Presence Of NO Donor Research Articles

Host-adaptive traits in the plant-colonizing Pseudomonas donghuensis P482 revealed by transcriptomic responses to exudates of tomato and maize

Pseudomonads are metabolically flexible and can thrive on different plant hosts. However, the metabolic adaptations required for host promiscuity are unknown. Here, we addressed this knowledge gap by employing RNAseq and comparing transcriptomic responses of Pseudomonas donghuensis P482 to root exudates of two plant hosts: tomato and maize. Our main goal was to identify the differences and the common points between these two responses. Pathways upregulated only by tomato exudates included nitric oxide detoxification, repair of iron-sulfur clusters, respiration through the cyanide-insensitive cytochrome bd, and catabolism of amino and/or fatty acids. The first two indicate the presence of NO donors in the exudates of the test plants. Maize specifically induced the activity of MexE RND-type efflux pump and copper tolerance. Genes associated with motility were induced by maize but repressed by tomato. The shared response to exudates seemed to be affected both by compounds originating from the plants and those from their growth environment: arsenic resistance and bacterioferritin synthesis were upregulated, while sulfur assimilation, sensing of ferric citrate and/or other iron carriers, heme acquisition, and transport of polar amino acids were downregulated. Our results provide directions to explore mechanisms of host adaptation in plant-associated microorganisms.

The Unexpected Role of Calcium-Activated Potassium Channels: Limitation of NO-Induced Arterial Relaxation.

BackgroundMultiple studies have shown that an NO‐induced activation of vascular smooth muscle BK channels contributes to the NO‐evoked dilation in many blood vessels. In vivo, NO is released continuously. NO attenuates vessel constrictions and, therefore, exerts an anticontractile effect. It is unknown whether the anticontractile effect of continuously present NO is mediated by BK channels.Methods and ResultsThis study tested the hypothesis that BK channels mediate the vasodilatory effect of continuously present NO. Experiments were performed on rat and mouse tail and rat saphenous arteries using isometric myography and FURA‐2 fluorimetry. Continuously present NO donors, as well as endogenous NO, attenuated methoxamine‐induced vasoconstrictions. This effect was augmented in the presence of the BK channel blocker iberiotoxin. Moreover, the contractile effect of iberiotoxin was reduced in the presence of NO donors. The effect of the NO donor sodium nitroprusside was abolished by an NO scavenger and by a guanylyl cyclase inhibitor. In addition, the effect of sodium nitroprusside was reduced considerably by a protein kinase G inhibitor, but was not altered by inhibition of H2S generation. Sodium nitroprusside attenuated the intracellular calcium concentration response to methoxamine. Furthermore, sodium nitroprusside strongly reduced methoxamine‐induced calcium influx, which depends entirely on L‐type calcium channels. It did not affect methoxamine‐induced calcium release.ConclusionsIn summary, this study demonstrates the following: (1) continuously present NO evokes a strong anticontractile effect on rat and mouse arteries; (2) the iberiotoxin‐induced augmentation of the effect of NO is associated with an NO‐induced reduction of the effect of iberiotoxin; and (3) NO evoked a reduction of calcium influx via L‐type calcium channels.

Location and Number of Astrocytes Determine Dopaminergic Neuron Survival and Function Under 6-OHDA Stress Mediated Through Differential BDNF Release.

While astrocytes throughout the CNS share many common traits, they exhibit significant differences in function and number among brain regions. The aim of the present study is to assess the effect of region-specificity and number of astrocytes on the survival of dopaminergic neurons under stress, and to understand the possible mechanism by which these astrocytes extend neuroprotection to dopaminergic neurons. Purified astrocytes obtained from forebrain, midbrain, and hindbrain region were characterized through FACS and immunofluorescence. Co-culture experiments (using trans-wells) were then performed to measure the effect of region-specificities and numbers of astrocytes on primary midbrain culture under 6-OHDA stress. Cell survival augmented with an increase in astrocyte seeding number and total cell survival was comparable among the different region-specific astrocytes for all numbers. However, striking differences were observed in dopaminergic neuronal (TH) cell survival in the presence of midbrain astrocytes in comparison to forebrain and hindbrain astrocytes at all seeding numbers. At 75μM 6-OHDA insult, while cell survival was comparable in purified astrocytes from the different brain regions, a distinct increase in BDNF secretion (significantly higher than its constitutive release) was noted for midbrain astrocytes compared to forebrain and hindbrain astrocytes. The TH immunopositive population decreased when TrkB inhibitor was added to the co-culture under 6-OHDA toxicity, suggesting that BDNF released by co-cultured astrocytes plays a key role in the survival of dopaminergic neurons. This BDNF release decreased in presence of NO inhibitor and increased in the presence of NO donor (DETA/NO). We conclude that the BDNF released from astrocytes under 6-OHDA toxicity is mediated through NO release through both autocrine and paracrine signaling, and this BDNF release is primarily responsible for the differential effect of region-specific astrocytes on TH neuron survival under these conditions.

Platelet hyperaggregability in obesity: is there a role for nitric oxide impairment and oxidative stress?

Epidemiological evidence has shown that platelet activation markers are consistently elevated in obesity, contributing to its prothrombotic state. In order to improve the understanding of the regulation of platelet function in obesity, the aim of this study was to investigate the l-arginine-nitric oxide (NO) pathway in obese adults without other cardiovascular risk factor. Seventeen obese (body mass index [BMI] 35.9±1.0kg/m(2) ) and eighteen age-matched normal weight subjects (BMI 22.0±0.6kg/m(2) ) were included in this study. l-arginine influx was measured with incubation of l-[(3) H]-arginine. NO synthase (NOS) and arginase activities were determined by the citrulline assay and the conversion of l-[(14) C]-arginine to [(14) C]-urea, respectively. Cyclic guanosine monophosphate (cGMP) content was evaluated by enzyme-linked immunosorbent assay. In addition, the study analyzed: platelet aggregation; intraplatelet antioxidant enzymes, via superoxide dismutase (SOD) and catalase activities; and systemic levels of l-arginine, fibrinogen, and C-reactive protein (CRP). Obese patients presented a significant decrease of platelet l-arginine influx, NOS activity, and cGMP levels, along with platelet hyperaggregability. On the presence of NO donor, platelet aggregation was similar between the groups. The fibrinogen and CRP systemic levels were significantly higher and SOD activity was reduced in obesity. No significant differences were observed in plasma levels of l-arginine and intraplatelet arginase and catalase activities between groups. The diminished NO bioavailability associated with inflammatory status and impaired enzymatic antioxidant defence may contribute to future cardiovascular complications in obesity.

An emerging role for gasotransmitters in the control of breathing and ionic regulation in fish

Three gases comprising nitric oxide, carbon monoxide and hydrogen sulphide, collectively are termed gasotransmitters. The gasotransmitters control several physiological functions in fish by acting as intracellular signaling molecules. Hydrogen sulphide, first implicated in vasomotor control in fish, plays a critical role in oxygen chemoreception owing to its production and downstream effects within the oxygen chemosensory cells, the neuroepithelial cells. Indeed, there is emerging evidence that hydrogen sulphide may contribute to oxygen sensing in both fish and mammals by promoting membrane depolarization of the chemosensory cells. Unlike hydrogen sulphide which stimulates breathing in zebrafish, carbon monoxide inhibits ventilation in goldfish and zebrafish whereas nitric oxide stimulates breathing in zebrafish larvae while inhibiting breathing in adults. Gasotransmitters also modulate ionic uptake in zebrafish. Though nothing is known about the role of CO, reduced activities of branchial Na(+)/K(+)-ATPase and H(+)-ATPase activities in the presence of NO donors suggest an inhibitory role of NO in fish osmoregulation. Hydrogen sulphide inhibits Na(+) uptake in zebrafish larvae and contributes to lowering Na(+) uptake capacity in fish acclimated to Na(+)-enriched water whereas it stimulates Ca(2+) uptake in larvae exposed to Ca(2+)-poor water.

Label-free proteomic analysis to confirm the predicted proteome of Corynebacterium pseudotuberculosis under nitrosative stress mediated by nitric oxide.

BackgroundCorynebacterium pseudotuberculosis biovar ovis is a facultative intracellular pathogen, and the etiological agent of caseous lymphadenitis in small ruminants. During the infection process, the bacterium is subjected to several stress conditions, including nitrosative stress, which is caused by nitric oxide (NO). In silico analysis of the genome of C. pseudotuberculosis ovis 1002 predicted several genes that could influence the resistance of this pathogen to nitrosative stress. Here, we applied high-throughput proteomics using high definition mass spectrometry to characterize the functional genome of C. pseudotuberculosis ovis 1002 in the presence of NO-donor Diethylenetriamine/nitric oxide adduct (DETA/NO), with the aim of identifying proteins involved in nitrosative stress resistance.ResultsWe characterized 835 proteins, representing approximately 41% of the predicted proteome of C. pseudotuberculosis ovis 1002, following exposure to nitrosative stress. In total, 102 proteins were exclusive to the proteome of DETA/NO-induced cells, and a further 58 proteins were differentially regulated between the DETA/NO and control conditions. An interactomic analysis of the differential proteome of C. pseudotuberculosis in response to nitrosative stress was also performed. Our proteomic data set suggested the activation of both a general stress response and a specific nitrosative stress response, as well as changes in proteins involved in cellular metabolism, detoxification, transcriptional regulation, and DNA synthesis and repair.ConclusionsOur proteomic analysis validated previously-determined in silico data for C. pseudotuberculosis ovis 1002. In addition, proteomic screening performed in the presence of NO enabled the identification of a set of factors that can influence the resistance and survival of C. pseudotuberculosis during exposure to nitrosative stress.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1065) contains supplementary material, which is available to authorized users.

Regulation of the functional and mechanical properties of platelet and red blood cells by nitric oxide donors

The effect of NO donors (sodium nitroprusside, S-nitrosoglutathione, dinitrosyl-iron complexes) on the functional and mechanical properties of human platelets and red blood cells has been investigated. It has been established by atomic force microscopy that NO donor-induced platelet disaggregation is accompanied by changes in the elastic properties of cells. It has been shown that, in the presence of NO donors, the detergent-induced hemolysis of red blood cells is delayed, and the elasticity modulus of these cells decreases. The results obtained indicate that NO donors regulate the structural and functional properties of platelets and red blood cells.

Excess no predisposes mitochondrial succinate–cytochrome c reductase to produce hydroxyl radical

Mitochondria-derived oxygen-free radical(s) are important mediators of oxidative cellular injury. It is widely hypothesized that excess NO enhances O 2 •− generated by mitochondria under certain pathological conditions. In the mitochondrial electron transport chain, succinate–cytochrome c reductase (SCR) catalyzes the electron transfer reaction from succinate to cytochrome c. To gain the insights into the molecular mechanism of how NO overproduction may mediate the oxygen-free radical generation by SCR, we employed isolated SCR, cardiac myoblast H9c2, and endothelial cells to study the interaction of NO with SCR in vitro and ex vivo. Under the conditions of enzyme turnover in the presence of NO donor (DEANO), SCR gained pro-oxidant function for generating hydroxyl radical as detected by EPR spin trapping using DEPMPO. The EPR signal associated with DEPMPO/ •OH adduct was nearly completely abolished in the presence of catalase or an iron chelator and partially inhibited by SOD, suggesting the involvement of the iron–H 2O 2-dependent Fenton reaction or O 2 •−-dependent Haber–Weiss mechanism. Direct EPR measurement of SCR at 77 K indicated the formation of a nonheme iron–NO complex, implying that electron leakage to molecular oxygen was enhanced at the FAD cofactor, and that excess NO predisposed SCR to produce •OH. In H9c2 cells, SCR-dependent oxygen-free radical generation was stimulated by NO released from DEANO or produced by the cells following exposure to hypoxia/reoxygenation. With shear exposure that led to overproduction of NO by the endothelium, SCR-mediated oxygen-free radical production was also detected in cultured vascular endothelial cells.

Polymorphisms in MT1a gene coding region are associated with longevity in Italian Central female population

Metallothioneins (MTs) play a pivotal role in zinc-related cell homeostasis because of their high affinity for zinc, which is in turn fundamental for immune response and antioxidant activity. MTs regulate zinc homeostasis by binding zinc and releasing zinc at the occurrence for immune response. The zinc release by MT is very limited in chronic inflammation and ageing. Some polymorphisms of MTs gene, in particular MT1a sub-isoform, may affect this release that is a problem still unresolved in ageing. The screening in the present paper of two polymorphisms in MT1a gene has revealed for the first time that the polymorphism corresponding to a A/C (Asp/Thr) transition at 647 nt position in the Mt1a coding region is the more involved in the longevity, at least in old women, rather than the other corresponding to A/G (Lys/Arg) transition at 1,245 nt position. Concomitantly, for the +647 MT1a polymorphism, old and very old female with Asp/Asp genotype (called C-carriers) display higher zinc release by MT (detected by Zinpyr-1 fluorescent probe in presence of NO donor), low MT levels and reduced IL-6 plasma concentrations, suggesting its involvement in longevity and in lower inflammatory status. This fact is confirmed by the analysis of haplotypes in which the allele Asp is more involved in longevity. Therefore the +647 MT1a polymorphism more affects MTs induction and zinc release, which are indispensable to undertake the inflammatory status under control and subsequently to reach healthy longevity.

Nitric oxide protects against oxidative stress under heat stress in the calluses from two ecotypes of reed

Calluses from two ecotypes of reed (Phragmites communis Trin.) plant (dune reed [DR] and swamp reed [SR]), which show different sensitivity to heat stress, were used to study plant acclimations to heat stress. SR callus suffered more oxidative damage than DR callus, and DR callus maintained higher relative growth rate and cell viability and lower ion leakage than SR callus under heat stress. Application of two nitric oxide donors, sodium nitroprusside (SNP) and S-nitroso-N-acetylpenicillamine (SNAP), dramatically alleviated heat stress induced ion leakage increase, growth suppression and cell viability decrease in both calluses under heat stress. H2O2 and MDA contents were decreased and the activities of superoxide dismutase, catalase, ascorbate peroxidase and peroxidase increased in both calluses in the presence of NO donors under heat stress. The potassium salt of 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (cPTIO), a specific NO scavenger, arrested NO donors mediated protective effects. Moreover, measurement of the rate of NO release showed that NO production increased significantly in DR callus while kept stable in SR callus under heat stress. Pretreatment with cPTIO had no effect on SR callus under heat stress while it significantly enhanced oxidative stress in DR callus compared with that of heat stress alone. These results suggest that NO can effectively protect both calluses from oxidative stress induced by heat stress and that NO might act as a signal in activating active oxygen scavenging enzymes under heat stress and thus confer DR callus thermotolerance.

Nitric Oxide Inhibits Peroxidase Activity of Cytochrome c· Cardiolipin Complex and Blocks Cardiolipin Oxidation

The increased production of NO during the early stages of apoptosis indicates its potential involvement in the regulation of programmed cell death through yet to be identified mechanisms. Recently, an important role for catalytically competent peroxidase form of pentacoordinate cytochrome c (cyt c) in a complex with a mitochondria-specific phospholipid, cardiolipin (CL), has been demonstrated during execution of the apoptotic program. Because the cyt c.CL complex acts as CL oxygenase and selectively oxidizes CL in apoptotic cells in a reaction dependent on the generation of protein-derived (tyrosyl) radicals, we hypothesized that binding and nitrosylation of cyt c regulates CL oxidation. Here we demonstrate by low temperature electron paramagnetic resonance spectroscopy that CL facilitated interactions of ferro- and ferri-states of cyt c with NO and NO(-), respectively, to yield a mixture of penta- and hexa-coordinate nitrosylated cyt c. In the nitrosylated cyt c.CL complex, NO chemically reacted with H(2)O(2)-activated peroxidase intermediates resulting in their reduction. A dose-dependent quenching of H(2)O(2)-induced protein-derived radicals by NO donors was shown using direct electron paramagnetic resonance measurements as well as immuno-spin trapping with antibodies against protein 5,5-dimethyl-1-pyrroline N-oxide-nitrone adducts. In the presence of NO donors, H(2)O(2)-induced oligomeric forms of cyt c positively stained for 3-nitrotyrosine confirming the reactivity of NO toward tyrosyl radicals of cyt c. Interaction of NO with the cyt c.CL complex inhibited its peroxidase activity with three different substrates: CL, etoposide, and 3,3'-diaminobenzidine. Given the importance of CL oxidation in apoptosis, mass spectrometry analysis was utilized to assess the effects of NO on oxidation of 1,1'2,2'-tertalinoleoyl cardiolipin. NO effectively inhibited 1,1'2,2'-tertalinoleoyl cardiolipin oxidation catalyzed by the peroxidase activity of cyt c. Thus, NO can act as a regulator of peroxidase activity of cyt c.CL complexes.

Truncated hemoglobin GlbO from Mycobacterium leprae alleviates nitric oxide toxicity

As a consequence of reductive genome evolution, the obligate intracellular pathogen Mycobacterium leprae has minimized the repertoire of genes implicated in defense against reactive oxygen and nitrogen species. Genes for multiple hemoglobin types coexist in mycobacterial genomes, but M. leprae has retained only glbO, encoding a group-II truncated hemoglobin. Mycobacterium tuberculosis GlbO has been involved in oxygen transfer and respiration during hypoxia, but a role in protection from nitric oxide (NO) has not been documented yet. Here, we report that the in vitro reaction of oxygenated recombinant M. leprae GlbO with NO results in an immediate stoichiometric formation of nitrate, concomitant with heme-protein oxidation. Overexpression of GlbO alleviates the growth inhibition of Escherichia coli hmp (flavohemoglobin gene) mutants in the presence of NO-donors, partly complementing the defect in Hmp synthesis. A promoter element upstream of glbO was predicted in silico, and confirmed by using a glbO::lacZ transcriptional fusion in the heterologous Mycobacterium smegmatis system. The glbO::lacZ fusion was expressed through the whole growth cycle of M. smegmatis, and moderately induced by NO. We propose that M. leprae, by retaining the unique truncated hemoglobin GlbO, may have coupled O 2 delivery to the terminal oxidase with a defensive mechanism to scavenge NO from respiratory enzymes. These activities would help to sustain the obligate aerobic metabolism required for intracellular survival of leprosy bacilli.

Nitric Oxide–Induced Accumulation of Lipofuscin-Like Materials Is Caused by Inhibition of Cathepsin S

To determine whether nitric oxide (NO) is involved in accumulation of lipofuscin-like material (LFM) in retinal pigment epithelial (RPE) cells and if this formation is related to NO-mediated modification of cathepsin S (cat S). RPE cell cultures were fed once every day with porcine photoreceptor outer segments (POS) in the presence of NO-donor [S-nitroso-N-acetylpenicillamine (SNAP) or NOC18] for 2 weeks. LFM autofluorescence within the cells was measured by fluorophotometric flow cytometry (FACS). The activity of purified cat S was measured in the presence of NO-donor with or without dithiothreitol (DTT). The following results were observed. SNAP and NOC18 caused LFM accumulation in RPE cells in a dose-dependent manner, and this accumulation was reversed by the addition of NO-scavengers (hydroxycobalamin, carboxy-PTIO). Purified cat S activities were inhibited by NO-donors without DTT, but in the presence of DTT, NO-donors exhibited no inhibitory effect on its activity. Phagocytic challenge of RPE cells increased cat S activity, which was reduced by the addition of NO donors. These results indicated that cat S activity was inhibited by NO-donors and resulted in LFM accumulation in RPE cells. We conclude that NO-mediated inhibition of cat S was caused through protein modification of cat S and resulted in LFM accumulation.

Nitric Oxide Acts as an Antioxidant and Delays Programmed Cell Death in Barley Aleurone Layers

Nitric oxide (NO) is a freely diffusible, gaseous free radical and an important signaling molecule in animals. In plants, NO influences aspects of growth and development, and can affect plant responses to stress. In some cases, the effects of NO are the result of its interaction with reactive oxygen species (ROS). These interactions can be cytotoxic or protective. Because gibberellin (GA)-induced programmed cell death (PCD) in barley (Hordeum vulgare cv Himalaya) aleurone layers is mediated by ROS, we examined the effects of NO donors on PCD and ROS-metabolizing enzymes in this system. NO donors delay PCD in layers treated with GA, but do not inhibit metabolism in general, or the GA-induced synthesis and secretion of alpha-amylase. alpha-Amylase secretion is stimulated slightly by NO donors. The effects of NO donors are specific for NO, because they can be blocked completely by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide. The antioxidant butylated hydroxy toluene also slowed PCD, and these data support our hypothesis that NO is a protective antioxidant in aleurone cells. The amounts of CAT and SOD, two enzymes that metabolize ROS, are greatly reduced in aleurone layers treated with GA. Treatment with GA in the presence of NO donors delays the loss of CAT and SOD. We speculate that NO may be an endogenous modulator of PCD in barley aleurone cells.

Gossypol inhibits follicle-stimulating hormone- and epidermal growth factor-stimulated expansion of oocyte-cumulus complexes from porcine preovulatory follicles

The role of gossypol in the cumulus expansion of oocyte-cumulus complexes (OCC) isolated from large antral porcine follicles was investigated. Marked suppression of cumulus expansion stimulated with follicle-stimulating hormone (FSH) and epidermal growth factor (EGF) was observed in the presence of different concentrations of gossypol. Comparable inhibitory effects were obtained in the presence of NO donor, S-nitroso-N-acetylpenicillamine or sodium nitroprusside, suggesting that the inhibitory effect of gossypol may be mediated via NO generation. The inhibitory effect of gossypol on cumulus expansion of OCC was accompanied by inhibition of progesterone secretion of OCC and the decrease of [125I]EGF binding to granulosa cells.

Detection of thiol modification following generation of reactive nitrogen species: analysis of synaptic vesicle proteins

S-nitrosylation is an important means of regulating the activity of proteins. We have developed a method which allows unbiased identification of thiol modified proteins within a complex mixture following NO generation, by taking advantage of the fact that prior nitrosylation will block subsequent modification of cysteine residues with 1-biotinamido-4-[4′-(maleimidomethyl)-cyclohexane-carboxamido] butane (biotin-BMCC). Thiol modified proteins are reduced in intensity when revealed by blotting and overlay with avidin–horseradish peroxidase. In the case of a purified synaptic vesicle fraction we observe a high degree of enrichment of specific biotinylated proteins relative to homogenate. We find that thiol modification of proteins in the presence of NO donors is widespread, occurring in the majority of proteins that will react with biotin-BMCC. In a further development of this technique we have depleted the biotinylated proteins from solubilised synaptic vesicles using avidin–agarose and analysed the supernatants with a panel of antibodies. This has allowed us to identify SNARE proteins (soluble NSF attachment protein receptors) as potential targets for S-nitrosylation.

PEROXYNITRITE INACTIVATES PROSTACYCLIN SYNTHASE BY HEME–THIOLATE-CATALYZED TYROSINE NITRATION

Previous work has shown a sensitive inhibition of prostacyclin synthase activity by peroxynitrite as well as by superoxide in the presence of NO donors. Neither superoxide nor NO alone nor decomposed peroxynitrite is effective. The inhibition of activity was paralleled by a nitration of a tyrosine residue and both could be prevented by a stable substrate analog. The same IC50 value for peroxynitrite was also found for the cellular prostacyclin activity in endothelial and kidney mesangial cells, indicating that the antioxidant potential of the cell cannot prevent the inactivation. Aortic tissue shows a co-localization of prostacyclin synthase and nitrotyrosine staining after treatment of the tissue with 1 μM peroxynitrite. It can be speculated that this pathway of enzyme nitration is of pathophysiological significance.

Nitric Oxide Exposure Inhibits Endothelial NOS Activity But Not Gene Expression: A Role For Superoxide And Peroxynitrite • 1937

Recent studies have characterized a rebound pulmonary vasoconstriction with abrupt withdrawal of inhaled nitric oxide (NO) during therapy for pulmonary hypertension. Although it has been suggested that inhaled NO may down-regulate basal NO production, the exact mechanism of this rebound pulmonary hypertension remains unclear. The objectives of these studies were to determine the effect of NO exposure on endothelial NOS (eNOS) gene expression and enzyme activity. The addition of NO donors had no effect on eNOS mRNA or protein levels, in cultured pulmonary arterial endothelial cells, but did produce a significant decrease in enzyme activity. Although NO treatment induced protein kinase C (PKC) dependent eNOS phosphorylation, blockade of PKC activity did not protect against the effects of NO. Superoxide levels were elevated in the presence of NO-donors and when the xanthine oxidase inhibitor, allopurinol, or the superoxide scavenger, 4,5-dihydroxy-1-benzene-disulphonic acid, were co-incubated with NO donors, the inhibitory effects on eNOS activity could be partially alleviated. This suggests that NO stimulates xanthine oxidase causing an increase in cellular superoxide generation. It is known that the reaction between NO and superoxide produces peroxynitrite which can cause the nitrosylation of proteins and subsequent inactivation. To further evaluate the potential effect of peroxynitrite on eNOS activity we purified eNOS protein using a yeast expression system. The presence of peroxynitrite was found to significantly reduce the activity of the purified eNOS protein. These data suggest that peroxynitrite reacts with the eNOS protein resulting in enzyme inactivation. This may explain, at least in part, how NO causes NOS inhibition in vivo, and may delineate the mechanism of rebound pulmonary hypertension following withdrawal of inhaled NO.