Muscle Fibres Of Xenopus Laevis Research Articles

Myosin heavy chain isoform composition and stretch activation kinetics in single fibres of Xenopus laevis iliofibularis muscle.

Skeletal muscle is composed of specialized fibre types that enable it to fulfil complex and variable functional needs. Muscle fibres of Xenopus laevis, a frog formerly classified as a toad, were the first to be typed based on a combination of physiological, morphological, histochemical and biochemical characteristics. Currently the most widely accepted criterion for muscle fibre typing is the myosin heavy chain (MHC) isoform composition because it is assumed that variations of this protein are the most important contributors to functional diversity. Yet this criterion has not been used for classification of Xenopus fibres due to the lack of an effective protocol for MHC isoform analysis. In the present study we aimed to resolve and visualize electrophoretically the MHC isoforms expressed in the iliofibularis muscle of Xenopus laevis, to define their functional identity and to classify the fibres based on their MHC isoform composition. Using a SDS-PAGE protocol that proved successful with mammalian muscle MHC isoforms, we were able to detect five MHC isoforms in Xenopus iliofibularis muscle. The kinetics of stretch-induced force transients (stretch activation) produced by a fibre was strongly correlated with its MHC isoform content indicating that the five MHC isoforms confer different kinetics characteristics. Hybrid fibre types containing two MHC isoforms exhibited stretch activation kinetics parameters that were intermediate between those of the corresponding pure fibre types. These results clearly show that the MHC isoforms expressed in Xenopus muscle are functionally different thereby validating the idea that MHC isoform composition is the most reliable criterion for vertebrate skeletal muscle fibre type classification. Thus, our results lay the foundation for the unequivocal classification of the muscle fibres in the Xenopus iliofibularis muscle and for gaining further insights into skeletal muscle fibre diversity.

Effects of strain and insulin on myonuclear a-skeletal actin, myogenin and MyoD mRNA content determined by in situ hybridisation in cultured single muscle fibres of Xenopus laevis

Effects of strain and insulin on myonuclear a-skeletal actin, myogenin and MyoD mRNA content determined by in situ hybridisation in cultured single muscle fibres of Xenopus laevis

Twitch and Tetanic Tension during Culture of Mature Xenopus laevis Single Muscle Fibres

Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 µm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days (‘stable fibres’), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture (‘unstable fibres’). After 7–9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors of the long-term behaviour of the fibres.

Twitch and Tetanic Tension during Culture of Mature Xenopus laevis Single Muscle Fibres

Investigation of the mechanisms of muscle adaptation requires independent control of the regulating factors. The aim of the present study was to develop a serum-free medium to culture mature single muscle fibres of Xenopus laevis. As an example, we used the culture system to study adaptation of twitch and tetanic force characteristics, number of sarcomeres in series and fibre cross-section. Fibres dissected from m. iliofibularis (n = 10) were kept in culture at a fibre mean sarcomere length of 2.3 µm in a culture medium without serum. Twitch and tetanic tension were determined daily. Before and after culture the number of sarcomeres was determined by laser diffraction and fibre cross-sectional area (CSA) was determined by microscopy. For five fibres twitch tension increased during culture and tetanic tension was stable for periods varying from 8 to 14 days (‘stable fibres’), after which fibres were removed from culture for analysis. Fibre CSA and the number of sarcomeres in series remained constant during culture. Five other fibres showed a substantial reduction in twitch and tetanic tension within the first five days of culture (‘unstable fibres’). After 7–9 days of culture, three of these fibres died. For two of the unstable fibres, after the substantial force reduction, twitch and tetanic tension increased again. Finally at day 14 and 18 of culture, respectively, the tensions attained values higher than their original values. For stable fibres, twitch contraction time, twitch half-relaxation time and tetanus 10%-relaxation time increased during culture. For unstable fibres these parameters fluctuated. For all fibres the stimulus threshold fluctuated during the first two days, and then remained constant, even for the fibres that were cultured for at least two weeks. It is concluded that the present culture system for mature muscle fibres allows long-term studies within a well-defined medium. Unfortunately, initial tetanic and twitch force are poor predictors of the long-term behaviour of the fibres.

Effects of stretch on mature single muscle fibres of Xenopus laevis in long term culture are not explained by the Feng effect

Effects of stretch on mature single muscle fibres of Xenopus laevis in long term culture are not explained by the Feng effect

Influence of inorganic phosphate and pH on sarcoplasmic reticular ATPase in skinned muscle fibres of Xenopus laevis.

1. The influence of 30 mM inorganic phosphate (Pi) and pH (6.2-7.4) on the rate of ATP utilization was determined in mechanically skinned bundles of myofibrils from the iliofibularis muscle of Xenopus laevis at approximately 5 C. 2. BDM (2,3-butanedione monoxime; 10 mM) depressed isometric force production and actomyosin (AM) ATPase activity equally. Therefore sarcoplasmic reticular (SR) ATPase activity could be determined by extrapolation of the total ATPase activity to zero force. 3. The SR ATPase activity without added Pi at pH 7.1 was 42 +/- 2 % of the total ATPase activity. Addition of 30 mM Pi reduced SR ATPase activity slightly, by 9 +/- 5 %, and depressed force by 62 +/- 2 % and AM ATPase activity by 21 +/- 6 %. 4. At pH 6.2, force, SR ATPase activity and AM ATPase activity were reduced by 21 +/- 5, 61 +/- 5 and 10 +/- 4 % of their respective values at pH 7.1. 5. The SR ATPase activity at 30 mM Pi and pH 6.2 was reduced markedly to 20 +/- 6 % of the value under control conditions, suggesting that the maximum rate of Ca2+ uptake during muscle fatigue was strongly depressed. This reduction was larger than expected on the basis of the effects of Pi and pH alone.

Size Principle of Striated Muscle Cells

We have investigated the relationship between cross-sectional area (CSA) and maximum rate of oxygen consumption (VO2max, in nmol .mm-3 .s-1) of heart and skeletal muscle cells from different species. VO2max and CSA were determined for single muscle fibres of Xenopus laevis at 20°C and for cardiomyocytes in thin trabeculae dissected from the right ventricle of rats at 38°C. Succinate dehydrogenase activity was determined using a quantitative histochemical method to estimate VO2max in mammalian skeletal muscle fibres. Literature values of volume density of mitochondria were used to estimate VO2max in some mammalian cardiomyocytes. We found that an inverse relationship exists between VO2max (range 1.5 to 0.024 nmol . mm-3 . s-1) and cross-sectional area (0.0002 to 0.018 mm2, respectively): VO2max = constant/CSA, where the value of the constant equals 0.39±0.18 pmol . mm-1 .s-1 (mean ± S.D., n = 14). Such a relationship is predicted by a simple Hill-type model for oxygen diffusion in cilindrical cells, if it is assumed that muscle cells are evolved so that anoxic cores in muscle cells are prevented. This indicates that the product of endurance of muscle cells (which is proportional to VO2max) and force production (which is proportional to cross-sectional area) is limited by oxygen diffusion, and that adaptation of heart and skeletal muscle cells to increased workload is limited by the interstitial oxygen tension.

Twitch characteristics and energy metabolites of mature muscle fibres of Xenopus laevis in culture

Mature, high-oxidative, skeletal muscle fibres of Xenopus laevis were kept in culture in L-(Leibovitz's)-15 medium supplemented with creatine and antibiotics and some other additions. Single fibres were mounted at a fixed sarcomere length in a flow-through culture chamber which accommodates stimulus electrodes and a force transducer. Twitch characteristics were determined daily. Depending on culture conditions, fibres remained excitable electrically for up to two weeks at 20 degrees C when foetal calf serum and/or phosphate were added to the culture medium. During the second week, fibres lost phosphocreatine and ATP, but relatively small changes (if any) in total creatine, glycogen and protein contents, fibre volume and dry weight occurred. Succinate dehydrogenase activity decreased after 9 days-when ATP was reduced already. Fibres which were inexcitable electrically contracted normally when exposed to caffeine, indicating that excitation-contraction coupling failed and that the contractile apparatus was still functional.

Augmented force output in skeletal muscle fibres of Xenopus following a preceding bout of activity.

1. The effect of a brief period of activity on subsequent isometric tetanic force production was investigated in single muscle fibres of Xenopus laevis. 2. Following a train of ten tetani separated by 4 s intervals, tetanic force was significantly augmented by about 10%. The tetanic force augmentation persisted for at least 15 min and then slowly subsided. A similar potentiation was seen following trains of five and twenty tetani. 3. During the period of tetanic force potentiation, tetanic calcium was reduced by more than 30%, and intracellular pH was reduced from 7.15 +/- 0.07 to 7.03 +/- 0.11 (n = 4). 4. Fibre swelling was greatest at 1 min and then subsided over 15-20 min and possibly accounted for a small part of the observed force potentiation. 5. A reduction in the inorganic phosphate (P1) concentration of more than 40% was found in fibres frozen in liquid nitrogen at the peak of force potentiation compared with resting fibres. 6. It is concluded that the augmentation of tetanic force found after a brief preceding bout of activity is due to a reduction in inorganic phosphate. This mechanism may underlie the improved performance observed in athletes after warm-up.

Mechano‐sensitive linkage in excitation‐contraction coupling in frog skeletal muscle.

1. Single skeletal muscle fibres of Xenopus laevis were used to investigate the involvement of a mechano-sensitive link in excitation-contraction coupling (EC coupling). 2. Fibres were stimulated by intermittent tetani until tension fell to about 40% of its initial level. Fibres were then stressed either by briefly stretching the fibres to 120% of their resting length or by exposing them to hypotonic Ringer solution ([NaCl] reduced to 80%) for 5 min. 3. In six of thirty-five stretched fibres and in all fourteen fibres exposed to hypotonic solution, a long-lasting depression of tension ensued. Tetanic tension then recovered slowly, often taking more than 10 h to return to its initial level. 4. During the period of minimal tension production, 12 mM caffeine induced a maximum contracture; 190 mM K+ induced a contracture larger than previous or subsequent tetani, and perchlorate (1 mM) slightly augmented tetanic tension. 5. Neither protease inhibitors nor a protein synthesis inhibitor altered the long-lasting period of tension depression and slow recovery. A free-radical scavenger was also without effect. 6. It is concluded that there is a mechano-sensitive link involved in EC coupling which can be damaged easily in fatigued muscle fibres.

ATP utilization for calcium uptake and force production in skinned muscle fibres of Xenopus laevis.

1. A method has been developed to discriminate between the rate of ATP hydrolysis associated with calcium uptake into the sarcoplasmic reticulum (SR) and force development of the contractile apparatus in mechanically or saponin-skinned skeletal muscle fibres. The rate of ATP hydrolysis was determined in fibres of different types from the iliofibularis muscle of Xenopus laevis by enzymatic coupling of ATP re-synthesis to the oxidation of NADH. 2. The ATPase activity was determined before and after exposure of the preparations for 30 min to a solution containing 0.5% Triton X-100, which effectively abolishes the SR ATPase activity. The fibres were activated in a solution containing 5 mM caffeine to ensure that calcium uptake into the SR was maximal. 3. At saturating Ca2+ concentrations the actomyosin (AM) and SR ATPase activities in fast-twitch fibres, at 4.3 degrees C, amounted to 1.52 +/- 0.07 and 0.58 +/- 0.10 mumol s-1 (g dry wt)-1, respectively (means +/- S.E.M.; n = 25). The SR ATPase activity was 25% of the total ATPase activity. At submaximal calcium concentrations the AM ATPase activity varied in proportion to the isometric force. 4. The calcium sensitivity of the SR ATPase was larger than that of the AM ATPase and its dependence on [Ca2+] was less steep. The AM ATPase activity was half-maximal at a pCa of 6.11 (pCa = -log [Ca2+]) whereas the SR ATPase activity was half-maximal at a pCa of 6.62. 5. In Triton X-100-treated fibres, at different 2,3-butanedione monoxime (BDM) concentrations, the AM ATPase activity and isometric force varied proportionally. The SR ATPase activity determined by extrapolation of the total ATPase activity in mechanically skinned or saponin-treated fibres to zero force, was independent of the BDM concentration in the range studied (0-20 mM). The values obtained for the SR ATPase activity in this way were similar to those obtained with Triton X-100 treatment. 6. The AM ATPase activity in slow-twitch fibres amounted to 0.74 +/- 0.13 mumol s-1 (g dry wt)-1, i.e. about a factor of two smaller than in fast-twitch fibres. The SR ATPase activity amounted to 0.47 +/- 0.07 mumol s-1 (g dry wt)-1, i.e. rather similar to the value in fast-twitch fibres. The proportion of the total ATPase activity that was due to SR ATPase (40%) was larger than in fast-twitch fibres. 7. The temperature dependence of the AM and SR ATPase activities in fast-twitch fibres differed. In the temperature range 5-10 degrees C, the relative changes in AM and SR ATPase activities for a 10 degrees C temperature change (Q10) were 3.9 +/- 0.3 and 7.2 +/- 1.5, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Lactate efflux from fatigued fast-twitch muscle fibres of Xenopus laevis under various extracellular conditions.

1. Isolated, fast-twitch, low-oxidative muscle fibres from the iliofibularis muscle of Xenopus laevis were fatigued by intermittent tetanic stimulation at 20 degrees C in different Ringer solutions and the amount of lactate released was determined. 2. The rate of lactate efflux was constant during 10 min of intermittent stimulation while lactate in the fibres accumulated, and lactate efflux was not hampered by an unstirred layer surrounding the isolated muscle fibre. 3. The rate of lactate efflux at extracellular pH 7.2 was the same as that at pH 7.8, but depended on the type of buffer used; the highest efflux rate (mean +/- S.E.M., 7.4 +/- 2.2 mumol min-1 (g dry weight)-1, n = 8) was observed in bicarbonate-buffered Ringer solution. This rate was about 2.5 times higher than the rate in phosphate-buffered Ringer solution (2.9 +/- 1.3 mumol min-1 (g dry weight)-1, n = 8), indicating that lactate-bicarbonate exchange is the most important route for lactate extrusion in vivo. 4. The highest rate of lactate efflux corresponds to a rate of glycolytic ATP production which is only about 30% of the oxidative rate of ATP production (calculated from the maximum rate of oxygen consumption determined previously). 5. In the presence of 5 mM alpha-cyano-4-hydroxycinnamate (CHC) the lowest lactate efflux rate (1.5 +/- 0.6 mumol min-1 (g dry weight)-1, n = 16) was found. This rate was independent of the composition of the Ringer solution. Assuming that 5 mM CHC completely inhibits lactate transporters in the sarcolemma, the rate of lactate efflux in the presence of 5 mM CHC can be explained by passive diffusion, but only if most lactate is extruded via the T-tubules.

Metabolic changes with fatigue in different types of single muscle fibres of Xenopus laevis.

1. Peak isometric force of single fast (type 1) and slow (type 3) muscle fibres of Xenopus decreased when fibres were stimulated intermittently above their predicted sustainable duty cycle at 20 degrees C. Type 1 fibres could be fatigued to zero force. In most type 3 fibres force did not decrease below 50% of the original (P0) before activation failure, as indicated by irregular contractions. 2. Fibres were rapidly frozen at different force levels and analysed by high-performance liquid chromatography (HPLC) for ATP, IMP, phosphocreatine (PCr) and creatine (Cr). Lactate was determined enzymatically in type 1 fibres only. The relationships between force and PCr, and between force and ATP during fatigue were, apart from the range of values obtained, the same for both fibre types. When force had fallen to about 60-80% of original, PCr was fully reduced. At lower force levels, the ATP content-decreased, and a concomitant rise of IMP content was found. At zero force, ATP had fallen to about 25% of its value in rested type 1 fibres, and up to 200 mumol lactate (g dry weight)-1 had accumulated. 3. Recovery from fatigue was studied in fibres where force had fallen to 0.6 P0 (both fibre types) and 0.2 P0 (type 1 only). After 1 h of recovery ATP had in all cases returned to the level measured in rested fibres. In fibres fatigued to 0.6 P0, force almost returned to its original value. However, in type 1 fibres fatigued to 0.2 P0, it returned to only 0.3 P0. After 1 h of recovery the PCr/Cr ratio in type 1 fibres was lower (probability, P less than 0.05) than in control fibres, whereas in type 3 fibres it was not significantly different from controls. 4. The relationship between peak force and stimulus frequency, which had a sigmoid shape in fully rested fibres, was drastically changed by fatiguing stimulation. Immediately after fatiguing stimulation of type 1 fibres, force hardly increased with stimulus frequency, corresponding to the observation that calcium efflux from the sarcoplasmic reticulum was decreased at high stimulus frequencies. The force-frequency relationship of type 3 fibres was the same before and after intermittent stimulation.

Maximum rate of oxygen consumption and quantitative histochemistry of succinate dehydrogenase in single muscle fibres of Xenopus laevis.

Three different types of single living muscle fibre were dissected from the iliofibularis muscle of Xenopus laevis. The fibres were mounted in a glass chamber and their rate of oxygen consumption was determined as a function of twitch frequency at 20 degrees C. The rate of oxygen consumption increased with twitch frequency until it levelled off and reached a maximum. The maximum rate of oxygen consumption varied between fibres (0.019 to 0.161 nmol O2 s-1 mm-3) and was reached at different twitch frequencies (less than 0.2 to 5.7 stimuli s-1). After the determination of the maximum rate of oxygen consumption, the succinate dehydrogenase activity in cross sections of the fibre was determined by means of a quantitative histochemical method. A proportional relationship between the maximum rate of oxygen consumption and the succinate dehydrogenase activity was found. The maximum rate of oxygen consumption and the succinate dehydrogenase activity are also proportional to the volume density of mitochondria in the three fibre types reported by Smith and Ovalle (1973; J. Anat., Lond. 116, 1-24). It is concluded that quantitative histochemistry of succinate dehydrogenase reliably predicts the maximum rate of oxygen consumption of muscle fibres in Xenopus laevis and that the maximum rate of oxygen consumption of single muscle fibres is determined by the volume density of mitochondria.

ATPase activity of intact single muscle fibres of Xenopus laevis is related to the rate of force redevelopment after rapid shortening.

Five different fibre types have been recognized in the iliofibularis muscle of Xenopus laevis. The force-velocity and histochemical characteristics of these fibres vary considerably and differences are also found in their myosin composition. In this study a comparison was made between the rate of ATP hydrolysis estimated from the stable maintenance heat rate and the mechanical performance of fibres of type 1, 2 and 3. In the experiments, firstly the force-velocity relation of a fibre was determined, and subsequently, heat production during isometric tetanic contractions at 20 degrees C was measured. Force redevelopment following the fastest shortening used for the measurement of the force-velocity relationship was fitted to a single exponential. The rate of ATP hydrolysis, estimated from the heat production, was found to be roughly proportional to the rate of force redevelopment. Crossbridge attachment rate was determined by using a simulation of a four state model of the crossbridge cycle. It appears that crossbridge attachment rate is proportional to the in vivo actomyosin ATPase activity during an isometric tetanic contraction.

O-308 - “Longitudinally cut” muscle fibres of Xenopus laevis for the study of excitation-contraction coupling

O-308 - “Longitudinally cut” muscle fibres of Xenopus laevis for the study of excitation-contraction coupling

The influence of voltage conditioning on chloride currents in amphibian muscle membrane: the sigmoid conductance-voltage relation extended into the outward current region.

Sartorius muscle fibres of Xenopus laevis were depolarized in solutions of high K+ content and then voltage clamped in solutions in which K+ was replaced by Rb+ and(or) tetraethylammonium ion (TEA+) at pH 9. A three-microelectrode clamp system was used in which the bath was held at virtual ground using an operational amplifier in a current meter configuration. The holding potential was set at zero membrane current potential (resting potential), close to -25 mV. A two-pulse paradigm was used to test the effects of conditioning the membrane at voltages away from the resting potential on initial currents at positive test potentials. In the absence of TEA+ rapidly rising outward currents were generated at positive test potentials, following hyperpolarizing conditioning. These currents inactivated in time and obscured predicted chloride currents. When TEA+ was added to the solution (60 mequiv./L) the currents at positive potentials rose more slowly and declined either very slowly or not at all. Projection of these current waveforms, by curve fitting, to the instant of potential change gave a sigmoid dependence of test current on conditioning voltage that was predicted from earlier results. Predictably there is a test voltage at which the initial current is independent of conditioning potential: from the data it appears that this is not necessarily the resting potential, but the cause of the shift is not clear. The results also indicated that there is a component of outward current that is very small, apparently carried by cations ("delayed rectifier current"), that does not inactivate, even at potentials more positive than the resting potential.

Effects of SITS on chloride permeation in Xenopus skeletal muscle.

Small signal cable analysis has been used to estimate the resting membrane chloride conductance of sartorius muscle fibres of Xenopus laevis. Muscles were bathed in solutions in which potassium was replaced by rubidium at pH's 8.8, 7.04, and 5.25. The conductance ratio in these solutions was 12:5:1. The conductance was significantly reduced at all pH's by the addition of 4-acetamido-4′-isothiocyano-2,2′-stilbene disulphonic acid (SITS). Rubidium conductance was determined as the membrane conductance in a solution in which potassium was replaced by rubidium and chloride was replaced by sulphate. Rubidium conductance was not pH sensitive. Subtraction of rubidium conductance indicated that 0.2 mM SITS reduced chloride conductance by about 50%, regardless of pH, and 1 mM SITS reduced it by at least 75%. When 2 mM SITS was added to solutions containing 120 and 30 mM chloride (2.5 mM potassium) the membrane potential of the muscle became insensitive to changes of chloride concentration in the bathing medium.It is concluded that SITS exerts a blocking effect on chloride conductance and permeability in Xenopus muscal membrane by binding to a site in the chloride channel that is not itself sensitive to the external pH.

Latency-relaxation in single muscle fibres.

1. Latency relaxation and twitch tension were recorded simultaneously in single isolated muscle fibres of Xenopus laevis. 2. During low frequency (0.6 or 1 pulse/sec) repetitive stimulation, three successive phases of twitch tension were observed: negative staricase (a slight drop in tension), positive staircase (about 15% increase in tension) and fatigue. At the same time the amplitude of latency relaxation decreased monotonically, and near the peak of positive staircase, the amplitude decreased almost to an undetectable level. 3. The application of caffeine (0.1--1.5 mM) increased peak twitch tension by 15--200%, but decreased the latency relaxation amplitude by 30--93%. 4. The application of Ca-release inhibitors, deuterium oxide and dantrolene sodium, caused a 43--89% decline in peak twitch tension but no change in latency relaxation amplitude. 5. The lack of correlation between changes in peak twitch tension and latency relaxation amplitude suggests that latency relaxation is associated with the mechanism which triggers Ca2+ relase rather than with Ca2+ release itself.

The Effect of Calcium on Potassium Contractures of Single Slow Muscle Fibres of Xenopus Laevis

AbstractIsolated slow muscle fibres of Xenopus laevis were subjectcd to step increases in external potassium concentration at various external calcium concentrations. An increase in [Ca]o shifted the curve relating peak tension to log [K]o to higher K‐koncentrations. a decrease in [Ca]o caused a shift in the opposite direction. Similar effects were seen on the relation between inactivation and log [K]o+. The peak tension was but little affected by [Ca]o; the time‐course of contractures was altered in such a way that the rate of relaxation was increased in low [Ca]o and decreased in high [Ca]o. Evidence was obtained that the effect of altered [Ca]o occurs as rapidly as that of altered [K]o. Some observations were also made on contractures in Ca‐free solutions and in solutions with Na partially subatituted bv Ba.