Frog Atrial Cells Research Articles

Modulation of voltage-dependent facilitation of the T-type calcium current by sodium ion in isolated frog atrial cells.

Sodium ions have been reported to alter the permeation properties of L- and N-type Ca2+ channels. Here in frog atrial cardiomyocytes under whole-cell patch-clamp conditions, we have examined the effects of lowering the external Na+ concentration on the amplitude of T-type Ca2+ current, ICaT, and on the relief of its steady-state inactivation by large depolarizing prepulses, ICaT facilitation. A partial reduction in Na+ ion concentration did not significantly alter ICaT amplitude elicited at -50 mV. However, after a large depolarization, low- Na+ solutions enhanced the relief of inactivation and induced ICaT facilitation. This facilitation occurred independently of the divalent charge carrier, high intracellular Ca2+ buffering or the intracellular Na+ content. Its effects were additional to the beta-adrenergic effects mediated by a decrease of Gi/o-protein inhibitory tone. In Ca2+-free solution the very large T-type current, then carried by Na+ ions, showed only a weak relief of inactivation. In conclusion, ICaT facilitation--which, as previously reported, is modulated by the transient voltage-dependent relief of Gi-protein inhibitory tone--is further enhanced in a low-Na+ solution. In Ca2+-free solution, relief of inactivation due to re-openings dependent on the divalent charge carrier is improbable. It thus appears that for a short while after a large depolarization, external Na+ compete with Ca2+ ions on permeation-controlling sites, so as to modulate channel re-openings and thus the amplitude of voltage-facilitated ICaT independently of the control exerted by the inhibitory G-protein.

Progression of cardiac potassium current modification after brief exposure to reactive oxygen

We reported previously that singlet oxygen ( 1O 2), generated by illuminating the photosensitizer rose bengal (RB), suppressed the delayed rectifier potassium current (I K) in single frog atrial cells. Considering the brief lifetime of 1O 2, one might expect I K modification to reach a steady-state soon after a brief exposure to RB-generated 1O 2. Here we report that, contrary to expectations, tens of seconds can be required for I K to reach a new steady-state. We will use the term “progression” to refer to the component of current modification which occurs after cessation of illumination. To gain insight into the mechanism of progression, we investigated how its time course and magnitude were affected by (1) membrane potential during and following RB illumination, and (2) the level of I K activation during illumination. We found that conditions which favored the open state of the potassium channel also favored progression, increasing both its time course and magnitude. Illumination while I K was activated produced significant progression having a very slow time course (tens of seconds). By comparison, illumination when I K was not activated produced no progression; I K modification was completed during the 2 s illumination period. These findings suggest progression results from the kinetics of potassium channel state transitions rather than from a long-lived reactive intermediate produced during the initial 1O 2 exposure.

Calcium currents and contraction in frog atrial trabeculae

The properties of two types of calcium current (ICa(T) and ICa(L)) and their relationship with contraction were studied in isolated frog atrial trabeculae using the double mannitol gap technique. In Ringer solution containing TTX the calcium currents observed are similar to the ICa(T) and ICa(L) found in single frog atrial cells. The results obtained by using inorganic calcium channel blockers have shown that the phasic contraction appears only when ICa(T) and ICa(L) are simultaneously present. It is proposed that ICa(T) loads the sarcoplasmic reticulum (SR) while ICa(L) triggers Ca2+ release from the SR.

Force responses to rapid length changes in single intact cells from frog heart.

1. Force transients in response to step perturbations in length were recorded in intact atrial cells from frog heart at various temperatures (6-15 degrees C). Length changes of various sizes and in either direction, complete in 0.5 ms, were applied to single myocytes near slack length (initial sarcomere length 2.1-2.2 microns) just before the peak of an isometric twitch. The frequency response of the force transducers used was 2-4 kHz in Ringer solution. 2. An early quick force recovery phase was clearly observed after the elastic force response to the length step and before the start of much slower recovery processes. The quick recovery phase became progressively faster with larger shortening steps and was almost as fast as that originally described in intact frog skeletal muscle fibres (rate constants above 1000 s-1 in large releases at 10 degrees C). 3. The force-extension relation determined at the end of the length change (T1 curve) indicates that an instantaneous shortening of 0.5-0.6% of the initial cell length (L0) brings the force to zero. The force--extension relation determined at the end of the quick recovery phase (T2 curve) showed that the early recovery leads to an almost complete restoration of the original force with small stretches and releases (up to 0.3% L0) and that it becomes negligible in shortening steps of about 1.4% L0. 4. The results suggest that the mechanical properties of attached cross-bridges and the rate of transitions between attached cross-bridge states are approximately the same in frog atrial cells and fast skeletal muscle fibres.(ABSTRACT TRUNCATED AT 250 WORDS)

Membrane potential can influence the rate of membrane photomodification.

Though cellular photomodification has been shown to change cellular resting membrane potential, an effect of membrane potential on the rate of photomodification has never been reported. Here we demonstrate that the rate of photomodification of potassium channels in frog atrial cells is voltage dependent. The rate of potassium channel photomodification using negatively charged Rose Bengal as the photosensitizer is about 2.5 times greater at the resting membrane potential of -70 mV compared to +40 mV. Similar results are obtained using the positively charged photosensitizer methylene blue. On the other hand, the rate of photomodified increase of leak current in the same cells does not significantly change in this voltage range with Rose Bengal as photosensitizer, but demonstrates a voltage dependence like that of potassium current when methylene blue is the photosensitizer. These observations cannot be explained based on voltage-dependent partitioning of the sensitizer, as similar effects on potassium current were obtained using either a positively charged or negatively charged sensitizer.

Atrial Membranes Contain Nucleoside Diphosphate Kinase (NDPK) Activity: Its Role in Regulation of Muscarinic K+ Channels

Guinea pig or rabbit atrial muscarinic K+ channels in cell-free inside-out patches can be activated in the absence of extracellular agonist and cytoplasmic G nucleotides by intracellular ATP-Mg2+. This ATP-dependent activation is compatible with the existence of a membrane-bound nucleoside diphosphate kinase (NDPK), which directly phosphorylates GK-bound GDP. We show that this ATP-dependent activation is also possible in frog atrial cells, and that atrial membranes of frog and guinea pig contain NDPK activity. The relative order of different nucleoside triphosphates (NTPs) as phosphate donors parallels the observed efficiency of these nucleotides in activation of the channels. Thus, atrial membranes contain NDPK activity, which can be responsible for the ATP-dependent activation of muscarinic K+ channels, seen in patches of atrial cells. Under physiological conditions, NDPK can act as a GTP supply in the immediate vicinity of the G protein to ensure reliable signal transduction.

Dual action of prajmalium on the Ca currents in frog isolated cardiomyocytes

The effects of N-n-propylajmaline (prajmalium) on the Na and Ca currents of single frog atrial and ventricular cells were studied by means of the whole-cell patch-clamp technique. Prajmalium (10 −9 to 10 −6 m) depressed the Na current ( I Na) in a dose- and use-dependent manner. In the same range of concentrations, prajmalium induced a dual effect on the high ( I CaL) and low ( I CaT) threshold Ca currents (the latter being only present in atrial cells). At a low concentration (10 −9 m), prajmalium increased both Ca currents while at high concentrations (10 −6 m) it depressed them. Prajmalium appeared very potent on I CaT although this current is generally reported to be barely sensitive to agonists and drugs. The action of the drug was also accompanied by a shortening in the half-time of inactivation of the Ca currents and a slight hyperpolarizing shift of their availability curves. The increase in I CaL by prajmalium was not prevented by prazosin (10 −7 m) nor by propranolol (10 −6 m), and it was also observed after I CaL had been fully stimulated by isoproterenol (10 −7 m). Nifedipine (10 −6 m), however, was able to prevent or block the prajmalium-induced increase in I CaL. Some similarities between the actions of prajmalium and dihydropyridine agonists on Ca currents are discussed.

Modification of cardiac ionic currents by photosensitizer-generated reactive oxygen

The effects of reactive oxygen species (ROS) generated by light and the photosensitizer Rose Bengal on ionic currents in single frog atrial cells were investigated. The excitatory inward sodium and calcium currents were both suppressed by ROS as was the outward, delayed rectifier potassium current. The inactivation kinetics of the sodium current were slowed markedly whereas the kinetics of calcium current inactivation were much less affected and potassium current activation was not changed. The sodium current-voltage relationship was shifted in the depolarizing direction by ROS whereas the voltage-dependencies of both the calcium and potassium currents were not affected. In addition to suppressing the time- and voltage-dependent sodium, calcium, and potassium currents, ROS enhanced a time-independent current which was outwardly directed at positive membrane potentials. However, the induction of this time-independent current required longer ROS exposure than was required to significantly suppress the other currents. The rapid onset of ROS-induced suppression of calcium and potassium currents followed by a later enhancement of a time-independent current can explain ROS-induced changes in action potential duration. Brief ROS exposure increased action potential duration whereas longer exposure reduced action potential duration.

Modulation of Ca currents in isolated frog atrial cells studied with photosensitive probes. Regulation by cAMP and Ca 2+: A common pathway?

We have studied the regulation of cardiac Ca current by intracellular cyclic AMP (cAMP) and Ca 2+, using photosensitive, caged compounds and the whole-cell, patch-clamp technique in isolated frog atrial cells. Although both low voltage activated (LVA) and high voltage activated (HVA) Ca channels were found to be present in these cells, only the HVA Ca currents were sensitive to modulation by isoproterenol or dihydropyridines (DHPs). The application of extracellular isoproterenol, as well as the photorelease of intracellular cAMP or Ca 2+ at micromolar and submicromolar concentrations, respectively, had no effect on LVA Ca currents. In contrast, these agents: (i) increased the amplitude of currents through HVA channels, carried by either Ca 2+ or Ba 2+ with a similar time-course, (ii) slowed the decay of the current when Ba 2+ was the permeating ion, and (iii) modulated the agonist effect of the DHP Bay-K 8644. The strong similarities between the effects of cAMP and Ca 2+ suggest that both of these intracellular messengers might eventually lead to the phosphorylation of HVA Ca channels. It is possible that Ca-dependent phosphorylation of the channels may account for the potentiation of Ca current induced by repetitive stimulation.

Effects of aluminium on electrical and mechanical properties of frog atrial muscle

1. The effects of aluminium on membrane ionic currents were studied in single cardiac myocytes. Most of the work was done on frog atrial cells, but some experiments were also carried out on single cells isolated from rabbit ventricles and atria. 2. The effects of aluminium on the force of contraction of frog atrial trabeculae were also investigated. 3. Aluminium was prepared from AlCl3 as a stock 0.5 M solution which has a pH of 3.5. Before each experiment, this solution was added to the control solution, to give a final concentration of 20-100 micrograms ml-1 aluminium (0.75-3.75 mM AlCl3). The solutions were brought to a pH of 7.4 or 7.6. at which they consist of a mixture of amorphous aluminium hydroxides and a very small amount of soluble ionic aluminium complexes: free aluminium cations (less than 10 pM), aluminohydroxide anions (less than 8 microM). The addition of this suspension reduced the peak inward calcium currents in single rabbit atrial and ventricular cells and in frog atrial cells. In the latter, the peak current was reduced (at + 10 mV) to 45% of control (mean of 9 cells). This effect was reversible upon washout, and was obtained at all membrane potentials, with no shift of the calcium current voltage relationship along the voltage axis. 4. Aluminium also reduced the time-dependent potassium current IK. This reduction was observed at all membrane potentials. For example, at + 10 mV, the mean reduction of IK (n = 9) was to 69% of the control amplitude. This effect, which was very difficult to reverse, was not due to IK rundown. The fully activated current-voltage relationships (obtained by standard 'tail' analysis) showed that the effect of aluminium was due mainly to a decrease in conductance and not to a shift in the activation range of IK. The mean voltage of half activation was shifted by 8 mV in the depolarizing direction (n = 5). 5. The background potassium current IK1 was also slightly but consistently changed in a complex fashion, with an outward shift at membrane potentials positive to -60 mV. For example, at a membrane potential of -40mV, the mean shift was by 22 + 4pA. At more negative potentials, there was an inward shift in the current amplitudes. For example, for steps to -I00 mV the current elicited was larger (more inward) by 53 pA (mean value, n = 10). The reversal potential was slightly shifted (<10 mV) in the hyperpolarizing direction. 6. The force of contraction of frog atrial trabeculae was altered by aluminium in a complex manner, which showed marked seasonal variation. During most of the year, 50-100,ug ml-1 aluminium caused a biphasic change, with an early small and consistent decrease, followed by a large increase in twitch amplitude. For a short period corresponding to the (local) winter months the sensitivity to aluminium was greatly enhanced. Aluminium lOOupgml-1 totally abolished contraction (n = 5), while a lower concentration (20,ug ml- 1) produced a sustained reduction in the force of contraction. Similar biphasic and seasonal responses have been reported to be induced by lanthanum. 7. The biphasic changes in twitch amplitude were independent of the transmembrane sodium gradient. Aluminium produced the same effects when 90% of the extracellular sodium was replaced by lithium. Caffeine (5 mM) attenuated or even inverted the positive inotropic effect of aluminium. These results imply that aluminium alters the release of calcium from intracellular, caffeine-sensitive stores. This could be effected either by augmenting the amount released during each activation, and/or by increasing the loading of stores prior to release.

Phenytoin preferentially inhibits L-type calcium currents in whole-cell patch-clamped cardiac and skeletal muscle cells

The effect of the anticonvulsant diphenylhydantoin (phenytoin) was tested on the inward calcium currents of whole-cell patch-clamped cells from rat and human muscles and from frog atrium. A concentration of 10 μM phenytoin was required to obtain a threshold inhibitory effect and, even with high concentrations (100 μM), the inhibition was not complete. In skeletal muscle (rat and human cells in culture), phenytoin (30 μM) exerted a more potent effect on the high-threshold calcium current (I Ca,L inhibition: 53 ± 6% mean ± SD n−1) rather than on the low-threshold one (I Ca,T inhibition: 16 ± 10%). Similar results were obtained on dissociated frog atrial cells. These data are to be contrasted with those previously reported on neuronal cells, where specific inhibition of I Ca,T was reported. Thus, the action of phenytoin appears to be different in muscle and nerve so that phenytoin does not appear to be a specific inhibitor of I Ca,T.

Reversible activation of the ATP-dependent potassium current with dialysis of frog atrial cells by micromolar concentrations of GDP

We studied the effects of internal and external solutions on potassium currents in frog atrial cells. Experiments were carried out in whole cell recording in the presence of tetrodotoxin and cobalt in the bath to suppress the inward currents. In the absence of pyruvate and glucose in the external solution, a time-independent current increased progressively in a few minutes till the death of the cell. This current had the properties of the ATP-sensitive potassium current IK(ATP) in mammalian cells. In the presence of pyruvate and glucose in the external solution, the membrane current stayed low for 30 min. Addition of guanosine monophosphate (GMP, 40 microM), guanosine triphosphate (GTP, 40 to 1000 microM), adenosine diphosphate (ADP, 40 microM) or adenosine triphosphate (ATP, 3000 microM) to the internal solution had no major effect on the current amplitude. In contrast, addition of GDP (20 or 40 microM) produced a loss of rectification in a few minutes. The current activated by GDP was time independent as was the current observed in the absence of glucose and pyruvate. It was sensitive to cesium and barium, it was blocked when ATP was added to GDP in the internal solution, and it was suppressed by the sulphonylurea glibenclamide (1 microM). We suggest that GDP produced a local depletion of ATP, by displacement of the equilibrium between ATP, GDP, ADP and GTP. This hypothesis is supported by the fact that the current activated by GDP was rapidly suppressed when adding GTP in excess to the internal solution.

Action of cromakalim on potassium membrane conductance in isolated heart myocytes of frog

1. The effects of cromakalim on membrane currents were studied at 20 degrees C in frog atrial and ventricular cells in patch clamp recording using the whole cell configuration. 2. When cromakalim (1 microM) was applied in the external medium, a time-independent current was activated in a few minutes. Cromakalim induced a weak increase of inward membrane currents recorded during hyperpolarization and a large increase of outward membrane currents recorded during depolarization. 3. The current voltage relationship of the cromakalim-induced current reversed near EK and rectified in the outward direction. 4. The cromakalim-activated current was inhibited by external application of cesium (20 mM), barium (1.8 mM), tolbutamide (1 mM) and glibenclamide (1 microM). 5. The effects of cromakalim were insensitive to a cytosolic increase in adenosine 5'-triphosphate (ATP, 3-5 mM). Cromakalim had no effects when applied in the cell. 6. Our results confirm that cromakalim activates an IK(ATP)-like conductance and suggest that the effects of the drug are due to an action on the external side of the membrane and are independent of the ATP cell content.

Single delayed rectifier channels in frog atrial cells. Effects of beta-adrenergic stimulation

The patch-clamp technique with two pipettes was used to record single delayed K+ channels (cell-attached electrode) and to control the potential and the composition of the intracellular compartment (whole-cell electrode). With 30 microM cAMP in the cell and physiological potassium concentrations inside and outside the patch, a channel carrying an outward current was characterized. Its open probability was very low and the channel was recorded in only 5% of patches under control conditions. Increasing intracellular cAMP increased the probability of finding a channel in a patch 10-fold. The channel had the characteristics expected of a delayed rectifier channel. The time-course of its ensemble average resembled the whole-cell current in the same cell. The current-voltage relationship exhibited inward rectification, with a slope conductance of 20 pS in the linear portion and a reversal potential close to EK. Both the open- and the closed-time distributions were described by the sum of two exponentials, suggesting a complicated gating scheme involving two closed states and two open states. The beta-adrenergic stimulation did not change the conductance of the channel, but increased its probability of opening.

Combined force and voltage measurement in rapidly superfused guinea pig heart cells

We describe the construction and use of a setup that allows the rapid exchange of the solution surrounding an isolated guinea pig heart cell while simultaneously measuring the isometric force and membrane potential (Em). Cells were stably attached, by means of poly-L-lysine, to a force transducer which was adapted from one previously used for a study of frog atrial cells [N. Shepherd and F. Kavaler.Am. J. Physiol. 251 (Cell Physiol. 20): C653-C661, 1986]. The modified transducer is simple to construct and use and can be readily added to existing patch-clamp setups. The strength of attachment of a cell to the transducer exceeded the strength of the gigaseal in all of the experiments. The membrane potential was measured by means of patch electrodes and a high-impedance voltage follower. Rapidly changing extracellular K concentration [( K]o) from 5.4 to 10.8 mM caused a positive change of Em by 16.5 +/- 1.4 mV with a half-time (t1/2) of 27 +/- 4 ms. Replacing calcium in the perfusate by magnesium instantly abolished the contraction and shortened the action potential. Twitch tension returned stepwise to the control value on return of calcium to the perfusate. Our initial observations show that the patch electrode can be used successfully in conjunction with the isometric force transducer and rapid extracellular solution changes for studies of excitation and contraction coupling in isolated mammalian heart cells.

Intracellular magnesium affects I(K) in single frog atrial cells.

Voltage-clamp experiments on single frog (Rana pipiens) atrial cells using whole cell recording techniques revealed that the addition of MgCl2 to the 150 mM KCl patch pipette solution influenced the voltage- and time-dependent potassium current (IK). After rupture of the membrane patch under the tip of the pipette, IK increased with time when the pipette solution was magnesium free, but decreased slightly when the solution contained 1.5 mM MgCl2. More dramatic decreases in IK occurred when the solution contained 3.0 or 10 mM MgCl2. In addition to suppressing the magnitude of IK, the activation rate of this current was enhanced by 10 mM MgCl2 but was not affected by 1.5 or 3 mM MgCl2. Other chloride salts containing mono-, di-, or trivalent cations were used to demonstrate that the effects of MgCl2 on IK were not related to alterations in ionic strength, osmolality, or chloride concentration produced by adding MgCl2 to the pipette solution. Our results suggest that changes in the intracellular magnesium concentration influence IK as the pipette solution exchanges with the intracellular fluid.

Existence of two calcium currents recorded at normal calcium concentrations in single frog atrial cells

A low threshold calcium current (ICaLT) was found in Cs +-loaded frog atrial cells in addition to the classical (high threshold) calcium current (ICaHT), and was investigated at physiological Ca 2+ concentrations using the whole-cell patch-clamp technique. The threshold potentials were approximately −60 mV for ICaLT and −40 mV for ICaHT. The amplitude and time course of ICaLT were almost unaffected by exchanging Ca 2+ for Ba 2+ or Sr 2+, while those of ICaLT were modified. ICaLT was inhibited by Ni 2+ (40 × 10 −6 M) but was not affected by Cd 2+ (20 × 10 −6 M) while ICaHT was inhibited by Cd 2+ and only slightly reduced by Ni 2+ at the same concentrations. Co 2+ (10 −3 M) inhibited both types of calcium currents while La 3+ (5 × 10 −6 M) had a greater blocking effect on ICaHT. ICaLT was neither modified by dihydropyridines (nisoldipine, Bay K) nor by adrenergic agonists (adrenaline, noradrenaline, isoprenaline), in contrast with the effects of these agents on ICaHT. Angiotensin II (40 × 10 −9 M) increased and atrial natriuretic factor (0.1 × 10 −6 M) decreased ICaLT while ICaHT, was not modified by these two substances.

Are Ba 2+ and Sr 2+ ions transported by the Na +Ca 2+ exchanger in frog atrial cells?

Ba 2+ and Sr 2+ ions are widely used to replace Ca 2+ ions for the study of Ca 2+ channel currents in electrophysiological experiments. Using the double sucrose gap technique, we investigated the effects of Sr 2+ and Ba 2+ ions on the Na +Ca 2+ exchange activity in frog atrial fibres where it is the major relaxation mechanism. With either Sr 2+ or Ba 2+ ions instead of Ca 2+ in the extracellular bath, Na-free contractures reversibly developed but with different kinetics. Voltage clamp experiments showed that the tonic tension recorded in the presence of Sr 2+ or Ba 2+ was markedly increased following the addition of monensin, a Na + ionophore known to increase the intracellular Na + activity. In Na-free solutions (Li-substituted), it was possible to induce contractures by substituting Sr 2+ or Ba 2+ ions for extracellular Ca 2+. These contractures could be relaxed by reintroducing Na + or Ca 2+ ions in the extracellular medium. Taken together, these results suggest that Sr 2+ and Ba 2+ ions can interact with the Na +Ca 2+ exchange mechanism and potentially participate not only in Na +-cation but also in Ca 2+-cation exchanges on either side of the sarcolemmal membrane.

Effects of halothane on membrane potentials and membrane ionic currents in single bullfrog atrial cells

Halothane exerts negative inotropic and negative chronotropic actions on the isolated heart in experimental animals. In order to assess directly the actions of halothane in myocardium, we studied the effects of halothane on membrane potentials and transmembrane ionic currents in single isolated frog atrial cells obtained by the enzymatic dissociation method. The results show: (a) that the action potential is prolonged and its plateau phase and overshoot are depressed, but the resting potential remains unchanged; (b) that there is a significant inhibition of a time- and voltage-dependent outward K+ current and a slow inward Ca2+ current, with a slight decrease of a fast inward Na+ current following halothane (1.0-4.0%) application; and that halothane has no effect on another K+ current, time-independent current.

Component of whole cell Ca current due to electrogenic Na-Ca-exchange in cardiac myocytes

A carrier mechanism that mediates an exchange of Na+ for Ca2+ across the cell membrane is believed to be an important regulator of intracellular Ca2+ and therefore the contractile strength of the heart. Considerable evidence indicates that there is an unequal transfer of electrical charge during this exchange which implies that the carrier may contribute substantially to the net flow of ionic current across the sarcolemmal membrane. To detect such a component of current, nondialyzed single frog atrial cells were transiently Ca2+-loaded by augmenting Ca2+ entry through membrane Ca2+ channels by exposure to either the beta-adrenergic agonist, isoproterenol, or to BAY K 8644, a Ca2+ channel agonist. A direct comparison of whole-cell Ca2+ currents during exposure to these compounds in Na+-containing and Na+-free solutions revealed the existence of an additional component of inward current in Na+-containing solutions which temporally overlaps with Ca2+ channel-mediated currents. The dependence of this component of inward current on intracellular Ca2+ and extracellular Na+ and its resemblance to previously characterized carrier-mediated currents (creep currents) in this preparation suggest that it is associated with electrogenic Na+-Ca2+-exchange activity. These experiments support earlier predictions that under some conditions a component of the slow inward current in the heart may actually represent Ca2+ efflux mediated by an electrogenic Na+-Ca2+-exchange carrier.