Functional role of T-type calcium channels in tumour growth and progression: prospective in cancer therapy

T-type Ca²⁺ channels represent a specific channel family overexpressed in different types of tumours. Their involvement in controlling the proliferation, angiogenesis and invasion of tumour cells, has been partially clarified. The article by Zhang et al. in this issue of BJP provides the first evidence of anti-tumoural effects of endostatin (ES) in U87 glioma cells. He demonstrated that ES or mibefradil (a L/T-type calcium channel blocker), reduces the proliferation and migration of U87 glioma cells in a T-type Ca²⁺ channel-dependent manner. However, the difference in the blocking effect of mibefradil on T-type calcium channel expression as compared with its ability to inhibit proliferation and migration, supports the idea of a broader T/L-type-independent effect of the mibefradil blocker. Overall, these findings provide new insights for the future development of a novel class of anti-T-type calcium channel blockers in the therapy of glioblastoma.

LINKED ARTICLE

This article is a commentary on Zhang et al., pp. 1247–1260 of this issue. To view this paper visit http://dx.doi.org/10.1111/j.1476-5381.2012.01852.x     

T型钙通道在心肌肥厚大鼠心肌细胞钙内流中的作用

目的研究T型钙通道在心肌细胞钙离子内流中的作用及其对心脏兴奋收缩耦联的可能影响。方法测定选择性T型钙通道阻滞剂米贝拉地尔对培养的SD乳大鼠心室肌细胞和二肾一夹心肌肥厚大鼠心室肌细胞[Ca2+]i的影响。结果血管紧张素Ⅱ(AngⅡ)刺激使乳大鼠心室肌舒张期细胞[Ca2+]i增高,收缩期细胞[Ca2+]i降低,[Ca2+]i上升和下降的时间延长。米贝拉地尔1.25～5μmol·L-1浓度依赖性降低AngⅡ引起的细胞[Ca2+]i变化。在心肌肥厚模型大鼠,咖啡因刺激后,[Ca2+]i增幅和最高[Ca2+]i明显降低。而米贝拉地尔25mg·kg-1·d-1(灌胃给药7～9周)组加入咖啡因刺激后细胞内[Ca2+]i增幅和最高[Ca2+]i明显增高。结论T型钙通道异常开放可以引起心肌细胞内钙超载。阻断T型钙通道,可能通过改善肌浆网摄取及释放钙的功能而抑制心肌细胞钙超载。     

Investigational calcium channel blockers for the treatment of hypertension

Introduction: Voltage-gated Ca²⁺ channels are the primary route of Ca²⁺ entry in vascular smooth muscle cells, playing a key role in the regulation of arterial tone and blood pressure. Since the 60´s, L-type Ca²⁺ channel blockers (CCBs) have been widely used for the treatment of hypertension.

Areas covered: T-type Ca²⁺ channels regulate vascular tone in small-resistance vessels and aldosterone secretion, and N-type channels expressed in sympathetic nerve terminals regulate the release of neurotransmitters. We performed a literature search in MEDLINE, PubMed and ClinicalTrials.gov to identify eligible studies published between January 2001 and March 2016 and reviewed the antihypertensive and renoprotective effects of four CCBs with different pharmacological profiles: azelnidipine (L-type), cilnidipine (L-/N-type) and benidipine and efonidipine (L-/T-type CCBs). Despite similar blood pressure lowering effects, L/N- and L/T-type CCBs, compared with L-type CCBs, decreased intraglomerular pressure, improved renal hemodynamics and provided a greater decrease in proteinuria even in patients already treated with renin-angiotensin-aldosterone inhibitors.

Expert opinion: Dual L/N- and L/T-type CCBs may exhibit therapeutic advantages over L-type blockers in hypertensive patients with chronic kidney disease. Because clinical trials supporting these advantages present important biases, further large-scale, long-term comparative trials are needed to confirm that these differences translate into improved clinical outcomes.     

Activators of voltage-dependent L-type calcium channels

The L class of voltage-dependent Ca²⁺ channels provides an important pathway for Ca²⁺ entry into a variety of excitable cells. Many drugs have been shown to be blockers of this channel including the clinically available nifedipine, verapamil, and diltiazem. An increasing number of compounds are now being recognized as activators of L-type Ca²⁺ channels. The best characterized of these are certain 1,4-dihydropyridines, typified by Bay K 8644, which act as partial agonists of the channel. The benzoylpyrrole group of molecules, which includes FPL 64176, have proven to be highly efficacious L channel agonists. Certain naturally occurring substances, ranging from toxins to endogenous ligands, have also been proposed as activators of this channel. Activators of L-type Ca²⁺ channels have proven to be valuable tools with which to study the structure and function of these channels and could lead to the development of new therapeutic entities. © 1994 Wiley-Liss, Inc.     

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     

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.     

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.     

Quantitative analysis of vascular to cardiac selectivity of L- and T-type voltage-operated calcium channel antagonists in human tissues

1. Classical L-type voltage-operated calcium channel (VOCC) antagonists dilate blood vessels, depress myocardial contractility and slow cardiac conduction. 2. We compared four L-type VOCC antagonists and a novel tetralol derivative, mibefradil, reportedly 10-fold more selective for T- (transient) over L-type VOCC in two in vitro assays of human tissue, namely isolated small arteries from the aortic vasa vasorum in a myograph and right atrial trabeculae muscle under isometric force conditions. 3. In arteries contracted with K+ (62 mmol/L), the relaxation pIC50 values for the VOCC antagonists felodipine, nifedipine, amlodipine, verapamil and mibefradil were 8.30, 7.78, 6.64, 6.26 and 6.22, respectively. In atrial trabeculae, the pIC50 values to inhibit the inotropic response to a submaximal concentration of isoprenaline (6 nmol/L) for felodipine, nifedipine, verapamil, amlodipine and mibefradil were 7.21, 6.95, 6.91, 5.94 and 4.61, respectively. 4. Taking the anti-log (pIC50 vessel-pIC50 atrium) the vascular relaxation to cardiac depression potency ratios for mibefradil, felodipine, nifedipine, amlodipine and verapamil were 41, 12, 7, 5 and 0.22, respectively. 5. We conclude that, in human tissue assays, perhaps T- over L-type VOCC selectivity confers the most favourable vascular selectivity on mibefradil. Alternatively, splice variants of L-type VOCC in the vasculature (CaV1.2b) may be more sensitive to mibefradil than the splice variants in the heart (CaV1.2a).     

Combination of calcium channel blockers and beta-blockers for patients with exercise-induced angina pectoris: beneficial effect of calcium channel blockers largely determined by their effect on heart rate.

The combination of calcium channel blockers and beta-blockers is more effective for the treatment of exercise-induced angina pectoris than beta-blocker monotherapy. As ischemia in exercise-induced angina is essentially preceded by an increase in heart rate, calcium channel blockers with a negative chronotropic property may perform better for this purpose than nonchronotropic compounds. A 335-patient, 10-week, double-blind, parallel-group comparison of amlodipine 5 mg and 10 mg, diltiazem 200 mg and 300 mg, and mibefradil 50 mg and 100 mg treatment added to baseline beta-blocker treatment was performed. Exercise testing (ETT) was performed by bicycle ergometry. All of the calcium channels blockers significantly delayed the onset of 1 mm ST-segment depression on ETT (p < 0.001 for any treatment vs. baseline). In addition, mibefradil, in both low- and high-dose treatments, produced the largest delays (low dose: different from diltiazem and amlodipine by 24.1 and 29.8 seconds, respectively, p < 0.003 and < 0.001; high dose: different from diltiazem and amlodipine by 33.7 and 37.0 seconds, respectively, p < 0.001 and < 0.001). A stepwise logistic regression analysis revealed that this beneficial effect of calcium channel blockers was largely dependent on their effect on heart rate. Serious symptoms of dizziness likewise occurred significantly more frequently on mibefradil (p < 0.05 vs. diltiazem) and urged no fewer than 19 patients on mibefradil to withdraw from the trial. The authors conclude that calcium channel blockers with a negative chronotropic property provide a better delay of ischemia in patients with exercise-induced angina, but the concomitant risk of intolerable dizziness may reduce this benefit.     

Combination of calcium channel blockers and β-adrenoceptor blockers for patients with exercise-induced angina pectoris: a double-blind parallel-group comparison of different classes of calcium channel blockers

AIMS: The combination of calcium channel blockers and beta-adrenoceptor blockers is more effective for the treatment of exercise-induced angina pectoris than beta-adrenoceptor blocker monotherapy. As ischaemia in exercise-induced angina is preceded by increase in heart rate, calcium channel blockers with negative chronotropic properties may perform better for this purpose than nonchronotropic compounds. METHODS: A 335 patient double-blind parallel-group study comparing 14 day treatment with amlodipine 5 and 10 mg, with diltiazem 200 and 300 mg, and mibefradil 50 and 100 mg added to baseline beta-adrenoceptor blocker treatment was performed. Exercise testing (ETT) was performed by bicycle ergometry. RESULTS: Although none of the calcium channel blockers improved duration of exercise or amount of workload, all significantly delayed onset of 1 mm ST-segment depression on ETT (P<0.001 for any treatment vs baseline). In addition, mibefradil, both low and high dose treatment, produced the longest delays (low dose: different from diltiazem and amlodipine by 24.1 and 29.8 s, respectively, P<0. 003 and <0.001; high dose: different from diltiazem and amlodipine by 33.7 and 37.0 s, respectively, P<0.001 and <0.001). These effects were linearly correlated with the reduction in rate pressure product (RPP). Serious symptoms of dizziness occurred significantly more frequently on mibefradil (P<0.05), and 19 patients on mibefradil withdrew from trial. CONCLUSIONS: Calcium channel blockers with negative chronotropic properties provide greater delay of ischaemia in patients with exercise-induced angina, but the concomitant risk of intolerable dizziness attenuates this benefit.     

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.     

Cardiomyocyte Ca<Superscript>2+</Superscript> overload in atrial tachycardia: is blockade of L-type Ca<Superscript>2+</Superscript> channels a promising approach to prevent electrical remodeling and arrhythmogenesis?

Electrical remodeling paradigm has important implications for the understanding of atrial fibrillation (AF) and improvement of current treatment. Cardiomyocyte Ca²⁺ overload is generally accepted as the initiating signal for the tachycardia-induced changes in atrial electrical properties (electrical remodeling). The precise role of cardiomyocyte Ca²⁺ overload in AF-related ion channel alterations that contribute to AF maintenance is not fully understood. Clinically, patients with AF are often treated with Ca²⁺ channel blockers such as verapamil to control their ventricular rate and to improve the success rate of cardioversion procedures. However, verapamil may produce an increased L-type Ca²⁺ channel current (I_Ca,L) that may reinforce Ca²⁺ overload thereby promoting AF in the atrium. Ca²⁺ channel blockers which target T-type Ca²⁺ channels in addition to I_Ca,L (for instance, efonidipine) may be more efficient at preventing Ca²⁺ overload and arrhythmogenic electrical remodeling, but the potential benefits of these drugs have usually been tested in experimental models where drug administration preceded the initiation of electrical remodeling. Studies in animal models with established atrial tachycardia remodeling and in patients with AF are clearly warranted to prove the efficacy of Ca²⁺ channel blockers that additionally target T-type Ca²⁺ channels.     

T-type calcium channel blockers: a patent review (2012–2018)

ABSTRACT

Introduction: T-type calcium channels are attractive targets for potential treatment of epilepsy inflammatory or neuropathic pain, insomnia, Parkinson’s disease, and cancer. Three isoforms having different biophysical functions are expressed in peripheral and central nerve. Since the withdrawal of mibefradil, the first compound marketed for selective T-type calcium channel blockade, extensive efforts have been made to identify more selective T-type calcium channel blockers.

Areas covered: This review covers the 43 patents describing ‘organic small molecules as T-type calcium channel blockers’-published since 2012. The most recent similar patent review was published in 2011. Information from a recent review article and relevant research papers has been included, as well as biological data and clinical trial results where available.

Expert opinion: Triazinone derivatives, carbazole compounds, and aryl triazole/imidazole amide derivatives display potent blockade activity α1H, α1G, and pan T-type calcium channel subtypes, respectively, though the specificity of the letter is still unsatisfactory. Nonetheless, improvements seen in the efficacy of compounds targeting α1H T-type calcium channels indicate significant progress. Ongoing clinical trials are for the candidates Z944 (Phase II) and ACT-709478 (Phase II) appear promising. These studies may lead to a new generation of inhibitors with higher selectivity, improved physicochemical properties, and reduced side effects.     

Role of T-type Ca2+ Channels in the Spontaneous Phasic Contraction of Pregnant Rat Uterine Smooth Muscle

Although extracellular Ca²⁺ entry through the voltage-dependent Ca²⁺ channels plays an important role in the spontaneous phasic contractions of the pregnant rat myometrium, the role of the T-type Ca²⁺ channels has yet to be fully identified. The aim of this study was to investigate the role of the T-type Ca²⁺ channel in the spontaneous phasic contractions of the rat myometrium. Spontaneous phasic contractions and [Ca²⁺]_i were measured simultaneously in the longitudinal strips of female Sprague-Dawley rats late in their pregnancy (on day 18~20 of gestation: term=22 days). The expression of T-type Ca²⁺ channel mRNAs or protein levels was measured. Cumulative addition of low concentrations (<1 µM) of nifedipine, a L-type Ca²⁺ channel blocker, produced a decrease in the amplitude of the spontaneous Ca²⁺ transients and contractions with no significant change in frequency. The mRNAs and proteins encoding two subunits (α1G, α1H) of the T-type Ca²⁺ channels were expressed in longitudinal muscle layer of rat myometrium. Cumulative addition of mibefradil, NNC 55-0396 or nickel induced a concentration-dependent inhibition of the amplitude and frequency of the spontaneous Ca²⁺ transients and contractions. Mibefradil, NNC 55-0396 or nickel also attenuated the slope of rising phase of spontaneous Ca²⁺ transients consistent with the reduction of the frequency. It is concluded that T-type Ca²⁺ channels are expressed in the pregnant rat myometrium and may play a key role for the regulation of the frequency of spontaneous phasic contractions.     

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.     

Effects of T-type calcium channel blockers and thalidomide on contractions of rat vas deferens

Background and purpose:

In rat vas deferens, nerve mediated-contractions to a single electrical stimulus consist of an early purinergic and a later adrenergic component with differing sensitivities to L-type calcium channel blockers. We have investigated the effects of the T-type calcium channel blockers mibefradil and (1S, 2S)-2-[2-[[3-(1H-benzimidazol-2-yl)propyl]methylamino]ethyl]-6-fluoro-1,2,3,4-tetrahydro-1-(1-methylethyl)-2-naphthalenyl cyclopropanecarboxylic dihydrochloride (NNC 55-0396) against contractions in rat vas deferens. In addition, the actions of thalidomide were examined.

Experimental approach:

Prostatic and epididymal portions of rat vas deferens were stimulated with a single electrical stimulus every 5 min, and mouse whole vas deferens was stimulated with 40 pulses at 10 Hz every 5 min.

Key results:

Both mibefradil and NNC 55-0396 (100 µM) produced inhibition of contractions of epididymal portions (42 ± 13%, n= 7, and 43 ± 4%, n= 15, of control respectively). However, both agents produced small inhibitions of responses in prostatic portions, presumably by L-type calcium channel block. Thalidomide (100 µM) inhibited contractions in epididymal (55 ± 4% of control, n= 17) but not in prostatic portions of rat vas deferens. Thalidomide (10–100 µM) also inhibited contractions in mouse vas deferens.

Conclusions and implications:

The T-type calcium channel blockers mibefradil and NNC 55-0396 block particularly the adrenoceptor-mediated, nifedipine-resistant response to nerve stimulation in rat vas deferens, and this may suggest that this component involves T-type calcium channels. In addition, thalidomide has actions that resemble those of the T-type calcium channel blockers, in that it blocks nifedipine-resistant contractions in epididymal portions.     

T-type and L-type calcium channel blockers exert opposite effects on renin secretion and renin gene expression in conscious rats

1. This study aimed to investigate and to compare the effects of pharmacological T-type calcium channel and of L-type calcium channel blockade on the renin system. To this end, male healthy Sprague-Dawley rats were treated with the T-channel blocker mibefradil or with the L-channel blocker amlodipine at doses of 5 mg kg⁻¹, 15 mg kg⁻¹ and 45 mg kg⁻¹ per day for four days and their effects on plasma renin activity (PRA) and kidney renin mRNA levels were determined.
2. Whilst amlodipine lowered basal systolic blood pressure at 5 mg kg⁻¹, mibefradil had no effect on basal blood pressure in the whole dose range examined. Amlodipine dose-dependently induced up to 7 fold elevation of PRA and renin mRNA levels. Mibefradil significantly lowered PRA and renin mRNA levels at 5 mg kg⁻¹ and moderately increased both parameters at a dose of 45 mg kg⁻¹, when PRA and renin mRNA levels were increased by 100% and 30%, respectively. In primary cultures of renal juxtaglomerular cells neither amlodipine nor mibefradil (0.1–10 μM) changed renin secretion.
3. In rats unilateral renal artery clips (2K-1C) mibefradil and amlodipine at doses of 15 mg kg⁻¹ day⁻¹ were equally effective in lowering blood pressure. In contrast mibefradil (5 mg kg⁻¹ and 15 mg  kg⁻¹ day⁻¹) significantly attenuated the rise of PRA and renin mRNA levels, whilst amlodipine (15 mg kg⁻¹) additionally elevated the rise of PRA and renin mRNA levels in response to renal artery clipping.
4. These findings suggest that T-type calcium channel blockers can inhibit renin secretion and renin gene expression in vivo, whilst L-type calcium channel blockers act as stimulators of the renin system. Since the inhibitory effect of T-type antagonists is apparent in vivo but not in vitro, one may infer that the effect on the renin system is indirect rather than directly mediated at the level of renal juxtaglomerular cells.

     

Voltage and pH dependent block of cloned N-type Ca2+ channels by amlodipine

1. Two types of Ca²⁺ channel α₁-subunits were co-expressed in Xenopus oocytes with the Ca²⁺ channel α₂- and β₁-subunits. The Ba²⁺ current through the α_1Cα₂β and the α_1Bα₂β channels had electrophysiological and pharmacological properties of L- and N-type Ca²⁺ channels, respectively.
2. Amlodipine had a strong blocking action on both the L-type and N-type Ca²⁺ channels expressed in the oocyte. The potency of the amlodipine block on the N-type Ca²⁺ channel was comparable to that on the L-type Ca²⁺ channel. At −100 mV holding potential, the IC₅₀ values for amlodipine block on the L-type and N-type Ca²⁺ channel were 2.4 and 5.8 μM, respectively.
3. The blocking action of amlodipine on the N-type Ca²⁺ channel was dependent on holding potential and extracellular pH, as has been observed with amlodipine block on the L-type Ca²⁺ channel. A depolarized holding potential and high pH enhanced the blocking action of amlodipine.
4. The time course of block development by amlodipine was similar for L-type and N-type Ca²⁺ channels. However, it was slower than the time course of block development by nifedipine for the L-type Ca²⁺ channel.

     

Changes of cardiac calcium homeostasis in spontaneously hypertensive rats

1 The aim of the present study was to assess the alterations in cardiac Ca²⁺ homeostasis induced by hypertension using electrically paced right ventricular strips from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR). 2 Basal contractile force was higher in SHR than in WKY. Similarly, the β-adrenoceptor agonist isoprenaline (10 n m –10 μm ) induced a concentration-dependent positive inotropic effect that was higher in SHR than in WKY, which was in turn inhibited by the β-adrenoceptor antagonist propranolol (1 μm ) in both strains. 3 Preincubation of strips with the L-type Ca²⁺ channel blockers, nifedipine (1 μm ) or verapamil (10 μm ), markedly inhibited the isoprenaline response, the inhibition being higher in SHR than in WKY. However, this inhibition was minor by the T-type Ca²⁺ channel blocker mibefradil (10 μm ). 4 Bay K 8644 (10 n m –10 μm ), a L-type Ca²⁺ channel activator induced a concentration-dependent positive inotropic effect, that was greater in SHR than WKY. 5 Nifedipine and verapamil (both 0.1 n m –10 μm ) inhibited in a concentration-dependent way the inotropic effect induced by 0.3 μm isoprenaline or 1 μm Bay K 8644. The inhibition was higher in SHR than in WKY. Mibefradil (0.1 n m –10 μm ) only clearly inhibited the isoprenaline and Bay K 8644 inotropic effects at 10 μm in both strains. 6 The inhibitor of the sarcoplasmic reticulum Ca²⁺ release, ryanodine (10 n m –10 μm ), was a more effective depressor of isoprenaline-induced response in SHR than in WKY. 7 These results suggest that cardiac Ca²⁺ homeostasis in SHR ventricular strips is altered compared with those of WKY, showing an increased Ca²⁺ entry through L-type Ca²⁺ channels and release from sarcoplasmic reticulum; the participation of T-type Ca²⁺ channels are irrelevant in this tissue.     

State-dependent verapamil block of the cloned human Ca(v)3.1 T-type Ca(2+) channel

Verapamil is a potent phenylalkylamine antihypertensive believed to exert its therapeutic effect primarily by blocking high-voltage-activated L-type calcium channels. It was the first clinically used calcium channel blocker and remains in clinical use, although it has been eclipsed by other calcium channel blockers because of its short half-life and interactions with other channels. In addition to blocking L-type channels, it has been reported to block T-type (low-voltage activated) calcium channels. This type of cross-reactivity is likely to be beneficial in the effective control of blood pressure. Although the interactions of T channels with a number of drugs have been described, the mechanisms by which these agents modulate channel activity are largely unknown. Most calcium channel blockers exhibit state-dependence (i.e., preferential binding to certain channel conformations), but little is known about state-dependent verapamil block of T channels. We stably expressed human Ca(v)3.1 T-type channels in human embryonic kidney 293 cells and studied the state-dependence of the drug with macroscopic and gating currents. Verapamil blocked currents at micromolar concentrations at polarized potentials similar to those reported for L-type channels, although unlike for L-type currents, it did not affect current time course. The drug exhibited use-dependence and significantly slowed the apparent recovery from inactivation. Current inhibition was dependent on potential. This dependence was restricted to negative potentials, although all data were consistent with verapamil binding in the pore. Gating currents were unaffected by verapamil. We propose that verapamil achieves its inhibitory effect via occlusion of the channel pore associated with an open/inactivated conformation of the channel.