MPP⁺-induced degeneration is potentiated by dicoumarol in cultures of the RCSN-3 dopaminergic cell line. Implications of neuromelanin in oxidative metabolism of dopamine neurotoxicity

We have tested the idea that oxidative metabolism of dopamine may be involved in MPTP toxicity using the RCSN-3 cell line derived from the substantia nigra of an adult rat. Treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (10 μM), MPTP combined with 40 μM dicoumarol (an inhibitor of DT-diaphorase) and dicoumarol alone, did not induce toxicity in RCSN-3 cells after 72 h incubation. The lack of toxicity MPTP-treated RCSN-3 cells may be explained by the fact that they are unable to metabolize MPTP to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridinium ion (MPP⁺) as determined by HPLC. Incubation for 72 h with 100 μM MPP⁺ induced 6.6 ± 1.4% cell death of RCSN-3 cells compared to 3.5 ± 0.4 observed in control cells. However, when the cells were treated with 100 μM MPP⁺ and 40 μM dicoumarol, cell death increased 4-fold compared to that of cells treated solely with MPP⁺ (27 ± 2%; P < 0.001). Underthese conditions, a significant increase in DNA fragmentation (3-fold compared to MPP⁺ alone; P < 0.01) and in calpain activation (P < 0.05 compared to control) was evident. The inhibition of DT-diaphorase by dicoumarol supports the idea that oxidative metabolism of dopamine is involved in MPP⁺ toxicity in RCSN-3 cells.