Abstract
In the present study, we examined the direct cytotoxic effects of cocaine on fetal cardiac myocytes. Cocaine treatment of cultured fetal rat (21 days) myocardial cells (FRMCs) induced a time- and concentration-dependent increase in apoptotic cells in FRMCs. Cocaine induced surface exposure of phosphatidylserine in FRMCs at 12-h treatment and increased apoptotic cells up to 96 h. Corresponding DNA fragmentation induced by cocaine in these cells was demonstrated in situ by terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling assay and by electrophoresis of labeled DNA fragments, showing the characteristic apoptotic ladders. The pD2 and maximum increase of cocaine-induced apoptosis in FRMCs were 4.3 and 3.2-fold, respectively. Both caspase-9 and caspase-3 inhibitors (Z-LEHD-FMK and Ac-DEVD-CHO, respectively) blocked cocaine-induced apoptosis. In addition, cyclosporin A inhibited cocaine-induced apoptosis in a concentration-dependent manner with an IC50 value of 0.1 μM. The maximum of 86% inhibition was obtained with 3 μM cyclosporin A. Cocaine induced the release of cytochromec from the mitochondria and increased its levels in the cytosol by 3.1-fold. In accordance, the level of cytochromec in the mitochondria fraction decreased by ∼60%. Cocaine-induced translocation of cytochrome c was inhibited by cyclosporin A. The results indicate that cocaine has a direct cytotoxic effect on fetal cardiomyocytes by inducing apoptosis in the cells. Furthermore, the release of cytochrome cfrom the mitochondria and its subsequent activation of caspase-9 and caspase-3 play a key role in cocaine-induced apoptosis.
Footnotes
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Send reprint requests to: Lubo Zhang, Ph.D., Center for Perinatal Biology, Department of Pharmacology, Loma Linda University School of Medicine, Loma Linda, CA 92350. E-mail: lzhang{at}som.llu.edu
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↵1 This work was supported in part by National Institutes of Health Grants HL54094 and HL57787, American Heart Association Grant-in-Aid 96007560, and Loma Linda University School of Medicine.
- Abbreviations:
- FRMC
- fetal rat myocardial cell
- FITC
- fluorescein isothiocyanate
- TUNEL
- terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling
- Received June 21, 1999.
- Accepted August 17, 1999.
- The American Society for Pharmacology and Experimental Therapeutics
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