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Comparative aspects of structure and action of molluscan neuropeptides.

A number of neuropeptides were isolated from the ganglia and muscles of molluscs, and their actions were examined. Diverse neuropeptides, in addition to several classical neurotransmitters, were suggested to be involved in the regulation of the anterior byssus retractor muscle of Mytilus. A wide structural variety of members of the Mytilus inhibitory peptide family was observed in each of the genera Mytilus, Achatina and Helix. Gly-Trp-NH2, the C-terminal dipeptide fragment of the neuropeptide AGPWamide, showed a more potent action than the parent peptide in all of the muscles examined. Peptides related to some molluscan neuropeptides were found to be distributed interphyletically. Some neuropeptides containing a D-amino acid residue were found in Achatina and Mytilus. These aspects of molluscan neuropeptides are thought not to be exceptional.

Amino Acid Sequence↗

Skinned smooth muscle: time course of force and ATPase activity during contraction cycle.

The time course of ATPase activity and force has been determined during contraction and relaxation in skinned (hyperpermeable) anterior byssus retractor muscle, ABRM, of Mytilus edulis and compared with corresponding measurements on skinned taenia coli of guinea-pigs. Following a calcium-induced contraction, lowering the [Ca++] to 10(-8) M rapidly reduces ATPase activity within 2 min to resting levels while force declines only to about 30-50% of maximal tension within the same time. Thus slow relaxation is due to a 'catch-like-state' which is common to different kinds of smooth muscles and can be reduced with cAMP in ABRM and by Pi in taenia coli.

Adenosine Triphosphatases↗

Response of fouling brown mussel, Perna perna (L.), to chlorine.

Perna perna (L.), the edible brown mussel, is very widely distributed in the tropical and subtropical regions and is commonly found in rocky shores. Apart from being a candidate for commercial cultivation, P. perna is also a common pest organism in cooling water systems of coastal power stations. Therefore, a lethal and sublethal response of this mussel to commonly used antifouling biocides is of considerable interest to the industry. Mortality pattern (LT(50) and LT(100)) and physiological activities (oxygen consumption, filtration rate, foot activity index, and byssus thread production) of different size groups (9-34 mm shell lengths) of P. perna were studied in the laboratory under different residual chlorine concentrations (0.25, 0.50, 0.75, and 1.00 mg/L for sublethal responses and 1, 2, 3, and 5 mg/L for mortality). Results showed that exposure time for 100% mortality of mussels significantly decreased with increasing residual chlorine concentration. For example, mussels of 9 mm size group exposed to 1 mg/L chlorine residual took 384 h (16 days) to reach 100% mortality, whereas those exposed to 5 mg/L chlorine took 84 h (4 days). The effect of mussel size on mortality was significant between 1 mg/L and 5 mg/L residual chlorine, with larger mussels showing greater resistance than smaller ones. For example, at 2 mg/L residual chlorine, 9 mm and 34 mm size group mussels took 228 h (10 days) and 304 h (13 days), respectively, to achieve 100% mortality. All size groups of P. perna showed progressive reduction in physiological activities, when chlorine residuals were gradually increased from 0 to 1 mg/L. Reduction in physiological activities was strongly correlated with the residual level. A comparison of present data with data available for other common fouling organisms suggests that P. perna is relatively less tolerant to chlorine than Perna viridis (L.) and Brachidontes striatulus (Hanley), which also cause fouling problems in tropical coastal waters.

Animals↗

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle.

The effects of orthovanadate (V(i)), inorganic phosphate (P(i)) and 2,3-butanedione monoxime (BDM) on tension, force transients and the catch state (passive tension maintenance) were investigated in saponin-skinned fibre bundles of the anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis at pH 6.7. During maximal Ca(2+) activation isometric force was depressed by V(i) (0.03-10 mM), P(i) (10 mM) and BDM (50 mM). Force transients following quick stretches (0.1-0.3% of fibre length) were accelerated substantially by 1 mM V(i), 10 mM P(i) or 50 mM BDM. These compounds also accelerated force responses in experiments in which ATP was released rapidly from caged ATP by flash photolysis at both pCa 4.7 (force rise) and at pCa>8 (force decline). The effects on the catch state were investigated in two types of experiments: (1) Ca(2+) removal after maximal Ca(2+) activation and (2) rapid ATP release during high-force rigor at pCa>8. In both cases rapid relaxation was followed by slow relaxation (slower than 2% of initial force per min). This later slow relaxation (catch) was insensitive to V(i) (1-10 mM), P(i) (10 mM) and BDM (50 mM) but was accelerated by 0.12 mM cAMP. Complete relaxation to almost zero force was attained by changing pH from 6.7 to 7.7 (pCa>8). We conclude that catch depends on cAMP- and pH-sensitive structures linking the myofilaments and not on the force-generating actomyosin cross-bridges that are sensitive to V(i), P(i) and BDM.

Adenosine Triphosphate↗

No effect of twitchin phosphorylation on the rate of myosin head detachment in molluscan catch muscle: are myosin heads involved in the catch state?

Phosphorylation of twitchin is known to abolish the catch state of anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis. To investigate the role of myosin head involvement in force maintenance during catch, the effect of twitchin phosphorylation on myosin head detachment was studied in saponin-skinned fibre bundles of ABRM. The detachment rate of myosin heads was deduced from two types of experiments: (1) force decay after stepwise stretch of maximally Ca2+-activated fibre bundles (pCa 4.5) and (2) force decay from high-force rigor, the former induced by a stepwise increase in ATP concentration elicited by photolysis of caged ATP (pCa<8). The rate of detachment was not affected by thiophosphorylation or phosphorylation of twitchin by 0.12 mM cAMP in the presence of the phosphatase inhibitor cyclosporine A (1 microM). Conversely, measurements of the rate of stretch-induced delayed force increase (stretch activation) and of the force increase following an ATP step in low-force rigor (pCa 4.5) suggest that the rate of myosin head attachment decreases after twitchin phosphorylation. We conclude that catch is not due to myosin heads remaining attached to actin filaments, but depends on myofilament interconnections that break down when twitchin is phosphorylated.

Actin Cytoskeleton↗

Force responses of skinned molluscan catch muscle following photoliberation of ATP.

Isometric force responses following flash photolysis of caged-ATP were measured from skinned preparations of the catch muscle anterior byssus retractor of Mytilus (ABRM). When fibres were transferred from Ca(2+)-free to Ca(2+)-containing rigor solution (pCa < 4) the force remained low, but flash photolysis produced an extended force increase (half-time, 0.30 +/- 0.07 s, n = 6). When Ca(2+)-activated fibres were transferred to a Ca(2+)-free rigor solution, their force remained at a high level. Flash photolysis produced a rapid force decay (half-time, 0.28 +/- 0.06 s, n = 9) to about 19% of the initial Ca(2+)-activated force. In the presence of 0.5 mM MgADP, the force increase was slowed down by a factor of 3 and the force decay by a factor of 5. These effects of MgADP on crossbridge kinetics are comparable to those observed in vertebrate smooth muscle and are thought to cause "latch", a catch-like state (Fuglsang et al. J Muscle Res Cell Motil 14:666-677, 1993). They are consistent with a model implicating competition between MgADP and MgATP for the nucleotide-binding site on crossbridges. Considering the relatively fast force responses induced by caged-ATP photolysis, even in the presence of MgADP, it appears unlikely that the detachment of crossbridges from the rigor state can account for catch-related processes. In view of the low myosin ATPase under maximal activating conditions (0.6 s-1, Butler et al. Biophys J 75:1904-1914, 1998), neither crossbridge attachment nor detachment of rigor crossbridges seems to be the rate-limiting processes of the crossbridge cycle.

Adenosine Diphosphate↗

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.

Animals↗

Twitchin purified from molluscan catch muscles regulates interactions between actin and myosin filaments at rest in a phosphorylation-dependent manner.

Twitchin, also called mini-titin, is structurally related to the giant elastic protein connectin/titin, and has been found in not only striated but also smooth muscles of bivalves. Many bivalve smooth muscles such as byssus retractor muscles and the opaque part of adductor muscles are known as catch muscles that can maintain high passive tension with little expenditure of energy after they have actively contracted. Twitchin is phosphorylated when this high-tension state (catch state) ceases. Our recent studies revealed that the catch tension is due to interactions between thick and thin filaments in the presence of MgATP at low free Ca2+ concentrations, which can be visualized in vitro under a light microscope (Yamada et al., 2001 Proc Natl Acad Sci USA 98: 6635-6640). We also found that twitchin is essential for the interactions of the catch state in mussel (Mytilus galloprovincialis) catch muscles. In the presence of twitchin, actin filaments bound to purified myosin filaments when twitchin was dephosphorylated by Ser/Thr protein phosphatase 2B, while they did not when it was phosphorylated by cAMP-dependent protein kinase. In the current study we demonstrate the same essential components of the catch state for another bivalve that exhibits catch, i.e., Japanese oyster (Crassostrea gigas).

Actins↗

Competitive inhibition by dimethylsulfoxide of molluscan and vertebrate acetylcholinesterase.

Anticholinesterase-like effects of dimethylsulfoxide (DMSO) were demonstrated on a variety of invertebrate muscles. The excitatory effects of acetylcholine (ACh) on the isolated preparations of the Geukensia demissa heart and anterior byssus retractor muscle (ABRM), and of the Busycon contrarium radula protractor muscle, were potentiated by DMSO (1-5 microliters/ml; 1 microliter/ml = 14 mM). The negative chronotropic effects of ACh, but not of 4-ketoamyltrimethylammonium, were potentiated by DMSO (1-5 microliters/ml) on the isolated heart of the oyster Crassostrea virginica. These four muscles have acetylcholinesterase enzymes of high activity. In contrast, Mercenaria mercenaria hearts have weak cholinesterase activity, and the effects of ACh on this isolated myocardium were not potentiated by DMSO (2-20 microliters/ml). DMSO (0.1-15 microliters/ml) was a competitive inhibitor of both a crude preparation of oyster heart acetylcholinesterase (AChE) (the Km increased 24-fold with DMSO at 15 microliters/ml; the I50 was 1.3 microliters/ml DMSO when [ACh] = Km) and a purified Electrophorus AChE (the Km increased 4.5-fold when DMSO was 10 microliters/ml; the I50 was 10 microliters/ml DMSO near [ACh] = Km). The same doses of DMSO were needed to potentiate the pharmacological effects of ACh on the oyster heart, as to inhibit the AChE of this tissue.

Acetylcholinesterase↗

The ultrastructure of the byssal apparatus of a mussel. V. Localization of collagenic and elastic components in the threads.

Ultrastructural and cytochemical studies have been carried out on the proximal part of byssus threads (TPP) in an attempt to localize collagenic and elastic components. The results show that TPP autoclaving followed by hot alkali treatment causes the extraction of about two-thirds of hydroxyproline and the parallel removal of most of the matrix, leaving filaments unaffected. Moreover the results of the staining reactions signaletic for elastic tissues indicate that TPP filaments contain glycoproteins with a reactivity similar to that of many invertebrate elastic tissues. On the basis of these morphological findings, it seems reasonable to suggest that collagen may be located in TPP matrix, while filaments could be responsible for the elastic properties.

Animals↗

Lethal and sublethal toxicity of 4-nonylphenol to the common mussel (Mytilus edulis L.).

The toxicity of nonylphenol to the common mussel (Mytilus edulis L.) has been determined in both semistatic and continuous flow test systems. The LC50 values obtained were for 96 h, 30 mg litre(-1); 360 h, 0.5 mg litre(-1); and 850 h, 0.14 mg litre(-1). Sublethal effects, manifested as decreased byssus strength and change of scope for growth, were obtained at a concentration as low as 0.056 mg litre(-1). Fertilization and early developmental success were not affected at the highest concentration tested (0.2 mg litre(-1)).

Journal Article↗

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. 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. Isolated resting muscle fibres revealed a lower energy charge than freshly-dissected ones. 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. The energy charges of the guanosine, uridine and cytidine systems did not show changes parallel to the adenosine system. 5. The levels of cyclic AMP were only changed under the influence of serotonin.

Animals↗

Purification and properties of caldesmon-like protein from molluscan smooth muscle.

In this comparative study, the heat-stable protein content of scallop muscles was reinvestigated. The hCaD-like protein was prepared and its properties carefully examined. The heat-stable high-molecular-mass caldesmon-like (hCaD-like) protein is only present in the catch (smooth) muscle and it is completely absent in the striated muscle of scallop. The isolated scallop hCaD-like protein cosediments with F-actin, binds to myosin significantly and inhibits the ATPase activity of acto-myosin. A partial cDNA clone from a Mytilus anterior byssus retractor muscle (ABRM)-related protein showed strong homology with the hCaD gizzard sequence. This allowed identification of the heat-stable 100-110 kDa protein doublet band isolated in this study as a caldesmon-like molecule.

Amino Acid Sequence↗

The relaxing effect of SKF 38393 on the catch contraction of Mytilus smooth muscle.

The relaxing effect of SKF 38393 on the catch contraction in the anterior byssus retractor muscle (ABRM) of Mytilus and the effect of SKF 38393 on the cyclic AMP (cAMP) levels in the ABRM were investigated. The catch contraction was relaxed by SKF 38393 in a dose-dependent manner. The dose-response curves of SKF 38393 were shifted in parallel to the right by butaclamol (3 X 10(-6) and 6 X 10(-6) M) and haloperidol (3 X 10(-6) and 3 X 10(-5) M). Their pA2 values were 5.69 +/- 0.05 and 5.80 +/- 0.07. The cAMP levels in the ABRM were not altered by SKF 38393 at a concentration (10(-5) M) sufficient to relax. These findings indicate that the dopamine receptors of the ABRM are D-1 like receptors but somewhat different from those of the vertebrates.

2,3,4,5-Tetrahydro-7,8-dihydroxy-1-phenyl-1H-3-ben↗

Organic Ca(2+)-antagonist-resistant response to FMRF-NH2 on the molluscan smooth muscle.

1. FMRF-amide (10(-7)-10(-5) M) contracted molluscan anterior byssus retractor muscle in a concentration-dependent fashion. 2. The concentration-response curve of FMRF-amide was shifted rightward by an analogue of FMRF-amide, FMRf-amide ([D-Phe4]FMRF-amide, putative FMRF-amide receptor antagonist) in a parallel manner (pA2 = 4.87 +/- 0.04). 3. Although a contractile response to KCl was reduced by the organic Ca2+ antagonists (verapamil, diltiazem and high concentration of nifedipine and nicardipine). FMRF-amide-induced contraction was not markedly reduced by them. 4. In the Ca(2+)-free medium, FMRF-amide-induced contraction was diminished. The response was also reduced by TMB-8 (10(-4) M), suggesting that FMRF-amide-induced contraction might be partly dependent on intracellular Ca2+. 5. An inorganic Ca(2+)-antagonist, MnCl2, markedly reduced the FMRF-amide- and KCl-induced contraction. The results show that FMRF-amide-induced contraction might be dependent on extracellular Ca2+. 6. These findings suggest that FMRF-amide-induced contraction might be mediated through an action on FMRF-amide receptors and not through the activation of organic Ca2+ antagonist-sensitive Ca2+ channels.

Animals↗

Actions of a small cardioactive peptide from Mytilus, APNFLAYPRLamide, on central neurones of Helix aspersa.

1. Mytilus small cardioactive peptide (SCP) was originally isolated from the anterior byssus retractor muscle of Mytilus edulis, with a primary structure, APNFLAYPRLamide. The mechanisms of action of this peptide were examined on identified central neurones of the snail, Helix aspersa, using intracellular recordings and a two electrode voltage clamp. 2. 50 microM APNFLAYPRLamide could elicit a long term excitation on F2 neurones, in normal saline, Na(+)-free saline, Ca(2+)-free saline or in 10 mM Co2+ saline. This indicates that the excitatory effect of APNFLAYPRLamide involved an increase in membrane conductance to both Na+ and Ca2+. 3. 10 microM APNFLAYPRLamide potentiated the Ca2+ inward current of F2 neurones while reversibly reducing the ACh-induced membrane current of these neurones. Forskolin had identical effects to those of APNFLAYPRLamide. This indicates that the action of APNFLAYPRLamide may be through a second messenger. In contrast APNFLAYPRLamide potentiated a cholinergic EPSP evoked in F2 neurones. 4. YPRLamide was inactive even at higher concentrations, for example, 100 microM, while LAYPRLamide exhibited activity but with a lower potency. This indicates that LAYPRLamide is the minimum structure required for activating this class of SCP-like peptide receptor. However, the mode of action of APNFLAYPRLamide on the Na+ current of F2 neurones requires further investigation.

Acetylcholine↗

Pharmacology of FMRFamide in Mytilus catch muscle.

In the anterior byssus retractor muscle (ABRM) of Mytilus, low concentrations of FMRFamide (10(-8)-10(-7) M) relax ACh-induced catch-tension, whereas high concentrations (greater than 10(-7) M) cause contraction. To study the structure-activity relations of these actions, a number of peptide analogs of FMRFamide were screened for their biological activities on the ABRM. The structure-activity relations for contraction were different from those for relaxation. Among the peptides tested, FMR-[D-Phe]-amide and gamma 1-MSH substantially antagonized FMRFamide contractions; but only gamma 1-MSH was even slightly antagonistic to FMRFamide-induced relaxation. Relaxations produced by 10(-7) M FMRFamide, or by 10(-5) M FMRFamide-relating relaxing peptides, were markedly depressed by treating the muscle first with 10(-5) M FMRFamide or with 10(-5) M FMRFamide-related contractile peptides. However, contractile agents that are structurally unrelated to FMRFamide, such as 3 X 10(-5) M SCPB and 2 X 10(-2) M caffeine, showed little or no such after-effect on the relaxation. Relaxations in response to submaximal serotonin, dopamine and repetitive electrical pulses of stimulation were not affected by a pretreatment with 10(-5) M FMRFamide. These results suggest that the ABRM of Mytilus has at least two pharmacologically distinct classes of receptors which are capable of being activated by FMRFamide.

Acetylcholine↗

Serotonin and dopamine as regulators of adenylate cyclase and relaxation in a smooth muscle of the mussel Mytilus edulis.

Serotonin and dopamine show similar dose-response relationships when they relax tonically contracted intact muscle fibres of the anterior byssus retractor muscle of Mytilus edulis. In saponin-treated muscle fibers, only serotonin or high doses of dopamine relax tonic contraction. In a membrane fraction, enriched in adenylate cyclase activity, serotonin and dopamine increase the rate of cAMP production. Serotonin is a far more potent activator of adenylate cyclase than dopamine.

Adenylyl Cyclases↗