Tracey A. O'Donnell

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.

P2X purinoceptor‐induced sensitization of ferret vagal mechanoreceptors in oesophageal inflammation

1 Using an in vitro single unit recording technique we studied the changes in mechanical and chemical sensitivity of vagal afferent fibres in acute oesophagitis, with particular attention to inflammatory products such as purines. 2 Histologically verified oesophagitis was induced by oesophageal perfusion of 1 mg ml−1 pepsin in 150 mM HCl in anaesthetized ferrets for 30 min on two consecutive days. Controls were infused with 154 mM NaCl. 3 The number of action potentials evoked in oesophageal mucosal afferents by mucosal stroking with calibrated von Frey hairs (10–1000 mg) was stimulus dependent. In oesophagitis responsiveness was reduced across the range of stimuli compared with controls. 4 Topical application of the P2X purinoceptor agonist αβ‐methylene ATP had no direct excitatory effect on afferents. In oesophagitis, but not in controls, there was a significant increase in responses to stroking with von Frey hairs during superfusion with αβ‐methylene ATP (1 μM). 5 Mucosal afferents responded directly to one or more chemical stimuli: 26 % (5/19 afferents) responded in controls, and 47 % (7/15 afferents) in oesophagitis. There were no differences in responsiveness to bradykinin (1 μM), prostaglandin E2 (100 μM), 5‐hydroxytryptamine (100 μM), capsaicin (1 mM) or hydrochloric acid (150 mM) between control and oesophagitis groups. 6 We conclude that a sensitizing effect of a P2X purinoceptor agonist on mechanosensory function is induced in oesophagitis. This effect is offset by a decrease in basal mechanosensitivity.

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

•  Obesity occurs when energy intake exceeds expenditure, and the excess energy is stored as fat. •  We show that, after a 14 h food deprivation or 12 weeks consumption of a high‐fat diet, gastric vagal afferent responses to mechanical stimulation in the presence of the satiety peptide leptin are altered. •  Leptin has an excitatory effect on gastric mucosal vagal afferents, which is abolished after food restriction or prolonged excess. •  In contrast, leptin has an inhibitory effect on gastric tension‐sensitive afferents, but only after food restriction or energy excess conditions. •  These changes in the response to leptin in the stomach, after food restriction or prolonged high‐fat feeding, occur in such a manner as to facilitate an increase in food intake in both conditions.

GABAB receptor‐mediated effects on vagal pathways to the lower oesophageal sphincter and heart

GABAB receptors influencing vagal pathways to the lower oesophageal sphincter and heart were investigated. In urethane‐anaesthetized ferrets, the GABAB agonist baclofen (7 μmol kg−1 i.v.) increased basal lower oesophageal sphincter (LOS) pressure. This was reversed by antagonism with CGP35348 (100 μmol kg−1 i.v.). Baclofens effect was abolished by vagotomy, suggesting a central action, yet it was ineffective when given centrally (3–6 nmol i.c.v.). Peripheral vagal stimulation (10 Hz, 5 s duration) caused LOS inhibition, followed by excitation, then prolonged inhibition. Bradycardia was also evoked during stimulation. Bradycardia and LOS responses were abolished after chronic supranodose vagotomy, indicating that they were due to stimulation of vagal pre‐ganglionic neurones, not antidromic stimulation of afferents. Baclofen (1–10 μmol kg−1) reduced bradycardia and enhanced LOS excitation, which was also seen in animals pretreated with atropine (400 μg kg−1 i.v.) and guanethidine (5 mg kg−1 i.v.), but not in those pretreated with L‐NAME (100 mg kg−1 i.v.). Effects of baclofen (7 μmol kg−1 i.v.) on vagal stimulation‐induced LOS and cardiac responses were unchanged by the GABAB antagonists CGP35348 or CGP36742 (up to 112 μmol kg−1 i.v.), but were reversed by CGP62349 (ED50 37 nmol kg−1 i.v.) or CGP54626 (ED50 100 nmol kg−1 i.v.). Responses of isolated LOS strips to electrical stimulation, capsaicin, NK‐1, NK‐2 and nicotinic receptor agonists were all unaffected by baclofen (200 μ M). We conclude that baclofen reduces vagal output at two peripheral sites: one presynaptically on pre‐ganglionic neurones (CGP35348‐insensitive), and another (CGP35348‐sensitive) that could not be identified. This demonstrates heterogeneity of GABAB receptors through differential sensitivity to antagonists.

Inhibition of mechanosensitivity in visceral primary afferents by GABAB receptors involves calcium and potassium channels

GABA(B) receptors inhibit mechanosensitivity of visceral afferents. This is associated with reduced triggering of events that lead to gastro-esophageal reflux, with important therapeutic consequences. In other neuronal systems, GABA(B) receptor activation may be linked via G-proteins to reduced N-type Ca(2+) channel opening, increased inward rectifier K(+) channel opening, plus effects on a number of intracellular messengers. Here we aimed to determine the role of Ca(2+) and K(+) channels in the inhibition of vagal afferent mechanoreceptor function by the GABA(B) receptor agonist baclofen. The responses of three types of ferret gastro-esophageal vagal afferents (mucosal, tension and tension mucosal receptors) to graded mechanical stimuli were investigated in vitro. The effects of baclofen (200 microM) alone on these responses were quantified, and the effects of baclofen in the presence of the G-protein-coupled inward rectifier potassium channel blocker Rb(+) (4.7 mM) and/or the N-type calcium channel blocker omega-conotoxin GVIA (0.1 microM). Baclofen inhibition of mucosal receptor mechanosensitivity was abolished by both blockers. Its inhibitory effect on tension mucosal receptors was partly reduced by both. The inhibitory effect of baclofen on tension receptors was unaffected. The data indicate that the inhibitory action of GABA(B) receptors is mediated via different pathways in mucosal, tension and tension mucosal receptors via mechanisms involving both N-type Ca(2+) channels and inwardly rectifying K(+) channels and others.

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.

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 NG-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.

Altered gastric vagal mechanosensitivity in diet-induced obesity persists on return to normal chow and is accompanied by increased food intake

Background and aims:Gastric vagal afferents convey satiety signals in response to mechanical stimuli. The sensitivity of these afferents is decreased in diet-induced obesity. Leptin, secreted from gastric epithelial cells, potentiates the response of vagal afferents to mechanical stimuli in lean mice, but has an inhibitory effect in high-fat diet (HFD)-induced obese mice. We sought to determine whether changes in vagal afferent function and response to leptin in obesity were reversible by returning obese mice consuming a HFD to standard laboratory chow diet (SLD).Methods:Eight-week-old female C57BL/6 mice were either fed a SLD (N=20) or HFD (N=20) for 24 weeks. A third group was fed a HFD for 12 weeks and then a SLD for a further 12 weeks (RFD, N=18). An in vitro gastro-oesophageal vagal afferent preparation was used to determine the mechanosensitivity of gastric vagal afferents and the modulatory effect of leptin (0.1–10 nM) was examined. Retrograde tracing and quantitative RT–PCR were used to determine the expression of leptin receptor (LepR) messenger RNA (mRNA) in whole nodose and specific cell bodies traced from the stomach.Results:After 24 weeks, both the HFD and RFD mice had increased body weight, gonadal fat mass, plasma leptin, plasma insulin and daily energy consumption compared with the SLD mice. The HFD and RFD mice had reduced tension receptor mechanosensitivity and leptin further inhibited responses to tension in HFD, RFD but not SLD mice. Mucosal receptors from both the SLD and RFD mice were potentiated by leptin, an effect not seen in HFD mice. LepR expression was unchanged in the whole nodose, but was reduced in the mucosal afferents of the HFD and RFD mice.Conclusion:Disruption of gastric vagal afferent function by HFD-induced obesity is only partially reversible by dietary change, which provides a potential mechanism preventing maintenance of weight loss.

Modulation of murine gastric vagal afferent mechanosensitivity by neuropeptide W.

Neuropeptide W (NPW) is an endogenous ligand for the receptors GPR7 and GPR8 and is involved in central regulation of energy homeostasis. NPW in the periphery is found in gastric gastrin (G) cells. In the stomach, energy intake is influenced by vagal afferent signals, so we aimed to determine the effect of NPW on mechanosensitive gastric vagal afferents under different feeding conditions.

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.