Chronic Stimulation of D1 Dopamine Receptors in Human SK-N-MC Neuroblastoma Cells Induces Nitric-oxide Synthase Activation and Cytotoxicity*

Jun Chen, Christophe Wersinger and Anita Sidhu {ddagger}

From the Department of Pediatrics, Georgetown University, Washington, D. C. 20007

Received for publication, March 26, 2003 , and in revised form, May 5, 2003.


    ABSTRACT
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Elevated synaptic levels of dopamine may induce striatal neurodegeneration in L-DOPA-unresponsive parkinsonism subtype of multiple system atrophy (MSA-P subtype), multiple system atrophy, and methamphetamine addiction. We examined the participation of dopamine and D1 dopamine receptors in the genesis of postsynaptic neurodegeneration. Chronic treatment of human SK-N-MC neuroblastoma cells with dopamine or H2O2 increased NO production and accelerated cytotoxicity, as indexed by enhanced nitrite levels and cell death. The antioxidant sodium metabisulfite or SCH 23390, a D1 dopamine receptor-selective antagonist, partially blocked dopamine effects but together ablated dopamine-mediated cytotoxicity, indicating the participation of both autoxidation and D1 receptor stimulation. Direct activation of D1 dopamine receptors with SKF R-38393 caused cytotoxicity, which was refractory to sodium metabisulfite. Dopamine and SKF R-38393 induced overexpression of the nitric-oxide synthase (NOS) isoforms neuronal NOS, inducible NOS (iNOS), and endothelial NOS in a protein kinase A-dependent manner. Functional studies showed that ~60% of total NOS activity was due to activation of iNOS. The NOS inhibitor N(G)-nitro-L-arginine methyl ester and genistein, wortmannin, or NF-{kappa}B SN50, inhibitors of protein tyrosine kinases phosphatidylinositol 3-kinase and NF-{kappa}B, respectively, reduced nitrite production by dopamine and SKF R-38393 but were less effective in attenuating H2O2-mediated effects. In rat striatal neurons, dopamine and SKF R-38393, but not H2O2, accelerated cell death through increased expression of neuronal NOS and iNOS but not endothelial NOS. These data demonstrate a novel pathway of dopamine-mediated postsynaptic oxidative stress and cell death through direct activation of NOS enzymes by D1 dopamine receptors and its associated signaling pathways.


    INTRODUCTION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Striatonigral neurodegeneration is implicated in the clinical expression of several human disorders and addictive states involving dopamine neuronal dysfunction, such as MSA,1 L-DOPA-unresponsive parkinsonism subtype of multiple system atrophy (MSA-P subtype) (1), secondary dopamine dysfunction in Huntington's disease (HD), and methamphetamine (METH)-induced neurotoxicity (2, 3), in which dopaminergic transmission is interrupted by progressive loss of striatal neurons bearing postsynaptic D1 and D2 dopamine receptors (4). Moreover, striatonigral neurodegeneration was also observed in monkeys treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine or 3-nitropropionic acid (5). Recent studies implicate the participation of both reactive oxygen species (ROS) and reactive nitrogen species (RNS) in the pathophysiology of striatonigral neurodegeneration (58) through mechanisms that remain undefined.

Because striatal neurons do not produce dopamine or express the dopamine transporter (DAT), the mechanism of dopamine-mediated neurotoxicity in such neurons occurs through extracellularly present dopamine in the synapse (9, 10). Normally, dopamine released into the synapse is rapidly cleared and recycled by the DATs of presynaptic neurons. In 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-treated animals, MSA-P, and in methamphetamine-induced neurotoxicity, however, large amounts of dopamine accumulate extracellularly in the synapse, which upon spontaneous autoxidation, produces ROS and RNS (11). Moreover, in METH addictive states, elevated synaptic levels of dopamine accumulate not only through blockage of DAT up-take activity but also by presynaptic depletion of dopamine from storage vesicles secondary to a blockage of the vesicular monamine transporter, with enhanced release of dopamine into the synapse, an effect also produced by MPP+ (1-methyl-4-phenylpyridinium), the bioactive metabolite of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine.

Synaptic dopamine stimulates its cognate D1 and D2 dopamine receptors, localized on postsynaptic neurons; prolonged stimulation of these receptors causes their desensitization (12, 13). In addition to stimulation of adenylyl cyclase, activation of D1 dopamine receptors induces the expression of several early immediate genes, such as c-fos, c-jun, JunB, and zif-268 (14), which in turn act as positive modulators of genes that promote ROS and RNS production when overexpressed, including NF-{kappa}B and NOS (15, 16). In particular, iNOS is capable of producing large amounts of nitric oxide (NO), which can rapidly reach cytotoxic levels (17, 18). Although a link between D1 receptor stimulation and the generation of ROS or RNS has never been demonstrated, recent studies indicate that blockage of D1 dopamine receptors with selective antagonists has strong neuroprotective and anti-parkinsonian effects (19, 20), supporting the concept of co-participation of D1 receptors in the maintenance and/or pathogenesis of postsynaptic neurodegeneration and oxidative stress.

To investigate the overall role of extracellular dopamine in degeneration of striatonigral neurons and of D1 dopamine receptors in particular, studies were conducted in both rat primary neuronal striatal cultures and in human SK-N-MC neuroblastoma cells. The latter was chosen as a postsynaptic striatal cell model system because it endogenously expresses the D1 dopamine receptor and contains the appropriate receptor-linked dopaminergic signaling machinery, a feature lacking in transfected cells (21). Moreover, the molecular properties of these cells, which include lack of expression of D2-like and D5 dopamine receptors (21, 22), the absence of DAT (23), a hallmark protein of presynaptic nigrostriatal neuronal projections, and negligible tyrosine hydroxylase (TH) activity (24), are reminiscent of D1 dopamine receptor-expressing striatal neurons. In this report we provide evidence to show that dopamine is a strong oxidant and that 50% of its potency is entirely attributable to the direct stimulation of D1 dopamine receptors and subsequent activation of specific signaling pathways that are modulated by these receptors. Dopamine caused a marked overexpression of NOS, primarily iNOS and nNOS, with concomitant increases in NO levels, oxidative stress, and cytotoxicity. These results highlight a previously unknown role of D1 dopamine receptors in neurodegeneration and provide a heuristic framework in which to view the mechanisms underlying, and treatment of, striatal neurodegeneration.


    EXPERIMENTAL PROCEDURES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Materials—Human SK-N-MC neuroblastoma cells were obtained from the American Type Culture Collection (Manassas, VA). Nu-serum was purchased from Collaborative Biomedical Products (Bedford, MA). RPMI 1640 medium without phenol red, dopamine, hydrogen peroxide (H2O2), L-NAME, sodium metabisulfite (SMBS), indatraline (INDT), sodium nitrite, SKF R-38393, and SCH 23390 were purchased from Sigma. KT5720, wortmannin, genistein, mPKCi, NF-{kappa}B SN50, and 1400W were from Calbiochem-Novabiochem Co.

Cell Culture, Drug Treatment, and Cell Viability—SK-N-MC neuroblastoma cells were grown in 12-well culture plates (seeding density 1.0 x 105/well) in RPMI 1640 medium without phenol red supplemented with 10% (v/v) Nu-serum, antibiotics and 2 mM L-glutamine in a humidified atmosphere of 95% air, 5% CO2 at 37 °C until 90% confluent. Cells were then serum-starved overnight with serum-free RPMI medium and incubated with drugs for 16 h. Control cells were treated with an equal concentration of solvent (0.2% H2O). After incubation, 0.3 ml of medium was removed to measure nitrite concentration. Cell viability in 12-well plates was evaluated by counting viable cells in a Neubauer cell using the trypan blue exclusion test whereby viable cells exposed to trypan blue for no more than 15 min exclude the dye, as described by the manufacturer's (Sigma) protocol. Values from each treatment were expressed as percentage of survival relative to control.

Rat Primary Striatal Neuronal Cultures—Striata from 18-day-old rat embryos were isolated, and cells were dissociated by mechanical disruption, counted, and grown (600–800 cells seeded/mm2) in neurobasal medium supplemented with 2% (v/v) B-27 supplement and 50 µM {beta}-mercaptoethanol on glass cover-slips precoated with poly-L-ornithine (15% w/v in PBS; Sigma) and laminin (3 µg/ml in PBS; Sigma) for 6 days, as described by Brewer et al. (25). Neurons were exposed for 16 h to 5 µM dopamine, SKF R-38393, or H2O2 with or without pretreatment for 30 min with either 20 µM SMBS or 1 µM D1-selective antagonist SCH 23390. Control cells were treated with an equal concentration of solvent (0.2% H2O). Neuronal cell viability was measured by the trypan blue exclusion method.

Nitrite and NOS Activity Measurements—NOS activity was measured by assessing nitrite levels, a stable byproduct of NO, by a modification of the method of Dawson et al. (26). Briefly, 0.3 ml of Griess reagent (1 part 0.1% naphthylethylenediamine dihydrochloride in H2O and 1 part 1% sulfanilamide in 5% H3PO4), and 0.3 ml of culture medium from treated cells (see above) were mixed. After 30 min of incubation at 45 °C, the absorbance at 550 nm was determined. The concentration was determined from a standard curve using NaNO2 at a range of 0–10 µM. Results were expressed as nM/1.0 x 105 cells or -fold increase in nitrite levels after drug treatment over distilled water-treated control. Cell viability was assessed by the trypan blue exclusion method.

NOS activity was measured by estimating the conversion of [3H]arginine to [3H]citrulline with an enzyme assay kit from Calbiochem. Briefly, SK-N-MC cells were harvested and homogenized in homogenization buffer (25 mM Tris-HCl, pH 7.4, 1 mM each, EDTA and EGTA). Cell extracts (5–10 µg of protein) were incubated with 16.13 µM [3H]arginine (Amersham Biosciences; 62 Ci/mmol) in reaction buffer for 30 min, and total NOS activity was measured as per the manufacturer's instruction. Cells that were not treated with SKF R-38393 were used as controls. To estimate iNOS activity, assays were conducted in the presence of 1 µM selective iNOS inhibitor 1400W, and the residual activity, presenting contribution by both eNOS and nNOS, was subtracted from total NOS activity, obtained in the absence of any inhibitor.

Plasmids and Transfection—Human dopamine transporter (hDAT) cDNA construct (a gift from H. B. Niznik and F. Liu) was subcloned into pcDNA3.1 (Invitrogen). SK-N-MC cells (60% confluent) were transiently transfected by the DEAE-dextran/chloroquine method as described by Pristupa et al. (27) and Lee et al. (28). Briefly, cells were washed with Dulbecco's PBS and RPMI 1640, and the DNA/DEAE-dextran/medium mixture was added (2 µg of DNA/well). 100 µM chloroquine (Sigma) was added into the wells to increase the transfection efficiency. After 3 h of incubation at 37 °C and 5% CO2 with gentle swirling of the wells every 15 min, the cells were exposed to 10% Me2SO in Dulbecco's PBS for exactly 1 min, washed twice with RPMI 1640 plus 10% Nu-serum, and grown for further 48 h in RPMI plus 10% Nuserum. Mock-transfected cells were transfected with a pcDNA3.1 plasmid that lacked a DNA insert.

[3H]Dopamine Uptake—[3H]DA uptake was measured as described by Lee et al. (28). Briefly, 2 days after transfection, culture medium was removed, and wells were rinsed twice with 1 ml of uptake buffer (5 mM Tris, 7.5 mM HEPES, 120 mM NaCl, 5.4 mM KCl, 1.2 mM CaCl2, 1.2 mM MgSO4,1mM ascorbic acid, 5 mM glucose, pH 7.1). Cells were incubated in triplicate with 20 nM [3H]dopamine (PerkinElmer Life Sciences NET-131; 31.6 Ci/mmol) in uptake buffer for 10 min at room temperature. Nonspecific uptake was defined in the presence of 10 µM DAT blocker indatraline. Wells were rinsed twice with 1 ml of uptake buffer, cells were counted using trypan blue, and radioactivity incorporated into cells was measured by liquid scintillation counting after protein hydrolysis in 1 ml of 0.1 N NaOH for 1 h at 37 °C.

Protein Preparation and Immunoblot Analysis—Cell homogenates were prepared as previously described (29), and protein concentrations were determined by the Bradford method (30). Immunoblot analysis was performed essentially as previously described (31). Briefly, homogenate protein (50 µg/lane) was loaded onto 8% SDS-PAGE and transferred overnight to polyvinylidene difluoride membranes (Micron Separations Inc., Westboro, MA). The membranes were blocked with 5% nonfat dried milk in Tris-buffered saline with Tween 20 (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, and 0.05% Tween 20) followed by an overnight incubation at 4 °C with rabbit anti-nNOS, anti-iNOS, and anti-eNOS polyclonal antibodies (1:1000, Santa Cruz Biotechnology, Santa Cruz, CA). After extensive washing, the blots were incubated for 2 h with horseradish peroxidase-linked donkey anti-rabbit IgG (1:5000). Enhanced chemiluminescence (ECL) was carried out using Renaissance Chemiluminescence Reagent Plus (PerkinElmer Life Sciences). The blots were stripped and reprobed overnight with goat anti-actin monoclonal antibodies (1:1000, Santa Cruz Biotechnology) followed by incubation for 2 h with horseradish peroxidase-conjugated donkey anti-goat IgG (1:5000; Santa Cruz Biotechnology) and ECL.

Immunocytochemistry—Six-day-old rat primary striatal cultures were fixed with 3% (v/v) paraformaldehyde and permeabilized with 0.1% Triton X-100 in Dulbecco's PBS, as described by Prou et al. (32). Cells were incubated with primary rabbit polyclonal antisera (1:500; Santa Cruz Biotechnology) against either nNOS, iNOS, or eNOS for 12 h at 4 °C and for 2 h at room temperature with Alexa® Fluor® 568-conjugated goat anti-rabbit secondary antibodies (1:500; Molecular Probes, Eugene, OR). Cells were analyzed with a Nikon Eclipse E800 fluorescent microscope.

Data Analysis—Results are expressed as the means ± S.E. of at least three independent experiments. Within an experiment, each data point was determined in triplicate. All statistical analyses were performed using Instat Statistical Software (Graphpad, Sorrento Valley, CA). Statistical comparisons were performed with both one-factor analysis of variance and Scheffe's F test. Values of p < 0.05 or less were considered as statistically significant.


    RESULTS
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Dopamine Enhances Free Radical Production and Cell Death—Because dopamine spontaneously autoxidizes to generate free radicals, its efficacy as an oxidant was compared with another oxidant, H2O2, which also undergoes spontaneous oxidation. SK-N-MC cells (90% confluence) were incubated for 16 h with increasing concentrations of either dopamine or H2O2, and free radical production was indexed by measuring nitrite levels, a stable byproduct of NO released into the growth medium (Fig. 1A). Although both compounds increased nitrite levels, dopamine-induced increase in nitrite production was 2-fold higher compared with H2O2. To assess if the dopamine effects were partly or entirely mediated via D1 dopamine receptors, we stimulated these receptors directly with increasing concentrations of the D1 receptor agonist, SKF R-38393. Similar to dopamine and H2O2, SKF R-38393 caused an increase in nitrite production that was comparable in magnitude to that obtained with H2O2 but was significantly (p < 0.01) less than that seen with dopamine (Fig. 1A). These data indicate that direct stimulation of D1 dopamine receptors induces production of NO. Because both dopamine and H2O2, but not SKF R-38393, are rapidly autoxidized, we measured the t1/2 of nitrite production (Fig. 1B) and found there were no significant differences between dopamine (~5 h) and H2O2 or SKF R-38393 (~6 h), indicating that the higher levels of nitrite production induced by dopamine were not linked to different rates of autoxidation of dopamine and H2O2.



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FIG. 1.
DA, H2O2, and D1 receptor agonist SKF R-38393 induced nitrite generation and cytotoxicity in SK-N-MC cells. A, dose response of DA, H2O2, and SKF R-38393 induced nitrite production in SK-N-MC cells. SK-N-MC cells were subcultured into 12-well plates and treated with increasing doses of DA, H2O2, or SKF R-38393 for 16 h. Nitrite production in the medium was determined using Greiss reagent as described under "Experimental Procedures." The values are expressed as the average ± S.E. of triplicate experiments. B, time course of DA, H2O2, and SKF R-38393 induced nitrite production in SK-N-MC cells. Cells were treated with 200 µM DA, H2O2, or SKF R-38393. Nitrite levels in the medium were determined for 20 h after drug treatment. The values are the average ± S.E. of triplicate determinations. C, DA, H2O2, and SKF R-38393 induced cytotoxicities in SK-N-MC cells. Cultures were treated with increasing doses of DA, H2O2, or SKF R-38393 for 16 h. Cell viability was determined using the trypan blue exclusion test. The values are expressed as the average ± S.E. of triplicate experiments.

 

NO is a physiologically relevant retrograde neurotransmitter and, as such, is not necessarily cytotoxic, except when produced in large amounts. It was, thus, essential to estimate whether such enhanced production of NO was associated with enhanced cytotoxicity toward the SK-N-MC cells. SK-N-MC cultures were exposed for 16 h with increasing doses of dopamine, H2O2, and SKF R-38393, and cell death was measured by trypan blue staining, whereby viable cells exclude the dye (Fig. 1C). The results show that at all doses tested, production of NO by these compounds accelerated cell death relative to untreated cells. Moreover, dopamine was persistently more cytotoxic than either H2O2 or SKF R-38393, consistent with its ability to produce larger amounts of NO than either of these two compounds.

Dopamine Effects Are Extracellularly Mediated—Striatal degeneration is necessarily mediated by extracellular dopamine, since such neurons do not express TH and do not produce any dopamine. Therefore, it was important to show that the cytotoxic effects of dopamine on SK-N-MC cells were extracellularly mediated. We measured [3H]DA uptake in SK-N-MC cells transfected with the hDAT DNA and compared our findings to cells transfected with the pcDNA3.1 vector. In mock-transfected cells, there was no measurable uptake of [3H]dopamine, consistent with the absence of hDAT in these cells (Fig. 2A). In hDAT-transfected cells, there was a large increase in [3H]DA uptake (p < 0.001), which was completely blocked by 10 µM specific DAT blocker, INDT. In mock-transfected SK-N-MC cells challenged with 50 µM dopamine, the additional presence of INDT (10 µM) had no effect on either nitrite production (Fig. 2B) or cell viability (Fig. 2C), providing further evidence that dopamine was not taken up into these cells and that the observed toxicity of dopamine was entirely extracellularly mediated.



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FIG. 2.
Nitrite production and cytotoxicity induced by dopamine are linked to extracellular dopamine in SK-N-MC cells. A, [3H]DA uptake analysis in mock-transfected, hDAT DNA-transfected, and in the absence or presence of the DAT antagonist, INDT. [3H]DA uptake was measured as described under "Experimental Procedures." The values are expressed as the average ± S.E. of triplicate experiments. ***, p < 0.001, compared with the control. B, nitrite production induced by DA in the absence and presence of INDT. Results shown are the average ± S.E. of three experiments. C, cytotoxicity of DA in the absence or presence of INDT. Determination of cell survival was performed by counting cells using the trypan blue exclusion assay in triplicate experiments. In B and C, INDT (10 µM) was added 15 min before DA (50 µM) treatment. p < 0.05 (*) and p < 0.001 (***), compared with DA treatment.

 

Dopamine Effects Are Partially Mediated via D1 Dopamine Receptors—To further dissect the nature of dopamine-mediated production of nitrite and cell death, SK-N-MC cells were treated with dopamine in the absence or presence (200 µM) of the antioxidant SMBS, the D1-selective antagonist SCH 23390 (10 µM), or both. In the presence of SMBS, dopamine-induced nitrite production and cell death were significantly (p < 0.01) decreased by ~50% (Fig. 3A). In the presence of both SCH 23390 and SMBS, these cytotoxic effects of dopamine were virtually abrogated. These data indicate that dopamine actions occur through two distinct pathways, one involving D1 dopamine receptors and the other due to its properties as an oxidant.



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FIG. 3.
DA effects are mediated by the activation of D1 dopamine receptors and free radical mechanism. A, the nitrite production and cytotoxicity induced by DA (50 µM) was partially blocked by a co-treatment with the D1 receptor-selective antagonist SCH 23390 and the antioxidant SMBS and almost completely blocked by a co-treatment with both SCH 23390 and SMBS. SCH 23390 (10 µM) and SMBS (200 µM) were added 15 min before DA (50 µM) treatment. p < 0.01 (**) and p < 0.001 (***), compared with DA treatment. B, nitrite production and cytotoxicity of H2O2 (50 µM) was blocked by the antioxidant SMBS but not by the D1 receptor-selective antagonist SCH 23390. SCH 23390 (10 µM) and SMBS (200 µM) were added 15 min before H2O2 (50 µM) treatment. **, p < 0.01, compared with H2O2 treatment. C, the nitrite generation and cytotoxic effects of SKF R-38393 (50 µM) was blocked by the D1 receptor-selective antagonist SCH 23390 but not by the antioxidant SMBS. SCH 23390 (10 µM) and SMBS (200 µM) were added 15 min before SKF R-38393 (50 µM) treatment. The values shown are the average ± S.E. of triplicate experiments. p < 0.05 (*) and p < 0.01 (**), compared with SKF R-38393 treatment.

 

We next conducted parallel studies using H2O2 and found that in the presence of SMBS both nitrite production and cell death were significantly (p < 0.01) reduced to near control levels (Fig. 3B). When these studies were performed in the presence of SCH 23390, the ability of H2O2 to increase nitrite production and cell death was completely unaffected. These data show that unlike dopamine, H2O2 mediates its effects solely as an oxidant and not through activation of the D1 dopamine receptor.

Similar cytotoxicity studies were also conducted by directly stimulating D1 receptors with SKF R-38393 in the presence or absence of SMBS or SCH 23390. Unlike the results obtained with dopamine or H2O2, SMBS failed to modulate either SKF R-38393-induced nitrite production or cell death, consistent with lack of oxidation of this agonist. However, both SKF R-38393-generated nitrite production and cell death were blocked upon co-incubation of cells with SCH 23390 (Fig. 3C), confirming the direct participation of D1 dopamine receptors in the SKF R-38393-induced production of NO associated with enhanced cell death.

D1 Dopamine Receptors Increase NOS Expression—To define the mechanisms by which D1 receptors and dopamine increase production of NO, we measured the expression levels of the three NOS isozymes. Both dopamine and H2O2 caused increased expression of nNOS, iNOS, and eNOS in a dose-dependent manner (Fig. 4, A, B, and C, respectively). At concentrations of 50 µM, dopamine elicited a small but significantly (p < 0.05) higher increase in nNOS expression than H2O2, and both compounds increased eNOS expression by equivalent amounts. The largest difference between these compounds (50 µM) was observed with iNOS, where dopamine induced a significantly (p < 0.01) higher (>2-fold) increase in expression of this enzyme compared with H2O2. Similarly, at a concentration of 100 µM, dopamine caused a significantly (p < 0.01) higher (~2-fold) increase in iNOS expression relative to H2O2.



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FIG. 4.
Increased NOS expression in DA-, H2O2-, and SKF R-38393-treated SK-N-MC cells. A, immunoblot analysis of nNOS expression in SK-N-MC cells after 1 h of DA or H2O2 treatment for the indicated doses (0, 10, 50, 100 µM). B, immunoblot analysis of iNOS expression in SK-N-MC cells after 1 h of DA or H2O2 treatment. C, immunoblot analysis of eNOS expression in SK-N-MC cells after 1 h of DA or H2O2 treatment. The values are expressed as the average ± S.E. of triplicate experiments. In A, B, and C, p < 0.05 (*) and p < 0.01 (**), compared with DA treatment. D, SKF R-38393 induced an increased expression of nNOS, iNOS, and eNOS in SK-N-MC cells that was blocked by D1 receptor-selective antagonist SCH 23390. SCH 23390 (10 µM) was added 15 min before the SKF R-38393 treatment. Cells were incubated with the indicated input doses of SKF R-38393 for 1 h. *, p < 0.05, compared with SKF R-38393 treatment.

 

When we concurrently examined the effects of SKF R-38393, we found significant increases in nNOS, iNOS, and eNOS protein levels at all concentrations of the agonist tested (Fig. 4D). Moreover, these increases in NOS expression were significantly (p < 0.05) reduced by SCH 23390, attesting to the ability of the D1 receptor to activate the NOS enzymes.

We also verified the ability of the NOS inhibitor, L-NAME, to block the activation of NOS. In the presence of L-NAME (50 µM), dopamine-mediated nitrite production was reduced by ~50% (Fig. 5A), with a comparable increase in cell viability compared with cells not treated with L-NAME (Fig. 5B). By contrast, H2O2-mediated effects were much less sensitive to L-NAME, and in the presence of this NOS inhibitor, nitrite levels were reduced by only 15%, accompanied by only a modest increase in cell viability (Fig. 5). The SKF R-38393 effects were almost completely blocked by L-NAME, with restoration of both nitrite levels and cell viability to near control levels (Fig. 5), suggesting that SKF R-38393 mediates its effects almost entirely through the activation of the NOS enzymes. By contrast, at least part of the effects elicited by dopamine and virtually all of that induced by H2O2 occur through mechanisms that do not require the participation of the NOS enzymes.



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FIG. 5.
The oxidative stress and cytotoxic effects of DA are blocked by the NOS inhibitor, L-NAME. A, the nitrite production induced by DA, H2O2, or SKF R-38393 was ablated by L-NAME. B, the cytotoxic effects induced by DA, H2O2, or SKF R-38393 (50 µM) treatments were blocked by L-NAME. In A and B, L-NAME (50 µM) was added 15 min before 50 µM DA, H2O2, or SKF R-38393 treatment. The values are expressed as the average ± S.E. of triplicate experiments. *, p < 0.05, compared with DA treatment. #, p < 0.05, compared with H2O2 treatment. {Delta}, p < 0.05, compared with SKF R-38393 treatment.

 

Specific Dopaminergic-linked Signal Transduction Pathways Are Activated—The D1 dopamine receptor modulates several signal transduction pathways, including the activation of adenylyl cyclase (33), phospholipase C (34), protein kinase C (35), and inhibition of Na+/K+-ATPase and Na+/H+-antiporter activities (36, 37). To identify the signaling pathway participating in the cytotoxic response of D1 receptors, we treated cells with selective inhibitors of various kinases (Table I). The effects of both dopamine and SKF R-38393 proceeded in part through the activation of protein kinase A, a cAMP-dependent enzyme, consistent with the ability of the D1 dopamine receptor to increase, after activation, the intracellular steady-state levels of cAMP. By contrast, the effects of H2O2 were refractory to the protein kinase A inhibitor, KT5720, indicating a lack of direct activation of protein kinase A by H2O2 in our conditions or a lack of activating effect of H2O2 directly on the D1 sites. Nearly 70–80% of dopamine- and SKF R-38393-mediated nitrite production was sensitive to the presence of genistein, an inhibitor of tyrosine kinases, whereas H2O2 effects were ~50% ablated by this compound (Table I). Similarly, ~60% of dopamine- and SKF R-38393-mediated nitrite production was attenuated by the presence of wortmannin, an inhibitor of phosphatidylinositol 3-kinase, whereas H2O2 effects were somewhat more resistant to this compound. Interestingly, the presence of mPKCi, a highly selective PKC inhibitor, was found to further increase dopamine-, SKF R-38393-, and H2O2-mediated production of nitrite (by ~50, ~80, and~60%, respectively) above the stimulated levels (Table I), suggesting that PKC activation may suppress the production of free radicals by these compounds.


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TABLE I
Effect of kinase or NF-{kappa}B inhibitor on nitrite production induced by dopamine, H2O2, or SKF R-38393 treatment of SK-N-MC neuroblastoma cells

Nitrite production was expressed as nitrite concentration induced by the drug treatment minus nitrite concentration measured in control cells (41.4 ± 4.7 nM/105 cells, n = 18) using the method described under "Experimental Procedures." Data are shown as the mean ± S.E. of increase in nitrite concentration. The inhibitors were added 15 min before treatment with 50 µM each of either dopamine, H2O2 or SKF R-38393 for 16 h.

 

We also examined the participation of the nuclear factor NF-{kappa}B, a positive modulator of iNOS, in mediating D1-induced cytotoxicity and found that in the presence of NF-{kappa}B SN50, which inhibits the translocation of NF-{kappa}B from the cytoplasm to the nucleus, ~70% of dopamine and ~60% of the SKF R-38393-attributable effects were blocked (Table I). By contrast a much smaller effect (~40%) of NF-{kappa}B SN50 was seen on H2O2-mediated events.

Functional Activation of iNOS upon Stimulation of D1 Dopamine Receptors—To identify the specific NOS isozyme activated upon D1 dopamine receptor stimulation, the enzymatic activity of iNOS and the combined activity of eNOS/nNOS were studied by measuring the conversion of [3H]arginine to [3H]citrulline in the presence or absence of the highly selective iNOS inhibitor, 1400W (Ki = 7 nM). Lysates prepared from SK-N-MC cells treated with 50 µM SKF R-38393 were used in the assays, and total activity was calculated after subtracting basal values corresponding to vehicle-treated cells (Fig. 6). SKF R-38393 induced a significant (p < 0.01) increase in total NOS activity in a D1 dopamine receptor-dependent manner, and in the presence of SCH 23390, the evoked response was reduced by 80%. In the presence of 1400W (1 µM), SKF R-38393-evoked response was significantly (p < 0.05) reduced by ~60%, suggesting that in these cells stimulation of D1 dopamine receptors primarily activates iNOS. By contrast, the 1400W-insensitive response, representing the combined eNOS/nNOS activity, constitutes ~40% of the total NOS activity. That these measured activities were entirely due to NOS is evidenced by the ability of L-NAME to reduce (by 80%) the production of [3H]citrulline to levels similar to that seen in the presence of SCH 23390 (Fig. 6).



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FIG. 6.
The stimulation of D1 dopamine receptors induced activation of NOS. SK-N-MC neuroblastoma cells were treated for 1 h with 50 µM SKF R-38393, and NOS activity was determined by measuring the conversion of [3H]arginine to [3H]citrulline, as described under "Experimental Procedures." Where indicated, L-NAME (1 mM) or 1400W (1 µM) or SCH 23390 (10 µM) was added 15 min before the addition of SKF R-38393. In all assays, NOS activity was calculated after subtracting basal values, obtained from cells treated with vehicle alone (16.9 fmol/µg protein/min). The values are expressed as the means ± S.E. of quadruplicate experiments. *, p < 0.05, compared with SKF R-38393 treatment.

 

Immunolabeling of nNOS in Rat Primary Striatal Neuronal Cultures—To assess the physiological relevance of our findings in SK-N-MC cells, cultures of rat primary striatal neurons were challenged with 5 µM each of dopamine, H2O2, or SKF R-38393 for 1 h followed by immunostaining with nNOS antiserum (Fig. 7). In untreated cultures, low levels of basal nNOS immunoreactivity (15.1 ± 2.3% of total cells) were seen in the neuronal cell bodies. Treatment with dopamine significantly increased nNOS immunolabeling (50.0 ± 2.6% of total cells, p < 0.05), which was reduced equally (to 30% of total cells, p < 0.05) by both SCH 23390 (1 µM) and SMBS (20 µM) (Fig. 7). Co-incubation of dopamine-treated cultures with both SCH 23390 and SMBS blocked the dopamine induction of nNOS expression (data not shown). Treatment with H2O2 did not affect nNOS expression compared with the basal immunoreactivity detected in untreated neuronal cultures regardless of pretreatment with SCH 23390 or SMBS (13.2–14.0% of total cells)



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FIG. 7.
Immunocytochemical analysis of nNOS labeling in dopamine-, H2O2-, and SKF R-38393-treated striatal neurons. Rat primary striatal neuronal cultures were treated for 1 h with 5 µM either dopamine, H2O2, SKF R-38393, or vehicle (0.2% H2O; untreated) in the presence or absence of SCH 23390 (1 µM) or SMBS (20 µM). Pretreatment with SCH 23390 or SMBS occurred 15 min before adding either dopamine, H2O2, or SKF R-38393. Data are representative of three experiments conducted in duplicate.

 

Incubation with SKF R-38393 substantially increased nNOS immunoreactivity (34.5 ± 4.3% of total cells, p < 0.05), which was reversed by SCH 23390 (15.7 ± 3.8% p < 0.05). By contrast, SMBS was devoid of any effect on SKF R-38393-induced increase in nNOS staining, and 33.7 ± 2.4% of the total neurons were immunopositive for nNOS. The ability of D1 dopamine receptors to activate nNOS in neurons is also seen in the functional activation of NOS activity where at least 40% of the activity was 1400W-insensitive (Fig. 6).

Taken together, these data clearly show that both dopamine and to a lesser extent SKF R-38393 increase nNOS expression in rat primary striatal neurons and are entirely consistent with our findings in SK-N-MC cells with these agonists. The effect of dopamine occurs through both its autoxidation (~50% of the effect) and an activation of D1 receptors (~50% of the effect); this later effect was mimicked by the selective D1-like agonist SKF R-38393. In contrast, H2O2 fails to affect nNOS expression in striatal neurons.

Immunolabeling of iNOS in Rat Primary Striatal Neuronal Cultures—We next examined iNOS immunolabeling in neurons and found that in untreated cultures there was little or no iNOS immunostaining (<6% of total cells were immunopositive for iNOS) (Fig. 8). Upon treatment with dopamine, however, there was a strong and significant (p < 0.05) increase (53.9 ± 3.2% of total cells) in iNOS immunoreactivity in striatal neurons. This induction of iNOS expression in neurons was significantly (p < 0.05) reduced, but not completely blocked, by both SCH 23390 and SMBS. Moreover, the iNOS immunoreactivity was almost identical in both dopamine plus SCH 23390-treated and dopamine plus SMBS-treated neurons (30.2 ± 1.7 and 33.1 ± 1.9% of total cells, respectively). Co-incubation of dopamine-treated cultures with SCH 23390 and SMBS together abrogated the dopamine induction of iNOS expression (data not shown). Treatment with H2O2 resulted in little or no increases in iNOS immunoreactivity, and only 7–8% of the total cells were immunopositive for iNOS (Fig. 8).



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FIG. 8.
Immunocytochemical analysis of iNOS labeling in dopamine-, H2O2-, and SKF R-38393-treated striatal neurons. Rat primary striatal neuronal cultures were treated for 1 h with 5 µM either dopamine, H2O2, SKF R-38393, or vehicle (0.2% H2O; untreated) in the presence or absence of SCH 23390 (1 µM) or SMBS (20 µM). Pretreatment with SCH 23390 or SMBS occurred 15 min before adding either dopamine, H2O2, or SKF-38393. Data are representative of three experiments conducted in duplicate.

 

In SKF R-38393-challenged cultures, iNOS staining was also significantly increased to 33.8 ± 2.7% of total cells (p < 0.05), although the increase was much less than that obtained with dopamine (Fig. 8). Such induction of iNOS expression by SKF R-38393 was completely reversed by pretreatment with SCH 23390 (to 10.3 ± 4.5% of total cells, p < 0.05) but was unaffected by pretreatment with SMBS (34.5 ± 2.5%). This finding is similar to that seen in SK-N-MC cells, where D1 dopamine receptor-mediated stimulation of NOS function was sensitive to SCH 23390 and where 60% of the activity was due to iNOS activation (Fig. 6).

These observations indicate that both dopamine and SKF R-38393 induce overexpression of iNOS in striatal neurons in an SCH 23390-dependent manner, with only the dopamine treatment sensitive to SMBS, in accordance with our findings in SK-N-MC cells, where dopamine effects occur through two pathways, autoxidation and activation of D1 receptors. By contrast, H2O2 had no effect in the induction of iNOS expression.

eNOS Immunostaining in Rat Striatal Neuronal Cultures— Weak basal eNOS immunoreactivity was seen in untreated cultures of striatal neurons, and ~12% of the total neurons were immunopositive for eNOS (Fig. 9). This basal level of eNOS immunoreactivity was not significantly changed upon treatment of neurons with dopamine, H2O2, or SKF R-38393 irrespective of whether or not SCH 23390 or SMBS was present. These data suggest that eNOS is only a very minor participant in both the 1400W-insensitive response of Fig. 6 and, thus, in the neurotoxicity induced by the dopaminergic agonists.



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FIG. 9.
Immunocytochemical analysis of eNOS labeling in dopamine-, H2O2-, and SKF R-38393-treated striatal neurons. Rat primary striatal neuronal cultures were treated for 1 h with 5 µM either dopamine, H2O2, SKF R-38393, or vehicle (0.2% H2O; untreated) in the presence or absence of SCH 23390 (1 µM) or SMBS (20 µM). Pretreatment with SCH 23390 or SMBS occurred 15 min before adding either dopamine, H2O2, or SKF R-38393. Data are representative of three experiments conducted in duplicate.

 

Cytotoxicity in Striatal Neurons—To further test the physiological relevance of our findings in SK-N-MC neuronal cells, we examined the cytotoxic effects of dopamine, H2O2, and SKF R-38393 on striatal neurons, as indexed by cell viability assays (Fig. 10). Incubation (16 h) of striatal neurons with dopamine induced higher neuronal cell death compared with H2O2 (p < 0.05), whereas SKF-R38393 induced almost similar levels of neurotoxicity as H2O2. Co-incubation of neurons with L-NAME (5 µM) ablated SKF-R38393-mediated neuronal death and decreased (by ~50%) dopamine-induced neuronal death without altering H2O2-mediated neuronal death (Fig. 10). The dopamine-mediated neurotoxicity was only partly sensitive to SMBS and SCH 23390, but together these reagents completely blocked dopamine-induced neuronal cell death (data not shown). Together these combined data suggest that in neurons dopamine effects are also mediated through two distinctive pathways, one that is dependent on its autoxidation and one that occurs through stimulation of the D1 dopamine receptor. Interestingly, H2O2-mediated toxicity does not proceed through activation of the NOS enzymes, somewhat different from our findings in SK-N-MC neuroblastoma cells. More importantly, however, is that these results emphasize that H2O2, which is widely used as a substitute for dopamine as a source of free radicals, may not be an appropriate oxidant of choice.



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FIG. 10.
Cell viability of primary neuronal cultures. Rat primary striatal neuronal cultures were treated for 16 h with 5 µM either dopamine, H2O2, SKF R-38393, or vehicle (0.2% H2O; untreated) in the presence or absence of L-NAME (5 µM). Pretreatment with L-NAME occurred 15 min before adding either dopamine, H2O2, or SKF-38393. The number of viable cells were 562,800 ± 7,100 cells in the controls. Data are representative of three experiments conducted in duplicate. *, p < 0.05 compared with control, untreated cells; ¶, p < 0.05 compared with cells not treated with L-NAME from within the same group.

 


    DISCUSSION
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
The mechanistic basis of postsynaptic striatal degeneration is ill-defined but is primarily observed in neurological states such as MSA-P, Huntington's disease, and as a secondary consequence of 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine exposure or METH addiction, where presynaptic neurons are depleted of dopamine stored in vesicles, resulting in increased release and sustained accumulation of extracellular dopamine in the synapse. Such an increase in synaptic dopamine efflux is accompanied by elevated ROS production through autoxidation of extracellular dopamine to dopamine quinone, superoxide, and the hydroxyl radical, ·OH.

Our findings highlight a previously unknown role of the D1 dopamine receptor in the activation of nNOS and iNOS and in the genesis of oxidative stress-induced degeneration, a role that may be central to the specific and selective loss of postsynaptic striatal neurons observed in the above-mentioned neurological conditions. Chronic stimulation of D1 sites has been shown by us (31, 38) and others (3942) to lead to the functional desensitization of the receptor with rapid loss of functional activity. The studies presented here show that there may be additional consequences of chronic increases in extracellular dopamine levels, which can cause profound cytotoxicity. Although many studies have demonstrated that dopamine autoxidation can induce production of NO and RNS, our studies directly link the chronic stimulation of D1 receptors to activation of NOS enzymes, with production of NO. Because rat striatal primary neurons also show activation of NOS in specific response to D1 receptor stimulation in a manner reminiscent to that in SK-N-MC cells, our findings in this human cell line are likely to be of physiological relevance. Although NO acts as a retrograde neurotransmitter in brain, its overproduction leads to oxidative stress through formation of RNS (43) such as peroxynitrite after combination with superoxide (44). The peroxynitrite in turn nitrosylates tyrosine residues on proteins and modifies lipids and DNA, causing cellular damage and eventual apoptosis (45).

In both striatal neurons and in SK-N-MC cells the expression of iNOS and nNOS was substantially increased in a D1 dopamine receptor-dependent manner, with no change observed for eNOS, consistent with the expression of eNOS predominantly in the endothelia of brain vascular tissues and glial cells. These data suggest that in both SK-N-MC cells and in brain there is minimal contribution by eNOS toward the cytotoxic events mediated by dopamine or SKF R-38393. Moreover, in SK-N-MC cells, there is a nearly 2-fold higher activation of iNOS enzymatic activity as compared with the combined activity of eNOS and nNOS, suggesting that this enzyme has a prominent role in mediating the effects of D1 receptor-induced cytotoxicity. In contrast to our findings in SK-N-MC, where H2O2 induced activation of NOS enzymes, albeit to a lesser extent than that seen with either dopamine or SKF R-38393, there did not appear to be any activation of the NOS enzymes by the oxidant in rat neuronal striatal cultures. Altogether, our results imply that the co-participation of nNOS and iNOS but not eNOS by both dopamine autoxidation and activation of D1 receptors mediates dopamine-induced neurotoxicity.

Interestingly, our results show that H2O2-induced cytotoxicity seems to involve signal transduction mechanisms distinct from induction of the NOS isoenzymes. Thus, even in SK-N-MC neuroblastoma cells, only a very modest activation of iNOS was seen at high concentrations, whereas in neurons, this response was completely absent. Moreover, in SK-N-MC cells H2O2 was much less cytotoxic than dopamine. Our results demonstrate that H2O2 may not be an appropriate substitute for studying dopamine-mediated cytotoxicity. This is a critical observation, especially given the fact that H2O2 is widely used to reproduce dopamine cytotoxicity due to the autoxidation of dopamine with generation of either H2O2 and/or ·OH.

Another interesting aspect of our findings is that we obtain dopamine cytotoxicity in cell lines at concentrations far below that used in most studies found in the literature. Thus, in many cultured cell lines, the toxic use of dopamine at concentrations of 300–500 µM is not uncommon. Although the concentrations of dopamine we used in our studies with SK-N-MC cells are higher than that seen in normal striatum, where dopamine content is believed to be ~1–10 µM (46, 47), our values of 50 µM are close to the pathological system, where dopamine content in striatum has been reported to reach as high as 50 µM (4648). The reason why we were able to use lower concentrations of dopamine is partly linked to the fact that SK-N-MC cells were serum-starved overnight and cell treatment occurred in a serum-free medium. Moreover, all of our studies on SK-N-MC cells were conducted in RPMI 1640 medium, a sodium pyruvate-free medium, and earlier studies by Wersinger (49) showed that pyruvate can act as a scavenger of both free radicals, H2O2 and hydroperoxides, and byproducts of oxidant-induced lipid peroxidation.

In neurons we were able to use even lower levels of dopamine (5 µM) than that used in SK-N-MC cells. The reasons for this are not immediately clear but may be related to differences in cell biology between neurons and SK-N-MC cells. Thus, most cell lines are tumor or transformed cells, which actively proliferate, overexpressing many of the trophic proteins and growth factors implicated in cell proliferation, thereby rendering them less sensitive to oxidative insults. By contrast, neurons only maturate and differentiate in primary cultures and are less well protected against oxidant-induced cytotoxicity due to very low levels of detoxification systems and intracellular antioxidants. Indeed, even in brain, neurons are known to derive most of their anti-oxidant and growth factors from glia and astrocytes.

In brain, NO produced by iNOS and nNOS has been demonstrated to be central to nitrative and oxidative mechanisms of striatal neurodegeneration. However, there is still much debate regarding the specific identity of the participatory NOS enzymes. Altered nNOS, but not iNOS, expression has been shown to contribute to disease progression in HD R6/2 transgenic mice, with increased expression during the presymptomatic stage of the disease (50). Quinolinic acid-induced striatal neurotoxicity, resembling the alterations observed in HD, was found to be mediated by activated nNOS (5153). Moreover, nNOS knock-out mice were protected from striatal N-methyl-D-aspartate-mediated excitotoxicity (54). By contrast, activated iNOS was found to mediate 3-nitropropionic acid-induced (55), N-methyl-D-aspartate-induced (56), and malonate-induced (57) neurodegenerative lesions of the rat striatum, various animal models of HD. iNOS-derived NO, mostly from activated microglia, has also been shown to be implicated in the chronic inflammatory reactions associated with HD and in the progression of this disease (58). Excessive production of NO upon overstimulation of N-methyl-D-aspartate receptors is implicated in calcium deregulation and neurodegeneration of striatal neurons due to increased intracellular calcium release from the mitochondrial pool through an NO-activated mitochondrial permeability transition pore (59). Intracellular calcium stabilizes the binding of calmodulin to nNOS or eNOS, initiating NO synthesis (60).

Because iNOS tightly binds to calmodulin, the activity of iNOS is not modulated by intracellular calcium. However, once activated, iNOS-derived NO production can last for several hours until the enzyme is degraded (61). The main controlling mechanism in iNOS-derived NO production occurs through transcriptional regulation of the enzyme after activation of several transcription factors (62). These include NF-{kappa}B, interferon regulatory factor (IRF-1), signal transducers and activators of transcription 1{alpha} (STAT1{alpha}), octamer binding factor, hypoxia-inducible factor, nuclear factor inteukin-6, cAMP response element (CRE), activating protein-1 (AP-1), and CAAT/enhancer-binding protein (C/EBP{beta}) (17, 6370). Although the precise mechanism by which D1 dopamine receptors stimulate iNOS transcription remains to be elucidated, our findings with the various kinase inhibitors suggests that one or more of the signaling intermediates activated by these receptors may be involved. Consequently, protein kinase A, tyrosine kinase, phosphatidylinositol 3-kinase/Akt cascade, and NF-{kappa}B may all contribute to the induction of iNOS synthesis. Our results with mPKCi suggest that in SK-N-MC cells, PKC may act to suppress iNOS activation, since its inhibition caused large increases in nitrite levels. The effect of PKC in modulating iNOS expression is either positive or negative and dependent on specific PKC isoforms expressed in cells (7173). The delineation of the participatory proteins and pathways modulating D1 dopamine receptor-mediated induction of NOS and NO release may have some potential in the therapeutic prevention of striatal neurodegeneration. Thus, although our findings need to be confirmed in animal models, our results would imply that, at least in diseases where there is accumulation of extracellular dopamine such as L-DOPA-MSA-P, MSA, METH addiction, and HD, blockade of D1 dopamine receptors may prove to be beneficial in therapy.


    FOOTNOTES
 
* This study was supported in part by National Institutes of Health Grants NS-34914 and NS-41555 and a National Alliance for Research on Schizophrenia and Depression Investigator Award. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

{ddagger} To whom correspondence should be addressed: Laboratory of Molecular Neurochemistry, The Research Bldg., Rm. W222, 3970 Reservoir Rd., NW, Washington, D. C. 20007. Tel.: 202-687-0282; Fax: 202-687-0279; E-mail: sidhua{at}georgetown.edu.

1 The abbreviations used are: MSA, multiple system atrophy; MSA-P, parkinsonism subtype of MSA; HD, Huntington's disease; ROS, reactive oxygen species; RNS, reactive nitrogen species; DAT, dopamine (DA) transporter; hDAT, human DAT; TH, tyrosine hydroxylase; NOS, nitric-oxide synthase; iNOS, inducible NOS; eNOS, endothelial NOS; nNOS, neuronal NOS; SMBS, sodium metabisulfite; L-NAME, N(G)-nitro-L-arginine methyl ester; INDT, indatraline; METH, methamphetamine; PBS, phosphate-buffered saline. Back


    ACKNOWLEDGMENTS
 
We thank Stephane Pere for excellent technical assistance.



    REFERENCES
 TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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