Crypt and Villus Enterochromaffin Cells are Distinct Stress Sensors in the Gut.

The crypt-villus structure of the small intestine serves as an essential protective barrier, with its integrity monitored by the gut's sensory system. Enterochromaffin (EC) cells, which are rare sensory epithelial cells that release serotonin (5-HT), surveil the mucosal environment and signal both within and outside the gut. However, it remains unclear whether EC cells in intestinal crypts and villi respond to different stimuli and elicit distinct responses. In this study, we introduce a new reporter mouse model to observe the release and propagation of serotonin in live intestines. Using this system, we show that crypt EC cells exhibit two modes of serotonin release: transient receptor potential A1 (TRPA1)-dependent tonic serotonin release that controls basal ionic secretion, and irritant-evoked serotonin release that activates gut sensory neurons. Furthermore, we find that a thick protective mucus layer prevents TRPA1 receptors on crypt EC cells from responding to luminal irritants such as reactive electrophiles; if this mucus layer is compromised, then crypt EC cells become susceptible to activation by luminal irritants. On the other hand, villus EC cells detect oxidative stress through TRPM2 channels and co-release serotonin and ATP to activate nearby gut sensory fibers. Our work highlights the physiological importance of intestinal architecture and differential TRP channel expression in sensing noxious stimuli that elicit nausea and/or pain sensations in the gut.

Activation of pruritogenic TGR5, MrgprA3, and MrgprC11 on colon-innervating afferents induces visceral hypersensitivity.

Itch induces scratching that removes irritants from the skin, whereas pain initiates withdrawal or avoidance of tissue damage. While pain arises from both the skin and viscera, we investigated whether pruritogenic irritant mechanisms also function within visceral pathways. We show that subsets of colon-innervating sensory neurons in mice express, either individually or in combination, the pruritogenic receptors Tgr5 and the Mas-gene-related GPCRs Mrgpra3 and Mrgprc11. Agonists of these receptors activated subsets of colonic sensory neurons and evoked colonic afferent mechanical hypersensitivity via a TRPA1-dependent mechanism. In vivo intracolonic administration of individual TGR5, MrgprA3, or MrgprC11 agonists induced pronounced visceral hypersensitivity to colorectal distension. Coadministration of these agonists as an "itch cocktail" augmented hypersensitivity to colorectal distension and changed mouse behavior. These irritant mechanisms were maintained and enhanced in a model of chronic visceral hypersensitivity relevant to irritable bowel syndrome. Neurons from human dorsal root ganglia also expressed TGR5, as well as the human ortholog MrgprX1, and showed increased responsiveness to pruritogenic agonists in pathological states. These data support the existence of an irritant-sensing system in the colon that is a visceral representation of the itch pathways found in skin, thereby contributing to sensory disturbances accompanying common intestinal disorders.

Structure-Activity Studies Reveal the Molecular Basis for GABAB-Receptor Mediated Inhibition of High Voltage-Activated Calcium Channels by α-Conotoxin Vc1.1.

α-Conotoxins are disulfide-bonded peptides from cone snail venoms and are characterized by their affinity for nicotinic acetylcholine receptors (nAChR). Several α-conotoxins with distinct selectivity for nAChR subtypes have been identified as potent analgesics in animal models of chronic pain. However, a number of α-conotoxins have been shown to inhibit N-type calcium channel currents in rodent dissociated dorsal root ganglion (DRG) neurons via activation of G protein-coupled GABAB receptors (GABABR). Therefore, it is unclear whether activation of GABABR or inhibition of α9α10 nAChRs is the analgesic mechanism. To investigate the mechanisms by which α-conotoxins provide analgesia, we synthesized a suite of Vc1.1 analogues where all residues, except the conserved cysteines, in Vc1.1 were individually replaced by alanine (A), lysine (K), and aspartic acid (D). Our results show that the amino acids in the first loop play an important role in binding of the peptide to the receptor, whereas those in the second loop play an important role for the selectivity of the peptide for the GABABR over α9α10 nAChRs. We designed a cVc1.1 analogue that is >8000-fold selective for GABABR-mediated inhibition of high voltage-activated (HVA) calcium channels over α9α10 nAChRs and show that it is analgesic in a mouse model of chronic visceral hypersensitivity (CVH). cVc1.1[D11A,E14A] caused dose-dependent inhibition of colonic nociceptors with greater efficacy in ex vivo CVH colonic nociceptors relative to healthy colonic nociceptors. These findings suggest that selectively targeting GABABR-mediated HVA calcium channel inhibition by α-conotoxins could be effective for the treatment of chronic visceral pain.

Cyclic analogues of α-conotoxin Vc1.1 inhibit colonic nociceptors and provide analgesia in a mouse model of chronic abdominal pain.

BACKGROUND AND PURPOSE: Patients with irritable bowel syndrome suffer from chronic visceral pain (CVP) and limited analgesic therapeutic options are currently available. We have shown that α-conotoxin Vc1.1 induced activation of GABAB receptors on the peripheral endings of colonic afferents and reduced nociceptive signalling from the viscera. However, the analgesic efficacy of more stable, cyclized versions of Vc1.1 on CVP remains to be determined. EXPERIMENTAL APPROACH: Using ex vivo colonic afferent preparations from mice, we determined the inhibitory actions of cyclized Vc1.1 (cVc1.1) and two cVc1.1 analogues on mouse colonic nociceptors in healthy and chronic visceral hypersensitivity (CVH) states. Using whole-cell patch clamp recordings, we also assessed the inhibitory actions of these peptides on the neuronal excitability of colonic innervating dorsal root ganglion neurons. In vivo, the analgesic efficacy of these analogues was assessed by determining the visceromotor response to colorectal distension in healthy and CVH mice. KEY RESULTS: cVc1.1 and the cVc1.1 analogues, [C2H,C8F]cVc1.1 and [N9W]cVc1.1, all caused concentration-dependent inhibition of colonic nociceptors from healthy mice. Inhibition by these peptides was greater than those evoked by linear Vc1.1 and was substantially greater in colonic nociceptors from CVH mice. cVc1.1 also reduced excitability of colonic dorsal root ganglion neurons, with greater effect in CVH neurons. CVH mice treated with cVc1.1 intra-colonically displayed reduced pain responses to noxious colorectal distension compared with vehicle-treated CVH mice. CONCLUSIONS AND IMPLICATIONS: Cyclic versions of Vc1.1 evoked significant anti-nociceptive actions in CVH states, suggesting that they could be novel candidates for treatment of CVP. LINKED ARTICLES: This article is part of a themed section on Recent Advances in Targeting Ion Channels to Treat Chronic Pain. To view the other articles in this section visit http://onlinelibrary.wiley.com/doi/10.1111/bph.v175.12/issuetoc.

Enterochromaffin Cells Are Gut Chemosensors that Couple to Sensory Neural Pathways.

Dietary, microbial, and inflammatory factors modulate the gut-brain axis and influence physiological processes ranging from metabolism to cognition. The gut epithelium is a principal site for detecting such agents, but precisely how it communicates with neural elements is poorly understood. Serotonergic enterochromaffin (EC) cells are proposed to fulfill this role by acting as chemosensors, but understanding how these rare and unique cell types transduce chemosensory information to the nervous system has been hampered by their paucity and inaccessibility to single-cell measurements. Here, we circumvent this limitation by exploiting cultured intestinal organoids together with single-cell measurements to elucidate intrinsic biophysical, pharmacological, and genetic properties of EC cells. We show that EC cells express specific chemosensory receptors, are electrically excitable, and modulate serotonin-sensitive primary afferent nerve fibers via synaptic connections, enabling them to detect and transduce environmental, metabolic, and homeostatic information from the gut directly to the nervous system.

TRPV1 Channels and Gastric Vagal Afferent Signalling in Lean and High Fat Diet Induced Obese Mice.

AIM: Within the gastrointestinal tract vagal afferents play a role in control of food intake and satiety signalling. Activation of mechanosensitive gastric vagal afferents induces satiety. However, gastric vagal afferent responses to mechanical stretch are reduced in high fat diet mice. Transient receptor potential vanilloid 1 channels (TRPV1) are expressed in vagal afferents and knockout of TRPV1 reduces gastro-oesophageal vagal afferent responses to stretch. We aimed to determine the role of TRPV1 on gastric vagal afferent mechanosensitivity and food intake in lean and HFD-induced obese mice. METHODS: TRPV1+/+ and -/- mice were fed either a standard laboratory diet or high fat diet for 20wks. Gastric emptying of a solid meal and gastric vagal afferent mechanosensitivity was determined. RESULTS: Gastric emptying was delayed in high fat diet mice but there was no difference between TRPV1+/+ and -/- mice on either diet. TRPV1 mRNA expression in whole nodose ganglia of TRPV1+/+ mice was similar in both dietary groups. The TRPV1 agonist N-oleoyldopamine potentiated the response of tension receptors in standard laboratory diet but not high fat diet mice. Food intake was greater in the standard laboratory diet TRPV1-/- compared to TRPV1+/+ mice. This was associated with reduced response of tension receptors to stretch in standard laboratory diet TRPV1-/- mice. Tension receptor responses to stretch were decreased in high fat diet compared to standard laboratory diet TRPV1+/+ mice; an effect not observed in TRPV1-/- mice. Disruption of TRPV1 had no effect on the response of mucosal receptors to mucosal stroking in mice on either diet. CONCLUSION: TRPV1 channels selectively modulate gastric vagal afferent tension receptor mechanosensitivity and may mediate the reduction in gastric vagal afferent mechanosensitivity in high fat diet-induced obesity.

Neuropeptide W modulation of gastric vagal afferent mechanosensitivity: Impact of age and sex.

AIM: Gastric vagal afferents are activated in response to mechanical stimulation, an effect attenuated by neuropeptide W (NPW) in 20-week-old female mice. In this study we aimed to determine whether there were age and sex dependent effects of NPW on gastric vagal afferent mechanosensitivity. METHODS: An in vitro gastro-oesophageal preparation was used to determine the effect of NPW on gastric vagal afferent mechanosensitivity from 8 and 20-week-old male and female C57BL/6 mice. Retrograde tracing and laser capture microdissection were used to selectively collect gastric vagal afferent cell bodies. Expression of NPW in the gastric mucosa and its receptor, GPR7, in gastric vagal afferent cell bodies was determined using quantitative RT-PCR. RESULTS: NPW inhibited gastric tension sensitive vagal afferents from 20-week-old male and female mice, but not 8-week-old mice. In contrast, NPW inhibited the mechanosensitivity of gastric mucosal vagal afferents in 8-week-old male and female mice, but not 20-week-old mice. NPW mRNA expression in the gastric mucosa was higher in 20-week-old male mice compared to 8-week-old male mice. GPR7 mRNA expression in vagal afferent neurons innervating the gastric muscular layers was higher in 20-week-old mice compared to 8-week-old mice in both sexes. CONCLUSION: The inhibitory effect of NPW on gastric tension sensitive and mucosal vagal afferents is age but not sex-dependent. These findings suggest that the physiological role of NPW varies depending on the age of the mice.

Diet-dependent modulation of gastro-oesphageal vagal afferent mechanosensitivity by endogenous nitric oxide.

Neuronal nitric oxide (NO) plays an important role in gastric motor activity and modulates the mechanosensitivity of gastro-oesophageal vagal afferents. Effects of NO on food intake are dependent on feeding status. We sought to determine the effect of NO on gastro-oesophageal vagal afferent activity in the normally fed and food-restricted states and the second messenger pathways mediating these effects. Eight week old female C56BL/6 mice were fed ad libitum or food restricted for 14 h. An in vitro preparation was used to determine the functional effects of NO and the second messenger pathways involved. Expression of NO signal transduction molecules in vagal afferents was determined by reverse-transcription polymerase chain reaction (RT-PCR). Endogenous NO and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) inhibited vagal mucosal afferent responses to tactile stimuli in mice fed ad libitum. After a 14 h fast endogenous NO and SNAP potentiated tension and mucosal afferent responses to mechanical stimulation. The excitatory effect of NO was blocked by the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase inhibitor apocynin. After a 14 h fast expression of NADPH oxidase 2 (NOX2) mRNA in whole nodose ganglia was significantly reduced and the excitatory effect of NO on gastro-oesophageal vagal afferents was lost. Under fasting conditions the inhibitory effect of NO was blocked with the hyperpolarisation-activated cyclic nucleotide-gated (HCN) channel blocker ivabradine and mRNA expression of HCN3 in the nodose ganglia was elevated. In conclusion, the role of NO in the peripheral modulation of gastro-oesophageal vagal afferents is dynamic and dependent on feeding status.

Gastric vagal afferent modulation by leptin is influenced by food intake status.

Energy intake is strongly influenced by vagal afferent signals from the stomach, and is also modulated by leptin. Leptin may be secreted from gastric epithelial cells, so we aimed to determine the direct effect of leptin on gastric vagal afferents under different feeding conditions. Female C57BL/6 mice were fed standard laboratory diet, high-fat diet or were food restricted. The expression of leptin receptor (Lep-R) and its signal transduction molecules in vagal afferents was determined by retrograde tracing and reverse-transcription polymerase chain reaction, and the relationship between leptin-immunopositive cells and gastric vagal afferent endings determined by anterograde tracing and leptin immunohistochemistry. An in vitro preparation was used to determine the functional effects of leptin on gastric vagal afferents and the second messenger pathways involved. Leptin potentiated vagal mucosal afferent responses to tactile stimuli, and epithelial cells expressing leptin were found close to vagal mucosal endings. After fasting or diet-induced obesity, potentiation of mucosal afferents by leptin was lost and Lep-R expression reduced in the cell bodies of gastric mucosal afferents. These effects in diet-induced obese mice were accompanied by a reduction in anatomical vagal innervation of the gastric mucosa. In striking contrast, after fasting or diet-induced obesity, leptin actually inhibited responses to distension in tension receptors. The inhibitory effect on gastric tension receptors was mediated through phosphatidylinositol 3-kinase-dependent activation of large-conductance calcium-activated potassium channels. The excitatory effect of leptin on gastric mucosal vagal afferents was mediated by phospholipase C-dependent activation of canonical transient receptor potential channels. These data suggest the effect of leptin on gastric vagal afferent excitability is dynamic and related to the feeding state. Paradoxically, in obesity, leptin may reduce responses to gastric distension following food intake.

Diet-induced adaptation of vagal afferent function.

Afferent signals from the stomach play an important role in inhibition of food intake during a meal. The gastric hormone ghrelin can influence gastric satiety signalling by altering the sensitivity of gastric vagal afferents. Changes in diet, including food restriction and high fat diet (HFD) alter satiety signalling. We hypothesised that the function of gastric vagal afferent endings are affected by both a period of food restriction and a high fat diet, and that the inhibitory effect of ghrelin on vagal afferents is influenced by the different feeding conditions. We found that both fasting and HFD reduced the responses of gastric vagal tension receptors to distension, but not responses of mucosal receptors to mucosal contact. We traced vagal afferents anterogradely to their terminals in the mucosa where we found they were in close apposition to ghrelin-containing cells. Ghrelin receptor mRNA was expressed in vagal afferent cell bodies of the nodose ganglia, and increased in response to caloric restriction, but decreased in HFD mice. In control mice, ghrelin decreased the sensitivity of tension but not mucosal receptors. After caloric restriction or high fat diet, ghrelin inhibited mucosal receptors, and the inhibition of mechanosensitive tension receptors was enhanced. Therefore, both caloric restriction and HFD decrease mechanosensory vagal afferent signals, and augment the inhibitory effect of ghrelin on vagal afferents, but different mechanisms mediate the short- and longer-term changes.

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.

Nitric oxide as an endogenous peripheral modulator of visceral sensory neuronal function.

Nitric oxide (NO) plays important roles in CNS and smooth muscle function. Here we reveal an additional function in peripheral sensory transmission. We hypothesized that endogenous NO modulates the function of gastrointestinal vagal afferent endings. The nonselective NO synthase (NOS) inhibitor N(G)-nitro-L-arginine methyl ester hydrochloride increased responses to tactile mechanical stimuli of mucosal afferent endings in two species, in some cases severalfold. This was mimicked by a neuronal NOS inhibitor but not an endothelial NOS inhibitor. NOS inhibitors did not affect the responsiveness of smooth muscle afferent endings, suggesting that the endogenous source of NO is exclusively accessible to mucosal receptors. The role of the NO-soluble guanylyl cyclase (sGC)-cGMP pathway was confirmed using the sGC inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-1-one and the cGMP phosphodiesterase 5' inhibitor sildenafil. The first enhanced and the second inhibited mechanosensory function. Exogenous NO, from the donor S-nitroso-N-acetylpenicillamine, significantly reduced mechanosensitivity of both types of ending. Up to one-third of stomach-projecting afferent neurons in the nodose ganglia expressed neuronal NOS (nNOS). However, anterograde-traced vagal endings were nNOS negative, indicating NOS is not transported peripherally and there are alternative sources of NO for afferent modulation. A subpopulation of enteroendocrine cells in the gut mucosa were nNOS positive, which were found anatomically in close apposition with mucosal vagal afferent endings. These results indicate an inhibitory neuromodulatory role of epithelial NO, which targets a select population of vagal afferents. This interaction is likely to play a role in generation of symptoms and behaviors from the upper gastrointestinal system.

Opioid modulation of ferret vagal afferent mechanosensitivity.

Despite universal use of opioids in the clinic to inhibit pain, there is relatively little known of their peripheral actions on sensory nerve endings, where in fact they may be better targeted with more widespread applications. Here we show differential effects of mu-, kappa-, and delta-opioids on mechanosensitive ferret esophageal vagal afferent endings investigated in vitro. The effects of selective agonists [d-Ala(2),N-Me-Phe(4),Gly-ol(5)]-enkephalin (DAMGO), 2-(3, 4-dichlorophenyl)-N-methyl-N-[(1S)-1phenyl-2-(1-pyrrolidinyl) ethyl] acetamide hydrochlorine (ICI 199441), and (+)-4-[(alphaR)-alpha-((2S,5R)-4-allyl-2,5-dimethyl-1-piperazinyl)-3-methoxybenzyl]-N,N-diethylbenzamide (SNC-80), respectively, on mechanosensory stimulus-response functions were quantified. DAMGO (10(-7) to 10(-5) M) reduced the responses of tension receptors to circumferential tension (1-5 g) by up to 50%, and the responses of mucosal receptors to mucosal stroking (10-1,000 mg von Frey hair) by >50%. DAMGO effects were reversed by naloxone (10(-5) M). Tension/mucosal (TM) receptor responses to tension and stroking were unaffected by DAMGO. ICI 199441 (10(-6) to 10(-5) M) potently inhibited all responses except TM receptor responses to tension, and SNC-80 (10(-5) to 10(-3) M) had no effect other than a minor inhibition of mucosal receptor responses to intense stimuli at 10(-3) M. We conclude that mu- and kappa-opioids have potent and selective peripheral effects on esophageal vagal afferents that may have applications in treatment of disorders of visceral sensation.

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.

Peripheral versus central modulation of gastric vagal pathways by metabotropic glutamate receptor 5.

Metabotropic glutamate receptors (mGluR) are classified into group I, II, and III mGluR. Group I (mGluR1, mGluR5) are excitatory, whereas group II and III are inhibitory. mGluR5 antagonism potently reduces triggering of transient lower esophageal sphincter relaxations and gastroesophageal reflux. Transient lower esophageal sphincter relaxations are mediated via a vagal pathway and initiated by distension of the proximal stomach. Here, we determined the site of action of mGluR5 in gastric vagal pathways by investigating peripheral responses of ferret gastroesophageal vagal afferents to graded mechanical stimuli in vitro and central responses of nucleus tractus solitarius (NTS) neurons with gastric input in vivo in the presence or absence of the mGluR5 antagonist 2-methyl-6-(phenylethynyl)pyridine (MPEP). mGluR5 were also identified immunohistochemically in the nodose ganglia and NTS after extrinsic vagal inputs had been traced from the proximal stomach. Gastroesophageal vagal afferents were classified as mucosal, tension, or tension-mucosal (TM) receptors. MPEP (1-10 microM) inhibited responses to circumferential tension of tension and TM receptors. Responses to mucosal stroking of mucosal and TM receptors were unaffected. MPEP (0.001-10 nmol icv) had no major effect on the majority of NTS neurons excited by gastric distension or on NTS neurons inhibited by distension. mGluR5 labeling was abundant in gastric vagal afferent neurons and sparse in fibers within NTS vagal subnuclei. We conclude that mGluR5 play a prominent role at gastroesophageal vagal afferent endings but a minor role in central gastric vagal pathways. Peripheral mGluR5 may prove a suitable target for reducing mechanosensory input from the periphery, for therapeutic benefit.

Metabotropic glutamate receptors inhibit mechanosensitivity in vagal sensory neurons.

BACKGROUND AND AIMS: Inhibitory G-protein-coupled receptors have demonstrated potential in treatment of gastroesophageal reflux disease (GERD) through actions on vagal afferent signaling. Metabotropic glutamate receptors (mGluR) belong to this receptor family and have great pharmacologic and molecular diversity, with 8 subtypes. We investigated mGluR in the vagal system of humans and other species. METHODS: Expression of mGluR1-8 in human, dog, ferret, and rodent nodose ganglia was investigated by reverse-transcription polymerase chain reaction. mGluR1-8 immunohistochemistry was performed in combination with retrograde tracing of vagal afferents from ferret proximal stomach to nodose ganglia. Transport of mGluR peripherally was investigated by vagal ligation, followed by immunohistochemistry. Glutamate receptor pharmacology of ferret and rodent gastroesophageal vagal afferents was investigated by testing single fiber responses to graded mechanical stimuli during drug application to their peripheral endings. RESULTS: Messenger RNA for several mGluR was detected in the nodose ganglia of all species. Retrograde tracing indicated that ferret gastric vagal afferents express mGluR protein. Accumulation of immunoreactivity proximal to a ligature showed that mGluR were transported peripherally in the vagus nerves. Glutamate (1-30 mumol/L with kynurenate 0.1 mmol/L) concentration dependently inhibited vagal afferent mechanosensitivity. This was mimicked by selective group II and III mGluR agonists but not by a group I agonist. Conversely, a group III mGluR antagonist increased mechanosensitivity to intense stimuli. CONCLUSIONS: Both exogenous and endogenous glutamate inhibits mechanosensitivity of vagal afferents. Group II (mGluR2 and 3) and group III mGluR (mGluR4, 6, 7, 8) are novel targets for inhibition of vagal signaling with therapeutic potential in, for example, GERD.

Modulation of gastro-oesophageal vagal afferents by galanin in mouse and ferret.

The neuropeptide galanin is found in the central and peripheral nervous systems. It may have excitatory or inhibitory actions via three subtypes of G-protein-coupled receptor, and it modulates the mechanosensitivity of somatic sensory fibres. We aimed to determine if galanin also modulates vagal afferent mechanosensitivity, and to localize endogenous sources. The responses of ferret and mouse gastro-oesophageal vagal afferents to graded mechanical stimuli were investigated in vitro. The effects of galanin and/or the galanin receptor antagonist galantide on these responses were quantified. Immunohistochemistry for galanin was performed in ferret and mouse proximal stomach and nodose ganglion. In ferrets, retrograde labelling of gastric afferents to the nodose ganglion was combined with immunohistochemistry. When exposed to galanin (1-10 nM), 18/31 ferret and 12/15 mouse gastro-oesophageal afferents (tension, mucosal and tension/mucosal receptors) showed inhibition of mechanosensitivity. Four of 31 ferret afferents showed potentiation of mechanosensitivity, and 9/31 were unaffected (2/15 and 1/15 in mouse, respectively). Galanin effects were reversed after washout or by galantide (10-30 nM). Galantide given alone increased mechanosensitivity. Galanin immunoreactivity was found in nodose neurones, including those innervating the stomach in ferret. Enteric neurones were also galanin immunoreactive, as were endings associated with myenteric ganglia and smooth muscle. We conclude that galanin potently modulates mechanosensitivity of gastro-oesophageal vagal afferents with either facilitatory or inhibitory actions on individual afferent fibres. Both intrinsic and extrinsic (vagal) neurones contain galanin and are therefore potential sources of endogenous galanin.