scholarly journals Measurement of calcium transients and slow calcium current in myotubes.

1994 ◽  
Vol 103 (1) ◽  
pp. 107-123 ◽  
Author(s):  
J García ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Calcium Current ◽  
Calcium Transient ◽  
Calcium Currents ◽  
Calcium Transients ◽  
Patch Clamp Technique ◽  
Slow Increase ◽  
Adult Skeletal Muscle ◽  
Calcium Indicator ◽  
Cultured Myotubes

The purpose of this study was to characterize excitation-contraction (e-c) coupling in myotubes for comparison with e-c coupling of adult skeletal muscle. The whole cell configuration of the patch clamp technique was used in conjunction with the calcium indicator dye Fluo-3 to study the calcium transients and slow calcium currents elicited by voltage clamp pulses in cultured myotubes obtained from neonatal mice. Cells were held at -80 mV and stimulated with 15-20 ms test depolarizations preceded and followed by voltage steps designed to isolate the slow calcium current. The slow calcium current had a threshold for activation of about 0 mV; the peak amplitude of the current reached a maximum at 30 to 40 mV a and then declined for still stronger depolarizations. The calcium transient had a threshold of about -10 mV, and its amplitude increased as a sigmoidal function of test potential and did not decrease again even for test depolarizations sufficiently strong (> or = 50 mV) that the amplitude of the slow calcium current became very small. Thus, the slow calcium current in myotubes appears to have a negligible role in the process of depolarization-induced release of intracellular calcium and this process in myotubes is essentially like that in adult skeletal muscle. After repolarization, however, the decay of the calcium transient in myotubes was very slow (hundreds of ms) compared to adult muscle, particularly after strong depolarizations that triggered larger calcium transients. Moreover, when cells were repolarized after strong depolarizations, the transient typically continued to increase slowly for up to several tens of ms before the onset of decay. This continued increase after repolarization was abolished by the addition of 5 mM BAPTA to the patch pipette although the rapid depolarization-induced release was not, suggesting that the slow increase might be a regenerative response triggered by the depolarization-induced release of calcium. The addition of either 0.5 mM Cd2+ + 0.1 mM La3+ or the dihydropyridine (+)-PN 200-110 (1 microM) reduced the amplitude of the calcium transient by mechanisms that appeared to be unrelated to the block of current that these agents produce. In the majority of cells, the decay of the transient was accelerated by the addition of the heavy metals or the dihydropyridine, consistent with the idea that the removal system becomes saturated for large calcium releases and becomes more efficient when the size of the release is reduced.

scholarly journals Relationship of calcium transients to calcium currents and charge movements in myotubes expressing skeletal and cardiac dihydropyridine receptors.

1994 ◽  
Vol 103 (1) ◽  
pp. 125-147 ◽  
Author(s):  
J García ◽  
T Tanabe ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Calcium Channel ◽  
Calcium Current ◽  
Calcium Release ◽  
Voltage Dependence ◽  
Calcium Transient ◽  
Calcium Currents ◽  
Calcium Transients ◽  
Charge Movement ◽  
The Relationship

In both skeletal and cardiac muscle, the dihydropyridine (DHP) receptor is a critical element in excitation-contraction (e-c) coupling. However, the mechanism for calcium release is completely different in these muscles. In cardiac muscle the DHP receptor functions as a rapidly-activated calcium channel and the influx of calcium through this channel induces calcium release from the sarcoplasmic reticulum (SR). In contrast, in skeletal muscle the DHP receptor functions as a voltage sensor and as a slowly-activating calcium channel; in this case, the voltage sensor controls SR calcium release. It has been previously demonstrated that injection of dysgenic myotubes with cDNA (pCAC6) encoding the skeletal muscle DHP receptor restores the slow calcium current and skeletal type e-c coupling that does not require entry of external calcium (Tanabe, Beam, Powell, and Numa. 1988. Nature. 336:134-139). Furthermore, injection of cDNA (pCARD1) encoding the cardiac DHP receptor produces rapidly activating calcium current and cardiac type e-c coupling that does require calcium entry (Tanabe, Mikami, Numa, and Beam. 1990. Nature. 344:451-453). In this paper, we have studied the voltage dependence of, and the relationship between, charge movement, calcium transients, and calcium current in normal skeletal muscle cells in culture. In addition, we injected pCAC6 or pCARD1 into the nuclei of dysgenic myotubes and studied the relationship between the restored events and compared them with those of the normal cells. Charge movement and calcium currents were recorded with the whole cell patch-clamp technique. Calcium transients were measured with Fluo-3 introduced through the patch pipette. The kinetics and voltage dependence of the charge movement, calcium transients, and calcium current in dysgenic myotubes expressing pCAC6 were qualitatively similar to the ones elicited in normal myotubes: the calcium transient displayed a sigmoidal dependence on voltage and was still present after the addition of 0.5 mM Cd2+ + 0.1 mM La3+. In contrast, the calcium transient in dysgenic myotubes expressing pCARD1 followed the amplitude of the calcium current and thus showed a bell shaped dependence on voltage. In addition, the transient had a slower rate of rise than in pCAC6-injected myotubes and was abolished completely by the addition of Cd2+ + La3+.

scholarly journals Calcium transients associated with the T type calcium current in myotubes.

1994 ◽  
Vol 104 (6) ◽  
pp. 1113-1128 ◽  
Author(s):  
J García ◽  
K G Beam
Keyword(s):  
Calcium Channel ◽  
Calcium Current ◽  
Time Course ◽  
Calcium Transient ◽  
Voltage Sensor ◽  
Calcium Currents ◽  
Calcium Transients ◽  
Patch Clamp Technique ◽  
In Cells

Immature skeletal muscle cells, both in vivo and in vitro, express a high density of T type calcium current and a relatively low density of the dihydropyridine receptor, the protein thought to function as the Islow calcium channel and as the voltage sensor for excitation-contraction coupling. Although the role of the voltage sensor in eliciting elevations of myoplasmic, free calcium (calcium transients) has been examined, the role of the T type current has not. In this study we examined calcium transients associated with the T type current in cultured myotubes from normal and dysgenic mice, using the whole cell configuration of the patch clamp technique in conjunction with the calcium indicator dye Fluo-3. In both normal and dysgenic myotubes, the T type current was activated by weak depolarizations and was maximal for test pulses to approximately -20 mV. In normal myotubes that displayed T type calcium current, the calcium transient followed the amplitude and the integral of the current at low membrane potentials (-40 to -20 mV) but not at high potentials, where the calcium transient is caused by SR calcium release. The amplitude of the calcium transient for a pulse to -20 mV measured at 15 ms after depolarization represented, on average, 4.26 +/- 0.68% (n = 19) of the maximum amplitude of the calcium transient elicited by strong, 15-ms test depolarizations. In dysgenic myotubes, the calcium transient followed the integral of the calcium current at all test potentials, in cells expressing only T type current as well as in cells possessing both T type current and the L type current Idys. Moreover, the calcium transient also followed the amplitude and time course of current in dysgenic myotubes expressing the cardiac, DHP-sensitive calcium channel. Thus, in those cases where the transient appears to be a consequence of calcium entry, it has the same time course as the integral of the calcium current. Inactivation of the T type calcium current with 1-s prepulses, or block of the current by the addition of amiloride (0.3-1.0 mM) caused a reduction in the calcium transient which was similar in normal and dysgenic myotubes. To allow calculation of expected changes of intracellular calcium in response to influx, myotubes were converted to a roughly spherical shape (myoballs) by adding 0.5 microM colchicine to culture dishes of normal cells. Calcium currents and calcium transients recorded from myoballs were similar to those in normal myotubes.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals A novel calcium current in dysgenic skeletal muscle.

1989 ◽  
Vol 94 (3) ◽  
pp. 429-444 ◽  
Author(s):  
B A Adams ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Charge Carrier ◽  
Calcium Channel ◽  
Calcium Current ◽  
Primary Cultures ◽  
High Sensitivity ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Whole Cell Patch Clamp ◽  
Voltage Dependent

The whole-cell patch-clamp technique was used to study voltage-dependent calcium currents in primary cultures of myotubes and in freshly dissociated skeletal muscle from normal and dysgenic mice. In addition to the transient, dihydropyridine (DHP)-insensitive calcium current previously described, a maintained DHP-sensitive calcium current was found in dysgenic skeletal muscle. This current, here termed ICa-dys, is largest in acutely dissociated fetal or neonatal dysgenic muscle and also in dysgenic myotubes grown on a substrate of killed fibroblasts. In dysgenic myotubes grown on untreated plastic culture dishes, ICa-dys is usually so small that it cannot be detected. In addition, ICa-dys is apparently absent from normal skeletal muscle. From a holding potential of -80 mV. ICa-dys becomes apparent for test pulses to approximately -20 mV and peaks at approximately +20 mV. The current activates rapidly (rise time approximately 5 ms at 20 degrees C) and with 10 mM Ca as charge carrier inactivates little or not at all during a 200-ms test pulse. Thus, ICa-dys activates much faster than the slowly activating calcium current of normal skeletal muscle and does not display Ca-dependent inactivation like the cardiac L-type calcium current. Substituting Ba for Ca as the charge carrier doubles the size of ICa-dys without altering its kinetics. ICa-dys is approximately 75% blocked by 100 nM (+)-PN 200-110 and is increased about threefold by 500 nM racemic Bay K 8644. The very high sensitivity of ICa-dys to these DHP compounds distinguishes it from neuronal L-type calcium current and from the calcium currents of normal skeletal muscle. ICa-dys may represent a calcium channel that is normally not expressed in skeletal muscle, or a mutated form of the skeletal muscle slow calcium channel.

Caffeine attenuates contraction-induced diminutions of the intracellular calcium transient in mouse lumbrical muscle ex vivo

2019 ◽  
Vol 97 (5) ◽  
pp. 429-435 ◽  
Author(s):  
Ian C. Smith ◽  
Rene Vandenboom ◽  
A. Russell Tupling
Keyword(s):  
Skeletal Muscle ◽  
Intracellular Calcium ◽  
Calcium Release ◽  
Ex Vivo ◽  
Calcium Transient ◽  
Calcium Transients ◽  
Inotropic Effects ◽  
Intracellular Calcium Transient ◽  
Lumbrical Muscle ◽  
Lumbrical Muscles

The amount of calcium released from the sarcoplasmic reticulum in skeletal muscle rapidly declines during repeated twitch contractions. In this study, we test the hypothesis that caffeine can mitigate these contraction-induced declines in calcium release. Lumbrical muscles were isolated from male C57BL/6 mice and loaded with the calcium-sensitive indicator, AM-furaptra. Muscles were then stimulated at 8 Hz for 2.0 s in the presence or absence of 0.5 mM caffeine, at either 30 °C or 37 °C. The amplitude and area of the furaptra-based intracellular calcium transients and force produced during twitch contractions were calculated. For each of these measures, the values for twitch 16 relative to twitch 1 were higher in the presence of caffeine than in the absence of caffeine at both temperatures. We conclude that caffeine can attenuate contraction-induced diminutions of calcium release during repeated twitch contractions, thereby contributing to the inotropic effects of caffeine.

Low-Voltage Activated T-Type Calcium Currents Are Differently Expressed in Superficial and Deep Layers of Guinea PigPiriform Cortex

1998 ◽  
Vol 79 (2) ◽  
pp. 808-816 ◽  
Author(s):  
Jacopo Magistretti ◽  
Marco de Curtis
Keyword(s):  
Guinea Pig ◽  
Calcium Current ◽  
Low Voltage ◽  
Piriform Cortex ◽  
Neuronal Firing ◽  
Calcium Currents ◽  
Patch Clamp Technique ◽  
Voltage Range ◽  
Deep Layers ◽  
Group 2

Magistretti, Jacopo and Marco de Curtis. Low-voltage activated T-type calcium currents are differently expressed in superficial and deep layers of guinea pig piriform cortex. J. Neurophysiol. 79: 808–816, 1998. A variety of voltage-dependent calcium conductances are known to control neuronal excitability by boosting peripheral synaptic potentials and by shaping neuronal firing patterns. The existence and functional significance of a differential expression of low- and high-voltage activated (LVA and HVA, respectively) calcium currents in subpopulations of neurons, acutely isolated from different layers of the guinea pig piriform cortex, were investigated with the whole cell variant of the patch-clamp technique. Calcium currents were recorded from pyramidal and multipolar neurons dissociated from layers II, III, and IV. Average membrane capacitance was larger in layer IV cells [13.1 ± 6.2 (SD) pF] than in neurons from layers II and III (8.6 ± 2.8 and 7.9 ± 3.1 pF, respectively). Neurons from all layers showed HVA calcium currents with an activation voltage range positive to −40 mV. Neurons dissociated from layers III and IV showed an LVA calcium current with the biophysical properties of a T-type conductance. Such a current displayed the following characteristics: 1) showed maximal amplitude of 11–16 pA/pF at −30 mV, 2) inactivated rapidly with a time constant of ∼22 ms at −30 mV, and 3) was completely steady-state inactivated at −60 mV. Only a subpopulation of layer II neurons (group 2 cells; circa 18%) displayed an LVA calcium current similar to that observed in deep layers. The general properties of layer II-group 2 cells were otherwise identical to those of group 1 neurons. The present study demonstrates that LVA calcium currents are differentially expressed in neurons acutely dissociated from distinct layers of the guinea pig piriform cortex.

scholarly journals Calcium currents in a fast-twitch skeletal muscle of the rat.

1983 ◽  
Vol 82 (4) ◽  
pp. 449-468 ◽  
Author(s):  
P L Donaldson ◽  
K G Beam
Keyword(s):  
Skeletal Muscle ◽  
Leakage Current ◽  
Calcium Current ◽  
Outward Current ◽  
Extracellular Calcium ◽  
Calcium Currents ◽  
Slow Inward Current ◽  
Delayed Rectifier ◽  
Inward Current ◽  
Time To Peak

Slow ionic currents were measured in the rat omohyoid muscle with the three-microelectrode voltage-clamp technique. Sodium and delayed rectifier potassium currents were blocked pharmacologically. Under these conditions, depolarizing test pulses elicited an early outward current, followed by a transient slow inward current, followed in turn by a late outward current. The early outward current appeared to be a residual delayed rectifier current. The slow inward current was identified as a calcium current on the basis that (a) its magnitude depended on extracellular calcium concentration, (b) it was blocked by the addition of the divalent cations cadmium or nickel, and reduced in magnitude by the addition of manganese or cobalt, and (c) barium was able to replace calcium as an inward current carrier. The threshold potential for inward calcium current was around -20 mV in 10mM extracellular calcium and about -35 mV in 2 mM calcium. Currents were net inward over part of their time course for potentials up to at least +30 mV. At temperatures of 20-26 degrees C, the peak inward current (at approximately 0 mV) was 139 +/- 14 microA/cm2 (mean +/- SD), increasing to 226 +/- 28 microA/cm2 at temperatures of 27-37 degrees C. The late outward current exhibited considerable fiber-to-fiber variability. In some fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it was primarily a time-independent, nonlinear leakage current. In other fibers it appeared to be the sum of both leak and a slowly activated outward current. The rate of activation of inward calcium current was strongly temperature dependent. For example, in a representative fiber, the time-to-peak inward current for a +10-mV test pulse decreased from approximately 250 ms at 20 degrees C to 100 ms at 30 degrees C. At 37 degrees C, the time-to-peak current was typically approximately 25 ms. The earliest phase of activation was difficult to quantify because the ionic current was partially obscured by nonlinear charge movement. Nonetheless, at physiological temperatures, the rate of calcium channel activation in rat skeletal muscle is about five times faster than activation of calcium channels in frog muscle. This pathway may be an important source of calcium entry in mammalian muscle.

scholarly journals Dihydropyridine Receptors as Voltage Sensors for a Depolarization-evoked, IP3R-mediated, Slow Calcium Signal in Skeletal Muscle Cells

2002 ◽  
Vol 121 (1) ◽  
pp. 3-16 ◽  
Author(s):  
Roberto Araya ◽  
José L. Liberona ◽  
J. César Cárdenas ◽  
Nora Riveros ◽  
Manuel Estrada ◽  
...  
Keyword(s):  
Skeletal Muscle ◽  
Calcium Release ◽  
Calcium Transient ◽  
Voltage Sensor ◽  
Calcium Transients ◽  
Early Gene ◽  
Calcium Signal ◽  
Calcium Signals ◽  
Transfected Cells ◽  
Voltage Dependent

The dihydropyridine receptor (DHPR), normally a voltage-dependent calcium channel, functions in skeletal muscle essentially as a voltage sensor, triggering intracellular calcium release for excitation-contraction coupling. In addition to this fast calcium release, via ryanodine receptor (RYR) channels, depolarization of skeletal myotubes evokes slow calcium waves, unrelated to contraction, that involve the cell nucleus (Jaimovich, E., R. Reyes, J.L. Liberona, and J.A. Powell. 2000. Am. J. Physiol. Cell Physiol. 278:C998–C1010). We tested the hypothesis that DHPR may also be the voltage sensor for these slow calcium signals. In cultures of primary rat myotubes, 10 μM nifedipine (a DHPR inhibitor) completely blocked the slow calcium (fluo-3-fluorescence) transient after 47 mM K+ depolarization and only partially reduced the fast Ca2+ signal. Dysgenic myotubes from the GLT cell line, which do not express the α1 subunit of the DHPR, did not show either type of calcium transient following depolarization. After transfection of the α1 DNA into the GLT cells, K+ depolarization induced slow calcium transients that were similar to those present in normal C2C12 and normal NLT cell lines. Slow calcium transients in transfected cells were blocked by nifedipine as well as by the G protein inhibitor, pertussis toxin, but not by ryanodine, the RYR inhibitor. Since slow Ca2+ transients appear to be mediated by IP3, we measured the increase of IP3 mass after K+ depolarization. The IP3 transient seen in control cells was inhibited by nifedipine and was absent in nontransfected dysgenic cells, but α1-transfected cells recovered the depolarization-induced IP3 transient. In normal myotubes, 10 μM nifedipine, but not ryanodine, inhibited c-jun and c-fos mRNA increase after K+ depolarization. These results suggest a role for DHPR-mediated calcium signals in regulation of early gene expression. A model of excitation-transcription coupling is presented in which both G proteins and IP3 appear as important downstream mediators after sensing of depolarization by DHPR.

scholarly journals Involvement of TRPC in the abnormal calcium influx observed in dystrophic (mdx) mouse skeletal muscle fibers

2002 ◽  
Vol 158 (6) ◽  
pp. 1089-1096 ◽  
Author(s):  
Clarisse Vandebrouck ◽  
Dominique Martin ◽  
Monique Colson-Van Schoor ◽  
Huguette Debaix ◽  
Philippe Gailly
Keyword(s):  
Skeletal Muscle ◽  
Transient Receptor Potential ◽  
Calcium Influx ◽  
Surface Membrane ◽  
Muscle Fibers ◽  
Mdx Mouse ◽  
Mdx Mice ◽  
Patch Clamp Technique ◽  
Adult Skeletal Muscle ◽  
Skeletal Muscle Fibers

Duchenne muscular dystrophy results from the lack of dystrophin, a cytoskeletal protein associated with the inner surface membrane, in skeletal muscle. The absence of dystrophin induces an abnormal increase of sarcolemmal calcium influx through cationic channels in adult skeletal muscle fibers from dystrophic (mdx) mice. We observed that the activity of these channels was increased after depletion of the stores of calcium with thapsigargin or caffeine. By analogy with the situation observed in nonexcitable cells, we therefore hypothesized that these store-operated channels could belong to the transient receptor potential channel (TRPC) family. We measured the expression of TRPC isoforms in normal and mdx adult skeletal muscles fibers, and among the seven known isoforms, five were detected (TRPC1, 2, 3, 4, and 6) by RT-PCR. Western blot analysis and immunocytochemistry of normal and mdx muscle fibers demonstrated the localization of TRPC1, 4, and 6 proteins at the plasma membrane. Therefore, an antisense strategy was used to repress these TRPC isoforms. In parallel with the repression of the TRPCs, we observed that the occurrence of calcium leak channels was decreased to one tenth of its control value (patch-clamp technique), showing the involvement of TRPC in the abnormal calcium influx observed in dystrophic fibers.

scholarly journals Spatiotemporal dynamics of calcium transients during embryogenesis of Drosophila melanogaster

2019 ◽  
Author(s):  
Olga Markova ◽  
Sébastien Senatore ◽  
Pierre-François Lenne
Keyword(s):  
Calcium Signaling ◽  
Fluorescent Protein ◽  
Calcium Transient ◽  
Calcium Transients ◽  
Waveform Analysis ◽  
Neural Cells ◽  
Calcium Indicator ◽  
Neuronal Tissues ◽  
Different Characteristics

Calcium signaling plays a crucial role in the physiology of the organs but also in various aspects of the organogenesis of the embryo. High versatility of calcium signaling is encoded by the dynamic variation of intracellular calcium concentration. While the dynamics of calcium is important, little is known about it throughout the embryogenesis of the largest class of animals, insects. Here, we visualize calcium dynamics throughout embryogenesis of Drosophila using a fluorescent protein-based calcium indicator, GCaMP3, and report calcium transients in epithelium and neuronal tissues. Local calcium transients of varying duration were detected in the outer epithelium, trachea and neural cells. In addition, gap-junction-dependent calcium waves were identified at stage 16 in the outer epithelium and in the trachea at stage 17. Calcium transient waveform analysis revealed different characteristics as a function of the duration, location and frequency. Detailed characterization of calcium transients during embryogenesis of Drosophila will help us better understand the role of calcium signaling in embryogenesis and organogenesis of insects.

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