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Identification of unique release kinetics of serotonin from guinea-pig and human enterochromaffin cells

•  Enterochromaffin (EC) cells are enteroendocrine cells that synthesise ∼95% of the bodys total serotonin (5‐HT). •  Although 5‐HT release from EC cells plays a number of important physiological roles, primary EC cells have not been studied at the single cell level. •  This study provides the first functional characterisation of single primary guinea‐pig and human EC cells. •  EC cells release 5‐HT from large dense core vesicles in a calcium‐dependent manner with kinetics surprisingly resembling release from synaptic vesicles. •  3D modelling indicates that the quantity of 5‐HT released per vesicle fusion event is physiologically relevant to GI tract function in terms of the concentrations needed to activate local 5‐HT receptors. •  These findings represent significant advances in our understanding of EC cell function and will be of broad interest to researchers in endocrine cell biology, gastroenterology, neuroscience, exocytosis and glucose control.

CrossTalk opposing view: 5‐HT is not necessary for peristalsis

Large quantities of 5-hydroxytryptamine (5-HT) are synthesized in the gut, in enterochromaffin (EC) cells of the mucosa (via tryptophan hydroxylase-1) and in a small proportion in enteric neurons (via tryptophan hydroxylase-2). It has been well established that 5-HT can be released from the mucosa by mechanical stimulation, including contractile activity of the gut and compression of the gut wall (Bertrand, 2006). Numerous 5-HT receptors are expressed on enteric neurons and extrinsic afferent endings, and exogenous 5-HT has potent effects on neuronal excitability and transmitter release (Gershon, 2000). Despite this, the evidence that 5-HT is necessary for peristalsis or CMMCs in the lower GI-tract is all circumstantial. Extensive evidence now suggests that these motor patterns occur independent of 5-HT.

The neurochemical changes in the innervation of human colonic mesenteric and submucosal blood vessels in ulcerative colitis and Crohn's disease.

Neurogenic inflammation involves vasodilation, oedema and sensory nerve hypersensitivity. Extrinsic sensory nerves to the intestinal wall mediate these effects and functional subsets of these extrinsic nerves can be characterized by immunohistochemical profiles. In this study such profiles were examined in samples from patients with inflammatory bowel disease (IBD), in particular ulcerative colitis (UC) and Crohns disease (CD).

IS SEROTONIN IN ENTERIC NERVES REQUIRED FOR DISTENSION-EVOKED PERISTALSIS AND PROPULSION OF CONTENT IN GUINEA-PIG DISTAL COLON?

Recent studies have shown genetic deletion of the gene that synthesizes 5-HT in enteric neurons (tryptophan hydroxylase-2, Tph-2) leads to a reduction in intestinal transit. However, deletion of the Tph-2 gene also leads to major developmental changes in enteric ganglia, which could also explain changes in intestinal transit. We sought to investigate this further by acutely depleting serotonin from enteric neurons over a 24-h period, without the confounding influences induced by genetic manipulation. Guinea-pigs were injected with reserpine 24h prior to euthanasia. Video-imaging and spatio-temporal mapping was used to record peristalsis evoked by natural fecal pellets, or slow infusion of intraluminal fluid. Immunohistochemical staining for 5-HT was used to detect the presence of serotonin in the myenteric plexus. It was found that endogenous 5-HT was always detected in myenteric ganglia of control animals, but never in guinea-pigs treated with reserpine. Interestingly, peristalsis was still reliably evoked by either intraluminal fluid, or fecal pellets in reserpine-treated animals that also had their entire mucosa and submucosal plexus removed. In these 5-HT depleted animals, there was no change in the frequency of peristalsis or force generated during peristalsis. In control animals, or reserpine treated animals, high concentrations (up to 10 μM) of ondansetron and SDZ-205-557, or granisetron and SDZ-205-557 had no effect on peristalsis. In summary, acute depletion of serotonin from enteric nerves does not prevent distension-evoked peristalsis, nor propulsion of luminal content. Also, we found no evidence that 5-HT3 and 5-HT4 receptor activation is required for peristalsis, or propulsion of contents to occur. Taken together, we suggest that the intrinsic mechanisms that generate peristalsis and entrain propagation along the isolated guinea-pig distal colon are independent of 5-HT in enteric neurons or the mucosa, and do not require the activation of 5-HT3 or 5-HT4 receptors.

By what mechanism does ondansetron inhibit colonic migrating motor complexes: does it require endogenous serotonin in the gut wall?

5‐HT3 antagonists, such as ondansetron (Zofran), retard colonic transit and provide effective relief of symptoms of chronic diarrhea and diarrhea‐predominant irritable bowel syndrome (IBS), but the mechanism by which ondansetron retards transit is unclear. What is clear is that the frequency of colonic migrating motor complexes (CMMCs) is reduced by ondansetron, which could account for reduced transit. Our aim was to determine whether an acute depletion of 5‐HT from enteric neurons would inhibit spontaneous CMMCs; and determine whether the sensitivity of ondansetron to reduce CMMC frequency would change in a 5‐HT‐depleted preparation.

Control of intrinsic pacemaker frequency and velocity of colonic migrating motor complexes in mouse

The mechanisms that control the frequency and propagation velocity of colonic migrating motor complexes (CMMCs) in mammals are poorly understood. Previous in vitro studies on whole mouse colon have shown that CMMCs occur frequently (~every 1–3 min) when the colon is devoid of all fecal content. Consequently, these studies have concluded that the generation of CMMCs and the frequency which they occur does not require the presence of fecal content in the lumen. However, in these studies, stimuli have always been unavoidably applied to these empty colonic preparations, facilitating recordings of CMMC activity. We tested whether CMMCs still occur in empty whole colonic preparations, but when conventional recording methods are not used. To test this, we used video imaging, but did not utilize standard recording methods. In whole isolated colons containing multiple endogenous fecal pellets, CMMCs occurred frequently (1.9 ± 0.1/min) and propagated at 2.2 ± 0.2 mm/s. Surprisingly, when these preparations had expelled all content, CMMCs were absent in 11/24 preparations. In the remaining preparations, CMMCs occurred rarely (0.18 ± 0.02/min) and at reduced velocities (0.71 ± 0.1 mm/s), with reduced extent of propagation. When conventional recording techniques were then applied to these empty preparations, CMMC frequency significantly increased, as did the extent of propagation and velocity. We show that in contrast to popular belief, CMMCs either do not occur when the colon is free of luminal contents, or, they occur at significantly lower frequencies. We believe that previous in vitro studies on empty segments of whole mouse colon have consistently demonstrated CMMCs at high frequencies because conventional recording techniques stimulate the colon. This study shows that CMMCs are normally absent, or infrequent in an empty colon, but their frequency increases substantially when fecal content is present, or, if in vitro techniques are used that stimulate the intestine.

Neurally mediated propagating discrete clustered contractions superimposed on myogenic ripples in ex vivo segments of human ileum.

Narrow muscle strips have been extensively used to study intestinal contractility. Larger specimens from laboratory animals have provided detailed understanding of mechanisms that underlie patterned intestinal motility. Despite progress in animal tissue, investigations of motor patterns in large, intact specimens of human gut ex vivo have been sparse. In this study, we tested whether neurally dependent motor patterns could be detected in isolated specimens of intact human ileum. Specimens (n = 14; 7-30 cm long) of terminal ileum were obtained with prior informed consent from patients undergoing colonic surgery for removal of carcinomas. Preparations were set up in an organ bath with an array of force transducers, a fiberoptic manometry catheter, and a video camera. Spontaneous and distension-evoked motor activity was recorded, and the effects of lidocaine, which inhibits neural activity, were studied. Myogenic contractions (ripples) occurred in all preparations (6.17 ± 0.36/min). They were of low amplitude and formed complex patterns by colliding and propagating in both directions along the specimen at anterograde velocities of 4.1 ± 0.3 mm/s and retrogradely at 4.9 ± 0.6 mm/s. In five specimens, larger amplitude clusters of contractions were seen (discrete clustered contractions), which propagated aborally at 1.05 ± 0.13 mm/s and orally at 1.07 ± 0.09 mm/s. These consisted of two to eight phasic contractions that aligned with ripples. These motor patterns were abolished by addition of lidocaine (0.3 mM). The ripples continued unchanged in the presence of this neural blocking agent. These results demonstrate that both myogenic and neurogenic motor patterns can be studied in isolated specimens of human small intestine.

High-resolution colonic motility recordings in vivo compared with ex vivo recordings after colectomy, in patients with slow transit constipation.

The pathogenesis of slow transit constipation (STC) remains poorly understood, with intrinsic and extrinsic abnormalities implicated. Here, we present high‐resolution colonic manometry recordings from four STC patients recorded before total colectomy, and subsequently, ex vivo, after excision.

Peristalsis and propulsion of colonic content can occur after blockade of major neuroneuronal and neuromuscular transmitters in isolated guinea pig colon

We recently identified hexamethonium-resistant peristalsis in the guinea pig colon. We showed that, following acute blockade of nicotinic receptors, peristalsis recovers, leading to normal propagation velocities of fecal pellets along the colon. This raises the fundamental question: what mechanisms underlie hexamethonium-resistant peristalsis? We investigated whether blockade of the major receptors that underlie excitatory neuromuscular transmission is required for hexamethonium-resistant peristalsis. Video imaging of colonic wall movements was used to make spatiotemporal maps and determine the velocity of peristalsis. Propagation of artificial fecal pellets in the guinea pig distal colon was studied in hexamethonium, atropine, ω-conotoxin (GVIA), ibodutant (MEN-15596), and TTX. Hexamethonium and ibodutant alone did not retard peristalsis. In contrast, ω-conotoxin abolished peristalsis in some preparations and reduced the velocity of propagation in all remaining specimens. Peristalsis could still occur in some animals in the presence of hexamethonium + atropine + ibodutant + ω-conotoxin. Peristalsis never occurred in the presence of TTX. The major finding of the current study is the unexpected observation that peristalsis can occur after blockade of the major excitatory neuroneuronal and neuromuscular transmitters. Also, the colon retained an intrinsic polarity in the presence of these antagonists and was only able to expel pellets in an aboral direction. The nature of the mechanism(s)/neurotransmitter(s) that generate(s) peristalsis and facilitate(s) natural fecal pellet propulsion, after blockade of major excitatory neurotransmitters, at the neuroneuronal and neuromuscular junction remains to be identified.