Exposure of Toluene Diisocyanate Induces DUSP6 and p53 through Activation of TRPA1 Receptor.

Toluene diisocyanate (TDI), a major intermediate agent used in the manufacturing industry, causes respiratory symptoms when exposed to the human body. In this study, we aimed to determine the molecular mechanism of TDI toxicity. To investigate the impact of TDI exposure on global gene expression, we performed transcriptomic analysis of human bronchial epithelial cells (BEAS-2B) after TDI treatment. Differentially expressed genes (DEGs) were sorted and used for clustering and network analysis. Among DEGs, dual-specificity phosphatase 6 (DUSP6) was one of the genes significantly changed by TDI exposure. To verify the expression level of DUSP6 and its effect on lung cells, the mRNA and protein levels of DUSP6 were analyzed. Our results showed that DUSP6 was dose-dependently upregulated by TDI treatment. Thereby, the phosphorylation of ERK1/2, one of the direct inhibitory targets of DUSP6, was decreased. TDI exposure also increased the mRNA level of p53 along with its protein and activity which trans-activates DUSP6. Since TRPA1 is known as a signal integrator activated by TDI, we analyzed the relevance of TRPA1 receptor in DUSP6 regulation. Our data revealed that up-regulation of DUSP6 mediated by TDI was blocked by a specific antagonist against TRPA1. TDI exposure attenuated the apoptotic response, which suggests that it promotes the survival of cancerous cells. In conclusion, our results suggest that TDI induces DUSP6 and p53, but attenuates ERK1/2 activity through TRPA1 receptor activation, leading to cytotoxicity.

Dysfunctional TRPM8 signalling in the vascular response to environmental cold in ageing.

Ageing is associated with increased vulnerability to environmental cold exposure. Previously, we identified the role of the cold-sensitive transient receptor potential (TRP) A1, M8 receptors as vascular cold sensors in mouse skin. We hypothesised that this dynamic cold-sensor system may become dysfunctional in ageing. We show that behavioural and vascular responses to skin local environmental cooling are impaired with even moderate ageing, with reduced TRPM8 gene/protein expression especially. Pharmacological blockade of the residual TRPA1/TRPM8 component substantially diminished the response in aged, compared with young mice. This implies the reliance of the already reduced cold-induced vascular response in ageing mice on remaining TRP receptor activity. Moreover, sympathetic-induced vasoconstriction was reduced with downregulation of the α2c adrenoceptor expression in ageing. The cold-induced vascular response is important for sensing cold and retaining body heat and health. These findings reveal that cold sensors, essential for this neurovascular pathway, decline as ageing onsets.

TRPA1: Pharmacology, natural activators and role in obesity prevention.

Transient receptor potential ankyrin 1 (TRPA1) channel is a calcium permeable, non-selective cation channel, expressed in the sensory neurons and non-neuronal cells of different tissues. Initially studied for its role in pain and inflammation, TRPA1 has now functionally involved in multiple other physiological functions. TRPA1 channel has been extensively studied for modulation by pungent compounds present in the spices and herbs. In the last decade, the role of TRPA1 agonism in body weight reduction, secretion of hunger and satiety hormones, insulin secretion and thermogenesis, has unveiled the potential of the TRPA1 channel to be used as a preventive target to tackle obesity and associated comorbidities including insulin resistance in type 2 diabetes. In this review, we summarized the recent findings of TRPA1 based dietary/non-dietary modulation for its role in obesity prevention and therapeutics.

Photosensitive and Photoswitchable TRPA1 Agonists Optically Control Pain through Channel Desensitization.

Transient receptor potential ankyrin 1 (TRPA1) channel, as a nonselective ligand-gated cation channel robustly in dorsal root ganglion sensory neurons, is implicated in sensing noxious stimuli and nociceptive signaling. However, small-molecule tools targeting TRPA1 lack temporal and spatial resolution, limiting their use for validation of TRPA1 as a therapeutic target for pain. In our previous work, we found that 4,4'-(diazene-1,2-diyl)dianiline (AB1) is a photoswitchable TRPA1 agonist, but the poor water solubility and activity hinder its further development. Here, we report a series of specific and potent azobenzene-derived photoswitchable TRPA1 agonists (series 1 and 2) that enable optical control of the TRPA1 channel. Two representative compounds 1g and 2c can alleviate capsaicin-induced pain in the cheek model of mice through channel desensitization but not in TRPA1 knockout mice. Taken together, our findings demonstrate that photoswitchable TRPA1 agonists can be used as pharmacological tools for study of pain signaling.

Allicin, a dietary trpa1 agonist, prevents high fat diet-induced dysregulation of gut hormones and associated complications.

Scope. Given the global epidemic of diabesity (co-existence of both diabetes and obesity), novel approaches that target gut hormone secretion and their modulation may offer the dual benefits of increased efficacy and limited side effects. In the present study, we tested the hypothesis that agonism of Transient Receptor Potential Ankyrin 1 (TRPA1), using a dietary activator, has a modulatory role in high fat diet (HFD)-induced dysregulation of post-prandial gut hormone responses and prevention of metabolic alterations. Methods and results. The effect of HFD on TRPA1 expression in different parts of the gut using immunohistochemistry, western blotting and RT-PCR was studied. Dietary TRPA1 agonist, Allicin Rich Garlic Juice (ARGJ), was co-administered along with HFD in mice for three months and various metabolic health parameters, relative gut hormone levels and inflammation were observed. The HFD caused substantial reduction in gut TRPA1 expression along with dysregulation in post-prandial normalization of gut hormone levels, particularly GLP-1, precipitating hunger phenotype, altered glucose homeostasis, hepatic inflammation and fat accumulation. TRPA1 agonism through ARGJ co-supplementation prevented HFD-induced dysregulation in post-prandial normalization of gut hormone levels and averted metabolic and inflammatory complications in peripheral tissues. Conclusion. Our findings provide evidence that ARGJ (diet-based TRPA1 agonism) can be employed as a feasible strategy, as nutraceuticals or food, to prevent HFD-induced metabolic complications.

ERK Phosphorylation Regulates the Aml1/Runx1 Splice Variants and the TRP Channels Expression during the Differentiation of Glioma Stem Cell Lines.

The identification of cancer stem cells in brain tumors paved the way for new therapeutic approaches. Recently, a role for the transcriptional factor Runx1/Aml1 and the downstream ion channel genes in brain cancer development and progression has been suggested. This study aimed to explore the expression and the role of Runx1/Aml1, its Aml1b and Aml1c splice variants and the downstream TRPA1 and TRPV1 ion channels in undifferentiated and day-14 differentiated neural stem cells (NSCs and D-NSCs) and glioblastoma stem cells (GSCs and D-GSCs) lines with different proneural (PN) or mesenchymal (MES) phenotype. Gene and protein expression were evaluated by qRT-PCR, cytofluorimetric, western blot and confocal microscopy analyses. Moreover, by western blot, we observed that ERK phosphorylation enhances the Aml1b and Aml1c protein expression during glioma differentiation. Furthermore, the agonists of TRPA1 and TRPV1 channels stimulated apoptosis/necrosis in GSCs and D-GSCs as evaluated by Annexin V and PI staining and cytofluorimetric analysis. Finally, by qRT-PCR, the modulation of Wnt/ß catenin, FGF, and TGFß/SMAD signaling pathways in PN- and MES-GSCs was reported. Overall, our results provide new evidence regarding Runx1/Aml1 isoform overexpression and modulation in TRP channel expression during gliomagenesis, thus offering new directions for glioblastoma therapy.

The TRPA1 Agonist Cinnamaldehyde Induces the Secretion of HCO3- by the Porcine Colon.

A therapeutic potential of the TRPA1 channel agonist cinnamaldehyde for use in inflammatory bowel disease is emerging, but the mechanisms are unclear. Semi-quantitative qPCR of various parts of the porcine gastrointestinal tract showed that mRNA for TRPA1 was highest in the colonic mucosa. In Ussing chambers, 1 mmol·L-1 cinnamaldehyde induced increases in short circuit current (ΔIsc) and conductance (ΔGt) across the colon that were higher than those across the jejunum or after 1 mmol·L-1 thymol. Lidocaine, amiloride or bumetanide did not change the response. The application of 1 mmol·L-1 quinidine or the bilateral replacement of 120 Na+, 120 Cl- or 25 HCO3- reduced ΔGt, while the removal of Ca2+ enhanced ΔGt with ΔIsc numerically higher. ΔIsc decreased after 0.5 NPPB, 0.01 indometacin and the bilateral replacement of 120 Na+ or 25 HCO3-. The removal of 120 Cl- had no effect. Cinnamaldehyde also activates TRPV3, but comparative measurements involving patch clamp experiments on overexpressing cells demonstrated that much higher concentrations are required. We suggest that cinnamaldehyde stimulates the secretion of HCO3- via apical CFTR and basolateral Na+-HCO3- cotransport, preventing acidosis and damage to the epithelium and the colonic microbiome. Signaling may involve the opening of TRPA1, depolarization of the epithelium and a rise in PGE2 following a lower uptake of prostaglandins via OATP2A1.

TRP channels in cancer pain.

Chronic pain is a common symptom experienced during cancer progression. Additionally, some patients experience bone pain caused by cancer metastasis, which further complicates the prognosis. Cancer pain is often treated using opioid-based pharmacotherapy, but these drugs possess several adverse effects. Accordingly, new mechanisms for cancer pain management are being explored, including transient receptor potential channels (TRPs). TRP ion channels are expressed in several tissues and play a key role in pain detection, especially TRP vanilloid 1 (TRPV1) and TRP ankyrin 1 (TRPA1). In the present review, we describe the role of TRPV1 and TRPA1 involved in cancer pain mechanisms. Several studies have revealed that the administration of TRPV1 or TRPA1 agonists/antagonists and TRPV1 or TRPA1 knockdown reduced sensitivity to nociception in cancer pain models. TRPV1 was also found to be involved in various models of cancer-induced bone pain (CIBP), with TRPV1 expression reportedly enhanced in some models. These studies have demonstrated the TRPV1 or TRPA1 association with cancer pain in models induced by tumour cell inoculation into the bone cavity, hind paw, mammary fat pad, and sciatic nerve in mice or rats. To date, only resiniferatoxin, a TRPV1 agonist, has been evaluated in clinical trials for cancer pain and showed preliminary positive results. Thus, TRP channels are potential targets for managing cancer-related pain syndromes.

The Agonist Action of Alkylphenols on TRPA1 Relates to Their Effects on Membrane Lipid Order: Implications for TRPA1-Mediated Chemosensation.

The Transient Receptor Potential Ankyrin 1 cation channel (TRPA1) is a broadly-tuned chemosensor expressed in nociceptive neurons. Multiple TRPA1 agonists are chemically unrelated non-electrophilic compounds, for which the mechanisms of channel activation remain unknown. Here, we assess the hypothesis that such chemicals activate TRPA1 by inducing mechanical perturbations in the plasma membrane. We characterized the activation of mouse TRPA1 by non-electrophilic alkylphenols (APs) of different carbon chain lengths in the para position of the aromatic ring. Having discarded oxidative stress and the action of electrophilic mediators as activation mechanisms, we determined whether APs induce mechanical perturbations in the plasma membrane using dyes whose fluorescence properties change upon alteration of the lipid environment. APs activated TRPA1, with potency increasing with their lipophilicity. APs increased the generalized polarization of Laurdan fluorescence and the anisotropy of the fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH), also according to their lipophilicity. Thus, the potency of APs for TRPA1 activation is an increasing function of their ability to induce lipid order and membrane rigidity. These results support the hypothesis that TRPA1 senses non-electrophilic compounds by detecting the mechanical alterations they produce in the plasma membrane. This may explain how structurally unrelated non-reactive compounds induce TRPA1 activation and support the role of TRPA1 as an unspecific sensor of potentially noxious compounds.

Atractylodin Produces Antinociceptive Effect through a Long-Lasting TRPA1 Channel Activation.

Atractylodin (ATR) is a bioactive component found in dried rhizomes of Atractylodes lancea (AL) De Candolle. Although AL has accumulated empirical evidence for the treatment of pain, the molecular mechanism underlying the anti-pain effect of ATR remains unclear. In this study, we found that ATR increases transient receptor potential ankyrin-1 (TRPA1) single-channel activity in hTRPA1 expressing HEK293 cells. A bath application of ATR produced a long-lasting calcium response, and the response was completely diminished in the dorsal root ganglion neurons of TRPA1 knockout mice. Intraplantar injection of ATR evoked moderate and prolonged nociceptive behavior compared to the injection of allyl isothiocyanate (AITC). Systemic application of ATR inhibited AITC-induced nociceptive responses in a dose-dependent manner. Co-application of ATR and QX-314 increased the noxious heat threshold compared with AITC in vivo. Collectively, we concluded that ATR is a unique agonist of TRPA1 channels, which produces long-lasting channel activation. Our results indicated ATR-mediated anti-nociceptive effect through the desensitization of TRPA1-expressing nociceptors.

Discovery of natural TRPA1 activators through pharmacophore-based virtual screening and a biological assay.

Transient receptor potential cation channel subfamily A member 1 (TRPA1), a member of the transient receptor potential family, detects a wide range of environmental stimuli, such as low temperature, abnormal pH, and reactive irritants. TRPA1 is of great interest as a target protein in fields related to pharmaceuticals and foods. In this study, a library of natural products was explored to identify TRPA1 activators by pharmacophore screening of known TRPA1 agonists and biological assays for agonist activity. The study identified six natural compounds as novel TRPA1 agonists. The discovery of these compounds may prove useful in elucidating the TRPA1 activation mechanism.

Sanguinarine is an agonist of TRPA1 channel.

Sanguinarine, a benzyl isoquinoline alkaloid extracted from the root of Papaveraceae plants, shows extensive pharmacological activities including anti-microbial, anti-trypanosoma, anti-tumor, anti-platelet, anti-hypertensive effects, as well as inhibition of osteoclast formation. Here we demonstrate that TRPA1 channel (Transient receptor potential cation channel, member A1) is a potential target for sanguinarine. Electrophysiological recordings show that sanguinarine activates TRPA1 channel potently with an EC50 0.09 (0.04-0.13) µM, but has no effects on other examined TRP channels. Sanguinarine increases the intracellular calcium levels and upregulates the excitability of mouse dorsal root ganglion (DRG) neurons in vitro significantly. Plantar injection of sanguinarine evokes nociceptive behaviors similar to that elicited by allyl isothiocyanate (AITC), a classic agonist of TRPA1. Both the enhancement of excitability of DRG neurons and the nociceptive behaviors can be attenuated by treatment of TRPA1 channel antagonist HC030031 or knockout of trpa1 gene. Taken together, our data demonstrate that sanguinarine is a potent and relatively selective agonist of TRPA1 channel.

A Non-covalent Ligand Reveals Biased Agonism of the TRPA1 Ion Channel.

The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.

The transient receptor potential A1 ion channel (TRPA1) modifies in vivo autonomous ureter peristalsis in rats.

AIMS: The current study aimed to explore the expression of transient receptor potential A1 ion channels (TRPA1) in the rat ureter and to assess if TRPA1-active compounds modulate ureter function. METHODS: The expression of TRPA1 in rat ureter tissue was studied by immunofluorescence. The TRPA1 distribution was compared to calcitonin gene-related peptide (CGRP), α-actin (SMA1), anoctamin-1 (ANO1), and c-kit. For in vivo analyses, a catheter was implanted in the right ureter of 50 rats. Ureter peristalsis and pressures were continuously recorded by a data acquisition set-up during intraluminal infusion of saline (baseline), saline plus protamine sulfate (PS; to disrupt the urothelium), saline plus PS with hydrogen sulfide (NaHS) or cinnamaldehyde (CA). Comparisons were made between rats treated systemically with vehicle or a TRPA1-antagonist (HC030031). RESULTS: TRPA1-immunoreactive nerves co-expressed CGRP and were mainly located in the suburothelial region of the ureter. Immunoreactivity for TRPA1 was also encountered in c-kit-positive but ANO1-negative cells of the ureter suburothelium and wall. In vivo, HC030031-treated rats had elevated baseline peristaltic frequency (p < 0.05) and higher intraluminal pressures (p < 0.01). PS increased the frequency of ureter peristalsis versus baseline in vehicle-treated rats (p < 0.001) but not in HC030031-treated rats. CA (p < 0.001) and NaHS (p < 0.001) decreased ureter peristalsis. This was counteracted by HC030031 (p < 0.05 and p < 0.01). CONCLUSIONS: In rats, TRPA1 is expressed on cellular structures considered of importance for peristaltic and mechanoafferent functions of the ureter. Functional data indicate that TRPA1-mediated signals regulate ureter peristalsis. This effect was pronounced after mucosal disruption and suggests a role for TRPA1 in ureter pathologies involving urothelial damage.

New natural agonists of the transient receptor potential Ankyrin 1 (TRPA1) channel.

The transient receptor potential (TRP) channels family are cationic channels involved in various physiological processes as pain, inflammation, metabolism, swallowing function, gut motility, thermoregulation or adipogenesis. In the oral cavity, TRP channels are involved in chemesthesis, the sensory chemical transduction of spicy ingredients. Among them, TRPA1 is activated by natural molecules producing pungent, tingling or irritating sensations during their consumption. TRPA1 can be activated by different chemicals found in plants or spices such as the electrophiles isothiocyanates, thiosulfinates or unsaturated aldehydes. TRPA1 has been as well associated to various physiological mechanisms like gut motility, inflammation or pain. Cinnamaldehyde, its well known potent agonist from cinnamon, is reported to impact metabolism and exert anti-obesity and anti-hyperglycemic effects. Recently, a structurally similar molecule to cinnamaldehyde, cuminaldehyde was shown to possess anti-obesity and anti-hyperglycemic effect as well. We hypothesized that both cinnamaldehyde and cuminaldehyde might exert this metabolic effects through TRPA1 activation and evaluated the impact of cuminaldehyde on TRPA1. The results presented here show that cuminaldehyde activates TRPA1 as well. Additionally, a new natural agonist of TRPA1, tiglic aldehyde, was identified and p-anisaldehyde confirmed.

Non-electrophilic TRPA1 agonists, menthol, carvacrol and clotrimazole, open epithelial tight junctions via TRPA1 activation.

Activation of the transient receptor potential A1 channel (TRPA1) by electrophilic agonists was reported to induce the opening of tight junctions (TJs). Because compounds that increase TJ permeability can be paracellular permeability enhancers, we investigated the effect of non-electrophilic TRPA1 activators, including food ingredients (menthol and carvacrol) and medication (clotrimazole), on epithelial permeability. We show that all three compounds induced increase of the permeability of fluorescein isothiocyanate-conjugated dextran (4 kDa) and decrease of transepithelial electrical resistance, accompanied by Ca2+ influx and cofilin activation in epithelial MDCK II monolayers. These phenotypes were attenuated by pretreatment of a TRPA1 antagonist, suggesting TRPA1-mediated opening of TJs. These results suggest that non-electrophilic TRPA1 activators with established safety can be utilized to regulate epithelial barriers.

Upper Airway Cough Syndrome in Pathogenesis of Chronic Cough.

Cough is one of the most important defensive reflexes. However, extensive non- productive cough is a harmful mechanism leading to the damage of human airways. Cough is initiated by activation of vagal afferents in the airways. The site of their convergence is particularly the nucleus of the solitary tract (nTS). The second-order neurons terminate in the pons, medulla and spinal cord and there is also the cortical and subcortical control of coughing.Upper airway cough syndrome (UACS) - previously postnasal drip syndrome - is one of the most common causes of chronic cough together with asthma and gastroesophageal reflux. The main mechanisms leading to cough in patients with nasal and sinus diseases are postnasal drip, direct irritation of nasal mucosa, inflammation in the lower airways, upper airway inflammation and the cough reflex sensitization. The cough demonstrated by UACS patients is probably due to hypersensitivity of the upper airways sensory nerve or lower airways sensory nerve, or a combination of both. Further studies are needed to clarify this mechanism.

Transient receptor potential ankyrin-1 causes rapid bronchodilation via nonepithelial PGE2.

Transient receptor potential ankyrin-1 (TRPA1) is a ligand-gated cation channel that responds to endogenous and exogenous irritants. TRPA1 is expressed on multiple cell types throughout the lungs, but previous studies have primarily focused on TRPA1 stimulation of airway sensory nerves. We sought to understand the integrated physiological airway response to TRPA1 stimulation. The TRPA1 agonists allyl isothiocyanate (AITC) and cinnamaldehyde (CINN) were tested in sedated, mechanically ventilated guinea pigs in vivo. Reproducible bronchoconstrictions were induced by electrical stimulation of the vagus nerves. Animals were then treated with intravenous AITC or CINN. AITC and CINN were also tested on isolated guinea pig and mouse tracheas and postmortem human trachealis muscle strips in an organ bath. Tissues were contracted with methacholine, histamine, or potassium chloride and then treated with AITC or CINN. Some airways were pretreated with TRPA1 antagonists, the cyclooxygenase inhibitor indomethacin, the EP2 receptor antagonist PF 04418948, or tetrodotoxin. AITC and CINN blocked vagally mediated bronchoconstriction in guinea pigs. Pretreatment with indomethacin completely abolished the airway response to TRPA1 agonists. Similarly, AITC and CINN dose-dependently relaxed precontracted guinea pig, mouse, and human airways in the organ bath. AITC- and CINN-induced airway relaxation required TRPA1, prostaglandins, and PGE2 receptor activation. TRPA1-induced airway relaxation did not require epithelium or tetrodotoxin-sensitive nerves. Finally, AITC blocked airway hyperreactivity in two animal models of allergic asthma. These data demonstrate that stimulation of TRPA1 causes bronchodilation of intact airways and suggest that the TRPA1 pathway is a potential pharmacological target for bronchodilation.

Identification of a new class of non-electrophilic TRPA1 agonists by a structure-based virtual screening approach.

Recent work has gradually been clarifying the binding site of non-electrophilic agonists on the transient receptor potential A1 (TRPA1). This study searched for non-electrophilic TRPA1 agonists by means of in silico drug discovery techniques based on three-dimensional (3-D) protein structure. First, agonist-bound pocket structures were explored using an advanced molecular dynamics simulation starting from the cryo-electron microscopic structure of TRPA1, and several pocket structures suitable for virtual screening were extracted by structure evaluation using known non-electrophilic TRPA1 agonists. Next, 49 compounds were selected as new non-electrophilic agonist candidates from a library of natural products comprising 10,555 compounds by molecular docking toward these pocket structures. Measurement of the TRPA1 agonist activity of these compounds showed notable TRPA1 activation with three compounds (decanol, 2-ethyl-1-hexanol, phenethyl butanoate). Decanol and 2-ethyl-1-hexanol, which are categorized as fatty alcohols, in particular have a novel chemical scaffold for TRPA1 activation. The results of this study are expected to be of considerable use in understanding the molecular mechanism of TRPA1 recognition by non-electrophilic agonists.

A randomized clinical trial on the acute therapeutic effect of TRPA1 and TRPM8 agonists in patients with oropharyngeal dysphagia.

BACKGROUND: Oropharyngeal dysphagia (OD) treatment is moving away from compensatory strategies toward active treatments that improve swallowing function. The aim of this study was to assess the acute therapeutic effect of TRPA1/M8 agonists in improving swallowing function in OD patients. METHODS: Fifty-eight patients with OD caused by aging, stroke, or neurodegenerative disease were included in a three-arm, quadruple-blind, randomized clinical trial (NCT02193438). Swallowing safety and efficacy and the kinematics of the swallow response were assessed by videofluoroscopy (VFS) during the swallow of 182 ± 2 mPa·s viscosity (nectar) boluses of a xanthan gum thickener supplemented with (a) 756.6 µmol/L cinnamaldehyde and 70 µmol/L zinc (CIN-Zn) (TRPA1 agonists), (b) 1.6 mmol/L citral (CIT) (TRPA1 agonist), or (c) 1.6 mmol/L citral and 1.3 mmol/L isopulegol (CIT-ISO) (TRPA1 and TRPM8 agonists). The effects on pharyngeal event-related potentials (ERP) were assessed by electroencephalography. KEY RESULTS: TRPA1 stimulation with either CIN-Zn or CIT reduced time to laryngeal vestibule closure (CIN-Zn P = .002, CIT P = .023) and upper esophageal sphincter opening (CIN-Zn P = .007, CIT P = .035). In addition, CIN-Zn reduced the penetration-aspiration scale score (P = .009), increased the prevalence of safe swallows (P = .041), and reduced the latency of the P2 peak of the ERP. CIT-ISO had no positive effect on biomechanics or neurophysiology. No significant adverse events were observed. CONCLUSIONS AND INFERENCES: TRPA1 stimulation with CIN-Zn or CIT improves the swallow response which, in the case of CIN-Zn, is associated with a significant improvement in cortical activation and safety of swallow. These results provide the basis for the development of new active treatments for OD using TRPA1 agonists.