Expression of nitric oxide synthases and effects of L-arginine and L-NMMA on nitric oxide production and fluid transport in collagenous colitis

Abstract

Background and aims: Luminal nitric oxide (NO) is greatly increased in the colon of patients with collagenous and ulcerative colitis. To define the source and consequence of enhanced NO production we have studied expression of NO synthase (NOS) isoforms and nitrotyrosine in mucosal biopsies from these patients. In addition, effects on colonic fluid transfer caused by manipulating the substrate of NOS were studied in patients with collagenous colitis.

Patients: Eight patients with collagenous colitis, nine with active ulcerative colitis, and 10 with uninflamed bowel were included.

Methods: Expression of NOS isoforms was quantified by western blotting. Inducible NOS (iNOS) and nitrotyrosine were localised by immunohistochemistry. Modulation of NOS activity by topical N(G)-monomethyl-L-arginine (L-NMMA) or L-arginine was assessed during perfusion of whole colon. Plasma and perfusate nitrite/nitrate (NOx) was measured by Griess' reaction.

Results: Both in collagenous and ulcerative colitis, expression of iNOS was 10(2)-10(3) higher (p<0.001) than in uninflamed bowel and localised primarily to the epithelium. Endothelial NOS was evenly expressed in all groups while neuronal NOS was undetectable. Nitrotyrosine was markedly expressed in active ulcerative colitis but rarely detected in collagenous colitis and never in uninflamed bowel. In collagenous colitis, the output of NOx was markedly increased compared with uninflamed bowel (283 (58) v <37 nmol/min; p<0.01) and fluid was net secreted. L-NMMA reduced the output of NOx by 13-66% (95% confidence intervals) and secretion of fluid by 25-109% whereas L-arginine increased the output of NOx by 3-39% and secretion of fluid by 15-93%.

Conclusions: In collagenous colitis, as opposed to ulcerative colitis, upregulation of iNOS occurs in the absence of nitrotyrosine formation and mucosal damage. Excess generation of NO may be the primary cause of diarrhoea in this condition.

Figure 1

Expression of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) in mucosal biopsies from patients with collagenous colitis, active ulcerative colitis, or uninflamed bowel. Expression was analysed by western blotting and quantified by densitometry relative to a reference, which was defined as 1.0. For internal control of loading, samples were also blotted against anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH) antibody. In the scatterplots, the logarithmic y axes define individual values with group means displayed as horizontal lines.

Figure 2

Localisation of inducible nitric oxide synthase (iNOS) in mucosal biopsies from human colon analysed by immunohistochemistry and counterstained with haematoxylin. (A) Uninflamed mucosa stained with iNOS IgG antibody (see text). No reaction was observed. (B) Biopsy section from a collagenous colitis patient showing a thickened collagenous band beneath the epithelium (open arrow). The reaction product (red) of iNOS antibody is localised at the luminal border of the epithelial cells (see arrows at surface and crypts) and to a minor degree in lamina propria mononuclear cells (arrowhead). (C) Biopsy from a patient with severe ulcerative colitis at endoscopy showing disturbed mucosal architecture and pronounced infiltration of inflammatory cells. The reaction product of the iNOS antibody is localised primarily in the surface epithelial cells (arrow). A discrete reaction is observed in adjacent neutrophils and in mononuclear cells of the lamina propria. In areas with less intense reaction this is primarily localised in the apical cytoplasm (arrowheads).

Figure 3

Localisation of nitrotyrosine in mucosal biopsies from human colon analysed by immunohistochemistry and counterstained with haematoxylin. (A) Uninflamed mucosa stained with nitrotyrosine antibody (see text). No reaction was observed. (B) Biopsy section from a patient with collagenous colitis. The reaction product (red) of the nitrotyrosine antibody is observed within mononuclear cells of the lamina propria (arrowheads). (C) Biopsy section from a patient with ulcerative colitis showing disturbed mucosal architecture and pronounced infiltration of inflammatory cells. The reaction product of the nitrotyrosine antibody is localised in the epithelium (arrows) in association with neutrophils and in lamina propria inflammatory cells (arrowheads)