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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 ([Ca 2+ ] 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 [Ca 2+ ] i , which was abolished by the removal of extracellular Ca 2+ in mouse DRG neurons. The [Ca 2+ ] i responses to econazole were suppressed by a TRPA1 blocker but not by a TRPV1 blocker. Attenuation of the econazole-induced [Ca 2+ ] i responses was observed in the TRPA1 (-/-) mouse DRG neurons but was not significant in the TRPV1 (-/-) neurons. Econazole increased the [Ca 2+ ] i in HEK293 cells expressing TRPA1 (TRPA1-HEK) but not in those expressing TRPV1, although at higher concentrations, it induced Ca 2+ mobilization from intracellular stores in untransfected naïve HEK293 cells. Miconazole, which is a structural analog of econazole, also increased the [Ca 2+ ] 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., (© 2024. The Author(s).)

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 EC 50 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 IC 50 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., (© 2024. The Author(s).)

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 β-methoxyacrylate group and a strobilurin analogue bearing modified β-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., (© The Author(s) 2024. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

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.

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.

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.

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 Ca 2+ concentration ([Ca 2+ ] i ) was measured using a Ca 2+ -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 [Ca 2+ ] i increases did not affect [Ca 2+ ] 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 [Ca 2+ ] i increases induced by KCl and voltage-gated Ca 2+ 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 Ca 2+ channels., Competing Interests: We have no conflict-of-interest to declare., (© 2023 The Authors.)

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., (© The Author(s) 2022. Published by Oxford University Press on behalf of Society for Molecular Biology and Evolution.)

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 [Ca 2+ ] i increases that were inhibited by removal of extracellular Ca 2+ or blockers of TRPA1 but not by those of TRPV1 and TRPM8. Persulfate failed to evoke [Ca 2+ ] 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 [Ca 2+ ] i increases. Moreover, in HEK 293 cells expressing mouse TRPV1 (mTRPV1-HEK), a high concentration of persulfate also evoked [Ca 2+ ] i increases. Similar [Ca 2+ ] 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 [Ca 2+ ] 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., (Copyright © 2019 Elsevier B.V. All rights reserved.)

T-type Ca 2+ 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 Ca 2+ channels and TRPA1 in mouse sensory neurons. Low concentration of KCl at 15 mM (15K) evoked increases of intracellular Ca 2+ concentration ([Ca 2+ ] i ), which were suppressed by selective T-type Ca 2+ channel blockers. RT-PCR showed that mouse sensory neurons expressed all subtypes of T-type Ca 2+ channel. The magnitude of 15K-induced [Ca 2+ ] 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 [Ca 2+ ] i responses to 15K in neurons sensitive to AITC, but failed to inhibit 40 mM KCl-induced [Ca 2+ ] i increases even in AITC-sensitive neurons. TRPV1 blockers did not inhibit the 15K-induced [Ca 2+ ] i increase regardless of the sensitivity to capsaicin. [Ca 2+ ] i responses to TRPA1 agonist were enhanced by co-application with 15K. These pharmacological data suggest the possibility of functional interaction between T-type Ca 2+ channels and TRPA1 in sensory neurons. Since TRPA1 channel is activated by intracellular Ca 2+ , we hypothesize that Ca 2+ entered via T-type Ca 2+ channel activation may further stimulate TRPA1, resulting in an enhancement of nociceptive signaling. Thus, T-type Ca 2+ channel may be a potential target for TRPA1-related pain., (© 2017 Wiley Periodicals, Inc.)

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 Ca 2+ concentration ([Ca 2+ ] 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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

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-1β 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-1β 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., (Copyright © 2017 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

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).

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.

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., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

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., (Copyright © 2014 Elsevier Inc. All rights reserved.)

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.

The transient receptor potential A1 (TRPA1) receptor is a member of the TRP family and an excitatory nonselective cation channel. An increasing body of evidence suggests that TRPA1 acts as a nociceptor for various chemicals and physical stimuli. Thus, many TRPA1 antagonists have been developed as analgesic agents. Recently, we found that AP18, a mammalian TRPA1 antagonist, does not inhibit heterologously expressed western clawed frog TRPA1 (fTRPA1). Here, we show that fTRPA1 is also insensitive to A967079, one of the most potent mammalian TRPA1 antagonists. Neither heterologously nor endogenously expressed fTRPA1 was inhibited by A967079 upon activation by TRPA1 agonists. Mutant channel analyses revealed that two specific amino acid residues located within the putative fifth transmembrane domain were involved in the inhibitory action of A967079. Our findings and previous reports based on species differences in the sensitivity to TRPA1 antagonists provide novel insights into the structure-function relationship of TRPA1 and supply useful information in the search for new analgesic medicines targeting TRPA1.

Hydrogen sulfide (H2 S), a toxic volcanic gas, functions as a gaseous physiological and pathophysiological molecule. Recently we have shown that H2 S elicits acute pain through the activation of transient receptor potential ankyrin 1 (TRPA1), which is expressed mainly in primary nociceptive neurons. We also demonstrated enhancement of H2 S-induced TRPA1 activation and pain under inflammatory acidic conditions, but the underlying mechanism has not been elucidated. Here, we attempted to clarify this mechanism by using endogenously TRPA1-expressing RIN-14B, a rat pancreatic islet cell line. For this purpose, the intracellular Ca(2+) concentration ([Ca(2+) ]i )], reactive oxygen species (ROS), and intracellular pH (pHi ) were measured with fluorescent imaging techniques. The intracellular H2 S concentration was assayed by the methylene blue method. To clarify the cellular function of H2 S, 5-hydroxytryptamine (5-HT) secretion was analyzed. In RIN-14B, the increase of [Ca(2+) ]i and the release of 5-HT induced by NaHS, an H2 S donor, were enhanced under inflammatory acidic conditions. Transition of H2 S into cells was enhanced at pH 6.8. H2 S failed to increase the intracellular ROS level and only slightly decreased pHi . These results suggest that H2 S directly activates TRPA1 and that its increment of diffusion into cells may be involved in the potentiation of TRPA1 activation under external acidic conditions. Thus, our study supports the pathophysiological functions of H2 S in inflammatory pain., (Copyright © 2013 Wiley Periodicals, Inc.)

Background: Cadmium (Cd) is an environmental pollutant and acute exposure to it causes symptoms related to pain and inflammation in the airway and gastrointestinal tract, but the underlying mechanisms are still unclear. TRPA1 is a nonselective cation channel expressed in sensory neurons and acts as a nociceptive receptor. Some metal ions such as Ca, Mg, Ba and Zn are reported to modulate TRPA1 channel activity. In the present study, we investigated the effect of Cd on cultured mouse dorsal root ganglion neurons and a heterologous expression system to analyze the effect of Cd at the molecular level. In addition, we examined whether Cd caused acute pain in vivo., Results: In wild-type mouse sensory neurons, Cd evoked an elevation of the intracellular Ca concentration ([Ca2+]i) that was inhibited by external Ca removal and TRPA1 blockers. Most of the Cd-sensitive neurons were also sensitive to cinnamaldehyde (a TRPA1 agonist) and [Ca2+]i responses to Cd were absent in TRPA1(-/-) mouse neurons. Heterologous expression of TRPA1 mutant channels that were less sensitive to Zn showed attenuation of Cd sensitivity. Intracellular Cd imaging revealed that Cd entered sensory neurons through TRPA1. The stimulatory effects of Cd were confirmed in TRPA1-expressing rat pancreatic cancer cells (RIN-14B). Intraplantar injection of Cd induced pain-related behaviors that were largely attenuated in TRPA1(-/-) mice., Conclusions: Cd excites sensory neurons via activation of TRPA1 and causes acute pain, the mechanism of which may be similar to that of Zn. The present results indicate that TRPA1 is involved in the nociceptive or inflammatory effects of Cd.

Serotonin (5-HT) released from descending fibers plays important roles in spinal functions such as locomotion and nociception. 5-HT2A and 5-HT3 receptors are suggested to contribute to spinal antinociception, although their activation also contributes to neuronal excitation. In the neonatal spinal cord, DL-p-chloroamphetamine (pCA), a 5-HT releaser, inhibited both A fiber-evoked monosynaptic reflex potential (MSR) and C fiber-evoked slow ventral root potential (sVRP). The pCA-mediated inhibition was reversed by ketanserin (a 5-HT2A receptor antagonist) and tropisetron (a 5-HT3 receptor antagonist). Bath-applied 5-HT also inhibited MSR and sVRP; in this case, the actions of 5-HT were antagonized by ketanserin, but not by tropisetron. The pCA-evoked inhibition of sVRP was reduced by bicuculline (a GABAA receptor antagonist) and strychnine (a glycine receptor antagonist). Furthermore, ketanserin inhibited the pCA-evoked release of gamma-aminobutyric acid (GABA) and glycine, while tropisetron inhibited the pCA-evoked release of 5-HT. These results suggest that 5-HT released by pCA activates 5-HT2A receptors, which in turn stimulates the release of GABA/glycine and thereby blocks the spinal nociceptive pathway. 5-HT3 receptors may be involved in the facilitation of 5-HT release via a positive feedback process., (Copyright © 2013 Elsevier B.V. All rights reserved.)

2-Aminoethoxydiphenyl borate (2-APB) is widely used as a pharmacological tool for analysis of cellular Ca(2+) regulation. In this study, we found that external acid potentiated neural cell death induced by 2-APB in rat pheochromocytoma 12 (PC12) cells. 2-APB induced cell death in half of the PC12 cells within 30min at pH 6.6 but not at pH 7.4. The extent of the 2-APB-induced cell death increased in a dose-, time- and pH-dependent manner. Ca(2+)-imaging revealed that 2-APB increased [Ca(2+)](i) in PC12 cells at pH 6.6. Removal of extracellular Ca(2+) and chelation of intracellular Ca(2+) inhibited the 2-APB-induced cell death. Antagonists of the store-operated Ca(2+) (SOC) channel (SKF96365 and ruthenium red) blocked both 2-APB-induced cell death and Ca(2+) influx, but those for transient receptor potential channels (BCTC, TRIM and BTP2), acid-sensing ion channels (amiloride) and proton-sensing G-protein-coupled receptors (U73122) did not. These results suggest that 2-APB induces neural cell death via Ca(2+) overload through SOC channel activation under acidic pH., (Copyright © 2012 Elsevier Ireland Ltd. All rights reserved.)

Transient receptor potential ankyrin 1 (TRPA1) and TRP vanilloid 1 (V1) perceive noxious temperatures and chemical stimuli and are involved in pain sensation in mammals. Thus, these two channels provide a model for understanding how different genes with similar biological roles may influence the function of one another during the course of evolution. However, the temperature sensitivity of TRPA1 in ancestral vertebrates and its evolutionary path are unknown as its temperature sensitivities vary among different vertebrate species. To elucidate the functional evolution of TRPA1, TRPA1s of the western clawed (WC) frogs and green anole lizards were characterized. WC frog TRPA1 was activated by heat and noxious chemicals that activate mammalian TRPA1. These stimuli also activated native sensory neurons and elicited nocifensive behaviors in WC frogs. Similar to mammals, TRPA1 was functionally co-expressed with TRPV1, another heat- and chemical-sensitive nociceptive receptor, in native sensory neurons of the WC frog. Green anole TRPA1 was also activated by heat and noxious chemical stimulation. These results suggest that TRPA1 was likely a noxious heat and chemical receptor and co-expressed with TRPV1 in the nociceptive sensory neurons of ancestral vertebrates. Conservation of TRPV1 heat sensitivity throughout vertebrate evolution could have changed functional constraints on TRPA1 and influenced the functional evolution of TRPA1 regarding temperature sensitivity, whereas conserving its noxious chemical sensitivity. In addition, our results also demonstrated that two mammalian TRPA1 inhibitors elicited different effect on the TRPA1s of WC frogs and green anoles, which can be utilized to clarify the structural bases for inhibition of TRPA1.

Antibody-dependent cellular cytotoxicity (ADCC) is dependent on the fucose content of oligosaccharides bound to monoclonal antibodies (MAbs). As MAbs with a low fucose content exhibit high ADCC activity, it is important to control the defucosylation levels (deFuc%) of MAbs and to analyze the factors that affect deFuc%. In this study, we observed that the deFuc% was inversely related to culture medium osmolality for MAbs produced in the rat hybridoma cell line YB2/0, with r (2) values as high as 0.92. Moreover, deFuc% exhibited the same correlation irrespective of the type of compound used for regulating osmolality (NaCl, KCl, fucose, fructose, creatine, or mannitol) at a culture scale ranging from 1 to 400 L. We succeeded in controlling MAb deFuc% by maintaining a constant medium osmolality in both perfusion and fed-batch cultures. In agreement with these observations, reverse transcription PCR analyses revealed decreased transcription of genes involved in glycolysis, GDP-fucose supply, and fucose transfer under hypoosmotic conditions.

The functional difference of thermosensitive transient receptor potential (TRP) channels in the evolutionary context has attracted attention, but thus far little information is available on the TRP vanilloid 1 (TRPV1) function of amphibians, which diverged earliest from terrestrial vertebrate lineages. In this study we cloned Xenopus tropicalis frog TRPV1 (xtTRPV1), and functional characterization was performed using HeLa cells heterologously expressing xtTRPV1 (xtTRPV1-HeLa) and dorsal root ganglion neurons isolated from X. tropicalis (xtDRG neurons) by measuring changes in the intracellular calcium concentration ([Ca(2+)](i)). The channel activity was also observed in xtTRPV1-expressing Xenopus oocytes. Furthermore, we tested capsaicin- and heat-induced nocifensive behaviors of the frog X. tropicalis in vivo. At the amino acid level, xtTRPV1 displays ∼60% sequence identity to other terrestrial vertebrate TRPV1 orthologues. Capsaicin induced [Ca(2+)](i) increases in xtTRPV1-HeLa and xtDRG neurons and evoked nocifensive behavior in X. tropicalis. However, its sensitivity was extremely low compared with mammalian orthologues. Low extracellular pH and heat activated xtTRPV1-HeLa and xtDRG neurons. Heat also evoked nocifensive behavior. In oocytes expressing xtTRPV1, inward currents were elicited by heat and low extracellular pH. Mutagenesis analysis revealed that two amino acids (tyrosine 523 and alanine 561) were responsible for the low sensitivity to capsaicin. Taken together, our results indicate that xtTRPV1 functions as a polymodal receptor similar to its mammalian orthologues. The present study demonstrates that TRPV1 functions as a heat- and acid-sensitive channel in the ancestor of terrestrial vertebrates. Because it is possible to examine vanilloid and heat sensitivities in vitro and in vivo, X. tropicalis could be the ideal experimental lower vertebrate animal for the study of TRPV1 function.

The beta subunits of voltage-dependent calcium channels are known to modify calcium channel currents through pore-forming alpha1 subunits. The beta3 subunit is expressed in the adrenal gland and participates in forming various calcium channel types. We performed a series of experiments in beta3-null mice to determine the role of the beta3 subunit in catecholamine release from the adrenal chromaffin system. Protein levels of N-type channel forming CaV2.2 and L-type forming CaV1.2 decreased. The beta3-null mice showed a decreased baroreflex, suggesting decreased sympathetic tonus, whereas plasma catecholamine levels did not change. Pulse-voltage stimulation revealed significantly increased amperometrical currents in beta3-null mice, while patch-clamp recordings showed a significant reduction in Ca(2+)-currents due to reduced L- and N-type currents, indicating facilitated exocytosis. A biochemical analysis revealed increased InsP3 production. In conclusion, our results indicate the importance of the beta3 subunit in determining calcium channel characteristics and catecholamine release in adrenal chromaffin cells., (Copyright 2010. Published by Elsevier Inc.)

Functional changes of the enteric nervous system have been observed under inflammatory states of inflammatory bowel disease increasing the endotoxin level. The aim of the present study was to determine the effect of lipopolysaccharides (LPS) on myenteric neuron-glia interaction in vitro. We examined the increase of the intracellular Ca(2+) concentration ([Ca(2+)](i)) and the release of interleukin-1beta (IL-1beta) or prostaglandin E(2) (PGE(2)) and COX-2 expression in myenteric plexus cells from the rat intestine induced by LPS. LPS potentiated BK-induced [Ca(2+)](i) increases in both myenteric neurons and enteric glial cells, which were suppressed by a B1R antagonist. Only in enteric glial cells, a B1R agonist increased [Ca(2+)](i). The effects of LPS were blocked by pretreatment with an interleukin-1 receptor antagonist or by reducing the density of enteric glial cells in culture. LPS prompted the release of IL-1beta from enteric glial cells. The augmenting effects of IL-1beta on the BK-induced neural [Ca(2+)](i) increase and PGE(2) release from enteric glial cells were abolished by a phospholipase A(2) (PLA(2)) inhibitor and a COX inhibitor, and partly suppressed by a COX-2 inhibitor. IL-1beta up-regulated the COX-2 expression in enteric glial cells. LPS promotes IL-1beta secretion from enteric glial cells, resulting in augmentation of the neural response to BK through PGE(2) release via glial PLA(2) and COX-2. The alteration of the regulatory effect of glial cells may be the cause of the changes in neural function in the enteric nervous system in inflammatory bowel disease., ((c) 2009 Wiley-Liss, Inc.)

Methylsalicylate (MS) is a naturally occurring compound that is used as a major active ingredient of balms and liniments supplied as topical analgesics. Despite the common use of MS as a pain reliever, the underlying molecular mechanism is not fully understood. Here we characterize the action of MS on transient receptor potential V1 (TRPV1). In human embryonic kidney 293 cells expressing human TRPV1 (hTRPV1), MS evoked increases of [Ca(2+)](i), which declined regardless of its continuous presence, indicative of marked desensitization. TRPV1 antagonists dose-dependently suppressed the MS-induced [Ca(2+)](i) increase. MS simultaneously elicited an inward current and increase of [Ca(2+)](i) in the voltage-clamped cells, suggesting that MS promoted Ca(2+) influx through the activation of TRPV1 channels. MS reversibly inhibited hTRPV1 activation by polymodal stimuli such as capsaicin, protons, heat, anandamide, and 2-aminoethoxydiphenyl borate. Because both the stimulatory and inhibitory actions of MS were exhibited in capsaicin- and allicin-insensitive mutant channels, MS-induced hTRPV1 activation was mediated by distinct channel regions from capsaicin and allicin. In cultured rat sensory neurons, MS elicited a [Ca(2+)](i) increase in cells responding to capsaicin. MS significantly suppressed nocifensive behavior induced by intraplantar capsaicin in rats. The present data indicate that MS has both stimulatory and inhibitory actions on TRPV1 channels and suggest that the latter action may partly underlie the analgesic effects of MS independent of inhibition of cyclooxygenases in vivo.

Bipolar vomeronasal sensory neurons (VSNs) in the vomeronasal organ (VNO) are believed to detect pheromones in most mammals. The vomeronasal sensory epithelium (VSE) is composed of VSNs and supporting cells. There are morphological differences in VNOs between species. Many electrophysiological experiments have been performed on rodent VSEs but few on other mammals. We therefore investigated voltage-gated channel properties of cells in the porcine VSE using slice whole-cell voltage-clamp techniques. In immunohistochemical study of the porcine VSE, most PGP9.5-immunoreactive cells were found between the middle and basal region, and negative cells were distributed in the apical to middle region. Depolarizing pulses to epithelial cells from -90mV produced transient inward Na+ channel currents and sustained outward K+ channel currents with various amplitudes. The distribution of cells having high and low Na+ current densities was mostly consistent with the histological distribution of VSNs and supporting cells, respectively. The half-inactivation voltage of voltage-gated Na+ channels in supporting cells was 26mV more negative than that in VSNs. Voltage-gated K+ channel currents in both cell types were suppressed by tetraethylammonium to the same extent. VSNs possessed TTX-sensitive voltage-gated Na+ channels and Ni2+ -sensitive T-type Ca2+ channels. These results suggest that the histological distribution of porcine vomeronasal epithelial cells is more similar to the dog and goat than to rodents, and that the electrophysiological characteristics of porcine vomeronasal epithelial cells are similar to those of rodents. It is also suggested that porcine VSNs detecting pheromones generate action potentials through these channels.

Transient receptor potential V1 (TRPV1) is a nonselective cation channel expressed in nociceptors and activated by capsaicin. TRPV1 detects diverse stimuli, including acid, heat, and endogenous vanilloids, and functions as a molecular integrator of pain perception. Herein we demonstrate a novel regulatory role of extracellular Na(+) ([Na(+)](o)) on TRPV1 function. In human embryonic kidney 293 cells expressing porcine TRPV1, low [Na(+)](o) evoked increases of [Ca(2+)](i) that were suppressed by TRPV1 antagonists and facilitated responses to capsaicin, protons, heat, and an endovanilloid. [Na(+)](o) removal simultaneously elicited a [Ca(2+)](i) increase and outward-rectified current with a reversal potential similar to those of capsaicin. Neutralization of the two acidic residues which confer the proton sensitivity to TRPV1 resulted in a reduction of low [Na(+)](o)-induced responses. In primary culture of porcine sensory neurons, the removal of [Na(+)](o) produced a [Ca(2+)](i) increase and current responses only in the cells responding to capsaicin. Low [Na(+)](o) evoked a [Ca(2+)](i) increase in sensory neurons of wild type mice, but not TRPV1-null mice, and in human embryonic kidney 293 cells expressing human TRPV1. The present results suggest that [Na(+)](o) negatively regulates the gating and polymodal sensitization of the TRPV1 channel. [Na(+)](o) surrounding several proton-sensitive sites on the extracellular side of the pore-forming loop of the TRPV1 channel may play an important role as a brake to suppress the excessive activity of this channel under physiological conditions.

Neurogenic components play a crucial role in inflammation and nociception. Mustard oil (MO) is a pungent plant extract from mustard seed, horseradish and wasabi, the main constituent of which is allylisothiocyanate. We have characterized the action of MO on transient receptor potential V1 (TRPV1), a key receptor of signal transduction pathways in the nociceptive system, using fura-2-based [Ca(2+)](i) imaging and the patch-clamp technique in a heterologous expression system and sensory neurons. In human embryonic kidney (HEK) 293 cells expressing porcine TRPV1 (pTRPV1), MO evoked increases of [Ca(2+)](i) in a concentration-dependent manner. A high concentration of MO elicited irreversible cell swelling. Capsazepine, ruthenium red and iodoresiniferatoxin dose-dependently suppressed the MO-induced [Ca(2+)](i) increase. MO elicited outward rectified currents in pTRPV1-expressing HEK 293 cells with a reversal potential similar to that of capsaicin. [Ca(2+)](i) responses to MO were completely abolished by the removal of external Ca(2+). MO simultaneously elicited an inward current and increase of [Ca(2+)](i) in the same cells, indicating that MO promoted Ca(2+) influx through TRPV1 channels. In cultured porcine dorsal root ganglion (DRG) neurons, MO elicited a [Ca(2+)](i) increase and inward current. Among DRG neurons responding to MO, 85% were also sensitive to capsaicin. The present data indicate that MO is a novel agonist of TRPV1 channels, and suggest that the action of MO in vivo may be partly mediated via TRPV1. These results provide an insight into the TRPV1-mediated effects of MO on inflammation and hyperalgesia.

To clarify the role of lipid rafts in 2-chloro-2'-deoxyadenosine (2CdA; Cladribine)-induced apoptosis, the effects of disruption of lipid rafts by methyl-beta-cyclodextrin (MbetaCD) and filipin on 2CdA-induced apoptosis were investigated in four human acute lymphoblastic leukemia (ALL) cell lines comprised of T cells (MOLT-4, Jurkat) and B cells (NALM, BALL-1). The disruption of lipid rafts significantly inhibited 2CdA-induced apoptosis, indicating the crucial role of lipid rafts in the induction of apoptosis in leukemia cells. These reagents significantly inhibited 2CdA-induced elevation of the intracellular calcium concentration ([Ca(2+)](i)) in MOLT-4 cells, and 2CdA-induced apoptosis was partly inhibited by the Ca(2+) chelators BAPTA-AM and EGTA, and the L-type Ca(2+) channel blocker nifedipine. On the other hand, they had no effects on the cellular uptake of 2CdA. These results indicated that lipid rafts partly contributed to 2CdA-induced apoptosis by regulating Ca(2+) influx via the plasma membrane.

Carrier (electron or hole) injection from a scanning tunneling microscope tip causes various surface modifications on the molecular scale. We report that injection into C60 close-packed layers forms a ring-shaped distribution of C60 polymers. This can be explained on the basis of the radial propagation and energy dissipation of carriers. Subsequent electron or hole injections enlarge the ring, showing that both carriers can induce both polymerization and depolymerization. Furthermore, we demonstrate visualization of carrier scattering by injecting carriers into C60 layers with grain boundaries.

5-Hydroxytryptamine (5-HT) is one of the major chemical mediators released in injured and inflamed tissue and is capable of inducing hyperalgesia in vivo. However, the cellular mechanisms of 5-HT-induced hyperalgesia remain unclear. Transient receptor potential V1 (TRPV1) plays a pivotal role in nociceptive receptors. In the present study, we determined whether 5-HT changes TRPV1 functions in cultured dorsal root ganglion (DRG) neurons isolated from neonatal rats, using Ca(2+) imaging and whole-cell patch-clamp techniques. In more than 70% of DRG neurons, 5-HT potentiated the increases of [Ca(2+)](i) induced by capsaicin, protons and noxious heat. Capsaicin-induced current and depolarizing responses, and proton-induced currents were also augmented by 5-HT. RT-PCR analysis revealed the expression of 5-HT(2A) and 5-HT(7) receptors in rat DRG neurons. Agonists for 5-HT(2A) and 5-HT(7) receptors mimicked the potentiating effect of 5-HT, and their antagonists decreased it. In DRG ipsilateral to the complete Freund's adjuvant-injected inflammation side, expression levels of 5-HT(2A) and 5-HT(7) mRNAs increased, and the potentiating effect of 5-HT was more prominent than in the contralateral control side. These results suggest that the PKC- and PKA-mediated signalling pathways are involved in the potentiating effect of 5-HT on TRPV1 functions through the activation of 5-HT(2A) and 5-HT(7) receptors, respectively. Under inflammatory conditions, the increases of the biosynthesis of these 5-HT receptors may lead to further potentiation of TRPV1 functions, resulting in the generation of inflammatory hyperalgesia in vivo.

Adenosine is one of the most important neuromodulators in the CNS, both under physiological and pathological conditions. In the isolated spinal cord of the neonatal rat in vitro, acute hypercapnic acidosis (20% CO2, pH 6.7) reversibly depressed electrically evoked spinal reflex potentials. This depression was partially reversed by 8-cyclopentlyl-1,3-dimethylxanthine (CPT), a selective A1 adenosine receptor antagonist. Isohydric hypercapnia (20% CO2, pH 7.3), but not isocapnic acidosis (5% CO2, pH 6.7), depressed the reflex potentials, which were also reversed by CPT. An ecto-5'-nucleotidase inhibitor did not affect the hypercapnic acidosis-evoked depression. An inhibitor of adenosine kinase, but not deaminase, mimicked the inhibitory effect of hypercapnic acidosis on the spinal reflex potentials. Accumulation of extracellular adenosine and inhibition of adenosine kinase activity were caused by hypercapnic acidosis and isohydric hypercapnia, but not isohydric acidosis. These results indicate that the activation of adenosine A1 receptors is involved in the hypercapnia-evoked depression of reflex potentials in the isolated spinal cord of the neonatal rat. The inhibition of adenosine kinase activity is suggested to cause the accumulation of extracellular adenosine during hypercapnia.

In the present study, we cloned a porcine orthologue of transient receptor potential V1 (pTRPV1) and heterologously expressed it in human embryonic kidney (HEK) 293 cells to characterize its pharmacological properties. At the amino acid level, pTRPV1 was highly homologous (83-90%) to other orthologues of TRPV1. The expression of receptors was examined with current and [Ca2+]i responses to capsaicin using whole-cell patch-clamp and fura-2 ratio imaging techniques, respectively, and by immunostaining with an anti-TRPV1 antibody. The receptors were characterized by changes in [Ca2+]i in response to various vanilloid agonists, low pH and heat and by the effects of TRPV1 antagonists on them. The various TRPV1 agonists activated pTRPV1 in a dose-dependent manner in the order of potency of resiniferatoxin (RTX) > olvanil > capsaicin > phorbol 12-phenylacetate 13-acetate 20-homovanillate (PPAHV), phorbol 12,13-dinonanoate 20-homovanillate (PDNHV). Isovelleral and scutigeral had no effect. Endogenous vanilloids (anandamide > 15 (s)-HPETE >> NADA), low pH and noxious heat (>42 degrees C) activated pTRPV1. Comparison of amino acid sequences with various mammalian TRPV1 homologues suggested some novel putative vanilloid recognition sites. TRPV1 antagonists, iodoRTX, ruthenium red and capsazepine suppressed capsaicin-induced responses. Similar to human TRPV1, but not rodent TRPV1, capsazepine was effective in blocking pH- and heat-induced responses. Similar pharmacological profiles were observed in cultured porcine dorsal root ganglion neurons. We discuss putative amino acid residues related to pharmacological differences among mammalian TRPV1 homologues.

We characterized ATP-induced changes in intracellular Ca2+ concentration ([Ca2+]i) and membrane current in cultured rat myenteric neurons using ratiometric Ca2+ imaging with fura-2 and the whole cell patch-clamp technique, respectively. Neuronal cells were functionally identified by [Ca2+]i responses to high K+ and nicotine, which occurred only in cells positive for neuron-specific protein gene product 9.5 immunoreactivity. ATP evoked a dose-dependent increase of [Ca2+]i that was greatly decreased by the removal of extracellular Ca2+ concentration ([Ca2+]o). The amplitude of the [Ca2+]i response to ATP was reduced by half in the presence of voltage-dependent Ca2+ channel blockers. In [Ca2+]o-free solution, ATP produced a small transient rise in [Ca2+]i similar to that induced by P2Y agonists. At -60 mV, ATP evoked a slowly inactivating inward current that was suppressed by the removal of extracellular Na+ concentration. The current-voltage relation for ATP showed an inward rectification with the reversal potential of about 0 mV. The apparent rank order of potency for the purinoceptor agonist-induced increases of [Ca2+]i was ATP > or = adenosine 5'-O-3-triphosphate > or = CTP > or = 2-methylthio-ATP > benzoylbenzoyl-ATP. A similar potency order was obtained with current responses to these agonists. P2 antagonists inhibited inward currents induced by ATP. Ca2+ and Mg2+ suppressed the ATP-induced current, and Zn2+, Cu2+, and protons potentiated it. RT-PCR and immunocytochemical studies showed the expression of P2X2 receptors in cultured rat myenteric neurons. These results suggest that ATP mainly activates ionotropic P2X2 receptors, resulting in a [Ca2+]i increase dependent on [Ca2+]o in rat myenteric neurons. A small part of the ATP-induced [Ca2+]i increase may be also mediated via a P2Y receptor-related mechanism.

The inhibitory effects of morphine and alpha2-adrenoceptor agonists on slow ventral root potentials (slow VRP) following ipsilateral dorsal root stimulation in neonatal rat spinal cord were compared with the analgesic effects of these drugs on formalin and capsaicin tests in neonatal rats. Morphine, (D-Phe2, D-Pen5)-enkephalin (DPDPE), dexmedetomidine, clonidine and xylazine showed concentration-related inhibition of slow VRP. The order of potency was dexmedetomidine>morphine=DPDPE>clonidine>xylazine. The inhibitory effects of opioid agonists and alpha2-adrenoceptor agonists were abolished by naloxone, an opioid antagonist, and atipamezole, an alpha2-adrenoceptor antagonist, respectively. There was no cross antagonism. Morphine, dexmedetomidine and xylazine dose-dependently inhibited body movement induced by formalin or capsaicin. The order of potency was dexmedetomidine>morphine>xylazine. Although morphine and dexmedetomidine inhibited formalin- and capsaicin-induced body movement in the same dose range, xylazine inhibited formalin-induced body movement at lower concentrations than capsaicin-induced one. The inhibitory potency for slow VRP by these drugs seems to be correlated with that for capsaicin-induced body movement but not that for formalin-induced one. Dexmedetomidine and morphine in combination inhibited slow VRP and body movement induced by capsaicin in an additive manner. It is suggested that the antinociceptive effects of dexmedetomidine and morphine but not xylazine on the capsaicin test are mainly due to spinal effects and that there is no synergistic interaction between dexmedetomidine and morphine in the neonatal rat.

We investigated the endogenous control through vesicular contents of voltage-dependent Ca2+ channels (VDCCs) in cultured porcine adrenal chromaffin cells. To examine paracrine regulation of VDCCs, catecholamine release was monitored amperometrically together with patch-clamp recording under culture conditions at different cell densities. A depolarizing pulse evoked Ca(2+)- (ICa) and Ba(2+)-currents (IBa) in Ca(2+)- and Ba(2+)-containing solutions, respectively. In cells cultured at high density, stop-flow of the external solution decreased the I(Ba) concomitant with a sustained increase of amperometric current (Iamp), but not in cells at low density, suggesting the endogenous modulation of VDCCs in a paracine fashion. The degree of the prepulse facilitation was similar regardless of the flow condition. Application of noradrenaline (NA), ATP, methionine-enkephalin (ENK) or protons decreased IBa. The extent of the prepulse facilitation of the endogenous VDCC inhibition was similar to those induced by NA and ATP. GDPbetaS, pertussis toxin (PTX), blockers for alpha-adrenoceptors and P2-purinoceptors significantly reduced the endogenous VDCC inhibition. These results suggest that VDCCs are regulated by vesicular substances in a paracrine fashion, at least by noradrenaline and ATP, through activation of alpha-adrenoceptors and P2-purinoceptors, respectively, in porcine adrenal chromaffin cells.

The involvement of mitochondrial Na+-Ca2+ exchange in Ca2+ responses to ATP was examined in rat pheochromocytoma (PC) 12 cells. Intracellular Ca2+ ([Ca2+]i) and Na+ concentrations ([Na+]i) were measured using fura-2 and SBFI, respectively. ATP caused concentration-dependent increases in [Ca2+]i and [Na+]i. High concentrations of ATP elicited a Ca2+ transient followed by a slow recovery of [Ca2+]i (a sustained phase) in 77% of PC12 cells. The sustained phase of Ca2+ response appeared only when the peak Ca2+ transient exceeded 500 nM. FCCP, a protonophore, greatly enhanced Ca2+ responses to ATP only in cells with the sustained phase but not without this phase. The sustained phase was decreased by clonazepam and CGP37157, mitochondrial Na+-Ca2+ exchange inhibitors, and extracellular Na+ removal but not by cyclosporin A, an inhibitor of permeability transition pores. The reintroduction of Na+ 3.5 min after ATP stimulation in the absence of Na+ caused Na+ concentration-dependent increases in [Ca2+]i and [Na+]i. The increase in [Na+]i was correlated with that in [Ca2+]i. FCCP caused a great increase in [Ca2+]i 4.5 min after ATP stimulation in the absence of extracellular Na+ but not in its presence, indicating that mitochondria retain Ca2+ in the absence of Na+. These results suggest that ATP causes a large increase in [Ca2+]i which was sequestered in mitochondria and that the sustained phase of Ca2+ response to ATP are mainly due to the release of mitochondrial Ca2+ through Na+-Ca2+ exchangers in PC12 cells.

Mammalian homologues of the Drosophila canonical transient receptor potential (trp) protein (TRPC) have been implicated to function as receptor-operated Ca(2+) channels (ROCs) or store-operated Ca(2+) channels (SOCs). To determine the role of TRPC5 protein in neural cells, TRPC5 was recombinantly expressed in rat pheochromocytoma cells (PC12) and changes in intracellular Ca(2+) concentration ([Ca(2+)](i)) and Na(+) concentration ([Na(+)](i)) were analyzed. TRPC1 and TRPC3 mRNAs were endogenously expressed in PC12 cells. In TRPC5-expressed cells (TRPC5-cells), the resting [Ca(2+)](i) and [Na(+)](i) were significantly higher than those in control cells. The [Ca(2+)](i) increases induced by bradykinin and uridine 5'-triphosphate were significantly larger in TRPC5-cells. TRPC5 expression did not change in store-operated Ca(2+) entry elicited by thapsigarigin. TRPC5-cells showed larger inward current and increase of [Na(+)](i) in response to BK than control cells. These results suggest that TRPC5 channels expressed in PC12 cells function as ROCs activated by G-protein/phospholipase C coupled receptors, but not as SOCs.

To clarify the apoptotic and survival signal transduction pathways in activated vascular endothelial cells exposed to oxidative stress, the effects of inhibitors of signal transduction on hydrogen peroxide (H(2)O(2))-induced apoptosis in bovine aortic vascular endothelial cells (BAEC) were examined. Treatment of BAEC with 1 mM H(2)O(2) caused increases of DNA fragmentation, p53 expression, Bax/Bcl-2 ratio, and the activities of caspases 3 and 9. The increases of DNA fragmentation, Bax/Bcl-2 ratio, and caspase activities were abrogated by BAPTA-AM (an intracellular Ca(2+) chelator) and N-acetyl-L-cysteine (an antioxidant), and augmented by wortmannin [a phosphatidylinositol 3-kinase (PI3K) inhibitor]. The increase of the intracellular Ca(2+) concentration ([Ca(2+)](i)) observed in H(2)O(2)-stimulated cells was unaffected by wortmannin, suggesting that the potentiating effect of wortmannin on the apoptosis was not due to an alteration of [Ca(2+)](i). H(2)O(2) increased the levels of PI3K activity and Akt phosphorylation. Both were attenuated by wortmannin and, to a lesser extent, by genistein (a tyrosine kinase inhibitor) and suramin (a growth factor receptor inhibitor), but not affected by BAPTA-AM. These results suggest that H(2)O(2) induces Ca(2+)-dependent apoptosis and Ca(2+)-independent survival signals such as redox-regulated activation of PI3K/Akt, which is partly mediated by the activation of growth factor receptors in BAEC.

We investigated the effects of dexamethasone, cortisol and aldosterone on responses to nicotine and muscarine in guinea-pig isolated adrenal medullary cells. Nicotine-induced inward currents were reversibly inhibited by these steroids in a dose-dependent and non-competitive manner. These steroids inhibited an increase in [Ca2+](i) in response to nicotine but not muscarine. Muscarine-induced catecholamine secretion was inhibited by cortisol and aldosterone but not dexamethasone. Phorbol 12-myristate 13-acetate, a protein kinase C activator, caused catecholamine secretion which was inhibited by cortisol and aldosterone but not dexamethasone. These results suggest that catecholamine secretion induced by cholinoceptor stimulation is inhibited by steroids via two distinct mechanisms; one is the inhibition of nicotinic receptors, another is the inhibition of protein kinase C activation in guinea-pig adrenal medullary cells.

To clarify the signaling pathways of oxidative stress-induced apoptosis in bovine aortic endothelial cells (BAEC), we treated cells with 1 mM H2O2 and investigated the roles of protein kinase C delta (PKC delta) and Ca2+ in the accumulation of p53 associated with apoptosis. The treatment of cells with H2O2 caused the accumulation of p53, which was inhibited by rottlerin (a PKC delta inhibitor) but not by BAPTA-AM (an intracellular Ca2+ chelator). PKC delta itself was activated through the phosphorylation at tyrosine residues. H2O2 induced the release of cytochrome c and the activation of caspases 3 and 9, and these apoptotic signals were inhibited by rottlerin and BAPTA-AM. These results suggest that PKC delta contributes to the accumulation of p53 and that Ca2+ plays a role in downstream signals of p53 leading to apoptosis in H2O2-treated BAEC.

A gene encoding a cholesterol oxidase from Brevibacterium sterolicum nov. sp. ATCC21387 was isolated by an expression cloning method and highly expressed by a recombinant strain Escherichia coli MM294/pnH10. The purified cholesterol oxidase was a typical flavoprotein with a molecular mass of 46.5 kDa, absorption peaks at 280, 360, and 450 nm. Optimum pH and temperature were found at pH 6.5 and 55 degrees C, respectively. The enzyme acted on 3beta-hydroxysteroids such as cholesterol, pregnenolone, and beta-sitosterol at high rates, but on dehydro-epi-androsterone to a lesser degree. The molecular and catalytic properties were different from those of cholesterol oxidase I, which was initially discovered in B. sterolicum nov. sp. ATCC21387. The new enzyme, designated cholesterol oxidase II, was distinguished by its high affinity toward cholesterol (K(m)=30 microM).

The effects of parathyroid hormone (PTH) on tension and intracellular Ca level ([Ca ] ) were examined in ring preparations of rat mesenteric artery using isometric tension recording and the fura-2 method, respectively. The PTH (30 n ) elicited relaxation in arterial rings precontracted by phenylephrine regardless of the presence or absence of endothelium. In the endothelium-denuded arterial rings precontracted by 3 micro M of phenylephrine or 60 m of potassium chloride (KCl), PTH-related protein and PTH produced concentration-dependent relaxation to the same extent, but inhibited contraction induced by phenylephrine more effectively than that induced by KCl. Phenylephrine-induced tonic contraction was changed to a phasic one with decreased peak tension in the presence of PTH. Similar changes were observed with extracellular Ca removal or methoxyverapamil plus SK&F96365, respective of voltage-gated and receptor-operated Ca channel inhibitors. Phenylephrine evoked a concentration-dependent contraction concomitant with an increase in [Ca ]. PTH reduced both responses to the same extent. In a Ca -free solution, PTH inhibited a phasic contraction and a transient increase in [Ca ] in response to phenylephrine but not caffeine. Reverse transcriptase-polymerase chain reaction showed that PTH and PTH receptors were expressed in the rat mesenteric artery. In this tissue, PTH increased cyclic adenosine monophosphate (cAMP) levels. These results suggest that the inhibitory effect of PTH on alpha -adrenoceptor-mediated contraction results from the inhibition of Ca influx through receptor-operated and voltage-gated Ca channels, and Ca release from Ca stores, probably via increased cAMP in the rat mesenteric artery.

In voltage-clamped guinea-pig chromaffin cells, muscarine (50 microM) or caffeine (30 mM) produced a transient intracellular Ca(2+) concentration ([Ca(2+)](i)) increase, catecholamine release and an outward K(+) current mediated through Ca(2+) released from internal Ca(2+) stores at a holding potential of -40 mV. Caffeine followed by muscarine failed to evoke these responses, while muscarine followed by caffeine was effective in producing about 30% of [Ca(2+)](i) increase and catecholamine secretion. In cells dialyzed with inositol 1,4,5-trisphosphate (IP(3)), caffeine failed to produce the [Ca(2+)](i) increase. Intracellular application of cyclic adenosine 5'-diphosphate-ribose (cADP-ribose) or 8-bromo cADP-ribose exerted no effect on the resting [Ca(2+)](i) and the caffeine-induced [Ca(2+)](i) increase. These results suggest that IP(3)-sensitive stores are functionally divided into two subpopulations, sensitive and insensitive to caffeine, and it is unlikely that cADP-ribose plays a role as a Ca(2+) releaser in guinea-pig adrenal chromaffin cells.