The effects of the myosin-II inhibitor N-benzyl-p-toluene sulphonamide on fatigue in mouse single intact toe muscle fibres.

AIM: This study determined whether fatigue in skeletal muscle is primarily due to the repeated elevations of myoplasmic free calcium concentration ([Ca(2+)](i)) or to metabolite accumulation. METHODS: We examined the effects of N-benzyl-p-toluene sulphonamide (BTS) which is a potent and specific inhibitor of fast muscle myosin-II on the development of fatigue in mouse flexor digitorum brevis (FDB) muscle fibres. Single intact FDB fibres were micro-injected with indo-1 to monitor changes in [Ca(2+)](i) and stimulated repeatedly for a maximum of 150 tetani or until force declined to 40%. RESULTS: BTS markedly reduced tetanic force but had no effect on the tetanic [Ca(2+)](i) transients. When fatigue was induced in the presence of BTS, the reduction in [Ca(2+)](i) and force transients occurred much more slowly than in the absence of BTS. The extent of force depression was similar after induction of fatigue in fibres exposed to Tyrode only or to BTS and force recovered to the same extent. CONCLUSION: The results suggest that the decrease in tetanic [Ca(2+)](i) and force caused during fatigue are due mainly to accumulated metabolic changes.

Effects of concentric and eccentric contractions on phosphorylation of MAPK(erk1/2) and MAPK(p38) in isolated rat skeletal muscle.

1. Exercise and contractions of isolated skeletal muscle induce phosphorylation of mitogen-activated protein kinases (MAPKs) by undefined mechanisms. The aim of the present study was to determine exercise-related triggering factors for the increased phosphorylation of MAPKs in isolated rat extensor digitorum longus (EDL) muscle. 2. Concentric or eccentric contractions, or mild or severe passive stretches were used to discriminate between effects of metabolic/ionic and mechanical alterations on phosphorylation of two MAPKs: extracellular signal-regulated kinase 1 and 2 (MAPK(erk1/2)) and stress-activated protein kinase p38 (MAPK(p38)). 3. Concentric contractions induced a 5-fold increase in MAPK(erk1/2) phosphorylation. Application of the antioxidants N-acetylcysteine (20 mM) or dithiothreitol (5 mM) suppressed concentric contraction-induced increase in MAPK(erk1/2) phosphorylation. Mild passive stretches of the muscle increased MAPK(erk1/2) phosphorylation by 1.8-fold, whereas the combination of acidosis and passive stretches resulted in a 2.8-fold increase. Neither concentric contractions, nor mild stretches nor acidosis significantly affected phosphorylation of MAPK(p38). 4. High force applied upon muscle by means of either eccentric contractions or severe passive stretches resulted in 5.7- and 9.5-fold increases of phosphorylated MAPK(erk1/2), respectively, whereas phosphorylation of MAPK(p38) increased by 7.6- and 1.9-fold (not significant), respectively. 5. We conclude that in isolated rat skeletal muscle an increase in phosphorylation of both MAPK(erk1/2) and MAPK(p38) is induced by mechanical alterations, whereas contraction-related metabolic/ionic changes (reactive oxygen species and acidosis) cause increased phosphorylation of MAPK(erk1/2) only. Thus, contraction-induced phosphorylation can be explained by the combined action of increased production of reactive oxygen species, acidification and mechanical perturbations for MAPK(erk1/2) and by high mechanical stress for MAPK(p38).

Contraction and intracellular Ca(2+) handling in isolated skeletal muscle of rats with congestive heart failure.

A decreased exercise tolerance is a common symptom in patients with congestive heart failure (CHF). This decrease has been suggested to be partly due to altered skeletal muscle function. Therefore, we have studied contractile function and cytoplasmic free Ca(2+) concentration ([Ca(2+)](i), measured with the fluorescent dye indo 1) in isolated muscles from rats in which CHF was induced by ligation of the left coronary artery. The results show no major changes of the contractile function and [Ca(2+)](i) handling in unfatigued intact fast-twitch fibers isolated from flexor digitorum brevis muscles of CHF rats, but these fibers were markedly more susceptible to damage during microdissection. Furthermore, CHF fibers displayed a marked increase of baseline [Ca(2+)](i) during fatigue. Isolated slow-twitch soleus muscles of CHF rats displayed slower twitch contraction and tetanic relaxation than did muscles from sham-operated rats; the slowing of relaxation became more pronounced during fatigue in CHF muscles. Immunoblot analyses of sarcoplasmic reticulum proteins and sarcolemma Na(+),K(+)-ATPase showed no difference in flexor digitorum brevis muscles of sham-operated versus CHF rats. In conclusion, functional impairments can be observed in limb muscle isolated from rats with CHF. These impairments seem to mainly involve structures surrounding the muscle cells and sarcoplasmic reticulum Ca(2+) pumps, the dysfunction of which becomes obvious during fatigue.

Changes in mitochondrial Ca2+ detected with Rhod-2 in single frog and mouse skeletal muscle fibres during and after repeated tetanic contractions.

The present study investigated mitochondrial Ca2+ uptake and release in intact living skeletal muscle fibres subjected to bouts of repetitive activity. Confocal microscopy was used in conjunction with the Ca2+-sensitive dye Rhod-2 to monitor changes in mitochondrial Ca2+ in single Xenopus or mouse muscle fibres. A marked increase in the mitochondrial Ca2+ occurred in Xenopus fibres after 10 tetani applied at 4 s intervals. The mitochondrial Ca2+ continued to increase with increasing number of tetani. After the end of tetanic stimulation, mitochondrial Ca2+ declined to 50% of the maximal increase within 10 min and thereafter took up to 60 min to return to its original value. Depolarization of the mitochondria with FCCP greatly attenuated the rise in the mitochondrial Ca2+ evoked by repetitive tetanic stimulation. In addition, FCCP slowed the rate of decay of the tetanic Ca2+ transient which in turn led to an elevation of resting cytosolic Ca2+. Accumulation of Ca2+ in the mitochondria was accompanied by a modest mitochondrial depolarization. In contrast to the situation in Xenopus fibres, mitochondria in mouse toe muscle fibres did not show any change in the mitochondrial Ca2+ during repetitive stimulation and FCCP had no effect on the rate of decay of the tetanic Ca2+ transient. It is concluded that in Xenopus fibres, mitochondria play a role in the regulation of cytosolic Ca2+ and contribute to the relaxation of tetanic Ca2+ transients. In contrast to their important role in Xenopus fibres, mitochondria in mouse fast-twitch skeletal fibres play little role in Ca2+ homeostasis.

Functional significance of Ca2+ in long-lasting fatigue of skeletal muscle.

Repeated activation of skeletal muscle causes fatigue, which involves a reduced ability to produce force and slowed contraction regarding both the speed of shortening and relaxation. One important component in skeletal muscle fatigue is a reduced sarcoplasmic reticulum (SR) Ca2+ release. In the present review we will describe different types of fatigue-induced inhibition of SR Ca2+ release. We will focus on a type of long-lasting failure of SR Ca2+ release which is called low-frequency fatigue, because this type of fatigue may be involved in the muscle dysfunction and chronic pain experienced by computer workers. Paradoxically it appears that the Ca2+ released from the SR, which is required for contraction, may actually be responsible for the failure of SR Ca2+ release during low-frequency fatigue. We will also discuss the relationship between gross morphological changes in muscle fibres and long-lasting failure of SR Ca2+ release. Finally, a model linking muscle cell dysfunction and muscle pain is proposed.

Vacuole formation in fatigued skeletal muscle fibres from frog and mouse: effects of extracellular lactate.

Isolated, living muscle fibres from either Xenopus or mouse were observed in a confocal microscope and t-tubules were visualized with sulforhodamine B. Observations were made before and after fatiguing stimulation. In addition, experiments were performed on fibres observed in an ordinary light microscope with dark-field illumination. In Xenopus fibres, recovering after fatigue, t-tubules started to show dilatations 2-5 min post-fatigue. These swellings increased in size over the next 10-20 min to form vacuoles. After 2-3 h of recovery the appearance of the fibres was again normal and force production, which had been markedly depressed 10-40 min post-fatigue, was close to control. Vacuoles were not observed in mouse fibres, fatigued with the same protocol and allowed to recover. In Xenopus fibres, fatigued in normal Ringer solution and allowed to recover in Ringer solution with 30-50 mM L-lactate substituting for chloride (lactate-Ringer), the number and size of vacuoles were markedly reduced. Also, force recovery was significantly faster. Replacement of chloride by methyl sulphate or glucuronate had no effect on vacuolation. Resting Xenopus fibres exposed to 50 mM lactate-Ringer and transferred to normal Ringer solution displayed vacuoles within 5-10 min, but to a smaller extent than after fatigue. Vacuolation was not associated with marked force reduction. Mouse fibres, fatigued in 50 mM lactate-Tyrode (L-lactate substituting for chloride in Tyrode solution) and recovering in normal Tyrode solution, displayed vacuoles for a limited period post-fatigue. Vacuolation had no effect on force production. The results are consistent with the view that lactate, formed during fatigue, is transported into the t-tubules where it attracts water and causes t-tubule swelling and vacuolation. This vacuolation may be counteracted in vivo due to a gradual extracellular accumulation of lactate during fatigue.

Isometric force and endurance in soleus muscle of thyroid hormone receptor-alpha(1)- or -beta-deficient mice.

The specific role of each subtype of thyroid hormone receptor (TR) on skeletal muscle function is unclear. We have therefore studied kinetics of isometric twitches and tetani as well as fatigue resistance in isolated soleus muscles of R-alpha(1)- or -beta-deficient mice. The results show 20-40% longer contraction and relaxation times of twitches and tetani in soleus muscles from TR-alpha(1)-deficient mice compared with their wild-type controls. TR-beta-deficient mice, which have high thyroid hormone levels, were less fatigue resistant than their wild-type controls, but contraction and relaxation times were not different. Western blot analyses showed a reduced concentration of the fast-type sarcoplasmic reticulum Ca(2+)-ATPase (SERCa1) in TR-alpha(1)-deficient mice, but no changes were observed in TR-beta-deficient mice compared with their respective controls. We conclude that in skeletal muscle, both TR-alpha(1) and TR-beta are required to get a normal thyroid hormone response.

Frog skeletal muscle fibers recovering from fatigue have reduced charge movement.

Following prolonged exercise, muscle force production is often impaired. One possible cause of this force deficit is impaired intracellular activation. We have used single skeletal muscle fibers from the lumbrical muscle of Xenopus laevis to study the effects of fatigue on excitation-contraction coupling. Fatigue was induced in 13 intact fibers. Five fibers recovered in normal Ringer only (fatigued-only fibers). The remaining eight fibers were subjected to a brief hypotonic treatment (F-H fibers) that is known to prolong the effects of fatigue. Intramembrane charge movement, changes in intracellular calcium concentration ([Ca2+]i) and force transients were measured in a single Vaseline gap chamber under voltage clamp. In F-H fibers, membrane capacitance was reduced. Confocal microscopy showed that this was not due to closure of the transverse tubules. The amount of normalized intramembrane charge was reduced from 21.0 +/- 2.8 nC/microF (n = 10) in rested fibers to 12.2 +/- 1.1 nC/microF in F-H fibers. However, the voltage dependence of intramembrane charge movement was unchanged. In F-H fibers, force production was virtually abolished. This was the consequence of the greatly reduced [Ca2+]i accompanying a depolarizing pulse. In recovering fatigued-only fibers, while the maximal available charge was not significantly smaller (18.3 +/- 1.1 nC/ microF), both calcium and force were reduced, albeit to a lesser extent than in F-H fibers. The data are consistent with a model where fatigue reduces the number of voltage sensors in the t-tubules and, in addition, alters the coupling between the remaining functional voltage sensors and the calcium channels of the sarcoplasmic reticulum.

The use of caged adenine nucleotides and caged phosphate in intact skeletal muscle fibres of the mouse.

The effects of 1-(2-nitrophenyl)ethyl ester of ATP (NPE-caged ATP), NPE-caged ADP, NPE-caged phosphate (Pi) and desoxybenzoinyl phosphate (desyl-caged Pi) on mouse skeletal muscle function were studied. All these caged compounds, when microinjected into intact single mouse muscle fibres, reduced the myoplasmic calcium during a tetanus (tetanic [Ca2+]i) and reduced force. Flash photolysis partially reversed this reduction of tetanic [Ca2+]i and force. In fibres fatigued by repeated tetani, flash photolysis of NPE-caged ATP, ADP and Pi, also caused a transient recovery of tetanic [Ca2+]i, and force. Because photolytic release of ATP, ADP and Pi produced comparable effects it seems that the mechanism of action is the reduction in concentration of the caged compound rather than the release of the biologically active molecule. Experiments on mechanically skinned rat skeletal muscle fibres with intact T-tubular/sarcoplasmic reticulum coupling showed that 1 mM NPE-caged ATP had no effect on depolarization-induced force. This result suggests that the depressant effects of the NPE-caged compounds are neither on voltage-activated Ca2+ release from the sarcoplasmic reticulum nor on myofibrillar function. Thus all the caged compounds tested inhibit excitation-contraction coupling in muscle by an unknown mechanism and this limits their value as sources of biologically important molecules. This inhibitory effect was smallest for desyl-caged Pi and under conditions of maximal activation photolytic release of Pi caused a direct inhibition of the contractile proteins. This inhibition amounted to a 1% reduction of maximum force with an increase of [Pi] of about 0.3 mM. The mean rate of force decline under these conditions was 55 s-1, which reflects the rate of cross-bridge cycling during a maximal tetanus.

Vacuole formation in fatigued single muscle fibres from frog and mouse.

Force recovery from fatigue in skeletal muscle may be very slow. Gross morphological changes with vacuole formation in muscle cells during the recovery period have been reported and it has been suggested that this is the cause of the delayed force recovery. To study this we have used confocal microscopy of isolated, living muscle fibres from Xenopus and mouse to visualise transverse tubules (t-tubules) and mitochondria and to relate possible fatigue-induced morphological changes in these to force depression. T-tubules were stained with either RH414 or sulforhodamine B and mitochondrial staining was with either rhodamine 123 or DiOC6(3). Fatigue was produced by repeated, short tetanic contractions. Xenopus fibres displayed a marked vacuolation which started to develop about 2 min after fatiguing stimulation, reached a maximum after about 30 min, and then receded in about 2 h. Vacuoles were never seen during fatiguing stimulation. The vacuoles developed from localised swellings of t-tubules and were mostly located in rows of mitochondria. Mitochondrial staining, however, showed no obvious alterations of mitochondrial structure. There was no clear correlation between the presence of vacuoles and force depression; for instance, some fibres showed massive vacuole formation at a time when force had recovered almost fully. Vacuole formation was not reduced by cyclosporin A, which inhibits opening of the non-specific pore in the mitochondrial inner membrane. In mouse fibres there was no vacuole formation or obvious changes in mitochondrial structure after fatigue, but still these fibres showed a marked force depression at low stimulation frequencies ('low-frequency fatigue'). Vacuoles could be produced in mouse fibres by glycerol treatment and these vacuoles were not associated with any force decline. In conclusion, vacuoles originating from the t-tubular system develop after fatigue in Xenopus but not in mouse fibres. These vacuoles are not the cause of the delayed force recovery after fatigue.

Effects of CO2-induced acidification on the fatigue resistance of single mouse muscle fibers at 28 degrees C.

The role of reduced muscle pH in the development of skeletal muscle fatigue is unclear. This study investigated the effects of lowering skeletal muscle intracellular pH by exposure to 30% CO2 on the number of isometric tetani needed to induce significant fatigue. Isolated single mouse muscle fibers were stimulated repetitively at intervals of 4-2.5 s by using 80-Hz, 400-ms tetani at 28 degrees C in Tyrode solution bubbled with either 5 or 30% CO2. Stimulation continued until tetanic force had fallen to 40% of the initial value. Exposure to 30% CO2 caused a significant fall in intracellular pH of approximately 0.3 pH unit but did not cause any significant changes in initial peak tetanic force. During the course of repetitive stimulation, intracellular pH fell by approximately 0.3 pH unit in both normal and acidified fibers. The number of tetani needed to reduce force to 40% of the initial value was not significantly different in 5 and 30% CO2 Tyrode. The sole effect of acidosis was to reduce the rate of relaxation of force, especially in fatigued fibers. It is concluded that, at 28 degrees C, acidosis per se does not accelerate the development of fatigue during repeated tetanic stimulation of isolated mouse skeletal muscle fibers.

Mechanisms underlying reduced maximum shortening velocity during fatigue of intact, single fibres of mouse muscle.

1. The mechanism behind the reduction in shortening velocity in skeletal muscle fatigue is unclear. In the present study we have measured the maximum shortening velocity (V0) with slack tests during fatigue produced by repeated, 350 ms tetani in intact, single muscle fibres from the mouse. We have focused on two possible mechanisms behind the reduction in V0: reduced tetanic Ca2+ and accumulation of ADP. 2. During fatigue V0 initially declined slowly, reaching 90 % of the control after about forty tetani. The rate of decline then increased and V0 fell to 70 % of the control in an additional twenty tetani. The reduction in isometric force followed a similar pattern. 3. Exposing unfatigued fibres to 10 microM dantrolene, which reduces tetanic Ca2+, lowered force by about 35 % but had no effect on V0. 4. In order to see if ADP might increase rapidly during ongoing contractions, we used a protocol with a tetanus of longer duration bracketed by standard-duration tetani. V0 in these three tetani were not significantly different in control, whereas V0 was markedly lower in the longer tetanus during fatigue and in unfatigued fibres where the creatine kinase reaction was inhibited by 10 microM dinitrofluorobenzene. 5. We conclude that the reduction in V0 during fatigue is mainly due to a transient accumulation of ADP, which develops during contractions in fibres with impaired phosphocreatine energy buffering.

Mechanisms underlying the reduction of isometric force in skeletal muscle fatigue.

A decline of isometric force production is one characteristic of skeletal muscle fatigue. In fatigue produced by repeated short tetani, this force decline can be divided into two components: a reduction of the cross-bridges' ability to generate force, which comes early; and a reduction of the sarcoplasmic reticulum Ca2+ release, which develops late in fatigue. Acidification due to lactic acid accumulation has been considered as an important cause of the reduced cross-bridge force production. However, in mammalian muscle it has been shown that acidification has little effect on isometric force production at physiological temperatures. By exclusion, in mammalian muscle fatigue, the reduction of force due to impaired cross-bridge function would be caused by accumulation of inorganic phosphate ions, which results from phosphocreatine breakdown. The reduction of sarcoplasmic reticulum Ca2+ release in late fatigue correlates with a decline of ATP and we speculate that the reduced Ca2+ release is caused by a local increase of the ADP/ATP ratio in the triads.

Mechanisms underlying the slow recovery of force after fatigue: importance of intracellular calcium.

Recovery of force production after an intense bout of activity may sometimes take several days, especially at low activation frequencies ('low frequency fatigue'). This slow recovery can also be observed in isolated muscle and single muscle fibres. The origin of the force deficit is failure of excitation-contraction coupling at the level of the triads. The most likely cause of the failure is an elevated intracellular Ca2+ level, but the site of action of Ca2+ is unclear. Available evidence does not support the involvement of Ca2+-activated proteases. Ca2+-induced damage to mitochondria or swelling of t-tubules do not seem to be causative factors. Other mechanisms are discussed, including possible detrimental effects of Ca2+-activated lipases, calmodulin, and reactive oxygen species.

The effect of intracellular pH on contractile function of intact, single fibres of mouse muscle declines with increasing temperature.

1. The effect of altered intracellular pH (pHi) on isometric contractions and shortening velocity at 12, 22 and 32 degrees C was studied in intact, single fibres of mouse skeletal muscle. Changes in pHi were obtained by exposing fibres to solutions with different CO2 concentrations. 2. Under control conditions (5% CO2), pHi (measured with carboxy SNARF-1) was about 0.3 pH units more alkaline than neutral water at each temperature. An acidification of about 0.5 pH units was produced by 30% CO2 and an alkalinization of similar size by 0% CO2. 3. In acidified fibres tetanic force was reduced by 28% at 12 degrees C but only by 10% at 32 degrees C. The force increase with alkalinization showed a similar reduction with increasing temperature. Acidification caused a marked slowing of relaxation and this slowing became less with increasing temperature. 4. Acidification reduced the maximum shortening velocity (V0) by almost 20% at 12 degrees C, but had no significant effect at 32 degrees C. Alkalinization had no significant effect on V0 at any temperature. 5. In conclusion, the effect of pHi on contraction of mammalian muscle declines markedly with increasing temperature. Thus, the direct inhibition of force production by acidification is not a major factor in muscle fatigue at physiological temperatures.

Slowed relaxation in fatigued skeletal muscle fibers of Xenopus and Mouse. Contribution of [Ca2+]i and cross-bridges.

Slowing of relaxation is an important characteristic of skeletal muscle fatigue. The aim of the present study was to quantify the relative contribution of altered Ca2+ handling (calcium component) and factors downstream to Ca2+ (cross-bridge component) to the slowing of relaxation in fatigued fibers of Xenopus and mouse. Two types of Xenopus fibers were used: easily fatigued, type 1 fibers and fatigue resistant, type 2 fibers. In these Xenopus fibers the free myoplasmic [Ca2+] ([Ca2+]i) was measured with indo-1, and the relaxation of Ca2(+)-derived force, constructed from tetanic [Ca2+]i records and in vivo [Ca2+]i-force curves, was analyzed. An alternative method was used in both Xenopus and mouse fibers: fibers were rapidly shortened during the initial phase of relaxation, and the time to the peak of force redevelopment was measured. These two methods gave similar results and showed proportional slowing of the calcium and cross-bridge components of relaxation in both fatigued type 1 and type 2 Xenopus fibers, whereas only the cross-bridge component was slowed in fatigued mouse fibers. Ca2+ removal from the myoplasm during relaxation was markedly less effective in Xenopus fibers as compared to mouse fibers. Fatigued Xenopus fibers displayed a reduced rate of sarcoplasmic reticulum Ca2+ uptake and increased sarcoplasmic reticulum Ca2+ leak. Some fibers were stretched at various times during relaxation. The resistance to these stretches was increased during fatigue, especially in Xenopus fibers, which indicates that longitudinal movements during relaxation had become less pronounced and this might contribute to the increased cross-bridge component of relaxation in fatigue. In conclusion, slowing of relaxation in fatigued Xenopus fibers is caused by impaired Ca2+ handling and altered cross-bridge kinetics, whereas the slowing in mouse fibers is only due to altered cross-bridge kinetics.

The role of ATP in the regulation of intracellular Ca2+ release in single fibres of mouse skeletal muscle.

1. Single fibres were dissected from mouse flexor brevis muscle and injected with indo-1 and the P3-1 (2-nitrophenyl)ethyl ester of ATP (caged ATP). Myoplasmic calcium concentration ([Ca2+]i) and force were monitored during single tetani or tetani repeated until force was reduced to about 30% of control values. In vitro experiments showed that an intense, brief ultraviolet illumination (a flash) photolysed 12% of the caged ATP to ATP. 2. Fibres that had been injected with caged ATP showed concentration-dependent changes. High concentrations of caged ATP caused a reduction in [Ca2+]i during tetani (tetanic [Ca2+]i), a reduction in force in unfatigued tetani and the fibres fatigued more rapidly when stimulated repeatedly. 3. Photolytic release of ATP in unfatigued fibres caused a concentration-dependent increase in tetanic [Ca2+]i and in force. 4. When ATP was released by photolysis in a fibre fatigued by repeated tetani, it produced a concentration-dependent increase in tetanic [Ca2+]i and force. The increase in tetanic [Ca2+]i was small (63 nM per 100 microM increase in ATP) and could explain some, but not all, the increase in force. However, taking into account the fact that control flashes in the absence of caged ATP caused a small decrease in tetanic [Ca2+]i, we believe that the increase in force may be explained by the increase in tetanic [Ca2+]i. There was no evidence of changes in the sarcoplasmic reticulum Ca2+ pump rate after photolysis of caged ATP. 5. Caged ATP affects some site(s) involved in excitation-contraction coupling and the consequences are similar to muscle fatigue. When a small fraction of this caged ATP is photolysed to ATP, the consequences of fatigue are partially reversed. These observations suggest that site(s) which either bind ATP or depend on ATP hydrolysis have a key role in excitation-contraction coupling and in muscle fatigue.

Effects of repetitive tetanic stimulation at long intervals on excitation-contraction coupling in frog skeletal muscle.

1. Single skeletal muscle fibres of Xenopus frogs were used to investigate the possibility that excitation-contraction (E-C) coupling can be impaired under conditions of elevated intracellular free Ca2+ ([Ca2+]i). 2. Fibres were stimulated with a train of up to 200 tetani at 10 or 20s intervals; this long-interval stimulation (LIS) scheme was chosen to minimize fatigue. After LIS, fibres were exposed to hypotonic Ringer solution for 5 min. At the end of LIS, force was about 90% of the original and the hypotonic challenge did not result in any force depression. 3. Caffeine, terbutaline and 2,5-di(tert-butyl)-1,4-benzohydroquinone increased both basal and tetanic [Ca2+]i. In ten out of thirteen fibres, the presence of any of these drugs during LIS resulted in a force reduction to about 10% of the control when fibres were returned to normal Ringer solution after the hypotonic challenge. Force production was severely depressed for at least 20 min and then recovered to control levels within 120 min. 4. Neither protease inhibitors nor a scavenger of reactive oxygen species prevented the impairment of E-C coupling. 5. It is concluded that after a period of elevated [Ca2+]i, E-C coupling in frog skeletal muscle becomes sensitive to the mechanical stress induced by exposure to hypotonic solution. The underlying molecular basis for this remains unclear.

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.

Reversible depression of action potentials and force production in frog single muscle fibres by calmodulin-inhibitors.

The effects of the calmodulin-inhibitors trifluoperazine, thioridazine and zaldaride maleate on the responses to electrical stimulation in isolated frog skeletal muscle fibres were investigated. All three drugs initially reduced the amplitude of the action potentials but potentiated twitch force. This was followed by a total loss of action potentials and force production. However, the resting membrane potential was not changed. The effects were completely reversible upon removal of the drugs. These results suggest that an intact calmodulin system is required for normal function of the sarcolemmal sodium channels of frog skeletal muscle.