Molluscan Muscle Research Articles

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.

Intracellular Buffering Capacity in Molluscan Muscle: Superfused Muscle versus Homogenates

In situ estimates of intracellular pH (pHi) in isolated bands of radula protractor muscles from the whelk Busycon canaliculatum were made using phosphorus nuclear magnetic resonance spectroscopy (31P-NMR). The 31P-NMR spectra contained distinct resonances for arginine phosphate and the α, β, and γ phosphates of ATP. In addition, initial spectra displayed a small inorganic phosphate (Pi) peak, which disappeared upon continued superfusion under aerobic conditions. Addition of 2 mM 2-deoxyglucose to the medium resulted in the generation of an additional resonance corresponding to 2-deoxyglucose-6-phosphate (2DGP). Using the chemical shift differences of both the Pi and 2DGP peaks relative to the internal reference arginine phosphate, the resting aerobic pH, was found to be 7.29 ± 0.07 SD (n = 3) and 7.32 ± 0.04 SD (n = 5), respectively. Intracellular buffering capacity (β) was estimated in situ using NH₄Cl prepulse and weak acid loading techniques. The β values were 30 ± 3.9 SD (n = 7) and 31 ± 3.8 SD (n = 7) μ mol·pH⁻¹·g intracellular water⁻¹, respectively. A third method, by titration of tissue homogenates, yielded a β value of 85.8 ± 17.5 SD (n = 8) μ mol·pH⁻¹·g intracellular water⁻¹. This large discrepancy can be partially explained by the rapid hydrolysis of arginine phosphate immediately after homogenization, generating free inorganic phosphate, an excellent buffer in the physiological pH range. The chemical nature of intracellular buffers in B. canaliculatum radula protractor muscle was examined through crude acid fractionation and subsequent amino acid analysis. The total histidine content in this muscle accounted for 72% of the in situ buffering capacity. Unlike some vertebrate systems where large amounts of histidine-containing dipeptides function as buffers, most of the histidine is associated with the protein fraction of the tissue.

Ca2+-sensitive transition in the molecular conformation of molluscan muscle myosins.

Filament assemblies of myosin molecules purified from scallop adductor muscles were stabilized by Ca2+ in the presence of ATP or ADP. Electron micrographs showed that the tail part of monomeric myosin molecules was folded in the absence of Ca2+, but was extended in the presence of Ca2+ at physiological ionic strength.

Effects of Mytilus inhibitory peptides on mechanical responses of various molluscan muscles

1. Effects of the Mytilus inhibitory peptides (MIPs), Ala 2-MIP and Ser 2-MIP, on mechanical responses of various molluscan muscles were examined. 2. In the ABRM of Mytilus edulis, all contractions in response to repetitive electrical pulses of stimulation, ACh, FMRFamide and caffeine were inhibited by MIPs. Contraction by 400 mM K + ASW was invariably not affected, but that by 100 mM K + ASW was slightly inhibited in some muscles. 3. In the pedal retractor muscle of Mytilus edulis, contractions in response to repetitive electrical pulses of stimulation and ACh were also inhibited by MIPs. 4. In the heart of Meretrix lusoria, its activity was inhibited by MIPs. Ala 2-MIP was found to be more potent than Ser 2-MIP. The ACh-antagonist benzoquinonium did not change the inhibitory effects of the peptides. In the heart of Tapes japonica, both of the peptides showed weak inhibitory effect at 100 nM. At 10 μM, however, Ala 2-MIP showed inhibitory effect followed by excitatory effect, and Ser 2-MIP showed excitatory effect and then brought about a transient cystolic arrest. 5. Twitch and tetanic contractions of the penis retractor muscle of Achatina fulica were inhibited by MIPs. Ala 2-MIP was found to be more potent than Ser 2-MIP. 6. MIPs seem to have an inhibitory effect on many molluscan muscles. There may exist a family of MIP-related peptides in molluscs.

Effects of the catch-relaxing peptide on molluscan muscles

1. Effects of the catch-relaxing peptide (CARP) on contractile responses were examined in the anterior byssus retractor muscle (ABRM) of Mytilus edulis and in various other molluscan muscles. 2. In the ABRM, phasic contraction in response to repetitive electrical pulses of stimulation was potentiated by low concentrations of CARP and inhibited by high concentrations. The inhibitory effect reached maximum in 2–10 min after applying the peptide. Contractions of the muscle in response to acetylcholine (ACh) and 100 mM K + artificial seawater (ASW) were also potentiated by low concentrations of CARP and inhibited by high concentrations. Contractions in response to FMRFamide and caffeine were simply inhibited by CARP. 3. In the radula protractor and retractor muscles of Rapana thomasiana and Fusinus perplexus, contractions in response to electrical pulse, ACh and glutamate were inhibited by CARP. 4. In the herats of Mytilus edulis, Merelrix lusoria, Tapes japonica and Rapana thomasiana, their activities were inhibited by CARP. The ACh-antagonist benzoquinonium did not affect the inhibitory effect of the peptide. 5. The results of the present study suggest that CARP has modulatory effects on contractions in many molluscan muscles and that its principal modulatory effect is one of inhibition.

Structures and actions of Mytilus inhibitory peptides

Two congeneric peptides that inhibit contraction of the anterior byssus retractor muscle of Mytilus edulis were isolated from the pedal ganglia of the mussel. Their structures were determined to be H-Gly-Ser-Pro-Met-Phe-Val-NH2 and H-Gly-Ala-Pro-Met-Phe-Val-NH2. These hexapeptides also showed inhibitory action on contractions in several other molluscan muscles, such as the cardiac muscle of Meretrix lusoria and the penis retractor muscle of Achatina fulica.

Transient-kinetic studies of the adenosine triphosphatase activity of scallop heavy meromyosin.

Fluorescence stopped-flow experiments were performed to elucidate the elementary steps of the ATPase mechanism of scallop heavy meromyosin in the presence and in the absence of Ca2+. ATP binding and hydrolysis, as monitored by the change in tryptophan fluorescence, appear to be Ca2+-insensitive, whereas both Pi release and ADP release are markedly suppressed in the absence of Ca2+. Rate constants for Pi release are 0.2 s-1 and 0.002 s-1 and for ADP release are 6 s-1 and 0.01 s-1 in the presence and in the absence of Ca2+ respectively. Ca2+ binding to the specific site of the regulatory domain is rapid and its release occurs at 25 s-1, consistent with the time scale of a twitch of the striated adductor muscle. Nucleotide binding is a multi-step process requiring a minimum of three states. In such a model Ca2+ controls the rate of conformational changes at the active site in both the forward and the reverse direction, leading to a large dependence of the rate of nucleotide release, but a lesser effect on the overall equilibrium position. The kinetic trapping of nucleotides and Pi at the active site, in the absence of Ca2+, appears to be a fundamental step in suppressing the interaction of the myosin head with the thin filaments in relaxed molluscan muscle.

Paramyosin and the catch mechanism.

We propose here the formation by molluscan and notochord muscles in the catch state of three-dimensional, entangled network structures composed of bent and sometimes entwined paramyosin thick filaments including myosin intermediate filaments and disordered actin thin filaments; in the relaxed state the three forms lie in parallel. The intact forms of bivalve (Andonta pacifica, Heude) muscle paramyosin are those of 120 and 95 kDa (beta-paramyosin). The 102 kDa form (alpha-paramyosin) is the proteinase cleavage product of 120 kDa paramyosin. Paramyosin could be phosphorylated in vitro by cyclic AMP-dependent protein kinase. The amino acid phosphorylated was at the serine residue. Paramyosin from muscles treated with acetylcholine (catch state) was phosphorylated to a greater extent than that of untreated muscles (normal state) and even more so in the case of serotonin-treated muscles (relaxed state). Actin markedly inhibited the phosphorylation of paramyosin in vitro.

Electromechanical uncoupling in a molluscan muscle examined by the sucrose gap technique. The effect of calcium antagonist and agonist agents.

Membrane potential and tension of Busycon radular protractor muscles were studied by sucrose gap methods. Excitation-contraction (EC) coupling was examined in response to acetylcholine (ACh) and high K which depolarized the fibres and induced tension, but without action potential firing. Potassium depolarization did not follow predictions expected from the Nernst equation at low and very high K levels, and maximum tension was found at about 100 mM K. EC coupling was very sensitive to [Ca]0. Ca-free media eliminated K- and ACh-induced tension but with normal depolarization, showing full electromechanical uncoupling. Ionophore A23187 enhanced K- and ACh-induced responses and X-537A enhanced ACh responses, demonstrating acute dependence of activation on [Ca]0 in this muscle. The calcium antagonists nifedipine and nisoldipine reduced tension in the muscle only at very high concentrations, and both agents slightly reduced K- and ACh-induced depolarization. Verapamil reduced K- and ACh-induced tension but paradoxically it enhanced the depolarizing actions of these agents leading to electromechanical uncoupling. Abscisic acid (ABA) enhanced ACh- and K-induced tension and simultaneously enhanced their depolarizing actions. Ionophores and ABA appear to enhance calcium influx which may secondarily influence sodium influx. Calcium antagonists have no consistent actions on this muscle, suggesting that calcium channel activity of the radular protractor may be different from that seen in mammalian visceral muscles.

Changes of nucleotide contents and of energy charge induced by contraction, catch and relaxation in smooth molluscan muscle fibres. An analysis using reversed-phase ion-pair high-performance liquid chromatography

1. 1. The content of 14 different nucleotides, including cAMP, in isolated muscle fibres of the anterior byssus retractor muscle of Mytilus edulis was analysed. The nucleotide levels were determined after the muscle fibres performed phasic, tonic, or tetanic contractions and after serotonin-induced relaxation of tonic contraction. 2. 2. Isolated resting muscle fibres revealed a lower energy charge than freshly-dissected ones. 3. 3. During active force development adenosine energy charge decreased to stay at the same low level during catch, tetanus and serotonin-induced relaxation of catch respectively. 4. 4. The energy charges of the guanosine, uridine and cytidine systems did not show changes parallel to the adenosine system. 5. 5. The levels of cyclic AMP were only changed under the influence of serotonin.

Neurohumoral control of ionic pumping in molluscan muscle—II. evidence for an electrogenic sodium pump in radular protractor muscle of Busycon canaliculatum

Abstract 1. A sucrose gap technique was used to study the effects of brief periods of superfusion with solutions in which the potassium content of artificial sea water was reduced or omitted. 2. Stepwise reduction in bath potassium had a complex effect, culminating in the response to potassium-free solution. This was composed of a rapid initial hyperpolarizing phase, overtaken by a slower depolarizing phase, which was accompanied by force. 3. Readmission of bath potassium induced a transient after-hyperpolarization. 4. There was a high degree of individual variability in RPM preparations from different animals. This was particularly evident in cases in which either the hyperpolarizing phase or the depolarizing phase predominated, in the response to zero-potassium, but the muscles from any one animal showed reproducible responses. 5. The RPM behaved as predicted on Nernst equation grounds, to the extent that initial hyperpolarization showed stepwise increases with stepwise reduction in [K + ] 0 , but as the steps approached zero-potassium there was a stepwise increase in the slower depolarizing response, suggesting reduction in electrogenic Na-K exchange. 6. In Na-free solution the depolarizing phase of the response to zero-K was abolished, leaving only an enhanced hyperpolarizing phase. 7. Abrupt chilling had a depolarizing effect. 8. There was only a slight increase in resistance during the action of zero K.

Catch-relaxing peptide isolated from Mytilus pedal ganglia

A peptide that relaxes catch tension of the anterior byssus retractor muscle of Mytilus edulis was purified from pedal ganglion extracts of the mussel. Its primary structure was determined to be H-Ala-Met-Pro-Met-Leu-Arg-Leu-NH 2. This peptide was found to have not only catch-relaxing action on the byssus retractor muscle but also modulatory actions on contractions in various molluscan muscles.

ChemInform Abstract: Water‐Soluble Active Ester Method. Part 3. Synthesis of FMRFamide, a Molluscan Neuropeptide.

AbstractThe peptide (I), prepared by the title method, has different effects on molluscan muscles.

Myosin rod phosphorylation and the catch state of molluscan muscles.

"Catch" is a prolonged state of tension in molluscan smooth muscles shown by mechanical measurements to be associated with the level of protein phosphorylation. Myosin isolated from these muscles is unusual in being phosphorylated in the rod portion by an endogenous kinase, like certain nonmuscle myosins. These findings suggest that the myosin rod is a target for phosphorylation and that this reaction may control the transition from catch to relaxation.

Multiple receptor sites for a molluscan peptide (FMRFamide) and related peptides of Helix.

1. The membrane actions of some tetrapeptide amides and heptapeptide amides chemically related to the molluscan neuropeptides Phe-Met-Arg-Phe-NH2 (FMRFamide) and p-Glu-Asp-Pro-Phe-Leu-Arg-Phe-NH2 (pQDPFLRFamide) were tested and compared on identified neurones of Helix aspersa. 2. The C-terminal sequence -Phe-NH2 was an important requirement for each of the four different actions studied: slow increase in K conductance (gK), fast increase in gK, increase in Na conductance (gNa) and decrease in gK. 3. The response of some neurones involved a combination of such actions. 4. The tetrapeptide amides FMRFamide, Phe-Leu-Arg-Phe-NH2 (FLRFamide) and Phe-Ile-Arg-Phe-NH2 (FIRFamide) were more potent than the heptapeptide amides at producing the slow increase in gK and also produced the increase in gNa, not seen at all with the heptapeptide amides. 5. The heptapeptide amides induced the fast increase in gK which was not observed with the tetrapeptide amides. 6. Evidence is presented that each of the tetrapeptide amides tested acts on the same receptor type which mediates the increase in gNa and on which the heptapeptides were inactive. 7. The results are interpreted in terms of multiple receptors each of which however appears to require the C-terminal sequence Phe-Met(or Leu)-Arg-Phe-NH2, as has been shown for some molluscan muscle preparations which react to these peptide amides (Price & Greenberg, 1980).

Excitation-contraction coupling in non-spiking smooth muscle in the gill ofAplysia

1. Excitation-contraction coupling (E-C Coupling) was investigated in the non-spiking longitudinal efferent vein muscle (LEVM) from theAplysia gill. The sucrose-gap method was used to study the E-C coupling in response to acetylcholine (ACh) and to elevated external potassium (Ko). 2. LEVMs from mature and oldAplysia were used in this study. The response to high Ko and ACh was not significantly different in the two groups. 3. Depolarization increased directly with the concentration of either Ko (50 to 500 mM) or ACh (1 μM to 1 mM). It preceded the development of tension, which increased with depolarization. These stimulants did not evoke action potentials. 4. In the presence of MnCl2 (10–20 mM) for 30 min, ACh-contracture was reduced to near zero; in contrast K-contracture was only reduced to 60% of the control level; and depolarization was unaffected. In a nominally Ca-free medium for 30 min, ACh-contracture was reduced to zero, whereas K-concentration persisted at 16% of the control level. Hence, ACh and high Ko apparently mobilize different Ca stores to initiate E-C coupling: In the non-spiking LEVM, ACh-contracture primarily involves influx of Ca, whereas K-contracture appears to rely on primarily cellular Ca. The results are contrasted to those in a spiking molluscan muscle. 5. We propose that mobilizing both stores of calcium to initiate E-C coupling insures synchronized contraction of LEVM fibers. The similarity of activation in vitro, by Ko and ACh, and in situ, by neuronal firing, is shown and discussed.

Physiology and motor innervation of the supralateral radular retractor muscles of the pulmonate snail,Planorbarius corneus

1. Some electrophysiological properties of a multifunctional molluscan muscle, the supralateral radular retractor (SLR), were examined. The resting membrane potential was 62.3±10.5 mV and was dependent upon permeabilities to K+, Na+ and Cl−. Current-voltage relationships for SLR muscle fibres were linear, and the fibres had input resistances of 22.5±5.6 MΩ. Indirect evidence suggested that they were not electrically coupled. 2. Miniature EJPs occurred spontaneously in the muscle. Action potentials in the identified motoneurones elicited monosynaptic, chemical EJPs in muscle fibres. The majority of fibres were polyneuronally innervated by up to four motor axons. Action potentials did not normally occur in the muscle fibres, and contraction was proportional to the degree of depolarization produced by EJPs. 3. Contractions elicited in the SLRs by the stimulation of motoneurones were bilaterally symmetrical, and were temporally and spatially characteristic of the type of neurone excited. Temporal differences in contraction between motor units were determined mainly by differences in initial EJP amplitude. The relatively small degree of facilitation shown by all motor units had little effect on the speed of contraction. A linear summation model adequately described the growth of facilitation at terminals of one type of motoneurone, but not at those of the other two types of motoneurones examined.

Identification of paramyosin as schistosome antigen recognized by intradermally vaccinated mice.

Mice immunized intradermally with extracts of Schistosoma mansoni in combination with the adjuvant BCG are significantly protected against subsequent infection with living larval forms of the parasite. Remarkably, these vaccinated animals produce antibodies predominantly against a single parasite protein of molecular weight 97 kilodaltons (Sm-97). A complementary DNA that encodes about half of the Sm-97 molecule has now been cloned and sequenced. Analysis of the deduced amino acid sequence reveals a protein containing periodic repeats of hydrophobic amino acids characteristic of an alpha-helical coiled-coil structure. The deduced amino acid composition of the cloned gene and several properties of the native protein are similar to that of paramyosin, an alpha-helical protein that forms the core for myosin filaments in invertebrate muscle. Paramyosin was isolated from Schistosoma mansoni adult worms and antibodies to Sm-97 were shown to react with this molecule as well as with a known paramyosin from molluscan muscle.

Nucleotide pools and energy charges of smooth molluscan muscles analysed by reversed-phase, ion-pair, high-performance liquid chromatography

A method is described for the analysis of the 5′ mono-, di- and triphosphates of adenosine, guanosine, uridine and cytidine, as well as uridinediphosphate-glucose and cyclic AMP. Separation is achieved by reversed-phase ion-pair chromatography with linear gradient elution. Application of this method to the analysis of nucleotides in smooth molluscan muscles is described, including the determination of cAMP-levels and the calculation of energy charges for all of the four nucleotide systems.

Studies on the extracts from the muscle of molluscs. IX. Biosynthesis of D-rhodoic acid in abalone.

The capacity for the biosynthesis of D-rhodoic acid from taurine and pyruvic acid was studied in the tissues of abalone Halioris discus hannai using radioactive tracer methods. D-Rhodoic acid, a newly isolated marine natural product, has an interesting structure analogous to octopine, alanopine and strombine all of which are referred to as opines. Rhodoic acid has been shown to be one of the predominant nitrogenous components of muscle extractives of this abalone. Opines are generally considered to be end products of anaerobic glycolysis of molluscs. Muscle and mid-gut gland homogenates were incubated with 10μCi of radioactive taurine or pyruvate, with gentle shaking at 30°C for 1h. D-Rhodoic acid was isolated by ion exchange chromatography and recrystallized from aqueous ethanol Incorporation of radioactivity into n-rhadoic acid from both taurine and pyruvate was observed in the muscle, but not in the mid-gut gland. It is likely that this biosynthesis is mediated by the action of rhodoic acid dehydrogenase which catalyzes the reductative condensation reaction: taurine+pyruvate+NAAH ?? D-rhodoic acid+NAD++H2O.Since D-rhodoic acid is the most commonly observed opine in abalone Haliotis discus hannai, it can be assumed that this dehydrogenase has an important physiological role such as the main-tenance of cytoplasmic redox balance in the muscle tissue during periods of elevated glycolytic flux.