Studies of the halothane-cooling contractures of skeletal muscle

The characteristics of transient contractures elicited by rapid cooling of frog or mouse muscles perfused in vitro with solutions equilibrated with 0.5-2.0% halothane are reviewed. The data indicate that these halothane-cooling contractures are dose dependent and reproducible, and their amplitude is larger in muscles containing predominantly slow-twitch type fibers, such as the mouse soleus, than in muscles in which fast-twitch fibers predominate, such as the mouse extensor digitorum longus. The halothane-cooling contractures are potentiated in muscles exposed to succinylcholine. The effects of Ca2+-free solutions, of the local anesthetics procaine, procainamide, and lidocaine, and of the muscle relaxant dantrolene on the halothane-cooling contractures are consistent with the proposal that the halothane-cooling contractures result from synergistic effects of halothane and low temperature on Ca sequestration by the sarcoplasmic reticulum. Preliminary results from skinned rabbit muscle fibers support this proposal. The halothane concentrations required for the halothane-cooling contractures of isolated frog or mouse muscles are comparable with those observed in serum of patients during general anesthesia. Accordingly, fascicles dissected from muscle biopsies of patients under halothane anesthesia for programmed surgery develop large contractures when rapidly cooled. The amplitude of these halothane-cooling contractures declined with the time of perfusion of the muscle fascicles in vitro with halothane-free physiological solutions. It is suggested that the halothane-cooling contractures could be used as a simple experimental model for the investigation of the effects of halothane on Ca homeostasis and contractility in skeletal muscle and for study of drugs of potential use in the management of the contractures associated with the halothane-induced malignant hyperthermia syndrome.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Organ culture of frog muscle: maintenance of mass, enzyme levels, and contractile force

This paper describes an organ culture system that maintains frog sartorius muscles in good condition for 5 days. In the absence of serum and insulin, muscles maintained at approximately 93% of resting length atrophied with significant decreases in dry weight, protein content, and contractile force, and in the levels of activity of citrate synthase, lactate dehydrogenase, and creatine kinase. Inclusion of 1.0 mU/ml of insulin in the culture medium prevented the decreases in muscle mass, twitch tension, and citrate synthase activity and minimized the decreases in lactate dehydrogenase, creatine kinase, and tetanic tension. Inclusion of 10% serum, in addition to 1 mU/ml insulin, in the medium did not have clear cut additional benefits. Stretching muscles to 110% of resting length (L0) resulted in marked deterioration with decreases in total protein, enzyme levels, and contractile force. Keeping muscles at approximately 93% L0 was as effective as maintenance at L0 in preventing atrophy and loss of contractile force and enzyme activities. This organ culture procedure, which maintains frog sartorius muscle in good condition without serum for at least 5 days, may provide a useful model for studying the regulatory mechanisms responsible for a variety of adaptations in muscle.     

The properties of the extraocular muscles of the frog. III. Morphological, mechanical and pharmacological properties of the isolated retractor bulbi muscle.

Some morphological, physiological, and pharmacological properties of the retractor bulbi muscle of the frog were tested. The enzyme-histochemical investigation shows that the retractor bulbi muscle contains twitch muscle fibres only. Two types of twitch muscle fibres, which are especially different in their diameter and in the content of mitochondria, build the muscle in an irregular arrangement; tonic muscle fibres were not observed. On the average, the isolated retractor bulbi muscle has at room temperature a contraction time of 26 ms, a half-relaxation time of 28 ms, a fusion frequency of 75 stimuli/s, and a twitch-tetanus ratio of 0.28. The fatigability of this muscle is higher than in oculorotatory eye muscles but lower than in skeletal muscles of the frog. An increase of the extracellular K+-concentration elicits in retractor bulbi muscles a quickly transient contracture; the mechanical threshold of the muscle fibres is found in a range between 20 and 25 mM K+ in Ringer solution. Similar short-lasting contractures, which are probably caused by twitch fibres, rich in mitochondria, are also evoked by application of depolarizing drugs like acetylcholine. The properties of the retractor bulbi muscle are compared with those of the sartorius muscle of the frog, which likewise contains twitch muscle fibres only.     

The effect of acid-base balance on fatigue of skeletal muscle

H+ ions are generated rapidly when muscles are maximally activated. This results in an intracellular proton load. Typical proton loads in active muscles reach a level of 20-25 mumol X g-1, resulting in a fall in intracellular pH of 0.3-0.5 units in mammalian muscle and 0.6-0.8 units in frog muscle. In isolated frog muscles stimulated to fatigue a proton load of this magnitude is developed, and at the same time maximum isometric force is suppressed by 70-80%. Proton loss is slowed when external pH is kept low. This is paralleled by a slow recovery of contractile tension and seems to support the idea that suppression results from intracellular acidosis. Nonfatigued muscles subjected to similar intracellular proton loads by high CO2 levels show a suppression of maximal tension by only about 30%. This indicates that only a part of the suppression during fatigue is normally due to the direct effect of intracellular acidosis. Further evidence for a component of fatigue that is not due to intracellular acidosis is provided by the fact that some muscle preparations (rat diaphragm) can be fatigued with very little lactate accumulation and very low proton loads. Even under these conditions, a low external pH (6.2) can slow recovery of tension development 10-fold compared with normal pH (7.4). We must conclude that there are at least two components to fatigue. One, due to a direct effect of intracellular acidosis, acting directly on the myofibrils, accounts for a part of the suppression of contractile force. A second, which in many cases may be the major component, is not dependent on intracellular acidosis. This component seems to be due to a change of state in one or more of the steps of the excitation-contraction coupling process. Reversal of this state is sensitive to external pH which suggests that this component is accessible from the outside of the cell.     

Characterization of contractile function and expression of muscarinic receptors,G proteins and adenylate cyclase in cultured tracheal smooth muscle of Swine

Smooth muscle cells lose their contractile function and phenotype very rapidly when placed in culture. During organ culture of smooth muscle strips, phenotype is lost more slowly. In the present studies, we established an organ culture model to study contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase in different serum concentrations in tracheal smooth muscle from swine. The results show that contractile function and the amounts of M(3) receptors, G proteins and adenylyl cyclase were maintained for up to 5 days in culture. The expression of M(2) receptors was significantly decreased in culture when compared to freshly isolated muscles. Maximal isometric tension was significantly increased in cultured muscles compared with freshly isolated muscles. Different serum concentrations did not significantly affect contractile function and expression of muscarinic receptors, G proteins and adenylyl cyclase. In conclusion, our studies suggest that cultured smooth muscle might be used as a model to study the regulation of contractile function of smooth muscle by various signal transduction pathways.     

Ouabain potentiation and Ca release from sarcoplasmic reticulum in cardiac and skeletal muscle cells

In this article, we describe a possible mechanism of ouabain potentiation in heart based on the following findings in cardiac and skeletal muscles of various species. (1) In heart ventricle muscles of frog and guinea pig, the ouabain potentiation is produced without an effect on Ca influx. In both frog and cat heart ventricle muscles, ouabain potentiates the rapid cooling contracture with or without caffeine in a Ca-deprived medium. It follows, therefore, that the ouabain potentiation is produced by an "intracellular" mechanism. (2) In crab single muscle fibers, contractile responses such as twitch, potassium-induced contracture, caffeine-induced contracture, and water-induced contracture are remarkably potentiated if ouabain is present within the fibers by microinjection, whereas the situation is reversed if the drug is given extracellularly. (3) The ouabain potentiated the Ca release from fragmented sarcoplasmic reticulum (FSR) isolated from cat, guinea pig, and frog heart and from skeletal muscles as a result of the procedures used, such as changing the ionic environment. (4) In frog, cat, and guinea pig heart ventricle muscles, a reduction of contractility as a result of pretreatment with urea--Ringer's was completely cancelled by ouabain almost without influencing the membrane depolarization. Based on these findings and others, the deduction was made that the positive inotropic effect of cardiac glycosides on the heart is brought about by potentiation of contraction - Ca release from the intracellular store sites, namely the sarcoplasmic reticulum.     

Contractile activation in scorpion striated muscle fibers. Dependence on voltage and external calcium

Excitation-contraction coupling was characterized in scorpion striated muscle fibers using standard microelectrode techniques as employed in studies on vertebrate skeletal muscle. The action potential of scorpion muscle consists of two phases of regenerative activity. A relatively fast, overshooting initial spike is followed by a prolonged after- discharge of smaller, repetitive spikes. This after-discharge is accompanied by a twitch that relaxes promptly upon repolarization. Twitches fail in Na-free, tetrodotoxin (TTX)-containing, or Ca-free media. However, caffeine causes contractures in muscles paralyzed by Na- and Ca-free solutions. Experiments on muscle fibers voltage-clamped at a point with two microelectrodes in Na-free or TTX-containing media indicate that: (a) the strength-duration relation for threshold contractions has a shape similar to that in frog muscle, but mean values are displaced approximately 20 mV in the positive direction; (b) tetracaine exerts a parallel effect on strength-duration curves from scorpion and frog; (c) contractile activation in scorpion is abolished in Ca-free media; and (d) the contractile threshold is highly correlated with the occurrence of inward Ca current for pulses of all durations. Thus, the voltage dependence of contractile activation in scorpion and frog muscle is similar. However, the preparations differ in their dependence on extracellular Ca for contraction. These results are discussed in relation to possible mechanisms coupling tubular depolarization to Ca release from the sarcoplasmic reticulum in vertebrate and invertebrate skeletal muscle.     

Denaturation time of actomyosin exposed to different chemicals]

The frog sceleton muscle actomyosin denaturation time dependence on the concentration of salts (NaCl and CaCl2) and organic chemicals (carbohydrates, narcotics and alcohols) was investigated. The following effects were detected: phase change in denaturation time associated with the rise of concentration of chemicals under study; actomyosin stabilization effect; coincidence of concentrations giving rise to protein stability with those increasing the survival time of isolated frog sceleton muscles in vitro (literature data); denaturation effect of alcohols used, both in high and very low concentrations.     

The properties of the extraocular muscles of the frog. I. Mechanical properties of the isolated superior oblique and superior rectus muscles.

The mechanical properties of two extraocular muscles (superior oblique and superior rectus muscles) of the frog were studied and compared with those of a frog's skeletal muscle (iliofibularis muscle) which contains the same types of muscle fibres as the oculorotatory muscles. The extraocular muscles are very fast twitching muscles. They exhibit a smaller contraction time, a smaller half-relaxation time, a higher fusion frequency, and a lower twitch-tetanus ratio than the skeletal muscles. The maximum isometric tetanic tension produced per unit cross-sectional area is lower in the extraocular muscles than in skeletal muscles. However, the extraocular muscles show a higher fatigue resistance than the skeletal muscles. With respect to the dynamic properties there are some differences between the various oculorotatory muscles of the frog. The superior rectus muscle exhibits a faster time-course of the contraction, a higher fusion frequency, and a higher fatigability than the superior oblique muscle. An increase of the extracellular K+-concentration evokes sustained contractures not only in the extraocular muscles but also in the iliofibularis muscle; between these muscles there are no striking differences in the mechanical threshold of the whole muscle preparation. The mechanical threshold depends on the Ca++-concentration of the bathing solution and it is found in a range between 12.5 and 17.5 mM K+ in a normal Ringer solution containing 1.8 mM Ca++. The static-mechanical properties of the extraocular muscles of the frog and the dependence of the active developed tension on the muscle extension are very similar to those which are known to exist in the extraocular muscles of other vertebrates. In tetanic activated frog's oculorotatory muscles a linear relationship exists between length and tension. A variation of the stimulation frequency does not change the slope of this curve but causes parallel shifts of the curve. The peculiar properties of the extraocular muscles of the frog are discussed with respect to the muscle fibre types in these muscles and to the diameter of the muscle fibres.     

Entry of cyclic nucleotides into frog muscles in vivo and in vitro

The radioactive isotopes of cyclic nucleotides were used to study the possibility of their income to the isolated frog muscle from the environment as well as their distribution between blood plasma and muscle after injection to an animal. It is shown that the intracellular levels of nucleotides may be considerably higher after soaking the isolated muscle in 10(-6) M cGMP or 10(-4) cAMP solutions. After the cAMP injection into the lymphatic sac its content in blood and especially in muscles is high for a long time. In this case the label loss in muscles proceeds slower than in blood. A conclusion is drawn that conduction of studies on the artificial increase of the intracellular level of cyclic nucleotides by means of their injection to an animal is appropriate.     

Carbonic anhydrase inhibition affects contraction of directly stimulated rat soleus

We have studied the contractile parameters of directly stimulated isolated rat soleus muscles incubated in media containing the carbonic anhydrase inhibitors chlorzolamide (5.10(-4)M) or cyanate (10(-2)M). Both inhibitors caused a decrease in isometric twitch and tetanic (5s) tensions and an increase in muscle relaxation time. It is speculated that among the three types of skeletal muscle carbonic anhydrase it may be the enzyme associated with the sarcoplasmatic reticulum whose inhibition caused the observed changes in contractile parameters.     

Experiments on the role of potassium in the blocking of neuromuscular transmission by curare and other drugs

1. Experiments with perfused frog muscles and with isolated frog muscles immersed in Ringer's solution have failed to show any effect of curare in liberating potassium from muscle tissue. This makes it difficult to suppose that the paralytic effect of curare can be attributed to cation exchange between curare and K whereby a labile potassium compound needed for stimulation is removed from the neuromuscular junction. 2. Similar negative results were obtained with dihydro-β-erythroidine and myanesin. 3. A small liberation of K from perfused muscle does result from treatment with acetylcholine. This is probably due to the contracture of the muscle since the effect is largely eliminated by previous treatment of the muscle with curare. The amount of potassium lost in this way from perfused muscles is too small to detect when muscles are analyzed after immersion in Ringer's solution with and without acetylcholine. It is concluded that there is no significant cation exchange between acetylcholine and K in muscle, but only a small loss of K due to the contracture produced by the acetylcholine.     

The nature of the changes in the activity of water-soluble enzymes in action on the muscles of damaging agents. VI. Glyceraldehyde-3-phosphate dehydrogenase binding and enzyme adsorption by F-actin in ghost muscle fiber]

A possibility of binding glyceraldehyde-3-phosphate dehydrogenase (GAPhDG) in frog (Rana temporaria L.) skeletal muscles was studied by measuring its solubilization in 0.15 M KCl and by its presence in isolated actomyosin. Using a 0.15 M KCl solution, more GAPhDG was extracted from intact muscles and muscles treated with heat at 38, 42 and 46 degrees C for 15 min than in a non-electrolyte medium. Actomyosin isolated from muscles reveals GAPhDG activity which cannot be removed by actomyosin reprecipitation. In myosin-, troponin- and tropomyosin-free single glycerinated muscle fibres (ghost fibres) GAPhDG absorption to F-actin was shown. It is suggested that under thermal injure of muscle cells, the increase in GAPhDG binding with thermolabile proteins of actomyosin complex may occur.     

Contractile effects of 16-methyl analogues of PGE2 on the circular and longitudinal muscles of the guinea-pig isolated colon.

The mechanical effects of 16-methyl analogues of PGE2, mainly 16,16-dimethyl PGE2, on circular and longitudinal muscles of the guinea-pig isolated proximal colon were investigated. In circular muscle strips, PGE2 100 nM produced an initial contraction followed by relaxation, while 16(R)-methyl PGE2 and 16,16-dimethyl PGE2 (1 nM - 1 microM) produced sustained contractions. In longitudinal muscle strips, PGE2 and 16-methyl analogues of PGE2 produced only contractions. The contractile responses of both muscle strips to 16,16-dimethyl PGE2 were not influenced by atropine or tetrodotoxin, indicating that these analogues act directly on the muscles, but were eliminated by the omission of extracellular Ca ions or in the presence of 1 mM lanthanum ions. However, verapamil, a Ca channel blocker, did not block the contractile response to the methyl analogues in circular muscle strips, although it completely inhibited the contractile response of longitudinal muscle strips. These results suggest that the contractile effect of 16-methyl analogues of PGE2 on the circular muscle may be due to an increased influx of Ca ions mainly via receptor-sensitive and partly voltage-sensitive Ca channels, while the contractile effect of the analogues on the longitudinal muscle may be due to an increase in influx of Ca ions via voltage-sensitive Ca channels.     

Comparative study of D-glyceraldehyde-3-phosphate dehydrogenase from frog and pike skeletal muscles]

The purified preparations of glyceraldehyde-3-phosphate dehydrogenase isolated from frog and pike skeletal muscles were found homogenous under polyacrylamide gel electrophoresis. Their amino acid composition is similar to that of glyceraldehyde-3-phosphate dehydrogenase from other animal species. The interaction kinetics for frog and pike glyceraldehyde-3-phosphate dehydrogenase SH-groups with 5,5'-dithio-bis-(2-nitrobenzoate) (DTNB) were studied. A negative correlation between the thermal stability of the enzyme preparations from pig, pike, lamprey and frog muscles and the reactivity of their SH-groups with respect to DTNB was observed. NAD at saturating concentrations was found to protect the enzyme from lower vertebrates muscles against thermal inactivation in a lesser degree than does the pig muscle enzyme. The weaker protective effect of NAD was observed for lamprey and frog enzyme preparations, which are characterized by a low SH-group reaction ability. Frog and pike apoenzymes are considerably more resistant to trypsin proteolysis than the pig apoenzyme.     

Contractile effects of 4-aminopyridine on isolated frog rectus abdominis muscles

The contractile effects of 4-aminopyridine (4-AP) on isolated frog rectus abdominis muscles were examined, and compared with KCl-induced contractures. 4-AP (1-40 mM) caused slowly developing, concentration-dependent contractures which were not modified by (+)-tubocurarine (2.7-13.3 microM). The contractures were prolonged and very slowly relaxed (greater than 30 min) on washout. KCl-induced contractures developed more rapidly and relaxation was equally rapid, both occurring within 90 s of application and washout, respectively. KCl contractures were slightly but significantly (P less than 0.05) attenuated by (+)-tubocurarine in concentrations that blocked carbachol contractures. In calcium-free Ringer's solution, KCl (10-120 mM) responses were completely abolished, but 4-AP concentration-response curves were shifted to the right three- to four-fold. The results show that 4-AP causes contracture of the frog rectus abdominis. It is suggested that at the lower concentration employed (less than 10 mM), 4-AP increases extracellular calcium entry into the muscle, while larger concentrations produce contractures by a direct intracellular mechanism. 4-AP contractures were independent of postjunctional nicotinic cholinoceptor activation.     

Calcium-independent phospholipase A2 modulates cytosolic oxidant activity and contractile function in murine skeletal muscle cells.

Phospholipase A2 (PLA2) activity supports production of reactive oxygen species (ROS) by mammalian cells. In skeletal muscle, endogenous ROS modulate the force of muscle contraction. We tested the hypothesis that skeletal muscle cells constitutively express the calcium-independent PLA2 (iPLA2) isoform and that iPLA2 modulates both cytosolic oxidant activity and contractile function. Experiments utilized differentiated C2C12 myotubes and a panel of striated muscles isolated from adult mice. Muscle preparations were processed for measurement of mRNA by real-time PCR, protein by immunoblot, cytosolic oxidant activity by the dichlorofluorescein oxidation assay, and contractile function by in vitro testing. We found that iPLA2 was constitutively expressed by all muscles tested (myotubes, diaphragm, soleus, extensor digitorum longus, gastrocnemius, heart) and that mRNA and protein levels were generally similar among muscles. Selective iPLA2 blockade by use of bromoenol lactone (10 microM) decreased cytosolic oxidant activity in myotubes and intact soleus muscle fibers. iPLA2 blockade also inhibited contractile function of unfatigued soleus muscles, shifting the force-frequency relationship rightward and depressing force production during acute fatigue. Each of these changes could be reproduced by selective depletion of superoxide anions using superoxide dismutase (1 kU/ml). These findings suggest that constitutively expressed iPLA2 modulates oxidant activity in skeletal muscle fibers by supporting ROS production, thereby influencing contractile properties and fatigue characteristics.     

Muscular fatigue and rate of tension development.

The effects of long-term fatigue upon maximal force and peak rate of tension development (PRTD) (dF/dt max) are studied in man (elbow flexors), in the rat (pseudo-isolated gastrocnemius muscle) and in the frog (isolated sartorius muscle). The muscles are fatigued by voluntary anisometric anisotonic contractions against an elastic resistance in man, and by maximal tetanic contractions in the frog and the rat. In man, the excitation level of the muscle is controlled by the integrated surface EMG of the biceps brachii. In the animals, the muscles are stimulated by a neurostimulator. The PRTD and the maximal isometric force are measured during fatigue tests. In man, frog and rat, the maximal voluntary isometric torque or the maximal force and the PRTD decrease initially more or less rapidly according to the power developed during the fatigue process, and then less rapidly. The relationship between PRTD and maximal force is linear in the animals and curvilinear in man. The variations of maximal force and PRTD are discussed in relation to the level of excitation of the muscles and of the composition in different motor units types and their spatio-temporal recruitment. From a biomechanical point of view, it seems necessary to study the behavior of the series elastic component during the evolution of long term fatigue.     

Localization of Mg2+-ATPase in the membranes of the skeletal muscles and myocardium of the frog Rana temporaria

Using differential centrifugation in sucrose density gradient, from muscles of the frog fractions were obtained which contain fragments of sarcolemma, as well as membranes of T-system tubules and sarcoplasmic reticulum. In isolated membrane fractions, studies were made on the activity of cation-stimulated ATPases (Na+, K+-, Ca2+, Mg2+- and Mg2+-ATPases). Enzymic and electrophoretic analyses showed that the highest content of Mg2+-ATPases is typical of the fractions which are located on the surface of 35% sucrose. The data obtained indicate that Mg2+-ATPase is the enzyme which is specific for the membranes of T-system tubules in skeletal muscles of not only birds but amphibians as well. From cardiac muscle of the frog, membrane fraction was isolated which is similar (with respect to its predominant content of Mg2+-ATPase) to the membranes of T-system tubules. It is suggested that the presence of Mg2+-ATPase in these membranes is a common property of phasic striated muscle fibers in all mature vertebrate animals.