Role of nitric oxide and nitric oxide synthases in experimental models of denervation and reinnervation

Nitric oxide (NO) is a short-living free molecule synthesized by three different isoforms of nitric oxide synthases (NOS)—neuronal NOS, endothelial NOS, and inducible NOS—associated with neuromuscular transmission, muscle contractility, mitochondrial respiration, and carbohydrate metabolism in skeletal muscle. Neuronal NOS is constitutively expressed at the muscle fiber sarcolemma linked to the dystrophin-glycoprotein complex and concentrated at the neuromuscular endplate. There is increasing evidence that altered expression of neuronal NOS plays a role in muscle fiber damage in neuromuscular diseases such as dystrophinopathies and denervating disorders. Although there have been some previous conflicting results on the neuronal NOS expression pattern in denervated muscle fibers, it is now well established that denervation is associated with a down-regulation and disappearance of sarcolemmal neuronal NOS at synaptic/extrasynaptic or both sites. As NO has been shown to induce collapse and growth arrest on neuronal growth cones, down-regulation of sarcolemmal neuronal NOS may contribute to axonal regeneration and attraction to muscle fibers aiming at the formation of new motor endplates providing reinnervation and reconstitution of NOS expression. As NO serves as a retrograde messenger, it may trigger structural downstream events responsible for neuromuscular synaptogenesis and preventing polyneural innervation. Nevertheless, decreased NO production in denervation reduces the cytoprotective scavenger function of NO for superoxide anions promoting oxidative stress that is likely to be involved in muscle fiber damage and death. However, the multifaced role of NOS and NO under physiological and pathological conditions remains poorly understood on the basis of the current knowledge. Microsc. Res. Tech. 55:181–186, 2001. © 2001 Wiley-Liss, Inc.     

一氧化氮电化学传感器及其在生物医学中的应用

一氧化氮（NO）是一种含有可以调控不断生长的生物学过程的非共享电子对的气体自由基，它由一氧化氮合成酶家族的L-精氨酸所形成。NO在人体内分布广泛，是帮助机体抵抗心血管疾病与其他疾病的信号分子。缺乏NO可能导致糖尿病、心血管疾病与其他疾病，而补充NO可预防和逆转此类疾病。NO是非常小的分子，十分活泼，半衰期短，它可以进入细胞，并向周围的细胞发出交流信号。但是，要准确检测其在生物体中的含量很困难。目前直接用于一氧化氮检测的方法不多，电化学方法尤其是电化学传感器是应用广泛的一类方法，由于其操作简单，灵敏度高，选择性好，已成为现代生物医学中研究一氧化氮的重要工具。本文主要综述近年来NO电化学传感器的研制及其在生物医学中的应用。     

Nitric oxide synthase expression and function in embryonic and adult cardiomyocytes.

Nitric oxide (NO) is an important signalling molecule that plays a relevant role in different cell systems, among them the adult heart. The effects of NO are primarily mediated through modulation of Ca(2+) homeostasis, myofibrillar contractility, and metabolic regulation in cardiomyocytes. Recent evidence also suggests an important role of NO for cardiomyogenesis by modulating proliferation and differentiation and regulating cardiac function. In the embryonic, but also the healthy and diseased, adult mammalian heart, the inducible (iNOS) and the endothelial (eNOS) nitric oxide synthases (NOS) are detected. However, the expression pattern of NO and its function differ during development. Furthermore, under pathophysiological conditions NOS expression can also change and cause impairment of cardiac performance and cytotoxic effects. The present review focuses on the role and function of NO during cardiomyogenesis, the mechanisms responsible for eNOS availability, and the paracrine effects of NO generated by cardiomyocytes.     

Neuronal nitric oxide synthase immunoreactive neurons in the mammalian retina

The development of immunocytochemistry has led to a better understanding of synaptic transmission carried out by neuroactive substances in the mammalian brain, including the retina. In the mammalian retina, nitric oxide (NO) is widely accepted as a neuromodulator. Histochemistry based on NADPH-d and immunocytochemistry based on nitric oxide synthase (NOS) have been used to identify the presence of nitric oxide in the mammalian retina. Certain types of amacrine cells and a class of displaced amacrine cells have been labeled consistently in all mammalian retinae studied to date. Other cell types showing NADPH-d reactivity or NOS immunoreactivity varied between species. NADPH-d reactive or NOS immunoreactive amacrine cells may serve as a source of NO for amacrine, bipolar, and ganglion cells in the inner retina, whereas interplexiform cells, bipolar cells, and horizontal cells may serve as a source of NO for the outer retina of mammals.     

Changes of inducible nitric oxide synthase in aortic cells during the development of hypertension: Effect of angiotensin II

Nitric oxide (NO) generation by inducible nitric oxide synthase (iNOS) in the vascular smooth muscle cells (VSMC), may play a role in blood vessel tone regulation. Lipopolysaccharide (LPS) induced iNOS activity and subsequent nitrite production by cultured aortic VSMC, from SHR with an established chronic blood pressure elevation (adult SHR) or during the period preceding the development of hypertension (young SHR) and from age-matched normotensive Wistar (W) rats were compared. Angiotensin II (Ang II) effect was also evaluated. Both basal LPS-induced iNOS activity and nitrite accumulation were significantly lower in young SHR VSMC compared to young W rat cells. In contrast, adult hypertensive and normotensive rat cells did not differ in NO generation. Besides, young SHR cells exhibited a significant smaller iNOS activity and nitrites than adult SHR cells. After 24h-incubation with Ang II, both variables were markedly reduced in all groups. The proportional reduction of iNOS activity and nitrites by Ang II was not different between hypertensive and normotensive rat cells, at any age. However, this Ang II inhibitory effect was greater in both adult SHR and W cells than in VSMC from young rats. In conclusion, a reduced LPS-induced iNOS activity and NO generation was observed in VSMC form spontaneously hypertensive rats before the raise of blood pressure, but not in adult hypertensive rat cells. Additionally, an inhibitory effect of angiotensin II on these variables is described. We can speculate that the impairment in vascular smooth muscle NO production precedes the development of hypertension in SHR and may play a pathophysiologic role in the early blood pressure elevation in genetically hypertensive rats.     

Region‐specific localization of NOS isoforms and NADPH‐diaphorase activity in the intratesticular and excurrent duct systems of adult domestic cats (Felis catus)

Nitric oxide (NO) is produced by nitric oxide synthases (NOSs) and plays an important role in all levels of reproduction from the brain to the reproductive organs. Recently, it has been discovered that all germ cells and Leydig cells in the cat testis exhibit stage‐dependent nuclear and cytoplasmic endothelial (eNOS) and inducible (iNOS)‐NOS immunoreactivity and cytoplasmic nicotinamide adenine dinucleotide phosphate‐diaphorase (NADPH‐d) reactivity. As a continuation of this finding, in this study, cellular localization of NADPH‐d and immunolocalization and expression of all three NOS isoforms were investigated in the intratesticular (tubuli recti and rete testis), and excurrent ducts (efferent ductules, epididymal duct and vas deferens) of adult cats using histochemistry, immunohistochemistry and western blotting. NADPH‐d activity was found in the midpiece of the spermatozoa tail and epithelial cells of all of ducts, except for nonciliated cells of the efferent ductules. Even though the immunoblotting results revealed similar levels of nNOS, eNOS and iNOS in the caput, corpus and cauda segments of epididymis and the vas deferens, immunostainings showed cell‐specific localization in the efferent ductules and region‐ and cell‐specific localization in the epididymal duct. All of three NOS isoforms were immunolocalized to the nuclear membrane and cytoplasm of the epithelial cells in all ducts, but were found in the tail and the cytoplasmic droplets of spermatozoa. These data suggest that NO/NOS activity might be of importance not only for the functions of the intratesticular and excurrent ducts but also for sperm maturation. Microsc. Res. Tech. 79:192–208, 2016. © 2016 Wiley Periodicals, Inc.     

Nitric oxide averts hypoxia-induced damage during reoxygenation in rat heart

Nitric oxide (NO), synthesized by the hemoproteins NO synthases (NOS), is known to play important roles in physiological and pathological conditions in the heart, including hypoxia/reoxygenation (H/R). This work investigates the role that endogenous NO plays in the cardiac H/R-induced injury. A follow-up study was conducted in Wistar rats subjected to 30 min of hypoxia, with or without prior treatment using the nonselective NOS inhibitor L-NAME (1.5 mM). The rats were studied at 0 h, 12 h, and 5 days of reoxygenation, analysing parameters of cell, and tissue damage (lipid peroxidation, apoptosis, and protein nitration), as well as in situ NOS activity and NO production (NOx). The results showed that after L-NAME administration, in situ NOS activity was almost completely eliminated in all the experimental groups, and consequently, NOx levels fell. Contrarily, the lipid peroxidation level and the percentage of apoptotic cells rose throughout the reoxygenation period. These results reveal that NOS inhibition exacerbates the peroxidative and apoptotic damage observed before the treatment with L-NAME in the hypoxic heart, pointing to a cardioprotective role of NOS-derived NO against H/R-induced injury. These findings could open the possibility of future studies to design new therapies for H/R-dysfunctions based on NO-pharmacology.     

Reactivity of Triphenyl Phosphorothionate in Lubricant Oil Solution

Investigating the thermo-oxidative reactivity of anti-wear additives in lubricant oil solution at high temperature can significantly contribute to an understanding of the mechanism of thermal film and tribofilm formation on metal surfaces. In this study, the reactivity of triphenyl phosphorothionate (TPPT) in lubricant oil solution at high temperature (423 and 473 K) has been studied by Fourier transform infrared spectroscopy (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy. The results show that the TPPT molecule was highly thermally stable and did not completely decompose in oil solution even upon heating at 423 K for 168 h and at 473 K for 72 h. The degradation of the TPPT molecule, which turned out to be a first-order reaction, started taking place after 6 h at both temperatures, leading to the breakage of the P=S bond with the formation of triphenyl phosphate. During these heating experiments, no oil-insoluble compounds were detected. The oxidation of the base oil as a result of the prolonged heating demonstrated that the TPPT molecule did not effectively act as oxidation inhibitor.     

Importance of anionic reactive intermediates for lubricant component reactions with friction surfaces

To understand the chemical behaviour of lubricant components during boundary lubrication, a general concept of negative ionradical reactive intermediates formation for these components has been proposed. The concept is based upon the ionisation mechanism of these compounds caused by the action of electrons of law energy (1–4 eV). Electrons of such energy (exoelectrons) are spontaneously emitted from most fresh surfaces formed during friction. The principal thesis of the model is that lubricant components form anions which are then chemisorbed on the positively charged areas of rubbing surfaces. The formation of negative ions and their decomposition process are simulated by electron attachment mass spectrography. This type of mass spectrography uses electrons of an energy range similar to those of exoelectrons. The proposed model encompasses the following major stages: (a) low-energy electron emission process and creation of positively charged spots, (b) action of emitted electrons with lubricant components (anion and radical formation), (c) reactions of anions with metal surfaces forming a film protecting the surface from wear, (d) cracking of chemical bonds producing other radicals. The model explains many lubrication phenomena of hydrocarbons, oxygen-containing compounds, and many types of chemicals used as antiwear and extreme-pressure additives.     

Gas-phase basicities of polyfunctional molecules. Part 1: Theory and methods

The experimental and theoretical methods of determination of gas-phase basicities, proton affinities and protonation entropies are presented in a tutorial form. Particularities and limitations of these methods when applied to polyfunctional molecules are emphasized. Structural effects during the protonation process in the gas-phase and their consequences on the corresponding thermochemistry are reviewed and classified. The role of the nature of the basic site (protonation on non-bonded electron pairs or on pi-electron systems) and of substituent effects (electrostatic and resonance) are first examined. Then, linear correlations observed between gas-phase basicities and ionization energies or substituent constants are recalled. Hydrogen bonding plays a special part in proton transfer reactions and in the protonation characteristics of polyfunctional molecules. A survey of the main properties of intermolecular and intramolecular hydrogen bonding in both neutral and protonated species is proposed. Consequences on the protonation thermochemistry, particularly of polyfunctional molecules are discussed. Finally, chemical reactions which may potentially occur inside protonated clusters during the measurement of gas-phase basicities or inside a protonated polyfunctional molecule is examined. Examples of bond dissociations with hydride or alkyl migrations, proton transport catalysis, tautomerization, cyclization, ring opening and nucleophilic substitution are presented to illustrate the potentially complex chemistry that may accompany the protonation of polyfunctional molecules.     

Age-related changes of NADPH-diaphorase-positive neurons in the rat inferior colliculus and auditory cortex

Nitric oxide (NO) has been implied in age-related changes of the central nervous system (CNS) and the central auditory pathway. The present study was conducted to investigate whether the number of NO-producing cells and their morphometric characteristics in the inferior colliculus (IC) and the auditory cortex (AC) are changed with the increasing age of the subjects. IC and AC sections of adult and senile Wistar rats were studied using the histochemical detection of NADPH-diaphorase activity (NADPH-d), a marker for neurons containing nitric oxide synthase (NOS). Our results showed a decreased area of the somas of NADPH-d-positive neurons in the dorsal cortex (DC) of the IC and a diffuse loss of NADPH-d-positive neurons in the senile IC and primary cortical auditory area (Te1). However, an increased number of NO-producing cells have been shown by other authors in different parts of the ageing auditory pathway and CNS. It seems that age-related changes in NADPH-d-positive cells may follow a region-specific route. These changes may be related to hearing impairments with increasing age.     

多参数动态气体校准仪的设计与实现

为产生ppb～ppm级微量浓度的高精准校准气体,设计了MFC-2000多参数动态气体校准仪,采用了具有强大功能模块的嵌入式微处理器MC9S12XDT512,完成了气体校准仪的结构及软硬件设计,提出了将臭氧发生器及光度计嵌入在仪器内部,实现了高精度的各种浓度校准气体的产生。试验结果表明,该仪器运行稳定,可靠性好,可以产生100 ppb～6 ppm级的臭氧、二氧化硫等标准气体,并可将产生的臭氧标气直接与NO零气反应产生特定浓度的NO2标气,为监测仪器的校准提供了更为简便的手段。     

Nanoscopic aspects of radiobiological damage: Fragmentation induced by secondary low-energy electrons

Low-energy electrons (LEEs) are produced in large quantities in any type of material irradiated by high-energy particles. In biological media, these electrons can fragment molecules and lead to the formation of highly reactive radicals and ions. The results of recent experiments performed on biomolecular films bombarded with LEEs under ultra-high vacuum conditions are reviewed in the present article. The major type of experiments, which measure fragments produced in such films as a function of incident electron energy (0.1-45 eV), are briefly described. Examples of the results obtained from DNA films are summarized along with those obtained from the fragmentation of elementary components of the DNA molecule (i.e., thin solid films of H(2)O, DNA bases, sugar analogs, and oligonucleotides) and proteins. By comparing the results of these different experiments, it is possible to determine fundamental mechanisms that are involved in the dissociation of biomolecules and the production of single- and double-strand breaks in DNA, and to show that base damage is dependent on the nature of the bases and on their sequence context. Below 15 eV, electron resonances (i.e., the formation of transient anions) play a dominant role in the fragmentation of all biomolecules investigated. These transient anions fragment molecules by decaying into dissociative electronically excited states or by dissociating into a stable anion and a neutral radical. These fragments usually initiate other reactions with nearby molecules, causing further chemical damage. The damage caused by transient anions is dependent on the molecular environment.     

Nitric oxide: relation to integrity, injury, and healing of the gastric mucosa

Nitric oxide (NO) plays a multifaceted role in mucosal integrity. The numerous functions of NO and the double-edged role played by NO in most of them provide a great complexity to the NO action. The three enzymatic sources of NO, neuronal NO-synthase (nNOS), endothelial NOS (eNOS), and inducible NOS (iNOS), have been characterised in the gastrointestinal tract. The protective properties of the NO derived from constitutive NO-synthases (eNOS and nNOS) have already been well established. Less clear is the role assigned to iNOS. The simplistic initial view of low levels of NO synthesised by constitutive NOS being protective while exaggerated NO levels after iNOS induction leading irremediably to cytotoxicity is being questioned by new evidence. As initially reported for constitutive NOS, iNOS activity may be associated to reduced leukocyte-endothelium interaction and platelet aggregation as well as protection of mucosal microcirculation. Moreover, iNOS activity may be important to resolve inflammation by increasing apoptosis in inflammatory cells. It is entirely possible that a low level of expression of iNOS will reflect a positive host-defense response to challenge, but that exaggerated or uncontrolled expression of iNOS itself becomes detrimental. There is no doubt about the protective role of NO in physiological conditions. However, when the mucosa is threatened, the role of NO becomes multiple and the final effect will probably depend on the nature of the insult, the environment involved, and the interaction with other mediators.     

Distribution of nitric oxide synthase immunoreactivity in the mouse brain

Nitric oxide (NO) is a gaseous intercellular messenger with a wide range of neural functions. NO is synthesized by activation of different isoforms of nitric oxide synthases (NOS). At present NOS immunoreactivity has been described in mouse brain in restricted and definite areas and no detailed mapping studies have yet been reported for NOS immunoreactivity. We have studied the distribution of neuronal NOS-containing neurons in the brain of three months male mice, using a specific commercial polyclonal antibody against the neuronal isoform of nitric oxide synthase (nNOS). Neuronal cell bodies exhibiting nNOS immunoreactivity were found in several distinct nuclei throughout the brain. The neurons that were positively stained exhibited different intensities of reaction. In some brain areas (i.e., cortex, striatum, tegmental nuclei) neurons were intensely stained in a Golgi-like fashion. In other regions, immunoreactive cells are moderately stained (i.e., magnocellular nucleus of the posterior commissure, amygdaloid nucleus, interpeduncular nucleus, lateral periaqueductal gray) or weakly stained (i.e., vascular organ of the lamina terminalis, hippocampus, inferior colliculus, reticular nucleus). In the mouse, the NO-producing system appears well developed and widely diffused. In particular, nNOS immunoreactive neurons seem chiefly present in several sensory pathways like all the nuclei of the olfactory system, as well as in many regions of the lymbic system. These data suggest a widespread role for the NO system in the mouse nervous system.     

Mass spectrometric characterization of high-valent metal-oxo, -peroxo and -peroxy intermediates of relevance in oxidation processes

The coupling of mass spectrometry with ionization techniques like electrospray ionization (ESI) or matrix-assisted matrix-assisted laser desorption-ionization (MALDI) offers many advantages over other well-established spectroscopic techniques employed for the investigation of intermediates or short-lived species in condensed-phase. In this review we describe some of the applications of mass spectrometry, in particular of ESI-MS to the detection and characterization of high-valent metal-oxo, -peroxo and -peroxy derivatives, crucial intermediates in the oxyfunctionalization of organic substrates. In addition, by utilizing gas-phase ion-molecule reactions and MS/MS experiments, information on the intrinsic reactivity of the short-lived intermediates may be obtained. The combined use of ESI-MS in association with other spectroscopic techniques and theoretical calculations is discussed as well.     

Surface and Tribological Chemistry of Water and Carbon Dioxide on Copper Surfaces

The tribological chemistry of carbon dioxide and water vapor is studied on copper surfaces at high pressures, with a view to understand the gas-phase lubrication of copper–copper sliding contacts. The adsorption and film formation properties are studied on vapor-deposited copper films in an ultrahigh vacuum chamber using a quartz crystal microbalance. The nature of the reactively formed film is studied after reaction by ex situ X-ray photoelectron spectroscopy (XPS). Carbon dioxide adsorbs reversibly on copper, while water vapor adsorbs both reversibly and irreversibly, where XPS reveals that the irreversibly formed film consists of a mixture of cuprous oxide/hydroxide. Measuring the thickness of the cuprous oxide/hydroxide film as a function of water vapor pressure and temperature reveals that its thickness varies between about 6 and 14 Å and is proportional to the total amount of water adsorbed on the surface. This results in a cuprous oxide/hydroxide film that is thinner at higher temperatures. Measurements of the friction coefficient as a function of temperature and pressure in the presence of water vapor shows that it correlates with film thickness, reaching a limiting value of ~0.35 for thicker films.     

Thermochemistry, bonding, and reactivity of Ni+ and Ni2+ in the gas phase

In this review, we present a general overview on the studies carried out on Ni(+-)- and Ni(2+)-containing systems in the gas phase since 1996. We have focused our attention in the determination of binding energies in parallel with an analysis of the structure and bonding of the complexes formed by the interaction of Ni(+) with one ligand, or in clusters where this metal ion binds several identical or different ligands. Solvation of Ni(2+) by different ligands is also discussed, together with the theoretical information available of doubly charged Ni-containing species. The final section of this review is devoted to an analysis of the gas-phase uni- and bimolecular reactivity of Ni(+) and Ni(2+) complexes.     

Improvement and experimental validation of a multi-zone model for combustion and NO emissions in CNG fueled spark ignition engine

This article reports the experimental and theoretical results for a spark ignition engine working with compressed natural gas as a fuel. The theoretical part of this work uses a zero-dimensional, multi-zone combustion model in order to predict nitric oxide (NO) emission in a spark ignition (SI) engine. The basic concept of the model is the division of the burned gas into several distinct zones for taking into account the temperature stratification of the burned mixture during combustion. This is especially important for accurate NO emissions predictions, since NO formation is strongly temperature dependent. During combustion, 12 products are obtained by chemical equilibrium via Gibbs energy minimization method and nitric oxide formation is calculated from chemical kinetic by the extended Zeldovich mechanism. The burning rate required as input to the model is expressed as a Wiebe function, fitted to experimentally derived burn rates. The model is validated against experimental data from a four-cylinder, four-stroke, SI gas engine (EF7) running with CNG fuel. The calculated values for pressure and nitric oxide emissions show good agreement with the experimental data. The superiority of the multizone model over its two-zone counterpart is demonstrated in view of its more realistic in-cylinder NO emissions predictions when compared to the available experimental data.