Madusha Peiris

Rapid, Opioid-sensitive Mechanisms Involved in Transient Receptor Potential Vanilloid 1 Sensitization

TRPV1 is a nociceptive, Ca2+-selective ion channel involved in the development of several painful conditions. Sensitization of TRPV1 responses by cAMP-dependent PKA crucially contributes to the development of inflammatory hyperalgesia. However, the pathways involved in potentiation of TRPV1 responses by cAMP-dependent PKA remain largely unknown. Using HEK cells stably expressing TRPV1 and the μ opioid receptor, we demonstrated that treatment with the adenylate cyclase activator forskolin significantly increased the multimeric TRPV1 species. Pretreatment with the μ opioid receptor agonist morphine reversed this increased TRPV1 multimerization. FRET analysis revealed that treatment with forskolin did not cause multimerization of pre-existing TRPV1 monomers on the plasma membrane and that intracellular pools of TRPV1 exist mostly as monomers in this model. This suggests that increased TRPV1 multimerization occurred from an intracellular store of inactive TRPV1 monomers. Treatment with forskolin also caused an increase in TRPV1 expression on the plasma membrane not resulting from increased TRPV1 expression, and this rapid TRPV1 translocation was inhibited by treatment with morphine. Thus, potentiation of TRPV1 responses by cAMP-dependent PKA involves plasma membrane insertion of functional TRPV1 multimers formed from an intracellular store of inactive TRPV1 monomers. This potentiation occurs rapidly and can be dynamically modulated by activation of the μ opioid receptor under conditions where cAMP levels are raised, such as with inflammation. Increased translocation and multimerization of TRPV1 channels provide a cellular mechanism for finetuning of nociceptive responses that allow for rapid modulation of TRPV1 responses independent of transcriptional changes.

A model of experimental autoimmune encephalomyelitis (EAE) in C57BL/6 mice for the characterisation of intervention therapies

Multiple sclerosis (MS) and its different forms are studied in the animal model experimental autoimmune encephalomyelitis (EAE). Relapsing-remitting MS, the most common form of the disease can be induced in mice where clinical symptoms fluctuate in severity over time. However, the animal model does not experience periods of recovery where clinical signs are absent, unlike the human disease. We have developed a novel model of relapsing-remitting EAE in C57BL/6 mice immunised with myelin oligodendrocyte glycoprotein (MOG) peptide and Quil A as adjuvant. These animals have relapses that are followed by periods of recovery, during which time the animals do not exhibit illness. Furthermore, administration of the PPARgamma agonist pioglitazone prior to a predicted relapse prevents the expected development of symptoms in a dose-dependent fashion. Immune cell infiltration into white matter of the CNS and decreased production of inflammatory cytokine IFN-gamma in treated animals were also observed. Our model will be a valuable tool in assessing intervention therapies for RR-MS sufferers.

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.

TRPM8 on mucosal sensory nerves regulates colitogenic responses by innate immune cells via CGRP

TRPM8 is the molecular sensor for cold; however, the physiological role of TRPM8+ neurons at mucosal surfaces is unclear. Here we evaluated the distribution and peptidergic properties of TRPM8+ fibers in naive and inflamed colons, as well as their role in mucosal inflammation. We found that Trpm8−/− mice were hypersusceptible to dextran sodium sulfate (DSS)-induced colitis, and that Trpm8−/− CD11c+ DCs (dendritic cells) showed hyperinflammatory responses to toll-like receptor (TLR) stimulation. This was phenocopied in calcitonin gene–related peptide (CGRP) receptor-deficient mice, but not in substance P receptor-deficient mice, suggesting a functional link between TRPM8 and CGRP. The DSS phenotype of CGRP receptor-deficient mice could be adoptively transferred to wild-type (WT) mice, suggesting that CGRP suppresses the colitogenic activity of bone marrow–derived cells. TRPM8+ mucosal fibers expressed CGRP in human and mouse colon. Furthermore, neuronal CGRP contents were increased in colons from naive and DSS-treated Trpm8−/− mice, suggesting deficient CGRP release in the absence of TRPM8 triggering. Finally, treatment of Trpm8−/− mice with CGRP reversed their hyperinflammatory phenotype. These results suggest that TRPM8 signaling in mucosal sensory neurons is indispensable for the regulation of innate inflammatory responses via the neuropeptide CGRP.

Distinct afferent innervation patterns within the human proximal and distal esophageal mucosa.

Little is known about the mucosal phenotype of the proximal human esophagus. There is evidence to suggest that the proximal esophagus is more sensitive to chemical and mechanical stimulation compared with the distal. This may have physiological relevance (e.g., in prevention of aspiration of gastroesophageal refluxate), but also pathological relevance (e.g., in reflux perception or dysphagia). Reasons for this increased sensitivity are unclear but may include impairment in mucosal barrier integrity or changes in sensory innervation. We assessed mucosal barrier integrity and afferent nerve distribution in the proximal and distal esophagus of healthy human volunteers. In 10 healthy volunteers baseline proximal and distal esophageal impedance was measured in vivo. Esophageal mucosal biopsies from the distal and proximal esophagus were taken, and baseline transepithelial electrical resistance (TER) was measured in Ussing chambers. Biopsies were examined immunohistochemically for presence and location of calcitonin gene-related peptide (CGRP)-immunoreactive nerve fibers. In a further four healthy volunteers we investigated for colocalization of CGRP and protein gene product (PGP) 9.5 immunoreactivity in nerve fibers. Baseline impedance was higher in the proximal than in the distal esophagus [2,936 Ω (SD578) vs. 2,229 Ω (SD821); P = 0.03], however, baseline TER was not significantly different between them. Mucosal CGRP-immunoreactive nerves were found in the epithelium of both proximal and distal esophagus, but were located more superficially in the proximal mucosa compared with the distal [11.5 (SD7) vs. 21.7 (SD5) cell layers from lumen, P = 0.002] 19% of proximal, and 10% of distal mucosal PGP-immunoreactive fibers colocalized with CGRP. PGP-immunoreactive fibers were also significantly closer to the luminal surface in the proximal compared with the distal esophagus (P < 0.001). We conclude that mucosal barrier integrity is similar in proximal and distal esophagus, but proximal mucosal afferent nerves are in a more superficial location. The enhanced sensitivity to reflux-evoked symptoms of the proximal esophagus most likely has an anatomical basis.

Ex vivo study of human visceral nociceptors

Objective 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. Design 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 (TRPV4) modulation on mechanical responses. Results 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 TRPV4 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. Conclusions Functionally distinct subpopulations of human visceral afferents can be demonstrated and could provide a platform technology to further study nociception in human tissue.

Peripheral KV7 channels regulate visceral sensory function in mouse and human colon

Background Chronic visceral pain is a defining symptom of many gastrointestinal disorders. The KV7 family (KV7.1–KV7.5) of voltage-gated potassium channels mediates the M current that regulates excitability in peripheral sensory nociceptors and central pain pathways. Here, we use a combination of immunohistochemistry, gut-nerve electrophysiological recordings in both mouse and human tissues, and single-cell qualitative real-time polymerase chain reaction of gut-projecting sensory neurons, to investigate the contribution of peripheral KV7 channels to visceral nociception. Results Immunohistochemical staining of mouse colon revealed labelling of KV7 subtypes (KV7.3 and KV7.5) with CGRP around intrinsic enteric neurons of the myenteric plexuses and within extrinsic sensory fibres along mesenteric blood vessels. Treatment with the KV7 opener retigabine almost completely abolished visceral afferent firing evoked by the algogen bradykinin, in agreement with significant co-expression of mRNA transcripts by single-cell qualitative real-time polymerase chain reaction for KCNQ subtypes and the B2 bradykinin receptor in retrogradely labelled extrinsic sensory neurons from the colon. Retigabine also attenuated responses to mechanical stimulation of the bowel following noxious distension (0–80 mmHg) in a concentration-dependent manner, whereas the KV7 blocker XE991 potentiated such responses. In human bowel tissues, KV7.3 and KV7.5 were expressed in neuronal varicosities co-labelled with synaptophysin and CGRP, and retigabine inhibited bradykinin-induced afferent activation in afferent recordings from human colon. Conclusions We show that KV7 channels contribute to the sensitivity of visceral sensory neurons to noxious chemical and mechanical stimuli in both mouse and human gut tissues. As such, peripherally restricted KV7 openers may represent a viable therapeutic modality for the treatment of gastrointestinal pathologies.

The extracellular matrix glycoprotein tenascin‐X regulates peripheral sensory and motor neurones

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.

Effects of Obesity and Gastric Bypass Surgery on Nutrient Sensors, Endocrine Cells, and Mucosal Innervation of the Mouse Colon

Background: Nutrient-sensing receptors located on enteroendocrine (EEC) cells modulate appetite via detection of luminal contents. Colonic ‘tasting’ of luminal contents may influence changes to appetite observed in obesity and after weight loss induced by bariatric surgery. We assessed the effects of obesity and gastric bypass-induced weight loss on expression of nutrient-sensing G-protein coupled receptors (GPCRs), EEC and enterochromaffin (EC) cells and mucosal innervation. Methods: qPCR and immunohistochemistry were used to study colonic tissue from (a) chow-fed/lean, (b) high-fat fed/obese, (c) Roux-en-Y gastric bypass surgery (RYGB), and (d) calorie restriction-induced weight loss mice. Results: Expression of GPR41, GPR43, GPR40, GPR120, GPR84, GPR119, GPR93 and T1R3 was increased in obese mice. Obesity-induced overexpression of GPR41, 40, 84, and 119 further increased after RYGB whereas GPR120 and T1R3 decreased. RYGB increased TGR5 expression. L-cells, but not EC cells, were increased after RYGB. No differences in mucosal innervation by protein gene product (PGP) 9.5 and GLP-1R-positive nerve fibers were observed. Stimulation of colonic mucosa with GPR41, GPR40, GPR85, GPR119, and TGR5 agonists increased cell activation marker expression. Conclusions: Several nutrient-sensing receptors induced activation of colonic EEC. Profound adaptive changes to the expression of these receptors occur in response to diet and weight loss induced by RYGB or calorie restriction.