Nitric oxide plays a key role in adaptive control of locomotion in cat

Diverse roles in cellular functions have been ascribed to nitric oxide (NO), and its involvement in induction of long-term depression in cerebellar Purkinje cells has been demonstrated. Manipulations of NO concentration or its synthesis in cerebellar tissues therefore provide a means for investigating roles of NO in cerebellar functions at both cellular and behavioral levels. We tested adaptive control of locomotion to perturbation in cats, and found that this form of motor learning was abolished by application of either an inhibitor of NO synthase or a scavenger of NO to the cerebellar cortical locomotion area. This finding supports the view that NO in the cerebellum plays a key role in motor learning.

Inhibition of NF-κB DNA binding and nitric oxide induction in human T cells and lung adenocarcinoma cells by selenite treatment

NF-κB is a major transcription factor consisting of 50(p50)- and 65(p65)-kDa proteins that controls the expression of various genes, among which are those encoding cytokines, cell adhesion molecules, and inducible NO synthase (iNOS). After initial activation of NF-κB, which involves release and proteolysis of a bound inhibitor, essential cysteine residues are maintained in the active reduced state through the action of thioredoxin and thioredoxin reductase. In the present study, activation of NF-κB in human T cells and lung adenocarcinoma cells was induced by recombinant human tumor necrosis factor α or bacterial lipopolysaccharide. After lipopolysaccharide activation, nuclear extracts were treated with increasing concentrations of selenite, and the effects on DNA-binding activity of NF-κB were examined. Binding of NF-κB to nuclear responsive elements was decreased progressively by increasing selenite levels and, at 7 μM selenite, DNA-binding activity was completely inhibited. Selenite inhibition was reversed by addition of a dithiol, DTT. Proportional inhibition of iNOS activity as measured by decreased NO products in the medium (NO2− and NO3−) resulted from selenite addition to cell suspensions. This loss of iNOS activity was due to decreased synthesis of NO synthase protein. Selenium at low essential levels (nM) is required for synthesis of redox active selenoenzymes such as glutathione peroxidases and thioredoxin reductase, but in higher toxic levels (>5–10 μM) selenite can react with essential thiol groups on enzymes to form RS–Se–SR adducts with resultant inhibition of enzyme activity. Inhibition of NF-κB activity by selenite is presumed to be the result of adduct formation with the essential thiols of this transcription factor.

Opposite effects of nitric oxide and nitroxyl on postischemic myocardial injury

Recent experimental evidence suggests that reactive nitrogen oxide species can contribute significantly to postischemic myocardial injury. The aim of the present study was to evaluate the role of two reactive nitrogen oxide species, nitroxyl (NO−) and nitric oxide (NO⋅), in myocardial ischemia and reperfusion injury. Rabbits were subjected to 45 min of regional myocardial ischemia followed by 180 min of reperfusion. Vehicle (0.9% NaCl), 1 μmol/kg S-nitrosoglutathione (GSNO) (an NO⋅ donor), or 3 μmol/kg Angeli’s salt (AS) (a source of NO−) were given i.v. 5 min before reperfusion. Treatment with GSNO markedly attenuated reperfusion injury, as evidenced by improved cardiac function, decreased plasma creatine kinase activity, reduced necrotic size, and decreased myocardial myeloperoxidase activity. In contrast, the administration of AS at a hemodynamically equieffective dose not only failed to attenuate but, rather, aggravated reperfusion injury, indicated by an increased left ventricular end diastolic pressure, myocardial creatine kinase release and necrotic size. Decomposed AS was without effect. Co-administration of AS with ferricyanide, a one-electron oxidant that converts NO− to NO⋅, completely blocked the injurious effects of AS and exerted significant cardioprotective effects similar to those of GSNO. These results demonstrate that, although NO⋅ is protective, NO− increases the tissue damage that occurs during ischemia/reperfusion and suggest that formation of nitroxyl may contribute to postischemic myocardial injury.

The heme-copper oxidases of Thermus thermophilus catalyze the reduction of nitric oxide: Evolutionary implications

We show that the heme-copper terminal oxidases of Thermus thermophilus (called ba3 and caa3) are able to catalyze the reduction of nitric oxide (NO) to nitrous oxide (N2O) under reducing anaerobic conditions. The rate of NO consumption and N2O production were found to be linearly dependent on enzyme concentration, and activity was abolished by enzyme denaturation. Thus, contrary to the eukaryotic enzyme, both T. thermophilus oxidases display a NO reductase activity (3.0 ± 0.7 mol NO/mol ba3 × min and 32 ± 8 mol NO/mol caa3 × min at [NO] ≈ 50 μM and 20°C) that, though considerably lower than that of bona fide NO reductases (300–4,500 mol NO/mol enzyme × min), is definitely significant. We also show that for ba3 oxidase, NO reduction is associated to oxidation of cytochrome b at a rate compatible with turnover, suggesting a mechanism consistent with the stoichiometry of the overall reaction. We propose that the NO reductase activity of T. thermophilus oxidases may depend on a peculiar CuB+ coordination, which may be revealed by the forthcoming three-dimensional structure. These findings support the hypothesis of a common phylogeny of aerobic respiration and bacterial denitrification, which was proposed on the basis of structural similarities between the Pseudomonas stutzeri NO reductase and the cbb3 terminal oxidases. Our findings represent functional evidence in support of this hypothesis.

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.

Carbon monoxide and nitric oxide as coneurotransmitters in the enteric nervous system: Evidence from genomic deletion of biosynthetic enzymes

Nitric oxide (NO) and carbon monoxide (CO) seem to be neurotransmitters in the brain. The colocalization of their respective biosynthetic enzymes, neuronal NO synthase (nNOS) and heme oxygenase-2 (HO2), in enteric neurons and altered intestinal function in mice with genomic deletion of the enzymes (nNOSΔ/Δ and HO2Δ/Δ) suggest neurotransmitter roles for NO and CO in the enteric nervous system. We now establish that NO and CO are both neurotransmitters that interact as cotransmitters. Small intestinal smooth muscle cells from nNOSΔ/Δ and HO2Δ/Δ mice are depolarized, with apparent additive effects in the double knockouts (HO2Δ/Δ/nNOSΔ/Δ). Muscle relaxation and inhibitory neurotransmission are reduced in the mutant mice. In HO2Δ/Δ preparations, responses to electrical field stimulation are nearly abolished despite persistent nNOS expression, whereas exogenous CO restores normal responses, indicating that the NO system does not function in the absence of CO generation.

Requirement for nitric oxide activation of p21ras/extracellular regulated kinase in neuronal ischemic preconditioning

The mechanisms underlying neuronal ischemic preconditioning, a phenomenon in which brief episodes of ischemia protect against the lethal effects of subsequent periods of prolonged ischemia, are poorly understood. Ischemia can be modeled in vitro by oxygen-glucose deprivation (OGD). We report here that OGD preconditioning induces p21ras (Ras) activation in an N-methyl-d-aspartate receptor- and NO-dependent, but cGMP-independent, manner. We demonstrate that Ras activity is necessary and sufficient for OGD tolerance in neurons. Pharmacological inhibition of Ras, as well as a dominant negative mutant Ras, block OGD preconditioning whereas a constitutively active form of Ras promotes neuroprotection against lethal OGD insults. In contrast, the activity of phosphatidyl inositol 3-kinase is not required for OGD preconditioning because inhibition of phosphatidyl inositol 3-kinase with a chemical inhibitor or with a dominant negative mutant does not have any effect on the development of OGD tolerance. Furthermore, using recombinant adenoviruses and pharmacological inhibitors, we show that downstream of Ras the extracellular regulated kinase cascade is required for OGD preconditioning. Our observations indicate that activation of the Ras/extracellular regulated kinase cascade by NO is a critical mechanism for the development of OGD tolerance in cortical neurons, which may also play an important role in ischemic preconditioning in vivo.

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.

Role of the arginine-nitric oxide pathway in the regulation of vascular smooth muscle cell proliferation

The objective of this study was to elucidate the mechanisms by which nitric oxide (NO) inhibits rat aortic smooth muscle cell (RASMC) proliferation. Two products of the arginine-NO pathway interfere with cell growth by distinct mechanisms. NG-hydroxyarginine and NO appear to interfere with cell proliferation by inhibiting arginase and ornithine decarboxylase (ODC), respectively. S-nitroso-N-acetylpenicillamine, (Z)-1-[N-(2-aminoethyl)-N-(2-aminoethyl)-amino]-diazen-1-ium-1,2-diolate, and a nitroaspirin derivative (NCX 4016), each of which is a NO donor agent, inhibited RASMC growth at concentrations of 1–3 μM by cGMP-independent mechanisms. The cytostatic action of the NO donor agents as well as α-difluoromethylornithine (DFMO), a known ODC inhibitor, was prevented by addition of putrescine but not ornithine. These observations suggested that NO, like DFMO, may directly inhibit ODC. Experiments with purified, recombinant mammalian ODC revealed that NO inhibits ODC possibly by S-nitrosylation of the active site cysteine in ODC. DFMO, as well as the NO donor agents, interfered with cellular polyamine (putrescine, spermidine, spermine) production. Conversely, increasing the expression and catalytic activity of arginase I in RASMC either by transfection of cells with the arginase I gene or by induction of arginase I mRNA with IL-4 resulted in increased urea and polyamine production as well as cell proliferation. Finally, coculture of rat aortic endothelial cells, which had been pretreated with lipopolysaccharide plus a cytokine mixture to induce NO synthase and promote NO production, caused NO-dependent inhibition of target RASMC proliferation. This study confirms the inhibitory role of the arginine-NO pathway in vascular smooth muscle proliferation and indicates that one mechanism of action of NO is cGMP-independent and attributed to its capacity to inhibit ODC.

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.

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.

Opposite actions of nitric oxide on cholinergic synapses: which pathways?

Nitric oxide (NO) produced opposite effects on acetylcholine (ACh) release in identified neuroneuronal Aplysia synapses depending on the excitatory or the inhibitory nature of the synapse. Extracellular application of the NO donor, SIN-1, depressed the inhibitory postsynaptic currents (IPSCs) and enhanced the excitatory postsynaptic currents (EPSCs) evoked by presynaptic action potentials (1/60 Hz). Application of a membrane-permeant cGMP analog mimicked the effect of SIN-1 suggesting the participation of guanylate cyclase in the NO pathway. The guanylate cyclase inhibitor, methylene blue, blocked the NO-induced enhancement of EPSCs but only reduced the inhibition of IPSCs indicating that an additional mechanism participates to the depression of synaptic transmission by NO. Using nicotinamide, an inhibitor of ADP-ribosylation, we found that the NO-induced depression of ACh release on the inhibitory synapse also involves ADP-ribosylation mechanism(s). Furthermore, application of SIN-1 paired with cGMP-dependent protein kinase (cGMP-PK) inhibitors showed that cGMP-PK could play a role in the potentiating but not in the depressing effect of NO on ACh release. Increasing the frequency of stimulation of the presynaptic neuron from 1/60 Hz to 0.25 or 1 Hz potentiated the EPSCs and reduced the IPSCs. In these conditions, the potentiating effect of NO on the excitatory synapse was reduced, whereas its depressing effect on the inhibitory synapse was unaffected. Moreover the frequency-dependent enhancement of ACh release in the excitatory synapse was greatly reduced by the inhibition of NO synthase. Our results indicate that NO may be involved in different ways of modulation of synaptic transmission depending on the type of the synapse including synaptic plasticity.

Nitric oxide inhibits creatine kinase and regulates rat heart contractile reserve.

Cardiac myocytes express both constitutive and cytokine-inducible nitric oxide syntheses (NOS). NO and its congeners have been implicated in the regulation of cardiac contractile function. To determine whether NO could affect myocardial energetics, 31P NMR spectroscopy was used to evaluate high-energy phosphate metabolism in isolated rat hearts perfused with the NO donor S-nitrosoacetylcysteine (SNAC). All hearts were exposed to an initial high Ca2+ (3.5 mM) challenge followed by a recovery period, and then, either in the presence or absence of SNAC, to a second high Ca2+ challenge. This protocol allowed us to monitor simultaneously the effect of SNAC infusion on both contractile reserve (i.e., baseline versus high workload contractile function) and high-energy phosphate metabolism. The initial high Ca2+ challenge caused the rate-pressure product to increase by 74 +/- 5% in all hearts. As expected, ATP was maintained as phosphocreatine (PCr) content briefly dropped and then returned to baseline during the subsequent recovery period. Control hearts responded similarLy to the second high Ca2+ challenge, but SNAC-treated hearts did not demonstrate the expected increase in rate-pressure product. In these hearts, ATP declined significantly during the second high Ca2+ challenge, whereas phosphocreatine did not differ from controls, suggesting that phosphoryl transfer by creatine kinase (CK) was inhibited. CK activity, measured biochemically, was decreased by 61 +/- 13% in SNAC-treated hearts compared to controls. Purified CK in solution was also inhibited by SNAC, and reversal could be accomplished with DTT, a sulfhydryl reducing agent. Thus, NO can regulate contractile reserve, possibly by reversible nitrosothiol modification of CK.

Excitotoxicity in the lung: N-methyl-D-aspartate-induced, nitric oxide-dependent, pulmonary edema is attenuated by vasoactive intestinal peptide and by inhibitors of poly(ADP-ribose) polymerase.

Excitatory amino acid toxicity, resulting from overactivation of N-methyl-D-aspartate (NMDA) glutamate receptors, is a major mechanism of neuronal cell death in acute and chronic neurological diseases. We have investigated whether excitotoxicity may occur in peripheral organs, causing tissue injury, and report that NMDA receptor activation in perfused, ventilated rat lungs triggered acute injury, marked by increased pressures needed to ventilate and perfuse the lung, and by high-permeability edema. The injury was prevented by competitive NMDA receptor antagonists or by channel-blocker MK-801, and was reduced in the presence of Mg2+. As with NMDA toxicity to central neurons, the lung injury was nitric oxide (NO) dependent: it required L-arginine, was associated with increased production of NO, and was attenuated by either of two NO synthase inhibitors. The neuropeptide vasoactive intestinal peptide and inhibitors of poly(ADP-ribose) polymerase also prevented this injury, but without inhibiting NO synthesis, both acting by inhibiting a toxic action of NO that is critical to tissue injury. The findings indicate that: (i) NMDA receptors exist in the lung (and probably elsewhere outside the central nervous system), (ii) excessive activation of these receptors may provoke acute edematous lung injury as seen in the "adult respiratory distress syndrome," and (iii) this injury can be modulated by blockade of one of three critical steps: NMDA receptor binding, inhibition of NO synthesis, or activation of poly(ADP-ribose) polymerase.

Pathogenesis of influenza virus-induced pneumonia: involvement of both nitric oxide and oxygen radicals.

The role of nitric oxide (NO) in the pathogenesis of influenza virus-induced pneumonia in mice was investigated. Experimental influenza virus pneumonia was produced with influenza virus A/Kumamoto/Y5/67(H2N2). Both the enzyme activity of NO synthase (NOS) and mRNA expression of the inducible NOS were greatly increased in the mouse lungs; increases were mediated by interferon gamma. Excessive production of NO in the virus-infected lung was studied further by using electron spin resonance (ESR) spectroscopy. In vivo spin trapping with dithiocarbamate-iron complexes indicated that a significant amount of NO was generated in the virus-infected lung. Furthermore, an NO-hemoglobin ESR signal appeared in the virus-infected lung, and formation of NO-hemoglobin was significantly increased by treatment with superoxide dismutase and was inhibited by N(omega)-monomethyl-L-arginine (L-NMMA) administration. Immunohistochemistry with a specific anti-nitrotyrosine antibody showed intense staining of alveolar phagocytic cells such as macrophages and neutrophils and of intraalveolar exudate in the virus-infected lung. These results strongly suggest formation of peroxynitrite in the lung through the reaction of NO with O2-, which is generated by alveolar phagocytic cells and xanthine oxidase. In addition, administration of L-NMMA resulted in significant improvement in the survival rate of virus-infected mice without appreciable suppression of their antiviral defenses. On the basis of these data, we conclude that NO together with O2- which forms more reactive peroxynitrite may be the most important pathogenic factors in influenza virus-induced pneumonia in mice.