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. 1998 Feb;42(2):180–187. doi: 10.1136/gut.42.2.180

Increased expression of an inducible isoform of nitric oxide synthase and the formation of peroxynitrite in colonic mucosa of patients with active ulcerative colitis

H Kimura 1, R Hokari 1, S Miura 1, T Shigematsu 1, M Hirokawa 1, Y Akiba 1, I Kurose 1, H Higuchi 1, H Fujimori 1, Y Tsuzuki 1, H Serizawa 1, H Ishii 1
PMCID: PMC1727005  PMID: 9536941

Abstract

Background—Increased production of reactive metabolites of oxygen and nitrogen has been implicated in chronic inflammation of the gut. The object of this study was to examine the magnitude and location of nitric oxide synthase (NOS) activity and peroxynitrite formation in the colonic mucosa of patients with ulcerative colitis in relation to the degree of inflammation. 
Subjects—Thirty three patients with active ulcerative colitis (17 with mild or moderate inflammation, 16 with severe inflammation). 
Methods—Inducible NOS activity was determined in the colonic mucosa by measuring the conversion of L-arginine to citrulline in the absence of calcium. The localisation of NOS and nitrotyrosine immunoreactivity was assessed immunohistochemically using the labelled streptavidin biotin method. 
Results—Inducible NOS activity increased in parallell with the degree of inflammation of the mucosa. Expression of inducible NOS was found not only in the lamina propria, but also in the surface of the epithelium. Peroxynitrite formation as assessed by nitrotyrosine staining was frequently observed in the lamina propria of actively inflamed mucosa. 
Conclusions—Nitric oxide and peroxynitrite formation may play an important role in causing irreversible cellular injury to the colonic mucosa in patients with active ulcerative colitis. 



Keywords: nitric oxide; peroxynitrite; nitric oxide synthase; ulcerative colitis; colonic mucosa

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Figure 1 .

Figure 1

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Detection of inducible nitric oxide synthase (iNOS) protein in the biopsy specimen of colonic mucosa by Western blotting with anti-iNOS antibody. Lane a, iNOS was not detected in homogenate of histologically normal mucosa from a control subject. Lane b, in the active UC mucosa with severe inflammation (grade 4), anti-iNOS antibody labelled a single major band which migrated at the 130 kDa position. Lane c, only a weak band was detected in the UC mucosa with mild inflammation (grade 2). 


Figure 2 .

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Inducible nitric oxide synthase (iNOS) activity in colonic mucosa. iNOS activity was determined in biopsy specimens from endoscopically normal (quiescent) mucosa and actively inflamed mucosa by monitoring the conversion of L-arginine to citrulline. Enzyme activities associated with mild or moderate inflammation (grade 2 or 3) and severe inflammation (grade 4 or 5) were compared. Values are expressed as means and SEM. *p<0.05 compared with quiescent mucosa. †p<0.05 compared with colonic mucosa with grade 2 or 3 inflammation. 


Figure 3 .

Figure 3

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Immunohistochemical study of inducible nitric oxide synthase (iNOS) activity in actively inflamed ulcerative colitis mucosa. Localisation of iNOS immunoreactivity was examined using the labelled streptavidin biotin method with antibodies against iNOS (macNOS). The biopsy specimen was obtained from actively inflamed mucosa grade 4. (A) Significant iNOS reactivity was demonstrated in the surface epithelium of the colonic mucosa as well as in the lamina propria. (B) A negative control picture without primary antibody is also shown.(Original magnification × 100.) 


Figure 4 .

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Immunohistochemical study of inducible nitric oxide synthase (iNOS) activity in actively inflamed ulcerative colitis mucosa. Localisation of iNOS immunoreactivity was examined using the labelled streptavidin biotin method employing antibodies against iNOS (macNOS). The biopsy specimen was obtained from actively inflamed mucosa with grade 5 inflammation. In this patient, iNOS reactivity was confirmed in the surface epithelium of the colonic mucosa (A). A negative control picture without primary antibody is shown (B). (Original magnification × 400.) 


Figure 5 .

Figure 5

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Relation between inducible nitric oxide synthase (iNOS) enzyme activity and the site of iNOS immunoreactivity in the inflamed colonic mucosa of patients with active ulcerative colitis. Values are expressed as means and SEM. *p<0.05 compared with colonic mucosa without iNOS immunoreactivity. †p<0.05 compared with the colonic mucosa with positive iNOS immunoreactivity observed in both lamina propria and epithelial cells. 


Figure 6 .

Figure 6

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Immunohistochemical study of nitrotyrosine activity in actively inflamed ulcerative colitis mucosa. Localisation of nitrotyrosine immunoreactivity was examined using the labelled streptavidin biotin method with antibodies against nitrotyrosine. The biopsy specimen was obtained from actively inflamed mucosa of grade 4 inflammation. Nitrotyrosine immunoreactivity was observed in the lamina propria of colonic mucosa (A). A negative control picture without primary antibody is shown (B). (Original magnification × 400.) 


Figure 7 .

Figure 7

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Immunohistochemical study of nitrotyrosine activity in actively inflamed ulcerative colitis mucosa. Localisation of nitrotyrosine immunoreactivity was examined using the labelled streptavidin biotin method with antibodies against nitrotyrosine. The biopsy specimen was obtained from grade 5 actively inflamed mucosa. In this patient, nitrotyrosine immunoreactivity was observed not only in the lamina propria, but also in the surface epithelium (A). A negative control picture without primary antibody is shown (B). (Original magnification × 400.) 


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