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 thr 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 Ca2+ ionophore (A23187)-induced NO production in mouse N1E-115 neuroblastoma cells, at a concentration of Less than 1 mu M. In contrast, no apparent inhibitory effect on Ca2+/CaM-dependent protein kinase II activity was observed in cultured hippocampal neurons up to 5 mu M. These results suggest that the inhibitory effect of DY-9760e on CaM-dependent NOS activities underlies neuroprotective effects of the agent. BIOCHEM PHARMACOL 60;5:693-699, 2000. (C) 2000 Elsevier Science Inc.