An analysis of volume changes in the T-tubes of frog skeletal muscle exposed to sucrose

The volume responses of the T-system of frog skeletal muscle to isotonic solutions of altered ionic composition, as studied by electron microscopic examination of osmium-fixed tissue, have been re-investigated using aldehyde fixatives.1. In confirmation of the earlier work we find that the T-tubes swell in frog sartorius muscles which have been soaked in Ringer solution in which the sodium chloride concentration has been reduced to 40 mM and tonicity maintained with sucrose before fixation in osmium.2. The swelling does not occur in muscles similarly treated, but fixed with acrolein or glutaraldehyde.3. Swelling of the T-tubes, which has been reported for osmium-fixed muscle following exposure to Ringer in which chloride is replaced by acetate, or reduced and sulphate substituted, does not occur when fixation is by acrolein.4. Analysis of the data from the earlier work with sucrose substitution shows that the degree of T-tube swelling is proportional to the sucrose concentration of the soaking medium, whether the solution be isotonic or anisotonic.5. It is concluded that the increase in T-tube volume arises in muscles which have been exposed to certain impermeant solutes during fixation by osmium, and a possible mechanism to account for the effect is described.6. The relevance of these observations to the hypothesis that the sarcoplasmic reticulum is an extracellular compartment in muscle and to the proposal that the T-tubes may represent an intermediate compartment for potassium fluxes is discussed.     

Stereologic analysis of dystrophic chicken muscle.

Stereologic methods have been used to estimate the volume and surface densities of sarcoplasmic reticulum (SR) and T tubules of normal and dystrophic chicken pectoralis muscle fibers. The surface and volume densities of the T system in dystrophic muscle fibers showed large increases compared with normal muscle fibers; the surface and volume densities of the SR showed large decreases. In addition, the SR and T system in dystrophic fibers undergo changes in shape. The tubules of the free SR become much narrower; the T system becomes dilated and vesiculated. Dystrophic fibers, on the average, are much larger than normal but maintain the same sarcolemmal surface/fiber volume ratio as normal fibers. Alterations in the surface and volume densities of the dystrophic sarcotubular system may account for some of the altered contractile properties of these muscles.     

Effect of cyclopiazonic acid, an inhibitor of the sarcoplasmic reticulum Ca-ATPase, on skeletal muscles from normal and mdx mice

AIM: In this study, we investigated Ca2+ loading by the sarcoplasmic reticulum in skeletal muscle from mdx mice, an animal model of human Duchenne's muscular dystrophy, at two stages of development: 4 and 11 weeks. METHOD: Experiments were conducted on fast- (extensor digitorum longus, EDL) and slow- (soleus) twitch muscles expressing different isoforms of Ca2+-ATPase, which is responsible for the uptake of Ca2+ by the sarcoplasmic reticulum. RESULTS: In sarcoplasmic reticulum vesicles, the ATP-dependent activity and sensitivity to cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum Ca2+-ATPase, were similar in mdx and normal EDL muscle. Furthermore, in chemically-skinned fibres from both normal and mdx muscles, the presence of CPA induced a decrease in Ca2+ uptake by the sarcoplasmic reticulum. However, the sensitivity to CPA was lower in mdx EDL muscle than in normal muscle. In addition, in EDL muscle from 4-week-old mdx mice, the expression of the slow Ca2+-pump isoform (SERCA2a) was significantly increased, without any accompanying change in slow myosin expression. In contrast, the expression and function of the Ca2+-ATPase in mdx soleus muscles at 4- and 11-weeks of development did not differ from those in age-matched controls. CONCLUSION: These findings show that in dystrophic muscle, where the Ca2+ homeostasis was perturbed, the Ca2+ handling by the sarcoplasmic reticulum was altered in fast-twitch muscle, and this was associated with the expression of the slow isoform of SERCA. In these muscles, reduced Ca2+ uptake could then contribute to an elevated concentration of Ca2+ in the cytosol, and also to Ca2+ depletion of the sarcoplasmic reticulum.     

"Giant" muscle fibres in skeletal muscle of normal pigs

Observations made during a growth and development study of the semitendinosus and trapezius muscles of 49 purebred Large White pigs between birth and 128 days of age revealed the presence of giant fibres. The occurrence, histochemical and ultrastructural properties of these giant fibres were investigated. A high proportion of the pigs (85 per cent) contained giant fibres in their muscles but these giant fibres usually represented less than 1 per cent of the total myofibre population. Giant fibres possessed enhanced adenosine triphosphatase activity and a high capacity for oxidative metabolism (indicated by succinate dehydrogenase activity) which was reflected ultrastructurally by the greatly heightened electron density of myofibrils and by an abnormally high percentage of mitochondria and lipid droplets. These deviations from normal muscle fibre composition, together with the reduced percentage volume of sarcoplasmic reticulum, were consistent with changes seen in functionally over-loaded muscle. It appears that giant fibre anomalies occur through increased activity stimulated in occasional muscle fibres, perhaps by a structural defect, such as an inadequate amount of sarcoplasmic reticulum, which causes hyper-contractile activity within the fibres and associated compensatory adaptations. Giant fibres did not appear to represent fibres undergoing degenerative changes.     

Structure and molecular organisation of the sarcoplasmic reticulum of skeletal muscle fibers

Activation of muscle contraction is a rapid event that is initiated by depolarization of the plasma membrane and transverse (T) tubules, which following transduction in the interior of the muscle cell, activate the release of calcium from the sarcoplasmic reticulum (SR). Pioneer studies using electron microscopy defined the organization of the sarcoplasmic reticulum and the details of the junctions between sarcoplasmic reticulum and T tubules, which are essential for translating the electrical signal on the plasma membrane to calcium release from the sarcoplasmic reticulum. Molecular biology and biochemistry studies have revealed the presence of several proteins located on the sarcoplasmic reticulum, some of which participate together with the ryanodine receptors to the assembly of a large multi-protein complex, while others, like the calcium pumps, have independent localization and activities. As a whole, the current view of this system contemplates the existence of a high level of structural organization in the sarcoplasmic reticulum with respect to the localization of ryanodine receptors and other proteins. In this review we shall summarize studies on the expression and possible functional significance of the ryanodine receptor type 3 in mammalian skeletal muscles and recent studies aimed to dissect the mechanisms that establish the organization of the SR in striated muscles.     

Freeze-fractured sarcoplasmic reticulum in adult and embryonic fast and slow muscles

Summary There is evidence to suggest that 8 nm calcium transport particles in the sarcoplasmic reticulum are involved in the regulation of twitch properties in adult muscles. We have studied ultrastructural characteristics of the sarcoplasmic reticulum in relation to previously defined physiological changes that take place in the normal course of development. The fast twitch posterior latissimus dorsi (PLD) and the slow tonic anterior latissimus dorsi (ALD) of the chicken were compared using the procedure of freeze-fracture. In the adult PLD, the sarcoplasmic reticulum was composed of longitudinal tubules, which gave rise to fenestrated cisternae at the centre of the H band and to terminal cisternae that form triads regularly at each A-I junction. In most of the fibres (85%), 8 nm intramembrane particles were closely packed in the concave fracture face (P-face). In the ALD, a tubular network with an open circular pattern extended the entire length of the A band and usually throughout the I band as well. Dyads or triads, which were infrequent, were often oriented obliquely. The density of intramembrane particles was low in the majority of the fibres, but there was a significant minority population (30%) in which particle density was relatively high. At 10 daysin ovo, when speed of contraction in both the ALD and PLD is slow, there was a circular configuration of sarcoplasmic reticulum components in both muscles, and particle density was low. Surprisingly, at 18 daysin ovo, when the rate of tension development and relaxation have reached nearly adult values in the fast PLD, this muscle, like the ALD, continued to exhibit a circular arrangement of sarcoplasmic reticulum tubules. The density of P-face particles, although greater than at 10 days, was still low relative to the adult PLD. Estimated values for the 18-day PLD were similar to those calculated for the adult slow muscle. Our observations, along with those of other investigators, suggest that abundant intramembrane particles may be related to the fast twitch properties of the adult PLD. However, they indicate that neither the pattern of membranes typical of the adult fast muscle nor the high content of calcium transport particles is required for the differentiation of fast twitch characteristics.     

Effect of cyclopiazonic acid,an inhibitor of the sarcoplasmic reticulum Ca‐ATPase,on skeletal muscles from normal and mdx mice

Aim: In this study, we investigated Ca²⁺ loading by the sarcoplasmic reticulum in skeletal muscle from mdx mice, an animal model of human Duchenne's muscular dystrophy, at two stages of development: 4 and 11 weeks. Method: Experiments were conducted on fast‐ (extensor digitorum longus, EDL) and slow‐ (soleus) twitch muscles expressing different isoforms of Ca²⁺‐ATPase, which is responsible for the uptake of Ca²⁺ by the sarcoplasmic reticulum. Results: In sarcoplasmic reticulum vesicles, the ATP‐dependent activity and sensitivity to cyclopiazonic acid (CPA), an inhibitor of the sarcoplasmic reticulum Ca²⁺‐ATPase, were similar in mdx and normal EDL muscle. Furthermore, in chemically‐skinned fibres from both normal and mdx muscles, the presence of CPA induced a decrease in Ca²⁺ uptake by the sarcoplasmic reticulum. However, the sensitivity to CPA was lower in mdx EDL muscle than in normal muscle. In addition, in EDL muscle from 4‐week‐old mdx mice, the expression of the slow Ca²⁺‐pump isoform (SERCA2a) was significantly increased, without any accompanying change in slow myosin expression. In contrast, the expression and function of the Ca²⁺‐ATPase in mdx soleus muscles at 4‐ and 11‐weeks of development did not differ from those in age‐matched controls. Conclusion: These findings show that in dystrophic muscle, where the Ca²⁺ homeostasis was perturbed, the Ca²⁺ handling by the sarcoplasmic reticulum was altered in fast‐twitch muscle, and this was associated with the expression of the slow isoform of SERCA. In these muscles, reduced Ca²⁺ uptake could then contribute to an elevated concentration of Ca²⁺ in the cytosol, and also to Ca²⁺ depletion of the sarcoplasmic reticulum.     

Immunogold localization of inositol 1,4,5-trisphosphate receptors and characterization of ultrastructural features of the sarcoplasmic reticulum in phasic and tonic smooth muscle

Summary Although agonist stimulation leads to an increase in inositol 1,4,5-trisphosphate (InsP₃) and decreased calcium in peripherally and centrally located sarcoplasmic reticulum in smooth muscle, the distribution of InsP₃ receptors is unknown. InsP₃ receptor and the calcium binding protein, calsequestrin were localized by immunolabelling in a tonic and a phasic smooth muscle. InsP₃ receptor labelling was predominatly localized at the cell periphery, where most of the sarcoplasmic reticulum is localized in vas deferens (phasic muscle). Elements of central sarcoplasmic reticulum, where present, were also labelled. Distribution of calsequestrin in vas deferens was similar to that of the InsP₃ receptor. In aorta (tonic muscle) the InsP₃ receptor labelling was proportional to sarcoplasmic reticulum distribution: predominantly central. No labelling of sections or immunoblots was observed with the anti-calsequestrin antibody in aorta. InsP₃ and caffeine, but not cyclic ADP-ribose, released intracellular Ca²⁺ in permeabilized vas deferens and aorta. The ultrastructure of the sarcoplasmic reticulum, investigated in stereo views of semi-thick and thin sections of osmium ferricyanide stained tissue, is shown to have several distinctive features, such as fenestrated sheets (single or in stacks), as well as numerous regions of continuity between central and peripheral sarcoplasmic reticulum, suggesting a single compartment within the smooth muscle cell. Regions of the sarcoplasmic reticulum were closely apposed to and often ensheathed mitochondria. We conclude that InsP₃ receptors are present in both the central and the peripheral sarcoplasmic reticulum of tonic and phasic smooth muscle, consistent with electron probe analysis results showing calcium release from both regions.     

Myosin polymorphism in muscles of the toadfish,Opsanus tau

The superfast swimbladder muscle of Opsanus tau differs from the corresponding fast skeletal muscle not only by its greater sarcoplasmic reticulum and parvalbumin content but also by a genuine myosin LC2. The study of light chains has been extended here to other striated muscles. Myosins from fast (trunk muscle), superfast (swimbladder muscle), slow (lateral-line muscle) and cardiac (ventricle muscle) were compared, using one- and two-dimensional polyacrylamide gel electrophoresis. The results showed that the light chains all appear distinct in isoelectric point and molecular weight, except for the two LC1 and two LC3 from fast and superfast muscles. Striated muscles from the toadfish Opsanus tau exhibited at least four isoenzymic forms of the myosin, related to the light chains and corresponding to different physiological properties.     

Scanning electron microscopic observations on muscle cells of experimental mitochondrial myopathy produced by 2, 4-dinitrophenol

The morphological changes of the skeletal muscle cells of the rat experimental myopathy induced by 2, 4-dinitrophenol were examined by scanning electron microscopy in comparison with the ultrastructure of normal muscle cells. Specimens were prepared by the Aldehyde-Osmium-DMSO-Osmium method which permits the three-dimensional demonstration of intracellular structures under SEM. In the specimen prepared by the method, myofibrils having been completely dissolved, intracellular membranous structures such as the sarcoplasmic reticulum, T-tubules and mitochondria were clearly demonstrated in three dimensions. In the experimental mitochondrial myopathy, large accumulations of mitochondria were observed at the subsarcolemmal region. Mitochondria in the perinuclear and intermyofibrillar region showed swelling and occasionally accompanied abnormal concentric cristae. The sarcoplasmic reticulum which showed regular network in normal muscle cells entirely disappeared in the mitochondrial myopathy. Although the mitochondrial changes obtained in this study were almost identical to those previously reported by transmission electron microscopy, the changes in the sarcoplasmic reticulum have not been described in previous works.     

Effects of age on sarcoplasmic reticulum properties and histochemical composition of fa stand slow-twitch rat muscles

Calcium release activity of sarcoplasmic reticulum and enzyme-histochemical properties were investigated in extensor digitorum longus (e.d.l.) and soleus muscles in young (4 months) and old (24 months) male rats. With age, the caffeine threshold concentration for calcium release from the sarcoplasmic reticulum of soleus skinned muscle fibres showed only minor modifications. On the other hand, in e.d.l. skinned muscle fibres, the caffeine threshold concentration decreased significantly (P < 0.05). The histochemical fibre type composition changed with age both in soleus and in e.d.l. muscles, showing a common transformation toward a more oxidative histochemical profile. In fact, in aged soleus, a significant (P < 0.05) increase was observed of type 1 fibres to represent almost the totality of the muscle fibres (more than 98%), while types 2C and 2A were reduced in proportion. In aged e.d.l. the percentage of type 1 (P < 0.05), 2A and 2X (a recently identified fourth component of the fast-twitch muscle types) fibres increased, with a reduction of type 2B (P < 0.01) fibres. The present results suggest that the changes in contractile properties of aged muscles may be related to the changes not only in fibre composition but also in the mechanism of calcium release from sarcoplasmic reticulum.     

Sarcoplasmic reticulum function in slow- and fast-twitch skeletal muscles from mdx mice

The aim of the present study was to establish whether alterations in sarcoplasmic reticulum function are involved in the abnormal Ca(2+) homeostasis of skeletal muscle in mice with muscular dystrophy ( mdx). The properties of the sarcoplasmic reticulum and contractile proteins of fast- and slow-twitch muscles were therefore investigated in chemically skinned fibres isolated from the extensor digitorum longus (EDL) and soleus muscles of normal (C57BL/10) and mdx mice at 4 and 11 weeks of development. Sarcoplasmic reticulum Ca(2+) uptake, estimated by the Ca(2+) release following exposure to caffeine, was significantly slower in mdx mice, while the maximal Ca(2+) quantity did not differ in either type of skeletal muscle at either stage of development. In 4-week-old mice spontaneous sarcoplasmic reticulum Ca(2+) leakage was observed in EDL and soleus fibres and this was more pronounced in mdx mice. In addition, the maximal Ca(2+)-activated tension was smaller in mdx than in normal fibres, while the Ca(2+) sensitivity of the contractile apparatus was not significantly different. These results indicate that mdx hindlimb muscles are affected differently by the disease process and suggest that a reduced ability of the Ca(2+)-ATPase to load Ca(2+) and a leaky sarcoplasmic reticulum membrane may be involved in the altered intracellular Ca(2+) homeostasis.     

Local calcium signals induced by hyper-osmotic stress in mammalian skeletal muscle cells

Strenuous activitiy of skeletal muscle leads to temporary osmotic dysbalance and isolated skeletal muscle fibers exposed to osmotic stress respond with characteristic micro-domain calcium signals. It has been suggested that osmotic stress targets transverse tubular (TT) dihydropyridine receptors (DHPRs) which normally serve as voltage-dependent activators of Ca release via ryanodine receptor (RyR1s) of the sarcoplasmic reticulum (SR). Here, we pursued this hypothesis by imaging the response to hyperosmotic solutions in both mouse skeletal muscle fibers and myotubes. Ca fluctuations in the cell periphery of fibers exposed to osmotic stress were accompanied by a substantial dilation of the peripheral TT. The Ca signals were completely inhibited by a conditioning depolarization that inactivates the DHPR. Dysgenic myotubes, lacking the DHP-receptor-alpha1-subunit, showed strongly reduced, yet not completely inhibited activity when stimulated with solutions of elevated tonicity. The results point to a modulatory, even though not essential, role of the DHP receptor for osmotic stress-induced Ca signals in skeletal muscle.     

Phosphate transport into the sarcoplasmic reticulum of skinned fibres from rat skeletal muscle

The rate, magnitude and pharmacology of inorganic phosphate (Pi) transport into the sarcoplasmic reticulum were estimated in single, mechanically skinned skeletal muscle fibres of the rat. This was done, indirectly, by using a technique that measured the total Ca2+ content of the sarcoplasmic reticulum and by taking advantage of the 1:1 stoichiometry of Ca2+ and Pi transport into the sarcoplasmic reticulum lumen during Ca--Pi precipitation- induced Ca2+ loading. The apparent rate of Pi entry into the sarcoplasmic reticulum increased with increasing myoplasmic [Pi] in the 10 mm--50 mm range at a fixed, resting myoplasmic pCa of 7.15, as judged by the increase in the rate of Ca--Pi precipitation-induced sarcoplasmic reticulum Ca2+ uptake. At 20 mm myoplasmic [Pi] the rate of Pi entry was calculated to be at least 51 m s–1 while the amount of Pi loaded appeared to saturate at around 3.5 mm (per fibre volume). These values are approximations due to the complex kinetics of formation of different species of Ca--Pi precipitate formed under physiological conditions. Phenylphosphonic acid (PhPA, 2.5 mm inhibited Pi transport by 37% at myoplasmic pCa 6.5 and also had a small, direct inhibitory effect on the sarcoplasmic reticulum Ca2+ pump (16%). In contrast, phosphonoformic acid (PFA, 1 mm) appeared to enhance both the degree of Pi entry and the activity of the sarcoplasmic reticulum Ca2+ pump, results that were attributed to transport of PFA into the sarcoplasmic reticulum lumen and its subsequent complexation with Ca2+. Thus, results from these studies indicate the presence of a Pi transporter in the sarcoplasmic reticulum membrane of mammalian skeletal muscle fibres that is (1) active at physiological concentrations of myoplasmic Pi and Ca2+ and (2) partially inhibited by PhPA. This Pi transporter represents a link between changes in myoplasmic [Pi] and subsequent changes in sarcoplasmic reticulum luminal [Pi]. It might therefore play a role in the delayed metabolic impairment of sarcoplasmic reticulum Ca2+ release seen during muscle fatigue, which should occur abruptly once the Ca--Pi solubility product is exceeded in the sarcoplasmic reticulum lumen     

Calsequestrin expression and calcium binding is increased in streptozotocin-induced diabetic rat skeletal muscle though not in cardiac muscle

Altered mechanisms of Ca2+ transport may underlie the contractile dysfunctions that have been frequently reported to occur in diabetic cardiac and skeletal muscle tissues. Calsequestrin, a high-capacity Ca2+-binding protein, is involved in the regulation of the excitation-contraction-relaxation cycle of both skeletal and cardiac muscle fibres. We have investigated the expression of calsequestrin and Ca2+ binding in cardiac and skeletal muscle from streptozotocin-induced diabetic rat. Immunoblotting of microsomal membranes from normal and streptozotocin-induced diabetic muscle revealed no significant changes in heart, but an increase in the relative abundance of calsequestrin and calsequestrin-like proteins in skeletal muscle. In analogy, the overall Ca2+-binding capacity of sarcoplasmic reticulum vesicles from diabetic skeletal muscle was drastically increased. The expression of fast muscle marker proteins was not affected, indicating that no relevant fibre transformation occurred in streptozotocin-treated rat muscles. The up-regulation of the high-capacity Ca2+-binding element calsequestrin might represent a compensatory mechanism of diabetic skeletal muscle. An increased Ca2+-buffering capacity of the sarcoplasmic reticulum lumen might counteract elevated cytosolic Ca2+ levels in diabetes thereby preventing Ca2+-dependent myo-necrosis.     

The contractile properties,histochemistry, ultrastructure and electrophysiology of the cricothyroid and posterior cricoarytenoid muscles in the rat

Summary The contractile, histochemical, morphological and electrophysiological properties of two rat laryngeal muscles, the cricothyroid and posterior cricoarytenoid, have been measured. Both muscles act during respiration to maintain upper airway patency and an even distribution of air in the lungs. The cricothyroid and posterior cricoarytenoid are fast-twitch muscles, having contraction times of 3.4 and 7.2 ms respectively, high myosin ATPase activity, abundant sarcoplasmic reticulum (with average volumes of 9% and 15%, respectively, of the fibre volume) and T-system membrane (with average areas of 0.4 and 0.5 µm² µ^–3 of fibre). The large areas of T-tubule membrane are reflected in the average specific membrane capacities of 6.5 µF cm^–2 to 10.5 µF cm^–2, which are high considering the small diameter of the fibres (20–30 µm). Of the two muscles, the posterior cricoarytenoid has the faster contraction time and the more abundant sarcoplasmic reticulum content. In addition, the posterior cricoarytenoid is less resistant to fatigue and demonstrates lower succinic dehydrogenase activity. The fatigability of this muscle, coupled with its general lack of functional reserve, suggest that its failure may contribute to upper airway obstruction during respiratory distress.     

Structure and some contractile properties of fast and slow muscles of the chicken

1. A comparative study has been made of the structure of the posterior and anterior latissimus dorsi muscles (P.L.D. and A.L.D.) of the chicken. Some of the contractile properties of these muscles, particularly of the A.L.D. muscle, have also been examined.2. In confirmation of earlier work, it is found that structural differences between these two muscles mainly reside in the organization of the T-system and the sarcoplasmic reticulum.3. In the P.L.D. muscle the T-system and sarcoplasmic reticulum are tubular networks of regular pattern, with frequent regions of contact between them in the form of the familiar triads. As described earlier, two series of triads are present in each sarcomere, near the level of the A-I boundary.4. The presence in the A.L.D. muscle of both T-system and sarcoplasmic reticulum has been established here by the combined use of electron microscopic and histochemical techniques. The appearance of these structures is considerably less regular in this than in the P.L.D. muscle, and the area of contact between them relatively small: in terms of contact area per sarcomere, about 15% or less of that in the P.L.D. muscle. Regions of contact are mainly in the form of dyads, but a few triads are also present.5. Contractures of the A.L.D. muscle in high K fluids have been recorded and their modification due to variation of the external concentration of Ca, K and Mg examined. The K contracture consists of two phases: an initial transient and a later maintained phase. The response of these phases to variation in the external medium is markedly different.6. Application of Ca-free fluids causes contracture tension of the late phase to subside within a few minutes whilst that of the initial phase declines completely only after several hours. High Mg concentrations depress the later but not the early phase of contracture.7. Tentative interpretations of both the morphological and physiological differences described have been proposed.     

Significance of the increase in glucose 6-phosphatase activity in skeletal muscle cells of the mouse by starvation

The effects of starvation on glucose 6-phosphatase (G6Pase; EC 3.1.3.9., D-glucose 6-phosphate phosphohydrolase) and glycogen phosphorylase (EC 2.4.1.1.) activities, and on glycogen content, were studied in skeletal muscles (m. rectus femoris) of mice. In the muscle cells from fed animals, the cytochemical reaction product for G6Pase activity was observed in moderate amounts in the terminal cisternae of sarcoplasmic reticulum and in small amounts in the nuclear envelope, and was rare or absent in the intermyofibrillar sarcoplasmic reticulum. After 4 days of starvation, however, the reaction product became abundant in all of the terminal cisternae, intermyofibrillar sarcoplasmic reticulum, and nuclear envelope. Biochemical G6Pase and glycogen phosphorylase a (active form) activities were higher in the muscles of starved mice than in those of fed animals. The glycogen content decreased markedly in the muscles of starved mice. The results suggest that the role of the increased G6Pase in skeletal muscle cells of starved mice is to release glucose into the blood by hydrolyzing glucose 6-phosphate produced through the increased phosphorylase activity.     

Early ultrastructural changes in skeletal muscle exposed to the local anaesthetic bupivacaine (Marcaine).

The local anaesthetic drug bupivacaine is known to produce a degeneration in mammalian muscle fibres which is followed by regeneration. In an attempt to define the site of action of this drug and the cell type responsible for the subsequent fibre regeneration, first lumbircal muscles of rats were exposed in vitro to concentrations of 10(-2), 5 X 10(-3), and 10(-3)M bupivacaine for periods ranging from 5 min to 3 h. An electron microscopic study of muscles exposed to 10(-2) and 5 X 10(-3)M bupivacaine revealed an initial supercontraction followed by evidence of severe and probably irreversible injury to plasma membrane, sarcoplasmic reticulum and mitochondria. Myonuclei showed signs of injury early, but satellite cells were found relatively resistant. The basal lamina remained intact throughout and myelinated axons did not suffer damage. However, both endothelial cells of capillaries and fibrocytes showed degenerative changes. Muscles exposed to 10(-3)M bupivacaine showed little change after incubation for 3 h. It is concluded that increased intracellular levels of Ca++ ions may play a part in the pathogenesis of muscle injury induced by bupivacaine and that their source may be the sarcoplasmic reticulum. The role of the satellite cell in the regeneration that follows this injury was not clearly established.     

Rapid cooling-induced contractures in rat skinned skeletal muscle fibres originate from sarcoplasmic reticulum Ca2+ release through ryanodine and inositol trisphosphate receptors

Previous reports have shown that cooling striated muscles induces contractile responses that are related to Ca2+ release from the sarcoplasmic reticulum. However, the effect of cooling has generally been studied in the presence of pharmacological agents that potentiate rapid cooling-induced contractures. The present study shows that in saponin-skinned rat skeletal muscle preparations, a drop in temperature from 22 degrees C to 2 degrees C per se induces a contracture which relaxes on return to 22 degrees C. In fast-twitch fibres, rapid cooling-induced contractures are fully blocked by ryanodine, an inhibitor of ryanodine receptors. By contrast, in slow-twitch fibres, ryanodine partially inhibits the rapid cooling-induced contractile response, leaving a residual tension that dissipates after application of inositol 1,4,5-trisphosphate (InsP3). At low concentrations, heparin, an inhibitor of InsP3 receptors, decreases rapid cooling-induced contractures in both types of muscle. The present results suggest that in skeletal muscle, rapid cooling-induced contractures are due to both ryanodine-sensitive and InsP3-sensitive Ca2+ release from the sarcoplasmic reticulum.