The contribution of the sarcoplasmic reticulum Ca2+-transport ATPase to caffeine-induced Ca2+ transients of murine skinned skeletal muscle fibres

The present study was carried out to investigate the contribution of the Ca²⁺-transport ATPase of the sarcoplasmic reticulum (SR) to caffeine-induced Ca²⁺ release in skinned skeletal muscle fibres. Chemically skinned fibres of balb-C-mouse EDL (extensor digitorum longus) were exposed for 1 min to a free Ca²⁺ concentration of 0.36 μM to load the SR with Ca²⁺. Release of Ca²⁺ from the SR was induced by 30 mM caffeine and recorded as an isometric force transient. For every preparation a pCa/force relationship was constructed, where pCa = −log₁₀ [Ca²⁺]. In a new experimental approach, we used the pCa/force relationship to transform each force transient directly into a Ca²⁺ transient. The calculated Ca²⁺ transients were fitted by a double exponential function: Y ₀ + A ₁⋅exp (−t/t ₁) + A ₂⋅exp(t/t ₂), with A ₁ < 0 < A ₂, t ₁ < t ₂ and Y ₀, A ₁, A ₂ in micromolar. Ca²⁺ transients in the presence of the SR Ca²⁺-ATPase inhibitor cyclopiazonic acid (CPA) were compared to those obtained in the absence of the drug. We found that inhibition of the SR Ca²⁺-ATPase during caffeine-induced Ca²⁺ release causes an increase in the peak Ca²⁺ concentration in comparison to the control transients. Increasing CPA concentrations prolonged the time-to-peak in a dose-dependent manner, following a Hill curve with a half-maximal value of 6.5 ± 3 μM CPA and a Hill slope of 1.1 ± 0.2, saturating at 100 μM. The effects of CPA could be simulated by an extended three-compartment model representing the SR, the myofilament space and the external bathing solution. In terms of this model, the SR Ca²⁺-ATPase influences the Ca²⁺ gradient across the SR membrane in particular during the early stages of the Ca²⁺ transient, whereas the subsequent relaxation is governed by diffusional loss of Ca²⁺ into the bathing solution. Received: 2 February 1996/Accepted: 1 April 1996     

Effects of cyclopiazonic acid on Ca2+ regulation by the sarcoplasmic reticulum in saponin-permeabilized skeletal muscle fibres

 The effects of the sarcoplasmic reticulum (SR) Ca²⁺ pump inhibitor cyclopiazonic acid (CPA) were studied in saponin-permeabilized frog skeletal muscle fibres. Release of Ca²⁺ from the SR was triggered by brief (2 s) applications of 40 mM caffeine at 2-min intervals. Changes in [Ca²⁺] within the fibre were monitored continuously using Fura-2 fluorescence. At a bathing [Ca²⁺] of 100 nM, introduction of 20 μM CPA induced a slow release of Ca²⁺ from the SR. The following one to two caffeine-induced Ca²⁺ transients were markedly increased in amplitude and duration. Thereafter, the caffeine-induced Ca²⁺ transients decreased progressively and were barely detectable 6–7 min after introduction of CPA. However, increasing the bathing [Ca²⁺] or increasing the Ca²⁺ loading period resulted in a partial recovery of the caffeine-induced Ca²⁺ transients, suggesting that pump inhibition is incomplete, even in the presence of 100 μM CPA. The slow Ca²⁺ efflux induced by CPA was insensitive to ryanodine, but absent following abolition of SR Ca²⁺ pump activity by ATP withdrawal. These results suggest that the caffeine-induced Ca²⁺ transient reflects a balance between efflux via the SR Ca²⁺ channel and reuptake by the Ca pump. Ca²⁺ release upon addition of CPA may result from inhibition of SR Ca²⁺ uptake, which reveals a tonic Ca²⁺ efflux that is independent of the Ca²⁺ release channels. Received: 26 November 1997 / Received after revision: 12 January 1998 / Accepted: 13 January 1998     

Effects of thapsigargin and cyclopiazonic acid on sarcoplasmic reticulum Ca2+ uptake,spontaneous force oscillations and myofilament Ca2+ sensitivity in skinned rat ventricular trabeculae

Thapsigargin (TG) and cyclopiazonic acid (CPA) have been reported to be potent inhibitors of the sarcoplasmic reticulum (SR) Ca²⁺ uptake in isolated SR vesicles and cells. We have examined the effect of TG and CPA on (1) the Ca²⁺ uptake by the SR in saponin-skinned rat ventricular trabeculae, using the amplitude of the caffeine-induced contraction to estimate the Ca²⁺ content loaded into the SR, (2) the spontaneous Ca²⁺ oscillations at pCa 6.6 using force oscillation as the indicator, and (3) the myofilament Ca²⁺ sensitivity in Triton X-100-treated preparations. Inhibition of Ca²⁺ loading by TG and CPA increased with time of exposure to the inhibitor over 18–24 min. TG and CPA produced half inhibition of Ca²⁺ loading at 34.9 and 35.7 μM respectively, when 18–24 min were allowed for diffusion. The spontaneous force oscillations were more sensitive to the inhibitors: 10 μM TG and 30 μM CPA both abolished the oscillations in this time. The myofilament Ca²⁺ sensitivity was not affected by 10 and 300 μM TG or CPA. The results show that the concentrations of TG and CPA necessary to inhibit the SR Ca²⁺ uptake of skinned ventricular trabeculae are much higher than the reported values for single intact myocytes. One reason for this may be slow diffusion of the inhibitors into the multicellular trabecula preparation. Received: 28 July 1995/Received after revision: 11 December 1995/Accepted: 18 December 1995     

The role of sarcoplasmic reticulum and sarcoplasmic reticulum Ca2+-ATPase in the smooth muscle tone of the cat gastric fundus

Circular smooth muscle strips isolated from cat gastric fundus were studied in order to understand whether the sarcoplasmic reticulum (SR) and SR Ca²⁺-ATPase could play a role in the regulation of the muscle tone. Cyclopiazonic acid (CPA), a specific inhibitor of SR Ca²⁺-ATPase, caused a significant and sustained increase in muscle tone, depending on the presence of extracellular Ca²⁺. Nifedipine and cinnarizin only partially suppressed the CPA-induced tonic contraction. Bay K 8644 antagonized the relaxant effect of nifedipine in CPA-contracted fundus. Nitric-oxide-releasing agents sodium nitroprusside and 3-morpholino-sydnonimine completely suppressed the CPA-induced tonic contraction. The blockers of Ca²⁺-activated K⁺ channels, tetraethylammonium, charybdotoxin and/or apamin, decreased the contractile effect of CPA. Vanadate increased the tone but did not change significantly the effect of CPA. CPA exerted its contractile effect even when Ca²⁺ influx was triggered through the Na⁺/Ca²⁺ exchanger and the other Ca²⁺ entry pathways were blocked. Thapsigargin, another specific SR Ca²⁺-ATPase inhibitor, also increased the muscle tone. The effect of thapsigargin was completely suppressed by sodium nitroprusside and 3-morpholino-sydnonimine and partially by nifedipine. In conclusion, under conditions when the SR Ca²⁺-ATPase is inhibited, the tissue develops a strong tonic contraction and a large part of this is mediated by Ca²⁺ influx presumably via nifedipine-sensitive Ca²⁺ channels. This study suggests the important role of SR Ca²⁺-ATPase in the modulation of the muscle tone and the function of SR as a “buffer barrier” to Ca²⁺ entry in the cat gastric fundus smooth muscle. Received: 10 August 1995/Received after revision: 9 November 1995/Accepted: 10 November 1995     

Effects of Mg2+ on Ca2+ handling by the sarcoplasmic reticulum in skinned skeletal and cardiac muscle fibres

The influence of myoplasmic Mg²⁺ (0.05–10 mM) on Ca²⁺ accumulation (net Ca²⁺ flux) and Ca²⁺ uptake (pump-driven Ca²⁺ influx) by the intact sarcoplasmic reticulum (SR) was studied in skinned fibres from the toad iliofibularis muscle (twitch portion), rat extensor digitorum longus (EDL) muscle (fast twitch), rat soleus muscle (slow twitch) and rat cardiac trabeculae. Ca²⁺ accumulation was optimal between 1 and 3 mM Mg²⁺ in toad fibres and reached a plateau between 1 and 10 mM Mg²⁺ in the rat EDL fibres and between 3 and 10 mM Mg²⁺ in the rat cardiac fibres. In soleus fibres, optimal Ca²⁺ accumulation occurred at 10 mM Mg²⁺. The same trend was obtained with all preparations at 0.3 and 1 M Ca²⁺. Experiments with 2,5-di-(tert-butyl)-1,4-benzohydroquinone, a specific inhibitor of the Ca²⁺ pump, revealed a marked Ca²⁺ efflux from the SR of toad iliofibularis fibres in the presence of 0.2 M Ca²⁺ and 1 mM Mg²⁺. Further experiments indicated that the SR Ca²⁺ leak could be blocked by 10 M ruthenium red without affecting the SR Ca²⁺ pump and this allowed separation between SR Ca²⁺ uptake and SR Ca²⁺ accumulation. At 0.3 M Ca²⁺, Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the toad iliofibularis and rat EDL fibres and between 1 and 10 mM Mg²⁺ in the rat soleus and trabeculae preparations. At higher [Ca²⁺] (1 M), Ca²⁺ uptake was optimal with 1 mM Mg²⁺ in the iliofibularis fibres and between 1 and 3 mM Mg²⁺ in the EDL fibres. In the soleus and cardiac preparations Ca²⁺ uptake was optimal between 1 and 10 mM Mg²⁺. The results of this study demonstrate that SR Ca²⁺ accumulation is different from SR Ca²⁺ uptake and that these two important determinants of muscle function are differently affected by Mg²⁺ in different muscle fibre types.     

Effects of ryanodine on skinned skeletal muscle fibers of the rabbit

The mechanism(s) of ryanodine-induced contracture of skeletal muscle were studied in skinned fibers from soleus (SL) and adductor magnus (AM) (slow- and fast-twitch skeletal muscles) of rabbits. Pieces of SL or AM were homogenized (sarcolemma disrupted). Single fibers were dissected from the homogenate and mounted on photodiode force transducers. At concentrations 1–50 M, ryanodine slightly but significantly increased the submaximal Ca²⁺-activated tension development of the contractile proteins in skinned fibers of AM but not of SL. Ryanodine in uptake phase or release phase increased caffeine-induced tension transients in the SR of both muscle types; however, no dose-response relation was found. Ryanodine 1 M decreased, however, the second control tension transients in a dose-dependent manner. The depression was nearly irreversible and activity-dependent. The concentrations of ryanodine that inhibited the second control tension transients by 50% were 10 M and 5 M for SL and AM, respectively, following ryanodine administration in the release phase, and 100 M and 30 M, respectively, for these preparations after the drug was present in the uptake phase. The quantity of calcium released from the SR by Triton X-100 and caffeine in the second control tension transient was unchanged by ryanodine at all concentrations tested when compared with that of the absence of ryanodine. The present findings suggest that the ability of ryanodine to increase immediate calcium release from the SR, and in AM but not SL, to increase the sensitivity of the contractile proteins to Ca²⁺ underlies the contracture caused by this agent in intact skeletal muscles. The delayed decreased Ca²⁺ efflux by caffeine, as evidenced by depression of tension transient with no change in the calcium content may be responsible for the decreased twitch tension caused by this agent.     

Effect of chloride on Ca2+ release from the sarcoplasmic reticulum of mechanically skinned skeletal muscle fibres

 The effect of intracellular Cl^– on Ca²⁺ release in mechanically skinned fibres of rat extensor digitorum longus (EDL) and toad iliofibularis muscles was examined under physiological conditions of myoplasmic [Mg²⁺] and [ATP] and sarcoplasmic reticulum (SR) Ca²⁺ loading. Both in rat and toad fibres, the presence of 20 mM Cl^–in the myoplasm increased Ca²⁺ leakage from the SR at pCa (i.e. –log₁₀ [Ca²⁺]) 6.7, but not at pCa 8. Ca²⁺ uptake was not significantly affected by the presence of Cl^–. This Ca²⁺-dependent effect of Cl^– on Ca²⁺ leakage was most likely due to a direct action on the ryanodine receptor/Ca²⁺ release channel, and could influence channel sensitivity and the resting [Ca²⁺] in muscle fibres in vivo. In contrast to this effect, acute addition of 20 mM Cl^– to the myoplasm caused a 40–50% reduction in Ca²⁺ release in response to a low caffeine concentration both in toad and rat fibres. One possible explanation for this latter effect is that the addition of Cl^– induces a potential across the SR (lumen negative) which might reduce Ca²⁺ release via several different mechanisms. Received: 20 October 1997 / Received after revision: 1 December 1997 / Accepted: 2 December 1997     

Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum ca2+ pump blockers: thapsigargin and cyclopiazonic acid

The effect of thapsigargin (TG) and cyclopiazonic acid (CPA) on the mechanical activity of the rat pulmonary artery were investigated. In chemically (-escin)-skinned arterial strips, application of TG (0.1–1 M) or CPA (0.5–10 M) prior and throughout the loading procedure of the internal Ca²⁺ stores (0.3 M free Ca²⁺ ions for 8–10 min) concentration dependently inhibited the subsequent contractile response induced by noradrenaline (NA, 10 M) or caffeine (25 mM). In intact strips repeatedly incubated in a Ca²⁺-containing solution (2.5 mM for 10 min), followed by incubation in a Ca²⁺-free solution 12 min before NA-stimulation, TG and CPA not only inhibited the NA-induced contraction but also increased the tension which appeared during the exposure time to Ca²⁺. The two phenomena developed with similar time courses. The increase in tension during the readmission of Ca²⁺ ions was not antagonized by verapamil (10 M) or nifedipine (1 M) but was blocked by La³⁺ (50 M) and Co²⁺ (1 mM) ions. The amplitude of the verapamil-insensitive TG (or CPA)-induced contraction was dependent on the external [Ca²⁺] [0.1–10 mM, concentration for half maximal effect (EC₅₀) =0.85 mM], not modified by the reduction of the external [Na⁺] (from 130 to 10 mM) and decreased by depolarization of the strip using K⁺-rich (30–120 mM) solutions. Under the latter condition, 38±9 and 83±4% reduction (n=5) was observed in the presence of 60 and 120 mM K⁺ respectively. This contraction was also concentration dependently inhibited by the tyrosine kinase inhibitors genistein (0.5–50 M) and tyrphostin (2–50 M). Sr²⁺ ions, which contracted both depolarized intact and skinned strips, failed to replace Ca²⁺ ions in the verapamil-insensitive contraction induced by TG or CPA (n=4). Finally, TG (1 M) and CPA (10 M) did not modify the pCa tension relationship in skinned strips (n=5). These results show that the main action of TG and CPA in rat pulmonary artery is to prevent the refilling of the internal Ca²⁺ store. TG and CPA also seem to facilitate a Ca²⁺ influx through a specific verapamil-insensitive pathway. The biophysical and molecular characteristics of this pathway remain to be elucitated, although it appears to involve a tyrosine kinase activity.     

Ca2+ uptake by cardiac sarcoplasmic reticulum ATPase in situ strongly depends on bound creatine kinase

The role of creatine kinase (CK) bound to sarcoplasmic reticulum (SR), in the energy supply of SR ATPase in situ, was studied in saponin-permeabilised rat ventricular fibres by loading SR at pCa 6.5 for different times and under different energy supply conditions. Release of Ca²⁺ was induced by 5 mM caffeine and the peak of relative tension (T/T _max) and the area under isometric tension curves, S _T, were measured. Taking advantage of close localisation of myofibrils and SR, free [Ca²⁺] in the fibres during the release was estimated using steady state [Ca²⁺]/tension relationship. Peak [Ca²⁺] and integral of free Ca²⁺ transients (S[Ca²⁺]_f) were then calculated. At all times, loading with 0.25 mM adenosine diphosphate, Mg²⁺ salt (MgADP) and 12 mM phosphocreatine (PCr) [when adenosine triphosphate (ATP) was generated via bound CK] was as efficient as loading with both 3.16 mM MgATP and 12 mM PCr (control conditions). However, when loading was supported by MgATP alone (3.16 mM), T/T _max was only 40% and S[Ca²⁺]_f 31% of control (P < 0.001). Under these conditions, addition of a soluble ATP-regenerating system (pyruvate kinase and phosphoenolpyruvate), did not increase loading substantially. Both S _T and S[Ca²⁺]_f were more sensitive to the loading conditions than T/T _max and peak [Ca²⁺]. The data suggest that Ca²⁺ uptake by the SR in situ depends on local ATP/ADP ratio which is effectively controlled by bound CK. Received: 23 January 1996/Received after revision: 19 April 1996/Accepted: 3 May 1996     

Calcium-induced inactivation of calcium release from the sarcoplasmic reticulum of skeletal muscle

The ability of myofilament space Ca²⁺ to modulate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle was investigated. Single fibers of the frog Rana pipiens belindieri were manually skinned (sarcolemma removed). Following a standard load and pre-incubation in varying myoplasmic Ca²⁺ concentrations, SR Ca²⁺ release was initiated by caffeine. Ca²⁺ release rates were calculated from the changes in absorbance of a Ca²⁺ sensitive dye, antipyrylazo III. An apparent dissociation constant (K _d) for dye-Ca²⁺ binding of 8000 M² was determined by comparing the buffering action of the dye with that of ethylenebis(oxonitrilo)tetraacetate (EGTA) using the contractile proteins of the skinned fiber as a measure of free Ca²⁺. This value for K _d was used in the calculation of Ca²⁺ release rates. As the myoplasmic space Ca²⁺ was increased from pCa 7.4, Ca²⁺ release rates declined sharply such that at pCa 6.9 the calculated release rate was 72±3% (mean ± SEM) of control (pCa 8.4). Further increases in myoplasmic Ca²⁺ from pCa 6.9 to pCa 6.1 did not result in a further decline in release rate. The effect of a decreased driving force on Ca²⁺ ions was investigated to determine whether it could account for the change in release rates observed. At pCa 6.9, where the greatest degree of inactivation occurred, the measured effects of a change in driving force could account for at most 40% of the observed inactivation. Varying concentrations of Ba²⁺ and Sr²⁺ in the myofilament space had no inactivating effect on the SR Ca²⁺ release rates. The ability of myofilament Ca²⁺ to inhibit SR Ca²⁺ release at concentrations normally encountered during muscle activation suggests a role for released Ca²⁺ as a modulator of the SR Ca²⁺ channel.     

Force and force transients in skeletal muscle fibres of the frog skinned by freeze-drying

A freeze-drying method is described by which single skinned skeletal muscle fibres or fibre bundles can readily be obtained. Skinned fibre segments of the ileofibularis and semitendinous muscles of the frog — activated by means of a rapid increase in the Ca-concentration — showed very stable and reproducible contractions. Complete activation occurred at a Ca-concentration of 1.6·10^–6 M and the mid-point of the pCa-tension curve occurred at 6.3·10^–7 M. Addition of phosphate (10^–2 M) had a depressing effect on the speed of the Ca-activated tension development as well as on the maximum tension reached.Addition of caffeine (10^–2 M) had no effect on the tension generation, indicating that the sarcoplasmic reticulum, if present, was not active. The force responses due to rapid length changes applied to the Ca-activated fibre preparations were found to be qualitatively similar to the force responses on intact tissue. This skinning technique might be employed on human biopsies, enabling the measurement of physiological parameters such as for example force and shortening velocity.     

Modulation of the ryanodine receptor sarcoplasmic reticular Ca2+ channel in skinned fibers of fast- and slow-twitch skeletal muscles from rabbits

This study was performed to compare skinned fibers from rabbit adductor magnus (AM) and soleus (SL) muscles with regard to the influence of caffeine, Ca²⁺ and Mg²⁺ on the depressive effects of ryanodine (RYA) on the caffeine-induced tension transients. Single skinned fibers were immersed in solution: to load Ca²⁺ into, and release Ca²⁺ from the SR (a load-release cycle). Three cycles were sequentially performed in each skinned fiber: (1) a control (no RYA) (2) a conditioning period in which activation was car ried out in the presence of ryanodine plus various con centrations of the modulators, i.e. caffeine, Ca²⁺ or Mg²⁺, and (3) a test (no RYA) which monitored the release activity retained after the conditioning cycle. The depressive effect of RYA was found to be a function of [ryanodine], [caffeine], or [Ca²⁺], and an inverse function of [Mg²⁺], where [caffeine] denotes concentration. The half-maximal effects of RYA in AM (5 M RYA) and SL (10 M RYA), respectively, occurred at a pCa₅₀ of 5.32 versus 5.43 without caffeine, or pCa₅₀ of 7.24 versus 6.88 and pMg₅₀ of 3.29 versus 3.61 with 25 mM caffeine, at a [caffeine] of 4.96 versus 7.29 mM, and at a [ryanodine] of 31.0 versus 101.6 M. Thus, the RYA depression in skinned muscle fibers is modulated by caffeine, Ca²⁺, and Mg²⁺ in both muscle types, and AM is at least two- to fourfold more sensitive than SL.     

Cross-bridge stiffness in Ca2+-activated skinned single muscle fibres

Tension transients, in response to small and rapid length changes (completed within 40 s), were obtained from skinned single frog muscle fibres incubated in activating solutions with varying concentrations of Ca²⁺. The first 2 ms of these transients were described by a linear model in which the fibre is regarded as a rod composed of infinitesimally small, identical segments containing a mass, one undamped elastic element and in the case of relaxed fibres two damped elastic elements in series, or in the case of activated fibres three such elastic elements in series. The stiffness of activated fibres, expressed in elastic constants or apparent elastic constants, increased with increasing concentrations of Ca²⁺. All the damped elastic constants that were necessary to describe the tension responses of activated fibres were proportional to isometric tension. However, the undamped elastic constant did not increase linearly with increasing isometric tension. Equatorial X-ray diffraction patterns were obtained from single frog muscle fibres under similar conditions as under which the tension transients were obtained. The filament spacing (d ₁₀)of Ca²⁺-activated single frog muscle fibres decreased with increasing isometric force, whereas the intensity ratio (I ₁₁/I₁₀)increased linearly with increasing isometric force. From experiments in which dextran (MW 200000 Da) was added, it followed that such a change in filament spacing would modify passive stiffness. The d ₁₀value of relaxed fibres decreased and stiffness increased with increasing concentrations of the polymer dextran, whereas I ₁₁/I₁₀remained constant. The relation of stiffness and filament spacing with concentration of dextran was used to eliminate the effect of decreased filament spacing on stiffness of activated fibres. After correction for changes in filament spacing the undamped complicance C ₁, normalized to tension, was not constant, but increased with increasing isometric tension. If we assume that isometric tension is proportional to the number of force generating cross-bridges, this means that only part of the undamped complicance of activated fibres is located in the crossbridges.     

Calcium sensitivity and myofibrillar protein isoforms of rat skinned skeletal muscle fibres

We investigated the calcium sensitivity for tension generation of different fibre types and the possible correlation between calcium sensitivity and the presence of distinct regulatory protein and myosin light chain (MLC) isoforms in rat skinned skeletal muscle fibres. Fibre types 1, 2A and 2B were identified by electrophoretic analysis of myosin heavy chain (MHC) isoforms. Fibres showing more than one MHC isoform were discarded. Type 1 fibres from the soleus showed a higher pCa (–log₁₀ [Ca], where [ ] denotes concentration) threshold and a lower slope of pCa/tension curve than type 2 extensor digitorum longus (EDL) fibres; between type 2 fibres, type 2B showed the higher slope of pCa/tension curve. Type 1 fibres from different muscles showed similar calcium sensitivities when containing only the slow set of regulatory proteins and MLC; when both slow and fast isoforms were present, calcium sensitivity shifted toward fast type fibre values. Type 2A fibres from different muscles showed a similar calcium sensitivity, independently of the set (purely fast or mixed) of regulatory proteins and MLC. It is suggested that when both fast and slow isoforms of regulatory proteins and of MLC are present in a muscle fibre, calcium sensitivity is dictated mainly by the fast isoforms.     

Nandrolone decanoate treatment affects sarcoplasmic reticulum Ca(2+) ATPase function in skinned rat slow- and fast-twitch fibres

The effects of anabolic-androgenic steroid administration on the function of the sarcoplasmic reticulum (SR) pump were investigated in chemically skinned fibres from the extensor digitorum longus (EDL) and soleus muscles of sedentary rats. Twenty male rats were divided into two groups, one group received an intramuscular injection of nandrolone decanoate (15 mg kg^–1) weekly for 8 weeks, the second received similar weekly doses of vehicle (sterile peanut oil). Compared with control muscles, nandrolone decanoate treatment reduced SR Ca²⁺ loading in EDL and soleus fibres by 49% and 29%, respectively. In control and treated muscles, the rate of Ca²⁺ leakage depended on the quantity of Ca²⁺ loaded. Furthermore, for similar SR Ca²⁺ contents, the Ca²⁺ leakage rate was not significantly modified by nandrolone decanoate treatment. Nandrolone decanoate treatment thus affects Ca ²⁺ uptake by the SR in a fibre-type dependent manner.     

Compared properties of the contractile system of skinned slow and fast rat muscle fibres

Chemically skinned fibres from soleus and plantaris rat muscles were used to compare the contractile properties of slow and fast muscles. The maximal isometric tension appeared larger in plantaris than in soleus fibres. The apparent Ca²⁺ threshold for activation was lower in slow than in fast fibres while Ca²⁺ concentrations required to obtain either the maximal tension or half maximal tension (pCa₅₀) were lower in fast than in slow fibres. This apparent difference in Ca²⁺ sensitivity will be discussed. As could be expected from other studies, a faster force development in plantaris than in soleus fibres occurred. However, one interesting new result showed that in soleus, the kinetics of the tension development estimated by the t _max parameter were slightly dependent on the Ca²⁺ concentration whereas the t ₅₀ parameter changed significantly with the Ca²⁺ concentration. In plantaris, both t _max and t ₅₀ parameters were found to depend strongly on the Ca²⁺ concentration. Finally, the plantaris muscle showed a greater caffeine sensitivity than the soleus muscle. All the results suggested that the Ca-regulatory mechanism in the slow fibres was essentially different from that in the fast fibres.     

Alteration of calcium sensitivity of skinned frog skeletal muscle fibres by inositol triphosphate and calmodulin antagonists

We investigated the influence of inositol triphosphate (IP₃), trifluoperazine (TFP), and perhexiline on the calcium sensitivity of freeze-dried frog semitendinosus muscle fibres. Further, the effect of IP₃ on calcium release from the sarcoplasmic reticulum (SR) of frog semitendinosus fibres skinned by saponin was studied. IP₃ decreased the calcium sensitivity of freeze-dried frog skeletal muscle fibres and failed to induce a calcium release from SR of saponin-skinned fibres. Freeze-dried frog skeletal muscle fibres were strongly sensitized for calcium by TFP and perhexiline.     

Ruthenium red affects the contractile apparatus but not sarcoplasmic reticulum Ca2+ release of skinned papillary muscle

Ruthenium red has been shown to have a positive inotropic effect on isolated perfused hearts. The cellular mechanism of this action is not clear. Ruthenium red is able to block the Ca²⁺ release channel in isolated sarcoplasmic reticulum (SR) vesicle and reconstituted channel preparations. However, the effect of ruthenium red on SR Ca²⁺ release has not been studied in skinned cardiac muscle preparations. In the present study we investigated the actions of ruthenium red on both the characteristics of force generation by the contractile apparatus and Ca²⁺ release from the SR in chemically skinned rat papillary muscle. Ruthenium red (2 and 10 M) significantly increased the Ca²⁺ sensitivity of the contractile apparatus (decreasing Ca²⁺ required for the half-maximal response from 1.56±0.04 M to 1.46±0.05 M) but had no effect on the maximal Ca²⁺-activated force in triton X-100 treated fibers. This result may suggest one explanation for the positive inotropic effect of ruthenium red on the heart. On the other hand, ruthenium red had no significant effect on either caffeine-induced Ca²⁺ release or Ca²⁺-induced Ca²⁺ release from the SR in saponin-skinned muscle fibers. Lack of a blocking effect on SR Ca²⁺ release by ruthenium red in skinned fibers suggests that the SR Ca²⁺ channels in intact preparations have characteristics that are different from those of either vesicular or reconstituted channel preparations.     

The cation selectivity of the sarcoball Ca2+ channel in frog muscle fibres

 We have measured single-channel currents from sarcoplasmic reticulum (SR) blebs (sarcoballs) of frog skeletal muscle fibres using conventional patch-clamp electrodes with excised patches. With both the pipette and bath solutions containing 50 mM Ca(gluconate)₂ the slope conductance of the single channels was 39.2 pS for the most commonly seen state, with a reversal potential of –0.4 mV. The cation selectivity of this channel was investigated by replacing the bathing solution with either gluconate or HEPES salts of selected cations. The Goldman permeability ratios, calculated from the reversal potentials, were found to be P(Ca²⁺)/P(K⁺)=2.4, P(Ca²⁺)/ P(Na⁺)=2.7, P(Ca²⁺)/P(Tris⁺)=3.1, P(Ca²⁺)/P(Mg²⁺)=1.0 and P(Ca²⁺)/P(Ba²⁺)=1.1. Each value for the monovalent ions was found to be less than the corresponding value reported for the SR ryanodine receptor channel from skeletal and cardiac muscle. Single-channel activity could be recorded when the preparation was bathed in symmetrical 50 mM Mg(gluconate)₂ solutions, and these channels had a similar conductance and open probability to that measured when the preparation was bathed in symmetrical Ca(gluconate)₂ solution. The channel activity in symmetrical 50 mM Ca(gluconate)₂ solution was insensitive to bath-applied caffeine (5 mM) and ryanodine (10 μM). The results are in agreement with the conclusion that the sarcoball Ca²⁺ channel is not the ryanodine receptor release channel, but possibly a form of the SR Ca²⁺-ATPase which is uncoupled from the catalytic events of the pump and acts as a passive ion channel. Received: 13 February 1998 / Received after revision: 6 April 1998 / Accepted: 7 April 1998     

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. Accepted: 6 June 2000