Mark J.S. Miller

Role of inducible nitric oxide synthase expression and peroxynitrite formation in guinea pig ileitis

BACKGROUND & AIMSnInflammatory bowel disease is characterized by increased synthesis of nitric oxide. The aim of this study was to determine if inducible NO synthase (iNOS) was responsible for tissue injury, potentially via peroxynitrite formation, in the guinea pig model of gut inflammation.nnnMETHODSnInflammation was induced in guinea pig ileum by intraluminal administration of the hapten trinitrobenzene sulfonic acid in 50% ethanol. iNOS gene expression was assessed by reverse-transcriptase polymerase chain reaction and Western blotting, immunohistochemistry was determined by its localization, and activity was inhibited with the specific inhibitor aminoguanidine administered via the drinking water for 7 days. Nitration of tyrosines was assessed by immunohistochemistry.nnnRESULTSnIn control animals, iNOS gene expression was minimal to absent, whereas, in hapten, inflammation-marked iNOS gene expression was evident from day 1 to 7. Nitrotyrosine and iNOS immunohistochemistry were colocalized, and positive staining was most intense in epithelia and neurons. Inhibition of NO formation prevented nitrotyrosine formation. Aminoguanidine inhibited the inflammatory response and restored morphology.nnnCONCLUSIONSnThe colocalization of tyrosine nitration with iNOS immunoreactivity suggests that iNOS may be responsible for tissue injury and the formation of NO-dependent nitrating species, potentially peroxynitrite. Inhibition of iNOS may afford a new therapeutic approach to the treatment of inflammatory bowel disease.

Nitric oxide inhibition causes intrauterine growth retardation and hind-limb disruptions in rats.

OBJECTIVEnOur purpose was to determine the effects of nitric oxide synthase inhibition on maternal and fetal health in the last third of pregnancy.nnnSTUDY DESIGNnPregnant rats were treated from gestational day 13 to day 19 or 20 with the nitric oxide synthase inhibitor NG-nitro-L-arginine methyl ester, which was administered in the drinking water ad libitum. Control animals received the inactive enantiomer NG-nitro-D-arginine methyl ester or no treatment. Maternal blood pressure, blood chemistry studies, and placenta and pup size were determined. A separate group of rats received nitroprusside sodium in conjunction with NG-nitro-L-arginine methyl ester.nnnRESULTSnNG-nitro-L-arginine methyl ester caused a dose-dependent reduction in placenta and pup size. Amniotic fluid levels of cyclic guanosine monophosphate were significantly reduced at 0.1 mg/ml but not at higher doses. Hemorrhagic necrosis of fetal hind limbs occurred only with treatment with NG-nitro-L-arginine methyl ester and was prevented by coadministration of nitroprusside sodium. Maternal blood pressure and blood and urine chemistry studies were unaffected by NG-nitro-L-arginine methyl ester.nnnCONCLUSIONnChronic reductions of nitric oxide production in the last third of pregnancy result in significant intrauterine growth retardation and hemorrhagic disruptions of hind limbs. Maternal complications were minimal and did not mimic preeclampsia.

Potential Role of Nitric Oxide in a Model of Chronic Colitis in Rhesus Macaques

BACKGROUND/AIMSnExcess nitric oxide formation, via the inducible NO synthase isoform, has been implicated in the pathogenesis of experimental and clinical inflammatory bowel disease. The aim of this study was to assess the site, enzyme source, and magnitude of NO production in juvenile rhesus macaques with idiopathic colitis.nnnMETHODSnNO production was assessed systemically from plasma and urine levels of reactive nitrogen intermediates and locally by the formation of [3H]citrulline from [3H]arginine and reduced nicotinamide adenine dinucleotide phosphate (NADPH) diaphorase histochemistry. Inducible NO synthase gene expression was assessed by reverse-transcription polymerase chain reaction.nnnRESULTSnPlasma and urine levels of reactive nitrogen intermediates were greater in colitic animals than in control monkeys by 13- and 5-fold, respectively. NADPH diaphorase activity in normal animals was confined to the myenteric plexus. In colitis, staining was also apparent in crypt abscesses and superficial epithelial and mucosal bands. Gene expression for inducible NO synthase was only found in colitic specimens. Colonic [3H]citrulline formation was markedly elevated in colitic specimens, and the inducible isoform accounted for 58% of total activity.nnnCONCLUSIONSnIt is proposed that excess NO, formed via the inducible form of NO synthase, contributes to the mucosal inflammation and symptoms of this idiopathic colitis model.

Nitric oxide release in response to gut injury.

We evaluated nitric oxide release in several models of intestinal inflammation through luminal nitrite concentrations. In anesthetized rabbits, piglets, and guinea pigs, luminal lavages were collected from loops of normal or injured small intestine. Lavages were analyzed spectrophotometrically for nitrite (Griess reagent) and protein. Myeloperoxidase (MPO) content of intestinal segments was used as an index of granulocyte infiltration and intestinal inflammation. Acute ileal inflammation was induced by luminal acetic acid + casein in rabbits and luminal deoxycholate in neonatal piglets and adult rabbits. Chronic ileitis was induced in guinea pigs by intraluminal trinitrobenzenesulfonic acid. In each model nitrite levels in ileal lavages were significantly greater than control loops/animals. Increased luminal protein and intestinal MPO activity paralleled the changes in nitrite levels. To determine whether nitric oxide production influenced mucosal repair, segments of ileum were perfused with the L-arginine antagonist NG-nitro-L-arginine methyl ester (L-NAME, 10 mg/ml) after acute acetic acid + casein exposure. L-NAME administration reversed the decline in epithelial permeability characteristic of epithelial restitution, causing an increase in epithelial permeability which was readily reversible. These results suggest that nitrite production is a useful index of gut injury and that nitric oxide may contribute to the functional repair of the epithelial barrier under acute conditions.

Bacteria rapidly colonize and modulate healing of gastric ulcers in rats

The stomach is generally regarded as an environment that is not conducive to bacterial colonization. In this study, we examined the possibility that this changes significantly when an ulcer has formed and that colonization of ulcers interferes with the normal healing process. Gastric ulcers were induced by serosal application of acetic acid. The relationship between ulcer healing and bacterial colonization was examined. The effects of antibiotics, induction of Lactobacilluscolonization, and selective colonization with an antibiotic resistant strain of Escherichia coli on ulcer healing were examined. Within 6-12 h of their induction, gastric ulcers were colonized by a variety of bacteria, with gram-negative bacteria predominating. Suppression of colonization with antibiotics resulted in marked acceleration of healing. Induction of Lactobacillus colonization also accelerated ulcer healing. The beneficial effects of antibiotics were reversed through selective colonization with antibiotic-resistant E. coli. Bacterial colonization occurred irrespective of the method used to induce the ulcer. This study demonstrates that colonization of gastric ulcers in rats occurs rapidly and significantly impairs ulcer healing. This effect appeared to be primarily attributable to gram-negative bacteria.The stomach is generally regarded as an environment that is not conducive to bacterial colonization. In this study, we examined the possibility that this changes significantly when an ulcer has formed and that colonization of ulcers interferes with the normal healing process. Gastric ulcers were induced by serosal application of acetic acid. The relationship between ulcer healing and bacterial colonization was examined. The effects of antibiotics, induction of Lactobacillus colonization, and selective colonization with an antibiotic resistant strain of Escherichia coli on ulcer healing were examined. Within 6-12 h of their induction, gastric ulcers were colonized by a variety of bacteria, with gram-negative bacteria predominating. Suppression of colonization with antibiotics resulted in marked acceleration of healing. Induction of Lactobacillus colonization also accelerated ulcer healing. The beneficial effects of antibiotics were reversed through selective colonization with antibiotic-resistant E. coli. Bacterial colonization occurred irrespective of the method used to induce the ulcer. This study demonstrates that colonization of gastric ulcers in rats occurs rapidly and significantly impairs ulcer healing. This effect appeared to be primarily attributable to gram-negative bacteria.

Casein: a milk protein with diverse biologic consequences.

Summary The consequences of bovine milk consumption are diverse, some of which are potentially deleterious. Although certain cultures shun cows milk or milk-based products, Western societies consume large quantities of cows milk. Although there are stronger similarities between bovine whey proteins and human whey proteins, the quantity and nature of casein in cows milk differ markedly from human milk. We propose that the consequences of diets based on bovine casein should be more closely evaluated and certainly expanded beyond the simplistic approach of growth. What is good for the goose may be good for the gander, but what is good for the cow could be harmful to the human.

Potential role of histamine monochloramine in a rabbit model of ileitis

Histamine chloramines, derived from the chlorination of histamine by granulocyte-derived oxidants, are potential mediators of intestinal injury and dysfunction in states of atopy or inflammation. We assessed the ability of histamine monochloramine to increase epithelial permeability in rabbit distal small intestine and determined whether the conditions for histamine chloramine formation are favorable in a rabbit model of ileitis. Epithelial permeability, quantified by the blood-to-lumen clearance of 51Cr-labeled ethylenediaminetetraacetic acid, was enhanced by luminal perfusion with either histamine or histamine monochloramine (10 microM), although the latter was twice as effective (p less than 0.05). In a rabbit model of ileitis induced by a luminal solution of acetic acid (200 mM) and casein (10 mg/ml) there was a marked increase in epithelial permeability and in the release into the lumen of histamine, myeloperoxidase, 6-keto-prostaglandin F1 alpha and protein. These results suggest that the conditions are favorable for histamine chloramine formation and that histamine and histamine chloramine may impair the integrity of the epithelial barrier.

EXAGGERATED INTESTINAL HISTAMINE RELEASE BY CASEIN AND CASEIN HYDROLYSATE BUT NOT WHEY HYDROLYSATE

Loops of rabbit distal small intestine received luminal acetic acid (pH 4.0) alone or in combination with bovine casein, casein hydrolysate, or whey hydrolysate. Blood-to-lumen movement of 51Cr-labeled ethylenediaminetetraacetic acid (EDTA) (an index of epithelial permeability) and loop fluid histamine levels were quantified after 45 min. Luminal acetic acid caused a marked increase in 51Cr-EDTA accumulation which was not modified by the addition of bovine casein or hydrolysates by of casein or whey. However, acetic acid-induced histamine release was potentiated by casein and casein hydrolysate (six- and four-fold respectively) but was not altered by whey hydrolysate. Casein hydrolysate-dependent histamine release was evident in naloxone-pretreated rabbits, suggesting that beta-casomorphins were not solely responsible. We conclude that luminal casein or casein hydrolysate, but not whey hydrolysate. can activate intestinal mast cells under conditions of enhanced epithelial permeability. This effect appears to involve components other than beta-casomorphins.