Nitration of soluble proteins in organotypic culture models of Parkinson's disease

• Published in: Neurochemistry international Vol. 52; no. 3; pp. 487 - 494
• Main Authors: Larsen, Trine R., Söderling, Ann-Sofi, Caidahl, Kenneth, Roepstorff, Peter, Gramsbergen, Jan Bert
• Format: Journal Article
• Language: English
• Published: Oxford Elsevier Ltd 01.02.2008; Elsevier
• Subjects: 1-Methyl-4-phenylpyridinium; 1-Methyl-4-phenylpyridinium - pharmacology; 3-Nitrotyrosine; alpha-synuclein; Animals; Biological and medical sciences; Cell Survival - drug effects; Cell Survival - physiology; Degenerative and inherited degenerative diseases of the nervous system. Leukodystrophies. Prion diseases; Dopamine; Dopamine - metabolism; dopaminergic-neurons; Dose-Response Relationship, Drug; Enzyme Inhibitors - pharmacology; free 3-nitrotyrosine; Fundamental and applied biological sciences. Psychology; induced neurotoxicity; Mass spectrometry; Medical sciences; Mice; Mice, Inbred C57BL; Molecular Biology; Molekylärbiologi; Neurochemistry - methods; Neurology; neuronal nitric-oxide; Neurosciences; Neurotoxins - pharmacology; Neurovetenskaper; Nitric oxide; Nitric Oxide Donors - pharmacology; Nitric Oxide Synthase - antagonists & inhibitors; Nitric Oxide Synthase - metabolism; Nitro Compounds - metabolism; Organ Culture Techniques; oxidative stress; Oxidative Stress - physiology; Parkinson Disease - metabolism; Parkinson Disease - physiopathology; prevents nitration; Proteins - metabolism; Pterins - pharmacology; Reactive Nitrogen Species - metabolism; Rotenone; Substantia Nigra - metabolism; Substantia Nigra - physiopathology; tetrahydrobiopterin precursor sepiapterin; Tyrosine - analogs & derivatives; Tyrosine - analysis; Tyrosine - metabolism; Tyrosine 3-Monooxygenase - metabolism; tyrosine-hydroxylase; Uncoupling Agents - pharmacology; Vertebrates: nervous system and sense organs; O 2; NO; Dopamine; l-NAME; MPP; AMPT; SIN-1; Rotenone; LDH; NOS; PD; TH; SNOG; Nitric oxide; 3-NT; 1-Methyl-4-phenylpyridinium; ONOO; 3-Nitrotyrosine; HBSS; MPTP; DOPA; DA; Mass spectrometry; Nitration; Nervous system diseases; Organotypic culture; Parkinson disease; Catecholamine; Protein; Cerebral disorder; Central nervous system disease; Neurotransmitter; Degenerative disease; Models; Extrapyramidal syndrome
• ISSN: 0197-0186; 1872-9754
• DOI: 10.1016/j.neuint.2007.08.008

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 μM MPP +, but significant DA depletions occurred at much lower concentrations of MPP + (1 μM). Chronic MPP + or rotenone treatments (3 weeks) caused maximum protein nitration by 1 μM (twofold) or 10 nM (fourfold), respectively. Co-treatment with the nitric oxide synthase inhibitor l-NAME (300 μM) prevented protein nitration by MPP +, but did not protect against MPP +-induced DA depletion or inhibition of TH activity. Acute incubation with 100 μM 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.