The relationship among intracellular sodium activity, calcium, and strophanthidin inotropy in canine cardiac Purkinje fibers

The role of sodium and calcium ions in strophanthidin inotropy was studied by measuring simultaneously the electrical, mechanical, and intracellular sodium ion activities in electrically driven cardiac Purkinje fibers under conditions that change the intracellular sodium or calcium level (tetrodotoxin, strophanthidin, high calcium, and norepinephrine). Tetrodotoxin (TTX; 1-5 X 10(-6)M) shifted the action potential plateau to more negative values, shortened the action potential duration, and decreased the contractile tension and the intracellular sodium ion activity (aiNa). The changes in tension and in aiNa caused by TTX appear to be related since they had similar time courses. Strophanthidin (2-5 X 10(-7)M) increased tension and aiNa less in the presence of TTX, and, for any given value of aiNa, tension was less than in the absence of TTX. Increasing extracellular calcium (from 1.8 to 3.3-3.6 mM) or adding norepinephrine (0.5-1 X 10(-6)M) increased tension and decreased aiNa less in the presence than in the absence of TTX. When two of the above procedures were combined, the results were different. Thus, during the increase in aiNa and tension caused by strophanthidin in the presence of TTX, increasing calcium or adding norepinephrine increased tension markedly but did not increase aiNa further. In a TTX-high calcium or TTX-norepinephrine solution, adding strophanthidin increased both tension and aiNa, and the increase in tension was far greater than in the presence of TTX alone. The results indicate that: (a) the contractile force in Purkinje fibers is affected by a change in aiNa; (b) a decrease in aiNa by TTX markedly reduces the inotropic effect of strophanthidin, possibly as a consequence of depletion of intracellular calcium; (c) increasing calcium influx with norepinephrine or high calcium in the TTX-strophanthidin solution produces a potentiation of tension development, even if aiNa does not increase further; and (d) when the calcium influx is already increased by high calcium or norepinephrine, strophanthidin has its usual inotropic effect even in the presence of TTX. In conclusion, the positive inotropic effect of strophanthidin requires that an increase in aiNa be associated with suitable calcium availability.     

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.     

Fluctuations in membrane current driven by intracellular calcium in cardiac Purkinje fibers

Spontaneous oscillatory fluctuations in membrane potential are often observed in heart cells, but their basis remains controversial. Such activity is enhanced in cardiac Purkinje fibers by exposure to digitalis or K-free solutions. Under these conditions, we find that voltage noise is generated by current fluctuations that persist when membrane potential is voltage clamped. Power spectra of current signals are not made up of single time-constant components, as expected from gating of independent channels, but are dominated by resonant characteristics between 0.5 and 2 HZ. Our evidence suggests that the periodicity arises from oscillatory variations in intracellular free Ca that control ion movements across the surface membrane. The current fluctuations are strongly cross-correlated with oscillatory fluctuations in contractile force, and are inhibited by removing extracellular Ca or exposure to D600. Chelating intracellular Ca with injected EGTA also abolishes the current fluctuations. The oscillatory mechanism may involve cycles of Ca (or Sr) movement between sarcoplasmic reticulum and myoplasm, as previously suggested for skinned cardiac preparations. Our experiments in intact cells indicate that changes in surface membrane potential can modulate cytoplasmic Ca oscillations in frequency and perhaps amplitude as well. A two-way interaction between surface membrane potential and intracellular Ca stores may be a common feature of heart, neuron, and other cell types.     

Control of action potential duration by calcium ions in cardiac Purkinje fibers

It is well known that cardiac action potentials are shortened by increasing the external calcium concentration (Cao). The shortening is puzzling since Ca ions are thought to carry inward current during the plateau. We therefore studied the effects of Cao on action potentials and membrane currents in short Purkinje fiber preparations. Two factors favor the earlier repolarization. First, calcium-rich solutions generally raise the plateau voltage; in turn, the higher plateau level accelerates time- and voltage-dependent current changes which trigger repolarization. Increases in plateau height imposed by depolarizing current consistently produced shortening of the action potential. The second factor in the action of Ca ions involves iK1, the background K current (inward rectifier). Raising Cao enhances iK1 and thus favors faster repolarization. The Ca-sensitive current change was identified as an increase in iK1 by virtue of its dependence on membrane potential and Ko. A possible third factor was considered and ruled out: unlike epinephrine, calcium-rich solutions do not enhance slow outward plateau current, ikappa. These results are surprising in showing that calcium ions and epinephrine act quite differently on repolarizing currents, even though they share similar effects on the height and duration of the action potential.     

Multiple effects of calcium antagonists on plateau currents in cardiac Purkinje fibers

We studied the influence of Mn, La, and D600 on action potentials and plateau currents in cardiac Purkinje fibers. The Ca antagonists each abolished the second inward current, but they failed to act selectively. Voltage clamp experiments revealed two additional effects: decrease of slow outward current (iotachi) activation, and increase of net outward time-independent plateau current. These effects occurred at inhibitor concentrations used in earlier studies, and were essential to the reconstruction of observed Ca antagonist effects on electrical activity. The inhibitory influence of Mn, La, and D600 on iotachi suggested that iotachi activation might depend upon prior Ca entry. This hypothesis was not supported, however, when [Ca]omicron was varied: elevating [Ca]omicron enhanced Ca entry, but iotachi was nevertheless depressed. Thus, the results suggested instead that Ca antagonists and Ca ions have rather similar effects on iotachi, possibly mediated by changes in membrane surface charge.     

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.     

Intracellular sodium ion activity: reliable measurement and stimulation-induced change in cardiac Purkinje fibers

Recently Na+-selective microelectrodes (NaSM) have been used to measure quantitatively small changes in intracellular sodium ion activity (aiNa) and to determine a precise time course of comparatively rapid change in aiNa. In such studies, accurate measurement of aiNa requires the following criteria: (i) NaSM should have a fast response time and (ii) an NaSM and a conventional voltage microelectrode should measure the same membrane potential. These criteria were evaluated by measuring aiNa when membrane potential of cardiac Purkinje fibers was suddenly hyperpolarized and depolarized by changing stimulation rate. The NaSM coated with a conductive silver paint had fast response times so that rapid changes in aiNa could be reliably measured. The cardiac Purkinje fibers stimulated at a constant rate generated uniform membrane voltage and the NaSM and conventional microelectrode measured virtually the same membrane potential. This result is somewhat different from that reported under voltage-clamp condition by other investigators. The aiNa of the fibers increased as the stimulation rate was increased over the range of 0.5-3 Hz. In fibers stimulated at 1 Hz, cessation of stimulation was immediately followed by an exponential decline of aiNa with an average time constant of 53 +/- 9 s (SD, n = 8), or rate constant of 0.020 +/- 0.004/s. Restimulation of the fibers produced an exponential rise of aiNa with an average time constant of 65 +/- 12 s (n = 8). Similar results were obtained in fibers stimulated at 2 Hz.(ABSTRACT TRUNCATED AT 250 WORDS)     

Voltage-dependent block by tetrodotoxin of the sodium channel in rabbit cardiac Purkinje fibers.

The two-microelectrode, voltage-clamp technique was applied to rabbit cardiac Purkinje fibers to study the interaction of tetrodotoxin (TTX) with the slowly inactivating Na current. Binding of TTX to rested, inactivated, and activated channels was estimated by measuring the relative decrease of current at the beginning (rested and inactivated channels) and the end (activated channels) of a 1 s depolarizing clamp to -45 mV. The accelerated decline of the Na current in the presence of a submaximal dose of TTX was interpreted as an increase in blocking efficiency upon depolarization. The experiments show that activated as well as inactivated channels are more sensitive to TTX than are rested channels. The dissociation equilibrium constants for the three states are 3.5 X 10(-6) M for the rested, 0.94 X 10(-6) M for the activated, and 0.75 X 10(-6) M for the inactivated channels. The time course of activation block was dependent on TTX concentration. Rate constants for association and dissociation of the activated state are 1.3 X 10(6) M-1 X s-1 and 1.5 s-1, respectively.     

The interrelationship of cesium, intracellular sodium activity, and pacemaker potential in cardiac Purkinje fibers

The actions of cesium (Cs) on intracellular sodium activity (aiNa), membrane potentials, and force were studied in sheep cardiac Purkinje and myocardial fibers superfused in vitro. In Purkinje fibers, Cs (2 mM) decreased diastolic depolarization, aiNa (-6.7%, p less than 0.005), and force (-28.0%, p less than 0.01). The effects of 4 and 8 mM Cs were more pronounced. In quiescent fibers, Cs (2-4 mM) also decreased aiNa (-17.3%, p less than 0.005) and induced an initial hyperpolarization (+5.6 +/- 1.3%, p less than 0.005) followed by a return toward control. Diastolic depolarization was almost abolished by driving the fibers at 180/min (diastole was very short) but still Cs decreased aiNa (-15.4%). Tetrodotoxin decreased aiNa (-16.2%, p less than 0.025) and reduced the Cs-induced fall in aiNa (-2.2%, p less than 0.05). In zero [K]o, Cs decreased aiNa and caused repolarization. In 0.1 mM strophanthidin, Cs did not decrease aiNa any longer and affected the membrane potential little. In quiescent myocardial fibers, Cs (4 mM) decreased aiNa (-12.6%, p less than 0.05) and transiently hyperpolarized (+2.1%). Rubidium (2 mM) decreased aiNa and resting potential in Purkinje fibers and in myocardial fibers and also decreased diastolic depolarization in Purkinje fibers. Thus, cesium and rubidium decrease aiNa and modify the membrane potential but not through a block of the inward pacemaker current If.     

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.     

Ionic mechanisms of pacemaker activity in cardiac Purkinje fibers.

Rhythmic activity in cardiac Purkinje fibers can be analyzed by using the voltage clamp technique to study pacemaker currents. In normally polarized preparations, pacemaker activity can be generated by two distinct ionic mechanisms. The standard pacemaker potential (phase 4 depolarization) involves a slow potassium current, IK2. Following action potential repolarization, the IK2 channels slowly deactivate and thus unmask a steady background inward current. The resulting net inward current causes the slow pacemaker depolarization. Epinephrine accelerates the diastolic depolarization by promoting more complete and more rapid deactivation of IK2 over the pacemaker range of potentials. The catecholamine acts rather selectively on the voltage dependence of the gating mechanism, without altering the basic character of the pacemaker process. The nature of the pacemaker depolarization is altered by intoxication with high concentrations of cardiac glycosides or aglycones. These compounds promote spontaneous impulses in Purkinje fibers by a mechanism that supersedes the ordinary IK2 pacemaker. The digitalis-induced depolarization is generated by a transient inward current that is either absent or very small in untreated preparations. The transient inward current is largely carried by sodium ions. Its unusual time course probably reflects an underlying subcellular event, the oscillatory release of calcium ions from intracellular stores.     

Membrane currents following activity in canine cardiac Purkinje fibers.

Recent experiments in canine Pukinje fibers (Gadsby and Cranefield, 1979) have shown that following a period of sodium loading in K+-free solution a slowly decaying outward current is observed. This current has been attributed to the activity of the electrogenic Na+-K+ exchange pump. In the present paper we show that similar slowly decaying outward currents are observed following prolonged periods of overdrive with action potentials or with brief depolarizing voltage clamp pulses. The dependent of the prolonged outward current on the duration and frequency of the preceding period of overdrive and on the potential following overdrive is reported. We also present results which indicate that a large portion of this current can be induced by phasic Na+ loading through the fast-inward channel.     

Relationships between voltage and tension in sheep cardiac Purkinje fibers

The two-microelectrode technique of voltage clamping sheep cardiac Purkinje fibers was used to examine the changes in contraction which occur during trains of voltage clamps. (A "train" is defined as a series of voltage clamps delivered at a particular rate, beginning after a rest long enough that the effects of previous stimulation have died away.) Contractions showed striking staircases, or progressive changes in peak isometric tension, during trains. Short clamps, clamps to voltages more negative than --20 or --30 mV, or holding potentials less negative than the resting potential favored negative staircases, while long clamps, clamps to positive voltages, and holding potentials near the resting potential each favored positive staircases. The staircase behavior appeared to be due to changes in the initial rate of recovery of the ability to contract. The changes in staircase behavior as a function of clamp voltage suggested that the relationship between peak tension and clamp voltage should depend on the experimental design. When the steady-state contraction was plotted as a function of clamp voltage, voltage-tension relations like those recently reported for working ventricle were obtained, with a threshold between --30 and - -40 mV and a steep relation between tension and voltage. When the first contraction after a rest was plotted, the threshold voltage was more negative, the curve was flatter, and the peak tensions at inside positive voltages were reduced.     

Low conductance sodium channels in canine cardiac Purkinje cells.

Low conductance sodium (Na) channels have been observed in nerve, skeletal muscle, and cardiac cells. In cardiac tissues the higher amplitude, more commonly observed Na channel was first investigated in detail by Cachelin et al. (Cachelin, A.B., J.E. de Peyer, S. Kokubun, and H. Reuter, 1983, J. Physiol. (Lond.), 340:389-402). They also reported low amplitude Na channel events. We have studied this low conductance Na channel in single canine cardiac Purkinje cells using cell-attached patches. Patch pipette solutions contained either 140 or 280 mM NaCl, and cells were bathed in a solution of 150 mM KCl to bring their resting potential close to zero. In 140 mM Na+, during steps to -50 mV, the lower and higher openings had amplitudes of 0.57 +/- 0.2 and 1.2 +/- 0.2 pA (means +/- SD of Gaussian fits). In 280 mM Na+ at -50 mV, amplitudes were 0.72 +/- 0.2 and 1.55 +/- 0.2 pA. Over a substantial voltage range, the lower events had amplitudes of about one-third that of the higher events. The frequency of the low conductance openings varied in different patches from zero to 22% of total openings. Histograms of open durations and latencies at several voltages suggested no difference in kinetics between the two channel events. The behavior of the low conductance channels was more consistent with a second population of channels rather than a second open state.     

Slow inactivation of a tetrodotoxin-sensitive current in canine cardiac Purkinje fibers.

We used the two-microelectrode voltage clamp technique and tetrodotoxin (TTX) to investigate the possible occurrence of slow inactivation of sodium channels in canine cardiac Purkinje fibers under physiologic conditions. The increase in net outward current during prolonged (5-20 s) step depolarizations (range -70 to +5 mV) following the application of TTX is time dependent, being maximal immediately following depolarization, and declining thereafter towards a steady value. To eliminate the possibility that this time-dependent current was due to inadequate voltage control of these multicellular preparations early during square clamp pulses, we also used slowly depolarizing voltage clamp ramps (range 5-100 mV/s) to ensure control of membrane potential. TTX-sensitive current also was observed with these voltage ramps; the time dependence of this current was demonstrated by the reduction of the peak current magnitude as the ramp speed was reduced. Reducing the holding potential within the voltage range of sodium channel inactivation also decreased the TTX-sensitive current observed with identical speed ramps. These results suggest that the TTX-sensitive time-dependent current is a direct measure of slow inactivation of canine cardiac sodium channels. This current may play an important role in modulating the action potential duration.     

Extracellular [K+] fluctuations in voltage-clamped canine cardiac Purkinje fibers.

Membrane currents and extracellular [K+] were measured in canine Purkinje strands during voltage-clamp steps to plateau or diastolic potentials. Extracellular [K+] increased during step depolarizations and decreased during step hyperpolarizations. On hyperpolarization, the largest fraction of the K+ depletion occurred during the initial 500 ms of the voltage-clamp step and was correlated with a potassium depletion current, the id. A slower component of the depletion also occurred on hyperpolarization and had a time constant consistent with cylindrical diffusion of potassium within the Purkinje strands. On depolarization, there is an accumulation of K+ that is correlated with the plateau current ix. On termination of depolarizing test pulses, the K+ accumulation decays with a time course similar to the ix tail current. Surprisingly, no accumulation of K+ occurred during the arrhythmogenic transient inward current, TI, suggesting that the selectivity of this current should be reevaluated.     

Photoalteration of calcium channel blockade in the cardiac Purkinje fiber.

Organic compounds that block calcium channel current (calcium antagonists) are important tools for the characterization of this channel. However, the practically irreversible nature of this block restricts the usefulness of this group of drugs. In this paper, we investigate the influence of light on calcium channel blockade by several organic compounds. Our results show that inhibition of calcium channel current by two dihydropyridine derivatives that contain an o-nitro moiety (nisoldipine and nifedipine) can be rapidly reversed by illumination. The energy range important to this reaction is for light wavelengths between 320 and 450 nm. Calcium channel inhibition by two other dihydropyridine derivatives (nicardipine and nitrendipine) as well as by D600, is not modulated by illumination. These results indicate that the photosensitivity of certain dihydropyridine calcium channel blockers make these compounds useful as reversible blockers of this channel.