1,25(OH)2D3 stimulates Mg2+
uptake into MDCT cells: modulation by extracellular Ca2+
and Mg2+
Gordon
Ritchie1,
Dirk
Kerstan1,
Long-Jun
Dai1,
Hyung Sub
Kang1,
Lucie
Canaff2,
Geoffrey N.
Hendy2, and
Gary A.
Quamme1
1 Department of Medicine, University Hospital, University of
British Columbia, Vancouver, British Columbia V6T 1Z3; and
2 Departments of Medicine, Physiology and Human Genetics, McGill
University and Royal Victoria Hospital, Montreal, Quebec, Canada H3A
1A1
 |
ABSTRACT |
The distal convoluted
tubule plays a significant role in renal magnesium conservation.
Although the cells of the distal convoluted tubule possess the vitamin
D receptor, little is known about the effects of
1
,25-dihydroxyvitamin D [1,25(OH)2D3] on
magnesium transport. In this study, we examined the effect of
1,25(OH)2D3 on distal cellular magnesium uptake
and the modulation of this response by extracellular Ca2+
and Mg2+ in an immortalized mouse distal convoluted tubule
(MDCT) cell line. MDCT cells possess the divalent cation-sensing
receptor (CaSR) that responds to elevation of extracellular
Ca2+ and Mg2+ concentrations to diminish
peptide hormone-stimulated Mg2+ uptake. Mg2+
uptake rates were determined by microfluorescence in
Mg2+-depleted MDCT cells. Treatment of MDCT cells with
1,25(OH)2D3 for 16-24 h stimulated basal
Mg2+ uptake in a concentration-dependent manner from basal
levels of 164 ± 5 to 210 ± 11 nM/s, representing a 28 ± 3% change. Pretreatment with actinomycin D or cycloheximide
abolished
1,25(OH)2D3-stimulated.Mg2+
uptake (154 ± 18 nM/s), suggesting that
1,25(OH)2D3 stimulates Mg2+ uptake
through gene activation and protein synthesis. Elevation of
extracellular Ca2+ inhibited
1,25(OH)2D3-stimulated Mg2+ uptake
(143 ± 5 nM/s). Preincubation of the cells with an antibody to
the CaSR prevented the inhibition by elevated extracellular Ca2+ of 1,25(OH)2D3-stimulated
Mg2+ uptake (202 ± 8 nM/s). Treatment with an
antisense CaSR mRNA oligodeoxynucleotide also abolished the effects of
extracellular Ca2+ on
1,25(OH)2D3-responsive Mg2+ entry.
This showed that elevated extracellular calcium modulates 1,25(OH)2D-mediated responses through the CaSR. In summary,
1,25(OH)2D3 stimulated Mg2+ uptake
in MDCT cells, and this is dependent on de novo protein synthesis.
Elevation of extracellular Ca2+, acting via the CaSR,
inhibited 1,25(OH)2D3-stimulated
Mg2+ entry. These data indicate that
1,25(OH)2D3 has important effects on the
control of magnesium entry in MDCT cells and these responses can be
modulated by extracellular divalent cations.
1
,25-dihydroxyvitamin D; calcium/magnesium-sensing receptor; adenosine 3',5'-cyclic monophosphate measurements; intracellular
magnesium determinations; magnesium uptake; fluorescence
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INTRODUCTION |
THE HORMONALLY ACTIVE
METABOLITE of vitamin D, 1,25-dihydroxyvitamin D
[1,25(OH)2D3], has an important role in
regulating mineral metabolism at the level of the intestine, bone, and
kidney (1, 32, 51). It is clear that
1,25(OH)2D3 increases intestinal calcium and
magnesium absorption and regulates bone formation, but the actions of
1,25(OH)2D3 on the kidney are not as well
characterized (1, 31, 51). On balance, clinical and
experimental observations support the notion that
1,25(OH)2D3 increases calcium and magnesium reabsorption within the kidney (1, 19, 25, 26, 33, 36, 38,
45). Nevertheless, some studies reported little, if any, affect
on renal divalent cation conservation (9, 13, 30, 39, 40).
This discrepancy may be due to associated vitamin D-induced changes in
plasma calcium and magnesium concentrations that affect renal divalent
cation handling. An extracellular
Ca2+/Mg2+-sensing receptor (CaSR) that
modulates calcium and magnesium reabsorption in the loop of Henle and
distal convoluted tubule has been identified along the length of the
kidney tubule (27, 43). The distal convoluted tubule plays
an important role in renal magnesium conservation as it has final
control of urinary magnesium excretion (43). We have
shown that elevated extracellular Ca2+ or Mg2+
inhibits hormone-mediated Mg2+ uptake in a distal
convoluted tubule cell line (3). Our evidence is that
hypercalcemia and hypermagnesemia diminish distal reabsorption, in
part, through CaSR inhibition of peptide hormone receptor-mediated responses (3). In the present study, we investigated
whether 1,25(OH)2D3 stimulates
Mg2+ uptake into distal convoluted tubule cells and whether
elevated extracellular Ca2+ or Mg2+ may inhibit
these responses. Our notion is that 1,25(OH)2D3
plays a role in magnesium conservation at both the intestinal and renal level and that the latter is modulated by the prevailing extracellular Ca2+ and Mg2+ concentrations through the CaSR.
Accordingly, increases in 1,25(OH)2D3 may lead
to enhanced magnesium conservation, no change, or increased urinary excretion, depending on the magnitude of changes in plasma calcium and magnesium concentration.
To date, there are no experiments reporting the direct effects of
vitamin D metabolites on cellular magnesium transport. In the present
studies, we used mouse distal convoluted tubule (MDCT) cells to
investigate the actions of 1,25-(OH)2D3 on
Mg2+ uptake rates. MDCT cells have been extensively used to
study cellular mechanisms of calcium and magnesium absorption within the distal tubule (23, 43). Parathyroid hormone (PTH),
glucagon, and arginine vasopressin (AVP) stimulate Mg2+
uptake in MDCT cells (16, 17). In a recent study, we
showed that MDCT cells also possess a polyvalent cation-sensing
mechanism that is responsive to extracellular Mg2+ and
Ca2+ (4). We documented the expression of the
parathyroid/kidney Ca2+-sensing receptor (i.e., CaSR) in
MDCT cells and suggested that it is responsible, either entirely or in
part, for the extracellular polyvalent cation-sensing mechanism in
these cells (4). We also found that activation of this
mechanism with either extracellular Ca2+, Mg2+,
or the polyvalent cation neomycin inhibited PTH-, glucagon-, and
AVP-stimulated cAMP release (4) as well as
hormone-stimulated Mg2+ uptake in MDCT cells
(3). These studies indicated that this cation-sensing mechanism in immortalized MDCT cells plays an
important role in modulating hormone-mediated intracellular signals in
response to changes in extracellular Mg2+ as well as
Ca2+. Yang et al. (54) have reported that MDCT
cells also express 1,25(OH)2D3- and
PTH-responsive 25(OH)D 24-hydroxylase (54), and Friedman
and Gesek (22) have shown that
1,25(OH)2D3 accelerates PTH-dependent
Ca2+ uptake, suggesting that vitamin D receptors are
present in this cell line and play a role in mineral metabolism
(22). Accordingly, these cells provide a useful model by
which to determine the effects of 1,25(OH)2D3
on Mg2+ handling in the distal convoluted tubule, which is
difficult to study in situ or dissect out for functional measurements.
The present studies demonstrate that
1,25(OH)2D3 stimulates Mg2+ uptake
in MDCT cells, providing evidence that the metabolite might affect
magnesium absorption in the intact distal tubule. Moreover, elevation
of divalent cations, acting through the CaSR, modulates
1,25(OH)2D3-mediated Mg2+ entry in
this cell line.
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MATERIALS AND METHODS |
Basal DMEM and Ham's F-12 media were from GIBCO
Laboratories, Grand Island, NY. FCS was from Flow Laboratories
(McLean, VA). Mag-fura 2-acetoxymethyl ester (AM) was
obtained from Molecular Probes (Eugene, OR).
1,25(OH)2D3 and other materials were from Sigma
(St. Louis, MO).
Cell preparation.
Immortalized MDCT cells were kindly provided by Dr. P. A. Friedman. They were cultured in DMEM-Ham's F-12 media, 1:1,
supplemented with 10% FCS, 1 mM glucose, 5 mM L-glutamine,
50 U/ml penicillin, and 50 µg/ml streptomycin in a humidified
environment of 5% CO2-95% air at 37°C. For cAMP
determinations, the MDCT cells were cultured to confluence in 24-well
plastic dishes. Sixteen hours before the cAMP measurements were made,
the culture medium was changed to one containing 0.2% BSA rather than
FCS. For the fluorescence studies, confluent cells were washed three
times with PBS containing 5 mM EGTA, trypsinized, and seeded on glass
coverslips. Aliquots of harvested cells were allowed to settle onto
sterile glass coverslips in 100-mm Corning tissue culture dishes, and
the cells were grown to confluence over 4-6 days in supplemented
media as described above. The normal media contained 0.6 mM magnesium
and 1.0 mM calcium. In the experiments indicated, the cells were
cultured in nominally magnesium-free media (<0.01 mM) for 16 h
before study (0.2% BSA replaced FCS during this period). Other
constituents of the magnesium-free media were identical to those of the
complete media.
cAMP measurements.
cAMP was determined in confluent MDCT cell monolayers cultured in
24-well plates in DMEM-Ham's F-12 media without serum, as previously
reported (4). After addition of various hormones, MDCT
cells were incubated at 37°C for 5 min in the presence of 0.1 mM
IBMX. cAMP was extracted with 5% trichloroacetic acid, which was
removed with ether acidified with 0.1 N HCl. The aqueous phase was
dried, dissolved in Tris-EDTA buffer, and then cAMP was measured with a
radioimmunoassay kit (Diagnostic Products, Los Angeles, CA).
Cytoplasmic Mg2+
measurements.
Coverslips were mounted within a perfusion chamber, and the
attached subconfluent cells were incubated with 5 µM mag-fura 2-AM
dissolved in pluronic acid F-127 (0.125%; Molecular Probes) for
determination of intracellular Mg2+ concentration
([Mg2+]i) in media for 20 min at 37°C.
Cells were subsequently washed three times with a buffered salt
solution containing (in mM) 145 NaCl, 4.0 KCl, 0.8 Ka2HPO4, 0.2 KH2PO4,
1.0 CaCl2, 5.0 glucose, and 20 HEPES-Tris, at pH
7.4. The MDCT cells were incubated for an additional 20 min to
allow for complete deesterification and washed once before measurement
of fluorescence.
Epifluorescence microscopy was used to monitor changes in the mag-fura
2 fluorescence of the MDCT cell monolayer. The chamber was mounted on
an inverted Nikon Diaphot-TMD microscope with a Fluor ×100 objective,
and, under oil immersion, fluorescence within a single cell was
monitored over the course of the study. Fluorescence was recorded at
1-s intervals by using a dual-excitation-wavelength spectrofluorometer
(Delta-scan, Photon Technologies, Princeton, NJ) with excitation for
mag-fura 2 at 335 and 385 nm (chopper speed set at 100 Hz) and emission
at 505 nm. Media changes were made without interruption in recording.
The free [Mg2+]i was calculated from the
ratio of the fluorescence at the two excitation wavelengths as
previously described (15) by using a dissociation constant
(Kd) of 1.4 mM for the mag-fura
2-Mg2+ complex. The minimum (Rmin) and maximum
(Rmax) ratios were determined for the cells at the end of
each experiment by using 20 µM digitonin. Rmax for
mag-fura 2 was found by the addition of 50 mM MgCl2 in the
absence of Ca2+, and Rmin was obtained by
removal of Mg2+ and addition of 100 mM EDTA, pH 7.2. The
excitation spectrum of the cellular mag-fura 2 under these conditions
was similar to that of free mag-fura 2 in the same solutions.
Antibody block of the CaSR in MDCT cells.
In those experiments where the CaSR was blocked with a specific
antibody, MDCT cells were Mg2+ depleted for 16 h and
then incubated with the antibody for 1.5-2 h at 36°C. A mouse
monoclonal antibody (ADD) raised against a peptide comprising residues
214-236 of the CaSR was used (20). The antibody,
which was provided by Drs. P. K. Goldsmith and A. M. Spiegel
(National Institutes of Health) and K. V. Rogers (NPS Pharmaceuticals), has been extensively characterized with respect to
specificity for the parathyroid/kidney CaSR (24). The
antibody was diluted to a final concentration of 1.6 µg/ml in a
buffer solution containing 0.02% BSA. The control cells were incubated with mouse IgG. The cells were washed twice in an appropriate buffer
solution, and intracellular cAMP content or microfluorescence experiments were performed.
Antisense oligodeoxynucleotides designed to the CaSR in MDCT
cells.
A second approach was used to block extracellular
Ca2+/Mg2+ sensing in MDCT cells.
Oligonucleotide (ODN) antisense methodology, a strategy designed to
bind specifically and efficiently to the complementary sequence of a
targeted mRNA, has been extensively used in in vitro systems.
Twenty-base pair phosphorothioate-derivatized sense and antisense CaSR
ODNs were synthesized on an automated solid-phase synthesizer by using
standard phosphoramide chemistry. The sequence of the sense ODN was
5'-GAGAAGGCAGAGCCATGGCATGG-3', and that of the antisense ODN was
5'-CCATGCCATGGCTCTGCCTTCTC-3'. The MDCT cells were incubated in buffer
containing 0.1% BSA with the ODN, 1.0 nmol/ml, for 48-72 h before
fluorescence measurements were performed. Cationic transfection agents,
such as lipofectamine, were avoided because they interfere with
Mg2+ transport, presumably because of membrane damage.
Protein was extracted from the cells for Western blot analysis.
Western blots.
Cells were lysed in triple detergent buffer [50 mM Tris · HCl
(pH 8.0), 150 mM NaCl, 0.02% NaN3, 0.1% SDS, 1 mM EDTA,
100 µg/ml polymethylsulfonyl fluoride, 2 µg/ml leupeptin, 2 µg/ml aprotonin, 0.1% NP-40, and 0.5% sodium deoxycholate] for 5 min at
0°C. The cell lysates were spun at 1,200 g for 2 min at
4°C, and the supernatants were stored at
80°C. Aliquots were
electrophoresed through 8% SDS-polyacrylamide gels and blotted onto
polyvinylidene difluoride membranes (Bio-Rad). Membranes were rinsed in
TBST (10 mM Tris · HCl, pH 8.0, 150 mM NaCl, 0.05% Tween 20),
blocked with 5% dried milk powder in TBST for 1-2 h, and
incubated with ADD, the antibody against the CaSR (24). As
a control, immunoblotting was carried out as described above with the
antiserum preabsorbed for 1 h with the peptide (10 µg/ml)
against which it was raised. Blots were also incubated with a
-tublin monoclonal antibody (Cedarlane Laboratories, Hornby, ON).
Statistical analysis.
Representative tracings of fluorescent intensity ratios are given,
and all results are means ± SE where indicated. Significance was
determined by Tukey's analysis of variance where indicated, and
comparisons among groups of data were made by using Student's t-test. A probability of P < 0.05 was taken
to be statistically significant.
 |
RESULTS |
1,25(OH)2D3 stimulates
Mg2+ entry in MDCT cells.
MDCT cells were treated with 1,25(OH)2D3 for
16 h before experimentation. During this time they were incubated
in media without magnesium to deplete them of intracellular
Mg2+ (16). The cells were then placed in 1.5 mM MgCl2, and [Mg2+]i was
monitored by fluorescence. Figure
1 shows a typical result of a
fluorescence experiment of MDCT cells pretreated with
1,25(OH)2D3 compared with control cells.
The rate of entry into control cells, measured over the initial
500 s, was 164 ± 5 nM/s, n = 6. Pretreatment with 1,25(OH)2D3 increased Mg2+
uptake by 59 ± 4% to 260 ± 15 nM/s, n = 5. Next, we determined that vitamin D-stimulated Mg2+ uptake
required a minimum of 3 h for the largest response and was
maintained for 24 h. We have previously shown that the channel blocker nitrendipine inhibits Mg2+ uptake into MDCT cells
(36). Nitrendipine (50 µM) inhibited Mg2+
uptake to a similar extent in
1,25(OH)2D3-treated cells (25 ± 5 nM/s,
n = 4) as in control cells (24 ± 5 nM/s,
n = 5), indicating that the vitamin D metabolite
1,25(OH)2D3 stimulated Mg2+ entry
and not intracellular Mg2+ release or inhibition of
Mg2+ efflux. 1,25(OH)2D3 increased
Mg2+ uptake in a concentration-dependent manner, with
half-maximal responses (185 ± 3 nM/s) occurring at
~10
9 M (Fig. 2). This
study indicates that 1,25(OH)2D3 stimulates Mg2+ entry into MDCT cells.

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Fig. 1.
1,25-Dihydroxyvitamin D [1,25(OH)2D3]
stimulates Mg2+ uptake in Mg2+-depleted
mouse distal convoluted tubule (MDCT) cells. MDCT cells were cultured
in Mg2+-free media (<0.01 mM) for 16 h. Where
indicated, 10 7 M 1,25(OH)2D3 was
added to the buffer solution from a stock ethanol solution during the
16-h Mg2+-depletion period. Fluorescence studies were
performed in buffer solutions in the absence of external magnesium,
and, where indicated, MgCl2 (1.5 mM final concentration)
was added to observe changes in intracellular Mg2+
concentration ([Mg2+]I). The buffer solutions
contained (in mM) 145 NaCl, 4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 1.0 CaCl2, 5.0 glucose, and 10 HEPES-Tris, pH 7.4, with and
without 1.5 mM MgCl2. Fluorescence was measured at 1 data
point/s with 25-point signal averaging, and the tracing was smoothed
according to methods previously described (17).
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Fig. 2.
Concentration dependence of 1,25(OH)2D3
stimulation of Mg2+ entry in MDCT cells. Values are
means ± SE for 3-6 cells. MDCT cells were treated with the
given 1,25(OH)2D3 concentrations for 16 h
before fluorescence determinations. The rate of Mg2+
influx, as determined by
d([Mg2+]i)/dt, was measured with
the given 1,25(OH)2D3 concentrations by using
fluorescence techniques described in the legend for Fig. 1.
d([Mg2+]i)/dt values were
determined over the first 500 s of fluorescence measurements.
Significantly different from control values,*P < 0.05.
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Steroid hormones may mediate cellular responses through nongenomic or
genomic actions (10). Nongenomic effects of
1,25(OH)2D3 include rapid changes in
phosphoinositide metabolism, increases in intracellular
Ca2+ concentration, elevation of cAMP levels, and
activation of protein kinase C (reviewed in Ref. 10). We
have reported that many of these signaling pathways may influence
hormone-mediated Mg2+ uptake in MDCT cells (16,
17). Accordingly, we tested the acute (<20 min) effects of
vitamin D metabolites on the mean Mg2+ entry rate. The
acute treatment of MDCT cells for 20 min with 1,25(OH)2D3 had no effect on Mg2+
uptake (178 ± 17 nM/s, n = 3), which suggested
that MDCT cells do not affect transport by nongenomic mechanisms.
Genomic mechanisms involve changes in gene expression, requiring some
hours before de novo protein synthesis. We determined this by using the
transcriptional and translational inhibitors actinomycin D and
cycloheximide, respectively. Because of the toxicity of actinomycin D,
we measured the effect of the inhibitor for a 3- to 4-h incubation
period. 1,25(OH)2D3 was equally effective in
stimulating Mg2+ entry after 3 h of incubation as it
was at 16 h (Fig. 3A).
The MDCT cells were initially Mg2+ depleted by incubating
them in magnesium-free media for 16 h before the addition of
1,25(OH)2D3, with and without actinomycin (5 µg/ml). The 1,25(OH)2D3-treated
Mg2+-depleted cells were incubated for a further 4-5
h; then, Mg2+ uptake was performed by microfluorescence.
Actinomycin D did not alter basal Mg2+ uptake in cells that
had been Mg2+ depleted for 16 h (18). In
those cells treated with actinomycin D, 10
7 M
1,25(OH)2D3 failed to stimulate
Mg2+ entry, indicating that the vitamin D metabolite
increases uptake by mechanisms involving transcriptional processes
(Fig. 3A). Next, we determined the effect of a translational
inhibitor, cycloheximide. Cycloheximide (1.0 µg/ml) was added with
10
7 M 1,25(OH)2D3 for 16 h
before fluorescence studies. 1,25(OH)2D3 did
not stimulate Mg2+ uptake (155 ± 18 nM/s,
n = 4) in those cells treated with cycloheximide, indicating that translational processes were also involved in this
response (Fig. 3B). Accordingly, these results indicate that 1,25(OH)2D3 stimulates Mg2+ entry
by genomic mechanisms involving de novo synthesis of transport proteins.

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Fig. 3.
1,25(OH)2D3-stimulated Mg2+
uptake requires transcriptional/translational processes. Values are
means ± SE. A: MDCT cells were Mg2+
depleted for 16 h, and 10 7 M
1,25(OH)2D3 was then added with and without
actinomycin D (5.0 µg/ml) as indicated for 4-5 h before
fluorescence determinations. Mg2+ uptake was determined
with microfluorescence according to methods described in the legend for
Fig. 1. B: MDCT cells were treated with 10 7 M
1,25(OH)2D3 for 16 h before the
determination of Mg2+ uptake. In those cells indicated,
cycloheximide (Cyclo; 1.0 µg/ml) was added with
1,25(OH)2D3 16 h before Mg2+
uptake and cAMP determinations. Actinomycin D and cyloheximide were
added from stock solutions of DMSO; the final DMSO concentration did
exceed 0.01% (vol/vol), which had no effect on Mg2+
uptake. Significantly different from control values, *P < 0.01.
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Acute effects of polyvalent cations on
1,25(OH)2D3-stimulated
Mg2+ uptake.
We have shown that elevation of extracellular polyvalent cations,
neomycin, Mg2+, and Ca2+ inhibit PTH-,
glucagon-, and AVP-mediated cAMP formation and glucagon- and
AVP-stimulated Mg2+ entry but not basal uptake rates in
MDCT cells (3, 4). MDCT cells were treated with
10
7 M 1,25(OH)2D3 for 16 h
before the addition of polyvalent cations and determination of
Mg2+ uptake. Acute (5-min) addition of 50 µM neomycin or
elevation of 5.0 mM extracellular Mg2+ or 5.0 mM
Ca2+did not affect Mg2+ uptake into
1,25(OH)2D3-treated, Mg2+-depleted
cells (Fig. 4). Accordingly, the acute
activation of the CaSR does not alter
1,25(OH)2D3-stimulated Mg2+ entry
in the cells pretreated with the vitamin D metabolite 16 h before
experimentation.

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Fig. 4.
Summary of the acute effects of cation-sensing receptor (CaSR)
activation on 1,25 (OH)2D3-stimulated
Mg2+ uptake. Values are means ± SE for 3-5
cells. Measurement d([Mg2+]i)/dt
was performed in Mg2+-depleted cells treated with or
without 1,25(OH)2D3 for 16 h before
performance of fluorescence. Mg2+ uptake was determined
with 1.5 mM extracellular Mg2+ in the absence or presence
of 50 µM extracellular neomycin (Neo); 5.0 mM Mg2+ or 5.0 mM Ca2+ was added 5 min before determination of
Mg2+ uptake. In the studies that tested high levels of
extracellular Mg2+, 5.0 rather than 1.5 mM
MgCl2 was used. The studies were performed as described in
the legend for Fig. 1. The Mg2+ uptake rate was determined
over 500 s after addition of 1.5 or 5.0 mM Mg2+.
Significantly different from control values,*P < 0.05.
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Chronic effects of elevated extracellular
Ca2+ on
1,25(OH)2D3-stimulated Mg2+ uptake.
Next, we determined the effects of chronic elevation of extracellular
Ca2+ on 1,25(OH)2D3-stimulated
Mg2+ uptake. A buffer solution containing high
CaCl2 (5.0 mM) was added with
1,25(OH)2D3 for 16-20 h before
fluorescence measurements were made. In support of our earlier
findings, high extracellular Ca2+ for 16-20 h did not
alter basal Mg2+ uptake (165 ± 7 nM/s,
n = 4) (Fig. 5). However,
high extracellular Ca2+ inhibited
1,25(OH)2D3-stimulated Mg2+ uptake
(166 ± 6 nM/s, n = 4). Accordingly, the addition
of high extracellular Ca2+ with
1,25(OH)2D3 diminished vitamin D-mediated
Mg2+ entry. These findings suggest that signaling via the
CaSR may alter 1,25(OH)2D3-induced synthesis of
proteins involved in Mg2+ transport.

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Fig. 5.
Chronic activation of CaSR-sensing inhibits
1,25(OH)2D3-stimulated Mg2+ uptake.
Values are means ± SE for 3-5 cells. MDCT cells were treated
with 10 7 M 1,25(OH)2D3 with and
without 5.0 mM CaCl2 for 16 h before microfluorescence
measurements. Mg2+ uptake was determined with 1.5 mM
extracellular Mg2+ in the absence or presence of 5.0 mM
Ca2+ as indicated. We have previously shown that high
extracellular Ca2+ does not affect basal Mg2+
uptake; i.e., the entry pathway is selective for Mg2+
(43). The studies were performed as described in the
legend for Fig. 1. The Mg2+ uptake rate was determined over
500 s after addition of 1.5 mM MgCl2. Significantly
different from control values,*P < 0.01.
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CaSR antibody blocks extracellular
Ca2+ inhibition of
1,25(OH)2D3-stimulated
Mg2+ uptake.
If elevation of extracellular Ca2+ inhibits
1,25(OH)2D3-stimulated transport through
CaSR-mediated transcriptional processes, then blocking this receptor
should abolish the chronic effects of extracellular calcium. To do
this, MDCT cells were incubated with a specific CaSR antibody. To
substantiate that the CaSR was blocked, we measured the effect of
extracellular Ca2+ and Mg2+ on PTH-mediated
cAMP formation in control cells and in those pretreated with the CaSR
antibody. PTH stimulated intracellular cAMP accumulation by about
threefold (72 ± 8 pmol · mg
protein
1 · 5 min
1) above control
values (25 ± 1 pmol · mg
protein
1 · 5 min
1). The presence of
5.0 mM Ca2+ or 5.0 mM Mg2+ abolished
PTH-stimulated cAMP formation (33 ± 2 and 29 ± 1 pmol · mg protein
1 · 5 min
1, respectively) (Fig.
6). Preincubation of MDCT cells with the antibody prevented the effect of extracellular Ca2+
(60 ± 5 pmol · mg protein
1 · 5 min
1) or Mg2+ (72 ± 8 pmol · mg
protein
1 · 5 min
1) on PTH-sensitive
cAMP release (72 ± 8 pmol · mg
protein
1 · 5 min
1). Accordingly,
the antibody blocked the activation of the CaSR and the inhibition of
hormone-mediated cAMP formation. With this knowledge, we determined
whether antibody blockade of the CaSR would inhibit the effect of
elevated Ca2+ on
1,25(OH)2D3-stimulated Mg2+ uptake.
We depleted MDCT cells of Mg2+ for 16 h before
experimentation and incubated them with ADD antiserum to the CaSR for
2 h. The cells were then treated with
1,25(OH)2D3 with and without 5.0 mM
CaCl2 for an additional 4-5 h before microfluorescence studies were performed. MDCT cells treated with the antibody responded normally to 1,25(OH)2D3 (210 ± 11 nM/s,
n = 5) regardless of the presence of elevated
extracellular Ca2+ (202 ± 8 nM/s, n = 4) (Fig. 7). This study indicates that
extracellular Ca2+ chronically inhibits
1,25(OH)2D3-mediated Mg2+ uptake
through the activation of CaSR-mediated mechanisms.

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Fig. 6.
CaSR antibody blocks extracellular polyvalent cation modulation of
parathyroid hormone (PTH)-mediated cAMP formation. Values are
means ± SE for 10 observations. In the studies indicated, MDCT
cells were preincubated with 1.6 µg/ml of a mouse monoclonal antibody
(ADD) or 1.6 µg/ml mouse IgG in a buffer solution containing 0.02%
BSA and (in mM) 0.5 MgCl2, 1.0 CaCl2, 145 NaCl,
4.0 KCl, 0.8 K2HPO4, 0.2 KH2PO4, 5.0 glucose, and 20 HEPES-Tris, pH 7.4, for 2 h before measurement of intracellular PTH-stimulated cAMP.
Where indicated, either 5.0 mM CaCl2 or 5.0 mM
MgCl2 was added for 5 min before PTH and cAMP
concentrations were measured with radioimmunoassay after a further
5-min incubation period. Significantly different from control
values,*P < 0.01.
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Fig. 7.
Specific antibody to the CaSR blocks extracellular Ca2+
inhibition of 1,25(OH)2D3-stimulated
Mg2+entry. Values are means ± SE for 3-5 cells.
MDCT cells were Mg2+ depleted for 16 h and incubated
with CaSR mouse monoclonal antibody ADD (1.6 µg/ml) for 2 h. The
cells were then treated with 10 7 M
1,25(OH)2D3 with and without 5.0 mM
CaCl2 where indicated, and Mg2+ uptake
measurement was performed with microfluorescence. The Mg2+
uptake rate was determined over 500 s after addition of 1.5 mM
MgCl2. Significantly different from control
values,*P < 0.05.
|
|
Antisense ODN to the CaSR blocks extracellular
Ca2+ inhibition of
1,25(OH)2D3-stimulated
Mg2+ uptake.
To substantiate the antibody studies, we used antisense ODN technology
to block CaSR receptor expression and its function. The MDCT cells were
loaded with sense or antisense CaSR ODNs. Cells with antisense ODN
expressed little CaSR as determined by Western analysis (Fig.
8). In the representative experiment
shown, the CaSR-to-
-tublin densitometry ratio was 2.77 for no ODN;
2.6 for sense ODN; and 0.17, for antisense ODN, the last value
representing 6.5% of the sense ODN ratio. Extracellular
Ca2+ did not inhibit PTH-mediated cAMP formation in MDCT
cells treated with antisense ODN to the CaSR (67 ± 1 pmol · mg protein
1 · 5 min
1), whereas the response in cells with sense ODN was
similar to that observed with control cells (32 ± 2 pmol · mg protein
1 · 5 min
1) (Fig. 9).
Accordingly, antisense ODN blocks expression and function of the CaSR.
We used this model to test whether extracellular Ca2+
diminishes 1,25(OH)2D3-stimulated
Mg2+ uptake through the CaSR. Mg2+ uptake in
sense ODN-treated cells was 115 ± 2% (n = 5) of
control cells (Fig. 10).
1,25(OH)2D3 stimulated Mg2+ entry
by 189 ± 50% (n = 4) in the sense CaSR
ODN-treated cells in the presence of normal extracellular
Ca2+ concentration (1.0 mM). Elevated extracellular
Ca2+ (5.0 mM) inhibited
1,25(OH)2D3-stimulated Mg2+ uptake
(87 ± 27%, n = 6) in these cells. In contrast,
1,25(OH)2D3-stimulated Mg2+ uptake
was 172 ± 31% (n = 6) in cells loaded with
antisense ODN in the presence of normal Ca2+ and 181 ± 26% (n = 9) in cells incubated with 5.0 mM
Ca2+. Accordingly, the antisense CaSR ODN inhibits CaSR
expression and mitigates the effect of extracellular Ca2+
on 1,25(OH)2D3-stimulated Mg2+
uptake. These studies, using antibody or antisense ODN technology, clearly indicate that extracellular Ca2+ chronically
inhibits 1,25(OH)2D3-mediated Mg2+
uptake through activation of CaSR-mediated mechanisms.

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|
Fig. 8.
Antisense oligodeoxynucleotides (ODN) to the CaSR
diminish receptor expression in MDCT cells. Western analysis of the
CaSR was performed in MDCT cells treated with sense or antisense ODNs
at concentrations of 1 nmol/ml for 36-42 h before experimentation.
Cell extracts (50 µg protein) were subjected to SDS-PAGE on a
4-12% gradient gel. The blot was stained with CaSR mouse
monoclonal antibody, and an identical blot was stained with -tubulin
antibody. Lane 1, control MDCT cells (no ODN); lane
2, sense ODN (CaSR sense); lane 3, antisense ODN (CaSR
antisense). The CaSR bands were demonstrated to be specific after the
blot was stained with the same antiserum preincubated with the peptide
against which it was raised (data not shown). Western blots were
performed twice with similar results.
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|

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Fig. 9.
Antisense CaSR ODN diminish CaSR inhibition of PTH-mediated cAMP
formation. Values are means ± SE for 6 individual observations.
In the studies indicated, MDCT cells were loaded with sense or
antisense ODN 72 h before measurement of intracellular
PTH-stimulated cAMP. Where shown, 5.0 mM CaCl2 was added
for 5 min before PTH and cAMP concentrations were measured with
radioimmunoassay after a further 5-min incubation period. Significantly
different from control values,*P < 0.01.
|
|

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Fig. 10.
Antisense ODN to the CaSR diminishes extracellular
Ca2+ inhibition of
1,25(OH)2D3-stimulated Mg2+entry.
Values are means ± SE for 4-8 cells. MDCT cells were loaded
with sense (S) or antisense (AS) ODN designed to the CaSR for 72 h
before fluorescence studies. The cells were Mg2+
depleted for 16 h, treated with 10 7 M
1,25(OH)2D3 for 3-4 h with 5.0 mM
CaCl2. The Mg2+ uptake rate was determined over
500 s after addition of 1.5 mM MgCl2.
|
|
 |
DISCUSSION |
The role of 1,25(OH)2D3 in renal
magnesium reabsorption.
Little is known about the effects of
1,25(OH)2D3 on tubular magnesium
transport. On balance, urinary clearance data from clinical observations and experimental studies indicate that
1,25(OH)2D3 may stimulate renal magnesium
handling (1, 13, 25, 26, 30, 33, 36, 38, 39, 45).
There have been no micropuncture studies performed to localize
the actions of 1,25(OH)2D3 on magnesium transport. The data concerning the effects of
1,25(OH)2D3 on renal calcium absorption are
more substantial but inconclusive (5, 7-9, 31, 49).
The most convincing data demonstrating that 1,25(OH)2D3 may have a direct effect on calcium
transport were obtained by using isolated cells. Bindels et al.
(5) and Van Baal et al. (50) showed that
1,25(OH)2D3 increased calbindin D28k protein levels and stimulated transcellular calcium
absorption in primary cultures of rabbit distal connecting tubule and
cortical collecting duct cells. The maximal transport response occurred at ~48 h posttreatment, suggesting that the response involved initiation of transcriptional processes (5). The response
to 1,25(OH)2D3 was independent of PTH and not
additive to PTH-stimulated calcium transport. The above-mentioned
authors also have biochemical evidence, as they showed that
1,25(OH)2D3 increased calbindin D28k mRNA and protein content without a change in
Na+/Ca2+ exchange or
Ca2+-ATPase RNA or protein in rabbit distal connecting
tubule and cortical collecting duct cell preparations
(50). Recently, Hoenderop et al. (29) have
identified an apical Ca2+ channel in
1,25(OH2)D3-responsive cells that is
responsible for calcium absorption in this segment. They have reported
that 1,25(OH)2D3 stimulates Ca2+
transport in rabbit cortical collecting duct cells
(29). The second study to clearly show effects of
vitamin D metabolites on calcium transport was performed in MDCT cells
by Friedman and Gesek (22). These co-workers reported that
1,25(OH)2D3 did not alter basal
Ca2+ uptake but accelerated PTH-dependent calcium entry
rates. This response was rapid, concentration dependent, significant at
2 h, maximal by 5 h, and mediated by translational processes
because it was inhibited by cycloheximide (22). The
reasons for the discrepancies between the above-mentioned two reports
are not known; examples may be the different cell types or the
techniques used to measure calcium transport in the two separate
studies. Nevertheless, it is clear that
1,25(OH)2D3 increases calcium-binding protein
in distal tubules, suggesting that it has significant actions on basal
or hormone-mediated calcium transport.
1,25(OH)2D3 increases divalent cation-binding
proteins, such as calbindin D9K or calbindin
D28k, in distal tubule cells, including convoluted segment,
connecting tubule, and collecting duct cells (34, 35, 46,
48). In a preliminary report, we have shown that
1,25(OH)2D3 stimulates calbindin
D9K in the MDCT cell line (14) The calbindin D
group is thought to be involved, either directly or indirectly, in
epithelial calcium transport (6-9, 21, 31, 49, 53). Experimental evidence also supports the notion that these proteins bind
magnesium, and some investigators have postulated that they are
involved in epithelial magnesium transport (2, 12, 28).
The present studies with MDCT cells, a widely used model of the distal
convoluted tubule, supports the notion that
1,25(OH)2D3 may affect renal magnesium
conservation. The hormonally active vitamin D metabolite
1,25(OH)2D3 increases Mg2+ entry
rates in MDCT cells in a concentration-dependent fashion (Fig. 2). The
response involves de novo protein synthesis, as it was sensitive to
actinomycin D and cycloheximide, inhibitors of transcription and
translation, respectively (Fig. 3). The evidence supports the notion
that 1,25(OH)2D3 stimulates the synthesis of
proteins that are involved with Mg2+ entry, perhaps
as yet uncharacterized Mg2+ channels
(42). As the distal convoluted tubule is the
terminal nephron segment providing final control of renal magnesium
excretion, the influence of 1,25(OH)2D3 may
play an important part in magnesium balance.
Effect of extracellular
Mg2+/Ca2+
sensing on 1,25(OH)2D3-mediated
Mg2+ uptake.
The extracellular CaSR plays an important role in mineral balance by
controlling, among other things, parathyroid gland function and renal
calcium and magnesium reabsorption (11, 27, 42). The CaSR
mediates a host of intracellular signaling pathways. The CaSR has been
best characterized in parathyroid gland cells (11). In the
parathyroid, CaSR regulates PTH secretion and activates multiple
intracellular pathways involving activation of G
-coupled proteins
and changes in gene expression among others (11). The ways
that CaSR activation influences salt and water transport in the kidney
are presently being addressed. Wang et al. (52) showed
that the elevation of extracellular calcium inhibits NaCl transport in
the thick ascending limb by release of cytochrome P-450
metabolites. We have demonstrated in immortalized MDCT cells that the
CaSR initiates intracellular Ca2+ transients and inhibits
PTH-, glucagon-, and AVP-mediated cAMP formation and magnesium
transport, probably by activating G
i-proteins (3,
4, 17). Finally, Sands et al. (47) reported that extracellular calcium inhibits AVP-stimulated cAMP induction of water
transport in the inner medullary collecting duct, again likely through
G
i-protein mediation. Accordingly, the CaSR may modulate
epithelial cell functions in many cell types by different intracellular
signaling pathways.
Elevation of extracellular Ca2+ inhibits
1,25(OH)2D3-stimulated Mg2+
uptake in MDCT cells (Fig. 5). The argument for modulation by the CaSR
of 1,25(OH)2D3-induced
transcriptional/translational processes involved with Mg2+
transport is persuasive. First, we show that
1,25(OH)2D3-mediated Mg2+ uptake is
responsive to actinomycin D and cycloheximide (Fig. 3). The acute,
short-term treatment of MDCT cells with
1,25(OH)2D3 does not elicit changes in
Mg2+ entry rates, so that a minimum of 3 h was
required; most of the present studies were preformed after 16 h of
1,25(OH)2D3 treatment. Second, acute elevation
of extracellular Ca2+ does not affect
1,25(OH)2D3-stimulated Mg2+ uptake
(Fig. 4). Third, blockade of the CaSR with a specific antibody or
prevention of CaSR expression with an antisense ODN abolished the
action of high extracellular Ca2+ on
1,25(OH)2D3-responsive Mg2+
transport (Figs. 7 and 10). Taken together, these data indicate that
1,25(OH)2D3-stimulated Mg2+ entry
into MDCT cells is modulated by extracellular polyvalent cations. Thus
the presence and magnitude of any 1,25(OH)2D3
responses are dependent on the existing divalent cation concentration.
Role of 1,25(OH)2D3 in overall renal
magnesium balance.
1,25(OH)2D3 increases intestinal calcium and
magnesium absorption and renal conservation, leading to positive
divalent cation balance. Elevation of serum calcium and magnesium
concentrations, in turn, diminish renal absorption through the actions
of the CaSR on electrolytes in the loop and distal convoluted tubule and water absorption in the medullary collecting ducts (27, 41,
42, 44, 47). Activation of the CaSR modulates the excessive
absorption of divalent cations, providing a negative-feedback control
to effect normal mineral balance. This may also explain the discrepant
reports in the literature concerning the effects of vitamin D
metabolites on renal magnesium transport.
 |
ACKNOWLEDGEMENTS |
We thank Dr. Peter A. Friedman for providing the MDCT cell line and
Drs. Kimberly V. Rogers (NPS Pharmaceuticals), Allen Spiegel, and Paul
Goldsmith (Metabolic Diseases Branch, National Institute of Diabetes
and Digestive and Kidney Diseases) for the ADD antibody.
 |
FOOTNOTES |
H. S. Kang is a Postdoctoral Fellow of the Korean Science and
Engineering Foundation, and L. Canaff is the recipient of a Doctoral
Fellowship of the Medical Research Council of Canada. This work was
supported by Medical Research Council of Canada Grants MT-5793 (to
G. A. Quamme) and MT-9315 (to G. N. Hendy) and grants from
the Kidney Foundation of Canada (to G. A. Quamme and G. N. Hendy).
Address for reprint requests and other correspondence: G. A. Quamme, Dept. of Medicine, Univ. Hospital, Koerner Pavilion, 2211 Wesbrook Mall, Vancouver, BC, Canada V6T 1Z3 (E-mail:
quamme{at}interchange.ubc.ca).
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.
Received 14 August 2000; accepted in final form 13 December 2000.
 |
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