Introduction

Top Abstract Introduction Experimental Procedures Results Discussion References

The ryanodine receptor (RyR), which functions as a Ca²⁺ release channel of sarcoplasmic reticulum (SR), is postulated to play a key role in excitation-contraction (EC) coupling in skeletal muscle (Shoshan and Ashley, 1998). Depolarization of the sarcolemma leads to stimulation of Ca²⁺ release from SR by being transmitted down to the RyR from the transverse tubules through dihydropyridine receptors. Following excitation, released Ca²⁺ is taken up into the lumen of SR by Ca²⁺-ATPase and stored mainly by binding to calsequestrin (Wang et al., 1998). Different species and different excitable cells express various RyR isoforms. In mammals, RyR-1 and RyR-2 appear to be expressed predominantly in skeletal muscle and heart, respectively, whereas RyR-3 is expressed in brain, smooth muscle, and epithelial cells (McPherson and Campbell, 1993; Furuichi et al., 1994; Giannini and Sorrentino, 1995). Furthermore, RyR-1 is expressed in sea urchin eggs and may be responsible for Ca²⁺ signaling in fertilization (McPherson et al., 1992). These reports suggest that RyRs play important roles not only in skeletal muscle but also in nonmuscle cells. In spite of the significance, the detailed regulatory mechanisms of the RyR remain unclear.

A number of thiol reagents, the reactive oxygen species, H₂O₂ (Oba et al., 1998), emodin (Cheng and Kang, 1998), S-nitrosoglutathione (Xu et al., 1998), N-ethylmaleimide (Aghdasi et al., 1997), and glutathione disulfide (Zable et al., 1997) act as stimulators of Ca²⁺ release channel of SR. These reports suggested that oxidation, alkylation, or nitrosylation of sulfhydryl groups on Ca²⁺ release channels of SR regulate the channel activation. It was reported that oxidation of sulfhydryl groups to disulfide caused stimulation of Ca²⁺ release from SR, contraction of skinned muscle fibers (Abramson and Salama, 1989), and alteration in high-affinity [³H]ryanodine binding to its receptor (Stoyanovsky et al., 1997). It is still unclear whether oxidation-reduction or disulfide interchange of sulfhydryls on the Ca²⁺ release channel plays a important role in EC coupling. But an increasing body of evidence suggests that such a mechanism could be an important element. Although there are a few articles that indicate the relationship between Ca²⁺ release from SR and sulfhydryl groups, the detailed pharmacological properties of Ca²⁺ release from SR through sulfhydryl modification have not been revealed.

It has been shown that xestoquinone (XQN) (Fig. 1), having a unique pentacyclic quinone structure, is a novel leading compound for valuable cardiotonic agents because of its unique mechanism of a positive inotropic effect on cardiac muscle (Kobayashi et al., 1991a,b). It also has been reported that XQN activated skeletal muscle actomyosin ATPase by modification of the specific sulfhydryl group in the myosin head (Sakamoto et al., 1995). Herein, we present the first report indicating that XQN powerfully induces Ca²⁺ release from the heavy fraction of fragmented sarcoplasmic reticulum (HSR) through modification of the crucial sulfhydryls.

View larger version (13K): [in this window] [in a new window]   Fig. 1.   Chemical structure of XQN.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Materials. XQN was isolated from the Okinawan sea sponge Xestospongia sapra as described previously (Nakamura et al., 1985). Briefly, the fresh sea sponge was extracted with methanol, and the methanol extract was chromatographed on silica gel columns to yield pure XQN. This compound was dissolved in diethyl sulfoxide and was stocked at 80°C. The substances we used were purchased from the sources indicated: ryanodine (S.B. Penick, New York, NY), ⁴⁵CaCl₂ (0.7 Ci/mmol; NEN Life Science Products, Boston, MA), and [³H]ryanodine (60 Ci/mmol; NEN Life Science Products). All other chemicals were of analytical grade.

Preparation of SR Vesicles from Skeletal Muscle. For all studies, HSR was prepared from rabbit white skeletal muscle as previously reported (Seino et al., 1991) with slight modification. White skeletal muscle was homogenized in 5 volumes of 5 mM Tris-maleate (pH 7.0) and centrifuged at 5000g for 15 min. The supernatant was further centrifuged at 12,000g for 30 min. The pellet fraction was suspended in a solution containing 90 mM KCl and 5 mM Tris-maleate (pH 7.0) and centrifuged at 70,000g for 40 min. The obtained HSR was suspended in the same solution containing 0.3 M sucrose at 80°C until use. The protein concentration was determined by the method of Bradford (1976) with BSA as a standard. The light fraction of fragmented sarcoplasmic reticulum (LSR) was prepared from rabbit white skeletal muscle as described by (Seino et al., 1991) with some modification.

⁴⁵Ca²⁺ Release Experiments. The ⁴⁵Ca²⁺ release from the vesicular HSR or LSR passively preloaded with ⁴⁵Ca²⁺ was measured at 0°C as described previously (Seino et al., 1991) with slight modification. After a 12-h preincubation of 20 mg/ml HSR or LSR suspension with 5 mM ⁴⁵CaCl₂ in a solution containing 90 mM KCl and 5 mM 3-(N-morpholino)propanesulfonic acid (MOPS)-Tris (pH 7.0) at 0°C, HSR or LSR suspension (5 µl) was diluted with 100 volumes (500 µl) of an ice-cold reaction medium containing 0.5 mM CaCl₂, 8.02 to 0.451 mM ethylene glycol bis(-aminoethyl ether)-N,N,N'N',-tetraacetic acid (EGTA), 90 mM KCl, and 50 mM MOPS-Tris (pH 7.0) in the presence or absence of the test substance. For measurement of the amount of ⁴⁵Ca²⁺ in HSR at time zero, HSR or LSR suspension was diluted with the reaction medium containing 5 mM LaCl₃ and 5 mM MgCl₂. At an appropriate time, 5 mM LaCl₃ and 5 mM MgCl₂ were added into the reaction medium to stop ⁴⁵Ca²⁺ release (Lattanzio et al., 1987). The reaction mixture was then filtered through a Millipore filter (HAMP type, 0.45-µm pore size; Millipore, Bedford, MA) and washed with 5 ml of an ice-cold solution containing 5 mM LaCl₃, 5 mM MgCl₂, 90 mM KCl, and 50 mM MOPS-Tris (pH 7.0). The amount of ⁴⁵Ca²⁺ remaining in HSR or LSR vesicles was measured by counting the radioactivity on the washed filters with a liquid scintillation counter. ⁴⁵Ca²⁺ release experiments were carried out at least in triplicate with preparations obtained from three different rabbits.

[³H]Ryanodine Binding Assay. [³H]ryanodine binding to HSR was examined as described previously (Furukawa et al., 1994) with slight modification. HSR (200 µg/ml) was incubated with 1 nM [³H]ryanodine at 37°C for 1 h in a solution containing 0.3 M sucrose, 0.3 M KCl, 100 µM CaCl₂, 20 mM Tris-HCl (pH 7.4), and (p-amidinophenyl) methanesulfonyl fluoride hydrochloride. The amount of [³H]ryanodine bound was determined by membrane filtration through Whatman filters (GF/B). Nonspecific binding was determined in the presence of 10 µM unlabeled ryanodine.

Free Ca²⁺ Concentration. The free Ca²⁺ concentration was maintained with Ca²⁺-EGTA buffer (0.55 mM CaCl₂ plus 8.02-0.451 mM EGTA) and was estimated with a microcomputer program that took into account the binding constant for Ca²⁺-EGTA; pH; and the concentrations of K⁺, Mg²⁺, and nucleotides (Sillen and Martell, 1964, 1971).

Statistical Analysis. Data are expressed as means ± S.E. Statistical comparisons were made with Student's t test for paired data. P < .05 was considered significant.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

⁴⁵Ca²⁺ Release from SR Vesicles. Because in the presence of Mg · ATP and an ATP-regenerating system Ca²⁺ release is immediately followed by reuptake, we have examined the release of passively loaded Ca²⁺ in the absence of Mg · ATP (Kim et al., 1983). The effect of XQN and caffeine on Ca²⁺ release from HSR or LSR were studied in ⁴⁵Ca²⁺ release experiments under conditions in which the Ca²⁺ pump does not work at 0°C. The ⁴⁵Ca²⁺ releasing activity of caffeine (1 mM) was significant in HSR (28.1% ± 0.3% of total Ca²⁺ loaded into HSR), whereas this was slightly observed in LSR (10.9% ± 1.7% of total Ca²⁺ loaded into LSR). However, XQN (30 µM) induced ⁴⁵Ca²⁺ release both from HSR (26.0 ± 0.2% of total Ca²⁺ loaded into HSR) and LSR (27.6 ± 0.5% of t total Ca²⁺ loaded into LSR) (data not shown). Figure 2 shows the concentration-response curve for XQN and caffeine in ⁴⁵Ca²⁺ efflux. ⁴⁵Ca²⁺ release from HSR was accelerated markedly by XQN and caffeine in a concentration-dependent manner. Based on the EC₅₀ value, XQN is 10 times more potent than caffeine. As shown in Fig. 3, ⁴⁵Ca²⁺ release induced by caffeine (1 mM) was not changed by dithiothreitol (DTT) (1 mM). However, the amount of ⁴⁵Ca²⁺ release induced by XQN (30 µM) was markedly inhibited by it (30 µM, 1 mM). Figure 4 shows the effects of typical inhibitors of Ca²⁺ release on XQN and caffeine-induced ⁴⁵Ca²⁺ release. Interestingly, ⁴⁵Ca²⁺ release induced by caffeine (1 mM) was inhibited by those blockers of Ca²⁺-induced Ca²⁺ release such as Mg²⁺ (Fig. 4A), procaine (Fig. 4B), and ruthenium red (Fig. 4C), respectively, in a concentration-dependent manner, whereas ⁴⁵Ca²⁺ release that induced by XQN (30 µM) was not inhibited by them. As shown in Fig. 5, XQN (10 µM) and caffeine (1 mM) potentiated ⁴⁵Ca²⁺ release from HSR with a bell-shaped profile of Ca²⁺ dependence, whereas both the patterns were different from each other. The sensitivity of Ca²⁺ for the channels increased in the presence of caffeine at concentrations of pCa 7 and 8, whereas it was not changed at pCa 6 to 3. However, XQN shifted that upward in a wider range of pCa 7 to 3. 

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Concentration-dependent acceleration of 45Ca2+ release from HSR vesicles by XQN and caffeine. 45Ca2+ release was measured at pCa 7 as described in Experimental Procedures. The amount of released Ca2+ was calculated from the decrease in the 45Ca2+ content in HSR vesicles during 1 min after dilution. Each value was obtained by subtracting the amount of released 45Ca2+ measured in the absence of the test substances from that measured in the its presence. Caffeine (); XQN (). Values are means ± S.E. (n = 3).

View larger version (57K): [in this window] [in a new window]   Fig. 3.   Effect of DTT, a thiol-reducing agent, on 45Ca2+ release induced by XQN and caffeine. 45Ca2+ release from HSR for 1 min was measured. Experimental protocols were the same as described in Fig. 2. The concentrations of XQN and caffeine were 30 µM and 1 mM, respectively. Values are means ± S.E. (n = 3). *P < .01.

View larger version (26K): [in this window] [in a new window]   Fig. 4.   Effect of typical inhibitors for Ca2+-induced Ca2+ release on 45Ca2+ release induced by XQN and caffeine at concentration of pCa 7. Concentration-dependent effect of Mg2+ (A), ruthenium red (B), and procaine (C) on XQN and caffeine-induced Ca2+ release was investigated. Values are described as difference between 45Ca2+ release in the presence and in the absence of XQN and caffeine. Experimental protocols were the same as those described in Fig. 2. 1 mM caffeine (); 30 µM XQN (). Values are means ± S.E. (n = 3).

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Ca2+ dependence of 45Ca2+ release induced by XQN and caffeine from HSR. 45Ca2+ release from HSR for 1 min was measured. Experimental protocols were the same as described in Fig. 2 except for free Ca2+ concentrations (pCa 8 to 3). Control (), 1 mM caffeine (), 30 µM XQN (). Values are means ± S.E. (n = 3). *P < .05, **P < .01.

View larger version (79K): [in this window] [in a new window]   Fig. 6.   Interrelations among the Ca2+-releasing activities of XQN, MBED, and caffeine. 45Ca2+ release from HSR for 1 min was measured. Experimental protocols were the same as described in Fig. 4. The concentration of XQN, caffeine, and MBED were 30 µM, 30 mM, and 1 µM, respectively. Values are means ± S.E. (n = 3). *P < .01.

[³H]Ryanodine Binding to HSR. [³H]ryanodine binding to HSR was examined in the presence or absence of XQN. XQN caused a concentration-dependent decrease in [³H]ryanodine binding to HSR (Fig. 7). Figure 8 shows a typical saturation curve (A) and its corresponding Scatchard plots (B) of [³H]ryanodine binding to HSR in the presence or absence of XQN (5 µM). XQN was shown to decrease [³H]ryanodine binding noncompetitively. The B_max value was decreased from 28.3 to 19.4 pmol/mg by adding XQN, whereas the K_D value was unaffected (10.02 nM for control and 9.95 nM with XQN). XQN (30 µM) induced a marked decrease in [³H]ryanodine binding to HSR in a wider range of pCa 7 to 4 (Fig. 9). Moreover, we investigated the effect of DTT on XQN-induced [³H]ryanodine binding to HSR to demonstrate whether XQN affected [³H]ryanodine binding to HSR through sulfhydryl modification. [³H]Ryanodine binding to HSR was decreased 90% by XQN (30 µM), and the effect of XQN on [³H]ryanodine binding was abolished by treatment with DTT (1 mM) (Fig. 10).

View larger version (17K): [in this window] [in a new window]   Fig. 7.   Concentration-dependent effects of XQN on [3H]ryanodine binding to HSR. HSR (200 µg/ml) was incubated with 1 nM [3H]ryanodine in the presence of various concentrations of XQN for 1 h at 37°C. Specific binding was derived by subtracting nonspecific binding determined in the presence of 10 µM unlabeled ryanodine. Values are means ± S.E. (n = 3).

View larger version (19K): [in this window] [in a new window]   Fig. 8.   A typical saturation curve (A) and its corresponding Scatchard plots (B) of [3H]ryanodine binding to HSR in the presence or absence of XQN. HSR (200 µg/ml) was incubated with 1 to 100 nM [3H]ryanodine for 1 h at 37°C in the presence () or absence () of 5 µM XQN. Values are means ± S.E. (n = 3).

View larger version (15K): [in this window] [in a new window]   Fig. 9.   Ca2+ dependence of [3H]ryanodine binding to HSR in the presence or absence of XQN. HSR (200 µg/ml) was incubated with 1 nM [3H]ryanodine at various concentrations of free Ca2+ for 1 h at 37°C in the presence () or absence () of 30 µM XQN. Specific binding was derived by subtracting nonspecific binding determined in the presence of 10 µM unlabeled ryanodine. Values are means ± S.E. (n = 3).

View larger version (85K): [in this window] [in a new window]   Fig. 10.   Effect of DTT on XQN-induced [3H]ryanodine binding. HSR (200 µg/ml) was incubated with 1 nM [3H]ryanodine in the presence of XQN, DTT, or both XQN and DTT for 1 h at 37°C. Specific binding was derived by subtracting nonspecific binding determined in the presence of 10 µM unlabeled ryanodine. The concentrations of XQN and DTT were 30 µM and 1 mM, respectively. Values are means ± S.E. (n = 3). *P < .01.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The fundamental idea that sulfhydryl-to-disulfide conversion of cysteine residues on various ion channels serves as a regulatory mechanism of their activity is becoming increasingly documented and persuasive. Regulatory sulfhydryls have been demonstrated in K⁺ channels (Lei et al., 1992), N-methyl-D-aspalate receptor-channel complex (Ruppersberg et al., 1991), the inositol 1,4,5-triphosphate receptor (Kaplin et al., 1994), and the RyR (Zaidi et al., 1989; Salama et al., 1992). The RyR generally known as a Ca²⁺-releasing channel may be the physiological mechanism of EC coupling in skeletal muscle (Shoshan et al., 1998). Recently, it has been reported that sulfhydryl groups have important roles in modulation of the activity of skeletal muscle Ca²⁺ release channel (Aghdasi et al., 1997; Stoyanovsky et al., 1997). However, the detailed pharmacological properties of Ca²⁺ release from SR through sulfhydryl modifications are not known yet.

XQN, isolated from sea sponge, has been indicated to act on substances possessing sulfhydryl groups (Sakamoto et al., 1995). It has been reported that bastadins, isolated from another class of sponge, convert ryanodine-insensitive leak states into ryanodine-sensitive channels that recognize [³H]ryanodine with high affinity through their modulatory actions on the FKBP12/RyR-1 complex (Mack et al., 1994). In the present study, we found that XQN caused a concentration-dependent ⁴⁵Ca²⁺ release from HSR and that the Ca²⁺-releasing potency of XQN was 10 times more potent than that of caffeine. XQN-induced ⁴⁵Ca²⁺ release from HSR was completely inhibited in the presence of DTT but that of caffeine was not affected by it. Furthermore, the effect of XQN on [³H]ryanodine binding to HSR was abolished in the presence of DTT. Scatchard analysis of [³H]ryanodine binding to HSR reveals that XQN decreases it by decreasing B_max but does not affect the change in dissociation rate of bound [³H]ryanodine, suggesting that XQN binds to sulfhydryl of Ca²⁺ release channels to cause a decrease in [³H]ryanodine binding to HSR noncompetitively. In general, [³H]ryanodine binding with high affinity to saturable and Ca²⁺-dependent sites in the Ca²⁺ release channel is enhanced by channel activators, whereas it is decreased by channel inhibitors (Pessah et al., 1987; Michalak et al., 1988). In this study, we found XQN markedly decreased [³H]ryanodine binding to HSR in a Ca²⁺-independent manner. Pessah et al. (1997) have shown that bastadins, another class of sponge toxins, enhance the number of high-affinity binding sites of [³H]ryanodine to SR. However, it has been reported that thimerosal, a thiol-oxidizing reagent, stimulates Ca²⁺ release from skeletal muscle SR with an EC₅₀ value of ~200 µM but inhibits [³H]ryanodine binding by decreasing B_max without affecting K_D (Abramson et al., 1995). These observations suggest that the pharmacological properties of XQN on HSR are similar to those of thimerosal and that XQN is approximately seven times more potent than thimerosal on the basis of EC₅₀. Recently, it has been reported that nanomolar naphthoquinone enhances occupancy of [³H]ryanodine, whereas low micromolar naphthoquinone inhibits the binding of [³H]ryanodine to SR membrane (Feng et al., 1999).

Procaine, ruthenium red, and Mg²⁺ have been used extensively as the inhibitors of Ca²⁺-induced Ca²⁺ release (Smith et al., 1988; McPherson and Campbell, 1993; Kawano, 1998). For example, procaine has been shown to interact selectively with a closed state of the channel rather than with an open state (Zahradnikova and Palade, 1993). We examined the effects of those inhibitors on ⁴⁵Ca²⁺ release induced by XQN and caffeine. Interestingly, caffeine-induced ⁴⁵Ca²⁺ release was inhibited by these blockers in a concentration-dependent manner, whereas XQN-induced ⁴⁵Ca²⁺ release was not inhibited by them. Furthermore, ⁴⁵Ca²⁺ release from HSR was potentiated by caffeine in a narrower range of pCa, whereas it was significantly stimulated by XQN in a wider range of pCa, demonstrating that the profile of Ca²⁺-dependence of XQN-induced ⁴⁵Ca²⁺ release was different from that of caffeine. Recently, it was reported that a functionally important interaction between RyR-1 and triadin exists that partially involves redox cycling of hyperreactive sulfhydryls in response to channel activation and inactivation (Liu and Pessah, 1994). Triadin has been shown to decrease [³H]ryanodine binding to HSR and openings of the RyR incorporated into the planar lipid bilayers (Ohkura et al., 1998). However, we found that XQN caused ⁴⁵Ca²⁺ release not only from HSR but also LSR, which had far fewer RyRs than HSR. On the basis of these observations, it is suggested that there are three possibilities: XQN causes Ca²⁺ release through sulfhydryl modification of 1) novel Ca²⁺ release channels; 2) the RyR or novel Ca²⁺ release channels having changed gating properties; or 3) regulatory protein, including triadin.

It has been reported that there are several effector-binding domains in the Ca²⁺ release channel (Shoshan et al., 1998). MBED, a derivative of bromo eudistomin D, induced a contraction of chemically skinned fiber from skeletal muscle, and has been shown to elicit Ca²⁺ release from the Ca²⁺ store site in isolated myocytes (Seino et al., 1990). MBED, which binds to the same site as that of caffeine, does not alter the maximal ⁴⁵Ca²⁺ release induced by caffeine, whereas adenosine triphosphate, which binds to different site from that of caffeine, further increases that induced by caffeine (Seino et al., 1990). In the present study, the interrelation among the stimulatory effects of XQN, caffeine, and MBED was examined. The maximum response of ⁴⁵Ca²⁺ release to caffeine was additively increased by XQN, whereas it was not increased by MBED. These observations suggest that XQN acts on a different site from that of caffeine and/or MBED in the Ca²⁺ release channels or novel Ca²⁺ release channels as well as RyRs.

In summary, it has been demonstrated that sulfhydryl groups are involved in both the XQN effect on Ca²⁺ release from HSR on ryanodine binding to HSR. XQN may be a useful pharmacological tool to elucidate the function of sulfhydryls in Ca²⁺ release channels in skeletal muscle.