Normal Levels Of Nitric Oxide Research Articles

The eNOS isoform exhibits increased expression and activation in the main olfactory bulb of nNOS knock-out mice.

The main olfactory bulb (MOB) is a neural structure that processes olfactory information. Among the neurotransmitters present in the MOB, nitric oxide (NO) is particularly relevant as it performs a wide variety of functions. In this structure, NO is produced mainly by neuronal nitric oxide synthase (nNOS) but also by inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS). The MOB is considered a region with great plasticity and the different NOS also show great plasticity. Therefore, it could be considered that this plasticity could compensate for various dysfunctional and pathological alterations. We examined the possible plasticity of iNOS and eNOS in the MOB in the absence of nNOS. For this, wild-type and nNOS knock-out (nNOS-KO) mice were used. We assessed whether the absence of nNOS expression could affect the olfactory capacity of mice, followed by the analysis of the expression and distribution of the NOS isoforms using qPCR and immunofluorescence. NO production in MOB was examined using both the Griess and histochemical NADPH-diaphorase reactions. The results indicate nNOS-KO mice have reduced olfactory capacity. We observed that in the nNOS-KO animal, there is an increase both in the expression of eNOS and NADPH-diaphorase, but no apparent change in the level of NO generated in the MOB. It can be concluded that the level of eNOS in the MOB of nNOS-KO is related to the maintenance of normal levels of NO. Therefore, our findings suggest that nNOS could be essential for the proper functioning of the olfactory system.

Setting the pace: CD4 T cell-intrinsic Arginase 1 orchestrates Th1 induction and contraction

Abstract CD4 T cell-intrinsic engagement of the complement receptor CD46 controls nutrient influx and the metabolic reprogramming events that are essential for both the initiation and contraction of human Th1 responses, characterized by IFN-g and IL-10 production respectively. Here, we demonstrate that CD46 also orchestrates T cell arginine metabolism by upregulating the arginine transporter CAT-1 and, unexpectedly, Arginase 1 (Arg1). Arg1 has been well characterized in macrophages where it is associated with the IL-10 secreting M2 type, but its expression or function in T cells has not been described. Surprisingly, in CD4 T cell, Arg1 seems to restrain IL-10 production and contraction: T cells isolated from four patients with rare Arg1 deficiency mount strong Th1 responses but display significantly increased IL-10 switching and early contraction when compared to healthy control cells. Similarly, Arg1fl/fl CD4-cre+ mice infected with influenza virus are characterized by an enhanced Th1 response that contracts more rapidly, resulting in viral control and significantly reduced lung pathology. Unexpectedly, both Arg1-deficient mouse and human T cells produce normal levels of nitric oxide (NO) and polyamines. Metabolic profiling rather revealed that T cells lacking Arg1 have enhanced glycolysis, reduced TCA-cycle intermediates, and engage an “alternative” glutamine pathway often utilized by cancer cells. Normalization of these metabolic perturbations, through the targeting of specific metabolic enzymes, restored typical Th1 induction/contraction. Overall, these data unveil an unexpected intrinsic role for Arginase 1 as a pacemaker of the Th1 lifecycle, which could be harnessed for the amelioration Th1-driven pathologies.

Periodic Assessment of Antenatal and Post Natal Serum Endothelin-1 (ET-1) and Nitric Oxide (NO) Levels in Hypertensive Disorders of Pregnancy (HDP)

Hypertensive Disorders of Pregnancy (HDP) is an independent risk factor of cardiovascular (CVS) disease with endothelial dysfunction postulated to be the pathophysiology. Endothelin-1 (ET-1), a potent vasoconstrictor, has been identified as a pivotal mediator in HDP. Disturbances in nitric oxide (NO) bioavailability found in endothelial dysfunction may increase susceptibility to cardiovascular diseases such as hypertension. The study aims to determine serial ET-1 and NO levels in patients with HDP and its role in persistent endothelial dysfunction. Thirty-six pregnant women from the following categories (i) normal pregnant women (Control) (ii) chronic hypertension during pregnancy (CH) and (iii) pregnancy induced hypertension (PIH) participated in this study. Blood pressure indices measurements and sample collection were done at antepartum (32 weeks) and postpartum (8 weeks and 12 weeks). ET-1 and serum NO were measured using the Human ET-1 (Endothelin-1) ELISA Kit and Nitric Oxide (total) detection kit respectively. Results: Serum ET-1 was significantly higher in patients with CH (55.3 pg/ml) and PIH (35.6 pg/ml) compared to Control (11.8 pg/ml) during antenatal until 3 months postpartum (CH 38.3 pg/ml, PIH 29.5 pg/ml, Control 1.9 pg/ml). This was accompanied by significantly lower levels of serum NO in HDP patients. Persistently higher than normal levels of ET-1 and lower than normal levels of NO up to 3 months postpartum in patients with history of HDP indicate presence of persistent endothelial dysfunction despite BP normalisation in PIH patients. Long term NO/ET-1 imbalance may account for the increased CVS disease risk.

Measurement of nitric oxide in exhaled air in primary school children in Ružomberok.

Nowadays, there are a number of respiratory illnesses that directly affect children. Exhaled nitric oxide measurement (FeNO) is one of the modern methods of investigation used for diagnosing and controlling allergic eosinophilic airway inflammation. It is a simple, painless and time-saving method for the patient. Because of its non-invasiveness and simplicity, it is a valuable diagnostic method, especially in younger children. Measurement of FeNO is a recognized biomarker and an accurate quantitative indicator in the detection, assessment and monitoring of airway inflammation, including asthma. However, only a few studies assess the relationship between FeNO and level of air pollution. The aim of our study is to determine the relationship between FeNO in third-grade students of primary schools and the level of air pollution in Ruzomberok. 146 children aged 8-10 years were included in the research. This is the first stage of a wider research project involving FeNO measurements in children at different times of the year. The goal of the research is to determine the concentration of FeNO in the studied group of children in the autumn when air pollution is lower than in second stage which is observed in winter. The study showed elevated values of FeNO>20 ppb in 11% of the tested children and correct values <20 ppb in 89% of them. Level of average air pollution in survey days for PM10 ranged from 23 to 40 μg/m3 and for NO2 – from 28 to 44 μg/m3. The study shows that the significant majority of children have normal levels of nitric oxide in the exhaled air when air pollution is low. Analysis of the second phase of FeNO measurements in winter, when air pollution was higher, will allow to compare the results and assess the correlation between the FeNO value and air pollution.

Age-related changes in vascular responses to angiotensin-(1-7) in female mice.

The vasodilatory effect of angiotensin-(1-7) seems to vary between sexes, and estradiol (E2) can modulate the magnitude of the Ang-(1-7) vasodilatory response in female rats. However, there are few studies addressing the influence of sex on the age-related vasodilatory effect of Ang-(1-7). Here, we evaluated the vasodilatory response to Ang-(1-7) on vascular ageing. Ang-(1-7) dose–response curves were determined in mice aortic rings from males (old and young) and females (E2 treated/non-treated old and young) mounted in an isolated organ chamber. Abdominal aortic rings were used for protein expression analysis and determination of reactive oxygen species (ROS) and nitric oxide (NO) production. Our results showed that the Ang-(1-7) vasodilatory effect was absent in aorta from old females, contrasting with a full response in vessels from young females. The Ang-(1-7) vasodilatory effect was restored by E2 replacement in old females. A robust increase in Mas receptor, SOD2, NRF-2 and NOX2 expression was observed in aorta from old females, which was normalized by E2. This effect of E2 was also associated with lower production of ROS and normal levels of NO. In conclusion, our data demonstrated that pathways involved in the Ang-(1-7) vasodilatory response in female mice is affected by hormonal changes in ageing and rescued by E2.

Single-Walled Carbon Nanotubes Induce Cell Death and Transcription of TNF-α in Macrophages Without Affecting Nitric Oxide Production

Single-walled carbon nanotubes (SWCNTs) are potent nanomaterials that have diverse shapes and features. The utilization of these molecules for drug delivery is being investigated; thus, it is important to determine whether they alter immune responses against pathogens. In this study, we show that macrophages treated with a mixture of lipopolysaccharide and SWCNTs produced normal levels of nitric oxide and inducible nitric oxide synthase mRNA. However, these treatments induced cell death, presumably via necrosis. In addition, treating cells with SWCNTs induced the expression of tumor necrosis factor-α mRNA, a potent pro-inflammatory cytokine. These results suggest that SWCNTs may influence immune responses, which could result in unexpected effects following their administration for the purpose of drug delivery.

Pluronic nanoparticles do not modulate immune responses mounted by macrophages

Nanomaterials have been developed for the target delivery of medicine because they show the characteristics of selective emission or controlled release. Although accumulated data suggest the efficacy of these materials for the target delivery, it still remains to be determined whether they modulate immune responses to pathogens or foreign materials. In this study, we examined whether Pluronic nanoparticles (NPs), a type of nanomaterial, alter immune responses mediated by macrophages. When RAW 264.7 cells (RAW cells) were treated with Pluronic NPs in the presence or absence of lipopolysaccharides (LPS), they produced normal levels of nitric oxide (NO). Furthermore, the treatment with Pluronic nanomaterials did not induce cytotoxicity with or without LPS. Further, LPS-stimulated RAW cells expressed comparable levels of proinflammatory cytokine genes, such as interleukin (IL)-1 β, IL-6 and tumor necrosis factor (TNF)-α, with or without treatment by Pluronic NPs. These data suggest that Pluronic NPs do not modulate immune responses mediated by macrophages. Open image in new window

α-Tocopherol mitigates ethanol induced malformations and cell damage in the eye and brain of the chick embryo

The goal of this study is to investigate the ability of vitamin E in the active form α-tocopherol to mitigate the ethanol induced damaging effects and malformations in the developing chick embryo. Fertilized eggs were divided into five groups; the control group, and treated groups. The experimental groups were injected, in the air sac, before incubation with a single dose of 100μl saline solution, 100μl of 10% ethanol in saline solution, a mixture of 100μl of 10% ethanol and 200ppm vitamin E or 10% ethanol and 400ppm vitamin E. Treatment of developing chick embryos with 10% ethanol resulted in growth retardation and malformations in the eye, brain, limbs and other body parts. Ethanol exerted its degenerative effects probably via increasing membrane fluidity leading to membrane damage and significantly increased levels of lipid peroxidation. Ethanol also induced significant reduction in nitric oxide levels resulting in reduced body weight of the treated embryo probably due to restricted bold flow. Ethanol significantly increased glutathione level as a defense response. Vitamin C levels were significantly decreased after ethanol treatment due to oxidation or utilization. Vitamin E in the active form α-tocopherol partially mitigated the ethanol damaging effects either by exerting its antioxidant properties leading to a significant reduction of lipid peroxidation levels, retaining normal levels of nitric oxide or maintaining normal levels of endogenous antioxidants, glutathione and vitamin C.

Nitric oxide in primary ciliary dyskinesia

Nitric oxide is continually synthesised in the respiratory epithelium and is upregulated in response to infection or inflammation. Primary ciliary dyskinesia (PCD) is characterised by recurrent sinopulmonary infections due to impaired mucociliary clearance. Despite chronic infections, nasal nitric oxide in such patients is markedly reduced and is used as a screening test for this condition. These low levels were first described >15 yrs ago but the underlying mechanisms have yet to be fully elucidated. We review epithelial nitric oxide synthesis, release and measurement in the upper airways with particular reference to PCD. The key hypotheses that have been proposed to explain the low nitric oxide levels in this condition are explored and the potential benefits of augmenting airway nitric oxide levels are considered. Further work in these patients clarifying both whether the respiratory epithelium is able to biosynthesise normal levels of nitric oxide and the role played by abnormalities in the anatomy of the paranasal sinuses is essential. While nitric oxide augmentation is unlikely to be beneficial in common PCD phenotypes, it has potential in the treatment of secondary dyskinesias and may also improve treatment of bacterial infections, particularly where biofilms are implicated.

Analytical method for determination of nitric oxide in zebrafish larvae: Toxicological and pharmacological applications

Zebrafish are currently used at various stages of the drug discovery process and can be a useful and cost-effective alternative to some mammalian models. Nitric oxide (NO) plays an important role in physiology of zebrafish. The availability of appropriate analytical techniques to quantify the NO is crucial for studying its role in physiological and pathological conditions. This work aimed at establishing a high-performance liquid chromatography method for determination of NO levels in zebrafish larvae. Attempts were also made to assess the normal levels of NO at the first days postfertilization and the possible changes under pathological conditions. The method validation was quantitatively evaluated in terms of sensitivity, specificity, precision, accuracy, linearity, and recovery. NO levels from zebrafish larvae at the first days postfertilization and larvae challenged to N(G)-nitro-l-arginine methyl ester, sodium nitroprusside, Escherichia coli lipopolysaccharide, and copper sulfate were analyzed. The samples were derivatized with 2,3-diaminonaphthalene, and fluorescence detection was used for the indirect determination of NO. The method showed a good performance for all validation parameters evaluated and was efficient to monitor changes in NO concentration under physiological and pathophysiological conditions. This method might represent a powerful tool to be applied in NO studies with zebrafish larvae.

Nitric Oxide-Dependent Neovascularization Role in the Lower Extremity Disease

Peripheral arterial occlusive disease (PAD) describes vascular disorders associated with ischemia and PAD affects about 8 million people in the United States. Moreover, PAD's prevalence can increase dramatically if cardiovascular disease is present. In healthy individuals reducing blood flow through the lower extremity is followed by a physiological process to limit ischemia in the distal tissue. This process is called revascularization and impairing revascularization results in PAD. Studies suggest nitric oxide (NO) maybe involved in the ischemia-dependent hindlimb revascularization process. NO is increased in the ischemic hindlimb and eliminating NO impairs the revascularization process. Moreover, restoring NO improves hindlimb revascularization. NO may be acting through its effects on vascular tone, cell migration, or extracellular matrix degradation. The present review illustrates nitric oxide's critical role in the ischemia-induced hindlimb revascularization. Thus, restoring normal NO levels in diseased arteries may represent a viable therapeutic avenue by supplementing exogenous NO or employing therapeutic strategies to either increase NO synthesis and its messengers or decrease NO catabolism.

Review of Hemoglobin-Induced Myocardial Lesions

Over 100 preclinical studies in several small and large animal species were performed to evaluate the safety and efficacy of diaspirin cross-linked hemoglobin (DCLHb; Baxter Healthcare Corp.) as an oxygen therapeutic. During the preclinical evaluation of DCLHb, myocardial lesions were observed following the administration of DCLHb to certain species. These lesions were characterized as minimal to moderate, focal-to-multifocal myocardial degeneration and/or necrosis that were scored using a severity scale of minimal to marked in relative severity. The lesions were typically observed 24-48 h after single topload infusions of DCLHb into rhesus monkeys or pigs at doses as low as 200 or 700 mg/kg, respectively. Dogs, sheep, and rats did not develop these lesions after single-dose administrations of DCLHb. The left ventricular myocardium, typically near the base of or including the papillary muscles, was the most severely affected region, followed by the intraventricular septum and the right ventricle. The left and right atria were usually not affected. In a study in rhesus monkeys, morphometric analysis revealed that these lesions comprised less than 3% of the total myocardium. Although increases in serum enzyme activities (AST, CK, LDH) were observed after infusion of DCLHb, myocardial-related isoenzymes did not increase. ECG analysis and echocardiography were not altered by these lesions, and there was no observable adverse effect on myocardial function. Polymerization of DCLHb reduced, but did not eliminate, the incidence and severity of the lesions. However, infusion of hemoglobin solutions with reduced reaction rates with nitric oxide (NO) resulted in a significant decrease in lesion incidence and severity, while administration of L-NAME, an NO synthase inhibitor, resulted in the appearance of lesions that were indistinguishable from those induced by hemoglobin, suggesting that reduction in normal NO levels is an important mechanistic factor. Overall, the presence of myocardial lesions represents a histopathologic finding that must be considered during the preclinical testing and development of new HBOCs.

Diabetes-induced nitrative stress in the retina, and correction by aminoguanidine.

Aminoguanidine inhibits the development of retinopathy in diabetic animals, but the mechanism remains unclear. Inasmuch as aminoguanidine is a relatively selective inhibitor of the inducible isoform of nitric oxide synthase (iNOS), we have investigated the effects of hyperglycemia on the retinal nitric oxide (NO) pathway in the presence and absence of aminoguanidine. In vivo studies utilized retinas from experimentally diabetic rats treated or without aminoguanidine for 2 months, and in vitro studies used bovine retinal endothelial cells and a transformed retinal glial cell line (rMC-1) incubated in 5 mm and 25 mm glucose with and without aminoguanidine (100 microg/mL). NO was detected as nitrite and nitrate, and nitrotyrosine and iNOS were detected using immunochemical methods. Retinal homogenates from diabetic animals had greater than normal levels of NO and iNOS (p < 0.05), and nitrotyrosine was greater than normal, especially in one band immunoprecipitated from retinal homogenates. Oral aminoguanidine significantly inhibited all of these increases. Nitrotyrosine was detected immunohistochemically only in the retinal vasculature of non-diabetic and diabetic animals. Retinal endothelial and rMC-1 cells cultured in high glucose increased NO and NT, and aminoguanidine inhibited both increases in rMC-1 cells, but only NT in endothelial cells. Hyperglycemia increases NO production in retinal cells, and aminoguanidine can inhibit this abnormality. Inhibition of diabetic retinopathy by aminoguanidine might be mediated in part by inhibition of sequelae of NO production.

A defect in HIV-1 transgenic murine macrophages results in deficient nitric oxide production

HIV transgenic mice bearing multiple copies of a noninfectious (Deltagag/pol) proviral DNA were tested for the systemic production of nitric oxide (NO). Serum levels of NO metabolites were reduced about 50% in HIV transgenic mice compared with nontransgenic sibling mice. This difference persisted when NO production was induced with peritoneal injections of bacterial endotoxin (LPS). Peritoneal inflammatory macrophages, but not resident peritoneal macrophages, derived from HIV-1 transgenic mice and activated in vitro with LPS and IFN-gamma (or tumor necrosis factor alpha and IFN-gamma) also produced about 50% less NO than did macrophages harvested from nontransgenic littermates. Isogenic, transgenic mice bearing mutated nef or vpr genes had normal serum levels of NO metabolites and their macrophages produced normal levels of NO when stimulated. An explanation for the reduced NO response of HIV[Vpr+Nef+] macrophages was not apparent from measured levels of iNOS expression, viral gene expression, or arginase activity in activated macrophages. Inhibition of nitric oxide synthase (NOS) isoforms with L-NAME or aminoguanidine blocked time-dependent increases in HIV gene expression in activated macrophages cultured ex vivo. Inhibition with L-NAME occurred despite high levels of NO generated by iNOS, and exogenously supplied NO induced HIV gene expression only weakly, suggesting that cNOS had the greater influence on proviral gene induction. This system is presented as a model of HIV-1 proviral gene expression and dysfunction in macrophages.

The role of nitric oxide in the formation of keloid and hypertrophic lesions

Keloid and hypertrophic scars are a type of scarring pathology which is characterised by excess collagen deposition produced during the wound healing process. The mechanism by which this occurs is not understood and although hypertrophic scars can regress spontaneously, keloids do not, and currently no effective treatment exists. In this paper we hypothesise that nitric oxide, a free radical molecule synthesised by numerous mammalian cells, is involved in the formation of these scars. We suggest that the excess collagen production in keloid lesions can be attributed to higher than normal levels of nitric oxide, as the free radical is a known stimulus for fibroblast collagen synthesis. Furthermore, we propose that the basal epidermis is a source of this additional nitric oxide and we discuss this in relation to known histological characteristics of keloid and hypertrophic lesions.

Evidence for different subgroups of difficult asthma in children

BACKGROUNDChildren with difficult asthma experience frequent symptoms despite treatment with high dose inhaled steroids. Persistent symptoms may result from persistent airway inflammation which can be monitored by measuring exhaled nitric...

Lead-Induced Hypertension

An elevation of mean blood pressure was found in rats treated with low lead (0.01% lead acetate) for 3 months, as contrasted to paired Sprague-Dawley control rats. In these rats, measurement of plasma and urine endothelins-1 and -3 revealed that plasma concentration and urinary excretion of endothelin-3 increased significantly after 3 months (plasma: lead group, 31.8+/-2.2, versus controls, 23.0+1.7 pg/mL, P<.001; urinary excretion: lead group, 46.6+11.7, versus controls, 35.6+6.7 pg/24 h, P<.05), whereas endothelin-1 was unaffected. Plasma and urinary nitric oxide (NO) and cyclic GMP concentrations were not significantly changed. However, assay of plasma and kidney cortex malondialdehyde by high-pressure liquid chromatography, as a measure of reactive oxygen species, was elevated in lead-treated rats compared with that in control rats (plasma: lead group, 4.74+1.27, versus controls, 2.14+.49 micromol/L, P<.001; kidney cortex: lead group, 28.75+3.46, versus controls, 16.38+2.37 nmol/g wet weight, P<.001). There was increased NO synthase activity in lead-treated rat brain cortex and cerebellum. In lead-treated rat kidney cortex, the endothelial constitutive NO synthase protein mass was unaffected, whereas the inducible NO synthase protein mass was increased. These data suggest a balance between increased NO synthesis and degradation (by reactive oxygen species) in lead-treated rats, which results in normal levels of NO. Thus, the hypertension may be related to an increase in the pressure substances, endothelin-3 and reactive oxygen species, rather than to an absolute decrease in nitric NO.