CRAYFISH SKELETAL MUSCLE REQUIRES BOTH INFLUX OF EXTERNAL Ca 2+ AND Ca 2+ RELEASE FROM INTERNAL STORES FOR CONTRACTION

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1 J. exp. Biol. 181, (1993) Printed in Great Britain The Company of Biologists Limited CRAYFISH SKELETAL MUSCLE REQUIRES BOTH INFLUX OF EXTERNAL Ca 2+ AND Ca 2+ RELEASE FROM INTERNAL STORES FOR CONTRACTION HIDEKI USHIO, SHUGO WATABE* Laboratory of Marine Biochemistry, Faculty of Agriculture, University of Tokyo, Tokyo 113, Japan and MASAMITSU IINO Department of Pharmacology, Faculty of Medicine, University of Tokyo, Tokyo 113, Japan Accepted 8 April 1993 Summary The isometric tension and membrane potential of single skeletal muscle fibres from the flexor muscle of the carpopodite in the meropodite of crayfish Procambarus clarkii (Girard) were studied to determine whether crayfish muscle contraction requires Ca 2+ release from the sarcoplasmic reticulum. Contraction elicited by brief extracellular electrical stimulation was reduced by the removal of Ca 2+ or by the addition of 25 mol l 1 nicardipine in crayfish Ringer s solution. Addition of 30 mol l 1 ryanodine with 1mmol l 1 caffeine induced a transient contracture, the peak tension of which was 10 30% of that of the high-k + induced contracture and which declined to the pretreatment level in 20 60min. After ryanodine caffeine treatment, 30mmol l 1 caffeine failed to induce contraction, suggesting that intracellular Ca 2+ stores had been exhausted by the treatment. Extracellular electrical stimulation also failed to induce contraction after ryanodine caffeine treatment, although the resting potential was not changed. These results suggest that Ca 2+ release from the sarcoplasmic reticulum, together with Ca 2+ influx via nicardipine-sensitive Ca 2+ channels, is essential to the contraction of crayfish leg muscle fibres after a brief membrane depolarization. Introduction Ca 2+ release from the sarcoplasmic reticulum (SR) is important in the regulation of vertebrate skeletal and cardiac muscle contraction (Endo, 1977). There is a regular array of structures known as feet at the junction between the terminal cisternae of the SR and the transverse tubules (Block et al. 1988). These correspond to the Ca 2+ release channel of the SR (Inui et al. 1987a,b; Lai et al. 1988). Ryanodine, a plant alkaloid, binds to this channel and at low concentrations fixes it in an open state (Fleischer et al. 1985; Rousseau *To whom correspondence should be addressed. Key words: crayfish, Procambarus clarkii, skeletal muscle, sarcoplasmic reticulum, ryanodine, Ca 2+ release, Ca 2+ channel.

2 96 H. USHIO, S. WATABE and M. IINO et al. 1987; Nagasaki and Fleischer, 1988). Using ryanodine as a marker, the Ca 2+ release channel has been purified (Hymel et al. 1988; Imagawa et al. 1987; Inui et al. 1987a,b) and sequenced (Takeshima et al. 1989). The Ca 2+ release channel is believed to face onto dihydropyridine-binding proteins in the transverse tubule membranes (Block et al. 1988). The two important proteins associated with the foot are also present in crayfish skeletal muscle. Formelova et al. (1990) isolated ryanodine-binding proteins from crayfish abdominal muscle and showed that they exhibited a Ca 2+ conductance in planar lipid bilayers. Recently, Krizanova et al. (1990) have isolated dihydropyridine-binding proteins from crayfish abdominal muscle and found that the properties of these proteins were similar to those of their mammalian counterparts. Furthermore, feet structures are also observed in crayfish muscle (Loesser et al. 1992). In crustacean skeletal muscle fibres, electrical stimulation triggers local, nonpropagatable depolarization (Fatt and Katz, 1953a,b), which subsequently induces an inward Ca 2+ current (Hagiwara and Naka, 1964; Hagiwara et al. 1964). The Ca 2+ influx has been reported to be essential (Gainer, 1968; Matsumura, 1978) or sufficient to (Atwater et al. 1974) give rise to contraction of crustacean muscles. It has been shown that Ca 2+ release from the SR is necessary for contraction of barnacle muscle fibres (Ashley and Lea, 1978). Mounier and Goblet (1987) investigated the effects of caffeine and procaine on the contraction of crab leg muscle fibres and proposed that Ca 2+ release from internal stores is required for contraction of these muscle fibres. However, procaine affects both K + and Ca 2+ currents in crustacean muscle membranes (Hagiwara et al. 1969), and further evidence is clearly necessary to confirm the proposal of Mounier and Goblet (1987). The purpose of this study was to examine the effects of pharmacological agents known to affect excitation contraction coupling in vertebrate skeletal muscle, such as ryanodine, caffeine, nicardipine and diltiazem, on the contraction of single crayfish muscle fibres in search of the source of Ca 2+ for their contraction. Materials and methods Single skeletal muscle fibres were prepared from the proximal flexor muscle of the carpopodite in the meropodite segment of the crayfish Procambarus clarkii (Girard) leg. These fibres were 5 8mm long and were shaped like elliptical cylinders, m along the major axis and m along the minor axis. Excised muscle fibres were put in a chamber at 10 C filled with a crayfish Ringer s solution (see below). The fibres were also observed in a transmission electron microscope by conventional methods as described in Brandt et al. (1965). Solutions The crayfish Ringer s solution contained 205mmol l 1 NaCl, 5.4mmol l 1 KCl, 13.5mmol l 1 CaCl 2, 5.6mmol l 1 MgCl 2 and 2.4mmol l 1 Hepes (ph7.2). The high- K + solution contained 132.4mmol l 1 sodium propionate, 8mmol l 1 NaCl, 70mmol l 1 potassium propionate, 13.5mmol l 1 calcium propionate and 5.6mmol l 1 MgCl 2 to keep the [K + ][Cl ] product constant. In experiments to elicit action potentials, a solution

3 Ca 2+ release from crayfish sarcoplasmic reticulum 97 containing tetraethylammonium (TEA + ), i.e. 107mmol l 1 TEACl, 105mmol l 1 NaCl, 5.4mmol l 1 KCl, 13.5mmol l 1 CaCl 2, 5.6mmol l 1 MgCl 2 and 2.4mmol l 1 Hepes (ph7.2), was used. In the Ca 2+ -free solution, CaCl 2 in the crayfish Ringer s solution was replaced by the same concentration of MgCl 2. All chemicals were of reagent grade from Sigma (St Louis), except that ryanodine and nicardipine were obtained from Wako Chemicals (Tokyo) and Yamanouchi Pharmaceutical (Tokyo), respectively. Measurement of tension and membrane potential Single fibres were stretched to 1.25 times their resting length. For extracellular stimulation, rectangular current pulses of 20ms duration were passed through platinum electrodes placed parallel to the fibre. Isometric tension was measured with a strain gauge transducer and displayed using a pen recorder. To measure membrane potential, a microelectrode filled with 3mol l 1 KCl (resistance approximately 10 M ) was inserted near one end of the fibre. In the experiments using intracellular stimulation, the second microelectrode was inserted about 30 m from the recording electrode and currents of 1.2 A were passed through an electric isolator. Changes in the membrane potential were monitored on an oscilloscope. Both tension and membrane potential signals were digitized using an analog-to-digital converter in a personal computer (PC-9801 VM, NEC, Tokyo) and stored on floppy disks for later analyses. Results Effect of Ca 2+ removal Single fibres excised from the proximal area of the carpopodite flexor muscle had a sarcomere length of about 8 m (range 7 10 m) under the electron microscope. The peak tension elicited by extracellular electrical stimulation of the single fibres was about 4 9mNmm 2 with a mean ± S.E.M. of 6.2±0.6mNmm 2 (N=9). The high-k + solution (70mmol l 1 K + ) induced a transient contracture, which attained a peak tension of about 135mNmm 2 in about 10s (N=4). When a fibre was immersed in Ca 2+ -free solution, the electrically evoked contraction faded rapidly (Fig. 1A). The contraction was promptly restored by washing with normal crayfish Ringer s solution. The membrane potential of the fibres in the Ca 2+ -free solution was the same as that before such treatment, i.e. it lay within the range 75 to 80mV (N=24). Effects of Ca 2+ antagonists Calcium antagonists such as nicardipine and diltiazem bind to L-type Ca 2+ channels and block the Ca 2+ current (Bean, 1984; Kanaya et al. 1983; Sanguinetti and Kass, 1984). Since dihydropyridine-binding proteins or L-type Ca 2+ channels have been isolated from crayfish skeletal muscle, we examined the effects of these Ca 2+ antagonists on the contraction of crayfish leg muscle fibres. Following the application of 25 moll 1 nicardipine in crayfish Ringer s solution, the peak tension elicited by extracellular stimulation was slightly and transiently enhanced and then gradually reduced until the

4 98 H. USHIO, S. WATABE and M. IINO A 0.1mN 0mmoll 1 Ca 2+ 2min B 25 moll 1 nicardipine Fig. 1. Effects of the removal of Ca 2+ (A) and the addition of 25 mol l 1 nicardipine (B) on the contraction of a single crayfish muscle fibre induced by extracellular electrical stimulation. contraction was abolished after 15 20min (Fig. 1B). The resting membrane potential after this treatment was in the range 77 to 81mV (N=4), which was not significantly different from the level in normal crayfish Ringer s solution. Diltiazem had no effect on the contraction elicited by extracellular electrical stimulation. It has been reported that diltiazem binds to Ca 2+ channels when they are in an inactivated state (Kanaya et al. 1983). In high-k + solution, a contracture was induced in crayfish fibres in the presence of diltiazem to see whether diltiazem is effective when Ca 2+ channels are inactivated. However, even at concentrations up to 100 mol l 1, diltiazem had no effect on the subsequent contraction (data not shown). Effects of caffeine and ryanodine Contractions elicited by extracellular electrical stimulation were enhanced by the addition of 1mmol l 1 caffeine; the peak tension was about 10 times greater than that in normal crayfish Ringer s solution (Fig. 2A). Irregular contractures were sometimes observed in the presence of caffeine (Fig. 2A). They were due to local contractures, visible under a binocular microscope, propagating from one end of the fibre to the other with gradual attenuation. Caffeine at a concentration of 30mmol l 1 induced a strong contracture so that the connection between the fibre and the tendon broke (data not shown). Removal of 1mmol l 1 caffeine restored contractions induced by extracellular electrical stimulation to pretreatment levels (Fig. 2A). Addition of 30 mol l 1 ryanodine with 1mmol l 1 caffeine initially enhanced the contraction induced by extracellular stimulation and then induced a contracture, the tension of which was 20 30% of that of the high-k + -induced contracture (Fig. 2B). This contracture was transient and subsided to the pretreatment level in about 20 30min. Following the ryanodine caffeine treatment, no contraction was observed in response to extracellular stimulation (Fig. 2A). The application of 30mmol l 1 caffeine also failed to

5 Ca 2+ release from crayfish sarcoplasmic reticulum 99 A 0.5 mn 1mmoll 1 caffeine 1mmoll 1 caffeine + 30 moll 1 ryanodine 30mmoll 1 caffeine B C 2min 2.5 mn 70 mmoll 1 K + 70 mmoll 1 K + Fig. 2. (A) The effects of successive treatment with 1mmol l 1 caffeine and 30 mol l 1 ryanodine plus 1mmol l 1 caffeine on the contraction induced by extracellular electrical stimulation and the aftereffect of the ryanodine treatment on the contraction induced by 30mmol l 1 caffeine. Contractures induced in 70mmol l 1 K + before (B) and after (C) the ryanodine caffeine treatment were recorded for comparison. Arrowheads mark irregular contractures during caffeine application. induce a contracture after ryanodine caffeine treatment (Fig. 2A). However, a high-k + - induced contracture was observed even after the treatment (Fig. 2C), although the peak tension was about half and the decay of tension was slower than those of the control high- K + -induced contracture (Fig. 2B). The high-k + -induced contracture rose to 50% of the peak tension in 2.50±0.41s (N=4) in the control condition. The corresponding duration after ryanodine caffeine treatment was 13.53±1.89s (N=4), and the difference between these two values was significant (P<0.01, t-test). The resting membrane potential after the ryanodine caffeine treatment was in the range 74 to 79mV (N=9), which is not significantly different from that in normal Ringer s solution. Application of 30 mol l 1 ryanodine without caffeine also induced a transient contracture, the tension of which was about half of that during ryanodine caffeine treatment and which decayed more slowly. After ryanodine treatment, extracellular electrical stimulation and 30mmol l 1 caffeine also failed to induce contraction (data not shown). Action-potential-induced contractions in the presence of TEA + Neither extracellular nor intracellular stimulation induced action potentials in the crayfish leg muscle fibres. In the presence of TEA +, however, the fibres produced action potentials accompanied by strong contractions. The action potential had a plateau phase lasting ms and a peak tension of about 100mNmm 2, which was attained about 300ms after the stimulus (Fig. 3A,B, top traces).

6 100 H. USHIO, S. WATABE and M. IINO A mV B mN Fig. 3. Effects of the removal of Ca 2+ from the crayfish Ringer s solution on the action potential induced by intracellular stimulation (using a 1.2 A current) in the presence of TEA + (A) and the accompanying development of tension (B). The short horizontal bars in A represent the 0mV level. Numbers represent the time (min) after the removal of Ca s When the fibres were immersed in Ca 2+ -free TEA + -containing solution, both action potential and contraction were reduced gradually and disappeared in 10 30min (Fig. 3). The depolarization remaining after 30min in Ca 2+ -free condition was mostly a passive response to intracellular stimulation. The slow time course of the effect of Ca 2+ withdrawal on the action potential duration could be explained by the difficulty of removing Ca 2+ from the multibranched cleft and transverse tubule systems (Brandt et al. 1965; Hoyle et al. 1973). Addition of 25 mol l 1 nicardipine also reduced both the action potential duration and the contraction (Fig. 4). Action potentials in the presence of TEA + had considerably longer time courses after ryanodine caffeine treatment (Fig. 5A,B). The duration of this prolonged action potential was not influenced by tetrodotoxin at concentrations up to 500 mol l 1 (data not shown). Although contractions elicited by extracellular stimulation were abolished by ryanodine caffeine treatment in the absence of TEA + (Fig. 2), a prolonged action potential during the treatment in the presence of TEA + was followed by a slow rise in tension (Fig. 5B, lower trace). Peak tension was attained after about 10s. In Ca 2+ -free TEA + -containing solution, both the action potential and the rise in tension were almost abolished (Fig. 5C). Discussion Our results clearly show that the electrically evoked contraction of single crayfish muscle fibres is greatly diminished after treatment with ryanodine, with or without

7 Ca 2+ release from crayfish sarcoplasmic reticulum 101 A mV B mN Fig. 4. Effects of addition of 25 mol l 1 nicardipine on the action potential induced by intracellular stimulation in the presence of TEA + (A) and the accompanying development of tension (B). The short bars in A represent the 0mV level. Numbers represent the time (min) after the addition of nicardipine. 0.5s simultaneous caffeine application. It has been shown in vertebrate skeletal muscle that ryanodine binds to the Ca 2+ -release channels of the SR when the channels are open and fixes them in an open state (Fleischer et al. 1985; Rousseau et al. 1987; Nagasaki and Fleischer, 1988), resulting in the inhibition of Ca 2+ release from the SR. The same effect was observed in the crayfish muscle fibres: caffeine blocked the release of Ca 2+ after ryanodine treatment (Fig. 2). These results indicate that electrically evoked contraction depends critically on Ca 2+ release from the SR. To determine whether ryanodine treatment had any effect on the membrane potential and/or on the voltage-dependent Ca 2 + channel, we studied the effect of ryanodine on action-potential-induced contractions. Crayfish muscle fibres do not usually generate action potentials, but these can be induced when K + currents are suppressed by TEA + (Fatt and Ginsborg, 1958; Hagiwara et al. 1964; Hencek and Zachar, 1977). Such action potentials are resistant to tetrodotoxin but are sensitive to nicardipine (Fig. 4 ), suggesting that the inward currents are mainly carried by Ca 2 +. After ryanodine treatment, the resting membrane potential was unchanged and the action potential duration was prolonged, causing enhanced Ca 2 + i n flux. However, the development of tension was slower and smaller (Fig. 5). Similarly, high-k + -induced depolarization produced slower and smaller forces after ryanodine treatment (Fig. 2). These results c o n firm that Ca 2 + i n flux is essential (Fatt and Katz, 1953a,b), but is not sufficient, for contraction following brief depolarizations. Under such circumstances, Ca 2 + r e l e a s e from the SR is important for the initiation of rapid tension development. However, when

8 102 H. USHIO, S. WATABE and M. IINO A B 50mV 2mN C Fig. 5. Effects of ryanodine caffeine treatment on action potential and tension development in the presence of TEA +. The action potentials (top traces) and the development of tension (bottom traces) were recorded before ryanodine caffeine treatment (A), after ryanodine caffeine treatment in the presence of Ca 2+ (B) and after ryanodine caffeine treatment in the absence of Ca 2+ (C). The bars in the upper traces represent the 0mV potential level. there is a prolonged depolarization, Ca 2 + i n flux alone may induce contraction, albeit very slowly (Fig. 5 B ). The prolongation of the action potential in the ryanodine-treated fibres could be explained by a reduction in the release of Ca 2+. The inactivation of inward Ca 2+ current is modulated by cytosolic Ca 2+ concentration in vertebrate cardiac (Lee et al. 1985), vertebrate smooth (Jmari et al. 1986) and insect skeletal (Ashcroft and Stanfield, 1981) muscles and in other excitable cells (Eckert and Chad, 1984). If this is also the case in crayfish muscle fibres, the inhibition of Ca 2+ release from the SR by ryanodine caffeine treatment will delay inactivation of Ca 2+ channels, thus prolonging the duration of membrane depolarization. In addition, part of the Ca 2+ -activated outward K + current is resistant to TEA + (Mounier and Vassort, 1975a,b). This K + current must contribute to repolarization, and the reduced rise in cytoplasmic Ca 2+ concentration due to ryanodine caffeine treatment is expected to delay repolarization. Confirmation of our hypothesis requires further studies on the kinetics of both Ca 2+ and K + currents after ryanodine treatment using a voltage-clamp method. The ryanodine-binding protein has been isolated from crayfish muscle and shown to have Ca 2+ channel activity (Formelova et al. 1990). Our results demonstrate that 0.5s

9 Ca 2+ release from crayfish sarcoplasmic reticulum 103 ryanodine has a profound effect on the Ca 2+ release mechanism. Goblet and Mounier (1986) showed that a low concentration of Ca 2+ (about 10 7 mol l 1 ) induced Ca 2+ release from the SR of crab leg muscles. Mounier and Goblet (1987) demonstrated, using the same type of muscle, that the tension development induced by voltage-clamp was inhibited by procaine, an inhibitor of Ca 2+ -induced Ca 2+ release from the SR (Endo, 1985). Similarly, the Ca 2+ -induced Ca 2+ release blocker induced a large decrease in intracellular Ca 2+ transients following step voltage changes in crayfish muscle fibres (Gyorke and Palade, 1992). These results indicate the presence of a Ca 2+ -induced Ca 2+ release mechanism in the crayfish muscle and its involvement in excitation contraction coupling. Since Ca 2+ influx is essential for the initiation of contraction, it is possible that the inward Ca 2+ current induces a secondary Ca 2+ release from the SR through a Ca 2+ - induced Ca 2+ release mechanism. Crustacean muscle fibres can be classified into two broad groups: the fast phasic muscle fibres with short sarcomeres and the slow tonic ones with long sarcomeres. Both the shape and the disposition of intracellular membrane systems are different in the two groups (Abbott and Parnas, 1965; Fahrenbach, 1967; Jahromi and Atwood, 1967; Eastwood et al. 1982; Franzini-Armstrong et al. 1986). In lobster fast phasic muscle, Crowe and Baskin (1981) showed that the transverse tubules have a surface area about 50% greater than that of the SR and suggested that the large quantities of Ca 2+ that enter the sarcoplasm from the extracellular space through the sarcolemma and the tubules of this muscle are sufficient to activate the contractile machinery. In contrast, fibres of the slow tonic type have less well developed tubular systems (Brandt et al. 1965). The crayfish fibres used in the present study belong to the latter type according to Govind et al. (1981) on the basis of their sarcomere lengths (about 8 m). Therefore, the role of the SR in excitation contraction coupling can vary according to the type of muscle fibre considered. The different effects of nicardipine and diltiazem on electrically evoked contraction were unexpected. Both drugs have a profound effect on Ca 2+ currents in vertebrate cardiac cells (Lee and Tsien, 1983; Sanguinetti and Kass, 1984). It will be interesting to determine whether crayfish Ca 2+ channels lack binding sites for diltiazem. The expenses of the present study were supported in part by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science and Culture of Japan. References ABBOTT, B. C. AND PARNAS, I. (1965). Electrical and mechanical responses in deep abdominal extensor muscles of crayfish and lobster. J. gen. Physiol. 48, ASHCROFT, F. M. AND STANFIELD, P. R. (1981). Calcium dependence of the inactivation of calcium currents in skeletal muscle fibers of an insect. Science 213, ASHLEY, C. C. AND LEA, T. J. (1978). Calcium fluxes in single muscle fibres measured with a glass scintillator probe. J. Physiol., Lond. 282, ATWATER, I., ROJAS, E. AND VERGARA, J.(1974). Calcium influxes and tension development in perfused single barnacle muscle fibres under membrane potential control. J. Physiol., Lond. 243, BEAN, B. P. (1984). Nitrendipine block of cardiac calcium channels: high-affinity binding to the inactivated state. Proc. natn. Acad. Sci. U.S.A.81,

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