Enhanced Nitric Oxide Production Research Articles

Vascular endothelial cell morphology and alignment regulate VEGF-induced endothelial nitric oxide synthase activation.

Nitric oxide (NO) production by endothelial nitric oxide synthase (eNOS) inhibits platelet and leukocyte adhesion while promoting vasorelaxation in smooth muscle cells. Dysfunctional regulation of eNOS is a hallmark of various vascular pathologies, notably atherosclerosis, often associated with areas of low shear stress on endothelial cells (ECs). While the link between EC morphology and local hemodynamics is acknowledged, the specific impact of EC morphology on eNOS regulation remains unclear. Morphological differences between elongated, aligned ECs and polygonal, randomly oriented ECs correspond to variations in focal adhesion and cytoskeletal organization, suggesting differing levels of cytoskeletal prestress. However, the functional outcomes of cytoskeletal prestress, particularly in the absence of shear stress, are not extensively studied in ECs. Some evidence suggests that elongated ECs exhibit decreased immunogenicity and enhanced NO production. This study aims to elucidate the signaling pathways governing VEGF-stimulated eNOS regulation in the aligned EC phenotype characterized by elongated and aligned cells within a monolayer. Using anisotropic topographic cues, bovine aortic endothelial cells (BAECs) were elongated and aligned, followed by VEGF treatment in the presence or absence of cytoskeletal tension inhibitors. Phosphorylation of eNOS ser1179, AKT ser437 and FAK Tyr397 in response to VEGF challenge were significantly heightened in aligned ECs compared to unaligned ECs. Moreover this response proved to be robustly tied to cytoskeletal tension as evinced by the abrogation of responses in the presence of the myosin II ATPase inhibitor, blebbistatin. Notably, this work demonstrates for the first time the reliance on FAK phosphorylation in VEGF-mediated eNOS activation and the comparatively greater contribution of the cytoskeletal machinery in propagating VEGF-eNOS signaling in aligned and elongated ECs. This research underscores the importance of utilizing appropriate vascular models in drug development and sheds light on potential mechanisms underlying vascular function and pathology that can help inform vascular graft design.

Extracellular ATP- and adenosine-mediated purinergic signaling modulates inducible nitric oxide synthase (iNOS) gene expression, enzyme activity and nitric oxide production in common carp (Cyprinus carpio) head kidney macrophages

Inducible nitric oxide (NO) synthase (iNOS) is a key immune mediator for production of inflammatory mediator NO from l-arginine. Tight regulation of iNOS expression and enzyme activity is critical for proper NO productions under inflammation and infection conditions. However, the regulatory mechanism for iNOS expression and enzyme activity in fish remains largely unknown. Here, we show that extracellular ATP treatment significantly up-regulates iNOS gene expression and enzyme activity, and consequently leads to enhanced NO production in Cyprinus carpio head kidney macrophages (HKMs). We further show that the extracellular ATP-induced iNOS enzyme activity and NO production can be attenuated by pharmacological inhibition of the ATP-gated P2X4 and P2X7 receptors with their respective specific antagonists, but enhanced by overexpression of P2X4 and P2X7 receptors in grass carp ovary cells. In contrast, adenosine administration significantly reduces iNOS gene expression, enzyme activity and NO production in carp HKMs, and these inhibitory effects can be reversed by pharmacological inhibition of adenosine receptors with the antagonist XAC. Furthermore, LPS- and poly(I:C)-induced iNOS gene expression, enzyme activity, and NO production are significantly attenuated by blockade of P2X4 and P2X7 receptors with their respective specific antagonists in carp HKMs, while overexpression of P2X and P2X7 receptors results in enhanced iNOS gene expression, enzyme activity and NO production in LPS- and poly(I:C)-treated grass carp ovary cells. Taken together, we firstly report an opposite role of extracellular ATP/adenosine-mediated purinergic signaling in modulating iNOS-NO system activity in fish.

Co-release of nitric oxide and L-arginine from poly (β-amino ester)-based adhesive reprogram macrophages for accelerated wound healing and angiogenesis in vitro and in vivo

Recently, insufficient angiogenesis and prolonged inflammation are crucial challenges of chronic skin wound healing. The sustained release of L-Arginine (L-Arg) and nitric oxide (NO) production can control immune responses, improve angiogenesis, enhance re-epithelialization, and accelerate wound healing. Here, we aim to improve wound healing via the controlled release of NO and L-Arg from poly (β-amino ester) (PβAE). In this regard, PβAE is functionalized with methacrylate poly-L-Arg (PAMA), and the role of PAMA content (50, 66, and 75 wt%) on the adhesive properties, L-Arg, and NO release, as well as collagen deposition, inflammatory responses, and angiogenesis, is investigated in vitro and in vivo. Results show that the PAMA/ PβAE could provide suitable adhesive strength (~25 kPa) for wound healing application. In addition, increasing the PAMA content from 50 to 75 wt% results in an increased release of L-Arg (approximately 1.4–1.7 times) and enhanced NO production (approximately 2 times), promoting skin cell proliferation and migration. The in vitro studies also show that compared to PβAE hydrogel, incorporation of 66 wt% PAMA (PAMA 66 sample) reveals superior collagen I synthesis (~ 3–4 times) of fibroblasts, controlled pro-inflammatory and improved anti-inflammatory cytokines secretion of macrophages, and accelerated angiogenesis (~1.5–2 times). In vivo studies in a rat model with a full-thickness skin defect also demonstrate the PAMA66 sample could accelerate wound healing (~98 %) and angiogenesis, compared to control (untreated wound) and Tegaderm™ commercial wound dressing. In summary, the engineered multifunctional PAMA functionalized PβAE hydrogel with desired NO and L-Arg release, and adhesive properties can potentially reprogram macrophages and accelerate skin healing for chronic wound healing.

Fenugreek extract improves diabetes-induced endothelial dysfunction via the arginase 1 pathway.

Endothelial dysfunction (ED) is an initiating trigger and key factor in vascular complications, leading to disability and mortality in individuals with diabetes. The research concerning therapeutic interventions for ED has gained considerable interest. Fenugreek, a commonly used edible plant in dietary consumption, has attracted significant attention due to its management of diabetes and its associated complications. The research presented in this study examines the potential therapeutic benefits of fenugreek in treating ED and investigates the underlying mechanism associated with its effects. The analysis on fenugreek was performed using 70% ethanol extract, and its chemical composition was analyzed using ultrahigh-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry (UPLC-Q-TOF/MS). In total, we identified 49 compounds present in the fenugreek extract. These compounds encompass flavonoids, saponins, and phospholipids. Then, the models of ED in streptozotocin-induced diabetic mice and high glucose-induced isolated rat aortas were established for research. Through vascular function testing, it was observed that fenugreek extract effectively improved ED induced by diabetes or high glucose. By analyzing the protein expression of arginase 1 (Arg1), Arg activity, Arg1 immunohistochemistry, nitric oxide (NO) level, and the protein expression of endothelial nitric oxide synthase (eNOS), p38 mitogen-activated protein kinase (p38 MAPK), and p-p38 MAPK in aortas, this study revealed that the potential mechanism of fenugreek extract in anti-ED involves the downregulation of Arg1, leading to enhanced NO production. Furthermore, analysis of serum exosomes carrying Arg activity indicates that fenugreek may decrease the activity of Arg transported by serum exosomes, potentially preventing the increase in Arg levels triggered by the uptake of serum exosomes by vascular endothelial cells. In general, this investigation offers valuable observations regarding the curative impact of fenugreek extract on anti-ED in diabetes, revealing the involvement of the Arg1 pathway in its mechanism.

Disturbances in Nitric Oxide Cycle and Related Molecular Pathways in Clear Cell Renal Cell Carcinoma.

It is important to note that maintaining adequate levels of nitric oxide (NO), the turnover, and the oxidation level of nitrogen are essential for the optimal progression of cellular processes, and alterations in the NO cycle indicate a crucial step in the onset and progression of multiple diseases. Cellular accumulation of NO and reactive nitrogen species in many types of tumour cells is expressed by an increased susceptibility to oxidative stress in the tumour microenvironment. Clear cell renal cell carcinoma (ccRCC) is a progressive metabolic disease in which tumour cells can adapt to metabolic reprogramming to enhance NO production in the tumour space. Understanding the factors governing NO biosynthesis metabolites in ccRCC represents a relevant, valuable approach to studying NO-based anticancer therapy. Exploring the molecular processes mediated by NO, related disturbances in molecular pathways, and NO-mediated signalling pathways in ccRCC could have significant therapeutic implications in managing and treating this condition.

Isolation of Latilactobacillus curvatus with Enhanced Nitric Oxide Synthesis from Korean Traditional Fermented Food and Investigation of Its Probiotic Properties.

Nitric oxide (NO) is a free radical associated with physiological functions such as blood pressure regulation, cardiovascular health, mitochondrial production, calcium transport, oxidative stress, and skeletal muscle repair. This study aimed to isolate Latilactobacillus curvatus strains with enhanced NO production from the traditional Korean fermented food, jangajji, and evaluate their probiotic properties for industrial purposes. When cells were co-cultured with various bacterial stimulants, NO production generally increased, and NO synthesis was observed in the range of 20-40 mg/mL. The selected strains of Lat. curvatus were resistant to acid and bile conditions and with variable effectiveness (1-14%) in adhering to Caco-2 cells. Most bacterial strains can inhibit the growth of various pathogens. In addition, they are capable of reducing cholesterol levels via assimilation of cholesterol at 10-50%. Among the selected NO synthases from Lat. curvatus strains, the strain JBCC38 showed the highest capacity to scavenge ABTS (30.1%) and DPPH radicals (39.4%). Moreover, these strains exhibited immunomodulatory properties. The production of TNF-α and IL-6 in the macrophages treated with various bacterial stimulants was induced in all the selected strains.

Pharmacokinetic effect of Astragalus membranaceus and Panax notoginseng saponins on arginine absorption and nitric oxide production in healthy subjects

Background: To the best of our knowledge, there are no clinical trials conducted with Astragalus and ginseng extracts on nitric oxide (NO) levels in humans. Therefore, this study aimed to examine whether the standardized intake of Astragalus membranaceus and Panax notoginseng saponins (APS) could increase NO production by enhancing arginine absorption and reducing levels of asymmetric dimethylarginine (AMDA).Methods: A clinical trial involving healthy adult participants aged between 20 to 80 years was conducted as a randomized, double-blind, cross-over study. The participants received 5 g of arginine powder and one capsule of APS or placebo twice, with a wash-out period between each administration. Plasma and urine were collected for testing, and 24 subjects were included for analysis after excluding six subjects with great individual differences.Results:It was found that after APS supplementation, the area under the curve (AUC) of arginine significantly increased by 17.3% (p = 0.041), the maximum concentration (Cmax) increased by 11.1%, and the Arg/ADMA ratio significantly increased by 167.1% (p = 0.007). Moreover, urinary nitrate and cGMP levels increased by 20.8% and 18.9%, respectively.Conclusions: APS showed increases in arginine absorption, decrease ADMA levels, and enhance NO production. With these findings, the addition of APS to arginine supplements could be advantageous for pre-workout and cardiovascular health.Keywords:Astragalus membranaceus and Panax notoginseng saponins (APS), arginine, asymmetric dimethylarginine (ADMA), nitric oxide (NO), cyclic guanosine monophosphate (cGMP)Clinical trial registration: NCT05024123

Development of nitric oxide generators to produce high-dose nitric oxide for inhalation therapy

BackgroundSeveral nitric oxide (NO) generating devices have been developed to deliver NO between 1 part per million (ppm) and 80 ppm. Although inhalation of high-dose NO may exert antimicrobial effects, the feasibility and safety of producing high-dose (more than 100 ppm) NO remains to be established. In the current study, we designed, developed, and tested three high-dose NO generating devices. MethodsWe constructed three NO generating devices: a double spark plug NO generator, a high-pressure single spark plug NO generator, and a gliding arc NO generator. The NO and NO2 concentrations were measured at different gas flows and under various atmospheric pressures. The double spark plug NO generator was designed to deliver gas through an oxygenator and mixing with pure oxygen. The high-pressure and gliding arc NO generators were used to deliver gas through a ventilator into artificial lungs to mimic delivering high-dose NO in the clinical settings. The energy consumption was measured and compared among the three NO generators. ResultsThe double spark plug NO generator produced 200 ± 2 ppm (mean ± SD) of NO at gas flow of 8 L/min (or 320 ± 3 ppm at gas flow of 5 L/min) with electrode gap of 3 mm. The nitrogen dioxide (NO2) levels were below 3.0 ± 0.1 ppm when mixing with various volumes of pure oxygen. The addition of a second generator increased the delivered NO from 80 (with one spark plug) to 200 ppm. With the high-pressure chamber, the NO concentration reached 407 ± 3 ppm with continuous air flow at 5 L/min when employing the 3 mm electrode gap under 2.0 atmospheric pressure (ATA). When compared to 1 ATA, NO production was increased 22% at 1.5 ATA and 34% at 2 ATA. The NO level was 180 ± 1 ppm when connecting the device to a ventilator with a constant inspiratory airflow of 15 L/min, and NO2 levels were below 1 (0.93 ± 0.02) ppm. The gliding arc NO generator produced up to 180 ± 4 ppm of NO when connecting the device to a ventilator, and the NO2 level was below 1 (0.91 ± 0.02) ppm in all testing conditions. The gliding arc device required more power (in watts) to generate the same concentrations of NO when compared to double spark plug or high-pressure NO generators. ConclusionsOur results demonstrated that it is feasible to enhance NO production (more than 100 ppm) while maintaining NO2 level relatively low (less than 3 ppm) with the three recently developed NO generating devices. Future studies might include these novel designs to deliver high doses of inhaled NO as an antimicrobial used to treat upper and lower respiratory tract infections.

Nitric oxide contribution to endothelium-mediated vasodilation is attenuated in postmenopausal compared to premenopausal females

Background: Hypertension prevalence increases in females following menopause. Poor endothelial microvascular function increases risk of hypertension. Endothelial-mediated vasodilation is dependent on nitric oxide (NO). Estrogen enhances NO production; therefore, reduced estrogen levels in postmenopausal females may result in a reduced contribution of NO to endothelial-mediated vasodilation, although studies are limited. Herein, we aimed to investigate the contribution of NO to endothelial-mediated vasodilation in premenopausal and postmenopausal females. We hypothesized that postmenopausal females would have attenuated endothelium-mediated vasodilation and reduced contribution of NO compared to premenopausal females. Methods: Ten premenopausal (27±3 yrs) and eleven postmenopausal (59±4 yrs) normotensive females were studied. Resting brachial blood pressures were measured using a standard arm cuff. Brachial arterial catheters were placed for continuous blood pressure measurement and pharmacologic infusions. Forearm blood flow (FBF) was measured using venous occlusion plethysmography at baseline (saline) and during infusion of an endothelium-dependent vasodilator (acetylcholine; 1.0, 2.0, 4.0, and 8.0 μg/100mL tissue/min) with and without a NO synthase inhibitor (L-NG-monomethylarginine [L-NMMA]; 1.0 mg/min) co-infusion. Forearm vascular conductance (FVC) was calculated as FBF/mean arterial pressure✕100. Data were analyzed using a two-way repeated-measures ANOVA. Results: Resting systolic blood pressure (SBP) but not diastolic blood pressure (DBP) was greater in postmenopausal compared to premenopausal females (SBP: 121±15 vs. 103±9 mmHg, respectively; P<0.01; DBP: 72±11 vs. 65±5 mmHg, respectively; P=0.07). FBF responses to acetylcholine did not differ between premenopausal and postmenopausal females (Group Main Effect: P=0.11). When correcting for blood pressure, premenopausal females exhibited greater FVC responses to acetylcholine compared to postmenopausal females (Group Main Effect: P=0.04). In premenopausal females, FBF and FVC responses to acetylcholine were reduced with co-infusion of L-NMMA compared to acetylcholine infusion only (L-NMMA Main Effect: P=0.02 and P=0.01, respectively). In postmenopausal females, the FBF and FVC responses to acetylcholine were unaffected by L-NMMA (L-NMMA Main Effect: P=0.79 and P=0.76, respectively). Conclusion: Compared to premenopausal females, postmenopausal females have impaired endothelial microvascular function. Attenuated NO contribution during endothelium-mediated vasodilation in postmenopausal women may partially contribute to the increased hypertension risk in this population. AHA 14PRE18040000, AHA 898649, NIH HL083947, NIH HL148144, NCATS UL1 TR000135 This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Utilizing the Off-Target Effects of T1R3 Antagonist Lactisole to Enhance Nitric Oxide Production in Basal Airway Epithelial Cells.

Human airway sweet (T1R2 + T1R3), umami (T1R1 + T1R3), and bitter taste receptors (T2Rs) are critical components of the innate immune system, acting as sensors to monitor pathogenic growth. T2Rs detect bacterial products or bitter compounds to drive nitric oxide (NO) production in both healthy and diseased epithelial cell models. The NO enhances ciliary beating and also directly kills pathogens. Both sweet and umami receptors have been characterized to repress bitter taste receptor signaling in healthy and disease models. We hypothesized that the sweet/umami T1R3 antagonist lactisole may be used to alleviate bitter taste receptor repression in airway basal epithelial cells and enhance NO production. Here, we show that lactisole activates cAMP generation, though this occurs through a pathway independent of T1R3. This cAMP most likely signals through EPAC to increase ER Ca2+ efflux. Stimulation with denatonium benzoate, a bitter taste receptor agonist which activates largely nuclear and mitochondrial Ca2+ responses, resulted in a dramatically increased cytosolic Ca2+ response in cells treated with lactisole. This cytosolic Ca2+ signaling activated NO production in the presence of lactisole. Thus, lactisole may be useful coupled with bitter compounds as a therapeutic nasal rinse or spray to enhance beneficial antibacterial NO production in patients suffering from chronic inflammatory diseases such as chronic rhinosinusitis.

Obesity- and Sex-Related Disturbances in Arginine and Nitric Oxide Kinetics

Abstract Objectives Arginine (Arg) is considered a conditionally essential amino acid (AA) in the state of inflammation because of its role in nitric oxide (NO) production. NO is emerging as a regulator of energy metabolism and body composition. Although a growing interest exists in supplementation of Arg and citrulline (Cit) because of their potential anti-obesity effects, there is no consensus on the physiological effects of obesity on Arg metabolism and the role of sex. Methods 163 subjects were stratified based on their BMI (20–30 vs > 30 kg/m2) into normal-to-overweight (n = 83, age 49.5 [44.4, 54.5] y, BMI = 25.2 [24.36, 25.8] kg/m2) and obese (n = 80, age: 54.0 [5.07, 57.3] y, BMI = 38.2 [36.9, 39.6] kg/m2) groups. In the overnight fasted state, we collected from each subject arterialized blood for analysis of concentrations of plasma AA, C-reactive protein (CRP as a marker of systemic inflammation), and glucose. We subsequently administered a pulse of AA stable tracers containing Arg, Cit, and ornithine (Orn) and measured their whole-body productions (WBP), calculated their clearance, and the conversions of Arg to Cit (a marker of NO synthesis), Cit to Arg (de novo Arg production), and Arg to Orn (a surrogate of urea production). We measured body composition by DXA. Statistics by ANCOVA adjusted for age, sex, and lean body mass. Data are mean [95% CI]. Significance at P < 0.05. Results The obese subjects were characterized by higher CRP (P < 0.0001) but comparable fasting glucose levels (P = 0.0884) as the normal-to-overweight subjects. We found reduced plasma Cit concentration (P < 0.0001) in the obese group and upregulated whole-body clearance of Arg (P = 0.0002), Cit (P < 0.0001), and Orn (P = 0.0002) but no differences in their WBP. We observed elevated production of NO (P < 0.0001) in the obese but reduced de novo Arg synthesis (P = 0.0007). Females had lower plasma Arg and Orn concentrations, WBP, and clearance of Arg, Cit, and Orn than the males (p < 0.05) but comparable Cit concentration and NO production. Obese females had 3.5-fold higher NO production (P = 0.0020) than obese males concomitant with a 40% decrease in de novo Arg production (P = 0.0059). Conclusions Plasma Cit is lower in obesity despite enhanced NO production and is related to reduced de novo Arg synthesis. Arg might be considered an essential amino acid in obese women. Funding Sources None.

Leptin Enhances Nitric Oxide Production and Decreases Blood Flow in Rat Skeletal Muscle

Objective: There are a few studies related to nitric oxide (NO) and leptin interaction in the regulation of physiological events in skeletal muscle. Therefore, the aim of the present study is to investigate the interaction of leptin and NO in response to blood flow and nitric oxide synthase (NOS) distribution on rat skeletal muscle. Materials and Methods: Twenty-four adult-male Wistar albino rats were divided into 4 groups: control (C), Leptin (LP) (50 μg/kg), L-NG-nitroarginine methyl ester (LN) (a non-specific nitric oxide synthase inhibitor, 10 mg/kg,) and L-NAME+Leptin (LN+LP) administrated groups. Drugs were administered via the right jugular vein. Hemodynamic parameters: mean arterial pressure, heart rate and blood flow were recorded during the experiment and at the end blood samples for biochemical analyses of leptin and nitrite/nitrate levels and gastrocnemius muscle tissues from the right hindlimb for NOS distribution were taken. Results: Leptin infusion after L-NAME administration significantly decreased heart rate and blood flow (p<0.05, p<0.001). In addition, there was no change in the mean arterial pressure in the leptin group and leptin-administered L-NAME group. Leptin levels were the highest in the LP+LN group (p<0.01). Decreased nitrate/nitrate levels with L-NAME administration (p<0.01 vs C) reached control values by leptin infusion. Both endothelial NOS (eNOS) and neuronal NOS (nNOS) distributions were observed in the skeletal muscle cells of the leptin group. Conclusion: Although NO synthesis is inhibited by L-NAME, it is concluded that leptin partially enhances NO production and leptin uses NO as a mediator in its physiological effects.

Design of biodegradable 3D-printed cardiovascular stent

A 3D cardiovascular stent based on the combination of 2-hydroxy ethyl methacrylate (HEMA) and methacrylate graphene oxide (MeGO) and loaded with resveratrol (RSL) was developed to enhance the mechanical properties and efficiency in recovery of endothelial cell function. A 3D stent model based on the combination of HEMA-MeGO was printed through the extrusion method conducted at a speed of 2 mm/s with a layer height of 0.16 mm, 100% infill density with a 27-gauge needle in a Carbopol support bath. The corresponding rheological properties of HEMA-MeGO hydrogel inks and the mechanical properties of 3D-printed stents were examined. The effects of RSL (∼1 mM) loaded in 3D stents on viability, nitric oxide (NO) production, and downregulation of oxidative stress from the human umbilical vein endothelial cells (HUVECs) were evaluated using MTS, Griess, and DCFDA assays, respectively. The percentage volume change of stents after 24 h made of either HEMA or HEMA-0.35MeGO were 36.7 ± 4.7% and 7.7 ± 1.6%, respectively. The remaining fractions of 3D stents after 21 days and corresponding first-order degradation rate were 95.9 ± 0.5% and 0.30 ± 0.10 × 10−4/hr for HEMA, whereas those were 98.2 ± 0.3% and 0.11 ± 0.04 × 10−4/hr for HEMA-0.35MeGO. The addition of MeGO significantly enhanced the stiffness of hydrogel inks for 3D stents, displaying the Young's modulus of 2.2 ± 0.1 kPa and 4.1 ± 0.02 kPa for HEMA and HEMA-0.35MeGO (F-ratio = 2.99 and p-value < 0.001 for 21 days), respectively. MTS assay revealed that 3D stent loaded with RSL (∼1 mM) exerted no cytotoxic effects on the HUVECs. The controlled release of RSL from the stent enhanced NO production, lowered TNF-α level, and alleviated H2O2-induced oxidative stress in HUVECs. The 3D-printed biodegradable stent based on HEMA-MeGO and loaded with RSL displayed excellent biocompatibility and mechanical properties. RSL released from 3D cardiovascular stents efficiently recovered the endothelial function.

Improved immune-enhancing activity of egg white protein ovotransferrin after enzyme hydrolysis.

Ovotransferrin (OTF), an egg protein known as transferrin family protein, possess strong antimicrobial and antioxidant activity. This is because OTF has two iron binding sites, so it has a strong metal chelating ability. The present study aimed to evaluate the improved immune-enhancing activities of OTF hydrolysates produced using bromelain, pancreatin, and papain. The effects of OTF hydrolysates on the production and secretion of pro-inflammatory mediators in RAW 264.7 macrophages were confirmed. The production of nitric oxide (NO) was evaluated using Griess reagent and the expression of inducible nitric oxide synthase (iNOS) were evaluated using quantitative real-time polymerase chain reaction (PCR). And the production of pro-inflammatory cytokines (tumor necrosis factor [TNF]-α and interleukin [IL]-6) and the phagocytic activity of macrophages were evaluated using an ELISA assay and neutral red uptake assay, respectively. All OTF hydrolysates enhanced NO production by increasing iNOS mRNA expression. Treating RAW 264.7 macrophages with OTF hydrolysates increased the production of pro-inflammatory cytokines and the phagocytic activity. The production of NO and pro-inflammatory cytokines induced by OTF hydrolysates was inhibited by the addition of specific mitogen-activated protein kinase (MAPK) inhibitors. In conclusion, results indicated that all OTF hydrolysates activated RAW 264.7 macrophages by activating MAPK signaling pathway.

Ferulic Acid Derivatives and Avenanthramides Modulate Endothelial Function through Maintenance of Nitric Oxide Balance in HUVEC Cells.

Wholegrain oats contain a variety of phenolic compounds thought to help maintain healthy vascular function, through the maintenance of local levels of the vasodilator nitric oxide (NO). Thus, the full molecular mechanisms involved are not yet clear. With this work we aim to understand the possible cellular mechanisms by which avenanthramides and ferulic acid derivatives, present in oats, may help maintain a healthy vascular function through the modulation of the NO pathway. Primary Human Umbilical Vein Endothelial Cells (HUVEC) were exposed to ferulic acid, isoferulic acid, hydroferulic acid, ferulic acid 4-O-glucuronide, isoferulic acid 3-O-sulfate, dihydroferulic acid 4-O-glucuronide, avenanthramide A, avenanthramide B and avenanthramide C (1 μM) or vehicle (methanol) for 24 h. Apocynin and Nω-Nitro-L-arginine (L-NNA) were additionally included as controls. NO and cyclic GMP (cGMP) levels, superoxide production and the activation of the Akt1/eNOS pathway were assessed. The statistical analysis was performed using one-way ANOVA followed by a Tukey post-hoc t-test. Apocynin and all phenolic compounds increased NO levels in HUVEC cells (increased DAF2-DA fluorescence and cGMP), and significantly reduced superoxide levels. Protein expression results highlighted an increase in the Akt1 activation state, and increased eNOS expression. Overall, our results indicated that the glucuronide metabolites do not enhance NO production through the Akt1/eNOS pathway, thus all compounds tested are able to reduce NO degradation through reduced superoxide formation.

Atg32-dependent mitophagy sustains spermidine and nitric oxide required for heat-stress tolerance in Saccharomycescerevisiae.

In Saccharomyces cerevisiae, the selective autophagic degradation of mitochondria, termed mitophagy, is critically regulated by the adapter protein Atg32. Despite our knowledge about the molecular mechanisms by which Atg32 controls mitophagy, its physiological roles in yeast survival and fitness remains less clear. Here, we demonstrate a requirement for Atg32 in promoting spermidine production during respiratory growth and heat-induced mitochondrial stress. During respiratory growth, mitophagy-deficient yeast exhibit profound heat-stress induced defects in growth and viability due to impaired biosynthesis of spermidine and its biosynthetic precursor S-adenosyl methionine. Moreover, spermidine production is crucial for the induction of cytoprotective nitric oxide (NO) during heat stress. Hence, the re-addition of spermidine to Atg32 mutant yeast is sufficient to both enhance NO production and restore respiratory growth during heat stress. Our findings uncover a previously unrecognized physiological role for yeast mitophagy in spermidine metabolism and illuminate new interconnections between mitophagy, polyamine biosynthesis and NO signaling.

C1q/TNF-related protein-9 ameliorates hypoxia-induced pulmonary hypertension by regulating secretion of endothelin-1 and nitric oxide mediated by AMPK in rats

Injury/dysfunction of the endothelium of pulmonary arteries contributes to hypoxia-induced pulmonary hypertension (HPH). We investigated whether C1q/tumor necrosis factor-related protein-9 (CTRP9), a newly identified cardiovascular agent, has protective roles in the development of HPH. HPH was induced in adult male rats by chronic hypobaric hypoxia. CTRP9 overexpression by adeno-associated virus (AAV)-CTRP9 transfection attenuated the increases in right ventricular systolic pressure, right ventricular hypertrophy index, and pulmonary arterial remodeling of rats under hypoxia. Importantly, CTRP9 overexpression improved endothelium-dependent vasodilation in pulmonary arterioles in HPH rats. CTRP9 overexpression enhanced expression of phosphorylated 5′-adenosine monophosphate-activated protein kinase (p-AMPK) and phosphorylated endothelial nitric oxide synthase (p-eNOS), and reduced phosphorylated extracellular signal-regulated protein kinase (p-ERK1/2) expression in pulmonary microvascular endothelial cells (PMVECs) of HPH rats. In cultured PMVECs, CTRP9 not only preserved the decrease of AMPK and eNOS phosphorylation level and nitric oxide (NO) production induced by hypoxia, but also blocked the increase in hypoxia-induced ERK1/2 phosphorylation level and endothelin (ET)-1 production. Furthermore, the effects of CTRP9 were interrupted by inhibitors or knockdown of AMPK. CTRP9 enhances NO production and reduces ET-1 production by regulating AMPK activation. CTRP9 could be a target for HPH.

The Nitric Oxide (NO) Donor Sodium Nitroprusside (SNP) and Its Potential for the Schizophrenia Therapy: Lights and Shadows.

Schizophrenia is a severe psychiatric disorder affecting up to 1% of the worldwide population. Available therapy presents different limits comprising lack of efficiency in attenuating negative symptoms and cognitive deficits, typical features of schizophrenia and severe side effects. There is pressing requirement, therefore, to develop novel neuroleptics with higher efficacy and safety. Nitric oxide (NO), an intra- and inter-cellular messenger in the brain, appears to be implicated in the pathogenesis of schizophrenia. In particular, underproduction of this gaseous molecule is associated to this mental disease. The latter suggests that increment of nitrergic activity might be of utility for the medication of schizophrenia. Based on the above, molecules able to enhance NO production, as are NO donors, might represent a class of compounds candidates. Sodium nitroprusside (SNP) is a NO donor and is proposed as a promising novel compound for the treatment of schizophrenia. In the present review, we intended to critically assess advances in research of SNP for the therapy of schizophrenia and discuss its potential superiority over currently used neuroleptics.

Hsps70 and 90 protect the heart of hyperthyroid rats via nitric oxide production and VEGF inhibition of apoptosis

Background. Changes occur in the heart's contractile and metabolic demands during altered thyroid states. The changes may be associated with alterations in the cellular signaling of vascular endothelial growth factors (VEGF) and expressions of heat shock proteins (Hsp). Aim. This study examined the effects of thyroid dysfunction on VEGF, hsp70, and hsp 90 concentrations in the heart tissues of dysthyroid rats. Methods. Wistar rats were allocated into control, hypothyroid (Carbimazole-treated), and hyperthyroid (Levothyroxine-treated) groups randomly (n=7). Thyroid function test, body weight changes, serum creatinine kinase (CK-MB), cardiac troponin I (cTnI) and troponin T (cTnT), NO, VEGF, Hsp70, and Hsp 90 were determined using standard methods. Results. There was a significant increase (P<0.05) in NO, VEGF, Hsp70, and Hsp 90 levels in the hyperthyroid group and reduced expression in the hypothyroid group compared to the control. Carbimazole treatment led to a significant increase in lipid peroxidation and CK-MB level, with a substantial decrease in the superoxide dismutase (SOD) activity in the hypothyroid group. Conclusion. Increased expression of Hsp70 and 90 in hyperthyroid rats' heart tissue play essential roles in protecting the heart from oxidative damage and cardiovascular derangements via enhancement of nitric oxide production and apoptosis inhibition by VEGF.

Alcohol hangover induces nitric oxide metabolism changes by impairing NMDA receptor-PSD95-nNOS pathway

Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.