David Coyle1,2, Anne Marie O'Donnell3, Nicolae Corcionivoschi3, John Gillick3,4, Prem Puri3. 1. National Children's Research Centre, Our Lady's Children's Hospital, Crumlin Rd., Dublin 12, Ireland. daithiocool@gmail.com. 2. Department of Paediatric Surgery, Temple Street Children's University Hospital, Temple St., Dublin 1, Ireland. daithiocool@gmail.com. 3. National Children's Research Centre, Our Lady's Children's Hospital, Crumlin Rd., Dublin 12, Ireland. 4. Department of Paediatric Surgery, Temple Street Children's University Hospital, Temple St., Dublin 1, Ireland.
Abstract
BACKGROUND: Rho-kinase (ROCK) is the primary effector protein in the RhoA pathway, which regulates Ca(2+)-independent smooth muscle contraction in the human bowel. This pathway has been reported to be hyper-activated in the aganglionic bowel of EDNRB-null (-/-) rats compared to the ganglionic bowel from EDNRB (+/+) rats. We hypothesised that ROCK expression is up-regulated in human aganglionic bowel and designed this study to investigate ROCK 1 and ROCK 2 expression in Hirschsprung's disease (HSCR) and controls. MATERIALS AND METHODS: Full-length specimens were collected following pull-through surgery for HSCR (n = 9). Colonic controls (n = 6) were obtained during colostomy closure from patients with anorectal malformations. Distribution of ROCK 1/2 expression was evaluated using double-labelled immunofluorescence and confocal microscopy. ROCK1/2 protein expression was assessed in mucosa and tunica muscularis using western blot analysis. RESULTS: There was strong expression of both ROCK 1 and ROCK 2 in interstitial cells of Cajal (ICCs) and ganglia. ROCK 1 expression was reduced in aganglionic bowel compared to HSCR ganglionic bowel and controls in both mucosa and tunica muscularis. ROCK 2 expression was similar in the colon of children with HSCR and controls. CONCLUSIONS: This is the first report of strong ROCK expression in colonic ICCs. Although the rat model of aganglionic bowel suggests that Ca(2+)-independent smooth muscle contraction involving ROCK is hyper-activated, our data indicate ROCK 1 expression is decreased in aganglionic bowel and ROCK 2 expression is unaltered in children with HSCR.
BACKGROUND: Rho-kinase (ROCK) is the primary effector protein in the RhoA pathway, which regulates Ca(2+)-independent smooth muscle contraction in the humanbowel. This pathway has been reported to be hyper-activated in the aganglionic bowel of EDNRB-null (-/-) rats compared to the ganglionic bowel from EDNRB (+/+) rats. We hypothesised that ROCK expression is up-regulated in human aganglionic bowel and designed this study to investigate ROCK 1 and ROCK 2 expression in Hirschsprung's disease (HSCR) and controls. MATERIALS AND METHODS: Full-length specimens were collected following pull-through surgery for HSCR (n = 9). Colonic controls (n = 6) were obtained during colostomy closure from patients with anorectal malformations. Distribution of ROCK 1/2 expression was evaluated using double-labelled immunofluorescence and confocal microscopy. ROCK1/2 protein expression was assessed in mucosa and tunica muscularis using western blot analysis. RESULTS: There was strong expression of both ROCK 1 and ROCK 2 in interstitial cells of Cajal (ICCs) and ganglia. ROCK 1 expression was reduced in aganglionic bowel compared to HSCR ganglionic bowel and controls in both mucosa and tunica muscularis. ROCK 2 expression was similar in the colon of children with HSCR and controls. CONCLUSIONS: This is the first report of strong ROCK expression in colonic ICCs. Although the rat model of aganglionic bowel suggests that Ca(2+)-independent smooth muscle contraction involving ROCK is hyper-activated, our data indicate ROCK 1 expression is decreased in aganglionic bowel and ROCK 2 expression is unaltered in children with HSCR.
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