Increased Nitric Oxide Production and Inducible Nitric Oxide Synthase Activity in Colonic Mucosa of Patients with Active Ulcerative Colitis and Crohn's Disease

It is postulated that an enhanced production ofnitric oxide by inflamed intestine plays a role in thepathophysiology of active inflammatory bowel disease. Inthis study, systemic NO_x concentrations and colonic nitric oxide synthase activity weredetermined in patients with ulcerative colitis orCrohn's disease. The relationship between these twoparameters and disease activity, as well as differences in nitric oxide synthase activity betweenulcerative colitis and Crohn's disease, were areas ofspecific focus. Patients with active ulcerative colitisand Crohn's disease had significantly elevated plasma NO_x concentrations; a positivecorrelation was found between NO_x values andinducible nitric oxide synthase activities in the activemucosa of these patients. In active ulcerative colitis,levels of inducible nitric oxide synthase were significantlyelevated in both normal and inflamed mucosa, althoughinducible nitric oxide synthase activity was higher inthe latter. These colonic inducible nitric oxidesynthase activities correlated well with the results ofendoscopic and histologic grading of inflammation. Therewas no increase in constitutive nitric oxide synthaseactivity in patients with active ulcerative colitis. However, constitutive nitric oxidesynthase activity was significantly increased in theinflamed mucosa in patients with Crohn's disease. InCrohn's disease, elevated inducible nitric oxidesynthase activity was found in both normal and inflamedmucosa, with no significant difference between thetissues. Such differences in nitric oxide production inthe colonic mucosa possibly reflect the significant differences in the pathophysiology andcharacteristic clinical features between ulcerativecolitis and Crohn's disease.     

Nitric Oxide Synthase (NOS) Expression in the Myenteric Plexus of Streptozotocin-Diabetic Rats

Nitric oxide (NO) is an important inhibitoryneurotransmitter in the gut. Alterations in NO mediatedresponses have been described in diabetic animals. Thepresence of nitric oxide synthase (NOS) reflects the potential for NO synthesis and is found inneurons in the myenteric plexus. The aim of this studywas to determine changes in nitric oxide synthase (NOS)expression in the myenteric plexus of thegastrointestinal tract of diabetic rats at three months ofstreptozotocin-induced diabetes, compared to age matchedcontrols, using immunohistochemistry. Diabetic animalsshowed a decrease in NOS expression in the antrum, with 59.1 ± 7.3% of neurons beingpositive for NOS in diabetes compared to 81.2 ±4.7% in controls (P < 0.05). NOS expression induodenum, ileum, and colon of diabetic animals was notstatistically different from controls. Decreased expression of NOS inantrum may contribute to altered gastric emptyingobserved in diabetics.     

Relationship Between Site of Disease and Familial Occurrence in Crohn's Disease

Concordance in the extent of disease among thefamily members of patients with Crohn's disease has notbeen widely investigated. Furthermore, the relationshipbetween the site of the disease and familial occurrence has never been studied. Our aim wasto evaluate the familial occurrence of Crohn's diseasein the various sites. Nine hundred thirty-four patientswith Crohn's disease, observed consecutively in two gastrointestinal departments, wereinvestigated to determine first-degree familialincidence (in both Crohn's disease and ulcerativecolitis). Whenever two or more members were attendingthe same clinic, only one was regarded as a propositus.The analysis, therefore, was carried out on 882patients. The exact site of the disease was determinedin all patients either at diagnosis or during thefollow-up by colonoscopy and by small bowel enema. Therate of concordance in the extent of disease andfamilial occurrence in the various sites was evaluatedand the difference was calculated by chi-square test. Sixty-one propositi were identified among allthe patients. Forty-nine had familial occurrence for thesame disease (concordant patients), whereas 12 had atleast one relative with ulcerative colitis (discordant patients). In 44 propositi with only onerelative affected, the rates of concordance in theextent of the disease were 84, 68, 18, and 0%respectively, for the ileum, the ileum-right colon, theileum-total colon, and the colon. The number of propositiin the various sites was as follows: 4 of 162 (2.4%)patients with the disease located in the colon, 1 of 9(11%) with the jejunum site, 24 of 380 (6.3%) with the ileum site, 16 of 165 (9.7%) with the ileumand right colon site, and 16 of 164 (9.7%) with theileum and total colon site. The chi-square values ofpropositi distribution among other sites and the colon was, respectively, as follows: jejunum,2.2 (N.S.); ileum, 3.4 (P = 0.06); ileum and rightcolon, 7.4 (P = 0.006); and ileum and total colon, 7.4(P = 0.006). This study shows a pronounced concordancein the site of the disease for family members withCrohn's disease and suggests that familial occurrence inCrohn's disease is less frequent when the disease islocated in the colon rather than elsewhere.     

Nitric Oxide as a Modulator of Intestinal Water and Electrolyte Transport

The role of nitric oxide in intestinal fluid andelectrolyte secretion depends upon whether theconditions under study are physiological orpathophysiological. In physiological conditions,endogenous nitric oxide seems to be a proabsorptive molecule,based on the findings that nitric oxide synthaseinhibitors reverse net fluid absorption to net secretionin mice, rats, guinea pigs, rabbits, and dogs. This proabsorptive mode involves the enteric nervoussystem, the suppression of prostaglandin formation, andthe opening of basolateral K⁺ channels.However, in some pathophysiological states nitric oxidesynthase may be produced at higher concentrations thatare capable of evoking net secretion. Thus nitric oxidesynthase contributes to the diarrheal response intrinitrobenzene sulfonic acid-induced ileitis in guinea pigs and is the mediator of the laxative actionof several intestinal secretagogues including castoroil, phenolphthalein, bisacodyl, magnesium sulfate, bilesalts, senna, and cascara in the rat. Corresponding with the in vivo results, nitric oxide-donatingcompounds or nitric oxide itself stimulate chloridesecretion in the guinea pig and rat intestine in vitro.Exceptions are the diarrhea produced by bacterial enterotoxins in the rat, in which nitric oxideseems to have a proabsorptive role, and the mouse ileumin vitro, in which nitric oxide-donating compoundsproduce a net proabsorptive effect on basal ion transport. Several endogenous secretagogues(substance P, 5-hydroxytryptamine, interleukin-1beta),which are important mediators of the inflammatory boweldiseases, act, at least in part, through the liberation of nitric oxide. Clinical studies have shownthat nitric oxide is elevated in several inflammatorybowel diseases and other secretory conditions includingulcerative colitis, Crohn's disease, toxic megacolon, diverticulitis, infectious gastroenteritis, andinfantile methemoglobinemia. However, the determinationof nitric oxide in secretory diarrhea per se does notgive conclusive information on the nitric oxide contribution to clinical secretorydiarrhea.     

Small Intestinal Transit Time and Intraluminal pH in Ileocecal Resected Patients with Crohn's Disease

The pH and transit times of the gut areimportant for the delivery of active drug from severaltablets used in the treatment of Crohn's disease (CD).Many patients with CD undergo an ileocecal resection, which might influence small intestinal pH andtransit time. The effect of ileocecal resection on thesevariables has not previously been studied. IntraluminalpH and transit time were measured in nineileocecal-resected CD patients and 13 healthy volunteers usingpH-sensitive radiocapsules. Small intestinal transittime (SITT) was significantly shorter inileocecal-resected patients (5.2 hr, controls 8.0 hr).The pH levels of the small intestine were identical inpatients and controls, whereas cecal pH was 0.9 pH unitshigher in resected CD patients. The time spent with pHhigher than 5.5, 6.0, 6.5, and 7.0 was significantly shorter in patients than in controls. There wasno correlation between the SITT and the length ofresected ileum or between the SITT and the time elapsedsince the resection. We conclude that ileocecal resection decreases the SITT and the time withpH higher than 5.5-7.0. The study indicates that thisreduction of the SITT is mainly due to the resection ofthe ileocecal valve and is, to a certain extent, independent of the length of resected ileum. Anileocecal resection might therefore affect the deliveryof active drug from tablets with pH-dependentdelivery.     

Mechanism of Acid Secretory Changes in Rat Stomach After Damage by Taurocholate Role of Nitric Oxide, Histamine, and Sensory Neurons

The present study was performed to investigatethe mechanism underlying the acid stimulatory responsein the stomach after damage under the inhibition ofnitric oxide (NO) production byN^G-nitro-L-arginine methyl ester (L-NAME). A rat stomach wasmounted in an ex vivo chamber, perfused with saline, andthe potential difference (PD) and acid secretion weremeasured before and after the application of 20 mM taurocholate (TC) for 30 min. Exposure of thestomach to TC caused a PD reduction and a decrease ofacid secretion. Pretreatment with L-NAME did not affectbasal acid secretion but significantly enhanced the acid secretion in the stomach after damagewith TC, without any effect on the PD response. Thiseffect of L-NAME was antagonized by simultaneousadministration of L-arginine but not D-arginine. The luminal appearance of NO was significantlyincreased in the stomach after exposure to TC, and thischange was completely blocked in the presence of L-NAMEor when EGTA was applied together with TC. The enhanced acid secretory response to TC in thepresence of L-NAME was inhibited by pretreatment withcimetidine, FPL-52694 (a mast cell stabilizer), orspantide (a substance P antagonist) or by chemical ablation of capsaicinsensitive sensory neurons.Mucosal exposure to TC increased histamine output in thelumen and decreased the number of metachromaticallystaining cells in the stomach, and these changes were also significantly prevented by FPL-52694,spantide, or sensory deafferentation. These resultssuggest that 1) damage in the stomach may activate theacid stimulatory pathway in addition to the NO-dependent inhibitory mechanism, but the latter effectovercomes the former, resulting in a decrease in acidsecretion, 2) the acid stimulation in the damagedstomach may be mediated by histamine released from the mucosal mast cell which may interact withcapsaicin-sensitive sensory nerves, and 3) L-NAMEunmasks the acid stimulatory response by suppressing theinhibitory mechanism.     

Osteoporosis and Determinants of Bone Density in Patients with Crohn's Disease

Low bone mineral density (BMD) is common inpatients with Crohn's disease; however, the pathogenesisof bone loss and risk factors for osteoporosis are notestablished. Our aim was to evaluate the clinical, dietary, and nutritional determinants of BMD inCrohn's disease. A cross-sectional analysis of 117patients with Crohn's disease was undertaken. Allpatients underwent a clinical and dietary evaluation including assessment of nutritional state andlife-style. BMD was measured at the hip and lumbar spineby dual-energy x-ray absorptiometry; and z scoresobtained by comparison with age- and sex-matched normal values for the healthy UK population.Multiple regression analysis was used to assessassociations between BMD and potential risk factors,allowing for possible confounding variables. Thirteen(11%) patients had osteoporosis (z score<–2), with osteopenia (z score <–1,>–2) in a further 34 (29%). Patients withjejunal disease had significantly lower BMD at the spine(P = 0.03) and femoral neck (P = 0.02) than those with disease atother sites. Mean BMD was significantly lower at the hipof patients with previous bowel resection (diff in means= 0.53, 95% CI-0.97, –0.08, P = 0.02), but type of surgery was not significant. Active disease,menstrual history, diet, level of physical activity, andsmoking were not associated with low bone mass. At thelumbar spine, body weight (P < 0.0001), male sex (P < 0.0001), and currentprednisolone use (P < 0.02) were independentlypredictive of low bone mass. Body weight (P <0.0001), male sex (P < 0.0001), and cumulativesteroid dose (P = 0.02) were predictive at the femoralneck. The major determinants of BMD in Crohn's diseaseare body weight, current steroid use, and cumulativesteroid dose. Men with Crohn's disease are at greatest risk of osteoporosis, with jejunal involvementand previous bowel resection also contributing to thelow bone mineral density.     

Immunoglobulin G Subclass Distribution of Anti-Endothelial Cell Antibodies (AECA) in Patients with Ulcerative Colitis or Crohn's Disease

Anti-endothelial cell antibodies have beendescribed in sera from patients with inflammatory boweldisease. The aim of this study was to determine, byELISA, the IgG subclass distribution of anti-endothelial cell antibodies, in patients with ulcerativecolitis (N = 28) or Crohn's disease (N = 82) as comparedwith blood donors (N = 95). Thirty-six percent ofulcerative colitis and 23% of Crohn's disease patients were positive for at least one of the IgGanti-endothelial cell subclasses. Interestingly, thepattern of IgG anti-endothelial cell subclass observedin the two inflammatory bowel diseases differs. InCrohn's disease, the IgG1 anti-endothelial cellantibody level was significantly increased (P < 0.05)while IgG2 and IgG4 anti-endothelial cell antibodylevels were decreased (P < 0.0001 and P < 0.01, respectively) as compared to ulcerative colitispatients. The immunoglobulin G₃anti-endothelial cell antibody level was decreased inboth ulcerative colitis and Crohn's disease patients ascompared to healthy blood donors. No relationship wasdetected between disease activity of ulcerative colitisor Crohn's disease patients and anti-endothelial cellIgG subclasses. Finally, the disparity of IgGanti-endothelial cell subclass distribution in these twoinflammatory bowel diseases suggests that the ability toactivate effector mechanisms is not identical, andhence, deals with the concept of distinctivepathogenetic mechanisms in these two diseases.     

Adrenomedullin, a Vasodilator Peptide Implicated in Hemodynamic Alterations of Liver Cirrhosis Relationship to Nitric Oxide

This prospective cohort study was aimed atinvestigating the role of adrenomedullin, a potentvasodilator peptide, in liver cirrhosis and itsrelationship with nitric oxide and cytokines. Overall,66 consecutive patients with liver cirrhosis and 15 controlsmatched for age and sex distribution were included.Adrenomedullin levels in patients with cirrhosis werehigher than in controls [28.1 (23.5-34.8) vs 21.9 (21.1-26.4) pmol/liter, P = 0.002]. Child classA patients had adrenomedullin levels similar to those ofcontrols, but lower than patients in class B and C,respectively (P = 0.01). Patients with ascites showed more elevated adrenomedullin levels thanpatients without (P = 0.001). Adrenomedullin levels hadsignificant correlations with aldosterone (r = 0.55; P< 0.001), plasma renin activity (r = 0.49; P < 0.001) and nitrates-nitrites levels (r= 0.52; P < 0.001). Weak correlations were found withtumor necrosis factor-alpha and interleukin-6. Thisstudy shows that high levels of adrenomedullin in liver cirrhosis correlate with featuresassociated with plasma volume expansion, and suggeststhat, in late stages of cirrhosis, adrenomedullin mightcontribute to vasodilatation by increasing thegeneration of nitric oxide.     

Role of Nitric Oxide Mechanisms in Control of Pyloric Motility and Transpyloric Flow of Liquids in Conscious Dogs

The role of nitric oxide (NO) mechanisms incontrol of pyloric function and transpyloric flow wereinvestigated in six conscious dogs. Antropyloroduodenalmotility, transpyloric flow, and gastric emptying were measured 15 min after intravenousinjection of 100 ml of either saline, L-arginine (50mg/kg), L-NNA (5 mg/kg), or L-arginine (50 mg/kg)followed by L-NNA (5 mg/kg). Infusion of L-NNA wasassociated with retardation of gastric emptying (65± 6%) in the first 30 min, in comparison to thesaline (90 ± 3%) or L-arginine (90 ± 2%).This effect was prevented by infusion of L-arginineprior to L-NNA, after which 89 ± 3% of the liquidemptied in 30 min. There was a significant reduction (P< 0.05) in the number and volume of flow pulses, andan increase in pyloric tone (P < 0.05) after L-NNA in comparison to the other three testconditions. There were no differences, however, in thenumber of antropyloric or isolated pyloric pressurewaves under the four conditions. Our findings suggestthat NO mechanisms influence gastric emptying andtranspyloric flow of nonnutrient liquids by altering thepyloric tone, thus increasing resistance toflow.     

Gastrosparing Effect of New Antiinflammatory Drug Amtolmetin Guacyl in the Rat (Involvement of Nitric Oxide)

The effect of the nonsteroidal antiinflammatorydrug (NSAID) amtolmetin guacyl (AMG) on the gastricmucosa was studied in the rat by means of histologicaland functional techniques. AMG administered at 50-300 mg/kg intragastrically was virtuallydevoid of gastrolesive properties after either acute orrepeated treatment. By contrast, its metabolite,tolmetin (TOL, 15-60 mg/kg, intragastrically) caused dose-dependent gastric damage after bothtreatments. Light and electron microscopy revealed thatAMG induced minimal changes in the surface epitheliumlayer, without signs of vasocongestion or leukocytes adherence. AMG (50 mg/kg intragastrically) didnot change basal gastric potential difference (PD),whereas acetylsalicylic acid and ibuprofen induced fallsin PD of 22 and 27 mV, respectively. AMG (50 mg/kg intragastrically) reduced by 60% the fall in PDinduced by 50% ethanol; this inhibition was dependent onthe incubation time, and was maximal when AMG was given4 hr before ethanol. AMG (100 mg/kg intragastrically) induced an increase in NO synthase type 2(NOS2) activity, which was significantly different fromcontrol values, when AMG was administered 4 hr beforethe test. The metabolites of AMG, tolmetin, MED 5, and guaiacol were ineffective. Pharmacokineticanalysis of the residence time of AMG in the differentareas of the gastrointestinal tract, revealed that AMGremains in the gastrointestinal tract at least for 4 hr, the time necessary for a maximalinduction of NOS2 and for maximal protection againstethanol-induced damage. In conclusion, these dataindicate that the nonsteroidal antiinflammatory drugamtolmetin guacyl is devoid of gastrolesive properties;this gastrosparing effect seems to involve theproduction of nitric oxide, which can counteract thedamaging effects due to prostaglandin inhibition. Thepresence in the stomach of the native molecule ofamtolmetin guacyl seems to be necessary for theprotective effect observed.     

Prevalence of Upper Gastrointestinal Lesions and Helicobacter pylori Infection in Crohn's Disease

Crohn's disease can affect the upper gut withreported variable frequency, although concurrentHelicobacter pylori infection has been reported to below. We prospectively investigated the prevalence of esophageal, gastric, and duodenal lesions andHelicobacter pylori infection in 67 Crohn's disease, 41ulcerative colitis patients, and 43 controls. Symptoms,esophagogastroduodenoscopy, and multiple biopsies were performed on all patients consecutively.Endoscopic lesions were found in 63% of Crohn's diseasepatients, with a Helicobacter pylori prevalence of 28%. Granulomas were found in three patients. Twenty-two percent of the ulcerative colitis patients hadlesions, with a 29% prevalence of Helicobacter pyloriinfection. Half of the controls had pathologicalendoscopy, and Helicobacter pylori was positive in 40% of the cases. Subjective symptoms did notpredict the presence of endoscopic lesions orHelicobacter pylori infection in inflammatory boweldisease patients. Chronic gastritis and duodenitis arecommon in Crohn's disease patients, and the majorityare not associated with Helicobacter pyloriinfection.     

Increased Gastric Bicarbonate Secretion in Portal Hypertensive Anesthetized Rats (Role of Prostaglandins and Nitric Oxide)

Gastric bicarbonate secretion might be modifiedin portal hypertension as a consequence of theintramucosal increase in prostaglandins and nitric oxidecontent. Therefore, we studied gastric bicarbonate secretion in control and portal hypertensiverats and investigated the role of prostaglandins andnitric oxide. Basal gastric bicarbonate secretion wasstudied in rats, using a gastric pH back-titration technique, two weeks after partial portal veinligation or a sham operation. The effects of thefollowing drugs were investigated: the prostaglandinsynthase inhibitor indomethacin (5 mg/kg intravenously), prostaglandin (PGE₂) (1 mg/kgintravenously), the nitric oxide synthase inhibitorsN^G-nitro-L-arginine methyl ester (LG NAME, 5mg/kg intravenously) andN^G-monomethyl-L-arginine (L-NMMA, 50 mg/kgintravenously), and the nitric oxide donor nitroprusside (5mmol/liter in the gastric perfusate). Plasma leakage inthe gastric wall was also measured after Evans blue dyeinjection in portal hypertensive and sham-operated rats pretreated by indomethacin (5 mg/kg,intravenously) and L-NAME (5 mg/kg, intravenously).Basal bicarbonate secretion was significantly increasedin portal hypertensive rats as compared to controls. After indomethacin, the bicarbonate secretionwas significantly reduced to a similar level in bothgroups. PGE₂ increased bicarbonate secretionsignificantly more in portal hypertensive rats than insham-operated rats. The NO synthase inhibitor L-NMMAsignificantly increased bicarbonate secretion in portalhypertensive rats only, while the other inhibitor,L-NAME, increased it significantly more in portalhypertensive than in the sham-operated rats. Plasma leakagein portal hypertensive rats, which was increased in thebasal condition as compared to control, was furtherenhanced by indomethacin but not by L-NAME pretreatment. The nitric oxide donor significantly reducedbicarbonate secretion in portal hypertensive rats toreach a similar level as in sham-operated rats. Basalgastric bicarbonate secretion is increased in portal hypertensive rats. This could be due to anenhanced prostaglandin mucosal level. Nitric oxide,which reduces bicarbonate secretion, may contribute tolimiting prostaglandin-induced bicarbonateoverproduction.     

Mucosal Expression of Interleukin-6 and Interleukin-8 Messenger RNA in Ulcerative Colitis and in Crohn's Disease

To elucidate the possible role ofproinflammatory cytokines in inflammatory bowel disease,the expression and localization of interleukin (IL)-6and IL-8 mRNAs were examined in colonic biopsy specimens obtained from 10 patients with activeulcerative colitis (UC), 5 with inactive UC, 6 withCrohn's disease (CD), and 5 normal controls. In situhybridization with digoxigenin-labeled probes andimmunohistochemistry for both cytokines were performed. The IL-6mRNA expression was enhanced in the inflamed mucosa in4 of 6 CD patients, while that of UC patients stayed atbaseline. In contrast, IL-8 mRNA expression was apparently augmented (P = 0.044) in 7 of 10active UC and 3 of 6 CD patients (NS). The cell countpositive for IL-8 mRNA per unit area was definitelyincreased in moderate/severe UC when compared to mild UC (53.1 ± 14.4/mm² vs 9.0± 5.1/mm², P = 0.028) according to thedegree of inflammation. IL-6 mRNA positive cells in CDwere preferentially located in deeper lamina propriathan IL-8 mRNA positive cells in UC. Interestingly, IL-8 mRNA wasexpressed in the mucosal epithelial cells in one UCpatient. The patients treated by corticosteroids tendedto show suppressed expression of each mRNA, except one patient with intractable UC. Our data suggestenhanced expression of mucosal IL-6 mRNA in CD and ofIL-8 mRNA in UC by infiltrating mononuclear cells,indicating the distinct participation of each cytokine in the pathogenesis of UC and CD. Moreover,intestinal epithelial cells in UC occasionally exhibitIL-8 mRNA.     

Elevated Serum Vascular Endothelial Growth Factor in Children and Young Adults with Crohn's Disease

Vascular endothelial growth factor (VEGF) is acytokine released by fibroblasts, epithelial cells, andleukocytes that potentiates vascular permeability andgrowth of new capillaries. Because of these multiple effects, VEGF has been postulated to play arole in the pathogenesis of autoimmune disease, as wellas in wound healing. We hypothesized that VEGF waspotentially important in mediating the vascularpermeability and angiogenesis seen in Crohn's disease, andtherefore that VEGF would be increased in the serum ofchildren with Crohn's disease. Serum was obtained from73 children and young adults with Crohn's disease, 47 with ulcerative colitis, and 29 controls.VEGF levels were measured by enzyme-linked immunosorbentassay. Mean VEGF levels were significantly higher inpatients with Crohn's disease (436.4 ± 37.2pg/ml) than in ulcerative colitis (306 ± 41.1pg/ml) or control (167.8 ± 29.6 pg/ml) patients.Serum VEGF also correlated significantly with diseaseactivity, being elevated in patients withmoderate/severe Crohn's disease and ulcerative colitis. Weconclude that serum VEGF is released by inflamed tissuesin children with Crohn's disease. This multifunctionalcytokine could promote inflammation by increasing vascular permeability or promote wound healingby mediating capillary growth.     

Vascular Endothelial Growth Factor (VEGF) in Crohn's Disease Increased Production by Peripheral Blood Mononuclear Cells and Decreased VEGF165 Labeling of Peripheral CD14+ Monocytes

Recently, increased serum levels of vascularendothelial growth factor (VEGF) have been shown inpatients with inflammatory bowel disease. The origins ofthe circulating VEGF are still not described. Monocytes play an important role in the inflammatoryprocess. VEGF binding to monocytes mediates monocyterecruitment and activation. The present studyinvestigates the VEGF production of peripheral bloodmononuclear cells and the ability of peripheral monocytesto bind VEGF₁₆₅ in patients with Crohn'sdisease. Nineteen patients with Crohn's disease and 10healthy volunteers were studied. VEGF₁₆₅labeling of CD14⁺ monocytes was measured using two-color flow cytometry.Density of VEGF labeling was expressed as the meanfluorescence intensity (MFI). Furthermore, VEGF levelswere determined in culture supernatants of unstimulated peripheral blood mononuclear cells. VEGF inculture supernatants was measured using a solid-phaseenzyme-linked immunosorbent assay. There was asignificantly decreased VEGF₁₆₅ labeling ofmonocytes of patients with active Crohn's disease (MFI:369.9 ± 121.6, N = 7, P < 0.002) compared topatients with inactive disease (MFI: 457.7 ±74.5, N = 6) and healthy controls (MFI: 542.9 ±96.2, N = 10). Unstimulated peripheral blood mononuclear cellsof patients with active Crohn's disease producedsignificantly higher amounts of VEGF (1142.6 ±483.9 pg/ml, N = 12, P < 0.001) compared withperipheral blood mononuclear cells of healthy volunteers(113.4 ± 101.8 pg/ml, N = 10). VEGF production byperipheral blood mononuclear cells of patients withactive disease was significantly increased compared to patients with quiescent disease (261.6± 254.8 pg/ml, N = 7, P < 0.001). Inconclusion, our data describe peripheral bloodmononuclear cells as one of the origins of the elevatedVEGF serum levels in patients with active Crohn's disease.Furthermore, a decrease in VEGF₁₆₅ bindingsites on peripheral monocytes of patients with activeCrohn's disease has been shown. The study underlines theimportant role of VEGF in Crohn's disease.     

Factors Involved in Upregulation of Inducible Nitric Oxide Synthase in Rat Small Intestine Following Administration of Nonsteroidal Anti-inflammatory Drugs

We investigated the functional mechanisms underlying the expression of inducible nitric oxide (NO) synthase (iNOS) in the rat small intestine following the administration of nonsteroidal anti-inflammatory drugs (NSAIDs) and found a correlation with the intestinal ulcerogenic properties of NSAIDs. Conventional NSAIDs (indomethacin, dicrofenac, naproxen, and flurbiprophen), a selective cyclooxygenase (COX)-1 inhibitor (SC-560) and a selective COX-2 inhibitor (rofecoxib) were administered p.o., and the intestinal mucosa was examined 24 hours later. Indomethacin decreased prostaglandin E₂ (PGE₂) production in the intestinal mucosa and caused intestinal hypermotility and bacterial invasion as well as the upregulation of iNOS expression and NO production, resulting in hemorrhagic lesions. Other NSAIDs similarly inhibited PGE₂ production and caused hemorrhagic lesions with intestinal hypermotility as well as iNOS expression. Hypermotility in response to indomethacin was prevented by both PGE₂ and atropine but not ampicillin, yet all these agents inhibited not only bacterial invasion but also expression of iNOS as well, resulting in prevention of intestinal lesions. SC-560, but not rofecoxib, caused a decrease in PGE₂ production, intestinal hypermotility, bacterial invasion, and iNOS expression, yet this agent neither increased iNOS activity nor provoked intestinal damage because of the recovery of PGE₂ production owing to COX-2 expression. Food deprivation totally attenuated both iNOS expression and lesion formation in response to indomethacin. In conclusion, the expression of iNOS in the small intestine following administration of NSAIDs results from COX-1 inhibition and is functionally associated with intestinal hypermotility and bacterial invasion. This process plays a major pathogenic role in the intestinal ulcerogenic response to NSAIDs.     

Endotoxin Actions on Myoelectric Activity, Transit, and Neuropeptides in the Gut (Role of Nitric Oxide)

The lipopolysaccharide (endotoxin) ofgram-negative bacteria has systemic effects in animalsand man. Our aim was to investigate the effects of E.coli lipopolysaccharide on motility and transit through the small intestine in rats and to analyzeplasma and tissue concentrations of intestinalneuropeptides. When lipopolysaccharide (20–160g/kg) was administered intravenously, the migratingmyoelectric complex was replaced by spike burstsaccompanied by rapid transit. Tissue concentrations ofsubstance P and neurokinin A decreased, while plasmalevels of calcitonin gene-related peptide increased.N-Nitro-L-arginine, N-L-arginine methyl ester,dexamethasone, or indomethacin prevented these changesin myoelectric activity and tissue contents ofneuropeptides. All of these compounds, exceptindomethacin, prevented the increased rate of transit. Thus,lipopolysaccharide changes motility through the nitricoxide and arachidonic pathways, resulting in rapidtransit through the gut.     

Distribution of Nitric Oxide Synthase and Secretory Role of Exogenous Nitric Oxide in the Isolated Rat Pancreas

Summary Background. Pancreatic production and in vivo effects of nitric oxide (NO) have been shown by several studies. In order to examine the direct actions of the NO donor sodium nitroprusside (SNP), this study used in vitro specimens of the rat pancreas where the distribution of neuronal nitric oxide synthase (NOS) and the secretory effects of SNP and the cyclic GMP (cGMP) analog 8-bromo cyclic GMP (8-Br cGMP) were investigated. Methods. NO containing pancreatic nerves were visualized by NOS immunohistochemistry. Basal and stimulated amylase output from rat pancreatic segments was measured by an on-line fluorimetric method Stimulation was achieved by either acetylcholine (ACh) or electrical field stimulation (EFS). Intracellular free calcium concentration ([Ca²⁺]_i) was measured in dispersed pancreatic acinar cells. Results. NOS containing nerves were demonstrated in the vicinity of pancreatic acini and blood vessels. SNP and 8-Br cGMP inhibited both basal and EFS evoked amylase output but failed to inhibit ACh induced amylase output. Basal [Ca²⁺]_i was decreased by both SNP and 8-Br cGMP but neither SNP nor 8-Br cGMP influenced the ACh evoked increase in [Ca²⁺]_i. Conclusion. NO is well distributed in the rat exocrine pancreas. Exogenous nitric oxide may have a dual action in the isolated rat pancreas: Inhibition of basal amylase secretion in acinar cells and inhibition of ACh release from intrinsic nere terminals. Both effects seem to be calcium dependent and possibly mediated by cGMP.     

Luminal Calcium in Regulation of Nitric Oxide Release and Acid Secretion in Rat Stomachs After Damage

We investigated the role of luminalCa²⁺ in the regulation of nitric oxide (NO)release and acid secretion in the rat stomach followingdamage by sodium taurocholate (TC). Under urethaneanesthesia, a rat stomach was mounted in an exvivo chamber,perfused with saline, and transmucosal potentialdifference (PD), luminal pH, acid secretion, and luminalcontents of Ca²⁺ and NO were measured beforeand after exposure to 20 mM TC for 30 min, with orwithout coapplication of EGTA (5 mM) and/orCaCl₂ (10 mM). Mucosal exposure to TC causeda reduction in PD and a decrease of acid secretion, witha concomitant increase of NO as well as Ca²⁺ content in thegastric lumen. The increase of NO release as well as thereduced acid response were attenuated by pretreatmentwith L-NAME or coapplication of EGTA, and the latterinhibited the luminal increase of Ca²⁺. Thechanges caused by L-NAME were antagonized bycoadministration of L-arginine, while those induced byEGTA were partially antagonized by coinstillation ofCaCl₂. Neither treatment tested had any effect on gastric PDresponses to TC. These results suggest that: (1) damagein the stomach increases the release of Ca²⁺as well as NO in the lumen; (2) acid secretion decreases in response to damage by both an NO- andCa²⁺-dependent mechanism; and (3) theincrease of luminal Ca²⁺ is an adaptiveresponse of the stomach to damage and may play animportant role in increasing NO production and hence in regulating acidsecretion.