Helen M. Cox

Immunoprecipitation and characterization of a binding protein specific for the peptide, intestinal trefoil factor

Recombinant rat intestinal trefoil factor (rITF) and human spasmolytic polypeptide (hSP) were irreversibly cross-linked to specific binding sites in solubilized rat intestinal epithelial membranes and human adenocarcinoma cells. Analysis of the immunoprecipitates by immunoblotting identified a cross-linked protein complex of approximately 45 kDa, which under reducing conditions appeared as a approximately 28-kDa band and the latter displayed ligand-stimulated phosphorylation of a tyrosine, but not a threonine or serine, residue in the binding complex. [125I]rITF was used to localize binding sites by autoradiography of frozen sections from rat gastrointestinal tissues. A high density of specific [125I]rITF binding sites was present within gastric, colonic, and jejunal mucosal glands. Unlabeled hSP partially inhibited [125I]rITF binding at a concentration of 1 microM when compared with the same concentration of unlabeled rITF. These studies support earlier observations for the existence of trefoil binding sites in the gastrointestinal tract and further suggest that hSP has affinity for the mucosal rITF binding site.

Effect of Ectopic Expression of Rat Trefoil Factor Family 3 (Intestinal Trefoil Factor) in the Jejunum of Transgenic Mice

To further examine the function of the trefoil factor family (TFF), the expression of which is up-regulated at sites of injury, we have produced transgenic mice that chronically express rat TFF3 within the jejunum (using a rat fatty acid-binding protein promoter). The expression of rat TFF3 was limited to the villi of the jejunum and had no effect on base-line morphology. Rat TFF3 expression did result, however, in a reduced sensitivity to indomethacin (85 mg/kg subcutaneously), which only caused a 29% reduction in villus height in transgenics versus 51% reduction in controls (p < 0.01). Indomethacin increased initial intestinal epithelial cell proliferation and migration, but the presence of rat TFF3 caused no additional change in proliferation (bromodeoxyuridine), cell migration ([3H]thymidine and bromodeoxyuridine), apoptosis (terminal deoxyuridine nucleotidyl nick end labeling), or E-cadherin immunostaining. In vitrostudies following changes in resistance of intestinal strips in Ussing chambers (voltage-clamp technique) showed increased base-line resistance in the rat TFF3-expressing region (326 ± 60versus 195 ± 48 ohm·cm2 in controls,p < 0.05) and reduced the fall in resistance following HCl exposure by about 40% (p < 0.01). Overexpression of TFF3 stabilizes the mucosa against noxious agents, supporting its role in mucosal protection/repair. It may therefore provide a novel approach to the prevention and/or treatment of intestinal ulceration.

Modulation of epidermal growth factor effects on epithelial ion transport by intestinal trefoil factor.

1 The direct epithelial effects of epidermal growth factor (EGF) and its modulation by intestinal trefoil factor (ITF) have been studied in a human colonic adenocarcinoma cell line called Colony‐29 (Col‐29). 2 When grown in culture as confluent monolayers and voltage‐clamped in Ussing chambers, these epithelia responded with an increase in short circuit current (SCC) to basolateral as well as to apically applied EGF although the latter responses (at 10 nM) were only 25% of those observed following basolateral peptide. 3 Recombinant rat ITF (added to the basolateral surface) did not alter basal SCC levels, but it did enhance the electrogenic effects of basolateral EGF. The EC50 values for EGF‐induced ion transport were 0.25 nM in control, and 0.26 nM in ITF pretreated Col‐29 epithelia. A significant increase in the size of EGF responses (0.1 nM −10 nM) was observed in the presence of 10 nM ITF and the half‐maximal concentration for this modulatory effect of ITF was 7.6 nM. 4 The EGF‐induced increases in SCC were partially inhibited (50%) by piretanide pretreatment, indicating that C1− secretion is involved. EGF responses either in the presence or absence of ITF were also significantly reduced (84% and 66% respectively) by the cyclo‐oxygenase inhibitor, piroxicam, therefore implicating prostaglandins as mediators of EGF‐stimulated anion secretion. 5 We conclude that in confluent Col‐29 epithelia, basolateral EGF stimulates a predominantly prostaglandin‐dependent increase in C1− secretion that is enhanced by basolateral ITF, and that these two peptides may interact in normal and damaged mucosa to alter the local apical solute and fluid environment.

Functional characterization of receptors with affinity for PYY, NPY, [Leu31,Pro34]NPY and PP in a human colonic epithelial cell line.

1 . Confluent epithelial layers of a human adenocarcinoma cell line called Colony‐6 have been shown to respond to nanomolar concentrations of vasoactive intestinal polypeptide (VIP), peptide YY (PYY), neuropeptide Y (NPY) and somatostatin (Som). 2 . The VIP‐induced increase in basal short‐circuit current (SCC) was attenuated by basolateral application of Som, PYY or NPY, and also by the Y1receptor agonist [Leu31, Pro34]NPY, as well as pancreatic polypeptide (PP). High concentrations (0.1–3.0 μm) of NPY(2–36) were effective but the C‐terminal fragment NPY(13–36) (0.1‐1.0 μm) and desamidoNPY (0.6 μm) were not active. A rank order of agonist EC50 values was: PYY > NPY >[Leu31, Pro34]NPY > PP > NPY(2–36) > > NPY (13–36) 3 . Receptors for all these peptides were preferentially located within the basolateral domain. Apical addition of PP (1 μm) and Som (100 nm) had no effect upon basal SCC while apical VIP (10 nm) responses were 18, and apical PYY (100 nm) were 27% the size of respective basolateral controls (100%). 4 . Cross‐desensitization was observed between [Leu31, Pro34]NPY (1 μm) and both PYY (100 nm) and PP (1 μm) and between PYY and NPY(2–36) (1 μm), but was not significant between PYY (100 nm) and PP (1 μm). We suggest that either these cells express a single new Y‐receptor with an unusual phenotype or that two Y‐receptor populations exist in Colony‐6 cells.

Calcitonin gene-related peptide receptors in human gastrointestinal epithelia.

1 The secretory responses to calcitonin gene‐related peptide (CGRP) receptor agonists have been characterized in two human adenocarcinoma cell lines, namely HCA‐7 and Colony‐29 (Col‐29) epithelia. These cells form polarized epithelial layers when grown on permeable supports and allow changes in electrogenic ion transport in response to agonists to be monitored continuously. 2 α‐CGRP (rat and human sequences), rat β‐CGRP and human [Tyr0]CGRP applied to the basolateral surface were found to be full agonists, causing prolonged increases in short‐circuit current. Concentration‐response curves exhibited EC50 values of 0.6‐1.5 nm in HCA‐7 cells. The same agonists were less effective in Col‐29 epithelia, the EC50 values ranging from 1 to 10 nm in these cells. [Cys(ACM)2,7]CGRP was effective in both cell lines and was more potent in HCA‐7 cells. 3 CGRP receptors were preferentially located on the basolateral surface in both cell types. Addition of rα‐CGRP to the apical domain produced significantly smaller secretory responses (8.1% in HCA‐7 and 29.2% in Col‐29) compared with those produced following basolateral application (100%). 4 In both cell lines rα‐CGRP‐elevated short‐circuit current was inhibited by the loop diuretic piretanide (200μm) and by somatostatin (100 nm). Pretreating epithelia with the cyclo‐oxygenase inhibitor, piroxicam (5μm) had no significant effect upon CGRP responses in either cell line. 5 Rat α‐CGRP (0.2 nm) responses in HCA‐7 epithelia were inhibited by the C‐terminal fragment CGRP(8–37) (1 μm). Pretreatment of Col‐29 cells with CGRP(8–37) did not, however, alter the size or profile of responses to rα‐CGRP (1 nm). 6 We conclude that high‐affinity CGRP receptors exist on the basolateral surface of both cell lines, however they differ in their sensitivity to CGRP(8–37) and agonist orders of potency. Thus different CGRP receptor subtypes appear to predominate in these two epithelial cell types.

Pharmacological characterisation of neurokinin receptors mediating anion secretion in rat descending colon mucosa

SummarySubstance P(SP), neurokinin A (NKA), neurokinin B (NKB), [Sar9, Met (O2)11]-SP (SMSP), senktide, [βAla8]-NKA(4–10) and neuropeptide γ (NPγ) all stimulate secretory responses in rat descending colon mucosa under voltage clamp conditions. Secretory responses (measured as short circuit current under voltage clamp conditions) were transient and those evoked by SP, SMSP, NKA and senktide were significantly reduced by pretreating tissues with the chloride channel blocker, diphenylamine carboxylate (DPC). Concentration-response curves showed varying degrees of sensitivity to tetrodotoxin (TTX). Senktide-induced secretion was virtually abolished by TTX, while NPγ and [βAla8]-NKA(4–10) were not significantly altered. Rightward shifts of concentration-response curves were observed for SMSP, NKA and SP in TTX treated preparations compared with controls. NKA response curves in the presence of TTX were further inhibited by MEN10,207 and CP-96,345. GR71251, GR82334 and CP-96,345 all inhibited SMSP secretory responses with pA2 values of 5.8, 6.5 and 6.9 respectively. In conclusion three types of neurokinin receptor exist in preparations of rat colon mucosa and their relative location within neuronal and epithelial surfaces are discussed.

Structure-activity studies of peptide YY(22–36): , a potent antisecretory peptide in rat jejunum

Peptide YY (PYY) and its homologous peptide, neuropeptide Y (NPY), are known to exhibit potent antisecretory effects in the intestine. To determine the structural requirements to elicit antisecretory effects, we have synthesized several analogs of the PYY active site, PYY(22-36), and compared their binding affinities and antisecretory potencies in rat jejunum. These investigations revealed that the hydroxyl groups of Ser23 and Thr32, as well as the imidazole group of His26, are important for activity in the intestine. N-alpha-acetylation of PYY(22-36) increased both the binding affinity and antisecretory potency. Structure-activity studies with N-alpha-Ac-PYY(22-36) showed that substitution of His26 with parachlorophenylalanine (pCl-Phe) or Tyr36 with N-Me-Tyr reduced receptor affinity, while replacement of Tyr27 with Phe increased the activity substantially. Furthermore, acylation of the alpha-NH2 group with hydrophobic groups, myristic and naphthaleneacetic acids, substantially reduced the antisecretory potencies but not the binding affinities. Further modification of N-alpha-Ac-[Phe27]PYY(22-36) may lead to the development of more potent agonist compounds, which may provide a framework for the design of a new class of antidiarrheal drugs.

The gene encoding mouse intestinal trefoil factor: structural organization, partial sequence analysis and mapping to murine chromosome 17q.

Trefoil peptides, a growing family of secretory molecules, have been identified mainly in the gastrointestinal tract of humans, rodents and amphibians. In the present study, the nucleotide sequence of a large portion (81%) of the gene encoding murine intestinal trefoil factor (mITF) and its whole genomic organization were determined. The mITF gene contains three exons distributed over 5 kb of genomic DNA. The genomic sequence is highly conserved, as compared with that of the rat and human ITF, and contains several AP-1-binding sites, the consensus binding site for the transcription factor Sp1, and a sequence homologous to a heat-shock element. Fluorescence in situ hybridization was used to assign ITF to chromosome 17 of the murine genome, a region syntenic with the trefoil gene cluster on human chromosome 21q22.3.

Inhibition of cyclic AMP-dependent chloride secretion by PP receptors and alpha 2-adrenoceptors in a human colonic epithelial cell line.

Abstract The effects of a number of agonists which inhibit intestinal chloride secretion were investigated in Colony-1 (Col-1) cells, a subpopulation derived from the HCA-7 human adenocarcinoma cell line. Neither peptide YY (PYY) or somatostatin 14–28 (SRIF) reduced short-circuit current (SCC) in Col-1 epithelial layers stimulated with vasoactive intestinal polypeptide (VIP), suggesting that their respective receptors are either absent in this cell line, or are not functionally coupled. A second member of the neuropeptide Y family, pancreatic polypeptide (PP), decreased VIP-elevated SCC with an EC50 of 25.6 nM. Maximal PP responses were unaffected by prior addition of PYY, indicating that Col-1 cells may express a PP specific, Y4-like receptor. The α2-adrenoceptor agonist clonidine also attenuated VIP-stimulated SCC (EC50 342 nM) through the α2A receptor subtype, since clonidine responses were inhibited by yohimbine and rauwolscine but not altered by previous addition of prazosin. Col-1 cells responded to both apical and basolateral addition of VIP or clonidine; to an extent, this lack of sidedness reflects the ability of drugs to permeate through the Col-1 epithelial layers.Both PP and clonidine also inhibited SCC in unstimulated Col-1 cells or those pretreated with 3-isobutyl-1-methylxanthine (IBMX) or a submaximal concentration of forskolin, agents which both directly elevate intracellular cAMP. After a maximal concentration of forskolin (10 μM), which increased SCC to a significantly greater extent than either VIP or IBMX, the effects of both agonists were negligible. The absence of PP and clonidine responses under these conditions may have implications for the mechanisms by which these agonists inhibit chloride secretion in Col-1 epithelia. In addition carbachol reduced SCC stimulated by 10 μM forskolin, in contrast to control carbachol responses which consisted of a rapid decrease followed by a transient elevation in SCC; this observation suggests that Col-1 cells may also be a useful model for studying the interactions between Ca2+- and cAMP-dependent mechanisms involved in epithelial ion transport.

The functional investigation of a human adenocarcinoma cell line, stably transfected with the neuropeptide Y Y1 receptor

1 The human adenocarcinoma cell line, HT‐29, has been stably transfected with the cDNA sequence for the rat neuropeptide Y (NPY) Y1 receptor, and three Y1 clones (Y1‐4, Y1‐7 and Y1‐16) have been isolated which express high levels of specific [125I]‐PYY binding. We have studied the functional responses or lack of responses to peptide YY (PYY) and its analogues in the three transfected clones and HT‐29 wild type (wt) cells. 2 Vasoactive intestinal polypeptide (VIP) produced long‐lasting increases in short‐circuit current (SCC) in both HT‐29 wt cells and the Y1 clones. VIP EC50 values were 8.4–11.7 nM in all four cases. The elevation in SCC after a maximal concentration of VIP (30 nM) was significantly greater in Y1‐7 cells than in either HT‐29 wt epithelia or the other Y1 cell lines. 3 PYY (100 nM) and human pancreatic polypeptide (hPP; 1 μm) were ineffective in HT‐29 wt cells under either basal or stimulated conditions. In contrast, basolateral additions of PYY reduced both basal and VIP‐stimulated SCC in all three Y1 clones. After VIP, the PYY EC50 values (in nM) were 18.6 in Y1‐4, 8.0 in Y1‐7 and 52.5 in Y1‐16. hPP (1 μm) produced only small and transient responses in each transfected cell type. 4 The Y1 receptor agonist, [Leu31, Pro34] NPY (1 μm) was also effective in the three Y1 cell lines. In the Y1‐7 clone the EC50 value for the effect of this peptide was 149 nM, 18.6 fold less potent than PYY. 5 PYY and the Y1‐selective non‐peptide antagonist, BIBP 3226 displaced [125I]‐PYY binding from Y1‐7 cell membranes with Ki values of 2.0 and 3.1 nM respectively. In the Y1‐7 clone, BIBP 3226 fully inhibited the reductions in VIP‐stimulated SCC induced by 30 nM PYY, with an IC50 of 27.2 nM and 30 nM BIBP 3226 caused a parallel rightward shift on the PYY concentration‐response curve, with an approximate pKB of 8.0. 6 HT‐29 clones stably expressing the Y1 receptor therefore show responses to PYY and its analogues that are characteristic of that subtype, and the Y1‐7 clone in particular will be useful in the assessment of novel Y1‐specific drugs. This approach will also allow the functional study of NPY Y1 receptors with selected mutations.