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Background & Aims: 5-Hydroxytryptamine (5-HT) is a neurotransmitter and paracrine signaling molecule in the gut. Paracrine signaling by enterochromaffin cells (EC), which release 5-HT, has not been studied in neonates. Our aim was to compare 5-HT disposition in the intestinal mucosa of neonatal and adult guinea pigs. Methods: 5-HT was locally measured in vitro from intestinal segments using a diamond microelectrode and continuous amperometry. The serotonin transporter (SERT) was measured using immunohistochemical and Western blot techniques. 5-HT intestinal content was measured using immunohistochemistry and high-performance liquid chromatography with electrochemical detection. Results: An oxidation current, reflective of local 5-HT release, was recorded with the microelectrode near the mucosal surface, and this current was larger in neonatal than in adult tissues. Mechanically stimulating the mucosa with a fine glass probe evoked an additional current in adult but not neonatal tissues. Oxidation currents were reduced by tetrodotoxin and were blocked in calcium-free solutions. Fluoxetine (1 μM) potentiated oxidation currents in adult but not neonatal tissues. SERT levels were lower in neonatal vs adult tissues. There was no difference in 5-HT content between neonates and adults but 5-hydroxyindole acetic acid/5-HT ratios were higher in adults. EC cell counts showed no difference in cell number, but EC cells were found in the crypts in neonatal and along the villi in adult tissues. Conclusions: SERT expression is low in neonates, and this is associated with high levels of free mucosal 5-HT and reduced metabolism. Postnatal maturation of 5-HT signaling may be important for development of neurohumoral control of intestinal motor reflexes.

Background & Aims: We have previously shown that orphanin FQ (OFQ) preferentially stimulates muscle contraction in the rat colon. However, the mechanism of action of OFQ remains unclear. Methods: We studied the effects of OFQ on muscle contractions and inhibitory junction potentials (IJPs) in rat colon. The site of action of OFQ was also investigated by in situ hybridization of OFQ receptors. Results: OFQ (10 −10 to 10 −6 mol/L) caused circular muscle contractions that were blocked by tetrodotoxin (10 −7 mol/L), suggesting the contractions were nerve mediated. Suramin (a nonselective P 2 -purinoceptor antagonist; 10 −4 mol/L) and reactive blue 2 (a P 2Y -purinoceptor antagonist; 3 × 10 −5 mol/L), but not pyridoxalphosphate-6-azophenyl-2',4' disulfonic acid (PPADS; a P 2X -purinoceptor antagonist; 3 × 10 −5 mol/L), abolished OFQ-induced colonic contractions. Focal stimulation of interganglionic fiber tracts evoked biphasic IJPs in colonic circular muscle cells. Suramin and reactive blue 2 inhibited the peak amplitude of the IJP, whereas PPADS had no effect. Cumulative addition of OFQ (10 −10 to 10 −6 mol/L) significantly inhibited the IJPs. In situ hybridization revealed that OFQ receptor messenger RNA was expressed in the colonic myenteric plexus but not in the smooth muscle cells, suggesting that the site of action of OFQ is neuronal. Conclusions: These results suggest that OFQ causes muscle contractions by inhibiting purinergic inhibitory motorneurons in the rat colon. GASTROENTEROLOGY 2000;119:1054-1063

BACKGROUND & AIMS: Adenosine triphosphate (ATP) acting at P2 receptors mediates some fast excitatory postsynaptic potentials (fEPSPs) in myenteric neurons of guinea pig ileum. The present studies investigate the distribution of purinergic fEPSPs along the length of the gut and characterize the P2-receptor subtype mediating fEPSPs. METHODS: Conventional intracellular electrophysiological methods were used to record from myenteric neurons in vitro. RESULTS: At a membrane potential of -97 +/- 1 mV, the amplitude (25 +/- 1 mV; n = 307) of fEPSPs was similar along the gut. Hexamethonium (100 micromol/L) inhibited fEPSPs in the gastric corpus by 98% +/- 1% (n = 31) and in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 42%-55%. In the presence of hexamethonium, suramin (100 micromol/L) or the P2X antagonist pyridoxal phosphate-6-azophenyl-2',4'-disulfonic acid (PPADS, 10 micromol/L) reduced the control fEPSP amplitude in the duodenum, ileum, taenia coli, proximal colon, and distal colon by 71%- 84%. The pharmacology of the purinergic fEPSPs was investigated in detail in the ileum. Noncholinergic fEPSPs were concentration- dependently (1-30 micromol/L) inhibited by PPADS (50%-inhibitory concentration, 3 micromol/L). In addition, alpha,beta-methylene 5'- adenosine triphosphate (1 micromol/L) also reduced purinergic fEPSPs. CONCLUSIONS: Fast EPSPs mediated in part through P2X receptors are prominent in myenteric neurons along the small and large intestines but are rare in the gastric corpus. (Gastroenterology 1997 Nov;113(5):1522-34)

Background & Aims 5-hydroxytryptamine receptor (5-HT 4 R) agonists promote gastrointestinal motility and attenuate visceral pain, but concerns about adverse reactions have restricted their availability. We tested the hypotheses that 5-HT 4 receptors are expressed in the colonic epithelium and that 5-HT 4 R agonists can act intraluminally to increase motility and reduce visceral hypersensitivity. Methods Mucosal expression of the 5-HT 4 R was evaluated by reverse-transcriptase polymerase chain reaction and immunohistochemical analysis of tissues from 5-HT 4 R(BAC)-enhanced green fluorescent protein mice. Amperometry, histology, and short-circuit current measurements were used to study 5-HT, mucus, and Cl − secretion, respectively. Propulsive motility was measured in guinea pig distal colon, and visceromotor responses were recorded in a rat model of colonic hypersensitivity. 5-HT 4 R compounds included cisapride, tegaserod, naronapride, SB204070, and GR113808. Results Mucosal 5-HT 4 receptors were present in the small and large intestines. In the distal colon, 5-HT 4 receptors were expressed by most epithelial cells, including enterochromaffin and goblet cells. Stimulation of 5-HT 4 Rs evoked mucosal 5-HT release, goblet cell degranulation, and Cl − secretion. Luminal administration of 5-HT 4 R agonists accelerated propulsive motility; a 5-HT 4 R antagonist blocked this effect. Bath application of 5-HT 4 R agonists did not affect motility. Oral or intracolonic administration of 5-HT 4 R agonists attenuated visceral hypersensitivity. Intracolonic administration was more potent than oral administration, and was inhibited by a 5-HT 4 R antagonist. Conclusions Mucosal 5-HT 4 receptor activation can mediate the prokinetic and antinociceptive actions of 5-HT 4 R agonists. Colon-targeted, intraluminal delivery of 5-HT 4 R agonists might be used to promote motility and alleviate visceral pain, while restricting systemic bioavailability and resulting adverse side effects.

BACKGROUND/AIM: Enterochromaffin (EC) cells are the best characterized subset of enteric endocrine cells and are the main source of 5-hydroxytryptamine (5-HT) in the gut. Previously we have shown an important immuno-endocrine axis in the gut, where CD4+ T cells play a key role in generation of EC cell hyperplasia and in up-regulation of 5-HT production in infection-induced gut inflammation (Gut 2007; 56: 949). However, the role of EC cells/5HT in regulation of inflammation or in host defense in enteric infection remains to be determined. The aim of this study was to investigate the role of 5-HT in the development of infection-induced gut inflammation and in host defense in a model of enteric infection. METHODS: Tryptophan hydroxylase1-deficient mice (TPH1-/-) which lack 5-HT in gut and wild type (TPH1+/+) mice were infected with nematode, Trichuris muris and sacrificed on various days post-infection (pi) to study the worm expulsion, macrophage infiltration by immunohistochemistry, myeloperoxidase (MPO) activity, and cytokines (IL-β,IL-4 and IL13) in colon by ELISA. Role of 5-HT in production of pro-inflammatory mediators was further studied by culturing macrophages harvested from peritoneal cavity of naive TPH1+/ + mice with 5-HT, and assessing IL-1β production after LPS stimulation. RESULTS: Worm expulsion from the intestine was significantly delayed in TPH1-/mice as compared to TPH1+/+ mice in T. muris infection (worm numbers: 24.2±3.8 vs. 40±6.7 in TPH1+/+ vs. TPH1-/mice on day 21 pi; p

capsaicin-sensitive afferents was clearly demonstrated, as capsaicin pretreatment prevented the development of cross-sensitization and the associated bladder mastocytosis. Furthermore, acute bladder afferent sensitization was noted at 1 hr, while sub-acutely, the degree of bladder mast cell migration was directly and temporally associated with increases in bladder permeability and decreases in bladder voiding intervals (irritative voiding). A causal role of mast cells could not be established. We hypothesize that pelvic organ mast cell infiltration, mediator release, and consequential afferent modulation are the primary mechanisms responsible for chronic pelvic organ sensitization. Methods: Intra-colonic TNBS (or vehicle) was administered to mast cell deficient mice (W/Wv) and wild type (WT) littermates. Eight days later, colonic and bladder mast cells were quantified, and under urethane anesthesia, bladder voiding intervals and permeability were measured using saline cystometry and fluorescein, respectively. Animal weights were recorded daily throughout the experiment. Results: Animal weights were decreased following intra-colonic TNBS beginning as early as day 2 and peaking at 15-20% on day 4 (p

The effects of surgical interruption of the myenteric plexus (myectomy), extrinsic denervation of a length of small intestine, or transection and reanastomosis of the intestinal wall on migration of phase III of the migrating myoelectric complex was studied in guinea pigs. In addition, the recovery of phase III migration and the regrowth of intestinal nerves and muscle across the reanastomosis was studied at various times up to 60 days after surgery. At 6–9 days after surgery, phase III did not migrate past the myectomy during 50%–60% of recorded migrating myoelectric complexes and transection and reanastomosis of the intestinal wall blocked aboral progression of phase III in 90% of cases. Extrinsic denervation did not alter phase III migration through the denervated segment. Phase III migration past the reanastomosis recovered with time after surgery; 80% recovery occurred by 60 days after surgery. Immunoreactivities for vasoactive intestinal peptide, gastrin-releasing peptide, and somatostatin were used as markers for intestinal nerves that were cut by transection. Immunoreactivities for vasoactive intestinal peptide and gastrinreleasing peptide are contained in myenteric neurons that project in an oral to anal direction to other myenteric ganglia and to the circular muscle. Immunoreactivity for somatostatin is contained in nerve fibers projecting aborally to other myenteric ganglia. At 7–15 days after surgery, there were accumulations of immunoreactivities for vasoactive intestinal peptide, gastrin-releasing peptide, and somatostatin in nerve fibers on the oral side of the reanastomosis, but nerve fibers containing these peptides were not observed in myenteric ganglia or circular muscle close to the anal edge. At 23–28 days, immunoreactivities for vasoactive intestinal peptide, gastrin-releasing peptide, and somatostatin nerve fibers were traced across the reanastomosis and nerve terminals were detected in ganglia and muscle close to the lesion on the anal side. Nerve fibers traversed the lesion in all cases at 57–60 days and vasoactive intestinal peptide-, gastrin-releasing peptide-, and somatostatin-immunoreactive nerve terminals were detected in the first two to three rows of myenteric ganglia on the anal side. Regrowth of intestinal muscle followed a similar timecourse to that observed for nerves. These data suggest that interruption of the myenteric plexus alone does not completely block phase III migration. In addition, recovery of phase III migration past a reanastomosis is associated with a restoration of both nervous and mechanical connections.