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TRPA1, also known as the wasabi receptor, is a non-selective cation ion channel that plays an essential role in the detection of noxious substances and irritants. Hylobius abietis, the pine weevil, is a pest that causes substantial damage to the forestry industry. The structure of the pine weevils TRPA1 (HaTRPA1) is so far unknown. If the structure were to be determined, an agonist could be developed, which could be used as a repellent against the pine weevil. Membrane proteins are naturally expressed at lower levels and can be highly unstable in certain conditions. Heterologous expression and different protocols must often be attempted and evaluated to produce a high-yield, stable membrane protein sample. The aim of this study is to optimize and evaluate different protocols to express and purify HaTRPA1, including using a truncated version of HaTRPA1 and a cleavable green fluorescent protein tag. HaTRPA1 was expressed through large-scale fermentation in Pichia pastoris. The cells were broken, and the membrane was isolated and washed. The protein was solubilized using Fos-choline 14, purified using Immobilized metal ion affinity chromatography (IMAC), cleavage with the tobacco etch virus protease, reverse IMAC, and separated using size exclusion chromatography. Steps throughout the expression and purification were evaluated using SDS-PAGE. The protocols were attempted for two constructs of HaTRPA1, the full-length construct and a truncated version labelled C2. The results showed that degradation was the major issue with the expression and purification of HaTRPA1. Both the full-length construct and the truncated C2 showed substantial degradation. A decrease in degradation was observed when the induction time during expression was reduced. Implementing a urea wash in the membrane preparation was beneficial in reducing the amount of degradation. The conclusion of this paper was that there is little difference in stability between the two constructs of HaTRPA1. To preve, Have you ever put too much wasabi on a piece of sushi? The painful sensation you feel that causes instant regret is generated by the so-called Transient Receptor Potential Channel, subfamily A, member 1 (TRPA1). The protein TRPA1 plays an essential role in detecting irritants and noxious substances, like our dear friend wasabi. While wasabi may be harmless, there are many substances that can be highly damaging, and our bodies need a way to tell us to stay away from them. Therefore, once TRPA1 is activated by a noxious substance, a signal is sent to our nervous system to generate a painful sensation. While human TRPA1 has been the subject of many studies, it is also worth studying TRPA1 in other organisms. Hylobius abietis, or the pine weevil, is a major pest in the commercial forest industry. It eats the bark of seedlings causing them to die. The pine weevil is responsible for millions of euros in damages each year. Currently, the fight against the pine weevil has involved plenty of harmful insecticides, and with a greater focus on environmentally friendly forestry, there is an interest in other alternatives. If the 3D structure of H. abietis TRPA1 was resolved, it would be possible to develop an activator of TRPA1 that would trigger a similar response in the pine weevil as our overindulgence in wasabi caused. This study attempted different conditions and protocols to produce a substantial amount of TRPA1 from the pine weevil for structural studies. Two different constructs were studied: the full-length construct and a truncated version. Both constructs of the protein were tagged with a green fluorescent protein to enable tracking of the protein throughout the purification and expression. The protein was expressed in the yeast species Pichia pastoris. Afterwards, the protein was solubilized and purified under different conditions to find the optimal protocol.

Osteosarcoma is a highly malignant, painful cancer with poor treatment opportunities and a bad prognosis. Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are non-selective cation channels that have been of great interest in cancer, as their expression is increased in some malignancies. In our study we aim to characterize the expression and functionality of the TRPA1 and TRPV1 channels in human and mouse osteosarcoma tissues and in a mouse cell line. TRPA1/Trpa1 and TRPV1/Trpv1 mRNA expressions were demonstrated by PCR gel electrophoresis and RNAscope in situ hybridization. The function of these channels was confirmed by their radioactive 45Ca2+ uptake in response to the TRPA1 agonist, Allyl-isothiocyanate (AITC), and TRPV1 agonist, capsaicin, in K7M2 cells. An ATP-based K2M7 cell viability luminescence assay was used to determine cell viability after AITC or capsaicin treatments. Both TRPA1/Trpa1 and TRPV1/Trpv1 were expressed similarly in human and mouse osteosarcoma tissues, while Trpa1 transcripts were more abundantly present in K7M2 cells. TRPA1 activation with 200 µM AITC induced a significant 45Ca2+ influx into K7M2 cells, and the antagonist attenuated this effect. In accordance with the lower Trpv1 expression, capsaicin induced a moderate 45Ca2+ uptake, which did not reach the level of statistical significance. Both AITC and capsaicin significantly reduced K7M2 cell viability, demonstrating EC50 values of 22 µM and 74 µM. The viability-decreasing effect of AITC was significantly but only partially antagonized by HC-030031, but the action of capsaicin was not affected by the TRPV1 antagonist capsazepine. We provide here the first data on the functional expression of the TRPA1 and TRPV1 ion channels in osteosarcoma, suggesting novel diagnostic and/or therapeutic perspectives.

Osteosarcoma is a highly malignant, painful cancer with poor treatment opportunities and a bad prognosis. Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are non-selective cation channels that have been of great interest in cancer, as their expression is increased in some malignancies. In our study we aim to characterize the expression and functionality of the TRPA1 and TRPV1 channels in human and mouse osteosarcoma tissues and in a mouse cell line. TRPA1/Trpa1 and TRPV1/Trpv1 mRNA expressions were demonstrated by PCR gel electrophoresis and RNAscope in situ hybridization. The function of these channels was confirmed by their radioactive 45Ca2+ uptake in response to the TRPA1 agonist, Allyl-isothiocyanate (AITC), and TRPV1 agonist, capsaicin, in K7M2 cells. An ATP-based K2M7 cell viability luminescence assay was used to determine cell viability after AITC or capsaicin treatments. Both TRPA1/Trpa1 and TRPV1/Trpv1 were expressed similarly in human and mouse osteosarcoma tissues, while Trpa1 transcripts were more abundantly present in K7M2 cells. TRPA1 activation with 200 µM AITC induced a significant 45Ca2+ influx into K7M2 cells, and the antagonist attenuated this effect. In accordance with the lower Trpv1 expression, capsaicin induced a moderate 45Ca2+ uptake, which did not reach the level of statistical significance. Both AITC and capsaicin significantly reduced K7M2 cell viability, demonstrating EC50 values of 22 µM and 74 µM. The viability-decreasing effect of AITC was significantly but only partially antagonized by HC-030031, but the action of capsaicin was not affected by the TRPV1 antagonist capsazepine. We provide here the first data on the functional expression of the TRPA1 and TRPV1 ion channels in osteosarcoma, suggesting novel diagnostic and/or therapeutic perspectives.

Osteosarcoma is a highly malignant, painful cancer with poor treatment opportunities and a bad prognosis. Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are non-selective cation channels that have been of great interest in cancer, as their expression is increased in some malignancies. In our study we aim to characterize the expression and functionality of the TRPA1 and TRPV1 channels in human and mouse osteosarcoma tissues and in a mouse cell line. TRPA1/Trpa1 and TRPV1/Trpv1 mRNA expressions were demonstrated by PCR gel electrophoresis and RNAscope in situ hybridization. The function of these channels was confirmed by their radioactive 45Ca2+ uptake in response to the TRPA1 agonist, Allyl-isothiocyanate (AITC), and TRPV1 agonist, capsaicin, in K7M2 cells. An ATP-based K2M7 cell viability luminescence assay was used to determine cell viability after AITC or capsaicin treatments. Both TRPA1/Trpa1 and TRPV1/Trpv1 were expressed similarly in human and mouse osteosarcoma tissues, while Trpa1 transcripts were more abundantly present in K7M2 cells. TRPA1 activation with 200 µM AITC induced a significant 45Ca2+ influx into K7M2 cells, and the antagonist attenuated this effect. In accordance with the lower Trpv1 expression, capsaicin induced a moderate 45Ca2+ uptake, which did not reach the level of statistical significance. Both AITC and capsaicin significantly reduced K7M2 cell viability, demonstrating EC50 values of 22 µM and 74 µM. The viability-decreasing effect of AITC was significantly but only partially antagonized by HC-030031, but the action of capsaicin was not affected by the TRPV1 antagonist capsazepine. We provide here the first data on the functional expression of the TRPA1 and TRPV1 ion channels in osteosarcoma, suggesting novel diagnostic and/or therapeutic perspectives.

Every living cell is surrounded by a cell membrane that is made up of amphipathicphospholipids, separating the aqueous solutions inside and outside the cell with ahydrophobic barrier. This compartmentalization is a prerequisite for life, but so arethe many molecules that are floating in the membrane, altering its properties andconnecting the inside with the outside. An important group are the transportproteins, that open ways for molecules and ions, that would normally be too large,too charged, or too polar to pass the membrane. The transport proteins are eitheractive transporters – like pumps – or passive transporters – like channels. In thisthesis, I put the spotlight on two types of channel proteins: Transient ReceptorPotential ion channels, that let ions pass when activated by temperature or pungentchemicals, and Aquaporins (AQP), that are mainly responsible for letting watercross the membrane.In my work, I have made an effort to study the structure of one TRP member inparticular, known as TRPA1 from pine weevil (Hylobius abietis). I had to find thebest possible conditions to solubilize the protein in detergents, and I have alsoinvestigated other tools such as nanodiscs to keep the protein stable in solution. Onemajor hurdle has always been the low yields, and it was therefore that a GFP-tag(Green fluorescent Protein) was added to the protein construct, to facilitate thetracking of the protein and evaluation of purification methods. Coupled with flowcytometry, a method for measuring fluorescence and scattering of individual cells,this proved very useful in designing an expression and purification protocol.The purified protein was used for Cryo-EM (Electron Microscopy), but theprotein was difficult to freeze on grids with a good homogeneous spread ofindividual particles. The use of SRCD (Synchrotron Radiation Circular Dichroism)proved more successful, and confirmed the secondary structure of the protein, andgave information on the temperature stability of the pro

Osteosarcoma is a highly malignant, painful cancer with poor treatment opportunities and a bad prognosis. Transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1) receptors are non-selective cation channels that have been of great interest in cancer, as their expression is increased in some malignancies. In our study we aim to characterize the expression and functionality of the TRPA1 and TRPV1 channels in human and mouse osteosarcoma tissues and in a mouse cell line. TRPA1/Trpa1 and TRPV1/Trpv1 mRNA expressions were demonstrated by PCR gel electrophoresis and RNAscope in situ hybridization. The function of these channels was confirmed by their radioactive 45Ca2+ uptake in response to the TRPA1 agonist, Allyl-isothiocyanate (AITC), and TRPV1 agonist, capsaicin, in K7M2 cells. An ATP-based K2M7 cell viability luminescence assay was used to determine cell viability after AITC or capsaicin treatments. Both TRPA1/Trpa1 and TRPV1/Trpv1 were expressed similarly in human and mouse osteosarcoma tissues, while Trpa1 transcripts were more abundantly present in K7M2 cells. TRPA1 activation with 200 µM AITC induced a significant 45Ca2+ influx into K7M2 cells, and the antagonist attenuated this effect. In accordance with the lower Trpv1 expression, capsaicin induced a moderate 45Ca2+ uptake, which did not reach the level of statistical significance. Both AITC and capsaicin significantly reduced K7M2 cell viability, demonstrating EC50 values of 22 µM and 74 µM. The viability-decreasing effect of AITC was significantly but only partially antagonized by HC-030031, but the action of capsaicin was not affected by the TRPV1 antagonist capsazepine. We provide here the first data on the functional expression of the TRPA1 and TRPV1 ion channels in osteosarcoma, suggesting novel diagnostic and/or therapeutic perspectives.

Chemotherapy-induced peripheral neuropathy is a frequent, disabling side effect of anticancer drugs. Oxaliplatin, a platinum compound used in the treatment of advanced colorectal cancer, often leads to a form of chemotherapy-induced peripheral neuropathy characterized by mechanical and cold hypersensitivity. Current therapies for chemotherapy-induced peripheral neuropathy are ineffective, often leading to the cessation of treatment. Transient receptor potential ankyrin 1 (TRPA1) is a polymodal, non-selective cation-permeable channel expressed in nociceptors, activated by physical stimuli and cellular stress products. TRPA1 has been linked to the establishment of chemotherapy-induced peripheral neuropathy and other painful neuropathic conditions. Sigma-1 receptor is an endoplasmic reticulum chaperone known to modulate the function of many ion channels and receptors. Sigma-1 receptor antagonist, a highly selective antagonist of Sigma-1 receptor, has shown effectiveness in a phase II clinical trial for oxaliplatin chemotherapy-induced peripheral neuropathy. However, the mechanisms involved in the beneficial effects of Sigma-1 receptor antagonist are little understood. We combined biochemical and biophysical (i.e. intermolecular Förster resonance energy transfer) techniques to demonstrate the interaction between Sigma-1 receptor and human TRPA1. Pharmacological antagonism of Sigma-1R impaired the formation of this molecular complex and the trafficking of functional TRPA1 to the plasma membrane. Using patch-clamp electrophysiological recordings we found that antagonists of Sigma-1 receptor, including Sigma-1 receptor antagonist, exert a marked inhibition on plasma membrane expression and function of human TRPA1 channels. In TRPA1-expressing mouse sensory neurons, Sigma-1 receptor antagonists reduced inward currents and the firing of actions potentials in response to TRPA1 agonists. Finally, in a mouse experimental model of oxaliplatin neuropathy, systemic treatment with

The role of TRPA1 in the thermosensitivity of the corneal cold thermoreceptor nerve endings was studied in young and aged mice. The contribution of the TRPA1-dependent activity to basal tearing and thermally-evoked blink was also explored. The corneal cold thermoreceptors' activity was recorded extracellularly in young (5-month-old) and aged (18-month-old) C57BL/6WT (WT) and TRPA1-/- knockout (TRPA1-KO) mice at basal temperature (34 °C) and during cooling (15 °C) and heating (45 °C) ramps. The blink response to cold and heat stimulation of the ocular surface and the basal tearing rate were also measured in young animals using orbicularis oculi muscle electromyography (OOemg) and phenol red threads, respectively. The background activity at 34 °C and the cooling- and heating-evoked responses of the cold thermoreceptors were similar in WT and TRPA1-KO animals, no matter the age. Similar to the aged WT mice, in the young and aged TRPA1-KO mice, most of the cold thermoreceptors presented low frequency background activity, a low cooling threshold, and a sluggish response to heating. The amplitude and duration of the OOemg signals correlated with the magnitude of the induced thermal change in the WT but not in the TRPA1-KO mice. The basal tearing was similar in the TRPA1-KO and WT mice. The electrophysiological data suggest that the TRPA1-dependent nerve activity, which declines with age, contributes to detecting the warming of the ocular surface and also to integrating the thermally-evoked reflex blink.

The current study was supported by grants from The Academy of Finland (EM) and Pirkanmaa Regional Fund of the Finnish Cultural Foundation (MS) and by the competitive research funding (VTR funding) by Tampere University Hospital (EM).

To maintain adequate oxygen levels in the body, which is essential for a healthy life, the respiratory and cardiovascular systems play vitally important roles. When the oxygen content is insufficient, i.e., when hypoxia is loaded, respiratory and cardiovascular systems respond to restore, compensate, or adapt to hypoxia, e.g., by increasing ventilation and blood flow to maintain oxygen transport to vital organs. Traditionally, it has been thought that hypoxia is detected solely by carotid and aortic bodies, i.e., by peripheral chemoreceptors, and information from the peripheral chemoreceptors is transmitted to respiratory and cardiovascular centers in the brainstem whose respiratory and cardiovascular neural outputs are regulated. However, recent progress in neurophysiology has clarified that there are hypoxia-sensors not only in the periphery but also in the central nervous system. Hypoxia also affects the vascular system causing atherosclerosis and pulmonary hypertension and impairs blood glucose regulation that also facilitates atherosclerosis. The effects of hypoxia on vital organs and tissues vary depending on the modality of hypoxia exposure, i.e., acute, chronically sustained, or intermittent hypoxia. Although these issues have been vigorously investigated, the underlying mechanisms are yet to be unraveled. Likewise, long-range consequences for organ and tissue functions affected by hypoxia have not been fully elucidated. In the article collection of this Research Topic, a series of studies report the latest and most notable pathophysiological findings that are categorized into four areas: respiratory control, glucose metabolism, pulmonary hypertension, and sympathetic nervous system activation. The articles attempt to clarify many of the unsolved issues summarized below. (Introduction)

Gentle touch such as stroking of the skin produces a pleasant feeling, which is detected by a rare subset of sensory neurons that express Mas-related G protein-coupled receptor B4 (MrgprB4) in mice. We examined small populations of MrgprB4-positive neurons in the trigeminal ganglion and the dorsal root ganglion, and most of these were sensitive to transient receptor potential ankyrin 1 (TRPA1) agonist but not TRPV1, TRPM8, or TRPV4 agonists. Deficiency of MrgprB4 did not affect noxious pain or itch behaviors in the hairless plantar and hairy cheek. Although behavior related to acetone-induced cold sensing in the hind paw was not changed, unpleasant sensory behaviors in response to acetone application or sucrose splash to the cheek were significantly enhanced in Mrgprb4-knockout mice as well as in TRPA1-knockout mice. These results suggest that MrgprB4 in the trigeminal neurons produces pleasant sensations in cooperation with TRPA1, rather than noxious or cold sensations. Pleasant sensations may modulate unpleasant sensations on the cheek via MrgprB4.

To maintain adequate oxygen levels in the body, which is essential for a healthy life, the respiratory and cardiovascular systems play vitally important roles. When the oxygen content is insufficient, i.e., when hypoxia is loaded, respiratory and cardiovascular systems respond to restore, compensate, or adapt to hypoxia, e.g., by increasing ventilation and blood flow to maintain oxygen transport to vital organs. Traditionally, it has been thought that hypoxia is detected solely by carotid and aortic bodies, i.e., by peripheral chemoreceptors, and information from the peripheral chemoreceptors is transmitted to respiratory and cardiovascular centers in the brainstem whose respiratory and cardiovascular neural outputs are regulated. However, recent progress in neurophysiology has clarified that there are hypoxia-sensors not only in the periphery but also in the central nervous system. Hypoxia also affects the vascular system causing atherosclerosis and pulmonary hypertension and impairs blood glucose regulation that also facilitates atherosclerosis. The effects of hypoxia on vital organs and tissues vary depending on the modality of hypoxia exposure, i.e., acute, chronically sustained, or intermittent hypoxia. Although these issues have been vigorously investigated, the underlying mechanisms are yet to be unraveled. Likewise, long-range consequences for organ and tissue functions affected by hypoxia have not been fully elucidated. In the article collection of this Research Topic, a series of studies report the latest and most notable pathophysiological findings that are categorized into four areas: respiratory control, glucose metabolism, pulmonary hypertension, and sympathetic nervous system activation. The articles attempt to clarify many of the unsolved issues summarized below. (Introduction)

To maintain adequate oxygen levels in the body, which is essential for a healthy life, the respiratory and cardiovascular systems play vitally important roles. When the oxygen content is insufficient, i.e., when hypoxia is loaded, respiratory and cardiovascular systems respond to restore, compensate, or adapt to hypoxia, e.g., by increasing ventilation and blood flow to maintain oxygen transport to vital organs. Traditionally, it has been thought that hypoxia is detected solely by carotid and aortic bodies, i.e., by peripheral chemoreceptors, and information from the peripheral chemoreceptors is transmitted to respiratory and cardiovascular centers in the brainstem whose respiratory and cardiovascular neural outputs are regulated. However, recent progress in neurophysiology has clarified that there are hypoxia-sensors not only in the periphery but also in the central nervous system. Hypoxia also affects the vascular system causing atherosclerosis and pulmonary hypertension and impairs blood glucose regulation that also facilitates atherosclerosis. The effects of hypoxia on vital organs and tissues vary depending on the modality of hypoxia exposure, i.e., acute, chronically sustained, or intermittent hypoxia. Although these issues have been vigorously investigated, the underlying mechanisms are yet to be unraveled. Likewise, long-range consequences for organ and tissue functions affected by hypoxia have not been fully elucidated. In the article collection of this Research Topic, a series of studies report the latest and most notable pathophysiological findings that are categorized into four areas: respiratory control, glucose metabolism, pulmonary hypertension, and sympathetic nervous system activation. The articles attempt to clarify many of the unsolved issues summarized below. (Introduction)

The TRPA1 ion channel is activated by electrophilic compounds through the covalent modification of intracellular cysteine residues. How non-covalent agonists activate the channel and whether covalent and non-covalent agonists elicit the same physiological responses are not understood. Here, we report the discovery of a non-covalent agonist, GNE551, and determine a cryo-EM structure of the TRPA1-GNE551 complex, revealing a distinct binding pocket and ligand-interaction mechanism. Unlike the covalent agonist allyl isothiocyanate, which elicits channel desensitization, tachyphylaxis, and transient pain, GNE551 activates TRPA1 into a distinct conducting state without desensitization and induces persistent pain. Furthermore, GNE551-evoked pain is relatively insensitive to antagonist treatment. Thus, we demonstrate the biased agonism of TRPA1, a finding that has important implications for the discovery of effective drugs tailored to different disease etiologies.

To maintain adequate oxygen levels in the body, which is essential for a healthy life, the respiratory and cardiovascular systems play vitally important roles. When the oxygen content is insufficient, i.e., when hypoxia is loaded, respiratory and cardiovascular systems respond to restore, compensate, or adapt to hypoxia, e.g., by increasing ventilation and blood flow to maintain oxygen transport to vital organs. Traditionally, it has been thought that hypoxia is detected solely by carotid and aortic bodies, i.e., by peripheral chemoreceptors, and information from the peripheral chemoreceptors is transmitted to respiratory and cardiovascular centers in the brainstem whose respiratory and cardiovascular neural outputs are regulated. However, recent progress in neurophysiology has clarified that there are hypoxia-sensors not only in the periphery but also in the central nervous system. Hypoxia also affects the vascular system causing atherosclerosis and pulmonary hypertension and impairs blood glucose regulation that also facilitates atherosclerosis. The effects of hypoxia on vital organs and tissues vary depending on the modality of hypoxia exposure, i.e., acute, chronically sustained, or intermittent hypoxia. Although these issues have been vigorously investigated, the underlying mechanisms are yet to be unraveled. Likewise, long-range consequences for organ and tissue functions affected by hypoxia have not been fully elucidated. In the article collection of this Research Topic, a series of studies report the latest and most notable pathophysiological findings that are categorized into four areas: respiratory control, glucose metabolism, pulmonary hypertension, and sympathetic nervous system activation. The articles attempt to clarify many of the unsolved issues summarized below. (Introduction)

To maintain adequate oxygen levels in the body, which is essential for a healthy life, the respiratory and cardiovascular systems play vitally important roles. When the oxygen content is insufficient, i.e., when hypoxia is loaded, respiratory and cardiovascular systems respond to restore, compensate, or adapt to hypoxia, e.g., by increasing ventilation and blood flow to maintain oxygen transport to vital organs. Traditionally, it has been thought that hypoxia is detected solely by carotid and aortic bodies, i.e., by peripheral chemoreceptors, and information from the peripheral chemoreceptors is transmitted to respiratory and cardiovascular centers in the brainstem whose respiratory and cardiovascular neural outputs are regulated. However, recent progress in neurophysiology has clarified that there are hypoxia-sensors not only in the periphery but also in the central nervous system. Hypoxia also affects the vascular system causing atherosclerosis and pulmonary hypertension and impairs blood glucose regulation that also facilitates atherosclerosis. The effects of hypoxia on vital organs and tissues vary depending on the modality of hypoxia exposure, i.e., acute, chronically sustained, or intermittent hypoxia. Although these issues have been vigorously investigated, the underlying mechanisms are yet to be unraveled. Likewise, long-range consequences for organ and tissue functions affected by hypoxia have not been fully elucidated. In the article collection of this Research Topic, a series of studies report the latest and most notable pathophysiological findings that are categorized into four areas: respiratory control, glucose metabolism, pulmonary hypertension, and sympathetic nervous system activation. The articles attempt to clarify many of the unsolved issues summarized below. (Introduction)

Background: Knowledge of exact signalling events during migraine attacks is lacking. Various substances are known to trigger migraine attacks in patients and calcitonin gene-related peptide antagonising drugs are effective against migraine pain. Here, we investigated the signalling pathways involved in three different mouse models of provoked migraine and relate them to calcitonin gene-related peptide and other migraine-relevant targets. Methods: In vivo mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim-induced migraine were applied utilising tactile sensitivity to von Frey filaments as measuring readout. Signalling pathways involved in the three models were dissected by use of specific knockout mice and chemical inhibitors. In vivo results were supported by ex vivo wire myograph experiments measuring arterial dilatory responses and ex vivo calcitonin gene-related peptide release from trigeminal ganglion and trigeminal nucleus caudalis from mice. Results: Glyceryl trinitrate-induced hypersensitivity was dependent on both prostaglandins and transient receptor potential cation channel, subfamily A, member 1, whereas cilostazol- and levcromakalim-induced hypersensitivity were independent of both. All three migraine triggers activated calcitonin gene-related peptide signalling, as both receptor antagonism and antibody neutralisation of calcitonin gene-related peptide were effective inhibitors of hypersensitivity in all three models. Stimulation of trigeminal ganglia and brain stem tissue samples with cilostazol and levcromakalim did not result in release of calcitonin gene-related peptide, and vasodilation following levcromakalim stimulation was independent of CGRP receptor antagonism. Conclusion: The mouse models of glyceryl trinitrate-, cilostazol- and levcromakalim- induced migraine all involve calcitonin gene-related peptide signalling in a complex interplay between different cell/tissue types. These models are useful in the study of migraine me

Female mosquitoes employ a diverse array of sensory cues when finding hosts for blood feeding. Among these cues, the mosquito’s ability to detect body heat while host seeking has been appreciated for decades. This thermosensory system is largely understood to detect convective air currents around warm surfaces that activate thermosensory neurons in the mosquito antenna. Because these convective currents occur quite physically close to the warm surface, we chose to investigate whether mosquitoes can also sense infrared radiation emitted from hosts as well, which could function at a much greater distance. To do this we developed a novel behavioral paradigm that exposes mosquitoes to infrared cues and records their behavioral responses. Using this new paradigm, we found that mosquitoes detect infrared while exposed to other host cues and use this sense to navigate towards host objects. Furthermore, this sense is influenced by both the IR intensity of the source and environmental conditions. Lastly, we found that mosquitoes lacking the thermally activated ion channel TRPA1 completely lose their ability to detect thermal infrared. This work provides the first demonstration that IR is a cue used by mosquitoes for host seeking.

Over the past decade, the transient receptor potential ankyrin 1 (TRPA1) receptor has been extensively studied because of its wide distribution across the human body and its roles in many aspects of sensation, some of which are physiological and others of which are pathophysiological. A member of the TRP channel family, TRPA1 is most commonly found in somatosensory neurons present in nociceptors, especially the dorsal root ganglia and trigeminal ganglia, but its expression has been reported in tissues as disparate as the cells of the inner ear, non-neuronal cells in the small and large intestines and lungs, and in the beta islet cells of the pancreas. Physiologically, TRPA1’s primary role is as a nociceptive chemosensor detecting exogenous agonists that are irritants, such as AITC from wasabi, and endogenous agonists that are indicators of oxidative stress, such as H2O2. However, when TRPA1 becomes dysregulated, its physiological roles can become distorted. Research has demonstrated that TRPA1 is involved in many pathologies including chronic pain and inflammation, neuropathic pain, itch, and respiratory diseases, but to date, few TRPA1-targeting therapeutics have been successful, and it is evident that greater understanding of the receptor is required to address this need. Recently, the discovery of optovin revealed that a synthetic photoreactive compound could confer photosensitivity onto vertebrate TRPA1, but much is still unknown about photo-dependent TRPA1 activation. One way to approach this question is through identification of additional chemotypes that act similarly to optovin. In this dissertation, I describe the process of identifying such compounds and the characterization of their properties. Analysis of data from a large-scale, behavior-based chemical screen conducted in larval zebrafish established a small subset of compounds that induced a strong behavioral response to light. Structural analysis showed that the hit compounds were in fact structurally

TRPA1 - also known as the ‘wasabi receptor’ - is activated by pungent natural products from mustard and allium plants and is responsible for the sinus clearing or eye stinging pain that one experiences when eating horse radish or chopping an onion. More importantly, TRPA1 is a receptor for an astoundingly broad class of volatile environmental irritants and endogenous pro-algesic agents that elicit or exacerbate tissue damage, pain, and neurogenic inflammation. Many of these irritants are chemically reactive electrophiles that activate TRPA1 through a fascinating mechanism involving covalent modification of the channel’s cytoplasmic N-terminus, a phenomenon that is not well understood at the biochemical or structural level. TRPA1 also functions as a ‘receptor-operated’ channel that is activated downstream of phospholipase C signaling pathways, such as those involved in the transduction of pruritic signals. Together, these actions make TRPA1 a major contributor to a broad range of chemo-nociceptive actions include pain and itch.In this work we have exploited natural product pharmacology, functional recording, and structural methods to understand how TRPA1 is activated by a range of stimuli (reactive and non-reactive, direct and indirect). Collectively, these data draw a detailed mechanistic picture of how this important chemo-nociceptive ion channel detects physiological stimuli, transduces this information into neuronal excitation, and is regulated by cellular events. In light of TRPA1’s well recognized role in acute and persistent pain and itch, our findings are of both investigational and therapeutic import. Beyond TRPA1, they are of general relevance to illuminating the gating mechanisms other sensory TRP channels, several of which are also regulated downstream of metabotropic receptors and/or by cellular calcium.

Over the past decade, the transient receptor potential ankyrin 1 (TRPA1) receptor has been extensively studied because of its wide distribution across the human body and its roles in many aspects of sensation, some of which are physiological and others of which are pathophysiological. A member of the TRP channel family, TRPA1 is most commonly found in somatosensory neurons present in nociceptors, especially the dorsal root ganglia and trigeminal ganglia, but its expression has been reported in tissues as disparate as the cells of the inner ear, non-neuronal cells in the small and large intestines and lungs, and in the beta islet cells of the pancreas. Physiologically, TRPA1’s primary role is as a nociceptive chemosensor detecting exogenous agonists that are irritants, such as AITC from wasabi, and endogenous agonists that are indicators of oxidative stress, such as H2O2. However, when TRPA1 becomes dysregulated, its physiological roles can become distorted. Research has demonstrated that TRPA1 is involved in many pathologies including chronic pain and inflammation, neuropathic pain, itch, and respiratory diseases, but to date, few TRPA1-targeting therapeutics have been successful, and it is evident that greater understanding of the receptor is required to address this need. Recently, the discovery of optovin revealed that a synthetic photoreactive compound could confer photosensitivity onto vertebrate TRPA1, but much is still unknown about photo-dependent TRPA1 activation. One way to approach this question is through identification of additional chemotypes that act similarly to optovin. In this dissertation, I describe the process of identifying such compounds and the characterization of their properties. Analysis of data from a large-scale, behavior-based chemical screen conducted in larval zebrafish established a small subset of compounds that induced a strong behavioral response to light. Structural analysis showed that the hit compounds were in fact structurally

TRPA1 - also known as the ‘wasabi receptor’ - is activated by pungent natural products from mustard and allium plants and is responsible for the sinus clearing or eye stinging pain that one experiences when eating horse radish or chopping an onion. More importantly, TRPA1 is a receptor for an astoundingly broad class of volatile environmental irritants and endogenous pro-algesic agents that elicit or exacerbate tissue damage, pain, and neurogenic inflammation. Many of these irritants are chemically reactive electrophiles that activate TRPA1 through a fascinating mechanism involving covalent modification of the channel’s cytoplasmic N-terminus, a phenomenon that is not well understood at the biochemical or structural level. TRPA1 also functions as a ‘receptor-operated’ channel that is activated downstream of phospholipase C signaling pathways, such as those involved in the transduction of pruritic signals. Together, these actions make TRPA1 a major contributor to a broad range of chemo-nociceptive actions include pain and itch.In this work we have exploited natural product pharmacology, functional recording, and structural methods to understand how TRPA1 is activated by a range of stimuli (reactive and non-reactive, direct and indirect). Collectively, these data draw a detailed mechanistic picture of how this important chemo-nociceptive ion channel detects physiological stimuli, transduces this information into neuronal excitation, and is regulated by cellular events. In light of TRPA1’s well recognized role in acute and persistent pain and itch, our findings are of both investigational and therapeutic import. Beyond TRPA1, they are of general relevance to illuminating the gating mechanisms other sensory TRP channels, several of which are also regulated downstream of metabotropic receptors and/or by cellular calcium.

Female mosquitoes employ a diverse array of sensory cues when finding hosts for blood feeding. Among these cues, the mosquito’s ability to detect body heat while host seeking has been appreciated for decades. This thermosensory system is largely understood to detect convective air currents around warm surfaces that activate thermosensory neurons in the mosquito antenna. Because these convective currents occur quite physically close to the warm surface, we chose to investigate whether mosquitoes can also sense infrared radiation emitted from hosts as well, which could function at a much greater distance. To do this we developed a novel behavioral paradigm that exposes mosquitoes to infrared cues and records their behavioral responses. Using this new paradigm, we found that mosquitoes detect infrared while exposed to other host cues and use this sense to navigate towards host objects. Furthermore, this sense is influenced by both the IR intensity of the source and environmental conditions. Lastly, we found that mosquitoes lacking the thermally activated ion channel TRPA1 completely lose their ability to detect thermal infrared. This work provides the first demonstration that IR is a cue used by mosquitoes for host seeking.

Introduction: Thermo transient receptor potential (thermoTRP) channels are some of the most intensely pursued therapeutic targets of the past decade. They are considered promising targets of numerous diseases including chronic pain and cancer. Modulators of these proteins, in particular TRPV1-4, TRPM8 and TRPA1, have reached clinical development, but none has been approved for clinical practice yet. Areas covered: The therapeutic potential of targeting thermoTRP channels is discussed. The discussion is centered on our experience and on available data found in SciFinder, PubMed, and ClinicalTrials.gov database from the past decade. This review focuses on the therapeutic progress concerning this family of channels, including strategies to improve their therapeutic index for overcoming adverse effects. Expert opinion: Although thermoTRPs are pivotal drug targets, translation to the clinic has faced two key problems, (i) unforeseen side effects in Phase I trials and, (ii) poor clinical efficacy in Phase II trials. Thus, there is a need for (i) an enhanced understanding of the physiological role of these channels in tissues and organs and (ii) the development of human-based pre-clinical models with higher clinical translation. Furthermore, progress in nanotechnology-based delivery strategies will positively impact thermoTRP human pharmacology.

Bacterial keratitis is a devastating, sight-threatening infection of the cornea. However, in order to truly grasp how bacterial keratitis ensues, it is first imperative to understand how the healthy eye is maintained. Only then can we truly decipher the intricacies of how a cornea becomes susceptible to potentially dangerous microorganisms.Bacteria surround us and inhabit almost every niche in our environment and on our bodies. Thus, the question arose: are bacteria present on the ocular surface, and if so, what role do they play in maintaining health? This is the first question I sought to address in my dissertation. I used bacterial labeling techniques to be able to identify live microbes and gain a spatial understanding of the bacteria on the mouse ocular surface. Interestingly, I found that live bacteria are rarely present on the cornea. This was in stark contrast to the conjunctiva, which does host bacterial inhabitants, including long filamentous bacteria. The cornea’s ability to prevent a microbiome is dependent on IL-1R and MyD88 signaling, an important finding to demonstrate the role of this pathway during homeostasis and not just as a response to pathogens.It is fortunate that the cornea is an expert at preventing bacterial colonization considering that it is constantly exposed to the environment and all of the surrounding microbes. Thus, I wanted to gain a further understanding of how the cornea prevents a microbiome on its surface. Tear fluid has previously been shown to be an important factor towards bacterial defense of the ocular surface. Several constituents that make up basal tears are antimicrobial and impede bacterial virulence. One such component is DMBT1, a glycoprotein that inhibits bacterial twitching. Twitching motility is a surface movement important for epithelial traversal and virulence. How DMBT1 inhibits Pseudomonas aeruginosa twitching, a common cause of contact lens related corneal infections, was investigated. It was found that N-glyc

Bacterial keratitis is a devastating, sight-threatening infection of the cornea. However, in order to truly grasp how bacterial keratitis ensues, it is first imperative to understand how the healthy eye is maintained. Only then can we truly decipher the intricacies of how a cornea becomes susceptible to potentially dangerous microorganisms.Bacteria surround us and inhabit almost every niche in our environment and on our bodies. Thus, the question arose: are bacteria present on the ocular surface, and if so, what role do they play in maintaining health? This is the first question I sought to address in my dissertation. I used bacterial labeling techniques to be able to identify live microbes and gain a spatial understanding of the bacteria on the mouse ocular surface. Interestingly, I found that live bacteria are rarely present on the cornea. This was in stark contrast to the conjunctiva, which does host bacterial inhabitants, including long filamentous bacteria. The cornea’s ability to prevent a microbiome is dependent on IL-1R and MyD88 signaling, an important finding to demonstrate the role of this pathway during homeostasis and not just as a response to pathogens.It is fortunate that the cornea is an expert at preventing bacterial colonization considering that it is constantly exposed to the environment and all of the surrounding microbes. Thus, I wanted to gain a further understanding of how the cornea prevents a microbiome on its surface. Tear fluid has previously been shown to be an important factor towards bacterial defense of the ocular surface. Several constituents that make up basal tears are antimicrobial and impede bacterial virulence. One such component is DMBT1, a glycoprotein that inhibits bacterial twitching. Twitching motility is a surface movement important for epithelial traversal and virulence. How DMBT1 inhibits Pseudomonas aeruginosa twitching, a common cause of contact lens related corneal infections, was investigated. It was found that N-glyc

TRPA1 is a chemosensory ion channel that functions as a sentinel for structurally diverse electrophilic irritants. Channel activation occurs through an unusual mechanism involving covalent modification of cysteine residues clustered within an amino-terminal cytoplasmic domain. Here, we describe a peptidergic scorpion toxin (WaTx) that activates TRPA1 by penetrating the plasma membrane to access the same intracellular site modified by reactive electrophiles. WaTx stabilizes TRPA1 in a biophysically distinct active state characterized by prolonged channel openings and low Ca2+ permeability. Consequently, WaTx elicits acute pain and pain hypersensitivity but fails to trigger efferent release of neuropeptides and neurogenic inflammation typically produced by noxious electrophiles. These findings provide a striking example of convergent evolution whereby chemically disparate animal- and plant-derived irritants target the same key allosteric regulatory site to differentially modulate channel activity. WaTx is a unique pharmacological probe for dissecting TRPA1 function and its contribution to acute and persistent pain.

Piperine, the principle pungent compound in black peppers, is known to activate the capsaicin receptor TRPV1 ion channel. How piperine interacts with the channel protein, however, remains unclear. Here we show that piperine binds to the same ligand-binding pocket as capsaicin but in different poses. There was no detectable detrimental effect when T551 and E571, two major sites known to form hydrogen bond with capsaicin, were mutated to a hydrophobic amino acid. Computational structural modeling suggested that piperine makes interactions with multiple amino acids within the ligand binding pocket, including T671 on the pore-forming S6 segment. Mutations of this residue could substantially reduce or even eliminate piperine-induced activation, confirming that T671 is an important site. Our results suggest that the bound piperine may directly interact with the pore-forming S6 segment to induce channel opening. These findings help to explain why piperine is a weak agonist, and may guide future efforts to develop novel pharmaceutical reagents targeting TRPV1.

The cation channel TRPA1 transduces a myriad of noxious chemical stimuli into nociceptor electrical excitation and neuropeptide release, leading to pain and neurogenic inflammation. Despite emergent evidence that TRPA1 is regulated by the membrane environment, it remains unknown whether this channel localizes in membrane microdomains or whether it interacts with cholesterol. Using total internal reflection fluorescence microscopy and density gradient centrifugation we found that mouse TRPA1 localizes preferably into cholesterol-rich domains and functional experiments revealed that cholesterol depletion decreases channel sensitivity to chemical agonists. Moreover, we identified two structural motifs in transmembrane segments 2 and 4 involved in mTRPA1-cholesterol interactions that are necessary for normal agonist sensitivity and plasma membrane localization. We discuss the impact of such interactions on TRPA1 gating mechanisms, regulation by the lipid environment, and role of this channel in sensory membrane microdomains, all of which helps to understand the puzzling pharmacology and pathophysiology of this channel., SCOPUS: ar.j, info:eu-repo/semantics/published

[Background] Diabetic peripheral neuropathy is a common long-term complication of diabetes. Accumulating evidence suggests that vascular impairment plays important roles in the pathogenesis of diabetic peripheral neuropathy, while the mechanism remains unclear. We recently reported that transient receptor potential ankyrin 1 (TRPA1) is sensitized by hypoxia, which can contribute to cold hypersensitivity. In this study, we investigated the involvement of TRPA1 and vascular impairment in painful diabetic peripheral neuropathy using streptozotocin-induced diabetic model mice. [Results] Streptozotocin-induced diabetic model mice showed mechanical and cold hypersensitivity with a peak at two weeks after the streptozotocin administration, which were likely to be paralleled with the decrease in the skin blood flow of the hindpaw. Streptozotocin-induced cold hypersensitivity was significantly inhibited by an antagonist HC-030031 (100 mg/kg) or deficiency for TRPA1, whereas mechanical hypersensitivity was unaltered. Consistent with these results, the nocifensive behaviors evoked by an intraplantar injection of the TRPA1 agonist allyl isothiocyanate (AITC) were enhanced two weeks after the streptozotocin administration. Both streptozotocin-induced cold hypersensitivity and the enhanced AITC-evoked nocifensive behaviors were significantly inhibited by a vasodilator, tadalafil (10 mg/kg), with recovery of the decreased skin blood flow. Similarly, in a mouse model of hindlimb ischemia induced by the ligation of the external iliac artery, AITC-evoked nocifensive behaviors were significantly enhanced three and seven days after the ischemic operation, whereas mechanical hypersensitivity was unaltered in TRPA1-knockout mice. However, no difference was observed between wild-type and TRPA1-knockout mice in the hyposensitivity for current or mechanical stimulation or the deceased density of intraepidermal nerve fibers eight weeks after the streptozotocin administration. [Conclusion] Th

H2S has excitatory and inhibitory effects on Ca2+ signals via transient receptor potential ankyrin 1 (TRPA1) and ATP-sensitive K+ channels, respectively. H2S converts intracellularly to polysulfides, which are more potent agonists for TRPA1 than H2S. Under inflammatory conditions, changes in the expression and activity of these H2S target channels and/or the conversion of H2S to polysulfides may modulate H2S effects. Effects of proinflammatory cytokines on H2S-induced Ca2+ signals and polysulfide production in RIN14B cells were examined using fluorescence imaging with fura-2 and SSP4, respectively. Na2S, a H2S donor, induced 1) the inhibition of spontaneous Ca2+ signals, 2) inhibition followed by [Ca2+](i) increase, and 3) rapid [Ca2+](i) increase without inhibition in 50% (23/46), 22% (10/46), and 17% (8/46) of cells tested, respectively. IL-1 beta augmented H2S-induced [Ca2+](i) increases, which were inhibited by TRPA1 and voltage-dependent L-type Ca2+ channel blockers. However, IL-1 beta treatment did not affect [Ca2+](i) increases evoked by a TRPA1 agonist or high concentration of KCl. Na2S increased intracellular polysulfide levels, which were enhanced by IL-1 beta treatment. A NOS inhibitor suppressed the increased polysulfide production and [Ca2+](i) increase in IL-1 beta-treated cells. These results suggest that IL-1 beta augments H2S-induced [Ca2+](i) increases via the conversion of H2S to polysulfides through NO synthesis, but not via changes in the activity and expression of target channels. Polysulfides may play an important role in the effects of H2S during inflammation.

The cellular and systemic effects induced by bacterial lipopolysaccharides (LPS) have been solely attributed to the activation of the Toll-like receptor 4 (TLR4) signalling cascade. However, recent studies have shown that LPS activates several members of the Transient Receptor Potential (TRP) family of cation channels. Indeed, LPS induces activation of the broadly-tuned chemosensor TRPA1 in sensory neurons in a TLR4-independent manner, and genetic ablation of this channel reduced mouse pain and inflammatory responses triggered by LPS and the gustatory-mediated avoidance to LPS in fruit flies. LPS was also shown to activate TRPV4 channels in airway epithelial cells, an effect leading to an immediate production of bactericidal nitric oxide and to an increase in ciliary beat frequency. In this review, we discuss the role of TRP channels as sensors of bacterial endotoxins, and therefore, as crucial players in the timely detection of invading gram-negative bacteria.

H2S has excitatory and inhibitory effects on Ca2+ signals via transient receptor potential ankyrin 1 (TRPA1) and ATP-sensitive K+ channels, respectively. H2S converts intracellularly to polysulfides, which are more potent agonists for TRPA1 than H2S. Under inflammatory conditions, changes in the expression and activity of these H2S target channels and/or the conversion of H2S to polysulfides may modulate H2S effects. Effects of proinflammatory cytokines on H2S-induced Ca2+ signals and polysulfide production in RIN14B cells were examined using fluorescence imaging with fura-2 and SSP4, respectively. Na2S, a H2S donor, induced 1) the inhibition of spontaneous Ca2+ signals, 2) inhibition followed by [Ca2+](i) increase, and 3) rapid [Ca2+](i) increase without inhibition in 50% (23/46), 22% (10/46), and 17% (8/46) of cells tested, respectively. IL-1 beta augmented H2S-induced [Ca2+](i) increases, which were inhibited by TRPA1 and voltage-dependent L-type Ca2+ channel blockers. However, IL-1 beta treatment did not affect [Ca2+](i) increases evoked by a TRPA1 agonist or high concentration of KCl. Na2S increased intracellular polysulfide levels, which were enhanced by IL-1 beta treatment. A NOS inhibitor suppressed the increased polysulfide production and [Ca2+](i) increase in IL-1 beta-treated cells. These results suggest that IL-1 beta augments H2S-induced [Ca2+](i) increases via the conversion of H2S to polysulfides through NO synthesis, but not via changes in the activity and expression of target channels. Polysulfides may play an important role in the effects of H2S during inflammation.

Background: Chemical intolerance (CI) is a widespread occupational and public health problem characterized by symptoms that reportedly result from low-levels of chemical exposure. The mechanisms behind CI are unknown, however modifications of the chemical senses (rather than toxic processes) have been suggested as key components. The aim of this study was to investigate whether individuals with self-reported CI report more sensory irritation during masked acrolein exposure compared to controls without CI. Methods: Individuals with CI (n = 18) and controls without CI (n = 19) were exposed in an exposure chamber. Each participant took part in two exposure conditions – one with heptane (the masking compound), and one with heptane and acrolein at a dose below previously reported sensory irritation thresholds. The exposures lasted for 60 min. Symptoms and confidence ratings were measured continuously throughout the exposure as were measurements of electrodermal activity and self-reported tear-film break-up time. Participants were blind to exposure condition. Results: Individuals with CI, compared with controls reported greater sensory irritation in the eyes, nose and throat when exposed to acrolein masked with heptane. There was no difference during exposure to heptane. Conclusions: Masked exposure to acrolein at a concentration below the previously reported detection threshold is perceived as more irritating by individuals with CI compared with controls. The results indicate that there is altered trigeminal reactivity in those with CI compared to controls., Special issue.

A frequent empirical observation is that cold-induced counter-irritation may attenuate itch. The aim of this randomized, single-blinded, exploratory study was to evaluate the counter-irritation effects of cold-stimulation and topical application of transient receptor potential TRPA1/M8-agonists (trans-cinnamaldehyde/Lmenthol, respectively), on histamine-induced itch, wheals and neurogenic inflammation in 13 healthy volunteers. Histamine 1% was applied to the volar forearms using skin prick-test lancets. Recorded outcomeparameters were itch intensity, wheal reactions, and neurogenic inflammation (measured by laser-speckle perfusion-imaging). Homotopic thermal counter-irritation was performed with 6 temperatures, ranging from 4°C to 37°C, using a 3 × 3-cm thermal stimulator. Chemical “cold-like” counter-irritation was conducted with 40% L-menthol and 10% trans-cinnamaldehyde, while 5% doxepin was used as a positive antipruritic control/comparator. Cold counter-irritation stimuli from 4°C to 22°C inhibited itch in a stimulus-intensitydependent manner (p < 0.05) and, to a lesser extent, also wheal reactions and neurogenic inflammation. Chemical “cold-like” counter-irritation with both Lmenthol and trans-cinnamaldehyde had antipruritic efficacy similar to doxepin (p < 0.05). Cold-induced counter-irritation had an inhibitory effect on histaminergic itch, suggesting that agonists of cold transduction receptors could be of potential antipruritic value.

According to the International Association for the Study of Pain (IASP) pain is characterized as an “unpleasant sensory and emotional experience associated with actual or potential tissue damage”. The TRP super-family, compressing up to 28 isoforms in mammals, mediates a myriad of physiological and pathophysiological processes, pain among them. TRP channel might be constituted by similar or different TRP subunits, which will result in the formation of homomeric or heteromeric channels with distinct properties and functions. In this review we will discuss about the function of TRPs in pain, focusing on TRP channles that participate in the transduction of noxious sensation, especially TRPV1 and TRPA1, their expression in nociceptors and their sensitivity to a large number of physical and chemical stimuli.

A frequent empirical observation is that cold-induced counter-irritation may attenuate itch. The aim of this randomized, single-blinded, exploratory study was to evaluate the counter-irritation effects of cold-stimulation and topical application of transient receptor potential TRPA1/M8-agonists (trans-cinnamaldehyde/Lmenthol, respectively), on histamine-induced itch, wheals and neurogenic inflammation in 13 healthy volunteers. Histamine 1% was applied to the volar forearms using skin prick-test lancets. Recorded outcomeparameters were itch intensity, wheal reactions, and neurogenic inflammation (measured by laser-speckle perfusion-imaging). Homotopic thermal counter-irritation was performed with 6 temperatures, ranging from 4°C to 37°C, using a 3 × 3-cm thermal stimulator. Chemical “cold-like” counter-irritation was conducted with 40% L-menthol and 10% trans-cinnamaldehyde, while 5% doxepin was used as a positive antipruritic control/comparator. Cold counter-irritation stimuli from 4°C to 22°C inhibited itch in a stimulus-intensitydependent manner (p < 0.05) and, to a lesser extent, also wheal reactions and neurogenic inflammation. Chemical “cold-like” counter-irritation with both Lmenthol and trans-cinnamaldehyde had antipruritic efficacy similar to doxepin (p < 0.05). Cold-induced counter-irritation had an inhibitory effect on histaminergic itch, suggesting that agonists of cold transduction receptors could be of potential antipruritic value.

NADH:quinone oxidoreductase (Complex I) is the first enzyme of the respiratory chain and is involved in energy conservation generating an electro-chemical gradient across a membrane. The enzyme can be divided into a membrane spanning domain and a hydrophilic domain, which protrudes from the membrane. In the hydrophilic domain electrons from NADH oxidation are transported via a wire of iron-sulfur (Fe-S ) clusters to quinone, which is reduced. While the membrane domain is responsible for proton translocation to maintain the proton motive force, which is important for ATP synthesis. Large protein complexes like complex I have evolved from an assembly of discrete functional building blocks of which there are extant homologs. Two very different protein families, the Mrpantiporter and membrane bound [NiFe]-hydrogenases contain subunits which are homologous to complex I subunits. Part one of this work aimed to better understand the functional relationship between the related protein subunits of complex I, Mrp-antiporter and [NiFe]-hydrogenases. This knowledge will help us to elucidate the proton translocation pathway in complex I. First we compared the functional differentiation of complex I antiporter-like subunits with transporter subunits of the Hyc and Hyf hydrogenases and the 11-subunit complex I. For that we tested if the different subunits could rescue the growth of two salt sensitive Bacillus subtilis strain, which each lacked one of the two large Mrp-antiporter subunits (MrpA/MrpD). The 11-subunit complex I subunits could restore the growth in a similar manner as the complex I subunits, whereas the hydrogenase subunits could substitute equally well for the two MrpA and MrpD. We confirmed that 11-subunit complex I is a bona fide complex I. and that the hydrogenase subunits have intermediate forms of the antiporter-like subunits. Secondly we examined the functional relationship of the two homologous proteins MrpA from the Mrp-antiporter and NuoL from complex I. We

Tutkielmassa selvitettiin mantelitumakkeen osallisuutta neuropaattisen kivun säätelyssä sekä mantelitumakkeen patologisten muutosten aiheuttamaa laskevien kipua muuntelevien ratojen herkistymistä. Tutkielman kokeellisessa osassa selvitettiin aiheuttaako neuropaattiseen kiputilaan johtava hermovaurio mantelitumakkeessa oksidatiivista stressiä sekä TRPA1-reseptorin ilmentymistä, mikä johtaisi sentraaliseen sensitisaatioon. Neuropaattisen kivun mallina käytettiin SNI-tekniikalla (spared nerve injury) operoituja rottia, joiden mantelitumakkeeseen annosteltiin TRPA1-antagonistia. Lääkeaineen vaikutusta ja kipukäyttäytymisen muutosta tutkittiin ärsyttämällä koe-eläinten operoitua raajaa monofilamenteilla ja mittaamalla PWT-käyttäytymismallin (paw withdrawal threshold) mukaisia vasteita. Oikeaan mantelitumakkeeseen annosteltu TRPA1-antagonisti vähensi mekaanista hypersensitiviteettiä SNI-rotilla, mutta ei tuottanut muutosta terveillä yksilöillä. Tulos viittaa siihen että neuropaattinen kiputila aiheuttaa mantelitumakkeessa TRPA1:n ilmentymistä, mikä osallistuu mekaanisen hypersensitiviteetin muodostumiseen.

In der vorliegenden Arbeit wurde anhand von zwei elektrophysiologischen Tiermodellen die Rolle des TRPA1-Rezeptorkanals sowie die Wirkung von Metaboliten des Stickstoffmonoxidstoffwechsels bei der meningealen Nozizeption als Grundlagen der Kopfschmerzentstehung untersucht. In einem in vivo Präparat an der narkotisierten Ratte wurde die Aktivität von sekundären Neuronen im spinalen Trigeminuskern mit mechanorezeptivem afferenten Zustrom aus den Meningen untersucht. Die TRPA1-Rezeptoragonisten Nitroxyl (Angeli´s Salt), Acrolein und Senföl wurden dabei peripher auf die freigelegte Dura mater encephali als auch zentral auf die Medulla oblongata appliziert. Acrolein wurde zudem direkt in das Ganglion trigeminale injiziert. Die Ergebnisse zeigten generell keine signifikanten Änderungen der Spontanaktivität und der mechanisch evozierten Aktivität. Lediglich bei topischer Applikation von 300 µM Acrolein auf die Dura mater oder den Hirnstamm zeigte sich tendenziell eine Zunahme der Spontanaktivität. Die gleiche Behandlung der Dura mater bewirkte einen signifikanten Anstieg des meningealen Blutflusses, welcher sowohl durch den TRPA1-Antagonisten HC030031 als auch durch den CGRP-Rezeptorantagonisten CGRP8-37 gehemmt wurde. Um die lokale Wirkung von Nitroxyl auf die primären Afferenzen in der Dura mater zu untersuchen, wurden in vitro Einzelfaserableitungen an Halbschädelpräparaten der Maus (C57Bl6) durchgeführt. Zur Überprüfung der TRPA1-Spezifität wurden die Experimente an TRPA1-Knockout-Mäusen und an TRPA1/TRPV1-Doppelknockout-Mäusen wiederholt. Die Ergebnisse zeigten einen kurzen heftigen Anstieg der neuronalen Aktivität gefolgt von einer Desensibilisierung der afferenten Fasern. Die Aktivierung schien jedoch nicht TRPA1-abhängig zu sein. Ein Vergleich der Intensität des Aktivitätsanstieges zwischen den Genotypen zeigte, dass dieser bei der Doppelknockoutgruppe statistisch signifikant stärker ausfiel als bei der Wildtypgruppe. Insgesamt bestätigen die Ergebnisse der vorlieg, The aim of the present study was to examine the role of TRPA1 receptor channels and nitric oxide metabolites in meningeal nociception underlying the generation of headaches in two electrophysiological animal models. In vivo the activity of spinal trigeminal neurons with afferent input from the dura mater was recorded in anesthetized rats. The TRPA1 agonists nitroxyl (Angeli´s Salt), acrolein and mustard oil were applied to the exposed dura mater as well as to the medulla oblongata. Acrolein was also injected directly into the trigeminal ganglion. The results showed generally no significant changes in spontaneous activity or in mechanically evoked activity. Only after topical application of 300 µM acrolein onto the dura mater or the brain stem the spontaneous activity tended to increase. The same treatment of the dura mater caused a significant increase in meningeal blood flow, which was inhibited by the TRPA1 antagonist HC030031 or the CGRP receptor antagonist CGRP8-37. To investigate the local effects of nitroxyl on the primary meningeal afferents, single fiber recordings were performed using half-skull preparations of mice (C57Bl6) in vitro. To verify if the observed effects depend on TRPA1 the experiments were repeated with TRPA1 knockout mice and TRPA1/TRPV1 double-knockout mice. The results showed a short-lasting vigorous increase in neuronal activity followed by a desensitization of the afferent fibers. However, the activation was not TRPA1-dependent. The increase in activity was statistically stronger for the TRPA1/TRPV1 double-knockout group compared to the wild-type group. Overall, the results of this study do not confirm the assumption that activation of trigeminal TRPA1 receptor channels triggers the generation of headaches or contributes to its aggravation. The activation of primary meningeal afferents by TRPA1 agonist causing increased meningeal blood flow does not significantly increase the activity of trigeminal second order neurons in vivo. Instead

Drosophila third-instar larvae exhibit changes in their behavioral responses to gravity and food as they transition from feeding to wandering stages. Using a thermal gradient encompassing the comfortable range (18°C to 28°C), we found that third-instar larvae exhibit a dramatic shift in thermal preference. Early third-instar larvae prefer 24°C, which switches to increasingly stronger biases for 18°C-19°C in mid- and late-third-instar larvae. Mutations eliminating either of two rhodopsins, Rh5 and Rh6, wiped out these age-dependent changes in thermal preference. In larvae, Rh5 and Rh6 are thought to function exclusively in the light-sensing Bolwig organ. However, the Bolwig organ was dispensable for the thermal preference. Rather, Rh5 and Rh6 were required in trpA1-expressing neurons in the brain, ventral nerve cord, and body wall. Because Rh1 contributes to thermal selection in the comfortable range during the early to mid-third-instar stage, fine thermal discrimination depends on multiple rhodopsins.

Drosophila third-instar larvae exhibit changes in their behavioral responses to gravity and food as they transition from feeding to wandering stages. Using a thermal gradient encompassing the comfortable range (18°C to 28°C), we found that third-instar larvae exhibit a dramatic shift in thermal preference. Early third-instar larvae prefer 24°C, which switches to increasingly stronger biases for 18°C-19°C in mid- and late-third-instar larvae. Mutations eliminating either of two rhodopsins, Rh5 and Rh6, wiped out these age-dependent changes in thermal preference. In larvae, Rh5 and Rh6 are thought to function exclusively in the light-sensing Bolwig organ. However, the Bolwig organ was dispensable for the thermal preference. Rather, Rh5 and Rh6 were required in trpA1-expressing neurons in the brain, ventral nerve cord, and body wall. Because Rh1 contributes to thermal selection in the comfortable range during the early to mid-third-instar stage, fine thermal discrimination depends on multiple rhodopsins.

The ability to sense and accommodate to an ever-changing environment is crucial for the survival of living organisms. Transient Receptor Potential (TRP) ion channels comprise a large superfamily of cation conducting membrane proteins that function as molecular sensors in diverse sensory processes including perception of light, taste, smell, sound, touch and temperature. The TRP Ankyrin 1 (TRPA1) ion channel is a unique member of the mammalian TRP superfamily, containing a large N-terminal ankyrin repeat domain (ARD) which constitutes half of the entire protein. TRPA1 responds to a variety of unrelated noxious stimuli such as chemicals, temperature and mechanical stress. It seems convincing that TRPA1 acts as a noxious chemical sensor and also plays a role in the detection of warm temperatures in non-mammalian species. The role of mammalian TRPA1 as a cold sensor is, however, controversial. The mammalian TRPA1 has been implicated in acute and inflammatory pain conditions and has been proposed as an important target for analgesics. If TRPA1 ion channels are intrinsically chemo-, thermo- and mechanosensitive proteins, regardless of species, remains to be shown. The overall of aim of this thesis was to investigate possible inherent thermo- and chemosensitive properties of human TRPA1 (hTRPA1) and the malaria mosquito Anopheles gambiae TRPA1 (AgTRPA1), and the role of the N-terminal ARD, containing suggested key temperature elements as well as cysteines targeted by thiol reactive oxidants and electrophiles known to activate TRPA1. Difficulties to express and purify large amounts of proteins have hampered structural and functional studies of TRPs but were here overcome by heterologous expression in the yeast Pichia pastoris. Single-channel electrophysiological recordings of purified hTRPA1 reconstituted into artificial lipid bilayers indicated that cold- and chemosensitivity are inherent channel properties recognized by structures outside the N-terminal ARD. Surprisingly

Itch is defined as an unpleasant sensation that evokes a desire to scratch. In contrast to acute itch that is transient, chronic itch is a persistent, debilitating condition for which there are few treatment options. Chronic itch accompanies a number of skin diseases and systemic conditions, as well as a variety of neurological disorders. However, little is known about the molecules and cell types that mediate acute or chronic itch in primary sensory neurons and skin.We have established an essential role for the ion channel, TRPA1, in multiple forms of both acute and chronic itch. We have shown that TRPA1 is required for neuronal activation and itch behaviors in mice downstream of acute exogenous and endogenous itch compounds. Similarly, TRPA1 is required for itch behavior and itch-evoked expressional changes in both sensory neurons and skin in a mouse model of dry skin. We have also identified a novel itch-causing compound: Thymic Stromal Lymphopoietin (TSLP). Numerous studies suggest that the epithelially-derived cytokine TSLP acts as a master switch that triggers both the initiation and maintenance of the chronic itch disease atopic dermatitis. Our work demonstrates that TSLP activates sensory neurons directly and leads to acute itch behaviors. TRPA1 is required for both TSLP-evoked neuronal activation and itch behaviors. Taken together, our work shows that TRPA1 is a master regulator of itch signaling.

Itch is defined as an unpleasant sensation that evokes a desire to scratch. In contrast to acute itch that is transient, chronic itch is a persistent, debilitating condition for which there are few treatment options. Chronic itch accompanies a number of skin diseases and systemic conditions, as well as a variety of neurological disorders. However, little is known about the molecules and cell types that mediate acute or chronic itch in primary sensory neurons and skin.We have established an essential role for the ion channel, TRPA1, in multiple forms of both acute and chronic itch. We have shown that TRPA1 is required for neuronal activation and itch behaviors in mice downstream of acute exogenous and endogenous itch compounds. Similarly, TRPA1 is required for itch behavior and itch-evoked expressional changes in both sensory neurons and skin in a mouse model of dry skin. We have also identified a novel itch-causing compound: Thymic Stromal Lymphopoietin (TSLP). Numerous studies suggest that the epithelially-derived cytokine TSLP acts as a master switch that triggers both the initiation and maintenance of the chronic itch disease atopic dermatitis. Our work demonstrates that TSLP activates sensory neurons directly and leads to acute itch behaviors. TRPA1 is required for both TSLP-evoked neuronal activation and itch behaviors. Taken together, our work shows that TRPA1 is a master regulator of itch signaling.

IntroductionThe present paper summarizes research using animal models to investigate the roles of thermosensitive transient receptor potential (TRP) channels in somatosensory functions including touch, temperature and pain. We present new data assessing the effects of eugenol and carvacrol, agonists of the warmth-sensitive TRPV3, on thermal, mechanical and pain sensitivity in rats.MethodsThermal sensitivity was assessed using a thermal preference test, which measured the amount of time the animal occupied one of two adjacent thermoelectric plates set at different temperatures. Pain sensitivity was assessed as an increase in latency of hindpaw withdrawal away from a noxious thermal stimulus directed to the plantar hindpaw (Hargreaves test). Mechanical sensitivity was assessed by measuring the force exerted by an electronic von Frey filament pressed against the plantar surface that elicited withdrawal.ResultsTopical application of eugenol and carvacrol did not significantly affect thermal preference, although there was a trend toward avoidance of the hotter surface in a 30 vs. 45°C preference test for rats treated with 1 or 10% eugenol and carvacrol. Both eugenol and carvacrol induced a concentration-dependent increase in thermal withdrawal latency (analgesia), with no significant effect on mechanosensitivity.ConclusionsThe analgesic effect of eugenol and carvacrol is consistent with previous studies. The tendency for these chemicals to increase the avoidance of warmer temperatures suggests a possible role for TRPV3 in warmth detection, also consistent with previous studies. Additional roles of other thermosensitive TRP channels (TRPM8 TRPV1, TRPV2, TRPV4, TRPM3, TRPM8, TRPA1, TRPC5) in touch, temperature and pain are reviewed.

Hydrogen sulfide (H2S) reportedly acts as a gasotransmitter because it mediates various cellular responses through several ion channels including ATP-sensitive K+ (K-ATP) channels and transient receptor potential (TRP) A1 channels. H2S can activate both K-ATP, and TRPA1 channels at a similar concentration range. In a single cell expressing both channels, however, it remains unknown what happens when both channels are simultaneously activated by H2S. In this study, we examined the effects of H2S on RIN14B cells that express both K-ATP and TRPA1 channels. RIN14B cells showed several intracellular Ca2+ concentration ([Ca2+](i)) responses to NaHS (300 mu M), an H2S donor, i.e., inhibition of spontaneous Ca2+ oscillations (37%), inhibition followed by [Ca2+](i) increase (24%), and a rapid increase in [Ca2+](i) (25%). K-ATP channel blockers, glibenclamide or tolbutamide, abolished any inhibitory effects of NaHS and enhanced NaHS-mediated [Ca2+](i) increases, which were inhibited by extracellular Ca2+ removal, HC030031 (a TRPA1 antagonist), and disulfide bond-reducing agents. NaHS induced 5-hydroxytryptamine (5-HT) release from RIN14B cells, which was also inhibited by TRPA1 antagonists. These results indicate that H2S has both inhibitory and excitatory effects by opening K-ATP and TRPA1 channels, respectively, in RIN14B cells, suggesting potential bidirectional modulation of secretory functions. (C) 2015 Elsevier B.V. All rights reserved.