Generation of NO by probiotic bacteria in the gastrointestinal tract

Probiotic bacteria elicit a number of beneficial effects in the gut but the mechanisms for these health promoting effects are not entirely understood. Recent in vitro data suggest that lactobacilli can utilise nitrate and nitrite to generate nitric oxide, a gas with immunomodulating and antibacterial properties. Here we further characterised intestinal NO generation by bacteria. In rats, dietary supplementation with lactobacilli and nitrate resulted in a 3-8 fold NO increase in the small intestine and caecum, but not in colon. Caecal NO levels correlated to nitrite concentration in luminal contents. In neonates, colonic NO levels correlated to the nitrite content of breast milk and faeces. Lactobacilli and bifidobacteria isolated from the stools of two neonates, generated NO from nitrite in vitro, whereas S. aureus and E. coli rapidly consumed NO. We here show that commensal bacteria can be a significant source of NO in the gut in addition to the mucosal NO production. Intestinal NO generation can be stimulated by dietary supplementation with substrate and lactobacilli. The generation of NO by some probiotic bacteria can be counteracted by rapid NO consumption by other strains. Future studies will clarify the biological role of the bacteria-derived intestinal NO in health and disease.     

A mammalian functional nitrate reductase that regulates nitrite and nitric oxide homeostasis

Inorganic nitrite (NO(2)(-)) is emerging as a regulator of physiological functions and tissue responses to ischemia, whereas the more stable nitrate anion (NO(3)(-)) is generally considered to be biologically inert. Bacteria express nitrate reductases that produce nitrite, but mammals lack these specific enzymes. Here we report on nitrate reductase activity in rodent and human tissues that results in formation of nitrite and nitric oxide (NO) and is attenuated by the xanthine oxidoreductase inhibitor allopurinol. Nitrate administration to normoxic rats resulted in elevated levels of circulating nitrite that were again attenuated by allopurinol. Similar effects of nitrate were seen in endothelial NO synthase-deficient and germ-free mice, thereby excluding vascular NO synthase activation and bacteria as the source of nitrite. Nitrate pretreatment attenuated the increase in systemic blood pressure caused by NO synthase inhibition and enhanced blood flow during post-ischemic reperfusion. Our findings suggest a role for mammalian nitrate reduction in regulation of nitrite and NO homeostasis.     

Gastrointestinal nitric oxide generation in germ-free and conventional rats

Nitric oxide (NO) is a central mediator of various physiological events in the gastrointestinal tract. The influence of the intestinal microflora for NO production in the gut is unknown. Bacteria could contribute to this production either by stimulating the mucosa to produce NO, or they could generate NO themselves. Using germ-free and conventional rats, we measured gaseous NO directly in the gastrointestinal tract and from the luminal contents using a chemiluminescence technique. Mucosal NO production was studied by using an NO synthase (NOS) inhibitor, and to evaluate microbial contribution to the NO generation, nitrate was given to the animals. In conventional rats, luminal NO differed profoundly along the gastrointestinal tract with the greatest concentrations in the stomach [>4,000 parts per billion (ppb)] and cecum (approximately 200 ppb) and lower concentrations in the small intestine and colon (< or =20 ppb). Cecal NO correlated with the levels in incubated luminal contents. NOS inhibition lowered NO levels in the colon, without affecting NO in the stomach and in the cecum. Gastric NO increased greatly after a nitrate load, proving it to be a substrate for NO generation. In germ-free rats, NO was low (< or =30 ppb) throughout the gastrointestinal tract and absent in the incubated luminal contents. NO also remained low after a nitrate load. Our results demonstrate a pivotal role of the intestinal microflora in gastrointestinal NO generation. Distinctly compartmentalized qualitative and quantitative NO levels in conventional and germ-free rats reflect complex host microbial cross talks, possibly making NO a regulator of the intestinal eco system.     

A new technique to determine hydrogen excreted by gnotobiotic rats

A new system, that allowed the monitoring of hydrogen (H2) excretion by gnotobiotic rats without affecting their defined microbial status, was developed. The system consists of an isolator containing a chamber for an experimental animal, and a life-support system (LSS), with a sampling port outside the isolator connected to it. H2 accumulation in the system was measured by analysing a defined volume of gas after removal. H2 concentrations were determined with an electrochemical cell or by gas chromatography. To validate this technique, H2 excretion by germ-free (GF) and mono-associated rats fed a chemically defined diet was measured after oral application of lactulose. Mono-associated rats had been obtained by colonizing GF rats with a H2-producing Clostridium perfringens type A strain isolated from human faeces of a healthy volunteer. Application of 50 mg lactulose to the mono-associated rats resulted in a significant increase in H2 excretion. The net H2 excretion was 7.82+/-1.28 ml H2 in 12 h corresponding to a net maximal rate of 1.1+/-0.3 ml H2/h. In contrast, in experiments with GF rats, less than 0.13 ml H2 were detectable within 12 h. The technique presented is a useful tool for studying bacterial H2 metabolism in vivo under gnotobiotic conditions.     

The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora

The activities of four bacterial biotransformation enzymes (beta-glucosidase, beta-glucuronidase, nitrate reductase and nitroreductase) were measured in the caecal contents of conventional flora rats or germ-free rats contaminated with a mixed, human faecal flora and compared with activities present in a fresh human stool preparation. Both the conventional flora rats and the rats inoculated with a human flora exhibited an enzyme profile generally similar to that of human faeces, although the conventional rat flora exhibited negligible nitrate reductase activity. The enzyme profile remained essentially unaltered in both human flora preparations following supplementation of the diet with pectin, whereas the conventional rat flora responded to this plant cell wall carbohydrate with a significant increase in nitrate reductase activity. The results demonstrate that enzymic activities of the human faecal microflora can be simulated in rats associated with a mixed population of human intestinal bacteria.     

The use of rats associated with a human faecal flora as a model for studying the effects of diet on the human gut microflora

The activities of four bacterial biotransformation enzymes (β-glucosidase, β-glucuronidase, nitrate reductase and nitroreductase) were measured in the caecal contents of conventional flora rats or germ-free rats contaminated with a mixed, human faecal flora and compared with activities present in a fresh human stool preparation. Both the conventional flora rats and the rats inoculated with a human flora exhibited an enzyme profile generally similar to that of human faeces, although the conventional rat flora exhibited negligible nitrate reductase activity. The enzyme profile remained essentially unaltered in both human flora preparations following supplementation of the diet with pectin, whereas the conventional rat flora responded to this plant cell wall carbohydrate with a significant increase in nitrate reductase activity. The results demonstrate that enzymic activities of the human faecal microflora can be simulated in rats associated with a mixed population of human intestinal bacteria.     

Nitrate Reduction to Nitrite,Nitric Oxide and Ammonia by Gut Bacteria under Physiological Conditions

The biological nitrogen cycle involves step-wise reduction of nitrogen oxides to ammonium salts and oxidation of ammonia back to nitrites and nitrates by plants and bacteria. Neither process has been thought to have relevance to mammalian physiology; however in recent years the salivary bacterial reduction of nitrate to nitrite has been recognized as an important metabolic conversion in humans. Several enteric bacteria have also shown the ability of catalytic reduction of nitrate to ammonia via nitrite during dissimilatory respiration; however, the importance of this pathway in bacterial species colonizing the human intestine has been little studied. We measured nitrite, nitric oxide (NO) and ammonia formation in cultures of Escherichia coli, Lactobacillus and Bifidobacterium species grown at different sodium nitrate concentrations and oxygen levels. We found that the presence of 5 mM nitrate provided a growth benefit and induced both nitrite and ammonia generation in E.coli and L.plantarum bacteria grown at oxygen concentrations compatible with the content in the gastrointestinal tract. Nitrite and ammonia accumulated in the growth medium when at least 2.5 mM nitrate was present. Time-course curves suggest that nitrate is first converted to nitrite and subsequently to ammonia. Strains of L.rhamnosus, L.acidophilus and B.longum infantis grown with nitrate produced minor changes in nitrite or ammonia levels in the cultures. However, when supplied with exogenous nitrite, NO gas was readily produced independently of added nitrate. Bacterial production of lactic acid causes medium acidification that in turn generates NO by non-enzymatic nitrite reduction. In contrast, nitrite was converted to NO by E.coli cultures even at neutral pH. We suggest that the bacterial nitrate reduction to ammonia, as well as the related NO formation in the gut, could be an important aspect of the overall mammalian nitrate/nitrite/NO metabolism and is yet another way in which the microbiome links diet and health.     

Decreased leukocyte recruitment by inorganic nitrate and nitrite in microvascular inflammation and NSAID-induced intestinal injury

Nitric oxide (NO) generated by vascular NO synthases can exert anti-inflammatory effects, partly through its ability to decrease leukocyte recruitment. Inorganic nitrate and nitrite, from endogenous or dietary sources, have emerged as alternative substrates for NO formation in mammals. Bioactivation of nitrate is believed to require initial reduction to nitrite by oral commensal bacteria. Here we investigated the effects of inorganic nitrate and nitrite on leukocyte recruitment in microvascular inflammation and in NSAID-induced small-intestinal injury. We show that leukocyte emigration in response to the proinflammatory chemokine MIP-2 is reduced by 70% after 7 days of dietary nitrate supplementation as well as by acute intravenous nitrite administration. Nitrite also reduced leukocyte adhesion to a similar extent and this effect was inhibited by the soluble guanylyl cyclase inhibitor ODQ, whereas the effect on emigrated leukocytes was not altered by this treatment. Further studies in TNF-α-stimulated endothelial cells revealed that nitrite dose-dependently reduced the expression of ICAM-1. In rats and mice subjected to a challenge with diclofenac, dietary nitrate prevented the increase in myeloperoxidase and P-selectin levels in small-intestinal tissue. Antiseptic mouthwash, which eliminates oral nitrate reduction, markedly blunted the protective effect of dietary nitrate on P-selectin levels. Despite attenuation of the acute immune response, the overall ability to clear an infection with Staphylococcus aureus was not suppressed by dietary nitrate as revealed by noninvasive IVIS imaging. We conclude that dietary nitrate markedly reduces leukocyte recruitment to inflammation in a process involving attenuation of P-selectin and ICAM-1 upregulation. Bioactivation of dietary nitrate requires intermediate formation of nitrite by oral nitrate-reducing bacteria and then probably further reduction to NO and other bioactive nitrogen oxides in the tissues.     

Active secretion and protective effect of salivary nitrate against stress in human volunteers and rats

Up to 25% of the circulating nitrate in blood is actively taken up, concentrated, and secreted into saliva by the salivary glands. Salivary nitrate can be reduced to nitrite by the commensal bacteria in the oral cavity or stomach and then further converted to nitric oxide (NO) in vivo, which may play a role in gastric protection. However, whether salivary nitrate is actively secreted in human beings has not yet been determined. This study was designed to determine whether salivary nitrate is actively secreted in human beings as an acute stress response and what role salivary nitrate plays in stress-induced gastric injury. To observe salivary nitrate function under stress conditions, alteration of salivary nitrate and nitrite was analyzed among 22 healthy volunteers before and after a strong stress activity, jumping down from a platform at the height of 68 m. A series of stress indexes was analyzed to monitor the stress situation. We found that both the concentration and the total amount of nitrate in mixed saliva were significantly increased in the human volunteers immediately after the jump, with an additional increase 1 h later (p<0.01). Saliva nitrite reached a maximum immediately after the jump and was maintained 1 h later. To study the biological functions of salivary nitrate and nitrite in stress protection, we further carried out a water-immersion-restraint stress (WIRS) assay in male adult rats with bilateral parotid and submandibular duct ligature (BPSDL). Intragastric nitrate, nitrite, and NO; gastric mucosal blood flow; and gastric ulcer index (UI) were monitored and nitrate was administrated in drinking water to compensate for nitrate secretion in BPSDL animals. Significantly decreased levels of intragastric nitrate, nitrite, and NO and gastric mucosal blood flow were measured in BPSDL rats during the WIRS assay compared to sham control rats (p<0.05). Recovery was observed in the BPSDL rats upon nitrate administration. The WIRS-induced UI was significantly higher in the BPSDL animals compared to controls, and nitrate administration rescued the WIRS-induced gastric injury in BPSDL rats. In conclusion, this study suggests that stress promotes salivary nitrate secretion and nitrite formation, which may play important roles in gastric protection against stress-induced injury via the nitrate-dependent NO pathway.     

Ethyl nitrite is produced in the human stomach from dietary nitrate and ethanol,releasing nitric oxide at physiological pH: potential impact on gastric motility

Background. Nitric oxide (^∙NO), a ubiquitous molecule involved in a plethora of signaling pathways, is produced from dietary nitrate in the gut through the so-called nitrate–nitrite–NO pathway. In the stomach, nitrite derived from dietary nitrate triggers a network of chemical reactions targeting endogenous and exogenous biomolecules, thereby producing new compounds with physiological activity.Objective. The aim of this study was to ascertain whether compounds with physiological relevance are produced in the stomach upon consumption of nitrate- and ethanol-rich foods.Design. Human volunteers consumed a serving of lettuce (source of nitrate) and alcoholic beverages (source of ethanol). After 15 min, samples of the gastric headspace were collected and ethyl nitrite was identified by GC–MS. Wistar rats were used to study the impact of ethyl nitrite on gastric smooth muscle relaxation at physiological pH.Result. Nitrogen oxides, produced from nitrite in the stomach, induce nitrosation of ethanol from alcoholic beverages in the human stomach yielding ethyl nitrite. Ethyl nitrite, a potent vasodilator, is produced in vivo upon the consumption of lettuce with either red wine or whisky. Moreover, at physiological pH, ethyl nitrite induces gastric smooth muscle relaxation through a cGMP-dependent pathway. Overall, these results suggest that ethyl nitrite is produced in the gastric lumen and releases ^∙NO at physiological pH, which ultimately may have an impact on gastric motility. Systemic effects may also be expected if ethyl nitrite diffuses through the gastric mucosa reaching blood vessels, therefore operating as a ^∙NO carrier throughout the body.Conclusion. These data pinpoint posttranslational modifications as an underappreciated mechanism for the production of novel molecules with physiological impact locally in the gut and highlight the notion that diet may fuel compounds with the potential to modulate gastrointestinal welfare.     

Characterization of the rat oral microbiome and the effects of dietary nitrate

The nitrate–nitrite–NO pathway to nitric oxide (NO) production is a symbiotic pathway in mammals that is dependent on nitrate reducing oral commensal bacteria. Studies suggest that by contributing NO to the mammalian host, the oral microbiome helps maintain cardiovascular health. To begin to understand how changes in oral microbiota affect physiological functions such as blood pressure, we have characterized the Wistar rat nitrate reducing oral microbiome. Using 16S rRNA gene sequencing and analysis we compare the native Wistar rat tongue microbiome to that of healthy humans and to that of rats with sodium nitrate and chlorhexidine mouthwash treatments. We demonstrate that the rat tongue microbiome is less diverse than the human tongue microbiome, but that the physiological activity is comparable, as sodium nitrate supplementation significantly lowered diastolic blood pressure in Wistar rats and also lowers blood pressure (diastolic and systolic) in humans. We also show for the first time that sodium nitrate supplementation alters the abundance of specific bacterial species on the tongue. Our results suggest that the changes in oral nitrate reducing bacteria may affect nitric oxide availability and physiological functions such as blood pressure. Understanding individual changes in human oral microbiome may offer novel dietary approaches to restore NO availability and blood pressure.     

A note: gut bacteria produce components of a locust cohesion pheromone

AIMS: Faecal pellets from germ-free locusts were used as culture media to determine the ability of locust gut bacteria to synthesize phenolic components of the locust cohesion pheromone. METHODS AND RESULTS: Inoculation of germ-free faecal pellets with Pantoea agglomerans, a species commonly isolated from locusts, resulted in the release of large amounts of guaiacol and small amounts of phenol, both of which are components of the locust cohesion pheromone. Two other locust-derived species, Klebsiella pneumoniae pneumoniae and Enterobacter cloacae, also produced guaiacol from germ-free faecal pellets, but the opportunistic locust pathogen, Serratia marcescens, did not. The most likely precursor for guaiacol is the plant-derived vanillic acid, which is present in large amounts in the faeces of both conventional and germ-free locusts. CONCLUSIONS: These observations are consistent with previous ones, that locust gut bacteria are responsible for the production of components of the locust cohesion pheromone. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings illustrate how an insect can adapt to make use of a common bacterial metabolite produced by one or more of its indigenous gut bacterial species. This observation has implications for our appreciation of insect gut microbiota interactions.     

Dietary nitrite induces occludin nitration in the stomach

The clinical implications of the nitrate–nitrite–nitric oxide pathway have been extensively studied in recent years. However, the physiological impact of bioactive nitrogen oxides produced from dietary nitrate has remained largely elusive. Here, we report a hitherto unrecognized nitrite-dependent nitrating pathway that targets tight junction proteins in the stomach. Inorganic nitrate, nitrite or saliva obtained after the consumption of lettuce were administered by oral gavage to Wistar rats. The enterosalivary circulation of nitrate was allowed to occur for 4?h after which the animals were euthanized and the stomach collected. Nitrated occludin was detected by immunoprecipitation in the gastric epithelium upon inorganic nitrite administration (p??NO production rates from inorganic and salivary nitrite under simulated gastric conditions, suggests that competing reactions at acidic pH determine the production of nitrating agents (^?NO₂) or other, more stable, oxides. Accordingly, it is shown in vitro that salivary nitrite yields higher steady state concentrations of ^?NO (0.37?±?0.01?μM) than sodium nitrite (0.12?±?0.03?μM). Dietary-dependent reactions involving the production of nitrogen oxides should be further investigated as, in the context of occludin nitration, the consumption of green leafy vegetables (with high nitrate content), if able to modulate gut barrier function, may have important implications in the context of leaky gut disorders.     

Evaluation of nitric oxide production by lactobacilli

Six strains of Lactobacillus fermentum and Lactobacillus plantarum were investigated for nitric oxide (NO) production. First, the potential presence of NO synthase was examined. None of the strains of L. fermentum and L. plantarum examined produced NO from L-arginine under aerobic conditions. Interestingly, all L. fermentum strains expressed strong L-arginine deiminase activity. All L. fermentum strains produced NO in MRS broth, but the NO was found to be chemically derived from nitrite, which was produced by L. fermentum from nitrate present in the medium. Indeed all L. fermentum strains express nitrate reductase under anaerobic conditions. Moreover, one strain, L. fermentum LF1, had nitrate reductase activity under aerobic conditions. It was also found that L. fermentum strains JCM1173 and LF1 possessed ammonifying nitrite reductase. The latter strain also had denitrifying nitrite reductase activity at neutral pH under both anaerobic and aerobic conditions. The LF1 strain is thus capable of biochemically converting nitrate to NO. NO and nitrite produced from nitrate by lactobacilli may constitute a potential antimicrobial mechanism. studied in a rat acute liver injury model (Adawi et al. 1997). The results indicate that Lactobacillus plantarum DSM 9842 may possess NOS (Adawi et al. 1997). However, NO production from L-arginine has not been investigated in pure cultures of L. plantarum. According to the results of a 15N enrichment experiment, traces of (NO2-+NO3-)-N (total oxidised nitrogen: TON), which seemed to be formed by the resting cells of Lactobacillus fermentum IFO3956, appeared to be derived from L-arginine (Morita et al. 1997). Therefore, it was suggested that L. fermentum may possess a NOS. However, NO produced from L-arginine was not directly measured and a NOS inhibitor test was not performed by Morita et al. (1997). It is known that L-arginine deiminase (ADI) in bacteria may convert L-arginine to NH4+ (Cunin et al. 1986), which may be further oxidised to TON via nitrification by bacteria. Therefore, 15N enrichment experiments could not definitely conclude that L. fermentum possess NOS to convert L-arginine directly to NO. In this study, six Lactobacillus strains belonging to L. plantarum and L. fermentum were measured for NO production in MRS broth. The metabolism of nitrate and L-arginine by the Lactobacillus cell suspensions was also studied. The possibility that NO and nitrite production by lactobacilli may be a potential probiotic trait is also discussed.     

Increase in gastric pH reduces hypotensive effect of oral sodium nitrite in rats

The new pathway nitrate-nitrite-nitric oxide (NO) has emerged as a physiological alternative to the classical enzymatic pathway for NO formation from l-arginine. Nitrate is converted to nitrite by commensal bacteria in the oral cavity and the nitrite formed is then swallowed and reduced to NO under the acidic conditions of the stomach. In this study, we tested the hypothesis that increases in gastric pH caused by omeprazole could decrease the hypotensive effect of oral sodium nitrite. We assessed the effects of omeprazole treatment on the acute hypotensive effects produced by sodium nitrite in normotensive and L-NAME-hypertensive free-moving rats. In addition, we assessed the changes in gastric pH and plasma levels of nitrite, NO(x) (nitrate+nitrite), and S-nitrosothiols caused by treatments. We found that the increases in gastric pH induced by omeprazole significantly reduced the hypotensive effects of sodium nitrite in both normotensive and L-NAME-hypertensive rats. This effect of omeprazole was associated with no significant differences in plasma nitrite, NO(x), or S-nitrosothiol levels. Our results suggest that part of the hypotensive effects of oral sodium nitrite may be due to its conversion to NO in the acidified environment of the stomach. The increase in gastric pH induced by treatment with omeprazole blunts part of the beneficial cardiovascular effects of dietary nitrate and nitrite.     

Dietary nitrate increases gastric mucosal blood flow and mucosal defense

Salivary nitrate from dietary or endogenous sources is reduced to nitrite by oral bacteria. In the acidic stomach, nitrite is further reduced to bioactive nitrogen oxides, including nitric oxide (NO). In this study, we investigated the gastroprotective role of nitrate intake and of luminally applied nitrite against provocation with diclofenac and taurocholate. Mucosal permeability ((51)Cr-EDTA clearance) and gastric mucosal blood flow (laser-Doppler flowmetry) were measured in anesthetized rats, either pretreated with nitrate in the drinking water or given acidified nitrite luminally. Diclofenac was given intravenously and taurocholate luminally to challenge the gastric mucosa. Luminal NO content and nitrite content in the gastric mucus were determined by chemiluminescence. The effect of luminal administration of acidified nitrite on the mucosal blood flow was also investigated in endothelial nitric oxide synthase-deficient mice. Rats pretreated with nitrate or given nitrite luminally had higher gastric mucosal blood flow than controls. Permeability increased more during the provocation in the controls than in the nitrate- and nitrite-treated animals. Dietary nitrate increased luminal NO levels 50 times compared with controls. Nitrate intake also resulted in nitrite accumulation in the loosely adherent mucous layer; after removal of this mucous layer, blood flow was reduced. Nitrite administrated luminally in endothelial nitric oxide synthase-deficient mice increased mucosal blood flow. We conclude that dietary nitrate and direct luminal application of acidified nitrite decrease diclofenac- and taurocholate-induced mucosal damage. The gastroprotective effect likely involves a higher mucosal blood flow caused by nonenzymatic NO production. These data suggest an important physiological role of nitrate in the diet.     

Mono-association of mice with non-cultivable,intestinal, segmented,filamentous bacteria

A technique is described so that mice mono-associated with non-cultivable, segmented filamentous bacteria (SFB's) can be produced for the first time. As SFB donors, mice were used which had an intestinal microflora consisting of both SFB's and bacteria of the genus Clostridium. Recipients were germ-free mice. It was demonstrated that the intraileal inoculation method was more effective than the orogastric route. Therefore, intestinal homogenates of donor mice were treated with filtered ethanol, diluted and administered intraileally to recipient mice. Evidence is presented that cage mates of the recipient mice were mono-associated with SFB's. The availability of these animals, i.e. in vivo monocultures of SFB's, allows taxonomic and functional characterization of SFB's, which was as yet not possible.Abbreviation SFB Segmented filamentous bacterium     

Oxidation of iron-nitrosyl-hemoglobin by dehydroascorbic acid releases nitric oxide to form nitrite in human erythrocytes

The reaction of deoxyhemoglobin with nitric oxide (NO) or nitrite ions (NO 2 (-)) produces iron-nitrosyl-hemoglobin (HbNO) in contrast to the reaction with oxyhemoglobin, which produces methemoglobin and nitrate (NO 3 (-)). HbNO has not been associated with the known bioactivities of NO. We hypothesized that HbNO in erythrocytes could be an important source of bioactive NO/nitrite if its oxidation was coupled to the ascorbic acid (ASC) cycle. Studied by absorption and electron paramagnetic resonance (EPR) spectroscopy, DHA oxidized HbNO to methemoglobin and liberated NO from HbNO as determined by chemiluminescence. Both DHA and ascorbate free radical (AFR), the intermediate between ASC and DHA, enhanced NO oxidation to nitrite, but not nitrate; nor did either oxidize nitrite to nitrate. DHA increased the basal levels of nitrite in erythrocytes, while the reactions of nitrite with hemoglobin are slow. In erythrocytes loaded with HbNO, HbNO disappeared after DHA addition, and the AFR signal was detected by EPR. We suggest that the ASC-AFR-DHA cycle may be coupled to that of HbNO-nitrite and provide a mechanism for the endocrine transport of NO via hemoglobin within erythrocytes, resulting in the production of intracellular nitrite. Additionally, intracellular nitrite and nitrate seem to be largely generated by independent pathways within the erythrocyte. These data provide a physiologically robust mechanism for erythrocytic transport of NO bioactivity allowing for hormone-like properties.     

Nitrate reductase activity of bacteria in saliva of term and preterm infants

The salivary glands of adults concentrate nitrate from plasma into saliva where it is converted to nitrite by bacterial nitrate reductases. Nitrite can play a beneficial role in adult gastrointestinal and cardiovascular physiology. When nitrite is swallowed, some of it is converted to nitric oxide (NO) in the stomach and may then exert protective effects in the gastrointestinal tract and throughout the body. It has yet to be determined either when newborn infants acquire oral nitrate reducing bacteria or what the effects of antimicrobial therapy or premature birth may be on the bacterial processing of nitrate to nitrite. We measured nitrate and nitrite levels in the saliva of adults and both preterm and term human infants in the early weeks of life. We also measured oral bacterial reductase activity in the saliva of both infants and adults, and characterized the species of nitrate reducing bacteria present. Oral bacterial conversion of nitrate to nitrite in infants was either undetectable or markedly lower than the conversion rates of adults. No measurable reductase activity was found in infants within the first two weeks of life, despite the presence of oral nitrate reducing bacteria such as Actinomyces odontolyticus, Veillonella atypica, and Rothia mucilaginosa. We conclude that relatively little nitrite reaches the infant gastrointestinal tract due to the lack of oral bacterial nitrate reductase activity. Given the importance of the nitrate-nitrite-NO axis in adults, the lack of oral nitrate-reducing bacteria in infants may be relevant to the vulnerability of newborns to hypoxic stress and gastrointestinal tract pathologies.     

Characterization of Tn5 mutants deficient in dissimilatory nitrite reduction in Pseudomonas sp. strain G-179, which contains a copper nitrite reductase.

Tn5 was used to generate mutants that were deficient in the dissimilatory reduction of nitrite for Pseudomonas sp. strain G-179, which contains a copper nitrite reductase. Three types of mutants were isolated. The first type showed a lack of growth on nitrate, nitrite, and nitrous oxide. The second type grew on nitrate and nitrous oxide but not on nitrite (Nir-). The two mutants of this type accumulated nitrite, showed no nitrite reductase activity, and had no detectable nitrite reductase protein bands in a Western blot (immunoblot). Tn5 insertions in these two mutants were clustered in the same region and were within the structural gene for nitrite reductase. The third type of mutant grew on nitrate but not on nitrite or nitrous oxide (N2O). The mutant of this type accumulated significant amounts of nitrite, NO, and N2O during anaerobic growth on nitrate and showed a slower growth rate than the wild type. Diethyldithiocarbamic acid, which inhibited nitrite reductase activity in the wild type, did not affect NO reductase activity, indicating that nitrite reductase did not participate in NO reduction. NO reductase activity in Nir- mutants was lower than that in the wild type when the strains were grown on nitrate but was the same as that in the wild type when the strains were grown on nitrous oxide. These results suggest that the reduction of NO and N2O was carried out by two distinct processes and that mutations affecting nitrite reduction resulted in reduced NO reductase activity following anaerobic growth with nitrate.