Inhibitory Effects of Calcium Channel Antagonists on Motor Dysfunction Induced by Intracerebroventricular Administration of Paraquat

Abstract: This study reports the effects of Ca²⁺ channel blockers (Ca antagonists) on intraneuronal Ca²⁺ ([Ca²⁺]_i) movements and on the disturbance of rotarod performance produced in rats by intracerebroventricular administration of paraquat. Paraquat (50 nmol) produced a decrement in rotarod performance which was present at 30 min. and maximal at 60 min. and was not associated with overt behavioural changes; larger doses of paraquat (100-400 nmol intracerebroventricularly) produced paresis and convulsions which severely disrupted rotarod behaviour. The disruption of rotarod performance after paraquat (50 nmol intracerebroventricularly) was significantly reduced by giving Ca antagonists (flunarizine, verapamil and nicardipine) not only intraperitoneally 15 min. after paraquat but also intracerebroventricularly immediately before paraquat. The order of pharmacological potency was flunarizine ≥ verapamil > nicardipine. In contrast, intracerebroventricular administration of Bay K 8644, a Ca agonist, enhanced the disruption of rotarod performance caused by paraquat (50 nmol). In in vitro studies, paraquat markedly potentiated the rapid increase in [Ca²⁺]_i levels evoked by 50 mM KCl in rat brain synaptosomal fraction, although paraquat alone produced a small prolonged rise in [Ca²⁺]_i levels which had a slow onset. The above results suggest that paraquat induced neurotoxicity is associated with increased [Ca²⁺]_i levels in brain neuronal cells, and that paraquat might effect on membrane activity instability.     

Direct inhibition,but indirect sensitization of pacemaker activity to sympathetic tone by the interaction of endotoxin with HCN‐channels

In critically ill patients regulation of heart‐rate is often severely disturbed. Interaction of bacterial endotoxin (lipopolysaccharide, LPS) with hyperpolarization‐activated cyclic nucleotide‐gated cation‐(HCN)‐channels may interfere with heart‐rate regulation. This study analyzes the effect of LPS, the HCN‐channel blocker ivabradine or Ca²⁺‐channel blockers (nifedipine, verapamil) on pacemaking in spontaneously beating neonatal rat cardiomyocytes (CM) in vitro. In vivo, the effect of LPS on the heart‐rate of adult CD1‐mice with and without autonomic blockade is analyzed telemetrically. LPS (100 ng/mL) and ivabradine (5 μg/mL) reduced the beating‐rate of CM by 20.1% and 24.6%, respectively. Coincubation of CM with both, LPS and ivabradine, did not further reduce the beating‐rate, indicating interaction of both compounds with HCN‐channels, while coincubation with Ca²⁺‐channel blockers and LPS caused additive beating‐rate reduction. In CD1‐mice (containing an active autonomic‐nervous‐system), injection of LPS (0.4 mg/kg) expectedly resulted in increased heart‐rate. However, if the autonomic nervous system was blocked by propranolol and atropine, in line with the in vitro data, LPS induced a significant reduction of heart‐rate, which was not additive to ivabradine. The in vivo and in vitro results indicate that LPS interacts with HCN‐channels of cardiomyocytes. Thus, LPS indirectly sensitizes HCN‐channels for sympathetic activation (tachycardic‐effect), and in parallel directly inhibits channel activity (bradycardic‐effect). Both effects may contribute to the detrimental effects of septic cardiomyopathy and septic autonomic dysfunction.     

An examination of the anticonvulsant properties of voltage-sensitive calcium channel inhibitors in amygdala kindled seizures

Voltage-sensitive calcium (VSC) channels may contribute to epileptogenesis. A systematic examination of the anticonvulsant efficacy of different classes of VSC channel inhibitors, however, is lacking in chronic seizure models. The present study evaluated representatives from three different classes of VSC channel inhibitors for their protection against amygdala kindled seizures. Adult male rats (n=12) were kindled to stage 5 seizures (GS), and a threshold intensity required to evoke a GS was determined. The Ca⁺⁺-channel inhibitors (verapamil 0, 10, 20, 40 mg/kg; nimodipine 0, 5, 25, 50 mg/kg; nitrendipine 0, 25, 50, 100 mg/kg and flunarizine 0, 20, 40, 80 mg/kg) were administered 60–90 min prior to amygdala stimulation at the established threshold. None of the drugs altered threshold for inducing a seizure. Verapamil, a phenylalkylamine, and the dihydropyridines nimodipine and nitrendipine were without effect on kindled seizures. The diphenylalkylamine, flunarizine, was found to be the most efficacious, reducing AD duration and duration of clonic seizure activity by more than 60% in most animals. Flunarizine also decreased the severity of behavioral seizures, with 40% of the animals displaying Stage 1–2 seizures only. It is concluded that some VSC Ca⁺⁺-channel inhibitors do possess anticonvulsant potential. Thus influx of extracellular calcium through VSC channels may contribute to the expression of kindled seizures.Although the research described in this article has been supported by the United States Environmental Protection Agency (through contract 68-02-4450 to Mantech Environmental Technology Incorp.), it has not been subjected to Agency review and therefore does not necessarily reflect the views of the Agency and no official endorsement should be inferred. Mention of tradenames or commercial products does not constitute endorsement or recommendation for use.     

Effects of flunarizine on spontaneous synaptic currents in rat neocortex

Flunarizine, a non-selective blocker of voltage-dependent Ca²⁺ and Na⁺ channels, is clinically effective against several neurological disorders, including epilepsy, migraine, and alternating hemiplegia of childhood. We examined the effects of flunarizine on spontaneous post-synaptic currents in acute brain slices maintained in vitro using patch-clamp electrophysiology. Flunarizine significantly attenuated the amplitude of spontaneous currents in pyramidal neurons from juvenile rat neocortex. Flunarizine had no effect on miniature spontaneous events recorded in the presence of tetrodotoxin, a blocker of voltage-dependent sodium channels. In high (9 mM) extracellular potassium, flunarizine reduced the amplitude and frequency of the spontaneous currents. Additionally, dimethyl sulfoxide, the solvent used in our experiments, reduced the amplitude of spontaneous currents, but only in high extracellular potassium. Our data suggest that the clinical activity of flunarizine may in part be a consequence of reducing spontaneous synaptic currents in the neocortex, especially under conditions of heightened neuronal activity.     

Effects of Terbutaline on α-Adrenergic Responses and Ca2+ Influx in Isolated Rabbit Aorta

Effects of terbutaline applied in vivo or in vitro on α-adrenergic receptors in the rabbit aorta in normal and Ca²⁺–free solution, and on basal, high potassium-, and phenylephrine-stimulated Ca²⁺ uptake into aorta were investigated. Three day terbutaline administration (25 mg/kg, subcutaneously three times daily) to rabbits increased the pK_B for phentolamine in aorta rings (control 7.3 + 0.2, n = 9; terbutaline 7.8 +0.2, n = 15). It also depressed phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free but not those in normal Krebs solution. It did not significantly depress the basal, or phenylephrine-evoked Ca²⁺ influx into aorta rings, but decreased high potassium-induced Ca²⁺–influx (control 0.58 + 0.05 umoles/g aorta; n = 3, terbutaline 0.41 +0.06 umoles/g aorta, n = 3). In vitro application of 50 μM terbutaline did not significantly alter phenylephrine-stimulated contractions of aorta rings in Ca²⁺–free Krebs solution or significantly depress basal or phenylephrine-induced Ca²⁺ influx into aortas, but did decrease high potassium-stimulated Ca²⁺–influx. Thus, 3-day terbutaline administration increased the affinity of α-adrenergic receptors for phentolamine and had a tendency to increase contractions of aorta rings to phenylephrine. It also decreased high potassium-stimulated Ca²⁺ influx, and depressed phenylephrine-induced contractions in Ca²⁺–free Krebs solution, while in vitro terbutaline application also decreased potassium-induced Ca²⁺ influx.     

Matrine compromises mouse sperm functions by a [Ca2+]i-related mechanism

Matrine, a bioactive alkaloid widely used in Chinese medicine, inhibits mouse sperm functions in vitro. In this study, we investigated the reproductive toxicity of matrine to male mice in vivo. C57BL/6J mice were administered with daily doses of 0, 1, 10 and 50 mg/kg matrine by intraperitoneal injection for 30 days. The results showed that matrine did not affect testis size, testis weight, sperm count and sperm viability, but it significantly inhibited total motility, progressive motility, linear velocity, capacitation and the progesterone-induced acrosome reaction of mouse sperm. Furthermore, the intracellular Ca²⁺ concentration ([Ca²⁺]_i), a key regulator of sperm function, was reduced in sperm of matrine-exposed mice. The current and gene expression of the sperm specific Ca²⁺ channel, CatSper, which modulates Ca²⁺ influx in sperm, were decreased in testes of matrine-exposed mice. These results indicate that matrine inhibits mouse sperm functions by a [Ca²⁺]_i-related mechanism via CatSper channel.     

Atypical antipsychotic profile of flunarizine in animal models

Rationale Flunarizine is known as a calcium channel blocker commonly used in many countries to treat migraine and vertigo. Parkinsonism has been described as one of its side-effects in the elderly, which is in agreement with its recently characterized moderate D₂ receptor antagonism.Objectives To perform a pre-clinical evaluation of flunarizine as a potential antipsychotic.Methods We evaluated the action of orally administered flunarizine in mice against hyperlocomotion induced by amphetamine and dizocilpine (MK-801) as pharmacological models of schizophrenia, induction of catalepsy as a measure for extrapyramidal symptoms and impairment induced by dizocilpine on the delayed alternation task for working memory.Results Flunarizine robustly inhibited hyperlocomotion induced by both amphetamine and dizocilpine at doses that do not reduce spontaneous locomotion (3–30 mg/kg). Mild catalepsy was observed at 30 mg/kg, being more pronounced at 50 mg/kg and 100 mg/kg. Flunarizine (30 mg/kg) improved dizocilpine-induced impairment on the delayed alternation test.Conclusions These results suggest a profile comparable to atypical antipsychotics. The low cost, good tolerability and long half-life (over 2 weeks) of flunarizine are possible advantages for its use as an atypical antipsychotic. These results warrant clinical trials with flunarizine for the treatment of schizophrenia.     

IBUDILAST REDUCES INTRACELLULAR CALCIUM ELEVATION INDUCED BY IN VITRO ISCHAEMIA IN GERBIL HIPPOCAMPAL SLICES

1. A microfluorometry was carried out to investigate the effect of 3-isobutyryl-2-isopropylpyrazolo[1,5-a]pyridine (ibudilast) on changes in levels of intracellular calcium concentration ([Ca²⁺]_i) induced by in vitro ischaemia in the CA1 field of gerbil hippocampal slices. 2. When slices, loaded with a calcium ion sensitive dye (rhod-2) were exposed to a glucose-free physiological medium equilibrated with a 95% N₂/5% CO₂ gas mixture (standard in vitro ischaemia), a large [Ca²⁺]_i elevation was detected approximately 5 min after the beginning of in vitro ischaemia. 3. When slices were perfused with the in vitro ischaemic medium containing 43 μmol/L ibudilast, a [Ca²⁺]_i elevation was still observed; however, the extent of the increase in [Ca²⁺]_i was significantly depressed in all subregions of the hippocampal slices. 4. The extent of this inhibitory effect of ibudilast on the in vitro ischaemia-induced [Ca²⁺]_i elevation was in a similar range as those of Ca²⁺ blockers, including (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cycloheptan-5,10-imine maleate (MK-801), flunarizine and dantrolene. 5. Similar [Ca²⁺]_i increases in the CA1 field were induced by a Ca²⁺-free in vitro ischaemia, a high concentration of KCl or by specific agonists for glutamate receptor subtypes (N-methyl-d -aspartate (NMDA), (s)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) and kainate); these increases were also depressed with 43 μmol/L ibudilast present in the perfusion medium. 6. These results indicate that ibudilast may act by depressing the Ca²⁺ accumulation during and shortly after ischaemia, a possible pharmacological action of ibudilast that leads to the amelioration of ischaemic injury in the central nervous system.     

Robust anti-arrhythmic efficacy of verapamil and flunarizine against dofetilide-induced TdP arrhythmias is based upon a shared and a different mode of action

BACKGROUND AND PURPOSE

The high predisposition to Torsade de Pointes (TdP) in dogs with chronic AV-block (CAVB) is well documented. The anti-arrhythmic efficacy and mode of action of Ca²⁺ channel antagonists, flunarizine and verapamil against TdP were investigated.

EXPERIMENTAL APPROACH

Mongrel dogs with CAVB were selected based on the inducibility of TdP with dofetilide. The effects of flunarizine and verapamil were assessed after TdP and in different experiments to prevent dofetilide-induced TdP. Electrocardiogram and ventricular monophasic action potentials were recorded. Electrophysiological parameters and short-term variability of repolarization (STV) were determined. In vitro, flunarizine and verapamil were added to determine their effect on (i) dofetilide-induced early after depolarizations (EADs) in canine ventricular myocytes (VM); (ii) diastolic Ca²⁺ sparks in RyR2^R4496+/+ mouse myocytes; and (iii) peak and late I_Na in SCN5A-HEK 293 cells.

KEY RESULTS

Dofetilide increased STV prior to TdP and in VM prior to EADs. Both flunarizine and verapamil completely suppressed TdP and reversed STV to baseline values. Complete prevention of TdP was achieved with both drugs, accompanied by the prevention of an increase in STV. Suppression of EADs was confirmed after flunarizine. Only flunarizine blocked late I_Na. Ca²⁺ sparks were reduced with verapamil.

CONCLUSIONS AND IMPLICATIONS

Robust anti-arrhythmic efficacy was seen with both Ca²⁺ channel antagonists. Their divergent electrophysiological actions may be related to different additional effects of the two drugs.     

Effects of NaF on the expression of intracellular Ca2+ fluxes and apoptosis and the antagonism of taurine in murine neuron

Sodium fluoride (NaF) has been shown to be cytotoxic and produces inflammatory responses in humans. However, the cellular mechanisms underlying the neurotoxicity of fluoride are unclear. The present study aims to define a possible mechanism of NaF-induced neurotoxicity with respect to apoptosis and intracellular Ca²⁺ fluxes. Meanwhile, the cytoprotective role of taurine in intervention, the toxic effects of NaF on neurons, is also investigated. The primary mouse hippocampal neurons were incubated with 5.0, 10.0, 15.0, 20.0, and 40.0?mg NaF/L in vitro and Kunming mice were exposed to 0.7, 2.8, and 11.2?mg NaF/kg and 7.5 and 15.0?mg taurine/kg in vivo. Intracellular Ca²⁺ fluxes and apoptosis were assayed. Compared with the control, the significant differences of intracellular Ca²⁺ concentration and apoptotic peaks were found in 5.0–40.0?mg NaF/L groups in vitro (p?<?0.01) and in the groups of 0.7–11.2?mg NaF/kg in vivo (p?<?0.01). Instantaneously, taurine can minimize F-induced neurotoxicity significantly at doses of 7.5 and 15.0?mg/kg (p?<?0.01). The present study herein suggested that NaF could increase intercellular Ca²⁺ concentration leading to apoptosis. Meanwhile, taurine could minimize neurotoxicity caused by fluoride through decreasing intercellular Ca²⁺ concentration and cell apoptosis.     

Effect of p-p′-DDE Administered in Vivo and in Vitro on Ca2+ Binding and Ca2+-Mg2+-ATPase Activity in Egg Shell Gland Mucosa of Ducks

Abstract: Thinning of the egg shell is produced by p-p′-DDT and DDE in several species of birds. A study was made of the effect of DDE administered in vitro and in vivo on the Ca²⁺ binding and Ca²⁺-Mg²⁺-activated ATPase of a homogenate of the egg shell gland of ducks (Anas platyrhynchos var.). The concentration of Ca²⁺ was 1×10^?4 and that of MgATP 1×10^?3M. In vitro, DDE in concentrations of 2–16 μg/ml of incubation medium inhibited the Ca²⁺-Mg²⁺-activated ATPase in a concentration-dependent manner, whereas Mg²⁺-activated ATPase was not affected by these concentrations. The Ca²⁺ binding by the homogenate was reduced by DDE in the same concentrations. The sodium azide sensitive Ca²⁺ binding was most sensitive. In vivo, DDE administered in a concentration of 40 mg/kg dry weight of the food for 45 days reduced the egg shell index by 18% in comparison to controls. After 45 days of treatment the DDE concentration in the egg shell gland mucosa was 1.20±0.16 μg/g of wet weight, while no DDE was detected in the controls. The Ca²⁺-Mg²⁺-activated ATPase was reduced by 32%, whereas the Mg²⁺-ATPase was not changed. The Ca²⁺ binding by the homogenate was reduced by 29%, the sodium azide sensitive part being most vulnerable. DDE increased the total Ca content of the egg shell gland mucosa by 44%. Since Ca is transported against a concentration gradient between blood plasma and the lumen of the shell gland, it is suggested that DDE, by inhibiting the Ca²⁺-Mg²⁺-activated ATPase, decreased the Ca translocation over the egg shell gland mucosa.     

Comparison of the calcium entry blockers nimodipine and flunarizine on human cerebral and temporal arteries: role in cerebrovascular disorders

Summary The effect of the calcium entry blockers flunarizine and nimodipine on isolated human cerebral and temporal arteries has been studied.Flunarizine induced only weak relaxation of precontracted temporal arteries in contrast to the response seen in cerebral arteries. Nimodipine invariably induced strong relaxation of both types of vessel. The effect of the calcium entry blockers on potassium (K⁺)-, noradrenaline (NA)- and 5-hydroxytryptamine (5-HT)-induced contraction was also examined. In general, the K^–-induced contraction was inhibited by both calcium entry blockers, nimodipine being more potent than flunarizine, the cerebral artery being more sensitive. The response to K⁺ consisted of two phases; the second, slowly developing phase of contraction was more sensitive to either blocker than the initial, fast phase of contraction.Flunarizine was significantly more potent in inhibiting NA-induced contraction of the human cerebral than of the temporal artery, and there was no difference in its action on 5-HT-induced contraction of either artery. The same pattern was found for nimodipine, which was more potent in every aspect. Both calcium entry blockers induced a parallel shift in calcium-induced contraction studied by application of calcium to vessels preincubated in calcium free medium. Flunarizine was more potent on cerebral than on temporal arteries and there was no difference between the two vessels in this action of nimodipine. However, the latter was more potent than flunarizine in preventing calcium-induced contraction.The clinical implications of the two agents are discussed in relation to cerebrovascular disorders.     

Inhibition of rat brain mitochondrial calcium transport by chlordecone.

Assessment of ⁴⁵Ca²⁺ transport in rat brain mitochondria following in vitro and in vivo exposures to the neurotoxic insecticide chlordecone revealed a locus for chlordecone's mitochondrial impairment in the coupling reactions of oxidative phosphorylation. Mitochondrial ⁴⁵Ca²⁺ transport supported by either 2 mm succinate (free energy supplied via electron transport) or 800 μm ATP (free energy supplied via the Mg²⁺ ATPase reaction) was inhibited by low concentrations of chlordecone (10^?8–10^?5m). Administration of a tremorigenic dose (40 mg/kg, po) of chlordecone to rats similarly inhibited both succinate- and ATP-supported ⁴⁵Ca²⁺ uptake in brain mitochondria in vitro. Studies on the subcellular distribution of [¹⁴C]chlordecone in rat brain following in vivo administrations were compared to those on the mitochondrial uptake of [¹⁴C]chlordecone in vitro. Administration of the 40-mg/kg tremorigenic dose of chlordecone produced mitochondrial chlordecone concentrations which were comparable to the tissue concentrations achieved by exposing mitochondria to a 2 × 10^?6m concentration of chlordecone in vitro. These data lend further support for mitochondrial inhibition as a mechanism for chlordecone toxicity.     

A novel Na+/Ca2+ channel blocker, NS-7, suppresses hypoxic injury in rat cerebrocortical slices

The substance 4-(4-fluorophenyl)-2-methyl-6-(5-piperidinopentyloxy) pyrimidine hydrochloride (NS-7) has been developed recently as a cerebroprotective compound with Na⁺ and Ca²⁺ channel blocking action. In the present study, the effect of NS-7 in an in vitro model of hypoxic injury was examined and the possible involvement of Na⁺ and Ca²⁺ channels in the hypoxic injury subsequently determined. When slices of rat cerebral cortex were exposed to hypoxia/glucose deprivation followed by reoxygenation and restoration of the glucose supply, marked leakage of lactate dehydrogenase (LDH) occurred 3–6 h after reoxygenation. This hypoxia/reoxygenation-induced injury was blocked almost completely by the removal of extracellular Ca²⁺ or by chelating intracellular Ca²⁺ with 1,2-bis(o-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid tetra(acetoxymethyl)ester (BAPTA/AM). In addition, combined treatment with the N-type Ca²⁺ channel blocker ω-conotoxin GVIA and the P/Q-type Ca²⁺ channel blocker ω-agatoxin IVA significantly reduced LDH leakage, although neither of these Ca²⁺ channel blockers alone, nor nimodipine, an L-type Ca²⁺ channel blocker, was effective. On the other hand, several Na⁺ channel blockers, including tetrodotoxin, local anaesthetics and antiepileptics, significantly reduced the hypoxic injury. NS-7 (3–30 μM) concentration-dependently inhibited LDH leakage caused by hypoxia/reoxygenation, but had no influence on the reduction of tissue ATP content and energy charge during hypoxia and glucose deprivation. It is suggested that blockade of Na⁺ and Ca²⁺ channels is implicated in the cerebroprotective action of NS-7. Received: 10 March 1998 / Accepted: 19 April 1998     

NEUROPROTECTION IN VITRO AND IN VIVO BY CELL MEMBRANE-PERMEANT Ca2+ CHELATORS

1. A method of attenuating excitotoxic and hypoxic/ ischaemic neurodegeneration in vitro and in vivo using cell-permeant Ca²⁺-chelating agents is described. 2. The mechanism of neuroprotection may depend on both pre- and post-synaptic effects on intracellular Ca²⁺ dynamics. 3. This method is unique, because it targets Ca²⁺ ions, the presumed triggers of neurodegeneration, rather than a specific cellular receptor.     

Effects of Mercuric Chloride and Methyl Mercury on Cholinergic Neuromuscular Transmission in the Guinea-Pig Ileum

Abstract: The effects of mercuric chloride (HgCl₂) and methyl mercury (MeHg) were examined on basal mechanical activity and electrically-induced neurogenic cholinergic contractions (twitch contractions) in longitudinal muscle-myenteric plexus strips from guinea-pig distal ileum. Both compounds at 0.3-3 μM slightly enhanced the amplitude of twitch contractions in ~50% preparations. This effect was probably due to facilitation of acetylcholine (ACh) release since 0.1 and 1 μM mercurials increased electrically-evoked tritium outflow from [³H]choline preloaded muscle layer with attached myenteric plexus. Conversely, higher mercury concentrations inhibited twitch contractions (HgCl₂ IC₅₀=21.3±6.4 μM; MeHg IC₅₀=45.1±5.5 μM), as well as contractions to exogenous ACh (0.1 μM) in resting preparations, and concomitantly increased the basal tone. The former effects possibly reflected an antimuscarinic activity of mercury, while the latter was related to alterations of calcium homeostasis in the effector cells. Indeed, the effect of HgCl₂ on basal tone was antagonized by the Ca²⁺ entry blocker nifedipine (3, 10, 30 nM), indicating Hg-induced facilitation of Ca²⁺ influx through voltage-dependent channels. On the whole, our results suggest that cholinergic neuromuscular transmission and Ca²⁺-dependent mechanisms underlying smooth muscle contractility are targets for mercury toxicity in the intestine.     

Compared effects of calcium entry blockers on calcium-induced tension in rat isolated cerebral and peripheral resistance vessels

Summary The effects of the calcium entry blockers verapamil (V), diltiazem (D), nifedipine (NF) and nicardipine (NC) have been studied on calcium concentration-effect curves elicited in depolarized (K⁺, 40 mmol/l) and in serotonin-exposed (6 mol/l) rat middle cerebral arteries (RMCA) in order to compare the relative potencies of the blockers against these two calcium channel activating mechanisms. In control conditions, Ca²⁺ sensitivity expressed as pD₂ and maximal active wall tension (AWT) were not significantly different in depolarized and in 5-HT-exposed vessels: pD₂: 3.39 ±0.08 vs 3.50 ± 0.06 and AWT: 0.93 ± 0.15 mN · mm^–1 vs 0.90 ± 0.16 mN · mm^–1 respectively. V, D, NF and NC displaced Ca²⁺ control curves to the right and depressed the maximum contractile response in the two experimental conditions, which suggests a noncompetitive type of antagonism. All the blockers were more potent inhibitors of Ca²⁺-induced contractions in depolarized than in serotonin-exposed middle cerebral arteries. The IC₅₀ values (concentration of blockers producing a 50% inhibition of maximal control contractile response) were (nmol/l) : V = 20, D = 120, NF = 0.4, NC = 1 and V = 400, D = 10000, NF = 20, NC = 7 in depolarized and serotonin-exposed arteries respectively. From these IC₅₀ values, the relative order of potency of the CEB's was not the same in the two experimental conditions suggesting that while serotonin and K⁺ both promote the entry of Ca²⁺ into vascular smooth muscle cells of RMCA, they either activate a different gating mechanism associated with a single common channel or perhaps distinct channels. Comparison of the results obtained in this study for depolarized rat middle cerebral arteries with those previously obtained in depolarized rat mesenteric resistance arteries (RMRA) revealed that while Ca²⁺-induced contractile responses were inhibited in a similar non-competitive manner by the four CEB's, the respective IC₅₀ values showed that potencies and rank of relative potency of the blockers were different in the two types of vessels. D and NC were equally potent in both preparations (IC₅₀ ratio = 2.5 and 3 respectively) but RMCA were more sensitive to V and NF than RMRA (IC₅₀ ratio = 6.5 and 11 respectively). These results are discussed and it is proposed that regional differencies in the conformation and/or the activation of the voltage-gated Ca²⁺ channels may exist in different vascular beds. Send offprint requests to J. L. Freston     

Induction of apoptosis by antimycin A in differentiated PC12 cell line

Antimycin A (AMA) is an inhibitor of mitochondrial electron transport chain via binding to mitochondrial complex III. This inhibition increases the production of reactive oxygen species (ROS). The aim of the present study was to investigate the effect of AMA on PC12 cells in vitro. Results of the nuclear morphology and the flow cytometer indicated that AMA efficiently induced PC12 cell apoptosis. Moreover, the levels of ROS and Ca²⁺ increased in the early stage of cell apoptosis induced by AMA treatment. All of Ca²⁺ chelators, L‐type Ca²⁺ channel blockers and inhibitors of Ca²⁺ released from endoplasmic reticulum and ROS scavenger, were used in this experiment. It was found that the Ca²⁺ chelators and ROS scavengers, in particular, could delay AMA‐induced PC12 cell apoptosis. In conclusion, the present study found that AMA induced PC12 cell apoptosis through ROS and Ca²⁺. Copyright © 2013 John Wiley & Sons, Ltd.     

Effects of Methyl Mercury on Protein Synthesis in Vitro

Abstract: Methyl mercury has been shown to interact with protein synthesis in vivo and in vitro. In the present paper a brain postmitochondrial supernatant was used for studies in vitro. Inorganic mercury (Hg²⁺) was shown to be a more potent inhibitor of protein synthesis than methyl mercury, puromycin or cycloheximide. The inhibitory effect of methyl mercury was potentiated by puromycin. It is thus possible that methyl mercury causes disintegration of polysomes in brain cells.     

Effects of acute and chronic ethanol administration on whole mouse brain synaptosomal calcium influx

Whole brain synaptosomes, isolated from pair-fed acute in vitro (sucrose-Sustacal diet for 10 days), acute in vivo [4.5g/kg, 20% (w/v) i.p. ethanol pretreatment] and chronic in vivo (ethanol-Sustacal diet for 10 days) female, Swiss-Webster mice, were challenged in vitro with ethanol (80 mM, final concentration) in either an incubation medium (12-min exposure), a depolarizing medium (2-min exposure with 74 mM KCI) or a nondepolarizing medium (2-min exposure with 5 mM KCl). Depolarizing and nondepolarizing media also contained ⁴⁵Ca²⁺ (2μCi/μmole). The results showed (1) a significant enhancement of ⁴⁵Ca²⁺ influx when synaptosomes isolated from acute in vitro and acute in vivo mice groups were challenged in vitro by ethanol (80 mM) in the depolarizing medium (74 mM KCl), (2) a significant enhancement by 80 mM ethanol of ⁴⁵Ca²⁺ accumulation by nondepolarized synaptosomes isolated from the acute in vitro mouse group, (3) a significant increase in ⁴⁵Ca²⁺ accumulation in synaptosomes from acute in vivo mice as compared with acute in vitro mice without an in vitro ethanol challenge, and (4) a significant decrease in ⁴⁵Ca²⁺ accumulation by synaptosomes isolated from chronic in vivo mice as compared to acute in vitro synaptosomes. The results presented here demonstrate the ability of ethanol to significantly increase calcium accumulation into whole brain synaptosomes and that tolerance to this phenomenon occurs in parallel with behavioral tolerance to the sedative action of ethanol. These ethanol-induced changes in calcium accumulation may be involved in the production of sedation and tolerance to sedation.