Effects of tetracaine on sarcoplasmic calcium release in mammalian skeletal muscle fibres.
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1. Single muscle fibres were dissociated enzymatically from the extensor digitorum communis muscle of rats. The fibres were mounted into a double Vaseline gap experimental chamber and the events in excitation-contraction coupling were studied under voltage clamp conditions in the presence and absence of the local anaesthetic tetracaine. 2. Changes in intracellular calcium concentration ([Ca2+]i) were monitored using the calcium sensitive dyes antipyrylazo III and fura-2 and the rate of calcium release (Rrel) from the sarcoplasmic reticulum (SR) was calculated. Tetracaine decreased the maximal attained [Ca2+]i and suppressed, in a dose-dependent manner, both the early peak and the steady level of Rrel in the voltage range examined. 3. The concentration dependence of the effects on the two kinetic components of Rrel were almost identical with a half-effective concentration (K50) of 70 and 71 microM and a Hill coefficient (nH) of 2.7 and 2.3 for the peak and the steady level, respectively. Furthermore, the drug did not alter the peak to steady level ratio up to a concentration (50 microM) that caused a 35 +/- 5 % reduction in calcium release. Higher concentrations did suppress the ratio but the degree of suppression was voltage independent. 4. Tetracaine (50 microM) neither influenced the total available intramembrane charge nor altered its membrane potential dependence. It shifted the transfer function, the normalized SR permeability versus normalized charge to the right, indicating that similar charge transfer caused a smaller increase in SR permeability. 5. To explore the site of action of tetracaine further the ryanodine receptor (RyR) calcium release channel of the SR was purified and reconstituted into planar lipid bilayers. The reconstituted channel had a conductance of 511 +/- 14 pS (n = 8) in symmetric 250 mM KCl that was not affected by tetracaine. Tetracaine decreased the open probability of the channel in a concentration-dependent manner with K50 = 68 microM and nH = 1.5. 6. These experiments show that tetracaine suppresses SR calcium release in enzymatic isolated mammalian skeletal muscle fibres. This effect is due, presumably, to the decreased open probability of the RyR in the presence of the drug. Since both the inactivating peak and the steady level of Rrel were equally affected by tetracaine, our observations suggest that there is a tight coupling between these kinetic components of SR calcium release in mammalian skeletal muscle. (+info)
Increased calcium entry into dystrophin-deficient muscle fibres of MDX and ADR-MDX mice is reduced by ion channel blockers.
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1. Single fibres were enzymatically isolated from interosseus muscles of dystrophic MDX mice, myotonic-dystrophic double mutant ADR-MDX mice and C57BL/10 controls. The fibres were kept in cell culture for up to 2 weeks for the study of Ca2+ homeostasis and sarcolemmal Ca2+ permeability. 2. Resting levels of intracellular free Ca2+, determined with the fluorescent Ca2+ indicator fura-2, were slightly higher in MDX (63 +/- 20 nM; means +/- s.d.; n = 454 analysed fibres) and ADR-MDX (65 +/- 12 nM; n = 87) fibres than in controls (51 +/- 20 nM; n = 265). 3. The amplitudes of electrically induced Ca2+ transients did not differ between MDX fibres and controls. Decay time constants of Ca2+ transients ranged between 10 and 55 ms in both genotypes. In 50 % of MDX fibres (n = 68), but in only 20 % of controls (n = 54), the decay time constants were > 35 ms. 4. Bath application of Mn2+ resulted in a progressive quench of fura-2 fluorescence emitted from the fibres. The quench rate was about 2 times higher in MDX fibres (3.98 +/- 1.9 % min-1; n = 275) than in controls (2.03 +/- 1.4 % min-1; n = 204). The quench rate in ADR-MDX fibres (2.49 +/- 1.4 % min-1; n = 87) was closer to that of controls. 5. The Mn2+ influx into MDX fibres was reduced to 10 % by Gd3+, to 19 % by La3+ and to 47 % by Ni2+ (all at 50 microM). Bath application of 50 microM amiloride inhibited the Mn2+ influx to 37 %. 6. We conclude that in isolated, resting MDX muscle fibres the membrane permeability for divalent cations is increased. The presumed additional influx of Ca2+ occurs through ion channels, but is well compensated for by effective cellular Ca2+ transport systems. The milder dystrophic phenotype of ADR-MDX mice is correlated with a smaller increase of their sarcolemmal Ca2+ permeability. (+info)
Relationship between L-type Ca2+ current and unitary sarcoplasmic reticulum Ca2+ release events in rat ventricular myocytes.
(11/4498)
1. The time courses of Ca2+ current and Ca2+ spark occurrence were determined in single rat ventricular myocytes voltage clamped with patch pipettes containing 0.1 microM fluo-3. Acquisition of line-scan images on a laser scanning confocal microscope was synchronized with measurement of Cd2+-sensitive Ca2+ currents. In most cells, individual Ca2+ sparks were observed by reducing Ca2+ current density with nifedipine (0.1-8 microM). 2. Ca2+ sparks elicited by depolarizing voltage-clamp pulses had a peak [Ca2+] amplitude of 289 +/- 3 nM with a decay half-time of 20.8 +/- 0.2 ms and a full width at half-maximum of 1.40 +/- 0.03 microm (mean +/- s. e.m., n = 345), independent of the membrane potential. 3. The time between the beginning of a depolarization and the initiation of each Ca2+ spark was calculated and data were pooled to construct waiting time histograms. Exponential functions were fitted to these histograms and to the decaying phase of the Ca2+ current. This analysis showed that the time constants describing Ca2+ current and Ca2+ spark occurrence at membrane potentials between -30 mV and +30 mV were not significantly different. At +50 mV, in the absence of nifedipine, the time constant describing Ca2+ spark occurrence was significantly larger than the time constant of the Ca2+ current. 4. A simple model is developed using Poisson statistics to relate macroscopic Ca2+ current to the opening of single L-type Ca2+ channels at the dyad junction and to the time course of Ca2+ spark occurrence. The model suggests that the time courses of macroscopic Ca2+ current and Ca2+ spark occurrence should be closely related when opening of a single L-type Ca2+ channel initiates a Ca2+ spark. By comparison with the data, the model suggests that Ca2+ sparks are initiated by the opening of a single L-type Ca2+ channel at all membrane potentials encountered during an action potential. (+info)
Release of Ca2+ from the sarcoplasmic reticulum increases mitochondrial [Ca2+] in rat pulmonary artery smooth muscle cells.
(12/4498)
1. The Ca2+-sensitive fluorescent indicator rhod-2 was used to measure mitochondrial [Ca2+] ([Ca2+]m) in single smooth muscle cells from the rat pulmonary artery, while simultaneously monitoring cytosolic [Ca2+] ([Ca2+]i) with fura-2. 2. Application of caffeine produced an increase in [Ca2+]i and also increased [Ca2+]m. The increase in [Ca2+]m occurred after the increase in [Ca2+]i, and remained elevated for a considerable time after [Ca2+]i had returned to resting values. 3. The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP), which causes the mitochondrial membrane potential to collapse, markedly attenuated the increase in [Ca2+]m following caffeine application and also increased the half-time for recovery of [Ca2+]i to resting values. 4. Activation of purinoceptors with ATP also produced increases in both [Ca2+]i and [Ca2+]m in these smooth muscle cells. In some cells, oscillations in [Ca2+]i were observed during ATP application, which produced corresponding oscillations in [Ca2+]m and membrane currents. 5. This study provides direct evidence that Ca2+ release from the sarcoplasmic reticulum, either through ryanodine or inositol 1,4, 5-trisphosphate (InsP3) receptors, increases both cytosolic and mitochondrial [Ca2+] in smooth muscle cells. These results have potential implications both for the role of mitochondria in Ca2+ regulation in smooth muscle, and for understanding how cellular metabolism is regulated. (+info)
Phospholamban-to-SERCA2 ratio controls the force-frequency relationship.
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The force-frequency relationship (FFR) describes the frequency-dependent potentiation of cardiac contractility. The interaction of the sarcoplasmic reticulum Ca2+-adenosinetriphosphatase (SERCA2) with its inhibitory protein phospholamban (PLB) might be involved in the control of the FFR. The FFR was analyzed in two systems in which the PLB-to-SERCA2 ratio was modulated. Adult rabbit cardiac myocytes were transduced with adenovirus encoding for SERCA2, PLB, and beta-galactosidase (control). After 3 days, the relative PLB/SERCA2 values were significantly different between groups (SERCA2, 0.5; control, 1.0; PLB, 4.5). SERCA2 overexpression shortened relaxation by 23% relative to control, whereas PLB prolonged relaxation by 39% and reduced contractility by 47% (0.1 Hz). When the stimulation frequency was increased to 1.5 Hz, myocyte contractility was increased by 30% in control myocytes. PLB-overexpressing myocytes showed an augmented positive FFR (+78%), whereas SERCA2-transduced myocytes displayed a negative FFR (-15%). A more negative FFR was also found in papillary muscles from SERCA2 transgenic mice. These findings demonstrate that the ratio of phospholamban to SERCA2 is an important component in the control of the FFR. (+info)
Cyclosporin A treatment alters characteristics of Ca2+-release channel in cardiac sarcoplasmic reticulum.
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Chronic treatment with cyclosporin A (CsA) has been reported (H. S. Banijamali, M. H. ter Keurs, L. C. Paul, and H. E. ter Keurs. Cardiovasc. Res. 27: 1845-1854, 1993; I. Kingma, E. Harmsen, H. E. ter Keurs, H. Benediktsson, and L. C. Paul. Int. J. Cardiol. 31: 15-22, 1991) to induce reversible alterations of contractile properties in rat hearts. To define the molecular mechanisms underlying the physiological alterations, the Ca2+-release channel (CRC) and Ca2+-ATPase from sarcoplasmic reticulum in rats were examined. Ryanodine binding to whole homogenates of rat hearts shows time- and dose-dependent alterations in CRC properties by CsA. On 3 wk of treatment with 15 mg CsA. kg body wt-1. day-1, 1) maximal ryanodine binding (Bmax) decreased, 2) the dissociation constant of ryanodine (Kd) increased, 3) caffeine sensitivity of CRC increased, and 4) ruthenium red sensitivity of CRC decreased. On the other hand, Bmax and Kd of ryanodine binding in rat skeletal muscles were not changed. Ryanodine-sensitive oxalate-supported Ca2+ uptake in whole homogenates was lower in CsA-treated rat hearts than in control hearts, whereas total Ca2+ uptake in the presence of 500 M ryanodine was not changed. Functional experiments with rapamycin and Western blot analysis suggest that the CsA-induced alteration of ryanodine binding is due at least in part to an upregulation of calcineurin. The heart muscle-specific alterations of CRC could be responsible for the previously reported contractile changes of CsA-treated rat hearts. (+info)
Acute estrogen administration relaxes vascular smooth muscle by decreasing intracellular Ca2+ concentration ([Ca2+]i). In the present study, we examined the hypothesis that this reduction in [Ca2+]i is mediated in part by enhanced Ca2+ efflux. Coronary artery smooth muscle cells were isolated from gonad-intact, sexually mature female pigs. The [Ca2+]i response to endothelin-1 was measured using fluo 3 and confocal microscopy. 17beta-Estradiol (E2beta), but not 17alpha-estradiol or triamcinolone acetonide, caused a concentration-dependent (IC50 = 10 nM) decrease in the [Ca2+]i response to endothelin-1. This decrease was blocked by the specific estrogen receptor antagonist ICI-182780. Under conditions in which Ca2+ influx and sarcoplasmic reticulum Ca2+ reuptake were blocked, E2beta still decreased [Ca2+]i. The response was blocked by extracellular lanthanum. These data indicate that E2beta decreases [Ca2+]i in coronary artery smooth muscle by affecting Ca2+ efflux via a receptor-mediated mechanism. (+info)
Ryanodine and the left ventricular force-interval and relaxation-interval relations in closed-chest dogs: insights on calcium handling.
(16/4498)
OBJECTIVE: Although the myocardial force-interval and relaxation-interval relations are considered to be mechanical expressions of myocardial Ca2+ handling, correlation of these phenomena with altered Ca2+ kinetics in the intact state is limited. Thus, I sought to determine the impact of selective impairment of physiologic sarcoplasmic reticulum Ca2+ release, achieved by the use of the drug ryanodine, on these relations in the intact animal. METHODS: Twelve dogs instrumented with left ventricular manometers and piezoelectric dimension crystals were studied before and after ryanodine (4 micrograms/kg intravenously). End-systolic elastance was measured at paced heart rates of 120-180 bpm to determine the force-frequency response. Mechanical restitution and relaxation restitution were determined by measuring contractile (single beat elastance) and relaxation (peak negative dP/dt) responses for beats delivered at graded extrasystolic intervals, with normalized responses expressed as a function of extrasystolic interval. RESULTS: Ryanodine accelerated mechanical restitution (time constant 60.3 +/- 3.9 versus 81.7 +/- 10.1 ms, p < 0.05) and reduced maximal contractile response (107.5 +/- 2.1 versus 122.1 +/- 5.7%, p < 0.05), slowed early relaxation restitution (time constant 65.5 +/- 13.8 versus 36.8 +/- 3.8 ms, p < 0.05) without changing late relaxation restitution kinetics, and amplified the force-frequency response (end-systolic elastance, 180 bpm, 19.4 +/- 4.3 versus 11.4 +/- 1.2 mm Hg/ml, p < 0.05). CONCLUSIONS: These findings suggest that in the intact animal, Ca2+ handling by the sarcoplasmic reticulum is a primary determinant of mechanical restitution and early relaxation restitution, but not late relaxation restitution. Conversely, ryanodine induced augmentation of the force-frequency response indicates a central role for sarcolemmal Ca2+ influx in producing frequency potentiation. (+info)