Nitric Oxide Production Capacity Research Articles

Genomic capacities for Reactive Oxygen Species metabolism across marine phytoplankton.

Marine phytoplankton produce and scavenge Reactive Oxygen Species, to support cellular processes, while limiting damaging reactions. Some prokaryotic picophytoplankton have, however, lost all genes encoding scavenging of hydrogen peroxide. Such losses of metabolic function can only apply to Reactive Oxygen Species which potentially traverse the cell membrane outwards, before provoking damaging intracellular reactions. We hypothesized that cell radius influences which elements of Reactive Oxygen Species metabolism are partially or fully dispensable from a cell. We therefore investigated genomes and transcriptomes from diverse marine eukaryotic phytoplankton, ranging from 0.4 to 44 μm radius, to analyze the genomic allocations encoding enzymes metabolizing Reactive Oxygen Species. Superoxide has high reactivity, short lifetimes and limited membrane permeability. Genes encoding superoxide scavenging are ubiquitous across phytoplankton, but the fractional gene allocation decreased with increasing cell radius, consistent with a nearly fixed set of core genes for scavenging superoxide pools. Hydrogen peroxide has lower reactivity, longer intracellular and extracellular lifetimes and readily crosses cell membranes. Genomic allocations to both hydrogen peroxide production and scavenging decrease with increasing cell radius. Nitric Oxide has low reactivity, long intracellular and extracellular lifetimes and readily crosses cell membranes. Neither Nitric Oxide production nor scavenging genomic allocations changed with increasing cell radius. Many taxa, however, lack the genomic capacity for nitric oxide production or scavenging. The probability of presence of capacity to produce nitric oxide decreases with increasing cell size, and is influenced by flagella and colony formation. In contrast, the probability of presence of capacity to scavenge nitric oxide increases with increasing cell size, and is again influenced by flagella and colony formation.

Abstract 13076: A Rare Stopgain Variant in SLC25A2 Associates With Low Nitric Oxide and Poor Clinical Outcome in Hypoplastic Left Heart Syndrome

Introduction: Mitochondrial respiration defects accompanied by low nitric oxide (NO) are associated with heart failure in congenital heart disease (CHD) patients with hypoplastic left heart syndrome (HLHS). Here we investigated the genetic etiology of low NO in patients with CHD. Hypothesis: Mutations in mitochondrial-related genes involved in NO metabolism contributes to the low NO associated risk for heart failure and need for heart transplantation in HLHS. Methods and Results: We performed a case-control association study among 588 white CHD cases including 41 with HLHS, and 24,143 white controls from the genome aggregation database (gnomAD). A rare conserved stopgain variant (rs562886845) in SLC25A2 encoding mitochondrial ornithine transporter was significantly enriched among the HLHS patients with poor clinical outcome (Group II, Table 1), with 15.8% carrying this mutation, yielding odds ratio of 17.07 ( p = 5.23 х 10 -4 ) in comparison to CHD patients without left ventricular outflow tract obstruction or 7.63 ( p =2.68 х 10 –3 ) in comparison to gnomAD control cohort. Significantly, SLC25A2 is the only mitochondrial transporter exporting asymmetric dimethylarginine, a natural NO synthase inhibitor. The three HLHS patients with this SLC25A2 variant showed very low nasal NO (nNO), a proxy for endogenous NO production capacity. All three patients had received heart transplant, with two being heterozygous and one homozygous for this variant. Significantly, the HLHS patient with the homozygous mutation had nNO at background levels (2.8 nl/min at 7 years of age as compared to >200 nl/min expected nNO). Conclusions: Our findings uncovered an essential role for SLC25A2 in determining NO production capacity in patients with HLHS. As all three HLHS patients with this variant and very low nNO survived with heart transplant, this variant should be further investigated as a genetic marker for predicting the need for early heart transplant in HLHS.

More Data “In Support” of the Ross

The report from Abeln and colleagues1 in this issue of The Annals of Thoracic Surgery is to be commended for its completeness of follow-up and overall outstanding results with survival and freedom from reoperation at 15 years. Furthermore 69% of autograft reoperations were valve-sparing operations. Perhaps the most important facet of this report is that it provides further compelling evidence that the Ross-Yacoub procedure is, to quote Dr David, “the best operation for young and middle age adults with aortic stenosis.”2 The fundamental driving force for this clinical advantage is that the autograft is alive—the leaflets retain the normal aortic valve’s capacity for nitric oxide production (antithrombotic) and contractile and neurohumoral responsiveness (valve performance and durability).

Cord blood-derived endothelial colony-forming cell function is disrupted in congenital diaphragmatic hernia.

Vascular growth is necessary for normal lung development. Although endothelial progenitor cells (EPCs) play an important role in vascularization, little is known about EPC function in congenital diaphragmatic hernia (CDH), a severe neonatal condition that is associated with pulmonary hypoplasia. We hypothesized that the function of endothelial colony-forming cells (ECFCs), a type of EPC, is impaired in CDH. Cord blood (CB) was collected from full-term CDH patients and healthy controls. We assessed CB progenitor cell populations as well as plasma vascular endothelial growth factor (VEGF) and stromal cell-derived factor 1α (SDF1α) levels. CB ECFC clonogenicity; growth kinetics; migration; production of VEGF, SDF1α, and nitric oxide (NO); vasculogenic capacity; and mRNA expression of VEGF-A, fms-related tyrosine kinase 1 (FLT1), kinase insert domain receptor (KDR), nitric oxide synthase (NOS) 1-3, SDF1, and chemokine (C-X-C motif) receptor 4 (CXCR4) were also assessed. Compared with controls, CB ECFCs were decreased in CDH. CDH ECFCs had reduced potential for self-renewal, clonogenicity, proliferation, and migration. Their capacity for NO production was enhanced but their response to VEGF was blunted in CDH ECFCs. In vivo potential for de novo vasculogenesis was reduced in CDH ECFCs. There was no difference in CB plasma VEGF and SDF1α concentrations, VEGF and SDF1α production by ECFCs, and ECFC mRNA expression of VEGF-A, FLT1, KDR, NOS1-3, SDF1, and CXCR4 between CDH and control subjects. In conclusion, CB ECFC function is disrupted in CDH, but these changes may be caused by mechanisms other than alteration of VEGF-NO and SDF1-CXCR4 signaling.

L-Arginine Attenuates Cardiac Dysfunction, But Further Down-Regulates α-Myosin Heavy Chain Expression in Isoproterenol-Induced Cardiomyopathy.

In view of previously reported increased capacity for nitric oxide production, we suggested that l-arginine (ARG), the nitric oxide synthase (NOS) substrate, supplementation would improve cardiac function in isoproterenol (ISO)-induced heart failure. Male Wistar rats were treated with ISO for 8days (5mg/kg/day, i.p.) or vehicle. ARG was given to control (ARG) and ISO-treated (ISO+ARG) rats in water (0.4g/kg/day). ISO administration was associated with 40% mortality, ventricular hypertrophy, decreased heart rate, left ventricular dysfunction, fibrosis and ECG signs of ischaemia. RT-PCR showed increased mRNA levels of cardiac hypertrophy marker atrial natriuretic peptide, but not BNP, decreased expression of myosin heavy chain isoform MYH6 and unaltered expression of pathological MYH7. ISO increased the protein levels of endothelial nitric oxide synthase, but at the same time it markedly up-regulated mRNA and protein levels of gp91phox, a catalytical subunit of superoxide-producing NADPH oxidase. Fibrosis was markedly increased by ISO. ARG treatment moderately ameliorated left ventricular dysfunction, but was without effect on cardiac hypertrophy and fibrosis. Combination of ISO and ARG led to a decrease in cav-1 expression, a further increase in MYH7 expression and a down-regulation of MYH6 that inversely correlated with gp91phox mRNA levels. Although ARG, at least partially, improved ISO-impaired basal left ventricular systolic function, it failed to reduce cardiac hypertrophy, fibrosis, oxidative stress and mortality. The protection of contractile performance might be related to increased capacity for nitric oxide production and the up-regulation of MYH7 which may compensate for the marked down-regulation of the major MYH6 isoform.

Estrogen attenuates lipopolysaccharide-induced nitric oxide production in macrophages partially via the nongenomic pathway

Steroid hormones exert genotropic effects through members of the nuclear hormone receptor family. In the present study, we examined the effects of 17β-estradiol (E2) on nitric oxide (NO) production following lipopolysaccharide (LPS) stimulation and investigated the mechanisms in mouse bone marrow-derived macrophages (BMMs). E2 alone did not affect NO production. In contrast, E2 inhibited LPS-induced production of NO in BMMs. Using a cell-impermeable E2 conjugated to BSA (E2–BSA), which has been used to investigate the nongenomic effects of estrogen, we found that the increase in NO production induced by LPS was also attenuated. In addition, the intracellular estrogen receptor blocker, ICI 182780, only partially antagonized the total effects of E2 on LPS-stimulated NO production capacity. E2 also attenuated the LPS activation of p38 mitogen-activated protein kinase (MAPK) but not that of extracellular-regulated protein kinase 1/2 (ERK1/2) and c-Jun NH2-terminal kinase (JNK). This attenuation was not abrogated by ICI 182780. Moreover, the p38 inhibitor, SB 203580, greatly reduced the LPS-induced NO production, and the remaining NO levels were no longer regulated by E2. Additionally, E2–BSA inhibited LPS-mediated changes in p38 MAPK activation to the same extent as E2. Moreover, E2 and E2–BSA inhibited LPS-induced activation of nuclear factor-kappa B (NF-κB) and activator protein 1 (AP-1). This inhibitory effect of E2 was only partially antagonized by ICI 182780. Taken together, these results suggest that E2 has an inhibitory effect on LPS-induced NO production in BMMs through inhibition of p38 MAPK phosphorylation, and blockade of NF-κB and AP-1 activation. These effects are mediated at least in part via a nongenomic pathway.

Glioma Stem Cell Proliferation and Tumor Growth Are Promoted by Nitric Oxide Synthase-2

Malignant gliomas are aggressive brain tumors with limited therapeutic options, and improvements in treatment require a deeper molecular understanding of this disease. As in other cancers, recent studies have identified highly tumorigenic subpopulations within malignant gliomas, known generally as cancer stem cells. Here, we demonstrate that glioma stem cells (GSCs) produce nitric oxide via elevated nitric oxide synthase-2 (NOS2) expression. GSCs depend on NOS2 activity for growth and tumorigenicity, distinguishing them from non-GSCs and normal neural progenitors. Gene expression profiling identified many NOS2-regulated genes, including the cell-cycle inhibitor cell division autoantigen-1 (CDA1). Further, high NOS2 expression correlates with decreased survival in human glioma patients, and NOS2 inhibition slows glioma growth in a murine intracranial model. These data provide insight into how GSCs are mechanistically distinct from their less tumorigenic counterparts and suggest that NOS2 inhibition may be an efficacious approach to treating this devastating disease.

Both plant and bacterial nitrate reductases contribute to nitric oxide production in Medicago truncatula nitrogen-fixing nodules.

Nitric oxide (NO) is a signaling and defense molecule of major importance in living organisms. In the model legume Medicago truncatula, NO production has been detected in the nitrogen fixation zone of the nodule, but the systems responsible for its synthesis are yet unknown and its role in symbiosis is far from being elucidated. In this work, using pharmacological and genetic approaches, we explored the enzymatic source of NO production in M. truncatula-Sinorhizobium meliloti nodules under normoxic and hypoxic conditions. When transferred from normoxia to hypoxia, nodule NO production was rapidly increased, indicating that NO production capacity is present in functioning nodules and may be promptly up-regulated in response to decreased oxygen availability. Contrary to roots and leaves, nodule NO production was stimulated by nitrate and nitrite and inhibited by tungstate, a nitrate reductase inhibitor. Nodules obtained with either plant nitrate reductase RNA interference double knockdown (MtNR1/2) or bacterial nitrate reductase-deficient (napA) and nitrite reductase-deficient (nirK) mutants, or both, exhibited reduced nitrate or nitrite reductase activities and NO production levels. Moreover, NO production in nodules was found to be inhibited by electron transfer chain inhibitors, and nodule energy state (ATP-ADP ratio) was significantly reduced when nodules were incubated in the presence of tungstate. Our data indicate that both plant and bacterial nitrate reductase and electron transfer chains are involved in NO synthesis. We propose the existence of a nitrate-NO respiration process in nodules that could play a role in the maintenance of the energy status required for nitrogen fixation under oxygen-limiting conditions.

Phenylhydrazine-induced anemia causes nitric-oxide-mediated upregulation of the angiogenic pathway in Notothenia coriiceps

Antarctic icefishes possess several cardiovascular characteristics that enable them to deliver oxygen adequately in the absence of hemoglobin (Hb). To gain insight into mechanisms driving development of these cardiovascular characteristics of icefish, we chemically induced severe anemia in a red-blooded notothenioid, Notothenia coriiceps. After 10 days of treatment with phenylhydrazine HCl, the hematocrit and Hb concentration of N. coriiceps decreased by >90% and >70%, respectively. Anemic fish exhibited a significantly higher concentration of nitric oxide (NO) metabolites in their plasma compared with that of control animals, indicating that corporeal levels of NO are higher in anemic animals than in control fish. The activity of nitric oxide synthase (NOS) was measured in brain, retina, pectoral muscle and ventricle of control and anemic animals. With the exception of retina, no significant differences in NOS activities were observed, indicating that the increase in plasma NO metabolites is due to loss of Hb, which normally plays a major role in the degradation of NO, and not due to an overall increase in the capacity for NO production. To determine whether loss of Hb can stimulate remodeling of the cardiovascular system, we measured expression of HIF-1alpha, PHD2 and VEGF mRNA in retinae of control and anemic fish. Expression of all three genes was higher in anemic animals compared with control N. coriiceps, suggesting a causative relationship between loss of Hb and induction of angiogenesis that probably is mediated through nitric oxide signaling.

Regional heterogeneity of substance P-induced endothelium-dependent contraction, relaxation, and -independent contraction in rabbit pulmonary arteries

Aims The present study examined whether substance P (SP)-induced endothelium-dependent TXA 2-mediated contraction (EDC), nitric oxide (NO)-mediated relaxation (EDR), and endothelium-independent contraction (EIC) are different between the rabbit proximal and distal intrapulmonary arteries. Main methods The helically cut strips of isolated proximal and distal arteries were fixed vertically between hooks in organ bath, and changes in isometric tension were measured. Key findings SP-induced EDC was greater in the distal than proximal arteries, and EDR was greater in the proximal than distal arteries. However, under the complete blockade of NK 2 receptors and NO production, SP (10 − 9 –3 × 10 − 7 M)-induced EDC did not differ between proximal and distal arteries. Under the complete blockade of NK 2 receptors and TXA 2 production, SP (3 × 10 − 10 –3 × 10 − 8 M)-induced EDR was greater in the proximal than distal arteries. Neither contraction induced by U-46619, a TXA 2 agonist, nor relaxation by sodium nitroprusside, an NO donor, was different between both portions of the arteries. Both ionomycin (10 − 8 M)- and l-arginine (1 mM)-induced EDRs were also significantly greater in the proximal than distal arteries. Under the blockade of NK 1 receptors and NO and TXA 2 production, SP (10 − 7 M)-induced EIC was greater in the distal than proximal arteries. In summary, the capacity for NO production is higher in the proximal than distal arteries, resulting in SP-induced higher EDR and lower EDC in the proximal arteries. Significance These regional differences in responses to SP may play important roles in maintaining the homogenous distribution of blood flow in the lung.

Heart mitochondrial nitric oxide synthase is upregulated in male rats exposed to high altitude (4,340 m)

Male rats exposed for 21 days to high altitude (4,340 m) responded with arrest of weight gain and increased hematocrit and testosterone levels. High altitude significantly (58%) increased heart mitochondrial nitric oxide (NO) synthase (mtNOS) activity, whereas heart cytosolic endothelial NOS (eNOS) and liver mtNOS were not affected. Western blot analysis found heart mitochondria reacting only with anti-inducible NOS (iNOS) antibodies, whereas the postmitochondrial fraction reacted with anti-iNOS and anti-eNOS antibodies. In vitro-measured NOS activities allowed the estimation of cardiomyocyte capacity for NO production, a value that increased from 57% (sea level) to 79 nmol NO.min(-1).g heart(-1) (4,340 m). The contribution of mtNOS to total cell NO production increased from 62% (sea level) to 71% (4340 m). Heart mtNOS activity showed a linear relationship with hematocrit and a biphasic quadratic association with estradiol and testosterone. Multivariate analysis showed that exposure to high altitude linearly associates with hematocrit and heart mtNOS activity, and that testosterone-to-estradiol ratio and heart weight were not linearly associated with mtNOS activity. We conclude that high altitude triggers a physiological adaptive response that upregulates heart mtNOS activity and is associated in an opposed manner with the serum levels of testosterone and estradiol.

Increased exhaled nitric oxide in active pulmonary tuberculosis due to inducible NO synthase upregulation in alveolar macrophages.

Nitric oxide (NO) plays an important role in resistance to Mycobacterium tuberculosis infection. Our aim was to determine whether inducible NO synthase (iNOS) expression and generation of reactive nitrogen intermediates (RNI) by alveolar macrophages (AM) are increased in patients infected with M. tuberculosis. NO levels in the exhaled air of 19 active pulmonary tuberculosis (TB) and 14 control subjects were measured using a chemiluminescence NO analyser. The expression of iNOS on AM was studied by labelling AM with anti-mac iNOS polyclonal antibody analysed with a flow cytometer. The spontaneous generation of RNI by cultured AM was also measured. Data are presented as mean+/-SEM. The level of NO in exhaled air was higher in patients with active TB (16.2+/-1.2 parts per billion (ppb)) compared to control subjects (6.5+/-0.9 ppb), p<0.0001. Exhaled NO decreased with anti-TB treatment. Compared to control subjects (29.0+/-4.5 fluorescence intensity (FI)), iNOS expression on AM was upregulated in TB patients (86.3+/-12.5 FI) p<0.001 and the capacity for spontaneous generation of nitrite was enhanced. Nitrite production was inhibited by N(G)-monomethyl-L-arginine (L-NMMA), a competitive inhibitor of iNOS. The expression of iNOS on AM was related to the concentration of exhaled NO (r=0.66, p<0.001) and the nitrite generation capacity of AM (r(s)=0.77, p<0.001). We conclude that the increase in exhaled nitric oxide observed in patients with active pulmonary tuberculosis is due to an upregulation of inhaled NO synthase expression in alveolar macrophages which have an enhanced capacity for nitric oxide production.

Protection of mice previously infected with Plasmodium vinckei against subsequent Salmonella enteritidis infection is associated with nitric oxide production capacity.

When mice previously cured of a Plasmodium vinckei infection were subsequently infected with Salmonella enteritidis the course of bacterial infection was significantly retarded, showing increased survival duration as compared with control infections in naive mice. Moreover, on stimulation with lipopolysaccharide and/or interferon-gamma, spleen cells from malaria-cured mice showed an increased capacity to produce tumor necrosis factor, interleukin 6, and reactive nitrogen intermediates as compared with spleen cells from naive mice. However, no significant variation in the capacity of spleen cells to release reactive oxygen intermediates was observed between previously malarious and naive mice. The most significant increases were observed in the capacity for reactive nitrogen intermediate production after P. vinckei malaria. These results suggest that the observed protection of mice against salmonellosis in the convalescent phase after malaria may be mediated by nitric oxides.

Ontogeny of nitric oxide in the pulmonary vasculature

The vasodilator molecule nitric oxide is critically involved in the successful cardiopulmonary transition from fetal to postnatal life. It is produced in the pulmonary endothelium by the endothelial isoform of the enzyme nitric oxide synthase. The expression of endothelial nitric oxide synthase in the lung increases dramatically during late gestation, optimizing the capacity for nitric oxide production at the time of birth. Studies in cultured cell models indicate that the developmental upregulation may be mediated by estrogen, and that the expression of the enzyme is also upregulated by oxygen. Pulmonary endothelial nitric oxide synthase expression is diminished in models of congenital diaphragmatic hernia and neonatal pulmonary hypertension induced by fetal ductal ligation. Thus, there is normally a marked developmental upregulation in endothelial nitric oxide synthase expression in the lung during late fetal life, and attenuated expression of the enzyme may contribute to the pathophysiology of a variety of forms of neonatal pulmonary vascular disease.

Nitric oxide stimulates guanylate cyclase and regulates sodium transport in rabbit proximal tubule.

The proximal tubule contains the target for nitric oxide (NO), soluble guanylate cyclase, and has the capacity for NO production. Inhibition of renal NO synthesis reduces fractional excretion of lithium, suggesting an inhibitory effect of NO on proximal tubule Na+ transport. The present studies determined direct effects of donors of NO in rabbit proximal tubule. In both freshly isolated proximal tubule segments and in primary cultures of proximal tubule cells, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP) caused dose-dependent increases in guanosine 3',5'-cyclic monophosphate (cGMP). SNAP was more potent than SNP in stimulating cGMP; this was associated with an enhanced production of nitrite, the stable end-product of NO. In rabbit proximal tubule cells, SNP or SNAP (10(-3) M) significantly inhibited the activity of the apical Na+/H+ exchanger, determined by assay of amiloride-sensitive 22Na+ uptake (% inhibition: SNP, 34.90 +/- 5.52%, P < 0.001; SNAP, 30.77 +/- 8.20%, P < 0.002). To determine the role of cGMP in mediating these effects, proximal tubule cells were incubated with the membrane-permeable analogue, 8-bromoguanosine 3',5'-cyclic monophosphate (8-BrcGMP). Na+/H+ exchange was significantly inhibited by 8-BrcGMP (10(3)M) (% inhibition: 32.40 +/- 9.06%: P < 0.05). The inhibitor of soluble guanylate cyclase, LY-83583, caused partial inhibition of SNP-stimulated cGMP generation and partly blocked the inhibitory effect of SNP on Na+/H+ exchange. Protein kinase A (PKA) activity was not stimulated by SNP, indicating that potential cross-activation of PKA by cGMP did not mediate the effects of NO donors. These data indicate that NO stimulates soluble guanylate cyclase in rabbit proximal tubule and causes inhibition of Na-/H+ exchange. This is at least partly mediated by generation of cGMP. We conclude that NO is an important autocrine or paracrine factor directly regulating Na+ transport in the proximal tubule.

Release of endothelium derived nitric oxide in relation to pressure and flow

Endothelium derived nitric oxide (NO) is an important modulator of resting vascular tone. The aim of the study was to investigate the extent by which the rate of NO release is modulated by the two determinants of vascular conductance: pressure and flow. DESIGN AND EXPERIMENTAL MATERIAL: Porcine macrovascular endothelial cells cultured on microcarrier beads were used as a model in which the rate of NO release was determined photometrically. Columns packed with endothelial cell covered beads were perfused at different flow rates (2, 10, 20 ml.min-1) and perfusion pressures (ranging from 6 to 200 mm Hg). Release of endothelial cell derived NO was continuously quantified under basal and ATP stimulated conditions using a specific difference spectrophotometric assay. Increasing flow flow from 2 to 20 ml.min-1 enhanced the basal NO release from endothelial cells fivefold. ATP (10(-4) M) augmented the NO release from endothelial cells more than 10-fold at each flow level studied. The ATP induced NO release rapidly increased by a factor of 5.8 when flow was enhanced from 2 to 20 ml.min-1 (greater than 180 pmol.min-1.mg endothelial cell protein). Raising perfusion pressure from 6 to 200 mm Hg in endothelial cells did not affect the rate of basal NO release. (1) The rate of NO release from endothelial cells increases when flow is enhanced. (2) Endothelial cells possess a high capacity for NO production, permitting a rapid adjustment of NO release to changes in flow. (3) The rate of NO release is not causally related to changes in perfusion pressure.