A postsynaptic effect of tyramine in ventricular muscle

Summary Tachyphylaxis and susceptibility to pretreatment with reserpine demonstrate that tyramine increases contractility of the guinea-pig papillary muscle in concentrations up to 10^–4 M by means of an indirect sympathomimetic effect.In higher concentrations (up to 3×10^–3 M) tyramine causes an increase in force of contraction which is characterized by a slowing of relaxation of the isometric contraction curve. Pretreatment with reserpine reveals that this positive inotropic effect is composed of an indirect sympathomimetic and a direct post-synaptic effect; the latter is not subject to tachyphylaxis and produces by itself (at 3×10^–3 M) an increase in force of contraction which amounts to about 50% of the maximum of the indirectly produced inotropic effect.The postsynaptically-induced positive inotropic effect of tyramine is not antagonized by propranolol (5×10^–6 M). It is unlikely, therefore, that it is brought about by stimulation of adrenergic -receptors.The height of the isometric contraction curve in reserpine-pretreated muscles is increased by tyramine as a result of an increase in the rate of force development despite a marked concentration-dependent increase in relaxation time (t ₂).The direct inotropic effect of tyramine is correlated with a prolongation of the duration of the action potential (AP) which amounts to 70% at 3×10^–3 M. In addition to its influence on the duration of the AP, tyramine slows the rate of depolarization and decreases the membrane resting potential.     

Differential responses to carbachol, sodium nitroprusside and 8-bromo-guanosine 3′,5′-monophosphate of canine atrial and ventricular muscle

1 The relation between force of contraction and cyclic nucleotide levels during muscarinic receptor stimulation, and after administration of sodium nitroprusside was assessed in canine isolated atrial and ventricular muscle.

2 The pD₂ value (negative logarithm of ED₅₀) for carbachol to decrease force of atrial contraction was similar to that required to inhibit adenosine 3′,5′-monophosphate (cyclic AMP)-mediated positive inotropic responses in ventricular muscle.

3 The cyclic AMP level of atrial muscle did not significantly change during carbachol-induced negative inotropic action, whilst the guanosine 3′,5′-monophosphate (cyclic GMP) level was elevated immediately after administration.

4 Sodium nitroprusside elevated cyclic GMP levels (without changing cyclic AMP levels) both in atrial and ventricular muscle. The force of atrial contraction was significantly reduced by the drug, whilst ventricular contractile force was unaffected.

5 8-Bromo-cyclic GMP markedly decreased contractile force in atrial muscle. In contrast, similar concentrations of 8-bromo-cyclic GMP had no effect on ventricular contractile force.

6 The positive inotropic action of phenylephrine on canine cardiac muscle, which is mediated through β-adrenoceptors, was unaffected either by sodium nitroprusside or by 8-bromo-cyclic GMP.

7 The present results suggest that the effect of muscarinic receptor stimulation in canine atrial and ventricular muscle is related to different changes in intracellular cyclic nucleotide metabolism. The direct myocardial depressant action on atrial muscle seems to be related to an elevation of cyclic GMP level, whilst a reduction of cyclic AMP may be responsible for the indirect action (`accentuated antagonism') in both atrial and ventricular muscle.

     

Effects of quinidine,procaine amide,and N-propyl-ajmaline on skeletal muscle

Summary Membrane and action potentials of rat diaphragm fibres were measured in vitro as affected by quinidine, procaine amide, and N-propyl-ajmaline in concentrations of 10^–5 to 10^–4 g/ml.All three drugs had immediate effects on the action potential; the effects progressed the faster, the higher the concentration: the de- and repolarization were slowed, the duration increased, the overshoot decreased. Later (e.g., 1 h after application of 10^–5 g/ml quinidine) the resting potential started to decrease and the fibres became inexcitable.Electrical threshold was increased and the refractory period was prolonged by all three drugs, most effectively by N-propyl-ajmaline, least by procaine amide. All drugs abolished myotonic symptoms produced by reduction of extracellular chloride.Quinidine and N-propyl-ajmaline (10^–5 to 10^–4 g/ml) produced a transitory increase of contraction force, on prolonged drug action the muscle went into contracture. Procaine amide always had a negative inotropic effect. All drugs increased contraction time.This work was supported by the Deutsche Forschungsgemeinschaft.     

Action of dimethindene on the electrophysiological and mechanical properties of atrial and ventricular myocardium of guinea-pig

The effect of dimethindene (DMI) on transmembrane potentials and contractile force was studied in atrial and ventricular myocardium of guinea-pigs. DMI reduced the maximum rate of depolarization (Vmax) without changing the resting potential in both preparations. In ventricular myocardium, DMI shortened the action potential duration and exerted a negative inotropic effect. In atrial muscle, the drug prolonged the action potential duration and induced a positive inotropic effect which could be antagonized with neither the alpha-blocker phentolamine, nor the beta-blocker pindolol, H1-blocker mepyramine and H2-blocker cimetidine. DMI had no effect on the slow action potentials induced by caffeine in K+-depolarized myocardial preparations. The drug consistently shifted the sodium inactivation curve to more negative membrane potentials. The results suggest that DMI has a quinidine-like membrane-stabilizing property, which may be due to its fast Na+ channel blocking activity. The differential inotropic action of the drug in atrial and ventricular myocardium is discussed.     

Possible mechanisms for inotropic actions of vanadate in isolated guinea pig and rat heart preparations

Summary The mechanism of inotropic actions of vanadate was studied in isolated, electrically stimulated atrial and ventricular muscle preparations of rat or guinea-pig heart. Vanadate produced a negative inotropic effect in guinea-pig left atrial preparations associated with a marked shortening of the action potential plateau. In guinea-pig papillary muscle, or rat atrial or ventricular muscle preparations, vanadate produced a positive inotropic effect, which was not affected by either propranolol, phentolamine or metiamide. The positive inotropic effect was observed when action potential duration was either increased or decreased. Inotropic concentrations of vanadate failed to significantly alter the ouabain-sensitive ⁸⁶Rb⁺-uptake, an estimate of sodium pump activity, or tissue concentration of cyclic AMP in electrically stimulated preparations. In partially depolarized rat atrial preparations in which fast sodium channels were inactivated in the presence of a high concentration of K⁺ (22 mmol/l), vanadate restored electrical activity (calcium-dependent action potentials) and the contraction, similar to isoproterenol. This action of vanadate was abolished by Mn²⁺, a slow channel inhibitor, but not by tetrodotoxin. The characteristic of vanadate- and isoproterenol-restored preparations, however, were substantially different. Moreover, vanadate failed to restore the contraction or action potential in partially depolarized guinea pig atrial preparations unlike isoproterenol. These results indicate that vanadate may either enhance or inhibit slow channels in cardiac muscle.     

Studies on the positive inotropic effect of dopamine in the guinea-pig heart

Summary The positive inotropic effect of dopamine has been studied in isolated ventricular strips of guinea-pig heart.The concentration-inotropic response curve for dopamine was significantly shifted to the right by pretreatment with reserpine.In preparations obtained from animals pretreated with reserpine (2.5 mg/kg, 24 h prior to the experiment) the dose-response curve was not significantly affected by haloperidol, a dopamine vascular receptor antagonist (10^–6–3×10^–6 M).The inotropic effect of dopamine was antagonized by practolol (3×10^–7–10^–6 M), but not by phentolamine (3×10^–6–10^–5 M); moreover the alpha-adrenoceptor blocking drug (10^–5 M) did not affect the curve for dopamine in the presence of practolol (3×10^–7 M).In preparations in which fast sodium channels were blocked by K⁺-rich medium, slow electrical responses (calcium-mediated action potentials) as well as contractions were induced by high concentrations of dopamine (10^–4–3×10^–4 M); again these responses were unaffected by phentolamine or haloperidol, but were blocked by practolol.It was concluded that in the guinea-pig ventricular muscle dopamine induced a positive inotropic effect through both indirect and direct action, and that the latter is due to the activation of beta-adrenoceptors.     

Positive inotropic response to 5-HT in human atrial but not in ventricular heart muscle

Summary The effects of 5-hydroxytryptamine (5-HT) on force of contraction (F_C), action potential (AP) and calcium current (I_Ca) were studied in human right atrial and left ventricular heart muscle. 5-HT exerted a concentration-dependent increase in F_C in multicellular atrial preparations; the EC₅₀ was approximately 3 × 10^–7 mol/l. Maximal increases in F_C (252±58% of control values; means ± SEM, n=6) were obtained at 5-HT 10^–5 mol/l. At this concentration, I_Ca was increased four- to sevenfold in enzymatically isolated atrial myocytes. In contrast, ventricular preparations did not respond to 5-HT; F_C, AP and I_Ca remained unaffected. In the same preparations, F_C was increased by isoprenaline three- to fourfold. These results confirm the observation that 5-HT induces a positive inotropic effect in the human atrium, possibly mediated by activation of the adenylyl cyclase — cyclic AMP system. Our study demonstrates, however, the complete lack of functional 5-HT receptors, with respect to changes in F_C, in the human ventricle. Since the positive inotropic effect of 5-HT in the human heart is obviously restricted to the atrium, our findings question the concept of developing 5-HT receptor agonists for the treatment of heart failure. Correspondence to: H. Nawrath at the above address     

Differential electrophysiologic and inotropic effects of phenylephrine in atrial and ventricular heart muscle preparations from rats

Summary Stimulation of ₁-adrenoceptors evokes a different pattern of inotropic responses in atrial and ventricular heart muscle preparations from rats. The inotropic effects are accompanied by different changes in membrane potential. In an attempt to clarify the question whether or to which extent these events are causally related, the effects of phenylephrine on force of contraction, transmembrane potential, Ca²⁺ current (I_Ca) and K⁺ currents were comparatively studied in either tissue.In atrial preparations, phenylephrine 10 mol/l caused an increase in force of contraction, a marked prolongation of the action potential duration and a depolarization of the membrane at rest. In the ventricle, however, the addition of phenylephrine 10 mol/l produced first a decline in force of contraction associated with a hyperpolarization of the membrane and a reduction in the action potential duration. These changes were followed by an increase in force,of contraction and a slight prolongation of the action potential, whereas the resting membrane potential remained increased. The hyperpolarization was eliminated in the presence of ouabain 100 mol/l.In enzymatically isolated atrial and ventricular myocytes, the whole-cell voltage clamp technique was used to study membrane currents on exposure to phenylephrine. Phenylephrine 30 mol/l did not affect the magnitude of I_Ca in either cell type. Transient and steady state K⁺ outward currents, however, were significantly diminished to a similar extent in atrial and in ventricular myocytes.It is concluded that the positive inotropic effect of ₁-adrenoceptor stimulation in the rat atrium is related to an increase in action potential duration and a decrease in resting membrane potential due to a decrease in K⁺ currents. In the ventricle, phenylephrine additionally activates the Na⁺/K⁺ pump thereby hyperpolarizing the membrane. The rapid onset of pump stimulation seems to overwhelm, in the beginning, the phenylephrine-induced decrease in K⁺ conductance and therefore to evoke a transient negative inotropic effect.It is assumed that phenylephrine can alter the intracellular Ca²⁺ concentration due to changes in the action potential duration. The way how Ca 2+ enters the cell remains speculative, since direct changes of Ica were not detected. The more complicated changes in membrane potential in the ventricle suggest that also other mechanisms for the positive inotropic response to phenylephrine must be considered. Send offprint requests to H. Nawrath at the above address     

Modification by glibenclamide of the electrophysiological consequences of myocardial ischaemia in dogs and rabbits

Summary Glibenclamide has been shown to block ATP-dependent K⁺ channels in the heart and prevent the shortening of cardiac action potentials caused by hypoxia in vitro. The present study examines the ability of glibenclamide to modify the effect of acute ischaemia on monophasic action potential duration in pentobarbital-anaesthetized rabbits, and on monophasic action potential duration and ventricular fibrillation threshold in pentobarbital-anaesthetized dogs. Left ventricular endocardial monophasic action potential duration was measured using a contact electrode catheter, and ventricular fibrillation threshold was measured by the single pulse method. Ischaemia was produced in rabbits by occluding the circumflex coronary for 5 min and in dogs by occluding the left anterior descending coronary artery for 40 min. In rabbits, glibenclamide (0.3 – 3 mg/kg, i. v.) had no effect on baseline monophasic action potential duration, but attenuated action potential shortening during ischaemia in a dose-related manner. In dogs, monophasic action potential duration did not shorten during ischaemia in the vehicle group, but tended to increase in the glibenclamide group (0.5 mg/kg, i. v.) both before and during ischaemia (7 ± 5% and 14 ± 8%, respectively, NS). Likewise, ventricular effective refractory period was significantly increased by glibenclamide prior to ischaemia (5 ± 1%). Ventricular fibrillation threshold tended to increase during 40 min of ischaemia in vehicle-treated dogs (40 ± 29%, NS), but was unchanged during ischaemia in the glibenclamide-treated dogs. Consistent with the increase in ventricular effective refractive period and monophasic action potential duration in dogs, glibenclamide (10^–7 – 10^–5 mol/l) significantly increased action potential duration in normoxic canine Purkinje fibres to a maximum of 19 – 2% at 95% repolarization. These data suggest that the opening of ATP-dependent K⁺ channels contributes to action potential shortening duration ischaemia in rabbits, but do not provide evidence that glibenclamide, at the dose studied, reduces the susceptibility to electrically-induced ventricular fibrillation in dogs subjected to acute ischaemia.Send offprint requests to Jeffrey Smallwood at the above address     

Actions of the phosphodiesterase inhibitor zardaverine on guinea-pig ventricular muscle

Summary The positive inotropic action of the phosphodiesterase inhibitor zardaverine was investigated in guinea-pig heart muscle. In right papillary muscles, 1–30 M zardaverine reversibly increased the force of contraction in a concentration-dependent manner. This effect was accompanied by a shortening of contraction and relaxation times. Resting membrane potential was unchanged, whereas action potential amplitude was significantly increased and duration was reduced. In papillary muscles partially depolarised with 22 mM K⁺, zardaverine (10 and 30 LM) restored slow action potentials, which were not influenced by cimetidine, propranolol or prazosin but were blocked by the calcium channel blocker (+)-nitrendipine or the muscarinic agonist carbachol. cAMP-specific phosphodiesterase III, isolated from guinea-pig ventricular muscle was inhibited by zardaverine as was cAMP-specific phosphodiesterase IV, isolated from dog trachea (IC₅₀s: 0.5 and 0.8 M, respectively). The results suggest that the observed positive inotropic and electrophysiological effects result from an inhibition of cellular phosphodiesterase.Send offprint requests to C. Schudt at the above address     

Potent inhibitory action of chlorethylclonidine on the positive inotropic effect and phosphoinositide hydrolysis mediated via myocardial alpha1-adrenoceptors in the rabbit ventricular myocardium

Summary The influence of the alpha_lb-adrenoceptor-selective antagonist chlorethylclonidine on the alpha₁-adrenergic positive inotropic effect and the phosphoinositide hydrolysis induced by phenylephrine was investigated in the rabbit ventricular myocardium. Pretreatment of membrane fractions derived from the rabbit ventricular muscle with 10^–5 mol/l chlorethylclonidine decreased the specific binding of [³H]prazosin (at a saturating concentration of 10^–9 mol/l) from the control value of 11.27±0.48 to 4.18±1.87 fmol/mg protein. The inhibition by adrenaline of the binding of [³H]prazosin (slope factor and affinity) was not affected by chlorethylclonidine. The positive inotropic effect of phenylephrine (in the presence of 3 × 10^–7 mol/l bupranolol) was inhibited by chlorethylclonidine in a concentration-dependent manner (10^–7–10^–5 mol/l) and abolished by 10^–5 mol/l chlorethylclonidine. The concentration of chlorethylclonidine to inhibit the phenylephrine-induced maximum response to 50% was 2.4 × 10^–6 mol/l. The accumulation of [³H]inositol monophosphate and [³H]inositol trisphosphate induced by 10^–5 mol/l phenylephrine was inhibited by chlorethylclonidine in the same concentration range. These findings indicate that the myocardial alpha₁-adrenoceptors mediating a positive inotropic effect in the rabbit ventricular myocardium may belong to the chlorethylclonidine-sensitive alpha_1b-subtype, and that the subcellular mechanism of action involve phosphoinositide hydrolysis. Send offprint requests to M. Endoh at the above address     

Electromechanical effects of anthopleurin-A (AP-A) on rabbit ventricular muscle: influence of driving frequency, calcium antagonists, tetrodotoxin, lidocaine and ryanodine

1 Anthopleurin-A (AP-A 5 × 10^-9 M, 1 × 10^-8 M) caused a prolongation of action potential duration (APD) and an increase of contractile force in rabbit isolated ventricular muscle preparations.

2 The prolongation of APD and the positive inotropic effect of AP-A (1 × 10^-8 M) were augmented by lowering the driving frequency from 2.0 to 0.2 Hz, resulting in an apparent negative staircase of contractile force in this frequency range. When the preparation was driven at an extremely low frequency (0.017 Hz), AP-A did not increase the contractile force, but caused a considerable prolongation of APD.

3 Verapamil (1 × 10^-6 M) and nifedipine (1 × 10^-6 M) had no apparent influence on the APD prolongation by AP-A (5 × 10^-9 M, 1 × 10^-8 M). The positive inotropic effect of AP-A was also relatively well maintained even in the presence of these calcium antagonistic drugs when the preparation was driven at a lower frequency (0.2 Hz).

4 Tetrodotoxin (TTX 2 × 10^-6 M) and lidocaine (1 × 10^-4 M) markedly inhibited both the APD prolongation and the positive inotropic effect of AP-A (1 × 10^-8 M).

5 In the presence of ryanodine (2 × 10^-6 M), an agent which is known to interfere with calcium release from the intracellular activator pool, AP-A (1 × 10^-8 M) failed to cause its positive inotropic effect in spite of the marked prolongation of APD.

6 These results suggest that the effects of AP-A on cardiac muscle are primarily mediated by the fast sodium inward current. Thus, delayed inactivation of sodium inward current may cause APD prolongation, and probably induces an alteration of intracellular calcium kinetics reflected by an increase of contractile force.

     

Antiarrhythmic effects of a benzothiazine derivative (SD-3211) and its stereoisomer (SA3212) in anaesthetized rats and isolated perfused rat hearts compared with bepridil

Summary The antiarrhythmic effects of a new calcium channel blocking agent (SD-3211) and its stereoisomer with additional sodium channel blocking activity (SA3212), were compared with those of a known antiarrhythmic drug (bepridil), using the left coronary artery ligation- and reperfusion-associated arrhythmia models both in isolated rat hearts and in anaesthetized rats.Isolated and perfused rat hearts were subjected to regional ischaemia for 15 min and subsequent reperfusion for 5 min. SD-3211 and SA3212 showed dose-dependently prolongations of the time interval between coronary ligation and first appearance of ventricular premature beats, reductions in the number of total ventricular premature beats during the ligation period and reductions in the incidence of reperfusion-induced ventricular fibrillation. The values of the negative logarithm of IC₅₀ (mol/l) of SD-3211, SA3212 and bepridil were 7.97, 7.41 and 6.64 for the reduction of ventricular premature beats during ligation and 6.43, 7.49 and 6.17 for the reduction of ventricular fibrillation during reperfusion, respectively. In a separate study on force of concentration and coronary flow in perfused heart paced at 340–360 beats/min SD-3211 caused a significant negative inotropic effect between 10^–7 and 10^–6 mol/l. SA3212 at the concentration of < 10^–6 mol/l did not result in any significant change in force of contraction. The coronary flow was increased dose-dependently by SA3212, while it was first increased and then reduced in the presence of higher concentration of SD-3211 (> 10^–7 mol/l). Hearts of aneasthetized rats were also subjected to regional ischaemia for 7 min and subsequent reperfusion. SD-3211, even at the lowest dose tested (25 mg/kg), had a marked protective effect against the ligation-associated arrhythmias. The highest dosage of SA3212 tested (100 mg/kg) also reduced them. SA3212, even at the lowest dosage (25 mg/kg), resulted in a significant reduction of the incidence of reperfusion-induced ventricular fibrillation, while only the highest dosage of SD-3211 (100 mg/kg) reduced it. As for the protective effect against ligation-associated ventricular premature beats SD-3211 is about seven times as potent as bepridil, and for the reduction in the incidence of reperfusion-induced ventricular fibrillation SA3212 is about fourteen times as potent as bepridil. Significant falls in systolic blood pressure and heart rate were observed at the higher doses of SD-3211 (50 and 100 mg/kg).Thus, SD-3211 affords substantial protection against ischaemia-induced ventricular antiarrhythmias partly through the negative inotropic and chronotropic effects, and reduction of afterload. The antiarrhythmic action of SA3212 against reperfusion-induced ventricular fibrillation may be partly explained by depression of automaticity together with a reduction of the inward sodium current.Send offprint requests to M. Fukuchi at the above address     

Effects of the Anemonia sulcata Toxin (ATX II) on Intracellular Sodium and Contractility in Rat and Guinea-Pig Myocardium

Abstract: The effects of the Anemonia sulcata toxin ATX II on action potentials and contractility of isolated papillary muscles and single myocytes from rat and guinea-pig hearts have been studied. ATX II prolonged the action potential in both rat and guinea-pig papillary muscle. Although it produced a positive inotropic effect in guinea-pig papillary muscle, it failed to do so in rat papillary muscle. However, in single rat and guinea-pig ventricular cells, it both prolonged the action potential and had a positive inotropic effect. We suggest that ATX II does not cause a positive inotropic effect in rat papillary muscle, because it induces Ca²⁺ overload. In single cells the positive inotropic effect was reduced by ?50% when the contractions were triggered by voltage clamp pulses of constant duration rather than by action potentials. This suggests that the inotropic effect of ATX II is in part the result of the prolongation of the action potential. The intracellular Na⁺ activity (aⁱ_Na) in single ventricular cells was measured with the Na⁺-sensitive fluorescent dye SBFI. After exposure of the cells to ATX II, aⁱ_Na was increased by a maximum of 1.9 ± 0.3 and 2.2 ± 0.3 mM in rat and guinea-pig cells, respectively. It is suggested that the positive inotropic effect of ATX II is also in part the result of the rise in aⁱ_Na.     

Evidence for adenosine receptor-mediated isoprenaline-antagonistic effects of the adenosine analogs PIA and NECA on force of contraction in guinea-pig atrial and ventricular cardiac preparations

Summary The effects of the adenosine agonists (–)-N⁶-phenylisopropyladenosine (PIA) and 5-N-ethylcarboxamideadenosine (NECA) on force of contraction, adenylate cyclase activity and normal as well as slow action potentials were studied in guinea-pig isolatedatrial (left auricles) andventricular preparations (papillary muscles).Inauricles PIA and NECA exerted concentration-dependent negative inotropic effects with similar potenticies (mean EC₅₀:0.05 mol l^–1 for PIA and 0.03 mol l^–1 for NECA). Similar results were obtained in the presence of isoprenaline.Inpapillary muscles PIA and NECA alone had no effect on force of contraction but produced negative inotropic effects in the presence of isoprenaline (mean EC₅₀:0.19 mol l^–1 for PIA and 0.10 mol l^–1 for NECA).In both preparations, the negative inotropic effects of PIA and NECA in the presence of isoprenaline were antagonized by the adenosine receptor antagonist 8-phenyltheophylline.In both preparations, PIA and NECA did not affect adenylate cyclase activity, both in the absence and presence of isoprenaline.Inauricles the negative inotropic effects of both nucleosides were accompanied by shortening of the action potential. This effect was also observed in the presence of isoprenaline. Inpapillary muscles the adenosine analogs did not detectably alter the shape of the normal action potential. Ca²⁺-dependent slow action potentials elicited in potassium-depolarized preparations also remained unaltered in the presence of PIA or NECA alone. However, the isoprenaline-induced enhancement of the maximal rate of depolarization of slow action potentials was attenuated by PIA or NECA.It is concluded that in guinea-pig atrial and ventricular cardiac preparations the adenosine analogs PIA and NECA exert isoprenaline-antagonistic effects on force of contraction via adenosine receptors the existence of which can thus be shown in a functional way. These receptors are not detectably coupled to the adenylate cyclase. The negative inotropic effect in theauricle is most likely due to a shortening of the action potential resulting from an activation of potassium channels, which in turn indirectly reduces the Ca²⁺ influx during the action potential. In theventricle the adenosine receptor is either not linked to these potassium channels or adenosine-sensitive potassium channels do not exist in the ventricle. Instead the activation of the receptor causes a decrease of the slow Ca²⁺ inward current but this effect is observed only when the slow Ca²⁺ inward current had previously been enhanced by a cyclic AMP-dependent mechanism.     

Electrophysiological actions of Mexiletine (Köl 173) on canine Purkinje fibres and ventricular muscle

1 The effects of mexiletine (Kö1173) were investigated in canine isolated cardiac Purkinje fibres and ventricular muscle with microelectrodes. Some Purkinje fibres were depolarized by mechanical stretch to induce spontaneous activity with slow upstroke velocity. The preparations were stimulated at rates of 1, 2, 3 and 4 Hz. The drug concentrations tested were 0.4, 2 and 10 μg/ml in Tyrode solution (KCl = 5.4 mM).

2 The `therapeutic' drug concentration (2 μg/ml) shortened action potential duration and effective refractory period of Purkinje fibres, the effect being pronounced at lower stimulation rates. In ventricular fibres, action potential duration changes were not consistent while the effective refractory period was prolonged.

3 In depolarized Purkinje fibres showing automatic activity, the drug (0.4 or 2 μg/ml) depressed phase 4 depolarization and reduced the firing rate without changing maximum diastolic potential. However, when depolarized Purkinje fibres were electrically driven at a constant rate, the maximum diastolic potential became more negative with a concomitant decrease of pacemaker slope and increase of maximum rate of rise (_max) of action potentials.

4 Moderate (2 μg/ml) to high (10 μg/ml) concentrations of the drug depressed _max in Purkinje fibres stimulated at 2 Hz by 12 and 42% respectively and depressed `membrane responsiveness'. The decrease in _max depended upon the stimulation rate, being minimum at the lowest (1 Hz) and maximum at the highest (4 Hz) stimulation rate.

5 The drug (2 μg/ml) improved _max of the earliest propagated premature action potentials by shifting the takeoff potential to more negative levels in both Purkinje and ventricular fibres.

6 Membrane conductance in fibres mounted in a single sucrose gap chamber was increased by the drug (2 μg/ml) in both fibre types in normal and in Na⁺-deficient solutions. This increase was attributed to an increase in membrane K⁺ permeability produced by the drug.

7 All these effects are similar to those of lignocaine, diphenylhydantoin or aprindine, and can explain the antiarrhythmic action of mexiletine.

     

Effect of sodium pentobarbital on the transmembrane action potential and the slow inward current of guinea pig ventricular myocytes

Barbiturates, which are widely used in clinical anesthesia, exert a negative inotropic effect on the myocardium. To investigate the mechanism for the negative inotropism, we studied the effect of sodium pentobarbital (SP) on the action potential and the slow inward current recorded from guinea pig ventricular myocytes. We found that SP at 0.6, 1.0, and 1.6 microM decreased action potential duration at 50% repolarization by 17 +/- 6, 25 +/- 11, and 29 +/- 8%, respectively (p less than 0.05, n = 4). At the concentration range of 0.6-1.6 microM, resting potential and action potential amplitude were unaffected. Voltage-clamp studies in ventricular myocytes demonstrated that SP at 0.6, 1.0, and 1.6 microM reduced the peak slow inward current by 30 +/- 4, 36 +/- 14, and 67 +/- 5%, respectively (p less than 0.005, n = 4). In conclusion, SP, a frequently used anesthetic, decreases the slow inward current in guinea pig ventricular myocytes at clinically relevant concentrations.     

Über den Einfluß von Mangan-Ionen auf die positiv inotrope Wirkung von Adrenalin,Theophyllin und Digitoxigenin an isolierten Meerschweinchenvorhöfen

Summary The influence of bivalent manganese ions (Mn⁺⁺) on the positive inotropic effect of adrenaline, theophylline, and digitoxigenin was studied in isolated, electrically driven left guinea-pig auricles in phosphate-free Tyrode's solutions with different extracellular calcium concentrations ([Ca]_e; 0.45; 1.8; 7.2 mM).Mn⁺⁺ (0.1–50 mM) exerted a dose-dependent negative inotropic effect which was dependent on [Ca]_e: Raising [Ca]_e decreased the inhibitory action of Mn⁺⁺. The negative inotropic effect of Mn⁺⁺ was exclusively due to a decrease in the rate of tension development; the time to peak tension and the duration of contraction remained unchanged.In a solution containing 0.45 mM Ca, pretreatment with 0.1 mM Mn⁺ significantly diminished the positive inotropic effect of adrenaline (10^–9-10^–5 g/ml) and theophylline (5×10^–6-10^–3 g/ml), but did not influence the effect of digitoxigenin (2×10^–7-2×10^–6 g/ml). The depression of the positive inotropic effect of adrenaline and theophylline with higher concentrations of Mn⁺ (0.35–2.25 mM, producing a negative inotropic effect of about 50%) was influenced by the [Ca]_e. The effect of Mn⁺ was most evident at 0.45 mM Ca, less pronounced (but significant) at 1.8 mM Ca and was not observed at 7.2 mM Ca. With the same concentrations of Mn⁺, however, the positive inotropic effect of digitoxigenin was only slightly decreased at 0.45 mM Ca, was not changed at 1.8 mM Ca and was increased at 7.2 mM Ca.As Mn⁺ selectively blocks the inward movement of Ca ions across the membrane of the myocardial cell during depolarisation, it is tentatively concluded from these experiments that the positive inotropic effect of adrenaline and theophylline may be due at least partially to an increase of the Ca influx during the excitation process, whereas the positive inotropic effect of digitoxigenin seems to be independent of this mechanism.

Herrn Dr. K. Löffelholz aus unserem Institut sind wir für die fluorometrische Adrenalinbestimmung zu Dank verpfiichtet. Außerdem danken wir Fräulein G. Rose für ihre Hilfe bei der Durchführung der Experimente.     

Electrophysiologic and anticholinergic effects of pirmenol enantiomers in guinea-pig myocardium

Summary Since it has been reported that several class I drugs stereoselectively block sodium channels, potassium channels and muscarinic receptors in cardiac tissues, electrophysiologic and anticholinergic effects of enantiomers of pirmenol, a class I antiarrhythmic drug, were examined. Both (+) and (–) pirmenol depressed the maximum upstroke velocity (V_max) of the action potential in a concentration-dependent manner in guinea-pig papillary muscles driven at 1.0 Hz, and there was no significant difference in the potency of the class I effect between the enantiomers. The onset rates of use-dependent block (UDB) of (V_max) at 2.0 Hz for 10 µmol/l (+) and (–) pirmenol were 0.30±0.03 and 0.29±0.01 per action potential, and the recovery time constants from UDB for (+) and (–) pirmenol were 27.0±2.7 and 27.7±1.9 s, respectively, indicating no difference in the binding and unbinding kinetics to the sodium channel between the enantiomers. Both (+) pirmenol and (–) pirmenol prolonged action potential duration (APD) at low concentrations (1–10 µmol/l) and shortened it at high concentrations (30–100 µmol/l) Again, there was little difference with respect to the effects on APD between the enantiomers. However, in the isolated guinea-pig left atria (–) pirmenol more potently antagonized the negative inotropic effect of carbachol than (+) pirmenol, and the pA₂ values for (+) and (–) pirmenol were 6.41 and 6.71, respectively. The functional study was supported by the radioligand binding experiments using [³H]N-methyl scopolamine ([³H]NMS) in guinea-pig left atrial membranes. Specific ([³H]NMS) binding was competitively displaced by the enantiomers, and the apparent dissociation constant for (+) pirmenol (1.95±0.23 µmol/l) was significantly greater than that for (–) pirmenol (0.90±0.18 µmol/l). These results suggest that pirmenol enantiomers stereoselectively interact with cardiac muscarinic receptors but not so with cardiac sodium and potassium channels. Correspondence to M. Kanno at the above address     

Activation of muscarinic receptors elicits inotropic responses in ventricular muscle from rats with heart failure through myosin light chain phosphorylation

Background and purpose:

Muscarinic stimulation increases myofilament Ca²⁺ sensitivity with no apparent inotropic response in normal rat myocardium. Increased myofilament Ca²⁺ sensitivity is a molecular mechanism promoting increased contractility in failing cardiac tissue. Thus, muscarinic receptor activation could elicit inotropic responses in ventricular myocardium from rats with heart failure, through increasing phosphorylation of myosin light chain (MLC).

Experimental approach:

Contractile force was measured in left ventricular papillary muscles from male Wistar rats, 6 weeks after left coronary artery ligation or sham surgery. Muscles were also frozen, and MLC-2 phosphorylation level was quantified.

Key results:

Carbachol (10 µmol·L⁻¹) evoked a positive inotropic response only in muscles from rats with heart failure approximating 36% of that elicited by 1 µmol·L⁻¹ isoproterenol (20 ± 1.5% and 56 ± 6.1% above basal respectively). Carbachol-evoked inotropic responses did not correlate with infarction size but did correlate with increased left ventricular end diastolic pressure, heart weight/body weight ratio and lung weight, primary indicators of the severity of heart failure. Only muscarinic receptor antagonists selective for M₂ receptors antagonized carbachol-mediated inotropic effects with the expected potency. Carbachol-evoked inotropic responses and increase in phosphorylated MLC-2 were attenuated by MLC kinase (ML-9) and Rho-kinase inhibition (Y-27632), and inotropic responses were abolished by Pertussis toxin pretreatment.

Conclusion and implications:

In failing ventricular muscle, muscarinic receptor activation, most likely via M₂ receptors, provides inotropic support by increasing MLC phosphorylation and consequently, myofilament Ca²⁺ sensitivity. Enhancement of myofilament Ca²⁺ sensitivity, representing a less energy-demanding mechanism of inotropic support may be particularly advantageous in failing hearts.