Received for publication June 22, 2007.
Revised August 3, 2007.
Accepted for publication August 27, 2007.

Mitochondrial Complex I Inhibitor Rotenone-elicited Dopamine Redistribution from Vesicles to Cytosol in Human Dopaminergic SH-SY5Y Cells

Abstract

Parkinson's disease is a chronic neurodegenerative disorder characterized by loss of dopaminergic neurons in the substantia nigra. Rotenone, a pesticide, produces selective degeneration of dopaminergic neurons and motor dysfunction in rats. To determine the mechanisms underlying rotenone-induced neuronal death, we investigated whether intracellular dopamine plays a role in rotenone (0.1-0.4 µM)-induced apoptosis, using an in vitro model of human dopaminergic SH-SY5Y cells. The forty percent decrease of dopamine content by inhibition of dopamine synthesis suppressed rotenone-induced apoptosis. On the other hand, the thirty percent increase of dopamine content by inhibition of dopamine metabolism enhanced rotenone-induced apoptosis. Depletion of intracellular dopamine using reserpine (0.1-10 µM) also prevented rotenone-induced apoptosis, and this effect was counteracted by dopamine (10-100 µM) replenishment. Inhibition of dopamine reverse transport increased cytosolic dopamine and enhanced rotenone-induced apoptosis. We examined the intracellular localization of dopamine in rotenone-treated cells immunocytochemically and quantitatively. Rotenone induced dopamine redistribution from vesicles to the cytosol. In this process, rotenone stimulated reactive oxygen species and protein carbonylation, and decreased an antioxidant, glutathione. Addition of an antioxidant, N-acetylcysteine (3 mM), prevented dopamine being expelled from vesicles and inhibited rotenone-induced apoptosis. Our findings demonstrate rotenone-generated reactive oxygen species to be involved in dopamine redistribution to the cytosol, which in turn may play a role in rotenone-induced apoptosis of dopaminergic cells.

Key words: Parkinson's disease, apoptosis, degeneration, dopamine, dopaminergic, neuronal death