The novel
calmodulin (CaM) antagonist
DY-9760e (3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-
indazole dihydrochloride 3.5 hydrate) with an apparent
neuroprotective effect in vivo preferentially inhibits
neuronal nitric oxide synthase (nNOS), Ca2+/CaM-dependent
protein kinase IIalpha (CaMKIIalpha), and
calcineurin in vitro. In the present study, we investigated the molecular mechanism underlying its
neuroprotective effect with the gerbil transient forebrain
ischemia model, by focusing on its inhibition of these Ca2+/CaM-dependent
enzymes. Post-ischemic
DY-9760e treatment (5 mg/kg, i.p.) immediately after 5-min
ischemia significantly reduced the delayed neuronal death in the hippocampal CA1 region. CaMKIIalpha was transiently autophosphorylated immediately after reperfusion with concomitant sustained decrease in its total amounts in the Triton X-100-soluble fractions.
Calcineurin activity, accessed by the phosphorylation state of
dopamine- and cAMP-regulated phosphoprotein of Mr 32,000 (DARPP-32) at Thr34, was elevated at 6 h after reperfusion. Post-treatment of
DY-9760e had no effects on both CaMKIIalpha and DARPP-32 phosphorylation at 6 h after reperfusion. However,
DY-9760e significantly inhibited
nitrotyrosine formation, as a
biomarker of NO, and in turn,
peroxynitrite (ONOO-) production. These results suggest that
DY-9760e primarily inhibits Ca2+/CaM-dependent neuronal NOS, without any effects on
CaMKII and
calcineurin, and the inhibition of NO production possibly accounts for its neuroprotective action in brain ischemic injury.