Colonic TRPV4 overexpression is related to constipation severity.

BACKGROUND: Chronic constipation is prevalent and involves both colon sensitivity and various changes in intestinal bacteria, particularly mucosa-associated microflora. Here we examined regulatory mechanisms of TRPV4 expression by co-culturing colon epithelial cell lines with intestinal bacteria and their derivatives. We also investigated TRPV4 expression in colon epithelium from patients with constipation. METHODS: Colon epithelial cell lines were co-cultured with various enterobacteria (bacterial components and supernatant), folate, LPS, or short chain fatty acids. TRPV4 expression levels and promoter DNA methylation were assessed using pyrosequencing, and microarray network analysis. For human samples, correlation coefficients were calculated and multiple regression analyses were used to examine the association between clinical background, rectal TRPV4 expression level and mucosa-associated microbiota. RESULTS: Co-culture of CCD841 cells with P. acnes, C. perfringens, or S. aureus transiently decreased TRPV4 expression but did not induce methylation. Co-culture with clinical isolates and standard strains of K. oxytoca, E. faecalis, or E. coli increased TRPV4 expression in CCD841 cells, and TRPV4 and TNF-alpha expression were increased by E. coli culture supernatants but not bacterial components. Although folate, LPS, IL-6, TNF-alpha, or SCFAs alone did not alter TRPV4 expression, TRPV4 expression following exposure to E. coli culture supernatants was inhibited by butyrate or TNF-alphaR1 inhibitor and increased by p38 inhibitor. Microarray network analysis showed activation of TNF-alpha, cytokines, and NOD signaling. TRPV4 expression was higher in constipated patients from the terminal ileum to the colorectum, and multiple regression analyses showed that low stool frequency, frequency of defecation aids, and duration were associated with TRPV4 expression. Meanwhile, incomplete defecation, time required to defecate, and number of defecation failures per 24 h were associated with increased E. faecalis frequency. CONCLUSIONS: Colon epithelium cells had increased TRPV4 expression upon co-culture with K. oxytoca, E. faecalis, or E. coli supernatants, as well as TNFα-stimulated TNFαR1 expression via a pathway other than p38. Butyrate treatment suppressed this increase. Epithelial TRPV4 expression was increased in constipated patients, suggesting that TRPV4 together with increased frequency of E. faecalis may be involved in the pathogenesis of various constipation symptoms.

Transient Receptor Potential Ankyrin 1 in Taste Nerve Contributes to the Sense of Sweet Taste in Mice.

Transient receptor potential (TRP) channels play a significant role in taste perception. TRP ankyrin 1 (TRPA1) is present in the afferent sensory neurons and is activated by food-derived ingredients, such as Japanese horseradish, cinnamon, and garlic. The present study aimed to investigate the expression of TRPA1 in taste buds, and determine its functional roles in taste perception using TRPA1-deficient mice. In circumvallate papillae, TRPA1 immunoreactivity colocalised with P2X2 receptor-positive taste nerves but not with type II or III taste cell markers. Behavioural studies showed that TRPA1 deficiency significantly reduced sensitivity to sweet and umami tastes, but not to salty, bitter, and sour tastes, compared to that in wild-type animals. Furthermore, administration of the TRPA1 antagonist HC030031 significantly decreased taste preference to sucrose solution compared to that in the vehicle-treated group in the two-bottle preference tests. TRPA1 deficiency did not affect the structure of circumvallate papillae or the expression of type II or III taste cell and taste nerve markers. Adenosine 5'-O-(3-thio)triphosphate evoked inward currents did not differ between P2X2- and P2X2/TRPA1-expressing human embryonic kidney 293T cells. TRPA1-deficient mice had significantly decreased c-fos expression in the nucleus of the solitary tract in the brain stem following sucrose stimulation than wild-type mice. Taken together, the current study suggested that TRPA1 in the taste nerve contributes to the sense of sweet taste in mice.

Intragastric administration of AMG517, a TRPV1 antagonist, enhanced activity-dependent energy metabolism via capsaicin-sensitive sensory nerves in mice.

Transient receptor potential vanilloid 1 (TRPV1), a nociceptive cation channel, is known to play roles in regulating the energy metabolism (EM) of the whole body. We previously reported that TRPV1 antagonists such as AMG517 enhanced EM in mice, however, these mechanisms remain unclear. The aim of this study was to explore the mechanisms underlying the enhancement of EM by AMG517, a selective TRPV1 antagonist, in mice. Respiratory gas analysis indicated that intragastric administration of AMG517 enhanced EM along with increasing locomotor activity in mice. Next, to clarify the possible involvement with afferent sensory nerves, including the vagus, we desensitized the capsaicin-sensitive sensory nerves of mice by systemic capsaicin treatment. In the desensitized mice, intragastric administration of AMG517 did not change EM and locomotor activity. Therefore, this study indicated that intragastric administration of AMG517 enhanced EM and increased locomotor activity via capsaicin-sensitive sensory nerves, including vagal afferents in mice.

Sensory nerve supports epithelial stem cell function in healing of corneal epithelium in mice: the role of trigeminal nerve transient receptor potential vanilloid 4.

In order to understand the pathobiology of neurotrophic keratopathy, we established a mouse model by coagulating the first branch of the trigeminal nerve (V1 nerve). In our model, the sensory nerve in the central cornea disappeared and remaining fibers were sparse in the peripheral limbal region. Impaired corneal epithelial healing in the mouse model was associated with suppression of both cell proliferation and expression of stem cell markers in peripheral/limbal epithelium as well as a reduction of transient receptor potential vanilloid 4 (TRPV4) expression in tissue. TRPV4 gene knockout also suppressed epithelial repair in mouse cornea, although it did not seem to directly modulate migration of epithelium. In a co-culture experiment, TRPV4-introduced KO trigeminal ganglion upregulated nerve growth factor (NGF) in cultured corneal epithelial cells, but ganglion with a control vector did not. TRPV4 gene introduction into a damaged V1 nerve rescues the impairment of epithelial healing in association with partial recovery of the stem/progenitor cell markers and upregulation of cell proliferation and of NGF expression in the peripheral/limbal epithelium. Gene transfer of TRPV4 did not accelerate the regeneration of nerve fibers. Sensory nerve TRPV4 is critical to maintain stemness of peripheral/limbal basal cells, and is one of the major mechanisms of homeostasis maintenance of corneal epithelium.

Keratin 13 gene is epigenetically suppressed during transforming growth factor-ß1-induced epithelial-mesenchymal transition in a human keratinocyte cell line.

Epithelial-mesenchymal transition (EMT) is a biological event in which epithelial cells lose their polarity and cell-cell adhesions and concomitantly acquire mesenchymal traits, and is thought to play an important role in pathological processes such as wound healing and cancer progression. In this study, we evaluated transforming growth factor (TGF)-ß1-treated human keratinocyte HaCaT cells as an in vitro model of EMT. HaCaT cells were changed into an elongated fibroblast-like morphology, which is indicative of EMT in response to TGF-ß1. Phalloidin staining demonstrated the formation of actin stress fibers in TGF-ß1-treated cells. Quantitative RT-PCR analysis revealed that TGF-ß1 increased the mRNA levels of EMT transcription factors (SNAI2, TWIST1, and ZEB1) and mesenchymal markers (CDH2, VIM, and FN1), while it decreased the transcripts of epithelial phenotypic genes (CLDN1, OCLN, KRT5, KRT15, KRT13, and TGM1). Furthermore, we found that KRT13 was drastically suppressed through the reduction of RNA polymerase II occupancy of its promoter, which was accompanied by a decrease in active histone marks (H3K4me3 and H3K27ac) and an increase in a repressive mark (H3K27me3) during EMT. These findings indicate that the TGF-ß1-induced EMT program regulates a subset of epithelial and mesenchymal marker genes, and that KRT13 is transcriptionally suppressed through the modulation of the chromatin state at the KRT13 promoter in HaCaT cells.

10-oxo-12(Z)-octadecenoic acid, a linoleic acid metabolite produced by gut lactic acid bacteria, enhances energy metabolism by activation of TRPV1.

Gut microbiota can regulate the host energy metabolism; however, the underlying mechanisms that could involve gut microbiota-derived compounds remain to be understood. Therefore, in this study, we investigated the effects of KetoA [10-oxo-12(Z)-octadecenoic acid]-a linoleic acid metabolite produced by gut lactic acid bacteria-on whole-body energy metabolism and found that dietary intake of KetoA could enhance energy expenditure in mice, thereby protecting mice from diet-induced obesity. By using Ca2+ imaging and whole-cell patch-clamp methods, KetoA was noted to potently activate transient receptor potential vanilloid 1 (TRPV1) and enhance noradrenalin turnover in adipose tissues. In addition, KetoA up-regulated genes that are related to brown adipocyte functions, including uncoupling protein 1 (UCP1) in white adipose tissue (WAT), which was later diminished in the presence of a ß-adrenoreceptor blocker. By using obese and diabetic model KK-Ay mice, we further show that KetoA intake ameliorated obesity-associated metabolic disorders. In the absence of any observed KetoA-induced antiobesity effect or UCP1 up-regulation in TRPV1-deficient mice, we prove that the antiobesity effect of KetoA was caused by TRPV1 activation-mediated browning in WAT. KetoA produced in the gut could therefore be involved in the regulation of host energy metabolism.-Kim, M., Furuzono, T., Yamakuni, K., Li, Y., Kim, Y.-I., Takahashi, H., Ohue-Kitano, R., Jheng, H.-F., Takahashi, N., Kano, Y., Yu, R., Kishino, S., Ogawa, J., Uchida, K., Yamazaki, J., Tominaga, M., Kawada, T., Goto, T. 10-oxo-12(Z)-octadecenoic acid, a linoleic acid metabolite produced by gut lactic acid bacteria, enhances energy metabolism by activation of TRPV1.

Lack of TRPV2 impairs thermogenesis in mouse brown adipose tissue.

Brown adipose tissue (BAT), a major site for mammalian non-shivering thermogenesis, could be a target for prevention and treatment of human obesity. Transient receptor potential vanilloid 2 (TRPV2), a Ca(2+)-permeable non-selective cation channel, plays vital roles in the regulation of various cellular functions. Here, we show that TRPV2 is expressed in brown adipocytes and that mRNA levels of thermogenic genes are reduced in both cultured brown adipocytes and BAT from TRPV2 knockout (TRPV2KO) mice. The induction of thermogenic genes in response to ß-adrenergic receptor stimulation is also decreased in TRPV2KO brown adipocytes and suppressed by reduced intracellular Ca(2+) concentrations in wild-type brown adipocytes. In addition, TRPV2KO mice have more white adipose tissue and larger brown adipocytes and show cold intolerance, and lower BAT temperature increases in response to ß-adrenergic receptor stimulation. Furthermore, TRPV2KO mice have increased body weight and fat upon high-fat-diet treatment. Based on these findings, we conclude that TRPV2 has a role in BAT thermogenesis and could be a target for human obesity therapy.

Role of Thermo-Sensitive Transient Receptor Potential Channels in Brown Adipose Tissue.

Brown and beige adipocytes are a major site of mammalian non-shivering thermogenesis and energy dissipation. Obesity is caused by an imbalance between energy intake and expenditure and has become a worldwide health problem. Therefore modulation of thermogenesis in brown and beige adipocytes could be an important application for body weight control and obesity prevention. Over the last few decades, the involvement of thermo-sensitive transient receptor potential (TRP) channels (including TRPV1, TRPV2, TRPV3, TRPV4, TRPM4, TRPM8, TRPC5, and TRPA1) in energy metabolism and adipogenesis in adipocytes has been extensively explored. In this review, we summarize the expression, function, and pathological/physiological contributions of these TRP channels and discuss their potential as future therapeutic targets for preventing and combating human obesity and obesity-related metabolic disorders.

Lysophosphatidic acid-induced itch is mediated by signalling of LPA5 receptor, phospholipase D and TRPA1/TRPV1.

KEY POINTS: Lysophosphatidic acid (LPA) is an itch mediator, but not a pain mediator by a cheek injection model. Dorsal root ganglion neurons directly respond to LPA depending on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1). LPA-induced itch-related behaviours are decreased in TRPA1-knockout (KO), TRPV1KO or TRPA1TRPV1 double KO mice. TRPA1 and TRPV1 channels are activated by intracellular LPA, but not by extracellular LPA following LPA5 receptor activation with an activity of Ca2+ -independent phospholipase A2 and phospholipase D. Intracellular LPA interaction sites of TRPA1 are KK672-673 and KR977-978 (K: lysine, R: arginine). ABSTRACT: Intractable and continuous itch sensations often accompany diseases such as atopic dermatitis, neurogenic lesions, uremia and cholestasis. Lysophosphatidic acid (LPA) is an itch mediator found in cholestatic itch patients and it induces acute itch and pain in experimental rodent models. However, the molecular mechanism by which LPA activates peripheral sensory neurons remains unknown. In this study, we used a cheek injection method in mice to reveal that LPA induced itch-related behaviours but not pain-related behaviours. The LPA-induced itch behaviour and cellular effects were dependent on transient receptor potential ankyrin 1 (TRPA1) and vanilloid 1 (TRPV1), which are important for itch signal transduction. We also found that, among the six LPA receptors, the LPA5 receptor had the greatest involvement in itching. Furthermore, we demonstrated that phospholipase D (PLD) plays a critical role downstream of LPA5 and that LPA directly and intracellularly activates TRPA1 and TRPV1. These results suggest a unique mechanism by which cytoplasmic LPA produced de novo could activate TRPA1 and TRPV1. We conclude that LPA-induced itch is mediated by LPA5 , PLD, TRPA1 and TRPV1 signalling, and thus targeting TRPA1, TRPV1 or PLD could be effective for cholestatic itch interventions.

Stimulation-dependent gating of TRPM3 channel in planar lipid bilayers.

The transient receptor potential melastatin (TRPM)-3 channel is critical for various physiologic processes. In somatosensory neurons, TRPM3 has been implicated in temperature perception and inflammatory hyperalgesia, whereas in pancreatic ß-cells the channel has been linked to glucose-induced insulin release. As a typical representative of the TRP family, TRPM3 is highly polymodal. In cells, it is activated by heat and chemical agonists, including pregnenolone sulfate (PS) and nifedipine (Nif). To define the nuances of TRPM3 channel activity and its modulators, we succeeded in incorporating the TRPM3 protein into planar lipid bilayers. We found that phosphatidylinositol-4,5-bisphosphate (PIP2) or clotrimazole is necessary for channel opening by PS. Unlike PS, the presence of Nif alone sufficed to induce TRPM3 activity and demonstrated distinct gating behavior. We also performed an extensive thermodynamic analysis of TRPM3 activation and found that TRPM3 exhibited slight temperature sensitivity in the bilayers. In the absence of other agonists TRPM3 channels remained closed upon heat-induced stimulation, but opened in the presence of PIP2, although with only a low open-probability profile. Together, our results elucidate the details peculiar to TRPM3 channel function in an isolated system. We confirmed its direct gating by PS and PIP2, but found a lack of the strong intrinsic temperature sensitivity common to other thermosensitive TRP channels.

TRPM2, a Susceptibility Gene for Bipolar Disorder, Regulates Glycogen Synthase Kinase-3 Activity in the Brain.

Bipolar disorder (BD) is a psychiatric disease that causes mood swings between manic and depressed states. Although genetic linkage studies have shown an association between BD and TRPM2, a Ca(2+)-permeable cation channel, the nature of this association is unknown. Here, we show that D543E, a mutation of Trpm2 that is frequently found in BD patients, induces loss of function. Trpm2-deficient mice exhibited BD-related behavior such as increased anxiety and decreased social responses, along with disrupted EEG functional connectivity. Moreover, the administration of amphetamine in wild-type mice evoked a notable increase in open-field activity that was reversed by the administration of lithium. However, the anti-manic action of lithium was not observed in the Trpm2(-/-) mice. The brains of Trpm2(-/-) mice showed a marked increase in phosphorylated glycogen synthase kinase-3 (GSK-3), a key element in BD-like behavior and a target of lithium. In contrast, activation of TRPM2 induced the dephosphorylation of GSK-3 via calcineurin, a Ca(2+)-dependent phosphatase. Importantly, the overexpression of the D543E mutant failed to induce the dephosphorylation of GSK-3. Therefore, we conclude that the genetic dysfunction of Trpm2 causes uncontrolled phosphorylation of GSK-3, which may lead to the pathology of BD. Our findings explain the long-sought etiologic mechanism underlying the genetic link between Trpm2 mutation and BD. SIGNIFICANCE STATEMENT: Bipolar disorder (BD) is a mental disorder that causes changes in mood and the etiology is still unknown. TRPM2 is highly associated with BD; however, its involvement in the etiology of BD is still unknown. We show here that TRPM2 plays a central role in causing the pathology of BD. We found that D543E, a mutation of Trpm2 frequently found in BD patients, induces the loss of function. Trpm2-deficient mice exhibited mood disturbances and impairments in social cognition. TRPM2 actively regulates the phosphorylation of GSK-3, which is a main target of lithium, a primary medicine for treating BD. Therefore, abnormal regulation of GSK-3 by hypoactive TRPM2 mutants accounts for the pathology of BD, providing the possible link between BD and TRPM2.

Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium.

Trpm7 is a divalent cation-permeable channel that has been reported to be involved in magnesium homeostasis as well as cellular adhesion and migration. We generated urothelium-specific Trpm7 knock-out (KO) mice to reveal the function of Trpm7 in vivo. A Trpm7 KO was induced by tamoxifen and was confirmed by genomic PCR and immunohistochemistry. By using patch clamp recordings in primary urothelial cells, we observed that Mg(2+)-inhibitable cation currents as well as acid-inducible currents were significantly smaller in Trpm7 KO urothelial cells than in cells from control mice. Assessment of voiding behavior indicated a significantly smaller voided volume in Trpm7 KO mice (mean voided volume 0.28 ± 0.08 g in KO mice and 0.36 ± 0.04 g in control mice, p < 0.05, n = 6-8). Histological analysis showed partial but substantial edema in the submucosal layer of Trpm7 KO mice, most likely due to inflammation. The expression of proinflammatory cytokines TNF-α and IL-1ß was significantly higher in Trpm7 KO bladders than in controls. In transmission electron microscopic analysis, immature intercellular junctions were observed in Trpm7 KO urothelium but not in control mice. These results suggest that Trpm7 is involved in the formation of intercellular junctions in mouse urothelium. Immature intercellular junctions in Trpm7 knock-out mice might lead to a disruption of barrier function resulting in inflammation and hypersensitive bladder afferent nerves that may affect voiding behavior in vivo.

Identification of significant amino acids in multiple transmembrane domains of human transient receptor potential ankyrin 1 (TRPA1) for activation by eudesmol, an oxygenized sesquiterpene in hop essential oil.

Transient receptor potential ankyrin 1 (TRPA1) is a calcium-permeable non-selective cation channel that is activated by various noxious or irritant substances in nature, including spicy compounds. Many TRPA1 chemical activators have been reported; however, only limited information is available regarding the amino acid residues that contribute to the activation by non-electrophilic activators, whereas activation mechanisms by electrophilic ligands have been well characterized. We used intracellular Ca(2+) measurements and whole-cell patch clamp recordings to show that eudesmol, an oxygenated sesquiterpene present at high concentrations in the essential oil of hop cultivar Hallertau Hersbrucker, could activate human TRPA1. Gradual activation of inward currents with outward rectification by eudesmol was observed in human embryonic kidney-derived 293 cells expressing human TRPA1. This activation was completely blocked by a TRPA1-specific inhibitor, HC03-0031. We identified three critical amino acid residues in human TRPA1 in putative transmembrane domains 3, 4, and 5, namely threonine at 813, tyrosine at 840, and serine at 873, for activation by ß-eudesmol in a systematic mutational study. Our results revealed a new TRPA1 activator in hop essential oil and provide a novel insight into mechanisms of human TRPA1 activation by non-electrophilic chemicals.

Activation of TRPV2 negatively regulates the differentiation of mouse brown adipocytes.

Transient receptor potential vanilloid 2 (TRPV2) acts as a Ca(2+)-permeable non-selective cation channel that has been reported to be sensitive to temperature, mechanical force, and some chemicals. We recently showed that TRPV2 is critical for maintenance of the thermogenic function of brown adipose tissue in mice. However, the involvement of TRPV2 in the differentiation of brown adipocytes remains unexplored. We found that the expression of TRPV2 was dramatically increased during the differentiation of brown adipocytes. Non-selective TRPV2 agonists (2-aminoethoxydiphenyl borate and lysophosphatidylcholine) inhibited the differentiation of brown adipocytes in a dose-dependent manner during the early stage of differentiation of brown adipocytes. The inhibition was rescued by a TRPV2-selective antagonist, SKF96365 (SKF). Mechanical force, which activates TRPV2, also inhibited the differentiation of brown adipocytes in a strength-dependent manner, and the effect was reversed by SKF. In addition, the inhibition of adipocyte differentiation by either TRPV2 ligand or mechanical stimulation was significantly smaller in the cells from TRPV2KO mice. Moreover, calcineurin inhibitors, cyclosporine A and FK506, partially reversed TRPV2 activation-induced inhibition of brown adipocyte differentiation. Thus, we conclude that TRPV2 might be involved in the modulation of brown adipocyte differentiation partially via a calcineurin pathway.

Ambient temperature affects the temperature threshold for TRPM8 activation through interaction of phosphatidylinositol 4,5-bisphosphate.

Cold sensation is an important and fundamental sense for animals and it is known to be affected by ambient temperature. Transient Receptor Potential Melastatin 8 (TRPM8), a nonselective cation channel expressed in a subset of peripheral afferent fibers, acts as a cold sensor, having an activation threshold of ∼28°C. Although the cold temperature threshold of TRPM8 is affected by menthol or pH, ambient temperature has not been reported to affect it. Because the cold temperature threshold was thought to be unchanged by alterations in ambient temperature, the relativity of temperature sensing in different ambient temperatures could not be understood at the level of molecular function of thermosensitive TRP channels. Here, we show that ambient temperature changed the temperature threshold for activation of human and rat TRPM8 in a heterologous expression system and cold responses in mouse DRG neurons. Moreover, reducing the level of cellular phosphatidylinositol 4,5-bisphosphate (PIP2) attenuated changes in the cold temperature threshold after alterations in ambient temperature. A single amino acid mutation at position 1008 in the C terminus of TRPM8 (arginine to glutamine) also attenuated changes in the cold temperature threshold induced by ambient temperature. These findings suggest that ambient temperature does affect the temperature threshold for TRPM8 activation through interaction of PIP2.

Extracellular zinc ion regulates transient receptor potential melastatin 5 (TRPM5) channel activation through its interaction with a pore loop domain.

The transient receptor potential melastatin 5 (TRPM5) channel is a monovalent cation channel activated by intracellular Ca(2+). Expression of this channel is restricted to taste cells, the pancreas and brainstem, and is thought to be involved in controlling membrane potentials. Its endogenous ligands are not well characterized. Here, we show that extracellular application of Zn(2+) inhibits TRPM5 activity. In whole-cell patch-clamp recordings, extracellular application of ZnCl2 inhibited step-pulse-induced TRPM5 currents with 500 nM free intracellular Ca(2+) in a dose-dependent manner (IC50 = 4.3 µM at -80 mV). ZnSO4 also inhibited TRPM5 activity. Extracellular application of ZnCl2 inhibited TRPM5 activation at several temperatures. Furthermore, inhibition by 30 µM ZnCl2 was impaired in TRPM5 mutants in which His at 896, and Glu at 926 and/or Glu at 939 in the outer pore loop were replaced with Gln. From these results, we conclude that extracellular Zn(2+) inhibits TRPM5 channels, and the residues in the outer pore loop of TRPM5 are critically involved in the inhibition.

Role of transient receptor potential vanilloid 4 activation in indomethacin-induced intestinal damage.

Gastrointestinal ulcers and bleeding are serious complications of nonsteroidal anti-inflammatory drug (NSAID) use. Although administration of antibiotics and Toll-like receptor 4 knockdown mitigate NSAID-induced enteropathy, the molecular mechanism of these effects is poorly understood. Intestinal hyperpermeability is speculated to trigger the initial damage due to NSAID use. Transient receptor potential vanilloid 4 (TRPV4) is a nonselective cation channel expressed throughout the gastrointestinal tract epithelium that is activated by temperature, extension, and chemicals such as 5,6-epoxyeicosatrienoic acid (5,6-EET). The aim of this study was to investigate the possible role of TRPV4 in NSAID-induced intestinal damage. TRPV4 mRNA and protein expression was confirmed by RT-PCR and immunochemistry, respectively, in mouse and human tissues while TRPV4 channel activity of the intestinal cell line IEC-6 was assessed by Ca(2+)-imaging analysis. TRPV4 activators or the NSAID indomethacin significantly decreased transepithelial resistance (TER) in IEC-6 cells, and indomethacin-induced TER decreases were inhibited by specific TRPV4 inhibitors or small-interfering RNA TRPV4 knockdown, as well as by the epoxygenase inhibitor N-(methylsulfonyl)-2-(2-propynyloxy)-benzenehexanamide, which decreased 5,6-EET levels. In TRPV4 knockout mice, indomethacin-induced intestinal damage was significantly reduced compared with WT mice. Taken together, these results show that TRPV4 activation in the intestinal epithelium caused epithelial hyperpermeability in response to NSAID-induced arachidonic acid metabolites and contributed to NSAID-induced intestinal damage. Thus, TRPV4 could be a promising new therapeutic target for the prevention of NSAID-induced intestinal damage.

Activation and inhibition of thermosensitive TRP channels by voacangine, an alkaloid present in Voacanga africana, an African tree.

Voacangine (1) is an alkaloid found in the root bark of Voacanga africana. Our previous work has suggested that 1 is a novel transient receptor potential vanilloid type 1 (TRPV1) antagonist. In this study, the agonist and antagonist activities of 1 were examined against thermosensitive TRP channels. Channel activity was evaluated mainly using TRP channel-expressing HEK cells and calcium imaging. Herein, it was shown that 1 acts as an antagonist for TRPV1 and TRPM8 but as an agonist for TRPA1 (EC50, 8 µM). The compound competitively blocked capsaicin binding to TRPV1 (IC50, 50 µM). Voacangine (1) competitively inhibited the binding of menthol to TRPM8 (IC50, 9 µM), but it showed noncompetitive inhibition against icilin (IC50, 7 µM). Moreover, the compound selectively abrogated chemical agonist-induced TRPM8 activation and did not affect cold-induced activation. Among these effects, the TRPM8 inhibition profile is unique and noteworthy, because to date no studies have reported a menthol competitive inhibitor of TRPM8 derived from a natural source. Furthermore, this is the first report of a stimulus-selective TRPM8 antagonist. Accordingly, 1 may contribute to the development of a novel class of stimulus-selective TRPM8 blockers.

TRPM2 contributes to antigen-stimulated Ca²âº influx in mucosal mast cells.

Food allergy (FA) is a common allergic disease without any currently available effective drug therapies. Mucosal mast cells (MMCs) play a particularly important role in FA, and the increase in their cytosolic Ca(2+) concentration ([Ca(2+)]cyt) is considered to be a principal component of the degranulation process. However, the mechanisms governing Ca(2+) influx remain poorly understood in MMCs. Recent reports have highlighted the functions of the transient receptor potential melastatin 2 (TRPM2) channel in immunocytes, including its role in monocyte chemokine production and macrophage phagocytic activity. Although TRPM2 gene expression has been demonstrated in mast cells, the significance of such expression remains virtually unknown. In this study, we found that antigen-stimulated degranulation was significantly reduced in mucosal-type bone marrow-derived mast cells (mBMMCs) prepared from TRPM2-knockout (TRPM2-KO) mice (TRPM2-KO mBMMCs) and was suppressed following the administration of three TRPM2 inhibitors with different chemical structures, including econazole, flufenamic acid (FFA), and 2-aminoethoxydiphenyl borate. Furthermore, the antigen-stimulated increase in [Ca(2+)]cyt was significantly decreased in TRPM2-KO mBMMCs and was also suppressed by the TRPM2 inhibitors econazole and FFA. In addition, thapsigargin-induced increase in [Ca(2+)]cyt was significantly decreased in TRPM2-KO mBMMCs. These results suggest that TRPM2 may participate in antigen-induced extracellular Ca(2+) influx and subsequent degranulation. In addition, TRPM2 inhibitors were shown to improve food allergic reactions in a mouse model. Together, these results suggest that TRPM2 inhibitors suppress MMC degranulation via regulation of the increase in [Ca(2+)]cyt. Thus, TRPM2 may play a key role in degranulation by modulating intracellular Ca(2+) in MMCs.

Involvement of TRPA1 activation in acute pain induced by cadmium in mice.

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.