N,N'-Bis(2-mercaptoethyl)isophthalamide Binds Electrophilic Paracetamol Metabolites and Prevents Paracetamol-Induced Liver Toxicity.

Paracetamol overdosing may cause liver injury including fulminant liver failure due to generation of the toxic metabolites, N-acetyl-p-benzoquinone imine (NAPQI) and p-benzoquinone (p-BQ). Herein, the chelating agent, N,N'-Bis(2-mercaptoethyl)isophthalamide (NBMI), was examined for its potential ability to entrap NAPQI and p-BQ and to prevent paracetamol-induced liver injury. Both NBMI and the conventional paracetamol antidote N-acetylcysteine (NAC) were investigated with regard to their abilities to scavenge the NAPQI and p-BQ in a Transient Receptor Potential Ankyrin 1-dependent screening assay. Stoichiometric evaluations indicated that NBMI was able to entrap these metabolites more efficiently than NAC. Furthermore, oral administration of either NBMI (680 mg/kg) or NAC (680 mg/kg) prevented the development of the characteristic liver necrosis and elevation of serum alanine aminotransferase in a mouse model for paracetamol-induced liver injury. In summary, these results show that NBMI is able to entrap the toxic metabolites NAPQI and p-BQ and to prevent paracetamol-induced liver injury in mice.

The antipyretic effect of paracetamol occurs independent of transient receptor potential ankyrin 1-mediated hypothermia and is associated with prostaglandin inhibition in the brain.

The mode of action of paracetamol (acetaminophen), which is widely used for treating pain and fever, has remained obscure, but may involve several distinct mechanisms, including cyclooxygenase inhibition and transient receptor potential ankyrin 1 (TRPA1) channel activation, the latter being recently associated with paracetamol's propensity to elicit hypothermia at higher doses. Here, we examined whether the antipyretic effect of paracetamol was due to TRPA1 activation or cyclooxygenase inhibition. Treatment of wild-type and TRPA1 knockout mice rendered febrile by immune challenge with LPS with a dose of paracetamol that did not produce hypothermia (150 mg/kg) but is known to be analgetic, abolished fever in both genotypes. Paracetamol completely suppressed the LPS-induced elevation of prostaglandin E2 in the brain and also reduced the levels of several other prostanoids. The hypothermia induced by paracetamol was abolished in mice treated with the electrophile-scavenger N-acetyl cysteine. We conclude that paracetamol's antipyretic effect in mice is dependent on inhibition of cyclooxygenase activity, including the formation of pyrogenic prostaglandin E2, whereas paracetamol-induced hypothermia likely is mediated by the activation of TRPA1 by electrophilic metabolites of paracetamol, similar to its analgesic effect in some experimental paradigms.-Mirrasekhian, E., Nilsson, J. L. Å., Shionoya, K., Blomgren, A., Zygmunt, P. M., Engblom, D., Högestätt, E. D., Blomqvist, A. The antipyretic effect of paracetamol occurs independent of transient receptor potential ankyrin 1-mediated hypothermia and is associated with prostaglandin inhibition in the brain.

The N-terminal Ankyrin Repeat Domain Is Not Required for Electrophile and Heat Activation of the Purified Mosquito TRPA1 Receptor.

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.

Human TRPA1 is a heat sensor displaying intrinsic U-shaped thermosensitivity.

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.

Quantitative sensory testing of temperature thresholds: Possible biomarkers for persistent pain?

OBJECTIVE: To investigate the reproducibility of thermal thresholds, as measured by repeated quantitative sensory testing (QST) in healthy controls, and to asses if temperature sensitivity differs between healthy controls and a cohort of patients with persistent pain. SUBJECTS: A total of 54 healthy controls were compared with 25 consecutive patients selected for pain rehabilitation by multidisciplinary assessment teams. METHODS: Heat and cold detection and pain thresholds in the forearm and neck were determined by QST. Reproducibility was evaluated by 2 consecutive tests 6-9 months apart. RESULTS: Thermal detection and pain thresholds were reproducible in a subgroup of 20 healthy controls. The patients had slightly increased heat and cold detection thresholds, but significantly lower thresholds for cold and heat pain. The most clear-cut differences between patients and healthy controls were observed for cold pain thresholds. Calculation of the differences between thermal detection and pain thresholds (delta values) further strengthened the differences between patients and healthy controls. CONCLUSION: Thermal detection and pain thresholds are reproducible over time, allowing longitudinal assessment of sensory function using QST. Although increased sensitivity to cold pain was the most prominent finding in this cohort of patients with persistent pain, calculation of the differences between thermal detection and pain thresholds may prove superior in detecting sensory alterations.

Human TRPA1 is intrinsically cold- and chemosensitive with and without its N-terminal ankyrin repeat domain.

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).

H2S and NO cooperatively regulate vascular tone by activating a neuroendocrine HNO-TRPA1-CGRP signalling pathway.

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.

TRPA1.

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.

TRPV4-mediated calcium influx and ciliary activity in human native airway epithelial cells.

The transient receptor potential vanilloid 4 (TRPV4) is a calcium permeable ion channel expressed in airway epithelial cells. Based on studies of cell lines and animals, TRPV4 has been suggested to play a role in the regulation of ciliary beat frequency (CBF). Whether the same is true for human ciliated epithelial cells is not known. Therefore, the aim was to examine the expression and function of TRPV4 in human native nasal epithelial cells. Expression of TRPV4 mRNA in nasal epithelial cells and in the cell lines BEAS2B and 16HBE was confirmed by quantitative real-time PCR. A marked apical TRPV4 immunoreactivity was observed in nasal epithelial cells using immunocytochemistry. Responses to pharmacological modulation of TRPV4 were assessed with calcium imaging and CBF measurements. The TRPV4 agonist GSK1016790A produced concentration-dependent calcium responses in TRPV4-expressing HEK293, BEAS2B and 16HBE cells, and the TRPV4 antagonist HC067047 caused a rightward shift of the GSK1016790A concentration-response curves. Nasal epithelial cells responded to the TRPV4 agonist GSK1016790A with increased intracellular calcium signals and increased CBF, followed by cessation of ciliary beating and cell death. These effects were prevented or inhibited by the TRPV4 antagonist HC067047, the TRP channel blocker ruthenium red or removal of extracellular calcium. We conclude that TRPV4 is expressed in human primary nasal epithelial cells and modulates epithelial calcium levels and CBF. Thus, TRPV4 may participate in mucociliary clearance and airway protection. However, exaggerated activation of TRPV4 may result in epithelial cell death.

Monoacylglycerols activate TRPV1--a link between phospholipase C and TRPV1.

Phospholipase C-mediated hydrolysis of phosphatidylinositol 4,5-bisphosphate generates diacylglycerol, inositol 1,4,5-trisphosphate and protons, all of which can regulate TRPV1 activity via different mechanisms. Here we explored the possibility that the diacylglycerol metabolites 2-arachidonoylglycerol and 1-arachidonoylglycerol, and not metabolites of these monoacylglycerols, activate TRPV1 and contribute to this signaling cascade. 2-Arachidonoylglycerol and 1-arachidonoylglycerol activated native TRPV1 on vascular sensory nerve fibers and heterologously expressed TRPV1 in whole cells and inside-out membrane patches. The monoacylglycerol lipase inhibitors methylarachidonoyl-fluorophosphonate and JZL184 prevented the metabolism of deuterium-labeled 2-arachidonoylglycerol and deuterium-labeled 1-arachidonoylglycerol in arterial homogenates, and enhanced TRPV1-mediated vasodilator responses to both monoacylglycerols. In mesenteric arteries from TRPV1 knock-out mice, vasodilator responses to 2-arachidonoylglycerol were minor. Bradykinin and adenosine triphosphate, ligands of phospholipase C-coupled membrane receptors, increased the content of 2-arachidonoylglycerol in dorsal root ganglia. In HEK293 cells expressing the phospholipase C-coupled histamine H1 receptor, exposure to histamine stimulated the formation of 2-AG, and this effect was augmented in the presence of JZL184. These effects were prevented by the diacylglycerol lipase inhibitor tetrahydrolipstatin. Histamine induced large whole cell currents in HEK293 cells co-expressing TRPV1 and the histamine H1 receptor, and the TRPV1 antagonist capsazepine abolished these currents. JZL184 increased the histamine-induced currents and tetrahydrolipstatin prevented this effect. The calcineurin inhibitor ciclosporin and the endogenous "entourage" compound palmitoylethanolamide potentiated the vasodilator response to 2-arachidonoylglycerol, disclosing TRPV1 activation of this monoacylglycerol at nanomolar concentrations. Furthermore, intracerebroventricular injection of JZL184 produced TRPV1-dependent antinociception in the mouse formalin test. Our results show that intact 2-arachidonoylglycerol and 1-arachidonoylglycerol are endogenous TRPV1 activators, contributing to phospholipase C-dependent TRPV1 channel activation and TRPV1-mediated antinociceptive signaling in the brain.

Fatty acid amide hydrolase-dependent generation of antinociceptive drug metabolites acting on TRPV1 in the brain.

The discovery that paracetamol is metabolized to the potent TRPV1 activator N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z-eicosatetraenamide (AM404) and that this metabolite contributes to paracetamol's antinociceptive effect in rodents via activation of TRPV1 in the central nervous system (CNS) has provided a potential strategy for developing novel analgesics. Here we validated this strategy by examining the metabolism and antinociceptive activity of the de-acetylated paracetamol metabolite 4-aminophenol and 4-hydroxy-3-methoxybenzylamine (HMBA), both of which may undergo a fatty acid amide hydrolase (FAAH)-dependent biotransformation to potent TRPV1 activators in the brain. Systemic administration of 4-aminophenol and HMBA led to a dose-dependent formation of AM404 plus N-(4-hydroxyphenyl)-9Z-octadecenamide (HPODA) and arvanil plus olvanil in the mouse brain, respectively. The order of potency of these lipid metabolites as TRPV1 activators was arvanil = olvanil>>AM404> HPODA. Both 4-aminophenol and HMBA displayed antinociceptive activity in various rodent pain tests. The formation of AM404, arvanil and olvanil, but not HPODA, and the antinociceptive effects of 4-aminophenol and HMBA were substantially reduced or disappeared in FAAH null mice. The activity of 4-aminophenol in the mouse formalin, von Frey and tail immersion tests was also lost in TRPV1 null mice. Intracerebroventricular injection of the TRPV1 blocker capsazepine eliminated the antinociceptive effects of 4-aminophenol and HMBA in the mouse formalin test. In the rat, pharmacological inhibition of FAAH, TRPV1, cannabinoid CB1 receptors and spinal 5-HT3 or 5-HT1A receptors, and chemical deletion of bulbospinal serotonergic pathways prevented the antinociceptive action of 4-aminophenol. Thus, the pharmacological profile of 4-aminophenol was identical to that previously reported for paracetamol, supporting our suggestion that this drug metabolite contributes to paracetamol's analgesic activity via activation of bulbospinal pathways. Our findings demonstrate that it is possible to construct novel antinociceptive drugs based on fatty acid conjugation as a metabolic pathway for the generation of TRPV1 modulators in the CNS.

Repeated intranasal TLR7 stimulation reduces allergen responsiveness in allergic rhinitis.

BACKGROUND: Interactions between Th1 and Th2 immune responses are of importance to the onset and development of allergic disorders. A Toll-like receptor 7 agonist such as AZD8848 may have potential as a treatment for allergic airway disease by skewing the immune system away from a Th2 profile. OBJECTIVE: To evaluate the efficacy and safety of intranasal AZD8848. METHODS: In a placebo-controlled single ascending dose study, AZD8848 (0.3-600 µg) was given intranasally to 48 healthy subjects and 12 patients with allergic rhinitis (NCT00688779). In a placebo-controlled repeat challenge/treatment study, AZD8848 (30 and 60 µg) was given once weekly for five weeks to 74 patients with allergic rhinitis out of season: starting 24 hours after the final dose, daily allergen challenges were given for seven days (NCT00770003). Safety, tolerability, pharmacokinetics, and biomarkers were monitored. During the allergen challenge series, nasal symptoms and lavage fluid levels of tryptase and α2-macroglobulin, reflecting mast cell activity and plasma exudation, were monitored. RESULTS: AZD8848 produced reversible blood lymphocyte reductions and dose-dependent flu-like symptoms: 30-100 µg produced consistent yet tolerable effects. Plasma interleukin-1 receptor antagonist was elevated after administration of AZD8848, reflecting interferon production secondary to TLR7 stimulation. At repeat challenge/treatment, AZD8848 reduced nasal symptoms recorded ten minutes after allergen challenge up to eight days after the final dose. Tryptase and α2-macroglobulin were also reduced by AZD8848. CONCLUSIONS: Repeated intranasal stimulation of Toll-like receptor 7 by AZD8848 was safe and produced a sustained reduction in the responsiveness to allergen in allergic rhinitis. TRIAL REGISTRATION: NCT00688779 and NCT00770003 as indicated above.

TRPA1 contributes to the acute inflammatory response and mediates carrageenan-induced paw edema in the mouse.

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.

Effect of mucosal TRPV1 inhibition in allergic rhinitis.

Transient receptor potential vanilloid-1 (TRPV1) has been implicated as a mediator of itch in allergic rhinitis. To address this possibility, we synthesized a TRPV1 blocker (SB-705498) for nasal administration in patients with seasonal allergic rhinitis. The pharmacological activity of SB-705498 was confirmed on human TRPV1-expressing HEK293 cells, using fluorometric calcium imaging, and in patients with allergic rhinitis subjected to nasal capsaicin challenges. The effect of SB-705498 was studied in patients with seasonal allergic rhinitis subjected to daily allergen challenges for 7 days, using a double-blind, placebo-controlled, randomized and cross-over design. SB-705498 was delivered by nasal lavage 2 min. before each allergen challenge. Primary end-point was total nasal symptom score on days 5-7. Nasal peak inspiratory flow (nPIF) and eosinophil cationic protein (ECP) content in nasal lavages were also monitored. Daily topical applications of SB-705498 at a concentration that inhibited capsaicin-induced nasal symptoms had no effect on total symptom score, nPIF and ECP levels in allergen-challenged patients with seasonal allergic rhinitis. The individual symptoms, nasal itch or sneezes, were also not affected. These findings may indicate that TRPV1 is not a key mediator of the symptoms in allergic rhinitis. However, additional studies, using drug formulations with a prolonged duration of action, should be conducted before TRPV1 is ruled out as a drug target in allergic rhinitis.

TRPA1 mediates spinal antinociception induced by acetaminophen and the cannabinoid Δ(9)-tetrahydrocannabiorcol.

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.

TRPV1 and TRPA1 stimulation induces MUC5B secretion in the human nasal airway in vivo.

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.

TRPV1 in brain is involved in acetaminophen-induced antinociception.

BACKGROUND: Acetaminophen, the major active metabolite of acetanilide in man, has become one of the most popular over-the-counter analgesic and antipyretic agents, consumed by millions of people daily. However, its mechanism of action is still a matter of debate. We have previously shown that acetaminophen is further metabolized to N-(4-hydroxyphenyl)-5Z,8Z,11Z,14Z -eicosatetraenamide (AM404) by fatty acid amide hydrolase (FAAH) in the rat and mouse brain and that this metabolite is a potent activator of transient receptor potential vanilloid 1 (TRPV(1)) in vitro. Pharmacological activation of TRPV(1) in the midbrain periaqueductal gray elicits antinociception in rats. It is therefore possible that activation of TRPV(1) in the brain contributes to the analgesic effect of acetaminophen. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that the antinociceptive effect of acetaminophen at an oral dose lacking hypolocomotor activity is absent in FAAH and TRPV(1) knockout mice in the formalin, tail immersion and von Frey tests. This dose of acetaminophen did not affect the global brain contents of prostaglandin E(2) (PGE(2)) and endocannabinoids. Intracerebroventricular injection of AM404 produced a TRPV(1)-mediated antinociceptive effect in the mouse formalin test. Pharmacological inhibition of TRPV(1) in the brain by intracerebroventricular capsazepine injection abolished the antinociceptive effect of oral acetaminophen in the same test. CONCLUSIONS: This study shows that TRPV(1) in brain is involved in the antinociceptive action of acetaminophen and provides a strategy for developing central nervous system active oral analgesics based on the coexpression of FAAH and TRPV(1) in the brain.

In vitro TRPV1 activity of piperine derived amides.

A series of natural and synthetic piperine amides were evaluated for activity on the human TRPV1 expressed in HEK293 cells. The agonistic effect of piperine amides was mainly dependent on the length of the carbon chain. Structural changes of double bonds and stereochemistry in the aliphatic chain of these compounds did not change their potency or efficacy, indicating that increased rigidity or planarity of the piperine structure does not affect the activity. The opening of the methylenedioxy ring or changes in the heterocyclic ring of the piperine molecule reduced or abolished activity. Furthermore, inactive compounds did not display functional antagonistic activity.

Distribution and function of the hydrogen sulfide-sensitive TRPA1 ion channel in rat urinary bladder.

OBJECTIVES: To investigate the distribution of the transient receptor potential (TRP) A1 ion channel in the rat urinary bladder, and to study the effects of hydrogen sulfide (H(2)S) and known TRPA1 activators on micturition in conscious rats and on heterologously expressed ion channels. METHODS: The expression of TRPA1 in urinary bladder was studied with fluorescence immunohistochemistry and real-time PCR in female Sprague-Dawley rats. Cystometric investigations were performed in conscious animals subjected to intravesical administration of sodium hydrogen sulfide (NaHS, donor of H(2)S), allyl isothiocyanate (AI), and cinnamaldehyde (CA). Fluorometric calcium imaging was used to study the effect of NaHS on human and mouse TRPA1 expressed in CHO cells. RESULTS: TRPA1 immunoreactivity was found on unmyelinated nerve fibres within the urothelium, suburothelial space, and muscle layer as well as around blood vessels throughout the bladder. All TRPA1 immunoreactive nerves fibres also expressed TRPV1 immunoreactivity and vice versa. TRPA1 was also detected in urothelial cells at both transcriptional and protein levels. AI increased micturition frequency and reduced voiding volume. CA and NaHS produced similar changes in urodynamic parameters after disruption of the urothelial barrier with protamine sulfate. NaHS also induced calcium responses in TRPA1-expressing CHO cells, but not in untransfected cells. CONCLUSIONS: The expression of TRPA1 on C-fibre bladder afferents and urothelial cells together with the finding that intravesical TRPA1 activators initiate detrusor overactivity indicate that TRPA1 may have a role in sensory transduction in this organ. The study also highlights H(2)S as a TRPA1 activator potentially involved in inflammatory bladder disease.

Pungent products from garlic activate the sensory ion channel TRPA1.

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.