Search Results (59)

Background/Objectives: Preterm infants are at risk of developing the chronic lung condition of bronchopulmonary dysplasia (BPD), with associated alveolar simplification and airway hyperreactivity. Inhibition of S-nitrosoglutathione (GSNO) reductase has been shown to rescue airway hyperreactivity in a murine model of BPD. Here, we investigate the effects of early treatment with N6022, a pharmacologic GSNO reductase inhibitor. Methods: Newborn C57BL/6 mice were exposed to either 21% (control) or 60% oxygen (BPD model) for 5 days after birth. Pups simultaneously received either subcutaneous saline or varying doses of N6022 for 5 days during hyperoxia exposure. Pups were then recovered in room air to 3 weeks postnatal age. H&E-stained lungs were analyzed for alveolar simplification and airway tethering. In vivo airway reactivity to inhaled methacholine was measured using a flexiVent system. In separate littermates, lungs were immediately harvested after 5 days of hyperoxia for protein quantification via automated capillary Westerns. Results: Alveolar simplification and decreased airway tethering were noted in the 60% + saline group. Pups treated with N6022 during hyperoxia displayed dose-dependent improvements in alveolar simplification and airway tethering. Similarly, hyperoxia-exposed pups had increased airway reactivity, as measured by elevated respiratory system resistance and elastance responses to methacholine. Treatment with 10 mg/kg/day N6022 during hyperoxia resulted in decreased resistance and elastance responses. TGF-β expressions were elevated in the 60% + saline group and attenuated in the 60% + N6022 groups. Conclusions: Early exposure to GSNO reductase inhibitors such as N6022 can prevent hyperoxia-induced alveolar simplification and airway hyperreactivity, with lasting effects even after cessation of treatment. Full article

Onychomycosis is a therapeutically challenging fungal infection. Systemic antifungals are limited by adverse effects and drug interactions, while topical therapies may fail to achieve therapeutic nail bed concentrations. Nitric oxide (NO), a small, diffusible free radical with broad-spectrum antimicrobial activity, offers a novel approach to overcoming these barriers. We assessed the penetration and subsequent efficacy of a nitric oxide–releasing gel (NORG) in the treatment of onychomycosis. Ex vivo human nail models assessed NORG’s transungual penetration and antifungal activity via colorimetric, immunohistochemical, and microbiological assays. NORG eradicated Trichophyton mentagrophytes completely (0 CFU/g), outperforming terbinafine (3.58 ± 0.2 log₁₀ CFU/g). In an ex vivo infection model, NORG achieved fungal clearance within 14 days, continuing to Day 30 treatment end, with no regrowth during 21 days of incubation post-treatment. Clinical data from patients with onychomycosis who received topical NORG therapy show that NORG penetrated the nail plate and nail bed, as evidenced by s-nitrosothiol accumulation and progressive discoloration. The NORG formulation demonstrates in vitro efficacy; controlled trials are warranted to fully assess clinical efficacy and safety of this NORG formulation in humans, and establish optimal treatment protocols. Full article

Inorganic nitrite contributes to the nitrosation of biomolecules and exerts antioxidant effects. The proton pump inhibitor omeprazole has pro-oxidant effects, inhibits the formation of nitroso species in the stomach, and abrogates the blood pressure-lowering effects of orally administered nitrite. Here, we examine whether a two-week treatment with nitrite leads to tissue nitrosation that scales with local thiol concentrations and whether oral nitrite treatment can prevent the pro-oxidant effects of omeprazole. Male Sprague–Dawley rats received daily doses of omeprazole 10 mg/kg i.p. (or vehicle) and sodium nitrite 15 mg/kg by gavage (or water) for 14 days. Animals were euthanized 6 h after the last nitrite dose, and blood and tissues (brain, heart, and liver) were collected for biochemical analyses. We found that nitrite treatment increased liver nitrite and total nitroso species (RxNO) concentrations approximately eight-fold (with minor increases in other organs), and omeprazole treatment attenuated these effects. Nitrite treatment selectively elevated non-protein thiol concentrations in the liver, but not in animals also receiving omeprazole. Tissue thiol elevation was associated with increased nitrosation of hepatic proteins, which was prevented by omeprazole. Nitrite upregulated mRNA expression of microsomal glutathione S-transferase-1 (Mgst1) and decreased superoxide and hydrogen peroxide production, especially in rats co-treated with omeprazole. While omeprazole increased liver xanthine oxidoreductase (XOR), nitrite treatment attenuated this effect. These results demonstrate that oral nitrite treatment robustly elevates nitrite and RxNO concentrations in the liver, and these effects are associated with increased hepatic glutathione production and an upregulation of Mgst1 expression, counteracting the pro-oxidant effects induced by omeprazole. Full article

Protein S-nitrosylation is a selective post-translational modification in which a nitrosyl group is covalently attached to the reactive thiol group of cysteine, forming S-nitrosothiol. This modification plays a pivotal role in modulating physiological and pathological cardiovascular processes by altering protein conformation, activity, stability, and other post-translational modifications. It is instrumental in regulating vascular and myocardial systolic and diastolic functions, vascular endothelial cell and cardiomyocyte apoptosis, and cardiac action potential and repolarization. Aberrant S-nitrosylation levels are implicated in the pathogenesis of various cardiovascular diseases, including systemic hypertension, pulmonary arterial hypertension, atherosclerosis, heart failure, myocardial infarction, arrhythmia, and diabetic cardiomyopathy. Insufficient S-nitrosylation leads to impaired vasodilation and increased vascular resistance, while excessive S-nitrosylation contributes to cardiac hypertrophy and myocardial fibrosis, thereby accelerating ventricular remodeling. This paper reviews the S-nitrosylated proteins in the above-mentioned diseases and their impact on these conditions through various signaling pathways, with the aim of providing a theoretical foundation for the development of novel therapeutic strategies or drugs targeting S-nitrosylated proteins. Full article

S-nitrosoglutathione (GSNO), a promising S-nitrosothiol, has been recognized for its ability to modulate vascular tone through its vasodilatory, antiplatelet, and antiproliferative effects. However, data on its vasodilatory effects in human vessels remain limited, and its mechanisms of action have yet to be fully elucidated. In this study, we aimed to investigate the vasorelaxant effect of GSNO and its underlying mechanisms, with particular focus on the soluble guanylate cyclase (sGC)/nitric oxide (NO) pathway and potassium channels in isolated human saphenous veins (SVs) obtained from patients undergoing coronary artery bypass grafting (CABG). GSNO (10⁻⁸–10⁻⁴ M) produced concentration-dependent relaxations in SV rings precontracted with phenylephrine. These relaxations were unaffected by NO synthase inhibition with L-NAME (10⁻⁴ M, 30 min) or NO scavenging with PTIO (10⁻⁴ M, 30 min), but were significantly reduced by the sGC inhibitor, ODQ (10⁻⁵ M, 30 min). Inhibition of ATP-sensitive (glibenclamid; 10⁻⁵ M, 30 min.), high-conductance Ca²⁺-activated (charybdotoxin; 10⁻⁷ M, 30 min), small-conductance Ca²⁺-activated (apamin; 10⁻⁶ M, 30 min), or voltage-dependent (4-aminopyridine; 10⁻³ M, 30 min) potassium channels did not alter the maximum relaxant responses to GSNO. Furthermore, pretreatment with GSNO (10⁻⁴ M, 30 min) significantly attenuated both the contractile response and sensitivity to phenylephrine. Collectively, these findings demonstrate that GSNO exerts acute vasorelaxant and modulatory effects in human SV primarily via cGMP-dependent mechanisms, highlighting its potential as a local therapeutic agent for preventing graft spasm in CABG. Full article

Protein S-nitrosation, a redox post-translational modification elicited by nitric oxide (NO), is essential for modulating diverse protein functions and signaling pathways. Dysregulation of S-nitrosation is implicated in various pathological processes, including oxygen-glucose deprivation/reperfusion (OGD/R) injury, a widely used model for ischemia-reperfusion diseases. The dynamic changes in S-nitrosothiols (SNOs) during ischemia-reperfusion highlight the need for theranostic strategies to monitor and modulate SNO levels based on pathological progression. However, to date, no theranostic strategies have been reported for addressing dysregulated SNO in disease models, particularly in OGD/R conditions. Here, we report the development of a selective probe P-EHC, which could specifically react with SNOs to release EHC, not only exhibiting turn-on fluorescence with high quantum yield and good water solubility but also demonstrating macrophage migration inhibitory factor (MIF) inhibitory activity. In an OGD/R model of SH-SY5Y cells, we observed elevated SNO levels by using live-cell confocal imaging. Treatment of P-EHC significantly reduced intracellular reactive oxygen species (ROS), lowered total NO_x species, and improved cell viability in the OGD/R model. In summary, the simplicity and versatility of P-EHC suggest its broad applicability for monitoring SNO in various biological models and therapeutic contexts, particularly in ischemia-reperfusion diseases. Full article

Nitric oxide (NO) is a multifunctional signaling molecule in plants, playing key roles in germination, microbial symbiosis, and nodule formation. However, its instability requires innovative approaches, such as using nanoencapsulated NO donors, to prolong its effects. This study evaluated the impact of treating soybean (Glycine max) seeds with the NO donor S-nitrosoglutathione (GSNO), encapsulated in polymeric nanoparticles, on the germination, nodulation, and plant growth. Seeds were treated with free GSNO, chitosan nanoparticles with/without NO (NP CS-GSNO/NP CS-GSH, where GSH is glutathione, the NO donor precursor), and alginate nanoparticles with/without NO (NP Al-GSNO/NP Al-GSH). Chitosan nanoparticles (positive zeta potential) were smaller and released NO faster compared with alginate nanoparticles (negative zeta potential). The seed treatment with NP CS-GSNO (1 mM, related to GSNO concentration) significantly improved germination percentage, root length, number of secondary roots, and dry root mass of soybean compared with the control. Conversely, NP CS-GSH resulted in decreased root and shoot length. NP Al-GSNO enhanced shoot dry mass and increased the number of secondary roots by approximately threefold at the highest concentrations. NP CS-GSNO, NP Al-GSNO, and NP Al-GSH increased S-nitrosothiol levels in the roots by approximately fourfold compared with the control. However, NP CS-GSNO was the only treatment that increased the nodule dry mass of soybean plants. Therefore, our results indicate the potential of chitosan nanoparticles to improve the application of NO donors in soybean seeds. Full article

Nitrogen fixation in legume nodules is crucial for plant growth and development. Therefore, this study aims to investigate the effects of nitric oxide [S-nitrosoglutathione (GSNO)] and silicon [sodium metasilicate (Si)], both individually and in combination, on soybean growth, nodule formation, leghaemoglobin (Lb) synthesis, and potential post-translational modifications. At the V1 stage, soybean plants were treated for 2 weeks with 150 µM GSNO, and Si at concentrations of 1 mM, 2 mM, and 4 mM. The results showed that NO and Si enhance the nodulation process by increasing phenylalanine ammonia-lyase activity and Nod factors (NIP2-1), attracting rhizobia and accelerating nodule formation. This leads to a greater number and larger diameter of nodules. Individually, NO and Si support the synthesis of Lb and leghaemoglobin protein (Lba) expression, ferric leghaemoglobin reductases (FLbRs), and S-nitrosoglutathione reductase (GSNOR). However, when used in combination, NO and Si inhibit these processes, leading to elevated levels of S-nitrosothiols in the roots and nodules. This combined inhibition may potentially induce post-translational modifications in FLbRs, pivotal for the reduction of Lb³⁺ to Lb²⁺. These findings underscore the critical role of NO and Si in the nodulation process and provide insight into their combined effects on this essential plant function. Full article

Cardiovascular diseases (CVDs) are often associated with impaired nitric oxide (NO) bioavailability, a critical pathophysiological alteration in CVDs and an important target for therapeutic interventions. Recent studies have revealed the potential of inorganic nitrite and nitrate as sources of NO, offering promising alternatives for managing various cardiovascular conditions. It is now becoming clear that taking advantage of enzymatic pathways involved in nitrite reduction to NO is very relevant in new therapeutics. However, recent studies have shown that nitrite may be bioactivated in the acidic gastric environment, where nitrite generates NO and a variety of S-nitrosating compounds that result in increased circulating S-nitrosothiol concentrations and S-nitrosation of tissue pharmacological targets. Moreover, transnitrosation reactions may further nitrosate other targets, resulting in improved cardiovascular function in patients with CVDs. In this review, we comprehensively address the mechanisms and relevant effects of nitrate and nitrite-stimulated gastric S-nitrosothiol formation that may promote S-nitrosation of pharmacological targets in various CVDs. Recently identified interfering factors that may inhibit these mechanisms and prevent the beneficial responses to nitrate and nitrite therapy were also taken into consideration. Full article

S-nitrosothiols are endogenous, bioactive molecules. S-nitrosothiols are implicated in many diseases, including sepsis. It is currently cumbersome to measure S-nitrosothiols clinically. We have previously developed an instrument to measure tissue S-nitrosothiols non-invasively using ultraviolet light. We have performed a prospective case control study of controls and children with sepsis admitted to the PICU. We hypothesized that tissue S-nitrosothiols would be higher in septic patients than controls. Controls were patients with no cardiopulmonary instability. Cases were patients with septic shock. We measured S-nitrosothiols, both at diagnosis and after resolution of shock. A total of 44 patients were enrolled: 21 controls and 23 with sepsis. At baseline, the controls were younger [median age 5 years (IQR 0, 9) versus 11 years (IQR: 6, 16), p-value = 0.012], had fewer comorbidities [7 (33.3%) vs. 20 (87.0%), p-value < 0.001], and had lower PELOD scores [0 (IQR: 0, 0) vs. 12 (IQR: 11, 21), p-value < 0.001]. S-nitrosothiol levels were higher in sepsis cohort (1.1 ppb vs. 0.8 ppb, p = 0.004). Five patients with sepsis had longitudinal measures and had a downtrend after resolution of shock (1.3 ppb vs. 0.9 ppb, p = 0.04). We dichotomized patients based on S-nitrosothiol levels and found an association with worse clinical outcomes, but further work will be needed to validate these findings. Full article

Nitrite is a nitric oxide (NO) metabolite, which may be bioactivated to generate NO in vivo and supplement endogenous NO formation, especially in cardiovascular and metabolic diseases. However, it is not known whether treatment with oral nitrite results in the accumulation of NO metabolites in different organs. Moreover, treatment with omeprazole, an inhibitor of gastric acid secretion, severely affects the gastric formation of S-nitrosothiols induced with oral nitrite treatment. However, no previous study has examined whether omeprazole affects the nitrite-induced accumulation of NO metabolites in different organs. This study examined in rats the effects of oral sodium nitrite treatment (15 mg/kg via gavage for 1 or 7 days) associated with omeprazole (10 mg/kg or vehicle) on nitrite and nitrate and nitrosylated species (RXNO) concentrations (measured using ozone-based chemiluminescence methods) assessed in the plasma, aorta, heart, liver, brain, and muscle. While our results showed that NO metabolite accumulation in different organs is not uniform, we found that the skeletal muscle, the heart, and the liver accumulate NO metabolites, particularly RXNO. This response was significantly attenuated by omeprazole in the heart and in the skeletal muscle. Together, these findings may indicate that the skeletal muscle, the heart, and the liver are major reservoir sites for NO metabolites after oral nitrite treatment, with major increases in nitrosylated species. Full article

Inflammation is an important component of the etiopathology of depression that uses oxidative and nitrosative stress (O&NS) and elevated inflammatory markers. SARS-CoV-2 infection is also associated with abnormal inflammatory processes, which may impair effective treatment of depression in COVID-19 survivors. In the presented study, thirty-three hospitalized patients with major depressive disorder (MDD) were started on antidepressant treatment, and twenty-one were re-evaluated after 4–6 weeks. The control group consisted of thirty healthy volunteers. All participants underwent neuropsychiatric evaluation, biochemical blood and urine analyses. The results of the research demonstrated positive correlations of the Hamilton Depression Rating Scale (HAM-D) scores with serum catalase (CAT) and urinary S-Nitrosothiols levels, and the Beck Depression Inventory (BDI) scores with serum reduced glutathione (GSH) and superoxide dismutase (SOD) levels. Depressed patients with a history of COVID-19 prior to the treatment had higher urinary nitric oxide (NO) levels and lower serum glutathione peroxidase (GPx) levels. In the control group, COVID-19 survivors had higher levels of urinary N-formylkynurenine (NFK). Our results suggest that the antidepressant treatment has a modulating effect on O&NS, reduces depressive symptoms and improves cognitive functions The present study does not indicate that clinical response to antidepressant treatment is associated with COVID-19 history and baseline SARS-CoV-2 antibody levels. Nevertheless, further research in this area is needed to systematize antidepressant treatment in COVID-19 survivors. Full article

We recently developed a combination of four chemiluminescence-based assays for selective detection of different nitric oxide (NO) metabolites, including nitrite, S-nitrosothiols (SNOs), heme-nitrosyl (heme-NO), and dinitrosyl iron complexes (DNICs). However, these NO species (NOx) may be under dynamic equilibria during sample handling, which affects the final determination made from the readout of assays. Using fetal and maternal sheep from low and high altitudes (300 and 3801 m, respectively) as models of different NOx levels and compositions, we tested the hypothesis that sample handling introduces artifacts in chemiluminescence assays of NOx. Here, we demonstrate the following: (1) room temperature placement is associated with an increase and decrease in NOx in plasma and whole blood samples, respectively; (2) snap freezing and thawing lead to the interconversion of different NOx in plasma; (3) snap freezing and homogenization in liquid nitrogen eliminate a significant fraction of NOx in the aorta of stressed animals; (4) A “stop solution” commonly used to preserve nitrite and SNOs leads to the interconversion of different NOx in blood, while deproteinization results in a significant increase in detectable NOx; (5) some reagents widely used in sample pretreatments, such as mercury chloride, acid sulfanilamide, N-ethylmaleimide, ferricyanide, and anticoagulant ethylenediaminetetraacetic acid, have unintended effects that destabilize SNO, DNICs, and/or heme-NO; (6) blood, including the residual blood clot left in the washed purge vessel, quenches the signal of nitrite when using ascorbic acid and acetic acid as the purge vessel reagent; and (7) new limitations to the four chemiluminescence-based assays. This study points out the need for re-evaluation of previous chemiluminescence measurements of NOx, and calls for special attention to be paid to sample handling, as it can introduce significant artifacts into NOx assays. Full article

Nitrite (O=N-O⁻, NO₂⁻) and nitrate (O=N(O)-O⁻, NO₃⁻) are ubiquitous in nature. In aerated aqueous solutions, nitrite is considered the major autoxidation product of nitric oxide (^●NO). ^●NO is an environmental gas but is also endogenously produced from the amino acid L-arginine by the catalytic action of ^●NO synthases. It is considered that the autoxidation of ^●NO in aqueous solutions and in O₂-containing gas phase proceeds via different neutral (e.g., O=N-O-N=O) and radical (e.g., ONOO^●) intermediates. In aqueous buffers, endogenous S-nitrosothiols (thionitrites, RSNO) from thiols (RSH) such as L-cysteine (i.e., S-nitroso-L-cysteine, CysSNO) and cysteine-containing peptides such as glutathione (GSH) (i.e., S-nitrosoglutathione, GSNO) may be formed during the autoxidation of ^●NO in the presence of thiols and dioxygen (e.g., GSH + O=N-O-N=O → GSNO + O=N-O⁻ + H⁺; pK_a^HONO, 3.24). The reaction products of thionitrites in aerated aqueous solutions may be different from those of ^●NO. This work describes in vitro GC-MS studies on the reactions of unlabeled (¹⁴NO₂⁻) and labeled nitrite (¹⁵NO₂⁻) and RSNO (RS¹⁵NO, RS¹⁵N¹⁸O) performed in pH-neutral aqueous buffers of phosphate or tris(hydroxyethylamine) prepared in unlabeled (H₂¹⁶O) or labeled H₂O (H₂¹⁸O). Unlabeled and stable-isotope-labeled nitrite and nitrate species were measured by gas chromatography–mass spectrometry (GC-MS) after derivatization with pentafluorobenzyl bromide and negative-ion chemical ionization. The study provides strong indication for the formation of O=N-O-N=O as an intermediate of ^●NO autoxidation in pH-neutral aqueous buffers. In high molar excess, HgCl₂ accelerates and increases RSNO hydrolysis to nitrite, thereby incorporating ¹⁸O from H₂¹⁸O into the SNO group. In aqueous buffers prepared in H₂¹⁸O, synthetic peroxynitrite (ONOO⁻) decomposes to nitrite without ¹⁸O incorporation, indicating water-independent decomposition of peroxynitrite to nitrite. Use of RS¹⁵NO and H₂¹⁸O in combination with GC-MS allows generation of definite results and elucidation of reaction mechanisms of oxidation of ^●NO and hydrolysis of RSNO. Full article

Soluble guanylyl cyclase (GC1) and oxido-reductase thioredoxin (Trx1) form a complex that mediates two NO signaling pathways as a function of the redox state of cells. Under physiological conditions, reduced Trx1 (rTrx1) supports the canonical NO-GC1-cGMP pathway by protecting GC1 activity from thiol oxidation. Under oxidative stress, the NO-cGMP pathway is disrupted by the S-nitrosation of GC1 (addition of a NO group to a cysteine). In turn, SNO-GC1 initiates transnitrosation cascades, using oxidized thioredoxin (oTrx1) as a nitrosothiol relay. We designed an inhibitory peptide that blocked the interaction between GC1 and Trx1. This inhibition resulted in the loss of a) the rTrx1 enhancing effect of GC1 cGMP-forming activity in vitro and in cells and its ability to reduce the multimeric oxidized GC1 and b) GC1’s ability to fully reduce oTrx1, thus identifying GC1 novel reductase activity. Moreover, an inhibitory peptide blocked the transfer of S-nitrosothiols from SNO-GC1 to oTrx1. In Jurkat T cells, oTrx1 transnitrosates procaspase-3, thereby inhibiting caspase-3 activity. Using the inhibitory peptide, we demonstrated that S-nitrosation of caspase-3 is the result of a transnitrosation cascade initiated by SNO-GC1 and mediated by oTrx1. Consequently, the peptide significantly increased caspase-3 activity in Jurkat cells, providing a promising therapy for some cancers. Full article