Intracellular dopamine oxidation mediates rotenone-induced apoptosis in PC12 cells.
OBJECTIVE
To study the role of dopamine (DA) in rotenone-induced neurotoxicity in PC12 cells.


METHODS
Cell viability was assessed by detecting the leakage of lactate dehydrogenase (LDH) into the medium. Apoptosis rate was measured by flow cytometry. Caspase-3-like activity was measured by fluorescence assay using the probe Ac-DEVD-AMC. The level of intracellular hydrogen peroxide and other peroxides in PC12 cells were quantified by loading cells with 2'-7'-Dichlorodihydrofluorescein diacetate (DCFH-DA) in fluorescence assay. Lactic acid was measured spectrophotometrically. The DA levels in PC12 cells were determined by HPLC-ECD.


RESULTS
A 48-h incubation of PC12 cells with rotenone caused an apoptotic cell death and elevated intracellular reactive oxygen species (ROS) and lactic acid accumulation. Intracellular DA depletion with reserpine significantly attenuated rotenone-induced ROS accumulation and apoptotic cell death. No change was found in rotenone-induced ROS accumulation when cells were co-treated with deprenyl. Brief treatment with reserpine at the end of rotenone treatment had no effect on rotenone-induced neurotoxicity. However, when cells were first incubated with deprenyl, a monoamine oxidase-B inhibitor for 30 min then co-incubated with rotenone plus deprenyl, a brief treatment with reserpine enhanced cell injury.


CONCLUSIONS
Rotenone-induced apoptosis in PC12 cells was mediated by intracellular dopamine oxidation.
Liu
Hua-qing
HQ
Department of Pharmacology, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Zhu
Xing-zu
XZ
Weng
En-qi
EQ
D000818:Animals; D017209:Apoptosis / Q000187:drug effects*; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D007770:L-Lactate Dehydrogenase / Q000378:metabolism; D019344:Lactic Acid / Q000378:metabolism; D008996:Monoamine Oxidase Inhibitors / Q000494:pharmacology; D010084:Oxidation-Reduction; D016716:PC12 Cells; D051381:Rats; D017382:Reactive Oxygen Species / Q000378:metabolism; D012110:Reserpine / Q000494:pharmacology; D012402:Rotenone / Q000633:toxicity*; D012642:Selegiline / Q000494:pharmacology*
D008996:Monoamine Oxidase Inhibitors; D017382:Reactive Oxygen Species; D012402:Rotenone; D012642:Selegiline; D019344:Lactic Acid; D012110:Reserpine; D007770:L-Lactate Dehydrogenase; C505410:Casp3 protein, rat; D053148:Caspase 3; D020169:Caspases; D004298:Dopamine