Reactions of peroxynitrite in the mitochondrial matrix

Superoxide radical (O2-) and nitric oxide (NO) produced at the mitochondrial inner membrane react to form peroxynitrite (ONOO-) in the mitochondrial matrix. Intramitochondrial ONOO- effectively reacts with a few biomolecules according to reaction constants and intramitochondrial concentrations. The second-order reaction constants (in M(-1) s(-1)) of ONOO- with NADH (233 +/- 27), ubiquinol-0 (485 +/- 54) and GSH (183 +/- 12) were determined fluorometrically by a simple competition assay of product formation. The oxidation of the components of the mitochondrial matrix by ONOO- was also followed in the presence of CO2, to assess the reactivity of the nitrosoperoxocarboxylate adduct (ONOOCO2-) towards the same reductants. The ratio of product formation was about similar both in the presence of 2.5 mM CO2 and in air-equilibrated conditions. Liver submitochondrial particles supplemented with 0.25-2 microM ONOO- showed a O2- production that indicated ubisemiquinone formation and autooxidation. The nitration of mitochondrial proteins produced after addition of 200 microM ONOO- was observed by Western blot analysis. Protein nitration was prevented by the addition of 50-200 microM ubiquinol-0 or GSH. An intramitochondrial steady state concentration of about 2 nM ONOO- was calculated, taking into account the rate constants and concentrations of ONOO- coreactants.     

Biphasic modulation of fatty acid synthase by hydrogen peroxide in Saccharomyces cerevisiae

Taking into account published contradictory results concerning the regulation of fatty acid synthase (Fas) by H(2)O(2), we carried out a systematic study where two methods of H(2)O(2) delivery (steady-state and bolus addition) and the effect of a wide range of H(2)O(2) concentrations were investigated. A decrease in Fas activity was observed for cells exposed to 100 and 150μM H(2)O(2) in a steady-state, while a bolus addition of the same H(2)O(2) concentrations did not alter Fas activity. Similar results were observed for the mRNA levels of FAS1, the gene that encodes Fas subunit β. However, the exposure to a steady-state 50μM H(2)O(2) dose lead to an increase in FAS1 mRNA levels, showing a biphasic modulation of Fas by H(2)O(2). The results obtained emphasize that cellular effects of H(2)O(2) can vary over a narrow range of concentrations. Therefore, a tight control of H(2)O(2) exposure, which can be achieved by exposing H(2)O(2) in a steady-state, is important for cellular studies of H(2)O(2)-dependent redox regulation.     

Identification of a critical lipid ratio in raft-like phases exposed to nitric oxide: An AFM study

Lipid rafts are discrete, heterogeneous domains of phospholipids, sphingolipids, and sterols that are present in the cell membrane. They are responsible for conducting cell signaling and maintaining lipid-protein functionality. Redox-stress-induced modifications to any of their components can severely alter the mechanics and dynamics of the membrane causing impairment to the lipid-protein functionality. Here, we report on the effect of sphingomyelin (SM) in controlling membrane permeability and its role as a regulatory lipid in the presence of nitric oxide (NO). Force spectroscopy and atomic force microscopy imaging of raft-like phases (referring here to the coexistence of “liquid-ordered” and “liquid-disordered” phases in model bilayer membranes) prepared from lipids: 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC):SM:cholesterol (CH) (at three ratios) showed that the adhesion forces to pull the tip out of the membrane increased with increasing SM concentration, indicating decreased membrane permeability. However, in the presence of NO radical (1 and 5 μM), the adhesion forces decreased depending on SM concentration. The membrane was found to be stable at the ratio POPC:SM:CH (2:1:1) even when exposed to 1 μM NO. We believe that this is a critical ratio needed by the raft-like phases to maintain homeostasis under stress conditions. The stability could be due to an interplay existing between SM and CH. However, at 5 μM NO, membrane deteriorations were detected. For POPC:SM:CH (2:2:1) ratio, NO displayed a pro-oxidant behavior and damaged the membrane at both radical concentrations. These changes were reflected by the differences in the height profiles of the raft-like phases observed by atomic force microscopy imaging. Malondialdehyde (a peroxidation product) detection suggests that lipids may have undergone lipid nitroxidation. The changes were instantaneous and independent of radical concentration and incubation time. Our study underlines the need for identifying appropriate ratios in the lipid rafts of the cell membranes to withstand redox imbalances caused by radicals such as NO.     

Cdk2 nitrosylation and loss of mitochondrial potential mediate NO-dependent biphasic effect on HL-60 cell cycle

Nitric oxide (NO), a multifaceted signaling molecule, regulates a wide array of cell functions, including proliferation, differentiation, cytostasis, and apoptosis, which depend on the cell type and redox status. This study systematically explores the effects of NO donors on promyelocytic HL-60 cell proliferation and apoptosis. The NO donor DETA-NO modulated the HL-60 cell cycle in a biphasic manner. DETA-NO in lower concentrations (1–100 μM) had a proliferative effect as investigated by [³H]thymidine incorporation, BrdU labeling, and cell cycle analysis, whereas cells treated with higher concentrations (250 μM–1 mM) showed cytostasis, apoptosis, mitochondrial membrane potential loss, caspase-3 activity, and dUTP nick-end labeling. The proliferative effect of DETA-NO was NO dependent and redox sensitive, as the effect was abolished by cPTIO and DTT pretreatment, respectively. Expression of various cell cycle regulators such as Cdk2, cyclin B, and cyclin E was significantly augmented in cells treated with 10–50 μM DETA-NO. The proliferative effect of NO was blocked by roscovitine, a Cdk2 inhibitor. S-nitrosylation of Cdk2 and an increase in the Cdk2-associated kinase activity was observed for the first time in DETA-NO-treated cells. This study demonstrates that the DETA-NO-mediated biphasic effect was dependent on Cdk2 nitrosylation/activation and the loss of mitochondrial potential at low and high concentrations, respectively.     

trans-[Ru(NO)(NH3)4(py)](BF4)3.H2O encapsulated in PLGA microparticles for delivery of nitric oxide to B16-F10 cells: Cytotoxicity and phototoxicity

The NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (py=pyridine) was loaded into poly-lactic-co-glycolic acid (PLGA) microparticles using the double emulsification technique. Scanning electron microscopy (SEM) and dynamic light scattering revealed that the particles are spherical in shape, have a diameter of 1600nm, and have low tendency to aggregate. The entrapment efficiency was 25%. SEM analysis of the melanoma cell B16-F10 in the presence of the microparticles containing the complex trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O (pyMP) showed that the microparticles were adhered to the cell surface after 2h of incubation. The complex with concentrations lower than 1x10(-4)M did not show toxicity in B16-F10 murine cells. The complex in solution is toxic at higher concentrations (>1x10(-3)M), with cell death attributed to NO release following the reduction of the complex. pyMP is not cytotoxic due to the lower bioavailability and availability of the entrapped complex to the medium and its reducing agents. However, pyMP is phototoxic upon light irradiation. The phototoxicity strongly suggests that cell death is due to NO release from trans-[Ru(NO)(NH(3))(4)(py)](3+). This work shows that pyMP can serve as a model for a drug delivery system carrying the NO donor trans-[Ru(NO)(NH(3))(4)(py)](BF(4))(3).H(2)O, which can release NO locally at the tumor cell by irradiation with light only.     

A system for exposing molecules and cells to biologically relevant and accurately controlled steady-state concentrations of nitric oxide and oxygen

Nitric oxide (NO) plays key roles in cell signaling and physiology, with diverse functions mediated by NO concentrations varying over three orders-of-magnitude. In spite of this critical concentration dependence, current approaches to NO delivery in vitro result in biologically irrelevant and poorly controlled levels, with hyperoxic conditions imposed by ambient air. To solve these problems, we developed a system for controlled delivery of NO and O(2) over large concentration ranges to mimic biological conditions. Here we describe the fabrication, operation and calibration of the delivery system. We then describe applications for delivery of NO and O(2) into cell culture media, with a comparison of experimental results and predictions from mass transfer models that predict the steady-state levels of various NO-derived reactive species. We also determined that components of culture media do not affect the steady-state levels of NO or O(2) in the device. This system provides critical control of NO delivery for in vitro models of NO biology and chemistry.     

Accommodation of NO in the active site of mammalian and bacterial cytochrome c oxidase aa3

Following different reports on the stoichiometry and configuration of NO binding to mammalian and bacterial reduced cytochrome c oxidase aa₃ (CcO), we investigated NO binding and dynamics in the active site of beef heart CcO as a function of NO concentration, using ultrafast transient absorption and EPR spectroscopy. We find that in the physiological range only one NO molecule binds to heme a₃, and time-resolved experiments indicate that even transient binding to Cu_B does not occur. Only at very high (∼ 2 mM) concentrations a second NO is accommodated in the active site, although in a different configuration than previously observed for CcO from Paracoccus denitrificans [E. Pilet, W. Nitschke, F. Rappaport, T. Soulimane, J.-C. Lambry, U. Liebl and M.H. Vos. Biochemistry 43 (2004) 14118-14127], where we proposed that a second NO does bind to Cu_B. In addition, in the bacterial enzyme two NO molecules can bind already at NO concentrations of ∼ 1 μM. The unexpected differences highlighted in this study may relate to differences in the physiological relevance of the CcO-NO interactions in both species.     

Mitochondrial metabolic states and membrane potential modulate mtNOS activity

The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, l-arginine (about 310 μM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their K_M values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H₂O₂ production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.     

S-nitrosoglutathione incorporated in poly(ethylene glycol) matrix: potential use for topical nitric oxide delivery.

Incorporation of nitric oxide (NO) donors in non-toxic polymeric matrices can be a useful strategy for allowing topical NO delivery. We have incorporated the NO-donor S-nitrosoglutathione (GSNO) into a liquid poly(ethylene glycol) (PEG)/H2O matrix through the S-nitrosation of GSH by a NO/O2 gas mixture. Kinetic measurements of GSNO decomposition associated with NO release were performed at 25, 35, and 45 degrees C in the dark and under irradiation with UV/Vis light, lambda>480 nm and lambda=333 nm. NO release from the liquid matrix to the gas phase was confirmed by mass spectrometry. The PEG/H2O matrix stabilizes GSNO leading to expressive reductions in the initial rates of thermal and photochemical NO release, compared to aqueous GSNO solution. This matrix effect is assigned to diffusional constrains imposed on the escape of the NO and GS radicals formed in the solvent cage. This effect allows the storage of PEG-GSNO formulations for extended periods (more than 65 days at freezer) with negligible decomposition. PEG-GSNO formulation seems therefore to be applicable in topical NO delivery and GSNO displays potential as a percutaneous absorption enhancer. Moreover, the rate of NO release can be locally increased by irradiation with visible light.     

Mitochondrial ryanodine‐sensitive Ca2+ channels of rat liver

To examine ryanodine‐sensitive Ca²⁺ channels in mitochondria of rat hepatocytes and their role in energy state of the cells via investigation of the ryanodine effect on mitochondrial membrane potential. Oxygen consumption was measured by polarography using the Clark electrode. The substrates of oxidation such as pyruvate (5mM), α‐ketoglutarate (5mM), or succinate (5mM) were used. Oxidative phosphorylation was stimulated by the addition of adenosine diphosphate (200nM). Mitochondrial membrane potential was measured using a voltage‐sensitive fluorescent probe tetramethylrhodamine‐methyl‐ester (0.1μM) and was analyzed by a flow cytometer. To evaluate the intact mitochondria, we used carbonil cyanide m‐chlorophenyl hydrazone (CCCP, 10μM). Changes in the ionized calcium concentration in rat liver mitochondria were measured using a fluorescent probe Fluo‐4 AM. Effect of ryanodine on oxygen consumption of rat liver mitochondria depends on the oxidation substrate and the incubation time. Oxidation of pyruvate in the presence of ryanodine (0.05μM) decreased the membrane potential of rat liver mitochondria by 38.4%. At higher concentrations, ryanodine (0.1μM or 1μM) led to decrease of membrane potential by 51.7% and 42.8%, respectively. In contrast, oxidation of α‐ketoglutarate in the presence of ryanodine (0.05μM) increased mitochondrial membrane potential by 16.8%. However, at higher concentrations, ryanodine (0.1μM or 1μM) triggered a decreasing of membrane potential by 42.5% and 31.0%, respectively. Therefore, ryanodine at various concentrations (0.05μM, 0.1μM, or 1μM) causes differential effects on Ca²⁺ concentration in the mitochondria matrix under oxidation of pyruvate or α‐ketoglutarate. The data suggest the presence of ryanodine receptors in mitochondrial membrane of rat hepatocytes. Their inhibition with higher concentrations of ryanodine leads to decreasing of intra‐mitochondrial Ca²⁺ concentration and affecting the energy state of mictochondria in hepatocytes.     

Formation of peroxynitrite from reaction of nitroxyl anion with molecular oxygen.

Peroxynitrite (ONOO(-)/ONOOH) is generally expected to be formed in vivo from the diffusion-controlled reaction between superoxide (O(2)) and nitric oxide ((*)NO). In the present paper we show that under aerobic conditions the nitroxyl anion (NO(-)), released from Angeli's salt (disodium diazen-1-ium-1,2,2-triolate, (-)ON=NO(2)(-)), generated peroxynitrite with a yield of about 65%. Simultaneously, hydroxyl radicals are formed from the nitroxyl anion with a yield of about 3% via a minor, peroxynitrite-independent pathway. Further experiments clearly underline that the chemistry of NO(-) in the presence of oxygen is mainly characterized by peroxynitrite and not by HO( small middle dot) radicals. Quantum-chemical calculations predict that peroxynitrite formation should proceed via intermediary formation of (*)NO and O(2), probably by an electron-transfer mechanism. This prediction is supported by the fact that H(2)O(2) is formed during the decay of NO(-) in the presence of superoxide dismutase (Cu(II),Zn-SOD). Since the nitroxyl anion may be released endogenously by a variety of biomolecules, substantial amounts of peroxynitrite might be formed in vivo via NO(-) in addition to the "classical" ( small middle dot)NO + O(2)() pathway.     

Template-directed synthesis of oligonucleotides under eutectic conditions

Summary One of the most important sets of model prebiotic experiments consists of reactions that synthesize complementary oligonucleotides from preformed templates under nonenzymatic conditions. Most of these experiments are conducted at 4°C using 0.01–0.1 M concentrations of activated nucleotide monomer and template (monomer equivalent). In an attempt to extend the conditions under which this type of reaction can occur, we have concentrated the reactants by freezing at –18°C, which is close to the NaCl–H₂O eutectic at –21°C.The results from this set of experiments suggest that successful syntheses can occur with poly(C) concentrations as low at 5×10^–4 M and 2MeImpG concentrations at 10^–3 M. It was also anticipated that this mechanism might allow the previously unsuccessful poly(A)-directed synthesis of oligo(U)s to occur. However, no template effect was seen with the poly(A) and ImpU system. The failure of these conditions to allow template-directed synthesis of oligo(U)s supports the previously proposed idea that pyrimidines may not have been part of the earliest genetic material.Because of the low concentrations of monomer and template that would be expected from prebiotic syntheses, this lower temperature could be considered a more plausible geologic setting for template-directed synthesis than the standard reaction conditions.     

Nitric oxide affects IL-6 expression in human peripheral blood mononuclear cells involving cGMP-dependent modulation of NF-κB activity

Interleukin 6 (IL-6) and nitric oxide (NO) are important mediators of the inflammatory response. We report that in human peripheral blood mononuclear cells (PBMCs), NO exerts a biphasic effect on the expression of IL-6. Using sodium nitroprusside (SNP) and S-nitrosoglutathione (GSNO) as NO-donating compounds, we observed that both mRNA and protein levels of IL-6 increased at lower (≤10μM) and decreased at higher (>100μM) concentrations of NO donors. Changes in the expression of IL-6 correlated with changes in the activity of NF-κB, which increased at lower and decreased at higher concentrations of both NO donors as shown by the electrophoretic mobility shift assay (EMSA). The effects of NO on NF-κB activity were cGMP-dependent because they were reversed in the presence of ODQ, the inhibitor of soluble guanylyl cyclase (sGC), and KT5823, the inhibitor of cGMP-dependent protein kinase (PKG). Moreover, the membrane permeable analog of cGMP (8-Br-cGMP) mimicked the effect of the NO donors. These observations show that NO, depending on its concentration, may act in human PBMCs as a stimulator of IL-6 expression involving the sGC/cGMP/PKG pathway.     

Reactivity of nitric oxide with the [4Fe-4S] cluster of dihydroxyacid dehydratase from Escherichia coli

Although the NO (nitric oxide)-mediated modification of iron-sulfur proteins has been well-documented in bacteria and mammalian cells, specific reactivity of NO with iron-sulfur proteins still remains elusive. In the present study, we report the first kinetic characterization of the reaction between NO and iron-sulfur clusters in protein using the Escherichia coli IlvD (dihydroxyacid dehydratase) [4Fe-4S] cluster as an example. Combining a sensitive NO electrode with EPR (electron paramagnetic resonance) spectroscopy and an enzyme activity assay, we demonstrate that NO is rapidly consumed by the IlvD [4Fe-4S] cluster with the concomitant formation of the IlvD-bound DNIC (dinitrosyl-iron complex) and inactivation of the enzyme activity under anaerobic conditions. The rate constant for the initial reaction between NO and the IlvD [4Fe-4S] cluster is estimated to be (7.0+/-2.0)x10(6) M(-2) x s(-1) at 25 degrees C, which is approx. 2-3 times faster than that of the NO autoxidation by O2 in aqueous solution. Addition of GSH failed to prevent the NO-mediated modification of the IlvD [4Fe-4S] cluster regardless of the presence of O2 in the medium, further suggesting that NO is more reactive with the IlvD [4Fe-4S] cluster than with GSH or O2. Purified aconitase B [4Fe-4S] cluster from E. coli has an almost identical NO reactivity as the IlvD [4Fe-4S] cluster. However, the reaction between NO and the endonuclease III [4Fe-4S] cluster is relatively slow, apparently because the [4Fe-4S] cluster in endonuclease III is less accessible to solvent than those in IlvD and aconitase B. When E. coli cells containing recombinant IlvD, aconitase B or endonuclease III are exposed to NO using the Silastic tubing NO delivery system under aerobic and anaerobic conditions, the [4Fe-4S] clusters in IlvD and aconitase B, but not in endonuclease III, are efficiently modified forming the protein-bound DNICs, confirming that NO has a higher reactivity with the [4Fe-4S] clusters in IlvD and aconitase B than with O2 or GSH. The results suggest that the iron-sulfur clusters in proteins such as IlvD and aconitase B may constitute the primary targets of the NO cytotoxicity under both aerobic and anaerobic conditions.     

The reaction of nitric oxide with ubiquinol: kinetic properties and biological significance

The reaction of nitric oxide (*NO) with ubiquinol-0 and ubiquinol-2, short-chain analogs of coenzyme Q, was examined in anaerobic and aerobic conditions in terms of formation of intermediates and stable molecular products. The chemical reactivity of ubiquinol-0 and ubiquinol-2 towards *NO differed only quantitatively, the reactions of ubiquinol-2 being slightly faster than those of ubiquinol-0. The ubiquinol/*NO reaction entailed oxidation of ubiquinol to ubiquinone and reduction of *NO to NO-, the latter identified by its reaction with metmyoglobin to form nitroxylmyoglobin and indirectly by measurement of nitrous oxide (N2O) by gas chromatography. Both the rate of ubiquinone accumulation and *NO consumption were linearly dependent on ubiquinol and *NO concentrations. The stoichiometry of *NO consumed per either ubiquinone formed or ubiquinol oxidized was 1.86 A 0.34. The reaction of *NO with ubiquinols proceeded with intermediate formation of ubisemiquinones that were detected by direct EPR. The second order rate constants of the reactions of ubiquinol-0 and ubiquinol-2 with *NO were 0.49 and 1.6 x 10(4) M(-1)s(-1), respectively. Studies in aerobic conditions revealed that the reaction of *NO with ubiquinols was associated with O2 consumption. The formation of oxyradicals - identified by spin trapping EPR- during ubiquinol autoxidation was inhibited by *NO, thus indicating that the O2 consumption triggered by *NO could not be directly accounted for in terms of oxyradical formation or H2O2 accumulation. It is suggested that oxyradical formation is inhibited by the rapid removal of superoxide anion by *NO to yield peroxynitrite, which subsequently may be involved in the propagation of ubiquinol oxidation. The biological significance of the reaction of ubiquinols with *NO is discussed in terms of the cellular O2 gradients, the steady-state levels of ubiquinols and *NO, and the distribution of ubiquinone (largely in its reduced form) in biological membranes with emphasis on the inner mitochondrial membrane.     

Membrane conductance in trained and untrained subjects using either steady state or single breath measurements of NO transfer.

The aim of this work was to define the relationship between membrane conductance for NO (Dm) and physical activity by using either the steady state NO transfer (T(LNO)SS) or the single breath method (T(LNO)SB), making the hypothesis that NO transfer is only limited by the membrane. Alterations in T(LNO)SS with lung volume during tidal ventilation were measured in six subjects at rest and during steady exercise at 30, 60, and 80% of maximal aerobic power (MAP). A fast responding chemoluminescent NO analyser was used. Two calculation methods were used by sampling NO: (1) at mid-tidal volume, (2) in the middle of the alveolar plateau. T(LNO)SB at rest and maximal oxygen consumption (V(.-)O(2)max) were also measured in 18 other subjects. At rest T(LNO)SS with method 2 was 192% of the value given by method 1. T(LNO)SS with method 1 increased by 50% with 80% MAP as it did not change with method 2. Method 2 seemed inaccurate. T(LNO)SB at rest, which is closely related to Dm, was correlated to age and V(.-)O(2)max, T(LNO)SB=182-1.2 age+24.3 V(.-)O(2) max(l min(-1)) (p<0.01, r(2)=0.72). The T(LNO)SS and T(LNO)SB versus lung volume relationships suggest an influence of the breathing pattern on Dm. Dm can be estimated either by these two NO transfer methods, however the use of the T(LNO)SS method is highly sensitive to the alveolar sampling level. Dm increase during exercise is a function of MAP. Dm at rest decreases with age as it increases with MAP.     

Stable polymer bilayers for protein channel recordings at high guanidinium chloride concentrations

The use of chaotropic reagents is common in biophysical characterization of biomolecules. When the study involves transmembrane protein channels, the stability of the protein channel and supporting bilayer membrane must be considered. In this letter, we show that planar bilayers composed of poly(1,2-butadiene)-b-poly(ethylene oxide) diblock copolymer are stable and leak-free at high guanidinium chloride concentrations, in contrast to diphytanoyl phosphatidylcholine bilayers, which exhibit deleterious leakage under similar conditions. Furthermore, insertion and functional analysis of channels such as α-hemolysin and MspA are straightforward in these polymer membranes. Finally, we demonstrate that α-hemolysin channels maintain their structural integrity at 2 M guanidinium chloride concentrations using blunt DNA hairpins as molecular reporters.     

Sodium azide as indirect nitric oxide donor: researches on the rat aorta isolated segments

A comparative investigations of heme-containing enzymes inhibitors NaN3 and NaCN effects on the rat aorta isolated segments tone has shown that NaN3 in the range of very low concentrations from 10(-9) to 10(-6) M displays pharmacological activity characteristic of nitric oxide (NO) donors, which is inhibited by NaCN. The value of vasodilatation, caused by NaN3, was also decreased in the presence of soluble guanylate cyclase inhibitor ODQ (10(-5) M). It was found that H2O2 injection to physiological solution containing NaN3 and horseradish peroxidase or catalase lead to NO2- accumulation in it, which was blocked by NaCN. The nonenzymic NaN3 oxidization by hydrogen peroxide was not found in control experiments. NaN3 physiological activity dependent on NO-donating properties of this traditional inhibitor of heme-containing enzymes is discussed.     

The Influence of Selenium on Root Growth and Oxidative Stress Induced by Lead in Vicia faba L. minor Plants

The effect of selenium (Se) on Vicia faba L. minor roots subjected to lead (Pb) stress was studied by investigating root growth, root viability, and antioxidant enzyme activity. The experiments were carried out on plants grown for 2 weeks on Hoagland medium supplied with 50 μM Pb in the form of lead nitrate Pb(NO(3))(2) and/or Se concentrations of 1.5 and 6 μM in the form of sodium selenite Na(2)SeO(3). It was shown that Pb reduced the root growth and caused serious damage in the roots, which was accompanied by metal accumulation in these tissues. The exposition of roots to Pb led to significant changes in the biochemical parameters: the MDA and T-SH content and glutathione peroxidase (GSH-Px) activity increased but the guaiacol peroxidase (GPOX) activity decreased. Moreover, Pb intensified O(2)(·-) production in the roots. Selenium at a lower concentration alleviated Pb toxicity which was accompanied by a decreased O(2)(·-) production in the apical parts of roots and increased the T-SH content and GPOX activity. However, higher Se concentration intensified MDA and T-SH accumulation and GPOX and GSH-Px activity in Pb-treated plant roots. At low concentration, Se improved cell viability whereas at high concentration it was pro-oxidant and enhanced the lipid peroxidation and cell membrane injury.     

Low micromolar intravascular cell-free hemoglobin concentration affects vascular NO bioavailability in sickle cell disease: a computational analysis

In sickle cell disease, the changes in RBC morphology destabilize the red blood cell (RBC) membrane and lead to hemolysis. Several experimental and clinical studies have associated intravascular hemolysis with pulmonary hypertension in sickle cell disease. Cell-free hemoglobin (Hb) from intravascular hemolysis has high affinity for nitrixc oxide (NO) and can affect the NO bioavailability in the sickle cell disease, which may eventually lead to pulmonary hypertension. To study the effects of intravascular hemolysis related cell-free Hb concentrations on NO bioavailability, we developed a two-dimensional mathematical model of NO biotransport in 50-μm arteriole under steady-state sickle cell disease conditions. We analyzed the effects of flow-dependent NO production and axial and radial transport of NO, a recently reported much lower NO-RBC reaction rate constant, and cell-free layer thickness on NO biotransport. Our results show that the presence of cell-free Hb concentrations as low as 0.5 μM results in an approximately three- to sevenfold reduction in the predicted smooth muscle cell NO concentrations compared with those under physiological conditions. In addition, increasing the diffusional resistance for NO in vascular lumen from cell-free layer or reducing NO-RBC reaction rate did not improve the NO bioavailability at the smooth muscle cell layer significantly for cell-free Hb concentrations ≥1 μM. These results suggest that lower NO bioavailability due to low micromolar cell-free Hb can disturb NO homeostasis and cause insufficient bioavailability at the smooth muscle cell layer. Our results supports the hypothesis that hemolysis-associated reduction in NO bioavailability may play a role in the development of pathophysiological complications like pulmonary hypertension in sickle cell disease that are observed in several clinical and experimental studies.