Actions of mibefradil, efonidipine and nifedipine block of recombinant T- and L-type Ca channels with distinct inhibitory mechanisms

We compared detailed efficacy of efonidipine and nifedipine, dihydropyridine analogues, and mibefradil using recombinant T- and L-type Ca2+ channels expressed separately in mammalian cells. All these Ca2+ channel antagonists blocked T-type Ca2+ channel currents (I(Ca(T))) with distinct blocking manners: I(Ca(T)) was blocked mainly by a tonic manner by nifedipine, by a use-dependent manner by mibefradil, and by a combination of both manners by efonidipine. IC50s of these Ca2+ channel antagonists to I(Ca(T)) and L-type Ca2+ channel current (I(Ca(L))) were 1.2 micromol/l and 0.14 nmol/l for nifedipine; 0.87 and 1.4 micromol/l for mibefradil, and 0.35 micromol/l and 1.8 nmol/l for efonidipine, respectively. Efonidipine, a dihydropyridine analogue, showed high affinity to T-type Ca2+ channel.     

Blockade of T-type voltage-dependent Ca2+ channels by benidipine, a dihydropyridine calcium channel blocker, inhibits aldosterone production in human adrenocortical cell line NCI-H295R

Benidipine, a long-lasting dihydropyridine calcium channel blocker, is used for treatment of hypertension and angina. Benidipine exerts pleiotropic pharmacological features, such as renoprotective and cardioprotective effects. In pathophysiological conditions, the antidiuretic hormone aldosterone causes development of renal and cardiovascular diseases. In adrenal glomerulosa cells, aldosterone is produced in response to extracellular potassium, which is mainly mediated by T-type voltage-dependent Ca2+ channels. More recently, it has been demonstrated that benidipine inhibits T-type Ca2+ channels in addition to L-type Ca2+ channels. Therefore, effect of calcium channel blockers, including benidipine, on aldosterone production and T-type Ca2+ channels using human adrenocortical cell line NCI-H295R was investigated. Benidipine efficiently inhibited KCl-induced aldosterone production at low concentration (3 and 10 nM), with inhibitory activity more potent than other calcium channel blockers. Patch clamp analysis indicated that benidipine concentration-dependently inhibited T-type Ca2+ currents at 10, 100 and 1000 nM. As for examined calcium channel blockers, inhibitory activity for T-type Ca2+ currents was well correlated with aldosterone production. L-type specific calcium channel blockers calciseptine and nifedipine showed no effect in both assays. These results indicate that inhibition of T-type Ca2+ channels is responsible for inhibition of aldosterone production in NCI-H295R cells. Benidipine efficiently inhibited KCl-induced upregulation of 11-beta-hydroxylase mRNA and aldosterone synthase mRNA as well as KCl-induced Ca2+ influx, indicating it as the most likely inhibition mechanism. Benidipine partially inhibited angiotensin II-induced aldosterone production, plus showed additive effects when used in combination with the angiotensin II type I receptor blocker valsartan. Benidipine also partially inhibited angiotensin II-induced upregulation of the above mRNAs and Ca2+ influx inhibitory activities of benidipine for aldosterone production. T-type Ca2+ channels may contribute to additional benefits of this drug for treating renal and cardiovascular diseases, beyond its primary anti-hypertensive effects from blocking L-type Ca2+ channels.     

Differential blocking action of dihydropyridine Ca2+ antagonists on a T-type Ca2+ channel (alpha1G) expressed in Xenopus oocytes

Recent reports show that efonidipine, a dihydropyridine Ca2+ antagonist, has blocking action on T-type Ca2+ channels, which may produce favorable actions on cardiovascular systems. However, the effects of other dihydropyridine Ca2+ antagonists on T-type Ca2+ channels have not been investigated yet. Therefore, in this study, we examined the effects of dihydropyridine compounds clinically used for treatment of hypertension on a T-type Ca2+ channel subtype, alpha1G, expressed in Xenopus oocytes. These effects were compared with those on T-type Ca2+ channel. Rabbit L-type (alpha1Calpha2/deltabeta1a) or rat T-type (alpha1G) Ca2+ channel was expressed in Xenopus oocytes by injection of cRNA for each subunit. The Ba currents through expressed channels were measured by conventional 2-microelectrode voltage-clamp methods. Twelve DHPs (amlodipine, barnidipine, benidipine, cilnidipine, efonidipine, felodipine, manidipine, nicardipine, nifedipine, nilvadipine, nimodipine, nitrendipine) and mibefradil were tested. Cilnidipine, felodipine, nifedipine, nilvadipine, minodipine, and nitrendipine had little effect on the T-type channel. The blocks by drugs at 10 microM were less than 10% at a holding potential of -100 mV. The remaining 6 drugs had blocking action on the T-type channel comparable to that on the L-type channel. The blocking actions were also comparable to that by mibefradil. These results show that many dihydropyridine Ca2+ antagonists have blocking action on the alpha1G channel subtype. The action of dihydropyridine Ca2+ antagonists in clinical treatment should be evaluated on the basis of subtype selectivity.     

Efonidipine hydrochloride: a dual blocker of L- and T-type ca(2+) channels

T-type Ca(2+) channels have properties different from those of the L-type and are involved in cardiac pacemaking and regulation of blood flow, but not in myocardial contraction. Efonidipine is an antihypertensive and antianginal drug with dihydropyridine structure that was recently found to block both L- and T-type Ca(2+) channels. In isolated myocardial and vascular preparations, efonidipine has potent negative chronotropic and vasodilator effects but only a weak negative inotropic effect. In experimental animals and patients, reduction of blood pressure by the drug was accompanied by no or minimum reflex tachycardia leading to improvement of myocardial oxygen balance and maintenance of cardiac output. Efonidipine increased glomerular filtration rate without increasing intraglomerular pressure. By relaxing both the afferent and efferent arterioles, efonidipine markedly reduced proteinuria. Thus, efonidipine, an L- and T-type dual Ca(2+) channel blocker, appears to have an ideal profile as an antihypertensive and antianginal drug with organ-protective effects in the heart and kidney.     

Identification of R(-)-isomer of efonidipine as a selective blocker of T-type Ca2+ channels

Efonidipine, a derivative of dihydropyridine Ca(2+) antagonist, is known to block both L- and T-type Ca(2+) channels. It remains to be clarified, however, whether efonidipine affects other voltage-dependent Ca(2+) channel subtypes such as N-, P/Q- and R-types, and whether the optical isomers of efonidipine have different selectivities in blocking these Ca(2+) channels, including L- and T-types. To address these issues, the effects of efonidipine and its R(-)- and S(+)-isomers on these Ca(2+) channel subtypes were examined electrophysiologically in the expression systems using Xenopus oocytes and baby hamster kidney cells (BHK tk-ts13). Efonidipine, a mixture of R(-)- and S(+)-isomers, exerted blocking actions on L- and T-types, but no effects on N-, P/Q- and R-type Ca(2+) channels. The selective blocking actions on L- and T-type channels were reproduced by the S(+)-efonidipine isomer. By contrast, the R(-)-efonidipine isomer preferentially blocked T-type channels. The blocking actions of efonidipine and its enantiomers were dependent on holding potentials. These findings indicate that the R(-)-isomer of efonidipine is a specific blocker of the T-type Ca(2+) channel.     

Effect of efonidipine on TGF-β1-induced cardiac fibrosis through Smad2-dependent pathway in rat cardiac fibroblasts

Transforming growth factor beta-1 (TGF-β1) plays a critical role in progression of cardiac fibrosis, which may involve intracellular calcium change. We examined effects of efonidipine, a dual T-type and L-type calcium channel blocker (CCB), on TGF-β1-induced fibrotic changes in neonatal rat cardiac fibroblast. T-type and L-type calcium channel mRNAs were highly expressed in cultured cardiac fibroblasts. TGF-β1 (5 ng/mL) significantly increased Smad2 phosphorylation and [(3)H]-leucine incorporation, which were attenuated by pretreatment with efonidipine (10 μM). Neither R(-)efonidipine (10 μM), selective T-type CCB, nor nifedipine (10 μM), selective L-type CCB, efficaciously inhibited both TGF-β1-induced Smad2 phosphorylation and [(3)H]-leucine incorporation. However, both were markedly attenuated by combination of R(-)efonidipine and nifedipine, EDTA, or calcium-free medium. Pretreatment with Smad2 siRNA significantly attenuated [(3)H]-leucine incorporation induced by TGF-β1. These data suggest that efonidipine elicits inhibitory effects on TGF-β1- and Smad2-dependent protein synthesis through both T-type and L-type calcium channel-blocking actions in cardiac fibroblasts.     

Proliferation of human peripheral blood mononuclear cells during calcium entry blockade. Role of protein kinase C.

To evaluate the role of protein kinase C (PKC) and intracellular calcium and particularly Ca(2+)-uptake in the initiation of lymphocyte mitogenesis, the proliferation of human peripheral blood mononuclear cells (PBMC) was investigated during calcium entry blockade with nifedipine (an L-type calcium channel blocker) and mibefradil (an L- and T-type calcium channel blocker with a higher selectivity for T-type channels). The rate of [3H]-thymidine, [3H]-uridine and [3H]-leucine incorporation into control and concanavalin A-stimulated PBMC cultured for 3 days in the presence or absence of the calcium channel blockers nifedipine or mibefradil (1, 10 or 50 microM) is assayed. Nifedipine and mibefradil concentration-dependently reduced cell number and [3H]-thymidine incorporation or de novo DNA synthesis in control and concanavalin A-stimulated PBMC, as well as de novo RNA and protein synthesis. The proliferative response of nifedipine- or mibefradil-treated cells was restored by addition of phorbol-12-myristate-13-acetate (PMA), an exogenous PKC activator. Our data show that PBMC treated with the Ca2+ channel blockers nifedipine or mibefradil are still capable of proliferating in response to PMA. However, in PKC-depleted cells, the proliferative response of PBMC was suppressed.     

A selective T-type Ca2+ channel blocker R(−) efonidipine

Recently, novel compound R(-) efonidipine was reported to selectively block low-voltage-activated (LVA or T-type) Ca(2+) channels in peripheral organs. We examined how R(-) efonidipine acts on T-type and high-voltage-activated (HVA) Ca(2+) channels in mammalian central nervous system (CNS) neurons. Furthermore, we compared the effects of R(-) efonidipine with those of flunarizine and mibefradil on both T-type and HVA Ca(2+) channels in rat hippocampal CA1 neurons by using the nystatin perforated-patch clamp technique. Flunarizine and mibefradil nonselectively inhibited both T-type and HVA Ca(2+) channels, though the dose-dependent blocking potency of flunarizine on T-type Ca(2+) channels was slightly stronger than that of mibefradil. In contrast, R(-) efonidipine inhibited only T-type Ca(2+) channels and did not show any effect on HVA Ca(2+) channels. The inhibitory actions of R(-) efonidipine or flunarizine were similar on both Ba(2+) and Ca(2+) current components passing through T-type Ca(2+) channels. In addition, flunarizine but not R(-) efonidipine inhibited voltage-dependent Na(+) channels and Ca(2+)-activated K(+) channels. Thus, it appears that R(-) efonidipine is a selective blocker for T-type Ca(2+) channels. It could be used as a pharmacological tool in future studies on T-type Ca(2+) channels.     

Pathophysiological significance of T-type Ca2+ channels: role of T-type Ca2+ channels in renal microcirculation

Since conventional Ca(2+) antagonists, with predominant blockade of L-type voltage-dependent Ca(2+) channels, elicit preferential dilation of afferent arterioles, they might ostensibly aggravate glomerular hypertension. Recently, novel Ca(2+) antagonists, with inhibitory action on L-/T-type Ca(2+) channels, have been reported to dilate both afferent and efferent arterioles. The present review attempted to characterize the renal action of these Ca(2+) antagonists and evaluated the consequences following the treatment with these agents. In contrast to conventional Ca(2+) antagonists (e.g., nifedipine), novel antagonists (e.g., benidipine, efonidipine) potently dilated afferent and efferent arterioles; their action on efferent arterioles appeared to be mediated by the T-type Ca(2+) channel blockade, probably through the inhibition of the intracellular Ca(2+) release. The comparison of the anti-proteinuric action in subtotally nephrectomized rats showed that efonidipine exerted more prominent action than nifedipine. Furthermore, Ca(2+) antagonists with T-type Ca(2+) inhibitory action inhibited renin/aldosterone release and proinflammatory process. Finally, patients with chronic renal disease given a 48-week efonidipine treatment showed reduced proteinuria, and this effect was seen even when mean arterial blood pressure failed to become less than 100 mmHg. Collectively, T-type Ca(2+) channel blockade provides beneficial action in renal injury. Various mechanisms serve to protect against renal injury, including systemic/glomerular hemodynamic action and non-hemodynamic mechanisms.     

A comparison between calcium channel blocking drugs with different potencies for T- and L-type channels in preventing atrial electrical remodeling

Calcium overload plays a key role in the development of atrial electrical remodeling. The effect of an L-type Ca channel blocker in preventing this remodeling has been reported to be short lasting, partly due to down-regulation of this channel and persisting Ca entry through the T-type Ca channel. To prove if efonidipine, a dual L- and T-type Ca channel blocker exerts a greater effect than an L-type Ca channel blocker verapamil, 21 dogs underwent rapid atrial pacing at 400 bpm for 14 days, pretreatment with efonidipine in 7 (E), verapamil in 7 (V), and none in 7 (C). We measured the atrial effective refractory period (ERP) serially during 14 days of rapid pacing. In response to rapid pacing, ERP decreased progressively in C. In contrast, in E and V, ERP remained greater than ERP in C (P < 0.01) on days 2 through 7. However, on the 14th day, ERP in V decreased to the level seen in C, whereas ERP in E remained significantly longer than ERPs in C or V (P < 0.01). The blockade L-type Ca channel alone is not sufficient, but the addition of a T-type Ca channel blockade shows a more sustained effect to prevent atrial electrical remodeling.     

Effects of Ca channel blockers on Ca loading induced by metabolic inhibition and hyperkalemia in cardiomyocytes

The effects of the L-type (nifedipine and verapamil) and the T-type (mibefradil) Ca²⁺ channel blockers on the increase in intracellular Ca²⁺ concentration ([Ca²⁺]_i) induced by NaCN metabolic inhibition and hyperkalemia were examined in chicken cardiomyocytes using fluorescence imaging with Fura-2. NaCN induced a slow and sustained rise in [Ca²⁺]_i, which was not affected by pretreating the cells for 5 min with nifedipine, verapamil, or mibefradil at 100 nM or 10 μM. Pretreatment of the cells with 10 μM nifedipine, verapamil, or mibefradil for 5 min remarkably inhibited the K⁺-induced increase in [Ca²⁺]_i. These inhibitory effects diminished after 48-h pretreatment with nifedipine or verapamil but not with mibefradil. Ryanodine also induces an increase in [Ca²⁺]_i, and this effect was enhanced by 48-h pretreatment of the cells with 10 μM verapamil but not with 10 μM mibefradil. We conclude that the NaCN-induced increase in [Ca²⁺]_i is independent of the Ca²⁺ influx though the L-type or T-type Ca²⁺ channels. Chronic inhibition of the L-type Ca²⁺ channels but not T-type channels may enhance the ryanodine receptor-mediated Ca²⁺ release, which may be responsible for the development of tolerance to their inhibitory effects on K⁺-induced increase in [Ca²⁺]_i.     

The R(-)-enantiomer of efonidipine blocks T-type but not L-type calcium current in guinea pig ventricular myocardium

In guinea pig ventricular cardiomyocytes, the R(-)-enantiomer of efonidipine concentration-dependently blocked T-type Ca2+ current with 85% inhibition at 1 microM. In contrast, R(-)-efonidipine (1 microM) had no effect on the L-type Ca2+ current and Ca2+ transient in cardiomyocytes and contractile force in papillary muscles. Thus, R(-)-efonidipine is a highly selective blocker of the T-type Ca2+ current in native myocardia.     

Pathophysiological significance of T-type Ca2+ channels: T-type Ca2+ channels and drug development

T-type Ca(2+) channels are present in cardiovascular, neuronal, and endocrine systems; and they are now receiving attention as novel therapeutic targets. Many drugs and compounds non-specificaly block T-type Ca(2+) channels. Certain dihydropyridine compounds, such as efonidipine, have blocking activity on both L-type and T-type Ca(2+) channels which possibly underlies their excellent clinical profiles such as minimum reflex tachycardia and renal protection. Selective inhibitors of T-type Ca(2+) channels, such as non-hydrolyzable mibefradil and R(-)-efonidipine, are powerful pharmacological tools for further studies and may lead to the development of novel therapeutic strategies.     

Mibefradil-induced inhibition of proliferation of human peripheral blood mononuclear cells

To evaluate the role of intracellular calcium and particularly Ca2+-uptake in the initiation of lymphocyte mitogenesis, the effect of mibefradil, which blocks both L- and T-type calcium channels with a more selective blockade of T-type channels, on the proliferation of human peripheral blood mononuclear cells (PBMCs) is compared with the effect of nifedipine, which blocks only the L-type calcium channel. The rate of [3H]thymidine incorporation into control and concanavalin A-stimulated PBMCs in the presence or absence of the calcium channel blockers mibefradil or nifedipine (1, 10, or 50 microM), and of the intracellular calcium antagonist 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (TMB-8; 1, 10, 25, or 50 microM) was assayed in the cells cultured for 3 days. The cellular cytotoxicity and the cell number in growing cultures also was determined in mibefradil- or nifedipine-treated control or stimulated cells. Restoration of the proliferative response in mibefradil- or nifedipine-treated cells was investigated by addition of exogenous interleukin-2. Interleukin-2-receptor expression in the cells was monitored by using anti-activated T-cell antigen (Tac) antibody, and the interleukin-2 production in the cell supernatants of the cultures was determined by an enzyme-amplified sensitive immunoassay. Mibefradil and nifedipine concentration-dependently reduced the cell number and the [3H]thymidine incorporation or the de novo DNA synthesis in control and concanavalin A-stimulated human PBMCs. Mibefradil exhibited a more pronounced inhibition of the proliferation of human PBMCs than did nifedipine. The inhibitory effect of mibefradil or nifedipine on DNA synthesis was dependent on the timing of treatment with the drugs. The inhibitory effect of mibefradil or nifedipine on the lymphoproliferative response was nearly abolished if the drugs were added 20 h after cell stimulation. A markedly reduced inhibitory effect was found when mibefradil or nifedipine was added 1-7 h after cell stimulation. However, regardless of time of addition, TMB-8 caused a persistent inhibition of the proliferation of human PBMCs. The inhibitory effect of mibefradil or nifedipine on the proliferation of human PBMCs is nearly abolished by addition of the calcium channel activator Bay K 8644. The proliferative response of mibefradil- or nifedipine-treated cells is restored by addition of exogenous interleukin-2. The normal expression of interleukin-2 receptors was preserved, whereas the interleukin-2 production was blocked in the presence of mibefradil or nifedipine. Our data show that mibefradil has a more pronounced inhibitory effect on the proliferation of human PBMCs than nifedipine and that this inhibitory effect on DNA synthesis is dependent on the timing of treatment with both drugs.     

Non-L-type voltage-dependent calcium channels control vascular tone of the rat basilar artery

1. Constriction of cerebral arteries is considered to depend on L-type voltage-dependent calcium channels (VDCCs); however, many previous studies have used antagonists with potential non-selective actions. Our aim was to determine the expression and function of VDCCs in the rat basilar artery. 2. The relative expression of VDCC subtypes was assessed using quantitative polymerase chain reaction and immunohistochemistry. Data were correlated with physiological studies of vascular function. Domains I-II of the T channel subtypes expressed in the rat basilar artery were cloned and sequenced. 3. Blockade of L-type channels with nifedipine had no effect on vascular tone. In contrast, in the presence of nifedipine, hyperpolarization of short arterial segments produced relaxation, whereas depolarization of quiescent segments evoked constriction. 4. The mRNA and protein for L- and T-type VDCCs were strongly expressed in the main basilar artery and side branches, with Ca(V)3.1 and Ca(V)1.2 the predominant subtypes. 5. T-Type VDCC blockers (i.e. 1 micromol/L mibefradil, 10 micromol/L pimozide and 100 micromol/L flunarizine) decreased intracellular calcium in smooth muscle cells, relaxed and hyperpolarized arteries, whereas nickel chloride (100 micromol/L) had no effect. In contrast with nifedipine, 10 micromol/L nimodipine produced hyperpolarization and relaxation. 6. When arteries were relaxed with 10 micromol/L U73122 (a phospholipase C inhibitor) in the presence of nifedipine, 40 mmol/L KCl evoked depolarization and constriction, which was significantly reduced by 1 micromol/L mibefradil. 7. Sequencing of domains I-II revealed splice variants of Ca(V)3.1, which may impact on channel activity. 8. We conclude that vascular tone of the rat basilar artery results from calcium influx through nifedipine-insensitive VDCCs with pharmacology consistent with Ca(V)3.1 T-type channels.     

Different effects of L-type and T-type calcium channel blockers on the hypnotic potency of triazolam and zolpidem in rats.

We examined the effects of an L-type Ca2+ channel blocker, nilvadipine (0.5 and 2.0 mg/kg), and that of a T-type Ca2+ channel blocker, flunarizine (10.0 and 40.0 mg/kg), on the hypnotic potency of both a benzodiazepine (BZ)-hypnotic, triazolam (1.0 mg/kg), and a non-BZ hypnotic, zolpidem (20.0 mg/kg), in rats. The polysomnogram was recorded for 6 h after administration of the vehicle solution alone, or after one of the Ca2+ channel blockers, with or without one of the hypnotics. Both Ca2+ channel blockers prolonged the increased total time of non-rapid eye movement (non-REM) sleep induced by either hypnotic. In the case of triazolam, however, the non-REM sleep-enhancing effect induced by nilvadipine was greater than that induced by flunarizine. These findings indicate that the hypnotic action of triazolam is potentiated more strongly by an L-type Ca2+ channel blocker than by a T-type Ca2+ channel blocker.     

钙通道不同分型与亚基和抗高血压药物的关系

该文阐述电压依赖性钙通道不同分型与亚型和抗高血压药物的关系。(1)传统的L型电压依赖性钙通道阻断剂舒张肾入球小动脉,但对肾出球小动脉无作用。第3代新的双氢吡啶类钙通道阻断剂(manidipine,nilvadipine,benzin-damine和efonidipine)能同时作用L及T型钙通道,对肾出球小动脉也能舒张,故对肾性高血压有效,并起保护肾脏作用。(2)L型钙通道的主要组成α1c亚基,在高血压时表达增加,使钙通道数量增多,从而加速高血压的发展,故能使α1c亚基数目恢复正常的药物,有望用于临床治疗高血压。     

Mibefradil (Ro 40-5967): the first selective T-type Ca2+ channel blocker

Mibefradil is a novel Ca2+ antagonist acting on both L- and T-type Ca2+ channels, with a ten-fold selectivity for T-type Ca2+ channels. It belongs to a chemical class different from other Ca2+ antagonists (tetralol derivative), and binds to a new receptor site on the L-type Ca2+ channel, where it does not affect dihydropyridine (DHP) binding but appears to overlap the verapamil and fantofarone sites. In vitro and in vivo studies indicate that mibefradil has a high selectivity for the coronary vasculature over the peripheral vasculature and the myocardium. It has no relevant negative inotropic effects in various animal models, in normotensive patients, and patients with hypertension or angina pectoris. Instead, treatment with mibefradil slightly decreases heart rate and improves cardiac function. Clinical studies confirm that mibefradil is an effective antihypertensive and anti-ischaemic drug, which may be beneficial in the treatment of heart failure. Its excellent pharmacological and safety profile combined with high bioavailability makes it a promising new drug. Many of the unique pharmacological properties of mibefradil may derive from its selective block of T-type Ca2+ channels.     

Mibefradil- and ω-conotoxin GVIA-induced inhibition of noradrenaline release from the sympathetic nerves of the human heart

Segments of human right atrial appendages preincubated with [3H]noradrenaline and superfused with physiological salt solution containing desipramine and corticosterone were used to determine the effects of mibefradil, ω-conotoxin (ω-CTx) GVIA and nifedipine on tritium overflow evoked by transmural electrical stimulation. Mibefradil (which predominantly blocks T-type, and at lower potency also N-type, Ca²⁺ channels) at concentrations of 0.3–3μM reduced the electrically evoked tritium overflow in a reversible and concentration-dependent manner (IC_50%: 1μM), whereas 0.1–10μM nifedipine (a selective blocker of L-type channels) was ineffective. The evoked tritium overflow was almost abolished by 0.2μM ω-CTx GVIA (a selective blocker of N-type channels). It is concluded that noradrenaline release from cardiac sympathetic nerves is triggered by Ca²⁺-influx via N-type, but not L-type, Ca²⁺ channels and that the inhibitory effect of mibefradil at clinically relevant concentrations on noradrenaline release is probably due to its blocking action on N-type Ca²⁺ channels. This property of mibefradil is unique among the therapeutically applied Ca²⁺ channel blockers and may contribute to the slight negative chronotropic effect of the drug in vivo. Received: 24 September 1997 / Accepted: 3 November 1997