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W J Lederer

Publications and source records attributed to W J Lederer.

160 records · Page 9Linked to original sources
PubMed1981

The dependence of sodium pumping and tension on intracellular sodium activity in voltage-clamped sheep Purkinje fibres.

1. Intracellular Na activity (aiNa) was measured in sheep cardiac Purkinje fibres using a recessed-tip Na+-sensitive micro-electrode. The membrane potentials was controlled with a two-micro-electrode voltage clamp. Tension was measured simultaneously. 2. Removing external K produced a rise of aiNa and both twitch and tonic tension. On adding 4-10 mM-[Rb]0 to reactivate the Na-K pump aiNa and tension declined. An electrogenic Na pump current transient accompanied the fall of aiNa. 3. The half-time of decay of the electrogenic Na pump current transient was similar to that of aiNa, (mean tNa0.5/tI0.5 = 0.97 +/- 0.03 (S.E.M.; n = 28)). Following the re-activation of the Na-K pump, the electrogenic Na pump current transient was linearly related to aiNa. 4. The duration of exposure to K-free, Rb-free solutions was varied to change the level of aiNa. On subsequently re-activating the Na-K pump with 10 mM-[Rb]0, the ratio of the charge extruded to the total change of aiNa was constant. It is concluded that the fraction of Na extruded electrogenically is unaffected by changes of aiNa. About 26% of the total Na extrusion appeared as charge transfer. 5. The relationship between tonic tension and aiNa was usually different during Na-K pump inhibition in a K-free, Rb-free solution compared with the relationship during Na-K pump re-activation. In general, a given aiNa was associated with a greater level of tonic tension during Na-K pump inhibition compared with that during pump re-activation. A similar hysteresis was often seen between twitch tension and aiNa.

Animals↗
PubMed1981

The effects of rubidium ions and membrane potentials on the intracellular sodium activity of sheep Purkinje fibres.

1. Intracellular Na activity, aiNa, was measured in voltage-clamped sheep cardiac Purkinke fibres. 2. Increasing [Rb]0 from 0 to 4 mM in K-free solutions (at a fixed membrane potential) decreased aiNa. Further increases of [Rb]0 (up to 20 mM) had little or no effect. 3. Following exposure to Rb-free, K-free solution, the addition of a test concentration of Rb produced an exponential decrease of aiNa. The rate constant of decay of aiNa increased with increasing [Rb]0 over the measured range (0-20 mM). 4. The accompanying electrogenic Na pump current transient decayed with the same rate constant as aiNa over the range of [Rb]0 examined. During this decay the electrogenic Na pump current was a linear function of aiNa. Increasing [Rb]0 increased the steepness of the dependence of the electrogenic current on aiNa. 5. A constant fraction of the net Na efflux was electrogenic. This fraction was not affected by varying [Rb]0 over the range 0-20 mM. 6. Using a simple model, it is shown that the dependence of steady-state aiNa on [Rb]0 is half-saturated by less than 1 mM-[Rb]0. The rate constant of decay of aiNa and the slope of the relationship between electrogenic Na pump current and aiNa are, however, better fitted with a lower affinity for Rb (K0.5 = 4 mM-[Rb]0). 7. Depolarizing the membrane potential with the voltage clamp decreased aiNa; hyperpolarization increased it. These effects persisted in the presence of 10(-5) M-strophanthidin. An effect of membrane potential on the net passive Na influx can account for the observations. 8. The effects of membrane potential on the net passive Na influx were examined by measuring the maximum rate of rise of aiNa at different holding potentials after inhibiting the Na-K pump in a K-free, Rb-free solution. Depolarization decreased the Na influx. 9. Using the constant field equation, the net passive Na influx was used to estimate the apparent Na permeability coefficient, PNa. This was between 0.8 x 10(-8) and 1.5 x 10(-8) cm sec-1.

Animals↗
PubMed1980

Characterization of the electrogenic sodium pump in cardiac Purkinje fibres.

1. The Na pump is examined in sheep cardiac Purkinje fibres using a two micro-electrode voltage clamp technique.2. After reducing the external K concentration, [K](o), to zero for 2 min or more, subsequent addition of an ;activator cation' (known to activate the Na pump in other preparations) produces a transient increase of outward current. This outward current transient is abolished by 10(-5)M-strophanthidin (cf. Gadsby & Cranefield, 1979a).3. It is concluded that this transient increase of outward current is a result of a transient stimulation of the sodium pump by the raised [Na](i) following exposure to 0-K(o). Although this current transient may reflect the activity of an electrogenic Na pump, it is difficult to use K as the activator cation to establish this point. This is due to the extracellular K depletion that occurs during Na pump reactivation and the subsequent change that this K depletion produces in the current-voltage relationship of the Purkinje fibre.4. Rb(o) or Cs(o) have been used instead of K(o) to reactivate the Na pump when examining the transient increase of outward current. On adding either of these cations after exposing a preparation to a solution without such ;activator cations', the outward current transient is relatively voltage independent over a wide range of potentials (-90 to +10 mV). It is concluded that, following the addition of Rb(o) or Cs(o), the transient increase of outward current is a direct measure of the transient increase of the electrogenic Na pump current.5. Increasing [Rb](o) or [Cs](o) over the range of 0-40 mM increases the rate of decay of the electrogenic Na pump current transient. Using a simple model (cf. Rang & Ritchie, 1968), it is shown that the decay rate constant of the electrogenic Na pump current transient is a good measure of the degree of activation of the external site of the Na pump. At a given concentration of activator cation, Rb(o) produces a greater activation of the Na pump than does Cs(o). The K(0.5) for Rb(o) is 6.3 mM and for Cs(o) is 14.2 mM. Li(o) activates the Na pump more weakly than Rb(o) and Cs(o).6. The coupling ratio of the Na pump is shown to be independent of Rb(o) or Cs(o) over the range 2-40 mM. Furthermore, consistent with the results of Gadsby & Cranefield (1979a), the coupling ratio is independent of Na(i) over the range considered.7. The Q(10) for the electrogenic Na pump current transient varies between 1.6 and 2.3 over the range of temperature 26-46 degrees C.8. A maximum Na pump current of about 0.78 muA cm(-2) is obtained. Assuming a coupling ratio of 3Na/2K, the rate of Na ion transport into the cell is estimated to be about 23 p-mole cm(-2) sec(-1). Assuming a Na pump turnover of 150 sec(-1), we estimate that there are about 1000 Na pump sites per mum(2) of cell surface.9. We conclude that the electrogenic Na pump current transient provides a good measure of the activity of the Na pump when Rb or Cs are used as ;activator cations'. This measure can be used in the intact preparation to investigate the relationship between Na pump rate and other cellular events such as the regulation of tension (Eisner & Lederer, 1980).

Animals↗
PubMed1980

The relationship between sodium pump activity and twitch tension in cardiac Purkinje fibres.

1. Sheep cardiac Purkinje fibres were studied under voltage clamp conditions to examine the relationship between the Na pump rate and twitch tension.2. The Na pump activity was measured following a period of decreased Na pump rate and increased internal Na activity, Na(i), by examining the electrogenic Na pump current transient produced by reactivating the Na pump (see Eisner & Lederer, 1980).3. Various concentrations of Cs or Rb were used to reactivate the Na pump in the absence of extracellular K, K(o). Results from such experiments showed that these ;activator cations' produced a monotonic increase in Na pump rate with increasing concentration while producing monotonic decreases in steady-state twitch tension. A given concentration of Rb was more potent than the same concentration of Cs in its effects on both Na pump rate and tension. Nevertheless, varying [Rb](o) or [Cs](o) produced the same relationship between Na pump activity and tension.4. After the preparation was exposed to low K(o) (below 4 mM), the Na pump was reactivated with 10 mM-Rb(o) (0-K(o)). The area under the resulting electrogenic Na pump current transient gives a measure of the increase of [Na](i) that occurred when the preparation was exposed to the test solution compared to the steady-state Na(i) in 10 mM-Rb(o). Increasing the duration of exposure to low K(o), further augments the twitch tension achieved at the end of the test period and the area under the electrogenic Na pump current transient. Similarly, for equal periods of exposure, the lower the [K](o) in the test solution, the greater the increase of twitch tension at the end of the test period and the greater the area under the electrogenic Na pump current transient.5. The relationship between tension and the area under the electrogenic Na pump current transient is the same for a variable duration exposure to 0-K(o) or for a constant exposure to various low K(o). It is concluded that a rise in [Na](i) is the rate limiting step linking Na pump activity and twitch tension.6. In an experiment similar to the one described in (4), the fibre was exposed to a test solution containing a variable concentration of one of the activator cations of the Na pump for a fixed period. The effects of different cations were compared with the effects of a test solution containing no activator cation. Increasing the concentration of a particular activator cation from zero reduced the twitch tension augmentation in the test solution and also reduced the area under the electrogenic Na pump current transient on subsequently reactivating the Na pump with 10 mM-Rb(o). It is concluded that the ability of a test solution to reduce the area of the electrogenic Na pump current transient reflects the ability of the test solution to activate the Na pump. Equivalent concentrations of the activator cations that are required to activate the Na pump are: 2 mM-K(o) = 2 mM-Rb(o) = 6 mM-Cs(o) = 6 mM-NH(4o) = 22 mM-Li(o). Tl(o) was more effective than K(o). The order of these cations to reduce twitch tension is found to be the same as that to reduce the area under the electrogenic Na pump current transient.7. We conclude that the effects of the activator cations on twitch tension are determined by their effects on the Na pump and thereby on [Na](i).

Animals↗
PubMed1979

Inotropic and arrhythmogenic effects of potassium-depleted solutions on mammalian cardiac muscle.

1. We have investigated the electrical and mechanical effects of reducing the bathing K concentration, K0, over the range from 4-0 mM in guinea-pig papillary muscle and in sheep Purkinje fibres. 2. In papillary muscle, reducing K0 to zero produces a negative shift in the resting potential and an initial increase in action potential duration. An increase of twitch tension ensues, followed by a reduction in actin potential duration and, eventually, an increase in tonic tension. This increase in tonic tension is often accompanied by a decrease of twitch tension. Finally, transient depolarizations and after contractions are produced. 3. In voltage clamped Purkinje fibres, K0 reduction decreases the slope conductance at the more negative potentials and reduces the pace-maker current, iK2. Twitch tension increases rapidly and voltage dependent tonic tension develops. After even very short exposures to very low K0 (1 mM and below), an oscillatory transient inward current and accompanying aftercontraction can be seen. The oscillatory transient inward current and aftercontraction are similar to those described for cardiotonic steroid intoxication by Kass, Lederer, Tsien & Weingart (1978). 4. Prolonged exposure to 0 K0 leads to the development of a slow current 'creep'. This current is activated by depolarization and has a reversal potential of -6.7 +/- 3.5 mV. The development of this creep current is accompanied by an increasing 'creep' in tonic tension with the same time course. On repolarization both the current creep and creep in tension recover with time courses still similar to each other. 5. Fluctuations appear in both the tension and current records during exposure to low K0. The tension and current fluctuations have similar principal frequencies (about 1 Hz). 6. Ca0 removal, substituting Ba0 for Ca0 or adding Mn0 (2 mM) can each remove the transient inward current, aftercontraction, fluctuations of current and tension, and creep current as well as the increase of twitch and tonic tension. 7. Replacing Ca0 by Sr0 leads to an increased inotropic effect of low K0 with altered kinetics and appears to abolish the transient inward current, aftercontraction and fluctuations of current and tension. 8. It is concluded that Ca1 plays a central role in the inotropic and arrhythmogenic effects of low K0. Possible mechanisms of Ca1 control are discussed in light of the results that have been presented.

Animals↗
PubMed1979

The role of the sodium pump in the effects of potassium-depleted solutions on mammalian cardiac muscle.

1. Mammalian Purkinje fibres and ventricular muscle are significantly affected by exposure to low K solutions (Eisner & Lederer, 1979). Such exposure produces two classes of effects. ;Early' effects, developing over tens of seconds include (in ventricular muscle) a more negative resting potential and a lengthening of the action potential. In Purkinje fibres the principal ;early' effect is a decrease in slope conductance. ;Late' effects develop over minutes. In ventricular muscle such effects include a shortening of the action potential, an increase in twitch and tonic tension, and the development of transient depolarizations and aftercontractions. The late effects in Purkinje fibres are the increase in twitch tension and voltage dependent tonic tension, the development of transient depolarizations and the underlying oscillatory transient inward currents, the appearance of aftercontractions accompanying the transient depolarizations or transient inward currents, and the development of a slow ;creep' in both current and tension.2. The rate of development of early effects is consistent with the time taken to change the bathing K concentration, K(o). However the time course of onset of the late effects (including the positive inotropy) is too slow to be explained by the time taken to change K(o).3. The late effects of reducing K(o) from 4 to 0 mM can be prevented by including appropriate concentrations of the activator cations of the Na pump (Tl, Rb, Cs, NH(4) or Li) in the 0 K(o) bathing solution. Similarly the late effects of 0 K(o), once established, can be reversed by adding these cations to the 0 K(o) superfusing solution.4. The order of potency of these cations to remove the effects of 0 K(o) was found to be: Tl > K approximately Rb > NH(4) approximately Cs > Li. This is similar to the order of efficacy shown to activate the external K site of the Na pump in nerve and other tissue (Rang & Ritchie, 1968).5. Strophanthidin (10(-5)M) produces qualitatively similar electrical and mechanical effects as those seen in 0 K(o). However, the effects of strophanthidin are not reversed by the activator cations. Furthermore, in the presence of strophanthidin (10(-5)M), these cations do not reverse the effects of 0 K(o).6. In voltage-clamped Purkinje fibres, returning to a solution of 4 mM-K(o) after exposure to 0 K(o) produces a transient increase in outward current. Similarly, during exposure to 0 K(o) the addition of activator cations also produces a transient increase of outward current. The ability of these ions to develop this outward transient current is correlated with their ability to remove the inotropic and arrhythmogenic effects of 0 K(o).7. The transient outward current produced by activator cations in 0 K(o) is blocked by strophanthidin (10(-5)M). We conclude that the outward current transient reflects activation of an electrogenic Na pump. Furthermore, we find that, as in other tissues, the activator cations can substitute for K(o) in activating the Na-K pump.8. The reversal of inotropic and arrhythmogenic effects of 0 K(o) by activator cations indicates that such effects result from Na pump blockade. No additional explanation (e.g. Ca/K exchange) need be invoked.

Animals↗
PubMed1978

Role of calcium ions in transient inward currents and aftercontractions induced by strophanthidin in cardiac Purkinje fibres.

1. Under the influence of strophantidin, Purkinje fibres exhibit transient inward current (TI) which contributes to arrhythmogenic activity. Voltage-clamp experiments were carried out to study the role of Ca ions in this phenomenon. 2. The amplitude of TI varied directly with the extracellular Ca concentration, CaO. Magnesium ions had an antagonistic effect. 3. TI was closely associated with a phasic increase in force ("aftercontraction"). Like TI, the aftercontraction was evoked by a preceding action potential or by the break of a strong depolarizing pulse. 4. TI and the aftercontraction displayed similar wave forms although peak current preceded peak force by 50--100 msec. Both transients were enhanced by increasing the strength or duration of the preceding depolarization pulse. Both events were slowed as the potential level following the pulse was displaced in the negative direction. 5. TI and the aftercontraction could be evoked in the absence of cardiotonic steroids by strongly elevating CaO. 6. Additional experiments were carried out to test the hypothesis the TI reflects an influx of Ca2+ ions. Moninhibited TI but the developed and removal of the inbibition lagged far behind the effects on the slow inward current. 7. TI could be suppressed and eventually inverted by varying the membrane potential in the positive direction. The inversion potential averaged -5mV and was not consistent with a Ca-specific pathway. The aftercontraction was more closely related to the phasic conductance change underlying the current than to thecurrent flow itself. 8. The results are consistent with the idea that an oscillatory release of Ca from an intracellular store is the primary event underlying both the aftercontraction and the conductance change which generates TI. Digitalis intoxication or very high CaO may promote such events by elevating intracellular Ca levels.

Animals↗
PubMed1976

Transient inward current underlying arrhythmogenic effects of cardiotonic steroids in Purkinje fibres.

1. Voltage-clamp experiments were carried out in calf Purkinje fibres to determine the basis of transient depolarizations (TDs) associated with digitalis-induced arrhythmias. 2. Under the influence of strophanthidin, depolarizing clamp pulses were followed by a transient inward current (TI) which was small or absent in untreated preparations. The TI also appeared in the wake of a train of action potentials. 8. The TI can help generate spontaneous depolarizations in preparations showing the low voltage oscillations which often occur with advanced digitalis toxicity. 8. The TI can help generate spontaneous depolarizations in preparations showing the 'low voltage oscillation' which often occur with advanced digitais toxicity. It was designated TI because its magnitude and timing were appropriate to account for the TD. 3. Longitudinal voltage non-uniformity during the TI was determined with two voltage-recording micro-electrodes. Although the non-uniformity was not severe, the TI wave form was observed when the voltage difference signal was used to measure membrane current density. 4. Over the diastolic range of potential, the strophanthidin-induced TI appeared superimposed upon the normal pace-maker mechanism, the decay of a potassium current, iK2. The TI could be dissociated from iK2, however, by means of its unusual kinetic properties. 5. TIs could also be recorded at holding potentials positive to -55 mV, i.e. outside the range where iK2 deactivation occurs. 6. The TI amplitude showed a slow and strongly sigmoid dependence on the duration of the preceding depolarizing pulse. Stronger depolarizing reduced the TI amplitude, while slowing and exaggerating the sigmoid time-dependence. 7. Two clamp pulses in close succession gave additive effects in evoking a subsequent TI. This finding and the sigmoid time-dependence fit with previous observations that TDs are most prominent following a series of closely spaced action potentials.

Action Potentials↗
PubMedDate not supplied

Real-time confocal microscopy and calcium measurements in heart muscle cells: towards the development of a fluorescence microscope with high temporal and spatial resolution.

Preliminary experiments and characterization of a modified confocal fluorescence microscope have been carried out and are presented in this article. We have made use of commercially available hardware and software (having modified and extended the system) and are continuing the process of making modifications to this system to enable us to carry out investigations in living and mobile cells. We report on identified problems in measuring intracellular calcium in myocardial cells, important lessons that have been learned and new findings regarding myocardial cells. Specifically, we have found that myocardial cellular organelles (e.g. nuclei and mitochondria) are sharply defined unlike images obtained with standard epifluorescence microscopes using intracellular indicators. Furthermore, we show that the organelles can accumulate or largely exclude certain indicators relative to the concentration in the cytosol. Additionally, we have examined the use of the new calcium indicator fluo-3 in contracting heart cells and have more clearly defined certain limitations in its use in this preparation. We are working on modifications of the present equipment to enable the system to work with an ultraviolet laser as a light source. The confocal microscope offers the prospect of extraordinarily good spatial and temporal resolution under specific conditions in heart cells.

Animals↗