Effects of cytosolic Ca2+ on the Ca2+ content of the sarcoplasmic reticulum in saponin-permeabilized rat ventricular trabeculae

 Rat ventricular trabeculae were mounted for isometric tension recording, and then permeabilized with saponin. The Ca²⁺ concentration ([Ca²⁺]) within the permeabilized preparation (cytosolic [Ca²⁺]) was monitored continuously using Indo-1 and the integrals of Ca²⁺ transients resulting from brief caffeine application used as an index of the sarcoplasmic reticulum (SR) Ca²⁺ content. The relationship between SR Ca²⁺ content and cytosolic [Ca²⁺] was studied within the reported physiological range (i.e. 50–250 nmol · l^–1 Ca²⁺). Increasing cytosolic [Ca²⁺] from 50 nmol · l^–1 to 250 nmol · l^–1 increased the steady-state SR Ca²⁺ content about threefold. However, increasing [Ca²⁺] above 250 nmol · l^–1 typically resulted in spontaneous SR Ca²⁺ release, with no further increase in SR Ca²⁺ content. The SR Ca²⁺ content increased only slowly when cytosolic [Ca²⁺] was increased; it was unchanged 20 s after a rapid increase in cytosolic [Ca²⁺], but increased progressively to a new steady-state level during the following 1–2 min. In a parallel series of experiments using intact papillary muscles, increasing extracellular [Ca²⁺] (from 0.5 to 5 mmol · l^–1) significantly increased twitch tension within 20 s of the solution change. These results support previous suggestions that the SR Ca²⁺ content may increase when diastolic cytosolic [Ca²⁺] rises during inotropic interventions such as increased stimulus rate or extracellular [Ca²⁺]. However, the rate at which SR Ca²⁺ responds to changes in cytoplasmic [Ca²⁺] within the diastolic range does not appear rapid enough to explain the early potentiation of twitch tension in intact preparations after an increase in extracellular [Ca²⁺]. Received: 26 August 1997 / Accepted: 28 October 1997     

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     

Differential effects of arginine,glutamate and phosphoarginine on Ca<Superscript>2+</Superscript>-activation properties of muscle fibres from crayfish and rat

The effects of two amino acids, arginine which has a positively charged side-chain and glutamate which has a negatively charged side-chain on the Ca²⁺-activation properties of the contractile apparatus were examined in four structurally and functionally different types of skeletal muscle; long- and short-sarcomere fibres from the claw muscle of the yabby (a freshwater decapod crustacean), and fast- and slow-twitch fibres from limb muscles of the rat. Single skinned fibres were activated in carefully balanced solutions of different pCa (-log₁₀[Ca²⁺]) that either contained the test solute (“test”) or not (“control”). The effect of phosphoarginine, a phosphagen that bears a nett negative charge, was also compared to the effects of arginine. Results show that (i) arginine (33-36 mmol l^-1) significantly shifted the force–pCa curve by 0.08–0.13 pCa units in the direction of increased sensitivity to Ca²⁺-activated contraction in all fibre types; (ii) phosphoarginine (9–10 mmol l^-1) induced a significant shift of the force–pCa curve by 0.18–0.24 pCa units in the direction of increased sensitivity to Ca²⁺ in mammalian fast- and slow-twitch fibres, but had no significant effects on the force–pCa relation in either long- or short-sarcomere crustacean fibres; (iii) glutamate (36–40 mmol l^-1), like arginine affected the force–pCa relation of all fibre types investigated, but in the opposite direction, causing a significant decrease in the sensitivity to Ca²⁺-activated contraction by 0.08–0.19 pCa units; (iv) arginine, phosphoarginine and glutamate had little or no effect on the maximum Ca²⁺-activated force of crustacean and mammalian fibres. The results suggest that the opposing effects of glutamate and arginine are not related to simply their charge structure, but must involve complex interactions between these molecules, Ca²⁺ and the regulatory and other myofibrillar proteins.     

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     

Early effects of denervation on sarcoplasmic reticulum properties of slow-twitch rat muscle fibres

 The Ca²⁺ release activity of the sarcoplasmic reticulum (SR) in chemically skinned single slow-twitch fibres from control, 2-day and 7-day denervated rat soleus muscle was studied. Histochemical fibre type composition of the whole muscle, electrophysiological properties and the Ca²⁺ sensitivity of tension development by single muscle fibres were also studied. All the data were correlated with contractile properties of the in vitro muscle. In the 2-day denervated muscle the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake decreased from the control values of 0.384 ± 0.030 μmol (mg fibre protein)^–1 and 19.8 ± 1.9 nmol min^–1 (mg fibre protein)^–1, respectively, to 0.210 ± 0.016 μmol (mg fibre protein)^–1 and 13.5 ± 0.9 nmol min^–1 (mg fibre protein)^–1; the calculated amount of Ca²⁺ released upon stimulation by caffeine decreased from the control value of 0.148 to 0.078 μmol (mg fibre protein)^–1. In the 7-day denervated muscle, the SR Ca²⁺ capacity and the rate of Ca²⁺ uptake increased to 0.517 ± 0.06 μmol (mg fibre protein)^–1 and 21.6 ± 2.3 nmol min^–1 (mg fibre protein)^–1, respectively; the calculated amount of Ca²⁺ released increased to 0.217 μmol (mg fibre protein)^–1. Both contraction time and tension of the isometric twitch decreased in 2-day denervated and increased in 7-day denervated muscles. Electrophysiological and histochemical changes, as well as changes in the Ca²⁺ sensitivity of the muscle fibres did not show any apparent correlation with mechanical changes. It is therefore concluded that the SR plays a prominent role in the early changes of contraction time and tension following denervation. Received: 15 October 1996 / Received after revision: 28 March 1997 / Accepted: 8 April 1997     

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 tetracaine and procaine on skinned muscle fibres depend on free calcium

Summary The local anaesthetics, tetracaine and procaine have previously been found to block, induce or potentiate Ca²⁺ release from the sarcoplasmic reticulum (SR) of skeletal muscle depending on the preparation, experimental conditions and design. We now show that low concentrations of tetracaine and procaine block SR Ca²⁺ release whereas high concentrations induce release from the SR of amphibian and mammalian skinned fibres. Both actions depend on pCa, such that a shift in pCa can alter their effect from blocking to releasing Ca²⁺. In skinned fibres with Ca²⁺-loaded SR, tetracaine (1mm) produced a tonic contraction with a time to half-peak of 15–20 s and a magnitude reaching 80% of maximum force. Ca²⁺ release by tetracaine or procaine occured at pCa 6.5 and was not blocked by Ruthenium Red (RR) (25 m). This action of tetracaine was attributed to SR Ca²⁺ release rather than to a displacement of bound Ca²⁺ because fibres lacking a functional SR due to pre-treatment with quercetin (100 m), A 23187 (100 g ml^–1) or Triton X-100 (1%) did not contract after additions of tetracaine. Lower concentrations of tetracaine (0.5mm) and procaine (10mm) blocked contractions due to caffeine (at pCa 6.73), sulphydryl oxidizing agents, or Ca²⁺-induced Ca²⁺ release (CICR). The inhibition of CICR as a function of pCa was difficult to measure quantitatively since lowering pCa to elicit CICR twitches was sufficient to initiate tetracaine-induced tonic contractions.Experiments with isolated SR vesicles showed that 1mm tetracaine inhibited CICR, over a wide range of pCa but 3–5mm tetracaine induced rapid Ca²⁺ release. The opposite effects of tetracaine and procaine depend mostly on their concentration in SR vesicles and/or pCa in skinned fibres. Blockade of release seems to occur via the CICR pathway, and induction of release through an increase in SR membrane permeability.Abbreviations SR sarcoplasmic reticulum - HEPES N-2-hydroxy-ethylpiperazine-N¹-2-ethanesulphonic acid - EGTA ethylene glycol bis (-aminoethyl ether)-N,N,N¹,-N¹-tetraacetic acid - CICR Ca²⁺-induced Ca²⁺ release - MOPS morpholinopropane sulphonic acid - RR Ruthenium Red     

The effect of extracellular Ca2+ concentration on the negative staircase of Ca2+ transient in field-stimulated rat ventricular cells

We performed experiments using the Ca²⁺ indicator dye, fura-2 to investigate the effect of extracellular Ca²⁺ concentration ([Ca²⁺]_o) on sarcoplasmic reticulum (SR) Ca²⁺ release and loading in single rat ventricular cells. In normal Tyrode solution (1.8 mM [Ca²⁺]_o) repetitive stimulation (0.5 Hz) resulted in a gradual decrease in calcium transients (the negative staircase phenomenon) without being accompanied by a gradual decrease in diastolic intracellular Ca²⁺ concentration. The rate of the slow decline in calcium transient was faster in lower [Ca²⁺]_o. However, the peak of the first calcium transient was relatively invariant over a wide range of [Ca²⁺]_o (0.5–5 mM). The size of the calcium transient elicited by field stimulation was proportional to that induced by 10 mM caffeine, applied following the field stimulation. These results suggest that the size of calcium transients depends mainly on the Ca²⁺ content of the SR. The quiescent period favoured the replenishment of the SR and this effect was promoted further by increasing the driving force for Ca²⁺ entry across the sarcolemma during this period. We conclude that in low [Ca²⁺]_o, short stimulation interval may limit Ca²⁺ influx across the sarcolemma during the quiescent period to cause a gradual reduction in calcium content of the SR and thus the calcium transient.     

Replacement of troponin-I in slow-twitch skeletal muscle alters the effects of the calcium sensitizer EMD 53998

 We extracted troponin-I (TnI) from skinned rat and rabbit soleus muscle fibres using a modification of the method described by Strauss et al. (FEBS Lett 310:229–234, 1992) for replacement of TnI in cardiac preparations. Incubation of soleus muscle fibres with 10 mmol/l vanadate virtually completely abolished the Ca²⁺dependence of force. Immunoblot analysis revealed that more than 80% of TnI had been extracted from the preparations. The Ca²⁺dependence of force was restored by incubation with a complex of cardiac TnI (cTnI) and troponin-C (cTnC). We examined the effects of the Ca²⁺-sensitizing compound EMD 53998 on isometric tension in native porcine cardiac and rabbit soleus skinned fibres as well as soleus in which the endogenous slow skeletal TnI (ssTnI) had been replaced by cTnI (soleus–cTnI). It was found that 10 μmol/l EMD 53998 in native soleus increased maximum Ca²⁺-activated force to 120±1.4% of control. In soleus–cTnI fibres, maximum force was increased to only 105±0.9%, which was similar to the effect observed in cardiac muscle (108±0.6%). In cardiac muscle, 10 μmol/l EMD 53998 induced a leftward shift of the pCa-tension relation by 0.65 log units. In native soleus, ΔpCa was only 0.40. Again, the effect of EMD 53998 on soleus–cTnI (ΔpCa=0.56) more closely resembled the response found in cardiac muscle than that observed in native soleus muscle. The apparent TnI-isoform dependence of the effects elicited by EMD 53998 suggests that its actions are modulated by the regulatory proteins of the thin filament. Received: 15 December 1997 / Accepted: 16 March 1998     

Measurement of SR Ca2+ content in the presence of caffeine in permeabilised rat cardiac trabeculae

 This study was designed to measure the Ca²⁺ content of rat cardiac sarcoplasmic reticulum (SR) after equilibration with normal diastolic levels of Ca²⁺ (100 nM), in the absence and presence of caffeine. Measurements of [Ca²⁺] based on Fura-2 fluorescence were made from a limited bath volume (230 nl) containing individual saponin-permeabilised rat cardiac trabeculae. Injection of caffeine (5–40 mM) into this volume caused an initial release of Ca²⁺ from the SR, but within 30 s the SR was able to re-accumulate a significant proportion of the Ca²⁺. Ca²⁺ re-accumulation into the SR could be prevented by removal of ATP to inhibit the SR Ca²⁺ pump. Incubation of the preparation in an ATP-containing solution containing caffeine (5–40 mM) and 100 nM Ca²⁺ indicated that the SR’s ability to retain Ca²⁺ depends inversely on the dose of caffeine. The relative Ca²⁺ content of the SR after preincubation with caffeine was 86.7±3.5% at a caffeine concentration of 5 mM, 62.5±5.1% at 10 mM caffeine, 37.8±8.1% at 20 mM caffeine and 7.1±1.9% at 40 mM caffeine. Measurement of the SR Ca²⁺ release in the presence of different BAPTA concentrations was used to calculate (1) the Ca²⁺-binding capacity of the preparation (equivalent to 245±10 μM BAPTA) and (2) the Ca²⁺ content of the SR accessed by caffeine after equilibration with 100 nM Ca²⁺ (186±11 μmol/l cell volume or 5.6 mmol/l SR volume). Received: 9 June 1998 / Received after revision: 29 July 1998 / Accepted: 31 July 1998     

The sarcolemmal mechanisms involved in the control of diastolic intracellular calcium in isolated rat cardiac trabeculae

 We performed experiments using the calcium indicator Indo-1 to determine the relative roles of the sarcolemmal mechanisms involved in the regulation of diastolic intracellular calcium concentration ([Ca²⁺]_i) in trabeculae from the rat heart. Ryanodine was used to eliminate sarcoplasmic reticulum (SR) function. In the functional absence of the SR, 76.8 ± 3.9% of the calcium was extruded by the Na-Ca exchange carrier in the [Ca²⁺]_i range of diastolic concentration ± 200–400 nM. This was assessed by measuring the recovery of [Ca²⁺]_i from small perturbations in the presence and absence of extracellular sodium. The steady-state relationship between [Ca²⁺]_o and [Ca²⁺]_i was linear over the range of 1–40 mM, a 20-fold increase of [Ca²⁺]_o produced a 1.97-fold ± 0.13-fold increase in [Ca²⁺]_i (n = 5). In the absence of extracellular sodium raising [Ca²⁺]_o had a variable effect. In some preparations there was little change of [Ca²⁺]_i while in others the response was almost as large as in control conditions. We conclude that the Na-Ca exchanger contributes ≈ 77% of sarcolemmal calcium extrusion following small perturbations in [Ca²⁺]_i and that this fraction does not diminish as the [Ca²⁺]_i declines. In addition we have shown a sodium-independent entry of calcium into quiescent cardiac muscle under resting conditions. Received: 16 May 1996 / Received after revision and accepted: 28 June 1996     

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 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     

A study of the mechanisms of excitation–contraction coupling in frog skeletal muscle based on measurements of [Ca<Superscript>2+</Superscript>] transients inside the sarcoplasmic reticulum

[Ca²⁺] transients inside the sarcoplasmic reticulum (SR) were recorded in frog skeletal muscle twitch fibers under voltage clamp using the low affinity indicator Mag Fluo 4 (loaded in its AM form) with the purpose of studying the effect on Ca²⁺ release of extrinsic Ca²⁺ buffers (i.e. BAPTA) added at high concentration to the myoplasm. When the extrinsic Ca²⁺ buffer is added to the myoplasm, part of the released Ca²⁺ binds to it, reducing the Ca²⁺ signal reported by a myoplasmic indicator. This, in turn, hinders the quantification of the amount of Ca²⁺ released. Monitoring release by measuring [Ca²⁺] inside the SR avoids this problem. The application of extrinsic buffers at high concentration reduced the resting [Ca²⁺] in the SR ([Ca²⁺]_SR) continuously from a starting value close to 400 μM reaching the range of 100 μM in about half an hour. The effect of reducing resting [Ca²⁺]_SR on the Ca²⁺ permeability of the SR activated by voltage clamp depolarization to 0 mV was studied in cells where the myoplasmic [Ca²⁺] ([Ca²⁺]_myo) transients were simultaneously recorded with Rhod2. The Ca²⁺ release flux was calculated from [Ca²⁺]_myo and divided by [Ca²⁺]_SR to obtain the permeability. Peak permeability was significantly reduced, from 0.026?±?0.005 ms^?1 at resting [Ca²⁺]_SR?=?372?±?5 μM to 0.021?±?0.004 ms^?1 at resting [Ca²⁺]_SR?=?120?±?16 μM (n?=?4, p?=?0.03). The time averaged permeability was not significantly changed (0.009?±?0.003 and 0.010?±?0.003 ms^?1, at the higher and lower [Ca²⁺]_SR respectively). Once the cells were equilibrated with the high buffer intracellular solution, the change in [Ca²⁺]_SR (Δ[Ca²⁺]_SR) in response to voltage clamp depolarization (0 mV, 200 ms) in 20 mM BAPTA was significantly lower (Δ[Ca²⁺]_SR?=?30.2?±?3.5 μM from resting [Ca²⁺]_SR?=?88.8?±?13.6 μM, n?=?5) than in 40 mM EGTA (Δ[Ca²⁺]_SR?=?72.2?±?10.4 μM from resting [Ca²⁺]_SR?=?98.2?±?15.6 μM, n?=?4) suggesting that a Ca²⁺ activated component of release was suppressed by BAPTA.     

The effect of extracellular tonicity on the anatomy of triad complexes in amphibian skeletal muscle

Ultrastructural features of tubular–sarcoplasmic (T–SR) triad junctions and measures of cell volume following graded increases of extracellular tonicity were compared under physiological conditions recently shown to produce spontaneous release of intracellularly stored Ca²⁺ in fully polarized amphibian skeletal muscle fibres. The fibres were fixed using solutions of equivalent tonicities prior to processing for electron microscopy. The resulting anatomical sections demonstrated a partially reversible cell shrinkage corresponding to substantial increases in intracellular solute or ionic strength graded with extracellular tonicity. Serial thin sections through triad structures confirmed the presence of geometrically close but anatomically isolated transverse (T-) tubular and sarcoplasmic reticular (SR) membranes contrary to earlier suggestions for the development of luminal continuities between these structures in hypertonic solutions. They also quantitatively demonstrated accompanying decreases in T–SR distances, increased numbers of sections that showed closely apposed T and SR membranes, tubular luminal swelling and reductions in luminal volume of the junctional SR, all correlated with the imposed increases in extracellular osmolarity. Fully polarized fibres correspondingly showed elementary Ca²⁺-release events (‘sparks’, in 100 mM-sucrose-Ringer solution), sustained Ca²⁺ elevations and propagated Ca²⁺ waves (≥350–500 mM sucrose) following exposure to physiological Ringer solutions of successively greater tonicities. These were absent in hypotonic, isotonic or less strongly hypertonic (∼50 mM sucrose-Ringer) solutions. Yet exposure to hypotonic solutions also disrupted T–SR junctional anatomy. It increased the tubular diameters and T–SR distances and reduced their area of potential contact. The spontaneous release of intracellularly stored Ca²⁺ thus appears more closely to correlate with the expected changes in intracellular solute strength or a reduction in absolute T–SR distance rather than disruption of an optimal anatomical relationship between T and SR membranes taking place with either increases or decreases in extracellular tonicity. This revised version was published online in July 2006 with corrections to the Cover Date.     

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.     

Cross-bridge movement in rat slow skeletal muscle as a function of calcium concentration

A single fibre bundle from rat soleus muscle was chemically skinned with saponin and the transfer of myosin heads from the thick filaments to the thin filaments at a sarcomere length of 2.4 μm was measured as a function of Ca²⁺ concentration using an x-ray diffraction method at 4–7 °C. In the relaxed state, the 1,0 spacing was 42.08 nm. The spacing showed no significant decrease when the Ca²⁺ concentration was below the threshold (−log₁₀ [Ca²⁺] or pCa 5.8). No significant transfer of the myosin heads occurred when the Ca²⁺concentration was below the threshold (pCa 5.8). When the muscle was maximally activated at pCa 4.4, the spacing decreased to 40.35 nm. During the maximum isometric contraction at pCa 4.4, 54.9 ± 6.5% (±SE of the mean) of the myosin heads were transferred to the thin filaments. The transfer of the myosin heads was approximately proportional to relative tension. These results suggest that myosin heads of both fast-twitch and slow-twitch skeletal muscles transferred on the common movement as a function of Ca²⁺ concentration. Received: 1 December 1995/Received after revision and accepted: 20 May 1996     

Q-type Ca2+ channels are located closer to secretory sites than L-type channels: functional evidence in chromaffin cells

 This study uses a new strategy to investigate the hypothesis that, of the various Ca²⁺ channels expressed by a neurosecretory cell, a given channel subtype is coupled more tightly to the exocytotic apparatus than others. The approach is based on the prediction that the degree of inhibition of the secretory response by various Ca²⁺ channel blockers will differ at low (0.5 mM) and high (5 mM) extracellular Ca²⁺ concentrations ([Ca²⁺]_o). So, at low [Ca²⁺]_o the K⁺-evoked catecholamine release from superfused bovine chromaffin cells was depressed 60–70% by 2 μM ω-agatoxin IVA (P/Q-type Ca²⁺ channel blockade), by 3 μM ω-conotoxin MVIIC (N/P/Q-type Ca²⁺ channel blockade), or by 3 μM lubeluzole (N/P/Q-type Ca²⁺ channel blockade); in high [Ca²⁺]_o these blockers inhibited the responses by only 20–35%. At 1–3 μM ω-conotoxin GVIA (N-type Ca²⁺ channel blockade) or 3 μM furnidipine (L-type Ca²⁺ channel blockade), secretion was inhibited by 30 and 50%, respectively; such inhibitory effects were similar in low or high [Ca²⁺]o. Combined furnidipine plus ω-conotoxin MVIIC, ω-agatoxin IVA or ω-conotoxin GVIA exhibited additive blocking effects at both Ca²⁺ concentrations. The results suggest that Q-type Ca²⁺ channels are coupled more tightly to exocytotic active sites, as compared to L-type channels. This hypothesis if founded in the fact that external Ca²⁺ that enters the cell through a Ca²⁺ channel located near to chromaffin vesicles will saturate the K⁺ secretory response at both [Ca²⁺]_o, i.e. 0.5 mM and 5 mM. In contrast, Ca²⁺ ions entering through more distant channels will be sequestered by intracellular buffers and, thus, will not saturate the secretory machinery at lower [Ca²⁺]_o. Received: 23 September 1997 / Received after revision: 29 October 1997 / Accepted: 30 October 1997     

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.     

Ca-induced Ca release from the sarcoplasmic reticulum of isolated myofibrillar bundles of barnacle muscle fibres

Isometric tension and aequorin light were recorded from isolated myofibrillar bundles (diameter 0.2 mm) of barnacle muscle fibres to examine Ca release from the sareoplasmic reticulum (SR). Transfer of a bundle from a pCa 6.7 solution to a pCa 5.8 solution, both buffered with 0.1mM EGTA, resulted in a phasic increase in myofibrillar free Ca²⁺ which was superimposed on a slow rise to a steady level and a fast rise in tension. The peak of the free Ca²⁺ response was higher than the free Ca²⁺ in the bulk solution. Treatment of the bundle with the detergent Brij to destroy the SR membranes abolished the phasic rise in Ca²⁺ and considerably reduced the amplitude of contraction. A second challenge of a bundle to the pCa 5.8 solution without prior reloading of the SR Ca store gave a much reduced phasic component. When a pCa 5.8 solution with 1.0 mM EGTA buffering was used, the phasic rise in myofibrillar free Ca²⁺ could not be detected and the rise in tension was four times slower than with 0.1 mM EGTA. The results are consistent with the operation of a Ca-induced Ca release mechanism in the SR membrane of this crustacean muscle.