Rate Of NO Generation Research Articles

Highly Efficient Electrosynthesis of Nitric Oxide for Biomedical Applications

AbstractNitric 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.

Evaluation of NOx emissions characteristics in a CO2-Free micro-power system by implementing a perforated plate

Ammonia (NH3), as a carbon-free fuel with a high hydrogen content, has been considered as a promising candidate to be applied for transportation, propulsion, and power generation sectors. However, due to the presence of nitrogen atoms, direct combustion of NH3 features a high NOx emission, which could hinder its wide application. Thus it is important to mitigate or minimize these emissions. For this, a three-dimensional (3D) computational model is developed by considering a detailed chemical-kinetic mechanism. The effects of 1) the perforated plate porosity σ, 2) the perforated plate thickness t1, 3) the perforated orifice off-z axis distance d, and 4) inlet thermodynamic pressure Pin are evaluated. Numerical results indicate that σ plays an important role in determining the NO formation. This is the combined effect of the strong species preferential diffusion and low flame temperature within the recirculation zone. Furthermore, varying t1 is shown to affect the NO generation rate due to the varied recirculation zone. Although the flow field is affected by d to some degree, it has a negligible effect on the NO generation. Finally, the emission behavior is found to have a strong dependence on Pin. Approximately 50% reduction of NO emission is achieved as the inlet pressure is varied from 1 to 3 atm. The present work opens up an applicable way to enable low-NOx ammonia-fueled power systems, and some of the findings could be applied for hydrocarbon systems design.

NO emission performance assessment on a perforated plate-implemented premixed ammonia-oxygen micro-combustion system

• The flow field is found to vary greatly downstream of the perforated plate. • The conjugate heat transfer affects NO formation by relocating the flame. • The preferential diffusion is intensified due to the two-dimensionality flow. • The coupling effect of these factors above determines the NO generation rate. The present work examines the NO x emission characteristics of a premixed micro-combustion system with a perforated plate implemented. For this, a three-dimensional (3D) computational model involving a detailed chemical-kinetic mechanism for ammonia-oxygen combustion in the micro-combustor is developed. The model is first validated with the experimental measurements available in the literature before conducting comprehensive analyses. It is found that implementing a perforated plate in the micro-combustion system creates a flow recirculation zone downstream characterized by a low flame temperature and combustion speed. Meanwhile, the conjugate heat transfer between the combustion products and the inner combustor walls is shown to play a key role in the NO generation by relocating the flame in the axial direction and thus changing the chemical reaction rate. Furthermore, the preferential diffusion caused by the variation in the mass diffusivity of different species and the two-dimensionality flow is identified to vary significantly in comparison with the case in the absence of the perforated plate, especially in the vicinity of the recirculation zone. This diffusion effect results in the considerable drop in the N/O atomic ratio, primarily due to the reduction and increase of O 2 and H 2 O, together with less available N 2 , and consequently affecting the NO generation rate. This work confirms that the flow field, the conjugate heat transfer as well as the preferential diffusion effect could be regarded as the potential mechanisms leading to the NO x emission variation in the recirculation zones.

INFLUENCE OF FERROMAGNETIC NANOCOMPOSITE (FERROPLAT) ON HUMAN BREAST CANCER CELLS OF DIFFERENT MALIGNANCY DEGREES: PRO/ANTIOXIDANT BALANCE AND ENERGY METABOLISM

To study the effect of Ferroplat (FrP) on the indexes of pro/antioxidant balance and energy metabolism in breast cancer cells of different malignancy degree and different sensitivity to drug therapy. The study was carried out on breast cancer cells of low (T47D, MCF-7) and high malignancy degree (MCF-7/DDP (cisplatin-resistant), MDA-MB-231, MDA-MB-468) using cell culture techniques, immunocytochemical, biochemical, biophysical methods, flow cytometry and polarography. We established that the addition of FrP to the culture medium reduces the activity of glucose-6-phosphate dehydrogenase (G6PDH), superoxide dismutase (SOD) and the level of non-protein thiols by 32-41% (p<0.05). At the same time, there was an increase of the total level of ROS and the rate of NO generation by inducible NO synthase by 1.7-2.5times (p<0.05). This testifies that FrP disturbs the antioxidant balance in cells, resulting in their death. Also, the use of FrP led to a decrease in the rate of oxygen absorption in MCF-7and T47D cells by 26% and 25%, respectively (p<0.05). In cells of high malignancy degree this index decreased by 38-40% under the influence of FrP. Incubation of MCF-7 and T47D cells with the indicated agent also reduced the content of phospholipid cardiolipin by 15-16% (p < 0.05), and in MDA-MB-231, MCF-7/DDP, MDA-MB-468cells- by 29%, 30% and 32%, respectively. In addition, the effect of FrP caused a decrease in the levels of Mg2+ and lactate in MCF-7 and T47D cells by 21-29% and 14-24%, respectively, whereas in MDA-MB-231, MDA-MB-468, MCF-7/DDP cells- by 34-38% and 32-35%, respectively. In this case, the agent raised the level of glucose in the cells of low malignancy degree by 20-23% (p<0.05), and in the cells of high malignancy degree and with the phenotype of drug resistance- by 31-36%. However, the nanocomposite did not affect the activity of lactate dehydrogenase in all studied breast cancer cells. The study has shown that FrP has an effect on the pro/antioxidant balance and energy metabolism of cancer cells. In addition, the denoted effect of FrP was more pronounced in the breast cancer cells with a high malignancy degree and the phenotype of drug resistance.

268 - Quantitation of peroxynitrite production by macrophages

Reaction of peroxynitrite with coumarin boronic acid [1] was used for quantitation of peroxynitrite produced by activated murine macrophages (RAW 264.7) in cell culture. Highly fluorescent 7-hydroxycoumarin produced in reaction leads to fluorescence increase, affording real-time detection. Quiescent cells exhibit low rate of fluorescence increase which is further decreased in the presence of catalase, while catalase does not significantly affect the rate for activated cells. Specificity of the assay is supported by the dependence of the rate on the presence of arginine in the medium, inhibition by N6-(1-iminoethyl) L-lysine (inhibitor of iNOS) and by ebselen (peroxynitrite scavenger). The rate of peroxynitrite generation by stimulated RAW 264.7 was estimated as ~0.5 amol/(cell·s) that can be compared with NO generation rate of ~ 1.7 amol/(cell·s) determined earlier for the same cells [2]. Under the activation scheme used in this work (gamma-interferon + LPS), human THP-1 derived macrophages do not produce any peroxynitrite detectable by coumarin boronic acid. The level of iNOS mRNA in activated THP-1 derived macrophages does not exceed the level in control cells. 1 Zielonka, J.; Sikora, A.; Hardy, et al. Boronate probes as diagnostic tools for real time monitoring of peroxynitrite and hydroperoxides. Chemical Research in Toxicology. (2012), 25 (9), 1793–1799. 2 Woldman, Y. Y., Eubank, T. D., Mock, A. J., et al. Detection of nitric oxide production in cell cultures by luciferine-luciferase chemiluminescence. Biochemical and Biophysical Research Communications (2015), 465 232-238

INFLUENCE OF EXOGENOUS LACTOFERRIN ON THE OXIDANT/ ANTIOXIDANT BALANCE AND MOLECULAR PROFILE OF HORMONE RECEPTOR-POSITIvE AND -NEGATIvE HUMAN BREAST CANCER CELLS IN VITRO

To investigate the mechanisms of cytotoxic activity and pro-/antioxidant effect of lactoferrin on hormone receptor-positive and receptor-negative breast cancer cells in vitro. The study was performed on receptor-positive (MCF-7, T47D) and receptor-negative (MDA-MB-231, MDA-MB-468) human breast cancer cell lines. Immunocytochemical staining, flow cytometry, low-temperature electron paramagnetic resonance, and the Comet assay were used. Upon treatment with lactoferrin, the increased levels of reactive oxygen species (ROS) (p < 0.05), NO generation rate by inducible NO-synthase (p < 0.05) and the level of "free" iron (p < 0.05) were observed. Moreover, the effects of lactoferrin were more pronounced in receptor-negative MDA-MB-231and MDA-MB-468cells. These changes resulted in increased expression of proapoptotic Bax protein (p<0.05), reduced expression of the antiapoptotic Bcl-2 protein (p < 0.05) and level of not-oxidized mitochondrial cardiolipin (1.4-1.7-fold, p< 0.05). This, in turn, caused an increase in the percentage of apoptotic cells (by 14-24%, p < 0.05). Cytotoxic effects of lactoferrin were accompanied by an increase in the percentage of DNA in the comet tail and blocking cell cycle at G2/M phase, especially in receptor-negative cell lines. The study showed that exogenous lactoferrin causes a violation of an antioxidant balance by increasing the level of ROS, "free" iron and NO generation rate, resalting in the blocking of cell cycle at G2/M-phase and apoptosis of malignant cells.

Blood redox state and survival in patients with rectal cancer

&lt;p&gt;The cells of an organism have a definite concentration of electrons that characterizes their stable redox status. Electrons are transported to the oxygen and create the potential energy to realize the cell functions such as growth&lt;br /&gt;and apoptosis. Imbalance in the redox state of cells initiates the development and progression of the most socially significant pathologies. The aim of the research was to study the blood redox state in patients with rectal cancer (RC ) (the levels of ceruloplasmin (CP), transferrin (TF) and “free iron” (“FI”), superoxyd-, NO-generating activity of neutrophils and platelets) and its impact on оverall survival (ОS ). Venous blood samples of 60 patients with RC (T2-4N0-2M0G2) and 20 donors were studied. The defining levels of CP, TF, “FI” in blood was performed by electron paramagnetic resonance (EPR) at the temperature of liquid nitrogen (T=77 K ). The blood redox state in patients with RC are formed by: 2 times reduced CP activity in comparison with the&lt;br /&gt;reference sample; 3 times reduced TF content and 10 times increased “FI” levels; slightly increased superoxide generating activity of neutrophils and 8–14 times increased superoxide generating activity of platelets; 3.7 and 4.3 times reduced NO generation rate of neutrophils and platelets, respectively. It was found that the survival in patients with RC was significantly influenced by the level of CP activity (p&amp;lt;0.044), levels of “FI” (p&amp;lt;0.026), superoxide generating activity of NADP·H-oxidase of neutrophils (p&amp;lt;0.043). It was found that the adenocarcinoma progression in patients with RC (T2-4N0-2M0G2) was ccompanied bychange in the blood redox state by reducing CP activity and level of TF that led to the emergence of “FI” and the increase of its level. Besides, the changes of superoxyde- and NO-generating activity NADP∙H-oxidase and iNOS of neutrophils and platelets significantly influenced the imbalance of the blood redox state in the patients. The state of redox forming blood components in patients correlated with the оverall 5-year survival. It was found that the CP&lt;/p&gt;&lt;p&gt;activity, the “FI” level and the superoxide-generating activity of NADP·H-xidase of neutrophils and platelets hadsignificant effect on ОS .&lt;/p&gt;

Detection of nitric oxide production in cell cultures by luciferin–luciferase chemiluminescence

A chemiluminescent method is proposed for quantitation of NO generation in cell cultures. The method is based on activation of soluble guanylyl cyclase by NO. The product of the guanylyl cyclase reaction, pyrophosphate, is converted to ATP by ATP sulfurylase and ATP is detected in a luciferin–luciferase system. The method has been applied to the measurement of NO generated by activated murine macrophages (RAW 264.7) and bovine aortic endothelial cells. For macrophages activated by lipopolysaccharide and γ-interferon, the rate of NO production is about 100 amol/(cell·min). The rate was confirmed by the measurements of nitrite, the product of NO oxidation. For endothelial cells, the basal rate of NO generation is 5 amol/(cell·min); the rate approximately doubles upon activation by bradykinin, Ca2+ ionophore A23187 or mechanical stress. For both types of cells the measured rate of NO generation is strongly affected by inhibitors of NO synthase. The sensitivity of the method is about 50 pM/min, allowing the registration of NO generated by 102–104 cells. The enzyme-linked chemiluminescent method is two orders of magnitude more sensitive than fluorescent detection using 4-amino-5-methylamino-2′,7′-difluorofluorescein (DAF-FM).

PHARMACOLOGICAL EFFECT OF AMINOFERROCENE IN MICE WITH L1210 LEUKEMIA

To study the cytostatic and some biological effects of aminoferrocene using mice with L1210 lymphoid leukemia. Experiments were performed on BDF1 male mice (DBA/2, female × C57Bl/6, male) with transplantable L1210 lymphoid leukemia. Determination of antitumor activity of Benzyl-Fc Boron (Bn), it was injected intraperitoneally 6 times daily, starting on day 2 after L1210 leukemia cell transplantation. Doses of Bn such as 26; 260 and 2600 μg/kg were used. The determination of intracellular content of cardiolipin, thiols, reactive oxygen species (ROS) and also analysis of Annexin V positivity and mitochondrial transmembrane potential (JC-1 staining) were performed with use of flow cytometry. The levels of "free iron" complexes, transferrin active forms and the rate of NO generation were measured by EPR-specroscopy. Six daily injections of Bn at a dose of 26 μg/kg resulted in an increased survival of mice with L1210 leukemia by 28% (p < 0.05). Bn led to an increase of apoptotic cells number and ROS amount in leukemia cells. Besides, Bn caused a decrease of cardiolipin and nonprotein thiol compounds content. The membrane electrochemical potential of cell mitochondria was decreased also after Bn administration. Studies using EPR-spectroscopy revealed a significant increase in a level of "free iron", content of transferrin active species and generation rate of NO by inducible NO-synthase in L1210 cells after aminoferrocene administration. Our data indicate that Benzyl-Fc Boron can be promising candidate for realizing a new strategy of anticancer therapy with the use of ROS-inducing agents.

Quantitative determination of nitric oxide production in haemocytes: Nitrite reduction activity as a potential pathway of NO formation in haemolymph of Galleria mellonella larvae

The generation of nitric oxide by Galleria mellonella larvae haemocytes has been investigated. For this purpose, a fluorescent method, specific for detection of NO, has been developed. The method is based on the application of fluorescence probe DAF-FM diacetate and nitronyl nitroxyl radical, NNR, which accelerates the NO-dependent formation of fluorescence product, DAF-FM triazole. The key feature of the method is the registration and analysis of differential kinetics, namely, the difference of kinetics obtained in samples with NNR and without NNR. This approach allows us to exclude any other kinetic processes not related to triazole formation. The differential kinetics were calibrated versus NO generation rate and the resulting low limit of method sensitivity was obtained as about 0.4–0.5nM/min. The generation of nitric oxide by the haemocytes of insects treated with LPS in vivo has been detected at a rate of 0.5–0.7nM/min. However, the production of NO in haemocyte suspensions treated with both the substrate, l-arginine, and the inhibitor, l-NAME, of NOS, has not been detected within method sensitivity. These data provide only the upper level of NO generation by haemocytes but cannot be used to draw definite conclusions about NOS as a source of NO. Meanwhile, it is known, that NO can be formed by NOS independent mechanism. Indeed, we have observed a significant increase in NO generation in the samples of haemocytes intracellularly loaded with nitrite. Moreover, adding nitrite to lysed haemocytes results in the higher NO generation rate. After addition of 500μM nitrite, the rates of NO generation in the samples are determined to be 2 and 20–30nM/min, respectively. The nitrite/nitrate content of haemocytes and lymph were found to be 5 and 50μM, respectively. The detected nitrite reduction activity of haemocytes allowed us to estimate the generation rate of nitric oxide as 0.05–0.5nM/min from endogenous nitrite. It is thus assumed that the observed nitrite reduction activity in haemocytes is dominant in the increased NO production during immune response of the G. mellonella larvae.

Reactive oxygen and nitrogen species regulate inducible nitric oxide synthase function shifting the balance of nitric oxide and superoxide production

Inducible NOS (iNOS) is induced in diseases associated with inflammation and oxidative stress, and questions remain regarding its regulation. We demonstrate that reactive oxygen/nitrogen species (ROS/RNS) dose-dependently regulate iNOS function. Tetrahydrobiopterin (BH 4)-replete iNOS was exposed to increasing concentrations of ROS/RNS and activity was measured with and without subsequent BH 4 addition. Peroxynitrite (ONOO −) produced the greatest change in NO generation rate, ∼95% decrease, and BH 4 only partially restored this loss of activity. Superoxide ( O 2 - ) greatly decreased NO generation, however, BH 4 addition restored this activity. Hydroxyl radical ( OH) mildly decreases NO generation in a BH 4-dependent manner. iNOS was resistant to H 2O 2 with only slightly decreased NO generation with up to millimolar concentrations. In contrast to the inhibition of NO generation, ROS enhanced O 2 - production from iNOS, while ONOO − had the opposite effect. Thus, ROS promote reversible iNOS uncoupling, while ONOO − induces irreversible enzyme inactivation and decreases both NO and O 2 - production.

Electrocatalytic Nitrite Reduction to Nitrogen Oxide by a Synthetic Analogue of the Active Site of Cu‐Containing Nitrite Reductase Incorporated in Nafion Film

AbstractThe electrocatalytic reduction of nitrite to NO by [CuMe2bpa(H2O)(ClO4)]+ (1), which is a model for the active site of copper‐containing nitrite reductase, incorporated in Nafion film was investigated. The Cu complex in the Nafion matrix exhibits an intense band at 267 nm and a broad band around 680 nm, assigned to d–d and ligand field transitions, respectively. The 77‐K EPR spectrum of 1 in the Nafion matrix reveals the typical axial signals (g//=2.28, g$\rm{_\perp}$=2.08, A//=13.3 mT) of a tetragonal Cu2+ chromophore. The redox potential, which is related to the Cu+/Cu2+ couple, was −146 mV (ΔE=72 mV) at pH 5.5. The redox reaction of 1 in Nafion was not dependent on pH and was a diffusion‐controlled process. The electronic structure and redox properties of 1 in the negatively charged polymer matrix were almost the same as those in aqueous solution. In the presence of nitrite, an increase in the cathodic current was observed in the cyclic voltammogram of 1 in the Nafion matrix. The current increase was dependent on the nitrite concentration and pH in solution. Upon reaching −400 mV, a linear generation of NO was observed for the 1/Nafion film coated electrode. The relationship between the rate of NO generation and the nitrite concentration in solution was analyzed with the Michaelis–Menten equation, where Vmax=45.1 nM s−1 and Km=15.8 mM at pH 5.5. The Cu complex serves the function of both the catalyst and electron transport in the Nafion matrix. The sensitivity of the electrode was estimated to be 3.23 μA mM−1 in the range of 0.1–0.4 mM nitrite.

Electroreduction of nitrite to nitrogen oxide by a copper-containing nitrite reductase model complex incorporated into collagen film

The electrocatalytic reduction of nitrite to NO by CuMe 2bpaCl 2, which is a model for the active site of copper-containing nitrite reductase, incorporated into collagen film was investigated. The 77-K EPR spectrum of CuMe 2bpaCl 2 in the collagen matrix revealed the typical axial signals ( g // = 2.26, g ⊥ = 2.05, A // = 16.4 mT) of a tetragonal Cu 2+ chromophore. The redox potential, which is related to the Cu +/Cu 2+ couple, was − 63 mV ( ∆E = 72 mV) at pH 5.5. In the presence of nitrite, an increase in the cathodic current was observed in the cyclic voltammogram of CuMe 2bpaCl 2 in the collagen matrix. Upon reaching − 300 mV, a linear generation of NO was observed for the CuMe 2bpaCl 2/collagen film-coated electrode. The relationship between the rate of NO generation and the nitrite concentration in solution was analyzed using the Michaelis–Menten equation, where V max = 3.16 nM s − 1 and K m = 1.1 mM at pH 5.5. The current increase and the reaction rate were dependent on the pH of the solution. The mechanism of nitrite reduction by the copper complex in the collagen matrix was the same mechanism as that of the enzyme in aqueous solution.

Flame structure and NO generation in oxy-fuel combustion at high pressures

A numerical study of oxy-fuel combustion has been carried out in the pressure range of 0.1–3 MPa with methane as the fuel and carbondioxide-diluted oxygen with trace amount of nitrogen (termed here as c_air) as the oxidant. The flame structure and NO generation rate have been calculated using the flamelet model with the detailed GRI 3.0 mechanism for two oxygen concentrations of 23.3% and 20% by weight in the oxidant at a strain rate of 40 s −1 (corresponding to a scalar dissipation rate of 1 s −1). It is observed that, for the reference case of 23.3 wt.% of oxygen, as the pressure increases, the peak temperature of the flame increases rapidly up to a pressure of 0.5 MPa, and more gradually at higher pressures. The concentrations of important intermediate radicals such as CH 3, H and OH decrease considerably with increasing pressure while NO concentration follows the same trend as the temperature. Reducing the oxygen concentration to 20% by weight leads to an order of magnitude reduction in NO concentration. Also, for pressures greater than 0.3 MPa, the NO concentration decreases with increasing pressure in spite of the increasing peak flame temperatures. This can be attributed to the increasing domination of recombination reactions leading to less availability of the intermediate radicals H and OH which are necessary for the formation of NO by the thermal route. It is concluded that a stable, low NO x oxy-fuel flame can be obtained at high pressures at slightly increased dilution of oxygen.

모형가스터빈 연소기에서 분무각 변화에 따른 연료농도 및 화염온도 분포

KIVA3V를 이용하여 모형 가스터빈 연소기에서 Jet-A의 분무와 연소에 관한 수치해석을 하였다. 연소기 형상은 6개의 희석홀을 가지며, 스월효과를 고려하였다. 연소 특성을 해석하기 위해 열적 NO 발생을 고려하였다. 다양한 분무각에 대해서 Jet-A 증기, 화염온도 분포와 NO 발생량에 대하여 비교하였다. 분무각이 커질수록 1차 영역에서 연료 증기가 비교적 고르게 나타나게 되며 증발이 더욱 빠르게 되고, 주변 기체와 연료 증기 사이의 혼합이 증가한다. 그 결과 고온부가 넓게 나타나게 되고, 이에 의해 열적 NO 발생이 증가한다. Jet-A spray and combustion were numerically analyzed in annular type combustor model using KIVA3V. The combustor geometry have 6 dilute holes. Swirl effect and thermal NO were considered in this investigation to analyze mixing and combustion characteristics. Fuel vapor, flame temperature, NO generation were investigated for various spray angle. As increase of spray angle, Jet-A vapor appeared uniformly in primary zone and evaporation rate was increased. Mixing between fuel vapor and ambient gas was enhanced as increase of spray angle. As a result, high temperature region appeared widely and thermal NO generation rate was increased.

Nitrite Reductase Activity of Hemoglobin S (Sickle) Provides Insight into Contributions of Heme Redox Potential Versus Ligand Affinity

Hemoglobin A (HbA) is an allosterically regulated nitrite reductase that reduces nitrite to NO under physiological hypoxia. The efficiency of this reaction is modulated by two intrinsic and opposing properties: availability of unliganded ferrous hemes and R-state character of the hemoglobin tetramer. Nitrite is reduced by deoxygenated ferrous hemes, such that heme deoxygenation increases the rate of NO generation. However, heme reactivity with nitrite, represented by its bimolecular rate constant, is greatest when the tetramer is in the R quaternary state. The mechanism underlying the higher reactivity of R-state hemes remains elusive. It can be due to the lower heme redox potential of R-state ferrous hemes or could reflect the high ligand affinity geometry of R-state tetramers that facilitates nitrite binding. We evaluated the nitrite reductase activity of unpolymerized sickle hemoglobin (HbS), whose oxygen affinity and cooperativity profile are equal to those of HbA, but whose heme iron has a lower redox potential. We now report that HbS exhibits allosteric nitrite reductase activity with competing proton and redox Bohr effects. In addition, we found that solution phase HbS reduces nitrite to NO significantly faster than HbA, supporting the thesis that heme electronics (i.e. redox potential) contributes to the high reactivity of R-state deoxy-hemes with nitrite. From a pathophysiological standpoint, under conditions where HbS polymers form, the rate of nitrite reduction is reduced compared with HbA and solution-phase HbS, indicating that HbS polymers reduce nitrite more slowly.

Hemoglobin-stabilized tetranitrosyl binuclear iron complex with pyridine-2-yl in aqueous solutions

The tetranitrosyl iron complex with pyridine-2-yl [Fe2(SC5H4N)2(NO)4] (1) has higher NO-donating activity in 3% aqueous solutions of DMSO (pH 7.0, 25 °C) than the organic NO donor, viz., adduct of NO with diethylenetriamine (NO-adduct). The NO concentration was determined by the spectrophotometric method based on the formation of an NO complex with hemoglobin (Hb). The apparent first-order rate constants of the studied reactions are (6.15±0.6)·10−1 s−1 and (0.8±0.08)·10−1 s−1 for complex 1 and the NO-adduct, respectively, at an Hb concentration of 2·10−1 mol L−1 and the ratio [NO donor]/[Hb] = 10. The effect of Hb and [NO donor]/[Hb] ratio on the rate of NO generation from a solution of complex 1 was studied. For a fourfold decrease in the concentration of complex 1 the reaction rate constant decreases to 0.5·10−4 s−1, whereas the fourfold increase in the Hb concentration results in the stabilization of complex 1.

Do nitric oxide donors mimic endogenous NO-related response in plants?

Huge advances achieved recently in elucidating the role of NO in plants have been made possible by the application of NO donors. However, the application of NO to plants in various forms and doses should be subjected to detailed verification criteria. Not all metabolic responses induced by NO donors are reliable and reproducible in other experimental designs. The aim of the presented studies was to investigate the half-life of the most frequently applied donors (SNP, SNAP and GSNO), the rate of NO release under the influence of light and reducing agents. At a comparable donor concentration (500 microM) and under light conditions the highest rate of NO generation was found for SNAP, followed by GSNO and SNP. The measured half-life of the donor in the solution was 3 h for SNAP, 7 h for GSNO and 12 h for SNP. A temporary lack of light inhibited NO release from SNP, both in the solution and SNP-treated leaf tissue, which was measured by the electrochemical method. Also a NO, selective fluorescence indicator DAF-2DA in leaves supplied with different donors showed green fluorescence spots in the epidermal cells mainly in the light. SNP as a NO donor was the most photosensitive. The activity of PAL, which plays an important role in plant defence, was also activated by SNP in the light, not in the dark. S-nitrosothiols (SNAP and GSNO) also underwent photodegradation, although to a lesser degree than SNP. Additionally, NO generation capacity from S-nitrosothiols was shown in the presence of reducing agents, i.e. ascorbic acid and GSH, and the absence of light. The authors of this paper would like to polemicize with the commonly cited statement that "donors are compounds that spontaneously break down to release NO" and wish to point out the fact that the process of donor decomposition depends on the numerous external factors. It may be additionally stimulated or inhibited by live plant tissue, thus it is necessary to take into consideration these aspects and monitor the amount of NO released by the donor.

Mitochondrial function and mitochondria-induced apoptosis in an overstimulated rat ovarian cycle

Female rats were treated with FSH (40 IU/kg) on the first and second diestrus days (D1 and D2) and with LH (40 IU/kg) on the proestrus (P) day to synchronize and maximize ovarian changes. Follicle area increased by 50% from D1 to P, and the estrus (E) phase showed multiple corpora lutea and massive apoptosis. Increased oxygen uptakes (42-102%) were determined in ovary slices and in isolated mitochondria in active state 3 along the proliferation phase (D1-D2-P) that returned to initial values in the E phase. Mitochondrial content and the electron transfer activities of complexes I and IV were also maximal in the P phase (20-79% higher than in D1). Production of NO by mitochondrial nitric oxide synthase (mtNOS), biochemically determined, and the mtNOS functional activity in regulating state 3 oxygen uptake were also maximal at P and 79-88% higher than at D1. The moderately increased rate of NO in the proliferative phase is associated with mitochondrial biogenesis, whereas the high rate of NO generation by mtNOS at phase P appears to trigger mitochondria-dependent apoptosis. The calculated fraction of ovary mitochondria in state 3 was at a minimal value at the P phase. Mitochondrial oxidative damage, with increased thiobarbituric acid-reactive substances and protein carbonyls, indicates progressive mitochondrial dysfunction between phases P and E. The roles of mitochondria as ATP provider, as a source of NO to signal for mitochondrial proliferation and mitochondria-dependent apoptosis, and as a source of O(2)(-) and H(2)O(2) appear well adapted to serve the proliferation-apoptosis sequence of the ovarian cycle.

Cytoprotective effects of nitrite during in vivo ischemia-reperfusion of the heart and liver

Nitrite represents a circulating and tissue storage form of NO whose bioactivation is mediated by the enzymatic action of xanthine oxidoreductase, nonenzymatic disproportionation, and reduction by deoxyhemoglobin, myoglobin, and tissue heme proteins. Because the rate of NO generation from nitrite is linearly dependent on reductions in oxygen and pH levels, we hypothesized that nitrite would be reduced to NO in ischemic tissue and exert NO-dependent protective effects. Solutions of sodium nitrite were administered in the setting of hepatic and cardiac ischemia-reperfusion (I/R) injury in mice. In hepatic I/R, nitrite exerted profound dose-dependent protective effects on cellular necrosis and apoptosis, with highly significant protective effects observed at near-physiological nitrite concentrations. In myocardial I/R injury, nitrite reduced cardiac infarct size by 67%. Consistent with hypoxia-dependent nitrite bioactivation, nitrite was reduced to NO, S-nitrosothiols, N-nitros-amines, and iron-nitrosylated heme proteins within 1-30 minutes of reperfusion. Nitrite-mediated protection of both the liver and the heart was dependent on NO generation and independent of eNOS and heme oxygenase-1 enzyme activities. These results suggest that nitrite is a biological storage reserve of NO subserving a critical function in tissue protection from ischemic injury. These studies reveal an unexpected and novel therapy for diseases such as myocardial infarction, organ preservation and transplantation, and shock states.