Amphibian Skeletal Muscle Fibres Research Articles

Cardiac glycosides inhibit detubulation in amphibian skeletal muscle fibres exposed to osmotic shock.

It has recently been suggested that the 'vacuolation' of the transverse tubular system that follows the imposition of an osmotic shock is a component process in the eventual 'detubulation' of amphibian skeletal muscle. However, such a hypothesis requires net fluid transfers from the intracellular space into the lumina of the transverse tubules against the prevailing transmembrane osmotic gradients. The present experiments tested the effects of cardiac glycosides on the consequences of established osmotic protocols known reliably to achieve high levels of both detubulation and vacuolation in Rana temporaria sartorius muscle. Tubular isolation (detubulation) was assessed through electrophysiological observations of the abolition or otherwise of the after-depolarisation components of muscle action potentials. Vacuolation was assessed by stereological estimation of the volume fraction of muscle that was occupied by fluorescence-labelled vacuoles observed using confocal microscopy. Introduction of ouabain in the osmotic shock solutions sharply reduced such measures of vacuolation from 48.5 +/- 3.6% (mean +/- SEM; n = 70) to 12.1 +/- 2.7% (n = 190) of the total fibre volume. This was accompanied by sharp reductions in the incidence of detubulation (detubulation index reduced from 96.3 +/- 2.6% to 0.0 +/- 0.0%). The presence of ouabain was critical at the osmotic shock stage in the procedures at which the hypertonic glycerol-containing solutions were replaced by isotonic Ca(2+)-Mg(2+)-Ringer solutions. Finally, the alternative cardiac glycosides, strophanthidine and digoxin, exerted similar effects. These findings support a scheme in which the osmotic shock initiates a metabolically dependent fluid expulsion. This distends the transverse tubules into vacuoles that in turn lead to fibre detubulation.

The influence of perchlorate ions on complex charging transients in amphibian striated muscle.

1. The effects of perchlorate ions on intramembrane charge movements were examined under different conditions of ryanodine receptor (RyR) modification in intact voltage-clamped amphibian skeletal muscle fibres studied in the gluconate-containing solutions previously reported to emphasize the features of q gamma at the expense of those of the q beta charge. 2. The introduction of graded increases in perchlorate concentration to the experimental solutions selectively shifted the threshold of appearance of the q gamma 'hump' currents to more negative test potentials at which they actually appeared in the absence of prior q beta transients at perchlorate concentrations of 4.0-8.0 mM. Such findings suggested that the delayed (q gamma) transitions can take place independently of any previous exponential (q beta) decay. 3. These kinetic effects were accompanied by hyperpolarizing shifts in the transition potentials (V*) of the steady-state voltage dependences of either the overall or the isolated q gamma charge. These shifts were graded with concentration and reached their maximum effects at 4.0-8.0 mM perchlorate. However, both the total charge (Qmax) and the steepness factor (k) remained conserved at values consistent with a system that included significant contributions from the steeply voltage-sensitive q gamma component (overall charge: Qmax approximately 19-21 nC microF-1, k approximately 7-9 mV; q gamma component alone: Qmax approximately 10-12 nC microF-1, k approximately 4-6 mV). This contrasts with earlier reports on the effects of perchlorate in fibres that were studied in sulphate- or methanesulphonate-containing extracellular solutions. 4. Perchlorate (8.0 mM) restored the 'hump' waveform associated with q gamma charge movements that had previously been obliterated by the prior application of fully effective (0.1 mM) concentrations of either ryanodine or daunorubicin. 5. Perchlorate similarly reversed the positive shift in the transition potential of the q gamma component that was brought about by such RyR modification (from V* approximately -40 mV back to V* approximately -60 mV). In contrast, the values of either Qmax (overall charge, 19-21 nC microF-1; q gamma component, 10-13 nC microF-1) or k (overall charge, 7-9 mV; q gamma component, 4-6 mV) remained conserved through all these experimental manoeuvres. 6. The inclusion of perchlorate also reversed the action of 2 mM tetracaine and restored delayed q gamma transients to an extent that was graded with concentration (0.5-8.0 mM perchlorate). There was an accompanying recovery of the steeply voltage-dependent steady-state (q gamma) component consistent with a competitive interaction between these agents upon the q gamma intramembrane charge. 7. The present findings suggest that perchlorate exerts a specific action upon the q gamma charge in independent transitions that are driven by the tubular membrane field. Its interactions with the known RyR inhibitors that nevertheless conserve both the charge and its voltage sensitivity suggest a primary action upon the RyR that in turn exerts reciprocal actions upon the voltage sensor.

The effect of temperature on charge movement repriming in amphibian skeletal muscle fibers.

Cut twitch muscle fibers, mounted in a triple Vaseline-gap chamber, were used to study the effects of temperature on intramembranous charge movement and, in particular, on the repriming of charge 1 (the intramembranous charge that normally moves in the potential range between -100 and +40 mV). Changing the holding potential from -90 to 0 mV modified the voltage distribution of charge movement but not the maximum movable charge. Temperature changes between 16 and 5 degrees C did not modify the fiber linear capacitance, the maximum nonlinear intramembranous charge, or the voltage distribution of charge 1 and charge 2 (the intramembranous charge moving in the membrane potential range between approximately -4 and -160 mV). We used a pulse protocol designed to study the repriming time course of charge 1, with little contamination from charge 2. The time course of charge movement repriming at 15 degrees C is described by a double exponential with time constants of 4.2 and 25 s. Repriming kinetics were found to be highly temperature dependent, with two rate-limiting steps having Q10 (increase in rate of a process by raising temperature 10 degrees C) values of 1.7 and 7.1 above and below 11.5 degrees C, respectively. This is characteristic of processes with a high energy of activation and could be associated with a conformational change of the voltage sensor or with the interaction between the voltage sensor and the calcium release channel.

Ryanodine interferes with charge movement repriming in amphibian skeletal muscle fibers

Cut twitch muscle fibers mounted in a triple Vaseline-gap chamber were used to study the effects of ryanodine on intramembranous charge movement, and in particular on the repriming of charge 1. Charge 1 repriming was measured either under steady-state conditions or by using a pulse protocol designed to study the time course of repriming. This protocol consisted of repolarizing the fibers to -100 mV from a holding potential of 0 mV, and then measuring the reprimed charge moving in the potential range between -40 and +20 mV. Ryanodine at a high concentration (100 microM) did not affect the maximum amount of movable charge 1 and charge 2, or their voltage dependence. This indicates that the alkaloid does not interact with the voltage sensor molecules. However, ryanodine did reduce the amount of reprimed charge 1 by approximately 60% suggesting the possibility of a retrograde interaction between ryanodine receptors and voltage sensors.

Dose-dependent activation and block by bisG10, a K + channel blocker, of mouse and frog skeletal muscle K ATP channels

The effects of a K + channel blocker, bisG10, were examined on ATP-sensitive K + (K ATP) channels in membrane patches excised from mammalian and amphibian skeletal muscle fibres using the patch-clamp technique. At micromolar concentrations, bisG10, added on the intracellular side, induced a strong, reversible, flickery block of K ATP channels. BisG10, added on the extracellular side, was about 100-fold less potent at inhibiting channel activity. At 10 nM, intracellular bisG10 increased K ATP channel activity. This activation was independent of the presence of internal ATP or Mg 2+. The inhibitory effect of bisG10 most likely arose from open-channel block whereas activation could result from more complex, indirect interactions.

Ultrastructure of sarcoballs on the surface of skinned amphibian skeletal muscle fibres.

The formation of sarcoballs on the surface of skinned fibres from semitendinosus muscles of Xenopus laevis, and the sarcoplasmic reticulum content of the structures, have been studied using conventional electron microscopic techniques and immunoelectron microscopy. Examination of the fibres showed many membrane-bound blebs projecting from the surface in areas where vesicles of internal membranes (including sarcoplasmic reticulum, T-tubules and mitochondria) were clustered in interfilament spaces. The blebs varied in size from 1 micron to 150 microns and those with diameters > 10 microns are referred to as sarcoballs. Small blebs were often seen in close association with each other and might have fused during sarcoball formation. The interior of the sarcoball was filled with foam-like material made up of vesicles with diameters of 100 nm to 1.0 microns. The sarcoplasmic reticulum membrane content of the sarcoballs was evaluated using two monoclonal antibodies, one to the Ca2+ ATPase of the sarcoplasmic reticulum and the second to ryanodine receptor calcium release channels in the junctional-face membrane. The antibodies bound to some components of the surface and interior of the sarcoball, but not to mitochondrial-like structures and tubular vesicles. The results show that a large component of the sarcoball and its surface is derived from sarcoplasmic reticulum and suggest that mitochondria and T-tubules might also contribute membranes to the structures. Our hypothesis is that (a) blebs bud out from the surface of the skinned fibre following fusion of internal vesicles that are extruded along interfilament channels during unrestrained contractures, (b) blebs grow into sarcoballs by additional fusion of internal membrane vesicles and fusion of adjacent blebs, and (c) the sarcoball is a foam-like structure composed of bathing medium and membrane lipid (containing membrane proteins).

Heterogeneity of T-tubule geometry in vertebrate skeletal muscle fibres.

Average dimensions of transverse tubules were obtained from electron micrographs of thin sections of mammalian and amphibian skeletal muscle fibres and the effect of transverse tubule geometry on the electrical characteristics of the fibres has been considered. The preparations examined were toad sartorius, mouse soleus, rat extensor digitorum longus, soleus and sternomastoid muscles. The T-tubule dimensions varied considerably between the different preparations and the average volume to surface ratio of the transverse tubule in amphibian fibres (8.1 nm) was generally greater than that in mammalian fibres (3.0-6.2 nm). The small volume to surface ratio of the mammalian transverse tubule would tend to reduce the electrical space constant of the transverse tubule system and reduce the rate of cross-sectional activation of the fibres during a twitch contraction. The area of transverse tubule membrane in junctional contact with the sarcoplasmic reticulum was determined and was found to be greater in mammalian fibres than in amphibian fibres. The relative areas of junctional contact, along a unit length of transverse tubule, were the same in rat extensor digitorum longus and soleus fibres.

Indentations in the terminal cisternae of amphibian and mammalian skeletal muscle fibers

Indentations (hillocks and dimples) in the terminal cisternae of mammalian and amphibian skeletal muscle fibers were studied using freeze-fracture and serial thin-section techniques. The structures were seen in all muscles and had a regular separation from each other and from the T-tubule. Indentations were smaller than fenestrations and formed concavities in, but not macromolecular pores through, the terminal cisternae. The average numbers of indentations in rat muscles (measured along the length of the terminal cisternae, within 150 nm of the triadic junction) varied from 0.9 per micrometer in soleus fibers to 9.6 per micrometer in posterior cricoarytenoid fibers. The average numbers in amphibian sartorius fibers varied from 1.6 to 3.6 per micrometer in muscles from different species. The regular alignment of the indentations along the triad, as well as a close correlation between their numbers and the contractile properties of the muscle, suggest that they function in contractile activation and may represent sites of calcium release from the terminal cisternae.

Branched skeletal muscle fibers not associated with dysfunction.

We report the occurrence of large clefts and branches in some amphibian vertebrate skeletal muscle fibers. Such fibers can be isolated intact from various skeletal muscles of fully mature animals and apparently are normal in other respects. Electron micrographs showed no signs of degeneration in most fibers. Electrical and mechanical studies revealed that all branches were excitable and able to contract. Branches evidently can arise in the absence of abnormal influences such as experimentally induced overload. The apparent cause may be either fusion or splitting of fibers.

Time courses of late after-potentials following tetanus or single shock in skeletal muscle fibers.

Characteristics of the late after-potential (LAP) of amphibian skeletal muscle fibers were studied in low chloride solutions. The LAP elicited by tetanus was larger in magnitude and slower in its course than the LAP elicited by single spike. The results are in quantitative agreement with predictions based on the theory that the LAP is produced by a potassium accumulation in the transverse tubular system and its time course determined by a diffusion process of the potassium from the tubular lumen into the outside.

Effects of membrane potential on mechanical activation in skeletal muscle.

The effect of subthreshold depolarization on mechanical threshold was investigated in tetrodotoxin-poisoned mammalian and amphibian skeletal muscle fibers using a two-microelectrode voltage-clamp technique. Mechanical threshold was determined with a 2-ms test pulse. The immediate effect of depolarization was inhibition of the mechanical system. The consequent increase in the test pulse threshold was linearly related to the size of the depolarization and there was, on the average, a 10% increase in threshold for a 10-mV depolarization in mammalian fibers. The duration of the inhibitory period was also related to the size of the depolarization. Inhibition was interrupted by the onset of activation (seen as a reduction in the test pulse threshold), and in rat soleus fibers this occurred within 100 ms with a 20-mV depolarization, inhibition decayed within 10 ms. The decay of activation after brief conditioning pulses was initially rapid (on the average, the test pulse threshold recovered to 80% of its control value within 1 ms) and then slow (full recovery took 100-500 ms). After long conditioning pulses, activation often decayed into a period of inhibition. When depolarization (of 20 mV or more) was maintained for several seconds, the fibers became inactivated. Rat extensor digitorum longus and sternomastoid fibers were strongly inactivated by depolarization to -40 mV and the test pulse to +40 mV did not cause contraction.

Calcium transients in isolated amphibian skeletal muscle fibres: detection with aequorin.

1. Single twitch muscle fibres isolated from frogs and toads were microinjected with the Ca2+-sensitive bioluminescent protein aequorin. The fibres contracted normally and emitted flashes of light (aequorin responses) in response to stimulation for many hours thereafter. 2. No luminescence was detected from healthy fibres at rest. 3. The aequorin diffused from the site of injection at a rate consistent with a diffusion coefficient of 5 x 10(-8) cm2/sec. 4. During trains of isometric contractions there was a progressive reduction in both the amplitude and the rate of decline of the aequorin response, an observation consistent with the theory that Ca is redistributed from sites of release to sites of sequestration under such circumstances. 5. In isometric tetani light emission continued to rise long after the plateau of force had been achieved. This and the fact that the amplitude of the tetanic aequorin response increased steeply with increasing stimulus frequency suggest that in tetani the sarcoplasmic [Ca2+] may normally be above the level required to saturate the contractile apparatus. 6. Both in twitches and in tetani the amplitude of the aequorin response increased slightly and then decreased substantially as the fibre was stretched progressively beyond slack length. 7. In potassium contractures the luminescent and mechanical responses first became detectable at about the same [K+], but for equivalent force luminescence was less intense than in twitches. The aequorin response was biphasic in solutions of high [K+]. 8. Exposure of the fibre to Ca2+-free solutions had no influence on either the mechanical or the luminescent responses in twitches. In Ca2+-free solutions tetanic aequorin responses tended not to be maintained as well as normally, suggesting that intracellular Ca stores do become somewhat depleted. 9. In twitches the amplitude of the aequorin response probably reflects the amount of Ca2+ liberated into the cytoplasm rather than a [Ca2+] in equilibrium with the myofilaments. Changes in the rate of decay of the aequorin response may reflect changes in the rate of Ca sequestration by the sarcoplasmic reticulum. 10. In K+-contractures and during the plateaus of tetani the aequorin signal changes slowly enough so that it seems unlikely that substantial gradients of [Ca2+] exist at the sarcomere level. Under such circumstances the amplitude of the aequorin response probably does reflect the [Ca2+] in equilibrium with the myofilaments.