The reaction of nitrogen monoxide with the haemocyanins of the crayfish Astacus leptodactylus and the snail Helix pomatia.

The rate of the reaction of Astacus leptodactylus methaemocyanin with NO follows the Henderson-Hasselbalch equation with a pKa of 5.85, suggesting that one imidazole ligand of Cu was exchanged for NO. The reaction is blocked by F- as a bridging ligand. The same imidazole residue might be responsible for the decomposition of nitrosylhaemocyanin, [Cu1NO+CuII], with an unlocated binding site for NO, into methaemocyanin and NO, as the rate increase with pH. NO could react preferentially with CuA of Helix pomatia methaemocyanin, CuA'IICuBII, as it possibly has only two histidine ligands instead of the three of CuA in Astacus haemocyanin. This difference might explain the higher formation rate and the much greater stability of Helix nitrosylhaemocyanin. The fast reaction is governed by a pKa of 6.80, probably of a bridging mu-aquo ligand. With F- or a mu-hydroxo bridging ligand a low reaction rate was still observed, in contrast with Astacus methaemocyanin. Helix nitrosylhaemocyanin was transformed by N3- into methaemocyanin with the liberation of N2 and N2O. This methaemocyanin could almost quantitatively be regenerated with H2O2. Helix nitrosylhaemocyanin was only partially regenerated by a direct treatment with H2O2 and almost quantitatively by HONH2 in a similar fairly fast reaction, followed by a much slower one.     

The reaction of nitrite with the haemocyanin of Astacus leptodactylus.

The reaction of nitrite at pH 5.7 with deoxyhaemocyanin of Astacus leptodactylus yielded methaemocyanin in two one-electron steps, as nitrite was reduced to NO. This methaemocyanin could be almost fully regenerated by an anaerobic treatment with HONH2, in contrast with the methaemocyanin prepared with H2O2. A destruction of active sites on treating oxyhaemocyanin with HONH2 explains the partial regeneration of methaemocyanin under air, as traces of H2O2 are formed in the autoxidation of HONH2. The reaction rate of nitrite with deoxyhaemocyanin is almost 15 times that with oxyhaemocyanin. The slope of -1.0 for the logarithm of the pseudo-first-order rate constants plotted against pH indicates that HNO2 is the reacting species. Methaemocyanin was e.p.r.-undetectable, but a binuclear signal was observed at g = 2 on binding nitrite to methaemocyanin. This signal disappeared with a pKa of 6.50, suggesting that a mu-aquo bridging ligand, which can be replaced by nitrite, is deprotonated to a mu-hydroxo bridging ligand, which resists substitution by nitrite. The intensity of this triplet e.p.r. signal allowed the determination of the association constant of nitrite to the active site of Astacus methaemocyanin and yielded a value of 237 M-1 at pH 5.7. The interpretation by some authors of nitrosylhaemocyanin as a nitrite derivative of semimethaemocyanin is contradicted by this rapid reaction of nitrite with copper(I) in deoxyhaemocyanin and in semi-methaemocyanin and by the low binding constant of nitrite to the active site of methaemocyanin.     

The reaction of hydroxyurea with oxyhaemocyanin and methaemocyanin of the crayfish Astacus leptodactylus and the snail Helix pomatia.

The reaction of hydroxyurea with the oxyhaemocyanins of Astacus leptodactylus and Helix pomatia yielded methaemocyanins which could be regenerated with hydroxylamine. Hydroxyurea did not react with Astacus methaemocyanin, but quantitatively regenerated Helix methaemocyanin under N2. The reaction of hydroxyurea with Helix haemocyanin at pH 5.7 under air thus led to a steady state, with an oxyhaemocyanin/methaemocyanin ratio of 2.05:1.     

The reaction of nitrogen monoxide and of nitrite with deoxyhaemocyanin and methaemocyanin of Helix pomatia.

Deoxyhaemocyanin, treated with NO under strictly anaerobic conditions, yielded methaemocyanin and N2O in a fast reaction. In a further slow reaction this methaemocyanin lost its triplet electron paramagnetic resonance (EPR) signal at g = 4 and yielded a nitrosyl derivative with a characteristic g = 2 Cu(II) EPR signal, indicating the binding of a single NO per copper pair. Thus under strictly anaerobic conditions deoxyhaemocyanin and methaemocyanin, treated with NO, gave the same derivative as shown by circular dichroism and EPR spectra. Methaemocyanin yielded, moreover, reversibly a nitrite derivative, characterized by a triplet signal at g = 4 with 7 hyperfine lines.     

Electron paramagnetic resonance studies on Pseudomonas nitrosyl nitrite reductase. Evidence for multiple species in the electron paramagnetic resonance spectra of nitrosyl haemoproteins.

The e.p.r. spectra of reduced 14NO- and 15NO-bound Pseudomonas nitrite reductase have been investigated at pH 5.8 and 8.0 in four buffer systems. At pH 8.0, absorption spectra indicated that only the haem d1 was NO-bound, but, although quantification of the e.p.r. signals in all cases accounted for NO bound the the haem d1 in both subunits of the enzyme, the precise form of the signals varied with buffer and temperature. A rhombic species, with gx = 2.07, gz = 2.01 and gy = 1.96, represented in the low-temperature spectra seen in all the buffers was converted at high temperatures (approx. 200K) into a form showing a reduced anisotropy. Hyperfine splitting on the gz component of this rhombic signal indicated a nitrogen atom trans to NO and it is proposed that histidine provides the endogenous axial ligand for haem d1. At pH 5.8, absorption spectra indicated NO binding to both haems c and d1 and e.p.r. quantifications accounted for NO-bound haems c and d1 in both enzyme subunits. The e.p.r. spectra at pH 5.8 were generally similar to those at pH 8.0 with respect to g-values and hyperfine coupling constants, but were broader with less well defined hyperfine splittings. As at pH 8, rhombic signals present in spectra at low temperatures were converted to less anisotropic forms at high temperatures. The results are discussed in relation to work on model nitrosyl-protohaem complexes [Yoshimura, Ozaki, Shintani & Watanabe (1979) Arch. Biochem, Biophys. 193, 301-313]. No. e.p.r. signal was observed from oxidized NO-bound Pseudomonas nitrite reductase at pH 6.0, over the temperature range 6-100K.     

Differential hydrogen ion titrations of the histidine residues in Helix pomatia haemocyanin

The properties of the histidine residues in Helix pomatia haemocyanin have been studied by differential hydrogen ion titrations. In oxy-and deoxyhaemocyanin 31 × 10^?5 histidine residues per g protein are titrated in contrast to 35 × 10^?5 residues in apohaemocyanin. The difference corresponds to a stoichiometry of one histidine residue per copper atom bound. Even in apohaemocyanin about 6 × 10^?5 histidine residues per g protein are not titrated in their normal pH region.In the presence of sufficient calcium to displace the dissociation completely out of the titration region the titration curve of apohaemocyanin could he linarized according to the model of Linderstrøm—Lang. In oxy- and deoxyhaemocyanin, however, a distinct deviation from linearity was found under the same conditions. In the absence of calcium the effect of the dissociation adds up to this deviation.The electrostatic interaction factors were determined for the protein at 0.1 M KCl and for the dissociation products: halves and tenths at 1.0 M KCl. The electrostatic interaction factor for the wholes and the halves are much smaller than the values calculated from the Linderstrøm—Lang equation, using the radius of the equivalent sphere either obtained from electron microscopy or from the partial specific volume. This is probably due to solvent penetration. For the tenths at 1.0 M KCl. this effect is small,     

Intermediates detected by visible spectroscopy during the reaction of nitrite with deoxyhemoglobin: the effect of nitrite concentration and diphosphoglycerate

The reaction of nitrite with deoxyhemoglobin (deoxyHb) results in the reduction of nitrite to NO, which binds unreacted deoxyHb forming Fe(II)-nitrosylhemoglobin (Hb(II)NO). The tight binding of NO to deoxyHb is, however, inconsistent with reports implicating this reaction with hypoxic vasodilation. This dilemma is resolved by the demonstration that metastable intermediates are formed in the course of the reaction of nitrite with deoxyHb. The level of intermediates is quantitated by the excess deoxyHb consumed over the concentrations of the final products formed. The dominant intermediate has a spectrum that does not correspond to that of Hb(III)NO formed when NO reacts with methemoglobin (MetHb), but is similar to metHb resulting in the spectroscopic determinations of elevated levels of metHb. It is a delocalized species involving the heme iron, the NO, and perhaps the beta-93 thiol. The putative role for red cell reacted nitrite on vasodilation is associated with reactions involving the intermediate. (1) The intermediate is less stable with a 10-fold excess of nitrite and is not detected with a 100-fold excess of nitrite. This observation is attributed to the reaction of nitrite with the intermediate producing N2O3. (2) The release of NO quantitated by the formation of Hb(II)NO is regulated by changes in the distal heme pocket as shown by the 4.5-fold decrease in the rate constant in the presence of 2,3-diphosphoglycerate. The regulated release of NO or N2O3 as well as the formation of the S-nitroso derivative of hemoglobin, which has also been reported to be formed from the intermediates generated during nitrite reduction, should be associated with any hypoxic vasodilation attributed to the RBC.     

Nitric oxide-forming reaction between the iron-N-methyl-D-glucamine dithiocarbamate complex and nitrite

The objective of this study was to elucidate the origin of the nitric oxide-forming reactions from nitrite in the presence of the iron-N-methyl-D-glucamine dithiocarbamate complex ((MGD)(2)Fe(2+)). The (MGD)(2)Fe(2+) complex is commonly used in electron paramagnetic resonance (EPR) spectroscopic detection of NO both in vivo and in vitro. Although it is widely believed that only NO can react with (MGD)(2)Fe(2+) complex to form the (MGD)(2)Fe(2+).NO complex, a recent article reported that the (MGD)(2)Fe(2+) complex can react not only with NO, but also with nitrite to produce the characteristic triplet EPR signal of (MGD)(2)Fe(2+).NO (Hiramoto, K., Tomiyama, S., and Kikugawa, K. (1997) Free Radical Res. 27, 505-509). However, no detailed reaction mechanisms were given. Alternatively, nitrite is considered to be a spontaneous NO donor, especially at acidic pH values (Samouilov, A., Kuppusamy, P., and Zweier, J. L. (1998) Arch Biochem. Biophys. 357, 1-7). However, its production of nitric oxide at physiological pH is unclear. In this report, we demonstrate that the (MGD)(2)Fe(2+) complex and nitrite reacted to form NO as follows: 1) (MGD)(2)Fe(2).NO complex was produced at pH 7.4; 2) concomitantly, the (MGD)(3)Fe(3+) complex, which is the oxidized form of (MGD)(2)Fe(2+), was formed; 3) the rate of formation of the (MGD)(2)Fe(2+).NO complex was a function of the concentration of [Fe(2+)](2), [MGD], [H(+)] and [nitrite].     

Assessments of the chemistry and vasodilatory activity of nitrite with hemoglobin under physiologically relevant conditions

Hypoxic vasodilation involves detection of the oxygen content of blood by a sensor, which rapidly transduces this signal into vasodilatory bioactivity. Current perspectives on the molecular mechanism of this function hold that hemoglobin (Hb) operates as both oxygen sensor and a condition-responsive NO reactor that regulates the dispensing of bioactivity through release of the NO group from the beta-cys93 S-nitroso derivative of Hb, SNO-Hb. A common path to the formation of SNO-Hb involves oxidative transfer of the NO-group from heme to thiol. We have previously reported that the reaction of nitrite with deoxy-Hb, which furnishes heme-Fe(II)NO, represents one attractive route for the formation of SNO-Hb. Recent literature, however, posits that the nitrite-reductase reaction of Hb might produce physiological vasodilatory effects through NO that evades trapping on heme-Fe(II) and may be stored before release as Fe(III)NO. In this article, we briefly review current perspectives in NO biology on the nitrite-reductase reaction of Hb. We report in vitro spectroscopic (UV/Vis, EPR) studies that are difficult to reconcile with suggestions that this reaction either generates a heme-Fe(III)NO reservoir or significantly liberates NO. We further show in bioassay experiments that combinations of nitrite and deoxy-Hb--under conditions that suppress SNO-Hb formation--exhibit no direct vasodilatory activity. These results help underscore the differences between physiological, RBC-regulated, hypoxic vasodilation versus pharmacological effects of exogenous nitrite.     

Kinetics of the co-operative and non-co-operative reaction of Helix pomatia haemocyanin with oxygen

The kinetics of the reaction of Helix pomatia haemocyanin with oxygen have been studied under conditions where ligand binding is co-operative (n = 4.5). The dissociation of oxygen from oxyhaemocyanin in the presence of sodium dithionite and the combination of deoxyhaemocyanin with oxygen were studied by the stopped-flow technique. The combination with oxygen, as well as the dissociation of oxyhaemocyanin, are clearly autocatalytic. The initial rate constant for oxygen combination to the fully deoxygenated state is 0.2 to 0.3 × 10⁶m^?1 s^?1; during the course of the reaction the rate constant increases to a value higher than 10⁶m^?1s^?1.The initial rate of oxygen dissociation from fully saturated haemocyanin is 10 s^?1, increasing to about 30 s^?1 as the reaction proceeds. Thus, both the combination and the dissociation rate constants contribute to the co-operativity of oxygen binding.Temperature-jump relaxation experiments were carried out at fractional oxygen saturations larger than 0.7. The dependence of the relaxation rate upon the concentration of the reactants indicates the presence of one principal bimolecular process. The calculated combination and dissociation rate constants for this process are: 3.8 × 10⁶m^?1 s^?1 and 10 s^?1, respectively. Evidence is presented which shows that the transition from the T-state to the R-state of the protein is relatively slow. Both the T and R-state seem to be largely stabilized at the expense of intermediate states.Under other conditions, where oxygen binding is non-co-operative, temperature-jump and stopped-flow experiments reveal considerable kinetic heterogeneity.     

Stoichiometry of the reaction of oxyhemoglobin with nitrite.

During the reaction of oxyhemoglobin (HbO2) with nitrite, the concentration of residual nitrite, nitrate, oxygen, and methemoglobin (Hb+) was determined successively. The results obtained at various pH values indicate the following stoichiometry for the overall reaction: 4HbO2 + 4NO2- 4H+ leads to 4Hb+ + 4NO3- + O2 + 2H2 O (Hb denotes hemoglobin monomer). NO2- binds with methemoglobin noncooperatively with a binding constant of 340 M-1 at pH 7.4 and 25 degrees C. Thus, the major part of Hb+ produced is aquomethemoglobin, not methemoglobin nitrite, when less than 2 equivalents of nitrite is used for the oxidation.     

Nitric oxide-mediated immune complex-induced prostaglandin E(2) production by peripheral blood mononuclear cells of humans infected with Schistosoma mansoni.

Granuloma reaction around Schistosoma mansoni eggs is the prominent lesion in human schistosomiasis. Studies have suggested the involvement of a series of suppressive mechanisms in the control of this reaction. Using an in vitro model of granuloma formation, we have shown that immune complexes (IC) isolated from sera of chronic intestinal schistosomiasis patients were able to reduce granulomatous reaction developed against soluble egg antigen-conjugated polyacrylamide beads. In this system, the role of the l-arginine-nitric oxide (NO) pathway in the formation of prostaglandin E(2) (PGE(2)) by human peripheral blood mononuclear cells (PBMC) of patients infected with schistosomiasis was investigated using IC. Preincubation of PBMC with IC produced a significant increase of both nitrite and PGE(2) levels in the cell supernatant. This effect was inhibited by coincubation of cells with Nomega-nitro-l-arginine methyl ester (L-NAME), a NO synthase inhibitor, showing that the release of PGE(2) subsequent to IC stimulation was driven by NO. The inhibitory effect of L-NAME on PGE(2) release by IC-treated PBMC was reversed by sodium nitroprusside, a known NO donor. Our results indicate that NO could be an important second signal for the stimulation of PGE(2) production induced by IC in PBMC from human schistosomiasis patients.     

Electron-paramagnetic-resonance studies of the mechanism of leaf nitrite reductase. Signals from the iron–sulphur centre and haem under turnover conditions

Low-temperature e.p.r. spectra are presented of nitrite reductase purified from leaves of vegetable marrow (Cucurbita pepo). The oxidized enzyme showed a spectrum at g=6.86, 4.98 and 1.95 corresponding to high-spin Fe(3+) in sirohaem, which disappeared slowly on treatment with nitrite. The midpoint potential of the sirohaem was estimated to be -120mV. On reduction with Na(2)S(2)O(4) or Na(2)S(2)O(4)+Methyl Viologen a spectrum at g=2.038, 1.944 and 1.922 was observed, due to a reduced iron-sulphur centre. The midpoint potential of this centre was very low, about -570mV at pH8.1, decreasing with increasing pH. On addition of cyanide, which binds to haem, and Na(2)S(2)O(4), the iron-sulphur centre became further reduced. We think that this is due to an increased midpoint potential of the iron-sulphur centre. Other ligands to haem, such as CO and the reaction product NH(3), had similar but less pronounced effects, and also changed the lineshape of the iron-sulphur signal. Samples were prepared of the enzyme frozen during the reaction with nitrite, Methyl Viologen and Na(2)S(2)O(4) in various proportions. Signals were interpreted as due to the reduced iron-sulphur centre (with slightly different g values), a haem-NO complex and reduced Methyl Viologen. In the presence of an excess of nitrite, the haem-NO spectrum was more intense, whereas in the presence of an excess of Na(2)S(2)O(4) it was weaker, and disappeared at the end of the reaction. A reaction sequence is proposed for the enzyme, in which the haem-NO complex is an intermediate, followed by other e.p.r.-silent states, leading to the production of NH(4) (+).     

Kinetics of the reactions of trioxodinitrate and nitrite ions with cytochrome d in Escherichia coli.

The rate of reaction of trioxodinitrate with reduced cytochrome oxidase d in membrane particles from Escherichia coli at pH 7 and 25 degrees C depends linearly upon [HN2O3-] over the concentration range studied (up to 0.05 mM) and is also first-order in cytochrome d. The known rate of decomposition of trioxodinitrate to give NO- and NO2- is about 4.5-times faster than the rate of reaction of reduced cytochrome d with trioxodinitrate, implying that cytochrome d reacts directly with NO-, with a trapping ratio of between 0.20 and 0.25, rather than with trioxodinitrate. The implications of the facile formation of the NO(-)-nitrosyl complex of cytochrome d for the mechanism of denitrification are discussed with particular reference to the mechanism of N-N bond formation. The reaction of reduced cytochrome d with nitrite (a decomposition product of trioxodinitrate) under these conditions is much slower than that with trioxodinitrate. The kinetics show a biphasic dependence of initial rate upon nitrite concentration. The rate data at low [NO2-] are consistent with saturation of a high affinity site for nitrite, having Vmax = 4.29.10(-9) M s-1 and Km = 0.034 mM. The existence of two binding sites for nitrite is consistent with the suggestion that the cytochrome bd complex contains two cytochrome d haems.     

Superoxide fluxes limit nitric oxide-induced signaling

Independently, superoxide (O2-) and nitric oxide (NO) are biologically important signaling molecules. When co-generated, these radicals react rapidly to form powerful oxidizing and nitrating intermediates. Although this reaction was once thought to be solely cytotoxic, herein we demonstrate using MCF7, macrophage, and endothelial cells that when nanomolar levels of NO and O2- were produced concomitantly, the effective NO concentration was established by the relative fluxes of these two radicals. Differential regulation of sGC, pERK, HIF-1alpha, and p53 were used as biological dosimeters for NO concentration. Introduction of intracellular- or extracellular-generated O2- during NO generation resulted in a concomitant increase in oxidative intermediates with a decrease in steady-state NO concentrations and a proportional reduction in the levels of sGC, ERK, HIF-1alpha, and p53 regulation. NO responses were restored by addition of SOD. The intermediates formed from the reactions of NO with O2- were non-toxic, did not form 3-nitrotyrosine, nor did they elicit any signal transduction responses. H2O2 in bolus or generated from the dismutation of O2- by SOD, was cytotoxic at high concentrations and activated p53 independent of NO. This effect was completely inhibited by catalase, suppressed by NO, and exacerbated by intracellular catalase inhibition. We conclude that the reaction of O2- with NO is an important regulatory mechanism, which modulates signaling pathways by limiting steady-state levels of NO and preventing H2O2 formation from O2-.     

Low NO concentration dependence of reductive nitrosylation reaction of hemoglobin

The reductive nitrosylation of ferric (met)hemoglobin is of considerable interest and remains incompletely explained. We have previously observed that at low NO concentrations the reaction with tetrameric hemoglobin occurs with an observed rate constant that is at least 5 times faster than that observed at higher concentrations. This was ascribed to a faster reaction of NO with a methemoglobin-nitrite complex. We now report detailed studies of this reaction of low NO with methemoglobin. Nitric oxide paradoxically reacts with ferric hemoglobin with faster observed rate constants at the lower NO concentration in a manner that is not affected by changes in nitrite concentration, suggesting that it is not a competition between NO and nitrite, as we previously hypothesized. By evaluation of the fast reaction in the presence of allosteric effectors and isolated β- and α-chains of hemoglobin, it appears that NO reacts with a subpopulation of β-subunit ferric hemes whose population is influenced by quaternary state, redox potential, and hemoglobin dimerization. To further characterize the role of nitrite, we developed a system that oxidizes nitrite to nitrate to eliminate nitrite contamination. Removal of nitrite does not alter reaction kinetics, but modulates reaction products, with a decrease in the formation of S-nitrosothiols. These results are consistent with the formation of NO(2)/N(2)O(3) in the presence of nitrite. The observed fast reductive nitrosylation observed at low NO concentrations may function to preserve NO bioactivity via primary oxidation of NO to form nitrite or in the presence of nitrite to form N(2)O(3) and S-nitrosothiols.     

TEMPOL enhances the antihypertensive effects of sodium nitrite by mechanisms facilitating nitrite-derived gastric nitric oxide formation

Orally administered nitrite exerts antihypertensive effects associated with increased gastric nitric oxide (NO) formation. While reducing agents facilitate NO formation from nitrite, no previous study has examined whether antioxidants with reducing properties improve the antihypertensive responses to orally administered nitrite. We hypothesized that TEMPOL (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl) could enhance the hypotensive effects of nitrite in hypertensive rats by exerting antioxidant effects (and enhancing NO bioavailability) and by promoting gastric nitrite-derived NO generation. The hypotensive effects of intravenous and oral sodium nitrite were assessed in unanesthetized freely moving rats with L-NAME (N^ω-nitro-L-arginine methyl ester; 100 mg/kg; po)-induced hypertension treated with TEMPOL (18 mg/kg; po) or vehicle. While TEMPOL exerted antioxidant effects in hypertensive rats, as revealed by lower plasma 8-isoprostane and vascular reactive oxygen species levels, this antioxidant did not affect the hypotensive responses to intravenous nitrite. Conversely, TEMPOL enhanced the dose-dependent hypotensive responses to orally administered nitrite, and this effect was associated with higher increases in plasma nitrite and lower increases in plasma nitrate concentrations. In vitro experiments using electrochemical and chemiluminescence NO detection under variable pH conditions showed that TEMPOL enhanced nitrite-derived NO formation, especially at low pH (2.0 to 4.0). TEMPOL signal evaluated by electron paramagnetic resonance decreased when nitrite was reduced to NO under acidic conditions. Consistent with these findings, increasing gastric pH with omeprazole (30 mg/kg; po) attenuated the hypotensive responses to nitrite and blunted the enhancement in plasma nitrite concentrations and hypotensive effects induced by TEMPOL. Nitrite-derived NO formation in vivo was confirmed by using the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (C-PTIO), which blunted the responses to oral nitrite. Our results showed that TEMPOL promotes nitrite reduction to NO in the stomach and enhanced plasma nitrite concentrations and the hypotensive effects of oral sodium nitrite through mechanisms critically dependent on gastric pH. Interestingly, the effects of TEMPOL on nitrite-mediated hypotension cannot be explained by increased NO formation in the stomach alone, but rather appear more directly related to increased plasma nitrite levels and reduced nitrate levels during TEMPOL treatment. This may relate to enhanced nitrite uptake or reduced nitrate formation from NO or nitrite.     

Melatonin nitrosation promoted by NO*2; comparison with the peroxynitrite reaction

N-nitroso species have recently been detected in animal tissues. Protein N-nitrosotryptophan is the best candidate for this N-nitroso pool. N-nitrosation of N-blocked trytophan derivatives like melatonin (MelH) by N2O3 or peroxynitrite (ONOOH/ONOO- ) has been observed under conditions of pH and reagent concentrations similar to in vivo conditions. We studied the reaction of NO*2 with MelH. When NO*2 was synthesized by gamma-irradiation of aqueous neutral solutions of nitrate under anaerobic conditions, detected oxidation and nitration of MelH were negligible. In the presence of additional nitrite, when NO* was also generated, formation of 1-nitrosomelatonin increased with nitrite concentration. Nitrosation is not due to N2O3 but could proceed via successive additions of NO*2 and NO*. For comparison, peroxynitrite was infused into a solution of MelH under air leading to the same products as those detected in irradiated solutions but in different proportions. In the presence of additional nitrite, the formation of nitroderivatives increased significantly while N-formylkynuramine and 1-nitrosomelatonin were maintained at similar levels. Mechanistic implications are discussed.     

Oxidative Modulation of the Glutathione-redox Couple Enhances Lipopolysaccharide-induced Interleukin 12 P40 Production by a Mouse Macrophage Cell Line,J774A.1

Abstract

Reactions of salivary nitrite with components of wine were studied using an acidic mixture of saliva and wine. The formation of nitric oxide (NO) in the stomach after drinking wine was observed. The formation of NO was also observed in the mixture (pH 3.6) of saliva and wine, which was prepared by washing the oral cavity with wine. A part of the NO formation in the stomach and the oral cavity was due to the reduction of salivary nitrite by caffeic and ferulic acids present in wine. Ethyl nitrite produced by the reaction of salivary nitrite and ethyl alcohol in wine also contributed to the formation of NO. In addition to the above reactions, caffeic acid in wine could be transformed to the oxathiolone derivative, which might have pharmacological functions. The results obtained in this study may help in understanding the effects of drinking wine on human health.     

Effect of nitric oxide – releasing compounds on phytochrome – controlled germination of Empress tree seeds

Using different nitric oxide releasing compounds and appropriate controls we have obtained data strongly suggesting the involvement of nitric oxide in the phytochrome controlled germination of Paulownia tomentosa seeds. Direct detection of nitric oxide, under various experimental conditions, was performed by a spin-trapping technique combined with electron paramagnetic resonance (EPR) spectroscopy. The addition of methylene blue prevented light-induced and NO donors-potentiated germination of P. tomentosa seeds. This inhibition could be completely overcome by addition of gibberellin. The promotive effect of nitrite was pH dependent, maximally pronounced at the pH range where nitrite undergoes dismutation and liberates nitric oxide. Under these conditions, nitrite exerted its efficacy at the same concentrations at which nitric oxide releasing compounds such as sodium nitroprusside (SNP), S-nitroso acetylpenicillamine (SNAP), and 3-morpholinosydnonimine (SIN-1), were the most effective. Likewise, the potentiation of P. tomentosa seed germination could be achieved by chemical reduction of nitrite with Na2S2O4 during which liberation of nitric oxide could be detected.