Luminal Nitric Oxide Research Articles

Intravesical Nitric Oxide Production Discriminates Between Classic and Nonulcer Interstitial Cystitis

Interstitial cystitis (IC) is one of the most bothersome conditions in urological practice. There are 2 subtypes, classic and nonulcer IC, with similar symptoms but different outcomes with respect to clinical course and response to treatment. Histologically there are fundamental differences between the 2 subtypes, classic IC presenting a severe abnormality of the urothelium and characteristic inflammatory cell infiltrates while inflammation is scant in nonulcer IC. Regulation of urinary nitric oxide synthase activity has been proposed to be of importance for immunological responses in IC. We present evidence of a profound difference between the 2 subtypes concerning nitric oxide production, mirroring the differences in inflammatory response in IC. A total of 17 patients with both subtypes and active disease as well as patients with disease in remission were included in the study, all diagnosed according to National Institute for Diabetes and Digestive and Kidney Diseases criteria. Luminal nitric oxide was measured in the bladder of patients using a chemiluminescence nitric oxide analyzer. All patients with classic IC had high levels of NO. None of the other patients had any significant increase in NO levels in the bladder. The NO level in patients with classic IC was not related to symptoms but rather to the assignment to this specific subgroup of IC. The highest levels of NO were found in patients in the initial phase of classic IC. The difference in NO evaporation between classic and nonulcer IC allows for subtyping of cases meeting National Institute for Diabetes and Digestive and Kidney Diseases criteria without performing cystoscopy. The findings in the present series together with previous findings clearly demonstrate that the 2 subtypes of IC represent separate entities. This separation further emphasizes the need to subtype all cases included in all scientific matters, ensuring that the 2 subtypes are evaluated separately in clinical studies.

Luminal Nitric Oxide and Epithelial Expression of Inducible and Endothelial Nitric Oxide Synthase in Collagenous and Lymphocytic Colitis

Background: Colonic nitric oxide (NO) production in collagenous colitis (CC) has been studied in a small number of patients and found increased. The cellular source of NO is believed to be the colonic epithelial cells. The aim of this study was to investigate colonic NO levels in patients with CC lymphocytic colitis (LC), to compare with the histopathological status and with the clinical activity, and to assess the epithelial expression of inducible and endothelial nitric oxide (iNOS and eNOS). Methods: We included 19 patients with CC, 8 patients with LC and 15 controls. During colonoscopy, luminal gas was sampled and NO levels were measured using the chemiluminescence technique. Mucosal biopsies were obtained for routine histopathologic examination and immunohistochemical studies of iNOS and eNOS. Clincal activity, as measured by the mean frequency of daily bowel movements during the week prior to colonoscopy, was assessed. Results: Luminal NO levels, median (25-75 percentiles), in the patients with CC and LC were greatly increased compared to the controls, 1673 (145-8143) parts per billion (ppb) and 1838 (1065-2694) ppb versus 28 (20-46) ppb (P ≺ 0.005, both). A positive association was seen between NO levels and histopathological status as well as clinical activity. Strong expression of iNOS was seen in the surface epithelium in 5 of 6 patients with CC and in 2 of 5 patients with LC. Conclusions: The fact that luminal NO levels are related to histopathological status and correlate with clinical activity indicates that NO is involved in the pathophsiology of CC and LC. The epithelial cells are the most likely source of luminal NO.

Dietary nitrate generates potentially mutagenic concentrations of nitric oxide at the gastroesophageal junction

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. 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. 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. 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.

Increased rectal nitric oxide in children with active inflammatory bowel disease.

Luminal nitric oxide increases in ulcerative colitis and Crohn disease. The authors have previously used a minimally invasive method to demonstrate increased luminal nitric oxide in ulcerative colitis and Crohn disease of the colon. The aim of the current study was to determine whether this method could be applied to identify inflammatory activity in ulcerative colitis and Crohn disease in children. Thirty-six children (18 of whom had active disease) with inflammatory bowel disease localized to the colon were studied. The control group comprised 12 healthy children. To measure nitric oxide, a silicon catheter with an inflatable balloon was inserted into the rectum and inflated with 10 mL of nitric oxide-free air. After a 10-minute incubation time, the air was withdrawn and nitric oxide concentrations were immediately analyzed using a chemiluminescence technique. Children with active ulcerative colitis and Crohn disease of the colon had greatly increased luminal nitric oxide concentrations in the rectum (8,840 +/- 5,120 and 15,170 +/- 4,757 parts per billion [ppb], respectively) compared with controls (77 +/- 17 ppb) (P < 0.001). Children with nonactive ulcerative colitis or Crohn disease displayed low concentrations of rectal nitric oxide (356 +/- 110 and 188 +/- 55 ppb, respectively), which was not different from that of healthy controls. Rectal nitric oxide measurement is a feasible and useful method for monitoring disease activity in inflammatory bowel disease, especially in children.

Increased Rectal Nitric Oxide in Children With Active Inflammatory Bowel Disease

ABSTRACTBackgroundLuminal nitric oxide increases in ulcerative colitis and Crohn disease. The authors have previously used a minimally invasive method to demonstrate increased luminal nitric oxide in ulcerative colitis and Crohn disease of the colon. The aim of the current study was to determine whether this method could be applied to identify inflammatory activity in ulcerative colitis and Crohn disease in children.MethodsThirty‐six children (18 of whom had active disease) with inflammatory bowel disease localized to the colon were studied. The control group comprised 12 healthy children. To measure nitric oxide, a silicon catheter with an inflatable balloon was inserted into the rectum and inflated with 10 mL of nitric oxide–free air. After a 10‐minute incubation time, the air was withdrawn and nitric oxide concentrations were immediately analyzed using a chemiluminescence technique.ResultsChildren with active ulcerative colitis and Crohn disease of the colon had greatly increased luminal nitric oxide concentrations in the rectum (8,840 ± 5,120 and 15,170 ± 4,757 parts per billion [ppb], respectively) compared with controls (77 ± 17 ppb) (P &lt; 0.001). Children with nonactive ulcerative colitis or Crohn disease displayed low concentrations of rectal nitric oxide (356 ± 110 and 188 ± 55 ppb, respectively), which was not different from that of healthy controls.ConclusionRectal nitric oxide measurement is a feasible and useful method for monitoring disease activity in inflammatory bowel disease, especially in children.

Alpha-Phenyl-N-tert-butylnitrone provides protection from dextran sulfate sodium-induced colitis in mice.

Nuclear factor-kappaB (NF-kappaB)-dependent up-regulation of inflammatory cytokines and inducible nitric oxide (iNOS) occurs in inflammatory bowel disease. We investigated the effect of alpha-phenylN-tert-butylnitrone (PBN), a spin-trapping agent that inhibits NF-kappaB activity, on dextran sulfate sodium (DSS)-induced colonic mucosal injury and inflammation in mice. Acute colitis was induced with DSS in female BALB/c mice receiving 0, 0.3, 3, and 30 mg/kg i.p. PBN daily. Colonic mucosal inflammation was evaluated biochemically and histologically. Nitric oxide was evaluated as luminal nitrite/nitrite concentration by the Griess reaction and as immunoreactive nitrotyrosine in mucosal cells. Mucosal tumor necrosis factor-alpha (TNF-alpha) and interferon-gamma (IFN-gamma) were determined by immunoassay. Colonic mRNA expression for iNOS, TNF-alpha, and IFN-gamma was measured by reverse transcription-polymerase chain reaction, and NF-kappaB activation was evaluated by electrophoretic mobility shift assay. After DSS administration, mice showed increased luminal nitrite/nitrate, mucosal TNF-alpha and IFN-gamma, and mRNA for iNOS and these cytokines, in addition to decreased colonic length and increased inflammatory score, luminal hemoglobin, and colonic myeloperoxidase activity. PBN inhibited increases in luminal nitric oxide production, nitrotyrosine immunoreactivity, and mucosal TNF-alpha and IFN-gamma. Colonic iNOS, TNF-alpha, and IFN-gamma mRNA were suppressed by PBN, as was a DSS-induced increase in colonic NF-kappaB DNA-binding activity. NF-kappaB is essential to DSS-induced colitis, suggesting molecular approach targeting of NF-kappaB for treatment of inflammatory bowel disease.

Sources of Intra-Oesophageal Nitric Oxide Production Following Intraluminal Acid Exposure

Background: The aim of the present study was to assess luminal nitric oxide (NO) levels in the oesophagus during baseline and acidic conditions and to clarify the sources of such oesophageal NO formation. Methods: Healthy volunteers received an intra-oesophageal infusion of either HCl (100 mM) or NaCl (150 mM) on two separate study days. After a low nitrate diet, nitrate load or no dietary restrictions/pretreatment, direct intraluminal measurements of NO formation were performed using a tonometric technique. Endoscopy was performed and mucosal biopsies were taken and analysed by means of immunohistochemistry, Western blot and RT-PCR. Results: No intra-oesophageal NO was detected during baseline conditions with pH neutrality. During the infusion of HCl the NO levels rose dramatically to around 12000 ppb. This high rate of NO formation fell by 95% following deviation of saliva. NO formation after an acute nitrate load was almost doubled during acid perfusion compared to control. Immunohistochemistry demonstrated distinct staining for iNOS in the oesophageal squamous epithelial cells, and Western blot and RT-PCR confirmed the presence of iNOS. Conclusion: Two sources exist for intra-oesophageal NO formation, both dependent on the luminal acidity: 1) chemical reduction of salivary nitrite, a mechanism related to dietary intake of nitrate, and 2) NO formation within the oesophageal mucosal epithelium by enzymatic degradation of L-arginine. In the latter case, the NO synthase has antigenic characteristics, indicating the inducible isoform, although a functional behaviour suggests an unconventional subtype.

Ileal luminal nitric oxide synthase inhibitors and E. coli lipopolysaccharide effects in the anesthetized rat.

Some of the effects of bacterial toxins are mediated through the local production of nitric oxide (NO) or its products. This study examined if NO inhibition in the intestinal mucosa had effects on the responses to intravenous lipopolysaccharide (LPS) in anesthetized rats. Aminoguanidine (AMGU, 500 microM), a relatively selective inducible NO synthase (iNOS) inhibitor, or N(G)-nitro-L-arginine (NOLARG, 50 or 500 microM), a nonselective inhibitor of iNOS and constitutive NOS (cNOS), were perfused through the ileal lumen during intravenous LPS (17 mg/kg) or saline administration. Intestinal H20 transport, NO3- + NO2- (NOx) secretion, absorptive site mucosal blood flow (ASBF), blood pressure, plasma [NOx], tissue damage, and blood leukocytes were measured for 4 hr. LPS increased luminal NOx secretion. At 50 microM, luminal NOLARG attenuated the LPS-induced NOx secretion and increased blood pressure. There were no significant changes in lethality, plasma [NOx] or other parameters. At 500 microM, luminal NOLARG converted a nonlethal dose of LPS into a lethal dose, but AMGU did not increase lethality. The LPS-induced luminal NOx secretion was blocked by 500 microM intraluminal AMGU and NOLARG. Luminal NOx secretion also increased in control animals. This increase was blocked by 500 microM NOLARG but not AMGU. Luminal 500 microM NOLARG increased blood pressure, but AMGU did not. Luminal 500 microM NOLARG prevented the LPS-induced increase in plasma [NOx] and the decrease in leukocytes, but AMGU did not. Tissue damage occurred with intravenous LPS plus intraluminal 500 microM NOLARG. It was concluded that luminal AMGU inhibited mucosal iNOS. Luminal NOLARG inhibited mucosal cNOS and iNOS, and cNOS inhibition primed a lethal LPS effect. NOLARG, but not AMGU, was absorbed from the intestine and had systemic effects.

Jejunal luminal nitric oxide during severe hypovolemia and sepsis in anesthetized pigs.

Lowered gut blood perfusion and the associated intestinal mucosal barrier dysfunction is considered important in the pathophysiology leading to critical illness. Intestinal mucosal nitric oxide formation has been attributed a key role in the regulation of epithelial permeability and other properties of the intestinal mucosal barrier. This study was performed to delineate intestinal mucosal NO formation during hypovolemia or sepsis, both of which are associated with intestinal hypoperfusion. Seventeen pigs were subjected to 2 h of severe hypovolemia (bleeding induced) or sepsis (systemic infusion of live Escherichia coli) or no treatment (controls). Jejunal mucosal NO production was monitored by a tonometer. Mesenteric blood flow was measured as portal venous blood flow by an ultrasonic transit time flowmeter probe, and oxygen delivery and consumption were calculated from regional blood samples. Intestinal perfusion and oxygen delivery were reduced by the same order of magnitude in both groups. Jejunal mucosal NO production and oxygen consumption decreased markedly in the hypovolemia group but remained stable in the group subjected to septic shock. These data suggest that blood loss inhibits jejunal mucosal NO production as part of a general downregulation of nonvital organs. Sepsis represents a more complex stress condition with activation/maintenance of host defense mechanisms as reflected by maintained jejunal mucosal NO production despite reduced gut blood perfusion.

BACILLUS CALMETTE-GUERIN INDUCES LONG-TERM LOCAL FORMATION OF NITRIC OXIDE IN THE BLADDER VIA THE INDUCTION OF NITRIC OXIDE SYNTHASE ACTIVITY IN UROTHELIAL CELLS

Bladder instillation of bacillus Calmette-Guerin (BCG) is effective therapy for recurrent superficial bladder cancer and carcinoma in situ. BCG induces nitric oxide synthase activity in the bladder. Nitric oxide is formed from L-arginine by nitric oxide synthase. We investigated nitric oxide formation and its localization in bladder cancer patients treated with intravesical BCG instillation. The L-citrulline conversion assay was done to assess nitric oxide synthase activity in BCG treated T24 human bladder cancer cells and cultured normal human urothelial cells. Nitrite and nitrate in cell culture medium, urine and plasma were measured by capillary electrophoresis. Nitric oxide formation in the bladder was measured by chemiluminescence. A 24-hour treatment with BCG induced calcium independent nitric oxide synthase activity in T24 cells in a dose dependent manner. Nitrite and nitrate production by T24 cells also increased in a dose dependent manner after 24-hour BCG treatment. BCG treatment of cultured normal human urothelial cells resulted in the induction of calcium dependent and independent nitric oxide synthase activity. Nitrite in the urine of patients receiving BCG for the first time was increased 5-fold 24 hours after instillation. Furthermore, BCG increased luminal nitric oxide in the bladder. The increase was noted after a single treatment and sustained for 6 months. No changes in plasma nitrite or nitrate were observed after BCG treatment. BCG induces the local formation of nitric oxide in the bladder, whereas no evidence for systemic nitric oxide formation was noted. Increased nitric oxide production in the bladder is likely due to the induction of nitric oxide synthase activity in urothelial cells.

NITRIC OXIDE: A USEFUL GAS IN THE DETECTION OF LOWER URINARY TRACT INFLAMMATION

Purpose Luminal nitric oxide has been shown to be elevated in the bladder of patients with cystitis of various etiologies. We determine whether luminal nitric oxide can be used as a marker to differentiate inflammation, that is interstitial cystitis, from urgency, frequency, nocturia and pain due to noninflammatory disorders, such as outflow obstruction and neurogenic dysfunction. Materials and Methods We measured luminal nitric oxide in the bladder of patients with urgency due to detrusor instability (6), outflow obstruction (7), sensory urge (19) and interstitial cystitis (8), and controls without urgency symptoms (11). Nitric oxide-free air was incubated in the bladder for 5 minutes and analyzed in a chemiluminescence nitric oxide analyzer. Results There was a nearly 20-fold increase in mean bladder nitric oxide concentration in patients with interstitial cystitis (234 +/- 67 parts per billion) compared to those with detrusor instability (11 +/- 1), outflow obstruction (9 +/- 1) and sensory urgency (10 +/- 1), and controls (13 +/- 2). Conclusions Measurement of nitric oxide in air from the bladder is a simple, safe and fast method to differentiate urgency due to inflammation from neurogenic disorders or outflow obstruction. The simplicity of this method makes it potentially useful as a screening method for office use.

Increased luminal nitric oxide concentrations in the small intestine of patients with coeliac disease.

Inflammation is known to be associated with enhanced nitric oxide production. A role for nitric oxide in coeliac disease has been suggested because of increased expression of the inducible isoform of nitric oxide synthase in the small intestine of patients with untreated coeliac disease. During small bowel endoscopy in 11 control subjects, 10 patients with untreated coeliac disease and seven patients with treated coeliac disease, gas was aspirated from different parts of the upper gastrointestinal tract and immediately analysed using a chemiluminescence technique. Luminal nitric oxide concentrations were also quantified in 13 control subjects who had undergone colonoscopy. Jejunal luminal nitric oxide concentrations were more than 20 times higher in patients with coeliac disease than in normal control subjects (mean 755 +/- 173 ppb, range 215-1690 ppb, vs. mean 31 +/- 9 ppb, range 1-83 ppb, P < 0.001). Jejunal luminal nitric oxide levels in patients with treated coeliac disease (mean 54 +/- 18 ppb, range 3-126 ppb) did not differ from those of control subjects. This study shows that intraluminal jejunal nitric oxide concentrations are significantly increased in patients with untreated active coeliac disease.

Measurement of luminal nitric oxide in bladder inflammation using a silicon balloon catheter: a novel minimally invasive method

Objectives. Nitric oxide (NO) measured in the gaseous phase has been shown to be a marker of inflammation in the urinary bladder. The NO content of air incubated in the bladder can be measured in an NO analyzer. The aim of our study was threefold: to evaluate whether NO can be measured in air incubated in a catheter balloon, to determine the optimal time of incubation, and to find the most suitable type of catheter. Methods. The NO concentration in air introduced directly into the bladder and into the catheter balloon was measured in patients with and without bladder infections. The air was incubated for 5 to 60 minutes. NO concentration in the bladder of patients with interstitial cystitis was also analyzed. The diffusion rate of NO through silicon and latex catheters was studied. Results. Elevated NO levels were detected in the urinary bladder in patients with bladder inflammation due to infection or interstitial cystitis. A marked increase in NO concentration was found after just 5 minutes of incubation and continued to rise for up to 20 minutes, both in air taken directly from the bladder and from the catheter balloon. The NO diffusion rate into the balloons of silicon catheters was high; the recovery rate in latex catheters was poor. Conclusions. Measurement of NO concentration in a silicon balloon catheter inserted into the urinary bladder is a fast, convenient, and reliable method to detect inflammation.© 1999, Elsevier Science Inc.

Increased Luminal Nitric Oxide in Inflammatory Bowel Disease as Shown with a Novel Minimally Invasive Method

Background: The production of nitric oxide (NO) is increased in ulcerative colitis, as shown by bioassays of NO synthase activity in mucosal biopsy specimens.We wanted to develop a less invasive method for measurement of NO directly in the rectum in patients with inflammatory bowel disease (IBD). Methods: We studied 10 patients with active ulcerative colitis, 6 with active Crohn's disease, 6 with non-active ulcerative colitis, and 24 controls without inflammation A tonometer balloon catheter was inserted in the rectum and inflated with 5 ml of NO-free air. After 15 min of incubation the sample was extracted, and the NO concentration was immediately analysed with a chemiluminescence technique. Results: Patients with active disease had greatly increased concentrations of NO in the rectum as compared with controls and patients with non-active disease. Conclusions: During inflammation in the large intestine increased amounts of NO are released from the mucosa. Measurements of NO directly in the rectum could be of help in further understanding the role of this gas in IBD. Moreover, it is tempting to speculate that this minimally invasive method could be clinically useful as a diagnostic tool and in monitoring the effect of therapy.

Greatly increased luminal nitric oxide in ulcerative colitis

A role for nitric oxide (NO) in ulcerative colitis has been suggested because mucosal NO synthase activity, measured by indirect assays, is increased in this disorder. We directly measured luminal NO in the colons of twelve controls and six patients with active ulcerative colitis. During colonoscopy, gas was aspirated from different parts of the colon and immediately analysed by a chemiluminescence technique. NO concentrations were more than 100 times higher in the patients than in the controls. Luminal NO is rapidly and easily measurable and may be used to monitor inflammatory bowel disease.