Inhibition of potassium outward currents and pacemaker current in sheep cardiac Purkinje fibres by the verapamil derivative YS 035

Summary The electrophysiologic mode of action and potency of the verapamil derivative YS 035 (N,N-bis-(3,4-dimethoxyphenethyl)-N-methyl amine) were investigated in sheep cardiac Purkinje fibres. Action potential duration measured at a repolarization level of –60 mV (APD-60) and membrane currents recorded with the two-microelectrode voltage-clamp technique were evaluated. At 10 mol/l YS 035 APD-60 was increased to about 115% of reference. Prolongation measured as percentage of the respective control exhibited on the average no dependence on stimulation frequency (0.17–2 Hz). At 100 mol/l membrane became depolarized to about –50 mV and action potentials could no longer be elicited. Further study was focussed on effects on outward currents, mostly activated at a frequency of 0.05 Hz. Transient outward current (i_to) was completely blocked at 100 mol/l and half-maximal inhibition occurred at about 14 mol/l. Inwardly rectifying potassium current (i_K1) was reduced to 47% of reference at 100 mol/l. An initially activating outward current at positive membrane potentials (i_inst) was reduced to 73% at 100 mol/l. Time-dependent (delayed) outward current (i_K) was on the average not affected up to 100 ol/l. Besides inhibition of repolarizing outward currents YS 035 completely blocked pacemaker current (i_f) at 100 mol/l and half-maximal reduction was achieved at 5 mol/l. YS 035 (1–100 mol/l) did not clearly affect time constants of activation at selected test potentials (I_K: +35 mV; i_f: –90 mV) or inactivation (i_to: 0 mV). Voltage-dependent control mechanisms of currents (it_to, i_f) were not influenced by YS 035 but the amount of available current was reduced.In conclusion, the verapamil derivative YS 035 inhibited pacemaker current and potassium outward currents which correlated to a prolongation of cardiac action po tentials. Electrophysiological actions of the compound favour it to be tested in vivo as an antiarrhythmic drug candidate. Send offprint requests to U. Borchard at the above address     

Effects of the bradycardic agent ZD 7288 on membrane voltage and pacemaker current in sheep cardiac Purkinje fibres

The bradycardic mechanism of ZD 7288 (4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride) was investigated in sheep cardiac Purkinje fibres. The pacemaker i_f-current measured with the two-microelectrode voltage-clamp technique, as well as the diastolic depolarization rate and the frequency of spontaneously active fibres were evaluated.ZD 7288 did inhibit i_f-current. The i_f-amplitude recorded with a 0.8s-lasting test pulse from about –50 mV to –100 mV was reduced to 50% of control at 0.85 mol/l and to 5% of control at 10 mol/l. The threshold potential of i_f-activation was unaffected at a concentration of 1 mol/l ZD 7288. The time constant of i_f-activation at different test potentials was not changed by 1 mol/l ZD 7288. The drug was equally effective during i_f-activation with a 0.5 s-lasting test pulse applied at 0.05 Hz or 0.5 Hz. During long lasting (5 s) hyperpolarizing test pulses (–120 mV) the inhibition of i_f-current was removed.In constantly stimulated Purkinje fibres (0.5 Hz) the slope of the early diastolic depolarization was decreased by ZD 7288. The half-maximal effect occurred at 0.92 mol/l. There was strong correlation over the concentration range of 0.01 to 10 mol/l ZD 7288 between the decrease of the slope of early diastolic depolarization and inhibition of i_f-amplitude recorded with 0.8s-lasting test pulses to –100 mV. The correlation coefficient was r = 0.97.These results will explain the decrease in frequency of spontaneously active (about 0.6 Hz) Purkinje fibres. At 0.3 mol/l ZD 7288 spontaneous activity had stopped in 8 of 11 preparations. Complete recovery of drug-induced effects on the frequency was gained after 3 h of wash-out with drug-free solution. Correspondence to: U. Borchard at the above address     

Inhibition of pacemaker current by the bradycardic agent ZD 7288 is lost use-dependently in sheep cardiac Purkinje fibres

The inhibition of the pacemaker current (i _f) in sheep cardiac Purkinje fibres by ZD 7288 [4-(N-ethyl-N-phenylamino)-1,2-dimethyl-6-(methylamino)pyrimidinium chloride] is lost use-dependently. This disinhibition of i _f was investigated by using the two-microelectrode voltage-clamp technique. The pulse protocol consisted of a rest period (holding potential of about -50 mV, 1–10 mol/l ZD 7288) followed by a train of test pulses (potential negative to -100 mV, stimulation frequency 0.05 Hz). At the beginning of the first test pulse there was an immediate reduction of i _f but inhibition was lost during continued stimulation. Activation of i _f is sigmoidal and the early delay in current activation was prolonged from 33 ms (no ZD 7288) to 424 ms (10 mol/l ZD 7288). Therefore hardly any disinhibition occurred during short test pulses (0.5 s). During longer test pulses (5 s, -120 mV, 10 mol/l) disinhibition developed with a time constant of about 2 s. The inhibition of i _f by ZD 7288 was lost voltage-dependently. With 10 mol/l ZD 7288 the half-maximal disinhibition occurred at -92 mV and the slope factor of the disinhibition/voltage curve (Boltzmann relation) was 4.8 mV. The voltage-dependent disinhibition could be abolished largely by extracellular application of protease (0.5 mg/ml, 7 min). After prior disinhibition, reinhibition at the holding potential (about -50 mV) followed a bi-exponential time course indicating that inhibition may be produced by a fast (=0.7 min) and a slow component (=20–30 min). Increasing ZD 7288 concentration from 1 to 10 mol/l accelerated reinhibition, mainly by an increase of the amplitude (A) of the fast component. The ratio A _fast/A _sIow was 0.399 at 1 mol/l and 2.65 at 10 mol/1 ZD 7288. The reinhibition of i _f was unchanged by shifting the holding potential from -50 mV to -20 mV Trials to wash out the effects of 10 mol/l ZD 7288 gave two results. The inhibition of i _f was slightly reversed after a wash-out of 1.5 h with drug-free solution. A second effect of the drug, the fast reinhibition, could be completely removed by washout. In summary i _f is inhibited by ZD 7288 at membrane potentials at which the virtual i _f gate is closed. Disinhibition occurs during long-lasting hyperpolarization but will hardly be operative in unclamped fibres under physiological conditions.     

Renal failure in haemorrhagic shock in dogs: Salutary effects of the calcium entry blocker felodipine

Summary The efficacy of felodipine, a dihydropyridine calcium entry blocker to restore renal function was investigated in Wiggers model of haemorrhagic shock. Mongrel dogs were anaesthetized with sodium pentobarbital and subjected to haemorrhagic shock by allowing the animals to bleed into a reservoir. After maintaining the hypotensive state (mean blood pressure 40–45 mm Hg) for a period of 150 min, the blood was reinfused and the recovery of the various parameters were monitored for an additional 120 min. These studies were conducted in three different groups of dogs: (A) Solvent control, (B) Felodipine 0.01 mol/kg i. v., administered 10 min prior to reinfusion of the blood, and (C) Felodipine 0.01 mol/kg i. v., administered prior to haemorrhage. In all the three groups arterial blood pressure returned to similar basal levels following reinfusion. Felodipine administration prior to haemorrhage or before reinfusion (Group B and C) resulted in a 80–95% recovery in the renal blood flow, 60–65% in the glomerular filtration rate, 15–300% in the urine volume and 80–100% in the urinary sodium and potassium excretions. In the vehicle-treated control group, despite a 45% recovery in the renal blood flow, renal function was not restored following reinfusion.The observations made in these studies suggest that felodipine, an arteriolar dilator which also possesses natriuretic properties, could be clinically useful in the treatment of renal failure in haemorrhagic shock. Prevention of cellular calcium overload during ischaemia and reperfusion by this dihydropyridine derivative, may account for its ability to preserve vascular as well as tubular function. Send offprint requests to B. S. Jandhyala at the above address     

The relation between the current underlying pacemaker activity and beta-adrenoceptors in cardiac Purkinje fibres: A study using adrenaline,procaine, atenolol and penbutolol

Summary The pacemaker current — i _K2 — in cardiac Purkinje fibres was analysed using the voltage clamp technique described by Deck et al. (1964). (–)-Adrenaline (5.5 · 10^–6 M) causes the wellknown shift of the Hodkin-Huxley kinetics in the depolarizing direction. Procaine (7.3·10^–4 M) does not cause any further shift of s in the presence of adrenaline. Atenolol (3.8·10^–5 M) causes a backshift of the kinetics in the negative direction in the presence of adrenaline and procaine. The instantaneous current-voltage relationship ( ) is altered neither with adrenaline, nor with procaine or atenolol. The results exclude the possibility that the local anaesthetic side effect of many beta-adrenoceptor blocking agents may be involved in the backshift of the s-kinetics. The voltage dependence of the reciprocals of the time constants is shifted in a similar way as s by the sympathomimetic or blocking drugs. Following the application of (–)-adrenaline (5.5·10^–6 M) the (–)-isomere of penbutolol (1.7 and 3.5·10^–6 M) is about equally effective in shifting the kinetics back as the (+)-isomere (3.5·10^–5 M). In the presence of (–)-adrenaline, the (+)- and (–)-forms of penubutolol cause virtually no change of the instantaneous current-voltage relationship, . Thus, (–)-adrenaline and (+)- and (–)-penbutolol are aiming for the s-kinetics whose voltage dependence is controlled by the electric field near the i _K2-channel of the membrane and do not influence the number of the i _K2-channels. These findings suggest that the sympathomimetic or blocking agents influence the s-kinetics of the pacemaker current i _K2 by altering the electric field; the fully activated current-voltage relationship which is proportional to the number of the open i _K2-channels is not subject to any appreciable modification. The results conclusively show that the kinetics of the pacemaker current can be controlled by beta-adrenoceptors.     

Effects of cinnarizine on calcium and pressure-dependent potassium currents in guinea pig vestibular hair cells

In vestibular hair cells, K⁺ currents induced by rises in hydrostatic pressure have recently been demonstrated. These currents are inhibited by charybdotoxin, a blocker of Ca²⁺-dependent K⁺ channels. On the other hand, cinnarizine is a blocker of voltage-gated Ca²⁺ currents in hair cells and is used as a drug in conditions with vestibular vertigo. Our aim was to test in patch-clamp experiments (conventional whole-cell mode) whether cinnarizine, by reducing Ca²⁺ influx, inhibited Ca²⁺ and pressure-sensitive K⁺ currents in vestibular type-II hair cells of guinea pigs. A quantitatively similar inhibition of K⁺ currents was evoked by extracellular Ca²⁺ removal, cinnarizine (0.5 M), and the L-type Ca²⁺ channel blocker nifedipine (3 M). Cinnarizine abrogated increases of K⁺ currents induced by increases in the hydrostatic pressure (from 0.2 to 0.5 cm H₂O). At a higher concentration (1 M), cinnarizine elicited K⁺ current inhibitions larger than those elicited by Ca²⁺ removal. Moreover, it reduced K⁺ currents in the absence of Ca²⁺, in contrast to nifedipine. However, charybdotoxin abolished these effects of cinnarizine. We thus conclude that cinnarizine inhibits, by two mechanisms, pressure-induced currents that are sensitive to charybdotoxin and Ca²⁺. It reduces Ca²⁺ influx and exerts a Ca²⁺-independent inhibition, with a lower IC₅₀ than that required for Ca²⁺ channel blockade. These two actions may importantly contribute to its therapeutic effects.P. Düwel and T. Haasler contributed equally to this work.     

The action of 22,23-dihydrobufalin and other cardioactive steroids on contraction and active sodium/potassium transport of sheep cardiac Purkinje fibres

Summary The recent synthesis of bufenolides permits the introduction into the lactone moiety of a latently reactive group suitable to localize exactly the binding site of the lactone ring of cardioactive steroids on the sodium/potassium pump. For this purpose it is essential to demonstrate that the bufenolides are cardioactive. In the present paper the effect of the bufenolide 22,23-dihydrobufalin on contraction and active sodium/potassium transport is studied by means of electrophysiological methods in sheep cardiac Purkinje fibres. The results are compared to the corresponding actions of some other cardioactive steroids.22,23-dihydrobufalin exerts a reversible positive inotropic effect. Simultaneous measurements of intracellular sodium activity and membrane current in voltage clamped fibres reveal an inhibition of the sodium/potassium pump by 22,23-dihydrobufalin in the concentration range tested (10^–7 to 5·10^–5 mol/1). An apparent equilibrium dissociation constant K_D of about 10^–6 mol/l is derived for the drug — sodium/potassium pump interaction. The KD value for bufalin is smaller, whereas the value for ouabain is comparable. The K_D value estimated for dihydroouabain is slightly larger.It is concluded that 22,23-dihydrobufalin shares important characteristics with cardioactive steroids. Thus, bufenolides are probably useful tools for an exact localization of the lactone binding site on the cardiac sodium/potassium pump.This work was supported by the Deutsche Forschungsgemeinschaft (Forschergruppe Konzell)Send offprint requests to H. G. Glitsch at the above address     

The effect of calcium load and the calcium channel blocker verapamil on gentamicin nephrotoxicity in rats

Gentamicin (GM) is used against serious and life-threatening Gram negative infections. However its use is limited by the occurrence of nephrotoxicity. Reports on the interaction between GM nephrotoxicity and calcium (Ca²⁺) or Ca blockers are conflicting. Therefore, in the present work we assessed the effect of treatment of rats with graded doses of calcium carbonate, CaCO₃ (0.25, 0.5 or 1.0 g/kg) orally, or the Ca²⁺ channel blocker verapamil (1.75, 3.5 or 7.0 mg/ kg) intramuscularly (i.m.), on the nephrotoxicity induced by concomitant i.m. treatment with GM (80 mg /kg/day for 6 days). Nephrotoxicity was evaluated histopathologically by light microscopy and biochemically by measuring the concentrations of urea and creatinine in plasma, reduced glutathione (GSH), lipid peroxidation and superoxide dismutase (SOD) activity in kidney cortex. The results indicated that the administration of CaCO₃ produced a dose-dependent amelioration in the biochemical indices of nephrotoxicity in plasma and renal cortex, which was significant at the two higher doses used. The histological picture of the renal proximal tubules followed a similar pattern. Treatment with verapamil induced a dose-dependent potentiation in the biochemical parameters of nephrotoxicity that was significant only at the highest dose used (7 mg/kg). This dose also exacerbated the GM-induced histological necrosis. The above interactions may be clinically relevant in patients treated concurrently with these agents.     

Flurazepam: Effects on sodium and potassium currents in myelinated nerve fibres

The effect of flurazepam-HCl on single myelinated nerve fibres of the frog Rana esculenta was investigated. Flurazepam affected both Na and K currents: 0.25 mM of the drug decreased the peak Na inward current to about 50%. The initial increase and subsequent decay of the Na current was slowed down by a factor of 1.5 independent of membrane potential. The drug induced a slow phase in the recovery from Na inactivation and frequency dependence of the Na current block. The K current rose at a normal rate and was then inactivated to a sustained outward current. The time constant of block development (π_k) and the steady state block were potential-dependent. With 1 mM flurazepam, π_k decreased from 2.9 ms at E = 10 mV to 1.5 ms at E =90 mV, and the steady state block increased from 65% at E = - 20 mV to 81% at E = 90 mV. Recovery from the block proceeded faster at E =- 70 mV (π = 27 ms) than at E = - 120 mV (π = 89 ms). The effects of the drug on the K current were interpreted in terms of the reaction scheme proposed by Armstrong (J. Gen. Physiol. 54, 553; 1969).     

Induction of the oscillatory current by low concentrations of caffeine in sheep cardiac Purkinje fibres

Summary The actions of low concentrations of caffeine (0.5–2 mmol/1) on the transient inward oscillatory current (I_os) and the inward tail current (I_ex) were studied in sheep cardiac Purkinje fibres by means of a two microelectrode voltage clamp method. The following results were obtained. Caffeine: 1. induced an I_os when this current was not already present; 2. increased the amplitude (within limits) and consistently decreased the time to peak of an already present I_os; 3. increased I_ex upon which I_os may be superimposed; 4. shifted the depolarizing threshold for the appearance of I_os to more negative and the repolarizing threshold to less negative values; 5. increased the effects of strophanthidin of I_os and I_ex; 6. exaggerated the effects of high [Ca]_o which by itself mimicked some of the actions of caffeine; 7. had a small effect of I_os and I_ex in low [Ca]_o; 8. reversed the effect of norepinephrine on I_ex; 9. enhanced the effects of trains of clamps and of longer clamp steps on I_os and I_ex. It is concluded that low concentrations of caffeine facilitate the manifestations of calcium overload thereby inducing or exaggerating the oscillatory and tail currents and that these effects are modulated by the cellular calcium load but are not mediated through adrenergic mechanisms. Send offprint requests to M. Vassalle at the above address     

Use- and frequency-dependent blockade by UL-FS 49 of the if pacemaker current in sheep cardiac Purkinje fibres

The mechanism by which the bradycardiac agent UL-FS 49 blocks the if pacemaker current was investigated in sheep Purkinje fibres using the two microelectrode voltage-clamp technique. If was activated by 1 s pulses applied between -30 mV and -120 mV at 0.4 Hz in a modified Tyrode solution containing BaCl2 and MnCl2, and with TRIS replacing most of the Na+. UL-FS 49 caused an exponential decline of the if current amplitude during a train of pulses. Both the rate and extent of the if reduction increased with drug concentration, without there being a resting blockade. Recovery from blockade followed a single exponential time course during prolonged hyperpolarizations. The recovery rate was extremely slow and increased with more negative voltages, as did the extent of steady state recovery from blockade. A frequency-dependent reduction of the diastolic depolarization rate resulted from a use-dependent blockade of the pacemaker current.     

Effects of 17β-estradiol on action potentials and ionic currents in male rat ventricular myocytes

This study describes electrophysiological effects of estrogens in isolated male rat ventricular myocytes. According to the literature these cells do not express the nuclear estrogen receptor. Action potentials or membrane currents were recorded in the whole-cell configuration with standard techniques. Action potential durations (APD) measured at a level of 0 mV (APD 0) and –70 mV (APD –70) were prolonged by 17β-estradiol (0.5 Hz stimulation frequency, 24–26° C). Threshold concentration was 1 μmol/l. At the highest concentration used (30 μmol/l) no saturation of the response was reached and APD 0 was 162% and APD –70 was 230% of the respective control. The resting potential remained unaffected in most cells. The prolongation induced by 17β-estradiol developed rapidly and reached a steady state 10 min after start of hormone superfusion. Effects of estrogen were completely reversible during 10–15 min wash-out with hormone-free solution. The extent of prolongation (10 μmol/l 17β-estradiol) was frequency dependent. Expressed as percentage of the respective control APD 0 (or APD –70) was 115% (188%) at 0.05 Hz, 118% (163%) at 0.5 Hz and 99% (129%) at 5 Hz stimulation frequency. The response was stereoselective, because 30 μmol/l 17α-estradiol did not prolong action potentials (APD 0: 101%, APD –70: 104% of the respective control, 0.5 Hz stimulation frequency). The endogenous estrogens estrone and estriol were less effective than 17β-estradiol. With 30 μmol/l estrone (0.5 Hz stimulation frequency) APD 0 was 103% and ADP-70 148% of control and with 30 μmol/l estriol APD 0 was 135% and APD –70 137% of control. The prolongation of action potentials can be explained by inhibition of transient outward current which, in rat ventricle, is composed of fast (i _to,f) and slowly (i _to,s) inactivating components. At 30 μmol/l 17β-estradiol i _to,f was reduced to 50% and i _to,s to 43% of their maximal amplitudes. The voltage sensor of i _to,f or i _to,s was hardly affected. Additionally, 17β-estradiol decreased the calcium current (i _Ca,L) to 76% (10 μmol/l) and 38% at 30 μmol/l. The inwardly rectifying potassium current (i _K1) was reduced partly with 30 μmol/l 17β-estradiol and its amplitude was 72% of control at –90 mV (inward current flow) and 65% at –40 mV (outward current flow). These results show that 17β-estradiol is active in cardiac cells which do not express the nuclear estrogen receptor. The hormone exerts class III activity and reduces calcium inward current. These effects, however, occur in vitro with concentrations above the physiological level and therefore may be without significance in vivo. Received: 6 May 1997 / Accepted: 18 October 1997     

Effects of flunarizine on induced calcium transients as measured in fura-2-loaded neurons of the rat dorsal root ganglion

Summary The effect of the calcium entry blocker flunarizine on a high-potassium induced increase of intracellular free calcium was studied. The experiments were done with neurons isolated from rat dorsal root ganglia and loaded with the calcium-sensitive dye fura-2. The increase of calcium induced by 60 mmol/1 potassium was abolished after removal of extracellular calcium, was reversibly reduced by 50 mol/l cadmium (76% inhibition), 50 mol/1 nickel (25% inhibition) and 10 mol/1 nifedipine (18°10 inhibition), and reversibly increased after removal of extracellular sodium (26% increase). The potassium induced increase of intracellular calcium is, therefore, mediated by transmembrane calcium influx, probably to a large extent through cadmium-sensitive calcium channels. Flunarizine (5 min incubation followed 1 min wash-out) reduced the amplitude of the high-potassium induced calcium increase in a dose-dependent manner (K _d = 370 ± 100 nmol/l; mean ± SEM; n = 8), causing complete inhibition at a concentration of 10 mol/1 in the majority of cells. Flunarizine ( 1 mol/1) caused a reversible increase of the resting level of intracellular calcium in some cells, an effect which disappeared in the absence of extracellular calcium. The drug (1 mol/1 had no influence on the time course of recovery of intracellular calcium subsequent to a rise induced by high-potassium or by the calcium ionophore A23187. It is concluded that flunarizine acts as an inhibitor of depolarization-mediated calcium influx. At a concentration of 1 mol/1, the drug presumably has no effect on cellular calcium extrusion and/or sequestration mechanisms. Correspondence to L. Leybaert at the above address     

Effects of bepridil and lidocaine on the intravenctricular conduction in acutely ischaemic and infarcted canine myocardium

Summary Effects of bepridil, an antiarrhythmic and antianginal drug, on intraventricular conduction in acutely ischaemic and infarcted myocardium were examined in anaesthetized dogs, and compared with those of lidocaine. Bepridil at doses of 2 and 5 mg/kg markedly prolonged the conduction time of a premature excitation induced by a ventricular stimulation in the infarcted zone. The effect of bepridil was dependent on a coupling time of the stimulation. Bepridil showed a marked effect at a coupling time of 150 ms, while it showed no significant effect at a prolonged coupling time of 1 s. In other words, the effect of bepridil was interval-dependent. Lidocaine showed a similar interval-dependent effect, but the effect of lidocaine at a longer coupling time was less than that of bepridil. The premature stimulation produced severely delayed conduction which resulted in reentrant beats. Bepridil blocked these conductions, thereby preventing reentrant beats. In contrast to the depressant effect of bepridil in the infarcted myocardium, bepridil prevented the prolongation of conduction time during acute ischaemia. The alternation of the ST-T complex during acute ischaemia which is also an important arrhythmogenic factor was also attenuated by bepridil. Contrary to bepridil, lidocaine significantly enhanced the conduction delay and the alternation in the ST-T complex. In conclusion, bepridil as well as lidocaine showed an interval-dependent depression of the conduction in the infarcted zone of the heart, whereas during acute ischaemia bepridil in contrast to lidocaine attenuated the conduction delay and ST-T alternans. Send offprint requests to H. Hashimoto at the above address     

Efects of benzyltetrahydropalmatine ?

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The inhibitory effects of the novel calcium antagonist Goe 5438 on calcium-dependent processes of excitation and contraction of single cardiomyocytes

Summary The calcium-antagonistic properties of the novel compound Goe 5438 have been studied in single cardiomyocytes from embryonic (chicken) and adult (guinea-pig) ventricles, in part in comparison with the inhibitory effects of the 1,4-dihydropyridine calcium antagonist nimodipine. Both substances block spontaneous action potentials and contractions of embryonic heart cells at about 0.1 mol/l. In collagenase-dispersed ventricular cardiomyocytes of guinea-pigs, stereospecific inhibition of the slow calcium current (I _ca ) by Goe 5438 was observed at 10 mol/l by means of voltage-clamp experiments. The (+)-enantiomer of Goe 5438 elicited a stronger inhibition of the slow inward current than the (–)-enantiomer. The frequency dependence of the inhibitory effect of Goe 5438 as well as that of nimodipine could be shown to be negligible in measurements of I _Ca and contractions, whereas the inhibitory influence of verapamil, verified in the same experimental arrangement, exhibited a distinct frequency dependence. With respect to a possible potential dependence of the inhibitory effect of Goe 5438 and nimodipine, it could be shown that a hyperpolarization during the course of application of either calcium antagonist produced recovery of the calcium-dependent excitation neither in adult nor in embryonic cells. In adult cardiomyocytes, the dependence of I _Ca on the membrane potential was not altered by Goe 5438. It is concluded that the mode of action of Goe 5438 resembles that of 1,4-dihydropyridine calcium antagonists.Supported by the Austrian Science Research Fund, grant No. 4662A preliminary report of some findings has been presented at the 28th Spring Meeting of Deutsche Gesellschaft für Pharmakologie und Toxikologie, Mainz 1987 (Wagner and Koidl 1987) Send offprint requests to B. Koidl     

Inhibitory effects of NS-21, a novel drug for urinary incontinence, and its active metabolite, RCC-36, on L-type calcium currents in isolated guinea pig detrusor smooth muscle cells

The inhibitory effects of NS-21, a newly developed drug for the treatment of urinary frequency and urinary incontinence, and its active metabolite, RCC-36, on L-type Ca²⁺ currents (I_Ca) in guinea pig detrusor smooth muscle cells have been compared to those of terodiline by a whole-cell patch-clamp technique. Like terodiline (10 μM), both NS-21 (10 μM) and RCC-36 (10 μM) induced a sizeable decrease in I_Ca elicited from a holding potential of -60 mV without changing the current-voltage relationship. The three drugs shifted the inactivation curves for I_Ca in the hyperpolarizing direction by 13 to 20 mV but had no effect on the activation curves for I_Ca, resulting in a decrease in the calcium window current. The inhibitory effects of NS-21 and RCC-36 were greater than those of terodiline. The three drugs inhibited I_Ca in a concentration- and holding-potential-dependent manner. The IC₅₀ values at a holding potential of -60 mV were 7.9 μM for NS-21, 6.4 μM for RCC-36, and 5.9 μM for terodiline, and at -40 mV they were 1.3, 1.2, and 3.5 μM, respectively. The ratio calculated by dividing the IC₅₀ value at -60 mV by the value at -40 mV was 6.1, 5.3 and 1.7, respectively, indicating that the inhibitory effects of NS-21 and RCC-36 on I_Ca were more sensitive to voltage than those of terodiline. These results suggest that NS-21 and RCC-36 could be more effective in the treatment of urinary bladder ailments, such as urinary frequency and urinary incontinence. Received: 27 August 1996 / Accepted: 13 February 1997     

Compared effects of calcium entry blockers on calcium-induced tension in rat isolated cerebral and peripheral resistance vessels

Summary The effects of the calcium entry blockers verapamil (V), diltiazem (D), nifedipine (NF) and nicardipine (NC) have been studied on calcium concentration-effect curves elicited in depolarized (K⁺, 40 mmol/l) and in serotonin-exposed (6 mol/l) rat middle cerebral arteries (RMCA) in order to compare the relative potencies of the blockers against these two calcium channel activating mechanisms. In control conditions, Ca²⁺ sensitivity expressed as pD₂ and maximal active wall tension (AWT) were not significantly different in depolarized and in 5-HT-exposed vessels: pD₂: 3.39 ±0.08 vs 3.50 ± 0.06 and AWT: 0.93 ± 0.15 mN · mm^–1 vs 0.90 ± 0.16 mN · mm^–1 respectively. V, D, NF and NC displaced Ca²⁺ control curves to the right and depressed the maximum contractile response in the two experimental conditions, which suggests a noncompetitive type of antagonism. All the blockers were more potent inhibitors of Ca²⁺-induced contractions in depolarized than in serotonin-exposed middle cerebral arteries. The IC₅₀ values (concentration of blockers producing a 50% inhibition of maximal control contractile response) were (nmol/l) : V = 20, D = 120, NF = 0.4, NC = 1 and V = 400, D = 10000, NF = 20, NC = 7 in depolarized and serotonin-exposed arteries respectively. From these IC₅₀ values, the relative order of potency of the CEB's was not the same in the two experimental conditions suggesting that while serotonin and K⁺ both promote the entry of Ca²⁺ into vascular smooth muscle cells of RMCA, they either activate a different gating mechanism associated with a single common channel or perhaps distinct channels. Comparison of the results obtained in this study for depolarized rat middle cerebral arteries with those previously obtained in depolarized rat mesenteric resistance arteries (RMRA) revealed that while Ca²⁺-induced contractile responses were inhibited in a similar non-competitive manner by the four CEB's, the respective IC₅₀ values showed that potencies and rank of relative potency of the blockers were different in the two types of vessels. D and NC were equally potent in both preparations (IC₅₀ ratio = 2.5 and 3 respectively) but RMCA were more sensitive to V and NF than RMRA (IC₅₀ ratio = 6.5 and 11 respectively). These results are discussed and it is proposed that regional differencies in the conformation and/or the activation of the voltage-gated Ca²⁺ channels may exist in different vascular beds. Send offprint requests to J. L. Freston     

Effects of metoprolol on action potential and membrane currents in guinea-pig ventricular myocytes

Summary The effects of the beta-adrenoceptor antagonist metoprolol on action potentials and membrane currents were studied in single guinea-pig ventricular myocytes. The experiments were carried out using the nystatin-method of whole-cell technique. This method was used in order to prevent the run-down of the calcium current. Metoprolol at concentrations of 10–100 mol/l shortened action potential in a dose-dependent way. The drug only decreased resting membrane potential at a concentration of 100 mol/1 in two out of five cells. Under voltage-clamp conditions, metoprolol blocked the high threshold calcium current at concentrations of 30 and 100 mol/l to 82 ± 4% and 73 ± 5% from control, respectively. The drug decreased the inward rectifying potassium current in a concentration-dependent manner. This effect was evident for inward current at voltages negative to the apparent reversal potential and for outward current at voltages between –30 and –80 mV. This blocking effect on the inward rectifying potassium current can explain the effect on resting membrane potential. At voltages positive to –30 mV metoprolol increased a time-independent outward current. This metoprolol-enhanced outward current was blocked by barium and cesium. This result suggests that the metoprolol-enhanced current is carried by potassium. The current component enhanced by metoprolol was not sensitive to glibenclamide and tetraethylammonium applied externally, which suggests that the adenosine triphosphate-sensitive channel is not the target of metoprolol. The activation of this time-independent outward current by metoprolol and the blocking effects on the calcium current seem to explain the shortening in action potential induced by the drug. Send offprint requests to J. Sánchez-Chapula at the above address     

Effects of calcium entry blocking agents on 5-hydroxytryptamine- and noradrenaline-induced contractions of rat isolated jugular vein and aorta

Summary We calculated the contribution of the intracellular releasable calcium pool to the contractile responses induced by 5-hydroxytryptamine (5-HT) and noradrenaline (NA) by constructing time-response curves to the agonists in Ca²⁺-deficient medium in the isolated rat jugular vein and aorta. Biexponential curves were obtained compatible with a two compartment model. In the aorta the intracellular calcium pools are likely to be different for both 5-HT and NA. Moreover, we investigated the effect of maximally effective concentrations of calcium entry blocking agents (CEB's) on K⁺, 5-HT- and NA-induced contractions in Ca²⁺-containing medium. Only a moderate inhibiting effect of nifedipine, diltiazem, flunarizine and gallopamil on 5-HT and NA-induced Ca²⁺ influx could be observed; in contrast, K⁺-induced Ca ²⁺ influx could be antagonized completely. The calculated contribution of intracellular Ca ²⁺ to 5-HT-and NA-induced contractions, obtained from the experiments in Ca²⁺-free medium was much lower than that obtained after pretreatment with CEB's, leading to the conclusion that after CEB-pretreatment a Ca²⁺ influx component persists. This hypothesis was supported by the observation that contractions in Ca²⁺-free medium consist of a monophasic, fast response only, whereas after CEB-pretreatment a response similar to the control, including a slow, sustained component, was obtained. The Ca²⁺ influx component not affected by maximally effective concentrations of CEB's seems to represent an inflow of extracellular Ca²⁺ directly into the cytosol and not into an intracellular calcium store. Send offprint requests to M. A. M. Gouw at the above address