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Acid-sensing ion channels (ASICs) are strong candidates for mammalian mechanoreceptors. We investigated whether mouse acid-sensing ion channel-1b (ASIC1b) is sensitive to mechanical stimuli using oocyte electrophysiology, because ASIC1b is located in the mechanosensory stereocilia of cochlear hair cells. Hypotonic stimuli that induced membrane stretch of oocytes evoked no significant current in ASIC1b-expressing oocytes at pH 7.5. However, acid (pH 4.0 or 5.0)-evoked currents in the oocytes were substantially enhanced by the hypotonicity, showing mechanosensitivity of ASIC1b and possible mechanogating of the channel in the presence of other components. Interestingly, the ASIC1b channel was permeable to K(+) (a principal charge carrier for cochlear sensory transduction) and the affinity of the channel for amiloride (IC(50) (inhibition constant)=approximately 48.3 microM) was quite similar to that described for the mouse hair cell mechanotransducer current. Taken together, these data raise the possibility that ASIC1b participates in cochlear mechanoelectrical transduction.

We electrophysiologically investigated the effects of nafamostat mesilate (NM: 6-amidino-2-naphthyl p-guanidinobenzoate dimethanesulfonate) and its two metabolites, 6-amidino-2-naphthol (AN) and p-guanidinobenzoic acid (PGBA), on three distinct types of human acid-sensing ion channels (ASICs). Acid-evoked inward currents at a holding potential of -60mV in ASIC1a- and ASIC2a-expressing oocytes were decreased by extracellular application of NM in a concentration-dependent manner with IC(50) (inhibition constant) values of approximately 13.5 and 70.6microM, respectively. The NM application also produced concentration-dependent inhibition of the initial-phase transient component of biphasic ASIC3 currents with an IC(50) value of approximately 2.5microM. Application of AN showed weak blocking effects on the ASIC1a, ASIC2a, and transient ASIC3 currents with IC(50) values of approximately 1.2, 1.3, and 0.14mM, respectively, whereas PGBA was insensitive to their currents.

Taste receptor cells detect gustatory stimuli using a complex arrangement of ion channels, G protein-coupled receptors, and signaling cascades. Sour and salty tastes are detected by ion channels in the rat. Using a combination of homology screening and functional expression approaches, we screened a rat circumvallate papilla cDNA library and identified acid-sensing ion channel-2a (ASIC2a) and ASIC2b as candidates for the rat sour-sensing channels. In situ hybridization and reverse transcription-polymerase chain reaction experiments revealed that ASIC2a and ASIC2b transcripts were localized in taste bud cells. Immunohistochemistry and immunoprecipitation also revealed that both subunits were expressed in a subset of taste cells and that some of the cells expressed ASIC2a/ASIC2b heteromeric assemblies. Electrophysiological studies demonstrated that stimulation of acetic acid produced larger ASIC2 currents than did hydrochloric acid at the same pH. ASIC2a/ ASIC2b channels generated maximal inward currents at pH

We investigated whether amiloride-blockable proton-gated cation channels ASIC1a (acid-sensing ion channel-1a) and ASIC1b are expressed in the stereocilia of mouse cochlear hair cells. In-situ hybridization studies showed that ASIC1b transcripts, but not ASIC1a transcripts, were expressed in the inner and outer hair cells. Fluorescent immunohistochemical and immunogold electron microscopic analyses revealed that the ASIC1b channels were located at the insertions of the stereocilia into the hair cells. Our findings provide a novel molecular key to the understanding of cochlear physiology and pathophysiology.

The activation of nociceptors by protons plays a crucial role in the initiation and maintenance of acidosis-linked pain. Acid-sensing ion channel (ASIC) and transient receptor potential/vanilloid receptor subtype-1 (TRPV1) encode proton-activated cation channels expressed by nociceptors and the opening of these channels results in nociceptor excitation. Histological relations among ASIC clones and the colocalization of each ASIC subunit and TRPV1 within single sensory neurons were examined on serial sections of rat dorsal root ganglia (DRG) using in situ hybridization histochemistry. ASIC1a transcripts were expressed in 20-25% of the DRG neurons, and most of the neurons had small (<30 microm)-diameter cell bodies. ASIC1b transcripts and ASIC3 transcripts were expressed in approximately 10% and 30-35% of the DRG neurons, respectively, and the greater part of each population was located in small-to-medium (30-50 microm)-diameter cells. The ASIC1a transcripts and ASIC1b transcripts were basically localized in the distinct populations of the DRG neurons, while approximately 20% of the ASIC1a-positive neurons and approximately 10% of the ASIC1b-positive neurons expressed ASIC3 transcripts. TRPV1 transcripts were expressed in 35-40% of the DRG neurons, and most of the TRPV1-positive neurons had small-diameter cell bodies. Intense expression signals for ASIC1a transcripts were detected in 40-45% of the TRPV1-positive neurons. Neurons expressing both ASIC1b and TRPV1 transcripts were barely detected in the DRG. Approximately 30% of the TRPV1-positive neurons expressed ASIC3 transcripts, and the double-labeled neurons were comprised of both small-diameter and medium-diameter cells. Approximately 13% of the TRPV1-positive neurons expressed both ASIC1a and ASIC3 transcripts.

Acid-sensing ion channel-2a (ASIC2a) is a proton-gated cation channel, probably contributing to sour-taste detection in rat taste cells. We found the transcripts of ASIC2b in the circumvallate, foliate and fungiform papillae. Immunohistochemical analyses also revealed that ASIC2b, as well as ASIC2a, was expressed in a subpopulation of taste cells, and some of the cells displayed both ASIC2a and ASIC2b immunoreactivities. Coimmunoprecipitation studies with circumvallate papillae extracts indicated that ASIC2b associated with ASIC2a to form assemblies. Oocyte electrophysiology demonstrated that the ASIC2a/ASIC2b channel generated amiloride-insensitive currents at pH 2.0. These findings provide persuasive explanations for the amiloride insensitivity of acid-induced responses of rat taste cells. Acid-sensing ion channels (ASICs) also mediate acid-induced nociception in mammals. 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 TRPV1. 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 did not block capsaicin-evoked localized pain. Our results suggest that ASICs (ASIC1a and ASIC3) are leading acid sensors in human nociceptors and that TRPV1 participates in the nociception mainly under extremely acidic conditions.

Taste cells located in taste buds respond to gustatory stimuli using a complex arrangement of ion channels, receptor molecules and signaling cascades. Previous electrophysiological experiments have shown that sour taste (essentially a taste of protons) is mediated, at least in part, by apically located amiloride-sensitive channels in the rat. Here, the molecular cloning of sour-taste receptor genes is described. Using a combination of homology screening and functional expression approaches, we screened a rat circumvallate papilla cDNA library and identified acid-sensing ion channel-2a (ASIC2a) and ASIC2b, amiloride-sensitive proton-activated cation channels. In situ hybridization and reverse transcription-polymerase chain reaction experiments showed that ASIC2a and ASIC2b transcripts were localized in taste cells. Immunohistochemical and immunoprecipitation studies revealed that both channels were expressed in a subset of taste cells and that some of the cells expressed ASIC2a/ASIC2b heteromeric assemblies. Immunoelectron microscopic analyses demonstrated that some of the ASIC2a-immunopositive cells had the characteristics of type III cells, which make synaptic contacts with gustatory afferent neurons. According to our electrophysiological studies, stimulation by acetic acid generated larger inward currents in ASIC2a- or ASIC2a/ASIC2b-expressing oocytes than those induced by hydrochloric acid at the same proton concentration and these findings are in good agreement with the well-known taste phenomenon that acetic acid is more sour than hydrochloric acid at equal pH. Taken together, the present results strongly suggest that mucosal protons dissociated from sour-taste substances induce taste cell depolarization through the activation of the ASIC2a and ASIC2a/ASIC2b channels, which leads to transmitter release onto gustatory neurons.

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.

We have isolated a cDNA encoding a splice variant of ASIC (acid-sensing ion channel)-beta from the rat trigeminal ganglion. This clone, designated ASIC-beta2, showed a 342 base deletion just after the first transmembrane domain in ASIC-beta. RT-PCR experiments revealed that ASIC-beta2 was expressed exclusively in the trigeminal ganglion and dorsal root ganglion. In situ hybridization showed that ASIC-beta2 mRNA was concentrated in both small diameter and large diameter neurons and co-localized with ASIC-beta mRNA within single sensory neurons in the trigeminal ganglion. When expressed in Xenopus oocytes, ASIC-beta2 was inactive by itself. However, it associated with ASIC-beta to form heteromers, which display lower affinity for protons than ASIC-beta alone.

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.

We studied expression of an osmoprotective gene, sodium/myo-inositol cotransporter (SMIT) in Marmarou's animal model for human diffuse brain injury by in situ hybridization and immunohistochemistry. In rats with diffuse brain injury, transient upregulation of SMIT mRNA was exclusively observed in the lateral area of pyramidal tract in lower brainstem. The expression was induced at 1 h after injury, peaked at 24 h, and returned to almost control levels at 48 h. Upregulated expression was found mainly in small glia-like cells. By immunohistochemistry using antibodies to phosphorylated mitogen-activated protein (MAP) kinases, inductions of phosphorylated p44/42 MAP kinase were also observed after diffuse brain injury. Interestingly, the distribution patterns of induced phosphorylated p44/42 MAP kinase were completely coincident with those of upregulated SMIT mRNA after diffuse brain injury. These results suggest that diffuse brain injury induces local expression of SMIT by activation of p44/42 MAP kinase cascade. The confined SMIT induction may reflect regional differences of damage and/or cellular differences in sensitivity to neuropathological stresses caused by this injury.