Bisprasin, a Novel Ca Releaser with Caffeine-Like Properties from a Marine Sponge, Dysidea spp., Acts on Ca-Induced Ca Release Channels of Skeletal Muscle Sarcoplasmic Reticulum

Bisprasin, a unique bromotyrosine derivative containing a disulfide linkage, was isolated from a marine sponge of Dysidea spp. This compound caused a concentration-dependent (from 10 to 30 mM) increase in the Ca release from the heavy fraction of skeletal muscle sarcoplasmic reticulum (HSR) of rabbit skeletal muscle in the same way as does caffeine. The 50% effective concentrations of bisprasin and caffeine were approximately 18 mM and 1.2 mM, respectively, indicating that the Ca-releasing activity of bisprasin was approximately 70 times more potent than that of caffeine in HSR. The bell-shaped profile of Ca dependence for bisprasin was almost the same as that for caffeine. Typical blockers of Ca-induced Ca release channels, such as Mg, procaine, and ruthenium red, inhibited markedly bisprasinand caffeine-induced Ca release from HSR. This compound, like caffeine, significantly enhanced [H]ryanodine binding to HSR. Scatchard analysis of [H]ryanodine binding to HSR revealed that bisprasin and caffeine decreased the KD value without affecting the Bmax value, suggesting that both the drugs facilitate the opening of ryanodine receptor channels. The bisprasinand caffeine-induced increases in [H]ryanodine binding were further enhanced by adenosine-59-(b,g-methylene)triphosphate. These results suggest that the pharmacological properties of bisprasin are almost similar to those of caffeine, except for its 70-fold higher potency. Here, we present the first report on the pharmacological properties of bisprasin, which, like caffeine, induces Ca release from skeletal muscle SR mediated through the ryanodine receptor. The ryanodine receptor, which functions as a Ca 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 Ca 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 Ca 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 Ca 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 Received for publication June 25, 1999. 1 This work was supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports and Culture of Japan. ABBREVIATIONS: SR, sarcoplasmic reticulum; HSR, heavy fraction of fragmented skeletal muscle sarcoplasmic reticulum; LSR, light fraction of fragmented skeletal muscle sarcoplasmic reticulum; MBED, 9-methyl-7-bromoeudistomin D; MOPS, 3-(N-morpholino)propanesulfonic acid; AMP-PCP, adenosine-59-(b,g-methylene)triphosphate. 0022-3565/00/2922-0725$03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 292, No. 2 Copyright © 2000 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 292:725–730, 2000 725 at A PE T Jornals on Jne 0, 2017 jpet.asjournals.org D ow nladed from 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 Ca release from skeletal muscle SR mediated through the ryanodine receptor in the same way as does caffeine. Experimental Procedures 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). CaCl2 (0.7 Ci/mmol) and [H]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 Ca-induced Ca 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. The light fraction of fragmented skeletal muscle SR (LSR) was prepared from rabbit skeletal muscle as described by Seino et al. (1991). 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 at 10,000g for 30 min. The supernatant was centrifuged again at 70,000g for 50 min. The pellets were suspended in a solution containing 0.6 M KCl, 5 mM Tris-maleate (pH 7.0), and 0.3 M sucrose, and this suspension was centrifuged at 100,000g for 70 min. This suspension/centrifugation cycle was repeated twice. The resultant pellets were washed with a solution containing 0.1 M KCl and 5 mM Tris-maleate (pH 7.0) and resuspended in the same solution to provide LSR suspension. The obtained SR vesicles were stored at 280°C until use. The protein concentration was determined according to the method of Bradford (1976) with BSA as a standard. Ca Release Experiments. Ca release from the vesicular HSR passively preloaded with Ca 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 CaCl2 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 Ca 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 Ca 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 Ca remaining in HSR vesicles was measured by counting the radioactivity on the washed filters. The free Ca concentration was maintained by using Ca-EGTA buffer (0.5 mM CaCl2 plus various concentrations of EGTA) and was estimated by using a microcomputer program that took into account the binding constant for Ca-EGTA, pH, and the concentration of K, Mg, and nucleotides (Sillen and Martell, 1964, 1971). Binding Assays. [H]Ryanodine binding to HSR was examined as described previously (Furukawa et al., 1994) with slight modification. HSR (100 mg/ml) was incubated with 1 to 20 nM [H]ryanodine at 37°C for 2 h in a solution containing 0.3 M sucrose, 0.3 M KCl, 100 mM CaCl2, and 20 mM Tris-HCl (pH 7.4). The amount of [ H]ryanodine bound was determined by membrane filtration through Whatman filters (GF/B). Nonspecific binding was determined in the presence of 10 mM 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 KrebsRinger-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 6 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.