dizocilpine-maleate and diphenyliodonium

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 (EC50) were about 3 microM 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 Ca2+ elevation. The amount of 45Ca2+ influx induced by N-methyl-D-aspartate was also inhibited by diphenyliodonium in a dose-dependent manner; EC50 was about 3 microM. 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; EC50 was 5-8 microM. 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 Ca2+ influx. This conclusion is supported by the similarity of the stereochemical structures predicted by computer between diphenyliodonium and dizocilpine maleate.

Topics: Animals; Biphenyl Compounds; Calcium; Cells, Cultured; Dizocilpine Maleate; Excitatory Amino Acid Agonists; Excitatory Amino Acid Antagonists; Glutamic Acid; N-Methylaspartate; Neurons; Nitric Oxide Synthase; Onium Compounds; Rats; Rats, Wistar; Receptors, N-Methyl-D-Aspartate

Binding of [3H](+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-imi ne (MK-801) was significantly inhibited by the addition of several different compounds related to generation of nitric oxide (NO) at 100 microM in rat brain synaptic membranes. These included sodium nitroprusside, diphenyliodonium (DI), diphenyleneiodonium (DPI) and methylene blue. However, neither S-nitroso-N-acetylpenicillamine nor S-nitroso-L-glutathione inhibited binding at 100 microM. Both DI and DPI inhibited binding in a concentration-dependent manner at a concentration range of over 1 microM, while further addition of spermidine (SPD) significantly attenuated the potency of DPI to inhibit binding without affecting that of DI. In contrast, SPD induced significant potentiation of the ability of unlabelled MK-801 to displace [3H]MK-801 binding in a fashion sensitive to antagonism by the novel polyamine antagonist bis-(3-aminopropyl)nonanediamine. This novel polyamine antagonist also prevented the reversing effect of SPD on inhibition by DPI of [3H]MK-801 binding. Moreover, DPI competitively exacerbated the ability of SPD to potentiate [3H]MK-801 binding in the presence of both L-glutamic acid and glycine at maximally effective concentrations. On the other hand, SPD was effective in reversing the inhibition by DPI in cerebellar, but not hippocampal, synaptic membranes. These results suggest that both DI and DPI may modulate synaptic responses mediated by the N-methyl-D-aspartate receptor through inhibition of opening processes of the ion channel in a manner irrespective of generation of NO radicals in particular situations. Possible involvement of the polyamine domain in the inhibition by DPI is also suggested.

Topics: Animals; Biphenyl Compounds; Brain; Diamines; Dizocilpine Maleate; Drug Evaluation, Preclinical; Drug Synergism; Enzyme Inhibitors; Free Radicals; Male; Nitric Oxide Synthase; Onium Compounds; Radioligand Assay; Rats; Rats, Wistar; Spermidine; Synaptic Membranes