Rate Of NO Release Research Articles

The Antiplatelet Action of S-Nitroso Human Serum Albumin in Whole Blood.

Nitric oxide donors (NO-donors) have been shown to have therapeutic potential (e.g., ischemia/reperfusion injury). However, due to their release rate/antiplatelet properties, they may cause bleeding in patients. We therefore studied the antiplatelet effects of the two different NO-donors, i.e., S-NO-Human Serum Albumin (S-NO-HSA) and Diethylammonium (Z)-1-(N,N-diethylamino)diazen-1-ium-1,2-diolate (DEA-NONOate) in whole blood (WB) samples. WB samples were spiked with S-NO-HSA or DEA-NONOate (100 µmol/L or 200 µmol/L), and the NO release rate (nitrite/nitrate levels via HPLC) and antiplatelet efficacy (impedance aggregometry, platelet function analyzer, Cone-and-platelet analyzer, thrombelastometry) were assessed. S-NO-HSA had a significantly lower NO release compared to equimolar concentrations of DEA-NONOate. Virtually no antiplatelet action of S-NO-HSA was observed in WB samples, whereas DEA-NONOate significantly attenuated platelet function in WB. Impedance aggregometry measurements revealed that Amplitudes (slope: −0.04022 ± 0.01045 ohm/µmol/L, p = 0.008) and Lag times (slope: 0.6389 ± 0.2075 s/µmol/L, p = 0.0051) were dose-dependently decreased and prolonged by DEA-NONOate. Closure times (Cone-and-platelet analyzer) were dose-dependently prolonged (slope: 0.3738 ± 0.1403 s/µmol/L, p = 0.0174 with collagen/ADP coating; slope: −0.5340 ± 0.1473 s/µmol/L, p = 0.0019 with collagen/epinephrine coating) by DEA-NONOate. These results in WB further support the pharmacological potential of S-NO-HSA as an NO-donor due to its ability to presumably prevent bleeding events even at high concentrations up to 200 µmol/L.

Features of the decomposition of cationic nitrosyl iron complexes with N-ethylthiourea and penicillamine ligands in the presence of albumin

In this work, transformations of two cationic dinitrosyl iron complexes (DNICs), namely, mononuclear with N-ethylthiourea ligands [Fe(SC(NH2)(NHC2H5))2(NO)2]+Cl−∙[Fe(SC(NH2)(NHC2H5))Cl(NO)2]0 (complex 1) and binuclear with penicillamine ligands [Fe2(S(C(CH3)2CH(NH3)COOH))2(NO)4]SO4·5H2O (complex 2) have been investigated in an aqueous solution in the presence of bovine serum albumin (BSA) under anaerobic and aerobic conditions. The spectroscopic measurements have shown that the complexes bind to the protein, which leads to their stabilization. The resulting products are much more stable in the absence of oxygen. The fluorescence measurements indicated that complexes 1 and 2 interact with BSA with equilibrium constants K = 4.9∙105 M−1 and 5.3∙105 M−1, respectively. Complex 1 can bind to the surface due to weak intermolecular interactions, resulting in prolonged generation of NO. Also, the decay product of complex 1, both under anaerobic conditions and in the presence of oxygen, is coordinated with Cys34 and His39 in the hydrophobic pocket of albumin, as evidenced by the appearance of a shoulder at 370–410 nm. As a result of these interactions, a high molecular weight albumin-bound complex is formed, which then slowly decomposes in more than 40 h. Complex 2 and its products are adsorbed on the surface of the protein. However, in the presence of oxygen, protein does not significantly affect the NO release rate.

Combustion Characteristics, Kinetics, SO2 and NO Release of Low-Grade Biomass Materials and Briquettes

The influence of the briquetting process on SO2 and NO release characteristics, combustion properties and kinetic characteristics during biomass combustion was investigated. Two biomass (Wheat straw and Tree bulk) and two obtained briquettes were analysed. The briquetting process helps to prevent the release of SO2 and NO. The experimental results show that once the biomass is made into a briquette, when the reaction temperature is 900 ∘C, the sulphur release ratio for TB was reduced from 34.7% to 4.3% and for WS was reduced from 12.4% to 1.6%. When the reaction temperature increases to 1000 ∘C, the sulphur release ratio for TB was reduced from 73.4% to 30.4%, for WS it was reduced from 58.4% to 10.2%. SEM micrographs show that the compact structure of the TB-Briquette and WS-Briquette reduce the rate of SO2 and NO release during combustion. The thermogravimetry confirmed that the combustion performance of WS-Briquette is the best, while the TB-Briquette is the worst. According to the Coats-Redfern method, the fitting was performed at segments of 250 ∘C to 550 ∘C, and the correlation coefficient of the fitting degree was above 0.99. The effective collision rate of WS-Briquette is much higher than that of other briquettes. Compared to BR-1 and BR-2, trying to mix TB with WS to make a compound biomass briquette can enhance the combustion performance of TB-Briquette. The results may guide the upgrading of biomass briquettes technology and benefit the efficient application of biomass briquettes.

Effects of Glutathione and Histidine on NO Release from a Dimeric Dinitrosyl Iron Complex (DNIC).

Rates of NO release from synthetic dinitrosyl iron complexes (DNICs) are shown to be responsive to coordination environments about iron. The effect of biologically relevant cellular components, glutathione and histidine, on the rate of NO release from a dimeric, "Roussin's Red Ester", DNIC with bridging μ-S thioglucose ligands, SGlucRRE or [(μ-SGluc)Fe(NO)2]2 (SGluc = 1-thio-β-d-glucose tetraacetate), was investigated. From the Griess assay and X-band EPR data, decomposition of the product from the histidine-cleaved dimer, [(SGluc)(NHis)Fe(NO)2], generated Fe(III) and increased the NO release rate in aqueous media when compared to the intact SGlucRRE precursor. In contrast, increasing concentrations of exogenous glutathione generated the stable [(SGluc)(GS)Fe(NO)2]- anion and depressed the rate of NO release. Both of the cleaved, monomeric intermediates were characterized with ESI-MS, EPR, and FT-IR spectroscopies. On the basis of the Griess assay coupled with data from an intracellular fluorometric probe, both the monomeric DNICs and dimeric SGlucRRE diffuse into smooth muscle cells, chosen as appropriate archetypes of vascular relaxation, and release their NO payload. Ultimately, this work provides insight into tuning NO release beyond the design of DNICs, through the incubation with safe, accessible biological molecules.

Kinetic regularities of NO donation by binuclear dinitrosyl iron complexes with thiolate ligands based on thiophenol derivatives in the presence of red blood cells

The kinetics of nitrogen oxide release by binuclear dinitrosyl iron complexes (B-DNICs) with thiolate ligands based on thiophenol and its several oxy, amino, and nitro derivatives was studied using a suspension of red blood cells simulating the internal medium of a blood vessel. The NO donating ability of the complexes was estimated by the kinetic parameters of first-order equation, which described the formation of intra-erythrocytic methemoglobin. Three typical kinetic profiles of NO donation were distinguished: pseudosaturation donation and donation with prolonged and explosive profiles. In the case of first-type NO donation typical of complexes with thiophenol and its nitro derivatives, the curves display a fast coming to saturation long before the complete release of all NO groups contained in the structure of the starting complex into a solution. Such a type of donation is likely due to the formation of long-lived nitrosyl intermediates in the system. The prolonged type of NO donation shown by the complex with hydroxyphenyl ligand is characterized by virtually constant rate of NO release into a solution without pronounced transition to saturation during experiment (10–12 min). In the case of explosive-type donation characteristic of the complex with aminophenyl ligands, a considerable portion of NO was fast released into a solution within 1–3 min. All complexes under study caused hemolysis of a 0.2% suspension of red blood cells. The complex with aminophenyl ligands exhibited the highest hemolytic activity.

Rapid and continuous accumulation of nitric oxide-releasing liposomes in tumors to augment the enhanced permeability and retention (EPR) effect.

The modulation of blood flow to tumors is a prominent strategy for improving the tumor accumulation of nanomedicines, resulting from the enhanced permeability and retention (EPR) effect. We previously reported a promising EPR enhancer-a nitric oxide (NO) donor-containing liposome (NO-LP)-which showed enhanced accumulation in tumor tissue. Herein, we study NO-LP in greater detail to clarify its practical use as an EPR enhancer. NO-LP was found to have advantages as a NO donor, including the ability to maintain NO donation over long periods of time, and a constant rate of NO-release irrespective of the environmental pH. NO-LP showed rapid accumulation in tumor tissue after injection (1 h), and then accumulation was continuously enhanced until 48 h. Enhanced NO-LP accumulation was observed specifically in tumor, while the accumulation in other organs remained relatively unchanged. The results obtained show the promising features of NO-LP as an EPR enhancer.

Models for potential dendritic nitric oxide donors: crystal structures of two 2-nitroanilino precursors and nitric oxide-release behavior of the nitrosated derivatives.

Two molecular precursors to dendrimeric materials that could serve as slow and sustained NO-releasing therapeutic agents have been synthesized and characterized. N1,N4-Bis(2-nitrophenyl)butane-1,4-diamine, C16H18N4O4, (I), crystallizes in a lattice with equal populations of two molecules of different conformations, both of which possess inversion symmetry through the central C-C bond. One molecule has exclusively anti conformations along the butyl chain, while the other has a gauche conformation of the substituents on the first C-C bond. N2,N2-Bis[2-(2-nitroanilino)ethyl]-N1-(2-nitrophenyl)ethane-1,2-diamine, C24H27N7O6, (II), crystallizes with one unique molecule in the asymmetric unit. Neighboring pairs of molecules are linked into dimers via N-H...O amine-nitro hydrogen bonds. The dimers are assembled into layers that stack in an A-B-A-B sequence such that the repeat distance in the stacking direction is over 46 Å. Molecular NO-release agents N1,N4-bis(2-nitrophenyl)-N1,N4-dinitrosobutane-1,4-diamine, C16H16N6O6, (III), and N1-(2-nitrophenyl)-N2,N2-bis{2-[(2-nitrophenyl)(nitroso)amino]ethyl}-N1-nitrosoethane-1,2-diamine, C24H24N10O9, (IV), were prepared via treatment of (I) and (II), respectively, with NaNO2 and acetic acid. The release of NO from solid-phase samples of (III) and (IV) suspended in phosphate buffer was monitored spectroscopically over a period of 21 days. Although (IV) released a greater amount of NO, as expected due to it having three NO moieties for every two in (III), the (IV):(III) ratio of the rate and extent of NO release was significantly less than 1.5:1, suggesting that some combination of electronic, chemical, and/or steric factors may be affecting the release process.

Analytical Expression for the NO Concentration Profile Following NONOate Decomposition in the Presence of Oxygen.

We have derived an analytical expression for the time dependent NO concentration from NONOate donors in the presence of oxygen for the process of NO release from NO donors following autoxidation. This analytical solution incorporates the kinetics of the releases with the autoxidation and is used to fit the simulated NO concentration profile to the experimental data. This allows one to determine the NO release rate constant, k 1, the NO release stoichiometric coefficient, v NO, and the NO autoxidation reaction rate constant, k 2. This analytical solution also allows us to predict the real NO concentration released from NO donors under aerobic conditions, while v NO is reportedly two under aerobic conditions, it falls to lower values in the presence of oxygen.

Instantaneous NO release from ellipsoidal particles during char combustion in a hot gas with fluctuating temperature

We have established the instantaneous governing equations for NO release during char combustion of nonspherical particles theoretically. The instantaneous released NO mass variations, the instantaneous char oxidation reaction rates, and the instantaneous NO reduction reaction rates of char particles were computed numerically under different fluctuation amplitudes of gas temperature, kinetic parameters of char oxidation reaction and NO reduction reaction, and particle aspect ratios. The gas temperature fluctuation results in a faster NO release rate, a faster char oxidation reaction rate, and a faster NO reduction reaction rate during the whole char combustion processes. The activation energy of char oxidation reaction has obvious influence on the NO release and char combustion processes. The kinetic parameters of NO reduction reaction have no contribution to the conversion time. Under the same particle surface area, the conversion rate of char nitrogen to NO increases with the increase in the particle aspect ratio.

Characterisation of NO production and consumption: new insights by an improved laboratory dynamic chamber technique

Abstract. Biogenic NOx emissions from natural and anthropogenically influenced soils are currently estimated to amount to 9 Tg a−1, hence a significant fraction of global NOx emissions (45 Tg a−1). During the last three decades, a large number of field measurements have been performed to quantify biogenic NO emissions. To study biogenic NO emissions as a function of soil moisture, soil temperature, and soil nutrients, several laboratory approaches have been developed to estimate local/regional NO emissions by suitable upscaling. This study presents an improved and automated laboratory dynamic chamber system (consisting of six individual soil chambers) for investigation and quantification of all quantities necessary to characterise biogenic NO release from soil (i.e. net NO release rate, NO production and consumption rate, and respective Q10 values). In contrast to former versions of the laboratory dynamic chamber system, the four experiments for complete characterisation can now be performed on a single soil sample, whereas former studies had to be performed on four sub-samples. This study discovered that the sub-sample variability biased former measurements of net NO release rates tremendously. Furthermore, it was also shown that the previously reported variation of optimum soil moisture (i.e. where a maximum net NO release rates occur) between individual sub-samples is most likely a methodical artefact of former versions of the laboratory dynamic chamber system. A comprehensive and detailed methodical concept description of the improved laboratory dynamic chamber system is provided. Response of all quantities (necessary to characterise net NO release) to soil temperature and NO mixing ratio of the flushing airstream are determined by automatic monitoring of these variables during one single drying-out experiment with one single soil sample only. The method requires precise measurements of NO mixing ratio at the inlet and outlet of each soil chamber; finally, four pairs of inlet/outlet NO mixing ratios are sufficient to derive all necessary quantities. Soil samples from drylands exhibit particularly low NO production, but even lower NO consumption rates. However, with the improved laboratory dynamic chamber system those low levels can be quantified, as well as corresponding NO compensation point mixing ratios and respective Q10 values. It could be shown that the NO compensation point mixing ratio seems to be generally independent of gravimetric soil moisture content, but, particularly for dryland soils, strongly dependent on soil temperature. New facilities have been included into the improved system (e.g. for investigation of net release rates of other trace gases, namely CO2 and volatile organic compounds – VOCs). First, results are shown for net release rates of acetone (C3H6O), acetaldehyde (C2H4O) and CO2. This new system is thus able to simultaneously investigate potential mechanistic links between NO, multitudinous VOC and CO2.

Trans‐Dinitrosyl‐Substituted Hexamolybdate and Study of Its Controllable NO Release

AbstractOn the basis of the well‐developed N,N′‐dicyclohexylcarbodiimide (DCC) imidoylization strategy, the novel trans‐dinitrosyl‐substituted polyoxometalate (POM) (NBu4)4[Mo6O17(≡NO)2] was synthesized in moderate yield with high purity through a highly selective reaction route under mild reaction conditions. Its structure was elucidated by IR and UV/Vis spectroscopy, ESI‐MS, and single‐crystal X‐ray diffraction studies. Furthermore, its ability to undergo controlled NO release was confirmed by ESI‐MS, and a curve of the amount of NO released as a function of time within a simulated body fluid system was obtained by UV spectroscopy. The rate of NO release is controlled by changing the pH of the system. This is the first time that the decomposition of a POM has been used to produce therapeutic NO species. This study provides a new route to controlled‐release technology for NO storage and targeted delivery.

Complexes of ferriheme nitrophorin 4 with low-molecular weight thiol(ate)s occurring in blood plasma

Nitrophorins are proteins occurring in the saliva of the blood-sucking insect Rhodnius prolixus to carry NO as a vasodilator and blood-coagulation inhibitor into the victim's tissue. It was suggested that the rate of NO release can be enhanced by the blood-plasma component l-cysteine [J.M.C.Ribeiro, Insect Biochem. Mol. Biol. 26 (1996) 899–905]. However, the mechanism of the reaction is not clear. In the attempt to exploit the reaction in detail, complexes of nitrophorin 4 (NP4) with the thiols 2-mercaptoethanol, l-cysteine, and l-homocysteine and with HS− were formed and characterized under anaerobic conditions using absorption spectroscopy, X-ray crystallography, and EPR spectroscopy. In contrast to met-myoglobin, which is reduced by l-cysteine, all four compounds form low-spin FeIII complexes with NP4. The weak equilibration constants (167–5200M−1) neither support significant complexation nor the simple displacement of NO in vivo. Both amino acid based thiols form additional H-bonds with side chains of the heme pocket entry. Glutathione and l-methionine did not form a complex, indicating the specificity of the complexes with l-cysteine and l-homocysteine. Continuous wave EPR spectroscopy reveals the simultaneous existence of three low-spin systems in each case that are attributed to various protonation and/or conformational stages in the heme pocket. Electron nuclear double resonance (ENDOR) spectroscopy demonstrates that the thiol sulfurs are, at least in part, protonated. Overall, the results not only demonstrate the good accessibility of the NP4 heme center by biologically relevant thiols, but also represent the first structural characterization of a ferriheme protein in complex with l-cysteine l-homocysteine.

Novel heterocyclic NO-donors as potential anti-cancer agents

A comparative study involving two sets of novel nitric oxide donating compounds was investigated. All target compounds were designed and prepared using traditional synthetic routes that were optimised to allow the efficient incorporation of either a furoxan core or a S-nitrosothiol group. In each synthetic sequence the NO was locked into a five-membered heterocyclic ring linked to an aromatic portion. This synthetic strategy provided the scope to generate a range of products with subtle substituent differences in order to help rationalise any variations seen, between individual compounds, in either the rate of NO release or biological profile. The chemical stability of all compounds was compared under a range of different conditions to ascertain the ease with which each heterocyclic ring breaks open, which is clearly a prerequisite for NO release. Having gained an appreciation of the stability and required handling conditions for each set of NO-donors, the compounds were evaluated for their ability to inhibit the proliferation of a panel of ovarian cancer cell lines. The results suggest that subtle chemical modifications to the structure of these NO-donors, in additional to changing the actual type of NO-donating species, can greatly influence their ability to release NO and act as cytotoxic agents.

Gating NO Release from Nitric Oxide Synthase

We have investigated the kinetics of NO escape from Geobacillus stearothermophilus nitric oxide synthase (gsNOS). Previous work indicated that NO release was gated at position 223 in mammalian enzymes; our kinetics experiments include mutants at that position along with measurements on the wild type enzyme. Employing stopped-flow UV-vis methods, reactions were triggered by mixing a reduced enzyme/N-hydroxy-l-arginine complex with an aerated buffer solution. NO release kinetics were obtained for wt NOS and three mutants (H134S, I223V, H134S/I223V). We have confirmed that wt gsNOS has the lowest NO release rate of known NOS enzymes, whether bacterial or mammalian. We also have found that steric clashes at positions 223 and 134 hinder NO escape, as judged by enhanced rates in the single mutants. The empirical rate of NO release from the gsNOS double mutant (H134/I223V) is nearly as rapid as that of the fastest mammalian enzymes, demonstrating that both positions 223 and 134 function as gates for escape of the product diatomic molecule.

Localization of HydrophobicN-Diazeniumdiolates in Aqueous Micellar Solution

The interaction of phenyl-substituted zwitterionic N-diazeniumdiolates PhCH(2)N[N(O)NO](-)(CH(2))(2)NH(3)(+) (1) and PhCH(2)N[N(O)NO](-)(CH(2))(2)NH(2)(+)CH(2)Ph (2) with aqueous micellar solutions of prototypal surfactants was investigated by means of UV/vis and (1)H NMR spectroscopy in order to establish the localization of hydrophobic N-diazeniumdiolates in micelles as a model for the binding of the NO donors in biological membranes. In the presence of sodium dodecyl sulfate (SDS), significant shifts of the apparent pK(a) values of 1 and 2 were observed, suggesting strong electrostatic interaction between the diazeniumdiolates and the negatively charged SDS micelles. No effect on both pK(a) and rate of NO release was found in the presence of Triton X-100. The solubilization site of micellar bound N-diazeniumdiolates was established by (1)H NMR spectroscopic studies, taking advantage of the spectroscopic effects induced by CH-pi interactions. The spectra indicate that in alkaline solutions of SDS 1 resides preferably at the micellar surface within the interfacial region, whereas the more hydrophobic NO donor 2 penetrates into the apolar region of the micelle. This suggests hydrophobic interaction as the main driving force for micellar binding of 2 in alkaline solution. Similar studies in presence of Triton X-100 indicate that 1 and 2 are adsorbed within the poly(oxyethylene) layer of the micellar surface rather than penetrating the palisade layer of the micelles. In alkaline solutions of hexadecyltrimethylammonium bromide (CTAB), 1 and 2 bind to the cationic micellar aggregates, whereby the solubilization site strongly depends on the hydrophobicity of the substrate. Up to a moderate pH of 8, the hydrophobic NO donor 2 penetrates the hydrocarbon region of the micelles. As a result, the rate of NO release from 2 is noticeably inhibited by the micellar aggregates due to the higher local concentration of hydroxide ions along the micelle-water interface. From solubilization studies, guidelines for the development and application of future NONOates can be derived. The rate of NO release from micellar bound diazeniumdiolates is determined by the surface charge of the micelles. This ability to tune stability is significant for the design and selection of potential NO delivery systems (drug formulations).

Modulation of NO bioavailability by temporal variability of cell‐free layer width in the arterioles

The formation of a cell (erythrocyte)‐free layer near the vessel wall during blood flow is capable of attenuating NO consumption by the red blood cells and thus improves the preservation of NO in the smooth muscle for eliciting vasodilatory responses. This study utilized a transient NO transport mathematical model to predict how temporal variability exhibited by the layer width and its relevant transient physiological responses could modulate NO preservation by this diffusion barrier at high pseudoshear rates (291.5 ± 13.5 s−1). Data on cell‐free layer width was experimentally acquired from arterioles (48.4 ± 2.3 μm) of the rat cremaster muscle. We found that a time‐varying diffusion barrier could lead to a slight decrease (2% – 6%) in NO bioavailability and this effect was independent of transient changes in NO scavenging rate in the blood lumen. Conversely, transient changes in wall shear stress and accompanying NO release rate played a dominant role in reversing this decline such that an augmentation in NO bioavailability of between 3% – 15% was found. However this increase was not sufficient to induce any significant change in vascular tone. This study highlighted the importance of accounting for transient wall shear stress effect on NO release rate as mediated by the layer width temporal variability when assessing the physiological implication of the layer on NO bioavailability in arterioles.

PH-Dependent Mechanism of Nitric Oxide Release in Nitrophorins 2 and 4

Nitrophorins are NO carrier proteins that transport and release NO through a pH-dependent conformational change. They bind NO tightly in a low pH environment and release it in a higher pH environment. Experimental evidence shows that the increase in the NO dissociation equilibrium constant, K(d), is due mainly to an increase in the NO release rate. Structural and kinetic data strongly suggest that NPs control NO escape by modulating its migration from the active site to the solvent through a pH-dependent conformational change. NP2 and NP4 are two representative proteins of the family displaying a 39% overall sequence identity, and interestingly, NP2 releases NO slower than NP4. The proposal that NPs' NO release relies mainly on the NO escape rate makes NPs a very peculiar case among typical heme proteins. The connection between the pH-dependent conformational change and ligand release mechanism is not fully understood and the structural basis for the pH induced structural transition and the different NO release patterns in NPs are unresolved, yet interesting issues. In this work, we have used state of the art molecular dynamics simulations to study the NO escape process in NP2 and NP4 in both the low and high pH states. Our results show that both NPs modulate NO release by switching between a "closed" conformation in a low pH environment and an "open" conformation at higher pH. In both proteins, the change is caused by the differential protonation of a common residue Asp30 in NP4 and Asp29 in NP2, and the NO escape route is conserved. Finally, our results show that, in NP2, the conformational change to the "open" conformation is smaller than that for NP4 which results in a higher barrier for NO release.

Hydrophobic N‐Diazeniumdiolates and the Aqueous Interface of Sodium Dodecyl Sulfate (SDS) Micelles

Zwitterionic diazeniumdiolates of the form RN[N(O)NO(-)](CH(2))(2)NH(2) (+)R, where R=CH(3) (1), (CH(2))(3)CH(3) (2), (CH(2))(5)CH(3) (3), and (CH(2))(7)CH(3) (4) were synthesized by reaction of the corresponding diamines with nitric oxide. Spectrophotometrically determined pK(a)(O) values, attributed to protonation at the terminal oxygen of the diazeniumdiolate group, show shifts to higher values in dependence of the chain lengths of R. The pH dependence of the decomposition of NO donors 1-3 was studied in buffered solution between pH 5 and 8 at 22 degrees C, from which pK(a)(N) values for protonation at the amino nitrogen, leading to release of NO, were estimated. It is shown that the decomposition of these diazeniumdiolates is markedly catalyzed by anionic SDS micelles. First-order rate constants for the decay of 1-4 were determined in phosphate buffer pH 7.4 at 22 degrees C as a function of SDS concentration. Micellar binding constants, K(SM), for the association of diazeniumdiolates 1-3 with the SDS micelles were also determined, again showing a significant increase with increasing length of the alkyl side chains. The decomposition of 1-3 in micellar solution is quantitatively described by using the pseudo-phase ion-exchange (PIE) model, in which the degree of micellar catalysis is taken into account through the ratio of the second-order rate constants (k(2m)/k(2w)) for decay in the micelles and in the bulk aqueous phase. The decay kinetics of 1-3 were further studied in the presence of cosolvents and nonionic surfactants, but no effect on the rate of NO release was observed. The kinetic data are discussed in terms of association to the micelle-aqueous phase interface of the negatively charged micelles. The apparent interfacial pH value of SDS micelles was evaluated from comparison of the pH dependence of the first-order decay rate constants of 2 and 3 in neat buffer and the rate data obtained for the surfactant-mediated decay. For a bulk phase of pH 7.4, an interfacial pH of 5.7-5.8 was determined, consistent with the distribution of H(+) in the vicinity of the negatively charged micelles. The data demonstrate the utility of 2 and 3 as probes for the determination of the apparent pH value in the Stern region of anionic micelles.

Electrochemical analysis of the kinetics of nitric oxide release from two diazeniumdiolates in buffered aqueous solutions

We have investigated in this work the kinetics of NO-release from two commercially available and commonly used diazeniumdiolates (so-called NONOates), namely diethylammonium ( Z)-1-( N, N-diethylamino)-diazen-1-ium-1,2-diolate (DEA-NONOate) and ( Z)-1-[ N-(2-aminoepropyl)- N-(2-ammoniopropyl)amino]-diazen-1-ium-1,2-diolate (PAPA-NONOate) by electrochemical measurements. This choice was driven by the distinctive half-life of each compound (2–20 min at 37 °C for DEA- and PAPA-NONOate, respectively) that allow covering a relatively wide range of rate of NO-release. The release of NO from NONOate decomposition was followed using a home-made NO-sensitive electrochemical sensor. The electrochemical features of the starting NONOates and of the by-products of the fully decomposed molecules were also examined. Also, the electrochemical experimental results obtained under aerobic atmosphere were numerically fitted to model the production of NO and its degradation through its oxidation by molecular oxygen. All these information gathered together allows in predicting the real amount of NO released under real aerobic conditions.

Mitochondrial nitric oxide synthase, a voltage-dependent enzyme, is responsible for nitric oxide diffusion to cytosol

The mitochondrial metabolic state regulates the rate of mitochondrial NO production and release to the cytosol. Nitric oxide release of rat heart mitochondria decreased markedly from 2.2 to 1.2 nmol NO/min. mg protein in the state 4 to state 3 transition. The activity of mtNOS, responsible for NO release, is driven by the membrane potential and not by intramitochondrial pH changes. The release of NO by rat liver mitochondria showed an exponential dependence on membrane potential. A similar behavior was reported for heart mitochondrial H2O2 production. The fraction of heart cytosolic NO provided by diffusion from mitochondria is 90%. The intramitochondrial concentrations of L-arginine and NADPH are higher than their KM values, and the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. These data indicate that the redox state of the respiratory chain components regulates H2O2 production and mitochondrial membrane potential modulates NO release, and support the speculation that NO and H2O2 are a biological signal that reports a high mitochondrial energy charge to the cytosol. The marked regulation of mtNOS activity, as a voltage-dependent enzyme and at the physiological range of membrane potentials, makes mtNOS a highly sensitive enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.