Transient Receptor Potential A1 Agonists Research Articles

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.

Molecular basis of TRPA1 regulation in nociceptive neurons. A review.

Transient receptor potential A1 (TRPA1) is an excitatory ion channel that functions as a cellular sensor, detecting a wide range of proalgesic agents such as environmental irritants and endogenous products of inflammation and oxidative stress. Topical application of TRPA1 agonists produces an acute nociceptive response through peripheral release of neuropeptides, purines and other transmitters from activated sensory nerve endings. This, in turn, further regulates TRPA1 activity downstream of G-protein and phospholipase C-coupled signaling cascades. Despite the important physiological relevance of such regulation leading to nociceptor sensitization and consequent pain hypersensitivity, the specific domains through which TRPA1 undergoes post-translational modifications that affect its activation properties are yet to be determined at a molecular level. This review aims at providing an account of our current knowledge on molecular basis of regulation by neuronal inflammatory signaling pathways that converge on the TRPA1 channel protein and through modification of its specific residues influence the extent to which this channel may contribute to pain.

Allergen challenge sensitizes TRPA1 in vagal sensory neurons and afferent C-fiber subtypes in guinea pig esophagus.

Transient receptor potential A1 (TRPA1) is a newly defined cationic ion channel, which selectively expresses in primary sensory afferent nerve, and is essential in mediating inflammatory nociception. Our previous study demonstrated that TRPA1 plays an important role in tissue mast cell activation-induced increase in the excitability of esophageal vagal nodose C fibers. The present study aims to determine whether prolonged antigen exposure in vivo sensitizes TRPA1 in a guinea pig model of eosinophilic esophagitis (EoE). Antigen challenge-induced responses in esophageal mucosa were first assessed by histological stains and Ussing chamber studies. TRPA1 function in vagal sensory neurons was then studied by calcium imaging and by whole cell patch-clamp recordings in 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate (DiI)-labeled esophageal vagal nodose and jugular neurons. Extracellular single-unit recordings were performed in vagal nodose and jugular C-fiber neuron subtypes using ex vivo esophageal-vagal preparations with intact nerve endings in the esophagus. Antigen challenge significantly increased infiltrations of eosinophils and mast cells in the esophagus. TRPA1 agonist allyl isothiocyanate (AITC)-induced calcium influx in nodose and jugular neurons was significantly increased, and current densities in esophageal DiI-labeled nodose and jugular neurons were also significantly increased in antigen-challenged animals. Prolonged antigen challenge decreased esophageal epithelial barrier resistance, which allowed intraesophageal-infused AITC-activating nodose and jugular C fibers at their nerve endings. Collectively, these results demonstrated that prolonged antigen challenge sensitized TRPA1 in esophageal sensory neurons and afferent C fibers. This novel finding will help us to better understand the molecular mechanism underlying esophageal sensory and motor dysfunctions in EoE.

Identification of Molecular Determinants for a Potent Mammalian TRPA1 Antagonist by Utilizing Species Differences

The transient receptor potential A1 (TRPA1) receptor is a member of the TRP family and an excitatory nonselective cation channel. An increasing body of evidence suggests that TRPA1 acts as a nociceptor for various chemicals and physical stimuli. Thus, many TRPA1 antagonists have been developed as analgesic agents. Recently, we found that AP18, a mammalian TRPA1 antagonist, does not inhibit heterologously expressed western clawed frog TRPA1 (fTRPA1). Here, we show that fTRPA1 is also insensitive to A967079, one of the most potent mammalian TRPA1 antagonists. Neither heterologously nor endogenously expressed fTRPA1 was inhibited by A967079 upon activation by TRPA1 agonists. Mutant channel analyses revealed that two specific amino acid residues located within the putative fifth transmembrane domain were involved in the inhibitory action of A967079. Our findings and previous reports based on species differences in the sensitivity to TRPA1 antagonists provide novel insights into the structure-function relationship of TRPA1 and supply useful information in the search for new analgesic medicines targeting TRPA1.

TRPA1‐activated anion secretion is linked with PGE2 receptor EP4 in human and rat colon

The transient receptor potential A1 (TRPA1) channel is considered a chemosensor in several sensory tissues. In the present study, we investigated the secretory effect of potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colons using the Ussing flux chamber. The mucosal application of AITC (10−6–10−3 M) induced Cl− and HCO3 − secretion in a concentration‐dependent manner, while the serosal application induced weaker effect. AITC‐evoked anion secretion was attenuated by the pretreatment with cyclooxygenase inhibitor piroxicam and prostaglandin (PG) E2, but the secretion was not affected by tetrodotoxin, atropine, or extracellular Ca2+ depletion. These results indicated that TRPA1 activation induces anion secretion through PG synthesis independent of neural pathway in the colon. Further analysis also indicated that AITC‐evoked anion secretion is mainly mediated by EP4. In addition, RT‐PCR using isolated colonic crypts, in situ hybridization and immunohistochemistry supported the TRPA1 expression in epithelial cells. Therefore, we conclude that the activation of TRPA1 activity on the colonic epithelial cells is likely involved in host defense mechanism through rapid anion secretion.

Activation of TRPA1 by luminal stimuli induces EP4-mediated anion secretion in human and rat colon

In gastrointestinal (GI) physiology, anion and fluid secretion is an important function for host defense and is induced by changes in the luminal environment. The transient receptor potential A1 (TRPA1) channel is considered to be a chemosensor in several sensory tissues. Although the function of TRPA1 has been studied in GI motility, its contribution to the transepithelial ion transport system has rarely been discussed. In the present study, we investigated the secretory effect of the potential TRPA1 agonist allyl isothiocyanate (AITC) in rat and human colon using an Ussing chamber. The mucosal application of AITC (10(-6)-10(-3) M) induced Cl(-) and HCO(3)(-) secretion in a concentration-dependent manner, whereas the serosal application induced a significantly weaker effect. AITC-evoked anion secretion was attenuated by tissue pretreatment with piroxicam and prostaglandin (PG) E(2); however, this secretion was not affected by TTX, atropine, or extracellular Ca(2+) depletion. These experiments indicate that TRPA1 activation induces anion secretion through PG synthesis, independent of neural pathways in the colon. Further analysis also indicates that AITC-evoked anion secretion is mediated mainly by the EP(4) receptor subtype. The magnitude of the secretory response exhibited segmental heterogeneity in rat colon. Real-time PCR analysis showed the segmental difference was corresponding to the differential expression of EP(4) receptor and cyclooxygenase-1 and -2. In addition, RT-PCR, in situ hybridization, and immunohistochemical studies showed TRPA1 expression in the colonic epithelia. Therefore, we conclude that the activation of TRPA1 in colonic epithelial cells is likely involved in the host defense mechanism through rapid anion secretion.

Sustained TRPA1 activation in vivo

Transient receptor potential A1 (TRPA1) is a calcium permeable non-selective cation channel that is selectively localized to peptidergic C-fibres in the pain pathway. TRPA1 is highly conserved across the animal kingdom and it is able to detect a wide range of potentially toxic environmental chemicals. An unusual mechanism of TRPA1 activation was recently elucidated in which reactive agonists bind covalently to cysteines and lysine in the intracellular N-terminus. Despite a covalent activation mechanism, only transient TRPA1 activation is seen in the maintained presence of reactive agonists in whole-cell patch clamp experiments. I suggest that previous patch clamp studies are performed under conditions that do not fully mimic all aspects of TRPA1 activation. Here, I argue that compelling evidence exists for sustained TRPA1 activation in several chronic (neuropathic) pain-related pathophysiological conditions in vivo. I discuss briefly putative mechanisms that are likely to contribute to and maintain sustained TRPA1 agonist levels through increased production and/or decreased metabolism and inactivation. Chronic pain can be understood as a false alarm evoked by sustained and increased levels of endogenous TRPA1 agonists in various pathophysiological conditions.

TRPA1 Receptor Induced Relaxation of the Human Urethra Involves TRPV1 and Cannabinoid Receptor Mediated Signals, and Cyclooxygenase Activation

We studied whether TRPA1 agonists interact with sensory and inflammatory signals to relax human urethral smooth muscle. Urethral specimens were obtained perioperatively from 19 patients, and prepared for immunohistochemistry and functional experiments. The effects of allyl isothiocyanate, cinnamaldehyde and NaHS were studied in phenylephrine activated preparations combined with capsaicin, capsazepine, N omega-nitro-L-arginine, indomethacin or CP55940. TRPA1, cannabinoid 1 and cannabinoid 2 immunoreactivity was colocalized in nerve fibers of the human urethra. All TRPA1 agonists produced relaxation of phenylephrine contracted urethral preparations. Capsaicin increased relaxant responses to all TRPA1 agonists. It increased the mean +/- SEM -logIC50 of cinnamaldehyde and NaHS from 4.91 +/- 0.26 to 5.15 +/- 0.22 and 3.27 +/- 0.14 to 3.79 +/- 0.35, and the -logIC30 of allyl isothiocyanate from 3.11 +/- 0.24 to 3.41 +/- 0.26 (each p <0.05). Capsazepine in 5 preparations, indomethacin in 6 and CP55940 in 5 decreased cinnamaldehyde mediated relaxation by up to 39%, 88% and 89%, respectively. Nomega-nitro-L-arginine and urothelial removal had no effect on relaxation by cinnamaldehyde in 5 preparations. Relaxation to TRPA1 agonists in human urethral preparations seem to work in cooperation with TRPV1 mediated signals, are negatively coupled via cannabinoid receptor activation and involve cyclooxygenase products. Urothelial TRPA1 signals may not be important to regulate normal human urethral smooth muscle tone. This does not exclude a role in the initiation of afferent activity normally and in disease states.

Activation of TRPA1 channels by fenamate nonsteroidal anti-inflammatory drugs

Transient receptor potential A1 (TRPA1) forms nonselective cation channels implicated in acute inflammatory pain and nociception. The mechanism of ligand activation of TRPA1 may involve either covalent modification of cysteine residues or conventional reversible ligand-receptor interactions. For certain electrophilic prostaglandins, covalent modification has been considered as the main mechanism involved in their stimulatory effect on TRPA1. Because some nonsteroidal anti-inflammatory drugs (NSAIDs) are structural analogs of prostaglandins, we examined several nonelectrophilic NSAIDs on TRPA1 activation using electrophysiological techniques and intracellular Ca(2+) measurements and found that a selected group of NSAIDs can act as TRPA1 agonists. Extracellularly applied flufenamic, niflumic, and mefenamic acid, as well as flurbiprofen, ketoprofen, diclofenac, and indomethacin, rapidly activated rat TRPA1 expressed in Xenopus oocytes and human TRPA1 endogenously expressed in WI-38 fibroblasts. Similarly, the NSAID ligands activated human TRPA1 inducibly expressed in HEK293 cells, but the responses were absent in uninduced and parental HEK293 cells. The response to fenamate agonists was blocked by TRPA1 antagonists, AP-18, HC-030031, and ruthenium red. At subsaturating concentrations, the fenamate NSAIDs also potentiate the activation of TRPA1 by allyl isothiocyanate, cinnamaldehyde, and cold, demonstrating positive synergistic interactions with other well-characterized TRPA1 activators. Importantly, among several thermosensitive TRP channels, the stimulatory effect is specific to TRPA1 because flufenamic acid inhibited TRPV1, TRPV3, and TRPM8. We conclude that fenamate NSAIDs are a novel class of potent and reversible direct agonists of TRPA1. This selective group of TRPA1-stimulating NSAIDs should provide a structural basis for developing novel ligands that noncovalently interact with TRPA1 channels.

Nociceptive Signals Induce Trafficking of TRPA1 to the Plasma Membrane

Transient receptor potential A1 (TRPA1) ion channel senses a variety of noxious stimuli and is involved in nociception. Many TRPA1 agonists covalently modify the channel, which can lead to desensitization. The fate of modified TRPA1 and the mechanism of preserving its response to subsequent stimuli are not understood. Moreover, inflammatory signals sensitize TRPA1 by involving protein kinase A (PKA) and phospholipase C (PLC) through unknown means. We show that TRPA1-mediated nocifensive behavior can be sensitized in vivo via PKA/PLC signaling and by activating TRPA1 with the ligand mustard oil (MO). Interestingly, both stimuli increased TRPA1 membrane levels in vitro. Tetanus toxin attenuated the response to the second of two pulses of MO in neurons, suggesting that vesicle fusion increases functional surface TRPA1. Capacitance recordings suggest that MO can induce exocytosis. We propose that TRPA1 translocation to the membrane might represent one of the mechanisms controlling TRPA1 functionality upon acute activation or inflammatory signals.

Transient Receptor Potential A1 and Cannabinoid Receptor Activity in Human Normal and Hyperplastic Prostate: Relation to Nerves and Interstitial Cells

BackgroundIon channel transient receptor potential A1 (TRPA1) and cannabinoid (CB) receptors are involved in mechanoafferent signaling from the bladder and the urethra. ObjectiveTo characterize TRPA1-, CB1-, and CB2-receptor activities in the human prostate. Design, setting, and participantsProstate specimens were obtained from 12 patients undergoing radical prostatectomy. We studied expressions (n=6) of TRPA1, CB1, and CB2 receptors and effects of the TRPA1 agonists allyl isothiocyanate (AI), cinnamaldehyde (CA), sodium hydrogen sulfide (NaHS), and CP 55940 (a CB1/CB2 agonist) on prostatic preparations. MeasurementsWestern blot, immunohistochemistry, and functional experiments were performed. Results and limitationsWestern blot detected expected bands for CB1, CB2, and TRPA1. TRPA1 immunoreactivity was located on nerves that were positive for CB1, CB2, calcitonin gene-related peptide (CGRP), nitric oxide synthase (NOS), or vesicular acetylcholine transporter (VAChT). CB1 and CB2 immunoreactivity was found on nerves that were positive for NOS, VAChT, or CGRP. Adrenergic nerves were not immunoreactive for TRPA1, CB1, or CB2. In nodular hyperplasia, nerves containing the above markers were scarce or absent. TRPA1 immunoreactivity was detected in cyclic guanosine monophosphate–positive basal cells of the glandular epithelium. Basal or subepithelial TRPA1-immunoreactive cells contained vimentin and c-kit immunoreactivity. CA and NaHS relaxed precontracted preparations by 55±7% and 35±3% (n=6 for each). CP 55940, NaHS, AI, capsaicin, and CA decreased nerve contractions up to 27%, 80%, 47%, and 87%, respectively (n=6 for each). ConclusionsThe distribution and function of TRPA1 and CB receptors in prostatic tissue suggest a role for these receptors in mechanoafferent signals, epithelial homeostasis, emission, or inflammation of the human prostate.

Nitrooleic Acid, an Endogenous Product of Nitrative Stress, Activates Nociceptive Sensory Nerves via the Direct Activation of TRPA1

Transient Receptor Potential A1 (TRPA1) is a nonselective cation channel, preferentially expressed on a subset of nociceptive sensory neurons, that is activated by a variety of reactive irritants via the covalent modification of cysteine residues. Excessive nitric oxide during inflammation (nitrative stress), leads to the nitration of phospholipids, resulting in the formation of highly reactive cysteine modifying agents, such as nitrooleic acid (9-OA-NO(2)). Using calcium imaging and electrophysiology, we have shown that 9-OA-NO(2) activates human TRPA1 channels (EC(50), 1 microM), whereas oleic acid had no effect on TRPA1. 9-OA-NO(2) failed to activate TRPA1 in which the cysteines at positions 619, 639, and 663 and the lysine at 708 had been mutated. TRPA1 activation by 9-OA-NO(2) was not inhibited by the NO scavenger carboxy-PTIO. 9-OA-NO(2) had no effect on another nociceptive-specific ion channel, TRPV1. 9-OA-NO(2) activated a subset of mouse vagal and trigeminal sensory neurons, which also responded to the TRPA1 agonist allyl isothiocyanate and the TRPV1 agonist capsaicin. 9-OA-NO(2) failed to activate neurons derived from TRPA1(-/-) mice. The action of 9-OA-NO(2) at nociceptive nerve terminals was investigated using an ex vivo extracellular recording preparation of individual bronchopulmonary C fibers in the mouse. 9-OA-NO(2) evoked robust action potential discharge from capsaicin-sensitive fibers with slow conduction velocities (0.4-0.7 m/s), which was inhibited by the TRPA1 antagonist AP-18. These data demonstrate that nitrooleic acid, a product of nitrative stress, can induce substantial nociceptive nerve activation through the selective and direct activation of TRPA1 channels.

TRPA1 in bradykinin-induced mechanical hypersensitivity of vagal C fibers in guinea pig esophagus

Bradykinin (BK) activates sensory nerves and causes hyperalgesia. Transient receptor potential A1 (TRPA1) is expressed in sensory nerves and mediates cold, mechanical, and chemical nociception. TRPA1 can be activated by BK. TRPA1 knockout mice show impaired responses to BK and mechanical nociception. However, direct evidence from sensory nerve terminals is lacking. This study aims to determine the role of TRPA1 in BK-induced visceral mechanical hypersensitivity. Extracellular recordings of action potentials from vagal nodose and jugular neurons are performed in an ex vivo guinea pig esophageal-vagal preparation. Peak frequencies of action potentials of afferent nerves evoked by esophageal distension and chemical perfusion are recorded and compared. BK activates most nodose and all jugular C fibers. This activation is repeatable and associated with a significant increase in response to esophageal distension, which can be prevented by the B2 receptor antagonist WIN64338. TRPA1 agonist allyl isothiocyanate (AITC) activates most BK-positive nodose and jugular C fibers. This is associated with a transient loss of response to mechanical distensions and desensitization to a second AITC perfusion. Desensitization with AITC and pretreatment with TRPA1 inhibitor HC-030031 both inhibit BK-induced mechanical hypersensitivity but do not affect BK-evoked activation in nodose and jugular C fibers. In contrast, esophageal vagal afferent Adelta fibers do not respond to BK or AITC and fail to show mechanical hypersensitivity after BK perfusion. This provides the first evidence directly from visceral sensory afferent nerve terminals that TRPA1 mediates BK-induced mechanical hypersensitivity. This reveals a novel mechanism of visceral peripheral sensitization.

Transient Receptor Potential A1 (TRPA1) Activity in the Human Urethra—Evidence for a Functional Role for TRPA1 in the Outflow Region

BackgroundA role for the transient receptor potential (TRP) A1 ion channel in rat lower urinary tract (LUT) sensation and disease has been proposed, but in the human LUT no information on TRPA1 activity is available. ObjectivesTo investigate the distribution of TRPA1 in the human urethra and to study the effect of TRPA1 agonists on isolated urethral strip preparations. Design, settings, and participantsUrethral specimens were obtained preoperatively from 10 patients and were freshly prepared for Western blot, immunohistochemistry, and functional in vitro investigations. MeasurementsThe expression patterns of TRPA1 were studied with Western blot and immunohistochemistry. The effects of allyl isothiocyanate (AI), cinnamaldehyde (CA), and NaHS (donor of H2S) on tension of urethral strips were investigated in tissue baths. Results and limitationsTRPA1 immunoreactivity (-IR) was found in nerve fibres in the suburothelial space and was also located to nerve fibres of the muscle layer. Single TRPA1-IR nerves extended into the urothelium. A majority, but not all TRPA1-IR nerves also expressed immunoreactivity for CGRP or TRPV1. In the urothelium, TRPV1 was located to the outer layers whereas TRPA1 was observed in basal urothelial cells. Interspersed between strands of smooth muscle cells of the urethral wall, TRPA1- and vimentin-IR cells containing central nuclei and slender cytoplasmatic extensions were observed.In functional experiments, TRPA1-agonists had no contractile effect in urethral preparations. After precontraction with phenylephrine, AI, CA, and NaHS caused concentration-dependent relaxations of urethral strip preparations. ConclusionsThe localization of TRPA1 to nerves that also express TRPV1 and CGRP, and in urothelial cells and interstitial cells, as well as the findings that TRPA1 agonists can modify tone of urethral preparations, propose a role for TRPA1 in afferent and efferent sensory signaling of the human outflow region.