Frog Atrium Research Articles

Effects of dantrolene sodium on the excitation-contraction coupling of the mammalian and amphibian cardiac muscle

Dantrolene sodium at concentrations which inhibit the twitch tension of frog toe muscle and is without effect on resting and action potentials exhibited a positive inotropic action in mammalian and amphibian myocardium. In guinea-pig atrial muscle, twitch tension was initially decreased and thereafter gradually increased above the control. In frog atrial muscle, twitch tension was potentiated by dantrolene sodium without a prior phase of decreased tension. In both guinea-pig and frog atrial muscles, the action potential duration was prolonged by the drug. The amplitude of the slow response was augmented by dantrolene sodium in guinea-pig and frog atria that were partially depolarized by KCl. These results suggest that dantrolene sodium increases the slow inward Ca current which consequently potentiates the twitch tension. The transient decrease of twitch tension in the guinea-pig atrial muscle could be due to inhibition of calcium release from the sarcoplasmic reticulum as postulated for skeletal muscle. The possibilities that the drug acts either on the Ca permeability of the membrane of the sarcoplasmic reticulum or on the junctional portion between the sarcolemma and the subsarcolemmal cisternae are discussed.

The contribution of potassium accumulation to outward currents in frog atrium.

1. Voltage-clamp experiments on frog atrial muscle were designed to distinguish effects due to K accumulation in extracellular spaces from those due to activation of K conductance mechanisms in the membrane. 2. The set of instantaneous current-voltage relations obtained at various external K concentrations following depolarization to about -10 mV for several seconds was found to be quite different from that obtained before the depolarization. Hence the process of increasing the extracellular K concentration cannot account for all the time-dependent changes in outward current during depolarization. 3. Although the instantaneous current-voltage relations obtained at different values of external K concentration before prolonged depolarization show the cross-over phenomenon (Noble, 1965), those obtained at the end of the depolarization did not show this feature. It is concluded that the current-voltage relations for the channels conducting the time-dependent K current do not show cross-over. 4. These results were used to construct a model involving both K activation and K accumulation. This model successfully reproduces the appearance of a very slow component in outward current decay tails which, when subtracted by semi-exponential curve-stripping leaves a component with the real time constant of conductance change. The model does not however reproduce the appearance of a fast decaying component without adding a second conductance mechanism, or assuming non-exponential decay of a single conductance mechanism. 5. It is therefore suggested that i chi, fast is not a perturbation of i chi, slow or of iK1 by the process of K accumulation. This conclusion is reinforced by the results of experiments showing that the relative magnitude of i chi, fast is not greatly changed by substantially increasing the external K concentration in order to reduce the proportionate effect of K accumulation on the K concentration.

Properties of cardiac contractions in zero sodium solutions: Intracellular free calcium controls slow channel conductance

Guinea-pig, rat and frog atria bathed in zero sodium, contract in response to high intensity electrical stimuli. A transient positive inotropic response after sodium removal is followed by a decline and then leveling off of twitch tension. Abolition of sodium-calcium exchange in zero sodium medium results in a species-dependent decline in the rate of relaxation and an increase in diastolic tension. The characteristic positive inotropic effect of isoproterenol is reduced in rat and frog atria and abolished in guinea-pig atria bathed in zero sodium. Cyclic AMP generation in response to isoproterenol is reduced but not abolished in zero sodium, and time to peak tension is still shortened in response to the catecholamine. Addition of strontium ion to guinea-pig atria in zero sodium restores the ability of isoproterenol to increase twitch tension and to enhance slow channel conductance now carried by strontium. Chelation of calcium ion in the presence of strontium ion by the calcium chelator ethyleneglycol bis-(β-amino ethylether)- n,n,n′,n′ tetraacetic acid (EGTA) K A Ca K A Sr > 100 greatly increases the duration of slow action potentials carried by strontium. These data suggest that intracellular calcium accumulation, but not strontium accumulation, decreases slow channel conductance and the ability of isoproterenol to enhance slow channel conductance. It is concluded that sodium reduction tends to increase intracellular free calcium by decreasing the process of sodium-calcium exchange which normally contributes to relaxation. A large accumulation of intracellular calcium diminishes the ability of the tissue to respond to isoproterenol both by reducing the ability of the tissue to generate cyclic AMP and by directly inhibiting slow channel conductance.

Antiarrhythmic and electrophysiological properties of etmozin and its diethylamino analog

Effects of antiarrhythmic drug etmosin and its diethylamine analogue (etmosin DAA) were compared in dogs with the ventricular rhythm disturbances induced by coronary artery ligation according to Harris' method. As demonstrated, both drugs stopped ventricular rhythm disturbances, but etmosin DAA had a more rapid and prolonged effect. Electrophysiological properties of etmosin and etmosin DAA were studied by the method of potential fixation on trabeculae of frog atria. Both drugs proved to reduce rapid sodium inflow, etmosin DAA acting more intensively and longer. Taking into account the high antiarrhythmic activity of etmosin DAA it is believed that this drug had good prospects for further investigation.

Effect of creatine phosphate on the slow inward calcium current, action potential, and the contractile force of frog atrium and ventricle

The effects of creatine phosphate on frog heart muscle contraction have been studied further. It has been shown that after inhibition of mitochondrial oxidative phosphorylation by sodium cyanide the frog heart ventricle contraction can be maintained at a high level by addition of creatine phosphate. The effect of creatine phosphate on the contractile force and action potential is similar for frog heart ventricle and atrium. It has been directly demonstrated by using the voltage-clamp technique that creatine phosphate controls the slow inward calcium current through the surface membrane of frog atrium cells.

The measurement and dynamic implications of thin filament lengths in heart muscle.

1. The lengths of the thin filaments in amphibian and mammalian cardiac muscle have been determined from electron micrographs of serial transverse sections. Thin filament lengths in frog atrial trabeculae range from 0.8 to greater than 1.3 micrometers, with a maximum possible error of 0.14--0.15 micrometer. In rat atrial tissue the span is from 0.6 to more than 1.1 micrometer, whereas in rat papillary muscle the breadth of the distribution is much narrower, from 0.9 to greater than 1.1 micrometer. Double overlap of thin filaments should, therefore, exist over a wide range of sarcomere lenghts. Thin filaments from opposite halves of a sarcomere accommodate each other by flexing up to an angle of about 2 degrees and moving from the trigonal position among the thick filaments to the centre of the region between two thick filaments. Such rearrangement probably contributes to the internal resistance to shortening in the muscle. 2. Except for the variation in thin filament lengths, the over-all morphology of the cardiac sarcomere is generally similar to that found in skeletal muscle. Thick filaments in heart muscle are uniform in length, and their profiles change along their lengths. They are generally round in the M band, triangular adjacent to the M band, round again in the overlap region, and either round or triangular near the tapered tips. The M bridges in rat cardiac tissue link neighbouring thick filaments to form a symmetric hexagonal array, whereas in the frog atrium, the M bridge connexions are incomplete and often form isolated triangular clusters. 3. Computed sarcomere length-developed tension curves were calculated using the thin filament length distributions and the assumptions basic to the sliding filament theory of muscle contraction. The curves for atrial tissue have plateau regions approximately as wide as the one-half micron variation in thin filament length. 4. Work done against the internal loads during systole may be stored as potential energy and released during diastole to produce sarcomeric re-extension.

The Nature of the Initial Positive Inotropic Effect of K Depletion in Bullfrog Atrial Muscle

In frog atrium, K depletion exerts initial, and late positive inotropic effects. The initial effect on the membrane potential, current and tension components was studied using a double gap method under voltage clamped and unclamped conditions. In both cases, the presence of the initial effect was demonstrated. The effect was clearer in preparations in which a positive inotropic effect to toxic doses of ouabain (10(-6) M) had already been attained. This suggests that the initial phase is independent from Na-pump inhibition. Voltage clamp study revealed that the slow inward calcium current (ICa) was enhanced transiently after K depletion concomitant with an augmentation of ICa-dependent phasic tension. ICa-independent tonic tension was not altered at this initial phase. For the enhancement of ICa, two mechanisms appeared to be involved, one an enhancement of gCa and the other a faster and/or longer opening of the slow channel. These results indicated that an enhancement of ICa together with the well-known prolongation of action potential were the cause of the initial positive inotropic effect of K depletion.

Displacement of activator thresholds in cardiac muscle by protons and calcium ions.

1. The Na current threshold in sheep cardiac Purkinje fibres and in frog atrium is shifted in a positive direction by protons and Ca2+ ions. The titration curves for Purkinje fibres are consistent with a surface potential of -18 mV at pH 7.4 and 1.8 mM-Ca. 2. In Purkinje fibres, the pacemaker K current activation curve, s infinity, is shifted in a positive direction by Ca2+ ions. The results are consistent with a surface potential of -16 mV in normal physiological solutions. 3. The results on s inifinity during pH changes are unexpected. As also shown by Van Bogaert, Vereecke & Carmeliet (1975) the voltage shifts are usually in the opposite direction to that expected from titration of external surface negative charges. 4. Acid solutions reduce the magnitude of iK2 when fully activated. Alkalinity has little effect on iK2. 5. Acidification and alkalinization are both capable of arresting spontaneous activity in Purkinje fibres. The effects of acidity are usually irreversible. The effects of alkalinity are reversible.

The relationship between contractile tension and intracellular cyclic AMP level in frog atrium: An analysis by using NAF, verapamil and ouabain

1. The relationship between contractile tension and intracellular cyclic AMP level was studied in bullfrog atrium, modifying the external Ca ion concentration and using NaF, verapamil and ouabain. 2. Withdrawal of Ca led to a decrease of contractile tension and a significant increase of cyclic AMP level. 3. Likewise, application of verapamil (8 μg/ml) reduced contractile tension and enhanced the level of cyclic AMP. 4. NaF showed a marked positive inotropic effect on the muscles at a concentration of 10 −5 M, but a negative inotropic effect at 10 −2 M. 10 −5 M of NaF produced a transient increase and afterwards a later decrease of the intracellular cyclic AMP. Conversely, on the addition of 10 −2 M, the level of cyclic AMP remained elevated. The level of cyclic AMP was decreased with time of incubation with ouabain, while an augmentation in the contractile tension was observed. 5. These results are interpreted as indicating that the intracellular free Ca ions may regulate the intracellular level of myocardial cyclic AMP.

Slow inward current and cardiac contraction; Effects of adenine and guanine nucleotides and nucleosides on frog atrium in relation with those of adrenaline

Slow inward current and cardiac contraction; Effects of adenine and guanine nucleotides and nucleosides on frog atrium in relation with those of adrenaline

Two mechanisms for positive inotropism of low-K Ringer solution in bullfrog atrium

IT has long been known that a reduction of K concentration in external fluid immediately potentiates the twitch contraction of frog heart1. Microelectrode studies2,3 have revealed a hyperpolarisation of the membrane and a prolongation of the action potential, which was thought to be a cause of the potentiation of contraction. But, when the muscle is soaked in K-depleted fluid the membrane Na/K ATPase and the activity of the Na pump are inhibited4 and Na ions progressively accumulate within the cell5. In myocardium, the accumulation or rise of [Na]i produces a secondary increase of [Ca]i due to Na–Ca exchange diffusion and the consequent positive inotropic effect6,7. Thus, Ehara8 has demonstrated the presence of a delayed potentiating effect of low-K Ringer solution possibly due to Na-pump inhibition in the frog ventricle as well as its immediate inotropic effect. We have tried to clarify further the nature of these inotropic actions. We show here that, in the frog atrium, augmentation of Ca-inward current (Ica) and increase of Ica-independent tonic tension, related to Na–Ca exchange, are responsible for the immediate and delayed inotropic effects under voltage clamp, respectively.

Identification of the pace-maker current in frog atrium.

1. The nature and interactions of the membrane currents underlying induced pace-maker activity in frog atrial muscle have been investigated using a double sucrose gap technique. 2. The membrane current which controls the speed of the atrial pacemaker depolarization (the pace-maker current, ip), is shown to be an outward current activated within the plateau potential range of a normal action potential. The subsequent deactivation of ip at more negative potentials unmasks the depolarizing action of time-independent inward membrane currents so that a pace-maker potential can result. 3. The deactivation of ip over a limited potential range (between about -30 and -60 mV) can be reliably recorded by switching on the voltage clamp during an induced pace-maker depolarization. 4. Investigation of the time and voltage-dependent behaviour of ip over a much wider potential range is less straightforward. How ip can be separated from other components of outward current present in the decay tails following square voltage clamp depolarizations is described. 5. The majority of such current decay tails contain three components of outward current. It appears that two of these components, one of which is ip, are true Hodgkin-Huxley conductance systems chiefly carrying potassium ions. 6. The nature of the third current, which decays very slowly at moderate membrane potentials (about -40 mV), is discussed and reasons are briefly given for considering it to result from the accumulation of potassium ions in extracellular spaces. Preliminary evidence that potassium depletion occurs at potentials negative to the resting potential of the trabeculum is also presented. 7. Because of the obvious complexities involved, a quantitative analysis of the atrial outward currents is not attempted here but forms the subject of a following paper (Brown, Clark & Noble, 1976a).

Analysis of pace-maker and repolarization currents in frog atrial muscle.

1. A quantitative analysis of the time-dependent component of outward membrane current in atrial wall trabeculae from Rana catesbeiana and Rana ridibunda has been carried out using a double sucrose gap technique. 2. Separation of the different components of delayed outward current was hampered by the sigmoid onset of one of the outward current systems ixfast and by the development of potassium ion accumulation which prevented current activation from reaching a steady state at positive membrane potentials. Semilogarithmic analysis of positive current decay tails recorded immediately following square voltage clamp depolarizations was therefore used to separate the two membrane conductance components ixfast and ixslow and the third component, attributable to potassium ion accumulation, which was almost invariably present in the tails. 3. It is shown that inaccuracies in this method of semilogarithmic separation of components caused by visual assessment of the i3 (accumulation) line are minor compared with the large changes in the time constants of ixfast and more especially of ixslow which would result from ignoring the potassium accumulation component. 4. Such semilogarithmic separation of the three components of outward current gave separate activation curves for each of the two membrane conductance components, ixfast and ixslow. 5. Measurement of 'total' activation curves in which all components of outward current were represented could be made more easily and fairly reliably. The position and shape of these activation curves on the voltage axis were found to closely resemble those obtained by three component separation. It is therefore suggested that such a simplified analysis reflects the properties of the individual currents sufficiently well for it to be of use in preliminary studies of, for example, drug action. 6. The kinetic properties of the atrial outward currents have been investigated over a wide potential range. Because of the presence of potassium ion accumulation, an indirect method of obtaining the average value of 1/gamma for outward current decay at negative potentials had to be employed. 7. It is shown that some degree of inward-going rectification is associated with the outward current systems of frog atrium. 8. The possible reasons for the differences between the analysis presented here and those presented earlier by us (Brown & Noble, 1969a, b) and by Ojeda & Rougier (1974) are discussed.

Potassium influx in the frog atrium during the cardiac cycle.

A method for measuring inwardly directed transmembrane tracer flow during the cardiac cycle was developed and applied to a study of 42K influx in frog atrial trabeculae. A fine frog atrial fiber was suspended in a stream of nonisotopic perfusate into which a smaller tracer-containing bolus could be injected, subjecting the fiber to a brief, controlled exposure to the tracer at any desired point in the cardiac cycle. In an experiment, the tissue was exposed to a fixed number of radioactive pulses at a selected point in the cardiac cycle; a brief flush with nonradioactive perfusate removed ambient and extracellular label and an extended wash and removed the remaining intracellular tracer for radioassay. The same procedure was repeated at different points in the cycle, and the resulting tracer uptake at each point measured the relative influx of the particular ion. In this way, a characteristic and reproducible 42K influx profile was demonstrated which exhibited a marked drop below diastolic values during the first 500 msec or so of the action potential followed by a rise and an overshoot above resting values. The time of return to the resting level was somewhat uncertain but was tentatively placed in the vicinity of rapid repolarization. We suggest that the rise and the overshoot reflect the activity of the membrane Na+, K+-adenosinetriphosphatase.

Intracellular chloride activity in frog heart.

1. Chloride liquid ion exchanger micro-electrodes have been used to measure intracellular chloride activity (aiCl) in isolated pieces of frog sinus venosus, atrium and ventricle. 2. aiCl was found to be 35-0 +/- 3-2, 17-3 +/- 0-8 and 17-6 +/- 0-8 mM in sinus venosus, atrium and ventricle, respectively. 3. In atrium and ventricle aiCl is independent of whether the tissue is beating or non-beating. 4. In non-beating ventricle aiCl changes rapidly and reversibly when aOCl is reduced by replacing chloride with glucuronate. 5. ECl calculated from the measured aiCl has been compared with ECl calculated from the constant field equation assuming either constant chloride conductance or constant chloride permeability. 6. It is suggested that chloride is actively transported into the cells in all three kinds of frog heart tissue.

Kinetic analysis of the delayed outward currents in frog atrium. Existence of two types of preparation.

1. The outward currents underlying delayed rectification have been studied in frog auricular trabeculae with the double sucrose gap method.2. The results obtained in our experimental conditions allow us to discard the hypothesis of an accumulation of ions during the flow of the outward current.3. In most of the cases the delayed current is composed of two components similar to those previously described by Noble & Tsien (1969a, b).4. The first component I(1) is activated with a time constant of about 0.3 sec at the resting potential (E = - 70 mV) and of about 0.75 sec at E = - 5 mV. The steady-state value of the degree of activation (x(1))(infinity) varies from 0 at E = - 90 mV to 1 at E = + 20 mV. The reversal potential is near E = - 80 mV; this current seems to be mainly carried by potassium ions.5. The second component I(2) is very slowly activated, with a time constant of 3.5 sec at E = - 70 mV and of 5 sec at E = - 5 mV. The steady-state value of the degree of activation (x(2))(infinity) varies from 0 at E = - 40 mV to 1 at E = + 25 mV. The reversal potential is near E = - 60 mV; this current is less specific than the first component.6. In some preparations the delayed current is composed of only one component. This current is activated with a time constant of 0.6 sec at the resting potential and of 1.5 sec at E = - 5 mV. The steady-state value of the degree of activation is 0 at E = - 90 mV and 1 at E = + 30 mV. The reversal potential is between E = - 70 and E = - 60 mV.7. The role of these currents in the processes of repolarization of normal and prolonged action potentials, and in the pace-making activity is discussed.

Potentiation by endotoxin of responses associated with increases in calcium conductance.

Bacterial endotoxin at concentrations of 0.03 mug/ml enhanced the following responses, each of which reflects calcium influx: (1) contraction of spiral intestine retractor muscle of dogfish; (2) amplitude of postsynaptic potential of squid giant synapse as a function of presynaptic depolarization; (3) membrane responses of deep abdominal extensor muscle of crayfish, stimulated intracellularly; and (4) slow transient (calcium) current in trabeculae of frog atrium under voltage clamp. Corticosteroid antagonized the endotoxin effect on the smooth muscle, not on the frog atrial current. One mechanism of action of endotoxin may be to increase transmembrane calcium currents.

Pacemaker current in frog atrium.

PACEMAKER currents have been investigated by voltage clamp studies in Purkinje fibres1,2 but not in tissues from those regions of the heart where the natural pacemaker lies: the amphibian sinus and mammalian sino-atrial node. Repetitive activity can often be induced in normally quiescent frog atrial trabeculae by application of small depolarizing constant current pulses3. These currents impose on the atrial cells the low membrane potential characteristic of sinus muscle4, Moreover, the potential changes involved are very similar to those which may be recorded from spontaneously active sinus, successive action potentials being separated by phases of slow diastolic depolarization4 (Fig. 1a). It seems likely therefore that the membrane current controlling this diastolic depolarization in atrium will closely resemble that which generates the natural sinus pacemaker.

The frequency dependence of outward current in frog auricular fibres. An experimental and theoretical study.

The voltage-clamp technique has been applied to investigate the frequency-dependence of the membrane currents in isolated frog atria. Two stimulation programs were selected. The experimental results were compared with the predictions of a Hodgkin and Huxley type of empirical mathematical model and a close correspondence was observed. The mechanism responsible for the frequency-dependence of the action potential is discussed in connection with the reported results.

Kinetics of Na inactivation in frog atria.

1. In frog atria, time and voltage dependence of inactivation and reactivation of fast transient Na current were studied using the sucrose-gap voltage clamp technique. Experiments were done at 4–7° C. 2. Fast inward current inactivated by conditioning depolarizing clamps. The relation between steady-state inactivation and membrane potential is represented by an S-shaped curve similar to that observed in squid axon. At the resting level the availability of the Na-carrying system is about 60–70%. 3. During depolarizing clamps inactivation of Na current follows an approximately exponential time course with time constants between 1.5 and 8 msec. 4. Recovery from inactivation (reactivation) is much slower than development of inactivation. Time constants between 100 and 600 msec were observed during the recovery process following a repolarization or a hyperpolarization. 5. A ratio between the time constants of inactivation and reactivation in the order of 1∶50 was found when the two processes were studied at the same potential level. 6. In the frog atrial action potential the slow reactivation of the Na system is reflected by a relative refractory period reaching far beyond full repolarization. 7. Assuming that the slow course of Na reactivation is an inherent feature of Na kinetics, the inactivation variable (h) of the Hodgkin-Huxley theory (1952d) seems inadequate to describe the inactivation-reactivation process in frog atria. Formally fast inactivation and slow reactivation could be accounted for by replacing the variableh with two variables which both decrease on depolarization but largely differ in the rate constants.