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Vincent Jacquemond - One of the best experts on this subject based on the ideXlab platform.
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intracellular calcium signals measured with Indo 1 in isolated skeletal muscle fibres from control and mdx mice
The Journal of Physiology, 1999Co-Authors: Claude Collet, Bruno Allard, Yves Tourneur, Vincent JacquemondAbstract:1Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator Indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2The mean (±s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 ± 5 nm (n= 72) and 48 ± 7 nm (n= 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than ∼35 weeks, resting [Ca2+]i was significantly lower in mdx (16 ± 4 nm, n= 11) than in control fibres (71 ± 10 nm, n= 14). 3Changes in [Ca2+]i elicited by short (5–35 ms) depolarizing pulses from −80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5–2. For longer pulses, no significant difference could be detected. 4In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.
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Intracellular calcium signals measured with Indo‐1 in isolated skeletal muscle fibres from control and mdx mice
The Journal of Physiology, 1999Co-Authors: Claude Collet, Bruno Allard, Yves Tourneur, Vincent JacquemondAbstract:1Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator Indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2The mean (±s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 ± 5 nm (n= 72) and 48 ± 7 nm (n= 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than ∼35 weeks, resting [Ca2+]i was significantly lower in mdx (16 ± 4 nm, n= 11) than in control fibres (71 ± 10 nm, n= 14). 3Changes in [Ca2+]i elicited by short (5–35 ms) depolarizing pulses from −80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5–2. For longer pulses, no significant difference could be detected. 4In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.
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Measurements of intracellular Mg2+ concentration in mouse skeletal muscle fibers with the fluorescent indicator mag-Indo-1.
Biophysical journal, 1998Co-Authors: László Csernoch, Jean Claude Bernengo, Péter Szentesi, Vincent JacquemondAbstract:Measurements of intracellular free magnesium concentration ([Mg2+]i) were performed on enzymatically isolated skeletal muscle fibers from mice, using the fluorescent ratiometric indicator mag-Indo-1. An original procedure was developed to calibrate the dye response within the fibers: fibers were first permeabilized with saponin in the presence of a given extracellular magnesium concentration and were then embedded in silicone grease. The dye was then pressure microinjected into the saponin-permeabilized silicone-embedded fibers, and fluorescence was measured. The results show that for all tested [Mg2+], the value of the measured fluorescence ratio was higher than that found in aqueous solutions. Furthermore, the apparent binding curve that could be fit to the in vivo ratio data was shifted toward higher [Mg2+] by a factor of approximately 2. Using the in vivo calibration parameters, the mean resting [Mg2+]i was found to be 1.53 +/- 0.16 mM (n = 7). In an attempt to gain insight into the myoplasmic magnesium buffering capacity, we measured, together with mag-Indo-1 fluorescence, the current elicited by the application of carbamylcholine (CCh) to the endplate of isolated fibers, in the presence of a high extracellular magnesium concentration. The results show that, under these conditions, a change in [Mg2+]i displaying a time course and amplitude qualitatively consistent with the CCh-induced inward current can be measured.
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Indo-1 fluorescence signals elicited by membrane depolarization in enzymatically isolated mouse skeletal muscle fibers.
Biophysical journal, 1997Co-Authors: Vincent JacquemondAbstract:Indo-1 fluorescence signals were measured from one extremity of enzymatically isolated skeletal muscle fibers of mice. An original and simple method was developed to allow the measurements to be made under voltage-clamp control: the major part of a single fiber was embedded in silicone grease, so that only a short portion of one end of the fiber, from which the fluorescence measurements were taken, was in contact with the external solution. Membrane potential was held and varied by using a patch-clamp amplifier in whole-cell configuration with a single microelectrode, the tip of which was inserted across the silicone grease within the insulated portion of the fiber. In response to 100-ms depolarizing command pulses to voltages more positive than -40 mV (from a holding potential of -80 mV), clear changes in fluorescence were qualitatively observed to feature a time course of rise and decay expected from a change in intracellular calcium concentration ([Ca2+]i) due to voltage-dependent sarcoplasmic reticulum (SR) calcium release. Although the peak [Ca2+]i elicited by a 100-ms depolarization at 0 or +10 mV varied from fiber to fiber, it could clearly reach a value high enough to saturate Indo-1. The overall results show that this method represents an efficient way of measuring depolarization-induced [Ca2+]i changes in enzymatically dissociated skeletal muscle fibers.
V.ya. Ganitkevich - One of the best experts on this subject based on the ideXlab platform.
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Use of Indo-1 FF for measurements of rapid micromolar cytoplasmic free Ca2+ increments in a single smooth muscle cell
Cell calcium, 1998Co-Authors: V.ya. GanitkevichAbstract:Abstract A low-affinity fluorescent Ca2+ indicator Indo-1 FF was used to measure cytoplasmic Ca2+ increments in single smooth muscle cells isolated from the urinary bladder of the guinea-pig. The in vitro Kd of Indo-1 FF for Ca2+ measured at the microscope stage was 21 μM. Calibration parameters measured in the cell differed substantially from respective in vitro values suggesting that the properties of the cytoplasmic dye had been altered. Addition of proteins (aldolase or albumin) increased the in vitro F 405 F 495 ratio close to the range observed intracellularly. Emission spectra of Ca2+-free Indo-1 FF demonstrated a blue-shift of 29 nm with 10 mg/ml aldolase and 60 nm with 10 mg/ml albumin. The Kd value of Indo-1 FF for Ca2+ in vitro was not changed by addition of aldolase (up to 20 mg/ml) and was approximately doubled in the presence of 20 mg/ml albumin. Intracellular calibration either by skinning the cells with β-escine, ‘opening’ the cell or by intracellular perfusion of 100 μM free Ca2+ (40 mM DPTA-Ca2+ buffer) suggest that the affinity of intracellular Indo-1 FF for Ca2+ is not markedly changed. The Indo-1 FF concentration of 20 μM in the patch-pipette was found to be a reasonable compromise between acceptable signal-to-noise ratio and increased cytoplasmic Ca2+ buffering. This is because neither the amplitude nor the time-course of depolarization-induced micromolar Ca2+ increments were significantly changed during cell loading with this concentration of the dye. In contrast to Indo-1 loaded cells where rapid changes of [Ca2+]i were buffered, in Indo-1 FF loaded cells Ica evoked rapid (rate of rise 150 μM/s) and large (4–6 μM in 35–60 ms) increments of free Ca2+. This results suggest that [Ca2+]i increments in smooth muscle cells are fast and large.
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The amount of acetylcholine mobilisable Ca2+ in single smooth muscle cells measured with the exogenous cytoplasmic Ca2+ buffer, Indo-1
Cell calcium, 1996Co-Authors: V.ya. GanitkevichAbstract:Abstract Single smooth muscle cells from guinea pig urinary bladder were voltage clamped with patch electrodes containing 1 mM Indo-1. As Indo-1 entered the cell, Δ[Ca 2+ ] i in response to Ca 2+ , influx with I Ca (1 s steps to -10 mV) was progressively decreased. ΔF 410 was used as a measure of the Ca 2+ amount bound to Indo-1. Within less than 2 min after establishment of the whole-cell configuration, the fraction of Ca 2+ entering the cell with I Ca which binds to Indo-1 became constant, suggesting that Indo-1 completely overrides the endogenous Ca 2+ buffers. Under these conditions, ΔF 410 was satisfactorily fitted with the time integral of I Ca during 1 s long steps. Acetylcholine (ACh, 50 μM) was rapidly applied to Indo-1 loaded cells to induce IP 3 -induced Ca 2+ release (IICR), which peaked within about 1 s. From ΔF 410 in response to I Ca and ACh and from the time integral of I Ca the amount of Ca 2+ released during IICR was estimated to be 680 attomole (680 × 10 −18 mole), corresponding to 230 μM for 3 pl of accessible cytoplasmic volume.
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High cytoplasmic Ca2+ levels reached during Ca2+-induced Ca2+ release in single smooth muscle cell as reported by a low affinity Ca2+ indicator Mag-Indo-1
Cell calcium, 1996Co-Authors: V.ya. Ganitkevich, H. HircheAbstract:The low-affinity Ca2+ indicator Mag-Indo-1 was used to measure increments of ionised Ca2+ concentration in the cytoplasm of single smooth muscle cells isolated from guinea-pig urinary bladder. With 3.6 mM [Ca2+]o, depolarization steps to 0 mV were associated with a transient increase of fluorescence ratio (F410/F470) only when Ica triggered a Ca(2+)-induced Ca2+ release (CICR). [Ca2+]i transiently peaked to 3-5 microM and despite continuous Ca2+ influx the [Ca2+]i signal fell close to the baseline. Rapidly applied caffeine (10 mM) increased [Ca2+]l by 16 microM, the response was completely blocked by intracellular ryanodine (20 microM). With ryanodine intracellularly, Ica produced very small [Ca2+]i signals unless it was augmented by elevation of [Ca2+]0 to 10 mM and addition of 1 microM Bay K8644. Under these conditions, [Ca2+]l responded with a tonic elevation lasting as long as the depolarizing pulse. It is concluded that the low-affinity indicator Mag-Indo-1 reports predominantly Ca2+ release from SR in cytoplasm.
A Kusumi - One of the best experts on this subject based on the ideXlab platform.
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Comparison of two-photon excitation laser scanning microscopy with UV-confocal laser scanning microscopy in three-dimensional calcium imaging using the fluorescence indicator Indo-1.
Journal of microscopy, 1997Co-Authors: Y Sako, A Sekihata, Y Yanagisawa, M Yamamoto, Y Shimada, K Ozaki, A KusumiAbstract:Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6-17, which enhances the contrast by a factor of 6-21: (2) a lower rate of photobleaching by a factor of 2-4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations. probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.6-phi pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-phi pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.
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Comparison of two‐photon excitation laser scanning microscopy with UV‐confocal laser scanning microscopy in three‐dimensional calcium imaging using the fluorescence indicator Indo‐1
Journal of Microscopy, 1997Co-Authors: Y Sako, A Sekihata, Y Yanagisawa, M Yamamoto, Y Shimada, K Ozaki, A KusumiAbstract:Two-photon excitation laser scanning fluorescence microscopy (2p-LSM) was compared with UV-excitation confocal laser scanning fluorescence microscopy (UV-CLSM) in terms of three-dimensional (3-D) calcium imaging of living cells in culture. Indo-1 was used as a calcium indicator. Since the excitation volume is more limited and excitation wavelengths are longer in 2p-LSM than in UV-CLSM, 2p-LSM exhibited several advantages over UV-CLSM: (1) a lower level of background signal by a factor of 6-17, which enhances the contrast by a factor of 6-21: (2) a lower rate of photobleaching by a factor of 2-4; (3) slightly lower phototoxicity. When 3-D images were repeatedly acquired, the calcium concentration determined by UV-CLSM depended strongly on the number of data acquisitions and the nuclear regions falsely exhibited low calcium concentrations. probably due to an interplay of different levels of photobleaching of Indo-1 and autofluorescence, while the calcium concentration evaluated by 2p-LSM was stable and homogeneous throughout the cytoplasm. The spatial resolution of 2p-LSM was worse by 10% in the focal plane and by 30% along the optical axis due to the longer excitation wavelength. This disadvantage can be overcome by the addition of a confocal pinhole (two-photon excitation confocal laser scanning fluorescence microscopy), which made the resolution similar to that in UV-CLSM. These results indicate that 2p-LSM is preferable for repeated 3-D reconstruction of calcium concentration in living cells. In UV-CLSM, 0.18-mW laser power with a 2.6-phi pinhole (in normalized optical coordinate) gives better signal-to-noise ratio, contrast and resolution than 0.09-mW laser power with a 4.9-phi pinhole. However, since the damage to cells and the rate of photobleaching is substantially greater under the former condition, it is not suitable for repeated acquisition of 3-D images.
Pierre Viallet - One of the best experts on this subject based on the ideXlab platform.
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Interaction of a protein, BSA, and a fluorescent probe, Mag-Indo-1, influence of EDTA and calcium on the equilibrium.
Biophysical chemistry, 1999Co-Authors: Anne-cécile Ribou, Pierre Viallet, Jean Vigo, Jean-marie SalmonAbstract:Recent findings indicate that ion-chelator probes with tetracarboxylate structure bind proteins. It was suggested that these fluorescent probes are valuable tools to gain information on protein structure through the energy transfer from tryptophans to the bound probe. Here, the binding of the fluorescent probe Mag-Indo-1 to bovine serum albumin (BSA) was investigated. Mag-Indo-1 was reported previously to serve as a probe for magnesium cations (Kd = 2.8 x 10(-4) M for zero ionic strength) which can also interact with calcium cations (Kd = 7.5 x 10(-7) M). Probe complexation with protein results in a shift of the emission fluorescence spectrum of the probe from 480 to 457 nm. We used emission fluorescence techniques to monitor this interaction. Computational resolution of the complex fluorescence spectra and a new software to test the theoretical model were developed in our laboratory. This enabled us to calculate the number of interacting sites and the dissociation constants. The fluorescent probe Mag-Indo-1 binds at a singular site with high affinity (Kd = 1.8 x 10(-7) M) to bovine serum albumin (BSA). Since proteins are known to bind several compounds unspecifically, we have studied the influence of EDTA as a competitor of the probe. Our findings suggest that the BSA binding site is identical for both Mag-Indo-1 and EDTA. We found that EDTA binds the protein with Kd = 0.4 x 10(-3) M. We studied the influence of calcium and found that Mag-Indo-1 does not bind the calcium free Apo-protein anymore.
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Mag-Indo-1 protein interaction as a tool for probing the 3D conformation of protein subdomains: influence of the chemical microenvironment of the histidin residue(s) on the paramaters of the intera
Biomedical Diagnostic Guidance and Surgical-Assist Systems, 1999Co-Authors: Pierre Viallet, Tuan Vo-dinh, Terry Bunde, Jean Vigo, Anne-cécile Ribou, Jean-marie SalmonAbstract:Magnesium complexation with Mag-Indo-1 results in a shift of the emission fluorescence spectrum from 480nm to 417nm. Mag-Indo-1 is also able to bind calcium and zinc. These cationic interactions induce the same spectral shift but the fluorescence intensity and the dissociation constant are dependent of the nature of the cation. Furthermore Mag-Indo-1 bind also proteins through a specific interaction with some histidin residues. That interaction induces a characteristic spectral shift of the emission fluorescence spectra from 480 to 457nM. Emission and synchronous fluorescence techniques have been used to monitor that interaction with proteins such as turkey and hen white egg lysozymes. Using a method of resolution of complex fluorescence spectra, it has been possible to calculate the number of interaction sites and the correlative dissociation constants. Depending on the nature of the protein a quenching of the natural fluorescence of the protein was observed, associated with an energy transfer from some tryptophan(s) to Mag-Indo-1. Moreover the value of the dissociation constant was found dependent of the molecular microenvironment of the histidin residue. These results suggest that Mag-Indo-1 could be used as an intramolecular fluorescent ruler to explore the changes in 3D conformation resulting from genetically induced amino-acid substitutions and to monitor changes in the chemical microenvironment of specific sub-domains of these proteins.
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Mag-Indo-1 as a potential reporter of the 3D conformation of protein subdomains
Biomedical Sensing and Imaging Technologies, 1998Co-Authors: Pierre Viallet, Tuan Vo-dinh, Terry Bunde, Jean Vigo, Anne-cécile Ribou, Jean-marie SalmonAbstract:Mag-Indo-1 is a well known fluorescent probe. Magnesium complexation results in a shift of the emission fluorescence spectrum from 480 nm to 417 nm with an intensity proportional to the magnesium concentration in the range 0.6 to 30 mM. Although designed as a specific magnesium chelator, Mag-Indo-1 is also able to bind calcium and zinc. All these cationic interactions induced the same spectral shift but the fluorescence intensity and the dissociation constant are dependent of the nature of the cation. Furthermore Mag-Indo-1 can also bind proteins through a specific interaction with some histidin residues. That interaction induces a characteristic spectral shift of the emission fluorescence spectra from 480 to 457 nM. All these properties suggest that Mag-Indo-1 could be used to study the protein-cation binding. Emission and synchronous fluorescence techniques have been used to monitor that interaction with proteins such as bovine serum albumin, human serum albumin, turkey white egg lysozyme. Using a method of resolution of complex fluorescence spectra, it has been possible to calculate the number of interaction sites and the correlative dissociation constants. Depending on the nature of the protein a quenching of the natural fluorescence of the protein was observed, associated with an energy transfer from some tryptophan(s) to Mag-Indo-1. All these data were tentatively correlated with the available information on the 3D conformation of the proteins. These results suggest that Mag-Indo-1 could be used as an intramolecular fluorescent ruler to monitor the changes in 3D conformation of specific sub-domains of proteins.
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Intracellular compartmentalization of Indo-1 and Mag-Indo-1: consequences on intracellular calcium and magnesium sensing and imaging for biomedical applications
Biomedical Sensing Imaging and Tracking Technologies II, 1997Co-Authors: Pierre Viallet, Tuan Vo-dinh, Terry Bunde, Judith Pescot, Jean Vigo, Jean-marie SalmonAbstract:Indo-1 and Mag-Indo-1 are well known fluorescent probes which consist in two different cages of complexation covalently bound to the same fluorophor. The ionic rap of Indo-1 fit perfectly the size of the calcium ion and its sensitivity is well adapted to the basal concentration of calcium ion in the cytoplasm. Although Mag-Indo-1 was designed to complex magnesium, it has been demonstrated that it is a chelator more efficient for calcium than for magnesium. Moreover, it has been suggested recently that Fura 2, Indo-1, and Mag-Indo-1 might be partially located inside the membranes of intracellular organelles where the concentration of calcium is expected to be far higher than that of magnesium. All these data question again the use of the simple spectrofluorimetric methods to quantify the cytoplasmic concentration in magnesium. Our studies in solution have confirmed that Mag-Indo-1 is a potent chelator for calcium and also demonstrated that the fluorescence spectrum of the calcium-bound Mag-Indo-1 is quite similar to that of magnesium-bound Mag-Indo-1. Experiments have been carried out with 3T3 cells to quantify the amount of Mag- Indo-1 entrapped in the intracellular membranes and its consequences on the monitoring of the intracytoplasmic concentration of magnesium.
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Are intracellular ionic concentrations accessible using fluorescent probes? The example of Mag-Indo-1.
Cell Biology and Toxicology, 1994Co-Authors: Bruno Morelle, Jean-marie Salmon, Jean Vigo, Pierre VialletAbstract:The study of the physicochemical properties of Mag-Indo-1, a fluorescent probe used for intracellular magnesium measurements, has shown that in a biological environment the deprotonated form of this probe is in simultaneous equilibrium with a protonated form, a protein and a magnesium-bound form. The complex emission fluorescence spectrum emitted by a single living cell was analyzed using a computerized method, allowing the evaluation of the contribution of each species of the Mag-Indo-1 to the cellular fluorescence. This approach used to evaluate intracellular Mg2+ concentration has also shown the variability of the important participation of protein-bound Mag-Indo-1 to the cellular fluorescence. Thus the widely used ratioing method, unable to take into account this variability, cannot afford a reliable evaluation of [Mg2+]. Whatever the technique used for investigation (microfluorimetry, flow cytometry, etc.) the evaluation of [Mg2+]i using the fluorescent probe Mag-Indo-1 requires a method able to quantify, in complex fluorescence, the fluorescence intensity of the forms involved in the equilibrium with Mg2+.
Claude Collet - One of the best experts on this subject based on the ideXlab platform.
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Intracellular calcium signals measured with Indo-1 in isolated skeletal muscle fibres from control and mdx mice.
The Journal of physiology, 1999Co-Authors: Claude Collet, Bruno Allard, Yves Tourneur, V JacquemondAbstract:1. Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator Indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2. The mean (+/- s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 +/- 5 nM (n = 72) and 48 +/- 7 nM (n = 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than approximately 35 weeks, resting [Ca2+]i was significantly lower in mdx (16 +/- 4 nM, n = 11) than in control fibres (71 +/- 10 nM, n = 14). 3. Changes in [Ca2+]i elicited by short (5-35 ms) depolarizing pulses from -80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5-2. For longer pulses, no significant difference could be detected. 4. In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5. Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.
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intracellular calcium signals measured with Indo 1 in isolated skeletal muscle fibres from control and mdx mice
The Journal of Physiology, 1999Co-Authors: Claude Collet, Bruno Allard, Yves Tourneur, Vincent JacquemondAbstract:1Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator Indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2The mean (±s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 ± 5 nm (n= 72) and 48 ± 7 nm (n= 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than ∼35 weeks, resting [Ca2+]i was significantly lower in mdx (16 ± 4 nm, n= 11) than in control fibres (71 ± 10 nm, n= 14). 3Changes in [Ca2+]i elicited by short (5–35 ms) depolarizing pulses from −80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5–2. For longer pulses, no significant difference could be detected. 4In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.
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Intracellular calcium signals measured with Indo‐1 in isolated skeletal muscle fibres from control and mdx mice
The Journal of Physiology, 1999Co-Authors: Claude Collet, Bruno Allard, Yves Tourneur, Vincent JacquemondAbstract:1Intracellular free calcium concentration ([Ca2+]i) was measured with the fluorescent indicator Indo-1 in single skeletal fibres enzymatically isolated from the flexor digitorum brevis and interosseus muscles of control and dystrophic mdx C57BL/10 mice. Measurements were taken from a portion of fibre that was voltage clamped to allow detection of depolarization-induced changes in [Ca2+]i. 2The mean (±s.e.m.) initial resting [Ca2+]i from all control and mdx fibres tested was 56 ± 5 nm (n= 72) and 48 ± 7 nm (n= 57), respectively, indicating no significant overall difference between the two groups. However, when comparing a batch of control and mdx fibres obtained from mice older than ∼35 weeks, resting [Ca2+]i was significantly lower in mdx (16 ± 4 nm, n= 11) than in control fibres (71 ± 10 nm, n= 14). 3Changes in [Ca2+]i elicited by short (5–35 ms) depolarizing pulses from −80 to 0 mV showed similar properties in control and mdx fibres. After a 5 ms duration pulse the mean time constant of [Ca2+]i decay was, however, significantly elevated in mdx as compared to control fibres, by a factor of 1.5–2. For longer pulses, no significant difference could be detected. 4In response to 50 ms duration depolarizing pulses of various amplitudes the threshold for detection of an [Ca2+]i change and the peak [Ca2+]i reached for a given potential were similar in control and mdx fibres. 5Overall results show that mdx skeletal muscle fibres are quite capable of handling [Ca2+]i at rest and in response to membrane depolarizations.