Electromechanical characterization of the effects of racemic sotalol and its optical isomers on isolated canine ventricular trabecular muscles and Purkinje strands

In both isolated canine ventricular trabecular muscle and Purkinje strand preparations, dl-sotalol and its two optical isomers d- and l-sotalol produced a concentration-dependent increase in action potential duration while other transmembrane electrical characteristics were not significantly affected. The magnitude of the increase in action potential durations was greater in Purkinje strand preparations. In Purkinje strand preparations, the effect was rate dependent (i.e., the increase in duration was proportionately greater when stimulation frequency was slowed). From the concentration of each compound calculated to produce a 50% maximal increase in Purkinje fiber action potential durations, d-sotalol appeared to be one to three times more potent than either l-sotalol or the racemate. Each compound appeared to increase force development in ventricular trabecular muscle preparations stimulated at a frequency of 2 Hz. Increased force development was only observed in Purkinje strand preparations stimulated at slower rates (0.5-0.33 Hz). These results are unlike those produced by other beta-adrenergic blockers and suggest that the antiarrhythmic effects of sotalol are related primarily to its effect of action potential duration. The estimated potency ratios established for the effect of dl-sotalol and its optical isomers on both trabeculae and Purkinje fiber action potential durations (d greater than dl-l) may indicate that these effects are unrelated to the beta-adrenergic blocking properties of these compounds. The differential effect of sotalol on isolated trabeculae and Purkinje strand preparations may help to explain the clinically reported phenomenon of sotalol-induced torsade de pointes.     

Ryanodine modification of cardiac muscle responses to potassium-free solutions. Evidence for inhibition of sarcoplasmic reticulum calcium release

To test whether ryanodine blocks the release of calcium from the sarcoplasmic reticulum in cardiac muscle, we examined its effects on the aftercontractions and transient depolarizations or transient inward currents developed by guinea pig papillary muscles and voltage-clamped calf cardiac Purkinje fibers in potassium-free solutions. Ryanodine (0.1-1.0 microM) abolished or prevented aftercontractions and transient depolarizations by the papillary muscles without affecting any of the other sequelae of potassium removal. In the presence of 4.7 mM potassium and at a stimulation rate of 1 Hz, ryanodine had only a small variable effect on papillary muscle force development and action potential characteristics. In calf Purkinje fibers, ryanodine (1 nM-1 microM) completely blocked the aftercontractions and transient inward currents without altering the steady state current-voltage relationship. Ryanodine also abolished the twitch in potassium-free solutions, but it enhanced the tonic force during depolarizing voltage- clamp steps. This latter effect was dependent on the combination of ryanodine and potassium-free solutions. The slow inward current was not blocked by 1 microM ryanodine, but ryanodine did appear to abolish an outward current that remained in the presence of 0.5 mM 4- aminopyridine. Our observations are consistent with the hypothesis that ryanodine, by inhibiting the release of calcium from the sarcoplasmic reticulum, prevents the oscillations in intracellular calcium that activate the transient inward currents and aftercontractions associated with calcium overload states.     

The potassium current activated by 2-nicotinamidoethyl nitrate (nicorandil) in single ventricular cells of guinea pigs

Membrane currents through potassium channels activated by nicorandil, which has a potent coronary vasodilating action, have been studied in ventricular cells of guinea pigs by using the single pipette whole-cell clamp technique. In the presence of 0.1 mM nicorandil, the duration of the action potential was shortened from 196 to 145 ms. Nicorandil markedly increased outward currents at potentials positive to the resting potential. When the difference in the currents before and after the application of nicorandil were plotted against the membrane potential, the current-voltage relation reversed close to the potassium equilibrium potential. The difference current during depolarizing pulses showed no time-dependent relaxation. These results indicate that the current evoked by nicorandil is carried by K+ ions and has voltage-independent kinetics. Power-density spectra obtained in the presence of nicorandil were fitted well by a single Lorentzian curve with a corner frequency of 4.4 Hz. The amplitude of the single-channel unit current was estimated from the relation between the variance and the mean current, and was 0.27 +/- 0.1 pA (n = 7) at -35 mV. The estimated slope conductance was 4.6 +/- 1.7 pS. Nicorandil did not affect Ca2+ currents. It is concluded that nicorandil activates a small-conductance K+ channel without affecting the Ca2+ channel.     

Is cyclic AMP involved in the defibrillating effect of sotalol ?

Ventricular fibrillation induced in animals pretreated with sotalol, a class III antiarrhythmic agent, would spontaneously terminate and revert into a sinus rhythm. This phenomenon has been atributed to the class III action of this Drug, I.e., prolongation of myocardial action potential duration and effective refractory period. Since various observations suggested that these alone cannot explain the defibrillating phenomenon, we hypothesised that sotalol affeced ventricular intercellular synchronization by increasing intercellular coupling. Our recent experimental studies have shown that sotalol antagonized the cellular decoupling to guinea pig ventricular muscle strip caused by perfusion with either a hypoxic normal Tyrode's solution or an oxygenated high Ca2+ Tyrode's solution. We assumed that the most likely mechanism for the restoration of intercellular coupling would be by increasing intracellular cAMP concentration. In order to test this hypothesis, we studied the modification of this sotalol-induced recoupling by a cAMP dependent protein kinase inhibitor. The results clearly supported our assumption since the addition of Arg-Gly-Tyr-Ala-Leu-Gly (pure A- kinase inhibitor) prevented the aforementioned cellular recoupling action of sotalol in a dose-dependent manner. It can thus be concluded that changes in intracellular cAMP level are involved in the synchronizing /defibrillating effect of sotalol.     

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.     

Frequency- and voltage-dependent effects of disopyramide in canine Purkinje fibers

The voltage- and frequency-dependent blocking actions of disopyramide were assessed in canine Purkinje fibers within the framework of concentrations, membrane potentials, and heart rates which have relevance to the therapeutic actions of this drug. Vmax was used to assess the magnitude of sodium channel block. Disopyramide produced a concentration- and rate-dependent increase in the magnitude and kinetics of Vmax depression. Effects on activation time (used as an estimate of drug effect on conduction) were exactly analogous to effects on Vmax. A concentration-dependent increase in tonic block was also observed. Despite significant increases in tonic block at more depolarized potentials, rate-dependent block increased only marginally with membrane potential over the range of potentials in which propagated action potentials occur. Increases in extracellular potassium concentration accentuated drug effect on Vmax but attenuated drug effect on action potential duration. Recovery from rate-dependent block followed two exponential processes with time constants of 689 +/- 535 ms and 15.7 +/- 2.7 s. The latter component represents dissociation of drug from its binding site and the former probably represents recovery from slow inactivation. A concentration-dependent increase in the amplitude of the first component suggested that disopyramide may promote slow inactivation. There was less than 5% recovery from block during intervals equivalent to clinical diastole. Thus, depression of beats of all degrees of prematurity was similar to that of basic drive beats. Prolongation of action potential duration by therapeutic concentrations of drug following a long quiescent interval was minimal. However, profound lengthening of action potential duration occurred following washout of drug effect at a time when Vmax depression had reverted to normal, suggesting that binding of disopyramide to potassium channels may not be readily reversed. Variable effects on action potential duration may thus be attributed to a block of the window current flowing during the action potential being partially or over balanced by block of potassium channels. Purkinje fiber refractoriness was prolonged in a frequency-dependent manner. Disopyramide did not significantly alter the effective refractory period of basic beats but did increase the effective refractory period of sequential tightly coupled extra stimuli. The results can account for the antiarrhythmic actions of disopyramide during a rapid tachycardia and prevention of its initiation by programmed electrical stimulation.     

Frequency-dependent and independent effects of tetrodotoxin on Vmax in cardiac fibers

Superfusion with 3 microM tetrodotoxin (TTX) induced both a use-dependent and a frequency-independent depression of the rate rise of the action potential (Vmax) in dog Purkinje and guinea pig ventricular muscle fibers. The recovery from block was fast and exponential with a time constant of 225.4 +/- 7.1 ms in dog Purkinje fibers (n = 6). The onset kinetics of the frequency-dependent Vmax block was rapid, i.e. reached steady state after 3.0 +/- 0.3 beats in guinea pig ventricular muscle (n = 6). The rapid use-dependent interactions with sodium channel make TTX similar to antiarrhythmic drugs with fast kinetics i.e. lidocaine, mexiletine, and tocainide, but unlike antiarrhythmic drugs, TTX-induces a large frequency-independent Vmax block at the same concentrations.     

Inactivation and recovery of sodium currents in cerebellar Purkinje neurons: evidence for two mechanisms

We examined the kinetics of voltage-dependent sodium currents in cerebellar Purkinje neurons using whole-cell recording from dissociated neurons. Unlike sodium currents in other cells, recovery from inactivation in Purkinje neurons is accompanied by a sizeable ionic current. Additionally, the extent and speed of recovery depend markedly on the voltage and duration of the prepulse that produces inactivation. Recovery is faster after brief, large depolarizations (e.g., 5 ms at +30 mV) than after long, smaller depolarizations (e.g., 100 ms at -30 mV). On repolarization to -40 mV following brief, large depolarizations, a resurgent sodium current rises and decays in parallel with partial, nonmonotonic recovery from inactivation. These phenomena can be explained by a model that incorporates two mechanisms of inactivation: a conventional mechanism, from which channels recover without conducting current, and a second mechanism, favored by brief, large depolarizations, from which channels recover by passing transiently through the open state. The second mechanism is consistent with voltage-dependent block of channels by a particle that can enter and exit only when channels are open. The sodium current flowing during recovery from this blocked state may depolarize cells immediately after an action potential, promoting the high-frequency firing typical of Purkinje neurons.     

Subtypes of dorsal root ganglion neurons based on different inward currents as measured by whole-cell voltage clamp

Electrophysiological and pharmacological properties distinguished subtypes of adult mammalian dorsal root ganglion neurons (DRGn) in monolayer dissociated cell culture. By analogy of action potential waveform and duration, neurons with short duration (SDn) and long duration (LDn) action potentials resembled functionally distinct subtypes of DRGn in intact ganglia. Patch clamp and conventional intracellular recording techniques were combined here to elucidate differences in the ionic basis of excitability of subtypes of DRGn in vitro. Both SDn and LDn were quiescent at the resting potential. Action potentials of SDn were brief (less than 2 msec), sensitive to tetrodotoxin (TTX, 5-10 nM), exhibited damped firing during long depolarizations, and did not respond to algesic agents applied by pressure ejection. Action potentials of LDn were 2-6 msec in duration, persisted in 30 microM TTX, and fired repetitively during depolarizing current pulses or exposure to algesic agents (e.g., capsaicin, histamine and bradykinin). Whole-cell recordings from freshly dissociated neurons revealed two inward sodium currents (INa; variable with changes in sodium but not calcium concentration in the superfusate) in various proportions: a rapidly activating and inactivating, TTX-sensitive current; and, a slower, TTX (30 microM)-resistant INa. Large neurons, presumable SDn, had predominantly TTX-sensitive current and little TTX-resistant current. The predominant inward current of small neurons, presumably LDn, was TTX-resistant with a smaller TTX-sensitive component. By analogy to findings from intact ganglia, these results suggest that fundamentally different ionic currents controlling excitability of subtypes of DRGn in vitro may contribute to functional differences between subtypes of neurons in situ.     

Electrophysiological and pharmacological evidence that peripheral type benzodiazepine receptors are coupled to calcium channels in the heart

PK 11195, an antagonist of the peripheral type benzodiazepine receptor, does not affect either the duration of the action potential or the tension of the guinea pig papillary muscle. However, it antagonized the effects of the calcium channel blockers, nitrendipine, verapamil, diltiazem, and of BAY K8644, a calcium channel agonist in this heart preparation. On the other hand, PK 11195 does not change the increase in the action potential duration provoked by the potassium channel blocker tetraethylammonium. RO5-4864, an agonist of the peripheral type benzodiazepine receptor, decreased the tension of the guinea pig papillary muscle. The effect was reversed by increasing extracellular Ca2+ concentrations up to 4 mM. These results suggest that in the heart the peripheral type benzodiazepine receptors are coupled to calcium channels.     

Effect of sotalol on transmembrane ionic currents responsible for repolarization in cardiac ventricular myocytes from rabbit and guinea pig

The effects of sotalol, a β-adrenoceptor blocker and class III antiarrhythmic agent, on transmembrane ionic currents were examined in single rabbit and guinea pig ventricular myocytes using whole-cell voltage-clamp techniques. In neither of these species did 60 μM sotalol appreciably effect the inward rectifier, the transient outward or the inward calcium currents. In addition, sotalol did not elicit a slowly inactivating component of the sodium current as did 1 μg/ml veratrine. In guinea pig ventricular myocytes, sotalol also significantly depressed the outward delayed rectifier current. An outward delayed rectifier current was not observed in rabbit ventricular myocytes examined at room temperature; and, under these conditions sotalol did not lengthen action potential duration. Sotalol induced lengthening of cardiac action potential duration can, therefore, be explained by depression the outward delayed rectifier current.     

Effects of several sea anemone and scorpion toxins on excitability and ionic currents in the giant axon of the cockroach

The membrane effects of 4 sea anemone and 6 scorpion toxins have been studied under current clamp and voltage clamp conditions. Micromolar concentrations of the purified toxins were applied externally on single giant axons of the american cockroach. Periplaneta americana in a double oil-gap arrangement and the effects on the resting potential, action potential and underlying currents analysed. The 4 sea anemone toxins (Condylactis toxin, Anemonia toxin 2, Anthopleurin toxin A and Parasicyonis toxin) were found to considerably prolong the action potential. This effect is frequency dependent and long plateau spikes (100-500 ms in duration) are consistently seen for frequencies lower than 0.2 Hz. This effect is due to a considerable delay in the turning-off of the sodium current during square membrane depolarizations associated, for large concentrations, with a decrease in the potassium conductance. Toxin effects on the sodium current are not prevented by pretreatment with STX. From the 4 purified toxins extracted from the venom of the scorpion, Androctonus australis Hector, 3 (Mammal toxins 1 and 2 and crustacean toxin) were found to have sea anemone toxin like effects and to induce long duration plateau action potentials. As for sea anemone toxins, this effect is due to a lengthening of the falling phase of the sodium current associated with a small decrease in the potassium conductance. The 4th toxin (insect toxin or ITAaH) depolarizes the membrane and induces repetitive firing of short action potentials.(ABSTRACT TRUNCATED AT 250 WORDS)     

The involvement of sodium and calcium ions in the release of amino acid neurotransmitters from mouse cortical slices elicited by hyperforin

Hyperforin is currently considered to be the major active antidepressant constituent of the medicinal herb St. John's wort (Hypericum perforatum L.). The mechanism of action however, is still largely unknown, although the involvement of sodium and calcium has been recently inferred. In the present study hyperforin (5 microM) significantly potentiated the release of endogenous aspartate and glutamate from mouse cortical slices when stimulated by veratridine or potassium. Hyperforin (5 microM) also stimulated the release of aspartate, glutamate, serine, glycine and GABA when perfused on its own. Perfusion of the sodium channel blocker, tetrodotoxin (TTX) inhibited the effect of hyperforin, whereas removal of extracellular calcium potentiated the effect. Our observations suggests that hyperforin increases the overflow of neurotransmitters from mouse cerebral cortex possibly through facilitating the entry of sodium into the neurone which leads to the release of calcium from intracellular stores.     

Persistence of the electrophysiologic effects of mexiletine in canine Purkinje fibers alters the response to subsequent hypoxia

The persistence of cellular electropharmacologic effects of mexiletine on canine Purkinje fibers was studied utilizing standard microelectrode techniques and two different protocols. In the first, the tissue was exposed to hypoxic perfusion before and 30 min after perfusion with one of the following: mexiletine hydrochloride 6.25 microM solution, mexiletine hydrochloride 12.5 microM solution, or drug-free Tyrode's solution. With the higher concentration of mexiletine, depression of the maximal upstroke velocity (Vmax) persisted 30 min after drug washout and subsequent exposure to hypoxia did not result in the anticipated shortening of action potential duration but did prevent the restoration of normal Vmax. After perfusion with the lower concentration of mexiletine, Vmax was not depressed and hypoxic action potential duration shortening was not prevented. In the second protocol, Purkinje fibers were perfused with 12.5 microM mexiletine hydrochloride solution and then exposed to hypoxia after 15, 30, 45, or 60 min of perfusion with drug-free solution. Depression of maximal upstroke velocity and shortening of action potential duration persisted during washout, returning to control values by 45 min, although mexiletine was not detectable in the tissue bath after 10 min of washout. Hypoxia initiated at 15 or 30 min of washout failed to produce the anticipated shortening of action potential duration. At 45 and 60 min, action potential duration was shortened by hypoxia. We concluded that mexiletine depression of Vmax and shortening of action potential duration may persist in the absence of drug. Further shortening of action potential duration in response to hypoxia is prevented during this period. The persistence of Vmax depression is prolonged by hypoxia.     

Voltage-dependent changes in the permeability of nerve membranes to calcium and other divalent cations.

Transmitter release from depolarized nerve terminals seems to be preceded by a rise in the intracellular concentration of ionized calcium. In squid giant axons, depolarization promotes calcium entry by two routes: one that is blocked by tetrodotoxin and one that is insensitive to tetrodotoxin. The TTX-sensitive route seems to be the sodium channel of the action potential; but the TTX-insensitive route seems to be quite distinct from the sodium and potassium channels of the action potential. It is blocked by Mg-2+, Mn-2+ and Co-2+ ions and by the organic calcium antagonist D-600 and has many features in common with the mechanism that couples excitation to secretion.     

Electrical rhythmicity and spread of action potentials in longitudinal muscle of guinea pig distal colon

Using simultaneous intracellular recordings, we have characterized 1) electrical activity in the longitudinal muscle (LM) of isolated segments of guinea pig distal colon free to contract spontaneously and 2) extent of propagation of spontaneous action potentials around the circumference of the colon. In all animals, rhythmical spontaneous depolarizations (SDs) were recorded that are usually associated with the generation of action potentials. Recordings from pairs of LM cells, separated by 100 microm in the circumferential axis, revealed that each action potential was phase locked at the two electrodes (mean propagation velocity: 3 mm/s). However, at an increased electrode separation distance of 1 mm circumferentially, action potentials and SDs became increasingly uncoordinated at the two recording sites. No SDs or action potentials ever propagated from one circumferential edge to the other (i.e., 13 mm apart). When LM strips were separated from the myenteric plexus and circular muscle, rhythmically firing SDs and action potentials were still recorded. Atropine (1 microM) or tetrodotoxin (1 microM) either reduced the frequency of SDs or temporarily abolished activity, whereas nifedipine (1 microM) always abolished SDs and action potentials. Kit-positive interstitial cells of Cajal were present at the level of the myenteric plexus and circular and longitudinal muscle. In summary, SDs and action potentials in LM propagate over discrete localized zones, usually <1 mm around the circumference of the colon. Furthermore, in contrast to the classic slow wave, rhythmic depolarizations in LM appear to be generated by an intrinsic property of the smooth muscle itself and are critically dependent on opening of L-type Ca(2+) channels.     

Slow sodium inactivation in nerve after exposure to sulhydryl blocking reagents

Exposure to N-ethylmaleimide (NEM), a reagent that binds covalently to protein sulfhydryl groups, results in a specific reduction in sodium conductance in crayfish axons. Resting potential, the delayed rise in potassium conductance, and the selectivity of the sodium channel are unaffected. Sodium currents are only slightly increased by hyperpolarizing prepulses of up to 50 ms duration, but can be restored to about 70% of their value before treatment if this duration is increased to 300-800 ms. The time to peak sodium current and the time constant of decay of sodium tail currents are unaffected by NEM, suggesting that the sodium activation system remains unaltered. Kinetic studies suggest that NEM reacts with a "slow" sodium inactivation system that is present in normal axons and that may be seen after depolarization produced by lowered the holding potential or increasing the external potassium concentration. NEM also perturbs the fast h inactivation system, and in a potential-dependent manner. At small depolarizations tauh is decreased, while at strong depolarizations it is increased over control values. Experiments with structural analogs of NEM suggest that sulfhydryl block is involved, but do not rule out an action similar to that of local anesthetics, p- Chloromercuriphenylsulfonic acid (PCMBS), another reagent with high specificity for SH groups, also blocks sodium currents, but restoration with prolonged hyperpolarizations is not possible.     

Synaptic effects of the synthetic pyrethroid resmethrin in rat brain in vitro

Resmethrin (30 microM) induced release of transmitters was not affected by manipulation of the Na+ current with either choline or tetrodotoxin agents which readily reversed the effects of veratridine, deltamethrin and cypermethrin. Resmethrin (I50: 2.2 microM) inhibited the ATP dependent uptake of Ca2+ but deltamethrin and cypermethrin were much less effective. Resmethrin also displaced Ca2+ from crude synaptosomal membranes. The release promoting effects of resmethrin in rat brain in vitro are better explained by its effects on Ca2+ rather than through a specific effect on the Na+ channel. In contrast, the effects of deltamethrin and cypermethrin promote transmitter release by a Na+ dependent process.     

Mechanisms of action of voltage-gated sodium channel ligands

The voltage-gated sodium channels play a key role in the generation of action potential in excitable cells. Sodium channels are targeted by a number of modulating ligands. Despite numerous studies, the mechanisms of action of many ligands are still unknown. The main cause of the problem is the absence of the channel structure. Sodium channels belong to the superfamily of P-loop channels that also the data abowt includes potassium and calcium channels and the channels of ionotropic glutamate receptors. Crystallization of several potassium channels has opened a possibility to analyze the structure of other members of the superfamily using the homology modeling approach. The present study summarizes the results of several recent modelling studies of such sodium channel ligands as tetrodotoxin, batrachotoxin and local anesthetics. Comparison of available experimental data with X-ray structures of potassium channels has provided a new level of understanding of the mechanisms of action of sodium channel ligands and has allowed proposing several testable hypotheses.     

Calcium-activated cation channel in rat thyroid follicular cells

Using the patch-clamp single-channel current recording technique, a cation channel in the contraluminal membrane of rat thyroid follicular cells has been characterized. The channel has a unit conductance of about 35 pS and is equally permeable to sodium and potassium. The pattern of channel opening and closing is independent of the membrane potential. The channel is only operational when the ionized calcium concentration in the fluid which is in contact with the inside of the membrane is at least 1 microM. This conductance pathway can be classified as a calcium dependent non-selective cation channel and could explain stimulant-evoked depolarizations in the thyroid follicular cells.