isolated before the first nuclear displacement had a low frequency of further development, while microspores isolated immediately preceding the onset of mitosis were much less sensitive to lithium, and the result was only a small increase in the frequency of symmetrical mitosis. In microspores isolated after the first nuclear displacement, a 1-day exposure to lithium was sufficient to switch the developmental pathway from an asymmetrical to a symmetrical mitosis while still allowing limited further development. However, we have not optimized culturing conditions for embryogenesis and the furthest development observed after a 1-week culture was to four- or five-celled proembryo-like structures.     

Taxonomic character of plant species in absorbing and accumulating alkali and alkaline earth metals grown in temperate forest of Japan

Summary Absorption and accumulation of alkali (Li, Na, K, Rb, Cs) and alkaline earth (Mg, Ca, Sr, Ba) metals were investigated as taxonomic characteristics (in 62 plant species). Leaf and soil samples were collected from 9 sites in temperature forest in Japan and the above mentioned elements were analyzed. Considerable differences were found among species in their ability to accumulate alkali and alkaline earth metals. Very high concentrations of Li (45 ppm, D.W.), K (37×10³ ppm), Rb (159 ppm) and Cs (8.2 ppm) were detected inLastrea japonica which were about 412, 12, 27 and 6 times higher than those of the species with the lowest concentrations. Na content was high inAcer micranthum (358 ppm) which was 16 times higher than species with the lowest concentration. Other species containing high levels of alkali metals wereHydrangea macrophylla, Struthiopteris niponica, Clethra barbinervis. Mean discrimination ratio (D.R.) for all investigated plant species for Li, Na, Rb, and Cs to K were 1.7, 0.44, 0.9 and 1.8 respectively. High concentrations of alkaline earth metals Ca (36×10³ ppm), Sr (345 ppm), and Ba (241 ppm) were found in the leaves ofHydrangea paniculata which were about 31, 84, and 72 times higher than those for the species with the lowest concentration. Mg was very high inStruthiopteris niponica (83×10² ppm). Other species with high concentrations of alkaline earth metals belonged to the genus Viburnum. Mean D.Rs. for Mg, Sr, and Bavs Ca were 1.0, 0.7 and 0.08. Principal component analysis of interrelationships between the mineral content in leaf tissues indicated that these elements could be classified into 2 groups with respect to their accumulation behavior in plants. The alkali metals K, Li, Rb, and Cs behaved similarly in their accumulation in leaves but Na behaved independently. Alkaline earth metals Ca, Mg, Sr, and Ba were also found to behave similarly in their accumulation. Factors scores of 1st and 2nd components revealed three groups of plant species: alkaliphilic, alkaline earthphilic, and neutral (non-accumulators).     

Lithium Chloride Inhibits the Phosphorylation of Newly Synthesized Neurofilament Protein, NF-M, in Cultured Chick Sensory Neurons

The middle and high molecular weight members of the neurofilament triplet, NF-M and NF-H, undergo extensive posttranslational polyphosphorylation, a process requiring 24 h or more for completion. We have investigated ways of perturbing this process in intact cells and have found that phosphorylation of newly synthesized NF-M in cultured chick sensory neurons is inhibited by Li+. [35S]Methionine pulse-chase experiments were carried out with pure neuronal cultures, and the phosphorylation of newly synthesized NF-M was monitored by following the accompanying change, with chase time, in apparent size and charge of the polypeptide. Addition of LiCl to the medium inhibited this mobility shift in a dose-dependent manner over concentrations between 2 and 25 mM. Incorporation of 32P into NF-M, as well as NF-H, was also inhibited, whereas incorporation into the low molecular weight neurofilament protein, beta-tubulin, and total protein was unaffected. Protein synthesis was not altered. Exposure to 25 mM LiCl for up to 72 h was not toxic, and the inhibition of NF-M phosphorylation was completely reversible. When 25 mM Li+ was added after NF-M had become partially phosphorylated, further progression was blocked, but there was no net dephosphorylation or degradation of NF-M. Additional experiments suggest that this action of Li+ is probably not due to effects on second messenger levels or to effects on tubulin metabolism and assembly state presented in our accompanying article, but rather to interference by Li+ itself, with the phosphorylation of NF-M and NF-H by specific neurofilament kinase(s).     

Some aspects of the chemistry of lithium in soils

Summary A method for the determination of exchangeable lithium using 0.5M NH₄Cl is described. The range of exchangeable Li in the fifty Papua New Guinea (PNG) soils analyzed was 0.002 to 0.409 gg^–1 in contrast to five Australian soils which ranged from 0.032 to 0.830 gg^–1. The PNG soils were divided into hill and alluvial soils with average exchangeable Li contents of 0.062 and 0.263 gg^–1 respectively. No significant correlation between total and exchangeable Li was found in either group of soils althoughr=0.67 for the comined data and was significant at the 5% level. From the analysis of three profiles exchangeable Li was found to be at least twice as high (0.27 gg^–1) in surface soils as in subsurface samples (0.10 gg^–1). The average value of the deeper subsoil samples was 0.18 ppm.R mode cluster analysis of the data for village garden soils collected on a sampling grid showed that exchangeable Li was more strongly assoicated with Ca and Mg than with pH, 0.05M EDTA soluble Zn, 0.5M NaHCO₃ soluble P or exchangeable Na and K. Computer constructed isographs using the analyses of grid samples from a garden illustrated the association between Li, Ca and Mg and the inverse association with Na.The correlation coefficient between Ca and Li in the ash of three food plants (Gnetum gnemon, Hibiscus abelmoschus andStenochlaena plustris) while not significant on an individual basis, was significant when the data was combined suggesting that the association between these elements in the soil may reflect an association in the ash returned to the soil when the garden was cleared. The correlation coefficient between soil exchangeable Li and Li in plant ash was positive, but not significant.Adsorption experiments over a five-day period demonstrated that Li was strongly adsorbed from solution. On average 63–75% of the adsorbed Li was fixed in a form which was not exchangeable with 0.5M NH₄Cl or soluble in 0.05M EDTA.     

Measurement of Lithium-Induced Changes in Mouse Inositol(1)Phosphate Levels In Vivo

An anion-exchange HPLC mass assay was used to characterize Swiss-Webster mouse brain and peripheral tissue inositol(1)phosphate [Ins(1)P]levels. Ins(1)P was identified in all tissues studied but Ins(4)P could be identified only in brain, and then only as a part of a peak containing an additional, unidentified component. As a result, it was not possible to quantify Ins(4)P levels. Following a single subcutaneous dose of lithium (10 mmol/kg), brain Ins(1)P levels were maximally elevated after 6 h (corresponding to peak brain lithium concentrations) and were increased to levels 35- and 20-fold higher than in saline-treated animals in cholinergic agonist (pilocarpine)-stimulated and unstimulated animals, respectively. The ED50 for the lithium-induced accumulation of brain Ins(1)P 6 h after administration was 4-6 mmol/kg. The pilocarpine stimulation of lithium-induced brain Ins(1)P accumulation had an ED50 of 22 mg/kg, with maximal accumulation occurring 120 min after pilocarpine administration. Atropine reduced Ins(1)P levels, in both the absence and the presence of lithium, by 40%, indicating that cholinergic systems contribute a large (40%) component of basal brain phosphatidylinositol (PI) cycle activity. In peripheral tissues, there were lithium-induced accumulations of Ins(1)P in kidney, heart, and liver (but not testes) but these were less than that seen in the brain, suggesting that under basal (and pilocarpine-stimulated) conditions, the brain has a higher turnover of the PI cycle than the various peripheral tissues studied. These data support the hypothesis that lithium exerts its effects in vivo via modulation of the PI cycle.(ABSTRACT TRUNCATED AT 250 WORDS)     

Application of a fluoride selective electrode to the stoichiometric analysis of lithium carbonate for nuclear purposes

An improved version of a potentiometric method for the determination of lithium has been applied to the accurate, high precision analysis of lithium carbonate, a raw material for the solid-state synthesis of lithium metazirconate (Li₂ZrO₃). This is one of the most promising tritium breeders in fusion reactors. The analytical procedure consisted of a decomposition of the sample in hydrochloric acid followed by evaporation to dryness. The residual lithium chloride was then dissolved in ethanol and titrated with ammonium fluoride in water-ethanol (1 + 1). A lanthanum fluoride solid-state ion-selective electrode was used as end point detector. By increasing the analyte concentration and by dissolving the titrant in the mixed solvent, more accurate results were achieved with respect to the older procedure. A comparison of the results obtained by this method and those obtained by ion-exchange showed no significant difference (P = 0.05). The precision in terms of relative standard deviation was 0.3-0.4%.