Differentiation of HL-60 promyelocytic leukemia cells is accompanied by a modification of magnesium homeostasis

Magnesium homeostasis in HL-60 promyelocytic leukemia cells was compared to that in neutrophyl-like HL-60 cells obtained by 1.3% DMSO treatment. Magnesium homeostasis was studied by the characterization of magnesium efflux, the identification of intracellular magnesium pools, and the regulation of intracellular ionized Mg²⁺. In both undifferentiated and neutrophyl-like HL-60 cells, magnesium efflux occurred via the Na-Mg antiporter which was inhibited by imipramine and stimulated by db cAMP and forskolin. Receptor-mediated signals such as ATP, IFN-α, or PGE1, which can trigger cAMP-dependent magnesium efflux, were ineffective in undifferentiated HL-60 cells but induced 60–70% increase of magnesium efflux in neutrophyl-like HL-60 cells. Selective membrane permeabilization by the cation ionophore A23187 induced a large magnesium release when cells were treated with rotenone. In both cell populations, the addition of glucose to rotenone-treated cells restored magnesium release to the control level. Permeabilization by 0.005% digitonin provoked the release of 90% cell total magnesium in both cell types. Intracellular [Mg²⁺]_i was 0.15 and 0.26 mM in undifferentiated and neutrophyl-like HL-60 cells, respectively. Stimuli that triggered magnesium efflux, such as db cAMP in undifferentiated and IFN-α in neutrophyl-like HL-60 cells, induced a slow but consistent increase of [Mg²⁺]_i which was independent from Ca²⁺movements. Overall, these data indicate that magnesium homeostasis is regulated by receptor-mediated magnesium efflux which was modified during differentiation of HL-60 cells. Stimulation of magnesium efflux is paralleled by an increase of [Mg²⁺]_i which reflects a release of magnesium from the bound cation pool. J. Cell. Biochem. 71:441–448, 1998. © 1998 Wiley-Liss, Inc.     

Catecholamines-evoked cytosolic ca2+rise in endothelial cells from bovine adrenal medulla

The effects of catecholamines on intracellular Ca²⁺concentrations ([Ca²⁺]_i) in single acutely dissociated bovine adrenal medulla endothelial cells (BAMECs) were measured using the intracellular fluorescent probe Fluo-3 AM. 100 m epinephrine or norepinephrine induced a biphasic [Ca²⁺]_i rise with an initial peak followed by a delayed phase. 10 m phenylephrine (₁-adrenergic agonist) caused a [Ca²⁺]_i rise similar to that evoked by catecholamines. The increase in [Ca²⁺]_i induced by 10 m phenylephrine was reverted by 10 m phenoxybenzamine (-adrenergic antagonist). Neither isoproterenol (-adrenergic agonist) nor clonidine (₂-adrenergic agonist) induced [Ca²⁺]_i rise. The initial peak was insensitive to zero external Ca²⁺ and it was abolished after Ca²⁺ internal storages were emptied by 10 mM caffeine. The delayed phase was reduced to near zero by external Ca²⁺ removal. These results indicate that BAMECs possess ₁-adrenergic receptors associated to both the release of caffeine-sensitive intracellular Ca²⁺ stores and the entry of extracellular Ca²⁺ We suggest that chromaffin cell secretion may activate BAMECs in vivo through an increase in [Ca²⁺]_i which could induce the secretion of vasoactive factors allowing a rapid entry of hormones into the circulation. (Mol Cell Biochem 000: 000-000,1999)     

Cyclic AMP-dependent and -independent protein kinases in HeLa cells

Malate saturation isotherms for the NAD⁺ malic enzyme determined at widely differing, but saturating, concentrations (8, 80, 160 mm) of magnesium show the same response to malate concentration only when velocity is plotted against the concentration of free malate^2?. This identification of the ionized malic acid as the true substrate for this enzyme, together with the observation that the complex of Mg with malate has no influence on the reaction rate even at very high concentrations, indicates that the metal ion activator of the enzyme must also bind in the ionized form. A kinetic analysis shows that, with respect to magnesium and malate, the malic enzyme catalyzes a rapid equilibrium reaction of the intersecting type. Either Mg²⁺ or malate^2? may bind first but the fact that the K_m's for both Mg²⁺ and malate^2? are smaller than the respective K_i's suggests that, when either metal ion or malate is present on the enzyme, the other is bound more tightly than when it binds to the free enzyme. This demonstration of the nature of the true substrates for this enzyme has implications for studies of the NAD⁺ malic enzyme in which conditions influencing the amount of free magnesium and malate, e.g., changes in pH, addition of weak acid effectors etc., are involved.     

FMLP-, thapsigargin-, and H2O2-evoked changes in intracellular free calcium concentration in lymphocytes and neutrophils of type 2 diabetic patients

Type 2 diabetic (T2DM) patients are immune-compromised having a higher susceptibility to infections and long-term complications in different parts of the body contributing to increased morbidity and mortality. A derangement in the homeostasis of intracellular free calcium concentration [Ca²⁺]_i is known to be associated with several diseases in the body including T2DM. Both neutrophils and lymphocytes play active protective roles in host immune response to infection showing impairment in microbicidal functions including phagocytosis and chemotaxis which are calcium-dependent processes. This study evaluated the process of [Ca²⁺]_i mobilization from both neutrophils and lymphocytes taken from blood of both T2DM patients and healthy age-matched control subjects investigating the effect of N-formyl-methionyl-leucyl-phenylalanine (fMLP), thapsigargin (TG), and hydrogen peroxide (H₂O₂) on [Ca²⁺]_i homeostasis. This study employed isolated peripheral blood neutrophils and lymphocytes from 24 T2DM patients and 24 healthy volunteers. Either neutrophils or lymphocytes were stimulated separately with fMLP, TG, or H₂O₂. Induced changes in [Ca²⁺] in both neutrophils and lymphocytes were evaluated using spectrofluorometric methods. Stimulation of human neutrophils and lymphocytes with fMLP, TG, or H₂O₂ in the presence of [Ca²⁺]_o resulted in significant decreases in [Ca²⁺]_i mobilization from T2DM patients compared with healthy controls. These data indicate that neutrophils and lymphocytes from T2DM patients are less responsive to calcium mobilizing agents compared with granulocytes from healthy controls and this is possibly due to the hyperglycemia. The results suggest that agonist-evoked decrease in [Ca²⁺]_i in immune cells might be one of the possible mechanisms of impaired immunity in diabetic patients.     

Is cytoplasmic Ca2+ in lymphocytes elevated in cystic fibrosis

An increased cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) has been implicated in the pathogenesis of cystic fibrosis. We compared the [Ca²⁺]_i levels of normal and cystic fibrosis peripheral blood lymphocytes and Epstein-Barr virus-transformed lymphoblasts using quin 2, an internally trapped indicator. The [Ca²⁺]_i levels of normal and cystic fibrosis cells were not significantly different. The ionophore-releasable intracellular Ca²⁺ stores were also comparable in both types of individual.     

Are intracellular ionic concentrations accessible using fluorescent probes? The example of Mag-indo-1

The study of the physicochemical properties of Mag-indo-1, a fluorescent probe used for intracellular magnesium measurements, has shown that in a biological environment the deprotonated form of this probe is in simultaneous equilibrium with a protonated form, a protein and a magnesium-bound form. The complex emission fluorescence spectrum emitted by a single living cell was analyzed using a computerized method, allowing the evaluation of the Mag-indo-1 to the cellular fluorescence. This approach used to evaluate intracellular Mg²⁺ concentration has also shown the variability of the important participation of protein-bound Mag-indo-1 to the cellular fluorescence. Thus the widely used ratioing method, unable to take into account this variability, cannot afford a reliable evaluation of [Mg²⁺]. Whatever the technique used for investigation (microfluorimetry, flow cytometry, etc.) the evaluation of [Mg²⁺]_i using the fluorescent probe Mag-indo-1 requires a method able to quantify, in complex fluorescence, the fluorescence intensity of the forms involved in the equilibrium with Mg²⁺.Abbreviations [Ca²⁺]₁ intracellular calcium concentration - [Mg²⁺]_i intracellular magnesium concentration     

Lithium induced changes in intracellular free magnesium concentration in isolated rat ventricular myocytes

We have examined the effect of exposing isolated rat ventricular myocytes to lithium while measuring cytosolic free magnesium ([Mg²⁺]_i) and calcium ([Ca²⁺]_i) levels with the fluorescent, ion sensitive probes mag-fura-2 and fura-2. There was a significant rise in [Mg²⁺]_i after a 5 min exposure to a solution in which 50% of the sodium had been replaced by Li⁺, but not when the sodium had been replaced by bis-dimethylammonium (BDA). However, there were significant increases in [Ca²⁺]_i when either Na⁺ substitute was used. The possibility that Li⁺, which enters the cells, interferes with the signal from mag-fura-2 was eliminated as Li⁺ concentrations up to 10 mM had no effect on the dye's fluorescence signal. A possible explanation for these findings is that Li⁺ displaces Mg²⁺ from intracellular binding sites. Having considered the binding constants for Mg²⁺ and Li⁺ to ATP, we conclude that Li⁺ can displace Mg²⁺ from Mg-ATP, thus causing a rise in [Mg²⁺]_i. This work has implications for other studies where Li⁺ is used as a Na⁺ substitute.     

Magnesium homeostasis in cardiac cells

Several aspects of Mg²⁺ homeostasis were investigated in cultured chicken heart cells using the fluorescent Mg²⁺ indicator, FURAPTRA. The concentration of cytosolic Mg²⁺ ([Mg²⁺]_i) is 0.48 ± 0.03 mM (n = 31). To test whether a putative Na/Mg exchange mechanism controls [Mg²⁺]_i below electrochemical equilibrium, we manipulated the Na⁺ gradient and assessed the effects on [Mg²⁺]_i. When extracellular Na⁺ was removed, [Mg²⁺]_i increased; this increase was not altered in Mg-free solutions, but was attenuated in Ca-free solutions. A similar increase in [Mg²⁺]_i, which was dependent upon extracellular Ca²⁺, was observed when intracellular Na⁺ was raised by inhibiting the Na/K pump with ouabain. These results do not provide evidence for Na/Mg exchange in heart cells, but they suggest that Ca²⁺ can modulate [Mg²⁺]_i. In addition, removing extracellular Na⁺ caused a decrease in intracellular pH (pH_i), as measured by pH-sensitive microelectrodes, and this acidification was attenuated when Cat+ was also removed from the solution. These results suggest that Ca²⁺ and H⁺ interact intracellularly. Since changes in the Na⁺ gradient can also alter pH_i, we questioned whether pH can modulate [Mg²⁺]_i. pH_i was manipulated by the NH₄Cl prepulse method. NH₄ ⁺-evoked changes in pH_i, as measured by the fluorescent indicator BCECF, were accompanied by opposite changes in [Mg²⁺]_i; [Mg²⁺]_i changed by –0.16 mM/unit pH. These NH₄ ⁺-evoked changes in [Mg²⁺]_i were not caused by movements of Mg₂₊ or Ca²⁺ across the sarcolemma or by changes in cytosolic Ca²⁺. Additionally, pH_i was manipulated by changing extracellular pH (pH_o). When pH_o was decreased from 7.4 to 6.3, pH_i decreased by 0.64 units and [Mg₂₊]_i increased by 0.12 mM; in contrast, when pH_o was raised from 7.4 to 8.3, pH_i increased by 0.6 units and [Mg²⁺]_i did not change significantly. The results of our investigations suggest that Ca ²⁺ and H⁺ can modulate [Mg²⁺]_i, probably by affecting cytosolic Mg²⁺ binding and/or subcellular Mg²⁺ transport and that such redistribution of intracellular Mg²⁺ may play an important role in Mg²⁺ homeostasis in cardiac cells.     

Influence of age,sex, and dietary restriction on intracellular free calcium responses of CD4+ lymphocytes in rhesus monkeys (Macaca mulatta)

The influence of aging and dietary restriction on increase in intracellular free calcium ([Ca²⁺]_i) of CD4⁺ lymphocytes from Macaca mulatta was examined after stimulation with anti-CD3 mAb. We used a flow cytometric assay with the dye indo-1 and either direct or reciprocal immunofluorescent staining to dientify CD4⁺ cells. After stimulation with anti-CD3 mAb, intracellular free calcium responses were reduced in CD4⁺ lymphocytes from old male and female ad libitum fed monkeys compared to young and adult male or female monkeys. Old female monkeys had significantly lower [Ca²⁺]_i than did old male monkeys. The reduced responses were in part related to a decreased percentage of responding cells. Dietary restrition of males over a four-year period did not alter [Ca²⁺]_i response compared to ad libitum fed male monkeys. Female monkeys of all ages (which were restricted only for four months) also had similar [Ca²⁺]_i responses to ad libitum fed controls. Our data suggest that age-related changes in [Ca²⁺]_i responses are similar between humans and M. mulatta, and that over these intervals, no effects of caloric restrictions can be detected. © 1995 Wiley-Liss, Inc.     

Monitoring Receptor Mediated Regulation of Cytosolic Calcium in Single Pituitary Cells by Dual Excitation Microfluorimetry

Abstract

Receptor-mediated alterations in the cytosolic free calcium concentration, [Ca²⁺]_i are monitored with the intracellular fluorescent calcium probe fura 2 by dual excitation microfluori-metry. The calcium dependence on the excitation spectrum of fura 2 allows us to choose two wavelengths, λ₁ and λ₂, at which an increase in [Ca²⁺]_i causes either a rise or a fall in fluorescence; the ratio of fluorescence at λ₁ and λ₂ (R =Fλ₁/Fλ₂) is a measure of [Ca²⁺]_i. It appears essential for such measurements that the alteration between the two excitation wavelengths is done rapidly, to allow us to distinguish between effects on [Ca²⁺]_i and other effects on fluorescence. In addition, specific problems relating to the calibration of fura 2 measurements, such as its relative insensitivity at basal [Ca²⁺]_i, the role of intracellular viscosity on fura 2 fluorescence, and the difficulties encountered in establishing calibration constants have to be appreciated. In spite of these potential drawbacks, it appears that monitoring receptor-mediated [Ca²⁺]_i regulation in single cells will prove essential for the further comprehension of stimulus-secretion coupling in pituitary and probably many other cell types.     

Competitive blockage of the sodium channel by intracellular magnesium ions in central mammalian neurones

The aim of this study was to determine from macroscopic current analysis how intracellular magnesium ions, Mg _i ²⁺ , interfere with sodium channels of mammalian neurones. It is reported here that permeation across the sodium channel is voltage- and concentration-dependently reduced by Mg _i ²⁺ . This results in a general reduction of sodium membrane conductance and an outward sodium peak current at large positive potentials. 30 mM Mg _i ²⁺ leads to a negative shift of voltage dependence of sodium channel gating parameters, probably due to the surface potential change of the membrane. This shift alone is, however, insufficient to explain the reduction of outward sodium currents. The blockage by Mg _i ²⁺ is decreased upon increasing intracellular or extracellular Na⁺ concentration, which suggests that Mg?' interferes with sodium permeation by competitively occupying sodium channels. Using a kinetic model to describe the sodium permeation, the dissociation constant (at zero membrane potential) of Mg _i ²⁺ for the sodium channel has been calculated to be 8.65 ± 1.51 mM, with its binding site located at 0.26 ± 0.05 electrical distance from the inner membrane. This dissociation constant is smaller than that of Na _i ⁺, which is 83.76 ± 7.60 mM with its binding site located at 0.75 ± 0.23. The low dissociation constant of Mg _i ²⁺ reflects its high affinity for the sodium channel.     

Sodium block and depolarization diminish P2Z-dependent Caentry in human B lymphocytes

Despite a high Ca²⁺-permeability of the P2Z receptor in human B lymphocytes, extracellular ATP⁴has only a minor effect on global [Ca²⁺]_i. The aim of this study was to reveal the mechanisms responsible for this discrepancy. We investigated the relationship between ATP⁴⁻-application, Ca_i²-response, membrane current and membrane potential in two human B cell lines and in human tonsillar B cells. This was achieved by a combination of FACS- and voltage clamp measurements and the usage of appropriate voltage- and Ca²-sensitive fluorescent dyes. ATP⁴-induced changes in whole-cell current and [Ca²]_iwere blocked by extracellular as well as intracellular Na⁺. Under current clamp conditions, ATP⁴⁻-induced Na⁺-entry diminished the Ca²⁺entry via reduction of the driving force. A substantial increase in [Ca²⁺]_iinduced by ATP⁴⁻was only observed in Na⁺-free solutions.The pathway of signal transduction activated by ATP⁴via P2Z receptor of human B lymphocytes under physiological conditions seems not to operate by an increase in the global intracellular Ca₂-concentration, but rather by the depolarization of the cell membrane as a result of the Na⁺-influx.     

Open-channel block of Na+ channels by intracellular Mg2+

1. Macroscopic and single-channel currents through several types of cloned rat brain Na⁺ channels, expressed in Xenopus oocytes, were measured using the patch-clamp technique. 2. For all cloned channel types and for endogenous Na⁺ channels in chromaffin cells, intracellular Mg²⁺ blocks outward currents in a voltage-dependent manner similar to that in rat brain type II Na⁺ channel (Pusch et al. 1989). 3. A sodium-channel mutant (cZ-2) with long single-channel open times was used to examine the voltage-dependent reduction of single-channel outward current amplitudes by intracellular Mg²⁺. This reduction could be described by a simple blocking mechanism with half-maximal blockage at 0 mV in 1.8 mM intracellular Mg²⁺ and a voltage-dependence of e-fold per 39 mV (in 125 mM [Na] _i ); this corresponds to a binding-site at an electrical distance of 0.32 from the inside of the membrane. 4. At low Mg²⁺ concentrations and high voltages, the open-channel current variance is significantly elevated with respect to zero [Mg] _i . This indicates that Mg²⁺ acts as a fast blocker rather than gradually decreasing current, e.g. by screening of surface charges. Analysis of the open-channel variance yielded estimates of the block and unblock rate constants, which are of the order of 2 · 10⁸ M^–1 s^–1 and 3.6 · 10⁵ s^–1 at 0 mV for the mutant cZ-2. 5. A quantitative analysis of tail-currents of wild-type 11 channels showed that the apparent affinity for intracellular Mg²⁺ strongly depends on [Na] _i . This effect could be explained in terms of a multi-ion pore model. 6. Simulated action potentials, calculated on the basis of the Hodgkin-Huxley theory, are significantly reduced in their amplitude and delayed in their onset by postulating Mg²⁺ block at physiological levels of [Mg] _i .abbreviations [Na]_i intracellular Na⁺ concentration - [K] _i intracellular K⁺ concentration - [Mg] _i intracellular Mg²⁺ concentration - HEPES N-2-hydroxylethyl piperazine-N-2-ethanesulfonic acid - EGTA ethyleneglycol-bis-[\-amino-ethyl ether] N,N-tetra acetic acid - TEA tetraethylammonium     

Effect of thapsigargin on cytoplasmic Ca2+ and proliferation of human lymphocytes in relations to AIDS

The tumuour-promoting sesquiterpene lactone, thapsigargin, induced a dose-dependent increase of the cytoplasmic Ca²⁺ concentrations ([Ca²⁺]_i) in human lymphocytes from a resting level between 100 and 150 nM up to about 1 μM. Half-maximum response was found at about 1 nM of thapsigargin, full response at 100 nM. The effect of thapsigargin on [Ca²⁺], expected that of phytohaemagglutinin (PHA) which raised [Ca²⁺]_i to maximum 300 nM. In combination with phorbol 12-myristate 13-acetate (PMA), thapsigargin stimulated the proliferation of normal lymphocytes to the same extent as did PHA, whereas the thapsigargin /PMA treatment could not restore the defective proliferation of AIDS lymphocytes in spite of the increased [Ca²⁺]_i. Thapsigargin or PMA added separately had no stimulatory effects on cell profileration. The thapsigargin/PMA treatment caused an increase in interleukin-2 (IL-2) production of the lymphocytes, which was much higher than that caused by the PHA treatment, even in AIDS lymphocytes. Moreover, the thapsigargin/PMA treatment stimulated the expression of the IL-2 receptors on both normal and AIDS lymphocytes, similar to the effect of PHA. It is concluded that thapsigargin exerts its effects on lymphocyte proliferation by increasing [Ca²⁺]_i, and that the general defect of AIDS lymphocytes, rather than being ascribed to the initiating signal systems, is associated with later events related to DNA synthesis and proliferation.     

Effect of magnesium on calcium efflux in dialyzed squid axon

The Ca efflux mechanism located in the axolemma of the tropical squid Doritheutis plei is shown to be affected by the concentration of intracellular Mg (Mg_i). The removal of all of the Mg from, the experimental preparation causes an increase in Ca efflux. This effect seems to be more pronounced at low levels of internal ionized calcium and high levels of internal Na.     

Control of free cytoplasmic calcium by intracellular pH in rat lymphocytes

Activation of Na⁺-H⁺ exchange in rat thymocytes was found to be followed by an increase in free cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i). We determined whether the change in [Ca²⁺]_i was secondary to the uptake of Na⁺, or to the cytoplasmic alkalinization that result from activation of the antiport. Increasing intracellular [Na⁺] by treating the cells with ouabain or gramicidin failed to affect [Ca²⁺]_i. In contrast, procedures that increased the cytoplasmic pH, such as addition of monensin or NH₃, significantly elevated [Ca²⁺]_i. These results suggest an important role of cytoplasmic pH in the control of [Ca²⁺]_i in lymphocytes.     

Modulation of Ca2+ Influx in Leech Retzius Neurons. I. Effect of Extracellular pH

We investigated the cytosolic free Ca²⁺ concentration ([Ca²⁺]_i) of leech Retzius neurons in situ while varying the extracellular and intracellular pH as well as the extracellular ionic strength. Changing these parameters had no significant effect on [Ca²⁺]_i when the membrane potential of the cells was close to its resting value. However, when the cells were depolarized by raising the extracellular K⁺ concentration or by applying the glutamatergic agonist kainate, extracellular pH and ionic strength markedly affected [Ca²⁺]_i, whereas intracellular pH changes appeared to have virtually no effect. An extracellular acidification decreased [Ca²⁺]_i, while alkalinization or reduction of the ionic strength increased it. Correspondingly, [Ca²⁺]_i also increased when the kainate-induced extracellular acidification was reduced by raising the pH-buffering capacity. At low extracellular pH, the membrane potential to which the cells must be depolarized to evoke a detectable [Ca²⁺]_i increase was shifted to more positive values, and it moved to more negative values at high pH. We conclude that in leech Retzius neurons extracellular pH, but not intracellular pH, affects [Ca²⁺]_i by modulating Ca²⁺ influx through voltage-dependent Ca²⁺ channels. The results suggest that this modulation is mediated primarily by shifts in the surface potential at the extracellular side of the plasma membrane. Received: 23 January 2001/Revised: 15 June 2001     

Basic fibroblast growth factor increases intracellular magnesium concentration through the specific signaling pathways

Basic fibroblast growth factor (bFGF) plays an important role in angiogenesis. However, the underlying mechanisms are not clear. Mg²⁺ is the most abundant intracellular divalent cation in the body and plays critical roles in many cell functions. We investigated the effect of bFGF on the intracellular Mg²⁺ concentration ([Mg²⁺]_i) in human umbilical vein endothelial cells (HUVECs). bFGF increased [Mg²⁺]_i in a dose-dependent manner, independent of extracellular Mg²⁺. This bFGF-induced [Mg²⁺]_i increase was blocked by tyrosine kinase inhibitors (tyrphostin A-23 and genistein), phosphatidylinositol 3-kinase (PI3K) inhibitors (wortmannin and LY294002) and a phospholipase Cγ (PLCγ) inhibitor (U73122). In contrast, mitogen-activated protein kinase inhibitors (SB202190 and PD98059) did not affect the bFGF-induced [Mg²⁺]_i increase. These results suggest that bFGF increases the [Mg²⁺]_i from the intracellular Mg²⁺ stores through the tyrosine kinase/PI3K/PLCγ-dependent signaling pathways.     

Glucose-induced alterations of intracellular ionized magnesium in human lymphocytes

The intracellular ionic content of human erythrocytes may be altered by hyperglycaemia. Despite this, very little is known about the cellular mechanisms linking glucose and cellular magnesium homeostasis. We measured intracellular ionized magnesium in human lymphocytes, by means of a fluorimetric technique, total intracellular magnesium by means of atomic absorption spectrophotometry and intracellular ATP by means of HPLC. The incubation of lymphocytes with D-glucose in the absence of insulin was followed by a significant decrease in intracellular ionized magnesium; this effect did not occur when the cells were incubated with L-glucose. The effect of glucose on intracellular ionized magnesium was blocked by amphotericin B and the EC(50) of the effect of glucose on intracellular ionized magnesium was about 5 mmol/l of glucose. The increase of intracellular ionized magnesium in cells incubated in the absence of glucose was followed by a decrease in intracellular ATP. In a Na(+)-free medium the decrease of intracellular ionized magnesium in the presence of glucose was still present and the incubation of lymphocytes with glucose did not modify total intralymphocyte magnesium. By selective permeabilization of cell membranes, we established that glucose could not increase compartmentalized intracellular ionized magnesium. Our data supports the hypothesis that glucose per se induces a substantial decrease in intracellular ionized magnesium, which is probably due to an augmented binding of intracellular ionized magnesium to cellular ATP.     

Direct visualization of intracellular calcium in rat osteoblasts by energy-filtering transmission electron microscopy

Osteoblasts are the highly specialized bone cells responsible for matrix mineralization. Mineralization is a complex, incompletely understood, process involving intracellular calcium homeostasis. Rapid changes in ionized calcium concentration ([Ca²⁺]_i) occur in these cells, but the intracellular distribution of total calcium, which may be involved in matrix mineralization, remains unknown. We have therefore investigated the distribution of total calcium in osteoblasts either ex vivo from rapidly mineralizing neonatal rat bones or in the same cells cultured to confluence before they had entered the mineralization phase, and without stimulation for mineralized matrix formation. All cells were examined bone-untreated (controls) or following the addition of the ionophore ionomycin that induced a large and sustained increase in [Ca²⁺]_i. Cryomethods, quick-freezing and freeze-drying, and OsO₄ vapor fixation were employed to preserve the original calcium distribution, and the preservation was verified by secondary ion mass spectrometry (SIMS). Intracellular calcium distribution was identified by energy-filtering transmission electron microscopy (EELS). Scarce calcium signals were recorded from all osteoblasts maintained in buffer (controls). Ionomycin addition resulted in the accumulation of calcium in mitochondria, and more calcium was stored in the mitochondria of osteoblasts involved in mineralization than in those of osteoblasts before mineralization. Moreover, in the former, strong calcium signals were recorded around the junctions between mitochondria and the endoplasmic reticulum. Thus EELS allowed to obtain high-resolution total calcium maps in defined intracellular structures, but only at elevated calcium levels.