Dopamine-induced apoptosis is mediated by oxidative stress and Is enhanced by cyanide in differentiated PC12 cells.
Dopamine (DA) oxidation and the generation of reactive oxygen species (ROS) may contribute to the degeneration of dopaminergic neurons underlying various neurological conditions. The present study demonstrates that DA-induced cytotoxicity in differentiated PC12 cells is mediated by ROS and mitochondrial inhibition. Because cyanide induces parkinson-like symptoms and is an inhibitor of the antioxidant system and mitochondrial function, cells were treated with KCN to study DA toxicity in an impaired neuronal system. Differentiated PC12 cells were exposed to DA, KCN, or a combination of the two for 12-36 h. Lactate dehydrogenase (LDH) assays indicated that both DA (100-500 microM) and KCN (100-500 microM) induced a concentration- and time-dependent cell death and that their combination produced an increase in cytotoxicity. Apoptotic death, measured by Hoechst dye and TUNEL (terminal deoxynucleotidyltransferase dUTP nick end-labeling) staining, was also concentration- and time-dependent for DA and KCN. DA plus KCN produced an increase in apoptosis, indicating that KCN, and thus an impaired system, enhances DA-induced apoptosis. To study the mechanism(s) of DA toxicity, cells were pretreated with a series of compounds and incubated with DA (300 microM) and/or KCN (100 microM) for 24 h. Nomifensine, a DA reuptake inhibitor, rescued nearly 60-70% of the cells from DA- and DA plus KCN-induced apoptosis, suggesting that DA toxicity is in part mediated intracellularly. Pretreatment with antioxidants attenuated DA- and KCN-induced apoptosis, indicating the involvement of oxidative species. Furthermore, buthionine sulfoximine, an inhibitor of glutathione synthesis, increased the apoptotic response, which was reversed when cells were pretreated with antioxidants. DA and DA plus KCN produced a significant increase in intracellular oxidant generation, supporting the involvement of oxidative stress in DA-induced apoptosis. The nitric oxide synthase inhibitor N(G)-nitro-L-arginine methyl ester and the peroxynitrite scavenger uric acid blocked apoptosis and oxidant production, indicating involvement of nitric oxide. These results suggest that DA neurotoxicity is enhanced under the conditions induced by cyanide and involves both ROS and nitric oxide-mediated oxidative stress as an initiator of apoptosis.
Jones
D C
DC
Neurotoxicology Laboratory, Department of Medicinal Chemistry and Molecular Pharmacology, Purdue University, West Lafayette, IN 47907- 1333, USA.
Gunasekar
P G
PG
Borowitz
J L
JL
Isom
G E
GE
D000818:Animals; D017209:Apoptosis / Q000187:drug effects* / Q000502:physiology; D002454:Cell Differentiation; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004298:Dopamine / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009474:Neurons / Q000166:cytology* / Q000187:drug effects / Q000201:enzymology; D009569:Nitric Oxide / Q000378:metabolism; D018384:Oxidative Stress / Q000187:drug effects* / Q000502:physiology; D016716:PC12 Cells; D011190:Potassium Cyanide / Q000494:pharmacology*; D051381:Rats
D004791:Enzyme Inhibitors; D009569:Nitric Oxide; D007770:L-Lactate Dehydrogenase; D011190:Potassium Cyanide; D004298:Dopamine