Depletion and accumulation of potassium in the extracellular clefts of cardiac Purkinje fibers during voltage clamp hyperpolarization and depolarization

1. Voltage clamp hyperpolarization and depolarization elicited current records consistent with depletion and accumulation, respectively, of potassium in the extracellular clefts of cardiac Purkinje fibers. Hyperpolarization was shown to shift the reversal potential for the pacemaker current, ik2, a measure of Ek, to more negative potentials. Upon depolarization, a slowly increasing outward current was observed. Analysis of the tail currents elicited by hyperpolarization revealed that a time-dependent change in gx could not explain the time-dependent outward current. However, the tail currents were consistent with a shift of Ek to more positive potentials during the depolarization. 2. Alteration in potassium driving force over time results in a time-dependent ik1 even though the underlying conductance is time-independent [29]. This time-dependent current may contribute to the currents usually identified as ik2 and ix. 3. The potential at which ik2 reverses direction is altered by the clamp program used to elicit it and is obscured by the superimposition of a time-dependent current due to depletion. 4. Records consistent with the extracellular cleft potassium concentration being less than that of the bulk phase in the quiescent fiber were obtained. However, an unequivocal interpretation of these current reocrds could not be made. 5. These results suggest that conclusions based on the assumption that potassium driving force remains constant during a voltage clamp pulse may be in error. Thus, time-dependent currents cannot be assumed to result solely from time-dependent conductance changes.     

Norepinephrine and potassium fluxes in cardiac Purkinje fibers

Summary Dog ventricular Purkinje fibers, loaded with⁴²K, were mounted in a tissue bath located over a beta-detector and washed in Tyrode solution. Tissue radioactivity was measured at intervals and transmembrane potential recorded by means of microelectrodes. Relative potassium influx was estimated by re-exposing the fiber to radioactive solution for fixed periods and measuring the tissue radio-activity increase at the end of the uptake period. Potassium efflux was estimated by measuring tissue and effluent radioactivities. After control recordings, administration of norepinephrine caused the following changes during the period it was applied: 1. increase in potassium influx; 2. increase in potassium efflux in nonstimulated fibers; 3. a smaller increase in potassium efflux in fibers stimulated electrically at a constant rate; 4. increase in potassium content in fibers loaded to equilibrium with⁴²K; 5. a smaller increase in K uptake in fibers driven at a high rate; and 6. enhancement of potassium uptake even in the presence of high [K]₀. It is concluded that norepinephrine increases both influx and efflux in Purkinje fibers. The larger enhancement of the influx with respect to the efflux and the increase in potassium content suggests that norepinephrine stimulates active transport of potassium. This stimulatory action can be dissociated from the stimulation of automaticity.This investigation was supported by a grant from N. I. H. (Nr. HE-10097).     

Electrophysiological effects of acetylcholine in sheep cardiac Purkinje fibers

The addition of acetylcholine (ACh) in concentrations of 10^–7 to 10^–5 mol·l^–1 to normal Tyrode solution results in the following changes of the electrical activity in sheep cardiac Purkinje fibers: prolongation of the action potential and shift of the plateau to more positive values, hyperpolarization of the maximum diastolic potential (E _max), and increase of the rate of diastolic depolarization.Prolongation of the action potential and shift of the plateau in the positive direction were more pronounced in distal Purkinje fibers, at low frequencies of stimulation, and in the presence of a reduced Ca (<3.6 mmol·l^–1) or K extracellular concentration (<5.4 mmol·l^–1); the effects persisted in Cl free media or after addition of Mn (2–10 mmol·l^–1) or verapamil (1–5 mg·l^–1).The effect of Ach on the maximum diastolic potential (stimulation frequency 60/min) was dependent on K₀. At 5.4 mmol·l^–1,E _max increased by 2 mV; at higher K₀ concentrations the change was less pronounced or absent; at low K₀ concentrations (membrane potential arrested at the plateau level) the addition of Ach invariably caused a depolarization. In unstimulated preparations the effect of Ach was unpredictable.The increase in rate of diastolic depolarization by Ach in 3.4 or 2.7 mmol·l^–1 K was large enough to result in spontaneous activity. When the membrane was depolarized to the plateau level (K₀<1.35 mmol·l^–1) Ach frequently reduced the frequency of oscillations.The effect of Ach was dose-dependent; desensitization was absent. Similar results were obtained with carbachol (10^–6 mol·l^–1) or choline (5·10^–3 mol·l^–1). The effect of Ach could selectively be abolished by atropine (10^–8 to 10^–6 mol·l^–1); it was not modified by succinylcholine (8·10^–5 mol·l^–1), phentolamine (10^–6 mol·l^–1) or propranolol (10^–6 mol·l^–1). The results indicate that the electrophysiological changes are due to stimulation of muscarinic receptors.Except for the hyperpolarization ofE _max the results in sheep Purkinje fibers are different and even opposite to those observed in the sino-atrial node and atrial muscle. Possible mechanisms and functional significance of the results are discussed.Supported by F.G.W.O. Belgium 3.0087.74     

The effects of extracellular potassium,ouabain, and prostaglandins on intracellular potassium activity in sheep cardiac Purkinje fibers

(1) Intracellular K activity (a _K ⁱ ) of sheep heart Purkinje fibers was measured using K-selective microelectrodes (liquid ion exchanger).a _K ⁱ in the resting state with an extracellular K of 4 mmol·l^–1 was 112.9±6.1 mmol·l^–1 (n=47) for a membrane potential (V _M) of –73.3±0.9 mV.V _M deviated from the calculated potassium equilibrium potential (E _K=–93 mV). (2) When extracellular K was decreased to 2 mmol·l^–1 or increased to 6 and 10 mmol·l^–1 E _K changed from –114 to –84 and –73 mV, with little change ina _K ⁱ . (3)a _K ⁱ andV _M significantly decreased after administration of 10^–6 mol·l^–1 ouabain. (4) Prostaglandins (PGI₂ 10–100 g·l^–1 and PGE₂ 0.01–1 g·l^–1) decreaseda _K ⁱ without greatly changingV _M. The differences betweenV _M andE _K became smaller. These effects indicate an increase in K permeability and may explain the antiarrhythmic action of prostaglandins.This study was supported by the Deutsche Forschungsgemeinschaft (grant Wi 328). In preliminary form part of the data has been presented (Pflügers Arch. 384: R 13, 1980, and Proc. XXVIII. Int Congr Physiol Sci, Vol XIV: 279, 1980)     

Electrophysiological characterization of histamine receptor subtypes in sheep cardiac Purkinje fibers

The histamine-receptor-subtype-mediated effects on action potentials of electrically driven and spontaneously active isolated sheep cardiac Purkinje fibers were investigated using H₁-and H₂-selective agonists and antagonists.In electrically stimulated Purkinje fibers, histamine (3 mol/l) increased the action potential plateau height, decreased the action potential duration measured at a repolarization level of –60 mV and enhanced the pacemaker activity. These effects were abolished by the H₂-selective antagonist cimetidine (30 mol/l), but were not impaired by the H₁-selective antagonist dimetindene (0.3 mol/l).In spontaneously active Purkinje fibers, histamine (10 mol/l) increased the spontaneous rate by 24%, the slope of diastolic depolarization by 45% and shortened the duration of the diastole by 32% of the respective control measurements. These effects were blocked by 30 mol/l cimetidine, but remained unchanged in the presence of 0.3 mol/l dimetindene.Concentration-response curves of histamine were shifted to the right by approximately 2 logarithmic units in the presence of 30 mol/l cimetidine, but were not influenced in the presence of 0.3 mol/l dimetindene. The H₂-selective agonist impromidine (0.001–0.3 mol/l) had similar actions as histamine on spontaneously active Purkinje fibers, while the H₁-selective agonist 2-(2-pyridyl-)ethylamine was ineffective. It is concluded that the pronounced stimulatory action of histamine on spontaneous activity in sheep cardiac Purkinje fibers is exclusively mediated by H₂ receptors.Dedicated to Prof. Dr. E. Mutschler on the occasion of his 60th birthday.Supported by Ministerium für Wissenschaft und Forschung, Nordrhein-Westfalen, Projekt-Nr. 40008786.     

Existence of two transient outward currents in sheep cardiac Purkinje fibers

Voltage clamp analysis of the transient outward (positive dynamic) current was performed in sheep Purkinje fibers at a pulse frequency of 1/min. 4-aminopyridine (4-AP, 1 mM) suppressed most of the transient outward current, thus revealing the slow inward current,i _si, and an associated brief outward current,i _bo. The long lasting component of the current suppressed by 4-AP was labelledi _lo. In the presence of 4-AP,i _bo was suppressed either by caffeine 10 mM or when Sr was substituted for Ca, both conditions makingi _si clearly detectable. Mn ions suppressed bothi _si andi _bo. Current decay was a monoexponential process fori _bo (=12 ms) and a two exponential process fori _lo (₁=80–100 ms, ₂=250–400 ms). The peak amplitude-E _m relationships were different for the two currents. It was shown that the reversal potential ofi _lo was not measureble by the usual method probably because of the too fast activation-deactivation kinetics of the current.It is concluded that not one but two transient outward currents with different electrophysiological and pharmacological characteristics exist in the sheep Purkinje fiber. The reason of the caffeine-sensitivity ofi _bo is discussed.Supported by F.G.W.O. Belgium 3.0087.74.     

Effect of acetylcholine on time-independent currents in sheep cardiac Purkinje fibers

Voltage-clamp experiments were carried out in sheep Purkinje fibers in order to find an explanation for the prolongation of the action potential, the positive shift of the plateau, the hyperpolarization of the maximum diastolic potential and the increase in rate of diastolic depolarization, occurring in the presence of acetylcholine (Ach).In the presence of Ach the instantaneous current-voltage relation is shifted in the inward direction for potentials positive to –75 mV, while the opposite shift is obtained for more negative potentials; the results suggest a decrease in background conductance.The contribution of K, Cl, Na and Ca to the Ach sensitive current was studied by varying K₀ concentration or adding 20 mmol·l^–1 Cs, by omitting Cl or Na, and by changing the Ca concentration.In 20 mmol·l^–1 Cs the apparent reversal potential of the Ach sensitive current is –50 mV, as compared to –75 mV in normal Tyrode. The component of the Ach sensitive current, which is suppressed by Cs, shows inward going rectification. In different K₀ concentrations the reversal potential of the Ach sensitive current is changed; the shift obeys the theoretical change in equilibrium potential of K. The results are consistent with a decrease in K background current by Ach (inward and outward rectifier).In Cl free media the Ach sensitive current is not decreased excluding a major contribution of Cl ions. The Ach effect also persists in Na free media; the reversal potential of the Ach sensitive current is slightly shifted in the hyperpolarizing direction. These results indicate that active electrogenic pumps (Na or Na–Ca) do not play an important role; they are in accord with a reduction in inward Na background current by Ach. The shift of the current-voltage relation by Ach was greater the lower the Ca_o concentration; the mechanism is not clear.The inward shift of the current at –40 mV was dependent on the Ach concentration. Half-maximum effect was obtained at 3·10^–7 mol·l^–1 Ach; the Hill coefficient was 1.12.It is concluded that Ach interacts in a one to one reaction with a muscarinic receptor and reduces the background current mainly carried by K (inward and outward rectifier), and less by Na (and probably Ca).Supported by F.G.W.O. Belgium 3.0087.74     

Effects of acetylcholine on time-dependent currents in sheep cardiac Purkinje fibers

Voltage clamp experiments were carried out on sheep Purkinje fibers to determine the effect of Ach on the time-dependent currents.On the pacemaker current (i _{K 2}) Ach 10^–6 mol·l^–1 had the following effects: shift of the activation curve by a few mV in the depolarizing direction, without change in the rectifier ratio. The potential dependence of the time constants for activation and deactivation was influenced in a similar way as the activation curve.Ach had no effect on the positive dynamic current (i _qr ) or the late plateau outward current (i _x ).The slow inward current (i _si ) as well as the transient inward current (T.I.) were reduced in amplitude and slowed in time course by Ach.The changes in pacemaker current are important in explaining the increased rate of diastolic depolarization in the presence of Ach. The decrease of slow inward current by Ach cannot be made responsible for the plateau shift or the prolongation of the action potential.Supported by F.G.W.O. Belgium 3.0087.74     

Epinephrine and the pacemaking mechanisms at plateau potentials in sheep cardiac Purkinje fibers

1. In 1.35 mM [K+]0, sheep cardiac Purkinje fibers depolarized to about -40 mV. Whereas some fibers oscillated spontaneously at plateau potentials, others could be made to oscillate when polarized by intracellular currents. Pacemaker activity at plateau potentials (-50 to 0 mV) was distinct from that caused by the iK2 pacemaker at more negative potentials (-60 to -100 mV). 2. Epinephrine induced spontaneously occurring action potentials and increased pacemaker activity in depolarized Purkinje fibers. The ED50 for the positive chronotropic effect of epinephrine was about 5 x 10(-7) M. This concentration is similar to that reported for the effect of epinephrine on plateau amplitude (Carmeliet and Vereecke, 1969) and the slow inward current (isi, Reuter, 1974). 3. In voltage clamp experiments, epinephrine, increased the magnitude of isi and of an outward plateau current, ixi. It is concluded that epinephrine effects pacemaking at plateau potentials by increasing isi and without shifting the voltage dependence of these currents. The onset of pacemaker activity by epinephrine was preceded by membrane depolarization that results from an inward shift of the steady-state current-voltage relation. This current may flow through isi channels that are activated but not completely inactivated.     

Pace-maker current changes during intracellular pH transients in sheep cardiac Purkinje fibres

Intracellular acidosis, at constant extracellular pH, hyperpolarizes the resting potential and reduces the diastolic depolarization rate of cardiac Purkinje fibres. With alkaline pHi, the fibre depolarizes and spontaneous firing is observed. Intracellular pH transients induced either by superfusion with Tyrode buffered with 5% CO2/23 mM HCO3- or 16% CO2/61 mM HCO3-, or with solutions containing weak undissociated acids, transiently shifted the half-maximum activation potential E0.5 of the pace-maker current. Similar transients were observed when NH4Cl was added and subsequently withdrawn from the solution. Simultaneous pHi measurements demonstrate a close relation between the time course of the pHi and E0.5 variations. Acid pHi shifts E0.5 to more negative and alkaline pHi's to less negative potentials. These pace-maker current activation voltage shifts are interpreted as the direct consequence of fixed charges titration at the inside of the sarcolemma. Other effects, like the slowing-down and reduction of the pace-maker current by acid pHi, presumably result from other interactions of protons with the pace-maker channel.     

Alinidine modifies the pacemaker current in sheep Purkinje fibers

(1) The specific bradycardic agent alinidine reduces the slope of the diastolic depolarization in sinoatrial tissue and Purkinje fibers. In short Purkinje fibers of sheep, alinidine (28 M) decreased the pacemaker current by a dual action. The voltage dependence ofi _f activation was shifted in the hyperpolarizing direction by 7.8±0.6 mV (n=18,p<0.001) and the conductance of the fully activated current was reduced to 73±2% (n=18,p<0.001) of its control value. These effects were reversible and dose-dependent. (2) Ionophoretic injections of alinidine caused reversible reductions of the diastolic depolarization rate and simultaneous transient hyperpolarizing shifts of thei _f activation range. (3) Some prolongation of the action potential duration was observed at 28 M and more pronounced at higher concentration. This was presumably the consequence of a reduction by alinidine of outward repolarizing current carried by the background inward rectifier and plateau currenti _x. (4) The action of alinidine oni _f resulted in a slower activation of a reduced fraction of the pacemaker current at the maximal diastolic potential level. This explains the decrease of the diastolic depolarization rate observed in Purkinje fibers.     

The potassium component of membrane current in Purkinje fibers

Summary The current voltage relations of shortened sheep Purkinje fibers have been measured by means of the voltage clamp technique. In order to identify the potassium component of the membrane current the potassium concentration of the bathing fluid was varied. In potassium-free solution a negative membrane current is observed in the range of negative membrane potentials. It is assumed that this negative current is also flowing in high potassium, therefore, the potassium current should be greater than the positive current measured in sodium-free solution.In sodium-free as well as in sodium-containing solution the membrane shows anomalous rectification of the potassium current: the potassium current is minimal at –20 mV (10^–5A/cm²).The current voltage relation of the potassium current is almost independent of speed and direction of the change of the membrane potential. Only on depolarization in Tyrode's solution the membrane current at the negative potentials is smaller than on repolarization. It is not decided, whether under this condition the potassium equilibrium potential shifts or the negative current is larger than observed in potassium-free solution.On repolarization in Tyrode's solution the membrane current changes its sign at different membrane potentials depending on the speed of potential change: At 300 mV/s this potential is more negative than at 3 mV/s. This leads to the pacemaker potential following the action potential. Our findings are not consistent with earlier interpretations of the pacemaker potential. One has to assume that either the potassium equilibrium shifts or that the negative current shows an appropriate dynamic behavior.Using partial inactivation of the sodium system it is shown, that the potassium system is practically time-independent. This result is discussed with contrary findings of McAllister and Noble (1966).In the conclusion section a schematic review is given of the various components of membrane current of the Purkinje fiber.

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Zusammenfassung An verkürzten Schafs-Purkinje-Fäden wurden mit Hilfe der Spannungsklemme Strom-Spannungs-Beziehungen gemessen. Zur Identifikation der Kaliumkomponente des Membranstromes wurde die Kaliumkonzentration der Badelösung variiert. In Kalium-freier Lösung findet man im Bereich negativer Membranpotentiale einen negativen Membranstrom, von dem angenommen wurde, daß er auch bei höheren Kalium-Außenkonzentrationen fließt. Unter Berücksichtigung dieser negativen Strom-Komponente ist der Kaliumstrom größer als es bisher angenommen wurde. Sowohl in Natrium-freier als auch in Natrium-haltiger Badelösung zeigt die Membran anomale Gleichrichtung des K-Stromes: Der K-Strom hat bei –20 mV ein ausgeprägtes Minimum (10^–5 A/cm²).Die Kennlinie des K-Stromes ist im wesentlichen unabhängig von der Geschwindigkeit und Richtung der Membranpotentialänderung. Nur bei Depolarisation in Natrium-haltiger Badelösung ist im negativen Potentialbereich der Membranstrom kleiner als bei Repolarisation. Es ist nicht entschieden, ob unter dieser Bedingung der K-Strom verkleinert ist bei gleichzeitiger Verschiebung des K-Gleichgewischtspotentials, oder ob der negative Strom größer ist, als er in K-freier Lösung gefunden wurde.Bei Repolarisation in Natrium-haltiger Badelösung hängt das Potential, bei dem der Membranstrom sein Vorzeichen wechselt, von der Geschwindigkeit der Potentialänderung ab: Bei 300 mV/s liegt dieses Potential negativer als bei 3 mV/s. Dies führt bei ungeklemmter Faser zum Schrittmacherpotential. Die Befunde decken sich nicht mit den bisherigen Interpretationen des Schrittmacherpotentials. Zu ihrer Erklärung muß man annehmen, daß entweder Verschiebungen des K-Gleichgewichtes stattfinden, oder daß der negative Strom eine entsprechende Dynamik aufweist.Durch teilweise Inaktivierung des Natrium-Systems wird gezeigt, daß das K-System praktisch keine Zeitabhängigkeit aufweist. Dieses Ergebnis wird mit gegensätzlichen Befunden von McAllister u. Noble (1966) diskutiert. In der Schlußfolgerung wird ein Übersichtsschema über die verschiedenen Membranstromkomponenten der Purkinje-Faser gegeben.

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This work was supported by the Deutsche Forschungsgemeinschaft.     

Purkinje fibers of sheep papillary muscle: Occurrence of discontinuous fibers

Mapping of the peripheral specialized conducting system of the heart could contribute to an understanding of normal and abnormal impulse propagation. In this study, a sheep right anterior papillary muscle was sectioned serially and stained for Purkinje fibers with the periodic acid Schiff and hematoxylin procedure. The subendocardial Purkinje fibers entered the papillary muscle through its base and extended nearly to its apex. Except for one “mixed” fiber group, the subendocardial fibers did not connect to the deep Purkinje fibers, and both groups contained discontinuous Purkinje fibers. The fibers of the moderator band radiated in such a complex fashion that they could not be reconstructed exactly, but they generally disappeared in the ventricular wall through the base of the papillary muscle and false tendons. No direct connections between the subendocardial Purkinje fibers and those of the moderator band could be found within the boundaries of the papillary muscle, and this may form the basis for the relatively late activation of the papillary muscle as compared to the surrounding myocardial wall. The discontinuous fibers we observed are compatible with a current concept regarding the embryology of the ventricular specialized conducting system, and are not in conflict with normal impulse propagation.     

Membrane capacity of the cardiac Purkinje fibre

1. The basis for the relatively high membrane capacitance of the cardiac Purkinje fibre has been investigated.2. The capacitance measured by analysis of the cable response to a current step (square wave) was compared in the same fibres to the capacitance calculated from the foot of the propagated action potential. The square wave value for capacitance was 12.8 +/- 1.3 muF/cm(2) and that from the foot of the action potential, 2.4 +/- 0.5 muF/cm(2).3. The capacitative filling at the beginning of a voltage clamp in short Purkinje fibres was measured. The current-time course deviated from that predicted by a model membrane containing resistance and capacitance in parallel.4. The results obtained by both methods are consistent with two components to the membrane capacitance, with part in parallel with the membrane resistance (2.4 muF/cm(2)) and part (7 muF/cm(2)) in series with a resistor (300 Omega cm(2)).5. The value of the series resistor could be increased by decreasing the conductivity of the extracellular fluid.6. The possible anatomical basis for these findings is discussed.7. Implications of this model on the shape of the Purkinje fibre action potential and on the electrical triggering of contraction are considered.     

Temperature sensitivity of outward current in cardiac Purkinje fibers

1. In cardiac Purkinje fibers the temperature sensitivity of the membrane current flowing after 2 sec in response to depolarizing clamp steps was recorded. When the temperature was quickly lowered (30 sec) from 37 degrees C to 20 degrees C for a period of 2 min the outward current was markedly reduced. The effect was immediately reversed upon rewarming. The reduction in outward current on cooling was most pronounced between 30 degrees C and 20 degrees C. 2. In the range of anomalous rectification cooling to 20 degrees C shifted the i.v. relation in a negative direction by a constant amount of 20 nA. Outside this potential range (negative to -80 mV and positive to -45 mV) the slope conductance was reduced with a Q10 of about 1.3. 3. In the presence of dihydroouabain cooling did not further reduce the outward current in the potential range of anomalous rectification. However, negative to -80 mV and positive to -40 mV the slope conductance was reduced. The results support the view that part of the outward current is generated by an electrogenic sodium pump which is inhibited by cooling.     

The surface area of sheep cardiac Purkinje fibres

1. Measurements combining the techniques of point counting and line integration were performed on light and electron micrographs of Purkinje fibres from the sheep's heart. The measurements were aimed at determining membrane areas of importance for the cellular electrophysiology of this tissue.2. The mean volume fractions of the cells occupied by various constituents were: myofibrils, 0.234; mitochondria, 0.103; and nuclei, 0.009. The mean volume fraction of the fibres occupied by the interspaces between the tightly packed cells was 0.0023.3. The mean fractions of intercellular surface area occupied by junctional specializations were: nexus, 0.17; desmosome, 0.023; and fascia adherens, 0.014.4. The mean surface to volume ratio of the Purkinje cells and fibres was 0.46 mu(-1) which is 11.5 times the value of the surface to volume ratio of a long right circular cylinder 100 mu in diameter.5. There are two reasons for the increment in the surface to volume ratio of the fibre (when compared to that of a long right circular cylinder 100 mu in diameter): the multicellular composition of the fibres and the extensive folding of the surface of the cells.6. After correction for the intercellular nexal area the surface to volume ratio of a long cylindrical fibre 100 mu in diameter was 0.39 mu(-1), or about 10 times the value for a long right circular cylinder 100 mu in diameter. The surface to volume ratio of the tissue interspaces in the same fibre was 170 mu(-1).7. It was concluded that the total sarcolemmal area in this tissue is great enough so that the specific membrane capacitance could be about 1 muF/cm(2) and the specific membrane resistance 20,000 Omega cm(2).