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
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Abstract
Introduction
Methods
Results
Discussion
References

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

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

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
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Abstract
Introduction
Methods
Results
Discussion
References

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 MOmega ) 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
Top
Abstract
Introduction
Methods
Results
Discussion
References

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).

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.

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.

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.

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.

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).

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
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Abstract
Introduction
Methods
Results
Discussion
References

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.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

1.   Allen, D. G., D. A. Eisner, M. J. Lab, and C. H. Orchard. The effects of low sodium solutions on intracellular calcium concentration and tension in ferret ventricular muscle. J. Physiol. (Lond.) 345: 391-407, 1983[Abstract].

2.   Allen, D. G., J. Lännergren, and H. Westerblad. Muscle cell function during prolonged activity: cellular mechanisms of fatigue. Exp. Physiol. 80: 497-527, 1995[Abstract].

3.   Allen, D. G., and H. Westerblad. The effects of caffeine on intracellular calcium, force, and the rate of relaxation of mouse skeletal muscle. J. Physiol. (Lond.) 487: 331-342, 1995[Abstract].

4.   Baro, I., S. C. O'Neill, and D. A. Eisner. Changes of intracellular [Ca2+] during refilling of sarcoplasmic reticulum in rat ventricular and vascular smooth muscle. J. Physiol. (Lond.) 465: 21-41, 1993[Abstract].

5.   Bers, D. M. Excitation-Contraction Coupling and Cardiac Contractile Force. Norwell, MA: Kluwer, 1991.

6.   Caputo, C., and P. Bolaños. Effect of external sodium and calcium on calcium efflux in frog striated muscle. J. Membr. Biol. 41: 1-14, 1978[Medline].

7.   Carafoli, E. The transport of calcium across the inner membrane of mitochondria. In: Membrane Transport of Calcium, edited by E. Carafoli. London: Academic, 1982, p. 109-139.

8.   Castillo, E., H. Gonzalez-Serratos, H. Rasgado-Flores, and M. Rozycka. Na-Ca exchange studies in frog phasic muscle cells. Ann. NY Acad. Sci. 639: 554-557, 1991[Medline].

9.   DiPolo, R., and L. Beauge. Characterization of the reverse Na/Ca exchange in squid axons and its modulation by Cai and ATP. J. Gen. Physiol. 90: 505-525, 1987[Abstract].

10.   Donoso, P., and C. Hidalgo. Sodium-calcium exchange in transverse tubules isolated from frog skeletal muscle. Biochim. Biophys. Acta 978: 8-16, 1989[Medline].

11.   Duchen, M. R., and T. J. Biscoe. Relative mitochondria membrane potential and [Ca2+]i in type I cells isolated from the rabbit carotid body. J. Physiol. (Lond.) 450: 33-61, 1992[Abstract].

12.   García, M. C., A. F. Diaz, R. Godinez, and J. A. Sánchez. Effect of sodium deprivation on contraction and charge movement in frog skeletal muscle fibers. J. Muscle Res. Cell Motil. 13: 354-365, 1992[Medline].

13.   Gilbert, J. R., and G. Meissner. Sodium-calcium ion exchange in skeletal muscle sarcolemmal vesicles. J. Membr. Biol. 69: 77-84, 1982[Medline].

14.   Gonzalez-Serratos, H., D. W. Hilgemann, M. Rozycka, A. Gauthier, and H. Rasgado-Flores. Na-Ca exchange studies in sarcolemmal skeletal muscle. Ann. NY Acad. Sci. 779: 556-560, 1996[Medline].

15.   Hidalgo, C., F. Cifuentes, and P. Donoso. Sodium-calcium exchange in transverse tubule vesicles isolated from amphibian skeletal muscle. Ann. NY Acad. Sci. 639: 483-497, 1991[Medline].

16.   Hidalgo, C., M. E. González, and A. M. Garcia. Calcium transport in transverse tubules isolated from rabbit skeletal muscle. Biochim. Biophys. Acta 854: 279-286, 1986[Medline].

17.   Hilgemann, D. W. Regulation and deregulation of cardiac Na+-Ca2+ exchange in giant excised sarcolemmal membrane patches. Nature 344: 242-245, 1990[Medline].

18.   Hilgemann, D. W. The cardiac Na-Ca exchanger in giant membrane patches. Ann. NY Acad. Sci. 779: 136-158, 1996[Medline].

19.   Hilgemann, D. W., A. Collins, and S. Matsuoka. Steady-state and dynamic properties of cardiac sodium-calcium exchange. J. Gen. Physiol. 100: 933-961, 1992[Abstract].

20.   Hoya, A., and R. A. Venosa. Characteristics of Na+-Ca2+ exchange in frog skeletal muscle. J. Physiol. (Lond.) 486: 615-627, 1995[Abstract].

21.   Juel, C. Potassium and sodium shifts during in vitro isometric muscle contraction, and the time course of the ion-gradient recovery. Pflügers Arch. 406: 458-463, 1986[Medline].

22.   Kapùs, A., K. Szászi, K. Káldi, E. Ligeti, and A. Fonyó. Is the mitochondria Ca2+ uniporter a voltage-modulated transport pathway? FEBS Lett. 282: 61-64, 1991[Medline].

23.   Kimura, J., A. Noma, and H. Irisawa. Na-Ca exchange current in mammalian heart cells. Nature 319: 596-597, 1986[Medline].

24.   Michalak, M., K. Famulski, and E. Carafoli. The Ca2+-pumping ATPase in skeletal muscle sarcolemma. J. Biol. Chem. 259: 15540-15547, 1984[Abstract/Free Full Text].

25.   Mickelson, J. R., T. M. Beaudry, and C. F. Louis. Regulation of skeletal muscle sarcolemmal ATP-dependent calcium transport by calmodulin and cAMP-dependent protein kinase. Arch. Biochem. Biophys. 242: 127-136, 1985[Medline].

26.   Rousseau, E., J. Ladine, Q. Lui, and G. Meissner. Activation of the Ca2+ release channel of skeletal muscle sarcoplasmic reticulum by caffeine and related compounds. Arch. Biochem. Biophys. 267: 75-86, 1988[Medline].

27.   Westerblad, H., and D. G. Allen. Changes of myoplasmic calcium concentration during fatigue in single mouse muscle fibers. J. Gen. Physiol. 98: 615-635, 1991[Abstract].

28.   Westerblad, H., and D. G. Allen. The influence of intracellular pH on contraction, relaxation and [Ca2+]i in intact single fibers from mouse muscle. J. Physiol. (Lond.) 466: 611-628, 1993[Abstract].

29.   Westerblad, H., and D. G. Allen. The role of sarcoplasmic reticulum in relaxation of mouse muscle: effects of 2,5-di(tert-butyl)-1,4-benzohydroquinone. J. Physiol. (Lond.) 474: 291-301, 1994[Abstract].

30.   Wier, W. G. Cytoplasmic [Ca2+]i in mammalian ventricle: dynamic control by cellular processes. Annu. Rev. Physiol. 52: 467-485, 1990[Medline].


AJP Cell Physiol 274(4):C940-C946
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