Comparison of mibefradil and derivative NNC 55-0396 effects on behavior, cytochrome P450 activity, and tremor in mouse models of essential tremor

NNC 55-0396 [(1S,2S)-2-(2-(N-[(3-benzimidazol-2-yl)propyl]-N-methylamino)ethyl)-6-fluoro-1,2, 3,4-tetrahydro-1-isopropyl-2-naphtyl cyclopropanecarboxylate dihydrochloride], is a mibefradil derivative that retains potent in vitro T-type calcium channel antagonist efficacy. We compared the two compounds for behavioral toxicity, effects on cytochrome P450 activity, and efficacy against tremor in the γ-aminobutyric acid type A (GABA_A) receptor subunit α1-null mouse, and the harmaline tremor model of essential tremor in wild-type mice. NNC 55-0396 was better tolerated than mibefradil in the horizontal wire test of sedation/motor function, with 3/6 failing at 300 and 30 mg/kg respectively. To assess for a potential interaction with harmaline, mice were given the drugs, followed by harmaline or vehicle, and tested 30 min later in the inverted wire grid test. Mibefradil exacerbated, whereas NNC 55-0396 ameliorated harmaline-induced test deficits. In mouse liver microsomes, NNC 55-0396 was a less potent inhibitor of harmaline O-demethylation than mibefradil (K_i: 0.95 and 0.29 μM respectively), and also less potent at inhibiting testosterone 6-β-hydroxylation (K_i: 0.71 and 0.12 μM respectively). In the GABA_A α1-null model, NNC 55-0396 but not mibefradil, (each at 20 mg/kg), suppressed tremor while NNC 55-0396 at 12.5 mg/kg suppressed harmaline-induced tremor by half by 20–100 min, whereas mibefradil at the same dose did not significantly affect tremor. In contrast to mibefradil, NNC 55-0396 is well tolerated and suppresses tremor, and exerts less cytochrome P450 inhibition. These results suggest potential clinical utility for NNC 55-0396 or similar derivatives as a T-type calcium antagonist.     

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.     

T-type calcium channel antagonists suppress tremor in two mouse models of essential tremor

Essential tremor is a common disorder that lacks molecular targets for therapeutic development. T-type calcium channel activation has been postulated to underlie rhythmicity in the olivo-cerebellar system that is implicated in essential tremor. We therefore tested whether compounds that antagonize T-type calcium channel currents suppress tremor in two mouse models that possess an essential tremor-like pharmacological response profile. Tremor was measured using digitized spectral motion power analysis with harmaline-induced tremor and in the GABA_A receptor α1 subunit-null model. Mice were given ethosuximide, zonisamide, the neuroactive steroid (3β,5α,17β)-17-hydroxyestrane-3-carbonitrile (ECN), the 3,4-dihydroquinazoline derivative KYS05064, the mibefradil derivative NNC 55-0396, or vehicle. In non-sedating doses, each compound reduced harmaline-induced tremor by at least 50% (range of maximal suppression: 53-81%), and in the GABA_A α1-null model by at least 70% (range 70-93%). Because the T-type calcium channel Cav3.1 is the dominant subtype expressed in the inferior olive, we assessed the tremor response of Cav3.1-deficient mice to harmaline, and found that null and heterozygote mice exhibit as much tremor as wild-type mice. In addition, ECN and NNC 55-0396 suppressed harmaline tremor as well in Cav3.1-null mice as in wild-type mice. The finding that five T-type calcium antagonists suppress tremor in two animal tremor models suggests that T-type calcium channels may be an appropriate target for essential tremor therapy development. It is uncertain whether medications developed to block only the Cav3.1 subtype would exhibit efficacy.     

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.     

Colonic hydrogen sulfide-induced visceral pain and referred hyperalgesia involve activation of both Ca(v)3.2 and TRPA1 channels in mice

Luminal hydrogen sulfide (H(2)S), a gasotransmitter, causes colonic pain / referred hyperalgesia in mice, most probably via activation of T-type Ca(2+) channels. Here we analyzed the mechanisms for H(2)S-induced facilitation of colonic pain signals. Intracolonic administration of NaHS, an H(2)S donor, evoked visceral pain-like nociceptive behavior and referred hyperalgesia in mice, an effect abolished by NNC 55-0396, a selective T-type Ca(2+)-channel blocker, or by knockdown of Ca(v)3.2. AP18, a TRPA1 blocker, also prevented the NaHS-induced colonic pain and referred hyperalgesia. These findings demonstrate that H(2)S-induced colonic pain and referred hyperalgesia require activation of both Ca(v)3.2 and TRPA1 channels in mice.     

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.     

Inhibition of T-type and L-type calcium channels by mibefradil: physiologic and pharmacologic bases of cardiovascular effects

Ca2+ channel antagonists of the dihydropyridine, benzothiazepine, and phenylalkylamine classes have selective effects on L-type versus T-type Ca2+ channels. In contrast, mibefradil was reported to be more selective for T-type channels. We used the whole-cell patch-clamp technique to investigate the effects of mibefradil on T-type and L-type Ca2+ currents (I(CaT) and I(CaL)) recorded at physiologic extracellular Ca2+ in different cardiac cell types. At a stimulation rate of 0.1 Hz, mibefradil blocked I(CaT) evoked from negative holding potentials (HPs) (-100 mV to -80 mV) with an IC50 of 0.1 microM in rat atrial cells. This concentration had no effect on I(CaL) in rat ventricular cells (IC50: approximately3 microM). However, block of I(CaL) was enhanced when the HP was depolarized to -50 mV (IC50: approximately 0.1 microM). Besides a resting block, mibefradil displayed voltage- and use-dependent effects on both I(CaT) and I(CaL). In addition, inhibition was enhanced by increasing the duration of the step-depolarizations. Similar effects were observed in human atrial and rabbit sinoatrial cells. In conclusion, mibefradil combines the voltage- and use-dependent effects of dihydropyridines and benzothiazepines on I(CaL). Inhibition of I(CaL), which has probably been underestimated before, may contribute to most of the cardiovascular effects of mibefradil.     

Involvement of the endogenous hydrogen sulfide/Ca(v) 3.2 T-type Ca(2+) channel pathway in cystitis-related bladder pain in mice

BACKGROUND AND PURPOSE Hydrogen sulfide (H(2) S), generated by enzymes such as cystathionine-γ-lyase (CSE) from L-cysteine, facilitates pain signals by activating the Ca(v) 3.2 T-type Ca(2+) channels. Here, we assessed the involvement of the CSE/H(2) S/Ca(v) 3.2 pathway in cystitis-related bladder pain. EXPERIMENTAL APPROACH Cystitis was induced by i.p. administration of cyclophosphamide in mice. Bladder pain-like nociceptive behaviour was observed and referred hyperalgesia was evaluated using von Frey filaments. Phosphorylation of ERK in the spinal dorsal horn was determined immunohistochemically following intravesical administration of NaHS, an H(2) S donor. KEY RESULTS Cyclophosphamide caused cystitis-related symptoms including increased bladder weight, accompanied by nociceptive changes (bladder pain-like nociceptive behaviour and referred hyperalgesia). Pretreatment with DL-propargylglycine, an inhibitor of CSE, abolished the nociceptive changes and partly prevented the increased bladder weight. CSE protein in the bladder was markedly up-regulated during development of cystitis. Mibefradil or NNC 55-0396, blockers of T-type Ca(2+) channels, administered after the symptoms of cystitis appeared, reversed the nociceptive changes. Further, silencing of Ca(v) 3.2 protein by repeated intrathecal administration of mouse Ca(v) 3.2-targeting antisense oligodeoxynucleotides also significantly attenuated the nociceptive changes, but not the increased bladder weight. Finally, the number of cells staining positive for phospho-ERK was increased in the superficial layer of the L6 spinal cord after intravesical administration of NaHS, an effect inhibited by NNC 55-0396. CONCLUSION AND IMPLICATIONS Endogenous H(2) S, generated by up-regulated CSE, caused bladder pain and referred hyperalgesia through the activation of Ca(v) 3.2 channels, one of the T-type Ca(2+) channels, in mice with cyclophosphamide-induced cystitis.     

Pharmacological properties of Cav3.2, a low voltage-activated Ca2+ channel cloned from human heart

Three genes encoding T-type Ca2+ channels have been described but their correspondence to the various native T-type Ca2+ currents remains uncertain. In particular, Ca(V)3.2 (or alpha1H) was cloned from a human heart library, its message was found abundantly in cardiac tissue, and expressed Ca(V)3.2 was shown to conduct low voltage-activated currents, which inactivate rapidly and are sensitive to Ni2+ and mibefradil. These observations suggested that Ca(V)3.2 might encode native cardiac T-type Ca2+ channels but more information on the pharmacology of Ca(V)3.2 was needed to confirm this hypothesis. In the present study, we compare the pharmacology of Ca(V)3.2 expressed in HEK293 cells and of native T-type Ca2+ channels in guinea pig atrial myocytes ("native-T"). (1) Ca(V)3.2 and native-T are insensitive to TTX and to toxins selective for N-, P-, or Q-type Ca2+ channels (omega-CTx-GVIA, omega-Aga-IVA, omega-CTx-MVIIC). (2) The half-maximal blocking concentration (IC50) of mibefradil on Ca(V)3.2 is near that on native-T and the block is similarly voltage-dependent. (3) Ca(V)3.2 is five- to sixfold less sensitive than native-T to the 1,4-dihydropyridine (DHP) amlodipine, suggesting a difference in the DHP binding site. (4) Both channels display similar (but not identical) sensitivities to the inorganic blockers Ni2+ and Cd2+ and the IC50s are in the range of values found for T-type Ca2+ currents in other cell types. (5) Ni2+ shifts the voltage dependence of Ca(V)3.2 activation but not that of native-T. The many similarities between the two channels support the contention that Ca(V)3.2 encodes cardiac T-type Ca2+ channels. The slight differences may be due to species variations and/or to the choice of splice variant.     

Effects of T-type calcium channel blockers on a parkinsonian tremor model in rats

T-type calcium channels are strongly associated with the generation of rhythmic firing patterns in the CNS. Blockers of these channels may have therapeutic potential for treating various types of tremor. The present study aimed to study the effects of a range of T-type calcium channel blockers in a parkinsonian tremor model in rats. We tested the effects of several T-type calcium channel blockers, including zonisamide (ZNS), ethosuximide, lomerizine, amiloride, mibefradil, and NCC 55-0396, a mibefradil derivative, on tacrine-induced tremulous jaw movements (TJMs), an animal model of parkinsonian tremor. Among the tested drugs, only ZNS and NCC 55-0396 significantly suppressed TJMs when given at a non-sedating dose. The transitivity of drugs to the central nervous system (CNS) may at least partially explain their differential anti-TJM effects. However, further studies are necessary to reveal other factors, since ethosuximide failed to show anti-TJM effects despite being known to cross the blood brain barrier. The present results suggest that T-type calcium channels in the CNS may be a suitable target for developing new therapeutic strategies for treating parkinsonian tremor.     

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.     

Mibefradil potently blocks ATP-activated K(+) channels in adrenal cells

Mibefradil is a novel Ca(2+) channel antagonist that preferentially blocks T-type Ca(2+) channels in many cells. Using whole-cell and single-channel patch-clamp recording, we found that mibefradil also potently blocked an ATP-activated K(+) channel (I(AC)) expressed by adrenal zona fasciculata cells. I(AC) channels were inhibited by mibefradil with an IC(50) value of 0.50 microM, a concentration 2-fold lower than that required to inhibit T-type Ca(2+) channels under similar conditions in the same cells. The inhibition of I(AC) by mibefradil was independent of the membrane potential. Mibefradil also reversibly blocked, with similar potency, unitary I(AC) currents recorded in outside-out membrane patches. An analysis of dwell time histograms indicated the presence of two closed and one open state. Mibefradil (1 microM) increased the duration of the two closed time constants (tau(c1) and tau(c2)) from 2.30 +/- 0.18 and 27.9 +/- 4.7 ms to 4.32 +/- 0.61 and 62.5 +/- 13.8 ms, respectively, but did not alter the open time constant (tau(o)). Mibefradil also failed to reduce the size of the unitary I(AC) current. A voltage-gated A-type K(+) current was also inhibited by mibefradil at concentrations approximately 10-fold higher than those required to block I(AC) (IC(50) = 4.65 microM). These results identify mibefradil as a potent inhibitor of ATP-activated K(+) channels in adrenal zona fasciculata cells. It appears to function by stabilizing closed states of these channels. In contrast to its selective block of T-type Ca(2+) channels, mibefradil may be a potent but less-selective K(+) channel blocker. In this regard, the block of K(+) channels may produce some of the toxicity associated with mibefradil in cardiovascular pharmacology.     

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.     

State-dependent mibefradil block of Na+ channels

Mibefradil is a T-type Ca2+ channel antagonist with reported cross-reactivity with other classes of ion channels, including K+, Cl-, and Na+ channels. Using whole-cell voltage clamp, we examined mibefradil block of four Na+ channel isoforms expressed in human embryonic kidney cells: Nav1.5 (cardiac), Nav1.4 (skeletal muscle), Nav1.2 (brain), and Nav1.7 (peripheral nerve). Mibefradil blocked Nav1.5 in a use/frequency-dependent manner, indicating preferential binding to states visited during depolarization. Mibefradil blocked currents of all Na+ channel isoforms with similar affinity and a dependence on holding potential, and drug off-rate was slowed at depolarized potentials (k(off) was 0.024/s at -130 mV and 0.007/s at -100 mV for Nav1.5). We further probed the interaction of mibefradil with inactivated Nav1.5 channels. Neither the degree nor the time course of block was dependent on the stimulus duration, which dramatically changed the residency time of channels in the fast-inactivated state. In addition, inhibiting the binding of the fast inactivation lid (Nav1.5 ICM + MTSET) did not alter mibefradil block, confirming that the drug does not preferentially interact with the fast-inactivated state. We also tested whether mibefradil interacted with slow-inactivated state(s). When selectively applied to channels after inducing slow inactivation with a 60-s pulse to -10 mV, mibefradil (1 microM) produced 45% fractional block in Nav1.5 and greater block (88%) in an isoform (Nav1.4) that slow-inactivates more completely. Our results suggest that mibefradil blocks Na+ channels in a state-dependent manner that does not depend on fast inactivation but probably involves interaction with one or more slow-inactivated state(s).     

Ca(v)3.2 channel is a molecular substrate for inhibition of T-type calcium currents in rat sensory neurons by nitrous oxide

Although nitrous oxide (N(2)O; laughing gas) remains widely used as an anesthetic and analgesic in clinical practice, its cellular mechanisms of action remain inadequately understood. In this report, we examined the effects of N(2)O on voltage-gated Ca(2+) channels in acutely dissociated small sensory neurons of adult rat. At subanesthetic concentrations, N(2)O blocks low-voltage-activated, T-type Ca(2+) currents (T currents), but not high-voltage-activated (HVA) currents. This blockade of T currents was concentration dependent, with an IC(50) value of 45 +/- 13%, maximal block of 38 +/- 12%, and Hill coefficient of 2.6 +/- 1.0. No desensitization of the response or change in current kinetics was observed during N(2)O application. The magnitude of T current blockade by N(2)O does not seem to reflect any use- or voltage-dependent properties. In addition, T current blockade was not altered when intracellular GTP was replaced with guanosine 5'-(gamma-thio)triphosphate or guanosine 5'-0-(2-thiodiphosphate) suggesting a lack of involvement of G-proteins in the inhibition. N(2)O selectively blocked currents arising from the Ca(v)3.2 but not Ca(v)3.1 recombinant channels stably expressed in human embryonic kidney (HEK) cells in a concentration-dependent manner with an apparent affinity and potency similar to native dorsal root ganglion currents. Analogously, the block of Ca(v)3.2 T currents exhibited little voltage- or use-dependence. These data indicate that N(2)O selectively blocks T-type but not HVA Ca(2+) currents in small sensory neurons and Ca(v)3.2 currents in HEK cells at subanesthetic concentrations. Blockade of T currents may contribute to the anesthetic and/or analgesic effects of N(2)O.     

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.     

Potent inhibition of a recombinant low voltage-activated Ca(2+) channel by SB-209712

T-type Ca(2+) currents were recorded in 2 mM Ca(2+) from HEK293 cells stably expressing the low voltage-activated Ca(2+) channel sub-unit alpha(1I). These currents were inhibited by the known Ca(2+) channel antagonist mibefradil with an IC(50) close to 1 microM. SB-209712 (1,6,bis?1-[4-(3-phenylpropyl)piperidinyl]?hexane), a compound originally developed as a high voltage-activated Ca(2+) channel blocker, proved to be a more potent T-type channel antagonist, exhibiting an IC(50) in the region of 500 nM. The antagonism produced by SB-209712 was reversed following drug removal and the observed antagonism exhibited little or no voltage-dependence with respect to either holding or test potential. These data indicate that SB-209712 is amongst the most potent known non-peptide T-type channel antagonists and thus may have some use in understanding the role of these channels in cellular function.     

Mibefradil (Ro 40-5967) inhibits several Ca2+ and K+ currents in human fusion-competent myoblasts

1. The effect of mibefradil (Ro 40-5967), an inhibitor of T-type Ca2+ current (I(Ca)(T)), on myoblast fusion and on several voltage-gated currents expressed by fusion-competent myoblasts was examined. 2. At a concentration of 5 microM, mibefradil decreases myoblast fusion by 57%. At this concentration, the peak amplitudes of I(Ca)(T) and L-type Ca2+ current (I(Ca)(L)) measured in fusion-competent myoblasts are reduced by 95 and 80%, respectively. The IC50 of mibefradil for I(Ca)(T) and I(Ca)(L) are 0.7 and 2 microM, respectively. 3. At low concentrations, mibefradil increased the amplitude of I(Ca)(L) with respect to control. 4. Mibefradil blocked three voltage-gated K+ currents expressed by human fusion-competent myoblasts: a delayed rectifier K+ current, an ether-à-go-go K+ current, and an inward rectifier K+ current, with a respective IC50 of 0.3, 0.7 and 5.6 microM. 5. It is concluded that mibefradil can interfere with myoblast fusion, a mechanism fundamental to muscle growth and repair, and that the interpretation of the effect of mibefradil in a given system should take into account the action of this drug on ionic currents other than Ca2+ currents.     

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.     

Inhibition of recombinant low-voltage-activated Ca(2+) channels by the neuroprotective agent BW619C89 (Sipatrigine)

T-type Ca(2+) currents were recorded in 2 mM Ca(2+) from HEK 293 cells stably expressing recombinant low-voltage-activated Ca(2+) channel subunits. Current-voltage relationships revealed that these currents were low-voltage activated in nature and could be reversibly antagonised by mibefradil, a known T-type channel blocker. At a test potential of -25 mV alpha(1I)-mediated Ca(2+) currents were rapidly and reversibly inhibited by 1-100 microM BW619C89 (IC(50)=14 microM, Hill coefficient 1.3). In contrast to its actions on N-type Ca(2+) channels, a near IC(50) dose (10 microM) of BW619C89 produced no alterations in either the kinetics or voltage-dependence of T-type currents. In additional single dose experiments, currents mediated by rat alpha(1G), human alpha(1H) or human alpha(1I) channel subunits were also inhibited by BW619C89. Overall our data indicate that T-type Ca(2+) channels are more potently blocked by BW619C89 than either type-II Na(+) channels or N-type Ca(2+) channels. It seems, therefore, that inhibition of low-voltage-activated Ca(2+) channels is likely to contribute to the anticonvulsant and neuroprotective actions of this and related compounds.