Elsevier

Neuroscience

Volume 121, Issue 2, 6 October 2003, Pages 379-386
Neuroscience

The neuroprotective effect of a novel calmodulin antagonist, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1h-indazole dihydrochloride 3.5 hydrate, in transient forebrain ischemia

https://doi.org/10.1016/S0306-4522(03)00490-1Get rights and content

Abstract

A 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, potently inhibits CaM-dependent nitric oxide synthase in situ. In the present study, we determined whether DY-9760e inhibits nitric oxide (NO) production and protein nitration by peroxynitrite (ONOO) formation in the hippocampal CA1 region of gerbils after transient forebrain ischemia.

In freely moving gerbils, NO production after 10-minute forebrain ischemia was monitored consecutively with in vivo brain microdialysis. Pretreatment with DY-9760e (50 mg/kg i.p.) significantly decreased the increased levels of NOx (NO metabolites, NO2 plus NO3) immediately after, 24 h after cerebral ischemia-reperfusion to the control levels of sham-operated animals. Western blot and immunohistochemical analyses using an anti-nitrotyrosine antibody as a marker of ONOO formation indicated a marked increase in nitrotyrosine immunoreactivity in the pyramidal neurons of the CA1 region 2 h after reperfusion, and DY-9760e significantly inhibited increased nitrotyrosine immunoreactivity. Coincident with the inhibition of the NO production and protein tyrosine nitration, pretreatment with DY-9760e rescued the delayed neuronal death in the hippocampal CA1 region. These results suggest that the inhibitory effects of DY-9760e on the NO-ONOO pathway partly account for its neuroprotective effects in cerebral ischemia.

Section snippets

Surgical preparation

All experiments were performed on male Mongolian gerbils (SLC, Shizuoka, Japan) weighing 60–70 g. All experimental procedures were approved by the Committee of Animal Experiments at Kumamoto University School of Medicine. All efforts were made to minimize the number of animals used and their suffering.

Gerbils were administered 50 mg/kg DY-9760e i.p. (Daiichi Pharmaceutical Co., Tokyo, Japan) dissolved in 10% dimethyl sulfoxide (DMSO; Sigma, St. Louis, MO, USA) in saline (10 mg/ml) or 50 mg/kg NG

Inhibitory effect of DY-9760e on NO production after 10-min forebrain ischemia

The basal level of NOx in 10-minute dialysate samples from the hippocampal CA1 region were 1.29±0.36, 1.08±0.22 and 1.21±0.18 μM (sham, vehicle and DY-9760e, respectively), and were not significantly different. Under freely moving condition without halothane anesthesia, which may affect NOS activity (Zuo et al., 1999), we successfully performed brain microdialysis to monitor NO production beyond 24 h after transient forebrain ischemia (Fig. 1). First, the levels of NOx in the sham-operated

Discussion

Accumulating evidence suggests that both nNOS and inducible NOS (iNOS) have detrimental effects on neurons in the ischemic brain, whereas endothelial NOS (eNOS) activity has protective effects Huang et al., 1994, Huang et al., 1996, Iadecola et al., 1997, Samdani et al., 1997. Targeted disruption of the nNOS gene also attenuates hippocampal injury after transient global ischemia (Panahian et al., 1996). In the present study, the newly developed CaM antagonist, DY-9760e, showed a powerful

Acknowledgements

This study was supported in part by Grants-in Aid for Scientific Research (14370035) from the ministry of Education, Science, Sports and Culture of Japan (K.F.).

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