David C. Bulmer

Essential role for TRPV1 in stress‐induced (mast cell‐dependent) colonic hypersensitivity in maternally separated rats

Abstract  Irritable bowel syndrome is in part characterized by an increased sensitivity to colonic distension. Stress is an important trigger factor for symptom generation. We hypothesized that stress induces visceral hypersensitivity via mast cell degranulation and transient receptor ion channel 1 (TRPV1) modulation. We used the rat model of neonatal maternal separation (MS) to investigate this hypothesis. The visceromotor response to colonic distention was assessed in adult MS and non‐handled (NH) rats before and after acute water avoidance (WA) stress. We evaluated the effect of the mast cell stabilizer doxantrazole, neutralizing antiserum against the mast cell mediator nerve growth factor (NGF) and two different TRPV1 antagonists; capsazepine (non‐specific) and SB‐705498 (TRPV1‐specific). Immunohistochemistry was used to assess post‐WA TRPV1 expression in dorsal root ganglia and the presence of immunocytes in proximal and distal colon. Retrograde labelled and microdissected dorsal root ganglia sensory neurons were used to evaluate TRPV1 gene transcription. Results showed that acute stress induces colonic hypersensitivity in MS but not in NH rats. Hypersensitivity was prevented by prestress administration of doxantrazole and anti‐NGF. Capsazepine inhibited and SB‐705498 reversed poststress hypersensitivity. In MS rats, acute stress induced a slight increase in colonic mast cell numbers without further signs of inflammation. Post‐WA TRPV1 transcription and expression was not higher in MS than NH rats. In conclusion, the present data on stress‐induced visceral hypersensitivity confirm earlier reports on the essential role of mast cells and NGF. Moreover, the results also suggest that TRPV1 modulation (in the absence of overt inflammation) is involved in this response. Thus, mast cells and TRPV1 are potential targets to treat stress‐induced visceral hypersensitivity.

5-HT(2B) receptors modulate visceral hypersensitivity in a stress-sensitive animal model of brain-gut axis dysfunction.

Background  Irritable bowel syndrome (IBS) is associated with an enhanced perception to visceral stimuli and exaggerated stress response. The serotonergic neurotransmitter system has been strongly implicated as a key player in the manifestation of IBS symptomatology including visceral hypersensitivity. However the role of 5‐HT2B receptors in visceral pain, although speculated, is currently unclear. Thus we assessed the impact of a selective 5‐HT2B receptor antagonist, RS‐127445, on visceral hypersensitivity in a model of brain gut axis dysfunction the Wistar Kyoto (WKY) rat.

Selective α7 nicotinic acetylcholine receptor agonists worsen disease in experimental colitis

Background and purpose:  In various models vagus nerve activation has been shown to ameliorate intestinal inflammation, via nicotinic acetylcholine receptors (nAChRs) expressed on immune cells. As the α7 nAChR has been put forward to mediate this effect, we studied the effect of nicotine and two selective α7 nAChR agonists (AR‐R17779, (‐)‐spiro[1‐azabicyclo[2.2.2] octane‐3,5′‐oxazolidin‐2′‐one and GSK1345038A) on disease severity in two mouse models of experimental colitis.

Multiple roles for NaV1.9 in the activation of visceral afferents by noxious inflammatory, mechanical, and human disease-derived stimuli.

&NA; NaV1.9 regulates normal colonic afferent mechanosensation and is required for hypersensitivity to noxious inflammatory mediators and those derived from inflammatory bowel disease tissues. &NA; Chronic visceral pain affects millions of individuals worldwide and remains poorly understood, with current therapeutic options constrained by gastrointestinal adverse effects. Visceral pain is strongly associated with inflammation and distension of the gut. Here we report that the voltage‐gated sodium channel subtype NaV1.9 is expressed in half of gut‐projecting rodent dorsal root ganglia sensory neurons. We show that NaV1.9 is required for normal mechanosensation, for direct excitation and for sensitization of mouse colonic afferents by mediators from inflammatory bowel disease tissues, and by noxious inflammatory mediators individually. Excitatory responses to ATP or PGE2 were substantially reduced in NaV1.9−/− mice. Deletion of NaV1.9 substantially attenuates excitation and subsequent mechanical hypersensitivity after application of inflammatory soup (IS) (bradykinin, ATP, histamine, PGE2, and 5HT) to visceral nociceptors located in the serosa and mesentery. Responses to mechanical stimulation of mesenteric afferents were also reduced by loss of NaV1.9, and there was a rightward shift in stimulus–response function to ramp colonic distension. By contrast, responses to rapid, high‐intensity phasic distension of the colon are initially unaffected; however, run‐down of responses to repeat phasic distension were exacerbated in NaV1.9−/− afferents. Finally colonic afferent activation by supernatants derived from inflamed human tissue was greatly reduced in NaV1.9−/− mice. These results demonstrate that NaV1.9 is required for persistence of responses to intense mechanical stimulation, contributes to inflammatory mechanical hypersensitivity, and is essential for activation by noxious inflammatory mediators, including those from diseased human bowel. These observations indicate that NaV1.9 represents a high‐value target for development of visceral analgesics.

Human visceral afferent recordings: preliminary report

Background Conditions characterised by chronic visceral pain represent a significant healthcare burden with limited treatment options. While animal models have provided insights into potential mechanisms of visceral nociception and identified candidate drug targets, these have not translated into successful treatments in humans. Objective To develop an in vitro afferent nerve preparation using surgically excised freshly isolated human colon and vermiform appendix-mesentery tissues. Methods Non-inflamed appendix (n=18) and colon (n=9) were collected from patients undergoing right and left hemicolectomy. Electrophysiological recordings were made from mesenteric nerves and the tissue stimulated chemically and mechanically. Results Ongoing neuronal activity was sparse and where units occurred peak firing rates were: colon (2.0±0.4 spikes/s, n=4) and appendix (2.4±0.6 spikes/s, n=9). Afferent nerves innervating the appendix responded with a significant increase in activity following stimulation with inflammatory mediators (73±10.6 vs 3.0±0.3 spikes/s, n=6, p<0.001, inflammatory mediator vs baseline) and capsaicin (63±15.8 vs 2±0.3 spikes/s, n=3, p<0.001, capsaicin vs buffer). Afferent nerves innervating the colon responded with increased activity to blunt probing of the serosal surface. Conclusions This first-in-human study demonstrates afferent nerve recordings from human gut tissue ex vivo and shows that tissue may be stimulated both chemically and mechanically to study neuronal responses. Collectively, the results provide preliminary evidence to validate this in vitro human tissue model as one that may aid future disease mechanistic studies and candidate drug testing.

Mechanisms of activation of mouse and human enteroendocrine cells by nutrients

Objective Inhibition of food intake and glucose homeostasis are both promoted when nutrients stimulate enteroendocrine cells (EEC) to release gut hormones. Several specific nutrient receptors may be located on EEC that respond to dietary sugars, amino acids and fatty acids. Bypass surgery for obesity and type II diabetes works by shunting nutrients to the distal gut, where it increases activation of nutrient receptors and mediator release, but cellular mechanisms of activation are largely unknown. We determined which nutrient receptors are expressed in which gut regions and in which cells in mouse and human, how they are associated with different types of EEC, how they are activated leading to hormone and 5-HT release. Design and results mRNA expression of 17 nutrient receptors and EEC mediators was assessed by quantitative PCR and found throughout mouse and human gut epithelium. Many species similarities emerged, in particular the dense expression of several receptors in the distal gut. Immunolabelling showed specific colocalisation of receptors with EEC mediators PYY and GLP-1 (L-cells) or 5-HT (enterochromaffin cells). We exposed isolated proximal colonic mucosa to specific nutrients, which recruited signalling pathways within specific EEC extracellular receptor-regulated kinase (p-ERK) and calmodulin kinase II (pCAMKII), as shown by subsequent immunolabelling, and activated release of these mediators. Aromatic amino acids activated both pathways in mouse, but in humans they induced only pCAMKII, which was colocalised mainly with 5-HT expression. Activation was pertussis toxin-sensitive. Fatty acid (C12) potently activated p-ERK in human in all EEC types and evoked potent release of all three mediators. Conclusions Specific nutrient receptors associate with distinct activation pathways within EEC. These may provide discrete, complementary pharmacological targets for intervention in obesity and type II diabetes.

A distinct subset of submucosal mast cells undergoes hyperplasia following neonatal maternal separation: a role in visceral hypersensitivity?

We recently read with great interest the article published by Barreau et al ( Gut 2008; 57 :582–90) who report, as one of their primary findings, mast cell hyperplasia and increased colonic rat mast cell protease II (RMCPII) activity following neonatal psychological stress. The authors also persuasively demonstrate a potential role for mast cell-derived nerve growth factor in contributing to increased colonic neural density in this model. In the study we describe here, using a related maternal separation procedure1 we also observed mast cell hyperplasia. However, this was restricted to the colonic submucosa and characterised by RMCPII immunoreactivity and predominantly blue or blue/red positive cells following Alcian blue/safranin staining, without an associated change in mucosal mast cell (MMC) number. Maternal separation was carried out using a protocol previously described by our group and known to induce a number of behavioural and gastrointestinal (GI) effects,1 and adult male animals, at least 11 weeks of age, were used in subsequent studies. To identify mast cells we used sheep anti-RMCP II (1:500; Moredun, Midlothian, UK) and Alcian blue (1% in 0.7 mol/l HCl)/safranin (0.5% in 0.125 mol/l HCl) staining. Analysis of colonic supernatants for RMCPII release was carried out using an enzyme-linked immunosorbent assay (ELISA; Moredun). Similarly to Barreau et al we identified extensive RMCPII positive staining along the crypt mucosa axis (non-separated (NS), 49.5 (SEM 3.7) mast cells/mm2, n = 10, maternally separated (MS), 53.1 (SEM 2.5) mast cells/mm2, n = 15, p>0.05) in rat colon; though …

Anti-emetic and emetic effects of erythromycin in Suncus murinus: Role of vagal nerve activation, gastric motility stimulation and motilin receptors

Paradoxically, erythromycin is associated with nausea when used as an antibiotic but at lower doses erythromycin activates motilin receptors and is used to treat delayed gastric emptying and nausea. The aim of this study was to characterise pro- and anti-emetic activity of erythromycin and investigate mechanisms of action. Japanese House musk shrews (Suncus murinus) were used. Erythromycin was administered alone or prior to induction of emesis with abnormal motion or subcutaneous nicotine (10mg/kg). The effects of erythromycin and motilin on vagal nerve activity and on cholinergically mediated contractions of the stomach (evoked by electrical field stimulation) were studied in vitro. The results showed that erythromycin (1 and 5mg/kg) reduced vomiting caused by abnormal motion (e.g., from 10.3 ± 1.8 to 4.0 ± 1.1 emetic episodes at 5mg/kg) or by nicotine (from 9.5 ± 2.0 to 3.1 ± 2.0 at 5mg/kg), increasing latency of onset to emesis; lower or higher doses had no effects. When administered alone, erythromycin 100mg/kg induced vomiting in two of four animals, whereas lower doses did not. In vitro, motilin (1, 100 nM) increased gastric vagal afferent activity without affecting jejunal afferent mesenteric nerve activity. Cholinergically mediated contractions of the stomach (prevented by tetrodotoxin 1 μM or atropine 1 μM, facilitated by l-NAME 300 μM) were facilitated by motilin (1-100 nM) and erythromycin (10-30 μM). In conclusion, low doses of erythromycin have anti-emetic activity. Potential mechanisms of action include increased gastric motility (overcoming gastric stasis) and/ or modulation of vagal nerve pathways involved in emesis, demonstrated by first-time direct recording of vagal activation by motilin.

P2Y Receptors Sensitize Mouse and Human Colonic Nociceptors

Activation of visceral nociceptors by inflammatory mediators contributes to visceral hypersensitivity and abdominal pain associated with many gastrointestinal disorders. Purine and pyrimidine nucleotides (e.g., ATP and UTP) are strongly implicated in this process following their release from epithelial cells during mechanical stimulation of the gut, and from immune cells during inflammation. Actions of ATP are mediated through both ionotropic P2X receptors and metabotropic P2Y receptors. P2X receptor activation causes excitation of visceral afferents; however, the impact of P2Y receptor activation on visceral afferents innervating the gut is unclear. Here we investigate the effects of stimulating P2Y receptors in isolated mouse colonic sensory neurons, and visceral nociceptor fibers in mouse and human nerve-gut preparations. Additionally, we investigate the role of Nav1.9 in mediating murine responses. The application of UTP (P2Y2 and P2Y4 agonist) sensitized colonic sensory neurons by increasing action potential firing to current injection and depolarizing the membrane potential. The application of ADP (P2Y1, P2Y12, and P2Y13 agonist) also increased action potential firing, an effect blocked by the selective P2Y1 receptor antagonist MRS2500. UTP or ADP stimulated afferents, including mouse and human visceral nociceptors, in nerve-gut preparations. P2Y1 and P2Y2 transcripts were detected in 80% and 56% of retrogradely labeled colonic neurons, respectively. Nav1.9 transcripts colocalized in 86% of P2Y1-positive and 100% of P2Y2-positive colonic neurons, consistent with reduced afferent fiber responses to UTP and ADP in Nav1.9−/− mice. These data demonstrate that P2Y receptor activation stimulates mouse and human visceral nociceptors, highlighting P2Y-dependent mechanisms in the generation of visceral pain during gastrointestinal disease. SIGNIFICANCE STATEMENT Chronic visceral pain is a debilitating symptom of many gastrointestinal disorders. The activation of pain-sensing nerves located in the bowel wall and their sensitization to physiological stimuli, including bowel movements, underpins the development of such pain, and is associated with mediators released during disease. This work addresses the unstudied role of purine and pyrimidine nucleotides in modulating colonic nociceptors via P2Y receptors using a combination of electrophysiological recordings from human ex vivo samples and a detailed functional study in the mouse. This is the first report to identify colonic purinergic signaling as a function of P2Y receptor activation, in addition to established P2X receptor activity, and the results contribute to our understanding of the development of visceral pain during gastrointestinal disease.

Achieving translation in models of visceral pain.

The failure of drugs to modify pain end points in clinical trials for irritable bowel syndrome (IBS) highlights the knowledge gap that exists in the translation of efficacy in animal models of visceral pain into the clinic. Recent progress has been made towards improving the translation of visceral pain, particularly with regard to the activation of the sensory nerves which relay pain from the gut to the brain. This review will focus on studies which have identified the presence of an altered gastrointestinal and immune environment in IBS patients. The development of human gastrointestinal visceral afferent recordings has allowed direct comparison between sensory nerve studies in animals and human, as well as important advances in our understanding of the ion channels that underpin the changes in sensory nerve excitability.