Phenylephrine-Induced Cardiomyocyte Injury Is Triggered by Superoxide Generation through Uncoupled Endothelial Nitric-Oxide Synthase and Ameliorated by 3-[2-[4-(3-Chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyinda zole (DY-9836), a Novel Calmodulin Antagonist

Department of Pharmacology, Graduate School of Pharmaceutical Sciences, Tohoku University, Sendai, Japan.
Molecular pharmacology (Impact Factor: 4.13). 10/2008; 75(1):101-12. DOI: 10.1124/mol.108.050716
Source: PubMed


The pathophysiological relevance of endothelial nitric-oxide synthase (eNOS)-induced superoxide production in cardiomyocyte injury after prolonged phenylephrine (PE) exposure remains unclear. The aims of this study were to define the mechanism of O2(*) production by uncoupled eNOS and evaluate the therapeutic potential of a novel calmodulin antagonist 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole (DY-9836) to rescue hypertrophied cardiomyocytes from PE-induced injury. In cultured rat cardiomyocytes, prolonged exposure for 96 h to PE led to translocation from membrane to cytosol of eNOS and breakdown of caveolin-3 and dystrophin. When NO and O2(*) production were monitored in PE-treated cells by 4-amino-5-methylamino-2',7'-difluorofluorescein and dihydroethidium, respectively, Ca(2+)-induced NO production elevated by 5.7-fold (p < 0.01) after 48-h PE treatment, and the basal NO concentration markedly elevated (16-fold; p < 0.01) after 96-h PE treatment. On the other hand, the O2(*) generation at 96 h was closely associated with an increased uncoupled eNOS level. Coincubation with DY-9836 (3 microM) during the last 48 h inhibited the aberrant O2(*) generation nearly completely and NO production by 72% (p < 0.01) after 96 h of PE treatment and inhibited the breakdown of caveolin-3/dystrophin in cardiomyocytes. PE-induced apoptosis assessed by TdT-mediated dUTP nick-end labeling staining was also attenuated by DY-9836 treatment. These results suggest that O2(*) generation by uncoupled eNOS probably triggers PE-induced cardiomyocyte injury. Inhibition of abnormal O2(*) and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.

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    • "It has been shown that oxidative inhibition of cystathionine β-synthase occurs, leading to accumulation of intracellular homocysteine [37]. Previous studies have demonstrated the production and accumulation of reactive oxygen species PE in cardiomyocytes [38]. Therefore, it is possible that PE-induced increase of homocysteine concentrations may result from the oxidative inhibition of the cystathionine β-synthase, which will be determined in our future studies. "
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    • "In situ DNA fragmentation was assessed using a TUNEL assay as previously described [20]. Images were recorded after counterstaining with TO-PRO3 (nuclei marker), and cardiac myocytes were identified by staining with anti-sarcomeric actinin (green). "
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