 |
INTRODUCTION |
Magnesium, the second most abundant intracellular cation, plays an
important role in cellular function. Intracellular Mg2+ is
an essential cofactor for many enzymes, it modulates various transporters and ion channels, it regulates lipid- and
phosphoinositide-derived second messengers, and it influences DNA and
protein synthesis (1-5). Mg2+ is an important modulator of
intracellular free Ca2+ concentration
([Ca2+]i) and pHi, which are major
determinants of cell contraction, secretion, and motility (6-8).
Furthermore, increasing evidence suggests that Mg2+ plays a
major role in cell differentiation and proliferation by regulating
transphosphorylations, DNA synthesis, and intracellular signaling (4).
These physiological processes require that the intracellular free
Mg2+ concentration ([Mg2+]i) be
controlled within a narrow range. Mechanisms regulating [Mg2+]i have not been fully elucidated. Early
reports suggested that intracellular sequestration of Mg2+
does not contribute to [Mg2+]i homeostasis (9).
However, these conclusions were based on studies of total cell
Mg2+, not free [Mg2+]i, the
physiologically important form of Mg2+. Mg2+ is
not a static ion. It moves rapidly between intracellular compartments and across the plasma membrane (10). Mg2+ enters cells
along a concentration gradient and by facilitated diffusion (11) and is
extruded from cells via a Na+/Mg2+ exchanger
(11-13). The Na+/Mg2+ exchanger was first
demonstrated in the squid giant axon where Na+ entry is
coupled to Mg2+ moving out of the cell (14).
Na+-dependent Mg2+ transport is
inhibited by amiloride, quinidine, imipramine, and manganese, and it
may be regulated by cAMP, protein kinase C, and the
Na+/H+ antiporter (15-19). Although the
transporter has been functionally demonstrated, it has not yet been
cloned or characterized. A P-type ATPase has been cloned from bacteria
and has been implicated in Mg2+ transport; however, whether
such an enzyme exists in mammalian cells is unclear (20). Another
Mg2+ transporter, the Mg2+/H+
exchanger, has recently been fully characterized in plant cells but not
in mammalian cells (21).
Increasing evidence suggests that humoral factors influence
transmembrane kinetics of Mg2+ to regulate cellular actions
of these hormones by modulating signal transduction pathways. Small
changes in [Mg2+]i can have significant effects
on biological responses. Studies with perfused heart and liver, as well
as with cardiac myocytes and hepatocytes, have demonstrated that
Mg2+ fluxes are under the control of various hormones, such
as insulin, endothelin-1, norepinephrine, epinephrine, aldosterone, and
arginine vasopressin (22-27). We recently reported that
angiotensin II (Ang II),1 a
potent vasoactive agent, modulates [Mg2+]i in
vascular smooth muscle cells and in platelets (17, 28, 29). However,
very little is known about the mechanisms underlying Mg2+
regulation by Ang II, and it is unknown whether Ang II influences Mg2+ metabolism in kidney cells, a major site for Ang
II actions.
The present study was therefore undertaken to examine Ang II effects on
[Mg2+]i in Madin-Darby canine kidney (MDCK)
cells, using the fluorescent indicator dye mag-fura-2 (30). Regulatory
mechanisms whereby Ang II modulates [Mg2+]i were
also investigated. Our findings demonstrate that in MDCK cells Ang II
decreases [Mg2+]i via AT1 receptors.
These effects are not related to changes in
[Ca2+]i but are dependent on extracellular
Na+ and a quinidine- and imipramine-sensitive transporter.
These data suggest that rapid modulation of
[Mg2+]i is not simply a result of
Mg2+ redistribution from intracellular buffering sites by
Ca2+ and provide evidence for the existence of a
Na+-dependent, Ang II-regulated transporter for
Mg2+ in renally derived cells.
 |
EXPERIMENTAL PROCEDURES |
Materials--
Mag-fura-2AM, fura-2AM, mag-fura-2,
sodium-binding benzofuran isophthalate (acetoxymethyl ester),
and gramicidin were purchased from Molecular Probes (Eugene, OR).
PD123319, 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid
tetrakis(acetoxymethyl ester), Bay K 8644, and thapsigargin were purchased from Calbiochem (San Diego, CA). Irbesartan was a kind
gift from Bristol-Myers Squibb Co. All other reagents were from
Sigma and ICN Biomedicals Inc. (Irvine, CA).
Cell Culture--
MDCK cells were purchased from
the American Type Culture Collection (ATTC, Manassas, VA). Cells were
cultured in Dulbecco's modified Eagle's medium containing fetal calf
serum, L-glutamine, HEPES, penicillin, and streptomycin and
grown on round glass coverslips and maintained at 37 °C in a
humidified incubator (95% air, 5% CO2) as previously
described (31). At subconfluence, cells were rendered quiescent by
serum deprivation for 30 h prior to experimentation.
Measurement of Intracellular Free Mg2+ and
Ca2+ Concentrations--
The selective fluorescent probe
mag-fura-2AM was used to measure [Mg2+]i. Cells
were washed with modified Hanks' buffered saline solution containing
(in mmol/liter): 137 NaCl, 5.4 KCl, 4.2 NaHCO3, 3 Na2HPO4, 0.4 KH2PO4,
1.3 CaCl2, 0.5 MgCl2, 0.8 MgSO4, 10 glucose, Hepes, pH 7.4 and loaded with mag-fura-2AM (4 µmol/liter), which was dissolved in dimethyl sulfoxide with 0.02%
pluronic acid. Cells were incubated for 30 min at room temperature and
then washed with warmed buffer and incubated for a further 15 min to
ensure complete deesterification. Under these loading conditions, the ratiometric (343/380 nm) fluorescence cell images were homogeneous, indicating that there was no significant intracellular compartmentation of mag-fura-2. The coverslip containing cells was placed in a stainless
steel chamber and mounted on the stage of an inverted microscope
(Axiovert 135, Zeiss) as previously described (31).
Cells were exposed to increasing concentrations of Ang II
(10
12-10
6
mol/liter). In some experiments, cells were pretreated (15 min) with
10
5 mol/liter irbesartan (AT1
receptor antagonist) or PD123319 (AT2 receptor blocker).
[Mg2+]i was measured in multiple cells
simultaneously by fluorescence digital imaging (Attofluor Ratiovision,
Zeiss) using an emission wavelength of 520 nm and alternating
excitatory wavelengths of 343 and 380 nm (30). The Attofluor system was
calibrated by viewing mag-fura-2, tetrapotassium salt solutions
containing zero and saturating Mg2+ concentrations and
including these data in the ratio calculations for construction of a
standard curve relating Mg2+ concentration to the 343/380
ratio. The curve was derived from the equation
[Mg2+]i (in mmol/liter) = Kd [(R
Rmin)/(Rmax
R)] ×
(30), where R is the ratio of fluorescence at 343 and 380 nm, Rmax and Rmin are the ratios for
mag-fura free acid at 343 and 380 nm in the presence of saturating
Mg2+ and zero Mg2+, respectively, and
is
the ratio of fluorescence of mag-fura-2 at 380 nm in zero and
saturating magnesium. Kd is the dissociation
constant of mag-fura-2 for Mg2+ and is assumed to be 1.5 mmol/liter (30). Video images of fluorescence at 520 nm emission were
obtained using an intensified charge-coupled device camera system with
the output digitized to a resolution of 512 × 480 pixels. Images
of fluorescence ratios were obtained by dividing, pixel by pixel, the
343 nm image after background subtraction by the 380 nm image after
background subtraction.
For determination of the mag-fura-2 fluorescence spectrum,
mag-fura-2-loaded cells were exposed to graded Mg2+ buffers
or graded Ca2+ buffers. The buffers were identical in
composition with the Hanks' buffer as indicated above, apart from a
variable concentration of MgCl2/MgSO4 or
CaCl2. Cells were lysed by addition of 0.1% Triton X-100
(final concentration). Excitation spectra were recorded, and the
343:380 nm ratio was determined.
In some experiments [Ca2+]i was measured. The
method used has been previously described in detail (31) and was
similar to that for [Mg2+]i described above.
Briefly, cells were loaded with fura-2AM (4 µmol/liter) and incubated
at room temperature in the dark for 30 min.
[Ca2+]i was measured in washed cells by
fluorescence digital imaging using excitation wavelengths of 343/380 nm
and an emission wavelength of 520 nm. Fura 2-pentapotassium salt was
used for [Ca2+]i calibration, and the absolute
Ca2+ concentration was determined from the Grynkiewicz
formula as stated above for [Mg2+]i (32).
The Kd for the fura-2-Ca2+ complex was
assumed to be 224 nmol/liter (32).
Measurement of Intracellular Free Na+
Concentration--
[Na+]i was measured using the
Na+-selective fluorescent probe SBFI-AM (33). Cells were
washed with modified Hanks' buffered solution and loaded with SBFI-AM
(4 µmol/liter) for 90 min at room temperature. The loaded cells were
washed and incubated for a further 15 min. [Na+]i
was measured using an emission wavelength of 20 nm and alternating
excitatory wavelengths of 343 and 380 nm. [Na+]i
was calibrated by equilibrating [Na+]i with the
extracellular Na+ concentration using the monovalent cation
ionophore gramicidin D (1 µmol/liter) (33). Na+
calibration solutions were made from appropriate mixtures of high
Na+ and high K+ solutions. The former contained
90 mmol/liter sodium gluconate, 60 mmol/liter NaCl, 1.2 mmol/liter
CaCl2, 10 mmol/liter Hepes; the high K+
solution was identical except for complete replacement of
Na+ by K+. The reference standards were
adjusted to give final concentrations of 0-100 mmol/liter
Na+ or K+. Cells were exposed to buffers in
which the extracellular Na+ concentration
([Na+]e) was varied from 10 to 80 mmol/liter.
Thereafter, gramicidin D was added to clamp
[Na+]i to [Na+]e. The final
values of the 343/380 excitation ratio were plotted versus
[Na+]e. The calibration curve, which was linear
between 10 and 80 mmol/liter (r = 0.97;
n = 5), was used to obtain
[Na+]i.
Determination of Na+-dependent
Mg2+ Transport--
To determine the dependence of
[Mg2+]i on Na+,
[Mg2+]i responses to Ang II were measured in
Na+-free solution. Cells were preincubated in
Na+-free Hanks' buffer (Na+ isosmotically
replaced with N-methylglucamine) for 15 min before [Mg2+]i measurement. To further assess the
relationship between Mg2+ and Na+,
[Mg2+]i responses were determined in cells that
had been pre-exposed to imipramine or quinidine (0.5 mmol/liter),
putative inhibitors of the Na+/Mg2+ exchanger
(5, 10, 16, 17, 34-36). In some experiments, cells were pre-exposed to
benzamil (10
5 mol/liter), which selectively
inhibits the Na+/Ca2+ exchanger (37).
Determination of Ca2+-dependent
Mg2+ Transport--
Intracellular free Ca2+
concentration was manipulated with 10
5
mol/liter thapsigargin (endoplasmic reticular Ca2+-ATPase
inhibitor) (38) or BAPTA-AM (50 µmol/liter), a high capacity chelator
that selectively binds Ca2+ about 105 over
Mg2+ (39). In these experiments cells were exposed to
either agent for 10-15 min in Ca2+-free buffer (Hanks'
buffer without CaCl2) before Ang II stimulation.
Statistical Analysis--
Each experiment was repeated at least
three times using different cell preparations. Data were calculated as
the mean response per experiment and then as the mean of multiple
experiments. Results are presented as the mean ± S.E. and
compared by Student's t test or by analysis of variance
where appropriate. Tukey-Kramers' correction was used to compensate
for multiple testing procedures. The Ang II concentration (mol/liter)
eliciting 50% of the maximal response (EC50) or minimal
response (IC50) was determined from concentration-response curves, which were fitted by nonlinear regression. Maximal (or minimal)
responses to Ang II were expressed as Emax (or
Emin) (in mmol/liter). Sensitivity to Ang II was expressed
as pD2 =
log [EC50] (or pI2 =
log IC50) (in mol/liter). p < 0.05 was significant.
 |
RESULTS |
Mag-fura Is a Mg2+-sensitive Fluoroprobe--
The
specificity of mag-fura for Mg2+ within the physiological
Ca2+ range was studied by recording the fluorescence
spectrum of mag-fura-2 in graded Ca2+ and Mg2+
buffers. With increasing Mg2+ concentrations, the
mag-fura-2 excitation ratio increased linearly, whereas the mag-fura-2
fluorescence spectrum was not influenced by Ca2+ (Fig.
1A). To further demonstrate
the specificity of mag-fura-2 for Mg2+, cells were exposed
to the Ca2+ channel agonist Bay K 8644, which increases
[Ca2+]i but not [Mg2+]i
(37). Bay K 8644 did not alter mag-fura-2 fluorescence but
significantly increased fura-2 fluorescence (Fig. 1, B and C), indicating that elevations in
[Ca2+]i do not influence mag-fura-2 fluorescence,
at least within the range of concentrations tested.

View larger version (23K):
[in this window]
[in a new window]
|
Fig. 1.
Mag-fura-2 is a Mg2+-sensitive
fluoroprobe. A, line graphs demonstrate mag-fura-2
selectivity for Mg2+ in MDCK cells. Mag-fura-2-loaded cells
were lysed in graded Mg2+ or Ca2+ buffers.
Shown is the 343:380 nm fluorescence excitation ratio at different
Mg2+ and Ca2+ concentrations. B,
representative tracings of fluorescence signals for fura-2 and
mag-fura-2 in cells exposed to the Ca2+ channel agonist Bay
K 8644 (10 7 mol/liter). Arrows
indicate time of Bay K 8644 addition. C, bar graphs
demonstrate effects of Bay K 8644 on [Ca2+]i and
[Mg2+]i in MDCK cells. Data were derived from
fluorescence signals as demonstrated in B. Results are
means ± S.E. of four experiments, with each experimental field
comprising 15-26 cells. **, p < 0.01 versus basal counterpart.
|
|
Effects of Ang II on [Mg2+]i,
[Na+]i, and [Ca2+]i
in MDCK Cells--
Resting levels of Mg2+,
Na+, and Ca2+ in MDCK cells were 0.51 ± 0.02 mmol/liter (n = 135 cells), 17.6 ± 0.12 mmol/liter (n = 17 cells), and 84 ± 8.8 nmol/liter (n = 70 cells), respectively, consistent
with previously reported values (40). Fig.
2A demonstrates representative
fluorescence tracings of Ang II effects on
[Mg2+]i, [Na+]i, and
[Ca2+]i in cells loaded with mag-fura-2AM,
SBFI-AM, and fura-2AM, respectively. Ang II induced a small transient
followed by a rapid decrease in mag-fura-2 fluorescence. This effect
was sustained for up to 300 s following Ang II addition. SBFI-AM
and fura-2AM fluorescence were increased by Ang II, indicating
increased [Na+]i and
[Ca2+]i. Maximal [Na+]i and
[Ca2+]i responses, which occurred within 90 and
40 s, respectively, were sustained for up to 400 s following
Ang II addition. Minimum fluorescence values for
[Mg2+]i and maximum fluorescence values for
[Na+]i and [Ca2+]i were
used to generate full dose-response curves. As shown in Fig.
2B, Ang II decreased [Mg2+]i
(Emin = 0.38 ± 0.01 mmol/liter;
pI2 = 8.8 ± 0.2) and increased
[Na+]i (Emax = 53 ± 4 mmol/liter; pD2 = 8.5 ± 0.15) and
[Ca2+]i (Emax = 618 ± 43 nmol/liter; pD2 = 8.0 ± 0.2) in a dose-dependent manner.

View larger version (12K):
[in this window]
[in a new window]
|
Fig. 2.
A, representative fluorescence tracings
of MDCK cells loaded with mag-fura-2AM, SBFI-AM, or fura-2AM. Ang II
(10 7 mol/liter) was added at the time
indicated by the arrows. The y axis is the
343:380 nm fluorescence excitation ratio. B, line graphs
demonstrate effects of increasing concentrations of Ang II on
[Mg2+]i, [Na+]i, and
[Ca2+]i in MDCK cells. Each data point is the
mean ± S.E. of five to seven experiments, with each experimental
field comprising 8-17 cells. Data were derived from minimum (for
[Mg2+]i) and maximum (for
[Na+]i and [Ca2+]i)
fluorescence signals from tracings similar to those shown in
A.
|
|
Mg2+ Effects of Ang II Are Mediated via AT1
Receptors--
To ascertain the receptor subtype through which Ang II
mediates [Mg2+]i effects, cells were exposed to
the AT1 receptor antagonist irbesartan or the selective
AT2 receptor blocker PD123319. In cells pre-exposed to
irbesartan, Ang II failed to elicit changes in
[Mg2+]i, whereas in cells pretreated with
PD123319, Ang II effects were unaltered (Fig.
3). These data suggest that Ang II actions are mediated exclusively via the AT1 receptor
subtype.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 3.
Bar graphs demonstrate Ang II
(10 7 mol/liter) effects on
[Mg2+]i in MDCK cells in the
absence or presence of irbesartan (Irb)
(AT1 receptor antagonist) or PD123319
(PD12) (AT2 receptor blocker). Cells
were pre-exposed to either antagonist for 15 min prior to Ang II
addition. Results are means ± S.E. of four experiments, with each
experimental field comprising 6-11 cells.*, p < 0.01.
|
|
Ang II Modulation of [Mg2+]i Is
Na+-dependent--
Two approaches were used to
determine whether Ang II modulates [Mg2+]i via
Na+-dependent mechanisms. First, cells were
incubated in Na+-free buffer, and second, cells were
exposed to quinidine and imipramine, two different inhibitors of the
Na+/Mg2+
exchanger.

View larger version (9K):
[in this window]
[in a new window]
|
Fig. 4.
Representative fluorescence tracings of MDCK
cells loaded with mag-fura-2AM, SBFI-AM, or fura-2AM. Cells were
stimulated with Ang II (10 7 mol/liter) in the
presence (+Na+) or absence
(-Na+) of extracellular Na+. The
arrows indicate time of Ang II addition.
|
|
As shown in Figs. 4 and 5, exposing cells
to Na+-free buffer inhibited the Ang II-induced
[Mg2+]i response. [Na+]i
effects were also blunted in Na+-free medium but were not
completely abolished. In the absence of extracellular Na+,
the magnitude of the [Ca2+]i response was
unchanged, even though the time course was altered (Fig. 4). Taken
together, these results indicate that Ang II-induced mobilization of
intracellular Mg2+ does not directly involve an increase in
[Ca2+]i, whereas changes in
[Na+]i are required. The Na+
dependence of Ang II-elicited [Mg2+]i responses
suggests that a Na+/Mg2+ exchange mechanism may
be operational.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 5.
Bar graphs demonstrate effects of Ang II
(10 7 mol/liter) on MDCK cell
[Mg2+]i,
[Na+]i, and
[Ca2+]i in the presence or
absence of extracellular Na+. Na+-free
Hanks' buffer was prepared by replacing Na+ with
N-methylglucamine. Results are means ± S.E. of four to
six experiments, with each experimental field comprising many cells. +,
p < 0.05; **, p < 0.01 versus basal counterpart.
|
|
As demonstrated in Figs. 6 and 7, quinidine and imipramine abrogated Ang
II-elicited [Mg2+]i
effects. [Na+]i responses were reduced by both
agents but were not completely inhibited (Figs. 6 and 7), indicating
that in addition to the Na+/Mg2+ exchanger,
other mechanisms control [Na+]i, possibly the
Na+/Ca2+ exchanger. To investigate whether this
antiporter contributes to Na+ and Mg2+
regulation by Ang II, the effects of benzamil, a selective inhibitor of
the Na+/Ca2+ exchanger, were assessed. Benzamil
significantly inhibited Ang II-induced Na+ responses but
did not alter [Mg2+]i effects (Fig. 7). When
cells were pretreated with imipramine and benzamil together, Ang
II-induced [Na+]i responses were almost
completely abolished (Fig. 7). These data suggest that whereas
Mg2+ is regulated primarily via a
Na+-dependent Mg2+ transporter,
Na+ regulation by Ang II is mediated in large part via the
Na+/Ca2+ exchanger.

View larger version (13K):
[in this window]
[in a new window]
|
Fig. 6.
Line graphs demonstrate effects of Ang II on
[Mg2+]i and
[Na+]i in MDCK cells in the
absence and presence of quinidine (5 × 10 4
mol/liter), a putative inhibitor of the
Na+/Mg2+ exchanger. Cells were pretreated
with quinidine for 15-20 min prior to Ang II
(10 7 mol/liter) addition. Each data point is
the mean ± S.E. of four experiments, with each experimental field
comprising 7-11 cells. *, p < 0.05; **,
p < 0.01 versus Ang II counterpart.
|
|

View larger version (17K):
[in this window]
[in a new window]
|
Fig. 7.
Line graphs demonstrate effects of Ang II on
[Mg2+]i and
[Na+]i in MDCK cells in the
absence and presence of imipramine (5 × 10 4
mol/liter), the most effective inhibitor of the
Na+/Mg2+ exchanger (36). Cells were
pretreated with imipramine for 15-20 min prior to Ang II
(10 7 mol/liter) addition. In some experiments
cells were exposed to benzamil (10 5
mol/liter), a selective inhibitor of the
Na+/Ca2+ exchanger, or to imipramine + benzamil. Each data point is the mean ± S.E. of four to seven
experiments, with each experimental field comprising 7-26 cells. *,
p < 0.05; **, p < 0.01 versus Ang II counterpart.
|
|
Ang II Modulation of [Mg2+]i in MDCK
Cells Is Ca2+-independent--
To examine whether Ang II
influences [Mg2+]i by increasing
[Ca2+]i, cells were exposed to BAPTA, which
prevents [Ca2+]i elevation by clamping
[Ca2+]i at 40-50 nmol/liter. Cells were also
treated with thapsigargin, a potent endoplasmic reticular
Ca2+-ATPase inhibitor, which depletes reticular
Ca2+ stores by preventing Ca2+ reuptake. In the
presence of BAPTA, the Ang II-induced increase in
[Ca2+]i was attenuated by >80%, whereas the Ang
II-mediated decrease in [Mg2+]i was unchanged
(Fig. 8). Pre-exposure of cells to
thapsigargin in Ca2+-free buffer increased basal
[Ca2+]i and attenuated Ang II-stimulated
[Ca2+]i responses compared with cells in the
absence of thapsigargin (Fig. 8). These results indicate that
Ca2+ stores were diminished and that Ang II was unable to
mobilize intracellular Ca2+ and to significantly increase
[Ca2+]i. Thapsigargin did not alter basal
[Mg2+]i or Ang II-induced
[Mg2+]i responses (Fig. 6). Taken together, these
data suggest that Ang II modulates [Mg2+]i via
mechanisms that are not dependent on [Ca2+]i
elevations.

View larger version (11K):
[in this window]
[in a new window]
|
Fig. 8.
Bar graphs demonstrate effects of Ang II
(10 7 mol/liter) on
[Mg2+]i and
[Ca2+]i in MDCK cells in the
absence and presence of thapsigargin (Thaps)
(10 5 mol/liter) or BAPTA-AM (50 µmol/liter). Results are means ± S.E. of
four experiments, with each experimental field comprising many cells.
*, p < 0.05; **, p < 0.01.
|
|
 |
DISCUSSION |
The major findings of the present study indicate that 1) Ang II
decreases [Mg2+]i and increases
[Na+]i and [Ca2+]i in MDCK
cells in a dose-dependent manner, 2) these effects are
blocked by irbesartan but not by PD123319, 3) Ang II-mediated
[Mg2+]i effects are abrogated by quinidine and
imipramine and in Na+-free buffer, 4) Ang II elicits
significant [Mg2+]i responses even when
[Ca2+]i effects are blunted, and 5) benzamil
inhibits Ang II-induced [Na+]i responses but does
not influence [Mg2+]i. These data suggest that in
MDCK cells Ang II modulates [Mg2+]i via
Na+-dependent, Ca2+-independent
mechanisms that are regulated exclusively by AT1 receptors.
Furthermore, we demonstrate that the Na+/Ca2+
exchanger is a major regulator of [Na+]i, but not
of [Mg2+]i, in Ang II-stimulated cells.
Although Mg2+ is the most abundant intracellular divalent
cation, little is known about underlying mechanisms controlling
[Mg2+]i, and there is much controversy whether
cytosolic Mg2+ functions as a second messenger and whether
it is regulated by hormonal signaling. A recent editorial by Murphy
(41) on the "mysteries of magnesium homeostasis" concluded that the
challenge for future research will be to elucidate whether agonists
induce physiologically significant alterations in Mg2+
fluxes. We demonstrate here that Ang II, an important regulator of
renal function (42), modulates [Mg2+]i in
kidney-derived epithelial cells. [Mg2+]i was
measured by ratio-imaging microscopy and the indicator mag-fura-2AM,
which fluoresces upon binding with Mg2+ (30).
Although the affinity of mag-fura-2 for Ca2+ is greater
than the affinity for Mg2+, physiological
[Mg2+]i is 2-3 orders of magnitude greater than
[Ca2+]i. Thus interference due to
[Ca2+]i only becomes significant under extreme
perturbations when [Ca2+]i is elevated to
pharmacologically high levels. When [Ca2+]i is
increased to 1 µmol/liter, the Ca2+-mag-fura-2 complex is
<10% of the Mg2+-mag-fura-2 complex. In our previous and
current studies, [Ca2+]i never reached levels as
high as 1 µmol/liter, even when maximal doses of Ang II were used.
Furthermore, we demonstrate here that mag-fura-2 fluorescence is
unaffected by changes in [Ca2+]i, whereas it is
increased by Mg2+ in a concentrationdependent
manner. For these reasons, the fluorescence of cellular mag-fura-2 in
the present study is considered to be closely and selectively
associated with changes in [Mg2+]i. In
unstimulated cells, [Mg2+]i was 0.51 ± 0.02 mol/liter, which is in agreement with previously reported values in
MDCK cells (0.49 ± 0.03 mmol/liter) (40). Basal
[Mg2+]i is maintained within a narrow range by
intracellular buffering processes and by specific and highly regulated
Mg2+ influx pathways (40).
Ang II decreased [Mg2+]i in a
dose-dependent fashion. These effects were blocked by
irbesartan, but not by PD123319, indicating the exclusive role of
AT1 receptors in these processes. Similar findings have
been reported in vascular smooth muscle cells (17), but to our
knowledge, these are the first data demonstrating that AT1
receptors regulate [Mg2+]i in MDCK cells.
Mg2+ exchanges, or is cotransported, with other
ions, and it uses the electrochemical gradient of other ions to cross
the plasma membrane. Na+ may be the counterion exchanged
with Mg2+. The Na+/Mg2+ exchanger,
inhibitable by imipramine and quinidine, has been functionally
demonstrated in sublingual mucous acini, erythrocytes, hepatocytes,
thymocytes, cardiac cells, and vascular smooth muscle cells (13, 15,
16, 43-47). Gunther (13) reported that the membrane-spanning
Mg2+ transporter is bidirectional and that reversing the
Na+ gradient results in net Mg2+ influx. In our
experimental conditions, [Mg2+]i responses were
temporally associated with [Na+]i changes and
were completely abolished in Na+-free medium and by
quinidine and imipramine, one of the most effective inhibitors of the
antiporter, suggesting that Mg2+ modulation by Ang II is
dependent on extracellular Na+ and that the
Na+/Mg2+ exchanger is the major regulator of
[Mg2+]i, exchanging extracellular Na+
for intracellular Mg2+. The stoichiometry of this Ang
II-activated exchanger in MDCK cells appears to be complex, and the
exact coupling awaits clarification.
Inhibitors of the Na+/Mg2+ antiporter
abrogated [Mg2+]i responses but only partially
decreased [Na+]i effects. These findings suggest
that whereas the Na+/Mg2+ exchanger is the
primary transporter for Mg2+, it contributes only partially
to [Na+]i regulation by Ang II and that other
mechanisms also control [Na+]i. To this end we
investigated the role of the Na+/Ca2+ exchanger
by exposing cells to benzamil, a selective inhibitor of the
transporter. Benzamil significantly decreased
[Na+]i responses but did not influence
[Mg2+]i. Inhibition of both antiporters with
imipramine and benzamil blocked Ang II-stimulated
[Na+]i responses. These findings demonstrate that
the Na+/Ca2+ exchanger does not control
transmembrane Mg2+ fluxes in MDCK cells but that it plays
an important role in Na+ transport. Similar findings for
[Na+]i have been demonstrated in cardiomyocytes
and vascular smooth muscle cells (37).
Some studies failed to show that intracellular Mg2+
homeostasis is regulated by Na+-dependent
mechanisms and suggested that changes in [Mg2+]i
are due primarily to changes in Ca2+ (22). Because
Ca2+ and Mg2+ bind to many common intracellular
sites, an increase in [Ca2+]i could displace
Mg2+ from mutual binding sites, thereby altering
[Mg2+]i. In cardiomyocytes and isolated hearts,
[Mg2+]i regulation is not due to
Na+/Mg2+ exchange but appears to be secondary
to changes in [Ca2+]i, which is influenced by the
Na+/Ca2+ exchanger (22, 48). In sublingual
acini, muscarinic-induced Mg2+ mobilization is both
Na+- and Ca2+-dependent (34). In
MDCK cells, Quamme and Dai (40) reported that Mg2+ refill
into Mg2+-depleted cells is regulated by a
verapamil-dependent pathway. In the present study we
explored the role of [Ca2+]i on
[Mg2+]i regulation, by depleting endoplasmic
reticular Ca2+ stores with the Ca2+-ATPase
inhibitor thapsigargin and by clamping [Ca2+]i at
about 50 mmol/liter with the Ca2+ chelator BAPTA-AM. Under
these conditions, Ang II-induced [Ca2+]i
responses were blunted, but [Mg2+]i transients
were unaltered, indicating that in MDCK cells
[Mg2+]i regulation by Ang II is not dependent on
intracellular Ca2+ mobilization or on increased
[Ca2+]i. Possible Ca2+-insensitive
signaling cascades whereby Ang II influences
[Mg2+]i could be via protein kinase C and
the Na+/H+ exchanger, which we previously
demonstrated to be functionally linked to the
Na+/Mg2+ exchanger (17).
In conclusion, the present study demonstrates that Ang II mobilizes
intracellular Mg2+ in renally derived cells through
AT1 receptor-mediated pathways. Ang II decreases
[Mg2+]i by activating the
Na+/Mg2+ exchanger to promote transmembrane
Mg2+ transport in a Ca2+-independent fashion.
These processes may play an important role in
[Mg2+]i homeostasis and in
Mg2+-dependent intracellular signaling pathways
that do not require changes in [Ca2+]i. Moreover,
we demonstrate that the Na+/Ca2+ exchanger is a
major regulator of [Na+]i, but not of
[Mg2+]i, in Ang II-stimulated cells. Our results
support the notion that rapid modulation of
[Mg2+]i is not simply a result of
Mg2+ redistribution from intracellular buffering sites by
Ca2+ and provide evidence for the existence of a
Na+-dependent, hormonally regulated transporter
for Mg2+ in renal epithelial cells.