Reactive nitrogen species derived activation of rat liver microsomal glutathione S-transferase

The effect of reactive nitrogen species on rat liver microsomal glutathione S-transferase (MGST1) was investigated using microsomes and purified MGST1. When microsomes or the purified enzyme were incubated with peroxynitrite (ONOO(-)), the GST activity was increased to 2.5-6.5 fold in concentration-dependent manner and a small amount of the MGST1 dimer was detected. MGST1 activity was increased by ONOO(-) in the presence of high amounts of reducing agents including glutathione (GSH) and the activities increased by ONOO(-) or ONOO(-) plus GSH treatment were decreased by 30-40% by further incubation with dithiothreitol (DTT, reducing disulfide) or by sodium arsenite (reducing sulfenic acid). Furthermore, GSH was detected by HPLC from the MGST1 which was incubated with ONOO(-) plus GSH or S-nitrosoglutathione followed by DTT treatment. In addition, the MGST1 activity increased by nitric oxide (NO) donors such as S-nitrosoglutathione, S-nitrosocysteine or the non-thiol NO donor 1-hydroxy-2-oxo-3 (3-aminopropyl)-3-isopropyl was restored by the DTT treatment. Since DTT can reduce S-nitrosothiol and disulfide bond to thiol, S-nitrosylation and a mixed disulfide bond formation of MGST1 were suggested. Thus, it was demonstrated that MGST1 is activated by reactive nitrogen species through a forming dimeric protein, mixed disulfide bond, nitrosylation and sulfenic acid.     

Flavohemoglobin and nitric oxide detoxification in the human protozoan parasite Giardia intestinalis

Flavohemoglobins (flavoHbs), commonly found in bacteria and fungi, afford protection from nitrosative stress by degrading nitric oxide (NO) to nitrate. Giardia intestinalis, a microaerophilic parasite causing one of the most common intestinal human infectious diseases worldwide, is the only pathogenic protozoon as yet identified coding for a flavoHb. By NO amperometry we show that, in the presence of NADH, the recombinant Giardia flavoHb metabolizes NO with high efficacy under aerobic conditions (TN = 116 ± 10 s⁻¹ at 1 μM NO, T = 37 °C). The activity is [O₂]-dependent and characterized by an apparent K_M,O2 = 22 ± 7 μM. Immunoblotting analysis shows that the protein is expressed at low levels in the vegetative trophozoites of Giardia; accordingly, these cells aerobically metabolize NO with low efficacy. Interestingly, in response to nitrosative stress (24-h incubation with ?5 mM nitrite) flavoHb expression is enhanced and the trophozoites thereby become able to metabolize NO efficiently, the activity being sensitive to both cyanide and carbon monoxide. The NO-donors S-nitrosoglutathione (GSNO) and DETA-NONOate mimicked the effect of nitrite on flavoHb expression. We propose that physiologically flavoHb contributes to NO detoxification in G. intestinalis.     

The placental cholinergic system: localization to the cytotrophoblast and modulation of nitric oxide

Background

The human placenta, a non-neuronal tissue, contains an active cholinergic system comprised of acetylcholine (ACh), choline acetyltransferase (ChAT), acetylcholinesterase (AChE), and high affinity muscarinic receptors. The cell(s) of origin of placental ACh and its role in trophoblast function has not been defined. These studies were performed to define the cellular location of ACh synthesis (ChAT) in the human placenta and to begin studying its functional role.

Results

Using immunohistochemical techniques, ChAT was observed primarily within the cytotrophoblasts of preterm placentae as well as some mesenchymal elements. Similar intense immunostaining of the cytotrophoblast was observed for endothelium-derived nitric oxide synthase (eNOS) suggesting that ACh may interact with nitric oxide (NO)-dependent signaling pathways. The ability of carbamylcholine (CCh), an ACh analogue, to stimulate a rise in intracellular Ca⁺⁺ and NO production in trophoblasts was therefore tested using the BeWo^b30 choriocarcinoma cell as a model system. First, CCh significantly increased intracellular calcium as assessed by fluorescence microscopy. We then examined the ability of CCh to stimulate NO production by measuring total nitrite/nitrate production in conditioned media using chemiluminescence-based analysis. CCh, alone, had no effect on NO production. However, CCh increased measurable NO approximately 100% in the presence of 10 nM estradiol. This stimulatory effect was inhibited by 1 (micro)M scopolamine suggesting mediation via muscarinic receptors. Estradiol, alone, had no effect on total NO or eNOS protein or mRNA.

Conclusion

These data demonstrate that placental ChAT localizes to the cytotrophoblast and some mesenchymal cells in human placenta. It further suggests that ACh acts via muscarinic receptors on the trophoblast cell membrane to modulate NO in an estrogen-dependent manner.     

Amperometric measurements of nitric oxide in erythrocytes

In the recent years, there has been an increase in the development of new biosensors that could be helpful in the study of various physiological processes. In this study, we report the development of a new in vitro experimental design for real-time nitric oxide (NO) amperometric measurements in erythrocyte suspensions. To achieve this, we employed human erythrocyte suspensions in sodium chloride 0.9%, pH 7 (haematocrit 0.05%). The production of NO by erythrocytes was measured with a commercial NO sensor during stimulation by L-arginine, acetylcholine, choline, atropine and velnacrine maleate (10 microM of final concentrations). We also measured the nitrite and nitrate concentrations produced by erythrocyte suspensions stimulated with the above effectors by means of the Griess reaction method. We observed that there was a direct relation between the electric current produced by the NO sensor, and the NO standard concentrations, thereby leading to a good calibration curve. The in vitro erythrocytes produced significant amperometric NO values in response to a wide range of effectors and these results have the same variation profile of the nitrites and nitrates results achieved with the Griess method. In conclusion, the amperometric NO sensor constitutes a reliable method for direct, and real-time measurement in vitro of the NO production of erythrocyte suspensions, As such, it offers a potential diagnostic technique for the evaluation of diseases, and the therapeutic progression of diseases, related to intracellular NO metabolism.     

Production of nitric oxide by human salivary peroxidase and by bovine lactoperoxidase

Peroxidases catalyze the oxidation of nitrite to nitrate in the presence of hydrogen peroxide. Two pathways may occur: one entailing the intermediate formation of NO(2) and the other implying the generation of peroxynitrite. The products of nitrite (NO(2) (-) ) oxidation by salivary peroxidase (SPO) and commercial bovine lactoperoxidase (LPO) are studied by utilizing an electrochemical assay that allows the direct, continuous monitoring of NO and/or NO(2) and by HPLC to assess nitrates at the end of the reaction. Dialyzed saliva and LPO, in the presence of H(2) O(2) , convert nitrite into nitrate and form some NO, with a molar ratio of 10(3) . In our experimental conditions, no NO(2) was detectable among the products of nitrite oxidation. SCN(-) inhibits NO formation and so does I(-) , although at higher concentrations. No effects are observed with Cl(-) or Br(-) . We conclude that SPO and LPO transform NO(2) (-) into nitrate-forming small amounts of NO in the presence of H(2) O(2) as an intermediate or a by-product, synthesized through the peroxynitrite pathway.     

Fibrinogen-Dependent Signaling in Microvascular Erythrocyte Function: Implications on Nitric Oxide Efflux

Experimental evidence has shown that plasma fibrinogen plays a key role as a major cardiovascular risk factor, acting directly to trigger erythrocyte aggregation in occlusive vascular disease. However, due to the complex and hitherto unclear interaction between fibrinogen and the erythrocyte membrane, no study has yet evaluated the effects of fibrinogen, under physiological range values, on the erythrocyte nitric oxide (NO) mobilization. Taking into consideration the potential NO-derived molecules, we have raised the hypothesis that fibrinogen, under physiological conditions, may act to influence blood flow via erythrocyte NO modulation. In this in vitro study whole-blood samples were harvested from healthy subjects, erythrocyte suspensions were incubated in the absence (control aliquots) and presence of different fibrinogen concentrations and levels of NO, nitrite, nitrate and S-nitroglutathione (GSNO) were determined. Our results showed, when compared with control aliquots, that the presence of fibrinogen modulates the NO mobilization in erythrocytes by (1) decreasing erythrocyte NO efflux levels (P < 0.001); (2) increasing levels of intraerythrocytic NO oxidative metabolites, namely, nitrite (P < 0.0001) and nitrate (P < 0.0001); and (3) enhancing the formation of GSNO (P < 0.001). In conclusion, this study provides new insights into an unknown mechanism by which fibrinogen modulates the erythrocyte capacity to supply NO, the effects of which on inflammation profiles (generally associated with blood hyperviscosity and hyperaggregation) still need to be elucidated. Also, increased erythrocyte GSNO levels may be associated with platelet NO metabolism, its activation status and hypotension, which may be extremely relevant in the clinical setting as biomarkers.     

Nitric oxide modulates intensity of non-quantal acetylcholine release in myocardium of the right atrium of rat

The main parasympathetic neurotransmitter acetylcholine (ACh) is released in the myocardium from the intramural postganglionic parasympathetic nerve endings. The mechanism of non-quantal ACh release has been recently demonstrated in these neurons. Non-quantal ACh release does not depend on exocytosis of ACh-containing vesicles in response to nerve impulse activity but is assumed to be mediated by the high-affinity choline uptake system. The intensity of non-quantal ACh release in the myocardium correlates with the degree of manifestation of the effects of acetylcholinesterase inhibitors inducing the accumulation of non-quantal ACh in the myocardium. The present study deals with the influence of putative modulators of non-quantal ACh release: nitric oxide (NO) and ATP, on the intensity of cholinergic effects induced by organophosphorous acetylcholinesterase inhibitor paraoxon. Intracellular registration of bioelectrical activity in isolated right atrium preparations from rats was used. Under normal conditions, paraoxon (10^?7–10^?5 M) induced a marked decrease in the action potential (AP) duration at a level of 50 and 90% repolarization in the working right atrial myocardium and slowed down the sinus rhythm. ATP, which is known to suppress nonquantal ACh release in the neuromuscular junction, did not induce significant reduction or augmentation of the effects of paraoxon (5 × 10^?6 M). The NO donors, sodium nitroprusside (10^?5 M) and SNAP (10^?4 M), significantly reduced the paraoxon-induced AP shortening. Moreover, sodium nitroprusside decreased the negative chronotropic effect of paraoxon by 43.7%. On the contrary, NO synthase inhibitor L-NAME (10^?4 M), which is known to suppress endogenous NO production, augmented the AP shortening caused by paraoxon. It may be deduced that NO is a universal regulator of non-quantal ACh release intensity both in the myocardium and in the neuromuscular junction.     

Modulation of Erythrocyte Acetylcholinesterase Activity and Its Association with G Protein-Band 3 Interactions

Circulating acetylcholine, substrate of membrane acetylcholinesterase (AChE), is known to enhance the band 3 protein degree of phosphorylation. The purpose of this study was to verify whether the band 3 phosphorylation status is associated with a G protein and whether it is an influent factor on AChE enzyme activity. From blood samples of healthy donors, erythrocyte suspensions were prepared and incubated with AChE substrate (acetylcholine) and inhibitor (velnacrine), along with protein tyrosine kinase (PTK) and tyrosine phosphatase (PTP) inhibitors. AChE activity was determined by spectrophotometry and extract samples were analyzed by western blotting using primary antibodies to different G protein subunits. Our results with phosphorylated band 3 (PTP inhibitor) show an increase in erythrocyte AChE (p < 0.0001). A dephosphorylated band 3 state (PTK inhibitor) shows a significant decrease. We identified a potential linkage of protein subunits Gα_i1/2 and G_β with band 3 protein. Gα_i1/2 and G_β may be linked to the band 3 C-terminal site. Gα_i1/2 is associated with the band 3 N-terminal domain, except for the control and ACh aliquots. G_β is associated with both phosphorylated and dephosphorylated band 3 in the presence of velnacrine. We conclude that an erythrocyte G protein with subunits Gα_i1/2 and G_β is associated with band 3. AChE depends on the degree of band 3 phosphorylation and its association with Gα_i1/2 and G_β.     

Nitrate (NO3) and nitrite (NO2) are endocrine disruptors to downregulate expression of tyrosine hydroxylase and motor behavior through conversion to nitric oxide in early development of zebrafish

With a view to consider the increasing concern over nitrogen pollution in the aquatic environment, we investigated effects of nitrate (NO₃⁻) and nitrite (NO₂⁻) on the activity of dopaminergic neuron in zebrafish embryos and larvae. Both nitrate and nitrite exposure decreased the expression of tyrosine hydroxylase (TH) in dopaminergic neurons at 48 hpf. Only nitrite decreased the response to tactile stimulation at 72 hpf, whereas both nitrate and nitrite decreased the swimming activity at 6 dpf. When the embryos were exposed to nitrate or nitrite together with an estrogen receptor blocker (ICI 182,780), the decreases in TH expression and motor behavior caused by nitrate or nitrite alone were reversed suggesting the effects of nitrate and nitrite were mediated through estrogen receptor (ER). The result of co-incubation with an oxidoreductase inhibitor, diphenyleneiodonium, indicated the conversion to nitric oxide (NO) is likely to be responsible for the effects of nitrate and nitrite, which was further supported by the increased staining for NO after exposure. The present study demonstrates that nitrate and nitrite are neurotoxicants acting as an endocrine disruptor possibly through conversion to NO to downregulate the activity of dopaminergic neuron in early development of zebrafish.     

In vitro production of superoxide and nitric oxide (as nitrite and nitrate) by Mytilus galloprovincialis haemocytes upon incubation with PMA or laminarin or during yeast phagocytosis

The phagocytic process is one of the most important elements of the self-defence system in mammals as well as in molluscs. In mammalian phagocytes, superoxide participates in the innate defence system by combining with nitric oxide to generate peroxynitrite, a strong oxidant that possesses highly cytotoxic properties against bacteria. To evidence a role of nitric oxide in the self-defence system of the marine bivalve Mytilus galloprovincialis similar to the role observed in the mammalian defence system, we measured the generation of superoxide and nitrite/nitrate (the stable end products of nitric oxide) upon in vitro stimulation of M. galloprovincialis haemocytes with PMA, laminarin, LPS and by phagocytosis of Saccharomyces cerevisiae (yeast cells). We show that stimulation with PMA, laminarin and yeast cell phagocytosis promotes superoxide and nitrite/nitrate generation from M. galloprovincialis haemocytes. Inhibitors of NADPH oxidase and inhibitors of NO synthase decreased the nitrite/nitrate levels generated by M. galloprovincialis haemocytes showing that both NADPH oxidase and NO synthase pathways are involved in the self-defence system of M. galloprovincialis.     

Peroxynitrite and NO donors form colored nitrite adducts with sinapinic acid: potential applications

Sinapinic acid (3,5-dimethoxy-4-hydroxycinnamic acid, SA) reacted with peroxynitrous acid at neutral pH with a second-order rate constant of 812 M(-1)s(-1), to yield a red product (lambda(max), 532 nm). The identical colored product could be formed with acidified decomposed peroxynitrous acid solutions or nitrite at slower rates (0.1M HCl, 8.32 M(-1)s(-1); 10% acetic acid, 0.0004 M(-1)s(-1)). The red compound is thought to be O-nitrososinapinic acid (3,5-dimethoxy-4-nitrosooxycinnamic acid) which can be formed by reaction with either peroxynitrous acid or nitrous acid. The extinction coefficient of O-nitrososinapinic acid (ONSA) was estimated to be 8419 M(-1)cm(-1) at 510 nm in 10% acetic acid and 90% acetonitrile. ONSA was also formed via NO(+) transfer from S-nitrosoglutathione (GSNO). ONSA in turn can S-nitrosate low molecular weight thiols and protein thiols. SA was also shown to act as a peroxynitrite sink as it effectively prevented the oxidation of dihydrorhodamine under physiological conditions. The fact that O-nitrososinapinic acid is stable and can be used to S-nitrosate thiol containing amino acids, peptides, and proteins makes it a potentially useful reagent in the study of S-nitrosothiol biochemistry and physiology. In addition, the relatively high extinction coefficient of O-nitrososinapinic acid means that it could be utilized as an analyte for the spectroscopic detection of peroxynitrite or NO(+)-donors in the submicromolar range.     

Relaxant effects of Schisandra chinensis and its major lignans on agonists-induced contraction in guinea pig ileum

In this study, the herbal extracts of Schisandra chinensis were demonstrated to inhibit the contractions induced by acetylcholine (ACh) and serotonin (5-HT) in guinea pig ileum, and the 95% ethanol extract was more effective than the aqueous extract. Analysis with High Performance Liquid Chromatography (HPLC) indicated that schisandrin, schisandrol B, schisandrin A and schisandrin B were the major lignans of Schisandra chinensis, and the ethanol extract contained higher amount of these lignans than the aqueous extract. All four lignans inhibited the contractile responses to ACh, with EC₂₀ values ranging from 2.2 ± 0.4 μM (schisandrin A) to 13.2 ± 4.7 μM (schisandrin). The effectiveness of these compounds in relaxing the 5-HT-induced contraction was observed with a similar magnitude. Receptor binding assay indicated that Schisandra lignans did not show significant antagonistic effect on muscarinic M3 receptor. In Ca²⁺-free preparations primed with ACh or KCl, schisandrin A (50 μM) attenuated the contractile responses to cumulative addition of CaCl₂ by 37%. In addition, schisandrin A also concentration-dependently inhibited ACh-induced contractions in Ca²⁺-free buffer. This study demonstrates that Schisandra chinensis exhibited relaxant effects on agonist-induced contraction in guinea pig ileum, with schisandrin, schisandrol B, schisandrin A and schisandrin B being the major active ingredients. The antispasmodic action of schisandrin A involved inhibitions on both Ca²⁺ influx through L-type Ca²⁺ channels and intracellular Ca²⁺ mobilization, rather than specific antagonism of cholinergic muscarinic receptors.     

Kinetics and thiol requirements of iodothyronine 5'-deiodination are tissue-specific in common carp (Cyprinus carpio L.)

Iodothyronine deiodinases determine the biological activity of thyroid hormones. Despite the homology of the catalytic sites of mammalian and teleostean deiodinases, in-vitro requirements for the putative thiol co-substrate dithiothreitol (DTT) vary considerably between vertebrate species. To further our insights in the interactions between the deiodinase protein and its substrates: thyroid hormone and DTT, we measured enzymatic iodothyronine 5′-deiodination, Dio1 and Dio2 mRNA expression, and Dio1 affinity probe binding in liver and kidney preparations from a freshwater teleost, the common carp (Cyprinus carpio L.). Deiodination rates, using reverse T3 (rT3, 3,3′,5′-triiodothyronine) as the substrate, were analysed as a function of the iodothyronine and DTT concentrations. In kidney rT3 5′-deiodinase activity measured at rT3 concentrations up to 10 μM and in the absence of DTT does not saturate appreciably. In the presence of 1 mM DTT, renal rT3 deiodination rates are 20-fold lower. In contrast, rT3 5′-deiodination in liver is potently stimulated by 1 mM DTT. The marked biochemical differences between 5′-deiodination in liver and kidney are not associated with the expression of either Dio1 or Dio2 mRNA since both organs express both deiodinase types. In liver and kidney, DTT stimulates the incorporation of N-bromoacetylated affinity labels in proteins with estimated molecular masses of 57 and 55, and 31 and 28 kDa, respectively. Although primary structures are highly homologous, the biochemistry of carp deiodinases differs markedly from their mammalian counterparts.     

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.     

The effect of oxidative stress on endothelium-dependent and nitric oxide donor-induced relaxation: implications for nitrate tolerance.

Increased inactivation of nitric oxide (NO) by superoxide has been implicated in nitrate tolerance. Here, we set out to compare the inhibitory effect of superoxide on endothelium-dependent, acetylcholine (ACh)-mediated vascular relaxation with that on the endothelium-independent effects of glyceryl trinitrate (GTN) and another NO donor drug, S-nitrosoglutathione (GSNO). Rings of thoracic aorta from adult male Wistar rats (350-450 g) were precontracted with phenylephrine (approximately EC(90)) prior to cumulative additions (10 nM/L-10 microM/L) of GTN, GSNO, or ACh. Rings were then treated with the superoxide generator pyrogallol (300 micromol/L) alone or following pretreatment with the Cu/Zn superoxide dismutase inhibitor diethyldithiocarbamate (DETCA; 100 micromol/L), and cumulative additions of the vasodilators were repeated. All experiments were conducted in the presence of catalase (3000 U/ml) to prevent accumulation of hydrogen peroxide. Relaxation to ACh was abolished by pyrogallol-derived superoxide. Relaxation to GSNO was significantly inhibited by superoxide (P < 0.05, n = 8) and was more pronounced at lower GSNO concentrations. However, GTN was relatively resistant to inhibition by superoxide with modest inhibition only occurring in rings pretreated with DETCA prior to pyrogallol (P < 0.05; n = 8). In contrast to GSNO, the inhibitory effect was more pronounced with high concentrations of GTN, suggesting that the mechanism underlying superoxide-mediated inhibition is different for the two NO donor drugs. Further experiments showed that vascular responses to ACh were not inhibited (P > 0.05, n = 6) in aortic rings made tolerant to GTN (10 micromol/L, 2-h incubation) and that treatment of vessels with the antioxidant vitamin C (1 mmol/L) successfully prevented the development of tolerance. Taken together, these results suggest that superoxide is not a major factor in tolerance in vitro and imply that the protective actions of vitamin C are unrelated to its antioxidant activity in this setting.     

Nitrite and nitroglycerin induce rapid release of the vasodilator ATP from erythrocytes: Relevance to the chemical physiology of local vasodilation

Extracellular ATP released from circulating erythrocytes induces vasodilation by stimulating receptor-mediated endothelium NO/EDRF (endothelium-derived relaxing factor) production. We report that pre-stimulation of freshly isolated human erythrocytes with physiological nitrite (100 nM ) or pharmacological nitroglycerin (10 μM) concentrations resulted in >200% spike in ATP release, which was detected on resuspending the cells in fresh medium. The observed response was instantaneous following pre-stimulation but a delay of ∼20 s followed nitroglycerin pre-stimulation, reflecting the time required for prodrug activation within the erythrocyte to its vasoactive metabolites, and NO. The data provided here are consistent with ATP being a conveyor of a NO-induced vasodilatory signal from the erythrocyte to the endothelium. Extended erythrocyte pre-stimulation with the NO donors resulted in a dose-dependent decrease in extracellular ATP, which would attenuate the signal in intact vessels to prevent excessive vasodilation. Importantly, our study constitutes the first report of enhanced vasodilator (ATP) release following human erythrocyte pre-stimulation by an endogenous or pharmacological (nitroglycerin) NO donor. The relevance of our findings to the therapeutic effects of nitroglycerin as well as to nitrate tolerance is discussed.     

Simultaneous production of nitric oxide and peroxynitrite by plant nitrate reductase: in vitro evidence for the NR-dependent formation of active nitrogen species

We examined the ability of plant nitrate reductase (NR) to produce nitric oxide (NO) using in vitro assays. Electrochemical and fluorometric measurements both showed that NO is produced by corn NR in the presence of nitrite and NADH at pH 7. The NO production was inhibited by sodium azide, a known inhibitor for NR. During the reaction, absorbance of 2',7'-dichlorodihydrofluorescein increased markedly. This change was completely suppressed by sodium azide, glutathione or depletion of oxygen. We conclude that plant NR produces both NO and its toxic derivative, peroxynitrite, under aerobic conditions when nitrite is provided as the substrate for NR.     

Product formation and kinetic simulations in the pH range 1-14 account for a free-radical mechanism of peroxynitrite decomposition

The yields of nitrate and nitrite from decomposition of peroxynitrite in phosphate buffer at 37 degrees C were determined in the pH range 1-14. The NO(2)(-)/NO(3)(-) yields showed a stepwise variation with pH, with inflection points at approximately pH 3.1, 5.8, 6.8, 8.0, and 11.9. Nitrite formation increased strongly above pH 7 at the expense of nitrate, but above pH 12 nitrate again became the major product (80% at pH 14). At this pH, the Arrhenius parameters were E(a)=24.1+/-0.2kcal mol(-1) and A=(4.9+/-1.3)x10(12)s(-1). The yields of NO(2)(-), NO(3)(-), and O(2) measured at pH 5.8, 7.4, and 8.5 as a function of the initial peroxynitrite concentration (50-1000 microM) were linear only at pH 5.8. In the presence of carbon dioxide, oxygen production at pH 7.5 and pH 10 was found to be linear on the CO(2) concentration. The experimental observations were satisfactorily reproduced by kinetic simulations including principal component analyses. These data strongly suggest that the chemistry of peroxynitrite is exclusively mediated by z.rad;NO(2) and HO(z.rad;) radicals in the absence, and by z.rad;NO(2) and CO(3)(z.rad;-) radicals in the presence of CO(2).     

Endothelial modulation and tolerance development in the vasorelaxant responses to nitrate of rabbit aorta

We investigated the endothelial modulations in nitrate tolerance in isolated rabbit aorta. Nitrate tolerance was induced by a 72-h treatment with transdermal nitroglycerin (NTG, 0.4 mg/h) in conscious rabbits, which was verified by a 20-fold increase in the EC50 values [NTG tolerance (6.1 +/- 0.8) x 10(-7) M vs control (3.0 +/- 0.6) x 10(-8) M]. The relaxations to NTG in tolerant and nontolerant aortic strips were enhanced when their endothelia were denuded [E(-)]. In the presence of endothelium [E(+)], NTG-tolerant vessels were not tolerant to acetylcholine (ACh), which can release endothelial nitric oxide (NO), exogenous NO or 8-bromo (Br)-cGMP. In NTG-tolerant and nontolerant vessels with endothelium, concentration-response curves for NO were the same as those in endothelium-absent tolerant vessels. In both NTG-tolerant and nontolerant vessels, treatment with superoxide dismutase (SOD, 20 units/ml), an O2-. scavenger, unaffected the responses to NTG reduced in the presence of endothelium, but treatment with NG-nitro-L-arginine methyl ester (L-NAME, 10(-4) M), an NO synthase (NOS) inhibitor, reversed these reductions. Thus, our data did not indicate that an increased endothelial superoxide O2-. production contributes to nitrate tolerance. Our study suggested that (i) an impaired biotransformation process from NTG to NO is responsible for the occurrence of nitrate tolerance and (ii) vascular response to NTG enhanced by endothelial removal is related to blocked endothelial NO release.