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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.

The trefoil factor family (TFF) peptides 1 and 2 (TFF1 and 2) are expressed in mucus cells of the stomach, whereas TFF3 is localized in goblet cells of the intestine. In the present study, we aimed to determine whether phosphatidylinositol 3-kinase (PI3-K) or signal transducer and activator of transcription protein 6 (STAT6) is involved in the expression of goblet cell specific markers. TFF3 expression was analyzed by RT-PCR, Northern blot, and radioimmunoassay (RIA) in relation to cell growth in subclones of HT-29 cells including the CL.16E and methotrexate (MTX) cell lines, which both exhibit a phenotype of mucus-secreting intestinal cells. A 30-fold increase in TFF3 mRNA levels and a 10-fold increase in TFF3-cell content were observed between the early proliferative and the late confluency states. The levels of MUC2 and MUC3 mRNA were also increased in the course of the differentiation process. A three to fourfold increase in PI3-K and Akt activities was observed in early post-confluent cells as compared with pre-confluent cells. Exposure of pre- and post-confluent cells to LY294002, a specific PI3-K inhibitor, for 1-4 days profoundly reduced TFF3 and MUC2 expression. A marked reduction in mucin granules content was also observed in LY-treated cells. Inhibition of the mitogen-activated protein (MAP) kinase kinase (MEK) with PD98059 did not modify the course of differentiation of the goblet cell lines. Moreover, stable transfection of HT-29 CL.16E cells with a dominant negative form of STAT6 had no effect on TFF3 induction. Together, these data indicate that PI3-K promotes the expression of TFF3 and MUC2 and that the PI3-K/Akt pathway may play a pivotal role in intestinal goblet cell differentiation.

The development of intestinal goblet cell hyperplasia/hypertrophy during nematode infection involves the Th2 cytokines IL-4 and IL-13 via STAT6 activation. This is thought to play an important role in host protective immunity against the infection. In this study we demonstrate that IL-4 and IL-13 up-regulate the specific goblet cell product trefoil factor-3 (TFF3) from the mucus-producing HT-29 CL.16E and HT-29 cells selected by adaptation to methotrexate. Up-regulation of TFF3 mRNA and protein levels occurred in a time- and dose-dependent fashion and was accompanied by up-regulation of the goblet cell product mucin 2 (MUC2). Addition of actinomycin D before IL-4/IL-13 stimulation led to decreases in TFF3 mRNA levels similar to those observed in controls without IL-4/IL-13. Furthermore, IL-4-mediated increased TFF3 transcription required de novo protein synthesis. Stable transfection of HT-29 CL.16E cells with a truncated dominant-negative form of STAT6 produced a cell line that was unresponsive to IL-4/IL-13. Although only one consensus STAT6 binding site is contained in the TFF3 gene, located in the intron 1, it did not operate as an enhancer in the context of an SV40 promoter/luciferase construct. Thus, STAT6 activation mediates a transcriptional enhancement of TFF3 by induction of de novo synthesized protein in goblet cells.

Background & Aims: Colonic fermentation of carbohydrates is known to influence gastric and esophageal motility in healthy subjects. This study investigated the effects of colonic fermentation induced by oral administration of fructooligosaccharides (FOS) in patients with gastroesophageal reflux disease (GERD)., Methods: In the cross-over design used in the study, 9 patients with symptomatic GERD were administered a low-residue diet (i.e., 10 g fiber/day) during 2, 7-day periods, receiving either 6.6 g of FOS or placebo 3 times daily after meals. Each period was separated by a wash out of at least 3 weeks. On day 7, esophageal motility and pH were recorded in fasting conditions and after a test meal containing 6.6 g of FOS or placebo. Breath hydrogen concentrations (reflecting colonic fermentation) and plasma concentrations of glucagon-like peptide 1 (GLP-1), peptide YY, and cholecystokinin were monitored., Results: Compared with placebo, FOS led to a significant increase in the number of transient lower esophageal sphincter relaxations (TLESRs) and reflux episodes, esophageal acid exposure, and the symptom score for GERD. The integrated plasma response of GLP-1 was significantly higher after FOS than placebo., Conclusions: Colonic fermentation of indigestible carbohydrates increases the rate of TLESRs, the number of acid reflux episodes, and the symptoms of GERD. Although different mechanisms are likely to be involved, excess release of GLP-1 may account, at least in part, for these effects.

The vagus is involved in the cephalic phase of insulin secretion but its role in the meal absorption phase of insulin release remains to be defined. The aim of this study was therefore to evaluate the role of the vagus in the early and the late meal absorption phases of insulin secretion. In six pigs, venous insulin profiles were compared in intact animals, after ventral or dorsal vagal trunk section, and after section of both vagal trunks (truncal vagotomy). Since gastric emptying could be modified by vagotomy, it was recorded concomitantly by gamma scintigraphy. Semi-solid (porridge) and liquid (glucose 10%) meals were tested. Truncal vagotomy significantly increased insulin release compare to intact animals after glucose (63.8%) and porridge (174.4%) meals in the early and the late absorption phases of insulin secretion, respectively. For the glucose meal, this effect could be explained by a vagally mediated change in gastric emptying rate, since insulin concentrations for a similar amount of nutrient propelled to the duodenum were not different in intact and truncal vagotomized animals. In contrast, after the porridge meal, truncal vagotomy was associated with a second, later occurring increase in circulating insulin, which could not be explained by changes in gastric emptying rate. These results demonstrate for the first time an inhibitory role of the vagus in the late meal absorption phase of insulin release.

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-beta (IL-1-beta) 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.

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 aim of this study was to evaluate the nervous and humoral pathways involved in short-chain fatty acid (SCFA)-induced ileal brake in conscious pigs. The role of extrinsic ileal innervation was evaluated after SCFA infusion in innervated and denervated Babkin's ileal loops, and gastric motility was measured with strain gauges. Peptide YY (PYY) and glucagon-like peptide-1 (GLP-1) concentrations were evaluated in both situations. The possible involvement of absorbed SCFA was tested by using intravenous infusion of acetate. Ileal SCFA infusion in the intact terminal ileum decreased the amplitude of distal and terminal antral contractions (33 +/- 1.2 vs. 49 +/- 1.2% of the maximal amplitude recorded before infusion) and increased their frequency (1.5 +/- 0.11 vs. 1.3 +/- 0.10/min). Similar effects were observed during SCFA infusion in ileal innervated and denervated loops (amplitude, 35 +/- 1.0 and 34 +/- 0. 8 vs. 47 +/- 1.3 and 43 +/- 1.2%; frequency, 1.4 +/- 0.07 and 1.6 +/- 0.06 vs. 1.1 +/- 0.14 and 1.0 +/- 0.12/min). Intravenous acetate did not modify the amplitude and frequency of antral contractions. PYY but not GLP-1 concentrations were increased during SCFA infusion in innervated and denervated loops. In conclusion, ileal SCFA inhibit distal gastric motility by a humoral pathway involving the release of an inhibiting factor, which is likely PYY.

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.

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.

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.

Endocrine L-cells of the distal intestine synthesize both peptide YY (PYY) and proglucagon-derived peptides (PGDPs), whose release has been reported to be either parallel or selective. Here we compare the release mechanisms of PYY, glucagon-like peptide-1 (GLP-1), and oxyntomodulin-like immunoreactivity (OLI) in vivo. Anaesthetized rats were intraduodenally (ID) given either a mixed semi-liquid meal or oleic acid, or they received oleic acid or short chain fatty acids (SCFA) intracolonically (IC). The ID meal released the three peptides with a similar time-course (peak at 30 min); ID oleic acid produced a progressive release of PYY and OLI, while GLP-1 release was less. IC oleic acid or SCFA released smaller (but significant) amounts of PYY but no OLI or GLP-1. Hexamethonium inhibited most of the response to the ID meal and ID oleic acid, but did not change the PYY response to IC oleic acid. NG-nitro-l-arginine methyl ester (l-NAME, a nitric oxide synthase inhibitor) inhibited meal-induced PYY release and left OLI and GLP-1 unaffected. BW10 (a gastrin-releasing peptide antagonist) had no effect on the meal-induced release of either peptide. These results suggest a parallel initial release of PYY, OLI and GLP-1 after the ID meal, or oleic acid, by an indirect mechanism triggered in the proximal bowel, using nicotinic synapses, and involving nitric oxide release for PYY and an unknown mediator for PGDPs. For PYY there is a later phase of peptide release, probably induced by direct contact between nutrients and colonic L-cells.

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 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.

Exposure of the ileum to nutrients markedly inhibits several upper gastrointestinal functions. Hormonal peptides of the ileal wall, i.e. peptide YY (PYY), glucagon-like peptide-1 (GLP-1), and neurotensin (NT), are thought to play a role in this negative feedback mechanism. The present study was conducted to comparatively assess the secretion of PYY, GLP-1, and NT upon luminal infusion of a variety of individual luminal factors in the isolated vascularly perfused rat ileum preparation. PYY, GLP-1, and NT were measured in the portal effluent with specific RIAs. Glucose (250 mM) induced a pronounced release of the three peptides, whereas a physiological concentration of 5 mM did not induce peptide secretion. Peptone (5%, wt/vol) evoked a sustained release of PYY, GLP-1, and NT. Only NT secretion was increased upon luminal administration of 100 mM sodium oleate. Short chain fatty acids (20 mM) evoked an early and transient release of the three peptides. In contrast, taurocholate (20 mM) induced a sustained release of PYY, GLP-1, and NT, but the threshold concentration for peptide release was lower for NT than for PYY or GLP-1. Cellulose or pectin (0.5%, wt/vol) did not modify peptide secretion. In conclusion, glucose and peptone are potent stimulants of PYY, GLP-1, and NT release. Only NT is released upon oleic acid stimulation. Finally, taurocholate is a potent stimulant of the release of the three peptides. Overall, PYY, GLP-1, and NT may participate cooperatively in the ileal brake. As relatively high concentrations of the various stimulants were required to elicit peptide release, it seems likely that this mechanism operates in cases of maldigestion or malabsorption.

Receptors for insulin-like growth factors (IGF-I and IGF-II) are expressed in mammalian intestinal epithelium. No information on the presence of IGF receptors in intestinal endocrine cells is available. We tested for IGF-I receptors the endocrine cell line STC-1, which synthesizes and processes cholecystokinin (CCK) among other peptides, and assessed the effects of IGF-I on cell growth and CCK content. Cell monolayers in serum-free culture medium specifically bound [125I]IGF-I. Scatchard analysis was consistent with a single class of high affinity binding sites (KD = 0.91 nM; Bmax = 4,700 sites/cell). In competitive binding assays, unlabeled IGF-I, IGF-II and insulin displaced in a dose-dependent manner [125I]IGF-I binding with the following potencies (KI): IGF-I (0.74 nM) > IGF-II (3 nM) >> insulin (1 microM). Affinity cross-linking with [125I]IGF-I using disuccinimidyl suberate and SDS-PAGE under reducing conditions yielded a polypeptide band with apparent Mr 130,000, consistent with the alpha-subunit of the IGF-I receptor. IGF-I and IGF-II (0.3-30 nM) dose-dependently stimulated [3H]thymidine incorporation, with a maximal response of 110% above basal. IGF-II was approximately 10-fold less potent than IGF-I, suggesting a mediation through IGF-I receptors. In addition, the numbers of cells treated with 3 nM IGF-I amounted to 116, 130 and 159% of control values after 1, 2 and 4 days of incubation, respectively (p < 0.05). A significant increase in the cell CCK contents was observed after a 48-hour exposure to 3 or 30 nM IGF-I. These results demonstrate IGF-I receptor expression by the enteroendocrine cell line STC-1. IGF-I stimulates proliferation in short-term experiments, and increases intracellular levels of CCK.

Truncated glucagon-like peptide (GLP)-1 is a potent incretin. Its synthesis and secretion are modulated by food, but the influence of individual nutrients remains to be established. The hypothesis that protein hydrolysates (peptones) can directly regulate both GLP-1 secretion and proglucagon (PG) gene transcription was tested in this study, ex vivo in the isolated vascularly perfused rat intestine and in vitro in the murine enteroendocrine cell line STC-1. Peptones were albumin egg hydrolysate (AEH) and meat hydrolysate (MH). We demonstrate in these two models that peptones dose-dependently stimulate GLP-1 release, whereas isocaloric quantities of bovine serum albumin or of an amino acid mixture had no stimulatory effect. A strong and rapid increase of PG RNA level was observed in STC-1 cells treated with peptones (14-fold and 7-fold increase after 4 h of incubation with 3% wt/vol MH and AEH, respectively). Peptones also increased the PG RNA level in the colonic PG-expressing cell line GLUTag. In contrast, peptones did not modify the PG RNA level in two pancreatic glucagon-producing cell lines, namely, the RINm5F and INR1G9 cells. The peptone effect in STC-1 cells was completely abolished by blocking transcription before MH treatment. The stability of proglugacon transcripts was not modified by MH treatment, but nascent transcripts were more abundant in STC-1 cells preincubated with MH. Finally, MH treatment strongly stimulated (15-fold stimulation) the transcriptional activity of two PG gene promoter fragments (-1100 and -350 base pair) linked to the CAT reporter gene transiently transfected in STC-1 cells. Overall, peptones evoke an as yet undescribed release of GLP-1 when brought into contact with native intestinal L-cells or with STC-1 enteroendocrine cells. The increased transcription of the glucagon gene in the latter system suggests an important role of protein hydrolysates in the control of not only the secretion but also the synthesis of the incretin hormone.

Bombesin has been reported to stimulate cholecystokinin (CCK) secretion from rat duodeno-jejunal I-cells. Bombesin was shown to activate mitogen-activated protein kinases (MAPKs) in cell types such as Swiss 3T3 fibroblasts and rat pancreatic acinar cells. No information is available on whether MAPK is activated in intestinal endocrine cells upon bombesin stimulation. This was studied by using the CCK-producing enteroendocrine cell line STC-1. Bombesin stimulated markedly and transiently both p42(MAPK) and p44(MAPK), with a maximum at 2 min, and a decrease to basal levels within 10 min. As expected, bombesin stimulated MAPK kinase 1 (MEK-1) activity. Activation of protein kinase C (PKC) with PMA also stimulated p42(MAPK), p44(MAPK) and MEK-1. Treatment of cells with PD 098059 (at 10 microM or 30 microM), which selectively inhibits MEK phosphorylation, blocked bombesin-induced p42(MAPK) and p44(MAPK) activation for at least 90 min. However, PD 098059 inhibited bombesin- and PMA-stimulated CCK secretion during the first 15 min, but failed to significantly reduce CCK release at later times. Inhibition of PKC with staurosporine, or PKC down-regulation by prolonged treatment with PMA, both drastically decreased MEK-1, p42(MAPK) and p44(MAPK) activation upon bombesin stimulation. Additionally, PKC activation appeared to be required for both MAPK-dependent (early) and -independent (late) CCK responses to bombesin. It is concluded that the early CCK secretory response of STC-1 cells to bombesin involves MAPK pathway activation through a PKC-dependent mechanism, whereas the late phase of bombesin-induced CCK secretion, that also requires PKC, appears to result from a MAPK-independent process.

In neurons, synaptic vesicle exocytosis involves the formation of a core complex particle including syntaxin-1, synaptosomal-associated protein of 25 kDa (SNAP-25) and vesicle-associated membrane protein (VAMP)-2/synaptobrevin. The expression of these proteins was investigated in a panel of cell lines, including lines of endocrine and intestinal origin, by Western blotting and/or immunocytochemistry. The three core complex proteins were detected in the enteroendocrine, cholecystokinin (CCK)-secreting, cell lines STC-1 and GLUTag, and in the endocrine non-intestinal cell lines CA-77 and HIT-T15. In contrast, SNAP-25 and syntaxin-1 were undetected in the intestinal non-endocrine cell lines IEC-6, HT-29 and Caco-2, whereas a slight expression of VAMP-2 was documented in IEC-6 and HT-29 cells. Co-immunoprecipitation experiments indicated that syntaxin-1, SNAP-25 and VAMP-2 were present in a complex similar to that identified in brain. In the STC-1 cell line, treatment of streptolysin-O-permeabilized cells with tetanus toxin (Tetx) selectively cleaved VAMP-2 and VAMP-3/cellubrevin, and simultaneously abolished Ca2+-induced CCK secretion (IC50 approximately 12 nM). These results show that endocrine cell lines of intestinal origin express syntaxin-1, SNAP-25 and VAMP-2, and suggest a key role for a Tetx-sensitive protein (for example VAMP-2 and/or VAMP-3) in the CCK secretion by STC-1 cells.

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.

In rats, protein hydrolysates (peptones) stimulate cholecystokinin (CCK) release both in vivo and in a model of isolated vascularly perfused duodeno-jejunum. However, the mechanisms involved in peptone-induced stimulation of CCK cells are not well understood. In particular, the possibility that peptones may directly interact with CCK-producing cells to stimulate CCK release and gene transcription has not yet been examined. To test this hypothesis, we used the enteroendocrine cell line STC-1. Incubation of STC-1 cells for 2 h with albumin egg hydrolysate over the concentration range 0.01-1% (wt/ vol) caused a dose-dependent release of CCK, with a maximal increase at 1420% of the control value. In contrast, BSA (1%, wt/vol) or a mixture of amino acids (1%, wt/vol) induced a modest rise in CCK secretion. A dose-dependent, hydrolysate-specific, increase in the CCK steady state RNA level was also observed. It was detectable by 2-4 h of peptone treatment and sustained until 24-48 h. Peptones did not increase the CCK RNA level in the colonic CCK-producing cell line GLUTag or in nonintestinal CCK-expressing cell lines, namely the pancreatic cell line RINm5F and the medullar thyroid carcinoma cell line CA77. The peptone-induced increase in the CCK RNA level resulted from enhanced gene transcription, because labeled CCK transcripts from nuclear run-on incubations increased 3-fold when cells were incubated with peptones, whereas the level of beta-actin transcripts was not modified. Finally, peptones dose-dependently stimulated the transcriptional activity of an 800-bp fragment of CCK gene promoter transfected in STC-1 cells. These studies indicate that peptones specifically stimulate CCK secretion and gene transcription in the intestinal cell line STC-1, and that cis-acting elements conferring peptone inducibility are located in the first 800 bp of the 5'-flanking region of the CCK gene.

Unlabelled: The model of the isolated, vascularly perfused rat colon was assessed in the present study to investigate the nervous, hormonal, and local/paracrine pathways involved in colonic mucin secretion. A colonic loop was perfused via the superior mesenteric artery with a Krebs-Henseleit buffer containing 25% washed bovine erythrocytes at a rate of 2.5 ml/min. After a 10-min control period, each compound to be tested was infused intra-arterially for 30 min. Tissue samples from the proximal and midsegments of the perfused rat colon were then fixed and stained for mucus cell count. Intra-arterial administration of bethanechol evoked a concentration-dependent decrease in the number of stained mucus cells per crypt section over the range 2 x 10(-6) to 2 x 10(-4) M: 16.6 +/- 1.4 stained mucus cells per crypt in the midportion of the perfused rat colon (n = 5) with bethanechol 2 x 10(-4) M versus 28.8 +/- 1.5 for controls (n = 6). After infusion of 1.25 and 2.5 microM 16,16-dimethyl prostaglandin E2 (dmPGE2), the number of stained mucus cells per crypt section was significantly reduced: 21.6 +/- 0.6 (n = 6) and 20.6 +/- 1.4 (n = 7), respectively. An increase in the number of cavitated mucus cells was also observed (22.1 +/- 6.7 and 38.5 +/- 4.1% of cavitated mucus cells in the midsegment of the perfused rat colon with 1.25 and 2.5 microM dmPGE2, respectively, vs. 12.3 +/- 4.1% for controls). In contrast, prostaglandin F2alpha did not significantly affect mucus discharge from colonic cells. Peptide YY (10(-10), 10(-9) and 10(-8) M) induced a dose-dependent increase in the percentage of cavitated mucus cells (16.7 +/- 2.8, 23.1 +/- 4.2, and 31.2 +/- 3.4% of cavitated mucus cells in the midsegment, respectively). The proximal and midsegments of the perfused rat colon were equally sensitive to each secretagogue., Conclusion: In the isolated, vascularly perfused rat colon, mucus cells strongly respond to the well-known mucin secretagogues, bethanechol and dmPGE2. This approach has already led to the identification of a novel stimulant of mucin secretion: peptide YY. Our ex vivo model, in which goblet cells are submitted to well-defined luminal and blood-borne stimuli is, therefore, reliable to investigate the nervous, hormonal, and local/paracrine pathways involved in the colonic mucin secretion.

Peptide YY (PYY) is produced in endocrine L cells primarily localized in the distal bowel. These open-type L cells make contact with the intestinal chyme which may thus affect their secretory activity. The aim of the present study was to examine a large variety of luminal compounds found in colonic contents for their potential as PYY-releasing factors, using the isolated vascularly perfused rat colon. The release of PYY into the portal effluent was measured by a specific RIA. Luminal administration of 5 mM glucose or 0.5% (w/v) starch for 30 min did not induce significant release of PYY. Oleic acid (10 and 100 mM) also did not significantly increase PYY secretion. A pharmacological concentration of glucose (250 mM) and a mixture of amino acids (total concentration 250 mM) both induced PYY secretion (200% of basal). Pectin, a poly-galacturonic acid, evoked dose-dependent secretion of PYY-like immunoreactivity over the range 0.1-0.5% (w/v). The maximal response was observed after infusion of 0.5% pectin which induced a prompt and sustained release of PYY (300% of basal). Galacturonic acid itself (5%) produced marked PYY secretion. Gum arabic (0.5%) induced a gradual increase in portal PYY concentration (maximal response 250% of the basal value) whereas cellulose (0.5%) did not elicit PYY secretion. Luminal n-butyrate over the range 0.5-5 mM produced a dose-dependent release of PYY (maximal response 300% of the basal value with 5 mM n-butyrate). Increasing the concentration of n-butyrate to 100 mM provoked a gradual decrease in PYY secretion. Propionate was a less potent stimulant than n-butyrate, and acetate did not increase PYY secretion above the basal value. At a concentration of 2 or 20 mM, taurocholate, cholate and deoxycholate brought about PYY secretion while hyodeoxycholate was without effect. In conclusion, glucose and amino acids may mediate PYY release but only when they are present at high supraphysiological concentrations in the colon while oleic acid does not produce any PYY secretion. Physiological concentrations of fibers (pectin, gum arabic), short-chain fatty acids (n-butyrate, propionate) and bile salts (taurocholate, cholate, deoxycholate) are all potent stimulants of PYY release. Whether the release of PYY by luminal factors is coupled to water and electrolyte transfer via a local/paracrine pathway remains an open question which requires additional work with the isolated vascularly perfused colon preparation.

The effects of the differentiation-inducing agent sodium butyrate on cholecystokinin (CCK) expression was investigated in the pancreatic islet tumor cell line RIN 1056E, which contains high levels of CCK-like immunoreactivity (CCK-LI). Exposure to butyrate for 24 h dose-dependently inhibited cell proliferation and increased the cell content in CCK-LI over the concentration range 0.1-8 mM. With 2 mM butyrate, cell proliferation was decreased by 50% and CCK-LI content was increased by 300%, whereas the level of steady-state CCK mRNA was reduced by 75%. Cycloheximide (10 μg/mL) abolished the sodium butyrate-induced increase in CCK-LI content. This article reports the novel finding that butyrate exerts opposite effects on CCK mRNA and immunoreactivity. The butyrate-induced increase in cellular CCK-LI content is entirely dependent on continuing protein synthesis.

The effect of glucocorticoids on the expression of intestinal cholecystokinin (CCK) was investigated both in vivo and in cell culture systems. In vivo, 2-day administration of methylprednisolone to adult male rats induced a decrease in CCK-like immunoreactivity (CCK-L1) and CCK mRNA levels in mucosal extracts. In two CCK-producing cell lines, RIN 1056E and STC-1 of pancreatic and intestinal origin respectively, dexamethasone induced dose-dependent decreases in both CCK-L1 and steady-state CCK mRNA levels. The decrease in CCK mRNA was totally prevented by incubation of cells with an excess of RU 38486, a competitive inhibitor for the binding of glucocorticoids to their receptor. Actinomycin D, used to prevent RNA synthesis, did not modify CCK mRNA stability in dexamethasone-pretreated cells as compared with cells not exposed to dexamethasone. When cells were first incubated with actinomycin D, subsequent addition of dexamethasone left the steady-state CCK mRNA levels unaltered in both cell lines. Nuclear run-on assays performed in RIN 1056E cells showed that glucocorticoids decreased the rate of transcription of the CCK gene. In addition, cycloheximide, used to prevent protein synthesis, abolished the inhibitory effects of dexamethasone on steady-state CCK mRNA levels. These results demonstrate that glucocorticoids down-regulate CCK gene expression in the rat intestinal mucosa and in two CCK-producing cell lines. The effect is blocked by a glucocorticoid receptor antagonist. Inhibition of CCK gene expression may result from a decrease in the transcription rate, and probably involves one or several steps that depend on protein synthesis.

Bombesin-related peptides are expressed in the skin of batrachians and mammals. As gastrin-releasing peptide belongs to this family, we searched for the presence and distribution of gastrin-releasing peptide receptors (GRPr) in the skin of healthy human adults by immunohistochemistry, flow cytometry and electron microscopy. The results indicated that GRPr are expressed on nerves and vessels in the dermis, on eccrine sweat glands, sebaceous glands and erector pili muscle. Within epidermis, staining was localized only on basal and suprabasal layer cells, or in the whole epidermis, according to the samples studied. Interestingly, suprabasal epidermal dendritic cells occasionally showed a strong labelling. Some of these epidermal dendritic cells were identified as Langerhans' cells by immunoelectron microscopy studies. Flow cytometry analysis of crude epidermal cell suspensions resulted in the expression of GRPr on about 43% of the cells. Therefore, we investigated whether human GRPr could modulate Langerhans' cells antigen-presenting functions. For this purpose, we added increasing concentrations of GRP (10(-12) to 10(-5) M) to mixed epidermal cell lymphocyte reactions. Allogeneic T-cell proliferation was not significantly modified when added to GRP-pretreated epidermal cells. In conclusion, we demonstrated the presence of GRPr in human skin, suggesting that GRP may modulate epidermal cell functions but does not modify antigenic presentation.

Neurotensin (NT), peptide YY (PYY), and several peptides derived from proglucagon are promptly released from endocrine cells of the distal part of the gut after oral ingestion of a meal, thus suggesting that release of these peptides is partly under neural and/or hormonal control. Our previous studies conducted with a model of isolated vascularly perfused rat colon showed that colonic L cells are highly responsive to several transmitters of the gut and to the hormonal peptide GIP. To test the possibility that hormones produced by the proximal small intestine or transmitters of the enteric nervous system may also modulate the secretory activity of the ileal L cells, various intestinal regulatory peptides and neurotransmitters were administered intraarterially for 30 min in the isolated vascularly perfused rat ileum preparation. The secretory activity of the ileal N cells was comparatively assessed. The release of NT, PYY, and glucagon-like peptide-1 (GLP-1) in the portal effluent was measured with specific RIAs. The muscarinic cholinergic agonist bethanechol at a concentration of 10(-4) M provoked a biphasic release of PYY, GLP-1, and NT, consisting of an early peak followed by a sustained response. Similarly, bombesin (10(-7) M) induced a marked biphasic release of PYY and GLP-1. In contrast, the NT response was essentially monophasic, characterized by an early peak secretion. Tetrodotoxin did not modify the bombesin-induced release of PYY, GLP-1, and NT. The beta-adrenergic agonist isoproterenol at a concentration of 10(-6) M induced a transient rise in portal PYY and GLP-1 concentrations, whereas the effect on NT release was clearly biphasic. Calcitonin gene-related peptide (5 x 10(-8) M) induced a dramatic rise in PYY, GLP-1, and NT immunoreactivities in the portal effluent (peaks at 600%, 500%, and 550% of the basal values, respectively, 4 mi n after the start of infusion). Intraarterial infusion of GIP over the concentration range (0.5-3 nM) evoked a significant increase in portal concentration of the three peptides only at the threshold concentration of 3 nM. Secretin (50 pM) or cholecystokinin (50 pM) did not affect the release of ileal hormones. In conclusion, ileal L and N cells respond to a variety of transmitters of the gut. The pattern of peptide release depends on the cell type studied. The two cosynthesized peptides, PYY and GLP-1, appear to be cosecreted in the conditions of the present study.

Background: Peptide YY (PYY) is promptly released from endocrine cells of the distal part of the gut after food intake. To test the possibility that hormones produced by the proximal small intestine or transmitters of the enteric nervous system may take part in the early phase of meal-induced PYY release, various regulatory peptides and neurotransmitters of the gut were administered intra-arterially in the isolated, vascularly perfused rat colon., Methods: A colonic loop was perfused with a Krebs-Henseleit buffer containing 20% washed bovine erythrocytes via the superior mesenteric artery. The release of PYY in portal effluent was measured by radioimmunoassay., Results: Cholecystokinin and secretin produced a small release of PYY. In contrast, infusion of gastric inhibitory polypeptide (GIP) over the concentration range 0.25-1 nM for 30 min produced a dose-dependent secretion of PYY with a maximal response at 800% above basal. Tetrodotoxin (TTX) did not modify the GIP-induced PYY release. Bethanechol (10(-5) M, 10(-4) M) produced a PYY release that was maximal at the end of the 30-min infusion period. The beta-adrenergic agonist isoproterenol (10(-7) M, 10(-6) M) caused a prompt release of PYY, followed by a sustained release at a lower value. Calcitonin gene-related peptide (CGRP) (5.10(-9) M and 5.10(-8) M) induced a PYY release with kinetics similar to that found for isoproterenol. Finally, bombesin (10(-9)-10(-7) M) provoked a dose-dependent release of PYY, consisting of an early peak followed by a sustained response. TTX did not modify the bethanechol-, isoproterenol-, CGRP-, and bombesin-induced PYY secretion., Conclusion: The hormonal peptide GIP and several transmitters of the nervous enteric system may mediate the release of PYY through the occupation of receptors possibly located at the surface of the colonic L-cells.

Glucagon-like peptide-1 (GLP-1) is released from endocrine cells of the distal part of the gut after ingestion of a meal. GLP-1 secretion is, in part, under the control of hormonal and/or neural mechanisms. However, stimulation of the colonic L cells may also occur directly by the luminal contents. This was examined in the present study, using an isolated vascularly perfused rat colon. GLP-1 immunoreactivity was measured in the portal effluent after luminal infusion of a variety of compounds which are found in colonic contents (nutrients, fibers, bile acids, short-chain fatty acids (SCFAs)). Oleic acid (100 mM) or a mixture of amino acids (total concentration 250 mM), or starch (0.5%, w/v) did not increase GLP-1 secretion over basal value. A pharmacological concentration of glucose (250 mM) elicited a marked release of GLP-1 which was maximal at the end of infusion (400% of basal), while 5 mM glucose was without effect on secretion. Pectin evoked a dose-dependent release of GLP-1 over the range 0.1-0.5% (w/v) with a maximal response at 360% of basal when 0.5% pectin was infused. Cellulose or gum arabic (0.5%) did not modify GLP-1 secretion. The SCFAs acetate, propionate or butyrate (5, 20 and 100 mM) did not induce a significant release of GLP-1. Among the four bile acids tested, namely taurocholate, cholate, deoxycholate and hyodeoxycholate, the last one was the most potent at eliciting a GLP-1 response with a maximal release at 300% and 400% of the basal value when 2 and 20 mM bile acid were administered respectively.(ABSTRACT TRUNCATED AT 250 WORDS)

Isolated, vascularly perfused, rat duodenojejunum and ileum preparations were used to study the transport parameters of cholyltaurine (C-tau; taurocholate). After a bolus administration of C-tau containing trace amounts of [3H]C-tau into the lumen of the isolated vascularly perfused duodenojejunum, the rate of appearance of C-tau in the portal effluent was dose-dependent over the range 5-20 mM. At a concentration of 20 mM, the rate of absorption was constant over time and amounted to 1.25 nmol.min-1.cm-1; 2.6% of the luminal load of C-tau was recovered in the portal effluent over the 40-min experimental period. Intra-arterial infusion of ouabain (10(-3)M) did not modify significantly the absorption rate of C-tau. The C-tau absorption from the lumen of the isolated vascularly perfused ileum was 7-fold higher than that from the duodenojejunum. Total absorption of C-tau was dose-dependent over the range 1-20 mM and the estimated Km and Tmax of the absorptive area of the rat ileum were 11.5 mM and 17.7 nmol.min-1.cm-1, respectively. Intraarterial infusion of ouabain reduced by 84% the recovery of C-tau in the portal effluent. In conclusion, the absorption parameters of bile acids in the duodenojejunum and in the ileum of the ex vivo rat intestinal preparations are consistent with a passive diffusion process in the proximal small intestine and an active transport in the ileum. The isolated vascularly perfused duodenojejunum and ileum preparations are therefore appropriate models for studying bile acid absorption process and the coupling with the associated local metabolic, motor, secretory and vascular events.

Glucagon-like peptide-1 (GLP-1) is promptly released from endocrine cells of the distal part of the gut after oral ingestion of a meal. To test the possibility that hormones produced by the proximal small intestine or transmitters of the enteric nervous system may be involved in the early phase of meal-induced GLP-1 secretion, various intestinal regulatory peptides and neurotransmitters of the gut were administered intraarterially in the isolated vascularly perfused rat colon preparation. The release of GLP-1 in the portal effluent was measured by a specific RIA. Intraarterial infusion of glucose-dependent insulinotropic peptide (GIP) over the concentration range 0.25-1 nM evoked a dose-dependent release of GLP-1, with a maximal response of 350% of the basal value. Tetrodotoxin did not modify the GIP-induced release of GLP-1. Secretin or cholecystokinin did not stimulate the secretion of GLP-1. Bombesin (10(-9)-10(-7) M) provoked a dose-dependent release of GLP-1, consisting of an early peak, followed by a sustained response. Calcitonin gene-related peptide (5 x 10(-8) M) induced a dramatic rise of GLP-1 immunoreactivity in the portal effluent (peak at 800% of the basal value 10 min after the start of infusion). Similarly, the beta-adrenergic agonist isoproterenol at concentrations of 10(-7) and 10(-6) M provoked a pronounced release of GLP-1 (peak at 500% of the basal value with 10(-6) M isoproterenol). Finally, the muscarinic cholinergic agonist bethanechol at a concentration of 10(-4) M evoked a gradual increase in GLP-1 immunoreactivity, which reached a maximal value (900% over basal) at the end of the 30-min infusion period. The lowest concentration of bethanechol used in the present study (10(-5) M) did not increase portal GLP-1 immunoreactivity over the basal value. Tetrodotoxin did not modify the bethanechol-, isoproterenol-, calcitonin gene-related peptide-, or bombesin-induced GLP-1 release. In conclusion, the present study conducted with the isolated vascularly perfused rat colon shows that there are interactions between the two most potent incretins, GIP and GLP-1, probably through an enteroendocrine pathway. Additionally, several transmitters of the gut are potent stimulants of GLP-1 release and, therefore, represent potential tools in the treatment of the noninsulin-dependent diabetes mellitus.

In an attempt to clarify the question of an involvement of the inhibition of intestinal glucose absorption in the mechanism of action of Metformin, we used several experimental approaches: 1 glucose/lactate measurement in rat portal blood in vivo and 2 in the venous effluent of an isolated perfused rat intestinal segment; 3 metabolism of freshly isolated enterocytes in vitro and tissue distribution of 3H-labeled Metformin was investigated both in vivo and in vitro. Metformin applied intraluminally had no significant effect on portal glycaemia after a glucose load, but lactate increased, whereas in vivo only a high Metformin dosage reduced portal glucose appearance significantly. Although high Metformin concentrations were found in gut biopsies, precise histological analysis in the isolated intestine revealed that it was absent from enterocytes; however the drug accumulated in villous lacteals. Intrarterially applied Metformin decreased glucose absorption in the isolated perfused ileo-jejunal segment. These data suggested that vascular Metformin boosted intestinal anaerobic glucose metabolism. Biochemical measurements performed on freshly isolated enterocytes showed that even high Metformin levels did not interfere with cell respiration or with Na+/K+ ATPase activity. Thus, our data agree with other recent reports, suggesting that even at nontherapeutic concentrations Metformin has no relevant inhibitory effect on intestinal glucose absorption. The data are discussed in the frame of previous divergent observations. The results suggest however that Metformin of vascular origin stimulates glucose consumption by the intestine, which then increases lactate output from the gut.

Similar to neuronal cells, neuroendocrine cells express voltage-dependent ion channels and fire action potentials. Ca2+ influx through voltage-dependent Ca2+ channels couples changes in membrane potential to Ca(2+)-dependent cellular processes, such as hormone release. Using the patch-clamp technique, we studied the spontaneous electrical activity as well as voltage-dependent Ca2+ channels in cholecystokinin-producing pancreatic cells (RIN 1056E cell line), in prolactin-secreting pituitary cells (GH3 cell line), and in calcitonin-secreting cells of the thyroid (rMTC 44-2 cell line). All three cell types displayed spontaneous electrical activity, that is, they spontaneously produced action potentials. RIN 1056E cells, GH3 cells, and rMTC cells exhibited (various types of) voltage-dependent Ca2+ channels that were regulated by various neurotransmitters and hormones, such as somatostatin.

The cellular events associated with cAMP-dependent cholecystokinin (CCK) release were investigated with an X-ray induced rat pancreatic tumor cell line (RIN 1056 E). Forskolin dose-dependently stimulated the release of CCK. Agents that increase [Ca2+]i (thapsigargin, Bay K 8644, ionomycin) also stimulated the release of CCK. Conversely, absence of extracellular Ca2+ or cell treatment with various calcium channel blockers strongly reduced the forskolin-induced CCK release. Finally, the cAMP-kinase inhibitor H89, the calmodulin antagonist W7 and the Ca/calmodulin-dependent protein-kinase II inhibitor KN62 strongly inhibited the forskolin-evoked CCK secretion. We conclude that the release of CCK via a cAMP-dependent pathway is dependent on the activation of voltage-dependent calcium channels and may implicate protein kinase A, calmodulin and the Ca/calmodulin-dependent protein kinase II.

Neurotensin and neuromedin N are two biologically active related peptides which are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences which are thought to represent putative processing sites occur in the precursor molecule. Of these sites, the three that are closest to the C-terminus flank and separate neurotensin and neuromedin N. The fourth precedes a neuromedin N-like sequence. The present studies were aimed at determining the extent to which each of these four dibasic sites is cleaved and at identifying and quantifying the intermediate and mature products to which this cleavage gives rise in extracts from whole rat brain, hippocampus and globus pallidus. This was achieved by means of radioimmunoassays specific for sequences of the neurotensin/neuromedin N precursor that are adjacent to the dibasic processing sites used in combination with high pressure liquid chromatography and arginine-directed trypsin digestion of tissue extracts. In all tissue extracts, it was found that the three most C-terminal dibasic processing sites in the neurotensin/neuromedin N precursor are processed to a similar extent, whereas the dibasic site that precedes the neuromedin N-like sequence is processed to a lesser extent. As reported previously, the globus pallidus was shown to contain proportionally lower levels of neuromedin N than other brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)

Studies on the cholinergic regulation of intestinal L-cells have been focused on the release of enteroglucagon, but the signal transduction pathways were not defined. These were here investigated by using as index the release of immunoreactive glucagon-like peptide-1 (GLP-1) from the endocrine cell line STC-1, that has been shown to contain proglucagon mRNA transcripts. Abundant GLP-1 immunoreactivity was revealed in STC-1 cells at immunocytochemistry and by RIA. The cell content was 4927 +/- 689 pg/10(6) cells, as measured with antiserum 199D that recognizes specifically the C-terminal amidated forms of GLP-1. The secretion of GLP-1 over a 2-h incubation period amounted to 1.4 +/- 0.3% of the total GLP-1 cell content and was significantly increased by 10 microM forskolin and 100 nM 12-O-tetradecanoylphorbol 13-acetate to 206% and 574% of control values, respectively. The cholinergic agonist carbachol stimulated GLP-1 secretion in a concentration-dependent manner, maximal release was observed at 1 mM carbachol (228% of the control value). Binding of the muscarinic antagonist [N-methyl-]scopolamine ([3H]NMS) on cell homogenates was time dependent, specific, and saturable. Scatchard analysis revealed one class of receptors (Kd, 14 pM; binding capacity, 20 fmol/mg protein). Carbachol (0.1 microM to 1 mM) dose dependently displaced [3H] NMS binding and increased the intracellular calcium concentration without modification of adenylate cyclase activity. The order of potency of different antagonists, showing a preferential affinity for M1, M2, and M3 muscarinic receptor subtypes, to inhibit [3H]NMS binding, the carbachol-induced increase in intracellular calcium, and carbachol-stimulated GLP-1 secretion, was as follows: atropine (nonselective) > 4-diphenylacetoxy-N-methylpiperidine methiodide (M3) > pirenzepine (M1) > AF-DX 116 (M2). The results of the present study, therefore, demonstrate that secretion of GLP-1 induced by cholinergic agonist depends on muscarinic M3-subtype receptors in the endocrine intestinal cell line STC-1. This system may prove useful to study the cellular mechanisms of GLP-1 secretion.

Short term effects of indomethacin on intestinal permeability were studied on a model of rat isolated vascularly perfused terminal ileum. The objectives of this study were (a) to assess the effects of indomethacin on intestinal permeability and histology; (b) to assess the effects of prostaglandins, leukotrienes, and platelet activating factor (PAF) on the same parameters; (c) to evaluate the role of these inflammation mediators on indomethacin induced permeability modifications. Intravascular administration of 1.25 and 2.5 mM indomethacin induced a significant increase of 51Cr-EDTA transfer rate. Histological analysis showed only mucosal oedema. Pretreatment with 16,16 dimethyl-prostaglandin E2 did not reverse these changes. Intravascular administration of PAF, leukotrienes B4 and D4 provoked a significant rise in 51Cr-EDTA transfer rate and intraluminal protein leakage, with an intense vascocongestion of the mucosal capillaries. These changes were completely prevented by perfusion of the respective specific antagonists (BN52021 for PAF, LY255,583 for leukotriene B4 and MK571 for leukotriene D4). None of these three antagonists, however, or MK886, a selective 5'-lipo-oxygenase inhibitor, could reverse the indomethacin induced permeability changes. Indomethacin induced increased intestinal permeability at these high concentrations does not seem to be a result of changed prostanoid or PAF metabolism. Alternative mechanisms of the initial damage of non-steroid anti-inflammatory drugs should be sought.

Bile salts in the distal small intestine are strong stimulants of neurotensin (NT) release, but the underlying mechanisms are not known. They were investigated using an isolated vascularly perfused rat jejuno-ileum preparation. Luminal administration of crude ox bile extract (0.25-1.5%, wt/vol) produced a dose-dependent release of NT-like immunoreactivity (NT-LI), with a maximal effect after infusion of 1% bile extract (500% of basal). Pretreatment of the 1% bile extract with the bile salt-sequestering resin cholestyramine (2%, wt/vol) abolished NT-LI release. Taurocholate (TC), the major bile salt in rats, dose dependently increased the release of NT. The maximal secretion of NT-LI was observed after infusion of 20 mM TC (400% of basal). Taurodeoxycholate (20 mM) was as potent as TC in stimulating NT-LI release, but the threshold concentration of taurodeoxycholate for NT-LI secretion was lower than that of TC. Glycocholate and cholate were 2- to 3-fold less potent than TC in releasing NT-LI over the concentration range 5-20 mM. Luminal infusion of oleic acid (sodium salt; 100 mM) increased by 100% the level of NT-LI in the portal effluent, whereas 20 mM oleate had no effect. In contrast, the micellar form of oleic acid (20 and 100 mM) in bile extract (1%) or TC (20 mM) dose dependently reduced the release of NT-LI induced by bile extract or TC alone. Neither intraarterial tetrodotoxin (10(-6) M), EGTA (2 mM), verapamil (5 x 10(-5) M), nor atropine (10(-5) M) had any effect on TC-induced NT-LI release. These results show that the tauro-conjugated forms of cholic and deoxycholic acid are strong stimulants of NT-LI release. The N-cell response is blunted when bile salts are complexed in the lumen by oleic acid. Finally, bile salt-induced NT-LI release is not mediated by intramural nerves and is not dependent on the activation of calcium channels.

In the isolated vascularly perfused rat duodenojejunum, vascular infusion of bombesin (100 nM) provoked an early, transient (6 min) release of CCK (500% of basal), followed by a sustained response (400% of basal). The calcium chelator EGTA (2 mM) reduced the early peak and abolished the second phase of CCK release. A similar variation was evoked by verapamil (10 microM), whereas diltiazem (100 microM), nifedipine (50 microM), and omega-conotoxin (100 nM) had no significant effect. It is concluded that bombesin-induced CCK release from rat intestine is dependent on the availability of extracellular calcium and on the activation of calcium channels sensitive to blockers of the phenylalkylamine family.

1. In this study, the mechanisms involved in the release of neurotensin-like immunoreactivity (NTLI) by glucose were investigated with the isolated, vascularly perfused rat jejunoileum preparation. 2. Luminal infusion of glucose (1-250 mM) produced a sharp and sustained release of NTLI in the intestinal venous effluent. The first significant response was observed with 5 mM glucose and the release reached a maximum under 250 mM glucose with a plateau secretion at 500% of basal. 3. There was no significant difference in the ability of galactose and 3-O-methylglucose to release NTLI when compared to glucose, but alpha-methylglucose, mannose, 2-deoxyglucose and fructose did not stimulate NTLI release. 4. Luminal infusion of 5 mM phloridzin reduced the glucose-induced release of NTLI by 90%. Intra-arterial infusion of glucose (25 mM) or of phloretin (20 microM) had no significant effect on the glucose-evoked NTLI secretion. 5. Intra-arterial infusion of ouabain (1 mM) produced a dramatic increase (at about 1500% of basal) in portal NTLI although it drastically reduced intestinal absorption of glucose. 6. Intra-arterial infusion of tetrodotoxin (1 microM), atropine (10 microM), verapamil (50 microM) or nifedipine (50 microM) did not modify the glucose-induced NTLI secretion. 7. Intra-arterial infusion of forskolin (2-20 microM) evoked a prompt and well-sustained secretion of NTLI which was increased to a mean value of 800% of basal with the highest dose tested. 3-Isobutyl-1-methylxanthine (IBMX, 10-100 microM) also stimulated the secretion of NTLI (maximal increase at 725% of basal at 100 microM). In contrast, intra-arterial infusion of 4-beta-phorbol 12-myristate, 13-acetate (PMA, 0.05-0.5 microM) had no effect on NTLI release. 8. IBMX (10-100 microM) synergistically enhanced NTLI responses induced by 250 mM glucose; the integrated response of NTLI release was 3- to 5-fold higher than the sum of individual responses produced by the same stimulants given separately. 9. It is concluded that the carbohydrate-induced NTLI release is related to the active, sodium-dependent hexose transport, but not to the carbohydrate catabolic pathway. Furthermore, the intramural nerves and L-type calcium channels are not involved in the glucose-induced NTLI secretion. Finally, the secretory activity of the intestinal N cell seems to be mainly stimulated through a cAMP-dependent pathway.

Neurotensin and neuromedin N are two biologically active, related peptides that are encoded in the same precursor molecule. In the rat, the precursor consists of a 169-residue polypeptide containing in its C-terminal region one copy each of neurotensin and neuromedin N. Four Lys-Arg sequences, which are thought to represent putative processing sites, occur in the precursor molecule. Studies by others have shown that rat pheochromocytoma PC12 cells produced neurotensin and dramatically increased their neurotensin/neuromedin N precursor mRNA content in response to a combination of nerve growth factor, dexamethasone, forskolin, and Li+. Here, we investigated the effects of this combination of inducers on the posttranslational processing of the neurotensin/neuromedin N precursor in PC12 cells. Radioimmunoassays coupled to HPLC and arginine-directed tryptic cleavage of cell extracts were performed with five antisera specific for precursor sequences adjacent to basic doublets. Thus, mature neurotensin and neuromedin N represented less than 1% of the total precursor content in PC12 cells. The PC12 cell line may represent an interesting model with which one could transfect the recently cloned prohormone convertases PC1 and PC2, thereby allowing the study of the role of these enzymes in the processing of the neurotensin/neuromedin N precursor.