Rhythms produced by high-amplitude periodic stimulation of spontaneously beating aggregates of embryonic chick ventricular myocytes.
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Animals↗
Biomedical subjects
A Shrier
Publications and source records attributed to A Shrier.
High affinity forskolin inhibition of L-type Ca2+ current in cardiac cells.
The diterpene forskolin is widely known for its ability to directly activate adenylyl cyclase and consequently increase intracellular cAMP. In cardiac cells, one result is a cAMP-mediated increase in the L-type Ca2(+)-channel current (ICa). However, forskolin was also shown recently to affect a number of ionic channels in noncardiac cells by mechanisms that do not involve activation of adenylyl cyclase. The present study reveals such an effect of forskolin on cardiac Ca2+ channels. Indeed, under appropriate conditions, forskolin was found to cause an inhibition of ICa. Although the stimulation of adenylyl cyclase and ICa requires micromolar concentrations of forskolin, the inhibitory effect of forskolin was observed in the nanomolar range of concentrations, i.e., 2-3 orders of magnitude lower. This high affinity forskolin inhibition of ICa was observed when ICa was previously enhanced via a cAMP-dependent pathway, but not when ICa was at its basal level or when the current was elevated by the dihydropyridine Bay K 8644. The inhibitory effect occurred at a site of action remote from adenylyl cyclase, because forskolin similarly inhibited ICa that had been previously elevated by isoprenaline (a beta-adrenergic agonist) or directly by intracellular perfusion with cAMP. Under these conditions, forskolin was inhibitory when applied to either side of the cell membrane, but only in its lipid-soluble form. The inhibitory effect of forskolin appeared to be independent of membrane potential and was not accompanied by a change in the time constants of ICa activation and inactivation. This may indicate that forskolin mainly reduces the number of functional Ca2+ channels without changing the gating of individual channels. However, the reduction in ICa amplitude was not equally distributed among the different exponential components that constitute ICa, which suggests that forskolin also modifies the resting state of the channels. This novel high affinity forskolin inhibition of ICa may take place at some step in the pathway between cAMP and Ca2+ channel phosphorylation and/or at Ca2+ channels only after they have been phosphorylated.
3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗
Inactivation of A currents and A channels on rat nodose neurons in culture.
Cultured sensory neurons from nodose ganglia were investigated with whole-cell patch-clamp techniques and single-channel recordings to characterize the A current. Membrane depolarization from -40 mV holding potential activated the delayed rectifier current (IK) at potentials positive to -30 mV; this current had a sigmoidal time course and showed little or no inactivation. In most neurons, the A current was completely inactivated at the -40 mV holding potential and required hyperpolarization to remove the inactivation; the A current was isolated by subtracting the IK evoked by depolarizations from -40 mV from the total outward current evoked by depolarizations from -90 mV. The decay of the A current on several neurons had complex kinetics and was fit by the sum of three exponentials whose time constants were 10-40 ms, 100-350 ms, and 1-3 s. At the single-channel level we found that one class of channel underlies the A current. The conductance of A channels varied with the square root of the external K concentration: it was 22 pS when exposed to 5.4 mM K externally, the increased to 40 pS when exposed to 140 mM K externally. A channels activated rapidly upon depolarization and the latency to first opening decreased with depolarization. The open time distributions followed a single exponential and the mean open time increased with depolarization. A channels inactivate in three different modes: some A channels inactivated with little reopening and gave rise to ensemble averages that decayed in 10-40 ms; other A channels opened and closed three to four times before inactivating and gave rise to ensemble averages that decayed in 100-350 ms; still other A channels opened and closed several hundred times and required seconds to inactivate. Channels gating in all three modes contributed to the macroscopic A current from the whole cell, but their relative contribution differed among neurons. In addition, A channels could go directly from the closed, or resting, state to the inactivated state without opening, and the probability for channels inactivating in this way was greater at less depolarized voltages. In addition, a few A channels appeared to go reversibly from a mode where inactivation occurred rapidly to a slow mode of inactivation.
Animals↗
Characterization of potassium-dependent currents in protoplasts of corn suspension cells.
Protoplasts obtained from corn (Zea mays) suspension cells were studied using the whole cell patch-clamp technique. One time-independent current, as well as two time-dependent currents were identified. All three currents were reduced by tetraethylammonium (9 millimolar), a K(+) channel blocker. The time-independent current had a nearly linear current-voltage relationship and its reversal potential, defined as the voltage at which there is zero current, was highly dependent on the extracellular potassium concentration. One of the two time-dependent currents was activated, with rapid kinetics, by membrane hyperpolarization to potentials more negative than -100 millivolts. The second time-dependent current was activated with a sigmoidal time course by membrane depolarization to potentials more positive than -60 millivolts. It exhibited no inactivation and was carried primarily by potassium ions. These characteristics suggest that this latter current is caused by the voltage-dependent opening of delayed-rectifier K(+) channels. These three currents, which are not generated by the plasmalemma H(+)-ATPase, are likely to assist in the regulation of the cellular K(+) fluxes and membrane potential.
Journal Article↗
Interactive effects of isoprenaline, forskolin and acetylcholine on Ca2+ current in frog ventricular myocytes.
1. Calcium currents (ICa) were measured in single cells isolated from frog ventricle using the whole-cell patch-clamp technique and a perfused pipette. The dose-dependent stimulatory effects of isoprenaline (Iso, 0.1-100 microM) and forskolin (Fo. 0.1-50 microM) on ICa were determined in the presence and absence of acetylcholine (ACh, 10 microM) and/or threshold concentrations of Fo (0.2 microM) and Iso (0.05 microM), respectively. EC50 (i.e. concentration of Iso or Fo at which the response was 50% of the maximum) and Emax (i.e. maximal stimulation of Ica expressed as percentage increase in ICa with respect to control) were measured under each condition. 2. ACh increased EC50 for the stimulatory action of Iso on ICa from 0.84 to 3.72 microM while it reduced Emax from 658 to 185%. Thus, ACh mainly reduced the efficacy of Iso to stimulate ICa. 3. ACh increased EC50 for the stimulatory action of Fo on ICa from 2.06 to 10.26 microM but only slightly reduced Emax from 893 to 778%. Thus, ACh mainly reduced the potency of Fo to stimulate ICa. 4. Intracellular perfusion with 100 microM of hydrolysis-resistant GTP analogues, GTP-gamma-S [guanosine-5'-O-(3-thiotriphosphate)] and Gpp (NH)p (5'-guanylylimido-diphosphate), had no effect on basal ICa but reduced by greater than 50% the stimulatory effect of 2 microM-Iso on ICa. 5. In the presence of Gpp(NH)p or GTP-gamma-S, Fo (3 microM) reversibly increased ICa by 490%, as compared to a 717% increase in control (GTP) intracellular solution. Although ACh could still inhibit Fo-stimulated ICa, the degree of inhibition was significantly smaller than in the presence of GTP. 6. Extracellular perfusion with low concentrations of a combination of Iso (33 nM) and Fo (330 nM) enhanced ICa to a much greater extent than did either agent alone at 3 times higher concentrations. Thus, low concentrations of Iso and Fo appear to increase ICa in a synergistic fashion. 7. ICa stimulated by a combination of Iso and Fo appeared to be more resistant to inhibition by ACh than when stimulated by either alone. It was the efficacy, rather than the potency, of ACh to inhibit ICa that was reduced upon dual stimulation of ICa. 8. In the presence of 0.2 microM-Fo, EC50 and Emax for the effects of Iso on ICa were 0.27 microM and 619%, respectively. By comparison with the effects of Iso alone, Fo reduced EC50 approximately 3 times with no significant change in maximal stimulation.(ABSTRACT TRUNCATED AT 400 WORDS)
Acetylcholine↗
Repolarization current in embryonic chick atrial heart cells.
1. We have measured the delayed rectifier potassium current, IK, with the whole-cell patch-clamp technique from single cultured cells from the atria of 6- to 11-day-old chick embryonic hearts. 2. The IK component was activated with depolarizing voltage-clamp steps positive to -30 mV (holding potential in the -60 to -40 mV range). Maximum activation of the IK conductance occurred at +25 mV, based on deactivation, or tail current amplitudes upon return to the holding potential. Activation and tail current kinetics could both be described by single-exponential functions of time. 3. The IK kinetics were voltage dependent, with a maximum time constant, tau n, of approximately 2 s at V = -20 mV. 4. The IK reversal potential measurements suggest that this current is carried predominantly by potassium ions. 5. The IK results from single cells, or clusters of two or three cells, were comparable to our recent measurements of IK (IX2) in heart cell aggregates (Shrier & Clay, 1986). However, we did not obtain clear evidence in single cells for the IX1 repolarization current, in contrast to the aggregate results. 6. Computer simulations based on our IK measurements demonstrate that this component is sufficient to initiate repolarization of the action potential in single cells. However, it is not sufficient to reproduce the latter phase of repolarization for potentials negative to -30 mV. Addition of a relatively small IX1 component (2% in absolute terms compared to the aggregate work) is sufficient to account for this part of the action potential.
Action Potentials↗
Phase-locked rhythms in periodically stimulated heart cell aggregates.
We have studied the effect of injecting a periodic train of current pulses into spontaneously beating aggregates of embryonic chick ventricular heart cells. Over a range of stimulation frequencies around the intrinsic frequency of an aggregate we find one action potential for each stimulus with a fixed latency from each stimulus to the subsequent action potential. For a stimulation frequency higher (lower) than the intrinsic frequency, this corresponds to overdrive (underdrive). At high frequencies of stimulation dropped beats occur leading to complex rhythms analogous to various Wenckebach rhythms observed clinically. At higher stimulation frequencies one can obtain a complete suppression of action potential generation. At low frequencies of stimulation, there are rhythms containing escape beats. Almost every rhythm seen bears a striking resemblance to some cardiac arrhythmia. We present a simple classification scheme that predicts the order of appearance of all the classes of rhythms experimentally observed as one changes the stimulation frequency. We propose that this scheme can be used generally to describe the behavior of other biological oscillators.
Action Potentials↗
Phase resetting in a model of cardiac Purkinje fiber.
The phase-resetting response of a model of spontaneously active cardiac Purkinje fiber is investigated. The effect on the interbeat interval of injecting a 20-ms duration depolarizing current pulse is studied as a function of the phase in the cycle at which the pulse is delivered. At low current amplitudes, a triphasic response is recorded as the pulse is advanced through the cycle. At intermediate current amplitudes, the response becomes quinquephasic, due to the presence of supernormal excitability. At high current amplitudes, a triphasic response is seen once more. At low stimulus amplitudes, type 1 phase resetting occurs; at medium amplitudes, a type could not be ascribed to the phase resetting because of the presence of effectively all-or-none depolarization; at high amplitudes, type 0 phase resetting occurs. The modeling results closely correspond with published experimental data; in particular type 1 and type 0 phase resetting are seen. Implications for the induction of ventricular arrhythmias are considered.
Electric Conductivity↗
Universal bifurcations and the classification of cardiac arrhythmias.
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Animals↗
Prediction of complex atrioventricular conduction rhythms in humans with use of the atrioventricular nodal recovery curve.
Theoretical considerations indicate that complex patterns of atrioventricular conduction produced by rapid atrial stimulation can be predicted from changes in atrioventricular conduction produced by premature stimulation of the atrium. The purpose of this study was to evaluate the validity of this theoretical approach in seven patients undergoing electrophysiologic investigation. The atrioventricular nodal recovery curve was determined at two different basic cycle lengths. Subsequently, periodic atrial stimulation was delivered for 30 sec periods over a range of frequencies giving 11, Wenckebach, reverse Wenckebach, and 21 rhythms. The recovery curve data was then used to compute the response to periodic stimulation by an iterative technique. The conduction patterns actually seen during periodic atrial stimulation showed close agreement with the computed patterns. This work thus provides a unified explanation for the appearance of Wenckebach, reverse Wenckebach, alternating Wenckebach, and high grade block rhythms.
Atrioventricular Node↗
Repolarization currents in embryonic chick atrial heart cell aggregates.
Outward membrane currents in aggregates of atrial cells prepared from 7-12-d-old chick embryonic hearts were measured with the two microelectrode voltage-clamp technique. Two outward current components, Ix1 and Ix2, were found in the plateau potential range of the action potential. The Ix1 component is activated between -50 and -20 mV; the Ix2 component is activated between -15 and +20 mV. The Ix1 component inwardly rectifies, whereas Ix2 has an approximately linear current-voltage relation. These preparations lack a time-dependent pacemaker current component, even though they beat spontaneously with an interbeat interval of approximately 1 s. A mathematical model of electrical activity is described based on our measurements of time-dependent outward current, and measurements in the literature of inward current components.
Animals↗
Potassium channel blockade: A mechanism for suppressing ventricular fibrillation.
The suppression of ventricular fibrillation by antidysrhythmic drugs is well correlated with their ability to block potassium channels in nerve and cardiac membranes. Blockade of potassium channels reduces electrical inhomogeneities in action potential and conduction parameters that lead to ventricular fibrillation. These actions tend to effectively decrease the electrical size of the heart, which suggests a mechanism for antifibrillatory drug action. The receptor sites for antifibrillatory drug action (IK blockade) appear to be on the outside of the cardiac membrane whereas receptors for antiarrhythmic drug action (INa blockade) appear to be on the inside of the cardiac membrane.
4-Aminopyridine↗
Phase resetting of spontaneously beating embryonic ventricular heart cell aggregates.
The influence of isolated 20-ms duration current pulses on the spontaneous rhythm of embryonic chick ventricular heart cell aggregates was studied. A pulse could either delay or advance the time of occurrence of the next action potential, depending on whether it fell early or late in the cycle. As the stimulus amplitude was increased, the transition from delay to advance occurred over a narrower range of coupling intervals. At low-stimulus amplitudes the transition from delay to advance occurred in a smooth continuous fashion; at medium-stimulus amplitudes the transition was discontinuous; at high-stimulus amplitudes graded action potentials were seen. It was impossible to annihilate spontaneous activity in aggregates with a single stimulus. The phase-resetting response to hyperpolarizing pulses was qualitatively the reverse of that produced by depolarizing pulses. A very high-amplitude depolarizing or hyperpolarizing pulse could produce rapid repetitive activity. Theoretical aspects of these phenomena are discussed.
Animals↗
Single-channel analysis of fast transient potassium currents from rat nodose neurones.
Single channels that underlie the fast transient potassium current (IA) were recorded, using patch-clamp techniques, from cultured sensory neurones. The open channel conductance was approximately 22 pS, and was constant over most of the physiological voltage range; single-channel conductance decreased at more depolarized levels. Summing single-channel currents resulted in an average current whose kinetics were similar to the macroscopic IA. The inactivation of these currents, at the potentials we studied, was fitted with a single exponential with a time constant of approximately 30 ms. For the currents evoked by large depolarizing steps (to +40 mV), the mean channel open time equals approximately 30 ms. For currents evoked at less depolarized levels (to 0 mV), the mean open time equals approximately 15 ms, half the inactivation time constant.
Action Potentials↗
Effects of D-600 on sodium current in squid axons.
The effects of the calcium antagonist D-600 (methoxyverapamil) on the excitatory inward sodium current, INa, of internally perfused squid giant axons were studied under voltage-clamp conditions. We observed little or no effect of the drug when it was added to the external solution at concentrations of 10-200 microM. Furthermore, it did not produce a frequency, or use-dependent block of INa when repetitive voltage-clamp pulses were used at rates of 2-5 Hz. However, it did produce use-dependent blockade of INa when it was placed internally at a concentration of 200 microM. These results in conjunction with other studies suggest that D-600 is a selective blocker of calcium channels in squid axons when the drug is placed in the external solution. Its effects, when placed in the internal solution, are similar to those of permanently charged local anesthetic derivatives, which also produce use-dependent block of INa.
Animals↗
Phase resetting of the rhythmic activity of embryonic heart cell aggregates. Experiment and theory.
Injection of a current pulse of brief duration into an aggregate of spontaneously beating chick embryonic heart cells resets the phase of the activity by either advancing or delaying the time of occurrence of the spontaneous beat subsequent to current injection. This effect depends upon the polarity, amplitude, and duration of the current pulse, as well as on the time of injection of the pulse. The transition from prolongation to shortening of the interbeat interval appears experimentally to be discontinuous for some stimulus conditions. These observations are analyzed by numerical investigation of a model of the ionic currents that underlie spontaneous activity in these preparations. The model consists of: Ix, which underlies the repolarization phase of the action potential, IK2, a time-dependent potassium ion pacemaker current, Ibg, a background or time-independent current, and INa, an inward sodium ion current that underlies the upstroke of the action potential. The steady state amplitude of the sum of these currents is an N-shaped function of potential. Slight shifts in the position of this current-voltage relation along the current axis can produce either one, two, or three intersections with the voltage axis. The number of these equilibrium points and the voltage dependence of INa contribute to apparent discontinuities of phase resetting. A current-voltage relation with three equilibrium points has a saddle point in the pacemaker voltage range. Certain combinations of current-pulse parameters and timing of injection can shift the state point near this saddle point and lead to an interbeat interval that is unbounded . Activation of INa is steeply voltage dependent. This results in apparently discontinuous phase resetting behavior for sufficiently large pulse amplitudes regardless of the number of equilibrium points. However, phase resetting is fundamentally a continuous function of the time of pulse injection for these conditions. These results demonstrate the ionic basis of phase resetting and provide a framework for topological analysis of this phenomenon in chick embryonic heart cell aggregates.
Action Potentials↗
A comparative study of collagenase- and trypsin-dissociated embryonic heart cells: reaggregation, electrophysiology, and pharmacology.
The electrophysiological and pharmacological properties of aggregates prepared from cells of 7-day-old chick embryo heart ventricles depend on the enzyme used for cell dissociation. The mean beat rate of aggregates formed from trypsin-dissociated cells was about 53 beats/min whereas aggregates formed from collagenase-dissociated cells had a mean beat rate of more than twice this value. Spontaneous activity of most aggregates formed from trypsin-dissociated cells was inhibited by elevating external potassium or by adding tetrodotoxin to the medium. A similar response to potassium was seen in all aggregates formed from collagenase-dissociated cells. However, approximately half of the aggregates formed from collagenase-dissociated cells were tetrodotoxin insensitive. Intracellular microelectrode recordings demonstrated that aggregates formed from collagenase-dissociated cells typically had reduced action potential maximal upstroke velocities and depolarized threshold potentials in comparison to those recorded from aggregates formed from trypsin-dissociated cells. In the presence of tetrodotoxin the maximal upstroke velocity of aggregates formed from either collagenase- or trypsin-dissociated cells decreased markedly. In the case of the collagenase-treated cells, the spontaneous activity which persisted in the presence of tetrodotoxin was abolished by the slow channel blocker D-600. Computer simulation of membrane depolarization supports the view that aggregates formed from collagenase-treated cells have a reduced fast inward sodium current and a significant leakage current. Aggregates prepared from trypsin-dissociated cells display properties which more closely resemble those of intact 7-day embryonic ventricular tissue. We therefore conclude that, contrary to previous reports, collagenase is not the enzyme necessarily best suited for cell dissociation in all tissue culture studies.
Action Potentials↗
Comparison of the pacemaker properties of chick embryonic atrial and ventricular heart cells.
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Animals↗