Stimulation Of Nitric Oxide Production Research Articles

EFFICACY OF COSUPPLEMENTATION THERAPY WITH VITAMINS B9, B12, AND D ON ENDOTHELIAL DYSFUNCTION IN STREPTOZOTOCIN-INDUCED DIABETIC RATS

Objective: This study evaluated the effects of Vitamins D, B9, and B12 given individually or combined in ameliorating some biochemical parameters related to endothelial dysfunction in diabetic rats.Methods: A total of 50 Sprague-Dawley male rats were divided into five groups: Control, diabetic, diabetic received Vitamin D, diabetic received Vitamins B9 and B12, and diabetic received Vitamins B9, B12, and D. At the end of 6 weeks, the rats were sacrificed and a set of assays was carried out to determine: Fasting blood sugar (FBS), lipid profile, nitric oxide (NO), homocysteine (Hcy), malondialdehyde (MDA), and serum levels of Vitamins B9, B12, and D.Results: Diabetic rat received Vitamin D and diabetic rat received Vitamins B9 and B12 had a significant decline in the levels of FBS, lipid profile, and Hcy with reduced MDA (p<0.05) release but significant increase in NO level. On the same hand, diabetic rat received combined supplementation of Vitamins B9, B12, and D had more pronounced effect (p<0.00).Conclusion: Given these findings, the combined vitamins therapy had antiatherosclerotic effects by inhibiting lipid peroxidation and stimulating NO production, resulting in amelioration the endothelial dysfunction in diabetic rat.

Nitric oxide precursors prevent Al-triggered auxin flow inhibition in Triticum aestivum roots

Aluminum (Al) is an element widely distributed in soils, even though Al3+ is one of the most detrimental cations to plant growth. The effect of nitric oxide (NO) precursors on indole-3-acetic acid (IAA) flow towards roots upon Al treatment is herein reported using two Triticum aestivum (wheat) cultivars with recognized differential Al tolerance. Roots of Al-tolerant seedlings with no treatment (control) accumulated higher amounts of NO than Al-sensitive ones. The treatment with Al further stimulated NO production in root cells while root exposure to NO3−, L-arginine (Arg) or the NO donor S-nitrosoglutathione (GSNO) decreased both Al and lipid peroxide accumulation in both cultivars. Regardless of the cultivar, NO3−, Arg or GSNO prevented the blockage of IAA flow towards roots. Overall, the treatment of wheat roots with NO precursors prior to Al treatment effectively guarantees normal IAA flow towards roots, a condition that favors the organ’s growth and development.

Vasoprotective Effects of Vildagliptin in Mice Is Masked by Metformin—Overlapping Mechanisms Implied

Preclinical and small clinical studies have demonstrated the direct cardiovascular protective effects of dipeptidyl peptidase 4 inhibitors (DPP-4Is). However, cardiovascular benefits have not been proven in large clinical trials that utilized multiple antidiabetic agents. We hypothesized that the cardiovascular protective effects of the first-line antidiabetic agent metformin (Met) affects those of DPP-4Is, so we evaluated the vasoprotective effects of vildagliptin (Vil) in combination with Met in mice. Methods: We randomly assigned C57BL6 and diabetic db/db mice (8 weeks old) to treatment with vehicle, Vil (3 mg/kg/d), Met (400 mg/kg/d), or the combination of Vil and Met. The mice were subjected to femoral artery wire injury to induce arterial remodeling. Four weeks after the injury, the femoral arteries were collected. Human umbilical vein endothelial cells (HUVECs) were used in the in vitro experiments. Results: In wild type mice, the biological parameters were similar among the groups. Both Vil and Met treatment reduced neointimal area and vascular cell proliferation to a similar extent (40% reduction) compared to that of the vehicle, and combining the two drugs did not further reduce those values. Similarly, both Vil and Met treatment reduced neointimal area in db/db mice (50% reduction) without affecting biological parameters, but no additive effect was found. In wild type mice, cotreatment with the nitric oxide (NO) synthase inhibitor completely abolished the vascular effects of both Vil and Met, suggesting that Vil and Met had common mechanisms that are dependent on endothelial NO. In HUVECs, Vil did not directly affect NO production. In contrast, glucagon like peptide (GLP)-1 did stimulate NO production. Met also stimulated NO production. However, the combination of GLP-1 and Met did not increase NO production. Conclusions: Metformin masks the vasoprotective effects of vildagliptin, possibly because of overlapping mechanisms that are dependent on endothelial NO. Disclosure H. Kushima: None. Y. Mori: None. M. Koshibu: None. M. Hiromura: None. K. Kohashi: None. M. Terasaki: None. T. Hirano: Speaker's Bureau; Self; Novo Nordisk Inc., AstraZeneca.

Activation of the β-common receptor by erythropoietin impairs acetylcholine-mediated vasodilation in mouse mesenteric arterioles.

Clinically, erythropoietin (EPO) is known to increase systemic vascular resistance and arterial blood pressure. However, EPO stimulates the production of the potent vasodilator, nitric oxide (NO), in culture endothelial cells. The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to activate two receptor complexes, the homodimeric EPO (EPOR 2) and the heterodimeric EPOR/β‐common receptor (β CR). The purpose of this study was to investigate the contribution of each receptor to the vasoactive properties of EPO. First‐order, mesenteric arteries were isolated from 16‐week‐old male C57BL/6 mice, and arterial function was studied in pressure arteriographs. To determine the contribution of each receptor complex, EPO‐stimulating peptide (ESP), which binds and activates the heterodimeric EPOR/β CR complex, and EPO, which activates both receptors, were added to the arteriograph chamber 20 min prior to evaluation of endothelium‐dependent (acetylcholine, bradykinin, A23187) and endothelium‐independent (sodium nitroprusside) vasodilator responses. Only ACh‐induced vasodilation was impaired in arteries pretreated with EPO or ESP. EPO and ESP pretreatment abolished ACh‐induced vasodilation by 100% and 60%, respectively. EPO and ESP did not affect endothelium‐independent vasodilation by SNP. Additionally, a novel β CR inhibitory peptide (β IP), which was computationally developed, prevented the impairment of acetylcholine‐induced vasodilation by EPO and ESP, further implicating the EPOR/β CR complex. Last, pretreatment with either EPO or ESP did not affect vasoconstriction by phenylephrine and KCl. Taken together, these findings suggest that acute activation of the heterodimeric EPOR/β CR in endothelial cells leads to a selective impairment of ACh‐mediated vasodilator response in mouse mesenteric resistance arteries.

Abstract 288: Nitric Oxide Mediates Active Downregulation of Tissue Factor Expression in Human Pericytes

Tissue Factor (TF) is a transmembrane protein that not only expresses pro-coagulant enzymatic activity, but also mediates signal transduction by several second messenger pathways. It plays an obvious role in hemostasis and thrombosis, as well as modulating angiogenesis, inflammation, immune responses and progression of malignancy and metastasis. TF is normally expressed by a limited subset of cell types. It can also be induced on other cells by a plethora of mediators, mostly linked to inflammation and immune responses. By contrast, TF has only been shown to be actively downregulated in a single circumstance: we have reported that constitutive expression of TF by pericytes around dermal vessels disappears during angiogenesis associated with wound healing. This suggests that downregulation of pericyte TF may play a functional role in the process of angiogenesis. We previously reported that TF protein in cultured human pericytes is significantly reduced 8 hours after treatment with phorbol 12-myristate 13-acetate (PMA) (94.71% reduction + 2.18%, p<0.001). This loss of TF protein is facilitated by reduction of TF mRNA. Downregulation of TF protein and mRNA depends on Protein Kinase C. The goal of the current study was to identify a physiologic mediator of pericyte TF downregulation. Treatment of human pericytes with growth factors and cytokines involved in wound healing did not change TF expression. Co-culture of human pericytes with human microvascular endothelial cells in transwells in the presence of basic fibroblast growth factor (bFGF) resulted in downregulation of pericyte TF. However, conditioned media from endothelial cells grown in the presence of bFGF did not trigger a decrease of TF in pericytes. bFGF has been shown to stimulate NO production, and both bFGF and NO have been linked to angiogenesis. This led us to consider nitric oxide (NO) as a potential labile mediator of TF downregulation. TF protein was reduced 8 hours after treatment with DETA/NO (44% reduction + 6.1%, p<0.01). However, expression of TF mRNA was not reduced at this time, as it is during culture with PMA. Our working hypothesis is that TF downregulation in pericytes is partially mediated by endothelial cell-derived NO, but is also mediated by additional PKC-dependent pathways.

Necrotic amplification loop in acute pancreatitis: pancreatic stellate cells and nitric oxide are important players in the development of the disease

Necrotic amplification loop in acute pancreatitis: pancreatic stellate cells and nitric oxide are important players in the development of the disease

The Role of Quinine-Responsive Taste Receptor Family 2 in Airway Immune Defense and Chronic Rhinosinusitis.

Bitter (T2R) and sweet (T1R) taste receptors in the airway are important in innate immune defense, and variations in taste receptor functionality in one T2R (T2R38) correlate with disease status and disease severity in chronic rhinosinusitis (CRS). Quinine is a bitter compound that is an agonist for several T2Rs also expressed on sinonasal cells, but not for T2R38. Because of this property, quinine may stimulate innate immune defense mechanisms in the airway, and functional differences in quinine perception may be reflective of disease status in CRS. Demographic and taste intensity data were collected prospectively from CRS patients and non-CRS control subjects. Sinonasal tissue from patients undergoing rhinologic surgery was also collected and grown at an air-liquid interface (ALI). Nitric oxide (NO) production and dynamic regulation of ciliary beat frequency in response to quinine stimulation were assessed in vitro. Quinine reliably increased ciliary beat frequency and NO production in ALI cultures in a manner consistent with T2R activation (p < 0.01). Quinine taste intensity rating was performed in 328 CRS patients and 287 control subjects demonstrating that CRS with nasal polyps (CRSwNP) patients rated quinine as significantly less intense than did control subjects. Quinine stimulates airway innate immune defenses by increasing ciliary beat frequency and stimulating NO production in a manner fitting with T2R activation. Patient variability in quinine sensitivity is observed in taste intensity ratings, and gustatory quinine "insensitivity" is associated with CRSwNP status. Thus, taste tests for quinine may be a biomarker for CRSwNP, and topical quinine has therapeutic potential as a stimulant of innate defenses.

Regulation of inducible nitric oxide synthase by arabinoxylans with molecular characterisation from wheat flour in cultured human monocytes

SummaryThe immunomodulatory activity of the arabinoxylans (AXs) extracts from cereal sources has been reported to impart health benefits in terms of immune enhancement. This study investigated the effect of enzymatic extraction on extraction yield and structure of AXs from wheat flour pentosan fraction. Under the optimised conditions, the extraction yield of AXs reached up to 81.25%. Furthermore, the study determined whether water‐extracted AXs (WEAXs) and enzyme‐extracted AXs (E‐WEAXs) from wheat flour were able to differentially stimulate nitric oxide (NO) secretion through increased levels of inducible nitric oxide synthase (iNOS) in human U937 monocytes. The results indicated that AXs concomitantly induced (P &lt; 0.05) both NO and iNOS productions in U937 monocytes compared to untreated cells. Compared with WEAXs, E‐WEAXs resulted in a higher proportion of low Mw (1–10 KDa) AXs (49.51% vs. 19.11% in WEAXs), a higher A/X ratio (0.83 vs. 0.48 in WEAXs) and a higher yield (12.83 ± 0.35% vs. 7.54 ± 0.47% in WEAXs). Moreover, E‐WEAXs induced significantly (P &lt; 0.05) greater NO and iNOS production per million viable cells (61.8 ± 2.7 μm and 42.41 ± 3.83 ng respectively) than WEAXs (51.6 ± 2.6 μm and 33.46 ± 1.48 ng, respectively). The findings suggest AXs may heighten innate immune activity in the absence of infection or disease through an iNOS‐mediated stimulation of NO production. The immunomodulatory activity of the wheat‐derived AXs was enhanced by enzyme treatment, with low Mw and high A/X ratio associated with elevated NO/iNOS levels in human monocytes compared to water extraction.

Abstract P500: Activation of the Heterodimeric Erythropoietin /β-Common Receptor Impairs Acetylcholine Mediated Vasodilation in Mouse Mesenteric Arterioles

Erythropoietin (EPO) increases systemic vascular resistance and blood pressure. However, endothelial cells cultured in the presence of EPO demonstrate increased production of the potent vasodilator, nitric oxide (NO). The mechanism by which EPO causes vasoconstriction despite stimulating NO production may be dependent on its ability to differentially activate the two receptor complexes, the homodimeric EPO (EPOR 2 ) and the heterodimeric EPOR/β-common receptor (βCR). Objective: The purpose of this study was to investigate the contribution of the EPOR 2 and βCR receptor to the vasoactive properties of EPO. Methods: First order, mesenteric arteries isolated from 16-week old male C57BL/6 mice were cannulated and perfused using a pressure arteriography system. To determine the contribution of each receptor complex, arteries were incubated with EPO stimulating peptide (ESP) which binds and activates only the heterodimeric EPOR/βCR complex or EPO which activates both receptors, 20 min prior to evaluation of vasoconstrictor (phenylephrine and potassium chloride), endothelium-dependent (acetylcholine, bradykinin, A23187) and -independent (sodium nitroprusside) vasodilator responses. Additionally, we studied the effect of a novel βCR inhibitory peptide (βIP) which was developed in silico and validated by demonstrating that it selectively inhibits binding of ligands to the βCR. Results: Acetylcholine induced vasodilation was impaired in arteries pretreated with EPO or ESP by 100% and 60%, respectively. EPO and ESP did not affect endothelium-dependent vasodilation by Bradykinin or A23187, endothelium-independent vasodilation by sodium nitroprusside, or vasoconstriction by phenylephrine and KCl. The βIP prevented the impairment of acetylcholine-induced vasodilation by EPO and ESP. Conclusion: Together, our findings suggest that activation of the heterodimeric EPOR/βCR leads to selective impairment of ACh-mediated vasodilator response in mouse mesenteric resistance arteries. Thus the βCR might have a role in mediating hypertensive effects of EPO. Therapeutic inhibition of the βCR might prevent vascular complications of EPO without affecting erythropoiesis.

Metabolites of flavonoid compounds preserve indices of endothelial cell nitric oxide bioavailability under glucotoxic conditions

We hypothesized that metabolites of dietary flavonoids attenuate impairments in nitric oxide (NO) bioavailability evoked by glucotoxic conditions mimicking Type 1 or 2 diabetes. To test this, human aortic endothelial cells were treated with either vehicle control, quercetin-3-O-glucoronide, piceatannol or 3-(3-hydroxyphenyl)propionoic acid for 24 h. These are metabolites of quercetin, resveratrol and proanthocyanidin, respectively. Next, cells were exposed to control (5 mM) or high (25 mM) glucose conditions for 48 h, followed by insulin treatment (100 nM, 10 min) to stimulate NO production. In control glucose conditions NO production, phosphorylated to total endothelial nitric oxide synthase (p-eNOSser1177: eNOS), and phosphorylated to total Akt (p-AktSer473: Akt) were all increased by insulin stimulation. This response was abolished during high glucose conditions. Pretreatment of cells with flavonoid metabolites prior to high glucose challenge preserved insulin stimulated increases in NO production, p-AktSer473: Akt and p-eNOSSer1177: eNOS. These effects may be secondary to oxidative stress as pretreatment with all flavonoid metabolites prevented elevations in reactive oxygen and nitrogen species in response to high glucose. These data support the hypothesis that beneficial effects of flavonoids on endothelial cell function in the context of glucotoxicity, at least in part, are secondary to their metabolites.

Nitric-oxide generation induced by metals plays a role in their accumulation by Phallusia nigra hemocytes

Ascidians are good monitors for assessing water quality, since they filter large volumes of water; however, little is known about how xenobiotics, including metals, can affect ascidian hemocytes. Metals can be either toxic or beneficial to health, inducing many different responses. The response mechanism depends on the class of metals to which organisms are exposed: essential, nonessential, and borderline. To analyze the influence of metals from different classes on the protective mechanisms of an ascidian, we investigated the production of nitric oxide (NO) after exposure to various concentrations of Mg, Mn and Pb over different time periods. We also determined the amounts of each metal in the hemocytes. Our results indicated that especially Pb could stimulate NO production. Although Pb induced the highest NO production, cell viability was not severely altered in all Pb concentrations and time periods. Ascidians might serve as biomonitor for Pb, since their vanadocytes accumulate Pb.

Neuronal nitric oxide synthase mediates insulin- and oxidative stress-induced glucose uptake in skeletal muscle myotubes

Previously published studies strongly suggested that insulin- and exercise-induced skeletal muscle glucose uptake require nitric oxide (NO) production. However, the signal transduction mechanisms by which insulin and contraction regulated NO production and subsequent glucose transport are not known. In the present study, we utilized the myotube cell lines treated with insulin or hydrogen peroxide, the latter to mimic contraction-induced oxidative stress, to characterize these mechanisms. We found that insulin stimulation of neuronal nitric oxide synthase (nNOS) phosphorylation, NO production, and GLUT4 translocation were all significantly reduced by inhibition of either nNOS or Akt2. Hydrogen peroxide (H2O2) induced phosphorylation of nNOS at the same residue as did insulin, and also stimulated NO production and GLUT4 translocation. nNOS inhibition prevented H2O2-induced GLUT4 translocation. AMP activated protein kinase (AMPK) inhibition prevented H2O2 activation and phosphorylation of nNOS, leading to reduced NO production and significantly attenuated GLUT4 translocation. We conclude that nNOS phosphorylation and subsequently increased NO production are required for both insulin- and H2O2-stimulated glucose transport. Although the two stimuli result in phosphorylation of the same residue on nNOS, they do so through distinct protein kinases. Thus, insulin and H2O2-activated signaling pathways converge on nNOS, which is a common mediator of glucose uptake in both pathways. However, the fact that different kinases are utilized provides a basis for the use of exercise to activate glucose transport in the face of insulin resistance.

Time Window Is Important for Adenosine Preventing Cold-induced Injury to the Endothelium.

Cold cardioplegia is used to induce heart arrest during cardiac surgery. However, endothelial function may be compromised after this procedure. Accordingly, interventions such as adenosine, that mimic the effects of preconditioning, may minimize endothelial injury. Herein, we investigated whether adenosine prevents cold-induced injury to the endothelium. Cultured human cardiac microvascular endothelial cells were treated with adenosine for different durations. Phosphorylation and expression of endothelial nitric oxide synthase (eNOS), p38MAPK, ERK1/2, and p70S6K6 were measured along with nitric oxide (NO) production using diaminofluorescein-2 diacetate (DAF-2DA) probe. Cold-induced injury by hypothermia to 4°C for 45 minutes to mimic conditions of cold cardioplegia during open heart surgery was induced in human cardiac microvascular endothelial cells. Under basal conditions, adenosine stimulated NO production, eNOS phosphorylation at serine 1177 from 5 minutes to 4 hours and inhibited eNOS phosphorylation at threonine 495 from 5 minutes to 6 hours, but increased phosphorylation of ERK1/2, p38MAPK, and p70S6K only after exposure for 5 minutes. Cold-induced injury inhibited NO production and the phosphorylation of the different enzymes. Importantly, adenosine prevented these effects of hypothermic injury. Our data demonstrated that adenosine prevents hypothermic injury to the endothelium by activating ERK1/2, eNOS, p70S6K, and p38MAPK signaling pathways at early time points. These findings also indicated that 5 minutes after administration of adenosine or release of adenosine is an important time window for cardioprotection during cardiac surgery.

Synthesis and biological activity of new phthalimides as potential anti-inflammatory agents

The overproduction of nitric oxide (NO) plays an important role in a variety of pathophysiological processes, including inflammation. Therefore, the suppression of NO production is a promising target in the design of anti-inflammatory agents. In the present study, a series of phthalimide analogs was synthesized, and their anti-inflammatory activities were evaluated using lipopolysaccharide (LPS)-stimulated NO production in cultured murine macrophage RAW264.7 cells. A structure-activity relationship study showed that the free hydroxyl group at C-4 and C-6 and the bulkiness of the N-substituted alkyl chain are associated with biological activity. Among the series of phthalimide derivatives, compound IIh exhibited potent inhibitory activity, with an IC50 value of 8.7µg/mL. Further study revealed that the inhibitory activity of compound IIh was correlated with the down-regulation of the mRNA and protein expression of LPS-stimulated inducible nitric oxide synthase (iNOS). Compound IIh also suppressed the induction of the pro-inflammatory cytokines tumor necrosis factor-α and interleukin-1β in LPS-stimulated RAW 264.7 cells. The anti-inflammatory activity of compound IIh was also found to be associated with the suppression of the Toll-like receptor (TLR)4 signaling pathway by down-regulating the activation of interferon regulatory factor 3 (IRF-3) and interferon-β and signal transducer expression. These findings demonstrate that novel phthalimides might be potential candidates for the development of anti-inflammatory agents.

Effect of nitric oxide on renal autoregulation during hypothermia in the rat

Hypothermia-induced reduction of metabolic rate is accompanied by depression of both glomerular perfusion and filtration. The present study investigated whether these changes are linked to changes in renal autoregulation and nitric oxide (NO) signalling. During hypothermia, renal blood flow (RBF) and glomerular filtration rate (GFR) were reduced and urine production was increased, and this was linked with reduced plasma cGMP levels and increased renal vascular resistance. Although stimulation of NO production decreased vascular resistance, blood pressure and urine flow, intravenous infusion of the NO precursor L-arginine or the NO donor sodium nitroprusside did not alter RBF or GFR. In contrast, inhibition of NO synthesis by Nw-nitro-L-arginine led to a further decline in both parameters. Functional renal autoregulation was apparent at both temperatures. Below the autoregulatory range, RBF in both cases increased in proportion to the perfusion ±pressure, although, the slope of the first ascending limb of the pressure-flow relationship was lower during hypothermia. The main difference was rather that the curves obtained during hypothermia levelled off already at a RBF of 3.9 ± 0.3 mL/min then remained stable throughout the autoregulatory pressure range, compared to 7.6 ± 0.3 mL/min during normothermia. This was found to be due to a threefold increase in, primarily, the afferent arteriolar resistance from 2.6 to 7.5 mmHg min mL−1. Infusion of sodium nitroprusside did not significantly affect RBF during hypothermia, although a small increase at pressures below the autoregulatory range was observed. In conclusion, cold-induced rise in renal vascular resistance results from afferent arteriolar vasoconstriction by the autoregulatory mechanism, setting RBF and GFR in proportion to the metabolic rate, which cannot be explained by reduced NO production alone.

Immunostimulated Arginase II Expression in Intestinal Epithelial Cells Reduces Nitric Oxide Production and Apoptosis

Increased production of nitric oxide (NO) and subsequent local cytotoxicity to mucosal epithelial cells has been proposed as a putative mechanism involved in the development of necrotizing enterocolitis (NEC). Intestinal epithelial cells (IECs) metabolize L-arginine to either nitric oxide (NO) by NO synthase (NOS) or to L-ornithine and urea by arginase. L-ornithine is the first step in polyamine synthesis important for cell proliferation, while NO production can lead to apoptosis. We hypothesized that in IECs immunostimulation increases both NOS and arginase expression, and that arginase activity mitigates NO production and apoptosis. Rat intestinal epithelial cells (rIEC-6) were immunostimulated by either incubation with lipopolysaccharide (LPS) alone for 24 h or by incubation with conditioned media (CM) for 24 h. CM was obtained from RAW 264.7 cells (a macrophage cell line) treated with LPS (E. coli 0127:B8; 1 μg/ml) for 4 h. The rIEC-6 stimulated with LPS or with CM had significantly higher levels of inducible NOS (iNOS) protein, NO production, and arginase II protein than did the control cells. Direct LPS stimulation of rIEC-6 produced a less robust increase in iNOS expression and NO (represented as nitrite percent of control) than did CM stimulation. Inhibition of arginase using Nω hydroxyl-L-arginine (NOHA) further increased stimulated NO production in rIEC-6. Viable cell numbers were significantly lower in CM stimulated cells after 24 h than in controls, and inhibition of arginase activity with NOHA resulted in a further significant decrease in viable cell numbers. We conclude that immunostimulated arginase expression of rIEC-6 cells tempers cytokine-induced iNOS-derived NO production and apoptosis.

Nitric oxide induced by polyamines involves antioxidant systems against chilling stress in tomato (Lycopersicon esculentum Mill.) seedling.

Polyamines (PAs) and nitric oxide (NO) are vital signals in modulating plant response to abiotic stress. However, to our knowledge, studies on the relationship between NO and PAs in response to cold stress in tomato are limited. Accordingly, in this study, we investigated the effects of putrescine (Put) and spermidine (Spd) on NO generation and the function of Spd-induced NO in the tolerance of tomato seedling under chilling stress. Spd increased NO release via the nitric oxide synthase (NOS)-like and nitrate reductase (NR) enzymatic pathways in the seedlings, whereas Put had no such effect. Moreover, H2O2 might act as an upstream signal to stimulate NO production. Both exogenous NO donor (sodium nitroprusside (SNP)) and Spd enhanced chilling tolerance in tomato, thereby protecting the photosynthetic system from damage. Compared to chilling treatment alone, Spd enhanced the gene expressions of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), and their enzyme activities in tomato leaves. However, a scavenger or inhibitor of NO abolished Spd-induced chilling tolerance and blocked the increased expression and activity due to Spd of these antioxidant enzymes in tomato leaves under chilling stress. The results showed that NO induced by Spd plays a crucial role in tomato's response to chilling stress.

Aged garlic extract exerts endothelium-dependent vasorelaxant effect on rat aorta by increasing nitric oxide production

BackgroundClinical trials have shown that aged garlic extract (AGE) is effective in reducing blood pressure of hypertensive patients. However, the mechanisms involved remain to be elucidated. PurposeThe aim of the present study was to investigate the vasorelaxant effect of AGE on the aorta and its mechanism of action in order to clarify the blood pressure-lowering action of AGE. MethodsThe vasorelaxant effect was evaluated in isolated rat aortic rings. After aortic rings were contracted by 3 × 10−6M norepinephrine (NE) for 30min, AGE and other test drugs were added to the aortic rings. All results were expressed as percentages of the maximal NE-induced contraction. ResultsAGE induced the concentration–dependent vasorelaxation of isolated rat aortic rings that had been precontracted with norepinephrine. The effect of AGE was severely impaired in aortic rings lacking endothelium. In addition, the effect of AGE was inhibited by a nitric oxide synthase (NOS) inhibitor and a nitric oxide (NO) scavenger. Moreover, AGE treatment of aorta significantly increased the NO production. When various constituents of AGE were tested, the vasorelaxation of aorta was observed only in the presence of L-arginine, a substrate of NOS. ConclusionAGE causes endothelium-dependent vasorelaxation of aorta via stimulation of NO production and that L-arginine in AGE serves as a key agent for NOS-mediated NO production.

Nitric oxide and cytokine production by glial cells exposed in vitro to neuropathogenic schistosome Trichobilharzia regenti.

BackgroundHelminth neuroinfections represent a serious health problem, but host immune mechanisms in the nervous tissue often remain undiscovered. This study aims at in vitro characterization of the response of murine astrocytes and microglia exposed to Trichobilharzia regenti which is a neuropathogenic schistosome migrating through the central nervous system of vertebrate hosts. Trichobilharzia regenti infects birds and mammals in which it may cause severe neuromotor impairment. This study was focused on astrocytes and microglia as these are immunocompetent cells of the nervous tissue and their activation was recently observed in T. regenti-infected mice.ResultsPrimary astrocytes and microglia were exposed to several stimulants of T. regenti origin. Living schistosomulum-like stages caused increased secretion of IL-6 in astrocyte cultures, but no changes in nitric oxide (NO) production were noticed. Nevertheless, elevated parasite mortality was observed in these cultures. Soluble fraction of the homogenate from schistosomulum-like stages stimulated NO production by both astrocytes and microglia, and IL-6 and TNF-α secretion in astrocyte cultures. Similarly, recombinant cathepsins B1.1 and B2 triggered IL-6 and TNF-α release in astrocyte and microglia cultures, and NO production in astrocyte cultures. Stimulants had no effect on production of anti-inflammatory cytokines IL-10 or TGF-β1.ConclusionsBoth astrocytes and microglia are capable of production of NO and proinflammatory cytokines IL-6 and TNF-α following in vitro exposure to various stimulants of T. regenti origin. Astrocytes might be involved in triggering the tissue inflammation in the early phase of T. regenti infection and are proposed to participate in destruction of migrating schistosomula. However, NO is not the major factor responsible for parasite damage. Both astrocytes and microglia can be responsible for the nervous tissue pathology and maintaining the ongoing inflammation since they are a source of NO and proinflammatory cytokines which are released after exposure to parasite antigens.Electronic supplementary materialThe online version of this article (doi:10.1186/s13071-016-1869-7) contains supplementary material, which is available to authorized users.

Secondary Metabolites of the Endophytic Fungus Lachnum abnorme from Ardisia cornudentata.

Fractionation of an EtOAc-soluble fraction of the solid fermentate of an endophytic fungus, Lachnum abnorme Mont. BCRC 09F0006, derived from the endemic plant, Ardisia cornudentata Mez. (Myrsinaceae), resulted in the isolation of three new chromones, lachnochromonins D–F (1–3), one novel compound, lachabnormic acid (4), along with nine known compounds (5–13). Their structures were elucidated by spectroscopic analyses. Alternariol-3,9-dimethyl ether (6) was given the correct data as well as 2D spectral analyses for the first time. This is the first report of the isolation of one unprecedented compound 4 from Lachnum genus, while all known compounds were also found for the first time from Lachnum. The effects of some isolates (3, 4, 7–9, 10, and 13) on the inhibition of nitric oxide (NO) production in lipopolysaccharide (LPS)-activated RAW 264.7 murine macrophages were also evaluated. Several compounds exhibited weak inhibitory activity on lipopolysaccharide (LPS)-stimulated NO production in RAW 264.7 macrophages.