Rate Of Nitric Oxide Generation Research Articles

Nitric oxide transport in carotid bifurcation after different stent interventions: a numerical study

Stent restenosis and late thrombosis compromise endovascular stent implantation clinical benefit, and the mechanism is unclear. Since nitric oxide (NO) plays a pivotal role in maintaining vascular homeostasis, we believe that stenting can affect NO concentration in the host artery, thereby contributing to postoperative adverse events. We numerically investigated NO concentration after stenting based on the patient-specific carotid to verify this hypothesis. The simulation revealed that stent implantation caused blood flow disturbance, a low wall shear stress, and a significant decrease in NO on the luminal surface, especially in the region of the stented segment. Moreover, severe damage to the artery wall or low blood flow, leading to a low NO generation rate, would induce relatively low NO level in the stented segment. Additionally, we demonstrated that NO distribution might be affected by the combination of stent struts and carotid bifurcation geometry, while the host arterial configuration might play a leading role in the distribution of NO concentration. In conclusion, the carotid artery had a relatively low NO concentration level near stent struts, especially at the severely injured artery, low blood flow, long stenting, and complex host artery which might lead to a genesis/development of adverse events after that intervention.

Highly Efficient Electrosynthesis of Nitric Oxide for Biomedical Applications.

Nitric oxide (NO) constitutes one of the most versatile therapeutics for biomedical applications. The efficient and on-demand NO generation essentially dictates its concentration dependent therapeutic activity. Here, we report an electrochemical system employing a rationally designed single-iron atom based biomimetic catalyst (i.e., Fe SAC), for high-efficient and controlled NO generation. The Fe SAC shows superior catalytic ability in electrochemical reduction of nitrite, with maximal NO generation rate achieving 2.1 μM (min μg)-1 . Theoretical studies suggest the significant decrease of Gibbs-free energy of NO2 - adsorption on single-iron atom accounts for its high catalytic efficiency. Moreover, ample amount of NO can be controllably generated in a potential dependent manner. For antibacterial application, the generated NO overwhelmingly disrupts both gram-negative and gram-positive strains, highlighting a great promise to expedite NO research in both basic and applied sciences.

Systematic Exploration of a Catalytic Metal–Organic Framework/Polyurethane Composite for Medical Device Applications: Effects of MOF Particle Size, MOF Loading, and Polymer Concentration on Composite Material Activity

The metal–organic framework (MOF) CuBTTri, H3[(Cu4Cl)3(BTTri)8] (where H3BTTri = 1,3,5-tris(1H-1,2,3-triazol-5-yl)benzene), is a promising catalyst for the development of antithrombotic medical device materials via localized nitric oxide (NO) generation from endogenous S-nitrosothiols. This work evaluates the effects of three key parameters of CuBTTri-embedded polyurethane composite materials—MOF preparation/particle size, MOF loading, and polymer concentration—on the rate of NO generation. We discovered that CuBTTri preparation and particle size have a significant impact on NO generation. Specifically, hand-ground MOF particles (0.3 ± 0.1 µm diameter) generate NO at greater rates compared to larger as-prepared, raw MOF particles (0.4 ± 0.2 µm diameter) and smaller, filtered MOF particles (0.2 ± 0.1 µm diameter) for composite materials. This finding contradicts previous research for CuBTTri powder which found that the smaller the particles, the greater the catalytic rate. In examining the effects of MOF loading and polymer concentration, our data show that increasing these parameters generally results in increased rates of NO generation; though thresholds appear to exist in which increasing these parameters results in diminishing returns and impedes NO generation capacity for certain composite formulations. We found that polymer concentration is the key determinant of water absorptivity and statistically significant decreases in water uptake accompany statistically significant increases in NO generation. It was also found that formulations with relatively high MOF loadings and low polymer concentrations or low MOF loadings and high polymer concentrations inhibit the rate of NO generation. In summary, this research provides a framework for more strategic selections of key parameters when fabricating composite materials for medical device applications.

Nitric Oxide generating coating alters hematoma structure and soft tissue healing

IntroductionPeri-implantitis remains a major clinical problem due to the challenges in soft tissue integration on implants surfaces. During tissue healing processes, hematoma formation is the initial response to implant surfaces, which serves as a reservoir for growth factors and provides a natural scaffold for cell infiltration. However, little information is available regarding whether altering hematoma properties can benefit soft tissue healing and tissue integration. This study aims to modulate hematoma properties to create a friendly microenvironment for a strong soft tissue seal on implant surfaces. MethodsWe developed a polydopamine (PDAM) based coating capable of well-controlled nitric oxide (NO) generation by applying the copper (Cu) dose gradient. The incorporated copper with progressive raw concentrations of 10, 50 and 250µg/mL in the coating (PDAM@Cu coatings) served as a catalyst and reacted with endogenous NO donors in the blood. The resulting influence on clot structure and tissue healing microenvironment were analyzed along with its impact on the migration and proliferation of human gingival fibroblasts (HGFs). ResultsIt was noted that NO generation rates could be controlled using Cu from the coatings as a catalyst. As a result of this, the clot formation, structural properties, clot degradation, and growth factor release could be manipulated. Consequently, the NO generating surfaces of PDAM@Cu coating yielded a desired porous structure in clots and growth factor release profile, especially of platelet derived growth factors such as platelet-derived growth factor (PDGF) and vascular endothelial growth factor (VEGF). The NO generating surfaces of PDAM@Cu coating subsequently allowed increased migration and proliferation of HGFs. ConclusionIn this study, we developed a novel NO-generating strategy to regulate hematoma properties and growth factors release. The optimized coating could provide a favourable micro-environment for soft tissue healing and prompt cell migration and proliferation of HGFs. Therefore, PDAM surface modification with incorporation of Cu as a catalyst for NO generation could induce a functional implant surface for soft tissue integration.

The role of glutathione and glutathione peroxidase in regulating cellular level of reactive oxygen and nitrogen species

Glutathione (GSH) and GSH/glutathione peroxidase (GPX) enzyme system is essential for normal intracellular homeostasis and gets disturbed under pathophysiologic conditions including endothelial dysfunction. Overproduction of reactive oxidative species (ROS) and reactive nitrogen species (RNS) including superoxide (O2•−), and the loss of nitric oxide (NO) bioavailability is a characteristic of endothelial dysfunction. The GSH/GPX system play an important role in eliminating ROS/RNS. Studies have provided important information regarding the interactions of ROS/RNS with the GSH/GPX in biological systems; however, it is not clear how this cross talk affect these reactive species and GSH/GPX enzyme system, under physiologic and oxidative/nitrosative stress conditions. In the present study, we developed a detailed endothelial cell kinetic model to understand the relationship amongst the key enzyme systems including GSH, GPX, peroxiredoxin (Prx) and reactive species, such as hydrogen peroxide (H2O2), peroxynitrite (ONOO−), and dinitrogen trioxide (N2O3). Our simulation results showed that the alterations in the generation rates of O2•− and NO led to the formation of a wide range of ROS and RNS. Simulations performed by varying the ratio of O2•− to NO generation rates as well as GSH and GPX concentrations showed that the GPX reducing capacity was dependent on GSH availability, level of oxidative/nitrosative stress, and can be attributed to N2O3 levels, but not to H2O2 and ONOO−. Our results showed that N2O3 mediated switch-like depletion in GSH and the incorporation of Prx had no considerable effect on the ROS/RNS species other than ONOO− and H2O2. The analysis presented in this study will improve our understanding of vascular diseases in which the levels and oxidation states of GSH, GPX and/or Prx are significantly altered and pharmacological interventions show limited benefits.

Tumor-associated redox state in metastatic colorectal cancer.

To determine the major indicators of redox-formative molecules in the tissue of metastatic CRC(mCRC), stages Т2-4N0-2M0G2-3, namely the rate of superoxide radical (SR) generation, nitric oxide (NO) content, the activity of matrix metalloproteinases (MMP), lactoferrin (LF) content, and "free" iron and their association with some clinical and pathological characteristics of the patients. mCRC samples from 51patients were analyzed (stage II, 31patients; stage III, 20patients). The LF and "free" iron were assessed by electron paramagnetic resonance(EPR) at the temperature of 77°K. The rate of SR and NO generation was determined with spin traps (ТЕМРО-Н, diethyl dithiocarbamate). The activity of MMP-2 and-9 was measured by gelatin zymography using SDS-polyacrylamide gel electrophoresis. Ki-67expression was analyzed by immunofluorescence technique. In tumors with metastases into the regional lymph nodes (N1-2category), SR generation rate was 2.2-fold higher than in the tumors categorized as N0. In G3mCRC, SR generation rate was 1.7-fold higher than in G2-tumors (p < 0.05). The rate of SR generation correlated inversely with differentiation grade of the tumor (r=-0.61; p < 0.05). MMP-2 and -9 activities in mCRC tissue correlated with SR generation rate and NO level (r=0.44 ÷ 0.53, p < 0.05). The direct correlation between LF content and the stage of the disease (r=0.42) and "free" iron content (r=0.61) was demonstrated while the correlation between LF content and tumor differentiation grade was inverse (r=-0.57; p < 0.05). The altered tumor-associated redox state in mCRC tissue contributes to the increased cell proliferation and formation of aggressive phenotype of the tumor. The assays for the content of redox-formative components in mCRC may be used as additional prognostic markers of the course of the disease in CRC patients.

Influence of Oxidation–Reduction Layering on Fuel Nitrogen Oxide Emissions during a Char Grate-Fired Process

Previous studies have found that the nitric oxide (NO) trend above the fuel seam of a chain boiler is opposite that of a reciprocating grate boiler. We have researched the factors that influence fuel NO emissions during the char grate-fired process by changing the char/oxygen (O2) ratio in a small-scale one-dimensional fixed-bed system. With an increase in the char/O2 ratio compared to the mass loss of char, the advent of excess O2 is delayed and carbon monoxide (CO) is generated. As a result, oxidation–reduction layering of char occurs. Char grate firing leads to an increase in char mass of the complete oxidation layer, and a thicker ash layer covers the char surface. The reduction layer and low-O2 region of the oxidation layer favor NO reduction; therefore, NO emission is delayed or shows a double-peak trend. The ash layer covering and a long residence time at a high temperature increase the NO generation rate of the oxidation layer. However, the overall NO generation rate is controlled by the reduction...

Fe, oxidative and nitrosative metabolism in the Antarctic limpet Nacella concinna

The hypothesis of this work was that oxidative and nitrosative metabolism in the digestive gland (DG) of two limpet populations (intertidal and subtidal) of the Antarctic species Nacella concinna show different behavior when they were exposed to either intermittent (intertidal) or constant (subtidal) natural Fe. Total Fe content and labile Fe pool were higher in the DG of the subtidal compared to the intertidal population. However, no significant differences between populations were seen on the Fe atoms content of the isolated ferritin. Ascorbyl radical content was 2.0±0.4 and 6.5±0.8pmol/mg FW in the DG of the intertidal and subtidal animals, respectively. Lipid damage, assessed as content of thiobarbituric reactive substances, was different between the tissues of intertidal and subtidal samples, 491±102 and 1242±367pmol/mg FW, respectively. Catalase and superoxide dismutase activities showed no differences between the limpets. Nitric oxide (NO) content was 25±3 and 22±2pmol/mg FW in DG from intertidal and subtidal animals, respectively. NO synthase-like (NOS-like) activity was evaluated supplementing the samples with the enzyme co-factors, and the inhibitory effect of Nω-nitro-L-arginine methyl ester hydrochloride was tested. NO generation rate was 3.4±0.3 and 4.7±0.6pmol/minmg FW in DG from the intertidal and subtidal population, respectively. These results showed that the oxidative condition of the limpet population constantly covered by the Fe enriched water is more affected than the intertidal population. However, the nitrosative metabolism seems to be independent of the environmental high Fe content since similar NO steady state concentration and NOS-like activity were measured in both populations.

Measurement of Nitric Oxide (NO) Generation Rate by Chloroplasts Employing Electron Spin Resonance (ESR).

Chloroplasts are among the more active organelles involved in free energy transduction in plants (photophosphorylation). Nitric oxide (NO) generation by soybean (Glycine max, var ADM 4800) chloroplasts was measured as an endogenous product assessed by electron paramagnetic resonance (ESR) spin-trapping technique. ESR spectroscopy is a methodology employed to detect species with unpaired electrons (paramagnetic). This technology has been successfully applied to different plant tissues and subcellular compartments to asses both, NO content and generation. The spin trap MGD-Fe(2+) is extensively employed to efficiently detect NO. Here, we describe a simple methodology to asses NO generation rate by isolated chloroplasts in the presence of either L-Arginine or nitrite (NO2 (-)) as substrates, since these compounds are required for enzymatic activities considered as the possible sources of NO generation in plants.

MhNCED3 in Malus hupehensis Rehd. induces NO generation under osmotic stress by regulating ABA accumulation

Abscisic acid (ABA) biosynthesis has been widely characterized in plants, whereas the effects of ABA biosynthesis on nitric oxide (NO) generation in osmotic stress are less well understood. In this study, Malus hupehensis Rehd. 9-cis-epoxycarotenoid dioxygenase gene (MhNCED3) which is the key gene in ABA biosynthesis was transformed into wild type (WT) and 129B08/nced3 mutant (AtNCED3 deficient), respectively, and two transgenic Arabidopsis lines were obtained. The transgenic Arabidopsis lines displayed higher endogenous ABA content, NO generation rate, AtNIA1 transcript level and nitrate reductase (NR) activity than WT and 129B08/nced3 mutant. Ectopic expression of MhNCED3 reduced the electrolyte leakage and relieved Arabidopsis damage caused by 20% PEG on the growth and development. The ABA content, NO generation rate, AtNIA1 expression and NR activity increased after 20% PEG treatment, importantly, their increases amplitude relative to that in control were higher in two transgenic lines. Additionally, during the treatment for the four genotype Arabidopsis, the time of ABA contents reaching the highest peak was earlier than the time of NO generation, AtNIA1 expression and NR activity reaching their highest peak. These results show that NCED gene indirectly induced endogenous NO generation in osmotic-stressed Arabidopsis partially contributing to the up-regulation of AtNIA1 expression and NR activity.

Correlative Analyses of Nitric Oxide Generation Rates and Nitric Oxide Synthase Levels in Individual Cells Using a Modular Cell-Retaining Device

Nitric oxide (NO) is recognized as one of the major immune system agents involved in the pathogenesis and control of various diseases that may benefit from novel drug development, by exploiting NO signaling pathways and targets. This calls for detection of both intracellular levels of NO and expression of its synthesizing enzymes (NOS) in individual, intact, living cells. Such measurements are challenging, however, due to short half-life, low and fluctuating concentrations of NO, cellular heterogeneity, and inability to trace the same cells over time. The current study presents a device and methodology for correlative analysis of NO generation rates and NOS levels in the same individual cells, utilizing fluorescent imaging followed by immunohistochemistry (IHC). U937 promonocyte cell populations demonstrated significant heterogeneity in their baseline levels, in NO-generation kinetics, and in their response rates to stimuli. Individual cell analysis exposed cell subgroups which showed enhanced NO production upon stimulation, concomitantly with significant up-regulation of inducible NOS (iNOS) levels. Exogenous NO modulated the expression of iNOS in nondifferentiated cells within 1 h, in a dose-dependent manner, while treatment with lysophosphatidylcholine (LPC) enhanced the expression of iNOS, demonstrating a nondependence on NO production.

The detection of the nitrite reductase and NO-generating properties of haemoglobin by mitochondrial inhibition

Nitrite (NO₂⁻), now regarded as an endocrine reserve of nitric oxide (NO), is bioactivated by nitrite reductase enzymes to mediate physiological responses. In blood, haemoglobin (Hb) catalyses nitrite reduction through a reaction modulated by haem redox potential and oxygen saturation, resulting in maximal NO production around the Hb P₅₀. Although physiological studies demonstrate that Hb-catalysed nitrite reduction mediates cyclic guanosine monophosphate (cGMP)-dependent vasodilation, the NO-scavenging effects of Hb raise questions about how NO generated from this reaction escapes the Hb molecule to signal at distant targets. Here, we characterize the NO-generating properties of Hb using the cGMP-independent and NO-dependent inhibition of mitochondrial cytochrome c oxidase. Using a novel technique to measure respiratory inhibition of isolated rat mitochondria, we provide evidence that the reduction of nitrite by intact red blood cells (RBCs) and Hb generates NO, which inhibits mitochondrial respiration. We show that allosteric modulators, which reduce the haem redox potential and stabilize the R state of Hb, regulate the ability of this reaction to inhibit respiration. Finally, we find that the rate of NO generation increases with the rate of Hb deoxygenation, explained by an increase in the proportion of partially deoxygenated R-state tetramers, which convert nitrite to NO more rapidly. These data reveal redox and allosteric mechanisms that control Hb-mediated nitrite reduction and regulation of mitochondrial function, and support a role for Hb-catalysed nitrite reduction in hypoxic vasodilation.

Mitochondria generated nitric oxide protects against permeability transition via formation of membrane protein S-nitrosothiols

Mitochondria generated nitric oxide (NO) regulates several cell functions including energy metabolism, cell cycling, and cell death. Here we report that the NO synthase inhibitors (L-NAME, L-NNA and L-NMMA) administered either in vitro or in vivo induce Ca2+-dependent mitochondrial permeability transition (MPT) in rat liver mitochondria via a mechanism independent on changes in the energy state of the organelle. MPT was determined by the occurrence of cyclosporin A sensitive mitochondrial membrane potential disruption followed by mitochondrial swelling and Ca2+ release. In in vitro experiments, the effect of NOS inhibitors was dose-dependent (1 to 50µM). In addition to cyclosporin A, L-NAME-induced MPT was sensitive to Mg2+ plus ATP, EGTA, and to a lower degree, to catalase and dithiothreitol. In contrast to L-NAME, its isomer D-NAME did not induce MPT. L-NAME-induced MPT was associated with a significant decrease in both the rate of NO generation and the content of mitochondrial S-nitrosothiol. Acute and chronic in vivo treatment with L-NAME also promoted MPT and decreased the content of mitochondrial S-nitrosothiol. SNAP (a NO donor) prevented L-NAME mediated MPT and reversed the decrease in the rate of NO generation and in the content of S-nitrosothiol. We propose that S-nitrosylation of critical membrane protein thiols by NO protects against MPT.

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.

The New Chemical Biology of Nitrite Reactions with Hemoglobin: R-State Catalysis, Oxidative Denitrosylation, and Nitrite Reductase/Anhydrase

Because of their critical biological roles, hemoglobin and myoglobin are among the most extensively studied proteins in human history, while nitrite tops the list of most-studied small molecules. And although the reactions between them have been examined for more than 140 years, a series of unusual and critical allosterically modulated reactions have only recently been characterized. In this Account, we review three novel metal- and nitrite-catalyzed reaction pathways in the context of historical studies of nitrite and hemoglobin chemistry and attempt to place them in the biological framework of hypoxic signaling. Haldane first described the reaction between nitrite and deoxymyoglobin, forming iron-nitrosylated myoglobin, in his analysis of the meat-curing process more than a century ago. The reaction of nitrous acid with deoxyhemoglobin to form nitric oxide (NO) and methemoglobin was more fully characterized by Brooks in 1937, while the mechanism and unusual behavior of this reaction were further detailed by Doyle and colleagues in 1981. During the past decade, multiple physiological studies have surprisingly revealed that nitrite represents a biological reservoir of NO that can regulate hypoxic vasodilation, cellular respiration, and signaling. Importantly, chemical analysis of this new biology suggests a vital role for deoxyhemoglobin- and deoxymyoglobin-dependent nitrite reduction in these processes. The use of UV-vis deconvolution and electron paramagnetic resonance (EPR) spectroscopy, in addition to refined gas-phase chemiluminescent NO detection, has led to the discovery of three novel and unexpected chemistries between nitrite and deoxyhemoglobin that may contribute to and facilitate hypoxic NO generation and signaling. First, R-state, or allosteric, autocatalysis of nitrite reduction increases the rate of NO generation by deoxyhemoglobin and results in maximal NO production at approximately 50% hemoglobin oxygen saturation, which is physiologically associated with greatest NO-dependent vasodilation. Second, oxidative denitrosylation of the iron-nitrosyl product formed in the deoxyhemoglobin-nitrite reaction allows for NO formation and release in a partially oxygenated environment. Finally, the deoxyhemoglobin-nitrite reaction participates in a nitrite reductase/anhydrase redox cycle that catalyzes the anaerobic conversion of two molecules of nitrite into dinitrogen trioxide (N(2)O(3)). N(2)O(3) may then nitrosate proteins, diffuse across hydrophobic erythrocyte membrane channels such as aquaphorin or Rh, or reconstitute NO via homolysis to NO and NO(2)(*). Importantly, the nitrite reductase/anhydrase redox pathway also represents a novel mechanism of both anaerobic and metal-catalyzed N(2)O(3) formation and S-nitrosation and may thus play a vital role in NO-dependent signaling in a hypoxic and heme-rich environment. We consider how these reactions may contribute to physiological and pathological hypoxic signaling.

Plasma Membrane Cholesterol Content Affects Nitric Oxide Diffusion Dynamics and Signaling

Nitric oxide (NO) signaling is inextricably linked to both its physical and chemical properties. Due to its preferentially hydrophobic solubility, NO molecules tend to partition from the aqueous milieu into biological membranes. We hypothesized that plasma membrane ordering provided by cholesterol further couples the physics of NO diffusion with cellular signaling. Fluorescence lifetime quenching studies with pyrene liposome preparations showed that the presence of cholesterol decreased apparent diffusion coefficients of NO approximately 20-40%, depending on the phospholipid composition. Electrochemical measurements indicated that the diffusion rate of NO across artificial bilayer membranes were inversely related to cholesterol content. Sterol transport-defective Niemann-Pick type C1 (NPC1) fibroblasts exhibited increased plasma membrane cholesterol content but decreased activation of both intracellular soluble guanylyl cyclase and vasodilator-stimulated phosphoprotein (VASP) phosphorylation at Ser(239) induced by exogenous NO exposure relative to their normal human fibroblast (NHF) counterparts. Augmentation of plasma membrane cholesterol in NHF diminished production of both cGMP and VASP phosphorylation elicited by NO to NPC1-comparable levels. Conversely, decreasing membrane cholesterol in NPC1 resulted in the augmentation in both cGMP and VASP phosphorylation to a level similar to those observed in NHF. Increasing plasma membrane cholesterol contents in NHF, platelets, erythrocytes and tumor cells also resulted in an increased level of extracellular diaminofluorescein nitrosation following NO exposure. These findings suggest that the impact of cholesterol on membrane fluidity and microdomain structure contributes to the spatial heterogeneity of NO diffusion and signaling.

Estimation of Nitric Oxide Concentration in Blood for Different Rates of Generation: EVIDENCE THAT INTRAVASCULAR NITRIC OXIDE LEVELS ARE TOO LOW TO EXERT PHYSIOLOGICAL EFFECTS

Endothelium-derived nitric oxide (NO) is a potent vasodilator in the cardiovascular system. Several lines of experimental evidence suggest that NO or NO equivalents may also be generated in the blood. However, blood contains a large amount of hemoglobin (Hb) in red blood cells (RBCs). The RBC-encapsulated Hb can react very quickly with NO, which is only limited by the rate of NO diffusion into the RBCs. It is unclear what the possible NO concentration levels in blood are and how the NO diffusion coefficient (D) and the permeability (Pm) of RBC membrane to NO affect the level of NO concentration. In this study, a steady-state concentration experimental method combined with a spherical diffusion model are presented for determining D and Pm and examining the effect of NO generation rate (V0) and hematocrit (Hct) on NO concentration. It was determined that Pm is 4.5 +/- 1.5 cm/s and D is 3410 +/- 50 microm2/s at 37 degrees C. Simulations based on experimental parameters show that, when the rate of NO formation is as high as 100 nm/s, the maximal NO concentration in blood is below 0.012 nM at Pm = 4.5 cm/s and Hct = 45%. Thus, it is unlikely that NO is directly exported or generated from the RBC as an intravascular signaling molecule, because its concentration would be too low to exert a physiological role. Furthermore, our results suggest that, if RBCs export NO bioactivity, this would be through NO-derived species that can release or form NO rather than NO itself.

Mathematical model of nitric oxide convection and diffusion in a renal medullary vas rectum

In this study, the generation, convection, diffusion, and consumption of nitric oxide (NO) in and around a single renal medullary descending or ascending vas rectum in rat were modeled using CFD. The vascular lumen (with a core RBC-rich layer and a parietal layer), the endothelium, the pericytes and the interstitium were represented as concentric cylinders. We accounted for the generation of NO by vascular endothelial cells, and that by the epithelial cells of medullary thick ascending limbs (mTALs) and inner medullary collecting ducts (IMCDs), the latter via interstitial boundary conditions. Luminal velocity profiles were obtained by modeling blood flow dynamics. Our results suggest that convection (i.e., blood flow per se) does not significantly affect NO concentrations along the cortico-medullary axis, because the latter are mostly determined by the rate of NO production and that of NO consumption by hemoglobin. However, the shear stress-mediated effects of blood flow on NO generation rates, and therefore NO concentrations, were predicted to be important. Finally, we found that unless epithelial NO generation rates (per unit tubular surface area) are at least 10 times lower than endothelium NO generation rates, NO production by mTALs and IMCDs affects vascular NO concentrations, with possible consequences for medullary blood flow distribution.

Hemoglobin: Nitrite Reductase or Nitric Oxide Scavenger?

Most recent studies have reported that deoxyghemoglobin can reduce nitrite leading to nitric oxide (NO) generation to elicit vasodilation. However, questions remain concerning how NO escapes the trapping of abundant hemoglobin in red blood cells. Thus, controversy remains regarding whether hemoglobin functions an important nitrite reductase involved in hypoxic vasodilation or as a NO scavenger blocking NO-mediated vasodilation. To characterize the enzymatic role of hemoglobin, spectrophotometric measurements, Electron Paramagnetic Resonance (EPR) spectroscopy, chemiluminescence NO analyzer, and NO electrode studies were performed. Both EPR spectroscopy and spectophotometric studies showed formation of methemoglobin and nitrosylhemoglobin upon incubation of deoxyhemoglobin with nitrite. In the presence of excess of hemoglobin, no NO generation was detected. Thus, it is certain that hemoglobin can react with nitrite, however, NO generated was trapped by ferro-hemoglobin. Using 5 ml blood instead of hemoglobin, addition of 0.1 mM nitrite triggered NO generation with a rate of 20 pM/s. In blood pretreated with CO, addition of 0.1 mM nitrite triggered NO generation with a rate four times higher than that from untreated blood. Thus, hemoglobin functioned as an NO scavenger. We further investigated nitrite reduction in the presence of 0.2 g/ml liver, heart, or aortic ring tissue pieces in PBS solution. The NO generation rates from 0.1 mM nitrite were ~ 4.2 nM/s (liver), 3.1 nM/s (heart), or 1.7 nM/s (aortic ring), respectively. Overall, these results indicate that nitrite reduction to NO is widely present in different tissues and blood; however, hemoglobin when present in excess appears to function as a NO scavenger rather than as a source of free NO.

Evaluation of oxidative–antioxidative status and the l-arginine–nitric oxide pathway in asthmatic patients

Reactive nitrogen, oxygen species and oxidative stress are related to many pulmonary diseases. Nitric oxide (NO) may be involved in either the protection against or the induction of oxidative stress within various tissues. It is derived from the amino acid L-arginine by the action of NO synthase (NOS). L-arginine can also be metabolized by arginase with the production of ornithine and urea. Because of the competition between NOS and arginase for the same substrate, their activities are regulated reciprocally. Therefore, the rate of NO generation associated with oxidative stress is dependent on the relative activities of both NOS and arginase. The objective of this study is to investigate the L-arginine-NO pathway, evaluate oxidative-antioxidative status in the patients with asthma and demonstrate their reciprocal regulation. 30 voluntary asthmatic patients and 30 healthy control subjects with similar age range and sex were included in the study. A total of 10 ml venous blood was drawn, plasma and packed erythrocytes were prepared for the biochemical analyses. Plasma arginase activities and NO levels, and erythrocyte malondialdehyde and reduced glutathione levels were detected. Plasma malondialdehyde levels were significantly higher and glutathione levels were lower in patients with asthma than those of the control subjects (P < 0.001 and P < 0.01, respectively). Arginase activities were significantly lower and NO levels were higher in the patients than those of the controls (P < 0.001 for both). The negative correlation between arginase and NO levels in the patients was significant (r = -0.47; P < 0.01). There was also a positive correlation between malondialdehyde and NO levels in the patients (r = 0.51; P < 0.01). The results suggest that the L-arginine-NO pathway is involved in the pathophysiology of asthma; the arginase activities decrease which causes an increase in the L-arginine levels thereby up-regulation of NO production may contribute to the increase of oxidative stress in asthma.