Does exhaled nitric oxide predict asthma exacerbation in children?

Asthma is a chronic complex disorder of the respiratory tract that affects millions of people globally, a large percentage of which are children. Triggered by a host of factors such as allergens and changes in temperature, the pathophysiologic and clinical indices vary among patients and have contributed to difficulties in overall management of asthma. Shortly after exhaled nitric oxide (eNO) was discovered in higher concentrations in asthma patients, it was shown to be superior to other markers such as PEFR, FEV1 and sputum eosinophils in screening asthma patients. Studies have also noted promising results regarding the use of eNO to predict asthma exacerbation in adults while in children, asthma symptoms have been observed to be good predictors of asthma exacerbation. Currently however, the potential of eNO as a predictor of asthma exacerbation in children is yet to be examined. The objective of this study was to assess eNO potential to predict asthma exacerbation in children by examining the relationship between eNO and changes in pulmonary function, asthma symptoms and rescue medication use.^ The primary study "Air Toxics and Asthma in Children" (ATAC), recruited children aged 9 to 14 years with labile persistent asthma diagnosed at least one year earlier. The data obtained from 30 study participants, included exhaled nitric oxide concentration, PEFR, FEV1, asthma symptoms and frequency of emergency medication use.^ Descriptive statistics, Pearson's and Spearman's correlation tests were followed by a simple linear regression in which eNO was the independent (predictor) variable while FEV1, PEFR, asthma symptoms and frequency of emergency medication use were the dependent (outcome) variables.^ Results showed that eNO was associated with percent change in FEV1, day time wheeze, night time shortness of breath, but correlated only weakly with PEFR, amplitude percent of mean PEFR, FEV1, percent change in FEV1 and asthma symptoms.^ Further research is imperative to better define the role of eNO and understand intrinsic pathologic mechanisms towards asthma management in children.^

Determinación de la actividad de la óxido nítrico sintetasa endotelial y del factor NF-kB, en la piel del paciente con pseudoangiomatosis eruptiva : informe preliminar

La pseudoangiomatosis eruptiva se caracteriza por la aparición brusca de múltiples pápulas eritematosas, asintomáticas, rodeadas de un halo blanquecino, con remisión espontánea. Histológicamente se observa dilatación vascular con escaso infiltrado inflamatorio. Su etiología permanece incierta a pesar de ser relacionada con virus o picaduras de insectos. Basados en el compromiso vascular, el objetivo del trabajo fue investigar la actividad de la enzima endotelial oxido nítrico sintetasa (eNOS) y la expresión del factor NF-kB por inmunohistoquimica en un intento de esclarecer su patogenia. Material y métodos: Se estudiaron diez pacientes con diagnóstico clínico de pseudoangiomatosis eruptiva (PAE) que presentaron la dermatosis en forma epidémica. Se realizaron biopsias teñidas con Hematoxilina-Eosina y Tricrómico de Masson. Se efectuó estudio virológico de los pacientes Nª 4, 9 y 10 mediante determinaciones serológicas para echovirus, enterovirus, citomegalovirus, parvovirus B19 y hepatitis A, B y C. En cinco pacientes se obtuvo material para determinación de eNOS y NF-kB. Resultados: Todos los pacientes, 5 hombres y 5 mujeres presentaron pápulas eritematosas rodeadas por un halo blanquecino, especialmente en las extremidades, alrededor de las rodillas. Histológicamente mostraron vasos dilatados y células endoteliales prominentes con un infiltrado discreto perivascular. Todos los estudios serológicos fueron negativos. La actividad de eNOS fue significativamente menor comparada con la piel normal (p= 0,002) y la expresión de NF- ĸB fue fuertemente positiva en los vasos de la dermis papilar y reticular. Conclusiones: Todos los pacientes fueron afectados en verano, por lo que la picadura del mosquito debe ser considerada como un factor etiológico. La baja expresión de eNOS está relacionada con la vasodilatación y la expresión aumentada de NF-ĸB confirma que el proceso es de tipo inflamatorio.

Persistent inhibition of cell respiration by nitric oxide: Crucial role of S-nitrosylation of mitochondrial complex I and protective action of glutathione

Both reversible and irreversible inhibition of mitochondrial respiration have been reported following the generation of nitric oxide (NO) by cells. Using J774 cells, we have studied the effect of long-term exposure to NO on different enzymes of the respiratory chain. Our results show that, although NO inhibits complex IV in a way that is always reversible, prolonged exposure to NO results in a gradual and persistent inhibition of complex I that is concomitant with a reduction in the intracellular concentration of reduced glutathione. This inhibition appears to result from S-nitrosylation of critical thiols in the enzyme complex because it can be immediately reversed by exposing the cells to high intensity light or by replenishment of intracellular reduced glutathione. Furthermore, decreasing the concentration of reduced glutathione accelerates the process of persistent inhibition. Our results suggest that, although NO may regulate cell respiration physiologically by its action on complex IV, long-term exposure to NO leads to persistent inhibition of complex I and potentially to cell pathology.

Adenosine acts by A1 receptors to stimulate release of prolactin from anterior-pituitaries in vitro

Adenosine has been identified in the anterior pituitary gland and is secreted from cultured folliculostellate (FS) cells. To determine whether adenosine controls the secretion of anterior pituitary hormones in vitro, adenosine was incubated with anterior pituitaries. It stimulated prolactin (PRL) release at the lowest concentration used (10−10 M); the stimulation peaked at 10−8 M with a threefold increase in release and declined to minimal stimulation at 10−4 and 10−3 M. Follicle-stimulating hormone release was maximally inhibited at 10−8 M, whereas luteinizing hormone release was not significantly inhibited. Two selective A1 adenosine receptor antagonists (10−7 or 10−5 M) had no effect on basal PRL release, but either antagonist completely blocked the response to the most effective concentration of adenosine (10−8 M). In contrast, a highly specific A2 receptor antagonist (10−7 or 10−5 M) had no effect on basal PRL release or the stimulation of PRL release induced by adenosine (10−8 M). We conclude that adenosine acts to stimulate PRL release in vitro by activating A1 receptors. Since the A1 receptors decrease intracellular-free calcium, this would decrease the activation of nitric oxide synthase in the FS cells, resulting in decreased release of nitric oxide (NO). NO inhibits PRL release by activating guanylate cyclase that synthesizes cGMP from GTP; cGMP concentrations increase in the lactotrophs leading to inhibition of PRL release. In the case of adenosine, NO release from the FS cells decreases, resulting in decreased concentrations of NO in the lactotrophs, consequent decreased cGMP formation, and resultant increased PRL release.

Recombinant interleukin-1β dilates steelhead trout coronary microvessels : effect of temperature and role of the endothelium, nitric oxide and prostaglandins

© 2015. Published by The Company of Biologists Ltd. Acknowledgements We thank Wenjuan Xu and Xin Xu (Hein Lab) for their excellent instruction in microvessel techniques, Dr David Heeley (Biochemistry Department, MUN) for assistance with selecting an appropriate (non-vasoactive) protein stabilizer, Dr Zou (SFIRC, Aberdeen) for advice with regards to the use of rIL-1β and Gordon Nash (Gamperl Lab) for his assistance with the rIL-1β purification protocol. Funding This research was supported by a Natural Sciences and Engineering Research Council of Canada Discovery Grant [RGPIN249926] and Accelerator Supplement [RGPAS412325-2011] to A.K.G. a National Institutes of Health Grant [EY018420] to T.W.H., and a doctoral fellowship from Fundaçã o para a Ciência e a Tecnologia, Portugal [SFRH/BD/27497/2006] to I.A.S.F.C. Deposited in PMC for release after 12 months.

A molecular basis for nitric oxide sensing by soluble guanylate cyclase

Nitric oxide (NO) functions as a signaling agent by activation of the soluble isoform of guanylate cyclase (sGC), a heterodimeric hemoprotein. NO binds to the heme of sGC and triggers formation of cGMP from GTP. Here we report direct kinetic measurements of the multistep binding of NO to sGC and correlate these presteady state events with activation of enzyme catalysis. NO binds to sGC to form a six-coordinate, nonactivated, intermediate (kon > 1.4 × 108 M−1⋅s−1 at 4°C). Subsequent release of the axial histidine heme ligand is shown to be the molecular step responsible for activation of the enzyme. The rate at which this step proceeds also depends on NO concentration (k = 2.4 × 105 M−1⋅s−1 at 4°C), thus identifying a novel mode of regulation by NO. NO binding to the isolated heme domain of sGC was also rapid (k = 7.1 ± 2 × 108 M−1⋅s−1 at 4°C); however, no intermediate was observed. The data show that sGC acts as an extremely fast, specific, and highly efficient trap for NO and that cleavage of the iron-histidine bond provides the driving force for activation of sGC. In addition, the kinetic data indicate that transport or stabilization of NO is not necessary for effective signal transmission.

Targeted gene deletion of heme oxygenase 2 reveals neural role for carbon monoxide

Neuronal nitric oxide synthase (nNOS) generates NO in neurons, and heme-oxygenase-2 (HO-2) synthesizes carbon monoxide (CO). We have evaluated the roles of NO and CO in intestinal neurotransmission using mice with targeted deletions of nNOS or HO-2. Immunohistochemical analysis demonstrated colocalization of nNOS and HO-2 in myenteric ganglia. Nonadrenergic noncholinergic relaxation and cyclic guanosine 3′,5′ monophosphate elevations evoked by electrical field stimulation were diminished markedly in both nNOSΔ/Δ and HO-2Δ/Δ mice. In wild-type mice, NOS inhibitors and HO inhibitors partially inhibited nonadrenergic noncholinergic relaxation. In nNOSΔ/Δ animals, NOS inhibitors selectively lost their efficacy, and HO inhibitors were inactive in HO-2Δ/Δ animals.

NF-κB and AP-1 Activation by Nitric Oxide Attenuated Apoptotic Cell Death in RAW 264.7 Macrophages

A toxic dose of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO; 1 mM) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenuated by cellular preactivation with a nontoxic dose of GSNO (200 μM) or with lipopolysaccharide, interferon-γ, and NG-monomethyl-l-arginine (LPS/IFN-γ/NMMA) for 15 h. Protection from apoptosis was achieved by expression of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mechanisms leading to Cox-2 expression. LPS/IFN-γ/NMMA prestimulation activated nuclear factor (NF)-κB and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-κB and activator protein-1 (AP-1). NF-κB supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-κB site derived from the murine Cox-2 promoter, confirmed NF-κB activation after NO addition. An NF-κB decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-γ/NMMA but also inducible protection. The importance of AP-1 for Cox-2 expression and cell protection by low-level NO was substantiated by using the extracellular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfection of a dominant-negative c-Jun mutant further attenuated Cox-2 expression by low-level NO. Whereas cytokine-mediated Cox-2 induction relies on NF-κB activation, a low-level NO–elicited Cox-2 response required activation of both NF-κB and AP-1.

Direct nitric oxide signal transduction via nitrosylation of iron-sulfur centers in the SoxR transcription activator

Nitric oxide (NO) has diverse roles in intercellular communication and (at higher levels) in immune-mediated cell killing. NO reacts with many cellular targets, with cell-killing effects correlated to inactivation of key enzymes through nitrosylation of their iron-sulfur centers. SoxR protein, a redox-sensitive transcription activator dependent on the oxidation state of its binuclear iron-sulfur ([2Fe-2S]) centers, is also activated in Escherichia coli on exposure to macrophage-generated NO. We show here that SoxR activation by NO occurs through direct modification of the [2Fe-2S] centers to form protein-bound dinitrosyl-iron-dithiol adducts, which we have observed both in intact bacterial cells and in purified SoxR after NO treatment. Functional activation through nitrosylation of iron-sulfur centers contrasts with the inactivation typically caused by this modification. Purified, nitrosylated SoxR has transcriptional activity similar to that of oxidized SoxR and is relatively stable. In contrast, nitrosylated SoxR is short-lived in intact cells, indicative of mechanisms that actively dispose of nitrosylated iron-sulfur centers.

Nitric oxide inhibits tumor necrosis factor-α-induced apoptosis by reducing the generation of ceramide

Apoptosis triggered by death receptors proceeds after defined signal-transduction pathways. Whether signaling at the receptor level is regulated by intracellular messengers is still unknown. We have investigated the role of two messengers, ceramide and nitric oxide (NO), on the apoptotic pathway activated in human monocytic U937 cells by tumor necrosis factor-α (TNF-α) working at its p55 receptor. Two transduction events, the receptor recruitment of the adapter protein, TRADD, and the activation of the initiator caspase, caspase 8, were investigated. When administered alone, neither of the messengers had any effect on these events. In combination with TNF-α, however, ceramide potentiated, whereas NO inhibited, TNF-α-induced TRADD recruitment and caspase 8 activity. The effect of NO, which was cGMP-dependent, was due to inhibition of the TNF-α-induced generation of ceramide. Our results identify a mechanism of regulation of a signal-transduction pathway activated by death receptors.

No ·NO from NO synthase

The nitric-oxide synthase (NOS; EC 1.14.13.39) reaction is formulated as a partially tetrahydrobiopterin (H4Bip)-dependent 5-electron oxidation of a terminal guanidino nitrogen of l-arginine (Arg) associated with stoichiometric consumption of dioxygen (O2) and 1.5 mol of NADPH to form l-citrulline (Cit) and nitric oxide (·NO). Analysis of NOS activity has relied largely on indirect methods such as quantification of nitrite/nitrate or the coproduct Cit; we therefore sought to directly quantify ·NO formation from purified NOS. However, by two independent methods, NOS did not yield detectable ·NO unless superoxide dismutase (SOD; EC 1.15.1.1) was present. In the presence of H4Bip, internal ·NO standards were only partially recovered and the dismutation of superoxide (O2⨪), which otherwise scavenges ·NO to yield ONOO−, was a plausible mechanism of action of SOD. Under these conditions, a reaction between NADPH and ONOO− resulted in considerable overestimation of enzymatic NADPH consumption. SOD lowered the NADPH:Cit stoichiometry to 0.8–1.1, suggesting either that additional reducing equivalents besides NADPH are required to explain Arg oxidation to ·NO or that ·NO was not primarily formed. The latter was supported by an additional set of experiments in the absence of H4Bip. Here, recovery of internal ·NO standards was unaffected. Thus, a second activity of SOD, the conversion of nitroxyl (NO−) to ·NO, was a more likely mechanism of action of SOD. Detection of NOS-derived nitrous oxide (N2O) and hydroxylamine (NH2OH), which cannot arise from ·NO decomposition, was consistent with formation of an ·NO precursor molecule such as NO−. When, in the presence of SOD, glutathione was added, S-nitrosoglutathione was detected. Our results indicate that ·NO is not the primary reaction product of NOS-catalyzed Arg turnover and an alternative reaction mechanism and stoichiometry have to be taken into account.

Mutagenesis associated with nitric oxide production in transgenic SJL mice

We recently reported development of an experimental model for the study of nitric oxide (NO·) toxicology in vivo. SJL mice were injected with superantigen-bearing RcsX (pre-B-cell lymphoma) cells, which migrated to the spleen and lymph nodes, where their rapid growth induced activation of macrophages to produce large amounts of NO· over a period of several weeks. In the experiments described here, we used this model to investigate mutagenesis in splenocytes exposed to NO· during RcsX cell growth. Transgenic mice were produced by crossbreeding animals of the pUR288 transgenic C57BL/6 and SJL strains. RcsX cells were injected into F1 mice and NO· production was confirmed by quantification of urinary nitrate, the ultimate metabolite of NO·. Mutant frequency in the lacZ gene of the pUR288 plasmid was determined in DNA isolated from spleen (target) and kidney (nontarget) tissues. A significant elevation in mutant frequency was found in the spleen, but not in the kidney, of tumor-bearing mice. Furthermore, increases in mutant frequency in the spleen as well as NO· production were abrogated by administration of N-methylarginine, a NO· inhibitor, to mice following injection of RcsX cells. These results indicate that NO· had mutagenic activity in RcsX tumor-bearing mice and thus support a possible role for its involvement in the carcinogenic process.

Nitric oxide-related species inhibit evoked neurotransmission but enhance spontaneous miniature synaptic currents in central neuronal cultures

Nitric oxide (NO·) does not react significantly with thiol groups under physiological conditions, whereas a variety of endogenous NO donor molecules facilitate rapid transfer to thiol of nitrosonium ion (NO+, with one less electron than NO·). Here, nitrosonium donors are shown to decrease the efficacy of evoked neurotransmission while increasing the frequency of spontaneous miniature excitatory postsynaptic currents (mEPSCs). In contrast, pure NO· donors have little effect (displaying at most only a slight increase) on the amplitude of evoked EPSCs and frequency of spontaneous mEPSCs in our preparations. These findings may help explain heretofore paradoxical observations that the NO moiety can either increase, decrease, or have no net effect on synaptic activity in various preparations.

Poly(ADP-ribosyl)ation basally activated by DNA strand breaks reflects glutamate–nitric oxide neurotransmission

Poly(ADP-ribose) polymerase (PARP) transfers ADP ribose groups from NAD+ to nuclear proteins after activation by DNA strand breaks. PARP overactivation by massive DNA damage causes cell death via NAD+ and ATP depletion. Heretofore, PARP has been thought to be inactive under basal physiologic conditions. We now report high basal levels of PARP activity and DNA strand breaks in discrete neuronal populations of the brain, in ventricular ependymal and subependymal cells and in peripheral tissues. In some peripheral tissues, such as skeletal muscle, spleen, heart, and kidney, PARP activity is reduced only partially in mice with PARP-1 gene deletion (PARP-1−/−), implicating activity of alternative forms of PARP. Glutamate neurotransmission involving N-methyl-d-aspartate (NMDA) receptors and neuronal nitric oxide synthase (nNOS) activity in part mediates neuronal DNA strand breaks and PARP activity, which are diminished by NMDA antagonists and NOS inhibitors and also diminished in mice with targeted deletion of nNOS gene (nNOS−/−). An increase in NAD+ levels after treatment with NMDA antagonists or NOS inhibitors, as well as in nNOS−/− mice, indicates that basal glutamate-PARP activity regulates neuronal energy dynamics.

Reactive oxygen species and nitric oxide mediate plasticity of neuronal calcium signaling

Reactive oxygen species (ROS) and nitric oxide (NO) are important participants in signal transduction that could provide the cellular basis for activity-dependent regulation of neuronal excitability. In young rat cortical brain slices and undifferentiated PC12 cells, paired application of depolarization/agonist stimulation and oxidation induces long-lasting potentiation of subsequent Ca2+ signaling that is reversed by hypoxia. This potentiation critically depends on NO production and involves cellular ROS utilization. The ability to develop the Ca2+ signal potentiation is regulated by the developmental stage of nerve tissue, decreasing markedly in adult rat cortical neurons and differentiated PC12 cells.