Expression of nitric oxide synthase isoforms and detection of nitric oxide in rat placenta

Nitric oxide (NO) production in therat placenta was monitored and quantified by electron paramagneticresonance (EPR) spectroscopy with hemoglobin and anFe-N-(dithiocarboxy)sarcosine (DTCS) complex as NO-trappingreagents. Expression of nitric oxide synthase (NOS) isoformswas also examined by quantitative RT-PCR analysis. The EPR spectrum ofthe placenta with hemoglobin trapping showed a three-line hyperfinestructure (g = 2.008 and a = 1.66-mT). The EPR signal was diminished after the placenta was homogenized or the NOSinhibitor L-NAME was administered to pregnant rats.Therefore, the specific signal was definitely identified as beingderived from endogenous NO spin-trapped by hemoglobin, and the EPRspectrum showed that the NO adduct existed as a pentacoordinate -NOheme species. The EPR spectrum of the placenta with Fe-DTCS trapping showed a triplet signal (g = 2.038) derived from anNO-Fe-DTCS complex. The height of the triplet signal did not varysignificantly with gestational stage during the last few days ofgestation. At the gestational stages examined, the level of NOS II mRNAexpression was significantly higher than that of NOS III mRNA. NOS IIexpression in term (day 21.5) placenta was significantlyincreased compared with that in preterm (day 19.5) placenta(P < 0.01, n = 4 or 5). These resultssuggest that NOS II is the predominant producer of NO in the placentaand that NOS II-generated NO plays significant roles in the maintenanceof placental functions immediately before birth.

     

Electrochemical activation of electrodes for amperometric detection of nitric oxide

An open question in the literature of nitric oxide detection was investigated: does electrochemical activation account for the enhanced properties of certain presumed chemically-modified electrodes? Uniform electrodes of graphite, iridium, palladium, platinum, and ruthenium were exposed to potential cycling and then tested for amperometric response to nitric oxide to identify principles that govern electrochemical activation of nitric oxide electrodes. These electrodes were compared to similar electrodes that were not cycled. Only cycled graphite and ruthenium showed significantly increased responses. Graphite demonstrated enhanced performance after exposure to cycling potentials at which oxygen, CO₂, and soluble carbonates form, suggesting that erosion of the electrode enhanced its response by increasing the surface area accessible to nitric oxide. This may explain the performance of carbon fibers cycled to the same potentials in solutions containing metalloporphyrins. The response of ruthenium was enhanced after cycling to less extreme potentials at which soluble species do not form and at which a metallic conductive oxide, RuO₂, could lay down a stable, adherent layer on the electrode surface. Cycled ruthenium also exhibited a much greater increase in capacitance after cycling, consistent with the formation of a conductive surface layer.     

Bacterial nitric oxide synthesis.

The structure-function relationships in nitrite reductases, key enzymes in the dissimilatory denitrification pathway which reduce nitrite to nitric oxide (NO), are reviewed in this paper. The mechanisms of NO production are discussed in detail and special attention is paid to new structural information, such as the high resolution structure of the copper- and heme-containing enzymes from different sources. Finally, some implications relevant to regulation of the steady state levels of NO in denitrifiers are presented.     

Mammalian nitric oxide synthases.

The nitric oxide (NO) synthase family of enzymes generate NO from L-arginine, which acts as a biologic effector molecule in a broad number of settings. This report summarizes some of the current information regarding NO synthase structure-function, reaction mechanism, control of catalysis, and protein interactions.     

In vivo detection of nitric oxide distribution in mice

This paper discusses in vivo detection of nitric oxide (NO) distribution in endotoxin-treated mice using L-band (1.1 GHz) electron paramagnetic resonance spectroscopy (EPR) in combination with the hydrophilic NO trapping complex: N-methyl-D-glucamine dithiocarbamate and iron (MGD-Fe). MGD-Fe-NO complex is found in the upper abdomen (liver region), lower abdomen (kidney and urinary bladder) and head region of ICR mice. Experiments with nitric oxide synthase (NOS) inhibition and ¹⁵N-labeled L-arginine as NOS substrate verify the origin of trapped NO from L-arginine. However, contribution from a 'nonenzymatic' NO generation pathway can not be ruled out. This paper further examines potential artifacts, which may arise in experiments using dithiocarbamate-iron complexes as NO trapping agents.     

In vivo detection of nitric oxide distribution in mice

This paper discusses in vivo detection of nitric oxide (NO) distribution in endotoxin-treated mice using L-band (1.1 GHz) electron paramagnetic resonance spectroscopy (EPR) in combination with the hydrophilic NO trapping complex: N-methyl-D-glucamine dithiocarbamate and iron (MGD-Fe). MGD-Fe-NO complex is found in the upper abdomen (liver region), lower abdomen (kidney and urinary bladder) and head region of ICR mice. Experiments with nitric oxide synthase (NOS) inhibition and 15N-labeled L-arginine as NOS substrate verify the origin of trapped NO from L-arginine. However, contribution from a 'nonenzymatic' NO generation pathway can not be ruled out. This paper further examines potential artifacts, which may arise in experiments using dithiocarbamate-iron complexes as NO trapping agents.     

Direct chemiluminescence detection of nitric oxide in aqueous solutions using the natural nitric oxide target soluble guanylyl cyclase

Nitric oxide (NO) is a free radical involved in many physiological processes including regulation of blood pressure, immune response, and neurotransmission. However, the measurement of extremely low, in some cases subnanomolar, physiological concentrations of nitric oxide presents an analytical challenge. The purpose of this methods article is to introduce a new highly sensitive chemiluminescence approach to direct NO detection in aqueous solutions using a natural nitric oxide target, soluble guanylyl cyclase (sGC), which catalyzes the conversion of guanosine triphosphate to guanosine 3′,5′-cyclic monophosphate and inorganic pyrophosphate. The suggested enzymatic assay uses the fact that the rate of the reaction increases by about 200 times when NO binds with sGC and, in so doing, provides a sensor for nitric oxide. Luminescence detection of the above reaction is accomplished by converting inorganic pyrophosphate into ATP with the help of ATP sulfurylase followed by light emission from the ATP-dependent luciferin–luciferase reaction. Detailed protocols for NO quantification in aqueous samples are provided. The examples of applications include measurement of NO generated by a nitric oxide donor (PAPA-NONOate), nitric oxide synthase, and NO gas dissolved in buffer. The method allows for the measurement of NO concentrations in the nanomolar range and NO generation rates as low as 100 pM/min.     

N-acetylcysteine inhibits in vivo nitric oxide production by inducible nitric oxide synthase.

This in vivo study evaluates the effect of N-acetylcysteine (NAC) administration on nitric oxide (NO) production by the inducible form of nitric oxide synthase (iNOS). NO production was induced in the rat by the ip administration of 2 mg/100 g lipopolysaccharide (LPS). This treatment caused: (1) a decrease in body temperature within 90 min, followed by a slow return to normal levels; (2) an increase in plasma levels of urea, nitrite/nitrate, and citrulline; (3) the appearance in blood of nitrosyl-hemoglobin (NO-Hb) and in liver of dinitrosyl-iron-dithiolate complexes (DNIC); and (4) increased expression of iNOS mRNA in peripheral blood mononuclear cells (PBMC). Rat treatment with 15 mg/100 g NAC ip, 30 min before LPS, resulted in a significant decrease in blood NO-Hb levels, plasma nitrite/nitrate and citrulline concentrations, and liver DNIC complexes. PBMC also showed a decreased expression of iNOS mRNA. NAC pretreatment did not modify the increased levels of plasma urea or the hypothermic effect induced by the endotoxin. The administration of NAC following LPS intoxication (15 min prior to sacrifice) did not affect NO-Hb levels. These results demonstrate that NAC administration can modulate the massive NO production induced by LPS. This can be attributed mostly to the inhibitory effect of NAC on one of the events leading to iNOS protein expression. This hypothesis is also supported by the lack of effect of late NAC administration.     

Chitinous materials inhibit nitric oxide production by activated RAW 264.7 macrophages

Chitinous materials have been studied in wound healing and artificial skin substitutes for many years. Nitric oxide (NO) has been shown to contribute to cytotoxicity in cell proliferation during inflammation of wound healing. In this study, we examined the effect of chitin and its derivatives on NO production by activated RAW 264.7 macrophages. Chitin and chitosan showed a significantly inhibitory effect on NO production by the activated macrophages. Hexa-N-acetylchitohexaose and penta-N-acetylchitopentaose also inhibited NO production but with less potency. However, N-acetylchitotetraose, -triose, -biose, and monomer of chitin, N-acetylglucosamine and glucosamine had little effect on NO production by the activated cells. These results suggest that the promotive effect of chitinous material on wound healing be related, at least partly, to inhibit NO production by the activated macrophages.     

Electrode materials for biphenyl-based rectification devices

An ab initio approach was utilized to explore the electronic transport properties of 4′-thiolate-biphenyl-4-dithiocarboxylate (TBDT) sandwiched between two electrodes made of various materials X (X?=?Cu, Ag, and Au). Analysis of current–voltage (I–V) characteristics, rectification performance, transmission functions, and the projected density of states (PDOS) under various external voltage biases showed that the transport properties of these constructed systems are markedly impacted by the choice of electrode materials. Further, Cu electrodes yield the best rectifying behavior, followed by Ag and then Au electrodes. Interestingly, the rectification effects can be tuned by changing the torsion angle between the two phenyl rings, as well as by stretching the contact distances between the end group and the electrodes. For Cu, the maximum rectifying ratio increases by 37 % as the contact distance changes from 1.7 Å to 1.9 Å. This is due to an increase in coupling strength asymmetry between the molecule and the electrodes. Our findings are compared with the results reported for other systems. The present calculations are helpful not only for predicting the optimal electrode material for practical applications but also for achieving better control over rectifying performance in molecular devices.     

Cell-compatible array of three-dimensional tip electrodes for the detection of nitric oxide release

An array of electrodes on which cells could be grown directly was fabricated using silicon anisotropic etching and a thick-photoresist process and employed for the detection of nitric oxide (NO) released from a population of adherently growing human umbilical vein endothelial cells (HUVEC). The electrodes are tip-shaped and are 40 microm high of which only the top 15 microm are exposed Pt-tips. After electrochemical induced modification of the exposed Pt tips using Ni phthalocyanine the individual addressable electrode tips were sensitive and selective for the detection of NO at an applied constant potential of 750 mV. The silicon nitride insulation of the lower part of the tip electrodes prevented the death of the cells upon the application of the working potential at which NO was detected. It also helped to avoid the perturbation of the integrity of the sensing chemistry imparted on the electrode surface that could have resulted from the contact of the adherently growing cells with the active electrode surface. The release of nitric oxide from HUVEC was successfully monitored with different numbers of tip electrodes simultaneously connected as combined working electrode.     

Methods of nitric oxide detection in plants: A commentary

Over the last decade nitric oxide (NO) has been shown to influence a range of processes in plants. However, when, where and even if NO production occurs is controversial in several physiological scenarios in plants. This arises from a series of causes: (a) doubts have arisen over the specificity of widely used 4,5-diaminofluorescein diacetate (DAF-2DA)/4-amino-5-methylamino-2,7-difluorofluorescein (DAF-FM) dyes for NO, (b) no plant nitric oxide synthase (NOS) has been cloned, so that the validity of using mammalian NOS inhibitors to demonstrate that NO is being measured is debatable, (c) the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-l-oxyl-3-oxide (cPTIO) needs to be used with caution, and (d) some discrepancies between assays for in planta measurements and another based on sampling NO from the gas phase have been reported. This review will outline some commonly used methods to determine NO, attempt to reconcile differing results obtained by different laboratories and suggest appropriate approaches to unequivocally demonstrate the production of NO.     

A nitric oxide concentration clamp.

We report a new method of generating nitric oxide (NO) that possesses several advantages for experimental use. This method consists of a photolysis chamber where NO is released by illuminating photolabile NO donors with light from a xenon lamp, in conjunction with feedback control. Control of the photolysis light was achieved by selectively gating light projected through a shutter before the light was launched into a light guide that conveyed the light to the photolysis chamber. By gating the light in proportion to a sensor that reported nearly instantaneous concentration from the photolysis chamber, a criterion NO concentration could be achieved, which could be easily adjusted to higher or lower criterion levels. To denote the similarity of this process with the electrophysiological process of voltage clamp, we term this process a concentration "clamp." This development enhances the use of the fiber-optic-based system for NO delivery and should enable the execution of experiments where the in situ concentration of NO is particularly critical, such as in biological preparations.     

The natural substrate for nitric oxide synthase activity.

There has been little evidence to indicate that arginine is the natural substrate for generating nitric oxide synthase (NOS) activity. It is now shown that carnosine, which is widely distributed in tissues, is likely to be the true substrate. In tissue sections it gives a stronger NOS reaction than does arginine.     

Cadmium reduces nitric oxide production by impairing phosphorylation of endothelial nitric oxide synthase.

Cadmium (Cd) perturbs vascular health and interferes with endothelial function. However, the effects of exposing endothelial cells to low doses of Cd on the production of nitric oxide (NO) are largely unknown. The objective of the present study was to evaluate these effects by using low levels of CdCl2 concentrations, ranging from 10 to 1000 nmol/L. Cd perturbations in endothelial function were studied by employing wound-healing and MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) assays. The results suggest that a CdCl2 concentration of 100 nmol/L maximally attenuated NO production, cellular migration, and energy metabolism in endothelial cells. An egg yolk angiogenesis model was employed to study the effect of Cd exposure on angiogenesis. The results demonstrate that NO supplementation restored Cd-attenuated angiogenesis. Immunofluorescence, Western blot, and immuno-detection studies showed that low levels of Cd inhibit NO production in endothelial cells by blocking eNOS phosphorylation, which is possibly linked to processes involving endothelial function and dysfunction, including angiogenesis.     

Modeling pulmonary nitric oxide exchange.

Nitric oxide (NO) was first detected in the exhaled breath more than a decade ago and has since been investigated as a noninvasive means of assessing lung inflammation. Exhaled NO arises from the airway and alveolar compartments, and new analytical methods have been developed to characterize these sources. A simple two-compartment model can adequately represent many of the observed experimental observations of exhaled concentration, including the marked dependence on exhalation flow rate. The model characterizes NO exchange by using three flow-independent exchange parameters. Two of the parameters describe the airway compartment (airway NO diffusing capacity and either the maximum airway wall NO flux or the airway wall NO concentration), and the third parameter describes the alveolar region (steady-state alveolar NO concentration). A potential advantage of the two-compartment model is the ability to partition exhaled NO into an airway and alveolar source and thus improve the specificity of detecting altered NO exchange dynamics that differentially impact these regions of the lungs. Several analytical techniques have been developed to estimate the flow-independent parameters in both health and disease. Future studies will focus on improving our fundamental understanding of NO exchange dynamics, the analytical techniques used to characterize NO exchange dynamics, as well as the physiological interpretation and the clinical relevance of the flow-independent parameters.     

Effect of heterogeneous ventilation and nitric oxide production on exhaled nitric oxide profiles.

Elevated exhaled nitric oxide (NO) in the breath of asthmatic subjects is thought to be a noninvasive marker of lung inflammation. Asthma is also characterized by heterogeneous bronchoconstriction and inflammation, which impact the spatial distribution of ventilation in the lungs. Since exhaled NO arises from both airway and alveolar regions, and its level in exhaled breath depends strongly on flow, spatial heterogeneity in flow patterns and NO production may significantly affect the exhaled NO signal. To investigate the effect of these factors on exhaled NO profiles, we developed a multicompartment mathematical model of NO exchange using a trumpet-shaped central airway segment that bifurcates into two similarly shaped peripheral airway segments, each of which empties into an alveolar compartment. Heterogeneity in flow alone has only a minimal impact on the exhaled NO profile. In contrast, placing 70% of the total airway NO production in the central compartment or the distal poorly ventilated compartment can significantly increase (35%) or decrease (-10%) the plateau concentration, respectively. Reduced ventilation of the peripheral and acinar regions of the lungs with concomitant elevated NO production delays the rise of NO during exhalation, resulting in a positive phase III slope and reduced plateau concentration (-11%). These features compare favorably with experimentally observed profiles in exercise-induced asthma and cannot be simulated with single-path models. We conclude that variability in ventilation and NO production in asthmatic subjects impacts the shape of the exhaled NO profile and thus impacts the physiological interpretation.