W J Lederer

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.

The control of tonic tension by membrane potential and intracellular sodium activity in the sheep cardiac Purkinje fibre.

Intracellular Na activity (aiNa) was measured with recessed‐tip, Na‐selective micro‐electrodes in voltage‐clamped sheep cardiac Purkinje fibres. Tension was measured simultaneously. aiNa was increased reversibly either by exposing the preparation to K‐free, Rb‐free solution of by adding the cardioactive steroid strophanthidin. An increase of aiNa produced an increase of tonic tension which was larger at depolarized membrane potentials. At sufficiently negative membrane potentials, changes of aiNa (over the range 6‐30 mM) had no effect on tonic tension. Therefore, both an increase of aiNa and a depolarization are required to increase tonic tension. It is concluded that either a low level of aiNa or a large negative membrane potential is sufficient to maintain a low intracellular Ca concentration. Tonic tension was measured as a function of aiNa. At a given membrane potential the relationship can be described empirically by an equation of the form: tonic tension = b(aiNa)y, where y is a constant and b depends on membrane potential. In five experiments y was found to be 3.7 +/‐ 0.7 (mean +/‐ S.E.M.) over a range of potentials from ‐60 to ‐10 mV. Tonic tension was measured as a function of membrane potential. At a given aiNa the relationship can be described approximately as: tonic tension = k exp (aV), where a is a constant and k depends on aiNa. In five experiments a was found to be 0.06 +/‐ 0.01 mV‐1 (mean +/‐ S.E.M.). A depolarization of 10 mV increases tonic tension by the same amount as does an increase of aiNa that is equivalent to a 3.7 mV change of the Na equilibrium potential, ENa. Hence ENa is nearly 3 times more effective than membrane potential in controlling tonic tension. During a prolonged depolarization (several minutes) the initial increase of tonic tension decays gradually. This is associated with a fall of aiNa. The relationship between tonic tension and aiNa is similar to that seen when aiNa is increased by inhibiting the Na pump. It is concluded that the fall of aiNa is responsible for the decay of tonic tension. The changes of tonic tension reported in this paper are consistent with the effects of aiNa and membrane potential on a voltage‐dependent Na‐Ca exchange. The possibility that a voltage‐dependent Ca channel contributes to tonic tension is also discussed.

The quantitative relationship between twitch tension and intracellular sodium activity in sheep cardiac Purkinje fibres.

Tension was measured in voltage clamped sheep cardiac Purkinje fibres while simultaneously measuring the intracellular Na activity (aiNa) with a recessed‐tip, Na‐selective micro‐electrode. Inhibiting the Na‐K pump either by exposing the preparation to a K‐free solution or by adding the cardioactive steroid strophanthidin increased both aiNa and twitch tension and resulted in the development of tonic tension, after‐contractions and a transient inward current (ITI). The increase of twitch tension was present at lower aiNa than that required to produce the other phenomena. The relationship between the magnitude of the twitch tension and aiNa was always non‐linear. Twitch tension increased steeply with aiNa at first but the relationship flattened off at higher aiNa and tension eventually decreased. Over the steep range, the relationship between tension and aiNa could be represented as: twitch tension = b (aiNa)y where y had a mean value of 3.2. Changing membrane potential or [Ca2+]o changed b but had little effect on y. Mn (2 mmol/l) greatly decreased twitch tension but, at least initially, had little effect on tonic tension. The steep relationship between twitch tension and aiNa was seen, irrespective of whether the Na‐K pump was inhibited either by exposure to K‐free solution or to strophanthidin and whether the relationship was measured either when aiNa was increasing or after it had reached a steady state. The steep dependence of twitch tension on aiNa observed in the present work means that manoeuvres which produce even small changes of aiNa will have significant effects on contraction.

The role of intracellular sodium activity in the anti‐arrhythmic action of local anaesthetics in sheep Purkinje fibres.

The effects of lidocaine have been examined on the arrhythmogenic transient inward current (ITI) in voltage‐clamped sheep cardiac Purkinje fibres. Tension and intracellular Na activity (aiNa) were measured simultaneously. The addition of lidocaine (200‐300 microM) produced an immediate decrease of inward holding current and a gradual fall of aiNa. The relative magnitudes of the changes of current and aiNa were shown to be consistent with the outward shift of current representing principally a reduction of inward Na current. The Na pump was inhibited by reducing the external Rb concentration in a K‐free solution. This produced an after‐contraction and transient inward current (ITI) along with a rise of aiNa. The subsequent addition of lidocaine decreased the magnitude of ITI and the after‐contraction while decreasing aiNa. Tetrodotoxin (TTX) had qualitatively similar effects to lidocaine on inward holding current, aiNa, ITI and the after‐contraction. When aiNa was changed by (i) lidocaine, (ii) TTX or (iii) small changes of external Rb concentration, a hysteresis was seen in the relationship between aiNa and ITI or after‐contraction. The hysteresis was similar to that previously found between aiNa and contraction (Eisner, Lederer & Vaughan‐Jones, 1981). Despite this hysteresis, neither lidocaine nor TTX affected the relationship between magnitudes of ITI and the after‐contraction. It is suggested that the fall of aiNa is a major factor in the reduction of ITI by lidocaine. These results are discussed in relation to the anti‐arrhythmic actions of lidocaine.

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.

Thick slurry bevelling

A simple, inexpensive and rapid bevelling method is described. A settled slurry of 0.05 μm alumina powder in saline is used as the grinding surface. The bevelling process is continuous and reproducible over a wide range of electrode resistances.

Effects of membrane potential on intracellular calcium concentration in sheep Purkinje fibres in sodium-free solutions.

1. The intracellular Ca2+ concentration [( Ca2+]i) was measured in voltage‐clamped sheep cardiac Purkinje fibers while recording tension simultaneously. 2. When [Na+]i was elevated (by Na+‐K+ pump inhibition) depolarization produced an increase of tonic tension. 3. Replacement of external Na+ by Li+ or choline produced a contracture which then relaxed spontaneously. Following this relaxation, depolarization either had no effect on tonic tension or produced a small decrease. 4. When external Na+ was replaced by Ca2+, depolarization (over the range ‐120 to ‐20 mV) produced a decrease of tonic tension and [Ca2+]i. Hyperpolarization increased tonic tension and [Ca2+]i. 5. An after‐contraction and accompanying increase of [Ca2+]i were produced by repolarization in both Na+‐free and Na+‐containing solution. This eliminates the possibility that the stimulus for the after‐contraction is the increase of [Ca2+]i during the depolarization and suggests that the stimulus may be the change of membrane potential. 6. The increase of [Ca2+]i on hyperpolarization seen in Na+‐free solutions persisted in the presence of ryanodine. 7. These results show, in contrast to previous work, that in Na+‐free solutions tonic tension is still sensitive to membrane potential. The results support the hypothesis that, in Na+‐containing solutions, the increase of tonic tension on depolarization results from a voltage‐dependent Na+‐Ca2+ exchange. The reduction of tonic tension on depolarization in Na+‐free solutions may be due to the decrease of the electrochemical gradient for Ca2+ to enter the cell.