Gastroenterology

Gastroenterology

Volume 124, Issue 5, May 2003, Pages 1369-1380
Gastroenterology

Basic-alimentary tract
NF-κB activation by oxidative stress and inflammation suppresses contractility in colonic circular smooth muscle cells

https://doi.org/10.1016/S0016-5085(03)00263-4Get rights and content

Abstract

Background & Aims:

Transcription factor nuclear factor κB (NF-κB) plays a critical role in transcriptional changes in several diseases, including inflammation. The aim of this study was to investigate whether NF-κB is activated by inflammation and oxidative stress in colonic circular smooth muscle cells and whether that leads to suppression of their contractility.

Methods:

The experiments were performed on freshly dissociated single cells using electrophoretic mobility shift assay, Western immunoblotting, and immunofluorescence imaging.

Results:

The NF-κB DNA binding was ∼6-fold greater in cells from the inflamed colon vs. those from the normal colon. Supershift assay indicated that the antibodies to p65, p50, and c-Rel, but not that to p52, shifted the NF-κB band. Western immunoblotting and immunofluorescence imaging also demonstrated the presence of p65, p50, and c-Rel proteins in the cytoplasm and their translocation to the nucleus by H2O2-induced oxidative stress. H2O2 treatment degraded IκBβ, but not IκBα, to translocate NF-κB to the nucleus. Hydrogen peroxide concentration and time dependently activated NF-κB DNA binding and suppressed cell contraction to acetylcholine. NF-κB inhibitors significantly inhibited these effects. Inhibition of NF-κB prior to and during inflammation in intact dogs also reversed the suppression of contractility.

Conclusions:

Transcription factor NF-κB is activated in colonic circular muscle cells by inflammation and oxidative stress. This activation of NF-κB mediates the suppression of cell contractility.

Section snippets

Induction of colonic inflammation

Acute colonic inflammation was induced by mucosal exposure to ethanol and acetic acid as described previously.22, 32, 33 Briefly, on day 1, the dogs were given an intravenous injection of 30 μg/kg neostigmine to induce defecation. Then they were anesthetized with 150 mg IM Telazol (Elkins-Sinn, Inc., Cherry Hill, NJ). An intraluminal tube with side holes in the first 5-cm length was advanced to the ascending colon, about 50 cm from the anal margin. The colon was cleansed by flushing it with 700

Clinical symptoms and mucosal injury

All dogs developed diarrhea within 24 hours after the induction of inflammation. The mucosa appeared diffusely inflamed, and there were scattered ulcerations and exudates over the mucosal surface. The mean morphology score in the inflamed colon, 4.2 ± 0.4 (n = 4), was significantly greater than that in the normal colon, 0.4 ± 0.2 (n = 7).

NF-κB DNA binding in cells from the normal and the inflamed colon

The mean relative value of NF-κB DNA binding in smooth muscle cells from the inflamed colon, (lanes 4–6, Figure 1) was 6.2-fold greater than that in cells

Discussion

Our findings show that inflammation as well as oxidative stress with H2O2 activates NF-κB in canine colonic circular smooth muscle cells, resulting in suppression of their contractility. The nuclear transcription factor NF-κB in colonic smooth muscle cells is composed of subunits p65, p50, and c-Rel. Using EMSA and Western immunoblotting, we did not find any evidence for the presence of p52 in these cells. However, we cannot rule out the possibility that the human antibody for p52 that we used

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    Supported in part by NIDDK grant DK32346 (to S.K.S.).

    1

    The authors thank the late Dr. You-Gang Lin for his help with immunofluorescence imaging in this study.

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