Mitsunobu Yoshii

Psychotropic drug block voltage-gated ion channels in neuroblastoma cells ☆

Effects of neuroleptics and tricyclic antidepressants on voltage-gated ion channels were investigated in the neuroblastoma cell line N1E-115 using the whole cell variation of patch electrode voltage-clamp techniques. Imipramine, chlorpromazine and haloperidol in micromolar concentrations blocked sodium, calcium and potassium channel currents in a reversible and concentration-dependent manner. The order of potency was chlorpromazine greater than imipramine greater than haloperidol. These direct blocking actions on neuronal ion channels might play a role in clinical effects of these drugs.

Role of Nitric Oxide in the Epileptogenesis of EL Mice

Summary: Purpose: To understand the role of nitric oxide (NO) in the regulation of seizures, we measured the extracellular levels of the NO metabolites nitrite and nitrate as indices of NO generation in the parietal cortex, hippocampus, and temporal cortex of EL mice. Furthermore, alterations of neuronal, endothelial, and inducible nitric oxide synthetase (nNOS, eNOS, and iNOS, respectively) were observed to correlate them with epileptogenesis.

Differential block of sodium and calcium channels by chlorpromazine in mouse neuroblastoma cells

1. The effects of chlorpromazine on the voltage‐activated sodium and type I (transient) calcium channels were studied in cultured mouse neuroblastoma cells (N1E‐115) using the whole‐cell patch‐clamp technique. 2. Chlorpromazine (2‐10 x 10(‐6) M) blocked both the sodium channel current and the calcium channel current as carried by Ba2+ in a reversible and dose‐dependent manner. 3. The block was not associated with any change in the time course of the activation and inactivation of the sodium and calcium channel currents. 4. The dose‐response relationships for the block of these channels measured with a holding potential of ‐120 mV indicated a 1:1 binding stoichiometry with apparent dissociation constants of 2.5 +/‐ 10(‐6) M and 1.5 +/‐ 10(‐5) M for the sodium and calcium channels, respectively. 5. The block was dependent on the holding potential for both channel currents. The apparent dissociation constant for the sodium channel was decreased to 0.65 +/‐ 10(‐6) M when the membrane was held at ‐80 mV. The apparent dissociation constant for the calcium channel was decreased to 3.2 +/‐ 10(‐6) M when the membrane was held at ‐60 mV. 6. The steady‐state inactivation curve for the sodium channel was shifted by 12.4 +/‐ 1.8 mV to more negative potentials by exposure to 1 x 10(‐6) M‐chlorpromazine. The inactivation curve for the calcium channel was also shifted by 15.4 +/‐ 3.2 mV to more negative potentials by exposure to 1 x 10(‐5) M‐chlorpromazine. These results indicate a greater affinity of chlorpromazine for the inactivated state of the channels than for the resting state. 7. Chlorpromazine caused a marked use‐dependent block of the sodium channel current, as demonstrated by a cumulative increase of the block during a train of depolarizing pulses. The use‐dependent block was observed even with an interpulse interval as long as 2 s. 8. On the other hand, the block of the calcium channel current did not notably accumulate during a train of depolarizing pulses even when extremely prolonged (1 s) pulses were applied at a very short interpulse interval (200 ms). 9. The marked use dependence of the sodium channel block was due to a very slow repriming of the drug‐bound sodium channels from inactivation, whereas the lack of use dependence of the calcium channel block was due to a rapid repriming of the drug‐bound calcium channels.(ABSTRACT TRUNCATED AT 400 WORDS)

The anti-dementia drug nefiracetam facilitates hippocampal synaptic transmission by functionally targeting presynaptic nicotinic ACh receptors.

Nefiracetam, a pyrrolidone derivative developed as an anti-dementia drug, persistently potentiated currents through neuronal nicotinic acetylcholine (ACh) receptors (alpha7, alpha4beta2) expressed in Xenopus oocytes, and the potentiation was blocked by either the selective protein kinase C (PKC) inhibitors, GF109203X and staurosporine, or co-expressed active PKC inhibitor peptide. In primary cultures of rat hippocampal neurons, nefiracetam increased the rate of nicotine-sensitive miniature excitatory postsynaptic currents, without affecting the amplitude, and the increase was inhibited by GF109203X. In addition, the drug caused a marked increase in the glutamate release from electrically stimulated guinea pig hippocampal slices, and the effect was abolished by the nicotinic ACh receptor antagonists, alpha-bungarotoxin and mecamylamine. Nefiracetam induced a long-lasting facilitation of synaptic transmission in both the CA1 area and the dentate gyrus of rat hippocampal slices, and the facilitation was inhibited by alpha-bungarotoxin and mecamylamine. Such facilitatory action was still found in the hippocampus with selective cholinergic denervation. The results of the present study, thus, suggest that nefiracetam enhances activity of nicotinic ACh receptors by interacting with a PKC pathway, thereby increasing glutamate release from presynaptic terminals, and then leading to a sustained facilitation of hippocampal neurotransmission. This may represent a cellular mechanism underlying the cognition-enhancing action of nefiracetam. The results also provide the possibility that nefiracetam could be developed as a promising therapeutic drug for senile dementia or Alzheimers disease.

Maitotoxin-induced membrane current in neuroblastoma cells

Maitotoxin (MTX) is a potent marine toxin isolated from the toxic dinoflagellate, Gambierdiscus toxicus. We have examined the possibility of MTX activating calcium channels using cultured neuroblastoma cells (N1E-115). MTX (10 ng/ml) produced a depolarization of the membrane, which was prevented by the removal of Ca2+ from the external medium. Under voltage clamp conditions, membrane currents were recorded with 50 mM Ba2+ as a charge carrier through calcium channels. After application of MTX (1 ng/ml), an inward current necessary to hold the membrane at -90 mV increased progressively. This was followed by a gradual decrease of the transient inward Ba2+ current through type I calcium channels recorded at -30 mV which was eventually abolished. A similar tendency was observed in the long-lasting inward Ba2+ current through type II calcium channels, which was recorded at +10 mV. The MTX action was antagonized by calcium channel blockers such as verapamil (100 microM) and La3+ (1 mM). A high concentration of verapamil (500 microM) blocked both types of calcium channels persistently. After washout of verapamil but while the calcium channels were still blocked, MTX (1 ng/ml) induced a steady-state current. The MTX-induced current showed an inward-rectifying property with a reversal potential of approximately -30 mV. The results suggest that the MTX-induced current does not flow through calcium channels. Thus, MTX may create a pore in the membrane with pharmacological properties similar to those of calcium channels.

Evidence that variation in the peripheral benzodiazepine receptor (PBR) gene influences susceptibility to panic disorder

Panic disorder (PD) is the repeated sudden occurrence of panic attacks, episodes characterized by psychological symptoms. Peripheral benzodiazepine receptor (PBR) is closely associated with personality traits for anxiety tolerance, and that it holds promise as a biological marker of stressful conditions. We have performed association analyses using the polymorphism to determine the PBR in PD. We screened the subjects for sequence variations within the 5′ region, the coding region (exons 2–4), and the 3′ noncoding region. One novel missense variant in exon 4, derived from the nucleotide transition in codon 162 (CGT → CAT:485G > A) resulting in an arginine‐to‐histidine (Arg → His) change, was detected in these subjects. The 485G > polymorphism of the PBR gene was analyzed in 91 PD patients and 178 controls. The genotypic and allelic analyses of the 485G > A revealed significant differences between the panic patients and the comparison subjects (P = 0.021 and 0.014, respectively). The present study provides new and important evidence that variation in the PBR gene influences susceptibility to PD.

Cellular mechanisms underlying cognition-enhancing actions of nefiracetam (DM-9384)

We have studied cellular mechanisms underlying cognition-enhancing actions of nefiracetam (DM-9384), a newly developed cognitive enhancer, by biochemical experiments on cholinergic and GABAergic transmissions as well as electrophysiological experiments on neuronal Ca2+ channels. In behavioral experiments in rats, nefiracetam (3 mg/kg) ameliorated amnesia induced by basal forebrain (BF) lesion or treatment of scopolamine. Biochemical experiments revealed that nefiracetam increased uptake and release of transmitters in both cholinergic and GABAergic systems in rat brain. In electrophysiological studies, nefiracetam (1 microM) increased long-lasting (N/L-type) Ca2+ channel currents in NG108-15 cells. The nefiracetam action on Ca2+ channels was blocked by pertussis toxin (PTX). The results suggest that nefiracetam improves impaired memory by facilitating cholinergic and GABAergic transmissions in the brain. It is further suggested that PTX-sensitive G-proteins and Ca2+ channels associated with these G-proteins are responsible for the action of nefiracetam on neurotransmission.

Gating and permeation properties of two types of calcium channels in neuroblastoma cells

The gating and permeation properties of two types of calcium channels were studied in the neuroblastoma cell line N1E-115. Calcium channel currents as carried by Ba2+ (50 mM) were recorded using the whole-cell variation of the patch electrode voltage-clamp technique. The two types of calcium channels showed similar membrane potential dependence with respect to the steady-state activation and inactivation gating properties. However, the properties of the long-lasting type II channels were shifted approximately 30 mV in the depolarizing direction compared with those of the transient type I channels. Activation of type I channels developed with a sigmoidal time course which was described by m2 kinetics, whereas the activation of type II channels was described by a single exponential function. Tail current upon repolarization followed an exponential decay in either type of calcium channels. In comparison to type I channels, the activation process of type II channels was shifted approximately 30 mV in the positive direction, while the deactivation process showed a 60 mV shift in the positive direction. The rate constants of activation obtained from the activation and deactivation processes indicated that under comparable membrane potential conditions, type II channels close 2.4 times faster than type I channels upon repolarization. When external 50 mM Ba2+ was replaced with Ca2+ or Sr2+ on the equimolar basis, the amplitudes of transient and long-lasting currents were altered without a significant change in their time courses. The ion permeability ratios determined from the maximum amplitude of the inward current were as follows: Ba2+ (1.0) = Sr2+ (1.0) greater than Ca2+ (0.7) for type I channels, and Ba2+ (1.0) greater than Sr2+ (0.7) greater than Ca2+ (0.3) for type II channels. Replacement of Ba2+ with Ca2+ caused a 10-12 mV positive shift in the current-voltage relation for type II channels. However, the shift for type I channels was much less. This suggests that negative surface charges are present around type II channels. After correction for the surface charge effect on the ion permeation, there was no significant difference between the permeability ratios of these cations for the two channel types. It was concluded that the two types of calcium channels have many common properties in their gating and permeation mechanisms despite their differential voltage sensitivity and ion selectivity.

Differential inhibition of transient and long-lasting calcium channel currents by benzodiazepines in neuroblastoma cells

The effects of diazepam, nitrazepam, clonazepam, and Ro5-4864 on transient (type I) and long-lasting (type II) calcium channels associated with low-affinity benzodiazepine receptors were investigated using the whole-cell patch-clamp technique. Clonazepam (100 microM), a specific agonist for the central-type benzodiazepine receptor, reduced transient currents through the type I calcium channel by 40% without affecting long-lasting currents through the type II calcium channel. Diazepam and nitrazepam (100 microM), non-specific agonists for both the central- and peripheral-type benzodiazepine receptors, reduced both transient and long-lasting currents equally by 25-30%. A similar non-selective inhibition was observed by Ro5-4864 (1-10 microM), a specific agonist for the peripheral-type benzodiazepine receptor. It is concluded that the two calcium channel types are regulated differentially by two different kinds of benzodiazepines; central-type for type I channel and peripheral-type for both type I and type II channels.

Preferential block of skeletal muscle sodium channels by geographutoxin II, a new peptide toxin fromConus geographus

The effects of geographutoxin II (GTX II), a novel polypeptide toxin isolated from the marine snailConus geographus, on nerves and muscles were studied by current clamp and voltage clamp techniques. GTX II (5×10−7 M) abolished the action potential of the guinea pig skeletal muscle without change in the resting potential. However, action potentials of the crayfish giant axon, mouse neuroblastoma N1E-115 cell and guinea pig cardiac muscle were not affected by GTX II even at concentrations higher than 1×10−6 M. In the voltage clamped bullfrog skeletal muscle fiber, sodium currents were almost completely blocked by GTX II (1×10−6 M), and slowly recovered after washout. The time course of sodium currents was not appreciably altered by GTX II. These results suggest that GTX II selectively blocks skeletal muscle sodium channels in much the same way as tetrodotoxin.