Histamine-mediated potentiation of transient receptor potential (TRP) ankyrin 1 and TRP vanilloid 4 signaling in submucosal neurons in patients with irritable bowel syndrome.

Previously, we showed histamine-mediated sensitization of transient receptor potential (TRP) vanilloid 1 (TRPV1) in patients with irritable bowel syndrome (IBS). Sensitization of TRP ankyrin 1 (TRPA1) and TRP vanilloid 4 (TRPV4) are also involved in aberrant pain perception in preclinical models of somatic pain. Here, we hypothesize that in parallel with TRPV1, histamine sensitizes TRPA1 and TRPV4, contributing to increased visceral pain in patients with IBS. Rectal biopsies were collected from patients with IBS and healthy subjects (HS) to study neuronal sensitivity to TRPA1 and TRPV4 agonists (cinnamaldehyde and GSK1016790A) using intracellular Ca2+ imaging. In addition, the effect of supernatants of rectal biopsies on patients with IBS and HS was assessed on TRPA1 and TRPV4 responses in murine dorsal root ganglion (DRG) sensory neurons. Finally, we evaluated the role of histamine and histamine 1 receptor (H1R) in TRPA1 and TRPV4 sensitization. Application of TRPA1 and TRPV4 agonists evoked significantly higher peak amplitudes and percentage of responding submucosal neurons in biopsies of patients with IBS compared with HS. In HS, pretreatment with histamine significantly increased the Ca2+ responses to cinnamaldehyde and GSK1016790A, an effect prevented by H1R antagonism. IBS supernatants, but not of HS, sensitized TRPA1 and TRPV4 on DRG neurons. This effect was reproduced by histamine and prevented by H1R antagonism. We demonstrate that the mucosal microenvironment in IBS contains mediators, such as histamine, which sensitize TRPV4 and TRPA1 via H1R activation, most likely contributing to increased visceral pain perception in IBS. These data further underscore H1R antagonism as potential treatment for IBS. NEW & NOTEWORTHY We provide evidence for histamine-mediated transient receptor potential (TRP) ankyrin 1 and TRP vanilloid 4 sensitization in irritable bowel syndrome (IBS) via histamine 1 receptor (H1R) activation, most likely contributing to increased visceral pain perception. Our results reveal a general role of sensory TRP channels as histamine effectors in the pathophysiology of IBS and provide novel mechanistic insights into the therapeutic potential of H1R antagonism in IBS.

Axon reflexes evoked by transient receptor potential vanilloid 1 activation are mediated by tetrodotoxin-resistant voltage-gated Na+ channels in intestinal afferent nerves.

Capsaicin-sensitive nerves mediate axon vasodilator reflexes in the intestine, but the ion channels underlying action potential (AP) propagation are poorly understood. To examine the role of voltage-gated Na(+) channels underlying these reflexes, we measured vasomotor and electrophysiological responses elicited by capsaicin in guinea pig and mouse dorsal root ganglia (DRG) neurons, submucosal arterioles, and mesenteric arteries in vitro. Transient receptor potential vanilloid 1 (TRPV1) agonists dilated guinea pig ileal submucosal arterioles and were blocked by capsazepine and ruthenium red. In double-chamber baths, capsaicin-evoked activation of TRPV1 on proximal perivascular nerves in the left chamber evoked dilations of the distal segment of the submucosal arteriole in the right chamber. Dilations were tetrodotoxin (TTX) (1 microM)-resistant, but reducing extracellular Na(+) (10% solution) or applying the Na(v) 1.8 antagonist A-803467 [5-(4-chlorophenyl-N-(3,5-dimethoxyphenyl)furan-2-carboxamide] (1 microM) in the proximal chamber blocked capsaicin-evoked dilations in the distal chamber (88%; P = 0.01 and 75% and P < 0.02, respectively). In mouse mesenteric arteries, electrical field stimulation and capsaicin (2 microM) evoked dilations that were also TTX-resistant. In perforated patch-clamp recordings, APs in mouse and guinea pig capsaicin-sensitive DRG neurons were TTX-resistant but blocked by 10% extracellular Na(+). When capsaicin-evoked AP conduction was studied in in vitro ileal multiunit afferent nerve preparations, capsaicin responses were elicited in the presence of TTX, whereas distention-evoked responses were almost completely blocked by TTX. Together, these data provide evidence for TTX-resistant AP conduction in extrinsic sensory neurons that innervate guinea pig and mouse intestine and suggest this neural propagation is sufficient to mediate axon reflexes in the intestine.

Opportunity costs of gastrointestinal endoscopic training in Canada.

BACKGROUND: No data exist to define the opportunity costs related to instruction in endoscopic procedures in Royal College of Physicians and Surgeons of Canada-accredited teaching centres. Academic and institutional administrators expect staff to achieve acceptable performance standards. There is a need to measure some of the effects of training activity in the establishment of such standards. OBJECTIVE: To measure the effect of resident training in colonoscopy on real procedure times and, as a secondary goal, to estimate procedural losses related to the process of training. METHODS: Real procedure times for ambulatory colonoscopy in a single academic, hospital-based endoscopy unit were documented. Times for certified endoscopy instructors functioning solo were compared with times for procedures involving trainees at several levels of colonoscopic experience. Procedural reductions associated with resident training were estimated based on the parameters derived from the results. The analysis was executed retrospectively using prospectively collected data. RESULTS: Resident training prolonged procedure times for ambulatory colonoscopy by 50%. The trainee effect was consistent, although variable in degree, among a variety of endoscopy instructors. Such increased procedure times have the potential to reduce case throughput and endoscopist remuneration. CONCLUSIONS: Resident training in colonoscopy in a Canadian certified training program has significant negative effects on case throughput and endoscopist billings. These factors should be considered in any assessment of performance in similar training environments.

Nutritional profile of rodent diets impacts experimental reproducibility in microbiome preclinical research.

The lack of reproducibility of animal experimental results between laboratories, particularly in studies investigating the microbiota, has raised concern among the scientific community. Factors such as environment, stress and sex have been identified as contributors, whereas dietary composition has received less attention. This study firstly evaluated the use of commercially available rodent diets across research institutions, with 28 different diets reported by 45 survey respondents. Secondly, highly variable ingredient, FODMAP (Fermentable Oligo-, Di-, Mono-saccharides And Polyols) and gluten content was found between different commercially available rodent diets. Finally, 40 mice were randomized to four groups, each receiving a different commercially available rodent diet, and the dietary impact on cecal microbiota, short- and branched-chain fatty acid profiles was evaluated. The gut microbiota composition differed significantly between diets and sexes, with significantly different clusters in ß-diversity. Total BCFA were highest (p = 0.01) and SCFA were lowest (p = 0.03) in mice fed a diet lower in FODMAPs and gluten. These results suggest that nutritional composition of commercially available rodent diets impact gut microbiota profiles and fermentation patterns, with major implications for the reproducibility of results across laboratories. However, further studies are required to elucidate the specific dietary factors driving these changes.

Colon cleansing before colonoscopy: does oral sodium phosphate solution still make sense?

Oral sodium phosphate (NaP) solution has been withdrawn from the market in the United States but remains available for over-the-counter purchase for bowel preparation for colonoscopy in Canada. The present review summarizes recent data regarding the renal toxicity of oral NaP as well as its efficacy and tolerability relative to other preparations. Given the availability of effective alternatives to NaP solution, its use for colonoscopy preparation in Canada should be limited. Candidate patients for oral NaP solution should be assessed for eligibility and preparation instructions should adhere to the current recommendations for maximizing the safety of oral NaP.

Dietary therapies for functional bowel symptoms: Recent advances, challenges, and future directions.

BACKGROUND: Functional gastrointestinal symptoms in irritable bowel syndrome (IBS) and quiescent inflammatory bowel disease (IBD) cause significant morbidity and a reduction in quality of life. Multiple dietary therapies are now available to treat these symptoms, but supporting evidence for many is limited. In addition to a further need for studies demonstrating efficacy and mechanism of action of dietary therapies, the risk of nutritional inadequacy, alterations to the microbiome and changes in quality of life are key concerns requiring elucidation. Identifying predictors of response to dietary therapy is an important goal as management could be tailored to the individual to target specific dietary components, and thereby reduce the level of dietary restriction necessary. PURPOSE: This review discusses the available dietary therapies to treat symptoms in patients with IBS and patients with quiescent IBD suffering from IBS symptoms, with the aim to understand where current dietary evidence lies and how to move forward in dietary research in this field.

Sham acupuncture is as efficacious as true acupuncture for the treatment of IBS: A randomized placebo controlled trial.

BACKGROUND: Irritable bowel syndrome (IBS) patients increasingly seek out acupuncture therapy to alleviate symptoms, but it is unclear whether the benefit is due to a treatment-specific effect or a placebo response. This study examined whether true acupuncture is superior to sham acupuncture in relieving IBS symptoms and whether benefits were linked to purported acupuncture mechanisms. METHODS: A double blind sham controlled acupuncture study was conducted with Rome I IBS patients receiving twice weekly true acupuncture for 4 weeks (n=43) or sham acupuncture (n=36). Patients returned at 12 weeks for a follow-up review. The primary endpoint of success as determined by whether patients met or exceeded their established goal for percentage symptom improvement. Questionnaires were completed for symptom severity scores, SF-36 and IBS-36 QOL tools, McGill pain score, and Pittsburg Sleep Quality Index. A subset of patients underwent barostat measurements of rectal sensation at baseline and 4 weeks. KEY RESULTS: A total of 53% in the true acupuncture group met their criteria for a successful treatment intervention, but this did not differ significantly from the sham group (42%). IBS symptom scores similarly improved in both groups. Scores also improved in the IBS-36, SF-36, and the Pittsburg Sleep Quality Index, but did not differ between groups. Rectal sensory thresholds were increased in both groups following treatment and pain scores decreased; however, these changes were similar between groups. CONCLUSIONS & INFERENCES: The lack of differences in symptom outcomes between sham and true treatment acupuncture suggests that acupuncture does not have a specific treatment effect in IBS.

A role for interleukin 17A in IBD-related neuroplasticity.

BACKGROUND: Changes to the structure and function of the innervation of the gut contribute to symptom generation in inflammatory bowel diseases (IBD). However, delineation of the mechanisms of these effects has proven difficult. Previous work on sympathetic neurons identified interleukin (IL)-17A as a novel neurotrophic cytokine. Since IL-17A is involved in IBD pathogenesis, we tested the hypothesis that IL-17A contributes to neuroanatomical remodeling during IBD. METHODS: Immunohistochemistry for tyrosine hydroxylase was used to identify sympathetic axons in mice with dextran sulphate sodium (DSS)-induced colitis and controls. Axon outgrowth from sympathetic neurons in response to incubation in cytokines or endoscopic patient biopsy supernatants was quantified. KEY RESULTS: DSS-induced colitis led to an increase in tyrosine hydroxylase immunoreactivity in the inflamed colon but not the spleen. Colonic supernatants from mice with colitis and biopsy supernatants from Crohn's disease patients increased axon outgrowth from mouse sympathetic neurons compared to supernatants from uninflamed controls. An antibody that neutralized IL-17A blocked the ability of DSS-induced colitis and Crohn's disease supernatants to induce axon extension. CONCLUSIONS AND INFERENCES: These findings identify IL-17A as a potential mediator of neuroanatomical remodeling of the gut innervation during IBD.

Stress increases descending inhibition in mouse and human colon.

BACKGROUND: A relationship between stress and the symptoms of irritable bowel syndrome (IBS) has been well established but the cellular mechanisms are poorly understood. Therefore, we investigated effects of stress and stress hormones on colonic descending inhibition and transit in mouse models and human tissues. METHODS: Stress was applied using water avoidance stress (WAS) in the animal model or mimicked using stress hormones, adrenaline (5 nM), and corticosterone (1 µM). Intracellular recordings were obtained from colonic circular smooth muscle cells in isolated smooth muscle/myenteric plexus preparations and the inhibitory junction potential (IJP) was elicited by nerve stimulation or balloon distension oral to the site of recording. KEY RESULTS: Water avoidance stress increased the number of fecal pellets compared to control (p < 0.05). WAS also caused a significant increase in IJP amplitude following balloon distension. Stress hormones also increased the IJP amplitude following nerve stimulation and balloon distension (p < 0.05) in control mice but had no effect in colons from stressed mice. No differences were observed with application of ATP between stress and control tissues, suggesting the actions of stress hormones were presynaptic. Stress hormones had a large effect in the nerve stimulated IJP in human colon (increased >50%). Immunohistochemical studies identified alpha and beta adrenergic receptor immunoreactivity on myenteric neurons in human colon. CONCLUSIONS & INFERENCES: These studies suggest that WAS and stress hormones can signal via myenteric neurons to increase inhibitory neuromuscular transmission. This could lead to greater descending relaxation, decreased transit time, and subsequent diarrhea.

Inflammation-induced hyperexcitability of nociceptive gastrointestinal DRG neurones: the role of voltage-gated ion channels.

Gastrointestinal (GI) inflammation modulates the intrinsic properties of nociceptive dorsal root ganglia neurones, which innervate the GI tract and these changes are important in the genesis of abdominal pain and visceral hyperalgesia neurones exhibit hyperexcitability characterized by a decreased threshold for activation and increased firing rate, and changes in voltages-gated Na(+) and K(+) channels play a major role in this plasticity. This review highlights emerging evidence that specific subsets of channels and signalling pathways are involved and their potential to provide novel selective therapeutics targets for the treatment of abdominal pain.

Basic and clinical pharmacology of new motility promoting agents.

Recent research has provided new information about drugs that could be used to treat functional motility disorders. Promotility drugs accelerate gastric emptying or colonic transit and these properties may contribute to their efficacy in treating symptoms associated with gastroparesis, functional dyspepsia or constipation. 5-Hydroxytryptamine4 receptors are targets for drugs (tegaserod, renzapride) that treat symptoms in constipated irritable bowel syndrome patients and in gastroparesis. Drugs acting at motilin (erythromycin) and cholecystokinin-1 (dexloxiglumide) receptors accelerate gastric emptying. Dexloxiglumide might be useful in the treatment of functional dyspepsia particularly that associated with lipid intake. Alvimopan is a mu-opioid receptor antagonist that does not cross the blood brain barrier. Alvimopan is effective in treating postsurgical ileus and perhaps opiate-induced bowel dysfunction. Successes and failures of recent efforts to develop promotility agents revealed opportunities and challenges for developing new promotility drugs. The pharmacological properties of partial agonists might be exploited to develop effective promotility drugs. However, opposing actions of promotility agents on motility (increased contraction vs decreased accommodation) limit the clinical efficacy of drugs with these opposing actions. Selection of appropriate patient populations for evaluation of new drugs is also critical.

Concurrent psychological stress and infectious colitis is key to sustaining enhanced peripheral sensory signaling.

BACKGROUND: The development of postinfectious-irritable bowel syndrome is associated with psychological stress but this relationship is poorly understood. The mouse Citrobacter rodentium model enhances the postinfectious excitability of colonic nociceptors, which can be further amplified by water-avoidance stress (WAS). This study tested whether concurrent infectious colitis and chronic stress enhance and sustain nociceptor excitability more than stress after resolution of infection. METHODS: Male C57 mice were gavaged with C. rodentium. WAS (1 h/day) was performed at different time-points relative to the infection. After the final session of WAS, T9-T13 colonic-projecting DRG neurons were isolated, cultured overnight and patch-clamped to assess excitability. To investigate potential mechanisms, histological damage scores and colonic cytokine production were assessed. KEY RESULTS: WAS more than 30 days after C. rodentium infection produced no greater DRG excitability than WAS in uninfected mice. However, when overlapped with chronic stress (3 sessions of WAS; 7 days before, 9 days during and 9 days after C. rodentium or sham gavage), C. rodentium significantly enhanced DRG excitability vs saline-gavaged chronically stressed mice. Bodyweights and colonic damage scores were unchanged. Both WAS and C. rodentium gavage were found to significantly alter colonic cytokines at postinfection day 30. CONCLUSIONS & INFERENCES: Chronic stress and infectious colitis combine in an additive manner to heighten and prolong the sensitivity of visceral nociceptors. The effect relies on temporal coincidence of stress and infection, does not involve substantial exacerbation of inflammation, and may involve combined direct stress hormone and immune signaling on DRG neurons.

Demonstration of elevated levels of active cathepsin S in dextran sulfate sodium colitis using a new activatable probe.

BACKGROUND: Proteases play a major role in inflammatory diseases of the gastrointestinal tract. Activatable probes are a major technological advance, enabling sensitive detection of active proteases in tissue samples. Our aim was to synthesize an activatable probe for cathepsin S and validate its use in a mouse model of colitis. METHODS: We designed and synthesized a new fluorescent activatable probe, NB200, for the detection of active cathepsin S. Colitis was induced in C57BL/6 mice by the administration of 3% dextran sulfate sodium (DSS). Homogenized mouse colons, with or without the addition of the specific cathepsin S inhibitor MV026031, were incubated with NB200 in a fluorescent plate reader. KEY RESULTS: NB200 selectively detected purified cathepsin S and not other common inflammatory proteases. Homogenates of colon from mice with DSS colitis induced a significant fluorescent increase when compared to control animals (control vs DSS: p < 0.05 at 200 min and p < 0.01 at 220-240 min), indicating cathepsin S activation. The cathepsin S inhibitor abolished this increase in fluorescence (DSS vs DSS + MV026031: p < 0.05 at 140 min, p < 0.01 at 180 min, p < 0.001 at 200-240 min), which confirms cathepsin S activation. Cathepsin S activity correlated with the disease activity index (Spearman r = 0.77, p = 0.017). CONCLUSIONS & INFERENCES: Our investigation has demonstrated the utility of activatable probes for detecting protease activity in intestinal inflammation. Panels of such probes may allow 'signature' protease profiles to be established for a range of inflammatory diseases and disorders.

Dendritic distribution of motoneurons innervating the three heads of the trapezius muscle in the cat.

The organization of the nuclei and dendritic architecture of motoneurons innervating the three heads of the trapezius muscle, clavotrapezius (CT), acromiotrapezius (AT), and spinotrapezius (ST), have been examined by using intracellular staining techniques. CT, AT, and ST motoneurons were found in the spinal accessory nucleus and were arranged in three overlapping subnuclei. CT motoneurons were primarily found in C2 and C3. In contrast, most AT motoneurons were found in C3, C4, and C5 and ST motoneurons were found in C4, C5, and the rostral parts of C6. Most dendrites of CT motoneurons, located in rostral C2, extended dorsally and many of these dendrites spread medially and laterally to encompass all of lamina VIII and the dorsolateral part of lamina VII. When viewed in the horizontal plane these motoneurons had a stellate appearance. The dendritic tree structure of CT motoneurons changed abruptly between rostral C2 and mid-C2. The majority of dendrites of CT motoneurons located in the central and caudal parts of C2 projected rostrally and caudally to form a complex bundle of dendrites in the motoneuron nucleus. Small numbers of dendrites were also found ventromedial and dorsal to the soma. The dendritic trees of CT motoneurons in C3 and C4 and AT and ST motoneurons located in C4 and the rostral parts of C5 also followed this fusiform distribution pattern. The dendritic trees of AT and ST motoneurons in caudal C5 were not fusiform but instead had a complex distribution pattern which consisted of dendrites projecting in several directions. Many dendrites projected rostrally and caudally, and in addition, there were major dendritic projections ventrolateral and dorsolateral to the soma. These results indicate that each head of the trapezius muscle is innervated by two structurally dissimilar groups of motoneurons which occupy different spinal segments. Trapezius motoneurons at the same segmental level, regardless of which head of the trapezius muscle they innervated, have similar dendritic trees whose structure differs from those of neighbouring dorsal neck muscle motoneurons in C2, C3, and C4. Thus, the organization of motoneuron dendritic trees appears to be governed by several factors including the muscle innervated by the motoneuron and the transverse and segmental position of the motoneuron's soma.

A quantitative analysis of the geometry of cat motoneurons innervating neck and shoulder muscles.

The geometry of the somata and dendritic trees of motoneurons innervating neck and shoulder muscles was investigated by using intracellular injections of HRP. In general, these motoneurons did not belong to a homogeneous population of motoneurons. Differences in average primary dendritic diameter, number of primary dendrites, and other measures of dendritic tree size were found between different neck and shoulder motoneuron groups. Several indices of proximal dendritic tree size (number of primary dendrites, sum of dendritic diameters, Rall's dendritic trunk parameter, and the sum of dendritic holes) were weakly correlated with the diameter or surface area of the soma. Some of these correlations depended on the muscle supplied by the motoneuron. The total combined dendritic length ranged from 66,660 to 95,390 microns. There was a weak, but positive, correlation between the diameter of primary dendrites and combined dendritic length. This relationship varied from motoneuron to motoneuron. The diameters of all dendrites of three trapezius motoneurons were examined in detail. The total dendritic surface area examined ranged from 415,000 to 488,100 microns 2 and represented approximately 99% of the total neuronal surface area. Last-order dendrites showed a high degree (39.9%) of taper. Dendritic tapering, by itself, was a major factor in the decrease of the (sum of dendritic diameters)3/2 measured at progressively distal sites from the soma. Although few parent and daughter dendrites obeyed the "three-halves law," the average exponent was 1.57. The diameters of primary dendrites and dendritic surface area were weakly correlated. The correlation between dendritic diameter and combined dendritic length or surface area improved if the weighted average of the diameter of second-order dendrites was used as a measure of dendrite size. Second-order dendrites, whose branches terminated in different regions of the spinal cord, showed different relationships between dendritic diameter and combined dendritic length or surface area. Comparisons between the motoneurons examined in the present study and motoneurons innervating other muscles indicate that, although all spinal motoneurons share several common features (e.g., long dendrites, dendritic tapering), each motoneuron group has a set of unique features (e.g., soma shape, relationship between primary dendrite diameter and dendritic surface area). Thus, the rules governing motoneuron dendritic geometry are not fixed but depend on the species of the motoneuron.

Converging and diverging cholinergic inputs from submucosal neurons amplify activity of secretomotor neurons in guinea-pig ileal submucosa.

The organization of synaptic connections between guinea-pig ileal submucosal neurons was examined using intracellular recordings from single or pairs of submucosal neurons. Synaptic inputs were elicited by stimulating cholinergic neurons using pressure-pulse application of 5-hydroxytryptamine (5-HT) in ganglia adjacent to those where intracellular recordings were obtained. In addition, when pairs of intracellular recordings were obtained, one neuron was activated by intracellular stimulation and synaptic responses were recorded in the other neuron. Neurobiotin-filled microelectrodes were employed to characterize cells electrophysiologically and immunohistochemically. Recordings were obtained from 176 (173 S-type and three AH-type) neurons; 81% of cells were classified as vasoactive intestinal peptide (VIP) neurons. No fast excitatory postsynaptic potentials and only rare slow excitatory postsynaptic potentials were recorded following intracellular stimulation of paired S-type neurons. However, when paired intracellular recordings were obtained from neurons within the same ganglion and 5-HT was applied to an adjacent ganglion, this stimulation evoked synchronized fast excitatory postsynaptic potentials in 94% of pairs. In contrast, when cell bodies of VIP-VIP pairs were located in different ganglia, fast synaptic activation evoked by 5-HT stimulation was not synchronized in 87% of pairs. When intracellular recordings were obtained from a single neuron and two separate ganglia were stimulated by 5-HT pressure-pulse activation, fast excitatory postsynaptic potentials originating from both sources were recorded in the same VIP neuron. Morphological study of 34 S-type and three AH-type horseradish peroxidase-labeled neurons was conducted. AH-type neurons had multiple axonal branches with dense arborization of collaterals containing numerous varicosities in three to nine ganglia, whereas axons of S-type neurons exhibited relatively rare collaterals and varicosities within adjacent ganglia. These results demonstrate that cholinergic neurons provide both diverging and converging inputs to VIP neurons, providing a mechanism to enhance activation of VIP secretomotor neurons. The axonal projections of AH-type neurons suggest they are likely candidates to provide diverging inputs to multiple VIP neurons.

Effects of 5-HT3 receptor antagonists on 5-HT and nicotinic depolarizations in guinea-pig submucosal neurones.

1. Intracellular recordings were made from neurones of the guinea-pig submucosal plexus. The effects of several 5-hydroxytryptamine3 (5-HT3) receptor antagonists on depolarizations produced by ionophoretic application of 5-HT and acetylcholine, as well as on fast excitatory postsynaptic potentials (fast e.p.s.ps) produced by nerve stimulation were examined. 2. ICS 205-930, GR 38032F, MDL 72222, cocaine and curare all inhibited the fast e.p.s.p. as well as the depolarizations in response to 5-HT and acetylcholine (ACh) ionophoresis in a dose-dependent fashion. 3. IC50 values for ICS 205-930, GR 38032F, MDL 72222, cocaine and curare in inhibiting the 5-HT mediated depolarizations were 12 nM, 100 nM, 3 microM, 3 microM and 20 microM, respectively. 4. IC50 values for ICS 205-930, GR 38032F, MDL 72222, cocaine and curare in inhibiting the nicotinic depolarizations were 4 microM, 12 microM, 11 microM, 6 microM and 17 microM, respectively. Similar IC50 values were obtained for inhibition of the fast e.p.s.ps by these antagonists. 5. The nicotinic receptor blocker, hexamethonium, inhibited the nicotinic depolarization and the fast e.p.s.p. with IC50 values of 10 microM. Hexamethonium (10 microM-5 mM) did not alter the depolarization induced by 5-HT. 6. These results demonstrate that the pharmacological profile of 5-HT3 receptors present on submucosal neurones is identical to that of 5-HT3 receptors on myenteric neurones and, thus, provide evidence that the enteric neuronal 5-HT3 receptor forms a receptor subtype distinct from that characterized in other parts of the autonomic nervous system.