Identification Of Nitric Oxide Research Articles

A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish

A Highly Stable Two-Photon Ratiometric Fluorescence Probe for Real-Time Biosensing and Imaging of Nitric Oxide in Brain Tissues and Larval Zebrafish

Identification of nitric oxide (NO)-responsive genes under hypoxia in tomato (Solanum lycopersicum L.) root

Flooding periods, as one probable consequence of climate change, will lead more frequently to plant hypoxic stress. Hypoxia sensing and signaling in the root, as the first organ encountering low oxygen, is therefore crucial for plant survival under flooding. Nitric oxide has been shown to be one of the main players involved in hypoxia signaling through the regulation of ERFVII transcription factors stability. Using SNP as NO donor, we investigated the NO-responsive genes, which showed a significant response to hypoxia. We identified 395 genes being differentially regulated under both hypoxia and SNP-treatment. Among them, 251 genes showed up- or down-regulation under both conditions which were used for further biological analysis. Functional classification of these genes showed that they belong to different biological categories such as primary carbon and nitrogen metabolism (e.g. glycolysis, fermentation, protein and amino acid metabolism), nutrient and metabolites transport, redox homeostasis, hormone metabolism, regulation of transcription as well as response to biotic and abiotic stresses. Our data shed light on the NO-mediated gene expression modulation under hypoxia and provides potential targets playing a role in hypoxia tolerance. These genes are interesting candidates for further investigating their role in hypoxia signaling and survival.

Expression of Concern: Identification of nitric oxide as an endogenous activator of the AMP-activated protein kinase in vascular endothelial cells.

Expression of Concern: Identification of nitric oxide as an endogenous activator of the AMP-activated protein kinase in vascular endothelial cells.

PRIMARY CILIARY DYSKINESIA IMMOTILE CILIA SYNDROME

Primary ciliary dyskinesia (PCD) is a genetically heterogeneous hereditary disease characterized by recurrent respiratory tract infections, decreased fertility, and situs inversus in 50% of cases. The core of the syndrome is the disturbance of mucociliary clearance due to the lack or defect of cilia leading to their partial or complete immobility. There are some tests for diagnostic PCD with specific benefits and limitations, but there is still no diagnostic «gold standard» yet. Identification of nitric oxide and nasal clearance of dye or saccharin are widely used as screening tests. Clearance of 99Tc-labeled colloidal albumin, high-speed video microscopy and transmission electron microscopy, the cell culture study and genetic testing are methods for the verification. Late identification of PCD is reported worldwide. There are no methods to control the development of PCD complications. The important role is played by the long-term and constant follow up (including spirometry, evaluation of pulmonary clearance and X-ray scanning).

Detection of Nitric Oxide via Electronic Paramagnetic Resonance in Mollusks.

Electronic paramagnetic resonance (EPR) is an appropriate tool to identify free radicals formed in tissues under normal as well as stressful conditions. Since nitric oxide (NO) as a free radical has paramagnetic properties it can be detected by EPR. The use of spin traps highly improves the sensitivity allowing NO identification, detection and quantification at room temperature in vitro and in vivo conditions. NO production in animals is almost exclusively associated to an enzyme family known as Nitric Oxide Synthases (NOSs). The digestive glands of mollusks are a major target for oxidative disruption related to environmental stress. A simple EPR-methodology to asses both, the presence of NO and its rate of generation in tissues from different mollusk species, is reported here.

Identification of Nitric Oxide as a Diffusible Intermediate in Aqueous Nitrogen-Containing Solutions Exposed to Ultrasound

Identification of Nitric Oxide as a Diffusible Intermediate in Aqueous Nitrogen-Containing Solutions Exposed to Ultrasound

Linking cardiovascular disease, erectile dysfunction and preeclampsia: NO problem

Nitric oxide (NO) is a colourless free radical gas, which has diverse and potentially opposing biological effects, depending on the local environment. The identification of NO and discovery of the mechanism of signalling are interesting tales on their own, leading to the Nobel Prize in Physiology or Medicine (1998) awarded to Murad, Furchgott and Ignarro. This article focuses on two very different – and important – consequences of the effects of NO on blood vessels: erectile dysfunction and pre-eclampsia.

Nitric Oxide: A Regulator of Cellular Function in Health and Disease

Nitric oxide (NO) is a gaseous messenger molecule synthesized from L-arginine and molecular oxygen by three different NO synthases, that is, neuronal (nNOS), endothelial (eNOS), and inducible (iNOS) form [1]. Since its discovery in the early 1980s by the three Nobel Laureates Furchgott, Ignarro & Murad [2], NO has been widely recognised as an important signalling molecule in many physiological processes. The initial identification of NO as an endothelium-derived relaxing factor (EDRF) [3] generated great interest in its function in vascular biology. Over the following years, however, the focus on NO research rapidly expanded from the vascular system to its role in immunity and inflammation, the nervous system, pregnancy, aging, and cell death. Many studies suggest that excessive or abnormal production of NO plays a crucial role in neuronal cell death associated with neurodegenerative disorders such as Alzheimer's and Parkinson's diseases, as well as various conditions of vascular dysfunction. At physiological levels, NO is essential for neuronal function, differentiation and survival through activation of signalling pathways that include the cyclic guanosine monophosphate (cGMP)/soluble guanylyl cyclase pathway [4] and S-nitrosylation, in which NO reversibly binds to thiol groups of proteins [5]. The vast network of NO-associated signalling paradigms further includes acetylation/deacetylation and methylation/demethylation modifications and peroxynitrite formation (leading to nitrotyrosination of protein residues), as well as modulation of gene expression via epigenetic changes [5–8]. How controlled S-nitrosylation/nitrotyrosination of proteins and activation of the NO/cGMP signalling pathway promote cellular survival and induce epigenetic changes while uncontrolled signalling promotes cell death and dysfunction remains to be elucidated. The aim of this Special Issue is to gather information encapsulating the above signalling pathways. The articles published in this Special Issue largely cover (1) NO signalling in neuronal function and disease as well as (2) vascular targets in endothelial function and dysfunction, both of which involve the broad range of actions of this signalling molecule. In order to understand the contribution of NO to neuronal dysfunction, one has to consider that NO is a crucial molecule in cellular physiology. Numerous studies show the involvement of NO in neuronal development, plasticity, excitability, and transmission [8–12]. However, the common mechanisms across several neurodegenerative disorders relate to the neurotoxicity of NO and its downstream reactive nitrogen species (RNS). Enhanced nitrotyrosine immunoreactivity is evident in brains from patients with Alzheimer's and Parkinson's disease. Nitrated proteins are associated with β-amyloid deposition and nitrotyrosination of Tau protein and synaptophysin has been reported in brain samples from patients with Alzheimer's disease. The potential downstream signalling pathways of these posttranslational modifications are discussed in the review by S. A. Bradley and J. R. Steinert in this Issue. The cellular roles of NO in neurodegenerative disease, such as Alzheimer's, are reviewed by R. Balez and L. Ooi with a focus on neurotoxicity versus neuroprotection while the role of aberrant NO signalling in neurodevelopmental disorders such as Fragile X syndrome is investigated in the study by E. Lima-Cabello et al. Other topics of this issue focus on aspects of vascular NO signalling with particular interest in fetoplacental dysfunction caused by abnormal eNOS regulation (as discussed by A. Leiva et al.). Endothelial function and eNOS regulation are essential for healthy cardiovascular responses but are also critical for adaptations during pregnancy. Several diseases associated with vascular dysfunction such as atherosclerosis, diabetes mellitus, hypertension, or preeclampsia involve altered NO signalling [13–15]. The paper by A. Leiva et al. reviews the mechanisms leading to abnormal NO signalling, which include reduced bioavailability of L-arginine (NOS substrate) and tetrahydrobiopterin (BH4), abnormal calcium-calmodulin signalling, and activation and inhibition of eNOS activity through phosphorylation of Ser1177 or Thr495, respectively [16]. NO has also been reported to play a crucial role in the transition of fetoplacental endothelial cells from a mitogenic to a metabolic phenotype in the macrocirculation, compared with a change from a metabolic to a mitogenic phenotype in the microcirculation in response to insulin in gestational diabetes mellitus [17]. Thus, the actions of NO are not only important during vascular adaptations to pregnancy and related dysfunctions but also essential during vascular signalling induced by physical training resulting in elevated nitrite and nitrate levels as reported in the study by A. M. Jacomini et al. Together, this Special Issue highlights the tremendous diversity of NO signalling pathways with regard to its function in health and disease. All contributing publications have emphasised that NO represents an important signalling molecule and future research required in this field will extend the understanding of the broad actions of NO. Luis Sobrevia Lezanne Ooi Scott Ryan Joern R. Steinert

Identification of nitric oxide in mitochondria of myometrium cell

Identification of nitric oxide in mitochondria of myometrium cell

Dopant titrating ion mobility spectrometry for trace exhaled nitric oxide detection

Ion mobility spectrometry (IMS) is a promising non-invasive tool for the analysis of exhaled gas and exhaled nitric oxide (NO), a biomarker for diagnosis of respiratory diseases. However, the high moisture in exhaled gas always brings about extra overlapping ion peaks and results in poor identification ability. In this paper, p-benzoquinone (PBQ) was introduced into IMS to eliminate the interference of overlapping ion peaks and realize the selective identification of NO. The overlapping ions caused by moisture were titrated by PBQ and then converted to hydrated PBQ anions (C6H4(H2O)n). The NO concentration could be determined by quantifying gas phase hydrated nitrite anions (N(H2O)n), product ions of NO. Under optimized conditions, a limit of detection (LOD) of about 1.4 ppbv and a linear range of 10–200 ppbv were obtained for NO even in 100% relative humidity (RH) purified air. Furthermore, this established method was applied to measure hourly the exhaled NO of eight healthy volunteers, and real-time monitoring the exhaled NO of an esophageal carcinoma patient during radical surgery. These results revealed the potential of the current dopant titrating IMS method in the measurement of exhaled NO for medical disease diagnosis.

Identification of nitric oxide as an endogenous inhibitor of 26S proteasomes in vascular endothelial cells.

The 26S proteasome plays a fundamental role in almost all eukaryotic cells, including vascular endothelial cells. However, it remains largely unknown how proteasome functionality is regulated in the vasculature. Endothelial nitric oxide (NO) synthase (eNOS)-derived NO is known to be essential to maintain endothelial homeostasis. The aim of the present study was to establish the connection between endothelial NO and 26S proteasome functionality in vascular endothelial cells. The 26S proteasome reporter protein levels, 26S proteasome activity, and the O-GlcNAcylation of Rpt2, a key subunit of the proteasome regulatory complex, were assayed in 26S proteasome reporter cells, human umbilical vein endothelial cells (HUVEC), and mouse aortic tissues isolated from 26S proteasome reporter and eNOS knockout mice. Like the other selective NO donors, NO derived from activated eNOS (by pharmacological and genetic approach) increased O-GlcNAc modification of Rpt2, reduced proteasome chymotrypsin-like activity, and caused 26S proteasome reporter protein accumulation. Conversely, inactivation of eNOS reversed all the effects. SiRNA knockdown of O-GlcNAc transferase (OGT), the key enzyme that catalyzes protein O-GlcNAcylation, abolished NO-induced effects. Consistently, adenoviral overexpression of O-GlcNAcase (OGA), the enzyme catalyzing the removal of the O-GlcNAc group, mimicked the effects of OGT knockdown. Finally, compared to eNOS wild type aortic tissues, 26S proteasome reporter mice lacking eNOS exhibited elevated 26S proteasome functionality in parallel with decreased Rpt2 O-GlcNAcylation, without changing the levels of Rpt2 protein. In conclusion, the eNOS-derived NO functions as a physiological suppressor of the 26S proteasome in vascular endothelial cells.

The Identification of Nitric Oxide as Endothelium-Derived Relaxing Factor

The identification of endothelium-derived relaxing factor as nitric oxide (NO) dramatically altered the course of vascular biology, as well as other biomedical disciplines. The ubiquity of this natural product of cell metabolism and the complexity of its biochemistry provide a rich source of molecular mediators of phenotype in health and disease.

Identification of NO induced and capacitation associated tyrosine phosphoproteins in buffalo (Bubalus bubalis) spermatozoa

To acquire the fertilizing competence, spermatozoa must undergo a cascade of physiological and biochemical changes collectively defined as capacitation. Compelling evidence signifies that the global increase in protein tyrosine phosphorylation is the driving factor for capacitation. In our laboratory, we previously demonstrated that nitric oxide (NO) induces capacitation in buffalo sperm and is associated with an increase in protein tyrosine phosphorylation. The aim of the present study is to identify the proteins undergo tyrosine phosphorylation during NO induced buffalo sperm capacitation using 2-D immunoblotting and mass spectrometry. The percentage of progressively motile and capacitated sperm was more in presence of l-arginine. Along with known tyrosine phosphoproteins like ATP synthase subunit beta, pyruvate dehydrogenase E1 component subunit beta, GST mu 3, F-actin capping protein subunit beta 2, GPD2 and VDAC2, interestingly novel tyrosine phosphoprotein substrates such as actin, serine/threonine-protein phosphatase PP1-gamma catalytic subunit, and glutamine synthetase were also identified which might be specific to the NO induced signaling and also emphasizes the species specificity with respect to tyrosine phosphorylation of proteins during capacitation. In conclusion, this study forms an essential step in delineating the proteins undergo tyrosine phosphorylation in response to NO induced signaling pathways during capacitation of buffalo sperm.

Exhaled nitric oxide in the diagnosis and management of bronchial asthma: From bench to bedside?

The identification of nitric oxide in exhaled breath and its role as a surrogate marker of eosinophilic inflammation of the airways led to rapid development in the area of its measurement. Standardized measurement techniques have been developed as well as simple, cheap, and reliable equipments. Although a number of factors have been identified as affecting its assay, fractional exhaled nitric oxide (FENO) can reliably be used to distinguish eosinophilic from non-eosinophilic airway inflammation. Unlike the other indirect measures of airway inflammation such as FEV1 reversibility and bronchoprovocative tests, FENO is noninvasive and gives a direct measure of inflammation. There are sufficient data supporting its role in the diagnosis of asthma, monitoring of disease, prediction of relapse, and level of compliance. Measurement of FENO has therefore made the transition from the bench to the bedside. Of recent, there are attempts at devising interpretative strategies for its use in day to day clinical practice.

Basal internal anal sphincter tone, inhibitory neurotransmission, and other factors contributing to the maintenance of high pressures in the anal canal

Maintenance of the basal tone in the internal anal sphincter (IAS) is critical for rectoanal continence. Effective evacuation requires a fully functional rectoanal inhibitory reflex (RAIR)-mediated relaxation of the IAS via inhibitory neurotransmission (INT). Systematic studies examining the nature of the INT in different species have identified nitric oxide (NO) as the major inhibitory neurotransmitter. However, other mediators such as vasoactive intestinal polypeptide (VIP), ATP, and carbon monoxide (CO) may also play species-specific role under certain experimental conditions. Measurements of the intraluminal pressures in the IAS along with the force of the isolated IAS tissues are the mainstay in the basic studies for the molecular mechanisms underlying the basal tone and in the nature of the INT. The identification of NO as the inhibitory neurotransmitter has led to major advances in the diagnosis and treatment of a number of rectoanal motility disorders associated with the IAS dysfunction. Besides the IAS, the high pressures in the anal canal are affected by the external anal sphincter (EAS) function, and its malfunction may also lead to rectoanal incontinence. Different approaches including biofeedback have been attempted to improve the EAS function, with variable outcomes. There is a dire need for the innovative ways to improve the week high pressures zone in the anal canal. This viewpoint focuses on two studies that extend the above concept of multiplicity of inhibitory neurotransmitters (Neurogastroenterol Motil 2011 23 e11–25), and that high pressures in the anal canal can be improved by the EAS plication (Neurogastroenterol Motil 2011 23 70–5).

Cardiovascular Consequences When Nitric Oxide and Lipid Signaling Converge

The identification of nitric oxide ((*)NO) as an endogenously produced free radical mediator of endothelial-dependent relaxation and host defense has fundamentally changed concepts of cell signal transduction. Ligand-receptor oriented paradigms of cell signaling were originally centered on the concept of a high affinity and specific interaction between a ligand and its receptor, resulting in the activation of secondary signaling events such as gene expression or modulation of catalytic protein function. While (*)NO ligation of the heme iron of soluble guanylate cyclase is consistent with this perspective, the readily diffusible and broadly reactive (*)NO is increasingly appreciated to react with a vast array of target molecules that mediate paracrine vasodilator actions, inhibition of thrombosis and neointimal proliferation, and both pro- and antiinflammatory signaling reactions that are not affected by inhibitors of soluble guanylate cyclase. There is an expanding array of functionally significant "off target" collateral reactions mediated by (*)NO that are guanylate cyclase-independent and rather are dictated by anatomic distribution and the formation of secondary (*)NO-derived species. These reactions are a critical element of redox-regulated signaling and are addressed herein in the context of the oxidation of unsaturated fatty acids to vascular and inflammatory signaling mediators. Because of their abundance and the intrinsic reactivity of unsaturated lipid intermediates and eicosanoid metabolism enzymes with (*)NO and other oxides of nitrogen, lipid signaling mechanisms are a significant target for regulation by (*)NO in the vascular compartment. This convergence of (*)NO and lipid signaling pathways thus adds another level of regulation to physiological responses such as vasodilation, thrombosis, and inflammation. Herein, interactions between (*)NO and lipid signaling events are placed in the context of cardiovascular regulation.

Nitric oxide and carbon monoxide synthesizing enzymes and soluble guanylyl cyclase within neurons of adult human cardiac ganglia

Nitric oxide and carbon monoxide are diffusible gas messengers, synthesized by nitric oxide synthase or heme oxygenase 2, respectively, that can activate soluble guanylyl cyclase in adjacent cells. Nitric oxide and carbon monoxide neuromodulation in cardiac ganglia has been demonstrated. However, identification of nitric oxide or carbon monoxide in human cardiac ganglia needs to be confirmed as suggested from animal model studies. Immunohistochemistry was used to demonstrate neuronal nitric oxide synthase, heme oxygenase 2, and soluble guanylyl cyclase immunoreactivity within neurons of adult human cardiac ganglia. Nitric oxide synthase immunoreactivity was present in 37% of neurons within cardiac ganglia, heme oxygenase 2 immunoreactivity in 79%, and soluble guanylyl cyclase in 53%. Our findings support the hypothesis that nitric oxide and carbon monoxide are modulators of neurotransmission in cardiac ganglia and in neural control of the adult human heart.

Identification of Nitric Oxide as an Endogenous Activator of the AMP-activated Protein Kinase in Vascular Endothelial Cells

In endothelial cells, the AMP-activated protein kinase (AMPK) is stimulated by sheer stress or growth factors that stimulate release of nitric oxide (NO). We hypothesized that NO might act as an endogenous activator of AMPK in endothelial cells. Exposure of human umbilical vein endothelial cells (HUVECs) to NO donors caused an increase in phosphorylation of both Thr-172 of AMPK and Ser-1177 of endothelial nitric oxide synthase, a downstream enzyme of AMPK. NO-induced activation of AMPK was not affected by inhibition of LKB1, an AMPK kinase. In contrast, inhibition of calcium calmodulin-dependent protein kinase kinase abolished the effect of NO in HUVECs. NO-induced AMPK activation in HeLa S3 cells was abolished by either 1H-(1,2,4)-oxadiazole[4,3-a]quinoxalon-1-one, a potent inhibitor for guanylyl cyclase, or 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetrakis (acetoxymethyl ester) (BAPTA-AM), an intracellular Ca(2+) chelator, indicating that NO-induced AMPK activation is guanylyl cyclase-mediated and calcium-dependent. Exposure of HUVECs or isolated mice aortas to either calcium ionophore A23187 or bradykinin significantly increased AMPK Thr-172 phosphorylation, which was abolished by N-nitro-L-arginine methyl ester, an inhibitor of nitric oxide synthase. Finally, A23187- or bradykinin-enhanced AMPK activation was significantly greater in aortas from wild type mice than those in the aortas of endothelial nitric oxide synthase knock-out mice. Taken together, we conclude that NO might act as an endogenous AMPK activator.

Impact of androgen deficiency on sexual dysfunction

The identification of nitric oxide as the neurotransmitter involved in penile erection led to the introduction of phosphodiesterase type 5 inhibitors, which revolutionized the evaluation and management of erectile dysfunction. However, recent studies have indicated that erection is not simply a neurovascular phenomenon. Animal studies have suggested that testosterone promotes or regulates erectile function, and clinical studies on humans have corroborated this association. Administration of testosterone may ameliorate sexual dysfunction related to androgen deficiency, which is commonly associated with aging. High, but not universal, rates of sexual dysfunction among men with prostate cancer undergoing chemical castration suggest that the promotion of erections can occur even at very low levels of serum testosterone.

The discovery of nitric oxide and its role in vascular biology

Nitric oxide (NO) is a relative newcomer to pharmacology, as the paper which initiated the field was published only 25 years ago. Nevertheless its impact is such that to date more than 31,000 papers have been published with NO in the title and more than 65,000 refer to it in some way. The identification of NO with endothelium-derived relaxing factor and the discovery of its synthesis from L-arginine led to the realisation that the L-arginine: NO pathway is widespread and plays a variety of physiological roles. These include the maintenance of vascular tone, neurotransmitter function in both the central and peripheral nervous systems, and mediation of cellular defence. In addition, NO interacts with mitochondrial systems to regulate cell respiration and to augment the generation of reactive oxygen species, thus triggering mechanisms of cell survival or death. This review will focus on the role of NO in the cardiovascular system where, in addition to maintaining a vasodilator tone, it inhibits platelet aggregation and adhesion and modulates smooth muscle cell proliferation. NO has been implicated in a number of cardiovascular diseases and virtually every risk factor for these appears to be associated with a reduction in endothelial generation of NO. Reduced basal NO synthesis or action leads to vasoconstriction, elevated blood pressure and thrombus formation. By contrast, overproduction of NO leads to vasodilatation, hypotension, vascular leakage, and disruption of cell metabolism. Appropriate pharmacological or molecular biological manipulation of the generation of NO will doubtless prove beneficial in such conditions.