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.     

Possible mechanisms of positive inotropic action of synthetic human calcitonin gene-related peptide in isolated rat atrium

The effect of synthetic human calcitonin gene-related peptide (hCGRP) on the isolated and electrically driven left atria of rats were investigated. The peptide at concentrations of 3 X 10(-9)-3 X 10(-7) M produced positive inotropic effects on the left atria in a dose-dependent manner. Verapamil (10(-5) M) and adenosine (10(-4) M) reduced the positive inotropic effect of hCGRP at concentrations of 3 X 10(-9) and 3 X 10(-8) M, but not at that of 3 X 10(-7) M. Ouabain (5 X 10(-5) M) inhibited the effect of hCGRP in concentrations of 3 X 10(-7) and 3 X 10(-8) M, but not in that of 3 X 10(-9) M. Simultaneous pretreatment with verapamil (10(-5) M) and ouabain (5 X 10(-5) M) suppressed the positive inotropy by hCGRP at all concentrations tested. On the other hand, tetrodotoxin (10(-6) M) potentiated only the positive inotropic effect of 3 X 10(-7) M hCGRP. Metoprolol (10(-7) M) and theophilline (10(-3) M) did not affect the inotropic effect of hCGRP. These results suggest that the positive inotropic effect of hCGRP is not mediated by beta-adrenoceptors but by two distinct mechanisms of action, which was inhibited by verapamil but not by ouabain (facilitation of Ca++ influx in lower concentrations of hCGRP) and which was blocked by ouabain but not by verapamil and potentiated by tetrodotoxin (inhibition of Na+/Ca++ exchange mechanism at higher concentrations of hCGRP).     

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.     

Effects of ritanserin on transmembrane action potentials in canine Purkinje fibers.

Ritanserin has been reported to be a potential antiarrhythmic. We studied the cellular electrophysiologic effects of ritanserin in canine Purkinje fibers. Ritanserin produced significant depressant effects on transmembrane action potentials elicited in canine Purkinje fibers. At concentrations of 10 and 40 mg/liter, ritanserin decreased Vmax (the upstroke velocity) of action potential in a dose-dependent fashion and shortened the duration of fast response action potential. These concentrations of ritanserin also reduced the amplitude and duration of the slow response action potentials induced in Purkinje fibers treated with isoproterenol (10(-5) M) and high K+ (22 mM). These in vitro results suggest that the cellular electrophysiologic actions of ritanserin may be due to its direct actions on cardiac sodium and calcium channels, which, in turn, may account for its antiarrhythmic effects.     

Modulation of the effects of sotalol on Purkinje strand electromechanical characteristics

The modulation of the effects of sotalol (30 microM) by two sodium channel blockers, tetrodotoxin (0.07 microM) and lidocaine (50 microM), and by a potassium channel activator, nicorandil (30 microM), were examined. Sotalol alone greatly increased Purkinje fiber transmembrane action potential duration and, in some preparations, induced early after depolarizations. Concurrent with the changes in action potential duration, sotalol also increased isolated Purkinje strand developed force paced at slow rates (0.33 Hz). These sotalol-induced alterations of Purkinje strand electromechanical characteristics were similar to those produced by either veratrine (0.6 or 1.0 micrograms/mL) or by tetraethylammonium (10 mM). The effects of sotalol on action potential duration and force development were reversed by exposure to either tetrodotoxin or nicorandil. Lidocaine also reversed the effects of sotalol on action potential duration and developed force. The sotalol-induced increase in action potential duration and development of early after depolarizations may, therefore, be abated by combination with drugs that either block cardiac sodium channels or that increase membrane potassium conductance. Combination with such drugs may help prevent the adverse arrhythmogenic effects of sotalol.     

Effect of ethmozine on action potentials and myocardial contraction in guinea pigs

Ethmozine decreased the maximum rate of action potential rise (Vmax) in a dose-dependent manner. Using the Scatchard plot the apparent dissociation constant was calculated to be 1.52 X 10(-5) g/ml. Ethmozine also decreased the force of contraction in the concentration range between 1 X 10(-6) and 1 X 10(-4) g/ml with the apparent dissociation constant obtained from the Scatchard plot being equal to 1.48 X 10(-5) g/ml. The linear correlation coefficient between the decrease in Vmax and the decrease in the force of contraction was found to be equal to 0.998. Negative inotropic action of ethmozine was less pronounced when the stimulation frequency had been switched from 0.8 to 0.1 Hz. The decrease in Vmax under the action of ethmozine (3 X 10(-5) g/ml) was diminished from 56 +/- 7% (0.8 Hz) to only 3 +/- 8% (0.1 Hz). This was accompanied by the decrease in the negative inotropic effect: from 58 +/- 9% (0.8 Hz) to 16 +/- 15% (0.1 Hz). It was assumed that the negative inotropic action of ethmozine was mediated by the Na--Ca exchange, which was inhibited by the decrease of the intracellular Na+ concentration due to the blockade of sodium channels by ethmozine.     

Specific binding sites for atrial natriuretic peptide (ANP) in cultured mesenchymal nonmyocardial cells from rat heart

Specific binding sites for atrial natriuretic peptide (ANP) were studied in cultured mesenchymal nonmyocardial cells (NMC) from rat heart. Binding study using 125I-labeled synthetic rat (r) ANP revealed the presence of a single class of high-affinity binding sites for rANP in cultured NMCs derived from both atria and ventricles; the apparent dissociation constant (Kd) was approximately 0.2 - 0.3 nM and the number of maximal binding sites was approximately 190,000 - 300,000 sites/cell. rANP significantly stimulated intracellular cGMP formation of cardiac NMCs in a dose-dependent manner (1.6 X 10(-8) M - 3.2 X 10(-7) M). rANP had no effect on synthesis of prostaglandin I2 by cultured cardiac NMCs. The physiological significance of ANP action on cardiac tissue remains to be determined.     

Characteristics of accumulation of ephedrine in rabbit atria.

The characteristics of uptake of (not equal to)-(beta-14C)ephedrine were studied in isolated rabbit atria. Ephedrine was rapidly accumulated against the concentration gradient. From 5 X 10-7 to 10-2 M, uptake occurred at a uniform initial rate. Uptake was slightly inhibited by high concentrations of ouabain, cocaine, desipramine, lidocaine and phenethylamines, and by a reduction in the external Na+ concentration. Uptake was not, however, reduced by omission of K+ from the medium, by metabolic inhibitors or by a variety of drugs known to inhibit the extraneuronal uptake and binding of noradrenaline. Pretreatment of animals with 6-hydroxydopamine very significantly reduced the uptake of (not equal to)-(3H)metaraminol, but did not alter the uptake of ephedrine. It was concluded that the uptake of ephedrine in rabbit atria occurred predominantly in extraneuronal tissues possibly as a result of passive diffusion followed by binding.     

Relation between intracellular Na ion activity and tension of sheep cardiac Purkinje fibers exposed to dihydro-ouabain.

The intracellular Na ion activity (aiNa) and the contractile tension (T) of sheep cardiac Purkinje fibers were simultaneously measured employing recessed-tip Na+-selective glass microelectrodes and a mechano-electric transducer. The aiNa of 6.4 +/- 1.6 mM (mean +/- SD, n = 56) was obtained in fibers perfused with normal Tyrode's solution. The changes in aiNa and T were measured during and after the exposure of fibers to a cardiac glycoside, dihydro-ouabain (DHO) in concentrations between 5 X 10(-8) M and 10(-5) M. The exposure time to DHO was 15 min. Both aiNa and T did not change in fibers exposed to 5 X 10(-8) M DHO, and the threshold concentration for the effect of DHO appeared to be around 10(-7) M. In DHO concentrations greater than the threshold, the increases in aiNa and T strongly correlated during the onset of DHO effects. The recoveries of aiNa and T were variable and slow, being dependent on the DHO concentration. In those fibers which recovered from the effects of DHO, the time-course of aiNa recovery was similar to that of T recovery. In fibers exposed to DHO of 5 X 10(-6) M or greater, the apparent toxic effects were observed in both action potential and contraction after an initial increase in T. The fibers manifesting the apparent toxic effects has a aiNa of approximately 30 mM or greater. The results of this study indicate that the increase in aiNa is associated with the positive inotropic action of the cardiac glycoside.     

Blood pressure and cardiac tissue responses to prostacyclin (PGI2) in various species

The effect of prostacyclin (PGI2) on blood pressure and heart rate (in vivo) and on isolated heart tissue has been investigated in different species. Isolated cardiac tissue had limited responses to PGI2 tested at 10(-13) to 10(-5) M. Cultured neonatal rat heart cells did not respond to PGI2, neither did intact rat hearts or rabbit cardiac tissue. Guinea pig and rat atria showed limited dose-dependent responses to PGI2 at concentrations greater than 10(7) M. In rat atria, 10(-5) M PGI2 produced a limited elevation of tissue cAMP content. When given by intravenous injection or infusion, PGI2 produced hypotension in anaesthetized primates (three species), rat, rabbit, pig, and dog. As a vasodepressor in all species, PGI2 (on a weight basis) was more active than prostaglandins of the B or E type and, in most species tested, it was approximately five times more active than PGE2. Heart responses in intact animals were often paradoxical in that decreases in heart rate often accompanied blood pressure falls.     

Lidocaine alters the input resistance and evokes neural activity in crayfish sensory neurons

Lidocaine, a use-dependent Na(+) channel blocker, paradoxically evokes neural activation in the slowly adapting stretch receptor organ of crayfish at 5-10 mmol/l concentration. For elucidating the underlying mechanisms of this paradoxical effect, a series of conventional electrophysiological experiments were performed in the stretch receptor neurons of crayfish. In the presence of tetrodotoxin, lidocaine did not evoke impulse activity, however, a slowly developing and dose-dependent depolarization occurred in both the rapidly and slowly adapting stretch receptors. Similar effects were observed by perfusion of equivalent concentrations of benzocaine but not of procaine or prilocaine. Lidocaine did not evoke neural activity in the rapidly adapting neuron which fires action potential(s) in response to rapid changes in membrane potential. Slowly developing mode of the depolarization indicated the reason why only depolarization but not action potential responses were observed in the rapidly adapting neuron. The depolarizing effect of lidocaine was independent from any ionic channel or exchanger system. However, lidocaine and benzocaine but not procaine and prilocaine evoked a dose-dependent alteration in the input resistance of the neuron. It was proposed that the principal mechanism of the effect could stem from a change in the physical properties of the neuronal membrane.     

Lidocaine turns the surface charge of biological membranes more positive and changes the permeability of blood-brain barrier culture models

The surface charge of brain endothelial cells forming the blood-brain barrier (BBB) is highly negative due to phospholipids in the plasma membrane and the glycocalyx. This negative charge is an important element of the defense systems of the BBB. Lidocaine, a cationic and lipophilic molecule which has anaesthetic and antiarrhytmic properties, exerts its actions by interacting with lipid membranes. Lidocaine when administered intravenously acts on vascular endothelial cells, but its direct effect on brain endothelial cells has not yet been studied. Our aim was to measure the effect of lidocaine on the charge of biological membranes and the barrier function of brain endothelial cells. We used the simplified membrane model, the bacteriorhodopsin (bR) containing purple membrane of Halobacterium salinarum and culture models of the BBB. We found that lidocaine turns the negative surface charge of purple membrane more positive and restores the function of the proton pump bR. Lidocaine also changed the zeta potential of brain endothelial cells in the same way. Short-term lidocaine treatment at a 10 μM therapeutically relevant concentration did not cause major BBB barrier dysfunction, substantial change in cell morphology or P-glycoprotein efflux pump inhibition. Lidocaine treatment decreased the flux of a cationic lipophilic molecule across the cell layer, but had no effect on the penetration of hydrophilic neutral or negatively charged markers. Our observations help to understand the biophysical background of the effect of lidocaine on biological membranes and draws the attention to the interaction of cationic drug molecules at the level of the BBB.     

Regulation by calcium and calmodulin of adenylate cyclase from rabbit intestinal epithelium

The effect of calcium on adenylate cyclase from rabbit small intestine has been studied using a particulate preparation obtained from isolated epithelial cells. Both basal and vasoactive intestinal peptide-stimulated activities were inhibited by calcium concentrations in the micromolar range. In the presence of calmodulin, a biphasic response was obtained. At low calcium concentration (4 X 10(-9)-6 X 10(-8) M) the enzyme was activated up to 50%. As the Ca2+ concentration was increased, the enzyme was concomitantly inhibited. Half-maximal inhibition of calmodulin-dependent activity was obtained at 1 microM free Ca2+. The activation of the enzyme was also dependent on the concentration of Mg2+. At less than 1 microM Ca2+, the enzyme exhibited a biphasic response, being activated at below 3 mM Mg2+ and inhibited at higher concentrations. At Ca2+ concentrations that were inhibitory, the enzyme did not show the biphasic response to Mg2+. At concentrations above 3 mM, the maximal rate (Vmax) remained constant. Vmax was inversely proportional to the concentration of Ca2+ present. Calmodulin altered Vmax when acting on vasoactive intestinal peptide-stimulated enzyme. Calmodulin had no effect on the Km for hormone activation. The calmodulin-dependent activity was inhibited by incubation with trifluoperazine.     

Lidocaine block of cardiac sodium channels

Lidocaine block of cardiac sodium channels was studied in voltage-clamped rabbit purkinje fibers at drug concentrations ranging from 1 mM down to effective antiarrhythmic doses (5-20 μM). Dose-response curves indicated that lidocaine blocks the channel by binding one-to-one, with a voltage-dependent K(d). The half-blocking concentration varied from more than 300 μM, at a negative holding potential where inactivation was completely removed, to approximately 10 μM, at a depolarized holding potential where inactivation was nearly complete. Lidocaine block showed prominent use dependence with trains of depolarizing pulses from a negative holding potential. During the interval between pulses, repriming of I (Na) displayed two exponential components, a normally recovering component (τless than 0.2 s), and a lidocaine-induced, slowly recovering fraction (τ approximately 1-2 s at pH 7.0). Raising the lidocaine concentration magnified the slowly recovering fraction without changing its time course; after a long depolarization, this fraction was one-half at approximately 10 μM lidocaine, just as expected if it corresponded to drug-bound, inactivated channels. At less than or equal to 20 μM lidocaine, the slowly recovering fraction grew exponentially to a steady level as the preceding depolarization was prolonged; the time course was the same for strong or weak depolarizations, that is, with or without significant activation of I(Na). This argues that use dependence at therapeutic levels reflects block of inactivated channels, rather than block of open channels. Overall, these results provide direct evidence for the “modulated-receptor hypothesis” of Hille (1977) and Hondeghem and Katzung (1977). Unlike tetrodotoxin, lidocaine shows similar interactions with Na channels of heart, nerve, and skeletal muscle.     

A Quantitative study of histamine H2-receptor bockade by burimamide in isolated artica.

The onset of burimamide inhibition of histamine stimulation of rabbit atria is rapid, and a near steady-state blockade occurs at approximately 15 min ( larger than or equal to 90% complete). The blockade is reversible but requires several washings suggesting the disassociation is slow. The administration of histamine may accelerate the decay of the burimamide effect. Reciprocal plots (rate response versus histamine concentration) of dose-response curves are linear for both rabbit and guinea pig atria. In the presence of low concentrations of burimamide; (2.4 times 10-5 M), the displacement of curves suggests a competitive type of inhibition both for rabbit and guinea guinea pig atria. The apparent association constants calculated from these curves are: K1 (rabbit) 3.7 times 10-6M and K-1 (guinea pig) 6.7 times 10-6M. These results for guinea pig atria are in satisfactory agreement with the value obtained in another laboratory (2). At higher concentrations of burimamide, inhibition curves showed distinct evidence of departure from competitive character for both guinea pig and rabbit atria.     

Inhibition of the positive inotropic effect of anthopleurin-A (AP-A) by dantrolene

The effect of dantrolene on the positive inotropic effects (PIE) of three cardiotonic agents was assessed on rat and rabbit atria. Dantrolene (10^?5M) had no effect on contractile tension or on the PIE to isoproterenol (10^?10?10^?7M) or ouabain (10^?6?10^?4). The dose-response curve for the PIE of anthopleurin-A (AP-A) was shifted to the right in rat and rabbit atria by dantrolene (10^?4?10^?6M). The maximum effect and the concentration of AP-A required for it remained the same. The results suggest that the PIE of AP-A involves intracellular translocation of calcium, unlike those of isoproterenol and ouabain which require increased transmembrane calcium flux. This conclusion is supported by the observation that the exchange and distribution of the labile calcium involved in excitation-contraction coupling was unaffected by AP-A (10^?8M), by dantrolene (10^?6M) or by the combination. Therefore, AP-A may produce its cardiotonic effect by a action at an intracellular site, a mechanism unlike that of isoproterenol or ouabain.     

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.     

Effect of vanadate on cardiac contraction and adenylate cyclase.

Vanadate produces a positive inotropic effect on ventricular muscle from rat, rabbit, guinea pig and cat; a positive inotropic effect on the atria of rat and rabbit, but a negative inotropic effect on the atria of guinea pig and cat. The effects of vanadate are completely reversible and occur in a concentration range of 10^?5M to 10^?3M. In this same concentration range, vanadate also causes a marked activation of cardiac adenylate cyclase suggesting that the positive inotropic action might be due in part to an elevation of cyclic AMP levels. The effects of vanadate are not influenced by alprenolol, cimetidine, or mepyramine, indicating a lack of involvement of β-adrenergic or histamine H₂ and H₁ receptors.     

Direct actions of prostaglandins E1, A1, and F2α on myocardial contraction

The inotropic and chronotropic actions of prostaglandin (PG) types PGE₁, PGA₁, and PGF_2α were studied in isolated guinea pig right and left atria, and papillary muscles; rabbit atria; and toad ventricular strips in order to more completely define the cardiac contractile properties of PG. All three prostaglandins, in muscle bath concentrations of 10μg/ml, exerted positive inotropic and chronotropic actions on guinea pig atrium. These contractile effects were persistent after removal of PG from the muscle bath and appeared to limit the relative response to a subsequent dose of PG. The inotropic action of PGE₁ was present over a wide range of bath calcium concentrations (1.1 to 4.4 mM/L). Beta adrenergic receptor blockade, histamine blockade, and pretreatment with reserpine failed to significantly affect the inotropic actions of PG. Norepinephrine and histamine produced more potent inotropic and chronotropic effects on guinea pig atria than did PG and these contractile effects did not exhibit persistence or tachyphylaxis. The prostaglandins did not significantly affect dose response curves for norepinephrine inotropic and chronotropic actions. The prostaglandins had no effect on the force or frequency of contraction in rabbit atria. PGE₁ exerted a positive inotropic effect on toad ventricular myocardium whereas PGA₁ had no effect and PGF_2α had a negative inotropic action.     

SUN 1165: a new antiarrhythmic Na current blocker in ventricular myocytes of guinea-pig

1. We compared the effect of a new antiarrhythmic compound, SUN 1165, on Na and Ca channels in papillary muscles and enzymatically dispersed single ventricular cells of guinea-pig. Action potential and contractile force in papillary muscle were measured by the conventional microelectrode technique and a strain gauge. The membrane currents were measured in internally perfused and voltage clamped cells by a single suction pipette technique. 2. In papillary muscles, SUN 1165 depressed the maximum rate of rise of action potential (Vmax) in a concentration dependent manner (IC30 = 1.7 X 10(-5) M) more markedly (about six times) than the contractile force. 3. In single ventricular cells, the Na current (INa) was reduced by the drug in a concentration dependent manner (IC30 = 9.1 X 10(-6) M). 4. It showed frequency-dependent block and the steady-state inactivation curve was shifted to more negative potentials. 5. The recovery of INa from inactivation was prolonged by SUN 1165. 6. The Ca current (ICa) was also blocked by the drug in a concentration dependent manner but much less than INa (IC30 = 5.5 X 10(-5) M). 7. These results suggested that SUN 1165 causes a selective inhibition of Na channels in guinea-pig ventricular cells at the antiarrhythmic concentrations.