Patrick A. Hughes

The ion channel TRPA1 is required for normal mechanosensation and is modulated by algesic stimuli

BACKGROUND & AIMS The transient receptor potential (TRP) channel family includes transducers of mechanical and chemical stimuli for visceral sensory neurons. TRP ankyrin 1 (TRPA1) is implicated in inflammatory pain; it interacts with G-protein-coupled receptors, but little is known about its role in the gastrointestinal (GI) tract. Sensory information from the GI tract is conducted via 5 afferent subtypes along 3 pathways. METHODS Nodose and dorsal root ganglia whose neurons innnervate 3 different regions of the GI tract were analyzed from wild-type and TRPA1(-/-) mice using quantitative reverse-transcription polymerase chain reaction, retrograde labeling, and in situ hybridization. Distal colon sections were analyzed by immunohistochemistry. In vitro electrophysiology and pharmacology studies were performed, and colorectal distension and visceromotor responses were measured. Colitis was induced by administration of trinitrobenzene sulphonic acid. RESULTS TRPA1 is required for normal mechano- and chemosensory function in specific subsets of vagal, splanchnic, and pelvic afferents. The behavioral responses to noxious colonic distension were substantially reduced in TRPA1(-/-) mice. TRPA1 agonists caused mechanical hypersensitivity, which increased in mice with colitis. Colonic afferents were activated by bradykinin and capsaicin, which mimic effects of tissue damage; wild-type and TRPA1(-/-) mice had similar direct responses to these 2 stimuli. After activation by bradykinin, wild-type afferents had increased mechanosensitivity, whereas, after capsaicin exposure, mechanosensitivity was reduced: these changes were absent in TRPA1(-/-) mice. No interaction between protease-activated receptor-2 and TRPA1 was evident. CONCLUSIONS These findings demonstrate a previously unrecognized role for TRPA1 in normal and inflamed mechanosensory function and nociception within the viscera.

Selective role for TRPV4 ion channels in visceral sensory pathways.

BACKGROUND & AIMS Although there are many candidates as molecular mechanotransducers, so far there has been no evidence for molecular specialization of visceral afferents. Here, we show that colonic afferents express a specific molecular transducer that underlies their specialized mechanosensory function: the transient receptor potential channel, vanilloid 4 (TRPV4). METHODS We found TRPV4 mRNA is highly enriched in colonic sensory neurons compared with other visceral and somatic sensory neurons. TRPV4 protein was found in colonic nerve fibers from patients with inflammatory bowel disease, and it colocalized in a subset of fibers with the sensory neuropeptide CGRP in mice. We characterized the responses of 8 subtypes of vagal, splanchnic, and pelvic mechanoreceptors. RESULTS Mechanosensory responses of colonic serosal and mesenteric afferents were enhanced by a TRPV4 agonist and dramatically reduced by targeted deletion of TRPV4 or by a TRP antagonist. Other subtypes of vagal and pelvic afferents, by contrast, were unaffected by these interventions. The behavioral responses to noxious colonic distention were also substantially reduced in mice lacking TRPV4. CONCLUSIONS These data indicate that TRPV4 contributes to mechanically evoked visceral pain, with relevance to human disease. In view of its distribution in favor of specific populations of visceral afferents, we propose that TRPV4 may present a selective novel target for the reduction of visceral pain, which is an important opportunity in the absence of current treatments.

Post-inflammatory colonic afferent sensitisation: different subtypes, different pathways and different time courses.

Objective: Intestinal infection evokes hypersensitivity in a subgroup of patients with irritable bowel syndrome (IBS) long after healing of the initial injury. Trinitrobenzene sulfonic acid (TNBS)-induced colitis in rodents likewise results in delayed maintained hypersensitivity, regarded as a model of some aspects of IBS. The colon and rectum have a complex sensory innervation, comprising five classes of mechanosensitive afferents in the splanchnic and pelvic nerves. Their plasticity may hold the key to underlying mechanisms in IBS. Our aim was therefore to determine the contribution of each afferent class in each pathway towards post-inflammatory visceral hypersensitivity. Design: TNBS was administered rectally and mice were studied after 7 (acute) or 28 (recovery) days. In vitro preparations of mouse colorectum with attached pelvic or splanchnic nerves were used to examine the mechanosensitivity of individual colonic afferents. Results: Mild inflammation of the colon was evident acutely which was absent at the recovery stage. TNBS treatment did not alter proportions of the five afferent classes between treatment groups. In pelvic afferents little or no difference in response to mechanical stimuli was apparent in any class between control and acute mice. However, major increases in mechanosensitivity were recorded from serosal afferents in mice after recovery, while responses from other subtypes were unchanged. Both serosal and mesenteric splanchnic afferents were hypersensitive at both acute and recovery stages. Conclusions: Colonic afferents with high mechanosensory thresholds contribute to inflammatory hypersensitivity, but not those with low thresholds. Pelvic afferents become involved mainly following recovery from inflammation, whereas splanchnic afferents are implicated during both inflammation and recovery.

Sensory neuro-immune interactions differ between Irritable Bowel Syndrome subtypes

Objective The gut is a major site of contact between immune and sensory systems and evidence suggests that patients with irritable bowel syndrome (IBS) have immune dysfunction. Here we show how this dysfunction differs between major IBS subgroups and how immunocytes communicate with sensory nerves. Design Peripheral blood mononuclear cell supernatants from 20 diarrhoea predominant IBS (D-IBS) patients, 15 constipation predominant IBS (C-IBS) patients and 36 healthy subjects were applied to mouse colonic sensory nerves and effects on mechanosensitivity assessed. Cytokine/chemokine concentration in the supernatants was assessed by proteomic analysis and correlated with abdominal symptoms, and expression of cytokine receptors evaluated in colonic dorsal root ganglia neurons. We then determined the effects of specific cytokines on colonic afferents. Results D-IBS supernatants caused mechanical hypersensitivity of mouse colonic afferent endings, which was reduced by infliximab. C-IBS supernatants did not, but occasionally elevated basal discharge. Supernatants of healthy subjects inhibited afferent mechanosensitivity via an opioidergic mechanism. Several cytokines were elevated in IBS supernatants, and levels correlated with pain frequency and intensity in patients. Visceral afferents expressed receptors for four cytokines: IL-1β, IL-6, IL-10 and TNF-α. TNF-α most effectively caused mechanical hypersensitivity which was blocked by a transient receptor potential channel TRPA1 antagonist. IL-1β elevated basal firing, and this was lost after tetrodotoxin blockade of sodium channels. Conclusions Distinct patterns of immune dysfunction and interaction with sensory pathways occur in different patient groups and through different intracellular pathways. Our results indicate IBS patient subgroups would benefit from selective targeting of the immune system.

A novel role for TRPM8 in visceral afferent function

&NA; Transient receptor potential ion channel melastatin subtype 8 (TRPM8) is activated by cold temperatures and cooling agents, such as menthol and icilin. Compounds containing peppermint are reported to reduce symptoms of bowel hypersensitivity; however, the underlying mechanisms of action are unclear. Here we determined the role of TRPM8 in colonic sensory pathways. Laser capture microdissection, quantitative reverse transcription‐polymerase chain reaction (RT‐PCR), immunofluorescence, and retrograde tracing were used to localise TRPM8 to colonic primary afferent neurons. In vitro extracellular single‐fibre afferent recordings were used to determine the effect of TRPM8 channel activation on the chemosensory and mechanosensory function of colonic high‐threshold afferent fibres. TRPM8 mRNA was present in colonic DRG neurons, whereas TRPM8 protein was present on nerve fibres throughout the wall of the colon. A subpopulation (24%, n = 58) of splanchnic serosal and mesenteric afferents tested responded directly to icilin (5 μmol/L). Subsequently, icilin significantly desensitised afferents to mechanical stimulation (P < .0001; n = 37). Of the splanchnic afferents responding to icilin, 21 (33%) also responded directly to the TRPV1 agonist capsaicin (3 μmol/L), and icilin reduced the direct chemosensory response to capsaicin. Icilin also prevented mechanosensory desensitization and sensitization induced by capsaicin and the TRPA1 agonist AITC (40 μmol/L), respectively. TRPM8 is present on a select population of colonic high threshold sensory neurons, which may also co‐express TRPV1. TRPM8 couples to TRPV1 and TRPA1 to inhibit their downstream chemosensory and mechanosensory actions. TRPM8 was localised to high‐threshold visceral afferent neurons. On visceral afferent peripheral endings, TRPM8 activation affected TRPV1 and TRPA1 downstream chemosensory and mechanosensory actions.

TRPA1 contributes to specific mechanically activated currents and sensory neuron mechanical hypersensitivity

Non‐technical summary  Detecting mechanical stimuli is vital to determining our responses to environmental challenges. The speed required for this process suggests ion channels are opened in response to mechanical forces. A specialised membrane protein called the TRPA1 ion channel mediates chemical based pain; however its role in mechanical pain remains unresolved. Here we show that TRPA1 contributes to the detection of mechanical stimuli in a select set of pain sensing neurons. Furthermore, we also show that acute activation of this channel enhances the mechanical responsiveness of these neurons. Finally, we also show that increasing the expression of TRPA1 causes a further enhancement in the mechanical response. These findings suggest that a drug designed to block TRPA1 would be beneficial for the treatment of numerous pathological conditions associated with mechanical pain.

Immune Activation in Irritable Bowel Syndrome: Can Neuroimmune Interactions Explain Symptoms?

Irritable bowel syndrome (IBS) is a functional disorder of the gastrointestinal (GI) tract characterized by pain or discomfort from the lower abdominal region, which is associated with altered bowel habit. Despite its prevalence, there is currently a lack of effective treatment options for patients. IBS has long been considered as a neurological condition resulting from alterations in the brain gut axis, but immunological alterations are increasingly reported in IBS patients, consistent with the hypothesis that there is a chronic, but low-grade, immune activation. Mediators released by immune cells act to either dampen or amplify the activity of GI nerves. Release of a number of these mediators correlates with symptoms of IBS, highlighting the importance of interactions between the immune and the nervous systems. Investigation of the role of microbiota in these interactions is in its early stages, but may provide many answers regarding the mechanisms underlying activation of the immune system in IBS. Identifying what the key changes in the GI immune system are in IBS and how these changes modulate viscerosensory nervous function is essential for the development of novel therapies for the underlying disorder.

Localization and comparative analysis of acid-sensing ion channel (ASIC1, 2, and 3) mRNA expression in mouse colonic sensory neurons within thoracolumbar dorsal root ganglia.

Reducing colonic mechanosensitivity is an important potential strategy for reducing visceral pain. Mice lacking acid‐sensing ion channels (ASIC) 1, 2, and 3 show altered colonic mechanosensory function, implicating ASICs in the mechanotransduction process. Deletion of ASICs affects mechanotransduction in visceral and cutaneous afferents differently, suggesting differential expression. We determined relative expression of ASIC1, 2, and 3 in mouse thoracolumbar dorsal root ganglia (DRG) by quantitative reverse‐transcriptase polymerase chain reaction (RT‐PCR) analysis (QPCR) and specifically in retrogradely traced colonic neurons isolated via laser capture microdissection. Localization of ASIC expression in DRG was determined with fluorescence in situ hybridization (FISH) and retrograde tracing. QPCR of whole thoracolumbar DRG revealed and abundance of ASIC2 > ASIC1 > ASIC3. Similarly, FISH of all neurons in thoracolumbar DRG demonstrated that ASIC2 was expressed in the most (40 ± 1%) neurons, followed by ASIC3 (24 ± 1%), then ASIC1 (18 ± 1%). Retrograde tracing from the distal colon labeled 4 ± 1% of neurons in T10‐L1 DRG. In contrast to whole DRG, FISH of colonic neurons showed ASIC3 expression in 73 ± 2%, ASIC2 in 47 ± 0.5%, and ASIC1 in 30 ± 2%. QPCR of laser captured colonic neurons revealed that ASIC3 was the most abundant ASIC transcript, followed by ASIC1, then ASIC2. We conclude that ASIC1, 2, and 3 are expressed preferentially in colonic neurons within thoracolumbar DRG. In particular ASIC3, the least abundant in the general population, is the most abundant ASIC transcript in colonic neurons. The prevalence of ASIC3 in neurons innervating the colon supports electrophysiological data showing that it makes a major contribution to colonic mechanotransduction and therefore may be a target for the treatment of visceral pain. J. Comp. Neurol. 500:863–875, 2007.

TRP channels: new targets for visceral pain

How often do gastroenterologists advise patients to “avoid spicy food” without really knowing why? The answer may lie in the family of transient receptor potential (TRP) channels, which includes receptors for compounds found in common herbs and spices. This aim of this review is not really to answer this question, but it will shed light on why spices may cause symptoms. Instead, its aim is to explore the TRP family for potential targets that may in fact reduce gut symptoms. Chronic pain and discomfort of unknown origin in functional gastrointestinal disorders represent a large unmet need for treatment and consequent economic impact. There are also features of other gut disease which generate symptoms with obscure origins. In order to understand how symptoms are generated in the gut and transmitted to the central nervous system, we need to know at least three principles of extrinsic sensory nerve function—first, what types are there and what do they signal?; secondly, what is the molecular basis of sensory transduction?; and thirdly, how does all of this change in disease? These questions are a key focus of this article, with a particular emphasis on the role of TRP channels in each case. Although there are no drugs yet available for clinical use that target TRP channels, some of the early pointers are identified that hold promise for their use in pharmacotherapy of gastrointestinal sensory dysfunction. ### Subtypes of sensory fibres Peripheral endings of sensory afferent fibres can be classified into five subtypes in the mouse gastrointestinal tract according to the location of their mechanoreceptive fields.1 2 These are: mucosal, muscular (or tension receptor), muscular–mucosal (or tension–mucosal), serosal and mesenteric afferents (fig 1).1 2 3 4 Mucosal afferents respond exclusively to fine tactile stimulation of the luminal surface. Anatomically, they appear as bare endings in the lamina propria …

Involvement of metabotropic glutamate 5 receptor in visceral pain

&NA; Metabotropic glutamate 5 receptor (mGluR5) antagonists are effective in animal models of inflammatory and neuropathic pain. The involvement of mGluR5 in visceral pain pathways from the gastrointestinal tract is as yet unknown. We evaluated effects of mGluR5 antagonists on the colorectal distension (CRD)‐evoked visceromotor (VMR) and cardiovascular responses in conscious rats, and on mechanosensory responses of mouse colorectal afferents in vitro. Sprague–Dawley rats were subjected to repeated, isobaric CRD (12 × 80 mmHg, for 30 s with 5 min intervals). The VMR and cardiovascular responses to CRD were monitored. The mGluR5 antagonists MPEP (1–10 μmol/kg, i.v.) and MTEP (1–3 μmol/kg, i.v.) reduced the VMR to CRD dose‐dependently with maximal inhibition of 52 ± 8% (p < 0.01) and 25 ± 11% (p < 0.05), respectively, without affecting colonic compliance. MPEP (10 μmol/kg, i.v.) reduced CRD‐evoked increases in blood pressure and heart rate by 33 ± 9% (p < 0.01) and 35 ± 8% (p < 0.05), respectively. Single afferent recordings were made from mouse pelvic and splanchnic nerves of colorectal mechanoreceptors. Circumferential stretch (0–5 g force) elicited slowly‐adapting excitation of action potentials in pelvic distension‐sensitive afferents. This response was reduced 55–78% by 10 μM MTEP (p < 0.05). Colonic probing (2 g von Frey hair) activated serosal splanchnic afferents; their responses were reduced 50% by 10 μM MTEP (p < 0.01). We conclude that mGluR5 antagonists inhibit CRD‐evoked VMR and cardiovascular changes in conscious rats, through an effect, at least in part, at peripheral afferent endings. Thus, mGluR5 participates in mediating mechanically evoked visceral nociception in the gastrointestinal tract.