A simple technique to characterize proximal and peripheral nitric oxide exchange using constant flow exhalations and an axial diffusion model.

The most common technique employed to describe pulmonary gas exchange of nitric oxide (NO) combines multiple constant flow exhalations with a two-compartment model (2CM) that neglects 1) the trumpet shape (increasing surface area per unit volume) of the airway tree and 2) gas phase axial diffusion of NO. However, recent evidence suggests that these features of the lungs are important determinants of NO exchange. The goal of this study is to present an algorithm that characterizes NO exchange using multiple constant flow exhalations and a model that considers the trumpet shape of the airway tree and axial diffusion (model TMAD). Solution of the diffusion equation for the TMAD for exhalation flows >100 ml/s can be reduced to the same linear relationship between the NO elimination rate and the flow; however, the interpretation of the slope and the intercept depend on the model. We tested the TMAD in healthy subjects (n = 8) using commonly used and easily performed exhalation flows (100, 150, 200, and 250 ml/s). Compared with the 2CM, estimates (mean +/- SD) from the TMAD for the maximum airway flux are statistically higher (J'aw(NO) = 770 +/- 470 compared with 440 +/- 270 pl/s), whereas estimates for the steady-state alveolar concentration are statistically lower (CA(NO) = 0.66 +/- 0.98 compared with 1.2 +/- 0.80 parts/billion). Furthermore, CA(NO) from the TMAD is not different from zero. We conclude that proximal (airways) NO production is larger than previously predicted with the 2CM and that peripheral (respiratory bronchioles and alveoli) NO is near zero in healthy subjects.     

A single-breath technique with variable flow rate to characterize nitric oxide exchange dynamics in the lungs.

Current techniques to estimate nitric oxide (NO) production and elimination in the lungs are inherently nonspecific or are cumbersome to perform (multiple-breathing maneuvers). We present a new technique capable of estimating key flow-independent parameters characteristic of NO exchange in the lungs: 1) the steady-state alveolar concentration (C(alv,ss)), 2) the maximum flux of NO from the airways (J(NO,max)), and 3) the diffusing capacity of NO in the airways (D(NO,air)). Importantly, the parameters were estimated from a single experimental single-exhalation maneuver that consisted of a preexpiratory breath hold, followed by an exhalation in which the flow rate progressively decreased. The mean values for J(NO,max), D(NO,air), and C(alv,ss) do not depend on breath-hold time and range from 280-600 pl/s, 3.7-7.1 pl. s(-1). parts per billion (ppb)(-1), and 0.73-2.2 ppb, respectively, in two healthy human subjects. A priori estimates of the parameter confidence intervals demonstrate that a breath hold no longer than 20 s may be adequate and that J(NO,max) can be estimated with the smallest uncertainty and D(NO,air) with the largest, which is consistent with theoretical predictions. We conclude that our new technique can be used to characterize flow-independent NO exchange parameters from a single experimental single-exhalation breathing maneuver.     

Characterizing airway and alveolar nitric oxide exchange during tidal breathing using a three-compartment model.

Exhaled nitric oxide (NO) may be a useful marker of lung inflammation, but the concentration is highly dependent on exhalation flow rate due to a significant airway source. Current methods for partitioning pulmonary NO gas exchange into airway and alveolar regions utilize multiple exhalation flow rates or a single-breath maneuver with a preexpiratory breath hold, which is cumbersome for children and individuals with compromised lung function. Analysis of tidal breathing data has the potential to overcome these limitations, while still identifying region-specific parameters. In six healthy adults, we utilized a three-compartment model (two airway compartments and one alveolar compartment) to identify two potential flow-independent parameters that represent the average volumetric airway flux (pl/s) and the time-averaged alveolar concentration (parts/billion). Significant background noise and distortion of the signal from the sampling system were compensated for by using a Gaussian wavelet filter and a series of convolution integrals. Mean values for average volumetric airway flux and time-averaged alveolar concentration were 2,500 +/- 2,700 pl/s and 3.2 +/- 3.4 parts/billion, respectively, and were strongly correlated with analogous parameters determined from vital capacity breathing maneuvers. Analysis of multiple tidal breaths significantly reduced the standard error of the parameter estimates relative to the single-breath technique. Our initial assessment demonstrates the potential of utilizing tidal breathing for noninvasive characterization of pulmonary NO exchange dynamics.     

A new method for monitoring nitric oxide production using Teflon membrane microdialysis

The measurement of nitric oxide (NO) by electron spin resonance (ESR) is complicated by potentially toxic spin-trapping agents, which may affect the NO-producing cells per se and/or cause artifacts and systemic side effects. These problems can be addressed by preventing direct interaction between the agent and the biological system. In the present study, we utilized Teflon as a barrier between the spin trap and the living cell, since the material is permeable to gas only. Our aim was to investigate if NO could diffuse across the membrane in sufficient amounts to be trapped and quantified by ESR. We used standard microdialysis equipment and specially designed dialysis probes, or tubing, with Teflon membranes. Sodium nitroprusside was used as a NO donor and Fe-N-dithiocarboxysarcosine (Fe(DTCS)2) as a spin trap. NO readily diffuses through Teflon and could be quantified in concentrations considerably below 50 nM in a reproducible and accurate manner. In cell cultures of activated murine macrophages, NO synthesis from iNOS could be monitored and we noted a huge increase in NO concentration by superoxide dismutase. We conclude that spin trapping of NO by Fe(DTCS)2 across Teflon membranes is an attractive approach for quantifying and monitoring nitric oxide production without interfering with cell viability.     

A technique to estimate the rate of whole body nitric oxide formation in conscious mice.

OBJECTIVE: We have established a technique to estimate the total rate of nitric oxide (NO) formation in mice, based on inhalation of a stable oxygen isotope (18O(2)). Changes of NO production with age were also studied. METHODS: The experiments were performed in eight-week- (n=6) and eight-month-old (n=6-7), respectively, female (C57/Bl6xCBAca) mice. Pairs of conscious mice were kept in an air-tight closed system allowing breathing of a mixture containing 18O(2). The 18O(2)-technique was validated by L-NAME (10mg/kg) and lipopolysaccharide (LPS, 8 mg/kg) administration. The concentrations of O(2) and CO(2) in the system were controlled and plasma nitrate analyzed by GC/MS technique. RESULTS: NO formation was similar in young and old mice (young=7.68+/-1.47 vs. old = 6.25+/-1.49 micromol/kg/h, n.s.). Total NO production was reduced after L-NAME treatment in young animals by 91% and in old animals by 71% (p<0.05 for both), whereas LPS administration increased NO production (114+/-17%, p<0.05).Conclusion. NO formation is unaltered with age in mice. The 18O(2)-technique is a valid and specific technique to estimate whole body NO production in conscious mice.     

Endogenous nitric oxide modulates responses of tissue and airway resistance to vagal stimulation in piglets.

The role of endogenous nitric oxide (NO) in modulating the excitatory response of distal airways to vagal stimulation is unknown. In decerebrate, ventilated, open-chest piglets aged 3-10 days, lung resistance (RL) was partitioned into tissue resistance (Rti) and airway resistance (Raw) by using alveolar capsules. Changes in RL, Rti, and Raw were evaluated during vagal stimulation at increasing frequency before and after NO synthase blockade with N(omega)-nitro-L-arginine methyl ester (L-NAME). Vagal stimulation increased RL by elevating both Rti and Raw. NO synthase blockade significantly increased baseline Rti, but not Raw, and significantly augmented the effects of vagal stimulation on both Rti and Raw. Vagal stimulation also resulted in a significant increase in cGMP levels in lung tissue before, but not after, L-NAME infusion. In seven additional piglets after RL was elevated by histamine infusion in the presence of cholinergic blockade with atropine, vagal stimulation failed to elicit any change in RL, Rti, or Raw. Therefore, endogenous NO not only plays a role in modulating baseline Rti, but it opposes the excitatory cholinergic effects on both the tissue and airway components of RL. We speculate that activation of the NO/cGMP pathway during cholinergic stimulation plays an important role in modulating peripheral as well as central contractile elements in the developing lung.     

Role of nitric oxide in the airway response to exercise in healthy and asthmatic subjects.

A role of nitric oxide (NO) has been suggested in the airway response to exercise. However, it is unclear whether NO may act as a protective or a stimulatory factor. Therefore, we examined the role of NO in the airway response to exercise by using N-monomethyl-L-arginine (L-NMMA, an NO synthase inhibitor), L-arginine (the NO synthase substrate), or placebo as pretreatment to exercise challenge in 12 healthy nonsmoking, nonatopic subjects and 12 nonsmoking, atopic asthmatic patients in a double-blind, crossover study. Fifteen minutes after inhalation of L-NMMA (10 mg), L-arginine (375 mg), or placebo, standardized bicycle ergometry was performed for 6 min using dry air, while ventilation was kept constant. The forced expiratory volume in 1-s response was expressed as area under the time-response curve (AUC) over 30 min. In healthy subjects, there was no significant change in AUC between L-NMMA and placebo treatment [28.6 +/- 17.0 and 1.3 +/- 20.4 (SE) for placebo and L-NMMA, respectively, P = 0.2]. In the asthmatic group, L-NMMA and L-arginine induced significant changes in exhaled NO (P < 0.01) but had no significant effect on AUC compared with placebo (geometric mean +/- SE: -204.3 +/- 1.5, -186.9 +/- 1.4, and -318.1 +/- 1.2%. h for placebo, L-NMMA, and L-arginine, respectively, P > 0.2). However, there was a borderline significant difference in AUC between L-NMMA and L-arginine treatment (P = 0.052). We conclude that modulation of NO synthesis has no effect on the airway response to exercise in healthy subjects but that NO synthesis inhibition slightly attenuates exercise-induced bronchoconstriction compared with NO synthase substrate supplementation in asthma. These data suggest that the net effect of endogenous NO is not inhibitory during exercise-induced bronchoconstriction in asthma.     

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

Aminoguanidine renders inducible nitric oxide synthase knockout mice more susceptible to Salmonella typhimurium infection.

Aminoguanidine (AG), a nitric oxide synthase (NOS) inhibitor, has been widely used to study the role of inducible NOS (iNOS) in host defense against infections caused by various pathogens including Salmonella typhimurium. iNOS has been reported to play an important role in host defense against S. typhimurium infection both in vitro and in vivo. In this report we show those AG treatment lead to weight loss in both wild-type and iNOS knockout mice, and rendered them more susceptible to Salmonella infection. These results suggest that AG may have side effects other than the inhibition of iNOS, and that data obtained from studies using AG should be interpreted with caution.     

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.     

A new class of selective and potent inhibitors of neuronal nitric oxide synthase.

The synthesis and SAR of a series of 6-(4-(substituted)phenyl)-2-aminopyridines as inhibitors of nitric oxide synthase are described. Compound 3a from this series shows potent and selective inhibition of the human nNOS isoform, with pharmacokinetics sufficient to provide in vivo inhibition of nNOS activity.     

Role of endogenous nitric oxide in hyperoxia-induced airway hyperreactivity in maturing rats.

We sought to define the effects of maturation and hyperoxic stress on nitric oxide (NO)-induced modulation of bronchopulmonary responses to stimulation of vagal preganglionic nerve fibers. Experiments were performed on decerebrate, paralyzed, and ventilated rat pups at 6-7 days (n = 21) and 13-15 days of age (n = 23) breathing room air and on rat pups 13-15 days of age (n = 19) after exposure to hyperoxia (>/=95% inspired O(2) fraction for 4-6 days). Total lung resistance (RL) and lung elastance (EL) were measured by body plethysmograph. Vagal stimulation and release of acetylcholine caused a frequency-dependent increase in RL and EL in all animals. The RL response was significantly potentiated in normoxic animals by prior blockade of nitric oxide synthase (NOS) (P < 0.05). Hyperoxic exposure increased responses of RL to vagal stimulation (P < 0.05); however, after hyperoxic exposure, the potentiation of contractile responses by NOS blockade was abolished. The response of EL was potentiated by NOS blockade in the 13- to 15-day-old animals after both normoxic and hyperoxic exposure (P < 0.01). Morphometry revealed no effect of hyperoxic exposure on airway smooth muscle thickness. We conclude that NO released by stimulation of vagal preganglionic fibers modulates bronchopulmonary contractile responses to endogenously released acetylcholine in rat pups. Loss of this modulatory effect of NO could contribute to airway hyperreactivity after prolonged hyperoxic exposure, as may occur in bronchopulmonary dysplasia.     

Signalling and cellular specificity of airway nitric oxide production in pertussis

Bordetella pertussis , the aetiological agent of whooping cough (pertussis), causes selective destruction of ciliated cells of the human airway mucosa. In a hamster tracheal organ culture model, B. pertussis causes identical cytopathology as does tracheal cytotoxin (TCT), a glycopeptide released by the bacterium. The damage caused by B. pertussis or TCT has been shown to be mediated via nitric oxide (NO·). Using immunofluorescence detection of the cytokine-inducible NO synthase (iNOS; NOS type II), we determined that B. pertussis induced epithelial NO· production exclusively within non-ciliated cells. This epithelial iNOS activation could be reproduced by the combination of TCT and endotoxin. However, neither TCT alone nor endotoxin alone was capable of inducing epithelial iNOS. This result mirrors the synergistic activity of TCT and endotoxin exhibited in monolayer cultures of tracheal epithelial cells. Therefore, TCT and endotoxin are both important virulence factors of B. pertussis , combining synergistically to cause the specific epithelial pathology of pertussis.     

Regulation of pulmonary circulation by alveolar oxygen tension via airway nitric oxide.

The effects of airway (AH) and vascular hypoxia (VH) on the production of nitric oxide (NO; VNO) were tested in isolated buffer-perfused (BFL) and blood-perfused rabbit lungs (BLL). To produce AH and/or VH, the lung was ventilated with 1% O(2) gas, and/or the perfusate was deoxygenated by a membrane oxygenator located on the inlet limb to the pulmonary artery. We measured exhaled NO (VNO), accumulation of perfusate NOx, and pulmonary arterial pressure (Ppa) during AH (inspired O(2) fraction = 0.01) and/or VH (venous PO(2) = 26 Torr). In BFL, a pure AH without VH caused decreases in VNO and NOx accumulation with a rise in Ppa. However, neither VNO, NOx accumulation, nor Ppa changed during VH. Similarly, in BLL, only AH reduced VNO, although NOx accumulation was not measurable because of Hb. When alveolar PO(2) was gradually reduced from 152 to 0 Torr for 20 min, AH reduced VNO curvilinearly from 73.9 +/- 8 to 25.6 +/- 8 nl/min in BFL and from 26.0 +/- 2 to 5. 2 +/- 1 nl/min in BLL. This plot was analogous to that of a substrate-velocity curve for an enzyme obeying Michaelis-Menten kinetics. The apparent Michaelis-Menten constant for O(2) was calculated to be 23.2 microM for BLL and 24.1 microM for BFL. These results indicate that the VNO in the airway epithelia is dependent on the level of inspired O(2) fraction, leading to the tentative conclusion that epithelial NO synthase is O(2) sensitive over the physiological range of alveolar PO(2) and controls pulmonary circulation.     

A new method to characterize chemically and topographically nanopatterned surfaces

Surface chemistry of topographically patterned grooved samples with ridges of 150 nm width, adsorbed with self-assembled monolayers (SAMs) of alkanethiols on gold, have been characterized by near edge X-ray absorption fine structure (NEXAFS) spectroscopy. Analysis reveals that NEXAFS may discriminate between different chemistries adsorbed to the tops, sidewalls and grooves of the patterns.     

Expression of nitric oxide synthase-2 in the lungs decreases airway resistance and responsiveness.

Individuals with asthma have increased levels of nitric oxide in their exhaled air. To explore its role, we have developed a regulatable transgenic mouse capable of overexpressing inducible nitric oxide synthase in a lung-specific fashion. The CC10-rtTA-NOS-2 mouse contains two transgenes, a reverse tetracycline transactivator under the control of the Clara cell protein promoter and the mouse nitric oxide synthase-2 (NOS-2) coding region under control of a tetracycline operator. Addition of doxycycline to the drinking water of CC10-rtTA-NOS-2 mice causes an increase in nitric oxide synthase-2 that is largely confined to the airway epithelium. The fraction of expired nitric oxide increases over the first 24 h from approximately 10 parts per billion to a plateau of approximately 20 parts per billion. There were no obvious differences between CC10-rtTA-NOS-2 mice, with or without doxycycline, and wild-type mice in lung histology, bronchoalveolar protein, total cell count, or count differentials. However, airway resistance was lower in CC10-rtTA-NOS-2 mice with doxycycline than in CC10-rtTA-NOS-2 mice without doxycycline or wild-type mice with doxycycline. Moreover, doxycycline-treated CC10-rtTA-NOS-2 mice were hyporesponsive to methacholine compared with other groups. These data suggest that increased nitric oxide in the airways has no proinflammatory effects per se and may have beneficial effects on pulmonary function.