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Paracetamol, one of the most widely used pain-relieving drugs, is deacetylated to 4-aminophenol (4-AP) that undergoes fatty acid amide hydrolase (FAAH)-dependent biotransformation into N-arachidonoylphenolamine (AM404), which mediates TRPV1-dependent antinociception in the brain of rodents. However, paracetamol is also converted to the liver-toxic metabolite N-acetyl-p-benzoquinone imine already at therapeutic doses, urging for safer paracetamol analogues. Primary amine analogues with chemical structures similar to paracetamol were evaluated for their propensity to undergo FAAH-dependent N-arachidonoyl conjugation into TRPV1 activators both in vitro and in vivo in rodents. The antinociceptive and antipyretic activity of paracetamol and primary amine analogues was examined with regard to FAAH and TRPV1 as well as if these analogues produced acute liver toxicity. 5-Amino-2-methoxyphenol (2) and 5-aminoindazole (3) displayed efficient target protein interactions with a dose-dependent antinociceptive effect in the mice formalin test, which in the second phase was dependent on FAAH and TRPV1. No hepatotoxicity of the FAAH substrates transformed into TRPV1 activators was observed. While paracetamol attenuates pyrexia via inhibition of brain cyclooxygenase, its antinociceptive FAAH substrate 4-AP was not antipyretic, suggesting separate mechanisms for the antipyretic and antinociceptive effect of paracetamol. Furthermore, compound 3 reduced fever without a brain cyclooxygenase inhibitory action. The data support our view that analgesics and antipyretics without liver toxicity can be derived from paracetamol. Thus, research into the molecular actions of paracetamol could pave the way for the discovery of analgesics and antipyretics with a better benefit-to-risk ratio., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 The Author(s). Published by Elsevier Masson SAS.. All rights reserved.)

Temperature sensors are crucial for animals to optimize living conditions. The temperature response of the ion channel transient receptor potential A1 (TRPA1) is intriguing; some orthologs have been reported to be activated by cold and others by heat, but the molecular mechanisms responsible for its activation remain elusive. Single-channel electrophysiological recordings of heterologously expressed and purified Anopheles gambiae TRPA1 (AgTRPA1), with and without the N-terminal ankyrin repeat domain, demonstrate that both proteins are functional because they responded to the electrophilic compounds allyl isothiocyanate and cinnamaldehyde as well as heat. The proteins' similar intrinsic fluorescence properties and corresponding quenching when activated by allyl isothiocyanate or heat suggest lipid bilayer-independent conformational changes outside the N-terminal domain. The results show that AgTRPA1 is an inherent thermo- and chemoreceptor, and analogous to what has been reported for the human TRPA1 ortholog, the N-terminal domain may tune the response but is not required for the activation by these stimuli., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

Unlabelled: Photosensitization, an exaggerated sensitivity to harmless light, occurs genetically in rare diseases, such as porphyrias, and in photodynamic therapy where short-term toxicity is intended. A common feature is the experience of pain from bright light. In human subjects, skin exposure to 405 nm light induced moderate pain, which was intensified by pretreatment with aminolevulinic acid. In heterologous expression systems and cultured sensory neurons, exposure to blue light activated TRPA1 and, to a lesser extent, TRPV1 channels in the absence of additional photosensitization. Pretreatment with aminolevulinic acid or with protoporphyrin IX dramatically increased the light sensitivity of both TRPA1 and TRPV1 via generation of reactive oxygen species. Artificial lipid bilayers equipped with purified human TRPA1 showed substantial single-channel activity only in the presence of protoporphyrin IX and blue light. Photosensitivity and photosensitization could be demonstrated in freshly isolated mouse tissues and led to TRP channel-dependent release of proinflammatory neuropeptides upon illumination. With antagonists in clinical development, these findings may help to alleviate pain during photodynamic therapy and also allow for disease modification in porphyria patients., Significance Statement: Cutaneous porphyria patients suffer from burning pain upon exposure to sunlight and other patients undergoing photodynamic therapy experience similar pain, which can limit the therapeutic efforts. This study elucidates the underlying molecular transduction mechanism and identifies potential targets of therapy. Ultraviolet and blue light generates singlet oxygen, which oxidizes and activates the ion channels TRPA1 and TRPV1. The disease and the therapeutic options could be reproduced in models ranging from isolated ion channels to human subjects, applying protoporphyrin IX or its precursor aminolevulinic acid. There is an unmet medical need, and our results suggest a therapeutic use of the pertinent antagonists in clinical development., (Copyright © 2016 the authors 0270-6474/16/365264-15$15.00/0.)

We have purified and reconstituted human transient receptor potential (TRP) subtype A1 (hTRPA1) into lipid bilayers and recorded single-channel currents to understand its inherent thermo- and chemosensory properties as well as the role of the ankyrin repeat domain (ARD) of the N terminus in channel behavior. We report that hTRPA1 with and without its N-terminal ARD (Δ1-688 hTRPA1) is intrinsically cold-sensitive, and thus, cold-sensing properties of hTRPA1 reside outside the N-terminal ARD. We show activation of hTRPA1 by the thiol oxidant 2-((biotinoyl)amino)ethyl methanethiosulfonate (MTSEA-biotin) and that electrophilic compounds activate hTRPA1 in the presence and absence of the N-terminal ARD. The nonelectrophilic compounds menthol and the cannabinoid Δ(9)-tetrahydrocannabiorcol (C16) directly activate hTRPA1 at different sites independent of the N-terminal ARD. The TRPA1 antagonist HC030031 inhibited cold and chemical activation of hTRPA1 and Δ1-688 hTRPA1, supporting a direct interaction with hTRPA1 outside the N-terminal ARD. These findings show that hTRPA1 is an intrinsically cold- and chemosensitive ion channel. Thus, second messengers, including Ca(2+), or accessory proteins are not needed for hTRPA1 responses to cold or chemical activators. We suggest that conformational changes outside the N-terminal ARD by cold, electrophiles, and nonelectrophiles are important in hTRPA1 channel gating and that targeting chemical interaction sites outside the N-terminal ARD provides possibilities to fine tune TRPA1-based drug therapies (e.g., for treatment of pain associated with cold hypersensitivity and cardiovascular disease).

Nitroxyl (HNO) is a redox sibling of nitric oxide (NO) that targets distinct signalling pathways with pharmacological endpoints of high significance in the treatment of heart failure. Beneficial HNO effects depend, in part, on its ability to release calcitonin gene-related peptide (CGRP) through an unidentified mechanism. Here we propose that HNO is generated as a result of the reaction of the two gasotransmitters NO and H2S. We show that H2S and NO production colocalizes with transient receptor potential channel A1 (TRPA1), and that HNO activates the sensory chemoreceptor channel TRPA1 via formation of amino-terminal disulphide bonds, which results in sustained calcium influx. As a consequence, CGRP is released, which induces local and systemic vasodilation. H2S-evoked vasodilatatory effects largely depend on NO production and activation of HNO-TRPA1-CGRP pathway. We propose that this neuroendocrine HNO-TRPA1-CGRP signalling pathway constitutes an essential element for the control of vascular tone throughout the cardiovascular system.

The transient receptor potential ankyrin subtype 1 protein (TRPA1) is a nonselective cation channel permeable to Ca(2+), Na(+), and K(+). TRPA1 is a promiscuous chemical nocisensor that is also involved in noxious cold and mechanical sensation. It is present in a subpopulation of Aδ- and C-fiber nociceptive sensory neurons as well as in other sensory cells including epithelial cells. In primary sensory neurons, Ca(2+) and Na(+) flowing through TRPA1 into the cell cause membrane depolarization, action potential discharge, and neurotransmitter release both at peripheral and central neural projections. In addition to being activated by cysteine and lysine reactive electrophiles and oxidants, TRPA1 is indirectly activated by pro-inflammatory agents via the phospholipase C signaling pathway, in which cytosolic Ca(2+) is an important regulator of channel gating. The finding that non-electrophilic compounds, including menthol and cannabinoids, activate TRPA1 may provide templates for the design of non-tissue damaging activators to fine-tune the activity of TRPA1 and raises the possibility that endogenous ligands sharing binding sites with such non-electrophiles exist and regulate TRPA1 channel activity. TRPA1 is promising as a drug target for novel treatments of pain, itch, and sensory hyperreactivity in visceral organs including the airways, bladder, and gastrointestinal tract.

Transient receptor potential ankyrin 1 (TRPA1) is an ion channel involved in thermosensation and nociception. TRPA1 is activated by exogenous irritants and also by oxidants formed in inflammatory reactions. However, our understanding of its role in inflammation is limited. Here, we tested the hypothesis that TRPA1 is involved in acute inflammatory edema. The TRPA1 agonist allyl isothiocyanate (AITC) induced inflammatory edema when injected intraplantarly to mice, mimicking the classical response to carrageenan. Interestingly, the TRPA1 antagonist HC-030031 and the cyclo-oxygenase (COX) inhibitor ibuprofen inhibited not only AITC but also carrageenan-induced edema. TRPA1-deficient mice displayed attenuated responses to carrageenan and AITC. Furthermore, AITC enhanced COX-2 expression in HEK293 cells transfected with human TRPA1, a response that was reversed by HC-030031. This study demonstrates a hitherto unknown role of TRPA1 in carrageenan-induced inflammatory edema. The results also strongly suggest that TRPA1 contributes, in a COX-dependent manner, to the development of acute inflammation.

TRPA1 is a unique sensor of noxious stimuli and, hence, a potential drug target for analgesics. Here we show that the antinociceptive effects of spinal and systemic administration of acetaminophen (paracetamol) are lost in Trpa1(-/-) mice. The electrophilic metabolites N-acetyl-p-benzoquinoneimine and p-benzoquinone, but not acetaminophen itself, activate mouse and human TRPA1. These metabolites also activate native TRPA1 and, as a consequence, reduce voltage-gated calcium and sodium currents in primary sensory neurons. The N-acetyl-p-benzoquinoneimine metabolite L-cysteinyl-S-acetaminophen was detected in the mouse spinal cord after systemic acetaminophen administration. In the hot-plate test, intrathecal administration of N-acetyl-p-benzoquinoneimine, p-benzoquinone and the electrophilic TRPA1 activator cinnamaldehyde produced antinociception that was lost in Trpa1(-/-) mice. Intrathecal injection of a non-electrophilic cannabinoid, Δ(9)-tetrahydrocannabiorcol, also produced TRPA1-dependent antinociception in this test. Our study provides a molecular mechanism for the antinociceptive effect of acetaminophen and discloses spinal TRPA1 activation as a potential pharmacological strategy to alleviate pain.

Aim: Nasal transient receptor potential vanilloid 1 (TRPV1) stimulation with capsaicin produces serous and mucinous secretion in the human nasal airway. The primary aim of this study was to examine topical effects of various TRP ion channel agonists on symptoms and secretion of specific mucins: mucin 5 subtype AC (MUC5AC) and B (MUC5B)., Methods: Healthy individuals were subjected to nasal challenges with TRPV1 agonists (capsaicin, olvanil and anandamide), TRP ankyrin 1 (TRPA1) agonists (cinnamaldehyde and mustard oil) and a TRP melastatin 8 (TRPM8) agonist (menthol). Symptoms were monitored, and nasal lavages were analysed for MUC5AC and MUC5B, i.e. specific mucins associated with airway diseases. In separate groups of healthy subjects, nasal biopsies and brush samples were analysed for TRPV1 and MUC5B, using immunohistochemistry and RT-qPCR. Finally, calcium responses and ciliary beat frequency were measured on isolated ciliated epithelial cells., Results: All TRP agonists induced nasal pain or smart. Capsaicin, olvanil and mustard oil also produced rhinorrhea. Lavage fluids obtained after challenge with capsaicin and mustard oil indicated increased levels of MUC5B, whereas MUC5AC was unaffected. MUC5B and TRPV1 immunoreactivities were primarily localized to submucosal glands and peptidergic nerve fibres, respectively. Although trpv1 transcripts were detected in nasal brush samples, functional responses to capsaicin could not be induced in isolated ciliated epithelial cells., Conclusion: Agonists of TRPV1 and TRPA1 induced MUC5B release in the human nasal airways in vivo. These findings may be of relevance with regard to the regulation of mucin production under physiological and pathophysiological conditions., (© 2011 The Authors. Clinical Physiology and Functional Imaging © 2011 Scandinavian Society of Clinical Physiology and Nuclear Medicine.)

Garlic belongs to the Allium family of plants that produce organosulfur compounds, such as allicin and diallyl disulfide (DADS), which account for their pungency and spicy aroma. Many health benefits have been ascribed to Allium extracts, including hypotensive and vasorelaxant activities. However, the molecular mechanisms underlying these effects remain unknown. Intriguingly, allicin and DADS share structural similarities with allyl isothiocyanate, the pungent ingredient in wasabi and other mustard plants that induces pain and inflammation by activating TRPA1, an excitatory ion channel on primary sensory neurons of the pain pathway. Here we show that allicin and DADS excite an allyl isothiocyanate-sensitive subpopulation of sensory neurons and induce vasodilation by activating capsaicin-sensitive perivascular sensory nerve endings. Moreover, allicin and DADS activate the cloned TRPA1 channel when expressed in heterologous systems. These and other results suggest that garlic excites sensory neurons primarily through activation of TRPA1. Thus different plant genera, including Allium and Brassica, have developed evolutionary convergent strategies that target TRPA1 channels on sensory nerve endings to achieve chemical deterrence.

Wasabi, horseradish and mustard owe their pungency to isothiocyanate compounds. Topical application of mustard oil (allyl isothiocyanate) to the skin activates underlying sensory nerve endings, thereby producing pain, inflammation and robust hypersensitivity to thermal and mechanical stimuli. Despite their widespread use in both the kitchen and the laboratory, the molecular mechanism through which isothiocyanates mediate their effects remains unknown. Here we show that mustard oil depolarizes a subpopulation of primary sensory neurons that are also activated by capsaicin, the pungent ingredient in chilli peppers, and by Delta(9)-tetrahydrocannabinol (THC), the psychoactive component of marijuana. Both allyl isothiocyanate and THC mediate their excitatory effects by activating ANKTM1, a member of the TRP ion channel family recently implicated in the detection of noxious cold. These findings identify a cellular and molecular target for the pungent action of mustard oils and support an emerging role for TRP channels as ionotropic cannabinoid receptors.