Sulfhydryls associated with H2O2-induced
channel activation are on luminal side of ryanodine receptors
Toshiharu
Oba1,
Tatsuya
Ishikawa2, and
Mamoru
Yamaguchi3
Departments of 1 Physiology and
2 Pediatrics, Nagoya City University Medical
School, Mizuho-ku, Nagoya 467, Japan; and
3 Department of Veterinary Bioscience, Ohio
State University, Columbus, Ohio 43210
 |
ABSTRACT |
The mechanism underlying H2O2-induced
activation of frog skeletal muscle ryanodine receptors was studied
using skinned fibers and by measuring single Ca2+-release
channel current. Exposure of skinned fibers to 3-10 mM H2O2 elicited spontaneous contractures.
H2O2 at 1 mM potentiated caffeine contracture.
When the Ca2+-release channels were incorporated into lipid
bilayers, open probability (Po) and open time
constants were increased on intraluminal addition of
H2O2 in the presence of cis catalase,
but unitary conductance and reversal potential were not affected.
Exposure to cis H2O2 at 1.5 mM failed
to activate the channel in the presence of trans catalase.
Application of 1.5 mM H2O2 to the trans
side of a channel that had been oxidized by cis
p-chloromercuriphenylsulfonic acid (pCMPS; 50 µM) still led to an
increase in Po, comparable to that elicited by
trans 1.5 mM H2O2 without pCMPS.
Addition of cis pCMPS to channels that had been treated with or
without trans H2O2 rapidly resulted in
high Po followed by closure of the channel. These
results suggest that oxidation of luminal sulfhydryls in the
Ca2+-release channel may contribute to
H2O2-induced channel activation and muscle
contracture.
frog skeletal muscle; calcium-release channel; sulfhydryl
oxidation; p-chloromercuriphenylsulfonic acid
 |
INTRODUCTION |
THE CALCIUM-RELEASE channel/ryanodine receptor in the
sarcoplasmic reticulum (SR) plays a crucial role in triggering skeletal muscle contraction. However, the underlying mechanism(s) by which depolarization of the transverse tubular membrane opens the
Ca2+-release channel is not well understood (28). A
remarkable increase in intracellular Ca2+ concentration due
to disturbance of Ca2+ homeostasis would result in muscle
injury (6). Strenuous exercise increases production of oxygen free
radical species such as hydroxyl radicals, superoxide anion, and
H2O2 (7, 13, 14, 26, 31) and frequently elicits
muscle fatigue and damage (25, 32). However, it remains elusive whether
an increase in cytoplasmic free radicals during contractile activity
directly causes muscle damage. Recent observations that free radicals
are produced in vivo during contraction in cat skeletal muscles and
that production occurs before muscle fatigue and damage (22) strongly
suggest the possibility that free radicals function as a trigger for
muscle dysfunction. H2O2 has been reported to
cause a transient twitch potentiation in cardiac and skeletal muscles,
followed by muscle injury (15, 20). H2O2
releases Ca2+ from isolated SR vesicles and increases the
open probability (Po) of the
Ca2+-release channel when channels are incorporated into
planar lipid bilayers (4, 11, 20, 34). Such actions of
H2O2 seem to be exerted via oxidation of
sulfhydryl groups in the Ca2+-release channel, since an
increase in Po is reversed by dithiothreitol treatment. In this regard, various other sulfhydryl reagents have been
reported to have an ability to release Ca2+ from the SR (1,
2, 16, 29, 30). Because H2O2 easily crosses the
lipid membranes (3), it is not known which of the sulfhydryls located
in cytoplasmic or intraluminal sites contributes to the action of
H2O2. By investigating this issue using the
lipid bilayer method, we would be able to have important information about the molecular mechanism underlying the action of
H2O2 on an increase in Ca2+ release
from the SR and probably leading to muscle dysfunction. In the present
paper, we demonstrate that H2O2 activates the
Ca2+-release channel by oxidizing sulfhydryl residues
located on the intraluminal side of the Ca2+-release
channel. Furthermore, the effect of H2O2 is
maintained even in channels in which sulfhydryl residues on the
cytoplasmic side have been oxidized by pretreatment with an organic
sulfhydryl reagent, p-chloromercuriphenylsulfonic acid (pCMPS).
 |
MATERIALS AND METHODS |
Mechanical skinning and experimental protocol for effect of
H2O2 on Ca2+ release.
The method for mechanical skinning of single fibers from bullfrog
semitendinosus muscle fibers and the compositions of the solutions used
for experiments were as previously described (20). After single fibers
were skinned in a relaxing solution (solution L; in mM: 100 KCl, 4 MgCl2, 4 ATP, 1 EGTA, and 20 Tris-maleate; pH 7.0),
Ca2+ remaining in the SR was removed by challenging with 5 mM caffeine (solution F; in mM: 100 KCl, 1 MgCl2, 4 ATP, 5 caffeine, and 20 Tris-maleate; pH 7.0). The fibers were rinsed
with solution H (solution L + 3 mM EGTA) to remove
Ca2+ from the medium and then washed three times with
solution L to remove caffeine. Skinned fibers were actively
loaded with Ca2+ by immersion in solution U [in
mM: 100 KCl, 4 MgCl2, 4 ATP, 4 EGTA, 1.2 CaCl2
(0.175 µM free Ca2+), and 20 Tris-maleate; pH 7.0] for
2 min. The amount of Ca2+ accumulated by the SR was
estimated as the peak caffeine contracture induced by solution
F. The fiber was soaked in solution H for 30 s immediately
after the tension reached a plateau and then in solution L
three times for 3 min. Ca2+ was loaded again by immersing
the fiber in solution U for 2 min. After rinses with
solutions H and L, the fiber was treated with 1, 3, or
10 mM H2O2 to observe spontaneous contracture.
Free Ca2+ concentration in each solution was calculated
with apparent stability constants of 1.14 × 104 for
MgATP, 5.15 × 103 for CaATP, and 2.51 × 106
for CaEGTA according to the method of Fabiato and Fabiato (9). Caffeine
contracture was checked to determine whether the SR still sustained the
ability of Ca2+ uptake after H2O2
treatment. In fibers in which no contracture occurred upon application
of H2O2, caffeine contracture was induced after
15 min. In some experiments, 5 µM ruthenium red, a specific Ca2+-induced Ca2+ release (CICR) inhibitor, was
applied to skinned fibers simultaneously with 10 mM
H2O2 to elucidate whether
H2O2 acts on the ryanodine receptor through the
CICR mechanism.
Heavy SR membrane preparation for single-channel recording.
Membrane fractions enriched in terminal cisternae (heavy SR vesicles)
were prepared from leg muscles of bullfrog (Rana catesbiana) as
described elsewhere (16). Heavy SR vesicles were suspended in a small
amount of 100 mM KCl, 20 mM Tris-maleate (pH 6.8), 20 µM
CaCl2, and 0.3 M sucrose. The SR vesicles were quickly
frozen in liquid N2 and then stored at
50°C until use.
Protein concentration was determined by the biuret reaction using BSA
as a standard.
Bilayer method and single-channel data acquisition and analysis.
Single-channel recordings were performed by incorporating heavy SR
vesicles into planar lipid bilayers according to our previous method
(19, 20). Lipid bilayers consisting of a mixture of L-
-phosphatidylethanolamine,
L-
-phosphatidyl-L-serine, and
L-
-phosphatidylcholine (5:3:2 wt/wt/wt) in
n-decane (30 mg/ml) were formed across a hole 200 µm in
diameter in a polystyrene partition separating two compartments: the
cis (volume 3 ml) and the trans (volume 2.2 ml). The
cis/trans solutions consisted of 250/50 mM
CsCH3SO3 and 10 mM CsOH (pH 7.4 adjusted by
HEPES). SR vesicles (~2 µg/ml) were added to the cis chamber. After channel fusion was checked by occurrence of flickering currents, the cis solution was perfused with a new solution (20 ml) to prevent further incorporation of channels. The cytoplasmic surface of the ryanodine receptor faced the cis side, as
previously shown using application of ATP to the cis chamber
(20).
Frog skeletal muscle SR expresses two isoforms of the ryanodine
receptor (
- and
-isoforms) (23) with distinct Ca2+
dependencies (5, 20). In this experiment, we used only the Ca2+-release channel (termed
-isoform) that displayed a
bell-shaped curve of Po against cis
Ca2+ concentration, i.e., was activated maximally at pCa
4-5 and blocked at pCa 3. The
-isoform in frog skeletal muscle
shares epitopes in common with the mammalian skeletal muscle ryanodine
receptor (17, 23). Therefore, we routinely determined the isoform type before the start of each experiment by checking whether the channel was
sensitive to challenge with a high concentration of cis
Ca2+. The trans side was held at ground potential,
and the cis side was clamped at 0 mV using 1.5% agar bridges
in 3 M KCl and Ag-AgCl electrodes, unless otherwise noted. Experiments
were carried out at room temperature (18-22°C).
Single-channel current amplified by a patch-clamp amplifier (CEZ-2300,
Nihon-Kohden, Tokyo, Japan) was filtered at 0.5 kHz using a four-pole
low-pass Bessel filter and digitized at 2 kHz for analysis. The data
were analyzed in a manner that excludes transitions <2 ms in
duration, and thus fast channel transitions with dwell times <2 ms
are excluded from data analysis. Data were saved on the hard disk of a
NEC personal computer. The 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 QP-120J
software (Nihon-Kohden) (19, 20).
The results are presented as means ± SE. Statistical analysis was
performed with Wilcoxon's U-test or paired t-test.
P < 0.05 was regarded as significant.
Chemicals.
Stock solutions for catalase (50,000 U/ml; Sigma, St. Louis, MO) and
ruthenium red (1 mM; Sigma) were dissolved in ultrapure water and
stored at
20°C. H2O2 (30% stock solution;
Mitsubishi Gas, Tokyo, Japan) was dissolved in buffer solution.
Caffeine (0.5 M; Sigma) and pCMPS (10 mM; Sigma) were prepared in
ultrapure warm water just before each experiment. Other reagents were
of analytical grade.
 |
RESULTS |
Induction of contracture by H2O2 in
mechanically skinned fibers.
Application of 1 mM H2O2 to skinned fibers in
which Ca2+ had been actively accumulated by incubation with
ATP in the presence of Ca2+ for 2 min elicited no
spontaneous contraction for at least 15 min (Fig.
1A and Table
1). Amplitude of the contracture induced by
5 mM caffeine in such H2O2-treated fibers was
slightly increased (1.16-fold), from 479 ± 32 µN in controls to 555 ± 64 µN (n = 5; Table 2). On
the other hand, maximum rate of rise of caffeine contracture was
significantly elevated (3.3-fold), from 39.5 ± 5.2 µN/s in controls
to 126.9 ± 12.8 µN/s after treatment with H2O2 (P < 0.01). An increase in
H2O2 to 3 mM elicited spontaneously a tiny
transient contraction in three of five preparations examined (Fig.
1B and Table 1). Contracture induced by challenging with 5 mM
caffeine after the spontaneous contracture was returned to the resting
tension level was enhanced to an extent similar to that in 1 mM
H2O2-treated fibers, indicating that the
transient contracture is derived from almost complete reuptake of
Ca2+ released on exposure to H2O2
by the SR. Further increase in H2O2 to 10 mM
led to the occurrence of large spontaneous and repeated contractures in
all of the preparations examined, and the maximum tension amplitude
(406 ± 61 µN, n = 5) reached ~80% of caffeine contracture before H2O2 treatment. Maximum rate
of rise of the spontaneous contracture was elevated 4.2-fold from 11 µN/s in 3 mM H2O2 to 48 µN/s in 10 mM
H2O2, but the relative maximum rate of rise, as
estimated by dividing the maximum rate of rise of tension by the
maximum tension, was not different (Table 1). Fiber-to-fiber variations
were observed in both the time required to onset of tension development
after addition of H2O2 and the maximum tension
amplitude. When H2O2 was removed from external medium and then Ca2+ was actively reaccumulated by ATP
addition, mechanical parameters in caffeine contracture were almost the
same as those in controls without H2O2. Thus
fibers were not deteriorated by such repeated contractures.

View larger version (20K):
[in this window]
[in a new window]
|
Fig. 1.
Induction of H2O2-induced spontaneous
contraction in mechanically skinned fibers. After amount of
Ca2+ accumulated by sarcoplasmic reticulum (SR) in
solution U for 2 min was checked by challenging with 5 mM
caffeine (solution F), fiber was washed with solutions
H and L, as noted in MATERIALS AND METHODS.
Ca2+ was accumulated again, and then fibers were exposed to
1 (A), 3 (B) and 10 (C) mM
H2O2 to evaluate whether
H2O2 elicits release of Ca2+ from
SR. Tensions at application of H2O2 show
resting or 0 tension level. Caffeine at 5 mM was added to each fiber
after spontaneous tension returned to resting tension level, to check
whether SR is still functional after exposure to
H2O2. Fibers that did not respond to
H2O2 were challenged with 5 mM caffeine after
~15 min (A), and effects of H2O2 on
caffeine contracture were examined. All fibers were evaluated again to
test ability of SR to accumulate Ca2+ after washout of
H2O2. Each trace in A, B, and C
was obtained from a different fiber.
|
|
Exposure of skinned fibers to 10 mM H2O2 no
longer elicited spontaneous contracture in the presence of 5 µM
ruthenium red (Fig. 2). When exposed to 5 mM caffeine, such paralyzed fibers produced a large contracture with a
decreased maximum rate of rise. In fibers without
H2O2 treatment, 5 µM ruthenium red induced no
contracture on application of 5 mM caffeine (Fig. 2A).
Similar results were observed in three separate experiments.

View larger version (14K):
[in this window]
[in a new window]
|
Fig. 2.
Effects of ruthenium red (5 µM) on caffeine- and
H2O2-induced contractures. See MATERIALS
AND METHODS for experimental protocol. After Ca2+
accumulation by solution F was checked, fiber was rinsed with
solutions H and L, and then Ca2+ was
actively accumulated again by SR. In presence of 5 µM ruthenium red,
spontaneous contracture was no longer observed on addition of 10 mM
H2O2 (B), similar to that in a
control fiber without H2O2 (A). Note
occurrence of a decreased caffeine contracture in B. Fiber in
A differs from that in B.
|
|
Activation of Ca2+-release channel by intraluminal
H2O2.
When 1.5 mM H2O2 was applied to the cis
side of the bilayer in 10 µM Ca2+, Po
was increased 2.4-fold from 0.073 ± 0.018 in control to 0.173 ± 0.052 after 10 min (n = 7), consistent with our previous
study (20). Time required to activate the channel after exposure to H2O2 varied between 3 and 10 min (mean:
6.3 ± 0.9 min). On the other hand, addition of 1.5 mM
H2O2 to the trans side of the channel elicited a rapid increase in Po with shorter lag
time. Just 30 s was sufficient to initiate the earliest channel
activation, and 3.5 min was required for the latest channel activation
(n = 5). This suggests the possibility that
H2O2 exerts such an effect by acting
preferentially on the Ca2+-release channel from the
intraluminal side even after application to the cis side
because H2O2 can permeate membranes (3). To further evaluate this issue, we performed experiments using 200 units
of catalase, an enzyme that can hydrolyze H2O2
to H2O and O2. When the trans side of
the channel was pretreated with catalase, cis
H2O2 elicited no increase in
Po for at least 10 min. When 1.5 mM
H2O2 was added to the trans side of the
channel in the presence of cis catalase, Po
was increased immediately after application of
H2O2 and reached a maximum within several
minutes. As shown in Fig. 3, trans
H2O2 in the presence of the cis
catalase kept the Po high for at least 10 min until
the closure of the channel was observed by application of 2 µM
ruthenium red. We compared effects of trans
H2O2 on open time distribution, unitary
conductance, and reversal potential with those of application to the
cis side. A typical result is shown in Fig.
4. In this channel,
H2O2 at 1.5 mM was added to the cis
side in the presence of trans catalase. Po
did not increase during 10 min (0.089 at 8 min as shown in Fig.
4A) and was comparable with Po of
controls (Po = 0.081). Cis
H2O2 and trans catalase were washed
out, and then H2O2 was added to the
trans side in the presence of cis catalase. The
Po increased to 0.218 after 1 min, as shown in Fig.
4A, traces 5 and 6. After 10 min, a subsequent addition
of 2 µM ruthenium red to the cis side blocked the channel.
The open lifetime distribution was best fit by two exponentials
(
o1 and
o2), and time constants of the
mean open lifetime after application of H2O2 to
trans side were larger than those in controls
(P < 0.05; control,
o1 = 1.93 ± 0.34 ms,
o2 = 5.65 ± 1.35 ms, n = 7;
cis H2O2,
o1 = 2.30 ± 0.45 ms,
o2 =
6.73 ± 1.57 ms, n = 5; trans
H2O2,
o1 = 2.54 ± 0.33 ms,
o2 = 8.69 ± 1.27, n = 8), although no
significant difference was observed between channels in which
H2O2 was applied to cis and
trans sides. Closed time constants in each group were best fit
by two similar exponentials. The trans
H2O2 did not affect the single-channel
conductance (Fig. 4C; 820 pS in control and 835 pS in
trans H2O2). Our previous results
have demonstrated no effect of cis H2O2
on the unitary conductance (20). Reversal potential was also not
affected by trans H2O2. Similar results were obtained in three other single channels.

View larger version (25K):
[in this window]
[in a new window]
|
Fig. 3.
Time-dependent effects of H2O2 (1.5 mM) on open
probability (Po) of the Ca2+-release
channel in presence of contralaterally applied catalase (200 units).
, trans H2O2-induced increase in
Po in Ca2+-release channels in which
cis side was treated with catalase (n = 7). Note that
high Po was sustained for at least 10 min
throughout presence of H2O2, except after
exposure to 2 µM ruthenium red. , No activation of channel by
application of H2O2 to cis side in
presence of trans catalase (n = 5). Vertical bars,
SE. Time scales are in min after addition of 1.5 mM
H2O2 (middle) and 2 µM ruthenium red
(right).
|
|

View larger version (30K):
[in this window]
[in a new window]
|
Fig. 4.
Differential effects of H2O2 (1.5 mM) applied
to either cis or trans side of a
Ca2+-release channel in presence of contralateral catalase
(200 units). A: trace nos. go from top to bottom.
Traces 1 and 2, control channel activity activated at pCa 5 (Po = 0.081). Traces 3 and 4, channel activity (Po = 0.089) 8 min after
addition of H2O2 to cis side in
presence of 200 units catalase on trans side; 10 min later,
both H2O2 and catalase were removed by
replacement of cis and trans solutions, respectively.
Traces 5 and 6, activated channel activity
(Po = 0.218 after 1 min) induced by trans
H2O2 in presence of cis catalase.
Traces 7 and 8, inhibition of channel activity after
subsequent exposure of cis side to 2 µM ruthenium red. Dashed
and solid lines, open and closed channel levels, respectively.
B: open time histograms (top, control; middle,
cis H2O2 in presence of trans
catalase; bottom, trans H2O2 in
presence of cis catalase). C: unitary conductances in
control (820 pS) and in trans H2O2 and
cis catalase (835 pS). Reversal potentials were not different
from each other ( 20.0 mV).
|
|
It is of interest to determine whether trans
H2O2 reacts with sulfhydryls on the luminal
surface of the Ca2+-release channel because
H2O2 can cross the lipid membranes (3). To
evaluate this issue, we used an organic water-soluble and
sulfhydryl-specific reagent, pCMPS, and modulated sulfhydryls on the
intraluminal side of the Ca2+-release channel.
Intraluminally applied pCMPS at 50 µM did not alter the
Po for at least 10 min in all channels examined
(0.076 ± 0.031 in controls and 0.065 ± 0.030 in pCMPS-treated
channels, n = 8). A subsequent exposure of the pCMPS-treated
channel to trans 1.5 mM H2O2 failed to
increase Po in the presence of cis
catalase. Exposure of the Ca2+-release channel to these
reagents did not affect the open and closed lifetime durations. An
example of such experiments is shown in Fig.
5. As previously reported (19), an addition
of 50 µM pCMPS to the cis side of the
Ca2+-release channel remarkably increased the
Po, followed by almost complete inhibition. A
similar result is shown again in Fig. 6, left. In this channel, Po was increased
from 0.056 in controls (cis pCa 5) to 0.732 after exposure to
pCMPS for 30 s. The Ca2+-release channel was almost closed
after 1 min. These results strongly suggest that 1) there are
at least two distinct pCMPS-susceptible sulfhydryls that may contribute
to activation and inactivation (or deactivation) of the channel,
2) such sulfhydryls should be on sites near the cytoplasmic
surface of the channel, and 3) pCMPS does not cross the lipid
membranes of the SR. Therefore, pCMPS would be a favorable chemical to
examine whether the increase in the Po caused by
trans H2O2 is attributable to oxidation
of intraluminal sulfhydryls in the Ca2+-release channel.
Figure 6 shows that the channel inhibited by pCMPS slowly regained its
function in a stepwise fashion upon subsequent
intraluminal application of 1.5 mM H2O2. The
channel first showed a long-lived one-half subconductance state (90-s trace) and then three-fourths of the control current after 2-3 min
(120-s and 180-s traces). Thereafter, the channel fluctuated with a
full conductance (300-s trace), similar to the characteristic fluctuation pattern observed in the channel treated with trans H2O2 in the absence of pCMPS (compare with Fig.
3).

View larger version (29K):
[in this window]
[in a new window]
|
Fig. 5.
Effect of trans H2O2 on
Ca2+-release channel in which trans side has been
pretreated with 50 µM p-chloromercuriphenylsulfonic acid
(pCMPS). Trace nos. go from top to bottom. Trace 1, control channel activity (Po = 0.104) at pCa 5;
open time constants ( o) = 2.13 and 6.92 ms; closed
time constants ( c = 5.38 and 27.55 ms. Trace
2, channel activity after trans 50 µM pCMPS treatment
(Po = 0.090); o = 1.90 and 5.13 ms; c = 4.27 and 28.39 ms. Trace 3, channel
activity after addition of cis 200 units catalase
(Po = 0.071); o = 2.37 and 5.63 ms; c = 5.73 and 22.51 ms. Traces 4-6,
channel activity during application of 1.5 mM
H2O2 to trans side
(Po = 0.065, 0.067, and 0.067 at 2, 5, and 10 min, respectively); o = 2.16 and 5.21 ms, 2.32 and 7.52 ms, and 2.30 and 6.57 ms respectively; c = 5.04 and
23.52 ms, 5.59 and 31.69 ms, and 5.14 and 33.27 ms, respectively.
Chemicals were added sequentially. Dashed and solid lines, open and
closed channel levels, respectively.
|
|

View larger version (35K):
[in this window]
[in a new window]
|
Fig. 6.
Stepwise time-dependent activation by trans
H2O2 of Ca2+-release channel in
which channel activity has been almost completely inhibited by exposure
of cis side to 50 µM pCMPS. Trace nos. go from top to
bottom; left, traces 1-5; right, traces
6-9. Trace 1, channel activity in control
(Po = 0.056); o = 1.90 and 5.13 ms; c = 6.07 and 23.78 ms. Traces 2 and
3, channel activity after cis 50 µM pCMPS treatment
(Po = 0.732 at 30 s); o = 5.82 and
26.90 ms; c = 4.46 and 14.49 ms. The channel closed 1 min after pCMPS treatment. Application of 1.5 mM
H2O2 to trans side of such a
deactivated channel activated in a stepwise fashion (traces
4-7), and finally channel fluctuated with full conductance
(trace 8; o = 2.66 and 17.04 ms;
c = 5.12 and 32.60 ms). Trace 9, channel
activity after 2 µM ruthenium red treatment. Numbers at left
of each trace represent time after addition of each chemical. All
chemicals were applied sequentially. Dashed and solid lines, open and
closed channel levels, respectively.
|
|
In Ca2+-release channels that have been activated by
treatment with trans 1.5 mM H2O2 for 5 min (to Po = 0.270 from 0.034 in controls), the
Po was increased to 0.825 after 20 s of treatment of this channel with cis 5 µM pCMPS, and such a high
Po was sustained for ~2 min (Fig.
7). Open lifetime constants were also
increased from 1.29 and 3.84 ms in the control channel to 1.77 and 9.81 ms after application of pCMPS to the cis side. In addition,
pCMPS elicited a third long open time constant (113.72 ms). Thereafter, the channel suddenly closed, similar to what was observed in
cis 5 µM pCMPS without H2O2
pretreatment. However, the duration of the high-activation state
induced by pCMPS was significantly prolonged (2.7-fold;
P < 0.05) after preexposure to H2O2
(126 ± 27 s, n = 6), compared with that without
H2O2 treatment (47 ± 9 s, n = 7).

View larger version (40K):
[in this window]
[in a new window]
|
Fig. 7.
Effect of cis pCMPS on Ca2+-release channel that
was activated by applying 1.5 mM H2O2 to
trans side. A: channel activity in control
(Po = 0.034; o = 1.29 and 3.84 ms; c = 7.29 and 35.89 ms). B: channel
activity 1 min (Po = 0.106;
o = 1.51 and 4.75 ms; c = 7.87 and
29.87 ms) and 5 min (Po = 0.270;
o = 1.78 and 5.85 ms, c = 6.68 and
28.68 ms) after trans H2O2 treatment.
C: channel activation followed by inhibition after treatment of
cis side of channel with 5 µM pCMPS
(Po = 0.825 at 20 s; o = 1.77,
9.81, and 113.72 ms; c = 2.64 and 23.14 ms). Note that
channel activity was suddenly and almost completely inhibited at 2 min.
D: channel activity after 2 µM ruthenium red. All chemicals
were added sequentially. Dashed and solid lines, open and closed
channel levels, respectively.
|
|
 |
DISCUSSION |
Previous reports using skeletal and cardiac muscles have indicated that
H2O2 activates the Ca2+-release
channel by oxidizing sulfhydryls in the channel protein incorporated
into planar lipid bilayers (4, 10, 20) and facilitates Ca2+
release from the SR (20, 34). These experiments were done by applying
H2O2 to the cytoplasmic face of the
Ca2+-release channel. H2O2 has been
reported to easily cross a lipid membrane such as the SR membrane (3).
Therefore, it remains unclear whether only cytoplasmic sulfhydryls
contribute to the action of H2O2. Using the
bilayer method, we provide evidence that the site of action of
H2O2 is on sulfhydryls of the
Ca2+-release channel to which H2O2
is accessible from its luminal side. This is based on observations that
application of H2O2 to the trans side
increased Po even in the presence of cis
catalase, whereas cis H2O2 failed to
activate the channel in the presence of trans catalase (Fig.
3). When sulfhydryls in the cis side had been oxidized by
pretreatment with pCMPS, the channels still responded to trans
H2O2 in a stepwise fashion (Fig. 6). Such
activation of the Ca2+-release channel on addition of
H2O2 would contribute to potentiation of
caffeine-induced Ca2+-release or spontaneous tension
development in skinned fibers (Fig. 1). In skinned fibers 3 mM
H2O2 was required to elicit the spontaneous
tension, whereas in bilayers much less was enough to activate the
channel. This discrepancy would be explained by the presence of the
Ca2+-ATPase, intracellular sulfhydryl-reducing agents, and
free radical scavengers in skinned fibers. Considerable amounts of
Ca2+ released by H2O2 would be
taken up into the SR lumen, again by action of Ca2+-ATPase.
Thus it seems likely that higher amounts of
H2O2 are necessary to cause spontaneous
contraction in skinned fibers. As shown in Fig. 1, 1 mM
H2O2 increased the amplitude and maximum rate
of rise of caffeine contracture, suggesting a potentiating action of
low concentrations of H2O2 on the CICR. This is
consistent with the finding that exposure of skeletal muscles to
catalase decreases twitch tension (27). If the intraluminal space of the SR lacks sulfhydryl-reducing agents and free radical scavengers such as GSH and catalase, flux of H2O2 produced
during strenuous contractile activity (22) into the intraluminal space
of the SR from cytoplasm would effectively activate the
Ca2+-release channel and in turn lead to a sustained
increase in cytoplasmic Ca2+ concentration. Further
investigations will be required to elucidate these issues.
In the absence of catalase, intraluminal application of
H2O2 elicited the increase in
Po rapidly, compared with the case of cytoplasmic
addition. This finding also supports the above conclusion on the site
of action of H2O2. The skeletal muscle SR
Ca2+-release channel is well known to be modulated by many
regulatory ligands such as caffeine, ATP, Ca2+,
Mg2+, ryanodine, and ruthenium red (18). Although the exact
binding sites of these ligands remain to be determined, most of them
seem to be on the cytoplasmic face of the Ca2+-release
channel (21). The Ca2+-release channel of skeletal muscle
SR is a homotetramer (33), and each subunit in the
-isoform of frog
skeletal ryanodine receptor has 94 cysteines (23). An important role of
sulfhydryls in the modification of Ca2+-release channel
gating has been demonstrated using sulfhydryl-reacting reagents (1, 20,
24, 29). Effects of sulfhydryl-oxidizing and -alkylating reagents on
the Ca2+-release channel kinetics are summarized in Table
3. Generally, an increase in
Po produced by sulfhydryl oxidation on the channel is associated with prolonged open time duration, in agreement with Fig.
4 in this study. However, it is not known which of these cysteines
contributes to the channel modulation, although the present result
suggests the importance of luminal sulfhydryl(s). Reportedly, there are
at least three classes of functionally important sulfhydryls on the
Ca2+-release channel of rabbit skeletal muscles (2). There
may be many sulfhydryls that associate with the channel modulation in
the case of the frog skeletal muscle
-isoform we used here. As shown
in Figs. 5 and 6, an organic sulfhydryl reagent, pCMPS, when applied to
the cis side, but not to the trans side, rapidly activated the Ca2+-release channel, followed by a sudden
and complete inhibition, consistent with our previous result (19). This
strongly suggests the existence of distinct types of sulfhydryls
responsible for activation and inactivation (or deactivation) of the
channel. The Ca2+-release channel activation always
precedes its inhibition on addition of pCMPS (Figs. 6 and 7), thereby
indicating that binding of pCMPS to a high-affinity site contributes to
channel activation and binding to a low-affinity site contributes to
channel inhibition. These sulfhydryls are probably on the cytoplasmic
side because exposure of the intraluminal side of the
Ca2+-release channel to pCMPS has no effect (Fig. 5).
However, we do not completely rule out the possibility that chemical
modification of sulfhydryls by pCMPS alters the channel structure to an
open configuration and in turn such a conformational change permits some sulfhydryl buried in a lipophilic site of the channel to be
exposed to a location that can respond to pCMPS. In this regard, it is
very interesting that the
-isoform of frog skeletal muscle ryanodine
receptor has two cysteines in the M2 membrane-spanning region in the
model proposed by Takeshima et al. (33). One or both of the two
sulfhydryls may contribute to the channel inactivation or deactivation.
This possibility has more recently been proposed by Eager et al. (8),
who found that reactive disulfides (2,2'- and 4,4'-dithiodipyridine)
activate within 1 min, with an irreversible loss of sheep cardiac
Ca2+-release channel activity, when incorporated into lipid
bilayers.
The results shown in Figs. 3, 4, and 6 suggest that the third
sulfhydryl associated with the channel modification occurs on the
intraluminal side of this channel. Only two cysteines are between the
membrane spanning regions M3 and M4 as luminal sulfhydryls. One of them
may be attacked by H2O2 to activate the
Ca2+-release channel. However, we found that application of
pCMPS to intraluminal space failed to activate the
Ca2+-release channel and that the channel inactivated by
cis pCMPS was never activated on exposure to intraluminal pCMPS
(data not shown). It is reasonable to consider that trans pCMPS
oxidizes intraluminal cysteines to activate the channel as
H2O2 does, if these cysteines are associated
with the channel modification. Therefore, it seems unlikely that two
cysteines in the intraluminal loop region between M3 and M4 contribute
to the channel activation induced by trans
H2O2. Even after the channel had been closed by
application of cis pCMPS, exposure of the trans side to
H2O2 led to channel activation in a stepwise
fashion (Fig. 6). Although the underlying mechanism remains unclear,
the existence of one-half and three-fourths subconductance states as
shown in Fig. 6 favors the possibility that sulfhydryl residues
modified by trans H2O2 after
pretreatment with cis pCMPS may be four in a pore in a
homotetramer of the Ca2+-release channel protein (probably
one in each subunit). One of two cysteines in the M2 spanning region
may be associated with this response, although further studies will be
required to elucidate this hypothesis.
A recent observation by Quinn and Ehrlich (24), who used
methanethiosulfonate (MTS) compounds, which are specific sulfhydryl reagents, indicated that exposure of the cis side of the
Ca2+-release channel of rabbit skeletal muscle to these
compounds decreased single-channel current amplitude in a stepwise
fashion when these compounds were used as a probe for channel
inhibition. Inconsistent with their results, the 50 µM pCMPS we used
did not result in such a stepwise inhibition of the channel. This
discrepancy may be caused by use of different sulfhydryl probes. MTS
initially activated the channel and decreased channel conductance to
three-fourths and one-fourth 4 and 20 min after application,
respectively (24), whereas pCMPS activates with full
conductance, followed by a sudden closure of the channel only 2 min
after application (Fig. 7). Therefore, both sulfhydryl reagents may act
on distinct sites of the channel. Alternatively, the difference may
come from the different ryanodine isoforms used by the investigators
(rabbit vs. frog). In this regard, sheep cardiac
Ca2+-release channels are transiently activated within 1 min of addition of disulfides to cis side of the channel and
almost completely blocked several minutes later (8), similar to the
present results. Further study will be required to elucidate these
issues.
It has been reported that oxygen free radicals such as
H2O2 and superoxide anion are produced in
skeletal muscle fibers during repetitive contractions (7, 26). Emphasis
is placed on oxidative stress as a component of muscle fatigue and
dysfunction. Muscle fatigue may be mainly developed by a decrease in
Ca2+ release from the SR (12). In their in vivo experiment,
O'Neill et al. (22) demonstrated that hydroxy radical, which is
converted from H2O2 and superoxide anion
produced by the contracting muscle, is present before the onset of
muscle fatigue and progressively increases throughout the period of
contraction. In the present experiments, we found that 1-1.5 mM
H2O2 stimulates Ca2+ release from
the SR and activates the Ca2+-release channel by attacking
from the intraluminal side. However, a question arises as to whether
H2O2 reaches such high intracellular concentrations even during strenuous muscle activation in vivo. When
H2O2 at a concentration as low as 0.1 mM was
applied to the cis side of skeletal muscle
Ca2+-release channels, an increase in
Po was reported by Favero et al. (10). If the SR
lacks sulfhydryl-reducing agents such as GSH and catalase in the
intraluminal space, H2O2 entering the SR should
be allowed to act for a long time and to keep Ca2+-release
channels activated, which in turn may make fibers susceptible to
damage.
 |
ACKNOWLEDGEMENTS |
We thank Dr. H. Suzuki for reading the manuscript.
 |
FOOTNOTES |
This work was supported by Grants-in-Aid for Scientific Research
086700600 and 09470012 from the Ministry of Education, Science, Sports,
and Culture, Japan (to T. Oba) and by grants from the American Heart
Association (Central Ohio Heart Chapter), the Muscular Dystrophy Association of America, and the Ohio State
University Canine Research and Equine Research Fund (to M. Yamaguchi).
Address for reprint requests: T. Oba, Dept. of Physiology, Nagoya City
University Medical School, Mizuho-ku, Nagoya 467, Japan.
Received 13 August 1997; accepted in final form 15 December 1997.
 |
REFERENCES |
1.
Abramson, J. J.,
J. L. Trimm,
L. Weden,
and
G. Salama.
Heavy metals induce rapid calcium release from sarcoplasmic reticulum vesicles isolated from skeletal muscle.
Proc. Natl. Acad. Sci. USA
80:
1526-1530,
1983[Abstract].
2.
Aghdasi, B.,
J.-Z. Zhang,
Y. Wu,
M. B. Reid,
and
S. L. Hamilton.
Multiple classes of sulfhydryls modulate the skeletal muscle Ca2+ release channel.
J. Biol. Chem.
272:
3739-3748,
1997[Abstract/Free Full Text].
3.
Beckman, J. S.,
and
B. A. Freeman.
Antioxidant enzymes as mechanistic probes of oxygen-dependent toxicity.
In: Physiology of Oxygen Radicals, edited by A. E. Taylor,
S. Matalon,
and P. A. Ward. Bethesda, MD: Am. Physiol. Soc., 1986, p. 39-53. (Clin. Physiol. Ser.)
4.
Boraso, A.,
and
A. J. Williams.
Modification of the gating of the cardiac sarcoplasmic reticulum Ca2+-release channel by H2O2 and dithiothreitol.
Am. J. Physiol.
267 (Heart Circ. Physiol. 36):
H1010-H1016,
1994[Abstract/Free Full Text].
5.
Bull, R.,
and
J. J. Marengo.
Sarcoplasmic reticulum release channels from frog skeletal muscle display two types of calcium dependence.
FEBS Lett.
331:
223-227,
1993[Medline].
6.
Byrd, S. K.
Alterations in the sarcoplasmic reticulum: a possible link to exercise-induced muscle damage.
Med. Sci. Sports Exerc.
24:
531-536,
1992[Medline].
7.
Davies, K.,
A. Quintanilha,
G. Brooks,
and
L. Packer.
Free radicals and tissue damage produced by exercise.
Biochem. Biophys. Res. Commun.
107:
1198-1205,
1982[Medline].
8.
Eager, K. R.,
L. D. Roden,
and
A. F. Dulhunty.
Actions of sulfhydryl reagents on single ryanodine receptor Ca2+-release channels from sheep myocardium.
Am. J. Physiol.
272 (Cell Physiol. 41):
C1908-C1918,
1997[Abstract/Free Full Text].
9.
Fabiato, A.,
and
F. Fabiato.
Calculator programs for computing the composition of the solution containing multiple metals and ligands used for experiments in skinned muscle cells.
J. Physiol. Paris
74:
463-505,
1979.
10.
Favero, G.,
A. C. Zable,
and
J. J. Abramson.
Hydrogen peroxide stimulates the Ca2+ release channel from skeletal muscle sarcoplasmic reticulum.
J. Biol. Chem.
270:
25557-25563,
1995[Abstract/Free Full Text].
11.
Favero, T. G.,
A. C. Zable,
M. B. Bowman,
A. Thompson,
and
J. J. Abramson.
Metabolic end products inhibit sarcoplasmic reticulum Ca2+ release and [3H]ryanodine binding.
J. Appl. Physiol.
75:
1665-1672,
1995.
12.
Gyorke, S.
Effects of repeated tetanic stimulation on excitation-contraction coupling in cut muscle fibres of the frog.
J. Physiol. (Lond.)
464:
699-710,
1993[Abstract].
13.
Jackson, M. J.,
R. H. T. Edwards,
and
M. C. R. Symons.
Electron spin resonance studies of intact mammalian skeletal muscle.
Biochim. Biophys. Acta
845:
185-190,
1985.
14.
Jenkins, R. R.
Free radical chemistry: relationship to exercise.
Sports Med.
5:
156-170,
1988[Medline].
15.
Josephson, R. A.,
H. S. Silverman,
E. G. Lakatta,
M. D. Stern,
and
J. L. Zweier.
Study of the mechanisms of hydrogen peroxide and hydroxyl free radical-induced cellular injury and calcium overload in cardiac myocytes.
J. Biol. Chem.
266:
2354-2361,
1991[Abstract/Free Full Text].
16.
Koshita, M.,
and
T. Oba.
Caffeine treatment inhibits drug-induced calcium release from sarcoplasmic reticulum and caffeine contracture but not tetanus in frog skeletal muscle.
Can. J. Physiol. Pharmacol.
67:
890-895,
1989[Medline].
17.
Lai, F. A.,
Q.-Y. Liu,
L. Xu,
A. El-Hashem,
N. R. Kramarcy,
R. Sealock,
and
G. Meissner.
Amphibian ryanodine receptor isoforms are related to those of mammalian skeletal or cardiac muscle.
Am. J. Physiol.
263 (Cell Physiol. 32):
C365-C372,
1992[Abstract/Free Full Text].
18.
Meissner, G.
Ryanodine receptor/Ca2+ release channels and their regulation by endogenous effectors.
Annu. Rev. Physiol.
56:
485-508,
1994[Medline].
19.
Oba, T.,
M. Koshita,
and
D. F. Van Helden.
Modulation of frog skeletal muscle Ca2+-release channel gating by anion channel blockers.
Am. J. Physiol.
271 (Cell Physiol. 40):
C819-C824,
1996[Abstract/Free Full Text].
20.
Oba, T.,
M. Koshita,
and
M. Yamaguchi.
H2O2 modulates twitch tension and increases Po of Ca2+-release channel in frog skeletal muscle.
Am. J. Physiol.
271 (Cell Physiol. 40):
C810-C818,
1996[Abstract/Free Full Text].
21.
Ogawa, Y.
Role of ryanodine receptors.
Crit. Rev. Biochem. Mol. Biol.
29:
229-274,
1994[Abstract].
22.
O'Neill, C. A.,
C. L. Stebbins,
S. Bonigut,
B. Halliwell,
and
J. C. Longhurst.
Production of hydroxyl radicals in contracting skeletal muscle of cats.
J. Appl. Physiol.
81:
1197-1206,
1996[Abstract/Free Full Text].
23.
Oyamada, H.,
T. Murayama,
T. Takagi,
M. Iino,
N. Iwabe,
T. Miyata,
Y. Ogawa,
and
M. Endo.
Primary structure and distribution of ryanodine-binding protein isoforms of the bullfrog skeletal muscle.
J. Biol. Chem.
269:
17206-17214,
1994[Abstract/Free Full Text].
24.
Quinn, K. E.,
and
B. E. Ehrlich.
Methanethiosulfonate derivatives inhibit current through the ryanodine receptor/channel.
J. Gen. Physiol.
109:
255-264,
1997[Abstract/Free Full Text].
25.
Rajgura, S. U.,
G. S. Yeargans,
and
N. W. Seidler.
Exercise causes oxidative damage to rat skeletal muscle microsomes while increasing cellular sulfhydryls.
Life Sci.
54:
149-157,
1994[Medline].
26.
Reid, M. B.,
K. E. Haack,
K. M. Franchek,
P. A. Valberg,
L. Kobzik,
and
M. S. West.
Reactive oxygen in skeletal muscle. I. Intracellular oxidant kinetics and fatigue in vitro.
J. Appl. Physiol.
73:
1797-1804,
1992[Abstract/Free Full Text].
27.
Reid, M. B.,
F. A. Khawli,
and
M. R. Moody.
Reactive oxygen in skeletal muscle. III. Contractility of unfatigued muscle.
J. Appl. Physiol.
75:
1081-1087,
1993[Abstract].
28.
Rios, E.,
and
G. Pizarro.
Voltage sensor of excitation-contraction coupling in skeletal muscle.
Physiol. Rev.
72:
849-908,
1991.
29.
Salama, G.,
and
J. Abramson.
Silver ions trigger Ca2+ release by acting at the apparent physiological release site in sarcoplasmic reticulum.
J. Biol. Chem.
259:
13363-13369,
1984[Abstract/Free Full Text].
30.
Salama, G.,
J. J. Abramson,
and
G. K. Pike.
Sulfhydryl reagents trigger Ca2+ release from the sarcoplasmic reticulum of skinned rabbit psoas fibres.
J. Physiol. (Lond.)
454:
389-420,
1992[Abstract].
31.
Sjodin, B.,
Y. H. Westing,
and
F. S. Apple.
Biochemical mechanisms for oxygen free radical formation during exercise.
Sports Med.
10:
236-254,
1990[Medline].
32.
Sternbergh, W. C., III,
and
B. Adelman.
The temporal relationship between endothelial cell dysfunction and skeletal muscle damage after ischemia and reperfusion.
J. Vasc. Surg.
16:
30-39,
1992[Medline].
33.
Takeshima, H.,
S. Nishimura,
T. Matsumoto,
H. Ishida,
K. Kangawa,
N. Minamino,
H. Matsuo,
M. Ueda,
M. Hanaoka,
T. Hirose,
and
S. Numa.
Primary structure and expression from complementary DNA of skeletal muscle ryanodine receptor.
Nature
339:
439-445,
1989[Medline].
34.
Trimm, J. L.,
G. Salama,
and
J. J. Abramson.
Sulfhydryl oxidation induces rapid calcium release from sarcoplasmic reticulum vesicles.
J. Biol. Chem.
261:
16092-16098,
1986[Abstract/Free Full Text].
AJP Cell Physiol 274(4):C914-C921
0363-6143/98 $5.00
Copyright © 1998 the American Physiological Society