Hiroyasu Furuichi

Behavior of intracellular cyclic nucleotide and calcium in pentylenetetrazole-induced bursting activity in snail neurons

To elucidate the intracellular mechanism of the bursting activity which is characteristic of seizure discharge, the behavior of the intracellular cyclic nucleotide and the intracellular calcium during pentylenetetrazole (PTZ)-induced bursting activity in snail neurons was investigated. Cyclic AMP was increased about 3-fold by the incubation of ganglia with PTZ. The effect of PTZ on phosphodiesterase activity measured using either cyclic AMP or cyclic GMP as substrate showed a slight increase in cyclic AMP phosphodiesterase activity. The release of calcium from the lysosome fraction was increased by the incubation of ganglia with dibutyryl cyclic AMP. Protein kinase activity was stimulated by the incubation of ganglia with PTZ. Adenylate cyclase activity was stimulated by the incubation of ganglia with PTZ. These findings suggest that PTZ-induced bursting activity in snail neurons is initiated by an intracellular increase of cyclic AMP, which promotes calcium release from lysosomes and induces protein kinase activation.

Effect of phenytoin on intracellular calcium and intracellular protein changes during pentylenetetrazole-induced bursting activity in snail neurons

Effects of phenytoin (PHT) on the intracellular calcium and intracellular protein changes during pentylenetetrazole (PTZ)-induced bursting activity in the neurons of the Japanese land snail Euhadra peliomphala were examined. In the examination with a computer controlled electron probe X-ray microanalyzer, PHT clearly inhibited the intracellular calcium shift induced by PTZ as well as the calcium binding state change near the cell membrane. PHT also clearly inhibited the intracellular protein changes induced by PTZ. PHT, however, did not show any change in the transmembrane ionic currents such as the sodium current, calcium current and potassium current. These findings suggest that one of the main sites of anticonvulsant action of PHT is pathologically changed intracellular calcium movement and intracellular protein changes during seizure discharge.

Intracellular calcium concentration during pentylenetetrazol-induced bursting activity in snail neurons

To clarify the role of intracellular free calcium in the provocation of bursting activity, the intracellular calcium concentration was measured using calcium-sensitive microelectrodes during pentylenetetrazol (PTZ)-induced bursting activity in snail neurons. In the PTZ-sensitive neurons of the snail, Euhadra peliomphala, the intracellular calcium concentration was higher than that in the normal state during PTZ-induced bursting activity, but lower than that which induced the calcium-activated potassium conductance elevation. By application of 50 microM calcium ionophore, A23187, a slight increase in intracellular calcium concentration with a slight depolarization occurred. Then a greater increase of intracellular calcium concentration with bursting activity was observed. By application of 100 microM A23187, a rapid and intense increase of intracellular calcium concentration with hyperpolarization was observed. These findings suggest that, for the provocation of bursting activity, sustained elevation of the intracellular calcium concentration, higher than that in the normal state but lower than that which evokes calcium-activated potassium conductance elevation, is required.

Cellular calcium binding state change during pentylenetetrazole-induced bursting activity in snail neurons

According to the examination by a computer controlled electron probe X-ray microanalyzer, the calcium binding state in the cell membrane during pentylenetetrazole-induced bursting activity was different from that of normal neuronal membrane.

Evidence that Ca2+/calmodulin-dependent protein phosphorylation is involved in the opening process of potassium channels in identified snail neurons

The effect of Ca2+/calmodulin-dependent protein phosphorylation on K+ channels was examined in snail neurons, using several pharmacological agents, the voltage clamp method and the pressure injection technique. H-7, a general protein kinase inhibitor, reduced the delayed outward K+ current (IKD) which was suppressed by tetraethylammonium. Ca2+/calmodulin-dependent protein kinase II, when injected into neurons which had been treated with H-7, transiently restored the reduced IKD nearly to the pre-H-7 level. However, this restoration was blocked by W-7, a calmodulin inhibitor. In contrast, the catalytic subunit of cAMP-dependent protein kinase or protein kinase C injected into the H-7-treated neurons had little effect on the current. These findings suggest that Ca2+/calmodulin-dependent protein phosphorylation is involved in the opening process of K+ channels.

Intramembraneous particle change during pentylenetetrazole-induced bursting activity in snail neurons

The freeze fracture study of snail neurons showed that during bursting activity induced by pentylenetetrazole, the intramembraneous particles demonstrated a different pattern from that of normal neurons.

Cyclic AMP-dependent protein phosphorylation is involved in the development of negative slope resistance and a reduction of the potassium current induced by pentylenetetrazole in identified snail neurons

The role of cAMP-dependent protein phosphorylation in the pentylenetetrazole (PTZ)-induced bursting activity was examined in snail neurons, using the voltage clamp method in combination with the pressure injection technique. The cAMP-dependent protein kinase inhibitors, protein kinase inhibitor isolated from rabbit muscle and isoquinolinesulfonamide, inhibited the PTZ-induced negative slope resistance (NSR) in the steady state I-V curve. These inhibitors also suppressed the action of PTZ on the delayed outward potassium current (IKD). This suppression was transiently abolished by intracellular injection of the catalytic subunit of cAMP-dependent protein kinase. These findings suggest that cAMP-dependent protein phosphorylation may be involved in both the development of the NSR and a reduction of the IKD by PTZ, leading to depolarizing phase of a bursting cycle.

Stimulation of synapsin I phosphorylation in synaptosomes by convulsants

Pentylenetetrazole markedly enhanced synapsin I (previously referred to as protein I) phosphorylation and synaptosomal uptake of Ca2+. Picrotoxin and strychnine sulfate caused a slight increase in the phosphorylation and no significant increase in Ca2+ uptake. The data indicate that pentylenetetrazole appears to influence this process.