Anticonvulsive action of (±)-kavain estimated from its properties on stimulated synaptosomes and Na + channel receptor sites

Abstract

Kava pyrones are constituents of the intoxicating pepper ( Piper methysticum Forst.), which has been shown to be anticonvulsive. The question of how the excitability of neurons is affected was investigated by determining the interaction of (±)-kavain with epitopes (site 1, site 2) of voltage-dependent Na + channels and the action of (±)-kavain on 4-aminopyridine-stimulated synaptosomes as model of repetitive firing neurons. [ 3H]Saxitoxin and [ 3H]batrachotoxin were used for radioligand-binding assays performed with synaptosomal membranes. Glutamate released from 4-aminopyridine-stimulated cerebrocortical synaptosomes and the cytosolic concentrations of Na + and Ca 2+ ([Na +] i, [Ca 2+] i) were detected fluorometrically by using an enzyme-linked assay, sodium-binding benzofuranisophthalate (SBFI) and Fura-2, respectively. (±)-Kavain failed to compete with [ 3H]saxitoxin up to 400 μmol/l but dose-dependently suppressed binding of [ 3H]batrachotoxin with an IC 50 value of 88 μmol/l ( K i = 72 μmol/l) although displacement of [ 3H]batrachotoxin was restricted to 33% of control at 400 μmol/l (±)-kavain. In stimulated synaptosomes, 5 mmol/1 4-aminopyridine provoked an increase in [Na +] i and [Ca 2+] i by 9 mmol/l Na + and 235 nmol/l Ca 2+. Comparable to the reduction in [ 3H]batrachotoxin binding, 400 μmol/l (±)-kavain suppressed the increase in [Na +] i and [Ca 2+] i to 38 and 29% of control, respectively. Consistent with the increase in [Na +] i and [Ca 2+] i, 5 mmol/l 4-aminopyridine provoked glutamate release (rate: 38 pmol/s∗mg protein) which was dose-dependently diminished to 60% of control by 400 μmol/l (±)-kavain. KCl depolarization (40 mmol/l) provoked an increase in [Ca 2+] i and glutamate release almost identical to the responses elicited by 4-aminopyridine but 400 μmol/l (±)-kavain suppressed only the rate of glutamate release by 9% of control. The data suggest an interaction of (±)-kavain with voltage-dependent Na + and Ca 2+ channels, thereby suppressing the 4-aminopyridine-induced increase in [Na +] i, [Ca 2+] i and the release of endogenous glutamate.