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Alternative plasticizers have become more popular due to health concerns about phthalate esters. We demonstrated that phthalate esters enhanced skin sensitization to fluorescein isothiocyanate (FITC) in mouse contact hypersensitivity models. Alternative plasticizers have not been well studied as to their effect on the immune system. We previously found that diisopropyl adipate (DIPA), an aliphatic dicarboxylic acid ester, enhanced skin sensitization to FITC. Sebacate esters are also widely used as alternative plasticizers. Here we tested diisopropyl sebacate (DIPS), which has the same alcohol with an aliphatic dicarboxylic acid of longer chain, using BALB/c mice. The results showed that DIPS facilitated skin sensitization to FITC and increased FITC-presenting dendritic cell trafficking from the skin to draining lymph nodes. Furthermore, DIPS activated transient receptor potential ankyrin 1 (TRPA1). The latter feature has been commonly observed for phthalate esters and DIPA, which have adjuvant effects. In summary, the adjuvant effect of a sebacate ester was demonstrated in a mouse model.

The perception of innocuous warmth is a sensory capability that facilitates thermoregulatory, social, hedonic, and even predatory functions. It has long been recognized that innocuous warmth perception is triggered by activation of a subpopulation of specially tuned peripheral thermosensory neurons. In addition, there is growing evidence that thermotransduction by nonneuronal cells, such as skin keratinocytes, might contribute to or modulate our thermosensory experience. Yet, the precise molecular mechanisms underlying warmth transduction are only now being uncovered. Recent molecular genetics approaches have led to the identification of multiple candidate warmth-transducing molecules that appear to confer thermosensitivity upon innocuous warmth afferents and/or neighboring cell types. Most, but not all, of these candidate transducers are members of the transient receptor potential (TRP) ion channel family. Among the latter, evidence supporting a function in innocuous warmth sensation is strongest for TRPV1 and TRPM2 in mammals and for TRPA1 in nonmammalian species., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Background Diabetic peripheral neuropathy is a common long-term complication of diabetes. Accumulating evidence suggests that vascular impairment plays important roles in the pathogenesis of diabetic peripheral neuropathy, while the mechanism remains unclear. We recently reported that transient receptor potential ankyrin 1 (TRPA1) is sensitized by hypoxia, which can contribute to cold hypersensitivity. In this study, we investigated the involvement of TRPA1 and vascular impairment in painful diabetic peripheral neuropathy using streptozotocin-induced diabetic model mice. Results Streptozotocin-induced diabetic model mice showed mechanical and cold hypersensitivity with a peak at two weeks after the streptozotocin administration, which were likely to be paralleled with the decrease in the skin blood flow of the hindpaw. Streptozotocin-induced cold hypersensitivity was significantly inhibited by an antagonist HC-030031 (100 mg/kg) or deficiency for TRPA1, whereas mechanical hypersensitivity was unaltered. Consistent with these results, the nocifensive behaviors evoked by an intraplantar injection of the TRPA1 agonist allyl isothiocyanate (AITC) were enhanced two weeks after the streptozotocin administration. Both streptozotocin-induced cold hypersensitivity and the enhanced AITC-evoked nocifensive behaviors were significantly inhibited by a vasodilator, tadalafil (10 mg/kg), with recovery of the decreased skin blood flow. Similarly, in a mouse model of hindlimb ischemia induced by the ligation of the external iliac artery, AITC-evoked nocifensive behaviors were significantly enhanced three and seven days after the ischemic operation, whereas mechanical hypersensitivity was unaltered in TRPA1-knockout mice. However, no difference was observed between wild-type and TRPA1-knockout mice in the hyposensitivity for current or mechanical stimulation or the deceased density of intraepidermal nerve fibers eight weeks after the streptozotocin administration. Conclusion These results suggest that TRPA1 sensitization during diabetic vascular impairment causes cold, but not mechanical, hypersensitivity in the early painful phase of diabetic peripheral neuropathy. However, TRPA1 may play little or no role in the progression of diabetic peripheral neuropathy.

Cramp-fasciculation syndrome (CFS) is a rare muscle hyperexcitability syndrome that presents with muscle cramps, fasciculations, and stiffness, as well as pain, fatigue, anxiety, hyperreflexia, and paresthesias. Although familial cases have been reported, a genetic etiology has not yet been identified. We performed whole-exome sequencing followed by validation and cosegregation analyses on a father-son pair with CFS. Both subjects manifested other hypersensitivity-hyperexcitability symptoms, including asthma, gastroesophageal reflux, migraine, restless legs syndrome, tremor, cold hyperalgesia, and cardiac conduction defects. Most symptoms improved with carbamazepine, consistent with an underlying cation channelopathy. We identified a variant in the transient receptor potential ankyrin A1 channel (TRPA1) gene that selectively cosegregated with CFS and the other hypersensitivity-hyperexcitability symptoms. This variant (c.2755C>T) resulted in a premature stop codon at amino acid 919 (p.Arg919*) in the outer pore of the channel. TRPA1 is a widely distributed, promiscuous plasmalemmal cation channel that is strongly implicated in the pathophysiology of the specific hypersensitivity-hyperexcitability symptoms observed in these subjects. Thus, we have identified a novel TRPA1 variant that is associated with CFS as part of a generalized hypersensitivity-hyperexcitability disorder. These findings clarify the diverse functional roles of TRPA1, and underscore the importance of this channel as a potential therapeutic target., (© 2017 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

AMPK is a widely expressed intracellular energy sensor that monitors and modulates energy expenditure. Transient receptor potential ankyrin 1 (TRPA1) channel is a widely recognized chemical and thermal sensor that plays vital roles in pain transduction. In this study, we discovered a functional link between AMPK and TRPA1 in dorsal root ganglion (DRG) neurons, in which AMPK activation rapidly resulted in downregulation of membrane-associated TRPA1 and its channel activity within minutes. Treatment with two AMPK activators, metformin or AICAR, inhibited TRPA1 activity in DRG neurons by decreasing the amount of membrane-associated TRPA1. Metformin induced a dose-dependent inhibition of TRPA1-mediated calcium influx. Conversely, in diabetic db/db mice, AMPK activity was impaired in DRG neurons, and this was associated with a concomitant increase in membrane-associated TRPA1 and mechanical allodynia. Notably, these molecular and behavioral changes were normalized following treatment with AMPK activators. Moreover, high-glucose exposure decreased activated AMPK levels and increased agonist-evoked TRPA1 currents in cultured DRG neurons, and these effects were prevented by treatment with AMPK activators. Our results identify AMPK as a previously unknown regulator of TRPA1 channels. AMPK modulation of TRPA1 could thus serve as an underlying mechanism and potential therapeutic molecular target in painful diabetic neuropathy., (© 2017 by the American Diabetes Association.)

TRPA1 of insects and several tetrapod vertebrates except for those of rodents have been reported to be activated by noxious chemicals and also by high temperature with a relatively clear threshold. We previously analyzed the characteristics of two TRPA1 paralogs of zebrafish (zTRPA1a, b) and demonstrated that zTRPA1a is specialized for chemical sensing while zTRPA1b responds to thermal stimulations, that zTRPA1b responds to both cold and heat stimuli, and that heat stimulation gradually activates zTRPA1b without a clear threshold. In the medaka genome, a single TRPA1 (olTRPA1) gene is present. To examine if functional properties of olTRPA1 are similar to TRPA1 of land animals or either of zTRPA1a or zTRPA1b, we isolated a TRPA1 cDNA from medaka and performed functional analyses. OlTRPA1 showed a sensitivity to four noxious chemicals (allyl isothiocyanate, caffeine, carvacrol, methyl anthranilate). We observed that cold stimulation does not activate olTRPA1, but heat stimulation gradually activates olTRPA1 with an unclear threshold. Results suggested that a single TRPA1 functions as a chemical and thermal sensor in medaka, and that a gradual heat-activation without clear threshold might be a common feature for TRPA1 of fish living in water., (Copyright © 2017 Elsevier Inc. All rights reserved.)

It is known that transient receptor potential ankyrin 1 (TRPA1) channels, expressed by nociceptors, contribute to neuropathic pain. Here we show that TRPA1 is also expressed in Schwann cells. We found that in mice with partial sciatic nerve ligation, TRPA1 silencing in nociceptors attenuated mechanical allodynia, without affecting macrophage infiltration and oxidative stress, whereas TRPA1 silencing in Schwann cells reduced both allodynia and neuroinflammation. Activation of Schwann cell TRPA1 evoked NADPH oxidase 1 (NOX1)-dependent H 2 O 2 release, and silencing or blocking Schwann cell NOX1 attenuated nerve injury-induced macrophage infiltration, oxidative stress and allodynia. Furthermore, the NOX2-dependent oxidative burst, produced by macrophages recruited to the perineural space activated the TRPA1-NOX1 pathway in Schwann cells, but not TRPA1 in nociceptors. Schwann cell TRPA1 generates a spatially constrained gradient of oxidative stress, which maintains macrophage infiltration to the injured nerve, and sends paracrine signals to activate TRPA1 of ensheathed nociceptors to sustain mechanical allodynia.

A facile, convenient, and green method for the synthesis of BAgNPs by using basalin is proposed in this work. The capping of the basalin on the surface of BAgNP was confirmed by the zeta potential and FTIR findings. Further, we have injected prepared BAgNPs into oxaliplatin-treated mice to alleviate neuropathic pain. The aluminum levels in the DRG were reduced by the chelating activity of BAgNPs. Moreover, behavioral analyses also manifested that the accumulation of aluminum on the DRG might be a reason for neuropathic hyperalgesia. These findings also recommended that the chelation of aluminum by AgNPs could provide an effective remedy to alleviate the symptoms of oxaliplatin-induced neuropathic pain in cancer patients., (Copyright © 2017. Published by Elsevier B.V.)

All animals must detect noxious stimuli to initiate protective behavior, but the evolutionary origin of nociceptive systems is not well understood. Here we show that noxious heat and irritant chemicals elicit robust escape behaviors in the planarian Schmidtea mediterranea and that the conserved ion channel TRPA1 is required for these responses. TRPA1-mutant Drosophila flies are also defective in noxious-heat responses. We find that either planarian or human TRPA1 can restore noxious-heat avoidance to TRPA1-mutant Drosophila, although neither is directly activated by heat. Instead, our data suggest that TRPA1 activation is mediated by H 2 O 2 and reactive oxygen species, early markers of tissue damage rapidly produced as a result of heat exposure. Together, our data reveal a core function for TRPA1 in noxious heat transduction, demonstrate its conservation from planarians to humans, and imply that animal nociceptive systems may share a common ancestry, tracing back to a progenitor that lived more than 500 million years ago.

Chronic, persistent itch is a devastating symptom that causes much suffering. In recent years, there has been great progress made in understanding the molecules, cells, and circuits underlying itch sensation. Once thought to be carried by pain-sensing neurons, itch is now believed to be capable of being transmitted by dedicated sensory labeled lines. Members of the Mas-related G protein-coupled receptor (Mrgpr) family demarcate an itch-specific labeled line in the peripheral nervous system. In the spinal cord, the expression of other proteins identifies additional populations of itch-dedicated sensory neurons. However, as evidence for labeled-line coding has mounted, studies promoting alternative itch-coding strategies have emerged, complicating our understanding of the neural basis of itch. In this review, we cover the molecules, cells, and circuits related to understanding the neural basis of itch, with a focus on the role of Mrgprs in mediating itch sensation.

Dibutyl phthalate (DBP) is a plasticizer used for many consumer products including cosmetics. Potential health concerns regarding DBP include reproductive and developmental toxicity, endocrine disruption and neurotoxicity. DBP is a high priority chemical as to reduction of exposure of children to it. Through reproductive toxicity studies, monobutyl phthalate (MBP) has been proposed to be the active metabolite derived from DBP. We previously demonstrated that DBP activates transient receptor potential ankyrin 1 (TRPA1) cation channels expressed on sensory neurons. We have also shown that DBP enhanced skin sensitization in a fluorescein isothiocyanate (FITC)-induced contact hypersensitivity (CHS) mouse model. Through MBP formation by esterase in the skin, it is possible that MBP exerts a major effect on the biological activity we observed. To test this possibility, we directly compared DBP and MBP. A more than 40-fold higher concentration of MBP as compared with DBP was required for activation of TRPA1 in vitro. Unlike DBP, MBP did not enhance skin sensitization to FITC. These results demonstrated that DBP directly, i.e., not through its metabolite MBP, activates TRPA1 and enhances FITC-CHS. It is noteworthy that butyl benzoate, a related compound, activated TRPA1 and enhanced FITC-CHS.

Although both persistent itch and inflammation are commonly associated with allergic contact dermatitis (ACD), it is not known if they are mediated by shared or distinct signaling pathways. Here we show that both TRPA1 and TRPV1 channels are required for generating spontaneous scratching in a mouse model of ACD induced by squaric acid dibutylester (SADBE), a small molecule hapten, through directly promoting the excitability of pruriceptors. TRPV1 but not TRPA1 channels protect the skin inflammation, as genetic ablation of TRPV1 function or pharmacological ablation of TRPV1-positive sensory nerves promotes cutaneous inflammation in the SADBE-induced ACD. Our results demonstrate that persistent itch and inflammation are mediated by distinct cellular and molecular mechanisms in a mouse model of ACD. Identification of distinct roles of TRPA1 and TRPV1 in regulating itch and inflammation may provide new insights into the pathophysiology and treatment of chronic itch and inflammation in ACD patients.

Bortezomib (BTZ) is generally used as a chemotherapeutic agent for the treatment of multiple myeloma; however, one of the significant limiting complications of BTZ is painful peripheral neuropathy observed during BTZ therapy. There is a lack of drugs which can prevent and/or treat the painful symptoms induced by BTZ, as the underlying molecular mechanism leading to neuropathic pain remains largely unclear. In the present study, we examined engagement of proteinase-activated receptor 2 (PAR2) and transient receptor potential ankyrin 1 (TRPA1) in neuropathic pain induced by BTZ in rats. Our results demonstrated that systemic injection of BTZ increased mechanical pain and cold sensitivity as compared with control animals (P less than 0.05 vs control rats). Our data further showed that blocking respective PAR2 and TRPA1 attenuated mechanical pain and cold sensitivity observed in control rats and BTZ rats (P less than 0.05 vs vehicle control). Notably, the attenuating effect of blocking PAR2 and TRPA1 on mechanical pain and cold sensitivity was significantly less in BTZ rats than that in control rats. In addition, protein expression of PAR2 and TRPA1 was upregulated in the lumbar dorsal root ganglion of BTZ rats, and inhibition of PAR2 decreased the levels of TRPA1 and attenuated its downstream pathways (namely, PKCɛ and PKA). Overall, we revealed specific signaling pathways leading to neuropathic pain induced by chemotherapeutic BTZ and that blocking PAR2 and TRPA1 in sensory nerves is beneficial to improve neuropathic pain during BTZ intervention.

The cough reflex becomes hyperresponsive in acute and chronic respiratory diseases, but understanding the underlying mechanism is hampered by difficulty accessing human tissue containing both nerve endings and neuronal cell bodies. We refined an adult stem cell sensory neuronal model to overcome the limited availability of human neurones and applied the model to study transient receptor potential ankyrin 1 (TRPA1) channel expression and activation.Human dental pulp stem cells (hDPSCs) were differentiated towards a neuronal phenotype, termed peripheral neuronal equivalents (PNEs). Using molecular and immunohistochemical techniques, together with Ca 2+ microfluorimetry and whole cell patch clamping, we investigated roles for nerve growth factor (NGF) and the viral mimic poly I:C in TRPA1 activation.PNEs exhibited morphological, molecular and functional characteristics of sensory neurons and expressed functional TRPA1 channels. PNE treatment with NGF for 20 min generated significantly larger inward and outward currents compared to untreated PNEs in response to the TRPA1 agonist cinnamaldehyde (p<0.05). PNE treatment with poly I:C caused similar transient heightened responses to TRPA1 activation compared to untreated cells.Using the PNE neuronal model we observed both NGF and poly I:C mediated sensory neuronal hyperresponsiveness, representing potential neuro-inflammatory mechanisms associated with heightened nociceptive responses recognised in cough hypersensitivity syndrome., Competing Interests: Conflict of interest: Disclosures can be found alongside this article at erj.ersjournals.com, (Copyright ©ERS 2017.)

Optogenetics is a powerful research approach that allows localized optical modulation of selected cells within an animal via the expression of genetically encoded photo-excitable ion channels. Commonly used optogenetic techniques rely on the expression of microbial opsin variants, which have many excellent features but suffer from various degrees of blue spectral overlap and limited channel conductance. Here, we expand the optogenetics toolbox in the form of a tunable, high-conductance vertebrate cation channel, zTrpa1b, coupled with photo-activated channel ligands, such as optovin and 4g6. Our results demonstrate that zTrpa1b/ligand pairing offers high light sensitivity, millisecond-scale response latency in vivo, as well as adjustable channel off latency. Exogenous in vivo expression of zTrpa1b in sensory neurons allowed subcellular photo-activation, enabling light-dependent motor control. zTrpa1b/ligand was also suitable for cardiomyocyte pacing, as shown in experiments performed on zebrafish hearts in vivo as well as in human stem cell-derived cardiomyocytes in vitro. Therefore, zTrpa1b/optovin represents a novel tool for flexible, high-conductance optogenetics.

Hydroquinone (HQ) is one of the most frequently used and effective skin-lightening products to treat skin hyperpigmentation disorders, including postinflammatory hyperpigmentation, melasma and solar lentigines. HQ is also widely used in cosmetic products for skin whitening. However, HQ treatment can evoke substantial skin irritation, a side effect that remains poorly understood. Here we demonstrate that HQ is an activator of the peripheral irritant receptor transient receptor potential (TRP) cation channel member A1 (TRPA1). HQ failed to activate TRPV1, TRPV4 or TRPM8. HQ-induced TRPA1 activation was dependent on essential redox-sensitive cysteine and lysine residues within N-terminus of channel protein. HQ elicited Ca 2+ influx in a subpopulation of mouse sensory neurons sensitive to the TRPA1 agonist, mustard oil. HQ-induced neuronal responses were significantly reduced by TRPA1 inhibitors, and reduced in neurons isolated from Trpa1-deficient mice. In mice, intraplantar injection of HQ at clinically relevant concentrations elicited both acute pain and persistent mechanical hyperalgesia which were almost completely abolished by TRPA1 inhibitors. These findings identify TRPA1 as a molecular target for HQ and provide insights into the mechanism of HQ-induced skin irritation. These findings also suggest that selective TRPA1 antagonists may be useful to counteract HQ-induced skin irritation.

Although light is most commonly thought of as a visual cue, many animals possess mechanisms to detect light outside of the eye for various functions, including predator avoidance, circadian rhythms, phototaxis and migration. Here we confirm that planarians (like Caenorhabditis elegans , leeches and Drosophila larvae) are capable of detecting and responding to light using extraocular photoreception. We found that, when either eyeless or decapitated worms were exposed to near-ultraviolet (near-UV) light, intense wild-type photophobic behaviors were still observed. Our data also revealed that behavioral responses to green wavelengths were mediated by ocular mechanisms, whereas near-UV responses were driven by extraocular mechanisms. As part of a candidate screen to uncover the genetic basis of extraocular photoreception in the planarian species Schmidtea mediterranea , we identified a potential role for a homolog of the transient receptor potential channel A1 ( TRPA1 ) in mediating behavioral responses to extraocular light cues. RNA interference (RNAi) to Smed-TrpA resulted in worms that lacked extraocular photophobic responses to near-UV light, a mechanism previously only identified in Drosophila These data show that the planarian TRPA1 homolog is required for planarian extraocular-light avoidance and may represent a potential ancestral function of this gene. TRPA1 is an evolutionarily conserved detector of temperature and chemical irritants, including reactive oxygen species that are byproducts of UV-light exposure. Our results suggest that planarians possess extraocular photoreception and display an unconventional TRPA1-mediated photophobic response to near-UV light., Competing Interests: Competing interestsThe authors declare no competing or financial interests., (© 2017. Published by The Company of Biologists Ltd.)

Phospholipids occurring in cell membranes and lipoproteins are converted into oxidized phospholipids (OxPL) by oxidative stress promoting atherosclerotic plaque formation. Here, OxPL were characterized as novel targets in acute and chronic inflammatory pain. Oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (OxPAPC) and its derivatives were identified in inflamed tissue by mass spectrometry and binding assays. They elicited calcium influx, hyperalgesia and induced pro-nociceptive peptide release. Genetic, pharmacological and mass spectrometric evidence in vivo as well as in vitro confirmed the role of transient receptor potential channels (TRPA1 and TRPV1) as OxPAPC targets. Treatment with the monoclonal antibody E06 or with apolipoprotein A-I mimetic peptide D-4F, capturing OxPAPC in atherosclerosis, prevented inflammatory hyperalgesia, and in vitro TRPA1 activation. Administration of D-4F or E06 to rats profoundly ameliorated mechanical hyperalgesia and inflammation in collagen-induced arthritis. These data reveal a clinically relevant role for OxPAPC in inflammation offering therapy for acute and chronic inflammatory pain treatment by scavenging OxPAPC.

Neuropathic pain is a nerve disorder characterized by the dysregulation of ion channels in dorsal root ganglion (DRG) neurons. MicroRNAs (miRs) may be associated with the molecular mechanisms underlying the altered levels of ion channels; however, the molecular mechanisms remain widely unknown. To investigate these mechanisms, the present study conducted a genomic analysis of miR between a unilateral spared nerve injury (SNI) model and sham control. Differentially expressed miRs between the SNI and sham groups were selected for transfection of DRG cells, a polymerase chain reaction (PCR) array analysis was subsequently performed. A total of three significantly differently expressed genes were selected from the results of the PCR array and further analyzed by reverse transcription‑quantitative PCR. Genomic analysis revealed that Mus musculus miR‑449a (mmu‑miR‑449a) was reduced in the SNI groups compared with the sham controls. The PCR array indicated that mmu‑miR‑449a‑transfection reduced the mRNA expression levels of transient receptor potential cation channel subfamily A member 1 (TRPA1), and calcium‑activated potassium channel subunit α‑1 (KCNMA1) and increased the level of transmembrane phosphatase with tension homology (TPTE) in the DRG cells (P<0.05). qRT‑PCR analysis further indicated that mmu‑miR‑449a transfection caused similar alterations in the mRNA expression levels of TRPA1, KCNMA1 and TPTE in DRG cells, respectively (P<0.05). Therefore, mmu‑miR‑449a may ameliorate neuropathic pain by decreasing the activity of the channel proteins TRPA1 and KCNMA1 and increasing the levels of TPTE. mmu‑miR‑449a may be a potential therapeutic molecule for the alleviation of neuropathic pain.

Itch is a common symptom in patients with skin and systemic diseases, but the effective treatment is limited. Here, we evaluated the anti-itch effects of the botulinum toxin type A (BoNT/A) using acute and chronic dry skin itch mouse models, which were induced by compound 48/80, chloroquine, and a mixture of acetone-diethylether-water treatment, respectively. Pretreatment of intradermal BoNT/A exerted long-term inhibitory effects on compound 48/80-induced and chloroquine-induced acute itch on days 1, 3, 7, and 14, but not on day 21, in mice. Furthermore, a single injection of BoNT/A reduced the expression of the transient receptor potential cation channel, subfamily V, member 1 (TRPV1), and the transient receptor potential cation channel, subfamily A, member 1 (TRPA1) at both transcriptional and translational levels in the dorsal root ganglia (DRG) in mice. Pretreatment of BoNT/A also attenuated chronic itch induced by acetone-diethylether-water treatment and abolished the upregulation of TRPA1 in the DRG. Thus, it was suggested that downregulation of the expression of TRPA1 and TRPV1 in the DRG may contribute toward the long-term anti-itch effects of a single injection of BoNT/A in mice and BoNT/A treatment may serve as an alternative strategy for anti-itch therapy.

The molecular sensor of innocuous (painless) cold sensation is well-established to be transient receptor potential cation channel, subfamily M, member 8 (TRPM8). However, the role of transient receptor potential cation channel, subfamily A, member 1 (TRPA1) in noxious (painful) cold sensation has been controversial. We find that TRPA1 channels contribute to the noxious cold sensitivity of mouse somatosensory neurons, independent of TRPM8 channels, and that TRPA1-expressing neurons are largely non-overlapping with TRPM8-expressing neurons in mouse dorsal-root ganglia (DRG). However, relatively few TRPA1-expressing neurons (e.g., responsive to allyl isothiocyanate or AITC, a selective TRPA1 agonist) respond overtly to cold temperature in vitro, unlike TRPM8-expressing neurons, which almost all respond to cold. Using somatosensory neurons from TRPM8-/- mice and subtype-selective blockers of TRPM8 and TRPA1 channels, we demonstrate that responses to cold temperatures from TRPA1-expressing neurons are mediated by TRPA1 channels. We also identify two factors that affect the cold-sensitivity of TRPA1-expressing neurons: (1) cold-sensitive AITC-sensitive neurons express relatively more TRPA1 transcripts than cold-insensitive AITC-sensitive neurons and (2) voltage-gated potassium (K V ) channels attenuate the cold-sensitivity of some TRPA1-expressing neurons. The combination of these two factors, combined with the relatively weak agonist-like activity of cold temperature on TRPA1 channels, partially explains why few TRPA1-expressing neurons respond to cold. Blocking K V channels also reveals another subclass of noxious cold-sensitive DRG neurons that do not express TRPM8 or TRPA1 channels. Altogether, the results of this study provide novel insights into the cold-sensitivity of different subclasses of somatosensory neurons., (Copyright © 2017 IBRO. Published by Elsevier Ltd. All rights reserved.)

Key Points: Chloroquine (CQ) stimulates itch nerves and causes intense scratching in mice by activating the G-protein coupled receptor (GPCR) MrgprA3; it is not known how stimulation of MrgprA3 (or other GPCRs) leads to activation of the itch nerve terminals in the skin, but previous studies have found that transient receptor potential A1 (TRPA1) gene deletion blocks CQ-induced scratching. In the present study we used a novel dorsal skin-nerve preparation to evaluate mechanisms underlying CQ- and histamine-induced action potential discharge in itch nerve terminals. We found that CQ activation of the nerves requires the beta3 isoform of phospholipase C, but TRPA1 or other TRP channel are not required. Evidence is provided for a role for calcium-activated chloride channels such as TMEM16a in GPCR-activation of itch nerve terminals. The mechanism by which TRP channels participate in pruritogen-induced scratching may involve sites of action other than the primary afferent terminals., Abstract: Chloroquine (CQ) and histamine are pruritogens commonly used to study itch in the mouse. A novel skin-nerve preparation was used to evaluate chloroquine (CQ)- and histamine-induced activation of afferent nerves in the dorsal thoracic skin of the mouse. All CQ sensitive nerves were C-fibres, and were also sensitive to histamine. The response to CQ, but not histamine, was largely absent in mrgpr-cluster Δ -/- mice, supporting the hypothesis that CQ evokes itch largely via stimulation of MrgprA3 receptors. The CQ-induced action potential discharge was largely absent in phospholipase Cβ3 knockout animals. The CQ and histamine responses were not influenced by removal of TRPA1, TRPV1, TRPC3 or TRPC6, nor by the TRP channel blocker Ruthenium Red. The bouts of scratching in response to CQ were not different between wild-type and TRPA1-deficient mice. A selective inhibitor of the calcium-activated chloride channel TMEM16A, N-((4-methoxy)-2-naphthyl)-5-nitroanthranilic acid (MONNA), inhibited CQ-induced action potential discharge at itch nerve terminals and bouts of scratching by about 50%. Although TRPA1 and TRPV1 channels may be involved in the scratching responses to intradermal pruritogens, this is unlikely to be due to an effect at the nerve terminals, where chloride channels may play a more important role., (© 2017 The Authors. The Journal of Physiology © 2017 The Physiological Society.)

Mechanical pain serves as a base clinical symptom for many of the world's most debilitating syndromes. Ion channels expressed by peripheral sensory neurons largely contribute to mechanical hypersensitivity. Transient Receptor Potential A 1 (TRPA1) is a ligand-gated ion channel that contributes to inflammatory mechanical hypersensitivity, yet little is known as to the post-translational mechanism behind its somatosensitization. Here, we utilize biochemical, electrophysiological, and behavioral measures to demonstrate that metabotropic glutamate receptor-induced sensitization of TRPA1 nociceptors stimulates targeted modification of the receptor. Type 1 mGluR5 activation increases TRPA1 receptor agonist sensitivity in an AKA-dependent manner. As a scaffolding protein for Protein Kinases A and C (PKA and PKC, respectively), AKAP facilitates phosphorylation and sensitization of TRPA1 in ex vivo sensory neuronal preparations. Furthermore, hyperalgesic priming of mechanical hypersensitivity requires both TRPA1 and AKAP. Collectively, these results identify a novel AKAP-mediated biochemical mechanism that increases TRPA1 sensitivity in peripheral sensory neurons, and likely contributes to persistent mechanical hypersensitivity.

When exposed to microorganisms, animals use several protective strategies. On one hand, as elegantly exemplified in Drosophila melanogaster, the innate immune system recognizes microbial compounds and triggers an antimicrobial response. On the other hand, behaviors preventing an extensive contact with the microbes and thus reducing the risk of infection have been described. However, these reactions ranging from microbes aversion to intestinal transit increase or food intake decrease have been rarely defined at the molecular level. In this study, we set up an experimental system that allowed us to rapidly identify and quantify food intake decreases in Drosophila larvae exposed to media contaminated with bacteria. Specifically, we report a robust dose-dependent food intake decrease following exposure to the bacteria Erwinia carotovora carotovora strain Ecc15. We demonstrate that this response does not require Imd innate immune pathway, but rather the olfactory neuronal circuitry, the Trpa1 receptor and the evf virulence factor. Finally, we show that Ecc15 induce the same behavior in the invasive pest insect Drosophila suzukii., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

TRPA1 receptors are calcium-permeable ligand-gated channels expressed in primary sensory neurons and involved in inflammation and pain. Activation of these neurons might have analgesic effect. Suggested mechanism of analgesic effect mediated by TRPA1 activation is the release of somatostatin (SOM) and its action on sst 4 receptors. In the present study analgesic effect of TRPA1 activation on primary sensory neurons by organic trisulfide compound dimethyl trisulfide (DMTS) presumably leading to SOM release was investigated. Opening of TRPA1 by DMTS in CHO cells was examined by patch-clamp and fluorescent Ca 2+ detection. Ca 2+ influx upon DMTS administration in trigeminal ganglion (TRG) neurons of TRPA1 receptor wild-type (WT) and knockout (KO) mice was detected by ratiometric Ca 2+ imaging. SOM release from sensory nerves of murine skin was assessed by radioimmunoassay. Analgesic effect of DMTS in mild heat injury-induced mechanical hyperalgesia was examined by dynamic plantar aesthesiometry. Regulatory role of DMTS on deep body temperature (T b ) was measured by thermocouple thermometry with respirometry and by telemetric thermometry. DMTS produced TRPA1-mediated currents and elevated [Ca 2+ ] i in CHO cells. Similar data were obtained in TRG neurons. DMTS released SOM from murine sensory neurons TRPA1-dependently. DMTS exerted analgesic effect mediated by TRPA1 and sst 4 receptors. DMTS-evoked hypothermia and hypokinesis were attenuated in freely-moving TRPA1 KO animals. Our study has presented original evidence regarding analgesic action of DMTS which might be due to TRPA1-mediated SOM release from sensory neurons and activation of sst 4 receptors. DMTS could be a novel analgesic drug candidate., (Copyright © 2017 Elsevier Inc. All rights reserved.)

General anesthetics suppress CNS activity by modulating the function of membrane ion channels, in particular, by enhancing activity of GABA A receptors. In contrast, several volatile (isoflurane, desflurane) and i.v. (propofol) general anesthetics excite peripheral sensory nerves to cause pain and irritation upon administration. These noxious anesthetics activate transient receptor potential ankyrin repeat 1 (TRPA1), a major nociceptive ion channel, but the underlying mechanisms and site of action are unknown. Here we exploit the observation that pungent anesthetics activate mammalian but not Drosophila TRPA1. Analysis of chimeric Drosophila and mouse TRPA1 channels reveal a critical role for the fifth transmembrane domain (S5) in sensing anesthetics. Interestingly, we show that anesthetics share with the antagonist A-967079 a potential binding pocket lined by residues in the S5, S6, and the first pore helix; isoflurane competitively disrupts A-967079 antagonism, and introducing these mammalian TRPA1 residues into dTRPA1 recapitulates anesthetic agonism. Furthermore, molecular modeling predicts that isoflurane and propofol bind to this pocket by forming H-bond and halogen-bond interactions with Ser-876, Met-915, and Met-956. Mutagenizing Met-915 or Met-956 selectively abolishes activation by isoflurane and propofol without affecting actions of A-967079 or the agonist, menthol. Thus, our combined experimental and computational results reveal the potential binding mode of noxious general anesthetics at TRPA1. These data may provide a structural basis for designing drugs to counter the noxious and vasorelaxant properties of general anesthetics and may prove useful in understanding effects of anesthetics on related ion channels.

Background: Animal data have suggested that the transient receptor potential ankyrin-1 (TRPA1) ion channel plays a key role in promoting airway inflammation in asthma and may mediate effects of paracetamol on asthma, yet confirmatory human data are lacking. To study associations of TRPA1 gene variants with childhood asthma and total IgE concentration, and interactions between TRPA1 and prenatal paracetamol exposure on these outcomes., Methods: We analysed associations between 31 TRPA1 single nucleotide polymorphisms (SNPs) and current doctor-diagnosed asthma and total IgE concentration at 7.5 years in the Avon Longitudinal Study of Parents and Children (ALSPAC) birth cohort. We sought to confirm the most significant associations with comparable outcomes in the Prevention and Incidence of Asthma and Mite Allergy (PIAMA) and Generation R birth cohorts. In ALSPAC, we explored interactions with prenatal paracetamol exposure., Results: In ALSPAC, there was strong evidence for association between six SNPs and asthma: rs959974 and rs1384001 (per-allele odds ratio for both: 1.30 (95% CI: 1.15-1.47), p = 0.00001), rs7010969 (OR 1.28 (1.13-1.46), p = 0.00004), rs3735945 (OR 1.30 (1.09-1.55), p = 0.003), rs920829 (OR 1.30 (1.09-1.54), p = 0.004) and rs4738202 (OR 1.22 (1.07-1.39), p = 0.004). In a meta-analysis across the three cohorts, the pooled effect estimates confirmed that all six SNPs were significantly associated with asthma. In ALSPAC, TRPA1 associations with asthma were not modified by prenatal paracetamol, although associations with IgE concentration were., Conclusion: This study suggests that TRPA1 may play a role in the development of childhood asthma. (249 words)., (© 2016 The Authors Pediatric Allergy and Immunology Published by John Wiley & Sons Ltd.)

The thiazolidine ring is a biologically active chemical structure and is associated with many pharmacological activities. However, the biological molecules that can interact with the thiazolidine ring are not known. We show that thiazolidine causes sustained activation of the TRPA1 channel and chemically reacts with glutathione, and the chemical reactivity of thiazolidine ring is required for TRPA1 activation. Reducing agents reverse thiazolidine-induced TRPA1 activation, and mutagenesis studies show that nucleophilic cysteine residues in TRPA1 are critical, suggesting an activation mechanism involving thioreactive chemical reactions. In vivo studies show that thiazolidine induces acute pain and inflammation in mouse and these responses are specifically dependent on TRPA1. These results indicate that thiazolidine compounds can chemically react with biological molecules containing nucleophilic cysteines, thereby exerting biological activities., (Copyright © 2017 Elsevier Inc. All rights reserved.)

The possible pathogenic role and mechanism of Di-iso-nonyl phthalate (DINP) in allergic dermatitis is still controversial. This work has shown that oral exposure to DINP exacerbated allergic dermatitis tissue lesions in FITC-sensitized mice. The lesions was accompanied by an enhancement of TRPA1 expression and an increase in IgG1, IL-6 and IL-13 levels. This work also found that blocking TRPA1 by HC030031 effectively prevented the development of allergic dermatitis resulting from oral exposure to DINP and/or FITC-sensitized mice. This result is marked by the down regulation of IgG1 levels, a reduction in mast cell degranulation and a decrease in IL-6 and IL-13 levels. We also showed that blocking NF-κB inhibited TRPA1 expression, and that blocking TRPA1 had no significant effect on the activation of NF-κB or TSLP expression. This study helps in understanding the role DINP exposure plays in the development of allergic dermatitis and provides new insight into the mechanisms behind the DINP-induced adjuvant effect.

The Notch1 pathway plays important roles in modulating erythroid and megakaryocyte differentiation. To screen the Notch1-related genes that regulate differentiation fate of K562 and HEL cells, the expression of transient receptor potential ankyrin 1 (TRPA1) was induced by Notch1 receptor intracellular domain (N1IC), the activated form of Notch1 receptor. N1IC and v-ets erythroblastosis virus E26 oncogene homolog 1 (Ets-1) bound to TRPA1 promoter region to regulate transcription in K562 cells. Transactivation of TRPA1 promoter by N1IC depended on the methylation status of TRPA1 promoter. N1IC and Ets-1 suppressed the DNA methyltransferase 3B (DNMT3B) level in K562 cells. Inhibition of TRPA1 expression after Notch1 knockdown could be attenuated by nanaomycin A, an inhibitor of DNMT3B, in K562 and HEL cells. Functionally, hemin-induced erythroid differentiation could be suppressed by TRPA1, and the reduction of erythroid differentiation of both cells by N1IC and Ets-1 occurred via TRPA1. However, PMA-induced megakaryocyte differentiation could be enhanced by TRPA1, and the surface markers of megakaryocytes could be elevated by nanaomycin A. Megakaryocyte differentiation could be reduced by Notch1 or Ets-1 knockdown and relieved by TRPA1 overexpression. The results suggest that Notch1 and TRPA1 might be critical modulators that control the fate of erythroid and megakaryocyte differentiation.

Sleep is a highly conserved and essential behaviour in many species, including the fruit fly Drosophila melanogaster. In the wild, sensory signalling encoding environmental information must be integrated with sleep drive to ensure that sleep is not initiated during detrimental conditions. However, the molecular and circuit mechanisms by which sleep timing is modulated by the environment are unclear. Here we introduce a novel behavioural paradigm to study this issue. We show that in male fruit flies, onset of the daytime siesta is delayed by ambient temperatures above 29 °C. We term this effect Prolonged Morning Wakefulness (PMW). We show that signalling through the TrpA1 thermo-sensor is required for PMW, and that TrpA1 specifically impacts siesta onset, but not night sleep onset, in response to elevated temperatures. We identify two critical TrpA1-expressing circuits and show that both contact DN1p clock neurons, the output of which is also required for PMW. Finally, we identify the circadian blue-light photoreceptor CRYPTOCHROME as a molecular regulator of PMW, and propose a model in which the Drosophila nervous system integrates information encoding temperature, light, and time to dynamically control when sleep is initiated. Our results provide a platform to investigate how environmental inputs co-ordinately regulate sleep plasticity.

Transient Receptor Potential Vanilloid 1 (TRPV1) and Transient Receptor Potential Ankyrin 1 (TRPA1) expressed mainly by primary sensory neurons function as major nociceptive integrators. They are also present on the rat endometrium in an oestrogen-regulated manner. TRPV1 is upregulated in peritoneal and ovarian endometriosis patients, but there is no information about TRPA1 and their pathophysiological significances. In this study, patients undergoing laparoscopic surgery were investigated: severe dysmenorrhoea due to rectosigmoid deep infiltrating endometriosis ( n = 15), uterine fibroid-induced moderate dysmenorrhoea ( n = 7) and tubal infertility with no pain ( n = 6). TRPA1 and TRPV1 mRNA and protein expressions were determined by quantitative polymerase chain reaction and semi-quantitative immunohistochemistry from the endometrium samples taken by curettage. Results were correlated with the clinical characteristics including pain intensity. TRPA1 and TRPV1 receptors were expressed in the healthy human endometrium at mRNA and protein levels. Sparse, scattered cytoplasmic TRPA1 and TRPV1 immunopositivities were found in the stroma and epithelial layers. We detected upregulated mRNA levels in deep infiltrating endometriosis lesions, and TRPV1 gene expression was also elevated in autocontrol endometrium of deep infiltrating endometriosis patients. Histological scoring revealed significant TRPA1 and TRPV1 difference between deep infiltrating endometriosis stroma and epithelium, and in deep infiltrating endometriosis epithelium compared to control samples. Besides, we measured elevated stromal TRPV1 immunopositivity in deep infiltrating endometriosis. Stromal TRPA1 and TRPV1 immunoreactivities strongly correlated with dysmenorrhoea severity, as well TRPV1 expression on ectopic epithelial cells and macrophages with dyspareunia. Epithelial TRPA1 and stromal TRPV1 immunopositivity also positively correlated with dyschezia severity. We provide the first evidence for the presence of non-neuronal TRPA1 receptor in the healthy human endometrium and confirm the expression of TRPV1 channels. Their upregulations in rectosigmoid deep infiltrating endometriosis lesions and correlations with pain intensity suggest potential roles in pathophysiological mechanisms of the disease.

Recently, in vivo confocal microscopy is used to examine the human corneal nerve fibers morphology. Corneal nerve fiber architecture and its role are studied in healthy and pathological conditions. Corneal nerves of rats were studied by nonspecific acetylcholinesterase (NsAchE) staining. NsAchE-positive subepithelial (stromal) nerve fiber has been found to be insensitive to capsaicin. Besides, NsAchE-negative but capsaicin-sensitive subbasal nerve (leash) fibers formed thick mesh-like structure showing close interconnections and exhibit both isolectin B4- and transient receptor potential vanilloid channel 1- (TRPV1-) positive. TRPV1, TRPV3, TRPA (ankyrin) 1, and TRPM (melastatin) 8 are expressed in corneal nerve fibers. Besides the corneal nerve fibers, the expressions of TRPV (1, 3, and 4), TRPC (canonical) 4, and TRPM8 are demonstrated in the corneal epithelial cell membrane. The realization of the importance of TRP channels acting as polymodal sensors of environmental stresses has identified potential drug targets for corneal disease. The pathophysiological conditions of corneal diseases are associated with disruption of normal tissue innervation, especially capsaicin-sensitive small sensory nerve fibers. The relationships between subbasal corneal nerve fiber morphology and neurotrophic keratopathy in corneal diseases are well studied. The recommended treatment for neurotrophic keratopathy is administration of preservative free eye drops.

Astrocytes display complex morphologies with an array of fine extensions extending from the soma and the primary thick processes. Until the use of genetically encoded calcium indicators (GECIs) selectively expressed in astrocytes, Ca 2+ signaling was only examined in soma and thick primary processes of astrocytes where Ca 2+ -sensitive fluorescent dyes could be imaged. GECI imaging in astrocytes revealed a previously unsuspected pattern of spontaneous Ca 2+ transients in fine processes that has not been observed without chronic expression of GECIs, raising potential concerns about the effects of GECI expression. Here, we perform two-photon imaging of Ca 2+ transients in adult CA1 hippocampal astrocytes using a new single-cell patch-loading strategy to image Ca 2+ -sensitive fluorescent dyes in the cytoplasm of fine processes. We observed that astrocyte fine processes exhibited a high frequency of spontaneous Ca 2+ transients whereas astrocyte soma rarely showed spontaneous Ca 2+ oscillations similar to previous reports using GECIs. We exploited this new approach to show these signals were independent of neuronal spiking, metabotropic glutamate receptor (mGluR) activity, TRPA1 channels, and L- or T-type voltage-gated calcium channels. Removal of extracellular Ca 2+ almost completely and reversibly abolished the spontaneous signals while IP 3 R2 KO mice also exhibited spontaneous and compartmentalized signals, suggesting they rely on influx of extracellular Ca 2+ . The Ca 2+ influx dependency of the spontaneous signals in patch-loaded astrocytes was also observed in astrocytes expressing GCaMP3, further highlighting the presence of Ca 2+ influx pathways in astrocytes. The mechanisms underlying these localized Ca 2+ signals are critical for understanding how astrocytes regulate important functions in the adult brain. GLIA 2016;64:2093-2103., (© 2016 Wiley Periodicals, Inc.)

Transient receptor potential ankyrin 1 (TRPA1), a member of the TRP superfamily, exists in sensory neurons such as trigeminal neurons innervating the nasal cavity and vagal neurons innervating the trachea and the lung. Although TRPA1 has been proposed as an irritant receptor that, when stimulated, triggers bradypnea, precise locations of the receptors responsible have not been elucidated. Here, we examined the relative importance of TRPA1 located in the upper airway (nasal) and the lower airway (trachea/lungs) in urethane-anesthetized mice. To stimulate the upper and lower airways separately, two cannulas were inserted through a hole made in the trachea just caudal to the thyroid cartilage, one into the nasal cavity and the second into the lower trachea. A vapor of one of the TRPA1-agonists, allyl isothiocyanate (AITC), was introduced by placing a piece of cotton paper soaked with AITC solution into the airline. AITC decreased the respiratory frequency when applied to the upper airway (ca -30%) but not to the lower airway (ca -5%). No response was observed in TRPA1 knockout mice. Contribution of the olfactory nerve seemed minimal because olfactory bulbectomized wild-type mice showed a similar response to that of the intact mice. AITC-induced bradypnea seemed to be mediated, at least in part, by the trigeminal nerve because trigeminal ganglion neurons were activated by AITC as revealed by an increase in the phosphorylated form of extracellular signal-regulated kinase in the neurons. These data clearly show that trigeminal TRPA1 in the nasal cavity play an essential role in irritant-induced bradypnea., (© 2016 The Authors. Physiological Reports published by Wiley Periodicals, Inc. on behalf of The Physiological Society and the American Physiological Society.)

Multiple sclerosis is a chronic inflammatory, demyelinating degenerative disease of the central nervous system. Current treatments target pathological immune responses to counteract the inflammatory processes. However, these drugs do not restrain the long-term progression of clinical disability. For this reason, new therapeutic approaches and identification of novel target molecules are needed to prevent demyelination or promote repair mechanisms. Transient Receptor Potential Ankyrin 1 (TRPA1) is a nonselective cation channel with relatively high Ca 2+ permeability. Its pathophysiological role in central nervous system disorders has not been elucidated yet. In the present study, we aimed to assess the distribution of TRPA1 in the mouse brain and reveal its regulatory role in the cuprizone-induced demyelination. This toxin-induced model, characterized by oligodendrocyte apoptosis and subsequent primary demyelination, allows us to investigate the nonimmune aspects of multiple sclerosis. We found that TRPA1 is expressed on astrocytes in the mouse central nervous system. Interestingly, TRPA1 deficiency significantly attenuated cuprizone-induced demyelination by reducing the apoptosis of mature oligodendrocytes. Our data suggest that TRPA1 regulates mitogen-activated protein kinase pathways, as well as transcription factor c-Jun and a proapoptotic Bcl-2 family member (Bak) expression resulting in enhanced oligodendrocyte apoptosis. In conclusion, we propose that TRPA1 receptors enhancing the intracellular Ca 2+ concentration modulate astrocyte functions, and influence the pro or anti-apoptotic pathways in oligodendrocytes. Inhibition of TRPA1 receptors might successfully diminish the degenerative pathology in multiple sclerosis and could be a promising therapeutic target to limit central nervous system damage in demyelinating diseases. GLIA 2016;64:2166-2180., (© 2016 Wiley Periodicals, Inc.)

Drosophila third-instar larvae exhibit changes in their behavioral responses to gravity and food as they transition from feeding to wandering stages. Using a thermal gradient encompassing the comfortable range (18°C to 28°C), we found that third-instar larvae exhibit a dramatic shift in thermal preference. Early third-instar larvae prefer 24°C, which switches to increasingly stronger biases for 18°C-19°C in mid- and late-third-instar larvae. Mutations eliminating either of two rhodopsins, Rh5 and Rh6, wiped out these age-dependent changes in thermal preference. In larvae, Rh5 and Rh6 are thought to function exclusively in the light-sensing Bolwig organ. However, the Bolwig organ was dispensable for the thermal preference. Rather, Rh5 and Rh6 were required in trpA1-expressing neurons in the brain, ventral nerve cord, and body wall. Because Rh1 contributes to thermal selection in the comfortable range during the early to mid-third-instar stage, fine thermal discrimination depends on multiple rhodopsins., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

We previously found that transient receptor potential ankyrin 1 (TRPA1) in guinea pig tracheal epithelial cells was elevated after 14 days of cigarette smoke (CS) exposure. However, the mechanism underlying CS-induced TRPA1 expression remains unknown. Here, we explored whether cigarette smoke extract (CSE)-induced TRPA1 expression is related with modulation of HIF1α in A549 cells. Our results showed that CSE increased TRPA1 expression in A549 cells, decreased Iκ B, PHD2, and HDAC2, and increased ROS release and nuclear translocation of NF-κ B and HIF1α. Moreover, HIF1α siRNA and/or MG132 (a proteasome inhibitor) pretreatment significantly inhibited CSE-induced TRPA1 expression and HIF1α nuclear translocation in A549 cells. However, HIF1α siRNA pretreatment did not affect CSE-induced NF-κ B nuclear translocation, suggesting that CSE-induced TRPA1 expression in A549 cells is directly mediated by HIF1α, but not by NF-κ B. Similar to CSE treatment, treatment of A549 cells with LPS caused significant increases in nuclear translocation of NF-κ B and HIF1α mRNA expression, but did not alter TRPA1 mRNA expression. However, pretreatment with PHD2 siRNA did result in increased TRPA1 mRNA expression in LPS-treated A549 cells; an effect that was inhibited by SN50 (a NF-κ B inhibitor). It suggests a role for NF-κ B to indirectly regulate TRPA1 mRNA expression via modulating HIF1α mRNA transcription. In addition, treatment cells with HDAC2 siRNA plus 2%CSE resulted in increased HIF1α nuclear translocation and TRPA1 expression, which was significantly inhibited by MG132 and HIF1α siRNA. These results suggest that HDAC2 indirectly modulates TRPA1 expression by promoting the DNA-binding activity of HIF1α. These findings show that CSE increases TRPA1 expression in airway epithelial cells by directly activating HIF1α, and that this increase in TRPA1 expression is indirectly regulated via NF-κ B, PHD2 and HDAC2 modulation of HIF1α activity., (Copyright © 2016. Published by Elsevier Inc.)

Mammalian transient receptor potential ankyrin 1 (TRPA1) is a polymodal nociceptor that plays an important role in pain generation, but its role as a cold nociceptor is still controversial. Here, we propose that TRPA1 can sense noxious cold via transduction of reactive oxygen species (ROS) signalling. We show that inhibiting hydroxylation of a proline residue within the N-terminal ankyrin repeat of human TRPA1 by mutation or using a prolyl hydroxylase (PHD) inhibitor potentiates the cold sensitivity of TRPA1 in the presence of hydrogen peroxide. Inhibiting PHD in mice triggers mouse TRPA1 sensitization sufficiently to sense cold-evoked ROS, which causes cold hypersensitivity. Furthermore, this phenomenon underlies the acute cold hypersensitivity induced by the chemotherapeutic agent oxaliplatin or its metabolite oxalate. Thus, our findings provide evidence that blocking prolyl hydroxylation reveals TRPA1 sensitization to ROS, which enables TRPA1 to convert ROS signalling into cold sensitivity.

Purpose: Airway smooth muscle cell (ASMC) phenotypic modulation is one of the key factors contributing to asthma. Temperature changes may induce asthma, and these changes are known to be related to the temperature-sensitive transient receptor potential channels (TS-TRPs). The present study was designed to investigate the cellular functions of cold-sensitive channels, TRPM8 and TRPA1, in the phenotypic modulation of ASMCs., Methods: A rat asthma model was constructed and the expression of TS-TRPs in ASM was tested. Using the agonists and antagonists for both TRPM8 and TRPA1, the effects of cold-sensitive channels on the phenotypic modulation of ASMCs were evaluated by measurement of contractile protein expression and cell proliferation and migration. Signaling pathways and matrix metalloproteinase-2 (MMP-2) activity were assayed with Western blotting and gelatin zymography., Results: TRPM8 and TRPA1 were decreased in the ASM of the rat asthma model. Icilin and menthol, agonists for TRPM8 and TRPA1, inhibited ASMC proliferation and migration induced by fetal bovine serum (FBS) or platelet-derived growth factor (PDGF). Moreover, icilin reversed the FBS-induced inhibition of the expression of contractile phenotype markers, smooth muscle α-actin, and SM22α. Icilin also antagonized the activation of p38 and MMP-2 and the repression of p21 caused by FBS., Conclusions: Our findings show, for the first time, that the activation of TRPM8 and TRPA1 inhibits ASMC proliferative phenotype. These data suggest that TRPM8 and TRPA1 agonists may be promising new therapies for asthma.

Drosophila larval crawling is an attractive system to study rhythmic motor output at the level of animal behavior. Larval crawling consists of waves of muscle contractions generating forward or reverse locomotion. In addition, larvae undergo additional behaviors, including head casts, turning, and feeding. It is likely that some neurons (e.g., motor neurons) are used in all these behaviors, but the identity (or even existence) of neurons dedicated to specific aspects of behavior is unclear. To identify neurons that regulate specific aspects of larval locomotion, we performed a genetic screen to identify neurons that, when activated, could elicit distinct motor programs. We used 165 Janelia CRM-Gal4 lines-chosen for sparse neuronal expression-to ectopically express the warmth-inducible neuronal activator TrpA1, and screened for locomotor defects. The primary screen measured forward locomotion velocity, and we identified 63 lines that had locomotion velocities significantly slower than controls following TrpA1 activation (28°). A secondary screen was performed on these lines, revealing multiple discrete behavioral phenotypes, including slow forward locomotion, excessive reverse locomotion, excessive turning, excessive feeding, immobile, rigid paralysis, and delayed paralysis. While many of the Gal4 lines had motor, sensory, or muscle expression that may account for some or all of the phenotype, some lines showed specific expression in a sparse pattern of interneurons. Our results show that distinct motor programs utilize distinct subsets of interneurons, and provide an entry point for characterizing interneurons governing different elements of the larval motor program., (Copyright © 2016 Clark et al.)

We demonstrate a novel dual strategy against inflammation and pain through body-wide desensitization of nociceptors via TRPA1. Attenuation of experimental colitis by capsazepine (CPZ) has long been attributed to its antagonistic action on TRPV1 and associated inhibition of neurogenic inflammation. In contrast, we found that CPZ exerts its anti-inflammatory effects via profound desensitization of TRPA1. Micromolar CPZ induced calcium influx in isolated dorsal root ganglion (DRG) neurons from wild-type (WT) but not TRPA1-deficient mice. CPZ-induced calcium transients in human TRPA1-expressing HEK293t cells were blocked by the selective TRPA1 antagonists HC 030031 and A967079 and involved three cysteine residues in the N-terminal domain. Intriguingly, both colonic enemas and drinking water with CPZ led to profound systemic hypoalgesia in WT and TRPV1(-/-) but not TRPA1(-/-) mice. These findings may guide the development of a novel class of disease-modifying drugs with anti-inflammatory and anti-nociceptive effects.

Thermosensitive Transient Receptor Potential (TRP) channels are believed to respond to either cold or heat. In the case of TRP subtype A1 (TRPA1), there seems to be a species-dependent divergence in temperature sensation as non-mammalian TRPA1 is heat-sensitive whereas mammalian TRPA1 is sensitive to cold. It has been speculated but never experimentally proven that TRPA1 and other temperature-sensitive ion channels have the inherent capability of responding to both cold and heat. Here we show that redox modification and ligands affect human TRPA1 (hTRPA1) cold and heat sensing properties in lipid bilayer and whole-cell patch-clamp recordings as well as heat-evoked TRPA1-dependent calcitonin gene-related peptide (CGRP) release from mouse trachea. Studies of purified hTRPA1 intrinsic tryptophan fluorescence, in the absence of lipid bilayer, consolidate hTRPA1 as an intrinsic bidirectional thermosensor that is modified by the redox state and ligands. Thus, the heat sensing property of TRPA1 is conserved in mammalians, in which TRPA1 may contribute to sensing warmth and uncomfortable heat in addition to noxious cold.

Human transient receptor potential ankyrin channel 1 (TRPA1) is a polymodal sensor implicated in pain, inflammation and itching. An important locus for TRPA1 regulation is the cytoplasmic N-terminal domain, through which various exogenous electrophilic compounds such as allyl-isothiocyanate from mustard oil or cinnamaldehyde from cinnamon activate primary afferent nociceptors. This major region is comprised of a tandem set of 17 ankyrin repeats (AR1-AR17), five of them contain a strictly conserved T/SPLH tetrapeptide motif, a hallmark of an important and evolutionarily conserved contribution to conformational stability. Here, we characterize the functional consequences of putatively stabilizing and destabilizing mutations in these important structural units and identify AR2, AR6, and AR11-13 to be distinctly involved in the allosteric activation of TRPA1 by chemical irritants, cytoplasmic calcium, and membrane voltage. Considering the potential involvement of the T/SP motifs as putative phosphorylation sites, we also show that proline-directed Ser/Thr kinase CDK5 modulates the activity of TRPA1, and that T673 outside the AR-domain is its only possible target. Our data suggest that the most strictly conserved N-terminal ARs define the energetics of the TRPA1 channel gate and contribute to chemical-, calcium- and voltage-dependence.

In fruit flies Drosophila melanogaster, environmental cycles of light and temperature are known to influence behavioral rhythms through dedicated sensory receptors. But the thermosensory pathways and molecular receptors by which thermal cycles modulate locomotor activity rhythms remain unclear. Here, we report that neurons expressing warmth-activated ion channel Drosophila Transient Receptor Potential-A1 (dTRPA1) modulate distinct aspects of the rhythmic activity/rest rhythm in a light-dependent manner. Under light/dark (LD) cycles paired with constantly warm ambient conditions, flies deficient in dTRPA1 expression are unable to phase morning and evening activity bouts appropriately. Correspondingly, we show that electrical activity of a few neurons targeted by the dTRPA1(SH)-GAL4 driver modulates temperature-dependent phasing of activity/rest rhythm under LD cycles. The expression of dTRPA1 also affects behavior responses to temperature cycles combined with constant dark (DD) or light (LL) conditions. We demonstrate that the mid-day "siesta" exhibited by flies under temperature cycles in DD is dependent on dTRPA1 expression in a small number of neurons that include thermosensory anterior cell neurons. Although a small subset of circadian pacemaker neurons may express dTRPA1, we show that CRY-negative dTRPA1(SH)-GAL4 driven neurons are critical for the suppression of mid-thermophase activity, thus enabling flies to exhibit siesta. In contrast to temperature cycles in DD, under LL, dTRPA1 is not required for exhibiting siesta but is important for phasing of evening peak. Our studies show that activity/rest rhythms are modulated in a temperature-dependent manner via signals from dTRPA1(SH)-GAL4 driven neurons. Taken together, these results emphasize the differential influence of thermoreceptors on rhythmic behavior in fruit flies in coordination with light inputs., (© 2016 The Author(s).)

We identified and characterized the TRPA1 channel of Tropilaelaps mercedesae (TmTRPA1), one of two major species of honeybee ectoparasitic mite. Three TmTRPA1 isoforms with unique N-terminal sequences were activated by heat, and the isoform highly expressed in the mite's front legs, TmTRPA1b, was also activated by 27 plant-derived compounds including electrophiles. This suggests that the heat- and electrophile-dependent gating mechanisms as nocisensitive TRPA1 channel are well conserved between arthropod species. Intriguingly, one TmTRPA1 isoform, TmTRPA1a, was activated by only six compounds compared with two other isoforms, demonstrating that the N-terminal sequences are critical determinants for the chemical sensitivity. This is the first example of isoform-specific modulation of chemical sensitivity of TRPA1 channel in one species. α-terpineol showed repellent activity towards T. mercedesae in a laboratory assay and repressed T. mercedesae entry for reproduction into the brood cells with fifth instar larvae in hives. Thus, α-terpineol could be used as the potential compound to control two major honeybee ectoparasitic mites, T. mercedesae and Varroa destructor, in the apiculture industry., (© 2016 The Authors.)

The activity of TRPA1 and TRPV1 channels, their sensitivity to selective activators - allyl isothiocyanate (AITC) and capsaicin (Caps), especially their interaction were studied. The method of microfluorescent microscopy and Ca2+ sensitive dye fura- 2AM. Registration of changes in the concentration of intracellular Ca2+ was performed by using the ratio of fluorescence signals measured at two wavelengths (R = F1/ F2). Researches were conducted on cultured neurons of rat dorsal ganglia (DRG neurons). Application of AITC and Caps on soma of DRG neurons resulted in an increase in intracellular Ca2+. Consistent repeated Caps applications resulted in a significant reduction in the amplitude of Ca2+ transients ( desensitization of TRPV1 channels), which accounted 20,7% of initial value. Further application of selective TRPA1 channel agonist (AITC) resulted in restoration of sensitivity to capsaicin TRPV1 channels ( resensitization TRPV1 channels). Thus, we have established the presence of regulation of TRPV1 channel activity by TRPA1 channels.