INTRODUCTION

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THE CONTRACTION-RELAXATION cycle of skeletal muscle is regulated by the release and accumulation of Ca²⁺ by the sarcoplasmic reticulum (SR) (24). T-tubule depolarization causes the rapid release of Ca²⁺ from the SR store, via the Ca²⁺ release channels (CRC), for the initiation of contraction. Subsequently, cytosolic Ca²⁺ is rapidly decreased to allow muscle relaxation. The latter involves reuptake of the released Ca²⁺ via the SR Ca²⁺-ATPase, which is an energy-dependent process (23).

Loss of SR function, such as occurs during and after repeated intensive muscular contractions, leads to alterations in SR Ca²⁺ cycling, both lower Ca²⁺ release and Ca²⁺ uptake, and lower force (i.e., muscle fatigue) (2). Curiously, intrinsic reductions in Ca²⁺ uptake and Ca²⁺ release, after various fatigue protocols, appear to be qualitatively and quantitatively similar (20, 42). After eccentric exercise (20), the time course (in days) for both the reduction and recovery in SR Ca²⁺ uptake and Ca²⁺ release is also closely associated. Moreover, in other models of muscular stress, such as ischemia-reperfusion injury (28, 37), a similar trend is observed, namely an apparent coupling between the reductions in both SR Ca²⁺ uptake and Ca²⁺ release. Conceivably, the apparent coupling could be explained by a common mechanism involving specific sites on the Ca²⁺-ATPase and the CRC and causing structural modifications to these proteins. Alternately, the possibility exists that the coupling is artifact, because of limitations in the measurement assays. In general, Ca²⁺ release is measured after Ca²⁺ uptake and SR Ca²⁺ loading as part of the same assay.

Recently, we have also shown that 4 h of ischemia leads to a reduction in both SR Ca²⁺ uptake and Ca²⁺ release in both homogenates and isolated SR vesicles prepared from rat skeletal muscle (40). In the ischemic study, we measured Ca²⁺ release in vitro using silver nitrate (AgNO₃), which produced a biphasic Ca²⁺ release response. There was an initial, rapid rate of release, which we called phase 1, followed by a more prolonged and slower rate of release, which we called phase 2. Surprisingly, we showed that, if the Ca²⁺-ATPase was inhibited with cyclopiazonic acid (CPA) just before Ca²⁺ release was induced with AgNO₃, then the rate of Ca²⁺ release corresponding with phase 2 would be greatly reduced in both ischemic and control SR. To explain this effect, we hypothesized that SR Ca²⁺ release and Ca²⁺ uptake processes are tightly coupled such that Ca²⁺ uptake by the SR during net SR Ca²⁺ release exerts an influence on Ca²⁺ release and is important for maintaining normal Ca²⁺ release function.

Alternatively, AgNO₃ might induce Ca²⁺ release by direct interaction with the Ca²⁺-ATPase. Initial reports have indicated that AgNO₃ induces Ca²⁺ release from the SR by acting on the CRC and not on the Ca²⁺-ATPase (1, 31). On that basis, we, among many others (12, 20, 29, 41, 42), have used AgNO₃ to assay for Ca²⁺ release in vitro to assess CRC function after various perturbations. However, there are reports in the literature suggesting that the Ca²⁺-ATPase may be the primary pathway for Ca²⁺ release induced by AgNO₃ (14, 36). If this is the case, inhibition of the Ca²⁺-ATPase by CPA could be responsible for the reduction in Ca²⁺ release observed in phase 2. It is noteworthy that, in several previous reports utilizing AgNO₃ as the Ca²⁺-releasing agent, a two-phase response is clearly indicated but not used in the analyses (27, 29, 41).

Understanding the specific effects of AgNO₃ on SR Ca² release and Ca²⁺-ATPase has important implications. On the one hand, if AgNO₃ has effects on both Ca²⁺ cycling processes, the interpretation of the effects of perturbations such as exercise or ischemia would be biased. On the other had, if an intrinsic coupling does exist, this would represent an important advance in understanding the role of the SR in Ca²⁺ regulation.

In the present study, our objective was to examine the specific effects of AgNO₃ on the CRC and the Ca²⁺-ATPase to determine whether an intrinsic coupling does, in fact, exist between SR Ca²⁺ uptake and Ca²⁺ release or whether the effects that we have observed are specific only for AgNO₃. To examine this issue, we used CPA, a specific inhibitor of the SR Ca²⁺-ATPase and Ca²⁺ uptake (13, 33), to inhibit SR Ca²⁺ uptake during net Ca²⁺ release, induced by either AgNO₃ or 4-chloro-m-cresol (4-CMC), which is known to directly activate the CRC (17). Our findings indicate that AgNO₃, but not 4-CMC, induces Ca²⁺ release by acting on both the CRC and the Ca²⁺-ATPase.

	METHODS

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Animal description and care. Adult female Sprague-Dawley rats weighing between 225 and 275 g were housed in an environmentally controlled room (temperature 22-24°C, 40-60% relative humidity) with reversed light-dark cycles. Animals were fed ad libitum on laboratory chow and water until the time of the experiment. All experiments (muscle sampling) were initiated at approximately the same time each day to avoid large diurnal variations in muscle glycogen (6). Experimental protocols were approved by the Animal Care Committee of the University of Waterloo.

Sample preparation for SR assessment in vitro. Sprague-Dawley rats (n = 9) were weighed and anesthetized using an intraperitoneal injection of pentobarbital sodium (6 mg/100 g body wt). After anesthetization, the gastrocnemius muscle (both red and white portions), along with the entire tibialis anterior muscle, was excised from each limb and placed in ice-cold buffer to be used for preparation of muscle homogenates according to Heilmann et al. (16). Mixed gastrocnemius and tibialis anterior muscles were diluted ~1:5 (wt/vol) in buffer containing (in mM) 5 HEPES (pH 7.5), 250 sucrose, 0.2% sodium azide, and 0.2 phenylmethylsulfonyl fluoride [no dithiothreitol (DTT)] and mechanically homogenized with a polytron homogenizer (PT 3100) at 16,500 rpm, for 2 × 30-s bursts. Aliquots of muscle homogenate were then rapidly frozen in liquid nitrogen and stored at 70°C for later analysis of SR function.

SR Ca²+ release measurements. AgNO₃-induced Ca²⁺ release was measured in muscle homogenates according to the methods of Ruell et al. (29), using the Ca²⁺ fluorescent dye indo 1, with minor modifications as previously described (40). Fluorescence measurements were made on a spectrofluorometer (RatioMaster system, Photon Technology International) equipped with dual-emission monochromators. The measurement of cytoplasmic (buffer) free Ca²⁺ ([Ca²⁺]_f) using the indo 1 procedure is based on the difference in the maximal emission wavelengths between the Ca²⁺-bound form of indo 1 and the Ca²⁺-free form. The excitation wavelength was 355 nm, and the emission maxima were 485 and 405 nm for Ca²⁺-free (G) and Ca²⁺-bound (F) indo 1, respectively. The ratio (R) of F to G is used to calculate [Ca²⁺]_f. With the use of Felix software (Photon Technology International), the ionized Ca²⁺ concentration was calculated by the following equation (15)

(1)

where K_d is the equilibrium constant for the interaction between Ca²⁺ and indo 1, R_min is the minimum value of R at the addition of 250 µM EGTA, G_max is the maximum value of G at the addition of 250 µM EGTA, G_min is the minimum value of G at the addition of 1 mM CaCl₂, and R_max is the maximum value of R at the addition of 1 mM CaCl₂. The K_d value for the Ca²⁺-dye complex is 250 nM for muscle homogenates (15).

1

1

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Changes in free calcium ([Ca2+]f) levels during Ca2+ uptake and during phase 1 and phase 2 of Ca2+ release initiated by silver nitrate (AgNO3; A) and by 4-chloro-m-cresol (4-CMC; B). The tracing is from a typical homogenate assay.

SR Ca²+-ATPase activity measurements. Spectrophotometric (Schimadzu UV 160U) measurement of SR Ca²⁺-ATPase activity was performed on homogenates using procedures developed by Simonides and van Hardeveld (35), with minor modifications as previously described (38). Total (Mg²⁺-activated)-ATPase activity was measured in the presence of ~6-10 µM [Ca²⁺]_f to obtain maximal ATPase activity and in the presence of the Ca²⁺ ionophore A-23187. Basal activity was measured in the presence of 40 µM CPA, which completely inhibits SR Ca²⁺-ATPase activity (33). The difference between total and basal activities represents the Ca²⁺-activated ATPase activity. To assess the effect of both AgNO₃ and 4-CMC on SR Ca²⁺-ATPase activity, in 50% of the trials either 141 µM AgNO₃ or 5 µM 4-CMC were added to the cuvette after maximal Ca²⁺-ATPase activity was obtained and before basal activity was obtained. Both Ca²⁺-ATPase activity and basal activity were expressed in micromoles per gram protein per minute. On a given analytic day, samples from all conditions were analyzed in duplicate.

Data analysis. For Ca²⁺ release measurements with and without 40 µM CPA and 100 µM RR, a two-way ANOVA was used to discriminate between differences due to CPA or RR (with vs. without) and releasing agent (AgNO₃ vs. 4-CMC). Where an overall interaction between assay and group was found, post hoc analyses (Tukey's) were performed to determine specific assay and group effects. For AgNO₃-induced Ca²⁺ release measurements made in the presence of varying CPA concentrations, a one-way ANOVA was used to test for differences between means. Where significant differences were found, Tukey's post hoc tests were used to compare specific means. For all other measurements, two-tailed paired t-tests were used to test for differences between means. For all comparisons, statistical significance was accepted at P < 0.05. All data are expressed as means ± SE.

	RESULTS

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Ca²+ release with AgNO₃. With the addition of AgNO₃, Ca²⁺ release proceeded in two distinct phases: an initial, rapid rate of release that was complete within seconds (phase 1), followed by a slower, more prolonged rate of release (phase 2) (Fig. 1A), which lasted 2-3 min until Ca²⁺ release stopped and the buffer [Ca²⁺]_f plateaued at some level (data not shown). The effects of CPA were opposite on these two phases of Ca²⁺ release. In all conditions, inhibition of Ca²⁺-ATPase activity with CPA after loading of SR with Ca²⁺ increased (P < 0.05) the early, rapid rate of release and decreased (P < 0.05) the slower, more prolonged rate of release. The same results are obtained with isolated SR vesicles (data not shown). The percent change in Ca²⁺ release rate was altered with varying concentrations of CPA, and, therefore, the extent of SR Ca²⁺-ATPase inhibition, for phase 2 but not for phase 1. A typical response for one animal showing the effects of different CPA concentrations on Ca²⁺ release is shown in Fig. 2. On average, phase 2 Ca²⁺ release rate was reduced (P < 0.05) by 44, 57, and 72% with 2, 5, and 40 µM CPA, respectively, compared with no CPA (Fig. 3). However, the average increase in phase 1 Ca²⁺ release rate with CPA was similar across all CPA concentrations used.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent effects of cyclopiazonic acid (CPA) in muscle homogenates on Ca2+ release kinetics during a typical assay in phase 1 and phase 2.

View larger version (44K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent effects of CPA in muscle homogenates on AgNO3-induced Ca2+ release kinetics for overall group in phase 1 and phase 2. Values are means ± SE (n = 9). Significantly different from * No CPA, ** No and 2 µM CPA, and *** No, 2, and 5 µM CPA, P < 0.05.

Ca²+ release with 4-CMC. The biphasic Ca²⁺ release rate response induced by 4-CMC was similar to the AgNO₃-induced Ca²⁺ release rate response (Fig. 1B). However, the absolute release rate for phase 1 was approximately sixfold higher (P < 0.05) with 4-CMC compared with AgNO₃ (1,961 ± 112 vs. 314 ± 51 µmol · g protein¹ · min¹). There were no differences (P > 0.05) in the absolute rate of release for phase 2 between 4-CMC and AgNO₃ (281 ± 17.1 vs. 267 ± 39 µmol · g protein¹ · min¹, respectively). Unlike AgNO₃, 40 µM CPA after Ca²⁺ loading of the SR had no effect on the 4-CMC-induced rate of release corresponding with both phase 1 and phase 2 (Fig. 4).

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Phase-dependent effect of CPA on 4-CMC-induced Ca2+ release kinetics in homogenates. Values are means ± SE (n = 7).

Ca²+ release with RR. A typical response showing the effects of RR for both AgNO₃- and 4-CMC-induced Ca²⁺ release rate is shown in Fig. 5. With AgNO₃, RR had no effect on phase 1 rate of release (P > 0.05) (342 ± 68 vs. 323 ± 51 µmol · g protein¹ · min¹) and decreased phase 2 release rate (P < 0.05) by 65% (245 ± 34 vs. 105 ± 8.2 µmol · g protein¹ · min¹) (Fig. 5A). Unlike AgNO₃, RR reduced phase 1 rate (P < 0.05) by 59% (2,468 ± 279 vs. 1,004 ± 87 µmol · g protein¹ · min¹) and completely blocked phase 2 (see Fig. 5B) when 4-CMC was used to induce Ca²⁺ release.

View larger version (18K): [in this window] [in a new window]   Fig. 5.   Effects of ruthenium red (RR; 100 µM) in muscle homogenates on Ca2+ release kinetics initiated by AgNO3 (A) and by 4-CMC (B). The tracing is from a typical homogenate assay.

Ca²+-ATPase activity. Maximal Ca²⁺-ATPase activity was inhibited by 95.4 ± 1.9% in the presence of 141 µM AgNO₃ (Fig. 6). Ca²⁺-ATPase activity in the presence of AgNO₃ was not different (P > 0.05) from basal activity. Even concentrations as low as 5 µM AgNO₃ appeared to partially inhibit Ca²⁺-ATPase activity (data not shown). Surprisingly, we also found that 4-CMC inhibited Ca²⁺-ATPase activity by 97 ± 1.4%.

View larger version (17K): [in this window] [in a new window]   Fig. 6.   Effects of AgNO3 and 4-CMC on maximal Ca2+-ATPase activity. Values are means ± SE (n = 9). * Significantly different (P < 0.05) from basal activity and net AgNO3 and 4-CMC activity.

	DISCUSSION

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The purpose of this study was to determine whether SR Ca²⁺ uptake and Ca²⁺ release are intrinsically coupled or whether the apparent coupling that has been observed is specific to the Ca²⁺-releasing agent employed. We have demonstrated that AgNO₃ induces a biphasic Ca²⁺ release rate response measured in homogenates in vitro, as we have shown previously (40). We also confirmed our earlier finding, namely that Ca²⁺-ATPase inhibition with CPA administered after SR Ca²⁺ loading and before inducing Ca²⁺ release causes a reduction in Ca²⁺ release rate (phase 2). However, this effect is only specific for AgNO₃. If Ca²⁺ release is induced with 4-CMC, inhibition of the Ca²⁺-ATPase with CPA after Ca²⁺ loading has no effect on either phase of Ca²⁺ release. Moreover, blocking the CRC with RR had no effect on phase 1 and inhibited phase 2 by 65% when AgNO₃ was used. When 4-CMC was employed as the releasing agent in conjunction with RR, phase 1 was reduced by 59%, and phase 2 was completely blocked. These results suggest that AgNO₃, in contrast to 4-CMC, has a unique effect on Ca²⁺-ATPase activity, resulting in reversal of the pump and extrusion of Ca²⁺ into the cytosol.

Recently, Ikemoto and colleagues (18, 30) proposed a tight coupling between SR Ca²⁺ uptake and Ca²⁺ release and suggested that the early kinetic changes in SR luminal Ca²⁺ during Ca²⁺ release provided the signal for a coordinated response from the Ca²⁺- ATPase and subsequent increased rate of Ca²⁺ uptake. Theoretically, the rate of Ca²⁺ uptake during net Ca²⁺ release could also affect SR luminal Ca²⁺ and thus the rate of Ca²⁺ release. A tempting hypothesis was that this could explain both the effects of CPA on SR Ca²⁺ release that we observed previously (38) and the observation from the literature that reductions in Ca²⁺ uptake and Ca²⁺ release after exercise (20, 42) and ischemia-reperfusion are closely matched (28, 37). However, in this experiment, we wanted to examine whether such a relationship exists intrinsically after the SR was loaded with Ca²⁺ or whether the relationship is specific to AgNO₃.

To investigate this problem, it was first necessary to demonstrate that the reduction in AgNO₃-induced Ca²⁺ release (phase 2) due to inhibition of the Ca²⁺-ATPase with CPA is a general phenomenon and is not just specific for AgNO₃. In fact, we found that CPA had no effect on the rate of Ca²⁺ release for either phase when 4-CMC was employed as the Ca²⁺ releasing agent. Although this does not contradict the findings by Ikemoto and Yamamoto (18) or the possibility that SR Ca²⁺ release and Ca²⁺ uptake are coupled, it does suggest that the mechanism by which AgNO₃ and 4-CMC act to cause Ca²⁺ release from SR vesicles is different. These results with 4-CMC also suggest that, if the SR is preloaded with Ca²⁺, inhibition of Ca²⁺-ATPase activity and Ca²⁺ uptake per se do not impair Ca²⁺ release, in either phase 1 or phase 2, at least under the conditions of our assay.

It has been known for some time that heavy metals, such as Cu²⁺, Hg²⁺, and Ag⁺, can induce a rapid Ca²⁺ release from Ca²⁺-loaded SR vesicles, which is related to their binding affinity for sulfhydryl groups (1). In the case of Ag⁺, it has been reported that the rate of Ca²⁺ release is five to six times higher in heavy SR vesicles compared with light SR vesicles and that Ca²⁺ release could be blocked by procaine, tetracaine, and RR, now well-known inhibitors of the CRC (31). Based on these results, it was argued that heavy metals and specifically Ag⁺ (AgNO₃) induce Ca²⁺ release from SR vesicles by interacting with the CRC and not the Ca²⁺-ATPase (31). In addition, single-channel activity measurements demonstrated the ability of Ag⁺ to directly activate the CRC (25).

However, in the study by Salama and Abramson (31), the light SR vesicles, in which the Ca²⁺-ATPase comprises 90% of the total protein (19), released 90-100% of the accumulated Ca²⁺ in the presence of AgNO₃. Moreover, the Ca²⁺-ATPase comprises 55-65% of total heavy SR vesicle protein (19). It is also well known that the SR Ca²⁺-ATPase contains 24 cysteine residues (3) and is highly susceptible to sulfhydryl oxidation (34, 43). Given these considerations, it is difficult to rule out the involvement of the Ca²⁺-ATPase in the AgNO₃-induced Ca²⁺ release response in SR vesicles.

The most compelling evidence to date that Ag⁺ can induce Ca²⁺ release by interaction with the Ca²⁺-ATPase comes from a study by Gould et al. (14) in which they showed a rapid Ca²⁺ efflux induced by Ag⁺ from reconstituted vesicles that contained the purified Ca²⁺-ATPase as the only protein. As we have shown for homogenates in the present study, they also showed that Ag⁺ greatly inhibited Ca²⁺-ATPase activity in both reconstituted vesicles and SR vesicles. Evidence from other cell types, such as liver (44) and HL-60 (36), suggests that heavy metals (including Ag⁺) induce Ca²⁺ release from intracellular stores mainly due to inhibition of the Ca²⁺-ATPase in conjunction with a direct effect on the CRC. In liver cells, both RR and tetracaine have no effect on Ca²⁺ efflux induced by heavy metals (44).

Our results strongly support a dual action of AgNO₃ for inducing Ca²⁺ release, at least from muscle homogenates prepared from rat skeletal muscle. Similar to HL-60 cells, it appears that AgNO₃ induces a rapid Ca²⁺ efflux from muscle homogenates by simultaneously interacting with both the CRC and the Ca²⁺-ATPase of the SR. The effects of RR on the AgNO₃-induced Ca²⁺ release response provides evidence that AgNO₃ directly activates the CRC. However, only phase 2 was inhibited with RR, and the extent of inhibition was only 65% compared with 100% for 4-CMC-induced Ca²⁺ release. This suggests that only part of the AgNO₃-induced Ca²⁺ release response can be explained by activation of the CRC. Nonetheless, the CRC does contribute to AgNO₃-induced Ca²⁺ release as expected, and recent work using single-channel analysis confirms that AgNO₃ directly activates both the skeletal muscle CRC isoform (ryanodine receptor 1) and the cardiac isoform (ryanodine receptor 2) (G. G. Du, personal communication).

In previous studies, it was shown that 4 mM DTT completely blocked AgNO₃-induced Ca²⁺ release from skeletal muscle samples (29, 41). In these studies, DTT, as opposed to RR, was used to validate the Ca²⁺ release assay using AgNO₃ and to demonstrate the specificity of AgNO₃ for the CRC. However, DTT is a sulfhydryl-reducing agent that is not specific for any particular protein (5), and we have shown that DTT can alter the effects of skeletal muscle ischemia on measurements of SR Ca²⁺-ATPase activity (39).

In HL-60 cells, it was concluded that the apparent Ca²⁺ release induced by AgNO₃ was mainly due to inhibition of the Ca²⁺ pump with increased permeability for Ca²⁺ (10). This mechanism cannot explain the rapid AgNO₃-induced Ca²⁺ release response observed in the present study. Even though AgNO₃ completely inhibited Ca²⁺-ATPase activity, the rate of Ca²⁺ leak due to Ca²⁺-ATPase inhibition alone that we observed only represented ~12% of the AgNO₃-induced rate of release in phase 2. Moreover, CPA reduced the AgNO₃-induced rate of release corresponding to phase 2 by 72% and, importantly, had no effect on 4-CMC-induced Ca²⁺ release. In the presence of lower CPA concentrations, the extent of inhibition of Ca²⁺ release for phase 2 was also lower. Because CPA can inhibit the Ca²⁺-ATPase in both directions (forward and reverse) (10, 11) but cannot reduce the leak of calcium ions through the Ca²⁺-ATPase passive channel (10), it would appear that AgNO₃ triggers the reversal of the Ca²⁺-ATPase and thus Ca²⁺ release through this pathway. Interestingly, 4-CMC also inhibited Ca²⁺-ATPase activity. However, because Ca²⁺ release (phase 2) was completely blocked when 4-CMC was combined with RR, inhibition of Ca²⁺-ATPase would appear to be specific for the forward direction only.

Gould et al. (14) proposed a similar mechanism in reconstituted vesicles, suggesting that the Ca²⁺ binding sites on the Ca²⁺-ATPase could remain in a low-affinity state and cycle between the luminal and cytoplasmic side of the SR membrane, thus releasing Ca²⁺ into the cytoplasm (14). Other studies have also shown that the Ca²⁺-ATPase can be reversed under various conditions and can synthesize ATP from ADP and P_i (7-9).

The nature of the Ca²⁺ release response for phase 1 induced by AgNO₃ is unclear from our data. We have found that various concentrations of CPA increased the rate of release by a similar extent. If phase 1 reflects Ca²⁺ release solely from the CRC, this finding would be expected, providing the rate of Ca²⁺ release simply reflects a balance between the rate of release and reuptake of Ca²⁺ during release. On the other hand, because AgNO₃ completely inhibits Ca²⁺-ATPase activity, the increase in phase 1 Ca²⁺ release in the presence of CPA is not due to reduced Ca²⁺ uptake during release. Moreover, RR had no effect on phase 1 Ca²⁺ release induced by AgNO₃, whereas phase 1 Ca²⁺ release induced by 4-CMC was inhibited by 59% with RR. Thus the CPA and RR results do not support a role for either the Ca²⁺-ATPase or the CRC for the early, rapid rate of Ca²⁺ release induced by AgNO₃.

The fact that 4-CMC-induced release corresponding to phase 1 was not completely blocked by RR is consistent with the known mechanism for RR blockade of the CRC. RR blocks the channel conduction pore of an open CRC but does not inactivate the CRC or prevent the CRC from opening (4, 22). Therefore, despite RR being present at the start of each assay, addition of 4-CMC would activate the channel and induce Ca²⁺ release until RR moved into the channel conduction pore to block further release of Ca²⁺. The question might be asked as to whether the effects of AgNO₃ might be different with whole muscle homogenates as used in this experiment and enriched SR fractions that contain primarily only the Ca²⁺-ATPase. Measurement of the Ca²⁺-ATPase in whole homogenates is highly specific as a result of the selective inhibition of other cellular ATPases (29, 35). Moreover, we have shown that Ca²⁺ release induced from enriched SR preparations demonstrates a similar biphasic release as whole homogenates and is affected by CPA in a similar manner (unpublished observations). These observations would suggest that the effects of AgNO₃ do not depend on the type of preparation.

The results from the present study suggest that 4-CMC is a much better agent to assess CRC function in vitro compared with AgNO₃. First, 4-CMC is a much more potent activator of the CRC than AgNO₃, as reflected by the different rates of release corresponding to phase 1 for the two chemicals. A similar result was reported previously (27). As well, 4-CMC has a higher affinity for the CRC than caffeine (17). Second, and most importantly, 4-CMC is specific for the CRC, as shown previously (17), whereas AgNO₃ affects both the Ca²⁺-ATPase and the CRC. There is also evidence that Ag⁺ binds to sulfhydryl groups of the dihydropyridine receptor (26) and enhances ryanodine contracture of the mouse diaphragm (21). Thus interpretation of data is complicated at best when AgNO₃ is used to assess Ca²⁺ release function.

Perspectives. There are several implications that can be drawn from the results of these experiments. One obvious implication is the selection of the releasing agent. For most experiments, a releasing agent that is highly sensitive and specific for the CRC is desirable. It would appear the 4-CMC meets these criteria. Moreover, experiments with CPA clearly indicate that neither phase of Ca²⁺ release rate is affected by inhibition of Ca²⁺-ATPase activity when the SR vesicles are loaded with Ca²⁺. Our results also suggest that both phase 1 and phase 2 are distinct entities and should be assessed routinely. Finally, our findings would appear to have important implications to the interpretations of acute exercise experiments when AgNO₃ is used as the CRC releasing agent. Exercise-induced inhibition of Ca²⁺-ATPase activity could effectively result in alterations of Ca²⁺ release (increase in phase 1 and decrease in phase 2), not only because of the inhibition of the pump per se, which would affect Ca²⁺ loading into SR, but because of the reversal of pump function caused by AgNO₃. In this regard, the effect would be similar to that observed when CPA is used in conjunction with AgNO₃. As with any in vitro preparation, the issue of physiological relevance is important. Our experiments were performed on homogenates and in highly contrived environments. Under these conditions, Ca²⁺ uptake and Ca²⁺ release rates occur over a much longer time frame than that observed in vivo. However, the value of in vitro models is that specific factors can be studied in isolation and the underlying mechanisms for any observed effects can be examined.