Tadashi Tsuda

Relationship between large conductance calcium-activated potassium channel and bursting activity.

To elucidate the role of the large conductance calcium-activated potassium channel (BK(Ca) channel) in the production of bursting activity, which is characteristic of convulsions, effects of iberiotoxin (IbTX), a selective blocker of the BK(Ca) channel, on bursting activity, induced by various procedures were examined using primary cultured neurons from the cerebral cortex of mice. IbTX completely inhibited bursting activity induced by pentylenetetrazol (PTZ), caffeine, 1,4,5-inositol triphosphate (IP3) and direct forced increase of intracellular calcium. Inherent spontaneous bursting activity in the cerebral cortical neurons of the El mouse, which shows a high susceptibility to convulsions was also completely inhibited by IbTX. Apamin, a specific blocker of the small conductance calcium-activated potassium channel (SK(Ca) channel) showed no inhibition of bursting activity. These findings suggest that the BK(Ca) channel is essential for the production of bursting activity, and also suggest the possibility of clinical use of blocking agents of the BK(Ca) channel against intractable epilepsy.

Inhibitory effect of peony root extract on pentylenetetrazol-induced EEG power spectrum changes and extracellular calcium concentration changes in rat cerebral cortex

To elucidate the mechanism of anticonvulsant action of peony root and to determine the relative contributions of pure component substances, the water, water/acetone and methanol extracts of peony roots, paeoniflorin, albiflorin and pentagalloylglucose were studied in rats using the EEG power spectrum changes induced by pentylenetetrazol administration and the extracellular calcium and potassium concentration changes related to seizure activity. The water extract of peony roots, albiflorin and pentagalloylglucose given orally completely inhibited the EEG power spectrum changes as well as the extracellular calcium and potassium concentration changes related to seizure activity. The water/acetone and methanol extracts and paeoniflorin were relatively less potent. These findings suggested that the anticonvulsant action of peony roots is due primarily to albiflorin and the gallotannin fraction. Albiflorin and pentagalloylglucose appear to manifest their anticonvulsant action due to inhibition of the seizure-related decrease of extracellular calcium and consequent intracellular calcium increase.

Protective Effects of Peony Root Extract and Its Components on Neuron Damage in the Hippocampus Induced by the Cobalt Focus Epilepsy Model

Protective effects of peony root extract and its components on neuron damage in the CA1 area of the hippocampus induced by the cobalt focus epilepsy model were examined. Neuron damage in the CA1 area of the hippocampus and frequent spike discharges induced by application of metallic cobalt to the cerebral cortex of rats were completely prevented when peony root extract was continuously administered orally at 1 g/kg/day for 30 days prior to cobalt application. Component crude gallotannin fraction showed marked but incomplete protective action. A combination of crude gallotannin fraction and paeoniflorin showed complete protective action in the same way as peony root extract against neuron damage although use of paeoniflorin alone had no effect. These findings together with our previous reports indicate that peony root extract and its component, gallotannin, have excellent protective effects on neuron damage in addition to anticonvulsant action by prior oral administration.

Intracellular protein changes during pentylenetetrazole induced bursting activity in snail neurons

The intracellular protein changes during pentylenetetrazole (PTZ)-induced bursting activity (BA) which is characteristic of seizure discharge were investigated using microdisk electrophoresis with 5 identified neurons. The identified neurons of the snail, Euhadra peliomphala, were used. The PTZ-sensitive neurons which manifest marked BA by application of PTZ were examined. PTZ induced in PTZ-sensitive neurons: (1) a prominent increase of 5-7 kdalton protein and (2) peak separation into 3 peaks of 10-15 kdalton protein. In the 5-7 and 10-15 kdalton protein, a marked increase in radioactivity of 45Ca was observed after PTZ application. PTZ-non-sensitive neurons showed neither these protein changes nor 45Ca incorporation into these proteins. The above findings suggest that during PTZ-induced BA, intracellular protein changes occurred in relation to the intracellular calcium shift.

Gene mapping of SEZ group genes and determination of pentylenetetrazol susceptible quantitative trait loci in the mouse chromosome

Gene mapping of the newly discovered SEZ genes (seizure-related genes) in the mouse was performed by linkage analysis. SEZ6 was on chromosome 11, SEZ12 on chromosome 16, SEZ15 on chromosome 3 and SEZ17 (PTZ17) on chromosome 18. The mouse chromosomal locus related to high susceptibility to pentylenetetrazol (PTZ) was also determined by linkage analysis using the recombinant inbred mouse, BXD (C57BLxDBA). A significant level of PTZ susceptibility was found on chromosome 2. Chromosomal loci of the newly discovered SEZ genes were not coincident with the significant chromosomal loci to PTZ susceptibility. Since epilepsy is assumed to be a disease syndrome which is probably manifested by abnormal expression of multifocal genes, determination of the role of each chromosomal locus in the provocation of seizure activity is important.

Cytotoxic Activity toward KB Cells of 2-Substituted Naphtho[2,3-b]furan-4,9-diones and Their Related Compounds

We investigated the cytotoxic activity of 2-substituted naphtho[2,3-b]furan-4,9-diones. We have previously synthesized 33 types of 2-substituted and related compounds, and the cytotoxic activity of these compounds was then examined by a KB cell culture assay. 2-(3-Furanoyl)benzoic acids and 1,4-naphthoquinones had no activity. 2-Acetyl-4,9-dimethoxynaphtho[2,3-b]furan 4 showed low activity. However, parent naphtho[2,3-b]furan-4,9-dione 2 and most 2-substituted derivatives exhibited cytotoxic activity. The parent structure was therefore for cytotoxicity. 2-Formylnaphtho[2,3-b]furan-4,9-dione 11 had particularly potent activity (ED50=0.09 μg/ml).

Cloning and characterization of pentylenetetrazol-related cDNA, PTZ-17

cDNAs related to pentylenetetrazol-induced bursting activity in neurons were screened by a differential hybridization method using normal and pentylenetetrazol-treated primary cultured neurons from the cerebral cortex of mice. Twenty clones of candidate cDNA with expression increased or decreased by treatment with pentylenetetrazol were obtained. One of them, PTZ-17, was sequenced. Injection of PTZ-17 derived RNA into Xenopus oocytes showed a large calcium inward current with extracellular application of pentylenetetrazol.

Pentylenetetrazole-induced changes of the single potassium channel in primary cultured cerebral cortical neurons

To elucidate the behavior of the single ionic channels of cerebral cortical neurons during bursting activity, the effects of pentylenetetrazole (PTZ) on a single potassium channel of primary cultured cerebral cortical neurons from mice were examined. All of the examined 10-day-old primary cultured neurons of the cerebral cortex showed clear bursting activity after extracellular application of PTZ using whole-cell patch-clamp recording. Less than half of the examined single potassium channels, both outward and inward, of the 2- and 3-day-old as well as 6-10-day-old primary cultured cerebral cortical neurons showed the bursting-type open-close state by application of PTZ. The PTZ-sensitive single potassium channels were found in the voltage-dependent as well as calcium-activated channels.

Molecular Mechanism of Preventive Effect of Peony Root Extract on Neuron Damage

The molecular mechanism of the protective effects of peony root extract and its component substances on neuron damage induced by the cobalt focus epilepsy model and the EL mouse was investigated. Long-term administration of peony root extract for 30 days prior to metallic cobalt powder application to the cerebral cortex of mice resulted in increased expression of A20, an inhibitor gene of cell death. In the EL mouse, a hereditary epilepsy animal model with vulnerable neurons, increased expression of A20 was observed even without administration of peony root extract. Long-term administration of peony root extract to the EL mouse resulted in a marked increase of expression of A20. These results suggested that an increase in A20 expression is the main molecular mechanism of protective action of peony root extract on neuron damage.

Peony root extract upregulates transthyretin and phosphoglycerate mutase in mouse cobalt focus seizure

Cobalt focus is a seizure focus model in which cerebral neurons exhibit long-lasting severe spike discharges, followed by neuronal death. However, the neuronal death is prevented when peony root extract (PR) is administered prior to cobalt application. We tested the hypothesis that PR modulates the expression of neuroprotective proteins in the cerebrum of mouse cobalt focus by proteomic analysis using two-dimensional polyacrylamide gel electrophoresis and mass spectrometry to screen for differentially expressed proteins. Analyses revealed that transthyretin, a carrier protein for thyroid hormones and retinoids, and the brain form of phosphoglycerate mutase, a glycolytic enzyme, were upregulated in the cobalt-treated mouse cerebrum and further increased by PR administration in association with upregulation of neurogranin/RC3, a target of the transcriptional activation by thyroid hormones and retinoids. These findings suggest that PR-induced protection of mouse cerebral neurons involves neurotrophic events caused by thyroid hormones and/or retinoids and enhanced glycolysis.