The cation channel TRPM8 influences the differentiation and function of human monocytes.

Monocytes are mononuclear phagocytes that can differentiate to a variety of cell fates under the influence of their microenvironment and hardwired commitment. We found that inhibition of TRPM8 in human blood CD14+ monocytes during a critical 3-h window at the beginning of their differentiation into macrophages led to enhanced survival and LPS-driven TNFα production after 24 h. TRPM8 antagonism also promoted LPS-driven TNFα production in CD14+ monocytes derived from the intestinal mucosa. Macrophages that had been derived for 6 days under blockade of TRPM8 had impaired phagocytic capacity and were transcriptionally distinct. Most of the affected genes were altered in a way that opposed normal monocyte to macrophage differentiation indicating that TRPM8 activity promotes aspects of this differentiation programme. Thus, we reveal a novel role for TRPM8 in regulating human CD14+ monocyte fate and function.

Functional and anatomical deficits in visceral nociception with age: a mechanism of silent appendicitis in the elderly?

The ability to sense visceral pain during appendicitis is diminished with age leading to delay in seeking health care and poorer clinical outcomes. To understand the mechanistic basis of this phenomenon, we examined visceral nociception in aged mouse and human tissue. Inflamed and noninflamed appendixes were collected from consenting patients undergoing surgery for the treatment of appendicitis or bowel cancer. Supernatants were generated by incubating samples in buffer and used to stimulate multiunit activity in intestinal preparations, or single-unit activity from teased fibres in colonic preparations, of young and old mice. Changes in afferent innervation with age were determined by measuring the density of calcitonin gene-related peptide-positive afferent fibres and by counting dorsal root ganglia back-labelled by injection of tracer dye into the wall of the colon. Finally, the effect of age on nociceptor function was studied in mouse and human colon. Afferent responses to appendicitis supernatants were greatly impaired in old mice. Further investigation revealed this was due to a marked reduction in the afferent innervation of the bowel and a substantial impairment in the ability of the remaining afferent fibres to transduce noxious stimuli. Translational studies in human tissue demonstrated a significant reduction in the multiunit but not the single-unit colonic mesenteric nerve response to capsaicin with age, indicative of a loss of nociceptor innervation. Our data demonstrate that anatomical and functional deficits in nociception occur with age, underpinning the atypical or silent presentation of appendicitis in the elderly.

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.

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.

Superficial Esophageal Mucosal Afferent Nerves May Contribute to Reflux Hypersensitivity in Nonerosive Reflux Disease.

BACKGROUND & AIMS: Little is known about the causes of heartburn in patients with gastro-esophageal reflux disease. Visible epithelial damage is seldom associated with symptom severity, evidenced by the significant symptom burden in patients with nonerosive reflux disease (NERD) compared with patients with erosive reflux disease (ERD) or Barrett's esophagus (BE). We studied the distribution of mucosal nerve fibers in patients with NERD, ERD, and BE, and compared the results with those of healthy subjects. METHODS: We performed a prospective study of 13 patients with NERD, 11 patients with ERD, and 16 patients with BE undergoing endoscopic evaluation in the United Kingdom or Greece. Biopsies were obtained from the proximal and distal esophageal mucosa of patients with NERD, from the distal esophageal mucosa of patients with ERD, and the distal-most squamous epithelium of patients with BE. These were examined for the presence and location of nerve fibers that reacted with a labeled antibody against calcitonin gene-related peptide (CGRP), a marker of nociceptive sensory nerves. The results were compared with those from 10 healthy volunteers (controls). RESULTS: The distribution of CGRP-positive nerves did not differ significantly between the distal esophageal mucosa of controls (median, 25.5 cell layers to surface; interquartile range [IQR], 21.4-28.8) vs patients with ERD (median, 23 cell layers to surface; IQR, 16-27.5), or patients with BE (median, 21.5 cell layers to surface; IQR, 16.1-27.5). However, CGRP-positive nerves were significantly more superficial in mucosa from patients with NERD-both distal (median, 9.5 cell layers to surface; IQR, 1.5-13.3; P < .0001 vs ERD, BE, and controls) and proximal (median, 5.0 cell layers to surface; IQR, 2.5-9.3 vs median 10.4 cell layers to surface; IQR, 8.0-16.9; P = .0098 vs controls). CONCLUSIONS: Proximal and distal esophageal mucosa of patients with NERD have more superficial afferent nerves compared with controls or patients with ERD or BE. Acid hypersensitivity in patients with NERD might be partially explained by the increased proximity of their afferent nerves to the esophageal lumen, and therefore greater exposure to noxious substances in refluxate.

Acute colitis chronically alters immune infiltration mechanisms and sensory neuro-immune interactions.

OBJECTIVE: Little is understood regarding how disease progression alters immune and sensory nerve function in colitis. We investigated how acute colitis chronically alters immune recruitment and the impact this has on re-activated colitis. To understand the impact of disease progress on sensory systems we investigated the mechanisms underlying altered colonic neuro-immune interactions after acute colitis. DESIGN: Inflammation was compared in mouse models of health, acute tri-nitrobenzene sulphonic acid (TNBS) colitis, Remission and Reactivated colitis. Cytokine concentrations were compared by ELISA in-situ and in explanted colon tissue. Colonic infiltration by CD11b/F4-80 macrophage, CD4 THELPER (TH) and CD8 TCYTOTOXIC (TC) and α4ß7 expression on mesenteric lymph node (MLN) TH and TC was determined by flow cytometry. Cytokine and effector receptor mRNA expression was determined on colo-rectal afferent neurons and the mechanisms underlying cytokinergic effects on high-threshold colo-rectal afferent function were investigated using electrophysiology. RESULTS: Colonic damage, MPO activity, macrophage infiltration, IL-1ß and IL-6 concentrations were lower in Reactivated compared to Acute colitis. TH infiltration and α4ß7 expression on TH MLN was increased in Remission but not Acute colitis. IFN-γ concentrations, TH infiltration and α4ß7 expression on TH and TC MLN increased in Reactivated compared to Acute colitis. Reactivated explants secreted more IL-1ß and IL-6 than Acute explants. IL-6 and TNF-α inhibited colo-rectal afferent mechanosensitivity in Remission mice via a BKCa dependent mechanism. CONCLUSIONS: Acute colitis persistently alters immune responses and afferent nerve signalling pathways to successive episodes of colitis. These findings highlight the complexity of viscero-sensory neuro-immune interactions in painful remitting and relapsing diseases.

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.

Pharmacologic treatments for esophageal disorders.

The following, from the 12th OESO World Conference: Cancers of the Esophagus, includes commentaries on the role for ketamine and other alternative treatments in esophageal disorders; the use of linaclotide in the treatment of esophageal pain; the alginate test as a diagnostic criterion in gastroesophageal reflux disease (GERD); the use of baclofen in treatment of GERD; the effects of opioids on the esophagus; the use of antagonists on the receptor level in GERD; the effect of local formulation of drugs on the esophageal mucosa; and the use of electroencephalographic fingerprints to predict the effect of pharmacological treatment.

Immune derived opioidergic inhibition of viscerosensory afferents is decreased in Irritable Bowel Syndrome patients.

Alterations in the neuro-immune axis contribute toward viscerosensory nerve sensitivity and symptoms in Irritable Bowel Syndrome (IBS). Inhibitory factors secreted from immune cells inhibit colo-rectal afferents in health, and loss of this inhibition may lead to hypersensitivity and symptoms. We aimed to determine the immune cell type(s) responsible for opioid secretion in humans and whether this is altered in patients with IBS. The ß-endorphin content of specific immune cell lineages in peripheral blood and colonic mucosal biopsies were compared between healthy subjects (HS) and IBS patients. Peripheral blood mononuclear cell (PBMC) supernatants from HS and IBS patients were applied to colo-rectal sensory afferent endings in mice with post-inflammatory chronic visceral hypersensitivity (CVH). ß-Endorphin was identified predominantly in monocyte/macrophages relative to T or B cells in human PBMC and colonic lamina propria. Monocyte derived ß-endorphin levels and colonic macrophage numbers were lower in IBS patients than healthy subjects. PBMC supernatants from healthy subjects had greater inhibitory effects on colo-rectal afferent mechanosensitivity than those from IBS patients. The inhibitory effects of PBMC supernatants were more prominent in CVH mice compared to healthy mice due to an increase in µ-opioid receptor expression in dorsal root ganglia neurons in CVH mice. Monocyte/macrophages are the predominant immune cell type responsible for ß-endorphin secretion in humans. IBS patients have lower monocyte derived ß-endorphin levels than healthy subjects, causing less inhibition of colonic afferent endings. Consequently, altered immune function contributes toward visceral hypersensitivity in IBS.

Multiple roles for NaV1.9 in the activation of visceral afferents by noxious inflammatory, mechanical, and human disease-derived stimuli.

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.

Linaclotide inhibits colonic nociceptors and relieves abdominal pain via guanylate cyclase-C and extracellular cyclic guanosine 3',5'-monophosphate.

BACKGROUND & AIMS: Linaclotide is a minimally absorbed agonist of guanylate cyclase-C (GUCY2C or GC-C) that reduces symptoms associated with irritable bowel syndrome with constipation (IBS-C). Little is known about the mechanism by which linaclotide reduces abdominal pain in patients with IBS-C. METHODS: We determined the effects of linaclotide on colonic sensory afferents in healthy mice and those with chronic visceral hypersensitivity. We assessed pain transmission by measuring activation of dorsal horn neurons in the spinal cord in response to noxious colorectal distention. Levels of Gucy2c messenger RNA were measured in tissues from mice using quantitative reverse transcription polymerase chain reaction and in situ hybridization. We used human intestinal cell lines to measure release of cyclic guanosine-3',5'-monophosphate (cGMP) by linaclotide. We performed a post-hoc analysis of data from a phase III, double-blind, parallel-group study in which 805 patients with IBS-C were randomly assigned to groups given an oral placebo or 290 µg linaclotide once daily for 26 weeks. We quantified changes in IBS-C symptoms, including abdominal pain. RESULTS: In mice, linaclotide inhibited colonic nociceptors with greater efficacy during chronic visceral hypersensitivity. Intra-colonic administration of linaclotide reduced signaling of noxious colorectal distention to the spinal cord. The colonic mucosa, but not neurons, was found to express linaclotide's target, GC-C. The downstream effector of GC-C, cGMP, was released after administration of linaclotide and also inhibited nociceptors. The effects of linaclotide were lost in Gucy2c(-/-) mice and prevented by inhibiting cGMP transporters or removing the mucosa. During 26 weeks of linaclotide administration, a significantly greater percentage of patients (70%) had at least a 30% reduction in abdominal pain compared with patients given placebo (50%). CONCLUSIONS: We have identified an analgesic mechanism of linaclotide: it activates GC-C expressed on mucosal epithelial cells, resulting in the production and release of cGMP. This extracellular cGMP acts on and inhibits nociceptors, thereby reducing nociception. We also found that linaclotide reduces chronic abdominal pain in patients with IBS-C.

Peripheral neural targets in obesity.

Interest in pharmacological treatments for obesity that act in the brain to reduce appetite has increased exponentially over recent years, but failures of clinical trials and withdrawals due to adverse effects have so far precluded any success. Treatments that do not act within the brain are, in contrast, a neglected area of research and development. This is despite the fact that a vast wealth of molecular mechanisms exists within the gut epithelium and vagal afferent system that could be manipulated to increase satiety. Here we discuss mechano- and chemosensory pathways from the gut involved in appetite suppression, and distinguish between gastric and intestinal vagal afferent pathways in terms of their basic physiology and activation by enteroendocrine factors. Gastric bypass surgery makes use of this system by exposing areas of the intestine to greater nutrient loads resulting in greater satiety hormone release and reduced food intake. A non-surgical approach to this system is preferable for many reasons. This review details where the opportunities may lie for such approaches by describing nutrient-sensing mechanisms throughout the gastrointestinal tract.

Small bowel homing T cells are associated with symptoms and delayed gastric emptying in functional dyspepsia.

OBJECTIVES: Immune activation may have an important pathogenic role in the irritable bowel syndrome (IBS). While little is known about immunologic function in functional dyspepsia (FD), we have observed an association between cytokine secretion by peripheral blood mononuclear cells (PBMCs) and symptoms in IBS. Upper gastrointestinal inflammatory diseases are characterized by enhanced small bowel homing α4-, ß7-integrin, chemokine receptor 9 (CCR9) positive T lymphocytes. We hypothesized that increased cytokine release and elevated circulating small bowel homing T cells are linked to the severity of symptoms in patients with FD. Thus, we aimed to (i) compare cytokine release in FD and healthy controls (HCs), (ii) quantify "gut homing" T cells in FD compared with HC and patients with IBS, and (iii) correlate the findings to symptom severity and gastric emptying. METHODS: PBMC from 45 (Helicobacter pylori negative) patients with FD (Rome II) and 35 matched HC were isolated by density gradient centrifugation and cultured for 24 h. Cytokine production (tumor necrosis factor (TNF)-α, interleukin (IL)-1ß, IL-6, IL-10) was measured by enzyme-linked immunosorbent assay. CD4+ α4ß7+CCR9+ T cells were quantified by flow cytometry in FD, HC and 23 patients with IBS. Gastric emptying was measured by scintigraphy. Symptom severity was assessed utilizing the standardized Gastrointestinal Symptom Score. RESULTS: FD patients had significantly higher TNF-α (107.2 ± 42.8 vs. 58.7 ± 7.4 pg/ml), IL-1ß (204.8 ± 71.5 vs. 80.2 ± 17.4 pg/ml), and IL-10 (218 ± 63.3 vs. 110.9 ± 18.5 pg/ml) levels compared with HC, and enhanced gut homing lymphocytes compared with HC or IBS. Cytokine release and CD4+α4ß7+CCR9+ lymphocytes were correlated with the symptom intensity of pain, cramps, nausea, and vomiting. Delayed gastric emptying was significantly associated (r = 0.78, P = 0.021) with CD4+α4ß7+CCR9+ lymphocytes and IL-1ß, TNF-α, and IL-10 secretion. CONCLUSIONS: Cellular immune activation with increased small bowel homing T cells may be key factors in the clinical manifestations of H. pylori-negative FD.

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.

Roles of muscarinic receptor subtypes in small intestinal motor dysfunction in acute radiation enteritis.

Administration of abdominal radiotherapy results in small intestinal motor dysfunction. We have developed a rat radiation enteritis model that, after exposure in vivo, shows high-amplitude, long-duration (HALD) pressure waves in ex vivo ileal segments. These resemble in vivo dysmotility where giant contractions migrate both antegradely and retrogradely. Mediation of these motor patterns is unclear, although enteric neural components are implicated. After the induction of acute radiation enteritis in vivo, ileal segments were isolated and arterially perfused. TTX, hexamethonium, atropine, or the selective muscarinic antagonists pirenzepine (M(1)), methoctramine (M(2)), and 1,1-dimethyl-4-diphenylacetoxypiperidinium iodide (4-DAMP; M(3)) were added to the perfusate. The baseline mean rate per minute per channel of HALD pressure waves was 0.35 +/- 0.047. This was significantly reduced by TTX (83.3%, P < 0.01), hexamethonium (90.3%, P < 0.03), and atropine (98.4%, P < 0.01). The HALD pressure wave mean rate per minute per channel was significantly reduced by pirenzepine (81.1%, P < 0.03), methoctramine (96.8%, P < 0.001), and 4-DAMP (93.1%, P < 0.03) compared with predrug baseline data. As an indicator of normal motility patterns, the frequency of low-amplitude, short-duration pressure waves was also assessed. The mean rate per minute per channel of 5.15 +/- 0.98 was significantly increased by TTX (19%, P < 0.05) but significantly reduced by pirenzepine (35.1%, P < 0.02) and methoctramine (75%, P < 0.0003). However, the rate of small-amplitude pressure waves was not affected by hexamethonium, atropine, or the M(3) antagonist 4-DAMP. The data indicate a role for neuronal mechanisms and the specific involvement of cholinergic receptors in generating dysmotility in acute radiation enteritis. The effect of selective M(3) receptor antagonism suggests that M(3) receptors may provide specific therapeutic targets in acute radiation enteritis.

Ghrelin selectively reduces mechanosensitivity of upper gastrointestinal vagal afferents.

Ghrelin is a peptide released from gastric endocrine cells that has an orexigenic effect via a vagal pathway. Here we determine the effect of ghrelin on mechanosensitivity of upper-intestinal vagal afferent fibers in ferret and mouse. The responses of gastroesophageal vagal afferents to graded mechanical stimulation were determined in vitro before and during application of ghrelin to their peripheral endings. Three types of vagal afferent were tested: tension receptors responding to circumferential tension, mucosal receptors responding only to mucosal stroking, and tension/mucosal (TM) receptors in ferret esophagus that responded to both stimuli. In the mouse, ghrelin did not significantly affect the response of mucosal receptors to mucosal stroking with calibrated von Frey hairs. However, it significantly reduced responses of tension receptors to circumferential tension (P < 0.005; two-way ANOVA) by up to 40%. This inhibition was reversed by the ghrelin receptor antagonist [d-Lys-3]-growth hormone-releasing peptide (GHRP)-6. In the ferret, ghrelin significantly reduced the response of mucosal and TM receptors to mucosal stroking with calibrated von Frey hairs. Surprisingly, ghrelin did not significantly alter the response to circumferential tension in either tension or TM receptors. RT-PCR analysis indicated that both ghrelin and its receptor are expressed in vagal afferent cell bodies in mouse nodose ganglia. In conclusion, ghrelin selectively inhibits subpopulations of mechanically sensitive gastroesophageal vagal afferents; there is also potential for ghrelin release from vagal afferents. However, the subpopulation of afferents inhibited differs between species. These data have broad implications for ghrelin's role in food intake regulation and reflex control of gastrointestinal function.

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 alpha,beta-methylene adenosine 5'-triphosphate (alpha,beta-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 microm). Significantly fewer PN afferents (7%) responded to alpha,beta-meATP. Correspondingly, 36% of colonic thoracolumbar DRG neurones exhibited P2X3-LI compared with only 19% of colonic lumbosacral neurones. Capsaicin (3 microm) 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 alpha,beta-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.