Amanda J. Page

Different contributions of ASIC channels 1a, 2, and 3 in gastrointestinal mechanosensory function

Aims: Members of the acid sensing ion channel (ASIC) family are strong candidates as mechanical transducers in sensory function. The authors have shown that ASIC1a has no role in skin but a clear influence in gastrointestinal mechanotransduction. Here they investigate further ASIC1a in gut mechanoreceptors, and compare its influence with ASIC2 and ASIC3. Methods and results: Expression of ASIC1a, 2, and 3 mRNA was found in vagal (nodose) and dorsal root ganglia (DRG), and was lost in mice lacking the respective genes. Recordings of different classes of splanchnic colonic afferents and vagal gastro-oesophageal afferents revealed that disruption of ASIC1a increased the mechanical sensitivity of all afferents in both locations. Disruption of ASIC2 had varied effects: increased mechanosensitivity in gastro-oesophageal mucosal endings, decreases in gastro-oesophageal tension receptors, increases in colonic serosal endings, and no change in colonic mesenteric endings. In ASIC3-/- mice, all afferent classes had markedly reduced mechanosensitivity except gastro-oesophageal mucosal receptors. Observations of gastric emptying and faecal output confirmed that increases in mechanosensitivity translate to changes in digestive function in conscious animals. Conclusions: These data show that ASIC3 makes a critical positive contribution to mechanosensitivity in three out of four classes of visceral afferents. The presence of ASIC1a appears to provide an inhibitory contribution to the ion channel complex, whereas the role of ASIC2 differs widely across subclasses of afferents. These findings contrast sharply with the effects of ASIC1, 2, and 3 in skin, suggesting that targeting these subunits with pharmacological agents may have different and more pronounced effects on mechanosensitivity in the viscera.

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.

An in vitro study of the properties of vagal afferent fibres innervating the ferret oesophagus and stomach

1 A novel preparation of the oesophagus with attached vagus nerve from the ferret maintained in vitro was used to study the properties of single vagal afferent nerve fibres with identified receptive fields. 2 Recordings were made from three types of gastro‐oesophageal vagal afferent fibres that were classified on the basis of their sensitivity to mechanical stimulation. There were those responding to mucosal stroking (mucosal receptors), to circular tension (tension receptors) and those responding to mucosal stroking and circular tension, which we have termed tension/mucosal (TM) receptors. 3 The conduction velocities for mucosal, TM and tension receptor fibres were 6.38 ± 1.22 m s−1 (n= 22), 6.20 ± 1.49 m s−1 (n= 13) and 5.33 ± 0.86 m s−1 (n= 22), respectively. 4 Receptive fields of afferents showed random topographical distribution by fibre type and conduction velocity. They were found mainly distal but also occasionally proximal to the point of vagal dissection. 5 Twenty‐eight per cent of mucosal, 63 % of TM and 43 % of tension receptors responded to one or more drugs or chemical stimuli applied to the receptive field. 6 In conclusion, this experimental preparation provides evidence for the existence of three types of oesophageal vagal afferent fibre, namely mucosal, tension and the newly identified tension/mucosal receptors.

Acute effects of capsaicin on gastrointestinal vagal afferents.

Capsaicin is an important tool for investigation of thin afferent fibres, but its acute effects on subtypes of vagal afferent endings are unknown. In the gastrointestinal tract, these subtypes are: muscle endings (thought to be purely tension sensitive), mucosal endings (sensitive to stroking and chemical stimuli) and endings in the oesophagus with both properties. Acute capsaicin sensitivity was investigated in ferrets using in vivo and in vitro methods. Single-fibre activity was recorded from 63 vagal afferents: 12 Adelta-fibres, 15 C-fibres and 36 unclassified fibres with endings in the oesophagus (n=42), stomach (n=19) and duodenum (n=2). Responses to capsaicin occurred independently of motility changes and were therefore due to direct activation of the receptor ending. In the oesophagus in vivo, two of 10 tension receptors and one of one mucosal receptor responded to intraluminal application of 3.25 mM capsaicin. In the stomach and duodenum, five of 14 tension receptors and two of four mucosal receptors responded to close-systemic (32-164 nmol) capsaicin. In an in vitro gastro-oesophageal preparation, three of five tension, four of 21 mucosal and two of eight tension/mucosal receptors responded to topical application of 1mM capsaicin. Occurrence of responses was therefore unrelated to location of endings and isolation of tissue. Responsiveness was also unrelated to conduction velocity. Capsaicin caused desensitization of responses to further capsaicin application in 37% of afferents. It additionally caused cross-desensitization to mechanical stimuli, which was also seen in afferents that did not respond directly to capsaicin. In conclusion, capsaicin acutely activates all subtypes of gut vagal afferents in vivo and in vitro, although responsiveness is restricted to 30% of fibres and follows no specific pattern. Acute desensitization may be induced with or without a response.

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.

P2X purinoceptor‐induced sensitization of ferret vagal mechanoreceptors in oesophageal inflammation

1 Using an in vitro single unit recording technique we studied the changes in mechanical and chemical sensitivity of vagal afferent fibres in acute oesophagitis, with particular attention to inflammatory products such as purines. 2 Histologically verified oesophagitis was induced by oesophageal perfusion of 1 mg ml−1 pepsin in 150 mM HCl in anaesthetized ferrets for 30 min on two consecutive days. Controls were infused with 154 mM NaCl. 3 The number of action potentials evoked in oesophageal mucosal afferents by mucosal stroking with calibrated von Frey hairs (10–1000 mg) was stimulus dependent. In oesophagitis responsiveness was reduced across the range of stimuli compared with controls. 4 Topical application of the P2X purinoceptor agonist αβ‐methylene ATP had no direct excitatory effect on afferents. In oesophagitis, but not in controls, there was a significant increase in responses to stroking with von Frey hairs during superfusion with αβ‐methylene ATP (1 μM). 5 Mucosal afferents responded directly to one or more chemical stimuli: 26 % (5/19 afferents) responded in controls, and 47 % (7/15 afferents) in oesophagitis. There were no differences in responsiveness to bradykinin (1 μM), prostaglandin E2 (100 μM), 5‐hydroxytryptamine (100 μM), capsaicin (1 mM) or hydrochloric acid (150 mM) between control and oesophagitis groups. 6 We conclude that a sensitizing effect of a P2X purinoceptor agonist on mechanosensory function is induced in oesophagitis. This effect is offset by a decrease in basal mechanosensitivity.

Diet-induced adaptation of vagal afferent function

Non‐technical summary  Obesity is the result of a disruption in the maintenance of energy balance such that energy intake exceeds expenditure. Why this occurs is unknown. We show that after food deprivation or consumption of a high fat diet gastric vagal afferent responses to distension are reduced. In addition, the effect of the orexigenic peptide ghrelin is enhanced. Thus the satiety signal is reduced not only after food deprivation but also after a high fat diet. This reduction in satiety signalling may explain the increase in energy intake and disruption in maintenance of energy balance in obesity.

Acid sensing ion channels 2 and 3 are required for inhibition of visceral nociceptors by benzamil.

Abstract The Deg/ENaC family of ion channels, including ASIC1, 2 and 3, are candidate mechanotransducers in visceral and somatic sensory neurons, although each channel may play a different role in different sensory pathways. Here we determined which distinct populations of visceral sensory neurons are sensitive to the non‐selective Deg/ENaC blocker benzamil, and which ASIC channels are targets for benzamil by studying its actions in knockout mice. Single afferent fiber recordings were made in vitro from mouse high threshold colonic thoracolumbar splanchnic afferents and low threshold gastroesophageal vagal afferents. mRNA expression of ASIC subtypes was compared between colonic and gastroesophageal afferents by quantitative RT‐PCR of transcripts following laser capture microdissection of retrogradely labeled cell bodies. Mechanosensitivity of colonic afferents was potently reduced by benzamil (10−6–3 × 10−4 M), whereas gastroesophageal afferents were marginally inhibited. Inhibition of colonic afferent mechanosensitivity by benzamil was markedly diminished in ASIC2−/− and ASIC3−/− mice, but unchanged in ASIC1a−/−. Therefore ASIC2 and 3 are targets for benzamil to inhibit colonic afferent mechanosensitivity. Conversely, gastroesophageal afferents are less sensitive to benzamil, and its action depends less on ASIC expression. mRNA for ASIC3 showed higher and ASIC1a showed lower relative expression in colonic afferents from thoracolumbar dorsal root ganglia than in gastric afferents from nodose (vagal) ganglia. These data indicate that ASICs on colonic afferents present distinct pharmacological targets for visceral pain.

Potentiation of mouse vagal afferent mechanosensitivity by ionotropic and metabotropic glutamate receptors

Glutamate acts at central synapses via ionotropic (iGluR – NMDA, AMPA and kainate) and metabotropic glutamate receptors (mGluRs). Group I mGluRs are excitatory whilst group II and III are inhibitory. Inhibitory mGluRs also modulate peripherally the mechanosensitivity of gastro‐oesophageal vagal afferents. Here we determined the potential of excitatory GluRs to play an opposing role in modulating vagal afferent mechanosensitivity, and investigated expression of receptor subunit mRNA within the nodose ganglion. The responses of mouse gastro‐oesophageal vagal afferents to graded mechanical stimuli were investigated before and during application of selective GluR ligands to their peripheral endings. Two types of vagal afferents were tested: tension receptors, which respond to circumferential tension, and mucosal receptors, which respond only to mucosal stroking. The selective iGluR agonists NMDA and AMPA concentration‐dependently potentiated afferent responses. Their corresponding antagonists AP‐5 and NBQX alone attenuated mechanosensory responses as did the non‐selective antagonist kynurenate. The kainate selective agonist SYM‐2081 had minor effects on mechanosensitivity, and the antagonist UBP 302 was ineffective. The mGluR5 antagonist MTEP concentration‐dependently inhibited mechanosensitivity. Efficacy of agonists and antagonists differed on mucosal and tension receptors. We conclude that excitatory modulation of afferent mechanosensitivity occurs mainly via NMDA, AMPA and mGlu5 receptors, and the role of each differs according to afferent subtypes. PCR data indicated that all NMDA, kainate and AMPA receptor subunits plus mGluR5 are expressed, and are therefore candidates for the neuromodulation we observed.