Amphibian Skeletal Muscle Research Articles

Effect of extracellular calcium on contractile activation in guinea-pig ventricular muscle.

The problem of whether or not the alterations in twitch tension of mammalian cardiac muscle induced by changes in extracellular Ca concentration ([Ca]o) are due to the Na-Ca exchange process across the surface membrane and/or the changes in the amount of Ca in the sarcoplasmic reticulum has been re-examined by using thin bundles (70-120 micron diameter) dissected from guinea-pig papillary muscle. The observed time course of the change in the twitch tension due to a step change in [Ca]o was compared with that computed on a basis of the diffusion process of Ca ions in a circular cylinder and of the steady-state relation between [Ca]o and twitch tension. After a sudden decrease in [Ca]o from 2 mM to various lower concentrations, the isometric twitch tension of the thin bundles first fell rapidly and monotonically and then showed a much smaller and slower secondary fall. The correspondence of the observed time course of the rapid phase with the predicted time course and the observed half-time of the rapid phase ranging from 1.0 to 2.5 s indicate that the rapidity of the twitch response may be dominated by simple diffusion of Ca ions through the extracellular space. If so, the effective diffusion constant of Ca ions inside the bundles was 1.4 +/- 0.2 X 10(-6) cm2/s (mean +/- S.E., n = 9). The magnitude and direction of the step change in [Ca]o or the change at different stimulus frequencies gave rise to dissimilar time courses of the contractile change; the difference in the rapid time courses due to these factors could be explained by the simple diffusion models, but not in the much slower phase. The half-time for the Ca effect was the same as that for the rapid effect of Na ions in the external solutions. The time course of twitch decline due to [Ca]o decrease in the Na-free (Li) solution was identical to that predicted from the time course measured in the Na-rich solution and the steady-state relation between [Ca]o and tension in the Na-free solution. The half-time of Ca leak from the sarcoplasmic reticulum in the skinned cardiac muscle was 40-60 s in the presence of 10 mM-EGTA, much shorter than that of the Ca leak in the skinned amphibian skeletal muscle, but much longer than that of twitch responses due to step changes in [Ca]o in the intact cardiac muscle.(ABSTRACT TRUNCATED AT 400 WORDS)

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.

Effects of total lymphoid irradiation in refractory rheumatoid arthritis

subunits from Torpedo californica. Biochemistry 18:4465. 28. Lindstrom, J., S. Tzartos, and B. Gullick. (1982). Structure and function of acetylcholine receptors studied using monoclonal antibodies. N.Y. Acad. Sci. 377:1. 29. Lindstrom, J. et ai. (1983). Use of monoclonal antibodies to study acetylcholine receptors from electric organs, muscle and brain and the autoimmune response to receptor in myasthenia gravis. 48th Cold Spring Harbor Symposium: Molecular Neurobiology. (in press). 30. Merlie, J. P. et al. (1979). Degradation of acetylcholine receptor in diaphragms of rats with experimental autoimmune myasthenia gravis. J. Biol. Chem. 254:6328. 31. Mochly-Rosen, C. and S. Fuchs. (1981). Monoclonal anti-acetylcholine receptor antibodies directed against the cholinergic binding site. Biochemistry 20:5920. 32. Raftery, M. et al. (1980). Acetylcholine receptor: Complex of homologous subunits. Science 208:1454. 33. Reynolds, ,I. A. and A. Karlin. (1978). Molecular weight in detergent solution of acetylcholine receptor from Torpedo californica. Biochemistry 17:2035. 34. Sargent, P. B. et al. (1984). The structure and transmembrane nature of the acetylcholine receptor in amphibian skeletal muscle as revealed by crossreacting monoclonal antibodies. J. Cell Biol. (in press). 35. Toyka, K. V. et al. (1975). MG: Passive transfer from man to mouse. Science 190:397. 36. Toyka, K. V. el al. (1977) MG: Study of humoral immune mechanisms by passive transfer to mice. N. Engl. J. Med. 296:125. 37. Tzartos, S. ct al. (1983). Demonstration of a main immunogenic region on acetylcholine receptors from human receptor. FEBS Lett. 158:116. 38. Tzartos, S. and J. 51. Lindstrom. (1980). Monoclonal antibodies used to probe acetylcholine receptor structure: Localization of the main immunogenic region and detection of similarities between subunits. Proe. Natl. Acad. Sci. USA 77:755. 39. Tzartos, S. and J. Lindstrom. (1981). Production of characterization of monoclonal antibodies for use as probes of acetylcholine receptors, p. 69. In: R. Fellows and G. Eisenbarth (eds.), Monoclonal Antibodies in Endocrine Research. New York: Raven Press. 40. Tzartos, S. et ai. (1981). Mapping of surface structure of Electrophorus acetylcholine receptor using monoclonal antibodies. J. Biol. Chem. 256:8653. 41. Tzartos, S., 5I. Seybold, and J. Lindstrom. (1981). Specificity of antibodies to acetylcholine receptors in sera from myasthenia gravis patients measured by monoclonal antibodies. Proe. Natl. Acad. Sci. USA 79:188. 42. Vincent, A. (1980). Immunology of acetylcholine receptors in relation to myasthenia gravis. Physiol. Rev. 60:757. 43. Watters, D. and A. Maelicke. (1983). Organization of ligand binding sites at the acetylcholine receptor: A study with monoclonal antibodies. Biochemistry 22:1811.

Trifluoperazine and the rapid, Ca 2+-triggered damage of skeletal and cardiac muscle

1. [Ca 2+] i was raised experimentally in mammalian and amphibian skeletal and cardiac muscles by A23187, DNP, anoxia or the Ca 2+-paradox. 2. Trifluoperazine (TFP) at 10 −5M failed to protect against the characteristic and rapid damage triggered by elevated [Ca 2+] i; in any of the preparations. 3. It is concluded that calmodulin is not implicated in this rapid ultrastructural damage. 4. TFP alone also causes identical patterns of damage. It may be acting to raise [Ca 2+] i in skeletal and cardiac muscle cells.

Vascular smooth muscle energetics.

ATP utilization (delta approximately P) during an isometric contraction has been studied in terms of both measurements of oxygen consumption and lactate production as well as of the tissue nucleotide and metabolite levels. The contribution of breakdown of preformed ATP and phosphocreatine (PCr) pools to delta approximately P during contraction is minor compared to that made by metabolic synthesis of ATP. For tonic vascular smooth muscle (VSM), in fact, no change in ATP or PCr from resting levels can be measured. In contrast to amphibian skeletal muscle, a P:O of 3 can be demonstrated in VSM. In both tonic and phasic VASM, delta approximately P is biphasic with contraction duration, attaining a maximal value before that of isometric force and declining to a steady-state value approximately 60% of the maximal suprabasal rate during the maintenance of constant isometric force. The steady-state rate of ATP utilization per unit force maintained increases with extracellular Ca2+. Both the pre-steady-state temporal dependence and the steady-state dependence on Ca2+ are consistent with the hypothesis that myosin phosphorylation modulates the cross-bridge cycle rates. VSM metabolism, when viewed in terms of ATP synthesis, is primarily oxidative. However, even under fully oxygenated conditions, lactate is the major end product of glucose catabolism. Recent work has shown that aerobic lactate production is specifically coupled to Na-K transport in many, but not all, vascular tissues. Oxidative metabolism, on the other hand, is strongly related to active isometric force. The biochemical basis of this functional compartmentation was investigated at the level of substrate specificity.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

Sarcoplasmic reticulum of mechanically skinned slow fibers of amphibian skeletal muscle

Properties of the Ca uptake and the Ca release mechanisms of the sarcoplasmic reticulum (SR) of slow tonic fibers were studied and were compared to those of fast twitch fibers. Single fibers were prepared from iliofibularis muscle of Xenopus laevis, and skinned mechanically. The amount of Ca in the SR was estimated from the magnitude of contracture induced by application of a high dose of caffeine in the presence of an appropriate concentration of EGTA. All experiments were done at 4°C.

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.

O-97 - Properties of the contractile system of skinned slow fibers of amphibian skeletal muscle

O-97 - Properties of the contractile system of skinned slow fibers of amphibian skeletal muscle

Isometric contractile properties and instantaneous stiffness of amphibian skeletal muscle in the temperature range from 0 to 20 degrees C.

The isometric contractile properties of frog (Rana pipiens) and toad (Bufo bufo) sartorii have been studied over the temperature range from 0 to 20 degrees C. The isometric twitch tension was found to vary considerably between these two species and between muscles in the same species. Between 0 and 4 degrees C there was very little change in maximum isometric twitch tension. Between 4 and 12 degrees C several muscles from frog or toad showed a potentiation of twitch tension whereas others showed a decline. Over this temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature range the toad sartorii consistently demonstrated a greater potentiation. By 12 degrees C a steady decline in twitch tension in both muscles was seen as the temperature approached 20 degrees C. The maximum isometric tetanic tension recorded between 18 and 20 degrees C increased fractionally to an average of 1.504 +/- 0.029 (n = 4) for frog sartorii and to 1.377 +/- 0.008 (n = 5) for toad sartorii. The time to peak twitch tension and the half-relaxation time decreased markedly with an increase in temperature. Moreover, the half-relaxation time was reduced by a greater proportion than the time to peak twitch tension. Measurements of instantaneous stiffness by controlled velocity releases from the plateau of isometric tetani revealed that the large increase in isometric tetanus tension as the muscle was warmed was not accompanied by a corresponding increase in the total number of active cross-bridges. The possibility that a decreased availability of intracellular Ca2+ ions at the contractile sites contributing to the fall of isometric twitch tension at elevated temperatures is discussed. The possibility exists that at elevated temperatures a change inthe intrinsic contractile ability of the muscle occurs which produces an increased tension per cross-bridge.

Inhibitory effects of inhaled methyl methacrylate vapor on amphibian skeletal muscle.

The effect of inhaled vapors of methyl methacrylate (MMA) at a 2000 ppm concentration was studied on electrically stimulated in situ amphibian skeletal muscle preparations. Upon exposure to the vapor for 30 min, there was a significant fall in maximal twitch tension, a depression in elicited responses to a train of tetanizing stimuli and an upward and rightward shift in the strength-duration curve. Responses during a 30-minute post-exposure recovery period seldom reached sham control values.

AMP-deaminase activity in denervation atrophy of the amphibian skeletal muscle.

The substrate (AMP) and co-factor (ATP)-dependent kinetic parameters of AMP-deaminase have been studied in the contralateral and denervated gastrocnemius muscles of frog, Rana hexadactyla. An increasing in apparent affinity (Km) and maximal velocity (V) were found with denervated muscle enzyme as compared to the contralateral muscle enzyme. The activation energy (delta E) values were found to be decreased on denervation suggesting increased catalytic efficiency of denervated muscle enzyme.

Ruthenium-red staining of skeletal and cardiac muscles

The effects of ruthenium red (RR) on amphibian and mammalian skeletal muscles and mammalian myocardium were examined. In skeletal muscle cells, a discrete pattern of staining can be brought about within the lumina of the terminal cisternae (junctional sarcoplasmic reticulum [SR]) by sequential exposure to RR and OSO4. After prolonged immersion in RR solution, formation of pentalaminar segments ("zippering") occurs at various points along the longitudinal ("network") SR tubules. Zippering can be elicited in skeletal SR at any stage of preparation prior to postfixation with OSO4. By means of dispersive X-ray analysis, both ruthenium and osmium were seen to be deposited in skeletal muscle junctional SR, and ruthenium was detected in the myoplasm as well. In skeletal muscles whose T tubules were ruptured by exposure to glycerol, the pattern of SR staining and zippering resulting from ruthenium-osmium treatment was not affected. These findings indicate that RR is capable of passage across the sarcolemma of skeletal muscle and that this passage does not occur solely under conditions in which the plasma membrane is damaged. In contrast, RR does not opacify or modify any region of the SR of cardiac muscle. However, after this treatment, randomly distributed opaque bodies, composed of parallel lamellar structures, appear throughout the myocardial cells. A few of these bodies are associated with lipid droplets, but the rest are of unknown origin. The failure of the SR of cardiac muscle to stain after exposure to ruthenium dye (even though this material enters these cells) suggests that the chemical composition of cardiac SR is significantly different from that of skeletal muscle SR.

An intermediate type of muscle fibre in Xenopus laevis

AMPHIBIAN skeletal muscle is generally regarded as being composed of two distinct fibre types, twitch fibres and slow fibres, and various morphological and functional criteria have been used to classify fibres into one of these two categories (for review see ref. 1). However, the results of several investigations on frog and toad, including Xenopus, muscles suggest that this is an oversimplification2–5. Smith and Ovalle6 demonstrated five classes of fibres in hind limb muscles of Xenopus laevis using various ultrastructural and histochemical criteria. The mechanical performances of fibres of two of these classes (fast, fatigue-resistant twitch fibres (type 2) and slow fibres (type 5), respectively) have recently been described in detail7. In the study reported here fibres with a characteristic microscopic appearance have been isolated from a hind limb muscle of Xenopus laevis and it is shown that they have functional properties which are intermediate between those of twitch and slow fibres. The fine structure of these fibres conforms to the description of Smith and Ovalle for the ‘type 4’ fibre, a fibre type which also can morphologically be said to represent an intermediary type.

Verruculotoxin potentiation of twitch tension in skeletal muscle

Verruculotoxin [1-oxo-3-benzyl-octahydro-2H-pyrido(1,2-α)pyrazinel, a mycotoxin, was shown to act directly on both isolated mammalian and amphibian skeletal muscles to potentiate twitch tension. After 10 min exposure to a concentration of 0.4 m m, the twitch tensions of the rat diaphragm and frog sartorius muscles were enhanced to 152 and 150% of controls, respectively. In the isolated frog sartorius muscle, 10 min exposure to verruculotoxin (VTX, 0.4 m m) significantly increased the rate of contraction ( dP dt , 120%), contraction time (CT, 127%), and half-relaxation time ( 1 2 RT , 126%) without significantly changing the twitch duration; in the rat diaphragm preparation, 10 min exposure to VTX (0.4 m m) significantly increased both dP dt (141%) and CT (112%) without altering 1 2 RT and twitch duration. Of the C-15 substituted analogs tested, two analogs, fluoroverruculotoxin and methoxyverruculotoxin, enhanced twitch tension with a potency comparable to that of VTX, while chloroverruculotoxin increased twitch tension to only 120% of control. Hydroxyverruculotoxin and analogs lacking the C-3 benzyl group did not alter the twitch amplitude from control. VTX (0.4 m m, 10 min exposure) reduced twitch tension in the isolated cat papillary muscle to 33% of control. VTX (0.4 m m) was nondepolarizing in endplate and nonendplate regions. Thirty minute exposure to VTX irreversibly increased both the rising phase (sodium influx, 115% of control) and the repolarization phase (115% of control) of the muscle action potential. The results of this study indicate that VTX is a new muscle potentiator with properties similar to caffeine and the other Type A muscle potentiators.

Cellular Cl content and concentration of amphibian skeletal and heart muscle.

Toads (Bufo marinus) and frogs (Rana pipiens pipiens) were given intraperitoneal injections of Na36Cl and Na235SO4. After in vivo equilibration for 20--180 min, the animals were pithed, and their ventricular and semitendinosus muscles were excised. Measurements of total Cl (by titrimetry) and 36Cl (by radioassay) showed that specific radioactivities of plasma and mus les approached equality within 1 h after injection for toad skeletal and heart muscle and frog ventricles, indicating complete exchange of cellular Cl with 36Cl. From the simultaneously measured muscle water contents and 35SO4 spaces, intracellular Cl concentrations in vivo (in mumol/g cell water) for semitendinosus and ventricular muscles were calculated to be, respectively, 1.4 +/- 0.3 and 2.3 +/- 0.8 for Bufo and 1.7 +/- 0.7 and 4.8 +/- 2.4 for Rana. In view of these low values, active cellular Cl accumulation seems improbable, but cannot be rigorously excluded without simultaneous membrane potential and intracellular ion activity measurements. A high concentration of Cl in the sarcoplasmic reticulum of skeletal muscle is also inconsistent with these measurements.

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.

Isometric tension and instantaneous stiffness in amphibian skeletal muscle exposed to solutions of increased tonicity

The instantaneous elasticity and maximum isometric tetanic tension of isolated frog and toad sartorii have been measured in hypertonic Ringer solution. Although the mechanical response of contraction muscle continued to decrease as the tonicity of the bathing solution was increased to 1.26 X R, 1.52 X R, and 2.04 X R, a similar change in the instantaneous stiffness could not be shown. This finding was not expected on the basis of our current model of the cross-bridge mechanism which predicts that the instantaneous stiffness is a measure of the total number of tension-generating cross-bridges formed in a stimulated muscle. The compatability of our findings with an electrostatic theory of the cross-bridge mechanism proposed by Iwazumi (1970) is discussed.