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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2014 Apr 11;171(9):2321–2334. doi: 10.1111/bph.12572

Reversal of SIN-1-induced eNOS dysfunction by the spin trap, DMPO, in bovine aortic endothelial cells via eNOS phosphorylation

Amlan Das 1,§, Bhavani Gopalakrishnan 1,§, Lawrence J Druhan 2, Tse-Yao Wang 1, Francesco De Pascali 1, Antal Rockenbauer 3, Ira Racoma 1, Saradhadevi Varadharaj 1, Jay L Zweier 1,4, Arturo J Cardounel 2, Frederick A Villamena 1,5
PMCID: PMC3997273  PMID: 24405159

Abstract

Background and Purpose

Nitric oxide (NO) derived from eNOS is mostly responsible for the maintenance of vascular homeostasis and its decreased bioavailability is characteristic of reactive oxygen species (ROS)-induced endothelial dysfunction (ED). Because 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), a commonly used spin trap, can control intracellular nitroso-redox balance by scavenging ROS and donating NO, it was employed as a cardioprotective agent against ED but the mechanism of its protection is still not clear. This study elucidated the mechanism of protection by DMPO against SIN-1-induced oxidative injury to bovine aortic endothelial cells (BAEC).

Experimental Approach

BAEC were treated with SIN-1, as a source of peroxynitrite anion (ONOO−), and then incubated with DMPO. Cytotoxicity following SIN-1 alone and cytoprotection by adding DMPO was assessed by MTT assay. Levels of ROS and NO generation from HEK293 cells transfected with wild-type and mutant eNOS cDNAs, tetrahydrobiopterin bioavailability, eNOS activity, eNOS and Akt kinase phosphorylation were measured.

Key Results

Post-treatment of cells with DMPO attenuated SIN-1-mediated cytotoxicity and ROS generation, restoration of NO levels via increased in eNOS activity and phospho-eNOS levels. Treatment with DMPO alone significantly increased NO levels and induced phosphorylation of eNOS Ser1179 via Akt kinase. Transfection studies with wild-type and mutant human eNOS confirmed the dual role of eNOS as a producer of superoxide anion (O2−) with SIN-1 treatment, and a producer of NO in the presence of DMPO.

Conclusion and Implications

Post-treatment with DMPO of oxidatively challenged cells reversed eNOS dysfunction and could have pharmacological implications in the treatment of cardiovascular diseases.

Keywords: eNOS, DMPO, spin trapping, endothelial dysfunction, peroxynitrite, oxidative stress, reactive oxygen species, nitric oxide, cardiovascular diseases, endothelial cells

Introduction

Oxidative stress resulting from the overproduction of reactive oxygen species (ROS), leads to endothelial dysfunction which is implicated in the pathophysiology of several cardiovascular disorders such as atherosclerosis, hypercholesterolaemia, hypertension, type 2 diabetes and heart failure (Cai and Harrison, 2000; Heitzer et al., 2001; Vita, 2011). Excessive ROS generation within the vasculature may lead to the oxidation of low-density lipoproteins, decreased bioavailability of endothelium-derived NO (O'Donnell and Freeman, 2001) and formation of peroxynitrite (ONOO−), a highly reactive anion (Beckman and Koppenol, 1996). All these events contribute to the impaired function of the vascular endothelium.

Endothelium-derived NO is an important signalling molecule which regulates vessel homeostasis by inducing vascular smooth muscle relaxation, and inhibiting vascular smooth muscle hypertrophy, regulating platelet aggregation and leukocyte adhesion (Gross and Wolin, 1995). In the endothelium, NO is produced by a family of enzymes termed NO synthases (NOS; nomenclature follows Alexander et al., 2013) that catalyze the conversion of L-arginine to L-citrulline and NO. The reactive oxygen and nitrogen species generated during oxidative stress are known to induce eNOS dysfunction that leads to the uncoupling of the enzyme and results in the production of NOS-derived O2•− instead of NO (Wei et al., 2003). The resulting imbalance between NO and O2•− can contribute to the onset of a variety of cardiovascular diseases.

NOS activity is regulated by a complex cascade of phosphorylation/dephosphorylation at particular regulatory residues in the enzyme. Phosphorylation of eNOS Ser635 and Ser1179 leads to the activation of eNOS function while phosphorylation at Thr497 down-regulates it (Harris et al., 2001; Michell et al., 2001). Activation by eNOS Ser1179 phosphorylation results from the enhanced electron flux through the reductase domain and inhibition of calmodulin-dissociation from the enzyme in a calcium-independent fashion (Fulton et al., 1999; McCabe et al., 2000) and is mediated by several upstream kinases, among which PI3/Akt has been reported to play a crucial role in the regulation of enzyme activity (Fulton et al., 1999).

Most of the NO donor drugs are limited only by their NO delivering property and usually exhibit uncontrolled delivery of NO because of their instability in blood plasma (Wang et al., 2005). Nitrones can both scavenge reactive species (Villamena and Zweier, 2004; Villamena et al., 2005) and also exhibit controlled release of NO as a consequence (Locigno et al., 2005; Nash et al., 2012), and are therefore, more desirable and different from current drugs. To date, there are two major types of nitrones: the cyclic nitrones which include 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), 5-carbamoyl-5-methyl-1-pyrroline-N-oxide, 5-ethoxycarbonyl-5-methyl-1-pyrroline-N-oxide (EMPO), 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide and the linear nitrones α-phenyl-N-tert-butylnitrone (PBN), (disodium-[(tert-butylimino) methyl] benzene-1,3-disulfonate N-oxide (NXY-059) which have been used to detect free radicals (Figure 1). Nitrones are structurally simple molecules, yet they possess rich chemistries and biological properties that make them important pharmacological agents (Zuo et al., 2009; Das et al., 2012; Traynham et al., 2012; Villamena et al., 2012; Zamora and Villamena, 2013). Nitrone spin traps such as DMPO, PBN and its derivative, NXY-059 have shown pharmacological activity against ischaemia and reperfusion (I/R) injury in the heart and brain (Floyd, 2009; Zuo et al., 2009), neurodegeneration (Floyd et al., 2013) and cancer (Floyd et al., 2013; Floyd, 2009). NXY-059 was the first neuroprotective agent that reached a clinical trial phase in the USA (Floyd et al., 2013). Application of DMPO as a cardioprotective agent was previously reported (Tosaki et al., 1990) but its modes of action were unclear. DMPO also conferred protection against cardiac I/R injury in Langendorff rat heart preparations by salvaging the mitochondrial electron transport chain (METC) complexes and decreased ROS production (Zuo et al., 2009). Other experimental evidence suggests involvement of other mechanisms for cardioprotection by DMPO other than its direct spin trapping properties, such as induction of phase II enzymes via nuclear translocation of the transcription factor NRF-2 and suppression of mitochondria-dependent proapoptotic signalling in endothelial cells (Das et al., 2012). Increase in myocyte contraction via regulation of nitroso-redox levels and increase in sarcoplasmic reticulum Ca2+ handling have also been proposed (Traynham et al., 2012). Although the reversal of eNOS uncoupling via supplementation with tetrahydrobiopterin (BH4) has already been described (Forstermann, 2006), in the present study, we explored the mechanism of reversal of eNOS dysfunction by post-treatment of endothelial cells with DMPO, after treating the cells with the peroxynitrite donor, 3-morpholinosydnonimine (SIN-1), offering another mechanistic insight into the cardioprotective property of nitrones.

Figure 1.

Figure 1

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Chemical structures of various cyclic (above) and linear (below) nitrones.

Methods

Cell culture and maintenance

Bovine aortic endothelial cells (BAEC; Cell Systems, Kirkland, WA) were cultured using a T75 cm2 flask in DMEM medium with 1 g L−1 D-glucose and 4 mM L-glutamine, and supplemented with 10% FBS, 50 μg mL−1 pencillin, 50 μg mL−1 streptomycin, 2.5 mg L−1 endothelial cell growth supplement and 1% MEM-non-essential amino acids at 37°C in a humidified atmosphere of 5% CO2 and 20% O2. HEK293 were cultured under same conditions in DMEM media without endothelial cell growth supplement and MEM-non-essential amino acids. The medium was changed every 2–3 days and cells were subcultured once they reached 90–95% confluence or 80% for HEK293 cells.

SIN-1 and DMPO treatment of cells

BAEC or HEK293 cells were seeded at a density of 104 cells mL−1 in each well using a 24-well plate and grown to 70% confluency. Cells were treated with 500 μM SIN-1 for 2 h, and then post-incubated in plain medium without SIN-1 for additional 24 h, in the absence or presence of DMPO. Cell viability was assessed by MTT assay following the protocol described previously (Das et al., 2010). Cell viability was calculated as the percentage of inhibition by the following formula (Equation (1)):

graphic file with name bph0171-2321-m1.jpg 1

where At and As are absorbance of the sample and solvent alone respectively. Results were presented as mean ± SEM, where n = 6.

ROS detection by confocal microscopy

Cells with density of 104 cells mL−1 were seeded on sterile glass cover slips 6-well plate. Treated cells were fixed with 10% paraformaldehyde at room temperature for 10 min, washed with PBS three times and subsequently incubated with 25 μM of the fluorogenic probe 2,7-dichlorodihydrofluorescein diacetate (DCFH-DA) or 10 μM dihydroethidium (DHE) for 30 min at 37°C, followed by nuclei staining with DAPI (1 μM) for 30 min. Images were then captured by Olympus FluoView-1000 confocal microscope (Olympus, Shinjuku, Tokyo, Japan) using an excitation/emission filter of 543/602 nm for DHE, 488/535 nm for DCFH-DA and 405/422 nm for DAPI. Fluorescence intensities of 100 cells from different fields were calculated and results were presented as mean ± SEM of three independent experiments (n = 3).

ROS and NO detection by EPR spin trapping

Cells (104 cells mL−1) were grown to 70% confluency in 6-well plates. After SIN-1 treatment, cells were incubated with 150 μL of EMPO (25 mM) and 150 μL of methyl-β-cyclodextrin (Me-β-CD; 50 mM) and 10 μL of 10 μM CaI for 15 min (Šnyrychová, 2010). The supernatant (300 μL) was transferred to a quartz flat cell and spin adduct formation was detected at room temperature using Bruker EMX X-Band EPR spectrometer (Bruker Corp., Billerica, MA, USA). Experiment was repeated in the presence or absence of DMPO post-incubation. Instrument parameters were as follows: microwave frequency, 9.8 GHz; centre field, 3485 G; modulation amplitude, 0.2–1.0 G; microwave power, 10 mW; conversion time: 41 ms; time constant: 82 ms, sweep time: 42 s; sweep width: 120 G; receiver gain, 1 × 105 and using incremental sweep. Spectra were simulated using an automatic fitting programme (Rockenbauer and Korecz, 1996) where the pertinent hyperfine splitting constants of HO2•, HO• and C-centred adducts and their concentrations from the total area of each spectrum were obtained. Adducts were independently prepared using hypoxanthine-xanthine oxidase, Fe2+-H2O2 and Fe2+-H2O2-ethanol for HO2•, HO• and C-centred adducts respectively (Supporting Information Table S3). Spectra were obtained from three independent experiments (n = 3). Using the same cell cultures in 6-well plates, generation of NO in cells were detected by EPR spin trapping using the ferrous N-methyl-D-glucamine dithiocarbamate complex (Fe(MGD)2) as the spin trap following the published protocol (Gopalakrishnan et al., 2012). Cells were washed with PBS after treatment, then incubated for 15 min with PBS with CaCl2 and MgCl2, calcium ionophore A21387 (10 μM), FeSO4 (3.5 mM) and NH4MGD (20.8 mM; Shinobu et al., 1984) at a final volume of 500 μL. The supernatant (300 μL) was transferred to a quartz flat cell and spin adduct formation was detected at room temperature with EPR instrument parameters: microwave frequency, 9.8 GHz; centre field, 3426.5 G; modulation amplitude, 6 G; microwave power, 12 mW; conversion time: 10 ms; time constant: 20 ms, sweep time: 10 s; sweep width: 100 G; receiver gain, 1 × 105 and using incremental sweep. The two-dimensional spectra were integrated and baseline corrected using Bruker WinEPR data processing software (Bruker Corp.). Results are as mean ± SEM of three independent experiments (n = 3).

BAEC in a T75 cm2 culture flask were serum-starved overnight then endogenous L-[14N]-arginine was exchanged for L-[15N]-arginine by incubating cells with Tyrode's solution supplemented with 84 mg L−1 L-[15N]-arginine (98 atom% [15N], Isotec Sigma-Aldrich, St. Louis, MO) for 30 min at 37°C. After incubation, cells were washed with PBS, treated with Fe(MGD)2 as above and EPR spectra were acquired (Gopalakrishnan et al., 2012).

Determination of eNOS activity

eNOS activity was determined using the conversion of arginine to citrulline assay with [14C]arginine (12.1 GBq/mmol; Moravek Biochemicals, Brea, CA) as a substrate using the previously published protocol with a slight modification (Giraldez and Zweier, 1998). Cultured BAEC (104 cells mL−1) were grown to 70% confluency in T75 cm2 culture flask and subjected to SIN-1 treatment and DMPO post-treatment. After treatment, eNOS activity was measured by L-[14C]arginine to L-[14C]citrulline conversion in a total volume of 50 μL buffer containing 50 mM Tris-HCl, pH 7.4, 1 μM L-[14C]arginine (605 Bq per incubation sample), 1 mM NADPH, 0.6 mM Ca2+, 2.5 μM calmodulin and 10 μM BH4. After 10 min incubation at 37°C, the mixture was terminated by adding 400 μL of stop buffer (20 mM HEPES, pH 5.5, 2 mM EDTA, 2 mM EGTA). L-[14C]citrulline was separated by mixing the reaction mixtures with 100 μL of pre-equilibrated Dowex AG 50W-X8 (Na+ form) cation exchange resin (Bio-Rad Laboratories, Hercules, CA, USA), then passed through spin columns and quantitated by liquid scintillation counter. Results were presented as mean ± SEM of three independent experiments (n = 3).

Measurement of BH4 by HPLC

The total BH4 content was measured using HPLC analysis as described elsewhere (Hyland, 1985; Dumitrescu et al., 2007). Cells were grown in T75 cm2 flask up to 70% confluency. SIN-1-treated cells were washed once with PBS and lysed with lysis buffer containing 1 mM ascorbate, 1 mM DTT and 100 μM diethylenetriamine pentaacetic acid (DTPA), followed by sonication. The mixture was then centrifuged at 12,000 × g for 1 min at 4°C. The supernatant containing the protein was removed by filtration through a Microcom YM-3 spin column (Millipore Corp. Billerica, MA, USA). The filtrates were injected into an ESA HPLC system with a C-18 column (T3 4.6 × 150 mm 5 micron) and HPLC separation was performed as described earlier (Hyland, 1985; Dumitrescu et al., 2007) using a mobile phase comprising 50 mM potassium phosphate, 10 mM phosphate monobase, 12% acetonitrile, 6 mM citric acid, 1 mM DTT, 5 mM octyl sulfate, pH 3 and isocratic elution at 1.2 mL min−1. Detection parameters were: 35 and 500mV for electrochemical cell; fluorescence: excitation at 348 nm and emission at 444 nm; UV at 254 nm. Results were expressed as means ± SEM of three independent experiments (n = 3).

Western blot analysis

Western blot analysis was performed to determine the expression levels of p-eNOS, p-Akt, total (t)-Akt and t-eNOS proteins in untreated, SIN-1-treated and nitrone-post-incubated SIN-1-treated BAEC. Prior to treatment, cultured BAEC (104 cells mL−1) were grown to 70% confluency on 60 mm Petri-dishes, and 50 μg of protein from each set was used for Western blotting. In a separate experimental set, the membrane was incubated with rabbit polyclonal anti-p-eNOS (Ser1179) Ab (1:1000 dilution, Cell Signaling Technology, Danvers, MA, USA), mouse monoclonal anti-p-Akt (Ser473) Ab (1:500 dilution, Cell Signaling Technology), mouse monoclonal anti-t-eNOS Ab (1:1000 dilution, Santa Cruz Biotechnology, Paso Robles, CA, USA), rabbit polyclonal anti-t-Akt Ab (1:500 dilution, Cell Signaling Technology) and mouse monoclonal anti-β-actin Ab (1:1000 dilution, Santa Cruz Biotechnology) overnight at 4°C. The protein bands were visualized using a chemiluminiscence kit (Thermo Scientific, Waltham, MA, USA), and the bands were quantified densitometrically by Bio-Rad Chemi Doc (1708070) system using the software Quantity One (Bio-Rad).

Transient transfection of HEK293 cells with wild-type and mutant eNOS cDNAs

HEK cells were transfected with 5 μg μL of eNOS cDNA (wild-type and mutant where Ser1179 was changed to alanine) (Lin et al., 2003) using Lipofectamine™ 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer's instructions. The transfection efficiency was analysed using Western blot analysis of eNOS proteins and by measuring NO levels.

Data analysis

Data are presented as the mean (±SEM) of at least three independent experiments. Data were analysed by one-way anova, with the Student-Newman-Keul post hoc test, using Sigma plot 12.0 (Systat Software, Inc, San Jose, CA, USA). P values <0.05 were considered to be statistically significant.

Materials

The reagents used were supplied as follows: 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) by Invitrogen (Eugene, OR, USA); DAPI, DCFH-DA, DTPA, Me-β-CD by Sigma; DHE by Molecular Probes (Eugene, OR, USA); DMPO by Dojindo (Rockville, MD, USA); SIN-1 by Enzo Life Science (Farmingdale, NY, USA).

Results

Dose-and time-dependent cytoprotective effects from post-treatment by DMPO of SIN-1-challenged BAEC

Treatment of cells with varying concentrations of DMPO alone (25–500 μM) for 24 h did not result in loss of cell viability (Supporting Information Figure S1). However, cells challenged with 500 μM SIN-1, over a range of times, resulted in significant cytotoxicity (Supporting Information Figure S2). For instance, a 50% decrease in cell viability was observed after 2 h of SIN-1 treatment followed by 24 h of post-incubation in plain media (without SIN-1) (Supporting Information Figure S2). SIN-1 has been suggested to induce cell death via two mechanisms, production of ONOO− through reaction between NO and O2•− to finally form HO• (Ishii et al., 1999), and generation of H2O2 (Lomonosova et al., 1998). When SIN-1-challenged cells were post-incubated with DMPO, cell viability was increased, in both dose-and time-dependent fashion (Figures 2A and B). The greatest cytoprotection was obtained when SIN-1-treated cells were post-incubated with 100 μM of DMPO for 24 h, compared with pretreatment of BAEC with DMPO for 24 h before 2 h SIN-1 treatment, a procedure that did not significantly change cell viability (data not shown). We therefore used post-treatment with 100 μM of DMPO after SIN-1 challenge, in all subsequent experiments.

Figure 2.

Figure 2

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Effect of DMPO on SIN-1-induced cytotoxicity. (A) Dose-dependence, (B) Time-dependence. Cell viability was measured using the MTT assay. All data are means ± SEM, *<0.05 versus control (untreated cells), **<0.05 versus SIN-1-treated cells; one-way ANOVA, Tukey's test, n = 6.

Attenuation of ROS generation in SIN-1-treated BAEC by DMPO

To test if the cells generated O2− after treatment with SIN-1, EPR spin trapping was performed using EMPO/Me-β–CD as spin trap on SIN-1-treated BAEC at various times (0–24 h) of incubation. EPR results showed increased ROS intensities with increasing time of post-incubation along with the formation of a C-centred adduct which could be formed as a secondary product (Supporting Information Figure S3A and Table S1 for the simulation). The low-field peak intensity of the EPR spectra increased significantly in SIN-1-treated cells by up to 2.9-fold after 24 h of post-incubation (Supporting Information Figure S3B). EPR results were confirmed by confocal microscopy using DHE staining of SIN-1-treated cells at different time points. Treatment of cells with SIN-1 for 2 h (corresponding to 0 h of post-incubation) resulted in a significant increase of DHE-fluorescence specific for O2− and the signal persisted for up to 6 h of post-incubation in plain media, with fluorescence decreases upon further incubation for 12 and 24 h (Supporting Information Figure S4).

As H2O2 could be also formed from SIN-1 treatment via O2− dismutation (Lomonosova et al., 1998), we performed a time-dependent confocal microscopy study on SIN-1-treated BAEC by staining the cells with the ROS-specific fluorescent probe, CM-H2DCFDA (Figure S5). SIN-1-untreated BAEC showed no detectable DCF-fluorescence signal indicating that the cells did not produce O2•−-derived ROS. However, when BAEC were treated with SIN-1 for 2 h and incubated without SIN-1 for an additional 24 h, a time-dependent accumulation of DCF-fluorescence signal was detected (Supporting Information Figure S5).

When SIN-1-challenged cells were post-treated with 100 μM DMPO, DCF-fluorescence intensity was significantly decreased (Figures 3A and B) compared to a threefold increase in DCF-fluorescence in SIN-1-treated cells without DMPO post-treatment. Simulation of the EPR spectra also revealed that the concentration of O-centred adduct was significantly decreased with a concomitant decrease in the concentration of C-centred adducts when SIN-1-treated BAEC were post-incubated with DMPO for 24 h (Supporting Information Table S2 and Figures 3C and D).

Figure 3.

Figure 3

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Effect of DMPO on SIN-1-induced ROS generation in BAEC. (A) Confocal micrographs of ROS generation in BAEC treated with SIN-1 (500 μM) alone and those post-incubated with DMPO (100 μM). (B) Plot of average DCF-fluorescence obtained from 100 cells. All data are means ± SEM, *P < 0.05 versus control (untreated cells), **P < 0.05 versus SIN-1-treated cells; one-way ANOVA, Tukey's test, n = 3. (C) Representative X-band EPR spectra and simulated (dotted lines) of radical adducts formed in SIN-1-treated BAEC, with and without DMPO (100 μM) incubation using EMPO/Me-β–CD as spin trap (see Supporting Information Table S2 for complete simulated EPR parameters). (D) Plot of the concentrations of the HO2•, HO• and C-centred adducts from Figures 2C.

Restoration of NO bioavailability in SIN-1-treated BAEC by DMPO

As oxidative stress leads to decreased bioavailability of NO (Cai and Harrison, 2000), the effect of SIN-1 on NO production in BAEC was investigated. Using EPR and Fe(MGD)2 as spin trap to detect NO generation (Figures 4A and B), a significant decrease in NO production of almost 50% was observed when cells were treated with SIN-1 for 2 h and post-incubated in plain media for 24 h. The same observation was previously reported for BAEC upon treatment with SIN-1 or ONOO− (Kuzkaya et al., 2003). However, post-treatment with DMPO for 24 h resulted in the restoration of the NO levels by about 35%. Treatment of cells with the NOS inhibitor L-NAME, completely abolished the NO signal (Figure 4B), indicating that the NO was derived from enzymic activity of NOS.

Figure 4.

Figure 4

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Restoration of NO production and eNOS activity in SIN-1-treated BAEC by DMPO. (A) X-band EPR spectra of NO using Fe(MGD)2 spin trap, generated from SIN-1-treated BAEC and with or without post-incubation with DMPO (100 μM). (B) Plot of the relative low-field EPR peak intensity of Fe(MGD)2-NO. (C) eNOS activity as measured by conversion of arginine to citrulline from BAEC treated with SIN-1 with and without DMPO incubation. All data are means ± SEM, *P < 0.05 versus control (untreated cells), **P < 0.05 versus SIN-1-treated cells; one-way ANOVA, Tukey's test, n = 3.

Restoration of eNOS activity in SIN-1-treated BAEC by DMPO

eNOS activity was assessed using the L-[14C] arginine to L-[14C] citrulline conversion assay. BAEC were treated with SIN-1 for 2 h and post-incubated for another 24 h that resulted in significant inhibition of eNOS activity by ∼30%. However, post-treatment with DMPO for 24 h led to the complete restoration of eNOS activity (Figure 4C). This provides definitive evidence that the effects of DMPO on NO production are mediated at least in part through increased eNOS-derived NO production and not merely a consequence of scavenging SIN-1-derived superoxide.

Increased bioavailability of NO in BAEC treated with DMPO alone

As post-treatment with DMPO resulted in a significant increase in NO production and NOS activity in SIN-challenged cells, we investigated if the nitrone alone could increase NO generation in BAEC, without oxidatively challenging the cells. EPR studies revealed that treatment of cells with DMPO alone resulted in a time-dependent increase in cellular NO and a 40% increase in NO production was observed after 24 h of treatment (Figure 5).

Figure 5.

Figure 5

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Time-dependent increase in NO production in DMPO-treated BAEC. (A) X-band EPR spectra of NO using Fe(MGD)2 spin trap generated in BAEC during 0 to 24 h of incubation with DMPO (100 μM) and NOS inhibitor, L-NAME. (B) Plot of the relative low-field EPR peak intensity of Fe(MGD)2-NO. Results are means ± SEM, *P < 0.05 versus control (untreated cells); one-way ANOVA, Tukey's test, n = 3.

Determination of NO source: nitrone versus L-arginine

To determine if the source of the generated NO was from chemical decomposition of the nitrone (Locigno et al., 2005) or from eNOS-catalyzed conversion of L-arginine to L-citrulline, an isotope-labelling experiment using L-[15N]-arginine was carried out. Because of the difference in the nuclear spins of 14N (I = ½) and 15N (I = 1), it is possible to determine the source of NO formed (Xia and Zweier, 1997). A time-dependent formation of a doublet signal, characteristic of 15NO–Fe–MGD spectrum was observed (Figure 6A) after incubation of L-[14N]-arginine-depleted BAEC with L-[15N]-arginine for 24 h. Incubation of cells in the presence of DMPO and L-[15N]-arginine for 24 h gave a similar doublet spectrum demonstrating that most of the detected NO were generated from the 15N-labelled arginine (Figure 6B). This result suggests that the NO generated in BAEC upon DMPO treatment did not originate from the nitrone itself, but rather from the metabolism of L-arginine to L-citrulline, and thus, implied that NOS was the source of the NO.

Figure 6.

Figure 6

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Determination of the source of NO. (A) X-band EPR spectra of 15NO–Fe–MGD showing the characteristic doublet peak produced from BAEC supplemented with L-[15N] arginine at various incubation times. (B) EPR spectra of NO adducts formed from BAEC incubated alone in medium containing L-[14N] arginine; L-[15N] arginine containing medium; and L-[15N] arginine containing medium supplemented with DMPO. All experiments were performed at least three times.

Induction of eNOS-and Akt phosphorylation in BAEC by DMPO in the absence of SIN-1 treatment

Western blots were carried out to detect p-eNOS Ser1179 in BAEC treated with DMPO for 12 and 24 h. Levels of p-eNOS increased time-dependently after DMPO treatment, with a significant 2.2-fold increase after 12 h of incubation. The p-eNOS level was only slightly increased after 24 h of treatment (Figures 7A and B). As PKB or Akt is known to be an upstream regulator of eNOS and results in the phosphorylation of the enzyme (Fulton et al., 1999), p-Akt Ser473 levels were also measured in the presence of DMPO. DMPO treatment of cells resulted in a similar time-dependent increase in p-Akt levels. Incubation of cells with DMPO for 12 and 24 h resulted in 2.7-and 3.5-fold increases in p-Akt levels respectively (Figures 7A and C). Therefore, these observations showed that DMPO could increase NO bioavailability in un-challenged BAEC via phosphorylation of Akt and eNOS.

Figure 7.

Figure 7

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Effect of DMPO on the phosphorylation status of eNOS and AKT proteins in BAEC. (A) Western blot expression levels of p-eNOS, total eNOS, p-Akt and t-Akt in BAEC treated with 100 μM DMPO at 12 and 24 h incubation. Densitometric analysis of the expression levels of (B) p-eNOS (B) and (C) p-Akt. All data are means ± SEM, *P < 0.05 versus control (untreated cells), one-way ANOVA, Tukey's test, n = 3.

Inhibition of SIN-1 mediated down-regulation of p-eNOS and p-Akt in BAEC by DMPO

The phosphorylation levels of eNOS increased by 22% after 2 h of SIN-1 treatment (corresponding to 0 h of post-incubation with DMPO) but there was a time-dependent decrease in p-eNOS levels during further incubation of SIN-1-treated cells in plain media. After 24 h of post-incubation, the p-eNOS band intensity decreased by 55% compared with control cells (Figures 8A and B). However, when SIN-1-challenged cells were post-treated with DMPO for 24 h, p-eNOS levels were significantly restored (Figures 8C and D). In addition, we also observed significant restoration of p-Akt levels in SIN-1-treated BAEC upon post-treatment with DMPO (Figures 8C and E). These observations show that DMPO can increase NO bioavailability in SIN-1-challenged BAEC, via phosphorylation of Akt and eNOS.

Figure 8.

Figure 8

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Modulation of eNOS and Akt phosphorylation by DMPO in SIN-1-treated cells. (A) Western blot analysis; and (B) densitometric analysis of p-eNOS Ser1179 and t-eNOS expression levels in BAEC treated with SIN-1 (500 μM, 2 h) and post-incubated in plain media (without SIN-1) at various incubation times. Results are represented as mean ± SEM,*P < 0.05 versus control (untreated cells). (C) Western blot analysis of p-eNOS Ser1179, t-eNOS, p-Akt Ser473 and t-Akt expression in BAEC treated with SIN-1 (500 μM) alone and post-incubated with DMPO (100 μM). Densitometric analysis of the expression levels of (D) p-eNOS Ser1179 and (E) p-Akt from the above mentioned conditions. Results are represented as mean ± SEM, *P < 0.05 versus control (untreated cells), **P < 0.05 versus SIN-1-treated cells; one-way ANOVA, Tukey's test, n = 3.

SIN-1-mediated eNOS dysfunction is independent of BH4 depletion

Treatment of BAEC with SIN-1 for 2 h in the absence of DMPO resulted in the depletion of BH4 levels but upon incubation in plain media for additional 24 h, BH4 levels were restored in SIN-1-challenged cells (Supporting Information Figure S6). This indicated that BH4 was metabolically replenished after 24 h of incubation and that this occurred in the absence of DMPO. We also investigated the effect of DMPO on BH4 levels in cells, within an hour of exposure to SIN-1 and found no change in the BH4 levels, whiohc were similar to those in the absence of DMPO.

Dual roles of eNOS as target for both SIN-1 and DMPO

HEK293 cells were transfected with the cDNAs encoding both wild-type and mutant (S1179A) bovine eNOS. This mutant eNOS is resistant to phosphorylation and also does not exhibit any Akt-dependent increase in NO generation (Fulton et al., 1999). Our Western blot results show equal transfection efficiency for both wild-type and mutant eNOS (Supporting Information Figure S7B) but no NO generation from mutant eNOS and with NO generation in wild-type eNOS-transfected cells were observed (Supporting Information Figure S7A). When HEK293 transfected cells were were treated with SIN-1 for 2 h, formation of O2− was observed, using DHE staining, in the cells transfected with wild-type eNOS-plasmid, whereas in mutant eNOS-transfected cells, O2•− generation was much lower (Figure 9A–C). EPR spin trapping to detect ROS generation in mutant eNOS-transfected cells gave low levels of ROS even after treatment with SIN-1 or DMPO (Figure 9D), confirming the important role of eNOS Ser1179 in generation of these free radicals. Phosphorylation of eNOS Ser1179 at low Ca2+ concentrations is known to be critical for O2− generation (Chen et al., 2008).

Figure 9.

Figure 9

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Detection of ROS generation in wild-type and mutant eNOS cDNA transfected HEK293 cells in the presence of SIN-1. Confocal DHE and DAPI images of untreated and SIN-1-treated HEK293 cells transfected with cDNAs encoding (A) wild-type eNOS Ser1179 (B) mutant eNOS cDNA S1179A. (C) Plot of relative average DHE-fluorescence intensities (n = 100 cells). Data are represented as mean ± SEM, *P < 0.05 versus control (non-transfected cells) and **P < 0.05 versus SIN-1-treated wild-type eNOS Ser1179 transfected cells; one-way ANOVA, Tukey's test, n=3. (D) X-band EPR detection of radical generation using EMPO/Me-β–CD from mutant eNOS cDNA transfected HEK293 cells that are untreated (top) and treated with SIN-1 (middle), and post-incubated with DMPO (bottom). All experiments were performed in triplicate.

To investigate whether DMPO could attenuate SIN-1-mediated ROS generation in HEK293 cells transfected with wild-type or mutant eNOS-plasmid, confocal microscopy with DCF-DA staining was used to detect ROS generation. We found that treatment with SIN-1 gave a ∼4.4-fold increase in DCF-fluorescence intensity and was significantly reduced on post-treatment with DMPO (Supporting Information Figure S8A and B).

Discussion

Oxidative insult to eNOS can reverse its function from being an NO generator to a pro-oxidative enzyme, thereby exacerbating the degenerative processes that have been associated with endothelial dysfunction (Cai and Harrison, 2000). In these experiments, we have demonstrated how DMPO could reverse eNOS function from being pro-oxidative to that of its normal NO synthase function, by exploiting the ability of nitrones to regulate the nitroso-redox balance in the cell by scavenging ROS, increasing NO bioavailability and ultimately eNOS phosphorylation.

Post-treatment by DMPO of SIN-1-challenged BAEC conferred cytoprotection and restored NO production, while post-incubation of BAEC with SIN-1 for an additional 24 h in plain medium (in the absence of DMPO) gradually increased the oxidative burden on the cells. Production of ONOO− via formation of ROS and NO had been implicated in SIN-1 cytotoxicity to endothelial cells (Ishii et al., 1999). Although nitrones are known to react with ONOO−, where ONOO−/ONOOH mediates the formation of the spin adducts using DMPO as spin trap (Gatti et al., 1998; Nash et al., 2012), the possibility that DMPO may be converting SIN-1 into a NO donor, rather than a ONOO− source, via O2•− scavenging is unlikely as 2 h of SIN-1 incubation with cells would not allow ONOO− to persist because of its short half-life (1.9 s at pH 7.4) (Beckman et al., 1990) especially in a biological milieu. Furthermore, fluorescence studies showed that the half-time for the steady state ONOO− production from SIN-1 ranged from 14 to 26 min (Martin-Romero et al., 2004). One could envision that after 2 h incubation of cells with SIN-1, ONOO− is released into the cell and could have had reacted, already causing oxidative damage, perhaps to eNOS. Therefore, the formation of ROS adducts we observed during spin trapping was not a direct reaction of ONOO− with DMPO. These results clearly suggested that post-treatment of cells with DMPO attenuated ROS production via eNOS dysfunction, in SIN-1-treated endothelial cells.

The ROS-derived ONOO− limits bioavailability of the eNOS cofactor, BH4, resulting from either BH4 depletion or oxidation and thus results in eNOS dysfunction (Kuzkaya et al., 2003). Also, in BAEC, co-incubation of BH4 or ascorbic acid with SIN-1 can reverse the effect of SIN-1 on NO production, perhaps via replenishment of oxidized BH4 or reversal in the oxidation state of the redox-modified BH4 (Xia et al., 1998; Vasquez-Vivar et al., 2002; Kuzkaya et al., 2003). However, unlike ascorbic acid (Kuzkaya et al., 2003), DMPO did not reverse eNOS function through restoration of BH4 levels during initial and 24 h of SIN-1 treatments. So, the decrease in NO and increase in ROS we observed could have been the result of mechanisms, other than uncoupling of eNOS because of BH4 depletion.

Therefore, the role of eNOS activation via phosphorylation was investigated to explain the observed increase in NO production in DMPO-treated cells. Phosphorylation of eNOS Ser1179 is known to activate eNOS by stimulating the flux of electrons within the reductase domain (McCabe et al., 2000; Harris et al., 2001; Michell et al., 2001). Under oxidative stress conditions, eNOS generates O2•− and phosphorylation of eNOS Ser1179 is inhibited leading to decrease in NO production. How phosphorylation of eNOS Ser1179 is regulated to maintain the O2•−-NO balance under oxidative stress conditions is not completely understood (Xia et al., 1996) but it has been proposed that eNOS phosphorylation is redox-sensitive and is activated by the PI3K/Akt pathway (Dimmeler et al., 1999; Fulton et al., 1999) and by other kinases (Alhosin et al., 2013). Regulation of ATP levels and activation of other kinases such as p38 MAPK and JNK by DMPO, therefore, warrants further investigation. This study, nevertheless, demonstrates that DMPO could reverse eNOS dysfunction through decreased ROS production, increased NO bioavailability and increased phosphorylation of eNOS Ser1179 by activation of Akt.

Traditional antioxidants such as ascorbic acid and N-acetyl cysteine have been explored for their ability to prevent or reverse eNOS uncoupling in BAEC (Kuzkaya et al., 2003) and in diabetic rat hearts (Okazaki et al., 2011), through an increase in the BH4/BH2 ratio. Recently, lipophilic NO donor and lipophilic antioxidants, when introduced as a hybrid molecule and not as a mixture, exhibited protection from I/R injury in hearts, through significant reduction of infarct size with improved recovery of cardiac function (Rastaldo et al., 2012). However, the mechanism of this protection was not clear. Moreover, magnesium lithospermate B protected endothelium from hyperglycaemia-induced dysfunction via eNOS phosphorylation, and induction of phase II enzymes by nuclear translocation of NRF-2, while α-lipoic acid did not cause these effects (Kim et al., 2010).

Cardiometabolic disease is a growing health problem that derives from a combination of various risk factors such as hypertension, hyperglycaemia and elevated plasma triglyceride levels, leading to the development of type 2 diabetes and cardiovascular diseases. While the antioxidants mentioned above could be promising, their therapeutic application could be limited in the treatment of cardiometabolic diseases as unlike nitrones, their modes of action may only be limited to a few mechanisms (Zamora and Villamena, 2013). Findings presented in this present work and in our past studies (Durand et al., 2009; Zuo et al., 2009; Das et al., 2012; Traynham et al., 2012) showed that nitrones offered opportunities for their application in the treatment of cardiometabolic disorders such as hypertension and hyperglycaemia which are associated with endothelial dysfunction and depletion of cellular antioxidant pool. This is because as the nitrones not only scavenge ROS (Durand et al., 2008; 2009) but also act through NO donation (Locigno et al., 2005), by salvaging METC activity (Zuo et al., 2009), by increasing METC biogenesis (Zuo et al., 2009) by inhibiting mitochondrial polarization leading to down-regulation of proapoptotic signalling pathways, by inducing phase II enzyme activities via NRF-2 nuclear translocation (Das et al., 2012), increasing myocyte contraction via increased sarcoplasmic reticulum Ca2+ handling (Traynham et al., 2012) and, in this study, reversal of eNOS dysfunction.

Future work needs to investigate how DMPO activates Akt by immuno-spin trapping of the relevant proteins and role of nitrones as potential phosphatase inhibitors, as protein phosphatase 2A was also reported to decrease eNOS Ser1179 phosphorylation. Therefore, therapeutic agents that can target the cytosolic matrix and affect kinase-mediated phosphorylation processes in the cell may be able to up-regulate eNOS. Attempts have been made to conjugate cyclic nitrones to several target-specific groups in order to facilitate their delivery to specific subcellular compartments (Durand et al., 2003; 2009; 2010), as the poor target specificity of some of the natural antioxidants such as vitamin E, vitamin C, or lipoic acid limit their applications to the treatment of ROS-induced cardiovascular disorders (Willcox et al., 2008; Shay et al., 2009; Kizhakekuttu and Widlansky, 2010). Considering that eNOS is mostly localized near the cell membrane and that mitochondria and sarcoplasmic reticulum are mostly cytosolic, the use of nitrones that can specifically target these compartments would provide a way of identifying which targeted cellular event/s contributed the most to I/R injury and which target-specific nitrone will exhibit the most robust protection. Such studies would give valuable insights into the mechanisms of protection by nitrones. To date, targeting oxidative stress at the site/s of their origination still remains an attractive strategy for prevention and therapy of cardiovascular dysfunction (Munzel et al., 2010). Therefore, a better understanding of the sources of free radicals and the mechanisms of oxidative stress, along with development of more target-specific antioxidants with multifunctional action, such as those exhibited by nitrones, are needed.

Acknowledgments

This work was funded by the NIH National Heart, Lung, and Blood Institute grant RO1 HL81248.

Glossary

ACN

acetonitrile

BAEC

bovine aortic endothelial cells

BH4

tetrahydrobiopterin

CM-H2DCFDA

5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate, acetyl ester

DCFH-DA

2,7-dichlorodihydrofluorescein diacetate

DHE

dihydroethidium

DMPO

5,5-dimethyl-1-pyrroline-N-oxide

DTPA

diethylenetriamine pentaacetic acid

EMPO

5-ethoxycarbonyl-5-methyl-1-pyrroline-N-oxide

hfsc

hyperfine splitting constant

Me-β-CD

methyl-β-cyclodextrin

METC

mitochondrial electron transport chain

MGD

N-methyl-D-glucamine dithiocarbamate

NXY-059

disodium-[(tert-butylimino) methyl] benzene-1,3-disulfonate N-oxide

PBN

α-phenyl-N-tert-butylnitrone

SIN-1

3-morpholinosydnonimine

Conflict of interest

None.

Supporting information

Additional Supporting Information may be found in the online version of this article at the publisher*s web-site: http://dx.doi.org/10.1111/bph.12572

Figure S1 Cytotoxicity of DMPO alone on BAEC. BAEC viability after incubation with DMPO (0–500 μM) as determined by MTT assay. Data are represented as the mean ± SEM, n = 6.

Figure S2 Time-dependent cytotoxicity of SIN-1 on BAEC. Time-dependent cytotoxic effects of SIN-1 (500 μM) on BAEC as determined by MTT assay at various time points (A) 1–6 h. BAEC were treated with SIN-1 for 2 h and post-incubated with plain media (without SIN-1) at various time points (B) 0–24 h. Cell viability was assessed by MTT assay. Data are represented as the mean ± SEM, * P < 0.05 versus control (untreated cells), n = 6.

Figure S3 Detection of O2 − generation in SIN-1-treated BAEC by EPR. (A) BAEC were treated with SIN-1 for 2 h and postincubated in plain media (without SIN-1) at various time points (0–24 h), and ROS generation was monitored by X-band EPR spectrometer using EMPO and Me-β–CD as the spin traps. (B) A plot of relative ROS intensities for the untreated and SIN-1-treated BAEC, post-incubated in plain media at various time points (0–24 h). Data are represented as mean ± SEM, * P < 0.05 versus control (untreated cells), n = 3.

Figure S4 Detection of O2 •− generation in SIN-1-treated BAEC by fluorescence. (A) O2 •− generation in untreated BAEC, and cells treated with SIN-1 (500 μM) and post-incubated with plain media at various points were monitored by confocal microscopy using DHE. The figure represents the merged image of DHE-fluorescence (red) and DAPIfluorescence (blue), bar represents 10 microns. (B) A plot of relative DHE-fluorescence obtained from the average of 100 cells. Data are represented as mean ± SEM, * P < 0.05 versus control (untreated cells).

Figure S5 Detection of ROS generation in SIN-1-treated BAEC by fluorescence. (A) Generation of ROS in untreated BAEC, and cells treated with SIN-1 (500 μM) and postincubated with plain media at various time points were monitored by confocal microscopy using ROS-specific fluorescence dye CM-H2DCFDA. The figure represents the merged image of DCF-fluorescence (green) and DAPI-fluorescence (blue), bar represents 10 microns. (B) A plot of relative DCFfluorescence obtained from the average of 100 cells. Data are represented as mean ± SEM, * P < 0.05 (vs.) control (untreated cells).

Figure S6 Determination of BH4 depletion by SIN-1-mediated eNOS dysfunction. The levels of BH4 in SIN-1-treated BAEC alone and post-incubated with DMPO was measured to determine the extent of eNOS uncoupling. (A) The BH4 chromatograms obtained at retention time of ∼18 min. BAEC treated with SIN-1-post-incubated for 0 h and 24 h. (B) Respective BH4 chromatograms for BAEC alone, treated with SIN-1, and post-incubated with DMPO for 2 h. All experiments were performed at least three times.

Figure S7 Detection of NO in non-transfected and transfected HEK293 cells. (A) NO generation in non-transfected and wild-type/mutant eNOS cDNA transfected HEK293 cells was monitored by X-band EPR spectrometer using Fe(MGD)2 as a spin trap upon stimulation with CaI. From top to bottom: Spectrum 1 represents NO generated from non-transfected HEK293 cells as control; Spectrum 2 represents NO generated from HEK293 cells transfected with wild-type eNOS cDNA (Ser-1779); Spectrum 3 represents NO generated from HEK293 cells transfected with mutant eNOS cDNA (Ser-1779A); and Spectrum 4 represents NO generated from HEK293 cells transfected with wild-type eNOS cDNA (Ser-1779) and treated with the NOS inhibitor L-NAME. (B) Representative Western blot expression levels of eNOS protein from HEK293 cells transfected with cDNAs encoding wildtype eNOS S1179 and mutant eNOS cDNA S1179A and the corresponding loading control (β-actin). (C) A plot of relative NO intensities from non-transfected and transfected HEK cells. Data are represented as mean ± SEM, * P < 0.05 versus control (untreated non-transfected cells), ** P < 0.05 versus WT eNOS treated cells, n = 3.

Figure S8 Attenuation of SIN-1-mediated ROS generation in wild-type eNOS-plasmid transfected HEK293 cells by DMPO. (A) Confocal micrographs using DCF-DA for ROS detection of wild-type eNOS-cDNA transfected HEK293 cells treated with SIN-1 (500 μM) alone and with post-incubation with nitrone DMPO (100 μM). White bar corresponds to 10 microns. (B) Plot of relative DCF-fluorescence obtained from an average of 100 cells. Data are represented as mean ± SEM, * P < 0.05 versus control, ** P < 0.05 versus SIN-1-treated cells, n = 3.

Table S1 Simulated EPR parameters and concentrations of various adducts formed from SIN-1 treatment of BEAC alone as a function of time obtained by EPR spin trapping using EMPO/methylated-β-cyclodextrin as spin traps.

Table S2 Simulated EPR parameters and concentrations of various adducts formed from post-incubation with DMPO of SIN-1-treated BAEC obtained by EPR spin trapping using EMPO/methylated-β-cyclodextrin as spin traps.

Table S3 Simulated EPR parameters various adducts formed from EMPO/methylated-β-cyclodextrin as spin traps using independently generated HOO, HO and C-centred radicals from hypoxanthine-xanthine oxidase, H2O2-Fe2+ and H2O2-Fe2+-EtOH systems.

bph0171-2321-sd1.docx (7.9MB, docx)

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1 Cytotoxicity of DMPO alone on BAEC. BAEC viability after incubation with DMPO (0–500 μM) as determined by MTT assay. Data are represented as the mean ± SEM, n = 6.

Figure S2 Time-dependent cytotoxicity of SIN-1 on BAEC. Time-dependent cytotoxic effects of SIN-1 (500 μM) on BAEC as determined by MTT assay at various time points (A) 1–6 h. BAEC were treated with SIN-1 for 2 h and post-incubated with plain media (without SIN-1) at various time points (B) 0–24 h. Cell viability was assessed by MTT assay. Data are represented as the mean ± SEM, * P < 0.05 versus control (untreated cells), n = 6.

Figure S3 Detection of O2 − generation in SIN-1-treated BAEC by EPR. (A) BAEC were treated with SIN-1 for 2 h and postincubated in plain media (without SIN-1) at various time points (0–24 h), and ROS generation was monitored by X-band EPR spectrometer using EMPO and Me-β–CD as the spin traps. (B) A plot of relative ROS intensities for the untreated and SIN-1-treated BAEC, post-incubated in plain media at various time points (0–24 h). Data are represented as mean ± SEM, * P < 0.05 versus control (untreated cells), n = 3.

Figure S4 Detection of O2 •− generation in SIN-1-treated BAEC by fluorescence. (A) O2 •− generation in untreated BAEC, and cells treated with SIN-1 (500 μM) and post-incubated with plain media at various points were monitored by confocal microscopy using DHE. The figure represents the merged image of DHE-fluorescence (red) and DAPIfluorescence (blue), bar represents 10 microns. (B) A plot of relative DHE-fluorescence obtained from the average of 100 cells. Data are represented as mean ± SEM, * P < 0.05 versus control (untreated cells).

Figure S5 Detection of ROS generation in SIN-1-treated BAEC by fluorescence. (A) Generation of ROS in untreated BAEC, and cells treated with SIN-1 (500 μM) and postincubated with plain media at various time points were monitored by confocal microscopy using ROS-specific fluorescence dye CM-H2DCFDA. The figure represents the merged image of DCF-fluorescence (green) and DAPI-fluorescence (blue), bar represents 10 microns. (B) A plot of relative DCFfluorescence obtained from the average of 100 cells. Data are represented as mean ± SEM, * P < 0.05 (vs.) control (untreated cells).

Figure S6 Determination of BH4 depletion by SIN-1-mediated eNOS dysfunction. The levels of BH4 in SIN-1-treated BAEC alone and post-incubated with DMPO was measured to determine the extent of eNOS uncoupling. (A) The BH4 chromatograms obtained at retention time of ∼18 min. BAEC treated with SIN-1-post-incubated for 0 h and 24 h. (B) Respective BH4 chromatograms for BAEC alone, treated with SIN-1, and post-incubated with DMPO for 2 h. All experiments were performed at least three times.

Figure S7 Detection of NO in non-transfected and transfected HEK293 cells. (A) NO generation in non-transfected and wild-type/mutant eNOS cDNA transfected HEK293 cells was monitored by X-band EPR spectrometer using Fe(MGD)2 as a spin trap upon stimulation with CaI. From top to bottom: Spectrum 1 represents NO generated from non-transfected HEK293 cells as control; Spectrum 2 represents NO generated from HEK293 cells transfected with wild-type eNOS cDNA (Ser-1779); Spectrum 3 represents NO generated from HEK293 cells transfected with mutant eNOS cDNA (Ser-1779A); and Spectrum 4 represents NO generated from HEK293 cells transfected with wild-type eNOS cDNA (Ser-1779) and treated with the NOS inhibitor L-NAME. (B) Representative Western blot expression levels of eNOS protein from HEK293 cells transfected with cDNAs encoding wildtype eNOS S1179 and mutant eNOS cDNA S1179A and the corresponding loading control (β-actin). (C) A plot of relative NO intensities from non-transfected and transfected HEK cells. Data are represented as mean ± SEM, * P < 0.05 versus control (untreated non-transfected cells), ** P < 0.05 versus WT eNOS treated cells, n = 3.

Figure S8 Attenuation of SIN-1-mediated ROS generation in wild-type eNOS-plasmid transfected HEK293 cells by DMPO. (A) Confocal micrographs using DCF-DA for ROS detection of wild-type eNOS-cDNA transfected HEK293 cells treated with SIN-1 (500 μM) alone and with post-incubation with nitrone DMPO (100 μM). White bar corresponds to 10 microns. (B) Plot of relative DCF-fluorescence obtained from an average of 100 cells. Data are represented as mean ± SEM, * P < 0.05 versus control, ** P < 0.05 versus SIN-1-treated cells, n = 3.

Table S1 Simulated EPR parameters and concentrations of various adducts formed from SIN-1 treatment of BEAC alone as a function of time obtained by EPR spin trapping using EMPO/methylated-β-cyclodextrin as spin traps.

Table S2 Simulated EPR parameters and concentrations of various adducts formed from post-incubation with DMPO of SIN-1-treated BAEC obtained by EPR spin trapping using EMPO/methylated-β-cyclodextrin as spin traps.

Table S3 Simulated EPR parameters various adducts formed from EMPO/methylated-β-cyclodextrin as spin traps using independently generated HOO, HO and C-centred radicals from hypoxanthine-xanthine oxidase, H2O2-Fe2+ and H2O2-Fe2+-EtOH systems.

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