Details of a Researcher - Misawa, Hidemi

Papers - Misawa, Hidemi

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Human choline acetyltransferase mRNAs with different 5'-region produce a 69-kDa major translation product

Misawa H., Matsuura J., Oda Y., Takahashi R., Deguchi T.

Molecular Brain Research 44 ( 2 ) 323 - 333 1997

Research paper (scientific journal), Joint Work, Accepted, ISSN 0169-328X

Choline acetyltransferase (ChAT, EC 2.3.1.6) is the biosynthetic enzyme for acetylcholine. We have previously shown that multiple ChAT mRNA species with different 5'-noncoding regions are expressed in the rat and mouse. However, the diversity of ChAT mRNA species in human has not completely been elucidated. In this work N1- and N2-type ChAT cDNAs were cloned from a human brain cDNA library and the N-exon located in the human ChAT gene. Polymerase chain reaction analysis indicates that four species of ChAT mRNAs (R-, N1-, N2- and M-types) are produced in human brain and spinal cord. In all human transcripts, the ATG initiation codon in the rat, mouse and pig was replaced by ACG, which does not serve as an initiation codon for translation. In vitro translation and mammalian expression analyses revealed that N1-, N2- and R-type mRNAs give rise to a single 69 kDa enzyme, while M-type mRNA produces both 82 and 69 kDa enzymes. The translation efficiency of M-type mRNA was lower than that of the other mRNA species. Moreover, the translation efficiency of human ChAT mRNAs was considerably lower than that of rat ChAT mRNA, suggesting that the ATG codons for human ChAT are unfavorable for translation initiation compared with the initiation codon for rat ChAT. These results provide rational explanations for the previous reports that human ChAT protein purified from the brain acid placenta had 66-70 kDa molecular mass, and that ChAT activity in a single motor neuron of human was far lower than that of other vertebrates. Sequencing of monkey ChAT gene showed that the initiation ATG in rodent ChAT was also replaced by ACA in the monkey.

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Molecular characterization of the mouse vesicular acetylcholine transporter gene

Naciff Jorge M., Misawa Hidemi, Dedman John R.

NeuroReport 8 ( 16 ) 3467 - 3473 1997

Research paper (scientific journal), Joint Work, Accepted, ISSN 0959-4965

The organization of the mouse choline acetyltransferase (ChAT) gene has been previously analyzed. Here we show that the first intron of the mouse ChAT gene contains an uninterrupted open reading frame. It is in the same transcriptional orientation as ChAT and encodes the vesicular acetylcholine (ACh) transporter (VAChT), the protein responsible for the translocation of cytoplasmic ACh into synaptic vesicles. The sequence of this transporter is very similar to the VAChT from rat and human (99% and 95% identity, respectively). Reverse transcription-polymerase chain reaction (RT-PCR) analysis showed expression of mouse VAChT mRNA in spinal cord, brain (excluding the cerebellum) and brain stem, but not in peripheral tissues such as liver and kidney. Transgenic mouse analysis revealed that the 5'-flanking region of the mouse ChAT gene encompasses regulatory elements that allowed elevated expression of VAChT in the cholinergic system of transgenic mice.

A subtype of κ-opioid receptor mediates inhibition of high-affinity GTPase inherent in G_i1 in Guinea pig cerebellar membranes

Ueda Hiroshi, Misawa Hidemi, Fukushima Nobuyuki, Katada Toshiaki, Ui Michio, Satoh Masamichi

Journal of Neurochemistry 66 ( 2 ) 845 - 851 1996.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-3042

The κ-opioid receptor agonists including U-50,488H and dynorphin A (1-17) in ranges of 0.1-100 nM inhibited the hydrolysis of GTP to GDP (P_i release) inherent in GTP-binding proteins (G proteins) in guinea pig cerebellar membranes. U-50,488H inhibited only high-affinity GTPase activity, not low-affinity activity. The action of this agonist was found to be biphasic, and there was no inhibition at concentrations >1 μM. The inhibition was abolished by pretreatment with preactivated pertussis toxin (PTX) at concentrations >1 μg/ml but not with preactivated cholera toxin (30 μg/ml). Similar blockade of κ-receptor-mediated inhibition was also observed when membranes were pretreated with a low concentration (8 μM) of N-ethylmaleimide (NEM) at low temperature (4°C), which alkylates the cysteine residue to be ADP-ribosylated by PTX; but this treatment caused no significant change in κ-agonist binding. When purified G_i1, but not G_o, was reconstituted into membranes pretreated with NEM, the κ-receptor-mediated inhibition was recovered. These findings suggest that a subtype of κ-opioid receptor is coupled to inhibition of intrinsic activity of G_i1.

A subtype of opioid κ-receptor is coupled to inhibition of Gil-mediated phospholipase C activity in the guinea pig cerebellum

Misawa Hidemi, Ueda Hiroshi, Katada Toshiaki, Ui Michio, Satoh Masamichi

FEBS Letters 361 ( 1 ) 106 - 110 1995.03

Research paper (scientific journal), Joint Work, Accepted, ISSN 0014-5793

PLC activity was stimulated either by 1-100 μM of GTP or by 100-3,000 μM Ca²⁺ in lysed synaptosomal membranes of the guinea pig cerebellum. The κ-opioid receptor agonist selectively inhibited the PLC activity stimulated by 100 μM GTP, but not by 100-3,000 μM Ca²⁺. Pretreatment of membranes with PTX abolished such a κ-agonist-induced inhibition of PLC activity. The reconstitution of Gi1, but not of Go purified from porcine brains with PTX-treated membranes showed a complete recovery of the κ-agonist-inhibition of PLC activity. These findings suggest that a novel subtype κ-receptor mediates inhibition of PLC through inhibiting the intrinsic activity of PTX-substrate G-proteins.

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cDNA cloning and chromosomal localization of the human ciliary neurotrophic factor gene

Yokoji Hidehiro, Ariyama Takeshi, Takahashi Ryosuke, Inazawa Johji, Misawa Hidemi, Deguchi Takeo

Neuroscience Letters 185 ( 3 ) 175 - 178 1995.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0304-3940

Full-length cDNA for human ciliary neurotrophic factor (CNTF) was isolated from a human sciatic nerve cDNA library. Sequence analysis revealed that the longest cDNA was comprised of a 48-bp 5′-untranslated region, a 600-bp coding region and a 1207-bp 3′-untranslated region containing four ATTA pentamer motifs and a polyadenylation sequence. The transcription starting point was assigned at 81 bp upstream of the initiation methionine by 5′ RACE analysis. Using the cDNA and genomic DNA fragment including the entire intron region as mixed probes, the human CNTF gene was localized to the long arm of chromosome 11 at region q12 by fluorescence in situ hybridization.

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Coordinate expression of vesicular acetylcholine transporter and choline acetyltransferase in sympathetic superior cervical neurones

Misawa H., Takahashi R., Deguchi T.

NeuroReport 6 ( 7 ) 965 - 968 1995

Research paper (scientific journal), Joint Work, Accepted, ISSN 0959-4965

The neurotransmitter acetylcholine is synthesized by choline acetyltransferase (ChAT) and transported into synaptic vesicles by the vesicular acetylcholine transporter (VAChT). Recently it has been reported that the entire coding region of VAChT mRNA is located in the first intron of the ChAT gene. In this study, ChAT and VAChT mRNAs were analysed in cultured sympathetic neurones. Cholinergic differentiation factor/leukaemia inhibitory factor and ciliary neurotrophic factor induced strong expression of ChAT and VAChT mRNAs in parallel. RT-PCR analysis of ChAT mRNAs revealed that five types of ChAT transcripts which differed in the 5' non coding regions were increased. RT-PCR analysis of VAChT mRNA indicated that the cytokines induced only VAChT mRNA species which did not contain the R-exon, and not those containing the R-exon. The results indicate that ChAT and VAChT expressions are coordinately but differentially regulated in cultured sympathetic neurones.

Discrete acetylcholine release from neuroblastoma or hybrid cells overexpressing choline acetyltransferase into the neuromuscular synaptic cleft

Zhong Zhen Guo, Kimura Yasuhiro, Noda Mami, Misawa Hidemi, Higashida Haruhiro

Neuroscience Research 22 ( 1 ) 81 - 88 1995

Research paper (scientific journal), Joint Work, Accepted, ISSN 0168-0102

Neuroblastoma (clones NS-20Y, N1E-115, and Neuro2A) and neuroblastoma × glioma hybrid (NG108-15) cells were transfected with mouse choline acetyltransferase (ChAT) complementary DNA (cDNA) or vector DNA alone and stably transformed cell lines were established to examine their ability to secrete acetylcholine (ACh). Membrane potentials were recorded from either presynaptic neuroblastoma and hybrid cells or postsynaptic myotubes in co-culture. After transformation with ChAT, synapses were formed and miniature end-plate potentials (MEPPs) were recorded in myotubes co-cultured with Neuro2A and N1E-115 cells, while parental and mock-transfected control cells totally lacked this ability. The rate of synapse formation and/or MEPP frequency was higher in transformed NG108-15 hybrid and NS-20Y cells than that in the control cells. Action potentials of NS-20Y, Neuro2A or NG108-15 cells overexpressing ChAT were able to evoke end-plate potentials in myotubes, though the average quantum content of these cells was 0.04-0.14, which is as low as the control value. The results show that increased concentrations of ACh by ChAT cDNA transfection reveal a masked property in vesicular ACh release from Neuro2A and N1E-115 cells with no endogenous ChAT activity, or modify their secretory capacity upwardly from NG108-15 and NS-20Y cells with endogenous activity.

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Overexpression of choline acetyltransferase reconstitutes discrete acetylcholine release in some but not all synapse formation-defective neuroblastoma cells

Zhong ZG G., Misawa H., Furuya S., Kimura Y., Noda M., Yokoyama S., Higashid H.

Journal of Physiology - Paris 89 ( 3 ) 137 - 145 1995

Research paper (scientific journal), Joint Work, Accepted, ISSN 0928-4257

Secretion of acetylcholine (ACh) in neuroblastoma cells overexpressing choline acetyltransferase (ChAT) was examined. With transient transfection of ChAT cDNA, neuroblastoma cells, which have no endogenous ChAT and either adhere to myotubes or not, failed to form functional synapses, and thus no evidence for release of ACh was detected. Stable neuroblastoma cell lines overexpressing ChAT accumulated ACh inside the cell, and slowly released ACh to the outside of the cell in a calcium-independent fashion. However, after co-culturing them with rat muscle cells, these transformed cells adhered to myotubes and ACh was secreted in a discrete fashion into the synaptic cleft efficiently in some neuroblastoma cell lines but rather inefficiently in another cell line. The results show that the latent secretion machinery of ChAT overexpressing neuroblastoma cells either is competent or possess defect(s) in ACh release.

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A null mutation in the human CNTF gene is not causally related to neurological diseases

Takahashi Ryosuke, Yokoji Hidehiro, Misawa Hidemi, Hayashi Michiyuki, Hu Jianguo, Deguchi Takeo

Nature Genetics 7 ( 1 ) 79 - 84 1994.05

Research paper (scientific journal), Joint Work, Accepted, ISSN 1061-4036

We report a null mutation in the human ciliary neurotrophic factor gene (CNTF). The mutated allele shows a G to A transition producing a new splice acceptor site and the resulting mRNA species codes for an aberrant protein. Analysis of tissue samples and transfection of CNTF minigenes into cultured cells demonstrates that the mutated allele expresses only the mutated mRNA species. In 391 Japanese people tested, 61.9% were normal homozygotes, 35.8% heterozygotes and 2.3% mutant homozygotes. The distribution of the three genotypes is similar in healthy and neurological disease subjects, indicating that human CNTF deficiency is not causally related to neurological diseases.

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Calcium-independent release of acetylcholine from stable cell lines expressing mouse choline acetyltransferase cDNA

Misawa Hidemi, Takahashi Ryosuke, Deguchi Takeo

Journal of Neurochemistry 62 ( 2 ) 465 - 470 1994.02

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-3042

Stably transfected cells expressing mouse choline acetyltransferase (ChAT) cDNA were established, and the synthesis and release of acetylcholine (ACh) were examined. A cDNA clone coding for mouse ChAT was inserted into an expression vector (pEF321) containing a promoter for human elongation factor 1α to construct pEFmChAT. Neuronal (NG108-15, NS20Y, N1E115, and Neuro2A) and nonneuronal cell lines (L cells and NIH3T3) were transfected with pEFmChAT, and the cell lines that stably expressed high ChAT activity were selected. These cells expressed the 66-kDa ChAT protein and accumulated ACh mostly in the cytosol. The concentration of intracellular ACh in the cells increased upon raising the choline level in the medium. The cells continuously released ACh in a Ca²⁺-independent fashion. Neither high K⁺ nor calcium ionophore stimulated release of ACh from the cells.

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Transcriptional regulation of choline acetyltransferase gene by cyclic AMP

Misawa Hidemi, Takahashi Ryosuke, Deguchi Takeo

Journal of Neurochemistry 60 ( 4 ) 1383 - 1387 1993.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-3042

The effect of cyclic AMP on the gene expression of cholinc acetyltransferase (ChAT) was studied in NG108-15, mouse neuroblastoma and rat glioma hybrid cell lines. Addition of dibutyryl cyclic AMP to the culture medium increased both the ChAT mRNA level and ChAT activity twofold. Polymerase chain reaction analysis of the ChAT mRNA indicated that, among the multiple mRNA species, M-type mRNA was transcribed most efficiently, with or without the addition of dibutyryl cyclic AMP. The 5́ region of the mouse ChAT gene was ligated to the bacterial chloramphenicol acetyltransferase gene, and the expression of chloramphenicol acetyltransferase activity was determined by transaction analysis. Cyclic AMP derivatives enhanced the reporter gene expression in both transiently and stably transfected cells. DNA deletion analysis indicated that the intron region downstream of the M-type exon is necessary for the cyclic AMP responsiveness, and that cyclic AMP derivatives increase ChAT gene transcription mainly from M-type promoter. These results suggest that a cis-acting DNA element that confers the cyclic AMP responsiveness of the ChAT gene is present in the intron downstream of the M-type exon.

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Multiple mRNA species of choline acetyltransferase from rat spinal cord

Kengaku Mineko, Misawa Hidemi, Deguchi Takeo

Molecular Brain Research 18 ( 1-2 ) 71 - 76 1993

Research paper (scientific journal), Joint Work, Accepted, ISSN 0169-328X

A cDNA library directed by a specific primer was constructed from the rat spinal cord and screened with ³²P-labeled rat choline acetyltransferase cDNA which was recently isolated in this laboratory. Sequence analysis of 29 clones indicated that there are four types of cDNA (R1-, R2-, N1- and M-types). The nucleotide sequences in these cDNAs were identical in the coding region and the first 38 bp of the 5′-noncoding region, but differed in the 5′-noncoding region upstream of -38 bp. The R1-type was identical to the cDNA previously cloned from the rat spinal cord. The M and N1-type cDNAs both had sequences homologous to that of the cDNA previously obtained from the mouse spinal cord. Polymerase chain reaction analysis confirmed the presence of these 4 types of mRNA and found another type (N2-type) of transcript. The numbers of cDNA clones isolated and the relative amounts of polymerase chain reaction products for each type of mRNA suggested that the most abundant transcript was M-type. Sequencing of the genomic clone containing the 5′-region of choline acetyltransferase mRNA revealed that these five types of mRNA species were transcribed from three different promoter regions and produced by differential splicing of the 5′-noncoding exons.

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Gene expression of mouse choline acetyltransferase: Alternative splicing and identification of a highly active promoter region

Misawa Hidemi, Ishii Kayoko, Deguchi Takeo

Journal of Biological Chemistry 267 ( 28 ) 20392 - 20399 1992.10

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

Seven types of mRNA for choline acetyltransferase that differ in the 5′-noncoding region were identified in the mouse spinal cord by cDNA cloning and polymerase chain reaction. Among these transcripts, the M-type mRNA corresponding to the previously cloned mouse cDNA was most abundant in the spinal cord of mouse. A mouse genomic DNA clone containing the 5′-region of choline acetyltransferase mRNA was isolated and sequenced. Comparison of the sequences between the cDNAs and the genomic DNA revealed that the different mRNA species were transcribed from different promoter regions and produced by differential splicing. Two murine cholinergic cell lines, NS20Y and NG108-15, were shown to express the M-type mRNA almost exclusively, and were therefore used to study transcription of M-type mRNA. Fragments of the 5′-region of choline acetyltransferase gene were ligated with chloramphenicol acetyltransferase reporter gene and introduced into cultured cells. The fragment from -2752 to +46, which contained the M-type exon, a TATA-box like element upstream of the M-type exon, and the downstream intron, induced a significant expression of CAT activity in neuronal but not in non-neuronal cell lines. This result indicates that this region of choline acetyltransferase gene contains elements that regulate neuron-specific expression of choline acetyltransferase activity. However, there was no parallel correlation between reporter gene expression in the transfected cells and intrinsic choline acetyltransferase activity in these neuronal cell lines. Possible mechanisms that would explain this observation are discussed.

κ-Opioid agonist inhibits phospholipase C, possibly via an inhibition of G-protein activity

Misawa Hidemi, Ueda Hiroshi, Satoh Masamichi

Neuroscience Letters 112 ( 2-3 ) 324 - 327 1990.05

Research paper (scientific journal), Joint Work, ISSN 0304-3940

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Functional reconstitution of purified G_i and G_o with μ-opioid receptors in guinea pig striatal membranes pretreated with micromolar concentrations of N-ethylmaleimide

Ueda Hiroshi, Misawa Hidemi, Katada Toshiaki, Ui Michio, Takagi Hiroshi, Satoh Masamichi

Journal of Neurochemistry 54 ( 3 ) 841 - 848 1990

Research paper (scientific journal), Joint Work, Accepted, ISSN 0022-3042

Functional coupling between μ-opioid receptors and GTP-binding regulatory proteins (G proteins) was investigated in reconstituted membranes of the guinea pig striatum. Selective μ-opioid agonists stimulated low-K_m GTPase in striatal membranes, in a Na⁺-dependent manner. The same μ-opioid agonist {[D-Ala²,N-Me-Phe⁴,Gly⁵-ol]-enkephaIin (DAGO)} caused no stimulation when the membranes were exposed to islet-activating protein (IAP; pertussis toxin). There was also no DAGO stimulation in preparations pretreated with a lower concentration (5 μM) of N-ethylmaleimide (NEM), which abolished the ADP-ribosylation of purified G_i( (the G protein that mediates inhibition of adenylate cyclase) and G_o (a G protein of unknown function purified from bovine brain) by IAP. In addition, as the NEM treatment caused no change in the μ-agonist binding, NEM could probably substitute for IAP in inactivating native G proteins, without exhibiting effects on the receptor binding in mem-branes. The μ-agonist stimulation of low-K_m GTPase activity in NEM-treated membranes was recovered by reconstitution with purified G_i or G_o. The μ-agonist stimulation of low-K_m GTPase was additive when G_i and G_o were simultaneously reconstituted in NEM-treated membranes in amounts of 0.5 pmol/assay, which was required for maximal recovery, in either reconstitution experiment. The present findings provide the first evidence that the μ-opioid receptor may exist in at least two different forms, separately coupled to G₁ or G_o.

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The kyotorphin (tyrosine-arginine) receptor and a selective reconstitution with purified G(i), measured with GTPase and phospholipase C assays

Ueda H., Yoshihara Y., Misawa H., Fukushima N., Katada T., Ui M., Takagi H., Satoh M.

Journal of Biological Chemistry 264 ( 7 ) 3732 - 3741 1989

Research paper (scientific journal), Joint Work, Accepted, ISSN 0021-9258

We attempted to identify the kyotorphin receptor and the post receptor mechanisms mediated by GTP-binding proteins (G-proteins), using reconstitution techniques. The specific binding of [³H]kyotorphin in rat brain membranes was composed of high affinity (K(d) = 0.34 nM) and low affinity (K(d) = 9.07 nM) binding. As the high affinity binding disappeared in the presence of guanosine 5'-O-(3-thiotriphosphate) and MgCl₂, we investigated the kyotorphin receptor-mediated changes in membrane G-protein activity by measuring low K(m) GTPase activity. Kyotorphin produced a stimulation of low K(m) GTPase, and this stimulation was antagonized by Leu-Arg, a synthetic dipeptide which showed a potent displacement of [³H]kyotorphin binding, yet in itself had no effect on the low K(m) GTPase. The kyotorphin stimulation of low K(m) GTPase was abolished by pretreating membranes with islet-activating protein, pertussis toxin, and was recovered by reconstitution with purified G-protein, G(i), but not with G(o). Similar evidence of selective coupling of kyotorphin receptor to G(i) was obtained with the phospholipase C assay. Kyotorphin-induced stimulation of phospholipase C was also abolished by islet-activating protein-treatment and recovered by reconstitution with G(i) but not with G(o). These findings indicate that specific high and low affinity kyotorphin receptors exist in the rat brain and that the kyotorphin receptor is functionally coupled to stimulation of phospholipase C, through G(i). This study provides the first evidence of a selective involvement of G(i) in the receptor-mediated activation of phospholipase C.

The specific opioid κ-agonist U-50,488H inhibits low Km GTPase

Ueda Hiroshi, Misawa Hidemi, Fukushima Nobuyuki, Takagi Hiroshi

European Journal of Pharmacology 138 ( 1 ) 129 - 132 1987.06

Research paper (scientific journal), Joint Work, Accepted, ISSN 0014-2999

DAGO, a specific opioid μ-agonists stimulated the low Km GTPase activity of membranes of the guinea-pig striatum at 1-1000 nM and this effect was antagonized by naloxone 10-100 nM. On the other hand, U-50,488H, a specific opioid κ-agonist inhibited the low Km GTPase activity of membranes of the guinea-pig cerebellum at 1-100 nM. As this effect was antagonized by MR2266 but not by naloxone, the findings suggest that opioid μ- and κ-receptor stimulation is probably linked to the stimulation and inhibition of low Km GTPase, respectively.

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Purified opioid μ-receptor is of a different molecular size than δ- and κ-receptors

Ueda Hiroshi, Harada Hitoshi, Misawa Hidemi, Nozaki Masakatsu, Takagi Hiroshi

Neuroscience Letters 75 ( 3 ) 339 - 344 1987.04

Research paper (scientific journal), Joint Work, Accepted, ISSN 0304-3940

Opioid μ-receptors were solubilized from rat brains with 0.5% Triton X-100 in the presence of 0.32 M sucrose and then purified with 6-succinyl morphine-Affi-Gel 102 column. Purified materials showed a major band with mol.wt. 58,000 Da and a minor band with mol.wt. 41,000 Da. Affinity cross linking with [³H]DAGO, a selective μ-opioid agonist, to the neural membranes and purified materials revealed that opioid μ-binding protein has a mol.wt. of 58,000 Da, a value totally different from that of δ- (mol.wt. 18,500 Da) and κ- (mol.wt. 36,600 Da) binding protein.

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