Increased Asymmetric Dimethylarginine in Severe Falciparum Malaria: Association with Impaired Nitric Oxide Bioavailability and Fatal Outcome

Asymmetrical dimethylarginine (ADMA), an endogenous inhibitor of nitric oxide synthase (NOS), is a predictor of mortality in critical illness. Severe malaria (SM) is associated with decreased NO bioavailability, but the contribution of ADMA to the pathogenesis of impaired NO bioavailability and adverse outcomes in malaria is unknown. In adults with and without falciparum malaria, we tested the hypotheses that plasma ADMA would be: 1) increased in proportion to disease severity, 2) associated with impaired vascular and pulmonary NO bioavailability and 3) independently associated with increased mortality. We assessed plasma dimethylarginines, exhaled NO concentrations and endothelial function in 49 patients with SM, 78 with moderately severe malaria (MSM) and 19 healthy controls (HC). Repeat ADMA and endothelial function measurements were performed in patients with SM. Multivariable regression was used to assess the effect of ADMA on mortality and NO bioavailability. Plasma ADMA was increased in SM patients (0.85 µM; 95% CI 0.74–0.96) compared to those with MSM (0.54 µM; 95%CI 0.5–0.56) and HCs (0.64 µM; 95%CI 0.58–0.70; p<0.001). ADMA was an independent predictor of mortality in SM patients with each micromolar elevation increasing the odds of death 18 fold (95% CI 2.0–181; p = 0.01). ADMA was independently associated with decreased exhaled NO (r_s = −0.31) and endothelial function (r_s = −0.32) in all malaria patients, and with reduced exhaled NO (r_s = −0.72) in those with SM. ADMA is increased in SM and associated with decreased vascular and pulmonary NO bioavailability. Inhibition of NOS by ADMA may contribute to increased mortality in severe malaria.     

Evaluation of Clinical and Laboratory Variables as Prognostic Indicators in Hospitalised Gastrointestinal Colic Horses

The present prospective study included 106 horses referred to the Department of Large Animal Sciences, The Norwegian School of Veterinary Science, as non-responders to the initial colic treatment in general practise. In 14 of these cases a required surgical treatment was not performed due to economical or other reasons and were excluded from the study. Clinical and laboratory data were obtained at the arrival in the hospital. The outcome for all analyses was survival/non-survival. A multivariable logistic regression was performed. The analyses were used in medically (46 horses) and surgically treated cases (46 horses) separately. The same analyses were also run for all 92 horses in a simulated "field" situation, where only clinical variables and D-dimer values were included. The fraction of survivors was 78% in the medical and 48% in the surgical cases. In total 63% of the horses survived. In the final multivariable logistic regression model packed cell volume (PCV) was the only important predictor for medically treated cases, and heart rate and presence of hyperaemic or cyanotic mucous membranes were the predictors in the surgically treated cases as well as in the simulated "field" situation. In conclusion, traditional variables as heart rate, mucous membranes and PCV were the important predictors for the outcome in hospitalised colic cases.     

Reduced Nasal Nitric Oxide Production in Cystic Fibrosis Patients with Elevated Systemic Inflammation Markers

Background

Nitric oxide (NO) is produced within the respiratory tract and can be detected in exhaled bronchial and nasal air. The concentration varies in specific diseases, being elevated in patients with asthma and bronchiectasis, but decreased in primary ciliary dyskinesia. In cystic fibrosis (CF), conflicting data exist on NO levels, which are reported unexplained as either decreased or normal. Functionally, NO production in the paranasal sinuses is considered as a location-specific first-line defence mechanism. The aim of this study was to investigate the correlation between upper and lower airway NO levels and blood inflammatory parameters, CF-pathogen colonisation, and clinical data.

Methods and Findings

Nasal and bronchial NO concentrations from 57 CF patients were determined using an electrochemical analyser and correlated to pathogen colonisation of the upper and lower airways which were microbiologically assessed from nasal lavage and sputum samples. Statistical analyses were performed with respect to clinical parameters (lung function, BMI), laboratory findings (CRP, leucocytes, total-IgG, fibrinogen), and anti-inflammatory and antibiotic therapy. There were significant correlations between nasal and bronchial NO levels (rho = 0.48, p<0.001), but no correlation between NO levels and specific pathogen colonisation. In patients receiving azithromycin, significantly reduced bronchial NO and a tendency to reduced nasal NO could be found. Interestingly, a significant inverse correlation of nasal NO to CRP (rho = −0.28, p = 0.04) and to leucocytes (rho = −0.41, p = 0.003) was observed. In contrast, bronchial NO levels showed no correlation to clinical or inflammatory parameters.

Conclusion

Given that NO in the paranasal sinuses is part of the first-line defence mechanism against pathogens, our finding of reduced nasal NO in CF patients with elevated systemic inflammatory markers indicates impaired upper airway defence. This may facilitate further pathogen acquisition in the sinonasal area, with consequences for lung colonisation and the overall outcome in CF.     

Studies of Nitric Oxide Production in Rat Tissues in Postnatal Development by Electron Paramagnetic Resonance Spectroscopy

Biophysics - Paramagnetic complexes containing nitric oxide (NO) have been assayed in rat heart and liver tissues by EPR spectroscopy to investigate the time variation of NO production during...     

Aerobic Exercise Training Prevents the Onset of Endothelial Dysfunction via Increased Nitric Oxide Bioavailability and Reduced Reactive Oxygen Species in an Experimental Model of Menopause

Objective

Previous studies have shown that estrogen deficiency, arising in postmenopause, promotes endothelial dysfunction. This study evaluated the effects of aerobic exercise training on endothelial dependent vasodilation of aorta in ovariectomized rats, specifically investigating the role of nitric oxide (NO) and reactive oxygen species (ROS).

Methods

Female Wistar rats ovariectomized (OVX – n=20) or with intact ovary (SHAM – n=20) remained sedentary (OVX and SHAM) or performed aerobic exercise training on a treadmill 5 times a week for a period of 8 weeks (OVX-TRA and SHAM-TRA). In the thoracic aorta the endothelium-dependent and –independent vasodilation was assessed by acetylcholine (ACh) and sodium nitroprusside (SNP), respectively. Certain aortic rings were incubated with L-NAME to assess the NO modulation on the ACh-induced vasodilation. The fluorescence to dihydroethidium in aortic slices and plasma nitrite/nitrate concentrations were measured to evaluate ROS and NO bioavailability, respectively.

Results

ACh-induced vasodilation was reduced in OVX rats as compared SHAM (Rmax: SHAM: 86±3.3 vs. OVX: 57±3.0%, p<0.01). Training prevented this response in OVX-TRA (Rmax: OVX-TRA: 88±2.0%, p<0.01), while did not change it in SHAM-TRA (Rmax: SHAM-TRA: 80±2.2%, p<0.01). The L-NAME incubation abolished the differences in ACh-induced relaxation among groups. SNP-induced vasodilation was not different among groups. OVX reduced nitrite/nitrate plasma concentrations and increased ROS in aortic slices, training as effective to restore these parameters to the SHAM levels.

Conclusions

Exercise training, even in estrogen deficiency conditions, is able to improve endothelial dependent vasodilation in rat aorta via enhanced NO bioavailability and reduced ROS levels.     

Down-Regulation of Neuronal Nitric Oxide Synthase by Nitric Oxide After Oxygen-Glucose Deprivation in Rat Forebrain Slices

Abstract : The precise role that nitric oxide (NO) plays in the mechanisms of ischemic brain damage remains to be established. The expression of the inducible isoform (iNOS) of NO synthase (NOS) has been demonstrated not only in blood and glial cells using in vivo models of brain ischemia-reperfusion but also in neurons in rat forebrain slices exposed to oxygen-glucose deprivation (OGD). We have used this experimental model to study the effect of OGD on the neuronal isoform of NOS (nNOS) and iNOS. In OGD-exposed rat forebrain slices, a decrease in the calcium-dependent NOS activity was found 180 min after the OGD period, which was parallel to the increase during this period in calcium-independent NOS activity. Both dexamethasone and cycloheximide, which completely inhibited the induction of the calcium-independent NOS activity, caused a 40-70% recovery in calcium-dependent NOS activity when compared with slices collected immediately after OGD. The NO scavenger oxyhemoglobin produced complete recovery of calcium-dependent NOS activity, suggesting that NO formed after OGD is responsible for this down-regulation. Consistently, exposure to the NO donor ( Z )-1-[(2-aminoethyl)- N -(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NONOate) for 180 min caused a decrease in the calcium-dependent NOS activity present in control rat forebrain slices. Furthermore, OGD and DETA-NONOate caused a decrease in level of both nNOS mRNA and protein. In summary, our results indicate that iNOS expression down-regulates nNOS activity in rat brain slices exposed to OGD. These studies suggest important and complex interactions between NOS isoforms, the elucidation of which may provide further insights into the physiological and pathophysiological events that occur during and after cerebral ischemia.     

Nitric Oxide in Arthritis

Nitric oxide’s (NO) involvement in arthritis was first demonstrated when levels of nitrite, a stable endproduct of NO metabolism, were shown to be elevated in serum and synovial fluid samples of rheumatoid and osteoarthritis patients. NO production by chondrocytes, its involvement in various biochemical events of cartilage metabolism, and the in vivo suppression of experimental arthritis by NO synthase inhibitors further implicated NO in arthritis. However, a conclusive role for NO in the pathogenesis of arthritis remains to be defined, in contrast to the NO-cGMP signal transduction pathway of endothelium-mediated vasodilation. It appears that NO has limited modulating effects in cartilage metabolism, with evidence for both protective and deleterious effects. Recent developments that contribute to our understanding of NO’s role in arthritis are discussed.     

Role of Nitric Oxide in Methamphetamine Neurotoxicity: Protection by 7-Nitroindazole, an Inhibitor of Neuronal Nitric Oxide Synthase

Abstract: The role of nitric oxide (NO^•) in the neurotoxic effects of methamphetamine (METH) was evaluated using 7-nitroindazole (7-NI), a potent inhibitor of neuronal nitric oxide synthase. Treatment of mice with 7-NI (50 mg/kg) almost completely counteracted the loss of dopamine, 3,4-dihydroxyphenylacetic acid, and tyrosine hydroxylase immunoreactivity observed 5 days after four injections of 10 or 7.5 mg/kg METH. With the higher dose of METH, this protection at 5 days occurred despite the fact that combined administration of METH and 7-NI significantly increased lethality and exacerbated METH-induced dopamine release (as indicated by a greater dopamine depletion at 90 min and 1 day). Combined treatment with 4 × 10 mg/kg METH and 7-NI also slightly increased the body temperature of mice as compared with METH alone. Thus, the neuroprotective effects of 7-NI are independent from lethality, are not likely to be related to a reduction of METH-induced dopamine release, and are not due to a decrease in body temperature. These results indicate that NO^• formation is an important step leading to METH neurotoxicity, and suggest that the cytotoxic properties of NO^• may be directly involved in dopaminergic terminal damage.     

Regional Estimate of Nitric Oxide Emissions Following Woody Encroachment: Linking Imaging Spectroscopy and Field Studies

Woody encroachment, a spatially explicit process of land-cover change, is known to affect the biophysical and biogeochemical properties of ecosystems. However, little information is available on the impacts of woody encroachment on N oxide emissions from savanna regions. We combined hyperspectral remote sensing and field measurements to quantify spatial patterns and estimate regional fluxes of soil N oxide emissions as they covary with vegetation cover and soil type across a semiarid rangeland in north Texas. Soil nitric oxide (NO) emissions were highly correlated with Prosopis canopy cover, allowing the extrapolation of NO fluxes from hyperspectral observations of woody cover. NO emissions were highly variable, ranging from 0 to 550 kg NO-N km^–2 y^–1 across the region, with the lowest emissions from shallow clay soils and highest from deeper upland clay loams. An estimate of annual NO emissions based on remotely derived Prosopis cover, temperature, and precipitation was 160 kg NO-N km^–2 y^–1, almost twice that of the value derived from traditional averaging of field measurements. We conclude that relationships between NO emissions and remotely sensed structure and composition are advantageous for quantifying NO emissions at the regional scale. This study also provides new insight into the role of woody encroachment on biogeochemical processes that are highly variable and otherwise difficult to measure at the regional scale.     

Nitric Oxide Transport in Normal Human Thoracic Aorta: Effects of Hemodynamics and Nitric Oxide Scavengers

Despite the crucial role of nitric oxide (NO) in the homeostasis of the vasculature, little quantitative information exists concerning NO transport and distribution in medium and large-sized arteries where atherosclerosis and aneurysm occur and hemodynamics is complex. We hypothesized that local hemodynamics in arteries may govern NO transport and affect the distribution of NO in the arteries, hence playing an important role in the localization of vascular diseases. To substantiate this hypothesis, we presented a lumen/wall model of the human aorta based on its MRI images to simulate the production, transport and consumption of NO in the arterial lumen and within the aortic wall. The results demonstrated that the distribution of NO in the aorta was quite uneven with remarkably reduced NO bioavailability in regions of disturbed flow, and local hemodynamics could affect NO distribution mainly via flow dependent NO production rate of endothelium. In addition, erythrocytes in the blood could moderately modulate NO concentration in the aorta, especially at the endothelial surface. However, the reaction of NO within the wall could only slightly affect NO concentration on the luminal surface, but strongly reduce NO concentration within the aortic wall. A strong positive correlation was revealed between wall shear stress and NO concentration, which was affected by local hemodynamics and NO reaction rate. In conclusion, the distribution of NO in the aorta may be determined by local hemodynamics and modulated differently by NO scavengers in the lumen and within the wall.     

Inactivation of Tryptophan Hydroxylase by Nitric Oxide: Enhancement by Tetrahydrobiopterin

Abstract: Tryptophan hydroxylase, the initial and rate-limiting enzyme in the biosynthesis of the neurotransmitter serotonin, is inactivated by the nitric oxide generators sodium nitroprusside, diethylamine/nitric oxide complex, and S -nitroso- N -acetylpenicillamine. Physiological concentrations of tetrahydrobiopterin, the natural and endogenous cofactor for the hydroxylase, significantly enhance the inactivation of the enzyme caused by each of these nitric oxide generators. The substrate tryptophan does not have this effect. The chemically reduced (tetrahydro-) form of the pterin is required for the enhancement, because neither biopterin nor dihydrobiopterin is effective. The 6 S -isomer of tetrahydrobiopterin, which has little cofactor efficacy for tryptophan hydroxylase, does not enhance enzyme inactivation as does the natural 6 R -isomer. A number of synthetic, reduced pterins share with tetrahydrobiopterin the ability to enhance nitric oxide-induced inactivation of tryptophan hydroxylase. The tetrahydrobiopterin effect is not prevented by agents known to scavenge hydrogen peroxide, superoxide radicals, peroxynitrite anions, hydroxyl radicals, or singlet oxygen. On the other hand, cysteine partially protects the enzyme from both the nitric oxide-induced inactivation and the combined pterin/nitric oxide-induced inactivation. These results suggest that the tetrahydrobiopterin cofactor enhances the nitric oxide-induced inactivation of tryptophan hydroxylase via a mechanism that involves attack on free protein sulfhydryls. Potential in vivo correlates of a tetrahydrobiopterin participation in the inactivation of tryptophan hydroxylase can be drawn to the neurotoxic amphetamines.     

Inactivation of Nitric Oxide by Uric Acid

The 1980 identification of nitric oxide (NO) as an endothelial cell-derived relaxing factor resulted in an unprecedented biomedical research of NO and established NO as one of the most important cardiovascular, nervous and immune system regulatory molecule. A reduction in endothelial cell NO levels leading to “endothelial dysfunction” has been identified as a key pathogenic event preceding the development of hypertension, metabolic syndrome, and cardiovascular disease. The reduction in endothelial NO in cardiovascular disease has been attributed to the action of oxidants that either directly react with NO or uncouple its substrate enzyme. In this report, we demonstrate that uric acid (UA), the most abundant antioxidant in plasma, reacts directly with NO in a rapid irreversible reaction resulting in the formation of 6-aminouracil and depletion of NO. We further show that this reaction occurs preferentially with NO even in the presence of oxidants peroxynitrite and hydrogen peroxide and that the reaction is at least partially blocked by glutathione. This study shows a potential mechanism by which UA may deplete NO and cause endothelial dysfunction, particularly under conditions of oxidative stress in which UA is elevated and intracellular glutathione is depleted.     

Detection of Nitric Oxide and Superoxide Radical Anion by Electron Paramagnetic Resonance Spectroscopy from Cells using Spin Traps

Reactive nitrogen/oxygen species (ROS/RNS) at low concentrations play an important role in regulating cell function, signaling, and immune response but in unregulated concentrations are detrimental to cell viability^{1, 2}. While living systems have evolved with endogenous and dietary antioxidant defense mechanisms to regulate ROS generation, ROS are produced continuously as natural by-products of normal metabolism of oxygen and can cause oxidative damage to biomolecules resulting in loss of protein function, DNA cleavage, or lipid peroxidation³, and ultimately to oxidative stress leading to cell injury or death⁴. Superoxide radical anion (O₂•-) is the major precursor of some of the most highly oxidizing species known to exist in biological systems such as peroxynitrite and hydroxyl radical. The generation of O₂•- signals the first sign of oxidative burst, and therefore, its detection and/or sequestration in biological systems is important. In this demonstration, O₂•- was generated from polymorphonuclear neutrophils (PMNs). Through chemotactic stimulation with phorbol-12-myristate-13-acetate (PMA), PMN generates O₂•- via activation of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase⁵. Nitric oxide (NO) synthase which comes in three isoforms, as inducible-, neuronal- and endothelial-NOS, or iNOS, nNOS or eNOS, respectively, catalyzes the conversion of L- arginine to L-citrulline, using NADPH to produce NO⁶. Here, we generated NO from endothelial cells. Under oxidative stress conditions, eNOS for example can switch from producing NO to O₂•- in a process called uncoupling, which is believed to be caused by oxidation of heme⁷ or the co-factor, tetrahydrobiopterin (BH₄)⁸.There are only few reliable methods for the detection of free radicals in biological systems but are limited by specificity and sensitivity. Spin trapping is commonly used for the identification of free radicals and involves the addition reaction of a radical to a spin trap forming a persistent spin adduct which can be detected by electron paramagnetic resonance (EPR) spectroscopy. The various radical adducts exhibit distinctive spectrum which can be used to identify the radicals being generated and can provide a wealth of information about the nature and kinetics of radical production⁹.The cyclic nitrones, 5,5-dimethyl-pyrroline-N-oxide, DMPO¹⁰, the phosphoryl-substituted DEPMPO¹¹, and the ester-substituted, EMPO¹² and BMPO¹³, have been widely employed as spin traps--the latter spin traps exhibiting longer half-lives for O₂•- adduct. Iron (II)-N-methyl-D-glucamine dithiocarbamate, Fe(MGD)₂ is commonly used to trap NO due to high rate of adduct formation and the high stability of the spin adduct¹⁴.     

Low Mercury Concentration Produces Vasoconstriction,Decreases Nitric Oxide Bioavailability and Increases Oxidative Stress in Rat Conductance Artery

Mercury is an environmental pollutant that reduces nitric oxide (NO) bioavailability and increases oxidative stress, having a close link with cardiovascular diseases, as carotid atherosclerosis, myocardial infarction, coronary heart disease and hypertension. One of the main sites affected by oxidative stress, which develops atherosclerosis, is the aorta. Under acute exposure to low mercury concentrations reactive oxygen species (ROS) production were only reported for resistance vessels but if low concentrations of mercury also affect conductance arteries it is still unclear. We investigated the acute effects of 6 nM HgCl₂ on endothelial function of aortic rings measuring the reactivity to phenylephrine in rings incubated, or not, with HgCl₂ for 45 min, the protein expression for cyclooxygenase 2 (COX-2) and the AT1 receptor. HgCl₂ increased Rmax and pD2 to phenylephrine without changing the vasorelaxation induced by acetylcholine and sodium nitroprusside. Endothelial damage abolished the increased reactivity to phenylephrine. The increase of Rmax and pD2 produced by L-NAME was smaller in the presence of HgCl₂. Enalapril, losartan, indomethacin, furegrelate, the selective COX-2 inhibitor NS 398, superoxide dismutase and the NADPH oxidase inhibitor apocynin reverted HgCl₂ effects on the reactivity to phenylephrine, COX-2 protein expression was increased, and AT1 expression reduced. At low concentration, below the reference values, HgCl₂ increased vasoconstrictor activity by reducing NO bioavailability due to increased ROS production by NADPH oxidase activity. Results suggest that this is due to local release of angiotensin II and prostanoid vasoconstrictors. Results also suggest that acute low concentration mercury exposure, occurring time to time could induce vascular injury due to endothelial oxidative stress and contributing to increase peripheral resistance, being a high risk factor for public health.     

Nitric Oxide Synthase-Independent Generation of Nitric Oxide in Muscle Ischemia–Reperfusion Injury

Nitric oxide (NO) is an important molecule in many physiological or pathophysiological processes including ischemia--reperfusion injury. The enzymatic nitric oxide synthase (NOS)-dependent pathway was universally accepted as the source of NO in ischemia-reperfusion injury. However, generation of NO that is independent of NOS has also been identified in ischemia--reperfusion injury to both cardiac and skeletal muscle. This review summarizes the evidence for the generation NOS-independent NO in ischemia--reperfusion injury to cardiac and skeletal muscle.