Search

Objective: Barrett's metaplasia might develop if GORD causes oesophageal squamous cells to convert into columnar cells. Acid and bile exposures upregulate columnar differentiation genes like CDX2 in oesophageal squamous cells, but it is not known if such exposures downregulate squamous differentiation genes like SOX2. In addition to acid and bile, patients with GORD also have high oesophageal concentrations of nitric oxide (NO). This study aims to determine how acid, bile salts and NO affect genes that influence oesophageal cell phenotype., Design: Oesophageal squamous cells from patients with Barrett's oesophagus were exposed to acidic bile salts or NOC-9 (an NO donor). SOX2, p63 (squamous transcription factor) and CDX2 mRNAs were measured by quantitative RT-PCR. SOX2 and its regulatory Akt pathway proteins were evaluated by western blotting. S-nitrosylation by NO was blocked by dithiothreitol. Immunohistochemistry for SOX2 was performed on the oesophagus of rats with surgically induced GORD which were fed diets with and without nitrite supplementation., Results: In oesophageal squamous cells, NO profoundly decreased SOX2 protein and caused a significantly greater decrease in SOX2 mRNA than did acidic bile salts. NO also decreased p63 and increased CDX2 expression. NO caused S-nitrosylation of Akt, blocking its phosphorylation. Akt pathway inhibition by LY294002 or Akt siRNA reduced SOX2 mRNA. Rats fed with nitrite-supplemented diets exhibited weaker SOX2 oesophageal staining than rats fed with normal diets., Conclusions: In oesophageal squamous cells, NO blocks SOX2 expression through Akt S-nitrosylation. NO also increases CDX2 and decreases p63 expression. By triggering molecular events preventing squamous differentiation while promoting intestinal differentiation, NO might contribute to Barrett's pathogenesis., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/)

Over the last 3 decades, the incidence of esophageal adenocarcinoma has dramatically increased in Western countries; a similar increase may be observed in Asian countries in the near future. Esophageal adenocarcinoma arises from a sequential gastroesophageal reflux disease (GERD) spectrum from reflux erosive esophagitis, to Barrett's esophagus, and finally to esophageal adenocarcinoma. At present, gastric acid and bile are assumed to be primarily involved in the etiology of the GERD spectrum. We reported in 2002 that, at the gastroesophageal junction in humans, abundant amounts of nitric oxide (NO) are generated luminally through the entero-salivary re-circulation of dietary nitrate. Since then, we have carried out a series of experiments to demonstrate that NO diffuses into the adjacent epithelium at cytotoxic levels. This diffusion results in disruption of the epithelial barrier function, exacerbation of inflammation, acceleration of columnar transformation in the esophagus (Barrett's esophagus) via the induction of caudal-type homeobox 2, and the shifting of carcinogenic N-nitroso compound formation from the luminal to epithelial compartment. These results suggest that, in addition to conventionally recognized causative factors, luminal NO could also be involved in the pathogenesis of the GERD spectrum. In addition, we recently showed that there is a prominent gender-related difference in NO-related cytotoxicity in the esophagus and that estrogen attenuated the esophageal tissue damage via the estrogen receptor in female rats. The role of estrogen in attenuating the esophageal tissue damage in NO-related esophageal damage could explain the well-recognized male predominance in the GERD spectrum in humans., (© 2014 Journal of Gastroenterology and Hepatology Foundation and Wiley Publishing Asia Pty Ltd.)

Objective: There is a strong male predominance of oesophageal adenocarcinoma, which might be related to the higher prevalence of precursor lesions such as erosive reflux oesophagitis in men compared with women. This experiment investigated the gender difference in a reflux oesophagitis model of rats and explored the potential role of oestrogen in controlling oesophageal tissue damage., Design: An acid-reflux oesophagitis model was surgically produced in male and female rats, and ascorbic acid in the diet and sodium nitrite in the drinking water were administered to half of either group to provoke luminal exogenous nitric oxide (NO) as an exacerbating agent. Seven days after the surgery, the oesophagus was excised, and the injury area, myeloperoxidase activity and pro-inflammatory cytokine levels were measured. Furthermore, 17β-oestradiol was administered to ovariectomised female rats or male rats, which then underwent reflux oesophagitis surgery., Results: While there was no gender difference in oesophageal damage in the baseline model, oesophageal damage was more intensively observed in males than in females in the presence of exogenous NO administration. While oesophageal damage was increased in ovariectomised rats compared with sham ovariectomised, exacerbated oesophageal damage was attenuated by the replacement of 17β-oestradiol. In addition, exacerbated oesophageal damage in male rats was suppressed by 17β-oestradiol., Conclusion: This is the first study showing the prominent gender difference in the severity of oesophageal tissue damage in a gastro-oesophageal reflux disease-related animal model, highlighting the critical involvement of oestrogen in controlling gastro-oesophageal reflux disease-related oesophageal epithelial injury.

Objective: The high concentration of nitric oxide (NO) around the gastro-esophageal junction (GEJ) might play an important role in the development of Barrett's esophagus (BE), a precursor of Barrett's adenocarcimona. Although previous studies revealed that the expression of caudal-type homeobox 2 (CDX2), an important marker of BE, might be induced through Epidermal Growth Factor Receptor (EGFR), the roles of NO in this signal transduction remain unclear., Material and Methods: First, we investigated the expressions of EGFR, CDX2 and nitrotyrosine by immunohistochemical study for BE and squamous epithelium of human specimens. Second, we studied the effect of peroxynitrite, peroxynitrite stimulator, SIN-1, or NO donor, NOC7, on the expression of phosphorylated EGFR and CDX2 in KYSE30, an EGFR-rich human esophageal squamous cell carcinoma cell-line. Specific inhibitors for EGFR, AG1478 and small interfering RNA for EGFR (EGFR-siRNA) were employed to elucidate the role of EGFR in the induction of CDX2., Results: The immunohistochemical study revealed that the expressions of EGFR, CDX2 and nitrotyrosine in BE were stronger than those in squamous epithelium with positive correlations. Exposure to peroxynitrite, SIN-1 or NOC7 induced EGFR phosphorylation and CDX2 expression in dose- and time-dependent manners. Both EGFR phosphorylation and CDX2 induction were significantly diminished by AG 1478 and EGFR-siRNA., Conclusions: We revealed for the first time that extrinsic NO might directly induce CDX2 expression through EGFR phosphorylation. We suggested that NO had an important role in the development of BE from squamous epithelium around GEJ.

Exposure of the esophageal mucosa to refluxed gastroduodenal contents is recognized to be an important risk factor for Barrett's esophagus (BE). At the human gastroesophageal junction, nitric oxide is generated luminally through the enterosalivary recirculation of dietary nitrate, and in cases with gastroesophageal reflux, the site of luminal nitric oxide generation could shift to the distal esophagus. The aim of this study is to investigate whether exogenous luminal nitric oxide could promote the development of BE in rats. Sodium nitrite plus ascorbic acid were administered to a rat surgical model of BE, in which the gastroduodenal contents were refluxed into the esophagus to generate exogenous luminal nitric oxide in the esophagus by the acid-catalyzed chemical reaction between the 2 reagents. The emergence of BE was evaluated histologically in the early phase (several weeks) after the surgery with or without exogenous nitric oxide administration. To elucidate the histogenesis of BE, CDX2, MUC2 and MUC6 expressions were investigated immunohistochemically. Coadministration of sodium nitrite plus ascorbic acid significantly accelerated the timing of emergence and increased the area of BE compared with controls. Administration of either reagent alone did not show any promotive effects on BE formation. Immunohistochemically, the columnar epithelium thus induced was similar to the specialized intestinal metaplasia in human BE. The results of this animal model study suggest that exogenous luminal nitric oxide could be involved in the pathogenesis of the columnar transformation of the esophagus. Further studies in human are warranted.

Objective: Cytotoxic concentrations of nitric oxide are generated luminally at the gastroesophageal junction through the entero-salivary recirculation of dietary nitrate in humans. The site of luminal nitric oxide generation shifts to the lower esophagus when gastric acid is refluxed into the esophagus. The aim of this study was to investigate the influence of persistent administration of exogenous nitric oxide on esophageal damage., Material and Methods: 0.1% sodium nitrite and/or 1% ascorbic acid was administered in an established rat acid-refluxed esophagitis model. Co-administration of both reactants in this model is thought to induce high concentrations of nitric oxide luminally in the esophagus by an acid-catalyzed chemical reaction when refluxed gastric acid is present. The tissue damage was evaluated by a macroscopic lesion index and myeloperoxidase activity. Nitrotyrosin was assessed immunohistochemically as a footprint of peroxynitrite formation., Results: Co-administration of sodium nitrite and ascorbic acid induced a 4- to 5-fold increase in the esophageal damage compared with baseline reflux esophagitis, while the damage was unchanged when either of the reagents alone was given. Nitrotyrosine was strongly stained in the tissue from the co-administration. Treatment of superoxide scavengers efficiently prevented the exacerbation of esophageal damage by exogenous nitric oxide exposure, suggesting an essential role of superoxide in esophageal damage., Conclusions: Exogenous luminal nitric oxide greatly exacerbated the tissue damage of reflux esophagitis. Diffusion of the luminal nitric oxide into the adjacent superoxide-enriched inflamed tissue of the esophagus could lead to the production of the highly toxic agent peroxynitrite, thus causing exacerbation of the esophageal damage.

In human, high concentrations of nitric oxide are generated at the gastro-oesophageal junction through entero-salivary recirculation of dietary nitrate. Nitric oxide is known to have a high affinity for Fe-S cluster proteins. The aim of this study is to investigate whether nitric oxide arising from the lumen diffuses into the adjacent tissue where it can interact with Fe-S proteins both in a rat animal model and human. An electron paramagnetic resonance detectable complex, dinitrosyl dithiolato iron complex (DNIC), was used as a biomarker for the interaction between Fe-S proteins and nitric oxide. The generation of the complex was evaluated in resected gastric tissue of nitrite-administered rat or biopsy specimens from human after nitrate ingestion. The activity of aconitase, one of the Fe-S cluster proteins, was also determined. The signal of the complex was observed at the rat gastro-oesophageal junction where luminal generation of nitric oxide from nitrite was maximal, and the intensity increased in a dose- and time-dependent manner. The appearance of the complex was accompanied by a significant inhibition of the aconitase activity at that site. The complex appeared in biopsy specimens from the gastro-oesophageal junction in three of five men after nitrate ingestion. Since DNIC is considered to be a decomposition product when Fe-S cluster proteins interact with nitric oxide, the appearance of the signal provides direct evidence that nitric oxide arising from the lumen can destroy such proteins. DNIC formation may represent the cellular mechanism responsible for the high prevalence of disease at the gastro-oesophageal junction.

Background: In humans, high concentrations of nitric oxide are generated luminally at the gastro-oesophageal junction through enterosalivary recirculation of dietary nitrate., Aim: To investigate whether luminal nitric oxide can diffuse into the adjacent digestive tissue and alter tissue integrity., Methods: We designed an animal model using Wistar rats in which physiological concentrations of nitrite and acidified ascorbic acid were administered separately so that the two reactants first meet to form nitric oxide at the gastro-oesophageal junction. Luminal and tissue concentrations of nitric oxide were measured with an electrode and an electron paramagnetic resonance spectrometer, respectively. Concentrations of glutathione in the tissue were measured as a marker of nitrosative stress., Results: High concentrations of luminal nitric oxide were generated locally at the gastro-oesophageal junction of nitrite administered rats, reproducing a phenomenon observed in humans. High levels of nitric oxide were also detected largely in the superficial epithelium of the gastro-oesophageal junction. The concentration of tissue glutathione at the gastro-oesophageal junction was significantly lower in nitrite administered rats compared with control rats, whereas that in the distal stomach was similar in the two rat groups., Conclusions: Using an animal model, this study demonstrated that nitric oxide generated in the lumen diffuses into the adjacent gastric tissue to a substantial degree, leading to localised consumption of glutathione in the tissue. Nitrosative stress induced by this mechanism may be involved in the high prevalence of inflammation and metaplasia, and subsequent development of neoplastic disease at this site.

The integrity of gastric mucosa during endotoxemia is maintained by the balance of inflammatory mediators, such as prostanoids originated from cyclooxygenase-2 (COX-2) and nitric oxide (NO) from inducible nitric-oxide synthase (iNOS). Thus, we elucidated in vivo cross talk between prostanoids and NO in gastric mucosa during endotoxemia, using an iNOS-specific inhibitor, N-(3-(aminomethyl)benzyl)acetamidine (1400W); a nonspecific COX inhibitor, indomethacin; and a COX-2-specific inhibitor, N-(2-[cyclohexyloxy]-4-nitrophenyl)methanesulfonamide (NS-398). Gastric mucosal NO and prostaglandin E2 (PGE2), a predominant product of COX, expressed as mean +/- S.D. of five rats per group, were assayed by electron paramagnetic resonance spectrometry and enzyme immunoassay technique, respectively. The levels of NO and PGE2 increased gradually up to 6 h after administration of bacterial lipopolysaccharide (LPS) (NO: control, 0.35 +/- 0.16; 6 h, 13.3 +/- 3.3 nmol/g tissue/30 min; and PGE2: control, 288 +/- 16; 6 h, 806 +/- 15 pg/g tissue). Pretreatment with 1400W decreased the increase in NO level without any effect on the PGE2 level (NO, 4.0 +/- 0.4 nmol/g tissue/30 min; PGE2, 788 +/- 26 pg/g tissue). In contrast, treatment with indomethacin and NS-398 inhibited not only PGE2 level but also NO level in a dose-dependent manner without any significant effect on both iNOS and COX protein and mRNA expression. These results demonstrate that in the LPS-treated rat gastric mucosa, PGE2 enhances the release of NO after activation of iNOS, although NO produced by iNOS does not stimulate the release of PGE2 by COXs. The effect of COX activity on iNOS-NO pathway can be important in the regulation of gastric mucosal integrity in inflammatory states.

Nitric oxide (NO) is a potent inducer of apoptosis, and its cytotoxicity is closely related to mitochondrial dysfunction. In this study we investigated the effects of a F0F1-ATPase inhibitor, oligomycin, and a mitochondrial respiratory chain complex III inhibitor, antimycin A, on NO-induced apoptosis. We used a normal rat gastric-epithelium cell line, RGM-1, treated with a pure NO donor, NOC-1 -1-hydroxy-2-oxo-3,3-bis(2-aminoethyl)-1-triazene - in the presence or absence of oligomycin or antimycin A. Changes in the expressions of Bax or Bcl-2 proteins, release of cytochrome C from mitochondria into the cytosol, activation of caspase-3, and changes in the mitochondrial membrane potential (DeltaPsi) were measured with the use of Western blotting, c43 lorimetric assays, and a mitochondrial potential sensor, JC-1 dye. Treatment with NOC-18 induced dose-dependent apoptotic cell death in RGM-1 cells. Cell death was accompanied by mitochondrial depolarization, increases in Bax protein expression and cytochrome C leakage, and, subsequently, caspase-3 activation. Oligomycin and antimycin A prevented NO-induced apoptosis in a dose-dependent fashion by preventing cytochrome C release independent of Bcl-2 expression. However, neither compound affected the up-regulation of Bax protein. On the one hand, oligomycin treatment was not accompanied by a decline in DeltaPsi. On the other hand, antimycin A treatment decreased DeltaPsi regardless of NOC-18 treatment. The findings of this study suggest that various functional molecules that constitute the mitochondrial respiratory chain may contribute to cytochrome C release that occurs during NO-induced apoptosis.

Nitric oxide (NO) plays an important role in gastric mucosal injury in the human stomach. Exposure to excessive NO leads to apoptosis; however, the mechanism remains largely unknown in gastric epithelial cells. The apoptotic process is modulated by energy states in cells. This study investigated molecular mechanisms of NO-induced apoptosis in gastric epithelial cells and influence of high glucose on those mechanisms. Normal rat gastric mucosal epithelial (RGM-1) cells were cultured in media containing either 1000 (low) or 4500 mg/l (high) of D-glucose. When the cells were incubated with a chemical NO donor NOC18, apoptosis was induced in a dose-dependent manner. Intracellular adenosine triphosphate (ATP) levels significantly increased in the cells cultured with high glucose in comparison with the low-glucose condition. The cells with high ATP levels were more resistant to NO-induced apoptosis than the cells with low ATP levels. NO-induced apoptosis was followed by mitochondrial depolarization, upregulation of Bax protein, cytochrome C release from mitochondria to the cytosol and subsequent caspases activation. These results suggest that NO inhibition of mitochondrial respiratory system and acute ATP depletion initiate apoptotic signalling in gastric epithelial cells. High glucose may prevent NO-induced apoptosis by leading to high levels of intracellular ATP or other metabolic changes in this cell line.

Background: Saliva contains substantial concentrations of nitrite derived from the enterosalivary recirculation of dietary nitrate., Methods: We have investigated factors in gastric juice influencing the fate of nitrite in swallowed saliva. When nitrite (100 microM) is added to human gastric juice pH 1.5 or pH 2.5 at 37 degrees C containing physiological concentrations of thiocyanate (1 mM) and ascorbic acid (200 microM), it is converted to nitric oxide within a few seconds., Results: The reduction of nitrite to nitric oxide is slower at pH 3.5 and very little is generated at pH 4.5. The rate of nitric oxide generation at acid pH increases with increasing thiocyanate concentration. The concentration of nitric oxide generated in the above way is maintained until the ascorbic acid is depleted by the recycling of nitric oxide to nitrite. In gastric juice depleted of ascorbic acid, very little nitrite is reduced to nitric oxide at any pH., Conclusion: These studies indicate that in the healthy acid-secreting stomach most salivary nitrite will be reduced to nitric oxide at the gastro-oesophageal junction and gastric cardia where it first encounters gastric juice.

Background & Aims: Twenty-five percent of absorbed dietary nitrate is re-secreted in saliva, and 30% of this is reduced to nitrite by buccal bacteria. When saliva is swallowed, the acidic gastric juice reduces the nitrite to nitric oxide. The aim of this study was to examine the anatomic distribution of nitric oxide generation within the lumen of the upper gastrointestinal tract under basal conditions and after ingesting nitrate equivalent to that in salad portion., Methods: Using custom-made sensors, the dissolved luminal nitric oxide concentration and pH were measured at 1-cm increments for 2 minutes throughout the length of the stomach and distal esophagus in 15 Helicobacter pylori-negative healthy volunteers with and without ingestion of 2 mmol potassium nitrate. Serum nitrate and saliva nitrite concentrations were also monitored., Results: The nitrate ingestion increased mean (range) serum nitrate from 30 micromol/L (18-49) to 95 micromol/L (32-152), mean salivary nitrite from 36 micromol/L (19-153) to 252 micromol/L (32-600), and mean peak luminal nitric oxide concentration from 4.7 micromol/L (1.4-7.8) to 23.2 micromol/L (2.1-50) (P < 0.05 for each). After nitrate, the peak nitric oxide concentration occurred in 11 of the 15 (73%) subjects within 1 cm distal to the gastroesophageal pH step-up point. The mean nitric oxide concentration over the 1-cm segment immediately distal to the gastroesophageal pH step-up after nitrate was 7.5 micromol/L (range, 0.5-30.7) and was significantly higher than at all other sites. Nitric oxide concentrations greater than 50 micromol/L were observed at the precise location where neutral esophageal pH fell to acidic gastric pH., Conclusions: Luminal generation of nitric oxide from dietary nitrate via salivary nitrite is maximal at the gastroesophageal junction and cardia. The high concentrations of nitric oxide generated may contribute to the high incidence of mutagenesis and neoplasia at this site.

Background: Dietary nitrate increases saliva nitrite levels and swallowed saliva is the main source of nitrite entering the acidic stomach. In acidic gastric juice, this nitrite can generate potentially carcinogenic N-nitrosocompounds. However, ascorbic acid secreted by the gastric mucosa can prevent nitrosation by converting the nitrite to nitric oxide., Methods: To study the potential for N-nitrosocompound formation in a model simulating salivary nitrite entering the acidic stomach and the ability of ascorbic acid to inhibit the process. Concentrations of ascorbic acid, total vitamin C, nitrite, nitrosomorpholine, oxygen and nitric oxide were monitored during the experiments., Results: The delivery of nitrite into HCl containing thiocyanate resulted in nitrosation of morpholine, with the rate of nitrosation being greatest at pH 2.5. Under anaerobic conditions, ascorbic acid converted the nitrite to nitric oxide and prevented nitrosation. However, in the presence of dissolved air, the ascorbic acid was ineffective at preventing nitrosation. This was due to the nitric oxide combining with oxygen to reform nitrite and this recycling of nitrite depleting the available ascorbic acid. Further studies indicated that the rate of consumption of ascorbic acid by nitrite added to natural human gastric juice (pH 1.5) was extremely rapid with 200 micromol/l nitrite consumed 500 micromol/l ascorbic acid within 10 s., Conclusions: The rapid consumption of ascorbic acid in acidic gastric juice by nitrite in swallowed saliva indicates that the potential for acid nitrosation will be maximal at the GO junction and cardia where nitrite first encounters acidic gastric juice. The high incidence of mutagenesis and neoplasia at this anatomical location may be due to acid nitrosation arising from dietary nitrate.

Objective. Dilatation of the intercellular space (DIS) of the esophageal epithelium is recognized as one of the earliest histological changes in gastroesophageal reflux disease patients. At the human gastroesophageal junction, reactive nitrogen oxide species (RNOS) are generated luminally through the entero-salivary re-circulation of dietary nitrate. In cases with gastroesophageal reflux, the site of luminal RNOS generation may shift to the distal esophagus. The aim of this study was to investigate whether luminal RNOS exposure could be involved in the pathogenesis of DIS. Material and methods. Rat esophageal mucosa was studied with an Ussing chamber model. On the luminal side of the chamber, RNOS were generated by the acidification of physiologic concentrations of sodium nitrite (1.0 or 5.0 mM). Esophageal barrier function was assessed by means of electrophysiological transmembrane resistance and membrane permeability by means of 3H-mannitol flux. The dimensions of the intercellular spaces were assessed by using transmission electron microscopy. Results. Administration of acid plus sodium nitrite induced DIS of the esophageal epithelium, and this ultrastructural morphological change was accompanied by a concomitant decrease in the transmembrane resistance and an increase in the epithelial permeability. The DIS induced by luminal RNOS was also confirmed in an in vivo exposure model. Conclusions. The present animal study indicates that the RNOS generated by the acidification of salivary nitrite in the presence of refluxed gastric acid in the esophagus could be a luminal factor that is responsible for the induction of DIS. Further studies are warranted to investigate the clinical relevance of the present findings to the human situation. [ABSTRACT FROM AUTHOR]

Objective: The human gastro-oesophageal junction is exposed to abundant amounts of luminal reactive nitrogen oxide species (RNOS) derived from the enterosalivary recirculation of dietary nitrate. The aim of this study is to investigate the direct effects of luminal RNOS on the adjacent gastric barrier function using an ex vivo chamber model. Methods: A chamber model in which the rat gastric mucosal membrane was mounted between the two halves of a chamber was designed to simulate the microenvironment of the lumen and the adjacent mucosa of the gastro-oesophageal junction. On the mucosal side of the chamber, RNOS were generated by the acidification of physiological concentrations of sodium nitrite. The epithelial barrier function was evaluated by electrophysiological transmembrane resistance, and membrane permeability with [³H]mannitol flux. The expression of occludin was evaluated by immunohistochemistry and immunoblotting. Dinitrosyl dithiolato iron complex (DNIC) was also measured by means of electron paramagnetic resonance spectroscopy to confirm the diffusion of RNOS from the mucosal lumen into the mounted mucosa. Results: The administration of acidified nitrite to the mucosal lumen caused both a decrease in transmembrane resistance and an increase in epithelial permeability, suggesting a disturbance of the gastric barrier function. These changes were accompanied by a derangement of the expression of occludin. The diffusion of luminal RNOS into the mounted membrane was confirmed by showing the generation of DNIC within the tissue. Conclusions: Simulating the microenvironment of the human gastro-oesophageal junction, this study demonstrated that RNOS generated luminally at the human gastro-oesophageal junction can derange the barrier function of the adjacent tissue by disrupting the tight junction. [ABSTRACT FROM AUTHOR]

Background and aims: When saliva, with its high nitrite content derived from the enterosalivary recirculation of dietary nitrate, meets acidic gastric juice, the nitrite is converted to nitrous acid, nitrosative species, and nitric oxide. In healthy volunteers this potentially mutagenic chemistry is focused at the gastric cardia. We have studied the location of this luminal chemistry in Barrett's patients during acid reflux. Methods: Ten Barrett's patients were studied before and after administration of 2 mmol nitrate. Using microdialysis probes we measured nitrite, ascorbic acid, total vitamin C, and thiocyanate concentrations and pH simultaneously in the proximal oesophagus, Barrett's segment, hiatal sac, proximal stomach, and distal stomach. In a subgroup, real time nitric oxide concentrations were also measured. Results: During acid reflux, Barrett's segment was the anatomical site with maximal potential for acid catalysed nitrosation, with its median concentration of nitrite exceeding that of ascorbic acid in two of 10 subjects before nitrate and in four of nine after nitrate. Thiocyanate, which catalyses acid nitrosation, was abundant at all anatomical sites. On entering the acidic Barrett's segment, there was a substantial fall in nitrite and the lowest ascorbic acid to total vitamin C ratio, indicative of reduction of salivary nitrite to nitric oxide at this anatomical site. Episodes of acid reflux were observed to generate nitric oxide concentrations of up to 60 μM within the Barrett's segment. Conclusion: The interaction between acidic gastric refluxate and nitrite rich saliva activates potentially mutagenic luminal nitrosative chemistry within Barrett's oesophagus. [ABSTRACT FROM AUTHOR]

Background: In humans, high concentrations of nitric oxide are generated luminally at the gastro- oesophageal junction through enterosalivary recirculation of dietary nitrate. Aim: To investigate whether luminal nitric oxide can diffuse into the adjacent digestive tissue and alter tissue integrity. Methods: We designed an animal model using Wistar rats in which physiological concentrations of nitrite and acidified ascorbic acid were administered separately so that the two reactants first meet to form nitric oxide at the gastro-oesophageal junction. Luminal and tissue concentrations of nitric oxide were measured with an electrode and an electron paramagnetic resonance spectrometer, respectively. Concentrations of glutathione in the tissue were measured as a marker of nitrosative stress. n; Results: High concentrations of luminal nitric oxide were generated locally at the gastro-oesophageal junction of nitrite administered rats, reproducing a phenomenon observed in humans. High levels of nitric oxide were also detected largely in the superficial epithelium of the gastro-oesophageal junction. The concentration of I tissue glutathione at the gastro-oesophageal junction was significantly lower in nitrite administered rats compared with control rats, whereas that in the distal stomach was similar in the two rat groups. Conclusions: Using an animal model, this study demonstrated that nitric oxide generated in the lumen diffuses into the adjacent gastric tissue to a substantial degree, leading to localised consumption of glutathione in the tissue. Nitrosative stress induced by this mechanism may be involved in the high prevalence of inflammation and metaplasia, and subsequent development of neoplastic disease at this site. [ABSTRACT FROM AUTHOR]

Background: We have examined the suitability of microdialysis probes for examining nitrosative chemistry within localized regions of the lumen of the gastrointestinal tract. Chemical nitrosation occurs maximally at pH 2.5 in the presence of nitrite and thiocyanate and absence of ascorbic acid. Nitrite and thiocyanate are delivered into the stomach in saliva and ascorbic acid is secreted in gastric juice. Methods: We used a benchtop model to reproduce the nitrosative chemistry occurring in the human upper GI tract and assessed the ability of the microdialysis probes to measure it. Results: The microdialysis probes were reliable at measuring nitrite ascorbic acid, total vitamin C and thiocyanate in both aqueous solutions and human gastric juice over the full range of intragastric pH, i.e. 1.5, 2.5, 3.5, 5.0 and 7.0. The probes were also reliable at measuring these chemicals under conditions simulating the active interaction between nitrite and ascorbic acid. Under such conditions with ascorbic acid in excess the probes gave a more accurate assessment of the nitrite level than that obtained by directly sampling the gastric juice. This was due to the probes not being subject to artefactual measurement of nitric oxide as nitrite. This was prevented by the rapid diffusion of nitric oxide through the probe collecting tube. Conclusion: Microdialysis probes provide a reliable means of examining nitrosative chemistry within the lumen of the upper GI tract. In addition, they have the advantage of measuring this chemistry in very local regions and of simultaneously comparing the chemistry in different regions of the upper GI tract. [ABSTRACT FROM AUTHOR]

High concentrations of nitric oxide are generated at the gastro-oesophageal (GO) junction due to the reduction of salivary nitrite to nitric oxide by acidic gastric juice containing ascorbic acid. Salivary nitrite is derived from the enterosalivary recirculation of dietary nitrate. Aims: To determine whether nitric oxide generated in the above way will exert nitrosative stress on the adjacent epithelium. Methods: A benchtop model was constructed reproducing the chemistry occurring at the GO junction and incorporating an epithelial compartment maintained at pH 7.4 separated from the lumen by a thin hydrophobic barrier. The secondary amine morpholine was added to each compartment and N-nitrosomorpholine formation at 15 min measured. Results: Adding 100µM nitrite to the acidic (pH 1.5) luminal compartment in the absence of ascorbic acid generated 6.2±2.0 (mean±SE) N-nitrosomorpholine in that compartment and 2.2±0.1 µM in the epithelial compartment. When 100µM nitrite was added to the acidic luminal compartment (pH 1.5) containing ascorbic acid, all the nitrite was immediately converted to nitric oxide and no N-nitrosomorpholine was formed within that compartment. However, the nitric oxide rapidly diffused into the adjacent epithelial compartment (pH 7.4) where it generated very high concentrations of N-nitrosomorpholine (137±5.6µM). The addition of ascorbic acid or glutathione to the epithelial compartment only reduced this nitric oxide induced nitrosation within the epithelial compartment by 40%. Conclusion: Ascorbic acid in gastric juice prevents acid-catalysed nitrosation within the gastric lumen. However, in doing so it generates nitric oxide which exerts a far higher nitrosative stress on the adjacent epithelium. This mechanism may be relevant to the aetiology of mutagenesis at the GO junction. [ABSTRACT FROM AUTHOR]