Transient Receptor Potential A1 Antagonists Research Articles

Essential Oils from Monarda fistulosa: Chemical Composition and Activation of Transient Receptor Potential A1 (TRPA1) Channels.

Little is known about the pharmacological activity of Monarda fistulosa L. essential oils. To address this issue, we isolated essential oils from the flowers and leaves of M. fistulosa and analyzed their chemical composition. We also analyzed the pharmacological effects of M. fistulosa essential oils on transient receptor potential (TRP) channel activity, as these channels are known targets of various essential oil constituents. Flower (MEOFl) and leaf (MEOLv) essential oils were comprised mainly of monoterpenes (43.1% and 21.1%) and oxygenated monoterpenes (54.8% and 77.7%), respectively, with a high abundance of monoterpene hydrocarbons, including p-cymene, γ-terpinene, α-terpinene, and α-thujene. Major oxygenated monoterpenes of MEOFl and MEOLv included carvacrol and thymol. Both MEOFl and MEOLv stimulated a transient increase in intracellular free Ca2+ concentration ([Ca2+]i) in TRPA1 but not in TRPV1 or TRPV4-transfected cells, with MEOLv being much more effective than MEOFl. Furthermore, the pure monoterpenes carvacrol, thymol, and β-myrcene activated TRPA1 but not the TRPV1 or TRPV4 channels, suggesting that these compounds represented the TRPA1-activating components of M. fistulosa essential oils. The transient increase in [Ca2+]i induced by MEOFl/MEOLv, carvacrol, β-myrcene, and thymol in TRPA1-transfected cells was blocked by a selective TRPA1 antagonist, HC-030031. Although carvacrol and thymol have been reported previously to activate the TRPA1 channels, this is the first report to show that β-myrcene is also a TRPA1 channel agonist. Finally, molecular modeling studies showed a substantial similarity between the docking poses of carvacrol, thymol, and β-myrcene in the binding site of human TRPA1. Thus, our results provide a cellular and molecular basis to explain at least part of the therapeutic properties of these essential oils, laying the foundation for prospective pharmacological studies involving TRP ion channels.

Identification and characterization of saikosaponins as antagonists of transient receptor potential A1 channel.

Neuropathic pain is associated with an increased sensitivity to painful stimuli or abnormal sensitivity to otherwise innocuous stimuli. However, in addition to adverse effects, currently available drugs have shown limited response in patients with neuropathic pain, which provides a rationale to explore new drug classes acting on novel targets and with better efficacy and safety profiles. Here, we found that saikosaponins potently inhibit agonist-induced activation of the transient receptor potential A1 (TRPA1) channel, which has been reported to mediate neuropathic pain by sensing a variety of chemical irritants. Molecular docking and site-directed mutagenesis analyses suggested that saikosaponins bind to the hydrophobic pocket in TRPA1 near the Asn855 residue, which, when mutated to Ser, was previously associated with enhanced pain perception in humans. In support of these findings, saikosaponin D significantly attenuated agonist-induced nociceptive responses and vincristine-induced mechanical hypersensitivity in mice. These results indicate that saikosaponins are TRPA1 antagonists and provide a basis for further elaboration of saikosaponin derivatives for the development of new therapeutics for neuropathic pain.

Optimization of a Novel Quinazolinone-Based Series of Transient Receptor Potential A1 (TRPA1) Antagonists Demonstrating Potent in Vivo Activity.

There has been significant interest in developing a transient receptor potential A1 (TRPA1) antagonist for the treatment of pain due to a wealth of data implicating its role in pain pathways. Despite this, identification of a potent small molecule tool possessing pharmacokinetic properties allowing for robust in vivo target coverage has been challenging. Here we describe the optimization of a potent, selective series of quinazolinone-based TRPA1 antagonists. High-throughput screening identified 4, which possessed promising potency and selectivity. A strategy focused on optimizing potency while increasing polarity in order to improve intrinsic clearance culminated with the discovery of purinone 27 (AM-0902), which is a potent, selective antagonist of TRPA1 with pharmacokinetic properties allowing for >30-fold coverage of the rat TRPA1 IC50 in vivo. Compound 27 demonstrated dose-dependent inhibition of AITC-induced flinching in rats, validating its utility as a tool for interrogating the role of TRPA1 in in vivo pain models.

Mechanisms of the adenosine A2A receptor-induced sensitization of esophageal C fibers.

Clinical studies indicate that adenosine contributes to esophageal mechanical hypersensitivity in some patients with pain originating in the esophagus. We have previously reported that the esophageal vagal nodose C fibers express the adenosine A2A receptor. Here we addressed the hypothesis that stimulation of the adenosine A2A receptor induces mechanical sensitization of esophageal C fibers by a mechanism involving transient receptor potential A1 (TRPA1). Extracellular single fiber recordings of activity originating in C-fiber terminals were made in the ex vivo vagally innervated guinea pig esophagus. The adenosine A2A receptor-selective agonist CGS21680 induced robust, reversible sensitization of the response to esophageal distention (10-60 mmHg) in a concentration-dependent fashion (1-100 nM). At the half-maximally effective concentration (EC50: ≈3 nM), CGS21680 induced an approximately twofold increase in the mechanical response without causing an overt activation. This sensitization was abolished by the selective A2A antagonist SCH58261. The adenylyl cyclase activator forskolin mimicked while the nonselective protein kinase inhibitor H89 inhibited mechanical sensitization by CGS21680. CGS21680 did not enhance the response to the purinergic P2X receptor agonist α,β-methylene-ATP, indicating that CGS21680 does not nonspecifically sensitize to all stimuli. Mechanical sensitization by CGS21680 was abolished by pretreatment with two structurally different TRPA1 antagonists AP18 and HC030031. Single cell RT-PCR and whole cell patch-clamp studies in isolated esophagus-specific nodose neurons revealed the expression of TRPA1 in A2A-positive C-fiber neurons and demonstrated that CGS21682 potentiated TRPA1 currents evoked by allylisothiocyanate. We conclude that stimulation of the adenosine A2A receptor induces mechanical sensitization of nodose C fibers by a mechanism sensitive to TRPA1 antagonists indicating the involvement of TRPA1.

Effect of transient receptor potential A1 antagonists on post-operation pain in a rat model of incisional pain

Objective To investigate the effect of intrathecal and intraplantar injection of transient receptor potential A1 (TRPA1) antagonists on post-operation pain in a rat model of incisional pain.Methods Seventy-two adult male Sprague-Dawley rats weighing 200-250 g were randomly divided into 6 groups (n =12):control group (group C),incision group (Ⅰ group).intrathecal injection of DMSO group (DMSO1 group),intraplantar injection of DMSO group (DMSO2 group),intrathecal injection of HC-030031 group (H1 group),intraplantar injection of HC-030031 group (H2 group).Rats in Ⅰ,DMSO1,DMSO2,H1,and H2 groups were operated with incision.Intrathecal or intraplantar injections were performed 24 h after operation.The rats in DMSO1 and H1 groups were respectively intrathecally injected with 10％ DMSO (20 μl) and HC-030031 (50 μg).DMSO2 and H2 groups were respectively intraplantarlly injected with 10％ DMSO (40 μ l) and HC-030031 (100 μg).The paw withdrawal mechanical threshold (PWMT) was measured at 1 h before injection (T0),and 0.5 h (T1),1.0 h (T2),2.0 h (T3),4.0 h (T4) and 6.0 h (T5) after intrathecal or intraplantar injections.Four SD rats in each group were sacrificed at T4 and the segments of the spinal cord were obtained for determination of TRPA1 expression in the spinal cord by using Western blotting.Results As compared with C group [(34.4 ± 4.7) g] and Ⅰ group [(23.8 ±4.3) g],PWMT was significantly decreased and the expression of TRPA1 was up-regulated in DMSO1 and DMSO2 groups at T4 in spinal dorsal horn.The PWMT in H1 group [(28.3 ± 2.4) g] and H2 group [(29.1 ±4.2) g] was increased and the expression of TRPA1 was down-regulated at T4 as compared with DMSO1 group [(24.6 ±3.5) g] and DMSO2 group [(22.1 ±4.7) g].Conclusion Intrathecal or intraplantar administration with TRPA1 antagonists could attenuate incisional pain in rats. Key words: Incisional pain; Spinal cord; Transient receptor potential A1

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.

Ultraviolet Light Phototransduction Activates TRPA1 to Mediate Melanin Synthesis in Human Melanocytes

Human skin is constantly exposed to solar ultraviolet radiation (UVR), the most prevalent environmental carcinogen. Humans have the unique ability among mammals to respond to UVR by increasing their skin pigmentation, a protective process driven by melanin synthesis in epidermal melanocytes. However, the mechanism used by melanocytes to detect and respond to UVR is not well understood. Here we report that transient receptor potential A1 (TRPA1) ion channels expressed in human melanocytes are activated by UVR and mediate early melanin synthesis. We show that in human epidermal melanocytes (HEMs) physiological doses of UVR activate a retinal-dependent current mediated by TRPA1. The UVR photocurrent density was reduced by TRPA1 antagonists and abolished in HEMs expressing TRPA1-targeted miRNA. The TRPA1 photocurrent is UVA specific and requires G protein signaling, providing the first evidence for TRPA1 function in mammalian phototransduction. In HEMs, TRPA1 activation contributes to UVR-induced calcium responses to mediate downstream cellular effects. Remarkably, the UVR-induced and retinal-dependent early increase in cellular melanin content was significantly reduced in HEMs treated with TRPA1 antagonists and abolished in HEMs expressing TRPA1-targeted miRNA, suggesting that TRPA1 is required for early melanin synthesis. Our results show that TRPA1 is essential for a novel extra-ocular phototransduction pathway in human melanocytes that is activated by physiological doses of UVR and results in early melanin synthesis.

Tricyclic 3,4-dihydropyrimidine-2-thione derivatives as potent TRPA1 antagonists

The transient receptor potential A1 (TRPA1) channel has been implicated in a number of inflammatory and nociceptive processes, and antagonists of the TRPA1 receptor could offer a potential treatment for conditions such as inflammatory or neuropathic pain, airway disorders, and itch. In a high throughput screen aimed at the identification of TRPA1 antagonists, 4-phenyl-2-thioxo-1,2,3,4-tetrahydro-indeno[1,2-d]pyrimidin-5-one (1) was identified as a potent TRPA1 receptor antagonist. A series of analogous tricyclic 3,4-dihydropyrimidine-2-thiones has been prepared via the multi-component Biginelli reaction and subsequent derivatization. This has led to TRPA1 antagonists with potencies around 10nM for both rat and human derived TRPA1 receptors. The activity was shown to reside exclusively in the 4R-enantiomers.

Heavy metals zinc, cadmium and copper stimulate pulmonary C‐fibers via direct activation of TRPA1

Airway exposure to zinc dust and zinc‐containing ambient particulates can cause symptoms of airway irritation and inflammation. However, the underlying molecular and cellular mechanisms are largely unknown. Transient receptor potential A1 (TRPA1) is selectively expressed in a subpopulation of pulmonary C‐fiber afferents, and has been considered as a major irritant sensor in the lung and airways. Using whole‐cell patch‐clamp recording and Ca2+ imaging, we have demonstrated that application of ZnCl2 concentration‐dependently evoked inward current and Ca2+ transient in isolated vagal pulmonary sensory neurons; both responses were almost completely prevented after pretreatment with AP18, a selective TRPA1 antagonist. In anesthetized spontaneously breathing animals, intratracheal instillation of ZnCl2 (2 mM, 25 μl) evoked pronounced respiratory sensory irritation in wild‐type mice, and the effect was essentially absent in TRPA1‐deficient mice. In addition, our study showed that two other heavy metals cadmium and copper also stimulated pulmonary sensory neurons via direct activation of TRPA1. In summary, our results suggest that activation of TRPA1 in pulmonary C‐fiber sensory nerves may contribute to the respiratory toxicity induced by airway exposure to these three heavy metals. Funded in part by grants from NIH AI076714 and AHA 0835320N.

Heavy metals zinc, cadmium, and copper stimulate pulmonary sensory neurons via direct activation of TRPA1

Airway exposure to zinc dust and zinc-containing ambient particulates can cause symptoms of airway irritation and inflammation, but the underlying molecular and cellular mechanisms are largely unknown. Transient receptor potential A1 (TRPA1) is selectively expressed in a subpopulation of pulmonary C-fiber afferents and has been considered as a major irritant sensor in the lung and airways. Using whole cell patch-clamp recording and Ca(2+) imaging, we have demonstrated that application of ZnCl(2) concentration dependently evoked inward current and Ca(2+) transient in isolated vagal pulmonary sensory neurons; both responses were almost completely inhibited after pretreatment with AP18, a specific TRPA1 antagonist. In anesthetized spontaneously breathing animals, intratracheal instillation of ZnCl(2) (2 mM, 25 microl) induced pronounced respiratory depression in wild-type mice, and the effect was essentially absent in TRPA1-deficient mice. In addition, our study showed that two other heavy metals, cadmium and copper, also stimulated pulmonary sensory neurons via a direct activation of TRPA1. In summary, our results suggest that activation of TRPA1 in pulmonary C-fiber sensory nerves may contribute to the respiratory toxicity induced by airway exposure to these three heavy metals.

The involvement of the transient receptor potential A1 (TRPA1) in the maintenance of mechanical and cold hyperalgesia in persistent inflammation

This study investigated the role of TRPA1 in the development and maintenance of mechanical and cold hyperalgesia in persistent inflammation induced by Complete Freund’s Adjuvant (CFA) in mice. The intraplantar (i.pl.) injection of CFA induced a long lasting (28days) hyperalgesia for both mechanical and thermal (cold) stimuli. The intraperitoneal (i.p., 30–300mg/kg), intraplantar (i.pl., 100μg/site) or intrathecal (i.t., 10μg/site) injection of the TRPA1 selective antagonist HC-030031 significantly reduced the mechanical hyperalgesia evaluated by the von Frey hair test. The effect of HC-030031 was evidenced on the day after CFA injection and was kept throughout the test. However, the intracerebroventricular (i.c.v., 10μg/site) injection of HC-030031 did not interfere with CFA-induced hyperalgesia. Treatment with HC-030031 (300mg/kg, i.p.) completely inhibited the noxious cold hyperalgesia induced by tetrafluoroethane in mice that received CFA. The pre-treatment with the TRPA1 oligonucleotide antisense (AS-ODN, i.t.) consistently prevented both mechanical and cold hyperalgesia. Interestingly, both TRPA1 protein expression and mRNA were over-expressed in spinal cord and dorsal root ganglia (DRG) of mice treated with CFA, an effect that was fully prevented by the pre-treatment with the TRPA1 antagonist HC-030031. Collectively, the present results showed that TRPA1 present at either peripheral or spinal sites play a relevant role in the development and maintenance of both mechanical and cold hyperalgesia during CFA-induced inflammation. Thus, TRPA1 selective antagonists represent promising candidates to treat hyperalgesia in persistent inflammatory 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.

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.

Plasticity in intact Aδ- and C-fibers contributes to cold hypersensitivity in neuropathic rats

Cold hypersensitivity is a common sensory abnormality accompanying peripheral neuropathies and is difficult to treat. Progress has been made in understanding peripheral mechanisms underlying neuropathic pain but little is known concerning peripheral mechanisms of cold hypersensitivity. The aim of this study was to analyze the contribution of uninjured primary afferents to the cold hypersensitivity that develops in neuropathic rats. Rats with a lumbar 5 (L5) and L6 spinal nerve ligation (SNL, Chung model) but not sham, developed mechanical allodynia, evidenced by decreased paw withdrawal thresholds and increased magnitude of response to von Frey stimulation. Cold hypersensitivity also developed in SNL but not sham rats, evidenced by enhanced nociceptive behaviors induced by placement on a cold plate (6 °C) or application of icilin (a transient receptor potential M8 (TRPM8)/transient receptor potential A1 (TRPA1) receptor agonist) to nerve-injured hind paws. Single fiber recordings demonstrated that the mean conduction velocities of intact L4 cutaneous Aδ- and C-fibers were not different between naive and SNL rats; however, mechanical thresholds of the Aδ- but not the C-fibers were significantly decreased in SNL compared with naive. There was a higher prevalence of C-mechanoheat-cold (CMHC) fibers in SNL compared with naive, but the overall percentage of cold-sensitive C-fibers was not significantly increased compared with naive. This was in contrast to the numerous changes in Aδ-fibers: the percentage of L4 cold sensitive Aδ-, but not C-fibers, was significantly increased, the percentage of L4 icilin-sensitive Aδ-, but not C-fibers, was significantly increased, the icilin-induced activity of L4 Aδ-, but not C-fibers, was significantly increased. Icilin-induced activity was blocked by the TRPA1 antagonist Ruthenium Red. The results indicate plasticity in both Aδ- and C-uninjured fibers, but Aδ fibers appear to provide a major contribution to cold hypersensitivity in neuropathic rats.