Anticonvulsive action of (+/-)-kavain estimated from its properties on stimulated synaptosomes and Na+ channel receptor sites.
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. Gultamate released from 4-aminopyridine-stimulated cerebrocortical synaptosomes and the cytosolic concentrations of Na+ and Ca2+ ([Na+]i, [Ca+]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 mumol/l but dose-dependently suppressed binding of [3H]batrachotoxin with an IC50 value of 88 mumol/l (Ki = 72 mumol/l) although displacement of [3H]batrachotoxin was restricted to 33% of control at 400 mumol/l (+/-)-kavain. In stimulated synaptosomes, 5 mmol/l 4-aminopyridine provoked an increase in [Na+]i and [Ca2+]i by 9 mmol/l Na+ and 235 nmol/l Ca2+. Comparable to the reduction in [3H]batrachotoxin binding, 400 mumol/l (+/-)-kavain suppressed the increase in [Na+]i and [Ca2+]i to 38 and 29% of control, respectively. Consistent with the increase in [Na+]i and [Ca2+]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 mumol/l (+/-)-kavain. KCl depolarization (40 mmol/l) provoked an increase in [Ca2+]i and glutamate release almost identical to the responses elicited by 4-aminopyridine but 400 mumol/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 Ca2+ channels, thereby suppressing the 4-aminopyridine-induced increase in [Na+]i, [Ca2+]i and the release of endogenous glutamate.
Gleitz
J
J
University Clinics Ulm, Institute of Naturheilkunde, Germany. johannes.gleitz@medizin.uni-ulm.de
Friese
J
J
Beile
A
A
Ameri
A
A
Peters
T
T
D015761:4-Aminopyridine / Q000008:administration & dosage; D000818:Animals; D000927:Anticonvulsants / Q000378:metabolism / Q000494:pharmacology*; D001496:Batrachotoxins / Q000378:metabolism; D002118:Calcium / Q000378:metabolism; D002540:Cerebral Cortex / Q000187:drug effects* / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D018698:Glutamic Acid / Q000378:metabolism; D008297:Male; D011753:Pyrones / Q000378:metabolism / Q000494:pharmacology*; D051381:Rats; D017208:Rats, Wistar; D012530:Saxitoxin / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D015222:Sodium Channels / Q000187:drug effects* / Q000378:metabolism; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism
D000927:Anticonvulsants; D001496:Batrachotoxins; D011753:Pyrones; D015222:Sodium Channels; D012530:Saxitoxin; D018698:Glutamic Acid; D012964:Sodium; D015761:4-Aminopyridine; D002118:Calcium; C011889:kavain