Evidence for
Na+/Ca2+
exchange in intact single skeletal muscle fibers from the
mouse
Christopher D.
Balnave and
David G.
Allen
Department of Physiology and Institute of Biomedical Research,
University of Sydney, Sydney, New South Wales 2006, Australia
 |
ABSTRACT |
The myoplasmic free Ca2+
concentration
([Ca2+]i)
was measured in intact single fibers from mouse skeletal muscle with
the fluorescent Ca2+ indicator
indo 1. Some fibers were perfused in a solution in which the
concentration of Na+ was reduced
from 145.4 to 0.4 mM (low-Na+
solution) in an attempt to activate reverse-mode
Na+/Ca2+
exchange (Ca2+ entry in exchange
for Na+ leaving the cell). Under
normal resting conditions, application of
low-Na+ solution only increased
[Ca2+]i
by 5.8 ± 1.8 nM from a mean resting
[Ca2+]i
of 42 nM. In other fibers,
[Ca2+]i
was elevated by stimulating sarcoplasmic reticulum (SR)
Ca2+ release with caffeine (10 mM)
and by inhibiting SR Ca2+ uptake
with
2,5-di(tert-butyl)-1,4-benzohydroquinone
(TBQ; 0.5 µM) in an attempt to activate forward-mode
Na+/Ca2+
exchange (Ca2+ removal from the
cell in exchange for Na+ influx).
These two agents caused a large increase in
[Ca2+]i,
which then declined to a plateau level approximately twice the baseline
[Ca2+]i
over 20 min. If the cell was allowed to recover between exposures to
caffeine and TBQ in a solution in which
Ca2+ had been removed, the
increase in
[Ca2+]i
during the second exposure was very low, suggesting that
Ca2+ had left the cell during the
initial exposure. Application of caffeine and TBQ to a preparation in
low-Na+ solution produced a large,
sustained increase in
[Ca2+]i
of ~1 µM. However, when cells were exposed to caffeine and TBQ in a
low-Na+ solution in which
Ca2+ had been removed, a sustained
increase in
[Ca2+]i
was not observed, although
[Ca2+]i
remained higher and declined slower than in normal
Na+ solution. This suggests that
forward-mode
Na+/Ca2+
exchange contributed to the fall of
[Ca2+]i
in normal Na+ solution, but when
extracellular Na+ was low, a
prolonged elevation of
[Ca2+]i
could activate reverse-mode
Na+/Ca2+
exchange. The results provide evidence that skeletal muscle fibers possess a
Na+/Ca2+
exchange mechanism that becomes active in its forward mode when [Ca2+]i
is increased to levels similar to that obtained during contraction.
intracellular calcium concentration; sarcoplasmic reticulum; caffeine; 2,5-di(tert-butyl)-1,4-benzohydroquinone
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INTRODUCTION |
STRIATED MUSCLE CELLS contain a plasma membrane
Na+/Ca2+
exchange. In its forward mode, this electrogenic antiporter exchanges three extracellular Na+ for the
removal of one intracellular Ca2+,
whereas in its reverse mode, one
Ca2+ enters the cell in exchange
for three intracellular Na+, which
are extruded (10). Although
Na+/Ca2+
exchange has been investigated extensively in cardiac muscle cells,
where it has a central role in myoplasmic free
Ca2+ concentration
([Ca2+]i)
regulation (30),
Na+/Ca2+
exchange in skeletal muscle cells has received less attention.
The most direct evidence for a functioning
Na+/Ca2+
exchange in skeletal muscle has come from studies that have measured
the flux of
45Ca2+
across the plasma membrane. This method has been used to identify Na+/Ca2+
exchange activity both in plasma membrane vesicles isolated from frog
and rabbit skeletal muscle cells (10, 13, 15) as well as in intact frog
skeletal muscles (6, 20). In addition, the reduction or absence of
extracellular Na+ results in an
increase in twitch height and the amplitude of K+ and caffeine contractures in
single frog skeletal muscle fibers (8, 12). Therefore, it appears that
a
Na+/Ca2+
exchange mechanism does exist in skeletal muscle. However, the role of
Na+/Ca2+
exchange in the regulation of
[Ca2+]i
in skeletal muscle is largely unknown and has not been investigated in
intact mammalian skeletal muscle cells.
The present investigation provides evidence for the presence of a
functioning
Na+/Ca2+
exchange mechanism in single mouse skeletal muscle fibers. Reverse-mode Na+/Ca2+
exchange activity was low under normal resting conditions and only
became measurable when
[Ca2+]i
was increased substantially above 80 nM. Our results suggest that an
elevation of
[Ca2+]i
to levels similar to that obtained during tetanic stimulation can
activate forward-mode
Na+/Ca2+
exchange, which can contribute to the lowering of
[Ca2+]i.
 |
METHODS |
Single muscle fibers were dissected from the flexor brevis muscle of
male mice killed by rapid cervical dislocation, as described previously
(27). Briefly, fibers were attached by platinum clips to an Akers AE
801 force transducer (SensoNor) at one end and to a fixed stainless
steel rod at the other. Thus the fibers were held at their optimum
force-generating length (~800 µm) throughout the experiment. This
length was determined by increasing the length of the muscle fiber from
a slack length until tetanic force was maximal. Platinum-plate
stimulating electrodes ran along the length of the muscle fiber.
Although all experiments were performed on resting unstimulated fibers,
muscle force was examined at regular periods to verify the functional
integrity of the fibers. If a muscle fiber failed to produce
approximately normal tetanic force, the experiment was excluded.
Control and experimental results were obtained in the same fiber for
direct comparison.
Solutions.
Fibers were perfused with the following standard solution (in mM):
121.0 NaCl, 5.0 KCl, 1.8 CaCl2,
0.5 MgCl2, 0.4 NaH2PO4, 24.0 NaHCO3, and 5.5 glucose. This
solution was bubbled with 95% O2-5%
CO2, giving a pH of 7.3. Fetal
calf serum was also added to the solution (~0.2%). In some
experiments, CaCl2 was replaced with equimolar concentrations of
MgCl2 (0 Ca2+ solution). To stimulate
Ca2+ influx via
Na+/Ca2+
exchange, the majority of the Na+
was replaced with
N-methyl-D-glucamine
(NMG). The composition of this
low-Na+ (NMG) solution was (in mM)
121.0 NMG-Cl, 5.0 KCl, 1.8 CaCl2, 0.5 MgCl2, 0.4 NaH2PO4,
24.0 NMG-HCO3, and 5.5 glucose.
Caffeine and
2,5-di(tert-butyl)-1,4-benzohydroquinone
(TBQ; from a stock solution of 1 mM in dimethyl sulfoxide) were added
to the appropriate solution to produce final concentrations of 10 mM
and 0.5 µM, respectively. In some experiments, cyanide was added to
the stimulation solution. A stock solution of 300 mM NaCN was created
and combined with 2 M
N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic
acid (HEPES) immediately before experimentation to give a solution
containing 200 mM CN
and
667 mM HEPES. The
CN
/ HEPES mixture (pH
~7.4) was then added to the stimulation solution to give a final
CN
concentration of 2 mM.
All experiments were performed at room temperature (~22°C).
Measurement of
[Ca2+]i.
The fluorescent Ca2+ indicator
indo 1 was used to measure
[Ca2+]i
in single muscle fibers. Indo 1 was dissolved in 150 mM KCl and 10 mM
HEPES, pH 7.3, at a concentration of 10 mM. Microelectrodes (resistance
15-50 M
) were filled with ~0.5 µl of this solution, and
indo 1 was pressure-injected to an approximate concentration of
25-50 µM (3). On withdrawal of the microelectrode, muscle fibers
were allowed to recover for 30 min before experimentation began. This
was sufficient time for the dye to become homogeneously distributed
along the fibers.
The experimental chamber was positioned on the stage of a Nikon Diaphot
microscope with a ×20 dry objective. Indo 1 was excited by
illuminating the fibers with ultraviolet light of wavelength 360 nm. A
shutter prevented the fiber from being illuminated except during
experimental measurements. The emitted signals at 400 and 510 nm were
measured by photomultiplier tubes, and the background fluorescence was
removed. The amplified output from each photomultiplier tube then
entered an analog divide circuit where the ratio was taken. An in vivo
calibration (28) was performed to convert the indo 1 ratio to
[Ca2+]i.
Although the fluorescence system allows for continuous measurement of
the fluorescent signal, the cell was only illuminated for brief periods
(5 s every minute) to avoid photobleaching of the indo 1.
Statistics.
Unless otherwise stated, data are quoted as means ± SE. Paired
t-tests were used to determine
statistical significance with P < 0.05 taken as significant.
 |
RESULTS |
Reverse-mode
Na+/Ca2+
exchange at normal
[Ca2+]i.
In cardiac muscle, removal of extracellular
Na+ results in a large, rapid
influx of Ca2+ through the
Na+/Ca2+
exchanger (1). To investigate whether a similar phenomenon occurs in
mammalian skeletal muscle cells, single fibers were placed in the
low-Na+ solution for 10 min. The
effects of this protocol on the mean [Ca2+]i
from seven fibers are shown in Fig. 1. In
these experiments, the resting
[Ca2+]i
was 42 ± 7 nM, and the increase in
[Ca2+]i
in low-Na+ solution was 5.8 ± 1.8 nM. This increase is very small compared with the tetanic
[Ca2+]i
of ~1,000 nM and is much smaller than the equivalent increase in
cardiac ventricular muscle, which can exceed 1,000 nM in a few seconds
(1).

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Fig. 1.
Effect on myoplasmic free Ca2+
concentration
([Ca2+]i)
of reducing Na+ concentration in
perfusing solution. Mean
[Ca2+]i
from 7 fibers increased by 5.8 ± 1.8 nM over 10 min when
extracellular Na+ was replaced
with
N-methyl-D-glucamine
(NMG).
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With the assumption that
Na+/Ca2+
exchange is present in mammalian skeletal muscle, there are several
possible reasons why its activity in reverse mode is apparently so low.
1) It may be that the
Ca2+ that enters the myoplasm is
rapidly taken up by the sarcoplasmic reticulum (SR). This would be
consistent with the demonstration that the twitches and
K+ and caffeine contractures
increased in low-Na+ solutions (8,
12). 2) Not only does the
Na+/Ca2+
exchanger possess binding sites for ion transport, but modulation of
exchange activity results from binding cytoplasmic
Ca2+ to a regulatory site on the
exchanger (9, 18, 23). It has been shown that reverse-mode
Na+/Ca2+
exchange is dependent on
[Ca2+]i
(17, 19), and it may be that under normal resting conditions [Ca2+]i
is sufficiently low to cause inactivation of
Na+/Ca2+
exchange. We have investigated these two possibilities by modifying the
function of the SR with TBQ and caffeine.
SR Ca2+ uptake was inhibited with
TBQ (0.5 µM), which has been shown to cause a pronounced inhibition
of the SR Ca2+ pump rate (29).
Under these conditions, a 7- to 10-min application of
low-Na+ solution failed to produce
a significant rise in
[Ca2+]i
(4.4 ± 1.4 nM; n = 4). This result
suggests that the SR did not buffer an influx of
Ca2+ during the experiment
described in Fig. 1. Caffeine has been shown to elevate the
[Ca2+]i
by increasing the number and duration of open SR
Ca2+- release channels (26).
Addition of 10 mM caffeine to the solution containing normal
Na+ concentration increased
[Ca2+]i
from 48.1 ± 13.1 nM by 26 ± 12.3 nM in three experiments,
comparable to previous studies on this preparation (3). However, a
10-min application of low-Na+
solution in the continuing presence of caffeine failed to produce a
significant increase in
[Ca2+]i
(6.5 ± 3.9 nM).
Effect of elevating
[Ca2+]i
with TBQ and caffeine.
To elevate
[Ca2+]i
further and more effectively suppress the function of the SR, 0.5 µM
TBQ and 10 mM caffeine were applied together. Figure
2 shows the
[Ca2+]i
and force records from a representative fiber during a 100-Hz isometric
tetanus (350 ms in duration) and then during exposure to TBQ and
caffeine. In this experiment, TBQ was added to the solution 5 min
before caffeine to ensure sufficient inhibition of the SR
Ca2+ pump. Usually this caused a
large, rapid elevation of
[Ca2+]i
and force upon caffeine exposure (Fig. 2), although in some experiments
[Ca2+]i
took a minute or two to peak. In some experiments, TBQ and caffeine
were applied at the same time, and
[Ca2+]i
took 1-2 min to reach its maximum, the magnitude of which was generally much lower than the rapid peak obtained by prior treatment with TBQ. However, after a peak was reached, the decline in
[Ca2+]i
was similar in all fibers.

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Fig. 2.
[Ca2+]i
and force in a representative fiber during a 100-Hz tetanus and during
exposure to 0.5 µM
2,5-di(tert-butyl)-1,4-benzohydroquinone
(TBQ) and 10 mM caffeine. During a tetanus,
[Ca2+]i
and force increased rapidly over a few milliseconds. On exposure to TBQ
and caffeine,
[Ca2+]i
and force took a number of seconds to reach maximum. Decline in
[Ca2+]i
and force was also very slow after TBQ and caffeine exposure relative
to tetanic stimulation.
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Figure 3A
shows the characteristic elevation and subsequent decline in
[Ca2+]i
over a longer time course in a representative fiber. During the 30-min
exposure to TBQ and caffeine,
[Ca2+]i
declined relatively rapidly at high concentrations and then slowed as
the
[Ca2+]i
became lower. After ~20-min exposure to TBQ and caffeine,
[Ca2+]i
had leveled off once more at a concentration higher than the baseline
level. In 14 experiments, the normal baseline
[Ca2+]i
was 43.6 ± 3.9 nM. The peak
[Ca2+]i
(771.1 ± 207.8 nM) was almost 20-fold higher than baseline, whereas
the plateau level following the secondary decline was 89.0 ± 8.6 nM. The following experiments were performed to determine whether the
decline in
[Ca2+]i
following the initial peak is due, at least in part, to
Ca2+ efflux from the cell via
forward-mode
Na+/Ca2+
exchange.

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Fig. 3.
Time course of decline in
[Ca2+]i
after exposure to 0.5 µM TBQ and 10 mM caffeine.
A:
[Ca2+]i
in a representative fiber during a 30-min exposure to TBQ and caffeine.
After initial large increase,
[Ca2+]i
declined slowly and reached a new plateau level after ~20 min. When
TBQ and caffeine were washed out,
[Ca2+]i
returned to levels close to baseline.
B: mean
[Ca2+]i
during 60-min washout period after TBQ and caffeine exposure
(n = 14; error bars only shown for 3rd
minute after washout for clarity). Upon washout,
[Ca2+]i
dropped significantly below baseline
[Ca2+]i
(indicated by dotted line). Symbols represent means ± SE of
baseline
[Ca2+]i
and of peak
[Ca2+]i
after TBQ and caffeine exposure.
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Upon washout from TBQ and caffeine exposure, stimulation of SR
Ca2+ release and inhibition of SR
Ca2+ uptake are removed. If there
is less Ca2+ in the cell, then as
long as SR Ca2+ uptake is faster
than Ca2+ entry back into the
cell,
[Ca2+]i
should fall below the original resting level. This undershoot of
baseline
[Ca2+]i
has been observed previously in cardiac and smooth muscle cells (4).
Figure 3B shows the mean resting and
peak
[Ca2+]i
with error bars from 14 fibers exposed to TBQ and caffeine for 30 min.
Also shown is the mean
[Ca2+]i
throughout the 60-min washout period from TBQ and caffeine in these
fibers. Upon washout, the
[Ca2+]i
fell significantly below the normal baseline
[Ca2+]i
(indicated by the dashed line). This undershoot of
[Ca2+]i
was observed in every experiment, and
[Ca2+]i
gradually returned to normal over 60 min. This is the result that would
be predicted if Ca2+ was removed
from the cell during TBQ and caffeine exposure.
There are a number of mechanisms by which muscle cells regulate
[Ca2+]i,
some of which could contribute to the lowering of
[Ca2+]i
during exposure to TBQ and caffeine. One possibility is that mitochondria take up Ca2+. In
three fibers, 2 mM cyanide was used to block electron transport, consequently preventing mitochondrial
Ca2+ uptake. Cyanide was added to
the perfusing solution at least 5 min before exposure of the fiber to
TBQ and caffeine and was maintained in the perfusing solution during
TBQ and caffeine exposure. However, cyanide did not affect the increase
or the decline in [Ca2+]i
after the exposure.
Effect of removal of extracellular
Ca2+ during
washout.
If Ca2+ is extruded from the cell
during TBQ and caffeine exposure, then we would expect that the removal
of Ca2+ from the washout solution
should prevent the reaccumulation of Ca2+ by the cell. Figure
4 shows the average results from four
fibers that were exposed to TBQ and caffeine for 30 min and then
allowed to recover in a 0 Ca2+
solution. During the initial exposure to TBQ and caffeine, the [Ca2+]i
rose sharply to 1,265.8 ± 614.6 nM and then declined in the manner
described above. However, after 60-min washout in the 0 Ca2+ solution, TBQ and caffeine
exposure could only increase
[Ca2+]i
to 100.6 ± 24.1 nM. In the experiments shown in Fig. 4, this second
exposure to TBQ and caffeine was performed in the 0 Ca2+ solution. However, in two
fibers, the second TBQ and caffeine exposure was performed with normal
concentrations of Ca2+ in the
perfusing solution (data not included in Fig. 4). In these fibers, the
initial TBQ and caffeine exposure increased
[Ca2+]i
to 812.0 ± 216.7 nM, whereas the exposure after washout in 0 Ca2+ solution increased
[Ca2+]i
to only 71.1 ± 8.3 nM. In the experiments shown in Fig. 4, a third
TBQ and caffeine exposure was given after a further 60-min washout
period, this time in the standard perfusing solution containing normal
Ca2+ concentration. This brought
about an increase in
[Ca2+]i
to 804.0 ± 185.6 nM, followed again by the characteristic decline in
[Ca2+]i
described previously.

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Fig. 4.
Removal of Ca2+ from perfusing
solution during recovery of
[Ca2+]i
from exposure to 0.5 µM TBQ and 10 mM caffeine. Mean
[Ca2+]i
during 3 exposures to TBQ and caffeine
(n = 4; error bars not shown for
clarity). Upon washout from first exposure, fibers were perfused in 0 Ca2+ solution until washout from
second exposure, when fibers were once again perfused in standard
Ca2+-containing solution. First
and third TBQ and caffeine exposures produced a large increase in
[Ca2+]i,
but after recovery in 0 Ca2+
solution, the second exposure only increased
[Ca2+]i
by a small amount.
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Effect of reducing extracellular
Na+ and
elevating
[Ca2+]i.
Figure 4 suggests that Ca2+ is
removed from the cell during TBQ and caffeine exposure. To examine
whether forward-mode
Na+/Ca2+
exchange contributes to this Ca2+
efflux, Na+ was removed from the
perfusing solution either 5 min before or at the same time as the
addition of TBQ and caffeine. Figure
5A shows
the
[Ca2+]i
in an individual cell during exposure to TBQ and caffeine for 30 min in
normal Na+ solution and in
low-Na+ solution. Between
exposures, the fiber recovered in normal
Na+ solution. Both exposures
produced a large, rapid increase in [Ca2+]i.
However, in normal Na+ solution,
the
[Ca2+]i
recovered in the characteristic manner described previously, whereas in
low-Na+ solution, there was a
brief period of recovery of
[Ca2+]i
followed by an extended period when
[Ca2+]i
was maintained at a high level. Upon washout,
[Ca2+]i
returned to baseline after both exposures in this fiber. Figure 5B shows a magnified comparison of the
washout periods after TBQ and caffeine exposure in normal
Na+ and
low-Na+ solutions. Note that in
normal Na+ solution, where
[Ca2+]i
was raised under conditions that should
allow-Na+/Ca2+
exchange to operate normally, washout produced an undershoot in
[Ca2+]i
below the baseline level. However, in
low-Na+ solution, where
[Ca2+]i
was raised under conditions in which
Ca2+ efflux via
Na+/Ca2+
exchange should be inhibited,
[Ca2+]i
did not undershoot upon washout.

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Fig. 5.
Effect on
[Ca2+]i
of reducing Na+ concentration in
perfusing solution during exposure to 0.5 µM TBQ and 10 mM caffeine.
A:
[Ca2+]i
in an individual fiber exposed to TBQ and caffeine for 30 min in
standard solution (normal Na+
concentration), followed 60 min later by a second exposure to TBQ and
caffeine in low-Na+ solution.
[Ca2+]i
declined to a new plateau level in ~20 min during first exposure but
remained elevated at ~900 nM during second exposure.
B: magnified plot of 60-min washout
periods in low-Na+ (solid line)
and normal Na+ (dashed line)
solutions. In normal Na+ solution,
[Ca2+]i
dropped below baseline level (dotted line), whereas in
low-Na+ solution,
[Ca2+]i
remained above baseline level.
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In most respects, the fiber used to generate Fig. 5 was representative
of all five fibers exposed to TBQ and caffeine in
low-Na+ solution, i.e.,
[Ca2+]i
increased to ~1,000 nM over 5-10 min and then showed a further rise after 20 min (Fig. 6). However, upon
washout, the only experiment in which
[Ca2+]i
returned to the baseline level after 30 min at the high
[Ca2+]i
was the experiment displayed in Fig. 5. In the other experiments, there
was only a transient recovery of
[Ca2+]i,
followed by a gradual, steady increase in
[Ca2+]i.
After 30-min washout,
[Ca2+]i
increased to very high levels (Fig. 6).

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Fig. 6.
Effect of extracellular Ca2+ on
[Ca2+]i
during exposure to 0.5 µM TBQ and 10 mM caffeine in
low-Na+ solution.
Ca2+ was excluded from
low-Na+ (small dashed line) and
normal Na+ (long dashed line)
solutions, and mean
[Ca2+]i
was measured during exposure to TBQ and caffeine
(n = 3; error bars not shown for
clarity).
[Ca2+]i
declined back to a new plateau level in normal
Na+ solution, whereas in
low-Na+ solution,
[Ca2+]i
remained higher than control level and declined slowly. However, a
sustained elevation in mean
[Ca2+]i
was only observed during exposure to TBQ and caffeine in a
Ca2+-containing
low-Na+ solution (solid line)
(n = 5; error bars not shown for
clarity).
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A separate set of experiments was performed to resolve whether the
Ca2+ responsible for the large
increase and maintained high
[Ca2+]i
is extracellular or intracellular in origin. Three fibers were exposed
for 30 min to a 0 Ca2+ solution
containing TBQ and caffeine. This experiment was performed in both
normal Na+ and
low-Na+ solutions, and the average
results are presented in Fig. 6. Again, in normal
Na+ solution,
[Ca2+]i
increased and declined in the characteristic manner. In
low-Na+ solution,
[Ca2+]i
remained higher than in normal Na+
solution. However, there was no sign of the large, sustained increase
in
[Ca2+]i.
Instead,
[Ca2+]i
slowly declined during the 30-min exposure to caffeine and TBQ.
 |
DISCUSSION |
The results of the present investigation provide evidence that
forward-mode
Na+/Ca2+
exchange is active in mammalian skeletal muscle fibers when the [Ca2+]i
is raised to levels similar to that obtained during tetanic stimulation. Similar conclusions have been drawn from experiments in
which Ca2+ efflux was estimated by
measuring the decrease in radioactivity of muscles loaded with
45Ca2+
(20). These investigators reported that elevating
[Ca2+]i
by about one order of magnitude above the basal level increased [Ca2+]i
efflux by almost 50%.
Reverse-mode
Na+/Ca2+
exchange.
Studies of the regulation of the exchanger suggest that the
reverse-mode
Na+/Ca2+
exchange current might be rather small under physiological conditions. 1) The
[Ca2+]i
in resting muscles is 30-50 nM, and Hilgemann and co-workers (17,
19) showed that the steady-state outward current from the cardiac
Na+/Ca2+
exchanger was sensitive to
[Ca2+]i.
For instance, the half-maximal current in 18 mM
[Na+]i
occurred at a
[Ca2+]i
of 800 nM, so that at the resting
[Ca2+]i
it would be very small. 2) The
[Na+]i
in skeletal muscle cells is ~12.7 mM (21), and measurements of the
outward exchange current in skeletal muscle giant patches show that at
this
[Na+]i
the current is only 3-5% of the value at 90 mM
[Na+]i
(14). Although these considerations suggest that reverse-mode Na+/Ca2+
exchange might be relatively inactive under resting conditions, in
agreement with the present results, they do not explain why the extent
of ionic exchange in skeletal muscle is so much smaller than in cardiac
muscle. The magnitude of the exchange current in giant vesicles from
skeletal muscle (14) appears to be ~10 times smaller than that
recorded under equivalent conditions from cardiac muscle (18), and this
may contribute to the difference we have observed. However, there may
also be a further unidentified difference in the regulation of the
exchanger in skeletal muscle as compared with cardiac muscle.
In the present study, removal of extracellular
Ca2+ can prevent much of the
large, sustained elevation in
[Ca2+]i
observed during exposure to TBQ and caffeine in
low-Na+ solution. Therefore, it
appears that reverse-mode
Na+/Ca2+
exchange can be activated when extracellular
Na+ concentration is lowered and
[Ca2+]i
is increased to levels at least greater than that produced by 10 mM
caffeine application (~80 nM), and probably substantially greater
than this level.
Na+/Ca2+
exchange activity after elevating
[Ca2+]i.
[Ca2+]i
was elevated by inhibiting SR Ca2+
uptake with TBQ and stimulating SR
Ca2+ release with caffeine.
Stimulated force was higher than the force induced by TBQ and caffeine
exposure, even though the stimulated [Ca2+]i
transient was only about one-half as high (Fig. 2). However, [Ca2+]i
took a number of seconds to reach a peak after TBQ and caffeine exposure, whereas the stimulated
[Ca2+]i
transient peaked in milliseconds. Therefore, although force generated
by electrical stimulation was the result of the simultaneous and
coordinated activation of every sarcomere, the reduced force generated
by TBQ and caffeine exposure may have resulted from the development of
sarcomere inhomogeneities.
If we assume that Ca2+ release
from the SR does not inactivate during exposure to caffeine and TBQ,
the slow decline in
[Ca2+]i
must be due either to the removal of
Ca2+ from the cell or to uptake of
Ca2+ by an intracellular
compartment, since intracellular buffers would be rapidly saturated. If
one examines the latter possibility first, the main compartment
candidate would be the mitochondria. Mitochondria can accumulate
massive amounts of Ca2+ when
sufficient inorganic phosphate is available (7). To investigate this
possibility, it was necessary to inhibit the uniport system for
mitochondrial Ca2+ uptake. Most of
the known blockers of the uniport system also block the SR
Ca2+-release channels. However,
the mitochondrial membrane potential can be abolished by cyanide (11).
Without the membrane potential, the uniporter cannot mediate
Ca2+ uptake (22). Therefore, in
the present study, cyanide was used to inhibit mitochondrial
Ca2+ uptake. However, because
cyanide had no effect on either the increase or decline of
[Ca2+]i,
mitochondria probably do not contribute to the decline in [Ca2+]i
after TBQ and caffeine exposure. The evidence suggests that Ca2+ efflux is responsible for the
decline of
[Ca2+]i
after TBQ and caffeine exposure. Washout of TBQ and caffeine with a 0 Ca2+ solution prevented a large
increase in
[Ca2+]i
during the next exposure. This is consistent with the idea that
Ca2+ leaves the cell during the
first exposure, and because there was no
Ca2+ in the extracellular
solution, the fiber could not reaccumulate Ca2+ for the next exposure.
Two observations support the hypothesis that forward-mode
Na+/Ca2+
exchange contributes to the removal of
Ca2+ from the cell during TBQ and
caffeine exposure. 1) Upon washout, [Ca2+]i
undershot the baseline
[Ca2+]i,
indicating that there was less
Ca2+ in the cell. Preventing
Na+/Ca2+
exchange-induced Ca2+ efflux by
reducing extracellular Na+
abolished this undershoot of the baseline
[Ca2+]i
upon washout. 2) Conditions that
inhibit the operation of
Na+/Ca2+
exchange in its forward mode (i.e., removal of extracellular Na+) also inhibited the fall of
[Ca2+]i
after TBQ and caffeine exposure. Even when
Ca2+ entry through reverse-mode
Na+/Ca2+
exchange was prevented by the removal of extracellular
Ca2+ as well as
Na+,
[Ca2+]i
remained higher and declined slower than was the case in normal Na+ solution.
Raising
[Ca2+]i
to high levels for extended periods of time appears to produce
irreversible damage to the muscle, which may manifest itself as a
decrease in the integrity of the plasma membrane, since although
washout produced a partial and temporary recovery of
[Ca2+]i,
[Ca2+]i
soon increased once more to very high levels (Fig. 6).
Role of
Na+/Ca2+
exchange in skeletal muscle.
Membrane vesicle studies have confirmed that the plasma membrane of
rabbit and porcine skeletal muscle contain a
Ca2+ pump as well as a
Na+/Ca2+
exchange (24, 25). Although the rate of
Ca2+ transport by the
Ca2+ pump in membrane vesicles was
reported as ~10
nmol · mg
1 · min
1
and the Michaelis constant
(Km) was
0.3-1.0 µM (16, 24, 25), the transport rate of
Na+/Ca2+
exchange in membrane vesicles has been reported as 50-90
nmol · mg
1 · min
1
and the Km as
~3 µM (10, 15, 25). Therefore, the higher affinity for
Ca2+ of the
Ca2+ pump would make it more
suitable for regulating
[Ca2+]i
under normal resting conditions when the
[Ca2+]i
is low. However, as
[Ca2+]i
is increased, the Ca2+ pump soon
becomes saturated. The low-affinity, high transport capacity of the
Na+/Ca2+
exchange makes it the more appropriate mechanism for
Ca2+ removal at higher
[Ca2+]i.
Although
Na+/Ca2+
exchange may not be directly involved in excitation-contraction
coupling in skeletal muscle (12), it is still unclear whether the
exchanger may operate to remove
Ca2+ from the cell subsequent to a
contraction. Certainly, the rate of
Ca2+ transport of the skeletal
muscle
Na+/Ca2+
exchange has been reported as being up to 30 times slower (10) than the
cardiac muscle exchanger, and the surface area-to-volume ratio is also
smaller in the skeletal muscle cell (5). In the present study, the half
time of the fall in
[Ca2+]i
in Fig. 2, which may represent
Ca2+ extrusion via
Na+/Ca2+
exchange, is ~12 s. However, at the end of a tetanus,
[Ca2+]i
falls with two phases (2), with approximate half times of 25-50 ms
for the initial fast phase and 1 s for the slow phase. The fast phase
covers the same
[Ca2+]i
range as Fig. 2 but involves mechanisms other than SR pumping (e.g.,
parvalbumin uptake). Nevertheless, it is clear that the time course of
[Ca2+]i
lowering by the SR is at least an order of magnitude greater than that
attributable to
Na+/Ca2+
exchange. Therefore,
Na+/Ca2+
exchange may not be important during a single skeletal muscle contraction. However, it may become useful during intense muscular activity when
[Ca2+]i
is elevated for extended periods of time, such as during fatiguing stimulation (27).
 |
ACKNOWLEDGEMENTS |
This work was supported by the National Health and Medical Research
Council of Australia.
 |
FOOTNOTES |
Address for reprint requests: C. D. Balnave, University Laboratory of
Physiology, Univ. of Oxford, Parks Road, Oxford OX1 3PT, UK.
Received 30 September 1997; accepted in final form 15 December
1997.
 |
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