The effects of inorganic phosphate (Pi) on Ca2+ release from the sarcoplasmic reticulum (SR) were studied in mechanically skinned rat skeletal muscle fibers. Application of caffeine or T-tubule depolarization was used to induce Ca2+ release from the SR, which was detected using fura 2 fluorescence. Addition of Pi (1–40 mM) caused a reversible and concentration-dependent reduction in the caffeine-induced Ca2+ transient. This effect was apparent at low Pi concentration (<5 mM), which did not result in detectable precipitation of calcium phosphate within the SR. The inhibitory effect of Pi exhibited a marked dependence on free Mg2+ concentration. At 0.5 mM free Mg2+, 5 mM Pi reduced the caffeine-induced transient by 25.1 ± 4.1% (n = 13). However, at 1.5 mM free Mg2+, 5 mM Pi reduced the amplitude of caffeine-induced Ca2+ transients by 68.9 ± 3.1% (n = 10). Depolarization-induced SR Ca2+release was similarly affected. These effects of Pi may be important in skeletal muscle fatigue, if an inhibitory action of Pi on SR Ca2+ release is augmented by the rise in cytosolic Mg2+ concentration, which accompanies ATP breakdown.
- sarcoplasmic reticulum
in isolated skeletal muscle fibers, a marked reduction in sarcoplasmic reticulum (SR) Ca2+ release occurs during the latter stages of fatigue induced by repeated tetanic stimulation (39, 42). Although this has been the subject of many previous studies, the mechanism underlying failure of the SR Ca2+ release mechanism remains uncertain. It has been suggested that fatigue-induced changes in the cytosolic environment might directly influence the sensitivity of the SR Ca2+ channel. In particular, cytosolic levels of H+, Ca2+, adenine nucleotides, and Mg2+ are known to change during fatigue, and all of these factors influence the gating properties of the SR Ca2+channel (for review see Ref. 9). However, recent work has failed to establish a clear link between release failure and any of these intracellular factors.
In the latter stages of fatigue, intracellular ATP concentration ([ATP]i) declines to ∼25–50% of resting values. This is associated with an abrupt rise in [Mg2+]i from ∼0.8 mM to ∼1.6 mM due to reduced buffering by ATP (for review see Ref. 1). Because these changes in [ATP] and [Mg2+] are coincident with release failure, a causal relationship has been suggested. However, in experiments involving microinjection of Mg2+ in isolated skeletal muscle fibers, the reduction in tetanic force was insufficient to explain the effects of fatigue (40). The role of ATP remains similarly equivocal: flash photolysis of caged ATP partially restored SR Ca2+ release during late fatigue (2). However, further experiments suggested that the recovery of Ca2+ release reflects an artifact associated with photolysis of the caged compound (3). Furthermore, skinned fiber experiments have provided evidence that the decrease in [ATP] during fatigue is unlikely to be large enough to explain release failure, although a larger localized reduction is possible (2, 30). The role of intracellular acidosis has also been questioned: in isolated mammalian fibers, reducing the intracellular pH to levels reported in fatigue had little effect on SR Ca2+release at physiological temperatures (8, 43).
Another possibility is that changes in intracellular inorganic phosphate (Pi) levels may influence SR Ca2+release. During fatigue, the intracellular [Pi] increases progressively to 30–40 mM, as creatine phosphate (CrP) is depleted (10, 29). Experiments using skinned fibers have shown that Pi entry into the SR lumen and subsequent precipitation of calcium phosphate can influence the amount of releasable Ca2+ (19). However, although this is an attractive hypothesis, the reported effects of Pi on skinned fibers are inconsistent: depending on the experimental conditions, exposure to Pi may increase, decrease, or have little influence on releasable Ca2+ (19, 21, 32,36).
These discrepancies may reflect aspects of the experimental technique. In previous studies on skinned fibers, the SR was Ca2+loaded in the presence of Pi. However, Pi was then washed out completely, before caffeine was applied to assess the amount of Ca2+ available for release. This was necessary because Pi inhibits myofilament force production, which was used to assess the amount of released Ca2+(22). One of the potential difficulties with this approach is that calcium phosphate precipitates may redissolve following washout of Pi, leading to an increase in releasable Ca2+ (19). Perhaps more importantly, this approach will not reveal other effects, present only in the presence of Pi. For example, recent work on skinned fibers has shown that Pi can induce Ca2+ release from the SR by reversal of the Ca2+ pump (15). A direct action of Pi on the SR Ca2+ channel has also been proposed (18). Understanding the role of Pi in fatigue requires these complex and interdependent effects on SR Ca2+ regulation to be studied in the presence of Pi.
The present study addresses the effects of Pi on caffeine and depolarized-induced Ca2+ release in mechanically skinned rat skeletal muscle fibers. In contrast to previous studies, SR Ca2+ release was directly detected using fura 2, and Ca2+ regulation was assessed before, during, and after exposure to Pi. The results suggest that rising levels of cytosolic Pi reduce the amount of Ca2+ released from SR in response to caffeine or to depolarization of the T-tubule system. This effect of Pi exhibited a marked dependence on free [Mg2+] over the range that occurs within the cytosol during fatigue. The combined effects of Mg2+ and Pi may be of importance in the latter stages of fatigue if the increase in [Mg2+]iaugments an inhibitory effect of Pi on SR Ca2+release. The mechanisms underlying the inhibitory action of Pi are discussed in relation to its reported effects on the Ca2+ pump and the Ca2+ channel. The possible role of calcium phosphate precipitation within the SR is also considered.
Wistar rats (250–300 g) were killed by a blow to the head and cervical dislocation according to standard Schedule 1 procedures. The extensor digitorum longus (EDL) muscle was removed rapidly and placed in “relaxing” solution approximating the intracellular milieu (as described in Solution composition). Single muscle fibers or pairs of adjacent fibers were mechanically skinned with fine forceps and then attached between an isometric tension transducer (SensoNor, Norway) and a fixed point using monofilament snares (diameter 30 μm; Ethicon) within stainless steel tubes (Goodfellow Metals, UK). In most experiments, two muscle fibers were attached in parallel to increase the amount of light collected by the objective lens and to improve the signal-to-noise ratio of the fluorescence signal. However, qualitatively similar results were obtained for single fiber preparations. For experiments in which caffeine was used to activate Ca2+ release, skinning was done in the presence of a low [Ca2+] (40 nM), which inactivates the depolarization-induced Ca2+ release mechanism (26).
For experiments on depolarization-induced Ca2+ release, the dissection procedure was modified: the EDL muscle was removed and bathed in a Ringer solution (in mM: 150 sodium propionate, 5 potassium propionate, 2 calcium chloride, 1 magnesium chloride, and 5 HEPES, pH 7). The muscle was blotted with filter paper and immersed in mineral oil. Single fibers were carefully isolated and skinned under oil with fine forceps and mounted for isometric tension recording. The preparation was then transferred to the experimental solution and perfused for several minutes in the potassium salt of 1,6-diaminohexane-N,N,N′,N′-tetraacetic acid (K+-HDTA) solution before inducing the first response. All experiments were done at room temperature (22–24°C).
The apparatus for simultaneous measurement of isometric force and SR Ca2+ release is described in detail elsewhere (13). Briefly, the preparation was mounted close to the bottom of a shallow bath with a coverslip base. A Perspex column (5 mm diameter) was lowered to within a few micrometers of the muscle to minimize the volume of the solution above the preparation. Throughout the experimental protocols, preparations were perfused by pumping solution at 0.8 ml/min via a narrow duct (200 μm diameter) that passed through the center of the column. Waste solution was collected continuously at the column edge. This created a volume of solution between the coverslip and the base of the column of ∼6 μl. The perfusing solution was changed using a series of valves positioned above the column. With the use of this method, the solution within the bath was exchanged within 10–15 s. The comparatively slow solution exchange reflects mixing of solutions in the tubing between the valves and the column. Solutions for inducing Ca2+ release (i.e., with caffeine or Na+) were rapidly applied (20 ml/min) for a 1.5-s duration via a narrow plastic tube connected to the base of the column. The higher flow rate and the smaller dead space allowed a more rapid exchange of solutions within the bath. Previous measurements based on the quench of indo 1 fluorescence by caffeine under similar conditions have shown that the caffeine concentration within the bath typically increased to 50% of the concentration injected within 8–10 ms.
The bath was placed on the stage of a S200 Nikon Diaphot inverted microscope. The muscle was viewed via a ×40 Fluor objective (Fluor, oil immersion, Nikon), and the length was increased to ∼20% above slack length. In control experiments, it was found that length did not have a direct effect on SR Ca2+ regulation. The preparation was alternately illuminated with light of wavelengths 340 and 380 nm at a 50-Hz frequency using a spinning wheel spectrophotometer (Cairn Research, Faversham, Kent, UK). The average [Ca2+] within the visual field containing the preparation was indicated from the ratio of light intensities emitted at >500 nm. Light emitted from areas of the visual field not occupied by the muscle image was reduced using a variable rectangular diaphragm on the side port of the microscope.
All chemicals were purchased from Sigma unless otherwise stated. The basic solution used to mimic the intracellular milieu in experiments involving caffeine application contained (in mM) 5 ATP, 10 CrP (Fluka), 0.2 EGTA, 25 HEPES, 130 K+, 32 Na+, 0.5–1.5 free Mg2+, and 0.0015 fura 2 (Calbiochem). NaN3 (2 mM) was added routinely to inhibit any possible mitochondrial activity. The free [Ca2+] of each solution was adjusted to 100 nM by addition of CaCl2 (Molar stock, BDH). CrP and ATP were added as disodium salts and Pi and oxalate as dipotassium salts. The pH was adjusted to 7.0 by addition of KOH. The [K+] was adjusted to 130 mM by adding KCl. The [Cl−] ranged from 60 to 130 mM. However, in control experiments, changes in [Cl−] over this range had no apparent effect on the results (not shown). Caffeine was added directly to the experimental solution as required. In further control experiments, the effects of Pi were studied when potassium propionate or K+-HDTA was used in place of KCl. However, the effects of Pi were not influenced by changing the principal anion.
In all experiments involving depolarization-induced Ca2+release, the solutions were modified to avoid inclusion of Cl−. The preparations were perfused with a K+-HDTA solution containing (in mM) 126 K+, 37 Na+, 50 HDTA2− (Aldrich), 5 ATP, 10 CrP (Fluka), 0.1 EGTA, 90 HEPES, 2 azide, and 0.0015 fura 2 (Calbiochem), pH 7.0. [Mg2+] was altered to give a free concentration of 0.5 or 1 mM. The resealed T-system of the mechanically skinned fiber was depolarized by exposure to a solution in which K+-HDTA was replaced by Na+-HDTA. In experiments where [Ca2+] was measured in a restricted volume, 0.1 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid was used instead of EGTA.
Measurement of free [Mg2+] with furaptra (4 μM) showed that it was necessary to add a total of 4.7, 5.75, and 6.4 mM to obtain a free Mg2+ of 0.5, 1, or 1.5 mM, respectively. Computer modeling using React II (16) confirmed that most of the Mg2+ is bound to ATP, with a small amount (∼0.2 mM) bound to CrP. In Pi-containing solutions, [Mg2+] was increased to compensate for Mg2+ binding by Pi. For example, an extra 0.5 mM Mg2+ was required to maintain a constant [Mg2+] in the presence of 20 mM Pi. For other details and associated references relating to preparation of solutions see Ref. 13. In some experiments the free [Mg2+] was changed from 0.5 or 1 to 1.5 mM. In control experiments, the change in the fura 2 fluorescence ratio, in response to changes in [Ca2+] over the range 100–800 nM was not significantly different when the free [Mg2+] was 0.5 or 1.5 mM (not shown). This is consistent with previous data showing that the effect of Mg2+ on the apparent affinity of fura 2 for Ca2+ is small over this range (37). As in previous studies, high [Pi] (>10 mM) caused a slight decrease in both the 340 and 380 nm emission wavelengths. However, there was no significant effect of Pi on the fluorescence ratio.
Data recording and analysis.
In all experiments, the ratio and individual wavelength intensities and the isometric tension signals were low-pass filtered (−3 dB at 30 Hz) and digitized for later analysis using an IBM-compatible 80486 computer with a data translation 2801A card. All values are represented as means ± SE. Paired t-tests were used to assess significance. Values were considered significant if P< 0.05.
Effect of Pi on caffeine-induced SR Ca2+ release.
Figure 1 A shows a continuous record of the 340:380 nm fluorescence ratio from a mechanically skinned preparation comprising two fibers mounted in parallel. The preparation was perfused continuously with weakly Ca2+-buffered solutions in the presence of 1.5 μM fura 2 and a free [Mg2+] of 1 mM. As in all subsequent experiments, the resting level of fluorescence indicates a bathing [Ca2+] of 100 nM. Caffeine was applied briefly for 1.5 s at 2-min intervals, producing reproducible cycles of SR Ca2+ release and reuptake. Each caffeine application was associated with a transient increase in the fluorescence ratio due to Ca2+ release from the SR (this is referred to as a Ca2+ transient). Increasing the loading period beyond 2 min had no significant effect on the caffeine responses (not shown). This confirms that, at 100 nM [Ca2+], the SR Ca2+content reaches a steady state within 2 min. Under these conditions, caffeine application was sufficient to produce a maximal response, i.e., a more prolonged exposure to caffeine did not increase the amplitude of the Ca2+ or force transients.
After four control responses, 2 mM Pi was introduced to the perfusing solution, resulting in a small transient efflux of Ca2+ from the SR. Stepwise increases in [Pi] resulted in corresponding decreases in the amplitude of the caffeine-induced Ca2+ transient. At 10 mM Pi, the resting fluorescence ratio also decreased slightly, and this effect became more apparent at 20 and 40 mM Pi. On removal of Pi, the resting fluorescence ratio and the Ca2+transient amplitude returned to control levels. These effects of Pi were completely reversible, and several exposures were possible without any significant change in amplitude or time course of the Ca2+ transient following Pi removal.
The decrease in the resting fluorescence ratio at high [Pi] is consistent with calcium phosphate precipitation within the SR, which would be expected to stimulate and maintain SR Ca2+ uptake (35). The maintained decline in the fluorescence ratio suggests that continuous uptake via the Ca2+ pump can lower [Ca2+] within the muscle slightly below that of the surrounding medium, despite the inward diffusion of fresh solution. Previous studies on skinned fibers have shown that the transient increase in [Ca2+] associated with addition of Pi results from reversal of the SR Ca2+ pump (15).
Cumulative data illustrating the relationship between [Pi] and the amplitude of the Ca2+ and force transients are shown in Fig. 1 B. The abscissa indicates [Pi], and the ordinate indicates the mean steady-state amplitude (average of 2) of the caffeine-induced responses, expressed as a percentage of the mean control response. The amplitude of the Ca2+ and tension responses declined markedly as [Pi] was increased from 0 to 20 mM. However, a further increase in [Pi] to 40 mM had only a small additional inhibitory effect. It is also apparent that, when [Pi] was increased from 10 to 20 mM, the amplitude of the Ca2+transient declined more rapidly. This may have resulted from calcium phosphate precipitation within the SR and a consequent decrease in releasable Ca2+. Depression of the force responses by Pi reflects both reduced SR Ca2+ release and a direct depressive effect of Pi on the contractile proteins (22).
Dependence on free [Mg2+].
Previous studies suggest that [Mg2+] is 0.5–1 mM in resting fibers but increases to ∼1.6 mM during fatigue (e.g., Ref.40). Therefore, experiments were carried out to assess whether the influence of Pi on SR Ca2+ release is dependent on the free [Mg2+]. Figure2 A shows the effect of 5 mM Pi on caffeine-induced Ca2+ release at 0.5 mM (left) and 1.5 mM free Mg2+ (right), in the same muscle preparation. At 0.5 mM free Mg2+, addition of 5 mM Pi again caused a small transient increase in the fluorescence ratio. On average, the amplitudes of the Ca2+ transients were reduced by 25.1 ± 4.1% (n = 13), in the presence of 5 mM Pi. After removal of Pi, the Ca2+ and force responses returned to near control levels within two to three load and release cycles. In the presence of 1.5 mM free Mg2+(right), addition of 5 mM Pi again caused a small transient increase in the fluorescence ratio. This Pi-induced release of Ca2+ was not significantly different to that obtained at 0.5 mM free Mg2+. However, despite this, subsequent caffeine-induced Ca2+ transients were reduced by a much greater extent (68.9 ± 3.1%, n = 12). In the absence of Pi, increasing the free Mg2+ from 0.5 to 1.5 mM (compare Fig. 2 A, left and right) reduced the steady-state amplitude of the Ca2+ transient by 32.2 ± 3.2% (n = 7). The effect illustrated in Fig. 2 A was unaffected by extending the loading period beyond 2 min (not shown).
Figure 2 B shows the effect of [Mg2+] on responses obtained in the presence of 2, 5, and 10 mM Pi, using the protocol shown in Fig. 2 A. In each panel, the first transient is the control response to caffeine and the second is the steady-state response following equilibration with Pi. The third response is that obtained following washout of Pi. All responses were obtained from the same preparation. In the continuous presence of 0.5 mM Mg2+(left), increasing [Pi] resulted in a concentration-dependent decrease in the caffeine-induced Ca2+ transient. However, in the presence of 1.5 mM Mg2+ (right), this effect was much more pronounced at all [Pi].
Cumulative data showing the relationship between [Pi] and SR Ca2+ release at 0.5, 1, and 1.5 mM free Mg2+are shown in Fig. 3. The abscissa indicates [Pi] and the ordinate the mean steady-state amplitude (average of 2 in each preparation) of the caffeine-induced Ca2+ transients, expressed as a percentage of the control response. The control response is the mean of two to three Ca2+ transients preceding exposure to Pi. On average, the control responses differed by <10%. The graph shows that the relative decrease in the amplitude of the Ca2+transient was larger at 1.5 mM free [Mg2+]. However, for most points, there was no significant difference between the responses at 0.5 and 1 mM free Mg2+.
Threshold for precipitation of calcium phosphate within the SR.
Previous studies in skinned and intact fibers have attributed the inhibitory effects of Pi to calcium phosphate precipitation within the SR lumen, which may reduce the pool of Ca2+available for release (19). The protocol shown in Fig.4 was used to assess directly whether precipitation occurred at a [Pi] that depresses caffeine-induced Ca2+ release, using a method previously described for use in skinned cardiac preparations (35). Briefly, under control conditions, the releasable pool of SR Ca2+ reaches a steady state within 2 min (not shown). Thereafter, any Ca2+ leak from the SR is balanced by uptake via the SR Ca2+-ATPase, and net Ca2+ uptake is zero. Hence, when the perfusion is stopped, [Ca2+] within the bath remains constant for several minutes. However, the situation is quite different under conditions where precipitation of calcium phosphate occurs within the SR. Ca2+ uptake is maintained, because the constant entry of Pi into the SR, followed by precipitation with Ca2+, reduces the free luminal [Ca2+] to a level dictated by the solubility product. Because the preparation occupies a significant fraction of the bath, precipitation results in a sustained decrease in [Ca2+] within the solution when perfusion is stopped.
Figure 4 shows the protocol used to investigate the threshold for calcium phosphate precipitation within the SR. Under control conditions, when the flow was stopped in the absence of Pi, the baseline [Ca2+] remained constant. The preparation was then equilibrated with a range of [Pi] during continuous perfusion. When the flow was stopped in the presence of 1 or 2 mM Pi, there was no apparent change in [Ca2+] within the bath. However, when the bathing [Pi] was increased to 5 mM, a downward drift in the [Ca2+] was apparent when the flow was stopped. This effect was more pronounced at 10 or 20 mM Pi. These results suggest that the threshold for precipitation of calcium phosphate within the SR was ∼5 mM under these conditions. Even after prolonged exposures, a downward drift in the [Ca2+] was not observed at lower levels of Pi under these conditions. Indeed, in some preparations, precipitation was not apparent until the [Pi] reached 10 mM or above (not shown).
In control experiments designed to characterize this response (11), it has been shown that that the decrease in [Ca2+] that occurs on stopping the flow is abolished completely by inhibition of the SR Ca2+ pump by cyclopiazonic acid or by disruption of the SR membrane with Triton X-100 but is unaffected by SR Ca2+ channel blockers (e.g., ruthenium red).
Comparative effects of Pi and oxalate on Ca2+ uptake and release.
As considered above, Pi may have several distinct effects on SR Ca2+ regulation. Therefore, to assess the influence of calcium phosphate precipitation on releasable Ca2+, the effects of Pi were compared with oxalate. Oxalate is also known to precipitate (with Ca2+) within the SR and to stimulate Ca2+ uptake but would not be expected to induce pump reversal or to directly affect the SR Ca2+ channel.
Figure 5 A shows representative steady-state Ca2+ transients obtained from the same preparation in the constant presence of 1.5 mM free Mg2+, under control conditions (left) or in the presence of 2 mM Pi, 2 mM oxalate, 20 mM Pi, or 10 mM oxalate. As shown earlier, the caffeine response in the presence of 2 mM Pi was markedly reduced in amplitude. In this example, 2 mM oxalate reduced the amplitude of the Ca2+ transient to a similar extent. When [Pi] was increased to 20 mM, a further decrease in the Ca2+ transient occurred, while exposure to 10 mM oxalate effectively abolished the response.
Although oxalate and Pi both influence the caffeine-induced Ca2+ transient, differences in the underlying mechanisms were apparent when the flow was stopped under each condition (Fig.5 B). As in Fig. 4, there was no evidence of precipitation when the flow was stopped in the presence of 2 mM Pi. However, when the flow was stopped in the presence of 2 mM oxalate, a marked and prolonged reduction in [Ca2+] occurred within the bath. This difference probably reflects the lower solubility product of calcium oxalate. When [Pi] was increased to 20 mM, the decrease in fluorescence ratio was similar to that in the presence of 2 mM oxalate. Finally, following equilibration with 10 mM oxalate, an even more rapid and pronounced decrease in the fluorescence ratio occurred. The reduction in [Ca2+] in the presence of oxalate or high [Pi] was abolished by prior exposure to the Ca2+ pump inhibitor cyclopiazonic acid (not shown).
These results suggest that 1) the inhibitory action of oxalate on SR Ca2+ release can be explained by a reduction in releasable Ca2+ due to precipitation of calcium oxalate within the SR, 2) the inhibitory effects of low [Pi] (<5 mM) occurs via a mechanism that is unlikely to involve precipitation, and 3) when [Pi] is increased to high levels, calcium phosphate precipitation does occur, and this may contribute to a reduction in the caffeine-induced Ca2+ transient (Fig. 3).
Simultaneous withdrawal of Mg2+ and application of caffeine.
One interesting feature of the present data is that Pireduced the amplitude of the caffeine-induced Ca2+transient at low levels, below that reported to produce calcium phosphate precipitation within the SR at 100 nM bathing Ca2+ (Fig. 5). It is possible that the Pi-induced reduction in Ca2+ release at low [Pi] might be explained by activation of a Ca2+-efflux pathway and a consequent decline in luminal [Ca2+]. Indeed, as shown in this (e.g., Fig.2 A) and previous studies (15), introduction of Pi was associated with a transient release of Ca2+ due to pump reversal. However, this explanation seems unlikely, because the Pi-induced release is small, or even absent completely in the presence of CrP (15). Furthermore, in experiments involving measurement of net SR Ca2+ uptake following complete Ca2+ depletion, it was found that Pi had little effect on net Ca2+ uptake (and hence total content) when CrP was present in the bathing solution (11).
Another possibility is that Pi directly influences the sensitivity of the SR Ca2+ release mechanism to caffeine, either via a direct action on the channel or an indirect action involving a reduction in luminal [Ca2+]. Interpretation of the data shown in Figs. 1 and 2 is complicated by the fact that even high caffeine concentration does not fully activate the SR Ca2+ channel in EDL fibers at 1 mM free Mg2+(20). As a consequence, the caffeine-induced Ca2+ transient can be decreased by factors that inhibit the release mechanism (12). However, previous work has also shown that maximal Ca2+ release can be achieved by simultaneously applying high levels of caffeine and reducing [Mg2+] to micromolar levels (24, 25). Under these conditions, the amplitude of the caffeine-induced Ca2+ transient more accurately reflects the SR Ca2+ content, and any inhibitory influence on the SR Ca2+ release mechanism is minimized.
In Fig. 6 A, the preparation was continuously perfused with a solution containing 1.5 mM free Mg2+. Introduction of 2 mM Pi produced a large decrease in the amplitude of the caffeine-induced Ca2+transients to ∼50% of controls. However, when Mg2+ was absent from the caffeine solution (Fig. 6 B), addition of 2 mM Pi produced only a small (∼10%) decrease in the amplitude of the caffeine-induced Ca2+ transient. This suggests that 1) the small Ca2+ release on addition of 2 mM Pi has little steady-state influence on the SR Ca2+ content and 2) the effects of low [Pi] may involve a direct or indirect inhibitory action on the SR Ca2+ release mechanism.
Measurement of [Mg2+] using furaptra showed that the free [Mg2+] decreased to ∼50 μM within the preparation during an injection with a 0 mM Mg2+ (n = 4), 40 mM caffeine solution (not shown). Brief exposure to this level of Mg2+ (without caffeine application) did not cause loss of Ca2+ from the SR or development of rigor tension. Similarly, the descending phase of the Ca2+ and force transients was not prolonged when Mg2+ was absent from the caffeine solution. This suggests that SR Ca2+ reuptake is not compromised by this protocol (13).
Simultaneous application of caffeine and Pi.
If Pi directly inhibits the SR Ca2+ release mechanism via a cytosolic action, this effect might be expected to develop rapidly. The protocol shown in Fig.7 was used to investigate the rate at which Pi influences caffeine-induced Ca2+release. Representative steady-state caffeine responses were obtained while perfusing the preparation with a solution containing 1.5 mM free Mg2+. Brief applications of 10 mM caffeine (filled triangles) induced Ca2+ release from the SR, and a representative steady-state response is shown. Inclusion of 10 mM Pi (open triangles) in the caffeine-containing solution (but not the perfusing solution) caused a reduction in amplitude of the caffeine response (28.6 ± 5.2%, n = 5). Removal of Pi from the caffeine solution reversed this effect. With the use of this protocol, the [Pi] will not reach 10 mM within the fiber by the peak of the caffeine-induced Ca2+transient. Hence, the degree of inhibition is not as great as the steady-state effect seen in the constant presence of Pi.
Effects of Pi on depolarization-induced Ca2+ release.
The preceding experiments used caffeine to induce Ca2+release by directly activating the SR Ca2+ channel [ryanodine receptor (RyR)]. An alternative approach is to activate the RyR via the physiological mechanism. When fibers are skinned under oil, the T-tubules reseal and repolarize. In this state, depolarization of the T-system by substitution of K+-HDTA with Na+-HDTA induces Ca2+ release via the physiological pathway (26).
Figure 8 A shows fluorescence records from a mechanically skinned single fiber. The fiber was continuously perfused with K+-HDTA solution in the presence of 1.5 μM fura 2 and at a free [Mg2+] of 1.5 mM. The resting fluorescence ratio corresponds to a bathing cytosolic [Ca2+] of 100 nM. The T-system was depolarized by a brief (1.5-s) application of Na+-HDTA solution (vertical lines). This resulted in an increase in the fluorescence ratio, due to Ca2+ release from the SR, and an associated force response. The brief Na+ application was sufficient to produce a maximal response, i.e., a more prolonged exposure did not further increase the amplitude of the Ca2+ transient. The descending phase of the depolarization-induced Ca2+ and force transients was significantly briefer than responses induced by caffeine application. After five control responses (3 shown), 2 mM Pi was introduced in the perfusing solution. Subsequent Na+-HDTA-induced responses were decreased in amplitude. On withdrawal of Pi, depolarization-induced Ca2+release returned to control levels. The effects of Pi on depolarization-induced Ca2+ release were usually reversible. However, in some fibers, force and Ca2+responses did not return completely to control levels after Pi withdrawal. This was probably a result of “rundown” as reported previously (26).
Figure 8 B shows cumulative data illustrating the effect of 2 and 5 mM Pi on the amplitude of depolarization-induced SR Ca2+ release at 0.5 and 1.5 mM free Mg2+. The ordinate indicates the mean amplitude expressed as a percentage of the control. The control value was the average of two transients before and two transients after Pi exposure. The experimental values were the average of two steady-state responses in the presence of Pi. The graph shows a marked difference in the effect of Pi on the amplitude of depolarization-induced Ca2+ release at 0.5 or 1.5 mM free Mg2+.
This is the first study on skinned fibers to investigate Ca2+ regulation by the SR in the continued presence of Pi. The main finding is that Pi induces a concentration-dependent decrease in the amount Ca2+released from the SR in response to caffeine or depolarization of the T-tubules. This effect exhibited a marked dependence on the cytosolic free [Mg2+]. Previous studies have shown that Pi has a number of actions on the SR, including1) activation of a Ca2+ efflux pathway involving reversal of the SR Ca2+ pump, 2) precipitation of calcium phosphate within the SR, and 3) modulation of the SR Ca2+ release channel activation (15, 18,19). Direct measurement of [Ca2+] allowed these effects of Pi to be detected and the relative influence of each assessed.
The role of calcium phosphate precipitation with the SR.
It has been suggested that precipitation of calcium phosphate within the SR lumen may be the primary mechanism underlying fatigue-induced release failure in intact skeletal muscle (19). Precipitation occurs with a distinct threshold when the calcium phosphate solubility product is exceeded within the SR. In the present study, it was possible to establish the threshold for calcium phosphate precipitation from changes in the resting fluorescence ratio. Under conditions favoring precipitation, the continuous entry of Pi into the SR, followed by calcium phosphate precipitation, stimulates and maintains Ca2+ uptake. As in cardiac muscle (35), this results in a characteristic downward drift in the baseline fluorescence.
During constant perfusion, a downward drift in the baseline fluorescence ratio was only apparent at or above 10 mM Pi(Fig. 1). However, during perfusion, the constant inward diffusion of Ca2+ from the surrounding solution tends to obscure the effects of sustained Ca2+ uptake by the SR. Consequently, the threshold for calcium phosphate precipitation cannot be determined accurately during perfusion. In contrast, when the flow is stopped and the bath volume restricted, even a small maintained net uptake of Ca2+ by the SR reduces [Ca2+] over several minutes. The “stop-flow” experiments shown in Fig. 4 demonstrate that the threshold for calcium phosphate precipitation occurs when the bathing [Pi] is ∼5 mM; at lower [Pi] a downward drift in the baseline did not occur under these conditions, even after prolonged exposure. This is consistent with previous work showing that the threshold for precipitation is ∼5 mM Pi, when skinned EDL fibers are loaded at 100 nM Ca2+(19).
In the presence of higher [Pi], a more pronounced decline in the fluorescence ratio occurred when the flow was stopped (Fig. 4). A decrease in the fluorescence ratio occurred when the flow was stopped in the presence of oxalate (Fig. 5). Under these conditions, precipitation of calcium oxalate also reduced the amplitude of the caffeine-induced Ca2+ transient. Together, these results suggest that 1) at [Pi] ≥ 5 mM, calcium phosphate precipitation occurs within the SR, and 2) calcium phosphate precipitation probably contributes to the decrease in releasable Ca2+ at [Pi] ≥ 5 mM.
These experiments also reveal important differences between the actions of low oxalate concentration and [Pi]. In the example shown in Fig. 5, the amplitude of the caffeine-induced Ca2+transient decreased to a similar extent when either 2 mM oxalate or 2 mM Pi was present in the bathing medium. In the presence of 2 mM oxalate, a downward drift in the cytosolic [Ca2+] occurred when the flow was stopped. Hence, 2 mM oxalate may reduce the amount of Ca2+ released from the SR as a consequence of calcium oxalate precipitation. However, the [Ca2+] remained constant when the flow was stopped in the presence of <5 mM Pi. This suggests that the decrease in the caffeine-induced Ca2+ transient at <5 mM Pi cannot be explained by precipitation of calcium phosphate within the SR.
Role of Pi-induced reversal of the SR Ca2+ pump.
Introduction of Pi was associated with a small transient increase in the fluorescence ratio (e.g., Fig. 1). Previous work on skinned fibers has shown that Pi-induced Ca2+efflux is abolished by treatment with the Ca2+ pump inhibitor cyclopiazonic acid, mimicked by addition of ADP and insensitive to ruthenium red (15). This is consistent with early studies on isolated SR, demonstrating that Pi can induce Ca2+ release by reversal of the SR Ca2+pump (23).
Although Pi can cause a small transient efflux of Ca2+ from the SR, several lines of evidence suggest that this does not result in a large decrease in the steady-state SR Ca2+ content, under the conditions of the present study. First, sustained pump reversal requires a source of ADP (23). However, this study was carried out in the presence CrP, which markedly reduces the local accumulation of ADP via the creatine kinase reaction. This explains why the Pi-induced Ca2+ efflux is much smaller (or in some cases absent completely) in the presence of CrP (15). Second, when Ca2+ release was maximized by simultaneous withdrawal of Mg2+ and addition of caffeine, the amount of Ca2+ available for release from the SR was barely affected by 2 mM [Pi] (Fig. 6). This suggests that depletion of SR Ca2+ cannot explain the marked decrease in the caffeine-induced Ca2+ transient, when 2 mM Piwas added in the constant presence of 1.5 mM [Mg2+] (e.g., Fig. 2). Third, further experiments have shown that Pi has little effect on net Ca2+ reuptake following complete depletion of SR Ca2+, when CrP is present in the solutions (see Ref. 11 for discussion). Together, these results suggest that pump reversal and consequent depletion of SR Ca2+ cannot explain the marked decrease in the amplitude of the SR Ca2+ transient at low levels of Pi and in the presence of CrP.
Possible effects of Pi on the SR Ca2+ release mechanism.
As considered above, the present data suggest that reversal of the Ca2+ pump has little effect on the SR Ca2+content at low [Pi], whereas the decrease in the Ca2+ transient can be substantial (>50% at 2 mM). Precipitation of calcium phosphate may reduce the amount of Ca2+ available for release, but this effect is only apparent at levels of Pi ≥ 5 mM (Fig. 4). This introduces the possibility that Pi may have an additional inhibitory influence on SR Ca2+ release, which is independent from calcium phosphate precipitation or pump reversal.
As shown in Fig. 7, caffeine-induced Ca2+ release was reduced when Pi was included in the briefly applied caffeine-containing solution. The rapid onset of this effect suggests a cytosolic site of action rather than an effect involving Pientry into the SR, which occurs slowly over minutes (21). This effect cannot be explained by pump reversal, which would increase rather than decrease the amount of Ca2+ released on simultaneous application of caffeine and Pi. This result also suggests that prior depletion of SR Ca2+ is not required for release inhibition by Pi. On balance, the characteristics of this effect appear most consistent with cytosolic action of Pi, involving inhibition of Ca2+release.
The inhibitory action of Pi exhibited a marked dependence on [Mg2+] (Fig. 2) and was much more pronounced at 1.5 mM than at 1 mM free Mg2+. Experiments similar to those shown in Fig. 4 confirmed that the threshold for precipitation did not alter significantly following an increase in [Mg2+] from 1 to 1.5 mM (not shown). Hence, changes in the characteristics of calcium phosphate precipitation cannot explain the Mg2+ dependence of Pi inhibition. Furthermore, at 2 mM [Pi], the amplitude of the Ca2+ transient was restored close to control levels by decreasing [Mg2+] in the caffeine solution to micromolar levels (Fig. 6). This may be explained if reducing [Mg2+] disinhibits the SR Ca2+release mechanism, thereby reducing an inhibitory influence of Pi and allowing stored Ca2+ to be accessed. Interestingly, recent work on fast-twitch rat skeletal muscle has shown that reducing [ATP] from 8 to 0.5 mM has no effect on SR Ca2+ release at 1 mM Mg2+. However, in the presence of 3 mM Mg2+, the same decrease in [ATP] significantly reduced Ca2+ release induced by caffeine or depolarization (5). Hence, the SR Ca2+ channel may be more susceptible to inhibition by a variety of factors when [Mg2+] is increased above physiological levels.
Previously reported effects of Pi on the SR Ca2+ channel.
Previous studies have suggested that binding of Pi to a specific site on the SR Ca2+ channel can influence the gating properties. However, in isolated channels from skeletal muscle SR, Pi has only been shown to increase channel activity (18). Recent experiments on skinned fibers also suggested that Pi may stimulate Ca2+-induced Ca2+ release (CICR; Ref. 4). One possible explanation for this apparent discrepancy is the mechanism of channel activation. Increased gating of isolated channels was studied by introduction of Pi during activation with micromolar levels of Ca2+ (4, 18). In skinned fibers, CICR was induced by prolonged exposure to solutions containing ∼30 μM Ca2+ (4). Previous studies have not examined caffeine-induced Ca2+ release or the physiological depolarization-induced release mechanism in the presence of Pi.
Another consideration is that experiments on isolated channels and membrane fractions are typically carried out under conditions that differ substantially from skinned fiber studies or the in vivo state. Recent work suggests that the activity of the SR Ca2+channel is influenced by a number of associated proteins including FK506 binding protein, triadin, and calsequestrin (6, 7,27). Purification of SR Ca2+ channels or membrane fractions results in loss of these proteins to varying degrees, and this has been linked to alterations in channel gating characteristics and the response to physiological or pharmacological activators (7, 17, 27, 38). Hence, effects obtained on isolated channels or membrane fractions may not be the same in more intact preparations.
It is also not clear from the present data whether Piinhibits Ca2+ release by interacting directly with the Pi-binding site on the RyR, or by a nonspecific action at one or more of the other sites that modulate the sensitivity of the Ca2+ release mechanism to caffeine or depolarization. Further work is required to characterize the effects of Pion the SR Ca2+ channel in the in vivo state. One possible approach to this might be to study the effects of Pi on localized Ca2+ sparks in skinned or intact cells. Although apparently absent in mammalian skeletal muscle, Ca2+ sparks are readily detectable in amphibian muscle (34).
The possible role of Pi-induced release inhibition in fatigue.
The main finding in the present study is that Pi induces a concentration-dependent decrease in the amount of Ca2+released from the SR. Whatever the underlying mechanism, Pi-induced inhibition of SR Ca2+ release and the dependence on free Mg2+ may have implications for events during fatigue. When the free [Mg2+] is at normal levels found within the cytosol (∼0.5–1 mM), the inhibitory influence of Pi may be relatively small. However, in end-stage fatigue, when [ATP] begins to decline, the sudden rise in [Mg2+] to ∼1.6 mM would be expected to make the inhibitory action of Pi more pronounced.
One potential difficulty with this interpretation is that the inhibition of Ca2+ release appears larger than expected from the changes in tetanic [Ca2+] reported in intact cells. During fatiguing stimulation, the influence of Pi on the SR Ca2+ channel might be expected to follow a profile similar to that shown in Fig. 3, for 0.5–1 mM Mg2+. In end-stage fatigue, release inhibition by Pi would suddenly become more effective when [Mg2+] increased to ∼1.5 mM (bottom curve). This suggests that SR Ca2+ release should decrease by ∼80% of the control value in the presence of 20 mM Pi. However, in intact cells, the tetanic [Ca2+] declined by only 20–30%, before the final stage of release failure (39).
Several factors may explain the apparent difference between present data and Ca2+ measurements in intact cells. First, the resting [Pi] is 1–2 mM in fast-twitch skeletal muscle fibers. Hence, Pi may have a tonic inhibitory influence on SR Ca2+ release, which is present even in relaxed fibers. As a consequence, in intact fibers, the influence of Pi may begin from a level which corresponds to ∼80% of the maximum shown in Fig. 3. In this case, the relative Pi-induced reduction would be smaller, until the final increase in Mg2+.
Another consideration is that the response to caffeine (at 1 and 0.5 mM Mg2+) declines faster above 10 mM Pi (Fig. 3). As discussed earlier, this probably reflects precipitation of calcium phosphate within the SR. However, the present experiments were carried out in the presence of CrP, which minimizes Pi-induced Ca2+ efflux via the SR Ca2+ pump (15). Activation of this pathway, combined with the additional effects of CrP withdrawal (14), may contribute to the progressive rise in resting [Ca2+] during fatigue (39). Consequently, in intact cells, the SR Ca2+ content will actually decline as [Pi] increases, and Ca2+ is redistributed from the SR to the cytosol. As in cardiac muscle (35), such an effect would reduce the likelihood of calcium phosphate precipitation, or its probable influence. In support of this, it has been shown that precipitation is not apparent in skinned fibers in the absence of CrP, even when [Pi] is increased to >20 mM (11). This factor will also tend to reduce the relative decline in the caffeine-induced Ca2+ transient >10 mM Pi, bringing the present data in closer agreement with Ca2+measurements in intact cells.
In conclusion, this study demonstrates that the amount of Ca2+ released from the SR in response to caffeine or T-tubule depolarization is reduced in the continued presence of Pi. Inhibition of SR Ca2+ release by Pi was markedly potentiated when [Mg2+] was increased from ∼1 to 1.5 mM. These effects may be important in end-stage fatigue if the rise in the cytosolic [Mg2+], subsequent to net ATP hydrolysis, makes an inhibitory action of Pi more pronounced.
Financial support from the British Heart Foundation and the Wellcome Trust is acknowledged.
Address for reprint requests and other correspondence: D. S. Steele, School of Biomedical Sciences, Univ. of Leeds, Leeds LS2 9JT, UK (E-mail).
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- Copyright © 2001 the American Physiological Society