Kunitoshi Uchida

The TRPV4 Cation Channel Mediates Stretch-evoked Ca2+ Influx and ATP Release in Primary Urothelial Cell Cultures

Transient receptor potential channels have recently been implicated in physiological functions in a urogenital system. In this study, we investigated the role of transient receptor potential vanilloid 4 (TRPV4) channels in a stretch sensing mechanism in mouse primary urothelial cell cultures. The selective TRPV4 agonist, 4α-phorbol 12,13-didecanoate (4α-PDD) evoked Ca2+ influx in wild-type (WT) urothelial cells, but not in TRPV4-deficient (TRPV4KO) cells. We established a cell-stretch system to investigate stretch-evoked changes in intracellular Ca2+ concentration and ATP release. Stretch stimulation evoked intracellular Ca2+ increases in a stretch speed- and distance-dependent manner in WT and TRPV4KO cells. In TRPV4KO urothelial cells, however, the intracellular Ca2+ increase in response to stretch stimulation was significantly attenuated compared with that in WT cells. Stretch-evoked Ca2+ increases in WT urothelium were partially reduced in the presence of ruthenium red, a broad TRP channel blocker, whereas that in TRPV4KO cells did not show such reduction. Potent ATP release occurred following stretch stimulation or 4α-PDD administration in WT urothelial cells, which was dramatically suppressed in TRPV4KO cells. Stretch-dependent ATP release was almost completely eliminated in the presence of ruthenium red or in the absence of extracellular Ca2+. These results suggest that TRPV4 senses distension of the bladder urothelium, which is converted to an ATP signal in the micturition reflex pathway during urine storage.

Lack of TRPM2 Impaired Insulin Secretion and Glucose Metabolisms in Mice

OBJECTIVE TRPM2 is a Ca2+-permeable nonselective cation channel activated by adenosine dinucleotides. We previously demonstrated that TRPM2 is activated by coapplication of heat and intracellular cyclic adenosine 5′-diphosphoribose, which has been suggested to be involved in intracellular Ca2+ increase in immunocytes and pancreatic β-cells. To clarify the involvement of TRPM2 in insulin secretion, we analyzed TRPM2 knockout (TRPM2-KO) mice. RESEARCH DESIGN AND METHODS Oral and intraperitoneal glucose tolerance tests (OGTT and IPGTT) were performed in TRPM2-KO and wild-type mice. We also measured cytosolic free Ca2+ in single pancreatic cells using fura-2 microfluorometry and insulin secretion from pancreatic islets. RESULTS Basal blood glucose levels were higher in TRPM2-KO mice than in wild-type mice without any difference in plasma insulin levels. The OGTT and IPGTT demonstrated that blood glucose levels in TRPM2-KO mice were higher than those in wild-type mice, which was associated with an impairment in insulin secretion. In isolated β-cells, smaller intracellular Ca2+ increase was observed in response to high concentrations of glucose and incretin hormone in TRPM2-KO cells than in wild-type cells. Moreover, insulin secretion from the islets of TRPM2-KO mice in response to glucose and incretin hormone treatment was impaired, whereas the response to tolbutamide, an ATP-sensitive potassium channel inhibitor, was not different between the two groups. CONCLUSIONS These results indicate that TRPM2 is involved in insulin secretion stimulated by glucose and that further potentiated by incretins. Thus, TRPM2 may be a new target for diabetes therapy.

Intracellular alkalization causes pain sensation through activation of TRPA1 in mice

Vertebrate cells require a very narrow pH range for survival. Cells accordingly possess sensory and defense mechanisms for situations where the pH deviates from the viable range. Although the monitoring of acidic pH by sensory neurons has been attributed to several ion channels, including transient receptor potential vanilloid 1 channel (TRPV1) and acid-sensing ion channels (ASICs), the mechanisms by which these cells detect alkaline pH are not well understood. Here, using Ca2+ imaging and patch-clamp recording, we showed that alkaline pH activated transient receptor potential cation channel, subfamily A, member 1 (TRPA1) and that activation of this ion channel was involved in nociception. In addition, intracellular alkalization activated TRPA1 at the whole-cell level, and single-channel openings were observed in the inside-out configuration, indicating that alkaline pH activated TRPA1 from the inside. Analyses of mutants suggested that the two N-terminal cysteine residues in TRPA1 were involved in activation by intracellular alkalization. Furthermore, intraplantar injection of ammonium chloride into the mouse hind paw caused pain-related behaviors that were not observed in TRPA1-deficient mice. These results suggest that alkaline pH causes pain sensation through activation of TRPA1 and may provide a molecular explanation for some of the human alkaline pH-related sensory disorders whose mechanisms are largely unknown.

Unusual Pungency from Extra-Virgin Olive Oil Is Attributable to Restricted Spatial Expression of the Receptor of Oleocanthal

Oleocanthal, a major phenolic compound in extra-virgin olive oil with antiinflammatory properties, elicits an unusual oral pungency sensed almost exclusively in the throat. This contrasts with most other common oral irritants, such as cinnamaldehyde, capsaicin, and alcohol, which irritate mucus membranes throughout the oral cavity. Here, we show that this rare irritation pattern is a consequence of both the specificity of oleocanthal for a single sensory receptor and the anatomical restriction of this sensory receptor to the pharynx, within the oral cavity. We demonstrate, in vitro, that oleocanthal selectively activates the hTRPA1 channel in HEK 293 cells and that its ability to excite the trigeminal nervous system in rodents requires a functional TRPA1. Moreover, we similarly demonstrate that the over-the-counter analgesic, ibuprofen, which elicits the same restricted pharyngeal irritation as oleocanthal, also specifically excites rodent sensory neurons via TRPA1. Using human sensory psychophysical studies and immunohistochemical TRPA1 analyses of human oral and nasal tissues, we observe an overlap of the anatomical distribution of TRPA1 and the regions irritated by oleocanthal in humans. These results suggest that a TRPA1 (ANKTM1) gene product mediates the tissue sensitivity to oleocanthal within the oral cavity. Furthermore, we demonstrate that, despite the fact that oleocanthal possesses the classic electrophilic reactivity of many TRPA1 agonists, it does not use the previously identified activation mechanism via covalent cysteine modification. These findings provide an anatomical and molecular explanation for a distinct oral sensation that is elicited by oleocanthal and ibuprofen and that is commonly experienced around the world when consuming many extra-virgin olive oils.

1,8-cineole, a TRPM8 agonist, is a novel natural antagonist of human TRPA1.

BackgroundEssential oils are often used in alternative medicine as analgesic and anti-inflammatory remedies. However, the specific compounds that confer the effects of essential oils and the molecular mechanisms are largely unknown. TRPM8 is a thermosensitive receptor that detects cool temperatures and menthol whereas TRPA1 is a sensor of noxious cold. Ideally, an effective analgesic compound would activate TRPM8 and inhibit TRPA1.ResultsWe screened essential oils and fragrance chemicals showing a high ratio of human TRPM8-activating ability versus human TRPA1-activating ability using a Ca2+-imaging method, and identified 1,8-cineole in eucalyptus oil as particularly effective. Patch-clamp experiments confirmed that 1,8-cineole evoked inward currents in HEK293T cells expressing human TRPM8, but not human TRPA1. In addition, 1,8-cineole inhibited human TRPA1 currents activated by allyl isothiocyanate, menthol, fulfenamic acid or octanol in a dose-dependent manner. Furthermore, in vivo sensory irritation tests showed that 1,8-cineole conferred an analgesic effect on sensory irritation produced by TRPA1 agonists octanol and menthol. Surprisingly, 1,4-cineole, which is structurally similar and also present in eucalyptus oil, activated both human TRPM8 and human TRPA1.Conclusions1,8-cineole is a rare natural antagonist of human TRPA1 that has analgesic and anti-inflammatory effects possibly due to its inhibition of TRPA1.

Fish oil intake induces UCP1 upregulation in brown and white adipose tissue via the sympathetic nervous system

Brown adipose tissue (BAT) plays a central role in regulating energy homeostasis, and may provide novel strategies for the treatment of human obesity. BAT-mediated thermogenesis is regulated by mitochondrial uncoupling protein 1 (UCP1) in classical brown and ectopic beige adipocytes, and is controlled by sympathetic nervous system (SNS). Previous work indicated that fish oil intake reduces fat accumulation and induces UCP1 expression in BAT; however, the detailed mechanism of this effect remains unclear. In this study, we investigated the effect of fish oil on energy expenditure and the SNS. Fish oil intake increased oxygen consumption and rectal temperature, with concomitant upregulation of UCP1 and the β3 adrenergic receptor (β3AR), two markers of beige adipocytes, in the interscapular BAT and inguinal white adipose tissue (WAT). Additionally, fish oil intake increased the elimination of urinary catecholamines and the noradrenaline (NA) turnover rate in interscapular BAT and inguinal WAT. Furthermore, the effects of fish oil on SNS-mediated energy expenditure were abolished in transient receptor potential vanilloid 1 (TRPV1) knockout mice. In conclusion, fish oil intake can induce UCP1 expression in classical brown and beige adipocytes via the SNS, thereby attenuating fat accumulation and ameliorating lipid metabolism.

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.

Hypoxia-induced sensitization of transient receptor potential vanilloid 1 involves activation of hypoxia-inducible factor-1 alpha and PKC

&NA; The capsaicin receptor, transient receptor potential vanilloid 1 (TRPV1), acts as a polymodal detector of pain‐producing chemical and physical stimuli in sensory neurons. Hyperglycemia and hypoxia are two main phenomena in diabetes associated with several complications. Although many studies on streptozotocin‐induced diabetic rats indicate that early diabetic neuropathy is associated with potentiation of TRPV1 activity in dorsal root ganglion neurons, its underlying mechanism and distinctive roles of hyperglycemia and hypoxia have not been completely clarified. Here, we show that hypoxic and high glucose conditions (overnight exposure) potentiate the TRPV1 activity without affecting TRPV1 expression in both native rat sensory neurons and human embryonic kidney‐derived 293 cells expressing rat or human TRPV1. Surprisingly, hypoxia was found to be a more effective determinant than high glucose, and hypoxia‐inducible factor‐1 alpha (HIF‐1α) seemed to be involved. In addition, high glucose enhanced TRPV1 sensitization only when high glucose existed together with hypoxia. The potentiation of TRPV1 was caused by its phosphorylation of the serine residues, and translocation of protein kinase C (PKC)&egr; was clearly observed in the cells exposed to the hypoxic conditions in both cell types, which was inhibited by 2‐methoxyestradiol, a HIF‐1α inhibitor. These data suggest that hypoxia is a new sensitization mechanism for TRPV1, which might be relevant to diabetes‐related complications, and also for other diseases that are associated with acute hypoxia. TRPV1 was found to be sensitized upon in vitro overnight exposure to hypoxia and high glucose mainly by hypoxia in a PKCepsilon‐ and HIF‐1alpha‐dependent manner.

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.

Activation of Polycystic Kidney Disease-2-like 1 (PKD2L1)-PKD1L3 Complex by Acid in Mouse Taste Cells

Five basic tastes (bitter, sweet, umami, salty, and sour) are detected in the four taste areas where taste buds reside. Although molecular mechanisms for detecting bitter, sweet, and umami have been well clarified, those for sour and salty remain poorly understood. Several channels including acid-sensing ion channels have been proposed as candidate sour receptors, but they do not encompass all sour-sensing abilities in vivo. We recently reported a novel candidate for sour sensing, the polycystic kidney disease-2-like 1 (PKD2L1)-PKD1L3 channel complex. This channel is not a traditional ligand-gated channel and is gated open only after removal of an acid stimulus, called an off response. Here we show that off responses upon acid stimulus are clearly observed in native taste cells from circumvallate, but not fungiform papillae, of glutamate decarboxylase 67-green fluorescent protein (GAD67-GFP) knock-in mice, from which Type III taste cells can be visualized, using Ca2+ imaging and patch clamp methods. Off responses were detected in most cells where PKD2L1 immunoreactivity was observed. Interestingly, the pH threshold for acid-evoked intracellular Ca2+ increase was around 5.0, a value much higher than that observed in HEK293 cells expressing the PKD2L1-PKD1L3 complex. Thus, PKD2L1-PKD1L3-mediated acid-evoked off responses occurred both in HEK293 cells and in native taste cells, suggesting the involvement of the PKD2L1-PKD1L3 complex in acid sensing in vivo.