Effects of pH on spontaneous tension oscillation in skinned bovine cardiac muscle

Skinned cardiac muscle preparations exhibit spontaneous tension oscillations (spontaneous oscillatory contractions; SPOCs) in the absence of Ca²⁺, and in the presence of MgATP, MgADP and inorganic phosphate (Pi; ADP-SPOC). Similar oscillations occur in the presence of sub-micromolar concentrations of Ca²⁺ under normal activating conditions without MgADP and Pi (Ca-SPOC). In the study presented here, we investigated the effects of pH on both types of SPOC in skinned bovine cardiac ventricular muscle. First, a decrease in pH increased the MgADP concentration required to induce the half-maximal isometric tension that is obtained in the absence of Ca²⁺ and in the presence of MgATP (ADP-contraction). The inhibitory effect of Pi on ADP-contractions was not affected by pH. Second, ADP-SPOCs occurred upon the addition of Pi to the solution that resulted in ADP-contraction, and the relative amplitude and the period of the tension oscillation in the presence of 2 mM MgATP, 10 mM MgADP and 10 mM Pi were unchanged under all pH conditions examined (6.6, 7.0, 7.4). On the contrary, the relative amplitude and the period of the Ca-SPOCs were markedly diminished at pH 6.6. Finally, we constructed state diagrams showing the effects of pH on SPOC conditions. The state diagram shows that SPOCs occur less frequently under acidic conditions than at neutral pH. We suggest that the intermediate state of crossbridges that is required for SPOCs is more difficult to attain at a low pH. Received: 14 September 1998 / Received after revision: 23 February 1999 / Accepted: 10 March 1999     

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     

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     

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     

Regulation of smooth muscle delayed rectifier K+ channels by protein kinase A

We identified voltage-activated K⁺ channels in freshly dispersed smooth muscle cells from the circular layer of the canine colon in patch-clamp experiments using 200 nM charybdotoxin to suppress 270-pS Ca²⁺-activated K⁺ channels (BK channels). Three channel types were distinguished in symmetrical 140 mM KCl solutions: 19.5 ± 1.7 pS channels (K_DR1), 90.6 ± 5.4 pS channels (K_DR2) and 149 ± 4 pS intermediate-conductance Ca²⁺-activated K⁺ channels (IK channels). All three types showed an increase in open probability with membrane depolarization. Ensemble average current from K_DR1 channels inactivated with a time constant of 1.7 ± 0.1 s at +60 mV test potential, while K_DR2 and IK channels did not show inactivation. IK channels were activated by free cytoplasmic [Ca²⁺] (10⁻⁶M) but were insensitive to 4-aminopyridine (4-AP, 10 mM) and intracellular tetraethylammonium (TEA, 1 mM). K_DR1 channels were sensitive to 4-AP (10 mM) and intracellular TEA (1–10 mM) but not to Ca²⁺. K_DR2 channels did not have a consistent pharmacological profile, suggesting that this class may be comprised of several subtypes. At +40 mV membrane potential, the catalytic subunit of protein kinase A (PKA) increased the open probability of K_DR1 channels 3.4-fold and of K_DR2 channels 3.9-fold, but had no effect on IK channels. In the absence of Mg-ATP, PKA did not affect channel open probabilities. At physiological membrane potentials (−60 mV) only openings of K_DR1 channels could be induced by PKA, suggesting that these 4-AP-sensitive 20-pS K⁺ channels are primarily responsible for the cAMP-mediated hyperpolarization of colonic smooth muscle cells. Received: 20 June 1995/Received after revision: 25 January 1996/Accepted: 7 February 1996     

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     

Recombinant troponin I substitution and calcium responsiveness in skinned cardiac muscle

Using treatment with vanadate solutions, we extracted native cardiac troponin I and troponin C (cTnI and cTnC) from skinned fibers of porcine right ventricles. These proteins were replaced by exogenously supplied TnI and TnC isoforms, thereby restoring Ca²⁺-dependent regulation. Force then depended on the negative logarithm of Ca²⁺ concentration (pCa) in a sigmoidal manner, the pCa for 50% force development, pCa₅₀, being about 5.5. For reconstitution we used fast-twitch rabbit skeletal muscle TnI and TnC (sTnI and sTnC), bovine cTnI and cTnC or recombinant sTnIs that were altered by site-directed mutagenesis. Incubation with TnI inhibited isometric tension in TnI-extracted fibers in the absence of Ca²⁺, but restoration of Ca²⁺ dependence required incubation with both TnI and TnC. Relaxation at low Ca²⁺ levels and the steepness of the force/pCa relation depended on the concentration of exogenously supplied TnI in the reconstitution solution (range 20–150 μM), while Ca²⁺ sensitivity, i.e. the pCa₅₀, was dependent on the isoform, and also on the concentration of TnC in the reconstitution solution. At pH 6.7, skinned fibers reconstituted with optimal concentrations of sTnC and sTnI (120 μM and 150 μM, respectively) were more sensitive to Ca²⁺ than those reconstituted with cTnC and cTnI (difference in pCa₅₀ approx. 0.2 units). Rabbit sTnI was cloned and expressed in Escherichia coli using a high yield expression plasmid. We introduced point mutations into the TnI inhibitory region comprising the sequence of the minimal common TnC/actin binding site (-G¹⁰⁴-K-F-K-R-P-P-L-R-R-V-R¹¹⁵-). The four mutants produced by substitution of T for P¹¹⁰, G for P¹¹⁰, G for L¹¹¹, and G for K¹⁰⁵ were chosen, based on previous work with synthetic peptides showing that single amino acid substitution in this region diminished the capacity of these peptides to inhibit acto-S₁ ATPase or contraction of skinned fibers. Therefore, all amino acid residues of the inhibitory region are thought to contribute to biological activity of TnI. However, each of the recombinant TnIs could substitute for endogenous TnI. In combination with exogenous TnC, Ca²⁺ dependence could be restored when ^gly110sTnI, ^thr110sTnI or ^gly111sTnI was used for reconstitution. The mutant ^gly105sTnI, on the other hand, reduced the ability of skinned fibers to relax at low Ca²⁺ concentrations and it caused an increase in Ca²⁺ sensitivity. Received: 5 October 1995/Received after revision and accepted: 1 December 1995     

The effects of inorganic phosphate and arsenate on both passive muscle visco-elasticity and maximum Ca2+ activated tension in chemically skinned rat fast and slow twitch muscle fibres

The effects of adding either 25 mM inorganic phosphate (Pi) or its structural analogue arsenate (ASi) on both the maximum Ca²⁺ activated tension (Po) and passive muscle visco-elasticity (P₂ tension) were investigated at 10°C, using segments of single, chemically skinned rat muscle fibres. Whilst the results confirmed some previous findings on the effects of Pi on Po, they also showed that the addition of 25 mM ASi led to a large (∼50%) but completely reversible depression of Po in both the fast and slow twitch rat muscle fibres. Moreover, the depression of Po by ASi was greater at low than at high pH values. Examined in the presence of Dextran T-500, the passive tension and sarcomere length responses to a ramp stretch were found to be qualitatively and quantitatively similar to those previously reported in intact rat muscle fibres. Thus, the tension response to a ramp stretch, in the presence and absence of either 25 mM Pi or ASi, consisted of a viscous (P₁), a visco-elastic (P₂) and an elastic (P₃) tension. However, the addition of either 25 mM Pi or ASi led to ∼15–18% increase in the amplitude of the visco-elastic (P₂) tension but had little or no effect on the amplitudes of the other two tension components (viscous, P₁ and elastic, P₃ tensions). Furthermore, neither compound significantly altered the relaxation rate of the passive muscle visco-elasticity (P₂ tension). These results show that Po (arising from cycling cross-bridges) and passive muscle visco-elasticity (P₂ tension) are affected differently by both Pi and ASi and suggest that they may not share a common structural basis. The possibility that passive muscle visco-elasticity (P₂ tension) arises from the gap-(titin) filament (as suggested previously by Mutungi and Ranatunga, 1996b J Physiol 496: 827–837) and that Pi and ASi increase its amplitude by interacting with the PEVK region of the filament are discussed. This revised version was published online in July 2006 with corrections to the Cover Date.     

Sarcomere length-dependent Ca<Superscript>2+</Superscript> activation in skinned rabbit psoas muscle fibers: coordinated regulation of thin filament cooperative activation and passive force

In skeletal muscle, active force production varies as a function of sarcomere length (SL). It has been considered that this SL dependence results simply from a change in the overlap length between the thick and thin filaments. The purpose of this study was to provide a systematic understanding of the SL-dependent increase in Ca²⁺ sensitivity in skeletal muscle, by investigating how thin filament “on–off” switching and passive force are involved in the regulation. Rabbit psoas muscles were skinned, and active force measurements were taken at various Ca²⁺ concentrations with single fibers, in the short (2.0 and 2.4 μm) and long (2.4 and 2.8 μm) SL ranges. Despite the same magnitude of SL elongation, the SL-dependent increase in Ca²⁺ sensitivity was more pronounced in the long SL range. MgADP (3 mM) increased the rate of rise of active force and attenuated SL-dependent Ca²⁺ activation in both SL ranges. Conversely, inorganic phosphate (Pi, 20 mM) decreased the rate of rise of active force and enhanced SL-dependent Ca²⁺ activation in both SL ranges. Our analyses revealed that, in the absence and presence of MgADP or Pi, the magnitude of SL-dependent Ca²⁺ activation was (1) inversely correlated with the rate of rise of active force, and (2) in proportion to passive force. These findings suggest that the SL dependence of active force in skeletal muscle is regulated via thin filament “on–off” switching and titin (connectin)-based interfilament lattice spacing modulation in a coordinated fashion, in addition to the regulation via the filament overlap.     

Effects of external cadmium ions on excitation–contraction coupling in rat soleus fibres

 The actions of external Cd²⁺ on the twitch and tetanic contractions, action potentials and potassium (K⁺) contractures of rat soleus muscle fibre bundles have been investigated. Cd²⁺ at 1–1.5 mM did not significantly alter tetanic tension, but increased twitch tension and increased the duration and overshoot of action potentials. At ≥3 mM, Cd²⁺ (1) depressed tetanic contractions and initially potentiated but later depressed twitches, (2) abolished the action potential overshoot, and (3) shifted peak K⁺ contracture tension to more positive membrane potentials. Twitch and tetanic contractions, and action potentials remained depressed when Cd²⁺ was washed out of the bath. The effects of Cd²⁺ on the twitch, tetanus and action potential were mimicked by Zn²⁺, while La³⁺ and Co²⁺ at 3 mM – or Mg²⁺ and Ca²⁺ at 30 mM – depressed peak twitch and tetanic tension, but did not potentiate twitches. The results suggest that: (1) Cd²⁺ and Zn²⁺ potentiate twitch tension by prolonging action potential depolarisation; (2) Cd²⁺ depresses twitch and tetanic tension by reducing the action potential overshoot, and causing a positive shift in the voltage dependence of contraction; and (3) the irreversible depression of action potential amplitude in rat soleus muscle is a specific property of Cd²⁺ and Zn²⁺ that is not shared by Co²⁺, Mg²⁺ or Ca²⁺. Received: 14 April 1998 / Received after revision and accepted: 3 September 1998     

Caffeine-evoked contractures in single slow (tonic) muscle fibres of the frog (Rana temporaria and R. esculenta)

Single slow (tonic) muscle fibres were dissected from cruralis muscles of Rana temporaria and R. esculenta. Increasing concentrations of caffeine were applied in Ringer solution, and contractures were measured isometrically. Sigmoid caffeine concentration-response curves were obtained, the threshold value being near 1.2 mmol/l, and maximum contractures being obtained with 10 to 20 mmol/l concentrations of caffeine. Contracture solutions were modified by varying the Ca²⁺ concentration or by replacing Ca²⁺ with 1.8 mmol/l Mg²⁺, Ni²⁺, Co²⁺ or with 0.1–5.0 mmol/l La³⁺. The effects of low pH (5.3), K⁺ (6,10 and 95 mmol/l), adenosine (10 mmol/l) and gallopamil (D600; 30 μmol/l) were examined too. The caffeine threshold was lowered by Mg²⁺, K⁺, 0 .1 mmol/l La³⁺ and D600, while all other substances including 0.5–5.0 mmol/l La³⁺ increased it. The amplitude of contractures evoked by high caffeine concentrations was unaffected. Caffeine (1–40 mmol/l) was also pressure injected into slow fibres. The composition of the solution was modified in a number of ways, but a contractile response was not observed or measured. Extracellular application of caffeine from the same pipettes evoked local contractures. Similar injection experiments in twitch fibres revealed the same results. These observations suggest that an extracellular binding site seems to be involved in the initiation of caffeine-evoked contractures in intact frog muscle fibres. Possible reasons for the ineffectiveness of intracellular caffeine are discussed. Received: 2 September 1995/Received after revision: 22 December 1995/Accepted: 4 January 1996     

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.     

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     

Presence of two Ca2+ influx components in internal Ca2+-pool-depleted rat parotid acinar cells

The molecular mechanism(s) involved in mediating Ca²⁺ entry into rat parotid acinar and other non-excitable cells is not known. In this study we have examined the kinetics of Ca²⁺ entry in fura-2-loaded parotid acinar cells, which were treated with thapsigargin to deplete internal Ca²⁺ pools (Ca²⁺-pool-depleted cells). The rate of Ca²⁺ entry was determined by measuring the initial increase in free cytosolic [Ca²⁺] ([Ca²⁺]_i) in Ca²⁺-pool-depleted, and control (untreated), cells upon addition of various [Ca²⁺] to the medium. In untreated cells, a low-affinity component was detected with K _Ca = 3.4 ± 0.7 mM (where K _Ca denotes affinity for Ca²⁺) and V _max = 9.8 ± 0.4 nM [Ca²⁺]_i/s. In thapsigargin-treated cells, two Ca²⁺ influx components were detected with K _Ca values of 152 ±  79 μM (V _max = 5.1 ± 1.9 nM [Ca²⁺]_i/s) and 2.4 ±  0.9 mM (V _max = 37.6 ± 13.6 nM [Ca²⁺]_i/s), respectively. We have also examined the effect of Ca²⁺ and depolarization on these two putative Ca²⁺ influx components. When cells were treated with thapsigargin in a Ca²⁺-free medium, Ca²⁺ influx was higher than into cells treated in a Ca²⁺-containing medium and, while there was a 46% increase in the V _max of the low-affinity component (no change in K _Ca), the high-affinity component was not clearly detected. In depolarized Ca²⁺-pool-depleted cells (with 50 mM KCl in the medium) the high-affinity component was considerably decreased while there was an apparent increase in the K _Ca of the low-affinity component, without any change in the V _max. These results demonstrate that Ca²⁺ influx into parotid acinar cells (1) is increased (four- to five-fold) upon internal Ca²⁺ pool depletion, and (2) is mediated via at least two components, with low and high affinities for Ca²⁺. Received: 30 October 1995/Received after revisionand accepted: 13 December 1995     

RISC (Repolarization-induced stop of caffeine-contracture) is not due to store depletion in cultured murine skeletal muscle

 We have combined the patch-clamp technique with Fura-2 measurements to investigate whether RISC (repolarization-induced stop of caffeine-contracture) is a consequence of store depletion in cultured skeletal muscles of rats and mice. Weak depolarizations (–45 to –40 mV) of long duration induced a barely detectable Ca²⁺ transient. Even under these conditions, caffeine-activated Ca²⁺transients (CafTs) were terminated upon membrane repolarization (–70 mV) at all stages of CafT. Following the peak of the CafT, massive Ca²⁺ release was elicited by either flash-photolysis of caged Ca²⁺ or further depolarization to 0 mV, demonstrating the lack of store depletion. Thus, RISC is not due primarily to store depletion but to closure of the Ca²⁺ release channels possibly through a mechanical interaction with voltage sensors. RISC was not present in rat heart muscle, further supporting a role of direct interaction in skeletal muscle. Received: 23 April 1996 / Accepted: 12 July 1996     

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     

Effects of halothane on functionally skinned rabbit soleus muscle fibers: A correlation between tension transient and45Ca release

The mechanism(s) involved in the halothane-induced increase in skeletal muscle contraction was studied using functionally skinned soleus muscle fibers from rabbits: For the tension study, single functionally skinned fibers were individually mounted on two pairs of forceps, with one end attached to a photodiode tension transducer. Ca²⁺-activated tension development of the contractile proteins, and Ca²⁺ uptake and release from the sarcoplasmic reticulum (SR) using caffeine-induced tension transients were studied. To measure the amount of calcium, skinned fibers at 0.1 g/ml were used and 0.075 Ci⁴⁵Ca/ml was spiked in the solution 3 (pCa 6.5 and 1 mM [EGTA]) which promoted rapid loading of Ca²⁺. Halothane (1–3%) did not change the [Ca²⁺]-tension relationship; 2 and 3% halothane reduced the maximum Ca²⁺-activated tension by 6–7%. Halothane (1–3%) added to the solution 3, reduced⁴⁵Ca uptake by 3, 22 and 23%; however, the subsequent caffeine-induced tension transient and⁴⁵Ca release were increased by 10–40%. During the release phase only halothane increased both caffeine-induced tension transient and⁴⁵Ca release by 20–60%. The effects of halothane on the tension transient and on the⁴⁵Ca release were comparable. There was no dose-response relationship to the effects of halothane on the above parameters. It is concluded that halothane affects the SR by increasing its membrane permeability to Ca²⁺, resulting in an increase in myoplasmic [Ca²⁺] and thus in the twitch tension in skeletal muscle.     

Exogenous lysophosphatidylcholine increases non-selective cation current in guinea-pig ventricular myocytes

Whole cell, patch-clamp studies were performed to examine the effect of lysophosphatidylcholine (LPC) on the membrane current in guinea-pig ventricular myocytes. The addition of 10 μM LPC to the external solution induced a membrane current which had a reversal potential of 0 mV. When Na⁺, the main cation in the external solution, was replaced by either K⁺, N-methyl-D-glucamine (NMG) or 90 mM Ca²⁺, LPC induced a current with the reversal potential near 0 mV, indicating that the current passed through a Ca²⁺-permeable non-selective cation channel. The order of the cationic permeability calculated from the reversal potential of the current was Cs⁺ > K⁺ > NMG > Na⁺ > Ca²⁺. Cl⁻ did not pass through the LPC-induced channel. The LPC-induced current was not blocked by Gd³⁺ in the external solution, nor by the absence of Ca²⁺ in the pipette solution. In conclusion, LPC induces a Ca²⁺-permeable non-selective cation channel in guinea-pig ventricular myocytes. Received: 11 September 1995/Received after revision: 3 January 1996/Accepted: 12 February 1996     

Calcium-dependent, sustained enhancement of excitability during washout of aconitine in rat hippocampal slices

 Extracellular recording of the stimulus-evoked population spike was performed in the CA1 area of rat hippocampal slices in order to investigate delayed effects of the plant alkaloids aconitine and veratridine. Veratridine (1 μM and 10 μM) suppressed the orthodromic and antidromic population spike. After washout of the drug, only a partial recovery was obtained. Aconitine (1 μM) exerted the same inhibitory action as veratridine. However, after washout, the spike amplitude was enhanced compared with the control. This enhancement of the spike amplitude was dependent on the concentration of aconitine and was maintained during the observation period of at least 2 h. Lowering the Ca²⁺ concentration of the bathing medium from 2.5 mM to 1.25 mM during application of aconitine attenuated recovery and prevented the enhancement observed during washout of the drug. Application of aconitine in the presence of CdCl₂ as well as in the presence of inhibitors of protein kinase C and Ca²⁺/calmodulin-dependent protein kinase II prevented the increase in spike amplitude during washout with standard artificial cerebrospinal fluid. In contrast, the N-methyl-d-aspartate (NMDA) receptor antagonists D-AP5 and MK-801 as well as the non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione were ineffective in abolishing the aconitine-induced enhancement. These data support the conclusion that different modes of action are involved in the effects of aconitine but not veratridine. It is concluded that the aconitine-induced increase in neuronal activity is mediated by intracellular Ca²⁺-dependent mechanisms leading to an activation of Ca²⁺-dependent protein kinases. This effect is independent of Ca²⁺ entrance through NMDA and non-NMDA receptors. Received: 3 July 1996 / Accepted: 14 November 1996     

Effects of vanadate, phosphate and 2,3-butanedione monoxime (BDM) on skinned molluscan catch muscle

The effects of orthovanadate (V_i), inorganic phosphate (P_i) and 2,3-butanedione monoxime (BDM) on tension, force transients and the catch state (passive tension maintenance) were investigated in saponin-skinned fibre bundles of the anterior byssus retractor muscle (ABRM) of the bivalve mollusc Mytilus edulis at pH 6.7. During maximal Ca²⁺ activation isometric force was depressed by V_i (0.03–10 mM), P_i (10 mM) and BDM (50 mM). Force transients following quick stretches (0.1–0.3% of fibre length) were accelerated substantially by 1 mM V_i, 10 mM P_i or 50 mM BDM. These compounds also accelerated force responses in experiments in which ATP was released rapidly from caged ATP by flash photolysis at both pCa 4.7 (force rise) and at pCa>8 (force decline). The effects on the catch state were investigated in two types of experiments: (1) Ca²⁺ removal after maximal Ca²⁺ activation and (2) rapid ATP release during high-force rigor at pCa>8. In both cases rapid relaxation was followed by slow relaxation (slower than 2% of initial force per min). This later slow relaxation (catch) was insensitive to V_i (1–10 mM), P_i (10 mM) and BDM (50 mM) but was accelerated by 0.12 mM cAMP. Complete relaxation to almost zero force was attained by changing pH from 6.7 to 7.7 (pCa>8). We conclude that catch depends on cAMP- and pH-sensitive structures linking the myofilaments and not on the force-generating actomyosin cross-bridges that are sensitive to V_i, P_i and BDM.