ARTICLE
Auteur(s) : Monika
Schweigel1, Judith Kuzinski1, Carolin
Deiner2, Martin Kolisek2
1Research Unit Nutritional Physiology “Oskar
Kellner”, Research Institute for the Biology of Farm
Animals (FBN), Dummerstorf
2Department of Veterinary Physiology, Free
University of Berlin, Berlin, Germany
Regulation of the free intracellular magnesium concentration
([Mg2+]i) and of whole body Mg homeostasis
occurs through a variety of mechanisms involving pathways for Mg
uptake, Mg efflux, and Mg exchange between intracellular
compartments [2-5]. However, only a few Mg transport proteins,
namely, the TRPM6 and TRPM7 channels of the Melastatin-related
Transient Receptor Potential family and the mitochondrial channel
MRS2 (Mitochondrial RNA Splicing member 2), have been identified at
the molecular level so far [2, 6]. In addition, using a
differential gene expression approach, the group of Quamme [7-11]
described SLC41A1 and SLC41A2 (Solute Carrier family 41; subfamily
A; members 1 and 2), MagT1 (Magnesium Transporter 1), ACDP 2
(Ancient Conserved Domain Protein; subtype 2), and the protein
NIPA1 (Nonimprinted in Prader-Willi/Angelman) as putative Mg
transporters. Based on the transport characteristics reported to
date [9-13], it seems that only SLC41A1 mediates Mg efflux and that
none of these proteins is identical to the
Na+/Mg2+ exchanger. At a functional level,
the latter has been demonstrated to mediate Mg efflux in most cell
systems investigated [14-18]. In our previous work with isolated
ovine ruminal epithelial cells (REC) [5], we showed that a
Na+/Mg2+ exchanger is the main Mg efflux
mechanism in these cells, being responsible for about 98% of total
Mg release. In ruminating animals, the required Mg is absorbed from
the forestomachs by active transcellular mechanisms [19]. As shown
by using the Ussing-chamber technique [20], a Na-dependent
imipramine-sensitive mechanism is essentially involved in these
processes. Moreover, the REC Na+/Mg2+
exchanger is an important mechanism for maintaining cellular Mg
balance [5] by compensating for the marked Mg influx [37.5 to 42
μM/min) that is a characteristic of this Mg absorbing cell [21,
22]. In REC, as in other cell systems,
[Mg2+]i can be assumed to be a cofactor for
enzymes and signal-transduction proteins and to regulate
bioenergetics, ion transport, growth, and proliferation [23-26]. In
order to maintain [Mg2+]i in an optimal
physiological range, Mg transport systems have to be under tight
control. However, investigation of the modalities of the REC Mg
transporter operation, regulation and interactions among each other
is hindered by their unknown molecular identities.
Thus, the present study was performed to identify the main Mg
influx pathways in REC at a molecular level. In addition, we
investigated the effect of low and high extracellular Mg conditions
on the expression and functional activity of TRPM7, MagT1 and the
Na+/Mg2+ exchanger. This was done by using an
anti-Na+/Mg2+ exchanger antibody prepared in
our laboratory [27] and a combination of cell physiological
approaches (fluorescence-spectroscopic measurement of the
[Mg2+]i, flow cytometry, immunocytochemistry)
and molecular techniques (RT-PCR, Western blot). The preliminary
results were presented at the European Meeting on Magnesium and
published in short form [1].
Materials and methods
Materials
Medium 199, trypsin, glutamine, antibiotics (gentamycin, nystatin,
kanamycin, penicillin-streptomycin), and Dulbecco`s
phosphate-buffered saline (DPBS) were purchased from PAN Biotech
(Aidenbach, Germany). Fetal calf serum (FCS) and HyQTase were
obtained from Biochrom (Berlin, Germany) and Thermo Scientific
(USA), respectively. Mag-fura 2-AM and pluronic acid were from
Molecular Probes Inc. (Eugene, OR). All other chemicals were
purchased from Sigma (St. Louis, USA).
Cell Culture
Primary cultures of REC were prepared as described by Galfi
et al. [28]. 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.
Experimental design
Experiments were performed between 5 and 6 days after seeding. On
the day before a particular experiment, culture dishes were washed
twice with warm DPBS and divided into three groups. Thereafter, to
change the Mg status of cells, they were provided with fresh media
supplemented with 1.2 mM (control), 5 mM (high-Mg), or 0.12 mM
(low-Mg) Mg and then incubated at 38°C for 24 h. This
pre-incubation medium was a custom-made (Biochrom, Berlin, Germany)
Medium 199 with Earl’s salts containing no Ca/Mg or phenol red.
Before use, 1.36 mM glutamine, 20 mM HEPES, 1.2 mM Ca and Mg as
indicated above were added.
On the experimental day, some REC from each Mg group were loaded
with mag-fura 2 to determine their Mg transport activity (see
“Measurement of cytoplasmic Mg”), fixed with methanol for flow
cytometric analysis of Na+/Mg2+ exchanger
abundance (see “Flow cytometry”), or used to extract total protein
(see “Western blot analysis”).
Experiments to determine Mg efflux
The Mg efflux capacity was determined as the
[Mg2+]i decrease over a 20-min period.
Directly before the efflux experiment, REC were Mg-loaded by a
15-min incubation in divalent-free Hank’s balanced solution (HBS)
supplemented with 1.2 mM Ca, 20 mM HEPES, 1.36 mM L-glutamine
(HBSsup), and 5 mM Mg. Then, the remaining extracellular Mg was
removed by washing the cells twice in a custom-made (Biochrom,
Berlin, Germany) Na-free PBS (PBS-Na; No. 1825, but without Ca/Mg
and with the Na being substituted with N-methyl-D-glucamine; NMDG).
Thereafter, measurements were started by re-suspending REC in a
custom-made (Biochrom, Berlin, Germany), completely Mg-free Medium
199 with Hank’s salts supplemented with 1.36 mM glutamine, 20 mM
HEPES, 1.2 mM Ca.
Solution for Mg influx experiments
[Mg2+]i increase was measured in the PBS-Na
supplemented with 20 mM HEPES, 1.36 mM L-glutamine, and 5 mM Mg.
Measurement of cytoplasmic Mg
REC were rinsed twice with ice-cold divalent-free PBS, gently
detached by incubation with HyQTase (15 min, at 38°C),
centrifuged, washed twice in pre-incubation medium with the
respective [Mg] and once in HBS, and finally re-suspended in the
last-mentioned solution. For the determination of
[Mg2+]i, cells were loaded (25 min at
37°C) with 5 μM mag-fura 2-AM in the presence of pluronic
acid. After centrifugation, cells were suspended in HBSsup,
incubated for a further 30 min to allow for complete
de-esterification, and washed twice in HBSsup before measurement of
fluorescence. In influx experiments, these steps were performed
with HBS supplemented with HEPES and L-glutamine only.
Intracellular ion concentrations were determined by measuring
the fluorescence of the probe-loaded REC in a spectrofluorometer
(LS-50 B, Perkin-Elmer, Wiesbaden, Germany) by using the
fast-filter accessory, which allowed fluorescence to be measured at
20-ms intervals with excitation for mag-fura 2 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. [29] by
using a dissociation constant of 1.5 mM for the mag-fura-2/Mg
complex. The minimum (Rmin) and maximum
(Rmax) ratios were determined at the end of each
experiment by using digitonin. Rmax was measured after
the addition of 25 mM MgCl2 in the absence of Ca, and
Rmin was obtained by addition of 50 mM EDTA, pH 7.2, to
remove all Mg from the solution.
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 per well) and incubated overnight in an FCS-free
Medium 199. On the next day, the 24-h pre-incubation of REC with
media containing 1.2, 5, or 0.12 mM Mg was initiated as described
above. Afterwards, [cAMP]i was measured by use of an
enzyme-linked immunoassay system (Amersham Pharmacia Biotech, UK)
according to the protocol of the manufacturer.
RNA isolation and reverse-transcription polymerase chain
reaction (PCR)
Total ovine REC RNA was isolated by use of the NucleoSpin RNA II
kit (Macherey-Nagel, Düren, Germany) according to the
manufacturer’s protocol, and its integrity was examined with a
Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, USA).
Total RNA (100 ng) and oligo (dT)12 primers were used to
synthesize cDNA from poly A-containing mRNA with the iScript cDNA
synthesis kit (BioRad, Hercules, USA). The cDNA (TRPM6/7 and MagT1)
was amplified by PCR (peqSTAR, PEQLAB Biotechnologie, Erlangen,
Germany) with HotStar HiFidelity Polymerase (Quiagen, Hilden,
Germany).
The following gene-specific primers were used for PCR
amplification (the identity of the particular gene is denoted in
the name of each primer):
- – (1) bTRPM7-forw-(588-608); IVF-FU#501b12:
5’-TTGGCCAGAGTGAAGCAGTT-3’;
- – (2) bTRPM7- rev-(745-764); IVFFU#478b12:
5’-TTTCCAACAGTGCCATCATC-3’;
- – (3) hMagT1-forw-(540-559); IVF-FU#689b19:
5’-GCCGACAGAACTGATGTCAA-3’;
- – (4) hMagT1-rev-(749-768); IVF-FU#690b19:
5’-TATGGGCATATGGTGGTCCT-3’;
- – (5) hTRPM6-forw(P1)-(4356-4375); IVF-FU#774b21:
5’-TGGCAACTGAACAGGACATC-3;
- – (6) hTRPM6-rev(P1)-(4811-4830); IVF-FU#775b21:
5’-CGGAGAGGATTGATCCAAAA-3’;
- – (7) hTRPM6-forw(P2)-(4437-4456); IVF-FU#776b21:
5’-GGATGAACCCAAGGAAAAG-3;
- – (8) hTRPM6-rev(P2)-(4831-4850); IVF-FU#777b21:
5’-TAGCGAAGGGCCTGTATCTG-3’.
For all three genes, optimal amplification was obtained in a
reaction volume of 50 μL and with the following PCR program (35
cycles): denaturation at 94°C for 15 s, annealing at 53°C for
1 min (TRPM6) or at 55°C for 1 min (TRPM7 and MagT1), and
elongation at 72°C for 1 min.
Ethidium-bromide-stained PCR-products were electrophoretically
resolved on a 2% agarose gel and visualized in ChemilmagerTM 5500
(Alpha Innotech, San Leandro, USA). The PCR-amplified TRPM7, MagT1,
and TRPM6 fragments were sequenced (AGOWA, Berlin, Germany) for
identity-verification purposes.
Antibodies
The monoclonal mouse anti-Na+/Mg2+ exchanger
antibody (mab) used in this study was raised against the porcine
erythrocyte Na+/Mg2+ exchanger in our
laboratory and has been shown to react specifically with the
protein in REC [5, 27]. The hybridoma cultures were established in
RPMI1640 medium (PAN Biotech, Aidenbach, Germany) containing 10%
FCS. During days 2-3, hybridomas were stepwise switched to
serum-free medium for mab production (HyQ ADCF-Mab, HyClone, Bonn,
Germany). Culture supernatants containing the produced mabs were
sampled for 4-6 days. Thereafter, supernatants were purified and
concentrated by ultracentrifugation in Vivaspin Centrifugal
Concentrators (Vivaspin 15R, Vivascience, Hannover, Germany).
Centrifugation was performed at 3 000 × g for 30 min.
After dilution of the concentrated sample in PBS (1:1), a second
ultrafiltration step was performed. The IgG concentration was then
determined by using a Nanodrop photometer (Implen GmbH, München,
Germany) and set to about 1 mg/mL with PBS containing 0.5% bovine
serum albumin (BSA) and 0.02% Na-azide. Aliquots were subsequently
stored at -20°C until use. A rabbit anti-TRPM7 antibody
including a GH3 cell line protein lysate as control antigen
(ACC-047, lot AN-01) was obtained from Alamone Labs (Jerusalem,
Israel). The rabbit anti-MagT1-antibody was a gift from Dr. G.
Quamme (Department of Medicine, University of British Columbia,
Vancouver, Canada) and the mouse anti-β-actin antibody was
purchased from Sigma (St. Louis, MO, USA). For Western blotting,
secondary anti-mouse or anti-rabbit IgG antibodies coupled with
horseradish peroxidase (HRP; Sigma, St. Louis, MO) were used. Alexa
fluor 488-conjugated anti-mouse or anti-rabbit IgG antibodies
obtained from Invitrogen (Paisley, UK) or Molecular Probes (Eugene,
OR) were used for immunocytochemistry and flow cytometric
experiments.
Western blot analysis
For Western blot analysis, total proteins from washed REC were
extracted by use of the M-PER Mammalian Protein Extraction Reagent
(Pierce, Bonn, Germany), complemented with a protease inhibitor
cocktail (Pierce, Bonn, Germany). The protein concentration was
determined by means of the Bradford assay (Sigma, St. Louis, MO,
USA). Protein samples (40 to 80 μg) were separated by SDS
(10%)-polyacrylamide gel electrophoresis and subsequently blotted
to polyvinylidene fluoride (PVDF) membranes. After transfer,
membranes were blocked with a 3% solution of non-fat dry milk in
TRIS-buffered saline (pH 7.5) containing 0.05% Tween 20 (TBS-T) for
1 h. Following blocking, membranes were incubated at 4°C with
the primary anti-Na+/Mg2+ exchanger antibody
(1:200 dilution; 0.25 μg/mL) and with the mouse anti-β-actin
antibody (0.5 μg/mL, a gel-loading control) overnight, washed three
times for 10 min in TBS-T, incubated for 1 h with
HRP-conjugated secondary antibody (diluted 1:10,000), and finally
washed three times for 10 min in TBS-T. Bands were visualized
in Chemilmager TM 5500 (Alpha Innotech, San Leandro, CA, USA) with
the Rotilumin reagents (Roth, Karlsruhe, Germany) or ECL Plus
Western Blotting detection system (GE Healthcare/Amersham).
Relative intensities of the band-densities were determined by
analysis with NIH ImageJ 1.410 software (US National Institutes of
Health, Bethesda, MD, USA).
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 twice in PBS-EDTA and incubated for
1 h in a 200-fold dilution (0.5 μg/mL) of Alexa fluor
488-conjugated anti-mouse-IgGF(ab`)2 (Molecular Probes,
Eugene, OR). The anti-Na+/Mg2+ exchanger
antibody was omitted from control incubations. After a further two
washes 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 per
cell). TRPM7 and MagT1 abundance was determined in the same way,
but the specific (5 and 4 μg/mL) and secondary (Alexa fluor
488-conjugated anti-rabbit-IgG, 0.5 μg/mL) antibodies were used.
Flow cytometric analysis was performed as described previously [5].
Briefly, an argon-laser-equipped flow cytometer (Beckmann
Coulter-XL, Krefeld, Germany) was used to record emissions of
multiple fluorescence (green, orange, red) excited at 488 nm
(counting 5 000 cells). Particle size was calibrated by using
standard beads (Coulter). Cells of interest were identified by:
- – establishing a histogram on the basis of cell size and
granularity;
- – establishing the fluorescence histogram;
- – projecting the fluorescence into the size-granularity
histogram. Afterwards, the cells were gated, and the portion of
fluorescent cells and their fluorescence intensity were
automatically computed.
Immunocytochemistry
REC (2 × 106 cells/mL) were grown on sterile glass cover
slips (Neolab, Germany) for 24 to 48 h. Thereafter, the 24-h
pre-incubation was performed as described above. After being rinsed
twice with PBS, REC were fixed in methanol (10 min at -20°C).
If not otherwise stated, all the following steps were carried out
at room temperature. After two PBS washes, cells were permeabilized
in 0.3% Triton X-100 in PBS with 7% goat serum (Dianova, Hamburg,
Germany) for 10 min and again rinsed three times with PBS.
Non-specific binding of IgG was suppressed by incubation of
specimens with 7% goat serum in PBS for 20 min. Subsequently,
cells were rinsed with PBS (three times for 5 min) and then
incubated overnight at 4°C with the primary
anti-Na+/Mg2+ exchanger antibody (0.25 mg/mL
solved in PBS with 1% BSA; PBS-BSA). After being rinsed three times
with PBS, cells were incubated for 2 h with the secondary,
Alexa Fluor-488-labeled goat anti-mouse IgG1 (γ1)
antibody (1:200 in PBS-BSA). After three changes of PBS (5 min
each), nuclei were counterstained with 300 nM
4,6-diamidino-2-phenylindole (DAPI) in S-buffer (containing: 75 mM
KCl, 3 mM MgSO4.7H2O, 1 mM EGTA, 0.2 mM
dithiothreitole, 10 mM imidazol, 1 μg/mL aprotinin, 0.1 mM
phenylmethane sulfonyl-fluoride). Cover slips were then mounted
with 30 μL mounting medium (Dianova, Hamburg, Germany). Digital
images were acquired by using a fluorescence microscope Olympus
IX50 (Hamburg, Germany) and MetaMorph version 7.5.2.0 and
AutoDeblur version 1.4.1 software (Visitron Systems GmbH, Puchheim,
Germany).
Statistical analysis
If not otherwise stated, data are presented as means ± standard
error (SE). Significance was determined by Student’s t-test or the
paired t-test 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
[Mg2+]i of REC after a 24-h
pre-incubation in media with various extracellular
[Mg2+]
The basal [Mg2+]i of Mag-Fura 2-loaded REC
was measured in Mg-free media after a 24-h incubation of cells in
media with normal (1.2 mM), high (5 mM), or low (0.12 mM)
[Mg]. As demonstrated in figure 1,
[Mg2+]i was unaltered in low- and high-Mg REC
(0.49 ± 0.04 and 0.41 ± 0.02 mM) versus control (0.40 ± 0.03 mM).
This result indicates that mechanisms are present enabling cells to
maintain an optimal [Mg2+]i under various
conditions. Therefore, in the next experimental step, we determined
the efflux capacity of differently pre-incubated REC.
[Mg2+]i change in differently
pre-incubated REC
In contrast to other cell systems [30, 31], REC are known to have a
high basal Mg influx capability [21]. For this reason, there was no
need to load the cells with Mg by using the ionophore A 23187
as in our previous study [5]. To prepare REC for the following
efflux experiment, it was sufficient to incubate them for
15 min in high-Mg loading solution
([Mg2+]e = 5 mM). Figure 1 shows that in
control and treatment groups a significant
[Mg2+]i increase of about 158 ± 26 μM was
induced by this treatment.
Directly after loading, efflux experiments were started by
suspending REC in completely Mg-free but Na-containing media. The
Mg efflux capacity was measured as a decrease of the
[Mg2+]i over a 20-min period. Previously, the
latter has been demonstrated to reflect Na-dependent Mg extrusion
from REC [5]. Typical original traces of the
[Mg2+]i decrease observed during the
incubation of control, high-, and low-Mg pre-incubated REC in
totally Mg-free Na media are given in figure 2A. Clearly, the
[Mg2+]i decrease was faster and stronger
after pre-incubation in high-Mg medium and reduced in low-Mg REC,
when compared with control cells. Data from all experiments are
summarized in figure
2B. For low-Mg conditions only results from measurements
with detectable efflux (n = 10 of 12) were used for calculations.
Compared with control cells (4.1 ± 0.7 μM/min), REC
pre-incubated in low- and high-Mg medium showed reduced (2.8 ± 0.6
μM/min) and accelerated Mg extrusion rates (6.4 ± 0.9 μM/min),
respectively.
Basal intracellular [cAMP] and cAMP effect
on the [Mg2+]i change observed
in differently pre-incubated REC
An increase of the intracellular cAMP concentration
([cAMP]i) has been shown to stimulate the Na-dependent
Mg efflux in REC [5]. Therefore, a changed Mg efflux capacity of
REC after pre-incubation in media with different [Mg2+]
may result from the modulation of the basal [cAMP]i of
such cells. However, REC mean [cAMP]i amounted to 5.1 ±
0.5 fmol/μg protein (n = 36), and no influence of the extracellular
Mg status was detectable (figure 2C).
Next, we wished to know whether the pre-incubation in low- or
high-Mg solutions had any effect on the response of REC to
cAMP-stimulation. We therefore repeated the above-described efflux
experiments in the presence of the cell membrane-permeable cAMP
analog db-cAMP (100 μM). The data from these experiments are
summarized in figure
2B and show that the observed [Mg2+]i
decrease was always stronger after db-cAMP application and was
stimulated by 64 ± 18, 109 ± 39, and 304 ± 92% in control, high-,
and low-Mg REC, respectively.
Influence of [Mg2+]e
on the abundance of REC putative
Na+/Mg2+ exchanger
To test whether a distinct Na+/Mg2+ exchanger
protein expression could cause the changed efflux capacity, we used
Western analysis and flow cytometry (figure 3). As in previous
experiments with REC and other cell systems [5], our
anti-Na+/Mg2+ antibody labeled a single
protein with an apparent molecular weight of 70 kDa as the
candidate Na+/Mg2+ exchanger protein (figure 3A). Flow
cytometric analysis confirmed the presence of this protein in
primary cultured REC (figure 3C), with an
average of 80 ± 3% cells being positive for
Na+/Mg2+ exchanger.
Typical results of Western blot analysis of whole cell protein
extracts derived from differently pre-incubated REC are presented
in figure 3B
(inset). Downstream densitometric analysis, which is summarized in
figure 3B,
revealed that the amount of the detected protein was decreased (9.9
± 1.3%) after pre-incubation in the low-Mg medium or increased
(17.7 ± 2.6%) after pre-incubation in high-Mg medium compared with
control (REC pre-incubated in medium containing 1.2 mM Mg).
Densitometric analysis conducted for immunolabeled β-actin (42 kDa,
negative experimental control and loading control) did not show any
[Mg2+]e-dependent variation in its abundance
(figure 3B).
Flow cytometric analysis (figure 3C) confirmed that,
compared with control conditions, a decreased (-24.8 ± 3.7%) or
increased (36.2 ± 6.4%) Na+/Mg2+ exchanger
protein amount per single cell was observed after a 24-h incubation
of REC in low- and high-Mg medium, respectively.
Epithelial and cellular localization and abundance
of the putative Na+/Mg2+
exchanger
For a better understanding of the following data, the morphology of
the rumen epithelium is shown in figure 4A. It can be seen
that, compared with the single layer of renal or intestinal
epithelia, the rumen epithelium has a more complex multilayered
structure. Starting from the blood side, four distinct cell layers,
namely, the stratum (str.) basale, the str. spinosum, the str.
granulosum, and the keratinized str. corneum, can be distinguished
(figure 4A).
Three different REC fractions obtained by trypsination as described
in “materials and methods” and representing 1) mainly cells from
the stratum basale, 2) mainly cells from str. spinosum and lower
str. granulosum, and 3) mainly cells from stratum granulosum were
analyzed for Na+/Mg2+ exchanger abundance
using flow cytometry. The composition of the cell fractions was
evaluated by microscopy but it has to be mentioned that every
fraction contained some cornified cells not involved in epithelial
transport processes. As shown in figure 4B, the
Na+/Mg2+ exchanger was enriched in cells of
the stratum basale (45 ± 9% of positive cells). A progressive
reduction of the number of Na+/Mg2+
exchanger-positive REC was observed in the stratum spinosum (29 ±
5%) and stratum granulosum (24 ± 4%).
Next we investigated the distribution and abundance of the
putative REC Na+/Mg2+ exchanger protein by
immunocytochemistry, and examples of characteristic staining
patterns are given in figure 5. The
Na+/Mg2+ exchanger was localized in the cell
membrane or in its close vicinity, although cytoplasmic staining
was also observed (figure 5). The latter
seemed to increase if REC had been incubated in the high-Mg medium.
Corresponding to results from the functional, Western blot, and
flow cytometric experiments, the expression level of the putative
Na+/Mg2+ exchanger was considerably reduced
in low-Mg pre-incubated REC (figure 5A), and more
protein was labeled after pre-incubation in high-Mg medium (figure 5C).
Role of other Mg transport systems
To date, functional studies performed at the tissue and cell levels
[21, 22, 32] suggest the existence of at least two different Mg
uptake mechanisms in REC. Although their molecular identity is not
known, candidates are the recently described TRPM6, TRPM7, and
MagT1 Mg channels/transporters [4, 9, 33], which have been shown to
be influenced by Mg status [9, 34]. PCR revealed the TRPM7 and
MagT1 transcripts in REC (figure 6A). The results
were confirmed by sequencing the products. The sequences attained
were compared with the ovine (TRPM7) or human (MagT1) sequences
that had been used for the primer design, yielding an identity of
100 and 99.6%, respectively. In addition, flow cytometric analysis
showed the presence of TRPM7 in 93 ± 2% and of MagT1 in 80 ± 10% of
these cells. In contrast, we were not able to identify TRPM6
transcripts with specific primer pairs (PP1 and PP2; figure 6A) in REC. As a
control, PP1 and PP2 were used to detect TRPM6 transcripts in
Caco-2 (human epithelial colorectal adenocarcinoma) cells. In this
cell system the presence of the TRPM6 transcript tested positive
(figure 6A).
We next evaluated whether a pre-incubation in Mg-deficient or
high-Mg medium resulted in activity changes of Mg influx pathways.
To test this possibility, influx experiments were performed in the
absence of Na ([Na]i > [Na]e) and the
presence of 5 mM Mg ([Mg2+]i <
[Mg]e) in the extracellular solution. Under such
conditions, some of the Mg uptake is mediated via the
Na+/Mg2+ exchanger working in the reverse
mode [5, 32]. Known unspecific inhibitors of the
Na+/Mg2+ exchanger (imipramine [32]) and of
channel-mediated Mg influx (Co(III)hexaammine [2, 13]) were used to
differentiate between transport components. In addition, cell
samples were analyzed for the abundance of TRPM7 and MagT1 by using
Western analysis and flow cytometry.
The results of the functional experiments are given in figure 6B.
Irrespective of the [Mg] of the pre-incubation medium, the initial
[Mg2+]i of REC amounted to 0.68 ± 0.04 mM in
these experiments. In all three treatment groups, a significant
[Mg2+]i increase occurred during the 20-min
incubation period. However, compared with control cells (19.8 ± 1.4
μM/min), low- and high-Mg pre-incubated REC were both characterized
by an increased influx capacity amounting to 25.8 ± 1.7 and 26.4 ±
2.8 μM/min, respectively (figure 6B). Application of
imipramine (250 μM), Co(III)hex (1 mM), or of a combination of both
inhibitors led to a reduced [Mg2+]i increase
in all groups of REC, thereby reflecting the existence of
channel-mediated and of Na-dependent components of Mg transport. In
agreement with the observed changes in
Na+/Mg2+ exchanger expression, the imipramine
effect was lowest (-4.8 ± 2.0 μM/min) in REC pre-incubated in the
0.12 mM-Mg medium and highest (-13.4 ± 2.6 μM/min) in cells
pre-incubated in the high-Mg medium. However, the response of
low-Mg REC to imipramine was not significantly different from that
(-5.7 ± 2.3 μM/min) of control cells. In the latter, the
Co(III)Hex-induced decrease in the Mg uptake rate amounted to 2.5 ±
0.5 μM/min. Interestingly, the inhibitor effect was stronger in
both experimental groups, and the Mg uptake rate was reduced by
12.2 ± 2.8 and 7.7 ± 0.8 μM/min in low- and high-Mg pre-incubated
REC, respectively. No additive effects of Co(III)Hex and imipramine
were observed.
To evaluate the TRPM7 and MagT1 protein content in REC,
immunoblotting was performed. As shown in figure 6C, Western analysis
of total REC homogenate revealed major bands at the 160- to 170-kDa
and 38-kDa positions, corresponding to TRPM7 and MagT1,
respectively. A total protein lysate derived from GH3 cells
(Alamone Labs, Jerusalem, Israel) was used as a positive control.
Interestingly, pre-incubation in media with different
[Mg]e had a slight influence on the protein expression
of TRPM7 only (figure
6C). However, compared with control conditions, a decreased
or increased MagT1 (-35 ± 10%; 20.1 ± 9.5%) abundance was observed
after the 24-h incubation of REC in low- and high-Mg medium,
respectively. These latter results indicate the importance of
obtaining information on the functional activity of MagT1 in
control REC and in cells pre-incubated under low- or high-Mg
conditions. Goytain and Quamme [9] have shown that the
1,4-dihydropyridine analog nitrendipine is an effective inhibitor
of MagT1-related currents. Therefore, we studied the effect of this
blocker (50 μM) on REC Mg influx measured over a 20-min period
after reversing the transmembrane Na gradient ([Mg]e = 5
mM). The results are summarized in figure 7. Again, low- (36 ±
4 μM/min) and high-Mg (30 ± 2 μM/min) pre-incubated REC showed an
increased Mg uptake compared with the control cells (21 ± 0.6
μM/min). The inhibitor application reduced the Mg influx by 17 ± 3,
10 ± 1, and 16 ± 2 μM/min in low-Mg, control, and high-Mg REC,
respectively.
Discussion
The plasma membrane Na+/Mg2+ exchanger is
thought to be a key element in the regulation of both organism and
cellular Mg homeostasis [35-38]. This is supported by the results
of this study showing that the transporter functional activity and
its expression are quantitatively altered by extracellular Mg
deficiency or overload.
[Mg2+]i is regulated
by a changed Mg efflux capacity
The modulation of the extracellular [Mg2+] induced no
significant change of the REC basal [Mg2+]i
measured in Ca/Mg-free medium. It ranged from 0.40 ± 0.03 to 0.49 ±
0.04 mM and was thus consistent with the values of 0.37 ± 0.05 and
0.54 ± 0.08 mM seen in our previous studies [5, 21]. This has led
us to hypothesize that transmembrane Mg transport is adapted to
perpetuating the physiological [Mg2+]i.
The REC Na+/Mg2+ exchanger has been shown
to mediate 98% of Mg efflux [5], and its inhibition induces a
marked increase in the cytosolic [Mg2+] [32]. Therefore,
it represents a good candidate for playing an important role in
[Mg2+]i regulation. Our data show that the Mg
efflux capacity of REC has indeed been modulated by
[Mg]e changes. Cells pre-incubated in Mg-deficient and
high-Mg medium are characterized by a decreased (32%) and
accelerated (56%) Mg efflux rates, respectively, when compared with
controls. Interestingly, Feillet-Coudray et al. [39] observed
a decreased Mg efflux in erythrocytes obtained from mice fed a
Mg-deficient diet. To examine the regulation of Na-dependent Mg
efflux in the renal epithelial cell line NRK-52E, Ikari et al.
[40] cultured them for 1-2 days in media containing 5 mM Mg. In
agreement with our results, Mg extrusion in a Na-containing,
Mg-free medium was negligible in control NRK-52E cells but
significant in high-Mg cultured cells. An enhanced
Na+/Mg2+ exchanger activity was also observed
in a mutant cell line from mouse cortical tubular cells that were
selected to grow in media with an extremely high [Mg] of 100 mM
[38]. However, the reasons for these changes have not been
identified in these studies.
Dai et al. [41] have suggested that epithelial cells can
sense environmental [Mg2+] and alter Mg transport
through transcription- and translation-dependent processes to
maintain Mg balance. Epithelial cells from the renal tubule show an
intrinsic adaptation to diminished extracellular magnesium
involving an increased expression of pathways related to Mg uptake
and/or their translocation to the cell membrane [41, 11, 7]. Thus,
we investigated the influence of low and high medium
[Mg2+] on the expression of the 70-kDa protein
representing the candidate Na+/Mg2+
exchanger.
A change of the Na+/Mg2+
exchanger expression is part of the system regulating REC
Mg homeostasis
Our results clearly show that the Na+/Mg2+
exchanger protein is quantitatively altered following changes in
extracellular Mg. We used three different methods, namely
immunobloting, flow cytometry, and immunocytochemistry, and
obtained concordant results showing that the amount of
Na+/Mg2+ exchanger protein is elevated after
exposure of REC to high-Mg conditions and is reduced by incubating
them in the low-Mg solution. Flow cytometric analysis enables real
quantification of the effects and revealed a 36% and 25% increase
and decrease in the Na+/Mg2+ exchanger
abundance per single cell, respectively. As all methods give
similar findings, we conclude that a changed
Na+/Mg2+ exchanger expression is the main
mechanism in the homeostatic up- or down-regulation of Na-dependent
Mg transport. Excess extracellular Mg can be assumed to induce an
increased Mg efflux via the increased abundance of the
Na+/Mg2+ exchanger in the REC cell membrane
and an elevated activity of the protein. First, this protects cells
against Mg overload; and second, it generates a driving force to
maintain the Mg influx into this absorbing cell [42, 21]. The way
that this extracellular [Mg] sensing works is not yet clear. The
Ca/Mg-sensing receptor might be involved because an increased
functional activity of a Na-independent Mg efflux has been
demonstrated after its stimulation [43].
In addition to intrinsic mechanisms [9, 11, 41], extracellular
stimuli can affect Mg transport [34, 43-46]. Various hormones and
other factors are known to activate the
Na+/Mg2+-exchanger-related Mg efflux via
protein kinase C [47, 48], PI 3-kinase [49], or the cAMP-protein
kinase A [30, 50, 51] pathways. The last-mentioned
specifically triggers Na-dependent Mg release from REC [5] and
increases transcellular Mg absorption across the rumen epithelium
[20]. Measurements of REC [cAMP]i have shown that the
concentration of this second messenger is not changed after
modification of the transmembrane Mg gradient. In consequence, we
tested whether Na+/Mg2+ exchanger sensitivity
to intracellular cAMP was influenced by pre-incubation of REC in
media with reduced or elevated [Mg].
Sensitivity of the Na+/Mg2+
exchanger to an intracellular [cAMP] increase is
maintained
As in our previous studies [5, 32], we used db-cAMP, a cell
permeant cAMP analog to modulate the [cAMP]i of REC. The
applied dosage of 100 μM has been shown to increase the
[cAMP]i about 12-fold [5]. Independently of the [Mg] of
the pre-incubation medium, the stimulation of REC with db-cAMP
results in a markedly increased Mg efflux. In agreement with
investigations on erythrocytes [52], the stimulatory effect of cAMP
is lower in control (64%) and high-Mg (109%) REC, than in low-Mg
cells (304%). Following phosphorylation of the transport protein,
cAMP is known to augment the affinity of the
Na+/Mg2+ exchanger for intracellular Mg and
to stimulate Mg extrusion from non-loaded cells [30, 50, 52]. The
latter also provides an explanation for the lower 55% stimulation
seen after db-cAMP application of artificially (A23187) Mg-loaded
REC [5]. A strong cAMP-related stimulation of Mg efflux in
low-Mg pre-incubated REC might be of pathophysiological importance
if this leads to a further loss of intracellular Mg, e.g., by the
extrusion of Mg that has been mobilized from intracellular buffers
and organelles.
The keratinizing epithelium lining the rumen has a very complex
multilayer structure. In contrast to monolayer epithelia,
functional polarization and thus generation of transport gradients
is realized by dominant location of transport proteins to special
cell layers. Like the ruminal Na/K-ATPase [53], the
Na+/Mg2+ exchanger can be found in the cell
membrane of most non-keratinized REC but is particularly expressed
in the basal cells. As the basal cell layer corresponds to the
basolateral membrane of monolayer epithelia, this result is in
accordance with the postulated role of the ruminal
Na+/Mg2+ exchanger for transepithelial Mg
absorption. However, a profound investigation of the
Na+/Mg2+ exchanger localization in the rumen
epithelium is beyond the scope of the present study.
TRPM7 and MagT1 are the main Mg influx systems
existing in REC
In ruminating animals, most of the required Mg is absorbed from the
forestomachs by active transcellular mechanisms [19]. Therefore,
unsurprisingly, REC are equipped with effective Mg influx
mechanisms. These include an ion channel [21] and an
Mg-Cl-cotransport [22], both of which are well characterized, at
least at a functional level. However, until now the molecular
identity of the ruminal Mg influx mechanisms was unknown. To our
knowledge, this study shows for the first time, that TRPM7 and
MagT1 are expressed in REC at both the mRNA and the protein levels.
Our attempt to identify the TRPM6 transcript, which is known to be
expressed in the absorptive or re-absorptive epithelia (intestine,
colon and kidney; NCBI-AceView) was surprisingly unsuccessful. It
could therefore be assumed that TRPM6 is not expressed in ovine
REC. Both primer pairs (PP1 and PP2, figure 6A), however, have
been designed and tested (figure 6A) against human
TRPM6 because of the unknown sequence of the ovine TRPM6 and the
expected high sequential conservation across mammals similar to
those of TRPM7 and/or other TRP channels. Therefore a possibility
that PP1 and PP2 do not match the ovine TRPM6 template could be
also the reason. Taken together, at present, we cannot conclude
whether TRPM6 is or is not expressed in REC. Identification of the
ovine TRPM6 sequence and further experimentation will be necessary
before making the final conclusion.
Both, MagT1 and TRPM7 have been shown to be involved in Mg
uptake [6, 9, 13, 34, 54]. We therefore questioned whether the
influx capacity of REC exposed to Mg deficiency or oversupply was
changed. As in previous studies [5, 32], incubation of REC in
Na-free media containing 5 mM Mg resulted in a marked rise in
[Mg2+]i reflecting the influx of
extracellular Mg. A substantial part of this Mg uptake was
significantly inhibited by imipramine and Co(III)Hex. As in other
cells [51, 55, 56], imipramine has been shown to be a
Na+/Mg2+ exchanger inhibitor in REC [5, 32],
and Co(III)Hex is a known Mg channel blocker [13, 57]. Thus, a
considerable part of the Mg influx observed under these
experimental conditions resulted from the
Na+/Mg2+ exchanger working in the reverse
mode [5, 32] and from channel-mediated transport pathways.
Compared with control cells, both high- and low-Mg REC are
characterized by an increased Mg influx rate. In high-Mg
pre-incubated REC, the larger part (51%) of their elevated influx
rate resulted from facilitation of the imipramine-sensitive
Na+/Mg2+-exchanger-related transport
component. Similar imipramine effects ranging from 53% to 64% have
been reported for high-Mg (5 mM) pre-incubated or high-Mg (101 mM)
adapted renal epithelial cells [38, 56]. Such data are in agreement
with the increased abundance and activity of the
Na+/Mg2+ exchanger found after oversupplying
REC with Mg. In control and low-Mg cells however, Mg influx via
reverse operating Na+/Mg2+ exchangers
amounted to 29% and 18% respectively.
In REC pre-incubated in Mg-deficient medium, the elevation of
their Mg influx rate results mainly from a strong
Co(III)Hex-sensitive transport component. Recently, we showed that
Co(III)Hex specifically inhibits the TRPM7 channel [13]. Thus, our
findings suggest that an ion-channel-mediated mechanism, most
probably TRPM7, facilitates Mg influx into previously Mg-deprived
REC. TRPM7 is thought to be the main regulator of cellular Mg
homeostasis, and the destruction of the TRPM7 gene or its
down-regulation markedly decreases Mg accumulation, reduces the
basal [Mg2+]i, and is followed by growth
arrest and/or reduced viability of cells [4, 34, 58]. Elevated
TRPM7 activity after pre-incubation of REC in Mg-deficient media
should promote the replenishment of intracellular Mg stores and
thus help to avoid such negative consequences of intracellular Mg
deficiency.
Whether and how the REC Na+/Mg2+ exchanger
communicates with other Mg transport systems of the plasma
membrane, such as TRPM7 and MagT1, or of intracellular organelles,
remains unknown. He et al. [34] postulated a potential
cross-talk between TRPM7 and Na+/Mg2+
exchangers via changes of [Mg2+]i. Our own
results showing a reciprocal activity of Co(III)Hex- and
imipramine-sensitive influx components also point to a
communication between these proteins. Such a signaling partnership
between the Na+/Mg2+ exchanger and other ion
transport systems appears essential for cellular Mg
homeostasis.
It has been proposed that TRPM7 has a dominant role in
physiological situations in which a receptor-regulated Mg influx is
required, such as the Mg uptake needed for organismal Mg
homeostasis, cell growth, or neuronal functions [12]. Angiotensin
II and aldosterone, for example, have been shown to regulate TRPM7
mRNA and protein content in renal and vascular smooth muscle cells
[34, 59] and to stimulate angiotensin-II-mediated cell growth in
the latter [34]. This would agree with the finding, that, as shown
by immunoblot and flow cytometric analysis, the expression of REC
TRPM7 protein does not change depending on extracellular Mg, and
that its functional activity is low in non-stimulated control cells
in Mg equilibrium. In contrast to high- (- 29%) and low-Mg
(- 47%) REC, Co(III)Hex treatment reduces their Mg influx rate
by only 12%. Our results agree with data from a recent in vivo
study of Bruno et al. [59] who showed that Mg supplementation
increases the mRNA expression of TRPM7 but has no effect on its
protein content. Thus, other proteins, most probably MagT1, are
presumably responsible for the background level of Mg uptake under
static conditions. For MagT1, this is supported by the generally
high expression and activity levels of the protein observed in our
flow cytometry experiments and in functional studies with
nitrendipine, an inhibitor of MagT1 channels [9].
Conclusion
As expected for proteins relevant to the regulation of Mg
homeostasis and/or directed epithelial transport of the ion,
functional activity and/or expression of REC
Na+/Mg2+ exchangers, TRPM7 and MagT1 proteins
are quantitatively altered by extracellular Mg deficiency or
overload. Thus, we have identified these transport proteins as main
components of the specific intrinsic system appropriately adapting
Mg efflux and influx to Mg status. TRPM7 seems to be most important
for the replenishment of intracellular Mg stores after exposure to
low-Mg conditions. Independently of their Mg status, REC
Na+/Mg2+ exchanger activity can be modulated
by external stimuli via the 3`,5`-cyclic monophosphate
(cAMP)-protein kinase.
Acknowledgments
We express our thanks to Dr T. Viergutz (FBN Dummerstorf) for
helping with the flow cytometric measurements and to Zoran Nikolic
for performing the RT-PCR experiments. We gratefully acknowledge
the valuable technical assistance of Renate Brose (FBN Dummerstorf)
and Heike Pröhl (FBN Dummerstorf). This study was supported by a
research grant from the Deutsche Forschungsgemeinschaft (Schw 642,
MS) and from the Margarete-Markus foundation (Project Animal
Performance and Health, MK).
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