Introduction

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The ryanodine receptor, which functions as a Ca²⁺ release channel of sarcoplasmic reticulum (SR), is postulated to play a key role in excitation-contraction coupling in the muscle (McPherson and Campbell, 1993; Coronado et al., 1994; Sutko and Airey, 1996). Genes encoding ryanodine receptors have been referred to as ryanodine receptors 1, 2, and 3. Ryanodine receptors 1 and 2 appear to be expressed predominantly in skeletal muscle and heart, respectively, whereas ryanodine receptor 3 is expressed in brain, smooth muscle, and epithelial cells (McPherson and Campbell, 1993; Sutko and Airey, 1996). Several compounds, such as amentoflavone (Suzuki et al., 1999), 2-hydroxycarbazole (Tovey et al., 1998), bastadins (Mack et al., 1994), and 9-methyl-7-bromoeudistomin D (MBED; Seino et al., 1991), have been shown to induce Ca²⁺ release from skeletal muscle SR mediated by the ryanodine receptor. Our previous reports indicated that myotoxin a (Ohkura et al., 1995), puff adder lectin (Ohkura et al., 1996a), and quinolidomicin A₁ (Ohkura et al., 1996b) induced Ca²⁺ release from the heavy fraction of fragmented SR (HSR) with novel properties.

Numerous marine natural products have been isolated and given much attention as useful tools for pharmacological and biological studies because of their actions on the specific sites of functional proteins (Ohizumi, 1997; Moriya et al., 1998; Strachan et al., 1999). In the course of our survey of pharmacologically active substances from natural resources, we have devoted our attention to the occurrence of natural compounds possessing Ca²⁺ releasing activity from skeletal muscle SR, because these compounds are useful as chemical probes to elucidate the functional ryanodine receptor. Recently, we successfully isolated bisprasin (Fig. 1) from a marine sponge of Dysidea spp. collected at Palau. This compound is a unique brominated tyrosine-derived metabolite containing a disulfide linkage. Here, we present the first report on the pharmacological properties of bisprasin, which induces Ca²⁺ release from skeletal muscle SR mediated through the ryanodine receptor in the same way as does caffeine.

View larger version (5K): [in this window] [in a new window]   Fig. 1.   Chemical structure of bisprasin.

	Experimental Procedures

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Materials. Bisprasin was isolated from a marine sponge of Dysidea spp. Briefly, Dysidea spp. (500 g) was extracted with acetone/methanol (1:1). The extract was concentrated under reduced pressure, and the residue was partitioned between ethyl acetate and water. The ethyl acetate-soluble fraction (3.4 g) was chromatographed over silica gel with a stepwise gradient of chloroform/methanol as eluant. The fraction (1.2 g) eluted with chloroform/methanol (9:1) was subjected to the HPLC with methanol/water as eluant, resulting in the isolation of active compound (380 mg). The chemical structure of this active compound was elucidated to be bisprasin on the basis of physicochemical data such as NMR, mass, and infrared spectra (Arabshahi and Schmitz, 1987). We purchased ryanodine from S. B. Penick (New York, NY). Procaine was purchased from Sigma Chemical Co. (St. Louis, MO). ⁴⁵CaCl₂ (0.7 Ci/mmol) and [³H]ryanodine (60 Ci/mmol) were purchased from NEN Life Science Products. All other chemicals were of analytical grade.

Preparation of SR Vesicles from Skeletal Muscle. HSR enriched in Ca²⁺-induced Ca²⁺ release activity was prepared from rabbit skeletal muscle as previously reported (Seino et al., 1991) with slight modification. Male rabbits (Japanese White; weight, 3 kg) were anesthetized by i.v. injection of pentobarbital sodium, and the white muscle was removed. The animals used in this study were treated in accordance with the principles and guidelines of Tohoku University Council on Animal Care. All solutions used to prepare SR membranes included protease inhibitors 76.8 mM aprotinin and 0.83 mM benzamidine. White muscle was homogenized four times with a National MX-915C mixer in 5 volumes of 5 mM Tris-maleate (pH 7.0) for 30 s at 30-s intervals. The homogenate was centrifuged at 5000g for 15 min. The supernatant was filtered through the cheesecloth, and the filtrate was centrifuged again at 12,000g for 30 min. The pellets were 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 pellets were suspended in a solution containing 90 mM KCl, 5 mM Tris-maleate (pH 7.0), and 0.3 M sucrose.

⁴⁵Ca²⁺ Release Experiments. ⁴⁵Ca²⁺ release from the vesicular HSR passively preloaded with ⁴⁵Ca²⁺ was measured at 0°C as described previously (Nakamura et al., 1986) with slight modification. After a 12-h preincubation of 20 mg/ml HSR with 5 mM ⁴⁵CaCl₂ in a solution containing 90 mM KCl and 50 mM 3-(N-morpholino)propanesulfonic acid (MOPS)-Tris (pH 7.0) at 0°C, the suspension was diluted with 100 volumes of an ice-cold reaction medium containing 0.4 mM CaCl₂ with varying concentrations of EGTA, 90 mM KCl, and 50 mM MOPS-Tris (pH 7.0). For measurement of the amount of ⁴⁵Ca²⁺ in HSR at time 0, the suspension was diluted with the reaction medium containing 5 mM LaCl₃. At an appropriate time, 5 mM LaCl₃ was added to stop ⁴⁵Ca²⁺ release. The reaction mixture was then filtered with a Millipore filter (HAMP type, 0.45 mm pore size) and washed with 5 ml of a 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 vesicles was measured by counting the radioactivity on the washed filters.

Binding Assays. [³H]Ryanodine binding to HSR was examined as described previously (Furukawa et al., 1994) with slight modification. HSR (100 µg/ml) was incubated with 1 to 20 nM [³H]ryanodine at 37°C for 2 h in a solution containing 0.3 M sucrose, 0.3 M KCl, 100 µM CaCl₂, and 20 mM Tris-HCl (pH 7.4). 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.

Mechanical Response. The procedure for preparing the diaphragm and the technique of measurement of contractile response were performed as described previously (Ohizumi et al., 1986). Hemidiaphragm preparations were isolated from male mice (ddys; weight, 25-30 g) and mounted in an organ bath containing 5 ml of Krebs-Ringer-bicarbonate solution of 120 mM NaCl, 4.8 mM KCl, 1.2 mM CaCl₂, 1.3 mM MgSO₄, 1.2 mM KH₂PO₄, 25.2 mM NaHCO₃, and 5.8 mM glucose (pH 7.4) and were aerated with 95% O₂/5% CO₂ at 37°C. A resting tension of 1 g was applied to each preparation. Isometric contractions were measured by a force-displacement transducer and recorded on a polygraph. Preparations were stimulated directly with 5-ms pulses (supramaximal voltage) at a frequency of 0.1 Hz.

Statistical Analysis. The data are expressed as means ± S.E. Statistical comparisons were made by using Student's t test for unpaired data. The n number for statistical analysis presented the number of different preparations. P < .05 was considered significant.

	Results

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⁴⁵Ca²⁺ Release from SR Vesicles by Bisprasin and Caffeine. The effects of bisprasin and caffeine on ⁴⁵Ca²⁺ release from HSR vesicles were measured under the conditions in which the Ca²⁺ pump did not work. Figure 2 shows the time course of the change in ⁴⁵Ca²⁺ content in HSR after the addition of bisprasin. The ⁴⁵Ca²⁺ release was markedly accelerated by bisprasin over the concentration range of 10 to 100 µM. The concentration-response curve of ⁴⁵Ca²⁺ release from HSR for bisprasin and caffeine is shown in Fig. 3. ⁴⁵Ca²⁺ release was increased by bisprasin (EC₅₀ = 18 µM) and caffeine (EC₅₀ = 1.2 mM) in a concentration-dependent manner. In LSR, 2.1 ± 0.4 and 11.2 ± 2.5% of ⁴⁵Ca²⁺ release from HSR were induced by bisprasin (30 µM) and caffeine (1 mM), respectively.

View larger version (19K): [in this window] [in a new window]   Fig. 2.   The time course of stimulatory effects of bisprasin on 45Ca2+ release from HSR. The 45Ca2+ content in HSR vesicles was measured at 0°C by the Millipore filtration method. , control; , 10 µM bisprasin; , 20 µM bisprasin; , 30 µM bisprasin; , 100 µM bisprasin. **P < .01, statistically significant difference from the control.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent acceleration of 45Ca2+ release from HSR by bisprasin () and caffeine (). 45Ca2+ release was measured at a Ca2+ concentration of 0.1 µM. The amount of 45Ca2+ release 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 45Ca2+ release measured in the absence of bisprasin from that measured in its presence. Values are means ± S.E. (n = 3). **P < .01, statistically significant difference from the control.

Effects of Bisprasin and Caffeine on ⁴⁵Ca²⁺ Release from HSR over a Wide Range of Free Ca²⁺ Concentrations. ⁴⁵Ca²⁺ release was increased in a linear fashion at the free Ca²⁺ concentration from 0.1 to 1 µM (EC₅₀ = 0.25 µM), reached a maximal response at 3 µM, and decreased when the free Ca²⁺ concentration was increased further from 10 to 100 µM (Fig. 4). Bisprasin (20 µM) and caffeine (1 mM) caused a parallel left shift of the concentration-response curve for ⁴⁵Ca²⁺ release plotted against the external Ca²⁺ concentration (from 1 nM to 3 µM) without affecting the maximal response (Fig. 4). At a free Ca²⁺ concentration of 100 µM, bisprasin (20 µM) stimulated ⁴⁵Ca²⁺ release, but caffeine (1 mM) did not affect it.

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Effects of free Ca2+ concentration on the 45Ca2+ release from HSR in the absence or presence of bisprasin and caffeine. , control. , 1 mM caffeine. , 20 µM bisprasin. 45Ca2+ release was expressed as a percentage against a maximum release (100%) in the absence of bisprasin and caffeine at a Ca2+ concentration of 3 µM. Values are means ± S.E. (n = 3). *P < .05, **P < .01, statistically significant difference from the control.

Effects of Typical Inhibitors of Ca²⁺-Induced Ca²⁺ Release Channels on Bisprasin- and Caffeine-Induced ⁴⁵Ca²⁺ Release. ⁴⁵Ca²⁺ release induced by 20 µM bisprasin and 1 mM caffeine was inhibited by ruthenium red (Fig. 5), Mg²⁺ (Fig. 6), and procaine (Fig. 7) in a concentration-dependent manner. It is probable that under the present experimental conditions, there was a component refractory to the inhibition by procaine (Fig. 7).

View larger version (15K): [in this window] [in a new window]   Fig. 5.   Effects of ruthenium red on the 45Ca2+ release induced by bisprasin and caffeine from HSR. Data are expressed as the difference between 45Ca2+ release in the presence and in the absence of bisprasin and caffeine. Values are means ± S.E. (n = 3). , 1 mM caffeine. , 20 µM bisprasin. **P < .01, statistically significant difference from the control (100%).

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Effects of Mg2+ on the 45Ca2+ release induced by bisprasin and caffeine from HSR. Data are expressed as the difference between 45Ca2+ release in the presence and in the absence of bisprasin and caffeine. Values are means ± S.E. (n = 3). , 1 mM caffeine. , 20 µM bisprasin. *P < .05, **P < .01, statistically significant difference from the control (100%).

View larger version (14K): [in this window] [in a new window]   Fig. 7.   Effects of procaine on the 45Ca2+ release induced by bisprasin and caffeine from HSR. Data are expressed as the difference between 45Ca2+ release in the presence and in the absence of bisprasin and caffeine. Values are means ± S.E. (n = 3). , 1 mM caffeine. , 20 µM bisprasin. *P < .05, **P < .01, statistically significant difference from the control (100%).

[³H]Ryanodine Binding to HSR. [³H]Ryanodine binding to HSR was examined in the presence or absence of bisprasin and caffeine. As shown in Fig. 8, [³H]ryanodine binding was increased by bisprasin (10 and 20 µM) and caffeine (30 mM). The degree of enhancement by bisprasin (by approximately 25%) was comparable to that of caffeine (by 25%) and MBED (by 20%, Seino et al., 1991). Figure 9 shows a saturation curve (A and C) and a corresponding Scatchard plot (B and D) of [³H]ryanodine binding in the presence or absence of bisprasin (A and B) or caffeine (C and D). The K_D value was decreased from 8.33 ± 0.48 (n = 3) to 4.17 ± 0.52 (n = 3) and from 6.20 ± 0.88 (n = 3) to 4.80 ± 0.73 (n = 3) by adding bisprasin and caffeine, respectively, whereas the B_max value was unaffected. As shown in Fig. 10, bisprasin (20 µM)- and caffeine (30 mM)-induced increases in [³H]ryanodine binding to HSR were further increased with adenosine-5'-(,-methylene)triphosphate (AMP-PCP; 100 µM) by 20 and 30%, respectively.

View larger version (60K): [in this window] [in a new window]   Fig. 8.   Effects of bisprasin and caffeine on [3H]ryanodine binding to HSR. HSR (100 µg/ml) was incubated with 2.5 nM [3H]ryanodine in the presence of various concentrations of bisprasin for 2 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). **P < .01, statistically significant difference from the control.

View larger version (24K): [in this window] [in a new window]   Fig. 9.   A typical saturation curve (A and C) and its corresponding Scatchard plot (B and D) of [3H]ryanodine binding to HSR in the presence or absence of 20 µM bisprasin (A and B) and 3 mM caffeine (C and D). HSR (100 µg/ml) was incubated with 1 to 20 nM [3H]ryanodine for 2 h at 37°C in the presence or absence of 20 µM bisprasin and 3 mM caffeine. A and B: , control; , 20 µM bisprasin. C and D, , control; , 3 mM caffeine. Values are means ± S.E. (n = 3). *P < .05, statistically significant difference from the control.

View larger version (52K): [in this window] [in a new window]   Fig. 10.   Effects of AMP-PCP on the [3H]ryanodine binding by bisprasin and caffeine. HSR (100 µg/ml) was incubated with 2.5 nM [3H]ryanodine in the presence of various concentrations of bisprasin for 2 h at 37°C. Specific binding was derived by subtracting nonspecific binding determined in the presence of 10 µM unlabeled ryanodine. The concentrations of bisprasin, caffeine, and AMP-PCP were 20 µM, 30 mM, and 100 µM, respectively. Values are means ± S.E. (n = 3). *P < .05, **P < .01, statistically significant difference.

Effects of Bisprasin on Contractile Response of Isolated Mouse Hemidiaphragm. Tetrodotoxin (1 µM), a Na⁺ channel blocker, nearly abolished the contraction of hemidiaphragm induced by direct electrical stimulation, whereas bisprasin (30 µM) had no or little effect on it. KCl (50 mM)-induced contracture of hemidiaphragm was not affected by bisprasin (30 µM).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

The ryanodine receptor, which is generally known as a Ca²⁺-induced Ca²⁺ release channel of SR (Ebashi, 1991; Sutko and Airey, 1996), may be the machinery of excitation-contraction coupling in skeletal muscle (Ford and Podolsky, 1970; Endo, 1977). Ryanodine was reported to selectively bind to its receptor in an open state. (McPherson and Campbell, 1993). The Ca²⁺ channel has been purified using [³H]ryanodine as a specific ligand (Inui et al., 1987; Hymel et al., 1988; Wagenknecht et al., 1989). Not only ryanodine but also a variety of natural products, such as imperatoxin (Valdivia et al., 1992) and MBED (Seino et al., 1991), have attracted the attention of pharmacologists, physiologists, and biochemists because they act on their specific binding sites in the ryanodine receptor with high affinity. The function of Ca²⁺ release channels is inhibited by several inhibitors, such as procaine, Mg²⁺, ruthenium red, and spermine (Palade, 1987; McPherson and Campbell, 1993).

In our survey of natural products that exhibit Ca²⁺ releasing activity, we found that bisprasin, a unique brominated tyrosine derivative with a disulfide linkage from a marine sponge, caused a concentration-dependent increase in ⁴⁵Ca²⁺ release from HSR under the conditions in which the Ca²⁺ pump did not work. The potency of the Ca²⁺ releasing action of bisprasin from HSR was approximately 70 times greater than that of caffeine. The Ca²⁺ dependence of bisprasin-induced ⁴⁵Ca²⁺ release from HSR had a bell-shaped profile similar to that of caffeine. The ⁴⁵Ca²⁺ release induced by bisprasin and caffeine was significantly inhibited by typical blockers of Ca²⁺-induced Ca²⁺ release channels, including procaine. These results suggest that like caffeine, bisprasin causes Ca²⁺ release by affecting Ca²⁺-induced Ca²⁺ release channels in HSR.

[³H]Ryanodine was reported to selectively bind to Ca²⁺-induced Ca²⁺ release channels in an open state (McPherson and Campbell, 1993). In general, [³H]ryanodine binding is potentiated by channel activators such as caffeine, MBED, and adenine nucleotides, whereas it is decreased by blockers of Ca²⁺-induced Ca²⁺ release channels (Su and Chang, 1995; Ohkura et al., 1996b). [³H]Ryanodine binding experiments are useful for the study of the functional state of the channel (Coronado et al., 1994). Bisprasin, like caffeine (Seino et al., 1991), markedly enhanced [³H]ryanodine binding to HSR. Scatchard analysis of [³H]ryanodine binding revealed that bisprasin and caffeine decreased the K_D value without affecting the B_max value. Bisprasin- and caffeine-induced increase in [³H]ryanodine binding to HSR was further increased by AMP-PCP, an unhydrolyzable adenine nucleotide analog. These results suggest that bisprasin, like caffeine, makes it easy to open the Ca²⁺-induced Ca²⁺ release channels, resulting in inducing Ca²⁺ release from HSR.

It has been reported that in skeletal muscle, the twitch response to direct electrical stimulation is attributed to an increasing Na⁺ permeability of Na⁺ channels, whereas the KCl-induced contracture is due to an increase in Ca²⁺ permeability of Ca²⁺ channels. Tetrodotoxin, a Na⁺ channel blocker, nearly abolished the contraction of isolated mouse hemidiaphragm induced by direct electrical stimulation. However, even at high concentrations, bisprasin had no or little effect on it. KCl-induced contracture of hemidiaphragm was not affected by bisprasin. These results suggest that bisprasin might be a specific Ca²⁺ releaser that has no effect on Na⁺ or Ca²⁺ channels. On the other hand, it is well known that LSR has far fewer ryanodine receptors than HSR (Liu et al., 1994). Bisprasin, like caffeine, slightly caused ⁴⁵Ca²⁺ release from LSR, suggesting that both of the drugs are preferential Ca²⁺ releasers in HSR.

In conclusion, the pharmacological properties of bisprasin-induced Ca²⁺ release from HSR are very similar to those of caffeine, except for its 70-fold higher potency. This is the first report on the pharmacological properties of bisprasin, which is a caffeine-like Ca²⁺ releaser in HSR that may serve as a useful biochemical tool to clarify the regulatory mechanism of Ca²⁺-induced Ca²⁺ release in HSR.