Nitric Oxide Biology Research Articles

Exhaled nitric oxide: A test for diagnosis and control of asthma?

The fractional concentration of nitric oxide (FE(NO)) in exhaled breath is a noninvasive marker of airway inflammation in asthma. The precise role of FE(NO) in the asthma management algorithm has not been defined. However, there are compelling data for use of FE(NO) for diagnosing asthma, assessing control and severity, titrating inhaled corticosteroids, and detecting ongoing airway inflammation. This article reviews the biology of nitric oxide in airway pathology and its role in asthma.

Genetic polymorphisms associated with priapism in sickle cell disease

Priapism occurs in 30-45% of male patients with sickle cell disease (SCD), but the possible influence of genetic risk factors on the incidence of priapism is not well understood. We examined genetic polymorphisms in 199 unrelated, adult (>18 years), male patients with Hb SS and Hb Sbeta(0)-thalassaemia, 83 (42%) of whom reported a history of priapism. Candidate genes for association with priapism were identified based on their involvement in adhesion, coagulation, inflammation and cell signalling. Additionally, we examined genes involved in nitric oxide biology (NOS2, NOS3, SLC4A1), as well as polymorphisms in the klotho (KL) gene, which has previously been associated with priapism. Strong evidence of association was found for single nucleotide polymorphisms in transforming growth factor-beta receptor, type III (TGFBR3) (rs7526590; P = 0.00058), aquaporin (AQP1) (rs10244884; P = 0.00068), integrin alphav (ITGAV) (rs3768780; P = 0.00090), and the A1 subunit of coagulation factor XIII (F13A1) (hcv1860621; P = 0.00156). Associations with TGFBR3, AQP1, and ITGAV remained significant after adjusting for multiple testing, using the Benjamini-Hochberg procedure. Our data suggest that genes involved in the TGFbeta pathway, coagulation, cell adhesion and cell hydration pathways may be important in risk for priapism.

Deconstructing sickle cell disease: Reappraisal of the role of hemolysis in the development of clinical subphenotypes

Hemolysis, long discounted as a critical measure of sickle cell disease severity when compared with sickle vaso-occlusion, may be the proximate cause of some disease complications. New mechanistic information about hemolysis and its effects on nitric oxide (NO) biology and further examination of the subphenotypes of disease requires a reappraisal and deconstruction of the clinical features of sickle cell disease. The biology underlying clinical phenotypes linked to hemolysis may increase our understanding of the pathogenesis of other chronic hemolytic diseases while providing new insights into treating sickle cell disease. The pathophysiological roles of dysregulated NO homeostasis and sickle reticulocyte adherence have linked hemolysis and hemolytic rate to sickle vasculopathy. Nitric oxide binds soluble guanylate cyclase which converts GTP to cGMP, relaxing vascular smooth muscle and causing vasodilatation. When plasma hemoglobin liberated from intravascularly hemolyzed sickle erythrocytes consumes NO, the normal balance of vasoconstriction:vasodilation is skewed toward vasoconstriction. Pulmonary hypertension, priapism, leg ulceration and stroke, all subphenotypes of sickle cell disease, can be linked to the intensity of hemolysis. Hemolysis plays less of a role in the vaso-occlusive-viscosity complications of disease like the acute painful episode, osteonecrosis of bone and the acute chest syndrome. Agents that decrease hemolysis or restore NO bioavailability or responsiveness may have potential to reduce the incidence and severity of the hemolytic subphenotypes of sickle cell disease. Some of these drugs are now being studied in clinical trials.

The biology of nitric oxide and other reactive intermediates in systemic lupus erythematosus

Formation of reactive nitrogen and oxygen intermediates (RNI and ROI) is an essential part of the innate immune response. Markers of systemic RNI production are increased in the setting of systemic lupus erythematosus (SLE) activity. Several lines of evidence suggest mechanisms through which the activity of inducible nitric oxide synthase (iNOS) is pathogenic in SLE, including the ability of peroxynitrite (ONOO −, a product of iNOS activity) to modify proteins, lipids, and DNA. These modifications can alter enzyme activity and may increase the immunogenicity of self antigens, leading to a break in immune tolerance. In humans, observational data suggest that overexpression of iNOS and increased production of ONOO − lead to glomerular and vascular pathology. Therapies designed to target iNOS activity or scavenge ROI and RNI are in development and may provide the means to reduce the pathogenic consequences of ROI and RNI in SLE.

Modifier genes and sickle cell anemia

With the completion of the human genome project and HapMap, previously unknown genetic polymorphisms associated with disease have been observed. This review highlights genetic polymorphisms that have provided insight into the pathophysiology underlying the many phenotypes of sickle cell disease. The phenotypes of sickle cell disease are likely to be modulated by polymorphisms in genes that are involved in inflammation, cell-cell interaction, and nitric oxide biology. Case-control studies are beginning to define the relationships between single-nucleotide polymorphisms in candidate genes and the many subphenotypes of sickle cell anemia. A common theme emerging from these studies is that single-nucleotide polymorphisms in genes of the transforming growth factor-beta/bone morphogenetic protein and a few other genes such as Klotho are associated with several subphenotypes of sickle cell disease. Genomic medicine is merging with clinical practice as our understanding of the structure and variability of the human genome increases. Patients with diseases caused by identical mutations in a single gene - sickle cell anemia is a prime example - can have clinical courses very different from one another, and when environmental influences are removed the phenotypic heterogeneity of mendelian single-gene disorders is best explained by single-nucleotide polymorphisms in genes that modulate the disease phenotype. As this field expands, insights will be gained into complex epistatic factors that influence the clinical presentation of sickle cell disease, enabling physicians to better predict and manage the many complications of this disease.

The Metabolic Coupling of Arginine Metabolism to Nitric Oxide Generation by Astrocytes

Arginine, the only known precursor of nitric oxide, enters the brain parenchyma from the blood through the endothelial cells or from the cerebral spinal fluid through the ependymal cells. Astrocytes, whose processes abut the endothelium and ependymum, take up arginine through cationic amino acid transporters and release arginine through this transport system to the synapses that astrocytes shield. Some of these synapses are excitatory, and liberate glutamate into the synaptic cleft. Glutamate induces arginine release from astrocytes, making it available to the neuron. Neurons can take up arginine to be used in nitric oxide-mediated processes, such as neurotransmission. Thus, neural and nonneural cells act in concert to affect neuron physiology in an elegantly integrated system. This review focuses on the components of the interaction between astrocytes and neurons in nitric oxide biology.

Clinical biology of nitric oxide.

Nitric oxide is a pluripotential molecule that acts as both an autocrine and paracrine mediator of homoeostasis, and derangement of its metabolism can be linked with many pathophysiological events. This review provides a broad overview of the basic and clinical scientific aspects of nitric oxide.

Priapism in SCD: Clinical and Genetic Correlations.

Priapism is a complication of sickle cell disease (SCD) that occurs due to obstruction of the corpora cavernosa of the penis. We have studied priapism in relation to several clinical and genetic factors in 249 adult male patients with SCD, 92 (37%) of whom reported a positive history of priapism. The mean age of male patients without a history of priapism was 35.2 years (± 10.8 years) compared with a mean age of 36.4 years (± 11.3 years) in male patients with a positive history of priapism. Because of the possible relationship with nitric oxide biology, we examined the co-occurrence of priapism with proteinuria, leg ulcers and stroke. Of the males with a positive history of priapism, 20% also had a history of 2+ or greater proteinuria, compared to a presence of 2+ or greater proteinuria in only 10% of males without a history of priapism (p=0.03). Similarly, 34% of males with a positive history of priapism also had a history of leg ulcers, compared to the presence of leg ulcers in 22% of males without priapism (p=0.03). No statistically significant association between the occurrence of priapism and stroke was observed. In an effort to identify genetic risk factors for priapism, we examined 262 single nucleotide polymorphisms (SNPs) in a total of 56 genes, primarily involved in red blood cell adhesion and inflammation pathways. Chi Square tests of association were constructed for the genotypes of each SNP with two clinical categories: patients with a positive history of priapism and patients without a history of priapism. When the frequency of rare homozygotes was less than five individuals, we combined these rare homozygote individuals with heterozygote individuals for analysis. All p-values were uncorrected for multiple testing. We found associations with 12 SNPs in 8 genes: SLC4A2 (p=0.003); ITGAV (p=0.004 and p=0.02 for two different SNPs); F13A1 (p=0.004 and p=0.02 for two different SNPs); AQP1 (p=0.01 and p=0.04 for two different SNPs); TGFBR2 (p=0.01 and p=0.02 for two different SNPs); ADRB2 (p=0.03); MGC (p=0.04); and ARG2 (p<0.05). These genes are involved in a variety of functions, including adhesion, coagulation, signal transduction, NO biology and immune response. We examined 21 non-coding SNPs in the Klotho gene, but we did not find an association between priapism and Klotho, as was recently reported by Nolan and colleagues (2005). The only possible trend for association we observed in Klotho was at marker rs1888057 (p=0.07); we did not observe association with the SNP (rs2249358) (p=0.82) Nolan and colleagues found associated with priapism. These data support our over-arching hypothesis that genetic factors mediate the variability and risk of developing organ-specific complications of SCD. A better understanding of the genetic factors that contribute to the occurrence of complications such as priapism should ultimately lead to a better understanding of SCD pathophysiology as well as to improved treatment for patients with SCD.

The biology of exhaled nitric oxide (NO) in ischemia–reperfusion-induced lung injury: A tale of dynamism of NO production and consumption

The main objective of this paper is to review the potential diagnostic roles of exhaled nitric oxide (NO) in evaluating ischemia–reperfusion-induced lung injury associated with cardiac surgery. We shall start by elaborating on current clinical practice of cardiac surgery and to arrive at the conclusion that clinically important ischemia–reperfusion injury is a common scenario of many forms of these surgical procedures. We shall conclude this part by establishing the clinical need for biomarkers of inflammation in cardiothoracic surgery and by proposing that exhaled NO could be an important new addition to our anaesthetic monitoring repertoire based on our expertise with exhaled breath monitoring. We shall then take a closer look at mechanisms of ischemia–reperfusion injury and will propose the role of reactive oxygen and nitrogen species as mediators and biomarkers of acute lung injury. This analysis will provide a good opportunity to highlight major potential mechanisms of altered NO production and bioactivity of NO. We shall conclude that multiple relevant mechanisms may either lead to increased production of NO or enhance consumption of NO, leaving us with the paradigm that NO maybe used either as a positive or negative biomarker of inflammation. In order to explore this dilemma further, we will investigate the predominant effect of oxidative stress on NO bioactivity in cell culture models of ischemia–reperfusion injury. We will then turn to animal models of ischemia–reperfusion injury to elucidate the ultimate effects of this condition on lung NO production and concentrations of NO in the lung. Finally, we shall complete this journey by highlighting the human relevance of these observations by reviewing our own experience at Harefield Hospital, UK, and that of others, regarding exhaled NO in ischemia–reperfusion injury associated with cardiac surgery and lung transplantation.

C-Nitroso Compounds: Synthesis, Physicochemical Properties and Biological Activities

Because of the chemical and physical properties of nitric oxide, its effective use and delivery for therapeutic application represents a significant challenge. Accordingly, current understanding of nitric oxide biology largely stems from the use of nitric oxide prodrugs and adducts whose biological activities are based on their ability to release nitric oxide or a redox-related species. Among the structurally diverse ensemble of nitric oxide donor compounds reported to date are the C-nitroso compounds. These compounds have only recently been investigated with respect to their potential as nitric oxide donors, although they have been known and studied for over 120 years. Here, we consider the synthesis and physico-chemical properties of the C-nitroso compounds and the available data regarding their biological activities. Synthetic methods reviewed include direct substitution of H by NO, oxidative approaches, and the addition of various oxides of nitrogen across multiple bonds. The electronic spectra of C-nitroso compounds and the mechanism and thermodynamics of monomer-dimer equilibration are described. The physico-chemical and biological properties of two related classes of compounds, the diazetine dioxides and the furoxans, are also described.

Detection of Human Red Blood Cell-bound Nitric Oxide

Major disparities in reported levels of basal human nitric oxide metabolites have resulted in a recent literature focusing almost exclusively on methods. We chose to analyze triiodide chemiluminescence, drawn by the prospect of identifying why the most commonly employed assay in nitric oxide biology typically yielded lower metabolite values, compared with several other techniques. We found that the sensitivity of triiodide was greatly affected by the auto-capture of nitric oxide by deoxygenated cell-free heme in the reaction chamber. Potential contaminants and signal losses were also associated with standard sample purification procedures and the chemistry involved in nitrite removal. To inhibit heme nitric oxide auto-capture, we added potassium ferricyanide to the triiodide reagent, reasoning this would provide a more complete detection of any liberated nitric oxide. From human venous blood samples, we established nitric oxide levels ranging from 0.000178 to 0.00024 mol nitric oxide/mol hemoglobin. We went on to find significantly elevated nitric oxide levels in venous blood taken from diabetic patients in comparison to healthy controls (p < 0.0001). We concluded that the lack of signals reported of late by several groups using triiodide chemiluminescence for the detection of hemoglobin-bound nitric oxide may not represent levels on the border of assay sensitivity but rather underestimated values because of methodological limitations. We therefore stress the need for assay systems to be developed that differentiate between individual nitric oxide metabolite species and overcome the limitations we outline, allowing accurate conclusions to be drawn regarding physiological nitric oxide metabolite levels.

Organogenic nodule formation from hop internodes. Reactive oxygen species and expression of lipoxygenase and extracellular-regulated kinases

From Cell Biology of Nitric Oxide and Cell Death in Plants Yalta, Ukraine, 8–11 September 2004

Association of Single Nucleotide Polymorphisms in Klotho with Priapism in Sickle Cell Anemia.

The phenotypic heterogeneity of sickle cell anemia (HbSS) is likely to be accounted for by multiple genetic modifiers. Priapism, a common vasoocclusive complication of HbSS, may reflect sickle vasculopathy. We hypothesized that the likelihood of developing priapism, and other vascular complications of sickle cell disease, may be influenced by genetic heterogeneity in genes that modulate inflammation, oxidant injury, nitric oxide (NO) biology, vasoregulation, cell-cell interaction and hemostasis. Accordingly, we studied patients with HbSS with or without coincident α thalassemia and examined the association of 129 single nucleotide polymorphisms (SNPs) in 44 candidate genes with priapism. One hundred forty-eight patients had at least one episode of priapism and were compared with 529 controls. Validated SNPs in the candidate genes were first selected from a public database. Genotypic counts were compared between cases and controls using multiple logistic regression. For each SNP, odds ratio (OR) and 95% confidence intervals (CI) were calculated. Pairwise linkage disequilibrium between each pair of SNP loci was evaluated by using a maximum likelihood method to infer phase for dual heterozygotes and was expressed as r2. In our first analysis, we considered a SNP to have an association with a phenotype when the p-value was equal to or less than 0.01, unless there was more than one SNP in a gene showing an association, when the p-value for significance was set at 0.05. When a SNP met these criteria, we further studied the gene with as many informative SNPs as possible. Haplotypes were reconstructed by using Bayesian methods as implemented in the PHASE package. A sliding window approach was used to assess evidence for association between haplotype and priapism. Patients with HbSS-α thalassemia were less likely to have priapism than patients with HbSS (p<0.05). Two SNPs in Klotho gene (KL), rs657049 and rs211239, were associated with priapism by genotypic analysis (OR: 1.74, 95% CI 1.03–2.96 and OR: 1.74, 95% CI: 1.16–2.63, respectively). Since it is likely that the control group contains patients who might ultimately develop priapism, this may be an underestimate of the true association. Seventeen SNPs were used to reconstruct the haplotype of KL gene for cases and controls separately. Haplotype association analysis was performed using sliding windows of 2, 3, and 4 SNPs separately, and p-values were calculated after 10,000 permutations. We found that haplotypes around rs211239 (5th SNP among 17 SNPs by physical location) were associated with priapism in different windows, (1) 2 SNPs: window 3-4 and 4-5, p: 0.001 and 0.01 respectively, (2) 3 SNPs: window 2-3-4, 4-5-6 and 6-7-8, p: 0.004, 0.01, and 0.05, respectively, (3) 4 SNPs: window 1-2-3-4, 3-4-5-6, and 4-5-6-7, p: 0.03, 0.002, 0.02, respectively. KL encodes a membrane protein that regulates many vascular functions, including endothelial NO release and a variant protein may alter NO biology. We conclude that polymorphisms in a limited number of genes, or in linkage disequilibrium with functionally important genes, may set an overall risk for some vasoocclusive complications of HbSS. Identifying potential genetic modifiers, like KL, will permit these genes to be evaluated in biological studies and for networks of genotype-phenotype interactions to be modeled.

Nitric oxide reductases of prokaryotes with emphasis on the respiratory, heme–copper oxidase type

The investigation of respiratory N-oxide reduction as part of a biogeochemical process sustained by prokaryotes has roots over a century ago and has laid the groundwork for microbial nitric oxide (NO) biology and recognizing that NO is of bioenergetic importance as an electron acceptor in anaerobic environments. NO is an obligatory respiratory substrate of nitrate- and nitrite-denitrifying prokaryotes that release nitrous oxide or dinitrogen as products. We witness currently a broadening of the scope of NO functionality and an increase in awareness that other heme-based NO-metabolizing systems contribute to the overall capability of the prokaryotic cell to cope with NO both in anaerobic and aerobic environments, including the pathogen–host interface. NO reduction of newly recognized physiological importance is catalyzed by the pentaheme nitrite reductase, cytochrome c′, flavohemoglobin and flavorubredoxin. Respiratory NO reductases are heme–nonheme Fe proteins that can be classified either in a short-chain group, which are complexes with cytochrome c, or a long-chain group, which have a fused quinol oxidase domain. Even though NORs are not proton pumps, both reductase groups are structural homologues of heme–copper oxidases. As a unique case, the short-chain NOR of Roseobacter denitrificans acts on oxygen, based on a heme b 3-Cu B center. In turn, certain heme–copper oxidases have significant turnover rates with NO. NOR mechanisms have been proposed from oxidase active site chemistry. Besides being a respiratory substrate, NO is also a signaling molecule that triggers gene expression of the principal components of NO respiration by members of the Crp–Fnr superfamily of transcription regulators.

Cardioprotective effects of nitroparacetamol and paracetamol in myocardial infarction

Study objectives: The biology of nitric oxide has been extensively studied for many years. Currently, researchers are investigating the possibility that slow-release nitric oxide donors (nitric oxide–donating drugs) may produce biological effects quite different from classic nitric oxide donors (eg, sodium nitroprusside). The precise role of nitric oxide in many cardiovascular diseases remains unclear. In our current study, we aim to determine whether nitroparacetamol (nitric oxide donor) exhibits cardioprotective effects. Methods: Male Wistar rats weighing between 220 and 250 g were used. Treatment groups included vehicle, nitroparacetamol, paracetamol, and ramipril. These drugs were given by the intraperitoneal route at doses of 5 mL/kg/day, 5 mg/kg/day, 15 mg/kg/day, and 0.5 mg/kg/day, respectively. Drug treatment was started 1 week before surgery. Myocardial infarction was induced by permanent ligation of the left anterior descending coronary artery and mortality rate at 2 days after myocardial infarction induction was compared. Infarction size was also compared between these groups. Results: Mortality rates for vehicle (n=60), nitroparacetamol (n=60), paracetamol (n=60), and ramipril (n=60) groups were 38%, 25%, 20%, and 28% respectively. A corresponding reduction in infarction size in the groups with better survival rate was noted. Infarction size for the vehicle group was 48% (±6%) of the left ventricle and for the ramipril group was 38% (±3%). For the nitroparacetamol and paracetamol groups, infarction size was 30% (±8%) and 32% (±6%) of the left ventricle, respectively. These were statistically significant when compared with the vehicle group. Nitroparacetamol and paracetamol administration in this way did not have any discernible effects on blood pressure in these animals. Conclusion: The study is underpowered to show statistical significance of the difference in mortality rate obtained. Nevertheless, the observation that paracetamol and nitroparacetamol produce a lower mortality rate than ramipril, an angiotensin-converting enzyme inhibitor, indicates the potential clinical significance of the cardioprotective effects of these drugs. The significant reduction in infarction size further reinforces this conclusion.

EPR quantification of vascular nitric oxide production in genetically modified mouse models

With increasing use of genetically modified mice to study endothelial nitric oxide (NO) biology, methods for reliable quantification of vascular NO production by mouse tissues are crucial. We describe a technique based on electron paramagnetic resonance (EPR) spectroscopy, using colloid iron (II) diethyldithiocarbamate [Fe(DETC) 2], to trap NO. A signal was seen from C57BL/6 mice aortas incubated with Fe(DETC) 2, that increased 4.7-fold on stimulation with calcium ionophore A23187 [3.45 ± 0.13 vs 0.73 ± 0.13 au (arbitrary units)]. The signal increased linearly with incubation time ( r 2=0.93), but was abolished by addition of N G-nitro- l-arginine methyl ester ( l-NAME) or endothelial removal. Stimulated aortas from eNOS knockout mice had virtually undetectable signals (0.14 ± 0.06 vs 3.17 ± 0.21 au in littermate controls). However, the signal was doubled from mice with transgenic eNOS overexpression (7.17 ± 0.76 vs 3.37 ± 0.43 au in littermate controls). We conclude that EPR is a useful tool for direct NO quantification in mouse vessels.

Tetrahydrobiopterin: Mediator of Endothelial Protection

During the past twenty years, the essential role of endothelial cells in preservation of vascular homeostasis has been well established.1,2⇓ Protection of the vascular endothelium against harmful influences of circulating substances including excessive levels of lipids has been a major therapeutic approach in prevention of atherosclerosis. While endothelial dysfunction, defined as a loss of biologically active nitric oxide (NO) produced in the endothelium, has been recognized as a prime target for prevention and reversal of atherosclerotic process, identification of exact molecular mechanisms responsible for the loss of endothelial NO have been more difficult to determine. In the current issue of Arteriosclerosis, Thrombosis, and Vascular Biology , Alp et al3 report a series of elegant studies on genetically modified mice supporting the concept that NO synthase cofactor, tetrahydrobiopterin (Figure 1), is an important vascular protective molecule. The beneficial effect of tetrahydrobiopterin in apolipoprotein E (ApoE)-deficient mice appears to be mediated by preservation of endothelial nitric oxide synthase (eNOS) activity and production of nitric oxide in endothelial cells. The major steps of tetrahydrobiopterin biosynthesis. GTP=guanosine 5′-triphosphate. See page 445 In the early 1990s it was recognized that in the presence of suboptimal concentrations of tetrahydrobiopterin, activation of purified neuronal nitric oxide synthase (nNOS) causes “uncoupling of NOS” with subsequent increase …

Probing fundamental mechanisms of nitric oxide reactions with metal centers

Abstract Studies in this laboratory have been concerned with mapping the chemical properties and mechanisms of NO interactions with hemes and other metal centers. These are models relevant to the mammalian biology of nitric oxide, an important bioregulatory molecule. Presented here will be an overview of flash photolysis kinetics investigations of ferri- and ferro-heme nitrosyl formation in model complexes and several heme proteins. Also described will be ongoing studies of reductive nitrosylation mechanisms involving the reactions of NO with water-soluble Fe(III) porphyrins and ferri-heme proteins and of several Cu(II) model complexes.

3rd International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide

3rd International Conference on the Biology, Chemistry, and Therapeutic Applications of Nitric Oxide

Hypoxia increases nitric oxide concentrations that are not completely inhibited by l-NMMA

Introduction. Recent investigations have demonstrated a wealth of knowledge about the biology of nitric oxide (NO) and the synthases (NOS) that generate it, under both physiologic and pathophysiologic conditions. In this study, we investigated the generation of NO under conditions of hypoxia in the absence of confounding functions such as blood loss or hypovolemia. Methods. Rats were subjected to either 30 or 60 min of hypoxia and sacrificed immediately or after 1 or 24 h of recuperation. Splenocyte proliferative ability and NO concentrations were determined in whole splenocytes and purified T-cell populations, with and without the use of 20 mg/kg l-NMMA, a potent NOS inhibitor. RT-PCR was performed to determine the presence or absence of inducible NOS transcription after hypoxia and hypoxia followed by reoxygenation with and without l-NMMA. Results. Hypoxia followed by reoxygenation stimulated NO production in whole splenocytes, which correlated with a decrease in splenocyte proliferation. T-cell stimulation and NO production were not upregulated under these conditions. Additionally, NO generation occurred following hypoxia, even in the presence of l-NMMA. Conclusion. These results demonstrate that NO production after hypoxia may be due to an alternate, O 2-independent pathway. Further investigations into the generation of NO through alternate pathways may identify treatments for hypoxia-related injury.