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
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ABSTRACT |
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
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INTRODUCTION |
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.
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MATERIALS AND METHODS |
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).
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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RESULTS |
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
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 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 ( 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.
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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. and  P < 0.05 and
P < 0.01, respectively.
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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.
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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.
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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.
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DISCUSSION |
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.
 |
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