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. 2001 Apr;54(4):293–297. doi: 10.1136/jcp.54.4.293

The physiological expression of inducible nitric oxide synthase (iNOS) in the human colon

P Roberts 1, G Riley 1, K Morgan 1, R Miller 1, J Hunter 1, S Middleton 1
PMCID: PMC1731394  PMID: 11304846

Abstract

Background—Inducible nitric oxide synthase (iNOS) is expressed in the colonic epithelium in both inflammatory bowel disease and colorectal cancer. Nitric oxide (NO), the product of this enzyme, has been implicated in the pathogenesis of both conditions. However, there are conflicting data on whether iNOS is expressed in the normal, uninflamed human colon.

Aims—To evaluate the expression of iNOS in histologically normal, non-inflamed human colonic mucosa.

Patients/Methods—Reverse transcription polymerase chain reaction (RT-PCR), immunoblotting, and immunohistochemistry were used to investigate the expression of iNOS in 17 histologically normal specimens obtained at colectomy performed for colorectal neoplasia. In addition, 16 endoscopic mucosal biopsies, taken from normal individuals, were also evaluated. Eleven surgical specimens and 16 endoscopic biopsies from patients with refractory ulcerative colitis were used as inflammatory controls.

Results—All types of specimens expressed iNOS mRNA. Immunoblotting revealed a protein of approximately 130 kDa consistent with iNOS in mucosal extracts of 77% of normal individuals, and 85% of diseased controls. Immunolabelling localised this protein to the surface epithelium in most of the normal specimens and also to the crypt epithelium and inflammatory cells in the diseased controls.

Conclusions—These findings provide evidence that iNOS is often expressed in the surface epithelium of non-inflamed human colon, suggesting that it is induced by local luminal factors, such as bacterial lipopolysaccharide (endotoxin). The resultant NO produced at this site might act as an oxidative barrier, reducing bacterial translocation and providing a means of defence against pathogenic microorganisms.

Key Words: inducible nitric oxide synthase • ulcerative colitis • nitric oxide • colorectal carcinoma

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Figure 1 Qualitative RT-PCR analysis of inducible nitric oxide synthase (iNOS) expression in surgical specimens showing the ubiquitous presence of a 424 bp DNA product indicating iNOS expression. MW, molecular weight markers (indicating 400 bp left lane). Lanes 1–6, normal tissue; lanes 7, 9, and 10, diseased specimens; lane 8, negative control.

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Figure 2 Example of kinetic PCR analysis of inducible nitric oxide synthase (iNOS) expression in a diseased (left hand set) and a normal specimen (right hand set). Densitometrical analysis revealed the expected increase of iNOS product in diseased tissue compared with normal tissue. PCR cycle lengths: G3PDH, 30–34; iNOS, 41–45. G3PDH, glyceraldehyde-3-phosphate dehydrogenase.

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Figure 3 Immunoblotting of surgical mucosal specimens with primary anti-inducible nitric oxide synthase (iNOS) antibody showing band of approximately 130 kDa expressed in normal mucosa, consistent with full length iNOS protein expression. Lanes 1 and 2, normal mucosa; lane S, iNOS recombinant protein standard (smaller sized bands indicating iNOS degradation on storage). Lane P shows immunoreactivity of iNOS inhibited by coincubation with the immunising peptide.

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Figure 4 Immunolocalisation of inducible nitric oxide synthase (iNOS) in surgical specimens showing surface epithelial staining in the normal tissue, and more extensive labelling in the diseased tissue. (A) Non-immune serum, normal mucosa; (B) iNOS expression, normal mucosa; (C) control (primary antibody quenched with 2x concentration of immunising peptide), normal mucosa; (D) iNOS expression, diseased mucosa. Original magnification x120 (A, C, and D,); original magnification x240 (B).

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