THE MAXIMUM SHORTENING VELOCITY OF HOLOTHURIAN MUSCLE AND EFFECTS OF TONICITY CHANGE ON IT

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1 J. exp. Biol. 9, (1985) 3 \ Printed in Great Britain The Company of Biologists Limited 1985 THE MAXIMUM SHORTENING VELOCITY OF HOLOTHURIAN MUSCLE AND EFFECTS OF TONICITY CHANGE ON IT BY T. TSUCHIYA* Bermuda Biological Station for Research, Ferry Reach 1-15, Bermuda Accepted 8 May 1985 SUMMARY 1. The maximum shortening velocity of the longitudinal retractor muscle of the holothurian, Isostichopus badionotus, was measured using a 'slack test' in solutions of normal, increased and reduced osmotic strength. 2. At normal osmotic strength, the average maximum shortening velocity was 0-44 ± 0-04 LoS" 1 (N=14) which is fast compared to other smooth muscles. This is unexpected considering the slow locomotion of these animals. 3. Raising the osmotic strength by 55 % by addition of sucrose reduced the shortening velocity by more than 50 %. Raising the osmotic strength by 52 % with NaCl caused a 35 % reduction in shortening velocity. 4. Lowering the osmotic strength by 19% or 39%, by reducing NaCl concentration, did not significantly affect shortening velocity. 5. The shortening velocity was found to be proportional to the volume of the muscle preparation. INTRODUCTION Holothurians have five longitudinal retractor muscles which run along the inner surface of the body and these muscles are supposed to be for locomotion. A. V. Hill (192) and Levin & Wyman (1927) suggested that there were fundamental similarities between mechanical properties of these smooth muscles and vertebrate skeletal muscle, although the rate of contraction was much slower in the holothurian muscle (see review by Takahashi, 19). In the present study, the maximum shortening velocity of holothurian smooth muscle was determined by the 'slack test' (Edman, 1979) and the effect of osmotic strength was examined. MATERIALS AND METHODS Preparations The holothurians Isostichopus badionotus were collected from the seashore near Bermuda Biological Station immediately before dissection. They were cut open and Present address: Department of Physiology, School of Medicine, Teikyo University, Itabashi-ku, Tokyo 173, Japan. Key words: Holothurian muscle, shortening velocity, tonicity change.

2 32 T. TSUCHIYA 1 SM H, P, H 2 In o Fig. 1. Schematic diagram showing the analysis of records (A, B) and experimental arrangement (C). (A) The upper and lower traces represent the length and tension changes respectively. The tension falls to zero when the muscle is quickly released by AL during plateau of isometric tension and redevelops after a short delay, At, which is the duration of unloaded shortening. (B) AL is plotted against At and the maximum shortening velocity is calculated from the slope of the line. (C) Aj, arm of servo-motor; A 2, arm of tension transducer; Hi, H 2, hooks attached to both ends of a preparation by silk thread; I, inlet for bath solution; O, outlet for bath solution; P, preparation; SM, servo-motor; TT, tension transducer. Table 1. Composition of solutions Solution IT l-28tna l-s2tna l-28tsu ISSTSu 0-81TNa 0-1TNa NaCl (mmol I" 1 ) KC1 (mmol r 1 ) CaCl 2 (mmol P 1 ) MgCl 2 (mmol 1 NaHCO 3 ') (mmol P 1 ) Sucrose (mmol P 1 ) Osmolality (mosmol kg" 1 ) the longitudinal retractor muscles were excised and then split lengthwise. A strip mm long was mounted horizontally in afluidchamber between an arm attached to a servo-motor and an arm attached to a tension transducer (Shinko; U-gauge, compliance, 1 ^mg" 1 ) (Fig. 1). The servo-motor was used to apply length steps and the tension was recorded on an ink writing oscillograph. An appropriate position clamp was achieved by feed-back control using a signal from a displacement transducer, which was installed inside the motor. All experiments were performed at room temperature (24-27 C).

3 Shortening velocity of holothurian muscle 33 o L lo" 4 Concentration of ACh (mol 1 Fig. 2. Relationship between the peak height of contracture tension and acetylcholine (ACh) concentration. Solutions Solutions were as shown in Table 1. The solutions were named according to the osmolality relative to the standard solution (IT solution) and to the constituent responsible for the tonicity change, for example, l*28tna represents an osmolality 1-28 times that of the standard solution, using NaCl. The osmolality (mosmolkg" 1 water) of salt solutions was measured directly with a freezing point osmometer (Advanced Instruments Inc. Model 3L). The osmolality of the sucrose solution was calculated according to the formulae provided by Dydyriska & Wilkie (193) and that of the solutions containing both salt and sucrose was taken to be the sum of measured and calculated values. Measurement of shortening velocity To determine the shortening velocity of an unloaded fibre preparation, a contracture was first induced using acetylcholine (ACh). During the plateau phase, the arm of the servo-motor was moved to allow the fibre to slacken (20 ms for completion of a length step) and the time taken for redevelopment of tension (At) was plotted against the size of the length step (AL) (Fig. 1 A,B). Data points showed a close fit to a straight line, using the least-squares methods. Length steps were within the range of 0-32 of a fibre length (Lo). Measurement of fibre volume Relative volume was estimated from change in weight. Volume could not be estimated from diameter because diameter and cross-sectional area were not constant

4 34 T. TSUCHIYA D 0-5 s Fig. 3. Redevelopment of tension after quick release of muscle preparation during plateau of isometric tension in three different length steps (A, D, 019L«; B, E, 0-23 Lo; C, F, 0-3 L«) and at two different tonicities of the extracellular medium (A, B, C, in IT solution; D, E, F, in l-28tsu solution). along the length of the strip preparation. The muscle preparations were equilibrated for 20 min in the standard, hypertonic or hypotonic solutions. They were then lightly blotted with filter paper, wrapped with aluminium foil to avoid evaporation and weighed. RESULTS Tension development by acetylcholine For all the measurements of maximum shortening velocity, tension was developed by application of acetylcholine (ACh), which is generally believed to be the transmitter in the neuromuscular system of echinoderms (R. B. Hill, 1970; Pentreath & Cobb, 1972; Suzuki, 1982). The isometric tension showed very gradual relaxation during prolonged application of ACh. Tension was reduced by only 5 % after 45 s. A dose-response study of the effects at s showed that ACh concentrations above 3 X ~ s mol I" 1 produced maximum tension (Fig. 2). This dose is a little lower than that for the muscle of another holothurian (Sugi et al. 1982). An ACh concentration of ~ 5 moll~ 1 was adopted as the standard for further experiments, rather than 3x ~ s moll~ 1, because the muscle could break with repeated full activation. Effects of acetylcholine concentration on Vm and the dependence on time after the activation To measure the velocity of free load shortening (Vm), the muscle preparation was

5 Shortening velocity of holothurian muscle a 0-2 u (A s GO jj jj Duration of unloaded shortening (s) Fig. 4. (A) Plots of length change versus duration of unloaded shortening in three different concentrations of ACh (, "*moir l ; O, 5 X KT'moir 1 ;, ~! moll" 1 ). The maximum shortening velocity was calculated from the slope of the fitted straight line. (B) Plots of length change versus duration of unloaded shortening at two different times (, s; O, 45 s) after the application of ACh. quickly released during the plateau tension induced by 5 mol 1 1 ACh. The tension fell to zero at the onset of release and redeveloped after a delay (Fig. 3). It is clearly shown that the duration of unloaded shortening increases with an increase of step length (A, 0 19 Lo; B, 0 23 Lo; C, 0 3 Lo). The step lengths were plotted against the durations of unloaded shortening (Fig. 4). The data showed a good fit to a straight line, and the slope gave the shortening velocity. The velocity was constant at ACh concentrations greater than 5 X ~ moll~ 1 (Fig. 4A). Whereas isometric tension showed only a very gradual reduction as the ACh exposure was prolonged, the maximum shortening velocity was more strongly affected. After45 s of ACh, the velocity was only 50 % of the value at s (Fig. 4B). Shortening velocities were therefore measured s after the activation by ACh for further experiments. The averaged maximum shortening velocity of 14preparationswasO - 44 ± 0-04 Los" 1. Effects of hypertonicity on the maximum shortening velocity Shortening velocity was first measured in the standard solution. The preparation was then equilibrated for 20 min in hypertonic solution and measurements were made in that solution.

6 3 t a 2 o-i a. u Duration of unloaded shortening (s) Fig. 5. Plots of length change versus duration of unloaded shortening in increased osmotic strength made by addition of sucrose (A,, in IT; O, in l-28tsu;, in l-55tsu) or NaCl (B,, in IT; O, in l-28tna;, in l-52tna). In solutions made hypertonic with sucrose, the duration of the unloaded shortening was longer for a given length step, and the isometric tension was lower, than in the standard solution (compare Fig. 3D-F with Fig. 3A-C). The relationship between the amplitude of the length steps and the duration of unloaded shortening is illustrated in Fig. 5A, in which the maximum shortening velocities in IT (the standard solution), l-28tsu and l-55tsu solutions are 0-Los" 1, 0-38Los" 1 and 0-22 Los" 1 respectively; i.e. with relative values of 1:0-79:0-4. Average relative values were 0-78 (S.D. 0-1; N = 4) for 1-28TSu, and 0-42 (S.D. 0-05,TV = 4) for 1-55TSu. The strong effect of the hypertonic sucrose solution upon the maximum shortening velocity, and the observation that the rate of the tension redevelopment is slower in hypertonic solution (Fig. 3), suggests that the turnover rate of crossbridges may be slower in hypertonic solution. Maximum shortening velocity was less affected by hypertonic solutions in which tonicity was raised with NaCl rather than sucrose. In Fig. 5B the maximum

7 Shortening velocity of holothurian muscle s 0-2 CO c 0-2 Duration of unloaded shortening (s) 0-4 Fig.. Plots of length change versus duration of unloaded shortening in reduced oemotic strength., in IT; O, in 0-81TNa;, in 0-1TNa. shortening velocities in IT, 1-28TNa and 1-52TNa solutions are 0-47 Lo s" 1,0-47 Lo s" 1 and 0-30 Lo s" 1 respectively; with relative values of 1:1:0-4. Average relative shortening velocities were 0-91 (S.D. 0-l,N = 4) for l-28tna and 05 (S.D. 0-05, AT =4) for l-52tna. Effects of hypotonicity on the maximum shortening velocity After measurement of shortening velocity in the standard solution, the preparation was equilibrated for 20min in hypotonic solution. Shortening velocity was little affected. In Fig., the shortening velocities in IT, 0-81TNa and 0-1TNa solutions are 0-58LOS" 1, 0-54LOS" 1 and 0-7Los -1 respectively; with relative values of 1:0-93:1-1. The average relative shortening velocities were 0-98 (S.D. O04,./V = 4) ino-81tnaand 1-05 (S.D. 0-15, N = 4) in 0-1TNa. Tension changes in hypertonic and hypotonic solution The tension induced by ~ 5 mol I" 1 ACh was not significantly altered in solutions made hypertonic by addition of NaCl, or in hypotonic solution. In hypertonic sucrose solutions, however, tension decreased significantly both in l-28tsu and in l-55tsu (Fig. 7A). It was observed that in a few preparations appreciable tension developed in hypertonic or hypotonic solution without ACh and these fibres were not used for the measurements of tension or shortening velocity. Volume changes in hypertonic and hypotonic solutions Preparation volume was first measured in the standard solution, then in the hypertonic or hypotonic solutions and finally again in the standard solution. A steady value

8 38 T. TSUCHIYA was attained within 20min after a solution change. There was a large reduction in volume in solutions made hypertonic with sucrose, while solutions made hypertonic with NaCl had less effect and hypotonic solutions produced no appreciable change (Fig. 7B) a o-9 o Relative osmotic strength 1-5 Fig. 7. (A) Relationship between relative tension and relative osmotic strength :, in IT; O, osmotic strength was increased or reduced by changing NaCl concentration;, osmotic strength was increased by addition of sucrose. Each symbol and bar represents the mean and s.d. of four experiments. (B) Relationship between relative volume and relative osmotic strength :, in IT; O, osmotic strength was increased or reduced by changing NaCl concentration; D, osmotic strength was increased by addition of sucrose. Each symbol and bar represents the mean and s.d. of five measurement in different preparations.

9 Shortening velocity of holothurian muscle 39 DISCUSSION The average maximum shortening velocity of the longitudinal retractor muscle of Isostichopus badionotus was 0-44Los" 1. In cat muscle (Close & Hoh, 197), it is 22-8 Lo s" 1. In smooth muscles of pig carotid artery (Herlihy & Murphy, 1974), dog trachea (Stephens, Kroeger & Mehta, 199), rabbit urinary bladder (Uvelius, 197) and guinea pig taenia coli (Mashima, Okada & Okumura, 1979), the velocities are 0-12, 0-17, 0-23 and 0-53 LOs" 1 respectively. For invertebrate smooth muscle, the shortening velocities have been found to be Los" 1 for anterior byssus retractor muscle of Mytilus (Lowy & Millman, 193; Tameyasu & Sugi, 197) and 0-42L0S" 1 for the foot muscle of Mytilus (Ishii & Takahashi, 1981). Thus, the shortening velocity of holothurian muscle is fast compared with that of other smooth muscles, and not slow as might be expected from the slow locomotion of these animals. Therefore, it is conceivable that the fast contraction of this muscle is not utilized in the locomotion of this animal, but in some other behaviour. In striated muscle, the maximum shortening velocity, which is much faster than that in smooth muscle, has been shown to decrease remarkably in hypertonic solution and to increase appreciably in hypotonic solution (Edman & Hwang, 1977). If a fundamental mechanism of contraction is common to striated and smooth muscles, it may be that similar phenomena are observed in smooth muscles. Little information is at present available about the effects of hypertonic solution on the contractile tension and the shortening velocity in smooth muscles except for Mytilus (Sugi, Yamaguchi & Tanaka, 1977; Tameyasu, 1978). An essential step in the analysis of the maximum shortening velocity is tofind out whether the tension level before the release affects the velocity. In holothurian muscle, shortening velocity is apparently not related to the tension at ACh concentrations above 5 X ~ mol I" 1 (Fig. 4). Shortening velocity is, however, related to volume (Figs 5,, 7). In striated fibres it is suggested that the effect of volume upon velocity could be by change in ionic strength (Edman & Hwang, 1977). But it has recently been demonstrated that the maximum shortening velocity decreases under constant ionic strength in osmotically compressed skinned fibres of frog, suggesting that the decrease in the shortening velocity is a direct effect of the decrease in inter-filament spacing (Tsuchiya, 1984). The reduction of velocity during ACh exposure (Fig. 4B) is reminiscent of the catch phenomenon in the anterior byssus retractor of Mytilus (Tameyasu & Sugi, 197; Sugi & Tsuchiya, 1979). A similar phenomenon has also been reported in striated muscle (Julian & Sollins, 1972). The mechanism responsible for these phenomena is still incompletely understood, though ADP has been found to slow down the cycling rate of ATP hydrolysis of crossbridge movement (Giith, Kuhn, Tsuchiya & Ruegg, 1981). Further analysis of holothurian muscle or smooth muscle may be possible using glycerinated or chemically-skinned preparations, thus enabling independent control of inter-filament spacing and ionic environment as in skeletal muscle (Tsuchiya, 1984). The author would like to thank the staff of the Bermuda Biological Station for Research for providing facilities. This work was supported by a grant, the Fairfield

10 40 T. TSUCHIYA Maxwell Fellowship, through the Bermuda Biological Station for Research. He also wishes to express his sincere thanks to Professor R. B. Hill for kind guidance about the Bermuda Biological Station and to Professor H. Sugi for affording an opportunity of working in Bermuda. REFERENCES CLOSE, R. & HOH, J. F. Y. (197). Force-velocity properties of kitten muscles. J. Phytiol., Land. 192, DYDYNSKA, M. & WILKIE, D. R. (193). The osmotic properties of striated muscle fibres in hypertonic solutions. J. Physiol, bond. 19, EDMAN, K. A. P. (1979). The velocity of unloaded shortening and its relation to sarcomere length and isometric force in vertebrate muscle fibres..?. Physiol., Land. 291, EDMAN, K. A. P. & HWANG, J. C. (1977). The force-velocity relationship in vertebrate muscle fibres at varied tonicity of the extracellular medium. J. Physiol., Land. 29, GOTH, K., KUHN, H. J., TSUCHIYA, T. & ROEGG, J. C. (1981). Length dependent state of activation - length change dependent kinetics of crossbridges in skinned insect flight muscle. Btophys. struct. Mech. 7, HILL, A. V. (192). The viscous elastic properties of smooth muscle. Proc. R. Soc. B 0, 8-5. HILL, R. B. (1970). Effects of some postulated neurohumors on rhythmicity of the isolated cloaca of a holothurian. Physiol. Zool. 43, HEJUJHY, J. & MURPHY, R. A. (1974). Force-velocity and series elastic characteristics of smooth muscle from the hog carotid artery. Circulation Res. 34, ISHII, N. & TAKAHASHI, K. (1981). Mechanical properties and fine structure of the pedal retractor muscle of Mytilus eduhs. Comp. Biochem. Physiol. 70A, JULIAN, F. J. & SOLLJNS, M. R. (1972). Regulation of force and speed of shortening in muscle contraction. Symp. quant. Biol. 37, LEVIN, A. & WYMAN, J. (1927). The viscous elastic properties of muscle. Pmc. R. Soc. B 1, LOWY, J. & MILLMAN, B. M. (192). The contractile mechanism of the anterior byssus retractor muscle of Mytilus edulis. Phil. Trans. R. Soc. Ser. B 24, 5-1. MASHIMA, H., OKADA, T. & OKUMUBA, H. (1979). The dynamics of contraction in the guinea pig taenia coli. Jap. J. Physiol. 29, PENTREATH, V. W. & COBB, J. L. S. (1972). Neurobiology of echinodermata. Biol. Rev. 47, STEPHENS, N. L., KKOEGER, E. & MEHTA, J. A. (199). Force-velocity characteristics of respiratory airway smooth muscle. J. appl. Physiol. 2, SUGI, H., SUZUKI, S., TSUCHIYA, T., GOMI, S. & FUJIEDA, N. (1982). Physiological and ultrastructural studies on the longitudinal retractor muscle of a sea cucumber Stichopus japonicus. I. Factors influencing the mechanical response. J. exp. Biol. 97, 1-1. SUGI, H. & TSUCHIYA, T. (1979). The change in the load-sustaining ability and in the series elasticity inmytilus smooth muscle during isotonic shortening. J. Physiol., Land. 288, 35. SUGI, H., YAMAGUCHI, T. & TANAKA, H. (1977). The effect of hypertonic solutions on contracture tension and volume in Mytilus smooth muscle. Comp. Biochem. Physiol. 58A, SUZUKI, S. (1982). Physiological and cytochemical studies on activator calcium in contraction by smooth muscle of a sea cucumber, Isostichopus badionotus. Cell Tissue Res. 222, 24. TAKAHASHI, K. (19). Muscle physiology. lnphysiologyofechinoderm, (ed. R. A. Boolootian), pp New York: Interscience Publishers. TAMEYASU, T. (1978). The effect of hypertonic solutions on the rate of relaxation of contracture tension in Mytilus smooth muscle. J. exp. Biol. 74, TAMEYASU, T. & SUGI, H. (197). The series elastic component and the force-velocity relation in the anterior byssal retractor muscle of Mytilus edulis during active and catch contractions. J. exp. Biol. 4, TSUCHIYA, T. (1984). Changes in mechanical properties in osmotically compressed skinned muscle fibers of frog. In Contractile Mechanisms in Muscle, (edsg. H. Pollack & H. Sugi), pp New York: Plenum Publishing Corporation. UVELJUS, B. (197). Isometric and isotonic length-tension relations and variations in cell length in longitudinal smooth muscle from rabbit urinary bladder. Acta physiol. scand. 97, 1-12.