Characterization of the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells as an NG-hydroxyl-L-arginine-nitric oxide adduct.

The nature of an L-arginine-derived relaxing factor released from vascular smooth muscle cells cultured on microcarrier beads and stimulated for 20 h with interleukin 1 beta was investigated. Unlike the unstable relaxation elicited by authentic nitric oxide (NO) in a cascade superfusion bioassay system, the effluate from vascular smooth muscle cells induced a stable relaxation that was susceptible to inhibition by oxyhemoglobin. Three putative endogenous NO carriers mimicked this stable relaxing effect: S-nitroso-L-cysteine, low molecular weight dinitrosyl-iron complexes (DNICs), and the adduct of NG-hydroxy-L-arginine (HOArg) with NO. Inactivation of S-nitroso-L-cysteine by Hg2+ ions or trapping of DNICs with agarose-bound bovine serum albumin abolished their relaxing effects, whereas that of the vascular smooth muscle cell effluate remained unaffected. In addition, neither S-nitrosothiols nor DNICs were detectable in the effluate from these cells, as judged by UV and electron spin resonance (ESR) spectroscopy. The HOArg-NO adduct was instantaneously generated upon reaction of HOArg with authentic NO under bioassay conditions. Its pharmacological profile was indistinguishable from that of the vascular smooth muscle cell effluate, as judged by comparative bioassay with different vascular and nonvascular smooth muscle preparations. Moreover, up to 100 nM HOArg was detected in the effluate from interleukin 1 beta-stimulated vascular smooth muscle cells, suggesting that sufficient amounts of HOArg are released from these cells to spontaneously generate the HOArg-NO adduct. This intercellular NO carrier probably accounts for the stable L-arginine-derived relaxing factor released from cytokine-stimulated vascular smooth muscle cells and also from other NO-producing cells, such as macrophages and neutrophils.

Nitric oxide in the contractile action of bradykinin, oxytocin, and prostaglandin F2 α in the estrogenized rat uterus

Experiments were performed on uteri from estrogen-primed female rats. Bradykinin (BK) (10−8 M) significantly augmented biosynthesis of prostaglandin F2 α (PGF2α) and prostaglandin E2 (PGE2), and this synthesis was completely blocked by NG-monomethyl l-arginine (NMMA) (300 μM), a competitive inhibitor of nitric oxide synthase (NOS). Blockade of prostaglandin synthesis by indomethacin caused rapid dissipation of isometric developed tension (IDT) induced by BK. Blockade of NOS with NMMA had similar but less marked effects. Combining the two inhibitors produced an even more rapid decay in IDT, suggesting that BK-induced NO release maintains IDT by release of prostanoids. The decline of frequency of contraction (FC) was not significantly altered by either indomethacin or NMMA but was markedly accelerated by combination of the inhibitors, which suggests that PGs maintain FC and therefore FC decline is accelerated only when PG production is blocked completely by combination of the two inhibitors of PG synthesis. The increase in IDT induced by oxytocin was unaltered by indomethacin, NMMA or their combination indicating that neither NO nor PGs are involved in the contractions induced by oxytocin. However, the decline in FC with time was significantly reduced by the inhibitor of NOS, NMMA, suggesting that FC decay following oxytocin is caused by NO released by the contractile process. In the case of PGF2α, NMMA resulted in increased initial IDT and FC. The decline in FC was rapid and dramatically inhibited by NMMA. Receptor-mediated contraction by BK, oxytocin, and PGF2α is modulated by NO that maintains IDT by releasing PGs but reduces IDT and FC via cyclic GMP.

NF-κB and AP-1 Activation by Nitric Oxide Attenuated Apoptotic Cell Death in RAW 264.7 Macrophages

A toxic dose of the nitric oxide (NO) donor S-nitrosoglutathione (GSNO; 1 mM) promoted apoptotic cell death of RAW 264.7 macrophages, which was attenuated by cellular preactivation with a nontoxic dose of GSNO (200 μM) or with lipopolysaccharide, interferon-γ, and NG-monomethyl-l-arginine (LPS/IFN-γ/NMMA) for 15 h. Protection from apoptosis was achieved by expression of cyclooxygenase-2 (Cox-2). Here we investigated the underlying mechanisms leading to Cox-2 expression. LPS/IFN-γ/NMMA prestimulation activated nuclear factor (NF)-κB and promoted Cox-2 expression. Cox-2 induction by low-dose GSNO demanded activation of both NF-κB and activator protein-1 (AP-1). NF-κB supershift analysis implied an active p50/p65 heterodimer, and a luciferase reporter construct, containing four copies of the NF-κB site derived from the murine Cox-2 promoter, confirmed NF-κB activation after NO addition. An NF-κB decoy approach abrogated not only Cox-2 expression after low-dose NO or after LPS/IFN-γ/NMMA but also inducible protection. The importance of AP-1 for Cox-2 expression and cell protection by low-level NO was substantiated by using the extracellular signal-regulated kinase inhibitor PD98059, blocking NO-elicited Cox-2 expression, but leaving the cytokine signal unaltered. Transient transfection of a dominant-negative c-Jun mutant further attenuated Cox-2 expression by low-level NO. Whereas cytokine-mediated Cox-2 induction relies on NF-κB activation, a low-level NO–elicited Cox-2 response required activation of both NF-κB and AP-1.

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 regulates vascular cell adhesion molecule 1 gene expression and redox-sensitive transcriptional events in human vascular endothelial cells.

Decreased nitric oxide (NO) activity, the formation of reactive oxygen species, and increased endothelial expression of the redox-sensitive vascular cell adhesion molecule 1 (VCAM-1) gene in the vessel wall are early and characteristic features of atherosclerosis. To explore whether these phenomena are functionally interrelated, we tested the hypothesis that redox-sensitive VCAM-1 gene expression is regulated by a NO-sensitive mechanism. In early passaged human umbilical vein endothelial cells and human dermal microvascular endothelial cells, the NO donor diethylamine-NO (DETA-NO, 100 microM) reduced VCAM-1 gene expression induced by the cytokine tumor necrosis factor alpha (TNF-alpha, 100 units/ml) at the cell surface level by 65% and intracellular adhesion molecule 1 (ICAM-1) gene expression by 35%. E-selectin gene expression was not affected. No effect on expression of cell adhesion molecules was observed with DETA alone. Moreover, DETA-NO suppressed TNF-alpha-induced mRNA accumulation of VCAM-1 and TNF-alpha-mediated transcriptional activation of the human VCAM-1 promoter. Conversely, treatment with NG-monomethyl-L-arginine (L-NMMA, 1 mM), an inhibitor of NO synthesis, augmented cytokine induction of VCAM-1 and ICAM-1 mRNA accumulation. By gel mobility shift analysis, DETA-NO inhibited TNF-alpha activation of DNA binding protein activity to the VCAM-1 NF-kappa B like binding sites. Peroxy-fatty acids such as 13-hydroperoxydodecanoeic acid (linoleyl hydroperoxide) may serve as an intracellular signal for NF-kappa B activation. Using thin layer chromatography, DETA-NO (100 microM) suppressed formation of this metabolite, suggesting that DETA-NO modifies the reactivity of oxygen intermediates in the vascular endothelium. Through this mechanism, NO may function as an immunomodulator of the vessel wall and thus mediate inflammatory events involved in the pathogenesis of atherosclerosis.

Nitric oxide inhibits the release of norepinephrine and dopamine from the medial basal hypothalamus of the rat.

Previous research indicates that norepinephrine and dopamine stimulate release of luteinizing hormone (LH)-releasing hormone (LHRH), which then reaches the adenohypophysis via the hypophyseal portal vessels to release LH. Norepinephrine exerts its effect via alpha 1-adrenergic receptors, which stimulate the release of nitric oxide (NO) from nitricoxidergic (NOergic) neurons in the medial basal hypothalamus (MBH). The NO activates guanylate cyclase and cyclooxygenase, thereby inducing release of LHRH into the hypophyseal portal vessels. We tested the hypothesis that these two catecholamines modulate NO release by local feedback. MBH explants were incubated in the presence of sodium nitroprusside (NP), a releaser of NO, and the effect on release of catecholamines was determined. NP inhibited release of norepinephrine. Basal release was increased by incubation of the tissue with the NO scavenger hemoglobin (20 micrograms/ml). Hemoglobin also blocked the inhibitory effect of NP. In the presence of high-potassium (40 mM) medium to depolarize cell membranes, norepinephrine release was increased by a factor of 3, and this was significantly inhibited by NP. Hemoglobin again produced a further increase in norepinephrine release and also blocked the action of NP. When constitutive NO synthase was inhibited by the competitive inhibitor NG-monomethyl-L-arginine (NMMA) at 300 microM, basal release of norepinephrine was increased, as was potassium-evoked release, and this was associated in the latter instance with a decrease in tissue concentration, presumably because synthesis did not keep up with the increased release in the presence of NMMA. The results were very similar with dopamine, except that reduction of potassium-evoked dopamine release by NP was not significant. However, the increase following incubation with hemoglobin was significant, and hemoglobin, when incubated with NP, caused a significant elevation in dopamine release above that with NP alone. In this case, NP increased tissue concentration of dopamine along with inhibiting release, suggesting that synthesis continued, thereby raising the tissue concentration in the face of diminished release. When the tissue was incubated with NP plus hemoglobin, which caused an increase in release above that obtained with NP alone, the tissue concentration decreased significantly compared with that in the absence of hemoglobin, indicating that, with increased release, release exceeded synthesis, causing a fall in tissue concentration. When NO synthase was blocked by NMMA, the release of dopamine, under either basal or potassium-evoked conditions, was increased. Again, in the latter instance the tissue concentration declined significantly, presumably because synthesis did not match release. Therefore, the results were very similar with both catecholamines and indicate that NO acts to suppress release of both amines. Since both catecholamines activate the release of LHRH, the inhibition of their release by NO serves as an ultra-short-loop negative feedback by which NO inhibits the release of the catecholamines, thereby reducing the activation of the NOergic neurons and decreasing the release of LHRH. This may be an important means for terminating the pulses of release of LHRH, which generate the pulsatile release of LH that stimulates gonadal function in both male and female mammals.

The killing of Leishmania major by human macrophages is mediated by nitric oxide induced after ligation of the Fc epsilon RII/CD23 surface antigen.

Serum IgE concentrations and the expression of the low-affinity receptor for IgE (Fc epsilon RII/CD23) are increased in cutaneous leishmaniasis or after immune challenge with Leishmania antigens. In vitro, the ligation of CD23 by IgE-anti-IgE immune complexes (IgE-IC) or by anti-CD23 monoclonal antibody (mAb) induces nitric oxide (NO) synthase and the generation of various cytokines by human monocytes/macrophages. The present study shows that IgE-IC, via CD23 binding, induce intracellular killing of Leishmania major in human monocyte-derived macrophages through the induction of the L-arginine:NO pathway. This was demonstrated by increased generation of nitrite (NO2-), the stable oxidation product of NO, and by the ability of NG-monomethyl-L-arginine to block both NO generation and parasite killing. A similar NO-dependent effect was observed with interferon gamma-treated cells. Tumor necrosis factor alpha is involved in this process, since both the induction of NO synthase and the killing of parasites caused by anti-CD23 mAb were inhibited by an anti-tumor necrosis factor alpha mAb. Treatment of noninfected CD23+ macrophages with IgE-IC provided protection against subsequent in vitro infection of these cells by Leishmania major promastigotes. Thus, IgE-IC promote killing of L. major by inducing NO synthase in human macrophages.

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.

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.

L-arginine may mediate the therapeutic effects of low protein diets.

We have previously shown beneficial effects of dietary protein restriction on transforming growth factor beta (TGF-beta) expression and glomerular matrix accumulation in experimental glomerulonephritis. We hypothesized that these effects result from restriction of dietary L-arginine intake. Arginine is a precursor for three pathways, the products of which are involved in tissue injury and repair: nitric oxide, an effector molecule in inflammatory and immunological tissue injury; polyamines, which are required for DNA synthesis and cell growth; and proline, which is required for collagen production. Rats were fed six isocaloric diets differing in L-arginine and/or total protein content, starting immediately after induction of glomerulonephritis by injection of an antibody reactive to glomerular mesangial cells. Mesangial cell lysis and monocyte/macrophage infiltration did not differ with diet. However, restriction of dietary L-arginine intake, even when total protein intake was normal, resulted in decreased proteinuria, decreased expression of TGF-beta 1 mRNA and TGF-beta 1 protein, and decreased production and deposition of matrix components. L-Arginine, but not D-arginine, supplementation to low protein diets reversed these effects. These results implicate arginine as a key component in the beneficial effects of low protein diet.

Lipoxin A4 stable analogs inhibit leukocyte rolling and adherence in the rat mesenteric microvasculature: Role of P-selectin

Three different stable lipoxin A4 (LXA4) analogs (i.e., 16-phenoxy-LXA4-Me, 15-cyclohexyl-LXA4-Me, and 15-R/S-methyl-LXA4-Me) were studied for their ability to modulate leukocyte-endothelial cell interactions in the rat mesenteric microvasculature. Superfusion of the rat mesentery with 50 μmol/liter NG-nitro-l-arginine methyl ester (l-NAME) caused a significant, time-dependent increase in leukocyte rolling (56 ± 8 cells/min; P < 0.01 vs. control) and leukocyte adherence (12.5 ± 1.2 cells/100 μm length of venule; P < 0.01 vs. control) after 120 min of superfusion. Concomitant superfusion of the rat mesentery with 10 nmol/liter of each of three lipoxin analogs consistently and markedly attenuated l-NAME-induced leukocyte rolling to 10 ± 4 (P < 0.01), 4 ± 1 (P < 0.01), and 32 ± 7 (P < 0.05) cells/min, and adherence to 4 ± 0.8 (P < 0.01), 1.1 ± 0.4 (P < 0.01), and 7 ± 0.7 (P < 0.05) cells/100 μm length of venule (16-phenoxy-LXA4-Me, 15-cyclohexyl-LXA4-Me, and 15-R/S- methyl-LXA4-Me, respectively). No alterations of systemic blood pressure or mesenteric venular shear rates were observed in any group. Immunohistochemical up-regulation of P-selectin expression on intestinal venular endothelium was significantly increased (P < 0.01) after exposure to l-NAME, and this was significantly attenuated by these lipoxin analogs (P < 0.01). Thus, in vivo superfusion of the rat mesentery with stable lipoxin analogs at 10 nmol/liter reduces l-NAME-induced leukocyte rolling and adherence in the mesenteric rat microvasculature by attenuating P-selectin expression. This anti-inflammatory mechanism may represent a novel and potent regulatory action of lipoxins on the immune system.

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.

Effects of anesthesia on functional activation of cerebral blood flow and metabolism

Functional brain mapping based on changes in local cerebral blood flow (lCBF) or glucose utilization (lCMRglc) induced by functional activation is generally carried out in animals under anesthesia, usually α-chloralose because of its lesser effects on cardiovascular, respiratory, and reflex functions. Results of studies on the role of nitric oxide (NO) in the mechanism of functional activation of lCBF have differed in unanesthetized and anesthetized animals. NO synthase inhibition markedly attenuates or eliminates the lCBF responses in anesthetized animals but not in unanesthetized animals. The present study examines in conscious rats and rats anesthetized with α-chloralose the effects of vibrissal stimulation on lCMRglc and lCBF in the whisker-to-barrel cortex pathway and on the effects of NO synthase inhibition with NG-nitro-l-arginine methyl ester (l-NAME) on the magnitude of the responses. Anesthesia markedly reduced the lCBF and lCMRglc responses in the ventral posteromedial thalamic nucleus and barrel cortex but not in the spinal and principal trigeminal nuclei. l-NAME did not alter the lCBF responses in any of the structures of the pathway in the unanesthetized rats and also not in the trigeminal nuclei of the anesthetized rats. In the thalamus and sensory cortex of the anesthetized rats, where the lCBF responses to stimulation had already been drastically diminished by the anesthesia, l-NAME treatment resulted in loss of statistically significant activation of lCBF by vibrissal stimulation. These results indicate that NO does not mediate functional activation of lCBF under physiological conditions.

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.

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.