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

doi:10.1016/S0006-2952(00)00370-1

Biochemical Pharmacology

Volume 60, Issue 5, 1 September 2000, Pages 693-699

https://doi.org/10.1016/S0006-2952(00)00370-1 Get rights and content

Abstract

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 Ca²⁺ ionophore (A23187)-induced NO production in mouse N1E-115 neuroblastoma cells, at a concentration of less than 1 μM. In contrast, no apparent inhibitory effect on Ca²⁺/CaM-dependent protein kinase II activity was observed in cultured hippocampal neurons up to 5 μM. These results suggest that the inhibitory effect of DY-9760e on CaM-dependent NOS activities underlies neuroprotective effects of the agent.

Section snippets

Reagents

DY-9760e was synthesized by the Daiichi Pharmaceutical Co. [5]. The following chemicals and reagents were obtained from the indicated sources: l-[2,3-³H]arginine, DuPont-New England Nuclear; W-7, N-(6-aminohexyl)- 5-chloro-1-naphthalenesulfonamide, the Seikagaku Co.; calmodulin, Sigma; and HUVEC, Clonetics. nNOS was purified from rat cerebellum by the method of Bredt and Snyder [14]. The purity of nNOS was judged to be greater than 90% by SDS–PAGE, as shown in Fig. 1. The specific activity of

Inhibition of purified nNOS by DY-9760e and W-7

Since we have recently observed an inhibitory action of DY-9760e on various Ca²⁺/CaM-dependent enzymes [5], its effect on purified nNOS activity was examined. The 150-kDa nNOS purified from rat cerebellum was homogeneous by SDS–PAGE, as shown in Fig. 1A, and the ec₅₀ for CaM was 6 nM as reported previously [14]. In the presence of 10 nM CaM, the ic₅₀ for DY-9760e was 40 μM, which was similar to that for W-7 (ic₅₀ = 62 μM), a prototype CaM antagonist [22](Fig. 1B). The K_i values of DY-9760e and

Discussion

We previously reported that DY-9760e inhibits the various CaM-dependent enzymes with a competitive manner for CaM [5]. In addition, the drug has a neuroprotective effect in transient focal brain ischemia [6]. To analyze the molecular mechanisms underlying its neuroprotective action, we investigated the effects of DY-9760e on NOS activity in vitro and in situ. As expected, DY-9760e inhibited both nNOS and eNOS in crude preparations, and the K_i value (K_i = 0.9 μM) for purified nNOS was similar to

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β-amyloid accumulation in neurovascular units following brain embolism
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Nitric oxide (NO) toxicity is in part mediated by generation of peroxynitrite with concomitant production of superoxide under pathological brain conditions such as ischemia and Alzheimer’s disease. The pathophysiological relevance of endothelial nitric oxide synthase (eNOS) to brain embolism-induced neurovascular injury has not been documented. We found that microsphere embolism (ME)–induced aberrant eNOS expression in vascular endothelial cells likely mediates blood-brain barrier (BBB) disruption via peroxynitrite formation and in turn causes brain edema. We also demonstrated that a mild ME model was useful for investigating the sequential events of neurovascular injury followed by β-amyloid accumulation and tau hyperphosphorylation. Indeed, immunoblotting of purified brain microvessels revealed that β-amyloid accumulation significantly increased one week after ME induction and remained elevated for twelve weeks in those animals. Moreover, we also confirmed that peroxynitrite formation and eNOS uncoupling–mediated superoxide generation in microvessels are inhibited by a novel calmodulin inhibitor. Thus, peroxynitrite formation via elevated eNOS is associated with endothelial cell injury with concomitant β-amyloid accumulation in microvessels of aged rats. In this review, we focus on the detrimental effects of eNOS expression following brain embolism and introduce an attractive model representing progressive Alzheimer’s disease pathology in brain.
Allosteric inhibition of rat neuronal nitric-oxide synthase caused by interference with the binding of calmodulin to the enzyme
2007, Biochimica et Biophysica Acta - General Subjects
A sigmoid-type dependence on the inhibitor concentration was observed in the cytochrome c reductase activity for peptide inhibitors (mastoparan and melittin), calmodulin antagonists (W-7 and tamoxifen) and monobutyltin in a reconstituted system comprised of recombinant rat neuronal nitric-oxide synthase (nNOS) and calmodulin (CaM). The increase in the concentration of CaM in the system induced a decrease in the inhibitory effect, indicating that the inhibitors might interfere with the interaction between nNOS and CaM. The changes in the fluorescence spectra of dansylated CaM caused by the addition of mastoparan, melittin and monobutyltin indicated complex formation between CaM and those compounds, which led to the decrease in the effective concentration of CaM available to nNOS. The sigmoid-type inhibition of mastoparan and melittin fit the theoretical equations quite well, assuming that two CaM molecules bind cooperatively to one nNOS homodimer. Monobutyltin, tamoxifen and W-7 were found to inhibit nNOS activity by binding to the CaM binding site of the nNOS homodimer, in addition to the binding of the inhibitors to calmodulin. These compounds inhibited the l-citrulline formation of nNOS from l-arginine, and the inhibitory effects were abrogated by raising the concentration of calmodulin. It became clear that the binding of calmodulin to nNOS can be interfered with in two ways: (1) via a decrease in the effective concentration of calmodulin caused by complex formation between the inhibitor and calmodulin, and (2) via the inhibition of the binding of calmodulin to nNOS caused by the occupation of the binding site by the inhibitor.
Cytoprotective effect 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) against ischemia/reperfusion-induced injury in rat heart involves inhibition of fodrin breakdown and protein tyrosine nitration
2005, Journal of Pharmacological Sciences
We here assessed the effects 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, on infarct size in the rat heart subjected to ischemia/reperfusion. Rats were subjected to a 30-min coronary occlusion followed by a 24-h reperfusion. DY-9760e was intravenously infused for 20 min, starting at 20 min after coronary occlusion. Treatment with DY-9760e (10 mg/kg) significantly reduced the infarct size in the risk area assessed by Evans Blue/TTC (triphenyltetrazolium chloride) staining. DY-9760e treatment also ameliorated contractile dysfunction of the left ventricle 72 h after reperfusion. DY-9760e significantly inhibited fodrin breakdown and caspase-3 activation. The inhibitory effect of DY-9760e on the fodrin breakdown was prominent in the rim rather than in the center of the risk area. DY-9760e also blocked protein tyrosine nitration associated with infarction. These results suggest that the cardioprotective effect of DY-9760e involved inhibition of calpain/caspase activation and protein tyrosine nitration.
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
2004, Journal of Pharmacological Sciences
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 IIα (CaMKIIα), 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. CaMKIIα 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 CaMKIIα 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.
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
2003, Neuroscience
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 NO_x⁻ (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.
Calmodulin and calmodulin-dependent kinase II mediate neuronal cell death induced by depolarization
2003, Brain Research
Citation Excerpt :
In fact, the L-type Ca2+ channel mediates the cell death in our system, as shown by the protective action of the specific L-type Ca2+ channel blocker. It is well known that NOS and calcineurin play important roles in neuronal cell death [4,5,8,28]. In those studies, l-NAME, NNA, and FK-506 potently suppress the glutamate/NMDA toxicity in neurons.
Depolarization has been known to play an important role in the neuronal damage that occurs following cerebral ischemia. In the present study, we investigated the roles of calmodulin (CaM) and CaM-dependent enzymes in depolarization-induced neuronal cell death. Treatment of primary cortical neurons with 10 μM veratridine, a voltage sensitive Na⁺ channel activator, induced cell death as indicated by lactate dehydrogenase leakage from neurons. CaM antagonists (calmidazolium, trifluoperazine, W-7, and W-5) inhibited cell death induced by veratridine in a concentration-dependent manner. CaM kinase II (CaMKII) inhibitors (KN-62, KN-93, and myristoylated autocamtide-2 related inhibitory peptide), but not inhibitors of nitric oxide synthase or calcineurin, prevented veratridine-induced neuronal cell death. Veratridine rapidly activated CaMKII in neurons, and CaM antagonists and a CaMKII inhibitor suppressed the CaMKII activation. These results suggest that the CaM–CaMKII pathway contributes to depolarization-evoked cell death in neurons.

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NeuroscienceInhibition 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

Abstract

Section snippets

Reagents

Inhibition of purified nNOS by DY-9760e and W-7

Discussion

Neurosci Lett

Eur J Pharmacol

Eur J Pharmacol

Neuropharmacology

J Biol Chem

J Biol Chem

Anal Biochem

Anal Biochem

Anal Biochem

Neurosci Lett

J Biol Chem

J Biol Chem

Mechanisms of nitric oxide-mediated neurotoxicity in primary brain cultures

J Neurosci

Immunophilins mediate the neuroprotective effects of FK506 in focal cerebral ischemia

Nature

A specific inhibitor of calcium/calmodulin-dependent protein kinase-II provides neuroprotection against NMDA- and hypoxia/hypoglycemia-induced cell death

J Neurosci

Maturation of neuroblastoma cells in the presence of dimethylsulfoxide

Proc Natl Acad Sci USA

Neuroscience
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