INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

SKELETAL MUSCLE SARCOPLASMIC RETICULUM (SR) is an intramembranous structure that regulates intracellular free Ca²⁺. At rest, nanomolar Ca²⁺ is maintained by a 115-kDa Ca²⁺-stimulated Mg²⁺-dependent ATPase that actively transports Ca²⁺ into the SR. After depolarization, the SR releases stored Ca²⁺, which diffuses through a Ca²⁺ channel [ryanodine receptor (RyR) 1] into the myoplasm to activate cross-bridge cycling and subsequent force production. This process is referred to as excitation contraction coupling.

Oxygen free radicals have been shown to mediate various pathological conditions in a variety of tissues, including skeletal muscle (16). Free radicals are molecular oxygen-derived intermediates generated either by electron reduction or energy activation (i.e., light) and have also been referred to as reactive oxygen species (ROS). Among these ROS, hydrogen peroxide (H₂O₂), singlet oxygen, hypochlorous acid (HOCl), superoxide radical, and the hydroxyl radical are included. In addition, reactive nitrogen species have also been implicated in modifying muscle cell function (20). Many papers have detailed the effects of one or more ROS on various aspects of skeletal muscle function (3, 5, 14, 17, 26, 29). We have shown that Ca²⁺ release channels/RyR display redox sensitivity to both H₂O₂ (14) and singlet oxygen (27, 31). Both reagents stimulate Ca²⁺ release from actively loaded vesicles and stimulate the activity of single Ca²⁺ channel reconstituted into a bilayer lipid membrane. Oba et al. (24) demonstrated activation of channel activity in lipid bilayers at 10 µM when the channel was under a controlled redox potential. Still, work in more complete or intact systems has not always supported data derived from isolated SR experiments. Originally, Andrade et al. (3) reported biphasic effects in that H₂O₂ (100-300 µM) increased force generation without any change in myoplasmic Ca²⁺, and longer exposures reduced force generation that was independent of myoplasmic Ca²⁺. Recently, they have shown that low concentrations (10¹⁰ to 10⁵ M) decreased mean tetanic Ca²⁺ and increased force by ~10% (4). Others have suggested that contractile proteins are sensitive to hydroxyl radical and superoxide but not to H₂O₂ (5). This is not surprising when the millimolar concentrations of H₂O₂ that were required to produce an effect in SR vesicle experiments are considered.

Prolonged exercise increases production of ROS (2, 8) and frequently elicits muscle fatigue and damage (13, 18). Unlike the ROS mentioned above, HOCl is not likely to be produced in muscle during rest or during contraction. Myeloperoxidase catalyzes the chlorination of H₂O₂ to produce HOCl in neutrophils. As part of the inflammatory response to injury, neutrophils migrate and adhere to endothelial tissues. HOCl and other neutrophil-generated ROS are intended to clear away damaged tissue and have been noted to increase dramatically after ischemia and reperfusion of both cardiac and skeletal muscle. Still, HOCl is likely to cross the sarcolemma and has been shown to be 10-20 times more effective in oxidizing proteins (15). HOCl is an extremely toxic oxidant that can react with a variety of cellular components, and its concentration has been reported to reach 200 µM in some tissues (10). Polymorphonuclear cells are released as a result of exercise with a concomitant elevation in MPO. After continuous bouts of exercise at 45, 60, and 75% of maximal O₂ uptake, plasma levels of MPO were raised almost twofold at each intensity level (6). A recent study demonstrated that exercise induced peripheral neutrophilia with an increase in the oxidative activity within 30 min during a single bout of exercise and peaked within 1-3 h postexercise (29). Although the reason(s) for neutrophil mobilization resulting from exercise remains unresolved, it is clear that MPO concentrations are elevated and thus may factor into acute changes in muscle function during prolonged exercise.

In our previous work, we showed that H₂O₂ activated the RyR at millimolar concentrations of H₂O₂ but did not modify the activity of the SR Ca²⁺ ATPase up to concentrations of 80 mM (14). In a subsequent paper (12), our laboratory demonstrated that HOCl inhibited the SR Ca²⁺ ATPase at concentrations in the micromolar range and that the modification of enzymatic activity was due to an oxidation of thiol groups located on the ATPase. Thus it was our hypothesis that HOCl will produce similar effects on the SR Ca²⁺ release channel/RyR to those observed with H₂O₂, but at more physically relevant concentrations.

	METHODS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

Preparation of SR vesicles. For all studies, SR vesicles were prepared from rabbit hindleg and back white skeletal muscle according to the method of MacLennan (22). The protein concentration was determined by absorption spectroscopy (19).

HOCl treatment of SR. HOCl is produced by the enzyme MPO, which is present in granules of neutrophils. Previous documentation by Winterbourne (30) indicated that the commercial product sodium hypochlorite yielded results identical to those obtained with HOCl produced by the MPO system. Thus, for our assays, all concentrations of HOCl were generated by diluting from stock concentrations of sodium hypochlorite (6.75 M; Sigma Chemical, St. Louis, MO).

Measurement of Ca²+ uptake. Ca²⁺ fluxes across SR vesicles were monitored by using a Ca²⁺-selective electrode interfaced to an IBM XT computer (1). The standard procedure was as follows. Ca²⁺ uptake into SR vesicles (0.2 mg/ml) was carried out in a buffer containing 100 mM KCl, 20 mM HEPES, 1 mM MgCl2, and 20 µM free Ca²⁺. Uptake was initiated by the addition of 0.5 mM Mg²⁺-ATP. Extravesicular Ca²⁺ concentration was recorded (10 Hz) as a function of time and stored in the computer. An analysis program displayed the data as a function of time and calculated the maximal Ca²⁺ uptake (in nmol/mg).

ATPase activity. Ca²⁺-independent, Ca²⁺-dependent, and A23187-stimulated Ca²⁺ ATPase activities were determined spectrophotometrically for untreated and HOCl-treated SR vesicle preparations (14). The standard assay buffer contained (in mM) 100 KCl, 20 HEPES, 1 MgCl, 1 EGTA, 3 NADH, 1 phosphoenol pyruvate, 0.5 Mg-ATP, 5 units of lactate dehydrogenase, and 5 units of pyruvate kinase at pH 7.0 in a 1-ml volume. This assay was carried out in a stepwise fashion. Ca2+-independent (Mg²⁺-dependent) ATPase activity was initiated by the addition of SR (0.1 mg) to the cuvette, and the absorbance changes at 340 nm were recorded for 2 min (22°C). To measure Ca²⁺-stimulated activity, Ca²⁺ was added to reach 8 µM (free), and again during absorbance five changes were monitored for 3 min. The Ca²⁺ ionophore A-23187 was added to prevent back inhibition that results from overloading of vesicles with Ca²⁺, and the reaction was run to completion. SR (1.0 mg/ml) was incubated at several different HOCl concentrations for 1 min before the assays were conducted. Ca²⁺ uptake and Ca²⁺ ATPase assays were carried out in identical buffers. The data are the average of three independent experiments and are expressed as a percentage of the control.

[³H]Ryanodine binding. The use of ryanodine, a plant alkaloid, has significantly enhanced our understanding of SR Ca²⁺-channel function. Ryanodine binds with nanomolar affinity to open SR Ca²⁺ channels (25). With few exceptions, compounds that stimulate Ca²⁺ release via opening of the Ca²⁺ channel [Ca²⁺ (µM), ATP, caffeine] stimulate the binding of ryanodine to its receptor, whereas compounds that inhibit Ca²⁺ release by closing the Ca²⁺ channel [Ca²⁺ (mM), Mg²⁺, ruthenium red] inhibit ryanodine binding. Briefly, SR membranes (500 µg/ml) were incubated at 37°C for 3 h in a medium containing 250 mM KCl, 15 mM NaCl, 15 nM [³H]ryanodine, and 20 mM PIPES, pH 7.1. The binding reaction was quenched by rapid filtration through a Whatman GF/B glass fiber filter, which was then rinsed with 5 ml of ice-cold buffer. The filters were placed in polytubes, filled with 2.5 ml of scintillation cocktail, shaken overnight, and counted the next day. The experiments were repeated at least five times on two different SR preparations, with essentially identical results. Nonspecific binding was measured in the presence of a 100-fold excess of unlabeled ryanodine and subtracted before specific binding was calculated. Details of individual experiments are described in the figure captions.

Initial rates of [³H]ryanodine binding. The initial rate of [³H]ryanodine binding was determined from time-dependent measurements at 3, 6, 9, and 12 min at 37°C. SR at 1.0 mg/ml was pretreated with 0.1-0.3 mM GSH for 10 min at room temperature in binding buffer containing (in mM) 250 KCl, 15 NaCl, and 20 PIPES, pH 7.1. The time-dependent reaction was initiated by dilution into an equal volume of binding buffer containing 8 nM [³H]ryanodine, 50 µM free Ca²⁺ (buffered with EGTA), and various concentrations of HOCl. For Ca²⁺-dependent measurements, Ca²⁺ was buffered with 50 µM EGTA to a free Ca²⁺ concentration as was calculated by WinMaxc. The binding reaction was quenched by rapid filtration through Whatman GF/B filters mounted on a 48-well Brandel Cell Harvester. Filters were rinsed twice with binding buffer containing 50 µM Ca²⁺. Scintillation vials were filled with scintillation fluid, shaken overnight, and counted the next day. The initial binding rate was calculated from a linear regression fit of four time-dependent measurements of bound ryanodine.

Single Ca²+-channel analysis. Single Ca²⁺ release channel activity was recorded and analyzed after reconstitution of a SR vesicle into a bilayer lipid membrane, as described previously (31). Free Ca²⁺ concentration was 3-5 µM, and experiments were carried out at 22°C. In a typical experiment, after channel reconstitution, 20 µM HOCl was added to the cis chamber, stirred, and the resultant channel activity was recorded for at least 1 min. Subsequently, 20 µM HOCl was added to the cis chamber, and activity was recorded again. For analysis, the data were passed through a Krohn-Hite low-pass filter (model 3202) at 1.5 kHz, digitized with a Scientific Solutions analog-to-digital converter, and analyzed by using the pCLAMP software package (version 5.0, Axon Instruments, Burlingame, CA).

Flourescent labeling of hyperreactive thiols with (7-diethylphenyl)-2-methylcourmarin. The fluorogenic sulfhydryl probe (7-diethylphenyl)-2-methylcourmarin (CPM) was used to evaluate the effects of HOCl on hyperreactive thiols on the RyR protein. CPM (100 µM) dissolved in DMSO was stored at 20°C. The maximum volume of DMSO added to SR membranes was limited to 0.5% (vol/vol) of the assay buffer to ensure that the solvent did not interfere with fluorescence. With the use of a 3-ml quartz cuvette, 100 µg/ml SR was added to a buffer containing (in mM) 250 KCl, 20 PIPES, and 15 NaCl. To simulate the closed state of the RyR, 1 mM CaCl₂ was added to the cuvette. The contents of the cuvette were constantly stirred. Fluorescence was measured by using a spectrofluorometer (SML 8000; SML Instrument, Urbana, IL) and was interfaced with an IBM computer recording system. Excitation and emission wavelengths were set at 397 and 465 nm (slit 16 nm), respectively. CPM was then added to give a final CPM concentration of 80 nM. For HOCl treatments, the cuvette containing SR, buffer, and Ca²⁺ were treated for 1 min with various concentrations of HOCl (30, 100, and 300 µM) at room temperature. CPM was then added as stated above, and fluorescence for all HOCl and SR experiments was measured over 360 s. CPM did not interact with the HOCl concentration. Fluorescence was expressed as counts per second vs. time in seconds. Raw data were fit to an exponential, and the rate constant was derived.

Materials. All reagents were analytical grade. HEPES was obtained from Research Organics (Cincinnati, OH). [³H]ryanodine was purchased from New England Nuclear. CPM was obtained from Molecular Probes. All other chemicals were obtained from Sigma Chemical.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

SR Ca²+ release. In Fig. 1, it is demonstrated that HOCl directly stimulates Ca²⁺ release from actively loaded SR vesicles. Ca²⁺ was released at concentrations of HOCl as low as 25 µM. The maximal release rate derived from the kinetic evaluation was 12.91 ± 0.51 nmol · mg¹ · min¹, whereas the EC₅₀ was 483 ± 12.8 µM. HOCl, at increasing concentrations, also increased the amount of Ca²⁺ release (Fig. 1). DMSO was utilized in an attempt to scavenge HOCl, and, at sufficiently high concentrations, DMSO inhibited HOCl-induced release (Fig. 2). When Ca²⁺ release was measured in the presence of the Ca²⁺ channel inhibitor ruthenium red (20 µM), no release of Ca²⁺ was observed with up to 2 mM HOCl (data not shown). The ability to block Ca²⁺ release with a specific channel inhibitor suggests that HOCl was affecting the Ca²⁺ release mechanism of SR and was not causing an increase in the SR Ca²⁺ permeability by nonspecific effects, such as lipid peroxidation.

View larger version (18K): [in this window] [in a new window]   Fig. 1.   Hypochlorous acid (HOCl) stimulated Ca2+ release. Sarcoplasmic reticulum (SR) vesicles at 0.2 mg/ml were actively loaded, as described in METHODS. On completion of Ca2+ uptake (free Ca2+ = 1-2 µM), release was initiated by adding the indicated concentration of HOCl. Ca2+ release rate () was calculated from the initial slope of the free Ca2+ vs. time. Ca2+ release amount () was calculated by subtracting the total free Ca2+ after release from the Ca2+ concentration at the end of uptake. Data are means of 4 independent experiments.

View larger version (9K): [in this window] [in a new window]   Fig. 2.   HOCl-stimulated Ca2+ release is inhibited by DMSO. SR vesicles at 0.2 mg/ml were actively loaded, as described in METHODS. On completion of Ca2+ uptake (free Ca2+ concentration = 1-2 µM), HOCl (concentration of 400 µM) was added to induce release. DMSO was added at various concentrations before HOCl-stimulated release was carried out. Ca2+-release rate was calculated from the initial slope of free Ca2+ concentration vs. time after the addition of HOCl. Data are means ± SD of 5 independent experiments.

Ca²+ transport and ATPase activity. In our laboratory's previous work (12), our laboratory demonstrated that HOCl at micromolar concentrations inhibited both ATPase activity and active accumulation of Ca²⁺ by the SR. Because those experiments only evaluated Ca²⁺ uptake (i.e., with a closed release channel) and ATPase activity, they were conducted with high levels of Mg²⁺ (5 mM) and longer incubation times of 10 min. In these experiments, to demonstrate that the initial rates of release were due to HOCl activation of the RYR, our laboratory conducted additional transport and ATPase studies (Fig. 3) at lower Mg²⁺ with incubation times of 1 min. The ATPase studies were conducted in the presence of A-23187, Ca²⁺-ionophore to ensure that maximal catalytic activity was not prone to "back inhibition" of the pump or stimulation of ATPase activity via opening of the RyR. Ca²⁺ uptake declined significantly up to a concentration of 200 µM. Conversely, A-23187-stimulated ATPase activity did not begin to decline until concentrations of 200 µM HOCl, which demonstrated the different sensitivities of the Ca²⁺ uptake and Ca²⁺ release processes to HOCl.

View larger version (12K): [in this window] [in a new window]   Fig. 3.   Effect of HOCl on A-23187-stimulated ATPase activity and the rate of Ca2+ uptake. SR (1 mg/ml) was incubated at different HOCl concentrations for 1 min before the ATPase and uptake assays were conducted. The final assay conditions for the uptake experiments were SR = 0.2 mg/ml; buffer (in mM): 100 KCl and 20 HEPES at pH 7.0, 1 Mg2+, 0.5 Mg-ATP. For ATPase experiments, all concentrations were similar except SR concentrations (0.1 mg/ml). Data are the averages of 3 independent experiments and are expressed as a percentage of control. , In the presence of A23187-stimulated ATPase activity = 1.2 µmol · mg1 · min1; , Ca2+ uptake (control = 69.1 nmol/mg).

[³H]Ryanodine binding studies. A more direct assay to monitor the interaction between the RyR and HOCl can be obtained through the use of the highly specific Ca²⁺ channel probe, ryanodine. Similar to what was observed with H₂O₂, HOCl caused a biphasic HOCl and SR interaction between [³H]ryanodine and its receptor (Fig. 4). [³H]Ryanodine binding was stimulated above 10 µM HOCl. Maximal receptor occupancy was observed at 100 µM. At concentrations >100 µM, binding of [³H]ryanodine to its receptor was inhibited. Lower binding was detected at 1 and 3 mM HOCl.

View larger version (10K): [in this window] [in a new window]   Fig. 4.   HOCl-stimulated high-affinity [3H]ryanodine binding in a concentration-dependent manner. [3H]ryanodine binding was carried out as follows. SR membranes (0.5 mg/ml) were incubated at 30°C for 3 h in a medium containing 250 mM KCl, 15 mM NaCl, 15 nM [3H]ryanodine, and 20 mM PIPES, pH 7.1. The assay buffer contained 50 µM Ca2+ (free) and various amounts of HOCl. The binding was quenched by rapid filtration. The filters were rinsed twice with 5 ml of buffer and counted. The data shown are the average of representative experiments performed in triplicate on different SR preparations. 100% bound = 1.02 pmol/mg SR protein.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   Effect of HOCl on time-dependent high-affinity [3H]ryanodine binding. [3H]ryanodine binding was carried out as follows. SR membranes (0.5 mg/ml) were incubated at 30°C in a medium containing 250 mM KCl, 15 mM NaCl, 15 nM [3H]ryanodine, and 20 mM PIPES, pH 7.1. The amount of bound [3H]ryanodine was determined at various time points while SR vesicles were exposure to 0 (), 30 (), or 300 () µM HOCl. The assay buffer contained 50 µM Ca2+ (free). The binding was quenched by rapid filtration. The filters were rinsed twice with 5 ml of buffer and counted. Data shown are the average of representative experiments performed in duplicate.

View larger version (12K): [in this window] [in a new window]   Fig. 6.   Initial rate of HOCl-stimulated high-affinity [3H]ryanodine binding in a concentration-dependant manner. [3H]ryanodine binding was carried out as follows. SR membranes (0.5 mg/ml) were incubated at 30°C over a time period of 0-12 min in a medium containing 250 mM KCl, 15 mM NaCl, 15 nM [3H]ryanodine, and 20 mM PIPES, pH 7.1. [3H]ryanodine binding was measured at 0, 3, 6, and 12 min at various concentrations of HOCl. The assay buffer contained 50 µM Ca2+ (free). The binding was quenched by rapid filtration. Data shown are the average of representative experiments performed in triplicate.

View larger version (13K): [in this window] [in a new window]   Fig. 7.   Effect of HOCl on Ca2+ dependence of ryanodine binding to SR vesicles. Ryanodine binding was carried out in a Ca2+-dependent manner over the range of 10 nM to 3 mM free Ca2+. Free Ca2+ concentrations were obtained by titrating Ca2+ with various amounts of chelating agent EGTA. Influence of HOCl was examined by adding 0 (), 30 (), and 300 () µM HOCl to the reaction medium. Data shown are results of 4 independent experiments. Typical error values between experiments were <5%.

View larger version (17K): [in this window] [in a new window]   Fig. 8.   The effects of DTT on HOCl-stimulated [3H]ryanodine binding. SR vesicles were incubated with 40 µM HOCl for 10 min, followed by the addition of either DMSO or DTT. Subsequently, [3H]ryanodine was added, and aliquots were assayed for binding until the indicated time when binding was terminated by rapid filtration.

Single-channel measurements. Stimulation of single-channel activity of a reconstituted SR Ca²⁺ release channel after exposure to micromolar concentrations of HOCl was observed in Fig. 9. At 20 µM HOCl, the open-state probability increased from 0.45 to 0.96, which almost locked the channel in an open state. When the concentration was increased to 40 µM, HOCl subsequently reduced the open-state probability to 0.01 This biphasic behavior confirms our observations of HOCl activation and inhibition of ryanodine binding.

View larger version (42K): [in this window] [in a new window]   Fig. 9.   HOCl modification of Ca2+ release channel activity. After fusion of a SR vesicle to a bilayer lipid membrane, the cis chamber was perfused with standard buffer (500 mM CsCl, no added Ca2+, free Ca2+ = 3-5 µM), and current was recorded as a function of time. A: control. B: 20 µM HOCl was added to the cis side. C: HOCl concentration was increased to 40 µM. Open-state probabilities (Po) for traces A-C are as follows: A, Po = 0.45; B, Po = 0.96; C, Po = 0.01. The time for each trace was 500 ms/trace. Current traces were recorded at a holding potential of +25 mV with respect to the trans side (ground) of the bilayer. These observations were made in 5 independent bilayer experiments. O, channel open; C, channel closed.

Hyperreactive thiols. RyR protein has been shown to have 101 cysteine residues per subunit (23), which can be partitioned into three classes of thiols based on the basis of their susceptibility to redox modification (28). Several of these cysteines have been classified as hyperreactive thiols and are suggested to be essential for normal channel function (21). The accessibility of the hyperreactive thiols is dependent on the conformational state of the RyR. When the channel is in the closed state, the hyperreactive thiols are accessible and can be labeled through the use of a fluorogenic sulfhydryl probe (CPM). To determine whether HOCl was oxidizing hyperreactive thiols, we exposed SR to various concentrations of HOCl before CPM was bound. Figure 10 demonstrates a concentration-dependent decrease in the rate of fluorescence development. This indicates that either hyperreactive thiols are oxidized by HOCl or, as in the case when the channel is in the "open state," hyperreactive thiols are no longer accessible to alkylation by CPM.

View larger version (28K): [in this window] [in a new window]   Fig. 10.   Effects of HOCl on ryanodine receptor hyperreactive thiols with the use of the fluorogenic sulfhydryl probe (7-diethylphenyl)-2-methylcourmarin (CPM). SR vesicles (100 µg/ml) were exposed to various concentrations (30, 100, and 300 µM) of HOCl for 1 min in a buffer containing (in mM) 250 KCl, 15 NaCl, 20 PIPES, pH 7.1. SR vesicles were induced into a closed-state conformation by the addition of 1 mM Ca2+ (control) and then exposed to HOCl. The development of fluorescence was then recorded as a function of time. CPM was added in 10 µl aliquots to yield a final concentration of 80 nM (in 3 ml). Fluorescence is expressed as CPM counts per second vs. time (in s). The raw data were fitted to an exponential curve, and the rate constant was derived from that fit.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

It is now clear that the RyR-channel function can be altered by a wide variety of oxidants. Our laboratory's previous work with H₂O₂ and RyR function demonstrated that oxidation of sulfhydryl groups promotes activation of the receptor. However, the concentrations required to induce activation were most likely unphysiological. In contrast, we demonstrate in this paper that HOCl, a strong oxidant derived from H₂O₂, produces similar effects at significantly lower concentrations. More importantly, we demonstrate that HOCl oxidizes critical thiols and stimulates the RyR protein.

The effects of HOCl on RyR function are almost identical to those observed with H₂O₂. Both oxidants stimulated Ca²⁺ release from actively loaded vesicles, induced a biphasic (stimulation followed by inhibition) effect on ryanodine binding, reduced the EC₅₀ for Ca²⁺ activation of the receptor, and are inhibited by reducing agents. The primary difference between the two sets of studies is that HOCl produced these effects on SR vesicles at much lower concentrations than peroxide.

Qualitatively similar effects were also observed at the level of single-channel analysis. HOCl activated the channel activity at 20 µM, a concentration consistent with activation of ryanodine binding. However, we observed inhibition at 40 µM, a concentration much lower than inhibition observed in the binding experiments. This enhanced sensitivity of the channel to various compounds is consistent with observations using H₂O₂ (14, 24) and singlet oxygen (31). It is possible that fusing of a SR vesicle to a bilayer lipid membrane modifies the RyR protein in some manner that enhances its sensitivity to channel activators and inhibitors.

Our data also demonstrate both time- and concentration-dependent effects. Lower concentrations of HOCl (<100 µM) activate ryanodine binding. Higher concentrations of HOCl (>300 µM) initially activate and with increasing time subsequently inhibit RyR (Fig. 5). Very high concentrations (10 mM) appear to only inhibit the RyR as demonstrated in Fig. 6. However, Ca²⁺ release is still stimulated by 12 mM concentrations of HOCl (Fig. 1). Unfortunately, the time frame over which ryanodine binding is measured does not permit acute and rapid time-dependent resolution (<1 min) when very high concentrations of HOCl are used. The time-dependent effects of higher concentrations of HOCl indicate distinct activation and inhibition sites in which the activation site is more sensitive to oxidation than the inhibition site. When it is considered that it is likely that HOCl rises from nanomolar to micromolar concentrations in the cell, activation of the channel would likely be observed before inhibition if the concentrations rise to a sufficiently high level. Neither the single-channel experiments nor the ryanodine binding experiments provide precise data that allows us to accurately predict the effects of HOCl under more physiological or in vivo conditions. However, the data do indicate that SR Ca²⁺ handling proteins can be modified by HOCl in a manner similar to other ROS.

Another difference noted was that HOCl also inhibited the SR Ca²⁺-ATPase, whereas H₂O₂ did not. This is not surprising because it has been previously published that HOCl interacts with the ATPase protein from both skeletal (12) and cardiac SR (10, 11). These studies indicated that SR Ca²⁺ ATPase was shown to have been inactivated by oxidation of thiol groups on the ATPase protein and that functionality could be restored via the application of the reducing agent DTT. However, it is clear from Fig. 3 that the RyR protein is more sensitive to oxidation than the ATPase protein. After a 1-min exposure to 200 µM HOCl, Ca²⁺ uptake declines dramatically to 10% of control. At this concentration, the A-23187-stimulated Ca²⁺-ATPase activity was unaffected.

In our laboratory's previous work, we suggested that the oxygen radical targets free SH groups on the RyR protein (14). To further understand the nature of the interaction between HOCl and RyR, we assayed for the reactivity with a fluorogenic probe to determine the accessibility of hyperreactive thiols. Exposure of SR vesicles to increasing concentrations of HOCl reduced the amount of CPM fluorescence, which possibly indicates that the hyperreactive thiol groups were targets of oxidation.

It is likely the thiol groups oxidized in our studies are similar to those described by Sun et al. (28). They demonstrated that oxidation of up to 10 SH groups does not modify RyR activity. Oxidation of an additional 10-15 SH groups reversibly stimulates RyR activity. Oxidation induced by HOCl up to 40 µM is reversed by the addition of DTT as demonstrated in Fig. 8. This work demonstrates that thiols critical for normal channel function were targets of HOCl-induced oxidation.

Although these data appear to suggest that HOCl might prove a better candidate than H₂O₂ for oxidation-induced damage to SR proteins, it is a difficult conclusion to make given the track record of H₂O₂. Our laboratory previously demonstrated biphasic effects on RyR function by using millimolar concentrations of H₂O₂ (14). It was also reported that H₂O₂ was without effect on caffeine-induced Ca²⁺ release from the SR of chemically skinned diaphragm muscle fibers (7). Yet the recent work of Andrade et al. (4) showed that exposure to physiologically relevant concentrations of H₂O₂ (10¹⁰ to 10⁶ M) modifies myofibrillar function independent of SR Ca²⁺ handling, and concentrations as low as 10 µM will increase resting Ca²⁺ and slow Ca²⁺ uptake.

Still, support exists that HOCl may modify SR and/or other muscle protein function via oxidation. Eley et al. (10) demonstrated that HOCl causes a rise in resting Ca²⁺ by inducing release from the SR. Exposure to DTT caused a rapid restoration of steady-state Ca²⁺ and Ca²⁺ transient amplitude in rabbit ventricular myocytes (11), leading them to conclude that these effects occur through the alteration in protein thiol redox status. In saponin-permeabilized skeletal muscle fibers, exposure to 20 µM HOCl inhibited caffeine-induced Ca²⁺ release (7).

This study and others provide strong evidence that SR proteins, particularly the RyR, are sensitive to oxidative damage. Still, it is not known to what extent oxidation plays in normal physiological muscle function during rest, contraction, or after contractile activity. Because HOCl would likely be generated extracellularly and during the latter stages of or after a bout of exercise, it is highly unlikely to participate on a time course similar to either superoxide or H₂O₂. Continued study should help elucidate the role of ROS, including HOCl, in reperfusion of ischemic muscle, vitamin E deficiency, muscular dystrophy, and other important pathological conditions that demonstrate cytotoxic damage (9).