Byssus Retractor Muscle Of Mytilus Research Articles

Stepwise Length Changes in Single Invertebrate Thick Filaments

Previous experiments on thick filaments of the anterior byssus retractor muscle of Mytilus and the telson-levator muscle of Limulus polyphemus have shown large, reversible length changes up to 23% and 66% of initial length, respectively, within the physiological tension range. Using nanofabricated cantilevers and newly developed high-resolution detection methods, we investigated the dynamics of isolated Mytilus anterior byssus retractor muscle thick filaments. Single thick filaments were suspended between the tips of two microbeams oriented perpendicular to the filament axis: a deflectable cantilever and a stationary beam. Axial stress was applied by translating the base of the deflectable nanolever away from the stationary beam, which bent the nanolever. Tips of flexible nanolevers and stationary beam were imaged onto a photodiode array to track their positions. Filament shortening and lengthening traces, obtained immediately after the motor had imposed stress on the filament, showed steps and pauses. Step sizes were 2.7nm and integer multiples thereof. Steps of this same size paradigm have been seen both during contraction of single sarcomeres and during active interaction between single isolated actin and myosin filaments, raising the question whether all of these phenomena might be related.

Further identification of bioactive peptides in the anterior byssus retractor muscle of Mytilus: Two contractile and three inhibitory peptides

1. Two contractile and three inhibitory peptides were newly isolated from the anterior byssus retractor muscles (ABRMs) of the bivalve mollusc Mytilus edulis by using the muscle as the bioassay system. 2. The structures of the two contractile peptides were GPFGTHIKamide (GPFG-8) and GPFGLNKHGamide (GPFG-9). The contractile response of the ABRM to the first-time application of GPFG-8 or GPFG-9 was of considerable size. The threshold concentrations of the peptides were around 10(-9) M. However, the contractile response to the second-time application was far smaller than that to the first-time application in both cases. Namely, the muscle showed tachyphylaxis to the peptides. 3. Two of the three inhibitory peptides were members of the Mytilus-inhibitory-peptide (MIP) family. Their structures were RAPLFIamide (MIP6) and RSPMFVamide (MIP7). The peptides, as well as the other MIPs previously identified, showed a potent inhibitory effect on phasic contraction of the ABRM in response to repetitive electrical stimulation. The remaining one was an MIP-related peptide (MIP-RP) having the sequence of MRYFVamide. The MIP-RP was less potent than the two MIPs in inhibiting the contraction of the ABRM.

Changes in sarcoplasmic metabolite concentrations and pH associated with the catch contraction and relaxation of the anterior byssus retractor muscle ofMytilus edulis measured by phosphorus-31 nuclear magnetic resonance

The sarcoplasmic concentrations of phosphorus metabolites and pH (pHin) were measured in the anterior byssus retractor muscle (ABRM) of Mytilus edulis by 31P nuclear magnetic resonance spectroscopy. During an active contraction induced by 10(-3) acetylcholine, the concentration of arginine phosphate ([Arg-P]in) decreased from the resting value of 7.47 +/- 0.26 (mean +/- SE, n = 8) to 6.67 +/- 0.29 (n = 6) mumol g-1, and that of inorganic phosphate (Pi) consistently increased from 0.84 +/- 0.06 (n = 7) to 1.61 +/- 0.12 (n = 5) mumol g-1. In the 'catch' state following the active contraction, these concentrations were close to their resting levels, indicating that the catch is an inactive state. 5-hydroxytryptamine caused a rapid relaxation of the catch, which was associated with a slight decrease in [Arg-P]in and an increase in pHin by ca 0.2 units. The sarcoplasmic concentration of ATP (mean, 1.6 mumol g-1) did not change throughout the contraction-relaxation cycle.

The 'catch' mechanism in molluscan muscle: an electron microscopy study of freeze-substituted anterior byssus retractor muscle of Mytilus edulis.

A method for quick-freezing muscles while observing their mechanical properties until the moment of freezing is described. This method was used to freeze the anterior byssus retractor muscle (ABRM) of Mytilus edulis. Intact muscle in the presence of sucrose as a cryoprotectant was freeze-substituted in acetone, fixed and embedded for electron microscopy. ABRM was frozen in a number of mechanical states including 'catch', the state of high passive tension particularly associated with some molluscan muscles. Transverse sections were examined to determine the distribution of filaments in the muscle cells. In the relaxed muscle thick and thin filaments are fairly randomly distributed. Groups of thin filaments and of thick filaments are often seen, and there is no obvious association between the two types of filaments. In contrast, in rigor muscles, both glycerol-extracted and intact, most of the thin filaments were found to lie in rings or rosettes around the thick filaments. In some places bridges between thick and thin filaments could be distinguished. In actively contracting muscle (phasic contraction) the appearance is intermediate between that of the relaxed and rigor muscles. Many thick filaments are surrounded by rosettes of thin filaments but many of the thin filaments are grouped and have no connections with thick filaments. The 'catch' state, left after a period of tonic contraction, is similar in its distribution of thick and thin filaments to the active state, many of the thin filaments lying between the thick. Frequently thick and thin filaments seem to be closer together than in other states of the muscle where a pronounced exclusion zone is present around the thick filaments. There is no evidence for association between the thick filaments. The different distribution of thin filaments in the different states is consistent with the previously described X-ray diffraction data if it is assumed that most of the contribution to the equatorial reflection at 12 nm comes from the groups of thin filaments. Our data support a model for catch in which there is a change in the association between thick and thin filaments, rather than one in which thick filaments are clumped.

Mg-ATPase activity and motility of native thick filaments isolated from the anterior byssus retractor muscle ofMytilus edulis

A method for isolating native thick filaments from the anterior byssus retractor muscle (ABRM) of Mytilus edulis is described. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed that the isolated thick filament preparation contained mainly paramyosin and myosin but almost no actin. Electron microscopy of negatively stained preparations showed that the isolated thick filaments were tapered at both ends and of various sizes, in the range 5-31 microns in length and 51-94nm in width in the central region. Central bare zones were observed in the smaller filaments, but were not clearly seen in the larger filaments. Mg-ATPase activity of the isolated thick filaments was activated by skeletal muscle F-actin in a Ca2+-dependent manner. The maximal activity was about 20 nmol min-1 mg-1 thick filaments (20 degrees C, pH7.0). Motility of the thick filaments attached to latex beads (diameter, 2 microns) was also studied using the native actin cables of the freshwater alga, Chara. In the presence of Mg-ATP and Ca2+, the beads moved along the actin cables at a maximal velocity of about 1 micron s-1. In the absence of Ca2+, almost no movement was observed. These results show that the isolated thick filaments are structurally intact and retain the essential mechanochemical characteristics of the ABRM myosin.

Pharmacology of relaxing response of mytilus smooth muscle to the catch-relaxing peptide

1. Pharmacological properties of relaxing action of the catch-relaxing peptide (CARP) on the anterior byssus retractor muscle of Mytilus were examined and compared with those of the actions of other relaxing stimuli. 2. Butaclamol (10 −5 M), a dopamine blocker, showed little effect on relaxing responses to CARP, serotonin and repetitive electrical pulses of stimulation. 3. Mersalyl (5 × 10 −4 M), a serotonin blocker, depressed but did not abolish relaxing response to CARP. Pre-treatment of the muscle with KCl-EGTA solution also depressed but did not abolish the response to CARP. 4. Propranolol (10 −3 M) depressed or abolished relaxing responses to serotonin, dopamine and repetitive electrical stimulation, whereas it enhanced the response to CARP. 5. Phenoxybenzamine (10 −4 M) enhanced relaxing responses to CARP, serotonin and repetitive electrical stimulation, but it showed little effect on the response to dopamine. 6. Simple exposure of the muscle to La 3+ (5 mM) showed little effect on relaxing response to CARP. 7. When the muscle was continuously immersed (for 30 min or more) in CARP (5 × 10 −8 M), relaxing response to repetitive electrical stimulation was depressed or abolished. 8. It is suggested that CARP relaxes catch tension of the ABRM by acting on postsynaptic sites in the muscle.

Reversible inhibition of acetylcholine-induced contraction by benextramine in mytilus smooth muscle

1. 1. The effects of alpha-adrenoceptor antagonists, benextramine, dibenamine, prazocin and yohimbine, on the contractions induced by ACh (acetylcholine) and DMPP (1,1-dimethyl-4-phenyl piperazinium iodide) in the anterior byssus retractor muscle of Mytilus (ABRM) were investigated. 2. 2. Benextramine (10 −5 and 3 × 10 −5M) and dibenamine (10 −5 and 3 × 10 −5M) inhibited the contraction induced by ACh (3 × 10 −7−10 −4M) and DMPP (3 × 10 −6−3 × 10 −4M). 3. 3. The inhibitory effects of both drugs were slowly reversible. 4. 4. Prazocin and yohimbine, at 3 × 10 −5M, had no effect on ACh- and DMPP-induced contractions. 5. 5. These results suggest that benextramine and dibenamine interact directly with the ACh receptor in the ABRM with non-covalent binding.

The anaerobic energy metabolism in the anterior byssus retractor muscle ofMytilus edulis during contraction and catch

The contribution of anaerobic metabolism in normoxic and hypoxic anterior byssus retractor muscles (ABRM) to ATP production during tonic and repeated phasic contractions under isotonic conditions was estimated by analysing changes in the levels of ATP, phospho-l-arginine,l-arginine, octopine, alanopine, strombine,l- andd-lactate, succinate,l-aspartate andl- andd-alanine.

Effects of temperature and metabolic inhibitors on contraction of anterior byssus retractor muscle of Mytilus

1. 1. Anterior byssus retractor muscle of Mytilus (ABRM) was stimulated to contract by ACh (acetylcholine) and effects of temperature (5–30°C), FDNB (1-fluoro 2,4 dinitro-benzene) and IAA (iodoacetic acid) on tension response were examined. 2. 2. Isometric tension was highest at the temperature range of 10–20°C and decreased at higher and lower temperature than that range. 3. 3. The rate of tension decay after washing of ACh was accelerated by the increase of temperature. 4. 4. Tension redevelopment after release of 1 % during contraction was much smaller at 5°C than at 20°C. 5. 5. Tension development by ACh and the rate of tension decay after washing of ACh were remarkably decreased by the treatment of FDNB or IAA. 6. 6. The above results were discussed from the viewpoint that energy metabolism might be related to catch.

Time course of tension decay after stretch and unloaded shortening velocity in molluscan catch muscle at various physiological states

1. 1. The anterior byssus retractor muscle of Mytilus was stimulated to contract with ACh, and the time courses of tension decay after an applied stretch (6% of l 0), as well as the unloaded shortening velocity, were examined at various states, i.e. (1) at the peak of initial tension development, (2) during the prolonged application of ACh, (3) after washout of ACh, (4) in the sucrose-hypertonic solution and (5) in the NaCl-hypertonic solution. 2. 2. The tension decay after stretch was resolved into three exponential components. 3. 3. The relationship between the amplitudes of three exponential components and the unloaded shortening velocity was examined in the above five states. It was shown that the intermediate component and the slow one were related to active and tonic contractions, respectively.

The site of action of dopamine and its related compounds in smooth muscle of Mytilus

1. The effect of butaclamol on the relaxation of catch contraction by dopamine, m-tyramine, p-tyramine and phenylethylamine was investigated in the anterior byssus retractor muscle of Mytilus. 2. The dose-response curves of dopamine, m-tyramine and p-tyramine were shifted in parallel to the right by butaclamol (3 × 10 −6 M) and their p A 2 values were 6.13 ± 0.04 against dopamine, 6.07 ± 0.04 against m-tyramine and 5.57 ± 0.08 against p-tyramine, but the curve of phenylethylamine was not shifted. 3. The dose-response curve of m-tyramine was shifted in parallel to the right by haloperidol (3 × 10 −6 and 3 × 10 −5 M) and the p A 2, value was 5.44 ± 0.06. 4. These findings suggest that dopamine, m-tyramine and p-tyramine act on the common dopamine recognition site, but phenyethylamine does not.

Stretch induced tension rise in a molluscan smooth muscle skinned by freeze drying

The anterior byssus retractor muscle (ABRM) ofMytilus edulis was skinned by freeze drying. Tension transients in response to quick length steps were recorded during isometric contraction induced in ATP salt solution containing 2×10−6 M Ca2+. These transients consisted of four phases similar to those described by Huxley (1974) in skeletal muscle. Under certain conditions (stretch amplitude not larger than 0.6% LO), and in particular in the presence of cyclic AMP, we observed a delayed tension rise following a quick stretch (stretch activation) which appears to be similar to the stretch activation of insect flight muscle (Jewell and Ruegg 1966).

Molluscan catch muscle: Regulation and mechanics in living and skinned anterior byssus retractor muscle ofMytilus edulis

Immediate stiffness and static elastic modulus were estimated in skinned and living molluscan smooth muscle during active contraction and catch state. The stiffness to tension ratio was found to be greater in catch than in active contraction. In skinned preparations, a catch like state could be reproduced in a Ca2+ free solution (pCa>8) which was mechanically not different from catch in living muscle. Addition of cAMP or the catalytic subunit of the cAMP dependent protein kinase type II caused a rapid relaxation of this catch like state. The results are discussed in terms of a dual intracellular regulatory mechanism of catch and active contraction.

Effects of 5-hydroxytryptamine, dopamine, and acetylcholine on accumulation of cyclic AMP and cyclic GMP in the anterior byssus retractor muscle of Mytilus edulis L. (Mollusca).

We investigated in vitro accumulation of adenosine 3',5'-monophosphate (induced by 5-hydroxytryptamine and dopamine) and of guanosine 3',5'-monophosphate (induced by acetylcholine) in the anterior byssus retractor muscle of Mytilus. The response to 5-hydroxytryptamine exceeded that induced by equimolar concentrations of dopamine. 1-methyl lysergic acid, a 5-hydroxytryptamine-blocking agent, diminished the 5-hydroxytryptamine-induced increase of cyclic AMP level. This parallels the effect of this amine on the contracted muscle. Acetylcholine, which causes a tonic contraction of the muscle, increased intracellular levels of cyclic GMP in a dose-dependent (max. 45-fold at 10(-4) M ACh) manner. The time course of the rise in cyclic GMP level was rapid and transient (peak concentration of cyclic GMP at 2 min). Mytolon was the most effective of all cholinergic blockers tested. It was concluded that cyclic nucleotides may play a role in the modulatory process of the transmitters. A direct relation to the relaxation-contraction process could not be established.

Degeneration of monoamine nerves in anterior byssus retractor muscle of Mytilus induced by 5,6-dihydroxytryptamine.

Preliminary ultrastructural studies on the effects of 5,6-Dihydroxytryptamine (5,6-DHT) on the anterior byssus retractor muscle (ABRM) of Mytilus show degeneration of 2 types of monoaminergic nerves after 10 days of drug treatment. One type contained large granular vesicles (560-1,680 A) possibly represent serotonergic and dopaminergic nerves, thought to innervate this muscle. Two other types of profiles seemed to be unaffected by the drug. One conforms to cholinergic nerves while the other has a predominance of large opaque vesicles (1,200-2,500 A). The significance of these findings is discussed in the light of recent observations on the neurotoxic effects of 5,6-DHT on vertebrate and molluscan nerves.

An X-Ray Diffraction Study of Contracting Molluscan Smooth Muscle

The living anterior byssus retractor muscle of Mytilus (ABRM), a smooth, "catch" muscle, has been studied by X-ray diffraction while relaxed and while tonically contracted. X-ray reflections were observed from the actin and paramyosin filaments and from the alpha-helical substructure of the paramyosin filaments. No differences in spacings or relative intensities were observed when the relaxed and contracting muscle patterns were compared. This result is consistent with a sliding filament mechanism involving an interaction between actin and paramyosin filaments.

Electrical and mechanical responses of chromatophore muscle fibers of the squid, Loligo opalescens, to nerve stimulation and drugs

The muscle fibers of brown and red chromatophores in the skin of the squid, Loligo opalescens, respond to motor nerve stimulation with non-propagating excitatory postsynaptic potentials (e.p.s.p.'s) of fluctuating amplitude. Depending on the strength of stimulation several size classes of e.p.s.p.'s are found, indicating polyneuronal innervation. Facilitation and summation are minimal even though the reversal potential of the e.p.s.p.'s is close to zero. Acetylcholine (ACh) and 5-hydroxytryptamine (5-HT) have no effect on membrane characteristics of the muscle fiber, but ACh greatly augments the “spontaneous” quantal release of transmitter [increase in the frequency of miniature postsynaptic potentials (m.p.s.p.'s)] and thereby causes tonic contraction (“miniature tetanus”). 5-HT reduces the frequency of miniature potentials and abolishes tonic contraction. Inhibition of cholinesterase by eserine does not affect the amplitude or time course of e.p.s.p.'s and of m.p.s.p.'s. High concentrations of cholinergic blocking agents (atropine, banthine) reduce the postsynaptic effects of nerve stimulation in some cases. The natural transmitter substance of the motoneurones may not be ACh. The action of 5-HT appears to be intracellular. Neighboring muscle fibers are electrically coupled through low resistance pathways. These are most likely provided by the close junctions that form part of the myo-muscular junctions. The specific membrane resistance of the regular muscle fiber membrane was found to range from 1,056 to 1,320 Ohm×cm2, that of the close junctions ranges from 12.8 to 22.6 Ohm×cm2. The area occupied by close junctions is small, however, and only 10% of the current injected into one cell passes into the next. Some of the e.p.s.p.'s observed in a given muscle fiber most likely represent the electrotonic spread of the e.p.s.p.'s of the neighbor fibers. Of the six classes of e.p.s.p.'s observed in some muscle fibers, only two may originate in these fibers themselves. Chromatophores in aged preparations often exhibit pulsations. These are caused by spike potentials arising within muscle fibers whose membranes have become electrically excitable. Each spike is preceded by a generator depolarization. The electrical coupling of neighboring muscle cells permits conduction of the spike potentials throughout the set of muscle fibers of a pulsating chromatophore. Altered conditions within such preparations also lead to tonic contractions and contractures that are not necessarily accompanied by electrical activity. Several arguments are presented in support of the hypothesis that the “tonic condition” of nerve terminals (characterized by enhanced spontaneous transmitter release) and of muscle fibers (characterized by inability to relax) is due to an abnormal condition of intracellular calcium (lack of Ca-binding by sarcoplasmic reticulum or other storage sites). No evidence could be found for an inhibitory innervation of the chromatophore muscles. The nerve-induced relaxation of tonically contracted muscle fibers is caused by the action of motoneurones. Preliminary experiments on muscle fibers of the anterior byssus retractor muscle of Mytilus support the hypothesis that the tonic behavior (“catch”) of other molluscan muscles is due to mechanisms similar to those found in the chromatophore muscles.

The contractile mechanism of the anterior byssus retractor muscle ofMytilus edulis

The mechanical properties of the living, isolated anterior byssus retractor muscle ofMytilus(ABRM) have been studied under isometric conditions, and during length changes at constant speed. Stimulation of the muscle produces two distinct types of response, characterized by their tension decay rates. The rate is high in a phasic response produced by repetitive stimulation, and low in a tonic response produced by direct current or acetylcholine (ACh) stimulation. After cessation of contractile activity (i.e. the ability to shorten and develop tension actively), in both phasic and tonic responses the decay of tension becomes exponential. The time constant of tension decay ranges from 1 to 7 s for the phasic response, and from 5 to 100 min for the tonic response. Application of 5 hydroxytryptamine (5HT) to a tonically contracted muscle converts the tonic tension decay rate to the phasic tension decay rate. The resistance of the muscle to extension is greater during tonic than during phasic stimulation, but much the same during and after tonic stimulation. This is interpreted to indicate that the system which is responsible for tonic contraction is in action during tonic stimulation. Nevertheless, there is no difference in either the shortening speed at zero load, or the rate of rise of tension (after a release) during tonic and phasic stimulation; and this is also true for the muscle’s undamped series compliance. In both phasic and tonic contractions, tension is developed actively from 0*2 to 1*5l0, wherel0is defined as the shortest length of the muscle at which resting tension can be detected. Maximum tension is developed nearl0and decreases on either side of this optimum. The shape of the isometric tension-length curves is similar for phasic and tonic (ACh) contractions, but about 20% more tension is usually developed in the latter case. When the unstimulated muscle is slowly extended abovel0two types of tension are produced: (1) true resting tension, probably due to inert elastic material in parallel with the contractile apparatus, and (2) a variable amount of tension identical to passive tension (i.e. the tension remaining after stimulation when the muscle’s ability to shorten and develop tension actively is over). At any particular muscle length, the sum of passive and active tension is nearly the same, although there may be large variations in the amount of passive tension present. In a twitch, as in a tonic response, tension decays slowly, and greatly outlasts the muscle’s ability to shorten and develop tension actively. When the muscle is stimulated with single shocks spaced 10 to 30 s apart, a high level of tension can be developed, and maintained for periods up to 20 h. Such an intermittent activation mechanism may operate vivo during tonic contraction of the smooth retractor and adductor muscles of lamellibranch molluscs, thus enabling tension to be maintained very economically. Tonic tension is, in fact, often maintained in the isolated (stretched abovel0) by `spontaneous ’ contractions occurring about once every 10 s. Each individual contraction, which resembles a twitch, is accompanied by one or more action potentials similar to those observed in twitches, or at the onset of repetitive stimulation. It is suggested that tension in theABRMis developed and maintained by a system similar to that in vertebrate striated muscle, theABRMsystem being specialized in that under certain conditions tension decays extremely slowly. A hypothesis in terms of a sliding filament contractile mechanism is put forward which postulates: (1) that contraction in theABRMis due to linkages formed between two kinds of filaments, and (2) that the breaking rate of these linkages (and thus the rate of tension decay) is governed by the concentration of a relaxant (probably 5HT), the single process of breaking of linkages being reflected by the exponential phase of tension decay. The results are interpreted on this hypothesis, and discussed with reference to another hypothesis (Johnson, Kahn & Szent-Györgyi 1959; Rüegg 1959, 1961a), which holds that in addition to a contractile (actomyosin) system, muscles like theABRMhave a second (paramyosin) system which becomes rigid during tonic contraction, and thus maintains the tension developed by the contractile system.