Shinya Ugawa

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.

Receptor that leaves a sour taste in the mouth.

The ability to detect taste stimuli results from the activation of taste receptors located in taste-bud cells. There are several gustatory transduction mechanisms, involving membrane receptors, guanine-nucleotide-binding proteins (G proteins), second messengers and ion channels, but genes encoding taste receptors have not yet been identified. Here we identify a complementary DNA that encodes a receptor for sour tastes.

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.

TRPM8 activation suppresses cellular viability in human melanoma.

The transient receptor potential melastatin subfamily (TRPM), which is a mammalian homologue of cell death-regulated genes in Caenorhabditis elegans and Drosophila, has potential roles in the process of the cell cycle and regulation of Ca(2+) signaling. Among this subfamily, TRPM8 (also known as Trp-p8) is a Ca(2+)-permeable channel that was originally identified as a prostate-specific gene upregulated in tumors. Here we showed that the TRPM8 channel was expressed in human melanoma G-361 cells, and activation of the channel produced sustainable Ca(2+) influx. The application of menthol, an agonist for TRPM8 channel, elevated cytosolic Ca(2+) concentration in a concentration-dependent manner with an EC(50) value of 286 microM in melanoma cells. Menthol-induced responses were significantly abolished by the removal of external Ca(2+). Moreover, inward currents at a holding potential of -60 mV in melanoma cells were markedly potentiated by the addition of 300 microM menthol. The most striking finding was that the viability of melanoma cells was dose-dependently depressed in the presence of menthol. These results reveal that a functional TRPM8 protein is expressed in human melanoma cells to involve the mechanism underlying tumor progression via the Ca(2+) handling pathway, providing us with a novel target of drug development for malignant melanoma.

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.

Neuropsin regulates an early phase of Schaffer‐collateral long‐term potentiation in the murine hippocampus

We found that neuropsin, an extracellular matrix serine protease, has a regulatory effect on Schaffer‐collateral long‐term potentiation (LTP) in the mouse hippocampus. Bath application of 1–170 n m recombinant neuropsin modulated early phase LTP in the Schaffer‐collateral pathway with a ‘bell‐shape’ dose–response curve. The maximum enhancing activity (134% of control LTP) was found at ∼ 2.5 nm. Bath application of a neutralizing antibody against neuropsin in the hippocampal slice resulted in a marked inhibition of the tetanus‐induced early phase of LTP. The in vivo continuous intraventricular infusion of an antisense oligonucleotide against neuropsin significantly reduced the amplitude of the tetanus‐induced early phase of LTP in vitro. Neuropsin did not directly change the N‐methyl d‐aspartate (NMDA) current. Thus, neuropsin appears to act as a regulatory molecule in the early phase of LTP via its proteolytic function on extracellular matrix rather than affecting NMDA receptor‐mediated calcium increase.

Expression of receptor-activity modifying protein (RAMP) mRNAs in the mouse brain

Receptor activity modifying proteins (RAMPs) comprise a family of accessory proteins for G protein-coupled receptors (GPCRs). They function as receptor modulators that determine the ligand specificity of receptors for calcitonin gene-related peptide (CGRP), amylin and adrenomedullin (ADM). Here we demonstrate for the first time the characteristic distributions of the RAMP family mRNAs in the brain. Northern blot analysis revealed that mRAMP 1 and 3 mRNAs were intensely expressed in the brain, but mRAMP2 mRNA less abundantly. In situ hybridization studies showed the heterogenous and unique distributions of mRAMP mRNAs; RAMP1 mRNA was widely expressed throughout the brain including the cerebral cortex, caudate putamen, amygdaloid complex, hippocampus, cerebellum and ependyma, mRAMP2 was most abundant in the hippocampus, cerebellum, pia mater and blood vessels, while mRAMP3 was specifically distributed in a variety of thalamic nuclei and the cerebellum. In addition, RAMP1 and -3 genes were also detected in the subfornical organ and area postrema, which are members of circumventricular organs lacking blood-brain barrier. The present results help in understanding the diversification and regulation of receptor functions for calcitonin family peptides, and potentially other GPCRs in the brain.

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.

Protons activate the δ-subunit of the epithelial Na channel in humans

The amiloride-sensitive epithelial Na+ channel (ENaC) controls Na+ transport into cells and across epithelia. So far, four homologous subunits of mammalian ENaC have been isolated and are denoted as α, β, γ, and δ. ENaCδ can associate with β and γ subunits and generate a constitutive current that is 2 orders of magnitude larger than that of homomeric ENaCδ. However, the distribution pattern of ENaCδ is not consistent with that of the β and γ subunits. ENaCδ is expressed mainly in the brain in contrast to β and γ subunits, which are expressed in non-neuronal tissues. To explain this discrepancy, we searched for novel functional properties of homomeric ENaCδ and investigated the detailed tissue distribution in humans. When human ENaCδ was expressed in Xenopus oocytes and Chinese hamster ovary cells, a reduction of extracellular pH activated this channel (half-maximal pH for an activation of 5.0), and the acid-induced current was abolished by amiloride. The most striking finding was that the desensitization of the acid-evoked current was much slower (by ∼10% 120 s later), dissociating from the kinetics of acid-sensing ion channels in the degenerin/epithelial Na+ channel family, which were rapidly desensitized during acidification. RNA dot-blot analyses showed that ENaCδ mRNA was widely distributed throughout the brain and was also expressed in the heart, kidney, and pancreas in humans. Northern blotting confirmed that ENaCδ was expressed in the cerebellum and the hippocampus. In conclusion, human ENaCδ activity is regulated by protons, indicating that it may contribute to the pH sensation and/or pH regulation in the human brain.