Distribution of ASIC4 transcripts in the adult wild-type mouse brain.

Acid-sensing ion channel 4 (ASIC4) belongs to the ASIC gene family of neuronal proton-gated cation channels, and is the least understood subtype among the members. Previous studies of ASIC4 expression in the mammalian central nervous system have shown that ASIC4 is abundantly expressed in the spinal cord and in various brain regions, such as the cerebral cortex, the hippocampus, and the cerebellum. However, the detailed distribution of ASIC4 transcripts in mammalian brains still remains to be elucidated. In the present study, radioactive in situ hybridization histochemistry with an ASIC4-specific cRNA probe was performed on wild-type mouse brains, followed by X-gal staining experiments with Asic4-lacZ reporter mice Asic4tm1a(KOMP)Mbp. It was found that ASIC4 mRNAs were widely expressed throughout the wild-type brain, but preferentially concentrated in the olfactory bulb, the piriform cortex, the caudate putamen, the preoptic area, the paraventricular nucleus, the medial habenular nucleus, the pretectal area, the lateral geniculate nucleus, the amygdaloid complex, the superior colliculus, the interpeduncular nucleus, and the granule cell layer of the ventral hippocampus, and these results were in agreement with the X-gal-positive reactions observed in the mutant brain. In addition, X-gal staining combined with immunohistochemistry identified intense signals for ASIC4 transcriptional activity in most of the choline acetyltransferase (ChAT)-positive principal neurons located in the basal forebrain cholinergic nuclei. Our data provide useful information to speculate possible roles of ASIC4 in diverse brain functions.

Acid inhibits TRPV4-mediated Ca²âº influx in mouse esophageal epithelial cells.

BACKGROUND: The transient receptor potential vanilloid 4 (TRPV4), a thermo-sensitive stretch-activated cation channel, is expressed in the skin stratified squamous epithelium, contributing to the acquisition of barrier function. Similarly, functional TRPV4 may be located in the stratified squamous epithelial lining of the esophagus, being involved in the pathogenesis of gastroesophageal reflux disease (GERD). Here we investigated the expression of TRPV4 in the mouse esophageal epithelium. METHODS: TRPV4 expression at the mRNA and protein levels was examined by reverse transcription-polymerase chain reaction (RT-PCR), in situ hybridization, and immunohistochemistry. A calcium imaging technique and ATP assay were used to evaluate the functionality of TRPV4 in freshly isolated esophageal epithelial cells. KEY RESULTS: Transcripts and proteins encoding TRPV4 were colocalized in the basal and intermediate layers of the esophageal epithelium. Both 4α-phorbol 12,13- didecanoate (4α-PDD), a selective agonist for TRPV4, and hypo-osmolar solution (160 mOsm) elevated the intracellular calcium concentration ([Ca(2+) ](i) ) in a subset of the isolated cells (70%). These [Ca(2+) ](i) increases were potently inhibited by ruthenium red (RuR), a TRPV4 channel antagonist, and were suppressed by extracellular protons (pH 5.0). Finally, application of 4α-PDD evoked ATP release in primary esophageal epithelial cells. CONCLUSIONS & INFERENCES: Acid-sensitive TRPV4 channels were mainly expressed in the esophageal epithelial cells of the basal and intermediate layers. Direct exposure of TRPV4-expressing cells to gastric acid, as would occur in cases of GERD, could influence their cellular functions, possibly aggravating the disease state.

Cloning and functional expression of ASIC-beta2, a splice variant of ASIC-beta.

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.

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.

Identification of coding single-nucleotide polymorphisms in human taste receptor genes involving bitter tasting.

T2Rs comprise a G-protein-coupled receptor superfamily that contains functionally defined bitter taste receptors. Here we report the tissue expressions and coding single-nucleotide polymorphisms (cSNPs) in human T2R genes (hT2R3, hT2R4, and hT2R5) on chromosome 7q31. We first demonstrated that hT2R3, hT2R4, and hT2R5 are actually expressed in the circumvallate papillae of the human tongue by reverse transcription-polymerase chain reaction (RT-PCR). We identified six cSNPs within the T2R receptor genes. The hT2R4 and hT2R5 contained four and one cSNPs that cause missense mutations, respectively, while hT2R3 included one silent nucleotide mutation. However, we could not find any nonsense mutations that resulted in a frameshift or a premature stop codon within the open reading frames. Genotype frequencies of each cSNP were in Hardy-Weinberg equilibrium. The identification of nucleotide diversity and amino acid polymorphisms in human T2R receptors could help clarify individual differences in the acceptability and sensitivity to bitter compounds.

A single amino acid substitution in MDEG2 specifically alters desensitization of the proton-activated cation current.

To clarify functional roles of MDEG2 (mammalian degenerin-2), a modulatory subunit of proton-activated cation channels, in MDEG1/MDEG2 heteromer, we replaced the Gly481 residue in MDEG2 with cysteine or phenylalanine and characterized them electrophysiologically. Expression of MDEG1 in Xenopus oocytes elicited proton-activated cation currents that were rapidly desensitized. Co-expression of MDEG1 and MDEG2 (or MDEG2-G481C) displayed similar current traces as MDEG1 alone. In contrast, co-expression of MDEG1 and MDEG2-G481F dramatically attenuated desensitization of the proton-activated currents. Interestingly, the G481F mutation in MDEG2 did not alter other channel properties including maximal whole-cell currents, ionic selectivity, pH-sensitivity and affinity for amiloride. Thus, Gly481 in MDEG2 specifically controls inactivation process of the MDEG1/MDEG2 channel.

Characterization of a mouse colonic system B(0+) amino acid transporter related to amino acid absorption in colon.

Previous experiments have shown that an amino acid transport system B(0+) transporter in cultured colonic epithelial cells mediates amino acid absorption. Here we describe the cloning and functional characterization of a system B(0+) transporter selectively expressed in the colon. Using the combination of an expressed sequence tag database search and RT-PCR approaches, we cloned a mouse colonic amino acid transporter, designated mCATB(0+). Northern blot analysis revealed that mCATB(0+) was selectively expressed in the large intestine. In situ hybridization showed the mCATB(0+) mRNA to be localized in absorptive epithelial cells. When expressed in Xenopus oocytes, mCATB(0+) exhibited a Na(+)-dependent stereoselective uptake and a broad specificity for neutral and cationic amino acids, which is characteristic of amino acid transport system B(0+). In vivo [(3)H]glycine uptake assay demonstrated that a system B(0+)-like transporter protein was expressed on the apical surface of the colonic absorptive cells. Our data suggest that a mouse colonic amino acid transporter mCATB(0+) may absorb amino acids from the intestinal contents in the colon.

Diffuse brain injury induces local expression of Na+/myo-inositol cotransporter in the rat 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.

A novel strategy for cancer therapy by mutated mammalian degenerin gene transfer.

Mammalian degenerin (MDEG) is a member of the amiloride-sensitive sodium ion channel family, and its site-directed active mutant (MDEG-G430F) induces massive Na+ influx into cells, leading to cell ballooning and cell bursting. We attempted a novel therapeutic approach for gastric cancers by transferring MDEG-G430F into cancer cells using tumor-specific promoters. In carcinoembryonic antigen (CEA)-producing gastric cancer cells, the level of cell death observed when MDEG-G430F was used with a CEA promoter was similar to that observed when using a potent nonspecific promoter such as the cytomegalovirus promoter. In an in vivo study, fusogenic liposome complexes containing MDEG-G430F driven by the CEA promoter were injected intraperitoneally into CEA-producing gastric cancer cells in a mouse peritoneal dissemination model. Although all 15 of the control mice were dead by 50 days postinoculation, 13 of the 15 mice treated with MDEG-G430F survived. These results indicate that transferring MDEG-G430F into cancer tissues using tumor-specific promoters can achieve striking and selective cancer cell death irrespective of the transcriptional efficiency of the promoters used in vivo, and suggest that this approach is a promising new strategy for cancer gene therapy.

Histochemical studies on glycosaminoglycans in Bruch's membrane of postnatal rat eyes.

Localization of glycosaminoglycans (GAG) in Bruch's membrane of postnatal rat eyeballs was examined histochemically. Fixed eyeballs from postnatal rats (ages 5 days and 8 weeks) were routinely processed and embedded in paraffin wax or Quetol 651 resin. Paraffin-embedded tissue sections were stained with hematoxylin and eosin or sensitized high iron diamine procedure in combination with selective methods such as GAG-degrading enzyme digestions and/or a chemical modification, and examined by light microscopy. Quetol 651-embedded ultrathin sections were stained with heavy metals and examined by electron microscopy. In rats at postnatal day 5, Bruch's membrane contained mainly chondroitin sulfate (CS) and heparan sulfate (HS). In contrast, at 8 weeks after birth the membrane included a large amount of dermatan sulfate (DS) and HS. According to electron microscopic findings, Bruch's membrane on day 5 consisted of only 3 layers without a central elastic layer. However, at 8 weeks after birth the membrane was constructed of 5 layers. These findings suggested that the difference in GAG molecular species in the membranes at 5 days and at 8 weeks after birth could be correlated with the development and maturation of the collagenous layer in Bruch's membrane. Moreover, maturation of Bruch's membrane may contributes to the architectural stabilization of the outer portions of the photoreceptor cells.

Retinal fascin: functional nature, subcellular distribution, and chromosomal localization.

PURPOSE: To investigate the functional properties, subcellular localization, and chromosomal location of retinal fascin. METHODS: Recombinant retinal fascin protein was prepared by using a baculovirus-insect expression system. Actin-binding and -bundling assays were performed with chick actin purified from skeletal muscle. Western blot analysis and immunohistochemistry were performed with a polyclonal antibody raised against bovine retinal fascin. A human retinal cDNA library was screened with an expressed sequence tag cDNA fragment. Chromosomal location was determined with fluorescent in situ hybridization. RESULTS: The actin-binding and actin-bundling activities of retinal fascin were demonstrated by high- and low-speed centrifugation assays. Formation of filamentous (F)-actin bundles by retinal fascin in vitro was also morphologically confirmed by fluorescence microscopy and electron microscopy. Immunohistochemical analysis revealed that retinal fascin protein was localized specifically in the outer and inner segments of the photoreceptor cells in the retina. Two splicing variants of human retinal fascin cDNA were also located. One clone encoded 492 amino acids, and the other encoded 516 amino acids. The gene encoding retinal fascin was localized to human chromosome 17, region q24 -25. CONCLUSIONS: These results suggest that retinal fascin may play a role in formation of unique morphologic structures of the photoreceptor cells and is a candidate gene for retinal degenerative disorders.

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 nM 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 approximately 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.

Regulatory mechanisms of TRAF2-mediated signal transduction by Bcl10, a MALT lymphoma-associated protein.

To elucidate the function of Bcl10, recently cloned as an apoptosis-associated gene mutated in MALT lymphoma, we identified its binding partner TRAF2, which mediates signaling via tumor necrosis factor receptors. In mammalian cells, low levels of Bcl10 expression promoted the binding of TRAF2 and c-IAPs. Conversely, excessive expression inhibited complex formation. Overexpressed Bcl10 reduced c-Jun N-terminal kinase activation and induced nuclear factor kappaB activation downstream of TRAF2. To determine whether overexpression of Bcl10 could perturb the regulation of apoptosis in vivo, we generated Bcl10 transgenic mice. In these transgenic mice, atrophy of the thymus and spleen was observed at postnatal stages. The morphological changes in these tissues were caused by acceleration of apoptosis in T cells and B cells. The phenotype of Bcl10 transgenic mice was similar to that of TRAF2-deficient mice reported previously, indicating that excessive expression of Bcl10 might deplete the TRAF2 function. In contrast, in the other organs such as the brain, where Bcl10 was expressed at high levels, no apoptosis was detected. The altered sensitivities to overexpressed Bcl10 may have been due to differences in signal responses to Bcl10 among cell types. Thus, Bcl10 was suggested to play crucial roles in the modulation of apoptosis associated with TRAF2.