Abstract
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 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 production were monitored in PE-treated cells by 4-amino-5-methylamino-2′,7′-difluorofluorescein and dihydroethidium, respectively, Ca2+-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 generation at 96 h was closely associated with an increased uncoupled eNOS level. Coincubation with DY-9836 (3 μM) during the last 48 h inhibited the aberrant 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 generation by uncoupled eNOS probably triggers PE-induced cardiomyocyte injury. Inhibition of abnormal and NO generation by DY-9836 treatment represents an attractive therapeutic strategy for PE/hypertrophy-induced cardiomyocyte injury.
Footnotes
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This work was supported by the Ministry of Education, Science, Sports and Culture of Japan [Grant 19390150] and the Smoking Research Foundation.
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ABBREVIATIONS: NOS, nitric-oxide synthase; eNOS, endothelial nitric-oxide synthase; nNOS, neuronal nitric-oxide synthase; iNOS, inducible nitric-oxide synthase; BH4, tetrahydrobiopterin; PE, phenylephrine; DMEM, Dulbecco's modified Eagle's medium; DAF-FM, 4-amino-5-methylamino-2′,7′-difluorofluorescein; DHE, dihydroethidium; TUNEL, TdT-mediated dUTP nick-end labeling; FBS, fetal bovine serum; PBS, phosphate-buffered saline; PAGE, polyacrylamide gel electrophoresis; TEMPOL, 4-hydroxy-2,2,6,6-tetramethylpiperidinyloxy; MES, 2-(N-morpholino)ethanesulfonic acid; l-NAME, Nω-nitro-l-arginine methyl ester; A23187, calcimycin; DY-9836, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxyindazole; DY-9760e, 3-[2-[4-(3-chloro-2-methylphenyl)-1-piperazinyl]ethyl]-5,6-dimethoxy-1-(4-imidazolylmethyl)-1H-indazole dihydrochloride 3.5 hydrate.
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↵ The online version of this article (available at http://molpharm.aspetjournals.org) contains supplemental material.
- Received July 17, 2008.
- Accepted October 24, 2008.
- The American Society for Pharmacology and Experimental Therapeutics
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