Norepinephrine induces apoptosis in neonatal rat endothelial cells via a ROS-dependent JNK activation pathway

Abstract

Our previous study demonstrated that norepinephrine (NE) induces endothelial apoptosis mainly through down-regulation of Bcl-2 protein and activation of the beta-adrenergic and caspase-2 pathways. However, whether reactive oxygen species (ROS) and mitogen-activated protein kinases (MAPKs) are involved in this signal transduction remains unknown. Endothelial cells cultured from neonatal rat heart were treated with 100 microM NE. Proteins of MAPKs and Bcl-2 family were assayed by Western blotting. Apoptosis was determined by terminal deoxynucleotidyl transferase-mediated nick end-labeling assay. ROS was analyzed with flow cytometry. Caspase activity was measured using specific fluorogenic substrates. Treatment with NE increased intracellular ROS level and extracellular signal-regulated kinase (ERK), c-Jun N-terminal kinase (JNK), and p38 phosphorylation. Whereas the phosphorylated form of Akt was decreased. The NE-induced apoptosis was abrogated by SP600125 (a specific inhibitor of JNK). Antioxidants such as vitamin C and N-acetyl cysteine inhibited NE-induced ROS production, JNK phosphorylation, caspase activation and apoptosis. Exogenously added superoxide dismutase or catalase markedly diminished NE-induced ROS production and cell death. In conclusions, our study is the first report documenting that NE induces apoptosis in neonatal rat endothelial cells via a ROS-dependent JNK activation pathway. Antioxidants may be useful in the prevention and management of NE-mediated endothelial apoptosis during heart failure.