Molluscan Smooth Muscle Research Articles

Molecular Characterization and Expression Pattern of Paramyosin in Larvae and Adults of Yesso Scallop.

Simple SummaryParamyosin is an important myofibrillar protein in smooth muscle in molluscs that is not present in vertebrate muscles. This study characterized its sequence feature and expression patterns in Yesso scallop Patinopecten yessoensis and revealed the unique phosphorylation sites in scallops. The mRNA and protein expression of paramyosin was mainly found in foot and smooth adductor muscle. At late larval stages, strong paramyosin mRNA signals were detected in the symmetric positions of anterior and posterior adductor muscles. The present findings support that paramyosin may serve as the most important component of smooth muscle assembly during muscle development and catch regulation in scallops.Paramyosin is an important myofibrillar protein in molluscan smooth muscle. The full-length cDNA encoding paramyosin has been identified from Yesso scallop Patinopecten yessoensis. The length of paramyosin molecule has been found to be 3715 bp, which contains an open reading frame (ORF) of 2805 bp for 934 amino acid residues. Characterization of P. yessoensis paramyosin reveals the typical structural feature of coiled-coil protein, including six α-helix (α1-α6) and one coil (η) structures. Multiple phosphorylation sites have been predicted at the N-terminus of paramyosin, representing the unique phosphorylation sites in scallops. The highest levels of mRNA and protein expression of paramyosin have been found in foot and the smooth adductor muscle. According to whole-mount in situ hybridization (WISH), strong paramyosin mRNA signals were detected in the symmetric positions of anterior and posterior adductor muscles at late larval stages. These findings support that paramyosin may serve as the most important components for myogenesis and catch regulation in scallops. The present findings will not only help uncover the potential function of myofibrillar proteins in molluscs but also provide molecular evidence to infer evolutionary relationships among invertebrates.

Mechanism and Function of the Catch State in Molluscan Smooth Muscle: A Historical Perspective.

Molluscan smooth muscles exhibit the catch state, in which both tension and resistance to stretch are maintained with very low rates of energy consumption. The catch state is studied mainly on the anterior byssus retractor muscle (ABRM) of a bivalve molluscan animal, Mytilus, which can easily be split into small bundles consisting of parallel fibers. The ABRM contracts actively with an increase in the intracellular free Ca ion concentration, [Ca2+]i, as with all other types of muscle. Meanwhile, the catch state is established after the reduction of [Ca2+]i to the resting level. Despite extensive studies, the mechanism underlying the catch state is not yet fully understood. This article briefly deals with (1) anatomical and ultrastructural aspects of the ABRM, (2) mechanical studies on the transition from the active to the catch state in the isotonic condition, (3) electron microscopic and histochemical studies on the intracellular translocation of Ca ions during the transition from the active to the catch state, and (4) biochemical studies on the catch state, with special reference to a high molecular mass protein, twitchin, which is known to occur in molluscan catch muscles.

Phosphorylation Properties of the N-Terminal Region of Twitchin from Molluscan Catch Muscle

Molluscan smooth muscles, such as the bivalve adductor muscles and the mussel anterior byssus retractor muscles (ABRM), exhibit a unique contraction called “catch”. Catch contraction is regulated through twitchin phosphorylation and dephosphorylation. Twitchin from the ABRM of the Mediterranean mussel, Mytilus galloprovincialis, is phosphorylated by cAMP-dependent protein kinase (PKA), and PKA phosphorylation sites are located in both the N- and C-terminal regions of the twitchin molecule. The D2 site, which is adjacently located to the C-terminus, participates in forming a myosin, actin, and twitchin complex that is thought to contribute towards the maintenance of tension in the catch state. In contrast, although it has been reported to interact with thin-filaments, the molecular function of the region including the D1 site has remained largely unstudied. Three additional PKA consensus sequences were identified near the D1 site; however, it was not known if these sites could be directly phosphorylated by PKA. Here, we performed phosphorylation assays to identify phosphorylation sites near the D1 site using recombinant protein variants (TWD1-SSSS, TWD1-AAAS, TWD1-AASA, TWD1-ASAA, TWD1-SAAA, and TWD1-AAAA). All variants, except TWD1-AAAA (where all phosphorylatable serine residues were replaced by alanines), were phosphorylated by PKA. The four phosphorylation sites were named D1-1, D1-2, D1-3, and D1-4 (the originally identified D1) in order from the N-terminus. Phosphorylation assays using a 1/12.5 weight ratio of PKA to each TWD1 variant revealed that D1-4 was the most rapidly phosphorylated, closely followed by D1-1. However, D1-2 and D1-3 were phosphorylated at a lower level under equivalent conditions and were not phosphorylated when PKA was incubated with each TWD1 variant at a 1/100 weight ratio. Furthermore, we observed that TWD1-SSSS was phosphorylated in a stepwise fashion. These findings contribute towards the elucidation of the function of the twitchin D1 region in the regulatory system of catch contraction.

Phosphorylation properties of twitchin from Yesso scallop catch and striated muscles

Molluscan smooth muscle exhibits a unique muscle contraction, called catch, where the tension is maintained for long periods with little energy consumption. Catch contraction is regulated by twitchin, also called mini-titin, a member of the titin/connectin family. Twitchin is also found in molluscan non-catch striated muscle. We isolated twitchins from catch and striated muscles in the Yesso scallop Mizuhopecten yessoensis. Both twitchins are ~500 kDa in size. Western blotting using the anti-twitchin kinase domain antibody revealed that both twitchins have a kinase domain. Both twitchins were phosphorylated by the catalytic subunit of cAMP-dependent protein kinase in vitro. Stoichiometric analysis revealed that 0.6 and 1.7 mol Pi were incorporated into catch and striated muscle twitchins, respectively. Phosphopeptide mapping revealed that catch muscle twitchin has one phosphorylation site, whereas striated muscle twitchin has two. cDNA cloning gave partial sequences of two Yesso scallop twitchin isoforms. One had two putative phosphorylation sites and the other had one.

Molluscan smooth catch muscle contains calponin but not caldesmon

We isolated Ca(2+)-regulated thin filaments from the smooth muscle of the mussel Crenomytilus grayanus and studied the protein composition of different preparations from this muscle: whole muscle, heat-stable extract, fractions from heat-stable extract, thin filaments and intermediate stages of thin filaments purification. Among the protein components of the above-listed preparations, we did not find caldesmon (CaD), although two isoforms of a calponin-like (CaP-like) protein, which along with CaD is characteristic of vertebrate smooth muscle, were present in thin filaments. Thus, CaD is not Ca(2+)-regulator of thin filaments of this muscle. On the other hand, the mussel CaP-like protein is also not such Ca(2+)-regulator since we have shown that this protein can be selectively removed from isolated mussel thin filaments without loss of their Ca(2+)-sensitivity. We suggest that thin filaments in the smooth catch muscle possess other type of Ca(2+)-regulation, different from that in vertebrate smooth muscles.

Filamin isoforms in molluscan smooth muscle

The role of filamin in molluscan catch muscles is unknown. In this work three proteins isolated from the posterior adductor muscle of the sea mussel Mytilus galloprovincialis were identified by MALDI-TOF/TOF MS as homologous to mammalian filamin. They were named FLN-270, FLN-230 and FLN-105, according to their apparent molecular weight determined by SDS-PAGE: 270kDa, 230kDa and 105kDa, respectively. Both FLN-270 and FLN-230 contain the C-terminal dimerization domain and the N-terminal actin-binding domain typical of filamins. These findings, together with the data from peptide mass fingerprints, indicate that FLN-270 and FLN-230 are different isoforms of mussel filamin, with FLN-230 being the predominant isoform in the mussel catch muscle. De novo sequencing data revealed structural differences between both filamin isoforms at the rod 2 segment, the one responsible for the interaction of filamin with the most of its binding partners. FLN270 but not FLN230 was phosphorylated in vitro by cAMP-dependent protein kinase. As for the FLN-105, it would be an N-terminal proteolytic fragment generated from the FLN-270 isoform or a C-terminally truncated variant of filamin. On the other hand, a 45-kDa protein that copurifies with mussel catch muscle filamins was identified as the mussel calponin-like protein. The fact that this protein coelutes with the FLN-270 isoform from a gel filtration chromatography suggests a specific interaction between both proteins.

Expression of several domains of twitchin and myorod in the ontogeny of the mussel Mytilus trossulus

The expression of MLCK- and PEVK-domains of twitchin, as well as the unique N-terminal domain of myorod in early development of the mussel Mytilus trossulus has been studied. The MLCK-domain of twitchin and the unique N-terminal domain of myorod appear at the early stages of development, whereas the PEVK-domain of twitchin is present only in muscles of adult mussel. The sizes of genes of the N-terminal domain of myorod, obtained at the blastula stage and from the adult animal are similar, but the proteins have significant differences in the amino acid sequences. Consequently, myorod and twitchin appear at early stages of larval mussels before the formation of "adult" muscles capable of catch contraction, and at these stages both proteins are isoforms, which differ from the isoforms of adult animals. It is possible that the MLCK-domain in the "larval" isoform of twitchin is necessary for regulating the formation of the contractile apparatus of molluscan smooth muscles, while the PEVK-domain is important for the regulation of the catch state in muscles of adult animals.

Myosin Kinase of Molluscan Smooth Muscle. Regulation by Binding of Calcium to the Substrate and Inhibition of Myorod and Twitchin Phosphorylation by Myosin

Major contractile proteins were purified from relaxed actomyosin extracted from molluscan catch muscle myofibrils using ammonium sulfate fractionation and divalent cation precipitation. A fraction of this actomyosin was precipitated and purified as a supramolecular complex composed of twitchin (TW), myosin (MY), and myorod (MR). Another TW-MR complex was obtained via the removal of myosin. These supramolecular complexes and filaments assembled from purified myosin contained an endogenous protein kinase that phosphorylated myosin and myorod. Significantly, the activity of this novel myosin-associated (MA) kinase was inhibited at calcium concentrations of >0.1 microM. After partial purification of the kinase, we established that the inhibition resulted from binding of calcium to the substrate (myosin) and not from the binding to the enzyme (kinase). No inhibition was observed when myorod was used as a substrate, although the latter is identical to the rod portion of myosin lacking the head domains. Phosphorylation sites of myorod were identified, three at the C-terminal tip and three at the N-terminal domain. In the presence of calcium, addition of myosin to the TW-MR complex resulted in inhibition of this phosphorylation, while in the absence of myosin, this inhibition was negligible. Added myosin also inhibited phosphorylation of twitchin by PKA-like kinase, the latter also present in the complex. The opposite was true with the TW-MY-MR complex; that is, phosphorylation of myosin was inhibited by twitchin and/or myorod. Thus, in parallel to the well-established direct activation by calcium, molluscan catch muscle myosin also regulated its own phosphorylation. Therefore, in addition to the established phosphorylation of twitchin by PKA-like kinase, phosphorylation of myosin and myorod by myosin-associated kinase appears to play an important role in the development of the catch state.

Catch muscle of bivalve molluscs contains myosin- and twitchin-associated protein kinase phosphorylating myorod

We have shown previously that myorod, a molluscan thick filament protein of unknown function, is phosphorylated by vertebrate smooth myosin light chain kinase (MLCK) in N-terminal unique region. The aim of the present study was to clarify whether such phosphorylation may occur in molluscan muscles. We detected three kinases endogenous to molluscan catch muscle, namely, to the complex of surface thick filament proteins that consists of twitchin, myosin, and myorod. The first kinase was a protein kinase A because it was inhibited by a specific inhibitor; the second one was associated with twitchin and phosphorylated myorod at its N-terminal unique region independently of Ca 2+; and the third kinase was bound to myosin and phosphorylated myorod as well as myosin in the C-terminal part of both proteins. The myosin-associated kinase was inhibited by micromolar concentration of calcium ions. This enzyme could be separated from myosin by chromatography, whereas the kinase associated with twitchin could not be separated from twitchin. Since twitchin has a MLCK-like domain, it is possible that this domain was responsible for myorod phosphorylation. Phosphorylation of myorod within the twitchin–myosin–myorod complex increased the actin-activated Mg 2+-ATPase activity of myosin. Taken together, these results indicate that phosphorylation of myorod by kinases associated with key proteins of catch contraction may contribute to the functional activity of myorod in molluscan smooth muscle.

Modulation of Actin-Myosin Interaction by N-terminal Unique Domain of Myorod of Molluscan Catch Muscle

Myorod, a thick filament protein of molluscan smooth muscle, is an alternative product of the myosin heavy chain gene. It contains the rod domain identical to that of the rod portion of the myosin molecule and a unique N-terminal domain (NMR). We previously reported that myorod is phosphorylated within NMR at Thr141 by vertebrate smooth muscle myosin light chain kinase (Sobieszek et al., Arch. Biochem. Biophys., 454: 197-205, 2006). To investigate whether phosphorylation of NMR may affect the actin-myosin interaction, two peptides were synthesized with sequence corresponding to this domain. One of two peptides included a phosphorylated Thr141 (NMR-P) and the other not (NMR-unP). We found that the latter peptide interacted with rabbit and molluscan F-actin causing an aggregation and sedimentation of F-actin at low-speed centrifugation while NMR-P had no effect on the distribution of F-actin in the supernatant and pellet fractions. Co-sedimentation of NMR-unP with isolated molluscan thin filaments revealed that in this case the interaction was Ca2+-dependent. NMR-unP slightly inhibited the Mg2+-ATPase activity of actomyosin reconstructed from molluscan myosin and rabbit F-actin. In contrast, NMR-P as well as intact phosphorylated myorod increased actomyosin Mg2+-ATPase activity of about 1.5-3 fold depending on the experimental conditions. This finding was supported by a 3-fold higher binding affinity of NMR-P for myosin filaments with comparison of that of NMR-unP. Taken together these results implicate that myorod, a thick filament protein of molluscan catch muscle, can modulate actin-myosin interaction in a phosphorylation-dependent manner.

Myosin Loop 2 Is Involved in the Formation of a Trimeric Complex of Twitchin, Actin, and Myosin

Molluscan smooth muscles exhibit a low energy cost contraction called catch. Catch is regulated by twitchin phosphorylation and dephosphorylation. Recently, we found that the D2 fragment of twitchin containing the D2 site (Ser-4316) and flanking immunoglobulin motifs (TWD2-S) formed a heterotrimeric complex with myosin and with actin in the region that interacts with myosin loop 2 (Funabara, D., Hamamoto, C., Yamamoto, K., Inoue, A., Ueda, M., Osawa, R., Kanoh, S., Hartshorne, D. J., Suzuki, S., and Watabe, S. (2007) J. Exp. Biol. 210, 4399-4410). Here, we show that TWD2-S interacts directly with myosin loop 2 in a phosphorylation-sensitive manner. A synthesized peptide, CAQNKEAETTGTHKKRKSSA, based on the myosin loop 2 sequence (loop 2 peptide), competitively inhibited the formation of the trimeric complex. Isothermal titration calorimetry showed that TWD2-S binds to the loop 2 peptide with a K(a) of (2.44 +/- 0.09) x 10(5) m(-1) with two binding sites. The twitchin-binding peptide of actin, AGFAGDDAP, which also inhibited formation of the trimeric complex, bound to TWD2-S with a K(a) of (5.83 +/- 0.05) x 10(4) m(-1) with two binding sites. The affinity of TWD2-S to actin and myosin was slightly decreased with an increase of pH, but this effect could not account for the marked pH dependence of catch in permeabilized fibers. The complex formation also showed a moderate Ca(2+) sensitivity in that in the presence of Ca(2+) complex formation was reduced.

Twitchin of mollusc smooth muscles can induce “catch”-like properties in human skeletal muscle: support for the assumption that the “catch” state involves twitchin linkages between myofilaments

Molluscan catch muscles can maintain tension with low or even no energy utilization, and therefore, they represent ideal models for studying energy-saving holding states. For many decades it was assumed that catch is due to a simple slowing of the force-generating myosin head cross-bridge cycles. However, recently evidences increased suggesting that catch is rather caused by passive structures linking the myofilaments in a phosphorylation-dependent manner. One possible linkage structure is the titin-like thick filament protein twitchin, which could form bridges to the thin filaments. Twitchin is known to regulate the catch state depending on its phosphorylation state. Here, we found that twitchin induces a catch-like stiffness in skinned human skeletal muscle fibres, when these fibres are exposed to this protein. Subsequent phosphorylation of twitchin reduces the stiffness. These findings support the assumption that catch of molluscan smooth muscle involves twitchin linkages between thick and thin filaments.

Molecular basis of the catch state in molluscan smooth muscles: a catchy challenge

The catch state (or 'catch') of molluscan smooth muscles is a passive holding state that occurs after cessation of stimulation. During catch, force and, in particular, resistance to stretch are maintained for long time periods with low (or no) energy consumption at basal intracellular free [Ca2+]. The catch state is initiated by Ca2+-stimulated dephosphorylation of the titin-like protein twitchin and is inhibited by cAMP-dependent phosphorylation of twitchin. In addition, catch is pH sensitive, but the reason for this is unknown. According to a traditional model, catch is due to slower cross-bridge cycles where myosin heads remain longer attached to the actin filaments after force generation, possibly caused by a hindered release of ADP from the myosin heads. However, this model was disproved by recent findings which showed that (i) inhibitors of myosin function, such as vanadate, do not affect catch force; (ii) factors which terminate the catch state do not accelerate myosin head detachment kinetics and (iii) a catch-like high resistance to stretch is still inducible when force development is prevented. Thus, catch probably involves passive linkage structures interconnecting the myofilaments (catch linkages). For example twitchin could (i) tie myosin heads to the thin filaments, (ii) mechanically lock them in a stretch resistant state or (iii) interconnect thick and thin filaments directly. However, it is questionable if these mechanisms are sufficient since twitchin seems to be about 15-times less abundant than myosin. Therefore, in addition, interconnections between thick filaments could exist, which could involve e.g. paramyosin or twitchin. Catch could even involve changes in the compliance of thick filaments. The function of myorod, found specifically in catch muscles in equal abundance with myosin, is not known. The suggestion is made here that catch linkages are present already during active contraction either as ratchet-like elements resisting stretch and not opposing shortening or in some kind of 'standby' mode ready to transform suddenly into the working mode by stretches or after Ca2+ removal following cessation of stimulation.

Small-angle X-ray diffraction studies of a molluscan smooth muscle in the catch state

Small-angle X-ray diffraction patterns from the anterior byssus retractor muscle of Mytilus edulis in the resting, active, and catch states were examined closely to elucidate the structural features of catch. The specimens were isometrically contracted by stimulation with acetylcholine. The specimens that produced strong tensions in both the active and catch states showed noticeable structural change in the thick filaments. Although the tension was weaker in the catch state than in the active state, the axial spacings of the 14.5 nm meridional reflection and its higher order reflections from the thick filaments were more elongated in the catch state than in the active state. This means that the thick filaments were stretched more strongly in the catch state than in the active state.

“Twitchin–actin linkage hypothesis” for the catch mechanism in molluscan muscles: Evidence that twitchin interacts with myosin, myorod, and paramyosin core and affects properties of actomyosin

“Twitchin–actin linkage hypothesis” for the catch mechanism in molluscan smooth muscles postulates in vivo existence of twitchin links between thin and thick filaments that arise in a phosphorylation-dependent manner [N.S. Shelud’ko, G.G. Matusovskaya, T.V. Permyakova, O.S. Matusovsky, Arch. Biochem. Biophys. 432 (2004) 269–277]. In this paper, we proposed a scheme for a possible catch mechanism involving twitchin links and regulated thin filaments. The experimental evidence in support of the scheme is provided. It was found that twitchin can interact not only with mussel myosin and rabbit F-actin but also with the paramyosin core of thick filaments, myorod, mussel thin filaments, “natural” F-actin from mussel, and skeletal myosin from rabbit. No difference was revealed in binding of twitchin with mussel and rabbit myosin. The capability of twitchin to interact with all thick filament proteins suggests that putative twitchin links can be attached to any site of thick filaments. Addition of twitchin to a mixture of actin and paramyosin filaments, or to a mixture of Ca 2+-regulated actin and myosin filaments under relaxing conditions caused in both cases similar changes in the optical properties of suspensions, indicating an interaction and aggregation of the filaments. The interaction of actin and myosin filaments in the presence of twitchin under relaxing conditions was not accompanied by an appreciable increase in the MgATPase activity. We suggest that in both cases aggregation of filaments was caused by formation of twitchin links between the filaments. We also demonstrate that native thin filaments from the catch muscle of the mussel Crenomytilus grayanus are Ca 2+-regulated. Twitchin inhibits the ability of thin filaments to activate myosin MgATPase in the presence of Ca 2+. We suggest that twitchin inhibition of the actin–myosin interaction is due to twitchin-induced switching of the thin filaments to the inactive state.

Myosin Cross-Bridge Kinetics and the Mechanism of Catch

Catch force in molluscan smooth muscle requires little, if any, energy input and is controlled by the phosphorylation state of the thick filament-associated mini-titin, twitchin. The kinetic parameters of myosin cross-bridge turnover in permeabilized catch muscle, and how they are potentially modified by the catch mechanism, were determined by single turnover measurements on myosin-bound ADP. Under isometric conditions, there are fast and slow components of cross-bridge turnover that probably result from kinetic separation of calcium-bound and calcium-free cross-bridge pools. The structure responsible for catch force maintenance at intermediate [Ca+2] does not alter the processes responsible for the fast and slow components under isometric conditions. Also, there is no measurable turnover of myosin-bound ADP during relaxation of catch force by phosphorylation of twitchin at pCa>8. The only effects of the catch link on myosin-bound ADP turnover are 1), a small, very slow extra turnover when catch force is maintained at very low [Ca+2] (pCa>8); and 2), attenuation of the shortening-induced increase in turnover at subsaturating [Ca+2]. These limited interactions between the catch link and myosin cross-bridge turnover are consistent with the idea that catch force is maintained by a thick and thin filament linkage other than the myosin cross-bridge.

Phosphorylation of myorod (catchin) by kinases tightly associated to molluscan and vertebrate smooth muscle myosins

Myorod, also known as catchin, a newly discovered component of molluscan smooth muscle thick filaments, is an alternative product of the myosin heavy chain gene. It contains a C-terminal rod part that is identical to that part of myosin and a unique N-terminal domain that is very small relative to the myosin head domain. The role of myorod in contraction or relaxation of this muscle type is unknown. In the present study we demonstrated that myorod was phosphorylated not only by a kinase endogenous to molluscan myosin and twitchin but also to vertebrate smooth muscle myosin light chain kinase (MLCK). The rates and maximal levels of phosphorylation were up to threefold higher than those observed by protein kinase A with clear optima at the physiological salt concentrations. Using a mild digestion with chymotrypsin we isolated an 11 kDa phosphopeptide and showed that the phosphorylation site was located at the N-terminal domain of myorod at Thr 141 position. The sequence around this site exhibited a high degree of similarity to that expected for the substrate recognition site of MLCK. The phosphorylation rates strongly depended on the ionic conditions indicating that this site could be readily sterically blocked during myorod polymerization. Another component of the thick filaments involved in regulation of the catch state, twitchin, was phosphorylated by MLCK and exhibited endogenous myorod kinase and MLCK activities. A possible role of these phosphorylation reactions in the regulation of molluscan smooth muscles is discussed.

Twitchin as a regulator of catch contraction in molluscan smooth muscle

Molluscan catch muscle can maintain tension for a long time with little energy consumption. This unique phenomenon is regulated by phosphorylation and dephosphorylation of twitchin, a member of the titin/connectin family. The catch state is induced by a decrease of intracellular Ca2+ after the active contraction and is terminated by the phosphorylation of twitchin by the cAMP-dependent protein kinase (PKA). Twitchin, from the well-known catch muscle, the anterior byssus retractor muscle (ABRM) of the mollusc Mytilus, incorporates three phosphates into two major sites D1 and D2, and some minor sites. Dephosphorylation is required for re-entering the catch state. Myosin, actin and twitchin are essential players in the mechanism responsible for catch during which force is maintained while myosin cross-bridge cycling is very slow. Dephosphorylation of twitchin allows it to bind to F-actin, whereas phosphorylation decreases the affinity of the two proteins. Twitchin has been also been shown to be a thick filament-binding protein. These findings raise the possibility that twitchin regulates the myosin cross-bridge cycle and force output by interacting with both actin and myosin resulting in a structure that connects thick and thin filaments in a phosphorylation-dependent manner.

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.