Protection by diphenyliodonium against glutamate neurotoxicity due to blocking of N-methyl- d-aspartate receptors

The protective effect of diphenyliodonium, known as an inhibitor of flavin enzymes including nitric oxide synthases, was examined against the neurotoxicity of excitatory amino acids on cultured spinal neurons of the rat. Diphenyliodonium reduced the neuronal damage induced by 15-min exposure to glutamate or N-methyl- d-aspartate in a dose-dependent manner; half effective concentrations (EC 50) were about 3 μM for both. Protection was only observed when diphenyliodonium was added into the exposure medium. Diphenyliodonium showed no effect on the toxicity induced by 24 h exposure to non- N-methyl- d-aspartate receptor agonists. Using a microfluorometry technique with Fura 2, we observed that diphenyliodonium reversibly inhibited the N-methyl- d-aspartate-evoked intracellular Ca 2+ elevation. The amount of 45Ca 2+ influx induced by N-methyl- d-aspartate was also inhibited by diphenyliodonium in a dose-dependent manner; EC 50 was about 3 μM. Furthermore, we examined the effect of diphenyliodonium on an opening activity of the N-methyl- d-aspartate receptors estimated by binding of dizocilpine maleate to membrane fractions from whole brain of adult rat and from cultured spinal neurons. Diphenyliodonium inhibited the binding of dizocilpine maleate dose-dependently; EC 50, was 5–8 μM. These results suggest that diphenyliodonium is a new antagonist to the N-methyl- d-aspartate receptors and that diphenyliodonium protects neurons against glutamate toxicity due to a direct blocking of the Ca 2+ influx. This conclusion is supported by the similarity of the stereochemical structures predicted by computer between diphenyliodonium and dizocilpine maleate.