S-nitrosothiols Regulate Nitric Oxide Production And Storage In Plants Through The Nitrogen Assimilation Pathway.

Nitrogen assimilation plays a vital role in plant metabolism. Assimilation of nitrate, the primary source of nitrogen in soil, is linked to the generation of the redox signal nitric oxide (NO). An important mechanism by which NO regulates plant development and stress responses is through S-nitrosylation, that is, covalent attachment of NO to cysteine residues to form S-nitrosothiols (SNO). Despite the importance of nitrogen assimilation and NO signalling, it remains largely unknown how these pathways are interconnected. Here we show that SNO signalling suppresses both nitrate uptake and reduction by transporters and reductases, respectively, to fine tune nitrate homeostasis. Moreover, NO derived from nitrate assimilation suppresses the redox enzyme S-nitrosoglutathione Reductase 1 (GSNOR1) by S-nitrosylation, preventing scavenging of S-nitrosoglutathione, a major cellular bio-reservoir of NO. Hence, our data demonstrates that (S)NO controls its own generation and scavenging by modulating nitrate assimilation and GSNOR1 activity.

Hdl Levels And Oxidizability During Myocardial Infarction Are Associated With Reduced Endothelial-mediated Vasodilation And Nitric Oxide Bioavailability.

Acute phase response modifies high-density lipoprotein (HDL) into a dysfunctional particle that may favor oxidative/inflammatory stress and eNOS dysfunction. The present study investigated the impact of this phenomenon on patients presenting ST-elevation myocardial infarction (STEMI). Plasma was obtained from 180 consecutive patients within the first 24-h of onset of STEMI symptoms (D1) and after 5 days (D5). Nitrate/nitrite (NOx) and lipoproteins were isolated by gradient ultracentrifugation. The oxidizability of low-density lipoprotein incubated with HDL (HDLaoxLDL) and the HDL self-oxidizability (HDLautox) were measured after CuSO4 co-incubation. Anti-inflammatory activity of HDL was estimated by VCAM-1 secretion by human umbilical vein endothelial cells after incubation with TNF-α. Flow-mediated dilation (FMD) was assessed at the 30(th) day (D30) after STEMI. Among patients in the first tertile of admission HDL-Cholesterol (<33 mg/dL), the increment of NOx from D1 to D5 [6.7(2; 13) vs. 3.2(-3; 10) vs. 3.5(-3; 12); p = 0.001] and the FMD adjusted for multiple covariates [8.4(5; 11) vs 6.1(3; 10) vs. 5.2(3; 10); p = 0.001] were higher than in those in the second (33-42 mg/dL) or third (>42 mg/dL) tertiles, respectively. From D1 to D5, there was a decrease in HDL size (-6.3 ± 0.3%; p < 0.001) and particle number (-22.0 ± 0.6%; p < 0.001) as well as an increase in both HDLaoxLDL (33%(23); p < 0.001) and HDLautox (65%(25); p < 0.001). VCAM-1 secretion after TNF-a stimulation was reduced after co-incubation with HDL from healthy volunteers (-24%(33); p = 0.009), from MI patients at D1 (-23%(37); p = 0.015) and at D30 (-22%(24); p = 0.042) but not at D5 (p = 0.28). During STEMI, high HDL-cholesterol is associated with a greater decline in endothelial function. In parallel, structural and functional changes in HDL occur reducing its anti-inflammatory and anti-oxidant properties.

Nitric Oxide-releasing Poly(vinyl Alcohol) Film For Increasing Dermal Vasodilation.

Pathological conditions associated with the impairment of nitric oxide (NO) production in the vasculature, such as Raynaud's syndrome and diabetic angiopathy, have stimulated the development of new biomaterials capable of delivering NO topically. With this purpose, we modified poly(vinyl-alcohol) (PVA) by chemically crosslinking it via esterification with mercaptosuccinic acid. This reaction allowed the casting of sulfhydrylated PVA (PVA-SH) films. Differential scanning calorimetry and X-ray diffractometry showed that the crosslinking reaction completely suppressed the crystallization of PVA, leading to a non-porous film with a homogeneous distribution of -SH groups. The remaining free hydroxyl groups in the PVA-SH network conferred partial hydrophylicity to the material, which was responsible for a swelling degree of ca. 110%. The PVA-SH films were subjected to an S-nitrosation reaction of the -SH groups, yielding a PVA containing S-nitrosothiol groups (PVA-SNO). Amperometric and chemiluminescence measurements showed that the PVA-SNO films were capable of releasing NO spontaneously after immersion in physiological medium. Laser Doppler-flowmetry, used to assess the blood flow in the dermal microcirculation, showed that the topical application of hydrated PVA-SNO films on the health skin led to a dose- and time-dependent increase of more than 5-fold in the dermal baseline blood flow in less than 10min, with a prolonged action of more than 4h during continuous application. These results show that PVA-SNO films might emerge as a new material with potential for the topical treatment of microvascular skin disorders.

Reduction Of Blood Nitric Oxide Levels Is Associated With Clinical Improvement Of The Chronic Pelvic Pain Related To Endometriosis.

The objective of this prospective study was to determine the plasma levels of nitric oxide (NO) in women with chronic pelvic pain secondary to endometriosis (n=24) and abdominal myofascial pain syndrome (n=16). NO levels were measured in plasma collected before and 1 month after treatment. Pretreatment NO levels (μM) were lower in healthy volunteers (47.0±12.7) than in women with myofascial pain (64.2±5.0, P=0.01) or endometriosis (99.5±12.9, P<0.0001). After treatment, plasma NO levels were reduced only in the endometriosis group (99.5±12.9 vs 61.6±5.9, P=0.002). A correlation between reduction of pain intensity and reduction of NO level was observed in the endometriosis group [correlation = 0.67 (95%CI = 0.35 to 0.85), P<0.0001]. Reduction of NO levels was associated with an increase of pain threshold in this group [correlation = -0.53 (-0.78 to -0.14), P<0.0001]. NO levels appeared elevated in women with chronic pelvic pain diagnosed as secondary to endometriosis, and were directly associated with reduction in pain intensity and increase in pain threshold after treatment. Further studies are needed to investigate the role of NO in the pathophysiology of pain in women with endometriosis and its eventual association with central sensitization.

Nitric Oxide Released From Luminal S-nitroso-n-acetylcysteine Increases Gastric Mucosal Blood Flow.

Nitric oxide (NO)-mediated vasodilation plays a key role in gastric mucosal defense, and NO-donor drugs may protect against diseases associated with gastric mucosal blood flow (GMBF) deficiencies. In this study, we used the ex vivo gastric chamber method and Laser Doppler Flowmetry to characterize the effects of luminal aqueous NO-donor drug S-nitroso-N-acetylcysteine (SNAC) solution administration compared to aqueous NaNO2 and NaNO3 solutions (pH 7.4) on GMBF in Sprague-Dawley rats. SNAC solutions (600 μM and 12 mM) led to a rapid threefold increase in GMBF, which was maintained during the incubation of the solutions with the gastric mucosa, while NaNO2 or NaNO3 solutions (12 mM) did not affect GMBF. SNAC solutions (600 μM and 12 mM) spontaneously released NO at 37 °C at a constant rate of 0.3 or 14 nmol·mL-1·min-1, respectively, while NaNO2 (12 mM) released NO at a rate of 0.06 nmol·mL-1·min-1 and NaNO3 (12 mM) did not release NO. These results suggest that the SNAC-induced GMBF increase is due to their higher rates of spontaneous NO release compared to equimolar NaNO2 solutions. Taken together, our data indicate that oral SNAC administration is a potential approach for gastric acid-peptic disorder prevention and treatment.

Assessment of ocular surface toxicity after topical instillation of nitric oxide donors

PURPOSE: To evaluate the ocular surface toxicity of two nitric oxide donors in ex vivo and in vivo animal models: S-nitrosoglutathione (GSNO) and S-nitroso-N-acetylcysteine (SNAC) in a hydroxypropyl methylcellulose (HPMC) matrix at final concentrations 1.0 and 10.0 mM. METHODS: Ex vivo GSNO and SNAC toxicities were clinically and histologically analyzed using freshly excised pig eyeballs. In vivo experiments were performed with 20 albino rabbits which were randomized into 4 groups (5 animals each): Groups 1 and 2 received instillations of 150 µL of aqueous HPMC solution containing GSNO 1.0 and 10.0 mM, respectively, in one of the eyes; Groups 3 and 4 received instillations of 150 µL of aqueous HPMC solution-containing SNAC 1.0 and 10.0 mM, respectively, in one of the eyes. The contralateral eyes in each group received aqueous HPMC as a control. All animals underwent clinical evaluation on a slit lamp and the eyes were scored according to a modified Draize eye test and were histologically analyzed. RESULTS: Pig eyeballs showed no signs of perforation, erosion, corneal opacity or other gross damage. These findings were confirmed by histological analysis. There was no difference between control and treated rabbit eyes according to the Draize eye test score in all groups (p>0.05). All formulations showed a mean score under 1 and were classified as non-irritating. There was no evidence of tissue toxicity in the histological analysis in all animals. CONCLUSION: Aqueous HPMC solutions containing GSNO and SNAC at concentrations up to 10.0 mM do not induce ocular irritation.

Enhanced Endothelium-dependent Relaxation Of Rat Pulmonary Artery Following β-adrenergic Overstimulation: Involvement Of The No/cgmp/vasp Pathway.

The aim of this study was to investigate whether β-adrenoceptor (β-AR) overstimulation induced by in vivo treatment with isoproterenol (ISO) alters vascular reactivity and nitric oxide (NO) production and signaling in pulmonary arteries. Vehicle or ISO (0.3mgkg(-1)day(-1)) was administered daily to male Wistar rats. After 7days, the jugular vein was cannulated to assess right ventricular (RV) systolic pressure (SP) and end diastolic pressure (EDP). The extralobar pulmonary arteries were isolated to evaluate the relaxation responses, protein expression (Western blot), NO production (diaminofluorescein-2 fluorescence), and cyclic guanosine 3',5'-monophosphate (cGMP) levels (enzyme immunoassay kit). ISO treatment induced RV hypertrophy; however, no differences in RV-SP and EDP were observed. The pulmonary arteries from the ISO-treated group showed enhanced relaxation to acetylcholine that was abolished by the NO synthase (NOS) inhibitor N(ω)-nitro-l-arginine methyl ester (l-NAME); whereas relaxation elicited by sodium nitroprusside, ISO, metaproterenol, mirabegron, or KCl was not affected by ISO treatment. ISO-treated rats displayed enhanced endothelial NOS (eNOS) and vasodilator-stimulated phosphoprotein (VASP) expression in the pulmonary arteries, while phosphodiesterase-5 protein expression decreased. ISO treatment increased NO and cGMP levels and did not induce eNOS uncoupling. The present data indicate that β-AR overactivation enhances the endothelium-dependent relaxation of pulmonary arteries. This effect was linked to an increase in eNOS-derived NO production, cGMP formation and VASP content and to a decrease in phosphodiesterase-5 expression. Therefore, elevated NO bioactivity through cGMP/VASP signaling could represent a protective mechanism of β-AR overactivation on pulmonary circulation.