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]: a new selective inhibitor of T-type calcium channels

Mibefradil is a Ca2+ channel antagonist that inhibits both T-type and high-voltage-activated Ca2+ channels. We previously showed that block of high-voltage-activated channels by mibefradil occurs through the production of an active metabolite by intracellular hydrolysis. In the present study, we modified the structure of mibefradil to develop a nonhydrolyzable analog, (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 (NNC 55-0396), that exerts a selective inhibitory effect on T-type channels. The acute IC(50) of NNC 55-0396 to block recombinant alpha(1)G T-type channels in human embryonic kidney 293 cells was approximately 7 microM, whereas 100 microM NNC 55-0396 had no detectable effect on high-voltage-activated channels in INS-1 cells. NNC 55-0396 did not affect the voltage-dependent activation of T-type Ca2+ currents but changed the slope of the steady-state inactivation curve. Block of T-type Ca2+ current was partially relieved by membrane hyperpolarization and enhanced at a high-stimulus frequency. Washing NNC 55-0396 out of the recording chamber did not reverse the T-type Ca2+ current activity, suggesting that the compound dissolves in or passes through the plasma membrane to exert its effect; however, intracellular perfusion of the compound did not block T-type Ca2+ currents, arguing against a cytoplasmic route of action. After incubating cells from an insulin-secreting cell line (INS-1) with NNC 55-0396 for 20 min, mass spectrometry did not detect the mibefradil metabolite that causes L-type Ca2+ channel inhibition. We conclude that NNC 55-0396, by virtue of its modified structure, does not produce the metabolite that causes inhibition of L-type Ca2+ channels, thus rendering it more selective to T-type Ca2+ channels.     

Mibefradil block of cloned T-type calcium channels

Mibefradil is a tetralol derivative chemically distinct from other calcium channel antagonists. It is a very effective antihypertensive agent that is thought to achieve its action via a higher affinity block for low-voltage-activated (T) than for high-voltage-activated (L) calcium channels. Estimates of affinity using Ba(2+) as the charge carrier have predicted a 10- to 15-fold preference of mibefradil for T channels over L channels. However, T channel IC(50) values are reported to be approximately 1 microM, which is much higher than expected for clinical efficacy because relevant blood levels of this drug are approximately 50 nM. We compared the affinity for mibefradil of the newly cloned T channel isoforms, alpha1G, alpha1H, and alpha1I with an L channel, alpha1C. In 10 mM Ba(2+), mibefradil blocked in the micromolar range and with 12- to 13-fold greater affinity for T channels than for L channels (approximately 1 microM versus 13 microM). When 2 mM Ca(2+) was used as the charge carrier, the drug was more efficacious; the IC(50) for alpha1G shifted to 270 nM and for alpha1H shifted to 140 nM, 4.5- and 9-fold higher affinity than in 10 mM Ba. The data are consistent with the idea that mibefradil competes for its binding site on the channel with the permeant species and that Ba(2+) is a more effective competitor than Ca(2+). Raising temperature to 35 degrees C reduced affinity (IC(50) 792 nM). Reducing channel availability to half increased affinity ( approximately 70 nM). This profile of mibefradil affinity makes these channels good candidates for the physiological target of this antihypertensive agent.     

Mechanism of myricetin stimulation of vascular L-type Ca2+ current

An in-depth analysis of the mechanism of the L-type Ca(2+) current [I(Ca(L))] stimulation induced by myricetin was performed in rat tail artery myocytes using the whole-cell patch-clamp method. Myricetin increased I(Ca(L)) in a frequency-, concentration-, and voltage-dependent manner. At holding potentials (V(h)) of -50 and -90 mV, the pEC(50) values were 4.9 +/- 0.1 and 4.2 +/- 0.1, respectively; the latter corresponded to the drug-apparent dissociation constant for resting channels, K(R), of 67.6 microM. Myricetin shifted the maximum of the current-voltage relationship by 10 mV in the hyperpolarizing direction but did not modify the threshold for I(Ca(L)) or the T-type Ca(2+) current. The Ca(2+) channel blockers nifedipine, verapamil, and diltiazem antagonized I(Ca(L)) in the presence of myricetin. Myricetin increased the time to peak of I(Ca(L)) in a voltage- and concentration-dependent manner. Washout reverted myricetin effect on both current kinetics and amplitude at V(h) of -90 mV while reverting only current kinetics at V(h) of -50 mV. At the latter V(h), myricetin shifted the voltage dependence of inactivation and activation curves to more negative potentials by 6.4 and 13.0 mV, respectively, in the mid-potential of the curves. At V(h) of -90 mV, myricetin shifted, in a concentration-dependent manner, the voltage dependence of the inactivation curve to more negative potentials with an apparent dissociation constant for inactivated channels (K(I)) of 13.8 muM. Myricetin induced a frequency- and V(h)-dependent block of I(Ca(L)). In conclusion, myricetin behaves as an L-type Ca(2+) channel agonist that stabilizes the channel in its inactivated state.     

A mibefradil metabolite is a potent intracellular blocker of L-type Ca(2+) currents in pancreatic beta-cells

It has been shown that mibefradil (Ro 40-5967) exerts a selective inhibitory effect on T-type Ca(2+) currents, although at higher concentrations it can antagonize high voltage-activated Ca(2+) currents. The action of mibefradil on Ca(2+) channels is use- and steady-state-dependent and the binding site of mibefradil on L-type Ca(2+) channels is different from that of dihydropyridines. By using conventional whole-cell and perforated patch-clamp techniques, we showed that mibefradil has an inhibitory effect on both T- and L-type Ca(2+) currents in insulin-secreting cells. However, the effect on L-type Ca(2+) currents was time-dependent and poorly reversible in perforated patch-clamp experiments. By using mass spectrometry, we demonstrated that mibefradil accumulates inside cells, and furthermore, a metabolite of mibefradil was detected. Intracellular application of this metabolite selectively blocked the L-type Ca(2+) current, whereas mibefradil exerted no effect. This study demonstrates that mibefradil permeates into cells and is hydrolyzed to a metabolite that blocks L-type Ca(2+) channels specifically by acting at the inner side of the channel.     

Characterization of mibefradil block of the human heart delayed rectifier hKv1.5

The goal of this study was to analyze the effects of mibefradil on a human cardiac K(+) channel (hKv1.5) stably expressed in Chinese hamster ovary cells using the whole-cell configuration of the patch-clamp technique. Mibefradil inhibited in a concentration-dependent manner the hKv1.5 current with a K(D) value of 0.78 +/- 0.05 microM and a Hill coefficient of 0.97 +/- 0.06. Block induced by mibefradil was voltage dependent, consistent with a value of electrical distance of 0.13. The apparent association (k) and dissociation (l) rate constants measured at +50 mV were found to be 7.3 +/- 0.5 x 10(6) M(-1).s(-1) and 4.3 +/- 0.1 s(-1), respectively. Block increased rapidly between -20 and +10 mV, coincident with channel opening and suggested an open channel block mechanism, which was confirmed by a slower deactivation time course resulting in a "crossover" phenomenon when tail currents recorded under control conditions and in the presence of mibefradil were superimposed. Shifts toward negative potentials of the maximum conductance and the activation curve were observed, confirming the voltage dependence of block. Mibefradil induced a significant use-dependent block when trains of depolarization at frequencies between 0.02 and 2 Hz were applied. In the presence of mibefradil, recovery of inactivation was faster than under control conditions, suggesting that mibefradil might compete with the inactivation gate of hKv1.5. These results indicate that mibefradil blocks hKv1.5 channels in a concentration-, voltage-, time- and use-dependent manner and the concentrations needed to observe these effects are in the therapeutic range.     

Differential effects of mibefradil, verapamil, and amlodipine on myocardial function and intracellular Ca(2+) handling in rats with chronic myocardial infarction

Mibefradil is a selective T-type Ca(2+) channel blocker that exerts a potent vasodilating but weak inotropic action. The present study compared mibefradil with traditional L-type Ca(2+) channel blockers in regard to the effects of chronic oral administration on hemodynamics, contractility, and intracellular Ca(2+) handling in failing myocardium from postinfarction rats. Male Wistar rats with ligation-induced myocardial infarction were assigned to placebo or treatment with mibefradil (10 mg/kg/day), verapamil (8 mg/kg/day), or amlodipine (4 mg/kg/day) by oral gavage starting 7 days before the induction of myocardial infarction. Six weeks after myocardial infarction, hemodynamic measurements were performed in conscious animals. In addition, isometric force and free [Ca(2+)](i) were determined in isolated left ventricular papillary muscles. Placebo-treated rats exhibited a decreased mean atrial pressure, an increased left ventricular end-diastolic pressure, and a reduced rate of pressure rise compared with sham-operated animals. Mibefradil treatment significantly improved all of these parameters, whereas both amlodipine and verapamil exerted only minor effects. beta-Adrenergic stimulation with isoproterenol (ISO) enhanced contractility and Ca(2+) availability in papillary muscles from sham-operated rats, whereas the ISO-induced inotropic effect in muscles from placebo-treated rats was severely blunted. Chronic mibefradil treatment significantly improved the inotropic response to ISO stimulation, although the Ca(2+)(i) availability appeared to be less than in muscles from placebo-treated animals. In contrast, both verapamil and amlodipine did not restore the inotropic and Ca(2+)(i) modulating effect of ISO in remodeled myocardium. Thus, T-type Ca(2+) current appears to be of pathophysiological relevance in postischemic reperfused myocardium.     

Potent inhibition of native TREK-1 K+ channels by selected dihydropyridine Ca2+ channel antagonists

Bovine adrenal zona fasciculata (AZF) cells express bTREK-1 background K+ channels that set the resting membrane potential. Whole-cell and single-channel patch-clamp recording were used to compare five Ca2+ channel antagonists with respect to their potency as inhibitors of native bTREK-1 K+ channels. The dihydropyridine (DHP) Ca2+ channel antagonists amlodipine and niguldipine potently and specifically inhibited bTREK-1 with IC50 values of 0.43 and 0.75 microM, respectively. The other Ca2+ channel antagonists, including the DHP nifedipine, the diphenyldiperazine flunarizine, and the cannabinoid anandamide were less potent, with IC50 values of 8.18, 2.48, and 5.07 microM, respectively. Additional studies with the highly prescribed antihypertensive amlodipine showed that inhibition of bTREK-1 by this agent was voltage-independent and specific. At concentrations that produced near complete block of bTREK-1, amlodipine inhibited voltage-gated Kv1.4 K+ and T-type Ca2+ currents in AZF cells by less than 10%. At the single-channel level, amlodipine reduced bTREK-1 open probability without altering the unitary conductance. The results demonstrate that selected DHP L-type Ca2+ channel antagonists potently inhibit native bTREK-1 K+ channels, whereas other Ca2+ channel antagonists also inhibit bTREK-1 at higher concentrations. Collectively, organic Ca2+ channel antagonists make up the most potent class of TREK-1 inhibitors yet described. Because TREK-1 K+ channels are widely expressed in the central nervous and cardiovascular systems, it is possible that some of the therapeutic or toxic effects of frequently prescribed drugs such as amlodipine may be due to their interaction with TREK-1 K+ rather L-type Ca2+ channels.     

Reversal of experimental neuropathic pain by T-type calcium channel blockers

Experimental nerve injury results in exaggerated responses to tactile and thermal stimuli that resemble some aspects of human neuropathic pain. Neuronal hyperexcitability and neurotransmitter release have been suggested to promote such increased responses to sensory stimuli. Enhanced activity of Ca(2+) current is associated with increased neuronal activity and blockade of N- and P-types, but not L-type, calcium channels have been found to block experimental neuropathic pain. While T-type currents are believed to promote neuronal excitability and transmitter release, it is unclear whether these channels may also contribute to the neuropathic state. Rats were prepared with L(5)/L(6) spinal nerve ligation, and tactile and thermal hypersensitivities were established. Mibefradil or ethosuximide was administered either intraperitoneally, intrathecally (i.th.), or locally into the plantar aspect of the injured hindpaw. Systemic mibefradil or ethosuximide produced a dose-dependent blockade of both tactile and thermal hypersensitivities in nerve-injured rats; responses of sham-operated rats were unchanged. Local injection of mibefradil also blocked both end points. Ethosuximide, however, was inactive after local administration, perhaps reflecting its low potency when compared with mibefradil. Neither mibefradil nor ethosuximide given i.th. produced any blockade of neuropathic behaviors. The results presented here suggest that T-type calcium channels may play a role in the expression of the neuropathic state. The data support the view that selective T-type calcium channel blockers may have significant potential in the treatment of neuropathic pain states.     

Actions of two main metabolites of propiverine (M-1 and M-2) on voltage-dependent L-type Ca2+ currents and Ca2+ transients in murine urinary bladder myocytes

The anticholinergic propiverine (1-methyl-4-piperidyl diphenylpropoxyacetate), which is used for the treatment of overactive bladder syndrome, has functionally active metabolites [M-1 (1-methyl-4-piperidyl diphenylpropoxyacetate N-oxide) and M-2 (1-methyl-4-piperidyl benzilate N-oxide)], but the site of actions of these metabolites is uncertain. Propiverine is rapidly absorbed after oral administration and is extensively biotransformed in the liver, giving rise to several active metabolites (M-1 and M-2). This study determines the effect of M-1 and M-2 on voltage-dependent nifedipine-sensitive inward Ca(2+) currents (I(Ca)) using patch-clamp techniques and fluorescent Ca(2+) imaging [after electrical field stimulation (EFS) and acetylcholine (ACh)] in the murine urinary bladder. In conventional whole-cell recording, propiverine and M-1 but not M-2 inhibited the peak amplitude of I(Ca) in a concentration-dependent manner at a holding potential of -60 mV (propiverine, K(i) = 10 microM; M-1, K(i) = 118 microM). M-1 shifted the steady-state inactivation curve of I(Ca) to the left at -90 mV by 7 mV. Carbachol (CCh) reversibly inhibited I(Ca). This inhibition probably occurred through muscarinic type 3 receptors, coupling with G-proteins, because nanomolar concentrations of 4-diphenylacetoxy-N-methyl-piperidine greatly reduced this inhibition, whereas pirenzepine or 11-([2-[(diethylamino)methyl]-1-piperdinyl]acetyl)-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one (AF-DX 116) at concentrations up to 1 microM was almost ineffective. In the presence of M-2, the CCh-induced inhibition of I(Ca) was blocked. In fluorescent Ca(2+) imaging, M-2 inhibited EFS-induced and ACh-induced Ca(2+) transients. These results suggest that M-1 acts, at least in part, as a Ca(2+) channel antagonist (as it inhibited I(Ca)), whereas M-2 has more direct antimuscarinic actions.     

Molecular pharmacology of human Cav3.2 T-type Ca2+ channels: block by antihypertensives, antiarrhythmics, and their analogs

Antihypertensive drugs of the "calcium channel blocker" or "calcium antagonist" class have been used to establish the physiological role of L-type Ca(2+) channels in vascular smooth muscle. In contrast, there has been limited progress on the pharmacology T-type Ca(2+) channels. T-type channels play a role in cardiac pacemaking, aldosterone secretion, and renal hemodynamics, leading to the hypothesis that mixed T- and L-type blockers may have therapeutic advantages over selective L-type blockers. The goal of this study was to identify compounds that block the Ca(v)3.2 T-type channel with high affinity, focusing on two classes of compounds: phenylalkylamines (e.g., mibefradil) and dihydropyridines (e.g., efonidipine). Compounds were tested using a validated Ca(2+) influx assay into a cell line expressing recombinant Ca(v)3.2 channels. This study identified four clinically approved antihypertensive drugs (efonidipine, felodipine, isradipine, and nitrendipine) as potent T-channel blockers (IC(50) < 3 microM). In contrast, other widely prescribed dihydropyridines, such as amlodipine and nifedipine, were 10-fold less potent, making them a more appropriate choice in research studies on the role of L-type currents. In summary, the present results support the notion that many available antihypertensive drugs block a substantial fraction of T-current at therapeutically relevant concentrations, contributing to their mechanism of action.     

Augmentation of Cav3.2 T-type calcium channel activity by cAMP-dependent protein kinase A

Ca2+ influx through T-type Ca2+ channels is crucial for important physiological activities such as hormone secretion and neuronal excitability. However, it is not clear whether these channels are regulated by cAMP-dependent protein kinase A (PKA). In the present study, we examined whether PKA modulates Cav3.2 T-type channels reconstituted in Xenopus oocytes. Application of 10 microM forskolin, an adenylyl cyclase stimulant, increased Cav3.2 channel activity by 40+/-4% over 30 min and negatively shifted the steady-state inactivation curve (V50=-61.4+/-0.2 versus -65.5+/-0.1 mV). Forskolin did not affect other biophysical properties of Cav3.2 channels, including activation curve, current kinetics, and recovery from inactivation. Similar stimulation was achieved by applying 200 microM 8-bromo-cAMP, a membrane-permeable cAMP analog. The augmentation of Cav3.2 channel activity by forskolin was strongly inhibited by preincubation with 20 microM N-[2-(4-bromocinnamylamino)ethyl]-5-isoquinoline (H89), and reversed by subsequent application of 500 nM protein kinase A inhibitor peptide. The stimulation of Cav3.2 channel activity by PKA was mimicked by serotonin when 5HT7 receptor was coexpressed with Cav3.2 in Xenopus oocytes. Finally, using chimeric channels constructed by replacing individual cytoplasmic loops of Cav3.2 with those of the Nav1.4 channel, which is insensitive to PKA, we localized a region required for the PKA-mediated augmentation to the II-III loop of the Cav3.2.     

Hydrogen sulfide as a novel nociceptive messenger

Hydrogen sulfide (H(2)S), an endogenous gasotransmitter, modulates various biological events such as inflammation in the mammalian body. The present study investigated possible involvement of H(2)S in peripheral nociceptive processing. Intraplantar (i.pl.) administration of NaHS, a H(2)S donor, produced prompt hyperalgesia in rats, accompanied by expression of Fos in the spinal dorsal horn. The H(2)S-evoked hyperalgesia was blocked by 5,5'-dithio-bis-(2-nitrobenzoic acid) (DTNB), an oxidizing agent, or ethosuximide and mibefradil, T-type Ca(2+) channel inhibitors. L-Cysteine, an endogenous source for H(2)S, given i.pl., also elicited hyperalgesia, an effect being abolished by DL-propargylglycine (PPG) and beta-cyanoalanine (BCA), inhibitors of cystathionine-gamma-lyase, a H(2)S synthesizing enzyme. PPG and/or BCA partially inhibited the hyperalgesia induced by i.pl. lipopolysaccharide, an effect being reversed by i.pl. NaHS. In the patch-clamp study using undifferentiated NG108-15 cells that express T-type, but not other types, of Ca(2+) channels, NaHS enhanced the currents through the T-type channels, an effect being blocked by DTNB. Thus, H(2)S appears to function as a novel nociceptive messenger through sensitization of T-type Ca(2+) channels in the peripheral tissues, particularly during inflammation.     

Inhibition of volume-regulated and calcium-activated chloride channels by the antimalarial mefloquine

We have used the whole-cell patch-clamp technique to study the effect of mefloquine (Lariam), a commonly used antimalarial drug, on the volume-regulated anion channel (VRAC) in cultured bovine pulmonary artery endothelial cells. We also examined its effects on other Cl(-) channels, i.e., the Ca(2+)-activated Cl(-) channel and the cystic fibrosis transmembrane conductance regulator, to assess the specificity of this compound for VRAC. At pH 7.4 mefloquine induced a fast and reversible block of the volume-sensitive chloride current (I(Cl,swell)), with an IC(50) value of 1.19 +/- 0.07 microM. The blocking efficiency increased with increasing extracellular pH (IC(50) value for pH 8.8 was 0.15 +/- 0.01 microM), indicating that this effect is mediated by the uncharged form of mefloquine. Ca(2+)-activated Cl(-) currents, I(Cl,Ca), activated by loading T84 cells via the patch pipette with 1 microM free Ca(2+) also were inhibited by mefloquine (IC(50) value 3.01 +/- 0.17 microM at pH 7.4). The cystic fibrosis transmembrane conductance regulator channel, transiently transfected in cultured bovine pulmonary artery endothelial cells, was not affected by 10 microM of the drug. This study describes for the first time effects of mefloquine on anion channels. Our data reveal a potent block of VRAC and Ca(2+)-activated Cl(-) channel at therapeutic concentrations. These results may contribute to a better understanding of the actions and side effects of this widely used antimalarial drug.     

Comparison of L-type calcium channel blockade by nifedipine and/or cadmium in guinea pig ventricular myocytes

We tested the assumption that nifedipine blocks L-type calcium current [I(Ca(L))] at +10 mV and unmasks Na(+)/Ca(2+) exchange-triggered contractions in guinea pig isolated ventricular myocytes. Voltage-clamp pulses elicited I(Ca(L)) at +10 mV and evoked contractions in myocytes superfused with Tyrode's solution (35 degrees C). Nifedipine blocked I(Ca(L)) with an IC(50) of 0.3 microM; this decreased to 50 nM at a holding potential of -40 mV, indicating preferential block of inactivated L-type Ca(2+) channels. Use-independent block of I(Ca(L)) increased with concentration (10-100 microM) and application time when nifedipine was rapidly applied (t(1/2) = approximately 0.2 s) during rest intervals (5-30 s). The fraction of use-dependent block of I(Ca(L)) diminished with increasing drug concentration. Nifedipine also accelerated I(Ca(L)) inactivation on the first test pulse. The combination of 30 microM nifedipine/30 microM Cd(2+) (Nif 30/Cd 30) was as effective as 100 microM nifedipine to suppress I(Ca(L)) on the first test pulse at +10 mV. The incidence of complete block of contractions, as for complete block of I(Ca(L)), increased as a function of nifedipine concentration and application time. Neither nifedipine nor Nif 30/Cd 30 affected Na(+)/Ca(2+) exchange current at +10 to +100 mV. Contractions at +100 mV, although as large as those at +10 mV, were delayed in onset and resistant to nifedipine or Nif 30/Cd 30. We conclude that nifedipine-sensitive I(Ca(L)) triggers contractions at +10 mV, whereas nifedipine-resistant Na(+)/Ca(2+) exchange current initiates those at +100 mV.     

Propafenone modulates potassium channel activities of vascular smooth muscle from rat portal veins

We have studied the effects of the class Ic antiarrhythmic propafenone on K+ currents in freshly isolated smooth muscle cells from rat portal veins and on the spontaneous contractions in whole tissues. Under Ca2+-free conditions, when cells were clamped at -80 mV (whole-cell configuration) depolarizing steps from -80 to +50 mV induced a family of K+ currents (I(Ktotal)) that mainly comprised the delayed rectifier current [I(K(V))], whereas when held at -10 mV only small-amplitude, noninactivating, currents (I(NI)) were recorded. Propafenone (10 microM) markedly inhibited I(Ktotal), but at potentials positive to +30 mV it also induced a noisy outwardly rectifying current [I(BK(Ca))] that was abolished by iberiotoxin (0.1 microM). Inhibition of I(Ktotal) by propafenone was concentration-dependent (EC50 = 0.059 +/- 0.009 microM). Propafenone also inhibited the transient outward current [I(K(A))] and ATP-sensitive potassium current [I(K(ATP))] induced by levcromakalim (10 microM). Inhibition of I(K(V)), I(K(A)), and I(K(ATP)) by propafenone was voltage-independent. In Ca(2+)-containing conditions propafenone inhibited I(K(V)) and I(BK(Ca)) and immediately abolished spontaneous outward transient K+ currents. In whole veins, propafenone behaved as the K(V) inhibitor 4-aminopyridine, increasing the amplitude and duration of spontaneous contractions. Propafenone also inhibited the inhibitory effects of the K(ATP) channel opener levcromakalim on spontaneous contractions. These results indicate that in vascular smooth muscle cells, propafenone inhibits K(V), K(A), BK(Ca), and K(ATP) channels. These actions correlated with its effects on mechanical activity in whole portal veins.     

Two structurally different T-type Ca 2+ channel inhibitors, mibefradil and pimozide, protect CA1 neurons from delayed death after global ischemia in rats

Recent in vitro evidence suggests that T-type Ca(2+) channels are implicated in the mechanisms of ischemia-induced delayed neuronal cell death. The aim of this work was to study the neuroprotective potential of mibefradil and pimozide, both T-type Ca(2+) channel inhibitors, in an in vivo rat model of global ischemia. We performed blinded and randomized placebo vs. treatment experiments using 57 animals to test mibefradil and fourteen animals to test pimozide. Each treated animal received a single stereotactic intraventricular injection of mibefradil or intraperitoneal injection of pimozide prior to transient global cerebral ischemia. The primary endpoint was the number of neurons surviving in the CA1 region 72 h after insult as evaluated by NeuN-labeled cell counts. All physiological variables monitored immediately before and after ischemic insult were equivalent between all groups. Surviving neurons in the CA1 region were significantly more frequent in the treated groups compared to the placebo group (mibefradil: 36.8 ± 2.8 cells in a 200 × 100 μm counting area vs. placebo: 25.2 ± 3.2 [P < 0.01]; pimozide: 39.4 ± 1.12 vs. placebo: 27.8 ± 0.7 [P < 0.0001]). Thus, administration of mibefradil or pimozide effectively prevents neuronal death after ischemia in a rat model of global ischemia. This study provides further support for a neuroprotective effect of T-type Ca(2+) current inhibition during ischemia.     

Inorganic mercury and methylmercury inhibit the Cav3.1 channel expressed in human embryonic kidney 293 cells by different mechanisms

Part of the neurotoxic effects of inorganic mercury (Hg(2+)) and methylmercury (MeHg) was attributed to their interaction with voltage-activated calcium channels. Effects of mercury on T-type calcium channels are controversial. Therefore, we investigated effects of Hg(2+) and MeHg on neuronal Ca(v)3.1 (T-type) calcium channel stably expressed in the human embryonic kidney (HEK) 293 cell line. Hg(2+) acutely inhibited current through the Ca(v)3.1 calcium channel in concentrations 10 nM and higher with an IC(50) of 0.63 +/- 0.11 microM and a Hill coefficient of 0.73 +/- 0.08. Inhibition was accompanied by strong deceleration of current activation, inactivation, and deactivation. The current-voltage relation was broadened, and its peak was shifted to a more depolarized membrane potentials by 1 microM Hg(2+). MeHg in concentrations between 10 nM and 100 microM inhibited the current through the Ca(v)3.1 calcium channel with an IC(50) of 13.0 +/- 5.0 microM and a Hill coefficient of 0.47 +/- 0.09. Low concentration of MeHg (10 pM to 1 nM) had both positive and negative effects on the current amplitude. Micromolar concentrations of MeHg reduced the speed of current activation and accelerated current inactivation and deactivation. The current-voltage relation was not affected. Up to 72 h of exposure to 10 nM MeHg had no significant effect on current amplitude, whereas 72-h-long exposure to 1 nM MeHg increased significantly current density. Acute treatment with Hg(2+) or MeHg did not affect HEK 293 cell viability. In conclusion, interaction with the Ca(v)3.1 calcium channel may significantly contribute to neuronal symptoms of mercury poisoning during both acute poisoning and long-term environmental exposure.     

Effects of methylmercury on human neuronal L-type calcium channels transiently expressed in human embryonic kidney cells (HEK-293)

Methylmercury (MeHg) disrupts the function of native, high voltage-activated neuronal Ca(2+) channels in several types of cells. However, the effects of MeHg on isolated Ca(2+) channel phenotypes have not been examined. The aim of the present study was to examine the action of MeHg on recombinant, neuronal L-type voltage-sensitive Ca(2+) channels. Human embryonic kidney cells (HEK-293) were transfected with human neuronal cDNA clones of the alpha(1C-1) subunit in combination with alpha(2b) and beta(3a) Ca(2+) channel subunits and the reporter jellyfish green fluorescent protein for transient expression. Current from expressed channels (I(Ba)) and their response to MeHg applied acutely were measured using whole-cell voltage-clamp recording techniques and Ba(2+) (5 mM) as charge carrier. Amplitude of I(Ba) in these cells was reduced by the dihydropyridine (DHP), nimodipine, and enhanced by Bay K8644 [S-(-)-1,4-dihydro-2,6-dimethyl-5-nitro-4-(2-[trifluoromethyl]phenyl)-3 pyridine carboxylic acid methyl ester]. MeHg (0.125-5.0 microM) caused a time- and concentration-dependent reduction in amplitude of the peak and sustained current through these channels. However, even at the highest concentration of MeHg tested, reduction of current amplitude by MeHg was incomplete. Washing with MeHg-free solution could not reverse its effects. The steady-state inactivation curve was unaltered by MeHg. Increasing the stimulation frequency or the extracellular Ba(2+) concentration each attenuated slightly the reduction in amplitude of I(Ba) by MeHg. In the presence of MeHg (5.0 microM), Bay K8644 still increased the remaining current, and nimodipine (10 microM) reduced residual current that was resistant to MeHg. Thus, although MeHg reduces the amplitude of recombinant, heterologously expressed L-type channel current, a portion of current is resistant to reduction by MeHg. Furthermore, DHP agonists and antagonists retain their ability to affect L-type Ca(2+) channel current even in the presence of MeHg.     

Regulation of membrane chloride currents in rat bile duct epithelial cells.

This study examines the conductive properties of the plasma membrane of cells isolated from the intrahepatic portion of bile ducts. Membrane Cl- conductance was measured in single cells using whole-cell patch clamp recording techniques and in cells in short-term culture using 36Cl and 125I efflux. Separate Ca(2+)- and cAMP-dependent Cl- currents were identified. Ca(2+)-dependent Cl- currents showed outward rectification of the current-voltage relation, time-dependent activation at depolarizing potentials, and reversal near the equilibrium potential for Cl-. Ionomycin (2 microM) increased this current from 357 +/- 72 pA to 1,192 +/- 414 pA (at +80 mV) in 5:7 cells, and stimulated efflux of 125I > 36Cl in 15:15 studies. Ionomycin-stimulated efflux was inhibited by the Cl- channel blocker 4,4'-diisothiocyano-2,2'-stilbene disulfonic acid (DIDS) (150 microM). A separate cAMP-activated Cl- current showed linear current-voltage relations and no time dependence. Forskolin (10 microM) or cpt-cAMP (500 microM) increased this current from 189 +/- 50 pA to 784 +/- 196 pA (at +80 mV) in 11:16 cells, and stimulated efflux of 36Cl > 125I in 16:16 studies. cAMP-stimulated efflux was unaffected by DIDS. Because the cAMP-stimulated Cl- conductance resembles that associated with cystic fibrosis transmembrane conductance regulator (CFTR), a putative Cl- channel protein, the presence of CFTR in rat liver was examined by immunoblot analyses. CFTR was detected as a 150-165-kD protein in specimens with increased numbers of duct cells. Immunoperoxidase staining confirmed localization of CFTR to bile duct cells but not hepatocytes. These findings suggest that Ca(2+)- and cAMP-regulated Cl- channels may participate in control of fluid and electrolyte secretion by intrahepatic bile duct epithelial cells, and that the cAMP-regulated conductance is associated with endogenous expression of CFTR. Abnormal ductular secretion may contribute to the pathogenesis of cholestatic liver disease in cystic fibrosis.     

Imatinib-mesylate blocks recombinant T-type calcium channels expressed in human embryonic kidney-293 cells by a protein tyrosine kinase-independent mechanism

The 2-phenylaminopyrimidine derivative imatinib-mesylate, a powerful protein tyrosine kinase (PTK) inhibitor that targets abl, c-kit, and the platelet-derived growth factor receptors, is rapidly gaining a relevant role in the treatment of several types of neoplasms. Because first generation PTK inhibitors affect the activity of a large number of voltage-dependent ion channels, the present study explored the possibility that imatinib-mesylate could interfere with the activity of T-type channels, a class of voltage-dependent Ca2+ channels that take part in the chain of events elicited by PTK activation. The effect of the drug on T-type channel activity was examined using the whole-cell patch-clamp technique with Ba2+ (10 mM) as the permeant ion in human embryonic kidney-293 cells, stably expressing the rat Ca(V)3.3 channels. Imatinib-mesylate concentrations, ranging from 30 to 300 microM, reversibly decreased Ca(V)3.3 current amplitude with an IC(50) value of 56.9 microM. By contrast, when imatinib-mesylate (500 microM) was intracellularly dialyzed with the pipette solution, no reduction in Ba2+ current density was observed. The 2-phenylaminopyrimidine derivative modified neither the voltage dependence of activation nor the steady-state inactivation of Ca(V)3.3 channels. The decrease in extracellular Ba2+ concentration from 10 to 2 mM and the substitution of Ca2+ for Ba2+ increased the extent of 30 microM imatinib-mesylate-induced percentage of channel blockade from 25.9 +/- 2.4 to 36.3 +/- 0.9% in 2 mM Ba2+ and 44.2 +/- 2.3% in 2 mM Ca2+. In conclusion, imatinib-mesylate blocked the cloned Ca(V)3.3 channels by a PTK-independent mechanism. Specifically, the drug did not affect the activation or the inactivation of the channel but interfered with the ion permeation process.