L. Ashley Blackshaw

Transient lower esophageal sphincter relaxation

RAVINDER K. MITTAL,* RICHARD H. HOLLOWAY,* ROBERTO PENAGINI, § L. ASHLEY BLACKSHAW, t and JOHN DENT* *Department of Internal Medicine, University of Virginia, Charlottesville, Virginia; *Gastrointestinal Medicine, Royal Adelaide Hospital, Adelaide, South Australia, Australia; and §Cattedra di Gastroenterologia, Istituto di Scienze Mediche, University of Milan, IRCCS Ospedale Maggiore, Milan, Italy

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.

Effect of the artificial sweetener, sucralose, on gastric emptying and incretin hormone release in healthy subjects

The incretin hormones, glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), play an important role in glucose homeostasis in both health and diabetes. In mice, sucralose, an artificial sweetener, stimulates GLP-1 release via sweet taste receptors on enteroendocrine cells. We studied blood glucose, plasma levels of insulin, GLP-1, and GIP, and gastric emptying (by a breath test) in 7 healthy humans after intragastric infusions of 1) 50 g sucrose in water to a total volume of 500 ml (approximately 290 mosmol/l), 2) 80 mg sucralose in 500 ml normal saline (approximately 300 mosmol/l, 0.4 mM sucralose), 3) 800 mg sucralose in 500 ml normal saline (approximately 300 mosmol/l, 4 mM sucralose), and 4) 500 ml normal saline (approximately 300 mosmol/l), all labeled with 150 mg 13C-acetate. Blood glucose increased only in response to sucrose (P<0.05). GLP-1, GIP, and insulin also increased after sucrose (P=0.0001) but not after either load of sucralose or saline. Gastric emptying of sucrose was slower than that of saline (t50: 87.4+/-4.1 min vs. 74.7+/-3.2 min, P<0.005), whereas there were no differences in t50 between sucralose 0.4 mM (73.7+/-3.1 min) or 4 mM (76.7+/-3.1 min) and saline. We conclude that sucralose, delivered by intragastric infusion, does not stimulate insulin, GLP-1, or GIP release or slow gastric emptying in healthy humans.

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.

Differential chemosensory function and receptor expression of splanchnic and pelvic colonic afferents in mice

Lumbar splanchnic (LSN) and sacral pelvic (PN) nerves convey different mechanosensory information from the colon to the spinal cord. Here we determined whether these pathways also differ in their chemosensitivity and receptor expression. Using an in vitro mouse colon preparation, individual primary afferents were tested with selective P2X and transient receptor potential vanilloid receptor 1 (TRPV1) receptor ligands. Afferent cell bodies in thoracolumbar and lumbosacral dorsal root ganglia (DRG) were retrogradely labelled from the colon and analysed for P2X3‐ and TRPV1‐like immunoreactivity (LI). Forty per cent of LSN afferents responded to α,β‐methylene adenosine 5′‐triphosphate (α,β‐meATP; 1 mm), an effect that was concentration dependent and reversed by the P2X antagonist pyridoxyl5‐phosphate 6‐azophenyl‐2′,4′‐disulphonic acid (PPADS) (100 μm). Significantly fewer PN afferents (7%) responded to α,β‐meATP. Correspondingly, 36% of colonic thoracolumbar DRG neurones exhibited P2X3‐LI compared with only 19% of colonic lumbosacral neurones. Capsaicin (3 μm) excited 61% of LSN afferents and 47% of PN afferents; 82% of thoracolumbar and 50% of lumbosacral colonic DRG neurones displayed TRPV1‐LI. Mechanically insensitive afferents were recruited by α,β‐meATP or capsaicin, and were almost exclusive to the LSN. Capsaicin‐responsive LSN afferents displayed marked mechanical desensitization after responding to capsaicin, which did not occur in capsaicin‐responsive PN afferents. Therefore, colonic LSN and PN pathways differ in their chemosensitivity to known noxious stimuli and their corresponding receptor expression. As these pathways relay information that may relate to symptoms in functional gastrointestinal disease, these results may have implications for the efficacy of therapies targeting receptor modulation.

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.

In vitro recordings of afferent fibres with receptive fields in the serosa, muscle and mucosa of rat colon

1 Colonic afferent fibres were recorded using a novel in vitro preparation. Fibres with endings in the colonic mucosa are described, along with those in muscle and serosa, and their responses to a range of mechanical and chemical luminal stimuli. 2 Mechanical stimuli were applied to the tissue, which included stretch, blunt probing of the mucosa and stroking of the mucosa with von Frey hairs (10‐1000 mg). Chemical stimuli were applied into a ring that was placed over the mechanoreceptive field of the fibre; these were distilled water, 154 and 308 mM NaCl, 100 μM capsaicin, 50 mM HCl, and undiluted and 50 % ferret bile. 3 Recordings were made from 52 fibres, 12 of which showed characteristics of having endings in the mucosa. Mucosal afferents were sensitive to a 10 mg von Frey hair and were generally chemosensitive to ≥ 1 chemical stimulus. 4 Ten fibres showed characteristics of having receptive fields in the muscular layer. These fibres responded readily to circumferential stretch, as well as to blunt probing. 5 Twenty‐seven fibres showed characteristics of having endings in the serosal layer. They adapted rapidly to circumferential stretch and responded to blunt probing of the serosa. Fifteen of 19 serosal fibres tested also responded to luminal chemicals. 6 Three fibres were unresponsive to all mechanical stimuli but were recruited by chemical stimuli. 7 This is the first characterization of colonic afferent fibres using an in vitro method and the first documentation of afferent fibres with their endings in the mucosa of the colon. These fibres are likely to be important in aspects of colonic sensation and reflex control.

Excitation of rat colonic afferent fibres by 5-HT3 receptors

The gastrointestinal tract contains most of the bodys 5‐hydroxytryptamine (5‐HT) and releases large amounts after meals or exposure to toxins. Increased 5‐HT release occurs in patients with irritable bowel syndrome (IBS) and their peak plasma 5‐HT levels correlate with pain episodes. 5‐HT3 receptor antagonists reduce symptoms of IBS clinically, but their site of action is unclear and the potential for other therapeutic targets is unexplored. Here we investigated effects of 5‐HT on sensory afferents from the colon and the expression of 5‐HT3 receptors on their cell bodies in the dorsal root ganglia (DRG). Distal colon, inferior mesenteric ganglion and the lumbar splanchnic nerve bundle (LSN) were placed in a specialized organ bath. Eighty‐six single fibres were recorded from the LSN. Three classes of primary afferents were found: 70 high‐threshold serosal afferents, four low‐threshold muscular afferents and 12 mucosal afferents. Afferent cell bodies were retrogradely labelled from the distal colon to the lumbar DRG, where they were processed for 5‐HT3 receptor‐like immunoreactivity. Fifty‐six percent of colonic afferents responded to 5‐HT (between 10−6 and 10−3 M) and 30 % responded to the selective 5‐HT3 agonist, 2‐methyl‐5‐HT (between 10−6 and 10−2 M). Responses to 2‐methyl‐5‐HT were blocked by the 5‐HT3 receptor antagonist alosetron (2 × 10−7 M), whereas responses to 5‐HT were only partly inhibited. Twenty‐six percent of L1 DRG cell bodies retrogradely labelled from the colon displayed 5‐HT3 receptor‐like immunoreactivity. We conclude that colonic sensory neurones expressing 5‐HT3 receptors also functionally express the receptors at their peripheral endings. Our data reveal actions of 5‐HT on colonic afferent endings via both 5‐HT3 and non‐5‐HT3 receptors.

Vagal afferent discharge from gastric mechanoreceptors during contraction and relaxation of the ferret corpus

Vagal afferent fibres with mechanoreceptive fields in the gastric corpus were studied electrophysiologically in the urethane-anaesthetized ferret in an attempt to assess how changes in muscle tension and length modulate receptor discharge. Single afferent units were spontaneously active and increased their firing on distension of the corpus. During vagally evoked isovolumetric contractions and relaxations, the afferent discharge closely followed changes in intracorpus pressure. Under near-isotonic conditions, changes in both intracorpus pressure and afferent discharge were attenuated to similar degrees despite large changes in corpus volume, although the latter had a modulating influence on afferent discharge. These mechanoreceptors appeared to behave as tension receptors but were also influenced by muscle length as would occur if the receptor were associated with the laminar intramuscular septa of connective tissue which may serve as intramuscular tendons in this tissue.