Prolonged exercise reduces Ca2+ release in rat skeletal muscle sarcoplasmic reticulum

Prolonged exercise decreased the rate of Ca⁺ release in sarcoplasmic reticulum (SR) vesicles isolated from rat muscle by 20–30% when release was initiated by 5, 10, and 20 M AgNO₃. [³H]Ryanodine binding was also depressed by 20% in SR vesicles isolated from the exercised animals. In contrast, the maximum amount of Ca²⁺ released by Ag⁺ remained unaffected by exercise. The passive permeability of SR vesicles and the rate of Ca²⁺ release in the presence of ruthenium red, a known inhibitor of the Ca²⁺ release mechanism, was not affected by prolonged exercise. These results suggest that exercise depressed Ca²⁺ release from SR by directly modifying the Ca²⁺ release channel. Current address: Department of Physics, Portland State University, Portland, OR 97207, USA     

Functional characterization of the Ca2+-gated Ca2+ release channel of vascular smooth muscle sarcoplasmic reticulum

The Ca²⁺-gated Ca²⁺ release channel of aortic sarcoplasmic reticulum (SR) was partially purified and reconstituted into planar lipid bilayers. Canine and porcine aorta microsomal protein fractions were solubilized in the detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane sulphonate (CHAPS) in the presence and absence of ³[H]-ryanodine and centrifuged through linear sucrose gradients. A single ³[H]-ryanodine receptor peak with an apparent sedimentation coefficient of 30 s was obtained. Upon reconstitution into planar lipid bilayers, the unlabelled 30 s protein fraction induced the formation of a Ca²⁺- and monovalent-ion-conducting channel (110 pS in 100 mM Ca²⁺, 360 pS in 250 mM K⁺). The channel was activated by micromolar Ca²⁺, modulated by millimolar adenosine triphosphate, Mg²⁺ and the Ca²⁺-releasing drug caffeine, and inhibited by micromolar ruthenium red. Micro- to millimolar concentrations of the plant alkaloid ryanodine induced a permanently closed state of the channel. Our results suggest that smooth muscle SR contains a Ca²⁺-gated Ca²⁺ release pathway, with properties similar to those observed for the skeletal and cardiac ryanodine receptor/Ca²⁺ release channel complexes.     

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.     

A possible role of sarcoplasmic Ca2+ release in modulating the slow Ca2+ current of skeletal muscle

Ca²⁺ channels are regulated in a variety of different ways, one of which is modulation by the Ca²⁺ ion itself. In skeletal muscle, Ca²⁺ release sites are presumably located in the vicinity of the dihydropyridine-sensitive Ca²⁺ channel. In this study, we have tried to investigate the effects of Ca²⁺ release from the sarcoplasmic reticulum on the L-type Ca²⁺ channel in frog skeletal muscle, using the double Vaseline gap technique. We found an increase in Ca²⁺ current amplitude on application of caffeine, a well-known potentiator of Ca²⁺ release. Addition of the fast Ca²⁺ buffer BAPTA to the intracellular solution led to a gradual decline in Ca²⁺ current amplitude and eventually caused complete inhibition. Similar observations were made when the muscle fibre was perfused internally with the Ca²⁺ release channel blocker ruthenium red. The time course of Ca²⁺ current decline followed closely the increase in ruthenium red concentration. This suggests that Ca²⁺ release from the sarcoplasmic reticulum is involved in the regulation of L-type Ca²⁺ channels in frog skeletal muscle.     

Measurement of Ca2+-release-dependent inward current reveals two distinct components of Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ current (L-type) and inward current caused by Ca²⁺ release from the sarcoplasmic reticulum and carried by electrogenic Na⁺/Ca²⁺ exchange have been measured in cultured atrial myocytes from hearts of adult guinea-pigs using whole-cell voltage clamp techniques. The pipette solution, used for internal dialysis of the cells, contained a high concentration, 60 mM or 25 mM, of citrate as a non-saturable low-affinity Ca²⁺-chelating compound. It has been shown previously that Ca²⁺-release-dependent inward current under these conditions is carried by electrogenic Na⁺/Ca²⁺ exchange. Furthermore, Ca²⁺-release-dependent inward current (the release signal) can be completely separated from triggering Ca²⁺ current if brief depolarizations for activating I _Ca are used. In the majority of cells that did not produce spontaneous Ca²⁺ release, conditions could be found that caused the release signal to be split into two components: an early component of variable amplitude and a late component of rather constant amplitude. The delay of the late component with regard to triggering I _Ca was inversely related to the amplitude of the first one. Below a certain amplitude of the first component, the second one failed to be elicited. This suggests the second component to be triggered by the first one. Weakly Ca²⁺-buffered cells produced spontaneous Ca²⁺ release, resulting in irregular transient inward currents at constant membrane-holding potential. Synchronization by trains of step depolarizations unmasked two components also in the spontaneous release signals. In none of the cells studied was any indication of more than two components of the release signal detected. The results are discussed in terms of two distinct compartments of sarcoplasmic reticulum with different properties of Ca²⁺ release.Supported by the Deutsche Forschungsgemeinschaft (FG Konzell)     

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     

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     

Effects of intracellular Ca2+ chelating compounds on inward currents caused by Ca2+ release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum (SR) of mammalian cardiac myocytes occuring either due to activation by a depolarization or the resulting transmembrane Ca²⁺ current (I _Ca), or spontaneously due to Ca²⁺ overload has been shown to cause inward current(s) at negative membrane potentials. In this study, the effects of different intracellular Ca²⁺ chelating compounds on I _Ca-evoked or spontaneous Ca²⁺-release-dependent inward currents were examined in dialysed atrial myocytes from hearts of adult guinea-pigs by means of whole-cell voltage-clamp. As compared to dialysis with solutions containing only a low concentration of a high affinity ethylene glycol-bis(-aminoethylether) N,N,N,N-tetraacetic acid (EGTA) like chelator (50–200 M), inward membrane currents (at –50 mV) due to evoked Ca²⁺ release, spontaneous Ca²⁺ release or Ca²⁺ overload following long-lasting depolarizations to very positive membrane potentials are prolonged if the dialysing fluid contains a high concentration of a low affinity Ca²⁺ chelating compound such as citrate or free adenosine 5-triphosphate (ATP). Without such a non-saturable Ca²⁺ chelator in the dialysing fluid, Ca²⁺-release-dependent inward currents are often oscillatory and show an irregular amplitude. With a low affinity chelator in a non-saturable concentration, discrete inward currents with constant properties can be recorded. We conclude that the variability in Ca²⁺-release-dependent inward current seen in single cells arises from spatial inhomogeneities of intracellular Ca²⁺ concentration ([Ca²⁺]_i) due to localized saturation of endogenous and exogenous high affinity Ca²⁺ buffers (e.g. [2]). This can be avoided experimentally by addition of a non-saturable buffer to the intracellular solution. This condition might be useful, if properties of Ca²⁺ release from the SR and/ or the resulting membrane current, like for example arrhythmogenic transient inward current, are to be investigated on the single cell level.     

Effect of Ca2+-independent myosin light chain kinase on different skinned smooth muscle fibers

In various skinned smooth muscle fiber preparations, (porcine carotid artery, rat tail artery, chicken gizzard and Taenia coli from guinea pig) a Ca²⁺-independent myosin light chain kinase (MLCK) initiated a contraction in absence of Ca²⁺. While the Ca²⁺ insensitive MLCK was effective on the vertebrate smooth muscles it did not act on the invertebrate skinned skeletal muscle preparation from Limulus and anterior byssus retractor muscle from Mytilus edulis. The results indicate that in vertebrate smooth muscles phosphorylation is sufficient for activation and that there is no obligatory role for an additional mechanism in initiation of contraction.     

Inositol trisphosphate enhances Ca2+ oscillations but not Ca2+-induced Ca2+ release from cardiac sarcoplasmic reticulum

The role of inositol 1,4,5-trisphosphate [Ins(1,4,5)P ₃] in excitation-contraction coupling in cardiac muscle is still unclear, although many laboratories are beginning to assume a critical role for this putative second messenger. Earlier studies from this laboratory [Nosek et al. (1986) Am J Physiol 250:C807] found that Ins(1,4,5)P ₃ enhanced spontaneous Ca²⁺ release and the caffeine sensitivity of Ca²⁺ release from myocardial sarcoplasmic reticulum (SR) and proposed an increase in the Ca²⁺ sensitivity of the release as a possible mechanism. In order to clarify the phyisological relevance of these actions of Ins(1,4,5)P ₃ and specifically to test the effect of Ins(1,4,5)P ₃ on the Ca²⁺ sensitivity of Ca²⁺ release, we compared the effects of Ins(1,4,5)P ₃ on Ca²⁺ oscillations and on Ca²⁺-induced Ca²⁺ release (CICR) from the SR in saponin-skinned rat papillary muscle. We found that: (a) 30 M Ins(1,4,5)P ₃ enhanced the Ca²⁺ oscillations (measured by tension oscillations) from the rat cardiac SR, consistent with the previous report on guinea pig tissue; (b) both GTP and GTP[S] enhanced Ca²⁺ oscillations. The effect was not additive to that of Ins(1,4,5)P ₃ indicating that two different Ca²⁺-release pools do not exist in cardiac SR; (c) 30 M Ins(1,4,5)P ₃ had no effect on the Ca²⁺ sensitivity of CICR; (d) Ins(1,4,5)P ₃ (up to 30 M) had no effect on SR Ca²⁺ loading. The studies were performed in the presence of Cd²⁺ or 2,3-bisphosphoglycerate, agents that inhibit Ins(1,4,5)P ₃ hydrolysis. These results suggest that: (a) two different mechanisms underlie Ca²⁺ oscillations and CICR, Ins(1,4,5)P ₃ influencing Ca²⁺ oscillations but not CICR; (b) Ins(1,4,5)P ₃ does not increase the Ca²⁺ sensitivity of Ca²⁺ release from the SR; (c) cardiac muscle is different from smooth muscle where Ca²⁺ release from the SR is dependent upon GTP; (d) the physiological role of Ins(1,4,5)P ₃ in excitation-contraction coupling in cardiac muscle is minimal. In contrast, Ins(1,4,5)P ₃ may play a pathological role in cardiac arrhythmogenesis by enhancing spontaneous Ca²⁺ ocsillations.     

Intracellular citrate induces regenerative calcium release from sarcoplasmic reticulum in guinea-pig atrial myocytes

Ca²⁺ release from the sarcoplasmic reticulum was studied in voltage-clamped guinea-pig atrial myocytes. Cells were dialysed with a pipette solution containing the Ca²⁺ indicator 1- [2-amino-5-(6-carboxyindol-2-yl) phenoxy]-2-(2-amino-5-methylphenoxy) ethane-N,N,N,N-tetraacetic acid](Indo-1, 100 M) and as main anion either chloride or the low-affinity Ca²⁺ buffer citrate. Intracellular Ca²⁺ transients (Ca_i transients) were elicited by depolarizations from a holding potential of –50 mV. In chloride-dialysed cells, Ca_i transients showed a bell-shaped dependence on the amplitude of the depolarizing pulse. In citratedialysed cells, membrane depolarizations were associated with a small rise in [Ca²⁺]_i. These small changes in [Ca²⁺]_i were either followed by a large Ca_i. transient or failed to induce large changes in [Ca²⁺]_i. The peak amplitude of the large Ca_i transient did not vary with the amplitude of the depolarizing pulse. These results demonstrate that in the presence of intracellular chloride, Ca²⁺ release in atrial cells is a graded process triggered by Ca²⁺ influx. Using citrate as the main intracellular anoin, Ca²⁺ release triggered by Ca²⁺ entry was no longer graded but occurred in a regenerative manner. The results are discussed in terms of two models in which citrate, affects the spatial distribution of [Ca²⁺]_i or the loading state of the sarcoplasmic reticulum.     

Potentiation of sarcoplasmic reticulum Ca2+ release by 2,3-butanedione monoxime in crustacean muscle

The effect of the chemical phosphatase 2,3-butanedione monoxime (BDM) on various aspects of excitation/contraction coupling in crustacean muscle was investigated. Despite having a depressant effect on vertebrate skeletal and cardiac muscle, BDM was a potentiator of contraction in crustacean muscle. At concentrations of 1–3 mM BDM caused an increase of potassium contractures in bundles of fibers isolated from crayfish muscle. At higher concentrations BDM caused oscillatory contractions by itself. In single voltage-clamped cut muscle fibers loaded with rhod-2, BDM (0.5–2 mM) potentiated the magnitude and duration of intracellular Ca²⁺ transients elicited by depolarization. At the same time BDM did not affect the rate of Ca²⁺ removal from the myoplasm under conditions where Ca²⁺ release was blocked by tetracaine. Nor did BDM increase Ca²⁺ entry; in fact it caused a decrease in the amplitude of the inward Ca²⁺ current (I _Ca). In microsomes isolated from lobster muscle, BDM also potentiated Ca²⁺ release induced by caffeine and at higher concentrations (above 3 mM) induced release by itself. At the same time it had little effect on Ca²⁺ uptake. These results indicate that BDM potentiates Ca²⁺ release in crustacean muscle possibly by dephosphorylation of the Ca²⁺-release channel.     

Caffeine-induced calcium release from isolated sarcoplasmic reticulum of rabbit skeletal muscle

The essential conditions for the Ca²⁺ releasing action of caffeine from isolated sarcoplasmic reticulum (SR) of rabbits were evaluated by an investigation into the effects of Ca²⁺, Mg²⁺, MgATP^2–, and ATP concentration, ionic strength, and degree of loading. The heavy fraction (4,500×g) of the reticulum was used. Except for the study on degree of loading, 0.2 mg protein·ml^–1 SR was loaded actively with 0.02 mM⁴⁵CaCl₂, resulting in >90 nmol·mg protein^–1 at steady state, and then the effects of various parameters with or without (control) caffeine were tested.It was found that (1) caffeine induces a transient, dosedependent release of Ca²⁺, (2) the absolute amount of Ca²⁺ released by caffeine increases with the Ca²⁺ load of the SR, (3) increasing the ionic strength () from 0.09 to 0.3 lowers the threshold concentration of caffeine, (4) the SR is refractory to a repeated challenge by a caffeine concentration causing maximal effect, (5) caffeine-induced Ca²⁺ release increases with increasing (a) external Ca²⁺ concentrations up to 5 M total Ca²⁺ (or 3 M free Ca²⁺) and (b) free ATP concentrations up to 0.45 mM, and (6) caffeine-induced Ca²⁺ release is not affected by changes of either the Mg²⁺ or the MgATP^2– concentration.     

腺相关病毒介导的PLB基因反义RNA对糖尿病大鼠心肌肌浆网

目的: 构建磷酸受纳蛋白(PLB)反义RNA腺相关病毒载体(rAAV-asPLB),建立糖尿病(DM)大鼠模型。直接心肌注射rAAV-asPLB,观察其对DM大鼠心肌PLB基因转录和蛋白表达的影响,以及对心肌肌浆网Ca²⁺-ATPase活性的作用。方法: 利用质粒辅助重组腺相关病毒系统试剂盒构建rAAV-asPLB。腹腔注射链脲佐菌素(STZ)诱导DM大鼠模型,将实验动物分为4组:正常组、DM组、盐水组和rAAV-asPLB组。盐水或rAAV-asPLB注射后6周,RT-PCR检测心肌PLB mRNA转录;Western blotting检测PLB蛋白表达水平;检测心肌肌浆网Ca²⁺-ATPase活性。结果: (1)成功构建rAAV-asPLB,诱导出DM大鼠模型。(2)DM组和盐水组PLB mRNA水平均高于正常组;rAAV-asPLB组PLB mRNA水平较DM组和盐水组明显降低。 (3)DM组和盐水组PLB 蛋白水平均高于正常组;rAAV-asPLB组PLB 蛋白水平较DM组和盐水组明显降低。(4)肌浆网Ca²⁺-ATPase活性在DM组和盐水组中较正常组明显降低,而rAAV-asPLB组较DM组和盐水组升高。结论: rAAV-asPLB抑制DM大鼠心肌PLB表达,增强Ca²⁺-ATPase活性。     

Myofibril and sarcoplasmic reticulum changes with exercise and growth

Summary The intent of this study was to observe the effects of different treadmill running programs upon selected biochemical properties of soleus muscle from young rats. Young 10 day litter-mates were assigned to endurance (E), sprint (S) and control (C) groups. Each was partitioned into either 21 or 51 day exercising groups and 10 day controls. For C the myofibril ATPase activity at 21 and 51 days were lower than 10 day activity (p0.05). In the 51 day E group ATPase activity (0.378±0.009 mol Pi·mg^–1·min^–1) was greater than at 10 and 21 days (0.307±0.006 and 0.323±0.008 mol Pi·mg^–1·min^–1) (p0.05). No change occurred in the S group from 10 to 21 and 51 days (p0.05). Both the 21 and 51 day S (0.318±0.011 and 0.399±0.010 mol Pi·mg^–1·min^–1) and E (0.323±0.008 and 0.378±0.009 mol Pi·mg^–1·min^–1) groups had higher activity compared to the C group (0.193±0.029 and 0.172±0.031 mol Pi·mg^–1·min^–1) (p0.05). Maturation (10–51 day) resulted in a lowered sarcoplasmic reticulum (SR) yield and Ca²⁺ binding (p0.05) while Ca²⁺ uptake ability did not change (p0.05). SR yield, Ca²⁺ binding and uptake were not altered with S training (p0.05). The E training resulted in greater Ca²⁺ uptake at 51 days compared to C and S (p0.05), with no change in Ca²⁺ binding (p0.05). The data suggest that E training alters the normal development pattern of young rat soleus muscle.Supported by grants A-6449 and A-0425 from the Natural Sciences and Engineering Research Council of Canada     

Effects of thapsigargin and cyclopiazonic acid on the sarcoplasmic reticulum Ca2+ pump of skinned fibres from frog skeletal muscle

Thapsigargin has been reported to inhibit ATP-dependent Ca²⁺ uptake by isolated sarcoplasmic reticulum (SR) vesicles of vertebrate skeletal muscle fibres at nanomolar concentrations. There have been no reports confirming this effect in skinned muscle fibre preparations. We have examined the ability of thapsigargin to inhibit the uptake of Ca²⁺ by the SR in mechanically skinned fibres of frog iliofibularis muscles, using the size of the caffeine-induced contracture to assess the Ca²⁺ content of the SR. The SR was first depleted of Ca²⁺ and then reloaded for 1 min at pCa 6.2 in the presence and absence of thapsigargin. When 5 min were allowed for diffusion, a thapsigargin concentration of at least 131 M was required to inhibit Ca²⁺ loading by 50%. In contrast, another SR Ca²⁺ uptake inhibitor, cyclopiazonic acid, was more effective, producing 50% inhibition at 7.0 M and total inhibition at 50 M. When cyclopiazonic acid (100 M) was applied after, rather than during, Ca²⁺ loading, the caffeine-induced contracture was not changed. Thapsigargin (300 M), on the other hand, caused some reduction in the peak amplitude of the caffeine-induced contracture when applied after Ca²⁺ loading. The poor effectiveness of thapsigargin in the skinned fibres, compared with in SR vesicles, is attributed to its slow diffusion into the skinned fibres, perhaps as a result of binding to myofibrillar components.     

Characterization of Ca2+- and Sr2+-activated tension in functionally skinned chicken fibers of normal and dystrophic skeletal and normal cardiac muscle

The Ca²⁺ and Sr²⁺ activation of tension in functionally skinned chicken fibers of normal and dystrophic skeletal and normal cardiac muscle were studied. The muscles studied can be separated into two groups based upon their Ca²⁺ and Sr²⁺ sensitivities: those which are significantly more sensitive to Ca²⁺ than to Sr²⁺, pectoralis and posterior latissimus dorsi (PLD), and those which show no Ca²⁺/Sr²⁺ sensitivity difference, cardiac and anterior latissimus dorsi (ALD). This suggests that there is more than one type of Ca²⁺ site involved in Ca²⁺ control of muscle contraction in different muscle types and suggests that ALD and cardiac muscle may be controlled by a different type of binding site than PLD and pectoralis muscle. Dystrophic ALD and PLD muscles showed little change in their Ca²⁺ and Sr²⁺ sensitivities from those of normal muscles in contrast to the pectoralis which showed a decrease in both Ca²⁺ and Sr²⁺ sensitivity (approaching that of PLD) with the onset of dystrophy. Similarly, upon SDS polyacrylamide gel electrophoresis, dystrophic ALD and PLD muscles showed no difference in contractile proteins from those of normal muscles, in contrast to pectoralis muscle where the appearance of a 36,000 dalton protein band correlated with the onset of dystrophy and the changes in the Ca²⁺/Sr²⁺ activation properties of this muscle. The contractile protein band pattern of normal and dystrophic PLD and dystrophic pectoralis muscle were similar including the presence of the 36,000 dalton protein.     

Ca2+ sensitizing effects of EMD 53998 after troponin replacement in skinned fibres from porcine atria and ventricles

Skinned fibres from porcine ventricles exhibited a higher Ca²⁺ sensitivity (pCa₅₀, i.e. -log₁₀ Ca²⁺ concentration required for half-maximal activation, for force generation) than atrial fibres. The thiadiazinone derivative EMD 53998 increased Ca²⁺ sensitivity and Ca²⁺ efficacy in both preparations. The drug effect depended on the isoform of troponin (Tn). Using the vanadate method TnI and TnC could be partly extracted and replaced by foreign tropin or by the TnI subunit of added foreign troponins. We investigated the relationship between pCa and force development before and after replacement of TnI with foreign troponin (bovine ventricular troponin, cTn, or rabbit skeletal muscle troponin, sTn) in the presence and absence of EMD 53998. Substitution with bovine cTn increased Ca²⁺ sensitivity to a value characteristic of bovine ventricular skinned fibres (pCa₅₀=5.4) and was further increased by EMD 53998. Substitution with sTn also increased Ca²⁺ sensitivity, but subsequent addition of EMD 53998 caused little further increase in Ca²⁺ sensitivity. Following extraction of TnI with vanadate, skinned fibres contracted in a Ca²⁺-independent manner and failed to relax at a pCa of 8. Relaxation could be induced, however, by bovine ventricular TnI and rabbit skeletal muscle recombinant TnI. This relaxation could be reversed by EMD 53998 (100 M). The Ca²⁺-independent force of contracted fibres could also be depressed by a TnI inhibitory peptide, (cTnI 137–148) and, in addition, this effect was antagonized by EMD 53998. These results suggest that EMD 53998 antagonizes the inhibitory action of TnI, possibly by interfering with the interaction of the TnI inhibitory region with actin.     

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     

Caffeine-induced oscillations of cytosolic Ca2+ in GH3 pituitary cells are not due to Ca2+ release from intracellular stores but to enhanced Ca2+ influx through voltage-gated Ca2+ channels

Caffeine, a well known facilitator of Ca²⁺-induced Ca²⁺ release, induced oscillations of cytosolic free Ca²⁺ ([Ca²⁺]_i) in GH₃ pituitary cells. These oscillations were dependent on the presence of extracellular Ca²⁺ and blocked by dihydropyridines, suggesting that they are due to Ca²⁺ entry through L-type Ca²⁺ channels, rather than to Ca²⁺ release from the intracellular Ca²⁺ stores. Emptying the stores by treatment with ionomycin or thapsigargin did not prevent the caffeine-induced [Ca²⁺]_i oscillations. Treatment with caffeine occluded phase 2 ([Ca²⁺]_i oscillations) of the action of thyrotropin-releasing hormone (TRH) without modifying phase 1 (Ca²⁺ release from the intracellular stores). Caffeine also inhibited the [Ca²⁺]_i increase induced by depolarization with high-K⁺ solutions (56% at 20 mM), suggesting direct inhibition of the Ca²⁺ entry through voltage-gated Ca²⁺ channels. We propose that the [Ca²⁺]_i increase induced by caffeine in GH₃ cells takes place by a mechanism similar to that of TRH, i.e. membrane depolarization that increases the firing frequency of action potentials. The increase of the electrical activity overcomes the direct inhibitory effect on voltage-gated Ca²⁺ channels with the result of increased Ca²⁺ entry and a rise in [Ca²⁺]_i. Consideration of this action cautions interpretation of previous experiments in which caffeine was assumed to increase [Ca²⁺]_i only by facilitating the release of Ca²⁺ from intracellular Ca²⁺ stores.