Roberts, Philip John; (2001) The physiological expression of inducible nitric oxide synthase (iNOS) in the human colon. Doctoral thesis (M.D), UCL (University College London).

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Abstract

Background: Inducible nitric oxide synthase (iNOS) is expressed in the colonic epithelium in both inflammatory bowel disease (IBD) and colorectal cancer. Nitric oxide (NO), the product of this enzyme has been implicated in the pathogenesis of both conditions. There is conflicting data, however, on whether iNOS is expressed in the normal, uninflamed human colon. Hypothesis: In preliminary work using a colonocyte cell line (HT-29), I was able to show expression of iNOS under bacterial lipopolysaccharide (LPS) stimulation. I postulated therefore, that normal human colonic mucosa might express iNOS in the same manner in the light of its close proximity to such stimulants as LPS. Patients and methods: RT-PCR, immunoblotting and immunohistochemistry were used to investigate iNOS expression in 17 histologically normal specimens obtained at colectomy performed for colorectal neoplasia. In addition, 14 normal mucosal biopsies were obtained endoscopically and evaluated. Twelve surgical specimens and 16 endoscopic biopsies from patients with active ulcerative colitis (UC) were used as inflammatory controls. Results: All specimens expressed iNOS mRNA, with increased product demonstrated in the inflamed compared to normals using kinetic analysis. Immunoblotting revealed a protein of approximately 130 kDa consistent with iNOS in mucosal extracts of 77% normals and 81% diseased controls. Immunolabelling localised this protein to the surface epithelium in the majority of the normal specimens and also to the crypt epithelium and inflammatory cells in the colitic specimens. Conclusions: My data provides evidence that iNOS is commonly expressed in the surface epithelium of the non-inflamed human colon, suggesting that it might be induced by local luminal factors, such as LPS (endotoxin). The resultant NO produced at this site may act as an oxidative barrier reducing bacterial translocation and providing a means of defence against pathogenic microorganisms.

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