Prolonged relaxation after stimulation of the clasping muscle of male frog, Rana japonica, during the breeding season.

We investigated the mechanical properties of the flexor carpi radialis muscle (FCR), a forelimb muscle used mainly for amplexus in the breeding season (February to March), of the male Japanese brown frog, Rana japonica. In the present experiment, the changes in force and stiffness of the FCR before, during, and after contraction were measured at 4 degrees C. The total time from the end of stimulation to the end of relaxation was about 30 min. The time course of this prolonged relaxation was fitted by two exponential decay processes. Stiffness decreased during prolonged relaxation, but stayed higher than force, when normalized to peak values. These mechanical properties of the FCR were different from those of the glutaeus magnus muscle (GM) in the hindlimb, used for jumping. When a quick release was applied to the FCR during relaxation, the force recovered gradually after a sudden decrease. The time course of this force recovery was fitted by a single exponential term, and the rate constant decreased as the prolonged relaxation proceeded. The possible involvement of active process(es) in the prolonged relaxation is discussed.

Sliding movements of molluscan and algal myosin attached to a magnetizable bead under a load controlled by electromagnet.

We developed an electromagnetic apparatus to perform a quick change in load in the motility system, using magnetizable beads on which myosin thick filaments from molluscan smooth muscle or green algae, Chara, myosin were attached. The quick change in load to beads (diameter 4.5 microm) was applied in the range of 0-85 pN. The movement of beads was recorded by a video-system and analyzed with special software. When the quick increase in load was applied during the movement of beads under no load, the beads showed a transient movement to the reverse direction before the steady slower movement to the normal direction. When the application of load was stopped, the beads showed a transient fast phase of movement. The change in load-sustaining ability was measured by a double load step. The backward velocity at the second constant test load was smaller when the first preceding step was increased, suggesting that the ability to sustain load was higher with a higher preceding step. These phenomena were observed both in molluscan thick filaments and in Chara myosin, and the time course of the movement of a bead was quite similar to those observed previously in frog single muscle fibers. This suggested that the velocity transients are the intrinsic properties induced by the interaction between actin and myosin, irrespective of the hexagonal lattice structure of filaments, the regular sarcomere structure, and myosin type, namely, that the molecule of myosin itself has the ability to adjust to mechanical circumstances.

Sources of activator calcium ions in the contraction of smooth muscles in Aplysia kurodai.

The physiological and pharmacological properties of contraction and the ultrastructure of buccal mass retractor muscle (I4) and gill-pinnule closure muscle (GPCM) in Aplysia kurodai were studied to learn more about the sources of activator Ca2+ in molluscan smooth muscle. Acetylcholine (ACh) and high K+-induced contractions were reduced by lowering the external Ca2+ concentration, and eliminated by the removal of extracellular Ca2+. Nifedipine appreciably reduced ACh- and high K+-induced contractions, while amiloride decreased only ACh-induced contractions and had no significant effect on high K+-induced contractions. When nifedipine and amiloride were applied together, either type of contraction was still appreciable. Serotonin (5-HT) could potentiate subsequent ACh- and high K+-induced contractions in I4; potentiated tension was significantly reduced by nifedipine and amiloride, whereas 5-HT inhibited ACh-and high K+-induced contractions in GPCM. The potentiating effects of 5-HT may be mediated by the activation of the Ca2+-channel to increase the influx from extracellular Ca2+. Caffeine caused contractions in Ca2+-free solution in both muscles. Electron microscopy revealed sarcolemmal vesicles underneath the plasma membrane in both muscle fibers. Electron microscopical cytochemistry demonstrated that pyroantimonate precipitates were localized in the sarcolemmal vesicles and in the inner surface of plasma membranes in the resting fibers. Present results indicate that the contractions of I4 and GPCM fibers are caused not only by Ca2+-influx but also by Ca2+ release from the intracellular storage sites, such as the sarcolemmal vesicles and the inner surface of plasma membranes.

Enhancement of twitch force by stretch in a nerve-skeletal muscle preparation of the frog Rana porosa brevipoda and the effects of temperature on it.

We investigated the mechanism of the enhancement of twitch force by stretch and the effects of temperature on it in nerve-skeletal muscle preparations of whole iliofibularis muscles isolated from the frog Rana brevipoda. When a preparation was stimulated indirectly and stretched, the twitch force after the stretch was enhanced remarkably in comparison to that observed before a stretch at low temperature. The enhanced force obtained by a stretch of 20% resting muscle length (l0) at low temperature was as high as the force obtained by direct stimulation. The phenomenon was not dependent on the velocity but on the amplitude of stretch. The enhanced force obeyed the length-force relationship when a stretch was long enough. The above results were observed when the frogs were kept at room temperature (20-22 degrees C). Measurements were also taken at low temperature (4 degrees C); when frogs were kept at low temperature for more than 2 months, twitch force obtained without stretch was considerably higher at l0. The amplitude of the action potential recorded extracellularly from the muscle surface increased remarkably after a stretch, but was same before and after a stretch when recorded from the nerve innervating muscle. The effects of temperature on twitch and tetanic force by direct or indirect stimulation without stretch were also studied as basic data of the stretch experiment. The results from this study suggest that stretch-induced force enhancement in a nerve-muscle preparation is caused by an increase in the transmission rate between nerve and muscle, and the amplitude of the enhanced force is determined by the length-force relationship of the muscle. The phenomenon is also strongly affected by the temperature at which the frogs are kept.

The influences of L(+)-lactate and pH on contractile performance in rabbit glycerinated skeletal muscle.

To know whether L(+)-lactate directly induces the decrease in muscle contractile performance, several parameters of cross-bridge function were measured at various concentrations of lactate and pH in glycerinated rabbit psoas and soleus muscles at three different temperatures (5, 20, 28 degrees C). At all pHs studied (pH 7.0, 6.5, 6.0, and 5.5), isometric tension, unloaded velocity of shortening, and stiffness of a fiber during active and resting state in the presence of 50 mM lactate were not virtually different from those in the absence of lactate, but pH had remarkable effects on these parameters. The active stiffness decreased only slightly, and the small resting stiffness appeared at low pH; they were not affected by the presence of lactate. The present results indicate that the lactate anions may not have marked influence on the interaction between actin and myosin, and the concomitant change in pH with the production of lactate may remarkably affect it, as far as they were examined under the existing conditions of the experimental solutions.

Development of an apparatus to control load by electromagnet for a motility system in vitro.

We developed an electromagnet to perform quick changes in load in the motility system consisting of myosin molecules attached to a magnetizable bead and actin filaments The electromagnet was combined with an inverted microscope and load could be quickly changed under optical observation. The magnetic field was generated by high electric current (6V, 0-125A) and the maximum field was 8,000 Oe. The maximum force exerted on a bead was 80pN at 2.5mm distance from a magnet. The change in force was 0.48% at the distance of 5.0mm from the magnet when a bead moved longitudinally for 30microm. The time to change load was about 20ms. The movements of a bead in water were recorded by video when step changes in magnetic field were applied and it was shown that a bead exactly followed the change in force. This apparatus is very much useful to analyze the transient changes in the movement of a bead, if the movement is relatively slow as in the interaction between actin and myosin from molluscan smooth muscle.

Altered mechanical properties in smooth muscle of mice with a mutated calponin locus.

The mechanical properties of smooth muscles in aorta and vas deferens were studied in mice with a mutated basic calponin locus to learn the physiological function of calponin. The intact smooth muscles were stimulated with high KCl and the force development was compared between calponin deficient (knockout, KO) mice and wild type (WT) ones. The isometric force induced by various concentrations of high KCl was lower in KO than in WT both in aorta and in vas deferens. The length-force relations were compared between KO and WT. The active isometric force in KO was significantly lower at most muscle lengths examined than in WT without the change in resting force both in aorta and in vas deferens. In vas deferens, the rate of force development after quick release in length at the peak force was significantly faster in KO than in WT. The above results show that the force development is lower and the rate of cross-bridge cycle is faster in KO mice than in WT ones, suggesting that calponin plays basic roles in the control of the contraction of smooth muscle.