Effects of the new cardiotonic agent sulmazole on the slow inward current of sheep cardiac Purkinje fibres.

The new cardiotonic agent 2-[(2-methoxy-4-methylsulfinyl)-phenyl]-1H-imidazo[4,5-b]pyridine (sulmazole, AR-L 115 BS) has marked positive inotropism but causes a depression in the plateau phase of the action potential of cardiac Purkinje fibres. This loss of plateau is known to occur with calcium antagonists which reduce contractility. In order to identify the mechanism underlying this possibly controversal effect the slow (calcium dependent) inward current (isi) was measured using the double microelectrode voltage clamp technique. In this current system, sulmazole was observed to have a slight effect on the inactivation parameter f infinity of isi by shifting it in hyperpolarizing direction. This increase in inactivation was considered when isi was determined. However, isi itself is reduced quite considerably and the linear instantaneous current voltage relationship is shifted to negative potentials. The kinetics of activation (d infinity) are not affected by sulmazole. From the more or less parallel shifts of isi we conclude that the reversal potential of isi is decreased which in turn strongly indicates an increase of intracellular calcium ion concentration. The reduction of isi by sulmazole is not the result of a specific membrane effect as in the case of some calcium antagonists. Sulmazole does not generate its positive inotropism by way of an increased slow inward current as do beta-adrenoceptor agonists but rather reduces the slow inward current by means of a negative shift of Eisi and a decrease in isi-driving force after it has affected intracellular calcium.

The response of the calcium dependent myocardial contractility to the new cardiotonic agent sulmazole.

In vitro and in vivo animal experiments to test the positive inotropism of 2-[(2-methoxy-4-methylsulfinyl)-phenyl]-1H-imidazo[4,5-b]pyridine (sulmazole, AR-L 115 BS) have shown increases of contractility of up to 220% in the mmol/l concentration range. Comparative studies designed to attribute to sulmazole mechanisms established for other positive inotropic drugs have been negative for digitalis and beta-adrenoceptor agonists but showed slight similarities to xanthine derivatives. To explore the yet unknown mode of action of sulmazole we investigated the interaction between sulmazole and non-humoral/non-drug interventions for positive inotropism in vitro. Peak tension obtained in solutions varying in calcium content between 0.36 and 7.2 mmol/l Ca2+ were normalized with respect to the tension measured at standard 1.8 mmol/l Ca2+ for control and sulmazole treated preparations (sheep interventricularis cordis muscle and guinea pig papillary muscle). Double logarithmic plots of normalized tension versus calcium concentration were linear and superimposable for control, 10(-4), and 10(-3) mol/l sulmazole. In depleted sodium solutions (choline substitution) normalized peak tension of control and 10(-4) mol/l sulmazole containing solutions did not significantly differ. In length-tension plots sulmazole behaved as did increased extracellular calcium. We assume that the positive inotropism of sulmazole is primarily due to a displacement of calcium at non-specific ligand sites within the protein matrix of the cytosol which effectively raises the apparent intracellular calcium activity.

Thallium determinations in fetal tissues and maternal brain and kidney.

The transfer of T1+ cations through the placental barrier of pregnant mice and rats was studied by comparing the thallium concentrations in the maternal brain and kidney and those in fetal tissue at times of 10 min to 50 h after dosage of the animals with 10 mg/kg body wt. T1(2)SO4. The quantitative determinations were performed with field desorption mass spectrometry after dilution of the homogenised tissue samples with enriched stable isotopes of thallium. The total sample quantity required for one complete assay is 1-3 micrograms, the analysis time for one determination about 30 min.

Determination of rubidium in human serum.

Field desorption mass spectrometry (FD-MS) combined with stable isotope dilution has been used to determine rubidium concentrations from human serum. Samples obtained from 110 healthy volunteers (50 males, 60 females) were examined. The rubidium concentrations found varied from 0.96 to 3.56 mumoles/l, the average value being 1.96 mumoles/l. The precision of the measurements within a batch was 0.8%. The time for 1 analysis, including sample preparation is about 30 min, the total sample consumption is 100-200 mul. The corresponding potassium concentrations were also determined from all these serum samples; a weak trend towards higher potassium levels with increasing rubidium concentration is found.

Effects of acrihellin on cardiac contractility in comparison to various inotropic interventions.

The inotropic effects of the new cardiotonic drug 3 beta,5,14-trihydroxy-19-oxo-5 beta-bufa-20,22-dienolide-3-(3-methylcrotonate) (D 12316, acrihellin) were investigated using twitch contractions time to peak and twitch lengths did not change. Peak tension increased by 140% on an average in 12 experiments at 10(-6) mol/l (acrihellin). In the dose-response curve half maximal positive inotropism occurred at approx. 10(-7) mol/l. Experiments on length tension relationships of resting and twitch tension confirmed that resting tension does not change at any stretch length after administering acrihellin. The position Lmax of maximal twitch contraction force also was not changed but in normalized length-tension cones the positive slope region showed an increase relative to maximal tension which is a feature of positive inotropic interventions. Measurements of contractility in various external calcium solutions between 0.45 mmol/l and 7.2 mmol/l gave evidence that the response of contraction force of fibres exposed to acrihellin to variation of external calcium differs from untreated fibres which respond more strongly. This behaviour of the cardiosteroid acrihellin was very similar to that of adrenaline (epinephrine) whereas digoxin and ouabain did not affect the response of twitch force to changes in external calcium.

Voltage clamp study of the action of the new cardiotonic compound acrihellin on fast and slow inward currents in cardiac Purkinje fibres.

The action of 3 beta,5,14-trihydroxy-19-oxo-5 beta-bufa-20,22-dienolide-3-(3-methylcrotonate) (D 12316, acrihellin) on the fast and slow inward currents of sheep cardiac Purkinje fibres was analyzed by means of a conventional double microelectrode voltage clamp technique. Acrihellin diminished action potential duration and the plateau phase. Effective refractory periods were shortened in relation to the decrease in action potential duration. The upstroke velocity was slightly decreased; a systematic study of dV/dtmax, however, did not reveal any specific local anaesthetic side-effect of the drug. The decrease in the plateau phase of the action potential was due to a reduction in time-dependent slow inward current. The kinetics and the reversal potential of the slow inward current was not affected by acrihellin, but only the current voltage relationship, which was decreased.

The influence of a new cardiotonic drug, acrihellin, on sinus node and the current underlying pacemaker activity of cardiac Purkinje fibres.

The spontaneous beating frequency of guinea pig sino-atrial preparations was observed under the influence of 3 beta, 5, 14-trihydroxy-19-oxo-5 beta-bufa-20,22-dienolide-3-(3-methylcrotonate (D 12316, acrihellin), a new cardiotonic drug. There was a slight acceleration of spontaneous frequency after administering rather high doses (10(-6) mol/l) of the drug. Voltage clamp experiments were performed in sheep cardiac Purkinje fibres. AcriheLlin strongly affected the current underlying pacemaker activity, ik2. The kinetic parameter s infinity of this current system was shifted in the hyperpolarizing direction by 4-13 mV. The amplitude of the pacemaker current in Purkinje fibres was reduced by up to 25% in voltage clamp experiments, but the reversal potential and the inward going rectification were left unchanged. After exposure to adrenaline (epinephrine) the well-known depolarizing shift of s infinity was partially restored by acrihellin. This can be interpreted as an antiadrenergic effect of the drug similar to that of beta-adrenoceptor antagonists. However, beta-blockers do not affect the pacemaker kinetics of fibres untreated with adrenaline as does acrihellin. This suggests that the interplay of acrihellin and adrenaline is not that of an agonist and antagonist at a membrane receptor but rather the overlapping of two separate membrane effects.

Tl+-ions: effects on the automaticity of sinoatrial tissue and dV/dtmax and iK2 of cardiac Purkinje fibres.

The disrhythmic effects of thallium were investigated in various cardiac tissues to determine the primary site of intoxication with respect to ensuing arrhythmias. In isolated cardiac tissue Lameijer and van Zwieten [1] had contended that arrhythmias arise from the sinus node after thallium poisoning. To test this hypothesis we administered concentrations of Tl+ between 10(-7) and 10(-4) M to guinea pig sinoatrial preparations, to guinea pig papillary muscles and to sheep cardiac Purkinje fibres. In sinoartial preparations thallium provoked increases and decreases of spontaneous beat frequency which were not linked to corresponding changes in contractile force. In conductive tissue, Purkinje fibres, the inactivation kinetics of the fast sodium current and the pacemaker current iK2 were investigated by voltage clamp experiments. Here, thallium was seen to be essentially without toxic effects which could account for arrhythmias. In ventricular muscle actions potentials and contractile force were recorded simultaneously. Here again, ventricular arrhythmias are not to be expected from thallium intoxication in rather high concentrations. The findings support the view that arrhythmogenic effects of thallium are restricted to the sinus node.

Tl+-ions: influence on cardiac contractility.

Attention has recently focussed on the heavy metal thallium as an environmental contaminant of increasing importance. From accidental or suicidal ingestions of thallium it has been known for many years that cardiovascular disorders regularly emerge, and for this reason, a variety of investigations of cardiological interest have been conducted. Amongst these, the effects of thallium on the contractile force of isolated myocardial tissues have been studied. Previous experiments were all carried out at concentrations far beyond those encountered during intoxication and yielded controversal data. We therefore reinvestigated the effects of thallium on myocardial tissue at levels between l0(-8) and 10(-3) M, thus covering the range of thallium concentrations encountered after uptake from a polluted environment through those seen after unintentional or intentional ingestion to levels at which previous studies were performed. Sheep interventricular cordis muscles were used at a stimulation frequency of 0.4 Hz showing three types of responses to thallium exposure. From a total of 32 experiments in 15% of all cases thallium caused a persistent increase in contractility which tended to decrease with time and thallium concentration but always remained greater than the control value. 50% of the experiments showed a progressive loss of contractile force with time and thallium concentration, despite transient increases in contractility which lasted for only 2-5 min after the application of each new thallium concentration. A combination of these types of reaction was observed in the remaining experiments in that a low thallium concentrations myocardial contractility increased considerably but then decreased progressively with time and thallium concentration. Guinea pig papillary muscles were used to test one thallium concentration only for up to 75 min. At 10(-8) M there was no effect, at 10(-7), 10(-6), 10(-5) M Tl+ there were positive inotropic transients followed by an inotropic decay; at 10(-4) M Tl+ only a progressive decrease of contractility was observed. The relationships between time and thallium concentration at different rates of stimulation were examined in two series of experiments at 0.1, 0.2, 0.4, 1.0, and 2.0 Hz. The effects of thallium were accelerated with increased beating rate and the decay of contraction also proceeded to markedly lower levels. In the rested state, thallium was also very effectual; this was illustrated in two series of experiments in which after 10 min intervals of quiescency 15 or more test stimuli were applied at different beating rates (0.1 to 2.0 Hz). The configurations of the resulting staircase phenomena were analyzed with respect to control behavior for each frequency of the test stimuli and for each thallium concentration. These results suggested an involvement of the slow inward current. The steady state values after quiescency showed a pronounced thallium-induced decay similar to that obtained at high constant stimulation rates...

A voltage clamp study of the effects of AR-L 115 BS on the pacemaker current of cardiac Purkinje fibres.

The new cardiotonic agent AR-L 115 BS was investigated by means of the double-micro-electrode voltage clamp technique on sheep cardiac Purkinje fibres. Clinical and pharmacological studies show that AR-L 115 BS increases heart rate as a side effect at medium to high therapeutic doses. The classical analysis of the pacemaker current was therefore performed to study the possible mechanism of this effect at a cellular level. The kinetic parameter s infinity and the reversal potential of the pacemaker current were shifted in the depolarizing direction after exposure to AR-L 115 BS. Peak values of the fully activated pacemaker current were either increased or diminished, while potassium leakage was slightly increased. These results are not related to the action of AR-L 115 BS on beta-adrenergic receptors but possibly due to enhanced intracellular calcium (see third paper in this series). Despite its tendency to increase heart rate, high concentrations of AR-L 115 BS should not be expected to promote arrhythmias in the Purkinje system since the electrophysiological effects tend to counteract each other.

The action of beta-adrenoceptor antagonist penbutolol on the pacemaker current of cardiac Purkinje fibres.

The action of beta-adrenoceptor antagonist penbutolol on the current underlying pacemaker activity in cardiac Purkinje fibres was analysed using the voltage clamp technique described by DECK et al. (1964). After the application of adrenalin, beta-blockers are able to counteract the well known shift of the s-kinetics of the pacemaker current. However, without any prior application of adrenalin the beta-blocker Penbutolol has no effect on these kinetics except for a small depression of the amplitude of the pacemaker current tails. The rectifier properties of the pacemaker current and the negative slope of the fully activated current voltage relationship of iK2 are unchanged. Penbutolol is able--even after a longer period of washout (about 60-90 min was necessary)--to protect the beta-adrenoceptors from the action of adrenalin (HASHIMOTO et al., 1979). These findings suggest that beta-blockers are competitive inhibitors of beta-stimulators and further support the notion that the pacemaker current in cardiac Purkinje fibres is controlled by beta-adrenoceptors.

Quantitative investigations on the diaplacental transfer of thallium by field desorption mass spectrometry.

The placental transfer of thallium cations in pregnant mice was investigated by determining the thallium concentrations in fetal and maternal tissue 0.5 to 24 h after application of thallium. The maternal dose of thallium was 8 mg/kg body weight throughout. Uterus and fetus were found not to differ from other organs like heart and liver in time course and magnitude of thallium uptake with an initial surge during the first few hours of exposure to thallium and a rapid decrease to steady 12 and 24 h values somewhat lower than those found in the kidney. Diaplacental transfer is therefore assumed comparatively rapid and a specific placental barrier for thallium does not seem to exist. For the determination of thallium concentrations Field Desorption Mass Spectrometry was utilized as a reliable, fast and sensitive method for the analysis of metal cations in biological material. This method does not require extensive pretreatment of the tissue and total sample amounts in the range of milligrams and less are sufficient for quantitative analysis.

Quantitative measurement of time-dependent thallium distribution in organs of mice by field desorption mass spectrometry.

The time-dependent distribution of the toxic heavy metal TI in mouse organs was determined after feeding 80, 130, and 160 mg/kg TI. Quantitative measurements were performed by field desorption mass spectrometry with stable isotope dilution. No pretreatment of the tissue samples, other than homogenizing and centrifugation, was necessary. The precision of the data was about +/- 10%. The main results are as follows: Heart, liver, kidney, and stomach showed an organ-specific initial uptake of TI during the first 2-3 h. This was followed by a period of washout to relatively low TI levels. Brain TI uptake was comparatively low and constant during the first 12 h. In the terminal stage (24 h) all organs, including the brain, contained increased TI levels of the same order of magnitude.

Effects of cryptand 2.2.1Py on the fast sodium current in cardiac Purkinje fibers.

For study of dose-dependent influence on sodium current, sheep heart Purkinje fibers were perfused with a cryptand (2.2.1Py) and measured for resting potential, overshoot, amplitude and duration of action potential, refractory period, and rate of rise. Concomitant investigation was made by voltage clamp. The cryptand was found to depress maximal rate rise with increase of concentration and induce shift toward negative potential with increase of dose. Also, effect on sodium current kinetics was observed but not on resting potential. The findings indicate that 2.2.1Py action probably is not primarily ionophores, negating the idea that cryptands promote transmembrane ion exchange.

Effect of cryptands on cardiac Purkinje fibres.

Cryptands are possibly useful synthetical models for passive ion carriers in biomembranes. Five mainly Na+ and K+ complex forming cryptands were studied in cardiac Purkinje fibres by means of standard microelectrode technique. Even at high concentrations the cryptands 22, 221 and 222 showed no effect. Possibly they are not lipophilic enough to accumulate within the membrane. The more lipophilic cryptands 222B and 222D caused a decrease of overshoot and maximal rate of rise (Vmax), a pronounced loss of plateau and a shortening of the action potential, while the resting potential was only slightly depolarized. 222B made some fibres inexcitable at normal resting potentials; only by increase in stimulation voltage a regular response was obtained. After about 20 min both cryptands (5 x 10(-5) mol/l) depolarized the fibres leading to irreversible inexcitability. The availability of the fast sodium current ("h infinity"-curve) was measured using Weidmann's (1955) technique. With 222B the h infinity-curve-showed a shift in the hyperpolarizing direction, and the maximal Vmax was diminished.