A review of HNS-32: a novel azulene-1-carboxamidine derivative with multiple cardiovascular protective actions

HNS-32 [N(1),N(1)-dimethyl-N(2)-(2-pyridylmethyl)-5-isopropyl-3,8-dimethylazulene-1- carboxamidine] (CAS Registry Number: 186086-10-2) is a newly synthesized azulene derivative. Computer simulation showed that its three dimensional structure is similar to that of the class Ib antiarrhythmic drugs, e.g., lidocaine or mexiletine. HNS-32 potently suppressed ventricular arrhythmias induced by ischemia due to coronary ligation and/or ischemia-reperfusion in dogs and rats. In the isolated dog and guinea pig cardiac tissues, HNS-32 had negative inotropic and chronotropic actions, prolonged atrial-His and His-ventricular conduction time and increased coronary blood flow. In the isolated guinea pig ventricular papillary muscle, HNS-32 decreased maximal rate of action potential upstroke (Vmax) and shortened action potential duration (APD). These findings suggest that HNS-32 inhibits inward Na+ and Ca2+ channel currents. In the isolated pig coronary and rabbit conduit arteries, HNS-32 inhibited both Ca2+ channel-dependent and -independent contractions induced by a wide variety of chemical stimuli. HNS-32 is a potent inhibitor of protein kinase C (PKC)-mediated constriction of cerebral arteries. It is likely to block both, Na+ and Ca2+ channels expressed in cardiac and vascular smooth muscles. These multiple ion channel blocking effects are largely responsible for the antiarrhythmic and vasorelaxant actions of HNS-32. This drug may represent a novel approach to the treatment of arrhythmias.     

Electrophysiological effects of a new antiarrhythmic substance Bonnecor (AWD 19-166, GS 015) on mammalian myocardium

A new antiarrhythmic acting drug (Bonnecor, AWD 19-166, GS 015, 3- Carbethoxyamino-5-dimethyl-amino-acetyl-iminodibenzyl -hydrochloride) was tested electrophysiologically on isolated sinus node preparations and right ventricular papillary muscle of the rabbit. Transmembrane potentials were measured using a standard microelectrode technique. The substance was tested within a concentration range between 0.1 and 10 mg/l (2.5 X 10(-7) to 2.5 X 10(-5) mol/l). AWD 19-166 exerted negative chronotropic effects due to decreasing of the slope of the diastolic depolarization in sinus node cells. In ventricular myocardium AWD 19-166 increased the threshold strength of stimulation, prolonged the duration of action potentials at 90% but shortened it at 25% repolarization. The maximum upstroke velocity was found to be depressed. The maximum overshoot potential was diminished inspite of a drug-induced hyperpolarization of the resting transmembrane potential. Analysis of membrane responsiveness due to premature stimulation showed a concentration-dependent delay in the restitution of the maximum upstroke velocity of the premature action potentials and both a flattening and a shift towards more negative potentials of the potential-responsiveness relationship. AWD 19-166 decreased both the maximum upstroke velocity and the duration of Ca-mediated action potentials in concentrations between 2 and 8 mg/l. It is concluded that AWD 19-166 is able to exert potent antiarrhythmic effects in influencing both the fast and slow channel activity.     

Inhibition of T-type and L-type Ca(2+) currents by aranidipine, a novel dihydropyridine Ca(2+) antagonist

The effects of aranidipine, a novel dihydropyridine Ca(2+) channel antagonist, on membrane currents in guinea pig ventricular myocytes and on action potentials in rabbit sinoatrial node tissue were examined. In myocytes, aranidipine (10 nmol/l to 1 micromol/l) concentration-dependently decreased T-type and L-type Ca(2+) currents. Aranidipine (1 micromol/l) had little effect on K(+) currents. In the sinoatrial node, 0.1 micromol/l aranidipine increased cycle length, and decreased +V(max) and the slope of the phase 4 depolarization. Thus, inhibition of both T-type and L-type Ca(2+) currents by aranidipine may partly explain its potent negative chronotropic activity.     

Cardiotoxic effects of fenfluramine hydrochloride on isolated cardiac preparations and ventricular myocytes of guinea-pigs

The cardiotoxic effects of fenfluramine hydrochloride on mechanical and electrical activity were studied in papillary muscles, Purkinje fibres, left atria and ventricular myocytes of guinea-pigs. Force of contraction (f(c)) was measured isometrically, action potentials and maximum rate of rise of the action potential (V(max)) were recorded by means of the intracellular microelectrode technique and the sodium current (I(Na)) with patch-clamp technique in the cell-attached mode. For kinetic analysis (S)-DPI-201-106-modified Na(+) channels from isolated guinea-pig ventricular heart cells were used. Fenfluramine (1 - 300 microM) produced negative chronotropic and inotropic effects; additional extracellular Ca(2+) competitively antagonized the negative inotropic effect. Fenfluramine concentration-dependently reduced V(max) and showed tonic blockade of sodium channels, shortened the action potential duration in papillary muscles and Purkinje fibres. In cell-attached patches, fenfluramine decreased I(Na) concentration-dependently (10 - 100 microM), frequency-independently (0.1 - 3 Hz; 30 microM). The h(infinity) curve was shifted towards hyperpolarizing direction. At 30 microM, fenfluramine blocked the sodium channel at all test potentials to the same degree, and neither changed the threshold and reversal potentials nor the peak of the curve. No effect on single channel availability, but a significant decrease in mean open times and increase in mean closed times was observed. Mean duration of the bursts decreased and number of openings per record increased with increasing drug concentration. It is concluded that the effect on I(Na) plays an important role in the cardiotoxicity of fenfluramine in addition to primary pulmonary hypertension and valvular disorders.     

Antiarrhythmic and cardiohemodynamic effects of a novel Ca(2+) channel blocker, AH-1058, assessed in canine arrhythmia models

The antiarrhythmic profile and cardiohemodynamic effect of a novel Ca(2+) channel blocker, 4-(5H-Dibenzo[a, d]cyclohepten-5-ylidene)-1-[(E)-3-(3-methoxy-2-nitro)phenyl-2-p ropeny l]piperidine hydrochloride (AH-1058), were analyzed using the epinephrine-, digitalis- and two-stage coronary ligation-induced canine ventricular arrhythmia models. Intravenous administration of AH-1058 (100 microg/kg) effectively suppressed each of the ventricular arrhythmias accompanied by weak hypotensive effects. The results contrast well with those of a typical Ca(2+) channel blocker, verapamil, which suppresses only the epinephrine-induced ventricular arrhythmia with severe hypotension. These results indicate that AH-1058 may possess a more selective inhibitory action on Ca(2+) channels in the heart than on those in the vessels. Furthermore, the antiarrhythmic actions of AH-1058 were slower in onset and longer-lasting, than those in our previous studies using other antiarrhythmic drugs, including Na(+) and Ca(2+) channel blockers. The antiarrhythmic effects of AH-1058 did not correlate with its plasma concentrations when administered either intravenously or orally. These results suggest that AH-1058 can become a long-acting Ca(2+) channel blocker with unique antiarrhythmic properties, and that AH-1058 may be used in certain pathological processes, for which selective inhibition of the cardiac Ca(2+) channels is essential.     

Electrophysiological effects of bunaftine, an antiarrhythmic drug, on action potential characteristics in ventricular muscle preparations

The electrophysiological effects of bunaftine were studied using the glass microelectrode technique. Bunaftine at 1, 5 and 10 mg/l produced a concentration-dependent depression of the maximum rate of rise of the action potential in guinea pig papillary muscle preparations without affecting the resting membrane potential and the action potential amplitude. The action potential duration (APD50, APD90) was significantly prolonged by the treatment with 1 and 5 mg/l bunaftine, while it was not changed by the treatment with 10 mg/l. The absolute refractory period (ARP) was also prolonged dose-dependently by the treatment of bunaftine; It was excessively prolonged (177% of control) at the concentration of 10 mg/l. Disopyramide produced similar electrophysiological changes to those of bunaftine except for the prolongation of ARP. ARP/APD90 ratio was significantly increased by bunaftine, but not by disopyramide. These electrophysiological effects of bunaftine and disopyramide observed in guinea pig ventricular preparations were similarly found in canine ventricular muscle preparations. These results indicate that the electrophysiological characteristics of bunaftine are similar to those of disopyramide in that the most prominent effect was the prolongation of ARP.     

HNS-32, a novel azulene-1-carboxamidine derivative, inhibits nifedipine-sensitive and -insensitive contraction of the isolated rabbit aorta

The vasorelaxant profile of a novel azulene-1-carboxamidine derivative, HNS-32 [N1,N1-dimethyl-N2-(2-pyridylmethyl)-5-isopropyl-3,8-dimethyl-azulene-1-carboxamidine, CAS 186086-10-2], was investigated in the isolated rabbit aorta precontracted with high KCl, noradrenaline (NA) or phorbol 12, 13-dibutyrate (PDBu) and compared with those of nifedipine and nitroglycerin. In preparations without endothelium, HNS-32 elicited concentration-dependent, full inhibition of contractions elicited by high KCI (80 mM), NA (3x10(-6) M) or PDBu (10(-6) M). In contrast, nifedipine inhibited only the contraction elicited by membrane depolarization with high KCl. Nitroglycerin also attenuated high-KCl-, NA- and PDBu-elicited contractions effectively, although full suppression was obtained only for NA-elicited contraction. Whilst the relaxant effect of HNS-32 was not affected by the presence of endothelium, the relaxant response to acetylcholine was endothelium dependent. Addition of excess Ca2+ restored both the HNS-32-reduced tension in muscle precontracted with high KCI and the nifedipine-mediated tension decrease. Relaxation elicited by HNS-32 was not affected by the adenylate cyclase inhibitor, 9-(tetrahydro-2'-furyl)adenine (SQ 22,536, 10(-4) M), the soluble guanylate cyclase inhibitor, 1H-(1,2,4)-oxadiazolo-(4,3-a)-quinoxalin-1-one (ODQ, 10(-5) M) or a cocktail of K+ channel blockers (glybenclamide 10(-6) M, tetraethylammonium 2x10(-3) M, apamin 10(-7) M, 4-aminopyridine 10(-4) M and Ba2+ 10(-5) M). These findings indicate that HNS-32 inhibits both L-type Ca2+ channel-dependent and -independent vascular contraction. Blockade of Ca2+ entry through L-type Ca2+ channels may be involved in the inhibitory effect of HNS-32 on the contraction due to membrane depolarization with high KCl. On the other hand, HNS-32 seems to inhibit Ca2+ channel-independent contraction via mechanism(s) other than elevation of cyclic nucleotides (cAMP and cGMP) and opening of K+ channels.     

Cardiovascular effects of orally administered HNS-32, an originally synthesized azulene-1-carboxamidine derivative,assessed in the in vivo rat model

HNS-32, an azulene-1-carboxamidine derivative, is an originally synthesized antiarrhythmic compound. Its cardiovascular effects after oral administration (1-10 mg/kg) were assessed using the pentobarbital-anesthetized in vivo rat model in comparison with those of verapamil (3 mg/kg, p.o.). Verapamil decreased the heart rate and mean blood pressure and prolonged the PR interval without changing the QRS width (n = 6). Similar results were observed for HNS-32 except that the QRS width was prolonged by the highest dose and the effects occurred slowly and lasted longer. These results suggest that HNS-32 is an orally active slowly-acting calcium plus sodium channel blocker.     

Comparison of cardiovascular effects of pirmenol with those of disopyramide in isolated canine heart preparations cross-circulated with a donor dog

To assess the cardiovascular profiles of pirmenol, a new antiarrhythmic drug, and to compare them with those of disopyramide, isolated canine sinoatrial node, papillary muscle and atrioventricular node preparations cross-circulated with a donor dog were used. Pirmenol injected intraarterially into the isolated preparations showed negative chronotropic and inotropic effects, which were comparable to those of disopyramide; and it also showed coronary vasodilator and negative dromotropic effects on atrio-His as well as His-ventricular conduction, which were significantly more potent than those of disopyramide. Similarly, pirmenol administered intravenously into the donor dog showed more potent negative dromotropic effects on the PQ interval and QRS width than disopyramide, while in the isolated preparations cross-circulated by the donor dog, pirmenol and disopyramide showed equipotent cardiodepressant effects. In the same preparation, pirmenol decreased coronary blood flow following a transient increase, while disopyramide only decreased coronary blood flow. Since the antiarrhythmic action of class I drugs is considered to result from inhibition of the fast inward current, which generates and propagates action potentials and also induces ventricular automaticity, our results suggest that pirmenol possesses an electrophysiologic effect typical to an efficacious class I agent such as disopyramide.     

Effects of AH-1058, a new antiarrhythmic drug, on experimental arrhythmias and cardiac membrane currents

AH-1058 is a newly synthesized antiarrhythmic agent. We investigated the antiarrhythmic and electrophysiological effects of AH-1058 in experimental arrhythmia models and isolated cardiomyocytes. In the ouabain-induced arrhythmia model of the guinea pig, pretreatment with AH-1058 (0.1-0.3 mg/kg, i.v.) delayed the appearance of premature ventricular complex (PVC) and ventricular fibrillation (VF) induced by intravenous infusion of ouabain. However, disopyramide (10 mg/kg, i.v.) delayed only that of PVC, and verapamil (1 mg/kg, i.v.) failed to affect the ouabain-induced ventricular arrhythmias. In the reperfusion-induced arrhythmia model of the rat, in which 5-min coronary occlusion and 10-min reperfusion were produced, AH-1058 (0.1-0.3 mg/kg, i.v.) inhibited the incidence of both ventricular tachycardia (VT) and VF, whereas disopyramide (5 mg/kg, i.v.) inhibited only reperfusion-induced VF. On the other hand, a higher dose of AH-1058 (1 mg/kg, i.v.) did not affect the aconitine-induced arrhythmias in rats, which were inhibited by disopyramide (5 mg/kg, i.v.). We also confirmed oral activity of AH-1058 in the reperfusion-induced arrhythmia model of the rat. AH-1058, at doses of 2-4 mg/kg, dose-dependently inhibited VT and VF. Electrophysiological experiments with patch-clamp techniques revealed that AH-1058 potently suppressed the L-type Ca2+ currents in isolated cardiomyocytes of the guinea pig. These results suggest that AH-1058 is a potent antiarrhythmic drug having a Ca2+ channel-blocking action. The antiarrhythmic profile of AH-1058 is different from that of disopyramide and verapamil.     

Chronotropic, inotropic, dromotropic and coronary vasodilator effects of bisaramil, a new class I antiarrhythmic drug, assessed using canine isolated, blood-perfused heart preparations.

The cardiovascular effects of a new class I antiarrhythmic drug, bisaramil, were examined using canine isolated, blood-perfused heart preparations. Bisaramil exerted negative chronotropic, inotropic and dromotropic effects as well as coronary vasodilator action, which are qualitatively the same as those of classical class I drugs. The selectivity of bisaramil for the intraventricular conduction vs the other cardiac variables was compared with that of disopyramide and flecainide. Bisaramil was the most selective for intraventricular conduction, while it was the least selective for ventricular muscle contraction. We conclude that bisaramil may become a useful antiarrhythmic drug with less cardiac adverse effects.     

Effects of disopyramide on SA nodal pacemaker activity and contractility in the isolated blood-perfused atrium of the dog.

The isolated blood-perfused preparations of canine atrium were suspended in a bath and perfused with arterial blood led from the carotid artery of the heparinized donor dog. Disopyramide caused dose-related negative chronotropic and inotropic effects in a dose range of 30-1000 microgram when injected directly into the cannulated sinus node artery of the isolated atrium. The order of potencies for inducing the negative chronotropic effect in isolated atrium preparations was verapamil greater than propranolol greater than lidocaine = quinidine greater than phenytoin greater than or equal to disopyramide greater than procainamide. On the other hand, the order of potencies for inducing the negative inotropic effect was verapamil = propranolol greater than lidocaine greater than or equal to phenytoin greater than disopyramide greater than procainamide greater than or equal to quinidine. When disopyramide (1 mg/kg or 3 mg/kg) was administered i.v. into the jugular vein of the donor dog, the systemic blood pressure of the donor dog was markedly decreased. However, the tension developed and sinus rate of the isolated atrium were only slightly decreased. Disopyramide produced greater suppression at higher frequencies and slightly depressed the calcium chloride-induced positive inotropic effects.     

Electrophysiological actions of a new antiarrhythmic drug, bisaramil, on isolated heart preparations.

Electrophysiological effects of bisaramil--a new antiarrhythmic drug under clinical trial--were investigated on isolated heart preparations, at a concentration range of 2.3-23 x 10(-6) M. Bisaramil dose dependently decreased the maximum rate of depolarization (Vmax), action potential amplitude (APA) and overshoot (OS) both in auricle and in papillary muscle of guinea-pig heart. There was no significant and obvious effect on the duration of the action potential and the resting membrane potential was also unchanged. Bisaramil slowed the spontaneous frequency of pacemaker cells in rabbit sinus node preparation due to its inhibitory effect on slow diastolic depolarization (SDD). Bisaramil was able to inhibit slow Ca(2+)-action potentials induced by isoprenaline on K(+)-depolarized papillary muscle. Results obtained with transmembrane current measurements revealed that bisaramil inhibited both fast Na(+)-current and slow Ca(2+)-current in frog sinoauricular fibres at the same concentration. Bisaramil with a mixed mode of the action seems to be a very promising drug.     

Class III antiarrhythmic drugs (amiodarone,bretylium and sotalol) on action potentials and membrane currents in rabbit sino-atrial node preparations

Summary Electrophysiological effects of class III antiarrhythmic drugs (amiodarone, bretylium and sotalol) were examined in spontaneously beating and voltage-clamped rabbit sino-atrial node preparations, using a two microelectrode technique. At 10^–6 mol/l these class III antiarrhythmic drugs prolonged the cycle length significantly, but did not affect the action potential duration. At high concentration (10^–4 mol/l), amiodarone and sotalol prolonged the action potential duration as well as the cycle length. Sotalol 10^–5 mol/l depolarized the maximum diastolic potential. Amiodarone 10^–4 mol/l and bretylium 10^–5 mol/l depressed the maximum rate of depolarization. At concentrations ranging from 10^– to 10^–4 mol/l, amiodarone induced dysrhythmia in 5 of 10 preparations and bretylium in 3 of 7 preparations, but sotalol in none of 5 preparations. In voltage-clamped sinoatrial node preparations, all the class III antiarrhythmic drugs decreased the slow inward current in a concentration-dependent manner. The steady-state outward and the hyperpolarization-activated inward currents were also reduced. Sotalol (10^–5 mol/1) decreased both the outward current and the hyperpolarization-activated inward current stronger than the slow inward current. In addition, amiodarone (3 × 10^–6 mol/l) depressed the inactivation curve for the slow inward current, but it did not shift the potential of half-maximum inactivation. The durgs also depressed the activation curve for the outward current in a concentration-dependent manner. However, the values of half-maximum activations were not influenced by these drugs as compared to control. These results suggest that the decreases in all three currents (the slow inward current, the outward current and the hyperpolarization-activated inward current) induced by class III antiarrhythmic drugs may be responsible for the negative chronotropic effect in rabbit sino-atrial node cells.     

4-Chloro-7-trifluoromethyl-10H- benzo[4,5]furo[3,2-b]indole-1-carboxylic acid (TBIC), a putative BK(Ca) channel opener with uterine relaxant activities

In the present study, we examined the uterine relaxant activity of 4-chloro-7-trifluoromethyl-10H-benzo[4,5]furo[3,2-b]indole-1-carboxylic acid (TBIC), a putative opener of the large conductance Ca(2+)-activated K(+) (BK(Ca)) channel. TBIC concentration-dependently inhibited spontaneous uterine contractions (EC(50) = 4.63 μmol/l; E(max) = 94.85 ± 1.85%; 100 μmol/l, n = 6). It also reduced contractions induced by oxytocin (EC(50) = 4.10 μmol/l; E(max) = 84.3 ± 3.83%; 100 μmol/l, n = 6), prostaglandin F(2)(α) (EC(50) = 2.14 μmol/l; E(max) = 73.70 ± 5.21%; 100 μmol/l, n = 6) and acetylcholine (EC(50) = 4.37 μmol/l; E(max) = 83.67 ± 4.82; 100 μmol/l, n = 6). TBIC decreased KCl (20 mmol/l) -induced contractions (EC(50) = 3.04 μmol/l; E(max) = 94.0 ± 3.12%; 100 μmol/l, n = 6) indicating its K(+) channel opening activity. BK(Ca) channel blockers penitrem A (100 nmol/l) and tetraethylammonium chloride (1 mmol/l) attenuated the inhibitory activities of TBIC (p < 0.001) but not other K(+) channel blockers such as barium chloride and glibenclamide (K(IR) and K(ATP) channel blockers, respectively). These results demonstrate the uterine relaxant effects of TBIC in a mechanism of action largely referable to the potentiation of the BK(Ca) channels. We have provided evidence for the potential use of TBIC as a tocolytic agent and support for the utility of BK(Ca) channel openers in pathophysiologic conditions involving smooth muscle hyperactivity.     

Comparative actions of cibenzoline and disopyramide on I(Kr) and I(Ks) currents in rat sino-atrial nodal cells

Modulation by class Ia antiarrhythmic drugs, cibenzoline and disopyramide, of the pacemaking activity and the underlying ionic currents in rat sino-atrial nodal cells was investigated using current-clamp and whole-cell patch-clamp techniques. Both drugs depressed the spontaneous activity and often caused sinus arrest. The negative chronotropic effect was significant at 10 microM cibenzoline and 30 microM disopyramide. The L-type Ca(2+) current (I(Ca)) and the hyperpolarization-activated inward current decreased by 69.7+/-3.2% and by 45.8+/-3.0% at 30 microM cibenzoline and by 51. 2+/-3.3% and by 48.3+/-2.7% at 100 microM disopyramide, respectively. The delayed rectifier K(+) current, which is composed of rapidly and slowly activated currents (I(Kr) and I(Ks)), also decreased. The IC(50) values of I(Kr) for cibenzoline and disopyramide were 8.8+/-1. 1 and 25.1+/-2.3 microM, respectively. In the presence of 5 microM E-4031 (1-[2-(6-methyl-2-pyridyl)ethyl]-4-(4-methylsulfonylaminobenzoyl) piperidine), the IC(50) values of I(Ks) for cibenzoline and disopyramide were 12.3+/-1.8 and 81.1+/-2.3 microM, respectively. The I(Ks) was completely blocked by 30 microM 293B (trans-6-cyano-4-(N-ethylsulphonyl-N-methtamino)-3-hydroxy-2 , 2-dimethyl-chromane). These results indicate that the ionic currents are more sensitive to cibenzoline than disopyramide in rat sino-atrial nodal cells, and that I(Ca) and I(Kr) make major contributions to pacemaking activity.     

Involvement of non-selective Ca2+ channels in the contraction induced by alkalinization of rat anococcygeus muscle cells

Intracellular pH is a modulator of cellular functions such as smooth muscle contraction. Changes in cytosolic Ca(2+) concentration ([Ca(2+)](c)) associated with contraction are brought about by Ca(2+) influx and release from the sarcoplasmic reticulum, and alterations in the intracellular pH can affect both processes. In this work, therefore, we have investigated the Ca(2+) influx pathway that contributes to the contraction induced by the alkalinizing agent NH(4)Cl in the rat anococcygeus smooth muscle. For this purpose, we measured the isometric tension in muscle preparations, and [Ca(2+)](c) was measured on isolated cells loaded with 5 micromol/l FURA2/AM by using the ratio 340/380 nm. NH(4)Cl (10 mmol/l) induced a larger increase in [Ca(2+)](c) (100%) when compared with the [Ca(2+)](c) increase induced by 0.1 micromol/l phenylephrine (57.0+/-12.3% n=4). Incubation of the muscle preparations for 1 min in Ca(2+)-free medium reduced the contractions induced by 10 mmol/l NH(4)Cl to 11.5+/-5.1% (n=5), when compared with the contractions induced in 2.5 mmol/l Ca(2+) solution (100%). After 3 min in Ca(2+) free medium, contractions stimulated with NH(4)Cl were almost abolished (0.6+/-0.4%, n=5). In the same way, incubation with 10 micromol/l 1-[beta-[3[(4-methoxyphenyl)propoxyl]-4-methoxy-phenetyl]-1H-imidazole hydrochloride (SKF96365), a non-selective Ca(2+) channels, reduced the contractions stimulated with NH(4)Cl to 47.6+/-6.7% (n=7). On the other hand, 1 micromol/l verapamil, a voltage-operated Ca(2+) channel blocker and 0.05 micromol/l calphostin C, a protein kinase-C inhibitor, did not alter the contractions induced by NH(4)Cl. On isolated cells, [Ca(2+)](c) was reduced to 72.2+/-1.7% (n=4) by 10 micromol/l SKF96365. Taken together, our results suggest that NH(4)Cl induces contraction of rat anococcygeus smooth muscle cells, as well as [Ca(2+)](c) increase due to Ca(2+) influx through non-selective Ca(2+) channels.     

The influence of verapamil on the amplitude and frequency of cholinergic-initiated contractions of isolated gastric muscularis muscle of Bufo marinus

In order to elucidate the role of Ca2+ in the acetylcholine-initiated amplitude and frequency of spontaneous contractions of isolated circular muscle strips from Bufo marinus stomach, we investigated the effects of omission of Ca2+ in the nutrient fluid and also the influence of verapamil, a calcium antagonist, on the amplitude and frequency of the initiated spontaneous contractions. The gastric muscularis muscle strips were prepared and mounted in an organ bath. The control preparation was challenged with a predetermined dose of acetylcholine (1.50 x 10(-5) mol/l) to elicit isometric contractions and there was no decline in the strength of contractions during the experimental period (2 h). Omission of Ca2+ in the nutrient solution prevented acetylcholine-initiated contractions. When the preparation was challenged with the concentration of acetylcholine in the presence of different concentrations of verapamil(10(-10)-10(-3) mol/l), verapamil concentration-dependently suppressed the amplitude of acetylcholine-initiated contractions. When the contractions were seen the frequency was identical. From this investigation, it was concluded that Ca2+ plays a significant part in acetylcholine-initiated spontaneous contractions of circular gastric muscularis muscle of B. marinus.     

Effect of cilnidipine on L- and T-type calcium currents in guinea pig ventricle and action potential in rabbit sinoatrial node

Cilnidipine, a dihydropyridine Ca(2+) channel antagonist, is known to have inhibitory effects on both L- and N-type Ca(2+) currents. In the present study, we examined the effect of cilnidipine on myocardial L- and T-type Ca(2+) currents and sinoatrial node action potential configuration. In voltage clamped guinea pig ventricular myocytes, cilnidipine concentration-dependently decreased L- and T-type Ca(2+) currents. In rabbit sinoatrial node tissue, cilnidipine increased cycle length through reduction of phase 4 depolarization slope. In conclusion, cilnidipine has inhibitory effects on T-type Ca(2+) current, which may contribute to its negative chronotropic potency.     

Interaction between calcium and slow channel blocking drugs on atrial rate

The relationship between extracellular calcium concentration and the chronotropic effect of prenylamine, verapamil and nifedipine was studied in isolated spontaneously beating rat atria. The three slow channel blocking drugs produced a concentration-dependent decrease in atrial rate, though with different relative potencies. The order of potency for decreasing atrial rate, independently of the calcium level (1.0, 3.0, 6.0 or 9.0 mmol/l) was: verapamil greater than nifedipine greater than prenylamine. Increasing calcium from 1.0 to 6.0 and 9.0 mmol/l increased atrial rate from 251 +/- beats . min-1 to 265 +/- 6 beats . min-1 and 285 +/- 9 beats . min-1 (mean +/- 1 standard error) respectively (P less than 0.05). Despite their positive chronotropic effect high calcium levels failed to reverse the negative chronotropic effect of the slow channel blockers. Furthermore, the negative chronotropic effect of both verapamil and nifedipine was enhanced at high calcium levels. Raising calcium from 1.0 to 6.0 mmol/l in the presence of verapamil (1 X 10(-7) mol/l) or nifedipine (3 X 10(-7) mol/l) increased 2-fold the negative chronotropic effect of the calcium channel blockers. In addition, the concentration-effect curves for verapamil and nifedipine shifted to the left by 0.50 +/- 0.14 and 0.50 +/- 0.16 log units, respectively, when calcium increased from 1.0 to 6.0 mmol/l. The data show that increasing calcium may produce positive or negative chronotropic effects depending on whether or not the calcium channels are blocked. This paradoxical effect of calcium ions can be produced either by opposite chronotropic effects on automatic cells or by shifting the pacemaker activity to a group of cells which respond in a different way to an increment of calcium.