Physiological Nitric Oxide Research Articles

Antioxidant and anti-glycation activities of Mandevilla velutina extract and effect on parasitemia levels in Trypanosoma cruzi experimental infection: in vivo, in vitro and in silico approaches

Ethnopharmacological relevanceMandevilla velutina (Mart. Ex Stadelm.) Woodson, known in Brazil as "infalível" and "jalapa", is a medicinal plant native from the Cerrado region (Brazilian Savannah). The underground organ (xylopodium) of this species is prepared as ethanolic extract or infusion and it is commonly used in traditional medicine to treat snake venom. Although, locals and indigenous populations from Cerrado have used M. velutina for the treatment of infection by Trypanosoma cruzi (Chagas’ disease). Aim of the studyThis study aimed to evaluate the in vitro antioxidant and anti-glycation activities of the crude hydroethanolic extract of M. velutina xylopodium. Besides, it aimed to evaluate its effect on parasitemia levels in in vivo T. cruzi experimental infection. In addition, this study aimed to determine possible interactions between the main compound of the extract and molecular targets associated with survival and virulence of T. cruzi in in silico approaches. Materials and MethodsDetermination of total polyphenols, flavonoids and steroidal aglycones content were performed. In addition, high performance liquid chromatography (HPLC) was carried out to identify main compounds of the extract. Antioxidant activity was determined by DPPH radical scavenging, ferric ion reducing power (FRAP), Thiobarbituric acid reactive species (TBARS) and Oxygen Radical Absorbance Capacity (ORAC) methods. Anti-glycation activity was demonstrated through relative mobility in electrophoresis (RME), determination of free amino groups and inhibition of AGEs formation. Determination of the action of extract in parasitemia levels was performed by T. cruzi experimental infection of mice and nitrite levels were measured in the serum of animals evaluated in this study. Molecular docking analyses of the main compound (Velutinol A) with DNA and molecular targets associated with survival and virulence of T. cruzi. ResultsPhytoconstituents evaluation exhibited the presence polyphenols, flavonoids and steroidal aglycone, and HPLC identified the major presence of Velutinol A. Antioxidant and anti-glycation evaluations showed that the extract present significant activity in all methods evaluated. In addition, extract reduced the number of trypomastigotes and increased the survival of treated animals. The treatment using extract showed an interference in the synthesis of physiological nitric oxide as an immune response to infection. In silico assays demonstrated interaction between Velutinol A and DNA and molecular targets of T. cruzi. ConclusionsThe results showed that the hydroethanolic extract of M. velutina xylopodium contains bioactive compounds including polyphenols, flavonoids and steroidal aglycones (mainly Velutinol A) of which may be responsible for the antioxidant, anti-glycation and anti-parasitic activity against T. cruzi. Trypanocidal activity of M. velutina compounds may be linked to their influence on NO synthesis during infection and/or their capacity to bind and inhibit molecules associated to virulence and survival of T. cruzi.

Nasal and tracheobronchial nitric oxide production and its influence on oxygenation in horses undergoing total intravenous anaesthesia

BackgroundThe present study aimed to investigate the effect of endotracheal intubation on nasal and tracheal endogenous NO concentrations, gas exchange and oxygenation in horses undergoing general anaesthesia. In many species a major part of physiological nitric oxide (NO) production takes place in the nasopharynx. Inhaled NO acts as a pulmonary vasodilator and regulates lung perfusion and endotracheal intubation bypasses the nasopharynx. Six horses were randomly assigned to either the “intubated” (INT) or the “non-intubated” (nINT) treatment group. Horses were premedicated with dexmedetomidine (5 μg/kg IV). Anaesthesia was induced with 2.5 mg/kg ketamine and 0.05 mg/kg diazepam IV, and it was maintained by administration of a triple-drip (100 mg/kg/h guaifenesin, 4 mg/kg/h ketamine, 7 μg/kg/h dexmedetomidine). The horses were spontaneously breathing room air. Heart rate, cardiac output, arterial blood pressure, pulmonary arterial blood pressures and respiratory rate were recorded during a 100-min anaesthesia period. Arterial, venous and mixed venous blood samples were taken every 10 minutes and analysed for partial pressure of oxygen (PO2) and carbon dioxide (PCO2), oxygen saturation and haemoglobin content. Standard oxygenation indices were calculated. Nasal and tracheal endogenous NO concentration was determined by chemiluminescence.ResultsCardiovascular variables, respiratory rate, PO2, PCO2, oxygen saturation, haemoglobin content, CaO2, O2ER, P(a-ET)CO2 and Qs/Qt did not differ significantly between the two treatment groups. The P(A-a)O2 was significantly higher in INT (6.1 ± 0.3 kPa) compared to nINT (4.9 ± 0.1 kPa) (p = 0.045), respectively. The nasal (8.0 ± 6.2 ppb) and tracheal (13.0 ± 6.3 ppb) endogenous NO concentration differed significantly in INT (p = 0.036), but not in nINT (nasal: 16.9 ± 9.0 ppb; tracheal: 18.5 ± 9.5 ppb) (p = 0.215).ConclusionEndotracheal intubation reduces the nasal and tracheal endogenous NO concentration. The influence on pulmonary gas exchange and oxygenation is negligible in horses breathing room air.

Efficacy and safety of inhaled nebulized sodium nitrite in asthmatic patients

BackgroundNitrite is a physiologic nitric oxide (NO) derivative that can be bioactivated to NO. NO has been shown to attenuate airway inflammation and enhance the anti-inflammatory effect of corticosteroids in the animal model of asthma. Here, we aimed to investigate the efficacy and safety of inhaled sodium nitrite as add-on therapy with inhaled corticosteroid (ICS) in adult patients with persistent asthma. MethodsIn protocol 1, 10 asthmatic patients were administered a single dose of nebulized 15-mg sodium nitrite to assess safety, effect on lung function, and pharmacokinetics of nitrite within 120 min. In protocol 2, 20 patients were randomly assigned to a nitrite (15 mg twice daily) group or a placebo group to assess the efficacy over 12 weeks. The primary outcome was the forced expiratory volume in 1 s (FEV1). The secondary outcomes were other lung function parameters, unplanned asthma-related visits at the emergency department (ED) or outpatient department (OPD), admission days, asthma control test (ACT), and safety. ResultsNebulized sodium nitrite had neither acute adverse effect nor effect on lung function test within 120 min. No blood pressure change was seen. At week 12, FEV1 increased in the nitrite group, whereas there was no change in the placebo group. There were 5 events of asthma exacerbation, 4 ED visits, and one unplanned OPD visit in the placebo group, but none of these was noted in the nitrite group. There was no change in ACT scores in both groups. No adverse event was reported during 12 weeks in the nitrite group. There was no change in methemoglobin levels and sputum inflammatory markers. ConclusionFrom our pilot trial, nebulized sodium nitrite is safe in asthmatic patients, and shows the potential to reduce asthma exacerbation compared with placebo.

Changes in the asymmetric dimethylarginine and endothelial nitric oxide synthase levels in pathogenesis of experimental diabetic retinopathy

Endothelial dysfunction associated with impaired nitric oxide excretion plays an important role in the onset and progression of diabetic retinopathy. It has been proven that a decrease in the activity of endothelial NO-synthase (еNО-S), the inhibitor of which is asymmetric dimethylarginine (ADMA), plays an important role in this. Objective: to study the level of asymmetric dimethylarginine and endothelial nitric oxide synthase at different stages of development of diabetic retinopathy in the experiment. The study was conducted in Wistar white rats of 180-200 g weight. According to the tasks, the animals were separated into 2 groups as follows: group 1 – 60 intact animals, group 2 – 60 animals with simulated diabetic retinopathy without further correction. Type 2 diabetes mellitus and diabetic retinopathy were simulated through intraperitoneal administration of Streptozotocin (Sigma, USA) diluted in 0.1M citrate buffer with pH=4.5. Streptozotocin dose of 55 mg per kg of animal weight was divided into two administrations. The streptozotocin intake was preceded by a 28-day high-fat diet. Our study showed impaired endothelial function in diabetic retinopathy, as evidenced by an increased ADMA level (p<0.001). We have determined a stepwise increase of asymmetric dimethylarginine level in blood of rats with simulated pathology which is apparent in its highest at phase 3. Pathogenetic effect of increased ADMA on еNО-S activity was verified at all experimental stages, Impairment of physiological nitric oxide synthesis in simulated pathology has been proved as evidenced by reduced activity of endothelial NO-synthase yet on the 30th day with further negative dynamics up to the 180th day (p<0.001 compared with the intact group findings).

Challenging development of storable particles for oral delivery of a physiological nitric oxide donor

Nitric oxide (NO) deficiency is often associated with several acute and chronic diseases. NO donors and especially S-nitrosothiols such as S-nitrosoglutathione (GSNO) have been identified as promising therapeutic agents. Although their permeability through the intestinal barrier have recently be proved, suitable drug delivery systems have to be designed for their oral administration. This is especially challenging due to the physico-chemical features of these drugs: high hydrophilicity and high lability. In this paper, three types of particles were prepared with an Eudragit® polymer: nanoparticles and microparticles obtained with a water-in-oil-in-water emulsion/evaporation process versus microparticles obtained with a solid-in-oil-in-water emulsion/evaporation process. They had a similar encapsulation efficiency (around 30%), and could be freeze-dried then be stored at least one month without modification of their critical attributes (size and GSNO content). However, microparticles had a slightly slower in vitro release of GSNO than nanoparticles, and were able to boost by a factor of two the drug intestinal permeability (Caco-2 model). Altogether, this study brings new data about GSNO intestinal permeability and three ready-to-use formulations suitable for further preclinical studies with oral administration.

Regulation of IL-10 and IL-17 mediated experimental autoimmune encephalomyelitis by S-nitrosoglutathione

In this study, we investigated IL-10 and IL-17 specific immunomodulatory potential of S-nitrosoglutathione (GSNO), a physiological nitric oxide carrier molecule, in experimental autoimmune encephalomyelitis (EAE). In active EAE model, GSNO treatment attenuated EAE severity and splenic CD4+ T cells isolated from these mice exhibited decreased IL-17 expression without affecting the IFN-γ expression compared to the cells from untreated EAE mice. Similarly, adoptive transfer of these cells to nave mice resulted in reduction in IL-17 expression in the spinal cords of recipient mice with milder EAE severity. CD4+ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, still expressed lower levels of IL-17 under TH17 skewing conditions, but expressed similar levels of IFN-γ under TH1 skewing condition. Interestingly, under both TH17 and TH1 skewing condition, CD4+ T cells isolated from GSNO treated EAE mice, as compared to untreated EAE mice, expressed higher levels of IL-10 and adoptive transfer of these TH17 and TH1 skewed cells seemingly exhibited milder EAE disease. In addition, adoptive transfer of CD4+ T cells from GSNO treated EAE mice to active EAE mice also ameliorated EAE disease with induction of spinal cord expression of IL-10 and reduction in of IL-17, thus suggesting the participation of IL-10 mechanism in GSNO mediated immunomodulation. GSNO treatment of mice passively immunized with CD4+ T cells either from GSNO treated EAE mice or untreated mice further ameliorated EAE disease, supporting efficacy of GSNO for prophylaxis and therapy in EAE. Overall, these data document a modulatory role of GSNO in IL-17/IL-10 axis of EAE and other autoimmune diseases.

Caveolin and Endothelial NO Signaling.

Pulmonary vascular diseases are associated with several factors including infection, cigarette smoking, abuse of dietary suppressants and drugs, prolonged exposure to high altitude, and other causes which in part induce significant oxidative stress resulting in endothelial cell injury, apoptosis, hyperproliferation, and vaso-occlusive disease. Maintenance of normal endothelial cell function is a critical role of endothelial nitric oxide synthase (eNOS) activity and physiologic nitric oxide (NO) signaling in the vascular wall. eNOS expression and activity is regulated by the membrane-associated scaffolding protein caveolin-1 (Cav-1), the main protein constituent of caveolae. This chapter summarizes the literature and highlights unanswered questions related to how inflammation-associated oxidative stress affects Cav-1 expression and regulatory functions, and how dysregulated eNOS enzymatic activity promotes endothelial dysfunction. Focus is given to how the conversion of eNOS from a NO-producing enzyme to a transient oxidant-generating system is associated twith Cav-1 depletion, endothelial cell injury, and pulmonary vascular diseases. Importantly, the vascular defects observed in absence of Cav-1 that give rise to injured or hyperproliferative endothelial cells and promote remodeled vasculature can be rescued by "re-coupling," inhibiting, or genetically deleting eNOS, supporting the notion that strict control of Cav-1 expression and eNOS activity and signaling is critical for maintaining pulmonary vascular homeostasis.

Effects of Physiological Nitric Oxide Donor on Oxidative Metabolism in Rat Blood.

The effects of exogenous dinitrosyl iron complexes with glutathione ligands on the parameters of oxidative metabolism in the rat blood are studied. The intensity of LPO, total antioxidant activity, and MDA level in the plasma are compared in rats receiving intraperitoneal injections of saline without and with dinitrosyl iron complexes in various concentrations (0.15, 0.30, 0.45, and 0.60 mM). Glutathione-containing dinitrosyl iron complexes are characterized by antioxidant effect that non-linearly depended of the dose with optimum at 0.30-0.45 mM.

Potential Role of Nitrobiolome in Autoimmune Disease: Efficacy of GSNO in EAE.

Abstract S-nitrosoglutathione (GSNO) is a physiological nitric oxide (NO) carrier molecule important in modulation of NO-signaling. Cellular GSNO homeostasis is maintained by its synthesis by nitric oxide synthase (NOS) as well as its catabolism by GSNO reductase (GSNOR). Treatment of EAE mice (wild-type /WT) with exogenous GSNO or GSNOR inhibitor (N6022) significantly alleviated the clinical disease and associated myelin damage. In addition, more profound alleviation of EAE disease was observed in mice deficient in GSNOR (GSNOR−/−). However, EAE disease was further exacerbated in mice deficient in inducible NOS (iNOS−/−) as compared to WT mice. As expected, the spleen levels of S-nitrosothiol proteins were higher in GSNOR−/− EAE mice but lower in iNOS−/− EAE mice compared to WT EAE mice indicating a negative correlation of spleen GSNO levels with EAE disease severities. Both GSNO and N6022 treated EAE mice had decreased TH17 and increased CD4+/CD25+/FOXP3− Treg differentiations without any significant alterations in other CD4+ T cell subset differentiation. Accordingly, CD4 T+ cells from GSNO or N6022 treated EAE mice produced lower amount of IL-17 and higher amount of IL-10 compared to the cells from untreated WT EAE mice. GSNOR−/− EAE mice also exhibited decreased T 17 and increased CD4+/CD25+/FOXP3− Treg differentiations. On the other hand, iNOS−/− EAE mice exhibited increased TH17, but decreased Tregs differentiations compared to WT EAE mice. In summary, these studies document that nitrobiolome (GSNO) participates in differentiation of autoreactive TH17 and Treg subsets during the course of EAE, and that GSNO as a potential therapeutic drug for multiple sclerosis. Supported in-part by VA(BX002829) and NIH(NS037766).

Can supplemental nitrate in cured meats be used as a means of increasing residual and dietary nitrate and subsequent potential for physiological nitric oxide without affecting product properties?

The effects of formulated sodium nitrate plus supplemental nitrate (SN) from celery juice powder on residual nitrite, residual nitrate, rancidity, microbial growth, color, sensory properties, and proximate composition of frankfurters, cotto salami and boneless ham during storage (1°C) were studied. The products were assigned one of two treatments, which were each replicated twice: control (156ppm sodium nitrite) or SN (156ppm sodium nitrite and 1718ppm sodium nitrate in combination with 2% VegStable 502). Sensory parameters and proximate composition were measured once for each replication. All other analytical measurements were conducted at regular intervals for 97–98days. The SN showed no increase in residual nitrite compared to the control. No changes (P>0.05) were observed in residual nitrate during storage for any of the products. The results showed that addition of SN did not significantly alter most physical, chemical or microbial properties of cured meat products during refrigerated storage, but some product dependent sensory effects were observed.

Curcumin and folic acid abrogated methotrexate induced vascular endothelial dysfunction.

Methotrexate, an antifolate drug widely used in rheumatoid arthritis, psoriasis, and cancer, is known to cause vascular endothelial dysfunction by causing hyperhomocysteinemia, direct injury to endothelium or by increasing the oxidative stress (raising levels of 7,8-dihydrobiopterin). Curcumin is a naturally occurring polyphenol with strong antioxidant and anti-inflammatory action and therapeutic spectra similar to that of methotrexate. This study was performed to evaluate the effects of curcumin on methotrexate induced vascular endothelial dysfunction and also compare its effect with that produced by folic acid (0.072 μg·g(-1)·day(-1), p.o., 2 weeks) per se and in combination. Male Wistar rats were exposed to methotrexate (0.35 mg·kg(-1)·day(-1), i.p.) for 2 weeks to induce endothelial dysfunction. Methotrexate exposure led to shedding of endothelium, decreased vascular reactivity, increased oxidative stress, decreased serum nitrite levels, and increase in aortic collagen deposition. Curcumin (200 mg·kg(-1)·day(-1) and 400 mg·kg(-1)·day(-1), p.o.) for 4 weeks prevented the increase in oxidative stress, decrease in serum nitrite, aortic collagen deposition, and also vascular reactivity. The effects were comparable with those produced by folic acid therapy. The study shows that curcumin, when concomitantly administered with methotrexate, abrogated its vascular side effects by preventing an increase in oxidative stress and abating any reduction in physiological nitric oxide levels.

Consistent antioxidant and antihypertensive effects of oral sodium nitrite in DOCA-salt hypertension

Hypertension is a common disease that includes oxidative stress as a major feature, and oxidative stress impairs physiological nitric oxide (NO) activity promoting cardiovascular pathophysiological mechanisms. While inorganic nitrite and nitrate are now recognized as relevant sources of NO after their bioactivation by enzymatic and non-enzymatic pathways, thus lowering blood pressure, mounting evidence suggests that sodium nitrite also exerts antioxidant effects. Here we show for the first time that sodium nitrite exerts consistent systemic and vascular antioxidant and antihypertensive effects in the deoxycorticosterone-salt (DOCA-salt) hypertension model. This is particularly important because increased oxidative stress plays a major role in the DOCA-salt hypertension model, which is less dependent on activation of the renin-angiotensin system than other hypertension models. Indeed, antihypertensive effects of oral nitrite were associated with increased plasma nitrite and nitrate concentrations, and completely blunted hypertension-induced increases in plasma 8-isoprostane and lipid peroxide levels, in vascular reactive oxygen species, in vascular NADPH oxidase activity, and in vascular xanthine oxidoreductase activity. Together, these findings provide evidence that the oral administration of sodium nitrite consistently decreases the blood pressure in association with major antioxidant effects in experimental hypertension.

Nitrite as Direct S-Nitrosylating Agent of Kir2.1 Channels.

Nitrite, a physiological nitric oxide (NO) storage form and an alternative way for NO generation, affects numerous biological processes through NO-dependent and independent pathways, including the S-nitrosylation of thiol-containing proteins. Mechanisms underlying these phenomena are not fully understood. The purpose of this study was to analyse in the rat heart (as prototype of mammalian heart) whether nitrite affects S-nitrosylation of cardiac proteins and the potential targets for S-nitrosylation. Rat hearts, perfused according to Langendorff, were exposed to nitrite. By Biotin Switch Method, we showed that nitrite treatment increased the degree of S-nitrosylation of a broad range of membrane proteins. Further analysis, conducted on subfractioned proteins, allowed us to identify a high level of nitrosylation in a small range of plasmalemmal proteins characterized by using an anti-Kir2.1 rabbit polyclonal antibody. We also verified that this effect of nitrite is preserved in the presence of the NO scavenger PTIO (2-phenyl-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide). Our results suggest, for the first time, that nitrite represents a direct S-nitrosylating agent in cardiac tissues and that inward-rectifier potassium ion channels (Kir2.1) are one of the targets. These observations are of relevance since they support the growing evidence that nitrite is not only a NO reserve but also a direct modulator of important functional cardiac proteins.

Preclinical Therapeutic Potential of a Nitrosylating Agent in the Treatment of Ovarian Cancer

This study examines the role of s-nitrosylation in the growth of ovarian cancer using cell culture based and in vivo approaches. Using the nitrosylating agent, S-nitrosoglutathione (GSNO), a physiological nitric oxide molecule, we show that GSNO treatment inhibited proliferation of chemoresponsive and chemoresistant ovarian cancer cell lines (A2780, C200, SKVO3, ID8, OVCAR3, OVCAR4, OVCAR5, OVCAR7, OVCAR8, OVCAR10, PE01 and PE04) in a dose dependent manner. GSNO treatment abrogated growth factor (HB-EGF) induced signal transduction including phosphorylation of Akt, p42/44 and STAT3, which are known to play critical roles in ovarian cancer growth and progression. To examine the therapeutic potential of GSNO in vivo, nude mice bearing intra-peritoneal xenografts of human A2780 ovarian carcinoma cell line (2×106) were orally administered GSNO at the dose of 1 mg/kg body weight. Daily oral administration of GSNO significantly attenuated tumor mass (p<0.001) in the peritoneal cavity compared to vehicle (phosphate buffered saline) treated group at 4 weeks. GSNO also potentiated cisplatin mediated tumor toxicity in an A2780 ovarian carcinoma nude mouse model. GSNO’s nitrosylating ability was reflected in the induced nitrosylation of various known proteins including NFκB p65, Akt and EGFR. As a novel finding, we observed that GSNO also induced nitrosylation with inverse relationship at tyrosine 705 phosphorylation of STAT3, an established player in chemoresistance and cell proliferation in ovarian cancer and in cancer in general. Overall, our study underlines the significance of S-nitrosylation of key cancer promoting proteins in modulating ovarian cancer and proposes the therapeutic potential of nitrosylating agents (like GSNO) for the treatment of ovarian cancer alone or in combination with chemotherapeutic drugs.

Abstract 1474: Hepatocytes either radioprotect or sensitize colorectal cancer cells through nitric oxide-induced oxygen sparing in hypoxic microenvironment

Abstract Purpose: Impaired tumor oxygenation remains a major obstacle to curative radiotherapy in advanced primary tumors and metastases. In this study, tailored to the liver metastases of colorectal cancer (CRC), we compared the ability of tumor cells and normal host hepatocytes to metabolically consume oxygen and induce hypoxia. Next, we examined the possibility to re-oxygenate and thereby radiosensitize human CRC cells through the physiological mediator nitric oxide (NO) that switches the mitochondrial oxygen metabolism in hepatocytes from consumption to sparing. Methods and Materials: To model tumor-relevant hypoxia, hepatocytes and tumor cells were incubated in a tissue-mimetic co-culture system with limited oxygenation, which was monitored by fluorescence and linked to hypoxic radioresponse. To activate NO production through inducible nitric oxide synthase (iNOS), hepatocytes (or tumor cells) were exposed to a cytokine mixture of IFN-γ, TNF-α, IL-1α and LPS, and iNOS expression was examined by RT-PCR, western blotting, and NO/nitrite production. Results: Primary mouse hepatocytes consumed 10 to 40-times more oxygen than mouse CT26 and human DLD-1, HT29, HCT116 and SW480 CRC cells, suggesting that hepatocytes are the major effectors of hypoxic conditioning. At cell densities of 0.6-1.2 mln/ml, hepatocytes induced radiobiologically-relevant hypoxia within 20 min, and thereby radioprotected tumor cells 2.0 to 2.5-times. Following exposure to cytokines, hepatocytes revealed significant activation of iNOS-mediated production of NO that targeted mitochondrial aconitase and complexes II, and induced a profound respiratory self-arrest at up to 90 min. As a result, the spared oxygen caused uniform re-oxygenation and radiosensitization in a panel of CRC cell lines (by 1.9 to 2.7-times). Next, both oxygen sparing and radiosensitization could be counteracted by aminoguanidine, a metabolic iNOS inhibitor. Contrasting, all CRC cell lines showed little if any iNOS expression and therefore could not contribute to the oxygen-sparing and radiosensitizing effects of NO-producing hepatocytes. Finally, the NO donor S-nitroso-N-acetylpenicillamine and the mitochondrial inhibitors rotenone, antimycine A and sodium azide were able to block the mitochondrial respiratory chain and to reverse both metabolic hypoxia and hypoxia-induced radioresistance. Conclusion: Hepatocytes may profoundly modulate the hypoxic microenvironment and either protect or sensitize CRC cells to radiation, dependent on the mitochondrial respiratory switch. Our study for the first time provides evidence that host liver cells possess intrinsic radiosensitizing potential through NO-induced oxygen sparing. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1474. doi:1538-7445.AM2012-1474

Modeling the Effect of Oxygen on the Amperometric Response of Immobilized Organoselenium-Based S-Nitrosothiol Sensors

Amperometric detection of S-nitrosothiols (RSNOs) at submicromolar levels in blood samples is of potential importance for monitoring endothelial function and other disease states that involve changes in physiological nitric oxide (NO) production. It is shown here that the elimination of dissolved oxygen from samples is critical when covalently attached diselenocystamine-based amperometric RSNO sensors are used for practical RSNO measurements. The newest generation of RSNO sensors utilizes an amperometric NO gas sensor with a thin organoselenium modified dialysis membrane mounted at the distal sensing tip. Sample RSNOs are catalytically reduced to NO within the dialysis membrane by the immobilized organoselenium species. In the presence of oxygen, the sensitivity of these sensors for measuring low levels of RSNOs (<μM) is greatly reduced. It is demonstrated that the main scavenger of the generated nitric oxide is not the dissolved oxygen but rather superoxide anion radical generated from the reaction of the reduced organoselenium species (the reactive species in the catalytic redox cycle) and dissolved oxygen. Computer simulations of the response of the RSNO sensor using rate constants and diffusion coefficients for the reactions involved, known from the literature or estimated from fitting to the observed amperometric response curves, as well as the specific geometric dimensions of the RSNO sensor, further support that nitric oxide and superoxide anion radical quickly react resulting in near zero sensor sensitivity toward RSNO concentrations in the submicromolar concentration range. Elimination of oxygen from samples helps improve sensor detection limits to ca. 10 nM levels of RSNOs.

Neisseria meningitidis induces platelet inhibition and increases vascular endothelial permeability via nitric oxide regulated pathways

Despite antibiotic therapy, infections with Neisseria meningitidis still demonstrate a high rate of morbidity and mortality even in developed countries. The fulminant septicaemic course, named Waterhouse-Friderichsen syndrome, with massive haemorrhage into the adrenal glands and widespread petechial bleeding suggest pathophysiological inhibition of platelet function. Our data show that N. meningitidis produces the important physiological platelet inhibitor and cardiovascular signalling molecule nitric oxide (NO), also known as endothelium-derived relaxing factor (EDRF). N. meningitidis -derived NO inhibited ADP-induced platelet aggregation through the activation of soluble guanylyl cyclase (sGC) followed by an increase in platelet cyclic nucleotide levels and subsequent activation of platelet cGMP- and cAMP- dependent protein kinases (PKG and PKA). Furthermore, direct measurement of horseradish peroxidase (HRP) passage through a vascular endothelial cell monolayer revealed that N. meningitidis significantly increased endothelial monolayer permeability. Immunfluorescence analysis demonstrated NO dependent disturbances in the structure of endothelial adherens junctions after co-incubation with N. meningitidis . In contrast to platelet inhibition, the NO effects on HBMEC were not mediated by cyclic nucleotides. Our study provides evidence that NO plays an essential role in the pathophysiology of septicaemic meningococcal infection.

Prodrug design for the potent cardiovascular agent Nω-hydroxy-l-arginine (NOHA): Synthetic approaches and physicochemical characterization

N(ω)-Hydroxy-L-arginine (NOHA)--the physiological nitric oxide precursor--is the intermediate of NO synthase (NOS) catalysis. Besides the important fact of releasing NO mainly at the NOS-side of action, NOHA also represents a potent inhibitor of arginases, making it an ideal therapeutic tool to treat cardiovascular diseases that are associated with endothelial dysfunction. Here, we describe an approach to impart NOHA drug-like properties, particularly by wrapping up the chemically and metabolically instable N-hydroxyguanidine moiety with different prodrug groups. We present synthetic routes that deliver several more or less highly substituted NOHA derivatives in excellent yields. Versatile prodrug strategies were realized, including novel concepts of bioactivation. Prodrug candidates were primarily investigated regarding their hydrolytic and oxidative stabilities. Within the scope of this work, we essentially present the first prodrug approaches for an interesting pharmacophoric moiety, i.e., N-hydroxyguanidine.

Redox Regulation of the NPR1-TGA1 System of Arabidopsis thaliana by Nitric Oxide

The role of reactive oxygen and nitrogen species in local and systemic defense reactions is well documented. NPR1 and TGA1 are key redox-controlled regulators of systemic acquired resistance in plants. NPR1 monomers interact with the reduced form of TGA1, which targets the activation sequence-1 (as-1) element of the promoter region of defense proteins. Here, we report the effect of the physiological nitric oxide donor S-nitrosoglutathione on the NPR1/TGA1 regulation system in Arabidopsis thaliana. Using the biotin switch method, we demonstrate that both NPR1 and TGA1 are S-nitrosylated after treatment with S-nitrosoglutathione. Mass spectrometry analyses revealed that the Cys residues 260 and 266 of TGA1 are S-nitrosylated and S-glutathionylated even at GSNO concentrations in the low micromolar range. Furthermore, we showed that S-nitrosoglutathione protects TGA1 from oxygen-mediated modifications and enhances the DNA binding activity of TGA1 to the as-1 element in the presence of NPR1. In addition, we observed that the translocation of NPR1 into the nucleus is promoted by nitric oxide. Taken together, our results suggest that nitric oxide is a redox regulator of the NPR1/TGA1 system and that they underline the importance of nitric oxide in the plant defense response.

S-nitrosoglutathione a physiologic nitric oxide carrier attenuates experimental autoimmune encephalomyelitis.

S-nitrosoglutathione (GSNO) is a physiological nitric oxide molecule which regulates biological activities of target proteins via s-nitrosylation leading to attenuation of chronic inflammation. In this study we evaluated the therapeutic efficacy of GSNO in experimental autoimmune encephalomyelitis (EAE), an animal model of multiple sclerosis. Oral administration of GSNO (0.5 or 1.0 mg/kg) reduced disease progression in chronic models (SJL and C57BL/6) of EAE induced with PLP((139-151)) or MOG((35-55)) peptides, respectively. GSNO attenuated EAE disease by reducing the production of IL17 (from Th(i) or Th17 cells) and the infiltration of CD4 T cells into the central nervous system without affecting the levels of Th1 (IFN gamma) and Th2 (IL4) immune responses. Inhibition of IL17 was observed in T cells under normal as well as Th17 skewed conditions. In vitro studies showed that the phosphorylation of STAT3 and expression of ROR gamma, key regulators of IL17 signaling, were reduced while phosphorylation of STAT4 or STAT6 and expression of T-bet or GATA3 remained unaffected, suggesting that GSNO preferentially targets Th17 cells. Collectively, GSNO attenuated EAE via modulation of Th17 cells and its effects are independent of Th1 or Th2 cells functions, indicating that it may have therapeutic potential for Th17-mediated autoimmune diseases.