Histamine- and pruritogen-induced itch is inhibited by a TRPM8 agonist: a randomized vehicle-controlled human trial.

BACKGROUND: Allergies often present challenges in managing itch and the effects of histamine. Cooling agents that act via transient receptor potential melastatin 8 (TRPM8) agonism have shown potential in itch management. However, animal studies on itch have limitations, as animals cannot communicate subjective events and their fur-coated skin differs from that of humans. Human studies offer more direct and reliable information. OBJECTIVES: To investigate the effects of a specific TRPM8 agonist gel (cryosim-1) on itch induced by various pruritogens in human skin. METHODS: Calcium imaging experiments determined the binding of cryosim-1 and histamine to their respective receptors. Thirty healthy volunteers underwent skin prick tests with pruritogens and a control vehicle. Itch and pain intensity were measured using a numerical rating scale (NRS) across 10 min. Participants were randomly assigned to pretreatments with vehicle or TRPM8 agonist gel. Tests were repeated at a later date, and skin moisture, transepidermal water loss and mechanical sensitivity were measured. RESULTS: The in vitro study confirmed that histamine is not a TRPM8 agonist and cryosim-1 does not act as an agonist or antagonist on the human histamine 1 receptor. The TRPM8 agonist gel significantly reduced the itch intensity for all pruritogens compared with the vehicle-only gel. It also reduced itch NRS and the integrated itch score. Mechanical sensitivity was also reduced. CONCLUSIONS: The specific TRPM8 agonist gel effectively suppressed human skin itch induced by various pruritogens. These versatile actions suggest that cooling agents may be promising treatments for multiple forms of itch stimuli.

Managing itching and the effects of histamine can be difficult for people with allergies. Cooling the skin or applying menthol provides some relief from itch, but the way they work is not fully understood. Cooling agents interact with a protein called TRPM8 (also known as the 'cold and menthol receptor') and have shown potential for the management of itch. However, much of the research has been done on animals and has limitations when compared with human studies. Antihistamine medications can help with histamine-induced itching, but they may not work for other causes of itch. This study investigated the effects of a specific TRPM8 agonist (a chemical that activates a receptor to produce a biologic response) gel called cryosim-1 on itch in human skin. To do this, we conducted tests on 30 healthy people using five different substances that cause itching. Participants rated the itch intensity and pain using a scale and we measured various aspects of their skin. The results showed that all substances caused significant itching compared to a control substance, but itchiness gradually decreased over time. Histamine and compound 48/80 also caused pain. However, when participants applied the TRPM8 activator gel before exposure, they experienced less itching and lower itch intensity versus the gel without the activator. There were no significant differences in pain between the TRPM8 activator and the gel without it. In summary, our findings showed that activating TRPM8 receptors with a specific substance effectively relieved itching caused by various irritants on human skin. This suggests its potential as a treatment for itch-related conditions. Further research is needed to understand its mechanisms better and evaluate its effectiveness in real-life situations.

Spontaneous activity of specific C-nociceptor subtypes from diabetic patients and mice: Involvement of reactive dicarbonyl compounds and (sensitized) transient receptor potential channel A1.

BACKGROUND: Diabetic metabolism causes changes of the chemical milieu including accumulation of reactive carbonyl species, for example, methylglyoxal (MGO). MGO activates chemosensitive TRPA1 on nociceptors, but the contribution to neuronal pathophysiology causing pain and hyperalgesia in diabetic neuropathy is not fully understood. METHODS: We employed single-nerve-fiber recordings in type 2 diabetes patients with (spDN) and without cutaneous pain (DN) and in streptozotocin-diabetic and healthy mice. In mice, we measured Ca++ transients in cultured DRG neurons and stimulated CGRP release from hairy skin. RESULTS: In diabetic patients, we recorded a large proportion of pathologically altered nerve C-fibers (79%). In spDN patients we found a higher percentage (72%) of spontaneously active C-nociceptors than in DN patients (15%). The proportion of spontaneous activity was highest among pathological fibers with mechanoinsensitive fiber properties which are particularly sensitive to MGO in contrast to mechanosensitive fibers. Mouse polymodal nociceptors, in contrast to purely mechanosensitive C-fibers, showed highest prevalence of TRPA1-related chemosensitivity. In diabetic mice about 37% of polymodal nociceptors developed spontaneous activity and exhibited significantly greater MGO responses, indicating sensitized TRPA1 receptors. Low-threshold mechanosensitive Aδ-fibers were vigorously activated by MGO but independently of TRPA1 activation. INTERPRETATION: Our translational findings suggest that TRPA1-expressing C-nociceptors, which in human correspond to mechanoinsensitive and in mice to polymodal nociceptors, are especially vulnerable to develop spontaneous activity. Those two different nociceptor classes might share the functional role as dicarbonyl-sensitive chemosensors and represent the critical nociceptor population that support the development of pain and hyperalgesia in diabetic neuropathy.

Regulation of TRPM8 channel activity by Src-mediated tyrosine phosphorylation.

The transient receptor potential melastatin type 8 (TRPM8) receptor channel is expressed in primary afferent neurons where it is the main transducer of innocuous cold temperatures and also in a variety of tumors, where it is involved in progression and metastasis. Modulation of this channel by intracellular signaling pathways has therefore important clinical implications. We investigated the modulation of recombinant and natively expressed TRPM8 by the Src kinase, which is known to be involved in cancer pathophysiology and inflammation. Human TRPM8 expressed in HEK293T cells is constitutively tyrosine phosphorylated by Src which is expressed either heterologously or endogenously. Src action on TRPM8 potentiates its activity, as treatment with PP2, a selective Src kinase inhibitor, reduces both TRPM8 tyrosine phosphorylation and cold-induced channel activation. RNA interference directed against the Src kinase diminished the extent of PP2-induced functional downregulation of TRPM8, confirming that PP2 acts mainly through Src inhibition. Finally, the effect of PP2 on TRPM8 cold activation was reproduced in cultured rat dorsal root ganglion neurons, and this action was antagonized by the protein tyrosine phosphatase inhibitor pervanadate, confirming that TRPM8 activity is sensitive to the cellular balance between tyrosine kinases and phosphatases. This positive modulation of TRPM8 by Src kinase may be relevant for inflammatory pain and cancer signaling.

Role of 5-HT1A and 5-HT3 receptors in serotonergic activation of sensory neurons in relation to itch and pain behavior in the rat.

Serotonin (5-hydroxytryptamine, 5-HT) released by platelets, mast cells, and immunocytes is a potent inflammatory mediator which modulates pain and itch sensing in the peripheral nervous system. The serotonergic receptors expressed by primary afferent neurons involved in these sensory functions are not fully identified and appear to be to a large extent species dependent. Moreover, the mechanisms through which 5-HT receptor activation is coupled to changes in neuronal excitability have not been completely revealed. Using a combination of in vitro (calcium and voltage imaging and patch-clamp) and in vivo behavioral methods, we used both male and female Wistar rats to provide evidence for the involvement of two 5-HT receptor subtypes, 5-HT1A and 5-HT3, in mediating the sustained and transient effects, respectively, of 5-HT on rat primary afferent neurons involved in pain and itch processing. In addition, our results are consistent with a model in which sustained serotonergic responses triggered via the 5-HT1A receptor are due to closure of background potassium channels, followed by membrane depolarization and action potentials, during which the activation of voltage-gated calcium channels leads to calcium entry. Our results may provide a better understanding of mammalian serotonergic itch signaling.

Neuronal microRNAs modulate TREK two-pore domain K+ channel expression and current density.

The TREK family of leak potassium channels has been found to play critical roles in nociception, sensitivity to general anaesthetics, neuroprotection, and memory. The three members of the family, TREK1, TREK2 and TRAAK establish the resting potential and modify the duration, frequency and amplitude of action potentials. Despite their apparent importance, the repertoire of regulatory interactions utilized by cells to control their expression is poorly understood. Herein, the contribution of miRNAs in the regulation of their post-transcriptional gene expression has been examined. Using different assays, miR-124 and to a lesser extent miR-128 and miR-183 were found to reduce TREK1 and TREK2 levels through specific binding to their 3'UTRs. In contrast, miR-9 which was predicted to bind to TRAAK 3'UTR, did not alter its expression. Expression of miR-124, miR-128 and miR-183 was found to mirror that of Trek1 and Trek2 mRNAs during brain development. Moreover, application of proinflammatory mediators in dorsal root ganglion (DRG) neurons revealed an inverse correlation between miR-124 and Trek1 and Trek2 mRNA expression. Voltage clamp recordings of TREK2-mediated currents showed that miR-124 reduced the sensitivity of TREK2-expressing cells to non-aversive warmth stimulation. Overall, these findings reveal a significant regulatory mechanism by which TREK1 and TREK2 expression and hence activity are controlled in neurons and uncover new druggable targets for analgesia and neuroprotection.Abbreviations: microRNA: miRNA; UTR: untranslated region; K2p channels: two-pore domain K+channels; DRG: dorsal root ganglion; CNS: central nervous system; FBS: fetal bovine serum; TuD: Tough Decoy; TREK: tandem P-domain weak inward rectifying K+ (TWIK)-related K+ channel 1; TRAAK: TWIK-related arachidonic acid K+.

Photosensitization in Porphyrias and Photodynamic Therapy Involves TRPA1 and TRPV1.

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.

The anthelminthic drug praziquantel is a selective agonist of the sensory transient receptor potential melastatin type 8 channel.

Praziquantel is the most effective anthelminthic drug for the treatment of schistosomiasis, an infectious disease caused by the platyhelminth Schistosoma mansoni. While praziquantel is known to trigger calcium influx into schisostomes, followed by spastic paralysis of the worms and tegumental disruption, the mechanism of action of the drug is not completely understood. Although relatively well tolerated, praziquantel has been reported to cause mild adverse effects, including nausea, abdominal pain and headaches. As a number of putative Transient Receptor Potential (TRP) channel genes have recently been predicted in S. mansoni, we sought to investigate the effect of praziquantel on three mammalian TRP channels, TRP melastatin type 8 (TRPM8), TRP vanilloid type 1 (TRPV1) and TRP ankyrin type 1 (TRPA1). Using calcium microfluorimetry and the patch clamp technique, we recorded the effect of praziquantel on HEK293T cells expressing recombinant TRPM8, TRPV1 or TRPA1, as well as on cultured dorsal root ganglion (DRG) neurons from wild type and TRPM8 null mutant mice. We discovered that praziquantel is a relatively potent and selective partial agonist of the mammalian and avian cold and menthol receptor TRPM8. The activation of cultured DRG neurons by clinically relevant concentrations of praziquantel is predominantly mediated by TRPM8. Our results may provide clues to a better understanding of praziquantel's mechanism of action and its adverse effects.

Transient Receptor Potential Ankyrin 1 (TRPA1) Modulation by 4-Hydroxynonenal (4-HNE) in Pancreatic Adenocarcinoma Cell Lines: Putative Roles for Therapies.

BACKGROUND: Transient receptor potential channels (TRP) are overexpressed in some pancreatic adenocarcinoma (PDAC) patients and cell lines, settling them as putative therapeutic targets in this disease. Reactive oxygen species (ROS), with levels increased in PDAC, modulate some members of the TRP family renamed "redox channels". Here, we investigate the direct effects of 4-hydroxinonenal (4-HNE) on TRPA1, natively expressed in PDAC cell lines and in association with cell migration and cell cycle progression. METHODS: We performed microfluorimetry experiments, while the activation of resident membrane channels was investigated using confocal microscopy. We applied a prospective molecular docking of 4-HNE using Autodock and AutoDock Tools4. Also, we simulated the diffusion of 4-HNE through the membrane from the extracellular space with the Permeability of Molecules across Membranes (PerMM) web server. The analysis of cell migration was performed using the wound healing assay, and cell cycle progression was acquired using a Beckman Coulter CytoFlex flow cytometer. RESULTS: Our results show, for the first time in PDAC, that 4-HNE diffuses through the cell membrane and rapidly activates Ca2+ uptake in PDAC cells. This process depends on TRPA1 activation, as 4-HNE forms a covalent binding with a pocket-like region within the intracellular N-terminal of the channel, shaped by the cysteine residues 621, 641, and 665. The activation of TRPA1 by 4-HNE inhibits cell migration and induces cell cycle arrest in the G2/M phase. CONCLUSIONS: Our study brings new insights into the effects of 4-HNE, highlighting the activation of the TRPA1 channel, a druggable, putative target for PDAC-expressing tumors.

Inhibition of TRPM8 function by prostacyclin receptor agonists requires coupling to Gq/11 proteins.

BACKGROUND AND PURPOSE: The TRPM8 ion channel is involved in innocuous cold sensing and has a potent anti-inflammatory action. Its activation by lower temperature or chemical agonists such as menthol and icilin induces analgesic effects, reversing hypersensitivity and reducing chronic pain. On the other hand, prostacyclin (PGI2) enhances pain and inflammation by activating the IP receptors. Due to the critical roles of TRPM8 and IP receptors in the regulation of inflammatory pain, and considering their overlapping expression pattern, we analysed the functional interaction between human TRPM8 and IP receptors. EXPERIMENTAL APPROACH: We transiently expressed human TRPM8 channels and IP receptors in HEK293T cells and carried out intracellular calcium and cAMP measurements. Additionally, we cultured neurons from the dorsal root ganglia (DRGs) of mice and determined the increase in intracellular calcium triggered by the TRPM8 agonist, icilin, in the presence of the IP receptor agonist cicaprost, the IP receptor antagonist Cay10441, and the Gq/11 inhibitor YM254890. KEY RESULTS: Activation of IP receptors by selective agonists (cicaprost, beraprost, and iloprost) inhibited TRPM8 channel function, independently of the Gs-cAMP pathway. The potent inhibition of TRPM8 channels by IP receptor agonists involved Gq/11 coupling. These effects were also observed in neurons isolated from murine DRGs. CONCLUSIONS AND IMPLICATIONS: Our results demonstrate an unusual signalling pathway of IP receptors by coupling to Gq/11 proteins to inhibit TRPM8 channel function. This pathway may contribute to a better understanding of the role of TRPM8 channels and IP receptors in regulating pain and inflammation.

TRPM8-dependent shaking in mammals and birds.

Removing water from wet fur or feathers is important for thermoregulation in warm-blooded animals. The "wet dog shake" (WDS) behavior has been largely characterized in mammals but to a much lesser extent in birds. Although it is known that TRPM8 is the main molecular transducer of low temperature in mammals, it is not clear if wetness-induced shaking in furred and feathered animals is dependent on TRPM8. Here, we show that a novel TRPM8 agonist induces WDS in rodents and, importantly, in birds, similar to the shaking behavior evoked by water-spraying. Furthermore, the WDS onset depends on TRPM8, as we show in water-sprayed mice. Overall, our results provide multiple evidence for a TRPM8 dependence of WDS behaviors in all tested species. These suggest that a convergent evolution selected similar shaking behaviors to expel water from fur and feathers, with TRPM8 being involved in wetness sensing in both mammals and birds.

Camphor activates and sensitizes transient receptor potential melastatin 8 (TRPM8) to cooling and icilin.

Camphor is known to potentiate both heat and cold sensations. Although the sensitization to heat could be explained by the activation of heat-sensitive transient receptor potential (TRP) channels TRPV1 and TRPV3, the camphor-induced sensitization to cooling remains unexplained. In this study, we present evidence for the activation of the cold- and menthol-sensitive channel transient receptor potential melastatin 8 (TRPM8) by camphor. Calcium transients evoked by camphor in HEK293 cells expressing human and rat TRPM8 are inhibited by the TRPM8 antagonists 4-(3-chloro-2-pyridinyl)-N-[4-(1,1-dimethylethyl)phenyl]-1-piperazinecarboxamide and 2-aminoethyl diphenylborinate. Camphor also sensitized the cold-induced calcium transients and evoked desensitizing outward-rectifying currents in TRPM8-expressing HEK293 cells. In the presence of ruthenium red (a blocker of TRPV1, TRPV3, and TRPA1), the camphor sensitivity of cultured rat dorsal root ganglion neurons was highest in a subpopulation of cold- and icilin-sensitive neurons, strongly suggesting that camphor activates native TRPM8. Camphor has a dual action on TRPM8: it not only activates the channel but also inhibits its response to menthol. The icilin-insensitive chicken TRPM8 was also camphor insensitive. However, camphor was able to activate an icilin-insensitive human TRPM8 mutant channel. The activation and sensitization to cold of mammalian TRPM8 are likely to be responsible for the psychophysical enhancement of innocuous cold and "stinging/burning" cold sensations by camphor.

Sensory neuron sodium channel Nav1.8 is essential for pain at low temperatures.

Sensory acuity and motor dexterity deteriorate when human limbs cool down, but pain perception persists and cold-induced pain can become excruciating. Evolutionary pressure to enforce protective behaviour requires that damage-sensing neurons (nociceptors) continue to function at low temperatures. Here we show that this goal is achieved by endowing superficial endings of slowly conducting nociceptive fibres with the tetrodotoxin-resistant voltage-gated sodium channel (VGSC) Na(v)1.8 (ref. 2). This channel is essential for sustained excitability of nociceptors when the skin is cooled. We show that cooling excitable membranes progressively enhances the voltage-dependent slow inactivation of tetrodotoxin-sensitive VGSCs. In contrast, the inactivation properties of Na(v)1.8 are entirely cold-resistant. Moreover, low temperatures decrease the activation threshold of the sodium currents and increase the membrane resistance, augmenting the voltage change caused by any membrane current. Thus, in the cold, Na(v)1.8 remains available as the sole electrical impulse generator in nociceptors that transmits nociceptive information to the central nervous system. Consistent with this concept is the observation that Na(v)1.8-null mutant mice show negligible responses to noxious cold and mechanical stimulation at low temperatures. Our data present strong evidence for a specialized role of Na(v)1.8 in nociceptors as the critical molecule for the perception of cold pain and pain in the cold.

The antimalarial artemisinin is a non-electrophilic agonist of the transient receptor potential ankyrin type 1 receptor-channel.

Artemisinin and its derivatives are the main therapeutic drugs against Plasmodium protists, the causative agents of malaria. While several putative mechanisms of action have been proposed, the precise molecular targets of these compounds have not been fully elucidated. In addition to their antimalarial properties, artemisinins have been reported to act as anti-tumour agents and certain antinociceptive effects have also been proposed. We investigated the effect of the parent compound, artemisinin, on a number of temperature-gated Transient Receptor Potential ion channels (so called thermoTRPs), given their demonstrated roles in pain-sensing and cancer. We report that artemisinin acts as an agonist of the Transient Receptor Potential Ankyrin type 1 (TRPA1) receptor channel. Artemisinin was able to evoke calcium transients in HEK293T cells expressing recombinant human TRPA1, as well as in a subpopulation of mouse dorsal root ganglion (DRG) neurons which also responded to the selective TRPA1 agonist allyl isothiocyanate (AITC) and these responses were reversibly abolished by the selective TRPA1 antagonist A967079. Artemisinin also triggered whole-cell currents in HEK293T cells transiently transfected with human TRPA1, as well as in TRPA1-expressing DRG neurons, and these currents were inhibited by A967079. Interestingly, using human TRPA1 mutants, we demonstrate that artemisinin acts as a non-electrophilic agonist of TRPA1, activating the channel in a similar manner to carvacrol and menthol. These results may provide a better understanding of the biological actions of the very important antimalarial and anti-tumour agent artemisinin.

TRPA1 and substance P mediate colitis in mice.

BACKGROUND & AIMS: The neuropeptides calcitonin gene-related peptide (CGRP) and substance P, and calcium channels, which control their release from extrinsic sensory neurons, have important roles in experimental colitis. We investigated the mechanisms of colitis in 2 different models, the involvement of the irritant receptor transient receptor potential of the ankyrin type-1 (TRPA1), and the effects of CGRP and substance P. METHODS: We used calcium-imaging, patch-clamp, and neuropeptide-release assays to evaluate the effects of 2,4,6-trinitrobenzene-sulfonic-acid (TNBS) and dextran-sulfate-sodium-salt on neurons. Colitis was induced in wild-type, knockout, and desensitized mice. RESULTS: TNBS induced TRPA1-dependent release of colonic substance P and CGRP, influx of Ca2+, and sustained ionic inward currents in colonic sensory neurons and transfected HEK293t cells. Analysis of mutant forms of TRPA1 revealed that TNBS bound covalently to cysteine (and lysine) residues in the cytoplasmic N-terminus. A stable sulfinic acid transformation of the cysteine-SH group, shown by mass spectrometry, might contribute to sustained sensitization of TRPA1. Mice with colitis had increased colonic neuropeptide release, mediated by TRPA1. Endogenous products of inflammatory lipid peroxidation also induced TRPA1-dependent release of colonic neuropeptides; levels of 4-hydroxy-trans-2-nonenal increased in each model of colitis. Colitis induction by TNBS or dextran-sulfate-sodium-salt was inhibited or reduced in TRPA1-/- mice and by 2-(1,3-dimethyl-2,6-dioxo-1,2,3,6-tetrahydro-7H-purin-7-yl)-N-(4-isopro-pylphenyl)-acetamide, a pharmacologic inhibitor of TRPA1. Substance P had a proinflammatory effect that was dominant over CGRP, based on studies of knockout mice. Ablation of extrinsic sensory neurons prevented or attenuated TNBS-induced release of neuropeptides and both forms of colitis. CONCLUSIONS: Neuroimmune interactions control intestinal inflammation. Activation and sensitization of TRPA1 and release of substance P induce and maintain colitis in mice.

The formalin test does not probe inflammatory pain but excitotoxicity in rodent skin.

The most widely used formalin test to screen antinociceptive drug candidates is still apostrophized as targeting inflammatory pain, in spite of strong opposing evidence published. In our rat skin-nerve preparation ex vivo, recording from all classes of sensory single-fibers (n = 32), 30 units were transiently excited by formaldehyde concentrations 1-100 mM applied to receptive fields (RFs) for 3 min, C and Aδ-fibers being more sensitive (1-30 mM) than Aß-fibers. From 30 mM on, ~1% of the concentration usually injected in vivo, all RFs were defunctionalized and conduction in an isolated sciatic nerve preparation was irreversibly blocked. Thus, formaldehyde, generated a state of 'anesthesia dolorosa' in the RFs in so far as after a quiescent interphase all fibers with unmyelinated terminals developed a second phase of vigorous discharge activity which correlated well in time course and magnitude with published pain-related behaviors. Sural nerve filament recordings in vivo confirmed that higher formalin concentrations (> 42 mM) have to be injected to the skin to induce this second phase of discharge. Patch-clamp and calcium-imaging confirmed TRPA1 as the primary transducer of formaldehyde (10 mM) effects on mouse sensory neurons. However, stimulated CGRP release from isolated skin of TRPA1+/+ and TRPA1-/- mice showed a convergence of the saturating concentration-response curves at 100 mM formaldehyde, which did not occur with nerve and trachea preparations. Finally, skin-nerve recordings from C and Aδ-fibers of TRPA1-/- mice revealed a massive reduction in formaldehyde (30 mM)-evoked discharge. However, the remaining activity was still biphasic, thus confirming additional unspecific excitotoxic actions of the fixative that diffuses along still excitable axons as previously published. The multiplicity of formaldehyde's actions requires extensive discussion and literature review, leading to a fundamental reevaluation of the formalin test.

Mini-review: The nociceptive sensory functions of the polymodal receptor Transient Receptor Potential Ankyrin Type 1 (TRPA1).

Over the last 17 years since its cloning in 2003, the receptor-channel TRPA1 has received increasing attention due to its polymodal features and prominent role in pain signaling in a variety of human disease states. While evidence has been accumulating for non-neuronal TRPA1 expression, it is the presence of this channel in nociceptive nerve endings which has taken centre stage, due to its potential clinical ramifications. As a consequence, we shall focus in this review on the sensory functions of TRPA1 related to its expression in the peripheral nervous system. While substantial research has been focused on the putative role of TRPA1 in detecting irritant compounds, noxious cold and mechanical stimuli, the current overall picture is, to some extent, still cloudy. The chemosensory function of the channel is well demonstrated, as well as its involvement in the detection of oxidative and nitrosative stress; however, the other sensory features of TRPA1 have not been fully elucidated yet. The current state of the experimental evidence for these physiological roles of TRPA1 in mammals, and particularly in humans, will be discussed in this review.

Functional expression of the transient receptor potential ankyrin type 1 channel in pancreatic adenocarcinoma cells.

The transient receptor potential ankyrin type 1 (TRPA1) channel belongs to the TRP superfamily of ion channels. TRPA1 is a membrane protein with multiple functions able to respond to noxious stimuli, reactive oxygen species, inflammatory cytokines or pungent substances, and it participates in pain signalling, taste, inflammation and various steps of the tumorigenic process. To date, no reports have addressed the expression and function of TRPA1 in pancreatic ductal adenocarcinoma (PDAC) cells. This work reports the endogenous expression of TRPA1 channels in human pancreatic adenocarcinoma cell lines and provides insights into the function of the TRPA1 protein in the Panc-1 cell line. This study reports that cell lines isolated from PDAC patients had different levels of TRPA1 expression. The channel activity in Panc-1 cells, as assessed with electrophysiological (whole-cell patch clamp) and microfluorimetry methods, showed that non-selective cationic currents were activated by allyl isothiocyanate (AITC) in Panc-1 cells and inhibited by the selective TRPA1 antagonist A-967079. The current elicited by the specific agonist was associated with a robust increase in intracellular Ca2+. Furthermore, siRNA-induced downregulation of TRPA1 enhanced cell migration in the wound healing assay, indicating a possible role of ion channels independent from pore function. Finally, TRPA1 activation changed the cell cycle progression. Taken together, these results support the idea of channel-dependent and independent role for TRPA1 in tumoral processes.

Psoralens activate and photosensitize Transient Receptor Potential channels Ankyrin type 1 (TRPA1) and Vanilloid type 1 (TRPV1).

BACKGROUND: PUVA (psoralen UVA) therapy is used to treat a variety of skin conditions, such as vitiligo psoriasis, eczema and mycosis fungoides, but it is frequently accompanied by phototoxicity leading to burning pain, itch and erythema. METHODS: We used a combination of calcium and reactive oxygen species (ROS) imaging, patch clamp and neuropeptide release measurement to investigate whether certain ion channels involved in pain and itch signalling could be responsible for these adverese effects of PUVA. RESULTS: Clinically used psoralen derivatives 8-methoxypsoralen (8-MOP) and 5-methoxypsoralen at physiologically relevant concentrations were able to activate and photosensitize two recombinant thermoTRP (temperature-gated Transient Receptor Potential) ion channels, TRPA1 (Transient Receptor Potential Ankyrin type 1) and TRPV1 (Transient Receptor Potential Vanilloid type 1). 8-MOP enhanced ROS production by UVA light, and the effect of 8-MOP on TRPA1 could be abolished by the antioxidant N-acetyl cysteine and by removal of critical cysteine residues from the N-terminus domain of the channel. Natively expressed mouse TRPA1 and TRPV1 both contribute to photosensitization of cultured primary afferent neurons by 8-MOP, while direct neuronal activation by this psoralen-derivative is mainly dependent on TRPV1. Both TRPA1 and TRPV1 are to a large extent involved in controlling 8-MOP-induced neuropeptide release from mouse trachea. CONCLUSIONS: Taken together our results provide a better understanding of the phototoxicity reported by PUVA patients and indicate a possible therapeutic approach to alleviate the adverse effects associated with this therapy. SIGNIFICANCE: Our work provides evidence for the involvement of thermoTRP channels TRPA1 and TRPV1 in the activation and photosensitization of peripheral nociceptors during PUVA (Psoralen UVA) therapy.

The M-channel blocker linopirdine is an agonist of the capsaicin receptor TRPV1.

Linopirdine is a well known blocker of voltage-gated potassium channels from the Kv7 (or KCNQ) family that generate the so called M current in mammalian neurons. Kv7 subunits are also expressed in pain-sensing neurons in dorsal root ganglia, in which they modulate neuronal excitability. In this study we demonstrate that linopirdine acts as an agonist of TRPV1 (transient receptor potential vanilloid type 1), another ion channel expressed in nociceptors and involved in pain signaling. Linopirdine induces increases in intracellular calcium concentration in human embryonic kidney 293 (HEK293) cells expressing TRPV1, but not TRPA1 and TRPM8 or in wild-type HEK293 cells. Linopirdine also activates an inward current in TRPV1-expressing HEK293 cells that is almost completely blocked by the selective TRPV1 antagonist capsazepine. At low concentrations linopirdine sensitizes both recombinant and native TRPV1 channels to heat, in a manner that is not prevented by the Kv7-channel opener flupirtine. Taken together, these results indicate that linopirdine exerts an excitatory action on mammalian nociceptors not only through inhibition of the M current but also through activation of the capsaicin receptor TRPV1.

The phospholipase C inhibitor U73122 is a potent agonist of the polymodal transient receptor potential ankyrin type 1 (TRPA1) receptor channel.

The aminosteroid U73122 is frequently used as a phospholipase C (PLC) inhibitor and as such was used to investigate PLC-dependent activation and modulation of the transient receptor potential ankyrin type 1 (TRPA1) receptor channel. However, U73122 was recently shown to activate recombinant TRPA1 directly, albeit this interaction was not further explored. Our aim was to perform a detailed characterization of this agonistic action of U73122 on TRPA1. We used Fura-2 calcium microfluorimetry and the patch clamp technique to investigate the effect of U73122 on human and mouse wild type and mutant (C621S/C641S/C665S) TRPA1 expressed in HEK293t cells, as well as native TRPA1 in primary afferent neurons from wild type and TRPV1 and TRPA1 null mutant mice. In addition, we measured calcitonin gene-related peptide (CGRP) release from skin isolated from wild-type and TRPA1 null mutant mice. Human and mouse TRPA1 channels were activated by U73122 in the low nanomolar range. This activation was only partially dependent upon modification of the N-terminal cysteines 621, 641, and 665. U73122 also activated a subpopulation of neurons from wild-type and TRPV1 null mutant mice, but this effect was absent in mice deficient of TRPA1. In addition, U73122 evoked marked calcitonin gene-related peptide (CGRP) release from skin preparations of wild type but not TRPA1 null mutant mice. Our results indicate that U73122 is a potent and selective TRPA1 agonist. This effect should be taken into account when U73122 is used to inhibit PLC in TRPA1-expressing cells, such as primary nociceptors. In addition, U73122 may present a novel lead compound for the development of TRPA1-targeting drugs.