Oxidation of NG-hydroxy-L-arginine by nitric oxide synthase: evidence for the involvement of the heme in catalysis

The involvement of the protoporphyrin IX heme iron of macrophage nitric oxide synthase (NOS) in the oxidation of NG-hydroxy-L-arginine (L-NHA) to nitric oxide (NO) and citrulline was investigated by carbon monoxide (CO) inhibition studies and binding difference spectroscopy. A CO:oxygen mixture (80:20) was found to inhibit the reaction by 33% with L-NHA as a substrate compared to 57% with L-arginine. Spectral perturbations were observed upon the addition of L-NHA to oxidized NOS, producing a type I binding difference spectrum with a maximum at 384 nm and minimum at 420 nm. In addition, L-NHA was incapable of reducing anaerobic oxidized NOS in the absence of NADPH. These studies support the involvement of the heme in the oxidation of L-NHA to NO and citrulline, indicating that the heme functions in both of the currently characterized oxidative steps of the NOS reaction.     

Effects of various imidazole ligands on heme conformation in endothelial nitric oxide synthase

We have evaluated the influence of a series of substituted imidazoles on the heme structure of endothelial nitric oxide synthase (eNOS). Optical, MCD, and EPR spectra reveal widely differing effects on heme spin state and geometry. 1-Substituted imidazoles always yield low-spin heme complexes, but the size of the 2- and 4-substituent influences their structural effects on the heme. Methyl substituents lead to low-spin complexes while the bulky phenyl group yields high-spin complexes. The only exception to this behavior is provided by 2-aminoimidazole. Although this compound has three functional groups which can serve as an axial ligand to the heme, its binding to eNOS leads to a pure high-spin complex. This result can only be interpreted as due to a direct binding of 2-aminoimidazole to the guanidine binding subdomain of L-arginine. MCD spectra also imply that an O-ligand is present in the low-spin resting eNOS, while EPR data reveal the presence of two low-spin heme complexes in resting eNOS and its imidazole complexes. EPR also distinguishes four different high-spin forms of eNOS generated by different imidazole analogues. This series of ligands promises to be useful in probing the subtle structural difference among the active sites of three NOS isozymes and in developing selective inhibitors to these important enzymes.     

Establishment of patterned thalamocortical connections does not require nitric oxide synthase

Subplate neurons are early-generated neurons that project into the overlying neocortex and are required for the formation of ocular dominance columns. A subset of subplate neurons express nitric oxide synthase (NOS) and produce nitric oxide (NO), a neuronal messenger thought to be involved in adult hippocampal synaptic plasticity and also in the establishment of certain specific connections during visual system development. Here, we examine whether the NOS-containing subplate neurons are involved in ocular dominance column formation in the ferret visual system. Ocular dominance columns form in ferrets between postnatal day 35 (P35) and P60. NOS expression in the visual subplate is low at birth, increases to a maximum at the onset of ocular dominance column formation, and falls thereafter. Nevertheless, blockade of NOS with daily injections of nitroarginine from P14 to P56 fails to prevent the formation of ocular dominance columns, although NOS activity is reduced by >98%. To test further a requirement for NOS in the patterning of connections during CNS development, we examined the cortical barrels in the somatosensory system of mice carrying targeted disruptions of NOS that also received injections of nitroarginine; cortical barrels formed normally in these animals. In addition, barrel field plasticity induced by whisker ablation at birth was normal in nitroarginine-injected NOS knock-out mice. Thus, despite the dynamic regulation of NOS in subplate neurons, NO is unlikely to be essential for the patterning of thalamocortical connections either in visual or somatosensory systems.     

Selective inhibition of human glial inducible nitric oxide synthase by interferon-beta: implications for multiple sclerosis

Nitric oxide generated from the inducible nitric oxide synthase (iNOS) has been implicated in the pathogenesis of multiple sclerosis. Because significant species- and cell-specific differences exist in the expression of iNOS, we used primary human glial cell cultures to screen for an inhibitor of iNOS expression. Remarkably, among numerous soluble factors tested, interferon-beta (IFN-beta) alone showed a selective and potent inhibition of interleukin-1beta/interferon-gamma (IL-1beta/IFN-gamma)-induced iNOS expression in astrocytes. Inhibition of iNOS may provide a mechanism by which IFN-beta can ameliorate inflammation and cytotoxicity in the central nervous system of patients with multiple sclerosis.     

Sperm nitric oxide and motility: the effects of nitric oxide synthase stimulation and inhibition

Nitric oxide (NO) is synthesized from L-arginine by a family of enzymes known as the nitric oxide synthases (NOS). We have recently shown a NOS similar to constitutive brain NOS (bNOS) and endothelial NOS (ecNOS) to be present in spermatozoa. The aim of this study is to investigate NO production by human spermatozoa and the effects of stimulation and inhibition of NOS. This was carried out using the Iso-NO, an isolated NO meter and sensor, which provides rapid, accurate and direct measurements of NO. Semen samples with normozoospermic and asthenozoospermic profiles were prepared using a direct swim-up technique. Basal concentrations of NO and stimulated NO production were measured after exposure to the calcium ionophore (A23187; 0.01-10 microM) a potent activator of constitutive NOS. NO production in human spermatozoa was significantly increased by the addition of A23187 30 seconds after stimulation. Furthermore, this response was greatly diminished by pre-incubating the samples with competitive inhibitors of L-arginine, the substrate for NOS, before treatment with calcium ionophore. In the presence of N(G)-nitro-L-arginine methyl ester (L-NAME), N(G)-nitro-L-arginine (L-NA) or N(G)-methyl-L-arginine (L-NMMA; all at 10 microM), NO production was inhibited with a rank order of potency L-NAME > L-NMMA > L-NA which is in accordance with the inhibition of an endothelial type of constitutive NOS.     

The versatile and complex enzymology of nitric oxide synthase

The biogenesis of nitric oxide is catalyzed by nitric oxide synthase (NOS) which forms L-citrulline and NO from L-arginine. Here we review the enzymology of NOS. We discuss its modular structure, its prosthetic groups and cofactors, and we provide a brief account of present knowledge regarding cellular targeting and regulation of the different isoforms. The various reactions which are catalyzed by NOS are reviewed, and an inventory of different inhibitor types is given. Special attention is paid to the role of the cofactor tetrahydrobiopterin (BH4) and of the dimeric structure, and to the possibility that the main product of NOS catalysis under some conditions may not be NO. Based on a number of recent observations, we postulate that neuronal NOS with one equivalent of BH4 per dimer (a state which may be physiologically relevant) catalyzes the concerted formation of peroxynitrite.     

Perivascular localization of nitric oxide synthase in the rat adenohypophysis: potential implications for function and cell-cell interaction

Effects of activation of protein kinase C (PKC) on N-methyl-D-aspartate) NMDA receptor function were analyzed by quantitative autoradiography using [3H]MK-801 in rat brain slices. The density of [3H]MK-801 binding was highest in hippocampus and high levels were found in cortex, striatum and thalamus. Levels in brainstem and molecular layer of cerebellum were low. The receptor binding was markedly decreased in almost all areas by addition of 2. 5 mM Mg2+. After activation of PKC by 100 nM phorbol-12, 13-dibutyrate (PDBu), [3H]MK-801 binding was increased in most areas, but binding levels were not changed in brainstem and cerebellum. The elevated [3H]MK-801 binding produced by PDBu was significantly inhibited by addition of Mg2+ except in inferior colliculus and cerebellum. These results suggest that activation of PKC potentiates NMDA receptor function in a region-specific manner in the rat brain.     

Comparative functioning of dihydro- and tetrahydropterins in supporting electron transfer, catalysis, and subunit dimerization in inducible nitric oxide synthase

The nitric oxide synthases (NOS) are the only heme-containing enzymes that require tetrahydrobiopterin (BH4) as a cofactor. Previous studies indicate that only the fully reduced (i.e., tetrahydro) form of BH4 can support NO synthesis. Here, we characterize pterin-free inducible NOS (iNOS) and iNOS reconstituted with eight different tetrahydro- or dihydropterins to elucidate how changes in pterin side-chain structure and ring oxidation state regulate iNOS. Seven different enzyme properties that are important for catalysis and are thought to involve pterin were studied. Only two properties were found to depend on pterin oxidation state (i.e., they required fully reduced tetrahydropterins) and were independent of side chain structure: NO synthesis and the ability to increase heme-dependent NADPH oxidation in response to substrates. In contrast, five properties were exclusively dependent on pterin side-chain structure or stereochemistry and were independent of pterin oxidation state: pterin binding affinity, and its ability to shift the heme iron to its high-spin state, stabilize the ferrous heme iron coordination structure, support heme iron reduction, and promote iNOS subunit assembly into a dimer. These results clarify how structural versus redox properties of the pterin impact on its multifaceted role in iNOS function. In addition, the data reveal that during NO synthesis all pterin-dependent steps up to and including heme iron reduction can take place independent of the pterin ring oxidation state, indicating that the requirement for fully reduced pterin occurs at a point in catalysis beyond heme iron reduction.     

Impact of surgery on nitric oxide in rats: evidence for activation of inducible nitric oxide synthase

We investigated the effect of euvolemic surgical preparation, on chemical indices of activity of the nitric oxide (NO) system, in anesthetized, acutely prepared rats. The urinary excretion of NO2+NO3 (UNOXV) and cGMP (UcGMPV) increased progressively during the experiment. Pretreatment with aminoguanidine or dexamethasone, inhibitors of inducible NO synthase (iNOS), prevented the increase in UNOXV and UcGMPV but had no impact on mean arterial pressure (BP), renal vascular resistance (RVR) or GFR. Since these variables did not change in the conscious rat, the increased UNOXV results from some aspect of the acute surgical preparation. When acutely prepared rats received L-NAME, a non-specific NOS inhibitor, BP and RVR increased but paradoxical increases in UNOXV and UcGMPV were also seen. Nonselective NOS inhibition (+L-NAME) was fatal in 50% of acutely prepared rats, causing cardiac contracture. The same dose of L-NAME produced no deaths in either conscious chronically catheterized rats or in acutely prepared rats, previously subjected to sterile surgery and acute L-NAME in the conscious state. These data indicate that acute, nonsterile surgery induces expression of iNOS, but that the additional NO generated has no obvious cardiovascular/renal actions. Acute UNOXV and UcGMPV do not predict total NO production, or "hemodynamically active" NO. Generalized NO inhibition in rats acutely stressed by surgery/anesthesia can be fatal.     

The nitric oxide pathway in pre-eclampsia: pathophysiological implications

Ageing constitutes a risk factor for magnesium deficit. Primary magnesium deficit originates from two etiological mechanisms: deficiency and depletion. Primary magnesium deficiency is due to insufficient magnesium intake. Dietary amounts of magnesium are marginal in the whole population whatever the age. Nutritional deficiencies are more pronounced in institutionalized than in free-living ageing groups. Primary magnesium depletion is due to dysregulation of factors controlling magnesium status: intestinal magnesium hypoabsorption, reduced magnesium bone uptake and mobilisation, sometimes urinary leakage, hyperadrenoglucocorticism by decreased adaptability to stress, insulin-resistance and adrenergic hyporeceptivity. Secondary magnesium deficit in ageing largely results from various pathologies and treatments common to elderly persons: i.e. non insulin dependent diabetes mellitus and use of hypermagnesuric diuretics. Magnesium deficit may participate in the clinical pattern of ageing: mainly neuromuscular, cardiovascular and renal symptomatologies. The consequences of hyperadrenoglucocorticism--whose non response to dexamethasone suppression test appears the simplest marker--may concern immunosuppression, muscle atrophy, centralization of fat mass, osteoporosis, hyperglycemia, hyperlipidemia, atherosclerosis, disturbances in mood and mental performances through accelerated hippocampal ageing particularly. Treatment of magnesium deficiency requires simple oral physiological magnesium supplementation. Treatment of the different types of magnesium depletion leads to a more or less specific control of pathophysiological disturbances of the required magnesium substrate. Open and double blind studies on the effects of the treatments of magnesium deficiency and of magnesium depletions in geriatic populations are too scarce. Further study is necessary to assess the accurate place of magnesium deficit in the physiopathology of ageing.     

Immunohistochemical localization of nitric oxide synthase in normal human skin: expression of endothelial-type and inducible-type nitric oxide synthase in keratinocytes

Nitric oxide (NO) is a critical mediator of various biological functions. NO is generated from L-arginine by nitric oxide synthase (NOS), which has three isoforms; endothelial-type NOS (eNOS) and brain-type NOS (bNOS) are constitutive enzymes, and inducible-type NOS (iNOS) is expressed after stimulation. We investigated the expression of NOS in normal human skin by an immunohistochemical technique and western blotting analysis. In human skin, epidermal keratinocytes and the outer root sheath were labeled with not only eNOS antibody but also with iNOS antibody. Both eNOS and iNOS protein in epidermal keratinocytes were confirmed by western blotting. eNOS immunoreactivity was observed in endothelial cells, fibroblasts, the arrector pili muscle, apocrine secretory gland, eccrine coiled duct, and eccrine secretory gland. bNOS immunoreactivity was observed in mast cells. No staining with anti-bNOS antibody was observed in any other cell type. Our present findings suggest that epidermal keratinocytes in normal human skin contain both eNOS and iNOS.     

The effect of ketotifen on nitric oxide synthase activity

1. We studied the effect of ketotifen, a second generation H1-receptor antagonist on nitric oxide synthase (NOS) activity in colonic mucosa and in renal tissues, and on rat renal haemodynamics in vivo. 2. Ketotifen (100 micrograms ml-1) increased human colonic NOS activity from 3.7 +/- 0.6 to 14.5 +/- 1.3 nmol g-1 min-1 (P < 0.005, ANOVA). In rat renal cortical and medullary tissues ketotifen increased NOS activity by 55% and 86%, respectively (P < 0.001). The stimulation of NOS activity was attenuated by NADPH deletion and by the addition of N omega nitro-L-arginine methyl ester (L-NAME) or aminoguanidine, but not by [Ca2+] deprivation. NOS activity was unaffected by two other H1-antagonists, diphenhydramine and astemizole, or by the structurally related cyproheptadine. Renal cortical NOS activity was also significantly stimulated 90 min after intravenous administration of ketotifen to anaesthetized rats. 3. Ketotifen administration to anaesthetized rats induced modest declines in blood pressure and reduced total renal, cortical and outer medullary vascular resistance. This is in contrast to diphenhydramine, which did not induce renal vasodilatation. 4. We conclude that ketotifen stimulates NOS activity by mechanisms other than H1-receptor antagonism. The association of this effect with therapeutic characteristics of ketotifen and the clinical implications of these findings are yet to be defined.     

Inhibition of inducible nitric oxide synthase by peroxisome proliferator-activated receptor agonists: correlation with induction of heme oxygenase 1

Genetic knock-out in mice of peroxisome proliferator-activated receptor-alpha (PPAR alpha) can prolong inflammation in response to leukotriene B4. Although cyclooxygenase 2 has been shown to be induced by PPAR activation, the effect of PPAR agonists on the key inflammatory enzyme systems of nitric oxide synthase (NOS) and stress proteins has not been investigated. The effect on these of naturally occurring eicosanoid PPAR agonists (leukotriene B4 and 8(S)-hydroxyeicosatetraenoic acid, which are PPAR alpha selective; PGA2, PGD2, PGJ2, and delta12PGJ2, which are PPAR gamma selective) and the synthetic PPAR alpha agonist Wy14,643 was examined in activated RAW264.7 murine macrophages. Leukotriene B4 and 8(S)-hydroxyeicosatetraenoic acid stimulated nitrite accumulation, indicative of enhanced NOS activity. PGA2, PGD2, PGJ2, delta12PGJ2, and Wy14,643 reduced nitrite accumulation, with delta12PGJ2 being the most effective. The mechanism behind this reduction was examined using Western blotting. Inhibition of nitrite accumulation was associated with a fall in inducible NOS protein and an induction of heme oxygenase 1, correlating both dose dependently and temporally. Other proteins examined (cyclooxygenase 2, heme oxygenase 2, heat shock protein 70, and glucose-regulated protein 78) were unaffected. The data suggest that naturally occurring PPAR agonists can inhibit the inducible NOS enzyme pathway. This inhibition may be mediated by modulation of the stress protein, heme oxygenase 1. Thus, the generation of eicosanoid breakdown products during inflammation may contribute to its eventual resolution by activation of the PPAR system. This system may thus represent a novel target for therapeutic intervention in inflammatory disease.     

Sleep and nitric oxide: effects of 7-nitro indazole, inhibitor of brain nitric oxide synthase

The in vitro amplification method for heterologous gene expression in mammalian cells is based on the stable transfection of cells with long, linear DNA molecules having several copies of complete expression units, coding for the gene of interest, linked to one terminal unit, coding for the selectable marker. DNA concatenamers containing additional expression units can also be prepared: we exploited this feature by co-polymerizing expression units coding for granulocyte colony-stimulating factor (G-CSF) with cassettes for dihydrofolate reductase (DHFR) and for neomycin (Nm) resistance, as selectable markers. We were thus able to obtain high level production of G-CSF in chinese hamster ovary (CHO) dhfr- cells by combining in vitro amplification to just one step of in vivo amplification. This approach required a considerably shorter time than the classical, stepwise amplification by methotrexate.     

Role of nitric oxide in rat nephrotoxic nephritis: comparison between inducible and constitutive nitric oxide synthase

Nitric oxide (NO), generated by inducible NO synthase (iNOS) in migrating macrophages, is increased in glomerulonephritis. This study investigates the effect of NO inhibition on rat nephrotoxic nephritis (NTN) to clarify the role of NO production in glomerular damage. NTN was induced in Sprague Dawley rats by an injection of an anti-glomerular basement membrane (GBM) antibody. Urinary nitrite excretion and nitrite release from kidney slices (5.47 +/- 1.19 versus 2.15 +/- 0.73 nmol/mg protein, NTN versus Control, P < 0.05) were increased in NTN on day 2. Glomerular macrophage infiltration and intercellular adhesion molecule (ICAM)-1 expression increased from day 2. iNOS expression was increased in interstitial macrophages. Glomerular endothelial cell NOS (ecNOS) expression evaluated by counting immunogold particles along GBM was suppressed (0.06 +/- 0.02 versus 0.35 +/- 0.04 gold/micron GBM, P < 0.0001). Glomerular damage developed progressively. NG-nitro-L-arginine methyl ester (L-NAME), which inhibits both iNOS and ecNOS and aminoguanidine (AG), a relatively selective inhibitor for iNOS, equally suppressed nitrite in urine and renal tissue. Glomerular ICAM-1 expression and macrophage infiltration were reduced by L-NAME, but not by AG. Expression of ecNOS was significantly increased by L-NAME (0.91 +/- 0.08, P < 0.0001 versus NTN), but slightly by AG (0.18 +/- 0.04). AG significantly and L-NAME slightly attenuated the glomerular damage at day 4. In conclusion, suppression of iNOS prevents glomerular damage in the early stage of NTN. Treatment by L-NAME reduces macrophage infiltration by suppression of ICAM-1 expression, which may be explained by an increase in ecNOS expression.     

Effect of a nitric oxide synthase inhibitor and a glucocorticosteroid on exhaled nitric oxide

Nitric oxide (NO) is produced by a variety of cells within the respiratory tract, including inflammatory epithelial cells. NO has been detected in the exhaled air of normal human subjects, and its concentration is raised in asthmatic patients. To study whether exhaled NO arises from the respiratory tract, we administered a NO synthase (NOS) inhibitor, NG-monomethyl-L-arginine (L-NMMA), by inhalation (490 mg) in a double-blind randomized manner in nine normal and six asthmatic subjects. Because exhaled NO may arise from an inducible isoform of NO synthase that may be inhibited by glucocorticosteroids, we also studied the effects of oral prednisolone (30 mg orally for 3 d) in seven normal and six asthmatic subjects in a separate double-blind crossover study with matched placebo. After nebulized L-NMMA, there was a significant fall in peak exhaled NO compared with saline control values, with a mean fall of 43.6 +/- 5.6% in normal subjects (p < 0.01) and of 39.7 +/- 6.5% (p < 0.01) in asthmatic subjects, which persisted for 4 h. There were no effects of L-NMMA inhalation on heart rate, blood pressure, or FEV1 in either normal or asthmatic patients. Administration of oral prednisolone (30 mg) resulted in a fall in exhaled NO concentrations in asthmatic subjects by 21.6 +/- 5.0% at 48 h (p < 0.01) but no significant change in normal subjects. These data suggest that NOS inhibitors may be safely given in normal and asthmatic patients and that the increased exhaled NO seen in asthmatic patients is likely to be caused by induction of inducible NOS.     

Apoptosis and expression of inducible nitric oxide synthase are mutually exclusive in renal mesangial cells

Nitric oxide (NO) is a multipurpose messenger molecule, important for blood vessel relaxation, neuronal communication, and antimicrobial activities. The generation of NO from L-arginine is catalyzed by NO synthase (NOS). An inducible form of NOS, iNOS, was first characterized in macrophages and then in many other tissues and cells, including renal mesangial cells. Mesangial cells play a crucial role in the regulation of the glomerular filtration rate as well as in the pathophysiology of certain forms of glomerulonephritis in which mesangial cells and macrophages produce NO in high amounts. Because reports have associated NO production with apoptotic cell death in macrophages and we recently demonstrated NO-mediated apoptosis in mesangial cells, we searched for the relationship between in situ iNOS induction and apoptosis by iNOS immunocytochemistry and terminal desoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) staining. RAW 264.7 macrophages exhibited homogeneous iNOS expression and apoptotic nuclei in the iNOS-containing cells upon stimulation with interferon-gamma and lipopolysaccharide. In contrast, stimulated rat mesangial cells stained heterogeneously for iNOS, depending on cell passage and iNOS-stimulating pathway. Mesangial cells expressing iNOS did not display signs of apoptosis and, vice versa, cells showing characteristic features of apoptosis did not stain for iNOS. Thus, our study suggests that mesangial cells react to stimulation by interleukin-1 and/or cAMP-elevating compounds with mutually exclusive responses, either by expression of iNOS or by undergoing programmed cell death.     

The interplay of nitric oxide and peroxynitrite with signal transduction pathways: implications for disease

Since the discovery that at least one form of endothelium derived relaxing factor is nitric oxide (NO), numerous studies have uncovered diverse roles for this free radical in a variety of physiological and pathophysiological processes. NO production, a process mediated by a family of enzymes termed NO synthases, has been detected in most cell types. Many of the effects of NO are thought to be mediated through its direct interaction with specific and defined cell signaling pathways. The nature of such interactions are highly dependent on the concentration of NO and cell type. Furthermore, specific NO derived reaction products, such as peroxynitrite, also have the potential to effect cell signal transduction events. As with NO, this can occur through diverse mechanisms and depends on concentration and cell type. It is perhaps not surprising that the reported effects of NO in different disease states are often conflicting. In this brief overview, a framework for placing these apparently disparate properties of NO will be described and will focus on the effects of NO and peroxynitrite on signaling pathways.     

Insulin down-regulates the inducible nitric oxide synthase pathway: nitric oxide as cause and effect of diabetes?

Evidence in this paper indicates that insulin can down-regulate the inducible nitric oxide synthase (iNOS) pathway in vivo. The iNOS pathway is up-regulated in diabetes-prone rats and mice and is associated with an autoimmune process. However, the results presented here indicate that macrophage nitric oxide (NO) production and iNOS mRNA expression are also elevated in rats or mice made diabetic by streptozotocin injection in which there is no primary autoimmune component. Insulin administration reduces NO production in autoimmune-prone and streptozotocin-induced diabetic rodents. Finally, insulin decreases macrophage NO production in normal hosts. These results indicate that the autoimmune paradigm is inadequate to explain increased NO in diabetes. As a potential mechanism to explain insulin-mediated regulation of NO production, TGF-1 may be involved because 1) macrophages from diabetic mice produce less TGF-beta1 than macrophages from normal hosts; 2) the circulating TGF-beta1 level is lower in diabetic mice; and 3) insulin administration increases circulating TGF-beta1 in normal mice. Together, these results provide evidence that increased NO in diabetes is not only a cause but also an effect of beta-cell destruction and results in part from a heretofore unrecognized immunomodulatory activity of insulin.