Spontaneous tension oscillation in skinned bovine cardiac muscle

 Skinned fibres from bovine ventricles exhibited spontaneous tension oscillations when MgADP and inorganic phosphate (Pi) were added to the solution bathing fibres in the relaxed state (ADP-SPOC). A similar type of oscillation was observed at intermediate concentrations of free Ca²⁺ in the absence of MgADP and Pi (Ca-SPOC). To investigate the correlation between ADP-SPOC and Ca-SPOC, we constructed two-dimensional state diagrams of cardiac muscle using different concentrations of Pi (0–20 mM) and free Ca²⁺ [pCa=around 5 (+Ca²⁺), pCa=5.15–6.9 and +EGTA (–Ca²⁺)], with varying concentrations of MgADP (0–10 mM), with 2 mM MgATP and 2 mM free Mg²⁺ maintaining ionic strength at 0.15±0.01 M, pH 7.0, 25 °C. The three-dimensional (pCa-Pi-MgADP) state diagram thus obtained was divided into three regions, i.e. the contraction region in which tension oscillation was undetectable, the spontaneous tension oscillation (SPOC) region and the relaxation region. We found that the regions of ADP-SPOC and Ca-SPOC were continuously connected by a single oscillation region sandwiched between the contraction and relaxation regions. The state diagram, which encompasses physiological conditions, shows that the probability of SPOC is higher in cardiac muscle than in skeletal muscle. From these results, we suggest that, despite distinct ionic conditions, the molecular state of cross-bridges during SPOC is common to both ADP-SPOC and Ca-SPOC. Received 19 February 1996 / Received after revision: 16 July 1996 / Accepted: 14 August 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     

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     

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     

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     

Effect of alkalinization of cytosolic pH by amines on intracellular Ca2+ activity in HT29 cells

The effect of secondary, tertiary and quaternary methyl- and ethylamines on intracellular pH (pH_i) and intracellular Ca²⁺ activity ([Ca²⁺]_i) of HT₂₉ cells was investigated microspectrofluorimetrically using pH- and Ca²⁺- sensitive fluorescent indicators, [i.e. 2′,7′-biscarboxyethyl-5(6)-carboxyfluorescein (BCECF) and fura-2 respectively]. Membrane voltage (V _m) was studied by the patch-clamp technique. Secondary and tertiary amines led to a rapid and stable concentration-dependent alkalinization which was independent of their pK _a value. Trimethylamine (20 mmol/l) increased pH_i by 0.78 ± 0.03 pH units (n = 9) and pH remained stable for the application time. Removal led to an undershoot of pH_i and a slow and incomplete recovery: pH_i stayed 0.26 ± 0.06 pH units more acid than the resting value. The quaternary amines, tetramethyl- and tetraethylamine were without influence on pH_i. All tested secondary and tertiary amines (dimethyl-, diethyl-, trimethyl-, and triethyl-amine) induced a [Ca²⁺]_i transient which reached a peak value within 10–25 s and then slowly declined to a [Ca²⁺]_i plateau. The initial Δ[Ca²⁺]_i induced by trimethylamine (20 mmol/l) was 160 ± 15 nmol/l (n = 17). The [Ca²⁺]_i peak was independent of the Ca²⁺ activity in the bath solution, but the [Ca²⁺]_i plateau was significantly lower under Ca²⁺-free conditions and could be immediately interrupted by application of CO₂ (10%; n = 6), a manoeuvre to acidify pH_i in HT₂₉ cells. Emptying of the carbachol- or neurotensin-sensitive intracellular Ca²⁺ stores completely abolished this [Ca²⁺]_i transient. Tetramethylamine led to higher [Ca²⁺]_i changes than the other amines tested and only this transient could be completely blocked by atropine (10⁻⁶ mol/l). Trimethylamine (20 mmol/l) hyperpolarized V _m by 22.5 ± 3.7 mV (n = 16) and increased the whole-cell conductance by 2.3 ± 0.5 nS (n = 16). We conclude that secondary and tertiary amines induce stable alkaline pH_i changes, release Ca²⁺ from intracellular, inositol-1,4,5-trisphosphate-sensitive Ca²⁺ stores and increase Ca²⁺ influx into HT₂₉ cells. The latter may be related to both the store depletion and the hyperpolarization. Received: 11 September 1995/Received after revision and accepted: 18 December 1995     

Regulation of force and shortening velocity by calcium and myosin phosphorylation in chemically skinned smooth muscle

 The phosphatase inhibitor okadaic acid (OA) was used to study the relationship between [Ca²⁺], rates of phosphorylation/dephosphorylation and the mechanical properties of smooth muscle fibres. Force/velocity relationships were determined with the isotonic quick release technique in chemically skinned guinea-pig taenia coli muscles at 22° C. In the maximally thiophosphorylated muscle neither OA (10 μM) nor Ca²⁺ (increase from pCa 9.0 to pCa 4.5) influenced the force-velocity relationship. When the degree of activation was altered by varying [Ca²⁺] in the presence of 0.5 μM calmodulin, both force and the maximal shortening velocity (V _max) were altered. At pCa 5.75, at which force was about 35% of the maximal at pCa 4.5, V _max was 55% of the maximal value. When OA was introduced into fibres at pCa 6.0, force was increased from less than 5% to 100% of the maximal force obtained in pCa 4.5. The relationship between the degree of myosin light chain phosphorylation and force was similar in the two types of activation; varied [OA] at constant [Ca²⁺] and at varied [Ca²⁺]. The relation between force and V _max when the degree of activation was altered with OA was almost identical to that obtained with varied [Ca²⁺]. The results show that Ca²⁺ and OA do not influence force or V _max in the maximally phosphorylated state and suggest that the level of myosin light chain phosphorylation is the major factor determining V _max. The finding that the relationship between force and V _max was similar when activation was altered with OA and Ca²⁺ suggests, however, that alterations in the absolute rates of phosphorylation and dephosphorylation at a constant phosphorylation level do not influence the mechanical properties of the skinned smooth muscle fibres. Received: 1 December 1995 / Received after revision: 20 June 1996 / Accepted 12 July 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     

The effect of Ca2+ on the structure of synthetic filaments of smooth muscle myosin

Using electron microscopy and negative staining we have studied the effect of Ca²⁺ on the structure of synthetic filaments of chicken gizzard smooth muscle myosin under conditions applied by Frado and Craig (1989) for demonstration of the influence of Ca²⁺ on the structure of synthetic filaments of scallop striated muscle myosin. The results show that Ca²⁺ induces the transition of compact, ordered structure of filaments with a 14.5 nm axial repeat of the myosin heads close to the filament backbone (characteristic of the relaxing conditions) to a disordered structure with randomly arranged myosin heads together with subfragments-2 (S-2) seen at a distance of up to 50 nm from the filament backbone. This order/disorder transition is much more pronounced in filaments formed of unphosphorylated myosin, since a substantial fraction of phosphorylated filaments in the relaxing solution is already disordered due to phosphorylation. Under rigor conditions some of the filaments of unphosphorylated and phosphorylated myosin retain a certain degree of order resembling those under relaxing conditions, while most of them have a substantially disordered appearance. The results indicate that Ca²⁺-induced movement of myosin heads away from the filament backbone is an inherent property of smooth muscle myosin, like molluscan muscle myosin regulated exclusively by Ca²⁺ binding, and can play a modulatory role in smooth muscle contraction.     

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     

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     

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     

Evidence for the existence of inositol (1,4,5)-trisphosphate- and ryanodine-sensitive pools in bovine endothelial cells. Ca2+ releases in cells with different basal level of intracellular Ca2+

In single bovine aortic endothelial (BAE) cells pre-loaded with Fura-2, Ca²⁺ transients in a Ca²⁺-free medium have been revealed, which evidently reflects Ca²⁺ release from intracellular stores. In cells with different levels of resting basal cytoplasmic Ca²⁺ ([Ca²⁺]_i) from about 50 to 110 nM, a biphasic dependence of the Ca²⁺ transients on resting [Ca²⁺]_i was shown and spontaneous Ca²⁺ oscillations were observed. At a [Ca²⁺]_i level over 110 nM, a pronounced rise in Ca²⁺ transients occurred and only single transients were observed. Ryanodine (10 μM) produced a transient [Ca²⁺]_i elevation, suggesting the presence of ryanodine receptors in intracellular store membranes. The results imply that both inositol 1,4,5-trisphosphate-sensitive Ca²⁺ release (IICR) and Ca²⁺-sensitive Ca²⁺ release (CICR) take place in BAE cells. Only IICR seems to be sufficient for generating baseline Ca²⁺ oscillations in BAE cells, whereas the ATP-induced (5–100 μM) Ca²⁺ response involves the CICR set in motion by an oscillatory IICR of high frequency. The completion of both the spontaneous and ATP-induced Ca²⁺ transients was associated with a [Ca²⁺]_i decrease to a level below the initial resting [Ca²⁺]_i (undershoot). Its depth biphasically depended on the resting [Ca²⁺]_i from 50 to 110 nM, suggesting that the lack of a Ca²⁺ leak from inositol 1,4,5-trisphosphate-sensitive stores is responsible for the undershoot in this range. The Ca²⁺ leak is concluded to play a key role in the initiation and termination of regenerative IICR both in spontaneous oscillations and in ATP-induced transients. Received: 13 November 1995/Received after revision and accepted 27 March 1996     

Attenuation of stimulated Ca2+ influx in colonie epithelial (HT29) cells by cAMP

In HT₂₉ colonic epithelial cells agonists such as carbachol (CCH) or ATP increase cytosolic Ca²⁺ activity ([Ca²⁺]_i) in a biphasic manner. The first phase is caused by inositol 1,4,5-trisphophate-(Ins P ₃-) mediated Ca²⁺ release from their respective stores and the second plateau phase is mainly due to stimulated transmembraneous Ca²⁺ influx. The present study was undertaken to examine the effect of increased adenosine 3′,5′-cyclic monophasphate (cAMP) (forskolin 10 μmol/l = FOR) on the Ca²⁺ transient in the presence of CCH (100 μmol/l). In unpaired experiments it was found that FOR induced a depolarization and reduced cytosolic Ca²⁺ ([Ca²⁺]_i, measured as the fura-2 fluorescence ratio 340/380 nm) significantly. Dideoxyforskolin had no such effect. The effect of FOR was abolished when the cells were depolarized by a high-K⁺ solution. In further paired experiments utilizing video imaging in conjunction with whole-cell patch-clamp, [Ca²⁺]_i was monitored separately for the patch-clamped cell and three to seven neighbouring cells. In the presence of CCH, FOR reduced [Ca²⁺]_i uniformly from a fluorescence ratio (345/380) of 2.9 ± 0.12 to 1.8 ± 0.07 in the patch-clamped cell and its neighbours (n = 48) and depolarized the membrane voltage (V _m) of the patch-clamped cells significantly and reversibly from −54 ± 7.4 to −27 ± 5.9 mV (n = 6). In additional experiments V _m was depolarized by 15–54 mV by various increments in the bath K⁺ concentration. This led to corresponding reductions in [Ca²⁺]_i. Irrespective of the cause of depolarization (high K⁺ or FOR) there was a significant correlation between the change in V _m and change in [Ca²⁺]_i. These data indicate that the cAMP-mediated attenuation of Ca²⁺ influx is caused by the depolarization produced by this second messenger. Received: 12 March 1996/Accepted: 2 April 1996     

Modulation of Ca2+ channels by intracellular Mg2+ ions and GTP in frog ventricular myocytes

Under conditions of low intracellular [Mg²⁺] ([Mg²⁺]_i), achieved by dialysis with pipette solutions containing ethylenediamine tetraacetic acid (EDTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA) and adenosine triphosphate (ATP) as chelator, calcium currents through the L-type calcium channels (I _Ca) were increased in frog ventricular myocytes. Total suppression of phosphorylation by depleting the cell of ATP with a cocktail of β,γ-methyleneadenosine 5′-triphosphate (AMP-PCP) 2-deoxyglucose and carboxylcyanide-M-chlorophenylhydrazone (CCCP) did not inhibit the increase in I _Ca in the Mg²⁺-deficient medium. Thus, the involvement of phosphorylation process in the increase in I _Ca was not likely. Effective suppression of this enhancement of I _Ca was achieved by the application of guanosine triphosphate (GTP). From the dose-response curve for GTP, the GTP concentration required for half-maximal inhibition (IC₅₀) was estimated to be 4.0 μM at pMg 6. This GTP-induced suppression of I _Ca is not due to the guanine nucleotide binding protein (G-protein) cascade, because both activators and inhibitors of G-protein, which are structural analogues of GTP, suppressed I _Ca similarly. Treatment with pertussis toxin (PTX) did not affect the inhibitory action of Mg²⁺ and GTP on I _Ca. GTP is therefore assumed to bind directly to the Ca²⁺ channel. Interaction of Mg²⁺ and GTP with the Ca²⁺ channel activated in the Mg²⁺-deficient medium was examined by comparing the dose/response curves for GTP at two different [Mg²⁺]. The IC₅₀ for GTP suppression was estimated to be 5.7 μM at pMg 6 and 6.9 μM at pMg 5. The results suggest strongly that Mg²⁺ and GTP independently bind and control Ca²⁺ channels. Received: 22 December 1995/Received after revision and accepted: 11 March 1996     

Length-dependent Ca2+ activation in cardiac muscle: some remaining questions

The steep relationship between systolic force and end diastolic volume in cardiac muscle (Frank–Starling relation) is, to a large extent, based on length-dependent changes in myofilament Ca²⁺ sensitivity. How sarcomere length modulates Ca²⁺ sensitivity is still a topic of active investigation. Two general themes have emerged in recent years. On the one hand, there is a large body of evidence indicating that length-dependent changes in lattice spacing determine changes in Ca²⁺ sensitivity for a given set of conditions. A model has been put forward in which the number of strong-binding cross-bridges that are formed is directly related to the proximity of the myosin heads to binding sites on actin. On the other hand, there is also a body of evidence suggesting that lattice spacing and Ca²⁺ sensitivity are not tightly linked and that there is a length-sensing element in the sarcomere, which can modulate actin–myosin interactions independent of changes in lattice spacing. In this review, we examine the evidence that has been cited in support of these viewpoints. Much recent progress has been based on the combination of mechanical measurements with X-ray diffraction analysis of lattice spacing and cross-bridge interaction with actin. Compelling evidence indicates that the relationship between sarcomere length and lattice spacing is influenced by the elastic properties of titin and that changes in lattice spacing directly modulate cross-bridge interactions with thin filaments. However, there is also evidence that the precise relationship between Ca²⁺ sensitivity and lattice spacing can be altered by changes in protein isoform expression, protein phosphorylation, modifiers of cross-bridge kinetics, and changes in titin compliance. Hence although there is no unique relationship between Ca²⁺ sensitivity and lattice spacing the evidence strongly suggests that under any given set of physiological circumstances variation in lattice spacing is the major determinant of length-dependent changes in Ca²⁺ sensitivity.     

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     

Effect of calmodulin on Ca2+-induced Ca2+ release of skeletal muscle from mutant mice expressing either ryanodine receptor type 1 or type 3

 We analyzed the effects of calmodulin (CaM) on Ca²⁺-induced Ca²⁺ release (CICR) in mouse skeletal muscle cells expressing only ryanodine receptor type 1 (RyR-1) or type 3 (RyR-3) following targeted disruption of one of the RyR genes. Under Mg²⁺-free conditions, CaM potentiated CICR via RyR-3 at low Ca²⁺ concentrations (pCa≥6) but inhibited CICR at high Ca²⁺ concentrations (pCa≤5). On the other hand, CaM potentiated CICR via RyR-1 between pCa 7 and pCa 5. Greater concentrations of CaM were required for potentiation of CICR at pCa 6 than for the inhibition at pCa 5 in the RyR-3-expressing cells. Similarly, higher concentrations of CaM were required for the potentiation of CICR via RyR-1 at pCa 6 than potentiation at pCa 5. In the presence of Mg²⁺ and β,γ-methyleneadenosine 5′-trisphosphate (AMPOPCP), the same differential effects of CaM on the CICR via the different subtypes of RyR were observed. These data suggest that multiple CaM-binding sites are involved in the differential effects on RyR-1 and RyR-3. These effects of CaM are important for the evaluation of the physiological roles of RyRs. Received: 5 May 1998 / Received after revision: 14 August 1998 / 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     

Fluorescence changes on contractile activation in TnCDANZ labeled skinned rabbit psoas fibers

The increase in fluorescence of dansylaziridine (DANZ) labeled troponin C (TnC_DANZ) substituted into skinned rabbit psoas fibers was determined as a function of the pCa. The fluorescence data are expressed as the ratio of two wavelength bands, one that sees the fluorescence of TnC_DANZ, and one that sees background fluorescence and scatter. The percent TnC replaced with TnC_DANZ was varied between 10 and 50% and, the fibers were randomly stretched, at the start of each experiment, between 10 and 50%. A large ratio increase accompanies increase in [Ca²⁺]. The pCa/force data are best fit by the Hill equation but the pCa/ratio data are best fit by a model in which Ca²⁺ binds in two phases. The position of the force curve on the pCa axis varies little between fibers, in contrast to that of the ratio or Δ-fluorescence curve. In accord with previous reports the Δ-fluorescence can be left of the force on the pCa axis (type I) or superimpose in part on the force (type II). Not described previously, we find curves in which the second phase of the ratio cross-over the pCa/force curve. This type III relationship is found only in fibers less than 3 weeks postmuscle harvest. We propose that the first, relatively invariant, phase of the biphasic pCa/ratio curve accompanies Ca²⁺ binding to either of the two low affinity sites on TnC_DANZ as it does for TnC in solution. The second, highly cooperative, phase of the ratio curve that accompanies muscle contraction and enhanced Ca²⁺ binding is initiated when sufficient Ca²⁺ is bound to overcome inhibitory systems. Loose coupling between the initial Ca²⁺ binding and the cooperative switch point may account for much of the variation in the shape and position of the pCa/ratio curve. There is evidence that, in the overlap zone, weakly attached myosin cross-bridges enhance cooperation between the regulatory units of the thin filaments. This revised version was published online in July 2006 with corrections to the Cover Date.