Yusuke Ishida

Amiloride-blockable acid-sensing ion channels are leading acid sensors expressed in human nociceptors

Many painful inflammatory and ischemic conditions such as rheumatoid arthritis, cardiac ischemia, and exhausted skeletal muscles are accompanied by local tissue acidosis. In such acidotic states, extracellular protons provoke the pain by opening cation channels in nociceptors. It is generally believed that a vanilloid receptor subtype-1 (VR1) and an acid-sensing ion channel (ASIC) mediate the greater part of acid-induced nociception in mammals. Here we provide evidence for the involvement of both channels in acid-evoked pain in humans and show their relative contributions to the nociception. In our psychophysical experiments, direct infusion of acidic solutions (pH > or = 6.0) into human skin caused localized pain, which was blocked by amiloride, an inhibitor of ASICs, but not by capsazepine, an inhibitor of VR1. Under more severe acidification (pH 5.0) amiloride was less effective in reducing acid-evoked pain. In addition, capsazepine had a partial blocking effect under these conditions. Amiloride itself neither blocked capsaicin-evoked localized pain in human skin nor inhibited proton-induced currents in VR1-expressing Xenopus oocytes. Our results suggest that ASICs are leading acid sensors in human nociceptors and that VR1 participates in the nociception mainly under extremely acidic conditions.

Amiloride-Insensitive Currents of the Acid-Sensing Ion Channel-2a (ASIC2a)/ASIC2b Heteromeric Sour-Taste Receptor Channel

Acid-sensing ion channel-2a (ASIC2a) is an amiloride-blockable proton-gated cation channel, probably contributing to sour-taste detection in rat taste cells. To isolate another subtype of the sour-taste receptor, we screened a rat circumvallate papilla cDNA library and identified ASIC2b, an N-terminal splice variant of ASIC2a. Reverse transcription-PCR analyses confirmed the expression of ASIC2b transcripts in the circumvallate papilla and, moreover, demonstrated its expression in the foliate and fungiform papillae. Immunohistochemical analyses revealed that ASIC2b, as well as ASIC2a, was expressed in a subpopulation of taste cells in the circumvallate, foliate, and fungiform papillae, and some of the cells displayed both ASIC2a and ASIC2b immunoreactivities. Subsequent coimmunoprecipitation studies with circumvallate papillae extracts indicated that ASIC2b associated with ASIC2a to form assemblies and, together with our immunohistochemical findings, strongly suggested that both ASIC2 subunits formed heteromeric channels in taste cells in the circumvallate, foliate, and fungiform papillae. Oocyte electrophysiology demonstrated that the ASIC2a/ASIC2b channel generated maximal inward currents at a pH of ≤2.0, which is in agreement with the in vivo pH sensitivity of rat taste cells, and that the amiloride sensitivity of the heteromer decreased with decreasing pH and was almost completely abolished at a pH of 2.0. These findings provide persuasive explanations for the amiloride insensitivity of acid-induced responses of rat taste cells.

Cellular localization of P2X7 receptor mRNA in the rat brain

P2X7 receptor is a member of the P2X family of ATP-gated ion channels. The cellular localization of P2X7 receptors in the central nervous system remains controversial because immunohistochemical staining patterns are inconsistent among antibodies. Here we examined the precise distribution of P2X7 mRNAs in the rat brain using isotopic in situ hybridization. P2X7-positive glial-like small cells were sporadically scattered in almost all areas of the brain. P2X7-positive glial-like small cells were also observed in nerve fiber tracts such as the anterior commissure, corpus callosum (CC), optic tract, and internal capsule. P2X7-positive neurons were found in the anterior olfactory nucleus, cerebral cortex, piriform cortex (Pir), lateral septal nucleus (LS), hippocampal pyramidal cell layers of CA1, CA3, CA4, pontine nuclei, external cuneate nucleus, and medial vestibular nucleus. P2X7 hybridization signals were also observed in the motor neurons of the trigeminal motor nucleus, facial nucleus, hypoglossal nucleus, and the anterior horn of the spinal cord. P2X7 mRNA was expressed in the ependymal cells around the olfactory ventricle, lateral ventricles (LV), third ventricle (3V), cerebral aqueduct (Aq), fourth ventricle (4V), and central canal. The P2X7 hybridization signal was also very strong in the area postrema (AP). The double staining experiments demonstrate that neurons, oligodendrocytes, and microglia expressed P2X7 receptor mRNAs. These findings suggest that P2X7 receptors may play a variety of roles in a wide range of cell types in the brain.

Differential Localizations of the Transient Receptor Potential Channels TRPV4 and TRPV1 in the Mouse Urinary Bladder

We studied the localization and physiological functions of the transient receptor potential (TRP) channels TRPV1 (TRP vanilloid 1) and TRPV4 (TRP vanilloid 4) in the mouse bladder, because both channels are thought to be mechanosensors for bladder distention. RT-PCR specifically amplified TRPV4 transcripts from the urothelial cells, whereas TRPV1 transcripts were barely detectable. ISH experiments showed that TRPV4 transcripts were abundantly expressed in the urothelium, whereas TRPV1 transcripts were not detectable in the urothelial cells. Immunoblotting and IHC studies showed that TRPV4 proteins were mainly localized at the basal plasma membrane domains of the basal urothelial cells. In contrast, TRPV1-immunoreactivities were found not in the urothelial cells but in the nerve fibers that innervate the urinary bladder. In Ca2+-imaging experiments, 4α-phorbol 12,13-didecanoate, a TRPV4 agonist, and hypotonic stimuli induced significant increases in intracellular calcium ion concentration ([Ca2+]i) in isolated urothelial cells, whereas capsaicin, a TRPV1 agonist, showed no marked effect on the cells. These findings raise the possibility that, in mouse urothelial cells, TRPV4 may contribute to the detection of increases in intravesical pressure related to the micturition reflex.

Vanilloid receptor subtype-1 (VR1) is specifically localized to taste papillae.

Pungent sensation of hot peppers is thought to be mediated by vanilloid receptor subtype-1 (VR1), which can be activated by capsaicin, but there is little information regarding its histological localization in the tongue. We examined the immunohistochemical distribution of VR1 in the rat tongue. Intensely labeled VR1-immunoreactive (VR1-IR) fibers were concentrated in the circumvallate, foliate, and fungiform papillae, while sparse VR1-IR fibers were scattered throughout the tongue. VR1-positive taste-bud cells were not observed. Many VR1-positive nerve fibers surrounded the furrows of the circumvallate and foliate papillae, forming plexuses. Some of these VR1-positive fibers penetrated the apical epithelium and the trench wall epithelium, while a few of them entered taste buds. These VR1 distribution patterns resembled those of substance P (SP) and the calcitonin gene-related peptide (CGRP). Double-labeling experiments revealed that most of the VR1-immunoreactivity coexisted with SP- or CGRP-immunoreactivity in the nerve terminals in the circumvallate and foliate papillae. On the other hand, in the fungiform papillae, most of the VR1-IR fibers were positive for SP, but fewer were also positive for CGRP. Although VR1-immunoreactivity was not observed in taste-bud cells, our findings that a large number of VR1-IR fibers concentrated in the taste papillae suggest that capsaicin easily reaches the VR1 nerve terminals because of its lipophilic nature.

P2X2‐ and P2X3‐positive fibers in fungiform papillae originate from the chorda tympani but not the trigeminal nerve in rats and mice

The subtype 2 and subtype 3 ionotropic purinergic receptors (P2X receptors) are crucial for gustation, but the distribution of these receptors in the geniculate ganglion (GG) and their colocalization in tongue papillae remain unknown. Here we investigated the expression and colocalization of P2X2 and P2X3 receptors in the GG and fungiform papillae in rats and mice by using in situ hybridization and immunohistochemistry. In both species, P2X2 transcripts and immunoreactivity were detected in approximately 50–60% of GG neuronal somata, whereas those of P2X3 were observed in almost all neurons. In each fungiform papilla, immunoreactivity for both receptors was mostly colocalized and was seen in nerve fibers and their bundles concentrated in the taste buds. Because it is well known that the P2X receptors are involved in not only taste but also nociception, we determined whether the expression originated from the chorda tympani nerve (CT, gustatory) or trigeminal nerve (somatosensory) by cutting the CT in both animals. Most P2X2 and P2X3 immunoreactivity in the fungiform papillae was abolished after transection, although the nerve fiber immunoreactivity of transient receptor potential V1 (a marker of somatosensory nerve fibers) remained unchanged, indicating that most fungiform papillae nerve fibers with P2X2 and P2X3 receptors were derived from CT. Taken together, these findings suggest that most P2X2 and P2X3 receptors in fungiform papillae are used for gustation rather than somatosensation. J. Comp. Neurol. 514:131–144, 2009.

Functional expression of transient receptor potential vanilloid 3 (TRPV3) in corneal epithelial cells: Involvement in thermosensation and wound healing

Transient receptor potential vanilloid 3 (TRPV3), a member of the calcium-permeable thermosensitive TRP (thermoTRP) subfamily of receptors, is an important cutaneous sensor that detects thermal and chemical stimuli. TRPV3 is activated by innocuous warm temperature stimuli (>33 degrees C) and a variety of physiologically active substances. While the corneal epithelium is known to respond to such stimuli, it is unknown whether TRPV3 is involved in this phenomenon. We show here that TRPV3 mRNA and protein are abundantly expressed in the epithelial cells of human and mouse cornea. Carvacrol, an agonist of TRPV3, elevated cytosolic Ca2+ concentration in both primary mouse corneal epithelial cells and cultured human corneal epithelial cells (HCE-T cells). The response to carvacrol was inhibited by ruthenium red, a TRPV channel antagonist. Moreover, repetitive agonist stimulation sensitized the response with gradually increasing amplitude, suggesting that the TRPV3 in the cornea has similar physiological and pharmacological characteristics to that in skin keratinocytes. Finally, a wound healing assay revealed that appropriate calcium ion influx via activated TRPV3 in corneal epithelial cells accelerated their proliferation. Thus, functional TRPV3 is present in corneal epithelial cells and may play a role not only in thermosensation, but also in the regulation of cell proliferation.

The 5-HT3 receptor is essential for exercise-induced hippocampal neurogenesis and antidepressant effects

Exercise has a variety of beneficial effects on brain structure and function, such as hippocampal neurogenesis, mood and memory. Previous studies have shown that exercise enhances hippocampal neurogenesis, induces antidepressant effects and improves learning behavior. Brain serotonin (5-hydroxytryptamine, 5-HT) levels increase following exercise, and the 5-HT system has been suggested to have an important role in these exercise-induced neuronal effects. However, the precise mechanism remains unclear. In this study, analysis of the 5-HT type 3A receptor subunit-deficient (htr3a−/−) mice revealed that lack of the 5-HT type 3 (5-HT3) receptor resulted in loss of exercise-induced hippocampal neurogenesis and antidepressant effects, but not of learning enhancement. Furthermore, stimulation of the 5-HT3 receptor promoted neurogenesis. These findings demonstrate that the 5-HT3 receptor is the critical target of 5-HT action in the brain following exercise, and is indispensable for hippocampal neurogenesis and antidepressant effects induced by exercise. This is the first report of a pivotal 5-HT receptor subtype that has a fundamental role in exercise-induced morphological changes and psychological effects.

TRPV3, a thermosensitive channel is expressed in mouse distal colon epithelium.

The thermo-transient receptor potential (thermoTRP) subfamily is composed of channels that are important in nociception and thermo-sensing. Here, we show a selective expression of TRPV3 channel in the distal colon throughout the gastrointestinal tract. Expression analyses clearly revealed that TRPV3 mRNA and proteins were expressed in the superficial epithelial cells of the distal colon, but not in those of the stomach, duodenum or proximal colon. In a subset of primary epithelial cells cultured from the distal colon, carvacrol, an agonist for TRPV3, elevated cytosolic Ca(2+)concentration in a concentration-dependent manner. This response was inhibited by ruthenium red, a TRPV channel antagonist. Organotypic culture supported that the carvacrol-responsive cells were present in superficial epithelial cells. Moreover, application of carvacrol evoked ATP release in primary colonic epithelial cells. We conclude that TRPV3 is present in absorptive cells in the distal colon and may be involved in a variety of cellular functions.

Distinct expression of cold receptors (TRPM8 and TRPA1) in the rat nodose-petrosal ganglion complex

TRPM8 and TRPA1 are cold-activated transient receptor potential (TRP) cation channels. TRPM8 is activated by moderate cooling, while TRPA1 is activated by extreme, noxious cold temperatures. These cold receptors are expressed in different subpopulations of primary afferent neurons. TRPA1 is co-expressed in a subpopulation of somatosensory neurons expressing TRPV1, which is activated by heat. However, the distribution and co-expression of these channels in the nodose-petrosal ganglion complex, which contains the jugular (JG), petrosal (PG), and nodose ganglia (NG) (mainly involved in putative somatic, chemo- and somato-sensation, and somato and visceral sensation, respectively), remain unknown. Here, we conducted in situ hybridization analysis of the rat nodose-petrosal ganglion complex using specific riboprobes for TRPM8, TRPA1, and TRPV1 to compare the features of the cranial sensory ganglia. Hybridization signals for TRPA1 were diffusely observed throughout these ganglia, whereas TRPM8 transcripts were seen in the JG and PG but not in the NG. We retrogradely labeled cranial nerve X with Fast Blue (fluorescent dye) and found TRPM8 transcripts in the jugular-vagal ganglion but not the NG neurons. TRPA1 transcripts were not detected in TRPM8-expressing neurons but were present in the subpopulation of TRPV1-expressing visceral sensory neurons. Taken together, these findings support that in the vagal system the expression of cold-activated TRP channels differs between nodose- and jugular-ganglion neurons suggesting different mechanisms of cold-transduction and that the TRPA1 distribution is consistent with its proposed function as a cold-sensing receptor in the visceral system.