The ion channel TRPA1 is required for normal mechanosensation and is modulated by algesic stimuli.
ABSTRACT: 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.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.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.These findings demonstrate a previously unrecognized role for TRPA1 in normal and inflamed mechanosensory function and nociception within the viscera.
Project description:Vagal afferent neurons relay important information regarding the control of the gastrointestinal system. However, the ionic mechanisms that underlie vagal activation induced by sensory inputs are not completely understood. We postulate that transient receptor potential (TRP) channels and/or two-pore potassium (K2p) channels are targets for activating vagal afferents. In this study we explored the distribution of these channels in vagal afferents by quantitative PCR after a capsaicin treatment to eliminate capsaicin-sensitive neurons, and by single-cell PCR measurements in vagal afferent neurons cultured after retrograde labeling from the stomach or duodenum. We found that TRPC1/3/5/6, TRPV1-4, TRPM8, TRPA1, TWIK2, TRAAK, TREK1, and TASK1/2 were all present in rat nodose ganglia. Both lesion results and single-cell PCR results suggested that TRPA1 and TRPC1 were preferentially expressed in neurons that were either capsaicin sensitive or TRPV1 positive. Expression of TRPM8 varied dynamically after various manipulations, which perhaps explains the disparate results obtained by different investigators. Last, we also examined ion channel distribution with the A-type CCK receptor (CCK-R(A)) and found there was a significant preference for neurons that express TRAAK to also express CCK-R(A), especially in gut-innervating neurons. These findings, combined with findings from prior studies, demonstrated that background conductances such as TRPC1, TRPA1, and TRAAK are indeed differentially distributed in the nodose ganglia, and not only do they segregate with specific markers, but the degree of overlap is also dependent on the innervation target.
Project description:The nerve growth factor (NGF) and glial cell line-derived neurotrophic factor (GDNF) families of growth factors regulate the sensitivity of sensory neurons. The ion channels transient receptor potential vanilloid 1 (TRPV1) and transient receptor potential channel, subfamily A, member 1 (TRPA1), are necessary for development of inflammatory hypersensitivity and are functionally potentiated by growth factors. We have shown previously that inflamed skin exhibits rapid increases in artemin mRNA with slower, smaller increases in NGF mRNA. Here, using mice, we show that, in inflamed colon, mRNA for both growth factors increased with a pattern distinct from that seen in skin. Differences were also seen in the pattern of TRPV1 and TRPA1 mRNA expression in DRG innervating inflamed skin and colon. Growth factors potentiated capsaicin (a specific TRPV1 agonist) and mustard oil (a specific TRPA1 agonist) behavioral responses in vivo, raising the question as to how these growth factors affect individual afferents. Because individual tissues are innervated by afferents with unique properties, we investigated modulation of TRPV1 and TRPA1 in identified afferents projecting to muscle, skin, and colon. Muscle and colon afferents are twice as likely as skin afferents to express functional TRPV1 and TRPA1. TRPV1 and TRPA1 responses were potentiated by growth factors in all afferent types, but compared with skin afferents, muscle afferents were twice as likely to exhibit NGF-induced potentiation and one-half as likely to exhibit artemin-induced potentiation of TRPV1. Furthermore, skin afferents showed no GDNF-induced potentiation of TRPA1, but 43% of muscle and 38% of colon afferents exhibited GDNF-induced potentiation. These results show that interpretation of afferent homeostatic mechanisms must incorporate properties that are specific to the target tissue.
Project description:There is a growing awareness of the role that TRP channels play in regulating sensory and motor functions in the gastrointestinal tract. In this study we used an in-vitro murine model of colonic peristaltic-like complexes (CPMCs) to evaluate the role of exogenous and endogenous TRPA1 signaling processes in regulating colonic motility. Using in-vitro recordings of intraluminal pressure to monitor the presence of CPMCs in colonic segments we performed a series of experiments on male CD1 mice (2 months of age) and found that CPMC activity was attenuated by TRPA1 agonists. Bath application of the TRPA1 antagonist HC-030031 had no effect upon basal CPMC activity whereas application of the synthetic TRPA1 agonist ASP7663 caused a reversible dose dependent decrease in CPMC frequency that was blocked by HC-030031. Cinnamaldehyde and 4-hydroxy-2-nonenal elicited long lasting decreases in CPMC frequency that were blocked by HC-030031 whereas the decreased CPMC activity invoked by AITC could not be blocked by HC-030031. Our results show that any potential mechanosensory function of TRPA1 doesn't involve contributing to distension induced colonic motor activity and that a role for TRPA1 in the colon is through regulating motility through exogenous and endogenous agonist induced inhibitory effects.
Project description:Glycoprotein 130 (gp130) is the signal transducing receptor subunit for cytokines of the interleukin-6 (IL-6) family, and it is expressed in a multitude of cell types of the immune and nervous system. IL-6-like cytokines are not only key regulators of innate immunity and inflammation but are also essential factors for the differentiation and development of the somatosensory system. Mice with a null mutation of gp130 in primary nociceptive afferents (SNS-gp130(-/-)) are largely protected from hypersensitivity to mechanical stimuli in mouse models of pathological pain. Therefore, we set out to investigate how neuronal gp130 regulates mechanonociception. SNS-gp130(-/-) mice revealed reduced mechanosensitivity to high mechanical forces in the von Frey assay in vivo, and this was associated with a reduced sensitivity of nociceptive primary afferents in vitro. Together with these findings, transient receptor potential ankyrin 1 (TRPA1) mRNA expression was significantly reduced in DRG from SNS-gp130(-/-) mice. This was also reflected by a reduced number of neurons responding with calcium transients to TRPA1 agonists in primary DRG cultures. Downregulation of Trpa1 expression was predominantly discovered in nonpeptidergic neurons, with the deficit becoming evident during stages of early postnatal development. Regulation of Trpa1 mRNA expression levels downstream of gp130 involved the classical Janus kinase family-signal transducer and activator of transcription pathway. Our results closely link proinflammatory cytokines to the expression of TRPA1, both of which have been shown to contribute to hypersensitive pain states. We suggest that gp130 has an essential role in mechanonociception and in the regulation of TRPA1 expression.
Project description:The functional roles of transient receptor potential (TRP) channels in the gastrointestinal tract have garnered considerable attention in recent years. We previously reported that daikenchuto (TU-100), a traditional Japanese herbal medicine, increased intestinal blood flow (IBF) via adrenomedullin (ADM) release from intestinal epithelial (IE) cells (Kono T et al. J Crohns Colitis 4: 161-170, 2010). TU-100 contains multiple TRP activators. In the present study, therefore, we examined the involvement of TRP channels in the ADM-mediated vasodilatatory effect of TU-100. Rats were treated intraduodenally with the TRP vanilloid type 1 (TRPV1) agonist capsaicin (CAP), the TRP ankyrin 1 (TRPA1) agonist allyl-isothiocyanate (AITC), or TU-100, and jejunum IBF was evaluated using laser-Doppler blood flowmetry. All three compounds resulted in vasodilatation, and the vasodilatory effect of TU-100 was abolished by a TRPA1 antagonist but not by a TRPV1 antagonist. Vasodilatation induced by AITC and TU-100 was abrogated by anti-ADM antibody treatment. RT-PCR and flow cytometry revealed that an IEC-6 cell line originated from the small intestine and purified IE cells expressed ADM and TRPA1 but not TRPV1. AITC increased ADM release in IEC cells remarkably, while CAP had no effect. TU-100 and its ingredient 6-shogaol (6SG) increased ADM release dose-dependently, and the effects were abrogated by a TRPA1 antagonist. 6SG showed similar TRPA1-dependent vasodilatation in vivo. These results indicate that TRPA1 in IE cells may play an important role in controlling bowel microcirculation via ADM release. Epithelial TRPA1 appears to be a promising target for the development of novel strategies for the treatment of various gastrointestinal disorders.
Project description:BACKGROUND AND PURPOSE: Capsiate is produced by 'CH-19 Sweet' (Capsicum annuun L.), a non-pungent cultivar of red pepper. Like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system and suppressing inflammation, but the underlying mechanisms for this are uncertain. We previously reported that capsiate could activate transient receptor potential vanilloid 1 (TRPV1), a capsaicin receptor. The purpose of the present study is to investigate whether capsinoids activate other TRP channels. EXPERIMENTAL APPROACH: Using Ca(2+) imaging and whole-cell patch-clamp methods, we analysed the response of TRP channels to three kinds of capsinoids, capsiate, dihydrocapsiate and nordihydrocapsiate, in HEK293T cells expressing TRP channels or in primary cultures of mouse dorsal root ganglion neurons. KEY RESULTS: We found that in both cell types TRP ankyrin 1 (TRPA1) had a slightly weaker response to capsinoids compared with TRPV1, with the capsiate EC(50) for TRPA1 activation being more than that for TRPV1 activation, and that the capsinoid-evoked action was blocked by a specific TRPA1 antagonist. TRPA1 was activated by capsinoids, but not by their degradation products. Amino acids known to participate in TRPA1 activation following cysteine covalent modification or zinc treatment were not involved in the activation of TRPA1 by capsinoid. CONCLUSIONS AND IMPLICATIONS: Taken together, these results indicate that capsinoids activate TRPA1 by an as yet unknown mechanism, and TRPA1 could be involved in physiological phenomena associated with capsinoid treatment.
Project description:The Transient Receptor Potential Ankyrin 1 (TRPA1) cation channel expressed on capsaicin-sensitive afferents, immune and endothelial cells is activated by inflammatory mediators and exogenous irritants, e.g., endotoxins, nicotine, crotonaldehyde and acrolein. We investigated its involvement in acute and chronic pulmonary inflammation using Trpa1 gene-deleted (Trpa1-/-) mice. Acute pneumonitis was evoked by intranasal Escherichia coli endotoxin (lipopolysaccharide: LPS) administration, chronic bronchitis by daily cigarette smoke exposure (CSE) for 4 months. Frequency, peak inspiratory/expiratory flows, minute ventilation determined by unrestrained whole-body plethysmography were significantly greater, while tidal volume, inspiratory/expiratory/relaxation times were smaller in Trpa1-/- mice. LPS-induced bronchial hyperreactivity, myeloperoxidase activity, frequency-decrease were significantly greater in Trpa1-/- mice. CSE significantly decreased tidal volume, minute ventilation, peak inspiratory/expiratory flows in wildtypes, but not in Trpa1-/- mice. CSE remarkably increased the mean linear intercept (histopathology), as an emphysema indicator after 2 months in wildtypes, but only after 4 months in Trpa1-/- mice. Semiquantitative histopathological scores were not different between strains in either models. TRPA1 has a complex role in basal airway function regulation and inflammatory mechanisms. It protects against LPS-induced acute pneumonitis and hyperresponsiveness, but is required for CSE-evoked emphysema and respiratory deterioration. Further research is needed to determine TRPA1 as a potential pharmacological target in the lung.
Project description:The activation of capsaicin-sensitive lung vagal (CSLV) afferents can elicit airway reflexes. Hypersensitivity of these afferents is known to contribute to the airway hypersensitivity during airway inflammation. Hydrogen sulfide (H2S) has been suggested as a potential therapeutic agent for airway hypersensitivity diseases, such as asthma, because of its relaxing effect on airway smooth muscle and anti-inflammatory effect. However, it is still unknown whether H2S affects airway reflexes. Our previous study demonstrated that exogenous application of H2S sensitized CSLV afferents and enhanced Ca2+ transients in CSLV neurons. The present study aimed to determine whether the H2S-induced sensitization leads to functional changes in airway reflexes and elevates the electrical excitability of the CSLV neurons. Our results showed that, first and foremost, in anesthetized, spontaneously breathing rats, the inhalation of aerosolized sodium hydrosulfide (NaHS, a donor of H2S; 5 mg/mL, 3 min) caused an enhancement in apneic response evoked by several stimulants of the CSLV afferents. This enhancement effect was found 5 min after NaHS inhalation and returned to control 30 min later. However, NaHS no longer enhanced the apneic response after perineural capsaicin treatment on both cervical vagi that blocked the conduction of CSLV fibers. Furthermore, the enhancing effect of NaHS on apneic response was totally abolished by pretreatment with intravenous HC-030031 (a TRPA1 antagonist; 8 mg/kg), whereas the potentiating effect was not affected by the pretreatment with the vehicle of HC-030031. We also found that intracerebroventricular infusion pretreated with HC-030031 failed to alter the potentiating effect of NaHS on the apneic response. Besides, the cough reflex elicited by capsaicin aerosol was enhanced by inhalation of NaHS in conscious guinea pigs. Nevertheless, this effect was entirely eliminated by pretreatment with HC-030031, not by its vehicle. Last but not least, voltage-clamp electrophysiological analysis of isolated rat CSLV neurons showed a similar pattern of potentiating effects of NaHS on capsaicin-induced inward current, and the involvement of TRPA1 receptors was also distinctly shown. In conclusion, these results suggest that H2S non-specifically enhances the airway reflex responses, at least in part, through action on the TRPA1 receptors expressed on the CSLV afferents. Therefore, H2S should be used with caution when applying for therapeutic purposes in airway hypersensitivity diseases.
Project description:Schistosomiasis, caused by parasitic flatworms of the genus Schistosoma, is a neglected tropical disease affecting hundreds of millions globally. Praziquantel (PZQ), the only drug currently available for treatment and control, is largely ineffective against juvenile worms, and reports of PZQ resistance lend added urgency to the need for development of new therapeutics. Ion channels, which underlie electrical excitability in cells, are validated targets for many current anthelmintics. Transient receptor potential (TRP) channels are a large family of non-selective cation channels. TRP channels play key roles in sensory transduction and other critical functions, yet the properties of these channels have remained essentially unexplored in parasitic helminths. TRP channels fall into several (7-8) subfamilies, including TRPA and TRPV. Though schistosomes contain genes predicted to encode representatives of most of the TRP channel subfamilies, they do not appear to have genes for any TRPV channels. Nonetheless, we find that the TRPV1-selective activators capsaicin and resiniferatoxin (RTX) induce dramatic hyperactivity in adult worms; capsaicin also increases motility in schistosomula. SB 366719, a highly-selective TRPV1 antagonist, blocks the capsaicin-induced hyperactivity in adults. Mammalian TRPA1 is not activated by capsaicin, yet knockdown of the single predicted TRPA1-like gene (SmTRPA) in S. mansoni effectively abolishes capsaicin-induced responses in adult worms, suggesting that SmTRPA is required for capsaicin sensitivity in these parasites. Based on these results, we hypothesize that some schistosome TRP channels have novel pharmacological sensitivities that can be targeted to disrupt normal parasite neuromuscular function. These results also have implications for understanding the phylogeny of metazoan TRP channels and may help identify novel targets for new or repurposed therapeutics.
Project description:In primates, C-fibre polymodal nociceptors are broadly classified into two groups based on mechanosensitivity. Here we demonstrate that mechanically sensitive polymodal nociceptors that respond either quickly (QC) or slowly (SC) to a heat stimulus differ in responses to a mild burn, heat sensitization, conductive properties and chemosensitivity. Superficially applied capsaicin and intradermal injection of ?-alanine, an MrgprD agonist, excite vigorously all QCs. Only 40% of SCs respond to ?-alanine, and their response is only half that of QCs. Mechanically insensitive C-fibres (C-MIAs) are ?-alanine insensitive but vigorously respond to capsaicin and histamine with distinct discharge patterns. Calcium imaging reveals that ?-alanine and histamine activate distinct populations of capsaicin-responsive neurons in primate dorsal root ganglion. We suggest that histamine itch and capsaicin pain are peripherally encoded in C-MIAs, and that primate polymodal nociceptive afferents form three functionally distinct subpopulations with ?-alanine responsive QC fibres likely corresponding to murine MrgprD-expressing, non-peptidergic nociceptive afferents.