Favero, Terence G., David Colter, Paul F. Hooper, and Jonathan J. Abramson. Hypochlorous acid inhibits Ca2+-ATPase from skeletal muscle sarcoplasmic reticulum. J. Appl. Physiol. 84(2): 425–430, 1998.—Hypochlorous acid (HOCl) is produced by polymorphonuclear leukocytes that migrate and adhere to endothelial cells as part of the inflammatory response to tissue injury. HOCl is an extremely toxic oxidant that can react with a variety of cellular components, and concentrations reaching 200 μM have been reported in some tissues. In this study, we show that HOCl interacts with the skeletal sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (ATPase), inhibiting transport function. HOCl inhibits sarcoplasmic reticulum Ca2+-ATPase activity in a concentration-dependent manner with a concentration required to inhibit ATPase activity by 50% of 170 μM and with complete inhibition of activity at 3 mM. A concomitant reduction in free sulfhydryl groups after HOCl treatment was observed, paralleling the inhibition of ATPase activity. It was also observed that HOCl inhibited the binding of the fluorescent probe fluorescein isothiocyanate to the ATPase protein, indicating some structural damage may have occurred. These findings suggest that the reactive oxygen species HOCl inhibits ATPase activity via a modification of sulfhydryl groups on the protein, supporting the contention that reactive oxygen species disrupt the normal Ca2+-handling kinetics in muscle cells.
- reactive oxygen species
skeletal muscle sarcoplasmic reticulum (SR) is an intramembranous structure that regulates intracellular free Ca2+. After muscle activation, the SR releases stored Ca2+, which diffuses into the myoplasm to activate cross-bridge cycling and subsequent force production. When intracellular Ca2+ is elevated, the SR Ca2+-adenosinetriphosphatase (ATPase) actively transports Ca2+back into the SR and muscle relaxation ensues. The 115-kDa Ca2+-stimulated Mg2+-dependent ATPase is the major SR protein that transports 2 mol of Ca2+ across the SR bilayer membrane with hydrolysis of 1 mol of ATP. The ATPase has been shown to contain 26 sulfhydryl (SH)-containing cysteine residues, 6 of which reside in a disulfide conformation, leaving 20 free SH groups (12). Reactive SH groups have been shown to be critical for pumping activity because oxidation or blocking of free SH groups with thiol- directed reagents leads to both a loss of ATPase activity and Ca2+ accumulation (12, 22). Thus the ATPase is sensitive to oxidants that can modify its function.
Reactive oxygen species (ROS) have drawn attention for their potential to disrupt normal muscle function by targeting specific proteins for modification. Molecular oxygen-derived intermediates are produced extensively in living tissues undergoing oxidative stress, such as ischemia, reperfusion, and vitamin deficiency (11). Reactive oxygen intermediates generated during repetitive muscle contraction have also been shown to promote muscle fatigue (23). These ROS include hydrogen peroxide (H2O2), singlet oxygen (1O2), hypochlorous acid (HOCl), the superoxide radical, and the hydroxyl radical (⋅ OH).
Several recent reports have indicated that ROS are produced in the active muscle cell (7, 23). Reid et al. (23) have shown that free radicals alter contractile parameters of fiber bundles isolated from rat diaphragm (23). In addition, myeloperoxidase (MPO), the enzyme responsible for HOCl production, is significantly elevated during and after submaximal exercise bouts (6). This growing body of work suggests that ROS are produced as a result of exercise and may be operative in skeletal muscle dysfunction associated with prolonged bouts of exercise.
The effect of ROS on skeletal muscle and sarcoplasmic reticulum function has been examined in a few studies. Peroxydisulfate and ⋅ OH have been shown to inhibit Ca2+ uptake into isolated SR vesicles while peroxydisulfate also increased the Ca2+ permeability of the SR membrane (24). In other work describing the effect of SH oxidizing reagents on SR Ca2+ release, H2O2and the highly reactive1O2were shown to stimulate Ca2+release from actively loaded SR vesicles and modify ryanodine binding to its receptor (9, 26). Similar observations have been reported in cardiac muscle (5). Ca2+ release induced by ROS was shown to be blocked by SH reducing agents and ruthenium red, indicating the SR Ca2+ release channel as a primary target for their interaction.
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). To begin to understand the emerging relationship between Ca2+regulation, ROS, and exercise, we evaluated the effects of the cellular oxidant HOCl on catalytic and functional activity of the SR Ca2+-ATPase.
MATERIALS AND METHODS
Preparation of SR vesicles.
For all studies, SR vesicles were prepared from rabbit hind leg and back white skeletal muscle according to the method of MacLennan (19). The protein concentration was determined by absorption spectroscopy (14).
HOCl treatment of SR.
HOCl is produced by the enzyme MPO, which is present in the granules of neutrophils. Previous documentation by Winterbourne (29) indicated that chemical analysis that used 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). Before all analyses, SR membranes were prepared and treated in the following manner: SR (100 μg/ml) were incubated for 10 min (22°C) with various final concentrations of HOCl (0.1, 0.2, 0.5, 1.0, and 3.0 mM).
A-23187-stimulated Ca2+-ATPase activity.
A-23187-stimulated Ca2+-ATPase activity was determined spectrophotometrically for untreated and HOCl-treated SR vesicle preparations by using a procedure previously documented (9, 18). (A-23187 is a Ca2+ ionophore.) The standard assay buffer contained 100 mM KCl, 20 mMN-2-hydroxyethylpiperazine-N′-2-ethanesulfonic acid (HEPES), 10 mM MgCl2, 1 mM ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid, 3 mM NADH, 1 mM phosphoenol pyruvate, 5 U lactate dehydrogenase, 5 U pyruvate kinase, and 0.5 mM Mg-ATP at pH 7.0 in a 1-ml volume. Ca2+-independent (Mg2+-dependent basal activity) 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 maximal Ca2+-stimulated activity, 8 μM Ca2+ (free) and the Ca2+ ionophore A-23187 (2 μg/ml) were added to the cuvette, and absorbance changes were monitored for 5 min or until the reaction was run to completion. A-23187 was added to remove enzyme back inhibition and to reduce the influence of potential alterations in membrane Ca2+permeability after the HOCl treatment.
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, 5 mM MgCl2, and 20 μM free Ca2+. Considering that net Ca2+ uptake is an equilibrium between Ca2+ entry into the vesicles and Ca2+ efflux through the release channel, the assays were conducted with 5 mM Mg2+ in the uptake medium. This is a concentration that is sufficient to inhibit oxidation-induced Ca2+ efflux through the release channel (1). 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 displays the data as a function of time and calculates the maximal Ca2+ uptake (nmol/mg).
ATPase and Ca2+ uptake recovery.
Restoration of Ca2+- ATPase activity with dithiothreitol (DTT) and glutathione (GSH) was measured as follows. Assays were conducted in the standard assay buffer as described in A-23187-stimulated Ca2+-ATPase activity measurements. SR (100 μg/ml) was treated with 3 mM HOCl for 10 min at room temperature. After treatment, 10 mM dimethyl sulfoxide (DMSO) was added to the incubation mixture to scavenge the remaining HOCl. The HOCl-treated SR was then added to the ATPase reaction buffer, which contained the reducing agents DTT or GSH (5 mM). The HOCl-treated SR was allowed to incubate in the reaction buffer for 2 min before the ATPase assay was initiated. The procedures for evaluating the recovery of Ca2+ uptake by DTT and GSH were performed in an identical manner. Note that neither the rate of ATPase activity nor the amount of Ca2+uptake was affected when the assays were conducted in the presence of up to 5% DMSO.
The SH content of the SR was estimated by measuring the increase in absorbance at 412 nm after reaction with 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) (15). SR (100 μg) was pretreated with various concentrations of HOCl as described inHOCl treatment of SR and subsequently diluted into the assay medium containing 50 mM tris(hydroxymethyl)aminomethane (pH 7.0), 1% sodium dodecyl sulfate (SDS), 1 mM EDTA, and 1 mM DTNB in a 1-ml cuvette. Absorbance changes were monitored for 8 min, a time point after which no additional absorbance changes could be observed. Free SH groups were calculated according to the Beers-Lambert relationship by using a molar extinction coefficient of 13,600.
Polyacrylamide gel electrophoresis (PAGE).
SDS-PAGE (5%) with 3% acrylamide in the stacking gel were prepared according to the method of Laemmli (16). SR vesicles (10 μg) were suspended in 0.03 ml of buffer containing 20 mM HEPES and 100 mM KCl, pH 7.0. These samples were incubated for 10 min with HOCl and then for 30 min in 50 μM fluorescein isothiocyanate (FITC). The samples were then solubilized with Laemmli sample buffer containing the reducing agent β-mercaptoethanol (5%) and were electrophoresed. The gels were stained overnight in 45% methanol, 10% acetic acid, and 0.10% Coomassie blue and were destained in an identical solution without Coomassie blue.
FITC labeling was conducted according to the procedures outlined by Luckin et al. (18). After HOCl treatment, SR fractions (10 μg in 30 ml) were incubated in buffer for 30 min at room temperature with 50 μM FITC. The reaction was stopped by the addition of sample buffer, and the proteins were electrophoresed. To visualize FITC-labeled proteins, the gels were fixed in 10% isopropanol for 1 h and then were photographed with ultraviolet (UV) illumination.
All reagents were analytic grade. HEPES and DTT were obtained from Research Organics (Cincinnati, OH). All other chemicals were obtained from Sigma Chemical.
The effect of HOCl treatment on Ca2+-stimulated ATPase activity is shown in Fig. 1. A significant decrease in ATPase activity was observed at increasing HOCl concentrations, with 170 μM required to inhibit ATPase activity by 50% (IC50).
Inhibition of Ca2+-ATPase activity resulted in a parallel reduction in the amount of Ca2+ accumulated by the SR (Fig.2). The reduction in Ca2+ uptake closely followed the concentration-dependent inhibition of ATPase activity, with an IC50 for Ca2+ uptake of 420 μM, a value higher than that observed for ATPase activity. Although this value is in the general range of the IC50 for ATPase activity, we believe the difference can be explained by the higher protein concentrations used in the Ca2+-uptake assays. To ensure the reduction in Ca2+ uptake was due to the loss of ATPase activity, control experiments were conducted to assess the effect of HOCl on SR Ca2+ permeability. After active Ca2+ loading, the SR vesicles were exposed to increasing concentrations of HOCl, and no increase in Ca2+ permeability was noted until HOCl reached 1.6 mM, a concentration far in excess of that required to significantly inhibit Ca2+-ATPase activity and uptake. Still, at 1.6 mM, the increase in Ca2+ permeability was very slow (in min) and most likely had little effect on SR Ca2+ uptake (data not shown).
Catalytic activity of the Ca2+-pump protein is critically dependent on free SH groups. Therefore, analysis of free SH groups was performed on SR membranes treated with HOCl. Almost 20 SH groups per ATPase molecule were measured before HOCl treatment, a value in close agreement with previously published data (Fig.3) (15, 22). After treatment with HOCl, we observed a concentration-dependent decline in the number of free SH groups per ATPase molecule with an IC50 of ∼400 μM. At 3 mM HOCl, seven free SH groups were unaffected by HOCl.
A decrease in the number of free SH groups that parallels inhibition of ATPase activity suggests that the SH groups may have been oxidized to disulfides by HOCl. Therefore, ATPase activity was measured after 3 mM HOCl exposure and subsequent incubation with the reducing agents DTT or GSH. As observed in Fig.4 A, treatment with HOCl completely inhibits ATPase activity, whereas subsequent incubation with either 5 mM DTT or 5 mM GSH resulted in a 55% recovery of the lost ATPase activity. Similarly, the loss of oxidant-induced Ca2+ uptake was partially reversed after treatment with both DTT and GSH (Fig.4 B). Control assays run in the presence and absence of DMSO or GSH/DTT did not alter the native activities of Ca2+ uptake or Ca2+-ATPase activity.
Mitchinson et al. (21) and Hidalgo et al. (13) have demonstrated that the FITC binding is localized to a specific lysine residue located in the adenine nucleotide binding site. To evaluate the possibility that HOCl-induced reduction in ATPase activity was due to modification of the adenine nucleotide binding site, we analyzed the binding of the fluorescent probe FITC to control and HOCl-treated SR vesicles. SR was treated with HOCl and subsequently exposed to FITC. Visualization of the bound ligand by UV illumination provides a qualitative measurement of the integrity of the adenine nucleotide binding site after exposure to the oxidant (21). In Fig. 5, we show the SDS-PAGE and accompanying UV illumination. Figure5 Ademonstrates that HOCl treatment does not significantly modify the protein concentration of the Ca2+-ATPase. However, UV illumination of the FITC-treated protein clearly shows a concentration-dependent decrease in fluorescence, and at 3 mM HOCl no fluorescence was detected (Fig. 5 B).
HOCl is a product of MPO, an enzyme present in the granules of polymorphonuclear leukocytes (PMN). This peroxidase catalyzes the conversion of H2O2and Cl− to HOCl, a molecule important in microbial action of neutrophils. As part of the inflammatory response to injury, PMN migrate and adhere to endothelial tissues. Once localized they can penetrate and invade the tissues. 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). PMN have been shown to be 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). Although the reason(s) for neutrophil mobilization resulting from exercise remains unresolved, it is clear MPO concentrations are elevated and thus may factor into acute changes in muscle function during prolonged exercise. In this study, we show that HOCl inhibits the skeletal SR Ca2+-ATPase, a result that will ultimately compromise myoplasmic Ca2+ regulation, a hallmark of muscle fatigue and a possible precursor to muscle damage (3).
The reduction in catalytic activity of the ATPase is consistent with reduction in free SH groups measured from SR vesicles. Several groups have shown the importance of SH groups in normal ATPase function (12,15). In fact, Kawakita and Yamashita (15) demonstrated that oxidation of only two thiols significantly inhibits ATPase and Ca2+ pumping activity of this enzyme. Our results indicate a qualitative agreement with this finding. The concentration of HOCl required to inhibit ATPase activity by 50% (IC50 = 170 μM HOCl; Fig. 1) would have oxidized approximately two thiol groups on the SR protein (Fig. 3). HOCl appears to oxidize free SH groups to disulfides, which disrupts enzyme activity. This is supported by the recovery experiments that used disulfide-reducing agents. Addition of either DTT or GSH after HOCl exposure resulted in a significant recovery of ATPase activity and Ca2+ uptake (Fig. 4,A andB). Although full restoration was not observed in the presence of the reducing agent, the partial recovery indicates that HOCl treatment causes disulfide formation. It is also likely that oxidation products that are formed are insensitive to reduction by reducing agents.
This work supports the results of Eley et al. (8), who perfused rat hearts with HOCl (100 μM) and observed both mechanical dysfunction and an inhibition in Ca2+-ATPase activity in isolated SR vesicles. In follow-up experiments, addition of DTT after a washout of HOCl induced a partial recovery of Ca2+-ATPase activity similar to that observed in Fig. 4. Their data suggest that HOCl is capable of penetrating cell membranes and targets SR proteins, inducing the type of damage we observe in our study.
Experiments have shown that depressions in SR ATPase activity and Ca2+ transport can be linked to conformational changes in the ATPase protein (13). Evaluation of ATPase activities after chronic stimulation and a prolonged bout of exercise has demonstrated that increased muscular activity is capable of altering the nucleotide binding site within the ATPase protein (17,18). The reduction in FITC binding after HOCl treatment, as visualized by UV illumination, suggests that a structural change has occurred in this region of the pump protein. Thus modification of SH groups present or near the nucleotide binding site or SH groups that determine the conformation of the binding site may be altered after exposure to HOCl.
Although cysteine appears to be the likely target for modification by HOCl, it may not be the only site of action. Other amino acids, such as histidine, tyrosine, and lysine, have been shown to interact with HOCl (27). However, considering that the ATPase protein represents ∼70% of the SR protein and its activity is critically dependent on SH group viability, it remains a likely candidate for attack by cellular oxidants. Second, cysteine has been shown to be 100 times more reactive to HOCl than any other amino acid (29).
Oxidation injury to cells has long been thought to be mediated by peroxidation of cell membranes and may be responsible in part for the inhibition in ATPase activity. Free radicals attack polyunsaturated fatty acids, a component of cell membranes producing hydroxy-conjugated dienes. These compounds undergo decomposition to several aldehyde products, the most common being malondialdehyde (MDA). Increases in plasma and skeletal muscle MDA levels have been noted after exhaustive exercise in rats (2, 7). However, reports have shown that disruption of cellular transport processes by oxidation may not always be associated with lipid peroxidation. Scherer and Deamer (24) were unable to correlate lipid peroxidation with oxidation-induced ATPase inhibition. In addition, Luckin et al. (18) found no difference in the rotational mobility of a lipophilic probe inserted into the bilayer membrane in SR vesicles isolated from exercised animals, whereas ATPase activity declined by 35%. This suggests that, whereas lipid peroxidation may have occurred as a result of exercise, the gross fluidity properties of the membrane appeared to be unchanged.
Attention is currently focused on proteins as the site of oxidative damage because proteins are more sensitive to oxidation and cellular damage appears to occur at lower levels of oxidative stress than does lipid peroxidation (10, 24). Because we did not measure lipid peroxidation as a function of decline in ATPase activity, we cannot totally rule it out as a contributing mechanism to the inhibition of ATPase activity. In addition, our ability to only partially restore ATPase activity after addition of reducing agents may be a result of modification of the supporting lipid membrane.
Comparisons of HOCl and H2O2have shown the latter to be much less reactive in protein and cellular damage. Our previous work with H2O2supported this contention because peroxide failed to inhibit ATPase activity at exposures up to 80 mM (3 min) (9). Although peroxide appears to be less reactive in the presence of iron, peroxide can generate highly toxic ⋅ OH, a species that has no physiological scavenger.
The effects of ROS on skeletal muscle function are receiving a great deal of attention. Recent reports suggest that several ROS are generated during reperfusion of ischemic muscle and during repetitive muscle contractions and exercise (7, 23). Evidence indicates that, regardless of the particular stress to the muscle cell, disruptions in its ability to effectively regulate Ca2+ may have dire consequences.
Ca2+-ATPase activity is obligatory for precise control of intracellular Ca2+, and regulation of free Ca2+ levels are required for maintaining structural integrity and functional viability in most cell types. This is especially true in skeletal muscle cells, where contraction and relaxation of myofibrillar proteins are critically dependent on Ca2+ concentrations. Intracellular free Ca2+ has been shown to rise during muscle fatigue induced by low-frequency electrical stimulation in single mouse fibers (28). Analysis of the SR Ca2+ pump after this fatigue protocol showed a twofold reduction in pumping capacity, a result that has also been observed after exhaustive exercise in animals (18).
Loss of Ca2+ homeostasis has been suggested to contribute to a decline in muscle function via several processes (3). With regard to metabolism, increases in resting free Ca2+ levels would stimulate glucose transport into skeletal muscle (30), inhibit glycogenolysis, stimulate pyruvate dehydrogenase, and depress mitochondrial function (20). Structural and morphological degradation after exercise has also been proposed to be the result of increases in the cytosolic Ca2+ concentration. Several Ca2+-dependent proteases have been found to be activated after exercise (3), and myofibrillar proteins have been degraded by lysosomal proteases (25).
The effects of oxidants on cellular and, in particular, muscle cell function are still being determined and described. Reperfusion of ischemic skeletal muscle and exercise-induced oxidative stress are two areas in which oxidant research may be fruitful. With regard to exercise, HOCl appears to induce modification of the SR Ca2+-ATPase, consistent with what is observed after prolonged exercise.
We acknowledge the support of the Medical Research Foundation of Oregon (to T. G. Favero), the Beta Beta Beta Undergraduate Research Fund (to P. F. Hooper), and the American Heart Association, Oregon Affiliate (to D. Colter).
Address for reprint requests: T. G. Favero, Dept. of Biology, Univ. of Portland, 5000 N. Willamette Blvd., Portland, OR 97203.
- Copyright © 1998 the American Physiological Society