Electron paramagnetic resonance analysis of nitrosylhemoglobin in humans during NO inhalation

The reactions of nitric oxide with hemoglobin play an important role in explaining the vascular biology of this free radical. It is perhaps surprising that the level of nitrosylhemoglobin (HbNO) in which NO is bound to the ferrous hemoglobin heme in whole human blood under basal and stimulated conditions is a matter of some controversy, with measurements ranging from <1 nm to close to 10 mum. In order to examine HbNO levels in human blood by using EPR spectroscopy, we have developed a regression-based spectral analysis technique that has a detection level of about 200 nm HbNO. We have utilized this methodology to detect the level of HbNO under basal conditions and during NO inhalation. The major findings of this study are as follows. (i) HbNO can be accurately detected and quantified in whole blood with a detection limit of approximately 200 nm. (ii) By using regression analysis, levels of HbNO as low as 0.5-1 mum can be deconvoluted into component species. (iii) HbNO is present at less than 200 nm at basal conditions in both arterial and venous blood and is formed at a level of 0.5-2.5 mum upon inhalation of 80 ppm NO. (iv) The levels of HbNO detected by EPR are remarkably close (within a factor of 2) to those detected by tri-iodide-based chemiluminescence and much smaller than those detected by photolysis chemiluminescence. (v) The half-time of HbNO in vivo is approximately 40 min.     

A spectrophotometric method for the direct detection and quantitation of nitric oxide, nitrite, and nitrate in cell culture media

A method for the spectrophotometric determination of nitric oxide, nitrite, and nitrate in tissue culture media is presented. The method is based on the nitric oxide-mediated nitrosative modification of sulfanilic acid that reacts with N-(1-naphthyl)ethylenediamine dihydrochloride forming an orange-colored product absorbing at 496 nm. Nitric oxide levels were determined in culture media from this absorbance measurement using chemiluminescence standardization. Extinction coefficients of 5400 and 6600 M(-1) cm(-1) were determined for the nitric oxide product in assay solutions containing 0.1 or 100 mM KPO4 buffer (pH 7.4), respectively, with a limit of detection of 1 microM. Acidification of these reactions (pH 2.4) generated a pink-colored product absorbing at 540 nm allowing for quantitation of total nitric oxide/nitrite levels using extinction coefficients of 38,000 and 36,900 M(-1) cm(-1), for the assay solutions described. The limit of detection of this assay was approximately 300 nM. Using the 100 mM KPO4 buffer system, nitrate levels were determined following reduction to nitrite using a copper-coated cadmium reagent with an extinction coefficient of 29,500 M(-1) cm(-1) and a detection limit of 0.5 microM. The utility of these assays was demonstrated in the standardization of nitric oxide-saturated cell culture media, and the release of nitric oxide by the NONOate compound DEA/NO.     

Measurement of circulating nitrite and S-nitrosothiols by reductive chemiluminescence

Considerable disparities in the reported levels of basal human nitrite and S-nitrosothiols (RSNO) in blood have brought methods of quantifying these nitric oxide (NO) metabolites to the forefront of NO biology. Ozone-based chemiluminescence is commonly used and is a robust method for measuring these species when combined with proper reductive chemistry. The goal of this article is to review existing methodologies for the measurement of nitrite and RSNO by reductive chemiluminescence. Specifically, we discuss in detail the measurement of nitrite and RSNO in biological matrices using tri-iodide and copper(I)/cysteine-based reduction methods coupled to chemiluminescence. The underlying reaction mechanisms, as well as the potential pitfalls of each method are discussed.     

Endogenous nitric oxide formation correlates negatively with circulating matrix metalloproteinase (MMP)-2 and MMP-9 levels in black subjects

Deficient formation of endogenous nitric oxide (NO) contributes to cardiovascular diseases, and this may be associated with increased circulating levels of matrix metalloproteinase-9 (MMP-9), as previously shown in white subjects. Because interethnic differences exist with respect to risk factors, prevalence, and severity of cardiovascular diseases, we designed this study to examine whether the circulating levels of nitrites (a marker of endogenous NO formation) are associated with the plasma levels of MMP-9 and MMP-2 in healthy black subjects. We studied 198 healthy subjects self-reported as blacks not taking any medications. Venous blood samples were collected and plasma and whole blood nitrite levels were measured using an ozone-based chemiluminescence assay. Plasma MMP-2 and MMP-9 levels were determined by gelatin zymography. We found a positive correlation between plasma MMP-9 and MMP-2 levels (P < 0.0001, rs = 0.556). Interestingly, we found a negative relationship between the plasma MMP-9 levels and the plasma or whole blood nitrites levels (P = 0.04, rs = -0.149; and P < 0.0001, rs = -0.349, respectively). In parallel, we found similar negative relationships between plasma MMP-2 levels and plasma or whole blood nitrites levels (P = 0.02, rs = -0.172; and P < 0.0001, rs = -0.454, respectively). This is the first study to show that endogenous nitric oxide formation correlates negatively with the circulating levels of both MMP-2 and MMP-9 in black subjects. Our findings suggest a mechanistic link between deficient NO formation and increased MMPs levels, which may promote cardiovascular diseases.     

Immunocytochemical localization of the urotensin-II receptor, UT, to rat and human tissues: relevance to function

We have examined whether differential expression of UT receptors in cardiovascular tissues from rats and humans may account for the diverse vascular actions reported for urotensin-II. We found UT immunoreactivity ubiquitously expressed in arterial and venous smooth muscle and cardiomyocytes in both species, however, compared to human, levels of UT immunoreactivity in rat vascular endothelial cells was below the level for detection. In rat skeletal muscle cells UT receptor localized to the sarcolemma, a pattern comparable to that for isoforms of nitric oxide synthase suggesting that urotensin-II mediated hindquarter vasodilatation may involve release of nitric oxide from skeletal muscle fibers.     

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.     

Characterization and application of the biotin-switch assay for the identification of S-nitrosated proteins

S-Nitrosation of protein cysteinyl residues has been suggested to be an important nitric oxide-dependent posttranslational modification. The so-called biotin-switch method has been developed to identify S-nitrosated proteins. This method relies on the selective reduction of S-nitrosothiols by ascorbate. In this study we have assessed the ability of ascorbate to reduce S-nitrosothiols and show that ascorbate is a very inefficient reducing agent. We show that higher concentrations of ascorbate and longer incubation times can significantly improve immunological detection of S-nitrosothiols. We have compared immunological detection of S-nitrosothiols with the level of intracellular S-nitrosothiols measured by tri-iodide chemiluminescence and show that the biotin-switch method is capable of detecting only high (nmol/mg protein) levels of intracellular S-nitrosothiols obtained after exposing cells to S-nitrosocysteine, but not the low levels observed during physiological nitric oxide formation. Preliminary proteomic analysis of protein S-nitrosothiols has identified elongation factor 2, heat shock protein 90 beta, and a 65-kDa macrophage protein homologous to human L-plastin as major nitrosation targets at high intracellular nitrosation levels in the murine macrophage-derived RAW 264.7 cell line. While the biotin-switch method may be a useful tool to aid in the positive identification of protein S-nitrosothiols, it cannot match the sensitivity of chemiluminescence-based methods and its use in proteomic studies likely suffers from selective detection of more easily reducible S-nitrosothiols.     

Effect of nitrate and l-arginine therapy on nitric oxide levels in serum, heart, and aorta of fetal hypothyroid rats

Reduced nitric oxide availability and a heterogeneous pattern of nitric oxide synthase activity in some tissues have been reported in hypothyroidism. This study aimed at determining the effects of oral nitrate and l-arginine administration on serum, heart, and aorta nitric oxide metabolite concentrations in fetal hypothyroid rats. In an experimental study, pregnant Wistar rats were administrated tap water or 0.02 % of 6-propyl-2-thiouracil in drinking water during pregnancy and their male pups were followed (n?=?8/group). In adult progeny, serum, heart, and aorta nitric oxide metabolite concentrations were measured by the Griess method after 1-week administration of sodium nitrate (500 mg/L) or l-arginine (2 %) in drinking water. Serum thyroid hormone and thyroid-stimulating hormone levels were also measured. Compared to controls, fetal hypothyroid progeny had significantly lower nitric oxide metabolite concentrations in heart (0.32?±?0.07 vs. 0.90?±?0.14 nmol/mg protein, p?=?0.004) and aorta (2.98±0.56 vs. 6.15±0.74 nmol/mg protein, p?=?0.011) tissues. Nitrate therapy restored heart nitric oxide metabolite levels decreased by fetal hypothyroidism, while l-arginine administration further decreased aorta nitric oxide metabolite levels. Sodium nitrate increased and l-arginine decreased serum nitric oxide metabolite levels in both control and fetal hypothyroid animals. In conclusion, nitrate therapy restores decreased heart nitric oxide metabolite levels, whereas l-arginine decreases aorta nitric oxide metabolite levels even further in fetal hypothyroid rats, findings relevant to the cardiovascular consequences of congenital hypothyroidism in adulthood.     

Evaluation of nitric oxide synthase activity and inhibition kinetics by chemiluminescence.

The binding affinity (K(I)) and inactivation rate (k(inact)) parameters of nitric oxide synthase (NOS) inhibitors are typically estimated by kinetic activity studies. Methods currently used in the estimation of these parameters frequently employ radiolabeled materials and require intensive sample preparation. We have devised a simple, reproducible, and sensitive method for the kinetic analysis of NOS activity and inhibition kinetics using chemiluminescence. We have used this method to characterize enzyme activity for purified murine macrophage nitric oxide synthase (NOS II). Using this method, we have also estimated the inhibitory parameters for a series of competitive antagonists and mechanism-based inactivators of NOS II. The estimated parameters are in agreement with those reported using other methods. We conclude that the chemiluminescence method can be used for kinetic studies of NOS activity and inhibition. This method represents a more efficient means for conducting kinetic studies of NOS inhibition.     

Measurement of nitric oxide levels in the red cell: validation of tri-iodide-based chemiluminescence with acid-sulfanilamide pretreatment

The tri-iodide-based chemiluminescence assay is the most widely used methodology for the detection of S-nitrosothiols (RSNOs) in biological samples. Because of the low RSNO levels detected in a number of biological compartments using this assay, criticism has been raised that this method underestimates the true values in biological samples. This claim is based on the beliefs that (i) acidified sulfanilamide pretreatment, required to remove nitrite, leads to RSNO degradation and (ii) that there is auto-capture of released NO by heme in the reaction vessel. Because our laboratories have used this assay extensively without ever encountering evidence that corroborated these claims, we sought to experimentally address these issues using several independent techniques. We find that RSNOs of glutathione, cysteine, albumin, and hemoglobin are stable in acidified sulfanilamide as determined by the tri-iodide method, copper/cysteine assay, Griess-Saville assay and spectrophotometric analysis. Quantitatively there was no difference in S-nitroso-hemoglobin (SNOHb) or S-nitroso-albumin (SNOAlb) using the tri-iodide method and a recently described modified assay using a ferricyanide-enhanced reaction mix at biologically relevant NO:heme ratios. Levels of SNOHb detected in human blood ranged from 20-100 nM with no arterial-venous gradient. We further find that 90% of the total NO-related signal in blood is caused by erythrocytic nitrite, which may partly be bound to hemoglobin. We conclude that all claims made thus far that the tri-iodide assay underestimates RSNO levels are unsubstantiated and that this assay remains the "gold standard" for sensitive and specific measurement of RSNOs in biological matrices.     

Nitric oxide generation by hemocytes of the mussel Mytilus galloprovincialis.

The phagocytic activity of Mytilus galloprovincialis hemocytes is thought to be associated with NADPH-oxidase activity of the plasma membrane, thus producing superoxide anions. Few studies, however, have been devoted to nitric oxide release by these haemocytes. We investigated NO generation in M. galloprovincialis in order to understand its role in the defensive mechanisms of these organisms. The presence of NO-synthase-like enzymatic activity in protein homogenates from M. galloprovincialis hemocytes was revealed by the conversion of radiolabelled L-arginine to L-citrulline. We observed partial inhibition of the luminol-dependent chemiluminescence of stimulated M. galloprovincialis hemocytes by both NO-synthase inhibitors and superoxide dismutase, indicating that peroxynitrite (which results from the reaction between nitric oxide and superoxide anions) partially mediated this chemiluminescence. Furthermore, we confirmed the production of nitric oxide by M. galloprovincialis by highlighting the nitric oxide-synthase-dependence of the nitrate and nitrite production of stimulated hemocytes.     

A chemiluminescense-based assay for S-nitrosoalbumin and other plasma S-nitrosothiols

The lack of a simple assay for the quantification of S-nitrosothiols in complex biological matrices has hampered our understanding of their contribution to normal physiology and pathophysiological states. In this paper we describe an assay based upon the release of nitric oxide by reaction with a mixture consisting of Cu(I), iodine and iodide with subsequent quantification by chemiluminescense. With this method we can detect levels of S-nitrosothiols down to 5 nM in plasma. Following alkylation of free thiols with N-ethylmaleimide, and removal of nitrite with acidified sulfanilamide, we were able to measure known amounts of S-nitrosoalbumin added to plasma or whole blood, with an inter-assay variation for plasma S-nitrosothiols of ～4%. Further studies showed that the mean concentration of circulating S-nitrosothiols in venous plasma of healthy human volunteers was 28 ± 7 nM.     

Acute effect of nitric oxide supplement on blood nitrate/nitrite and hemodynamic variables in resistance trained men

Nitric oxide dietary supplements are extremely popular within the sport and bodybuilding community. Most products contain l-arginine, for which there is no direct evidence that oral L-arginine increases circulating nitric oxide or blood flow. A new molecule (2-[nitrooxy]thyl 2-amino-3-methylbutanoate) is being marketed as a sport supplement for purposes of delivering "real nitric oxide" to the circulation. In the present study, we measured the acute effects of this supplement on blood nitrate/nitrite and hemodynamic variables. Ten resistance trained men (26 ± 4 years old; 8 ± 6 years of resistance exercise training) reported to the laboratory in random order after a 10-hour overnight fast on 2 occasions separated by 1 week and were provided the supplement (2-[nitrooxy]ethyl 2-amino-3-methylbutanoate) or placebo. Heart rate and blood pressure were recorded, and venous blood samples were collected before and at 5, 15, 30, and 60 minutes after complete breakdown of the supplement (5 minutes post intake) or placebo. Blood samples were assayed for plasma nitrate/nitrite. No interaction (p = 0.99), condition (p = 0.18), or time (p = 0.98) effects were noted for plasma nitrate/nitrite, with values remaining nearly identical across time for placebo (～27 μmol·L(-1)) and increasing a maximum of ～6.7% (from 32.9 to 35.1 μmol·L(-1)) at the 15-minute collection period for the supplement. In regards to hemodynamic variables, no interaction, condition, or time effects were noted for heart rate, systolic, or diastolic blood pressure (p > 0.05), with values near identical between conditions and virtually unchanged across time. These findings indicate that 2-(nitrooxy)ethyl 2-amino-3-methylbutanoate has a small effect on increasing circulating nitrate/nitrite and does not cause any change in hemodynamic variables within the 1 hour postingestion period in a sample of resistance trained men.     

A chemiluminescense-based assay for S-nitrosoalbumin and other plasma S-nitrosothiols

The lack of a simple assay for the quantification of S-nitrosothiols in complex biological matrices has hampered our understanding of their contribution to normal physiology and pathophysiological states. In this paper we describe an assay based upon the release of nitric oxide by reaction with a mixture consisting of Cu(I), iodine and iodide with subsequent quantification by chemiluminescense. With this method we can detect levels of S-nitrosothiols down to 5 nM in plasma. Following alkylation of free thiols with N-ethylmaleimide, and removal of nitrite with acidified sulfanilamide, we were able to measure known amounts of S-nitrosoalbumin added to plasma or whole blood, with an inter-assay variation for plasma S-nitrosothiols of ∼4%. Further studies showed that the mean concentration of circulating S-nitrosothiols in venous plasma of healthy human volunteers was 28 ± 7 nM.     

Elimination of matrix-based interferences to a fluorescent nitrite/nitrate assay by a simple filtration procedure

Pathophysiological levels of oxygen radical metabolites have been studied as indicators of trauma caused by burn insult. The 2, 3-diaminonaphthalene assay is routinely used in the determination of nitrite/nitrate levels in biological fluids and cellular extracts as one indicator of nitric oxide activity. Several laboratories, including ours, have noted matrix-based interferences resulting in decreased assay sensitivity during nitrite/nitrate analysis. We evaluated filtration using Millipore Ultrafree-MC 10,000 NMWL filters for the ability to eliminate matrix-based interferences from human serum and tissue culture medium, thereby restoring assay sensitivity.     

Nitric Oxide Production in Plants: Facts and Fictions

There is now general agreement that nitric oxide (NO) is an important and almost universal signal in plants. Nevertheless, there are still many controversial observations and opinions on the importance and function of NO in plants. Partly, this may be due to the difficulties in detecting and even more in quantifying NO. Here, we summarize major pathways of NO production in plants, and briefly discuss some methodical problems.Key Words: chemiluminescence, DAF-fluorescence, mitochondria, nitrate reductase, nitric oxide, nitric oxide synthase, NO detection, NO signaling     

A continuous, fluorescent, high-throughput assay for human dimethylarginine dimethylaminohydrolase-1

Inhibitors of human dimethylarginine dimethylaminohydrolase-1 (DDAH-1) are of therapeutic interest for controlling pathological nitric oxide production. Only a limited number of biologically useful inhibitors have been identified, so structurally diverse lead compounds are desired. In contrast with previous assays that do not possess adequate sensitivity for optimal screening, herein is reported a high-throughput assay that uses an alternative thiol-releasing substrate, S-methyl-L-thiocitrulline, and a thiol-reactive fluorophore, 7-diethylamino-3-(4'-maleimidylphenyl)-4-methylcoumarin, to enable continuous detection of product formation by DDAH-1. The assay is applied to query two commercial libraries totaling 4446 compounds, and two representative hits are described, including a known DDAH-1 inhibitor. This is the most sensitive DDAH-1 assay reported to date and enables screening of compound libraries using [S] = K (M) conditions while displaying Z' factors from 0.6 to 0.8. Therefore, this strategy now makes possible high-throughput screening for human DDAH-1 inhibitors in pursuit of molecular probes and drugs to control excessive nitric oxide production.     

Dietary nitrate increases gastric mucosal blood flow and mucosal defense

Salivary nitrate from dietary or endogenous sources is reduced to nitrite by oral bacteria. In the acidic stomach, nitrite is further reduced to bioactive nitrogen oxides, including nitric oxide (NO). In this study, we investigated the gastroprotective role of nitrate intake and of luminally applied nitrite against provocation with diclofenac and taurocholate. Mucosal permeability ((51)Cr-EDTA clearance) and gastric mucosal blood flow (laser-Doppler flowmetry) were measured in anesthetized rats, either pretreated with nitrate in the drinking water or given acidified nitrite luminally. Diclofenac was given intravenously and taurocholate luminally to challenge the gastric mucosa. Luminal NO content and nitrite content in the gastric mucus were determined by chemiluminescence. The effect of luminal administration of acidified nitrite on the mucosal blood flow was also investigated in endothelial nitric oxide synthase-deficient mice. Rats pretreated with nitrate or given nitrite luminally had higher gastric mucosal blood flow than controls. Permeability increased more during the provocation in the controls than in the nitrate- and nitrite-treated animals. Dietary nitrate increased luminal NO levels 50 times compared with controls. Nitrate intake also resulted in nitrite accumulation in the loosely adherent mucous layer; after removal of this mucous layer, blood flow was reduced. Nitrite administrated luminally in endothelial nitric oxide synthase-deficient mice increased mucosal blood flow. We conclude that dietary nitrate and direct luminal application of acidified nitrite decrease diclofenac- and taurocholate-induced mucosal damage. The gastroprotective effect likely involves a higher mucosal blood flow caused by nonenzymatic NO production. These data suggest an important physiological role of nitrate in the diet.     

Radiochemical HPLC detection of arginine metabolism: measurement of nitric oxide synthesis and arginase activity in vascular tissue.

Nitric oxide (NO) plays a key role in vascular homeostasis. Accurate measurement of NO production by endothelial nitric oxide synthase (eNOS) is critical for the investigation of vascular disease mechanisms using genetically modified animal models. Previous assays of NO production measuring the conversion of arginine to citrulline have required homogenisation of tissue and reconstitution with cofactors including NADPH and tetrahydrobiopterin. However, the activity and regulation of NOS in vivo is critically dependant on tissue levels of these cofactors. Therefore, understanding eNOS regulation requires assays of NO production in intact vascular tissue that do not depend on the addition of exogenous cofactors and have sufficient sensitivity and specificity. We describe a novel technique, using radiochemical detection of arginine to citrulline conversion, to measure NO production within intact mouse aortas, without exogenous cofactors. We demonstrate the presence of arginase activity in mouse aortas which has the potential to confound this assay. Furthermore, we describe the use of N-hydroxy-nor-L-arginine (nor-NOHA) to inhibit arginase and permit specific detection of NO production in intact mouse tissue. Using this technique we demonstrate a 2.4-fold increase in NO production in aortas of transgenic mice overexpressing eNOS in the endothelium, and show that this technique has high specificity and high sensitivity for detection of in situ NO synthesis by eNOS in mouse vascular tissue. These results have important implications for the investigation of NOS regulation in cells and tissues.     

Nitric Oxide and Blood Pressure in Mice Lacking Extracellular-Superoxide Dismutase

Nitric oxide is a major vasorelaxant and regulator of the blood pressure. The blood vessels contain several active sources of the superoxide radical, which reacts avidly with nitric oxide to form noxious peroxynitrite. There are large amounts of extracellular-superoxide dismutase (EC-SOD) in the vascular wall. To evaluate the importance of EC-SOD for the physiology of nitric oxide, here we studied the blood pressure in mice lacking the enzyme. In chronically instrumented non-anaesthetized mice there was no difference in mean arterial blood pressure between wild-type controls and EC-SOD mutants. Extensive inhibition of nitric oxide synthases with N -monomethyl- l -arginine however resulted in a larger increase in blood pressure, and infusion of the nitric oxide donor nitrosoglutathione caused less reduction in blood pressure in the EC-SOD null mice. We interpret the alterations to be caused by a moderately increased consumption of nitric oxide by the superoxide radical in the EC-SOD null mice. One role of EC-SOD may be to preserve nitric oxide, a function that should be particularly important in vascular pathologies, in which large increases in superoxide formation have been documented.