The post-ischemic administration of 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 calmodulin antagonist, prevents delayed neuronal death in gerbil hippocampus.
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.
Hashiguchi
Akihito
A
Department of Neurosurgery, Kumamoto University School of Medicine, Kumamoto-city, Japan.
Yano
Shigetoshi
S
Morioka
Motohiro
M
Hamada
Junichiro
J
Shirasaki
Yasufumi
Y
Kochi
Masato
M
Fukunaga
Kohji
K
D000818:Animals; D002545:Brain Ischemia / Q000188:drug therapy* / Q000378:metabolism / Q000473:pathology; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase; D002147:Calmodulin / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D005849:Gerbillinae; D006624:Hippocampus / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D007191:Indazoles / Q000008:administration & dosage*; D008297:Male; D009474:Neurons / Q000187:drug effects* / Q000378:metabolism / Q000473:pathology; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism
C109602:3-(2-(4-(3-chloro-2-methylphenyl)1-piperazinyl)ethyl)5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazol dihydrochloride 3.5 hydrate; D002147:Calmodulin; D007191:Indazoles; D017346:Protein Serine-Threonine Kinases; D054737:Calcium-Calmodulin-Dependent Protein Kinase Kinase