Abstract

The present study examined the role of nitric oxide in cocaine-induced apoptosis in bovine coronary artery endothelial cells (BCAECs). Cocaine produced a time-dependent decrease in cell viability and an increase in apoptosis in BCAECs, which were blocked by the nitric oxide donors DETA-NONOate (DETA-NO) andS-nitroso-N-acetyl-penicillamine. In accordance, cocaine decreased nitric oxide production in BCAECs at each time point of the study. Cocaine significantly increased caspase-3 activity that was blocked by the inhibitors of cytochromec release (cyclosporin A), caspase-3 (Ac-DEVD-CHO), and caspase-9 (Z-LEHD-FMK), respectively. In addition, cocaine activated caspase-9, which was blocked by cyclosporin A and Z-LEHD-FMK. Ac-DEVD-CHO only partially blocked cocaine-induced caspase-9 activity. DETA-NO (20 μM) blocked cocaine-mediated activation of both caspase-9 and caspase-3. Cocaine decreased Bcl-2 protein levels, which was partially blocked by Ac-DEVD-CHO and Z-LEHD-FMK, but not by DETA-NO. Furthermore, cocaine induced a translocation of Bax from the cytosol to the mitochondria in BCAECs, and increased Bax levels in mitochondria by 2.2-fold. In accordance, cytosolic Bax levels decreased about 42%. Neither Ac-DEVD-CHO nor DETA-NO affected cocaine-induced translocation of Bax. We conclude that cocaine-induced Bcl-2 protein down-regulation and Bax translocation to the mitochondria are upstream signals of caspase-9 activation that precedes caspase-3. Cocaine-induced attenuation of nitric oxide plays a key role in the activation of the caspase cascade in BCAECs.