Inhibition of VMAT-2 and DT-diaphorase induce cell death in a substantia nigra-derived cell Line - An experimental cell model for dopamine toxicity studies

We have induced intracellular dopamine oxidation to aminochrome in RCSN-3 cells derived from rat substantia nigra by inhibiting VMAT-2 with reserpine to increase free cytosolic dopamine concentration, to study aminochrome-dependent neurotoxicity in the absence of exogenous oxidizing agents such as metals, which may potentiate an aminochrome cytotoxic effect. The expression of VMAT-2 in RCSN-3 cells was determined by reverse transcriptase-polymerase chain reaction and immunocytochemistry. We observed double membrane bodies containing melanin when RCSN-3 cells were incubated with 100 mu M dopamine by using transmission electron microscopy. No significant difference in the cell death was observed when the cells were treated 100 mu M dopamine and 25 mu M reserpine in the absence or presence of 100 mu M dicoumarol, an inhibitor of DT-diaphorase. The lack of effect was due to the inhibitory action of 25 mu M reserpine on DT-diaphorase (K-i = 24 mu M). However, a significant increase in the cell death was observed when DT-diaphorase was inhibited when the cells were incubated with 1 mu M reserpine and 100 mu M dopamine for 12 h since at this concentration reserpine inhibits VMAT-2 but not DT-diaphorase. Under this condition, we observed (i) the formation of blebbing; (ii) chromatin condensation accompanied by the formation of massive patches in contact with the nuclear membrane; (iii) the smoothness of the cell's surface, that is, lack of surface microprojections; and (iv) mitochondrial damage characterized by disruption of cristae architecture, which remains closely packed; disorganization of the mitochondrial matrix due to separation of the outer membrane from the internal membrane and considerable enlargement of the intermembrane space; and disruption of the external mitochondrial membrane determined by transmission electron microscopy. These results support the proposed neuroprotective role of DT-diaphorase against aminochrome neurotoxicity, and it suggests that RCSN-3 cells incubated with reserpine and dopamine are an excellent and more physiological cellular experimental model to study the role of dopamine oxidation in neurotoxic effects of dopamine.