Ability Of Nitric Oxide Synthase Research Articles

Evidence for Nitric Oxide Synthase Activity in Staphylococcus xylosus Mediating Nitrosoheme Formation

Staphylococcus xylosus is used as a starter culture in fermented meat products and contributes to color formation by the reduction of nitrate to nitrite. Nitrite is a food additive that is chemically turned to nitric oxide (NO) in meat but its safety has been questioned. The objective of this study was to determine the ability of NO synthase (NOS) of S. xylosus C2a to produce NO. For this purpose, a nos deletion mutant (Δnos) in S. xylosus was constructed and NO production was evaluated in a test based on its ability to form nitrosomyoglobin and nitrosoheme. Production of NO was abrogated in the Δnos mutant under aerobic conditions and reduced about 35-40% comparing to the wild type C2a under limited oxygenation. This mutant was sensitive to oxidative stress. The expression of genes encoding catalase was modulated in the mutant with an up-regulation of katA and a down-regulation of katB and katC. The Δnos mutant displayed high colony pigmentation after prolonged growth on agar medium. Finally, the Δnos mutant showed no growth in minimal medium. Growth was not restored in the minimal medium by complementation with nos, but was restored by either addition of phenylalanine or complementation with pdt, a gene that encodes a prephenate dehydratase involved in phenylalanine biosynthesis and co-transcribed with nos. Our findings clearly demonstrate NOS-mediated NO production in S. xylosus, a meat-associated coagulase-negative Staphylococcus.

Effects of cytokine-induced macrophages on the response of tumor cells to banoxantrone (AQ4N)

Tumor-associated macrophages (TAMs) are found in many solid tumors and have often been shown to accumulate in the hypoxic regions surrounding areas of necrosis. TAMs are the major site of expression of nitric oxide synthase (NOS), a heme-containing homodimeric enzyme consisting of oxygenase and reductase domains. The latter has a high degree of sequence homology to cytochrome P450 reductase and a functional consequence of this is the ability of NOS, under hypoxic conditions, to activate the bioreductive drugs tirapazamine and RSU1069. Banoxantrone (AQ4N) is a bioreductive prodrug activated in hypoxia by an oxygen-dependent two-electron reductive process to yield the topoisomerase II inhibitor AQ4. A feature of this process is that the final product could potentially show bystander cell killing. Thus, in this study, we investigated the ability of inducible NOS (iNOS)-expressing TAMs to activate AQ4N and elicit toxicity in cocultured human tumor cells. Murine macrophages were induced to overexpress iNOS by treatment with a combination of cytokines, mixed with HT1080 and HCT116 human tumor cells, and the toxicity of AQ4N was determined under aerobic or hypoxic conditions. The aerobic toxicity of AQ4N toward tumor cells was not affected through coculturing with macrophages. However, under hypoxic conditions, the induction of iNOS activity in the macrophages was associated with an increase in AQ4N metabolism and a substantial increase in tumor cell toxicity, which was dependent on the proportion of macrophages in the culture. This study is the first demonstration of TAM-mediated prodrug activation to result in bystander killing of human tumor cells.

INFLUENCE OF SALT ON SUBCELLULAR LOCALIZATION OF NITRIC OXIDE SYNTHASE ACTIVITY AND EXPRESSION IN THE RENAL INNER MEDULLA

1. The aims of this study were: (i) to characterize the subcellular localization of nitric oxide synthase (NOS) 1 and NOS3 activity and expression within the cytosolic, plasma membrane and intracellular membrane subcellular fractions of the renal inner medulla of rats; and (ii) to determine whether NOS1 and NOS3 activity and expression in subcellular fractions of the renal inner medulla are regulated by dietary salt intake. Although the NOS system is important in maintaining Na(+) and water homeostasis, the identity of the NOS isoform that is sensitive to dietary Na(+) remains unclear. In addition, subcellular localization of both NOS1 and NOS3 has been shown to regulate enzymatic activity and influence the ability of NOS to produce nitric oxide (NO). 2. Renal inner medullae were dissected from male Sprague-Dawley rats and separated into cytosolic, plasma membrane and intracellular membrane fractions for measurement of NOS activity and western blot analysis. 3. On a normal-salt diet, NOS activity and NOS1 and NOS3 protein expression were present in all three subcellular fractions, although total NOS activity was enriched in the intracellular membrane fraction. In response to a high-salt diet, urinary nitrate/nitrite (NO(x)) increased. Despite an increase in NO(x) excretion, total NOS activity in the renal inner medullary homogenate was decreased. There were no detectable differences in NOS activity in the subcellular fractions. Expression of NOS1 protein was decreased in the cytoplasmic and plasma membrane fractions, although maintained in the intracellular membrane fraction, in response to high salt. Expression of NOS3 protein was unaffected by high salt. 4. In conclusion, we hypothesize that NOS1 localization in the intracellular membrane is important in increasing NO production to aid Na(+) and water homeostasis.

Seasonal periodicity of enteric nitric oxide synthesis and its regulation in the snail,Helix lucorum

Abstract.The snailHelix lucorumhas been used as a model to study the adaptation of a nitric oxide (NO)‐forming enteric neural network to the long‐term resting period of summer estivation or winter hibernation. Quantification of the NO‐derived nitrite established that NO formation is confined to the nitric oxide synthase (NOS)‐containing myenteric network of the mid‐intestine. In active snails but not in resting snails, NO production could be enhanced by the NOS substratel‐arginine (l‐ARG, 1 mM). We followed the enteric NO synthesis in a snail population kept at natural conditions for 1 year. Our findings indicate that NO synthesis was depressed in July during entry to the estivation, had a peak in autumn before hibernation, and finally was reduced during hibernation. Monoamines (histamine, serotonin, and adrenalin) could inhibit the NO liberation in active snails. Cofactors of NOS (β‐NADPH, β‐NAD, FAD, FMN, Ca2+, TH4) did not alter the low nitrite production in hibernating snails. We conclude that enteric NO synthesis inH. lucorumhas a regular seasonal periodicity following the annual physiological cycles of terrestrial snails. During estivation or hibernation, NOS activity is blocked. Monoamines, the levels of which are elevated during hibernation, can trigger decreased NOS activity. The reduced activity of NOS cannot be restored by the administration of NOS cofactors; therefore, their absence cannot be the cause of the temporarily blocked L‐ARG/NO conversion ability of NOS.

Early diabetes as a model for testing the regulation of juxtaglomerular NOS I.

Dysregulation of kidney nitric oxide synthase (NOS) I may alter renal hemodynamics in diabetes. Four types of studies were performed in anesthetized 1- to 2-wk-streptozotocin diabetic rats. 1) Glomerular filtration rate (GFR) was measured before and during NOS I blockade. Subsequent addition of nonspecific NOS blocker tested for residual NO from other isoforms. Acute systemic NOS I blockade reduced GFR only in diabetics. Nonspecific NOS blockade had no additional effect on NOS I-blocked diabetics. 2) Renal blood flow (RBF) was monitored for evidence that tubuloglomerular feedback (TGF) resets during 1 h of continuous activation with benzolamide. NOS I blockade was added to test for the role of NOS I in TGF resetting. During 1 h of TGF activation in controls, RBF initially declined and then returned to baseline. In diabetic and NOS I-blocked rats, RBF declined and remained low. 3) The ability of NOS I blockade to increase the homeostatic efficiency of TGF in diabetes was tested by micropuncture in free-flowing nephrons. The addition of NOS I blocker to the tubular fluid increased TGF efficiency in control and diabetic rats. 4) The influence of distal salt delivery on local NOS I activity was tested by micropuncture. Henle's loop was perfused at varying rates with NOS I blocker while single-nephron GFR (SNGFR) from the late proximal tubule was measured. In controls, NOS I blockade mainly reduced SNGFR when flow through Henle's loop was high. In diabetics, NOS I blockade reduced SNGFR independently of flow through Henle's loop. In conclusion, normally, salt delivered to the macula densa (MD) exerts immediate control over MD NOS I activity. In diabetes, there is ongoing overactivity of NOS I that is not regulated by MD salt.

Nitric oxide synthesis is blocked in the enteral nervous system during dormant periods of the snail Helix lucorum.

During dormancy of terrestrial snails, the whole neuromodulation of the nervous system is deeply modified. In this work we studied the adaptation of a previously described, putatively nitric oxide (NO) forming enteral network to the long-term resting periods of the snail Helix lucorum. The standard NADPH diaphorase (NADPHd) technique, which is an accepted method for histochemical NO synthase (NOS) detection, labeled the same enteric neurons of the midintestine in active or hibernated snails. Quantification of the NO-derived nitrite by the Griess reaction established that the nitrite formation is confined to the NADPHd-reactive network containing the midintestinal segment. In active snails, the nitrite formation could be enhanced by the NOS substrate L-arginine (10 microM-1 mM), but decreased by the known NOS inhibitors 1 mM N(omega)-nitro-L-arginine (NOARG) and 10 mM aminoguanidine (AG). Application of 1 mM L-arginine and 1 mM NOARG decreased the amplitude of the midintestinal muscle contractile activity, but did not affect the rectal motility. In dormancy, the nitrite formation was reduced in the NADPHd-reactive midintestinal network. Application of l-arginine could not provoke nitrite production and did not influence the midintestinal motility. Our findings indicate that NO is involved in the neural transmission to intestinal muscles of gastropods, but enteric release of NO is blocked during dormancy. The decreased NO synthesis is possibly due to an as yet undefined mechanism, by which the L-arginine/NO conversion ability of NOS could temporarily be inhibited in the long-term resting period of H. lucorum.

Oxidation of the Ketoxime Acetoxime to Nitric Oxide by Oxygen Radical-Generating Systems

Ketoximes undergo a cytochrome P450-catalyzed oxidation to nitric oxide and ketones in liver microsomes. In addition, nitric oxide synthase (NOS) can catalyze the oxidative denitration of the >CN-OH group of amidoximes. The objective of this work was to characterize the oxidation of a ketoxime (acetoxime) and to assess the ability of NOS to catalyze the generation of nitric oxide/nitrogen monoxide ( · NO) from acetoxime. Acetoxime was oxidized to NO−2 (and NO−3) by microsomes enriched with several P450 isoforms, including CYP2E1, CYP1A1, and CYP2B1. Nitric oxide was identified as an intermediate in the overall reaction. Superoxide dismutase and catalase significantly inhibited the reaction. Exogenous iron increased the microsomal generation of NO−2 from acetoxime, while metal chelators (desferrioxamine, EDTA, DTPA) inhibited it. A Fenton-like system (Fe2+ plus H2O2, pH 7.4) consumed acetoxime with production of NO−2 and NO−3, whereas oxidation by superoxide or by H2O2 was inefficient. The results presented suggest a role for hydroxyl radical-like oxidants in the oxidation of acetoxime to nitric oxide. O-Acetylacetoxime and O-tert-butylacetoxime were not oxidized by a Fenton system or by liver microsomes to any significant extent. Formation of the 5,5′-dimethyl-1-pyrroline-N-oxide/ · OH adduct by a Fenton system was significantly inhibited by acetoxime, while O-acetylacetoxime and O-tert-butylacetoxime were inactive. These results suggest that the · OH-dependent oxidation of acetoxime initially proceeds via abstraction of a hydrogen atom from its hydroxyl group, as opposed to the oxidation of its >CN- function. HepG2 cells with low levels of expression of P450 did not significantly produce NO−2 from acetoxime, while HepG2 cells expressing CYP2E1 did, and this generation was blocked by a CYP2E1 inhibitor. Acetoxime was inactive either as a substrate or as an inhibitor of iNOS activity. These results indicate that reactive oxygen species play a key role in the oxidation of acetoxime to · NO by liver microsomes by a mechanism involving H abstraction from the OH moiety by hydroxyl radical-like oxidants and suggest the possibility that acetoxime may be an effective producer of · NO primarily in the liver by a pathway independent of NOS.

Food intake and the kidney: The right amounts at the right times

Food intake and the kidney: The right amounts at the right times

Small for gestational age: a new insight?

Acidosis reduces the ability of nitric oxide synthase to generate nitric oxide (NO) from L-arginine (L-arg), even if dietary intake or circulating plasma levels of L-arg are normal. During systemic acidemia, therefore, vascular perfusion in one or more organs may be compromised. Arginine is also a powerful anabolic amino acid. If dietary sources of L-arg are lower than normal, or if the production of NO is reduced even without frank acidemia, then vascular perfusion, and growth, and tissue repair are likely to be compromised. Two conditions in which acidemia is reported to occur, namely slow fetal growth in utero (acidemia is severe) and loss of bone and muscle in microgravity (acidemia is modest), are compared with respect to the accompanying alteration in the balance between acidemia and NO production.

Effects of transition metals on nitric oxide synthase catalysis.

The biosynthesis of nitric oxide (NO) by the enzyme NO synthase (NOS) proceeds by the hydroxylation of L-arginine to form NG-hydroxy-L-arginine followed by the conversion of NG-hydroxy-L-arginine to L-citrulline and NO. The previously identified requirements of this relatively complicated reaction include several protein-bound cofactors: cytochrome P450-type heme, flavin mononucleotide (FMN), flavin adenine dinucleotide (FAD), and tetrahydrobiopterin (H4B). In addition to L-arginine, NOS also requires the substrates NADPH and molecular oxygen. The role of H4B in NOS catalysis has long been a subject of debate and uncertainty fueled, in part, by the failure to detect any dependence of the NOS reaction on nonheme iron, a cofactor integral to catalysis in every other H4B-dependent enzyme. Here we report the ability of NOS to bind transition metals stoichiometrically, and demonstrate that the rate of catalysis is enhanced by nonheme iron. We also show that other divalent transition metals, including Cu, Zn, Co, and Ni, inhibit NOS catalysis. Also, the addition of Cu2+ to NOS inhibits heme reduction, whereas the addition of Fe2+ does not. Overall, the results appear to connect NOS to the known H4B/nonheme iron-dependent hydroxylases, and suggest a similar, if not identical, step in the NOS reaction mechanism.

Nitric oxide synthase inhibitors produce phencyclidine-like behavioral effects in pigeons

The present study assessed the ability of nitric oxide synthase (NOS) inhibitors to produce PCP-like behavioral effects in pigeons. Food-restricted pigeons were trained to discriminate between PCP (1.0 mg/kg i.m.) from saline in a two-key operant procedure. NOS inhibitors 7-nitroindazole (7-NI) and N ω-nitro- l-arginine methyl ester ( l-NAME) produced PCP-like discriminative stimulus effects. 7-NI (17.8 mg/kg, i.m.) completely generalized to PCP. l-NAME (320-1000 mg/kg) produced partial generalization to PCP. d-NAME, the enantiomer of l-NAME, did not produce PCP-appropriate behavior. l-NAME was approximately 200-times more potent i.c.v., but did not fully generalize to PCP. Both NOS inhibitors were effective in producing catalepsy, which is an effect commonly produced by competitive and uncompetitive NMDA antagonists. 7-NI (32 mg/kg) produced catalepsy in all subjects, whereas l-NAME (3200 mg/kg) produced catalepsy in 50% of the subjects. d-NAME did not produce catalepsy. Pretreatment with l-arginine (32–3200 mg/kg) prevented the PCP-like discriminative stimulus and cataleptic effects of 7-NI (17.8-32 mg/kg), demonstrating that 7-NI produced PCP-like effects through blockade of NO synthesis. The current studies reveal that NOS inhibitors induced two behavioral actions, discriminative stimulus effects and catalepsy, that are very selective for NMDA antagonists in pigeons.