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Eduardo Ríos - One of the best experts on this subject based on the ideXlab platform.
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cicr and calcium dependent inactivation quantified through the response to artificial ca sparks in single Muscle cells
Biophysical Journal, 2010Co-Authors: Lourdes Figueroa, Eduardo Ríos, Vyacheslav M Shkryl, Jingsong Zhou, Atsuya Momotake, Graham C R Ellisdavies, Lothar A Blatter, Gustavo BrumAbstract:Local calcium stimuli (artificial sparks) generated by 2-photon breakdown of the cage NDBF-EGTA were applied to evoke Ca release from the SR in single Skeletal or cardiac Muscle cells undergoing fast Ca imaging with the low affinity dye fluo 4FF. The figure shows selected sequential images of the Ca transient generated by a Frog Skeletal Muscle fiber with permeabilized plasmalemma, in response to a spark (elicited outside the fiber to avoid photodamage). Two types of responses were observed: (i) an all-or-none wave -shown- that propagates over the entire cell and (ii) graded responses, which fail to propagate. Release analysis (Rios, JGP 1999; Figueroa, this meeting) separates SR release from simple diffusion of photo-released Ca into cells. The technique yields a sensitive measure of threshold [Ca2+] for release activation, which in the example (0.3 mM [Mg2+]cyto) was 1 μM, and can monitor inactivation by combining multiple stimuli. Modeling of these responses aims at describing quantitatively the properties of activation, as well as the roles of inactivation and depletion in the control of Ca release. Other details and acknowledgments are presented elsewhere (Figueroa, this meeting.)View Large Image | View Hi-Res Image | Download PowerPoint Slide
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a preferred amplitude of calcium sparks in Skeletal Muscle
Biophysical Journal, 2001Co-Authors: Eduardo Ríos, Natalia Shirokova, Michael D. Stern, Wolfgang G Kirsch, Gonzalo Pizarro, Adom GonzálezAbstract:In Skeletal and cardiac Muscle, calcium release from the sarcoplasmic reticulum, leading to contraction, often results in calcium sparks. Because sparks are recorded by confocal microscopy in line-scanning mode, their measured amplitude depends on their true amplitude and the position of the spark relative to the scanned line. We present a method to derive from measured amplitude histograms the actual distribution of spark amplitudes. The method worked well when tested on simulated distributions of experimental sparks. Applied to massive numbers of sparks imaged in Frog Skeletal Muscle under voltage clamp in reference conditions, the method yielded either a decaying amplitude distribution (6 cells) or one with a central mode (5 cells). Caffeine at 0.5 or 1 mM reversibly enhanced this mode (5 cells) or induced its appearance (4 cells). The occurrence of a mode in the amplitude distribution was highly correlated with the presence of a mode in the distribution of spark rise times or in the joint distribution of rise times and spatial widths. If sparks were produced by individual Markovian release channels evolving reversibly, they should not have a preferred rise time or amplitude. Channel groups, instead, could cooperate allosterically or through their calcium sensitivity, and give rise to a stereotyped amplitude in their collective spark.
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involvement of multiple intracellular release channels in calcium sparks of Skeletal Muscle
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Adom González, Natalia Shirokova, Michael D. Stern, Wolfgang G Kirsch, Gonzalo Pizarro, Gustavo Brum, Isaac N Pessah, Eduardo RíosAbstract:In many types of Muscle, intracellular Ca2+ release for contraction consists of brief Ca2+ sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from Frog Skeletal Muscle and evaluating their Ca2+ release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca2+ release current decays 10- to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark.
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calcium release flux underlying ca2 sparks of Frog Skeletal Muscle
The Journal of General Physiology, 1999Co-Authors: Eduardo Ríos, Adom González, Michael D. Stern, Gonzalo Pizarro, Natalia ShirokovaAbstract:An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Huser, and E. Rios. 1997. Proc. Natl. Acad. Sci. USA. 94:4176–4181) was modified and applied to sparks obtained by confocal microscopy in single Frog Skeletal Muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3–containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0.7 μm. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 μm, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.
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a damped oscillation in the intramembranous charge movement and calcium release flux of Frog Skeletal Muscle fibers
The Journal of General Physiology, 1994Co-Authors: Natalia Shirokova, Gonzalo Pizarro, Eduardo RíosAbstract:Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of Frog Skeletal Muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)
Natalia Shirokova - One of the best experts on this subject based on the ideXlab platform.
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a preferred amplitude of calcium sparks in Skeletal Muscle
Biophysical Journal, 2001Co-Authors: Eduardo Ríos, Natalia Shirokova, Michael D. Stern, Wolfgang G Kirsch, Gonzalo Pizarro, Adom GonzálezAbstract:In Skeletal and cardiac Muscle, calcium release from the sarcoplasmic reticulum, leading to contraction, often results in calcium sparks. Because sparks are recorded by confocal microscopy in line-scanning mode, their measured amplitude depends on their true amplitude and the position of the spark relative to the scanned line. We present a method to derive from measured amplitude histograms the actual distribution of spark amplitudes. The method worked well when tested on simulated distributions of experimental sparks. Applied to massive numbers of sparks imaged in Frog Skeletal Muscle under voltage clamp in reference conditions, the method yielded either a decaying amplitude distribution (6 cells) or one with a central mode (5 cells). Caffeine at 0.5 or 1 mM reversibly enhanced this mode (5 cells) or induced its appearance (4 cells). The occurrence of a mode in the amplitude distribution was highly correlated with the presence of a mode in the distribution of spark rise times or in the joint distribution of rise times and spatial widths. If sparks were produced by individual Markovian release channels evolving reversibly, they should not have a preferred rise time or amplitude. Channel groups, instead, could cooperate allosterically or through their calcium sensitivity, and give rise to a stereotyped amplitude in their collective spark.
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involvement of multiple intracellular release channels in calcium sparks of Skeletal Muscle
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Adom González, Natalia Shirokova, Michael D. Stern, Wolfgang G Kirsch, Gonzalo Pizarro, Gustavo Brum, Isaac N Pessah, Eduardo RíosAbstract:In many types of Muscle, intracellular Ca2+ release for contraction consists of brief Ca2+ sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from Frog Skeletal Muscle and evaluating their Ca2+ release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca2+ release current decays 10- to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark.
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calcium release flux underlying ca2 sparks of Frog Skeletal Muscle
The Journal of General Physiology, 1999Co-Authors: Eduardo Ríos, Adom González, Michael D. Stern, Gonzalo Pizarro, Natalia ShirokovaAbstract:An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Huser, and E. Rios. 1997. Proc. Natl. Acad. Sci. USA. 94:4176–4181) was modified and applied to sparks obtained by confocal microscopy in single Frog Skeletal Muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3–containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0.7 μm. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 μm, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.
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a damped oscillation in the intramembranous charge movement and calcium release flux of Frog Skeletal Muscle fibers
The Journal of General Physiology, 1994Co-Authors: Natalia Shirokova, Gonzalo Pizarro, Eduardo RíosAbstract:Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of Frog Skeletal Muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)
Stephen M. Baylor - One of the best experts on this subject based on the ideXlab platform.
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calcium sparks in Skeletal Muscle fibers
Cell Calcium, 2005Co-Authors: Stephen M. BaylorAbstract:Abstract Ca 2+ sparks monitor transient local releases of Ca 2+ from the sarcoplasmic reticulum (SR) into the myoplasm. The release takes place through ryanodine receptors (RYRs), the Ca 2+ -release channels of the SR. In intact fibers from Frog Skeletal Muscle, the temporal and spatial properties of voltage-activated Ca 2+ sparks are well simulated by a model that assumes that the Ca 2+ flux underlying a spark is 2.5 pA (units of Ca 2+ current) for 4.6 ms (18 °C). This flux amplitude suggests that 1–5 active RYRs participate in the generation of a typical voltage-activated spark under physiological conditions. A major goal of future experiments is to estimate this number more precisely and, if it is two or more, to investigate the communication mechanism that allows multiple RYRs to be co-activated in a rapid but self-limited fashion.
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model of sarcomeric ca2 movements including atp ca2 binding and diffusion during activation of Frog Skeletal Muscle
The Journal of General Physiology, 1998Co-Authors: Stephen M. Baylor, Stephen HollingworthAbstract:Cannell and Allen (1984. Biophys. J. 45:913–925) introduced the use of a multi-compartment model to estimate the time course of spread of calcium ions (Ca2+) within a half sarcomere of a Frog Skeletal Muscle fiber activated by an action potential. Under the assumption that the sites of sarcoplasmic reticulum (SR) Ca2+ release are located radially around each myofibril at the Z line, their model calculated the spread of released Ca2+ both along and into the half sarcomere. During diffusion, Ca2+ was assumed to react with metal-binding sites on parvalbumin (a diffusible Ca2+- and Mg2+-binding protein) as well as with fixed sites on troponin. We have developed a similar model, but with several modifications that reflect current knowledge of the myoplasmic environment and SR Ca2+ release. We use a myoplasmic diffusion constant for free Ca2+ that is twofold smaller and an SR Ca2+ release function in response to an action potential that is threefold briefer than used previously. Additionally, our model includes the effects of Ca2+ and Mg2+ binding by adenosine 5′-triphosphate (ATP) and the diffusion of Ca2+-bound ATP (CaATP). Under the assumption that the total myoplasmic concentration of ATP is 8 mM and that the amplitude of SR Ca2+ release is sufficient to drive the peak change in free [Ca2+] (Δ[Ca2+]) to 18 μM (the approximate spatially averaged value that is observed experimentally), our model calculates that (a) the spatially averaged peak increase in [CaATP] is 64 μM; (b) the peak saturation of troponin with Ca2+ is high along the entire thin filament; and (c) the half-width of Δ[Ca2+] is consistent with that observed experimentally. Without ATP, the calculated half-width of spatially averaged Δ[Ca2+] is abnormally brief, and troponin saturation away from the release sites is markedly reduced. We conclude that Ca2+ binding by ATP and diffusion of CaATP make important contributions to the determination of the amplitude and the time course of Δ[Ca2+].
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resting myoplasmic free calcium in Frog Skeletal Muscle fibers estimated with fluo 3
Biophysical Journal, 1993Co-Authors: A B Harkins, N Kurebayashi, Stephen M. BaylorAbstract:Fluo-3 is an unusual tetracarboxylate Ca2+ indicator. For recent lots supplied by Molecular Probes Inc. (Eugene, OR), FMAX, the fluorescence intensity of the indicator in its Ca(2+)-bound form, is approximately 200 times that of FMIN, the fluorescence intensity of the indicator in its Ca(2+)-free form. (For earlier lots, impurities may account for the smaller reported values of FMAX/FMIN, 36–40). We have injected fluo-3 from a high-purity lot into intact single fibers from Frog Muscle and measured the indicator's absorbance and fluorescence signals at rest (A and F, respectively) and changes in absorbance and fluorescence following action potential stimulation (delta A and delta F signals substantially lagged behind that of the myoplasmic free Ca2+ transient. Our analysis of fluo-3's signals from myoplasm therefore focused on information about the level of resting myoplasmic free [Ca2+] ([Ca2+]r). From A, delta A, and in vitro estimates of fluo-3's molar extinction coefficients, the change in the fraction of fluo-3 in the Ca(2+)-bound form during activity (delta f) was estimated. From delta f, delta F, and F, the fraction of the indicator in the Ca(2+)-bound form in the resting fiber (fr) was estimated by fr = (delta f x F/delta F) + (1-FMAX/FMIN)-1. Since FMAX/FMIN is large, the contribution of the second term to the estimate of fr is small. At 16 degrees C, the mean value (mean +/- S.E.) of fr was 0.086 +/- 0.004 (N = 15). From two estimates of the apparent dissociation constant of fluo-3 for Ca2+ in the myoplasm, 1.09 and 2.57 microM, the average value of [Ca2+]r is calculated to be 0.10 and 0.24 microM, respectively. The smaller of these estimates lies near the upper end of the range of values for [Ca2+]r in Frog fibers (0.02–0.12 microM) estimated by others with aequorin and Ca(2+)-selective electrodes. The larger of the estimates lies within the range of values (0.2–0.3 microM) previously estimated in this laboratory with fura red. We conclude that [Ca2+]r in Frog fibers is at least 0.1 microM and possibly as large as 0.3 microM.
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myoplasmic calcium transients in intact Frog Skeletal Muscle fibers monitored with the fluorescent indicator furaptra
The Journal of General Physiology, 1991Co-Authors: M Konishi, Stephen Hollingworth, A B Harkins, Stephen M. BaylorAbstract:Furaptra (Raju, B., E. Murphy, L. A. Levy, R. D. Hall, and R. E. London. 1989. Am. J. Physiol. 256:C540-C548) is a "tri-carboxylate" fluorescent indicator with a chromophore group similar to that of fura- 2 (Grynkiewicz, G., M. Poenie, and R. Y. Tsien. 1985. J. Biol. Chem. 260:3440-3450). In vitro calibrations indicate that furaptra reacts with Ca2+ and Mg2+ with 1:1 stoichiometry, with dissociation constants of 44 microM and 5.3 mM, respectively (16-17 degrees C; ionic strength, 0.15 M; pH, 7.0). Thus, in a Frog Skeletal Muscle fiber stimulated electrically, the indicator is expected to respond to the change in myoplasmic free [Ca2+] (delta[Ca2+]) with little interference from changes in myoplasmic free [Mg2+]. The apparent longitudinal diffusion constant of furaptra in myoplasm was found to be 0.68 (+/- 0.02, SEM) x 10(-6) cm2 s-1 (16-16.5 degrees C), a value which suggests that about half of the indicator was bound to myoplasmic constituents of large molecular weight. Muscle membranes (surface and/or transverse-tubular) appear to have some permeability to furaptra, as the total quantity of indicator contained within a fiber decreased after injection; the average time constant of the loss was 302 (+/- 145, SEM) min. In fibers containing less than 0.5 mM furaptra and stimulated by a single action potential, the calibrated peak value of delta[Ca2+] averaged 5.1 (+/- 0.3, SEM) microM. This value is about half that reported in the preceding paper (9.4 microM; Konishi, M., and S. M. Baylor. 1991. J. Gen. Physiol. 97:245-270) for fibers injected with purpurate-diacetic acid (PDAA). The latter difference may be explained, at least in part, by the likelihood that the effective dissociation constant of furaptra for Ca2+ is larger in vivo than in vitro, owing to the binding of the indicator to myoplasmic constituents. The time course of furaptra's delta[Ca2+], with average values (+/- SEM) for time to peak and half- width of 6.3 (+/- 0.1) and 9.5 (+/- 0.4) ms, respectively, is very similar to that of delta[Ca2+] recorded with PDAA. Since furaptra's delta[Ca2+] can be recorded at a single excitation wavelength (e.g., 420 nm) with little interference from fiber intrinsic changes, movement artifacts, or delta[Mg2+], furaptra represents a useful myoplasmic Ca2+ indicator, with properties complementary to those of other available indicators.
Adom González - One of the best experts on this subject based on the ideXlab platform.
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Effects of Sulfhydryl Inhibitors on Depolarization-Contraction Coupling in Frog Skeletal Muscle Fibers
2013Co-Authors: Carlo Caputo, Adom GonzálezAbstract:A B S TRACT We have studied the effects of the suffhydryl reagents on contractile responses, using either electrically stimulated single musde fibers or short Muscle fibers that were voltage-clamped with a two-microelectrode voltage-clamp technique that allows the fiber tension in response to membrane depolarization to be recorded. The sulfhydryl inhibitors para-chloromercuribenzoic acid (PCMB) and parahydroximercuriphenyl sulfonic acid (PHMPS), at concentrations from 0.5 to 2 mM, cause loss of the contractile ability; however, before this effect is completed, they change the fiber contractile behavior in a complex way. After relatively short exposure to the compounds, <20 rain, before the fibers lose their contractile capacity, secondary tension responses may appear after electrically elicited twitches or tetani. After losing their ability to contract in response to electrical stimulation, the fibers maintain their capacity to develop caffeine contractures, even after prolonged periods (120 min) of exposure to PHMPS. In fibers under voltage-clamp conditions, contractility is also lost; however, before this happens, long-lasting (i.e., minutes) episodes of spontaneous contractile activity may occur with the membrane polarized at-100 mV. After more prolonged exposure (> 30 min), the responses to membrane depolarization are reduced and eventually disappear. The agent DTI " at a concentration of 2 mM appears to protect the fibers from the effects of PCMB and PHMPS. Furthermore, after loss of the contractile responses by the action of PCMB or PHMPS, addition of 2 mM DTF causes recovery of tension development capacity
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a preferred amplitude of calcium sparks in Skeletal Muscle
Biophysical Journal, 2001Co-Authors: Eduardo Ríos, Natalia Shirokova, Michael D. Stern, Wolfgang G Kirsch, Gonzalo Pizarro, Adom GonzálezAbstract:In Skeletal and cardiac Muscle, calcium release from the sarcoplasmic reticulum, leading to contraction, often results in calcium sparks. Because sparks are recorded by confocal microscopy in line-scanning mode, their measured amplitude depends on their true amplitude and the position of the spark relative to the scanned line. We present a method to derive from measured amplitude histograms the actual distribution of spark amplitudes. The method worked well when tested on simulated distributions of experimental sparks. Applied to massive numbers of sparks imaged in Frog Skeletal Muscle under voltage clamp in reference conditions, the method yielded either a decaying amplitude distribution (6 cells) or one with a central mode (5 cells). Caffeine at 0.5 or 1 mM reversibly enhanced this mode (5 cells) or induced its appearance (4 cells). The occurrence of a mode in the amplitude distribution was highly correlated with the presence of a mode in the distribution of spark rise times or in the joint distribution of rise times and spatial widths. If sparks were produced by individual Markovian release channels evolving reversibly, they should not have a preferred rise time or amplitude. Channel groups, instead, could cooperate allosterically or through their calcium sensitivity, and give rise to a stereotyped amplitude in their collective spark.
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involvement of multiple intracellular release channels in calcium sparks of Skeletal Muscle
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Adom González, Natalia Shirokova, Michael D. Stern, Wolfgang G Kirsch, Gonzalo Pizarro, Gustavo Brum, Isaac N Pessah, Eduardo RíosAbstract:In many types of Muscle, intracellular Ca2+ release for contraction consists of brief Ca2+ sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from Frog Skeletal Muscle and evaluating their Ca2+ release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca2+ release current decays 10- to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark.
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calcium release flux underlying ca2 sparks of Frog Skeletal Muscle
The Journal of General Physiology, 1999Co-Authors: Eduardo Ríos, Adom González, Michael D. Stern, Gonzalo Pizarro, Natalia ShirokovaAbstract:An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Huser, and E. Rios. 1997. Proc. Natl. Acad. Sci. USA. 94:4176–4181) was modified and applied to sparks obtained by confocal microscopy in single Frog Skeletal Muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3–containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0.7 μm. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 μm, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.
Gonzalo Pizarro - One of the best experts on this subject based on the ideXlab platform.
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a preferred amplitude of calcium sparks in Skeletal Muscle
Biophysical Journal, 2001Co-Authors: Eduardo Ríos, Natalia Shirokova, Michael D. Stern, Wolfgang G Kirsch, Gonzalo Pizarro, Adom GonzálezAbstract:In Skeletal and cardiac Muscle, calcium release from the sarcoplasmic reticulum, leading to contraction, often results in calcium sparks. Because sparks are recorded by confocal microscopy in line-scanning mode, their measured amplitude depends on their true amplitude and the position of the spark relative to the scanned line. We present a method to derive from measured amplitude histograms the actual distribution of spark amplitudes. The method worked well when tested on simulated distributions of experimental sparks. Applied to massive numbers of sparks imaged in Frog Skeletal Muscle under voltage clamp in reference conditions, the method yielded either a decaying amplitude distribution (6 cells) or one with a central mode (5 cells). Caffeine at 0.5 or 1 mM reversibly enhanced this mode (5 cells) or induced its appearance (4 cells). The occurrence of a mode in the amplitude distribution was highly correlated with the presence of a mode in the distribution of spark rise times or in the joint distribution of rise times and spatial widths. If sparks were produced by individual Markovian release channels evolving reversibly, they should not have a preferred rise time or amplitude. Channel groups, instead, could cooperate allosterically or through their calcium sensitivity, and give rise to a stereotyped amplitude in their collective spark.
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involvement of multiple intracellular release channels in calcium sparks of Skeletal Muscle
Proceedings of the National Academy of Sciences of the United States of America, 2000Co-Authors: Adom González, Natalia Shirokova, Michael D. Stern, Wolfgang G Kirsch, Gonzalo Pizarro, Gustavo Brum, Isaac N Pessah, Eduardo RíosAbstract:In many types of Muscle, intracellular Ca2+ release for contraction consists of brief Ca2+ sparks. Whether these result from the opening of one or many channels in the sarcoplasmic reticulum is not known. Examining massive numbers of sparks from Frog Skeletal Muscle and evaluating their Ca2+ release current, we provide evidence that they are generated by multiple channels. A mode is demonstrated in the distribution of spark rise times in the presence of the channel activator caffeine. This finding contradicts expectations for single channels evolving reversibly, but not for channels in a group, which collectively could give rise to a stereotyped spark. The release channel agonists imperatoxin A, ryanodine, and bastadin 10 elicit fluorescence events that start with a spark, then decay to steady levels roughly proportional to the unitary conductances of 35%, 50%, and 100% that the agonists, respectively, promote in bilayer experiments. This correspondence indicates that the steady phase is produced by one open channel. Calculated Ca2+ release current decays 10- to 20-fold from spark to steady phase, which requires that six or more channels be open during the spark.
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calcium release flux underlying ca2 sparks of Frog Skeletal Muscle
The Journal of General Physiology, 1999Co-Authors: Eduardo Ríos, Adom González, Michael D. Stern, Gonzalo Pizarro, Natalia ShirokovaAbstract:An algorithm for the calculation of Ca2+ release flux underlying Ca2+ sparks (Blatter, L.A., J. Huser, and E. Rios. 1997. Proc. Natl. Acad. Sci. USA. 94:4176–4181) was modified and applied to sparks obtained by confocal microscopy in single Frog Skeletal Muscle fibers, which were voltage clamped in a two-Vaseline gap chamber or permeabilized and immersed in fluo-3–containing internal solution. The performance of the algorithm was characterized on sparks obtained by simulation of fluorescence due to release of Ca2+ from a spherical source, in a homogeneous three-dimensional space that contained components representing cytoplasmic molecules and Ca2+ removal processes. Total release current, as well as source diameter and noise level, was varied in the simulations. Derived release flux or current, calculated by volume integration of the derived flux density, estimated quite closely the current used in the simulation, while full width at half magnitude of the derived release flux was a good monitor of source size only at diameters >0.7 μm. On an average of 157 sparks of amplitude >2 U resting fluorescence, located automatically in a representative voltage clamp experiment, the algorithm reported a release current of 16.9 pA, coming from a source of 0.5 μm, with an open time of 6.3 ms. Fewer sparks were obtained in permeabilized fibers, so that the algorithm had to be applied to individual sparks or averages of few events, which degraded its performance in comparable tests. The average current reported for 19 large sparks obtained in permeabilized fibers was 14.4 pA. A minimum estimate, derived from the rate of change of dye-bound Ca2+ concentration, was 8 pA. Such a current would require simultaneous opening of between 8 and 60 release channels with unitary Ca2+ currents of the level recorded in bilayer experiments. Real sparks differ from simulated ones mainly in having greater width. Correspondingly, the algorithm reported greater spatial extent of the source for real sparks. This may again indicate a multichannel origin of sparks, or could reflect limitations in spatial resolution.
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a damped oscillation in the intramembranous charge movement and calcium release flux of Frog Skeletal Muscle fibers
The Journal of General Physiology, 1994Co-Authors: Natalia Shirokova, Gonzalo Pizarro, Eduardo RíosAbstract:Asymmetric membrane currents and calcium transients were recorded simultaneously from cut segments of Frog Skeletal Muscle fibers voltage clamped in a double Vaseline-gap chamber in the presence of high concentration of EGTA intracellularly. An inward phase of asymmetric currents following the hump component was observed in all fibers during the depolarization pulse to selected voltages (congruent to -45 mV). The average value of the peak inward current was 0.1 A/F (SEM = 0.01, n = 18), and the time at which it occurred was 34 ms (SEM = 1.8, n = 18). A second delayed outward phase of asymmetric current was observed after the inward phase, in those experiments in which hump component and inward phase were large. It peaked at more variable time (between 60 and 130 ms) with amplitude 0.02 A/F (SEM = 0.003, n = 11). The transmembrane voltage during a pulse, measured with a glass microelectrode, reached its steady value in less than 10 ms and showed no oscillations. The potential was steady at the time when the delayed component of asymmetric current occurred. ON and OFF charge transfers were equal for all pulse durations. The inward phase moved 1.4 nC/microF charge (SEM = 0.8, n = 6), or about one third of the final value of charge mobilized by these small pulses, and the second outward phase moved 0.7 nC/microF (SEM = 0.8, n = 6), bringing back about half of the charge moved during the inward phase. When repolarization intersected the peak of the inward phase, the OFF charge transfer was independent of the repolarization voltage in the range -60 to -90 mV. When both pre- and post-pulse voltages were changed between -120 mV and -60 mV, the equality of ON and OFF transfers of charge persisted, although they changed from 113 to 81% of their value at -90 mV. The three delayed phases in asymmetric current were also observed in experiments in which the extracellular solution contained Cd2+, La3+ and no Ca2+. Large increases in intracellular [Cl-] were imposed, and had no major effect on the delayed components of the asymmetric current. The Ca2+ transients measured optically and the calculated Ca2+ release fluxes had three phases whenever a visible outward phase followed the inward phase in the asymmetric current. Several interventions intended to interfere with Ca release, reduced or eliminated the three delayed phases of the asymmetric current.(ABSTRACT TRUNCATED AT 400 WORDS)
