- Reduction in thrombosis and bacterial adhesion with 7 day implantation of S-nitroso-N-acetylpenicillamine (SNAP)-doped Elast-eon E2As catheters in sheep
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Thrombosis and infection are two common problems associated with blood-contacting medical devices such as catheters. Nitric oxide (NO) is known to be a potent antimicrobial agent as well as an inhibitor of platelet activation and adhesion. Healthy endothelial cells that line the inner walls of all blood vessels exhibit a NO flux of 0.5-4 × 10-10 mol cm-2 min-1 that helps prevent thrombosis. Materials with a NO flux that is equivalent to this level are expected to exhibit similar anti-thrombotic properties. In this study, NO-releasing catheters were fabricated by incorporating S-nitroso-N-acetylpenicillamine (SNAP) in the Elast-eon E2As polymer. The SNAP/E2As catheters release physiological levels of NO for up to 20 days, as measured by chemiluminescence. Furthermore, SNAP is stable in the E2As polymer, retaining 89% of the initial SNAP after ethylene oxide (EO) sterilization. The SNAP/E2As and E2As control catheters were implanted in sheep veins for 7 days to examine the effect on thrombosis and bacterial adhesion. The SNAP/E2As catheters reduced the thrombus area when compared to the control (1.56 ± 0.76 and 5.06 ± 1.44 cm2, respectively). A 90% reduction in bacterial adhesion was also observed for the SNAP/E2As catheters as compared to the controls. The results suggest that the SNAP/E2As polymer has the potential to improve the hemocompatibility and bactericidal activity of intravascular catheters, as well as other blood-contacting medical devices (e.g., vascular grafts, extracorporeal circuits). This journal is
- Brisbois, Elizabeth J.,Davis, Ryan P.,Jones, Anna M.,Major, Terry C.,Bartlett, Robert H.,Meyerhoff, Mark E.,Handa, Hitesh
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- Formation of the distinct redox-interrelated forms of nitric oxide from reaction of dinitrosyl iron complexes (DNICs) and substitution ligands
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Release of the distinct NO redox-interrelated forms (NO+, NO, and HNO/NO-), derived from reaction of the dinitrosyl iron complex (DNIC) [(NO)2Fe(C12HaN)2]- (1) (C12H8N = carbazolate) and the substitution Iigands (S2CNMe2)2, [SC6H 4-O-NHC(O)(C5H4N)]2, ((PyPepS) 2), and P(C6H3,-3SiMe3-2-SH) 3 ([P(SH)3]), respectively, was demonstrated. In contrast to the reaction of (PyPepS)2 and DNIC 1 in a 1:1 stoichiometry that induces the release of an NO radical and the formation of complex [PPN][Fe(PyPepS)2] (4), the incoming substitution ligand (S 2CNMe2)2 triggered the transformation of DNIC 1 into complex [(NO)Fe(S2CNMe2J2] (2) along with N-nitrosocarbazole (3). The subsequent nitrosation of N-acetylpenicillamine (NAP) by N-nitrosocarbazole (3) to produce S-nitroso-A-acetylpenicillamine (SNAP) may signify the possible formation pathway of S-nitrosothiols from DNICs by means of transnitrosation of N-nitrosamines. Protonation of DNIC 1 by [P(SH)3] triggers the release of HNO and the generation of complex [PPN][Fe(NO)P(C6H3-3-SiMe3-2-S)3] (5). In a similar fashion, the nucleophilic attack of the chelating ligand P(C6H3,-3-SiMe3,-2-SNa)3 ([P(SNa)3]) on DNIC 1 resulted in the direct release of [NO] - captured by [(15NO)Fe(SPh)3]-, thus leading to [(15NO)- (14NO)Fe(SPh) 2]-. These results illustrate one aspect of how the incoming substitution ligands ((S2CNMe2)2 vs. (PyPepS)2 vs. [P(SH)3]/[P(SNa)3]) in cooperation with the carbazolate-coordinated ligands of DNIC 1 function to control the release of NO+, NO, or [NO]- from DNIC 1 upon reaction of complex 1 and the substitution ligands. Also, these results signify that DNICs may act as an intermediary of NO in the redox signaling processes by providing the distinct redox-interrelated forms of NO to interact with different NO-responsive targets in biological systems.
- Lu, Tsai-Te,Chen, Chih-Hao,Liaw, Wen-Feng
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- Kinetics of S-nitrosation of thiols in nitric oxide solutions
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The S-nitroso adducts of nitric oxide (NO) may serve as carriers of NO and play a role in cell signaling and/or cytotoxicity. A quantitative understanding of the kinetics of S-nitrosothiol formation in solutions containing NO and O2 is important for understanding these roles of S- nitroso compounds in vivo. Rates of S-nitrosation in aqueous solutions were investigated for three thiols: glutathione, N-acetylcysteine, and N- acetylpenicillamine. Nitrous anhydride (N2O3), an intermediate in the formation of nitrite from NO and O2, is the most likely NO donor for N- nitrosation of amines as well as for S-nitrosation of thiols, at physiological pH. This motivated the use of a competitive kinetics approach, in which the rates of thiol nitrosation were compared with that of a secondary amine, morpholine. The kinetic studies were carried out with known amounts of NO and O2 in solutions containing one thiol (400 μM) and morpholine (200-5700 μM) in 0.01 M phosphate buffer at pH 7.4 and 23 °C. It was found that disulfide formation, transnitrosation reactions, and decomposition of the S-nitrosothiol products were all negligible under these conditions. The rate of formation of S-nitrosothiols was expressed as k7[N2O3][RSH], where RSH represents the thiol. The rate constant for S- nitrosation relative to that for N2O3 hydrolysis (k4) was found to be k7/k4 = (4.15 ± 0.28) x 104, (2.11 ± 0.11) x 104, and (0.48 ± 0.04) x 104 M-1 for glutathione, N-acetylcysteine, and N-acetylpenicillamine, respectively. The overall (observed) rates of nitrosothiol formation reflect the fact that [N2O3] ∞ [NO]2[O2] and that [N2O3] also depends on [RSH] and the concentration of phosphate. Using a detailed kinetic model to account for these effects, the present results could be reconciled with apparently dissimilar findings reported previously by others.
- Keshive, Manish,Singh, Sukhjeet,Wishnok, John S.,Tannenbaum, Steven R.,Deen, William M.
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- Direct measurement of actual levels of nitric oxide (NO) in cell culture conditions using soluble NO donors
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Applying soluble nitric oxide (NO) donors is the most widely used method to expose cells of interest to exogenous NO. Because of the complex equilibria that exist between components in culture media, the donor compound and NO itself, it is very challenging to predict the dose and duration of NO cells actually experience. To determine the actual level of NO experienced by cells exposed to soluble NO donors, we developed the CellNO Trap, a device that allows continuous, real-time monitoring of the level of NO adherent cells produce and/or experience in culture without the need to alter cell culturing procedures. Herein, we directly measured the level of NO that cells grown in the CellNO Trap experienced when soluble NO donors were added to solutions in culture wells and we characterized environmental conditions that effected the level of NO in in vitro culture conditions. Specifically, the dose and duration of NO generated by the soluble donors S-nitroso-N-acetylpenicillamine (SNAP), S-nitrosoglutathione (GSNO), S-nitrosocysteine (CysNO) and the diazeniumdiolate diethyltriamine (DETA/NO) were investigated in both phosphate buffered saline (PBS) and cell culture media. Other factors that were studied that potentially affect the ultimate NO level achieved with these donors included pH, presence of transition metals (ion species), redox level, presence of free thiol and relative volume of media. Then murine smooth muscle cell (MOVAS) with different NO donors but with the same effective concentration of available NO were examined and it was demonstrated that the cell proliferation ratio observed does not correlate with the half-lives of NO donors characterized in PBS, but does correlate well with the real-time NO profiles measured under the actual culture conditions. This data demonstrates the dynamic characteristic of the NO and NO donor in different biological systems and clearly illustrates the importance of tracking individual NO profiles under the actual biological conditions.
- He, Weilue,Frost, Megan C.
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- Direct detection of S-nitrosothiols using planar amperometric nitric oxide sensor modified with polymeric films containing catalytic copper species
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The direct amperometric detection of S-nitrosothiol species (RSNOs) is realized by modifying a previously reported amperometric nitric oxide gas sensor with thin hydrophilic polyurethane films containing catalytic Cu(II)/(I) sites. Catalytic Cu(II)/(I)-mediated decomposition of S-nitrosothiols generates NO(g) in the thin polymeric film at the distal tip of the NO sensor. Three different species are examined to create the catalytic layer: (1) a lipophilic Cu(II)-ligand complex; (2) Cu(II)-phosphate salt; and (3) small (3-μm) metallic Cu0 particles. All three catalytic layers yield reversible amperometric response in proportion to the concentration of S-nitrosothiols (e.g., nitrosocysteine, nitrosoglutathione, S-nitroso-N- acetylcysteine, S-nitrosoalbumin) present in the aqueous test solution. Sensitivity toward the different RSNO species is dependent on the respective catalytic rates of decomposition of the RSNO species by reactive Cu(I), accessibility of the species into the polyurethane layer containing the catalyst, the level of reducing agents (ascorbate) used in solution to help generate reactive Cu(I) species, and the concentration of metal ion complexing agents present in the test solution (e.g., EDTA). Under optimized conditions, all RSNO species can be detected at ≤ μM levels, with sensor lifetimes of at least 10 days for the sensors based on Cu(II)-phosphate and Cu0 particles. It is further shown that the new RSNO sensors can be used to assess the "NO-generating" ability of fresh blood samples by effectively detecting the total level of reactive RSNO species present in such samples.
- Cha, Wansik,Lee, Youngmi,Oh, Bong Kyun,Meyerhoff, Mark E.
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- THROMBORESISTANT/BACTERICIDAL S-NITROSO-N-ACETYLPENICILLAMINE (SNAP)-DOPED NITRIC OXIDE RELEASE POLYMERS WITH ENHANCED STABILITY
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In an example of a method for making an NO-releasing polymeric composition, a discrete RSNO adduct is dissolved in a solvent to form a discrete RSNO adduct solution. A polymer material is soaked in the discrete RSNO adduct solution for a predetermined time to swell the polymer material and impregnate the polymer material with the discrete RSNO adduct.
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Paragraph 0100; 0101
(2015/09/23)
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- Rates of release of nitric oxide from HbSNO and internal electron transfer
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The discovery that hemoglobin (Hb) in erythrocytes contains a fraction of β-Cys-93 thiols as the nitrosylated derivative (HbSNO) led to the suggestion that this species is involved in transporting and releasing nitric oxide, which is the signal for local vasodilation. The release of NO from HbSNO requires an electron transfer to facilitate release and to regenerate the cysteine thiol via one-electron reduction in the absence of added thiols. An alternative mechanism, which has received much attention, transfers the nitrosyl group to an external thiol, which in turn would have to be reduced. The observed first order rate constant for the spontaneous oxidation of the ferrous heme of deoxy HbSNO is 1.0×10-4s-1 in the absence of thiols. Under the same conditions, native Hb is stable. The oxidation of HbSNO occurs with the same rate constant that can be derived for the rate reported for the formation of HbNO from HbSNO. These similarities suggest that both processes involve the same reaction: internal electron transfer and direct release of nitric oxide.
- Ship, Noam J.,Pezacki, John Paul,Kluger, Ronald
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- Equilibrium and kinetics studies of transnitrosation between S-nitrosothiols and thiols
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Using UV-vis spectrometrical measurements, equilibrium constants for NO transfer between S-nitroso-N-acetyl-penicillamine (SNAP) and different thiols as well as kinetic data for NO transfer from S-nitroso bovine serum albumin (BSANO) to thiols have been obtained. NO transfer from SNAP to other primary/secondary thiols are thermodynamically favorable, whereas other S-nitrosothiols exhibit similar NO transfer potential. The obtained Gibbs free energy, enthalpy and entropy data indicated that NO transfer reactions from SNAP to four thiols are exothermic with entropy loss. The kinetic behavior of BSANO/RSH transfer can be related to both the acidity of sulfhydryl group and the electronic structure in thiol.
- Wang, Kun,Wen, Zhong,Zhang, Wei,Xian, Ming,Cheng, Jin-Pei,Wang, Peng George
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p. 433 - 436
(2007/10/03)
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- Interrelationships between conformational dynamics and the redox chemistry of S-nitrosothiols
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An increasing number of biological roles are ascribed to S-nitrosothiol compounds. Their inherent instability in multicomponent solutions is recognized as forming the basis for their physiological effects, such as the release of nitric oxide or the posttr
- Arulsamy,Bohle,Butt,Irvine,Jordan,Sagan
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p. 7115 - 7123
(2007/10/03)
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- NO-Group transfer (transnitrosation) between S-nitrosothiols and thiols. Part 2
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The kinetics of NO-group transfer have been measured for the reaction between a nitrosothiol (HOCH2CH2SNO) and nine thiols, mostly based on the cysteine structure.The reaction is second-order and there is evidence for a steric effect for thiols containing 1,1-dimethyl substituents (penicillamine derivatives).Reaction occurs via the thiolate anion as shown by the pH-rate constant profile, and a full kinetic analysis for the reactions of two thiols (N-acetylcysteine and glutathione) is quantitatively in agreement with this mechanism.Variation of the nitrosothiol structure for reaction with N-acetylcysteine shows that electron-withdrawing substituents in the nitrosothiol promote reaction; there is a similarity with the corresponding reactions of alkyl nitrites.
- Barnett, D.Jonathan,Rios, Ana,Williams, D. Lyn H.
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p. 1279 - 1282
(2007/10/03)
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- Characterization of bronchodilator effects and fate of S-nitrosothiols in the isolated perfused and ventilated guinea pig lung
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In this study the effects of S-nitrosothiols, in particular S- nitrosoglutathione (GSNO), were evaluated with regard to their bronchodilating properties, both after infusion via the pulmonary circulation and after inhalation, in the isolated perfused and ventilated guinea pig lung. Infused GSNO induced bronchorelaxation of lungs that were precontracted with methacholine. During a 15-min period of single-passage perfusion with GSNO (10 μM), maximally 10% was taken up and/or degraded by the lung. A spontaneous breakdown of GSNO in the perfusion buffer was also observed, which was partially accompanied by the formation of nitrite. Low levels of nitric oxide (NO) were detected in the perfusion buffer when GSNO was present. This was due to the presence of contaminating transition metals, because EDTA and 2,2'-dipyridyl largely reduced the formation of NO. The NO- scavenging agents oxyhemoglobin and 2-(4-carboxyphenyl)-4,4,5,5- tetramethylimidazoline-1-oxyl 3-oxide abolished levels of NO in the buffer but did not abolish GSNO-induced bronchodilation. The effects of infused GSNO are therefore attributed to an action of the intact S-nitrosothiol and not to NO released from GSNO in the perfusion buffer. Similarly, perfusion with S- nitrosated glutathione isopropyl ester, cysteinyl glycine, N-acetyl-L- cysteine or N-acetyl-D,L-penicillamine, but not with nitrosated bovine serum albumin or sodium nitrite, was found to induce bronchodilation. Inhalation of nebulized GSNO induced bronchodilation of methacholine-precontracted lungs with a rapid onset of action, although it was a less potent bronchodilator than salbutamol. The results show that infused or inhaled S-nitrosothiols have bronchodilating properties in the isolated perfused and ventilated guinea pig lung.
- Bannenberg,Xue,Engman,Cotgreave,Moldeus,Ryrfeldt
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p. 1238 - 1245
(2007/10/03)
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- Preparation of Some Novel S-Nitroso Compounds as Potential Slow-release Agents of Nitric Oxide in vivo
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The optimum conditions for the preparation of N-acetyl-S-nitrosopenicillamine (SNAP) 3 were determined and applied to the synthesis of the corresponding N-formyl compound 7.The nitrosation of penicillamine dipeptides was investigated and bis-thionitroso compounds 13, 18, 26 and 27 were isolated.S,S'-Dinitroso dithiol 13 showed biological activity akin to that of glyceryl trinitrate in decreasing systemic arterial blood pressure in anaesthetized rats and rabbits.Concomitant inhibition of collagen- or ADP-induced platelet aggregation was observed.
- Moynihan, Humphrey A.,Roberts, Stanley M.
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p. 797 - 806
(2007/10/02)
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- New Thionitrites: Synthesis, Stability, and Nitric Oxide Generation
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In order to study the influence of substitutions at the α and β carbon atoms on the stability of the S-NO bond, water-soluble thionitrites RSNO have been synthesized by nitrosation of cysteamine and mercaptoethanol derivatives and characterized. 1H and 13C NMR spectroscopies have proven to be excellent probes for the nitrosation of thiols.In water, at physiological pH, the compounds decomposed into nitric oxide NO and the corresponding disulfides.The rate at which NO was released was very sensitive to modifications at the α and β carbon atoms.Tertiary thionitrites were more stable than primary thionitrites.The β-substituents decreased the rates of decomposition in the following order: OH > NHCOCH3 > NH3(+).S-Nitrosocysteamine derivatives were greatly stabilized at low pH.The compounds described here might be convenient and useful as vehicles for spontaneous generation of nitric oxide in biological systems, at rates that can be finely tuned and controlled over a wide range.
- Roy, Beatrice,Moulinet d'Hardemare, Amaury du,Fontecave, Marc
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p. 7019 - 7026
(2007/10/02)
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- Hydrolysis, Nitrosyl Exchange, and Synthesis of Alkyl Nitrites
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Alkyl nitrites undergo relatively slow hydrolysis in phosphate-buffered aqueous media under neutral conditions with small but significant dependence of reactivity on structure.However, rapid nitrosyl exchange with alcohols is observed, and equilibrium constants for this transformation exhibit remarkable correlation with equilibrium constants for nitrosyl exchange of alcohols with nitrosyl chloride and with nitrous acid. tert-Butyl nitrite has the greatest driving force for nitrosyl transfer among the 12 alkyl nitrites examined, and this capability is used for the synthesis of alkyl nitrites derived from steroidal alcohols and of alkyl thionitrites.
- Doyle, Michael P.,Terpstra, Jan W.,Pickering, Ruth A.,LePoire, Diane M.
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p. 3379 - 3382
(2007/10/02)
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- Kinetics and Mechanism of the Nitrosation of Alcohols, Carbohydrates, and a Thiol
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Rate constants have been determined by stopped-flow spectrometry for the nitrosation in water of the following alcohols and carbohydrates: methanol, ethanol, propan-l-ol, propan-2-ol, ethane-1,2-diol, propane-1,2,3-triol, mannitol, sucrose, and glucose, in some cases at 25o and others at 0o.In all cases the reactions were reversible, forming equilibrium concentrations of the corresponding alkyl nitrites.With a large excess of the alcohol present over the total nitrous acid concentration, the observed first order rate constants increased linearly with the alcohol concentration, and from the slope and the intercept the rate constants of the forward and reverse reactions were obtained.Both reactions were acid catalysed and also catalysed by chloride and bromide ion.The forward reaction is interpreted as one involving O-nitrosation by H2NO2+ (or NO+), NOCl or NOBr.For methanol, rate constants for attack by nitrosyl chloride and by nitrosyl bromide were 2.1 x 105 mol-1s-1, respectively.There was little difference between the rate constants for chloride ion and bromide ion attack for the reverse reaction, suggesting that the reactions for attack on the protonated alkyl nitrite are very rapid, possibly approaching the diffusion-controlled limit.There was a decrease in the overall equilibrium constant for the non-halide ion-catalysed pathway, along the series MeOH, EtOH, PriOH, and ButOH; the value for the latter was so small as to make the quantitative determination of the rate constants impossible by this method.This decrease was due almost entirely to a decreasing value for the forward rate constant along the series, which implies that a steric effect is operational.All the carbohydrates studied had very similar equilibrium constants of ca. 1.5.In contrast the nitrosation of N-acetylpenicillamine gave the thionitrite quantitatively at low acidities.Again the reaction was acid- and halide (and thiocyanate) ion-catalysed and the reactivity sequence NOCl > NOBr > NOSCN was established.For both the alcohols and the thiol, the forward rate constants are well below the diffusion controlled values and are several powers of ten less than the corresponding values for the reactions of aniline.The results are discussed in terms of the nucleophilicities of the oxygen and sulphur sites for the forward reaction and in terms of the protonation equilibria of the nitrites and thionitrite for the reverse reaction.
- Aldred, S. Elaine,Williams. D. Lyn H.,Garley, Michael
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p. 777 - 782
(2007/10/02)
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