Metabolism of 3-Nitrotyrosine Induces Apoptotic Death in Dopaminergic Cells

Materials.

Tyrosine, 3-nitrotyrosine, 2-aminobicyclo-(2,2,1)-heptane-2-carboxylic acid (BCH), pargyline, NSD1015 (3-hydroxybenzylhydrazine dihydrochloride), GBR12935 (1-[2-(diphenyl-methoxy)-ethyl]-4-(3-phenylpropyl)piperazine), and tyramine oxidase were purchased from Sigma (St. Louis, MO). The holoenzyme tyrosine (aromatic amino acid) decarboxylase from Streptococcus faecalis was purchased from Worthington (Freehold, NJ).

Cell culture.

The experiments were performed with the undifferentiated human teratocarcinoma NT2 and rat pheochromocytoma PC12 cell lines. NT2 cells were cultured in Optimem medium supplemented with 10% fetal bovine serum and 1% penicillin–streptomycin (containing 100 μm tyrosine). PC12 cells were cultured in RPMI-1640 medium supplemented with 10% heat-inactivated horse serum, 5% fetal calf serum, 5 mm l-glutamine, and 1% penicillin–streptomycin. The medium was refreshed every 2–3 d. For all experiments, PC12 cells were plated on collagen-coated surface for 48 h before additional treatment or until cells reached 60% confluence. Both cell lines were maintained at 37°C in 5% CO₂.

Cell treatment.

NT2 or PC12 cells (50–60% confluent) were treated with a range of 3-nitrotyrosine concentrations for 24, 48, or 72 h, in the presence or absence of several inhibitors. Chemical compounds used were the l-aromatic amino acid transporter inhibitor BCH (at a final concentration of 100 μm), the monoamine oxidase inhibitor pargyline (100 μm), the aromatic amino acid decarboxylase inhibitor NSD1015 (100 μm), the dopamine transporter inhibitor GBR12935 (10 μm), the caspase-1 inhibitor benzyloxycarbonyl-Try-Val-Ala-Asp-chloromethyl-ketone (Ac-YVAD-CMK) (50 μm; Alexis Biochemicals, San Diego, CA), the caspase-3 inhibitor Z-Asp-Glu-Val-fluoromethyl ketone (Z-DEVD-FMK) (50 μm; BD PharMingen, San Jose, CA), and the general caspases inhibitor N-benzyloxycarbonyl-Val-Ala-Asp-fluoromethyl ketone (Z-VAD-FMK) (50 μm; BD PharMingen, San Jose, CA). Cells were pretreated with cysteine (final concentration of 1 mm) or the inhibitors for 18 h before 3-nitrotyrosine treatment and re-added every 24 h.

Measurement of cell death with trypan blue exclusion method.

Cell viability during the treatment with 3-nitrotyrosine in the presence or absence of inhibitors was monitored using the trypan blue exclusion method. Numbers of alive and dead cells were determined using a hemocytometer under a light microscope. Healthy nuclei from viable cells appeared round and phase bright, whereas nuclei from dead or dying cells appeared blue and irregularly shaped. In all experiments, floating cells were collected and assayed using the procedure and criteria described above. All cells were counted, and results were expressed as ratio of dead cells versus total cells (alive + dead).

Annexin V and flow cytometry.

For apoptosis or necrosis detection, cells were stained with fluorescein isothiocyanate-labeled annexin V and propidium iodide (PI) (Vybrant apoptosis assay kit; Invitrogen, Carlsbad, CA). After incubations, floating as well as adherent cells that were later trypsinized were pooled and centrifuged for 5 min at 1400 rpm. Pelleted cells were washed with PBS, centrifuged again, and resuspended in 100 μl of 1× annexin-binding buffer (10 mm HEPES, 140 mm NaCl, and 2.5 mm CaCl₂, pH 7.4) yielding a cell density of 2 × 10⁶ cells/ml. A total of 10 μl of annexin V conjugate and 2 μl of 100 μg/ml PI working reagent were added to each 100 μl of cell suspension. The cells were incubated at room temperature for 30 min. After the incubation period, 400 μl of 1× annexin-binding buffer was added and the samples were kept on ice. The stained cells were analyzed by flow cytometry, and the percentage of cells was calculated using the Cellquest software. This method was able to distinguish among living (annexin V⁻/PI⁻), apoptotic (annexin V⁺/PI⁻), and necrotic (annexin V⁻/PI⁺) cells.

Postnatal rat midbrain cell culture model.

Midbrain cultures obtained as described previously (Przedborski et al., 1996) were maintained 2 weeks at 37°C in 5% CO₂, and then wells were randomly assigned to either treatment group or to the control group. 3-Nitrotyrosine or 3-nitrotyramine was added to the culture medium at a final concentration of 2 and 1 mm, respectively, for 70 h. Separate wells were pretreated for 24 h with 400 μm NSD1015 or 500 μm pargyline before treatment with 3-nitrotyrosine. Control cultures received PBS instead of 3-nitrotyrosine, and NSD1015 was re-added every 24 h. At the end of the treatment, the medium was aspirated and cultures were fixed in 0.1 m PBS containing 4% paraformaldehyde for 30 min at 4°C. After four washes with PBS, the cells were incubated in 0.1% Triton X-100 in PBS plus 5% normal goat serum for 1 h. The cells were then incubated overnight at 4°C with a mouse monoclonal anti-tyrosine hydroxylase antibody (1:800; Chemicon, Temecula, CA) in PBS containing 0.1% Triton X-100 and 3% normal goat serum. After four washes in PBS, cells were incubated with an Alexa-Fluor 488 secondary antibody (goat anti-mouse, 1:500; Invitrogen) in PBS containing 0.1% Triton X-100 and 3% normal goat serum for 1 h. Cultures were then rinsed four times in PBS, and coverslips were mounted on slides using DakoCytomation (Glostrup, Denmark) fluorescent mounting medium. Only cells with distinct immunoreactivity, clear neuronal shape, and a nucleus were considered for counting tyrosine hydroxylase-positive neurons.

Western blotting.

Cells were washed with PBS, solubilized in 100 μl of ice-cold lysis buffer [20 mm HEPES, 150 mm NaCl, 1 mm EGTA, 1.5 mm MgCl₂, 10% glycerol, 1% Triton X-100, protease inhibitors (1 mm phenylmethylsulfonyl fluoride, 1 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mm sodium pyrophosphate, and 50 mm sodium fluoride), 2 mm sodium orthovanadate, and 1 μm lactacystin), sonicated, and analyzed by Western blotting. The protein concentration was determined by a Bradford assay. Proteins (40 μg) were separated by a 10% SDS-page gel transferred overnight to polyvinylidene difluoride or nitrocellulose membranes at 20 V and probed with an affinity-purified polyclonal anti-3-nitrotyrosine antibody (1:5000 dilution) (Fries et al., 2003), a monoclonal anti-α-tubulin antibody (1:3000 dilution; clone B-5-1-2; Sigma), and a monoclonal anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:40,000 dilution; Chemicon). HRP-conjugated goat anti-rabbit IgG or HRP-conjugated goat anti-mouse IgG (1:5000 dilution) (Bio-Rad, Hercules, CA) was used for detection. Proteins were then visualized by enhanced chemiluminescence. For band quantification, fluorescent secondary antibodies were used suitable for the infrared imaging system, membranes were scanned, and fluorescence was measured (1:7500 dilution; Odyssey; LI-COR Biosciences, Lincoln, NE).

Immunofluorescence microscopy.

Cells were fixed with cold methanol for 20 min at −20°C and a mixture of cold acetone/methanol at 1:1 for 5 min at −20°C. The cells were blocked in PBS plus 0.3% Triton X-100 (PBS-T) containing 10% BSA and normal goat serum for 1 h at 25°C. After several washes with PBS-T, the cells were double labeled with polyclonal anti-3-nitrotyrosine (1:300) and monoclonal anti-α-tubulin (1:1000) antibodies left overnight at 4°C. Secondary antibodies were indocarbocyanine-conjugated goat anti-rabbit IgG (1:250 dilution; Jackson ImmunoResearch, West Grove, PA) and Alexa-Fluor 488-conjugated goat anti-mouse IgG (1:500 dilution; Invitrogen). Nuclear counterstaining was performed by using 4′-6-diamidino-2-phenylindole (1:10,000 dilution; Invitrogen). Fluorescent images were obtained using an inverted microscope 1X70 (Olympus Optical, Tokyo, Japan) or by confocal immunofluorescence microscopy.

Tyrosine decarboxylase activity assay.

Tyrosine decarboxylase activity was quantified by measuring the conversion of tyrosine to tyramine by HPLC. Tyrosine (0.4 mm) diluted in 0.1 m sodium acetate buffer at pH 5.5, the reported optimal pH for the catalytic activity of tyrosine decarboxylase (Epps, 1944), was incubated at 37°C with tyrosine decarboxylase (0.25 U/ml), in the absence or presence of different concentrations of 3-nitrotyrosine. Aliquots of the reaction mixture were taken over time, acidified to stop the enzymatic reaction, centrifuged at 10,000 rpm for 5 min, and then analyzed by HPLC equipped with a diode array detector.

Tyramine oxidase activity assay.

Tyramine oxidase activity was quantified by measuring the conversion of tyramine to 4-hydroxybenzaldehyde by HPLC. To obtain pure 3-nitrotyramine, 3-nitrotyrosine (1.5 mm) was incubated with 0.25 U/ml tyrosine decarboxylase at 37°C in 0.1 m sodium acetate buffer, pH 5.5, for 5 h. After acidification and centrifugation, the reaction mixture was analyzed by HPLC showing the formation of a single product presenting an absorbance at 365 nm. The identity of the nitrated derivative formed was confirmed by mass spectrometry as 3-nitrotyramine (mass/charge ratio of 183).

The reported optimal pH for the reaction of tyramine oxidase is 7.0 (Yamada et al., 1967). Tyramine (0.4 mm) diluted in 0.1 m sodium acetate at pH 6.9 was incubated at 37°C with tyramine oxidase (0.25 U/ml) in the absence or presence of different concentrations of 3-nitrotyramine. Aliquots were removed (80 μl) over time, acidified (addition of 8 μl of 1N HCl) to stop the enzymatic reaction, centrifuged at 10,000 rpm for 5 min, and then analyzed by HPLC.

HPLC detection of tyrosine and nitrotyrosine metabolites.

Aliquots of reaction mixtures were analyzed by a Hewlett Packard (Palo Alto, CA) HPLC system with a diode array detector using an octadodecyl silica gel reverse-phase column, 5 μm, 4.6 × 250 mm (Jupiter; Phenomenex, Torrance, CA) and 0.1% trifluoroacetic acid in ultra pure water (solvent A) and 100% acetonitrile (solvent B). The reaction mixture was eluting using an increasing linear gradient of solvent B from 0 to 15% in 10 min and then to 17% in 25 min, at a flow rate of 1 ml/min. The HPLC detector was set at 215, 275, and 365 nm. The HPLC system was calibrated with external standards at 275 and 365 nm to follow the nitrated products. Under these HPLC conditions, tyrosine, tyramine, 3-nitrotyrosine, 3-nitrotyramine, 4-hydroxybenzaldehyde, and 4-hydroxy-3-nitro-benzaldehyde were eluted at 11.1, 10.6, 14.1, 15, 13.6, and 22.2 min, respectively. Kinetic parameters were determined by Lineweaver–Burk plots of initial velocities obtained with a substrate concentration of 0.4 mm tyrosine and tyramine for tyrosine decarboxylase and tyramine oxidase, respectively.

HPLC detection of dopamine metabolites.

PC12 cells were lysed in mobile phase (citrate/phosphate buffer containing 2% methanol, pH 3), sonicated, and centrifuged. Supernatants were further centrifuged on 0.22 μm filter Eppendorf, preequilibrated with mobile phase. Fifty microliter aliquots were analyzed by HPLC with electrochemical detection set at −200, 0, 200, 300, and 400 V (Coularray; ESA, Chelmsford, MA). Chromatographic separation was achieved using an octadodecyl silica gel reverse-phase column, 5 μm, 4.6 × 250 mm (Jupiter; Phenomenex), providing complete separation of all analytes of interest, including, in order of elution, l-3,4-dihydroxyphenylalanine (l-DOPA), dopamine, and 3,4-dihydroxyphenylacetic acid (DOPAC). The residual pellets were resuspended in lysis buffer and sonicated, and protein concentrations were determined.

Quantification of 3-nitrotyrosine by HPLC-tandem mass spectrometry

Control and 3-nitrotyrosine treated cells were washed extensively with ice-cold PBS and harvested after application of trypsin in PBS. Cells were sonicated, and the content of free and protein 3-nitrotyrosine in lysates was analyzed by HPLC with on-line electrospray ionization tandem mass spectrometry (LC/ESI/MS/MS) using stable isotope dilution methodology on a triple quadrupole mass spectrometer (API 365; Applied Biosystems, Foster City, CA) with Ionics EP 10+ upgrade (Ionics, Concord, Ontario, CA) interfaced to a Cohesive Technologies (Franklin, MA) Aria LX Series HPLC multiplexing system, as described in detail previously (Brennan et al., 2002). Synthetic [¹³C₆]-labeled standard was used as internal standard for quantification of natural abundance 3-nitrotyrosine. Simultaneously, a universal labeled precursor amino acid, [¹³C₉,¹⁵N₁]tyrosine, was added, permitting potential intrapreparative formation of nitro[¹³C₉,¹⁵N]tyrosine to be routinely monitored and shown to be negligible (i.e., ≪5% of the level of the natural abundance product observed). Results are normalized to the content of the precursor amino acid tyrosine (i.e., nitrotyrosine/tyrosine, micromoles/moles), which was monitored within the same injection.