Isolated Vascularly Perfused Rat Colon Research Articles

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

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

Selective Involvement of Calcium and Calcium Channels in Stimulated Mucin Secretion from Rat Colon

Background: The cellular mechanisms involved in the mucin secretion of rat colon are unknown. The objective of the present study was thus to determine the role of extracellular calcium and of L-type calcium channels in rat intestinal mucin discharge. Methods: The experiments were conducted using the isolated vascularly perfused rat colon. Mucin secretion was evaluated using an enzyme-linked immunosorbent assay. Results: Intra-arterial bethanechol (200 μM) or luminal deoxycholate (5 mM) produced a significant mucin discharge (609% and 386% of controls, respectively). The colonic mucin output induced by these two secretagogues was significantly inhibited by arterial administration of EGTA (2 mM), verapamil (100 μM) or nifedipine (50 μM). In contrast, luminal EGTA (2 mM) had no inhibitory effect. Intra-arterial infusion of the calmodulin antagonist trifluoperazine (10 μM) also reduced mucin discharge induced by bethanechol or deoxycholate (304% and 223% of controls, respectively). Colonic mucin secretion was significantly stimulated after intra-arterial infusion of 3-isobutyl-methylxanthine (IBMX, 100 μM) or forskolin (2-20 μM). Stimulation by forskolin was unaffected by arterial EGTA, verapamil, nifedipine or trifluoperazine. Conclusion: In the isolated vascularly perfused rat colon, mucin discharge induced by bethanechol or deoxycholate requires extracellular calcium and the activation of voltage-dependent calcium channels of L-type. In contrast, forskolin does not appear to stimulate mucin release by increasing calcium entry.

Release of guanylin immunoreactivity from the isolated vascularly perfused rat colon.

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.

Release of Guanylin Immunoreactivity from the Isolated Vascularly Perfused Rat Colon

Release of Guanylin Immunoreactivity from the Isolated Vascularly Perfused Rat Colon

Mucin discharge from the isolated vascularity perfused rat colon in response to bile salts

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

Mucin secretion is modulated by luminal factors in the isolated vascularly perfused rat colon

BACKGROUNDMucins play an important protective role in the colonic mucosa. Luminal factors modulating colonic mucus release have been not fully identified.AIMTo determine the effect of some dietary compounds on mucus...

Effects of neurotransmitters, gut hormones, and inflammatory mediators on mucus discharge in rat colon.

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.

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

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

Colonic mucin discharge by a cholinergic agonist, prostaglandins, and peptide YY in the isolated vascularly perfused rat colon.

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. 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.

Luminal peptide YY-releasing factors in the isolated vascularly perfused rat colon.

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.

Studies on the viability of the isolated vascularly perfused rat colon.

The colon contains large numbers of endocrine cells. Insight into their physiological function is limited. This is due to the fact that no sufficient model of isolated endocrine colon cells is available. In the present study we introduce an isolated vascularly perfused colon model for in vitro studies. This model offers the advantage that it keeps the endocrine cells in their physiological orientation and environment. The gut mucosa is highly sensitive to ischemia. Therefore, a careful validation of its viability is crucial in gut organ preparations. This study demonstrates that, by utilizing an oxygenated vascular medium supplemented with 25% washed bovine erythrocytes, a perfusion of the colon is achieved for at least 1 h without obvious tissue injuries. During this time parameters such as perfusion pressure, venous lactate dehydrogenase release, glucose consumption, lactate output, oxygen consumption, perfusate loss by the preparation and morphology were analyzed. Dependent on stimulation, the endocrine L cells of the colon released glucagon-like peptide-I upon arterial perfusion of methacholine or gastrin-releasing peptide. In conclusion, a model for the isolated perfusion of the colon is introduced which is suitable for studies of endocrine colon cells.

Luminal glucagon-like peptide-1(7-36) amide-releasing factors in the isolated vascularly perfused rat colon.

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)

Luminal peptide YY releasing factors in the isolated vascularly perfused rat colon: [formula omitted], V. Dumoulin, J.A. Chayvialle and J.C. Cuber. INSERM U45, Pavillon Hbis, Hôpital Ed. Herriot, 69437 Lyon Cedex 03, France

Luminal peptide YY releasing factors in the isolated vascularly perfused rat colon: [formula omitted], V. Dumoulin, J.A. Chayvialle and J.C. Cuber. INSERM U45, Pavillon Hbis, Hôpital Ed. Herriot, 69437 Lyon Cedex 03, France

Regulation of glucagon-like peptide-1-(7-36) amide secretion by intestinal neurotransmitters and hormones in the isolated vascularly perfused rat colon

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.

Regulation of glucagon-like peptide-1-(7-36) amide secretion by intestinal neurotransmitters and hormones in the isolated vascularly perfused rat colon.

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.

Release of glucagon-like peptide I (GLP-I) and PYY in the isolated vascularly perfused rat colon

Release of glucagon-like peptide I (GLP-I) and PYY in the isolated vascularly perfused rat colon