Stimulus-dependent neuronal cell responses in SH-SY5Y neuroblastoma cells.

The aim of the present study was to elucidate the intracellular mechanisms that cause neuronal cell death following exposure to excitatory neurotransmitter­induced neurotoxicity, neurotoxins and oxidative stress. Human SH­SY5Y neuroblastoma cells were exposed to various stimuli, including glutamate, 6­hydroxydopamine (6­OHDA), and glucose oxidase, and cell viability was determined by MTT assay. Early apoptosis and necrosis were examined by Annexin V/propidium iodide double staining and flow cytometric analysis. Intracellular calcium ion concentration and mitochondrial membrane potential were assessed by Fluo­3a and JC­1 staining, respectively. In addition, protein expression of receptor­interacting protein (RIP) kinase 1 and RIP kinase 3 were evaluated by western blotting. Glutamate, 6­OHDA and glucose oxidase treatment decreased cell viability. Glutamate induced apoptosis and necrosis, whereas, 6­OHDA induced cell necrosis and glucose oxidase induced apoptosis. Furthermore, glutamate, 6­OHDA or glucose oxidase treatment significantly increased intracellular calcium concentrations (P<0.05). The effect of glutamate on mitochondrial membrane potential varied with high and low concentrations, whereas 6­OHDA and glucose oxidase significantly increased the mitochondrial membrane potential in the SH­SY5Y cells (P<0.05). Glutamate significantly upregulated expression levels of RIP kinase 1 (P<0.05), but not RIP kinase 3. These findings demonstrate that the response of SH­SY5Y cells varies with the stimuli. Furthermore, RIP kinase 1 may specifically regulate programmed necrosis in glutamate­mediated excitatory toxicity, but not in cell damage induced by either 6-OHDA or glucose oxidase.