Ca2+-mediated neuronal death in rat brain neuronal cultures by veratridine: protection by flunarizine

Neuronal cell degeneration was studied in vitro in primary rat brain neuronal cultures grown in serum-free, chemically defined, CDM R12 medium, by measuring lactate dehydrogenase (LDH) released in the culture medium. A Ca2+-dependent neuronal cell degeneration was observed after prolonged and transient exposure 30 microM veratridine. The release of LDH occurred gradually and could be completely prevented by 2 mM ethylene glycol bis (beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, 0.1 microM tetrodotoxin, and 1 microM flunarizine. Flunarizine was without effect on neuronal cell loss induced by 1 mM glutamate, 1 mM kainic acid, and 5 mM KCN. The lack of effect on neurotoxicity induced by 1 mM glutamate differentiates flunarizine from N-methyl-D-aspartate antagonists such as MK-801. The latter protected at nanomolar concentrations against glutamate-induced neuronal cell death but had a maximal effect only at 0.1 mM on the veratridine-induced released LDH. It is suggested that, besides the excitatory amino acid receptor pathway, prolonged opening of the veratridine-sensitive Na+ channel can be neurotoxic. The latter can be prevented by flunarizine. The role of Na+ channel blockers as therapeutic agents in cerebral ischemia is discussed.