Consequences of Citrobacter rodentium infection on enteroendocrine cells and the enteric nervous system in the mouse colon

We tested the hypothesis that Citrobacter rodentium infection leads to changes in the mucosal enteroendocrine signalling and the enteric nervous system and that the host's immune response contributes to these changes. Enteroendocrine cells, serotonin (5-HT) reuptake transporter (SERT), 5-HT release, and inducible nitric oxide synthase (iNOS) expression were assessed in the colon of infected wild-type or severe combined immunodeficient (SCID) mice. Immunoreactivity for iNOS and neuropeptides were examined in the submucosal and myenteric plexuses. Mice were orogastrically infected with C. rodentium and experiments were conducted during the injury phase (10 days) and the recovery phase (30 days). 5-HT and somatostatin enteroendocrine cells and SERT were significantly reduced 10 days after infection, with numbers returning to control values at 30 days. 5-HT release was increased at 10 days. Changes to the mucosal serotonin signalling system were not observed in SCID mice. iNOS immunoreactivity was increased in the submucosa and mucosa at 10 days and returned to baseline levels by 30 days. No differences were observed in neuropeptide or iNOS immunoreactivity in the enteric plexuses following infection. The host's immune response underlies changes to enteroendocrine cells, SERT expression and 5-HT release in C. rodentium infection. These changes could contribute to disturbances in gut function arising from enteric infection.     

Serotonin availability is increased in mucosa of guinea pigs with TNBS-induced colitis

5-HT released from enterochromaffin cells acts on enteric nerves to initiate motor reflexes. 5-HT's actions are terminated by a serotonin reuptake transporter (SERT). In this study, we tested the hypothesis that inflammation leads to altered mucosal 5-HT signaling. Colitis was induced by 2,4,6-trinitrobenzene sulfonic acid (TNBS), and experiments were conducted on day 6. 5-HT content, number of 5-HT-immunoreactive cells, and the proportion of epithelial cells that were 5-HT-immunoreactive increased twofold in colitis. The amount of 5-HT released under basal and stimulated conditions was significantly increased in colitis. SERT inhibition increased the 5-HT concentration in media bathing-stimulated control tissue to a level comparable to that of the stimulated colitis tissue. mRNA encoding SERT and SERT immunoreactivity were reduced during inflammation. Slower propulsion and reduced sensitivity to 5-HT-receptor antagonism were observed in colitis. These data suggest that colitis alters 5-HT signaling by increasing 5-HT availability while decreasing 5-HT reuptake. Altered 5-HT availability may contribute to the dysmotility of inflammatory bowel disease, possibly due to desensitization of 5-HT receptors.     

Proliferative capacity of enterochromaffin cells in guinea-pigs with experimental ileitis

Enterochromaffin (EC) cells regulate gut motility and secretion in response to luminal stimuli, via the release of serotonin (5-HT). Inflammatory bowel disease and other gastrointestinal disorders are associated with increased numbers of EC cells and 5-HT availability. Our aim was to determine whether proliferation of EC cells contributed to the hyperplasia associated with intestinal inflammation. Ileitis was induced in guinea-pigs by intraluminal injection of 2,4,6-trinitrobenzene sulphonic acid (TNBS). A single pulse of 5-bromo-2′-deoxyuridine (BrdU) was injected 1 or 24 h before the collection of tissue, 6 or 7 days after TNBS treatment. In the controls, the labelling index (percentage of BrdU-labelled EC cells) was less than 1%. Despite a significant increase in EC cells in the inflamed ileum, the labelling index was similar in the TNBS-treated animals to that of controls. An increased occurrence of EC cells in the BrdU-labelled zone accounted for the increase in EC cells in the inflamed ileum. Goblet cell numbers were also significantly increased in the inflamed ileum, indicating that cell hyperplasia was not limited to the enteroendocrine cell lineage. This study demonstrates that a small portion of EC cells retain some proliferative capacity; however, hyperplasia associated with ileitis is not attributable to the increased proliferation of EC cells and is not limited to one cell lineage. Therefore, EC cell hyperplasia most probably occurs at the level of the stem cell or recruitment from the progenitor pool. This work was supported by an operating grant from the Crohn’s and Colitis Foundation of Canada (CCFC; to Keith Sharkey and Dr. Gary Mawe, University of Vermont, USA). Keith Sharkey is an Alberta Heritage Foundation for Medical Research (AHFMR) Medical Scientist and the CCFC Chair in Inflammatory Bowel Disease Research. Jennifer O’Hara is an AHFMR graduate student.     

Serotonin availability in rat colon is reduced during a Western diet model of obesity

Constipation and slowed transit are associated with diet-induced obesity, although the mechanisms by which this occurs are unclear. Enterochromaffin (EC) cells within the intestinal epithelium respond to mechanical stimulation with the release of serotonin [5-hydroxytryptamine (5-HT)], which promotes transit. Thus our aim was to characterize 5-HT availability in the rat colon of a physiologically relevant model of diet-induced obesity. EC cell numbers were determined immunohistochemically in chow-fed (CF) and Western diet-fed (WD) rats, while electrochemical methods were used to measure mechanically evoked (peak) and steady-state (SS) 5-HT levels. Fluoxetine was used to block the 5-HT reuptake transporter (SERT), and the levels of mRNA for tryptophan hydroxylase 1 and SERT were determined by quantitative PCR, and SERT protein was determined by Western blot. In WD rats, there was a significant decrease in the total number of EC cells per crypt (0.86 ± 0.06 and 0.71 ± 0.05 in CF and WD, respectively), which was supported by a reduction in the levels of 5-HT in WD rats (2.9 ± 1.0 and 10.5 ± 2.6 μM at SS and peak, respectively) compared with CF rats (7.3 ± 0.4 and 18.4 ± 3.4 μM at SS and peak, respectively). SERT-dependent uptake of 5-HT was unchanged, which was supported by a lack of change in SERT protein levels. In WD rats, there was no change in tryptophan hydroxylase 1 mRNA but an increase in SERT mRNA. In conclusion, our data show that foods typical of a WD are associated with decreased 5-HT availability in rat colon. Decreased 5-HT availability is driven primarily by a reduction in the numbers and/or 5-HT content of EC cells, which are likely to be associated with decreased intestinal motility in vivo.     

Serotonin (5-HT) release and uptake measured by real-time electrochemical techniques in the rat ileum

Serotonin (5-HT) is released from the enterochromaffin cells and plays an important role in regulating intestinal function. Although the release of 5-HT is well documented, the contribution of the serotonin reuptake transporter (SERT) to the levels and actions of 5-HT in the intestine is unclear. This study aimed to demonstrate real-time SERT activity in ileal mucosa and to assess the effects of SERT inhibition using fluoxetine. Electrochemical recordings were made from the mucosa in full-thickness preparations of rat ileum using a carbon fiber electrode to measure 5-HT oxidation current and a force transducer to record circular muscle (CM) tension. Compression of the mucosa stimulated a peak 5-HT release of 12 +/- 6 microM, which decayed to 7 +/- 4 microM. Blockade of SERT with fluoxetine (1 microM) increased the peak compression-evoked release to 19 +/- 9 microM, and the background levels of 5-HT increased to 11 +/- 7 microM (P < 0.05, n = 7). When 5-HT was exogenously applied to the mucosa, fluoxetine caused a significant increase in the time to 50% and 80% decay of the oxidation current. Fluoxetine also increased the spontaneous CM motility (P < 0.05; n = 7) but did not increase the CM contraction-evoked 5-HT release (P > 0.05, n = 5). In conclusion, this is the first characterization of the real-time uptake of 5-HT into the rat intestine. These data suggest that SERT plays an important role in the modulation of 5-HT concentrations that reach intestinal 5-HT receptors.     

IFN-gamma and TNF-alpha decrease serotonin transporter function and expression in Caco2 cells

Recent studies have shown that mucosal serotonin (5-HT) transporter (SERT) expression is decreased in animal models of colitis, as well as in the colonic mucosa of humans with ulcerative colitis and irritable bowel syndrome. Altered SERT function or expression may underlie the altered motility, secretion, and sensation seen in these inflammatory gut disorders. In an effort to elucidate possible mediators of SERT downregulation, we treated cultured colonic epithelial cells (Caco2) with conditioned medium from activated human lymphocytes. Application of the conditioned medium caused a decrease in fluoxetine-sensitive [(3)H]5-HT uptake. Individual proinflammatory agents were then tested for their ability to affect uptake. Cells were treated for 48 or 72 h with PGE(2) (10 microM), IFN-gamma (500 ng/ml), TNF-alpha (50 ng/ml), IL-12 (50 ng/ml), or the nitric oxide-releasing agent S-nitrosoglutathione (GSNO; 100 microM). [(3)H]5-HT uptake was then measured. Neither PGE nor IL-12 had any effect on [(3)H]5-HT uptake, and GSNO increased uptake. However, after 3-day incubation, both TNF-alpha and IFN-gamma elicited significant decreases in SERT function. Neither TNF-alpha nor IFN-gamma were cytotoxic when used for this period of time and at these concentrations. These two cytokines also induced decreases in SERT mRNA and protein levels. By altering SERT expression, TNF-alpha and IFN-gamma could contribute to the altered motility and expression seen in vivo in ulcerative colitis or irritable bowel syndrome.     

Expression and function of 5-HT3 receptors in the enteric neurons of mice lacking the serotonin transporter

The actions of enteric 5-HT are terminated by 5-HT transporter (SERT)-mediated uptake, and gastrointestinal motility is abnormal in SERT -/- mice. We tested the hypothesis that adaptive changes in enteric 5-HT(3) receptors help SERT -/- mice survive despite inefficient 5-HT inactivation. Expression of mRNA encoding enteric 5-HT(3A) subunits was similar in SERT +/+ and -/- mice, but that of 5-HT(3B) subunits was fourfold less in SERT -/- mice. 5-HT(3B) mRNA was found, by in situ hybridization, in epithelial cells and enteric neurons. 5-HT evoked a fast inward current in myenteric neurons that was pharmacologically identified as 5-HT(3) mediated. The EC(50) of the 5-HT response was lower in SERT +/+ (18 microM) than in SERT -/- (36 microM) mice and desensitized rapidly in a greater proportion of SERT -/- neurons; however, peak amplitudes, steady-state current, and decay time constants were not different. Adaptive changes thus occur in the subunit composition of enteric 5-HT(3) receptors of SERT -/- mice that are reflected in 5-HT(3) receptor affinity and desensitization.     

Persistent alterations to enteric neural signaling in the guinea pig colon following the resolution of colitis

Functional changes induced by inflammation persist following recovery from the inflammatory response, but the mechanisms underlying these changes are not well understood. Our aim was to investigate whether the excitability and synaptic properties of submucosal neurons remained altered 8 wk post-trinitrobenzene sulfonic acid (TNBS) treatment and to determine whether these changes were accompanied by alterations in secretory function in submucosal preparations voltage clamped in Ussing chambers. Mucosal serotonin (5-HT) release measurements and 5-HT reuptake transporter (SERT) immunohistochemistry were also performed. Eight weeks after TNBS treatment, colonic inflammation resolved, as assessed macroscopically and by myeloperoxidase assay. However, fast excitatory postsynaptic potential (fEPSP) amplitude was significantly increased in submucosal S neurons from previously inflamed colons relative to those in control tissue. In addition, fEPSPs from previously inflamed colons had a hexamethonium-insensitive component that was not evident in age-matched controls. AH neurons were hyperexcitable, had shorter action potential durations, and decreased afterhyperpolarization 8 wk following TNBS adminstration. Neuronally mediated colonic secretory function was significantly reduced after TNBS treatment, although epithelial cell signaling, as measured by responsiveness to both forskolin and bethanecol in the presence of tetrodotoxin, was comparable with control tissue. 5-HT levels and SERT immunoreactivity were comparable to controls 8 wk after the induction of inflammation, but there was an increase in glucagon-like peptide 2-immunoreactive L cells. In conclusion, sustained alterations in enteric neural signaling occur following the resolution of colitis, which are accompanied by functional changes in the absence of active inflammation.     

p38 MAPK activation elevates serotonin transport activity via a trafficking-independent, protein phosphatase 2A-dependent process

Presynaptic, plasma membrane serotonin (5-hydroxytryptamine; 5-HT) transporters (SERTs) clear 5-HT following vesicular release and are regulated through trafficking-dependent pathways. Recently, we provided evidence for a trafficking-independent mode of SERT regulation downstream of adenosine receptor (AR) activation that is sensitive to p38 MAPK inhibitors. Here, we probe this pathway in greater detail, demonstrating elevation of 5-HT transport by multiple p38 MAPK activators (anisomycin, H(2)O(2), and UV radiation), in parallel with p38 MAPK phosphorylation, as well as suppression of anisomycin stimulation by p38 MAPK siRNA treatments. Studies with transporter-transfected Chinese hamster ovary cells reveal that SERT stimulation is shared with the human norepinephrine transporter but not the human dopamine transporter. Saturation kinetic analyses of anisomycin-SERT activity reveal a selective reduction in 5-HT K(m) supported by a commensurate increase in 5-HT potency (K(i)) for displacing surface antagonist binding. Anisomycin treatments that stimulate SERT activity do not elevate surface SERT surface density whereas stimulation is lost with preexposure of cells to the surface-SERT inactivating reagent, 2-(trimethylammonium)ethyl methane thiosulfonate. Guanylyl cyclase (1H-(1,2,4)-oxadiazolo[4,3-a]-quinoxalin-1-one) and protein kinase G inhibitors (H8, DT-2) block AR stimulation of SERT yet fail to antagonize SERT stimulation by anisomycin. We thus place p38 MAPK activation downstream of protein kinase G in a SERT-catalytic regulatory pathway, distinct from events controlling SERT surface density. In contrast, the activity of protein phosphatase 2A inhibitors (fostriecin and calyculin A) to attenuate anisomycin stimulation of 5-HT transport suggests that protein phosphatase 2A is a critical component of the pathway responsible for p38 MAPK up-regulation of SERT catalytic activity.     

PACAP 1-38 as neurotransmitter in the porcine antrum

The concentration of PACAP 1-38 in porcine antrum amounted to 15.4+/-7.9 and 20.3+/-8 pmol/g tissue in the mucosal and muscular layers. PACAP immunoreactive (IR) fibres innervated the muscular (co-localised with VIP) and submucosal/mucosal layers (some co-storing VIP and CGRP) including myenteric and submucosal plexus and blood vessels. Only myenteric nerve cell bodies contained PACAP-IR (co-storing VIP). In isolated perfused antrum, vagus nerve stimulation (8 Hz) and capsaicin (10(-5) M) increased PACAP 1-38 release. PACAP 1-38 (10(-9) M) increased substance P (SP), gastrin releasing peptide (GRP) and VIP release. PACAP 1-38 (10(-8) M) inhibited gastrin secretion and stimulated somatostatin secretion and motility dose-dependently. PACAP-induced motility was strongly inhibited by the antagonist PACAP 6-38 but also by atropine and substance P-antagonists (CP99994/SR48968) but PACAP 6-38 had no effect on vagus-induced secretion or motility. Conclusion: PACAP 1-38 may be involved in antral motility and secretion by interacting with cholinergic, SP-ergic, GRP-ergic and/or VIP-ergic neurones, and may also be involved in afferent reflex pathways.     

Density distribution of free fatty acid receptor 2 (FFA2)-expressing and GLP-1-producing enteroendocrine L cells in human and rat lower intestine,and increased cell numbers after ingestion of fructo-oligosaccharide

Glucagon-like peptide 1 (GLP-1) is a multifunctional hormone in glucose metabolism and intestinal function released by enteroendocrine L-cells. The plasma concentration of GLP-1 is increased by indigestible carbohydrates and luminal infusion of short-chain fatty acids (SCFAs). However, the triggers and modulators of the GLP-1 release remain unclear. We hypothesized that SCFAs produced by bacterial fermentation are involved in enteroendocrine cell proliferation and hormone release through free fatty acid receptor 2 (FFA2, also known as FFAR2 or GPR43) in the large intestine. Fructo-oligosaccharide (Fructo-OS), fermentable indigestible carbohydrate, was used as a source of SCFAs. Rats were fed an indigestible-carbohydrate-free diet (control) or a 5% Fructo-OS-containing diet for 28 days. FFA2-, GLP-1-, and 5-hydroxytryptamine (5-HT)-positive enteroendocrine cells were quantified immunohistochemically in the colon, cecum, and terminal ileum. The same analysis was performed in surgical specimens from human lower intestine. The coexpression of FFA2 with GLP-1 was investigated both in rats and humans. Fructo-OS supplementation in rats increased the densities of FFA2-positive enteroendocrine cells in rat proximal colon, by over two-fold, relative to control, in parallel with GLP-1-containing L-cells. The segmental distributions of these cells in human were similar to rats fed the control diet. The FFA2-positive enteroendocrine cells were GLP-1-containing L-cells, but not 5-HT-containing EC cells, in both human and rat colon and terminal ileum. Fermentable indigestible carbohydrate increases the number of FFA2-positive L-cells in the proximal colon. FFA2 activation by SCFAs might be an important trigger for produce and release GLP-1 by enteroendocrine L-cells in the lower intestine.     

Epithelial cells and their neighbors. II. New perspectives on efferent signaling between brain, neuroendocrine cells, and gut epithelial cells

Surface sensory enteroendocrine cells are established mucosal taste cells that monitor luminal contents and provide an important link in transfer of information from gut epithelium to the central nervous system. Recent studies now show that these cells can also mediate efferent signaling from the brain to the gut. Centrally elicited stimulation of vagal and sympathetic pathways induces release of melatonin, which acts at MT2 receptors to increase mucosal electrolyte secretion. Psychological factors as well mucosal endocrine cell hyperplasia are implicated in functional intestinal disorders. Central nervous influence on the release of transmitters from gut endocrine cells offers an exciting area of future gastrointestinal research with a clinical relevance.     

Mucosal mast cell secretion processes imaged using three-photon microscopy of 5-hydroxytryptamine autofluorescence

The secretion process of the mucosal mast cell line RBL-2H3 was imaged using infrared three photon excitation (3PE) of serotonin (5-hydroxytryptamine, 5-HT) autofluorescence, a measurement previously difficult because of the technical intractability of deep UV optics. Images of prestimulation 5-HT distributions were analyzed in loaded cell populations (those incubated in a 5-HT-rich medium overnight) and in unloaded populations and were found to be strictly quantifiable by comparison with bulk population high-performance liquid chromatography measurements. Antigenically stimulated cells were observed to characteristically ruffle and spread as granular 5-HT disappeared with no detectable granule movement. Individual cells exhibited highly heterogeneous release kinetics, often with quasi-periodic bursts. Neighboring granule disappearances were correlated, indicative of either spatially localized signaling or granule-granule interactions. In one-half of the granule release events, weak residual fluorescence was visible suggestive of leftover 5-HT still bound to the granule matrix. The terminal stages of secretion (>300 s) consisted primarily of unresolved granules and remainder 5-HT leakage from already released granules.     

Serotonin Regulation of Serotonin Uptake in RN46A Cells

1. Aim: The role of the serotonin transporter (SERT) is to remove serotonin (5-HT) from the synaptic space. In vitro studies have shown that 5-HT uptake via SERT is influenced by the availability of its substrate, 5-HT. We used RN46A cells, a line that expresses SERT, to investigate 5-HT regulation of 5-HT uptake and the intracellular signaling pathways involved. RN46A cells also express mRNAs for 5-HT receptors (5-HT_1A, 5-HT_1B, 5-HT_2A, and 5-HT_2C) and as cAMP and intracellular Ca²⁺ are modulated by different 5-HT receptors, we studied both pathways.2. Methods: 5-HT uptake was determined as imipramine-inhibitable uptake of [³H]5-HT, intracellular cAMP was measured by RIA and intracellular Ca²⁺ changes were determined using the ratiometric method of intracellular Ca²⁺ imaging.3. Results: For uptake experiments, cells were kept for 30 min either with or without 1 μM 5-HT in the medium before measuring uptake. Removal of 5-HT for 30 min significantly decreased [³H]5-HT uptake. The absence of 5-HT for 15 min failed to induce any changes in intracellular cAMP levels. Removal of 5-HT from the medium did not change intracellular Ca²⁺ levels either; however, adding 1 μM 5-HT after 5 min in 5-HT-free conditions rapidly increased intracellular Ca²⁺ levels in 50% of the cells. The remaining cells showed no changes in the intracellular Ca²⁺ levels.4. Conclusions: We have shown that in RN46A cells, that endogenously express SERT and mRNAs for several 5-HT receptors, changes in 5-HT levels influence 5-HT uptake rate as well as induce changes in intracellular Ca²⁺ levels. This suggests that 5-HT may utilize intracellular Ca²⁺ to regulate 5-HT uptake.     

Intestinal serotonin acts as paracrine substance to mediate pancreatic secretion stimulated by luminal factors

We recently demonstrated that luminal factors such as osmolality, disaccharides, and mechanical stimulation evoke pancreatic secretion by activating 5-hydroxytryptamine subtype 3 (serotonin-3, 5-HT3) receptors on mucosal vagal afferent fibers in the intestine. We hypothesized that 5-HT released by luminal stimuli acts as a paracrine substance, activating the mucosal vagal afferent fibers to stimulate pancreatic secretion. In the in vivo rat model, luminal perfusion of maltose or hypertonic NaCl increased 5-HT level threefold in intestinal effluent perfusates. Similar levels were observed after intraluminal 10(-5) M 5-HT perfusion. These treatments did not affect 5-HT blood levels. In a separate study, intraduodenal, but not intraileal, 5-HT application induced a dose-dependent increase in pancreatic protein secretion, which was not blocked by the CCK-A antagonist CR-1409. Acute vagotomy, methscopolamine, or perivagal or intestinal mucosal application of capsaicin abolished 5-HT-induced pancreatic secretion. In conscious rats, luminal 10(-5) M 5-HT administration produced a 90% increase in pancreatic protein output, which was markedly inhibited by the 5-HT3 antagonist ondansetron. In conclusion, luminal stimuli induce 5-HT release, which in turn activates 5-HT3 receptors on mucosal vagal afferent terminals. In this manner, 5-HT acts as a paracrine substance to stimulate pancreatic secretion via a vagal cholinergic pathway.     

Functional interactions between native Gs-coupled 5-HT receptors in HEK-293 cells and the heterologously expressed serotonin transporter

In HEK-293 cells, serotonin (5-hydroxytryptamine, 5-HT) was found to induce cAMP production showing pharmacological characteristics consistent with the 5-HT(7) receptor. The presence of 5-HT(7) (and 5-HT(6)) receptor mRNA was confirmed by RT-PCR. Stable HEK-293 cell lines expressing either wild-type or haemagglutinin (HA)-tagged human 5-HT transporter (SERT) were selected and SERT function was confirmed using [3H]5-HT transport. The presence of SERT caused a 10-fold reduction in the potency of 5-HT-induced cAMP production compared to control cells. Downstream signalling by 5-HT(6/7) receptors could be detected as 5-HT-induced protein kinase A activation and phosphorylation of MAP kinase and CREB using phospho-specific antibodies. SERT inhibitors reversed the reduction in potency of 5-HT-induced cAMP production caused by the presence of SERT, resulting in a concentration-dependent left shift in EC(50) values but also a progressive decrease in the maximal response. Thus, when antidepressants were used to block SERT activity, 5-HT receptor signalling was effectively clamped within a mid-range.     

Myristoylation of cGMP-dependent protein kinase dictates isoform specificity for serotonin transporter regulation

By transporting serotonin (5-HT) into neurons and other cells, serotonin transporter (SERT) modulates the action of 5-HT at cell surface receptors. SERT itself is modulated by several processes, including the cGMP signaling pathway. Activation of SERT by cGMP requires the cGMP-dependent protein kinase (PKG). Here we show that in HeLa cells lacking endogenous PKG, expression of PKGIα or PKGIβ was required for 8-bromoguanosine-3',5'-cyclic monophosphate (8-Br-cGMP) to stimulate SERT phosphorylation and 5-HT influx. Catalytically inactive PKG mutants and wild-type PKGII did not support this stimulation. However, a mutant PKGII (G2A) that was not myristoylated substituted for functional PKGI, suggesting that myristoylation and subsequent membrane association blocked productive interaction with SERT. PKG also influenced SERT expression and localization. PKGI isoforms increased total and cell surface SERT levels, and PKGII decreased cell surface SERT without altering total expression. Remarkably, these changes did not require 8-Br-cGMP or functional kinase activity and were also observed with a SERT mutant resistant to activation by PKG. Both PKGIα and PKGIβ formed detergent-stable complexes with SERT, and this association did not require catalytic activity. The nonmyristoylated PKGII G2A mutant stimulated SERT expression similar to PKGI isoforms. These results suggest multiple mechanisms by which PKG can modulate SERT and demonstrate that the functional difference between PKG isoforms results from myristoylation of PKGII.     

Desensitization of the peristaltic reflex induced by mucosal stimulation with the selective 5-HT4 agonist tegaserod

The intestinal peristaltic reflex induced by mucosal stimulation is mediated by mucosal release of serotonin (5-HT), which acts on 5-HT(4) receptors located on CGRP-containing afferent nerve terminals. Exposure of the colonic mucosa to the 5-HT(4) receptor agonist tegaserod in the range of 1 nM to 10 muM elicits a peristaltic reflex and stimulates colonic propulsion. The present study was designed to identify the 5-HT(4) receptor subtype mediating the reflex and determine whether functionally effective concentrations of tegaserod desensitize the reflex induced by mucosal stimulation. Exposure of rat colonic mucosa to tegaserod in the range of 5 nM to 5 muM for 5 or 10 min caused rapid time- and concentration-dependent desensitization of the peristaltic reflex induced by mucosal stroking, consistent with the operation of a rapidly desensitizing 5-HT(4b) receptor subtype. Desensitization was accompanied by a decrease in CGRP release. The rate of recovery of peristaltic response depended on the desensitizing concentration of tegaserod: ascending contraction and descending relaxation recovered within 15 min after 5-50 nM tegaserod, 30 min after 0.5 muM, and 60 min after 5 muM. Neither CGRP release nor the peristaltic reflex induced by muscle stretch was affected by 5-HT(4) receptor desensitization, providing further evidence that 5-HT does not mediate the reflex induced by muscle stretch. These results suggest in cases of increased 5-HT availability or prolonged exposure, such as colitis, that it is likely the peristaltic reflex will be blunted.