Hua-qing Liu1, Xing-zu Zhu, En-qi Weng. 1. Department of Pharmacology, Institute of Materia Medica, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai 201203, China.
Abstract
AIM: 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. CONCLUSION: Rotenone-induced apoptosis in PC12 cells was mediated by intracellular dopamine oxidation.
AIM: 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. CONCLUSION:Rotenone-induced apoptosis in PC12 cells was mediated by intracellular dopamine oxidation.
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