Fosinoprilate prolongs the action potential: reduction of iK and enhancement of the L-type calcium current in guinea pig ventricular myocytes.

OBJECTIVE: The aim was to study the effect of fosinoprilate, a new ACE inhibitor, on the action potential and plateau currents of cardiac muscle. METHODS: Whole cell patch technique was used to record action potentials (n = 6), the L-type iCa (iCaL; n = 5), in some cases (n = 4) also using Cs+ loaded pipettes; with 5 mM Co2+, the time dependent K+ current (IK) underlying delayed rectification was analysed in guinea pig ventricular myocytes (n = 3). RESULTS: Fosinoprilate prolonged the 50% repolarisation (APD50) from 440(SEM 50) ms to 485(48) ms (0.1 microM), to 525(46) ms (0.3 microM), to 632(58) ms (1 microM), and to 702(69) ms (3.0 microM). The APD90 was delayed from 510(63) ms to 540(45) ms (0.1 microM), to 583(42) ms (0.3 microM), to 702(62) ms (1.0 microM), and to 765(72) ms (3.0 microM). Higher concentrations (10-100 microM) caused early afterdepolarisations, very long action potentials, and irregular oscillations. ICaL was enhanced by up to 183%, showing a Kd of 0.2 microM; in contrast to the steady state activation (d infinity), the inactivation curve f infinity was shifted in the depolarising direction, considerably enlarging the Ca2+ window. Slow inactivation time course was unchanged, whereas the fast time constant (tau f) was accelerated. Fosinoprilate reduced the outward current during depolarising clamps from 1.7(0.2) nA to 1.41(0.11) nA with a 0.1 microM dose, and to 0.54(0.14) nA with a 1.0 microM dose; the tails were decreased from 0.39(0.03) nA to 0.27(0.03) nA with 0.1 microM and to 0.13(0.02) nA with 1.0 microM. Kinetics of IK were unaltered. Computer simulations based on these data using the OXSOFT-HEART program mimicked the results rather closely. CONCLUSIONS: The results suggest that fosinoprilate prolongs the plateau due to a partial block of iK and an extension of the Ca2+ window by 10 mV, causing a class III antiarrhythmic effect. High concentrations further open the Ca2+ window resulting in early afterdepolarisations and plateau oscillations and may cause an inward transport of Ca2+ ions by the Na-Ca exchange.

Fast and slow blockade of sodium channels by flecainide in rabbit cardiac Purkinje fibres.

The electrophysiological effects of flecainide were tested using the two-microelectrode voltage-clamp technique and Vmax-measurements in isolated rabbit cardiac Purkinje fibres. Flecainide predominantly unfolds its sodium-channel blocking action during the upstroke phase of the cardiac action potential, because its Vmax-depressant effects are independent of the duration of the depolarizing interval. Very long lasting depolarizations caused a second, very slow blocking activity. Starting from a steady-state block, recovery from block was tested and yielded a time constant of 7.3 s for a membrane potential of -105 mV. The strong blockade of sodium-channels combined with a delayed recovery behaviour of the drug-associated channels gives reasons for the observation of a marked use-dependent block. This block increased when the cycle length was shortened or the holding potential was less negative. Additional application of lidocaine in several concentrations did not significantly increase or attenuate the phasic block caused by flecainide alone. Under special conditions we investigated flecainide's depression and shift of the Vmax/Vm-relation and we observed that the concentration dependence of both parameters could be described by simple 1:1 binding reaction. The effects of flecainide are largely reversible often greater than or equal to 15 min. Flecainide could be characterized as an open channel blocker with a very slow inactivated channel blocking activity. For the qualitative description of the sodium-channel block by flecainide we used the "modulated-receptor hypothesis", whereas for reconstructions of the use-dependent action we applied the "guarded-receptor hypothesis", which enables computations of phasic block with the knowledge of only one forward and one reverse rate constant.

Pirmenol, a new antiarrhythmic drug with potassium- and sodium-channel blocking activity; a voltage-clamp study in rabbit Purkinje fibres.

The target of this study was to characterize the effect of pirmenol hydrochloride on the electrophysiological properties of cardiac cells. Action potential studies were carried out using the standard microelectrode technique in isolated rabbit Purkinje fibres. Information about the effect of pirmenol on the fast sodium current was obtained by Vmax-measurement. Furthermore the delayed rectifying current ix was studied by the two microelectrode voltage clamp technique. In concentrations of 0.5-5 mumol/l pirmenol caused a marked prolongation of the action potential duration in isolated rabbit Purkinje fibres. Measurements of the delayed rectifying current ix displayed a strong depression with a KD-value of 1 mumol/l pirmenol. The steady-state current voltage relation showed that pirmenol also caused a reduction of the steady-state sodium window current and/or of the slowly decaying components of the sodium current. In concentrations of greater than or equal to 10 mumol/l pirmenol the action potential duration was diminished again and Vmax was depressed in a use-dependent manner. Furthermore pirmenol caused a depression and a negative shift of the Vmax/Em-relation. Pirmenol blocked sodium channels which recovered from block with a time constant of 6.7 s at a holding potential of -105 mV. Similar to quinidine and sotalol the prolongation of the action potential duration under pirmenol is essentially caused by a diminution of the delayed rectifying current ix. The depression of Vmax is mainly independent from the action potential duration indicating the dominance of an open channel block. Pirmenol is a new drug with class Ia antiarrhythmic action.

Effects of flecainide on the electrophysiologic properties of isolated canine and rabbit myocardial fibers.

The electrophysiologic properties of flecainide, a new potent antiarrhythmic drug, are poorly defined. In this study, they were investigated by standard microelectrode technique in isolated cardiac muscle from rabbit and dog hearts. The concentrations of flecainide used were between 0.1 and 10.0 micrograms/ml. Flecainide produced a concentration-dependent decrease in maximal rate of rise of phase 0 of the action potential (Vmax), action potential amplitude and overshoot potential with an increase in the effective refractory period in ventricular muscle. Vmax was reduced by 52.5% after 1 microgram/ml of flecainide (p less than 0.001) and by 79.8% after 10.0 micrograms/ml (p less than 0.001). The corresponding values for Purkinje fibers were 18.6% (p less than 0.01) and 70.8% (p less than 0.001), respectively, but in these fibers the effective refractory period was shortened at the lower concentration and restored to control value at the higher concentration. The depression of Vmax by flecainide was frequency-dependent. The action potential duration was lengthened by flecainide in ventricular muscle and shortened in Purkinje fibers. At high concentrations (10 micrograms/ml), flecainide depressed slow channel-dependent fibers. Purkinje fiber automaticity induced by isoproterenol was slowed by flecainide. The data indicate that the overall electrophysiologic effects of flecainide in isolated cardiac muscle are complex with a major depressant action on Vmax that may account for its dominant antiarrhythmic effects. It is also possible that the differential effects of the compound on the action potential duration and refractoriness in ventricular muscle and Purkinje fibers contribute to the known arrhythmogenic potential of the drug.

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.

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 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.

Separation of the pace-maker and plateau components of delayed rectification in cardiac Purkinje fibres.

1. Experiments on sheep Purkinje fibres were designed to determine whether the current mechanisms responsible for delayed rectification at the pace-maker (negative to -50 mV) and plateau (positive to -50 mV) ranges of potential are kinetically separable and independent.2. Hyperpolarizations from the plateau range were shown to produce decay of a single component of outward current within the plateau range, but two components were evident when the hyperpolarizations entered the pace-maker range.3. The time courses of recovery of the two components were too similar at -25 mV to allow temporal resolution at this potential. Clear temporal resolution was, however, possible at potentials between -55 and -95 mV. An indirect method of resolving the two components at -25 mV was used.4. The kinetic properties of the two components correspond to those previously described for the pace-maker potassium current, i(K) (2), and the outward plateau current, i(x) (1) (Noble & Tsien, 1968, 1969a).5. The instantaneous (fully activated) current-voltage relation for i(K) (2) was reconstructed from the analysed current records. It was found that this relation shows a negative slope conductance at all potentials positive to -75 mV and that the current tends towards zero at zero membrane potential.6. The results are compared with those predicted by two reaction models of the i(K) (2) and i(x) (1) mechanisms. It is concluded that i(K) (2) and i(x) (1) are kinetically separable but that it is not possible with present techniques to decide whether they are controlled by the same or completely independent membrane structures. It is also shown that the instantaneous current-voltage relation calculated for i(K) (2) does not depend on whether the controlling mechanisms are assumed to be independent or linked.