Several inflammatory processes of the bowel are characterized by an accumulation of eosinophils at sites of inflammation. The mechanisms that govern mucosal infiltration with eosinophils are not fully understood. Eotaxin-3/CCL-26 belongs to a family of CC chemokines, which are potent chemoattractants for eosinophils. In this study, we hypothesized that intestinal epithelial cells could release eotaxin-3. We demonstrate that the T helper 2 type cytokines interleukin-4 or interleukin-13 increase eotaxin-3 mRNA levels and eotaxin-3 protein expression in the human intestinal epithelial cell lines HT-29 CL.19A and T84 in a dose-dependent manner. Addition of actinomycin-D prior to interleukin-4/-13 stimulation led to decreases in eotaxin-3 mRNA levels similar to those observed in controls without interleukin-4/-13. Interleukin-4 and interleukin-13 activated signal transducer and activator of transcription 6 which was found to bind the two canonical signal transducer and activator of transcription 6 binding sites located in the eotaxin-3 promoter. Experiments with the eotaxin-3 promoter luciferase constructs revealed that the most proximal signal transducer and activator of transcription 6 binding site located between positions -62 and -71 relative to the transcriptional start was necessary for full eotaxin-3 promoter activity. Importantly, we present evidence that the signal transducer and activator of transcription 6 is necessary and sufficient for interleukin-4 or interleukin-13 mediated eotaxin-3 gene up-regulation using HT-29 CL.19A cells expressing a dominant-negative signal transducer and activator of transcription 6. Overall, these results demonstrate that epithelial eotaxin-3 is up-regulated in the context of a T helper 2 mediated inflammatory bowel disease via the signal transducer and activator of transcription 6, thus suggesting that the intestinal epithelium actively participates in the recruitment of eosinophils at the site of inflammation.

Mainly composed of mucins, mucus secreted by goblet cells in the intestinal epithelium is critically involved in the protection of the gastrointestinal mucosa. The hypothesis that bile and some bile salts can induce mucus secretion was tested in the isolated perfused rat colon. Mucus release was evaluated using enzyme-linked immunosorbent assays and supported by histological analysis. Luminal administration of bile extract (1%) provoked mucus secretion in the rat colon. Deoxycholate (0.5–10 mM) induced a dose-dependent increase in rat colonic mucus release. Chenodeoxycholate (10 mM) and hyodeoxycholate (10 mM) also evoked mucus discharge, whereas 10 mM cholate, 10 mM ursodeoxycholate, or Tween-20 did not release mucus. Taurine-conjugated bile salts (deoxycholate, hyodeoxycholate, and chenodeoxycholate) were less potent mucus secretagogues than the corresponding unconjugated forms. The deoxycholate-induced mucus discharge was not altered by pharmacological blockers (tetrodotoxin, atropine), indomethacin, mast cell stabilizers (ketotifen, doxantrazole), H1 histamine receptor antagonist (pyrilamine), or 5-HT receptor antagonists (ketanserin, ondansetron, SDZ 205-557). Our findings suggest that some bile salts, especially in the unconjugated form, may provoke colonic mucus secretion, probably through a direct action on mucus-secreting cells.

The secretion of PYY by endocrine L cells of the terminal gut is under the control of nutrients, the autonomic nervous system and hormones. Catecholamines, and the non-specific beta-adrenergic agonist isoproterenol induce PYY secretion from rat isolated colon or ileum. Because beta3-adrenergic receptors now appear to mediate many of the effects of catecholamines in the gastrointestinal tract, we investigated the involvement of beta1-, beta2-, and beta3-adrenoceptor stimulation in PYY secretion from the isolated, vascularly perfused rat colon. Infusion of 10(-6) M isoproterenol induced a transient increase in PYY secretion (from 36+/-4 to 87+/-20 fmol/2 min; n=7, P<0.05), that was abolished by a previous infusion of the beta1- and beta2-adrenergic blocker (and partial beta3-agonist) alprenolol (10(-6) M). The beta1-adrenergic agonist dobutamine and the beta-2 agonist terbutaline also (both at 10(-5) M) significantly stimulated PYY secretion, from 29+/-1 to 79+/-12 fmol/2 min and from 19+/-1 to 73+/-13 fmol/2 min respectively (n=7, P<0.05). Neither of the beta3-adrenergic agonists tested (BRL 37 344 (10(-5), 10(-6) M) and SR 58 611A (10(-6) M)) significantly stimulated PYY secretion, thus confirming the exclusive involvement of beta1- and beta2-receptors in beta-adrenergic agonist induced hormone secretion.

BACKGROUND Mucins play an important protective role in the colonic mucosa. Luminal factors modulating colonic mucus release have been not fully identified. AIM To determine the effect of some dietary compounds on mucus discharge in rat colon. METHODS An isolated vascularly perfused rat colon model was used. Mucus secretion was induced by a variety of luminal factors administered as a bolus of 1 ml for 30 minutes in the colonic loop. Mucin release was evaluated using a sandwich enzyme linked immunosorbent assay supported by histological analysis. RESULTS The three dietary fibres tested in this study (pectin, gum arabic, and cellulose) did not provoke mucus secretion. Luminal administration of sodium alginate (an algal polysaccharide used as a food additive) or ulvan (a sulphated algal polymer) induced a dose dependent increase in mucin discharge over the concentration range 1–25 mg/l (p<0.05 for 25 mg/l alginate and p<0.05 for 10 and 25 mg/l ulvan). Glucuronic acid and galacturonic acid, which are major constituents of a variety of fibres, produced significant mucin secretion (p<0.05). Hydrogen sulphide and mercaptoacetate, two sulphides produced in the colonic lumen by microbial fermentation of sulphated polysaccharides, did not modify mucin secretion. Among the short chain fatty acids, acetate (5–100 mM) induced a dose dependent release of mucus (p<0.05 for 100 mM acetate). Interestingly, butyrate at a concentration of 5 mM produced colonic mucin secretion (p<0.05), but increasing its concentration to 100 mM provoked a gradual decrease in mucus discharge. Propionate (5–100 mM) did not induce mucin release. Several dietary phenolic compounds (quercetin, epicatechin, resveratrol) did not provoke mucus discharge. CONCLUSIONS Two algal polysaccharides (alginate and ulvan), two uronic acids (glucuronic acid and galacturonic acid), and the short chain fatty acids acetate and butyrate induce mucin secretion in rat colon. Taken together, these data suggest that some food constituents and their fermentation products may regulate the secretory function of colonic goblet cells.

The intestinal mucus layer plays an important role in the protection of the mucosa. This function is performed mainly by mucins, which are high molecular-weight glycoproteins produced by goblet cells. Luminal factors that govern mucin secretion are poorly known. The hypothesis that bile salts could induce colonic mucin discharge was thus tested in a model of isolated vascularly perfused rat colon. Methods : Ligated loops (I O-cm length) of rat colon were separated from adjacent tissue, transferred to a saline bath and perfused through the superior mesenteric artery with a Krebs Henseleit buffer containing 25% washed bovine erythrocytes, 3% bovine albumin, amino acids and glucose. After a lO-min control period, loops were filled with Iml of prewarmed saline containing stimulants. After 30-min period of stimulation, mucus was assayed in luminal content by enzyme-linked immunosorbent assay. Tissue samples from perfused rat colon were also immersed in Karnovsky's fixative for subsequent staining of mucus cells with alcian blue followed by the periodic acid schiff reagent. Results : Luminal administration of deoxycholate (I-lOmM) produced a dose-dependent release of mucins (maximal response at 550 % of control loops) in the isolated perfused rat colon. Hyodeoxycholate (IOmM) and Chenodeoxycholate (I0mM) also induced an increase in mucin discharge (response at 310 % and 290 % of controls,respectively) whereas IOmM cholate, ursodeoxycholate and tween-20 were without effect. Taurine conjugated bile salts (deoxycholate,hyodeoxycholate, and chenodeoxycholate) were less potent mucus secretagogues than unconjugated forms. Tetrodotoxin, atropine, indomethacin, a mast cells stabilizer (ketotifen), H I histamine antagonist(pyrilamine) and 5-HT receptors antagonists (ketanserin, SDZ, Zophren) did not significantly modify deoxycholate-induced mucin secretion. Conclusions : The present study conducted with the isolated vascularly perfused rat colon suggests that some bile salts may playa role in the regulation of colonic mucin secretion by way of a direct action on mucus cells

The secretion of PYY by endocrine L cells of the terminal gut is under the control of nutrients, the autonomic nervous system and hormones. Catecholamines, and the non-specific -adrenergic agonist isoproterenol induce PYY secretion from rat isolated colon or ileum. Because 3-adrenergic receptors now appear to mediate many of the effects of catecholamines in the gastrointestinal tract, we investigated the involvement of 1-, 2-, and 3- adrenoceptor stimulation in PYY secretion from the iso- lated, vascularly perfused rat colon. Infusion of 106 M isoproterenol induced a transient increase in PYY secre- tion (from 36 4t o 8720 fmol/2 min; n=7, P<0·05), that was abolished by a previous infusion of the 1- and

The intestinal peptide guanylin regulates the electrolyte/water transport in the intestinal epithelium. The aim of the present study was to investigate the mechanisms that modulate its secretion in the isolated vascularly perfused rat colon by using a specific guanylin RIA. Intraarterial infusion of bethanechol (10(-4) M) or bombesin (10(-7) M) elicited a significant 6-fold increase in the release of guanylin immunoreactivity (G-IR) in the lumen. Bombesin-stimulated G-IR secretion was strongly reduced by tetrodotoxin, whereas atropine had no effect. VIP (10(-7) M) induced a moderate release of G-IR, whereas substance P, calcitonin gene-related peptide, peptide YY, somatostatin, and neurotensin were without effect. Dimethyl-PGE2 (1.4 x 10(-5) M) or interleukin-1beta (2.5 x 10(-10) M) induced a 3-fold increase in G-IR in the lumen, whereas the degranulator compound bromolasalocid did not stimulate guanylin secretion. Forskolin (10(-5) M) or sodium nitroprusside (10(-4)-10(-3) M) induced a significant release of G-IR. In contrast, PMA (10(-7) M) or ionophore A23187 (10(-6) M) did not modify basal secretion of G-IR. Upon stimulation of guanylin release with bombesin or bethanechol, an increase in G-IR in the portal effluent was also detected. The release of G-IR in the portal effluent was 40-fold lower than that of G-IR into the luminal perfusate. Additionally, analysis with gel chromatography revealed that the immunoreactive material released in the lumen or in the portal effluent coeluted with the 15-amino acid peptide originally isolated from rat intestine. In conclusion, the present data suggest that the enteric nervous system and immune cells may modulate guanylin release from the rat colon. The release of guanylin in the lumen and portal effluent suggests that this peptide may exert both luminal/paracrine and hormonal effects.

Leptin is an adipocyte produced peptide that controls food intake and the energy expenditure.Many of its effects are mediated centrally via hypothalamic receptor but also peripheral effects of leptin such as gastroprotection has been recently described and found to contribute to action of CCK on the stomach.Leptin receptors and leptin were detected recently in the.pancreas.CCK was found to increase the plasma leptin but this leptin seems to originate mainly from stomach where it inhibits gastric secretion but its role control of pancreatic secretion has not been studied.The aim of this study was to evaluate the effect of leptin on pancreatic secretion in vivo in rats surgically with pancreatic fistulas and in vitro using dispersed pancreatic acini on exocrine.After preliminary administration of various doses ofleptin, the standard dose of (10 I-tglkg-h, s.c.), that inhibited gastric secretion for testing pancreatic secretion.The pancreatic secretory response to sham feeding, ordinary feeding and pancreatic juice diversion were significantly inhibited by this dose of leptin by about 29%, 60% and 82%, respectively.In isolated pancreatic acini, obtained from the rat by collagenase digestion, increasing concentrations of leptin (10•1~-1O.7M)added to incubation medium failed to affect significantly basal secretion but produced dose-dependent inhibition of amylase release stimulated by submaximal dose of caerulein (1O_ 12M) but not by urecholine (lO•5M).We conclude that leptin inhibits stimulated pancreatic secretion both in vivo and in vitro, and this effect could be mediated at least in part by its direct action on pancreatic acinar cells 1664

Melatonin is synthesized during the night by the pineal gland. Recently, melatonin binding sites have been identified in the gut. Despite few studies, the physiological role of melatonin in gut function remains unclear. The objective of the present study was to investigate the effects of melatonin in the regulation of intestinal motility by using the melatonin receptor antagonist S 22153 in rats. Twenty‐four male Wistar rats (400±25 g) were equipped with intraparietal electrodes along the small intestine. Rats were subjected to a 12:12 hr light:dark schedule. During the dark phase, intestinal migrating motor complexes (MMCs) frequency increased (P<0.05) by 20% in the duodenum and in the jejunum compared with daylight. This effect is due to a significant reduction in the irregular spiking activity (ISA) of MMCs. Concurrently, at night, the duration of the postprandial motor response is reduced by 30% in the duodenum and 50% in the jejunum and ileum. The administration of S 22153 (2 mg/kg sc) at night suppressed these nocturnal variations and restored the daylight values. In contrast, S 22153 was ineffective during daylight whatever the digestive state. Administration of melatonin (1 mg/kg iv) during the preprandial state, 3 hr after light onset, decreased (−80%) the duration of the ISA of MMCs at the three intestinal levels. During the satiety phase, melatonin administered 10 min before or 15 min after food onset induced the appearance of a transitory preprandial‐like motor profile in the entire small intestine. In contrast, when administered at the end of the meal it was ineffective. Preprandial and postprandial melatonin effects were prevented by S 22153 pretreatment. In conclusion, these findings reveal, first, that endogenous melatonin is physiologically involved in the pre‐ and postprandial changes of intestinal motility at night. Second, exogenous melatonin produces pharmacological effects on pre‐ and postprandial intestinal motility. In both cases, the action of melatonin corresponds to an inhibition of ISA and a reinforcement of the cyclic MMC pattern.

Postprandial release of peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) from L cells results from both nutrient transit in the ileal lumen and neural drive of endocrine cells. The adrenosympathetic system and its effectors have been shown to induce secretion of L cells in vivo or in vitro. Because these transmitters act through three receptors, beta, alpha1, alpha2, coupled to different intracellular pathways, we evaluated the responses of L cells to specific agonists, using the model of isolated vascularly perfused rat ileum. General stimulation of adrenergic receptors with epinephrine (10(-7) M) induced significant GLP-1 and PYY secretions (94+/-38 and 257+/-59 fmol/8 min respectively) which were abolished upon propranolol (10(-7) M) pretreatment and strongly decreased upon infusion with 10(-8) M prazosin. Blockade of alpha2-receptors with idazoxan (10(-8) M) did not alter epinephrine-induced peptide secretion. The beta-adrenergic agonist isoproterenol (10(-6) M) infused for 30 min induced a transient release of GLP-1 and PYY (integrated release over the 8 min of the peak secretion: 38+/-16 and 214+/-69 fmol for GLP-1 and PYY respectively, P<0.05). Because terbutaline but not dobutamine or BRL 37,344 (10(-5) M) induced significant GLP-1 and PYY secretions (135+/-30 and 305+/-39 fmol/8 min respectively), isoproterenol-induced secretions are suggested to result mainly from stimulation of the beta2-isoreceptor type. In contrast, the alpha1-agonist phenylephrine (10(-7) M) did not stimulate peptide release. When co-infused with 10(-6) M or 10(-7) M isoproterenol, 10(-7) M phenylephrine raised GLP-1 release to 174+/-53 and 108+/-28 fmol/8 min respectively (vs 38+/-16 and 35+/-10 fmol/8 min for isoproterenol alone, P<0.05) whereas PYY secretion was not significantly increased. Clonidine (10(-7) M), an alpha2-agonist, induced a moderate and delayed increase of GLP-1 and PYY but abolished the isoproterenol-induced peptide secretion. Our results showed that general stimulation of adrenergic receptors stimulates the secretory activity of ileal endocrine L cells. The net peptide secretion results from the activation of the beta2-isoreceptor type. Additionally, GLP-1 and PYY secretions are positively modulated by alpha1-receptor stimulation and inhibited by alpha2-receptor activation upon beta-receptor occupation.

The intestinal epithelium can be separated in two main compartments: the crypt and the villus, each with its own unique cellular composition. Crypts contain undifferentiated cells as their chief component, but also harbor enteroendocrine cells, Paneth cells, and goblet cells. It is generally accepted that the undifferentiated crypt cells act as the progenitor cells from which all cell types of intestinal epithelial cells originate. With the striking exception of Paneth cells, which remain at the crypt base, other crypt cell types appear to migrate toward the villus surface. During this migration, particularly in the upper portion of the crypt, many undifferentiated crypt cells acquire structural and functional characteristics of villus absorptive cells. The villus absorptive cell is the major cell type on the villus compartment, but goblet and enteroendocrine cells are also present. The function of one cell type is different from that of another, but at least three cell types share common features: they are capable of secreting regulatory peptides. These cell types include the enteroendocrine, Paneth, and goblet cells. The enteroendocrine cells have been subclassified on the basis of their peptide contents as revealed by immunohistochemistry. At least 16 different endocrine cell types have been identified. However, they constitute a small minority of the epithelial cells lining the gut wall. Enterochromaffin (EC) cells are located both in the epithelium of the small intestine and in the colonic epithelium. There is a consensus that EC cells constitute the majority of endocrine cells in the colon. One of the products stored in the secretory granules of these cells is 5-hydroxytryptamine (5-HT). Recent studies indicate that EC cells also synthesize guanylin, which once released into the lumen, participates in water and electrolyte homeostasis.

The neuropeptide bombesin and its mammalian homologue, gastrin-releasing peptide (GRP), enhance proliferation in some but not all human tumor cell lines. The pathophysiological relevance of the bombesin/GRP receptor (GRP-R), which is expressed in 30% of human colon tumor cell lines and in 24-40% of native tumors, has not been clearly assessed at this time. We studied the effects of bombesin in the recently characterized human colon carcinoma Isreco1 cell line. Competitive reverse transcription-PCR showed a high GRP-R mRNA level in Isreco1 cells, and binding studies confirmed the expression of bombesin/GRP-subtype receptors (Kd = 0.42 nM; Bmax = 18,000 sites/cell). Exposure to bombesin resulted in an increase of intracellular calcium concentrations. Bombesin (1 nM) induced cell spreading at 24 h (21.7+/-1.6% versus 6.4+/-0.8% in control cells; P<0.01) and markedly increased the formation of lamellipodia. In addition, adhesion of Isreco1 cells to collagen I-coated culture dishes was stimulated in the presence of 1 nM bombesin (69+/-6% versus 42+/-1% in control cells; P<0.01). Finally, bombesin significantly increased [3H]thymidine uptake by Isreco1 cells in a dose-dependent manner, with a first significant response at 0.1 nM and a maximal effect at 100 nM bombesin (192.2+/-9.7% of control). These results clearly indicate that bombesin exerts morphological, adhesive, and proliferative effects on Isreco1 cells, suggesting that expression of the bombesin/GRP-R may contribute to the malignant properties of colon carcinoma cells.

The neuropeptide galanin is widely distributed in the gastrointestinal tract and exerts several inhibitory effects, especially on intestinal motility and on insulin release from pancreatic beta-cells. The presence of galanin fibres not only in the myenteric and submucosal plexus but also in the mucosa, prompted us to investigate the regulatory role of galanin, and its mechanism of action, on the secretion of the insulinotropic hormone glucagon-like peptide-1 (GLP-1). Rat ileal cells were dispersed through mechanical vibration followed by moderate exposure to hyaluronidase, DNase I and EDTA, and enriched for L-cells by counterflow elutriation. A 6- to 7-fold enrichment in GLP-1 cell content was registered after elutriation, as compared with the crude cell preparation (929 +/- 81 vs 138 +/- 14 fmol/10(6) cells). L-cells then accounted for 4-5% of the total cell population. Bombesin induced a time-(15-240 min) and dose- (0.1 nM-1 microM) dependent release of GLP-1. Glucose-dependent insulinotropic peptide (GIP, 100 nM), forskolin (10 microM) and the phorbol ester 12-0-tetradecanoylphorbol-13-acetate (TPA, 1 microM) each stimulated GLP-1 secretion over a 1-h incubation period. Galanin (0.01-100 nM) induced a dose-dependent inhibition of bombesin- and of GIP-stimulated GLP-1 release (mean inhibition of 90% with 100 nM galanin). Galanin also dose-dependently inhibited forskolin-induced GLP-1 secretion (74% of inhibition with 100 nM galanin), but not TPA-stimulated hormone release. Pretreatment of cells with 200 ng/ml pertussis toxin for 3 h, or incubation with the ATP-sensitive K+ channel blocker disopyramide (200 microM), prevented the inhibition by galanin of bombesin- and GIP-stimulated GLP-1 secretion. These studies indicate that intestinal secretion of GLP-1 is negatively controlled by galanin, that acts through receptors coupled to pertussis toxin-sensitive G protein and involves ATP-dependent K+ channels.

The effect of potential mediators of mucus secretion was investigated in the isolated vascularly perfused rat colon by using a sandwich enzyme-linked immunosorbent assay for rat colonic mucin and by histochemical analysis. Bethanechol (100-200 microM), bombesin (100 nM), and vasoactive intestinal peptide (VIP, 100 nM) provoked a dramatic mucin discharge (maximal response at 900, 900, and 600% of control loops, respectively). VIP-stimulated mucin secretion was abolished by tetrodotoxin, whereas atropine was without effect. In contrast, both tetrodotoxin and atropine significantly decreased mucin release induced by bombesin. Isoproterenol or calcitonin gene-related peptide was without effect. Serotonin (1-5 microM) and peptide YY (10 nM) evoked mucin discharge, whereas glucagon-like peptide-1 did not release mucin. Finally, bromolasalocid (20 microM), interleukin-1beta (0.25 nM), sodium nitroprusside (1 mM), and dimethyl-PGE2 (2.5 microM) induced mucus discharge. The results demonstrated a good correlation between the immunological method and histological analysis. In conclusion, these findings suggest a role for the enteric nervous system, the enteroendocrine cells, and resident immune cells in mediation of colonic mucus release.

Peptones are potent stimulants of cholecystokinin (CCK) release in rats, both in vivo and ex vivo in a model of isolated vascularly perfused duodeno-jejunum preparation and in vitro in the intestinal CCK-producing cell line STC-1. The underlying mechanisms were here investigated with this cell line. Protein hydrolysates from various origins (meat, casein, soybean, and ovalbumin; 0.5-1%, wt/vol) dose dependently increased CCK release. Cephalosporin antibiotics, which mimic tripeptides, also stimulated the release of CCK over the concentration range 1-20 mM. The study of concentration dependence of cephalosporin uptake indicated a passive diffusion process at either pH 7.4 or pH 6.0, thus arguing against the involvement of a peptide transporter in CCK secretion. After pertussis toxin treatment (200 ng/ml; 5 h), the peptone- and cephalexin-induced CCK secretion was significantly reduced, suggesting the involvement of pertussis toxin-sensitive heterotrimeric G protein(s) in the secretory activity of STC-1 cells. Consistent with this was the identification by Western blot of G(i2)alpha, G(i3)alpha, and G(o)alpha immunoreactivities in STC-1 cell extracts. Additionally, peptones and cephalexin increased the cellular content in inositol phosphates, whereas a mild increase in cAMP content was restricted to peptone-treated cells. Protein kinase A or C inhibition did not modify peptone- or antibiotic drug-evoked CCK release. The extracellular Ca2+ chelator EGTA (500 microM) and the intracellular Ca2+ chelator BAPTA-AM [1,2-bis-(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid tetra(acetoxymethyl) ester; 20 microM] abolished the peptone- and antibiotic drug-induced CCK release. Nifedipine and verapamil (10 microM) reduced by about 50% the CCK secretion evoked by these two secretagogues. In conclusion, peptones and some cephalosporins are potent stimulants of CCK release in the STC-1 cell line. The cellular mechanisms involve pertussis toxin-sensitive G protein(s) and are dependent on Ca2+ availability. We suggest that the STC-1 cell line is a useful model to study the molecular basis of peptone-induced CCK secretion.