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.

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 protein calorie malnutrition on tuberculosis in mice

Infectious diseases and malnutrition represent major burdens afflicting millions of people in developing countries. Both conditions affect individuals in industrialized nations, particularly the aged, the HIV-infected, and people with chronic diseases. While malnutrition is known to induce a state of immunodeficiency, the mechanisms responsible for compromised antimicrobial resistance in malnourished hosts remain obscure. In the present study, mice fed a 2% protein diet and developing protein calorie malnutrition, in contrast to well-nourished controls receiving a 20% protein diet, rapidly succumbed to infection with Mycobacterium tuberculosis. Malnourished mice exhibited a tissue-specific diminution in the expression of interferon γ, tumor necrosis factor α, and the inducible form of nitric oxide synthase in the lungs, but not the liver. The expression of these molecules critical to the production of mycobactericidal nitrogen oxides was depressed in malnourished animals in the lungs specifically at early times (<14 days) after infection. At later times, levels of expression became comparable to those in well-nourished controls, although the bacillary burden in the malnourished animals continued to rise. Nevertheless, urinary and serum nitrate contents, an index of total nitric oxide (NO) production in vivo, were not detectably diminished in malnourished, mycobacteria-infected mice. In contrast to the selective and early reduction of lymphokines and the inducible form of nitric oxide synthase in the lung, a marked diminution of the granulomatous reaction was observed in malnourished mice throughout the entire course of infection in all tissues examined (lungs, liver, and spleen). Remarkably, the progressively fatal course of tuberculosis observed in the malnourished mice could be reversed by restoring a full protein (20%) diet. The results indicate that protein calorie malnutrition selectively compromises several components of the cellular immune response that are important for containing and restricting tuberculous infection, and suggest that malnutrition-induced susceptibility to some infectious diseases can be reversed or ameliorated by nutritional intervention.

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.

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.

Correlation of breath ammonia with blood urea nitrogen and creatinine during hemodialysis

We have spectroscopically determined breath ammonia levels in seven patients with end-stage renal disease while they were undergoing hemodialysis at the University of California, Los Angeles, dialysis center. We correlated these measurements against simultaneously taken blood samples that were analyzed for blood urea nitrogen (BUN) and creatinine, which are the accepted standards indicating the level of nitrogenous waste loading in a patient's bloodstream. Initial levels of breath ammonia, i.e., at the beginning of dialysis, are between 1,500 ppb and 2,000 ppb (parts per billion). These levels drop very sharply in the first 15–30 min as the dialysis proceeds. We found the reduction in breath ammonia concentration to be relatively slow from this point on to the end of dialysis treatment, at which point the levels tapered off at 150 to 200 ppb. For each breath ammonia measurement, taken at 15–30 min intervals during the dialysis, we also sampled the patient's blood for BUN and creatinine. The breath ammonia data were available in real time, whereas the BUN and creatinine data were available generally 24 h later from the laboratory. We found a good correlation between breath ammonia concentration and BUN and creatinine. For one of the patients, the correlation gave an R2 of 0.95 for breath ammonia and BUN correlation and an R2 of 0.83 for breath ammonia and creatinine correlation. These preliminary data indicate the possibility of using the real-time breath ammonia measurements for determining efficacy and endpoint of hemodialysis.

Involvement of Ureides in Nitrogen Fixation Inhibition in Soybean1

The sensitivity of N2 fixation to drought stress in soybean (Glycine max Merr.) has been shown to be associated with high ureide accumulation in the shoots, which has led to the hypothesis that N2 fixation during drought is decreased by a feedback mechanism. The ureide feedback hypothesis was tested directly by measuring the effect of 10 mm ureide applied by stem infusion or in the nutrient solution of hydroponically grown plants on acetylene reduction activity (ARA). An almost complete inhibition of ARA was observed within 4 to 7 d after treatment, accompanied by an increase in ureide concentration in the shoot but not in the nodules. The inhibition of ARA resulting from ureide treatments was dependent on the concentration of applied ureide. Urea also inhibited ARA but asparagine resulted in the greatest inhibition of nodule activity. Because ureides did not accumulate in the nodule upon ureide treatment, it was concluded that they were not directly inhibitory to the nodules but that their influence mediated through a derivative compound, with asparagine being a potential candidate. Ureide treatment resulted in a continual decrease in nodule permeability to O2 simultaneous with the inhibition of nitrogenase activity during a 5-d treatment period, although it was not clear whether the latter phenomenon was a consequence or a cause of the decrease in the nodule permeability to O2.

Reactive oxygen and nitrogen intermediates in the relationship between mammalian hosts and microbial pathogens

This review summarizes recent evidence from knock-out mice on the role of reactive oxygen intermediates and reactive nitrogen intermediates (RNI) in mammalian immunity. Reflections on redundancy in immunity help explain an apparent paradox: the phagocyte oxidase and inducible nitric oxide synthase are each nonredundant, and yet also mutually redundant, in host defense. In combination, the contribution of these two enzymes appears to be greater than previously appreciated. The remainder of this review focuses on a relatively new field, the basis of microbial resistance to RNI. Experimental tuberculosis provides an important example of an extended, dynamic balance between host and pathogen in which RNI play a major role. In diseases such as tuberculosis, a molecular understanding of host–pathogen interactions requires characterization of the defenses used by microbes against RNI, analogous to our understanding of defenses against reactive oxygen intermediates. Genetic and biochemical approaches have identified candidates for RNI-resistance genes in Mycobacterium tuberculosis and other pathogens.

Manganese oxide minerals: Crystal structures and economic and environmental significance

Manganese oxide minerals have been used for thousands of years—by the ancients for pigments and to clarify glass, and today as ores of Mn metal, catalysts, and battery material. More than 30 Mn oxide minerals occur in a wide variety of geological settings. They are major components of Mn nodules that pave huge areas of the ocean floor and bottoms of many fresh-water lakes. Mn oxide minerals are ubiquitous in soils and sediments and participate in a variety of chemical reactions that affect groundwater and bulk soil composition. Their typical occurrence as fine-grained mixtures makes it difficult to study their atomic structures and crystal chemistries. In recent years, however, investigations using transmission electron microscopy and powder x-ray and neutron diffraction methods have provided important new insights into the structures and properties of these materials. The crystal structures for todorokite and birnessite, two of the more common Mn oxide minerals in terrestrial deposits and ocean nodules, were determined by using powder x-ray diffraction data and the Rietveld refinement method. Because of the large tunnels in todorokite and related structures there is considerable interest in the use of these materials and synthetic analogues as catalysts and cation exchange agents. Birnessite-group minerals have layer structures and readily undergo oxidation reduction and cation-exchange reactions and play a major role in controlling groundwater chemistry.

Does a Low Nitrogen Supply Necessarily Lead to Acclimation of Photosynthesis to Elevated CO2?1

Long-term exposure of plants to elevated partial pressures of CO2 (pCO2) often depresses photosynthetic capacity. The mechanistic basis for this photosynthetic acclimation may involve accumulation of carbohydrate and may be promoted by nutrient limitation. However, our current knowledge is inadequate for making reliable predictions concerning the onset and extent of acclimation. Many studies have sought to investigate the effects of N supply but the methodologies used generally do not allow separation of the direct effects of limited N availability from those caused by a N dilution effect due to accelerated growth at elevated pCO2. To dissociate these interactions, wheat (Triticum aestivum L.) was grown hydroponically and N was added in direct proportion to plant growth. Photosynthesis did not acclimate to elevated pCO2 even when growth was restricted by a low-N relative addition rate. Ribulose-1, 5-bisphosphate carboxylase/oxygenase activity and quantity were maintained, there was no evidence for triose phosphate limitation of photosynthesis, and tissue N content remained within the range recorded for healthy wheat plants. In contrast, wheat grown in sand culture with N supplied at a fixed concentration suffered photosynthetic acclimation at elevated pCO2 in a low-N treatment. This was accompanied by a significant reduction in the quantity of active ribulose-1, 5-bisphosphate carboxylase/oxygenase and leaf N content.

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.

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.

Iron chelates bind nitric oxide and decrease mortality in an experimental model of septic shock.

The hydroxamic acid siderophore ferrioxamine B [FeIII(HDFB)+] and the iron complex of diethylenetri-aminepentaacetic acid [FeIII(DTPA)2-] protected mice against death by septic shock induced by Corynebacterium parvum + lipopolysaccharide. Although FeIII(DTPA)2- was somewhat more effective than FeIII(HDFB)+, the iron-free ligand H4DFB+ was significantly more effective than DTPA. The hydroxamic acid chelator has a much higher iron affinity than the amine carboxylate, allowing for more efficient formation of the FeIII(HDFB)+ complex upon administration of the iron-free ligand. Electrochemical studies show that FeIII(DTPA)2- binds NO stoichiometrically upon reduction to iron(II) at biologically relevant potentials to form a stable NO adduct. In contrast, FeIII(HDFB)+ is a stable and efficient electrocatalyst for the reduction of NO to N2O at biologically relevant potentials. These results suggest that the mechanism of protection against death by septic shock involves NO scavenging and that particularly effective drugs that operate a low dosages may be designed based on the principle of redox catalysis. These complexes constitute a new family of drugs that rely on the special ability of transition metals to activate small molecules. In addition, the wealth of information available on siderophore chemistry and biology provides an intellectual platform for further development.

Role of neuronal nitric oxide in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced dopaminergic neurotoxicity.

1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) causes nigrostriatal dopaminergic pathway damage similar to that observed in Parkinson disease (PD). To study the role of NO radical in MPTP-induced neurotoxicity, we injected MPTP into mice in which nitric oxide synthase (NOS) was inhibited by 7-nitroindazole (7-NI) in a time- and dose-dependent fashion. 7-NI dramatically protected MPTP-injected mice against indices of severe injury to the nigrostriatal dopaminergic pathway, including reduction in striatal dopamine contents, decreases in numbers of nigral tyrosine hydroxylase-positive neurons, and numerous silver-stained degenerating nigral neurons. The resistance of 7-NI-injected mice to MPTP is not due to alterations in striatal pharmacokinetics or content of 1-methyl-4-phenylpyridinium ion (MPP+), the active metabolite of MPTP. To study specifically the role of neuronal NOS (nNOS), MPTP was administered to mutant mice lacking the nNOS gene. Mutant mice are significantly more resistant to MPTP-induced neurotoxicity compared with wild-type littermates. These results indicate that neuronally derived NO mediates, in part, MPTP-induced neurotoxicity. The similarity between the MPTP model and PD raises the possibility that NO may play a significant role in the etiology of PD.

Polynitrosylated proteins: characterization, bioactivity, and functional consequences.

Chemical modification of proteins is a common theme in their regulation. Nitrosylation of protein sulfhydryl groups has been shown to confer nitric oxide (NO)-like biological activities and to regulate protein functions. Several other nucleophilic side chains -- including those with hydroxyls, amines, and aromatic carbons -- are also potentially susceptible to nitrosative attack. Therefore, we examined the reactivity and functional consequences of nitros(yl)ation at a variety of nucleophilic centers in biological molecules. Chemical analysis and spectroscopic studies show that nitrosation reactions are sustained at sulfur, oxygen, nitrogen, and aromatic carbon centers, with thiols being the most reactive functionality. The exemplary protein, BSA, in the presence of a 1-, 20-, 100-, or 200-fold excess of nitrosating equivalents removes 0.6 +/- 0.2, 3.2 +/- 0.4, 18 +/- 4, and 38 +/- 10, respectively, moles of NO equivalents per mole of BSA from the reaction medium; spectroscopic evidence shows the proportionate formation of a polynitrosylated protein. Analogous reaction of tissue-type plasminogen activator yields comparable NO protein stoichiometries. Disruption of protein tertiary structure by reduction results in the preferential nitrosylation of up to 20 thus-exposed thiol groups. The polynitrosylated proteins exhibit antiplatelet and vasodilator activity that increases with the degree of nitrosation, but S-nitroso derivatives show the greatest NO-related bioactivity. Studies on enzymatic activity of tissue-type plasminogen activator show that polynitrosylation may lead to attenuated function. Moreover, the reactivity of tyrosine residues in proteins raises the possibility that NO could disrupt processes regulated by phosphorylation. Polynitrosylated proteins were found in reaction mixtures containing interferon-gamma/lipopolysaccharide-stimulated macrophages and in tracheal secretions of subjects treated with NO gas, thus suggesting their physiological relevance. In conclusion, multiple sites on proteins are susceptible to attack by nitrogen oxides. Thiol groups are preferentially modified, supporting the notion that S-nitrosylation can serve to regulate protein function. Nitrosation reactions sustained at additional nucleophilic centers may have (patho)physiological significance and suggest a facile route by which abundant NO bioactivity can be delivered to a biological system, with specificity dictated by protein substrate.