Key points
Tenascin‐X (TNX) is an extracellular matrix glycoprotein with anti‐adhesive properties in skin and joints. Here we report the novel finding that TNX is expressed in human and mouse gut ...tissue where it is exclusive to specific subpopulations of neurones.
Our studies with TNX‐deficient mice show impaired defecation and neural control of distal colonic motility that can be rescued with a 5‐HT4 receptor agonist. However, colonic secretion is unchanged. They are also susceptible to internal rectal intussusception. Colonic afferent sensitivity is increased in TNX‐deficient mice. Correspondingly, there is increased density of and sensitivity of putative nociceptive fibres in TNX‐deficient mucosa.
A group of TNX‐deficient patients report symptoms highly consistent with those in the mouse model.
These findings suggest TNX plays entirely different roles in gut to non‐visceral tissues – firstly a role in enteric motor neurones and secondly a role influencing nociceptive sensory neurones
Studying further the mechanisms by which TNX influences neuronal function will lead to new targets for future treatment.
The extracellular matrix (ECM) is not only an integral structural molecule, but is also critical for a wide range of cellular functions. The glycoprotein tenascin‐X (TNX) predominates in the ECM of tissues like skin and regulates tissue structure through anti‐adhesive interactions with collagen. Monogenic TNX deficiency causes painful joint hypermobility and skin hyperelasticity, symptoms characteristic of hypermobility Ehlers Danlos syndrome (hEDS). hEDS patients also report consistently increased visceral pain and gastrointestinal (GI) dysfunction. We investigated whether there is a direct link between TNX deficiency and GI pain or motor dysfunction. We set out first to learn where TNX is expressed in human and mouse, then determine how GI function, specifically in the colon, is disordered in TNX‐deficient mice and humans of either sex. In human and mouse tissue, TNX was predominantly associated with cholinergic colonic enteric neurones, which are involved in motor control. TNX was absent from extrinsic nociceptive peptidergic neurones. TNX‐deficient mice had internal rectal prolapse and a loss of distal colonic contractility which could be rescued by prokinetic drug treatment. TNX‐deficient patients reported increased sensory and motor GI symptoms including abdominal pain and constipation compared to controls. Despite absence of TNX from nociceptive colonic neurones, neuronal sprouting and hyper‐responsiveness to colonic distension was observed in the TNX‐deficient mice. We conclude that ECM molecules are not merely support structures but an integral part of the microenvironment particularly for specific populations of colonic motor neurones where TNX exerts functional influences.
Key points
Tenascin‐X (TNX) is an extracellular matrix glycoprotein with anti‐adhesive properties in skin and joints. Here we report the novel finding that TNX is expressed in human and mouse gut tissue where it is exclusive to specific subpopulations of neurones.
Our studies with TNX‐deficient mice show impaired defecation and neural control of distal colonic motility that can be rescued with a 5‐HT4 receptor agonist. However, colonic secretion is unchanged. They are also susceptible to internal rectal intussusception. Colonic afferent sensitivity is increased in TNX‐deficient mice. Correspondingly, there is increased density of and sensitivity of putative nociceptive fibres in TNX‐deficient mucosa.
A group of TNX‐deficient patients report symptoms highly consistent with those in the mouse model.
These findings suggest TNX plays entirely different roles in gut to non‐visceral tissues – firstly a role in enteric motor neurones and secondly a role influencing nociceptive sensory neurones
Studying further the mechanisms by which TNX influences neuronal function will lead to new targets for future treatment.
Colonic enteroendocrine cells (EECs) store and release potent anorectic hormones that are key regulators of satiety. EECs express multiple nutrient sensing receptors, particularly for medium-chain ...fatty acids (MCFAs): GPR84 and FFAR4. Here we show a non-surgical approach with targeted colonic delivery of MCFA, which induces EEC and neuronal activation leading to anorectic effects.
A randomised, double-blind, placebo-controlled, cross-over study was performed in obese adults given combined GPR84 and FFAR4 agonists in colonic release capsules before meals. We measured serum hormones, energy intake and appetite perception. Cell type, activation by agonists and hormone/serotonin release were determined in human colonic explants. Mouse colonic afferent nerve responses to nutrients/mediators were recorded electrophysiologically.
Subjects receiving GPR84 and FFAR4 agonists had reduced overall calorific intake and increased postprandial levels of PYY versus placebo. Receptors including GPR84 and FFAR4 were coexpressed on human colonic EEC. Activation of GPR84 exclusively induced intracellular pERK, whereas FFAR4 selectively activated pCaMKII. Coactivation of GPR84 and FFAR4 induced both phosphoproteins, and superadditive release of GLP-1 and PYY. Nutrients and hormones convergently activated murine colonic afterent nerves via GLP-1, Y2 and 5-HT3 receptors.
Colonic GPR84 and FFAR4 agonists reduce energy intake and increase postprandial PYY in obese adults. Human colonic EECs coexpress these receptors, which activate cells via parallel intracellular pathways and synergistically evoke hormone release. Further synergism occurs in sensory nerve responses to MCFA and EEC mediators. Thus, synergistic activation of colonic endocrine cells via nutrient receptors is an important target for metabolic regulation.
NCT04292236.
Key points
Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability.
Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and ...inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown.
Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling.
These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics.
Voltage‐gated sodium channel NaV1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of NaV1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor‐specific NaV1.7 knockout mouse (NaV1.7Nav1.8) and selective small‐molecule NaV1.7 antagonist PF‐5198007. NaV1.7Nav1.8 mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both NaV1.7Nav1.8 and littermate controls. Loss, or blockade, of NaV1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve–gut preparations in mouse, or following antagonism of NaV1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage‐gated sodium channel α subunits revealed NaV1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of NaV1.7 (in NaV1.8‐expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective NaV1.7 antagonist PF‐5198007. Our data demonstrate that NaV1.7 (in NaV1.8‐expressing neurons) contributes to defined pain pathways in a modality‐dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of NaV1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.
Key points
Voltage‐gated sodium channels play a fundamental role in determining neuronal excitability.
Specifically, voltage‐gated sodium channel subtype NaV1.7 is required for sensing acute and inflammatory somatic pain in mice and humans but its significance in pain originating from the viscera is unknown.
Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for NaV1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling.
These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality‐specific manner and help to direct drug discovery efforts towards novel visceral analgesics.
Voltage-gated sodium channels play a fundamental role in determining neuronal excitability. Specifically, voltage-gated sodium channel subtype Na
1.7 is required for sensing acute and inflammatory ...somatic pain in mice and humans but its significance in pain originating from the viscera is unknown. Using comparative behavioural models evoking somatic and visceral pain pathways, we identify the requirement for Na
1.7 in regulating somatic (noxious heat pain threshold) but not in visceral pain signalling. These results enable us to better understand the mechanisms underlying the transduction of noxious stimuli from the viscera, suggest that the investigation of pain pathways should be undertaken in a modality-specific manner and help to direct drug discovery efforts towards novel visceral analgesics.
Voltage-gated sodium channel Na
1.7 is required for acute and inflammatory pain in mice and humans but its significance for visceral pain is unknown. Here we examine the role of Na
1.7 in visceral pain processing and the development of referred hyperalgesia using a conditional nociceptor-specific Na
1.7 knockout mouse (Na
1.7
) and selective small-molecule Na
1.7 antagonist PF-5198007. Na
1.7
mice showed normal nociceptive behaviours in response to intracolonic application of either capsaicin or mustard oil, stimuli known to evoke sustained nociceptor activity and sensitization following tissue damage, respectively. Normal responses following induction of cystitis by cyclophosphamide were also observed in both Na
1.7
and littermate controls. Loss, or blockade, of Na
1.7 did not affect afferent responses to noxious mechanical and chemical stimuli in nerve-gut preparations in mouse, or following antagonism of Na
1.7 in resected human appendix stimulated by noxious distending pressures. However, expression analysis of voltage-gated sodium channel α subunits revealed Na
1.7 mRNA transcripts in nearly all retrogradely labelled colonic neurons, suggesting redundancy in function. By contrast, using comparative somatic behavioural models we identify that genetic deletion of Na
1.7 (in Na
1.8-expressing neurons) regulates noxious heat pain threshold and that this can be recapitulated by the selective Na
1.7 antagonist PF-5198007. Our data demonstrate that Na
1.7 (in Na
1.8-expressing neurons) contributes to defined pain pathways in a modality-dependent manner, modulating somatic noxious heat pain, but is not required for visceral pain processing, and advocate that pharmacological block of Na
1.7 alone in the viscera may be insufficient in targeting chronic visceral pain.
EphB receptors tyrosine kinases and ephrinB ligands were first identified as guidance molecules involved in the establishment of topographical mapping and connectivity in the nervous system during ...development. Later in development and into adulthood their primary role would switch from guidance to activity-dependent modulation of synaptic efficacy. In sensory systems, they play a role in both the onset of inflammatory and neuropathic pain, and in the establishment of central sensitisation, an NMDA-mediated form of synaptic plasticity thought to underlie most forms of chronic pain. We studied wild type and EphB1 knockout mice in a range of inflammatory and neuropathic pain models to determine 1), whether EphB1 expression is necessary for the onset and/or maintenance of persistent pain, regardless of origin; 2), whether in these models cellular and molecular changes, e.g. phosphorylation of the NR2B subunit of the NMDA receptor, increased c-fos expression or microglial activation, associated with the onset of pain, are affected by the lack of functional EphB1 receptors. Differences in phenotype were examined behaviourally, anatomically, biochemically and electrophysiologically. Our results establish firstly, that functional EphB1 receptors are not essential for the development of normal nociception, thermal or mechanical sensitivity. Secondly, they demonstrate a widespread involvement of EphB1 receptors in chronic pain. NR2B phosphorylation, c-fos expression and microglial activation are all reduced in EphB1 knockout mice. This last finding is intriguing, since microglial activation is supposedly triggered directly by primary afferents, therefore it was not expected to be affected. Interestingly, in some models of long-term pain (days), mechanical and thermal hyperalgesia develop both in wild type and EphB1 knockout mice, but recovery is faster in the latter, indicating that in particular models these receptors are required for the maintenance, rather than the onset of, thermal and mechanical hypersensitivity. This potentially makes them an attractive target for analgesic strategies.
Abstract Background & Aims Histamine sensitizes the nociceptor TRPV1 and has been shown to contribute to visceral hypersensitivity in animals. We investigated the role of TRPV1 in irritable bowel ...syndrome (IBS) and evaluated if an antagonist of histamine receptor H1 (HRH1) could reduce symptoms of patients in a randomized placebo controlled trial. Methods Using live calcium imaging, we compared activation of submucosal neurons by the TRPV1 agonist capsaicin in rectal biopsies collected from 9 patients with IBS (ROME 3 critera) and 15 healthy subjects. Sensitization of TRPV1 by histamine, its metabolite imidazole acetaldehyde, and supernatants from biopsies was assessed by calcium imaging of mouse dorsal root ganglion neurons. We then performed a double-blind trial of patients with IBS (mean age, 31 years; range, 18–65 years; 34 female). After a 2 week run-in period, subjects were randomly assigned to groups given either the HRH1 antagonist ebastine (20 mg/day; n=28) or placebo (n=27) for 12 weeks. Rectal biopsies were collected, barostat studies were performed, and symptoms were assessed (using the validated gastrointestinal symptom rating scale) before and after the 12 week period. Patients were followed for an additional 2 weeks. Abdominal pain, symptom relief, and health-related quality of life were assessed on a weekly basis. The primary endpoint of the study was the effect of ebastine on the symptom score evoked by rectal distension. Results TRPV1 responses of submucosal neurons from patients with IBS were potentiated compared to those of healthy volunteers. Moreover, TRPV1 responses of submucosal neurons from healthy volunteers could be potentiated by their pre-incubation with histamine; this effect was blocked by the HRH1 antagonist pyrilamine. Supernatants from rectal biopsies from patients with IBS, but not from the healthy volunteers, sensitized TRPV1 in mouse nociceptive dorsal root ganglion neurons via HRH1; this effect could be reproduced by histamine and imidazole acetaldehyde. Compared to subjects given placebo, those given ebastine had reduced visceral hypersensitivity, increased symptom relief (ebastine 46% vs placebo 13%; P=.024) and reduced abdominal pain scores (ebastine 39±23 vs placebo 62±22, P=.0004). Conclusion In studies of rectal biopsies from patients, we found that HRH1-mediated sensitization of TRPV1 is involved in IBS. Ebastine, an antagonist of HRH1, reduced visceral hypersensitivity, symptoms, and abdominal pain in patients with IBS. Inihbitors of this pathway might be developed as a new treatment approach for IBS. ClinicalTrials.gov no: NCT01144832
The development of effective visceral analgesics free of deleterious gut-specific side effects is a priority. We aimed to develop a reproducible methodology to study visceral nociception in human ...tissue that could aid future target identification and drug evaluation.
Electrophysiological (single unit) responses of visceral afferents to mechanical (von Frey hair (VFH) and stretch) and chemical (bradykinin and ATP) stimuli were examined. Thus, serosal afferents (putative nociceptors) were used to investigate the effect of tegaserod, and transient receptor potential channel, vanilloid 4 (TRPV
) modulation on mechanical responses.
Two distinct afferent fibre populations, serosal (n=23) and muscular (n=21), were distinguished based on their differences in sensitivity to VFH probing and tissue stretch. Serosal units displayed sensitivity to key algesic mediators, bradykinin (6/14 units tested) and ATP (4/10), consistent with a role as polymodal nociceptors, while muscular afferents are largely insensitive to bradykinin (0/11) and ATP (1/10). Serosal nociceptor mechanosensitivity was attenuated by tegaserod (-20.8±6.9%, n=6, p<0.05), a treatment for IBS, or application of HC067047 (-34.9±10.0%, n=7, p<0.05), a TRPV
antagonist, highlighting the utility of the preparation to examine the mechanistic action of existing drugs or novel analgesics. Repeated application of bradykinin or ATP produced consistent afferent responses following desensitisation to the first application, demonstrating their utility as test stimuli to evaluate analgesic activity.
Functionally distinct subpopulations of human visceral afferents can be demonstrated and could provide a platform technology to further study nociception in human tissue.
NaV1.9 regulates normal colonic afferent mechanosensation and is required for hypersensitivity to noxious inflammatory mediators and those derived from inflammatory bowel disease tissues.
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.
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 Na(v)1.9(-/-) 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.
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