INTRODUCTION

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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 (P_o) 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 P_o 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 P_o 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

TOP 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--phosphatidylethanolamine, L--phosphatidyl-L-serine, and L--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).

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).

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION 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⁺ (P_o = 0.055) was not affected by application of 4.4 mM (0.02%) ethanol to the cytoplasmic side (P_o = 0.049) (Fig. 1). Exposure of ethanol-treated channels to 10 µM H₂O₂ markedly increased P_o to 0.293. Increase in H₂O₂ concentration to 100 µM caused a further increase in P_o 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 _O1 = 0.51 ms (a relative area; 86%) and _O2 = 2.52 ms (14%) in controls and _O1 = 0.48 ms (85%) and _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 (_O1 = 0.62 ms and _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,_C1, was increased and that of the longest,_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 P_o 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 P_o 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.

View larger version (46K): [in this window] [in a new window]   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.

View larger version (37K): [in this window] [in a new window]   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 (O1,O2, or O3) open and 3 (C1,C2, or C3) closed time constants and their relative areas as %. A, control; B: 4.4 mM EtOH; C: 4.4 mM EtOH + 10 µm H2O2.

View larger version (34K): [in this window] [in a new window]   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.

View larger version (19K): [in this window] [in a new window]   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.  and 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.

View larger version (45K): [in this window] [in a new window]   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 (P_o = 0.217 ± 0.011 before FKBP12 treatment to P_o = 0.068 ± 0.017, n = 5, P < 0.01). Five similar experiments were conducted; a sudden drop of P_o with some delay time on addition of FKBP12 was observed in two of the five channels examined, whereas with three channels, the P_o 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).

View larger version (32K): [in this window] [in a new window]   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 (P_o = 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 (P_o = 0.019 ± 0.006 or P_o = 0.026 ± 0.006, respectively). Ethanol application to such channels increased the P_o 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 P_o 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 P_o 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).

View larger version (47K): [in this window] [in a new window]   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

TOP 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 P_o 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 P_o (0.85) on exposure to 100 µM H₂O₂. On the other hand, the P_o 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 (P_o = 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 P_o 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 P_o 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). P_o 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 P_o 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 P_o 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 P_o 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.