Search

Background: Acetylcholine is the main excitatory neurotransmitter in the enteric nervous system (ENS) in all animal models examined so far. However, data for the human ENS is scarce., Methods: We used neuroimaging using voltage and calcium dyes, Ussing chamber, and immunohistochemistry to study fast synaptic neurotransmission in submucosal plexus neurons of the human gut., Key Results: Electrical stimulation of intraganglionic fiber tracts led to fast excitatory postsynaptic potentials (fEPSPs) in 29 submucosal neurons which were all blocked by the nicotinic antagonist hexamethonium. The nicotinic agonist DMPP mimicked the effects of electrical stimulation and had excitatory effects on 56 of 73 neurons. The unselective NMDA antagonist MK-801 blocked fEPSPs in 14 out of 22 neurons as well as nicotine evoked spike discharge. In contrast, the application of NMDA showed only weak effects on excitability or calcium transients. This agreed with the finding that the specific NMDA antagonist D-APV reduced fEPSPs in only 1 out of 40 neurons. Application of AMPA or kainite had no effect in 41 neurons or evoked spike discharge in only one out of 41 neurons, respectively. Immunohistochemistry showed that 98.7 ± 2.4% of all submucosal neurons (n = 6 preparations, 1003 neurons) stained positive for the nicotinic receptor (α 1 , α 2 or α 3 -subunit). Hexamethonium (200 µM) reduced nerve-evoked chloride secretion by 34.3 ± 18.6% (n = 14 patients), whereas D-APV had no effect., Conclusion & Inference: Acetylcholine is the most important mediator of fast excitatory postsynaptic transmission in human submucous plexus neurons whereas glutamatergic fEPSPs were rarely encountered., (© 2021 The Authors. Neurogastroenterology & Motility published by John Wiley & Sons Ltd.)

Background: Muscarinic receptor 1 positive allosteric modulators (M1PAMs) enhance colonic propulsive contractions and defecation through the facilitation of M1 receptor (M1R)-mediated signaling. We examined M1R expression in the colons of 5 species and compared colonic propulsion and defecation caused by the M1PAM, T440, the 5-HT 4 agonist, prucalopride, and the cholinesterase inhibitor, neostigmine, in rats and dogs., Methods: M1R expression was profiled by immunostaining and in situ hybridization. In vivo studies utilized male SD rats and beagle dogs. Colonic propulsive contractions were recorded by manometry in anesthetized rats. Gut contractions in dogs were assessed using implanted force transducers in the ileum, proximal, mid, and distal colons., Key Results: M1R was localized to neurons of myenteric and submucosal plexuses and the epithelium of the human colon. A similar receptor localization was observed in rat, dog, mouse, and pig. T440 enhanced normal defecation in rats in a dose-dependent manner. Prucalopride also enhanced defecation in rats, but the maximum effect was half that of T440. Neostigmine and T440 were similarly effective in enhancing defecation, but the effective dose of neostigmine was close to its lethal dose. In rats, all 3 compounds induced colonic contractions, but the associated propulsion was strongest with T440. In dogs, intestinal contractions elicited by T440 propagated from ileum to distal colon. Prucalopride and neostigmine also induced intestinal contractions, but these were less well coordinated. No loss of effectiveness of T440 on defecation occurred after 5 days of repeated dosing., Conclusion and Inferences: These results suggest that M1PAMs produce highly coordinated propagating contraction by actions on the enteric nervous system of the colon. The localization of M1R to enteric neurons in both animals and humans suggests that the M1PAM effects would be translatable to human. M1PAMs provide a potential novel therapeutic option for constipation disorders., (© 2020 John Wiley & Sons Ltd.)

Background: 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., Methods: Mice were injected with reserpine, 24 h prior to euthanasia to deplete neuronally synthesized 5-HT. Mechanical recordings were made from proximal and mid-distal regions of isolated whole mouse colon. Immunohistochemical staining for 5-HT was used to detect neuronal 5-HT., Key Results: Reserpine depleted all detectable 5-HT from enteric nerves. In whole colons, with mucosa and submucosal plexus removed, the frequency and amplitude of spontaneous CMMCs was not different between groups treated with or without reserpine. Surprisingly, in mucosa and submucosal plexus-free preparations, ondansetron was equally or significantly more effective at inhibiting CMMC frequency compared with control preparations (containing 5-HT). Reserpine pretreatment had no effect on the sensitivity of ondansetron to inhibit CMMCs., Conclusions & Inferences: Endogenous 5-HT in enteric neurons (or the mucosa) is not required for the spontaneous generation or propagation of CMMCs. Furthermore, the primary mechanism by which ondansetron inhibits CMMC frequency is not mediated via the mucosa, submucosal plexus or 5-HT in myenteric neurons., (© 2013 John Wiley & Sons Ltd.)

Background: Beneficial effects of ginger in the treatment of gastrointestinal (GI) problems and chemotherapy-induced nausea and vomiting are well accepted. In rodents, the action of ginger seems to be mediated by the inhibition of 5-HT3 receptors, which are established targets to combat emesis and irritable bowel syndrome., Methods: Heterologously expressed human 5-HT3 A or 5-HT3 AB receptors were characterized by means of Ca(2+) influx studies using HEK293 cells. Complementing Ca(2+) measurements in Fluo-4-AM-stained whole-mount preparations of the human submucous plexus were carried out. Furthermore, [3H]GR65630 binding assays were performed to reveal the mode of action of ginger and its pungent compounds., Key Results: We show for the first time that ginger extracts and its pungent arylalkane constituents concentration-dependently inhibit activation of human 5-HT3 receptors. Ginger extracts inhibited both receptors with increasing content of pungent compounds, confirming that these are part of ginger's active principle. Inhibition potencies of the arylalkanes 6-gingerol and 6-shogaol on both receptors were in the low micromolar range. A lipophilic ginger extract and 6-gingerol had no influence on 5-HT potency, but reduced the 5-HT maximum effect, indicating non-competitive inhibition. The non-competitive action was confirmed by [(3) H]GR65630 binding, showing that the ginger extract did not displace the radioligand from 5-HT3 A and 5-HT3 AB receptors. The potential relevance of the inhibitory action of ginger on native 5-HT3 receptors in the gut was confirmed in whole-mount preparations of the human submucous plexus. While a general neurotoxic effect of 6-gingerol was ruled out, it inhibited the 2-methyl-5-HT-mediated activation of 5-HT3 receptors residing on enteric neurons., Conclusions & Inferences: Our findings may encourage the use of ginger extracts to alleviate nausea in cancer patients receiving chemotherapy and to treat functional GI disorders., (© 2013 Blackwell Publishing Ltd.)

The role of the human enteric nervous system (ENS) in the control of the intestinal epithelium organization and proliferation is unknown. To address this issue, we developed a novel co-culture model, consisting of human submucosa containing the submucosal plexus and a human colonic epithelial monolayer. After 3 days in basal conditions (i.e. in absence of neuronal activation) epithelium disorganization and proliferation occurred. In contrast, electrical activation of submucosal neurones maintained monolayer organization and decreased cell proliferation. These effects were blocked by tetrodotoxin and a vasoactive intestinal peptide (VIP) receptor antagonist, and reproduced by VIP. In conclusion, our study suggests that the human ENS is involved in the control of epithelial cell proliferation.

Intracellular recording methods were used to investigate actions of glutamate on morphologically identified neurones in the myenteric and submucous plexuses of guinea-pig small intestine. Glutamate evoked a tetrodotoxin-resistant, slowly activating depolarizing response in most of the submucous neurones (86 of 125, 69%) and a smaller number of myenteric neurones (6 of 60, 10%). The depolarizing responses were restricted to S-type neurones with uniaxonal morphology. The group I metabotropic glutamate receptor (mGluRs) agonists quisqualate, 1S, 3R-ACPD and DHPG mimicked the depolarizing action of glutamate. A group I mGluRs antagonist, S-4-carboxyphenylglycine (S-4CPG), suppressed the glutamate responses with an IC50 of 357 microM at 30 microM glutamate. Group II or III mGluRs agonists did not produce depolarizing responses and group II or III mGluRs antagonists did not alter glutamate-evoked depolarization. The ionotropic glutamate receptor (iGluRs) agonists NMDA, AMPA, or kainate did not evoke depolarizing responses and glutamate-evoked depolarization was unaffected by the iGluRs antagonists D-APV, MK-801, or DNQX. No rapidly activating fast depolarizing responses reminiscent of fast excitatory postsynaptic potentials (EPSPs) were ever observed during application of glutamate or AMPA and stimulus-evoked fast EPSPs were unaffected by DNQX. The results suggest that the excitatory action of glutamate on enteric neurones is mediated by group I metabotropic glutamate receptors and that ionotropic glutamate receptors are not involved. The results also suggest that glutamate-mediated fast EPSPs may not be present in myenteric and submucous neurones in guinea-pig small bowel.