Secondary excitotoxicity contributes to dopamine-induced apoptosis of dopaminergic neuronal cultures

Dopamine (DA) and related catechols may contribute to selective degeneration of dopaminergic neurons in the substantia nigra in Parkinson's disease. To investigate whether DA induces apoptosis of dopaminergic neurons, we characterized the effects of various concentrations of exogenous DA on a substantia nigra/neuroblastoma hybrid cell line (MES 23.5 or MES). The hybrid MES cells were maintained in the presence of 50 microM glutamate in logarithmic growth on poly-D-lysine-precoated T-75 flasks and plated either onto petri dishes with glass coverslips for morphological studies or onto 6-well plates for quantification of apoptosis by flow cytometry. The results showed that DA exposure (0.5-20 microM) induced time- and dose-dependent apoptotic cell death of MES cells. To further analyze the mechanism responsible for DA-mediated apoptosis, we repeated the experiments at 20 microM DA in the presence or absence of 40 microM nomifensine, a DA re-uptake inhibitor, and 25 microM 2-amino-5-phosphonopentanoic acid (AP5), an N-methyl-D-aspartate (NMDA) receptor antagonist. The data indicate that both compounds significantly prevented DA-induced apoptosis of MES cells and that combination of AP5 and nomifensine provided greater protection against DA toxicity than AP5 alone. These results suggest for the first time that DA-induced apoptosis in dopaminergic neurons is partially attributable to increased vulnerability of these cells to non-toxic levels of excitatory amino acids, i.e., secondary excitotoxicity.