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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.

Spices in food and beverages and compounds in tobacco smoke interact with sensory irritant receptors of the transient receptor potential (TRP) cation channel family. TRPV1 (vanilloid type 1), TRPA1 (ankyrin 1) and TRPM8 (melastatin 8) not only elicit action potential signaling through trigeminal nerves, eventually evoking pungent or cooling sensations, but by their calcium conductance they also stimulate the release of calcitonin gene-related peptide (CGRP). This is measured as an index of neuronal activation to elucidate the chemo- and thermosensory transduction in the isolated mouse buccal mucosa of wild types and pertinent knockouts. We found that the lipophilic capsaicin, mustard oil and menthol effectively get access to the nerve endings below the multilayered squamous epithelium, while cigarette smoke and its gaseous phase were weakly effective releasing CGRP. The hydrophilic nicotine was ineffective unless applied unprotonated in alkaline (pH9) solution, activating TRPA1 and TRPV1. Also, mustard oil activated both these irritant receptors in millimolar but only TRPA1 in micromolar concentrations; in combination (1 mm) with heat (45 °C), it showed supraadditive, that is heat sensitizing, effects in TRPV1 and TRPA1 knockouts, suggesting action on an unknown heat-activated channel and mustard oil receptor. Menthol caused little CGRP release by itself, but in subliminal concentration (2 mm), it enabled a robust cold response that was absent in TRPM8-/- but retained in TRPA1-/- and strongly reduced by TRPM8 inhibitors. In conclusion, all three relevant irritant receptors are functionally expressed in the oral mucosa and play their specific roles in inducing neurogenic inflammation and sensitization to heat and cold.

Crotalphine is a structural analogue to a novel analgesic peptide that was first identified in the crude venom from the South American rattlesnake Crotalus durissus terrificus. Although crotalphine's analgesic effect is well established, its direct mechanism of action remains unresolved. The aim of the present study was to investigate the effect of crotalphine on ion channels in peripheral pain pathways. We found that picomolar concentrations of crotalphine selectively activate heterologously expressed and native TRPA1 ion channels. TRPA1 activation by crotalphine required intact N-terminal cysteine residues and was followed by strong and long-lasting desensitization of the channel. Homologous desensitization of recombinant TRPA1 and heterologous desensitization in cultured dorsal root ganglia neurons was observed. Likewise, crotalphine acted on peptidergic TRPA1-expressing nerve endings ex vivo as demonstrated by suppression of calcitonin gene-related peptide release from the trachea and in vivo by inhibition of chemically induced and inflammatory hypersensitivity in mice. The crotalphine-mediated desensitizing effect was abolished by the TRPA1 blocker HC030031 and absent in TRPA1-deficient mice. Taken together, these results suggest that crotalphine is the first peptide to mediate antinociception selectively and at subnanomolar concentrations by targeting TRPA1 ion channels.

Irritating effects of volatile general anesthetics on tracheal nerve endings and resulting spastic reflexes in the airways are not completely understood with respect to molecular mechanisms. Neuropeptide release and neurogenic inflammation play an established role.The basal and stimulated calcitonin gene-related peptide (CGRP) release from the isolated superfused mouse trachea was analyzed as an index of sensory neuron activation, applying irritant (desflurane and isoflurane) and nonirritant (sevoflurane) volatile anesthetics as stimuli. Various gas concentrations (0.5-, 1-, or 2-fold minimum alveolar concentration [MAC]) and different O2 atmospheres were used for tracheal stimulation at 38°C. Null mutants of the capsaicin receptor TRPV1 and of the chemoreceptor TRPA1, as well as double knockout mice, were used as tissue donors.Desflurane and, less so, isoflurane caused a concentration-dependent tracheal CGRP release, both saturating at 1 MAC (human), that is, 6% and 1.25%, respectively. With desflurane, the O2 concentration (25% or 94%) did not make a difference. Sevoflurane 1 MAC did not activate tracheal CGRP release. TRPV1 mice showed 75% reduced desflurane responses, and TRPA1 and double-null mutants showed no responses at all.Our results confirm the clinical experience that desflurane is more irritating than isoflurane at equal anesthetic gas concentration, whereas sevoflurane does not activate tracheobronchial sensory nerves to release neuropeptides and induce neurogenic inflammation. Both irritant receptor channels, TRPA1 more than TRPV1, are involved in mediating the adverse effects that may even extend to systemic proinflammatory sequelae.

Loss-of-function mutations in the enzyme 7-dehydrocholesterol reductase are responsible for the Smith-Lemli-Opitz syndrome, in which 7-dehydrocholesterol (7-DHC) levels are markedly increased in the plasma and tissues of patients. This increase in 7-DHC is probably associated with the painful and itchy photosensitivity reported by the majority of patients with Smith-Lemli-Opitz syndrome. To identify the molecular targets involved in the activation and photosensitization of primary afferents by 7-DHC, we focused on TRPA1 and TRPV1, two ion channels expressed in nociceptive nerve endings and previously shown to respond to ultraviolet and visible light under pathophysiological circumstances. Recombinant human TRPA1 is activated and photosensitized in the presence of 7-DHC. Prolonged preexposure to 7-DHC causes more pronounced photosensitization, and while TRPV1 contributes less to the acute effect, it too becomes highly photosensitive upon preincubation with 7-DHC for 1 to 15 hours. Dorsal root ganglion neurons in primary culture display acute sensitivity to 7-DHC in the dark and also light-evoked responses in the presence of 7-DHC, which are exclusively dependent on TRPA1 and TRPV1. Similarly, prolonged exposure of mouse dorsal root ganglion neurons to 7-DHC renders these cells photosensitive in a largely TRPA1- and TRPV1-dependent manner. Single-fiber recordings in mouse skin-nerve preparations demonstrate violet light-evoked activation and a sensitization to 7-DHC exposure. Vice versa, 7-DHC pretreatment of the isolated trachea leads to a TRPA1- and TRPV1-dependent increase of the light-induced calcitonin gene-related peptide release. Taken together, our results implicate TRPA1 and TRPV1 channels as potential pharmacological targets to address the 7-DHC-induced hypersensitivity to light in patients.

High concentrations of nicotine, as in the saliva of oral tobacco consumers or in smoking cessation aids, have been shown to sensitize/activate recombinant transient receptor potential vanilloid type 1 (rTRPV1) and mouse TRPA1 (mTRPA1) channels. By measuring stimulated calcitonin gene-related peptide (CGRP) release from the isolated mouse trachea, we established a bimodal concentration-response relationship with a threshold below 10 µ M (−)-nicotine, a maximum at 100 µ M, an apparent nadir between 0.5 and 10 mM, and a renewed increase at 20 mM. The first peak was unchanged in TRPV1/A1 double-null mutants as compared with wild-types and was abolished by specific nicotinic acetylcholine receptor (nAChR) inhibitors and by camphor, discovered to act as nicotinic antagonist. The nicotine response at 20 mM was strongly pH e -dependent, – five times greater at pH 9.0 than 7.4, indicating that intracellular permeation of the (uncharged) alkaloid was required to reach the TRPV1/A1 binding sites. The response was strongly reduced in both null mutants, and more so in double-null mutants. Upon measuring calcium transients in nodose/jugular and dorsal root ganglion neurons in response to 100 µ M nicotine, 48% of the vagal (but only 14% of the somatic) sensory neurons were activated, the latter very weakly. However, nicotine 20 mM at pH 9.0 repeatedly activated almost every single cultured neuron, partly by releasing intracellular calcium and independent of TRPV1/A1 and nAChRs. In conclusion, in mouse tracheal sensory nerves nAChRs are 200-fold more sensitive to nicotine than TRPV1/A1; they are widely coexpressed with the capsaicin receptor among vagal sensory neurons and twice as abundant as TRPA1. Nicotine is the major stimulant in tobacco, and its sensory impact through nAChRs should not be disregarded.

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.

The transcription factor Sox10 is expressed throughout Schwann cell development and has already been shown to be essential for specification and for the identity and further development of immature Schwann cells. Here, we show that Sox10 is also required in Schwann cells for establishing the myelinating state. This is concluded from the fact that a peripheral neuropathy develops in mice in which Sox10 is deleted by a Cre recombinase whose expression is under control of Krox20 regulatory elements. This neuropathy is characterized by altered marker gene expression along the peripheral nerve, decreased conductivity, and severe persistent hypomyelination. As the Cre recombinase is additionally active in boundary cap cells, we also analyzed the role of Sox10 during embryogenesis in establishment and maintenance of the boundary between central and peripheral nervous systems. Sox10 deletion did not affect establishment or survival of boundary cap cells but appeared to compromise barrier function as cells expressing oligodendrocyte and astrocyte markers were no longer restricted to the central nervous system, and instead found in peripheral nerves. We infer that in addition to its many roles in Schwann cells, Sox10 is also important for the integrity of the boundary between central and peripheral nervous systems. © 2012 Wiley Periodicals, Inc.

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 transcription factor Sox10 functions during multiple consecutive stages of Schwann-cell development in the peripheral nervous system (PNS). Although Sox10 continues to be expressed in mature Schwann cells of the adult peripheral nerve, it is currently unclear whether it is still functional. Here, we used a genetic strategy to selectively delete Sox10 in glia of adult mice in a tamoxifen-dependent manner. The tamoxifen-treated mice developed a severe peripheral neuropathy that was associated with dramatic alterations in peripheral nerve structure and function. Demyelination and axonal degeneration were as much evident as signs of neuroinflammation. Compound action potentials exhibited pathophysiological alterations. Sox10-deleted Schwann cells persisted in the peripheral nerve, but did not exhibit a mature, myelinating phenotype arguing that Sox10 is rather required for differentiation and maintenance of the differentiated state than for survival. Our report is the first evidence that Sox10 is still essentially required for Schwann-cell function in the adult PNS and establishes a useful model in which to study human peripheral neuropathies.

The Sox10 transcription factor is required to maintain as well as specify glial identity, adding new causes for the neuropathies associated with SOX10 mutations., Mutations in the transcription factor SOX10 cause neurocristopathies, including Waardenburg-Hirschsprung syndrome and peripheral neuropathies in humans. This is partly attributed to a requirement for Sox10 in early neural crest for survival, maintenance of pluripotency, and specification to several cell lineages, including peripheral glia. As a consequence, peripheral glia are absent in Sox10-deficient mice. Intriguingly, Sox10 continues to be expressed in these cells after specification. To analyze glial functions after specification, we specifically deleted Sox10 in immature Schwann cells by conditional mutagenesis. Mutant mice died from peripheral neuropathy before the seventh postnatal week. Nerve alterations included a thinned perineurial sheath, increased lipid and collagen deposition, and a dramatically altered cellular composition. Nerve conduction was also grossly aberrant, and neither myelinating nor nonmyelinating Schwann cells formed. Instead, axons of different sizes remained unsorted in large bundles. Schwann cells failed to develop beyond the immature stage and were unable to maintain identity. Thus, our study identifies a novel cause for peripheral neuropathies in patients with SOX10 mutations.

Chronic cough derives from inflammatory hypersensitivity of tracheobronchial nerve endings, most of which express the polymodal capsaicin receptor-channel transient receptor potential vanilloid (TRPV) type 1 and the secretory neuropeptide calcitonin gene-related peptide (CGRP). An isolated mouse trachea preparation was established to measure chemically and thermally stimulated CGRP release as an index for sensory transduction of potential cough-inducing stimuli. TRPV1 knockout mice were employed to assess the TRPV1 contribution to tracheal responsiveness and sensitization. Graded heat-induced CGRP release depended entirely on extracellular calcium and partly on TRPV1; knockout mice showed 60% less CGRP release at 45 degrees C (for 5 min) than wild-types. This heat response was facilitated by the TRPV1 agonist ethanol and the TRPV1-3 agonist 2-aminoethoxydiphenyl borate, effects that were reduced or absent in TRPV1(-/-), respectively. The TRPV1 antagonists ruthenium red and N-(4-t-butylphenyl)-4-(3-chloropyridin-2-yl) tetrahydropyrazine-1(2H)-carboxamide were ineffective on the basal heat response. A step increase of temperature from 22 to 40 degrees C caused a TRPV1-independent CGRP release that was doubled by bradykinin in wild-types but not TRPV1(-/-). Proton stimulation resulted in a bell-shaped concentration-response curve with threshold at pH 6.7 and a maximum at pH 5.7; responses were greatly reduced but not abolished in TRPV1(-/-). Coadministration of amiloride (30 microm), the blocker of acid-sensing ion channels, was ineffective in both TRPV1 genotypes. The data suggest that tracheal acid sensing mainly involves TRPV1 but not acid-sensing ion channels, whereas noxious heat responsiveness partly depends and (inflammatory) sensitization to heat largely depends on the capsaicin receptor in tracheal nerve endings. Lowering of their heat threshold to near body temperature may sustain hypersensitivity and neurogenic inflammation of the upper airways.

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 STATEMENTCutaneous 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.

Transient receptor potential (TRP) cation channel subfamily M member 3 (TRPM3), a member of the TRP channel superfamily, was recently identified as a nociceptor channel in the somatosensory system, where it is involved in the detection of noxious heat; however, owing to the lack of potent and selective agonists, little is known about other potential physiological consequences of the opening of TRPM3. Here we identify and characterize a synthetic TRPM3 activator, CIM0216, whose potency and apparent affinity greatly exceeds that of the canonical TRPM3 agonist, pregnenolone sulfate (PS). In particular, a single application of CIM0216 causes opening of both the central calcium-conducting pore and the alternative cation permeation pathway in a membrane-delimited manner. CIM0216 evoked robust calcium influx in TRPM3-expressing somatosensory neurons, and intradermal injection of the compound induced a TRPM3-dependent nocifensive behavior. Moreover, CIM0216 elicited the release of the peptides calcitonin gene-related peptide (CGRP) from sensory nerve terminals and insulin from isolated pancreatic islets in a TRPM3-dependent manner. These experiments identify CIM0216 as a powerful tool for use in investigating the physiological roles of TRPM3, and indicate that TRPM3 activation in sensory nerve endings can contribute to neurogenic inflammation. ispartof: Proceedings of the National Academy of Sciences of the United States of America vol:112 issue:11 pages:E1363-E1372 ispartof: location:United States status: published

This study establishes a mechanism for metabolic hyperalgesia based on the glycolytic metabolite methylglyoxal. We found that concentrations of plasma methylglyoxal above 600 nM discriminate between diabetes-affected individuals with pain and those without pain. Methylglyoxal depolarizes sensory neurons and induces post-translational modifications of the voltage-gated sodium channel Na(v)1.8, which are associated with increased electrical excitability and facilitated firing of nociceptive neurons, whereas it promotes the slow inactivation of Na(v)1.7. In mice, treatment with methylglyoxal reduces nerve conduction velocity, facilitates neurosecretion of calcitonin gene-related peptide, increases cyclooxygenase-2 (COX-2) expression and evokes thermal and mechanical hyperalgesia. This hyperalgesia is reflected by increased blood flow in brain regions that are involved in pain processing. We also found similar changes in streptozotocin-induced and genetic mouse models of diabetes but not in Na(v)1.8 knockout (Scn10(-/-)) mice. Several strategies that include a methylglyoxal scavenger are effective in reducing methylglyoxal- and diabetes-induced hyperalgesia. This previously undescribed concept of metabolically driven hyperalgesia provides a new basis for the design of therapeutic interventions for painful diabetic neuropathy.