Search PubMed⌕ Search

Biomedical subjects

D A Eisner

Publications and source records attributed to D A Eisner.

At least 37 records · Page 2Linked to original sources
PubMed2001

Altered cardiac sarcoplasmic reticulum function of intact myocytes of rat ventricle during metabolic inhibition.

Changes in the behavior of the sarcoplasmic reticulum (SR) in rat ventricular myocytes were investigated under conditions of metabolic inhibition using laser-scanning confocal microscopy to measure intracellular Ca(2+) and the perforated patch-clamp technique to measure SR Ca(2+) content. Metabolic inhibition had several effects on SR function, including reduced frequency of spontaneous releases of Ca(2+) (sparks and waves of Ca(2+)-induced Ca(2+) release), increased SR Ca(2+) content (79.4+/-5.7 to 115.2+/-6.6 micromol/L cell volume [mean+/-SEM; P:<0.001]), and, after a wave of Ca(2+) release, slower reuptake of Ca(2+) into the SR (rate constant of fall of Ca(2+) reduced from 8.5+/-1.1 s(-)(1) in control to 5.2+/-0.4 s(-)(1) in metabolic inhibition [P:<0.01]). Inhibition of L-type Ca(2+) channels with Cd(2+) (100 micromol/L) did not reproduce the effects of metabolic inhibition on spontaneous Ca(2+) sparks. These results are evidence of inhibition of both Ca(2+) release and reuptake mechanisms. Reduced frequency of release could be attributable to either of these effects, but the increased SR Ca(2+) content at the time of reduced frequency of spontaneous release of Ca(2+) shows that the dominant effect of metabolic inhibition is to inhibit release of Ca(2+) from the SR, allowing the accumulation of greater than normal amounts of Ca(2+). In the context of ischemia, this extra accumulation of Ca(2+) would present a risk of potentially arrhythmogenic, spontaneous release of Ca(2+) on reperfusion of the tissue.

Aniline Compounds↗
PubMed2001

Coordinated control of cell Ca(2+) loading and triggered release from the sarcoplasmic reticulum underlies the rapid inotropic response to increased L-type Ca(2+) current.

The aim of this study was to investigate how sarcoplasmic reticulum (SR) Ca(2+) content and systolic Ca(2+) are controlled when Ca(2+) entry into the cell is varied. Experiments were performed on voltage-clamped rat and ferret ventricular myocytes loaded with fluo-3 to measure intracellular Ca(2+) concentration ([Ca(2+)](i)). Increasing external Ca(2+) concentration ([Ca(2+)](o)) from 1 to 2 mmol/L increased the amplitude of the systolic Ca(2+) transient with no effect on SR Ca(2+) content. This constancy of SR content is shown to result because the larger Ca(2+) transient activates a larger Ca(2+) efflux from the cell that balances the increased influx. Decreasing [Ca(2+)](o) to 0.2 mmol/L decreased systolic Ca(2+) but produced a small increase of SR Ca(2+) content. This increase of SR Ca(2+) content is due to a decreased release of Ca(2+) from the SR resulting in decreased loss of Ca(2+) from the cell. An increase of [Ca(2+)](o) has two effects: (1) increasing the fraction of SR Ca(2+) content, which is released on depolarization and (2) increasing Ca(2+) entry into the cell. The results of this study show that the combination of these effects results in rapid changes in the amplitude of the systolic Ca(2+) transient. In support of this, the changes of amplitude of the transient occur more quickly following changes of [Ca(2+)](o) than following refilling of the SR after depletion with caffeine. We conclude that the coordinated control of increased Ca(2+) entry and greater fractional release of Ca(2+) is an important factor in regulating excitation-contraction coupling.

4-Aminopyridine↗
PubMed2001

The role of intracellular Ca buffers in determining the shape of the systolic Ca transient in cardiac ventricular myocytes.

We have studied the kinetics of decay of cardiac Ca transients elicited by either caffeine or electrical stimulation. The decay of the caffeine-evoked increase of intracellular Ca concentration ([Ca2+]i) could not be fit by a single exponential. A two exponential fit showed an initial rapid component. The rate of decay of total Ca was calculated from measured free Ca and the buffering properties of the cell. There was no initial rapid phase of decay of total Ca. We conclude that the rapid phase of decay of free Ca is due to a decrease of Ca buffering power at elevated [Ca2+]i. In contrast, the decay of the Ca transient, produced by voltage-clamp depolarization or field stimulation was fit by a single exponential. We suggest that these apparently simple kinetics arise because a tendency to saturation at elevated [Ca2+]i of the sarcoplasmic reticulum Ca-ATPase offsets the decrease of Ca buffering power. These data show the importance of Ca buffers as well as transporters in determining the kinetics of changes of [Ca2+]i.

Aniline Compounds↗
PubMed2001

The effects of exogenous calcium buffers on the systolic calcium transient in rat ventricular myocytes.

The aim of this work was to characterize the effects that two commonly used "caged" calcium buffers (NP-EGTA and nitr-5) have on the amplitude and time course of decay of the calcium transient. We made quantitative measurements of both free and total calcium using the measured buffering properties of the cell. Intracellular calcium concentration ([Ca(2+)](i)) was measured with fluo-3 in rat ventricular myocytes. Incorporation of the buffer NP-EGTA decreased both the amplitude and rate of decay of the caffeine response. The slowing could be quantitatively accounted for by the measured increased buffering. These effects were removed by photolysis of NP-EGTA. Similar results were obtained with nitr-5 except that the effects were not completely removed by photolysis. This was shown to be due to the persistence of a component of the increased buffering after photolysis. Both buffers decreased the amplitude of the systolic calcium transient. However, although nitr-5 produced a simple slowing of the decay, NP-EGTA resulted in an initial rapid phase of decay. This rapid phase of decay is attributed to calcium binding to NP-EGTA. This work represents the first quantitative analysis of the effects that extra buffering by a fast and a slow calcium chelator may have on the calcium transient.

Aniline Compounds↗
PubMed2000

The effect of acidosis on systolic Ca2+ and sarcoplasmic reticulum calcium content in isolated rat ventricular myocytes.

We have investigated the mechanisms responsible for the changes of systolic Ca2+ that occur in voltage-clamped rat ventricular myocytes during acidosis produced by application of the weak acid butyrate (30 mM). Intracellular pH regulation was inhibited with dimethylamiloride (bicarbonate-free solution). The application of butyrate produced an intracellular acidification of 0.33 pH units. This was accompanied by a decrease in systolic Ca2+ to about 50% of control. However, within 2 min, systolic Ca2+ returned to control levels. The decrease in systolic Ca2+ was accompanied by a decrease in the Na+-Ca2+ exchange current observed on repolarisation so that the calculated Ca2+ efflux on Na+-Ca2+ exchange was less than the entry on the L-type Ca2+ current. The magnitude of the Na+-Ca2+ exchange current recovered along with systolic Ca2+ until it equalled the Ca2+ entry on the L-type Ca2+ current. From the measurement of Ca2+ fluxes, it was calculated that, during acidosis, the cell gains 121.6+/-16.2 micromol l(-1) of Ca2+. This is equal to the measured increase of sarcoplasmic reticulum (SR) calcium content obtained by applying caffeine (20 mM) and integrating the resulting Na+-Ca2+ exchange current. We conclude that the recovery of the amplitude of the systolic Ca2+ transient is due to decreased SR calcium release, resulting in reduced Ca2+ efflux from the cell leading to increased SR calcium content.

Acidosis↗
PubMed2000

Integrative analysis of calcium cycling in cardiac muscle.

The control of intracellular calcium is central to regulation of contractile force in cardiac muscle. This review illustrates how analysis of the control of calcium requires an integrated approach in which several systems are considered. Thus, the calcium content of the sarcoplasmic reticulum (SR) is a major determinant of the amount of Ca(2+) released from the SR and the amplitude of the Ca(2+) transient. The amplitude of the transient, in turn, controls Ca(2+) fluxes across the sarcolemma and thence SR content. This control of SR content influences the response to maneuvers that modify, for example, the properties of the SR Ca(2+) release channel or ryanodine receptor. Specifically, modulation of the open probability of the ryanodine receptor produces only transient effects on the Ca(2+) transient as a result of changes of SR content. These interactions between various Ca(2+) fluxes are modified by the Ca(2+) buffering properties of the cell. Finally, we predict that, under some conditions, the above interactions can result in instability (such as alternans) rather than ordered control of contractility.

Biological Transport↗
PubMed2000

Modulation of CICR has no maintained effect on systolic Ca2+: simultaneous measurements of sarcoplasmic reticulum and sarcolemmal Ca2+ fluxes in rat ventricular myocytes.

1. The effects of modulating Ca2+-induced Ca2+ release (CICR) in single cardiac myocytes were investigated using low concentrations of caffeine (< 500 microM) in reduced external Ca2+ (0.5 mM). Caffeine produced a transient potentiation of systolic [Ca2+]i (to 800 % of control) which decayed back to control levels. 2. Caffeine decreased the steady-state sarcoplasmic reticulum (SR) Ca2+ content. As the concentration of caffeine was increased, both the potentiation of the systolic Ca2+ transient and the decrease in SR Ca2+ content were increased. At higher concentrations, the potentiating effect decayed more rapidly but the rate of recovery on removal of caffeine was unaffected. 3. A simple model in which caffeine produces a fixed increase in the fraction of SR Ca2+ which is released could account qualitatively but not quantitatively for the above results. 4. The changes in total [Ca2+] during systole were obtained using measurements of the intracellular Ca2+ buffering power. Caffeine initially increased the fractional release of SR Ca2+. This was followed by a decrease to a level greater than that under control conditions. The fraction of systolic Ca2+ which was pumped out of the cell increased abruptly upon caffeine application but then recovered back to control levels. The increase in fractional loss is due to the fact that, as the cytoplasmic buffers become saturated, a given increase in systolic total [Ca2+] produces a larger increase in free [Ca2+] and thence of Ca2+ efflux. 5. These results confirm that modulation of the ryanodine receptor has no maintained effect on systolic Ca2+ and show the interdependence of SR Ca2+ content, cytoplasmic Ca2+ buffering and sarcolemmal Ca2+ fluxes. Such analysis is important for understanding the cellular basis of inotropic interventions in cardiac muscle.

Algorithms↗
PubMed2000

Measurement of calcium entry and exit in quiescent rat ventricular myocytes.

The aim of this work was to obtain the first quantitative measurements of Ca2+ influx and efflux in quiescent cardiac cells. The relationship between free and total Ca2+ was obtained during a caffeine application. This buffering curve was then used to calculate changes of total Ca2+ from measurements of free cytosolic [Ca2+] ([Ca2+]i) made with Indo-1. The rate of Ca2+ removal from the cytoplasm was calculated by differentiating total Ca2+ with respect to time. The dependence of d(total Ca2+)/dt on [Ca2+]i was hyperbolic. Inhibition of either Na+-Ca2+ exchange (by addition of 10 mmol l(-1) NiCl2 or removal of external Na+) or the sarcolemmal Ca2+-activated adenosine triphosphatase (Ca2+-ATPase) (with carboxyeosin) decreased the calculated efflux. In both cases, the main effect was on the apparent maximum rate (Vmax) with little effect on the Michaelis-Menten constant (Km). These results suggest that the Na+-Ca2+ exchange and Ca2+-ATPase have very similar affinities for [Ca2+]i and that their fractional contributions do not change over the systolic range of [Ca2+]i. Ca2+ influx was quantified in two ways. The first method was to extrapolate the curve relating Ca2+ efflux to [Ca2+]i to zero [Ca2+]i. This gave a value of 4.49+/-0.54 micromol l(-1) s(-1) which was reduced to zero by either removal of external Ca2+ or addition of Ni2+. In other experiments external Ca2+ was removed and the maximum rate of fall of total Ca2+ calculated as 2.53+/-0.93 micromol l(-1) s(-1). This approach can be used to provide a quantitative analysis of the control of resting [Ca2+]i.

Animals↗
PubMed2000

Effects of mefloquine on cardiac contractility and electrical activity in vivo, in isolated cardiac preparations, and in single ventricular myocytes.

1. To examine the possible cardiotoxicity of the antimalarial drug mefloquine, increasing doses (0.3 - 30 mg kg(-1)) were given i.v. to anaesthetized guinea-pigs. Mefloquine did not alter ECG intervals significantly but gradually increased systolic blood pressure (at 3 mg kg(-1)) then had a depressor effect (at 10 mg kg(-1)). Death due to profound hypotension, probably resulting from cardiac contractile failure or AV block, occurred after either 10 mg kg(-1) (2/6) or 30 mg kg(-1) (4/6) mefloquine. 2. In isolated cardiac preparations mefloquine (3 - 100 microM) did not alter the effective refractory period but at the higher concentrations resting tension increased. Developed tension was reduced by 100 microM mefloquine in left atria (from 5.8+/-1.7 to 2.2+/-0.4 mN) whereas in papillary muscles although 30 microM mefloquine reduced developed tension (from 2. 6+/-0.5 to 1.1+/-0.1 mN) subsequent addition of 100 microM caused a marked, but not sustained, positive inotropic effect (from 1.2+/-0.1 to 3.8+/-0.8 mN). 3. In single ventricular myocytes, mefloquine (10 microM) shortened action potential duration (e.g. APD(90) from 285+/-29 to 141+/-12 ms) and reduced the amplitude of the systolic Ca(2+) transient. 4. These effects were accompanied by a decrease in the L-type Ca(2+) current. These results indicate that the main adverse effect of mefloquine on the heart is a negative inotropic action. This action can be explained by blockade of L-type Ca(2+) channels.

Animals↗
PubMed2000

The effects of low concentrations of caffeine on spontaneous Ca release in isolated rat ventricular myocytes.

We have investigated the effects on spontaneous SR Ca release of modulating the sarcoplasmic reticulum ryanodine receptor (RyR) with low (<0.5 mM) concentrations of caffeine. Experiments were performed on isolated rat ventricular myocytes. Intracellular Ca concentration was measured with Indo-1 or Fluo-3 in voltage-clamped cells. Spontaneous Ca release was produced by elevating external Ca to 5 mM. Caffeine application increased the frequency of spontaneous release. Both the magnitude of the spontaneous Ca transients and the integral of the resulting Na-Ca exchange current were decreased by caffeine. The combination of increased frequency of spontaneous release and decreased Ca efflux per event meant that the Ca efflux per unit time was unaffected by low concentrations of caffeine. The SR Ca content was reduced by caffeine. The extra Ca efflux calculated from the Na-Ca exchange current integrals occurring during the initial burst of spontaneous activity on application of caffeine accounted for this reduction of SR Ca content. In contrast to these maintained effects on spontaneous release, caffeine had only transient effects on stimulated Ca release produced by depolarizing pulses. We conclude that stimulation of the RyR results in spontaneous release at SR Ca contents lower than those at which release would normally occur. Therefore, the balance between normal and spontaneous Ca release can be shifted by modulation of the RyR. This will have important consequences for arrhythmogenesis due to spontaneous Ca release.

Animals↗
PubMed1999

The role of the sarcoplasmic reticulum as a Ca2+ sink in rat uterine smooth muscle cells.

1. The mechanisms responsible for removing calcium ions from the cytoplasm were investigated in single rat uterine myocytes using indo-1. 2. Trains of depolarizing voltage-clamp pulses increased [Ca2+]i. The rate of decay of [Ca2+]i was slowed by inhibition of the sarcoplasmic reticulum (SR) Ca2+-ATPase with cyclopiazonic acid (CPA). However, if the sarcolemmal Na+-Ca2+ exchanger and Ca2+-ATPase were inhibited then recovery of [Ca2+]i was abolished showing that the SR Ca2+-ATPase alone cannot produce decay of [Ca2+]i. 3. In another series of experiments, Ca2+ release from the SR was induced with carbachol in a Ca2+-free solution. Under these conditions responses to repeated applications of carbachol could be obtained. In the presence of CPA, however, only the first application was effective. This suggests that the SR Ca2+-ATPase sequesters a significant amount of Ca2+ into the SR. 4. CPA slowed the rate of decay of [Ca2+]i following carbachol addition by > 50 %. Again, however, after a brief transient fall, decay was abolished when the Na+-Ca2+ exchanger and sarcolemmal Ca2+-ATPase were inhibited. 5. These data show that, although the SR Ca2+-ATPase contributes to the decay of [Ca2+]i, it cannot function effectively in the absence of Ca2+ removal from the cell. These data are discussed in the context of the superficial buffer barrier model in which Ca2+ is taken up into the SR and then released very close to sarcolemmal Ca2+ extrusion sites, i.e. the SR acting in series with the surface membrane extrusion mechanisms. We also suggest that the amount of filling of the SR influences the rate of Ca2+ removal.

Animals↗
PubMed1999

The role of sarcolemmal Ca2+-ATPase in the regulation of resting calcium concentration in rat ventricular myocytes.

1. The aim of this work was to investigate the role of sarcolemmal Ca2+-ATPase in rat ventricular myocytes. We have measured intracellular Ca2+ concentration ([Ca2+]i) using indo-1. The actions of the ATPase inhibitor carboxyeosin were studied. 2. Carboxyeosin increased resting [Ca2+]i and the magnitude of the systolic Ca2+ transient and slowed the rate of its relaxation by 5 %. 3. Carboxyeosin increased the magnitude of the caffeine-evoked increase in [Ca2+]i and slowed its relaxation by 20 %. Removal of extracellular Na+ slowed the rate constant by 80 %. When Na+ was removed in a carboxyeosin-treated cell, the caffeine-evoked increase in [Ca2+]i did not decay. 4. Carboxyeosin increased the integral of the Na+-Ca2+ exchange current activated by caffeine. This is, in part, due to an increase in sarcoplasmic reticulum Ca2+ content. 5. When extracellular Na+ was removed, there was a transient increase in [Ca2+]i which then decayed. The rate of this decay was slowed by carboxyeosin by a factor of about four. The residual decay could be abolished with caffeine. 6. In the absence of extracellular Na+, increasing extracellular Ca2+ concentration ([Ca2+]o) elevated [Ca2+]i. In carboxyeosin-treated cells, [Ca2+]i was much more sensitive to [Ca2+]o. 7. These results demonstrate the role of a carboxyeosin-sensitive Ca2+-ATPase in the control of resting [Ca2+]i and the reduction in [Ca2+]i following an increase in [Ca2+]i.

Animals↗
PubMed1999

A novel, rapid and reversible method to measure Ca buffering and time-course of total sarcoplasmic reticulum Ca content in cardiac ventricular myocytes.

This paper outlines a simple method of estimating both the Ca-buffering properties of the cytoplasm and the time-course of changes of sarcoplasmic reticulum (s.r.) Ca concentration during systole. The experiments were performed on voltage-clamped ferret single ventricular myocytes loaded with the free acid of fluo-3 through a patch pipette. The application of caffeine (10 mM) resulted in a Na-Ca exchange current and a transient increase of the free intracellular Ca concentration ([Ca2+]i). The time-course of change of total Ca in the cell was obtained by integrating the current and this was compared with the measurements of [Ca2+]i to obtain a buffering curve. This could be fit with a maximum capacity for the intrinsic buffers of 114+/-18 micromol l-1 and Kd of 0.59+/-0.17 microM (n=8). During the systolic rise of [Ca2+]i, the measured changes of [Ca2+]i and the buffering curve were used to calculate the magnitude and time-course of the change of total cytoplasmic Ca and thence of both s.r. Ca content and Ca release flux. This method provides a simple and reversible mechanism to measure Ca buffering and the time-course of both total cytoplasmic and s.r. Ca.

Animals↗
PubMed1999

Strophanthidin-induced gain of Ca2+ occurs during diastole and not systole in guinea-pig ventricular myocytes.

We have investigated the effects of inhibiting the Na-K pump with strophanthidin on the intracellular Ca2+ concentration ([Ca2+]i), sarcoplasmic reticulum (s.r.) Ca2+ content and membrane currents. s. r. Ca2+ content was measured by integrating the Na-Ca exchange current resulting from application of 10 mM caffeine. The application of strophanthidin increased both diastolic and systolic [Ca2+]i. This was accompanied by an increase of s.r. Ca2+ content from a resting value of 17.9+/-1.5 micromol/l to 36.9+/-3.3 micromol/l (n=16) after 5 min. Systolic fluxes of Ca2+ into and out of the cell before and during strophanthidin application were also measured. Ca2+ efflux (measured as the integral of the Na-Ca exchange tail current) rose steadily in the presence of strophanthidin, while Ca2+ influx (the integral of the L-type Ca2+ current) was reduced. In spite of this, s.r. Ca2+ content rose substantially. In the presence of Cd2+ (100 microM), which inhibits the L-type Ca2+ current, strophanthidin had negligible effects on current suggesting that Ca2+ influx via Na-Ca exchange during depolarization does not account for the increase of s.r. Ca2+ content. This suggests that changes of Ca2+ flux during systole are not responsible for the strophanthidin-induced increase of s.r. Ca2+. We conclude that the primary mechanism by which the cardiac cell gains Ca2+ when the Na-K pump is inhibited is by a net influx during diastole.

Animals↗
PubMed1999

The effects of inhibition of the sarcolemmal Ca-ATPase on systolic calcium fluxes and intracellular calcium concentration in rat ventricular myocytes.

The effects of carboxyeosin, an inhibitor of the sarcolemmal Ca-ATPase, were studied on intracellular Ca and membrane currents in isolated rat ventricular myocytes. In the absence of carboxyeosin, 150-ms-duration depolarizing pulses from -40 to 0 mV resulted in an L-type Ca current on depolarization and a Na-Ca exchange "tail" current on repolarization. The calculated entry of Ca on the L-type current was 1.3 times greater than the efflux via the Na-Ca exchange. The addition of carboxyeosin (20 microM) resulted in either an increase of the Na-Ca exchange current or a decrease of the L-type Ca current such that the Ca entry and efflux were exactly equal. These results suggest that, under control conditions, a carboxyeosin-sensitive Ca-ATPase contributes about 24% of the total Ca efflux from the cell and, therefore, that the sarcolemmal Ca-ATPase has a significant role in regulation of sarcolemmal Ca fluxes.

Animals↗