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Am J Physiol Cell Physiol 274: C914-C921, 1998;
0363-6143/98 $5.00
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Vol. 274, Issue 4, C914-C921, April 1998

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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-alpha -phosphatidylethanolamine, L-alpha -phosphatidyl-L-serine, and L-alpha -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 (alpha - and beta -isoforms) (23) with distinct Ca2+ dependencies (5, 20). In this experiment, we used only the Ca2+-release channel (termed alpha -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 alpha -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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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.


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  		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.

                              
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  		Table 1.   H2O2-induced spontaneous contraction in mechanically skinned fibers

                              
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  		Table 2.   Effects of 1 mM H2O2 pretreatment on caffeine contracture in skinned fibers

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.


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  		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 (tau o1 and tau 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, tau o1 = 1.93 ± 0.34 ms, tau o2 = 5.65 ± 1.35 ms, n = 7; cis H2O2, tau o1 = 2.30 ± 0.45 ms, tau o2 = 6.73 ± 1.57 ms, n = 5; trans H2O2, tau o1 = 2.54 ± 0.33 ms, tau 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.


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  		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). bullet , 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. open circle , 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).


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  		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).


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  		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 (tau o) = 2.13 and 6.92 ms; closed time constants (tau c = 5.38 and 27.55 ms. Trace 2, channel activity after trans 50 µM pCMPS treatment (Po = 0.090); tau o = 1.90 and 5.13 ms; tau c = 4.27 and 28.39 ms. Trace 3, channel activity after addition of cis 200 units catalase (Po = 0.071); tau o = 2.37 and 5.63 ms; tau 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); tau o = 2.16 and 5.21 ms, 2.32 and 7.52 ms, and 2.30 and 6.57 ms respectively; tau 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.


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  		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); tau o = 1.90 and 5.13 ms; tau 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); tau o = 5.82 and 26.90 ms; tau 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; tau o = 2.66 and 17.04 ms; tau 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).


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  		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; tau o = 1.29 and 3.84 ms; tau c = 7.29 and 35.89 ms). B: channel activity 1 min (Po = 0.106; tau o = 1.51 and 4.75 ms; tau c = 7.87 and 29.87 ms) and 5 min (Po = 0.270; tau o = 1.78 and 5.85 ms, tau 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; tau o = 1.77, 9.81, and 113.72 ms; tau 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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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 alpha -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 alpha -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 alpha -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.

                              
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  		Table 3.   Effects of sulfhydryl-oxidizing and -alkylating reagents on Ca2+-release channel kinetics

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
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

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