Time Course Of Transients Research Articles

Characterization of spontaneous and action potential-induced calcium transients in developing myotubes in vitro.

We have investigated the onset and maturation of action potential- and calcium-induced calcium release from the sarcoplasmic reticulum during the differentiation of excitation-contraction coupling in skeletal muscle. Microfluorometry and video imaging of cultured myotubes loaded with the fluorescent calcium indicator fluo-3 revealed the dynamics, time course, and physiological properties of calcium transients as well as their changes during development. Spontaneous and stimulated contractions in well-differentiated myotubes are accompanied by brief (200-500 ms) increases in the concentration of free cytoplasmic calcium. These transients are modulated by sub-threshold concentrations of caffeine, resulting in a plateau of elevated calcium. Two novel types of calcium transients were observed in non-contracting myotubes. 1) Fast localized transients (FLTs) are radially restricted focal release events that occur spontaneously within the myoplasm at various densities and frequencies. 2) Upon addition of caffeine, propagating calcium waves are generated (35-70 microns/s velocity), which are accompanied by contractures. Aside from caffeine sensitivity, calcium waves and contraction-related sustained release events are similar in amplitude and duration, as well as in their inactivation and refractory properties. Thus, these transients may represent calcium-induced calcium release in quiescent and active myotubes, respectively. Following one calcium-induced calcium release event, myotubes become refractory to new calcium-induced transients; however, action potential-induced transients and FLTs are not blocked. This suggests that these transients occur by distinct release mechanisms and that dual modes of calcium release exist prior to the coupling of calcium release to excitation.

Effects of Ca 2+ -buffer concentration and stimulus interval on the voltage dependence and timecourse of calcium-release-dependent inward current in rabbit atrial myocytes

To investigate the kinetics of the inward Na-Ca exchange tail current activated by internal calcium in rabbit atrial cells, the whole-cell patch-clamp technique with intracellular perfusion was used. We recorded the inward phase of this current during repolarizations following a brief 2-5 ms depolarizing pulse to +40 mV from a holding potential of -70 mV. Peak activation of the current occurs about 10 ms from the beginning of the depolarizing pulse, and it decays spontaneously with a slow timecourse. The voltage dependence of the process that activates the inward current from -40 mV to +40 mV has a very steep slope between -40 and -20 mV and then virtually saturates between -10 mV and +40 mV. The voltage dependence of the process that activates the inward current is steeper than that which activates the sarcolemmal calcium current, iCa.L, and the timecourse of the current relaxation is much slower at low-frequency stimulation and when using low concentrations of Ca-buffer. The magnitude and timecourse of the calcium transients estimated by the inward tail current are smaller and faster, and the slow component of decay was abolished by the presence of high intracellular concentrations of Ca-buffer or by high frequency stimulation. These observations suggest that calcium release from the sarcoplasmic reticulum may be triggered by only a small fraction of the sarcolemmal calcium current.

The hepatocyte calcium oscillator

Hepatocytes stimulated with calcium-mobilising agonists generate free Ca transients whose frequency is modulated by hormone concentration. Importantly, the time-course of individual free Ca transients is independent of agonist dose but does change with agonist species. A receptor-controlled model in which protein kinase C provides negative feedback directed against the different receptors, or receptor-specific G proteins, has been proposed in order to explain the agonist-specificity of the falling phase of the free Ca spikes. Here we show further evidence, from mixing of hormones and from the effects of elevated cAMP, of receptor-specific information within the spikes.

Modulation of Ca2+ transients and contractile properties by beta‐adrenoceptor stimulation in ferret ventricular muscles.

1. The mechanism of modulation of Ca2+ transients and contraction by beta-adrenoceptor stimulation was studied in ferret ventricular muscles using aequorin to measure intracellular Ca2+. 2. Peaks of tension and light transients were increased by isoprenaline (10(-9) - 5 x 10(-7) M) which also abbreviated their time courses. 3. Time-to-peak tension was significantly shortened by 5 x 10(-9) M-isoprenaline and time-to-peak light was abbreviated by 10(-9) M-isoprenaline. 4. The time for the light to decay was shortened at 10(-9) M-isoprenaline. However, a higher concentration of isoprenaline (10(-8) M) was required for significant shortening of the half-relaxation time (TR50). 5. When isoprenaline was removed and beta-blocker (bupranolol, 1 microM) was applied, the time course of the light transients recovered but the time course of relaxation did not recover. 6. The relationship between [Ca2+]i and tension in tetanic contraction produced in the presence of ryanodine (5 microM) was shifted to the right by isoprenaline (10(-8) M). This was recovered by the replacement of isoprenaline with bupranolol (1 microM). 7. Isoprenaline (10(-7) M) added to the solution containing 20 mM [Ca2+]O and Bay K 8644 (1 microM), which produced maximal tension, caused a large light signal and enhancement of the initial phasic tension in tetanic contraction. However, the replacement of isoprenaline with bupranolol after immersing the preparation in 20 mM [Ca2+]O solution with Bay K 8644 and isoprenaline, did not significantly change the tension level, although the light signal decreased. Similar results were obtained in the ventricular muscle of young rats. 8. These results suggest that the dose dependence of modulation of the contractile element and sarcoplasmic reticulum (SR) by beta-adrenoceptor stimulation differs, and that additional factors, other than the faster Ca2+ uptake by SR and the decrease in Ca2+ sensitivity of the contractile element, might be involved in the shortening of the half-relaxation time by beta-adrenoceptor stimulation. In addition, beta-adrenoceptor stimulation does not produce a marked change in the maximal tension level.

Mechanical transients of single toad stomach smooth muscle cells. Effects of lowering temperature and extracellular calcium.

Smooth muscle's slow, economical contractions may relate to the kinetics of the crossbridge cycle. We characterized the crossbridge cycle in smooth muscle by studying tension recovery in response to a small, rapid length change (i.e., tension transients) in single smooth muscle cells from the toad stomach (Bufo marinus). To confirm that these tension transients reflect crossbridge kinetics, we examined the effect of lowering cell temperature on the tension transient time course. Once this was confirmed, cells were exposed to low extracellular calcium [( Ca2+]o) to determine whether modulation of the cell's shortening velocity by changes in [Ca2+]o reflected the calcium sensitivity of one or more steps in the crossbridge cycle. Single smooth muscle cells were tied between an ultrasensitive force transducer and length displacement device after equilibration in temperature-controlled physiological saline having either a low (0.18 mM) or normal (1.8 mM) calcium concentration. At the peak of isometric force, after electrical stimulation, small, rapid (less than or equal to 1.8% cell length in 3.6 ms) step stretches and releases were imposed. At room temperature (20 degrees C) in normal [Ca2+]o, tension recovery after the length step was described by the sum of two exponentials with rates of 40-90 s-1 for the fast phase and 2-4 s-1 for the slow phase. In normal [Ca2+]o but at low temperature (10 degrees C), the fast tension recovery phase slowed (apparent Q10 = 1.9) for both stretches and releases whereas the slow tension recovery phase for a release was only moderately affected (apparent Q10 = 1.4) while unaffected for a stretch. Dynamic stiffness was determined throughout the time course of the tension transient to help correlate the tension transient phases with specific step(s) in the crossbridge cycle. The dissociation of tension and stiffness, during the fast tension recovery phase after a release, was interpreted as evidence that this recovery phase resulted from both the transition of crossbridges from a low- to high-force producing state as well as a transient detachment of crossbridges. From the temperature studies and dynamic stiffness measurements, the slow tension recovery phase most likely reflects the overall rate of crossbridge cycling. From the tension transient studies, it appears that crossbridges cycle slower and have a longer duty cycle in smooth muscle. In low [Ca2+]o at 20 degrees C, little effect was observed on the form or time course of the tension transients.(ABSTRACT TRUNCATED AT 400 WORDS)

Force transient time course in heart muscle with high and low V 1 to V 3 myosin isoenzyme ratio

Values of mechanical and biochemical indicators of muscle performance measured in heart muscle having mostly the V1 isoenzyme of myosin are different than measurements of the same indicators made in muscle having mostly the V3 isoenzyme of myosin. It has been suggested that these differences in performance indicators might be attributable to subtle differences in myosin-actin crossbridge cycling kinetics between the V1 and the V3 isoforms of myosin. To investigate this, we derived information about myosin-actin cycling kinetics from the time course of force transients following rapid small amplitude length releases applied to chemically "skinned", isometrically contracting trabeculae from the hearts of normal (greater than = 90% V1) and propylthiouracil treated (greater than = 90% V3) rats. The rate constant for rapid force recovery measured in trabeculae from normal rats was twice that measured in trabeculae from treated rats (88.8 +/- 18.8 (n = 12) vs. 43.7 +/- 6.5 (n = 10)/s, mean +/- S.D.). We interpret this difference in rate constants as evidence that the kinetics of at least one step in the interaction of myosin with actin depends on the isoenzyme of myosin present in the heart.

Single cell measurements in research on calcium-mobilising purinoceptors.

The Ca-sensitive photoprotein aequorin has been used to record repetitive free Ca transients in single rat liver cells responding to either ATP or ADP. The time-course of individual transients is longer when ATP is the agonist. If both agonists operate through the same receptor, then curtailment of the receptors' activity occurs more rapidly when ADP is the agonist, and we infer that an agonist-occupied receptor linked to a GTP-liganded G protein is the moiety responsible for activating a phosphatidylinositol bisphosphate-specific phospholipase C. The alternative explanation is that ATP and ADP act through different receptors.

Optical recording of calcium and voltage transients following impulses in cell bodies and processes of identified leech neurons in culture

Optical methods were used to examine the spread of electrical potentials and the distribution and time course of calcium transients in individual identified nerve cells isolated from the leech. A photodiode array detected voltage transients by measuring absorbance changes of cells stained with the voltage-sensitive dye RH155 added to the bath. Calcium transients were recorded by measuring absorbance changes of the dye arsenazo III, which had been injected into the cells. In addition, Lucifer yellow was injected to outline the some and processes. Calcium changes resulting from individual action potentials were recorded from N, P, and Retzius cells without averaging. Signals from T cells and anterior pagoda (AP) cells were weaker but could be detected with averaging. These results are in accord with previous studies on calcium contributions to action potentials in these cells. For all cells, larger or wider action potentials gave bigger signals. Calcium changes from each of a train of action potentials were of equal amplitude, showing no sign of facilitation. Calcium transients from Retzius cells that had formed chemical synapses with P cells had properties similar to those of isolated cells. We were also able to detect responses from prolonged subthreshold depolarizations to -40 mV from a hyperpolarized membrane potential (-65 mV). These signals rose throughout the duration of the pulse (1-2 sec). With the photodiode array we mapped the distribution of the calcium signals. The amplitudes from each pixel are proportional to the amount of calcium entering that element in response to the stimulating pulse, if the simplifying assumption is made that the calcium buffering of the cytoplasm is uniform throughout the cell. The largest signals were detected over the axon stump left from the cell isolation procedure. Large signals were also detected from the soma. Weak signals were detected from the processes of some cells. From many Retzius cells, no signals at all were detected from the newly formed processes. Using the photodiode array, we also recorded voltage transients from the cells. Signals were recorded from all over the arborization of the neuron, with no obvious variation in time course, showing that the entire cell, including fine slender processes and broad growth cones, was essentially isopotential. Combining these observations with the measured distribution of calcium transients in the same cell suggests that the density of calcium channels in most cells is less in the outgrowing processes than in the soma or axon stump.

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

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.

Regional properties of calcium entry in barnacle neurons determined with Arsenazo III and a photodiode array

Calcium changes were simultaneously measured at many positions on individual neurons from the supraesophageal ganglion of the barnacle by detecting absorbance changes of the indicator dye Arsenazo III with a 10 X 10 photodiode array. These changes were correlated with positions on the stimulated cell determined from Lucifer yellow injections. Absorbance signals were found at all locations on the cells, demonstrating that calcium channels were distributed on the somata, axons, and neuropil processes. By comparing the amplitude of the signal with the membrane area in each region, we could measure the calcium entry per impulse per unit of surface in each part of the cell. Assuming that the properties of the calcium channels are the same in all regions, we determined that calcium channels were distributed uniformly along the commissural axon of one cell and were found at higher density in the neuropil. Because significant calcium changes are only detected when cells are depolarized above about -20 mV, the presence of absorbance signals indicates membrane depolarization above this level. We used this fact to show that calcium spikes propagate along the axon and into the neuropil of one cell, along the axon of another, and not at all in a third. Differences in time course of calcium transients were observed in different regions of cells. The recovery time course was faster at the edge of the cell body than in the center and faster in the neuropil than in the axon or soma. During trains of action potentials or during wide action potentials in tetraethylammonium (TEA) saline, the calcium signal reached a plateau in the neuropil while continuing to rise in the axon and soma.

Electric birefringence in concentrated solutions of tobacco mosaic virus.

AbstractA tentative and phenomenological analysis of negative electric birefringence, which has often been observed as an anomalous birefringence phenomenon in a concentrated solution of rodlike macromolecules, is presented. Tobacco mosaic virus (TMV) was used as a typical example for the investigation. It was found that if the applied electric field is sufficiently high, the steady‐state birefringence becomes becomes positive even at a very high concentration of TMV. From this finding and analysis of the time course of birefringence transients, it was suggested that the TMV (common strain, OM type), which originally has no inherent permanent dipole, behaves as if it possesses a permanent dipole perpendicular to its long axis. Supporting evidence was also obtained from birefringence experiments on concentrated solutions of the HR strain of TMV, which has an inherent permanent dipole along its long axis. Other possibilities, for example, the effects of the walls of electrodes or of polymerization of TMV molecules, were excluded.

The attenuation of passively propagating dendritic potentials in a motoneurone cable model.

1. The Rall model of the motoneurone, which consists of a lumped resistance and capacitance, representing the soma, in parallel with a number of distributed resistance-capacitance networks of finite and equal electrical length, representing equivalent dendritic cables, has been used to study the effects of varying electrical and geometrical parameters on the time course and amplitude of transients generated at different locations on the dendritic cables.2. An analytical solution has been obtained for the time course of the voltage transient generated at the point of current injection on the parallel combination of all dendritic cables, in terms of the distance from the soma to the current injection point, the electrotonic length of the equivalent dendritic cable, the dendritic to soma conductance ratio and the membrane time constant. The current applied is a current impulse, and the response to any synaptic current time course may be obtained from the analytical expression for the current impulse response. A smooth current time course of the form Te(-alphaT) has been used in computations.3. An analytical expression has been obtained for the early part of the voltage response at the point of current injection, when the current is applied to a fraction of the total dendritic cable. This response is in terms of all the cable parameters, and the assumed fraction of the dendritic cable which receives the synaptic current. Computations of this response have been carried out assuming a smooth time course of synaptic current.4. The computations of the peak amplitude of the voltage transient obtained from these expressions, together with similar computations for the peak amplitude of the voltage transient after propagation to the soma (Jack & Redman, 1971b), have been used to derive a set of attenuation curves for dendritic propagation. These curves give the ratio of the peak amplitude of the voltage transient at the synaptic location on the dendritic cable, and the peak amplitude after propagation to the soma, in terms of the electrotonic distance to the synaptic location, the time course of current injection, and the cable parameters for the motoneurone model.

An electrical description of the motoneurone, and its application to the analysis of synaptic potentials.

1. The Rall model of the motoneurone, which consists of a lumped resistance and capacitance, representing the soma, in parallel with a distributed resistance-capacitance network of finite length, representing the equivalent dendritic cable, has been used to investigate the effects of varying electrical and geometrical parameters on the time course of transients generated at the model soma.2. An analytical solution has been obtained for the voltage at the model soma, following a brief current injection at any point on the dendritic cable, in terms of the dendritic to soma conductance ratio, the electrotonic length of the cable, the membrane time constant, and the electrotonic distance between the point of current injection and the soma. This solution has been used to study the response at the soma to currents with a smooth time course, and to brief rectangular current pulses. Computations of these voltage transients are given to illustrate the effect of the above parameters on voltage time course.3. A method for determining the membrane time constant, the dendritic to soma conductance ratio, and the electrotonic length of the dendritic cable, is described. The method involves measurements from the decay time course of the transient at the soma following a brief current pulse being applied at the soma.4. A method is described whereby the time course of a synaptic potential, assumed to be generated by synaptic knobs located exclusively at the soma, may be used to determine the motoneurone parameters, and a parameter describing the time course of current injection.5. A method for estimating the distance between soma and origin of a non-somatic synaptic potential, once the parameters of the motoneurone are known, is described.

The propagation of transient potentials in some linear cable structures.

1. Analytical solutions have been given for the time course of voltage transients occurring in one-dimensional cable structures, with linear uniform membrane properties.2. These solutions show how the time course of the voltage transients generated at different distances are affected by variations in the time course of current injection (at one point on the cable) and by alterations in the length of the cable, with either sealed or open end terminations. These effects are illustrated with computed results.3. Simple measurements from the time course of a membrane potential displacement, occurring in a one-dimensional cable structure following a brief current injection across the membrane, are used to evaluate the parameters which describe the cable model. These parameters are the membrane time constant, the electrotonic length of the cable, the cable end termination, and in the case of a post-synaptic potential, the distance between the electrode and the active synapse.