Yoshiro Suzuki

Functional Role for Piezo1 in Stretch-evoked Ca2+ Influx and ATP Release in Urothelial Cell Cultures

Background: The Piezo1 channel was recently identified as a genuine mechanosensor in mammalian cells. Results: Urothelial cells exhibited a Piezo1-dependent increase in cytosolic Ca2+ concentrations in response to mechanical stretch stimuli, leading to ATP release. Conclusion: Piezo1 senses extension of the bladder urothelium, which is converted into an ATP signal. Significance: Inhibition of Piezo1 might provide a new treatment for bladder dysfunction. The urothelium is a sensory structure that contributes to mechanosensation in the urinary bladder. Here, we provide evidence for a critical role for the Piezo1 channel, a newly identified mechanosensory molecule, in the mouse bladder urothelium. We performed a systematic analysis of the molecular and functional expression of Piezo1 channels in the urothelium. Immunofluorescence examination demonstrated abundant expression of Piezo1 in the mouse and human urothelium. Urothelial cells isolated from mice exhibited a Piezo1-dependent increase in cytosolic Ca2+ concentrations in response to mechanical stretch stimuli, leading to potent ATP release; this response was suppressed in Piezo1-knockdown cells. In addition, Piezo1 and TRPV4 distinguished different intensities of mechanical stimulus. Moreover, GsMTx4, an inhibitor of stretch-activated channels, attenuated the Ca2+ influx into urothelial cells and decreased ATP release from them upon stretch stimulation. These results suggest that Piezo1 senses extension of the bladder urothelium, leading to production of an ATP signal. Thus, inhibition of Piezo1 might provide a promising means of treating bladder dysfunction.

Activation of transient receptor potential A1 by a non-pungent capsaicin-like compound, capsiate.

BACKGROUND AND PURPOSE Capsiate is produced by ‘CH‐19 Sweet’ (Capsicum annuun L.), a non‐pungent cultivar of red pepper. Like capsaicin, capsiate is thought to enhance energy metabolism by activating the sympathetic nervous system and suppressing inflammation, but the underlying mechanisms for this are uncertain. We previously reported that capsiate could activate transient receptor potential vanilloid 1 (TRPV1), a capsaicin receptor. The purpose of the present study is to investigate whether capsinoids activate other TRP channels.

Modulation of water efflux through functional interaction between TRPV4 and TMEM16A/anoctamin 1

Transient receptor potential vanilloid 4 (TRPV4), a calcium‐permeable channel, is highly expressed in the apical membrane of choroid plexus epithelial cells (CPECs) in the brain. The function of TRPV4 is unknown. Here, we show physical and functional interaction between TRPV4 and anoctamin 1 (ANO1) in HEK293T cells and CPECs. Chloride currents induced by a TRPV4 activator (GSK1016790A) were markedly increased in an extracellular calcium‐dependent manner in HEK293T cells expressing TRPV4 with ANO1, but not with ANO4, ANO6, or ANO10, the mRNAs of which were expressed in the choroid plexus. We also found physical interaction between TRPV4 and ANO1 in both HEK293T cells and choroid plexus. We observed that ANO1 was activated at a warm temperature (37°C) in HEK293T cells and that the heat‐evoked chloride currents were markedly enhanced after GSK1016790A application in CPECs. Simultaneous stimulation by warmth and hyposmosis induced chloride current activation in wild‐type, but not in TRPV4‐deficient, CPECs. Cell volume changes were induced by ANO1‐mediated chloride currents in parallel with membrane potential changes, and the cell volume was significantly decreased at negative membrane potentials by TRPV4‐induced ANO1 activation. Thus, physical and functional interactions between TRPV4 and ANO1 can modulate water transport in the choroid plexus.—Takayama, Y., Shibasaki, K., Suzuki, Y., Yamanaka, A., Tominaga, M. Modulation of water efflux through functional interaction between TRPV4 and TMEM16A/anoctamin 1. FASEB J. 28, 2238–2248 (2014). www.fasebj.org

Identification of a splice variant of mouse TRPA1 that regulates TRPA1 activity

Transient receptor potential ankyrin 1 (TRPA1) protein is a nonselective cation channel. Although many studies suggest that TRPA1 is involved in inflammatory and neuropathic pain, its mechanism remains unclear. Here we identify an alternative splice variant of the mouse Trpa1 gene. TRPA1a (full-length) and TRPA1b (splice variant) physically interact with each other and TRPA1b increases the expression of TRPA1a in the plasma membrane. TRPA1a and TRPA1b co-expression significantly increases current density in response to different agonists without affecting their single-channel conductance. Exogenous overexpression of Trpa1b gene in wild-type and TRPA1KO DRG neurons also increases TRPA1a-mediated AITC responses. Moreover, expression levels of Trpa1a and Trpa1b mRNAs change dynamically in two pain models (complete Freund’s adjuvant-induced inflammatory pain and partial sciatic nerve ligation-induced neuropathic pain models). These results suggest that TRPA1 may be regulated through alternative splicing under these pathological conditions.

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 Ca2+‐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 β‐adrenergic receptor stimulation is also decreased in TRPV2KO brown adipocytes and suppressed by reduced intracellular Ca2+ 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 β‐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.

Potential role of transient receptor potential (TRP) channels in bladder cancer cells

Transient receptor potential (TRP) channels play important roles in thermal, chemical, and mechanical sensation in various tissues. In this study, we investigated the differences in urothelial TRP channels between normal urothelial cells and bladder cancer cells. TRPV2 and TRPM7 expression levels and TRPV2 activator-induced intracellular Ca2+ increases were significantly higher, whereas TRPV4 expression and TRPV4 activator-induced intracellular Ca2+ increases were significantly lower in mouse bladder cancer (MBT-2) cells compared to normal mouse urothelial cells. The proliferation rate of MBT-2 cells overexpressing dominant-negative TRPV2 was significantly increased. In contrast, treatment with TRPV2 activators significantly decreased the proliferation rate. TRPM7-overexpressing MBT-2 cells proliferated more slowly, as compared to mock-transfected cells. Moreover, expression of dominant-negative TRPV2 significantly decreased plasma membrane Ca2+ permeability of MBT-2 cells as compared to that in mock-transfected cells. Increases in the expression of TRPV2 mRNA, immunoreactivity, and TRPV2 activator-induced intracellular Ca2+ were also observed in T24 human bladder cancer cells. These results suggested that TRPV2 and TRPM7 were functionally expressed in bladder cancer cells and served as negative regulators of bladder cancer cell proliferation, most likely to prevent excess mechanical stresses.

Trpm7 Protein Contributes to Intercellular Junction Formation in Mouse Urothelium

Background: Transient receptor potential melastatin 7 (Trpm7) is a Ca2+-permeable channel with a kinase domain that is implicated in cell migration and adhesion. Results: Urothelium-specific Trpm7 knock-out caused immature intercellular junctions, inflammation, and smaller voided volume. Conclusion: Trpm7 contributes to the intercellular junction formation in the urothelium. Significance: These findings might provide the first evidence for the significance of Trpm7 in bladder function in vivo. 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 Mg2+-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-1β 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.

Mouse Anaphylactic Hypotension Is Characterized by Initial Baroreflex Independent Renal Sympathoinhibition Followed by Sustained Renal Sympathoexcitation

Aim: The hemodynamic response to mouse systemic anaphylaxis is characterized by an initial hypertension followed by sustained hypotension. However, the defense mechanisms of the sympathetic nervous system against this circulatory disturbance is not known. Here, we investigated the renal sympathetic nerve activity (RSNA) response to mouse systemic anaphylaxis, along with the roles of carotid sinus baroreceptor, vagal nerves and the transient receptor potential vanilloid type 1 channel (TRPV1). Methods: Male ovalbumin-sensitized C57BL/6N mice were used under pentobarbital anesthesia. RSNA, systemic arterial pressure (SAP) and heart rate (HR) were continuously measured for 60 min after the antigen injection. Results: Within 3 min after antigen injection, RSNA decreased along with a transient increase in SAP. Thereafter, RSNA showed a progressive increase during sustained hypotension. In contrast, HR continuously increased. Sinoaortic denervation, but not vagotomy, significantly attenuated the renal sympathoexcitation and tachycardia from 30 and 46 min, respectively, after antigen. The responses of RSNA, SAP and HR to anaphylaxis were not affected by pretreatment with a TRPV1 inhibitor, capsazepine, or by genetic knockout of TRPV1. Conclusion: The mouse systemic anaphylaxis causes a biphasic RSNA response with an initial baroreflex-independent decrease and secondary increase. The antigen-induced sympathoexcitation and tachycardia at the late stage are partly mediated by carotid sinus baroreceptors. Either vagal nerve or TRPV1 does not play any significant roles in the RSNA and HR responses in anesthetized mice.

Publisher Correction: Identification of a splice variant of mouse TRPA1 that regulates TRPA1 activity

This corrects the article DOI: 10.1038/ncomms3399.

Hypotonicity-induced cell swelling activates TRPA1

Hypotonic solutions can cause painful sensations in nasal and ocular mucosa through molecular mechanisms that are not entirely understood. We clarified the ability of human TRPA1 (hTRPA1) to respond to physical stimulus, and evaluated the response of hTRPA1 to cell swelling under hypotonic conditions. Using a Ca2+-imaging method, we found that modulation of AITC-induced hTRPA1 activity occurred under hypotonic conditions. Moreover, cell swelling in hypotonic conditions evoked single-channel activation of hTRPA1 in a cell-attached mode when the patch pipette was attached after cell swelling under hypotonic conditions, but not before swelling. Single-channel currents activated by cell swelling were also inhibited by a known hTRPA1 blocker. Since pre-application of thapsigargin or pretreatment with the calcium chelator BAPTA did not affect the single-channel activation induced by cell swelling, changes in intracellular calcium concentrations are likely not related to hTRPA1 activation induced by physical stimuli.