Nociceptive transient receptor potential ankyrin 1 (TRPA1) in sensory neurons are targets of the antifungal drug econazole.

BACKGROUND: Econazole is a widely used imidazole derivative antifungal for treating skin infections. The molecular targets for its frequent adverse effects of skin irritation symptoms, such as pruritus, burning sensation, and pain, have not been clarified. Transient receptor potential (TRP) channels, non-selective cation channels, are mainly expressed in peripheral sensory neurons and serve as sensors for various irritants. METHODS: We investigated the effect of econazole on TRP channel activation by measuring intracellular calcium concentration ([Ca2+]i) through fluorescent ratio imaging in mouse dorsal root ganglion (DRG) neurons isolated from wild-type, TRPA1(-/-) and TRPV1(-/-) mice, as well as in heterologously TRP channel-expressed cells. A cheek injection model was employed to assess econazole-induced itch and pain in vivo. RESULTS: Econazole evoked an increase in [Ca2+]i, which was abolished by the removal of extracellular Ca2+ in mouse DRG neurons. The [Ca2+]i responses to econazole were suppressed by a TRPA1 blocker but not by a TRPV1 blocker. Attenuation of the econazole-induced [Ca2+]i responses was observed in the TRPA1(-/-) mouse DRG neurons but was not significant in the TRPV1(-/-) neurons. Econazole increased the [Ca2+]i in HEK293 cells expressing TRPA1 (TRPA1-HEK) but not in those expressing TRPV1, although at higher concentrations, it induced Ca2+ mobilization from intracellular stores in untransfected naïve HEK293 cells. Miconazole, which is a structural analog of econazole, also increased the [Ca2+]i in mouse DRG neurons and TRPA1-HEK, and its nonspecific action was larger than econazole. Fluconazole, a triazole drug failed to activate TRPA1 and TRPV1 in mouse DRG neurons and TRPA1-HEK. Econazole induced itch and pain in wild-type mice, with reduced responses in TRPA1(-/-) mice. CONCLUSIONS: These findings suggested that the imidazole derivatives econazole and miconazole may induce skin irritation by activating nociceptive TRPA1 in the sensory neurons. Suppression of TRPA1 activation may mitigate the adverse effects of econazole.

Linalyl acetate exerts analgesic effects by inhibiting nociceptive TRPA1 in mice.

Clary sage essential oil (CSEO) is utilized in perfumery, aromatherapy, and skincare. Linalyl acetate (LA), a primary component of CSEO, possesses sedative, anxiolytic, and analgesic properties. However, the mechanism of its analgesic action is not clearly understood. Transient receptor potential ankyrin 1 (TRPA1) channel, a non-selective cation channel, is mainly expressed in sensory neurons and serves as a sensor of various irritants. In this study, we investigated the effects of LA on TRPA1 channel using heterologous expression system and isolated sensory neurons. To detect channel activity, we employed Ca2+ imaging and the whole-cell patch-clamp technique. The analgesic action of LA was measured in a pain-related behavioral mouse model. In cells that heterologously expressed TRPA1, LA diminished [Ca2+]i and current responses to allylisothiocyanate (AITC) and carvacrol: exogenous TRPA1 agonists, and the inhibitory effects were more pronounced for the former than for the latter. Moreover, LA suppressed [Ca2+] i and current responses to PGJ2: an endogenous TRPA1 agonist. Similar inhibitory actions were observed in native TRPA1 channels expressed in mouse sensory neurons. Furthermore, LA diminished PGJ2-induced nociceptive behaviors in mice. These findings suggest that analgesic effects of LA exert through inhibition of nociceptive TRPA1, making it a potential candidate for novel analgesic development.

Strobilurin X acts as an anticancer drug by inhibiting protein synthesis and suppressing mitochondrial respiratory chain activity.

PURPOSE: Strobilurins act as antifungal agents by inhibiting the mitochondrial respiratory chain. The cytotoxic activity of strobilurins, focusing on its anticancer activities, has been reported. However, the mechanisms involved in these activities remain unclear. METHODS: The cytotoxic effects of strobilurin X isolated from the mycelium of Mucidula. venosolamellata were examined in human cancer cell lines (A549 and HeLa) and normal fibroblasts (WI-38). RESULTS: Strobilurin X significantly decreased the viability of A549 and HeLa cells compared to that in the WI-38 cells after 48 h of exposure. The EC50 values for cytotoxicity in the A549, HeLa, and WI-38 cells were 3.4, 5.4, and 16.8 µg/mL, respectively. Strobilurin X inhibited the mitochondrial respiratory chain and enhanced the release of lactate in the A549 cells. The IC50 value of strobilurin X against the mitochondrial respiratory chain complex III activity was 139.8 ng/mL. The cytotoxicity induced by strobilurin X was not completely rescued after adding uridine, methyl pyruvate, or N-acetyl cysteine. Furthermore, pharmacological approaches demonstrated that strobilurin X failed to modulate the mitogen-activated protein kinase family and phosphoinositide 3-kinase-Akt pathways; alternatively, it suppressed protein synthesis independent of uridine. CONCLUSION: Strobilurin X induced cytotoxicity by blocking the mitochondrial respiratory chain and suppressing protein synthesis. These findings may aid in the development of novel anticancer drugs using strobilurins.

Involvement of interaction of Cav3.2 and nociceptive TRPA1 in pathological pain transmission.

T-type Ca2+ channels and TRPA1 expressed in sensory neurons are involved in pain. We previously demonstrated a functional interaction of these channels under physiological conditions. Here we investigated the possible involvement of these channels in inflammatory pain condition. We also evaluated the relationship of these channels endogenously expressed in RIN-14B, a rat pancreatic islet tumor cell line. In dorsal root ganglion (DRG) neurons innervated inflammatory side, [Ca2+]i increases induced by 15 mM KCl (15K) were enhanced in neurons responded to AITC. This enhancement was not observed in genetically TRPA1-deficient neurons. The T-type and AITC-induced currents were larger in neurons of the inflammatory side than in those of the control one. In DRGs of the inflammatory side, the protein expression of Cav3.2, but not TRPA1, was increased. In RIN-14B, 15K-induced [Ca2+]i increases were decreased by blockers of T-type Ca2+ channel and TRPA1, and by TRPA1-silencing. Immunoprecipitation suggested the coexistent of these channels in sensory neurons and RIN-14B. In mice with inflammation, mechanical hypersensitivity was suppressed by blockers of both channels. These data suggest that the interaction of Cav3.2 with TRPA1 in sensory neurons is enhanced via the augmentation of the activities of both channels under inflammatory conditions, indicating that both channels are therapeutic targets for inflammatory pain.

Inhibition of melanoma cell proliferation by strobilurins isolated from mushrooms and their synthetic analogues.

Strobilurins A and X, isolated from Mucidula venosolamellata culture extracts, demonstrated potent inhibition of human melanoma G-361 cell proliferation. Strobilurin X exhibited milder inhibitory effects on human fibroblast cells (NB1RGB) compared to strobilurin A. Additional strobilurin-related compounds were isolated from the other mushroom species. Oudemansins A and B displayed weaker activities on G-361 cells than strobilurins A and B, respectively, emphasizing the importance of a conjugated double-bond structure. Among isolated compounds, strobilurin G showed the lowest IC50 value for G-361 cells. Additional strobilurins bearing various substituents on the benzene ring were synthesized. Synthetic intermediates lacking the methyl ß-methoxyacrylate group and a strobilurin analogue bearing modified ß-methoxyacrylate moiety showed almost no inhibitory activity against G-361 cells. The introduction of long or bulky substituents at the 4' position of the benzene ring of strobilurins enhanced the activity and selectivity, suggesting differential recognition of the benzene ring by G-361 and NB1RGB cells.

Possible role of transient receptor potential melastatin 4 channels in adrenergic contractions in mouse prostate smooth muscles.

Transient receptor potential melastatin 4 (TRPM4) cation channels are expressed in prostate glands. However, the precise role of these channels in prostate contractility remains unclear. In this study, we examined whether TRPM4 channels were involved in adrenergic contractions in the mouse prostate gland. Adrenergic contractile responses elicited by noradrenaline or electrical field stimulation of the sympathetic nerve were isometrically recorded, and the effects of 9-phenanthrol, a specific TRPM4 channel inhibitor, on those contractile responses were investigated in mouse ventral prostate preparations. 9-phenanthrol (10 or 30 µM) inhibited noradrenaline- and sympathetic nerve-evoked contractions in a concentration-dependent manner. A similar inhibitory effect was observed with another TRPM4 channel inhibitor, 4-chloro-2-(2-(naphthalene-1-yloxy) acetamido) benzoic acid (NBA; 10 µM). Inhibition by 9-phenanthrol and NBA were much greater at lower noradrenaline concentrations and lower stimulus frequencies than those of higher concentrations or frequencies. However, 9-phenanthrol did not inhibit the noradrenaline-induced contractile response when the membrane potential was decreased to approximately 0 mV in the 140 mM K+ medium. Moreover, 9-phenanthrol does not affect noradrenaline-induced increases in spontaneous contractions of cardiac atrial preparation. This agent inhibited noradrenaline-induced contractions in the posterior aorta preparation. However, the inhibitory effect was significantly weaker than that observed in the prostate gland. These results suggest that TRPM4 channels are involved in adrenergic contractions in the mouse prostate gland, possibly through membrane depolarization by their opening; therefore, they might be potential candidates for treating benign prostatic hyperplasia.

Inhibitory effects of linalool, an essential oil component of lavender, on nociceptive TRPA1 and voltage-gated Ca2+ channels in mouse sensory neurons.

Linalool, an essential oil component of lavender is commonly used in fragrances. It is known that linalool has anxiolytic, sedative, and analgesic actions. However, the mechanism of its analgesic action has not yet been fully clarified. Pain signals elicited by the activation of nociceptors on peripheral neurons are transmitted to the central nervous system. In the present study, we investigated the effects of linalool on transient receptor potential (TRP) channels and voltage-gated channels, both of which are important for pain signaling via nociceptors in somatosensory neurons. For detection of channel activity, the intracellular Ca2+ concentration ([Ca2+]i) was measured using a Ca2+-imaging system, and membrane currents were recorded using the whole-cell patch-clamp technique. Analgesic actions were also examined in vivo. In mouse sensory neurons linalool at concentrations that did not induce [Ca2+]i increases did not affect [Ca2+]i responses to capsaicin and acids, TRPV1 agonists, but suppressed those induced by allyl isothiocyanate (AITC) and carvacrol, TRPA1 agonists. Similar inhibitory effects of linalool were observed in cells that heterologously expressed TRPA1. Linalool attenuated the [Ca2+]i increases induced by KCl and voltage-gated Ca2+ currents but only slightly suppressed voltage-gated Na＋currents in mouse sensory neurons. Linalool diminished TRPA1-mediated nociceptive behaviors. The present data suggest that linalool exerts an analgesic action via the suppression of nociceptive TRPA1 and voltage-gated Ca2+ channels.

Evolutionary Tuning of Transient Receptor Potential Ankyrin 1 Underlies the Variation in Heat Avoidance Behaviors among Frog Species Inhabiting Diverse Thermal Niches.

Environmental temperature is a critical factor for all forms of life, and thermal tolerance defines the habitats utilized by a species. Moreover, the evolutionary tuning of thermal perception can also play a key role in habitat selection. Yet, the relative importance of thermal tolerance and perception in environmental adaptation remains poorly understood. Thermal conditions experienced by anuran tadpoles differ among species due to the variation in breeding seasons and water environments selected by parental frogs. In the present study, heat tolerance and avoidance temperatures were compared in tadpoles from five anuran species that spatially and temporally inhabit different thermal niches. These two parameters were positively correlated with each other and were consistent with the thermal conditions of habitats. The species difference in avoidance temperature was 2.6 times larger than that in heat tolerance, suggesting the importance of heat avoidance responses in habitat selection. In addition, the avoidance temperature increased after warm acclimation, especially in the species frequently exposed to heat in their habitats. Characterization of the heat-sensing transient receptor potential ankyrin 1 (TRPA1) ion channel revealed an amphibian-specific alternatively spliced variant containing a single valine insertion relative to the canonical alternative spliced variant of TRPA1, and this novel variant altered the response to thermal stimuli. The two alternatively spliced variants of TRPA1 exhibited different thermal responses in a species-specific manner, which are likely to be associated with a difference in avoidance temperatures among species. Together, our findings suggest that the functional change in TRPA1 plays a crucial role in thermal adaptation processes.

Identification of molecular targets for toxic action by persulfate, an industrial sulfur compound.

Persulfate salts are broadly used as industrial chemicals and exposure to them causes occupational asthma, occupational rhinitis and contact dermatitis. However, the mechanisms underlying these toxic actions are not fully elucidated. Transient receptor potential (TRP) vanilloid 1 (V1), ankyrin 1 (A1) and melastatin 8 (M8) are non-selective cation channels preferentially expressing sensory neurons. These channels are known to be involved in respiratory and skin diseases. In the present study, we investigated the effects of sodium persulfate on these TRP channels. In wild-type mouse sensory neurons, persulfate evoked [Ca2+]i increases that were inhibited by removal of extracellular Ca2+ or blockers of TRPA1 but not by those of TRPV1 and TRPM8. Persulfate failed to evoke [Ca2+]i responses in neurons from TRPA1(-/-) mice, but did evoke them in neurons from TRPV1(-/-) mice. In HEK 293 cells expressing mouse TRPA1 (mTRPA1-HEK), persulfate induced [Ca2+]i increases. Moreover, in HEK 293 cells expressing mouse TRPV1 (mTRPV1-HEK), a high concentration of persulfate also evoked [Ca2+]i increases. Similar [Ca2+]i responses were observed in HEK 293 cells expressing human TRPA1 and human TRPV1. Current responses were also elicited by persulfate in mTRPA1- and mTRPV1-HEK. Analysis using mutated channels revealed that persulfate acted on electrophilic agonist-sensitive cysteine residues of TRPA1, and it indirectly activated TRPV1 due to the external acidification, because of the disappearance of [Ca2+]i responses in acid-insensitive mTRPV1 mutant. These results demonstrate that persulfate activates nociceptive TRPA1 and TRPV1 channels. It is suggested that activation of these nociceptive channels may be involved in respiratory and skin injuries caused by exposure to this industrial sulfur compound. Thus, selective TRPA1 and TRPV1 channel blockers may be effective to remedy persulfate-induced toxic actions.

Involvement of nociceptive transient receptor potential channels in repellent action of pulegone.

Pulegone, one of avian repellents, is used to prevent the economic loss caused by birds. Chemical repellents often evoke unpleasant sensations and sensory irritation resulting in avoidance under some circumstances. It is recognized that some TRP channels expressing sensory neurons are related to nociception. Here we determined the molecular mechanisms of the repellent action of pulegone using isolated chicken sensory neurons and heterologous expression system. Pulegone increased the intracellular Ca2+ concentration ([Ca2+]i) in chicken sensory neurons. There were two types of neurons exhibiting different sensitivity to pulegone. One was responded to it at low concentrations and the other at high concentrations. Pharmacological analyses revealed that the former was predominantly mediated by TRP melastatin 8 (TRPM8), and the latter by both TRP ankyrin 1 (TRPA1) and TRPM8. An activation of both channels by pulegone was also determined using heterologously expression system. At high concentrations, pulegone suppressed chicken TRPM8 but not chicken TRPA1. The intraplantar injection of pulegone in chicks caused pain-related behaviors that were attenuated by TRPA1 antagonist. These results indicate that pulegone stimulates both TRPM8 and TRPA1 channel in chicken sensory neurons and suppresses the former but not the latter at high concentrations. Together, these data suggest that the molecular target for the repellent action of pulegone in avian species is nociceptive TRPA1.

Possible involvement of transient receptor potential ankyrin 1 in Ca2+ signaling via T-type Ca2+ channel in mouse sensory neurons.

T-type Ca2+ channels and TRPA1 are expressed in sensory neurons and both are associated with pain transmission, but their functional interaction is unclear. Here we demonstrate that pharmacological evidence of the functional relation between T-type Ca2+ channels and TRPA1 in mouse sensory neurons. Low concentration of KCl at 15 mM (15K) evoked increases of intracellular Ca2+ concentration ([Ca2+ ]i ), which were suppressed by selective T-type Ca2+ channel blockers. RT-PCR showed that mouse sensory neurons expressed all subtypes of T-type Ca2+ channel. The magnitude of 15K-induced [Ca2+ ]i increase was significantly larger in neurons sensitive to allylisothiocyanate (AITC, a TRPA1 agonist) than in those insensitive to it, and in TRPA1-/- mouse sensory neurons. TRPA1 blockers diminished the [Ca2+ ]i responses to 15K in neurons sensitive to AITC, but failed to inhibit 40 mM KCl-induced [Ca2+ ]i increases even in AITC-sensitive neurons. TRPV1 blockers did not inhibit the 15K-induced [Ca2+ ]i increase regardless of the sensitivity to capsaicin. [Ca2+ ]i responses to TRPA1 agonist were enhanced by co-application with 15K. These pharmacological data suggest the possibility of functional interaction between T-type Ca2+ channels and TRPA1 in sensory neurons. Since TRPA1 channel is activated by intracellular Ca2+ , we hypothesize that Ca2+ entered via T-type Ca2+ channel activation may further stimulate TRPA1, resulting in an enhancement of nociceptive signaling. Thus, T-type Ca2+ channel may be a potential target for TRPA1-related pain.

Membrane translocation of transient receptor potential ankyrin 1 induced by inflammatory cytokines in lung cancer cells.

Transient receptor potential ankyrin 1 (TRPA1) is known as one of the nociceptors expressed in sensory neurons. It also plays a role in non-neural cells in inflammatory sites. However, the regulatory mechanisms for the reactivity of TRPA1 in these cells under inflammatory conditions are not clear. To clarify these mechanisms, we examined the effects of inflammatory cytokines (interleukin [IL]-1α, IL-1ß and tumor necrosis factor α [TNFα]) on TRPA1 reactivity and expression in the endogenously TRPA1-expressing lung tumor cell line A549. Treatment with IL-1α, but not IL-1ß or TNFα, increased the number of cells responding to allyl isothiocyanate, a TRPA1 agonist, in a dose- and time-dependent manner. The IL-1α-induced increase of TRPA1 responsiveness was inhibited by an extracellular-regulated kinase (Erk) inhibitor (PD98059) but not by inhibitors of c-Jun kinase, p38 mitogen-activated protein kinase or phosphatidylinositol-3 kinase. Phosphorylation of Erk gradually increased at 24 h after its transient induction in cells treated with IL-1α. IL-1α increased the TRPA1 levels on biotinylated cell surface proteins. These results suggest that IL-1α enhances the translocation of TRPA1 to the plasma membrane via the activation of Erk in A549. TRPA1 may have a pathophysiological role in non-neural lung cells under inflammatory conditions.

CAPS1 RNA Editing Promotes Dense Core Vesicle Exocytosis.

Calcium-dependent activator protein for secretion 1 (CAPS1) plays a distinct role in the priming step of dense core vesicle (DCV) exocytosis. CAPS1 pre-mRNA is known to undergo adenosine-to-inosine RNA editing in its coding region, which results in a glutamate-to-glycine conversion at a site in its C-terminal region. However, the physiological significance of CAPS1 RNA editing remains elusive. Here, we created mutant mice in which edited CAPS1 was solely expressed. These mice were lean due to increased energy expenditure caused by physical hyperactivity. Electrophysiological and biochemical analyses demonstrated that the exocytosis of DCVs was upregulated in the chromaffin cells and neurons of these mice. Furthermore, we showed that edited CAPS1 bound preferentially to the activated form of syntaxin-1A, a component of the exocytotic fusion complex. These findings suggest that RNA editing regulates DCV exocytosis in vivo, affecting physical activity.

Carbonyl stress-induced 5-hydroxytriptamine secretion from RIN-14B, rat pancreatic islet tumor cells, via the activation of transient receptor potential ankyrin 1.

Methylglyoxal (MG), a highly reactive dicarbonyl substance, is known as an endogenous carbonyl stress-inducing substance related to various disease states. Irritable bowel syndrome (IBS) is one of the most frequently encountered gastrointestinal disorders and MG is considered to be its causal substance. An increased serum 5-hydroxytryptamine (5-HT) level is related to IBS symptoms and the majority of 5-HT originates from enterochromaffin (EC) cells in the intestine. Here we examine the mechanisms of MG-induced 5-HT secretion using RIN-14B cells derived from a rat pancreatic islet tumor since these cells are used as a model for EC cells. MG increased the intracellular Ca(2+) concentration ([Ca(2+)]i) and 5-HT secretion, both of which were inhibited by the removal of extracellular Ca(2+) and specific transient receptor potential ankyrin 1 (TRPA1) antagonists. MG elicited an inward current under voltage-clamped conditions. Prior application of MG evoked reciprocal suppression of subsequent [Ca(2+)]i responses to allylisothiocyanate, a TRPA1 agonist, and vice versa. Glyoxal, an analog of MG, also evoked [Ca(2+)]i and secretory responses but its potency was much lower than that of MG. The present results suggest that MG promotes 5-HT secretion through the activation of TRPA1 in RIN-14B cells. These results may indicate that TRPA1 is a promising target for the treatment of IBS and that the RIN-14B cell line is a useful model for investigation of IBS.

Evolution of Heat Sensors Drove Shifts in Thermosensation between Xenopus Species Adapted to Different Thermal Niches.

Temperature is one of the most critical environmental factors affecting survival, and thus species that inhabit different thermal niches have evolved thermal sensitivities suitable for their respective habitats. During the process of shifting thermal niches, various types of genes expressed in diverse tissues, including those of the peripheral to central nervous systems, are potentially involved in the evolutionary changes in thermosensation. To elucidate the molecular mechanisms behind the evolution of thermosensation, thermal responses were compared between two species of clawed frogs (Xenopus laevis and Xenopus tropicalis) adapted to different thermal environments. X. laevis was much more sensitive to heat stimulation than X. tropicalis at the behavioral and neural levels. The activity and sensitivity of the heat-sensing TRPA1 channel were higher in X. laevis compared with those of X. tropicalis The thermal responses of another heat-sensing channel, TRPV1, also differed between the two Xenopus species. The species differences in Xenopus TRPV1 heat responses were largely determined by three amino acid substitutions located in the first three ankyrin repeat domains, known to be involved in the regulation of rat TRPV1 activity. In addition, Xenopus TRPV1 exhibited drastic species differences in sensitivity to capsaicin, contained in chili peppers, between the two Xenopus species. Another single amino acid substitution within Xenopus TRPV1 is responsible for this species difference, which likely alters the neural and behavioral responses to capsaicin. These combined subtle amino acid substitutions in peripheral thermal sensors potentially serve as a driving force for the evolution of thermal and chemical sensation.

Endogenous 5-HT outflow from chicken aorta by 5-HT uptake inhibitors and amphetamine derivatives.

Chemoreceptor cells aggregating in clusters in the chicken thoracic aorta contain 5-hydroxytryptamine (5-HT) and have voltage-dependent ion channels and nicotinic acetylcholine receptors, which are characteristics typically associated with neurons. The aim of the present study was to investigate the effects of 5-HT uptake inhibitors, fluvoxamine, fluoxetine and clomipramine (CLM), and amphetamine derivatives, p-chloroamphetamine (PCA) and methamphetamine (MET), on endogenous 5-HT outflow from the isolated chick thoracic aorta in vitro. 5-HT was measured by using a HPLC system with electrochemical detection. The amphetamine derivatives and 5-HT uptake inhibitors caused concentration-dependent increases in endogenous 5-HT outflow. PCA was about ten times more effective in eliciting 5-HT outflow than MET. The 5-HT uptake inhibitors examined had similar potency for 5-HT outflow. PCA and CLM increased 5-HT outflow in a temperature-dependent manner. The outflow of 5-HT induced by PCA or 5-HT uptake inhibitors was independent of extracellular Ca(2+) concentration. The 5-HT outflow induced by CLM, but not that by PCA, was dependent on the extracellular NaCl concentration. These results suggest that the 5-HT uptake system of 5-HT-containing chemoreceptor cells in the chicken thoracic aorta has characteristics similar to those of 5-HT-containing neurons in the mammalian central nervous system (CNS).

Polysulfide evokes acute pain through the activation of nociceptive TRPA1 in mouse sensory neurons.

BACKGROUND: Hydrogen sulfide (H2S) is oxidized to polysulfide. Recent reports show that this sulfur compound modulates various biological functions. We have reported that H2S is involved in inflammatory pain in mice. On the other hand, little is known about the functional role of polysulfide in sensory neurons. Here we show that polysulfide selectively stimulates nociceptive TRPA1 and evokes acute pain, using TRPA1-gene deficient mice (TRPA1(-/-)), a heterologous expression system and a TRPA1-expressing cell line. RESULTS: In wild-type mouse sensory neurons, polysulfide elevated the intracellular Ca concentration ([Ca(2+)]i) in a dose-dependent manner. The half maximal effective concentration (EC50) of polysulfide was less than one-tenth that of H2S. The [Ca(2+)]i responses to polysulfide were observed in neurons responsive to TRPA1 agonist and were inhibited by blockers of TRPA1 but not of TRPV1. Polysulfide failed to evoke [Ca(2+)]i increases in neurons from TRPA1(-/-) mice. In RIN-14B cells, constitutively expressing rat TRPA1, polysulfide evoked [Ca(2+)]i increases with the same EC50 value as in sensory neurons. Heterologously expressed mouse TRPA1 was activated by polysulfide and that was suppressed by dithiothreitol. Analyses of the TRPA1 mutant channel revealed that cysteine residues located in the internal domain were related to the sensitivity to polysulfide. Intraplantar injection of polysulfide into the mouse hind paw induced acute pain and edema which were significantly less than in TRPA1(-/-) mice. CONCLUSIONS: The present data suggest that polysulfide functions as pronociceptive substance through the activation of TRPA1 in sensory neurons. Since the potency of polysulfide is higher than parental H2S and this sulfur compound is generated under pathophysiological conditions, it is suggested that polysulfide acts as endogenous ligand for TRPA1. Therefore, TRPA1 may be a promising therapeutic target for endogenous sulfur compound-related algesic action.

Molecular basis determining inhibition/activation of nociceptive receptor TRPA1 protein: a single amino acid dictates species-specific actions of the most potent mammalian TRPA1 antagonist.

The transient receptor potential ankyrin 1 (TRPA1) is a Ca(2+)-permeable, nonselective cation channel mainly expressed in a subset of nociceptive neurons. TRPA1 functions as a cellular sensor detecting mechanical, chemical, and thermal stimuli. Because TRPA1 is considered to be a key player in nociception and inflammatory pain, TRPA1 antagonists have been developed as analgesic agents. Recently, by utilizing species differences, we identified the molecular basis of the antagonistic action of A967079, one of the most potent mammalian TRPA1 antagonists. Here, we show a unique effect of A967079 on TRPA1 from diverse vertebrate species, i.e. it acts as an agonist but not as an antagonist for chicken and frog TRPA1s. By characterizing chimeric channels of human and chicken TRPA1s, as well as point mutants, we found that a single specific amino acid residue located within the putative fifth transmembrane domain was involved in not only the stimulatory but also the inhibitory actions of A967079. AP18, structurally related to A967079, exerted similar pharmacological properties to A967079. Our findings and previous reports on species differences in the sensitivity to TRPA1 antagonists supply useful information in the search for novel analgesic medicines targeting TRPA1.

Molecular mechanism of 2-APB-induced Ca²âº influx in external acidification in PC12.

2-Aminoethoxydiphenyl borate (2-APB) is used as a pharmacological tool because it antagonizes inositol 1,4,5-trisphosphate receptors and store-operated Ca(2+) (SOC) channels, and activates some TRP channels. Recently, we reported that 2-APB enhanced the increase in cytotoxic [Ca(2+)]i, resulting in cell death under external acidic conditions in rat pheochromocytoma cell line PC12. However, the molecular mechanism and functional role of the 2-APB-induced Ca(2+) influx in PC12 have not been clarified. In this study, to identify the possible target for the action of 2-APB we examined the pharmacological and molecular properties of [Ca(2+)]i and secretory responses to 2-APB under extracellular low pH conditions. 2-APB dose-dependently induced a [Ca(2+)]i increase and dopamine release, which were greatly enhanced by the external acidification (pH 6.5). [Ca(2+)]i and secretory responses to 2-APB at pH 6.5 were inhibited by the removal of extracellular Ca(2+) and SOC channel blockers such as SK&F96365, La(3+) and Gd(3+). PC12 expressed all SOC channel molecules, Orai 1, Orai 2 and Orai 3. When we used an siRNA system, downregulation of Orai 3, but not Orai 1 and Orai 2, attenuated both [Ca(2+)]i and secretory responses to 2-APB. These results suggest that 2-APB evokes external acid-dependent increases of [Ca(2+)]i and dopamine release in PC12 through the activation of Orai 3. The present results indicate that 2-APB may be a useful pharmacological tool for Orai channel-related signaling.

Heat and noxious chemical sensor, chicken TRPA1, as a target of bird repellents and identification of its structural determinants by multispecies functional comparison.

Nociceptive receptors enable animals to sense tissue-damaging stimuli, thus playing crucial roles in survival. Due to evolutionary diversification, responses of nociceptive receptors to specific stimuli can vary among species. Multispecies functional comparisons of nociceptive receptors help elucidate their evolutionary process and molecular basis for activation. The transient receptor potential ankyrin 1 (TRPA1) ion channel serves as a nociceptive receptor for chemical and thermal stimuli that is heat-activated in reptiles and frogs while potentially cold-activated in rodents. Here, we characterized channel properties of avian TRPA1 in chicken. Chicken TRPA1 was activated by noxious chemicals that also activate TRPA1 in other vertebrates. Regarding thermal sensitivity, chicken TRPA1 was activated by heat stimulation, but not cold, thus thermal sensitivity of avian TRPA1 does not coincide with rodent TRPA1, although both are homeotherms. Furthermore, in chicken sensory neurons, TRPA1 was highly coexpressed with TRPV1, another nociceptive heat and chemical receptor, similar to mammals and frogs. These results suggest that TRPA1 acted as a noxious chemical and heat receptor, and was coexpressed with TRPV1 in the ancestral terrestrial vertebrate. The acquisition of TRPV1 as a novel heat receptor in the ancestral terrestrial vertebrate is likely to have affected the functional evolution of TRPA1 regarding thermal sensitivity and led to the diversification among diverse vertebrate species. Additionally, we found for the first time that chicken TRPA1 is activated by methyl anthranilate (MA) and its structurally related chemicals used as nonlethal bird repellents. MA-induced responses were abolished by a TRPA1 antagonist in somatosensory neurons, indicating that TRPA1 acts as a MA receptor in chicken. Furthermore, TRPA1 responses to MA varied among five diverse vertebrate species. Utilizing species diversity and mutagenesis experiments, three amino acids were identified as critical residues for MA-induced activation of chicken TRPA1.