Selenium nanoparticles as catalysts for nitric oxide generation.

The nitric oxide prodrug, V-PYRRO/NO, protects against cadmium toxicity and apoptosis at the cellular level

Nitroso-caged upconversion luminescent prodrug: Near infrared light-activatable NO nano-donor for gas therapy

The metal–organic framework (MOF) CuBTTri, H3[(Cu4Cl)3(BTTri)8] (where H3BTTri = 1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene), is a promising catalyst for the development of antithrombotic medical device materials via localized nitric oxide (NO) generation from endogenous S-nitrosothiols. This work evaluates the effects of three key parameters of CuBTTri-embedded polyurethane composite materials—MOF preparation/particle size, MOF loading, and polymer concentration—on the rate of NO generation. We discovered that CuBTTri preparation and particle size have a significant impact on NO generation. Specifically, hand-ground MOF particles (0.3 ± 0.1 µm diameter) generate NO at greater rates compared to larger as-prepared, raw MOF particles (0.4 ± 0.2 µm diameter) and smaller, filtered MOF particles (0.2 ± 0.1 µm diameter) for composite materials. This finding contradicts previous research for CuBTTri powder which found that the smaller the particles, the greater the catalytic rate. In examining the effects of MOF loading and polymer concentration, our data show that increasing these parameters generally results in increased rates of NO generation; though thresholds appear to exist in which increasing these parameters results in diminishing returns and impedes NO generation capacity for certain composite formulations. We found that polymer concentration is the key determinant of water absorptivity and statistically significant decreases in water uptake accompany statistically significant increases in NO generation. It was also found that formulations with relatively high MOF loadings and low polymer concentrations or low MOF loadings and high polymer concentrations inhibit the rate of NO generation. In summary, this research provides a framework for more strategic selections of key parameters when fabricating composite materials for medical device applications.

BackgroundPrevious studies have suggested that peroxisome proliferator activated receptor-gamma (PPAR-γ)-mediated neuroprotection involves inhibition of microglial activation and decreased expression and activity of inducible nitric oxide synthase (iNOS); however, the underlying molecular mechanisms have not yet been well established. In the present study we explored: (1) the effect of the PPAR-γ agonist pioglitazone on lipopolysaccharide (LPS)-induced iNOS activity and nitric oxide (NO) generation by microglia; (2) the differential role of p38 mitogen-activated protein kinase (p38 MAPK), c-Jun NH(2)-terminal kinase (JNK), and phosphoinositide 3-kinase (PI3K) on LPS-induced NO generation; and (3) the regulation of p38 MAPK, JNK, and PI3K by pioglitazone.MethodsMesencephalic neuron-microglia mixed cultures, and microglia-enriched cultures were treated with pioglitazone and/or LPS. The protein levels of iNOS, p38 MAPK, JNK, PPAR-γ, PI3K, and protein kinase B (Akt) were measured by western blot. Different specific inhibitors of iNOS, p38MAPK, JNK, PI3K, and Akt were used in our experiment, and NO generation was measured using a nitrite oxide assay kit. Tyrosine hydroxylase (TH)-positive neurons were counted in mesencephalic neuron-microglia mixed cultures.ResultsOur results showed that pioglitazone inhibits LPS-induced iNOS expression and NO generation, and inhibition of iNOS is sufficient to protect dopaminergic neurons against LPS insult. In addition, inhibition of p38 MAPK, but not JNK, prevented LPS-induced NO generation. Further, and of interest, pioglitazone inhibited LPS-induced phosphorylation of p38 MAPK. Wortmannin, a specific PI3K inhibitor, enhanced p38 MAPK phosphorylation upon LPS stimulation of microglia. Elevations of phosphorylated PPAR-γ, PI3K, and Akt levels were observed with pioglitazone treatment, and inhibition of PI3K activity enhanced LPS-induced NO production. Furthermore, wortmannin prevented the inhibitory effect of pioglitazone on the LPS-induced NO increase.ConclusionWe demonstrate that pioglitazone protects dopaminergic neurons against LPS insult at least via inhibiting iNOS expression and NO generation, which is potentially mediated via inhibition of p38 MAPK activity. In addition, the PI3K pathway actively participates in the negative regulation of LPS-induced NO production. Our findings suggest that PPAR-γ activation may involve differential regulation of p38 MAPK and of the PI3K/Akt pathway in the regulation of the inflammatory process.

Effect of yeast saccharide treatment on nitric oxide accumulation and chilling injury in cucumber fruit during cold storage

Ca(2+) rise and nitric oxide (NO) generation are essential early steps in plant innate immunity and initiate the hypersensitive response (HR) to avirulent pathogens. Previous work from this laboratory has demonstrated that a loss-of-function mutation of an Arabidopsis (Arabidopsis thaliana) plasma membrane Ca(2+)-permeable inwardly conducting ion channel impairs HR and that this phenotype could be rescued by the application of a NO donor. At present, the mechanism linking cytosolic Ca(2+) rise to NO generation during pathogen response signaling in plants is still unclear. Animal nitric oxide synthase (NOS) activation is Ca(2+)/calmodulin (CaM) dependent. Here, we present biochemical and genetic evidence consistent with a similar regulatory mechanism in plants: a pathogen-induced Ca(2+) signal leads to CaM and/or a CaM-like protein (CML) activation of NOS. In wild-type Arabidopsis plants, the use of a CaM antagonist prevents NO generation and the HR. Application of a CaM antagonist does not prevent pathogen-induced cytosolic Ca(2+) elevation, excluding the possibility of CaM acting upstream from Ca(2+). The CaM antagonist and Ca(2+) chelation abolish NO generation in wild-type Arabidopsis leaf protein extracts as well, suggesting that plant NOS activity is Ca(2+)/CaM dependent in vitro. The CaM-like protein CML24 has been previously associated with NO-related phenotypes in Arabidopsis. Here, we find that innate immune response phenotypes (HR and [avirulent] pathogen-induced NO elevation in leaves) are inhibited in loss-of-function cml24-4 mutant plants. Pathogen-associated molecular pattern-mediated NO generation in cells of cml24-4 mutants is impaired as well. Our work suggests that the initial pathogen recognition signal of Ca(2+) influx into the cytosol activates CaM and/or a CML, which then acts to induce downstream NO synthesis as intermediary steps in a pathogen perception signaling cascade, leading to innate immune responses, including the HR.

This study determines the release of nitric oxide (NO) from the coronary circulation of Langendorff hearts of rabbits, subsequent to administration of glyceryl trinitrate (GTN) and SIN-1. NO was measured on-line in the coronary effluent by the oxyhaemoglobin technique. Infusion of either GTN (10-40 mumoles/l) or SIN-1 (0.1-2.3 mumoles/l) into the coronary inflow resulted in a concentration-dependent NO release into the coronary effluent and a decrease in the coronary vascular resistance. NO generation from SIN-1 was identical with and without passage of the coronary circulation whereas NO generation from GTN was only detected after passage of the coronary vascular bed. NO generation by both substances was in the same range as endogenous NO release by two endothelium-dependent vasodilators, bradykinin (0.05 mumoles/l) and substance P (0.05 mumoles/l). Oxyhaemoglobin used for the assay of NO, inhibited the relaxation by SIN-1, but did not reduce vessel relaxations induced by GTN or iloprost, a stable prostacyclin analogue. Removal of the coronary endothelium by trypsin or pretreatment with L-NG-Monomethylarginine (30 mumoles/l) did neither affect NO release from GTN and SIN-1 nor the vasodilatory effect of both substances. These data are the first to directly demonstrate endothelium-independent NO release from organic nitrates during passage of an intact organ circulation. They additionally suggest a subendothelial site of metabolic NO formation from GTN.

The influence of Twinline (SNN-6010), an elemental diet containing medium-chain triglycerides, on the generation of nitric oxide (NO) and superoxide (O2.-) has been examined in mouse peritoneal macrophages and polymorphonuclear leukocytes (PMN). When PMN and peritoneal macrophages obtained from untreated mice were cultured in medium containing 0.1% and 1% (v/v) Twinline for 48h and stimulated with phorbol myristate acetate or N-formyl-methionyl-leucyl-phenylalanine, their chemiluminescence and O2.- generation were strongly suppressed, as was NO generation from peritoneal macrophages. PMN and peritoneal macrophages obtained from mice fed Twinline for 30 days generated much smaller amounts of 02.- and NO compared with PMN and peritoneal macrophages from control mice. In conjunction with this suppressed NO generation, inducible NO synthase and its mRNA expression in peritoneal macrophages were suppressed by Twinline both in-vivo and ex-vivo. Although phagocytosis of PMN and peritoneal macrophages was not suppressed by Twinline; their candida-killing activity was markedly suppressed. These results indicate that Twinline suppresses the host-defence function of PMN and peritoneal macrophages by down-regulating their generation of reactive-oxygen intermediates and NO.

This paper reported an efficient method to significantly reduce nitrous oxide (N(2)O) and nitric oxide (NO) generation in anaerobic-aerobic (low dissolved oxygen) processes. It was found that by the use of waste-activated sludge alkaline fermentation liquid as the synthetic wastewater-carbon source, compared with the commonly used carbon source in the literature (e.g., acetic acid), the generation of N(2)O and NO was reduced by 68.7% and 50.0%, respectively, but the removal efficiencies of total phosphorus (TP) and total nitrogen (TN) were improved. Both N(2)O and NO were produced in the low dissolved oxygen (DO) stage, and the use of sludge fermentation liquid greatly reduced their generation from the denitrification. The presences of Cu(2+) and propionic acid in fermentation liquid were observed to play an important role in the reduction of N(2)O and NO generation. The analysis of the activities of denitrifying enzymes suggested that sludge fermentation liquid caused the significant decrease of both nitrite reductase activity to NO reductase activity ratio and NO reductase activity to N(2)O reductase activity ratio, which resulted in the lower generation of NO and N(2)O. Fluorescence in situ hybridization analysis indicated that the number of glycogen accumulating bacteria, which was reported to be relevant to nitrous oxide generation, in sludge fermentation liquid reactor was much lower than that in acetic acid reactor. The quantitative detection of the nosZ gene, encoding nitrous oxide reductase, showed that the use of fermentation liquid increased the number of bacteria capable of reducing N(2)O to N(2). The feasibility of using sludge fermentation liquid to reduce NO and N(2)O generation in an anaerobic-low DO process was finally confirmed for a municipal wastewater.

During three decades, an enormous number of studies have demonstrated the critical role of nitric oxide (NO) as a second messenger engaged in the activation of many systems including vascular smooth muscle relaxation. The underlying cellular mechanisms involved in vasodilatation are essentially due to soluble guanylyl-cyclase (sGC) modulation in the cytoplasm of vascular smooth cells. sGC activation culminates in cyclic GMP (cGMP) production, which in turn leads to protein kinase G (PKG) activation. NO binds to the sGC heme moiety, thereby activating this enzyme. Activation of the NO-sGC-cGMP-PKG pathway entails Ca(2+) signaling reduction and vasodilatation. Endothelium dysfunction leads to decreased production or bioavailability of endogenous NO that could contribute to vascular diseases. Nitrosyl ruthenium complexes have been studied as a new class of NO donors with potential therapeutic use in order to supply the NO deficiency. In this context, this article shall provide a brief review of the effects exerted by the NO that is enzymatically produced via endothelial NO-synthase (eNOS) activation and by the NO released from NO donor compounds in the vascular smooth muscle cells on both conduit and resistance arteries, as well as veins. In addition, the involvement of the nitrite molecule as an endogenous NO reservoir engaged in vasodilatation will be described.

This study investigated coronary perfusion pressure, nitric oxide (NO) and superoxide production, nitrotyrosine (NT) formation, and cardiac cell apoptosis in isolated hearts perfused with high glucose concentration. Coronary perfusion pressure; NO and superoxide anion generation; immunostaining for NT, inducible NO synthase (iNOS), and the constitutive type of NO synthase (NOS) eNOS; iNOS and eNOS mRNA expression by Western blot and RT-PCR; and apoptosis of cardiac cells were studied in hearts perfused for 2 h with solutions containing D-glucose at a concentration of 11.1 mmol/l (control), D-glucose at the concentration of 33.3 mmol/l (high glucose), or D-glucose (33.3 mmol/l) plus glutathione (0.3 mmol/l). Perfusion of isolated hearts in conditions of high glucose concentration caused a significant increase of coronary perfusion pressure (P < 0.001) and an increase of both NO and superoxide generation. However, superoxide production was 300% higher than baseline, whereas NO production was 40% higher (P < 0.001 for both). This effect was accompanied by the formation of NT, and an increase of iNOS expression. eNOS remained unchanged. At the end of the experiments, cardiac cell apoptosis was evident in hearts perfused with high glucose. The effects of high glucose were significantly prevented by glutathione. This study demonstrates that high glucose for 2 h is enough to increase iNOS gene expression and NO release in working rat hearts. Upregulation of iNOS and raised NO generation are accompanied by a marked concomitant increase of superoxide production, a condition favoring the production of peroxynitrite, a powerful pro-oxidant that can mediate the toxic effects of high glucose on heart by itself and/or via the formation of nitrotyrosine, as suggested by the detection of cell apoptosis.

UV-B irradiation induced production of secondary metabolites in plant cells. However, the mechanisms of UV-B-induced secondary metabolite production remained largely unknown. Here we report that UV-B treatment stimulated nitric oxide (NO) generation and camptothecin (CPT) production in Camptotheca acuminata cells. To investigate the origin of the UV-B-triggered NO and the role of NO in UV-B-induced CPT production, we assayed the responses of nitrate reductase (NR) and NO synthase (NOS) activities of the cells to UV-B exposure and examined the effects of NR and NOS inhibitors on CPT production in UV-B-treated cells. The data showed that UV-B irradiation enhanced NR activities in the cells. Pretreatment with NR inhibitors tungstate and okadaic acid not only suppressed the UV-B-triggered NR activities but also inhibited the UV-B-induced NO generation and CPT production in the cells. In contrast, UV-B irradiation had no effects on NOS activity of the cells and treatment of NOS inhibitor did not suppress UV-B-induced CAT production. Together, the results demonstrated that NR activity was essential for UV-B-triggered NO generation and that NR-mediated NO signaling was involved in UV-B-induced CPT production in C. acuminata cells.

Nitric oxide (NO)-based cancer therapy has attracted much attention in recent years owing to its broad effects on cancer. Low concentrations of NO stimulate cancer cell progression, while its higher levels induce cell apoptosis, and thus, it has motivated the development of probes for in situ NO release monitoring. In this work, a galactose-modified benzothiadiazole-based fluorescent probe (GalNONP/C) was synthesized as both a NO-responsive nanoprobe and NO prodrug carrier. The probe exhibited far-red emission in the range from 550 to 800 nm, and the response showed acidity preference. The galactose on the probe enabled selective targeting of hepatocellular carcinoma (HCC) cells by binding to the asialoglycoprotein receptor (ASGPR) on the cell surface. The probe also delivered low-molecular weight NO prodrug JS-K into cells and monitored the real-time release of the generated NO. Furthermore, in vivo NO imaging with tumor targeting was demonstrated in HCC orthotopic transplantation nude mice and liver sections. Compared with the control experiment using a probe without NO prodrug loading, higher fluorescence response of NO was detected in the cell (3.0 times) and liver slices of the HCC tumor model (2.7 times). This strategy may pave the way to develop nanoprobes for in situ NO monitoring and therapy evaluation in NO-related cancer therapy.

Continuous monitoring of cellular nitric oxide generation by spin trapping with an iron-dithiocarbamate complex

Aim Postoperative ileus (POI) is one of the most common complications after abdominal surgery, especially subsequent to intra-abdominal infection. The underlying mechanism remains unclear. This study is to investigate the role of HIF1α in the progression of POI. Methods Bioinformatics analysis was utilized to analyze the association between HIF1α and POI. A murine model with POI was generated by surgical intestinal manipulation (IM). Gastrointestinal transit (GIT), leukocyte infiltration and inflammatory mediators were measured. Targeting HIF1α with specific inhibitor (2ME2) was performed in both in vivo and in vitro experiment. Human samples were collected for further validation. Results Public RNA sequencing data demonstrated upregulation of HIF1α in murine intestinal muscularis after POI generation. This was confirmed by our murine models as well as human samples, which was accompanied by impaired GIT, higher inflammatory cytokines, inducible nitric oxide synthetase (iNOS) expression and nitric oxide (NO) generation. HIF1α-specific inhibitor 2ME2 reversed inflammatory response in macrophages, suppressed NO production, and eventually ameliorated GI motility. Conclusions HIF1α controls inflammatory response and NO generation in intestinal muscularis, Targeting HIF1α effectively improves GI motility and ameliorates POI development.Fig. 1Gastrointestinal motility injury and inflammatory cell infiltration in the intestinal muscularis after IM-induced POIa Gross pictures of the representational photo in the Sham and IM groups. b FITC-dextran distribution in the intestinal tract of mice 24 h after IM and the genomic center (GC) of FITC-dextran distribution in each experiment group. c Typical HE staining images in each group (×200, ×400). d Typical immunohistochemical staining images with MPO (×200, ×400). Fig. 2 Identification of differentially expressed genes (DEGs) in the GEO dataset GSE134942 a Volcano plot of DEGs. Log2|Foldchange| &amp;gt;1 and P &amp;lt; 0.05 were set as the cutoff values. The red plots represent upregulated DEGs, while the blue plots represent downregulated DEGs. b The top 20 GSEA pathways based on the adjusted P-values. (c–f) Representative results of GSEA analysis of the IM24h group genes. Fig. 3The upregulation of HIF1α was validated in animal models of postoperative ileusa HE staining of the whole layer of the small intestine and the small intestinal muscle layer after the elimination of mucosa. b Representative Western blotting images probed for HIF1α and iNOS extracted from the small intestinal muscularis. c Quantitative RT-PCR analysis of HIF1α, iNOS, IL1β, and COX-2 mRNA levels in murine intestinal muscularis. d The immunofluorescence images of the sham-operated and IM mice intestine. Fig. 4 Validation of HIF1α upregulation in the clinical specimens of patients with intestinal obstruction a Representative Western blotting images probed for HIF1α and iNOS extracted from muscularis in surgically resected intestinal segments. The control group represents the normal intestinal segments of gastric cancer patients that require gastrojejunostomy and small bowel resection. The ileus group represents ischemia intestinal segments from intestinal obstruction patients who underwent bowel anastomosis after the resection of the necrotic bowel. b Quantitative RT-PCR analysis of the HIF1α, iNOS, IL1β, IL6, and COX-2 mRNA levels in human intestinal muscularis. c The immunofluorescence images of the control and ileus clinical intestinal muscularis specimens. Fig. 5Intraperitoneal injection of HIF1α-specific inhibitor 2ME2 relieved gastrointestinal motility injury in vivoa A schematic diagram of the experiment in vivo. b, c FITC-dextran distribution in the intestinal tract of mice 1 day after IM and genomic center (GC) of FITC-dextran distribution in each experiment group. d Representative Western blotting images probed for HIF1α, iNOS, and COX2 extracted from the small intestinal muscularis. e Total NO extracted from the intestinal muscularis homogenate. Fig. 6 2ME2-induced alleviation of the inflammatory cytokines’ expression and NO generation in macrophages a Representative Western blotting images probed for HIF1α, iNOS, and COX2 extracted from 2ME2 treated-RAW264.7 cells subjected to LPS stimulation or hypoxia culture. b Total NO in the cell culture supernatant.