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Am J Physiol Gastrointest Liver Physiol 290: G56-G65, 2006. First published August 18, 2005; doi:10.1152/ajpgi.00014.2005
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MUCOSAL BIOLOGY

Characterization of the Na+-dependent Mg2+ transport in sheep ruminal epithelial cells

Monika Schweigel, Hi-Sung Park, Benjamin Etschmann, and Holger Martens

Department of Veterinary Physiology, Free University of Berlin, Berlin, Germany

Submitted 12 January 2005 ; accepted in final form 12 August 2005


   ABSTRACT
TOP
 ABSTRACT
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This study examines the routes by which Mg2+ leaves cultured ovine ruminal epithelial cells (REC). Mg2+-loaded (6 mM) REC were incubated in completely Mg2+-free solutions with varying Na+ concentrations, and the Mg2+ extrusion rate was calculated from the increase of the Mg2+ concentration in the incubation medium determined with the aid of the fluorescent probe mag-fura 2 (Na+ salt). In other experiments, REC were also studied for the intracellular free Mg2+ concentration ([Mg2+]i; using mag-fura 2), the intracellular Na+ concentration (using Na+-binding benzofuran isophthalate), the intracellular cAMP concentration ([cAMP]i; using an enzyme-linked immunoassay), and Na+/Mg2+ exchanger existence [using a monoclonal antibody (mAb) raised against the porcine red blood cell Na+/Mg2+ exchanger]. Mg2+-loaded REC show a Mg2+ efflux that was strictly dependent on extracellular Na+. The Mg2+ extrusion rate increased from 0.018 ± 0.009 in a Na+-free medium to 0.73 ± 0.3 mM·l cells–1·min–1 in a 145 mM Na+ medium and relates to extracellular Na+ concentration ([Na+]e) according to a typical saturation kinetic (Km value for [Na+]e = 24 mM; maximal velocity = 11 mM·l cells–1·min–1). Mg2+ efflux was reduced by imipramine (48%) and increased after application of dibutyryl-cAMP (55%) or PGE2 (17%). These effects are completely abolished in Na+-free media. Furthermore, an elevation of [cAMP]i led to an [Mg2+]i decrease that amounted to 375 ± 105 µM. The anti-Na+/Mg2+ exchanger mAb inhibits Mg2+ extrusion; moreover, it detects a specific 70-kDa immunoreactive band in protein lysates of ovine REC. The data clearly demonstrate that a Na+/Mg2+ exchanger is existent in the cell membrane of REC. The transport protein is the main pathway (97%) for Mg2+ extrusion and can be assumed to play a considerable role in the process of Mg2+ absorption as well as the maintenance of the cellular Mg2+ homeodynamics.

sheep rumen; magnesium efflux; sodium/magnesium exchanger; cobalt(III)hexaammine; mag-fura 2

IN RUMINATING ANIMALS, most of the required Mg2+ is absorbed from the forestomachs by active, transcellular mechanisms. It is therefore not surprising that ruminal epithelial cells (REC) are equipped with effective Mg2+ uptake mechanisms. These include an ion channel (34) and an Mg2+-Cl– cotransporter (35), which are both well characterized, at least at a functional level. In contrast, only few data are available regarding the Mg2+ efflux mechanism(s). In one study, experiments have been carried out with isolated rumen epithelium, showing that a low serosal Na+ concentration led to a significant reduction of mucosal-to-serosal Mg2+ fluxes and a slight increase in serosal-to-mucosal Mg2+ fluxes (25). It was suggested that Mg2+ absorption across rumen epithelium involves an Na+-dependent extrusion mechanism. Some further evidence for the existence of an Na+/Mg2+ exchanger in the cell membrane of REC comes from our finding of an imipramine-sensitive Mg2+ uptake mechanism, which was observed after reversing the transmembrane Na+ gradient [intracellular Na+ concentration ([Na+]i) > extracellular Na+ concentration ([Na+]e); see Refs. 36]. It was the aim of the present study to explore the routes by which Mg2+ leaves REC and in particular to clarify the role of a Na+/Mg2+ exchanger in this process.

To this purpose, we have measured the Mg2+ efflux from Mg2+-loaded isolated REC in a completely Mg2+-free incubation medium. The increase of the extracellular Mg2+ concentration ([Mg2+]e) was determined with the aid of the non-membrane-permeable fluorescent probe mag-fura 2 (Na+ salt). Mg2+ efflux was measured under basal conditions, after changing the [Na+]e and after stimulation with cAMP and extracellular PGE2. Transport inhibitors (imipramine and cobalt(III)hexaammine [Co(III)hex]) have been used to differentiate between Na+/Mg2+ exchanger-mediated Mg2+ efflux and other possible ways of Mg2+ extrusion. The data clearly show that a Na+/Mg2+ exchanger is the main Mg2+ efflux mechanism in sheep REC, responsible for ~98% of total Mg2+ release. Moreover, we present direct molecular evidence for the expression of a Na+/Mg2+ exchanger in ovine REC.

MATERIALS AND METHODS

Materials. Medium 199, trypsin, glutamine, antibiotics (gentamycin, nystatin, and kanamycin), and FCS were purchased from Sigma (St. Louis, MO). Dulbecco's PBS (DPBS) and collagen were obtained from Biochrom (Berlin, Germany). Mag-fura 2-AM, Mag-fura 2 (Na+ salt), Na+-binding benzofuran isopthalate (SBFI)-AM, A-23187 (calcimycin), and pluronic acid were from Molecular Probes (Eugene, OR). All other chemicals were purchased from Sigma.

Cell culture. Primary cultures of REC were prepared as described by Galfi et al. (13). Briefly, REC were isolated by fractional trypsination and grown in medium 199 containing 10% FCS, 1.36 mM glutamine, 20 mM HEPES, and antibiotics (50 mg/l gentamycin, 100 mg/l kanamycin) in an atmosphere of humidified air-5% CO2 at 38°C. Experiments were performed between 6 and 12 days after seeding.

Solutions for measuring the free intracellular Mg2+ concentration and [Na+]i. [Na+]i and/or intracellular Mg2+ concentration ([Mg2+]i) were measured in either a high-Na+ solution containing (in mM) 100 NaCl, 45 N-methyl-D-glucamine (NMDG)-Cl, 5 KCl, 1 CaCl2, 2 MgCl2, 10 HEPES, and 5 glucose, pH 7.4 or in a custom-made (Biochrom) Mg2+- and Na+-free PBS. To the latter, Mg2+ was added as appropriate before starting the experiment.

Solutions for measuring the Mg2+ efflux. All efflux solutions used were custom-made by Biochrom (Germany). Particularly, a completely Mg2+-free medium 199 (no. F0633, without Ca2+/Mg2+ and phenol red) and a completely Mg2+- and Na+-free PBS (no. 1825, but without Ca2+/Mg2+ and the Na+ substituted with NMDG) were produced. Experimental solutions with varying Na+ concentrations ([Na+]) were then prepared by diluting the Mg2+-free, 145 mM Na+-medium 199 with appropriate volumes of an Na+- and Mg2+-free PBS.

Solutions for Mg2+ loading. The high-K+ loading solution contained (in mM) 130 potassium gluconate, 15 KCl, 10 NaCl, 6 MgCl2, 5 glucose, 10 HEPES, and 0.006 A-23187, pH 7.1. The washing solution used to remove the ionophore has the same composition as the loading medium except that it contained 1% BSA (fraction V) and no A-23187.

Mg2+ loading of REC. Mg2+ loading was performed by incubating a 10% REC suspension for 30 min at 37°C in high-K+ loading solution containing 6 mM Mg2+ and 6 µM of the ionophore A-23187. For removal of the ionophore, the REC were incubated three times in ionophore-free washing solution for 10 min at 37°C (1st wash) or at room temperature (2nd and 3rd wash). Thereafter, remaining extracellular Mg2+ was removed by washing cells several times in Na+- and Mg2+-free NMDG medium at 4°C. Finally, they were resuspended in the same medium and stored at 4°C until the start of measurements.

Measurement of Mg2+ extrusion. Mg2+ efflux was calculated from the increase of Mg2+ concentration ([Mg2+]) in the incubation medium. Measurement of solutions with varying [Na+] was prepared by diluting an Mg2+-free medium 199 (Biochrom) containing 145 mM Na+ with appropriate volumes of a Na+- and Mg2+-free DPBS. A 3-ml fluorescence cuvette was prefilled with 1.5 ml freshly prepared measuring medium, and mag-fura 2 (Na+ salt) was added to give a final dye concentration of 1 µM. Samples were mixed thoroughly. Inhibitors or stimulators used in some experiments were also applied before starting the measurement by adding 0.5 ml of a 40% REC suspension in 0 Mg2+ DPBS. The dilutions were made to yield a 10% cell suspension and final [Na+] of 0, 2.5, 5, 10, 20, 50, 80, 145 mM. All measurements were performed under continuous stirring at 37°C. At the beginning and after different time points, fluorescence spectra of the mag-fura 2 (Na+ salt) were taken in a spectrofluorometer (LS-50 B; Perkin-Elmer) with excitation between 300 and 400 nm and emission at 515 nm.

The [Mg2+]e was determined from a calibration curve that was constructed on every experimental day by recording fluorescence spectra for [Mg2+]e between 0 and 35 mM. The data were then plotted as log(R – Rmin)/(Rmax – R) vs. log[Mg2+]e. R is the 340/380 fluorescence ratio of the sample, and Rmin and Rmax are the minimum and maximum fluorescence ratios determined in the 0 mM and 35 mM Mg2+ solutions, respectively. The Mg2+ concentration of the samples was calculated using a linear equation fitted to the data of the double log plot.

Measurement of cytoplasmic Mg2+ and Na+ by spectrofluorometry. For the determination of [Mg2+]i and of [Na+]i, cells were loaded with 5 µM mag-fura 2-AM and 10 µM SBFI-AM, respectively. Cells were subsequently washed two times in DPBS. REC were incubated for a further 30 min to allow for complete deesterification and washed two times before measurement of fluorescence. Intracellular ion concentrations were determined by measuring the fluorescence of the probe-loaded REC in a spectrofluorometer (LS-50 B; Perkin-Elmer) using the fast-filter accessory, which allowed fluorescence to be measured at 20-ms intervals with excitation for mag-fura 2 and SBFI at 340 and 380 nm and emission at 515 nm. All measurements were made at 37°C in a 3-ml cuvette containing 2 ml cell suspension (10% cytocrit) under stirring.

[Mg2+]i was calculated from the 340/380-nm ratio according to the formula of Grynkiewicz et al. (14) by using a dissociation constant of 1.5 mM for the mag-fura 2/Mg2+ complex. The minimum (Rmin) and maximum (Rmax) ratios were determined at the end of each experiment by using digitonin. Rmax was found by the addition of 25 mM MgCl2 in the absence of Ca2+, and Rmin was obtained by addition of 50 mM EDTA, pH 7.2, to remove all Mg2+ from the solution. SBFI signals were calibrated to ion concentrations by using the ionophore gramicidin (10 µM) to equilibrate intra- and extracellular [Na+]. The procedure was repeated for various [Na+] between 0 and 160 mM.

Determination of intracellular cAMP concentration. The intracellular cAMP concentration ([cAMP]i) was determined in REC (106 cells/ml) seeded in 96-well plates (100 µl/well) and incubated overnight in a FCS-free medium 199. On the experimental day, REC were provided with fresh medium. After addition of dibutyryl-cAMP (DBcAMP) or PGE2, REC were incubated at 37°C for 5 or 10 min, respectively. The [cAMP]i was then measured by use of a enzyme-linked immunoassay system (Amersham Pharmacia Biotech) according to the protocol of the manufacturer.

Western blot analysis. The monoclonal antibody (mAb) used in this study was raised against the porcine red blood cell Na+/Mg2+ exchanger isolated from porcine red blood cells in our laboratory. It is a special feature of the Na+/Mg2+ exchanger of porcine red blood cells that it can transport Mn2+ in place of Na+. Therefore, effective antibody-producing clones were selected by screening of hybridoma culture supernatants for their ability to inhibit 54Mn2+ influx as a measure of the Na+/Mg2+ exchange activity. With the use if this functional assay, a hybridoma clone was generated and subsequently used to detect the Na+/Mg2+ exchanger in REC. For Western blots, REC were solubilized in 20 µl Laemmli sample buffer (Bio-Rad, München, Germany) and loaded on polyacrylamide gels. Polypeptides resolved by electrophoresis were electroblotted on polyvinylidene difluoride membranes (Roth). After bathing 30 min in blocking solution [5% BSA in PBS with 0.3% Tween 20 (PBS-T)], blots were incubated for 12 h with the undiluted culture supernatants and subsequently washed three times in PBS-T. After this step, blots were incubated for 120 min in a 10,000-fold dilution of Anti-Mouse IgG antibody coupled with horseradish peroxidase (Sigma) and washed a further three times in PBS-T. Bands were visualized by chemiluminescence using Roti-Lumin (Roth, Karlsruhe, Germany) according to the manufacturer's instructions in a digital imaging system (Alpha Innotec, Montreal, ON, Canada).

Flow cytometry. Methanol-fixed REC were incubated overnight at 4°C with anti-Na+/Mg2+ mAb (5 µg/ml in 10 mM PBS with 0.2% BSA and 1 mM EDTA, pH 7.3). After being warmed to room temperature, cells were washed two times in PBS-EDTA and incubated for 1 h in a 200-fold dilution (0.5 µg/ml) of FITC-conjugated anti-mouse-IgGF(ab')2 (Sigma). The anti-Na+/Mg2+ exchanger antibody was omitted from control incubations. After washing a further two times in PBS-EDTA, quantitative analysis of cellular fluorescence was carried out by flow cytometry to analyze the cells simultaneously according to size, granularity, and Na+/Mg2+ exchanger abundance (portion of protein-expressing cells and relative fluorescence intensity/cell). Flow cytometric analysis was performed as described previously (27). Briefly, an argon laser-equipped flow cytometer (Coulter-XL) was used to record emissions of multiple fluorescence (green, orange, and red) excited at 488 nm (counting 5,000 cells). Particle size was calibrated using standard beads (Coulter). Cells of interest were identified 1) by establishing a histogram on the basis of cell size and granularity, 2) by establishing the fluorescence histogram, and 3) by projecting the fluorescence to the size-granularity histogram. Afterward, cells were gated, and the portion of fluorescent cells and their fluorescence intensity were automatically computed.

Statistical analysis. If not otherwise stated, data are presented as means ± SE. Significance was determined by Student's t-test or Tukey's ANOVA as appropriate. Correlations between variables were tested by calculating Pearson's product moment correlation coefficients. P < 0.05 was considered to be significant. All statistical calculations were performed by using SigmaStat (Jandel Scientific).


   RESULTS
TOP
 ABSTRACT
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Effect of changing the [Na+]e on Mg2+ efflux from REC. It is well known that the [Mg2+]i influences the Mg2+ efflux activity (44, 18, 10, 23). Therefore, the effect of the [Na+]e on Mg2+ extrusion was tested with Mg2+-loaded (6 mM) REC. Mg2+-loaded REC were suspended in absolutely Mg2+-free media with various [Na+], and the Mg2+ efflux was measured over a 15-min period (Fig. 1A). As demonstrated by the increase of [Mg2+]e, Mg2+ is transported out of the cells. The intensity of the Mg2+ efflux was clearly dependent on the [Na+] of the media and increased from 0.28 ± 0.14 mM·l cells–1·15 min–1 in 0 Na+ medium to 9.37 ± 4.6 mM·l cells–1·15 min–1 in 145 Na+ medium. After loading, the [Mg2+]i of REC was measured to be 0.72 ± 0.04 mM, which corresponds to a 369 ± 50 µM increase compared with [Mg2+]i (0.37 ± 0.05 mM) of non-loaded cells (Fig. 1B). After resuspension in Mg2+-free, Na+-containing medium, Mg2+-loaded REC show a decrease of the [Mg2+]i in parallel with a rise of the [Na+]i, which also reflects Na+-dependent Mg2+ extrusion (Fig. 1C).



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  		Fig. 1. Effect of variation in extracellular Na+ concentration ([Na+]e) on Mg2+ efflux from Mg2+-loaded sheep ruminal epithelial cells (REC). A: Mg2+ extrusion in the presence of various concentrations of extracellular Na+. Values are means ± SD of 3–6 single experiments. NMDG, N-methyl-D-glucamine. B: intracellular Mg2+ concentration ([Mg2+]i) of REC before and after Mg2+ loading (6 mM). Number of single experiments is shown in parenthesis. Values are means ± SE. *P < 0.05. C: representative original recordings of [Mg2+]i and intracellular Na+ concentration ([Na+]i) changes in Mg2+-loaded REC after resuspension in Mg2+-free, Na+-containing medium.

 
We then calculated the kinetics of the Na+/Mg2+ exchanger (Fig. 2). The analysis of the experimental data shows that the Mg2+ extrusion relates to [Na+]e according to Michaelis-Menten kinetics, and in most experiments a steady-state [Mg2+]e is reached after ~10 min. The Km value for extracellular Na+ amounts to 24 mM and maximal velocity was 11.1 mM·l cells–1·15 min–1.



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  		Fig. 2. Mg2+ efflux as a function of [Na+]e. The activation of the Mg2+ efflux by [Na+]e follows a simple Michaelis-Menten relationship with a Km of 24 mM. Vmax, maximal velocity.

 
Effect of imipramine and of Co(III)hex on Mg2+ efflux from REC. In previous experiments with REC, an imipramine-sensitive [Mg2+]i increase has been observed after reversing the transmembrane Na+ gradient (36). Therefore, we studied the effect of this unspecific Na+/Mg2+ exchange inhibitor on the Mg2+ efflux from Mg2+-loaded REC. Cells were incubated in a medium with an [Na+]e of 20 mM without or with 250 µM imipramine. Data are summarized in Fig. 3, which shows that inhibitor application reduced Mg2+ efflux from 4.95 ± 0.26 to 2.58 ± 1 mM·l cells–1·10 min–1. There was a considerable variation of the imipramine effect, which ranged from a 27 to a 69% inhibition. Furthermore, in some experiments, a second linear increase of the Mg2+ extrusion occurs (Fig. 3A). Because cell damage has been excluded as a reason for this observation by trypan blue staining, another mechanism should be responsible for this effect. As can be seen in Fig. 3, inset A, the rapid [Mg2+]e increase can be suppressed by Co(III)hex, a compound that has been shown to inhibit Mg2+ channels (24, 22). If Co(III)hex is given together with imipramine, only a slight additional reduction of the mean Mg2+ efflux rate occurs, but the variation is substantially reduced (Fig. 3).



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  		Fig. 3. Effect of imipramine and of cobalt(III)hexaammine [Co(III)hex] on Mg2+ efflux from Mg2+-preloaded sheep REC. Measurements were made in a completely Mg2+-free medium with 20 mM Na+ after addition of 500 µM Co(III)hex and/or 250 µM imipramine. Control experiments were done in the same medium without inhibitor application. Values are means ± SD of 3–6 single experiments. *P < 0.05 vs. control. Inset A: results from a typical experiment showing the second, Co(III)hex-inhibitable component of Mg2+ efflux.

 
Effect of an increase of [cAMP]i on the free [Mg2+]i and on the Mg2+ efflux rate. The cAMP-protein kinase A (PKA) pathway has been shown to have effects on several ruminal transport processes, e.g., Na+ and Cl– transport (12). Furthermore, an increase of [cAMP]i stimulated Na+-dependent Mg2+ efflux in a multitude of cell systems (17, 42). In contrast, nothing is known about regulation of ruminal Mg2+ absorption and of Mg2+ homeostasis in REC. Therefore, in a next series of experiments, we tested cAMP as a possible regulatory agent. DBcAMP, a cell-permeant cAMP analog, and PGE2, which is known to modulate adenylyl cyclase activity by interacting with stimulatory or inhibitory receptors, were used to modulate the [cAMP]i of REC. As shown in Fig. 4, REC [cAMP]i increased 12-fold and 8-fold after application of 100 µM DBcAMP and of 100 nM PGE2, respectively.



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  		Fig. 4. Influence of dibutyryl-cAMP (DBcAMP) and of PGE2 on the intracellular cAMP concentration of sheep REC. Bars represent means ± SD of 3 single experiments. **P < 0.01 vs. control.

 
Figure 5 shows the effect of DBcAMP and of PGE2 on the [Mg2+]i of REC. Mag-fura 2-loaded cells were preincubated in control solution (HEPES-buffered Na+ solution, [Na+]e = 100 mM, [Mg2+]e = 2 mM) or in the same solution with either 100 µM DBcAMP or 100 nM PGE2. Measurements were started after 5 min, and [Mg2+]i was then determined over 10 min. As in previous experiments (34, 36), an increase of the [Mg2+]i was observed. However, in REC exposed to 100 µM DBcAMP, the [Mg2+]i was well below that of the control cells during the whole time course of the experiments (Fig. 5A). The observed [Mg2+]i decrease amounted to 375 ± 105 µM. In contrast to the DBcAMP effect, PGE2 application led to a slight, nonsignificant [Mg2+]i increase (Fig. 5B). After a 5- or 15-min incubation in PGE2-containing media, the [Mg2+]i of REC was increased by 120 ± 40 and 145 ± 40 µM, respectively.



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  		Fig. 5. [Mg2+]i of REC after application of DBcAMP or of PGE2. Experiments were performed in high-Na+ solution ([Na+]e = 100 mM) after a 5-min preincubation with DBcAMP (A), with PGE2 (B), or without any stimulator (control). Extracellular Mg2+ concentration ([Mg2+]e) = 2 mM; pHe = 7.4. [Mg2+]i values obtained directly after starting the experiment (5 min) and at the end of the 10-min measuring period (15 min) ± SE are given; n, no. of experiments. *P < 0.05 vs. control.

 
To evaluate the role of transmembrane transport and/or intracellular Mg2+ pools for the observed changes in [Mg2+]i, further experiments were performed in the absence of extracellular Na+ and both in the presence and in the absence of extracellular Mg2+. The results of these experiments are presented in Fig. 6. No change of the [Mg2+]i was observed in Mg2+-free medium, irrespective of the presence of DBcAMP or of PGE2 in the extracellular solution. In contrast, with 2 mM Mg2+ in the extracellular medium, an [Mg2+]i increase occurs that amounted to 12.2 ± 2.2 µM/min. Figure 6 also shows that the Mg2+ influx rate was lowered to 8.0 ± 0.6 µM/min in DBcAMP-stimulated and increased to 24.2 ± 3.9 µM/min in PGE2-stimulated REC. As in previous investigations (35), this [Mg2+]i increase was concentration dependent and accompanied by a decrease of the [Na+]i (Fig. 7).



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  		Fig. 6. Effect of extracellular Mg2+ on cytoplasmatic Mg2+ responses to stimulation with cAMP or PGE2 in REC exposed to Na+-free medium. REC were suspended in 0 Na+ solution (Na+ replaced by NMDG) and stimulated with DBcAMP (100 µM) or with PGE2 (10 nM) in the presence or absence of 2 mM extracellular Mg2+. An [Mg2+]i increase only occurs in Mg2+-containing media, and it was modified by cAMP (reduction) and PGE2 (elevation). Values are means ± SE of 4–7 single experiments. *P < 0.05 and **P < 0.01 vs. control (Na+- and Mg2+-free medium). aP < 0.05 vs. control (Na+-free, Mg2+-containing medium).

 


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  		Fig. 7. Effect of variation in [Mg2+]e on [Mg2+]i and [Na+]i in REC. REC were exposed to Na+-free NMDG solution with 0, 2, 5, or 10 mM Mg2+. A: records from typical experiments performed in solutions with 0 and 5 mM extracellular Mg2+ are shown. B: means ± SE of 3–6 single experiments are given. *P < 0.05 vs. control (NMDG medium without Mg2+).

 
Next, we studied the effect of DBcAMP and of PGE2 on REC Mg2+ efflux. Mg2+-loaded (6 mM) REC were suspended in absolutely Mg2+-free media with a [Na+] of 20 mM. The Mg2+ efflux was measured over a 15-min period in the presence or absence of 100 µM DBcAMP or 100 nM PGE2, respectively. The Mg2+ extrusion rate was higher (Fig. 8A) after application of either DBcAMP (7.66 ± 0.97 mM·l cells–1·15 min–1) or PGE2 (5.76 ± 0,43 mM·l cells–1·15 min–1) compared with that in control medium (4.94 ± 0.26 mM·l cells–1·15 min–1). The stimulating effect of high [cAMP]i was completely abolished when REC were incubated in a Na+-free medium (Fig. 8B).



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  		Fig. 8. Stimulation of Mg2+ efflux from Mg2+-loaded sheep REC by DBcAMP and PGE2. A: magnitude of the mean Mg2+ efflux rate of control cells and of DBcAMP- or PGE2-treated REC. Measurements were made in a completely Mg2+-free medium with 100 mM Na+ after addition of 100 µM DBcAMP or of 100 nM PGE2. Control experiments were done in the same medium without stimulator application. Values are means ± SD of 3–6 single experiments. *P < 0.05 vs. control. B: response of Mg2+ efflux to the omission of extracellular Na+. For comparability, the small figure shows efflux curves obtained in high-Na+ medium.

 
Immunoblot of the REC Na+/Mg2+ exchanger. Before its use for Western blotting, we tested the ability of the produced mAb to inhibit Na+-dependent Mg2+ transport in the ruminant system. As shown in Fig. 9A, application of mAb-containing hybridoma supernatant to a high-K+ or high-Na+ transport medium led to a 50%-reduction and to a 23% increase of the REC [Mg2+]i, respectively, thereby reflecting inhibition of Na+-dependent Mg2+ transport. The results of the Western blot analyses are shown in Fig. 9B. As can be seen, in immunoblots of proteins from REC, the antibodies specifically labeled a protein with an apparent molecular mass of 70 kDa (Fig. 9B). In addition, anti-Na+/Mg2+ also stained a 70 kDa-protein in porcine red blood cell membranes used as positive control (Fig. 9B).



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  		Fig. 9. Expression of Na+/Mg2+ antiporter in sheep rumen epithelial cells as determined by Western analysis. A: functional test of the antibody effectiveness in the ruminant system. Changes of [Mg2+]i of REC incubated in high-K+ ([Na+]i ≥ [Na+]e) or high-Na+ ([Na+]i < [Na+]e) medium are measured. By use of these media, Mg2+ influx or Mg2+ efflux is promoted. Influx experiments were performed after a preincubation of REC in Ca2+-free media to reduce the [Mg2+]i. Addition of a blocking agent, e.g., the antibody, should decrease [Mg2+]i in the influx experiment (high-K+ solution) but should increase it in the efflux experiment (high-Na+ solution). As can be seen, these predicted effects occur if the monoclonal antibody (mAb) was added 5 min before starting the experiment. Values are means ± SE of 4 single experiments. *P < 0.05 vs. control. B: immunoblot to detect an Na+/Mg2+ exchanger in REC. Ovine REC lysates and porcine red blood cell (RBC) lysates were electrophoresed on a 12% polyacrylamide gel and blotted on polyvinylidene difluoride membranes. Membranes were stained with undiluted hybridoma supernatants, and bound antibodies were visualized using chemilimuniscence. In REC and RBC alike, a protein band at ~70 kDa is stained.

 
Flow cytometry. The results of the Western blot analysis were confirmed by flow cytometric measurements as a significant proportion of REC specifically bound the anti-Na+/Mg2+ exchanger mAb (Fig. 10). Moreover, the relative fluorescence intensity of single cells increased significantly (P < 0.05) from 5.20 ± 0.19 in control medium ([Mg2+]e = 1.2 mM) to 7.11 ± 0.44 after a 24-h incubation of REC in a high-Mg2+ medium ([Mg2+]e = 5 mM).



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  		Fig. 10. Flow cytometric analysis of the effect of high [Mg2+]e on the abundance of the Na+/Mg2+ antiporter. A: fluorescence histogram of REC (5,000 counts), foreward scatter (FSC), and side scatter (SSC) denote cell size and granularity. Gate c identifies single cells without cell debris and aggregates. B and C: fluorescence intensity of Na+/Mg2+ exchanger-positive REC. Cells were cultured in media with physiological [Mg2+] of 1.2 mM (B) or in media with high [Mg2+] of 5 mM (C). The number of fluorescence-positive cells (ordinate) was plotted against the fluorescence intensity per cell (channel number, abscissa). A shift of the channel number toward higher values is indicative of an increased Na+/Mg2+ exchanger expression/single cell. The fine-line peaks (a) represent controls (obtained by incubation of a cell aliquot only with fluorogenic antibodies), the bold-line peaks (b and c) represent Na+/Mg2+ exchanger-positive cells. REC incubated in high [Mg2+]e (Cc) shows a significant higher fluorescence than REC incubated under control conditions (Bb). *P < 0.05.

 

   DISCUSSION
TOP
 ABSTRACT
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The Na+/Mg2+ exchanger is the best characterized Mg2+ transporter of nonepithelial cells and tissues (18, 21, 23, 44, 45). In contrast, the functional properties of the epithelial Na+/Mg2+ exchanger, including its transport regulation by extra- and intracellular stimuli, have not been studied extensively so far. We have shown previously that cells isolated from the sheep rumen epithelium take up Mg2+ when incubated in a Mg2+-containing medium and that this influx is stimulated when the external medium contains no or very small amounts (10 mM) of Na+ (36). Moreover, this Mg2+ influx was accompanied by a decrease in the [Na+]i and inhibited by quinidine and imipramine. Such results point to a Na+/Mg2+ exchanger as the underlying mechanism, and this hypothesis is clearly confirmed by the results of this study.

Mg2+ loss from Mg2+-loaded REC incubated in Mg2+-free, high-Na+ medium occurs at a rate of 9.4 ± 4.7 mM·l cells–1·15 min–1. To characterize the mechanism(s) of this Mg2+ efflux, we tested its sensitivity to changes in [Na+]e and to the Na+ channel blocker imipramine, which is known to inhibit the Na+/Mg2+ exchanger in many cell types (18, 23, 37).

Na+ dependence and kinetics of Mg2+ efflux. The activity of the Na+/Mg2+ exchanger is known to be dependent on the transmembrane Na+ and Mg2+ gradients (10, 21, 23). All efflux experiments of this study were performed with REC that have been preloaded with Mg2+ in a medium containing 6 mM of the ion. The purpose of this treatment was to avoid any influence of [Mg2+]i differences on transporter activity. The [Mg2+] of the loading medium was selected because the Km for [Mg2+]i has been calculated to be 3–4 mM in other studies (19, 41). Moreover, results obtained by using this method are more comparable with those from other experiments. Resulting from the fact that the exchanger is quiescent at the physiological [Mg2+]i (18) and that, in contrast to REC, most cells have a low membrane permeability to Mg2+ (17, 31, 37, 45), Mg2+ loading is a prerequisite for efflux activation (37, 44, 46). As in other studies (17, 46), loading of REC with 6 mM Mg2+ results in a comparatively small [Mg2+]i increase of ~370 µM, indicating effective intracellular Mg2+ buffering. However, from the data reported in this study, we were not able to determine whether binding to cytosolic Mg2+ ligands or sequestration within intracellular organelles plays the main role in this process.

In line with data described by other authors (7, 17, 20, 21, 39) for a large variety of cell types (red blood cells, thymocytes, hepatocytes, myocytes, and neurons), REC showed a loss of Mg2+ that was dependent upon extracellular Na+ in a concentration-dependent manner. Moreover, corresponding changes of [Mg2+]i and of [Na+]i, namely decrease and increase, have been demonstrated. In totally Na+-free medium, only a negligible Mg2+ efflux of 0.27 ± 0.14 mM·l cells–1·15 min–1 has been observed. Modification of the transmembrane Na+ gradient by raising the extracellular [Na+] from 0 up to 145 mM resulted in an increased Mg2+ extrusion, and the calculated maximal velocity for this process amounted to 11 mM·l cells–1·15 min–1. This rate of Mg2+ loss is even greater than the rate reported under the same conditions for most of the other cell systems (3, 37, 46). This seems to reflect the capability of REC to maintain a normal [Mg2+]i of 0.6–1.2 mM in spite of disturbances induced by marked Mg2+ influx (37.5–42 µM/min), which is a characteristic of this Mg2+-absorbing cell (34, 35). This interpretation is substantiated by our finding of a rapid increase of the [Mg2+]i in Na+-free medium (Figs. 6 and 7) and after application of the anti-Na+/Mg2+ exchanger antibody (Fig. 9A). Similar high transport rates have been found only with Mg2+-loaded rat (16) and hamster (41, 44) red blood cells, which on the other hand show a reduced activity of other Na+-dependent mechanisms, e.g., Na+/H+ exchanger. The dissociation constant for extracellular Na+ is calculated to be 24 mM. This is within the range of 11–30 mM that has been reported in other studies (4, 9, 15, 16, 37).

Imipramine binds to the Na+ site of the Na+/Mg2+ exchanger, and competition between these two compounds slows Mg2+ efflux. By treatment of REC with imipramine, Mg2+ efflux was decreased in about the same degree (26 to 70%) as Mg2+ uptake via reverse Na+/Mg2+ exchange (36). Using imipramine concentrations between 10 and 500 µM, other investigators achieved a 33–85% inhibition of Mg2+ efflux (7, 18, 38, 43), which is in agreement with our findings. As in this study, the imipramine inhibition was always incomplete, and an even greater reduction of Mg2+ extrusion could be obtained by omitting extracellular Na+.

Collectively, imipramine sensitivity and strict dependence of Mg2+ extrusion from extracellular Na+ confirm that a Na+/Mg2+ exchanger is the main Mg2+ efflux mechanism in sheep REC and is responsible for ~98% of Mg2+ release.

Role of other transport systems. Normally, the transport saturates after 10–15 min, but, in some cases, a second steep rise of Mg2+ efflux occurs. After excluding cell damage as a cause for this phenomenon, we suspect that an additional mechanism could be activated at this time point. Its linear kinetic and the complete suppression of this component by Co(III)hex suggest that a channel-mediated Mg2+ loss is the underlying cause. So far, Co(III)hex has been found to inhibit specifically the cobalt resistence gene A Mg2+ transporter of bacteria (11, 24) and the mitochondrial RNA splicing factor 2p expressed in the inner membrane of yeast mitochondria (22). In one study, the Mg2+ uptake in brush-border membrane vesicles of fish enterocytes was reduced (2). Under normal conditions, such an Mg2+ channel will mediate potential-driven Mg2+ influx in REC (34). However, it seems possible that the artificial Mg2+ loading gives rise to a reversal of the electrochemical gradient ([Mg2+]i > [Mg2+]e, membrane depolarization), which in turn induces the observed Mg2+ efflux. The question arises why this component of Mg2+ loss appears with a delay and why it is not seen in every case. One explanation could be a regulation by [Mg2+]i, since it has been described for Mg2+-permeable channels of the long transient receptor protein (TRP) family, namely TRPM6 and TRPM7 (32, 33). It has been shown that these transport proteins are inhibited by elevation and activated by lowering of the [Mg2+]i (33). Preloading of REC with excess Mg2+ could block channel-mediated Mg2+ efflux (and influx) until it has been lowered to a threshold allowing channel activation.

Modulation of the Na+/Mg2+ exchanger activity by [cAMP]i and by PGE2. Mg2+ extrusion is activated by cAMP, prostaglandins, and cytokines in ascites (42), thymus (17), blood (43), heart, and liver (8) cells. In perfused heart and liver, a transient Mg2+ efflux can be induced by {beta}-adrenergic substances and by application of DBcAMP (8, 30). In this study, incubation of REC with PGE2 or with DBcAMP, a membrane-permeable analog of cAMP, resulted in an increased [cAMP]i and a 16–55% stimulation of the Mg2+ efflux, respectively. Moreover, our data show that the stimulating effect of cAMP corresponds to a modulation of Na+/Mg2+ exchanger activity, since DBcAMP was ineffective in Na+-free NMDG medium. These results are in line with others (43) showing that an increase in intracellular cAMP specifically triggers the Na+-dependent Mg2+ efflux. This effect has been explained by an augmented exchanger affinity for intracellular Mg2+ induced by phosphorylation of the transport protein (17, 42). In some cell types, such as cardiomyocytes and hepatocytes, a cAMP-induced intracellular Mg2+ mobilization, e.g., from intracellular organelles, has been shown to be responsible for the initiation of Mg2+ efflux. There was no indication of such a process playing a role in REC because cAMP constantly led to a decrease of the [Mg2+]i. This cAMP-induced reduction of [Mg2+]i has been demonstrated to activate a nonselective cation conductance expressed in the apical membrane of REC and, thereby, to play an important role in the regulation of ruminal Na+ transport (26). In contrast, REC respond to PGE2 stimulation with an [Mg2+]i elevation. The latter was more pronounced after inhibition of Mg2+ efflux by incubating the cells in a Na+-free media but completely suppressed in Mg2+-free solutions. Therefore, we propose that the elevated [Mg2+]i observed after stimulation with PGE2 was chiefly the result of an increased influx of Mg2+ from the extracellular space.

In the concentration used (100 nM), PGE2 clearly increases [cAMP]i, indicating the presence and activation of the stimulating PGE2 receptors E prostanoid (EP) 2 or EP 4 in REC (1, 28). There is no indication for an inhibitory effect of this PGE2 dosage on the adenylyl cyclase, as has been described by Wolf et al. (43) with PGE1 concentrations up to 20 nM in spleen lymphocytes. Nevertheless, the stimulating effect of PGE2 on Mg2+ efflux amounts to only 16% compared with the 55% seen after DBcAMP application. The parallel stimulation of both Mg2+ uptake and Mg2+ extrusion by PGE2 gives an explanation for this finding. In this study, Mg2+ efflux from REC suspended in a completely Mg2+-free medium has been calculated from the increase of the [Mg2+] in this medium. If a greater proportion of the secreted Mg2+ is reabsorbed by the cells, the efflux rate is underestimated. Considering the chemical gradient for Mg2+ ([Mg2+]e = 0.28 mM and [Mg2+]i = 0.72 mM) and the membrane potential (–25 mV, inside negative) of REC (34), an electrochemical driving force of –12 mV can be calculated to exist under our experimental conditions, which is large enough to allow at least an electrogenic, channel-mediated uptake of Mg2+ (34). A stimulation of Mg2+ uptake by PGE2 has been found in a study of Dai et al. (6) in immortalized renal epithelial cells of the mouse distal convoluted tubule. The effect was contributed to multiple intracellular signaling pathways, including cAMP-mediated PKA activation as well as stimulation of phospholipase C and of the protein kinase C (PKC) pathway. In rat heart myocytes and liver cells, intracellular cAMP formation stimulates Mg2+ efflux, and Mg2+ accumulation was related to changes in PKC activity (29). Further experiments at the whole epithelium level are necessary to confirm the stimulating effect of PGE2 on apical Mg2+ influx. Nevertheless, the suggestion of a regulation of REC Mg2+ transport is of general importance, and there is a need to investigate what hormones or other signals are involved.

Molecular evidence for the expression of an Na+/Mg2+ exchanger in REC. Using a Western blot assay, the mAb selected by a functional screening detected a protein with a molecular mass of ~70 kDa in REC and in the porcine red blood cell membranes used as positive control. Although the evidence concerning the molecular characteristics of a Na+/Mg2+ exchanger in REC remains incomplete, it seems likely that the protein detected in the Western blot experiments conducted in this study is indeed this protein. This assumption is particularly supported by the fact that the hybridoma supernatants successfully used in the aforementioned Western blot assays showed a specific blocking capability for Na+/Mg2+ exchange in earlier functional analyses. More importantly, as shown by flow cytometric experiments, the expression and/or translocation to the cell surface of the detected protein is influenced by [Mg2+]e as its abundance increased by 36.7% after exposing REC to high-Mg2+ solutions. Further supporting the hypothesis that the protein detected at 70 kDa in a Western blot assay represents the Na+/Mg2+ exchanger in REC is the fact that other exchangers, such as the Na+/H+ exchanger or the Na+/Ca2+ exchanger, have somewhat higher molecular weights of ~90–110 kDa (5, 40).

In conclusion, the present results demonstrate an Na+ gradient-dependent Mg2+ efflux activity in REC. This Mg2+ transport is consistent with Na+/Mg2+ antiport, as it is half-maximally activated by 24 mM [Na+]e, stimulated by elevated intracellular cAMP levels, and inhibited by imipramine. Although the Na+/Mg2+ exchanger involved in this activity remains to be clearly identified, it has an apparent molecular mass of ~70 kDa. This mechanism seems to perform 98% of the Mg2+ extrusion and is therefore most important for the regulation of [Mg2+]i as well as for Mg2+ extrusion in connection with transepithelial Mg2+ absorption.


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 ABSTRACT
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This study was supported by a research grant from the Deutsche Forschungsgemeinschaft (Schw 642) and a student gratification of the Schaumann Stiftung to H.-S. Park.


   ACKNOWLEDGMENTS
 
We thank Dr. T. Viergutz for carrying out the flow cytometric measurements. We gratefully acknowledge the valuable assistence of Dr. Almut Böttcher.


   FOOTNOTES
 

Address for reprint requests and other correspondence: M. Schweigel, Research Institute for the Biology of Farm Animals-FBN, Dept. of Nutritional Physiology "Oskar Kellner," Wilhelm-Stahl-Allee 2, 18196 Dummerstorf, Germany (e-mail: mschweigel{at}fbn-dummerstorf.de)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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