Inhibition of neuronal nitric oxide synthase activity by 3-[2-[4-(3-chloro-2-methylphenyl)- 1-piperazinyl]ethyl]-5, 6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate (DY-9760e), a novel neuroprotective agent, in vitro and in cultured neuroblastoma cells in situ.

DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5, 6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate, a novel calmodulin (CaM) antagonist, possesses neuroprotective activity. In the current study, we examined the effects of DY-9760e on nitric oxide synthase (NOS) activities in vitro and on calcium ionophore-induced NO production in situ. DY-9760e inhibited both neuronal NOS and endothelial NOS activities without affecting inducible NOS activity. It also inhibited purified neuronal NOS activity with a potency similar to that seen for purified CaM kinase II activity in vitro. Furthermore, DY-9760e significantly inhibited Ca(2+) ionophore (A23187)-induced NO production in mouse N1E-115 neuroblastoma cells, at a concentration of less than 1 microM. In contrast, no apparent inhibitory effect on Ca(2+)/CaM-dependent protein kinase II activity was observed in cultured hippocampal neurons up to 5 microM. These results suggest that the inhibitory effect of DY-9760e on CaM-dependent NOS activities underlies neuroprotective effects of the agent.