Crayfish Skeletal Muscle Research Articles

Crayfish Skeletal Muscle Requires Both Influx of External Ca2+ and Ca2+ Release from Internal Stores for Contraction

ABSTRACT The isometric tension and membrane potential of single skeletal muscle fibres from the flexor muscle of the carpopodite in the meropodite of crayfish Procambarus clarkii (Girard) were studied to determine whether crayfish muscle contraction requires Ca2+ release from the sarcoplasmic reticulum. Contraction elicited by brief extracellular electrical stimulation was reduced by the removal of Ca2+ or by the addition of 25 μmol l−1 nicardipine in crayfish Ringer’s solution. Addition of 30 μmol l−1 ryanodine with 1mmol l−1 caffeine induced a transient contracture, the peak tension of which was 10–30% of that of the high-K+induced contracture and which declined to the pretreatment level in 20–60min. After ryanodine–caffeine treatment, 30mmol l−1 caffeine failed to induce contraction, suggesting that intracellular Ca2+ stores had been exhausted by the treatment. Extracellular electrical stimulation also failed to induce contraction after ryanodine–caffeine treatment, although the resting potential was not changed. These results suggest that Ca2+release from the sarcoplasmic reticulum, together with Ca2+ influx via nicardipine-sensitive Ca2+ channels, is essential to the contraction of crayfish leg muscle fibres after a brief membrane depolarization.

Calcium-induced calcium release in crayfish skeletal muscle.

1. Cut crayfish skeletal muscle fibres were mounted in a triple Vaseline-gap voltage clamp with the Ca(2+)-sensing dye Rhod-2 allowed to diffuse in via the cut ends. Ca2+ currents across the surface/T-tubule membranes (ICa) were recorded simultaneously with changes in myoplasmic Ca2+ concentration (Ca2+ transients). 2. Excitation-contraction coupling in crayfish skeletal muscle fibres is abolished when calcium in the extracellular solution is replaced by Mg2+. 3. The amplitude of the Ca2+ transients elicited by voltage clamp pulses closely followed the amplitude of the peak calcium currents recorded simultaneously across the surface/T-tubule membranes. This included decreases in both parameters as the pulse potential approached ECa (reversal potential for Ca2+), as well as secondary Ca2+ transients accompanying large tail calcium currents occurring upon repolarization from very large depolarizations. 4. A large contribution of sarcoplasmic reticulum (SR) Ca2+ release to the Ca2+ transients was revealed by a large decrease in the transient caused by the calcium-induced calcium release (CICR) blockers procaine and tetracaine. 5. Short pulses which interrupted the calcium current while SR Ca2+ release was in progress at high rates caused the Ca2+ transient to stop rising nearly immediately after the end of the pulse in most fibres. In about 15% of the fibres the Ca2+ transients continued to rise, albeit at a slower rate, for 10-20 ms after the end of the pulse, as if released Ca2+ was able to elicit some further Ca2+ release from the SR for a while. 6. Even with fibres displaying little sign of continued release after termination of short pulses under control conditions, procaine accelerated the decay of Ca2+ transients elicited by short pulses, indicating that continued release was taking place even as the transient was declining. 7. These results suggest that CICR in crayfish fibres is more closely controlled by a small entry of Ca2+ via surface/T-tubule membrane Ca2+ current than by a larger amount of Ca2+ released from the SR. The limited positive feedback of released Ca2+ on further Ca2+ release allows CICR to remain graded (according to ICa) rather than all-or-none.

Peptide cotransmitter potentiates calcium channel activity in crayfish skeletal muscle

The activity of 2 types of Ca2+ channels (38 and 14 pS in 137 mM Ba2+) in the plasma membrane of the crayfish tonic flexor muscle is modulated by the peptide proctolin. This peptide serves as a cotransmitter in 3 of the 5 excitatory tonic flexor motoneurons and greatly enhances tension after depolarization by the conventional neurotransmitter. Proctolin alone has no effect on these channels, but renders them capable of sustained activity following depolarization. After depolarization induces activity, 5 x 10(-9) M proctolin increases the open probability of the larger channel up to 50-fold due to a marked decrease in the mean channel closed time. There is also at least a 4-fold increase in the percentage of patches with active channels for the large channel and a 2-fold increase for the small channel. Proctolin modulation appears to occur via an intracellular messenger, possibly cAMP. The peptide's effect on channel activity is dose dependent in a manner that parallels its effect on tension. These results indicate that the activation of these channels and the resulting influx of Ca2+ into the muscle fiber play a role in the potentiation of tension in this muscle.

Characterization of DHP binding protein in crayfish striated muscle

The dihydropyridine calcium channel blocker, [ 3H]PN 200-110, binds specifically also to crayfish muscle membranes, though with a binding capacity smaller than that measured with rabbit or human skeletal muscle membranes. [ 3H]PN 200-110 binding proteins from the crayfish T-tubules were solubilized and purified on WGA Sepharose or extracted from gel. The purified protein has a molecular mass of approximately 190 kDa under nonreducing conditions and was able to transport calcium after reconstitution. Polyclonal antibodies against crayfish T-tubules enriched with purified DHP-binding protein were shown to bind to DHP-binding protein from both the crayfish and the rabbit skeletal muscle, although not with the same intensity. Electron microscopy showed the presence of ovoid particles. Our results suggest that a voltage-dependent calcium channel may be present in crayfish skeletal muscle, which is homological with the L-type calcium channel in rabbit skeletal muscle.

Tissue Specificity of Tropomyosin from the Crayfish, Cambarus clarki

The molecular heterogeneity and tissue specificity of crustacean tropomyosin were investigated, using muscle and nonmuscle tissues from the crayfish, Cambarus clarki. In muscle, three types of tropomyosin isoforms were found on two-dimensional gel electrophoresis. One of them was specific to cardiac muscle, and the other two were shared by skeletal and visceral muscles. In nonmuscle tissues, four types of isoforms were found on two-dimensional gel electrophoresis and in immunoreplica tests using an antiserum against crayfish skeletal muscle tropomyosin. Two of them were common to the muscle isoforms, but the other two were not detected in muscles. Furthermore, nonmuscle tissues contained several peculiar isoforms, the electrophoretic mobilities of which were considerably higher than those of the other isoforms mentioned above. When tropomyosin was purified from the mid-gut gland, these isoforms with high mobilities were found in the crude tropomyosin preparation. These results showed that the crayfish tropomyosin was heterogeneous and that the isoforms were distributed in a tissue-specific manner, like vertebrate tropomyosin. However, the results did not coincide with those of our previous study on horseshoe crab tropomyosin, which showed molecular heterogeneity but no tissue specificity. In view of the difference in the isoform distributions between the two major groups (Crustacea and Merostomata) of Arthropoda, the significance of the tissue specificity of tropomyosin isoforms was discussed.

Postsynaptic fall in intracellular pH induced by GABA-activated bicarbonate conductance.

Synaptic inhibition mediated by gamma-aminobutyric acid (GABA) is known to involve opening of receptor-gated chloride channels. Recent evidence indicates that these channels also show a significant permeability to the physiologically important bicarbonate anion. In all the excitable cells studied to date, the intracellular pH (pHi) is higher than would be predicted from a passive distribution of H+ ions, and consequently there is an outwardly directed electrochemical driving force for HCO3-. In the presence of CO2/HCO3- therefore, activation of GABA-gated channels could give rise to a significant efflux of bicarbonate, leading to a fall in postsynaptic pHi. We have examined the influence of GABA on pHi in crayfish skeletal muscle and we find that in the presence of CO2, GABA induces a dramatic fall in pHi which is coupled to an alkalosis at the extracellular surface. This fall in pHi and the extracellular alkalosis are attributable to a GABA-activated, picrotoxin-sensitive HCO3--conductance. In view of the sensitivity of ion channels and intracellular ion concentrations to changes in pHi, a GABA-induced postsynaptic acidosis could prove to be important in the modulation of inhibitory transmission.

238) - Effects of the substitution of Ca2+ for Sr2+ on Ca2+ uptake and release by isolated sarcoplasmic reticulum and on excitation-contraction coupling in crayfish skeletal muscle

238) - Effects of the substitution of Ca2+ for Sr2+ on Ca2+ uptake and release by isolated sarcoplasmic reticulum and on excitation-contraction coupling in crayfish skeletal muscle

Lipid composition of isolated external and internal skeletal muscle membranes

1. 1. Total lipids, phospholipids and cholesterol were quantitatively determined in sarcolemma and sarcotubular membranes isolated from rabbit and crayfish skeletal muscle. 2. 2. No interspecies differences were found in the content of total lipids, sterols and phospholipids in the sarcolemma. 3. 3. There are slight quantitative interspecies differences in the composition of the sarcotubular membranes. The total lipids and phospholipids are higher in the crayfish and the sterols in the rabbit. 4. 4. The cholesterol to phospholipid ratio was found to be very close in both animals; namely 0·67 and 0·54 in the sarcolemma of the rabbit and crayfish respectively and 0·08 and 0·1 in the sarcotubular membranes of the rabbit and crayfish respectively.

Innervation and vascular supply of the crayfish opener muscle: scanning and transmission electron microscopy.

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were employed to study the innervation and vascular supply of crayfish skeletal muscle.

Superprecipitation of actomyosin extracted from crayfish skeletal muscle: the effect of heavy metal cations, EDTA and drugs.

AbstractActomyosin from crayfish skeletal muscle will superprecipitate in the presence of ATP without added Ca++ or Mg++. EDTA totally inhibits Ca++‐induced superprecipitation of actomyosin, but in some cases Mg++ will reverse this effect. It is suggested that Mg++ and not Ca++ is a prerequisite for superprecipitation with ATP alone. EDTA only partly inhibits heavy metal‐induced actomyosin superprecipitation.Heavy metal cations induce far greater actomyosin superprecipitation than Ca++, and they can even enhance previously formed Ca++‐induced precipitates. It is suggested that heavy metal cations may be more strongly bound to crayfish actomyosin than Ca++, and that they may displace Ca++ from existing actomyosin binding sites. Heavy metal potentiation of muscular contraction may thus involve direct action at myofibril binding sites.Caffeine and quinine are without effect either on the rate or extent of development of actomyosin precipitates in the presence of ATP, ATP plus Ca++, and ATP plus Zn++. These observations rule out any direct effect of these drugs at the myofibril level during drug‐induced potentiation of muscular contraction.

Localization of the intracellular site of action of caffeine on skeletal muscle

1. 1. Exposure of frog and crayfish skeletal muscle to 10 mM caffeine saline results in a rapid dilation of the sarcoplasmic reticular tubules. 2. 2. Prolonged exposure to caffeine leads to disintegration of the sarcoplasmic reticular membranes, and after 30 min treatment the transverse tubules rupture,' destroying the trids and dyads. 3. 3. Destruction of triads and dyads releases their bound calcium, causing protracted contractures, and the calcium pump of the sarcoplasmic reticulum is inactivated. 4. 4. Tension changes in muscles exposed to caffeine closely parallel the damage caused by caffeine to the sub-cellular structures involved in excitation-contraction] coupling.

EVIDENCE FOR ANION-PERMSELECTIVE MEMBRANE IN CRAYFISH MUSCLE FIBERS AND ITS POSSIBLE ROLE IN EXCITATION-CONTRACTION COUPLING.

Under certain conditions only, isolated crayfish skeletal muscle fibers change in appearance, becoming grainy, darkening, and seemingly losing their striations. These changes result from development of large vesicles on both sides of the Z-line. The longitudinal sarcoplasmic reticulum remains unaffected. The vesicles are due to swelling of a transverse tubular system (TTS) which is presumably homologous with the T-system tubules of other muscle fibers. The vesiculations occur during efflux of water or on reducing external K or Cl, but only when KCl can leave the fiber. They never result from osmotic, ionic, or electrical changes when KCl cannot leave. Inward currents, applied through a KCl-filled intracellular cathode, also cause the vesiculations. These are not produced when the cathode is filled with K-propionate, nor by outward or longitudinal currents. Thus the transverse tubules swell only when Cl leaves the cell. Accordingly, their membrane is largely or exclusively anion-permselective. These findings also indicate that the TTS forms part of a current loop, connecting with the exterior of the fiber probably through radial tubules (RT) possessing membrane of low conductivity. Thus, part of the current flowing inward across the sarcolemma during activity can return to the exterior through the membrane of the TTS. The structure and properties of the latter offer the possibility for an efficient electrical mechanism to initiate excitation-contraction coupling.