Elsevier

Regulatory Peptides

Volume 101, Issues 1–3, 15 September 2001, Pages 35-41
Regulatory Peptides

Secretion of the trefoil factor TFF3 from the isolated vascularly perfused rat colon

https://doi.org/10.1016/S0167-0115(01)00257-9Get rights and content

Abstract

The trefoil factor TFF3 is a peptide predominantly produced by mucus-secreting cells in the small and large intestines. It has been implicated in intestinal protection and repair. The mechanisms that govern TFF3 secretion are poorly understood. The aim of this study was, therefore, to evaluate the influence of neurotransmitters, hormonal peptides and mediators of inflammation on the release of TFF3. For this purpose, an isolated vascularly perfused rat colon preparation was used. After a bolus administration of 1 ml isotonic saline into the lumen, TFF3 secretion was induced by a 30-min intra-arterial infusion of the compounds to be tested. TFF3 was evaluated in the luminal effluent using a newly developed radioimmunoassay. TFF3 was barely detected in crude luminal samples. In contrast, dithiothreitol (DTT) treatment of the effluent revealed TFF3 immunoreactivity, which amounted to about 0.3 pmol min−1 cm−1 in the basal state. Gel chromatography of DTT-treated luminal samples revealed a single peak that co-eluted with the monomeric form of TFF3. TFF3 was not detected in the portal effluent. Bethanechol (10−6–10−4 M), vasoactive intestinal peptide (VIP, 10−8–10−7 M) or bombesin (10−8–10−7 M) induced a dose-dependent release of TFF3. In contrast, substance P evoked a modest release of TFF3, whereas calcitonin gene-related peptide (CGRP), somatostatin, neurotensin or peptide YY (PYY) did not modify TFF3 secretion. The degranulator compound bromolasalocid, 16,16-dimethyl PGE2 (dmPGE2) or interleukin-1-β (IL-1-β) also evoked a marked release of TFF3. In conclusion, TFF3 in the colonic effluent is present in a complex. This association presumably involves a disulfide bond. Additionally, the present results suggest a role for enteric nervous system and resident immune cells in mediation of colonic TFF3 secretion.

Introduction

The trefoil factor family comprises a small family of polypeptides that share one or more distinctive three-loop structures formed by intrachain disulfide bonding, based on a conserved motif of six cysteine residues called a trefoil domain (for reviews, see [1], [2], [3], [4]). There are three distinct members of the trefoil peptides identified in mammalian species: pS2 (TFF1), spasmolytic polypeptide (SP or TFF2) and intestinal trefoil factor (ITF or TFF3). These peptides are highly expressed in region-specific patterns throughout the digestive system. TFF1 and TFF2 are produced in the stomach [5], whereas TFF3 is primarily localized within goblet cells in the small and large bowels [6], [7], [8]. Several findings indicate that TFF3 is important in the protection against mucosal damage and in aiding in intestinal mucosal protection and epithelial repair. Indeed, targeted disruption of the mouse TFF3 gene leads to impaired healing and epithelial regeneration of the intestinal mucosa, and intracolonic administration of trefoil peptides protects against experimental colonic injury in mice and rats [9], [10]. Additionally, two recent studies indicate that TFF3 confers colonic epithelial resistance to apoptosis in an in vitro model [11] and suppresses the growth of colon carcinoma cells [12].

Despite the apparent importance of TFF3 in several aspects of epithelial barrier function, little is known about the regulation of TFF3 secretion. A recent study showed that short-chain fatty acids inhibit TFF3 release from the colon cancer LIM 1215 cell that has a goblet-enterocyte phenotype, thus suggesting that luminal factors may modulate TFF3 secretion [13]. In addition, TFF3-producing cells are located in the vicinity of endocrine cells, immune cells and enteric nerves, which could modulate TFF3 release. In the tumoral cell line HT-29, carbachol, vasoactive intestinal peptide (VIP) and somatostatin were shown to be potent TFF3 secretagogues, while constituents of the mucosal cytokine network had no effect [14]. The release of TFF3 from native goblet cells of the colon has not yet been studied. The aim of the present study was, therefore, to investigate the potential influence of various local transmitters, inflammatory mediators and hormonal peptides on the secretion of TFF3 from the rat colon.

To specifically address this question, we used an isolated, vascularly perfused rat colon preparation [15]. This model maintains the polarity of peptide secretion and allows separate sampling of the luminal and vascular effluents. Additionally, the TFF3-producing cells may be submitted to well-defined luminal, neural and blood-borne stimuli in a manner that eliminates influences potentially encountered in vivo.

Section snippets

Materials

BSA was purchased from Biovalori (Cassen, France). Azonutril 25, a mixture of amino acids, was obtained from Laboratoires Roger Bellon (Neuilly-sur-Seine, France). This solution consists of 3.4% (w/v) isoleucine, 9.3% leucine, 8.5% lysine, 6.3% methionine, 8.3% phenylalanine, 3.4% threonine, 1.7% tryptophan, 8.4% valine, 2.7% aspartic acid, 3.4% glutamic acid, 6.4% alanine, 16.8% arginine, 1% cysteine, 6% glycine, 3.4% histidine, 5.4% proline, 0.9% serine, 0.2% tyrosine, 2% citrulline, and 1.5%

Surgical procedure

The procedure to prepare an isolated vascularly perfused rat colon was described previously [15]. Male Wistar rats (250–350 g) were anesthetized with pentobarbital sodium (50 mg/kg, i.p.) and the abdomen was opened with a midline incision. The proximal and transverse colons were freed from their visceral fixations. The ileocolic veins and arteries were tied. The colonic loops (10 cm in length) were then rinsed twice with 10 ml isotonic saline, flushed with 20 ml air and ligated at both ends. A

Release of TFF3 in the colonic lumen by neurotransmitters

The mean release of TFF3 immunoreactivity was 106±10 pmol/30 min in the control group (n=10). Upon stimulation with 10−5 M bethanechol, a significant increase of TFF3 release was observed. The TFF3 response culminated at 550% of the basal control value upon intra-arterial infusion of 10−4 M bethanechol (Fig. 3). Atropine (10−5 M) blocked the secretion of TFF3 induced by 10−5 M bethanechol (data not shown). Vascular infusion of VIP (10−8 or 10−7 M) induced a moderate, dose-dependent release of

Discussion

Studies providing direct evidence for TFF3 release from the colonic epithelium are scarce. This work demonstrates that TFF3 is released from the colonic mucosa and that it is present in a complex. This association involves a disulfide bond. Additionally, we show that several transmitters of the enteric nervous system and compounds involved in the inflammatory process are potent stimulants of TFF3 release.

TFF3 is selectively expressed in goblet cells throughout the small and large intestines.

Acknowledgments

The “Fondation pour la Recherche Médicale” (France) and NHMRC (Australia) supported part of this study. The technical assistance of Henri Guignard is warmly acknowledged.

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