Search PubMed⌕ Search

Biomedical subjects

W G Wier

Publications and source records attributed to W G Wier.

At least 55 records · Page 3Linked to original sources
PubMed1992

Macroscopic and unitary properties of physiological ion flux through T-type Ca2+ channels in guinea-pig heart cells.

1. We sought to distinguish two types of Ca2+ channel in guinea-pig ventricular cells (T-type and L-type) and to characterize their respective gating and permeation properties when Ca2+ (1-10 mM) is the charge carrier, as is the case physiologically. 2. Na+ was removed from both the external and internal solutions to eliminate currents through Na+ channels and Na(+)-Ca2+ exchange. Major differences in the voltage dependence of steady-state inactivation were exploited to separate the two Ca2+ current components. 3. From a holding potential of -50 mV, only L-type channels were available to open with depolarization. When holding at -90 mV, T-type channels contributed an additional rapidly inactivating component superimposed upon the L-type current. Only the L-type channels thus identified were sensitive to the dihydropyridine Ca2+ channel blocker nitrendipine. 4. T-type currents, measured by taking the difference between the currents elicited from a holding potential of -90 mV and those elicited from -50 mV, peaked within 10 ms and decayed completely within 50-100 ms. 5. Macroscopic T-type currents were largest during depolarizing pulses between -40 and -30 mV (peak current density of 0.62 +/- 0.21 nA nF-1) and decreased at more positive potentials, becoming unmeasurably small above 0 mV. 6. Unitary currents recorded with similar ionic conditions and voltage protocols exhibited a single-channel conductance of 4-5 pS in 10 mM Ca2+. Ensemble average currents through a single channel reproduced accurately the time course of whole-cell T-type current. Permeation properties could not explain the absence of macroscopic T-type currents at positive test potentials, which must therefore be attributable to gating. 7. Convolution analysis was employed to clarify the single-channel basis of the rapidly decaying current waveform of T-type channels. The latencies to first opening and reopening, which reflect activation and deactivation, influenced the waveform most strikingly. Open times were sufficiently brief that they contributed little to shaping the average current. Thus, macroscopic inactivation largely reflects rate-limiting activation events. 8. The unitary current amplitudes and peak open probabilities measured for single T-type channels, when compared to the average macroscopic T-type current density, predict 10.6 functional channels per picofarad, or approximately 1700 T-type channels per typical ventricular myocyte.

Animals↗
PubMed1992

Macroscopic and unitary properties of physiological ion flux through L-type Ca2+ channels in guinea-pig heart cells.

1. We investigated the currents through L-type Ca2+ channels when Ca2+ (1-10 mM) was the charge carrier, as is the case physiologically. 2. Na+ was removed from both the external and internal solutions to eliminate currents through Na+ channels and Na(+)-Ca2+ exchange. 3. From a holding potential of -50 mV only L-type channels were available to open with depolarization. Macroscopic L-type currents were maximal during depolarizing pulses to +10 mV (peak current density of 4.7 +/- 0.3 nA nF-1). 4. During depolarizing steps as long as 180 ms, the decay of current through L-type channels was incomplete, in contrast to that of T-type current. 5. Unitary currents recorded with comparable ionic conditions and voltage protocols exhibited a single-channel conductance of 6.9 pS in 10 mM Ca2+. Ensemble average currents reproduced accurately the features of whole-cell L-type current, including the maintained component. 6. Convolution analysis was employed to clarify the single-channel basis of the complex current waveform of L-type channels. First openings underlie the peak, while the maintained pedestal is generated by multiple re-openings. As with T-type channels, single openings are brief and contribute little to the time course of the average current. 7. The prominent maintained component of macroscopic and ensemble average L-type current cannot be explained by simple Markov models in which current decay reflects the progressive entry of channels into an absorbing inactivated state. 8. We considered the possibility that the maintained component of current arises from the existence of multiple distinct gating patterns, one of which lacks inactivation. Individual sweeps were sorted among three patterns of gating (no openings, active-early and active-late). Patterns of activity are not randomly distributed; instead, they tend to cluster over time. 9. Most of the maintained current is attributable to the 'active-late' pattern of gating. Considered separately, this pattern can be well described by a simple Markov chain lacking an inactivated state. The 'active-early' gating pattern accounts entirely for the initial current transient, and for about one-third of the maintained component; thus, inactivation, even when present, must be reversible rather than absorbing. 10. The unitary current amplitudes and peak open probabilities measured for single L-type channels, when compared to the average macroscopic L-type current density, predict 170 functional channels per picofarad, or 28,000 L-type channels per typical ventricular myocyte.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Agonist-induced [Ca2+]i waves and Ca(2+)-induced Ca2+ release in mammalian vascular smooth muscle cells.

Focal application of vasopressin to cultured vascular smooth muscle cells (A7r5 cells) elicits first a localized increase of intracellular Ca2+ concentration ([Ca2+]i) and then a wave of elevated [Ca2+]i that propagates at constant velocity throughout the cell. The cellular mechanisms of such complex spatiotemporal patterns of [Ca2+]i are of interest because they are involved fundamentally in cellular signal transduction in many types of cells. Vasopressin evoked a [Ca2+]i transient even in the absence of extracellular Ca2+, and intracellular perfusion with heparin completely blocked the response to vasopressin stimulation. Therefore the initial response to vasopressin reflects release of Ca2+ from an intracellular myo-inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ store. We tested four hypotheses on how a localized increase in [Ca2+]i propagates as a [Ca2+]i wave throughout the entire cell: the hypotheses distinguished 1) whether IP3 or Ca2+ is the primary intracellular messenger that diffuses, and 2) whether positive feedback on the release of intracellular Ca2+ (Ca2+i) is involved (further release of Ca2+ through activation of phospholipase C by Ca2+ and increased production of IP3 or by Ca(2+)-induced Ca2+ release). The results of various experimental interventions, which included probing Ca2+i stores (heparin, caffeine, and ryanodine), were compared with predictions from mathematical models for intracellular diffusion, release, and uptake of Ca2+. We conclude that in A7r5 smooth muscle cells, which have been stimulated focally with vasopressin, Ca2+ is released initially by IP3. The localized increase in [Ca2+]i then propagates throughout the cell as a [Ca2+]i wave. Ca2+ activates its own release, through Ca(2+)-induced release of Ca2+, by diffusing to distant Ca(2+)-release sites.

Animals↗
PubMed1992

Spontaneous synchronous synaptic calcium transients in cultured cortical neurons.

The firing pattern displayed by neuronal aggregates is thought to play a key role in cortical development and physiology. In this study, we have employed optical recording of intracellular calcium to monitor activity of multiple neurons simultaneously in primary cortical cultures. With this approach, we have observed spontaneous synchronous calcium transients among adjacent cortical neurons. These transients appear to be mediated by prominent spontaneous synaptic excitation, as they are enhanced by picrotoxin, a blocker of inhibitory GABAergic transmission, and reduced by antagonism of glutamate receptors or addition of TTX. After picrotoxin treatment, the calcium transients exhibit regular frequency and amplitude, and occur in synchrony with bursts of excitatory synaptic potentials every 10-20 sec. Using electrical stimulation, we have identified a relative refractory period, extending up to 5 sec after a synchronous burst, that may play a role in cell synchronization. NMDA receptor antagonists or reduced extracellular calcium levels lower the amplitude of the calcium transients yet fail to alter their frequency, suggesting that intracellular calcium levels may not be a major determinant of burst frequency. In contrast, mild depolarization with kainic acid (0.5-1 microM) increased burst frequency up to fivefold, suggesting a critical dependence of rhythmic activity on membrane potential. Chronic blockade of electrical activity with TTX beginning a few days after plating of cultures dampens the amplitude and significantly increases the frequency of calcium transients in mature cultures. These studies demonstrate that aggregates of cultured cortical neurons express synchronous firing activity in vitro and that this network activity is dependent in part on neuronal firing during development.

2-Amino-5-phosphonovalerate↗
PubMed1991

Flux of Ca2+ across the sarcoplasmic reticulum of guinea-pig cardiac cells during excitation-contraction coupling.

1. A method has been developed for calculating the flux of Ca2+ across the sarcoplasmic reticulum (SR) during excitation-contraction coupling in mammalian heart cells. FSR will symbolize the net rate of movement of Ca2+, per litre of accessible cytoplasm, into or out of the sarcoplasmic reticulum. FSR has the units MS-1. 2. A theory of the cytoplasmic [Ca2+]i transient in mammalian heart cells is presented in which the [Ca2+]i transient results from the various cellular processes that tend to increase or decrease cytoplasmic [Ca2+]i. According to the theory, FSR can be calculated if all cellular processes that contribute to the [Ca2+]i transient (other than Ca2+ fluxes across the SR) are either eliminated or are known quantitatively. 3. To obtain the measurements required to apply this theory, [Ca2+]i transients and membrane currents were recorded in guinea-pig single ventricular myocytes subjected to whole-cell voltage clamp and internal perfusion. [Ca2+]i transients were recorded through the use of the Ca2+ indicator, Fura-2 (pentapotassium salt). 4. Ca2+ fluxes through the sodium-calcium exchanger were eliminated in all experiments, by perfusing the cells, internally and externally with Na(+)-free solutions. Ca2+ flux through the sarcolemmal L-type Ca2+ channel was measured as the verapamil-sensitive current. Influx of Ca2+ through all other voltage-dependent pathways was found to be negligible for the calculation of FSR over the time course of a single [Ca2+]i transient. 5. In the combined absence of Ca2+ current, Na(+)-Ca2+ exchange and fluxes across the SR (10 mM-caffeine), the net rate of removal of Ca2+ from the cytoplasm, which includes presumed contributions from sarcolemmal Ca(2+)-ATPase and mitochondrial Ca2+ transport, was found to be a negligible quantity in the calculation of FSR, over the time course of a single [Ca2+]i transient. 6. Calculation of FSR requires that the Ca(2+)-binding capacity of cytoplasm be known. [Ca2+]i transients recorded during measurable total Ca2+ influx into the cytoplasm (verapamil-sensitive current in the absence of fluxes across the SR) were compared with theoretical Ca2+ transients computed on the assumption that the entering Ca2+ could bind only to intracellular ligands (values for ligands taken from literature) and to Fura-2 (30 microM). The slope of the regression line relating calculated total change in [Ca2+]i to the measured total Ca2+ influx was 0.99, not different from the perfect theoretical slope of 1.0 (correlation coefficient, 0.81; standard deviation of slope, 0.14; n = 7).4+ the SR and FSR had a similar time course to that on depolarization. 10. The unidirectional efflux of Ca2+ from the SR, symbolized FSR, rel was calculated utilizing assumed characteristics of the Ca2+ pump of the SR. The value of FSR, rel was not affected by repolarization from voltage-clamp pulses greater than 150 ms in duration.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1991

Ryanodine does not affect calcium current in guinea pig ventricular myocytes in which Ca2+ is buffered.

Calcium current in mammalian ventricular muscle is altered in the presence of ryanodine. Previous studies performed on rat ventricular cells have shown a slowing of Ca2+ current inactivation and suggest the hypothesis that ryanodine, by reducing the release of Ca2+ from the sarcoplasmic reticulum, reduces the availability of Ca2+ for inactivation of Ca2+ current (Ca(2+)-dependent inactivation). Another hypothesis is that the effects of ryanodine on Ca2+ current are due to a mechanical connection of the ryanodine receptor with the L-type Ca2+ channel. To further test these hypotheses we examined the effect of ryanodine on Ca2+ current in single voltage-clamped guinea pig ventricular myocytes that contained Ca2+ indicator and Ca2+ buffer. We used fura 2 (pentapotassium salt) to confirm that the ryanodine we used was capable of abolishing Ca2+ release from the sarcoplasmic reticulum during the period in which it was present. We perfused the cells with 10 mM EGTA to block changes in intracellular Ca2+ concentration. In the absence of internal EGTA, Ca2+ currents displayed biexponential inactivation and Ca(2+)-dependent inactivation (steady-state inactivation curves turned up at positive potentials). Inactivation was slowed by ryanodine at 10 microM. In cells perfused internally with EGTA, however, ryanodine had no effects, and steady-state inactivation curves were not shifted to the right. We conclude that, in guinea pig ventricular myocytes, the effects of ryanodine on Ca2+ current are mediated by Ca2+ and thus the effects of ryanodine do not provide a basis on which to postulate a physical connection between the L-type Ca2+ channel and the ryanodine receptor (sarcoplasmic reticulum Ca2+ release channel).

Animals↗
PubMed1991

Intracellular calcium during excitation-contraction coupling in mammalian ventricle.

A rapid, transient, rise in cytoplasmic free calcium ion concentration ([Ca2+]i-transient) couples electrical excitation to contraction in muscle. Such [Ca2+]i-transients in muscle are actually subcellular spatio-temporal events that are determined dynamically by i) diffusional fluxes of Ca2+, ii) by the binding or unbinding of Ca2+ to ligands such as troponin c and calmodulin, and iii) by the various cellular processes, such as release of Ca2+ from sarcoplasmic reticulum, that produce fluxes of Ca2+ across the membranes bounding organelles or the cell. In heart muscle, a particularly large number of cellular processes contribute to the cytoplasmic [Ca2+]i-transient, compared with skeletal muscle. In addition, the actual change in cytoplasmic free [Ca2+]i is now known to be a small fraction of the total [Ca] transient (free plus bound) because most (98 to 99%) of the Ca2+ that enters the cytoplasm binds to ligands. In this article it is shown that under most physiological conditions the SR is the major determinant of the [Ca2+]i-transient in heart, that release of Ca2+ from the SR is induced by Ca2+ entering via L-type Ca(2+)-channels, and that the Na/Ca exchanger is the major route by which Ca2+ leaves the cell. The precise quantitative contribution of all these processes to the [Ca2+]i-transient still remains to be determined, however.

Animals↗
PubMed1990

Intracellular diffusion, binding, and compartmentalization of the fluorescent calcium indicators indo-1 and fura-2.

We studied intracellular binding and possible compartmentalization of the fluorescent Ca2+ indicators, indo-1 and fura-2, in single mammalian cardiac ventricular cells that had been loaded with indo-1 and fura-2 by exposure to the acetoxymethylester form of the indicators (indo-1/AM and fura-2/AM). Techniques similar to those used in experiments on fluorescence recovery after photobleaching (FRAP) were used. It was assumed that reversible binding in myoplasm would be evident as slowed recovery of fluorescence after photobleaching, and that irreversible binding of the indicators to immobile myoplasmic sites (or "compartmentalization" in organelles) would be evident as incomplete recovery. Through the use of a mask, one half of a cell was exposed to high-intensity ultraviolet (UV) light to bleach the indo-1 or fura-2 in only that part of the cell. Upon removal of the mask and termination of the high-intensity UV illumination, fluorescence recovered in the bleached half of the cell, indicating diffusion of indo-1 and fura-2. Mathematical modeling of the diffusional redistribution of the indicators indicated that in these cells the apparent diffusion coefficient for indo-1 is 1.57 x 10(-7) cm2 s-1 (SD 0.48 x 10(-7) cm2 s-1; n = 5 cells, 21 degrees C), and for fura-2 is 3.19 x 10(-7) cm2 s-1 (SD 1.85 x 10(-7) cm2 s-1; n = 6 cells, 21 degrees C). These values are approximately 6 and 3, respectively, times smaller than those expected for free diffusion in the myoplasm. In the bleached half of the cell the recovered level of fluorescence never reached the final level in the half not exposed to UV light. The extent of incomplete recovery was variable amongst the cells. Our analysis indicated that, under the conditions we used, approximately one-third of the intracellular dye is not diffusible in the myoplasm.

Animals↗
PubMed1990

Calcium waves in mammalian heart: quantification of origin, magnitude, waveform, and velocity.

A dual, digital, indo-1 fluorescence imaging system was used to obtain high-speed ratiometric images of [Ca2+]i waves in single voltage-clamped mammalian cardiac cells. The spatiotemporal origin of [Ca2+]i waves in depolarized cells was detected as the spontaneous appearance, over 100-300 ms, of domelike regions of elevated [Ca2+]i, approximately 20 microns in diameter and 300 nM at the center. Images of [Ca2+]i taken at 67-ms intervals during propagation of [Ca2+]i waves revealed that the [Ca2+]i wave front was 1) constant in shape, 2) spatially steep, typically rising from 500 to 1200 nM in about 10 microns, and 3) propagating at constant velocity, typically 100 microns/s at 22 degrees C. The observed spatial and temporal patterns of origin and propagation of [Ca2+]i waves are consistent with the hypothesis that [Ca2+]i waves arise from propagating Ca2(+)-induced release of Ca2+ mediated by diffusion of cytosolic Ca2+. The [Ca2+]i waves are smaller in peak magnitude and can occupy a larger fraction of the cell than thought previously on the basis of indirect observations.

Animals↗
PubMed1990

Cytoplasmic [Ca2+] in mammalian ventricle: dynamic control by cellular processes.

A quantitative reconstruction of [Ca2+]i transients is the desired goal, but that goal has yet to be reached. It will be reached by solving Equation 2., once an adequate mathematical description of all its terms is obtained. If computed [Ca2+]i transients match closely those recorded under many experimental conditions, then we can be confident that our understanding of the cellular processes that control [Ca2+]i is correct. The SL Ca2+ ATPase and the SL Ca2+ leak do not make an important contribution on a given beat. All the available data, physiologic and biochemical, indicate clearly that the Ca2+ fluxes via the SL Ca2(+)-ATPase and SL Ca2(+)-leak pathways are small in comparison to others. Over many beats, however, the fluxes through these pathways will contribute to loading of the SR with Ca2+. In the abnormal case of resting cardiac muscle, [Ca2+]i will be determined by the balance between Ca2+ influx via leak and Ca2+ efflux via Na/Ca exchange and SL Ca2+ ATPase. There is an emerging consensus that the amount of Ca2+ entering via Na/Ca exchange during normal activity is small. This consensus derives from direct observation of changes in [Ca2+]i attributable to Na/Ca exchange, from computations that utilize new quantitative data on the current-voltage relation of the exchanger and on the quantitative relationship of exchanger current to [Ca2+]i. Clearly, the efflux of Ca2+ via Na/Ca exchange on each beat is significant. From theory and the fact that SL Ca2+ pumping is small, the efflux of Ca2+ via the exchanger must equal the Ca2+ influx through SL Ca2+ channels, but experimental studies have not yet verified this quantitatively. All the studies, recent and older, indicate that the [Ca2+]i transient in all mammalian species is dominated by Ca2+ released from SR. Even in the rat, widely believed to be the species most dependent on SR, the Ca2+ current contributes measurable Ca2+ (24). Provided that the SR is not depleted by rest, it is the major cellular entity that determines the [Ca2+]i transient in mammalian ventricular tissue on a given beat. Quantitative knowledge of the flux of Ca2+ from it, required for evaluating theories of excitation-contraction coupling, still awaits determination.

Animals↗
PubMed1989

Sodium-calcium exchange in mammalian heart: current-voltage relation and intracellular calcium concentration.

Membrane currents and changes in intracellular calcium ion concentration ([Ca2+]i) have been recorded that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Single guinea-pig ventricular myocytes under voltage clamp were perfused internally with the fluorescent Ca2+-indicator, fura-2, and changes in [Ca2+]i and membrane current that resulted from Na-Ca exchange were isolated through the use of various organic channel blockers (verapamil, TTX), impermeant ions (Cs+, Ni2+), and inhibitors of sarcoplasmic reticulum (ryanodine). The I-V relation of Na-Ca exchange was obtained from the Ni2+-sensitive current elicited by ramp repolarization from +90 mV to -80 mV. Ramps were sufficiently rapid that little change in [Ca2+]i occurred during the ramp. The (constant) [Ca2+]i during the ramp was varied over the range 100 nM to 1000 nM by varying the amplitude and duration of a pre-pulse to the ramp. The reversal potential of the Ni2+-sensitive ramp current varied linearly with 1n[( Ca2+])i. The I-V relations at different [Ca2+]i over the range -60 mV to +140 mV were in reasonable accord with the predictions of a simple, simultaneous scheme of Na-Ca exchange, on the basis that only [Ca2+]i had changed. The relationship between [Ca2+]i and current at a constant membrane voltage was also in accord with this scheme. We suggest that Ca2+-fluxes through the exchanger during the cardiac action potential can be understood quantitatively by considering the binding of Ca2+ to the exchanger during the [Ca2+]i-transient and the effects of membrane voltage on the exchanger.

Animals↗
PubMed1989

Sodium-calcium exchange in guinea-pig cardiac cells: exchange current and changes in intracellular Ca2+.

1. Membrane currents and changes in [Ca2+]i attributable to the operation of an electrogenic Na-Ca exchange mechanism were recorded in single isolated guinea-pig ventricular myocytes under voltage clamp and internal perfusion with the Ca2+ indicator Fura-2. 2. Ionic currents that interfere with the measurement of Na-Ca exchange current were blocked through the use of caesium (Cs+), verapamil and tetrodotoxin (TTX). Entry of Ca2+ through surface membrane Ca2+ channels and release of Ca2+ from sarcoplasmic reticulum were blocked with verapamil and ryanodine, respectively. 3. In the presence of the blockers listed above, depolarization to positive membrane potentials elicited slow increases in [Ca2+]i and, after an instantaneous increase, a declining outward current. Repolarization elicited a decline in [Ca2+]i and, after an instantaneous increase, a declining inward current. The changes in [Ca2+]i and a major component of the current were abolished by nickel ions (Ni2+; 5 mM). 4. The reversal potential of the current abolished by Ni2+ (Ni2+-sensitive current) was determined at different levels of [Ca2+]i by ramp repolarizations from +80 to -80 mV (1-5 mV/ms). The reversal potential of the current increased linearly with log [Ca2+]i. As a result of the foregoing and other data, the Ni2+-sensitive current was taken to be Na-Ca exchange current (INaCa). 5. The relation between INaCa and [Ca2+]i (less than 1 microM) at constant voltage over the range of -80 to +60 mV was approximately linear. No evidence of saturation could be found; small deviations from linearity at high [Ca2+]i were in the direction expected for a minor contribution from Ca2+-activated non-specific cation current (Ehara, Noma & Ono, 1988). 6. When measured at the same [Ca2+]i, the peak INaCa upon repolarization to -80 to -140 mV seemed to approach a limiting value at very negative potentials. 7. Over the range of +40 to +160 mV INaCa (measured soon after depolarization and thus at the same [Ca2+]i) increased exponentially with clamp-pulse potential. These pulses (to potentials up to +160 mV) elicited a slow rise in [Ca2+]i with the peak at the end of the pulse also increasing exponentially with pulse potential. 8. Inward membrane currents with properties similar to those described above were also recorded in association with physiological [Ca2+]i transients, when Ca2+ channels and the sarcoplasmic reticulum were not blocked. 9. Some of the results are not consistent with certain predictions of a sequential step model, or with those of a simultaneous step model in which the internal binding site for Ca2+ is saturated, or with those of a model based only on thermodynamics.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Effects of activation on distribution of Ca2+ in single arterial smooth muscle cells. Determination with fura-2 digital imaging microscopy.

A rise in cytosolic free Ca2+ is the immediate trigger for contraction in mammalian vascular smooth muscle. We used the fluorescent calcium indicator fura-2 and digital imaging microscopy to study the spatial distribution of intracellular Ca2+ in arterial myocytes and the changes elicited by activation with norepinephrine (NE). Viable arterial myocytes were obtained from bovine tail arteries by enzymatic digestion. In modified Krebs' solution containing 1.8 mM Ca2+, these myocytes were relaxed and spindle-shaped. The cells contracted rapidly when exposed to NE or high-K+ solution ejected from a micropipette; they relaxed slowly when the activator was washed away. NE evoked a rise in Ca2+ concentration ([Ca2+]) in the cells within 100 msec, at a time when the cells had not yet begun to contract. Maximal [Ca2+] levels were attained within 600 msec, at which time the cells were substantially contracted. Digital analysis of images of cellular fura-2 fluorescence revealed that the intracellular [Ca2+] was relatively uniformly distributed prior to activation, with an average resting level of 111 +/- 14 nM (n = 6). During NE-evoked contractions, intracellular [Ca2+] increased, and the distribution of [Ca2+] became much more heterogeneous. On recovery from activation, the cells relaxed, usually attaining less than 90% of their original resting length. In contrast to the relatively uniform Ca2+ distribution observed prior to NE activation, discrete regions of elevated [Ca2+] were observed throughout the recovered cells. The large spatial variation of [Ca2+] after cell activation implies that Ca2+ was sequestered at localized sites in the cell during relaxation.

Animals↗
PubMed1988

Mechanism of release of calcium from sarcoplasmic reticulum of guinea-pig cardiac cells.

1. The mechanisms that control release of Ca2+ from the sarcoplasmic reticulum (SR) of guinea-pig ventricular cells were studied by observing intracellular calcium concentration ([Ca2+]i transients) and membrane currents in voltage-clamped guinea-pig ventricular myocytes perfused internally with Fura-2. 2. Sarcolemmal Ca2+ current was identified through the use of tetrodotoxin (TTX) and Ca2+ channel antagonists (verapamil) and agonists (Bay K 8644). 3. Changes in [Ca2+]i attributable to release of Ca2+ from the SR were identified through the use of ryanodine, which abolishes the ability of the SR to release Ca2+. Ryanodine-sensitive increases in [Ca2+]i could be elicited either by depolarization or by repolarization (from depolarizing pulses to relatively positive membrane potentials). 4. At appropriate voltages, it is the initial fast change in [Ca2+]i elicited by either depolarization or repolarization that is abolished by ryanodine, and is defined here as ryanodine sensitive. 5. The amplitude of the ryanodine-sensitive [Ca2+]i transient elicited by depolarization had a bell-shaped dependence on membrane potential with a maximum of about 500 nM at 10 mV, and with the upper minimum between 60 and 70 mV. Verapamil-sensitive current activated over approximately the same potential range as the [Ca2+]i transient, with a peak amplitude at 10 mV, and a reversal potential of 65 mV. 6. When a holding potential of -68 mV and TTX (30 microM) were used, the most negative pulse potential at which activation of an inward current occurred was -49 mV while changes in [Ca2+]i occurred at -43 mV. 7. Ryanodine-sensitive increases in [Ca2+]i elicited by repolarization (tail transients) were maximal for repolarization to 0 mV. Smaller changes in [Ca2+]i than maximal were elicited by repolarization to both more positive and more negative potentials than 0 mV. The peak amplitude of the verapamil-sensitive tail currents elicited by repolarization increased continuously as the membrane was repolarized to potentials more negative than 60 mV. 8. Increasing depolarizing pulse duration beyond 10-20 ms did not increase the amplitude of the [Ca2+]i transient, but prolonged it. 9. The experimental results are compared to the predictions of two theories on the mechanism of excitation-contraction coupling: Ca2+-induced release of Ca2+ (CICR), as it has been formulated from data in skinned cardiac cells, and a charge-coupled release mechanism (CCRM), as it has been formulated to explain excitation-contraction coupling in skeletal muscle. 10. Some of the results are clearly not consistent with certain features of a charge-coupled release mechanism.(ABSTRACT TRUNCATED AT 400 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
PubMed1988

[Ca2+]i in single isolated cardiac cells: a review of recent results obtained with digital imaging microscopy and fura-2.

The use of fluorescent Ca2+ indicators to observe [Ca2+]i transients in voltage-clamped single cells has many advantages over previous methods, such as the use of aequorin in multicellular preparations, for studying excitation-contraction coupling. In the studies reviewed in this article, [Ca2+]i in single isolated mammalian ventricular myocytes was observed through the use of the fluorescent Ca2+ indicator, fura-2. Individual cells, loaded with fura-2 either by internal perfusion or by exposure to fura-2/AM, were generally studied with the use of inverted microscopes equipped with ultraviolet epifluorescence illumination, intensified silicon intensifier target cameras (ISIT), and (or) a photomultiplier tube. Analysis of subcellular patterns of fura-2 fluorescence was performed by digital analysis of the images obtained with the ISIT camera. Variation of membrane voltage and exposure of cells to ryanodine (which was assumed to selectively block the release of Ca2+ from the sarcoplasmic reticulum) were used to investigate the cellular processes that determine the [Ca2+]i transient. The main results of these studies are the following. (1) In any population of enzymatically isolated heart cells, there are (i) mechanically quiescent cells in which [Ca2+]i is spatially uniform, constant over time, and relatively low; (ii) spontaneously contracting cells, which have a relatively elevated [Ca2+]i, but in which the spatial uniformity of [Ca2+]i is interrupted periodically by spontaneous, propagating waves of high [Ca2+]i; and (iii) cells that are hypercontracted (rounded up) and that have higher levels of [Ca2+]i than the other two types.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Sodium-calcium exchange in heart: membrane currents and changes in [Ca2+]i.

Recordings have been made of changes in intracellular calcium ion concentration ([Ca2+]i) that can be attributed to the operation of an electrogenic, voltage-dependent sodium-calcium (Na-Ca) exchanger in mammalian heart cells. Guinea pig ventricular myocytes under voltage clamp were perfused internally with fura-2, a fluorescent Ca2+-indicator, and changes in [Ca2+]i and membrane current that resulted from Na-Ca exchange were identified through the use of various organic channel blockers and impermeant ions. Depolarization of cells elicited slow increases in [Ca2+]i, with the maximum increase depending on internal [Na+], external [Ca2+], and membrane voltage. Repolarization was associated with net Ca2+ efflux and a decline in the inward current that developed instantaneously upon repolarization. The relation between [Ca2+]i and current was linear, and the slope was made steeper by hyperpolarization.

Animals↗
PubMed1987

Cellular and subcellular heterogeneity of [Ca2+]i in single heart cells revealed by fura-2.

Digital imaging of calcium indicator signals (fura-2 fluorescence) from single cardiac cells has revealed different subcellular patterns of cytoplasmic calcium ion concentration ([Ca2+]i) that are associated with different types of cellular appearance and behavior. In any population of enzymatically isolated rat heart cells, there are mechanically quiescent cells in which [Ca2+]i is spatially uniform, constant over time, and relatively low; spontaneously contracting cells, which have an increased [Ca2+]i, but in which the spatial uniformity of [Ca2+]i is interrupted periodically by spontaneous propagating waves of high [Ca2+]i; and cells that are hypercontracted (rounded up) and that have higher levels of [Ca2+]i than the other two types. The observed cellular and subcellular heterogeneity of [Ca2+]i in isolated cells indicates that experiments performed on suspensions of cells should be interpreted with caution. The spontaneous [Ca2+]i fluctuations previously observed without spatial resolution in multicellular preparations may actually be inhomogeneous at the subcellular level.

Animals↗