Carbachol induces TGF-alpha expression and colonic epithelial cell proliferation in sensory-desensitised rats.

OBJECTIVE/BACKGROUND: Signals for the expression of the peptide growth factors epidermal growth factor and transforming growth factor-alpha (TGFalpha) in the gastrointestinal mucosa are largely unknown. We have shown earlier that extrinsic afferents in the gastrointestinal tract induce TGFalpha expression in colonic mucosa via the deliberation of neurotransmitters substance P and calcitonin gene-related peptide. The aim of our present study was to determine the effects of carbachol on mucosal TGFalpha expression and epithelial cell proliferation in vivo. DESIGN/METHODS: Rats were divided in three groups. Group 1 was treated with vehicle only, group 2 received one single subcutaneous injection of 250 microg/kg of carbachol and animals in group 3 were sensory-desensitised prior to the injection of 250 microg/kg carbachol. TGFalpha expression and epithelial cell proliferation was evaluated by polymerase chain reaction, Western blot analysis and bromodeoxyuridine staining. RESULTS: Carbachol induced a significant increase in mucosal epithelial cell proliferation and TGFalpha expression. Sensory desensitisation did neither abolish the increased TGFalpha expression nor the increase in epithelial cell proliferation. CONCLUSION: Parasympathetic pathways are involved in the control of TGFalpha expression in gastrointestinal mucosa as well as in epithelial cell proliferation.

Substance P and calcitonin gene related peptide induce TGF-alpha expression in epithelial cells via mast cells and fibroblasts.

OBJECTIVE/BACKGROUND: Signals for the expression of EGF, TGFalpha and related proteins are largely unknown. Having shown earlier, that activation of the afferent sensory nervous system by capsaicin induced epithelial cell proliferation and TGFalpha expression in vivo the aim of this study was to demonstrate, that neurotransmitters of the afferent sensory nervous system induce TGFalpha expression via mast cells and fibroblasts. RESULTS: A significant increase in TGFalpha-mRNA and protein expression was detected in epithelial cells exposed to supernatants of CGRP-stimulated mast cells and SP-stimulated fibroblasts. Epithelial cell proliferation was only detected in epithelial cells exposed to supernatants of CGRP-stimulated mast cells but not in epithelial cells exposed to supernatants of SP-stimulated fibroblasts. TGFbeta mRNA expression was increased in epithelial cells exposed to fibroblasts but not to mast cells. No increase in EGF-receptor expression was detected in epithelial cells exposed to either fibroblast or mast cell supernatants. CONCLUSION: Neurotransmitters of the afferent sensory nervous system induce TGFalpha expression in epithelial cells mediated by mast cells and fibroblasts. Release of CGRP and SP from neuroeffector junctions is an important signal for the TGFalpha expression after mucosal injury. Mucosal expression of TGFalpha may contribute to the protective effects of the sensory nervous system after mucosal injury.

Increased duodenal expression of transforming growth factor-alpha and epidermal growth factor during experimental colitis in rats.

OBJECTIVE/BACKGROUND: Epidermal growth factor (EGF) and transforming growth factor alpha (TGFalpha) protect gastrointestinal mucosa against injury. Having shown earlier, that TGFalpha but not EGF is locally increasingly expressed after mucosal injury in the colon, we now wanted to explore the pattern of expression of EGF and TGFalpha in the remaining gastrointestinal tract and to infer from the pattern of expression, to possible signals for the induction of the growth factor expression and further mechanisms for mucosal protection. DESIGN/METHODS: The trinitrobenzene sulfonic acid/ethanol-induced model of colitis in rats was used. TGFalpha-mRNA and EGF-mRNA expression was evaluated in inflamed and noninflamed colon, in the ileum, jejunum, duodenum, stomach, and in the submandibulary glands. RESULTS: A significant increase of TGFalpha-mRNA and EGF-mRNA expressions was detected in the duodenal mucosa and a significant increase in TGFalpha-mRNA expression was observed in the inflamed colonic mucosa after mucosal injury in the colon within the first hours of colitis. CONCLUSION: The increased expression of EGF and TGFalpha in the duodenum may lead to neutralization of gastric acid and proteolytic enzymes in the upper gastrointestinal tract during the course of colitis. Possible signals for the increased expression of EGF and TGFalpha presumably are fasting, parasympathetic, or adrenergic parts of the enteric nervous system or yet unknown mechanisms.

Sensory neuropeptides and epithelial cell restitution: the relevance of SP- and CGRP-stimulated mast cells.

BACKGROUND: Calcitonin-gene-related peptide (CGRP) and substance P (SP) are neurotransmitters of extrinsic primary afferent neurons located within the dorsal root ganglia. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on intestinal mucosa was presumed. The mucosal protection partly depends on a CGRP-mediated modulation of mucosal blood flow. Limited data are available regarding CGRP- or SP-mediated effects on epithelial cell restitution. Having shown earlier that SP-stimulated fibroblasts but not CGRP-stimulated fibroblasts induce epithelial cell migration in vitro, the aim of this study was to explore whether mast cells mediate effects of SP and CGRP on epithelial cell restitution in vitro. METHODS: Mast cells (C57) were exposed to SP [10(-12)-10(-6 M)] and CGRP [10(-12)-10(-7 M)]. After a 24-h incubation period, the cell supernatants (conditioned media, CDM) were taken from mast cell cultures and directly applied to rat intestinal epithelial cell lines-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h prior to the experiments. Epithelial cell migration was assessed by counting cells across the wound edge and epithelial cell proliferation was measured using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide test. RESULTS: CGRP significantly induced epithelial cell migration and proliferation via mast cells when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralizing anti-transforming growth factor-beta (TGF-beta) had been added to the cell cultures. SP had no effects on epithelial cells following stimulation of mast cells. CONCLUSION: CGRP modulates epithelial cell restitution in vitro mediated by mast cells. The CGRP- and mast-cell-induced epithelial cell migration is TGF-beta dependent. This observation underlines an important role for extrinsic primary afferent neurons in mucosal defence and repair and in keeping the mucosal homeostasis. This knowledge leads to a better understanding of the interaction of the enteric nervous system and wound healing and may, in the future, lead to new therapeutic approaches to inflammatory diseases of the intestine.

Substance P induces intestinal wound healing via fibroblasts--evidence for a TGF-beta-dependent effect.

BACKGROUND: Substance P (SP) and calcitonin gene-related peptide (CGRP) are neurotransmitters of the afferent sensory nervous system. In experimental models of colitis in rats and rabbits, a protective role of SP and CGRP on the intestinal mucosa was presumed. In part, mucosal protection depends on a SP-mediated and CGRP-mediated modulation of mucosal blood flow after injury. We thought to explore whether there is a fibroblast-mediated effect of SP and CGRP on epithelial cell restitution in vitro. MATERIALS AND METHODS: Rat kidney fibroblast (NRK-49F) cell lines were exposed to CGRP or SP in various concentrations. After incubation, the cell culture supernatants were taken from the fibroblast cultures and were directly applied to IEC-18 or Caco-2 monolayers, which had been wounded with a razor blade 24 h before the experiments. Epithelial cell migration was assessed by counting cells across the wound edge. Epithelial cell proliferation was assessed using the 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium bromide (MTT) test. RESULTS: SP significantly induced epithelial cell migration and inhibited epithelial cell proliferation via stimulation of fibroblasts when supernatants were directly applied to epithelial cells in vitro. The effects on epithelial cell migration were abolished after neutralising anti-transforming growth factor-beta (TGF-beta) was added to the cell cultures. CGRP had no effect on epithelial cells via stimulation of fibroblasts. Neither CGRP nor SP had any effect on epithelial cell migration or proliferation when directly applied to epithelial cells. CONCLUSION: SP modulates epithelial cell restitution in vitro mediated by fibroblasts. The epithelial cell migration depends on a TGF-beta release from SP-stimulated fibroblasts. This observation underlines an important role for the sensory nervous system in mucosal defence and repair and in keeping mucosal homeostasis. Modulation of SP may be potentially useful for the treatment of various intestinal disorders characterised by injury and ineffective repair of the intestinal mucosa.