Spermine-nitric oxide complex (spermine NONOate), 6-(2-Hydroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine (MAHMA NONOate), lactate dehydrogenase, pyruvate kinase, glyceraldehyde-3-phosphate, 2-phosphoglycerate, and ATP luciferin-luciferase assay kit, were purchased from Sigma-Aldrich (Saint Louis, MO). 3-Morpholinylsydnoneimine Cl (SIN-1) and DL-threo-β-benzyloxyaspartic acid (DL-TBOA) were from Tocris Bioscience (Ellisville, MO). Synthetic peroxynitrite (ONOO−) was purchased from EMD-Calbiochem (La Jolla, CA). Thiol-labeling reagent Nα-(3-maleimidylpropionyl)biocytin was from Invitrogen (Carlsbad, CA). L-[3H]glutamate (51 Ci/mmol), was obtained from Amersham-GE Healthcare (Piscataway, NJ). 45CaCl2 (0.91 Ci/mmol) was purchased from PerkinElmer (Boston, MA). And all other salts and reagents were purchased from Sigma-Aldrich, and were of highest purity available.
Preparation of rat forebrain synaptosomes
Presynaptic nerve endings (synaptosomes) were isolated from forebrains of male Sprague-Dawley rats (150–200 g), according to the method of Hajos [37
] with modifications as outlined below. All animal procedures were approved by the Institutional Animal Use and Care Committee. Animals were euthanized by rapid decapitation; whole brains were rapidly removed and transferred into ice-cold sucrose medium. Cerebellum and brain stem were dissected, and remaining tissue was homogenized in 0.32 M sucrose/5 mM HEPES (pH 7.4) using a Teflon-glass homogenizer. The homogenate was centrifugated for 10-min at 900g (2°C) to remove nuclei and cell debris. The first pellet was discarded and supernatant was further centrifugated for 20 min at 9,000 (2°C). The resulting pellet (P2) was resuspended in 0.32 M sucrose, layered over 0.8 M sucrose, and additionally centrifugated for 20 min at 9,000g (2°C). The myelin-containing layer at the 0.32–0.8 M sucrose interface was aspirated, 0.8 M-sucrose supernatant containing synaptosomes was removed, and the 0.8 M-sucrose pellet containing predominantly mitochondria was discarded. Synaptosomal suspension in 0.8 M sucrose was slowly diluted with an equal volume of the phosphate-buffered basal medium containing (in mM): 125 NaCl, 5 KCl, 1.2 MgSO4
, 1 CaCl2
, 2.5 NaH2
, 7.5 Na2
, and 10 D-glucose (pH 7.4). The resulting suspension was centrifugated for 20 min at 9,000g (2°C). The final synaptosomal pellet was resuspended in the same phosphate-buffered basal solution and used in the subsequent experiments. In each experiment synaptosomal suspension (~1.5–2 mg protein/ml) was initially preincubated for 1 h at 37°C with constant agitation to restore normal metabolic status and transmembrane ion gradients.
Treatment with exogenous RNS donors
Synaptosomal suspensions were allowed to recover metabolically as described above and then were divided into several aliquots and treated for 30 min at 37°C in the dark with the NO donors, spermine NONOate (half-life at 37°C ~39 min), MAHMA NONOate (half-life at 37°C ~1.5 min), the peroxynitrite donor SIN-1 (half life at 37°C ~90–230 min), or the nitrosothiol compound S
-nitroso-L-cysteine. All agents were added from freshly prepared stock solutions to the final concentration of 100 μM, except for SIN-1, which was used at both 100 and 500 μM. 100 mM spermine NONOate and 100 mM MAHMA NONOate were prepared in 10 mM NaOH. 500 mM SIN-1 stock solution was prepared immediately before experiment in H2
O and stored on ice. 200 mM S
-nitroso-L-cysteine was freshly synthesized for each experiment from L-cysteine and NaNO2
under acidic conditions as previously described [34
]. All stock solutions were diluted 1,000-fold and tested for potential changes in pH (none found). Vehicles were routinely added to the control samples. In our previous work [34
] we performed control experiments with light-decomposed S
-nitroso-L-cysteine and found no significant effects on vesicular neurotransmitter release up to the concentration of 1 mM.
After the initial 30-min treatment with NO donors, we used two different experimental designs for studying the functional impact of NO on synaptosomal functions. In the first type of experiments, after NO treatment, synaptosomes were pelleted by 2-min centrifugation at 10,000 g in an excess of basal phosphate medium, washed twice with 2 mL of HEPES-buffered Basal medium containing (in mM) 135 NaCl, 3.8 KCl, 1.2 MgSO4, 1.3 CaCl2, 1.2 KH2PO4, 10 D-glucose, and 10 HEPES (pH 7.4), and used for the subsequent assays (see below). These experiments were conducted to reveal the lasting functional impact of NO after its removal. In the second type of experiments, synaptosomes were washed of extracellular NO donors as described above, and then were allowed to recover for one additional hour in the basal HEPES-buffered medium. The recovered synaptosomes were washed three additional times and then used for functional assays. This second experimental design was employed to study the reversibility of NO effects upon metabolic recovery. To study if functional recovery after the NO treatment can be accelerated, in some experiments we supplemented the recovery media with thiol-reducing reagents or metabolic substrates as indicated in the text and figure legends. The concentration of synaptosomal protein during all treatments was maintained between 0.9 and 1.4 mg/mL.
L-[3H]Glutamate release assay
To measure vesicular and non-vesicular glutamate release we employed a radiotracer assay. Synaptosomes were loaded with L-[3
H]glutamate (10 μCi/ml) at 37°C for 30 min with constant agitation. The high specific activity of [3
H]-tracer was critical to achieve significant labeling of the vesicular L-glutamate pool. To terminate loading and remove the extracellular isotope, 10 volumes of ice-cold sucrose stop solution (medium S) was added to the suspension, and synaptosomes were centrifuged for 20 min at 9,000g (2°C). The medium S contained (mM): 243 sucrose (isoosmotic replacement for 135 mM NaCl); 3.8 KCl, 1.2 MgSO4
, 1.2 KH2
, 10 HEPES, 10 D-Glucose (pH 7.4). The resulting pellets were stored on ice and resuspended in 8 mL ice-cold medium S immediately before neurotransmitter release measurements. This procedure prevents spontaneous synaptosome depolarization and neurotransmitter release during storage on ice [34
To start efflux assay, 400-μL aliquots of L-[3H]glutamate–loaded synaptosomes were dispensed into tubes, containing 4.5 ml of warm basal medium, or high K+, or Ca2+-free high K+ medium. In high K+ medium [K+]o was elevated to 43 mM (final [K+]o=40 mM) by equimolar replacement of Na+, leaving all other components the same as in basal medium. The composition of Ca2+-free high K+ medium was similar, except 1 mM CaCl2 was replaced with 1 mM MgCl2 and 50 μM EGTA was added. After 5 min incubation at 37°C, the glutamate efflux was terminated by a rapid vacuum filtration through the GF/B filters (Whatman-GE Healthcare, Florham Park, NJ). The filters were placed overnight into scintillation vials with 4 ml Ecoscint A scintillation cocktail (National Diagnostics, Atlanta, GA) and counted for radioactivity that remained in synaptosomes. Fractional release of L-[3H]glutamate was calculated in relationship to total isotope content in synaptosomes at time 0. In control experiments we determined that at the beginning of the experiment >85% of L-[3H]glutamate was contained inside synaptosomes and that nonspecific binding of L-[3H]glutamate to the filters was negligible (<1–2% of the measured values).
Measurements of intrasynaptosomal ATP content
Intrasynaptosomal levels of ATP were measured using commercially available luciferin-luciferase ATP assay kit (Sigma). Synaptosomes were treated with NO donors, and subjected to similar wash procedures as in the neurotransmitter release experiments. 100 μL of solution containing 100 mM perchloric acid plus 50 mM EDTA was added to 1-mL synaptosomal aliquots, and they were immediately boiled for 30 sec, followed by sedimentation of the denatured proteins (10,000 g at room temperature, 22°C). Supernatants were neutralized by adding 25 μL aliquots of 3 M KOH plus 1 M Tris. 25 μL of lysate samples or the freshly prepared ATP standards were added to 2 mL of luciferin-luciferase mix diluted 1:200 with the ATP assay dilution buffer (Sigma). ATP levels were quantified as light production in a TriCarb TR1900 scintillation counter (PerkinElmer, Waltham, MA), and compared to standards with known ATP content. ATP levels were then normalized to protein content in each sample as determined using standard bicinchoninic acid (BCA) assay kit and bovine serum albumin as a standard (Pierce-Thermo Scientific, Rockford, IL).
Chemiluminescence assay of intrasynaptosomal S-nitrosothiols
Total amount of protein and lipid-bound intrasynaptosomal NO and intrasynaptosomal RSNO levels were measured using triiodide-dependent reductive release of bound NO followed by detection of ozone-based chemiluminescence as described elsewhere [25
]. Briefly, synaptosomal samples were washed with phosphate-buffered basal medium, sedimented at 10,000 g, and then lysed in 2 mL of 4 mM phosphate buffer (pH 7.5) containing 100 μM EDTA and 10 mM NEM. Samples were then pretreated in the dark with acidified sulfanilamide (final concentration 10 mM, 100 mM sulfanilamide stock prepared in 1 N HCl) for 15 min to remove nitrite with or without HgCl2
(final concentration 4.9 mM) to evaluate for the presence of RSNOs. Hg2+
selectively decomposes RSNOs [25
] such that RSNO concentrations may be determined from the difference in NO signal with or without pretreatment of paired samples with HgCl2
. 400 μL of each sample were injected in a purge vessel that contained 4.5 ml of glacial acetic acid and 500 μL of an aqueous mixture of 450 mM potassium iodide and 100 mM iodine. The vessel was kept at 70°C, and the solution was constantly purged with nitrogen and changed every four injections. The amounts of NO released from the purge vessel were quantified by gas phase chemiluminescence (NOA 280, Sievers Instruments, Boulder, CO). Peak integration was performed, and the results were converted to NO concentrations using authentic NO as a standard, and then normalized to the protein content in each sample.
Biotin-switch assay of intrasynaptosomal nitrosothiols
In order to visualize the extent of protein S
-nitrosylation we employed a biotin-switch technique developed by Jaffrey et al. [23
] with several modifications as described below. Two main changes were as following. (1) We used NEM (final concentration ~23 mM) instead of the originally proposed MMTS. This modification allowed for more consistent blocking at room temperature (22°C) and strongly reduced nonspecific background biotinylation of the masked free thiols. (2) We used a different biotinylating agent, Nα-(3-maleimidylpropionyl)biocytin, that in our hands worked more consistently and produced lower background signal than the biotin-HPDP proposed in the original method. Synaptosomes were washed from NO donors three times with HEPES-buffered basal medium, and then incubated for 1 hour at 25°C in the dark in 1 mL of blocking solution, containing 1 volume of 0.25 M NEM, 9 volumes of HEN buffer pH 7.7, and 1 volume of 25% SDS. HEN buffer was composed of (mM): 250 HEPES, 1 EDTA, 0.1 neocuproine. To remove NEM, the proteins were precipitated by adding 2 mL of pre-chilled acetone for 15 min at −20°C, followed by sedimentation. After removing the acetone, the RSNOs were labeled for 1 hour at room temperature (22°C) in the dark with 0.5 mL of reducing/labeling solution with final concentration of 3 mM ascorbate and 1 mM Nα-(3-maleimidylpropionyl)biocytin in HEN buffer, pH 7.0. The samples were desalted with acetone one more time, pelleted and resuspended in the Basal medium. Small aliquots were used for determination of protein concentration by a colorimetric BCA assay kit (Pierce-Thermo Scientific), the rest was diluted with 2x Laemmli reducing buffer (BioRad) and used for Western blot analysis. The proteins were resolved by SDS-polyacrylamide gel electrophoresis (10%) and transferred on to Immun-Blot PVDF membrane (BioRad). After blocking with 5% milk in phosphate-buffered saline buffer, containing 0.05% Tween 20 (PBST), the membrane was incubated with polyclonal anti-biotin antibody (1:50,000 dilution, Bethyl laboratories Inc, Montgomery, TX) for 1 hour at 25°C, followed by five washes for 5 min with 1% milk PBST. The membrane was incubated for 1 hr with secondary anti-rabbit horseradish peroxidase-conjugated antibodies (GE Healthcare/Amersham Biosciences, Piscataway, NJ; 1:20,000 dilution), followed by four PBST washes. Chemiluminescence was detected using ECLplus kit (GE Healthcare-Amersham Biosciences) and CL-Xposure film (Pierce).
GAPDH activity assay
The activity of GAPDH was measured by monitoring the enzymatic reduction of NAD+ to NADH in the presence of the GAPDH substrate, glyceraldehyde-3-phosphate (GA-3-P). Washed and pelleted synaptosomes were lysed in 0.5 mL of ice-cold lysis buffer (4 mM phosphate buffer, 500 μM EDTA, Roche protease inhibitor cocktail, pH 7.5) and homogenized using hypodermal syringe. The lysates were clarified by 10 min centrifugation at 10,000 g (2°C). 50 μL of clarified lysates were added to 950 μL of the GAPDH reaction mix containing (in mM): 100 glycine, 100 KH2PO4, 5 EDTA, 1 NAD+, and 1.5 GA-3-P (pH 8.9, adjusted with NaOH). NAD+ and GA-3-P were added to the mix immediately before the assay. The GAPDH reaction was initiated by addition of synaptosomal lysates and carried on for 5 min at 25°C. Enzymatic production of NADH was measured as increase in the optical density at 340 nm using a ELx800 plate reader (Bio-Tek Instruments, Winooski, VT) and calculated using the NADH molar extinction coefficient of 6,300 cm−1 M−1. Total protein concentration in the samples was determined using BCA assay. Results were expressed as nmol NADH produced/mg total protein per 5 min.
Enolase activity assay
The neuron specific enolase (NSE) activity was assayed in a coupled enzymatic assay by monitoring the conversion of NADH to NAD+ resulting in a decrease of absorbance at 340 nm. The lysates were prepared in the same manner as for GAPDH assay (see above). 50 μL of clarified lysates was added to 950 μL of the NSE reaction mix containing (in mM): 100 HEPES, 25 MgSO4, 100 KCl, 0.2 NADH, 1.3 ADP, 2 2-phosphoglycerate, 10 U lactate dehydrogenase, 7 U pyruvate kinase, (pH 7.4, adjusted with NaOH). Reaction was carried on for 5 min at 25°C. Enzymatic production of NAD+ was measured as a decrease in the optical density at 340 nm using a ELx800 plate reader and calculated using the NADH molar extinction coefficient of 6,300 cm−1 M−1. Total protein concentration in the samples was determined using a BCA assay. Results were expressed as μmol NADH consumed/mg total protein per 5 min.
Ca2+ uptake via the voltage-gated Ca2+ channels
uptake was measured using 45
as a radiotracer [39
]. Synaptosomes were incubated with NO donors and subjected to the same procedures as in all other assays, and then pelleted in the excess of ice-cold sucrose stop solution S (for composition see above) and transferred onto ice. Before measurements the pellets were resuspended in the same sucrose solution (protein concentration 3–4 mg/mL). 100-μL aliquots of synaptosomes were injected into 900 μL of warm basal HEPES-buffered medium or 80 [K+
media, additionally containing 0.5 μCi/mL of 45
. Basal medium consisted of (mM) 135 NaCl, 5 KCl, 1.2 MgSO4
, 0.1 CaCl2
, 10 HEPES, 10 D-Glucose (pH 7.4). In high K+
was elevated to 88.9 mM (final concentration 80 mM) by equimolar replacement of Na+
. The uptake reaction was carried for 2 min at 37°C, and was terminated by vacuum filtration through the GF/B paper (Whatman) followed by two washes with ice-cold washing buffer containing (in mM): 125 LiCl, 5 KCl, 10 MgSO4
, 10 Tris (pH7.4). The Ca2+
uptake values were corrected for the nonspecific adsorption on filters. The rates of 45
uptake were calculated as V
is radioactivity (cpm) of a sample containing c
mg of protein, a
is a specific radioactivity of 45
related to the total content of Ca2+
incubation medium (cpm/nmol), and t
is the time of incubation.
All data are presented as mean values ±S.E. Statistical difference between experimental groups was calculated using Student’s t-test or one-way analysis of variance with a priory Newman-Keuls test for multiple comparisons. Probability values of less than 0.05 were considered significantly different. Origin 6.0 (OriginLab, Northampton, MA) and GraphPad Prism 5.0 (GraphPad Software, San Diego, CA) were used to perform statistical analyses.