Relation between intracellular Na ion activity and tension of sheep cardiac Purkinje fibers exposed to dihydro-ouabain.

The intracellular Na ion activity (aiNa) and the contractile tension (T) of sheep cardiac Purkinje fibers were simultaneously measured employing recessed-tip Na+-selective glass microelectrodes and a mechano-electric transducer. The aiNa of 6.4 +/- 1.6 mM (mean +/- SD, n = 56) was obtained in fibers perfused with normal Tyrode's solution. The changes in aiNa and T were measured during and after the exposure of fibers to a cardiac glycoside, dihydro-ouabain (DHO) in concentrations between 5 X 10(-8) M and 10(-5) M. The exposure time to DHO was 15 min. Both aiNa and T did not change in fibers exposed to 5 X 10(-8) M DHO, and the threshold concentration for the effect of DHO appeared to be around 10(-7) M. In DHO concentrations greater than the threshold, the increases in aiNa and T strongly correlated during the onset of DHO effects. The recoveries of aiNa and T were variable and slow, being dependent on the DHO concentration. In those fibers which recovered from the effects of DHO, the time-course of aiNa recovery was similar to that of T recovery. In fibers exposed to DHO of 5 X 10(-6) M or greater, the apparent toxic effects were observed in both action potential and contraction after an initial increase in T. The fibers manifesting the apparent toxic effects has a aiNa of approximately 30 mM or greater. The results of this study indicate that the increase in aiNa is associated with the positive inotropic action of the cardiac glycoside.     

Slow inward current and contraction of sheep cardiac Purkinje fibers

A "slow" inward current (Is) has been identified in ventricular muscle and Purkinje fibers of several mammalian species. The two- microelectrode voltage clamp technique is used to examine some of the relationships between Is and contraction of the sheep cardiac Purkinje fiber. "Tails" of inward current occurring on repolarization and extrapolation of Is recovery each show that the Is system may not inactivate completely during prolonged depolarization. The rate of recovery of Is after a depolarization is slow, and when a train of 300- ms clamps (frequency 1 s-1) is begun after a rest, Is is larger for the first clamp than it is for succeedings clamps. For the first clamp after a rest, the thresholds for Is and tension are the same and there is a direct correlation between peak tension and peak Is for clamp voltages between threshold and minus 40 mV. After a clamp, however, the ability to contract recovers much more slowly than does Is. Therefore, since Is may occur under certain conditions without tension, the realtionship between Is and tension must be indirect. Calcium entering the cell via this current may replenish or augment an intracellular calcium pool.     

4-Aminopyridine and the early outward current of sheep cardiac Purkinje fibers

We have studied the effects of the potassium-blocking agent 4-aminopyridine (4-AP) on the action potential and membrane currents of the sheep cardiac Purkinje fiber. 4-AP slowed the rate of phase 1 repolarization and shifted the plateau of the action potential to less negative potentials. In the presence of 4-AP, the substitution of sodium methylsulfate or methanesulfonate for the NaCl of Tyrode's solution further slowed the rate of phase 1 repolarization, even though chloride replacement has no effect on the untreated preparation. In voltage clamp experiments, 4-AP rapidly and reversibly reduced the early peak of outward current that is seen when the Purkinje fiber membrane is voltage-clamped to potentials positive to -20 mV. In addition, 4-AP reduced the steady outward current seen at the end of clamp steps positive to -40 mV. 4-AP did not appear to change the slow inward current observed over the range of -60 to -40 mV, nor did it greatly change the current tails that have been used as a measure of the slow inward conductance at more positive potentials. 4-AP did not block the inward rectifying potassium currents, IK1 and IK2. A phasic outward current component that was insensitive to 4-AP was reduced by chloride replacement. We conclude that the early outward current has two components: a chloride-sensitive component plus a 4-AP-sensitive component. Since a portion of the steady-state current was sensitive to 4-AP, the early outward current either does not fully inactivate or 4-AP blocks a component of time-independent background current.     

Effects of changes of intracellular pH on contraction in sheep cardiac Purkinje fibers

Intracellular pH (pHi) was measured with a pH-sensitive microelectrode in voltage-clamped sheep cardiac Purkinje fibers while tension was simultaneously measured. All solutions were nominally CO2/HCO3 free and were buffered with Tris. The addition of NH4Cl (5-20 mM) produced an initial intracellular alkalosis that was associated with an increase of twitch tension. At the same time, a component of voltage-dependent tonic tension developed. Prolonged exposure (greater than 5 min) to NH4Cl resulted in a slow recovery of pHi accompanied by a decrease of tension. Removal of NH4Cl produced a transient acidosis that was accompanied by a fall of force. In some experiments, there was then a transient recovery of force. If extracellular pH (pHo) was decreased, then pHi decreased slowly. Tension also fell slowly. An increase of pHo produced a corresponding increase of both force and pHi. The application of strophanthidin (10 microM) increased force and produced an intracellular acidosis. The addition of NH4Cl, to remove this acidosis partially, produced a significant increase of force. The above results show that contraction is sensitive to changes of intracellular but not extracellular pH. This pH dependence will therefore modify the contractile response to inotropic maneuvers that also affect pHi.     

Effect of diameter on membrane capacity and conductance of sheep cardiac Purkinje fibers

Membrane electrical properties were measured in sheep cardiac Purkinje fibers, having diameters ranging from 50 to 300 mum. Both membrane capacitance and conductance per unit area of apparent fiber surface varied fourfold over this range. Membrane time constant, and capacitance per unit apparent surface area calculated from the foot of the action potential were independent of fiber diameter, having average values of 18.8 +/- 0.7 ms, and 3.4 +/- 0.25 muF/cm2, respectively (mean +/- SEM). The conduction velocity and time constant of the foot of the action potential also appeared independent of diameter, having values of 3.0 +/- 0.1 m/s and 0.10 +/- 0.007 ms. These findings are consistent with earlier suggestions that in addition to membrane on the surface of the fiber, there exists a large fraction of membrane in continuity with the extracellular space but not directly on the surface of the fiber. Combining the electrical and morphological information, it was possible to predict a passive length constant for the internal membranes of about 100 mum and a time constant for chaning these membranes in a passive 100-mum fiber of 1.7 ms.     

An analysis of calcium effects on diastolic depolarization in sheep cardiac Purkinje fibers

The events by which [Ca]O modifies diastolic depolarization (DD) were analyzed in sheep cardiac Purkinje fibers perfused in vitro. Cs (2 mM) reduced diastolic depolarization (DD) at different [Ca]O and in 10.8 mM [Ca]O revealed an oscillatory potential (VOS) and the decay of a prolonged depolarization (Vex). In the presence of Cs, procedures that reduce Cai (a slower driving rate, lower [Ca]O or tetrodotoxin) abolished VOS and Vex and partially restored DD. In 10.8 mM [Ca]O and at all driving rates, Cs reduced DD slope, DD amplitude and VOS amplitude but had little effect on the VOS time to peak. In 10.8 mM [Ca]O, decreasing calcium overload by different means (2.6 microM TTX, 0.2 mM Cd) abolished VOS and decreased DD slope and amplitude. Substituting Na with Li induced marked aftercontractions but small VOS. In 10.8 mM [Ca]O, Li increased the amplitude of the aftercontractions and decreased that of VOS. Li also depolarized slightly the resting membrane and abolished the voltage undershoot (Emax) at the end of the action potential. In low [K]O, Li repolarized the resting membrane but the repolarization was maintained only in the presence of Ca. It is concluded that Ca overload causes both VOS and Vex which can either be masked by or can mask DD depending on the magnitude of DD and of Ca overload. VOS is apparently caused by an electrogenic Na-Ca exchange since Li-induced Ca overload increases the aftercontraction but decreases VOS.     

Electrogenic sodium extrusion in cardiac Purkinje fibers

Thin canine cardiac Purkinje fibers in a fast flow chamber were exposed to K-free fluid for 15 s to 6 min to initiate "sodium loading," then returned to K-containing fluid to stimulate the sodium pump. The electrophysiological effects of enhanced pump activity may result from extracellular K depletion caused by enhanced cellular uptake of K or from an increase in the current generated as a result of unequal pumped movements of Na and K, or from both. The effects of pump stimulation were therefore studied under three conditions in which lowering the external K concentration ([K]0) causes changes opposite to those expected from an increase in pump current. First, the resting potential of Purkinje fibers may have either a "high" value of a "low" (less negative) value: at the low level of potential, experimental reduction of [K]0 causes depolarization, whereas an increase in pump current should cause hyperpolarization. Second, in regularly stimulated Purkinje fibers, lowering [K]0 prolongs the action potential, whereas an increase in outward pump current should shorten it. Finally, lowering [K]0 enhances spontaneous "pacemaker" activity in Purkinje fibers, whereas an increase in outward pump current should reduce or abolish spontaneous activity. Under all three conditions, we find that the effects of temporary stimulation of the sodium pump are those expected from a transient increase in outward pump current, not those expected from K depletion.     

Effects of caffeine, tetracaine, and ryanodine on calcium-dependent oscillations in sheep cardiac Purkinje fibers

Membrane current and tension were measured in voltage-clamped sheep cardiac Purkinje fibers. Elevating the intracellular calcium concentration ([Ca2+]i) results in oscillations of membrane current and tension both at rest and during stimulation. During stimulation, an oscillatory transient inward current and an after contraction follow repolarization. We have examined the effects on the oscillations of changing the extracellular calcium concentration ([Ca2+]o) and of adding various drugs. In agreement with previous work, high concentrations of drugs that affect the sarcoplasmic reticulum, namely caffeine (10-20 mM), tetracaine (1 mM), and ryanodine (10 microM), abolish the oscillations. However, at lower concentrations, these three drugs have different effects on the oscillations. Caffeine (1-2 mM) decreases the oscillation amplitude but increases the frequency. Tetracaine (100-500 microM) has little effect on the magnitude of the oscillations but decreases their frequency. Ryanodine, at all concentrations used (0.1-10 microM), eventually abolishes the oscillations but, in doing so, decreases the magnitude, leaving the frequency unaffected. When [Ca2+]o was changed in order to vary [Ca2+]i, both the frequency and the magnitude of the oscillations always changed in the same direction. This suggests that these three drugs have effects in addition to just changing [Ca2+]i.     

Intracellular pH and cell-to-cell transmission in sheep Purkinje fibers.

Intracellular pH (pHi) is a significant modifier of cell-to-cell communication in some tissues but its role is uncertain in heart tissue. The present studies examined the effect of cytosolic protons on electrotonic spread and conduction velocity in cardiac Purkinje fibers. Cable analysis provided values for internal longitudinal resistance (ri) and pH-selective microelectrodes monitored pHi during CO2 and HCO3- alterations. Resting fibers developed changes in ri that were proportional to intracellular free proton concentration ([H+]i) during CO2 changes at constant [HCO3-]. However, the effects on ri were small between pHi 6.9-7.8 and predicted only a 2.2% increase in ri per 10 nM increase in [H+]i. Other findings suggested that titration of cytosolic protons may not directly produce the changes in ri: (a) For an equal change in [H+]i, the effects on ri were roughly three times greater (6.8% increase per 10 nM rise in [H+]i) if bicarbonate was lost during CO2 changes. (b) pH-associated changes in ri were preceded by a time delay (1-5 min) producing hysteresis in the [H+]i-ri relation during successive perturbations. (c) The same CO2 variations modified the direction and magnitude of ri differently during pacing than at rest. The cumulative results suggest that the action of protons on ri in the heart may be subordinate to another regulator or mediated by another pH-dependent substance or reaction.     

Na,K pump stimulation by intracellular Na in isolated, intact sheep cardiac Purkinje fibers

Regulation of the Na,K pump in intact cells is strongly associated with the level of intracellular Na+. Experiments were carried out on intact, isolated sheep Purkinje strands at 37 degrees C. Membrane potential (Vm) was measured by an open-tipped glass electrode and intracellular Na+ activity (aNai) was calculated from the voltage difference between an Na+-selective microelectrode (ETH 227) and Vm. In some experiments, intracellular potassium (aiK) or chloride (aCli) was measured by a third separate microelectrode. Strands were loaded by Na,K pump inhibition produced by K+ removal and by increasing Na+ leak by removing Mg++ and lowering free Ca++ to 10(-8) M. Equilibrium with outside levels of Na+ was reached within 30-60 min. During sequential addition of 6 mM Mg++ and reduction of Na+ to 2.4 mM, the cells maintained a stable aNai ranging between 25 and 90 mM and Vm was -30.8 +/- 2.2 mV. The Na,K pump was reactivated with 30 mM Rb+ or K+. Vm increased over 50-60 s to -77.4 +/- 5.9 mV with Rb+ activation and to -66.0 +/- 7.7 mV with K+ activation. aiNa decreased in both cases to 0.5 +/- 0.2 mM in 5-15 min. The maximum rate of aiNa decline (maximum delta aNai/delta t) was the same with K+ and Rb+ at concentrations greater than 20 mM. The response was abolished by 10(-5) M acetylstrophantidin. Maximum delta aNai/delta t was independent of outside Na+, while aKi was negatively correlated with aNai (aKi = 88.4 - 0.86.aNai). aCli decreased by at most 3 mM during reactivation, which indicates that volume changes did not seriously affect aNai. This model provided a functional isolation of the Na,K pump, so that the relation between the pump rate (delta aNai/delta t) and aiNa could be examined. A Hill plot allowed calculation of Vmax ranging from 5.5 to 27 mM/min, which on average is equal to 25 pmol.cm-2.s-1.K 0.5 was 10.5 +/- 0.6 mM (the aNai that gives delta aNai/delta t = Vmax/2) and n equaled 1.94 +/- 0.13 (the Hill coefficient). These values were not different with K+ or Rb+ as an external activator. The number of ouabain-binding sites equaled 400 pmol.g-1, giving a maximum Na+ turnover of 300 s-1. The Na,K pump in intact Purkinje strands exhibited typical sigmoidal saturation kinetics with regard to aNai as described by the equation upsilon/Vmax = aNai(1.94)/(95.2 + aNai(1.94)). The maximum sensitivity of the Na,K pump to aiNa occurred at approximately 6 mM.     

Cell-to-Cell diffusion of Procion Yellow in sheep and calf Purkinje fibers

Summary Sheep and calf Purkinje fibers (false tendons) were cut near one end and exposed to a solution containing no calcium and the dye Procion Yellow (M4RS, molecular weight near 700). Fifteen minutes later the damaged end was sealed by applying calcium ions (Tyrode solution). Traces of Procion Yellow were detected within the intracellular compartment at a distance of 2.4 mm from the site of damage when the preparations had been washed in dye-free solution for 4 hr. This indicates that the dye had diffused through about 20 cells in succession. There was no detectable uptake of Procion Yellow through intact surface membranes. Visual curve fitting to quantitative data on concentrationvs. distance gives an apparent diffusion coefficient (cell junctions and myoplasm in series) of 3×10^–8 cm² sec^–1, as against 1×10^–6 cm² sec^–1 in an agar gel. It is concluded that specialized contact areas between neighboring cardiac cells represent a considerable yet not an absolute hindrance to the movement of this particle.     

Membrane current following activity in canine cardiac Purkinje fibers

Membrane current following prolonged periods of rapid stimulation was examined in short (less than 1.5 mm) canine cardiac Purkinje fibers of radius less than 0.15 mm. The Purkinje fibers were repetitively stimulated by delivering trains of depolarizing voltage clamp pulses at rapid frequencies. The slowly decaying outward current following repetitive stimulation ("post-drive" current) is eliminated by the addition of 10(-5) M dihydro-ouabain. The post-drive current is attributed to enhanced Na/K exchange caused by Na loading during the overdrive. Depolarizing voltage clamp pulses initiated from negative (- 80 mV) or depolarized (-50 mV) holding potentials can give rise to post- drive current because of activation of tetrodotoxin-sensitive or D600- sensitive channels. The magnitude of the post-drive current depends on the frequency of voltage clamp pulses, the duration of each pulse, and the duration of the repetitive stimulation. The time constant of decay of the post-drive current depends on extracellular [K] in accordance with Michaelis-Menten kinetics. The Km is 1.2 mM bulk [K], [K]B. The mean time constant in 4 mM [K]B is 83 s. Epinephrine (10(-5) M) decreases the time constant by 20%. The time constant is increased by lowering [Ca]o between 4 and 1 mM. Lowering [Ca]o further, to 0.1 mM, eliminates post-drive current following repetitive stimulation initiated from depolarized potentials. The latter result suggests that slow inward Ca2+ current may increase [Na]i via Na/Ca exchange.     

Calcium- and voltage-activated plateau currents of cardiac Purkinje fibers

We have used the two-microelectrode voltage-clamp technique to investigate the components of membrane current that contribute to the formation of the early part of the plateau phase of the action potential of calf cardiac Purkinje fibers. 3,4-Diaminopyridine (50 microM) reduced the net transient outward current elicited by depolarizations to potentials positive to -30 mV but had no consistent effect on contraction. We attribute this effect to the blockade of a voltage-activated transient potassium current component. Ryanodine (1 microM), an inhibitor of sarcoplasmic reticulum calcium release and intracellular calcium oscillations in Purkinje fibers (Sutko, J.L., and J.L. Kenyon. 1983. Journal of General Physiology. 82:385-404), had complex effects on membrane currents as it abolished phasic contractions. At early times during a depolarization (5-30 ms), ryanodine reduced the net outward current. We attribute this effect to the loss of a component of calcium-activated potassium current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. At later times during a depolarization (50-200 ms), ryanodine increased the net outward current. This effect was not seen in low-sodium solutions and we could not observe a reversal potential over a voltage range of -100 to +75 mV. These data suggest that the effect of ryanodine on the late membrane current is attributable to the loss of sodium-calcium exchange current caused by the inhibition of sarcoplasmic reticulum calcium release and the intracellular calcium transient. Neither effect of ryanodine was dependent on chloride ions, which suggests that chloride ions do not carry the ryanodine-sensitive current components. Strontium (2.7 mM replacing calcium) and caffeine (10 mM), two other treatments that interfere with sarcoplasmic reticulum function, had effects in common with ryanodine. This supports the hypothesis that the effects of ryanodine may be attributed to the inhibition of sarcoplasmic reticulum calcium release.     

Influence of chloride, potassium, and tetraethylammonium on the early outward current of sheep cardiac Purkinje fibers

In voltage clamp studies of cardiac Purkinje fibers, a large early outward current is consistently observed during depolarizations to voltages more positive than -20 mV. After the outward peak of the current, the total membrane current declines slowly. Dudel et al. (1967. Pfluegers Arch. Eur. J. Physiol. 294:197--212) reduced the extracellular chloride concentration and found that the outward peak and the decline of the current were abolished. They concluded that the total membrane current at these voltages was largely determined by a time- and voltage-dependent change in the membrane chloride conductance. We reinvestigated the chloride sensitivity of this current, taking care to minimize possible sources of error. When the extracellular chloride concentration was reduced to 8.6% of control, the principal effect was a 20% decrease in the peak amplitude of the outward current. This implies that the membrane chloride conductance is not the major determinant of the total current at these voltages. The reversal potential of current tails obtained after a short conditioning depolarization was not changed by alterations in the extracellular chloride or potassium concentrations. We suspect that the tail currents contain both inward and outward components, and that the apparent reversal potential of the net tail current largely reflects the kinetics of the outward component, so that this experiment does not rule out potassium as a possible charge carrier. The possibility that potassium carries much of the early outward current was further investigated using tetraethylammonium, which blocks potassium currents in nerve and skeletal muscle. This drug substantially reduced the early outward current, which suggests that much of the early outward current is carried by potassium ions.     

Sheep cardiac Purkinje fibers: Configurational changes during the cardiac cycle

Previous attempts to study the cytoarchitecture of cardiac Purkinje fibers with the scanning electron microscope (SEM) have been limited by the surrounding dense connective tissue. In this study the connective tissue was removed by treatment with 8N HCl, after adult sheep hearts were fixed in diastole or systole and tissue taken for SEM and transmission electron microscopy (TEM). In SEM, Purkinje fibers freely anastomosed in false tendons and formed a subendocardial plexus. In systole, medium and small-sized Purkinje fibers formed deep clefts not observed in diastole. The clefts are thought to be due to sarcolemmal folding and fiber buckling and may therefore affect conduction. The myofibrils beneath the laterally apposed sarcolemmas of adjacent Purkinje cells when fixed in systole were often observed as tightly curved arches in series. Similar configurations with expanded arches were observed in diastole. The formation of arches by myofibrils is unique to Purkinje fibers and is interpreted as the mechanism responsible for their compliance to stretch. The significance of contraction in producing the observed geometric changes in Purkinje fibers and the implications of their cytoarchitecture with respect to conduction are discussed.