Cocaine induces apoptosis in fetal myocardial cells through a mitochondria-dependent pathway.
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 pD(2) 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 IC(50) value of 0.1 microM. The maximum of 86% inhibition was obtained with 3 microM cyclosporin A. Cocaine induced the release of cytochrome c from the mitochondria and increased its levels in the cytosol by 3.1-fold. In accordance, the level of cytochrome c in the mitochondria fraction decreased by approximately 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 c from the mitochondria and its subsequent activation of caspase-9 and caspase-3 play a key role in cocaine-induced apoptosis.
Xiao
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Center for Perinatal Biology, Department of Pharmacology and Physiology, Loma Linda University School of Medicine, Loma Linda, CA 92350, USA.
He
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Gilbert
R D
RD
Zhang
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D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D061945:Caspase Inhibitors; D002478:Cells, Cultured; D003042:Cocaine / Q000494:pharmacology*; D016572:Cyclosporine / Q000494:pharmacology; D003574:Cytochrome c Group / Q000378:metabolism; D053938:DNA Fragmentation; D004305:Dose-Response Relationship, Drug; D004347:Drug Interactions; D006321:Heart / Q000187:drug effects* / Q000196:embryology; D020287:In Situ Nick-End Labeling; D008929:Mitochondria, Heart / Q000502:physiology*; D010718:Phosphatidylserines / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D013997:Time Factors
D061945:Caspase Inhibitors; D003574:Cytochrome c Group; D010718:Phosphatidylserines; D016572:Cyclosporine; D003042:Cocaine