YASUSHI OHIZUMI, KIMIHIRO MATSUNAGA, KEIGO NAKATANI and JUN ICHI KOBAYASHI

Transcription

1 /98/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 285, No. 2 Copyright 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 285: , 1998 Potent Stimulation of Myofilament Force and ATPase Activity of Skeletal Muscle by Eudistomin M, a Novel Ca -Sensitizing Agent from a Caribbean Tunicate 1 YASUSHI OHIZUMI, KIMIHIRO MATSUNAGA, KEIGO NAKATANI and JUN ICHI KOBAYASHI Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences (Y.O., K.M., K.N.), Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980, Japan and Department of Pharmacognosy, Faculty of Pharmaceutical Sciences (J.K.), Hokkaido University, Sapporo 060, Japan Accepted for publication January 20, 1998 This paper is available online at ABSTRACT In the course of our survey of biologically active compounds from natural sources, eudistomins were isolated from a Caribbean tunicate Eudistoma olivaceum. In the present experiments, eudistomin M (Eud-M, 10-5 M) caused a concentration-dependent increase in the contractile response of skinned fibers from guinea pig skeletal psoas muscles to Ca. The superprecipitation and ATPase activity of myosin B from fast skeletal muscles of rabbit back and leg were potentiated by this compound ( 10-5 M) in a concentration-dependent manner. In skinned fibers, superprecipitation and the ATPase activity of myosin B, Eud-M shifted the concentration-response curve for Binding of Ca to the Ca -specific sites of troponin C alters the interactions between troponin C, troponin I and troponin T. This in turn alters the troponin I-actin and troponin I- and troponin T-tropomyosin interactions in a manner that strengthens the actin-myosin interaction and results in a remarkable enhancement of the actomyosin ATPase activity. Concomitant with these change is the well known shift of tropomyosin in the groove of F-actin (Huxley, 1971). The superprecipitation of actomyosin is generally accepted to be basically the same phenomenon in vitro as a contraction in skeletal muscle cells (Szent-Györgyi, 1951). Numerous marine natural products have been useful as tools for physiological and biological studies because of their actions on specific sites of functional protein (Ohizumi, 1997). In the course of our survey on biologically active substances from marine sources, much attention has been given to compounds affecting the contractile apparatus. Recently, we have isolated several natural products that affect myosin and Received for publication September 10, 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. Financial support from The Sagawa Foundation for Promotion of Cancer Research and The Nakatomi Foundation are also acknowledged. Ca to the upper direction. Ca -, K -EDTA- or Mg - ATPase was not affected by Eud-M. This compound had no effect on the ATPase activity of actomyosin reconstituted from actin and myosin in the presence or absence of troponin. However, the ATPase activity of actin-myosin-troponin-tropomyosin reconstituted system was increased significantly by Eud-M. These results suggest that Eud-M increases the Ca sensitivity of the contractile apparatus in skeletal muscles at least partially mediated through troponin-tropomyosin system and thus enhances the ATPase activity of myosin B and the contractile force of myofilament. actin functions, such as purealin which modulates myosin ATPase activity (Takito et al., 1986; Nakamura et al., 1987), xestoquinone which modulates the specific sulfhydryl groups of myosin (Kobayashi et al., 1991a, b; Sakamoto et al., 1995) and goniodomin A which induces modulation of actomyosin ATPase activity mediated through conformational change of actin (Furukawa et al., 1993). In further research into marine natural products, eudistomins having -carboline skeleton were isolated from a Caribbean tunicate (Kobayashi et al., 1984). In our structure-activity relationship studies of eudistomin derivatives we found that MBED induced Ca release from the skeletal muscle SR about 1000 times more potent than caffeine (Seino et al., 1991) and bound the same binding site the same as that of caffeine (Fang et al., 1993). In our continuous screening program for bioactive substances from natural resources, Eud-M (fig. 1) has been shown to potentiate the ATPase activity of myosin B. It is of interest whether the potentiation by Eud-M is due to the direct effect on the myosin molecules or to the modulatory effect on the interaction between actin and myosin. We present the first report indicating that Eud-M potentiates the contractile system of skeletal muscles. ABBREVIATIONS: Eud-M, eudistomin M; MBED, 9-methyl-7-bromoeudistomin D; SR, sarcoplasmic reticulum; EGTA, ethylene glycol bis ( -aminoethyl ether)-n, N -tetraacetic acid; Ms, methanesulfonate. 695

2 696 Ohizumi et al. Vol. 285 Fig. 1. Chemical structure of eudistomin M (Eud-M). Materials and Methods Materials. Eud-M was isolated from a Caribbean tunicate Eudistoma olivaceaum as previously reported (Kobayashi et al., 1984). In the biochemical experiment, fast skeletal muscles of male rabbit (3 kg) back and leg were used to obtain much amount of experimental materials. Myosin B, actin, myosin, tropomyosin and troponin were prepared as described by Szent-Györgyi (1951), Spudich and Watt (1971), Weeds and Taylor (1975), Ebashi et al. (1968) and Kohama (1979), respectively. In skinned fiber experiment, psoas muscles of male guinea pig ( g) and male rabbit (3 kg) were used (Endo and Kitazawa, 1978; Endo and Iino, 1980; Horiuti, 1986). Skinned fiber experiments. Psoas muscles of male guinea pig and male rabbit were excised and washed rapidly with a Ringer s solution containing (mm): NaCl, 150; KCl, 2; CaCl 2, 2; glucose, 5.5; and 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid, 5 (ph 7.4) and were immediately transferred into relaxing solution containing (mm): K-Ms, 74.7; Mg-Ms 2, 5.4; ATP, 4; EGTA, 10; and piperazine-n, N -bis(2-ethanesulfonic acid)-koh, 20 (ph 7.0). A small muscle bundle of 4-5 fibers (ca. 0.1 mm in diameter and ca. 3 mm in length) was dissected from the psoas muscle. One end of the fiber was secured to the tissue holder by a ligature and the other end was connected to a force-displacement transducer (Acers AE801; Horten, Norway; the compliance of tension measurement system being approximately 0.5 mm/g) for measurement of isometric contraction of the fiber at 20 to 23 C. Fibers were treated with the relaxing solution containing 50 g/ml saponin for 30 min and then with a 0.5% Triton X-100 solution for 15 min (Endo and Iino, 1980; Horiuti, 1986). Various solutions for skinned fiber experiments were prepared as described elsewhere (Kobayashi et al., 1991a). The maximal tension in response to high Ca concentration was similar to the values in the literature (Endo and Iino, 1980; Horiuti, 1986). The survival of the preparation was at least 5 hr. Superprecipitation assay. The superprecipitation was induced by adding 0.4 mm ATP in 0.3 mg/ml myosin B, 0.76 mm CaCl 2,1mM EGTA, 2 mm MgCl 2, 50 mm KCl and 20 mm Tris-HCl at ph 6.8 and 25 C, and the change in the absorbance at 660 nm was followed. Enzyme assay. The reaction mixture for each ATPase was follows (mm): 0.3 mg/ml myosin B; ATP, 2; EGTA, 1; MgCl 2, 2; CaCl 2, 0.76; KCl, 50; and Tris-HCl, 20 (ph 6.8) for myosin B ATPase; 0.1 mg/ml actin; 0.1 mg/ml myosin; ATP, 1; CaCl 2, 0.76; EGTA, 2; KCl, 50; MgCl 2 2; and Tris-HCl, 20 (ph 6.8); for the ATPase activity of actomyosin reconstituted from actin and myosin; 0.1 mg/ml actin; 0.1 mg/ml myosin; 0.1 mg/ml troponin; ATP, 2; EGTA, 1; MgCl 2, 2; KCl, 50; Tris-HCl, 20 (ph 6.8); for the ATPase activity of actomyosin reconstituted from actin, myosin and troponin; 0.1 mg/ml actin; 0.1 mg/ml myosin; 0.2 mg/ml troponin-tropomyosin complex; ATP, 2; EGTA, 1; MgCl 2, 2; KCl, 50; Tris-HCl, 20 (ph 6.8); for the ATPase activity of actomyosin reconstituted from actin, myosin and troponintropomyosin complex; 0.15 mg/ml myosin; ATP, 2; CaCl 2, 10; KCl, 500; and Tris-HCl, 50 (ph 7.4); for the Ca -ATPase activity of myosin, mg/ml myosin; ATP, 2; EDTA-Tris, 5; KCl, 500; Tris- HCl, 50; for the K -EDTA-ATPase activity of myosin; 1.5 mg/ml myosin; ATP, 2; MgCl 2, 5; KCl, 500; and Tris-HCl, 50 (ph 7.4); for the Mg -ATPase activity of myosin. The mixture preincubated in the absence of Eud-M and ATP at 30 C for 5 min, followed by the addition of Eud-M and further preincubation. Eud-M was dissolved in dimethyl sulfoxide and a final concentration of dimethyl sulfoxide did not exceed 1%. Less than 1% dimethyl sulfoxide had little effect on the ATPase activities. The reaction was started by the addition of ATP and stopped by adding an equal volume of cold 10% trichloroacetic acid. The amount of inorganic phosphate liberated during the 5 min incubation was determined by the method of Martin and Doty (1949). Statistical analysis of the data. The data are expressed as means S.E.M. Statistical comparisons were made by using Student s t test. P.05 was considered significant. Results Contractile response of skinned fibers. To measure the contractile force of skinned fibers under the direct influence of Ca concentration, the fibers were prepared from guinea pig and rabbit skeletal muscles by sufficient treatment with detergents to destroy the function of both the cell membrane and SR membrane. Caffeine (40 mm) did not cause any contraction of skinned fibers, suggesting destruction of SR membrane (Nakamura et al., 1986). Figure 2 shows the typically recording trace of contractile response of skinned fibers of guinea pig skeletal muscle before and after exposure to Eud-M (10-4 M) in the presence of Ca ( M). The effect of Eud-M was abolished after wash out. Also similar recording trace was obtained in rabbit skeletal muscle skinned fibers (data not shown). As shown in figure 3, Eud-M ( to M) produced a concentration-dependent enhancement of the contractile response of skinned fibers to Ca. At high Ca concentrations above M, Eud-M ( M) increased the contractile response of skinned fibers to Ca (fig. 4). The maximum response to Ca was increased by 20% by it (fig. 4). Superprecipitation of myosin B. The effect of Eud-M was examined on the superprecipitation of skeletal myosin B, an in vitro model reaction of muscle protein contraction, by monitoring the turbidity change. After the addition of ATP, clearing occurred and then the turbidity increased for 20 min. Eud-M at 10-6 M or more enhanced the increase in turbidity without affecting clearing. Figure 5 shows the representative trace of the effects of various concentrations of Eud-M on the superprecipitation of myosin B prepared from rabbit skeletal muscles. As shown in figure 6, Eud-M caused a concentration-dependent increase in the maximum turbid- Fig. 2. Typically recording trace of the contractile response of skinned fibers from guinea pig psoas muscles at the Ca concentration of M in the presence or absence of eudistomin M (Eud-M, 10-4 M). The fibers were treated with Eud-M 1.5 min before the application of the MCa. The activating solution was washed off with the relaxing solution at allows (W).

3 1998 Eudistomin M Enhances Contraction 697 Fig. 3. Effects of eudistomin M (Eud-M) on the contractile response of skinned guinea pig skeletal muscle fibers. Increase in contraction was expressed as a percentage against the control tension (14 g) at the Ca concentration of M. Values are means S.E.M. (n 4). Fig. 4. Ca dependence of contractile response of skinned guinea pig skeletal muscle fibers in the presence ( ) or absence ( ) of eudistomin M (Eud-M, M). Relative contraction was expressed as a percentage against each maximum tension (62 g, 100%) at the Ca concentration of 10-6 M. Values are means S.E.M. (n 4). Statistical significance is indicated in the figure: *P.05. Fig. 6. Effects of eudistomin M (Eud-M) on the superprecipitation of rabbit skeletal muscle myosin B. Increase in superprecipitation was expressed as a percentage against the control activity (ABS 660, 0.4) at a Ca concentration of M. The maximum change in the absorbance was determined 20 min after application. Values are mean S.E.M. (n 3). Fig. 7. Ca dependence of superprecipitation of rabbit skeletal muscle myosin B in the presence ( ) or absence ( ) of eudistomin M (Eud-M, M). Relative superprecipitation was expressed as a percentage against a maximum activity (ABS 660, 1.51, 100%) in the absence of Eud-M ataca concentration of 10-4 M. Values are means S.E.M. (n 3). Statistical significance is indicated in the figure: *P.05. Fig. 5. Typically recording traces of superprecipitation of rabbit skeletal muscle myosin B in the presence of various concentrations of eudistomin M (Eud-M). ity change 20 min after application. Eud-M ( M) enhanced the superprecipitation activity of myosin B in a concentration-dependent manner (figs. 5 and 6). The Ca concentration-activity relationship curve for superprecipitation was shifted to the upper direction by Eud-M ( M, fig. 7). Fig. 8. Effects of eudistomin M (Eud-M) on the ATPase activity of rabbit skeletal muscle myosin B. Increase in ATPase activity was presented as a percentage against the control activity (0.188 mol/mg/min) in the absence of Eud-M at the Ca concentration of M. Values are means S.E.M. (n 3). Myosin B ATPase and other enzymes. The ATPase activity of rabbit skeletal myosin B was measured in the presence of various concentrations of Eud-M. As shown in figure 8, Eud-M caused a concentration-dependent increase in the myosin B ATPase activity. In the Ca concentration-

4 698 Ohizumi et al. Vol. 285 activity relationship curve for myosin B ATPase, The maximum response to Ca ( M) was increased by Eud-M ( M, fig. 9). Eud-M also increased the ATPase activity of actomyosin reconstituted from actin, myosin and troponin-tropomyosin complex (table 1) and the Ca sensitivity was increased (data not shown). Furthermore, Eud-M did not affect the activities of Ca -, K -EDTA- or Mg -AT- Pase of myosin, ATPase of actomyosin reconstituted from actin and myosin in the presence or absence of troponin as well as SR Ca -ATPase (table 1). Discussion The ATPase activity is related functionally to the shortening velocity of unloaded muscle, whereas the isometric tension is related to the number of cross-bridge complexes (Barany, 1967; Barany and Close, 1971). The formation of force-generating cross-bridges depends not only on the presence of Mg -ATP, but also on the free energy change in ATP hydrolysis. For this process the energy is provided by the ATPase located in the cross-bridges which is activated upon complexation of myosin with actin (Lymn and Taylor, 1971; Eisenberg et al., 1980; Stein et al., 1981). The troponintropomyosin interaction is thought to be a crucial part of the protein interactions that regulate the actomyosin ATPase activity of skeletal muscles (Huxley, 1971; Morris and Lehrer, 1984; Ingraham and Swenson, 1985). It is well known that superprecipitation of skeletal natural actomyosin is an in vitro model reaction of muscle protein contraction (Szent- Györgyi, 1951). In the present experiment, Eud-M enhanced Ca -induced tension development of skinned fibers, superprecipitation and ATPase activity of myosin B. The concentration dependence of Eud-M in the tension development of skinned muscle fibers, superprecipitation and the ATPase activity of myosin B were closely correlated. These observations suggest that an increase in the ATPase activity of myosin B by Eud-M brings about the enhancement of superprecipitation of myosin B and tension development of skinned fibers. Contraction of skeletal muscle is switched on and off by Ca over the concentration range of 10-7 to 10-6 M. Troponin isaca -binding protein in thin filament of skeletal and TABLE 1 Effects of eudistomin M (10 4 M) on various ATPase activities from rabbit skeletal muscle Enzyme Change in Enzyme Activity (%) a Myosin K -EDTA-ATPase Ca -ATPase Mg -ATPase Myosin actin Myosin actin troponin Myosin actin troponin tropomyosin b SR Ca 2 -ATPase a Mean S.E.M. (n 3). b Significantly higher than the absence of eudistomin M, P.05. cardiac muscles. It is generally accepted that in skeletal muscles Ca binding to troponin results in shifting the position of tropomyosin on skeletal thin filament, leading to the contraction of muscle fibers. Troponin confers Ca sensitivity on the contractile system of skeletal muscle (Farah and Reinach, 1995; Gagne et al., 1997). It has been reported that Ca sensitizing substances increase skeletal or cardiac muscle contraction by increasing the responsiveness of the contractile proteins to Ca rather than by increasing the free Ca ion concentration (Strauss et al., 1994). In our experiments, Eud-M potentiated Ca -induced tension development of skinned fibers. The superprecipitation and AT- Pase activity of myosin B were stimulated by Eud-M in the same concentration range. Eud-M increased Ca sensitivity of skinned fibers, superprecipitation and ATPase activity of myosin B. The activities of Ca -, K -EDTA- or Mg -AT- Pase of myosin and ATPase of actomyosin reconstituted from actin and myosin were not affected by Eud-M, suggesting elimination of possible involvements of direct stimulation of myosin ATPase or actin-myosin interaction on the Eud-Minduced enhancement of superprecipitation and ATPase activity of myosin B. Eud-M significantly potentiates the ATPase activity of actin-myosin-troponin-tropomyosin reconstituted system. As previously reported, Eud-M did not cause Ca release from SR (Nakamura et al., 1986). These observations suggest that Eud-M increases Ca sensitivity of contractile protein system, resulting in stimulation of myosin B ATPase activity and thus enhances contractility of skinned fibers. It is also suggested that an increase in Ca sensitivity of the contractile protein system is caused at least partially mediated through troponin-tropomyosin complex. Eud-M even at high concentration of 10-4 M did not affect myosin ATPase or SR Ca -ATPase, suggesting a highly selective Ca -sensitizing agent. Eud-M has become an useful tool to study the molecular regulatory mechanism of Ca sensitivity of the contractile protein system. Fig. 9. The Ca dependence of the ATPase activity of rabbit skeletal muscle myosin B in the presence ( ) or absence ( ) of eudistomin M (Eud-M) ( M). Relative ATPase activity was expressed as a percentage against a maximum activity (0.322 mol/mg/min, 100%) in the absence of Eud-M at a Ca concentration of 10-4 M. Values are means S.E.M. (n 3). Statistical significance is indicated in the figure: *P.05. Acknowledgments The authors are indebted to the late Masaki Kobayashi and Ms. Hiromi Kobayashi for technical assistance. References Barany M (1967) ATPase activity of myosin correlated with speed of muscle sorting. J Gen Physiol 50: Barany M and Close RI (1971) The transformation of myosin in cross- innervated rat muscles. J Physiol 213: Ebashi S, Kohama A and Ebashi F (1968) Troponin. I. Preparation and physiological function. J Biochem 64: Eisenberg E, Hill TL and Chen Y (1980) Cross-bridge model of muscle contraction. Quantitative analysis. Biophys J 29: Endo M and Iino M (1980) Specific perforation of muscle cell membranes with preserved SR functions by saponin treatment. J Muscle Res Cell Motil 1:

5 1998 Eudistomin M Enhances Contraction 699 Endo M and Kitazawa T (1978) E-C coupling studies on skinned cardiac studies, in Biophysical Aspects of Cardiac Muscle (Morad M ed) pp , Academic Press, New York. Fang Y, Adachi M, Kobayashi J and Ohizumi Y (1993) High affinity binding 9-[ 3 H]methyl-7-bromoeudistomin D to the caffeine-binding site of skeletal muscle sarcoplasmic reticulum. J Biol Chem 268: Farah CS and Reinach FC (1995) The troponin complex and regulation of muscle contraction. FASEB J 9: Furukawa K-I, Sakai K, Watanabe S, Maruyama K, Murakami M, Yamaguchi K and Ohizumi Y (1993) Goniodomin A induces modulation of actomyosin ATPase activity mediated through conformational change of actin. J Biol Chem 268: Gagne SM, Li MX and Sykes BD (1997) Mechanism of direct coupling and induced structural change in regulatory calcium binding proteins. Biochemistry 36: Horiuti K (1986) Some properties of the contractile system and sarcoplasmic reticulum of skinned slow fibers from Xenopus muscle. J Physiol 373:1-23. Huxley HE (1971) Structural changes during muscle contraction. Biochem J 125: 85P. Ingraham RH and Swenson CA (1985) Interaction of troponin and tropomyosin: Spectroscopic and calorimetric studies. Biochemistry 24: Kobayashi J, Harbour GC, Gilmore J and Reinhart KL Jr (1984) Eudistomins A, D, G, H, I, J, M, N, O, P, and Q, bromo, hydroxy, pyrrolyl and iminoazepino -carbolines from the antiviral Caribbean tunicate Eudistoma olivaceum. J Am Chem Soc 106: Kobayashi M, Nakamura H, Kobayashi J and Ohizumi Y (1991a) Mechanism of inotropic action of xestoquinone, a novel cardiac agent isolated from a sea sponge. J Pharmacol Exp Ther 257: Kobayashi M, Muroyama A, Nakamura H, Kobayashi J and Ohizumi Y (1991b) Xestoquinone, a novel cardiotonic agent activates actomyosin ATPase to enhance contractility of skinned cardiac or skeletal muscle fiber. J Pharm Exp Ther 257: Kohama K (1979) Divalent cation binding properties of slow skeletal muscle troponins. J Biochem 86: Lymn RW and Taylor EW (1971) Mechanism of adenosin triphosphate hydrolysis by actomyosin. Biochemistry 10: Martin JB and Doty DM (1949) Determination of inorganic phosphate. Modification of the iso-butyl alcohol procedure. Anal Chem 21: Morris EP and Lehrer SS (1984) Troponin-tropomyosin interactions. Fluorescence studies of the binding of troponin, troponin T, and chymotryptic troponin T fragments to specifically labeled tropomyosin. Biochemistry 23: Nakamura Y, Kobayashi J, Gilmore J, Mascal M, Rinehart K, Nakamura H and Ohizumi Y (1986) Bromo-eudistomin D, a novel inducer of calcium from fragmented sarcoplasmic reticulum that causes contractions of skinned muscle fibers. J Biol Chem 26: Nakamura Y, Kobayashi M, Nakamura, Wu H, Kobayashi J and Ohizumi Y (1987) Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA-ATPase that enhanced the superprecipitation of actomyosin. Eur J Biochem 167:1-6. Ohizumi Y (1997) Application of physiologically active substances isolated from natural resources to pharmacological studies. Jpn J Pharmacol 73: Sakamoto H, Furukawa K-I, Matsunaga K, Nakamura H and Ohizumi Y (1995) Xestoquinone activates skeletal muscle actomyosin ATPase by modification of the specific sulfhydryl group in the myosin head probably distinct from sulfhydryl groups SH1 and SH2. Biochemistry 34: Seino A, Kobayashi M, Kobayashi J, Fang J, Ishibashi M, Nakamura H, Momose K and Ohizumi Y (1991) 9-Methyl-7-bromo-eudistomin D, a powerful radio-labelable Ca releaser having caffeine-like properties, acts on Ca -induced Ca release channels of sarcoplasmic reticulum. J Pharmacol Exp Ther 256: Spudich JA and Watt S (1971) Regulation of rabbit skeletal muscle contraction. Biochemical studies of the interaction of the tropomyosin-troponin complex with actin and the proteolytic fragments of myosin. J Biol Chem 246: Stein LA, Chock PB and Eisenberg E (1981) Mechanism of the actomyosin ATPase: effect of actin on the ATP hydrolysis step. Proc Natl Acad Sci USA 78: Strauss JD, Bletz C and Rüegg JP (1994) The calcium sensitizer EMD antagonizes phosphate-induced increase in energy cost of isometric tension in cardiac skinned fibers. Eur J Pharmacol 252: Szent-Györgyi A (1951) Chemistry of Muscular Contraction, 2nd ed, Academic Press, New York. Takito J, Nakamura H, Kobayashi J, Ohizumi Y, Ebisawa K and Nonomura Y (1986) Purealin, a novel stabilizer of smooth muscle myosin filaments that modulates ATPase activity of dephosphorylated myosin. J Biol Chem 261: Weeds AG and Taylor RS (1975) Separation of subflagment-1 isoenzymes from rabbit skeletal muscle myosin. Nature 257: Send reprint requests to: Dr. Yasushi Ohizumi, Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980, Japan.