Rotenone is an inhibitor of mitochondrial complex I that produces a model of Parkinson's disease (PD), in which neurons undergo dopamine release dysfunction and other features. In neurons, exocytosis is one of the processes associated with dopamine release and is dependent on Ca2+ dynamic changes of the cell. In the present study, we have investigated the exocytosis of dopamine and the involvement of Ca2+ in dopamine release in PC12 cells administrated with rotenone. Results demonstrated that rotenone led to an elevation of intracellular Ca2+ through Ca2+ influx by opening of the voltage-gated Ca2+ channel and influenced the soluble N-ethylmaleimide attachment protein receptor (SNARE) proteins expression (including syntaxin, vesicle-associated membrane protein 2 (VAMP(2)) and synaptosome-associated protein 25 (SNAP-25)); pretreatment with a blocker of L-type voltage-activated Ca2+ channels (nifedipine) decreased the intracellular dopamine levels and ROS formation, increased the cell viability and enhanced the neurite outgrowth and exocytosis of synaptic vesicles. These results indicated that the involvement of intracellular Ca2+ was one of the factors resulting in suppression of dopamine release suppression in PC12 cells intoxicated with rotenone, which was associated with the rotenone-induced dopamine neurotoxicity.

To investigate the effects of rotenone on dopamine transporter (DAT) and vesicular monoamine transporter 2 (VMAT2 ) in PC12 cells. PC12 cells were exposed to rotenone with different concentrations for 24 h. Then HE staining was used to observe the action of rotenone on the morphology of PC12 cells. The expression changes of TH,DAT and VMAT2 proteins in PC12 cells were observed with immunohistochemical staining. RT-PCR was employed to detect the mRNA expressions of DAT and VMAT2 in PC12 cells. Na+/K+-ATPase activity of the PC12 cells was measured biochemically. The results showed that the morphology of PC12 cells was affected by 1.0 mmol/L rotenone,and cytoblastema dyeing is uneven,circular cell body,cell nucleus anomalous and karyoplasm proportion increases. The number TH positive cells decreased in the experimental group compared with control group (P<0.05). Protein and mRNA expressions of DAT and VMAT2 significantly increased and decreased respectively in the PC12 cells administrated with rotenone,which presented a dose-dependent manner. The activity of Na+/K+-ATPase decreased significantly in the PC12 cells administrated with 1.0 mmol/L rotenone compared to the control group (P<0.05). These results suggest that the abnormal expressions of DAT and VMAT2 may be involved in the action of rotenone induced PC12 cells toxicity.

鱼藤酮作为广泛应用的杀虫剂可以导致多巴胺能神经元退变[1-2].鱼藤酮透过血脑屏障以及细胞膜,不依赖多巴胺转运体(dompamine transporter,DAT)就可以直接进入胞质选择性作用于线粒体而造成细胞死亡[3].

Objective To study the effects of rotenone on glutamate uptaking in primary cultured astrocytes.Methods The extracellular glutamate was detected by high performance liquid chromatography(HPLC),the uptaking ability of glutamate was detected with isotope lebeling method and the mRNA and protein expression of glutamate transporter were detected using RTPCR and Western blot,respectively.Results It was showed that the extracellular rotenone level was obviously risen,shown the uptaking function of astrocyte to glutamate was decreased,Meanwhile,the expression of GLAST(glutamate/aspartate transporter)mRNA and protein also decreased significantly in 1.0 and 2.0 μmol/L rotenone exposed groups,but the expression of glutamate transporter-1(GLT-1)enhanced obviously.Conclusions It was suggested that rotenone might reduce the uptaking ability of astrocyte to glutamate,induce the extracellular level of Glu risen,the down-regulation of GLAST may be the main reason of elevated extracellular Glu level induced by rotenone,while the increased expression of GLT-1 might represent a self-protective mechanism by nerve cells for limiting the neurotoxicity by Glu.

Rotenone, a mitochondrial complex-I inhibitor, has been verified to cause dopaminergic neurons degeneration in vivo and in vitro, and the substantia nigra pars compacta (SNc) and the striatum are the main target organs of rotenone in the rat brain. However, whether rotenone could cause damage to other regions in the brain has been unclear till now. To address this question, the rotenone-induced neurotoxicity in the hippocampal neurons was investigated in the present study. Rotenone (4mg/kg) was given to the male Sprague-Dawley rats per day for up to 4 weeks by using the osmotic minipumps. Results showed that neurodegeneration was formed and phosphorylated ERK1/2 (p-ERK1/2) was induced in the hippocampus of rats following rotenone treatment. In additionally, Ras, PKA and PKC were also activated and free [Ca(2+)](i) was increased in the cytoplasm of the hippocampus neurons. To determine how ERK cascade was activated, studies in the primary cultured hippocampus neurons were carried out in a further. Cell viability was reduced, and also apoptosis was induced in vitro following rotenone administration. Expressions of p-ERK1/2 were also enhanced evidently in the cultured neurons treated by rotenone. Free [Ca(2+)](i) was also increased in the cultured neurons induced by rotenone. However, this influx might not take main effect in ERK1/2 phosphorylation. In conclusion, Ras-Raf-1-MEK-ERK1/2 classic signal pathway, not by PKA/PKC alternative pathway may be the mainly contributor to the ERK1/2 phosphorylation. And also, Ras protein is the dominant activator in the ERK phosphorylation induced by rotenone.

Objective To investigate the effects of rotenone on the apoptosis and expression of extracellular signal regulated kinases 1 and 2 (ERK1/2) proteins in PC12 cells in order to find out correlation between ERK1/2 and rotenone-induced apoptosis of PC12 cells. Methods The model of PC12 cells treated by rotenone was established. The morphological changes of PC12 cells induced by rotenone were observed by microscope, and cell apoptosis rates were detected by flow cytometry. The expression and localization of ERK1/2 were determined by Western blot and immunohistochemical methods. Results After treatment with rotenone for 24 hours, PC12 cells showed evident morphological changes and higher apoptosis rates, and the changes became more evident as a higher-dose rotenone was given. Pretreatment with MEK inhibitor PD98059(20 μmol/L) for an hour had not changed cell apoptosis rates significantly compared with rotenone intoxication merely. The phosphorylated ERK1/2 expressions were significantly up-regulated at protein levels and mostly located in cytoplasm. Conclusions Rotenone could induce apoptosis of PC12 cells, and the expression of phosphorylated ERK1/2 proteins is enhanced by activation of MAPK/ERK signal cascade.

Objective To study degenerative neurons in striatum and hippocampus of rats induced by chronic rotenone intoxication.Methods The model of chronic rotenone intoxication was established in rats by the subcutaneous implantation of miniosmotic pumps. The general state of the rats were observed,and the degenerative neurons and normal neurons in the striatum and hippocampus were detected by Fluoro-JadeB combined with fluorescent Nissl staining.Results The noticeable syndromes like Parkinson’s disease appeared in the rotenone-intoxicateed rats,in whom ,there were a number of the positive degenerative neurons in the striaum and hippocampus.The above-mentioned changes were not found in the control group.Conclusions Compared with traditional rotenone subcutaneous injection,osmotic subcutaneous implantation is an ideal way to establish rotenone chronic intoxication model.Rotenone can cause neuronal degeneration in the striatum and hippocampus of rat.

Objective To investigate the mechanism of oxidative stress in rotenone neurotoxicity onto dopaminergic neuron.Methods High differentiated PC12 cells as dopaminergic neurons were treated by different concentrations of rotenone.Cell morphology was observed with inverted phase contrast microscope and transmission electron microscope.SOD and MDA were detected with biochemical assay.And the specific fluorescent probe(DCF-DA) was used to examine the ROS in PC12 cells.Results After being treated with rotenone for 24 hours,most of PC12 cells treated by rotenone became smaller and rounder.The structure and metabolism of mitochondrial changed.Cell proliferation inhibition increased and cell activity decreased significantly when treated by rotenone.SOD increased and MDA decreased.The intensity of fluorescence was more obvious in PC12 cells treated by rotenone compared with control group.Conclusions In vitro,rotenone should be neurotoxic to dopaminergic neuron.It was suggested that rotenone might affect the metabolism of oxidative stress in the pathogenesis of dopaminergic neuron.