scholarly journals Voltage-dependent Ca2+ Fluxes in Skeletal Myotubes Determined Using a Removal Model Analysis

2003 ◽  
Vol 123 (1) ◽  
pp. 33-52 ◽  
Author(s):  
R.P. Schuhmeier ◽  
W. Melzer
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Flux Ratio ◽  
Cyclopiazonic Acid ◽  
High Concentration ◽  
Fura 2 ◽  
Input Flux ◽  
Indicator Dye ◽  
Removal Model ◽  
Tonic Phase

The purpose of this study was to quantify the Ca2+ fluxes underlying Ca2+ transients and their voltage dependence in myotubes by using the “removal model fit” approach. Myotubes obtained from the mouse C2C12 muscle cell line were voltage-clamped and loaded with a solution containing the fluorescent indicator dye fura-2 (200 μM) and a high concentration of EGTA (15 mM). Ca2+ inward currents and intracellular ratiometric fluorescence transients were recorded in parallel. The decaying phases of Ca2+-dependent fluorescence signals after repolarization were fitted by theoretical curves obtained from a model that included the indicator dye, a slow Ca2+ buffer (to represent EGTA), and a sequestration mechanism as Ca2+ removal components. For each cell, the rate constants of slow buffer and transport and the off rate constant of fura-2 were determined in the fit. The resulting characterization of the removal properties was used to extract the Ca2+ input fluxes from the measured Ca2+ transients during depolarizing pulses. In most experiments, intracellular Ca2+ release dominated the Ca2+ input flux. In these experiments, the Ca2+ flux was characterized by an initial peak followed by a lower tonic phase. The voltage dependence of peak and tonic phase could be described by sigmoidal curves that reached half-maximal activation at −16 and −20 mV, respectively, compared with −2 mV for the activation of Ca2+ conductance. The ratio of the peak to tonic phase (flux ratio) showed a gradual increase with voltage as in rat muscle fibers indicating the similarity to EC coupling in mature mammalian muscle. In a subgroup of myotubes exhibiting small fluorescence signals and in cells treated with 30 μM of the SERCA pump inhibitor cyclopiazonic acid (CPA) and 10 mM caffeine, the calculated Ca2+ input flux closely resembled the L-type Ca2+ current, consistent with the absence of SR Ca2+ release under these conditions and in support of a valid determination of the time course of myoplasmic Ca2+ input flux based on the optical indicator measurements.

Simultaneous measurements of Ca2+ currents and intracellular Ca2+ concentrations in single skeletal muscle fibers of the frog

1987 ◽  
Vol 65 (4) ◽  
pp. 681-685 ◽  
Author(s):  
G. Brum ◽  
E. Stefani ◽  
E. Rios
Keyword(s):  
Time Course ◽  
Muscle Fibers ◽  
Optical Signal ◽  
Single Muscle ◽  
Drastic Reduction ◽  
Maximum Value ◽  
Input Flux ◽  
Voltage Dependent ◽  
The Relationship ◽  
Removal Model

The relationship between Ca2+ current amplitudes and myoplasmic Ca2+ transients was studied in single muscle fibers. Segments of muscle fibers were voltage-clamped in a double Vaseline gap chamber. Ca2+ transients were measured as an optical signal derived from the interaction between Ca2+ and the dye antipyrylazo III. The cells were maintained at −90 mV. Ca2+ currents were detected at pulse potentials to −50 mV, reached a maximum value at 0 mV, were reduced in size for larger depolarizations, and reversed at about 40 mV. Ca2+ transients were also detected at −50 Mv and progressively increased in size with larger pulse potentials up to 10 mV. Depolarizations to voltages greater than 10 mV did not further increase the size of the transient. The magnitude and time course of transients from 10 to 70 mV were almost identical. Ca2+ fluxes into the myoplasm (Ca2+ input fluxes) were calculated from the Ca2+ transients applying a removal model. The size of the input fluxes increased with depolarization up to 0 mV. Between 0 and 70 mV the peak input flux slightly increased, while the flux measured at 200 ms remained unchanged. In conclusion, Ca2+ transients and input fluxes were not reduced during pulses to large positive potentials, even though a drastic reduction of Ca2+ current occurred at these potentials. These observations make it very unlikely that a voltage-dependent Ca2+ entry is the triggering signal for contraction.

Decline of contractility during ischemia-reperfusion injury: actin glutathionylation and its effect on allosteric interaction with tropomyosin

2006 ◽  
Vol 290 (3) ◽  
pp. C719-C727 ◽  
Author(s):  
Frank C. Chen ◽  
Ozgur Ogut
Keyword(s):  
Reperfusion Injury ◽  
Posttranslational Modifications ◽  
Actin Polymerization ◽  
Time Course ◽  
Troponin I ◽  
Ischemia Reperfusion Injury ◽  
Ischemia Reperfusion ◽  
High Concentration ◽  
Cross Sectional ◽  
The Impact

The severity and duration of ischemia-reperfusion injury is hypothesized to play an important role in the ability of the heart subsequently to recover contractility. Permeabilized trabeculae were prepared from a rat model of ischemia-reperfusion injury to examine the impact on force generation. Compared with the control perfused condition, the maximum force (Fmax) per cross-sectional area and the rate of tension redevelopment of Ca2+-activated trabeculae fell by 71% and 44%, respectively, during ischemia despite the availability of a high concentration of ATP. The reduction in Fmax with ischemia was accompanied by a decline in fiber stiffness, implying a drop in the absolute number of attached cross bridges. However, the declines during ischemia were largely recovered after reperfusion, leading to the hypothesis that intrinsic, reversible posttranslational modifications to proteins of the contractile filaments occur during ischemia-reperfusion injury. Examination of thin-filament proteins from ischemic or ischemia-reperfused hearts did not reveal proteolysis of troponin I or T. However, actin was found to be glutathionylated with ischemia. Light-scattering experiments demonstrated that glutathionylated G-actin did not polymerize as efficiently as native G-actin. Although tropomyosin accelerated the time course of native and glutathionylated G-actin polymerization, the polymerization of glutathionylated G-actin still lagged native G-actin at all concentrations of tropomyosin tested. Furthermore, cosedimentation experiments demonstrated that tropomyosin bound glutathionylated F-actin with significantly reduced cooperativity. Therefore, glutathionylated actin may be a novel contributor to the diverse set of posttranslational modifications that define the function of the contractile filaments during ischemia-reperfusion injury.

scholarly journals Correlation between Charge Movement and Ionic Current during Slow Inactivation in Shaker K+ Channels

1997 ◽  
Vol 110 (5) ◽  
pp. 579-589 ◽  
Author(s):  
Riccardo Olcese ◽  
Ramón Latorre ◽  
Ligia Toro ◽  
Francisco Bezanilla ◽  
Enrico Stefani
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Ionic Current ◽  
K Channel ◽  
Charge Movement ◽  
Slow Inactivation ◽  
Gating Currents ◽  
Similar Time ◽  
K Channels ◽  
Recovery From Inactivation

Prolonged depolarization induces a slow inactivation process in some K+ channels. We have studied ionic and gating currents during long depolarizations in the mutant Shaker H4-Δ(6–46) K+ channel and in the nonconducting mutant (Shaker H4-Δ(6–46)-W434F). These channels lack the amino terminus that confers the fast (N-type) inactivation (Hoshi, T., W.N. Zagotta, and R.W. Aldrich. 1991. Neuron. 7:547–556). Channels were expressed in oocytes and currents were measured with the cut-open-oocyte and patch-clamp techniques. In both clones, the curves describing the voltage dependence of the charge movement were shifted toward more negative potentials when the holding potential was maintained at depolarized potentials. The evidences that this new voltage dependence of the charge movement in the depolarized condition is associated with the process of slow inactivation are the following: (a) the installation of both the slow inactivation of the ionic current and the inactivation of the charge in response to a sustained 1-min depolarization to 0 mV followed the same time course; and (b) the recovery from inactivation of both ionic and gating currents (induced by repolarizations to −90 mV after a 1-min inactivating pulse at 0 mV) also followed a similar time course. Although prolonged depolarizations induce inactivation of the majority of the channels, a small fraction remains non–slow inactivated. The voltage dependence of this fraction of channels remained unaltered, suggesting that their activation pathway was unmodified by prolonged depolarization. The data could be fitted to a sequential model for Shaker K+ channels (Bezanilla, F., E. Perozo, and E. Stefani. 1994. Biophys. J. 66:1011–1021), with the addition of a series of parallel nonconducting (inactivated) states that become populated during prolonged depolarization. The data suggest that prolonged depolarization modifies the conformation of the voltage sensor and that this change can be associated with the process of slow inactivation.

scholarly journals Amplification of the thapsigargin-evoked increase in the cytosolic free Ca2+ concentration by acetylcholine in acutely isolated mouse submandibular acinar cells

1996 ◽  
Vol 317 (3) ◽  
pp. 779-783 ◽  
Author(s):  
Peter. M. SMITH ◽  
Helen. E. REED
Keyword(s):  
Plasma Membrane ◽  
Time Course ◽  
Acinar Cells ◽  
Fold Increase ◽  
Extrusion Process ◽  
Significant Alteration ◽  
Atpase Inhibitor ◽  
Fura 2

The intracellular Ca2+ concentration was measured in single, acutely isolated, mouse submandibular acinar cells loaded with fura-2 AM. All experiments were performed in the absence of extracellular Ca2+ in order to eliminate Ca2+ influx. The microsomal ATPase inhibitor, thapsigargin, was used to release Ca2+ from intracellular stores and simultaneously prevent re-uptake into the stores. Sequential application of thapsigargin (2 μM) and the Ca2+ ionophore ionomycin (500 nM) indicated that thapsigargin was able to mobilize practically all intracellular Ca2+. Furthermore, in comparison with results obtained following inhibition of the plasma membrane Ca2+-ATPase by La3+ (2 mM), it may be shown that slowly unloading the intracellular Ca2+ stores using thapsigargin does not normally cause a massive, cytotoxic, increase in the cytosolic Ca2+ concentration, because Ca2+ is rapidly extruded from the cell across the plasma membrane. Application of a submaximal dose of acetylcholine (500 nM) during the rising phase of the response to thapsigargin caused a 3–4-fold increase in the amplitude of the rise in the cytosolic Ca2+ concentration without any significant alteration of the time course of the response. As thapsigargin alone is capable of mobilizing all releasable Ca2+, this increase in amplitude is most likely the result of inhibition of the Ca2+ extrusion process by acetylcholine.

Properties of neuroprotective cell-permeant Ca2+ chelators: effects on [Ca2+]i and glutamate neurotoxicity in vitro

1994 ◽  
Vol 72 (4) ◽  
pp. 1973-1992 ◽  
Author(s):  
M. Tymianski ◽  
M. P. Charlton ◽  
P. L. Carlen ◽  
C. H. Tator
Keyword(s):  
Time Course ◽  
Dependent Manner ◽  
Protective Effects ◽  
Tetraacetic Acid ◽  
Acetoxymethyl Ester ◽  
Neuroprotective Effects ◽  
Concentration Dependent Manner ◽  
Fura 2 ◽  
Glutamate Neurotoxicity

1. Cell-permeant Ca2+ chelators such as 1,2-bis-(2-amino-phenoxy)ethane- N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA-AM) protect neurons against excitotoxic and ischemic neuronal injury in vitro and in vivo. Here we provide the first steps toward characterizing the mechanisms by which these agents produce their neuroprotective effects. 2. Cultured mouse spinal neurons were simultaneously loaded with the Ca2+ indicator fura-2 and with one of three permeant chelators derived from the fast Ca2+ buffer BAPTA, or with ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid acetoxymethyl ester (EGTA-AM). Adding these chelators did not interfere with the fluorescence spectrum of fura-2 and had no effect on baseline [Ca2+]i. 3. The neurons were challenged with 250 microM L-glutamate for 50 min, producing a marked transient [Ca2+]i increase followed by a decay of [Ca2+]i to a lower “plateau.” About 80% of control neurons succumbed to this excitotoxic insult. Neurons that survived adjusted their plateau [Ca2+]i to lower levels than those that succumbed. 4. Neurons that were pretreated with permeant Ca2+ chelators became more resistant to these neurotoxic challenges. 5. We examined whether this reduction in glutamate neurotoxicity could be related to the given buffer's known Ca2+ affinity (Kd), its Ca2+ binding kinetics, and its ability to attenuate glutamate-induced [Ca2+]i increases. 6. Pretreatment of neurons with BAPTA analogues having Kds ranging from 100 to 3,600 microM 1) attenuated the amplitude and 2) lengthened the time constant describing the rise and decay of the glutamate-evoked [Ca2+]i transient. The magnitude of these effects paralleled the affinity of the chelator for Ca2+. 7. BAPTA-AM and its analogues dramatically attenuated the early neurotoxicity of glutamate, reducing cell deaths by up to 80%. However, in contrast with the graded effects of chelators having different Ca2+ affinities on Ca2+ transients, all BAPTA analogues were equally protective. These protective effects did not relate to the chelators' Ca2+ affinity within a Kd range of 100 nM (for BAPTA) to 3,600 nM (for 5,5'-dibromo BAPTA). 8. BAPTA-AM protected neurons in a concentration-dependent manner with 50% protection obtained with 10 microM, a concentration having no effect on the [Ca2+]i transient amplitude. 9. EGTA, a slow Ca2+ buffer with a similar Ca2+ affinity to BAPTA produced the same effects as BAPTA on [Ca2+]i transient kinetics. However, it was far less protective than BAPTA. 10. The time course of early glutamate neurotoxicity was altered by the BAPTA analogues, but not EGTA. BAPTA analogues caused a small increase in cell deaths in the first minutes of each experiment, followed by relative sparing from further neurodegeneration. 11. The ability of low Ca2+ affinity chelators such as 5,5'-dibromo BAPTA to protect neurons without markedly attenuating measured [Ca2+]i increases conflicts with the hypothesis that global elevations in [Ca2+]i are responsible for triggering neurotoxicity.(ABSTRACT TRUNCATED AT 400 WORDS)

Copper metabolism in the kidney of rats administered copper and copper-metallothionein

1998 ◽  
Vol 274 (4) ◽  
pp. F783-F790 ◽  
Author(s):  
Masaaki Kurasaki ◽  
Masashi Okabe ◽  
Shigeru Saito ◽  
Mika Suzuki-Kurasaki
Keyword(s):  
Time Course ◽  
Copper Metabolism ◽  
Degradation Process ◽  
High Concentration ◽  
Tubular Cells ◽  
Microscopic Studies

To gain a greater understanding of the mechanism of Cu metabolism in kidneys of rats, using autofluorescence of Cu-metallothioneins (Cu-MTs) we revealed the behavior of Cu-MT in the kidneys of rats administered Cu-MT. Yellow and orange fluorescent signals of Cu-MT were observed in the cortex. By microscopic studies, Cu-MT was dominant in the proximal convolute tubular cells of the cortex. A high concentration of Cu-MT presented in the lysosome-like organelles of the proximal convolute tubular adjacent to the glomeruli. During the time course after the injection, the orange signal in lysosome-like organelles gradually converted to a yellow signal, indicating that the Cu-MT was involved in a degradation process in lysosomes by oxidation, and the MT mRNA increased in the cortex, although the immunoreactivity of MT was almost constant in the same region. These results suggested that Cu bound to the injected MT was released in lysosomes and became a new inducer of MT biosynthesis in the cortex. In conclusion, the biosynthesis and degradation of Cu-MT occur repeatedly in the proximal convolute tubular cells.

Voltage Dependence of the Glycine Receptor–Channel Kinetics in the Zebrafish Hindbrain

1999 ◽  
Vol 82 (5) ◽  
pp. 2120-2129 ◽  
Author(s):  
Pascal Legendre
Keyword(s):  
Time Constant ◽  
Rise Time ◽  
Time Course ◽  
Voltage Dependence ◽  
Good Description ◽  
Glycine Receptor ◽  
Relative Amplitude ◽  
Time Constants ◽  
Low Concentration ◽  
Voltage Dependent

Electrophysiological recordings of outside-out patches to fast-flow applications of glycine were made on patches derived from the Mauthner cells of the 50-h-old zebrafish larva. As for glycinergic miniature inhibitory postsynaptic currents (mIPSCs), depolarizing the patch produced a broadening of the transient outside-out current evoked by short applications (1 ms) of a saturating concentration of glycine (3 mM). When the outside-out patch was depolarized from −50 to +20 mV, the peak current varied linearly with voltage. A 1-ms application of 3 mM glycine evoked currents that activated rapidly and deactivated biexponentially with time constants of ≈5 and ≈30 ms (holding potential of −50 mV). These two decay time constants were increased by depolarization. The fast deactivation time constant increased e-fold per 95 mV. The relative amplitude of the two decay components did not significantly vary with voltage. The fast component represented 64.2 ± 2.8% of the total current at −50 mV and 54.1 ± 10% at +20 mV. The 20–80% rise time of these responses did not show any voltage dependence, suggesting that the opening rate constant is insensitive to voltage. The 20–80% rise time was 0.2 ms at −70 mV and 0.22 ms at +20 mV. Responses evoked by 100–200 ms application of a low concentration of glycine (0.1 mM) had a biphasic rising phase reflecting the complex gating behavior of the glycine receptor. The time constant of these two components and their relative amplitude did not change with voltage, suggesting that modal shifts in the glycine-activated channel gating mode are not sensitive to the membrane potential. Using a Markov model to simulate glycine receptor gating behavior, we were able to mimic the voltage-dependent change in the deactivation time course of the responses evoked by 1-ms application of 3 mM glycine. This kinetics model incorporates voltage-dependent closing rate constants. It provides a good description of the time course of the onset of responses evoked by the application of a low concentration of glycine at all membrane potentials tested.

Separation and identification of multiple potassium currents regulating the pacemaker activity of insect neurosecretory cells (DUM neurons)

1995 ◽  
Vol 73 (1) ◽  
pp. 160-171 ◽  
Author(s):  
F. Grolleau ◽  
B. Lapied
Keyword(s):  
Time Course ◽  
Voltage Dependence ◽  
Outward Current ◽  
Potassium Currents ◽  
Neurosecretory Cells ◽  
Transient Current ◽  
Pacemaker Activity ◽  
Dum Neurons ◽  
K Current ◽  
A Current

1. Whole cell voltage-clamp studies performed in isolated adult neurosecretory cells identified as dorsal unpaired median (DUM) neurons of the terminal abdominal ganglion of the cockroach Periplaneta americana have allowed us to reveal a complex voltage-dependent outward current regulating the pacemaker activity. 2. The global outward current remaining after tetrodotoxin treatment was activated by depolarization above -50 mV, showing steep voltage dependence and outward rectification. 3. We used tail current analysis to determine the ionic selectivity of this outward current. The reversal potentials for two extracellular potassium concentrations (-92.7 and -65.4 mV for 3.1 and 10 mM, respectively) is consistent with the expected equilibrium potential for potassium ions. 4. Both peak and sustained components of the global outward K+ current were reduced by external application of 20 mM tetraethylammonium chloride, 10 nM iberiotoxin, 1 nM charybdotoxin (CTX) and 1 mM cadmium chloride. Subtraction of current recorded in CTX solution from that in control solution revealed an unusual biphasic Ca(2+)-dependent K+ current. The fast transient current resistant to 5 mM 4-aminopyridine (4-AP) is distinguished by its dependence on holding potential and time course from the late sustained current. 5. In addition, two other components of CTX-resistant outward K+ current could be separated by sensitivity to 4-AP, time course, and voltage dependence. Beside a calcium-independent delayed outwardly rectifying current, a 4-AP-sensitive fast transient current resembling the A-current has been also identified. It activates at negative potential (about -65 mV) and unlike the A-current of other neurons, it inactivates rapidly with complex inactivation kinetics. A-like current is half-inactivated at -63.5 mV and half-activated at -35.6 mV. 6. Our findings demonstrate for the first time in DUM neuron cell bodies the existence of multiple potassium currents underlying the spontaneous electrical activity. Their identification and characterization represent a fundamental step in further understanding the pacemaker properties of these insect neurosecretory cells.

Pi inhibits the SR Ca2+ pump and stimulates pump-mediated Ca2+ leak in rabbit cardiac myocytes

2000 ◽  
Vol 279 (2) ◽  
pp. H577-H585 ◽  
Author(s):  
G. L. Smith ◽  
A. M. Duncan ◽  
P. Neary ◽  
L. Bruce ◽  
F. L. Burton
Keyword(s):  
Steady State ◽  
Inorganic Phosphate ◽  
Time Course ◽  
Ventricular Myocytes ◽  
Cyclopiazonic Acid ◽  
Ruthenium Red ◽  
Uptake Capacity ◽  
Exponential Decline ◽  
Pump Rate ◽  
Uptake Process

Measurements of sarcoplasmic reticulum (SR) Ca2+ uptake were made from aliquots of dissociated permeabilized ventricular myocytes using fura 2. Equilibration with 10 mM oxalate ensured a reproducible exponential decline of [Ca2+] from 600 nM to a steady state of 100–200 nM after addition of Ca2+. In the presence of 5 μM ruthenium red, which blocks the ryanodine receptor, the time course of the decline of [Ca2+] can be modeled by a Ca2+-dependent uptake process and a fixed Ca2+leak. Partial inhibition of the Ca2+ pump with 1 μM cyclopiazonic acid or 50 nM thapsigargin reduced the time constant for Ca2+ uptake but did not affect the SR Ca2+leak. Addition of 10 mM inorganic phosphate (Pi) decreased the rate of Ca2+ accumulation by the SR and increased the Ca2+ leak rate. This effect was reversed on addition of 10 mM phosphocreatine. 10 mM Pi had no effect on Ca2+ leak from the SR after complete inhibition of the Ca2+ pump. In conclusion, Pi decreases the Ca2+ uptake capacity of cardiac SR via a decrease in pump rate and an increase in Ca2+ pump-dependent Ca2+ leak.

Regulation of contraction and relaxation by membrane potential in cardiac ventricular myocytes

2000 ◽  
Vol 278 (5) ◽  
pp. H1618-H1626 ◽  
Author(s):  
Gregory R. Ferrier ◽  
Isabel M. Redondo ◽  
Cindy A. Mason ◽  
Cindy Mapplebeck ◽  
Susan E. Howlett
Keyword(s):  
Membrane Potential ◽  
Sarcoplasmic Reticulum ◽  
Guinea Pig ◽  
Voltage Dependence ◽  
Ventricular Myocytes ◽  
Release Mechanism ◽  
Fura 2 ◽  
Regulation Of Contraction ◽  
Contraction And Relaxation ◽  
Sustained Responses

Control of contraction and relaxation by membrane potential was investigated in voltage-clamped guinea pig ventricular myocytes at 37°C. Depolarization initiated phasic contractions, followed by sustained contractions that relaxed with repolarization. Corresponding Ca2+ transients were observed with fura 2. Sustained responses were ryanodine sensitive and exhibited sigmoidal activation and deactivation relations, with half-maximal voltages near −46 mV, which is characteristic of the voltage-sensitive release mechanism (VSRM) for sarcoplasmic reticulum Ca2+. Inactivation was not detected. Sustained responses were insensitive to inactivation or block of L-type Ca2+ current ( I Ca-L). The voltage dependence of sustained responses was not affected by changes in intracellular or extracellular Na+ concentration. Furthermore, sustained responses were not inhibited by 2 mM Ni2+. Thus it is improbable that I Ca-L or Na+/Ca2+ exchange generated these sustained responses. However, rapid application of 200 μM tetracaine, which blocks the VSRM, strongly inhibited sustained contractions. Our study indicates that the VSRM includes both a phasic inactivating and a sustained noninactivating component. The sustained component contributes both to initiation and relaxation of contraction.

Sign in / Sign up

Export Citation Format

Share Document