Direct measurement of nitric oxide generation from nitric oxide synthase

Although nitric oxide synthase (NOS) is widely considered as the major source of NO in biological cells and tissues, direct evidence demonstrating NO formation from the purified enzyme has been lacking. It was recently reported that NOS does not synthesize NO, but rather generates nitroxyl anion (NO−) that is subsequently converted to NO by superoxide dismutase (SOD). To determine if NOS synthesizes NO, electron paramagnetic resonance (EPR) spectroscopy was applied to directly measure NO formation from purified neuronal NOS. In the presence of the NO trap Fe2+-N-methyl-d-glucamine dithiocarbamate, NO gives rise to characteristic EPR signals with g = 2.04 and aN = 12.7 G, whereas NO− is undetectable. In the presence of l-arginine (l-Arg) and cofactors, NOS generated prominent NO signals. This NO generation did not require SOD, and it was blocked by the specific NOS inhibitor N-nitro-l-arginine methyl ester. Isotope-labeling experiments with l-[15N]Arg further demonstrated that NOS-catalyzed NO arose from the guanidino nitrogen of l-Arg. Measurement of the time course of NO formation demonstrated that it paralleled that of l-citrulline. The conditions used in the prior study were shown to result in potent superoxide generation, and this may explain the failure to measure NO formation in the absence of SOD. These experiments provide unequivocal evidence that NOS does directly synthesize NO from l-Arg.

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.

Interaction between inducible nitric oxide synthase and cyclooxygenase-2 after cerebral ischemia

Focal cerebral ischemia is associated with expression of both inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2), enzymes whose reaction products contribute to the evolution of ischemic brain injury. We tested the hypothesis that, after cerebral ischemia, nitric oxide (NO) produced by iNOS enhances COX-2 activity, thereby increasing the toxic potential of this enzyme. Cerebral ischemia was produced by middle cerebral artery occlusion in rats or mice. Twenty-four hours after ischemia in rats, iNOS-immunoreactive neutrophils were observed in close proximity (<20 μm) to COX-2-positive cells at the periphery of the infarct. In the olfactory bulb, only COX-2 positive cells were observed. Cerebral ischemia increased the concentration of the COX-2 reaction product prostaglandin E2 (PGE2) in the ischemic area and in the ipsilateral olfactory bulb. The iNOS inhibitor aminoguanidine reduced PGE2 concentration in the infarct, where both iNOS and COX-2 were expressed, but not in the olfactory bulb, where only COX-2 was expressed. Postischemic PGE2 accumulation was reduced significantly in iNOS null mice compared with wild-type controls (C57BL/6 or SV129). The data provide evidence that NO produced by iNOS influences COX-2 activity after focal cerebral ischemia. Pro-inflammatory prostanoids and reactive oxygen species produced by COX-2 may be a previously unrecognized factor by which NO contributes to ischemic brain injury. The pathogenic effect of the interaction between NO, or a derived specie, and COX-2 is likely to play a role also in other brain diseases associated with inflammation.

Periplasmic superoxide dismutase protects Salmonella from products of phagocyte NADPH-oxidase and nitric oxide synthase

Superoxide dismutase (SOD) catalyzes the conversion of superoxide radical to hydrogen peroxide. Periplasmic localization of bacterial Cu,Zn-SOD has suggested a role of this enzyme in defense against extracellular phagocyte-derived reactive oxygen species. Sequence analysis of regions flanking the Salmonella typhimurium sodC gene encoding Cu,Zn-SOD demonstrates significant homology to λ phage proteins, reflecting possible bacteriophage-mediated horizontal gene transfer of this determinant among pathogenic bacteria. Salmonella deficient in Cu,Zn-SOD has reduced survival in macrophages and attenuated virulence in mice, which can be restored by abrogation of either the phagocyte respiratory burst or inducible nitric oxide synthase. Moreover, a sodC mutant is extremely susceptible to the combination of superoxide and nitric oxide. These observations suggest that SOD protects periplasmic or inner membrane targets by diverting superoxide and limiting peroxynitrite formation, and they demonstrate the ability of the respiratory burst and nitric oxide synthase to synergistically kill microbial pathogens in vivo.

Pharmacological and immunohistochemical evidence for a functional nitric oxide synthase system in rat peritoneal eosinophils

Eosinophil migration in vivo is markedly attenuated in rats treated chronically with the NO synthase (NOS) inhibitor Nω-nitro-l-arginine methyl ester (l-NAME). In this study, we investigated the existence of a NOS system in eosinophils. Our results demonstrated that rat peritoneal eosinophils strongly express both type II (30.2 ± 11.6% of counted cells) and type III (24.7 ± 7.4% of counted cells) NOS, as detected by immunohistochemistry using affinity purified mouse mAbs. Eosinophil migration in vitro was evaluated by using 48-well microchemotaxis chambers and the chemotactic agents used were N-formyl-methionyl-leucyl-phenylalanine (fMLP, 5 × 10−8 M) and leukotriene B4 (LTB4, 10−8 M). l-NAME (but not d-NAME) significantly inhibited the eosinophil migration induced by both fMLP (54% reduction for 1.0 mM; P < 0.05) and LTB4 (61% reduction for 1.0 mM; P < 0.05). In addition, the type II NOS inhibitor 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine and the type I/II NOS inhibitor 1-(2-trifluoromethylphenyl) imidazole also markedly (P < 0.05) attenuated fMLP- (52% and 38% reduction for 1.0 mM, respectively) and LTB4- (52% and 51% reduction for 1.0 mM, respectively) induced migration. The inhibition of eosinophil migration by l-NAME was mimicked by the soluble guanylate cyclase inhibitor 1H-[1,2,4] oxadiazolo [4,3,-a] quinoxalin-1-one (0.01 and 0.1 mM) and reversed by either sodium nitroprusside (0.1 mM) or dibutyryl cyclic GMP (1 mM). We conclude that eosinophils do express NO synthase(s) and that nitric oxide plays an essential role in eosinophil locomotion by acting through a cyclic GMP transduction mechanism.

Complex regulation of human inducible nitric oxide synthase gene transcription by Stat 1 and NF-κB

The human inducible nitric oxide synthase (hiNOS) gene is expressed in several disease states and is also important in the normal immune response. Previously, we described a cytokine-responsive enhancer between −5.2 and −6.1 kb in the 5′-flanking hiNOS promoter DNA, which contains multiple nuclear factor κβ (NF-κB) elements. Here, we describe the role of the IFN-Jak kinase-Stat (signal transducer and activator of transcription) 1 pathway for regulation of hiNOS gene transcription. In A549 human lung epithelial cells, a combination of cytokines tumor necrosis factor-α, interleukin-1β, and IFN-γ (TNF-α, IL-1β, and IFN-γ) function synergistically for induction of hiNOS transcription. Pharmacological inhibitors of Jak2 kinase inhibit cytokine-induced Stat 1 DNA-binding and hiNOS gene expression. Expression of a dominant-negative mutant Stat 1 inhibits cytokine-induced hiNOS reporter expression. Site-directed mutagenesis of a cis-acting DNA element at −5.8 kb in the hiNOS promoter identifies a bifunctional NF-κB/Stat 1 motif. In contrast, gel shift assays indicate that only Stat 1 binds to the DNA element at −5.2 kb in the hiNOS promoter. Interestingly, Stat 1 is repressive to basal and stimulated iNOS mRNA expression in 2fTGH human fibroblasts, which are refractory to iNOS induction. Overexpression of NF-κB activates hiNOS promoter–reporter expression in Stat 1 mutant fibroblasts, but not in the wild type, suggesting that Stat 1 inhibits NF-κB function in these cells. These results indicate that both Stat 1 and NF-κB are important in the regulation of hiNOS transcription by cytokines in a complex and cell type-specific manner.

Regulation of the expression of nitric oxide synthase and leishmanicidal activity by glycoconjugates of Leishmania lipophosphoglycan in murine macrophages.

Lipophosphoglycan (LPG) glycoconjugates from promastigotes of Leishmania were not able to induce the expression of the cytokine-inducible nitric oxide synthase (iNOS) by the murine macrophage cell line, J774. However, they synergize with interferon gamma to stimulate the macrophages to express high levels of iNOS. This synergistic effect was critically time-dependent. Preincubation of J774 cells with the LPG glycans 4-18 h before stimulation with interferon gamma resulted in a significant reduction in the expression of iNOS mRNA and of NO synthesis, compared with cells preincubated with culture medium alone. The regulatory effect on the induction of iNOS by LPG is located in the LPG phosphoglycan disaccharide backbone. Synthetic fragments of this backbone had a similar regulatory effect on NO synthesis. Further, the production of NO by activated macrophages in the present system was correlated directly with the leishmanicidal capacity of the cells. These data therefore demonstrate that LPG glycoconjugates have a profound effect on the survival of Leishmania parasites through their ability to regulate the expression of iNOS by macrophages.

Cytokine-treated human neutrophils contain inducible nitric oxide synthase that produces nitration of ingested bacteria.

Although the production of NO within rodent phagocytes is well-characterized, its production and function within human phagocytes are less clear. We show here that neutrophils within human buffy coat preparations stimulated with a mixture of interleukin 1, tumor necrosis factor alpha, and interferon gamma contain inducible NO synthase mRNA and protein, one of the enzymes responsible for NO production. The protein colocalizes with myeloperoxidase within neutrophil primary granules. Using an inhibitor of NO synthase, L-N-monomethyl arginine, we show that activity of this enzyme is required for the formation of nitrotyrosine around phagocytosed bacteria, most likely through the intermediate production of peroxynitrite, a reaction product of NO and superoxide anions.

Neuronal nitric oxide synthase and dystrophin-deficient muscular dystrophy.

Neuronal nitric oxide synthase (nNOS) in fast-twitch skeletal muscle fibers is primarily particulate in contrast to its greater solubility in brain. Immunohistochemistry shows nNOS localized to the sarcolemma, with enrichment at force transmitting sites, the myotendinous junctions, and costameres. Because this distribution is similar to dystrophin, we determined if nNOS expression was affected by the loss of dystrophin. Significant nNOS immunoreactivity and enzyme activity was absent in skeletal muscle tissues from patients with Duchenne muscular dystrophy. Similarly, in dystrophin-deficient skeletal muscles from mdx mice both soluble and particulate nNOS was greatly reduced compared with C57 control mice. nNOS mRNA was also reduced in mdx muscle in contrast to mRNA levels for a dystrophin binding protein, alpha 1-syntrophin. nNOS levels increased dramatically from 2 to 52 weeks of age in C57 skeletal muscle, which may indicate a physiological role for NO in aging-related processes. Biochemical purification readily dissociates nNOS from the dystrophin-glycoprotein complex. Thus, nNOS is not an integral component of the dystrophin-glycoprotein complex and is not simply another dystrophin-associated protein since the expression of both nNOS mRNA and protein is affected by dystrophin expression.

Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells leading to peroxynitrite-mediated cellular injury.

Besides synthesizing nitric oxide (NO), purified neuronal NO synthase (nNOS) can produce superoxide (.O2-) at lower L-Arg concentrations. By using electron paramagnetic resonance spin-trapping techniques, we monitored NO and .O2- formation in nNOS-transfected human kidney 293 cells. In control transfected cells, the Ca2+ ionophore A23187 triggered NO generation but no .O2- was seen. With cells in L-Arg-free medium, we observed .O2- formation that increased as the cytosolic L-Arg levels decreased, while NO generation declined. .O2- formation was virtually abolished by the specific NOS blocker, N-nitro-L-arginine methyl ester (L-NAME). Nitrotyrosine, a specific nitration product of peroxynitrite, accumulated in L-Arg-depleted cells but not in control cells. Activation by A23187 was cytotoxic to L-Arg-depleted, but not to control cells, with marked lactate dehydrogenase release. The cytotoxicity was largely prevented by either superoxide dismutase or L-NAME. Thus, with reduced L-Arg availability NOS elicits cytotoxicity by generating .O2- and NO that interact to form the potent oxidant peroxynitrite. Regulating arginine levels may provide a therapeutic approach to disorders involving .O2-/NO-mediated cellular injury.

Huntingtin-associated protein (HAP1): discrete neuronal localizations in the brain resemble those of neuronal nitric oxide synthase.

Huntington disease stems from a mutation of the protein huntingtin and is characterized by selective loss of discrete neuronal populations in the brain. Despite a massive loss of neurons in the corpus striatum, NO-generating neurons are intact. We recently identified a brain-specific protein that associates with huntingtin and is designated huntingtin-associated protein (HAP1). We now describe selective neuronal localizations of HAP1. In situ hybridization studies reveal a resemblance of HAP1 and neuronal nitric oxide synthase (nNOS) mRNA localizations with dramatic enrichment of both in the pedunculopontine nuclei, the accessory olfactory bulb, and the supraoptic nucleus of the hypothalamus. Both nNOS and HAP1 are enriched in subcellular fractions containing synaptic vesicles. Immunocytochemical studies indicate colocalizations of HAP1 and nNOS in some neurons. The possible relationship of HAP1 and nNOS in the brain is reminiscent of the relationship of dystrophin and nNOS in skeletal muscle and suggests a role of NO in Huntington disease, analogous to its postulated role in Duchenne muscular dystrophy.

Complementation analysis of mutants of nitric oxide synthase reveals that the active site requires two hemes.

For catalytic activity, nitric oxide synthases (NOSs) must be dimeric. Previous work revealed that the requirements for stable dimerization included binding of tetrahydrobiopterin (BH4), arginine, and heme. Here we asked what function is served by dimerization. We assessed the ability of individually inactive mutants of mouse inducible NOS (iNOS; NOS2), each deficient in binding a particular cofactor or cosubstrate, to complement each other by generating NO upon cotransfection into human epithelial cells. The ability of the mutants to homodimerize was gauged by gel filtration and/or PAGE under partially denaturing conditions, both followed by immunoblot. Their ability to heterodimerize was assessed by coimmunoprecipitation. Heterodimers that contained only one COOH-terminal hemimer and only one BH4-binding site could both form and function, even though the NADPH-, FAD-, and FMN-binding domains (in the COOH-terminal hemimer) and the BH4-binding sites (in the NH2-terminal hemimer) were contributed by opposite chains. Heterodimers that contained only one heme-binding site (Cys-194) could also form, either in cis or in trans to the nucleotide-binding domains. However, for NO production, both chains had to bind heme. Thus, NO production by iNOS requires dimerization because the active site requires two hemes.

Nitric oxide synthase content of hypothalamic explants: increase by norepinephrine and inactivated by NO and cGMP.

Release of luteinizing hormone (LH)-releasing hormone (LHRH), the hypothalamic peptide that controls release of LH from the adenohypophysis, is controlled by NO. There is a rich plexus of nitric oxide synthase (NOS)-containing neurons and fibers in the lateral median eminence, intermingled with terminals of the LHRH neurons. To study relations between NOS and LHRH in this brain region, we measured NOS activity in incubated medial basal hypothalamus (MBH). NOS converts [14C]arginine to equimolar quantities of [14C]citrulline plus NO, which rapidly decomposes. The [14C]citrulline serves as an index of the NO produced. NOS basal activity was suppressed by incubation of the tissue with an inhibitor of NOS, nitroarginine methyl ester (NAME) (10(-5) M). Furthermore, incubation of MBH explants for 30 min with norepinephrine (NE) increased NOS activity and the increase was prevented by prazosine (10(-5) M), an alpha 1-adrenergic receptor blocker; however, direct addition of NE to the tissue homogenate or to a preparation of MBH synaptosomes did not alter enzyme activity, which suggested that NE increased the content of NOS during incubation with the tissue. After purification of NOS, the increase in enzyme content induced by NE was still measurable. This indicates that within 30 min NE increased the synthesis of NOS in vitro. Incubation of MBH or the MBH homogenate with various concentrations of sodium nitroprusside (NP), a releaser of NO, reduced NOS activity at high concentrations (> or = 0.9 mM), which were associated with either a reduction of stimulation or a plateau of LHRH release. Finally, incubation of either MBH or the homogenate with cGMP, a major mediatior of NO action, at concentrations that increased LHRH release also reduced NOS activity. These results indicate that NO at high concentrations can inactivate NOS and that cGMP can also inhibit the enzyme directly. Therefore, the increased NOS activity induced by activation of alpha 1 receptors by NE is inhibited by NO itself and a principal product of its activity, cGMP, providing negative feedback on NOS. In central nervous system (CNS) infections with high concentrations of inducible NOS produced by glial elements, the high concentrations of NO and cGMP produced may suppress LHRH release, resulting in decreased gonadotropin and gonadal steroid release.

Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B.

The objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation products of NO (NO2- + N03-, designated as NOX-), cellular RNA was extracted for determination of iNOS mRNA levels by Northern blot analysis, and nuclear extracts were prepared for determination of NF-kappa B by electrophoretic mobility-shift assay. Inhibitors of calpain (alpha-N-acetyl-Leu-Leu-norleucinal; N-benzyloxycarbonyl-Leu-leucinal) and the proteasome (N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-leucinal) markedly inhibited or abolished the induction of iNOS in macrophages. The proteinase inhibitors interfered with lipopolysaccharide-induced NOX- production by macrophages, and this effect was accompanied by comparable interference with the appearance of both iNOS mRNA and NF-kappa B. Calpain inhibitors elicited effects at concentrations of 1-100 microM, whereas the proteasome inhibitor was 1000-fold more potent, producing significant inhibitory effects at 1 nM. The present findings indicate that the proteasome pathway is essential for lipopolysaccharide-induced expression of the iNOS gene in rat alveolar macrophages. Furthermore, the data support the view that the proteasome pathway is directly involved in promoting the activation of NF-kappa B and that the induction of iNOS by lipopolysaccharide involves the transcriptional action of NF-kappaB.