Nitration of soluble proteins in organotypic culture models of Parkinson's disease.
Protein nitration due to oxidative and nitrative stress has been linked to the pathogenesis of Parkinson's disease (PD), but its relationship to the loss of dopamine (DA) or tyrosine hydroxylase (TH) activity is not clear. Here we quantified protein-bound 3-nitrotyrosine (3-NT) by a novel gas chromatography/negative chemical ionization tandem mass spectrometry technique and DA and 3,4-dihydroxyphenylalanine (DOPA) by HPLC in tissues or medium of organotypic, mouse mesencephalon cultures after acute or chronic treatments with the peroxynitrite donor 3-morpholino-sydnonimine (SIN-1), the dopaminergic toxin 1-methyl-4-phenylpyridinium (MPP(+)) or the lipophilic complex I inhibitor rotenone. Incubation with SIN-1 (24 h) or MPP(+) treatments (48 h) caused dose-dependent protein nitration reaching a maximum of eightfold increase by 10 mM SIN-1 or twofold by 10 microM MPP(+), but significant DA depletions occurred at much lower concentrations of MPP(+) (1 microM). Chronic MPP(+) or rotenone treatments (3 weeks) caused maximum protein nitration by 1 microM (twofold) or 10nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 microM) prevented protein nitration by MPP(+), but did not protect against MPP(+)-induced DA depletion or inhibition of TH activity. Acute incubation with 100 microM SIN-1 inhibited TH activity, which could be blocked by co-treatment with the tetrahydrobiopterin precursor l-sepiapterin, but tissue DA depletions required higher doses of SIN-1 (>1 mM, 24 h) and longer survival. In conclusion, protein nitration and TH activity or DA depletion are not directly related in these models.
Larsen
Trine R
TR
Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Denmark. trlarsen@health.sdu.dk
Söderling
Ann-Sofi
AS
Caidahl
Kenneth
K
Roepstorff
Peter
P
Gramsbergen
Jan Bert
JB
D015655:1-Methyl-4-phenylpyridinium / Q000494:pharmacology; D000818:Animals; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D004298:Dopamine / Q000378:metabolism; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D008810:Mice, Inbred C57BL; D009448:Neurochemistry / Q000379:methods; D009498:Neurotoxins / Q000494:pharmacology; D020030:Nitric Oxide Donors / Q000494:pharmacology; D019001:Nitric Oxide Synthase / Q000037:antagonists & inhibitors / Q000378:metabolism; D009574:Nitro Compounds / Q000378:metabolism*; D009924:Organ Culture Techniques; D018384:Oxidative Stress / Q000502:physiology*; D010300:Parkinson Disease / Q000378:metabolism* / Q000503:physiopathology; D011506:Proteins / Q000378:metabolism*; D011622:Pterins / Q000494:pharmacology; D026361:Reactive Nitrogen Species / Q000378:metabolism; D013378:Substantia Nigra / Q000378:metabolism* / Q000503:physiopathology; D014443:Tyrosine / Q000031:analogs & derivatives* / Q000032:analysis / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000378:metabolism; D014475:Uncoupling Agents / Q000494:pharmacology
D004791:Enzyme Inhibitors; D009498:Neurotoxins; D020030:Nitric Oxide Donors; D009574:Nitro Compounds; D011506:Proteins; D011622:Pterins; D026361:Reactive Nitrogen Species; D014475:Uncoupling Agents; C002744:3-nitrotyrosine; D014443:Tyrosine; C016727:sepiapterin; D019001:Nitric Oxide Synthase; D014446:Tyrosine 3-Monooxygenase; D015655:1-Methyl-4-phenylpyridinium; D004298:Dopamine