SNAP, nerve growth factor (NGF), 1H-[1, 2, 4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ) and KT5823 were obtained from Wako Pure Chemical (Osaka, Japan). Trifluoroacetic acid (TFA), S-methyl methanethiosulfonate (MMTS), α-cyano-4-hydroxycinnamic acid (α-CHCA), bovine serum albumin (BSA), imipramine hydrochloride, dopamine, 8-bromoadenosine 3', 5'-cyclic monophosphate (8-Br-cAMP), 8-Br-cGMP, and glibenclamide were purchased from Sigma-Aldrich (St. Louis, MO, USA). Pituitary adenylate cyclase-activating polypeptide (PACAP) and N-[6-(biotinamido)hexyl]-3'-(2'-pyridyldithio)propionamide (biotin-HPDP) were supplied by Peptide Institute (Osaka, Japan) and Pierce Chemical (Rockford, IL, USA), respectively. Other chemicals were of reagent grade.
Preparation of S2 and P2 fractions from spinal cords
Male ddy mice (5 weeks old) were purchased from Shizuoka Laboratory Centre (Hamamatsu, Japan). The mice were housed under conditions of a 12-h light-12-h dark cycle, a constant temperature of 22 ± 2°C, and 60 ± 10% humidity. They received food and water ad libitum. All animal experiments were carried out in accordance with the National Institutes of Health guide for the care and use of laboratory animals and were approved by the Animal Experimentation Committee of Kansai Medical University.
Under anesthesia with pentobarbital (50 mg/kg), mouse spinal cords were quickly removed and homogenized twice for 30 s with a Polytron homogenizer containing 10 volumes of HEN buffer consisting of 250 mM HEPES (pH 7.7), 1 mM EDTA, and 0.1 mM neocuproine. The homogenate was centrifuged at 800 × g for 10 min, and the supernatant was recovered and then centrifuged at 10,000 × g for 20 min. After the resulting pellet had been dissolved in 10 volumes of HEN buffer, the resulting supernatant and this dissolved pellet were employed as S2 and P2 fractions, respectively.
S-Nitrosylation of proteins in vitro and biotin-switch method
S-Nitrosylated proteins were detected by the biotin-switch method as described by Jaffrey et al. . Briefly, S2 and P2 fractions of the spinal cord were incubated at room temperature without or with various concentrations of SNAP for 1 h in the dark. Then, SNAP was removed from the reaction mixture by cold acetone precipitation; and the pellets were subsequently dissolved in HENS buffer containing 25 mM HEPES, pH 7.7, 0.1 mM EDTA, 0.01 mM neocuproine, and 1% sodium dodecyl sulfate (SDS). The SNAP-treated fractions were blocked with fresh 4 mM MMTS for 20 min at 50°C. After 2 steps of acetone precipitation, the pellets were then resuspended in HENS buffer. For labeling, the samples were subjected to the biotin-switch assay, in which the sample was mixed with 1 mM ascorbic acid and 0.2-0.4 mM biotin-HPDP as final concentrations and kept for 1 h in the dark. Biotinylated proteins were resolved by non-reducing SDS-polyacrylamide gel electrophoresis (PAGE), and transferred to a polyvinylidene difluoride membrane, followed by immunoblotting with peroxidase-conjugated anti-biotin antibody (1:1000; Sigma-Aldrich). To confirm and quantify S-nitrosylated actin in the S2 fraction of the spinal cord or cell lysates, after the S-nitrosylated proteins had been immunoblotted with anti-biotin antibody, the same membrane was stripped to detect total actin with anti-actin antibody (1:5000; BD Bioscience, San Jose, CA, USA) by using Enhanced Chemiluminescence (GE Healthcare, Piscataway, NJ, USA). The intensity of S-nitrosylated actin was quantified by using ImageJ software and normalized by that of total actin.
Furthermore, to detect individual S-nitrosylated proteins including actin in S2 fractions of the spinal cord, we removed free biotin-HPDP by using an NAP-5 column (GE Healthcare) after the biotin-switch assay and incubated the eluate overnight at 4°C with 50 μl of a streptavidin-agarose slurry in 2 volumes of a neutralization buffer (20 mM HEPES, pH 7.5, 100 mM NaCl, 1 mM ETDA, and 0.5% Triton X-100). The beads were washed 4 times with 1 ml of the neutralization buffer supplemented with 500 mM NaCl. The adsorbed proteins were eluted with SDS-sample buffer at room temperature for 20 min. The eluted sample was then analyzed by SDS-PAGE, followed by immunoblotting with anti-actin antibody (1:5000).
Identification of S-nitrosylated proteins by mass spectrometry
Most of the sample eluted from streptavidin-agarose gels was applied to a 10% gel for SDS-PAGE and used for identification of S-nitrosylated proteins. After the gel had been stained by the Vorum silver staining protocol , proteins of interest were excised from the gel, destained, and in-gel digested as described by Katano et al. . The gel piece was dehydrated with acetonitrile and dried by using a Tomy CC-180 vacuum centrifuge concentrator (Tokyo, Japan). After reduction with 10 mM dithiothreitol and alkylation with 55 mM iodoacetamide, proteins in the gel were digested overnight at 37°C with 10 μg/ml trypsin (Promega, Madison, WI, USA) in 25 mM NH4HCO3. The digested peptides were extracted with 50% acetonitrile in 1% TFA. Peptides were desalted and concentrated by using a Ziptip μC18 (Millipore, Billerica, MA, USA) and eluted with the matrix solution (1 mg/ml α-CHCA in 70% acetonitrile and 1% TFA) onto a target plate. Matrix-assisted laser desorption/ionization reflection time-of-flight mass spectrometry (MALDI-TOF MS) was performed by using a Voyager DE-PRO (Applied Biosystems). All spectra were obtained in a positive reflector mode using an accelerating voltage of 20 kV. Database searches were carried out by using the MASCOT search program http://www.matrixscience.com and NCBI protein databases. The specified taxonomy was Mus musculus (house mouse), and the specified initial mass tolerance was 50 or 70 ppm.
Cell culture and S-nitrosylation in cells
Pheochromocytoma cell line PC12 cells were maintained in Dulbecco's modified Eagle medium supplemented with 5% fetal calf serum, 10% horse serum, and 50 U/ml penicillin and kept in a humidified environment of 95% air and 5% CO2 at 37°C. For the S-nitrosylation assay, PC 12 cells were cultured on 6-cm dishes for 2 d; and the medium was replaced with serum-free medium 12 h prior to experiments. After 5-min treatment of the cells with PACAP (10 nM) and/or SNAP (100, 300, 500 μM), the cells were disrupted in HEN buffer by sonication; and the lysate was then subjected to the biotin-switch assay.
Measurement of dopamine release from PC12 cells
PC12 cells were seeded on 24-well plates. After 2 days in culture, the cells were preincubated for 15 min in 190 μl of HEPES buffer (140 mM NaCl, 5 mM KCl, 2 mM CaCl2, 1.2 mM MgCl2, 10 mM glucose, and 10 mM HEPES, pH 7.4); and then the appropriate agents (10 μl) were added to the medium. Incubation was carried out 37°C for the desired times in the absence or presence of 10 μM imipramine, an inhibitor of dopamine reuptake. After incubation, the culture medium in each well was harvested; and perchloric acid in HEPES buffer was then added to each well for a final concentration of 3%. Culture media and cell lysates were adjusted to pH 4 by 1 M sodium acetate, and then the samples were centrifuged at 15,000 × g for 5 min. The supernatants of culture media and cell lysates were measured for dopamine released into the medium and cellular dopamine by using an HPLC column equipped with an Eicom electrochemical detector model 700 (Kyoto, Japan). HPLC was performed by using a reversed-phase C18 column (Eicom CA-50DS, 2.1 mm × 150 mm) with a phosphate-buffered mobile phase containing 20% methanol, 50 mg/L EDTA, and 0.5 mg/L sodium 1-octanesulfonate. Cellular dopamine content was around 7.2 ± 0.8 ng/well; and basal and PACAP-evoked release of dopamine into the culture media were 1.5-2 and 15-20% of cellular dopamine, respectively.
Cerebellum and dorsal root ganglia (DRG) of mice and PC12 cells were homogenized in 20 mM Tris-HCl (pH 7.4) containing 150 mM NaCl, 4 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 1 μg/ml pepstatin A, 2 μg/ml aprotinin, and 2 μg/ml leupeptin; and the supernatants (100 μg) obtained after centrifugation at 10,000 × g for 30 min were subjected to immunoblotting on a polyvinylidene difluoride membrane. After blocking with 5% (w/v) BSA in TBS-T buffer at room temperature for 1 h, the membrane was incubated overnight at 4°C with rabbit anti-cGKI (type I cGK) antibody (1:200; Santa Cruz Biotech., Santa Cruz, CA, USA) and then at room temperature for 1 h with horseradish peroxidase-conjugated anti-rabbit-IgG (1:20,000; GE Healthcare). The immunoreactivity was detected by using Enhanced Chemiluminescence.
Fluorescence images for actin in PC12 cells
PC12 cells (3 × 104 cells/well) were plated on poly-L-lysine-coated glass-bottomed dishes (35 mm) and caused to differentiate with NGF (50 ng/ml) in Dulbecco's modified Eagle medium supplemented with 1% fetal calf serum and 2% horse serum for 4 days. After the cells had been cultured overnight in serum-free medium, they were incubated without or with SNAP (10, 30, 100 μM) or cytochalasin B (10 μM) for 5 min. After fixation in 4% paraformaldehyde in 0.12 M sodium phosphate buffer, pH 7.4 for 10 min, incubation with 0.3% Triton X-100 in phosphate-buffered saline (PBS) for 15 min, 3 washings with PBS, and blocking with 2% normal goat serum and 1% BSA in PBS for 30 min, the PC12 cells were stained for actin. For this procedure, the cells were incubated for 2 h at room temperature with Alexafluora 488-phalloidin (1:500, Invitrogen, Eugeme, OR, USA) alone or with anti-actin monoclonal antibody (1:800), and then for 1 h with anti-mouse IgG-Alexafluora 546 antibody (1:500) in PBS. Digital images were captured by a Zeiss LSM510 laser-scanning confocal microscope (Oberkochen, Germany), and the fluorescence intensity was quantified by using ImageJ. More than 40 cells were quantified at each datum point, and 3 experiments were carried out for each analysis.
Data were presented as the mean ± SD or mean ± SEM. Data for dopamine release and S-nitrosylation of actin were analyzed by paired Student's t-test and Mann-Whitney U-test, respectively. Data for F-actin level were analyzed by one-way ANOVA and statistical significance was further examined by Dunnett's test using a Statview software program. P < 0.05 was considered significant.