Quantification and Localization of Intracellular Free Mg2+ in Bovine Chromaffin Cells

Magnesium is an essential element for all living systems. The quantification of free intracellular Mg2+ concentration ([Mg2+]i) is of utmost importance since changes in its basal value may be an indication of different pathologies due to abnormalities of Mg2+ metabolism. In this work we used 31P NMR and fluorescence spectroscopy to determine the resting [Mg2+]i in bovine chromaffin cells, a neuron-like cellular model, as well as confocal laser scanning microscopy to study the free Mg2+ spatial distribution in these cells. 31P NMR spectroscopy did not prove to be effective for the determination of [Mg2+]i in this particular case due to some special morphological and physiological properties of this cell type. A basal [Mg2+]i value of 0.551 ± 0.008 mM was found for these cells using fluorescence spectroscopy and the Mg2+-sensitive probe furaptra; this value falls in the concentration range reported in the literature for neurons from different sources. This technique proved to be an accurate and sensitive tool to determine the [Mg2+]i. lntraceilular free Mg2+ seems to be essentially localized in the nucleus and around it, as shown by confocal microscopy with the Mg2+-sensitive probe Magnesium Green. It was not possible to derive any conclusion about free Mg2+ localization inside the chromaffin granules and/or in the cytoplasm due to the lack of sufficient spatial resolution and to probe compartmentalization.


INTRODUCTION
The importance of the extracellular and intracellular magnesium ion (Mg2+) has become gradually recognized during the last century. Nowadays it is well known that an altered metabolism of Mg 2+ is involved in several pathologies. This cation is essential for all living systems, being the most abundant in tissues, Vol. 9, Nos. I-2, 2002 Quant([cation and Localization of lntraceHular Free Mg 2 in Bovine Chroma.ffin Cells exceeded only by the potassium ion (K+)/1/. Mg 2+ plays a vital role in several cellular regulatory processes, but it is unlikely that it can have a "trigger" function like the calcium ion (Ca2+) does, due to its concentrations inside and outside the cells, and due to its chemical properties. However, slow and small magnitude changes in Mg 2+ concentration can be important in the fine control and coordination of cellular activity/2/. Mg 2+ is involved in the synthesis of DNA and RNA, as well as in the maintenance of their conformation/3/. This cation has also been implicated in the control of membrane fluidity and permeability /4, 5/and has been directly associated with the secretion of hormones, like insulin/6/and prolactin/7/. Many of the actions of Mg 2+ result from its role as a co-factor of a wide range of enzymes, in the form of Mg-ATP/ADP or Mg-GTP/GDP complexes /2/. Mg 2+ activates virtually all the enzymes involved in the metabolism of phosphorylated compounds, as well as many enzymes of the glycolytic and tricarboxylic acid pathways/8/. Additionally, Mg 2+ is necessary for the optimal performance of the Na+/K+-ATPase and Ca 2+ pump/9/and it affects different transport systems, such as the Na+/K+/CI co-transport, the Na+/H + and CI" /HCO3" exchange/10/and several ion channels/11/. To help understand the role of intracellular Mg2+, its homeostasis and regulation, accurate measurements of total and free intracellular magnesium concentrations ([Mg+]i) were developed. The total intracellular magnesium concentration ([Mg2+]-) can be determined by atomic absorption spectrophotometry (AAS), inductively-coupled plasma atomic emission spectrophotometry, thin layer and ion chromatographies/12, 13, 14/. These methods require submitting the sample to several destructive steps before measuring the [Mg2+]r, which implies that there may be a source of sample contamination. Also, the invasive nature of these techniques may lead to errors in the study of Mg 2+ transport due to non-specific binding of Mg 2+ to cell membranes and other components, as well as additional ion transport during sample preparation/15/. However several methods can be used to determine [Mg2+] in intact cells, such as Mg+-sensitive microelectrodes, nuclear magnetic resonance (NMR) spectroscopy, fluorescence techniques and coupled assays using Mg2+-dependent enzymes /14, 16, 17, 18, 19/. The improvement of these methods to determine both total and free intracellular Mg 2+ concentrations indicates that only 5-10% of the total Mg 2+ is free, the remaining being bound to highly charged anionic ligands such as ATP, ADP, RNA, polyphosphates, proteins and citrate/20,21/. A substantial amount of the total Mg 2+ is distributed in the nucleus, endoplasmic reticulum, mitochondria and cytoplasm/16/. Bovine chromaffin cells, obtained from bovine adrenal medulla, are a good neuronal model because they are endocrine and sympathetic neuron-like cells, originated from the same precursor cells as those of the sympathetic ganglion cells, and constitute a convenient model to study a variety of neurological disorders L. P. ,t Imltezinho el aL MetaLBased Drugs /39,40/. These cells have a higher degree of intracellular complexity when compared with other types of cells; they contain chromaffin granules, which are highly heterogeneous organelles with an unusually high ionic strength and low pH value (-5.5/41/), a high concentration of ATP complexed with catecholamines, chromogranin A and some ions/42/.
In this work we used two different techniques to determine [Mg2+]i (3p NMR and fluorescence spectroscopies) in bovine chromaffin cells, and another one to study the spatial distribution of Mg 2+ inside these cells (confocal microscopy). The  Isolation and culture of bovine chromaffin cells Bovine adrenal glands were obtained from a local slaughterhouse and transported to the laboratory on ice. Bovine chromaffin cells were isolated from bovine adrenal glands and purified on a Percoll gradient as previously described/43/. Cells were then cultured in a 1:1 mixture DMEM/F-12 (1.56%) medium with 15 mM HEPES and 26 mM NaHCO3, supplemented with 5% of heat-inactivated fetal calf serum, penicillin (100 units/mL), streptomycin (100 lag/mL) and amphoteriein B (0.25 lag/mL), at 37 C, in a humidified CO2 (5%) and air (95%) atmosphere.
The cells were cultured up to a density of million cells/mL in 100 mm Petri dishes and maintained in culture for 3 days before the NMR experiments.
For the fluorescence experiments, the cell preparation was further purified by a Urografin gradient as described before/44, 45/and were used 2 days after plating. The cells were plated at a density of 0.8 million cells/era on square cover-slips (lcm2) previously coated with poly-L-lysine when fluorescence spectroscopy l'oi. 9, Nos. 1-2. 2002 Quant(ficution and Localization of lntracelhdar Free Mg: in Bovine Chroma.[fin Cells was used, and at a density of 0.15 million cells/mL/well on glass cover-slips (16 mm diameter), also coated with poly-L-lysine, in 12-well plates for confocal microscopy experiments.
3p NMR spectroscopy i) NMR sample preparation After 3 days in culture, bovine chromaffin cells were resuspended and centrifuged at 115 g (800 rpm) during 8 min at 25 C (Sigma 3K10). The pellet was resuspended in culture medium up to a volume of 500 laL.
Bovine chromaffin cells were immobilized in agarose gel threads, placed in a 10 mm NMR tube and perfused with oxygenated culture medium(5% CO2 95 % O2), at 37 C, pH 7.35. The immobilization was performed by mixing the 500 .tL of cell suspension (50 to 75 million cells) with 500 },tL of Krebs medium (in raM: NaCI 140, KCI 5, CaC12 2, MgC12 1, glucose 10, HEPES 20, pH 7.35) containing 2% of low-gelling temperature agarose, in a 1:1 proportion (final agarose concentration of 1%), at 37 C. The threads were formed by passing this cell mixture through a Teflon tubing with 0.5 mm internal diameter, partially submersed in ice. Once it was passed through the iced portion of the tubing, the mixture solidified and threads with 0.5 mm diameter with entrapped cells were formed into the 10 mm NMR tube. The immobilized cells were continuously perfused at approximately mL/min with culture medium, pH 7.35. This procedure ensures the maintenance of cell viability throughout the NMR experiments, as already demonstrated with another type of cells/46, 47/and with chromaffin cells in our lab (data not shown).

ii) NMR experiments
Proton decoupled 3P NMR spectra were acquired at 202. 3 MHz, 37  The spatial distribution of intracellular free magnesium ion (Mg2+i) was observed using the Mg 2+ specific fluorescent probe Magnesium Green, instead of furaptra, because it has the physical properties adequate to the technical characteristics of the available confocal equipment. This indicator exhibits a higher affinity for Mg 2+ (Kd ---1.0 raM) than does furaptra (Kd -1.9 raM) or mag-indo-1 (Kd --.2.7 mM); this indicator also binds Ca 2+ with moderate affinity (Kd for Ca 2+ in the absence of Mg 2+ ---6 IM), at 22C/55/. Upon binding Mg2+, Magnesium Green exhibits an increase in fluorescence emission intensity without a shift in wavelength/55/.
We used a MRC600 confocal imaging system (BioRad laboratories, Milan, Italy) linked to a Nikon Optiphot-2 fluorescence microscope. A krypton/argon mixed laser was used in combination with a 488 nm band pass filter (excitation) and a 585 long-pass filter (emission). The cells, adherent to coverslips, were incubated with Krebs medium supplemented with 1% BSA and 5 tM of the cell-permeant acetoxymethyl ester form of this Mg 2+ indicator (Magnesium Green-AMTM) during 45 min, in a humidified CO2 (5%) and air (95%) atmosphere, at 37 C. After this loading period, the medium was replaced by fresh Krebs medium containing 1% BSA and the cells were incubated for an additional period of 15 rain, under the same atmosphere conditions. Then the coverslips with the attached cells were washed three times with a Krebs medium containing 0.2% BSA and placed in a special perfusion camera in the confocal apparatus. This camera was specially made in order to allow the continuous perfusion of the cells with Krebs medium, at 37 C, during the time course of the confocal microscopy experiments, ensuring the maintenance of cellular viability. The flux rate of the medium surrounding the cells inside the camera was set to mL/min with a peristaltic pump (MasterFlex(R), Cole-Parmer Instrument Co., Illinois, USA). The images were acquired with the excitation and emission wavelengths fixed to 488 nm and 585 nm, respectively. Fluorescence images were treated using confocal assistant and PainShopPro sofiwares.

RESULTS AND DISCUSSION
P NMR experiments 3p NMR spectroscopy was used in order to determine the [MgZ+]i and also to check the cell viability. The spectra were acquired over time from chromaffin cells immobilised in agarose gel threads and perfused with the oxygenated DMEM/F-12 medium (as described in the Methods section) (Fig. 1).
The "P NMR chemical shift difference between the P, and P NMR resonances of ATP (AS,) is Py-ATP; VI-Granular P,.ATP; VII-Cytosolie P-ATP; VIII-Granular P-ATP.
in the literature/57/, the P, resonance of ATPeyt is hidden under the Pa signal of ATPg,.a,,, preventing an accurate measurement of A5,ts of ATPyt, thus not allowing the exact determination of free intraeellular Mg / concentration in the cytosol, using equations (1) and (2). Moreover, as the internal pH of the vesicles is low (when compared with the pH value of other intracellular organelles) and because of the association of ATP with the positively charged catecholamines within the granules, the P, and P!s NMR chemical shifts of ATPg,.,, are unusual/58, 59/. In consequence A, is higher in the chromaffin vesicle (tle value obtained by us was l.08 +_ 0.02 ppm (n=4)) than the theoretically expected value for ATP in the absence of Mg 2+ (10.82 ppm,/50, 51/), leading to negative [Mg2+]i values when equations (l) and (2) are used.
Therefore, one may conclude that this technique, which has been widely used with biomolecules/50, 60,

Confocal microscopy experiments
Confocal microscopy and the fluorescent indicator Magnesium Green were used to study the spatial distribution of the intracellular free Mg 2/ in bovine chromaffin cells. The image obtained with the cells previously loaded with Magnesium Green-AM TM and continuously perfused with Krebs medium (37 C) is shown in Fig. 2. The distribution of the probe inside the nucleus and in the cytoplasm, which is reflected by the probe emission fluorescence intensity observed in the cells' image, depends on the degree of incorporation of the fluorescent probe inside them. The image shows that the emission fluorescence intensity of the Magnesium Green-Mg 1+ complex is mainly located in the nucleus of the cells and heterogeneously Iocalised in the cytoplasm mostly around the nucleus, probably in the endoplasmic reticulum. The high fluorescence intensity in the nucleus can be due to a high free Mg 2+ concentration inside it and/or to an accumulation of the probe in this organelle. It is known that compartmentalization into cellular compartments and binding to proteins can be a problem in the use of fluorescent indicators in cells/65/.
The image of the cells does not have enough resolution to decide if the residual fluorescence from the peripheral part of the cytoplasm comes from the Magnesium Green-Mg 2+ complex inside the granules or from other organelles. Analysis of Fig. 2 does not allow one to draw any conclusion about the presence and distribution of free Mg 2+ in the granules.
It is also possible to observe that in the cell membrane and close to it the fluorescence emission is due to the fluorescence of the uncomplexed probe, indicating that no free Mg 2+ is located in this part of the cell, which is not surprising since Mg 2+ is usually bound to the negatively charged binding sites in the membrane (e.g. phospholipids, proteins). L. P. A.hntezinho et ai.

CONCLUSIONS
In this work the determination of [Mg+]i and its spatial localization in bovine chromaffin cells were carried out using three different methods.
3p NMR and fluorescence spectroscopic techniques were used in order to quantify the intracellular free Mg 2+ in these cells. The method developed by Gupta and his collaborators/48, 49/to determine the [Mg2+]i by P NMR proved to be useless in this cell type, although it has been successfully used in biomolecules/50, 60, 61, 62/and other cell types/47, 63/. This is due to the particular characteristics of these cells which contain two different pools of ATP, granular and cytosolic pools; the overlap of the P NMR resonances of ATPyt and ATPg,.,,, prevented the determination of [Mg2+]i in the cytosol, while specific ATPg,.an interactions and the unusually low intragranular pH value and high ionic strength did not allow the quantification of the intragranular free Mg 2+ using this method.
Fluorescence spectroscopy with the Mg2+-sensitive fluorescent probe furaptra proved to be a more accurate and sensitive technique to determine [Mg2+]i in these cells, giving similar values to the ones obtained for other cell types, as described in the literature.
Confocal microscopy using the Mg2+-sensitive fluorescent probe Magnesium-Green proved to be a useful tool to visualize the spatial distribution of Mg 2+ inside bovine chromaffin cells. However, due to some technical inherent limitations such as probe compartmentalization and insufficient spatial resolution, it was not possible to draw further conclusions about free Mg 2+ distribution in these cells. I'oI. 9, Nos. [1][2]2002 Quanti)qcation and Localization of htracellular Free Mg: in Bovine Chromq[]Tn Cells