The decomposition of peroxynitrite does not yield nitroxyl anion and singlet oxygen

In a recent article [Khan, A. U., Kovacic, D., Kolbanovsky, A., Desai, M., Frenkel, K. & Geacintov, N. E. (2000) Proc. Natl. Acad. Sci. USA 97, 2984–2989], the authors claimed that ONOO−, after protonation to ONOOH, decomposes into 1HNO and 1O2 according to a spin-conserved unimolecular mechanism. This claim was based partially on their observation that nitrosylhemoglobin is formed via the reaction of peroxynitrite with methemoglobin at neutral pH. However, thermochemical considerations show that the yields of 1O2 and 1HNO are about 23 orders of magnitude lower than those of ⋅NO2 and ⋅OH, which are formed via the homolysis of ONOOH. We also show that methemoglobin does not form with peroxynitrite any spectrally detectable product, but with contaminations of nitrite and H2O2 present in the peroxynitrite sample. Thus, there is no need to modify the present view of the mechanism of ONOOH decomposition, according to which initial homolysis into a radical pair, [ONO⋅ ⋅OH]cage, is followed by the diffusion of about 30% of the radicals out of the cage, while the rest recombines to nitric acid in the solvent cage.

Inhibition of gastric acid secretion by stress: A protective reflex mediated by cerebral nitric oxide

Moderate somatic stress inhibits gastric acid secretion. We have investigated the role of endogenously released NO in this phenomenon. Elevation of body temperature by 3°C or a reduction of 35 mmHg (1 mmHg = 133 Pa) in blood pressure for 10 min produced a rapid and long-lasting reduction of distension-stimulated acid secretion in the rat perfused stomach in vivo. A similar inhibitory effect on acid secretion was produced by the intracisternal (i.c.) administration of oxytocin, a peptide known to be released during stress. Intracisternal administration of the NO-synthase inhibitor, NG-nitro-l-arginine methyl ester (l-NAME) reversed the antisecretory effect induced by all these stimuli, an action prevented by intracisternal coadministration of the NO precursor, l-arginine. Furthermore, microinjection of l-NAME into the dorsal motor nucleus of the vagus nerve reversed the acid inhibitory effects of mild hyperthermia, i.v. endotoxin, or i.c. oxytocin, an action prevented by prior microinjection of l-arginine. By contrast, microinjection of l-NAME into the nucleus tractus solitarius failed to affect the inhibitory effects of hyperthermia, i.v. endotoxin, or i.c. oxytocin. Immunohistochemical techniques demonstrated that following hyperthermia there was a significant increase in immunoreactivity to neuronal NO synthase in different areas of the brain, including the dorsal motor nucleus of the vagus. Thus, our results suggest that the inhibition of gastric acid secretion, a defense mechanism during stress, is mediated by a nervous reflex involving a neuronal pathway that includes NO synthesis in the brain, specifically in the dorsal motor nucleus of the vagus.

Ascorbic acid acts as an inhibitory transmitter in the hypothalamus to inhibit stimulated luteinizing hormone-releasing hormone release by scavenging nitric oxide

Because ascorbic acid (AA) is concentrated in synaptic vesicles containing glutamic acid, we hypothesized that AA might act as a neurotransmitter. Because AA is an antioxidant, it might therefore inhibit nitric oxidergic (NOergic) activation of luteinizing hormone-releasing hormone (LH-RH) release from medial basal hypothalamic explants by chemically reducing NO. Cell membrane depolarization induced by increased potassium concentration [K+] increased medium concentrations of both AA and LH-RH. An inhibitor of NO synthase (NOS), NG-monomethyl-l-arginine (NMMA), prevented the increase in medium concentrations of AA and LH-RH induced by high [K+], suggesting that NO mediates release of both AA and LH-RH. Calcium-free medium blocked not only the increase in AA in the medium but also the release of LH-RH. Sodium nitroprusside, which releases NO, stimulated LH-RH release and decreased the concentration of AA in the incubation medium, presumably because the NO released oxidized AA to dehydro-AA. AA (10−5 to 10−3 M) had no effect on basal LH-RH release but completely blocked high [K+]- and nitroprusside-induced LH-RH release. N-Methyl-d-aspartic acid (NMDA), which mimics the action of the excitatory amino acid neurotransmitter glutamic acid, releases LH-RH by releasing NO. AA (10−5 to 10−3 M) inhibited the LH-RH-releasing action of NMDA. AA may be an inhibitory neurotransmitter that blocks NOergic stimulation of LH-RH release by chemically reducing the NO released by the NOergic neurons.

Nitric oxide and salicylic acid signaling in plant defense

Salicylic acid (SA) plays a critical signaling role in the activation of plant defense responses after pathogen attack. We have identified several potential components of the SA signaling pathway, including (i) the H2O2-scavenging enzymes catalase and ascorbate peroxidase, (ii) a high affinity SA-binding protein (SABP2), (iii) a SA-inducible protein kinase (SIPK), (iv) NPR1, an ankyrin repeat-containing protein that exhibits limited homology to IκBα and is required for SA signaling, and (v) members of the TGA/OBF family of bZIP transcription factors. These bZIP factors physically interact with NPR1 and bind the SA-responsive element in promoters of several defense genes, such as the pathogenesis-related 1 gene (PR-1). Recent studies have demonstrated that nitric oxide (NO) is another signal that activates defense responses after pathogen attack. NO has been shown to play a critical role in the activation of innate immune and inflammatory responses in animals. Increases in NO synthase (NOS)-like activity occurred in resistant but not susceptible tobacco after infection with tobacco mosaic virus. Here we demonstrate that this increase in activity participates in PR-1 gene induction. Two signaling molecules, cGMP and cyclic ADP ribose (cADPR), which function downstream of NO in animals, also appear to mediate plant defense gene activation (e.g., PR-1). Additionally, NO may activate PR-1 expression via an NO-dependent, cADPR-independent pathway. Several targets of NO in animals, including guanylate cyclase, aconitase, and mitogen-activated protein kinases (e.g., SIPK), are also modulated by NO in plants. Thus, at least portions of NO signaling pathways appear to be shared between plants and animals.

Inducible nitric oxide synthase: identification of amino acid residues essential for dimerization and binding of tetrahydrobiopterin.

Nitric oxide synthases (NOSs) require tetrahydrobiopterin (BH4) for dimerization and NO production. Mutation analysis of mouse inducible NOS (iNOS; NOS2) identified Gly-450 and Ala-453 as critical for NO production, dimer formation, and BH4 binding. Substitutions at five neighboring positions were tolerated, and normal binding of heme, calmodulin, and NADPH militated against major distortions affecting the NH2-terminal portion, midzone, or COOH terminus of the inactive mutants. Direct involvement of residues 450 and 453 in the binding of BH4 is supported by the striking homology of residues 448-480 to a region extensively shared by the three BH4-utilizing aromatic amino acid hydroxylases and is consistent with the conservation of these residues among all 10 reported NOS sequences, including mammalian NOSs 1, 2, and 3, as well as avian and insect NOSs. Altered binding of BH4 and/or L-arginine may explain how the addition of a single methyl group to the side chain of residue 450 or the addition of three methylenes to residue 453 can each abolish an enzymatic activity that reflects the concerted function of 1143 other residues.

Nitric oxide inhibits hypothalamic luteinizing hormone-releasing hormone release by releasing gamma-aminobutyric acid.

Nitric oxide synthase (NOS)-containing neurons, termed NOergic neurons, occur in various regions of the hypothalamus, including the median eminence-arcuate region, which plays an important role in controlling the release of luteinzing hormone-releasing hormone (LHRH). We examined the effect of NO on release of gamma-aminobutyric acid (GABA) from medial basal hypothalamic (MBH) explants incubated in vitro. Sodium nitroprusside (NP) (300 microM), a spontaneous releaser of NO, doubled the release of GABA. This release was significantly reduced by incubation of the tissue with hemoglobin, a scavenger of NO, whereas hemoglobin alone had no effect on the basal release of GABA. Elevation of the potassium concentration (40 mM) in the medium increased GABA release 15-fold; this release was further augmented by NP. Hemoglobin blocked the increase in GABA release induced by NP but had no effect on potassium-induced release, suggesting that the latter is not related to NO. As in the case of hemoglobin, NG-monomethyl-L-arginine (NMMA), a competitive inhibitor of NOS, had no effect on basal release of GABA, which indicates again that NO is not significant to basal GABA release. However, NMMA markedly inhibited the release of GABA induced by high potassium, which indicates that NO plays a role in potassium-induced release of GABA. In conditions in which the release of GABA was substantially augmented, there was a reduction in GABA tissue stores as well, suggesting that synthesis of GABA in these conditions did not keep up with release of the amine. Although NO released GABA, there was no effect of the released GABA on NO production, for incubation of MBH explants with GABA had no effect on NO release as measured by [14C]citrulline production. To determine whether GABA had any effect on the release of LHRH from these MBH explants, GABA was incubated with the tissue and the effect on LHRH release was determined. GABA (10(-5) or 10(-6) M) induced a 70% decrease in the release of LHRH, indicating that in the male rat GABA inhibits the release of this hypothalamic peptide. This inhibition in LHRH release induced by GABA was blocked by NMMA (300 microM), which indicates that GABA converts the stimulatory effect of NO on LHRH release into an inhibitory one, presumably via GABA receptors, which activate chloride channels that hyperpolarize the cell. Previous results have indicated that norepinephrine stimulates release of NO from the NOergic neurons, which then stimulates the release of LHRH. The current results indicate that the NO released also induces release of GABA, which then inhibits further LHRH release. Thus, in vivo the norepinephrinergic-driven pulses of LHRH release may be terminated by GABA released from GABAergic neurons via NO.

Calcium-independent activation of endothelial nitric oxide synthase by ceramide

The endothelial isoform of NO synthase (eNOS) is targeted to sphingolipid-enriched signal-transducing microdomains in the plasma membrane termed caveolae. Among the caveolae-targeted sphingolipids are the ceramides, a class of acylated sphingosine compounds that have been implicated in diverse cellular responses. We have explored the role of ceramide analogues in eNOS signaling in cultured bovine aortic endothelial cells (BAEC). Addition of the ceramide analogue N-acetylsphingosine (C2-ceramide; 5 μM) to intact BAEC leads to a significant increase in NO synthase activity (assayed by using the fluorescent indicator 4,5-diaminofluorescein) and translocation of eNOS from the endothelial cell membrane to intracellular sites (measured by using quantitative immunofluorescence techniques); the biologically inactive ceramide N-acetyldihydrosphingosine is entirely without effect. C2-ceramide-induced eNOS activation and translocation are unaffected by the intracellular calcium chelator 1,2-bis-o-aminophenoxyethane-N,N,N′,N′-tetraacetic acid (BAPTA). Using the calcium-specific fluorescent indicator fluo-3, we also found that C2-ceramide activation of eNOS is unaccompanied by a drug-induced increase in intracellular calcium. These findings stand in sharp contrast to the mechanism by which bradykinin, estradiol, and other mediators acutely activate eNOS, in which a rapid, agonist-promoted increase in intracellular calcium is required. Finally, we show that treatment of BAEC with bradykinin causes a significant increase in cellular ceramide content; the response to bradykinin has an EC50 of 3 nM and is blocked by the bradykinin B2-receptor antagonist HOE140. Bradykinin-induced ceramide generation could represent a mechanism for longer-term regulation of eNOS activity. Our results suggest that ceramide functions independently of Ca2+-regulated pathways to promote activation and translocation of eNOS, and that this lipid mediator may represent a physiological regulator of eNOS in vascular endothelial cells.

Peroxynitrite, the coupling product of nitric oxide and superoxide, activates prostaglandin biosynthesis

Peroxynitrite activates the cyclooxygenase activities of constitutive and inducible prostaglandin endoperoxide synthases by serving as a substrate for the enzymes’ peroxidase activities. Activation of purified enzyme is induced by direct addition of peroxynitrite or by in situ generation of peroxynitrite from NO coupling to superoxide anion. Cu,Zn-superoxide dismutase completely inhibits cyclooxygenase activation in systems where peroxynitrite is generated in situ from superoxide. In the murine macrophage cell line RAW264.7, the lipophilic superoxide dismutase-mimetic agents, Cu(II) (3,5-diisopropylsalicylic acid)2, and Mn(III) tetrakis(1-methyl-4-pyridyl)porphyrin dose-dependently decrease the synthesis of prostaglandins without affecting the levels of NO synthase or prostaglandin endoperoxide synthase or by inhibiting the release of arachidonic acid. These findings support the hypothesis that peroxynitrite is an important modulator of cyclooxygenase activity in inflammatory cells and establish that superoxide anion serves as a biochemical link between NO and prostaglandin biosynthesis.

Microtubule dysfunction by posttranslational nitrotyrosination of α-tubulin: A nitric oxide-dependent mechanism of cellular injury

NO2Tyr (3-Nitrotyrosine) is a modified amino acid that is formed by nitric oxide-derived species and has been implicated in the pathology of diverse human diseases. Nitration of active-site tyrosine residues is known to compromise protein structure and function. Although free NO2Tyr is produced in abundant concentrations under pathological conditions, its capacity to alter protein structure and function at the translational or posttranslational level is unknown. Here, we report that free NO2Tyr is transported into mammalian cells and selectively incorporated into the extreme carboxyl terminus of α-tubulin via a posttranslational mechanism catalyzed by the enzyme tubulin–tyrosine ligase. In contrast to the enzymatically regulated carboxyl-terminal tyrosination/detyrosination cycle of α-tubulin, incorporation of NO2Tyr shows apparent irreversibility. Nitrotyrosination of α-tubulin induces alterations in cell morphology, changes in microtubule organization, loss of epithelial-barrier function, and intracellular redistribution of the motor protein cytoplasmic dynein. These observations imply that posttranslational nitrotyrosination of α-tubulin invokes conformational changes, either directly or via allosteric interactions, in the surface-exposed carboxyl terminus of α-tubulin that compromises the function of this critical domain in regulating microtubule organization and binding of motor- and microtubule-associated proteins. Collectively, these observations illustrate a mechanism whereby free NO2Tyr can impact deleteriously on cell function under pathological conditions encompassing reactive nitrogen species production. The data also yield further insight into the role that the α-tubulin tyrosination/detyrosination cycle plays in microtubule function.

Nerve growth factor rapidly suppresses basal, NMDA-evoked, and AMPA-evoked nitric oxide synthase activity in rat hippocampus in vivo

In adult forebrain, nerve growth factor (NGF) influences neuronal maintenance and axon sprouting and is neuroprotective in several injury models through mechanisms that are incompletely understood. Most NGF signaling is thought to occur after internalization and retrograde transport of trkA receptor and be mediated through the nucleus. However, NGF expression in hippocampus is rapidly and sensitively regulated by synaptic activity, suggesting that NGF exerts local effects more dynamically than possible through signaling requiring retrograde transport to distant afferent neurons. Interactions have been reported between NGF and nitric oxide (NO). Because NO affects both neural plasticity and degeneration, and trk receptors can mediate signaling within minutes, we hypothesized that NGF might rapidly modulate NO production. Using in vivo microdialysis we measured conversion of l-[14C]arginine to l-[14C]citrulline as an accurate reflection of NO synthase (NOS) activity in adult rat hippocampus. NGF significantly reduced NOS activity to 61% of basal levels within 20 min of onset of delivery and maintained NOS activity at less than 50% of baseline throughout 3 hr of delivery. This effect did not occur with control protein (cytochrome c) and was not mediated by an effect of NGF on glutamate levels. In addition, simultaneous delivery of NGF prevented significant increases in NOS activity triggered by the glutamate receptor agonists N-methyl-d-aspartate (NMDA) and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA). Rapid suppression by NGF of basal and glutamate-stimulated NOS activity may regulate neuromodulatory functions of NO or protect neurons from NO toxicity and suggests a novel mechanism for rapidly mediating functions of NGF and other neurotrophins.

An NO derivative of ursodeoxycholic acid protects against Fas-mediated liver injury by inhibiting caspase activity

Caspases are key mediators in liver inflammation and apoptosis. In the present study we provide evidence that a nitric oxide (NO) derivative of ursodeoxycholic acid (UDCA), NCX-1000 ([2-(acetyloxy)benzoic acid 3-(nitrooxymethyl)phenyl ester]), protects against liver damage in murine models of autoimmune hepatitis induced by i.v. injection of Con A or a Fas agonistic antibody, Jo2. Con A administration causes CD4+ T lymphocytes to accumulate in the liver and up-regulates FasL expression, resulting in FasL-mediated cytotoxicity. Cotreating mice with NCX-1000, but not with UDCA, protected against liver damage induced by Con A and Jo2, inhibited IL-1β, IL-18, and IFN-γ release and caspase 3, 8, and 9 activation. Studies on HepG2 cells demonstrated that NCX-1000, but not UDCA, directly prevented multiple caspase activation induced by Jo2. Incubating HepG2 cells with NCX-1000 resulted in intracellular NO formation and a DTT-reversible inhibition of proapoptotic caspases, suggesting that cysteine S-nitrosylation was the main mechanism responsible for caspase inhibition. Collectively, these data suggest that NCX-1000 protects against T helper 1-mediated liver injury by inhibiting both the proapoptotic and the proinflammatory branches of the caspase superfamily.

l-Arginine-dependent suppression of apoptosis in Trypanosoma cruzi: Contribution of the nitric oxide and polyamine pathways

Until recently, a capacity for apoptosis and synthesis of nitric oxide (⋅NO) were viewed as exclusive to multicellular organisms. The existence of these processes in unicellular parasites was recently described, with their biological significance remaining to be elucidated. We have evaluated l-arginine metabolism in Trypanosoma cruzi in the context of human serum-induced apoptotic death. Apoptosis was evidenced by the induction of DNA fragmentation and the inhibition of [3H]thymidine incorporation, which were inhibited by the caspase inhibitor Ac-Asp-Glu-Val-aspartic acid aldehyde (DEVD-CHO). In T. cruzi exposed to death stimuli, supplementation with l-arginine inhibited DNA fragmentation, restored [3H]thymidine incorporation, and augmented parasite ⋅NO production. These effects were inhibited by the ⋅NO synthase inhibitor Nω-nitroarginine methyl ester (l-NAME). Exogenous ⋅NO limited DNA fragmentation but did not restore proliferation rates. Because l-arginine is also a substrate for arginine decarboxylase (ADC), and its product agmatine is a precursor for polyamine synthesis, we evaluated the contribution of polyamines to limiting apoptosis. Addition of agmatine, putrescine, and the polyamines spermine and spermidine to T. cruzi sustained parasite proliferation and inhibited DNA fragmentation. Also, the ADC inhibitor difluoromethylarginine inhibited l-arginine-dependent restoration of parasite replication rates, while the protection from DNA fragmentation persisted. In aggregate, these results indicate that T. cruzi epimastigotes can undergo programmed cell death that can be inhibited by l-arginine by means of (i) a ⋅NO synthase-dependent ⋅NO production that suppresses apoptosis and (ii) an ADC-dependent production of polyamines that support parasite proliferation.

Catalytic consumption of nitric oxide by 12/15- lipoxygenase: Inhibition of monocyte soluble guanylate cyclase activation

12/15-Lipoxygenase (LOX) activity is elevated in vascular diseases associated with impaired nitric oxide (⋅NO) bioactivity, such as hypertension and atherosclerosis. In this study, primary porcine monocytes expressing 12/15-LOX, rat A10 smooth muscle cells transfected with murine 12/15-LOX, and purified porcine 12/15-LOX all consumed ⋅NO in the presence of lipid substrate. Suppression of LOX diene conjugation by ⋅NO was also found, although the lipid product profile was unchanged. ⋅NO consumption by porcine monocytes was inhibited by the LOX inhibitor, eicosatetraynoic acid. Rates of arachidonate (AA)- or linoleate (LA)-dependent ⋅NO depletion by porcine monocytes (2.68 ± 0.03 nmol ⋅ min−1 ⋅ 106 cells−1 and 1.5 ± 0.25 nmol ⋅ min−1 ⋅ 106 cells−1, respectively) were several-fold greater than rates of ⋅NO generation by cytokine-activated macrophages (0.1–0.2 nmol ⋅ min−1 ⋅ 106 cells−1) and LA-dependent ⋅NO consumption by primary porcine monocytes inhibited ⋅NO activation of soluble guanylate cyclase. These data indicate that catalytic ⋅NO consumption by 12/15-LOX modulates monocyte ⋅NO signaling and suggest that LOXs may contribute to vascular dysfunction not only by the bioactivity of their lipid products, but also by serving as catalytic sinks for ⋅NO in the vasculature.

NCX-1000, a NO-releasing derivative of ursodeoxycholic acid, selectively delivers NO to the liver and protects against development of portal hypertension

Portal hypertension resulting from increased intrahepatic resistance is a common complication of chronic liver diseases and a leading cause of death in patients with liver cirrhosis, a scarring process of the liver that includes components of both increased fibrogenesis and wound contraction. A reduced production of nitric oxide (NO) resulting from an impaired enzymatic function of endothelial NO synthase and an increased contraction of hepatic stellate cells (HSCs) have been demonstrated to contribute to high intrahepatic resistance in the cirrhotic liver. 2-(Acetyloxy) benzoic acid 3-(nitrooxymethyl) phenyl ester (NCX-1000) is a chemical entity obtained by adding an NO-releasing moiety to ursodeoxycholic acid (UDCA), a compound that is selectively metabolized by hepatocytes. In this study we have examined the effect of NCX-1000 and UDCA on liver fibrosis and portal hypertension induced by i.p. injection of carbon tetrachloride in rats. Our results demonstrated that although both treatments reduced liver collagen deposition, NCX-1000, but not UDCA, prevented ascite formation and reduced intrahepatic resistance in carbon tetrachloride-treated rats as measured by assessing portal perfusion pressure. In contrast to UDCA, NCX-1000 inhibited HSC contraction and exerted a relaxing effect similar to the NO donor S-nitroso-N-acetylpenicillamine. HSCs were able to metabolize NCX-1000 and release nitrite/nitrate in cell supernatants. In aggregate these data indicate that NCX-1000, releasing NO into the liver microcirculation, may provide a novel therapy for the treatment of patients with portal hypertension.

Whole body nitric oxide synthesis in healthy men determined from [15N] arginine-to-[15N]citrulline labeling.

The rates of whole body nitric oxide (NO) synthesis, plasma arginine flux, and de novo arginine synthesis and their relationships to urea production, were examined in a total of seven healthy adults receiving an L-amino acid diet for 6 days. NO synthesis was estimated by the rate of conversion of the [15N] guanidino nitrogen of arginine to plasma [15N] ureido citrulline and compared with that based on urinary nitrite (NO2-)/nitrate (NO3-) excretion. Six subjects received on dietary day 7, a 24-hr (12-hr fed/12-hr fasted) primed, constant, intravenous infusion of L-[guanidino-15N2]arginine and [13C]urea. A similar investigation was repeated with three of these subjects, plus an additional subject, in which they received L-[ureido-13C]citrulline, to determine plasma citrulline fluxes. The estimated rates (mean +/- SD) of NO synthesis over a period of 24 hr averaged 0.96 +/- 0.1 mumol .kg-1.hr-1 and 0.95 +/- 0.1 mumol.kg-1.hr-1, for the [15N]citrulline and the nitrite/nitrate methods, respectively. About 15% of the plasma arginine turnover was associated with urea formation and 1.2% with NO formation. De novo arginine synthesis averaged 9.2 +/- 1.4 mumol. kg-1.hr-1, indicating that approximately 11% of the plasma arginine flux originates via conversion of plasma citrulline to arginine. Thus, the fraction of the plasma arginine flux associated with NO and also urea synthesis in healthy humans is small, although the plasma arginine compartment serves as a significant precursor pool (54%) for whole body NO formation. This tracer model should be useful for exploring these metabolic relationships in vivo, under specific pathophysiologic states where the L-arginine-NO pathway might be altered.