Jane H Thompson

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

BACKGROUND & AIMS Inflammatory 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. METHODS Inflammation 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. RESULTS In 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. CONCLUSIONS The 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.

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

BACKGROUND/AIMS Excess 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. METHODS NO 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. RESULTS Plasma 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. CONCLUSIONS It 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.

MATERNAL ADMINISTRATION OF INTERLEUKIN10 PREVENTS LNAME INDUCED FETAL ANOMALIES BUT NOT GROWTH RESTRICTION † 289

MATERNAL ADMINISTRATION OF INTERLEUKIN10 PREVENTS LNAME INDUCED FETAL ANOMALIES BUT NOT GROWTH RESTRICTION † 289

EXPERIMENTAL NECROTIZING ENTEROCOLITIS BASED ON THE INTERACTIONS BETWEEN FEEDING, FLORA AND DYSMOTILITY ♦ 506

EXPERIMENTAL NECROTIZING ENTEROCOLITIS BASED ON THE INTERACTIONS BETWEEN FEEDING, FLORA AND DYSMOTILITY ♦ 506

BENEFICIAL EFFECTS OF INTERLEUKIN10 IN A RAT MODEL OF FETAL GROWTH RESTRICTION AND DEMISE. † 290

BENEFICIAL EFFECTS OF INTERLEUKIN10 IN A RAT MODEL OF FETAL GROWTH RESTRICTION AND DEMISE. † 290

PRENATAL N G -NITRO-L-ARGININE METHYL ESTER (L-NAME) TREATMENT PRODUCES ASYMMETRIC FETAL GROWTH RETARDATION AND A PARADOXICAL INCREASE IN NITRIC OXIDE PRODUCTION. † 1490

PRENATAL N G -NITRO-L-ARGININE METHYL ESTER (L-NAME) TREATMENT PRODUCES ASYMMETRIC FETAL GROWTH RETARDATION AND A PARADOXICAL INCREASE IN NITRIC OXIDE PRODUCTION. † 1490

ONTOGENY OF INDUCIBLE NITRIC OXIDE SYNTHASE IN FETAL RATS. |[utrif]| 376

Nitric oxide (NO) formation is upregulated during pregnancy, but its role in fetal and perinatal development has not been clearly defined. Both constitutive (cNOS) and inducible (iNOS) isoforms contribute to NO production in the utero-placental unit. cNOS is normally responsible for NO synthesis under physiologic conditions, whereas iNOS expression is a hallmark of host defense mechanisms and autoimmune disorders. The purpose of this study was to determine if iNOS is expressed in the utero-placental unit and fetus.STUDY DESIGN: Timed pregnant rats were sacrificed on days 14, 16, 19 and 21 of gestation and days 1, 3 and 7 of life. Fetal heart, intestine, liver, lung, spleen and thymus and maternal colon, placenta and uterus were harvested. RNA was extracted using the guanidine thiocyanate extraction method and iNOS expression determined from total RNA using RT-PCR. Additionally, uterine and placental tissues were evaluated for iNOS expression by immunohistochemical techniques. RESULTS: At 14 and 16 days gestation, fetal tissue (total) was positive for iNOS. At 19 and 21 days gestation, when dissection allowed testing of individual organs, iNOS was present in fetal lung and heart, intestine, spleen and thymus, respectively but absent in fetal liver. Results were similar at 1, 3 and 7 days postnatal life. Placenta, uterus and maternal colon were positive for iNOS throughout gestation. Immunohistochemical techniques reveal that the placental sinusoids and uterine smooth muscle as predominant sites of iNOS expression.CONCLUSIONS: The inducible enzyme, iNOS, a cytotoxic-cytostatic agent of the immune system, is expressed in the fetal tissue from the second trimester through early post-natal life. We postulate that the fetus can afford to utilize iNOS as a source of NO because the likelihood of peroxynitrite formation is reduced because of its hypoxic environment. It appears that the expression of iNOS and increased NO release perse, are not synonymous with cell injury.

IS NITRIC OXIDE-DEPENDENT UTEROPLACENTAL FUNCTION REGULATED BY iNOS, NOT cNOS? AN ELECTROCHEMICAL AND GENE EXPRESSION EVALUATION. ▴ 1395

IS NITRIC OXIDE-DEPENDENT UTEROPLACENTAL FUNCTION REGULATED BY iNOS, NOT cNOS? AN ELECTROCHEMICAL AND GENE EXPRESSION EVALUATION. ▴ 1395

INDUCIBLE NITRIC OXIDE SYNTHASE REGULATES FETAL GREAT VESSEL CALIBER. 420

Background Nitric oxide plays a role in the regulation of fetal great vessels and ductus arteriosus caliber near the end of gestation but the enzyme source is unknown. Aims: To determine if inducible nitric oxide synthase (iNOS) is present in the cardiovascular system of fetal rats, and to compare the effects ofL-NG-(1-lminoethyl)lysine (L-NIL) a selective inhibitor of iNOS, with lipopolysacharide (LPS), sodium nitroprusside (SNP) and untreated controls near the end of gestation.Methods and Results iNOS inthe rat fetus was demonstrated in heart/great vessels and placenta using RT-PCR. Rats in the last week of pregnancy were treated for 5 d with L-NIL (1, 10, and 100 μg/ml in the drinking water). LPS 30 μg/kg od ip 5 d. SNP in mini-osmotic pumps to deliver 10 μg/kg/min. Control group was undisturbed. On day 21 of gestation, dams were anesthetized, fetuses delivered by cesarean section and rapidly frozen in isopentane chilled in liquid nitrogen. Cryostat sections(10μm) were used to reconstruct a computer generated 3D image of the great vessels and ductus arteriosus, calibers were measured with electronic calipers in mm. Table shows Mean±SEM Significant dose-dependent constriction of the great vessels and ductus arteriosus was observed with L-NIL while both LPS and SNP significantly dilated the vessels.Conclusions Nitric oxide, generated by inducible nitric oxide synthase plays a significant role in the control of major vessels and ductus arteriosus caliber in the fetal rat. In this regard nitrergic regulation of vascular tone in the fetus differs from the adult.