Sodium gradient-dependent transport of magnesium in rat
ventricular myocytes
Michiko
Tashiro1 and
Masato
Konishi1,2
1 Department of Physiology, The Jikei University School of
Medicine, Tokyo 105 - 8461, and 2 Department of Physiology,
Tokyo Medical University, Tokyo 160-8402, Japan
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ABSTRACT |
Cytoplasmic concentration of Mg2+
([Mg2+]i) was measured with a fluorescent
indicator furaptra in ventricular myocytes enzymatically dissociated
from rat hearts (25°C). To study Mg2+ transport across
the cell membrane, cells were treated with ionomycin in
Ca2+-free (0.1 mM EGTA) and high-Mg2+ (10 mM)
conditions to facilitate passive Mg2+ influx. Rate of rise
of [Mg2+]i due to the net Mg2+
influx was significantly smaller in the presence of 130 mM
extracellular Na+ than in its absence. We also tested the
extracellular Na+ dependence of the net Mg2+
efflux from cells loaded with Mg2+. After
[Mg2+]i was raised by ionomycin and high
Mg2+ to the level 0.5-0.6 mM above the basal value
(~0.7 mM), washout of ionomycin and lowering extracellular
[Mg2+] to 1.2 mM caused rapid decline of
[Mg2+]i in the presence of 140 mM
Na+. This net efflux of Mg2+ was completely
inhibited by withdrawal of extracellular Na+ and was
largely attenuated by imipramine, a known inhibitor of Na+/Mg2+ exchange, with 50% inhibition at 79 µM. The relation between the rate of net Mg2+ efflux and
extracellular Na+ concentration
([Na+]o) had a Hill coefficient of 2 and
[Na+]o at half-maximal rate of 82 mM. These
results demonstrate the presence of Na+ gradient-dependent
Mg2+ transport, which is consistent with
Na+/Mg2+ exchange, in cardiac myocytes.
Na+/Mg2+ exchange; cardiac muscle; antiport; magnesium; sodium
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INTRODUCTION |
CYTOPLASMIC FREE
CONCENTRATION of Mg2+
([Mg2+]i) in mammalian cardiac myocytes has
been estimated with various methods and appears to be in the range of
0.5-1.0 mM, well below electrochemical equilibrium across the cell
membrane (7, 19, 27, 31). Since it has been reported that
submillimolar [Mg2+]i significantly
influences many intracellular processes of cardiac muscles, including
adenylate cyclase activity (2), K+ channels
(see Refs. 1 and 25 for reviews),
excitation-contraction coupling (36), Ca2+
sensitivity of myofilaments (8), and Ca2+
binding to intracellular sites (10, 20),
[Mg2+]i must be tightly regulated by active
extrusion from the cell to balance any passive leak influx of
Mg2+. Na+/Mg2+ exchange may play an
essential role as an active Mg2+ extrusion pathway in many
types of cells (for review see Refs. 12 and 30), but
experimental evidence so far obtained in cardiac muscle is very
controversial. The concept of the existence of Na+/Mg2+ exchange has been supported by
measurements of the net Mg2+ fluxes by atomic absorption
spectroscopy (29) and also by measurements of
[Mg2+]i either by ion-selective
microelectrodes (14) or a fluorescent indicator
(17). However, other studies with improved ion-selective microelectrodes (6, 7) and fluorescent indicators
(6, 18, 26, 31) failed to provide any evidence for a
Na+ gradient-dependent Mg2+ efflux in cardiac muscle.
This study describes [Mg2+]i measurements
with a fluorescent indicator carried out to seek evidence for
Na+/Mg2+ exchange in cardiac myocytes. The
results show that Na+-dependent changes in
[Mg2+]i are unmasked after facilitation of
passive Mg2+ influx by an ionophore and strongly suggest
the existence of a Na+ gradient-dependent Mg2+ efflux.
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METHODS |
General.
All experiments were carried out on single ventricular myocytes
enzymatically isolated from male Wistar rats (250-300 g), as
previously described (18, 20). After enzymatic digestion with 0.2 mg/ml collagenase (collagenase S-1; Nitta Zerachin, Tokyo, Japan) and 0.04 mg/ml protease (type XIV; Sigma, St. Louis, MO) in the
presence of 0.6 mg/ml BSA (Sigma), cells were stored in 0.2 mM
CaCl2-containing Tyrode solution at 6°C until used. Cells were placed in an experimental chamber on the stage of an inverted microscope (Diaphot; Nikon, Tokyo, Japan) and superfused with normal
Tyrode solution containing (in mM) 138 NaCl, 5.9 KCl, 2.4 CaCl2, 1.2 MgCl2, 11.8 glucose, and 5 HEPES (pH
7.4). Only quiescent rod-shaped cells that gave an all-or-none response
to a 5-ms field stimulation were used for the experiments. After the
indicator was loaded by incubation of the cells with 4 µM furaptra-AM
for 10 min at room temperature and washout of the AM ester for at least
10 min, fluorescence measurements were carried out under continuous
flow of the perfusate at 25°C.
The apparatus, methods for fluorescence measurements, and analysis have
been described previously (22, 34, 35). Briefly, excitation light beams of 350 and 382 nm were switched at 100 Hz and
focused with a ×40 objective (CF Fluor 40, Nikon), and the emitted
fluorescence at 500 nm (40 nm full width at half-maximum) at
each excitation wavelength was measured from single myocytes. The
background fluorescence was estimated from the measurement before the
indicator loading (see below) and was subtracted from the total
fluorescence measured after the indicator loading to calculate
indicator fluorescence intensity at each excitation wavelength and the
ratio of the indicator fluorescence intensities (R). Basal
[Mg2+]i was calculated from the basal R of
furaptra measured at the beginning of each experiment.
Calibration of furaptra signals.
The ratio of furaptra fluorescence intensities measured with excitation
at 382 and 350 nm [R = F(382)/F(350)] was used as a
Mg2+-related signal. Slow drift of the optical instruments
(e.g., aging of the lamp) was corrected by occasional measurement of R
in a Ca2+-Mg2+-free buffer solution (in mM: 140 KCl, 10 NaCl, 1 EDTA, 1 EGTA, 0.025 furaptra, and 10 PIPES, pH 7.1) as
a standard. All values of the measured R were normalized to the
standard R value taken with identical optics and were converted to
[Mg2+]i with the standard equation
where Rmin and Rmax are the R values at
zero [Mg2+]i and saturating
[Mg2+]i, respectively, and
KD is the dissociation constant. We used parameter values previously estimated in smooth muscle cells of tenia
at 25°C: Rmin = 0.986, Rmax= 0.199 and
KD = 5.43 mM (34). The
calibration procedure also included a correction for the small offset
of R (+0.035) found in cardiac myocytes in a previous study (34). This offset was observed shortly after application
of an ionophore cocktail containing Br-A-23187 and other ionophores, and was attributed to an artifact unrelated to
[Mg2+]i. Direct effects of imipramine (200 µM) and ionomycin (10 µM) on the furaptra R were tested at 0-4
mM [Mg2+] in vitro and were found to be negligible. It
has been reported that equimolar substitution of Na+ by
N-methyl-D-glucamine (NMDG+) up to
40 mM has little influence on the furaptra R in solutions containing
0-4 mM [Mg2+] (35).
We found that ionomycin caused a slow decrease in the
cell autofluorescence (Fig. 1).
Cell autofluorescence excited at either 350 or 380 nm decreased
exponentially with time of ionomycin treatment (time constant for the
decay: 12.4 min at 350 nm; 21.8 min at 380 nm) and partially recovered
after ionomycin washout (time constant for the recovery: 15.0 min at
350 nm; 4.5 min at 380 nm). The analysis of
[Mg2+]i measurements from the cells treated
with ionomycin for 30-60 min (see Figs. 3-7) therefore
included correction of the cell autofluorescence; we calculated, at
each wavelength, the autofluorescence at any time during the
[Mg2+]i measurement run using the fitted
parameters of the exponential function for the decay and the recovery
(see legend to Fig. 1).

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Fig. 1.
Long-term observation of cell autofluorescence, cell
F( ), excited at 350 nm (A) and 382 nm (B).
Intensities of autofluorescence relative to values measured just before
ionomycin application (the first data points in A and
B) were plotted as a function of time during application and
washout of ionomycin. Ionomycin (10 µM) was added to the
high-Mg2+ solution (10 mM) containing either 130 mM
Na+ (5 cells) or 130 mM
N-methyl-D-glucamine (NMDG+; 5 cells) for the periods indicated by the horizontal bars. Cells were
then perfused with Ca2+-free Tyrode solution containing 130 mM Na+. Values of F(350) were 0.551±0.042
(Na+) and 0.544±0.052 (NMDG+) at time = 30 min, and were 0.645±0.047 (Na+) and 0.598±0.032
(NMDG+) at time = 50 min. Values of F(382) were
0.721±0.057 (Na+) and 0.763±0.073 (NMDG+) at
time = 30 min, and were 0.780±0.090 (Na+) and
0.773±0.052 (NMDG+) at time = 50 min. Because the
ionomycin effect was not significantly different in the presence and in
the absence of extracellular Na+, the data from the 10 cells were treated as a single data set; each symbol represents a mean
(±SE) value measured from the total of 10 cells. Solid lines are the
least-squares fit of the mean values by an exponential (plus constant)
function of the form F( ) = c + a × exp( t/ ), where t is
time after the first data point in the fitted range, and is a time
constant. Constants c and a give, respectively,
an offset and a scaling factor. At each wavelength ( ), four data
points between 0 and 30 min ( ) were fitted for the
decay during the ionomycin treatment, and three data points between 30 and 50 min (the last and two ) were
fitted for the recovery after washout.
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Solutions and chemicals.
Ca2+-free Tyrode solution contained 0.1 mM EGTA replacing
2.4 mM CaCl2 of normal Tyrode solution.
High-Mg2+ Tyrode solution contained 10 mM
MgCl2, 0 mM CaCl2, and 0.1 mM EGTA, with NaCl
concentration reduced to 128 mM. In one experiment, Mg2+
concentration was raised to 30 mM by simple addition of 20 mM MgCl2 to the high-Mg2+ Tyrode solution without
any osmotic compensation. High-K+ (55.9 mM) solutions with
various Na+ concentrations ([Na+]; 0-90
mM) were made by equimolar substitution of NaCl with potassium methanesulfonate and sodium methanesulfonate to keep the
[K+]×[Cl
] product constant. For
Na+-free conditions, Na+ was substituted by
equimolar NMDG+. Furaptra (tetrapotassium salt of mag-fura
2) and furaptra-AM (mag-fura 2-AM) were purchased from Molecular Probes
(Eugene, OR). EGTA was obtained from Sigma Chemical. Ionomycin (Sigma
Chemical) was dissolved from a 10 mM stock solution in DMSO
(DOTITE Spectrosol; Dojindo, Kumamoto, Japan).
Imipramine · HCl (Nacalai Tesque, Kyoto, Japan) was
directly dissolved in the perfusates. All other chemicals were reagent grade.
Curve fitting and statistical analysis.
Nonlinear least-squares fitting was carried out with the program Origin
(version 5.0J; Microcal Software, Northampton, MA) that uses the
Levenberg-Marquardt algorithm. Statistical values were given as
means ± SE. The two-tailed Student's t-test was used
for statistical comparison with the significance level set at
P < 0.05.
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RESULTS. |
All [Mg2+]i measurements were carried
out in Ca2+-free conditions (0.1 mM EGTA) to minimize any
complications caused by changes in cytoplasmic [Ca2+]
([Ca2+]i; see DISCUSSION) and
also to avoid Ca2+ overloading of the cells. Values of
basal [Mg2+]i thus measured in the
Ca2+-free Tyrode solution showed a Gaussian distribution
(not shown) with a mean value of 0.71 mM (±0.01 mM, n = 128).
Effect of extracellular Na+ on
[Mg2+]i in intact myocytes.
Figure 2 shows the results of our initial
experiments, in which [Mg2+]i was
continuously measured from the same myocytes. Removal of extracellular
Na+, with extracellular [Mg2+]
([Mg2+]o) increased to 10 mM, did not cause
any significant change in [Mg2+]i during a
period of 30 min (Fig. 2A). Following Na+-free
perfusion, cytoplasmic [Na+]
([Na+]i) is expected to fall rapidly to a low
level (3), dissipating the Na+ gradient across
the cell membrane. High [Mg2+]o was employed
so that the inward driving force of Mg2+ and consequently
Mg2+ influx should be enhanced. One myocyte treated with
even higher [Mg2+]o (30 mM) also showed no
clear increase in [Mg2+]i (Fig.
2B), indicating no supporting evidence for the
Na+ gradient-dependent Mg2+ transport.
However, absence of any clear change in
[Mg2+]i in these experiments can also be
explained if Mg2+ permeability of the cell membrane is very
low and therefore passive Mg2+ influx is so slow (even with
the increased driving force) that inhibition of Mg2+
extrusion fails to cause detectable changes in
[Mg2+]i in the time scale of
these measurements. We therefore carried out experiments in which
passive Mg2+ influx was greatly facilitated by the aid of
ionomycin, an ionophore for divalent cations.

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Fig. 2.
Cytoplasmic concentration of Mg2+
([Mg2+]i) measurement with extracellular
perfusion of the Na+-free solution containing 10 mM
Mg2+ (A) or 30 mM Mg2+
(B). Cells were initially perfused with
Ca2+-free Tyrode solution, and extracellular
Na+ and Mg2+ concentrations were changed for
the periods indicated by the horizontal bars. Values of means ± SE from 3 cells are plotted in A, whereas values from single
myocyte are plotted in B.
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Effect of extracellular Na+ on
ionomycin-induced rise of
[Mg2+]i.
Under Ca2+-free conditions, ionomycin is expected to
facilitate Mg2+ influx (37), because
Mg2+ is the only divalent cation present in the
extracellular space. Perfusion of the myocytes with 10 µM ionomycin
plus high [Mg2+]o (10 mM) caused a gradual
and nearly linear increase in [Mg2+]i,
probably due to the increased influx of Mg2+ (Fig.
3). Changes in
[Mg2+]i
(
[Mg2+]i) by 30 min treatment of ionomycin
were clearly smaller in the presence of 130 mM extracellular
Na+ than in its absence (Fig. 3). The rate of
Na+-dependent
[Mg2+]i
calculated as the difference between the values with or without extracellular Na+ at 30 min (Fig. 3) was, on
average, 0.76 mM/30 min or 0.42 µM/s. The ionomycin-induced
[Mg2+]i was significantly reduced by
lowering [Mg2+]o, as expected from the
extracellular origin of the increased [Mg2+]i
(Fig. 4). The rise of
[Mg2+]i was substantial only in the absence
of extracellular Na+ at normal
[Mg2+]o (1.2 mM) and was virtually absent at
0.4 mM [Mg2+]o independent of extracellular
Na+ (Fig. 4). These observations are qualitatively similar
to those in smooth muscle cells of guinea pig tenia (see Fig. 3 of Ref. 35) and are consistent with the hypothesis that
Mg2+ is extruded through a Na+
gradient-dependent pathway to cause smaller net influx of
Mg2+. This hypothesis was further tested by observation of
the Na+ dependence of net Mg2+ efflux in the
following series of experiments.

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Fig. 3.
Effects of extracellular Na+ on rise of
[Mg2+]i induced by ionomycin plus high
extracellular [Mg2+]. Changes in
[Mg2+]i are expressed as difference from
basal level ( [Mg2+]i) and plotted as a
function of time. Cells were initially perfused with
Ca2+-free Tyrode solution, and 10 µM ionomycin was
introduced with raised [Mg2+] (10 mM) either in the
presence of 130 mM Na+ (+Na, ) or in the absence of
extracellular Na+ ( Na, ) for the period
indicated by horizontal bar. All cells treated with ionomycin in the
absence of Na+ and a part of the cells (8/42) treated with
ionomycin in the presence of 130 mM Na+ ( )
were then reperfused with Ca2+-free Tyrode solution. Other
cells were perfused with solutions of different compositions for the
experiments shown in Figs. 5-7. Each symbol represents mean ± SE (n = 42 for , n = 8 for
, n = 12 for ).
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Fig. 4.
Extracellular Mg2+ concentration
([Mg2+]o) dependence of ionomycin-induced
[Mg2+]i. With
[Mg2+]o set at 0.4, 1.2, and 10 mM either in
the absence of Na+ (open bars) or in the presence of
130-140 mM Na+ (solid bars),
[Mg2+]i values at 30 min after application
of 10 µM ionomycin were obtained from the experiments of the type
shown in Fig. 3. Each column shows mean ± SE; from
left to right, n = 6, 6, 5, 6, 42, and 12.
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Net Mg2+ efflux from loaded myocytes.
After the [Mg2+]i was raised by treatment
with ionomycin and high [Mg2+]o (see above),
washout of ionomycin by the Ca2+-free Tyrode solution
containing 1.2 mM Mg2+ caused a rapid decrease in
[Mg2+]i toward the basal level, indicating
net Mg2+ efflux (Fig. 3). Figure
5 shows the results of experiments
designed to specifically study the rate of decline of
[Mg2+]i from the Mg2+-loaded
cells. Myocytes were initially loaded with Mg2+ by
treatment with ionomycin in the high-Mg2+ solution
containing 10 mM Mg2+ and 130 mM Na+, as
described above. Because the effects of ionomycin on cellular Mg2+ loading is quite variable from cell to cell, we
appropriately adjusted the concentration (9-12 µM) and treatment
time (25-60 min) of ionomycin to achieve similar Mg2+
loading: 0.4-0.7 mM (0.57±0.01 mM, n = 93) above
the basal level. Washout of ionomycin and reduction of extracellular
Mg2+ back to the normal level quickly decreased
[Mg2+]i in the presence of 140 mM
extracellular Na+ (open inverted triangles in Fig.
5A). On the other hand, [Mg2+]i
did not decrease, or even slightly increase, in the absence of
extracellular Na+ (open circles in Fig. 5A);
this small increase in [Mg2+]i is probably
due to the continued Mg2+ influx driven by membrane
potential. [Mg2+]i started to decrease after
reintroduction of 140 mM Na+ (open circles in Fig.
5A). The decay of [Mg2+]i in the
presence of extracellular Na+ was approximately linear
during the first 10 min but slowed thereafter as
[Mg2+]i approached the basal level. We
therefore analyzed the
[Mg2+]i over the
initial 10 min in the following analysis.

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Fig. 5.
Effect of extracellular Na+ and imipramine on
[Mg2+]i. A: after cells were
loaded with Mg2+ by 9-12 µM ionomycin + 10 mM
Mg2+, the perfusate was changed, at zero time, to one of
the test solutions containing 1.2 mM Mg2+ with various
Na+ concentrations for the period indicated by a horizontal
bar. Cells were then washed by Ca2+-free Tyrode solution
containing 1.2 mM Mg2+ and 140 mM Na+. Changes
in [Mg2+]i (ordinate) are expressed as the
difference from the level achieved at the end of loading: generally
1.2-1.3 mM [Mg2+]i (for
details, see text). The test solution contained 0 mM Na+
( , n = 8), 140 mM Na+ ( ,
n = 8), 0 mM Na+ + 200 µM imipramine
( , n = 3), or 140 mM
Na+ + 200 µM imipramine ( ,
n = 6). B: imipramine concentration
dependence of [Mg2+]i measured at 10 min
after application of the test solutions with the protocol shown in
A. Solid line indicates least-squares fit of
[Mg2+]i data ( ) as a
function of imipramine concentration ([X]) by the Hill-type curve
with parameters shown in the panel:
[Mg2+]i = min + (max min)
{[X]N/(K1/2N + [X]N)}, where max and min denote,
respectively, [Mg2+]i in the presence of a
saturating concentration of imipramine and in its absence, N
is the Hill coefficient, and K1/2 is imipramine
concentration that gives a midpoint value of
[Mg2+]i between min and max. In the range
of independent variable values ([X]), the program iterated the
fitting procedure until a set of 4 adjustable variables (max, min,
K1/2, and N) that gave the least-squares sum of
the error of [Mg2+]i values was found. The
2 dotted lines show the levels of average
[Mg2+]i in the absence of imipramine at 0 mM [Na+]o (top) and 140 mM
[Na+]o (bottom). In A
and B, each symbol represents mean±SE of 3-8 cells.
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We examined the effect of imipramine, a known inhibitor of
Na+/Mg2+ exchange in erythrocytes (11,
13), on the decay rate of [Mg2+]i
(closed symbols in Fig. 5A). Imipramine (200 µM) did not
significantly influence
[Mg2+]i in the
Na+-free solution (closed circles in Fig. 5A),
but markedly slowed the decay of [Mg2+]i in
the presence of 140 mM Na+ (closed inverted triangles in
Fig. 5A). The effect of imipramine was quickly reversed
after washout. The relation between imipramine concentration and
[Mg2+]i showed that > 90% of the
Na+ gradient-dependent net Mg2+ efflux was
inhibited by imipramine with a half-inhibitory concentration of ~80
µM (Fig. 5B).
In pilot experiments, we tested other ways that possibly influence the
net Mg2+ efflux. Amiloride, a poorly selective blocker of
Na+-related transporters, has been reported to inhibit
Na+/Mg2+ exchange at millimolar concentrations
in erythrocytes (13, 16). We found, however, that
fluorescence of amiloride at such high concentrations significantly
interfered with the optical measurements. Na+ substitution
by Li+, instead of NMDG+, also had serious
difficulties because of the direct interaction of Li+ on
furaptra; substitution of 30 mM Na+ by 30 mM
Li+ caused ~10% decrease in the furaptra R in the
solution containing 0-1 mM [Mg2+] (not shown).
Na+ dependence of the net Mg2+ efflux was
further studied by monitoring the decay of
[Mg2+]i at various extracellular
[Na+] ([Na+]o) levels between 0 and 140 mM (Fig. 6A). To
obtain additional information on the influence of membrane potential,
the experiments were repeated at high extracellular [K+]
([K+]o = 55.9 mM, Fig. 6B),
in which the cell membrane was expected to depolarize. At both normal
[K+]o and high
[K+]o, lowering
[Na+]o dose dependently reduced the
Mg2+ efflux from the loaded cells, as indicated by the
slower decline of [Mg2+]i (Fig. 6). Figure
7A shows a more complete
analysis of the relation between [Na+]o and
the rate of net Mg2+ efflux. At
[Na+]o of 0 and 50 mM, the values of
[Mg2+]i were significantly more negative
at high [K+]o (closed squares) than at normal
[K+]o (open squares), suggesting that net
Mg2+ efflux was facilitated by high
[K+]o or cell membrane depolarization.
(Statistical significance was also found between the
[Mg2+]i values at 90 mM
[Na+]o + 55.9 mM
[K+]o and those at 100 mM
[Na+]o + 5.9 mM
[K+]o.) Figure 7B displays the
Na+-dependent
[Mg2+]i
calculated, at each [K+]o level, by
subtraction of
[Mg2+]i values in the
absence of extracellular Na+ from those in its presence. At
normal [K+]o, half activation of the
Na+ gradient-dependent Mg2+ efflux occurred at
~80 mM [Na+]o. High
[K+]o appeared to cause the shift of the
curve toward lower [Na+]o, with no obvious
change in the slope (n
2).

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Fig. 6.
Effect of extracellular Na+ and K+ on
[Mg2+]i. Changes in
[Mg2+]i from the loaded level (ordinate) were
measured at 5.9 mM [K+]o (A) or
55.9 mM [K+]o (B). After cells
were loaded with Mg2+, one of the test solutions containing
1.2 mM Mg2+ with various Na+ concentrations
(marked in mM near the symbols) was introduced at zero time for 20 min.
Each symbol represents mean±SE of 4-9 cells.
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Fig. 7.
Analysis of [Mg2+]i from the
experiments of the type shown in Fig. 6. A: extracellular
Na+ dependence of [Mg2+]i
measured at 10 min after application of the test solutions containing
either 5.9 mM K+ ( ) or 55.9 mM
K+ ( ). Solid line indicates the
least-squares fit of the data at 5.9 mM [K+]o
by the Hill-type curve (see below). B:
Na+-dependent [Mg2+]i
calculated by subtraction of the values at zero
[Na+]o for each data set and plotted as a
function of [Na+]o. Solid line indicates the
least-squares fit of the data at 5.9 mM [K+]o
( ) by the Hill-type curve with parameters shown in the
panel: [Mg2+]i = max + (min max) · {[Na]oN/(K1/2N + [Na]oN)}, where min and max denote,
respectively, minimum and maximum values of Na+-dependent
[Mg2+]i, N is the Hill
coefficient, and K1/2 is
[Na+]o that gives a midpoint value of the
Na+-dependent [Mg2+]i between
min and max.
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DISCUSSION |
The present experiments used fluorescence signals of a
Mg2+ indicator furaptra to study Mg2+ transport
across the cell membrane. This method allows noninvasive measurements
of [Mg2+]i from single myocytes over a period
of hours. A difficulty of [Mg2+]i measurement
by furaptra lies in the calibration of fluorescence signals in terms of
[Mg2+]i, because properties of furaptra are
likely altered in cytoplasmic environments (21). The
present calibration method of furaptra fluorescence signals relied on
parameter values previously estimated in smooth muscle cells
(34) and gave a mean value of the basal [Mg2+]i of 0.71 mM. Although obtained in
Ca2+-free Tyrode solution, this value is probably also
applicable to the [Mg2+]i in normal Tyrode
solution containing 2.4 mM Ca2+, because our previous study
showed that removal of extracellular Ca2+ had little
influence on [Mg2+]i (18). The
value of 0.71 mM is consistent with estimates of a 0.5-1.0 mM
range with various methods (see introduction), but somewhat lower than
our previous estimate, on average 1.13 mM calibrated under identical
conditions in rat cardiac myocytes (34). The difference
may be due to cell-to-cell variation in the small number of myocytes
(n = 9) in the previous study and was not further
considered here.
Although
[Mg2+]i measured in the present
study was thought to reflect, in the most part, Mg2+ flux
across the cell membrane (Fig. 4), alteration of intracellular Mg2+ binding or Mg2+ sequestration by
organelles could also contribute to
[Mg2+]i. We therefore minimized the
changes in [Ca2+]i and cytoplasmic pH that
should affect Mg2+ buffering (23); changes in
[Ca2+]i are expected to be negligible and
changes in pH should also be minimal in Ca2+-free
conditions (9). The previous study from this laboratory (18) has shown that, after ~10 min perfusion with a 0.1 mM EGTA-containing solution, neither Ca2+ release from the
sarcoplasmic reticulum (by 25 mM caffeine) nor intracellular acidosis
of ~0.4 pH unit (by 5% CO2) has little influence on
[Mg2+]i measured with furaptra.
Ca2+-free conditions should also minimize Mg2+
uptake by mitochondria, as shown by electron probe microanalysis (4), and Mg2+ uptake by the sarcoplasmic
reticulum as a counter ion of Ca2+ release
(33). We also avoided experimental conditions that induce
Mg2+ release from intracellular organelles by stimulation
of cAMP production (29, 30) or muscarinic stimulation
(38). It is possible, however, that quantification of
transmembrane Mg2+ flux based on the measured
[Mg2+]i suffers, because of uncertainties
related to intracellular binding and sequestration of
Mg2+.
Absence of Na+-dependent changes in
[Mg2+]i in intact myocytes.
While removal of extracellular Na+ (which should inhibit
Na+ gradient-dependent Mg2+ extrusion) in the
presence of high [Mg2+]o (which should
increase leak influx of Mg2+) failed to show any
significant changes in [Mg2+]i (Fig. 2), a
large increase in cell membrane permeability to Mg2+
unmasks the Na+ gradient-dependent Mg2+
transport (Fig. 3). It is thus conceivable that very controversial findings reported concerning cardiac myocytes (see introduction) may
result from variable Mg2+ permeability of the cell membrane
in different experimental conditions (or cell conditions). Handy et al.
(17) reported that Na+ withdrawal in the
presence of 5 mM Mg2+ caused a small but clear increase in
[Mg2+]i (~28 µM/min) in rat ventricular
myocytes at 37°C. The difference in the results between the present
study and Handy et al. (17) could be due simply to the
difference in experimental temperature (37°C vs. 25°C), but other
experimental conditions that somehow alter the cell membrane
permeability to Mg2+ could also be involved.
Removal of extracellular Na+ should reverse the
transmembrane electrochemical gradient of Na+ and could
reverse the direction of Na+/Mg2+ exchange to
raise [Mg2+]i, i.e., Mg2+ influx
associated with Na+ efflux (15). The present
results, although providing no evidence for the reversal, do not
necessarily exclude the possible reversal of Na+
gradient-dependent Mg2+ transport. After removal of
extracellular Na+, [Na+]i
probably falls to a low level within several minutes (3), and transport may not be driven in the reversed direction by this low
[Na+]i. Therefore, it is likely that
extracellular Na+-dependent
[Mg2+]i shown in the present study is also
influenced by changes in [Na+]i, as noted in
our previous study (35).
Evidence for
Na+/Mg2+
exchange.
Extracellular Na+ suppressed net Mg2+ influx
into ionomycin-treated cells and facilitated net Mg2+
efflux from Mg2+-loaded cells. Imipramine markedly
inhibited net Mg2+ efflux only in the presence of
extracellular Na+. These results are most likely explained
by Na+ gradient-dependent Mg2+ efflux (or
Na+/Mg2+ exchange). The putative
Na+-Mg2+ exchange may play a role in long-term
regulation of [Mg2+]i to prevent
Mg2+ overloading of cardiac myocytes.
From the Na+ dependence of the ionomycin-induced
[Mg2+]i (Fig. 3), we could estimate
transmembrane Mg2+ flux using values assumed for a cell
surface-to-volume ratio of 0.63 µm
1 [surface area
1.23×104 µm2 (24); volume
1.95×104 µm3 (5)], a
cytoplasm-to-cell volume ratio of 0.5 (32), and a
cytoplasmic Mg2+ buffering capacity of 2.5 (23). With these values taken from the literature,
Na+-dependent suppression in
[Mg2+]i of 0.42 µM/s (see
RESULTS) would correspond to Mg2+ flux (net
efflux) of 0.083 pmol · cm
2 · s
1.
With the Na+-dependent
[Mg2+]i
value of 28 µM/min estimated by Handy et al. (17) in rat ventricular myocytes at 5 mM [Mg2+]o and
37°C, a calculation under otherwise identical conditions yields a
similar Mg2+ flux value of 0.092 pmol · cm
2 · s
1. A value of
Na+ gradient-dependent Mg2+ flux somewhat lower
than, but within the same order of magnitude of, the present estimate
was reported previously in smooth muscle cells of guinea pig tenia at
10 mM [Mg2+]o and 25°C [0.026
pmol · cm
2 · s
1
(35)]. Thus our present results are in reasonable
agreement with earlier measurements. An estimate of the Na+
gradient-dependent Mg2+ efflux could also be obtained from
the Na+ dependence of [Mg2+]i
decay from the loaded myocytes (Fig. 5). From the
Na+-dependent
[Mg2+]i of
0.69 mM/10 min (a value at 140 mM [Na+]o in
Fig. 7B), a calculated value for the Na+
gradient-dependent Mg2+ efflux would be 0.23 pmol · cm
2 · s
1 at 1.2 mM
[Mg2+]o and 25°C. A 2.7 times greater value
than that estimated above from the Na+ dependence of the
ionomycin-induced
[Mg2+]i (0.083 pmol · cm
2 · s
1) may be due
to lower [Mg2+]o (1.2 vs. 10 mM) and/or
higher [Mg2+]i in the loaded myocytes; note
that the rate of [Mg2+]i decay is
significantly slowed as [Mg2+]i approaches
the basal level (Fig. 5).
Handy et al. (17) reported an almost complete inhibition
of Na+-dependent
[Mg2+]i by 10 µM imipramine in rat ventricular myocytes. It is not clear if the
difference in temperature (37°C vs. 25°C) could entirely explain
the much higher concentration of imipramine (a half-inhibitory concentration of ~80 µM) required in the present study (Fig.
5B). Our estimate is, however, roughly comparable to
reported IC50 values of the agent for
Na+/Mg2+ exchange in human erythrocytes [25
µM (11)] or ferret erythrocytes [<500 µM
(13)]. The Hill coefficient of 2 to best explain the relation between imipramine concentration and
[Mg2+]i suggests the binding of two (or
more) imipramine molecules to a putative transporter molecule.
The Hill coefficient of 2 was also obtained for the relation between
[Na+]o and the rate of Mg2+
efflux (reflected in
[Mg2+]i), suggesting
two (or more) Na+ binding sites on the transporter. If
Mg2+ is extruded in exchange for two (or more)
Na+, Na+/Mg2+ exchange should carry
no net current that may be insensitive to cell membrane potential (or
net inward current that may be inhibited by cell membrane
depolarization). On the contrary, however, the leftward shift of the
relation between [Na+]o and the rate of
Mg2+ efflux by high [K+]o (Fig.
7B) suggest facilitation of Na+/Mg2+
exchange by cell membrane depolarization. These conflicting results are
puzzling and could be explained if high [K+]o
substantially lowers [Na+]i by facilitation
of Na+-K+-ATPase,
Na+/Mg2+ exchange being driven by the larger
driving force for Na+. Alternatively, extracellular
K+ may be directly involved in Mg2+ transport
(28) in addition to the effect on membrane potential and
[Na+]i. Further studies are required to
stoichiometrically determine the exchange in cardiac myocytes.
In conclusion, the present results demonstrate the existence of a
Na+ gradient-dependent Mg2+ efflux activity in
rat cardiac myocytes. Being half-maximally activated by ~80 mM
[Na+]o and inhibited by imipramine, this
Mg2+ transport is consistent with
Na+/Mg2+ exchange. Our results do not provide
clear evidence for reversal of Mg2+ transport, i.e.,
Na+ gradient-dependent Mg2+ influx, in our
experimental conditions.
 |
ACKNOWLEDGEMENTS |
We thank Prof. Satoshi Kurihara of the Department of Physiology of
the Jikei University School of Medicine for helpful comments and Prof.
J. Patrick Barron of the International Medical Communications Center of
Tokyo Medical University for reading the manuscript.
 |
FOOTNOTES |
This work was supported by a Grant-in-Aid for Scientific Research from
the Ministry of Education, Science, and Culture, Japan.
Present address of M. Tashiro: Dept. of Internal Medicine, The Jikei
Univ. School of Medicine, 3-25-8 Nishishinbashi, Minato-ku, Tokyo 105-8461, Japan.
Address for reprint requests and other correspondence: M. Konishi, Dept. of Physiology, Tokyo Medical Univ., 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan (E-mail:
mkonishi{at}tokyo-med.ac.jp).
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 April 2000; accepted in final form 6 July 2000.
 |
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