title: Overexpression of VMAT-2 and DT-diaphorase protects substantia nigra-derived cells against aminochrome neurotoxicity.
abstract: We tested the hypothesis that both VMAT-2 and DT-diaphorase are an important cellular defense against aminochrome-dependent neurotoxicity during dopamine oxidation. A cell line with VMAT-2 and DT-diaphorase over-expressed was created. The transfection of RCSN-3 cells with a bicistronic plasmid coding for VMAT-2 fused with GFP-IRES-DT-diaphorase cDNA induced a significant increase in protein expression of VMAT-2 (7-fold; P<0.001) and DT-diaphorase (9-fold; P<0.001), accompanied by a 4- and 5.5-fold significant increase in transport and enzyme activity, respectively. Studies with synaptic vesicles from rat substantia nigra revealed that VMAT-2 uptake of ³H-aminochrome 6.3 ± 0.4nmol/min/mg was similar to dopamine uptake 6.2 ± 0.3nmol/min/mg that which were dependent on ATP. Interestingly, aminochrome uptake was inhibited by 2μM lobeline but not reserpine (1 and 10μM). Incubation of cells overexpressing VMAT-2 and DT-diaphorase with 20μM aminochrome resulted in (i) a significant decrease in cell death (6-fold, P<0.001); (ii) normal ultra structure determined by transmission electron microscopy contrasting with a significant increase of autophagosome and a dramatic remodeling of the mitochondrial inner membrane in wild type cells; (iii) normal level of ATP (256 ± 11μM) contrasting with a significant decrease in wild type cells (121±11μM, P<0.001); and (iv) a significant decrease in DNA laddering (21 ± 8pixels, P<0.001) cells in comparison with wild type cells treated with 20μM aminochrome (269 ± 9). These results support our hypothesis that VMAT-2 and DT-diaphorase are an important defense system against aminochrome formed during dopamine oxidation.
authors.affiliation: Molecular and Clinical Pharmacology, Faculty of Medicine, University of Chile, Santiago 8380453, Chile.
authors.forename: Patricia
authors.initials: P
authors.lastname: Muñoz
authors.forename: Irmgard
authors.initials: I
authors.lastname: Paris
authors.forename: Laurie H
authors.initials: LH
authors.lastname: Sanders
authors.forename: J Timothy
authors.initials: JT
authors.lastname: Greenamyre
authors.forename: Juan
authors.initials: J
authors.lastname: Segura-Aguilar
mesh_terms: D000704:Analysis of Variance; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D004247:DNA / Q000187:drug effects / Q000378:metabolism; D004298:Dopamine / Q000378:metabolism*; D006801:Humans; D045563:Indolequinones / Q000378:metabolism / Q000633:toxicity*; D008120:Lobeline / Q000494:pharmacology; D018613:Microscopy, Confocal; D046529:Microscopy, Electron, Transmission; D008928:Mitochondria / Q000378:metabolism / Q000648:ultrastructure; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000235:genetics / Q000378:metabolism*; D009474:Neurons / Q000187:drug effects / Q000378:metabolism; D010084:Oxidation-Reduction; D018384:Oxidative Stress; D010300:Parkinson Disease / Q000378:metabolism; D051381:Rats; D011916:Rats, Inbred F344; D012110:Reserpine / Q000494:pharmacology; D013378:Substantia Nigra / Q000166:cytology / Q000378:metabolism*; D014162:Transfection; D050493:Vesicular Monoamine Transport Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D045563:Indolequinones; C493452:SLC18A2 protein, human; D050493:Vesicular Monoamine Transport Proteins; C091584:aminochrome 1; D012110:Reserpine; D004247:DNA; D008120:Lobeline; D016660:NAD(P)H Dehydrogenase (Quinone); D004298:Dopamine