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Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction
Biochimica et Biophysica Acta 1427 (1999) 24^32
Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction Kimihiro Matsunaga a , Keigo Nakatani a , Masami Ishibashi b , Jun'ichi Kobayashi c , Yasushi Ohizumi a; * a
c
Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan b Laboratory of Natural Product Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan Department of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Received 23 September 1998; received in revised form 4 December 1998; accepted 4 December 1998
Abstract Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration^contractile response curve for external Ca2 was shifted to the left in a parallel manner, suggesting an increase in Ca2 sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2 in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin^tropomyosin complex. Ca2 -, K -EDTA- or Mg2 -ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2 sensitivity of the contractile apparatus mediated through troponin^tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Amphidinolide B; Calcium ion sensitivity; Skinned ¢ber; Superprecipitation; Actomyosin ATPase ; Troponin^tropomyosin complex
1. Introduction The force of muscle contraction is produced by the interaction between actin and myosin molecules in a process that involves cross-bridge cycling coupled with the hydrolysis of ATP [1^3]. Actomyosin is a precise machine for transduction of chemical energy
* Corresponding author. Fax: +81-22-217-6850; E-mail: [email protected]
in ATPase molecules into mechanical work [4]. From the viewpoint of enzymology, myosin is an ATPase whose activity is stimulated by the interaction with actin. Although the binding sites involved in the interaction between myosin and actin molecules have been determined [5,6], the role of conformational changes and the interactions of actin and myosin in muscle contraction remain to be elucidated. Therefore, novel tools that provide information on conformational changes and interactions of contractile proteins will be useful [7].
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 1 7 5 - 5
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Numerous marine natural products have been useful as tools for physiological and biological studies because of their actions on speci¢c sites of functional proteins. In the course of our survey on bioactive substances from marine sources, much attention has been given to compounds a¡ecting the contractile apparatus. Recently, we have isolated several natural products that a¡ect myosin and actin functions, such as purealin which modulates myosin ATPase activity [8,9], xestoquinone, which modulates the speci¢c sulfhydryl groups SH1 and SH2 of myosin [10^ 12] and goniodomin A, which modulates actomyosin ATPase activity through an induced change in actin conformation [13]. In further research, we found that amphidinolide B (AL-B, Fig. 1) isolated from a marine dino£agellate Amphidinium sp., which are symbiotes of Okinawan marine £atworm Amphiscolops sp., increased actomyosin ATPase activity. Here, we present the ¢rst report indicating that AL-B activates the contractile system of skeletal muscles. 2. Materials and methods 2.1. Materials AL-B was isolated from the dino£agellate Amphidinium sp. as previously reported [14]. Brie£y, the dino£agellate was cultivated, collected and extracted with methanol/toluene (3:1) followed by partitioning between toluene and water. The toluene-soluble fraction was subjected to repeated silica gel £ash chromatography followed by reversed-phase HPLC resulting in isolation of AL-B. AL-B was dissolved in dimethyl sulfoxide (DMSO) and a ¢nal concentration of DMSO did not exceed 1%. Less than 1% DMSO had little e¡ect on the contraction and ATPase activities of contractile protein system. DMSO was added in all control experiments. The animals used in this study were treated in according with the principles and guidelines of Tohoku University Council on Animal Care. In the biochemical experiments, fast skeletal muscles of male rabbit (3 kg) back and leg were used to obtain large amounts of the di¡erent contractile protein preparations. Myo¢brils, natural actomyosin, myosin, actin, troponin and tropomyosin were prepared from rabbit skeletal muscles by the method of Perry and Corsi [15], Meng
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et al. [16], Weeds and Taylor [17], Hill et al. [18] and Morris and Lehrer [19], respectively. In skinned ¢ber experiments, psoas muscles of male guinea pig (250^ 300 g) and male rabbit (3 kg) were used [20^22]. All other reagents were of analytical grade. 2.2. Skinned ¢ber experiments The skinned ¢ber experiment was carried out as previously described [11]. 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; CaCl2 , 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-Ms2 , 5.4; ATP, 4; EGTA, 10; and piperazineN,N-P-bis(2-ethanesulfonic acid)-KOH, 20 (pH 7.0). A small muscle bundle of 4^5 ¢bers (ca. 0.1 mm in diameter and ca. 3 mm in length) was dissected from the psoas muscle. One end of the ¢ber was secured to the tissue holder by a ligature and the other end to a force-displacement transducer (Acers AE 801; Horten, Norway; the compliance of tension measurement system being approximately 0.5 mm/g) for measurement of isometric contraction of the ¢ber at 20^ 23³C. Fibers were treated with the relaxing solution containing 50 mg/ml saponin for 30 min and then with a 0.5% Triton X-100 solution for 15 min [21,22]. Various solutions having di¡erent ionic strengths (0.1^0.3) were prepared for the skinned ¢ber experiments as described elsewhere [10]. The maximal tension in response to high Ca2 concentration was similar to the values in the literature [21,22]. The survival of the preparation was at least 5 h. 2.3. Superprecipitation assay The superprecipitation of natural actomyosin was induced by adding 0.4 mM ATP in 0.3 mg/ml natural actomyosin, 0.76 mM CaCl2 , 1 mM EGTA, 2 mM MgCl2 , 50 mM KCl and 20 mM Tris^HCl at pH 6.8 and 25³C, and the change in the absorbance at 660 nm was followed. 2.4. Enzyme assay The reaction mixture for each ATPase was as fol-
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lows (mM). For the ATPase activity of natural actomyosin (0.3 mg/ml): ATP, 2; CaCl2 , 0.76; EGTA, 1; KCl, 50; MgCl2 , 2; and Tris^HCl, 20 (pH 6.8); for the ATPase activity of myo¢brils (0.3 mg/ml): ATP, 2; CaCl2 , 0.52; EGTA, 1; KCl, 50; MgCl2 , 2; and Tris^HCl, 20 (pH 6.8); for the ATPase activity of actomyosin reconstituted from actin (0.1 mg/ml) and myosin (0.1 mg/ml): ATP, 2; CaCl2 , 0.09; EGTA, 1; KCl, 50; MgCl2 , 2; and Tris^HCl, 20 (pH 6.8); for the ATPase activity of actomyosin reconstituted from actin (0.1 mg/ml), myosin (0.1 mg/ml), troponin (0.1 mg/ml) and tropomyosin (0.1 mg/ml): ATP, 2; CaCl2 , 0.09; EGTA, 1; KCl, 50; MgCl2 , 2; and Tris^HCl, 20 (pH 6.8); for the Ca2 ATPase activity of myosin (0.2 mg/ml): ATP, 2; CaCl2 , 10; KCl, 500; and Tris^HCl, 50 (pH 6.8); for the K -EDTA-ATPase activity of myosin (0.02 mg/ml): the same conditions as above except for EDTA, 5 instead of CaCl2 , 10; for the Mg2 -ATPase activity of myosin (2 mg/ml): ATP, 2; MgCl2 , 5; KCl, 500; and Tris^HCl, 50 (pH 6.8). The mixture was preincubated in the absence of AL-B and ATP at 30³C for 5 min, followed by the addition of AL-B and further preincubation for 5 min. 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 [23]. The dilution method was employed to investigate the reversibility of the e¡ect of AL-B. Myosin (0.1 mg/ml), actin (0.1 mg/ml), or actomyosin (0.1 mg each myosin and actin/ml) was preincubated with AL-B for 5 min and then diluted 10 times by the dilution medium. The dilution medium contained fresh actin or myosin (or both) at ¢nal concentrations of 0.1 and 0.01 mg/ml, respectively. Then, the reaction of actomyosin ATPase was carried out under the same conditions as described above. ALB increased the activity of actomyosin ATPase to near-maximum levels at 3U1036 M, but did not produced any e¡ect on the activity at 3U1037 M. Then, 3U1036 M AL-B was used for preincubation.
muscle by the method of Kim et al. [24]. The extravesicular Ca2 concentration of HSR suspension was measured with a Ca2 electrode as described previously [25]. 2.6. Statistical analysis of the data The data are expressed as means þ S.E. Statistical comparisons were made by using Student's t-test. P 6 0.05 was considered signi¢cant. 3. Results 3.1. Contractile response of skinned ¢bers In order to measure the contractile force of skinned ¢bers under the direct in£uence of Ca2 concentration, the ¢bers were prepared from guinea pig and rabbit skeletal muscle by su¤cient treatment with detergents to destroy the function of both the cell membrane and SR membrane. Ca¡eine (40 mM) did not cause any contraction of skinned ¢bers, suggesting destruction of SR membrane [26]. Fig. 2 shows the typical recording traces of contractile response of skinned ¢bers of guinea pig skeletal muscle before and after exposure to AL-B (3U1035 M) in the presence of Ca2 . A similar recording trace was obtained in rabbit skeletal muscle skinned ¢bers (data not shown). AL-B caused a concentration-dependent increase in contractile force of skinned ¢bers at the Ca2 concentration of 3U1037 M (Fig. 3). The contraction of the ¢ber was increased to 75% of the control (0.012 kN/m2 ) by 1035 M AL-B. As shown in Fig. 4, the AL-B-induced enhancement of developed contraction of skinned ¢bers was largely dependent on the free Ca2 concentration in the assay medium. The concentration^contractile response curve for ex-
2.5. Extravesicular Ca2+ concentration measurement The heavy fraction of fragmented sarcoplasmic reticulum (HSR) was prepared from rabbit skeletal
Fig. 1. Chemical structure of amphidinolide B (AL-B).
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Fig. 2. Typical recording traces of the contractile response of skinned ¢bers from guinea pig psoas muscles at the Ca2 concentration of 2.5U1037 M (A) and 3.5U1037 M (B) in the presence or absence of amphidinolide B (AL-B, 3U1035 M). DMSO was added in all control experiments.
ternal Ca2 was markedly shifted to the left in a parallel manner by 1035 M AL-B. The change in the EC50 value of Ca2 was 1.8U1037 M. 3.2. Superprecipitation of natural actomyosin The e¡ect of AL-B was examined on the superprecipitation of skeletal natural actomyosin, an in vitro model reaction of muscle protein contraction, by monitoring the turbidity change. After the addition of ATP, the turbidity increased for 30 s. Fig. 5 shows the typical recording traces of the e¡ects of various concentrations of AL-B on the superprecipitation of natural actomyosin prepared from rabbit skeletal muscles. As shown in Fig. 6, AL-B produced
a concentration-dependent enhancement of the superprecipitation activity of natural actomyosin. AL-B not only increased the initial velocity of superprecipitation and shortened the time required to attain 50% of a maximum level, but also elevated signi¢cantly the maximum steady level (Figs. 5 and 6). The Ca2 concentration^activity relationship curve for superprecipitation was shifted to the upper direction by AL-B (1035 M, Fig. 7). 3.3. Natural actomyosin ATPase and other enzymes In the Ca2 concentration^activity relationship curve for natural actomyosin ATPase, the response to Ca2 (1036 to 1034 M) was increased by AL-B
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Fig. 3. The log concentration^contractile response curve for amphidinolide B (AL-B) in the skinned ¢bers from guinea pig skeletal muscles. Increase in force was expressed as a percentage against the control tension with DMSO (0.012 kN/m2 ) in the absence of AL-B at a Ca2 concentration of 3U1037 M. Each point represents the mean þ S.E. from four experiments.
Fig. 4. The log concentration^contractile response curve for Ca2 in the presence (b, maximum tension: 0.017 kN/m2 ) or absence (a, maximum tension: 0.018 kN/m2 ) of amphidinolide B (AL-B, 1035 M) in the skinned ¢bers from guinea pig skeletal muscles. Relative force was expressed as a percentage against the maximum tension with DMSO (100%, 0.032 kN/m2 ) at a Ca2 concentration of 1036 M. Each point represents the mean þ S.E. from four experiments. Statistical signi¢cance compared between control and AL-B is indicated in the ¢gure: *P 6 0.05.
Fig. 5. Typical recording traces of superprecipitation of rabbit skeletal muscle natural actomyosin in the presence of various concentrations of amphidinolide B (AL-B). The concentrations of AL-B were 0 (a), 1037 (b), 1036 (c), 3U1036 (d) and 1035 M (e).
(1035 M, Fig. 8). In the Ca2 electrode experiment, the change in free Ca2 concentration was not detected in all EGTA bu¡ers in the presence or absence of AL-B, suggesting that AL-B did not a¡ect the Ca2 binding a¤nity of the bu¡er EGTA. The ATPase activity of rabbit skeletal natural actomyosin
Fig. 6. The log concentration^activity curve for amphidinolide B (AL-B) in the initial velocity (a) and in the time required to reach a half of the maximum level (b) of superprecipitation of rabbit skeletal natural actomyosin at a Ca2 concentration of 2U1036 M. Each point represents the mean þ S.E. from three experiments.
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Fig. 7. The log concentration^response curve for Ca2 in the presence (b) or absence (a) of amphidinolide B (AL-B, 1035 M). The relative initial velocity of superprecipitation was expressed as a percentage against a maximum activity (100%, 0.075 vA/s) in the absence of AL-B at a Ca2 concentration of 2.3U1036 M. Each point represents the mean þ S.E. from three experiments. Statistical signi¢cance compared between control and AL-B is indicated in the ¢gure: *P 6 0.05.
was measured in the presence of various concentrations of AL-B. AL-B caused a concentration-dependent increase in the natural actomyosin ATPase activity (Fig. 9). As shown in Fig. 10, AL-B caused a concentration-dependent increase in skeletal myo¢bril ATPase activity and the maximal response (about 50% of the control value, 0.113 Wmol/min per mg of myo¢brils) was obtained with 3^6U1036 M. This e¡ect was not a¡ected by the SH group protecting reagent, dithiothreitol (1033 M, data not shown). AL-B produced a concentration-dependent increase in the ATPase activity of actomyosin reconstituted from actin and myosin (Fig. 11). The actomyosin ATPase activity of the actin^myosin reconstituted system was increased by AL-B even in the presence of troponin and tropomyosin (Fig. 11), suggesting an important e¡ect of AL-B on the troponin^ tropomyosin system in the mechanism of increasing Ca2 sensitivity of skinned ¢bers. The actomyosin ATPase activities of the actin^myosin^troponin^tropomyosin reconstituted system were markedly increased by AL-B (1036 M) at Ca2 concentrations of 1038 M (from 2 to 10 nmol/min per mg, i.e., a 400% increase), 1037 M (from 5 to 11 nmol/min per mg, i.e., a 120% increase) and 3U1037 M (from 16 to 24 nmol/min per mg, i.e., a 50% increase). However, the stimulatory e¡ect of AL-B was not observed at a
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Fig. 8. The log concentration^response curve for Ca2 in the ATPase activity of natural actomyosin in the presence (b) or absence (a) of amphidinolide B (AL-B, 1035 M). Relative ATPase activity was expressed as a percentage against a maximum activity (100%, 0.286 Wmol/min per mg) in the absence of AL-B at a Ca2 concentration of 1034 M. Each point represents the mean þ S.E. from three experiments. Statistical signi¢cance compared between control and AL-B is indicated in the ¢gure: *P 6 0.05.
Ca2 concentration of 1036 M or more. The activity of Ca2 , K -EDTA- or Mg2 -ATPase of myosin was not a¡ected by it (1037 to 1035 M, data not shown). The reversibility was examined using the dilution method. If the e¡ect of AL-B on actin or myosin was irreversible, some stimulation might be
Fig. 9. The log concentration^response curve for amphidinolide B (AL-B) in the ATPase activity of natural actomyosin. Increase in the ATPase activity was expressed as a percentage against the control activity (0.219 Wmol/min per mg) in the absence of AL-B at a Ca2 concentration of 3U1036 M.
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suggesting that AL-B interacts with actin or myosin reversibly. In the Ca2 electrode experiment, even after application of AL-B (3U1035 M), an increase in extravesicular Ca2 concentration of HSR was not detected, suggesting inactivity on the Ca2 release channel. Furthermore, Ca2 -pumping ATPase activity (0.512 Wmol/min per mg) in HSR was not changed by AL-B (3U1035 M). These data suggest that AL-B is a selective activator of skeletal myo¢lament contraction and actomyosin ATPase. 4. Discussion Fig. 10. The log concentration^response curve for amphidinolide B (AL-B) in the ATPase activity of myo¢brils. Increase in the ATPase activity was expressed as a percentage against the control activity (0.113 Wmol/min per mg) in the absence of ALB at a Ca2 concentration of 3U1036 M.
found even after the dilution. As a result, there was no signi¢cant di¡erence among the ATPase activities of actomyosin consisting of AL-B-pretreated actin and myosin, actomyosin consisting of AL-B-pretreated actin and fresh myosin or actomyosin consisting of AL-B-pretreated myosin and fresh actin,
Fig. 11. The log concentration^response curve for amphidinolide B (AL-B) in the ATPase activity of actomyosin reconstituted from actin and myosin in the absence (a) or presence (b) of troponin^tropomyosin complex. Increase in the ATPase activity was expressed as a percentage against the control activity (a, 0.044 Wmol/min per mg; b, 0.021 Wmol/min per mg) in the absence of AL-B at a Ca2 concentration of 1037 M. Each point represents the mean þ S.E. from three experiments.
A widely accepted theory to explain the mutual sliding of actin and myosin ¢laments is the crossbridge theory of muscle contraction. The crossbridge mechanism was ¢rmly established and seemed to be a reasonable explanation for the relative sliding of the actin and myosin ¢lament [27^30]. The force of muscle contraction is produced by the interaction between actin and myosin molecules [31] in which chemical energy in ATP molecules is converted into mechanical work [4,29,32]. The superprecipitation of natural actomyosin is generally accepted to be basically the same phenomenon in vitro as a contraction in skeletal muscle cells [33]. In the present experiment, AL-B increased the contractile response of skinned ¢bers and superprecipitation of natural actomyosin. The ATPase activity of skeletal muscle myo¢bril, natural actomyosin or actomyosin reconstituted from actin and myosin was enhanced markedly by AL-B. It is not clear why the e¡ect of AL-B on natural actomyosin ATPase activity is poor. The detail of this mechanism is now under investigation. However, the Ca2 , K -EDTA- or Mg2 -ATPase activity of myosin was not a¡ected by AL-B, suggesting the elimination of the possible involvement of direct stimulation of myosin ATPases in the mechanism of actomyosin ATPase activation. These results suggest that AL-B-induced enhancement of the contraction of skinned ¢bers and superprecipitation of natural actomyosin is caused at least partially by increasing the interaction between actin and myosin directly. The troponin^tropomyosin interaction is thought to be a crucial part of the protein interactions which regulate the actomyosin ATPase activity of skeletal
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muscles [19,34,35]. Contraction of skeletal muscle is switched on and o¡ by Ca2 over the concentration range of 1037 to 1036 M. It is generally accepted that in skeletal muscles Ca2 ¢rst interacts with troponinC, resulting in a shift of the position of tropomyosin on skeletal thin ¢lament, leading to the contraction of muscle ¢bers. Troponin-C confers Ca2 sensitivity on the contractile system of skeletal muscle [36,37]. It has been reported that Ca2 sensitizers such as levosimendan [38] and MCI-154 [39] directly in£uence the responsiveness of the contractile protein to Ca2 by a¡ecting troponin-C. The ATPase activity of the actin^myosin^troponin^tropomyosin reconstituted system was enhanced by AL-B. AL-B increased Ca2 sensitivity in skinned ¢bers and enhanced the response of natural actomyosin to Ca2 in both superprecipitation and ATPase activity. These results suggest that AL-B is a¡ecting the regulatory proteins, resulting in an increase in Ca2 sensitivity to enhance the contractile response of myo¢lament. In conclusion, AL-B directly activates the interaction of actin and myosin and gives in£uence on the function of the troponin^tropomyosin system, resulting in an increase in actomyosin ATPase activity and thus enhances the contraction of myo¢lament. AL-B has become a useful tool for the investigation not only of the interaction between actin and myosin but also of the molecular regulatory mechanism by the troponin^tropomyosin complex. Acknowledgements We are indebted to Dr. Ken-Ichi Furukawa of this department, Dr. Hideshi Nakamura of Hokkaido University and the late Masaki Kobayashi of Mitsubishi Kasei Institute of Life Sciences for useful advice. We also thank Ms. Akiko Muroyama and Ms. Hiromi Kobayashi for technical assistance. This work was partially supported by a Grant-in-aid for Scienti¢c Research from the Ministry of Education, Science, Sports and Culture of Japan. Financial support through Grants-in-aid from the Tokyo Biochemical Research Foundation, the Sagawa Foundation for Promotion of Cancer Research and the Nakatomi Foundation are also acknowledged.
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References [1] R.W. Lymn, E.W. Taylor, Mechanism of adenosine triphosphate hydrolysis by actomyosin, Biochemistry 10 (1971) 4617^4624. [2] E. Eisenberg, T.L. Hill, Y. Chen, Cross-bridge model of muscle contraction. Quantitative analysis, Biophys. J. 29 (1980) 195^227. [3] L.A. Stein, P.B. Chock, E. Eisenberg, Mechanism of the actomyosin ATPase : e¡ect of actin on the ATP hydrolysis step, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1346^1350. [4] H. Sugi, Molecular mechanism of ATP-dependent actin-myosin interaction in muscle contraction, Jpn. J. Physiol. 43 (1993) 435^454. [5] I. Rayment, W.R. Rypniewski, K. Schmidt-Base, R. Smith, D.R. Tomchick, M.M. Bennimg, D.A. Winkelmann, G. Wesenberg, H.M. Holden, Three-dimensional structure of myosin subfragment-1: a molecular motor, Science 261 (1993) 50^58. [6] I. Rayment, H.M. Holden, M. Whittaker, C.B. Yohn, M. Lorenz, K.C. Holmes, R.A. Millingan, Structure of the actin-myosin complex and its implications for muscle contraction, Science 261 (1993) 58^65. [7] Y. Ohizumi, Application of physiologically active substances isolated from natural resources to pharmacological studies, Jpn. J. Pharmacol. 73 (1997) 263^289. [8] J. Takito, H. Nakamura, J. Kobayashi, Y. Ohizumi, K. Ebisawa, Y. Nonomura, Purealin, a novel stabilizer of smooth muscle myosin ¢laments that modulates ATPase activity of dephosphorylated myosin, J. Biol. Chem. 261 (1986) 13861^ 13865. [9] Y. Nakamura, M. Kobayashi, H. Nakamura, H. Wu, J. Kobayashi, Y. Ohizumi, Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA-ATPase that enhanced the superprecipitation of actomyosin, Eur. J. Biochem. 167 (1987) 1^6. [10] M. Kobayashi, H. Nakamura, J. Kobayashi, Y. Ohizumi, Mechanism of inotropic action of xestoquinone, a novel cardiotonic agent isolated from a sea sponge, J. Pharmacol. Exp. Ther. 257 (1991) 82^89. [11] M. Kobayashi, A. Muroyama, H. Nakamura, J. Kobayashi, Y. Ohizumi, Xestoquinone, a novel cardiotonic agent activates actomyosin ATPase to enhance contractility of skinned cardiac or skeletal muscle ¢bers, J. Pharmacol. Exp. Ther. 257 (1991) 90^94. [12] H. Sakamoto, K.-I. Furukawa, K. Matsunaga, H. Nakamura, Y. Ohizumi, Xestoquinone activates skeletal muscle actomyosin ATPase by modi¢cation of the speci¢c sulfhydryl group in the myosin head probably distinct from sulfhydryl groups SH1 and SH2 , Biochemistry 34 (1995) 12570^ 12575. [13] K.-I. Furukawa, K. Sakai, S. Watanabe, K. Maruyama, M. Murakami, K. Yamaguchi, Y. Ohizumi, Goniodomin A induces modulation of actomyosin ATPase activity mediated through conformational change of actin, J. Biol. Chem. 268 (1993) 26026^26031.
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[14] M. Ishibashi, Y. Ohizumi, M. Hamashima, H. Nakamura, Y. Hirata, T. Sasaki, J. Kobayashi, Amphidinolide B, a novel macrolide with potent antineoplastic activity from the marine dino£agellate Amphidinium sp., J. Chem. Soc. Chem. Commun. 14 (1987) 1127^1129. [15] S.V. Perry, A. Corsi, Extraction of proteins other than myosin from the isolation rabbit myo¢bril, Biochem. J. 68 (1958) 5^12. [16] Y. Meng, T. Yasunaga, T. Wakabayashi, Determination of the protein components of native thin ¢laments isolated from natural actomyosin: neblin and K-actinin are associated with actin ¢lament, J. Biochem. 118 (1995) 422^427. [17] A.G. Weeds, R.S. Taylor, Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin, Nature 257 (1975) 54^56. [18] L.E. Hill, J.P. Mehegan, C.A. Butters, L.S. Tobacman, Analysis of troponin-tropomyosin binding to actin, J. Biol. Chem. 267 (1992) 16106^16113. [19] E.P. Morris, S.S. Lehrer, Troponin-tropomyosin interactions. Fluorescence studies of the binding of troponin, troponin T, and chymotryptic troponin T fragments to specifically labeled tropomyosin, Biochemistry 23 (1984) 2214^ 2220. [20] M. Endo, T. Kitazawa, E-C coupling studies on skinned cardiac studies, in: M. Morad (Ed.), Biophysical Aspects of Cardiac Muscle, Academic Press, New York, 1978, pp. 307^327. [21] M. Endo, M. Iino, Speci¢c perforation of muscle cell membranes with preserved SR functions by saponin treatment, J. Muscle Res. Cell Motil. 1 (1980) 89^100. [22] K. Horiuti, Some properties of the contractile system and sarcoplasmic reticulum of skinned slow ¢bers from Xenopus muscle, J. Physiol. 373 (1986) 1^23. [23] J.B. Martin, D.M. Doty, Determination of inorganic phosphate, Anal. Chem. 21 (1949) 965^967. [24] D.H. Kim, S. T, N. Ohnishi, Ikemoto, Kinetic studies of calcium release from sarcoplasmic reticulum in vitro, J. Biol. Chem. 258 (1983) 9662^9668. [25] A. Seino, M. Kobayashi, J. Kobayashi, Y.-I. Fang, M. Ishibashi, H. Nakamura, K. Momose, Y. Ohizumi, 9-Methyl-7bromoeudistomin D, a powerful radio-labelable Ca releaser having ca¡eine-like properties, acts on Ca -induced Ca release channels of sarcoplasmic reticulum, J. Pharmacol. Exp. Ther. 256 (1991) 861^867.
[26] Y. Nakamura, J. Kobayashi, J. Gilmore, M. Mascal, K. Rinehart, H. Nakamura, Y. Ohizumi, Bromo-eudistomin D, a novel inducer of calcium from fragmented sarcoplasmic reticulum that causes contractions of skinned muscle ¢bers, J. Biol. Chem. 261 (1986) 4139^4142. [27] A.F. Huxley, Muscle structure and theories of contraction, Prog. Biophys. Biophys. Chem. 7 (1957) 255^318. [28] H.E. Huxley, The mechanism of muscular contraction, Science 164 (1969) 1356^1366. [29] K.C. Holmes, The actomyosin interaction and its control by tropomyosin, Biophys. J. 68 (1995) 2S^7S. [30] K.C. Holmes, Muscle proteins ^ their actions and interactions, Curr. Opin. Struct. Biol. 6 (1996) 781^789. [31] S.Y. Bershitsky, A.K. Tsaturyan, O.N. Bershitskaya, G.I. Mashanov, P. Brown, R. Burns, M.A. Ferenczi, Muscle force is generated by myosin heads stereospeci¢cally attached to actin, Nature 388 (1997) 186^190. [32] Y. Tonomura, Energy-transducing ATPases ^ Structure and Kinetics, Cambridge University Press, Cambridge, 1986, pp. 44^88. [33] A. Szent-Gyorgyi, Chemistry of Muscular Contraction, 2nd ed., Academic Press, New York, 1951. [34] H.E. Huxley, Structural changes during muscle contraction, Biochem. J. 125 (1971) 85P. [35] R.H. Ingraham, C.A. Swenson, Interaction of troponin and tropomyosin: Spectroscopic and calorimetric studies, Biochemistry 24 (1985) 5221^5225. [36] C.S. Farah, F.C. Reinach, The troponin complex and regulation of muscle contraction, FASEB J. 9 (1995) 755^767. [37] S.M. Gagne, M.X. Li, B.D. Sykes, Mechanism of direct coupling and induced structural change in regulatory calcium binding proteins, Biochemistry 36 (1997) 4386^4392. [38] H. Haikala, E. Nissinen, E. Etemadzadeh, I.-B. Linden, P. Pohto, Levosimendan increases calcium sensitivity without enhancing myosin ATPase activity and impairing relaxation, J. Mol. Cell. Cardiol. 24 (1992) S97. [39] Y. Abe, Y. Kitada, A. Narimasu, E¡ect of MCI-154, a cardiotonic agent, on regional contractile function and myocardial oxygen consumption in the presence and absence of coronary artery stenosis in dogs, J. Pharmacol. Exp. Ther. 256 (1993) 819^825.
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Amphidinolide B, a powerful activator of actomyosin ATPase enhances skeletal muscle contraction Kimihiro Matsunaga a , Keigo Nakatani a , Masami Ishibashi b , Jun'ichi Kobayashi c , Yasushi Ohizumi a; * a
c
Department of Pharmaceutical Molecular Biology, Faculty of Pharmaceutical Sciences, Tohoku University, Aoba, Aramaki, Aoba-ku, Sendai 980-8578, Japan b Laboratory of Natural Product Chemistry, Faculty of Pharmaceutical Sciences, Chiba University, Yayoi-cho, Inage-ku, Chiba 263-8522, Japan Department of Natural Product Chemistry, Graduate School of Pharmaceutical Sciences, Hokkaido University, Sapporo 060-0812, Japan Received 23 September 1998; received in revised form 4 December 1998; accepted 4 December 1998
Abstract Amphidinolide B caused a concentration-dependent increase in the contractile force of skeletal muscle skinned fibers. The concentration^contractile response curve for external Ca2 was shifted to the left in a parallel manner, suggesting an increase in Ca2 sensitivity. Amphidinolide B stimulated the superprecipitation of natural actomyosin. The maximum response of natural actomyosin to Ca2 in superprecipitation was enhanced by it. Amphidinolide B increased the ATPase activity of myofibrils and natural actomyosin. The ATPase activity of actomyosin reconstituted from actin and myosin was enhanced in a concentration-dependent manner in the presence or absence of troponin^tropomyosin complex. Ca2 -, K -EDTA- or Mg2 -ATPase of myosin was not affected by amphidinolide B. These results suggest that amphidinolide B enhances an interaction of actin and myosin directly and increases Ca2 sensitivity of the contractile apparatus mediated through troponin^tropomyosin system, resulting in an increase in the ATPase activity of actomyosin and thus enhances the contractile response of myofilament. ß 1999 Elsevier Science B.V. All rights reserved. Keywords: Amphidinolide B; Calcium ion sensitivity; Skinned ¢ber; Superprecipitation; Actomyosin ATPase ; Troponin^tropomyosin complex
1. Introduction The force of muscle contraction is produced by the interaction between actin and myosin molecules in a process that involves cross-bridge cycling coupled with the hydrolysis of ATP [1^3]. Actomyosin is a precise machine for transduction of chemical energy
* Corresponding author. Fax: +81-22-217-6850; E-mail: [email protected]
in ATPase molecules into mechanical work [4]. From the viewpoint of enzymology, myosin is an ATPase whose activity is stimulated by the interaction with actin. Although the binding sites involved in the interaction between myosin and actin molecules have been determined [5,6], the role of conformational changes and the interactions of actin and myosin in muscle contraction remain to be elucidated. Therefore, novel tools that provide information on conformational changes and interactions of contractile proteins will be useful [7].
0304-4165 / 99 / $ ^ see front matter ß 1999 Elsevier Science B.V. All rights reserved. PII: S 0 3 0 4 - 4 1 6 5 ( 9 8 ) 0 0 1 7 5 - 5
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Numerous marine natural products have been useful as tools for physiological and biological studies because of their actions on speci¢c sites of functional proteins. In the course of our survey on bioactive substances from marine sources, much attention has been given to compounds a¡ecting the contractile apparatus. Recently, we have isolated several natural products that a¡ect myosin and actin functions, such as purealin which modulates myosin ATPase activity [8,9], xestoquinone, which modulates the speci¢c sulfhydryl groups SH1 and SH2 of myosin [10^ 12] and goniodomin A, which modulates actomyosin ATPase activity through an induced change in actin conformation [13]. In further research, we found that amphidinolide B (AL-B, Fig. 1) isolated from a marine dino£agellate Amphidinium sp., which are symbiotes of Okinawan marine £atworm Amphiscolops sp., increased actomyosin ATPase activity. Here, we present the ¢rst report indicating that AL-B activates the contractile system of skeletal muscles. 2. Materials and methods 2.1. Materials AL-B was isolated from the dino£agellate Amphidinium sp. as previously reported [14]. Brie£y, the dino£agellate was cultivated, collected and extracted with methanol/toluene (3:1) followed by partitioning between toluene and water. The toluene-soluble fraction was subjected to repeated silica gel £ash chromatography followed by reversed-phase HPLC resulting in isolation of AL-B. AL-B was dissolved in dimethyl sulfoxide (DMSO) and a ¢nal concentration of DMSO did not exceed 1%. Less than 1% DMSO had little e¡ect on the contraction and ATPase activities of contractile protein system. DMSO was added in all control experiments. The animals used in this study were treated in according with the principles and guidelines of Tohoku University Council on Animal Care. In the biochemical experiments, fast skeletal muscles of male rabbit (3 kg) back and leg were used to obtain large amounts of the di¡erent contractile protein preparations. Myo¢brils, natural actomyosin, myosin, actin, troponin and tropomyosin were prepared from rabbit skeletal muscles by the method of Perry and Corsi [15], Meng
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et al. [16], Weeds and Taylor [17], Hill et al. [18] and Morris and Lehrer [19], respectively. In skinned ¢ber experiments, psoas muscles of male guinea pig (250^ 300 g) and male rabbit (3 kg) were used [20^22]. All other reagents were of analytical grade. 2.2. Skinned ¢ber experiments The skinned ¢ber experiment was carried out as previously described [11]. 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; CaCl2 , 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-Ms2 , 5.4; ATP, 4; EGTA, 10; and piperazineN,N-P-bis(2-ethanesulfonic acid)-KOH, 20 (pH 7.0). A small muscle bundle of 4^5 ¢bers (ca. 0.1 mm in diameter and ca. 3 mm in length) was dissected from the psoas muscle. One end of the ¢ber was secured to the tissue holder by a ligature and the other end to a force-displacement transducer (Acers AE 801; Horten, Norway; the compliance of tension measurement system being approximately 0.5 mm/g) for measurement of isometric contraction of the ¢ber at 20^ 23³C. Fibers were treated with the relaxing solution containing 50 mg/ml saponin for 30 min and then with a 0.5% Triton X-100 solution for 15 min [21,22]. Various solutions having di¡erent ionic strengths (0.1^0.3) were prepared for the skinned ¢ber experiments as described elsewhere [10]. The maximal tension in response to high Ca2 concentration was similar to the values in the literature [21,22]. The survival of the preparation was at least 5 h. 2.3. Superprecipitation assay The superprecipitation of natural actomyosin was induced by adding 0.4 mM ATP in 0.3 mg/ml natural actomyosin, 0.76 mM CaCl2 , 1 mM EGTA, 2 mM MgCl2 , 50 mM KCl and 20 mM Tris^HCl at pH 6.8 and 25³C, and the change in the absorbance at 660 nm was followed. 2.4. Enzyme assay The reaction mixture for each ATPase was as fol-
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lows (mM). For the ATPase activity of natural actomyosin (0.3 mg/ml): ATP, 2; CaCl2 , 0.76; EGTA, 1; KCl, 50; MgCl2 , 2; and Tris^HCl, 20 (pH 6.8); for the ATPase activity of myo¢brils (0.3 mg/ml): ATP, 2; CaCl2 , 0.52; EGTA, 1; KCl, 50; MgCl2 , 2; and Tris^HCl, 20 (pH 6.8); for the ATPase activity of actomyosin reconstituted from actin (0.1 mg/ml) and myosin (0.1 mg/ml): ATP, 2; CaCl2 , 0.09; EGTA, 1; KCl, 50; MgCl2 , 2; and Tris^HCl, 20 (pH 6.8); for the ATPase activity of actomyosin reconstituted from actin (0.1 mg/ml), myosin (0.1 mg/ml), troponin (0.1 mg/ml) and tropomyosin (0.1 mg/ml): ATP, 2; CaCl2 , 0.09; EGTA, 1; KCl, 50; MgCl2 , 2; and Tris^HCl, 20 (pH 6.8); for the Ca2 ATPase activity of myosin (0.2 mg/ml): ATP, 2; CaCl2 , 10; KCl, 500; and Tris^HCl, 50 (pH 6.8); for the K -EDTA-ATPase activity of myosin (0.02 mg/ml): the same conditions as above except for EDTA, 5 instead of CaCl2 , 10; for the Mg2 -ATPase activity of myosin (2 mg/ml): ATP, 2; MgCl2 , 5; KCl, 500; and Tris^HCl, 50 (pH 6.8). The mixture was preincubated in the absence of AL-B and ATP at 30³C for 5 min, followed by the addition of AL-B and further preincubation for 5 min. 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 [23]. The dilution method was employed to investigate the reversibility of the e¡ect of AL-B. Myosin (0.1 mg/ml), actin (0.1 mg/ml), or actomyosin (0.1 mg each myosin and actin/ml) was preincubated with AL-B for 5 min and then diluted 10 times by the dilution medium. The dilution medium contained fresh actin or myosin (or both) at ¢nal concentrations of 0.1 and 0.01 mg/ml, respectively. Then, the reaction of actomyosin ATPase was carried out under the same conditions as described above. ALB increased the activity of actomyosin ATPase to near-maximum levels at 3U1036 M, but did not produced any e¡ect on the activity at 3U1037 M. Then, 3U1036 M AL-B was used for preincubation.
muscle by the method of Kim et al. [24]. The extravesicular Ca2 concentration of HSR suspension was measured with a Ca2 electrode as described previously [25]. 2.6. Statistical analysis of the data The data are expressed as means þ S.E. Statistical comparisons were made by using Student's t-test. P 6 0.05 was considered signi¢cant. 3. Results 3.1. Contractile response of skinned ¢bers In order to measure the contractile force of skinned ¢bers under the direct in£uence of Ca2 concentration, the ¢bers were prepared from guinea pig and rabbit skeletal muscle by su¤cient treatment with detergents to destroy the function of both the cell membrane and SR membrane. Ca¡eine (40 mM) did not cause any contraction of skinned ¢bers, suggesting destruction of SR membrane [26]. Fig. 2 shows the typical recording traces of contractile response of skinned ¢bers of guinea pig skeletal muscle before and after exposure to AL-B (3U1035 M) in the presence of Ca2 . A similar recording trace was obtained in rabbit skeletal muscle skinned ¢bers (data not shown). AL-B caused a concentration-dependent increase in contractile force of skinned ¢bers at the Ca2 concentration of 3U1037 M (Fig. 3). The contraction of the ¢ber was increased to 75% of the control (0.012 kN/m2 ) by 1035 M AL-B. As shown in Fig. 4, the AL-B-induced enhancement of developed contraction of skinned ¢bers was largely dependent on the free Ca2 concentration in the assay medium. The concentration^contractile response curve for ex-
2.5. Extravesicular Ca2+ concentration measurement The heavy fraction of fragmented sarcoplasmic reticulum (HSR) was prepared from rabbit skeletal
Fig. 1. Chemical structure of amphidinolide B (AL-B).
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Fig. 2. Typical recording traces of the contractile response of skinned ¢bers from guinea pig psoas muscles at the Ca2 concentration of 2.5U1037 M (A) and 3.5U1037 M (B) in the presence or absence of amphidinolide B (AL-B, 3U1035 M). DMSO was added in all control experiments.
ternal Ca2 was markedly shifted to the left in a parallel manner by 1035 M AL-B. The change in the EC50 value of Ca2 was 1.8U1037 M. 3.2. Superprecipitation of natural actomyosin The e¡ect of AL-B was examined on the superprecipitation of skeletal natural actomyosin, an in vitro model reaction of muscle protein contraction, by monitoring the turbidity change. After the addition of ATP, the turbidity increased for 30 s. Fig. 5 shows the typical recording traces of the e¡ects of various concentrations of AL-B on the superprecipitation of natural actomyosin prepared from rabbit skeletal muscles. As shown in Fig. 6, AL-B produced
a concentration-dependent enhancement of the superprecipitation activity of natural actomyosin. AL-B not only increased the initial velocity of superprecipitation and shortened the time required to attain 50% of a maximum level, but also elevated signi¢cantly the maximum steady level (Figs. 5 and 6). The Ca2 concentration^activity relationship curve for superprecipitation was shifted to the upper direction by AL-B (1035 M, Fig. 7). 3.3. Natural actomyosin ATPase and other enzymes In the Ca2 concentration^activity relationship curve for natural actomyosin ATPase, the response to Ca2 (1036 to 1034 M) was increased by AL-B
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Fig. 3. The log concentration^contractile response curve for amphidinolide B (AL-B) in the skinned ¢bers from guinea pig skeletal muscles. Increase in force was expressed as a percentage against the control tension with DMSO (0.012 kN/m2 ) in the absence of AL-B at a Ca2 concentration of 3U1037 M. Each point represents the mean þ S.E. from four experiments.
Fig. 4. The log concentration^contractile response curve for Ca2 in the presence (b, maximum tension: 0.017 kN/m2 ) or absence (a, maximum tension: 0.018 kN/m2 ) of amphidinolide B (AL-B, 1035 M) in the skinned ¢bers from guinea pig skeletal muscles. Relative force was expressed as a percentage against the maximum tension with DMSO (100%, 0.032 kN/m2 ) at a Ca2 concentration of 1036 M. Each point represents the mean þ S.E. from four experiments. Statistical signi¢cance compared between control and AL-B is indicated in the ¢gure: *P 6 0.05.
Fig. 5. Typical recording traces of superprecipitation of rabbit skeletal muscle natural actomyosin in the presence of various concentrations of amphidinolide B (AL-B). The concentrations of AL-B were 0 (a), 1037 (b), 1036 (c), 3U1036 (d) and 1035 M (e).
(1035 M, Fig. 8). In the Ca2 electrode experiment, the change in free Ca2 concentration was not detected in all EGTA bu¡ers in the presence or absence of AL-B, suggesting that AL-B did not a¡ect the Ca2 binding a¤nity of the bu¡er EGTA. The ATPase activity of rabbit skeletal natural actomyosin
Fig. 6. The log concentration^activity curve for amphidinolide B (AL-B) in the initial velocity (a) and in the time required to reach a half of the maximum level (b) of superprecipitation of rabbit skeletal natural actomyosin at a Ca2 concentration of 2U1036 M. Each point represents the mean þ S.E. from three experiments.
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Fig. 7. The log concentration^response curve for Ca2 in the presence (b) or absence (a) of amphidinolide B (AL-B, 1035 M). The relative initial velocity of superprecipitation was expressed as a percentage against a maximum activity (100%, 0.075 vA/s) in the absence of AL-B at a Ca2 concentration of 2.3U1036 M. Each point represents the mean þ S.E. from three experiments. Statistical signi¢cance compared between control and AL-B is indicated in the ¢gure: *P 6 0.05.
was measured in the presence of various concentrations of AL-B. AL-B caused a concentration-dependent increase in the natural actomyosin ATPase activity (Fig. 9). As shown in Fig. 10, AL-B caused a concentration-dependent increase in skeletal myo¢bril ATPase activity and the maximal response (about 50% of the control value, 0.113 Wmol/min per mg of myo¢brils) was obtained with 3^6U1036 M. This e¡ect was not a¡ected by the SH group protecting reagent, dithiothreitol (1033 M, data not shown). AL-B produced a concentration-dependent increase in the ATPase activity of actomyosin reconstituted from actin and myosin (Fig. 11). The actomyosin ATPase activity of the actin^myosin reconstituted system was increased by AL-B even in the presence of troponin and tropomyosin (Fig. 11), suggesting an important e¡ect of AL-B on the troponin^ tropomyosin system in the mechanism of increasing Ca2 sensitivity of skinned ¢bers. The actomyosin ATPase activities of the actin^myosin^troponin^tropomyosin reconstituted system were markedly increased by AL-B (1036 M) at Ca2 concentrations of 1038 M (from 2 to 10 nmol/min per mg, i.e., a 400% increase), 1037 M (from 5 to 11 nmol/min per mg, i.e., a 120% increase) and 3U1037 M (from 16 to 24 nmol/min per mg, i.e., a 50% increase). However, the stimulatory e¡ect of AL-B was not observed at a
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Fig. 8. The log concentration^response curve for Ca2 in the ATPase activity of natural actomyosin in the presence (b) or absence (a) of amphidinolide B (AL-B, 1035 M). Relative ATPase activity was expressed as a percentage against a maximum activity (100%, 0.286 Wmol/min per mg) in the absence of AL-B at a Ca2 concentration of 1034 M. Each point represents the mean þ S.E. from three experiments. Statistical signi¢cance compared between control and AL-B is indicated in the ¢gure: *P 6 0.05.
Ca2 concentration of 1036 M or more. The activity of Ca2 , K -EDTA- or Mg2 -ATPase of myosin was not a¡ected by it (1037 to 1035 M, data not shown). The reversibility was examined using the dilution method. If the e¡ect of AL-B on actin or myosin was irreversible, some stimulation might be
Fig. 9. The log concentration^response curve for amphidinolide B (AL-B) in the ATPase activity of natural actomyosin. Increase in the ATPase activity was expressed as a percentage against the control activity (0.219 Wmol/min per mg) in the absence of AL-B at a Ca2 concentration of 3U1036 M.
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suggesting that AL-B interacts with actin or myosin reversibly. In the Ca2 electrode experiment, even after application of AL-B (3U1035 M), an increase in extravesicular Ca2 concentration of HSR was not detected, suggesting inactivity on the Ca2 release channel. Furthermore, Ca2 -pumping ATPase activity (0.512 Wmol/min per mg) in HSR was not changed by AL-B (3U1035 M). These data suggest that AL-B is a selective activator of skeletal myo¢lament contraction and actomyosin ATPase. 4. Discussion Fig. 10. The log concentration^response curve for amphidinolide B (AL-B) in the ATPase activity of myo¢brils. Increase in the ATPase activity was expressed as a percentage against the control activity (0.113 Wmol/min per mg) in the absence of ALB at a Ca2 concentration of 3U1036 M.
found even after the dilution. As a result, there was no signi¢cant di¡erence among the ATPase activities of actomyosin consisting of AL-B-pretreated actin and myosin, actomyosin consisting of AL-B-pretreated actin and fresh myosin or actomyosin consisting of AL-B-pretreated myosin and fresh actin,
Fig. 11. The log concentration^response curve for amphidinolide B (AL-B) in the ATPase activity of actomyosin reconstituted from actin and myosin in the absence (a) or presence (b) of troponin^tropomyosin complex. Increase in the ATPase activity was expressed as a percentage against the control activity (a, 0.044 Wmol/min per mg; b, 0.021 Wmol/min per mg) in the absence of AL-B at a Ca2 concentration of 1037 M. Each point represents the mean þ S.E. from three experiments.
A widely accepted theory to explain the mutual sliding of actin and myosin ¢laments is the crossbridge theory of muscle contraction. The crossbridge mechanism was ¢rmly established and seemed to be a reasonable explanation for the relative sliding of the actin and myosin ¢lament [27^30]. The force of muscle contraction is produced by the interaction between actin and myosin molecules [31] in which chemical energy in ATP molecules is converted into mechanical work [4,29,32]. The superprecipitation of natural actomyosin is generally accepted to be basically the same phenomenon in vitro as a contraction in skeletal muscle cells [33]. In the present experiment, AL-B increased the contractile response of skinned ¢bers and superprecipitation of natural actomyosin. The ATPase activity of skeletal muscle myo¢bril, natural actomyosin or actomyosin reconstituted from actin and myosin was enhanced markedly by AL-B. It is not clear why the e¡ect of AL-B on natural actomyosin ATPase activity is poor. The detail of this mechanism is now under investigation. However, the Ca2 , K -EDTA- or Mg2 -ATPase activity of myosin was not a¡ected by AL-B, suggesting the elimination of the possible involvement of direct stimulation of myosin ATPases in the mechanism of actomyosin ATPase activation. These results suggest that AL-B-induced enhancement of the contraction of skinned ¢bers and superprecipitation of natural actomyosin is caused at least partially by increasing the interaction between actin and myosin directly. The troponin^tropomyosin interaction is thought to be a crucial part of the protein interactions which regulate the actomyosin ATPase activity of skeletal
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muscles [19,34,35]. Contraction of skeletal muscle is switched on and o¡ by Ca2 over the concentration range of 1037 to 1036 M. It is generally accepted that in skeletal muscles Ca2 ¢rst interacts with troponinC, resulting in a shift of the position of tropomyosin on skeletal thin ¢lament, leading to the contraction of muscle ¢bers. Troponin-C confers Ca2 sensitivity on the contractile system of skeletal muscle [36,37]. It has been reported that Ca2 sensitizers such as levosimendan [38] and MCI-154 [39] directly in£uence the responsiveness of the contractile protein to Ca2 by a¡ecting troponin-C. The ATPase activity of the actin^myosin^troponin^tropomyosin reconstituted system was enhanced by AL-B. AL-B increased Ca2 sensitivity in skinned ¢bers and enhanced the response of natural actomyosin to Ca2 in both superprecipitation and ATPase activity. These results suggest that AL-B is a¡ecting the regulatory proteins, resulting in an increase in Ca2 sensitivity to enhance the contractile response of myo¢lament. In conclusion, AL-B directly activates the interaction of actin and myosin and gives in£uence on the function of the troponin^tropomyosin system, resulting in an increase in actomyosin ATPase activity and thus enhances the contraction of myo¢lament. AL-B has become a useful tool for the investigation not only of the interaction between actin and myosin but also of the molecular regulatory mechanism by the troponin^tropomyosin complex. Acknowledgements We are indebted to Dr. Ken-Ichi Furukawa of this department, Dr. Hideshi Nakamura of Hokkaido University and the late Masaki Kobayashi of Mitsubishi Kasei Institute of Life Sciences for useful advice. We also thank Ms. Akiko Muroyama and Ms. Hiromi Kobayashi for technical assistance. This work was partially supported by a Grant-in-aid for Scienti¢c Research from the Ministry of Education, Science, Sports and Culture of Japan. Financial support through Grants-in-aid from the Tokyo Biochemical Research Foundation, the Sagawa Foundation for Promotion of Cancer Research and the Nakatomi Foundation are also acknowledged.
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References [1] R.W. Lymn, E.W. Taylor, Mechanism of adenosine triphosphate hydrolysis by actomyosin, Biochemistry 10 (1971) 4617^4624. [2] E. Eisenberg, T.L. Hill, Y. Chen, Cross-bridge model of muscle contraction. Quantitative analysis, Biophys. J. 29 (1980) 195^227. [3] L.A. Stein, P.B. Chock, E. Eisenberg, Mechanism of the actomyosin ATPase : e¡ect of actin on the ATP hydrolysis step, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1346^1350. [4] H. Sugi, Molecular mechanism of ATP-dependent actin-myosin interaction in muscle contraction, Jpn. J. Physiol. 43 (1993) 435^454. [5] I. Rayment, W.R. Rypniewski, K. Schmidt-Base, R. Smith, D.R. Tomchick, M.M. Bennimg, D.A. Winkelmann, G. Wesenberg, H.M. Holden, Three-dimensional structure of myosin subfragment-1: a molecular motor, Science 261 (1993) 50^58. [6] I. Rayment, H.M. Holden, M. Whittaker, C.B. Yohn, M. Lorenz, K.C. Holmes, R.A. Millingan, Structure of the actin-myosin complex and its implications for muscle contraction, Science 261 (1993) 58^65. [7] Y. Ohizumi, Application of physiologically active substances isolated from natural resources to pharmacological studies, Jpn. J. Pharmacol. 73 (1997) 263^289. [8] J. Takito, H. Nakamura, J. Kobayashi, Y. Ohizumi, K. Ebisawa, Y. Nonomura, Purealin, a novel stabilizer of smooth muscle myosin ¢laments that modulates ATPase activity of dephosphorylated myosin, J. Biol. Chem. 261 (1986) 13861^ 13865. [9] Y. Nakamura, M. Kobayashi, H. Nakamura, H. Wu, J. Kobayashi, Y. Ohizumi, Purealin, a novel activator of skeletal muscle actomyosin ATPase and myosin EDTA-ATPase that enhanced the superprecipitation of actomyosin, Eur. J. Biochem. 167 (1987) 1^6. [10] M. Kobayashi, H. Nakamura, J. Kobayashi, Y. Ohizumi, Mechanism of inotropic action of xestoquinone, a novel cardiotonic agent isolated from a sea sponge, J. Pharmacol. Exp. Ther. 257 (1991) 82^89. [11] M. Kobayashi, A. Muroyama, H. Nakamura, J. Kobayashi, Y. Ohizumi, Xestoquinone, a novel cardiotonic agent activates actomyosin ATPase to enhance contractility of skinned cardiac or skeletal muscle ¢bers, J. Pharmacol. Exp. Ther. 257 (1991) 90^94. [12] H. Sakamoto, K.-I. Furukawa, K. Matsunaga, H. Nakamura, Y. Ohizumi, Xestoquinone activates skeletal muscle actomyosin ATPase by modi¢cation of the speci¢c sulfhydryl group in the myosin head probably distinct from sulfhydryl groups SH1 and SH2 , Biochemistry 34 (1995) 12570^ 12575. [13] K.-I. Furukawa, K. Sakai, S. Watanabe, K. Maruyama, M. Murakami, K. Yamaguchi, Y. Ohizumi, Goniodomin A induces modulation of actomyosin ATPase activity mediated through conformational change of actin, J. Biol. Chem. 268 (1993) 26026^26031.
BBAGEN 24761 4-3-99
32
K. Matsunaga et al. / Biochimica et Biophysica Acta 1427 (1999) 24^32
[14] M. Ishibashi, Y. Ohizumi, M. Hamashima, H. Nakamura, Y. Hirata, T. Sasaki, J. Kobayashi, Amphidinolide B, a novel macrolide with potent antineoplastic activity from the marine dino£agellate Amphidinium sp., J. Chem. Soc. Chem. Commun. 14 (1987) 1127^1129. [15] S.V. Perry, A. Corsi, Extraction of proteins other than myosin from the isolation rabbit myo¢bril, Biochem. J. 68 (1958) 5^12. [16] Y. Meng, T. Yasunaga, T. Wakabayashi, Determination of the protein components of native thin ¢laments isolated from natural actomyosin: neblin and K-actinin are associated with actin ¢lament, J. Biochem. 118 (1995) 422^427. [17] A.G. Weeds, R.S. Taylor, Separation of subfragment-1 isoenzymes from rabbit skeletal muscle myosin, Nature 257 (1975) 54^56. [18] L.E. Hill, J.P. Mehegan, C.A. Butters, L.S. Tobacman, Analysis of troponin-tropomyosin binding to actin, J. Biol. Chem. 267 (1992) 16106^16113. [19] E.P. Morris, S.S. Lehrer, Troponin-tropomyosin interactions. Fluorescence studies of the binding of troponin, troponin T, and chymotryptic troponin T fragments to specifically labeled tropomyosin, Biochemistry 23 (1984) 2214^ 2220. [20] M. Endo, T. Kitazawa, E-C coupling studies on skinned cardiac studies, in: M. Morad (Ed.), Biophysical Aspects of Cardiac Muscle, Academic Press, New York, 1978, pp. 307^327. [21] M. Endo, M. Iino, Speci¢c perforation of muscle cell membranes with preserved SR functions by saponin treatment, J. Muscle Res. Cell Motil. 1 (1980) 89^100. [22] K. Horiuti, Some properties of the contractile system and sarcoplasmic reticulum of skinned slow ¢bers from Xenopus muscle, J. Physiol. 373 (1986) 1^23. [23] J.B. Martin, D.M. Doty, Determination of inorganic phosphate, Anal. Chem. 21 (1949) 965^967. [24] D.H. Kim, S. T, N. Ohnishi, Ikemoto, Kinetic studies of calcium release from sarcoplasmic reticulum in vitro, J. Biol. Chem. 258 (1983) 9662^9668. [25] A. Seino, M. Kobayashi, J. Kobayashi, Y.-I. Fang, M. Ishibashi, H. Nakamura, K. Momose, Y. Ohizumi, 9-Methyl-7bromoeudistomin D, a powerful radio-labelable Ca releaser having ca¡eine-like properties, acts on Ca -induced Ca release channels of sarcoplasmic reticulum, J. Pharmacol. Exp. Ther. 256 (1991) 861^867.
[26] Y. Nakamura, J. Kobayashi, J. Gilmore, M. Mascal, K. Rinehart, H. Nakamura, Y. Ohizumi, Bromo-eudistomin D, a novel inducer of calcium from fragmented sarcoplasmic reticulum that causes contractions of skinned muscle ¢bers, J. Biol. Chem. 261 (1986) 4139^4142. [27] A.F. Huxley, Muscle structure and theories of contraction, Prog. Biophys. Biophys. Chem. 7 (1957) 255^318. [28] H.E. Huxley, The mechanism of muscular contraction, Science 164 (1969) 1356^1366. [29] K.C. Holmes, The actomyosin interaction and its control by tropomyosin, Biophys. J. 68 (1995) 2S^7S. [30] K.C. Holmes, Muscle proteins ^ their actions and interactions, Curr. Opin. Struct. Biol. 6 (1996) 781^789. [31] S.Y. Bershitsky, A.K. Tsaturyan, O.N. Bershitskaya, G.I. Mashanov, P. Brown, R. Burns, M.A. Ferenczi, Muscle force is generated by myosin heads stereospeci¢cally attached to actin, Nature 388 (1997) 186^190. [32] Y. Tonomura, Energy-transducing ATPases ^ Structure and Kinetics, Cambridge University Press, Cambridge, 1986, pp. 44^88. [33] A. Szent-Gyorgyi, Chemistry of Muscular Contraction, 2nd ed., Academic Press, New York, 1951. [34] H.E. Huxley, Structural changes during muscle contraction, Biochem. J. 125 (1971) 85P. [35] R.H. Ingraham, C.A. Swenson, Interaction of troponin and tropomyosin: Spectroscopic and calorimetric studies, Biochemistry 24 (1985) 5221^5225. [36] C.S. Farah, F.C. Reinach, The troponin complex and regulation of muscle contraction, FASEB J. 9 (1995) 755^767. [37] S.M. Gagne, M.X. Li, B.D. Sykes, Mechanism of direct coupling and induced structural change in regulatory calcium binding proteins, Biochemistry 36 (1997) 4386^4392. [38] H. Haikala, E. Nissinen, E. Etemadzadeh, I.-B. Linden, P. Pohto, Levosimendan increases calcium sensitivity without enhancing myosin ATPase activity and impairing relaxation, J. Mol. Cell. Cardiol. 24 (1992) S97. [39] Y. Abe, Y. Kitada, A. Narimasu, E¡ect of MCI-154, a cardiotonic agent, on regional contractile function and myocardial oxygen consumption in the presence and absence of coronary artery stenosis in dogs, J. Pharmacol. Exp. Ther. 256 (1993) 819^825.
BBAGEN 24761 4-3-99