Interdependent effects of inorganic phosphate and creatine phosphate on sarcoplasmic reticulum Ca²⁺ regulation in mechanically skinned rat skeletal muscle

Abstract

1. The effects of creatine phosphate (CP) and inorganic phosphate (P_i) on sarcoplasmic reticulum (SR) Ca²⁺ regulation were investigated in mechanically skinned muscle fibres from rat extensor digitorum longus (EDL) muscles. Changes in [Ca²⁺] were detected using fura-2 fluorescence, during continuous perfusion or when the solution surrounding the preparation was restricted to approximately 6 μl by stopping perfusion.

2. In solutions with 5 mm ATP and 10 mm CP, stopping the flow for 2-3 min had no effect on [Ca²⁺] within the bath. This suggests that SR Ca²⁺ uptake is balanced by an efflux under these conditions.

3. In solutions with CP, the introduction of P_i induced a small transient rise in [Ca²⁺], due to Ca²⁺ loss from the SR. Following equilibration with solutions containing P_i (≥ 5 mm), a maintained decrease in [Ca²⁺] occurred when the flow was stopped. This is consistent with calcium phosphate (Ca-P_i) precipitation within the SR, resulting in maintained Ca²⁺ uptake.

4. In the absence of CP, the [Ca²⁺] within the bath increased progressively when the flow was stopped. This rise in [Ca²⁺] was inhibited by an alternative ATP regenerating system comprising phosphoenolpyruvate (PEP) and pyruvate kinase (PK). Therefore, the loss of Ca²⁺ from the SR may result from local ADP accumulation and the consequent reversal of the SR Ca²⁺ pump.

5. In the absence of CP, the initial Ca²⁺ release associated with the introduction of P_i increased markedly. Following prolonged equilibration with solutions containing P_i, a rise in [Ca²⁺] occurred within the bath when the flow was stopped. Maintained Ca²⁺ uptake associated with Ca-P_i precipitation was not apparent at any level of P_i tested (1–60 mm), when CP was absent.

6. These results suggest that withdrawal of CP is associated with activation of a SR Ca²⁺ efflux pathway. This may involve reversal of the SR Ca²⁺ pump, due to local ADP accumulation. In the absence of CP, the dominant influence of P_i appears to involve further Ca²⁺ efflux via the SR Ca²⁺ pump. The possible relevance of these effects to skeletal muscle fatigue is considered.

In skeletal muscle, intermittent fatiguing stimulation results in a progressive depletion of [CP]_i and an increase in [P_i]_i to 30 mm or more (Dawson et al. 1980; Nagesser et al. 1993). Experiments on isolated membrane vesicles have provided evidence of a functional link between the SR Ca²⁺ pump and creatine kinase (CK; Korge et al. 1993). Local rephosphorylation of ADP by bound CK serves to maintain a low ADP/ATP ratio in the vicinity of the SR Ca²⁺ pump. Withdrawal of CP or pharmacological inhibition of CK activates a Ca²⁺ efflux pathway involving reversal of the Ca²⁺ pump and impairs net SR Ca²⁺ accumulation (Duke & Steele, 1999). This suggests that CP depletion may contribute to impaired SR Ca²⁺ reuptake which has been shown to occur during fatigue (e.g. Westerblad & Allen, 1993).

Experiments on skinned fibres have shown that P_i entry into the SR lumen and subsequent precipitation of Ca-P_i can influence the amount of Ca²⁺ released from the SR (Fryer et al. 1995). Indeed, it has been suggested that Ca-P_i precipitation may underlie the decrease in SR Ca²⁺ release that occurs in the final stages of fatigue (e.g. Posterino & Fryer, 1998). However, the reported effects of P_i in skinned fibres are inconsistent; depending on the experimental conditions, exposure to P_i may increase, decrease or have little influence on releasable Ca²⁺ (Stienen et al. 1993; Fryer et al. 1995, 1997; Posterino & Fryer, 1998). Some of the variability in these results may reflect the fact that P_i has other effects on SR Ca²⁺ regulation; in isolated SR Ca²⁺ channels, P_i has been reported to facilitate activation by Ca²⁺ (Fruen et al. 1994). In skinned fibres, P_i produced an apparent inhibition of the SR Ca²⁺-ATPase, particularly at low pH (Stienen et al. 1999). Experiments on isolated SR vesicles and skinned muscle fibres have shown that P_i can induce Ca²⁺ efflux from the SR by reversal of the Ca²⁺ pump (Hasselbach, 1978; Duke & Steele, 2000).

Work on skinned cardiac muscle suggests that the actions of P_i may also depend on the cytosolic [CP] (Steele et al. 1995; Smith et al. 2000). In the absence of CP, P_i induced an efflux of Ca²⁺ and a marked decrease in the SR Ca²⁺ content, without evidence of Ca-P_i precipitation. However, in the presence of CP, the P_i-induced Ca²⁺ efflux was less pronounced and precipitation of Ca-P_i was apparent. This influence of CP on the response to P_i may reflect the fact that ADP is required for reversal of the SR Ca²⁺ pump (Hasselbach, 1978). In the presence of CP, the local [ADP] is low due to rephosphorylation via the CK reaction. This should reduce P_i-induced efflux via the SR Ca²⁺ pump, and the higher luminal [Ca²⁺] may then favour precipitation. The possible influence of CP on Ca-P_i precipitation has not yet been investigated in skeletal muscle.

The aim of the present study was to investigate the effect of P_i on Ca²⁺ fluxes across the SR membrane and the possible influence of CP on Ca-P_i precipitation. Experiments were carried out on mechanically skinned skeletal muscle fibres and SR Ca²⁺ uptake and release were detected using fura-2 fluorescence. The results suggest that Ca-P_i precipitation occurs within the SR when the bathing [P_i] is ≥ 5 mm, and when CP is present in the cytosol. Withdrawal of CP resulted in the loss of Ca²⁺ from the SR and subsequent introduction of P_i induced a further, more pronounced Ca²⁺ efflux. Maintained stimulation of SR Ca²⁺ uptake, characteristic of Ca-P_i precipitation, was not apparent in the absence of CP. The underlying mechanisms and the relevance of these results to skeletal muscle fatigue are discussed.

METHODS

Male Wistar rats (250-300 g) were killed by a blow to the head and cervical dislocation according to standard UK Schedule 1 procedures. The extensor digitorium longus (EDL) muscle was removed rapidly and placed in a solution mimicking the intracellular milieu. Single muscle fibres were mechanically skinned with fine forceps and then attached between an isometric tension transducer (SensoNor, Norway) and a fixed support using monofilament snares (diameter 30 μm) within stainless steel tubes (Goodfellow Metals, UK). Two muscle fibres were attached in parallel to increase the amount of light collected by the objective, thereby improving the fura-2 fluorescence ratio signal.

Apparatus

The apparatus for simultaneous measurement of isometric force and SR Ca²⁺ release is detailed elsewhere (Duke & Steele, 1998a). Briefly, the mounted preparation was lowered close to the bottom of a shallow bath with a glass coverslip base. A Perspex column (5 mm diameter) was lowered to within a few micrometres of the muscle to minimise the volume of the solution above the preparation. Throughout the experiments, preparations were perfused by pumping solution at 0.8 ml min⁻¹ via a narrow duct (100 μm diameter) passing through the centre of the column. Waste solution was collected continuously at the column edge. This created a film 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. In experiments involving detection of Ca²⁺ uptake by the SR, a solution containing ATP was rapidly applied via a narrow plastic tube, which joined the main perfusion duct close 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. The volume of solution and the frequency of application were regulated using a syringe pump under computer control.

Light emitted from areas of the field not occupied by the muscle image was reduced using a variable rectangular diaphragm on the side port of the microscope. The bath was placed on the stage of a S200 Nikon Diaphot inverted microscope. The muscle was viewed via a x40 Fluor objective (oil immersion, Nikon) and muscle length was increased to approximately 20 % above slack length. In control experiments, it was found that length did not have a direct effect on SR Ca²⁺ regulation (not shown). The preparation was alternately illuminated with light of wavelengths 340 nm and 380 nm at 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 by the ratio of light intensities emitted at > 500 nm.

Solution composition and data recording

All chemicals were obtained from Sigma unless otherwise stated. The basic perfusing solution contained (mm): K⁺, 130; Na⁺, 32; Hepes, 25; BAPTA, 0.1; ATP, 5; creatine phosphate (CP), 10; free Mg²⁺, 1; fura-2, 2 μm. Two millimolar NaN₃ was added routinely to inhibit mitochondrial activity. Where necessary, the equilibrium concentrations of metal ions in the calibration solutions were calculated using the affinity constants for H⁺ and Ca²⁺ for EGTA as previously reported (Fabiato & Fabiato, 1979; Smith & Miller, 1985) using the Windows-based REACT program (Duncan et al. 1999). Correction for ionic strength, details of pH measurement, allowance for EGTA purity and the principles of the calculations are as described in Miller & Smith (1984).

Measurement of [Mg²⁺] with furaptra showed that an extra 0.2 mm Mg²⁺ was needed to compensate for binding to 10 mm CP, an extra 1 mm Mg²⁺ to compensate for binding to 40 mm P_i, an extra 0.5 mm Mg²⁺ for binding to 2 mm oxalate and 0.75 mm Mg²⁺ for binding to 5 mm ADP. Mg²⁺ was added as MgCl₂. The free [Ca²⁺] of each solution was adjusted (to 100 nm unless stated otherwise) by addition of CaCl₂ (BDH). CP and ATP were added as disodium salts, P_i was added as potassium dihydrogen orthophosphate and oxalate as a dipotassium salt. In some experiments PEP (5 mm, monosodium salt) and PK (50 U ml⁻¹) were added as an alternative regenerating system. pH was adjusted in all solutions to 7.0 by addition of KOH. In solutions lacking CP or ATP, [Na⁺] was maintained at a constant level by addition of NaCl. In all solutions, [K⁺] was adjusted to 130 mm by addition of KCl. The [Cl⁻] ranged from ∼60 to ∼130 mm. However, this difference in [Cl⁻] had no influence on the results. In further control experiments, the effects of P_i were studied when potassium propionate or potassium 1,6-diaminohexane-N,N,N ‘,N ‘-tetraacetic acid (K-HDTA) was used in place of KCl. However, the effects of P_i were the same when propionate or K-HDTA replaced Cl⁻ as the principal anion. The CK inhibitor 2,4-dinitro-fluorobenzene (DNFB) was added from a 50 mm stock solution. Cyclopiazonic acid (CPA) was added from a 20 mm stock made up in DMSO. The final [DMSO] never exceeded 0.1 %, and this concentration had no apparent influence on the results. All experiments were performed at room temperature (22-24 °C).

In all experiments, the ratio and individual wavelength intensities were low-pass filtered (-3 dB at 30 Hz) and digitised for later analysis using an IBM-compatible 80486 computer with a Data Translation 2801A card.

RESULTS

Effects of p_i on net SR Ca²⁺ uptake in the presence of 10 mm CP

Figure 1A shows a continuous record of the fura-2 fluorescence ratio from two mechanically skinned EDL muscle fibres attached in parallel. The resting fluorescence ratio corresponds to a free [Ca²⁺] of 100 nm. The preparation was perfused for 10 min with a solution containing 10 mm CP. When the volume of solution surrounding the preparation was restricted (to ∼6 μl) by stopping perfusion, [Ca²⁺] remained constant (left), suggesting that Ca²⁺ uptake is balanced by efflux. This is consistent with work showing that the SR Ca²⁺ content reaches a steady state within 2 min under these conditions (Duke & Steele, 1998b).

Figure 1. Effects of P_i on net SR Ca²⁺ uptake in the presence of 10 mm CP.

Typical records of fura-2 fluorescence ratio from a preparation comprising two mechanically skinned EDL muscle fibres. A, after 10 min equilibration in the presence of 10 mm CP, stopping perfusion had no effect on fluorescence ratio (left). During continuous perfusion, introduction of 20 mm P_i caused a transient SR Ca²⁺ efflux, followed by a sustained reduction in bathing [Ca²⁺] below control levels. After 3 min exposure to 20 mm P_i the flow was stopped. This resulted in a maintained decrease in bathing [Ca²⁺] due to net SR Ca²⁺ uptake (middle). The preparation was then exposed to a P_i-free solution for over 10 min. Subsequent addition of 2 mm oxalate caused a sustained reduction in bathing [Ca²⁺] below control levels, which was more pronounced when the flow was stopped (right). B, after a control ‘stop flow’ response (left) introduction of 20 mm P_i induced a transient release of Ca²⁺, followed by a small maintained decrease in the resting [Ca²⁺], which became more pronounced when the flow was stopped. When the flow was resumed, [Ca²⁺] returned rapidly to the lower steady-state level. After a further 10 min perfusion, a similar decrease in [Ca²⁺] occurred when the flow was stopped (middle). Following perfusion with a P_i-free solution for 10 min, [Ca²⁺] remained constant when the flow was stopped (right). C, a control response to introduction of P_i and stopping the flow was followed by addition of 20 μm CPA (left). This resulted in a slow transient release of Ca²⁺ from the SR and the [Ca²⁺] returned to the original control level. When the flow was then stopped, a further slow increase in [Ca²⁺] occurred. D, after exposure to Triton X-100 to disrupt the SR membrane, the [Ca²⁺] remained constant when the flow was stopped.

As shown previously by Duke & Steele (2000), the introduction of 20 mm P_i resulted in a small transient increase in the fluorescence ratio, due to Ca²⁺ release from the SR (Fig. 1A, middle). After the P_i-induced Ca²⁺ transient decayed, the baseline [Ca²⁺] fell below that recorded in the absence of P_i. After 3 min of exposure to 20 mm P_i, perfusion was stopped. This was followed by a further, maintained decrease in bathing [Ca²⁺], which approached a new steady-state level within 2–3 min. When perfusion was resumed, the bathing [Ca²⁺] returned to the lower baseline level. The same preparation was then perfused with a P_i-free solution for 10 min. Following removal of P_i, the baseline [Ca²⁺] returned to the control level.

As considered previously (Steele et al. 1996), the decrease in [Ca²⁺] shown in Fig. 1A is consistent with Ca-P_i precipitation within the SR. Precipitation occurs when the product of the luminal [P_i] and [Ca²⁺] exceeds the Ca-P_i solubility product. The continual entry of P_i followed by precipitation will reduce the free luminal [Ca²⁺], thereby maintaining SR Ca²⁺ uptake. During perfusion, P_i-induced stimulation of Ca²⁺ uptake produced only a small, steady-state decrease in [Ca²⁺] within the preparation. This may be explained if the constant inward diffusion of Ca²⁺ maintains the free [Ca²⁺], within the preparation, close to that of the perfusate. However, when flow was stopped and the bath volume restricted, P_i-stimulated SR Ca²⁺ uptake resulted in a pronounced and maintained decrease in [Ca²⁺].

Similar changes in [Ca²⁺] occurred in the presence of oxalate, which also enters the SR precipitates with Ca²⁺ (Fig. 1A, right). A lower [oxalate] (e.g. 2 mm) produced an effect comparable with that of 20 mm P_i. This may reflect the lower solubility product for calcium oxalate (Lide, 1995), although the threshold for calcium oxalate precipitation was not defined in this study. Unlike P_i, the introduction of oxalate was not associated with a transient release of Ca²⁺ from the SR.

Figure 1B shows the reproducibility and reversibility of responses obtained following the addition of P_i. The preparation was initially perfused with a solution containing 10 mm CP and again [Ca²⁺] remained constant, when the flow was stopped (left). Introduction of P_i produced an efflux of Ca²⁺ from the SR and [Ca²⁺] then decreased to a new lower level (middle). A characteristic decrease in [Ca²⁺] occurred within the preparation when the flow was stopped. [Ca²⁺] returned to the new steady-state level when perfusion was resumed. After approximately 10 min, the flow was again stopped and [Ca²⁺] decreased towards the same steady-state level, over a similar time course. The preparation was then exposed to a P_i-free solution for 10 min (not shown). Following removal of P_i, there was no apparent change in [Ca²⁺] within the bath, when the flow was stopped (right).

If the P_i-induced fall in resting [Ca²⁺] reflects maintained SR Ca²⁺ uptake, then inhibition of the SR Ca²⁺ pump should reduce or abolish this effect. In Fig. 1C, the introduction of 20 mm P_i again caused a Ca²⁺ efflux and a decrease in bathing [Ca²⁺], which was more pronounced when the flow was stopped. On restarting the flow, [Ca²⁺] within the bath rapidly returned to a lower baseline level. Subsequent introduction of the SR Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) was followed by a slow transient release of Ca²⁺ from the SR and the [Ca²⁺] returned to the original control level. As previously suggested, this probably reflects the loss of Ca²⁺ via a leak pathway, which is revealed by inhibition of the Ca²⁺ pump (Duke & Steele, 1998a). Following exposure to CPA, [Ca²⁺]increased slightly when the flow was stopped. This shows that the reduction in [Ca²⁺] in the presence of 20 mm P_i requires active Ca²⁺ uptake via the SR Ca²⁺ pump. The rise in [Ca²⁺], which occurred when the flow was stopped, may reflect the continued loss of Ca²⁺ from the SR via Ca²⁺ leak pathways, and its accumulation in the restricted bath volume. In Fig. 1D, the preparation was exposed to 2 % Triton X-100, which disrupts all membranes, including the SR. This resulted in a large Ca²⁺ release, confirming that the SR was not fully Ca²⁺ depleted by exposure to CPA (not shown). Following treatment with Triton X-100, stopping the flow had no influence on [Ca²⁺] in the presence or absence of P_i. Qualitatively similar results to those shown in Fig. 1 were obtained in six other preparations.

In further experiments, it was found that the decrease in [Ca²⁺] was not influenced by azide, confirming that mitochondrial Ca²⁺ accumulation does not contribute significantly to the ‘stop flow’ response. The decrease in [Ca²⁺] was not influenced by ruthenium red, suggesting that SR Ca²⁺ channel activation is not significant over the range of [Ca²⁺] used in this study. Similar results were obtained when potassium propionate or K-HDTA was used in place of KCl. The rate of decline of [Ca²⁺] slowed progressively and approached a new steady state after 1–2 min and several factors may influence the magnitude and time course of this response. First, net Ca²⁺ uptake would be expected to reduce progressively as [Ca²⁺] falls further below the K_m of the Ca²⁺ pump. Second, the initial rate of Ca²⁺ uptake may be influenced by the rate of P_i (or oxalate) entry into the SR, which appears to occur relatively slowly over minutes (Fryer et al. 1997). Finally, as in intact cells during fatigue, accumulation of metabolites may also influence net Ca²⁺ uptake, particularly in the absence of CP (see below).

Ca-p_i precipitation threshold in the presence of CP

As considered above, several factors may influence the magnitude and time course of the fall in [Ca²⁺], which occurs when the flow is stopped in the presence of P_i. However, the absence or presence of this response can be used to assess whether or not Ca-P_i precipitation occurs under any given condition. Precipitation of Ca-P_i should occur with a distinct threshold when the solubility product is exceeded within the SR. Figure 2 shows the protocol used to determine the apparent threshold for Ca-P_i precipitation in the presence of CP and 100 nm bathing Ca²⁺. The basic protocol was similar to that shown in Fig. 1. When the flow was stopped in the absence of P_i, [Ca²⁺] within the bath remained constant. A small transient Ca²⁺ release was apparent on the introduction of 1 or 2 mm P_i. However, when the flow was stopped, [Ca²⁺] remained constant, suggesting that 1–2 mm P_i was insufficient to produce Ca-P_i precipitation under these circumstances. In the presence of 5 mm P_i, a small maintained decrease in [Ca²⁺] was apparent when the flow was stopped. This suggests that the solubility product was exceeded at 5 mm P_i, resulting in maintained SR Ca²⁺ uptake. At 10, 20 and 40 mm P_i, the initial Ca²⁺ release, on addition of P_i, was also followed by a maintained decreased in the steady-state [Ca²⁺] during perfusion. Hence, at 10–40 mm P_i, steady-state SR Ca²⁺ uptake was sufficiently rapid to decrease [Ca²⁺] within the muscle, despite the constant inward diffusion of Ca²⁺ from the perfusate. As previously reported, the amplitude and time course of the P_i-induced Ca²⁺ efflux was concentration dependent over the range 1–40 mm (Duke & Steele, 2000). Similar results were obtained in eight other preparations.

Figure 2. Apparent threshold for Ca-P_i precipitation.

Effect of stopping bath perfusion following equilibration with solutions containing various [P_i]. At each [P_i], introduction of P_i resulted in a transient increase in Ca²⁺ due to SR Ca²⁺ release. At 1 and 2 mm P_i, the [Ca²⁺] remained constant when the flow was subsequently stopped. At 5 mm P_i, a small maintained decrease in [Ca²⁺] occurred when the flow was stopped. At higher [P_i], the initial [Ca²⁺] release was followed by a maintained decrease in the steady state [Ca²⁺]. The fall in [Ca²⁺] that occurred when the flow was stopped became progressively greater as the [P_i] was increased above 5 mm. All responses were obtained in the same mechanically skinned preparation. The [Ca²⁺] of the bathing solution was 100 nm and the free [Mg²⁺], 1 mm. The vertical bar indicates a change in ratio of 0.1 units. All responses were obtained in the same preparation.

Effects of CP withdrawal on SR Ca²⁺ regulation

Figure 3A shows the effects of CP withdrawal on SR Ca²⁺ regulation. The preparation was initially perfused with a solution containing 10 mm CP and the [Ca²⁺] remained constant when the flow was stopped (left). Subsequent withdrawal of CP resulted in a slow, transient Ca²⁺ efflux from the SR, as reported previously (Duke & Steele, 1999). After [Ca²⁺] returned to baseline levels, a marked increase in bathing [Ca²⁺] occurred, when perfusion was stopped (middle). Following the re-introduction of CP, [Ca²⁺] again remained constant when the flow was stopped (right).

Figure 3. Effects of 20 mm P_i on net SR Ca²⁺ uptake in the presence and absence of CP.

A, after 10 min equilibration with solution containing 10 mm CP, [Ca²⁺] remained constant when the flow was stopped (left). During constant perfusion, withdrawal of CP resulted in a transient increase in [Ca²⁺]. Once the [Ca²⁺] had returned to the control level, stopping the flow resulted in a maintained increase in [Ca²⁺] (middle). B, the preparation was exposed to solutions containing a range of [CP]. In each case, the flow was stopped after 15 min exposure to each [CP]. The increase in [Ca²⁺] associated with stopping the flow became apparent at 2 mm CP and increased progressively as the [CP] was reduced further.

The concentration dependence of this effect of CP is shown in Fig. 3B. The preparation was initially perfused with a solution containing 10 mm CP and [Ca²⁺] remained constant when the flow was stopped. The [CP] was then decreased in a stepwise manner and the preparation exposed to each [CP] for 15 min before the flow was stopped. In the presence of 5 mm CP, [Ca²⁺] remained constant when the flow was stopped. However, when the flow was stopped in the presence of 2 mm CP, a small rise in resting [Ca²⁺] was apparent. This rise in Ca²⁺ became progressively more pronounced when [CP] was further reduced to 1, 0.5 and 0 mm. Similar results were obtained in six other preparations.

Responses obtained in the presence of an alternative ATP regenerating system

On withdrawal of CP, rephosphorylation of ADP via the creatine kinase reaction will cease and the local [ADP] will rise. Previous studies on skinned fibres suggest that the Ca²⁺ efflux on removal of CP, results from a local rise in [ADP], which induces reversal of the SR Ca²⁺ pump (Duke & Steele, 1999). Therefore, the increase in [Ca²⁺] that occurs when the flow is stopped in the absence of CP, might reflect further accumulation of ADP in the restricted bath volume. This would be expected to alter the balance between Ca²⁺ uptake and efflux, resulting in a progressive loss of Ca²⁺ from the SR. If this interpretation is correct, then an alternative ATP regenerating system should inhibit the rise in [Ca²⁺].

In each panel of Fig. 4, the baseline record is the control response obtained when the flow was stopped in the presence of 10 mm CP. In Fig. 4A, the characteristic rise in [Ca²⁺] obtained when the flow was stopped in complete absence of CP is shown superimposed. As shown in Fig. 4B, this rise in Ca²⁺ was markedly reduced when an alternative regenerating system comprising PEP and PK was included in the zero CP solution. Inclusion of PEP without exogenous PK also partially inhibited the rise in [Ca²⁺] (Fig. 4C). This may reflect the presence of endogenous bound PK within the preparation. However, as expected, addition of PK in the absence of PEP was without effect (Fig. 4D). Similar results were obtained in four other preparations.

Figure 4. Effects of an alternative regenerating system on stop flow response.

A, effect of stopping flow on [Ca²⁺] in the absence of CP. B, stopping flow in the presence of an alternative regenerating system comprising PEP (5 mm) and PK (50 units ml⁻¹). C, stopping flow in the presence of PEP, but in the absence of PK. D, stopping flow in the presence of PK, but in the absence of PEP. In each example, the control response to stopping the flow in the presence of 10 mm CP is superimposed (lower trace). All responses were obtained from the same preparation.

Effects of P_i in the absence of CP

Figure 5A (upper left) shows a typical response to the introduction of 20 mm P_i in the presence of 10 mm CP. In this experiment, the free [Ca²⁺] was increased to 250 nm to increase the probability of Ca-P_i precipitation within the SR. Following the initial Ca²⁺ efflux, [Ca²⁺] decreased to a lower steady-state level. When the flow was stopped, a further maintained decrease in [Ca²⁺] occurred, consistent with precipitation of Ca-P_i within the SR. The flow was then re-started and the preparation equilibrated for 15 min with a solution lacking both P_i and CP. When the flow was then stopped, [Ca²⁺] within the bath increased as shown previously (upper right). When 20 mm P_i was introduced in the absence of CP, the initial transient rise in [Ca²⁺] was markedly increased (lower left). Furthermore, after the initial Ca²⁺ release, [Ca²⁺] returned to control levels; a decrease below the baseline level did not occur during constant perfusion, even when [P_i] was increased to 60 mm (not shown). In contrast to the effects of P_i, introduction of oxalate resulted in a maintained decrease in [Ca²⁺], consistent with calcium oxalate precipitation within the SR and consequent stimulation of the Ca²⁺ pump (lower right). Figure 5B shows the responses to 20 mm P_i in the presence and absence of CP, on an expanded time scale.

Figure 5. Effects of 20 mm P_i in the presence and absence of CP.

A, after 10 min equilibration in a solution containing 10 mm CP, introduction of 20 mm P_i caused a small transient rise in [Ca²⁺] and a maintained decrease in resting [Ca²⁺]. A further reduction in bathing [Ca²⁺] occurred when the flow was stopped (left). Following equilibration with a solution lacking CP, [Ca²⁺] increased when the flow was stopped (right). In the absence of CP, introduction of 20 mm P_i induced a larger transient increase in [Ca²⁺], which then declined to the control level. A maintained increase in [Ca²⁺] occurred when the flow was subsequently stopped (lower left). Introduction of 3 mm oxalate resulted in a maintained decrease in the resting [Ca²⁺], which became more pronounced when the flow was stopped (lower right). B, superimposed fluorescence records showing changes in [Ca²⁺] on exposure to 20 mm P_i in the presence and absence of 10 mm CP. All responses were obtained from the same preparation. In this example, the free [Ca²⁺] was increased to 250 nm to increase the possibility of Ca-P_i precipitation. All results were obtained from the same preparation.

In the absence of CP, the increase in bathing [Ca²⁺] was less pronounced when the flow was stopped in the presence of P_i, than in absence (compare Fig. 5A upper right and lower left). This might reflect a decrease in SR Ca²⁺ content, due to the larger P_i-induced Ca²⁺ efflux that occurs in the absence of CP. Figure 6 shows that the general characteristics of the P_i response could be mimicked by the addition of millimolar levels of ADP. A control response in the presence of 10 mm CP is shown (left). This was followed by a transient release of Ca²⁺ on withdrawal of CP and a maintained increase in [Ca²⁺] when the flow was stopped (middle). Subsequent introduction of 2 mm ADP resulted in a further transient increase in [Ca²⁺], which then returned to baseline levels. However, when the flow was then stopped in the presence of 5 mm ADP, the maintained rise in [Ca²⁺] was markedly reduced. This supports the suggestion that the release of SR Ca²⁺ by P_i may explain the smaller rise in [Ca²⁺] that occurred when the flow was stopped. Similar results were obtained in five other preparations.

Figure 6. Effects of ADP in the absence of CP.

After 10 min equilibration in the presence of 10 mm CP, [Ca²⁺] remained constant when the flow was stopped (left). Withdrawal of CP resulted in a transient Ca²⁺ efflux. In the continued absence of CP, the [Ca²⁺] increased markedly when the flow was stopped (middle). Subsequent introduction of 2 mm ADP resulted in a further transient Ca²⁺ efflux (right). However, when perfusion was then stopped, the Ca²⁺ efflux was markedly reduced relative to that obtained in the absence of CP and P_i.

Effects of creatine kinase inhibition

Figure 7A shows the effects of CK inhibition on SR Ca²⁺ regulation. In the presence of 10 mm CP, [Ca²⁺] remained constant when the flow was stopped (left). As shown previously (Duke & Steele, 1999), introduction of the CK inhibitor DNFB (50 μm) resulted in a slow transient Ca²⁺ efflux from the SR, similar to that observed on removal of CP (middle). After exposure to DNFB, a pronounced increase in [Ca²⁺] occurred when the flow was stopped (right).

Figure 7. Effects of 20 mm P_i following inhibition of CK.

A, following 10 min exposure to a solution containing 10 mm CP, the flow was stopped and the [Ca²⁺] remained constant (left). Subsequent addition of 50 μm DNFB caused a transient efflux of Ca²⁺ from the SR (middle). After 30 min exposure to DNFB, a maintained increase in bathing [Ca²⁺] occurred when the flow was stopped (right). B, a control response to the addition of P_i and stopping the flow in the presence of CP (left) was followed by a response to the introduction of P_i 30 min after the introduction of DNFB (right). C, superimposed fluorescence records showing the effects of 20 mm P_i in the presence of 10 mm CP, before and after exposure to 50 mm DNFB. All responses were obtained from the same preparation. In this example, the free [Ca²⁺] was increased to 250 nm to increase the possibility of Ca-P_i precipitation. All results were obtained from the same preparation. In C, the bar represents a change in ratio of 0.23 units.

Figure 7B shows the response to 20 mm P_i in the presence of 10 mm CP, before and after treatment with DNFB. As in Fig. 5, the [Ca²⁺] of the perfusing solution was 250 nm throughout. During perfusion in the presence of 10 mm CP, the introduction of 20 mm P_i induced a small transient Ca²⁺ efflux from the SR and a decrease in [Ca²⁺] to a new lower level. A further maintained fall in [Ca²⁺] occurred when the flow was stopped (left). However, when 20 mm P_i was introduced following inhibition of CK, the initial rise in [Ca²⁺] increased markedly (right). As in the complete absence of CP, [Ca²⁺] returned to baseline levels after the initial Ca²⁺ release. A similar response was obtained when DNFB was applied in the absence of CP (not shown). The superimposed responses to addition of P_i, before and after inhibition of CK with DNFB, are shown in Fig. 7C. As previously reported, the effects of DNFB were not reversible within 20 min. These results suggest that the effects of CP withdrawal can be mimicked by inhibition of CK. This also confirms that the effects of CP withdrawal reflect the abolition of ATP buffering via the CK reaction, rather than a direct effect of CP on the SR. Similar results were obtained in five other preparations.

Net SR Ca²⁺ uptake following depletion of SR Ca²⁺

In previous figures, CP was withdrawn or P_i introduced after the SR content had reached steady state. However, such experiments do not provide information regarding the relative decrease in SR Ca²⁺ content associated with CP withdrawal or addition of P_i. Therefore, the protocol shown in Fig. 8A was designed to investigate the effects of CP and P_i on net Ca²⁺ uptake following complete depletion of SR Ca²⁺. Simultaneous records of the fluorescence ratio (upper panel) and force (lower panel) are shown. During continuous perfusion with a solution containing 5 mm ATP and 10 mm CP, the introduction of 20 mm caffeine caused a large increase in fluorescence and force due to SR Ca²⁺ release. After 10 min exposure to caffeine, ATP was withdrawn to prevent re-accumulation of Ca²⁺ by the SR. ATP withdrawal caused the development of rigor and a further slow, transient efflux of Ca²⁺ remaining in the SR (see Duke & Steele, 1998). Caffeine was then removed for 5 min and perfusion stopped. Following this, the solution within the bath was rapidly replaced (within 10 ms) with one containing 5 mm ATP and the flow stopped. This resulted in relaxation from rigor (lower panel) and a maintained fall in bathing [Ca²⁺]. This decrease in [Ca²⁺] did not occur following disruption of the SR membrane with Triton X-100, or after treatment of the fibre with the pump inhibitor CPA (not shown). This confirms that the maintained decrease in [Ca²⁺] is due to ATP-activated SR Ca²⁺ uptake. The uptake of Ca²⁺ on introduction of ATP was reproducible. However, some deterioration in the myofilament response was generally apparent with repeated exposures to zero ATP solution (not shown).

Figure 8. Effects of CP and P_i on net SR Ca²⁺ uptake.

A, simultaneous records of fluorescence ratio (upper panel) and force (lower panel) from a muscle preparation comprising two mechanically skinned fibres. The preparation was initially perfused with a solution containing 5 mm ATP and 10 mm CP. Introduction of 20 mm caffeine into the perfusate resulted in Ca²⁺ release from the SR and an associated force response. SR Ca²⁺ uptake was then abolished by withdrawal of ATP. This resulted in the development of a rigor tension response and a further, transient SR Ca²⁺ efflux. Perfusion was stopped and the bath solution replaced rapidly with one containing 5 mm ATP. This resulted in relaxation from rigor and a maintained reduction in [Ca²⁺] due to net SR Ca²⁺ uptake. B, superimposed fluorescence ratio records of ATP-induced SR Ca²⁺ uptake under control conditions, or in the presence of 20 mm P_i (left). In the presence of CP, 20 mm P_i had little effect on SR Ca²⁺ uptake. However, when CP was absent from the perfusate, Ca²⁺ reuptake was reduced. When 20 mm P_i was present (but CP absent), Ca²⁺ uptake was further reduced and a slow increase in [Ca²⁺] followed the initial rapid decline (right). C, in the presence of 10 mm CP and 20 mm P_i, Ca²⁺ reuptake was markedly reduced following inhibition of CK with 50 μm DNFB. Responses shown in A and B were from the same preparation.

The protocol shown in Fig. 8A was modified to assess the effects of 20 mm P_i on SR Ca²⁺ uptake in the presence and absence of 10 mm CP. In all cases, P_i was introduced in the absence of ATP, immediately after removal of caffeine. Figure 8B (left) shows that in the presence of 10 mm CP, net Ca²⁺ uptake was similar in the presence and absence of 20 mm P_i. However, in the absence of CP (right), net SR Ca²⁺ uptake was reduced. In the absence of CP, 20 mm P_i further reduced net Ca²⁺ uptake by the SR. Furthermore, in the presence of 20 mm P_i, the initial decrease in [Ca²⁺] within the bath was followed by a slower rise. This may result from the loss of Ca²⁺ from the SR as ADP progressively accumulates within the bath, facilitating (with P_i) reversal of the SR Ca²⁺ pump. The slow rise in [Ca²⁺] is unlikely to reflect inhibition of the SR Ca²⁺ pump due to ATP depletion, because (i) the myofilaments have a higher sensitivity to ATP than the SR Ca²⁺ pump and re-development of rigor force did not occur during this period, and (ii) [ATP] is unlikely to decrease from 5 mm to < 100 μm (as required to inhibit SR Ca²⁺ uptake) within 20-30 s, due to SR Ca²⁺-ATPase activity alone. Similar results were obtained in nine other preparations.

Figure 8C shows that the effect of 20 mm P_i on net Ca²⁺ uptake in the absence of CP could be mimicked by exposure of the preparation to 50 μm DNFB. The control response shows ATP-induced SR Ca²⁺ uptake in the presence of 20 mm P_i and then the same effect after exposure to DNFB. Both responses were in the constant presence of 10 mm CP. Similar results were obtained in four other preparations.

While net Ca²⁺ uptake was reduced in the absence of CP and in the presence of P_i, there was little effect on the initial decrease in [Ca²⁺] following the introduction of ATP. One possible explanation for this is that the initial rate of decline may be influenced by the inward diffusion of ATP. Alternatively, pump reversal may occur when the luminal [Ca²⁺] has risen significantly.

DISCUSSION

In most previous studies on skinned preparations, the presence and influence of Ca-P_i precipitation was assessed indirectly from changes in the amount of Ca²⁺ available for release from the SR following washout of P_i. In the present study, measurement of cytosolic [Ca²⁺] allowed changes in SR Ca²⁺ regulation to be studied in the continued presence of P_i.

Ca²⁺ flux pathways across the SR membrane

Experiments on isolated SR vesicles have shown that Ca²⁺ transport across the SR membrane is initially coupled to ATP hydrolysis with a molar ratio of 2:1. However, as the luminal [Ca²⁺] rises, SR ATPase activity and Ca²⁺ uptake progressively decrease (Inesi & de Meis, 1989). Inhibition of pump activity by raised luminal [Ca²⁺] appears to result from the occupation of low affinity Ca²⁺ binding sites at the inner surface of the SR membrane. In the absence of precipitating anions, the Ca²⁺ content of the SR will reach a steady state, when uptake by the Ca²⁺ pump is balanced by Ca²⁺ efflux. Efflux can occur via ryanodine-sensitive Ca²⁺ channels, a Ca²⁺‘leak’ pathway, or in some circumstances by reversal of the SR Ca²⁺ pump (for review see Feher & Fabiato, 1990). Previous work on skinned skeletal muscle fibres suggests that in the presence of 100-200 nm Ca²⁺ and millimolar Mg²⁺, the steady-state Ca²⁺ leak is small and may occur via the SR Ca²⁺ channel (Kabbara & Stephenson, 1994) and via a ryanodine-insensitive Ca²⁺ efflux pathway (Duke & Steele, 1998a). In the presence of CP, the local [ADP] is low and reversal of the SR Ca²⁺ pump would not be expected to constitute a major Ca²⁺ efflux pathway.

Effects of P_i in the presence of CP

In the protocol shown in Fig. 1, the preparation was initially perfused with a solution containing 10 mm CP. Previous studies involving caffeine application in skinned fibres have shown that the SR Ca²⁺ content reaches a steady state within 2 min under these conditions (Duke & Steele, 1998b). Thereafter, Ca²⁺ uptake must equal efflux. When the flow was stopped for 3 min in the presence of CP, [Ca²⁺] remained constant, suggesting that the balance of Ca²⁺ fluxes was unaltered. However, the introduction of P_i during perfusion resulted in a transient, concentration-dependent increase in [Ca²⁺] within the preparation (e.g. Fig. 1). We have shown previously that P_i-induced Ca²⁺ release is insensitive to ryanodine or other modulators of the SR Ca²⁺ channel, blocked by the Ca²⁺ pump inhibitor CPA and mimicked by addition of ADP (Duke & Steele, 2000). These characteristics are consistent with work on isolated SR vesicles showing that P_i can induce Ca²⁺ efflux by reversal of the SR Ca²⁺ pump (e.g. Hasselbach, 1978).

At low levels of P_i (< 5 mm), [Ca²⁺] returned to baseline levels following the initial P_i-induced Ca²⁺ release (Fig. 2). The [Ca²⁺] also remained constant when the flow was stopped in the presence of low [P_i]. This suggests that, following the initial P_i-induced [Ca²⁺] release from the SR, a new equilibrium state is rapidly achieved, where Ca²⁺ uptake again equals efflux. However, in the presence of higher levels of P_i (and CP), a maintained decrease in the baseline [Ca²⁺] was apparent during perfusion. A further, more pronounced decrease in [Ca²⁺] occurred when the flow was stopped. This decrease in Ca²⁺ was mimicked by oxalate and abolished by treatment with CPA or disruption of the SR membrane with Triton X-100, confirming maintained SR Ca²⁺ uptake underlies the fall in [Ca²⁺] (Fig. 1). As previously considered, it seems likely that the maintained uptake of Ca²⁺ results from Ca-P_i precipitation within the SR lumen. When P_i enters the SR, precipitation will occur when the Ca-P_i solubility product is exceeded. The resulting decrease in luminal [Ca²⁺] disinhibits the Ca²⁺ pump and Ca²⁺ uptake persists due to the continual entry of P_i from the surrounding medium. This phenomenon is commonly utilised in studies on isolated SR to maintain Ca²⁺ uptake and prevent inhibition of the Ca²⁺-ATPase by rising luminal Ca²⁺. Under these conditions, the capacity of the SR may be limited only by the eventual rupture of the SR membrane by solid precipitates (Feher & Lipford, 1995).

The results shown in Fig. 2 suggest that precipitation of Ca-P_i and consequent stimulation of the SR Ca²⁺ pump occurred at a threshold of ∼5 mm bathing P_i when the [Ca²⁺] was 100 nm (Fig. 2). A similar threshold for precipitation has been reported previously in skinned EDL fibres under comparable Ca²⁺ loading conditions (Fryer et al. 1995). Although the level of bathing P_i required for precipitation would be expected to change depending on the Ca²⁺ loading conditions, the apparent threshold remained consistent under the conditions of this study.

While the P_i and oxalate both produced effects consistent with precipitation, the responses also differed in a number of important respects. First, the initial Ca²⁺ release on addition of P_i was not apparent on addition of oxalate (compare Fig. 1A, centre and right). This presumably reflects the fact that unlike P_i, oxalate does not induce a reversal of the SR Ca²⁺ pump. Second, precipitation was apparent at lower levels of oxalate, below that producing maintained Ca²⁺ uptake in the presence of P_i (Fig. 1). This is consistent with the fact that the solubility product for oxalate is lower than that of P_i (Lide, 1995), and the fact that oxalate does not deplete SR Ca²⁺ by pump reversal.

Effects of CP withdrawal on SR Ca²⁺ regulation

As shown in Fig. 3, withdrawal of CP during perfusion resulted in loss of Ca²⁺ from the SR. This is consistent with previous work on isolated SR and skinned fibres suggesting that local rephosphorylation of ADP via the creatine kinase reaction is important for efficient Ca²⁺ transport by the SR ATPase. Previously, we have shown that the loss of Ca²⁺ from the SR on withdrawal of CP can be inhibited by CPA, which blocks the reversal of the SR Ca²⁺ pump (Duke & Steele, 1999). This suggests that the Ca²⁺ efflux associated with CP withdrawal may involve reversal of the SR Ca²⁺ pump due to local ADP accumulation.

Previous studies have shown that the SR Ca²⁺ content declines to a lower steady-state level within 2-3 min of CP withdrawal (Duke & Steele, 1999). At this point, Ca²⁺ uptake must again balance efflux. However, even after much more prolonged exposure to solutions lacking CP, a slow increase in [Ca²⁺] was apparent when the flow was stopped (Fig. 3). The rise in [Ca²⁺] was detectable when [CP] was reduced to 2 mm, and increased progressively as the concentration was further reduced to zero (Fig. 3). One possible explanation for the rise in [Ca²⁺] is that the balance of Ca²⁺ fluxes is altered when the flow is stopped, due to further ADP accumulation in the limited bath volume. Hence, on stopping the flow, Ca²⁺ efflux via the pump may increase progressively, resulting in accumulation of Ca²⁺ within the bath. This interpretation is supported by the observation that the rise in Ca²⁺ was markedly reduced by an alternative ATP regenerating system (Fig. 4). The inability to abolish completely the rise in [Ca²⁺] associated with stopping the flow is consistent with work on isolated SR vesicles suggesting that (i) bound CK is functionally linked to the SR Ca²⁺ ATPase and (ii) endogenous bound CK is more efficient than exogenous enzyme systems at maintaining a low ATP/ADP ratio in the vicinity of the SR ATPase (Korge et al. 1993).

Effects of P_i in the absence of CP

As shown in Fig. 5, the effects of P_i were markedly altered when CP was absent from the cytosolic environment. First, as previously reported (Duke & Steele, 2000), the P_i-induced Ca²⁺ efflux was larger in the absence of CP. Second, during continuous perfusion, the bathing [Ca²⁺] did not fall below control levels in the presence of P_i. Finally, when perfusion was stopped in the presence of P_i, [Ca²⁺] progressively increased. Even at the highest [P_i] tested (40 mm), a maintained decrease in [Ca²⁺] consistent with precipitation did not occur when CP was absent from the solutions. This might be explained if precipitation of Ca-P_i occurs, but maintained stimulation of SR Ca²⁺ uptake does not result, when CP is absent from the solutions. However, maintained stimulation of the SR Ca²⁺ pump was apparent when oxalate was introduced in the absence of CP (Fig. 5). This may reflect the fact that (i) oxalate does not deplete SR Ca²⁺ by reversal of the SR Ca²⁺ pump and (ii) calcium-oxalate has a lower solubility product. The effects of oxalate suggest that the inability of P_i to stimulate and maintain Ca²⁺ uptake in solutions lacking CP reflects the absence of Ca-P_i precipitation within the SR.

Several factors may contribute to the apparent absence of Ca-P_i precipitation in solutions lacking CP. First, the Ca²⁺ efflux, associated with CP withdrawal, results in a decrease in the steady-state SR Ca²⁺ content, as assessed by caffeine application (Duke & Steele, 2000). This efflux also decreases the capacity of the SR to re-accumulate Ca²⁺, when ATP is re-introduced following Ca²⁺ depletion (compare Fig. 8B, left and right). It has also been shown that ADP is required for pump reversal by P_i (Hasselbach, 1978). Hence, in the presence of CP, the low [ADP] may limit pump reversal, thereby reducing SR Ca²⁺ depletion by P_i. This suggestion is supported by the observation that P_i had little effect on net Ca²⁺ uptake on re-introduction of ATP, following depletion of SR Ca²⁺ (Fig. 8B).

In the absence of CP, the higher local [ADP] may facilitate a larger and maintained P_i-induced Ca²⁺ efflux from the SR. Consistent with this, the initial P_i-induced Ca²⁺ efflux was more pronounced in the absence of CP (Fig. 5). ATP-activated Ca²⁺ uptake by the SR was also markedly reduced by P_i, in solutions lacking CP (Fig. 8B). Furthermore, in the presence of P_i, the initial rapid decrease in [Ca²⁺] was followed by a slow increase over 2-3 min. This may reflect a progressive increase in Ca²⁺ efflux via the SR Ca²⁺ pump, as ADP accumulates within the bath. Together, these results suggest that when CP is depleted and [P_i] rises, efflux via the SR Ca²⁺ pump will increase progressively and the free luminal [Ca²⁺] will decline. If the free SR [Ca²⁺] decreases sufficiently, the Ca-P_i solubility product may not be exceeded in the absence of CP.

Physiological significance and limitations of the present study

Under physiological conditions, it has been shown that cytosolic CP can be depleted rapidly during fatigue induced by repeated tetanic stimulation (e.g. Nagesser et al. 1993). The present study has shown that the effects of P_i are markedly influenced by CP. This apparent interdependence of CP and P_i may have a bearing on previous studies addressing the effects of P_i in intact and skinned preparations. For example, in most previous studies on skinned fibres, P_i was introduced in the presence of millimolar levels of CP (e.g. Fryer et al. 1995). This would tend to minimise efflux via the SR Ca²⁺ pump and facilitate Ca-P_i precipitation. Similarly, in intact skeletal muscle, injection of P_i into the cytosol of unfatigued fibres produced a maintained decrease in resting Ca²⁺ and acceleration of Ca²⁺ uptake, consistent with Ca-P_i precipitation (Westerblad & Allen, 1996). This result appears consistent with the present study, where P_i produced a maintained decrease in resting Ca²⁺ in the presence of CP. However, this contrasts with the observation that resting [Ca²⁺]increases progressively throughout fatiguing stimulation (e.g. Allen & Westerblad, 1991). This rise in [Ca²⁺] might be explained if Ca²⁺ is progressively redistributed from the SR to the cytosol due to pump reversal associated with CP depletion and rising levels of P_i.

One limitation of the present study is that two extreme conditions were examined; P_i was introduced in the complete absence or presence of CP. However, progression to a state of fatigue involves reciprocal changes in [P_i] and [CP] (Dawson et al. 1980). Therefore, as fatigue develops, the levels of P_i within the SR might be high enough to cause precipitation, before complete CP depletion occurs. This may depend upon a number of factors such as the SR Ca²⁺ load and the rate of P_i entry (Fryer et al. 1997). In support of the Ca-P_i precipitation hypothesis, recent experiments in intact fibres showed that the amount of Ca²⁺ released from the SR in response to caffeine or 4-chloro-m-cresol (4-CmC) is reduced in fatigue. This was interpreted as evidence that the precipitation of Ca²⁺ within the SR decreases the amount of Ca²⁺ available for release. However, the sensitivity of the SR Ca²⁺ channel to caffeine (Duke & Steele, 1989b) and 4-CmC (A. M. Duke & D. S. Steeles, unpublished observations) is markedly affected by cytosolic levels of ATP. The local [ATP] may fall substantially in the final stages of fatigue, which could contribute to the apparent reduction in releasable Ca²⁺. Furthermore, based on the present data, precipitation of Ca-P_i should result in a sudden decrease in resting Ca²⁺, as the solubility product is exceeded and the SR Ca²⁺ pump is stimulated. This does not appear to occur in intact cells during any stage of the fatigue process. The results of the present study suggest that the dominant influence of P_i, when CP is depleted, is the activation of Ca²⁺ efflux via pump reversal and that precipitation of Ca-P_i does not occur under these conditions. This does not support the suggestion that precipitation of Ca-P_i underlies the failure of the SR Ca²⁺ release mechanism in end-stage fatigue (e.g. Posterino & Fryer, 1998).

Conclusions

These experiments suggest that under the conditions of this study, precipitation of Ca-P_i occurs within the SR when the bathing [P_i] is ≥ 5 mm and when CP is present in the solutions. Both withdrawal of CP and subsequent addition of P_i resulted in the activation of a Ca²⁺ efflux pathway, which may involve the reversal of the SR Ca²⁺ pump. In the absence of CP, maintained SR Ca²⁺ uptake characteristic of Ca-P_i precipitation did not occur.