Crustacean Skeletal Muscle Research Articles

Molecular characterization of four genes highly expressed during megalopa stage in Chinese mitten crab, Eriocheir sinensis

The molecular markers to distinguish different larval stages have various applications in ecological studies. Using the differential display RT-PCR technique, we isolated and characterized four genes, which are expressed predominantly in the megalopa stage of Eriocheir sinensis. The four genes include two cuticular proteins with different domain organization (Ers-CP15 and Ers-CP34) and two skeletal-muscle-specific genes (Ers-SCP and Ers-ActinSK1). The two cuticular protein genes were expressed predominantly in the epidermis and their expression level was significantly higher in the megalopa stage (about 7.0-folds) than it was in the zoea stage. However, their high transcriptional level in zoea IV suggested that these two cuticular protein genes may not be a useful target to discriminate megalopa from zoea. Ers-SCP encoded the invertebratespecific sarcoplasmic calcium binding protein and Ers-ActinSK1 gene encoded the crustacean skeletal muscle actin. Expressions of these two genes were detected only in muscular tissues; leg muscle, claw muscle and thoracic muscle. This suggests that the increased transcription levels of two muscle-specific genes during the megalopa stage are mainly due to increased muscular tissues. Among its three isoforms, Ers-SCPa displayed the highest difference (22.4-folds) between megalopa and zoea suggesting Ers-SCPa is the most reliable marker to distinguish megalopa from zoea. Although Ers-SCPc and Ers-ActinSK1 also showed similar expression profiles to Ers-SCPa and Ers-SCPb, differences in their expression levels were not as high as Ers-SCPa and Ers-SCPb.

Rheb REGULATES CRAB MUSCLE ATROPHY

Moulting their shells and misplacing limbs, crabs continually depend on their versatile musculature to reshape their bodies. According to Donald Mykles and colleagues from Colorado State University and the University of Wisconsin – Stevens Point, USA, the massive claw muscle of blackback land crabs withers by as much as 78% before they can slip their shells off. However, Mykles and his colleagues made an interesting discovery in 2010. Instead of shutting down protein synthesis to allow the muscle to wither, the crabs ramp it up 11-fold by shutting down myostatin expression – which usually keeps protein synthesis in check. Knowing that myostatin regulates protein expression in mammals via the mTOR (metazoan target of rapamycin) signalling cascade, which controls mRNA translation, Mykles decided to investigate the mTOR pathway in blackback land crabs to find out whether it also regulates muscle rebuilding in crabs during moulting (p. 590).Stimulating crabs to moult, the team collected muscles from the animals and searched for the mRNA of four key mTOR signalling proteins – Rheb, mTOR, Akt and s6k. The crabs expressed all four genes, confirming that the mTOR pathway is active in moulting crab’s atrophied muscle.Next, the team measured the expression levels of each gene in crabs that had been induced to moult using methods that raise the animals’ moulting hormone (ecdysteroid, which regulates myostatin levels) over different time scales. They found that Rheb levels increased in the moulting crabs’ claws muscles and in the atrophied thoracic muscles of crabs that had lost a limb. Mykles says ‘Rheb is a key activator of mTOR. Its up-regulation by moulting indicates that Rheb plays a role in the stimulation of mTOR-mediated protein synthesis by ecdysteroids’.However, when the team investigated the mechanism by which Rheb is upregulated in the two muscles, there were differences. They found a strong correlation between the levels of Rheb in the crabs’ claws and the ecdysteroid levels, suggesting that ecdysteroid does regulate protein synthesis via mTOR in the claw muscle. Yet, when they compared the ecdysteroid levels and Rheb levels in the thoracic muscles – that wither in response to limb loss – the team found no correlation. Ecdysteroid hormone does not control muscle atrophy in response to limb loss.Analysing the expression patterns of Rheb and myostatin in atrophied claw and thoracic muscles, Mykles and colleagues also saw different expression relationships between the proteins in the two tissues. ‘If blackback land crab myostatin plays a role in both types of atrophy, any unifying mechanism must reconcile the differences between myostatin and Rheb expression in the two muscles’, the team says.Having shown that Rheb is a key component of the signalling pathway that regulates muscle atrophy in crabs, the team says that it is keen to build, ‘a mechanistic understanding of the interactions between ecdysteroids and the signalling pathways that control protein metabolism in crustacean skeletal muscle’.

Muscle-specific calpain is localized in regions near motor endplates in differentiating lobster claw muscles

Calpains are Ca 2+-dependent proteinases that mediate protein turnover in crustacean skeletal muscles. We used an antibody directed against lobster muscle-specific calpain (Ha-CalpM) to examine its distribution in differentiating juvenile lobster claw muscles. These muscles are comprised of both fast and slow fibers early in development, but become specialized into predominantly fast or exclusively slow muscles in adults. The transition into adult muscle types requires that myofibrillar proteins specific for fast or slow muscles to be selectively removed and replaced by the appropriate proteins. Using immunohistochemistry, we observed a distinct staining pattern where staining was preferentially localized in the fiber periphery along one side of the fiber. Immunolabeling with an antibody directed against synaptotagmin revealed that the calpain staining was greatest in the cytoplasm adjacent to synaptic terminals. In complementary analyses, we used sequence-specific primers with real-time PCR to quantify the levels of Ha-CalpM in whole juvenile claw muscles. These expression levels were not significantly different between cutter and crusher claws, but were positively correlated with the expression of fast myosin heavy chain. The anatomical localization of Ha-CalpM near motor endplates, coupled with the correlation with fast myofibrillar gene expression, suggests a role for this intracellular proteinase in fiber type switching.

Cloning of a muscle-specific calpain from the American lobsterHomarus americanus: expression associated with muscle atrophy and restoration during moulting

A cDNA (1977 bp) encoding a crustacean calpain (Ha-CalpM; GenBank accession no. AY124009) was isolated from a lobster fast muscle cDNA library. The open reading frame specified a 575-amino acid (aa) polypeptide with an estimated mass of 66.3 kDa. Ha-CalpM shared high identity with other calpains in the cysteine proteinase domain (domain II; aa 111-396) and domain III (aa 397-575), but most of the N-terminal domain (domain I; aa 1-110) was highly divergent. Domain II contained the cysteine, histidine and asparagine triad essential for catalysis, as well as two conserved aspartate residues that bind Ca(2+). In domain III an acidic loop in the C2-like region, which mediates Ca(2+)-dependent phospholipid binding, had an expanded stretch of 17 aspartate residues. Ha-CalpM was classified as a non-EF-hand calpain, as it lacked domain IV, a calmodulin-like region containing five EF-hand motifs. Northern blot analysis, relative reverse transcription-polymerase chain reaction (RT-PCR) and real-time PCR showed that Ha-CalpM was highly expressed in skeletal muscles, but at much lower levels in heart, digestive gland, intestine, integument, gill, nerve cord/thoracic ganglion and antennal gland. An antibody raised against a unique N-terminal sequence recognized a 62 kDa isoform in cutter claw and crusher claw closer muscles and a 68 kDa isoform in deep abdominal muscle. Ha-CalpM was distributed throughout the cytoplasm, as well as in some nuclei, of muscle fibers. Purification of Ha-CalpM showed that the 62 kDa and 68 kDa isoforms co-eluted from gel filtration and ion exchange columns at positions consistent with those of previously described Ca(2+)-dependent proteinase III (CDP III; 59 kDa). Ha-CalpM mRNA and protein did not change during the moulting cycle. The muscle-specific expression of Ha-CalpM and the ability of Ha-CalpM/CDP III to degrade myofibrillar proteins suggest that it is involved in restructuring and/or maintaining contractile structures in crustacean skeletal muscle.

Tubular localization of silent calcium channels in crustacean skeletal muscle fibers.

Ca2+-induced Ca2+ release (CICR) in the superficial abdominal flexor muscle of the crustacean Atya lanipes appears to be mediated by a local control mechanism similar to that of vertebrate cardiac muscle, but with an unusually high gain. Thus, Ca2+ influx increases sufficiently the local concentration of Ca2+ in the immediate vicinity of the sarcoplasmic reticulum Ca2+ release channels to trigger the highly amplified release of Ca2+ required for contraction, but is too low to generate a macroscopic inward current (i.e., the Ca2+ channels are silent). To determine the localization of the silent Ca2+ Channels, the mechanical, electrophysiological and ultrastructural properties of the muscle were examined before and after formamide treatment, a procedure that produces the disruption of transverse tubules of striated muscle. We found that tubular disruption decreased tension generation by about 90%; reduced inward current (measured as Vmax, the maximum rate of rise of Sr2+ action potentials) by about 80%; and decreased membrane capacitance by about 77%. The results suggest that ca. 80% of the silent Ca2+ channels are located in the tubular system. Thus, these studies provide further evidence to support the local control mechanism of CICR in crustacean skeletal muscle.

Physiology and excitation-contraction coupling in the intestinal muscle of the crayfish Procambarus clarkii.

The intestinal muscles of Procambarus clarkii are striated and yet they are specialized to produce slow peristaltic waves of contraction, not unlike those seen in vertebrate visceral smooth muscle. These muscles cannot be tetanized either by repetitive stimulation or by elevated potassium saline. The excitation-contraction (E-C) coupling mechanism was explored and compared with that known in crustacean skeletal muscle. Contraction is dependent on external Ca2+ which triggers the release of intracellular calcium from the sarcoplasmic reticulum (SR) via calcium-induced calcium release (CICR). Whereas contraction force is proportional to [Ca2+]o up to that in normal saline (13.4 mM), higher levels of Ca2+ reduce force. Ryanodine, which blocks calcium release from the SR, abolishes electrically stimulated contractions and CICR. Relaxation is achieved by removal of calcium from the cytosol in at least two ways, first by the re-loading of calcium into the SR by Ca2+-ATPases and second by the movement of calcium out of the cell by extruding it across the sarcolemma via Na+/Ca2+-exchangers. It is hypothesized that the inability of this muscle to show tetanus arises from inactivation of the voltage-gated calcium channels by high calcium. This is supported by the result that caffeine application causes an increase in tonus and size of phasic contractions by circumventing the sarcolemma and dumping SR calcium stores.

Muscle Restructuring in Crustaceans: Myofiber Death, Transfiguration and Rebirth

Research on the dimorphic claws of the snapping shrimp Alpheus has revealed moult-associated changes in structure and biochemical composition—including atrophy and biochemical modification—of claw muscle fibers during morphological transformation of a claw from a pincer to a snapper. Electrophysiology, SDS-PAGE gel electrophoresis, and immunocytochemistry were used to analyze changes in claw closer muscle function and composition during the transformation process. Remodification of closer muscle during claw transformation, involving the complete loss of a central section of fast-contracting fibers and their replacement through enlargement of existing slowly-contracting segments of the muscle, may mimic similar muscle modifications during initial claw development. Exposure of intact animals to environmental ecdysteroid hormones accelerated the rate of these changes. These processes appear to be a product of a remarkable trophic plasticity of crustacean skeletal muscle first discovered by Skinner.

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Using the whole cell voltage-clamp technique and a Cl free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 ± 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of I Ba could be detected (exogenous I Ba). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 ± 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous I Ba could be divided into two components: a “fast component” and a “slow component” probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous I Ba was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.

Expression of calcium channels in Xenopus oocyte injected with crab skeletal muscle fibre mRNA

Using the whole cell voltage-clamp technique and a C1 free and Na free Ba methane sulfonate solution, stage V and VI Xenopus oocytes demonstrated a Ba current (endogenous component) with a peak amplitude average of 6 nA (6 +/- 2 nA). When oocytes were injected with crustacean skeletal muscle mRNA, an additional component of IBa could be detected (exogenous IBa). The latter current could be distinguished from the native one by several electrophysiological means: a peak amplitude average of 90 nA (90 +/- 4 nA), activation potential threshold, steady state inactivation properties and sensitivity to Ca blockers. As shown by Jdaïâa and Guilbault in crustacean skeletal muscle fibres, exogenous IBa could be divided into two components: a "fast component" and a "slow component" probably passing through two types of Ca channels (fast and slow) since the peak Ba current voltage relationship was biphasic and the fast component of exogenous IBa was less sensitive than the slow to nifedipine. The features of the newly synthesized channels incorporated in the Xenopus oocyte membrane suggest that they may be associated with fast and slow channels, previously described in many preparations, particularly in crustacean skeletal muscle fibres.

Morphological Fiber Type Correlates of Physiological and Biochemical Properties in Crustacean Muscle

SYNOPSIS. Morphological fiber typing is an attempt to group functionally related skeletal muscle fibers based on similarity of structure. Such grouping should bring a functional order or classification, based on structure, to a diverse set of skeletal muscle fibers which are each specialized so that the organism can produce a wide range of forces and movements. Fiber typing using morphological criteria is an attempt to identify a group of similarly performing fibers by simple visual inspection without the need for physiological measurement on each and every cell. If the typing system is unambiguous the system can then be used to study plasticity, maintenance, and development of particular functional/ biochemical modifications. In such a case, the method may predict both function and perhaps even alterations occurring in fiber type properties. If on the other hand, the typing methods are somewhat more ambiguous, such that intermediate characteristics are difficult to interpret with relation to functional or biochemical alteration the typing loses its usefulness as a predictive tool for the study of regulatory events controlling maintenance and plasticity of the fibers. In the vertebrate case, particularly for mammalian and avian systems, morphological fiber typing is a potent tool for studying regulation of muscle fiber properties. Classical histochemical fiber typing based on the combination of myofibrillar ATPase, oxidative enzyme activities, and glycogen content predict physiological function fairly well. These methods have been improved and substantiated in large measure by immunohistochemical procedures based on differences in biochemical isozymes of both contractile and sarcoplasmic reticulum proteins between fast and slow contracting fibers. Additionally, electron microscopic evaluation of organelle content also separates fibers in accord with their physiological performance. Thus the methodology for typing muscle fibers morphologically is well-developed and correlates well with biochemical and physiological performance, and therefore is used extensively to study muscle fiber development and plasticity. Crustacean skeletal muscle also shows a wide range for single muscle fiber performance, and this performance can be matched to morphological type generally. Morphological typing in crustacean systems rests on two major differences between the extremes on the performance continuum. Slow contracting fibers have long sarcomere lengths and low myosin ATPase activities, while very fast contracting fibers have short sarcomere lengths and high myosin ATPase activities. These methods, while useful in the majority of cases, unfortunately do not always accurately predict physiological performance of every fiber typed into a particular group. This makes the methodology particularly difficult to use in studies attempting to predict changes from an individual fiber adapted to one type of performance into a different type of fiber. The ambiguity therefore has not allowed morphological typing by itself to be as useful a tool in studying the properties which maintain fiber type in crustacean systems. Thus in these cases morphological typing must be confirmed using physiological measurement for each muscle fiber. Recent work indicates that crustacean fibers may be more accurately typed using immunohistochemical methods based on specific biochemical differences found in muscle fibers. Such methodology is commonly in use for defining mammalian muscle fiber types, and now appears to be a useful tool for typing of crustacean fibers. Typing of protein isoforms should not be as ambiguous as some of the classic methods for crustacean muscle fiber typing, and may be the tool which allows further understanding of fiber development, plasticity, and maintenance in crustacean systems.

Calcium channels and excitation-contraction coupling of crustacean skeletal muscle fiber

1. 1. The simultaneous recordings of membrane potential, E m, and contraction produced by a sudden increase in the external K concentration show, in presence of TEA, two phases in the contraction and an overshoot in E m, the overshoot appearing only when Ca 2+ ions were present in extracellular solution. 2. 2. Time course of E m and contraction were not changed when Na + ions were replaced by Li + ions; only the amplitude of the contractions was reduced by a factor of about 0.5. This reduction probably corresponded to a blockage of the Na-Ca exchange mechanism. 3. 3. In the presence of Mn 2+ ions (10 mM) and Li + ions, known to block the inward Ca current and Na-Ca exchange respectively, the second component of contraction persisted. It was associated with the overshoot which was somewhat reduced but longer than in normal conditions. The same was obtained in Mn-free solution containing Sr 2+ ions instead of Ca 2+ ions. 4. 4. Results suggest that the diphasic contraction could be dependent on two Ca inward currents (fast current, I Ca1 and slow current, I Ca2); these two inward currents flowing by two kinds of Ca channels in crustacean muscle fibre (Zahradnik and Zachar, 1982, Gen. Physiol. Biophys. 1, 457–461; Jdaïâa and Guilbault, 1986, Gen. Physiol. Biophys. 5, 3–16). 5. 5. In the presence of Sr 2+ ions and in the absence of Ca 2+ ions or in the presence of Mn 2+ ions and Ca 2+ ions, as Ica ca1, the first component of contraction was lost, which suggests that the fast Ca channel is more selective to Ca 2+ ions than the slow Ca channel. 6. 6. To conclude, in crab muscle fibre, the contraction recorded in our normal conditions i.e. in the presence of Na + ions and Ca 2+ ions, could correspond to three components elicited by three Ca movements: two movements of Ca corresponding to I Ca1 and I Ca2 and the last corresponding to Na-Ca exchange.

Octopamine action on the contractile system of crustacean skeletal muscle.

1. In the opener muscle of walking legs of crayfish (Astacus leptodactylus) octopamine (OA) greatly enhances the contractions resulting from brief applications of L-glutamate or of elevated K-concentrations. Synephrine is as effective as OA. 2. In the case of potentiation of responses to high-K applications a presynaptic component of the OA action was excluded by first desensitising the muscle fibres to the action of the natural transmitter, using a high concentration (1 mM) of glutamate. 3. The Ca-antagonists Co, Ni and Mn (1 mM) reduced the effects of glutamate and of elevated K to about one-half. In preparations treated with OA, the same Ca-antagonists also depressed the potentiated contractural responses to glutamate and to elevated K, again to about one-half. 4. OA also enhanced contractions resulting from the application of caffeine. 5. With 5-hydroxytryptamine (5-HT) application, the same postsynaptic effects were obtained as described for OA, except that the 5-HT actions were much weaker. 6. With OA, maximal effects were obtained with concentrations of 5 x 10(-6)-10(-5) M; maximally effective concentrations of 5-HT were around 10(-5) M. 7. The lowest effective concentrations of OA were around 10(-8) M; those of 5-HT were around 10(-7) M. 8. In the same preparation, 5-HT is far more effective in enhancing transmitter release (presynaptic action) than OA, the lowest effective concentration being around 10(-11) M while no presynaptic effects of OA were seen at concentrations below 10(-8) M, in some cases even below 10(-5) M.

The innervation pattern of crustacean skeletal muscle. Morphometry and ultrastructure of terminals and synapses.

The innervation pattern of distal muscle fibers of the opener muscle of walking legs of crayfish (Astacus leptodactylus) was investigated using methylene-blue staining, cobalt infiltration, and electron microscopy. A quantitative analysis of the entire innervation of single muscle fibers was attempted. It was found that instead of the generally assumed parallel array of numerous excitatory and inhibitory terminals, innervation consists of only a few branched terminals. The branches of excitatory and inhibitory terminals lie side-by-side. Both types are characterized by numerous varicosities (see Fig. 9B). The aggregate length of excitatory as well as inhibitory terminals on one muscle fiber is, on the average, about 1,500 micrometer with a total of 152 varicosities spaced about 10 micrometer apart. The average diameter of the varicosities is 4.26 micrometer, that of the connecting thin segments about 0.5 micrometer. Total terminal surface of motor or inhibitory terminals amounts to about 10,000 micrometers2 per muscle fiber. There are approximately 2,000 motor synapses on each muscle fiber, but their average total area is only about 6% of the terminal membrane area, or 0.06% of the (idealized) muscle fiber surface. There are conspicuous differences in the postsynaptic specializations associated with excitatory and inhibitory terminals; these are described in detail. The results are discussed in a functional context and with regard to design and results of electrophysiological experiments.

Toxicité de l'étain: Action sur les propriétés électriques et mécaniques de la fibre musculaire squelettique

The toxicity of tin on the excitation-contraction coupling of skeletal muscle fibers was studied in “nonpolluted” and in “polluted” crabs. In fibers obtained from “non-polluted” crabs living in artificial seawater (ASW): (i) the resting membrane potential of muscle fibers decreased irreversibly from the control value of −67.0 ± 3.1 to −59.8 ± 3.9 mV after 10 min of exposure to tin (10 −4 m); (ii) the action potential rapidly (5 min) and irreversibly changed from an all-or-none type to a graded response in the presence of tin; (iii) the twitch tension decreased in 4 min from a mean value of 0.92 ± 0.29 kg·cm −2 in ASW to a new value of 0.50 ± 0.32 kg·cm −2 in ASW + tin. The crabs which were housed in ASW + tin did not survive beyond 15 days. In the fibers from these “polluted” crabs: (i) the resting membrane potential fell to −59.6 ± 2.4 mV after 18 hr of exposure of the crabs to ASW + tin; the value after 24 hr was −51.5 ± 3.9 mV and the membrane potential was stable at that value until the ninth day of life in ASW + tin; (ii) the action potentials were all of the graded type after 18 hr in ASW + tin; (iii) the twitch tension was 0.61 ± 0.21 kg·cm −2 after 24 hr. It was still 0.55 ± 0.30 after 9 days. The potassium contracture decreased from the control value of 2.7 ± 0.1 to 0.97 ± 0.52 kg·cm −2 after 4 days of life in ASW + tin. At the concentration of 10 −4 m, tin is a toxic lethal substance which depresses electrical and mechanical activity of the crustacean skeletal muscle fiber.

Effect of changing the composition of the bathing solutions upon the isometric tension-pCa relationship in bundles of crustacean myofibrils.

1. The relative isometric tension-pCa relationship has been determined for isolated bundles of barnacle myofibrils under a variety of ionic conditions using [Ca(2+)]-buffered solutions which also contained an ATP regenerating system (creatine phosphate and creatine kinase).2. The results are in better agreement with the ;consecutive' scheme of reaction rather than with the ;independent' alternative (Ashley & Moisescu, 1972) for the co-operative action of two Ca(2+) ions in the process of tension activation in crustacean skeletal muscle.3. Variations in the pH of the activating solutions did have a marked effect on the relative tension-Ca curve, although no effect was observed on the absolute maximum value for isometric tension. A shift in pH by 0.5 u. in the range 6.6-7.6 shifted the Ca(2+)-activation curve by 0.5 log u. towards lower free Ca(2+) concentrations.4. Changes in the free Mg(2+) concentration of the activating solutions in the millimolar range produced a pronounced shift of the relative tension-pCa curve along the pCa axis. Increasing [Mg(2+)] from 1 to 5 mM shifted the curve by about 0.7 log u. to higher free Ca(2+) concentrations, without significantly modifying its steepness.5. Changes in the MgATP concentration of the activating solutions in the range of 1-13 mM had no significant effect on the relative tension-pCa relationship.6. Varying the K(+) concentration in the activating solutions was also observed to have a marked effect upon the tension-pCa relationship in barnacle. An increase in the K(+) concentration from 90 to 170 mM shifted the curve by some 0.6 log u. towards higher free Ca(2+) concentrations.7. Cooling the standard activating solutions from room temperature to +4 degrees C made no apparent difference to the relative tension-pCa relationship, but decreased significantly the absolute tension responses.8. The results presented show that tonicity by itself has a marked effect upon the absolute steady-state tension levels in isolated bundles of myofibrils.9. Maximum isometric tension in this preparation was not simply related to ionic strength, or to the monovalent cation concentration, but it depended, as well, upon the anionic composition of the activating solution. In addition, a change in ionic strength of 25 mM over the range of 245-270 mM did not appear to modify the relative tension-pCa relationship.10. The effect of the physiologically occurring cations H(+), K(+), Mg(2+) upon the relative isometric tension-pCa relationship can be accounted for on the basis of a model of competitive inhibition between these cations and Ca(2+) for the functional unit for tension. This inhibitory effect appears to involve at least one H(+), one Mg(2+) and two K(+) per each Ca(2+) ion participating in the activation process of the functional unit for tension.

The effect of some organophosphorous and organochlorine compounds on calcium uptake by Sarcoplasmic reticulum isolated from insect and crustacean skeletal muscle

Relatively pure mitochondrial-free Sarcoplasmic reticulum (SR) was isolated from crab and cockroach skeletal muscle, and the SR calcium uptake was studied in control and insecticide-treated conditions by incubation in Ca45 media followed by millipore filtration and counting by liquid scintillation methods. All insecticides used (parathion, DDT, γBHC and tri-cresyl phosphate) at 1 mM concentrations caused a slight fall in the intrinsic Ca2+ content of the SR, but this Ca2+ loss was insufficient to explain the contracture induction effect of these agents.

The effect of caffeine and quinine on calcium uptake by sarcoplasmic reticulum isolated from crustacean skeletal muscle in relation to the disruption of excitation-contraction coupling

Calcium uptake of sarcoplasmic reticulum (SR) isolated fromCarcinus skeletal muscle was examined under control conditions and in the presence of 5 mM quinine and 10 mM caffeine. Quinine was without significant effect on SR intrinsic calcium but caffeine caused a release of about 17% of this calcium compartment. Both alkaloids depressed ATP-promoted calcium binding by the SR, caffeine inducing a greater depression of binding. In addition, both alkaloids were able to release calcium from previously loaded SR. It is concluded that caffeine-induced release of intrinsic calcium may contribute to the rise in myoplasmic calcium associated with caffeine contracture, but caffeine action on SR labile calcium is the major basis for contracture-induction. In the case of quinine, electron microscopic evidence suggests that this drug acts on the mitochondrial calcium compartment of the muscle fibre as well as on the SR.

The effect of parathion on crustacean skeletal muscle—I. The mechanical threshold and dependence on Ca 2+ ions

1. 1. The threshold for potassium-induced contractures in Carcinus skeletal muscle is 70 mM, tension being maximal above 100 mM KCl. 2. 2. Parathion, at a concentration of 0·5 mM, induced irreversible tonic contractures, these contractures being enhanced to well beyond peak tetanus tension by increasing external K +. 3. 3. In parathion-containing salines, the mechanical threshold of the muscle fibres was considerably lowered, but unlike the caffeine effect tension was always generated, even at low K + levels. 4. 4. The parathion/K + contractures were reduced at low and at very high Ca 2+ levels, although measureable tension was still developed in zero Ca 2+ salines. Potassium-induced contractures were abolished at low Ca 2+ levels but were not reduced at very high Ca 2+ levels. 5. 5. Parathion does not appear to activate muscle by a transient depolarization, and although it modifies the mechanical threshold its major action appears to be on the internal calcium control system of the muscle fibre.

The effect of parathion on crustacean skeletal muscle—II. Disruption of excitation-contraction coupling

1. 1. Parathion induces tonic contractures in crab skeletal muscle. These contractures are irreversible at concentrations greater than about 100 μM. 2. 2. The contracture-induction effect was examined in terms of disruption of the electromechanical coupling process at a number of possible stages, (a) membrane depolarization, (b) the reticular calcium control system, (c) actomyosin cross bridge formation and (d) structural changes in the fibres. 3. 3. It has been established that parathion does not activate muscle by a transient depolarization, but it does significantly depress reticular calcium uptake. In addition, parathion greatly modifies actomyosin superprecipitation, and fine-structural studies show great disruption of reticular organization, suggestive of massive calcium loss from the reticulum into the myoplasm.

Fine structure of a neurosecretory axon in a crustacean skeletal muscle

Un axone neuro-secreteur a extremites localisees dans le muscle squelettique du craneCarcinus est decrit. Ces extremites contiennent 6 sortes de vesicules ayant les diametres de 1300 a 3700 A. Il est suggere que es petites vesicules correspondent a la phase de retablisement de la membrane, pendant la decharge des granules euro-secreteurs. On suppose aussi que cet axone peutetre l'intermediaire d'une influence trophique du nerf sur le muscle.