From the
Department of Psychiatry and Behavioral
Neurosciences, the ||Center for Molecular Medicine
and Genetics, Wayne State University School of Medicine, and the
John D. Dingell Veterans Affairs Medical Center,
Detroit, Michigan 48201, and the ¶Center for
Molecular Recognition and Departments of Pharmacology and Psychiatry, Columbia
University College of Physicians and Surgeons, New York, New York 10032
Received for publication, April 25, 2003 , and in revised form, May 22, 2003.
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ABSTRACT |
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INTRODUCTION |
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The participation of ONOO in cellular or neuronal toxicity is an evolving and complicated issue. Real-time measures of intracellular nitration indicate that ONOO may not cross cell membranes in sufficient amounts to cause intracellular tyrosine nitration (13). This may be a reflection of preferential nitration of hydrophobic, transmembrane tyrosines by ONOO as compared with tyrosines in the aqueous phase (14). Mayer and colleagues (15) have argued on chemical and kinetic grounds that ONOO is altogether ineffective as a tyrosine nitrating species in vivo. ONOO is by no means the only nitrating species and a strong case can be made for nitrogen dioxide (NO2) as a more relevant nitrating reagent (13, 16).
The tyrosine-nitrating properties of ONOO and NO2 are not often considered within a context of cellular phenotype, but this could be extremely important in the case of DA neurons. ONOO and NO2 react with DA to form o-quinones and various nitro-catechols (1721). DA-mediated neurotoxicity is associated with increased formation of catechol-quinones, and quinones are known to modify cysteine residues in proteins (2224), including TH (25). Catechol-quinones have also been implicated in Parkinson's disease (2630).
The interaction of DA with reactive nitrogen species could have important consequences in DA neurons by determining the pathway of toxicity, yet the influence of DA on the protein-nitrating properties of ONOO and NO2 has not been considered. Given that tyrosine nitration of TH may be an early biochemical event in the DA neurodegeneration associated with Parkinson's disease (9), we have studied the effects of DA on nitration of TH. It is found presently that catechols prevent nitration of TH by ONOO and NO2. Using intact cells expressing the human DA transporter (31, 32) along with a green fluorescent protein-TH fusion protein as a reporter of real-time nitration (13), we also observe that intracellular tyrosine nitration is prevented by DA. These findings suggest that nitrotyrosine formation may be suppressed in DA neurons as long as catechol synthesis and storage are intact and point to catechol-quinones as early participants in DA neuronal damage.
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EXPERIMENTAL PROCEDURES |
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Cloning and Assay of THTH was expressed as a glutathione S-transferase fusion protein. The recombinant protein was purified by glutathione-agarose affinity chromatography and the glutathione S-transferase fusion tag was removed by thrombin cleavage, resulting in a highly purified TH preparation (>95% pure) as previously described (25, 33, 34). TH catalytic activity was assessed by the tritium release method as described by Lerner et al. (35). Protein was measured using the method of Bradford (36).
Preparation of ONOO and
NO2 and Treatment of
THONOO was synthesized by the quenched-flow
method of Beckman et al.
(37), and its concentration
was determined by the extinction coefficient 302 = 1670
M1cm1. The hydrogen
peroxide contamination of ONOO solutions was removed by
manganese dioxide chromatography and filtration
(37). The typical
concentration of stock ONOO solutions ranged between 300 and
400 mM. ONOO was added to TH with vigorous mixing
in 50 mM potassium phosphate buffer, pH 7.4, containing 100
µM DTPA, and incubations were carried out for 15 min at 30
°C. The volume of ONOO added to the enzyme samples was
always less than 1% (v/v) and did not influence pH. When tested, catechols
were added immediately prior to ONOO when tested. Upon
completion of incubation with ONOO and other additions,
enzyme samples were diluted with 10 volumes of 50 mM potassium
phosphate, pH 6, and assayed for catalytic activity or post-translational
modification (nitration or quinolation) after SDS-PAGE and transfer to
nitrocellulose (see below). NO2 was produced by reacting
horseradish peroxidase or myeloperoxidase (specified below) with hydrogen
peroxide (100 µM) in the presence of sodium nitrite
(10500 µM) as described by Espey et al.
(13). TH was exposed to
NO2-generating conditions with or without catechols for 60 min at
30 °C after which the enzyme was diluted with 10 volumes of 50
mM potassium phosphate, pH 6, and assayed for catalytic activity or
post-translational modification as described above for
ONOO.
Post-translation Modification of TH by Reactive Nitrogen Species and Catechol-quinonesFollowing treatment with ONOO or NO2 with or without catechols, TH was subjected to SDS-PAGE (62). Proteins were transferred to nitrocellulose, blocked in Tris-buffered saline containing Tween 20 (0.1% v/v) and 5% nonfat dry milk, and probed with a monoclonal antibody specific for nitrotyrosine (1:2000 dilution). After incubations with primary antibodies, blots were incubated with a goat anti-mouse secondary antibody conjugated with horseradish peroxidase, and immunoreactive protein bands were visualized with enhanced chemiluminescence. Catechol-quinone modification of TH was assessed in separate experiments by staining blots with NBT in the presence of 2 M potassium glycinate buffer pH 10 as described previously (38).
Modification of Cysteine Residues in TH by ONOO and NO2The effect of ONOO and NO2 with or without catechols on TH cysteine residues was determined with the use of the thiol-reactive biotinylation reagent PMAB as described previously (33). PMAB reacts selectively with reduced cysteines in proteins and does not react with cysteines that have been oxidized. This probe is not quantitative but allows a relative measure of the extent to which cysteine residues have been modified. Untreated TH or enzyme exposed to ONOO or NO2 with or without catechols as described above was diluted with 100 mM Tris-HCl, pH 8.5, for subsequent labeling with PMAB (50 µM). Proteins were labeled for 60 min at room temperature in the dark after which they were subjected to SDS-PAGE and blotting to nitrocellulose. PMAB reactivity was detected with horseradish-conjugated streptavidin and visualized with ECL.
Direct Real-time Evaluation of Tyrosine Nitration with Enhanced Green Fluorescent ProteinA fusion protein comprised of enhanced green fluorescent protein (eGFP), and TH was constructed by cloning the full-length cDNA of TH into the vector pEGFP-3C at its XhoI/BamHI restriction sites in the multiple cloning site. In this orientation, the eGFP fusion tag was upstream of the TH amino terminus, and the entire fusion protein complex had a molecular mass of 87 kDa. The pEGFP/TH fusion vector was stably transfected into HEK293/EM4 cells already stably expressing the human DA transporter (hDAT) as described previously (31, 32). Because hDAT-expressing cells were resistant to hygromycin and Geneticin (G418), cells were transfected with pEGFP/TH and pCMV/Zeo in a ratio of 10:1 (in LipofectAMINE 2000), and selection was carried out in zeocin (40 µg/ml) and by observing eGFP fluorescence with a fluorescence microscope. Both elements of the eGFP/TH fusion protein retained their respective functionality (i.e. fluorescence and TH catalytic activity). Cells were maintained in Dulbecco's modified Eagle's medium (high glucose) containing 10% fetal calf serum in an atmosphere of 5% CO2. The resulting stable transformants expressed eGFP/TH (for evaluation of tyrosine nitration in intact cells) and hDAT (for transport of DA into cells). Real-time evaluation of eGFP/TH tyrosine nitration was carried out as described by Espey et al. (13). Intact cells (1 x 106) were washed into phosphate-buffered saline (pH 7.4) containing DTPA (100 µM) and exposed to NO2 via incubation with PTIO and PAPA/NO (39) at 37 °C with or without pre-loading of cells with DA as previously described (32). The extent of intracellular tyrosine nitration caused by NO2 was monitored through measures of reductions in eGFP/TH fluorescence at 488em/512ex nm (13, 39) in an Aminco-Bowman Series 2 fluorescence spectrometer. Immediately after measures of fluorescence, intact cells were placed on ice, washed 3x with ice-cold phosphate-buffered saline, and sonicated in 60 µl of potassium phosphate buffer (pH 6). TH activity was subsequently measured in the cell supernatant after sedimentation of membranes by centrifugation at 40,000 x g for 15 min at 4 °C.
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RESULTS |
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In view of the fact that catechols prevent the nitration of free tyrosine caused by ONOO (20, 40), we tested their effects on ONOO-induced nitration of tyrosine residues in TH. Fig. 2A shows that ONOO (100 µM) caused extensive nitration of tyrosine residues in TH as measured by immunoblotting with a monoclonal antibody against nitrotyrosine. Catechol compounds were tested at a concentration of 20 µM, and each completely prevented the ONOO-induced nitration of TH. Although a careful titration of the concentration effects of the catechols on ONOO-induced nitration was not carried out, we have observed that molar ratios of about 1:5 (DA:ONOO) are sufficient to block the TH-nitrating properties of ONOO. The last lane in Fig. 2A shows that the DA aminochrome (formed by reacting DA with sodium periodate), like DA itself, also blocked ONOO-induced tyrosine nitration in TH. When a blot similar to the one in Fig. 2A was exposed to redox cycling staining, it was observed that the catechol compounds in the presence of ONOO converted TH to a redox cycling protein. Fig. 2B shows that DA and DOPAC produced the strongest redox cycling staining in TH after exposure to ONOO. DOPA was somewhat less potent than DA and DOPAC in this regard. These results with ONOO-DA interactions agree with previous studies showing that chemical or enzymatic conversion of DA to its aminochrome or o-quinone, respectively, modifies TH, converting the enzyme to a redox cycling quinoprotein (25).
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Because the effects on TH of tyrosine nitrating species other than ONOO have not been investigated, we used increasing concentrations of sodium nitrite to generate a range of concentrations of NO2 in the presence of constant levels of horseradish peroxidase (25 units) and hydrogen peroxide (100 µM). Fig. 3A shows that TH was quite sensitive to inhibition by NO2. TH was inhibited by 4050% at a nitrite concentration of 200 µM; when the nitrite concentration reached 500 µM, TH activity was inactivated by 6070%. Omission of any one or two of the components needed to generate NO2 prevented inhibition of TH, indicating that the enzyme was not inhibited by the peroxidase, nitrite, or hydrogen peroxide. Substitution of myeloperoxidase for horseradish peroxidase produced the same inhibition of TH catalytic activity (data not shown). The effects of NO2 on TH activity were statistically significant (p < 0.01, ANOVA). The effects of DA on the NO2-induced inhibition of TH are presented in Fig. 3B. Low concentrations of DA (520 µM) slightly enhanced the inhibition of TH activity caused by NO2. For example, TH activity was reduced to about 50% of control by NO2 in the absence of DA, whereas 20 µM DA increased the inhibitory effects of NO2 on TH to 40% of control. Higher concentrations of DA (50100 µM) did not further alter the NO2-induced reduction in TH activity. The overall effect of DA on the inhibition of TH by NO2 was statistically significant (p < 0.05, ANOVA). Equimolar concentrations of DOPA and DOPAC were also tested for effects on NO2-induced inhibition of TH, and the results are included in Fig. 3C. The inhibitory effects of NO2 were not altered by DA or DOPA but were significantly increased by DOPAC. Whereas DA and DOPA increased the inhibition of TH by NO2 from 50% to about 65%, DOPAC plus NO2 resulted in a near-total inactivation of TH (i.e. 95% inhibition).
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The effect of catechols on nitration of TH by NO2 was tested, and the results are presented in Fig. 4A. The NO2-generating conditions that caused inhibition of TH catalytic activity (Fig. 3 above) resulted in the nitration of tyrosine residues in the TH monomer (60 kDa). Omission of any one of the components needed to produce NO2 prevented tyrosine nitration in TH (data not shown). It can be seen in Fig. 4A that equimolar concentrations (20 µM) of DA, DOPA, and DOPAC prevented NO2-induced nitration of tyrosine residues in TH. In agreement with results using ONOO as the nitrating species (Fig. 2), exposure of TH to NO2 in the presence of any of the catechols converted the enzyme to a redox cycling quinoprotein, as shown in Fig. 4B.
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In view of results showing that catechol prevention of TH nitration by
ONOO or NO2 did not relieve the enzyme of
inhibition, we tested the effects of these treatments on the status of
cysteine residues in TH. In agreement with previous studies
(33), ONOO
lowered PMAB labeling of TH as shown in
Fig. 5 (middle lane of
each nitration condition), an indication of cysteine modification.
NO2 caused similar reductions in PMAB labeling as seen with
ONOO. When ONOO or NO2 was
combined with DA, the reduction in PMAB labeling was still observed. DOPA and
DOPAC produced the same effects on PMAB labeling as seen with DA in
combination with ONOO or NO2 (data not shown).
Digital scans of the data in Fig.
5 indicate that the nitrating species reduced PMAB labeling by
95% and the DA-quinones also reduced labeling to roughly the same
extent.
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Espey et al. (13) recently introduced a method to measure intracellular tyrosine nitration directly and in real-time based on the sensitivity of eGFP to nitration-induced reductions in fluorescence. We created stable transformants expressing an eGFP/TH fusion protein in HEK293/EM4 cells bearing the hDAT (31). This cell line was chosen for use presently because of its low endogenous content of DA and because the hDAT could be used to transport DA into intact cells. Exposure of these cells to NO2 via treatment with PTIO-PAPA/NO caused a significant reduction in eGFP fluorescence as shown in Fig. 6. When cells were preloaded with DA before exposure to NO2, the nitration-induced reduction in fluorescence was largely prevented (Fig. 6). The effect of DA on the NO2-induced reduction in eGFP fluorescence was significant (p < 0.05, Bonferroni's test). TH activity was almost completely inhibited after exposure of intact cells to NO2. It can also be seen in Fig. 6 that DA provided partial protection against NO2-induced inhibition of TH. In agreement with Pfeiffer et al. (4143) and Espey et al. (13), ONOO in concentrations up to 1000 µM, added as a bolus or by slow decomposition of SIN-1, did not cause intracellular tyrosine nitration as measured by reductions in eGFP/TH fluorescence, nor did it inhibit TH activity (data not shown). Finally, to test if ONOO could be playing a role in NO2-mediated nitration reactions, as a downstream by-product, cells were incubated with the ONOO scavenger methionine (20 mM) during PTIO-PAPA treatment. Fig. 6 shows that methionine did not prevent NO2-induced nitration, suggesting that ONOO was not playing a role under the present treatment conditions.
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DISCUSSION |
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TH is quite sensitive to inhibition by ONOO and NO2. Both reactive nitrogen species cause concentration-dependent reductions in TH catalytic function. The enzyme is also modified postranslationally by ONOO and NO2 as evidenced by tyrosine nitration and cysteine oxidation. Although it has been suggested that the cytotoxicity associated with ONOO and NO2 can be mediated by tyrosine nitration, the effects of these reactive nitrogen species are not often considered within the context of cellular phenotype. One case where this is particularly important is the DA neuron. These neurons are characterized, obviously, by their selective and high content of DA. Catechols react with ONOO and NO2 to form o-quinones and other radical species (17) and, in the process, inhibit the nitration of free tyrosine (20, 40). As an initial step in assessing the influence of the DA phenotype on protein nitration, it was important to determine if catechols could modify the nitration of TH caused by reactive nitrogen species. We observed that DA, its precursor DOPA, and its metabolite DOPAC prevented ONOO- and NO2-induced nitration of tyrosine residues in TH. Despite prevention of tyrosine nitration in TH, the catechols did not relieve the enzyme of inhibition. Considering that ONOO-induced nitration of TH has been linked specifically to the loss of TH activity (9, 48), it is interesting that TH could be inhibited in the presence of ONOO or NO2, despite the prevention of tyrosine nitration. In view of results showing that catechols prevent tyrosine nitration caused by ONOO or NO2 without relieving the enzyme from inhibition, the possibility that another post-translational modification was mediating TH inhibition was investigated.
Catechol-quinones are known to attack protein cysteinyls (22, 30) and form redox cycling sites after binding (38, 49). TH (25), tryptophan hydroxylase (50, 51), and the dopamine transporter (32, 53) are examples of proteins that can be modified by DA-quinones and aminochromes. Quinone modification of each of these important proteins has the added effect of reducing their functional activity. Either ONOO or NO2, when combined with DA, DOPA, or DOPAC, modified TH to a redox cycling quinoprotein. However, subtle differences were noted between ONOO- and NO2-generated quinones and their impact on TH. For instance, DA did not alter the ONOO-induced inhibition of TH but slightly increased the inhibition caused by NO2. DOPA provided some protection against ONOO-induced inhibition of TH but was without effect on NO2. Finally, DOPAC did not change TH inhibition by ONOO but significantly increased the effects of NO2 on TH activity. These varying effects probably reflect differences in the chemical interactions between ONOO or NO2 and individual catechols. Any such differences in this regard do not mitigate the importance of the common property shared by catechols, the ability to prevent nitration of tyrosine residues in proteins while causing quinolation of cysteines.
The quinone of DOPAC caused the greatest amount of redox cycling in TH, especially when generated by ONOO, and the DOPA quinone resulted in the lowest amount. The relationship between enzyme inhibition and redox cycling does not appear to be directly correlated. Redox cycling by substituted quinones is a very complex chemical process and is difficult to use as a direct index of cysteine modification in TH. The o-quinones of DA (and other catechols as well) are extremely volatile, and the reactivity of any particular catechol-quinone will be determined by its access to sulfhydryls and by its electrophilicity (54, 55). The redox potential of substituted quinones is also very difficult to predict from their structures (49, 56), and, as an illustration, it has been shown that the redox cycling potential of DOPA-quinone is lower than that of many other protein-bound quinones (38). Thus, it appears that the total number of cysteines that are modified by catechol-quinones is a better predictor of the extent of TH inhibition than is the extent of redox cycling caused by a bound quinone.
We have argued recently that the ONOO-induced inhibition of tryptophan hydroxylase (57) and TH (33, 34) is mediated by cysteine modification, not tyrosine nitration. The effects of NO2 on TH have not been investigated, so we tested it along with ONOO for effects on cysteine residues in TH using PMAB labeling. This sulfhydryl-specific reactant labels reduced cysteine residues in proteins and its reactivity is diminished by treatment of proteins, including TH, with cysteine oxidants like ONOO (33). In agreement with the effects of ONOO, NO2 also reduced PMAB labeling of TH, indicative of cysteine modification. What is more, treatment of TH with ONOO or NO2, in the presence of DA (conditions preventing tyrosine nitration), resulted in reduced PMAB labeling as well. These data reinforce results with redox cycling and establish that catechol-quinones derived from ONOO or NO2 attack cysteine residues in TH. This post-translational modification appears to be the mechanism by which TH is inhibited when tyrosine nitration has been prevented by the catechols.
Evidence for nitration of TH by ONOO in intact cells has been difficult to obtain. Ara et al. (9) treated PC12 cell lysates, not intact cells, with ONOO and showed that TH was nitrated at selected tyrosine residues. We have not been able to establish that TH is nitrated after treatment of intact PC12 cells with ONOO. Several factors could account for this failure and led us to consider an alternative approach to the problem. First, it does not appear that ONOO penetrates the plasma membrane of intact cells in sufficient concentrations to cause tyrosine nitration in cytoplasmic proteins (13). Second, ONOO is formed de novo from the reaction of nitric oxide with superoxide and high concentrations of these reactants must be maintained at or near a 1:1 stoichiometry to avoid secondary reactions that form species incapable of tyrosine nitration. An imbalance in this stoichiometry can lead to a quenching of nitration and oxidation reactions or may even lead to the formation of nitrosating species (15, 58). Third, intact PC12 cells contain very high catecholamine concentrations that could also quench ONOO-induced tyrosine nitration. Fourth, it is possible that only a small number of the TH molecules in PC12 cells are nitrated, and immunoprecipitation and immunoblotting are too insensitive to detect TH nitration. We used the method of Espey et al. (13) to monitor tyrosine nitration in intact cells by NO2 through measures of fluorescence reductions in an eGFP/TH fusion protein stably expressed in hDAT-bearing HEK293/EM4 cells. We chose this cell line because of its extremely low endogenous DA content and because the intracellular content of DA could be increased substantially via the hDAT. The use of fluorescence is also a far more sensitive way of measuring nitration than immunoblotting. It was observed that NO2 caused a significant reduction in eGFP/TH fluorescence and TH catalytic activity in intact cells. It does not appear that ONOO plays a role, possibly as a downstream by-product of NO2 generation, because the ONOO scavenger methionine did not prevent reductions in eGFP fluorescence or TH activity. The magnitude of the reduction in TH activity (near-total) was greater than the reduction in eGFP fluorescence (about 50%) and also stands in contrast to in vitro results where TH activity was inhibited by 50% upon exposure to NO2. The reasons for this difference are not immediately evident but could result from use of different methods of NO2 production (i.e. chemical versus enzymatic) or an attack on cellular TH by nitric oxide generated through PAPA/NO decomposition. Nitric oxide would not alter eGFP fluorescence (13) but could inhibit TH activity in vitro as well as in intact cells. The possibility that TH is modified by nitric oxide is currently under investigation. Pre-loading of cells with DA largely prevented the reduction in eGFP/TH fluorescence caused by NO2. Although DA provided partial protection against NO2-induced inhibition of TH activity, the enzyme remained inhibited. These results indicate that cellular DA can modulate tyrosine nitration. Furthermore, the loss of TH catalytic activity after exposure of intact cells to PTIO-PAPA/NO establishes that both elements of the eGFP/TH fusion protein had been modified by NO2 (i.e. eGFP fluorescence and TH activity, respectively).
The present results provoke a re-consideration of ONOO-mediated tyrosine nitration as an early event in the neurotoxic process in DA neurons. It appears that DA, its precursor DOPA, and its metabolite DOPAC shift the balance of influence of ONOO and NO2 toward the formation of o-quinones at the expense of tyrosine nitration. Although most intracellular DA is sequestered within synaptic vesicles, where it might be protected from attack by ONOO or NO2, TH is a cytoplasmic enzyme, and newly synthesized DA appears in the cytoplasm before it is transported into vesicles. The cytoplasmic availability of DA is not determined solely by TH, particularly under conditions thought to cause the production of ONOO in vivo. For example, methamphetamine (59) and MPTP (60, 61) cause extensive redistribution of DA from storage vesicles into the cytoplasm and extracellular space. Thus, ONOO- or NO2-induced tyrosine nitration in DA neurons could be suppressed by endogenous catechols, reducing the likelihood that this post-translational modification is an early occurring event in DA neurodegeneration.
Hastings and colleagues have shown that intrastriatal injections of DA (22, 23) or systemic injections of methamphetamine (8), both of which result in damage to DA nerve endings, cause substantial increases in the levels of cysteinyl-DA adducts in proteins. Postmortem Parkinson's tissue contains elevated levels of cysteinyl-catechol species (29, 52), and cerebrospinal fluid from individuals with Parkinson's disease contain antibodies that recognize quinone-modified proteins (28). We have shown that TH that has been modified by DA-quinones acquires the ability to cause redox cycling of iron (25), reinforcing the cytotoxic potential of catechol-quinones that was established by the influential studies of Graham (52, 54, 55). Taken together, evidence is mounting that DA, and its catechol precursors and metabolites, in the form of their o-quinones, may play an early and influential role in determining the viability of DA neurons under conditions of oxidative or nitrosative stress.
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FOOTNOTES |
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** To whom correspondence should be addressed: Dept. Psychiatry & Behavioral Neurosciences, School of Medicine, Wayne State University, 2125 Scott Hall, 540 E. Canfield, Detroit, MI 48201. Tel./Fax: 313-577-9737; E-mail: donald.kuhn{at}wayne.edu.
1 The abbreviations used are: ONOO, peroxynitrite; DA,
dopamine; hDAT, human dopamine transporter; DOPA, 3,4-dihydroxyphenylalanine;
DOPAC, 3,4-dihydroxyphenylacetic acid; DTPA, diethylenetriaminepentaacetic
acid; ECL, enhanced chemiluminescence; eGFP, enhanced green fluorescent
protein; NBT, nitro blue tetrazolium; NO2, nitrogen dioxide; PMAB,
(polyethylene oxide)-maleimide activated biotin; PTIO,
2-phenyl-4,4,5,5-tetramethylimidazole-1-oxyl 3-oxide; TH, tyrosine
hydroxylase; ANOVA, analysis of variance; MPTP,
1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; PAPA, propylamine
propylamine.
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ACKNOWLEDGMENTS |
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REFERENCES |
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