From the Department of Pediatrics, Georgetown University, Washington, D. C. 20007
Received for publication, March 26, 2003 , and in revised form, May 5, 2003.
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ABSTRACT |
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INTRODUCTION |
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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-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.
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EXPERIMENTAL PROCEDURES |
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Cell Culture, Drug Treatment, and Cell ViabilitySK-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 CulturesStriata from
18-day-old rat embryos were isolated, and cells were dissociated by mechanical
disruption, counted, and grown (600800 cells seeded/mm2) in
neurobasal medium supplemented with 2% (v/v) B-27 supplement and 50
µM -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 MeasurementsNOS 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 010
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 (510 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 TransfectionHuman 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 AnalysisCell 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.
ImmunocytochemistrySix-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 AnalysisResults 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.
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RESULTS |
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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 MediatedStriatal 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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Dopamine Effects Are Partially Mediated via D1 Dopamine
ReceptorsTo 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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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 ExpressionTo 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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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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Specific Dopaminergic-linked Signal Transduction Pathways Are
ActivatedThe 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 7080% 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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We also examined the participation of the nuclear factor NF-B, a
positive modulator of iNOS, in mediating D1-induced cytotoxicity and found
that in the presence of NF-
B SN50, which inhibits the translocation of
NF-
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-
B SN50 was seen on
H2O2-mediated events.
Functional Activation of iNOS upon Stimulation of D1 Dopamine
ReceptorsTo 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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Immunolabeling of nNOS in Rat Primary Striatal Neuronal CulturesTo 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 (13.214.0% of total cells)
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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 CulturesWe 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 78% of the total cells were immunopositive for iNOS (Fig. 8).
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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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Cytotoxicity in Striatal NeuronsTo 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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DISCUSSION |
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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 300500 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 110 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-B, interferon regulatory factor (IRF-1), signal transducers and
activators of transcription 1
(STAT1
), 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
) (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-
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.
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FOOTNOTES |
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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.
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ACKNOWLEDGMENTS |
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REFERENCES |
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