The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were st... more The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca 2 ϩ ] i) by inhibiting the release of calcium from the SR. The suppression of [Ca 2 ϩ ] i was dose-dependent, with no effect at or below 1 M and a 53 Ϯ 8% (mean Ϯ SEM, n ϭ 9, cut fibers) attenuation at 0 mV with 25 M of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 Ϯ 8%) and of the early peak component (46 Ϯ 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (I Ca) were left, essentially, unaltered. However, the inactivation of I Ca was slowed fourfold, and the conductance was reduced from 200 Ϯ 16 to 143 Ϯ 8 SF Ϫ 1 (n ϭ 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 Ϯ 10% (n ϭ 3) at 12 M. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30-50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.
The ATP-dependent Caz' transport in sarcoplasmic reticulum involves transitions between several s... more The ATP-dependent Caz' transport in sarcoplasmic reticulum involves transitions between several structural states of the CaZ*-ATPase, that occur without major changes in the secondary structure. The rates ofthese transitions are modulated by the lipid enviroIl]~ent and by interactions between ATPase molecules. Although the Ca*+-ATPdse restricts the rotational mobility of a population of lipids, there is no evidence f&r specific interaction of the CazC-ATPase with phospholipids. Fluorescence polarization and energy transfer (FET) studies. using site specific fluorescent indicators, combined with crystallographic, immunological and chemical modification data, yielded a structural model of Ca ZC-ATPase in which the binding sites of Ca*+ and ATP are tentatively identified. The temperature dependence of FET between fluorophores attached to different regions of the ATPase indicates the existence of'rigid' and 'flexible' regions within the molecule characterized. by different degrees of thermally induced structural fluctuations.
Publisher Summary It is an old goal in the physiology to determine the space distribution of Ca. ... more Publisher Summary It is an old goal in the physiology to determine the space distribution of Ca. In steady-state, this can be handled as parallel compartments, each of them exchanging ions directly with the outside only. To determine the number of compartments and their kinetic parameters, the curve of desaturation q (t)=∑Aie-λit is measured and the number of exponentials and their parameters are calculated. A method is chosen for the deconvolution of superposition of exponentials and for the determination of kinetic parameters, which one is less sensitive for stochastic errors and objectively gives the number of components. This chapter discusses the development of a Fourier transform method for the deconvolution and presents the results obtained using this method. The Fourier transform method is used for deconvolution because it can be used in the case of theoretically infinite number of components and it is not necessary to give an approximate value for the parameters.
Journal of Muscle Research and Cell Motility, 2015
ABSTRACT Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin t... more ABSTRACT Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin therapy. Although the pathomechanism of statin-induced myopathy is not completely understood, changes in calcium homeostasis and reduced coenzyme Q10 levels are hypothesized to play important roles. In our experiments, fluvastatin and/or coenzyme Q10 was administered chronically to normocholesterolaemic or hypercholaestherolaemic rats, and the modifications of the calcium homeostasis and the strength of their muscles were investigated. While hypercholesterolaemia did not change the frequency of sparks, fluvastatin increased it on muscles both from normocholesterolaemic and from hypercholesterolaemic rats. This effect, however, was not mediated by a chronic modification of the ryanodine receptor as shown by the unchanged ryanodine binding in the latter group. While coenzyme Q10 supplementation significantly reduced the frequency of the spontaneous calcium release events, it did not affect their amplitude and spatial spread in muscles from fluvastatin-treated rats. This indicates that coenzyme Q10 supplementation prevented the spark frequency increasing effect of fluvastatin without having a major effect on the amount of calcium released during individual sparks. In conclusion, we have found that fluvastatin, independently of the cholesterol level in the blood, consistently and specifically increased the frequency of calcium sparks in skeletal muscle cells, an effect which could be prevented by the addition of coenzyme Q10 to the diet. These results support theories favouring the role of calcium handling in the pathophysiology of statin-induced myopathy and provide a possible pathway for the protective effect of coenzyme Q10 in statin treated patients symptomatic of this condition.
The sarcoplasmic reticulum of rabbit skeletal muscle contains a small "proteolipid,&... more The sarcoplasmic reticulum of rabbit skeletal muscle contains a small "proteolipid," i.e., a protein which is soluble in acidic CHCl3/CH3OH. We propose the name sarcolipin for this small protein, to signify its lipid-like solubility and association with the sarcoplasmic reticulum. We have determined the following amino acid sequence for sarcolipin, using protein chemistry methods: M E R S T R E L C L N F T V V L I T V I L I W L L V R S Y Q Y. This 31-residue sequence includes a 19-residue hydrophobic segment which probably spans the sarcoplasmic reticulum membrane. The molecular weight calculated from the sequence, 3733, agrees with that measured by fast atom bombardment mass spectrometry, showing that sarcolipin contains no attached fatty acyl or other prosthetic groups.
Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea p... more Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC(50) = 297 +/- 12 microM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V(max) = 0.47 +/- 0.04 nmol s(-1), EC(50) = 258 +/- 21 microM, Hill coefficient = 3.0 +/- 0.54), and decreased the activity of the calcium pump (EC(50) = 253 +/- 4.7 microM, Hill coefficient = 1.62 +/- 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, thi...
Concentration-dependent eects of bimoclomol, the novel heat shock protein coinducer, on intracell... more Concentration-dependent eects of bimoclomol, the novel heat shock protein coinducer, on intracellular calcium transients and contractility were studied in Langendor-perfused guinea-pig hearts loaded with the¯uorescent calcium indicator dye Fura-2. Bimoclomol had a biphasic eect on contractility: both peak left ventricular pressure and the rate of force development signi®cantly increased at a concentration of 10 nM or higher. The maximal eect was observed between 0.1 and 1 mM, and the positive inotropic action disappeared by further increasing the concentration of bimoclomol. The drug increased systolic calcium concentration with a similar concentrationdependence. In contrast, diastolic calcium concentration increased monotonically in the presence of bimoclomol. Thus low concentrations of the drug (10 ± 100 nM) increased, whereas high concentrations (10 mM) decreased the amplitude of intracellular calcium transients. 2 Eects of bimoclomol on action potential con®guration was studied in isolated canine ventricular myocytes. Action potential duration was increased at low (10 nM), unaected at intermediate (0.1 ± 1 mM) and decreased at high (10 ± 100 mM) concentrations of the drug. 3 In single canine sarcoplasmic calcium release channels (ryanodine receptor), incorporated into arti®cial lipid bilayer, bimoclomol signi®cantly increased the open probability of the channel in the concentration range of 1 ± 10 mM. The increased open probability was associated with increased mean open time. The eect of bimoclomol was again biphasic: the open probability decreased below the control level in the presence of 1 mM bimoclomol. 4 Bimoclomol (10 mM ± 1 mM) had no signi®cant eect on the rate of calcium uptake into sarcoplasmic reticulum vesicles of the dog, indicating that in vivo calcium reuptake might not substantially be aected by the drug. 5 In conclusion, the positive inotropic action of bimoclomol is likely due to the activation of the sarcoplasmic reticulum calcium release channel in mammalian ventricular myocardium.
Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transp... more Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.
The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were st... more The effects of the muscle relaxant dantrolene on steps of excitation-contraction coupling were studied on fast twitch muscles of rodents. To identify the site of action of the drug, single fibers for voltage-clamp measurements, heavy SR vesicles for calcium efflux studies and solubilized SR calcium release channels/RYRs for lipid bilayer studies were isolated. Using the double Vaseline-gap or the silicone-clamp technique, dantrolene was found to suppress the depolarization-induced elevation in intracellular calcium concentration ([Ca 2 ϩ ] i) by inhibiting the release of calcium from the SR. The suppression of [Ca 2 ϩ ] i was dose-dependent, with no effect at or below 1 M and a 53 Ϯ 8% (mean Ϯ SEM, n ϭ 9, cut fibers) attenuation at 0 mV with 25 M of extracellularly applied dantrolene. The drug was not found to be more effective if injected than if applied extracellularly. Calculating the SR calcium release revealed an equal suppression of the steady (53 Ϯ 8%) and of the early peak component (46 Ϯ 6%). The drug did not interfere with the activation of the voltage sensor in as much as the voltage dependence of both intramembrane charge movements and the L-type calcium currents (I Ca) were left, essentially, unaltered. However, the inactivation of I Ca was slowed fourfold, and the conductance was reduced from 200 Ϯ 16 to 143 Ϯ 8 SF Ϫ 1 (n ϭ 10). Dantrolene was found to inhibit thymol-stimulated calcium efflux from heavy SR vesicles by 44 Ϯ 10% (n ϭ 3) at 12 M. On the other hand, dantrolene failed to affect the isolated RYR incorporated into lipid bilayers. The channel displayed a constant open probability for as long as 30-50 min after the application of the drug. These data locate the binding site for dantrolene to be on the SR membrane, but be distinct from the purified RYR itself.
The ATP-dependent Caz' transport in sarcoplasmic reticulum involves transitions between several s... more The ATP-dependent Caz' transport in sarcoplasmic reticulum involves transitions between several structural states of the CaZ*-ATPase, that occur without major changes in the secondary structure. The rates ofthese transitions are modulated by the lipid enviroIl]~ent and by interactions between ATPase molecules. Although the Ca*+-ATPdse restricts the rotational mobility of a population of lipids, there is no evidence f&r specific interaction of the CazC-ATPase with phospholipids. Fluorescence polarization and energy transfer (FET) studies. using site specific fluorescent indicators, combined with crystallographic, immunological and chemical modification data, yielded a structural model of Ca ZC-ATPase in which the binding sites of Ca*+ and ATP are tentatively identified. The temperature dependence of FET between fluorophores attached to different regions of the ATPase indicates the existence of'rigid' and 'flexible' regions within the molecule characterized. by different degrees of thermally induced structural fluctuations.
Publisher Summary It is an old goal in the physiology to determine the space distribution of Ca. ... more Publisher Summary It is an old goal in the physiology to determine the space distribution of Ca. In steady-state, this can be handled as parallel compartments, each of them exchanging ions directly with the outside only. To determine the number of compartments and their kinetic parameters, the curve of desaturation q (t)=∑Aie-λit is measured and the number of exponentials and their parameters are calculated. A method is chosen for the deconvolution of superposition of exponentials and for the determination of kinetic parameters, which one is less sensitive for stochastic errors and objectively gives the number of components. This chapter discusses the development of a Fourier transform method for the deconvolution and presents the results obtained using this method. The Fourier transform method is used for deconvolution because it can be used in the case of theoretically infinite number of components and it is not necessary to give an approximate value for the parameters.
Journal of Muscle Research and Cell Motility, 2015
ABSTRACT Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin t... more ABSTRACT Myalgia and muscle weakness may appreciably contribute to the poor adherence to statin therapy. Although the pathomechanism of statin-induced myopathy is not completely understood, changes in calcium homeostasis and reduced coenzyme Q10 levels are hypothesized to play important roles. In our experiments, fluvastatin and/or coenzyme Q10 was administered chronically to normocholesterolaemic or hypercholaestherolaemic rats, and the modifications of the calcium homeostasis and the strength of their muscles were investigated. While hypercholesterolaemia did not change the frequency of sparks, fluvastatin increased it on muscles both from normocholesterolaemic and from hypercholesterolaemic rats. This effect, however, was not mediated by a chronic modification of the ryanodine receptor as shown by the unchanged ryanodine binding in the latter group. While coenzyme Q10 supplementation significantly reduced the frequency of the spontaneous calcium release events, it did not affect their amplitude and spatial spread in muscles from fluvastatin-treated rats. This indicates that coenzyme Q10 supplementation prevented the spark frequency increasing effect of fluvastatin without having a major effect on the amount of calcium released during individual sparks. In conclusion, we have found that fluvastatin, independently of the cholesterol level in the blood, consistently and specifically increased the frequency of calcium sparks in skeletal muscle cells, an effect which could be prevented by the addition of coenzyme Q10 to the diet. These results support theories favouring the role of calcium handling in the pathophysiology of statin-induced myopathy and provide a possible pathway for the protective effect of coenzyme Q10 in statin treated patients symptomatic of this condition.
The sarcoplasmic reticulum of rabbit skeletal muscle contains a small "proteolipid,&... more The sarcoplasmic reticulum of rabbit skeletal muscle contains a small "proteolipid," i.e., a protein which is soluble in acidic CHCl3/CH3OH. We propose the name sarcolipin for this small protein, to signify its lipid-like solubility and association with the sarcoplasmic reticulum. We have determined the following amino acid sequence for sarcolipin, using protein chemistry methods: M E R S T R E L C L N F T V V L I T V I L I W L L V R S Y Q Y. This 31-residue sequence includes a 19-residue hydrophobic segment which probably spans the sarcoplasmic reticulum membrane. The molecular weight calculated from the sequence, 3733, agrees with that measured by fast atom bombardment mass spectrometry, showing that sarcolipin contains no attached fatty acyl or other prosthetic groups.
Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea p... more Concentration-dependent effects of thymol on calcium handling were studied in canine and guinea pig cardiac preparations (Langendorff-perfused guinea pig hearts, canine ventricular trabeculae, canine sarcoplasmic reticular vesicles and single ryanodine receptors). Thymol induced a concentration-dependent negative inotropic action in both canine and guinea pig preparations (EC(50) = 297 +/- 12 microM in dog). However, low concentrations of thymol reduced intracellular calcium transients in guinea pig hearts without decreasing contractility. At higher concentrations both calcium transients and contractions were suppressed. In canine sarcoplasmic reticular vesicles thymol induced rapid release of calcium (V(max) = 0.47 +/- 0.04 nmol s(-1), EC(50) = 258 +/- 21 microM, Hill coefficient = 3.0 +/- 0.54), and decreased the activity of the calcium pump (EC(50) = 253 +/- 4.7 microM, Hill coefficient = 1.62 +/- 0.05). Due to the less sharp concentration-dependence of the ATPase inhibition, thi...
Concentration-dependent eects of bimoclomol, the novel heat shock protein coinducer, on intracell... more Concentration-dependent eects of bimoclomol, the novel heat shock protein coinducer, on intracellular calcium transients and contractility were studied in Langendor-perfused guinea-pig hearts loaded with the¯uorescent calcium indicator dye Fura-2. Bimoclomol had a biphasic eect on contractility: both peak left ventricular pressure and the rate of force development signi®cantly increased at a concentration of 10 nM or higher. The maximal eect was observed between 0.1 and 1 mM, and the positive inotropic action disappeared by further increasing the concentration of bimoclomol. The drug increased systolic calcium concentration with a similar concentrationdependence. In contrast, diastolic calcium concentration increased monotonically in the presence of bimoclomol. Thus low concentrations of the drug (10 ± 100 nM) increased, whereas high concentrations (10 mM) decreased the amplitude of intracellular calcium transients. 2 Eects of bimoclomol on action potential con®guration was studied in isolated canine ventricular myocytes. Action potential duration was increased at low (10 nM), unaected at intermediate (0.1 ± 1 mM) and decreased at high (10 ± 100 mM) concentrations of the drug. 3 In single canine sarcoplasmic calcium release channels (ryanodine receptor), incorporated into arti®cial lipid bilayer, bimoclomol signi®cantly increased the open probability of the channel in the concentration range of 1 ± 10 mM. The increased open probability was associated with increased mean open time. The eect of bimoclomol was again biphasic: the open probability decreased below the control level in the presence of 1 mM bimoclomol. 4 Bimoclomol (10 mM ± 1 mM) had no signi®cant eect on the rate of calcium uptake into sarcoplasmic reticulum vesicles of the dog, indicating that in vivo calcium reuptake might not substantially be aected by the drug. 5 In conclusion, the positive inotropic action of bimoclomol is likely due to the activation of the sarcoplasmic reticulum calcium release channel in mammalian ventricular myocardium.
Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transp... more Ionomycin, a recently discovered calcium ionophore, inhibits the ATP-dependent active Ca2+ transport of rabbit sarcoplasmic reticulum vesicles at concentrations as low as 10(-8) to 10(-6) M. The effect is due to an increase in the Ca2+ permeability of the membrane which is also observed on liposomes. The inhibition of Ca2+ uptake is accompanied by an increase in the Ca2+-sensitive ATPase activity of sarcoplasmic reticulum vesicles.
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Papers by István Jóna