H2O2 and ethanol act synergistically to gate ryanodine receptor/calcium-release channel

Toshiharu Oba1, Tatsuya Ishikawa2, Takashi Murayama3, Yasuo Ogawa3, and Mamoru Yamaguchi4

Departments of 1 Physiology and 2 Pediatrics, Nagoya City University Medical School, Nagoya 467-8601; 3 Department of Pharmacology, Juntendo University School of Medicine, Tokyo 113-8421, Japan; and 4 Department of Veterinary Biosciences, College of Veterinary Medicine, The Ohio State University, Columbus, Ohio 43210


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We examined the effect of low concentrations of H2O2 on the Ca2+-release channel/ryanodine receptor (RyR) to determine if H2O2 plays a physiological role in skeletal muscle function. Sarcoplasmic reticulum vesicles from frog skeletal muscle and type 1 RyRs (RyR1) purified from rabbit skeletal muscle were incorporated into lipid bilayers. Channel activity of the frog RyR was not affected by application of 4.4 mM (0.02%) ethanol. Open probability (Po) of such ethanol-treated RyR channels was markedly increased on subsequent addition of 10 µM H2O2. Increase of H2O2 to 100 µM caused a further increase in channel activity. Application of 4.4 mM ethanol to 10 µM H2O2-treated RyRs activated channel activity. Exposure to 10 or 100 µM H2O2 alone, however, failed to increase Po. Synergistic action of ethanol and H2O2 was also observed on the purified RyR1 channel, which was free from FK506 binding protein (FKBP12). H2O2 at 100-500 µM had no effect on purified channel activity. Application of FKBP12 to the purified RyR1 drastically decreased channel activity but did not alter the effects of ethanol and H2O2. These results suggest that H2O2 may play a pathophysiological, but probably not a physiological, role by directly acting on skeletal muscle RyRs in the presence of ethanol.

12-kiloDalton FK506-binding protein; skeletal muscle sarcoplasmic reticulum; acetaldehyde; single-channel current


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

STRENUOUS EXERCISE PRODUCES reactive oxygen species (ROS), including hydroxyl radicals, superoxide anion, and H2O2 (9, 13, 33, 36), and frequently elicits skeletal muscle fatigue followed by muscle damage (32, 35). The recent observation that ROS is produced in vivo during contraction of cat skeletal muscle before fatigue and damage (30) suggests that ROS function as a trigger for muscle dysfunction. However, the mechanism(s) by which ROS bring about muscle dysfunction still remains unclear. H2O2 has been reported to cause a transient twitch potentiation in cardiac and skeletal muscles (14, 26). H2O2 at concentrations of 1 mM or more also releases Ca2+ from sarcoplasmic reticulum (SR) vesicles and increases open probability (Po) of the Ca2+-release channel/ryanodine receptor (RyR) incorporated into planar lipid bilayers (5, 25, 26, 39). Using much lower concentrations of H2O2 (0.1-0.2 mM), Favero et al. (11) reported an increase in Po in the Ca2+-release channel from rabbit skeletal muscle SR. More recently, however, Andrade et al. (3) reported that brief exposure of mouse hindlimb muscle to 0.1-0.3 mM H2O2 does not alter intracellular Ca2+ concentration during submaximal tetani. This discrepancy remains unresolved. It is of interest to reevaluate whether low concentrations of H2O2 can activate the Ca2+-release channel and whether H2O2 plays physiological or pathophysiological roles in muscle function.

Acute intoxication due to alcohol consumption produces reversible skeletal muscle dysfunction (acute alcoholic myopathy) (2, 15, 31, 37). The mechanism underlying ethanol-induced alterations of muscle function remains unknown, but a remarkable increase in intracellular Ca2+ concentration due to disturbance of Ca2+ homeostasis would result in muscle dysfunction. The Ca2+-induced Ca2+ release is potentiated by exposure of the SR in rabbit and frog skeletal muscle to ethanol, although ethanol alone does not induce the release of Ca2+ (27, 29). When SR vesicles were incorporated into lipid bilayers, ethanol at a concentration as low as 2.2 mM markedly increased Po of the Ca2+-release channel that had been activated by pretreatment with 2 mM caffeine (27). Mean open time of such channels was prolonged, and mean closed time was shortened by application of ethanol without change in single-channel conductance. Therefore, a site of action of ethanol might be expected to be on the Ca2+-release channel in the SR. However, the SR vesicles contain endogenous modulating proteins including FK506-binding protein (FKBP12), triadin, and calsequestrin (22, 28, 41). In addition, acetaldehyde may be produced via oxidation of ethanol by H2O2. These endogenous substances may modulate the action of ethanol and H2O2 on the Ca2+-release channel. It is also important to elucidate if binding sites of ethanol and H2O2 are on the RyR molecule by using purified RyR. In the present study, we demonstrate that H2O2, at low concentrations (10-100 µM), activates the Ca2+-release channel by acting synergistically with ethanol. This action was independent of the presence or the absence of FKBP12.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Heavy SR vesicle preparation from frog and rabbit skeletal muscles. A heavy fraction of SR vesicles enriched in terminal cisterna (HSR) was prepared from the leg muscles of Rana catesbeiana as described previously (16). HSR (20-25 mg/ml) was suspended in a small amount of 100 mM KCl, 20 mM Tris-maleate (pH 6.8), 20 µM CaCl2, and 0.3 M sucrose. RyR channel activity of SR vesicles from frog skeletal muscle shows two distinct Ca dependencies (7). We used Ca2+-release channels that were blocked by application of 1 mM cis Ca2+ in the present study, as in our previous paper (27). Type 1 RyR (RyR1) was purified from rabbit back muscle by sucrose gradients and Mono-Q anion exchange column chromatography (24). Preparations were rapidly frozen in liquid nitrogen and stored at -80°C until use. Our previous study shows that purified RyR1 was free from FKBP12 but retained the ability to bind FKBP12 (23).

Planar lipid bilayer experiments. Single-channel recordings were carried out by incorporating frog HSR vesicles or purified RyR1 channels into planar lipid bilayers according to our previous method (23, 25, 26). Lipid bilayers consisting of a mixture of L-alpha -phosphatidylethanolamine, L-alpha -phosphatidyl-L-serine, and L-alpha -phosphatidylcholine (5:3:2 wt/wt) in n-decane (40 mg/ml) were formed across a hole of 250 µm in diameter in a polystyrene partition separating cis and trans chambers. The cis (1 ml)/trans (1.5 ml) solutions consisted of 250/50 mM CsCH3SO3 and 10 mM CsOH (pH 7.4 adjusted by HEPES) for HSR experiments and 500/50 mM KCl, 20 mM HEPES-Tris (pH 7.4), and 0.1 mM CaCl2 for RyR1 experiments. HSR vesicles or RyR1 channels were added to the cis chamber. After confirming the channel incorporation by the occurrence of flickering currents, further incorporation of channels was prevented by adding an aliquot of 2.2 M CsCH3SO3 (pH 7.4 by HEPES) or 3 M KCl (pH 7.4 by HEPES-Tris) to the trans compartment. The trans side was held at ground potential, and the cis side was clamped at -40 mV using 1.5% agar bridges in 3 M KCl and Ag-AgCl electrodes. The cytoplasmic surface of the RyR almost faced the cis side, as determined by application of EGTA to the cis chamber (23). Experiments were carried out at room temperature (18-22°C).

Single-channel currents were amplified by a patch-clamp amplifier (Axopatch 1D, Axon Instrument) filtered at 1 kHz using an eight-pole low-path Bessel filter (model 900, Frequency Devices) and then digitized at 5 kHz for analysis. Data were saved on the hard disk of an IBM personal computer. Mean Po and lifetime of open and closed events of the Ca2+-release channel from records of >2 min were calculated by 50% threshold analysis using pClamp (version 6.0.4, Axon Instrument) software.

The results are presented as means ± SE. Statistical analysis was done with the ANOVA followed by Fisher's least-significant difference method. Values of P < 0.05 were regarded as statistically significant.

Chemicals. H2O2 (31% stock solution; Mitsubishi Gas Chemical, Tokyo, Japan) and ethanol (99.5%; Wako Pure Chemical, Osaka, Japan) were diluted to appropriate concentrations with ultrapure water (Barnstead, Boston, MA) immediately before application to cis solution. Ryanodine (10 mM stock solution; Wako) and ruthenium red (1 mM stock solution; Sigma, St. Louis, MO) were dissolved in ethanol and ultrapure water, respectively, and stored at -20°C. Acetaldehyde (98% stock solution; Sigma) was dissolved in ultrapure water just before use. Other reagents were of analytic grade. Recombinant human FK506-binding protein (FKBP12) was kindly supplied by Fujisawa Pharmaceutical (Osaka, Japan).


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INTRODUCTION
MATERIALS AND METHODS
RESULTS
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REFERENCES

Synergistic action of ethanol and H2O2 on channel activation of the RyR in frog skeletal muscle. When the frog RyRs were incorporated into planar lipid bilayers, channel activity in 1 µM cis Ca2+ (Po = 0.055) was not affected by application of 4.4 mM (0.02%) ethanol to the cytoplasmic side (Po = 0.049) (Fig. 1). Exposure of ethanol-treated channels to 10 µM H2O2 markedly increased Po to 0.293. Increase in H2O2 concentration to 100 µM caused a further increase in Po to 0.395. In the channel, the effect of 10 µM H2O2 on open times was examined further by analyzing the open time distributions. Time constants of the mean open lifetime were tau O1 = 0.51 ms (a relative area; 86%) and tau O2 = 2.52 ms (14%) in controls and tau O1 = 0.48 ms (85%) and tau O2 = 2.11 ms (15%) after addition of 4.4 mM ethanol (Fig. 2, A and B). Addition of 10 µM H2O2 to the channel that has been exposed to ethanol elicited a new, third time constant of 8.93 ms (5.5%) in addition to two time constants (tau O1 = 0.62 ms and tau O2 = 3.11 ms) similar to those for the ethanol treatment (Fig. 2C). When similar experiments were repeated, channels with such a long open time constant were observed in three of six preparations. Closed time constants in each group were best fit by three exponentials. When 10 µM H2O2 was added in the presence of 4.4 mM ethanol (Fig. 2C), the relative area of the shortest closed time constant,tau C1, was increased and that of the longest,tau C3, was decreased. Results for six different channels are summarized in Table 1. Numbers of open events (36 vs. 38/s), mean open time (1.6 vs. 1.9 ms), and mean closed time (20.6 vs. 21.9 ms) were similar between control and ethanol-treated channels. The increase in Po by exposure of ethanol-treated channels to 10 µM H2O2 (3.28-fold, from 0.083 ± 0.012 in ethanol to 0.272 ± 0.076, P < 0.05) was due to a 1.7-fold increase in numbers of open events, a 1.6-fold increase in mean open time, and a 23% decrease in mean closed time. Increase in H2O2 concentration from 10 to 100 µM caused an increase in Po to 0.371 ± 0.095 (P < 0.01 from ethanol-treated) with a further increase in numbers of open events and mean open time and a decrease in mean closed time (Table 1). All of these channels were open-locked at a subconductance level by 10 µM ryanodine and closed by subsequent application of 5 µM ruthenium red, as depicted in Fig. 1, D and E.


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Fig. 1.   Effect of ethanol and H2O2 on single Ca2+-release channel activity in frog heavy fraction sarcoplasmic reticulum (HSR). A: control channel activity in cis pCa 6.0. B: channel activity during application of 4.4 mM ethanol. C: channel activity in the presence of 10 and 100 µM H2O2. H2O2 was cumulatively added to cis chamber in the presence of ethanol. D: long-lasting subconductance open state in response to 10 µM ryanodine. E: complete blockade by 5 µM ruthenium red. Each open probability (Po) is indicated at right. Closed level of the channel is shown as a short line to the right of each current trace. Calibrations, 20 pA and 100 ms.



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Fig. 2.   Effects of ethanol and H2O2 on open and closed time distribution of single Ca2+-release channel activity. Open and closed time constants were calculated from corresponding data in Fig. 1. Data were gathered to analyze time constants for more than 2 min. Numbers for each trace indicate 2 or 3 (tau O1,tau O2, or tau O3) open and 3 (tau C1,tau C2, or tau C3) closed time constants and their relative areas as %. A, control; B: 4.4 mM EtOH; C: 4.4 mM EtOH + 10 µm H2O2.


                              
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Table 1.   Synergistic action of H2O2 and ethanol on the Ca2+-release channel in frog sarcoplasmic reticulum

Exposure to 100 µM H2O2 did not increase Po (0.068 ± 0.008, n = 5). Subsequent application of 4.4 mM ethanol to such 100 µM H2O2-treated channels again produced a marked increase in Po to 0.414 ± 0.066. Such activation of the channel was inhibited by exposure to 100 µM dithiothreitol (Po = 0.056 ± 0.012). An example of such experiments is depicted in Fig. 3. The increase in the Po was due to an increase in numbers of open events from 44/s in H2O2 to 137/s after ethanol addition and to a remarkable decrease in mean closed time from 13.54 ± 4.67 to 3.32 ± 0.51 ms. In the presence of 100 µM H2O2, ethanol produced an increase in mean open time (1.47 ± 0.41 ms in H2O2 to 2.80 ± 0.67 ms). Addition of 10 µM H2O2 alone to the Ca2+-release channel in frog SRs failed to increase Po (Po = 0.068 ± 0.016 in controls and 0.112 ± 0.055 in H2O2, n = 11, P > 0.05), as expected from above results. Again, channels treated with such low concentration of H2O2 were markedly activated to Po = 0.270 ± 0.061 in response to subsequent exposure to 4.4 mM ethanol (Fig. 4). The Po data summarized in Fig. 4 clearly indicate that ethanol and H2O2, irrespective of order of application, only when simultaneously added, synergistically activate the frog Ca2+-release channel.


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Fig. 3.   Effect of H2O2 and ethanol on single Ca2+-release channel activity in frog HSR. A: control channel activity in pCa 6.5. B: channel activity during application of 100 µM H2O2. C: channel activity during subsequent addition of 4.4 mM ethanol in the presence of 100 µM H2O2. D: inhibition of channel activity after subsequent addition of 100 µM dithiothreitol to the cis side of the chamber. See Fig. 1 legend for other notations.



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Fig. 4.   Effects of H2O2 or ethanol alone and their simultaneous addition on Po in frog Ca2+-release channel. H2O2 at 10 or 100 µM and 4.4 mM ethanol, if applied alone, failed to activate the Ca2+-release channel. When exposed to 10 or 100 µM H2O2 in the presence of 4.4 mM ethanol or oppositely when ethanol was added after treatment with H2O2, channel activity was significantly increased. star  and star star P < 0.05 and P < 0.01, respectively.

Effect of H2O2 and ethanol on the purified RyR1 channel free from FKBP12. The HSR vesicles used here bind with FKBP12, but the purified RyR1 was free from FKBP12 (23). Therefore, it is not clear whether the site(s) of action of ethanol and H2O2 is on the Ca2+-release channel itself, FKBP12, or some sites associated with interaction between the channel and FKBP12. To elucidate this issue, we examined the effect of ethanol and H2O2 on the Ca2+-release channel/RyR1 with or without FKBP12.

The FKBP12-free rabbit RyR1 channel failed to increase Po on addition of 4.4 mM ethanol (Fig. 5, A and B, Po = 0.020 in control in pCa 7 and after ethanol), consistent with results for frog HSR (Fig. 1) and previous observations [Fig. 5 in Oba et al. (27)]. Application of 100 µM H2O2 to such an ethanol-treated channel increased channel activity 4.4-fold to Po = 0.088 (Fig. 5C). This channel was also sensitive to ryanodine and ruthenium red (Fig. 5, D and E). Similar experiments were repeated using six different RyR1 channels. The increase in Po after addition of 100 µM H2O2 (Po = 0.037 ± 0.011 in ethanol to Po = 0.083 ± 0.009) was attributable to increases in numbers of open events (25 ± 11/s in ethanol to 35 ± 8/s) and mean open time (1.64 ± 0.31 ms in ethanol to 2.01 ± 0.73 ms) and a decrease in mean closed time (43.87 ± 19.91 ms in ethanol to 24.23 ± 10.30 ms). On the other hand, if added alone, H2O2 over a range of 100-500 µM affected no single-channel activity of the purified RyR1 (Po = 0.045 ± 0.010 in control to Po = 0.048 ± 0.017, 0.023 ± 0.005, and 0.057 ± 0.020 at 100, 200, and 500 µM H2O2, respectively, n = 9). Numbers of open events and mean open and closed times were similar among each group. In the presence of 500 µM H2O2, subsequent addition of 4.4 mM ethanol to the channel increased Po 3.2-fold, from 0.057 ± 0.020 in H2O2 to 0.181 ± 0.071 (P < 0.01).


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Fig. 5.   Effects of exposure of the single purified type 1 ryanodine receptor (RyR1)/Ca2+-release channel treated with or without FK506 binding protein (FKBP12) to ethanol and H2O2. Left, results for a FKBP12-free channel in pCa 7; right, results for a 1-µM FKBP-treated channel in pCa 5. A: control channel activity. B: effects of application of 4.4 mM ethanol to the FKBP12-free and FKBP-treated channels, respectively. C: activated channel activity during subsequent addition of 100 µM H2O2 in the presence of 4.4 mM ethanol. Each chemical was cumulatively added to cis chamber. Each Po is indicated at right side of corresponding traces. D: long-lasting subconductance open state in response to 10 µM ryanodine. E: complete blockade by 5 µM ruthenium red. See Fig. 1 legend for other notations.

Effect of H2O2 and ethanol on the FKBP12-bound RyR1 channel. When 1 µM FKBP12 was added to the cis chamber, RyR1 single-channel activity was inhibited drastically (Po = 0.217 ± 0.011 before FKBP12 treatment to Po = 0.068 ± 0.017, n = 5, P < 0.01). Five similar experiments were conducted; a sudden drop of Po with some delay time on addition of FKBP12 was observed in two of the five channels examined, whereas with three channels, the Po decreased gradually. These results suggest rebinding of FKBP12 to the channel protein. With the FKBP12-bound channel, numbers of open events (117 ± 19/s in controls to 42 ± 20/s) and mean open time (2.0 ± 0.6 ms in controls to 1.2 ± 0.1 ms) decreased, whereas mean closed time increased (5.9 ± 2.0 ms in controls to 22.9 ± 11.3 ms).

As shown in Fig. 5, right, the FKBP12-bound RyR1 failed to activate the channel on addition of 4.4 mM ethanol (Po = 0.031 in controls to 0.027). Ethanol did not affect numbers of open events or mean open and closed times, similar to results for FKBP-free channels. The ethanol-treated channel increased Po 5.0-fold, from 0.027 in ethanol to 0.136 after subsequent addition of 100 µM H2O2 (Fig. 5C, right). The results obtained with five different channels indicate that the H2O2-induced increase in Po (0.068 ± 0.017 in FKBP12 and 0.073 ± 0.044 after 4.4 mM to 0.181 ± 0.051) was due to an increase in numbers of open events (52 ± 33/s in ethanol to 86 ± 34/s) and a decrease in mean closed time (21.09 ± 7.04 ms in ethanol to 12.81 ± 5.89 ms). These channels were sensitive to ryanodine and ruthenium red, as shown in Fig. 5, D and E, right.

On the other hand, addition of 100 µM H2O2 to the Ca2+-release channel in the presence of 1 µM FKBP12 failed to alter the channel activity (Po = 0.052 in FKBP12 to Po = 0.038 as shown in Fig. 6). Similar results were obtained with three different channels. This was consistent with results observed with FKBP12-free channels.


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Fig. 6.   No effect of H2O2 on activity of a FKBP12-bound channel prepared from rabbit skeletal muscles. A: control channel activity. B: an inhibitory effect of 1 µM FKBP12. C: no channel activation after exposure to 100 µM H2O2. D: long-lasting subconductance open state in response to 10 µM ryanodine. E: complete blockade by 5 µM ruthenium red. See Fig. 1 legend for other notations.

Effect of acetaldehyde and H2O2 on the FKBP12-free purified RyR1 channel. Ethanol may be oxidized by an oxidant, H2O2, to produce acetaldehyde, a metabolic product of ethanol. If this were the case, the possibility could not be eliminated that acetaldehyde, not ethanol, activates the RyR1 channel by reacting together with H2O2 on the channel molecule. This issue was examined using the FKBP12-free purified RyR1 channel. Exposure of the cis side of the channel to 1 mM acetaldehyde reduced the channel activity in four of six preparations used (Po = 0.056 ± 0.021 in control to 0.023 ± 0.003 after acetaldehyde application, P < 0.05, n = 6). Subsequent addition of 100 or 500 µM H2O2, however, did not alter the channel activity (Po = 0.019 ± 0.006 or Po = 0.026 ± 0.006, respectively). Ethanol application to such channels increased the Po significantly to 0.081 ± 0.020 (P < 0.05), equivalent to the result obtained in the purified RyR1 channel (Fig. 5). The instance in which acetaldehyde showed the most dramatic inhibitory effect on the channel activity is depicted in Fig. 7. As shown in Fig. 7, A and B, acetaldehyde reduced the Po by decreasing the number of open events (35.1 ± 13.8/s in control to 11.7 ± 1.5/s) and by increasing the mean closed time (28.02 ± 9.44 ms in control to 49.62 ± 8.75 ms) without any effect on the mean open time (1.56 ± 0.21 ms in control to 1.70 ± 0.29). A 3.1-fold increase in the Po observed after exposure of the 500-µM H2O2-treated channel to 4.4 mM ethanol was due to an increase in the number of open events (13.3 ± 3.4 to 29.3 ± 6.5/s) and to a decrease in the mean closed time (57.31 ± 15.66 to 27.55 ± 6.19 ms) with no effect on the mean open time (1.84 ± 0.39 to 2.34 ± 0.67 ms) (see Fig. 7, C and D).


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Fig. 7.   Inhibition of purified FKBP12-free RyR1 channel by acetaldehyde. A: control channel activity. B: an inhibitory effect of 1 mM acetaldehyde. C: no channel activation after exposure of the acetaldehyde-treated channel to 100 or 500 µM H2O2 (Po = 0.031 and 0.039, respectively). D: activated channel activity after subsequent addition of 4.4 mM ethanol in the presence of 500 µM H2O2. E: long-lasting subconductance open state in response to 10 µM ryanodine. F: complete blockade by 5 µM ruthenium red. See Fig. 1 legend for other notations.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present results are the first to report that exposure of the skeletal muscle RyR to H2O2 at <10 µM activates the channel by acting together with ethanol. Such concentrations of H2O2 may correspond to physiological concentrations produced during exercise or in response to ischemic reperfusion. This study also provides evidence that H2O2 may pathophysiologically, but not physiologically, function as a Ca2+-release channel activator in vivo and that such H2O2-induced activation occurs regardless of the presence of FKBP12. These conclusions are based on observations that application of 10 µM H2O2 to the Ca2+-release channel in the frog skeletal HSR markedly activates the channel by acting synergistically with a low concentration of ethanol (4.4 mM or 0.02%) and that ethanol-treated RyR1 purified from rabbit skeletal muscle also is sensitive to H2O2 at a concentration similar to frog RyRs, independent of the presence of FKBP12. Previous reports have indicated that H2O2 at millimolar concentrations elicits the release of Ca2+ from the SR and increases Po of skeletal and cardiac RyRs (5, 25, 26, 39). On the basis of these observations, we previously concluded that H2O2 may cause oxidation of sulfhydryl groups on the Ca2+-release channels to release Ca2+ and in turn make fibers susceptible to damage (26). This study, however, demonstrates that H2O2 at concentrations <500 µM does not activate the purified RyR1 channel, unless ethanol is present also. This is consistent with a recent observation that brief exposure of mouse skeletal muscle to 100-300 µM H2O2 does not alter intracellular Ca2+ concentration during submaximal tetani (3), in addition to previous observations (5, 25, 26, 39). If added alone, considerable amounts of H2O2 are required to activate the Ca2+-release channel of the skeletal muscle (25, 26, and this study). One report, however, demonstrated that as little as 100 µM H2O2 alone activates the channel by oxidizing sulfhydryl groups in the skeletal muscle RyR (11). The discrepancy between this result of Abramson's group (11) and our finding remains to be resolved but may be due to different redox states of the channel molecules used for the experiments. Recently, multiple populations of RyR1 channel activity have been reported to depend on redox states with SR vesicles and purified molecules prepared from skeletal muscle, with activation by oxidants and inhibition by reductants (20, 23). Figure 10 of Favero et al. (11) shows an extremely high Po (0.85) on exposure to 100 µM H2O2. On the other hand, the Po in the present study was only 0.371 in frog SR (Fig. 4 and Table 1) and 0.181 in purified rabbit RyR1 with FKBP12, even in the presence of both 100 µM H2O2 and ethanol. Our channels, therefore, may be in more reduced state than those of Abramson's group (11). The discrepancy may be resolved by elucidating these issues with channel molecules in different redox states. Reportedly, sustained twitch tension in skeletal muscle, as occurs in strenuous exercise (repeated tetanus), could give rise to H2O2 production and accumulation (30), but physiological concentrations of H2O2 will not exceed several micromoles (19). Furthermore, free radical scavengers, such as catalase and superoxide dismutase and glutathione, occur endogenously in muscle cells (35). Our recent and present studies show that reduction of the Ca2+-release channel by dithiothreitol, a specific reducing reagent, inhibits channel activity (Ref. 23 and Fig. 3 in this study). Similar results have been observed by Marengo et al. (20). The present study, therefore, strongly suggests that H2O2 alone may not be able to function physiologically to produce skeletal muscle dysfunction, even if strenuous exercise elicits a sudden increase in H2O2 formation and maintains the relatively high intracellular concentration. This conclusion must await precise determination of intracellular H2O2 concentrations during and just after strenuous exercise and experiments under more physiological conditions.

H2O2 acts as an oxidant (26) and in turn may produce acetaldehyde, an ethanol metabolite. If so, the synergistic action of H2O2 and ethanol may be elicited by acetaldehyde in the presence of H2O2. This possibility, however, was excluded from observations that 1 mM acetaldehyde inhibited the purified RyR1 channel activity (Po = 0.056 in control to 0.023, n = 6, see Fig. 7) and that subsequent exposure of such channels to 100 µM H2O2 did not alter the channel activity. In addition, reapplication of 4.4 mM ethanol elicited a significant increase in the Po to 0.081 (see Fig. 7D). This activation by ethanol was similar to the response induced by ethanol and H2O2 without acetaldehyde. These effects strongly suggest that the synergistic action of H2O2 and ethanol on RyR1 channel activation is not due to oxidation of ethanol by H2O2. There is, to our knowledge, no report on the effect of acetadehyde on the RyR1 channel, and the inhibitory action of 1 mM acetaldehyde observed here is the first report. Ren et al. (34) recently reported that acetaldehyde inhibited myocardial contraction and caffeine-induced Ca2+ release. If a similar effect is observed between cardiac and skeletal RyRs, the inhibitory action of acetaldehyde observed here may explain, in part, the mechanism underlying acetaldehyde-induced inhibition of Ca2+ release from myocardial SR.

Here, ethanol alone had no effect on the Ca2+-release channel (Figs. 1, 4, and 5). This is consistent with our previous results that ethanol over a range of 2.2 (0.01%) to 217 mM (1%) does not increase Po of the Ca2+-release channel of frog skeletal RyRs incorporated into lipid bilayers (27). The ethanol concentration used here (4.4 mM or 0.02%) is almost equivalent to blood concentration of persons who have drunk 350 ml of 5% alcohol-containing beer (unpublished data: 0.020 ± 0.003%, 0.019 ± 0.003%, 0.017 ± 0.002%, 0.016 ± 0.002%, and <0.010% at 3, 10, 20, 30 min, and 1 h after drinking, respectively, n = 7). When pedaling exercise was performed at 60% maximal O2 consumption for 30 min after drinking, plasma myoglobin level and plasma creatine kinase activity were not altered during, just after, and 3 h after exercise, compared with control values obtained by the same persons before drinking. Nobody was considered drunk while pedaling, because blood alcohol levels were so low. These observations, therefore, indicate that such a low concentration of ethanol would not contribute to skeletal muscle dysfunction.

It would be very interesting to elucidate the mechanism by which ethanol markedly enhances the action of H2O2 on the Ca2+-release channel or the binding site(s) involved. Ethanol has been reported to potentiate the Ca2+-induced Ca2+ release mechanism in rabbit and frog HSRs when Ca2+ and caffeine were used as activators for the channel (27, 29). However, these studies do not explain whether the sites of action of ethanol and H2O2 are just on the Ca2+-release channel molecule, because HSR preparations contain many endogenous modulators of the channel, such as FKBP12, calsequestrin, and triadin (22). FKBP12 would be more important as a modulator for the Ca2+-release channel, although calsequestrin and triadin have been reported to alter activity of the Ca2+-release channel (12, 18, 28). Po and mean open time of the Ca2+-release channel in skeletal HSR are increased by treatment with FK506 to remove FKBP12 (3, 21). The channel activity is decreased after readdition of FKBP12 to the FBKP12-free channel (Refs. 4, 8, and Fig. 6 in this study). FKBP12 also may play a vital role in enabling the voltage sensors on the dihydropyridine receptor in the transverse-tubular membrane to activate the Ca2+-release channel (17). Therefore, FKBP12 is expected to be associated with physiological excitation-contraction coupling in skeletal muscle. Previous studies show that FKBP12 is removed from the Ca2+-release channel during purification (23, 40). Our previous data also show that the purified RyR retains the ability to bind FKBP12 (23). To determine if this modulatory protein alters channel gating kinetics after exposure of the ethanol-treated RyR to H2O2, we examined the effect of FKBP12 on synergistic action of this free radical and ethanol by using the FKBP12-free channel. In the present study, addition of 1 µM FKBP12 to the purified RyR1 channel typically caused marked decreases in Po and mean open time and an increase in mean closed time, consistent with previous results (4, 8). The FKBP12-bound channel was activated by an increase in cis Ca2+ concentration, but Po did not increase after treatment with 4.4 mM ethanol (0.068 to 0.073) or with 100 µM H2O2 in the absence of ethanol (Fig. 6), similar to data in frog HSR (Fig. 1 and Table 1). In addition to results in the FKBP12-free channel, our observation that subsequent application of 100 µM H2O2 to the ethanol-treated RyR1 led to channel activation suggests that a binding site(s) of this ROS is on the Ca2+-release channel itself. Furthermore, the presence of FKBP12 is not indispensable for the synergistic action of H2O2 and ethanol. This is a new finding in this study. We have reported that H2O2 is accessible from the luminal side of the Ca2+-release channel in frog RyR (25). As catalase was added to the cytoplasmic side in that experiment, luminal H2O2 could not function on both the cytoplasmic surface of the channel molecule and FKBP12. These results support our above conclusion on the binding site(s) of H2O2. H2O2 has the ability to oxidize the sulfhydryl group of cysteine and the Ca2+-release channel in frog HSRs (26). More recently, Eager and Dulhunty (10) demonstrate that there is at least one cysteine on the luminal side of the RyR to activate the channel in sheep heart. However, it still remains unknown if the cysteine residue for the channel activation observed here is just the same as they postulated.

Our single-channel observation that ethanol did not affect unitary conductance of the Ca2+-release channel suggests that ethanol is unlikely to enlarge the size of channel pore and in turn to increase the ionic flow through the pore when the gate opened. It would be very interesting to determine the site of action of ethanol on the RyR. However, we still cannot explain why ethanol, even at the low concentrations (2-4 mM) used previously (26) and here potentiates the Ca2+-induced Ca2+ release mechanism and markedly activates channel activity of skeletal RyRs from frog and rabbit on caffeine or H2O2 treatment. One of the acute effects of ethanol on the cell membrane includes an increase in membrane fluidity (38). If increased fluidity of the SR membrane is also produced in response to ethanol treatment, such membrane alteration may make the Ca2+-release channel easy to open. The observation that ethanol alone has no effect suggests there is little alteration of membrane structure on treatment with ethanol at the concentrations used here. Acute ethanol toxicity has been shown to cause membrane depolarization, and the effect is mediated by a specific inhibition of Na+-K+-ATPase (6). Thus ethanol may attack the protein molecule directly to change its tertiary structure. Further research will be required to elucidate which actions of ethanol are involved in potentiation of the Ca2+-induced Ca2+ release mechanism in the RyRs.

In conclusion, ingestion immediately before or during exercise of alcohol, even at a low amount, brings about a marked increase in Po of the RyR channel in skeletal muscle by acting with H2O2 at near-physiological concentration. This may be pathophysiologically very meaningful. Exercise-induced repetitive muscle contractions can release large amounts of Ca2+ from the SR and produce ROS. When drunk before or during strenuous exercise, alcohol markedly potentiates the Ca2+-induced Ca2+-release mechanism activated by Ca2+ and ROS such as H2O2. This would result in an unusual increase in the cytoplasmic concentration of Ca2+. This phenomenon may be associated with clinical cases where exercise causes serious muscle dysfunction in patients with chronic alcohol myopathy.


    ACKNOWLEDGEMENTS

This work was supported by grants-in-aid for Scientific Research (B)(2) (#09470012) and partially by grants-in-aid for Research in Nagoya City University, Japan (to T. Oba), and partially by grants-in-aid from the American Heart Association (Central Ohio Heart Chapter) and from the Muscular Dystrophy Association of America (to M. Yamaguchi).


    FOOTNOTES

Address for reprint requests and other correspondence: T. Oba, Dept. of Physiology, Nagoya City Univ. Medical School, Mizuho-ku, Nagoya 467-8601, Japan (E-mail: tooba{at}med.nagoya-cu.ac.jp).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 20 December 1999; accepted in final form 17 May 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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