Systemically administered trefoil factors are secreted into the gastric lumen and increase the viscosity of gastric contents

Abstract

Background and purpose: Trefoil factors (TFFs) secreted by mucus-producing cells are essential for the defence of the gastrointestinal mucosa. TFFs probably influence the viscoelastic properties of mucus, but this has not been demonstrated in vivo. We therefore studied the gastric secretion of systemically administered TFF2 and TFF3, and their influence on the viscosity of the secretions.

Experimental approach: Mice and rats under general anaesthesia were injected intravenously with human (h) TFF2, hTFF3 (5 mg kg(-1) to mice and 25 mg kg(-1) to rats), murine (m) (125)I-TFF3, or (125)I-hTFF3 (300,000 cpm, mice only). The appearance of TFFs in the gastric mucosa and luminal secretions was analysed by autoradiography, gamma-counting, and ELISA, and the viscosity by rheometry.

Key results: (125)I-mTFF3 and (125)I-hTFF3 were taken up by secretory cells of the gastrointestinal tract and detected at the gastric mucosal surface 15 min after injection. Stressing the stomach by carbachol (3.5 microg kg(-1)) and pyloric ligation significantly increased the uptake. Injected hTFF2, hTFF3, and mTFF3 were retrieved from the gastric contents after 4 h. In rats, an approximately seven-fold increase in the viscosity was detected after injection of TFF2 compared to the controls, whereas TFF3 did not increase the viscosity. In mice, TFF2 increased the viscosity approximately 4-fold.

Conclusions: These data indicate that systemically administered TFFs are transferred to the gastric lumen in a biologically active form.

Figure 1

Distribution of ¹²⁵I-TFF3 to the stomach and gastric secretions of mice. Values shown are the percentage of injected dose (% ID) in a box and whisker plot. ¹²⁵I-mTFF3 or ¹²⁵I-hTFF3 was injected i.v. into control mice (n=8), mice that had the stomach ligated (n=16) or catheterized (n=12). Half of the mice in the surgically manipulated groups received 3.5 μg kg⁻¹ carbachol s.c. (+carbachol) and half received saline. Specimens were sampled 180 min after injection and the radioactivity measured. (a) In the gastric tissue, all manipulations resulted in significantly increased % ID as compared to control mice. (b) In the gastric secretions, a significantly higher % ID was found after ligation than after catheterization. Carbachol caused a significantly increased accumulation of ¹²⁵I-mTFF3 in catheterized but not ligated mice. In both (a) and (b), the last two values compare the distribution of mouse and human peptide. Although the tissue binding was comparable, significantly more ¹²⁵I-mTFF3 than ¹²⁵I-hTFF3 was detected in the secretions. ^**P<0.01, ^***P<0.0001.

Figure 2

Viscosity of gastric secretions. Rats and mice were injected with hTFF2 (rats, n=18; mice, n=13), hTFF3 (rats, n=6) or 0.9% saline (control: rats, n=20; mice, n=7) i.v. and s.c. and the stomach was ligated. Four hours after injection, the stomach contents were collected and the viscosity measured. Viscosity is expressed in nm² s⁻¹ (individual values and median). ^**P<0.01, ^***P<0.001.

Figure 3

Retrieval of injected hTFF2 and hTFF3 in gastric secretions from mice (a–b) and in serum and gastric secretions from rats (c). hTFF2, hTFF3 or saline was injected i.v., and the stomach was ligated. Specimens were sampled after 4 h, and the TFF concentrations measured by ELISA for either hTFF2 or hTFF3. (a) hTFF2 in gastric secretions from mice. Box and whisker plots. (b) hTFF3 in gastric secretions of mice. Values shown are the number of samples in a specific concentration range. (c) hTFF2 or hTFF3 in gastric secretions (secr.) and serum from rats. Box and whisker plot. In all groups injected with hTFF2 or hTFF3, significantly higher concentrations of hTFF2 or hTFF3, respectively, were detected (P<0.001).

Figure 4

Localization of i.v. injected and endogenous TFF by autoradiography, in situ hybridization and immunohistochemistry on sections of stomach tissue from controls, and from ligated carbachol-treated mice. The accumulation of grains in the mucous neck cells and pyloric glands was considerably higher in ligated animals than in controls, as was the amount of grains in the lumen of the gastric glands and on the surface of the stomach. The localization of TFF mRNA and peptide correlated, except for TFF3, where peptide but not mRNA was found in the corpus fundus. (a) Accumulation of grains in neck cells, control, (b) higher amounts of grains in the ligated stomach. Comparable differences are seen between pyloric glands of controls (c) and ligated (d). (e) Binding of grains to the basal part of mucous neck cells 15 min after injection. (f) Marked accumulation of grains in the lumen of gastric pits and (g) in the surface mucus layer of the ligated stomach. (h) TFF1 mRNA in the gastric surface epithelial cells. (i) TFF2 mRNA in surface epithelial and mucous neck cells. (j) Negative in situ hybridization for TFF3 mRNA in the stomach. (k) TFF1 peptide in surface epithelia. (l) TFF2 peptide in mucous neck cells. (m) TFF3 peptide in mucous neck cells. Scale bars: 50 μm.